(Essentials in Ophthalmology) Gyan Prakash, Takeshi Iwata - Advances in Vision Research, Volume II - Genetic Eye Research in Asia and The Pacific (2019, Springer Singapore) PDF

Essentials in Ophthalmology
Series Editor: Arun D. Singh
Gyan Prakash
Takeshi Iwata Editors
Advances in
Vision Research,
Volume II
Genetic Eye Research in Asia and the Pacific
Series editor
Arun D. Singh
Essentials in Ophthalmology aims to promote the rapid and efficient transfer
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diagnostics, treatment options and procedures as well as patient management.
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Contributions to the series are peer reviewed by an editorial board.
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Gyan Prakash • Takeshi Iwata
Editors
Advances in Vision
Research, Volume II
Genetic Eye Research in Asia
and the Pacific
Editors
Gyan Prakash Takeshi Iwata
National Eye Institute National Institute of Sensory Organs
National Institutes of Health Tokyo Medical Center
Bethesda, MD, USA National Hospital Organization
Tokyo, Japan
ISSN 1612-3212 ISSN 2196-890X (electronic)

ISBN 978-981-13-0883-3 ISBN 978-981-13-0884-0 (eBook)
https://doi.org/10.1007/978-981-13-0884-0
Library of Congress Control Number: 2017937303
© Springer Nature Singapore Pte Ltd. 2019

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This book – Volume 2 is dedicated to the blind children of the
world and the next generation of researchers who are choosing
the field of vision research as their career to help understand
the biology of eye diseases
Foreword by Arun D. Singh
The Asian Eye Genetics Consortium (AEGC) was established in 2014 to

encourage and focus on eye disease research in Asia. With major population
distribution and growth expected to occur in Asia, it is another way to
approach public health aspects of ophthalmic diseases. As infectious, nutri-
tional, and other preventable causes of vision loss and blindness are gradually
overcome, less common entities such as genetic eye diseases become
relevant.
With advancements in DNA sequencing, dissemination of technology, and
ease of data sharing, genetic eye diseases lend themselves to exploration.
AEGC focuses on studies of heretofore neglected Asian populations. The
consortium under the able leadership of Gyan Prakash and Takeshi Iwata has
brought together contributors from around the world.
The present monograph (second of the series) represents collective work
of researchers from the Middle East, South East, and as far as New Zealand
including the developed and the developing nations. The topics covered range
from exfoliation syndrome, myopia, keratoconus, retinal dystrophies, and
retinoblastoma providing unique Asian perspective and challenges.
It is my sincere hope that readers will find as much pleasure reading this
volume as the editors and authors had in writing and editing it. If you find
“Genetic Eye Research in Asia and the Pacific” informative, it is because
(paraphrasing Isaac Newton) “we have seen further, by standing on the shoul-
ders of the giants.”
Cleveland Clinic Foundation Arun D. Singh

Cleveland, OH, USA
vii
Foreword by Peter Wiedemann
Asia as the most populated region in the world is consequently the most
affected with regard to eye diseases. For a long time, research has shown that
genetic variations are widely involved in ocular diseases. The lack of infor-
mation on the genetic basis of eye disease in the Asian population is pertinent,
and the Asian Eye Genetics Consortium (AEGC) was established in 2014 to
close this gap. Focusing and concentrating on research and patient care on
eye disease in Asia, this institution has now grown with a worldwide mem-
bership and numerous research collaborations. In a first book published in
this series “Advances in Vision Research Volume 1, Genetic Research in Asia
and the Pacific,” the editors Dr. Gyan Prakash from NIH, NEI, USA and Dr.
Takeshi Iwata from Japan tried to coordinate the findings from existing
research studies on eye disease in Asians, translate them into patient care, and
to identify areas for further research.
The doubling time for information in medicine is two and a half years. We
benefit from the rapid evolution of basic science in all fields related to biology
and medicine, and especially in relation to ophthalmology and vision. The
radical advance in the understanding of inherited eye disease has placed a
major responsibility on ophthalmologists in their future care of patients.
Genetic research findings must be translated to impact and improve patient
care at the community level.
Now, only one year after the first volume, the same editors completed this
second volume of AEGC Advances in Vision Research. This book is again a
milestone: An update on the AEGC and its scientific outreach is given in the
first part. The challenges and opportunities for genetic research in Asia and
Pacific are then described. Presenting a cross section of genetics research
from different Asian countries the editors have collated a masterful and actual
review of current knowledge and future demands. Key eye diseases are cov-
ered: retinal degenerations, retinoblastoma, glaucoma, myopia, and keratoco-
nus. Assimilating and presenting this wealth of discovery the editors are to be
congratulated for their focus on clinically relevant information. This makes
the book a major resource for researchers and clinicians.
Light and vision have always promoted human development; loss of sight
is among our most basic fears. Ninety percent of the global burden of eye
disease is shouldered by developing countries, many of them in Asia. We
believe there is a human right to sight and the editors must be congratulated
to start and build the AEGC initiative and draw up this report to enhance eye
care to people in this region. While some states of Asia have for a long time
ix
x Foreword by Peter Wiedemann
had established world-known research centers, the dispersion of genetic

research into many more countries is an encouraging signal. The unconfined
exchange of information between research institutions will benefit our
patients in the long term. The first volume of this series was dedicated to the
blind children of the world, their caregivers for the noble cause, and the vision
researchers around the world who are finding the solutions to treat the blind-
ness. By close cooperation of scientists and clinicians we will prevent com-
mon forms of blindness in the future and reduce the burden of blindness for
the benefit of our children.
International Council of Ophthalmology (ICO)

Peter Wiedemann
San Francisco, CA, USA
Leipzig University Eye Hospital
LeipzigGermany
Foreword by Gullapalli N. Rao
One of the greatest advances in eye research during the past quarter century
is in the area of genetics. This has led to greater understanding of the genetic
basis of many of the ocular conditions. The exploitation of this knowledge to
find ways of preserving eye health, preventing some forms of disease, and
modify the course of others are the exciting challenges that are subjects of
worldwide research currently. Genetics research has become an integral part
of most leading eye institutions in the world, and these have not only contrib-
uted to major breakthroughs but also to the growth of researchers through
their graduate programs.
In the recent past, the proliferation of interest and the establishment of
genetics research in Asian institutions is a welcome development. While
some parts of Asia have advanced rapidly, the dissemination of this practice
into many more countries is an encouraging sign. Better and more research
productivity is seen from these centers including greater number of people
completing their doctoral programs. This research capacity building augurs
well for the future of research in the continent.
The efforts of Drs. Gyan Prakash and Takeshi Iwata have provided a fur-
ther boost to the collaborative endeavor among different groups in the region,
which will further accelerate the research on this front. This second edition of
the Asian Eye Genetics Consortium book, Advances in Eye Research, pres-
ents a rich profile of the genetics research from different countries of Asia.
The progress in many of the countries since the last edition is impressive.
This is the result of increasing investment in research in many institutions of
the region. Entry of more younger generation scientists into eye research,
excellent research infrastructure, and increasing number of peer-reviewed
publications in respected journals are all positive signs for a vibrant research
culture. This is slowly flowing into the clinical education programs with more
clinicians engaged in research, both clinical and basic partnering with basic
scientists. Hopefully, this will percolate to medical schools in the region.
Translational research can only happen when a critical mass of clinicians get
into the research mold. Genetics research is a fertile ground that is conducive
for clinicians exposed to the culture of research.
xi
xii Foreword by Gullapalli N. Rao
The content of this volume is a rich blend of the manifestation of all the
above developments. The spectrum of topics provides an excellent profile of
genetics of eye problems in Asia and should be a “must” in all the ophthalmic
libraries of Asia.
My congratulations to Drs. Prakash and Iwata for this wonderful effort.
L. V. Prasad Eye Institute Gullapalli N. Rao

Hyderabad, India
Preface
We have come a long way from the humble beginning of Asian Eye Genetics
Consortium (AEGC) in 2014 to a very active research based collaboration in
vision sciences. The establishment of AEGC has succeeded in creating sev-
eral international collaborations to understand the biology of eye diseases,
training the next generation of research leaders, and application of scientific
knowledge in clinical care and treatment. More than one hundred and seventy
AEGC members from over twenty countries are regularly interacting to
expand our knowledge of eye diseases. In the past year, the consortium has
received numerous inquiries from other regions of the world, including Africa
and South America for similar research collaborations. At the fifth founding
anniversary of the AEGC at ARVO in Honolulu, HI, USA, on May 1st, 2018,
the members present at the meeting unanimously approved the expansion of
AEGC to Global Eye Genetics Consortium (GEGC, http://gegc.org) effective
immediately. GEGC will stand for “a research based consortium for advanc-
ing global vision sciences.”
A concerted global effort like GEGC has the potential to accelerate the
collaborative genetic eye research in generating useful new scientific data to
combat eye diseases. The GEGC is seeking to uncover new scientific oppor-
tunities and identify shared priorities to create unique international collabora-
tions in genetic eye research. The GEGC is creating opportunity to help
establish partnerships among scientists, governments, companies, and non-
government organizations from many countries to support international
research programs for eye diseases.
We have the great honor and privilege to bring out the second volume of
this work on research related to the eye diseases as we have assembled more
than one hundred leading researchers from the field of Human Genetics,
Ophthalmology, Molecular Biology, Biochemistry, Sensory Sciences, clini-
cal research, and non-governmental organizations to present the status of the
growing field of genetic eye research. Our hope is that the second volume
proves to be a major stimulus for all researchers, clinicians, clinical research-
ers, and allied eye health professionals with interest in eye diseases, and
accelerates high quality research in our understanding of eye diseases. We
were privileged to work with a group of authors who are recognized leaders
in their respective areas and who willingly gave their time to contribute to this
volume despite their busy schedules. We are forever in their debt.
This book would not have become a reality without the support, encour-
agement, and assistance of several peers and distinguished colleagues. Chieko
xiii
xiv Preface
Watanabe, Toshiro Mikami, and Selvakumar Rajendran from Springer pro-

vided the continuous support of the project. Dr. Arun Singh of Cleveland
Clinic, the series editor, provided the support for inclusion in his acclaimed
series. We are very grateful to the valuable support of the leadership, senior
management and many distinguished colleagues of National Eye Institute in
the USA and Tokyo Medical Center in Japan. Finally, and most importantly
we are truly indebted to our family members including, Dr. Savita Prakash,
Dr. Fumino Iwata, Dr. Shivaani Prakash, Gary Prakash, and many relatives
and friends for their encouragement and continued support throughout the
project. We are indebted to all those mentioned above and several others who
willingly helped us in our endeavors to put this volume together.
Bethesda, MD, USA Gyan Prakash

Tokyo, Japan Takeshi Iwata
Contents
1 Asian Eye Genetics Consortium (AEGC):

The First 5 Years............................................................................. 1
Gyan Prakash, Takeshi Iwata, Sundaram Natarajan,
and Paul N. Baird
2 A Bibliometric Analysis of AEGC Scientific Outreach............... 13
Pamela C. Sieving
3 Opportunity for Population-Based Eye Research
in Asia and the Middle East: An NGO Perspective..................... 23
Suzanne S. Gilbert, Thulasiraj Ravilla, and Leslie Louie
4 eyeGENE®: A Model for Advancing Research of Rare,
Inherited Eye Conditions Through Biobanking
and Data Sharing............................................................................ 29
R. S. Parrish, K. E. Goetz, and S. J. Tumminia
5 Inherited Ocular Disease in the New Zealand Māori:
Novel Genetic Mechanisms and Founder Effects......................... 41
Andrea L. Vincent
6 Genetics of Ocular Diseases in Malaysia...................................... 57
A. T. Liza-Sharmini and T. A. Kamalden
7 Challenges and Opportunities in Genetic Research
from the Perspective of a Tertiary Eye Care
Hospital in Bangladesh................................................................... 71
Nazmun Nahar, Mohammad Ibn Abdul Malek,
and Bipul Kumer De Sarker
8 Genetic Research on Ocular Health and Disease
in a Population from Nepal............................................................ 75
Matthew P. Johnson, Suman S. Thapa, Sandra Laston,
Kent L. Anderson, Bradford Towne, Janardan Subedi,
John Blangero, and Sarah Williams-Blangero
9 Genetic Eye Research in the Philippines....................................... 85
Patrick R. Ching, Edward Ryan A. Collantes,
Michelle D. Lingao, Patricia E. Cabrera,
and Leo D. P. Cubillan
xv
xvi Contents
10 Hereditary Eye Disease in Ningxia Hui

Autonomous Region of China........................................................ 93
Weining Rong, Huiping Li, and Xunlun Sheng
11 Ophthalmic Genetics in India: From Tentative
Beginnings in the 1980’s to Major Achievements
in the Twenty-First Century.......................................................... 113
Govindasamy Kumaramanickavel and M. J. Denton
12 Panel-Based Next-Generation Sequencing
for Inherited Retinal Degenerations in Koreans.......................... 121
Sang Jin Kim
13 Genetic Disease in Ophthalmology:
Healthcare and Research Opportunity in Bangladesh................ 131
A. H. M. Enayet Hussain and Khaleda Islam
14 Update on the Japan Eye Genetics Consortium (JEGC)............ 137
Takeshi Iwata
15 Genetics and Susceptibility of Retinal Eye
Diseases in India.............................................................................. 147
Sunita Mohan, Uthra Satagopan, Soumittra Nagasamy,
Sundaram Natarajan, and Govindasamy Kumaramanickavel
16 Unique Patient Populations in Asia
for Genetic Eye Research............................................................... 169
Himshikha Bhutani, Neel Kamal Sharma, and Akshay Anand
17 Retina Genes in Chinese................................................................. 177
Jingna He, Wai Kit Chu, Li Ma, Calvin C. P. Pang,
and Guy L. J. Chen
18 Leber Congenital Amaurosis in Asia............................................. 191
Sharola Dharmaraj, Anshuman Verma, P. Sundaresan,
and Chitra Kannabiran
19 The Genetics of Inherited Retinal Diseases in the Israeli
and Palestinian Populations: A Lesson from Populations
with High Rates of Consanguinity ................................................ 233
Mor Hanany and Dror Sharon
20 Occult Macular Dystrophy (Miyake’s Disease)............................ 249
Kazushige Tsunoda
21 Clinical Genetics of Vitelliform Macular Dystrophy:
An Asian Perspective...................................................................... 255
Sung Wook Park, Chang Ki Yoon, Dae Joong Ma,
Un Chul Park, and Hyeong Gon Yu
22 Adeno-Associated Virus (AAV)-Mediated Gene
Therapy for Leber Hereditary Optic Neuropathy....................... 273
Kunpeng Xie, Shuai Ming, Mingzhu Yang, Xuemin Jin,
and Bo Lei
Contents xvii
23 Stargardt Disease in Asian Population.......................................... 279

Xiao Liu, Yu (Yokokawa) Fujinami, Lizhu Yang,
Gavin Arno, Kaoru Fujinami
24 Retinoblastoma Genes in Chinese Studies.................................... 297
Bi Ning Zhang, Yuning Jiang, Wai Kit Chu,
Winnie W. Y. Lau, Simon T. C. Ko, Kwong Wai Choy,
Calvin C. P. Pang, Guy L. J. Chen, and Jason C. S. Yam
25 Genetics of Retinoblastoma: Basic Research
and Clinical Applications............................................................... 313
Usha Kim, K. Thirumalairaj, Aloysius Abraham,
Shanthi Radhakrishnan, B. Devarajan, V. R. Muthukkaruppan,
and A. Vanniarajan
26 Genotype-Phenotype Correlation
in Retinal Degenerations................................................................ 323
Sripriya Srivatsan, Mathavan Sinnakaruppan, Vikas Khetan,
Sundaram Natarajan, Sangeetha Srinivasan,
and Rajiv Raman
27 CYP1B1 Gene Mutation in Primary
Congenital Glaucoma..................................................................... 337
Rita S. Sitorus
28 Diabetic Retinopathy: Clinical, Genetic,
and Health Economics (An Asian Perspective)............................ 345
Siddhita Nare, Sunita Mohan, Uthra Satagopan,
Sundaram Natarajan, and Govindasamy Kumaramanickavel
29 Glaucoma Genes in East Asian Studies......................................... 357
Shi Yao Lu, Clement C. Y. Tham, Pancy O. S. Tam,
Shisong Rong, Calvin C. P. Pang, Guy L. J. Chen,
and Wai Kit Chu
30 Quantitative Trait for Glaucoma................................................... 373
Sarangapani Sripriya, Ferdina Sharmila, Suganya Kandeepan,
and Ronnie George
31 Genetics of Exfoliation Syndrome in Asians................................. 381
Prakadeeswari Gopalakrishnan, Aravind Haripriya,
Banushree Ratukondla, and Periasamy Sundaresan
32 Proteomics of Neurodegenerative Disorders of the Eye.............. 393
Kim Ramasamy, Krishnadas Ramasamy,
Dharmalingam Kuppamuthu,
and Jeya Maheshwari Jayapal
33 Genomic Approaches to Eye Diseases:
An Asian Perspective...................................................................... 403
Bharanidharan Devarajan, Ayyasamy Vanniarajan,
and Periasamy Sundaresan
xviii Contents
34 Myopia Genes in Asians................................................................. 417

Shumin Tang, Yu Meng Wang, Aziz K. W. Kam,
Tommy C. Y. Chan, Calvin C. P. Pang, Jason C. S. Yam,
and Guy L. J. Chen
35 Keratoconus Genes in Chinese...................................................... 435
Yu Meng Wang, Ka Wai Kam, Tommy C. Y. Chan, Alvin L.
Young, Vishal Jhanji, Guy L. J. Chen, and Calvin C. P. Pang
36 Granular Corneal Dystrophy Type 2: Prevalence
in South Korea, Molecular Pathogenesis,
and Therapeutic Approaches......................................................... 449
Hun Lee, Seung-il Choi, Kyung Eun Han, Tae-im Kim,
and Eung Kweon Kim
About the Editors.................................................................................... 461
Index......................................................................................................... 465
Asian Eye Genetics Consortium
(AEGC): The First 5 Years 1
Gyan Prakash, Takeshi Iwata, Sundaram Natarajan,
and Paul N. Baird
Abstract including Australia, Bangladesh, China, India,

The Asian Eye Genetics Consortium (AEGC, Indonesia, Israel, Iran, Japan, Malaysia, New
http://asianeyegenetics.org) was established in Zealand, Pakistan, the Philippines, Saudi
2014 to focus on genetic eye research in Asia, Arabia, Singapore, South Korea, Sri Lanka,
the most populated region of the world where Taiwan, Thailand, Turkey, the UAE, and the
limited data are available on genetic variation USA are now participating in the collaborative
in eye diseases. The consortium has brought a research and discussion. The current AEGC
collective thinking and ideas from the collaborations include interacting and collab-
researchers around the world who have inter- orating to develop programs to share, cata-
est in genetic eye research in the Asian region. logue, and research work to identify the
Over 100 eye researchers from eye institutions genetic aspects of eye diseases in Asia.
and hospitals from more than 20 countries
Keywords
AEGC · GEGC · NEI · TMC · NISO ·
Moorfields · AJRVO · AJFTLE · ARVO ·
APAO · IERG · Database · EyeGene · PFV
G. Prakash (*)
National Eye Institute, National Institutes of Health, 1.1 Origin of AEGC
Bethesda, MD, USA
e-mail: [email protected] The Asian Eye Genetics Consortium (AEGC)
T. Iwata traces its beginning in December 2012 when Dr.
National Institute of Sensory Organs, Tokyo Medical Deborah Carper, Former Deputy Director,
Center, National Hospital Organization, Tokyo, Japan
National Eye Institute (NEI) at National Institutes
S. Natarajan of Health (NIH) in the USA, introduced Dr. Gyan
Aditya Jyot Research in Vision and Ophthalmology,
Mumbai, India Prakash at NEI to Dr. Takeshi Iwata at National
Institute of Sensory Organs (NISO) at Tokyo
Aditya Jyot Foundation for Twinkling Little Eyes,
Mumbai, India Medical Center (TMC) and helped in developing
a research collaboration agreement between the
Aditya Jyot Eye Hospital, Mumbai, India
two institutions. Subsequent deliberations
P. N. Baird between the two organizations led to the develop-
University of Melbourne, Melbourne, Australia
© Springer Nature Singapore Pte Ltd. 2019 1

G. Prakash, T. Iwata (eds.), Advances in Vision Research, Volume II, Essentials in Ophthalmology,
https://doi.org/10.1007/978-981-13-0884-0_1
2 G. Prakash et al.
ment of a research collaboration in April 2014 training between the participating institutions are
signed by Dr. Paul Sieving, Director of NEI, and one of the key goals of the AEGC programs.
Dr. Yozo Miyake (on behalf of TMC) during the Such programs have helped in establishing new
World Ophthalmology Congress in Tokyo. The eye genetic laboratories in the Asian region and
signing of research collaboration between the training the interested researchers and clinicians
two institutions began a series of discussions in eye genetic research. In addition, the data shar-
leading to the establishment of AEGC in May ing is being planned by constructing a common
2014 at ARVO in Orlando, USA, by more than a database for AEGC to pool genotype-phenotype
dozen leading eye researchers from around the information. The AEGC members are working
world. on obtaining new research support grants and
corporate support to conduct whole genome
sequence DNA samples from the countries that
1.2 AEGC: Current Roles have limited or no funding now to support or con-
duct research locally.
The Asian Eye Genetics Consortium (AEGC) was Over 150 eye researchers from more than 20
established to focus on eye research in Asia, the countries have become AEGC members since
most populated region of the world where very the inception. The members are currently inter-
little has been explored for genetic eye diseases. acting and collaborating to develop programs to
The consortium has brought together many scien- share, catalogue, and work to identify the
tists and clinicians from around the world inter- genetic aspect of eye diseases in the Asian
ested in collaborative international eye research countries. The first AEGC book, Advances in
on the patients of Asian region. A concerted global Vision Research – Genetic Eye Research in Asia
effort like AEGC has the potential to accelerate and the Pacific – Volume I, edited by Dr. Gyan
the collaborative genetic eye research in generat- Prakash and Dr. Takeshi Iwata, was published
ing useful new scientific data to help in our under- in May 2017 by Springer (ISBN: 1612-3212).
standing of eye diseases. The AEGC is seeking to The volume contained the work of more than
uncover new scientific opportunities and identify 100 scientists from the USA, Australia, Europe,
shared priorities to create unique international and many Asian countries illustrated in 38
collaborations in genetic eye research. The AEGC chapters [1].
has created a wide opportunity to establish part-
nerships among scientists, governments, compa-
nies, and nongovernment organizations to support 1.3 AEGC Goals
research programs for understanding the biology
of eye diseases. The consortium has brought a AEGC has the following goals and plans:
collective thinking from the researchers around
the world who have interest in genetic eye research 1. Share genetic information in the Asian popu-
in the Asian region. The theme is going to be lation to rapidly isolate common disease-
expanded in other regions of the world with a goal associated variants.
of creating Global Eye Genetics Consortium 2. Establish system for accurate diagnosis and
(GEGC) soon. grouping of Asian eye diseases.
In the last 3 years, several researcher 3. Establish system for cost-effective genetic

exchanges have taken place providing and analysis.
strengthening research collaborations on genetic 4. Develop a research-oriented database to col-
eye research. The scholar’s and visitor’s pro- lect, diagnose and catalog eye diseases in
grams combined with laboratory and clinic-based Asia.
1 Asian Eye Genetics Consortium (AEGC): The First 5 Years 3
5. Support and foster collaboration among the retinal dystrophy in China. Dr. Calvin Pang from
Asian countries for the advancement of China talked about the molecular genetics of pol-
research that will provide genetic information ypoidal choroidal vasculopathy and age-related
in the Asian population. macular degeneration. Dr. Takeshi Iwata from
6. Collaborate with other international or
Japan gave the updates on expansion of AEGC,
regional organizations with similar goals. and Dr. Mridul Kumar Sarkar from Bangladesh
7. Organize and hold regional congresses and gave a talk on the genetics of congenital cataract
other educational and scientific activities to in Bangladesh. Dr. Paul Baird from Australia
promote goals of the consortium. moderated the session.
Currently, two countries – Japan and India –

have established AEGC country consortia. 1.4.2 A
EGC Session at Asia-Pacific
Japan Eye Genetics Consortium (JEGC) Academy of Ophthalmology,
started in 2011 to identify gene mutations respon- Singapore
sible for 37 hereditary retinal diseases including
hereditary optic neuropathy and hereditary glau- An AEGC session was held on March 3, 2017, at
coma in the Japanese population. JEGC has now APAO meeting in Singapore. More than eight
expanded to 30 university ophthalmology depart- speakers from several Asian countries gave
ments in Japan and collected over 2200 Japanese recent progress report of their genetic research.
DNA samples. The new genotype-phenotype Dr. Rajkumar Patil from Singapore talked about
database was launched on 2017 for collection on the prevalence of ocular genetic disorders in
phenotypic information for each gene mutation. Asia. Dr. Hyeong Gon Yu from South Korea
Additional details on the JEGC are available in gave an update on genetic characterization of
Chap. 11 submitted by Dr. Takeshi Iwata. Korean retinitis pigmentosa patients. Dr. Rita
Sitorus from Indonesia talked about the NDP
polymorphism in ROP. Dr. Paisan
1.4 pdates on AEGC Sessions
U Ruamviboonsuk from Thailand introduced the
and Meetings GWAS study for AMD and PCV patients in the
Thai populations. Dr. Liza Sharmini Ahmad
The following sessions and meetings have taken Tajudin from Malaysia informed that she had
place since the publication of AEGC’s first book, been collecting DNA from glaucoma patients in
Advances in Vision Research – Volume I. Malaysia and presented her work on the prog-
ress. Dr. Subhabrata Chakrabarti from India also
presented his genetic interaction of different loci
1.4.1 A
EGC Session at ARVO-Asia, in glaucoma patients. The last speaker, Dr.
Brisbane, Australia Periasamy Sundaresan from India, gave the mul-
tiplex cytokine analysis in the aqueous humor of
An AEGC session was held at ARVO-Asia on patients with primary angle glaucoma. The ses-
February 5, 2017, in Brisbane, Australia. Dr. sion was moderated by Drs. Paul Baird from
Zi-Bing Jin from China started the session by Australia, Sundaram Natarajan from India, and
presentation of sporadic patients with inherited Rajkumar Patil from Singapore.
4 G. Prakash et al.
1.4.3 AEGC Meeting and Poster vided an update on the AEGC book, volume I,
Presentation at ARVO, and plans for the proposed volume II. Dr. Paul
Baltimore, USA Baird provided the short- and long-term goals of
the AEGC organization and discussed various
The fourth annual AEGC meeting at ARVO was activities related to researcher exchange, webi-
held on May 9, 2017, to update the progress of nars, and other educational programs. Dr.
AEGC activities. Drs. Takeshi Iwata from Japan Natarajan from India led the discussion on
and Paul Baird from Australia moderated the research funding to support the AEGC programs.
session. Dr. Iwata provided the recent updates on The discussion was supplemented by Drs. Iwata,
the AEGC programs around the world. Dr. Prakash, and Baird and included discussion on
Fujinami from Japan and Dr. Santa Tumminia government funding from NIH-NEI, JSPS,
and Ms. Kerry Goetz from the USA discussed Australia, DBT India, and several other sources
the desired characteristics of the proposed AEGC such as industry (Macrogen, etc.), foundations
database. Dr. Gyan Prakash from the USA pro- (FEB, etc.), and others.
1.4.4 ARVO 2018 Poster sion at the ARVO meeting. Several new members
Presentation signed up and discussed collaborative research
ideas with their international counterparts. The
An invited poster presentation at ARVO 2018 in poster presentation led to several successful new
Baltimore drew significant attention and discus- alliances for research partnerships.
1.4.5 AEGC Meeting at Asia-Pacific attended the meeting. Dr. Takeshi Iwata from
Academy of Ophthalmology, Japan summarized the status of the consortium
Hong Kong, China structure, the research funding opportunities,
and the status of genotype-phenotype database.
An AEGC session was held during the APAO Dr. Graham Holder who recently moved from
meeting in Hong Kong on February 8, 2018, to Moorfields Eye Hospital to National University
discuss the progress of genetic research in the of Singapore is setting up a diagnostic lab with
AEGC member countries. A total of 30 people electroretinogram (ERG) and training of young
6 G. Prakash et al.
ophthalmologists. Dr. Muneeb Faiq from All service in the most populated province in China.
India Institute of Medical Sciences in India gave Dr. Govindasamy Kumaramanickavel from
a summary of the genetic research in India and Aditya Jyot Foundation for Twinkling Little
surrounding countries. Dr. Yudisianil E. Kamal Eyes in India provided an update on diagnostic
and Dr. Rita S. Sitorus from University of and sample collection at the first AEGC genetic
Indonesia gave a presentation on the genetic lab in Mumbai, India. Dr. Paul Baird from
polymorphism in hepatocyte growth factor and University of Melbourne in Australia summa-
cMET gene as predisposing factors for myopia. rized the talk and discussed the challenges in
Dr. Bo Lei from Henan Eye Institute in China coordinating research funding and DNA
introduced his work on inherited eye disease sequencing projects.
1.4.6 S
essions of Indian Chapter India, took a lead in inviting the scientists from
of AEGC around the world to stimulate a dialogue on
building an AEGC-dedicated lab in Mumbai.
In the last 3 years, various research meetings, AJFTLE was visited by distinguished research-
local AEGC sessions, and CMEs were conducted ers, including Prof. Shomi Bhattacharya, Head of
in India to develop internationally accepted regis- Molecular Genetics, Institute of Ophthalmology,
try for inherited eye disorders. Such programs University College, London; Prof. Sudha Iyengar,
have helped in establishing first AEGC eye Center for Clinical Investigation, Cleveland,
genetic laboratory in Mumbai and promoting eye Ohio; Dr. Takeshi Iwata, Division Director,
genetic research in India. National Institute of Sensory Organs, Japan;
AEGC meeting was held on November 12, Prof. Calvin Pang, Director of the CUHK
2014, to decide on the diseases to be addressed Ophthalmic Research Center; and Dr. Ram
through AEGC, to identify labs for genetic testing, Nagaraj, Professor, University of Colorado
and to create formats for clinical data collection Denver, USA.
and documentation. A data sharing is being planned Dr. Takeshi Iwata delivered the first Dr. A. P.
by constructing a common database for AEGC to J. Abdul Kalam Public Endowment Lecture on
pool genotype-phenotype information. The AEGC “December 14, 2016,” and was the recipient of
members are working on new research support the scroll of honor and plaque in India. Under his
grants and corporate support to whole genome guidance, Aditya Jyot Research in Vision and
sequence DNA samples from the countries that Ophthalmology (AJRVO), R&D unit of AJFTLE,
have limited or no funding support to conduct has started research on age-related macular
research locally at present. Also, AEGC session degeneration. The purpose of Dr. Takeshi Iwata’s
was organized at All India Ophthalmology Society visit to Mumbai, India was to stimulate various
(AIOS) annual meetings in 2014 and 2015. research activities related to AEGC through col-
In the last 3 years, Aditya Jyot Foundation for laboration. AJFTLE (India) and NISO (Japan)
Twinkling Little Eyes (AJFTLE) in Mumbai, have agreed for a collaborative research.
The 24th annual meeting of the Indian Eye Foundation, Madurai, Tamil Nadu, from July 28
Research Group (IERG) – ARVO India Chapter to 29, 2017. The AEGC component of AJFTLE
Meeting – was held at Aravind Medical Research research team was represented at IERG 2017.
Prof. Govindasamy Kumaramanickavel, tion in genetic research. He delivered an oration

Research Director at AJRVO, was conferred the on “A Journey in Darkness: Pathway to
prestigious D. Balasubramanian Award by Dr. Ophthalmic Genes” at the conference that
P. Namperumalsamy, Chairman Emeritus of included the establishment of AEGC chapter in
Aravind Eye Hospital, Madurai, for his contribu- India.
8 G. Prakash et al.
AJFTLE participated in the poster and presen-

tation session at IERG. Dr. S. Rajkumar, Lab
head, AJRVO, presented a poster on
“Identification of Potential Candidate Genes of
Persistent Fetal Vasculature (PFV), A Congenital
Ocular Anomaly Using Combinatorial Whole
Exome Sequencing and Gene Prioritization
Tools.” Ms. Siddhita Nare, Junior Research
Fellow, AJRVO, presented free paper on
“Screening of Genetic Variations in Different
Ocular Disorder Using Targeted Exome
Sequencing (TES) Panel.”
In AIOS 2018 Conference, Aditya Jyot con-
ducted Instructional Course on Ophthalmic
Genetics under the leadership of Prof.
Govindasamy Kumaramanickavel.
1.5 he First AEGC-Dedicated

T
Lab in India
In the year 2016, the AJFTLE in India established

a genetic unit – AJRVO – at Thane, Maharashtra,
headed by Prof. S. Natarajan and Prof. AJRVO Lab in Thane, India
Govindasamy Kumaramanickavel. This genetic The major focus of the AEGC – genetic unit of
laboratory, under the AJFTLE, is recognized by AJFTLE – is to study the genetic and epigenetic
the Government of India – Department of Science mechanisms of diabetic retinopathy, age-related
and Industrial Research – as small industrial macular degeneration, glaucoma, cataract, myo-
research organization. The lab was inaugurated pia, and inherited eye diseases through genomics
by Dr. Takeshi Iwata. and proteomics tools. The genetic laboratory is
equipped with all basic amenities and instruments • Ability to look at different aspects of disease
needed to perform in-house genetic analysis. such as country-level mutations.
AJFTLE is in the process of making a biobank • Identify rare disease/subtype/clinical feature
for all available clinical specimens obtained and build number of samples.
through surgical procedures during intervention. • Generation of large data sets through more
The samples including peripheral blood for DNA samples.
and RNA isolation, plasma, serum, aqueous, and • Ask a broader range of questions that might
vitreous for biomarker analysis and lens aspirate, already be possible.
trabecular meshwork, epiretinal membrane and • Opportunity to be involved with other groups
inner limiting membrane for epigenetic, pro- in the region.
teomic, and microRNA analysis have been col- • Apply for funding and other opportunities.
lected and stored at −80 °C. • Write papers with bigger impact.
To facilitate the best design of a database, a

1.6 AEGC Database series of meetings were held with experts from
around the world in the last 3 years to garner
Most of our knowledge that underpins retinal dis- experience from different data platforms that
ease has come from studies based in Europe and were being used. These included meetings with
North America; this is particularly evident in the Dr. Takeshi Iwata from Japan providing the
composition of groups involved in large interna- example of the Japanese exome database, Dr.
tional consortia. As a result, most of the molecu- Andrew Webster from Moorfields Eye Hospital
lar information that underpins retinal disease and detailing how data is handled in London, and Ms.
the genes involved in its etiology are derived Kerry Goetz and Dr. Santa Tumminia at ARVO
from these populations. Africa, South America, and at NEI, USA, to assess “EyeGene.” While
and Asia are underrepresented in this global these databases were exceptional and had their
knowledge base. However, Asia has a third of the own merits, it was clear that the AEGC would
global population, and as such information need to devise its own database setup. The main
gained from the study of retinal diseases in this considerations to take into account were as
region will greatly advance our understanding of follows:
its molecular mechanisms and the pathobiology
of eye diseases. • Who would undertake the data management?
To accomplish the goals of the AEGC, there is • What kind of database was needed?
a need for a comprehensive database that must • Would the database be at one site or mirrored
allow for all interested groups across the region at several sites and if so, where?
to be placed on an equal platform with the empha- • What kind of data should be collected?
sis being on high-quality data and sharing of this • What data would and could be shared?
data at a de-identified level. Therefore, the suc- • How to ensure reproducibility of the collected
cess of the AEGC (and future GEGC) is under- data.
pinned by the type of database that can be used to • How would harmonization/data cleaning/
share clinical and genetic data. quality control data be established?
There are many unique advantages of having a • Who would provide database/data query sup-
region-based database. These include: port to users?
• Better identification of mutations and genes The groups approached around the world all
involved in a retinal disease. spoke of an aspiration of having databases that
• Identify mutations that are novel and specific “could talk to each other,” and this underlying
to the Asian population. thread will be incorporated into a design for a
• Aid in the diagnosis of patients. database.
10 G. Prakash et al.
1.6.1 AEGC Infrastructure be shipped to a site that could undertake the

genetic studies. Through discussions around the
To underpin the database aspirations, it was clear region, it was noted that not all countries such as
that Internet access and digitalization would be India and China would be able to export tissue
essential to collect data from not only remote or overseas for such analysis. To overcome this hur-
rural sites but also major urban sites. Therefore, dle, Drs. Iwata and Baird indicated that there
establishing country leaders to identify locally could be a two-tier system where countries with
interested groups and facilitate the identification the capacity to undertake genomic sequencing
of patients in their country would be a powerful would be best placed to perform this in their
resource. Such undertakings of building nodes of home country, whereas countries with no such
local expertise have now begun with the estab- facilities could send these tissues overseas for
lishment of the AJFTLE at Thane, Maharashtra, analysis.
India, under the guidance of Drs. S. Natarajan
and G. Kumaramanickavel.
1.6.4 Outcomes
1.6.2 Clinical Data The outcomes of the database work are to:
Given that medical clinics are extremely busy and • Provide a catalogue of mutations in retinal
that medical staff are time constrained, there was disease across the region.
a consideration as what data should be collected. • Provide a diagnostic molecular tag for a
While data would need to be collected on an indi- patient to aid in diagnosis.
vidual for diagnosis and verification of the pheno- • Provide information to identify missing
type by an independent expert, it was also essential genetic information that could explain retinal
that the collecting clinician should not be overbur- disease.
dened with multiple fields of data entry that would • Provide insights into molecular mechanisms
need to be completed. Critically, it was observed of disease.
that many of the fields necessary for the database • Provide a mechanism to map mutations across
are part of a regular clinical treatment, and so the the region and within countries to look for
requirement would be to ensure that images or main mutational effects.
clinical data were collected at an appropriate • Provide opportunities to develop therapies for
image resolution for assessment and that the clini- different types of retinal disease.
cal data collected met international standards for
diagnostic characterization of the retinal disease.
Therefore, one of the goals of AEGC is to build 1.6.5 The Database Today
this level of excellence across the region.
Initially, it is being planned that the AEGC data-
base will be housed at the Tokyo Medical Center
1.6.3 Genetic Data under Dr. Iwata’s support. Each group will have
access to their own data but not to other groups.
While clinical data is readily available, the The administrator in Japan will have access to the
genetic data related to a patient may not be avail- data that will be de-identified. Each group will
able at the local level. This may come about have a unique identifier for their patient where
through lack of resources, expertise, manpower, they will be able to reidentify the patient and
etc. AEGC aims to facilitate the elucidation of hence provide an unequivocal diagnosis back to
molecular changes that occur in retinal diseases their patient. No identifying information will be
and feed this information back to the treating transmitted between sites. Local ethics will be
doctor. Genetic data to be assessed would need to obtained for all material.
1.6.6 Future Work we will begin to expand this concept to other

areas including Africa and South America. The
Initial trials will need to be conducted to ensure hope is that a unified system to benefit retinal
that the transport of samples, processing, and patients can be established aiding in developing
transmission of genetic data can be established. new treatments for this group of diseases.
Assessment of the mutation will need to be
undertaken either in Japan or in a host country
with capacity to undertake this analysis. In all Reference
cases the genetic data will need to be married to
the clinical data. 1. Prakash G, Iwata T. Genetic eye research in Asia and
the pacific, advances in vision research, volume 1,
It is envisaged that once the database is in in essentials in ophthalmology series (Editor: Singh
place, then samples and/or data will begin to be AD). Springer; 2017. ISBN:978-4-431-56509-3.
uploaded rapidly. Once the process is established,
A Bibliometric Analysis of AEGC
Scientific Outreach 2
Pamela C. Sieving
Abstract research, but Journal Impact Factors™ and num-

The Asian Eye Genetics Consortium (AEGC) bers of citations are not the primary focus.
encourages and facilitates research and clini- The earliest attempts to analyze a body of lit-
cal collaborations to understand the genetic erature include work by Cole and Eales, who
etiology and physiology of ophthalmic dis- looked at the literature of comparative anatomy
eases and work toward clinical care and treat- from 1540 to 1860 [1], noting the first “outcrop
ment. Bibliometric analysis of ophthalmic of publications” between 1540 and 1575; they
genetic publications in Asian countries pro- identified centers of scholarship such as Padua,
vides a tool to better understand research fretted over apparent periods of lack of interest or
strengths and challenges. progress in anatomical studies, and invented
combined “working years” as a metric: they
Keywords count 2100 working years, for the active publica-
Ophthalmology · Eye diseases · Bibliometrics tion lives of prominent anatomists between 1650
· Asia · Research collaboration · Research and 1700. In 1962, Raisig published a paper on
funding · Genetics “statistical bibliography in the health sciences,”
[2] identifying some of the key principles of bib-
liometric analysis several years before Pritchard
first used the term “bibliometrics” [3].
2.1 Introduction Earlier bibliometric studies in ophthalmology
and vision have sometimes attempted to look at
Bibliometric research, sometimes also termed the entire field (Guerin [4], Mandal [5], Ohba [6],
“scientometrics,” analyzes the metadata of publi- Schulz [7]) but more typically focused on a spe-
cations to identify significant elements of the cific country, such as Davis, for Australia [8];
research reported beyond the scientific content: Kumaragurupari, for India [9]; Pahor, for
who is publishing, where is the research done, Slovenia [10]; Rahman, for Japan [11]; Risal, for
how is it funded, where is it published, and what Nepal [12]; Wolfram, for Germany [13]; and
are the trends and challenges, the successes, and Yohendran [14], for the indigenous population of
the gaps. Citation impact is one element of this Australia, or region, such as Ragghianti [15], for
five South American countries [ref], and Sweileh
[16], for Arab-focused ophthalmology. Others
P. C. Sieving (*) have analyzed the literature of specific diseases
National Eye Institute, National Institutes of Health, or ophthalmic knowledge: Boudry [17], for eye
Bethesda, MD, USA

https://doi.org/10.1007/978-981-13-0884-0_2
14 P. C. Sieving
neoplasms; Cardona [18], for contact lens; Dai the JIF categories), and both country and institu-
[19], for traumatic optic neuropathy; Dong [20], tional elements of the authors’ affiliations.
for glaucoma; Fan [21], for cataract and corneal Citation counts, based on reference lists in all
refractive surgery; Mimouni [22], who compared journals assigned JIFs, and page views since
pediatric- and adult-focused ophthalmic research; 2013 identify and quantify one form of impact of
Pekel [23], for corneal transplantation; Royle journals, articles, authors, institutions, granting
[24], for macular disease in the United Kingdom; agencies, and specific grants.
Şenel [25], for Behçet disease; Wang [26], for A search was constructed to identify articles
ocular ischemic syndrome; and Wen [27], for meeting four criteria: ophthalmology and vision-
cataract research. related publications; by at least one author from
This chapter reports the findings of a study of an Asian country, not limited to AEGC-
ophthalmic genetic publications by researchers in participating members; with genetic content; and
Asian countries, using the World Health published from 2000 through 2017. While biblio-
Organization’s designation of countries in the graphic indices such as MEDLINE® provide sub-
Southeast Asia, the Western Pacific, and the ject indexing using a controlled taxonomy, this is
Eastern Mediterranean regions. In addition to the not available in WoS; instead, lists of terms likely
standard bibliometric analyses, the results may to be used in the titles of target articles were con-
help identify researchers with access to unique structed, based on terminology in the MEDLINE®
genetic populations, highlight opportunities for MeSH® taxonomy for eyes and eye diseases and
private and public funders to identify possible for genetics and subject “hedges” developed by
research facilities while helping researchers iden- the author. WoS automatically invokes lemmati-
tify potential funders, and encourage collabora- zation, expanding the search to include variations
tions beyond those already recognized. in spelling and usage (e.g., “oedema” for
“edema,” “eyes” as well as “eye”). Two parallel
searches were run: the first used a two-part struc-
2.2 Methods ture of eye and vision terminology + genetic ter-
minology; the second used genetic terminology +
The Web of Science™ (WoS) database (Clarivate the WoS classification of journals in the field of
Analytics, Philadelphia, PA, USA) was used both ophthalmology. The two searches were combined
to identify research publications authored by to eliminate duplicate citations, and the addi-
researchers from Asian countries and to conduct tional element of country-level author affiliations
bibliometric analysis on the publications. WoS added to the result. Only articles published from
provides citation-level access to research publi- 2000 through 2017 were included in the final data
cations in journals judged to be significant in set. Review of the data revealed citations from
their subject areas, as indicated by the assign- unrelated fields such as astronomy and oceanog-
ment of Journal Impact Factor™ (JIF) rankings. raphy in which vocabulary overlapped; those
Citations include author institutional affiliations citations were manually removed.
for all authors; this information is not consis-
tently provided by the more generally available
MEDLINE®/PubMed® database for most of the 2.3 Results
publication years studied, hence the choice of
WoS. WoS also consistently includes data for both Number of Publications The final data set for
funding agencies and grant numbers. In addition, analysis included 24,715 citations: 21,316 arti-
WoS provides point-and-click analytics for almost cles, 1559 meeting abstracts, 992 reviews, 615
every data field which can be captured from the letters, and 464 proceedings papers. 24,636 of the
individual article and its bibliographic citation: citations were to publications in English
language of publication, document type, broad (99.684%); other languages included Chinese,
subject groupings for journals (corresponding to
2 A Bibliometric Analysis of AEGC Scientific Outreach 15
Japanese, German, Korean, French, Portuguese, Table 2.1 Publications by AEGC member countries,
Romanian, Spanish, and Turkish. 2000–2017
Australia 1253
Bangladesh 7
Year of Publication Publications increased PR China 4655
Egypt 15
steadily by year, from 706 in 2000 to 2419 in
India 1046
2017. As a check on accuracy, a basic search was
Indonesia 22
run in MEDLINE®/PubMed® for citations Israel 43
indexed using the most relevant MeSH® taxon- Iran 24
omy (“eye diseases/ge” or “eye diseases, heredi- Japan 2194
tary”) for 2000 and 2016, the last year for which South Korea 960
subject indexing is complete; there were 410 Malaysia 90
articles in 2000 and 1091 in 2016, a similar rate New Zealand 134
of increase, providing additional confidence that Philippines 8
this dramatic increase in number of publications Singapore 365
Saudi Arabia 69
is genuine.
Switzerland 97
Taiwan 402
Thailand 68
Country Twenty-one countries are formal Turkey 50
members of the AEGC; their contributions ranged United Arab Emirates 5
from 4655 papers (People’s Republic of China) United States 1936
to (Cambodia). One hundred seven countries
were represented by at least one author on these
Table 2.2 Publications by Non-AEGC Asian countries
papers. The highest number of contributions by
authors in non-AEGC countries was those from Cambodia 2
Iraq 4
England (492), Germany (367), the Netherlands
Jordan 4
(249), Canada (208), France (180), and Pakistan Kuwait 2
(174). Eighteen countries contributed to only a Mongolia 3
single paper and 11 to two papers; in total, 44 Myanmar 1
countries contributed to five or fewer (Tables 2.1, Nepal 15
2.2, and 2.3). Pakistan 174
Papua New Guinea 2
Qatar 9
Journals Web of Science™ sorts journals into Sri Lanka 6
Vietnam 15
broad categories which are then used to compare
journals for which Journal Impact Factors have
been computed; the “Ophthalmology” category Experimental Ophthalmology (342, 1.384%),
for 2016 publications includes 59 journals Japanese Journal of Ophthalmology (323,
(Journal Citation Reports™, 2016). Of the 1.307%), Cornea (286, 1.158%), and American
24,715 papers in this data set, 9812 (39.713%) Journal of Ophthalmology (280, 1.133%)
were published in journals in this category: (Table 2.4).
Investigative Ophthalmology & Visual Science
(published 2440, 9.876%), Molecular Vision The remaining 14,903 articles were published in
(1175, 4.756%), Experimental Eye Research 150 non-ophthalmology categories; leading in
(477, 1.931%), International Journal of numbers of ophthalmic genetics published were
Ophthalmology (373, 1.931%), British Journal of these categories: biochemistry/molecular biology
Ophthalmology (367, 1.485%), Current Eye (3064, 12.401%), genetics/heredity (2002,
Research (348, 1.409%), Clinical and 8.103%), neurosciences (1395, 5.646%), cell
16 P. C. Sieving
Table 2.3 Publications by countries collaborating with Table 2.5 Non-vision journals
AEGC-based researchers (top 20 by publications)
PLoS One 713
England 492 Sci Rep 297
Germany 367 Biochem Biophys Res Commun 242
Netherlands 249 J Biol Chem 166
Canada 208 Am J Hum Genet 141
France 180 Mol Med Rep 134
Italy 126 Chin Med J 127
Spain 87 Am J Med Genet A 112
Scotland 80 Hum Mol Genet 111
Sweden 71 J Hum Genet 103
Belgium 61 PNAS 100
Denmark 60 Int J Clin Exp Pathol 93
Brazil 59 J Invest Dermatol 92
Austria 37 Oncotarget 88
Norway 37 Int Med 85
Poland 37 Gene 84
Northern Ireland 33 Int J Clin Exp Med 80
Wales 28 Neurosci Lett 77
Finland 25 J Dermatol 74
Greece 25 Am Pathol 72
Argentina 24
internal (620, 2.509%), and endocrinology/

Table 2.4 Vision journals metabolism (613, 2.481%) (Table 2.5).
IOVS 2440 The top 100 journals in number of these
Mol Vision 1175 papers published include 42 ophthalmology and
Exp Eye Res 477
vision journal; this accords with the c40% of arti-
Int J Ophthalmol 373
cles published in journals in the Ophthalmology
BJO 367
Curr Eye Res 348
WoS subject category. Three of the 42 are open
Clin Exp Ophthalmol 342 access titles, included in the Directory of Open
Jpn J Ophthalmol 323 Access Journals, (BMC Ophthalmology, Indian
Cornea 286 Journal of Ophthalmology, and International
Am J Ophthalmol 280 Journal of Ophthalmology). WoS identifies 9933
Graefes Arch Clin Exp Ophthalmol 276 of the articles as being available via some form of
Ophthalmology 246 open access, 40.2%; approximately 24.4% of
Ind J Ophthalmol 211 articles indexed by MEDLINE® are identified as
Eye 203
being free full text.
Arch Ophthalmol/JAMA
Ophthalmology 168
Ophthalmic Res 153
Authors 56,541 individual authors are credited
Ophthalmic Genet 142 on these papers; 500 of them authored at least 34
BMC Ophthalmology 114 papers each. In 2017, a total of 12,001 individual
Retina 113 authors were credited on the 2419 papers; in
2000, 2882 authors were credited on 706 papers.
biology (1288, 5.213%), medicine research, Wang Ying (Capital Medical University,
experimental (1279, 5.177%), multidisciplinary Beijing) authored 245 papers; David A. Mackey
(1220, 4.938%), oncology (879, 3.558%), clini- (University of Western Australia, Perth), 244; Liu
cal neurology (683, 2.764%), medicine, general Yu (Sun Yat-sen University, Guangzhou), 207;
Jamie E. Craig (Flinders University, Adelaide), Trust (8), Sigrid Juselius Foundation (9),
205; Shigeru Kinoshita (Kyoto Prefectural Muscular Dystrophy Association (9), Uehara
University of Medicine), 204, were the others Foundation (10), and Moorfields Eye Charity
with more than 200 publications in this set. (10).
Funding Agencies 933 funding agencies were Grants When the information is provided in the
acknowledged in these publications; WoS notes, acknowledgment sections of publications, WoS
however, that 12,135 (49.116%) of these papers includes grant numbers in the metadata for each
did not contain data for this field. This is likely citation, as does MEDLINE®; this directly search-
due to a change in practice by authors and in part able field in the database record allows funders
due to the increased pressure from funders to see and other researchers to easily identify publica-
acknowledgments in papers resulting from their tions resulting from funded research. A single
funded research. Many funders now also require grant from the National Basic Research Program
deposit of the manuscripts or the published of China 973 program of the papers in this data set;
papers in a freely accessible archive (such as funding from an internal mechanism at the
PubMed Central®, maintained by the US Chongqing Key Laboratory of Ophthalmology
National Institutes of Health and indexed in funded 77 papers; one from the Key Project of
PubMed®) or on the authors’ institutional or per- Natural Science Foundation (China) funded 57;
sonal Web site. The reader is referred to another, from the National Health and Medical
ROARMAP for a current compilation of funder Research Council (NHMRC) and Centre for
policies in this area (http://roarmap.eprints.org). Clinical Research Excellence Grant, Canberra,
Note: variations in style of authors’ attribution of was acknowledged in 50 papers. An NIH National
funding necessarily mean some are overlooked, Eye Institute grant funded 41 papers.
and only those funding at least eight publications
are included here; listed here are some of pro-
grams most active in supporting this research and Organizations Nearly 12,000 institutions are
the number of papers acknowledging each: identified in the author address information in
this data set. The University of Melbourne led
National Natural Science Foundation of China, with 884 publications, followed by the University
2875 (of those funding at least 8). of Sydney (781), Sun Yat-sen University/
National Eye Institute, 804. Zhongshan Ophthalmic Center (764), the
NIH and individual institutes (including NIDDK, University of Tokyo (628), the University of
NINDS, NCRR, NIGMS, NHLBI, NICHD, California system (566), Shanghai Jiao Tong
NIDCD, NIAMS, NHGRI, NIA, NIAID, and University (557), Kyoto University (554), the
NIGMS), 1325. National University of Singapore (538), the
Research to Prevent Blindness, 378. University of London (528), the Chinese
Ministry of Education, Culture, Sports, Science Academy of Sciences (522), the Centre for Eye
and Technology of Japan, 954. Research Australia (508), the University of
National Basic Research Program of China, 501. Western Australia (508), and Osaka University
Ophthalmic Research Institute of Australia, 130. (506). US National Eye Institute researchers
Australian Research Council, 285. authored 266 papers, and researchers at other
Wellcome Trust, 130. NIH institutes such as NCI 63 and NHGRI 62
Foundation Fighting Blindness, 74. contributed as well, for a total of 491 papers. A
Fight for Sight, 59. sampling of the other organizations represented
include Harvard University/Massachusetts Eye
Many small funds and charities receive credit: and Ear Infirmary 415; LV Prasad Eye Institute
March of Dimes (8), Maurice and Phyllis Paykel 312; Royal Victorian Eye and Ear Hospital,
18 P. C. Sieving
Table 2.6 Institutional affiliations/publications Highly Cited Papers The most cited paper in
University of Melbourne 884 this data set, by Zuchner et al. [28], was cited
University of Sydney 781 more than 800 times at the time of data collec-
Sun Yat-sen University/Zhongshan tion. Notably, the WoS record lacks funding
Ophthalmic Center 764 information, as many in the early years of the set
University of Tokyo 628 do; the article itself acknowledges funding from
University of California system 566 the Deutsche Forschungsgemeinschaft, NIH, the
Shanghai Jiao Tong University 557
University of Antwerp, and the Belgian Federal
Kyoto University 554
National University of Singapore 538
Office for Scientific, Technological and Cultural
University of London 528 Affairs. Its 11 authors have institutional affilia-
Chinese Academy of Sciences 522 tions, all universities or research institutions, in 7
Centre for Eye Research Australia 508 counties. The second most cited, by Heier et al.
University of Western Australia 508 [29], had been cited 683 times; it includes 14
Osaka University 506 authors, from 6 counties, both universities and
Harvard University/Massachusetts industries, and cites a total of 22 funding sources.
Eye and Ear Infirmary 415 More than 450 of the papers in this data set have
Royal Victorian Eye and Ear Hospital, 412
been cited at least 100 times. Forty-eight are
Melbourne
LV Prasad Eye Institute 312
ranked as “highly cited” by WoS.
US National Eye Institute 266
Moorfields Eye Hospital, London 181
Aravind Eye Hospital/Aravind Research 173 2.4 Discussion
Foundation, Madurai
Hong Kong Polytechnic University 105
The results section has provided the reader with
King Khaled Eye Specialist Hospital, Riyadh 34
both specific and indicative information about the
University of Sao Paulo 24
range of publications in the broad area of oph-
thalmic genetics for the most recent 17 years with
Melbourne, 412; Moorfields Eye Hospital, at least one author from an Asian country. That
London, 181; the University of Michigan 277; researcher may be an established senior clinician,
Aravind Eye Hospital/Aravind Research a graduate student, a recently returned fellow, or
Foundation, Madurai, 173; the Hong Kong postdoc who trained in an active lab in another
Polytechnic University 105; McGill University, hemisphere.
Montreal, 60; the University of Tübingen 53; the One use for this data is to compare countries,
Karolinska Institute, Stockholm, 48; the for example, a developing and a developed coun-
University of Zürich 42; Queen’s University try. Looking at India:
Belfast 38; the University of Helsinki 38; King Indian authors are identified on 2242 of the
Khaled Eye Specialist Hospital, Riyadh, 34; the 24,715 records for publications (0.9%). 1101 of
University of Paris 62; and the University of Sao those papers were published in journals classified
Paulo 24. This is a sampling rather than a system- as “ophthalmology” by Web of Science™,
atic listing, meant to indicate the geographic and 49.240%, a higher than average choice of oph-
institutional breadth of research efforts in ophthalmology and vision journals to disseminate
thalmic genetics (Table 2.6). their research. Citation studies for this data set
include an H-index of 57; average citations per
publication is 10.55; the citations were cited by a
Citations More than 80 of the 2017 publications total of 17,328 other articles (excluding self-
have already received at least five citations. Of citations, 16,410); the publications average 1245
those, only six had three or fewer authors. By citations per year. Yearly productivity: the num-
contrast, 17 of the most cited articles from 2000 ber of publications grew from 35 in 2000 to
had 3 or fewer authors. 242 in 2016 and 239 in 2017, with a jump from
164 in 2012 to 229 in 2015. Journals differed The most cited paper including Indian authors
from the pattern for the entire data set: 185 of the is by Fritsche et al. on seven new loci associated
papers appeared in the Indian Journal of with age-related macular degeneration (361 cita-
Ophthalmology (8.274%), 143 in Molecular tions). This paper is perhaps typical of high-
Vision (6.395%), 131 in IOVS (5.859%), 48 in powered genetic research: 136 authors from a
Eye (2.141%), and 47 in British Journal of total of 14 countries, funding by major US and
Ophthalmology (2.096%); only 3 of the top 20 British government agencies. The second most
journals were not classified as “ophthalmology” cited paper, at 316 cites, is by Bork and 16 addi-
titles. Non-vision journals in which Indian tional authors’ 2001 paper on Usher syndrome;
authors published included PLoS One, 52 articles five countries are represented.
(2.319%), followed by the American Journal of Equivalent data for Japan:
Human Genetics, 19 papers (0.847%), and Japanese authors are associated with 7728
Journal of Genetics, 17 (0.758%). The Indian papers in this data set, 31.268%. Publications per
Journal of Ophthalmology was conferred its first year have varied from 388 (2011) to 492 (2016),
Journal Impact Factor™ for the 2010 publication not varying more than 10% between successive
year; this additional quality designation may years. Citation studies: this set of 7728 papers
have persuaded Indian authors to publish in this has an H-index value of 133, with an average of
journal preferentially. Indian authors collabo- 19.55 citations to each publication. A total of
rated with researchers from the following coun- 98,301 papers have cited the 7728 publications;
tries: the USA (336, 15.027%), England (97, excluding self-citations, the total is 94,246; 7952
4.338%), Australia (82, 3.667%), People’s is the average number of citations per year.
Republic of China (70, 3.131%), the Netherlands Ophthalmic journals in which Japanese oph-
(49, 2.191%), Japan (36, 1.610%), Singapore thalmic researchers choose to publish are similar
(34, 1.521%); and researchers from 18 countries to those of the entire population in this data set,
(Canada, France, Italy, Saudi Arabia, Brazil, except that the Japanese Journal of
Belgium, Switzerland, Turkey, South Korea, Ophthalmology ranks higher: IOVS 953
Spain, Israel, Argentina, Malaysia, Pakistan, (12.332%), Japanese Journal of Ophthalmology
Northern Ireland, Hungary, South Africa, and 287 (3.714%), Experimental Eye Research 178
Sweden) collaborated with Indian researchers on (2.303%), Molecular Vision 175 (2.264),
at least ten publications. Fifty-five additional American Journal of Ophthalmology 154
countries contributed with 1–9 publications. (1.993%), and British Journal of Ophthalmology
Sixteen AEGC member countries were among 121 (1.566%). Non-ophthalmic journals: used
those collaborating on these publications. PLoS One 162 (2.096%), Biochemical and
Approximately 1000 funding agencies were Biophysical Research Communications 108
credited in these papers. The Indian Department (1.398%), Internal Medicine 83 (1.074%),
of Biotechnology funded 201 (8.989%), followed Journal of Biological Chemistry 78 (1.009%),
by the Indian Council of Scientific and Industrial Scientific Reports 68 (0.880%), and Journal of
Research (138) and Indian Council of Medical Human Genetics 59 (0.763%). The countries of
Research (78). The US National Eye Institute the collaborating authors are the USA (1162,
(58) and other National Institutes of Health insti- 15.036%), England (239, 3.093%), People’s
tutes and centers (56) provided 5.098% of the Republic of China (223, 2.886%), Germany (206,
funding for the research presented in these publi- 2.666%), France (133, 1.721%), Canada (102,
cations. Private funders included the Hyderabad 1.320%), South Korea (94, 1.216%), Australia
Eye Research Foundation (49), Research to (88, 1.139%), the Netherlands (84, 1.087%), and
Prevent Blindness (33), the Foundation Fighting Italy (79, 1.022%). Funding was led by the
Blindness (16), Alcon (15), the Champalimaud Ministry of Education, Culture, Sports, Science
Foundation (15), and the Wellcome Trust (8). and Technology of Japan, which funded 785 of
20 P. C. Sieving
these papers, 10.158%; the Japan Society for the age, but each provides access to a discrete set of
Promotion of Science, 237 (3.067%); the US journals; in addition, the metadata captured or
National Eye Institute 211 (2.731%); the US added during the indexing process varies between
National Institutes of Health other than NEI, 289 the files and may also vary over time. The choice
(3.734%), a total percentage similar to but one of Web of Science™ for this work was made
point higher than that for Indian publications; because of the more complete author affiliation
and the Takeda Science Foundation, 130, 1.682%. and funding information in this file, as well as
Other leading funders were Japanese governmen- point-and-click analytics for the fields of each
tal agencies and foundations. The most cited database record and citation information and
paper including Japanese authors is that of analysis. Nevertheless, the lack of subject index-
Zuchner and the second that of Heier, both ing means the research must rely on either jour-
already discussed. The most cited paper with a nal classification or natural language keyword
first author at a Japanese institution is that by searches. The former is incomplete, as this study
Yoshida [30]; it may be of interest to consider demonstrates that approximately 40% of ophthal-
that this paper was published in the first volume mic genetic articles identified with keywords
of a new journal; thus the paper was submitted were published in ophthalmology-classified jour-
without consideration of the Journal Impact nals. Natural language searching relies on diver-
Factor™ associated with the journal, yet it has gent and creative association of important
been cited nearly 500 times. concepts in a field with terms capturing those
concepts and yet involves creative discrimination
to eliminate terms that are associated with a dif-
2.5 Conclusions ferent domain.
Metadata for fields such as author affiliations
Bibliometric analysis of publications focused on and funding agencies are taken from the pub-
a knowledge domain or geographic region pro- lished articles and are subject to the vagaries of
vides a “where we have been/where we are” the authors. For example, funding by the US
snapshot of scholarly communication and scien- National Eye Institute is styled variously as NEI
tific achievement. Findings here confirm that NIH HHS, National Eye Institute, NEI, National
ophthalmic genetics is an increasingly active and Eye Institute National Institutes of Health, NIH
productive research front, with a rapidly increas- NEI, National Eye Institute NIH, NEI NIH, and
ing pool of researchers contributing to knowl- National Eye Institute NEI; the Ministry of
edge of ophthalmic genetics. The impact of the Education Culture Sports Science and Technology
increasing numbers and variety of funding bodies of Japan is also the Ministry of Education Culture
is leveraged by international collaborations. Sports Science and Technology Japan, Japanese
Opportunities to disseminate research findings Ministry of Education Culture Sports Science
extend beyond journals of primary interest to the and Technology and presumably the Ministry of
ophthalmic community, again providing incen- Education Culture Sports Science and
tives for collaborations outside the vision research Technology, with no designation of country;
community. “Ministry of Education Culture Sports Science
and Technology MEXT” introduces the acronym
Limitations MEXT, possibly sometimes misspelled as
As with all research, the validity of bibliometric MEST. Thus identifying a single funder to assign
studies varies with the completeness and accu- credit is an uncertain task. Similarly, while the
racy of the data on which they are based. There is most common variants on National Eye Institute
no perfectly complete bibliographic database of as a funding agency yielded 804 citations from
biomedical publications. MEDLINE®/PubMed® recognized variants, searching for the string EY*
and Web of Science™ have overlapping cover- (the truncated version of the common two-letter
identifier on NEI grant numbers) and a small set Brazil, Chile, Paraguay and Uruguay (1995–2004).
Arq Bras Oftalmol. 2006;69(5):719–23.
of variations on the National Eye Institute as the 16. Sweileh WM, Al-Jabi SW, Shanti YI, Sawalha AF,
funding agency retrieved 1355 citations in this Zyoud SH. Contribution of Arab researchers to oph-
data set. thalmology: a bibliometric and comparative analysis.
Springerplus. 2015;4:42–8.
17. Boudry C, Mouriaux F. Eye neoplasm research:

Conflict of Interest The author declares that she has no
a bibliometric analysis from 1966 to 2012. Eur
conflict of interest.
J Ophthalmol. 2015;25(4):357–65.
18. Cardona G, Sanz JP. Citation parameters of contact
lens-related articles published in the ophthalmic lit-
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Opportunity for Population-Based
Eye Research in Asia 3
and the Middle East: An NGO
Perspective
Suzanne S. Gilbert, Thulasiraj Ravilla,
and Leslie Louie
Abstract 3.1 Community Eye Health

Advances in genetics eye research are meeting
and exceeding expectation. Research collabo- As noted elsewhere in this volume, the past
rations, rapid sharing of findings, and inclu- 20 years have marked a considerable increase in
sion of emerging technologies offer organized efforts to reach the unreached with
tremendous opportunities. Beyond research- comprehensive eye care. The 1999 launch of
ers per se, there is also a largely untapped VISION 2020: The Right to Sight, the joint ini-
opportunity for expanding the reach of tiative of the World Health Organization (WHO)
population-based genetics research through and the International Agency for the Prevention
engagement with the eye care NGO sector. In of Blindness [1], signaled global recognition of
addition to the prospect of enabling rapid the scope of the unmet needs. This global initia-
engagement with larger populations, the NGO tive prioritized strategies required to address the
sector can be an ally in building the capacity needs and an advocacy platform to align minis-
for research among service providers. This tries of health, nongovernmental organizations,
chapter frames this opportunity and offers rec- and professional societies in the vision field.
ommendations to genetics researchers who WHO’s Universal Eye Health: A Global
seek to broaden their array of research options. Action Plan 2014–2019 [2] prioritizes three
objectives for member state action: (1) recognize
Keywords the need for generating evidence on the magni-
Capacity building · Training · Population tude and causes of visual impairment and eye
research · VISION 2020 care services and using these data for advocacy;
(2) encourage the development and implementa-
tion of integrated national eye health policies,
plans, and programs to enhance universal eye
health in line with WHO’s framework for
strengthening health systems to improve health
outcomes; and (3) address multi-sectoral engage-
S. S. Gilbert (*) · L. Louie ment and effective partnerships to strengthen eye
Seva Foundation, Innovation & Sight Program, health. Three key indicators include (1) the prev-
Berkeley, CA, USA alence and causes of visual impairment, (2) the
e-mail: [email protected]
number of eye care personnel, and (3) cataract
T. Ravilla surgery volume.
Aravind Eye Care System, Madurai, India

https://doi.org/10.1007/978-981-13-0884-0_3
24 S. S. Gilbert et al.
These priorities plus findings regarding causes for organizational sustainability of the service
of vision impairment [3] and projections through delivery programs including, where feasible,
2050 [4] prompt a call to action and priority to financial sustainability.
improve program effectiveness. A comprehen- Several service institutions and international
sive presentation of data and strategies is found in nongovernmental organizations that had achieved
the IAPB Vision Atlas [5]. program stability experimented with reaching out
Through international efforts to conduct to help underperforming eye hospitals to improve
community- based prevalence studies including [13]. Capacity building has become more sys-
the initiation of the Rapid Assessment of tematic and has grown into a leading strategy
Avoidable Blindness (RAAB) strategy [6, 7], the implemented by a growing number of organiza-
prevalence of conditions causing visual impair- tions. One such organized approach is the Global
ment and systematic strategies to address them Sight Initiative which today involves 100 eye
has become better known. The global RAAB hospitals in 16 countries [14].
Repository [8] now cross-references findings Classically, capacity building involves devel-
from more than 320 studies meeting the stringent oping a relationship between the more established
RAAB criteria. Population-based studies and dis- institution (often referred to as a “mentor” and the
aggregation of service data have produced break- institution seeking to improve the “mentee”). The
throughs in recognition of service inequities and process begins with an exploratory phase of needs
need for interventions for marginalized groups. A assessment, followed by engagement together to
case in point is the finding that vision impairment determine a strategic plan and an action plan.
occurs disproportionately in females [9] which Ongoing consultation to enact the plan focuses on
has promoted efforts to refine strategies to reach improving administrative systems, training key
women and girls [10, 11]. clinical and nonclinical staff, strengthening com-
munity outreach, and advising on facilities
improvements and equipment that will enhance
3.2 apacity Building to Scale
C service quality and volume. Throughout, the
Service Delivery groundwork is laid for monitoring of key perfor-
mance indicators through data collection, report-
Given the growth in visual impairment projected ing, and feedback. Initial results in scaling cataract
due to global demographic trends and insufficient surgery productivity reported by the Global Sight
service levels causing a threefold increase by Initiative are encouraging [15].
2050 in the number of people who are blind and A critical aspect of capacity building is to
visually impaired, [4] planners recognize the ensure increase in a geographic region’s Cataract
requirement to develop programs that are built to Surgical Rate (CSR, number of cataract surgeries
last. A systems development approach is increas- performed per one million population in a given
ingly being applied to build the platform for year). Though this metric focuses on cataract sur-
robust service delivery. The scope of the “oph- geries, the strategies to achieve this require reach-
thalmologist gap” due to lack of number of oph- ing out to larger number of patients. This,
thalmologists and their uneven distribution combined with the thrust for comprehensive eye
around the world further exacerbates the need care, ensures enhanced delivery of eye care. This
[12]. A growing number of service programs are achievement requires intelligence regarding com-
working to increase services within a sustainable munity eye health seeking behavior or lack
framework. This requires having the means to thereof. A landmark study conducted with
develop and manage highly skilled staff, to Aravind Eye Care System reported that even with
design and implement community-engaged inter- extensive community outreach to find people in
ventions, and to provide high-quality surgical need of eye care services, only 7% of people in
and clinical care that is sought by patients. As need of eye care came for the local, free screening
programs have become more robust, the push is camp [16]. This finding plus observations by LV
3 Opportunity for Population-Based Eye Research in Asia and the Middle East: An NGO Perspective 25
Prasad Eye Institute and other providers lead to retain any ophthalmologists, the hospital has 117
the development of community-based vision cen- specialist doctors on staff [19].
ters which provide primary prevention services Over time, SNC’s capacity building extended
and early referral to the corresponding eye hospi- into establishing postgraduate ophthalmology
tal as needed [17]. training programs, optometry training, and train-
ing for ancillary personnel. Coaching and assis-
tance was provided by Seva, Aravind, and LV
3.3 apacity Building Case:
C Prasad Eye Institute to initiate a public health
Sadguru Netra Chikitsalaya library to support all of these training activities.
in Rural Central India During 2010, SNC started mentoring other eye
hospitals to improve their systems and results
2001: Sadguru Netra Chikitsalaya (SNC) was a through the Global Sight Initiative.
350 bed eye hospital producing 20,000 cataract (End of Case)
surgeries per year. SNC had one full-time oph- Establishing in-house training programs is a
thalmologist and a large number of volunteer sur- vital component of capacity building, particu-
geons during 3 months of the year when 85% of larly for training Allied Ophthalmic Personnel
annual surgeries were conducted. Surgeries were (ophthalmic assistants, midlevel ophthalmic per-
provided free of charge with just 16% of the cata- sonnel, refractionists, surgical assistants, patient
ract surgeries being done with an intraocular counselors, ward staff, and other members of the
lens. It was difficult to hold professional staff ophthalmologist-led eye care team). The
given local hardships such as lack of electricity International Council of Ophthalmology, Aravind
and schools for children. Eye Care System, and Seva Foundation estab-
A series of systematic capacity building inputs lished an immersion course to enable eye hospi-
were made with SNC by Aravind Eye Care tals to establish or expand their in-house training
System, Seva Foundation, and Orbis over a mul- programs [20]. This “Eyexcel” workshop has
tiple year period to attract and retain staff, train prepared 121 eye hospital teams from 24 coun-
managers, and install management and informa- tries to improve their training practices.
tion systems. The immediate focus was to con- However, capacity building does not stop at
vert from intracapsular to extracapsular cataract service delivery and training. There also are
surgery with IOL implants and soon to manual efforts underway by NGOs to strengthen research
small incision and phaco techniques. practice at the population and clinical level.
In 2016, to promote year-round utilization of
services, SNC was coached in building a robust
community eye screening program which has 3.4 esearch Skill Building
R
grown to include 43 vision centers, increasing and Partnership
proportion of cataract patients who are female to Development
50% and establishing strong financial analytics
and management practices [18]. SNC’s growth is The NGO sector has been engaged in population-
not limited to cataract surgery which in 2015– based studies of the prevalence and causes of
2016 rose to 117,661. Between the 2 years, visual impairment as a tool for service program
2014/2015 and 2015/2016 alone, SNC’s specialty planning, development, and impact assessment.
surgeries in glaucoma, vitreoretina, cornea, pedi- There also is a tradition of operations research to
atric surgeries, and orbit and oculoplasty improve the use of scarce resources for service
increased 10% from 23,613 to 26,063.. In delivery such as staff, facilities, equipment, and
2015/2016, SNC was able to serve 40% of its supplies. This institutional research promotes
patients free of charge based on cross-subsidies inquisitiveness among staff who seek to reach
from paying patients. From not being able to more patients more efficiently [21, 22]. Research
into the outcomes of care are conducted to
26 S. S. Gilbert et al.
d etermine how service outcomes as experienced 3. Flaxman SR, Bourne RRA, Resnikoff S, et al. On
behalf of the Vision Loss Expert Group. Global
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have no conflict of interest. The three authors are Suzanne 13. Ravilla T. Delivering efficient eye care. Cataract

S. Gilbert, Thulasiraj Ravilla, and Leslie Louie. Refract Surg Today. 2007:77–9.
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eyeGENE®: A Model for Advancing
Research of Rare, Inherited Eye 4
Conditions Through Biobanking
and Data Sharing
R. S. Parrish, K. E. Goetz, and S. J. Tumminia
Abstract 4.1 Introduction

eyeGENE® is a genomic medicine initiative cre-
ated to facilitate research into the causes and The National Ophthalmic Disease Genotyping
mechanisms of inherited eye diseases and accel- and Phenotyping Network or eyeGENE® is a
erate pathways to treatments. Through collabo- genomic medicine initiative created by the
rations with clinics across the United States and National Eye Institute (NEI) at the National
Canada, eyeGENE® recruited approximately Institutes of Health (NIH), a part of the United
6400 participants. To date, eyeGENE® has States (US) Department of Health and Human
returned more than 5800 clinical genetic test Services. The primary goal of eyeGENE® is to
results to participants and has granted 22 facilitate research into the causes and mecha-
research studies controlled access to clinical and nisms of inherited eye diseases. To accomplish
genetic data, DNA samples, and/or individuals this, eyeGENE® established a patient registry, a
consented to participate in research and clinical genotype/phenotype data repository, and a DNA
trials. These studies have identified new genes, biobank. eyeGENE® enables access to genetic
improved our understanding of inherited eye diagnostic testing for affected individuals and
disease, and validated new molecular diagnostic their families. Additionally, eyeGENE® provides
technology. eyeGENE® can serve as a model for research access to clinical and genetic informa-
creating a research resource to advance the tion including images, DNA samples, and the
study of rare, inherited diseases. opportunity to recruit individuals to participate in
additional research studies and clinical trials.
Keywords eyeGENE® arose from vision community
Data sharing · Biobank · Biospecimens · needs and opportunities. Thirty years ago, clini-
Genetics · Clinical phenotype · Common data cians could do little more than identify an inher-
elements · Clinical data collection ited eye disease through standard direct
ophthalmoscopy, but scientific and technological
advances have brought potential treatments and
R. S. Parrish · K. E. Goetz (*)
Ophthalmic Genetics Visual Function Branch, hope to individuals impacted by these conditions.
National Eye Institute (NEI), National Institutes of To date, scientists have identified over 400 genes
Health (NIH), Bethesda, MD, USA related to eye disorders that are associated with
e-mail: [email protected] both common complex diseases as well as rare
S. J. Tumminia diseases [1]. This profusion of genetic informa-
Office of the Director, NEI, NIH tion emphasizes the significant strides made in
Bethesda, MD, USA

https://doi.org/10.1007/978-981-13-0884-0_4
30 R. S. Parrish et al.
understanding the molecular basis of human eye Over the next 2 years, the NEI funded a set of
diseases. These advances are paving the way for extramural vision community research grant sup-
the development of gene-based therapies to treat plements to build the national capacity for
ophthalmic genetic diseases that were once con- CLIA1-approved molecular genetic testing for
sidered untreatable. ocular diseases. Concurrently, the NEI developed
eyeGENE® has accrued over 6400 individuals a web-based application to recruit participants
and to date has returned clinical test results to from clinics across the United States and Canada.
roughly 75% of participants through their refer- The application was used to collect eye examina-
ring clinician. Even though the program offers tion information, images, demographic informa-
this knowledge to the participant as an indirect tion, and molecular test results. Also, an internal
benefit, the main goal of eyeGENE® is to facilitate Operations Working Group was created to man-
research. Toward achieving this goal, researchers age the day-to-day activities and an external
have used eyeGENE® to identify new eye disease- Steering Committee was formed to help oversee
causing genes, better understand various charac- progress [8]. In 2006, eyeGENE® was officially
teristics of inherited eye disease, and validate new launched. Through the next decade, eyeGENE®
molecular diagnostic technology [2–7]. Through became well-known as an international multi-
experience, the eyeGENE® Operations Working center clinical genetics research initiative and a
Group has learned valuable lessons about build- model of partnership between the US federal
ing a network to advance research of rare genetic government, eye healthcare providers, CLIA-
eye diseases. Here we share many key points in approved molecular diagnostic laboratories, pri-
patient accrual, biobanking, clinical data collec- vate industry, and extramural scientists who
tion, and research analysis. support a broad research constituency. As a vision
community resource, eyeGENE® provides
patients with greater access to diagnostic gene
4.2 eyeGENE®: Initial Concept testing and genetic information. Clinicians and
and Design researchers also have access to diagnostic genetic
testing, centralized specimen collection, and a
In the early 2000s, diagnostic DNA testing was shared database of de-identified
not widely accessible to the public, and there genotype/phenotype information. Overall, this
were no concerted efforts to identify individuals public-private partnership benefits eye healthcare
who might benefit from potential gene-based providers, patients, and researchers [9].
treatments. The NEI addressed these needs by eyeGENE® was designed as a two-stage initia-
convening meetings with domestic and interna- tive governed by human clinical protocols
tional experts in ophthalmic genetics, bioethics, approved by an NIH Institutional Review Board
genetic counseling, and regulatory requirements. (IRB). Stage 1 of the eyeGENE® initiative
These experts advocated for the establishment of (Clinical Trials.gov Identifier NCT00378742),
a vision community resource to bring together National Ophthalmic Genotyping and
individual genetic testing laboratories for the Phenotyping Network, Creation of DNA
common goals of patient care, diagnosis, Repository for Inherited Ophthalmic Diseases,
research, treatment, and education. In February accrued samples from individuals with inherited
2004, a concept for this resource was approved
by the National Advisory Eye Council, an exter-
Clinical Laboratory Improvement Amendments
1
nal group authorized under 42 U.S.C. 284a, sec- (CLIA) – The Centers for Medicare & Medicaid Services
tion 406 of the US Public Health Service Act to (CMS) regulates all laboratory testing (except research)
advise, assist, consult with, and make recommen- performed on humans in the U.S. through the Clinical
Laboratory Improvement Amendments (CLIA). The
dations to the Secretary of Health and Human objective of the CLIA program is to ensure quality labora-
Services and the NEI Director on matters related tory testing (https://www.cms.gov/Regulations-and-
to NEI activities and policies. Guidance/Legislation/CLIA/index.html?redirect=/clia)
4 eyeGENE®: A Model for Advancing Research of Rare, Inherited Eye Conditions Through Biobanking… 31
eye diseases, while Stage 2, Distribution of Data Network. As technology evolves, the molecular
and Biomaterials from the NEI, allows the alloca- testing partners have changed and so has the
tion of samples, data, and the contact of partici- funding mechanism; however, the molecular
pants recruited through Stage 1. diagnostic testing services are primarily con-
When the program began, eyeGENE® tracted outside of the NEI with commercial and
screened for 20 known disease-causing genes in academic CLIA-approved laboratories.
9 disease categories. Currently, several hundred eyeGENE® accrued participants by allowing
genes are tested through panels available through healthcare professionals (ophthalmologists,
eyeGENE® Network Laboratories. As molecular optometrists, neurologists, genetic counselors,
testing technology evolves, so does the screening etc.) with knowledge and access to the clinical
capabilities of the program. The eyeGENE® web- details of an affected individual’s inherited eye
site lists summary data and shows the number of condition to enroll the patient. Because of the
participants in each disease category and the challenges of sample collection, the constraints
breakdown of variants for genes tested [10]. The on international blood shipments, and financial
two largest disease categories are retinitis pig- considerations, enrollment was open to health-
mentosa with more than 2000 participants and care professionals in the United States or Canada.
Stargardt disease with over 1300 participants. More than 220 clinical organizations registered
Nine additional disease categories have more with eyeGENE® and enrolled one or more par-
than 100 participants, which is noteworthy given ticipants. The first enrollments were patients
the rarity of these conditions in the North referred from the ophthalmic genetics branch of
American population. the NEI Clinic but quickly grew to include
patients from large research hospitals and aca-
demic institutions and private practice.
4.3 Building a Network In large part, clinical organization registra-
and Participant Recruitment tions were fueled by patient interest. A strong
driving force for some patients to participate in
One of the first strategies of eyeGENE® was to the program was that they would receive “free”
build a network of research and clinical investi- diagnostic testing. That is, the onus to pay for
gators interested in diagnosing genetic eye dis- diagnostic testing, often multiple testing, was on
ease through molecular testing. To achieve this, the NEI, and not on the patient. In return, the
the NEI issued a Notice of NEI Administrative patient agreed to participate in research with the
Supplements to Enhance Diagnostic Genotyping option to withdraw at any time. The patient was
for Ophthalmic Diseases (https://grants.nih.gov/ responsible for the cost of their clinical eye exam
grants/guide/notice-files/NOT-EY-05-002.html). and blood draw.
The Notice announced the availability of supple- From 2006 to the end of 2015, eyeGENE®
mental funding to ongoing NEI-funded research enrolled more than 6400 participants.
projects to expand and enhance molecular diag- Collaborating with community clinicians and
nostic testing for ophthalmic diseases. The pur- large academic institutions allowed eyeGENE®
pose of these administrative supplements was to to cast a wide net in finding rare eye disease
build infrastructure for genotype/phenotype patient populations as shown in Fig. 4.1. eye-
resources that would be publicly available by GENE® participant contact information was used
assisting researchers who had an ongoing NEI- to map participants by enrolling clinical organi-
supported project related to genetic screening of zation. The NEI Clinic enrolled more than 1000
inherited eye conditions to build or expand their (16%) participants. While the NEI Clinic had the
laboratories’ capacity for clinical testing. CLIA- greatest reach of any organization, most partici-
approved laboratories that received this funding pants live relatively close to the clinic. Thirteen
from the NEI were the first molecular diagnostic other clinical organizations enrolled more than
testing facilities collaborating in the eyeGENE® 100 participants each and close to 3500 partici-
32
Fig. 4.1 Location of eyeGENE® participants in the continental United States orange points are participants enrolled by the 13 high-enrolling clinics (each
and parts of Canada and Mexico, based on contact information provided during enrolled 100 or more participants). The blue points are participants enrolled by
enrollment. The black points are participants enrolled by the NEI Clinic. The clinics that enrolled less than 100 participants
R. S. Parrish et al.
pants (54%) collectively. The remaining 30% of number of eligible patients due to the prevalence
participants were registered by clinics that of these diseases. These organizations were
enrolled less than 100 participants, and 85 of approved to use the NIH-issued consent forms
these only enrolled one participant. Building a and were not expected to undergo the often
large, diverse network of clinicians allowed lengthy and expensive IRB protocol submission
patients in every part of the United States, includ- process.
ing rural and remote areas, to participate in eye- While the flexibility of this consenting strat-
GENE®. One downside to this type of network is egy expanded eyeGENE®‘s reach, it also created
that the quality of clinical data is highly variable some difficulties. Verbally confirming the NIH
due to differing levels of experience with rare consents obtained by collaborating organizations
diseases and access to diagnostic equipment was time-consuming for NEI staff. The indepen-
among clinicians. dent IRB-approved protocols and consents
expired at different times throughout the year and
ensuring that the protocols were being main-
4.4 Informed Consent tained and that valid consents were being used
required vigilance from both the eyeGENE®
All eyeGENE® participants were consented in Coordinating Center and the Laboratory.
accordance with the ethical standards of the Incomplete and expired consents were received
responsible committee on human experimenta- regularly, requiring follow-up, which delayed the
tion (institutional and national) and with the ability of NEI staff to complete patient enroll-
World Medical Association’s (WMA) Declaration ment and provide genetic test results.
of Helsinki, as revised in 2008. When planning a During the consenting process, eyeGENE®
biobank, national and institutional regulations participants were given several options. In addi-
regarding informed consent of human subjects tion to consenting to allow their de-identified
should be consulted. Specific ethical consider- samples and data to be used for vision research,
ations regarding health databases and biobanks they could choose whether they wanted to receive
can be found in the WMA’s Declaration of Taipei their genetic test results or just participate in
[11]. research. Most participants opted to receive their
Two routes of consenting were proposed and genetic results. Participants were also given the
accepted by the NIH Combined Neuroscience option to be recontacted in the future.
IRB. The consenting method was dependent on One constant in research is change, and as
the anticipated patient enrollment volume from technology and the knowledge of human genetics
an approved registered clinical organization as expanded, so did the potential for research over-
well as the internal regulatory policies of the lap outside of strictly vision-related elements
enrolling institution. Clinical organizations plan- within the original purview of the eyeGENE®
ning to enroll large numbers of participants in protocol. For instance, some genes associated
eyeGENE® were likely to be engaged in addi- with a diagnosis covered by eyeGENE® may also
tional research on those populations. Therefore, be involved in pathways related to other, non-eye
to enroll more than ten participants per year, a related conditions for which some researcher
clinical organization was required to submit the may be interested. Also, whole-genome/whole-
eyeGENE® protocol for approval to their respec- exome or other next-generation sequencing tech-
tive institution’s IRB or a commercial IRB. More niques may identify genetic causes of eye disease
than 40 clinical organizations sought and received in a participant that may not be strictly related to
independent IRB approval of the eyeGENE® pro- their clinically determined eye condition, espe-
tocol. A second infrastructure was implemented cially since there is a high level of clinical and
for those enrolling less than ten patients per year genetic heterogeneity in the retinal eye disease
as it was recognized that many private practices population. For these reasons, eyeGENE® is
or small institutions might only see one or a small implementing a patient portal to keep partici-
pants engaged in the research endeavor. The por- future research needs. Instead of collecting DNA
tal includes an electronic consent to provide that had been extracted by an outside source, eye-
existing participants an opportunity to determine GENE® chose to collect blood and process it to
whether they wish to participate in a broader defi- DNA centrally using standardized procedures in
nition of vision research. A specific advantage of the eyeGENE® CLIA-certified laboratory. This
an electronic consent is that it can involve multi- ensured that the quality and quantity of DNA
media information and be interactive, which is banked would be comparable and adequate for
important for providing anonymity for partici- current and future needs. While saliva was con-
pants with vision impairments who may need to sidered, blood was chosen because of the higher
use assistive technology for reading. Additional DNA yield and it is relatively easy to access and
benefits are reduced staff time, cost-benefit effi- transport.
ciencies, tools for checking participant compre- To enroll in eyeGENE®, participants had to be
hension, and electronic records management. willing and able to provide a blood sample of
The portal is also a useful tool for administering acceptable quality and volume to yield more than
surveys to expand the resource. eyeGENE® par- 50 μg of DNA. To meet this requirement, eye-
ticipants have complained about the need for GENE® requested blood (24–30 mL from adults
more communication, and this measure is one and 7–15 mL from children) collected in K2EDTA
step to serve their desire to play a continued role tubes, which are commonly used when collecting
in the research enterprise. specimens to be used for PCR-based genetic test-
ing. To control for quality, receipt and initial pro-
cessing of the blood had to be completed by the
4.5 Building a Biobank eyeGENE® Laboratory within 72 h from the time
of collection as it was assumed that the longer the
When eyeGENE® was launched, biobanking was sample was in variable transport conditions, the
beginning to be recognized for its value to scien- more degraded the DNA would become. Samples
tific research. Prior to this time, many considered received after 72 h from the blood draw were
a biobank to be a freezer full of patient-derived subject to rejection. Whenever possible, a partici-
samples often for personal research projects. pant’s blood sample was aliquoted into two or
Biobanking has blossomed to a scientific field of more tubes for the initial processing of sample to
its own with organizations like the International cell lysate. Then the tubes were extracted to DNA
Society for Biological and Environmental in separate batches and the resulting DNA stored
Repositories (ISBER) and the National Cancer in separate freezers. One DNA aliquot is desig-
Institute, also part of the NIH, providing science- nated as the active aliquot for distribution and
based evidence to this expanding landscape. stored at −20 °C. Any other DNA aliquots are
Extensive planning is required during the cre- designated as long-term storage and stored at
ation of a biobank, and best practices are avail- −80 °C. Processing a participant’s sample in
able [12, 13]. During the planning phase, it is multiple batches and storing the resulting DNA
important to consider the purpose of the biobank, in separate locations greatly reduce the risk of
type of specimen(s) to be collected, specimen losing an entire sample to processing errors or
processing and storage conditions, and type(s) of equipment failure.
research utilization among other characteristics. During initial processing, 0.5–1 mL of blood
eyeGENE® created a DNA biobank for rare was stored in one of the original blood tubes at
inherited ophthalmic diseases to provide DNA 4 °C. These original blood samples are used dur-
coupled to de-identified phenotypic information ing Sample Confirmation Testing (SCT), an
to researchers. To accomplish this goal, eye- internal QA/QC procedure that compares STR
GENE® collected large quantities of high molec- markers in the blood to those in the corresponding
ular weight DNA that could be used immediately DNA [14]. This process is a vital step to ensure
for diagnostic testing purposes and be stored for no significant laboratory errors, including misla-
beling, pipetting, or other human errors, occurred. Sciences Corporation) to customize and imple-
As policy, only DNA aliquots that have been ment their web-based commercial laboratory
verified to match the original blood sample information management system (LIMS). The
through SCT may be shipped out of the biobank two teams analyzed the eyeGENE®
to a Network CLIA-certified molecular diagnos- Biorepository’s existing processes and systems
tic laboratory and/or to an approved researcher. and identified problems and workflow bottle-
Standard operating procedures (SOPs) were necks. This analysis led to the development of
created and revised for each process involved in detailed LIMS requirements and an overall proj-
specimen receipt, handling, extraction, and stor- ect development plan. Using eyeGENE® SOPs as
age. The eyeGENE® Working Group shares these a guide, existing Sampleminded LIMS tools were
SOPs with anyone who has an interest in building configured and new tools were developed as nec-
their own capacity and standardized processes. essary. Iterations of releases were tested by the
eyeGENE® team, and corrections and improve-
ments were made before launching the produc-
4.6 eyeGENE® Laboratory tion system.
Sample Tracking System Nearly all laboratory procedures are now suc-
cessfully being managed by the customized
Developing and maintaining records manage- Sampleminded LIMS, which has markedly
ment and computer-based sample tracking sys- increased the efficiency of the eyeGENE®
tems are best practices for all biorepositories. Laboratory workflow. In addition to guiding
Over time, the eyeGENE® system for records users through each procedure, the LIMS has
management and sample tracking evolved from a eliminated the need for paper procedure work-
simple paper logbook and a Microsoft Access sheets because it includes a detailed audit trail
database to a complex custom-built system, here- which automatically records the user, reagent
after referred to as the eyeGENE® Legacy sys- information (lot number and expiration date), and
tem, that was informally developed in-house. batch. Using a robust barcode scanning system,
The lack of formal workflow analysis led to a the LIMS has automated chain of custody and
system that had to be supplemented by several improved sample tracking by annotating data
Microsoft Excel logbooks and paper procedure from each workflow event and recording the
worksheets. The logbooks served to batch and location for every sample in the biorepository.
track samples through processing as well as The scanning system also helps prevent errors in
assign sample storage locations, while the proce- tube transfer and storage. A reduction in manual
dure worksheets documented the technician, data entry combined with the LIMS double-entry
reagent information, and samples in the batch. requirements and data validity checks has
Free-text notes were manually entered in a text improved data quality (e.g., the LIMS will not
box comment field for each sample listed in the allow two tubes to be assigned the same
Legacy system as a basic way to track chain of location).
custody, storage conditions, and freeze-thaw A LIMS is an effective tool for automating
cycles. This process was time-consuming, prone and improving workflow, accurately recording
to human error, and not suitable to query. In addi- process and sample-related data, and tracking
tion, sample storage locations needed to be man- samples. Both in-house developed and custom-
ually entered into the database, which had no ized commercial LIMS require significant time
data validation mechanism. This deficiency and resources, and it is imperative that require-
allowed entry of nonstandard formats, erroneous ments are carefully generated before building or
and duplicate locations for multiple samples. selecting and customizing a LIMS. Despite these
To improve laboratory workflow, records challenges, those seeking to develop a biobank
management, and sample tracking, eyeGENE® should consider incorporating a LIMS to enhance
contracted Sampleminded (now part of Exact efficiency and, more importantly, to reduce
errors. Finally, it is best to consider a LIMS dur- would end up being a poor design choice even
ing the initial planning phase due to the added though the issues could not have been anticipated
resources that are needed to extract data from one at the onset of development planning. Upon
system and import that data into another system. enrolling a patient, the referring healthcare pro-
vider would select the patient’s clinical diagno-
sis. A clinical capture report form would then be
4.7 Clinical Data Collection presented to complete enrollment. Each form dif-
fered based on the diagnosis and comprised
Often biobanks contain clinical data that corre- roughly 10 to 15 questions that experts, solicited
spond to samples stored in their freezers. by the eyeGENE® Steering Committee, decided
Sometimes biobanks are created specifically to represented the core clinical symptoms of that
support a hospital system with a vast collection disease presentation. These questions were
of longitudinal clinical records. For eyeGENE®, dubbed “minimal clinical criteria” and were used
the Legacy system was developed to allow refer- to validate the patient’s diagnosis and the
ring healthcare providers to enter clinical data requested genetic test(s). Meeting these criteria
about the eye health of a patient at the time of also served to justify the federal support for the
enrollment. The system was designed to support clinical genetic testing costs. The program started
a laboratory workflow that prohibited genetic with 9 disease categories and grew to more than
diagnostic testing of the patient’s sample until the 35 categories over the course of 10 years, and
required paperwork and a minimal amount of each time a new diagnosis was added, an expert
defined clinical data was received. was identified to add new minimal clinical crite-
Over time, the eyeGENE® Legacy system ria questions to the system.
began to require integration of the data corre- Two critical hurdles arose from using this
sponding to patient samples as described in the strategy. First, through the years over 300 ques-
prior section. This need for operational efficiency tions were created to describe the phenotypes of
eventually led to all the sample-related data eyeGENE® participants. With only 35 disease
becoming part of the Legacy system. To keep an categories, most retinal diseases, there was a
acceptable turnaround time from enrollment to great deal of overlap of descriptive terms. For
genetic testing to the return of genetic results, example, there were at least six distinct ques-
most of the system development resources were tions that asked the referring clinic to describe
diverted to enhancing features of the laboratory fundus appearance. Each question contained a
aspects of the Legacy system. Additionally, in different set of answer selections. The second
2010 with the research goal of eyeGENE® in problem was that the diseases included in eye-
mind, the requirements of the Legacy system GENE® were clinically heterogeneous, meaning
mandated one more crucial feature – researcher that there was clinical overlap among condi-
access to de-identified clinical and genetic data. tions. It is sometimes difficult to distinguish one
The combination of three major core functions, diagnosis from another based solely on clinical
i.e., clinical data capture, laboratory management symptoms. Through genetic testing, eyeGENE®
(LIMS features), and research access, coupled has found that the genes associated with a diag-
with advances in technology and other system nosis also presented with a significant amount
constraints, eventually led to the retirement of the of heterogeneity. For instance, the RDS gene can
Legacy system as it could no longer keep pace be associated with cone-rod dystrophy, pattern
with other, cheaper products available “out of the dystrophy, Stargardt disease, or retinitis pig-
box” or with some customization. mentosa. These issues are critical to note for
One fundamental decision in the development anyone wishing to collect this type of data as it
of the Legacy system was the decision that data directly impacts how or if data sets can be com-
collection would be organized by disease. This bined later.
Several institutions are currently establishing biguously identify measurements and observa-
similar systems using a disease category approach tions enabling interoperable data exchange. For
similar or identical to that originally used by eye- each concept, LOINC® contains other details,
GENE®. While it is gratifying and validating to such as synonyms, units of measure, and care-
know that the data collection by disease approach fully crafted descriptions. LOINC® is freely
used in eyeGENE® in 2006 was logical and prac- available as well as widely accepted and utilized.
tical as others are now implementing similar It is also translated internationally. The
methods, the issues and challenges described eyeGENE®-NLM Working Group also created
above should give pause to the vulnerabilities of panels of similar data elements in LOINC®. For
this approach. Creating a clinical (phenotype) instance, a panel was created for tonometry
capture tool that siloed data on questions related groups, including the type of tonometer used for
solely to diseases and clinical diagnoses with the exam along with elements to capture the
profound phenotypic and genotypic heterogene- intraocular pressure in each eye. These terms can
ity made research query, filtering, and analysis be found at the NIH CDE Repository under the
cumbersome and prone to error. Once the focus NEI heading [15].
of eyeGENE® moved from data collection to data Common data elements (CDEs) and ontolo-
sharing, the existing Legacy data infrastructure gies should be carefully selected when embark-
was too embedded to be changed. Instead, a new ing on a clinical data capture project to ensure
approach was necessary. reproducibility and scientific rigor. It is impor-
In 2015, as part of a larger NIH effort to stan- tant to take time to carefully consider the level of
dardize disparate data, eyeGENE® began work- detail that a project requires [16]. For example,
ing with the Lister Hill Center at the NIH National in some cases, research users may only need to
Library of Medicine (NLM) to distill the existing know the presumed clinical diagnosis, while in
eyeGENE® 300 clinical questions into a more other cases, the specific details of how that diag-
manageable set of questions. The goal was to cre- nosis was derived are needed. In some studies, it
ate a resource for clinical data collection to may be enough to know whether a patient had an
describe the phenotype of patients with inherited abnormal electroretinogram (ERG), whereas in
eye conditions to be used throughout the vision other studies, it will be important to know the
community, not simply eyeGENE®. This could specific ERG wave values and times. One final
allow for broad data sharing on a global scale. note on data elements is the concept of “flavors
The eyeGENE®-NLM Working Group began by of null.” Essentially, it is necessary to distinguish
combining like terms, expanding lists of potential between a negative response, an unknown
permissible values, and aligning overlapping or response, and a blank field. For example, if the
similar terms. Then each unique item was trans- data element prompts for “gross defects of the
formed into a structured LOINC® term, and the left eye,” potential answers should include none,
appropriate data type and parameters were unknown, and possibly “other” field where a
described. LOINC® is an international coding user can enter text if something is drastically dif-
system developed in 1994 by the Regenstrief ferent from the answer choice options
Institute to meet the growing need for common presented.
laboratory and clinical terminology. It sets uni- Use of CDEs and ontologies will allow users
versal standards for classifying and identifying to capture and compare clinical research data
clinical and laboratory observations and mea- methodically, lowering the barrier for compara-
surements related to electronic health records. tive data analysis [16]. Worldwide adoption of set
LOINC® enables the exchange and aggregation standards across all disease categories for record-
of clinical results for care delivery, outcomes ing clinical encounters will allow for better data
management, and research by providing a set of mining and cross comparison of data sets across
universal codes and structured names to unam- a variety of studies and disciplines.
4.8 Community Access GENE® Research Resource. A full list of studies

to eyeGENE® Data and/or as well as publications are listed on the eye-
Biospecimens GENE® website (eyegene.nih.gov). eyeGENE®
has been successful in aiding researchers to iden-
In 2010, eyeGENE® reached the milestone of tify novel genetic causes of retinal conditions,
1000 participants. This number was deemed suf- testing sporadic cases of retinal degeneration with
ficient to allow researchers access to the rare dis- new technologies, genotype-phenotype correla-
ease data and sample resources. Access was tion studies, molecular modeling of pathogenic
designed to be as broad as possible, including variants, recruiting for rare variant-induced plu-
international research laboratories and commer- ripotent stem cell studies leading to the develop-
cial entities. Requests can be made for a portion ment of therapies, enrollment of treatment trials,
of DNA samples stored within the eyeGENE® and development of machine learning technolo-
Biorepository, access to de-identified genetic and gies to diagnose retinal degenerations in rural
clinical data, and/or to request that the eyeGENE® areas, among others. The diversity of these studies
Coordinating Center contact the participants for demonstrates the breadth and reach of eyeGENE®
enrollment into additional studies. Initial recon- as a valuable resource to the vision community
tact with the eyeGENE® participant is made and individuals with inherited eye disease.
through the eyeGENE® Coordinating Center to
determine interest so participants do not feel
pressured into joining research studies. Interested 4.9 Conclusions
investigators must submit an application, includ-
ing a research proposal and IRB/ethics board eyeGENE® serves as a model for advancing
review or exemption, to be considered for research of rare, inherited eye conditions through
resource access. All researchers must sign a biobanking and data sharing. It creates a continu-
Material Transfer Agreement/Data Usage ous bench to bedside life cycle, with opportuni-
Agreement in which they agree to share all data ties for partnership and collaboration among
with eyeGENE® with the intention that second- organizations, both domestic to the United States
ary research outcomes will eventually be made and abroad. Experience has shown the value of
available to the public. data standardization, not just the use of ontolo-
In late 2015, as the eyeGENE® Legacy system gies but also of CDEs. eyeGENE® readily shares
was retired, a new solution for vision community protocols, policies, and procedures upon request.
data access was needed. The NEI partnered with Global interest in the program has led to a
the NIH Center for Information Technology to pilot of eyeGENE® International, an umbrella
utilize Biomedical Research Informatics organization of international efforts using similar
Computing System (BRICS; https://brics.cit.nih. protocols and operating under similar standards
gov/), a bioinformatics platform that supports and policies. Benefits to participating in eye-
end-to-end life cycle support of research. It GENE® International is the ability to leverage
includes a set of six software modules that pro- local investment, work on problems of local con-
vide both web-based functionality and down- cern through collaboration, have access to meta-
loadable tools to support data definition, data data, and increased patient participation in
contribution, and data access for continuous gene-based trials.
research use. BRICS coordinates the collection eyeGENE® has demonstrated a successful
and quality assurance of data in standardized dic- public-private partnership and has facilitated the
tionary formats (CDEs) that enable query and evolution of ophthalmic care by integrating
distribution of aggregate data for acceleration of molecular genetics into standard of care for eye
eyeGENE® research by the research community. health professionals. The Network’s clinical and
At the time of this writing, eyeGENE® has genetics expertise, evolution of technology
received 24 applications for access to the eye- coupled with a DNA biorepository, de-identified
database, and individuals affected by inherited with macular involvement. Invest Ophthalmol Vis
Sci. 2015;56(6):3889–95. https://doi.org/10.1167/
eye diseases are powerful tools for current inves- iovs.15-16778.
tigations in human genetics and genomics and for 5. Freund PR, Sergeev YV, MacDonald IM. Analysis
the development of gene-based personalized med- of a large choroideremia dataset does not suggest a
icine treatments. Now eyeGENE® intends to max- preference for inclusion of certain genotypes in future
trials of gene therapy. Mol Genet Genomic Med.
imize this expertise by focusing its efforts on 2016;4(3):344–58. https://doi.org/10.1002/mgg3.208.
enhancing the research enterprise through data 6. Alapati A, Goetz K, Suk J, Navani M, Al-Tarouti
accessibility and sharing. Maximizing research A, Jayasundera T, Tumminia SJ, Lee P, Ayyagari
efforts can be best achieved through data stan- R. Molecular diagnostic testing by eyeGENE®:
analysis of patients with hereditary retinal dystro-
dardization and a willingness by the vision com- phy phenotypes involving central vision loss. Invest
munity to commit to data sharing. Worldwide Ophthalmol Vis Sci. 2014;55(9):5510–21. https://doi.
adoption of set standards across all disease cate- org/10.1167/iovs.14-14359.
gories for recording clinical encounters will allow 7. Ge Z, Bowles K, Goetz K, Scholl HP, Wang F, Wang
X, Xu S, Wang K, Wang H, Chen R. NGS-based
for data mining and cross comparison of data sets molecular diagnosis of 105 eyeGENE probands with
across a variety of studies and disciplines. retinitis pigmentosa. Sci Rep. 2015;5:18287. https://
doi.org/10.1038/srep18287.
Acknowledgments The eyeGENE® Network is sup- 8. Blain D, Goetz KE, Ayyagari R, Tumminia SJ. eye-
ported by the Department of Health and Human Services/ GENE®: a vision community resource facilitat-
National Institutes of Health/National Eye Institute intra- ing patient care and paving the path for research
mural program under eyeGENE® – Protocol 06-EI-0236, through molecular diagnostic testing. Clin Genet.
which has been funded in part under Contract No. 2013;84(2):190–7.
HHS-N-263-2017-00001-C. 9. Tumminia SJ. Harnessing academia, government, and
industry. Invest Ophthalmol Vis Sci. 2012;53(5):2515–
21. https://doi.org/10.1167/iovs.12-9483q.
Compliance with Ethical Requirements Authors 10. eyeGENE® Summary Data. Available from: https://
Parrish, R., Goetz, K., and Tumminia S. declare that they eyegene.nih.gov/node/35. Accessed 2 Oct 2017.
have no conflict of interest. 11. WMA Declaration of Taipei on Ethical Considerations
All procedures followed were in accordance with the Regarding Health Databases and Biobanks. 2016.
ethical standards of the responsible committee on human Available from: https://www.wma.net/policies-post/
experimentation (institutional and national) and with the wma-declaration-of-taipei-on-ethical-considerations-
Helsinki Declaration of 1975, as revised in 2000 (5). regarding-health-databases-and-biobanks/. Accessed
Informed consent was obtained from all patients for being 2 Oct 2017.
included in the study. 12. Best Practices for Repositories: Collection, Storage,
Retrieval and Distribution of Biological Materials
for Research, 3rd ed. Available from: www.isber.org/
default.asp?page=BPR. Accessed 2 Oct 2017.
References 13. NCI Best Practices for Biospecimen Resources. 2016.
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2. Arno G, Agrawal SA, Eblimit A, et al. Mutations in Tumminia SJ. Sample confirmation testing: a short
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org/10.1016/j.ajhg.2016.10.008. Biopreserv Biobank. 2016 Apr;14(2):149–55. https://
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Wang K, Zhao L, Li H, Li Y, Sui R, Chen R. Mutations 15. NEI CDEs. Available from: https://cde.nlm.nih.gov/
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eration. Hum Genet. 2015;134(10):1069–78. https:// 16. Sheehan J, Hirschfeld S, Foster E, Ghitza U, Goetz K,
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mutated in early onset photoreceptor degeneration doi.org/10.1177/1740774516653238.
Inherited Ocular Disease
in the New Zealand Māori: Novel 5
Genetic Mechanisms and Founder
Effects
Andrea L. Vincent
Abstract Keywords
New Zealand is a small island nation at the Māori · Pacific peoples · Genetic testing ·
bottom of the South Pacific, with a population Inherited retinal disease · PDE6B ·
of 4.7 million, and the largest Polynesian pop- ADAMTSL4
ulation in the world. 15% identify as Māori
and 7% as Pacific peoples, and the spectrum
of inherited eye disease encountered in this
population varies from that seen in those iden- 5.1 Introduction
tifying as NZ European. Keratoconus is more
common, while primary open-angle glaucoma New Zealand is a small island nation situated at
is rare. A number of founder pathogenic vari- the bottom of the South Pacific, with an estimated
ants have been elucidated in autosomal reces- population of 4,793,700 (2017, Stats NZ, www.
sive ectopia lentis, and a common PDE6B stats.govt.nz). With migration, New Zealand has
variant caused up to 16% of autosomal reces- an increasing ethnically diverse population, with
sive inherited retinal disease in the Māori pop- 15% identifying as Māori, and also the largest
ulation. Although many of those with inherited concentration of Pacific peoples, recorded as
retinal disease remain genetically uncharac- 260,000 (7%) in the 2013 census (Stats NZ). The
terised, research to date shows a range of Māori people of New Zealand were the first
novel variants in many genes. Understanding inhabitants, with their arrival from Eastern
the population-specific genetic disease spec- Polynesia (Cook Islands, Tahiti, Hawai’i) esti-
trum and clinical phenotypes, as well as a mated to occur around the year 1280 [1]. Based
knowledge of regional ancestry and iwi (tribe), on genetic investigation, it is believed the Eastern
aids in simplifying the diagnostic process. Polynesians descended from Taiwanese
Aborigines, with subsequent mixing with
Melanesians and European ethnic groups [2, 3].
A. L. Vincent (*) Of Polynesians, the Māori represent a relatively
Department of Ophthalmology, New Zealand
National Eye Centre, Faculty of Medical and Health new population genetically (about 35 genera-
Science, University of Auckland, tions) and have the smallest signal of external
Auckland, New Zealand relationship, consistent with extensive genetic
Eye Department, Greenlane Clinical Centre, drift [2]. It has been hypothesised that in the
Auckland District Health Board, migration through the Pacific, the seafaring
Auckland, New Zealand ancestors would likely have been subject to mul-

https://doi.org/10.1007/978-981-13-0884-0_5
42 A. L. Vincent
tiple founder effects, and consistent with this, Māori/Pacific students was 26.9% compared with
small numbers of haplotypes and reduced genetic 12.9% in NZ European (p = 0.0014) [10] and
diversity have been demonstrated [4]. 31% of affected Māori/Pacific peoples reporting
This chapter will provide an overview of the a positive family history of keratoconus [11].
spectrum of inherited eye diseases observed in Keratoconus is a complex disorder with putative
the New Zealand population, specifically dis- genetic and environmental contributions [12].
cussing the findings and genetic associations The visual system homeobox gene 1 (VSX1) is
seen in the New Zealand Māori and Pacific peo- one gene implicated in this disease, but in our
ples, based on study data, publications, and study, no pathogenic variants were detected in 26
observations. Polynesian probands [13]. Another gene,
Identification of unique genetic variants in this ZNF469, is associated with the autosomal reces-
specific population adds a further layer of com- sive disorder Brittle cornea syndrome, and in
plexity in determination of pathogenicity. This genome-wide association studies (GWAS), com-
population is underrepresented in databases of mon variants in this gene play a role in determi-
human variation; therefore, the frequency of any nation of central corneal thickness and
allele of interest needs to be determined in an eth- keratoconus [14]. In a NZ population, of which
nically matched control population. 49% were Polynesian, a large number of hetero-
Investigation of genetic variants in these pop- zygous variants in ZNF429 were detected, includ-
ulations requires an understanding of, and sensi- ing in 12 of the 21 Polynesian probands [15].
tivity to, cultural beliefs and customs. Our studies Two of the variants (p.(R2129K), p.(G3415V))
have all been undertaken with institutional ethi- were absent or very rare in databases of human
cal approval, including review by the Auckland variation, and predicted to be harmful, but were
District Health Board Māori Research Review present in a higher frequency in the control
committee. Our research follows the principles of Polynesian population. Five probands carried
the Treaty of Waitangi and follows the tenets of two ZNF469 variants, including three Polynesian
the Declaration of Helsinki. All participants have families, but segregation was not complete in all
provided informed consent. families. In other populations, variants in this
gene have not been shown to be of significance to
disease pathogenesis. A larger study is required
5.1.1 Cornea to determine if variants in this gene may explain,
in part, the higher prevalence of keratoconus in
5.1.1.1 Keratoconus this population.
Keratoconus is a progressive corneal dystrophy
leading to a characteristic pattern of thinning and
bulging of the cornea (ectasia), with induced 5.1.2 Corneal Dystrophies
irregular astigmatism. Onset is usually in the
teens, and the incidence within the general popu- Many of the corneal dystrophies have been
lation is estimated at 1 in 2000 [5]. In NZ, kerato- genetically characterised [16], with a consensus
conus is disproportionately the major indication classification established, and updated, by the
for corneal transplantation, accounting for 41% International Committee for Classification of
of keratoplasties over a 10-year period and 67% Corneal Dystrophies (IC3D) [17].
of all paediatric surgeries [6]. This compares with In our studies probing the genetics of corneal
rates of 11.4–28.8% in the USA and France [7– dystrophies, the prevalence of disease is not
9]. The incidence is thought to be higher in the clearly different from the general population;
Māori and Pacific populations; based on corneal however our numbers are small. In individuals
topographic values, suspected keratoconus in of Polynesian ethnicity, probing the TGFB1,
5 Inherited Ocular Disease in the New Zealand Māori: Novel Genetic Mechanisms and Founder Effects 43
VSX1, and ZEB1 genes, we have not identified

the genetic cause for disease (stromal fleck
n = 1, posterior polymorphous dystrophy n = 3)
[13, 18–21].
5.1.3 Anterior Segment Dysgenesis
A NZ Māori woman had a historical diagnosis of

aniridia, and congenital glaucoma, but on presen-
tation had an enucleated right eye and extensive
surgeries to the remaining eye, making clear phe-
notyping difficult; however we excluded a PAX6
mutation. She subsequently gave birth to a son, Fig. 5.1 Autosomal recessive ectopia lentis. Slit lamp
affected with posterior embryotoxon and iris photo demonstrating lens subluxation in autosomal reces-
hypoplasia. A novel PITX2 missense variant, sive ectopia lentis in a patient homozygous for ADAMTSL4
c.2237G>A, p.(Arg746His)
c.344G>A; p.(Arg115His) at a highly conserved
residue in the homeobox domain, was identified,
which segregated with disease in the family [22]. introduced into a small community by European
It was predicted to be pathogenic, and not present sailors in the early 1800s.
in 100 ethnically matched alleles. This pathogenic
variant segregated with disease in the family.
5.1.5 Glaucoma
5.1.4 A
utosomal Recessive Ectopia Primary open-angle glaucoma is not common in
Lentis the NZ Māori population. In an assessment of the
demographics of glaucoma in 567 individuals in
Isolated, non-syndromic ectopia lentis attribut- NZ, Māori and Pacific peoples were underrepre-
able to mutations in the ADAMTSL4 gene sented for all types of glaucoma except primary
(AREL) has been observed in multiple popula- angle closure [26]. In our series of 115 POAG
tions, with a common 20 base pair deletion patients in which we screened known glaucoma
described in the European population, with evi- genes (MYOC, CYP1B1, OPTN and WDR36),
dence for a founder mutation over 4000 years ago none were from these ethnic groups [27]. A
[23]. In a cohort of 11 AREL patients, we identi- recent case report describes the first recorded
fied a recurrent population-specific variant, observation of pseudoexfoliation syndrome
c.2237G>A, p.(Arg746His) [24]. This variant (PXF) and glaucoma in a Māori male [28]. A pre-
was present in 81% (9/11) of probands with an vious study in Rarotonga, Cook Islands, observed
early onset (under the age of 5 years) of AREL PXF in 3 of 1000 subjects examined [29]. LOXL1
(Fig. 5.1). 77.8% (7/9) of these probands were variants were not studied in any of these reports.
Polynesian (Cook Island Māori n = 5, NZ Māori
n = 2), and the remainder were NZ European. All
individuals with this variant, regardless of ethnic- 5.1.6 Retinoblastoma
ity, shared a common haplotype, suggestive of a
founder effect. This pathogenic variant had previ- In a retrospective audit of retinoblastoma genet-
ously been reported once, in Turkey [25], and we ics seen through the Auckland Hospital
hypothesised that it is possible this variant trav- Ophthalmology service, for a 5-year period from
elled from the Northern Hemisphere and was 2003 to 2008, we identified 20 patients, including
44 A. L. Vincent
1 family, with a 100% mutation detection rate methodology, tested for known mutations in
[30]. Four individuals were Māori (20%), and known genes on a “disease” microarray, e.g.
germ line mutations accounted for 75% of their autosomal recessive retinitis pigmentosa (ARRP)
disease, and a further 3 patients were Pacific peo- or autosomal dominant RP (ADRP). These tradi-
ples (15%). Disease in two Māori patients and tionally have had a low yield in our population.
one Cook Island Māori patient was attributed to a More recently their technology has been updated
de novo germ line mutation. Although the to incorporate a next-generation sequencing
numbers are small, and therefore not statistically (NGS) strategy around the regions of known
significant, the higher percentage of this disease mutations, but still does not sequence the gene(s)
in these ethnic groups may indicate a predisposi- in its entirety. Specific genes have been geno-
tion to de novo mutations or heritable disease, as typed over the years at a variety of institutions
has been shown with US Latino patients with including the Carver Lab, Iowa, and the Molecular
retinoblastoma, compared to non-Latinos [31]. Vision Laboratory (MVL, formerly Casey Eye
Institute), Portland, Oregon, as well as the
Genomic Diagnostics Laboratory, Manchester
5.1.7 I nherited Retinal Dystrophies Centre for Genomic Medicine, Manchester, UK,
(IRD) for X-linked RP genes. Newer technologies such
as NGS targeted disease panels are available,
In 2009, the New Zealand Registry of Inherited with reducing prices, allowing more targeted and
Retinal and Optic Nerve Disease was established, comprehensive screening of candidate genes, e.g.
with all participating patients recruited from the the macular panel at MVL costs $500USD and
Ocular Genetic Clinic, at the Eye Department, fully sequences 13 genes, including ABCA4, and
Greenlane Clinical Centre, Auckland District includes the intronic regions. Retinal disease
Health Board. This is a tertiary referral clinic, panels incorporating hundreds of genes are avail-
with patients from all over NZ referred for assess- able but – at the time of writing – are still cost-
ment. All individuals in this public hospital clinic prohibitive for our public health system.
are extensively phenotyped: history, pedigree, The yield for a positive genetic diagnosis is
visual acuity, dilated slit lamp and fundal exami- low in the Māori and Pacific peoples’ cohort
nation, OCT, fundus photography and fundus using the microarray panels (discussed under
autofluorescence, and, if possible, electrophysi- individual diseases below). Using a NGS targeted
ology. Genetic testing is initiated at the end of the retinal disease panel (105 genes, Manchester
consultation if appropriate, and only at this time Centre for Genomic Medicine), we undertook
is the invitation to participate in the IRD database analysis in a cohort of 28 Māori and Pacific peo-
given and informed consent obtained to enrol. ples with IRD (Vincent et al., IOVS 2016:57;12
Participation means that if no genetic diagnosis is ARVO e-abstract 3157). The diagnosis in these
made at commercially available testing facilities, patients was autosomal recessive rod-cone dys-
the option to explore the underlying genetic cause trophy (ARRP n = 15), dominant RP (ADRP
for disease on a research basis is permitted. To n = 2), Leber congenital amaurosis (LCA n = 3),
date there are over 700 probands and family maculopathy (n = 4), or cone/cone-rod dystrophy
members. This is a valuable tool to determine the (CORD n = 4). 21 unique, pathogenic variants
incidence, nature, and spectrum of different IRD (12 novel) were observed, allowing a definitive
phenotypes and genotypes in New Zealand. genetic diagnosis in 16/28 (57%) cases.
Commercially available testing has evolved Homozygosity was seen for four (PDE6B, RD3,
over the years; however the cost of testing is the SPATA7, PROM1). All LCA and ADRP cases
main determinant in a publically funded eye were solved. Two ARRP cases had mutations in
clinic. Asper Ophthalmics in Estonia offers a ADRP genes. Sixty percent of ARRP cases
microarray platform, which uses an APEX-based remain unsolved [32].
5.1.8 L
eber Congenital Amaurosis/ Pro50Leu [33], was identified that segregated
Early-Onset Severe Retinal with disease in the family.
Dystrophy (LCA/EOSRD) A 19-year-old Samoan female (Patient #6)
described an onset of symptoms, particularly
Childhood blindness due to IRD carries a signifi- nyctalopia, from the age of 8 years. Reportedly
cant social and economic burden and is estimated her brother, mother, and maternal grandmother
to affect 3–4 children in 10,000. LCA and also had similar symptoms, in particular night
EOSRD are at the severe end of this spectrum of vision difficulties. On examination her BCVA
disease, with children presenting at birth, or in was 6/7.5 OU, with relatively featureless fundi
the first few years of life with poor vision, roving with peripheral atrophy, significant vessel attenu-
eye movements, and non-recordable retinal elec- ation, sparse pigment and optic disc pallor, and
trical responses. bilateral cystic maculopathy (Fig. 5.3c–f). The
Of the estimated 56–140 children affected presumed diagnosis was autosomal dominant
with LCA/EOSRD in NZ, we have identified 41 (AD) rod-cone retinal dystrophy. NGS identified
children from the database, for which 25 have no a rare novel missense mutation in PRPF31,
genetic diagnosis. The genetic cause for disease c.682G>C, p.(Ala228Pro), and predicted to be
is identified in 16 individuals from 14 families, pathogenic. A second pathogenic variation in
with mutations in CRB1 (n = 5 probands), RPE65 IMPG2 (c.331C>T, p.(Arg111*)) previously
(n = 2), and singletons for GUCY2D, CRX, LCA5, undescribed was also present. IMPG2 is associ-
SPATA7, TULP1, PROM1, and RD3. Four of ated with both recessive and dominant vitelliform
these probands are NZ Māori/Pacific peoples, macular dystrophy (VMD) [34].
with pathogenic variants observed in LCA5, RD3, Patient #7: A 28-year-old Maori male had
RPE65, and SPATA7, of which novel changes nyctalopia for all of his life, with subsequent
were identified in 75% using a NGS targeted restriction of visual fields and gradual deteriora-
panel (105 retinal genes) in 2013 (Vincent AL tion of central vision in his early 20s. His mother
et al., IOVS 2016.57:12 ARVO E-Abstract 3157) (Ngati Putenga Hauraki) was also affected. On
(Table 5.1 and Fig. 5.2). examination, BCVA was NPL OD from trauma,
6/21 OS, with a diagnosis of presumed AD rod-
cone dystrophy (Fig. 5.3g, h). The NGS panel
5.2 Rod-Cone Dystrophies identified a previously identified IMPDH1
disease-causing variant c.968A>G, p.
5.2.1 A
utosomal Dominant Rod- (Lys323Arg).
Cone Dystrophy (ADRP)
The NZ IRD database includes 48 families with 5.2.2 A

utosomal Recessive Rod-
presumed ADRP, including 4 Māori, 1 Tongan, 1 Cone Dystrophy (ARRP)
Niuean, and 1 Samoan. A genetic diagnosis has
been made in 26 (54%) of the entire ADRP There are 117 probands with presumed ARRP in
cohort. In the Māori/Pacific peoples’ cohort, the the NZ IRD database, which includes those with
genetic cause has been identified in three fami- sporadic disease, no other affected family mem-
lies, in IMPDH1, PRPF31, and GUCA1A bers, and no known consanguinity. The X-linked
(Table 5.1). RP genes have been excluded in many of the
Patient #5: A 32-year-old Māori male had an affected sporadic males. Thirty-one of the pro-
onset of his symptoms at primary school and by bands (26%) are of Māori or Pacific peoples’ eth-
age 27 had 6/60 vision OU (Fig. 5.3a, b). His nicity (Māori n = 17, Samoan n = 9, Cook Island
father was affected, and a previously reported Māori n = 3, Niuean n = 1, Tongan n = 1). The
pathogenic variant in GUCA1A c.149C > T, p. diagnostic yield in this cohort using the Asper
Table 5.1 Genetic variants identified in inherited retinal dystrophies in the NZ Māori and Pacific Peoples
Result Allele frequency Protein Prediction
PolyPhen-2 Mutation taster Previously
ID Ethnicity Gene cdna Zygosity Protein EVS Exac 1000G (score) (p-value) SIFT (score) reported
AR LCA
1 Māori LCA5 c.194delC het 0 0 0 N/A Disease N/A No
causing (1)
LCA5 c.103C>T het p.(Arg35*) 0 0.00002471 0 N/A Disease N/A Yes
causing (1)
2 Tongan RD3 c.127C>T Hom p.(Gln43*) 0 0 0 N/A Disease N/A No
causing (1)
3 Samoan SPATA7 c.738_739dupAA Hom 0 0 0 Probably Disease Deleterious No
damaging causing (1) (0.00)
(0.992)
4 Māori RPE65 IVS1+5G>A Hom Splice 4/13002 0.00009094 0.0002 Splice Yes
ADRP 0.001203
5 Māori GUCA1A c.149C>T het p.Pro50Leu 13/12993 0.00001652 0 Benign Tolerated
(0.32) (0.1)
6 Samoan PRPF31 c.682G>C het p.(Ala228Pro) 0 0 0 Probably Disease Deleterious No
damaging causing (0.03)
(1.0) (0.9999)
IMPG2 c.331C>T het p.(Arg111*) 0 0.00001652 <0.01 N/A Disease N/A No
causing (1)
7 Māori IMPDH1 c.968A>G het p.(Lys323Arg) 0 0 0 Benign Disease Deleterious Yes
(0.370) causing (0.00)
(0.999)
ARRP
8 Māori PDE6B c.2197G>C Hom p.(Ala733Pro) 0 0 0 Probably Disease Deleterious No
damaging causing (1) (0.00)
(0.99)
XLRP
9 Māori RP2 c.945_946insT Hemi p.(Asn316*) 0 0 0 Insertion, No
premature
termination
10 Samoan RPGR c.283G>A Hemi p.Gly95Arg 0 0 0 Probably Disease Deleterious No
(1.0) (0.999)
11 Māori RPGR c.1236_1239delAAGA Hemi p.(Glu414Glyfs*10) 0 0 0 Deletion No
premature
termination
C1QTNF5 c.583dupG het p.(Ala195Glyfs*8) 0.00% 0 0.001 Insertion,
premature
termination
12 Māori RPGR c.2360delA Hemi 0 0 0 No
ORF15
Maculopathy
13 Māori ABCA4 c.5584G>T Hom p.(Gly1862Cys) 0 0 0 Probably No
damaging
(1.0)
14 Māori CRX c.774T>G het p.(Tyr258*) 0 0 0 Premature No
termination
15 Māori RP1L1 c.133C>T. het p.(Arg45Trp) 1/12603 0.00002 0.00023 Probably Polymorphism Deleterious Yes
damaging (0.01)
(1.0)
Cone-rod
16 Māori BBS9 c.205C>A het p.(Leu69Ile) 0 0 0.00002 Probably Disease Deleterious No
(0.968) (0.999)
BBS9 c.1014_1015delinsTT het p.(Leu338_ 0 0 0 Unknown No
His339delinsPheTyr)
17 Māori PROM1 c.1354_1355insT Hom p.(Y452Lfs*13) 3/12253 0.00015 0.00245 Unknown Disease Yes
causing
Hom Homozygous, het Heterozygous, hemi Hemizygous, EVS Exome variant server, EXAC Exome aggregation consortium
48 A. L. Vincent
Fig. 5.2 Retinal images of an 18-year-old Māori female vessel attenuation, but no pigment, and (b) fundus
with Leber congenital amaurosis (Patient #4) autofluorescence showing a paramacular ring of

homozygous for RPE65 IVS1+5G>A. (a) Optos widefield hyperautofluorescence
fundus photo showing mottling of the retina with mild
ARRP microarray was zero, and a causative potentially simplify the diagnostic process, i.e.
genetic diagnosis has only been possible using genetic testing for the single PDE6B variant can
the NGS targeted disease panel as described in be initiated instead of a more expensive, multi-
our recent publication [32]. gene NGS panel or array.
A novel homozygous PDE6B variant, PDE is a protein that has a high concentration
c.2197G > C; p.(Ala733Pro), was initially identi- in the peripheral membrane of retinal photorecep-
fied in four probands (#8) on the NGS panel and tors and integral to the phototransduction cycle. It
subsequently in a further six probands, based on consists of two catalytic subunits, PDE6A and
similarity of the retinal phenotype and their iwi PDE6B, and two gamma inhibitory subunits. This
(tribe). Nearly all probands came from the protein reduces the level of cyclic guanine mono-
Northern region of the North Island (Iwi Ngā phosphate (cGMP) by hydrolysation, thereby
Puhi) or the Gisborne, East Cape region (Iwi resulting in channel closure in response to light
Ngāti Porou). Nearly 200,000 (30%) of NZ [35, 36]. Mutations of the PDE6B gene result in
Māori identify with these two iwi. Based on our accumulation of cGMP due to a dysfunctional
observed minor allele frequency of 0.0076, and PDE protein, leading to photoreceptor cell death
assuming Hardy Weinberg equilibrium, we [37, 38]. Although autosomal recessive rod-cone
would only expect 11 affected individuals, and dystrophy is the most common phenotype
we have already identified 10. However, the car- described in association with mutations in this
rier frequency is likely higher within certain iwi, gene, a CSNB (congenital stationary night blind-
and this PDE6B variant may account for 16% of ness) phenotype is also described. The majority of
recessive IRD in Māori. pathogenic variants occur within the C terminal
The phenotype across all affected individuals catalytic terminal domain, including p.Ala733Pro,
was fairly consistent, with onset usually described and lead to complete loss of enzymatic activity
around the age of 20 with nyctalopia, and the fun- and subsequent accumulation of cGMP. The intra-
dus appearance showing a fairly lacy pattern of cellular build-up of cGMP is known to cause pho-
mid-peripheral bone spicule pigmentation, and toreceptor toxicity.
often a bullseye maculopathy (Fig. 5.4). By understanding the pathophysiology, it is
A careful history, pedigree ascertainment, and feasible to target an aberrant process created by
knowledge of familial iwi in a NZ Māori patient the pathogenic variants in the PDE6B gene. Using
presenting with a rod-cone retinal dystrophy, and animal models, rd1 and rd10 mice, treated with
familiarity with the retinal appearance of the PARP inhibitors and cGMP analogues, Sahaboglu
PDE6B variant, should always be considered and et al. demonstrated a reduction in cGMP accumu-
Fig. 5.3 Clinical features of autosomal dominant rod- and fundus autofluorescence OD (d) and OCT images of
cone dystrophies. GUCA1A c.149C>T, p.Pro50Leu coexisting cystic maculopathy (e) OD and (f) OS, with loss
(Patient #5), Optos widefield photo OD (a) and fundus of photoreceptors outside the subfoveal region. IMPDH1
autofluorescence OD (b). PRPF31, c.682G>C, p. c.968A>G, p.(Lys323Arg) (Patient #7) Optos widefield
(Ala228Pro) (Patient #6) Optos widefield photo OD (c) photo OS (g) and fundus autofluorescence OS (h)
lation and increased photoreceptor survival, con- gene editing techniques, specific to the pde6b
firming in vivo neuroprotection [39]. p.(Ala733Pro), to utilise as a resource for high-
We are currently creating a zebrafish model of throughput drug screening.
disease, with morpholino and CRISPR/Cas9
50 A. L. Vincent
Fig. 5.4 Clinical imaging of the founder PDE6B nal atrophy, vessel attenuation, and sparing of the macula
c.2197G>C; p.(Ala733Pro) variant associated with auto- in a 45-year-old. Fundus autofluorescence with the Optos
somal recessive rod-cone dystrophy. Optos widefield pho- OD (c), OS (d), and Spectralis OD (e), OS (f), with a peri-
tos OD (a) and OS (b) demonstrating the characteristic macular hyperfluorescent ring and bullseye appearance
mid-peripheral dense bone spicule pigmentation and reti-
5.3 X-Linked Disease 5.3.2 X-Linked Retinoschisis
5.3.1 Choroideremia Of seven probands with X-linked retinoschisis

and mutation-positive RS1 genetic analysis, all
Of the seven probands with a clinical diagnosis of are of NZ European ethnicity (Vincent AL
choroideremia and a positive genetic test of the et al., IOVS 2017.58:8 ARVO E-Abstract
CHM1 gene, none are Māori or Pacific peoples. 2770).
5.3.3 X-Linked Congenital and an ORF15 deletion c.2630delA, which segre-

Stationary Night Blindness gated with disease in 18 members of the family
(#12). A number of obligate carrier females
The clinical features of a large pedigree of Māori showed significant disease manifestation. In this
ethnicity with incomplete CSNB have been family, keratoconus also segregated with disease;
described by Hope et al. [40] and attributable to however the high incidence of keratoconus in this
the previously undescribed CACNA1F population has previously been discussed
c.2267T>C, p.Ile756Thr (also known as p. (Vincent AL et al, IOVS 2017.58:8 ARVO
Ile745Thr) allele. All affected males demon- E-Abstract 2770).
strated severe nonprogressive visual impairment, In a further male proband (#11), with three
with congenital nystagmus, altered colour vision, affected daughters, two variants were detected. A
hyperopia, and normal fundi. Some of these heterozygous variant in C1QTNF5, c.583dupG,
males were also intellectually disabled. In this p.(Ala195Glyfs*8), is rare and predicted to be
family, the obligate carrier females all showed a damaging, and he was also hemizygous for
moderate to severe ocular phenotype, with RPGR c.283G>A p.(Gly95Arg). We were how-
decreased vision and congenital nystagmus, and ever unable to confirm segregation, the pheno-
were often highly myopic. In both male and type was not typical for late-onset retinal
females, the ERG showed a characteristic nega- dystrophy (LORD) described with C1QTNF5,
tive wave form with reduction of both rod and and as his sister’s son was also reportedly
cone responses. affected, the RPGR variant was thought to be the
Further individuals from the extended fam- causative allele.
ily have subsequently been identified and geno-
type confirmed, although they did not always
directly know of the familial association. A 5.4 Maculopathies and Cone/
careful history, knowledge of the phenotype, Cone-Rod Dystrophies
and identification of iwi usually will help target
the genetic testing strategy and diagnostic 5.4.1 ABCA4-Associated Disease
algorithm.
In the NZ IRD database, 81 probands have at
least 1 pathogenic variant or VUS in ABCA4 and
5.3.4 X
-Linked Rod-Cone Retinal a clinical picture consistent with disease. Six of
Dystrophy (XLRP) these probands identify as Māori, including two
sib pairs.
We have characterised 19 families with XLRP, A novel homozygous variant c.5584G>T, p.
attributable to mutations in the RP2 gene (n = 1) (Gly1862Cys) observed in one proband (#13)
and RPGR, including ORF15 (n = 18) (Vincent with a maculopathy, occurs in the last nucleotide
AL et al, IOVS 2017.58:8 ARVO E-Abstract of exon 39 and is predicted to be probably dam-
2770). Of these families, three were NZ Māori aging by PolyPhen-2 with the maximum score of
and one Samoan, and the disease-causing vari- 1.000. This variant does not occur in databases of
ants observed in this ethnic group were not previ- human variation and was also homozygous in his
ously reported and segregated with disease in the affected sibling. Both siblings had a similar man-
family. In RP2, the variant c.945_946insT is pre- ifestation of disease.
dicted to cause a premature termination of the This variant was not detected initially using
protein p.(Asn316*). the ABCA4 Asper microarray in 2010, but only
The other three variants were in RPGR; subsequently in 2015 with testing on the MVL
c.283G>A, p.(Gly95Arg), a missense variant in NGS macular panel, which sequences ABCA4 in
exon 10, c.1236_1239delAAGA, also resulting its entirety, including probing for deep intronic
in premature termination p.(Glu414GlyfsTer10) variants.
52 A. L. Vincent
5.4.2 Non-ABCA4 Maculopathies in latency for both photopic flash and flicker at
30 Hz. The pattern ERG was poorly defined.
At the end of 2016, we identified patients with Mutations in the CRX gene have been shown
maculopathies or cone-rod dystrophies (CORD), to be associated with autosomal dominant LCA
in which ABCA4 had been excluded as the cause type III and as well as AD cone-rod dystrophy
of disease or a pathogenic variant(s) identified in and maculopathy [41].
a gene consistent with the phenotype. Eighty
patients were diagnosed with a maculopathy and
22 with a CORD. Vitelliform macular dystro- 5.4.4 Occult Macular Dystrophy
phies (VMD) were the most common maculopa-
thy (25.0%) of which 50.0% were BEST1 A 48-year-old NZ Māori male (Patient #15)
positive. Interestingly none of the individuals noticed an onset of deterioration in his vision in
with VMD were Māori or Pacific people. The his late teens and was troubled by glare. His
CORDs showed higher proportions of males vision measured 6/60 OD and 6/48 OS, with a
(68.2%), Māori (27.3%), and Polynesian (9.1%) localised area of photoreceptor abnormality sub-
patients than the maculopathies. A high propor- foveally (Fig. 5.5b). Full-field ERG was unre-
tion of Māori and Polynesian patients also lacked markable, and a pattern ERG showed a reduced
genetic characterisation. Eleven patients were p50 wave, consistent with a localised macular
identified with RDS/PRPH2 mutations and abnormality. He was heterozygous for the most
exhibited wide phenotypic variability, yet once commonly reported pathogenic variant in RP1L1,
again none of these individuals were Māori or c.133C>T. p.(Arg45Trp), as was his affected sis-
Pacific peoples, adding further weight to a differ- ter (Fig. 5.5b). Occult macular dystrophy has
ent genetic spectrum of disease in these ethnic been reported predominantly in the Japanese
groups. population [42].
5.4.3 CRX-associated CORD 5.4.5 BBS9 Cord
Patient #14 had a novel heterozygous nonsense A 43-year-old NZ Māori male (Patient #16)
variant, c.774 T p.(Tyr258ter), in the CRX gene became aware of shadows in his vision in his
when analysed by the Manchester NGS retinal early 30s, with vision measuring 6/9 at the
panel. This change was not present in databases time, but deteriorating to 6/60 2 years later and
of human variation. This patient had simplex to 1/60 at age 43. Electrophysiology initially
inheritance with a late-onset (age 50) severe showed marked attenuation of cone function
cone-rod dystrophy, with vision 6/60 OU at age and mildly affected rod photoreceptor function.
55 years. Fundal photography showed paramacu- When repeated at age 43, rod function was now
lar greying and atrophy of retinal pigment epithe- barely recordable. The fundus appearance
lium with a bullseye pattern and a central area of showed widespread sharply demarcated areas
loss of photoreceptors on OCT and a central dark of atrophy and small islands of residual retina.
area with a hyperfluorescent ring on FAF The retinal arterioles are mildly attenuated, and
(Fig. 5.5a). both maculae have sub-retinal fibrosis
Electrophysiology was performed to ISCEV (Fig. 5.5c). The patient had no other systemic
standards. Rod-mediated function was identified features but described multiple relatives on
with reduced photoreceptor A wave amplitude, both sides of the family with polydactyly.
but relatively normal B wave. Cone-mediated Testing with the Manchester NGS retinal panel
function was identified but significantly reduced identified compound heterozygosity for two
in amplitude to about 50% normal and increased novel variants in BBS9: c.205C>A, p.
Fig. 5.5 Clinical images of four patients with maculopa- demonstrating sparse pigment clumping and sharply
thies/cone-rod dystrophies. (a). CRX Patient #14 with a demarcated areas of retinal atrophy (i), corresponding to
heterozygous CRX variant, c.774T p.(Tyr258*): macula areas of hypoautofluorescence (ii), and marked thinning
photo (i), fundus autofluorescence (ii) and OCT (iii). (b). of the outer retina and loss of photoreceptor architecture
Occult macular dystrophy Optos photo (i) OD in Patient (iii). (d). PROM1 Retinal phenotype of autosomal reces-
#15 with RP1L1 c.133C>T, p.(Arg45Trp), and fundus sive PROM1 CORD c.1354_1355insT, p.(Y452Lfs*13),
autofluorescence (ii) and OCT (iii) in his affected sister disease (Patient #17), with foveal granularity and peripap-
(all OD). (c). BBS9 Patient #16, a compound heterozygote illary atrophy (i), reduced macular autofluorescence (ii),
for two novel BBS9 variants, c.205C>A, p.(Leu69Ile), and and disturbance of the photoreceptor outer segments in
c.1014_1015delinsTT p.(Leu338_His339delinsPheTyr) the foveal and perifoveal regions (iii)
(Leu69Ile), and c.1014_1015delinsTT p. 5.5 Conclusions

(Leu338_His339delinsPheTyr), both of which
are rare and predicted to be pathogenic. His Local knowledge and characterisation of disease
parents were heterozygotes for a single change prevalence and genetic variation within the New
each, but other family members were not avail- Zealand Māori and Pacific peoples have identi-
able for clinical nor genetic characterisation. fied ethnic-specific variation in the spectrum of
inherited eye disease compared to the NZ
European population. Keratoconus is more fre-
5.4.6 PROM1 CORD quent but glaucoma less common. Several
founder mutations are identified in ADAMTSL4
At the age of 3 years, a NZ Māori male was noted associated with autosomal recessive ectopia len-
to have poor vision and was very light sensitive. tis and PDE6B in ARRP. Knowledge of these
Initially a myopic correction was prescribed. changes, the presenting phenotype, and the
When seen at age 7 years, his visual acuity was regional origin and iwi of the patient can simplify
6/18 OD, 6/21 OS, with fine granularity noted at the diagnostic algorithm, to inform targeted
each fovea (Fig. 5.5d). Electrophysiology showed genetic testing. As this population is not well rep-
reduced rod-mediated amplitudes and non- resented in databases of human variation, segre-
recordable cone function and pattern ERG. On gation and in silico analysis are paramount to
the NGS panel, a homozygous PROM1 patho- determining pathogenicity of any variant
genic variant c.1354_1355insT, p.(Y452Lfs*13), detected.
was present and confirmed heterozygously in his Although many of our probands have not
non-consanguineous parents. undergone state-of-the-art NGS gene panel inves-
54 A. L. Vincent
tigation, the unique and novel variants identified 10. Owens H, Gamble GD, Bjornholdt MC, Boyce

NK, Keung L. Topographic indications of emerg-
to date provide a strong argument to justify fur- ing keratoconus in teenage New Zealanders. Cornea.
ther investigation within the NZ Māori and 2007;26(3):312–8.
Pacific peoples and a high likelihood of identifi- 11. Jordan CA, Zamri A, Wheeldon C, et al. Computerized
cation of further novel genetic variation as the corneal tomography and associated features in a large
New Zealand keratoconic population. J Cataract
cause for their eye disease. Refract Surg. 2011;37(8):1493–501.
12. Burdon KP, Vincent AL. Insights into keratoco-

Informed Consent All procedures followed were in nus from a genetic perspective. Clin Exp Optom.
accordance with the ethical standards of the responsible 2013;96(2):146–54.
committee on human experimentation (institutional and 13. Vincent AL, Jordan C, Sheck L, et al. Screening the
national) and with the Helsinki Declaration of 1975, as visual system homeobox 1 gene in keratoconus and
revised in 2000. Informed consent was obtained from all posterior polymorphous dystrophy cohorts identifies
patients for being included in the study. a novel variant. Mol Vis. 2013;19:852–60.
Additional informed consent was obtained from all 14. Lu Y, Vitart V, Burdon KP, et al. Genome-wide asso-
patients for which identifying information is included in ciation analyses identify multiple loci associated with
this article. central corneal thickness and keratoconus. Nat Genet.
No animal studies were carried out by the authors for 2013;45(2):155–63.
this article. 15. Vincent AL, Jordan CA, Cadzow MJ, Merriman TR,
McGhee CN. Mutations in the zinc finger protein gene,
Compliance with Ethical Requirements Author ALV ZNF469, contribute to the pathogenesis of keratoco-
has no conflicts of interest to declare. nus. Invest Ophthalmol Vis Sci. 2014;55(9):5629–35.
16. Oliver VF, Vincent AL. The genetics and patho-

physiology of IC3D category 1 corneal dystro-
phies: a review. Asia Pac J Ophthalmol (Phila).
2016;5(4):272–81.
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Genetics of Ocular Diseases
in Malaysia 6
A. T. Liza-Sharmini and T. A. Kamalden
Abstract tion, while SNP of 2245G/A was found to be

Understanding the role of genetics in clinical associated with diabetic retinopathy, possibly
practice has evolved drastically in Malaysia. through NF-κβ-mediated pro-inflammatory
However, the understanding of genetics in pathway. Exome-wide association studies
ocular diseases is still at the infancy. In this (EWAS) on a pilot cohort of 20 premature
chapter, we summarize the publications on Malaysian infants, different loci of SNPs in
genetic studies involving Malaysian popula- LRP5, FZD4, ZNF408 (chromosome 10), and
tion on glaucoma, age-related macular degen- KIF 11 (chromosome 11) genes were identi-
eration, diabetic retinopathy, retinopathy of fied. No specific hot spot in RB1 gene in
prematurity, and retinoblastoma. Potential Malaysian children with retinoblastoma was
susceptibility genetic markers for primary found. In general, more exploration is needed
open-angle glaucoma (POAG) and primary in understanding the genetic influences in ocu-
angle-closure glaucoma (PACG) were identi- lar diseases in Malaysia.
fied through single gene analysis and genome-
wide association study (GWAS). TGF-ß Keywords
signaling pathway is the potential susceptibil- Ocular genetics · Malaysia · Glaucoma ·
ity gene for POAG. MYOC gene was also Retinopathies
identified in a large Malay family with
juvenile-onset open-angle glaucoma (JOAG).
Single nucleotide polymorphism (SNP) of
VEGF+405 may play a role in wet age-related 6.1 Introduction
macular degeneration in Malaysian popula-
Genetic and molecular research in Malaysia is
still at its infancy. The Malaysia Institute for
A. T. Liza-Sharmini (*)
Department of Ophthalmology, School of Medical Medical Research (IMR) was established in
Sciences, Universiti Sains Malaysia, 1900, but the cytogenetic service was only
Kota Bharu, Kelantan, Malaysia available in 1968 [1]. About 28 years later,
e-mail: [email protected] molecular diagnostic service was established
T. A. Kamalden in 1996. Currently, there are many genetics
University of Liverpool, Liverpool, UK laboratories in Malaysia that cater for research
Faculty of Medicine, Department of Ophthalmology, rather than diagnostic services. However, the
UM Eye Research Centre, University of Malaya, research activities have not been coordinated
Kuala Lumpur, Malaysia

https://doi.org/10.1007/978-981-13-0884-0_6
58 A. T. Liza-Sharmini and T. A. Kamalden
resulting in many laboratories conducting sim- 6.2 Glaucoma and Genetics

ilar tests and being counterproductive. Till in Malaysia
now, there is no laboratory in Malaysia that
caters for diagnostic service for ocular Glaucoma is a chronic progressive optic neuropa-
diseases. thy with special characteristics of structural and
Genetic studies on ocular diseases were almost functional damage. It is one of the major causes
nonexistent until about 20 years ago. This is due of irreversible blindness in Malaysia. Based on
to the emphasis given to other aspects such as in the Malaysia National Eye Survey II, glaucoma
the development of ophthalmic services, training contributed 7% of blindness and 3% of severe
of ophthalmologists and supporting staffs, and visual impairment [6]. Glaucoma is a complex
strategic planning for prevention of common disease with interplay between genetic predispo-
causes of blindness such as cataract. However, sition and environmental factors [7]. Formulating
with the increasing number of ophthalmologists the diagnosis of glaucoma is challenging without
and availability of training in molecular research consensus in structural and functional changes,
among clinicians, the interest in ocular genetic intraocular pressure (IOP), and other factors. For
has escalated. years, various definitions were adopted resulting
Malaysia is a multiracial country bordered by in difficulty in providing an evidence-based man-
Thailand and Indonesia. Malays, Chinese, and agement. However, since the formation of an
Indians are the main ethnic groups in Malaysia. expert panel in glaucoma, World Glaucoma
Malays and other indigenous people contributed Association in 2001, a series on consensus have
to 67.4%, Chinese 24.6%, Indian 7.3%, and oth- been developed to standardize diagnosis and
ers 0.7% [2]. This diversity has led to variation patients’ care.
in phenotype and genotype of ocular diseases. Glaucoma has all the characteristics of a com-
Most ocular diseases are complex, and under- plex trait including locus heterogeneity, poly-
standing the disease in a multiracial country like genic inheritance, phenocopies, and incomplete
Malaysia is challenging. penetrance [8, 9]. To complicate the matter fur-
Currently, knowledge on clinical presenta- ther, the types of glaucoma are usually deter-
tion and genetic predisposition on ocular dis- mined by various mechanisms that end up with a
ease in Chinese and Indians is abundant [3, 4]. similar final pathway, glaucomatous optic neu-
This is basically due to advancement of research ropathy. Furthermore, the exact pathogenesis of
from China and India. Due to genetic inheri- glaucoma is not well understood. Without a
tance, the findings can be extrapolated to doubt, genetic component has a role in glaucoma.
Chinese and Indians residing in Malaysia with First-degree relatives of primary open-angle
the assumption of minimal environmental effect glaucoma (POAG) patients have a tenfold higher
on these populations. There is minimal knowl- risk of developing POAG compared to general
edge on ocular genetics in Malays and other populations [10]. Family history is one of the risk
indigenous people in Malaysia. Malays contrib- factors for glaucoma particularly POAG, primary
ute to 5% of general population in the world [5]. angle-closure glaucoma (PACG), and juvenile-
In this chapter, we concentrate on the available onset open-angle glaucoma (JOAG) [11, 12].
genetic data of common ocular diseases such as
glaucoma, age- related macular degeneration
(ARMD), retinopathy of immaturity (ROP), and 6.3 Single Gene Analysis
retinoblastoma (RB) among the Malaysian
population. Identification of genetic association to glaucoma
begins with single gene analysis. Thus, so far,
there are various potential genetic loci which
6 Genetics of Ocular Diseases in Malaysia 59
have been identified as susceptibility gene of encodes olfactomedin-like domain [19]. In spite
glaucoma. Among these genetic loci, only three of vast research on MYOC in many types of glau-
candidate genes were identified: myocilin gene coma, the impact of MYOC remains for POAG
(MYOC), optineurin gene (OPTN), and WD40- and JOAG. However, MYOC mutations were
repeat 36 gene (WDR36) [13–15]. There is no only found in 2–4% of POAG patients and 8–30%
available genetic screening for OPTN and in JOAG patients [19, 20].
WDR36 in Malaysian population. As yet, there is no available data on the asso-
ciation between MYOC and POAG among
Malaysians. A screening of MYOC among a
6.3.1 Myocilin Gene (MYOC) large Malay family with JOAG was conducted
and 122 of the family members were thoroughly
MYOC consists of 3 exons separated by 2 introns examined [21]. A total of 32 probands were iden-
and a 5 kb promoter region, encoding for tified: 11 new cases and 22 known cases.
55–57 kDa myocilin protein with 504 amino Autosomal dominant inheritance pattern with
acids and an isoelectric point approximately 5.21 variable penetrance was observed in this family
[16]. It encodes a secretory trabecular meshwork- (Fig. 6.1). Penetrance in JOAG is age dependent
inducible glucocorticoid response (TIGR) myo- and mutation specific [19]. Disease-causing
cilin protein, which is expressed in many human mutations (DCM), Asn480Lys (C→A at position
tissues including the iris, ciliary body, and tra- 1440 in exon 3) and a synonymous polymor-
becular meshwork [17]. TIGR myocilin protein phisms IVS2 730 +35G>A (rs2032555), were
has an amino terminal signal sequence, an also detected in this family. Asn480Lys was
olfactomedin domain, a myosin-like domain, and found in all probands with JOAG except for two
a leucine zipper domain [18]. The majority of [21]. Additionally, six probands with Asn480Lys
genetic variations were found in exon 3 which but without the disease development were identi-
Fig. 6.1 Pedigree chart of a large Malay family with juvenile-onset open-angle glaucoma and distribution of Asn480Lys
of MYOC
fied. Other research has shown that Asn480Lys 491C>T (rs1800885) on 97 glaucoma patients
was also identified in European population and (POAG and NTG) and 100 controls [29]. Out of
southern Indian POAG patients [22, 23]. these 197 subjects, there were 123 Malay and 74
Similarly, non-synonymous polymorphism IVS2 Chinese subjects who resided in Malaysia.
730 +35G>A was found in Chinese and southern Based on univariate analysis and without con-
Indian patients with POAG [24, 25]. sidering the potential effect of racial stratifica-
Lys480Lys and IVS2 730 +35G>A were sus- tion, there was no significant association
ceptible markers for Malay patients with JOAG between five polymorphisms of ADRB2 and
with linkage disequilibrium of 0.619 [21]. susceptibility to POAG and NTG. However,
Probands with Lys480Lys were detected at using stratified Mantel-Haenszel meta-analysis,
mid- twenties with lower IOP at diagnosis, minor allele 79C>G (79G) reduced the risk of
while IVS2 730 +35G>A was detected in pro- POAG by 0.3-fold (95% CI 0.1, 0.7) and -20T>C
bands at early twenties but with higher IOP at (-20T) increased the risk of NTG by 2.0-fold
diagnosis. There was no association of this (95% CI 1.1, 3.7).
DCM and nonsynonymous polymorphism
with severity of JOAG. Based on linkage dis-
equilibrium (0.619), there is a possibility of 6.3.3 P
rostanoid Receptor Gene
combination effect of Lys480Lys and IVS2 730 (PTGFR)
+35G>A. However, this potential effect was not
investigated in this study. Currently, prostaglandin analog is a potent first-
line topical drug for treatment of glaucoma. It
has gained popularity over the past 20 years
6.3.2 B
eta-2 Adrenoreceptor Gene since its first introduction in 1998 [30].
(ADRB2) Prostaglandin analog acts on specific receptor:
prostanoid (FP) receptor. Human FP receptor is
Most of pressure-lowering drugs work through encoded by PTG2α gene known as
interaction with receptors found in the trabecular PTGFR. PTGFR is located at short arm of chro-
meshwork, uveoscleral pathway, and ciliary mosome 1(13.1) [31]. It consists of 4 exons and
body. Beta adrenoreceptor (ADRB) is believed to 3 introns spanning 43.3kb [32]. The first exon is
play an important role in aqueous humor produc- relatively short (165 bp) and comprises most of
tion and outflow, regulating IOP [26]. ADRB is the 5′-untranslated region (5′-UTR). Intron 1 is
divided into three major subtypes: beta-1 approximately 1.3 kb in size and may contain
(ADRB1), beta-2 (ADRB2), and beta-3 (ADRB3) part of the promoter region. The second exon
[27]. Their expression differs according to the (870 bp) consists of approximately 70 bp of
organ. For example, ADRB2 is predominantly untranslated region and encodes the remaining
found in the human iris, ciliary body, and blood 5′-UTR, and the rest of the second exon is then
vessels to optic nerve head [26]. Polymorphism translated. The translated region continues up to
and mutation of beta-2 adrenoreceptor (ADRB2) Leu266 near the end of transmembrane
gene may alter the aqueous production and poten- VI. However, it is interrupted by the large second
tially play an important role in pathogenesis of intron (4.3 kb) and the third intron (38.5 kb). A
glaucoma. ADRB2 gene is an intronless gene small third exon is approximately 70 bp in size.
located at chromosome 5q31-32 [28]. The fourth exon is quite large, spanning 3344 bp,
ADRB2 gene was screened using multiplex but only a small fragment is translated, and the
polymerase chain reaction (PCR) for polymor- rest is the 3′-untranslated region (3′-UTR).
phisms at beta upstream peptide -47T>C Screening of PTGFR gene was conducted on
(rs1042711), intronic polymorphism -20T>C 90 glaucoma patients and 90 control subjects.
(rs1801704) and exonic polymorphisms 46A>G Among these patients and subjects, there were
(rs1042713), and 79C>G (rs1042714) and 124 Malays and 56 Chinese patients. A total of 63
single nucleotide polymorphisms (SNPs) were [37, 38]. However, there is no available data for
identified with 1 SNP found in the exon, and the Malaysian population.
rest were intronic polymorphisms [29]. The Kodisvary et al. conducted a cross-sectional
majority of the intronic polymorphisms (43 study to find the association between PTGFR and
SNPs) were found near the intron 3. Exonic SNP acute angle-closure presentation (APAC) in PAC
rs3766331 was found within exon 4 [33]. A novel patients [35]. Her team attempted to answer what
SNP rs3766332 was found at the flanking region triggers APAC in a susceptible individual. Why
of exon 4 [34] (Hoh et al., 2007). Microsatellite certain susceptible individuals remain asymp-
instability (MSI) was also found in intron 3, tomatic and insidiously developed optic neuropa-
“CA” deletion and “TA” insertion [29]. thy? There is a possibility of the genetics makeup
Among the intronic SNPs, three SNPs were that is responsible in determination of ocular
found as potential susceptible markers for glau- biometry and inducing acute attack [39].
coma in Malaysian population: rs2146489, Shallower anterior chamber depth (ACD) and
rs11162505, and rs556817. Non-synonymous smaller anterior segment dimension increased the
polymorphism of rs2146489 was found to susceptibility to PACG [40, 41].
increase the predisposition to glaucoma 3.1 fold A total of 27 PAC patients and 30 age-matched
(95%CI 1.1, 8.8) in Malaysian population. SNPs controls were involved in this study. Out of 27
rs11162505 and rs556817, which are located sev- patients with PAC, 16 presented with history of
eral base pairs apart, conferred protective effect APAC [35]. Genetic screening for rs3766332
against glaucoma susceptibility in Malaysian A>T and rs3766331 A>G of PTGFR was con-
population. There was no evidence of linkage ducted using denaturing high-performance chro-
disequilibrium between these two intronic SNPs, matography (dHPLC) technique. Samples with
but it was shown that rs11162505 was in linkage heteroduplex peak were then subjected to Sanger
disequilibrium with rs554185. There was signifi- sequencing technique. There was a significant
cant association of GG (p = 2.0 × 10−4) and GA association between rs3766332TT and APAC
(p = 3.0 × 10−4) haplotypes of rs11162505 and among PAC patients [35]. Ocular biometry was
rs554185, respectively, with susceptibility to also evaluated in this study using A-scan ultraso-
glaucoma. nography, anterior chamber depth (ACD), lens
PTGFR gene was also screened in Malay and thickness, and axial length. However, there was
Chinese patients with primary angle-closure no significant association of these two SNPs with
(PAC) patients residing in Kelantan, a northeast- ocular biometry of PAC patients in this small
ern state in peninsular of Malaysia. Kodisvary study. The FP receptor plays an important role in
et al. [35] evaluated the potential association a cascade of inflammatory reaction; there is ele-
between ocular biometry in PAC patients and ment of inflammation in APAC. rs3766332TT is
PTGFR polymorphisms. Ocular biometry is a potential candidate genetic marker for APAC.
considered as one endophenotype for PACG. The A study conducted in Taiwanese patients with
potential role of endophenotypes of glaucoma APAC identified rs2664538 of MMP-9 gene as
further complicates the complexity of phenotype potential candidate markers [42]. Matrix metal-
in glaucoma. Endophenotypes are plausible loproteinase (MMP) plays a role in regulation of
quantitative traits or risk factors with strong cytoskeletal changes of the uveoscleral outflow
genetic components that are related but not part [43]. Binding of prostaglandin analog to FP
of the symptom of the glaucoma [36]. These receptor that is regulated by PTGFR gene is
include IOP, central corneal thickness (CCT), known to induce remodeling of extracellular
vertical cup to disc ratio (VCDR), cup and disc matrix (ECM) of the sclera at the uveoscleral and
area, and ocular biometry for angle-closure glau- trabecular meshwork [44]. Widening of collagen
coma. There was high heritability of endopheno- in uveoscleral pathway facilitates the outflow of
type of POAG; 0.55 for IOP, 0.48–0.66 for the aqueous and reduction of the intraocular pres-
VCDR, 0.75 for cup area, and 0.72 for disc area sure. There is a possibility of gene-gene interac-
Table 6.1 Summary of single gene screening conducted on glaucoma patients and control subjects in Malaysia
Gene Type of glaucoma Race Findings
MYOC JOAG Malays Asn480Lys
IVS2 730 +35G>A
ADRB2 POAG Malays 79C>G (79GG)
NTG Chinese -20T>C (-20T)
PTGFR POAG Malays rs2146489
NTG Chinese rs11162505
rs556817
PTGFR PACG Malays rs3766332
Chinese rs3766331
tion of PTGFR and MMP genes. However, the Based on the findings of stage 1 and stage 2 (rep-
sample size is rather small with high potential of lication study of six countries), three susceptible
selection bias. A larger sample size is definitely genetic markers of PACG were identified:
necessary to confirm this finding. A replication rs11024102 in PLEKHA7, rs3753841 in
study is also important. COL11A1, and rs1015213 located at intergenic
Based on the available single gene analysis in region between PCMTD1 and ST18 [45] . In this
the Malaysian population, there is a potential role study, a total 1917 PACG patients and 8943 con-
of PTGFR in susceptibility of POAG and trol subjects were involved. PLEKHA7 encodes
NTG. For intronic polymorphism of PTGFR, pleckstrin homology domain containing protein 7
rs3766332 is also a potential marker for which is involved in maintenance stability of
APAC. The ADRB2 gene may also play a role in adherence junctions [46]. Adherence junctions
susceptibility to open-angle glaucoma. However, are abundant in the eye that regulates paracellular
the sample size was rather small for the studies permeability [47]. COL11A1 encodes one of the
involving screening of PTGFR and ADRB2 in alpha-chains of type XI collagen. Mutation of
Malaysian population. In addition, only Malays COL11A1 is responsible for ocular, orofacial,
and Chinese were involved. MYOC mutation is auditory, and skeletal manifestation of Marshall
found to play a strong association in a Malay syndrome, Stickler syndrome, and Stickler-like
family with JOAG (Table 6.1). syndrome [48]. Variants in COL11A1 are
believed to play a role in fibrillar collagen matrix
formation resulting in shorter eyeball and over-
6.4 Genome-Wide Association crowding of the anterior chamber. In addition,
Studies COL11A1 was also expressed in human trabecu-
lar meshwork, which may play a role in the aque-
With advancement of technology in genetics, ous outflow in PACG patient [49]. The potential
screening of entire human genes is currently pos- role of intergenic SNP rs1015213 is still
sible in a shorter time and at an acceptable cost. unknown.
To maximize the outcome, collaborative work Currently, another five susceptible markers for
with various institutions involving multiple pop- PACG were identified in discovery stage of
ulations has been initiated. A group of investiga- GWAS study: rs3816415 in EPDR1, rs736893 in
tors in Malaysia were involved in this smart GLIS3, intergenic SNP rs3739821 in between
partnership under the leadership of investigators DPM2 and FAM102A, rs1258267 in CHAT, and
in Singapore Eye Research Institute (SERI), rs7494379 in FERMT2 [50]. In this study, a total
Singapore. of 10,503 PACG patients and 29,567 control sub-
Malay PACG patients residing in Malaysia jects from 24 countries were involved. Four cen-
were included in stage 1 genome-wide associa- ters in Malaysia were involved in this discovery
tion study (GWAS) involving five countries. stage and the data collection includes all races in
Malaysia. The mRNA expression of these mark- per allele odds ratio of 1.13 (95% confidence
ers on human ocular tissues including on the iris, interval 1.06–1.22, p = 0.00046). ABCC5 is also
ciliary body, trabecular meshwork, cornea, lens, found to be expressed in human ocular tissue
retina, choroid, and optic nerve head was con- including the iris, ciliary body, and lens [57].
ducted using RT-PCR technique. Protein expres- A two-stage exome chip study involving dis-
sion of these markers on ocular tissue was also covery and replication stage was conducted in
identified. In general, protein expression corrob- POAG patients and control patients [55]. Malay
orated well with mRNA expression except for patients with POAG residing in Malaysia were
GLIS3 [50]. included in the replication stage of this GWAS.
EPDR1 encodes glycosylated type II trans- CDKN2B-AS1 rs2157719 was identified as
membrane protein known as ependymin-related potential genetic marker in both stages with per
1 [51]. FERMT2 encodes protein called pleck- allele effect of 0.71 (pmeta = 2.81 × 10−33) involv-
strin homology domain containing family C ing 12,677 POAG patients and 36,526 controls.
member 1 (PLEKHC1), a component of extracel- CDC-TGFBR3 rs1192415 (OR 1.13,
lular matrix [52]. Both EPDR1 and FERMT2 pmeta = 1.6 × 10−8) and FNDC36 rs4894796 (OR
have a role in cell adhesion [51, 52]. This further 0.93, p = 1.40 × 10−5) were identified in the repli-
supports the role PLEKHA7 in cell adherence, cation stage involving 9133 POAG patients and
whereby the knowledge was obtained from the 26,780 controls. All three loci, CDKN2B-AS1,
earlier stage of GWAS [45]. CHAT encodes cho- CDC-TGFBR3, and FNDC36, contain genes
line acetyltransferase, an enzyme responsible for which may contribute to transforming growth
synthesis of acetylcholine neurotransmitter factor-β (TGF-β) signaling. TGF-β is postulated
which is important in pupillary constriction [53]. responsible for retinal ganglion cell death and
Pupillary dilatation may cause pupillary block glaucomatous optic nerve damage. TGF-β signal-
and APAC in susceptible individual. There is ing plays important in various diseases including
potential genetic predisposition through the vari- cataract and glaucoma.
ation of CHAT which increases the risk of Based on available GWAS in Malaysian popu-
PACG. However, the role of intergenic SNP lation, susceptibility markers for PACG seem to
rs3739821 and GLIS3 in susceptibility to PACG associate with gene controlling cell adherence
is still not clear [54]. junction and collagen formation, suggesting a
ACD has shown high heritability based on close connection with changes in anterior cham-
Guangzhou Twin Study [55]. ACD is considered ber structure and aqueous formation and outflow,
as endophenotype for PACG. In another GWAS while susceptibility markers for POAG are poten-
study, rs1401999 in ABCC5 was identified as tially related to TGFBR pathway. Currently, there
quantitative trait loci for ACD in the stage 1 is a group of researchers searching for suscepti-
involving 5308 participants [56]. ACD was mea- bility genetic markers for progression of POAG
sured using IOL master, and only the right eye and PACG in Malays.
measurement was obtained from Singapore
Malay Eye Study (SiMES), Singapore Indian
Eye Study (SINDI), and Beijing Eye Study 6.5 Age-Related Macular
(BES). Based on meta-analysis, the effect size Degeneration
was −0.045 mm ACD for per minor allele of
rs1401999 (p = 8.17 × 10−9). Stage 2 involved Age-related macular degeneration (AMD) is a
case-control study on 2422 PACG patients and major cause of irreversible blindness in the world.
9193 controls from seven countries using SNP AMD is a progressive disease which affects per-
array analysis. Malay patients residing in sons aged 50 and above. Genetic predisposition
Malaysia were included in this case-control was first shown to have strong association in
study. There was modest association between AMD based on the landmark genome-wide asso-
minor allele (C) of rs1401999 and PACG with ciation study (GWAS) by Klein in 2005 [57].
This was followed by several GWAS from differ- receptor of advanced glycation end product
ent population around the world. Among the (RAGE) [72, 73]. In the Malaysian cohort, gene
most influential genes associated with AMD are polymorphism 2245G/A was found to be associ-
complement factor H (CFH) [58, 59] and age- ated with DR among Malaysian patients possibly
related maculopathy susceptibility 2(ARMS2) through NF-κβ-mediated pro-inflammatory path-
and HtrA serine peptidase 1 (HTRA1) [60–62]. way [74, 75]. Conversely, Gly82Ser, 1704G/T,
More recently, a GWAS and exome-wide 2184A/G, -429T/C, and -374T/A gene polymor-
(EWAS) association studies have reported the phisms were reported to have a lack of associa-
genotypes of exudative AMD among East Asians tion with DR [76, 77].
[63]. In the study, in addition to three novel loci
of mutation, an uncommon mutation at CETP
(D442G) was found to be specific to East Asian 6.7 Retinoblastoma
cohort of exudative AMD patients. In a study of
135 patients with age-related macular degenera- Retinoblastoma is the commonest primary intra-
tion in Malaysia [64], VEGF+405 polymorphism ocular tumor in children less than 5 years old
was found to be associated with the wet type of [78]. About 14.5 new cases of retinoblastoma are
AMD. This association was also found in diagnosed yearly in Malaysia [79]. Several stud-
Tunisian patients with AMD [65]. Several studies ies have reported retinoblastoma gene mutations
are currently underway to determine the geno- in the Malaysian population [80–82]. In a meta-
type of AMD in the Malaysian population. analysis of 932 reported RB1 mutations, Valverde
et al. showed heterogenous distribution of muta-
tions across the RB1 gene from around the world
6.6 Diabetic Retinopathy [83]. Mohd Khairul et al. showed similar distri-
bution of retinoblastoma mutation in Malaysia
Diabetes mellitus (DM) is a major cause of mor- [84]. The SNP 153104 in RB1 intron 18 was
bidity and mortality in the world and is fast reported to be one of the common SNPs found in
becoming a global pandemic. In 2014, it is esti- Malaysian children with retinoblastoma [80].
mated that 387 million people have diabetes There is no “hot spot” of RB1 gene found in
worldwide, and this number will increase to 592 Malaysian children with retinoblastoma [80–82,
million by the year 2035. The current national 84]. In fact, there is no mutation or SNP found in
prevalence of DM in Malaysia is 16.6%, with an N and C-termini and pocket A and B domain of
estimated 3.2million people with DM, compared RB1 in a number of children with sporadic RB in
to 1.9 million in 2013 [66]. Diabetic retinopathy Malaysia [80–82]. There is possibility of varia-
is the third common cause of avoidable blind- tions at the intronic and promoter region of RB1
ness and was responsible for blindness in an esti- which is not screened in Malaysian population.
mated 5 million of the world population in 2002
by the World Health Organization [67]. In
Malaysia, DR is the leading cause of visual loss 6.8 Retinopathy of Prematurity
in the working age group. The prevalence of dia-
betic retinopathy in Malaysia is estimated at Retinopathy of prematurity (ROP) is a visually
36.8% [68], which is similar to worldwide prev- threatening disease affecting premature infants’
alence of 37% [69]. retina caused by abnormal retinal angiogenesis.
The role of genetic susceptibility in DM has Known major risk factors associated with ROP
been well known. High concordance between include low gestational age, low birth weight,
twins with DR has been reported previously [70, and high supplemental oxygen [85, 86]. The role
71]. Most genetic association studies in DR were of genetic influence in ROP has been proposed
performed using candidate gene approach. One [87] based on similarities in ROP clinical pheno-
of the genes implicated in DR is the gene for types with other genetic conditions such as Norrie
disease and familial exudative vitreretinopathy. 4. Kannabiran C. Molecular and functional genetics of
inherited eye disorders in India. Acta Ophthalmol
Genetic variants may also explain observed 2008;86.
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Informed Consent All procedures followed were in
coma onset and progression. Surv Ophthalmol.
accordance with the ethical standards of the responsible
2008;53(6):S3–S10.
committee on human experimentation (institutional and
13. Monemi S, Spaeth G, Da Silva A, Popinchalk S,

national) and with the Helsinki Declaration of 1975, as
Ilitchev E, Liebman J, Ritch R, Heon E, Crick RP,
revised in 2000 (5). Informed consent was obtained from
Child A, Sarfarazi M. Identification of a novel adult-
all patients for being included in the study.
onset primary open-angle glaucoma (POAG) on
5q22.1. Hum Mol Genet. 2005;14(6):725–33.
Animal Studies No animal or human studies were car- 14. Rezaie T, Child A, Hitchings R, Brice G, Miller L,
ried out by the authors for this article. Coca-Prados M, Heon E, Krupin T, Ritch R, Kreutzer
D, Crick RP, Sarfarazi M. Adult-onset primary open
Compliance with Ethical Requirements Author Liza- angle glaucoma caused by mutations in optineurin.
Sharmini AT has received speaker honorarium by Pfizer Science. 2002;295(5557):1077–9.
Malaysia Sdn Bhd, Novartis Malaysia, and Santen 15. Stone EM, Fingert JH, Alward WL, Nguyen TD,

Malaysia. She was involved in clinical trial study spon- Polansky JR, Sunden SL, Nishimura D, Clark AF,
sored by Allergan. Nystuen A, Nichols BE, Mackey DA, Ritch R,
Author Tengku Ain Kamalden declares that she has no Kalenak JW, Craven ER, Sheffield VC. Identification
conflict of interest. of a gene that causes primary open angle glaucoma.
Science. 1997;275(5300):668–70.
16. Richards JE, Lichter PR, Boehnke M, Uro JLA,

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Challenges and Opportunities
in Genetic Research 7
from the Perspective of a Tertiary
Eye Care Hospital in Bangladesh
Nazmun Nahar, Mohammad Ibn Abdul Malek,
and Bipul Kumer De Sarker
Abstract Keywords
Clinical programs in developing countries like Molecular research · Epidemiology · Retina ·
Bangladesh are focused more on common Glaucoma
conditions and less toward advanced research.
But the potential for molecular research is
considerable, with heritable factors linked in
both common and uncommon diseases such as 7.1 Introduction
primary open-angle glaucoma, primary angle-
closure glaucoma, diabetic retinopathy, vascu- Bangladesh is a small country, albeit with a huge
litis retinae and age-related macular populace (over 160 million and counting) [1].
degeneration, congenital glaucoma, and reti- The Ispahani Islamia Eye Institute and Hospital
nitis pigmentosa, among others. Less common is a tertiary referral center, with around 2000
ocular diseases also lend themselves to diag- patients being seen daily. Due to the huge burden
nosis where all investigations are not always of patients and the necessity of clinical manage-
available. This paper considers the epidemiol- ment of these patients, research thus far has taken
ogy of some common and uncommon ocular a backseat. Nationwide statistics are not reliably
conditions in glaucoma and retina at a tertiary available for most ocular diseases. Clinical pro-
referral center in Bangladesh, to present the grams in the background of constrained funding
magnitude of ocular disease as well as outline mean treatment has been limited to mostly cata-
the huge potential of genetic research. As the ract and more recently the emerging ocular dis-
technology for genetic analysis already being eases outlined in Vision 2020. Molecular research
available, emphasis should be placed on its in ophthalmology is at present negligible. While
use in ophthalmology, so that new therapeutic there is biomedical research going on in
modalities can be developed for certain dis- Bangladesh, ophthalmology as a whole has not
eases and to provide treatment for people who yet taken the step into the genetics of the origin
at present have no recourse for either diagno- and progression of ocular diseases. With this
sis or treatment. paper, we seek to demonstrate the magnitude of
disease in our country from the perspective of the
N. Nahar (*) · M. I. Abdul Malek · B. K. De Sarker retina and glaucoma departments of a tertiary
Ispahani Islamia Eye Institute and Hospital, referral center and the scope for research into the
Dhaka, Bangladesh genetics of such diseases.
e-mail: [email protected]; Ibn.
[email protected]; [email protected]

https://doi.org/10.1007/978-981-13-0884-0_7
72 N. Nahar et al.
7.2 Genetics associated with PLEKHA7 and COL11A1 genes.

In congenital glaucoma, there are mutations in
It is known that a wide variety of retinal diseases two genes causing autosomal recessive congeni-
have their origins in the genetic pattern passed tal glaucoma, CYP1B1 coding for cytochrome
down through successive generations. The use of P450 1B1 and LTBP2. Recently, a genome-wide
genetics to predict the course of disease and to association study has identified a locus on 15q24,
determine the risk and prognosis of certain dis- and the lysyl oxidase-like 1 (LOXL1) gene has
eases will no doubt be invaluable in the future. been strongly associated with both pseudoexfo-
In the field of retina, diabetic retinopathy (DR) liation syndrome and pseudoexfoliative glau-
is one of the major diseases, and heritable factors coma. Glaucoma is associated with anterior
have been implicated in 25–50% of patients who segment dysgenesis, Axenfeld-Rieger syndrome,
develop diabetic retinopathy, and genetic factors and aniridia caused by mutations in PITX2,
are known to influence the severity of diabetic PAX6, and FOXC1, and mutations in these genes
retinopathy [2]. Certain genes, including ALR2, cause dominantly inherited disease. In normal-
VEGF, and RAGE genes, have been implicated tension glaucoma (NTG), there is a duplication
in DR, but a consistent association has not been of the TBK1 gene that interacts with optineurin, a
proven in all populations [2]. Age-related macu- protein that is also a rare cause of NTG.
lar degeneration(ARMD), known as the leading
cause of blindness in a Western population, has a
complex multifactorial etiology, but a major risk 7.3 Epidemiology and Research
variant within the complement factor H gene Opportunities
(CFH) has been identified [3]. Idiopathic polyp-
oidal choroidal vasculopathy is closely associ- There is paucity of data on epidemiological stud-
ated with ARMD, with genes in LOC387715 ies of eye diseases in Bangladesh. Therefore, we
rs10490924 associated with PCV and its clinical have considered the patients seen at Islamia over
manifestations. HTRA1, CFH, and C2 were also the past year. Although not fully representative of
found to be associated with PCV [4]. Retinitis population patterns, an assumption about the
pigmentosa (RP) is a well-documented cause of magnitude of disease can be made from this data.
progressive visual loss, which has extensive Over the year spanning July 2016 to June 2017,
research into the underlying genetics [5]. 282,712 patients were seen at the Ispahani
Although RP is said to have no ethnic predilec- Islamia Eye Institute and Hospital. Of these, a
tion, the pathogenesis is thought to differ between total of 47,145 patients (17%) were seen in the
populations, and numerous genes have been vitreo-retina dept, with 23,406 patients (8%)
identified with nonsyndromic autosomal domi- being seen by the glaucoma department.
nant, autosomal recessive, and X-linked RP [5]. Primary angle-closure glaucoma (PACG) con-
Vasculitis retinae comprise a rare group of disor- stitutes 43.1% (10,095) of all patients, a statistic
ders that has been associated with numerous surprising as POAG is more common in neigh-
HLA alleles, associated with many autoimmune boring India [8, 9]. This merits further research
disease [6]. From Bangladesh Perspective, tuber- into the genetic roots of this disease in a
culosis is one of the most common causes of reti- Bangladeshi population. Primary open-angle
nal vasculitis; nevertheless, a large proportion of glaucoma constitutes 25.5% (5966), juvenile
patients are idiopathic [7]. open-angle glaucoma (JOAG) 1.4% (335),
Primary open-angle glaucoma (POAG) and normal- tension glaucoma (NTG) 2.4% (549),
primary angle-closure glaucoma (PACG) com- pseudoexfoliation 0.3% (69), and congenital
prise the majority of patients presenting to a glau- glaucoma 1.06% (248). The remainder are sec-
coma dept. More than 30 chromosomal loci and 4 ondary glaucomas (26.2%), with an associated
genes (MYOC, OPTN, NTF4, and WDR36) have secondary cause. Most of the patients had unilat-
been linked to POAG, whereas PACG has been eral blindness at the time of presentation,
7 Challenges and Opportunities in Genetic Research from the Perspective of a Tertiary Eye Care Hospital… 73
e mphasizing the need for a tool to screen those at der that commonly affects a South Asian popula-
potential risk. Since most of these diseases have tion, and the genetic component of these
genetic associations, a detailed analysis of their apparently idiopathic cases is an absolute neces-
gene patterns would render invaluable informa- sity, so as to identify cases at risk and develop
tion regarding the likelihood of glaucoma and the future therapies. Macular dystrophies are fre-
factors affecting its progression. These numbers quently diagnosed, but subclassification requires
are just the tip of the iceberg of the true situation accompanying electroretinogram/electroculo-
in the community as many patients remained gram, which at present is not available. A genetic
undiagnosed at community level. mapping of these patients would identify the spe-
Over a 1-year period from July 2016 to June cific underlying genetic defect and would aid in
2017, a large percentage (21%, number 9991) of the development of future gene therapy.
patients were DR, of which 42% (4296) was pro- Additionally, many rare diseases including famil-
liferative and nearly equal numbers of non- pro- ial exudative vitreoretinopathy, Coats’ disease,
liferative (30%, 3000) and advanced diabetic eye and intermediate and posterior uveitis including
disease (27%, 2695). The higher proportion of Vogt-Koyanagi-Harada (VKH) syndrome have a
severe cases indicates the pattern of a tertiary frequent presentation and have a genetic basis
referral center, where the worse cases are usually that requires further investigation.
referred. These patients can provide a rich pool
into the genetic analysis of the causative factors
of progression and additionally will give an 7.4 Challenges
insight into the genetic underpinnings of DR in a
Bangladeshi population. ARMD, as already men- Resources have been the main challenge till now,
tioned, is a leading cause of blindness in a since the volume of patients have meant that
Western population. In our retina department, patients who can be given immediate treatment
they comprised 4% (1785) of all patients, which have been prioritized more than cases which tend
may indicate a decreased tendency to present or a to have a progressive outcome and have no known
tendency to present only when the disease is therapies. Genetic research represents a beacon
advanced. The number was approximately split of hope for these patients, and their subsequent
between the dry and wet varieties, with IPCV generations, so that gene therapy tailored to these
making up a good number of these cases. The patients can be developed. The technology for
genetic variabilities relating to the progression of genetic analysis is already available; its use in
ARMD have been researched, but this represents ophthalmology is as yet negligible. Awareness
a chance for the genetics of progression in a and resources should be dedicated so that these
Bangladeshi population, so that the findings can analyses can be carried out, since there is so
be compared and contrasted with those in the much that can be gleaned about the nature of
Western world, where most of ARMD research these diseases from such research. We are look-
till date has focused. Retinitis pigmentosa has ing to raise the awareness of the ophthalmologi-
been extensively researched, with numerous dif- cal community in Bangladesh as a whole, so that
ferent alleles implicated, and comprises approxi- there can be a hope for these patients in the near
mately 2.5% of patients of our outpatients. A future.
focus on the genetics and phenotypic expression
of these diseases in a Bangladeshi population, Compliance with Ethical Requirements Authors
Nahar N, Malek MIA, and Sarker BKD declare that they
and the expression of both syndromic and non- have no conflict of interest or any financial relationship to
syndromic RP in these patients, is a potential area disclose. The study was cleared by the institutional ethical
for investigation. Vasculitis retinae, comprising review committee at the Ispahani Islamia Eye Institute
around 2% of our patients, is an idiopathic disor- and Hospital (although being retrospective in nature, ethi-
cal approval was not mandatory).
74 N. Nahar et al.
References 5. Fahim AT, Daiger SP, Weleber RG. Nonsyndromic

retinitis pigmentosa overview. In Pagon R, Adam M,
Ardinger H, Wallace S, Amemiya A, Bean L, Bird T,
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Ledbetter N, Mefford H, Smith R, Stephens K, edi-
Available at: http://data.worldbank.org/country/ban-
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gladesh. (2017). Accessed 25 Aug 2017.
org. Available at: http://www.genereviews.org/.
2. Simó-Servat O, Hernández C, Simó R. Genetics
Accessed 26 Aug 2017.
in diabetic retinopathy: current concepts and new
6. Monach PA, Merkel PA. Genetics of vasculitis. Curr
insights. Curr Genomics. 2013;14(5):289–99. https://
Opin Rheumatol. 2010;22(2):157–63.
doi.org/10.2174/13892029113149990008. Published
7. Habibullah M, Uddin MS, Islam S. Association of
online 2013 Aug
tuberculosis with vasculitis retinae. Mymensingh
3. Haddad S, Chen CA, Santangelo SL, Seddon
Med J. 2008;17(2):129–33.
JM. The genetics of age-related macular degenera-
8. George R, Ve RS, Vijaya L. Glaucoma in India:
tion: a review of progress to date. Surv Ophthalmol.
estimated burden of disease. J Glaucoma.
2006;51(4):316–63.
2010;19(6):391–7.
4. Chen H, Liu K, Chen LJ, Hou P, Chen W, Pang
9. Paul C, Sengupta S, Choudhury S, Banerjee S, Sleath
CP. Genetic associations in polypoidal choroidal vas-
BL. Prevalence of glaucoma in Eastern India: the
culopathy: a systematic review and meta-analysis.
Hooghly River glaucoma study. Indian J Ophthalmol.
Mol Vis. 2012;18:816–29.
2016;64(8):578–83.
Genetic Research on Ocular Health
and Disease in a Population 8
from Nepal
Matthew P. Johnson, Suman S. Thapa,
Sandra Laston, Kent L. Anderson, Bradford Towne,
Janardan Subedi, John Blangero,
and Sarah Williams-Blangero
Abstract cal mechanisms underlying the development

Ocular disease is a major public health concern and progression of cataract and help identify
worldwide, including Asia where large num- those individuals at high risk of disease. The
bers of individuals suffer from blindness and primary objective of an ongoing study in the
visual impairment. In South Asian countries Jirel ethnic group of eastern Nepal (the Jiri Eye
like Nepal, for example, age-related cataract is Study) is to define the genetic architecture of
a leading cause of blindness and visual impair- normal ocular trait variation and characterize
ment. Age-related cataract is influenced by a genetic factors influencing risk for common
complex interplay of non-genetic and genetic ocular diseases such as cataract. The Jirel pop-
risk factors. Identifying the genetic risk factors ulation has been the focus of genetic epidemio-
involved can help elucidate the causal biologi- logical studies for more than two decades, and
a well-documented extended pedigree has
M. P. Johnson (*) · S. Laston · J. Blangero been developed for the group making it
S. Williams-Blangero extremely powerful and informative for genetic
South Texas Diabetes and Obesity Institute, studies. All 1292 study participants discussed
Brownsville, TX, USA
in the work presented here belong to this single
Department of Human Genetics, School of Medicine, extended pedigree. Members of the Jirel popu-
University of Texas Rio Grande Valley,
lation underwent a comprehensive eye exami-
Brownsville, TX, USA
e-mail: [email protected] nation that included lens opacity grading in
accordance with the Lens Opacities
S. S. Thapa
Tilganga Institute of Ophthalmology, Classification System II (LOCS II). A variance
Kathmandu, Nepal components method was used to estimate heri-
K. L. Anderson tability of cataract. Of the 1292 participants,
Department of Ophthalmology, School of Medicine, 57.0% were female and 43.0% were male. The
UT Health San Antonio, San Antonio, TX, USA mean (SD, range) age at exam is 42.0 (16.7,
B. Towne 18–88) years. The prevalence of cataract (any
Department of Population and Public Health type) in individuals aged 40 years or older is
Sciences, Boonshoft School of Medicine,
25.8%, and additive genetic effects (48.3%)
Wright State University, Kettering, OH, USA
play a significant role in determining the risk
J. Subedi
of developing cataract in this population. The
Department of Sociology and Gerontology,
College of Arts and Science, Miami University, Jirel population is a powerful resource for the
Oxford, OH, USA study and identification of genetic mechanisms

https://doi.org/10.1007/978-981-13-0884-0_8
76 M. P. Johnson et al.
influencing variation observed in ocular health Nepalese population resides in the hill or moun-
and disease metrics. We anticipate that the Jiri tainous regions of the country that are difficult or
Eye Study will continue to make significant impossible to reach by motorized transport.
contributions to the genetics of ocular health Access to some areas is hampered seasonally
and disease in Asia. during periods such as the monsoon when fre-
quent landslides block major motorized thor-
Keywords oughfares and impede access to or delivery of
Cataract · Heritability · Pedigree · Nepal · ophthalmic care to those in need.
Epidemiology
8.2 Age-Related Cataract:

Clinical Features and Risk
8.1 Introduction Factors
Blindness and visual impairment (VI) are a Age-related cataract (hereon, cataract) is an
global health concern that has a significant nega- opacification of the natural crystalline lens,
tive impact on the human condition. Worldwide, which impedes vision by perturbing the transmis-
cataract is the leading cause of blindness and the sion and focus of light onto the retina. In a healthy
second leading cause of VI (prevalence rates of lens, the cellular and protein material is arranged
33.4% and 18.4%, respectively) [10]. The caus- in a structured manner that allows light to pass
ative factors underlying blindness and VI differ through the transparent lens unobstructed.
across global regions, especially between devel- However, by the fifth or sixth decade of life, the
oping and developed regions. South Asia is a cellular arrangement of the lens may start to lose
developing region that includes Afghanistan, its normal structure and proteins may start to
Pakistan, India, Bangladesh, Bhutan, and Nepal. aggregate into larger masses, thereby disrupting
Of 20 global regions examined, South Asia ranks lens transparency [37].
fourth highest for age-standardized prevalence of There are three main types of cataract, which
blindness (4.4%) and highest for prevalence of VI are defined by their clinical appearance and loca-
(23.6%) [34]. Cataract is the leading cause of tion within the lens [24, 37]. Nuclear cataract
blindness and the second leading cause of VI occurs when mature lens fibers at the center of
throughout these countries in South Asia [10]. In the lens undergo oxidative stress which alters
Nepal, the prevalence of VI across several admin- protein solubility and stability so that protein
istrative zones has been reported to range from unfolding and aggregation occur, resulting in
18.6 to 43.0%, of which 0.7 to 17.4% is at least brunescence and opalescence. Additional periph-
unilateral blindness [26–29, 36]. Consistent with eral layers of lens fibers surround central lens
what is observed throughout South Asia and fibers leading to increased stiffness of the central
across the world, cataract is a leading causes of fibers, i.e., hardening or sclerosis. Cortical cata-
blindness and VI in Nepal. ract is defined by perturbations to the structure of
The high prevalence of blindness and VI due mature fiber cells within the lens cortex (envelop-
to easily correctable diseases like cataract in ing the lens nucleus) that may result in a loss of
developing regions may, in part, be a conse- essential metabolites which in turn may promote
quence of the limited medical infrastructure and protein oxidation and precipitation. Over time,
low numbers of trained medical personnel avail- discrete opacities may then develop in the cortex
able to administer adequate ophthalmic care. In and extend in a “spoke-like” fashion toward the
countries like Nepal, the difficulty in delivering center of the lens. Posterior subcapsular cataract
adequate ophthalmic care is exacerbated by the is characterized by granular, plaque-like opaci-
remoteness of the areas in which most rural ties that form and accumulate in the central pos-
inhabitants live. Approximately 52% of the terior cortex underneath the posterior capsule.
8 Genetic Research on Ocular Health and Disease in a Population from Nepal 77
Cataract is a complex multifactorial disease extended pedigrees provide optimal opportuni-

with non-genetic and genetic risk factors. ties to decompose the genetic architecture of
Advancing age is a risk factor, and females are at common complex phenotypes such as cataract or
greater risk of developing cataract than males other multifactorial ocular diseases. These pedi-
[24]. Exposure to ultraviolet B radiation (sun- gree structures optimize the examination of
light) and cigarette smoke (a preventable, modifi- genetic information such as heritability, linkage,
able risk factor) has been demonstrated to and association within the cohort and are less
augment cataractogenesis, as has systemic dis- prone to stratification phenomena that mask true
eases such as type 2 diabetes mellitus and hyper- genetic signals or generate false-positive signals
tension [24]. Furthermore, cataract prevalence (a potential limitation with study designs utiliz-
rates differ among ethnic groups (individuals of ing samples of unrelated individuals).
Asian ancestry tend to exhibit higher rates of Of these three pedigree frameworks, the
cataract), which highlights a genetic component extended pedigree design provides the most
to cataract development and progression. power to differentiate between genetic and non-
genetic effects, observe a greater number of mei-
otic events to localize genomic regions
8.3 Age-Related Cataract: influencing the tested phenotype (linkage), and
Genetics assess the association of sequence-specific vari-
ants within linkage regions that harbor causal
Depending on the genetic model tested (additive, genes. This is because the key advantage of an
dominant) and cataract assessment (clinical or extended pedigree framework is the large number
digital grades), genetic mechanisms influencing of relative pairs, of both close and distant kin,
cataract risk have been estimated to contribute available for analysis. The power to detect link-
between 20% and 53% of the total phenotypic age is maximized when individuals are concen-
variation [15–18]. Despite this solid evidence for trated into as few families as possible, and the
genetic risk factors influencing age-related cata- greater the average pedigree size, the fewer the
ract susceptibility, few genetic susceptibility loci number of individuals needed to achieve the
have been identified (in contrast to the case for same power to detect linkage [2, 6].
earlier onset, Mendelian forms of the disease) With an emerging appreciation of the role of
[31]. Examples of cataract susceptibility loci that functional rare variants in common complex phe-
have reached genome-wide significance include notypes [5, 14, 35], an extended pedigree study
linkage regions mapped to chromosomes 1p14- design also offers an optimal enrichment strategy
p35 and 6p12-q12 in a cohort of Caucasian sib- to identify rare variants [38]. Mendelian mechan-
ling pairs [20] and regions of linkage ics suggests that an extended pedigree framework
disequilibrium (association) mapped to chromo- maximizes the chance of identifying rare variants
somes 3 (KCNAB1 SNP rs7615568) and 21 (SNP sufficiently propagated from the pedigree found-
rs11911275 upstream of CRYAA) in a multiethnic ers down through the family lineage(s). This phe-
Asian (Malays, Indians, Chinese) cohort [23]. nomenon allows for robust direct statistical
testing while minimizing the influence of spuri-
ous linkage disequilibrium [7, 21]. Pedigree-
8.4 Mapping Genomic Regions based sequencing approaches also offer a more
Influencing Ocular Disease cost-effective strategy to catalog rare variants
Susceptibility: The Power compared to a population-based (unrelated sam-
of the Pedigree ples) strategy. Sequencing key family members
(e.g., founders) allows for accurate pedigree-
Family-based study designs that include relative based imputation of non-sequenced family mem-
pairs (e.g., siblings), nuclear families (e.g., trios bers utilizing phased (Mendelian-transmitted)
or other parent-offspring configurations), and/or sequence information to infer near exact
descendent haplotypes. This provides a more the east and west by the Tamba Kosi and the
accurate determination of “whole” allele dosages Likhu Khola rivers, respectively. The region is
(i.e., 0, 1, or 2 copies of the rare allele) compared named for the Jirels, a Tibeto-Burman language-
to genome-wide association-based imputation speaking group.
utilizing population data.
The collection of extended pedigrees can be
challenging. However, in areas of stable popula- 8.5.2 Study Population
tions with limited migration rates, such as endog-
amous groups within South Asia [25], the Approximately 2500 members of the Jirel pop-
ascertainment of large extended pedigrees is less ulation have participated in prior studies span-
of a challenge. Extended pedigrees from geo- ning a period of approximately 30 years. These
graphically and culturally isolated areas offer studies include anthropological investigations
several significant advantages for disease gene [4, 42, 43], assessment of population structure
identification compared to traditional, less homo- [44–46], interrogating the genetic susceptibility
geneous populations. These include a limited to parasitic worm infection [49–52], and identi-
number of ancestors which minimize genetic and fying genetic mechanisms influencing growth
allelic heterogeneity and result in fewer suscepti- and development [40, 41]. The long-running
bility genes (with greater overall effect) and research conducted in the region has resulted in
greater statistical power to identify these genes the collection of extensive genealogical infor-
and reduced environmental “noise” which mini- mation on the Jirel people. All individuals who
mizes the confounding effects of non-genetic have previously participated in research studies
variables. These characteristics are of significant belong to a single extended pedigree which
benefit for complex disease gene identification makes this cohort an informative and extremely
[9, 22, 32] and have proven effective in the iden- powerful resource for genetic studies [6, 47].
tification of ophthalmic disease genes [3, 30]. There is very little gene flow (<1% per genera-
Following the field-based data collection tech- tion) into the population from other ethnic
niques for collecting large extended pedigrees in groups, and inbreeding is minimized due to cul-
stable isolate populations outlined in Williams- tural rules [44]. Based on the strength and util-
Blangero and Blangero [47], we have success- ity of this existing resource, we initiated a
fully established several cohorts of extended large-scale genetic epidemiological study of
pedigrees from populations in Alaska [19], Brazil ocular traits and disorders in the Jirel popula-
[48], and Nepal (the Jirels) [49]. tion: the Jiri Eye Study (JES). The objective of
JES is to characterize the genetic architecture
of normal ocular trait variation and genetic fac-
8.5 Genetic Epidemiological tors influencing risk for ocular disorders such
Studies in the Jirels of Nepal as cataract that are of major public health
importance.
8.5.1 Study Area
Nepal is divided into 14 administrative zones that 8.6 The Jiri Eye Study: Methods
are further subdivided into 75 districts. The Jiri
region is located within the Dolakha District of 8.6.1 Study Inclusion Criteria
Janakpur Zone and is approximately 190 km east
of Kathmandu, the capital city of Nepal. The criteria for individuals to participate in JES
Geographically, it is located at latitude 27°38’N are that they be in good health and are at least
and longitude 86°14′E, at an average elevation of 18 years of age. Sex is not a selection factor, and
approximately 2300 m above sea level, covering we anticipate slightly more female than male vol-
an area of approximately 230 km2 bounded on unteers based on prior recruiting experience.
Female volunteers who are pregnant defer their coherence tomography (anterior, posterior).
participation until at least 12 weeks postpartum. Pertinent to cataract, lens opacity grading is con-
ducted post-mydriasis (a mydriatic mixture of
1% tropicamide + 5% phenylephrine is adminis-
8.6.2 Subject Recruitment tered) by comparing lenticular opacities with a
standard set of images outlined in the Lens
The target recruitment goal is 2000 individuals. Opacities Classification System II (LOCS II)
Subject recruitment and examination activities [13]. For the purposes of this analysis, we are
take place during biannual visits to the field interested in the general underlying genetic archi-
research site in Jiri (optimal data collection times tecture (i.e., heritability) influencing cataract
are March/April and November/December). (any subtype) in the Jirel population. Therefore,
Prior to each field session, local recruitment staff the clinical phenotype is dichotomized into the
contact potential study participants in person to presence of cataract (nuclear, cortical, and/or
explain the purpose and benefits of the study, pro- posterior subcapsular) in one or both eyes and
vide them a copy of the Nepali-translated consent absence of cataract in both eyes.
form (the consent form is read to individuals who
are illiterate), obtain an individual’s verbal agree-
ment to participate in the study, and arrange an 8.6.5 Statistical Methods:
appointment date and time to attend the field Heritability Estimate
research clinic that coincides with a forthcoming
The heritability estimate of cataract in the Jirel
field site visit. Upon arrival to the clinic on their
designated day, individuals are given time to pedigree was determined using a variance com-
reread the consent form or have it reread to them ponents approach as implemented in SOLAR [1].
and to ask any questions they may have. Informed Our approach to estimate the narrow sense herita-
consent is documented by signature or thumb bility (additive genetic effects), defined as
print (for illiterate individuals). h2 = σ2a/(σ2a + σ2e), fosters maximum likelihood
methods that partition the observed phenotypic
covariance (σ2p) into its additive genetic and envi-
8.6.3 Questionnaire ronmental attributes. In its most simplistic, uni-
variate form, the observed covariance of a
A detailed interviewer-administered question- quantitative trait in a pedigree of arbitrary size (n)
naire is conducted in Nepali to collect self- is modeled as Ω = 2Φ × σ2a + In × σ2e, where Ω is
reported information about systemic health (e.g., the n × n covariance matrix, 2Φ is the n × n coef-
hypertension, diabetes), ocular health (e.g., pre- ficient of relationship structuring matrix, σ2a is
vious trauma), and lifestyle factors such as ciga- the variance in the observed trait due to additive
rette smoking history. genetic effects, In is the n × n identity structuring
matrix for an implied individual-specific environ-
mental component, and σ2e is the variance in the
8.6.4 Ophthalmic Examination observed trait due to random (unmeasured)
individual- specific environmental effects.
A comprehensive ophthalmic exam is conducted Specific to this study, this model is easily
for all participants in the JES. It includes an extended to estimate the heritability of a dichoto-
external examination of the face and eyes, visual mous trait where we employ the liability and
acuity assessment, slit-lamp biomicroscopy, threshold model according to which it is assumed
Goldmann applanation tonometry, gonioscopy, that dichotomous observations are underlain by a
fundus examination, fundus and optic disc pho- latent normally distributed liability and that indi-
tography, ocular biometry, Humphrey visual field viduals with liability values equal to or above a
assessment (SITA 24-2), pachymetry, and optical threshold on the liability scale express the
“affected” status and those falling below the 8.7.3 C

urrent Jirel Pedigree
threshold express the “unaffected” status [39]. Structure
The covariates age, sex and their interactions,
and smoking (never, former, current) are also The current Jirel pedigree framework is com-
included in the additive genetic model. No bias of prised of 17,343 pairings of relatives of varying
ascertainment correction is required since the degrees of relationship to each other. These rela-
Jirel family-based sample was originally obtained tive pair relationships include 709 parent-
through house-to-house sampling, and the JES offspring pairs, 669 sibling pairs, 117
recruitment selection is not contingent on a spe- grandparent-grandchild pairs, 1319 avuncular
cific ocular condition. pairs, 106 half-sibling pairs, 26 double first-
cousin pairs, 2508 third-degree relative pairs,
3480 fourth-degree relative pairs, 3801 fifth-
8.7 The Jiri Eye Study: Results degree relative pairs, 2758 sixth-degree relative
pairs, 1633 seventh-degree relative pairs, 196
8.7.1 Power of the Jirel Pedigree eighth-degree relative pairs, and 21 ninth-degree
relative pairs.
The theoretical framework underlying the ana-
lytical power calculation performed was devel-
oped by Blangero et al. [8] and took into account 8.7.4 Cataract Distribution
the exact structure of the Jirel pedigree. There is
80% power to detect an additive genetic effect The distribution of cataract (any of the three sub-
(heritability) accounting for as little as 6.5% of types) in one or both eyes is presented in
the total phenotypic variation observed in the Table 8.1. No individual younger than 40 years of
trait, in this case cataract. age was diagnosed with cataract; therefore, the
percentages presented in the table are calculated
for the subset of individuals in the 40+ years’ age
8.7.2 Current Recruitment group (n = 666).
To date, 1292 individuals (57.0% female) have

successfully participated in the study. The age (in 8.7.5 Cataract Heritability Estimate
years) distribution is as follows: mean = 42.0,
median = 40, standard deviation = 16.7, mini- The heritability of cataract modeled as a dichoto-
mum = 18, and maximum = 88. With respect to mous trait in the Jirel pedigree was significant
cigarette smoking, 310 (24.0%) individuals never (h2 = 0.493; p = 0.046; SE = 0.292). For the lia-
smoked, 749 (58.0%) individuals are former bility and threshold model (dichotomous obser-
smokers, and 233 (18.0%) individuals are current vations underlain by a latent normally distributed
smokers. Age-related ocular conditions generally
start to manifest from 40 years of age. Therefore, Table 8.1 Cataract distribution in the Jirel pedigree
pertinent to this age demographic in the Jirels,
n %
there are currently 666 individuals (53.5%
Age 40–49 5 0.8
female), and the age (in years) distribution is as 50–59 22 3.3
follows: mean = 55.6, median = 54, and standard 60–69 75 11.3
deviation = 10.9. The smoking summary relevant 70–79 61 9.2
to this age group includes 211 (31.7%) individu- 80–89 9 1.4
als who never smoked, 250 (37.5%) individuals Total 172 25.8
who are former smokers, and 205 (30.8%) indi- Sex Male 72 10.8
viduals who still currently smoke. Female 100 15.0
Total 172 25.8
liability), the nearest equivalent to a measure of this population. While the genetic contribution to
the explained phenotypic variance due to covari- cataract risk in the Jirels is similar to other
ates is the Kullback-Leibler divergence (KLD) reported heritability estimates, we must note that
[11]. The KLD measures the proportionate reduc- our estimate is for any type of cataract, whereas
tion in uncertainty to the covariates in the model the studies by Hammond et al. [17, 18] and
which, for cataract, is 0.623. Estimating herita- Congdon et al. [15, 16] report heritabilities
bility of cataract in the 40+ years’ age group between 20% and 53% for specific cataract sub-
alone (n = 666) did not significantly alter the types. Variation in heritability estimates for age-
result (h2 = 0.483; p = 0.048; SE = 0.292; related cataract may result from different study
KLD = 0.513). This result highlights evidence of designs (extended pedigree, twins, sibling pairs,
a genetic contribution (approx. 50%) to influence respectively), ethnic differences (South Asian,
the risk of age-related cataract in the Jirel popula- Caucasians, Caucasian and African American,
tion. Therefore, further investigation is warranted respectively), and possibly the mean age of indi-
to localize genomic regions (linkage) and iden- viduals presenting with cataract (~55 years,
tify common and/or rare sequence-specific vari- ~62 years, ~75 years, respectively). The subjec-
ants within these linkage regions (association) tive nature of our LOCS II cataract grading
that harbor causal genes influencing age-related scheme will also likely produce nuances not only
cataract development and progression. in our cataract heritability estimates but also Jirel
cataract prevalence rates when compared to other
studies.
8.8 Comment In conclusion, we have demonstrated for the
first time that the prevalence of age-related cata-
We report, for the first time, data on cataract from ract in the Jirel population of Nepal is high
the Jiri Eye Study, an extended pedigree-based (25.8%) and that additive genetic effects (48.3%)
genetic epidemiological investigation of the play a significant role in risk of developing this
determinants of ocular traits and disorders in the condition, which has a significant negative impact
Jirel ethnic group of eastern Nepal. The Jiri Eye in Nepal, other Asian countries, and around the
Study aims to document the distribution and world. The Jirel population is ideally suited for
prevalence of standard ocular biometry and ocu- genetic epidemiological studies due to its genetic
lar disease in addition to characterizing the isolation and deep genealogical relationships
genetic architecture of normal ocular trait varia- which augment the power to localize genomic
tion and genetic risk factors for ocular diseases of regions and identify sequence-specific variants in
global public health importance. causal genes influencing gene networks related to
We found that the crude prevalence of any ocular biology. With data from a comprehensive
type of age-related cataract (25.8%) for individu- eye examination (anterior, posterior) being con-
als aged 40 years or older in the Jirels is in gen- ducted in the Jirel population, coupled with an
eral agreement with rates seen in other Asian existing set of genome-wide SNP data and a pro-
countries (8.3–61.9%) [12, 53], including Nepal spective catalog of whole-exome sequence vari-
(10.2–31.5%) [33]. The preponderance of Jirel ants, the Jiri Eye Study will continue to make
females (15.0%) with cataract, compared to Jirel significant contributions to the understanding of
males (10.8%), also is in agreement with other the genetics of ocular health and disease in Asia.
reports documenting female sex to be a risk fac-
tor [24].
Our results suggest a significant genetic con- 8.9 Summary
tribution (48.3%) to risk for cataract in the Jirels
and an almost equal contribution (51.3%) of non- To our knowledge this is the first large-scale
genetic factors (age, sex, their interactions, and genetic epidemiological study of ocular traits and
cigarette smoking) influencing cataract risk in disorders conducted in Nepal, and it is also the
first study to estimate the prevalence of blind- References

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study participants. This study will impart knowl- 2. Almasy L, Blangero J. Human QTL linkage mapping.
edge about ocular health and disease in the Jirel Genetica. 2009;136(2):333–40.
ethnic group which, in turn, will help the imple- 3. Axenovich T, Zorkoltseva I, Belonogova N, et al.
Linkage and association analyses of glaucoma related
menting body of the Nepal Eye (health) Program, traits in a large pedigree from a Dutch genetically iso-
the Tilganga Institute of Ophthalmology, plan lated population. J Med Genet. 2011;48(12):802–9.
for the future development of eye care infra- 4. Blangero J. Population genetic approaches to pheno-
structure and public health programs. This ocu- typic microevolution in the Jirels of Nepal. PhD dis-
sertation., Case Western Reserve University; 1987.
lar health knowledge will not only help the Jirel 5. Blangero J. Localization and identification of human
people and the Municipality of Jiri but also the quantitative trait loci: king harvest has surely come.
Nepalese population in general. For the study Curr Opin Genet Dev. 2004;14(3):233–40.
participants themselves, an immediate benefit 6. Blangero J, Williams JT, Almasy L. Novel family-
based approaches to genetic risk in thrombosis.
includes provision of sight-correcting glasses, an J Thromb Haemost. 2003;1(7):1391–7.
effective and inexpensive strategy to improve 7. Blangero J, Göring HH, Kent JW, et al. Quantitative
vision and reduce its burden on the individual trait nucleotide analysis using Bayesian model selec-
and society. Overall, data from this project will tion. Hum Biol. 2005;77(5):541–59.
8. Blangero J, Diego VP, Dyer TD, et al. A kernel of
contribute to our understanding of the determi- truth: statistical advances in polygenic variance com-
nants of blindness and visual impairment in a ponent models for complex human pedigrees. Adv
novel Asian population, a necessary step if we Genet. 2013;81:1–31.
are to significantly reduce its negative impact on 9. Bourgain C, Genin E, Quesneville H, et al. Search for
multifactorial disease susceptibility genes in founder
human well-being. populations. Ann Hum Genet. 2000;64(3):255–65.
10. Bourne RR, Stevens GA, White RA, et al. Causes of
Acknowledgments This study is funded by the National vision loss worldwide, 1990–2010: a systematic anal-
Eye Institute of the National Institutes of Health (grant ysis. Lancet Glob Health. 2013;1(6):e339–49.
number EY024384 to MPJ, SWB). Sight-correcting 11. Cameron AC, Windmeijer FAG. An R-squared mea-
glasses, in part, were kindly donated by the Texas Lions sure of goodness of fit for some common nonlinear
Eyeglass Recycling Center, Midland, Texas. We thank regression models. J Econom. 1997;77(2):329–42.
Bashu Dev Adhikari, Pradeep Banjara, Bhim Bdr Jirel, 12. Chua J, Koh JY, Tan AG, et al. Ancestry, socioeco-
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with the Jiri Family Studies over the past 32 years. rare alleles contribute to low plasma levels of HDL
cholesterol. Science. 2004;305(5685):869–72.
Compliance with Ethical Requirements All proce- 15. Congdon N, Broman KW, Lai H, et al. Nuclear cat-
dures were in accordance with the ethical standards of and aract shows significant familial aggregation in an
approved by the University of Texas Rio Grande Valley older population after adjustment for possible shared
Institutional Review Board (IRB# 2016-093-05) and the environmental factors. Invest Ophthalmol Vis Sci.
National Health Research Council of Nepal (Reg. No. 2004;45(7):2182–6.
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Helsinki Declaration of 1975, as revised in 2000. Informed not posterior subcapsular, cataract shows significant
consent was obtained from all individuals prior to partici- familial aggregation in an older population after
pating in the study. All authors declare that they have no adjustment for possible shared environmental factors.
conflict of interest. Ophthalmology. 2005;112(1):73–7.
17. Hammond CJ, Snieder H, Spector TD, et al. Genetic magnitude and temporal trends, 1990–2010.
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Genetic Eye Research
in the Philippines 9
Patrick R. Ching, Edward Ryan A. Collantes,
Michelle D. Lingao, Patricia E. Cabrera,
and Leo D. P. Cubillan
Abstract can lead to more discoveries, collaborations,

The Philippines is a developing nation in and increased representation of Filipinos in
Southeast Asia and is home to over 100 differ- population-based genetic studies. Through
ent ethnolinguistic groups. As in other coun- evidence-based policies and programs, health-
tries, genetic eye diseases in the Philippines care providers and public health practitioners
are rare but collectively represent a significant with the involvement of government and
cause of blindness. Although specialized facil- industry can use genomic knowledge for
ities providing genetic services in the country health promotion and disease prevention.
exist, only a handful of genetic eye studies
have been done in the Filipino population. Keywords
Training more Filipino scientists and ophthal- Philippines · Genetics · Eye diseases · Public
mologists in the field of genetic eye research health
P. R. Ching
Departments of Biostatistics and Epidemiology,
Harvard TH Chan School of Public Health, 9.1 The Philippines
Boston, MA, USA
Center for Evidence-Based Imaging, Brigham and The Philippines is an archipelago in Southeast
Women’s Hospital, Boston, MA, USA
Asia. Located in the western Pacific Ocean, it is
E. R. A. Collantes (*) composed of 7641 islands [1] that belong to three
Massachusetts Eye and Ear, Harvard Medical School,
Manila, Philippines island groups: Luzon, Visayas, and Mindanao. It
is bounded by the Philippine Sea to the east,
Manila Doctors Hospital, Manila, Philippines
e-mail: [email protected] Celebes Sea to the south, Sulu Sea to the south-
west, and South China Sea to the north and west.
M. D. Lingao
Department of Ophthalmology and Visual Sciences, With Manila as the capital, the Philippines has a
University of the Philippines Manila, total land area of about 300,000 km2.
Manila, Philippines According to the World Bank [2], the
Asian Eye Institute, Makati, Philippines Philippines continues to be the 12th most pop-
P. E. Cabrera · L. D. P. Cubillan ulated country in the world with a population
National Institutes of Health, Philippine Eye of 100.98 million and population density of
Research Institute, University of the Philippines 337 persons/km2 in 2015 [3]. The average
Manila, Manila, Philippines
annual population growth rate is 1.7% during

https://doi.org/10.1007/978-981-13-0884-0_9
86 P. R. Ching et al.
the period of 2010–2015 [3]. Of the total popu- 9.2 Burden of Eye Disease
lation, 50.6% was male and the median age
was 24.3 years. The working age group belong- According to the Third National Survey of
ing to ages 15–64 years made up 63.4% of the Blindness in the Philippines conducted in 2001–
population; children below 15 years accounted 2002 [10], the nationwide prevalence of visual
for 31.8% and those aged 65 years and above impairment (visual acuity worse than 6/18 in the
comprised 4.7%. The average household size better eye) was 4.62%. Bilateral and unilateral
in 2015 was 4.4 persons [3]. low vision (visual acuity worse than 6/18 but
Through the years, health indicators have equal to or better than counting fingers at 3 m)
improved. In 2015, the average life expectancy had prevalence of 1.43% and 0.87%, respectively,
among Filipino men and women was 65 and with error of refraction as the leading cause
72 years, respectively (both sexes, 69 years) [4]. (53%). The prevalence of bilateral and unilateral
The crude birth rate (per 1000 population) was blindness (visual acuity worse than counting fin-
24.4 and the crude death rate (per 1000 popula- gers at 3 m) was 0.58% and 0.71%, respectively.
tion) was 5.9 [5]. Adult mortality rate (the prob- Cataract (62.1%) was the most common cause of
ability of dying between 15 and 60 years of age blindness, followed by error of refraction
per 1000 population) for men was 255 (from (10.3%), glaucoma (8%), retinopathy (4%), and
272 in 1990) and for women was 136 (from maculopathy (4%). In a more recent study among
154 in 1990) [5]. Maternal mortality ratio Filipinos in the working age group, it was found
decreased from 152 to 114 per 100,000 live births that the most common causes of blindness or
from 1990 to 2015. A decline in infant mortality severe visual impairment were diabetic retinopa-
rate was also seen from 41.1 deaths per 1000 live thy, pathologic myopia, hereditary retinal disor-
births in 1990 to 23.5 deaths per 1000 live births ders, glaucoma, and optic atrophy [11].
in 2013 [5]. In 2016, cataract (28.2%) and errors of refrac-
Total health expenditure (THE) increased tion (26.8%) were the two most common diagno-
from PHP530.3 billion in 2013 to PHP585.3 bil- ses at the ophthalmology outpatient department of
lion in 2014, a growth of 10.4% [6]. While the Philippine General Hospital (PGH), the larg-
4.6% of the country’s gross domestic product est tertiary state-owned training hospital in the
(GDP) went to THE, the Philippines spent country. Eye diseases with presumed genetic eti-
0.1% of its GDP for scientific research and ologies made up approximately 1% of the cases,
development in 2014 and 0.14% in 2016 [7]. with retinitis pigmentosa as the most common
Although the country remains among the fast- one. Other conditions seen include inherited and
est growing Southeast Asian economies, galactosemia cataract; optic nerve, iris, and cho-
21.9 million Filipinos were still living in pov- rioretinal colobomas; congenital fibrosis of extra-
erty in 2015 [8]. ocular muscles; congenital nystagmus; Peter’s
With over 100 distinct ethnolinguistic groups, anomaly; and ocular albinism, among others [12].
the Philippines is a diverse country – geographi- In the Philippines, as in other countries, genetic
cally, linguistically, and culturally. Foreign influ- eye diseases are rare but collectively represent a
ences come from various parts of Asia, Europe, significant portion of blinding eye conditions. The
and the Americas. According to a population- prevalence of inherited eye diseases in the country
based genetic study of Filipinos, it was deter- is not known because they are grouped under
mined that Philippine groups shared genetic broad categories in epidemiologic studies [10,
affinities with groups from South Asia and 13]. Genetic diseases as a whole are underre-
Australia [9]. There are however much closer ported in the country, partly because of the limited
genetic affinities with groups from Southeast number of trained geneticists who can provide
Asia and Taiwan. specific diagnoses and partly because of the high
9 Genetic Eye Research in the Philippines 87
cost of establishing molecular diagnoses. With the 9.3.2 Prevalence of Color Vision
current developments in treatment for genetic eye Deficiency in Filipino High
diseases, interest in obtaining molecular diagno- School Students
ses for these conditions is increasing. Referrals to
genetic specialists are also expected to increase. Color vision deficiency is one of the most com-
mon disorders of vision and can be divided into
congenital and acquired forms [15]. The most
9.3 enetic Eye Disease Studies
G common color vision defects, commonly (and
in Filipinos incorrectly) termed color blindness, are congeni-
tal [16]. A cross-sectional study was conducted
9.3.1 V
isual Screening in the Aeta among 1258 male Filipino high school students
Population to screen for color vision deficiency. The preva-
lence of color vision deficiency was found to be
A Philippine ethnolinguistic group called the 5.17% of the total number of students screened
Aeta are hunter-gatherers who have a pygmy with the deutan type, the most common type in
phenotype [9]. The Aeta reside in the northeast- this study population [17].
ern part of Luzon island where they mainly prac-
tice swidden agriculture [13]. DNA analysis
revealed that some Aeta groups are genetically 9.3.3 Juvenile Open–Angle
affiliated with the Indian population, while Glaucoma in Filipino Families
another group of Aeta is closer to Southeast Asian
and Oceanian groups. Glaucoma is the most common cause of irrevers-
There was an interest in studying the visual ible blindness worldwide [18]. It is projected that
function of this population because they had by the year 2020, open-angle glaucoma will
never been examined in the past and as a conse- affect 58.6 million individuals [19]. Juvenile
quence of their relative isolation had been outside open-angle glaucoma (JOAG) is a primary open-
the scope of standard medical care [14]. Visual angle glaucoma characterized by disease onset
screening in 225 individuals belonging to the before the age of 35, high intraocular pressure,
Aeta population was implemented, and a stan- and a normal appearance of the drainage angle at
dard ophthalmologic examination was performed gonioscopy. JOAG follows an autosomal domi-
including visual acuity, intraocular pressure, nant inheritance pattern [20]. Myocilin (MYOC)
gonioscopic, and dilated fundus examination. gene mutations lead to JOAG by causing the
The major causes of blindness were cataract and myocilin protein to become misfolded. The mis-
refractive error in 86% of patients. Other ocular folded protein is then sequestered in the endo-
conditions observed in this study population were plasmic reticulum (ER) in trabecular meshwork
pterygium, corneal scarring, penetrating trauma, (TM) cells eventually leading to ER stress-
exfoliation syndrome, and retinal disease. There induced TM cell death [21–23].
were no definite cases of primary open-angle Genetic studies have been conducted on
glaucoma and closed-angle glaucoma identified. Filipino families with JOAG. The MYOC gene
The absence of glaucoma in this study population was screened for mutations in a family with an
may be due to chance or, more interestingly, dif- autosomal dominant form of JOAG [24]. No
ferences in genetic susceptibility or environmen- MYOC mutations were found in this family.
tal factors that lower the risk for primary forms of Linkage analysis was then performed which
glaucoma in these populations. Visual function showed mapping of a novel genetic locus at
screening in other ethnolinguistic groups in the 5q22.1-q32 [25], necessitating further studies on
Philippines may shed light on these unique popu- this genomic region. More recently, a novel
lations and the various eye conditions they may MYOC nonstop mutation, c.1515A>G
or may not have. (p.*505Wext*42), was identified in another
Filipino family [26]. Functional studies suggest nonsense mutation in exon 18 of the RB1 gene
that this mutation causes sequestration of the was detected in one tumor sample, while a novel
MYOC protein in the ER similar to cells trans- missense mutation in exon 19 was identified in
fected with a known MYOC mutation c.734G>A another tumor. Further studies are needed to con-
(p.C245Y) [27]. firm if this novel mutation is disease causing.
9.3.4 E
ye Movements in X–Linked 9.3.6 C
ase Reports of Rare Ocular
Dystonia with Parkinsonism Disorders
X-linked dystonia-parkinsonism is an X-linked There have been numerous case reports on vari-
recessive syndrome of combined dystonia- ous genetic eye diseases as well as systemic syn-
parkinsonism [28]. The underlying genetic cause dromes with eye manifestations in Filipinos.
has not been fully elucidated although efforts to Several families with retinitis pigmentosa were
determine this are currently underway. All XDP screened for mutations in rhodopsin and periph-
cases described have so far been linked to Filipino erin/RDS genes [36]. No mutations were identi-
ancestry with an especially high prevalence in the fied in these patients, necessitating follow-up
island of Panay. The prevalence of XDP in the studies to screen for other genes which cause
Philippines is 0.31 per 100,000 and in Panay inherited retinal diseases. In patients with muco-
island 5.74 per 100,000 [29]. polysaccharidosis type II, it was found that 40%
XDP manifests predominantly as torsion dys- of them had errors of refraction, while 20% had
tonia, later combined with or sometimes replaced retinal pigmentary changes [37]. Patients clini-
with parkinsonism [29]. Eye movements in cally characterized as having Usher syndrome
patients with XDP have also been described [30, [38], Alport syndrome [39], Bardet-Biedl syn-
31]. XDP patients were found to have Parkinson- drome [40], Waardenburg syndrome type 1 [41],
like oculomotor dysfunctions with normal main and encephalocraniocutaneous lipomatosis [42]
sequence, reduced saccade and smooth pursuit have also been reported. With the establishment
gain and normal horizontal saccade latency. of molecular diagnostic facilities in the country,
Further studies on the prevalence of ocular move- it is with great hope that future case reports will
ment abnormalities and its correlation to disease be accompanied by a genetic diagnosis to get a
severity in XDP patients may aid in better charac- better understanding of the disease as well as the
terization of the disease as well as potential mark- possibility of offering gene-based treatments to
ers of disease status and prognosis. these patients.
9.3.5 Molecular Profiling 9.4 Genetic Services

of Retinoblastoma in the Philippines
Retinoblastoma (RB) is the cause of approxi- The Institute of Human Genetics-National

mately 4% of all pediatric malignancies world- Institutes of Health (IHG-NIH) at the University
wide [32]. As the most common intraocular of the Philippines (UP) Manila is the largest pro-
malignancy in children, the estimated incidence vider of genetic services in the country. It pro-
of RB in the Philippines is 237 in 100,000 [33]. vides clinical, diagnostic, and research services
There is a 7–8% familial incidence among RB including clinical genetics, cytogenetics, molec-
patients, similar to prevalence rates in other ular genetics, biochemical genetics, and new-
countries [34]. born screening. It has a Clinical Genetics Unit
Molecular profiling of RB tumor samples was (CGU) which provides comprehensive clinical
done in a cohort of Filipino patients [35]. A known evaluation, appropriate management, and genetic
counseling services to persons and families with tional linkages within and outside the country
or at risk for an inherited condition [43]. The that facilitate the sharing of technology and
CGU offers karyotyping, high-resolution band- expertise in genomics. In addition, active promo-
ing, and fluorescence in-situ hybridization anal- tion of genomics competencies among health
yses for different chromosomal disorders and professionals needs to be emphasized as it
neoplasms. remains essential in the translation of genomics
The Molecular Genetics Unit is involved in to clinical and public health practice.
research utilizing molecular-based tools and In the Philippines, there are only a few medi-
techniques for unique and common genetic dis- cal geneticists. While only PGH offers fellowship
eases among Filipinos. Focused on the diagnosis training in clinical genetics, only a handful of
and management of inherited metabolic disor- institutions have graduate programs for genetics,
ders, the Biochemical Genetics Unit conducts molecular medicine/biology, or biomedical
biochemical tests including urine metabolic informatics.
screen, urine organic acid analysis, quantitative Although the number of Filipino scientists
amino acid analysis, etc. It also houses the first involved in medical genetics, genomics, bioinfor-
Newborn Screening Center (NSC) in the country matics, and related fields has increased through
that provides an advanced newborn screening for the years, the need for more researchers trained
more than 20 disorders [43]. in these fields still exists. Experts in genetic epi-
Aside from IHG-NIH, there are other NSCs in demiology, quantitative genetics, and computa-
the country and institutions that offer cytogenetic tional biology can mentor students and young
testing and clinical genetics services [43]. The investigators, and this can lead to more genetic
Department of Ophthalmology and Visual knowledge and discoveries in ophthalmology and
Sciences at the PGH and the ocular genetics other areas of medicine. Geneticists, traditional
department at the Asian Eye Institute, a privately and genetic epidemiologists, biostatisticians, bio-
owned ambulatory eye center in the Philippines, informatics specialists, and public health practi-
provide comprehensive ophthalmic care, diag- tioners have much to learn from one another. In
nostic evaluation, testing, and counseling for this era of big data where rapid technologic
patients with genetic eye diseases. advances continue, Filipino researchers should
Additionally, the Philippine Genome Center also explore opportunities in other omics fields
(PGC) is a multidisciplinary institution that seeks like epigenomics, transcriptomics, proteomics,
to develop genomics research in the country. and metabolomics.
With health as one of its priorities, the PGC aims
to apply genomics to both clinical and public
health practice for population benefit. Some of 9.6 Genomics for Health
the projects in the health program include web- Promotion and Public Health
based genome library for influenza virus (H1N1)
and development of diagnostic kits for early Advances in genomic eye research must be trans-
diagnosis of infectious diseases and noncommu- lated to public health. As the knowledge of the dis-
nicable diseases [43]. ease biology of different medical conditions
increase, healthcare systems become better
equipped to modify their prevention strategies.
9.5 Education and Training Sound health policies must be guided by scientific
research and community consultation. Public
Strengthening the academic and research infra- health programs borne out of these must be con-
structure requires more collaboration among the tinuously assessed. While advances in public
academics, government, and private industries. health do not correspond to the developments in
As the PGC implements research program-driven genomics over the recent years, creation of p olicies
agenda on priority areas, it establishes institu- for genetic testing and other services should
nevertheless be emphasized. As Brand and col- 2009 to 14% in 2016 [45], better representation
leagues said, a conceptual shift in both medicine of groups of non-European descent is still nec-
and public health is needed with the increasing essary. Since genomic research infrastructure to
genome-based knowledge: genomics needs to diagnose and study genetic eye diseases is
understand how it can include public health aspects already present in the Philippines, the next logi-
in its work program while public health needs to cal step is conducting more robust investiga-
analyze how genomics changes the concepts of tions. Through international collaborations
public health [44]. especially in the Asia Pacific region, efficient
It is important to recognize that genomic generation of research findings with better
variation is vital in understanding disease, their accuracy, relevance, and generalizability
impact on health, and how we understand dif- including the vulnerable and underserved will
ferences in ancestral populations. Genome- be within reach.
wide association studies (GWAS) have played Results should be made available to health-
a major role in our understanding of variants care providers and the public since these will
involved in disease as well as understanding guide management of eye conditions, and,
ancestral differences. It is worth noting how- through shared decision-making, the latter will
ever that 81% of GWAS studies involve sub- be able to make better informed choices.
jects who are of European ancestry [45]. These Moreover, discoveries from these studies will
figures have improved throughout the years, help public health officials and lawmakers in
with representation from those of Asian ances- planning, implementing, and evaluating public
try participating in GWAS steadily increasing health policy. This underscores the need to
from 3% in 2009 to 14% in 2016. In order to strengthen the genetics, genetic epidemiology,
improve the diversity in genetic databases, var- and bioinformatics training in the country. As
ious Asian populations like the Philippines computerized and online systems, biobank, and
should be more involved. The infrastructure to other genomic infrastructure become more
diagnose and study genetic eye diseases is available, our researchers should take full
already present in the country. The next logical advantage of these to inform prediction, screen-
step is to conduct more robust research as well ing, prevention, and intervention strategies.
as establish relationships with international Knowledge from genetic and genomic studies
collaborators especially in the Asia Pacific should not only benefit developed nations; it
region. should improve the health of all people.
Conflict of Interest The authors declare that they have

9.7 Summary no conflict of interest.
To date, only a few genetic studies in eye dis-

eases have been conducted in the Philippines. References
Although only a few causal genetic variants
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Hereditary Eye Disease in Ningxia
Hui Autonomous Region of China 10
Weining Rong, Huiping Li, and Xunlun Sheng
Abstract ing, we confirmed the clinical diagnosis of

Ningxia Hui Autonomous Region is the main combined Marfan syndrome with X-linked
gathering region of Hui nationality in China. hypophosphatemia in a pedigree with com-
Because of unique geographical environment plex phenotypes. The homozygous mutation
and national characteristics, the incidence of of BEST1 gene was found in a consanguine-
hereditary eye disease is higher in Ningxia ous family with complicated phenotype, and
Region. From 2010 to 2017, our research team the diagnosis of autosomal recessive bestro-
collected 230 pedigrees and 268 sporadic phinopathy (ARB) was made.
patients with monogenic hereditary eye dis-
ease, including 210 pedigrees and 250 spo- Keywords
radic patients with hereditary retinal diseases. Ningxia · Ocular disease · Genetic · Gene ·
Among these pedigrees, 89 (42%) families Mutation · Consanguineous marriage
reported a family history of consanguinity. We
carried genetic research on 113 pedigrees and
154 sporadic patients with hereditary retinal
diseases. The disease-causing mutations were 10.1 Introduction
identified in 66 pedigrees and 69 sporadic
patients. Total 63 related disease-causing Ningxia Hui Autonomous Region is located in
genes were involved. Four new disease-caus- the northwest of China. Ningxia covers a total
ing genes were discovered through whole area of 66,400 square kilometers. The total popu-
exome sequencing combined with a series of lation is 6.82 million in 2017, of which the Hui
cell experiments and animal experiments, people accounts for 35.6%. The Hui nationality
including GUCA1A gene that causes central religion is Islam. Ningxia is one of the five minor-
areolar choroidal dystrophy, CCT2 gene that ity autonomous regions in China. It governs 5
causes Leber congenital amaurosis, CEP78 prefecture-level cities and 22 counties (or county-
gene that causes Usher syndrome, and PRPF4 level cities, districts).
gene that causes autosomal dominant retinitis Compared with other provinces of China, the
pigmentosa. Through whole exome sequenc- economy of Ningxia is relatively backward.
Many counties are located in remote mountain-
ous areas with low traffic and population mobil-
W. Rong · H. Li · X. Sheng (*) ity, little communication with the outside world,
Ningxia Eye Hospital, Ningxia People’s Hospital, and low cultural quality of the population. Early
Ningxia, China

https://doi.org/10.1007/978-981-13-0884-0_10
94 W. Rong et al.
marriage and consanguineous marriage are Marfan syndrome (MFS), Weill-Marchesani syn-
extremely serious. In 2000, a survey about con- drome, Axenfeld-Rieger syndrome, dominant
sanguineous marriage of rural Hui population in optic atrophy, recessive optic atrophy, and
Ningxia showed that the consanguineous mar- X-linked juvenile retinoschisis (XLRS).
riage rate was 46%. Therefore, the incidence of We carried genetic research on 113 pedigrees
hereditary diseases in Ningxia is very high. and 154 sporadic patients with hereditary eye dis-
Our study conformed to the Declaration of eases. The disease-causing mutations were iden-
Helsinki and was approved and prospectively tified in 66 pedigrees and 69 sporadic patients;
reviewed by the Ethics Committee of Ningxia the positive rate was 58% and 45%, respectively.
People’s Hospital. Total 63 related disease-causing genes were
Since 2002, we have carried out research on involved. About 60% of patients with retinitis
hereditary eye disease in Ningxia. In our study, all pigmentosa are recessive; the mutation rate of
procedures followed were in accordance with the USH2A gene was the highest, followed by
ethical standards of the Ethics Committee of MYO7A gene in RP pedigrees and C2orf71 gene
Ningxia People’s Hospital on human experimen- in sporadic RP patients. In other hereditary reti-
tation and with the Helsinki Declaration of 1975, nal diseases, ABCA4 gene mutation rate was the
as revised in 2000 (5). Informed consent was highest, followed by CRB1 gene. Seven muta-
obtained from all patients for being included in tions were detected in 7 families with Marfan
the study. After more than 10 years of research, syndrome, and 15 mutations on mitochondrial
we have made some achievements in the field of gene were detected in 15 Leber hereditary optic
hereditary retinal diseases. From 2010 to 2017, neuropathy (LHON) patients.
our research team collected 230 pedigrees and Four novel disease-causing genes were dis-
268 sporadic patients with monogenic hereditary covered through whole exome sequencing com-
eye disease, including 210 pedigrees and 250 spo- bined with a series of cell experiments and animal
radic patients with hereditary retinal diseases. experiments, including GUCA1A gene that
Among these pedigrees, 89 (42%) families causes central areolar choroidal dystrophy, CCT2
reported a family history of consanguinity. The gene that causes Leber congenital amaurosis,
consanguinity marriages in 72 families were CEP78 gene that causes Usher syndrome, and
between first cousins, and 9 families were between PRPF4 gene that causes autosomal dominant
second cousins; only 5 families were between half retinitis pigmentosa. Through whole exome
cousins matrimony (2). The families we collected sequencing, we confirmed the clinical diagnosis
mainly include the following diseases: retinitis of combined Marfan syndrome with X-linked
pigmentosa (RP), cone degeneration (COD), hypophosphatemia in a pedigree with complex
cone-rod degeneration (CORD), Leber congenital phenotypes. The homozygous mutation of
amaurosis (LCA), Stargardt disease (STGD), BEST1 gene was found in a consanguineous
bestrophinopathies including best vitelliform family with complicated phenotype, and the
macular dystrophy (BVMD) and autosomal reces- diagnosis of autosomal recessive bestrophinopa-
sive bestrophinopathy (ARB), occult macular thy (ARB) was made. We will describe the typi-
dystrophy (OMD), achromatopsia, congenital sta- cal cases one by one.
tionary night blindness (CSNB), choroideremia
(CHM), gyrate atrophy of the choroid and retina,
Usher syndrome, Bardet-Biedl syndrome, 10.2 C
entral Areolar Choroidal
Crouzon syndrome, ocular albinism, Oliver- Dystrophy (CACD)
McFarlane syndrome, central areolar choroidal
dystrophy (CACD), microcornea, Leber heredi- 10.2.1 Introduction
tary optic neuropathy, Sveinsson’s chorioretinal
atrophy, blepharophimosis-ptosis-epicanthus Central areolar choroidal dystrophy (CACD),
inversus syndrome (BPES), congenital cataract, first described in consanguineous marriage fam-
10 Hereditary Eye Disease in Ningxia Hui Autonomous Region of China 95
ily [1], is a clinically and genetically heteroge- tor degeneration. The choriocapillaris atrophy
neous pigmentary retinopathy characterized by following RPE degeneration is a result of a lack
fine, mottled depigmentation in the macular area. of growth factors secreted by the RPE, which are
Later, atrophy of the retinal pigment epithelium essential for choroidal vasculature homeostasis.
and choriocapillaris is observed [2]. It usually This may be more reasonable to explain the lipo-
shows progressive and profound visual loss fuscin accumulated beneath the retina.
between 30 years and 60 years of age [3, 4]. It
may be challenging to diagnose CACD in the
early stages of the disorder because of the non- 10.2.3 Genetic Aspects
specific depigmentation. Also, the late-onset
CACD due to PRPH2 mutation which occurs by All genes associated with CACD are
the seventh decade share common features with photoreceptor-specific. Autosomal dominant
age-related macular degeneration (AMD), CACD is most commonly caused by mutations in
including choroidal neovascular and drusen the peripherin-2 (PRPH2) gene and GUCY2D
lesion, and may easily be misdiagnosed [5–7]. gene [3, 9–12]. To date, eight different mutations
The relationship between genotype and phono- in the PRPH2 gene [5, 11, 13–17] have been
type of CACD is not fully elucidated. CACD identified to cause the CACD phenotype. The
consist of 8% hereditary retinal dystrophies [8]. most frequently detected mutation in PRPH2 is
Epidemiological study base on population is lim- R172W, which has been reported in different eth-
ited due to rare occurrence. nic groups. Recently, our investigation reveals
that a novel GUCA1A mutation p.R120L con-
tributes to CACD etiology in dominant trait in a
10.2.2 Pathology five-generation family (Fig. 10.1) [9]. Clinically
CACD mainly affects RPE and choriocapillaris,
The pathology of CACD is characterized by but GUCA1A mutation results in photoreceptor
absence of photoreceptors, RPE, and choriocapil- dystrophy initially, followed by RPE and chorio-
laris in the area of atrophy, which coincides with capillaris atrophy. The diverse pathogenic mech-
clinical findings.[2] Thinning to complete loss of anisms of GUCA1A mutation may imply that the
the outer nuclear layer in optical coherence photoreceptor degeneration which might be
tomography (OCT) examination can be observed neglected previously triggers the cascade of RPE
in the CACD process [4]. Previous studies and choriocapillaris defect in another two genes
showed that lipofuscin accumulated in the sub- of CACD. Similar to our cases, overexpressing
retinal space where RPE cells were distorted and mutated PRPH2 p.R172W also impaired photo-
expanded that resulted in choriocapillaris atro- receptors of mice retina and secondary RPE/cho-
phy, but overlying photoreceptors were partly riocapillaris degeneration [18].
atrophic. Recently, our study renewed this con-
cept. We found a novel mutation GUCA1A p.
R120L in a five-generation family with 10.2.4 Clinical
CACD. The overexpression of p.R120L mutation
of GUCA1A in zebrafish resulted in a significant Four clinical stages of CACD have been described
degeneration of both rod and cone cell; also the [2, 3]. In stage I CACD, slight and focal
RPE is impaired (Fig. 10.1) [9]. We propose a parafoveally pigmentary RPE changes can be

hypothesis that degeneration of photoreceptor observed, usually in the adolescent patient. Stage
was primary insult for CACD; overaccumulation II is characterized by oval-to-round, mildly atro-
of OS debris or other abnormal substances phic, hypopigmented area, which shows speckled
derived from those dying photoreceptors are FAF pattern on a fundus autofluorescence (FAF)
toxic to the RPE, which can lead to RPE impair- image, usually 1.5 to several disc diameters. In
ment; and RPE will in turn aggravate photorecep- stage III one or more patches of
96 W. Rong et al.
Fig. 10.1 Impairments in photoreceptors, the retinal pig- messenger RNA (mRNA) levels of photoreceptor-, cone-,
ment epithelium (RPE), and ocular vasculature induced and rod-specific transcripts in GUCA1Ap.R120L- and
by GUCA1A p.R120L in zebrafish. (a–d) GUCA1AWT+p.R120L-injected zebrafish compared
Immunofluorescent staining of rhodopsin (RHO) (red; a, with the GUCA1AWT-injected group. (h–j) Robust Zpr-2
b), peanut agglutinin (PNA) lectin (green; a, b), and Zpr-1 expression was detected in retinal frozen sections of 4-dpf
(red; c, d). RHO and PNA expressions were significantly larvae injected with GUCA1AWT (h), whereas Zpr-2
reduced in zebrafish overexpressing GUCA1Ap.R120L expression was diminished in the GUCA1Ap.R120L-
(a, b). Zpr-1 expression was only slightly reduced in the injected (i) and GUCA1AWT+p.R120L-injected (j)
GUCA1Ap.R120L-injected group (d) when compared groups. (k) Relative mRNA levels of RPE characteristic
with GUCA1AWT-injected zebrafish (c). (e–g) Relative transcripts in the GUCA1Ap.R120L- and GUCA1AWT+p.
well-circumscribed atrophy of the RPE and CACD causative gene GUCA1A, revealed in our
choriocapillaris appear outside the fovea. In stage study, encodes the guanylyl cyclase-activating
IV, well-defined chorioretinal atrophy affects the protein1 (GCAP1) which is essential in the visual
fovea and results in a markedly decreased visual cycle and highly expressed in the outer segment
acuity. There is no drusen and neovascular lesion (OS) layer of cones [21], which acts as a calcium
in typical CACD. But CACD is a clinically het- sensor in the recovery of photoreceptors from
erogeneous disease and diverse in the age of photon capture by activation of the retinal gua-
onset, progression, and phenotype expression, nylate cyclase 1 (retGC1). RetGC1 is a
even within one family [2, 17, 19]; in a large con- membrane- bound homodimer enzyme, which
sanguineous Tunisian family, CACD segregated converts guanosine 5’-triphosphate (GTP) to
in an autosomal dominant trait. Among 21 cGMP, permitting reopening sodium- and
affected family members, 6 patients had drusen voltage-gated calcium channels to recover from
deposits. The PRPH2 gene and the 17p13 locus the dark state. The abnormal calcium sensitivity
were not associated with this phenomenon [6] of the cyclase increases cGMP-gated dark cur-
The Arg142Trp mutation in PRPH2 is one of the rent in the rod outer segments; overloading of
factors predisposing to drusen development [7], calcium reshapes rod photoresponses and trig-
while the Tyr141Cys mutation is susceptible to gers photoreceptor death [22]. Both GUCA1A
secondary development of choroidal neovascular p.R120L mutations and GUCY2D p.V933A
membranes [20]. We reveal a novel GUCA1A mutation can lead to CACD. The molecular
mutation, p.R120L, in a family with variable mac- mechanism of these two mutations functioning in
ulopathies ranging from mild photoreceptor CACD pathology needs to be further
degeneration to CACD (Figs. 10.2 and 10.3). This investigated.
means more attention should be attach on photo-
receptor function examination on whom with
undetectable physical sign in CACD family. 10.2.6 Summary
Although CACD is a rare disease, the clinical and

10.2.5 Molecular Biology genetic diversity confuse us whether CACD rep-
resent a severe pattern of maculopathy or possess
The molecular mechanism of CACD is still not complete different pathogenic modes. Much
clear now. The peripherin encoded by PRPH2 work had to be done on the molecular
gene is a surface glycoprotein localized to the rim mechanism.
region of the disc membranes of both rod and
cone photoreceptor, which functions as an adhe-
sion molecule to play an essential role in the 10.3 L
eber Congenital Amaurosis
assembly, orientation, and structural stability of (LCA)
outer segment discs and to account for an
increased turnover of instable membranous seg- 10.3.1 Introduction
ments [17]. PRPH2 mutation may lead to less
efficient clearing away of the instable membra- Leber congenital amaurosis (LCA) is a heredi-
nous segments, and more OS debris accumulate tary early-onset congenital retinopathy that
beneath the retina, which is toxic to RPE. Another develops concomitantly with severe macular
Fig. 10.1 (continued) R120L-injected groups were cantly reduced in the GUCA1Ap.R120L-injected group.

decreased when compared with the GUCA1AWT-injected (n) The fluorescent intensity of EGFP calculated by
zebrafish. (l) No truncal vascular anomaly is revealed in ImageJ in the two groups. Results were obtained from
transgenic (flk1: enhanced green fluorescent protein technical triplicates. Error bars represent the SE. Error
(EGFP)) zebrafish injected with GUCA1Ap.R120L. (m) bars represent the SD. NS not significant. *P < 0.05;
Axial projections of confocal images from the two **P < 0.01; ***P < 0.001. Scale bar = 20 mm
injected groups. Fluorescent intensity of EGFP is signifi-
98 W. Rong et al.
Fig. 10.2 Pedigree of family DC and haplotype recon- given for all participants. Black bars represent the ances-
struction for the mapped region on chromosome 6 in fam- tral haplotype associated with the disease. *Individuals
ily DC. Filled and open symbols represent affected and from whom blood samples were collected. The mapped
unaffected members, respectively. The proband is indi- region flanked by markers D6S276 and D6S460 was
cated by the black arrow. Haplotypes for tested short tan- shared by all patients and was absent in all unaffected
dem repeat (STR) markers in the mapped region and those members
flanking it, and genotypes for GUCA1A c.359_360, are
degeneration [23].Clinical observations of LCA were second cousins, and their child (II-6, in
patients are heterogeneous, although LCA Fig. 10.4a) was diagnosed as blind with severe
patients are usually born almost or completely retinopathy at the age of 6 months. All of the
blind. Thus, the parents of LCA patients typically members of this family were examined by fundus
notice their child’s blindness within several imaging and optical coherence tomography
months after birth [23]. Nearly 20% of blind (OCT). Six members exhibited normal fundus
school children have been diagnosed with LCA, and OCT images (Fig. 10.4c), while two of the
and this disease represents a great burden on their six children were further diagnosed with LCA
quality of life [24]. Children with LCA are based on the presence of attenuated blood vessels
defined by a very low, or non-recordable, electro- and pallor fundus images (Fig. 10.4d). These
retinogram (ERG) during infancy, and the vision observations were made in combination with the
loss experienced in LCA patients is severe and habitual oculodigital signs of photophobia and
progressive. The variety of clinical manifesta- eye poking (Fig. 10.4e), clinical hallmarks of
tions, combined with the onset of macular atro- LCA. In addition, visual acuity tests performed
phy and pallor optic nerve head, are concomitant for the affected individuals revealed no fixation
with the symptoms of LCA. of reflection and no following of movement.
Except for the retinal dystrophy, both patients
have hearing disorders and language barrier. But
10.3.2 Clinical Features they had no polydactyly or renal cysts, the
hallmarks of Bardet-Biedl syndrome (BBS) and
The pedigree was recruited from Ningxia Eye ciliopathy.
Hospital, Ningxia People’s Hospital. The parents
Fig. 10.3 Fundus photography, fundus autofluorescence Macular RPE was slightly changed (h). (i–l) In patient
(FAF), fundus fluorescein angiography (FFA), and optical DC-III:14 with grade III maculopathy, a patch of circum-
coherence tomography (OCT) presentations for patients scribed chorioretinal atrophy outside the central fovea is
with different grades of maculopathy. (a–d) Patient indicated on a fundus photograph. Slight hypopigmenta-
DC-IV:6 with grade I maculopathy showed slight parafo- tion is found within this lesion (i). This area shows
veal hypopigmentation (a) with discrete increased FAF severely decreased to absent FAF (j), with choroidal ves-
(b) and hyperfluorescent parafoveal changes on FFA (c). sels visible on FFA (k). OCT demonstrated that the ONL
OCT revealed that the outer nuclear layer (ONL) and and IS/OS layer in the fovea had vanished. Macular RPE
inner segment/outer segment (IS/OS) layer are slightly was moderately changed (l). (m–p) In patient DC-III:18
reduced in the fovea. The retinal pigment epithelium with grade IV maculopathy, the fundus photograph pres-
(RPE) layer was relatively normal (d). (e–h) Patient ents a well-demarcated area of chorioretinal atrophy
DC-III:16 with grade II maculopathy showed pigmental involving the fovea (m), corresponding to an area of
disturbance in the fundus (e) and speckled changes of absent FAF (n). FFA shows a well-defined area of chorio-
increased fluorescence in the maculae, with a round area retinal atrophy with a few large choroidal vessels visible
of hypofluorescence seen on FAF and FFA (f, g). OCT (o). OCT shows a vanished ONL and IS/OS layer in the
showed that the ONL and IS/OS layer were almost van- fovea. Macular RPE degeneration was the severest of all
ished in the fovea and its surrounding maculae, with no (p). OD = right eye; OS = left eye
remarkable changes detected in the peripheral retina.
10.3.3 Genetic Research andc.1547G > A, were present in this CCT2, and
these represented unrivaled candidates in this
Further genetic analysis revealed that two novel LCA family (Fig. 10.4b).These mutations were
compound heterozygous mutations, c.1198A > G located in exon 12 and exon 15 of the CCT2
100 W. Rong et al.
Fig. 10.4 Whole exome sequencing (WES) and clinical showed a normal retina. (d) Clinical evaluations of the
diagnosis. (a) Recessive patterning of a consanguineous affected individual, II-3. Upper panel: Color fundus pho-
LCA family was performed, and compound heterozygous tographs showed severe degenerative changes. The optic
mutations were identified. Whole exome sequencing was disc was pale and the retinal blood vessels were attenu-
performed, and two point mutations in CCT2 were found, ated. A maculopathy with chorioretinal atrophy and
c.1198A >C and c.1547G >A, which resulted in the fol- aggregation of the pigment were also observed. Lower
lowing substitutions: p.Thr400Pro (p.T400P) and p. panel: An OCT examination showed the thinning and dis-
Arg516His (p.R516H), respectively.(b) CCT2 gene muta- organization of the retina. Both c.1198A >C (M1 in red)-
tions and corresponding amino acid substitutions in the and c.1547G >A (M2 in green)-carrying individuals (e.g.,
CCTβ protein were identified. Point mutations in exon 12 II-3 and II-6, respectively) exhibited retinal dystrophy and
(c.1198A >C) and exon 15 (c.1547G >A) resulted in the a macular degeneration phenotypes. (E) Facial and behav-
CCTβ missense changes, T400P and R516H, respectively. ioral features of the oculodigital phenomenon (eye pok-
(c) Clinical evaluations of the non-affected individual, ing) and sunken eyes (exophthalmos) of the affected
II-2. Upper panel: Color fundus photographs showed a individual, II-6
normal retina. Lower panel: An OCT examination also
mRNA transcript, respectively. The CCT chaper- the photoreceptor cells. In addition, levels of
one machinery was found to be affected by the Gβ1, a major target of the CCT complex, and a
structurally instable T400P and R516H mutant critical protein in the phototransduction pathway,
proteins that exhibited an aberrant affinity to the were decreased following the knockdown of
CCTγ subunit adjacent to CCTβ. Moreover, the CCT2 in a mouse photoreceptor cell line.
T400P- andR516H-carrying patient-derived T In conclusion, two novel compound heterozy-
cells and iPSCs exhibited less proliferation. The gous mutations in CCT2 were identified in asso-
T400P and R516H mutants also exhibited limited ciation with a family affected by LCA. These
rescue effects on cell proliferation compared to mutations affected CCTβ-associated chaperone
wild-type CCTβ. Both subunits, CCTβ and activity which has an essential role in retinal
CCTγ, were expressed in the cells in retinal gan- development and photoreceptor physiology.
glion cell layer and near the connecting cilium in
10.4 Usher Syndrome 10.4.3 Genetic Research
10.4.1 Introduction The homozygous variant that affects the canoni-

cal splicing acceptor site of exon 14 (c.1629-
Usher syndrome is a genetically heterogeneous 2A>G (p.G545Pfs*6)) of CEP78 was identified
disorder characterized by combined retinitis pig- in both patients with Usher syndrome in this con-
mentosa and sensorineural hearing loss. It is sanguineous family. It was not observed in any
caused by disruption of protein components in public control databases. Sanger sequencing also
the supramolecular Usher protein network. Usher confirmed the genotype-phenotype cosegrega-
syndrome has a prevalence ranging from 3.2 to tion in this family (Fig. 10.6). Genetic screening
6.2 in 100 000 [25–27], and it can be classified in additional 71 unsolved patients with Usher
into three major categories, namely, USH I, II, syndrome did not identify additional cases with
and III, depending on the age of onset and sever- biallelic CEP78 mutations. We collected the
ity. Until now, 13 genes have been associated patient blood and performed RT-PCR with
with Usher syndrome (10). However, mutations Sanger sequencing. Finally, we confirmed that
in these genes can only account for 70–80% of this variant indeed affects the pre-mRNA splicing
cases [28–30], suggesting additional disease- in vivo and leads to frameshifts and premature
causing loci are yet to be identified. stop codon.
In summary, our results identified a novel cili-
ary gene CEP78 involved in Usher syndrome.
10.4.2 Clinical Features Further studies on additional cases and animal
models would help to describe the CEP78-related
Patients in a consanguineous family were col- phenotype more precisely and reveal the ciliary
lected at the Ningxia Eye Hospital, Ningxia involvement in Usher protein network in photore-
People’s Hospital. The proband developed night ceptor cells and inner ear hair cells, thus pushing
blindness since early childhood. She also noticed forward more efficient disease management and
hypochromatopsia and mild hearing loss since treatment for Usher syndrome.
age 8. She suffered from visual field decrease and
central vision loss since age 10. At her latest visit
at age 35, her BCVAs dropped to 20/125 OD and 10.5 Retinitis Pigmentosa
20/200 OS. Hypochromatopsia was confirmed by
color vision test. Funduscopy features include 10.5.1 Introduction
attenuated vessels, waxy optic disc, and pigment
deposits in the mid-peripheral retina (Fig. 10.5a, Retinitis pigmentosa [RP (MIM 268000)], the
b). Macular region was also involved. Attenuated most common form of inherited retinal dystro-
outer nucleus layer, retinal pigment epithelium phies (IRDs), displays a prevalence ranging from
(RPE), and loss of ellipsoid zone were suggested 1/3500 to 1/5000 among different populations
by OCT presentations (Fig. 10.5c, d). Consistent [31–33]. Clinically, photoreceptor degeneration
with the fundus photography, FFA also revealed and pigment migration are characteristics of RP,
aberrant vascular arcades and speckled changes and symptoms include night blindness, a con-
of increased fluorescence in the mid-peripheral stricted visual field (VF), and sometimes even-
retina (Fig. 10.5e, f). Both scotopic and photopic tual loss of central vision [34]. At the cellular
ERG responses were abolished. She has an level, rod photoreceptors and/or the retinal pig-
affected brother with similar ophthalmological ment epithelium (RPE) are affected at the initial
findings as well as hearing loss. According to stage of the disease, and cone photoreceptors
these abnormalities, both patients in this family may be involved at a later stage. At the molecular
were clinically diagnosed with atypical Usher level, RP exhibits significant genetic heterogene-
syndrome phenotype.
102 W. Rong et al.
Fig. 10.5 Clinical manifestations of the patients with atypical Usher syndrome. Fundus images of OD (a) and OS (b).
Optical coherence tomography images of OD (c) and OS (d). Fundus autofluorescence results of OD (e) and OS (f)
ity involving at least 87 disease-causing genes first referred to ophthalmic examination at the age
(RetNet). of 15 for his poor night vision. After that, he devel-
oped VF restriction and impaired central vision. At
the latest visit when he was 58 years old, he
10.5.2 Clinical Features showed bilateral cataracts and a typical RP fundus,
including retinal degeneration with macular
A three-generation family (AD01) with autosomal involvement, waxy pallor of optic disc, narrowed
dominant retinitis pigmentosa (adRP) and a spo- vasculature, and peripheral pigment deposit
radic (S01) RP patient were recruited at the (Fig. 10.7c versus e). Optical coherence tomogra-
Ningxia Eye Hospital, Ningxia People’s Hospital. phy (OCT) revealed attenuated outer nuclear layer
The proband of Family AD01, AD01-II:3, was (ONL) and RPE at the maculae with complete loss
Fig. 10.6 Genetic findings in the patients with Usher splicing-altering effect of the variant c.1629-2A>G. In the
syndrome. (a) CEP78 mutations were identified in two probands’ mRNA, a10 bp-long region in CEP78 exon 14
unrelated consanguineous families. The variants cosegre- was deleted, while in the unaffected heterozygous carrier,
gate with the disease phenotype. Arrows indicate pro- wild-type/mutant hybrid sequence was observed
bands. (b) RT-PCR and Sanger sequencing confirmed the
of outer/inner segments (OS/IS), all of which were 37 years of age. At a recent visit when he was
consistent with his poor visual acuity (Fig. 10.7d 53 years old, he showed severely impaired VA,
versus f). Diffused loss of VF and diminished elec- significantly restricted VF, undetectable ERG
troretinogram (ERG) responses were also responses, and a typical RP fundus (Fig. 10.7g, h).
observed. Patients AD01-II:1 and AD01-II:6
developed night blindness at 27 and 24 years of
age, respectively. At the time of the last examina- 10.5.3 Genetic Research
tion when they were 66 and 47 years of age,
respectively, they showed phenotypes similar to We identified two heterozygous variants in
those of the proband, including poor night vision, PRPF4, including c.-114_-97del in a S01 RP
poor central visual acuity, severely reduced VF, patient and c.C944T (p.Pro315Leu), which cose-
typical RP fundus appearances, and nearly unde- gregate with disease phenotype in AD01 family
tectable ERG responses. The individual AD01- with adRP (Fig. 10.8). Both variants were absent
III:4 was only 9 years old at the time of the last in 400 unrelated controls. The c.-114_-97del,
examination, whereas the ages at onset of disease predicted to affect two transcription factor bind-
in this family ranged from 15 to 27. Funduscopy ing sites, was shown to downregulate the
and OCT examinations on AD01-III:4 detected no promoter activity of PRPF4 by a luciferase assay
remarkable findings. ERG test revealed moderate and was associated with a significant reduction of
defects including prolonged implicit time of PRPF4 expression in the blood cells of the
b-wave in scotopic ERG and decreased amplitudes patient. In fibroblasts from an affected individual
of a- and b-waves in photopic ERG compared with with the p.Pro315Leu variant, the expression lev-
age-matched controls. We therefore diagnosed the els of several tri-snRNP components, including
AD01-III:4 as suspected RP. The Simplex patient, PRPF4 itself, were upregulated, with altered
S01, developed night blindness at the age of 20. expression pattern of SC35, a spliceosome
Constricted VF and decreased VA were noticed by marker. The same alterations were also observed
104 W. Rong et al.
Fig. 10.7 Pedigrees and clinical evaluations of Family fected member (AD01-III:2) are provided as normal con-
AD01 and Sporadic Case S01. (a and b) The pedigree of trols. (e and f) Right eye fundus of the proband (AD01-II:3)
Family AD01 indicates a likely dominant inheritance of reveals typical RP degeneration (e), including a waxy
RP over three generations (a), while no other family mem- optic disc, attenuated retinal vessels, and numerous bone
bers are affected within the pedigree of Sporadic Case S01 spicule-like pigments. The white line indicates the layer
(b). The genotypes, age of final examination, and disease for OCT examination (f), which demonstrates macular
onset (inside parentheses) are given below the pedigree atrophy showing attenuated ONL and RPE with complete
symbols. Black-filled and blank symbols represent loss of OS and IS (denoted by yellow arrows). (g and h)
affected and unaffected status, respectively. (c and d) The fundus of case S01 shows typical RP appearance for
Fundus and OCT examination of the right eye of the unaf- both eyes
in cells overexpressing hPrp4Pro315Leu, sug- 10.6 M

arfan Syndrome Combined
gesting that they arose as a compensatory with X-Linked
response to a compromised splicing mechanism Hypophosphatemia
caused by hPrp4 dysfunction. Further, overex-
pression of hPrp4Pro315Leu, but not hPrp4WT, 10.6.1 Introduction
triggered systemic deformities in wild-type
zebrafish embryos with the retina primarily Marfan syndrome (MFS; MIM 154700), charac-
affected, and dramatically augmented death rates terized by complicated manifestations in multiple
in morphant embryos, in which orthologous organ systems with high degrees of clinical diver-
zebrafish prpf4 gene was silenced. We conclude sity, is one of the most common autosomal domi-
that mutations of PRPF4 cause RP via haploin- nant inherited connective tissue diseases with a
sufficiency and dominant-negative effects and prevalence of 1 in 5,000[35]. Cardinal MFS
establish PRPF4 as a new U4/U6-U5 snRNP features involve the ocular, skeletal, and cardio-
component associated with ADRP. vascular systems, of which ectopia lentis and aor-
tic aneurysm are given special clinical
significance [36]. Excessive liner growth of the
Fig. 10.8 Genetic analyses of variants identified in the colors. Residue 315 is located in the second blade. A close
PRPF4 gene and expression of Prpf4 in murine tissues. (a) view of residue 315 highlighting the WT (proline) and
PRPF4 gene (red-filled box) spanning 17.56 kb on chro- mutated (leucine) amino acids at the position is presented
mosome 9q32 (upper panel) contains 14 exons (middle in the two bottom boxes. (e)Orthologous protein sequence
panel). The two identified heterozygous variants, c.- alignment of PRPF4 from human (Homo sapiens), chim-
114_-97del and c.C944T (p.Pro315Leu), were indicated panzees (P. troglodytes), dogs (C. lupus), cows (B. taurus),
by a red box crossing the green line and a red line in the rats (R. norvegicus), chickens (G. gallus), zebrafish (D.
middle panel, respectively. The two red arrows denote the rerio), fruit flies (D. melanogaster), roundworms (C. ele-
transcription start site (TSS) and the translation start site gans), yeast (S. cerevisiae and S. pombe), and plant (A.
(ATG). (b and c) Sequencing chromatograms of the thaliana). Conserved residues are shaded. The mutated
affected member from Family S01 (B) and AD01 (c) residue 315 is boxed and indicated. (f) Expression of
showing the c.-114_-97del and c.C944T substitution, Prpf4 in multiple murine tissues including the lung, kid-
respectively (top). WT sequences of the unaffected family ney, brain, spleen, heart, liver, neural retina, and retinal
members were shown at the bottom. (d) The top view illus- pigmented epithelium (RPE) is shown. A 176 bp PCR
trates the predicted structure of the WD-40 repeat domain product of the murine Prpf4 (top panel) was detected in all
of hPrp4 (residues 229–521), which comprises seven tested tissues. PCR products (162 bp) of the murine Gapdh
highly repeated blades. Each blade is indicated by different were analyzed in parallel as a loading control
long bones and joint laxity are hallmarks for the wasting with normal or low
skeletal systems. X-linked hypophosphatemia 1,25- dihydroxyvitamin D concentrations [37].
(XLH;MIM 307800), presenting a prevalence of Low serum phosphate concentration and reduced
1 in 20,000, is the most common form of familial tubular resorption of phosphate corrected for glo-
hypophosphatemic rickets (FHR), which is a merular filtration rate (TmP/GFR) are character-
dominant disorder biochemically featured by istic for XLH [38].
hypophosphatemia caused by renal phosphate
106 W. Rong et al.
Fig. 10.9 Clinical presentation for family XLH01. (a) stenomelia in the digits of patient XLH01-II:2 (h), but not
Medial bowing in the patients XLH01-I:2, XLH01-II:1, in patient XLH01-I:2 (d) or XLH01-II:1 (f). Widening of
and XLH01-II:2. (b, c) Anterior segment photography both proximal and distal tibia metaphysis in patients
indicates dislocation of lens toward nasal superior side in XLH01-II:1(f, g) and XLH01-II:2 (h, i) are shown. Medial
both eyes. (D–i) Radiographic findings reveal dolicho- bowing is found in all three patients (e, g, i)
10.6.2 Clinical Features line phosphatase (ALP) level, and reduced

tubular resorption of phosphate corrected for glo-
In June 2011, the 8-year-old proband merular filtration rate (TmP/GFR) in this patient.
(XLH01-II:1) was first referred to our ophthal- Serum calcium, intact parathyroid hormone
mic clinic for blurred vision over the past 2 years. (iPTH), 25-hydroxyvitamin D3, and
She was born at term to a 34-year-old woman 1,25-dihydroxyvitamin D3 were within the nor-
after an uneventful full-term pregnancy and mal range. Her 41-year-old mother (XLH01-I:2)
delivery. Her best corrected visual acuities and 10-year-old brother (XLH01-II:2) had no
(BCVAs) reached 0.1 for the right eye and 0.25 remarkable ophthalmic or cardiac conditions, but
for the left eye. Slit-lamp test revealed disloca- showed similar skeletal abnormalities as demon-
tion of lens toward nasal superior side and hippus strated by physical and laboratorial tests with
in both eyes (Fig. 10.9b, c). Medical records exception of dolichostenomelia in their digits
included an atrioseptopexy at age 5 for her atrial (Fig. 10.9d, g). Her brother’s height was 113.0 cm
septal defect (ASD). Other than the repaired ASD (<−3 SD). Her mother was 145 cm in height and
presentation, ultrasonic cardiogram (UCG) indi- had osteomalacia, severe ostealgia, and difficul-
cated left-to-right shunt flow through a ventricu- ties in walking, but no remarkable change in
lar septal defect (VSD) with the diameter of serum ALP level. The proband was there after
4.50 mm. Slight mitral, tricuspid, and pulmonary treated with a surgical removal of the dislocated
regurgitations were also detected. Her calculated lens with implantation of an artificial intraocular
Z-score for aortic root was −0.54. Physical lens and received amblyopia training after sur-
examination on our patient also showed complex gery. At the 3-year follow-up, her BCVAs
skeletal problems. Her height was 117.0 cm improved to 0.4/0.5 (OD/OS). She also had fol-
[<−2standard deviation (SD)] and her weight low-up visits in the cardiology and pediatric
was 30.0 kg(>+1 SD) on admission. Radiographic departments and was supplemented with elemen-
examinations showed widening of both proximal tal phosphorus and calcitriol. Her ALP level was
and distal tibia metaphysis with medial bowing, back to normal at 2-year follow-up with no
but dolichostenomelia in her digits was also complication.
revealed (Fig. 10.9h). Laboratory analyses indi-
cated hypophosphatemia, elevated serum alka-
Fig. 10.10 PHEX and FBN1 mutations identified in the gene (right panel). (c) Orthologous protein sequence
current family. (a). Pedigree of the included family. The alignment of PHEX from seven species. Conserved resi-
PHEX and FBN1 genotypes for all included members dues are shaded. The mutated residue 792 is boxed and
were annotated. Black-filled, gray-filled, and blank sym- indicated. (d–e) Structural modeling of the wild-type and
bols represent X-linked hypophosphatemia, Marfan syn- mutant fibrillin-1. One hydrogen bond in the wide-type
drome, and unaffected status, respectively. (b). DNA protein was eliminated due to the substitution from cyste-
sequencing profiles for the identified disease-causing ine to phenylalanine at residue 792
mutations in the PHEX gene (left panel) and the FBN1
10.6.3 Genetic Research mutant fibrillin-1 (residues 584–950) was con-

structed on the basis of low-density lipoprotein
We performed whole exome sequencing (WES) receptor (protein data bank [PDB] ID: 1N7D) with
on patients XLH01-I:2, XLH01-II:1, and the sequence similarity of 0.38 (Fig. 10.10d, e).
XLH01-II:2. A recurrent heterozygous/hemizy- One hydrogen bond between residue792 and
gous mutation in the PHEX gene (c.871C>T Thr791 was eliminated due to the substitution
[NM_000444]; p.R291* [NP_000435]) was iden- from cysteine to phenylalanine, which signifi-
tified in all three affected individuals. This muta- cantly altered its tertiary structure and would fur-
tion was previously reported in a patient with ther change relevant protein properties.
XLH. In addition, a de novo heterozygous muta-
tion in the FBN1 gene (c.2375G>T [NM_000138])
was only called in the affected girl (Fig. 10.10a, b). 10.7 Autosomal Recessive
This mutation would cause the amino acid change Bestrophinopathy(ARB)
from the hydrophilic cysteine to the hydrophobic
phenylalanine at residue 792 (p.C792F 10.7.1 Introduction
[NP_000129]). Residue Cys792 was predicted to
be highly conserved among multiple species. Autosomal recessive bestrophinopathy (ARB)
Crystal structural modeling of the wild-type and was characterized by macular degeneration with
108 W. Rong et al.
Fig. 10.11 Clinical evaluations of two patients, II:13 with increased cup-to-disc ratio (CDR) was observed in
(upper left) and II:6 (upper right). (a–d), Ultrasound bio- both eyes of II:13(e, f) and left eye of II:6(h). (i–l), Optical
microscopy revealed shallow anterior chamber and angle coherence tomography (OCT) examination demonstrated
closure. (e–h), Color funduscopy indicated macular dramatic bilateral macular cystoid edema of II:13(i, j) and
degeneration with yellowish-white deposits (black moderate accumulation of fluid in the subretinal space
arrows) and retinal pigment epithelium (RPE) pigmentary with RPE detachment at the fovea in II:6(k, l). White
atrophy (white asterisk) in both patients. Optic disc pale arrows indicate fluid accumulation
scattered punctate deposits and accumulation of lation of fluid in the subretinal and/or intraretinal
fluid within and/or beneath the neurosensory ret- space at the fovea (Fig. 10.11). Moreover, a sig-
ina [39]. Homozygous BEST1 mutations were nificant reduction in the EOG light rise with
associated with ARB. moderately decreased ERG responses was
detected in all patients, implying a primary insult
to the RPE. Patient II:5 was unavailable for our
10.7.2 Clinical Features clinical reevaluation, but her medical records
indicated the development of bilateral ACG, pre-
We studied a consanguineous Chinese family mature cataract, and maculopathy by 40 years of
(Fig. 10.12a) with five individuals possessing age. A total of 13 other family members were
complex ocular phenotypes. Individuals I:1 and examined and had no remarkable ophthalmic
I:2 are first-degree cousins and had no history of findings.
eye diseases at any point in their lives. Four
patients (II:6, II:12, II:13, and III:1) manifested
angle-closure glaucoma (ACG) as demonstrated 10.7.3 Genetic Findings
by dark-room gonioscopy, ultrasound biomicros-
copy, increased intraocular pressure, and By next-generation sequencing and Sanger
increased cup-to-disc ratio. Consistent with sequencing, only one variant, c.752G_A (p.
ACG, all four patients had moderately shortened C251Y) in BEST1, was identified as a putative
axial lengths with mild hyperopia. They also had pathogenic mutation. Sanger sequencing con-
bilateral cortical cataracts, which by patient firmed the presence of this variant in all five
report developed before the age of 20 years patients in the homozygous state and in ten unaf-
(Fig. 10.11). Fundus examination revealed in all fected family members in the heterozygous state
four patients’ characteristic features of ARB (Fig. 10.12a). Of note, patient III:1 is homozy-
including retinal pigment epithelium (RPE) dis- gous for the mutation, suggesting that his unaf-
turbances and scattered punctate yellow-whitish fected father, who is not known to be related to
fleck deposits surrounding maculae and accumu- the family, also harbors this variant, presumably
Fig. 10.12 Analysis of mutation BEST1 c.752 G_A (p. sequencing showing homozygous and heterozygous c.752
C251Y) and haplotype reconstruction for the homozy- G_A mutation in II:6 (patient) and his son, III:7 (unaf-
gous region on chromosome 11 in the family. (a) fected), respectively. Reference sequences are given at the
Haplotype of the family. M _ BEST1 c.752 G_A. Affected bottom. Het, heterozygous; Hom., homozygous.
and unaffected members are indicated by filled and open Conservation analysis of residue p.C251 (boxed) of
symbols, respectively. The black arrow indicates the pro- bestrophin-1 across seven species was shown on the right
band. Genotypes are given for all participants (*). The panel. (c) Schematic structure of bestrophin-1.5. Known
black-filled bars represent ancestral haplotype associated mutations are indicated by different symbols according to
with the disease. The homozygous region (denoted by red diagnoses. The fifth and sixth transmembrane-spanning
boxes) flanking by markers D11S4109 and rs7130122 helices of bestrophin-1 (TM5 and TM6) are denoted by
was shared by all patients. The site of BEST1 c.752 G_A dashed lines. Note that p.C251Y is the only ARB-
was indicated by red lines underneath. (b) Sanger associated mutation located in the TM5 domain
owing to a founder effect. This variant is absent with BEST1 homozygous mutations: ARB, ACG,
in the 3 remaining unaffected family members hyperopia, and cataracts. Our extensive genetic
tested and in 100 unrelated controls, is evolution- analyses likely rule out other genetic components
arily conserved (Fig. 10.12b), and is predicted to in this family.
be functionally highly deleterious by the bioin-
formatic programs PolyPhen-2 (score _ 1) and Conflict of Interest The authors declare that they have
SIFT (score _ 0). p.C251Y is located in the fifth no conflict of interest.
transmembrane-spanning helix (TM5), 5 very
close to the extracellular surface (Fig. 10.12c). Compliance with Ethical Requirements In our study,
all procedures followed were in accordance with the ethi-
The cosegregation of p.C251Y with a coher- cal standards of the Committee of Ningxia People’s
ent ocular phenotype in all five patients strongly Hospital on human experimentation and with the Helsinki
suggests a new constellation of traits associated Declaration of 1975, as revised in 2010.
110 W. Rong et al.
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Ophthalmic Genetics in India:
From Tentative Beginnings 11
in the 1980’s to Major
Achievements in the Twenty-First
Century
Govindasamy Kumaramanickavel
and M. J. Denton
Abstract
The journey to map autosomal recessive reti- 11.1 Introduction
nitis pigmentosa (RP) genes both syndromic
and non-syndromic started in mid-1990 in During the mid- to late 1980s, while one of us
India. The potential of consanguineous, large M.J. Denton (MJD) was establishing a gene map-
sibship families was exploited with over- ping lab in the Prince of Wales Hospital (POW)
whelming results particularly using homozy- in Sydney New South Wales, the other
gosity mapping methodology. The project G. Kumaramanickavel (GK) was studying and
came to standstill due to limited funding and collecting RP families in Chennai. Our eventual
complicated logistics to work in field in India. meeting in early 1989 proved to be a watershed in
Even though the struggle started with multiple Asian or more specifically south Asian ophthal-
barriers and challenges, it was one of the most mic genetics as it initiated the first concerted
successful stories that a team of ophthalmolo- effort to identify the genes responsible for inher-
gists and vision scientists in India and New ited retinal diseases in India and indeed in Asia
Zealand enjoyed. These sincere efforts have and led to some major advances in our under-
led today towards promising gene therapy. standing of genetic retinal diseases. Here we
This is a narrative story of those yesteryears. describe how our collaboration started in the late
1980s and how it matured over the next two
Keywords decades into an important productive ophthalmic
Autosomal recessive · Consanguinity · research project. We also acknowledge a number
Linkage analysis · Ophthalmic genetics · of genetic and ophthalmic researchers whose
Retinitis pigmentosa assistance was vital to the overall success of the
project.
11.2 Beginnings: The Prince

of Wales Hospital
G. Kumaramanickavel (*) In 1984 the head of pathology at the POW
Narayana Nethralaya, Bangalore, India
Professor Sidney Bell asked MJD to set up the
M. J. Denton gene mapping lab in the pathology department.
University of Otago, Dunedin, New Zealand

https://doi.org/10.1007/978-981-13-0884-0_11
114 G. Kumaramanickavel and M. J. Denton
The aim of the lab was (1) to identify the chromo- However to MJD’s surprise [and everybody’s
somal location of human disease genes by apply- working in the field], it turned out that the genet-
ing the then revolutionary technique of Southern ics of XLRP was far more complex than origi-
blotting in conjunction with radiolabelled probes nally thought and might have been inferred from
to detect ‘marker RFLPs’ linked to the disease Shomi’s initial paper. It was apparent from the
genes which was at the time seen to be the first very first linkage analysis studies that in many of
step towards the actual identification of the dis- Halliday’s families the xlRP gene was some dis-
ease gene itself and (2) to carry out genetic diag- tance from the marker L128 and subsequent link-
noses on individuals and families in which age studies of all Halliday’s families with a series
disease genes were segregating. of X-linked probes to determine the genetic dis-
MJD had the good fortune to be working in a tance from L128 confirmed that the commonest
medical centre with a world-class collection of xlRP locus was not closely linked to L128.
X-linked retinitis pigmentosa (xlRP) pedigrees, Because many of Halliday’s pedigrees were large
which had been collected and documented by Dr. multigenerational families and excellent for gene
Francis Halliday over several decades in the mapping purposes, the xlRP work at the Prince of
Sydney area of Australia. At the time (early Wales led to important advances in knowledge of
1984), none of the genes responsible for any form the genetics of X-linked RP including the most
of RP including the X-linked form had been iden- accurate location of the most common form of
tified nor was the chromosomal location for any xlRP [2]at the time and the first clear evidence
RP gene known even approximately. Intriguingly from analyses carried out in conjunction with
it was assumed that there would be one gene for Professor Andreas Gal (who was then at the
each of the three forms of RP, xlRP and the domi- Institute of Human Genetics in Bonn, West
nant (adRP) and recessive forms (arRP). Given Germany) that the disease was genetically het-
the wonderful collection of families, it seemed erogeneous [3]. This fact was increasingly con-
very natural to commence a project aimed at firmed by subsequent work by many [4].
identifying the gene responsible for xlRP.
However shortly after the Sydney lab was
operational, Professor Shomi Bhattacharya who 11.3 A
utosomal Recessive RP,
was at the time stationed at the MRC unit in India’s Potential
Edinburgh headed by Ed Southern [who was a
leading researcher in the nascent field of human It was the early realization by MJD that xlRP was
genomics and who had developed the key tech- heterogeneous and the likelihood that the other
nique of Southern blotting which everyone at that forms of RP including the commonest form of the
time used to detect restriction fragment length disease, arRP, might also be heterogeneous that
polymorphisms (RFLPs) linked to disease loci] led MJD to first consider in the late 1980s the
published a landmark paper in Nature reporting possibility of studying Indian families with
that the location of the xlRP locus was closely arRP. In Australia, families ideal for locating and
linked to an X-linked marker locus on the short identifying recessive disease genes, i.e. consan-
arm of the X chromosome called LI28 [1]. guineous and with large sibships, are very rare,
Assuming that there would be only one xlRP but such families are relatively common in south
gene [something that nearly everybody assumed Asia. If the disease was to be heterogeneous as
at the time] and assuming that it would be closely MJD suspected, then combining linkage infor-
linked to L128, MJD geared up to confirm the mation from several small families might not be
location by linkage analysis of the Halliday’s feasible. The best strategy would be to study indi-
families and to start a major genetic diagnostic vidual pedigrees with sufficient members affected
service for XLRP patients, offering prenatal to provide a LOD [‘logarithm of the odds’] score
diagnosis and carrier detection. of 3, indicative of a close relationship between a
marker locus and a disease gene. And India is
11 Ophthalmic Genetics in India: From Tentative Beginnings in the 1980’s to Major Achievements… 115
probably the best source of such families in the attempt in India and probably anywhere in Asia
world thought MJD. to map recessive disease genes. During the visit
MJD was encouraged to visit India regarding GKM introduced MJD to his Professor in Madras
the prospects of initiating a recessive gene map- Medical College and to a prominent ophthalmol-
ping project by the Australian of the year ogist in Chennai, Prof NS Sundaram, who were
Professor Fred Hollows [5] who was director of very supportive. In discussion between us [MJD
the Ophthalmology Department at the POW and and GKM], it was agreed that the key to the proj-
who had devoted considerable energy and was ect success would be to collect a set of large mul-
well known for his efforts in organizing eye tigenerational arRP pedigrees each with multiple
camps in various countries including Eritrea and affected and sufficiently large to map the disease
Nepal. gene in that family.
Later in 1989, MJD revisited India to meet up
with GKM to initiate the building of a collabora-
11.4 Beginnings: Chennai tive network of researchers, to assist with the col-
lection of the arRP pedigrees. Fortuitously MJD
While MJD was building a gene mapping had an Indian student in his Sydney lab, Aarti
research group in Sydney and starting to contem- Chand, who was completing Honours BSc at the
plate the possibility of studying Indian families to University of NSW, who had contacts in Mumbai
locate arRP genes, GKM [who gained his MBBS and at AIIMS in Delhi and was keen to assist in
degree from Madras Medical College in 1982] any way she could. In Mumbai Aarti introduced
was completing his MD in Physiology [at Madras MJD to Dr. DK Gahlot who had a large RP clinic
Medical College, awarded his MD in 1986] and in the city and who had a large number of arRP
had started his postgraduate studies of inherited families and agreed to collaborate in the study.
retinal diseases and was collecting and ascertain- Aarti also arranged a meeting with members of
ing RP families in Tamil Nadu. MJD first learnt the Parsi community in which arRP was quite
of GKM’s work and pedigree collection through common as well as an investigatory visit to
a fortuitous meeting with another genetics Ahmedabad. In Delhi Aarti introduced MJD to
researcher at the University of New South Wales, Prof I C Verma [Professor of Human Genetics at
Dr. Edward Max Nicholls, who had a long time AIIMs] who was very supportive and organized a
interest in Indian genetics and happened to meet talk at AIIMs for MJD to stimulate interest in the
GKM in 1987 in Chennai, and on returning to potential of India for recessive gene mapping.
Sydney, he informed MJD of GKM’s work on After Mumbai and Delhi, Aarti and MJD visited
arRP and his growing pedigree collection and GKM in Chennai, and on GKM’s suggestion we
suggested that MJD contact him to raise the pos- visited the Sankara Nethralaya, to meet the direc-
sibility of jointly working with him on the genet- tor Dr. SS Badrinath to request his collaboration
ics of arRP. and approval of the project which he generously
Having made initial contact by mail and phone granted.
in 1988 with GKM and having received a copy of This was an important milestone in the initia-
GKMs pedigrees and agreed tentatively to a tion of the project as Nethralaya was one of the
meeting in early 1989 MJD made an application most, perhaps at the time and even now, presti-
for funding to the British RP Society [during gious and advanced ophthalmic hospitals in
1988] to initiate an Indian recessive gene map- India, and families collected through this institu-
ping project. The submission was successful, and tion would be very thoroughly ascertained and
with the funds provided, MJD made his first visit clinically assessed. Dr. Badrinath introduced us
to India in January 1989 and spent some time to Drs Mary and Chandran Abrahams, retina spe-
with GKM in Chennai discussing the prospects cialist couples, who showed us some of the
for commencing a major arRP gene mapping Nethralaya collection of RP pedigrees. After
project which would be the first systematic Nethralaya obtained approval from the Indian
Council of Medical Research, Government of sitated in many instances arduous bus or taxi
India, to conduct the study in collaboration with journeys to and from major urban centres and
MJD and GKM, MJD took up an appointment as from urban centres to remote villages. We [MJD
a Senior Medical Research Fellow at the and GKM] did all the final pedigree ascertain-
University of Otago in Dunedin, NZ, in the early ment in the patient’s home, ourselves, and all
1990s with the specific purpose of setting up a blood specimens were again taken by ourselves.
major recessive RP research project with GKM Pedigrees collected between 1990 and 1993
based in the Biochemistry Department of Otago included the first two families in which mutations
University. in the retinal epithelium were shown to cause
arRP. One of these was the family with the cele-
brated mutation in the RPE65 gene provided by
11.5 Collecting the Pedigrees both Sankara Nethralaya and Rama Murthy,
which we [MJD and GKM] collected in Chennai
During the next 2 years [approximately early and suburban Bangalore, respectively. The
1990–late 1991] after having established collabo- RPE65 gene was used in the first successful
ration with Nethralaya in Chennai and after MJD application of gene therapy in the field of oph-
had moved to the University of Otago, we (MJD thalmic genetics [see below].
and GKM) made a major effort to interest leading Those families that had not been clinically
ophthalmologists in south India to join the proj- examined in one of the collaborating centres had
ect. We made many visits to various important to be brought into one of the collaborating clinics
ophthalmic centres, and some of the collabora- for a thorough ophthalmological workup which
tors who agreed to join the project included Dr. included in most case ERGs. Virtually all the
Rama Murthy in Bangalore [whom we contacted pedigrees included in the project save one or two
at Dr. Badrinath’s recommendation]. At the 1991 Parsi families from Mumbai were collected in
All India Ophthalmic Society (AIOS) meet in south India. By late 1993 the collection consisted
Bangalore, MJD had gone to meet up with Dr. K of approximately 50 inbred arRP families each
Rama Murthy. He also met Dr. Joseph Mani, an with multiple affected and each large enough to
ophthalmologist stationed in Kerala who invited provide independent evidence (by linkage) of the
us to visit the Little Flower Hospital in Angamaly location of the arRP gene. This collection was
(Kerala) to investigate the potential of Kerala to certainly at the time one of the largest if not the
collect arRP pedigrees. Taking up his invite we largest single collections of large inbred arRP
visited Kerala in late 1991, where he met up pedigrees with multiple affected, suitable for
again with Dr. Mani and met Dr. Tony Fernadez gene mapping purposes anywhere in the world.
and Dr. JK Mukkadan who was the research A problem we had to surmount was collection
director at the Little Flower Hospital at the time. of blood specimens. We chose to collect blood
Visiting Kerala was quite fortuitous as it turned samples from the families on newborn blood spot
out that Dr. Mukkadan had a very impressive col- cards, which improved the compliance. Although
lection of arRP pedigrees. Several visits were this complicated the analysis of the DNA as it
also made to another leading ophthalmic centre, had to be extracted from the blood spot and the
the LV Prasad Eye Institute in Hyderabad, where quality was not as good as DNA extracted from
the director G N Rao also gave his blessing to the whole blood, however it simplified the actual col-
project. lection as carrying a set of blood cards was a lot
During the next few years of the project simpler in Indian conditions than carrying whole
approximately from 1991 to 1993, we [MJD and blood specimens. Logistically it was easier in
GKM] set out energetically to ascertain and col- many ways, which finally, looking back, turned
lect blood specimens from patients and families out to be one of the key points for the success of
provided by the collaborating centres. As we the project.
were working on a shoestring budget, this neces-
The collection was overall a quite arduous 11.6 D

NA Analysis, Linkage
process. Conditions were primitive in many and the Otago Lab
places of India in the early 1990s before the
transformative economic development of the past In 1992 MJD and GKM applied successfully to
25 years, and we had many memorable experi- the NZ Health Research Council for support
ences travelling round south India, some amusing including a fellowship for GKM to spend time in
and others not too funny. On one occasion just as NZ. And in 1993 The US Foundation Fighting
we were about to place a needle in the arm of a Blindness provided a large research grant to pro-
patient near Chennai, there was a power outage, vide further support for the project, for the col-
and the venesection had to be continued by torch lection of additional pedigrees in India and for
light. But there were lighter moments. On one the setting up of a gene mapping laboratory in
occasion MJD was returning from Kerala to Otago.
Chennai by train on the eve of Diwali when he And the following year, the US NIH provided
was awoken in Coimbatore, just after having another grant to collect and study a set of arRP
crossed the Kerala-Tamil Nadu border to an families collected in collaboration with the LV
unbroken cacophony of explosions what he Prasad in Hyderabad which were to be analysed
assumed must be a terrorist attack having not pre- in the gene mapping lab at the National Eye
viously experienced the intensity of the fireworks Institute, NIH, headed by Fielding Hejtmancik.
displays associated with the celebration of Diwali Because of the size of the pedigree collection
in Tamil Nadu. Sleeping from Coimbatore to and the resources needed in the early 1990s to
Chennai thereafter was simply impossible. On carry out linkage analyses on so many pedigrees,
another occasion in Agra, Aarti having asked the it made sense for us to collaborate with other
cost of a shoe shine assuming it would be for both large genetics laboratories such as Fielding’s at
shoes was surprised to find that the quoted price the NIH. Because MJD had worked productively
was for one shoe only! On another occasion hav- with Professor Andreas Gal on xlRP, it was
ing asked the driver of a cab in Kerala why he agreed to send samples from many of the families
was driving round bends on the wrong side of the for linkage studies to his lab in the Institute of
road, he retorted ‘not to worry it’s my lucky day!’ Human Genetics in the University of Bonn.
Another tout in Kerala requested some money for [Professor Gal is currently Professor Emeritus,
reciting one to ten backwards in Italian. On former Director of the Institute of Human
another occasion while we were on route to Genetics at the University Medical Center
Vellore from Bangaluru, we noticed a VW beetle Hamburg-Eppendorf.]
several metres above the ground in the branches The gene mapping lab in Otago was headed
of a tree. How it got there we had no idea and for most of the 1990s by Dr. Marion Maw who
assumed that it had parked on top of a small sap- was responsible for carrying out the linkage anal-
ling years ago and gradually been carried aloft by yses in Otago and supervising DNA analyses in
the growing tree. Another amusing scenario the lab. The setup was small, consisting for most
occurred in the Bangalore Club where MJD of the period of Marion and two research assis-
stayed many times as a guest of Dr. Rama Murthy. tants plus one or two honours students. GKM
Due to repeated bouts of gastric upset, mostly he arrived on his first visit to NZ in February 92 and
ordered toast and black tea for breakfast; a regu- was a NZ Health Research Council Fellow till
lar waiter familiar with his ‘tummy upsets’ would September 97. During this period each year
greet him in the morning, ‘toast and tea as usual GKM spent several months in the Otago lab and
sir!’ several months with MJD in continuing pedigree
collection in India. During this period in addition defects in the RPE cells and or in the vitamin A
to arRP pedigrees, some pedigrees with other cycle may cause arRP. Until this finding all the
genetic eye diseases were also collected, genes previously implicated in RP, since the early
including one with Oguchi disease and one with 1980s, had been genes expressed in the photore-
blepharophimosis. ceptor cell layer or the neuroretina. Because the
Between 1992 and 2000, molecular genetic RPE is a far simpler structure, the neuroretina
studies of these Indian families in Otago and in defects in the RPE are more amenable to treat-
Andreas Gal’s lab resulted in a number of impor- ment (via vit A administration, drugs, transplan-
tant results including: tation, gene therapy, etc.). Consequently by
opening up the possibility of the development of
1. Identification of the first case of arRP caused therapies for at least a proportion of patients with
by a single amino acid change in rhodopsin these diseases, this result transformed the outlook
[6]. for sufferers with inherited retinal dystrophies.
2. Identification of the gene on chromosome 2 The finding was discussed in an editorial review
responsible for the form of hereditary night in Nature Genetics issue of 17th October 1997
blindness known as Oguchi disease [7]. and reported in the New Scientist in the October 4
3. Identification of a new arRP gene on chromo- issue 1997. One very recent consequence was the
some 15 encoding for cellular retinaldehyde- report of the first successful use of gene therapy
binding protein, a protein involved in the for a genetic eye disease, carried out at Moorfields
visual cycle in the RPE [8]. Eye Hospital [2008], where patients received a
4. The finding that mutations in RPE65, a gene healthy copy of the gene RPE65. This was a land-
expressed in the retinal epithelium cell which mark scientific advance and was listed in
encodes a protein involved in the visual cycle, Discovery magazine as the 24th most important
are responsible for arRP [9]. scientific result of 20081.
5. Identification of a new arRP locus on chromo- The overall success of the project over the first
some 16p12.1–p12.3 [10]. decade is indicated by the fact that as a result of
6. Location of the gene responsible for blepharo- the study, a total of eight new disease genes had
phimosis syndrome shown to be on chromo- been located by 1999, including six new non-
some 7p [11]. syndromal arRP genes. [In 1999 these six genes
7 . The demonstration that mutations in the gene represented a significant fraction of all then
(PDE6A) encoding the alpha subunit of cGMP known non-syndromal arRP loci. (Note that in
phosphodiesterase (PDE) are probably a rare March 1999 there were 16 known loci in which
cause of arRP in Indian families [12]. mutations cause non-syndromal arRP, the 14
8. Evidence of a new arRP locus on chromo- arRP loci cited on the RetNet website, 11 March
some 2 [9]. 1999, plus the two unpublished loci from the
9. Evidence that mutations in the prominin University of Otago mentioned above).]

(mouse)-like 1 gene cause arRP [13]. After working with MJD and his team for a
while at the Otago, GKM wanted to move back to
Of the above results, the most important was India to run an ocular genetics laboratory and
the discovery in 1997 of the two new arRP counselling unit. On returning to India, he was
genes—cellular retinaldehyde-binding protein asked by Dr. Badrinath and Dr. HN Madhavan,
(CRABP) and RPE65—both of which are the then research director, to set up a gene map-
expressed in the retinal epithelium (RPE) and ping/molecular genetics lab at Nethralaya, which
both also involved in vitamin A metabolism. The he finally established with the support of MJD
two new arRP genes were reported in two back- and Dr. Peter Swarbrick from Otago University,
to-back papers published in the leading journal in
this field Nature Genetics in October 1997 [8, 9]. 1
See Discover magazine published on line 17 Dec 2008.
The discovery that these two genes were respon- http://discovermagazine.com/columns/top-100-stories-of-
sible for arRP provided the first evidence that 2008/
New Zealand. GKM later went to join Fielding’s Ott J, Bhattacharya SS, Chen JD, et al. Proc. Natl
Acad Sci USA. 1990;87:701–4.
laboratory at the National Eye Institute to learn 5. http://www.hollows.org/au/about-fred; Lewis,
the then modern fluorescent-based gene mapping Wendy. 2010. Australians of the Year. Pier 9 Press.
and sequencing technology. Setting up the lab at ISBN 978-1-74196-809-5.
Nethralaya was a great progress and addition to 6. Kumaramanickavel G, Maw M, Denton MJ, John
S, Srikumari CRS, Orth U, Oehlmann R, Gal
the institute. What proved a propitious move fru- A. Missense rhodopsin mutation in a family with
tioned, as the lab went on to be perhaps the most recessive RP. Nat Genet. 1994;8:10–1.
productive in the area of ophthalmic genetics in 7. Maw MA, John S, Jablonka S, Kumaramanickavel
India (with more than 50 publications) and one of G, Oehlmann R, Denton MJ, Gal A. Oguchi disease:
suggestion of linkage to markers on chromosome 2q,
the important ophthalmic genetic centres in the J Med Genetics. 1994;32:396–98; Maw, MA, Denton
world, where thousands of patients were coun- MJ. Oguchi disease, retinitis pigmentosa and the
selled and benefitted. What results emerged from phototransduction pathway, In: Lavail M, Anderson
the bedside to the bench finally reached the bed- RE, editors. Degenerative retinal degenerative dis-
ease: proceedings of the 7th international sympo-
side again! sium on retinal degeneration. New York: Plenum
Starting as a small and precarious research Publishing Corporation; 1996. p. 313–8; Maw MA,
project in 1990s, we went on to be become one of Kumaramanickavel G, Kar B, John S, Bridges R,
the most successful RP projects in the world. Denton M. Two Indian siblings with Oguchi disease
are homozygous for an arrestin mutation encod-
What started off in India finally came back to the ing premature termination, Hum Mutat. 1998;Suppl
country by the establishment of an ocular genet- 1:S317–19.
ics laboratory and counselling unit, in the gener- 8. Maw MA, Kennedy B, Knight A, Bridges R, Roth
ous effort to fight against blindness and provide KE, Mani EJ, Mukkadan JK, Nancarrow D, Crabb
JW, Denton MJ. Mutation of the gene encoding cel-
vision and light to the visually impaired patients. lular retinaldehyde binding protein in autosomal reti-
nitis pigmentosa. Nat Genet. 1997;17:198–200.
Conflict of Interest None of the authors have any propri- 9. Gu S-m, Thompson DA, Srikumari CR, Birgit L,
etary interests or conflicts of interest related to this Kamramanickavel G, Nicoletti A, Murthy KR,
submission. Denton MJ, Gal A. Mutations in RPE65 cause auto-
somal recessive childhood onset severe retinal dystro-
Compliance with Ethical Requirements The whole phy. Nat Genet. 1997;17:194–7.
journey and scientific endeavours were in complican with 10. Finckh U, Xu S, Kumaramanickavel G, Schurmann
the ethical requirements. M, Mukkadan JK, Fernandez ST, John S, Weber
JL, Denton MJ, Gal A. Homozygosity Mapping
of autosomal recessive retinitis Pigmentosa locus
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3. Wirth B, Denton MJ, Chen JD, Neugebauer M, Publishing Corporation; 1997. pp 237–44.
Halliday FB, van Schooneveld M, Donald J, Bleeker- 13. Maw MA, Corbeil D, Koch J, Helliwig A, Wilson-
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1988;2:263–6. Denton MJ. Mutation of the gene coding for prominin
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Panel-Based Next-Generation
Sequencing for Inherited Retinal 12
Degenerations in Koreans
Sang Jin Kim

Inherited retinal degenerations (IRDs) include
various forms of blinding retinal degeneration Inherited retinal degenerations (IRDs) are one
with genetic heterogeneity such as retinitis of the most common forms of inherited human
pigmentosa, cone-rod dystrophy, Stargardt diseases worldwide. IRDs include various forms
disease, etc. Establishing a molecular diagno- of retinal degeneration such as retinitis pigmen-
sis in IRD is important for proper diagnosis, tosa (RP), cone-rod dystrophy (CRD), Stargardt
genetic counseling, predicting prognosis, and disease, choroideremia, etc. IRDs often cause
clinical trials of retinal gene therapies. In this serious visual impairment including legal blind-
chapter, recent studies using gene panel-based ness and severe constriction of visual field.
next-generation sequencing in Korean patients Moreover, IRDs are associated with socioeco-
with IRDs will be presented. Targeted gene nomic problems such as psychosocial difficul-
panel sequencing seems to be an efficient ties, physical inactivity, depressive mood, and
approach to find genetic causes of IRDs. poor mental health [1, 2]. To prevent further
retinal damage and to improve visual function,
Keywords gene therapy has been pursued for a long period
Choroideremia · Cone-rod dystrophy · of time. Recently, many clinical trials of gene
Next-generation sequencing · Retinitis therapy have been performed for the treatment
pigmentosa · Stargardt-like macular dystro- of IRDs [3, 4]. Most of these clinical trials have
phy · Targeted sequencing been performed in Western countries. One of
the reasons for low number of clinical trials in
Asian countries may be due to lack of extensive
genetic studies to identify causal mutations in
Asian countries. Promising results from clinical
trials of retinal gene therapy for IRDs empha-
sizes the importance of genetic studies to find
target genes in IRDs and eligible patients with
IRDs. In this chapter, we described the panel-
S. J. Kim (*) based next- generation sequencing in Korean
Department of Ophthalmology, Samsung Medical patients with IRDs.
Center, Sungkyunkwan University School of
Medicine, Seoul, South Korea

https://doi.org/10.1007/978-981-13-0884-0_12
122 S. J. Kim
12.2 Epidemiology of RP in Korea MAK, TULP1, GUCA1A, GUCA1B, PRPH2,

EYS, RIMS1, IMPG1, ELOVL4, KLHL7, RP9,
RP is the most common form of IRDs. The IMPDH1, KIAA1549, RP1L1, ADAM9, RP1,
worldwide prevalence of RP has been reported to CNGB3, C8ORF37, KCNV2, TOPORS, PRPF4,
be 1 in 3000–7000 [5]. Recently, nationwide RBP3, CDHR1, RGR, PDE6C, BEST1, ROM1,
population- based studies on epidemiology of MYO7A, C1QTNF5, CACNA2D4, PDE6H,
retinitis pigmentosa in Koreans have been pub- RDH5, MVK, RPGRIP1, NRL, RDH12, SPATA7,
lished. In a population-based study from 2011 to TTC8, NR2E3, RLBP1, ARL2BP, CNGB1,
2014 in Korea, the incidence rate of RP for all PRPF8, AIPL1, PITPNM3, GUCY2D,UNC119,
ages was found to be 1.64 cases per 100,000 CA4, RGS9, USH1G, PRCD, FSCN2, PDE6G,
person-years [6]. The incidence of RP distribu- RAX2, CRX, PRPF31, IDH3B, MKKS, PRPF6,
tion showed one smaller peak observed in the C21ORF2, TIMP3, OFD1, RPGR, RP2,
20–24-year-old age group (1.24 cases/100,000 CACNA1F, and CHM [8–10].
person-years) and a larger peak observed in the Targeted gene panel sequencing was per-
65–69-year-old age group (3.26 cases/100,000 formed as previously described [8–10]. Genomic
person-years) [6]. The overall incidence was sim- DNA was obtained from peripheral blood of the
ilar in men and women (1.64 versus 1.63 patients and family members, if possible, after
cases/100,000 person-years for men and women, obtaining informed consent. Genomic DNAs
respectively) [6]. Another population-based within the target genes were captured using
retrospective cohort study from 2011 to 2014 SureSelect customized kit (Agilent Technologies,
found that the prevalence of RP in Korea is about Santa Clara, CA), and libraries were prepared
1 in 9000 for all ages and 1 in 6000 for those over using the SureSelect Target Enrichment System
40 years of age [7]. The mean age at diagnosis protocol. Targeted exome sequencing was per-
was 44.8 years and was 6 years earlier for male formed on Illumina HiSeq 2500 platform
than for female patients (41.9 versus 47.8 years) (Illumina, San Diego, CA). Sequencing reads
[7]. The standardized mortality ratio was 1.56 for were aligned to the Genome Reference
all ages, peaking at 2.61 in men aged 40–59, Consortium human genome (build37) using
which was mainly attributed to 6.6-fold higher Burrows-Wheeler Aligner with MEM algorithm.
suicide rates than the same age group in the general Aligned SAM/BAM files were sorted and indexed
male population [7]. by SAMTOOLS and duplicated reads were
removed by Picard tools. Secondary reads align-
ment was performed following known insertions/
12.3 Panel-Based Next- deletions (indels) from gold standard (dbSNP138,
Generation Sequencing Mills, 1000 Genome Project phase 1 indels) by
(NGS) (1): Methods Genome Analysis ToolKit (GATK). Base recali-
bration was performed and annotated by
Gene panel for IRDs usually consists of genes GATK. Single nucleotide variants (SNVs) and
known to cause IRDs. The gene panel needs to be insertion or deletion (indels) were calculated by
updated regularly to include newly identified Unified Genotyper in GATK. ANNOVAR was
genes associated with IRD. From 2013 to 2015, used to annotate called variants from variant call
we used a gene panel of 98 genes, which includes format.
DHDDS, RPE65, ABCA4, PRPF3, SEMA4A, To identify candidate variants, following crite-
PDC, CRB1, NEK2, FLVCR1, USH2A, ZNF513, ria were applied: (1) located in exonic region, (2)
C2ORF71, EFEMP1, FAM161A, SNRNP200, rare allele frequency (MAF <0.01 of The Exome
CNNM4, MERTK, CERKL, SAG, ARL6, IMPG2, Aggregation Consortium), (3) damaged in pre-
RHO, CLRN1, PDE6B, RAB28, PROM1, diction tools (SIFT, Polyphen2, GERP++), and
GPR125, WDR19, CNGA1, LRAT, PDE6A, (4) clinical significance (ClinVar) [8–10].
12 Panel-Based Next-Generation Sequencing for Inherited Retinal Degenerations in Koreans 123
Sanger sequencing was also performed to electroretinogram (ERG) according to the

examine the presence of variants of interest in the International Society for Clinical
proband and family members. Electrophysiology of Vision standards showed
slightly reduced amplitudes in both cone and rod
responses (Fig. 12.2) [8].
12.4 Panel-Based Next- Targeted exome sequencing data were gener-
Generation Sequencing (2): ated, covering 97.8% of targeted genes with a
Application in Korean sequencing coverage of ≥ 100X as well as 99.3%
Patients with IRDs of ≥ 50X. Targeted regions of PROM1 were fully
covered, with a mean depth ranging from 168X
12.4.1 Stargardt-Like Macular to 513X [8]. A heterozygous missense variant
Dystrophy 4 in PROM1 (NM_006017.1: c.1117C>T (p.
R373C)) was detected in the affected proband
Classical Stargardt disease (STGD1; OMIM# with a high read depth [8]. Sanger sequencing
248200) is caused by mutations of ABCA4 gene confirmed the heterozygous presence of the vari-
on chromosome 1p with autosomal recessive ant in the proband (Fig. 12.3) [8]. No suspicious
transmission [11]. Families with autosomal dom- pathogenic variant was identified from the other
inant inheritance have also been described, 97 genes [8]. From the characteristic retinal phe-
including Stargardt-like macular dystrophy 3 notype and PROM1 p.R373C mutation, the
(STGD3; OMIM# 600110) caused by mutations patient was diagnosed as STGD4.
in ELOVL4 and STGD4 (OMIM# 603786)
caused by mutation in PROM1 gene on chromo-
some 4 [12, 13]. 12.4.2 Choroideremia
STGD4 is characterized by bilateral bull’s eye
atrophy of macula and the presence of yellow Choroideremia is an X-linked disorder causing
flecks. STGD4 is caused by mutation in PROM1 progressive degeneration of the retina, retinal
gene encoding prominin-1, a 5-transmembrane pigment epithelium (RPE), and choroid [15].
glycoprotein also known as CD133 [13]. It was Affected patients show night blindness with pro-
suggested that prominin-1 was involved in photo- gressive peripheral vision loss and eventual cen-
receptor disk morphogenesis [13]. PROM1 tral vision loss [16]. Female carriers may show
c.1117C>T (p.R373C) missense mutation is patchy chorioretinal atrophy [17]. CHM, which
known to cause several types of autosomal domi- encodes Rab Escort Protein-1 (REP-1), is a gene
nant retinal degeneration, including STGD4, responsible for choroideremia. REP-1 facilitates
bull’s eye macular dystrophy (MCDR2), retinitis posttranslational modification of Rab proteins
pigmentosa, and cone-rod dystrophy [13, 14]. regulating intracellular trafficking [18]. Various
We identified PROM1 p.R373C mutation in a type of mutations in CHM have been identified
38-year-old Korean man with a family history of including various size of deletions, nonsense
visual impairment suggesting autosomal domi- mutations, missense mutations, frameshift muta-
nant inheritance (Fig. 12.1) [8]. Multimodal fun- tions, splice site defects, and deletion of the entire
dus imaging revealed bilateral atrophic macular gene, causing the truncation, loss of functional
lesions with small flecks in the posterior pole domain, or absence of REP-1 protein [19, 20].
with a Bull’s-eye pattern of hypo-autofluorescent Because choroideremia is frequently caused
macular lesions surrounded by hyper-by large deletion of CHM, NGS alone may not
autofluorescence [8]. Spectral-domain optical make a proper molecular diagnosis for a consid-
coherence tomography showed retinal pigment erable number of patients with choroideremia.
epithelium atrophy and photoreceptor layer Therefore, direct sequencing of CHM gene, RNA
defect (Fig. 12.1) [8]. Automated visual field test (cDNA) sequencing, or immunoblot to detect
showed bilateral central scotoma [8]. Full-field truncated or absent protein have been performed
124 S. J. Kim
Fig. 12.1 Pedigree (a),

fundus photographs (b),
fundus autofluorescence
photographs (c), and
spectral-domain optical
coherence tomography
(d) of the affected
proband with Stargardt-
like macular dystrophy
4. (Reprinted from Ann
Lab Med. 2017;37:536–
539 with permission)
in cases with suspicious ocular phenotypes [20]. differentiate choroideremia from other inherited
Recently, NGS-based approaches also have retinal degenerations.
been reported with successful diagnosis of cho- Recently, CHM mutations were identified in
roideremia [9, 21]. NGS-based approach may be two Korean families [9]. In one family, direct
particularly useful when it is difficult to clinically CHM sequencing was performed, and in the other
Fig. 12.2 Full-field standard electroretinogram in a Korean patient with Stargardt-like macular dystrophy 4. (Reprinted
from Ann Lab Med. 2017;37:536–539 with permission)
family, panel-based NGS sequencing was done FAF, and SD-OCT scans were similar to those of
first, followed by direct CHM sequencing [9]. proband A (Fig. 12.4) [9].
In family A, the proband was a 45-year-old In proband B, targeted sequencing of 98 can-
man with night blindness, visual field defect, and didate genes for retinal degenerations revealed no
decreased central vision [9]. The fundus exam suspicious variations [9]. However, exon 9 of
showed bilateral chorioretinal atrophy and areas CHM was not captured at all, suggesting exon 9
of RPE disruption with sparing of the central deletion (Fig. 12.5) [9]. Direct CHM sequencing
macula (Fig. 12.4) [9]. In fundus autofluores- was also performed in the probands of family A
cence (FAF) photographs, residual RPE tissue and family B. Fifteen coding exons and their
appeared as a well-demarcated hyperfluorescent flanking introns were amplified by PCR, and the
area (Fig. 12.4) [9]. Standard ERG showed resultant amplicons were sequenced on an ABI
almost extinguished cone and rod responses. 3730xl Genetic Analyzer (Applied Biosystems,
SD-OCT showed retinal thinning, choriocapil- Foster City, CA, USA) [9].
lary atrophy and abrupt transition to atrophic In the proband of family A, 9 base pair dele-
areas (Fig. 12.4) [9]. In family B, the proband tion in exon 3 and adjacent intron sequences was
was a 41-year-old man with night blindness and identified (c.184_189 + 3delTACCAGGTA)
visual field defect. Findings of the fundus exam, (Fig. 12.6) [9]. In the proband of family B, PCR
126 S. J. Kim
Fig. 12.3 DNA electropherograms of a fragment of sequence showing a heterozygous missense variant

PROM1 gene by Sanger sequencing in a Korean patient (NM_006017.1: c.1117C>T; p.R373C). (Reprinted from
with Stargardt-like macular dystrophy 4. Proband DNA Ann Lab Med. 2017;37:536–539 with permission)
product of exon 9 was not detected, suggesting showed severe color vision deficiency [10].
exon 9 deletion (Fig. 12.6) [9]. Sequencing Standard ERG revealed almost extinguished
analysis of other amplified products showed no light-adapted 3 ERG and 30 Hz flicker ERG, and
pathogenic variant in the proband of family B [9].automated visual field test showed central sco-
toma in both eyes [10]. Thus, she was clinically
diagnosed as having CRD.
12.4.3 Cone-Rod Dystrophy Targeted exome sequencing data were gener-
ated with 2 million sequencing reads, covering
CRD is a group of inherited retinal disorders 97.42% of 98 targeted genes with a sequencing
presenting primary loss of cone photoreceptors coverage of ≥ 100X [10]. Targeted regions
and subsequent or simultaneous loss of rod pho- of RAB28 were fully covered, with a mean
toreceptors. Patients with CRD show severely- depth ranging from 195X to 342X [10]. A
impaired central visual acuity and color vision novel homozygous missense variant
deficiency. Autosomal recessive, autosomal (NM_004249.3:c.68C>T (p.Ser23Phe)) was
dominant, and X-linked forms of CRD have been detected in the proband, which was confirmed by
reported. Among the variable genetic causes of Sanger sequencing [10]. Unaffected parents and
autosomal recessive CRD, ABCA4 mutations are an unaffected male sibling were heterozygous
the most common ranging from 24% to 65% carriers of the missense variant [10]. In addition,
[22–27]. no pathogenic variants were identified in the
Recently, a novel homozygous missense other 97 genes including 8 genes known to be
variant in exon 1 of RAB28 was identified in a associated with autosomal recessive CRD
11-year-old Korean patient with progressive (ABCA4, ADAM9, C8orf37, CERKL, EYS,
visual impairment [10]. BCVA in both eyes was PROM1, RPGRIP1, and TULP1) [10].
20/100 [10]. The fundus examination showed This RAB28 c.68C>T variant is thought to be
slightly atrophic fovea, and SD-OCT showed an a “likely pathogenic” variant in that (1) the vari-
ellipsoid zone defect in both macular areas ant is either absent or present at extremely low
(Fig. 12.7) [10]. The Ishihara color vision test frequency in several population databases, [2]
Fig. 12.4 Ocular phenotypes shown in the probands with autofluorescence photograph. (d, h) Pedigrees of the two
choroideremia and the pedigrees of family A (a–d) and families. (Reprinted from Ann Lab Med. 2017;37:438–
family B (e–h). (a, e) Fundus photograph. (b, f) Spectral- 442 with permission)
domain optical coherence tomography. (c, g) Fundus
128 S. J. Kim
Fig. 12.5 BAM file created by using IGV viewer of tar- contrary, CHM exon 9 is absent in proband B. (Reprinted
geted sequencing of 98 candidate genes including CHM. from Ann Lab Med. 2017;37:438–442 with permission)
CHM exons were present in control subjects. On the
Fig. 12.6 CHM variants identified in the probands of exon 9. Exon 9 deletion was detected in the proband of
family A and family B. (a) Chromatogram of family B. (Reprinted from Ann Lab Med. 2017;37:438–
c.184_189 + 3delTACCAGGTA (p.Tyr62_Gln63del) 442 with permission)
detected in the proband of family A. (b) PCR products of
multiple prediction algorithms such as SIFT and mutations from four European families have been
PolyPhen-2 support a deleterious effect, [3] seg- reported including nonsense mutations in exons 5
regation of the RAB28 mutation with the pheno- and 6, a splice donor site mutation in exon 2, and
type in the family was shown, [4] no pathogenic a missense mutation in exon 8 [29, 30].
variants were identified in genes known to be
associated with IRDs or AR-CRD, and [5] the
variant was located in a well-established func- 12.5 Conclusion
tional domain of the RAB28 protein [10].
RAB28 is a recently identified gene in autoso- In patients with IRDs, establishing a molecular
mal recessive CRD [28–30]. RAB28 encodes a diagnosis is important for proper diagnosis,
member of the Rab subfamily of the RAS-related genetic counseling, predicting prognosis, and
small GTPases [28]. Previously, only four RAB28 clinical trials of retinal gene therapy. Because it is
Fig. 12.7 Spectral domain optical coherence tomography images in a Korean cone-rod dystrophy patient with RAB28
mutation
health of people with retinitis pigmentosa. Optom Vis

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Compliance with Ethical Requirements Author Sang nationwide incidence of retinitis pigmentosa in South
Jin Kim declares that he has no conflict of interest.All pro- Korea: a population-based retrospective study from
cedures followed were in accordance with the ethical 2011 to 2014. BMJ Open. 2017;7:e015531.
standards of the responsible committee on human experi- 7. Na KH, Kim HJ, Kim KH, Han S, Kim P, Hann
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Genetic Disease
in Ophthalmology: Healthcare 13
and Research Opportunity
in Bangladesh
A. H. M. Enayet Hussain and Khaleda Islam
Abstract Keywords
Genetic eye diseases which may pass on from Ocular genetic disease · Autosomal recessive
parent to children through genes include a · Autosomal dominant · Health system ·
large number of ocular pathologies all of Primary healthcare · Consanguineous
which do not cause visual impairment. Global marriage
as well as country burden of the problem is
unknown. Consanguineous marriage is com-
mon in the country which is a predisposing
factor for the genetic eye diseases. Bangladesh 13.1 Introduction
is striving to develop reliable infrastructure of
eye healthcare delivery system from commu- Genetic eye diseases include a large number of
nity clinic providing preventive and promotive ocular pathologies that have in common the
care to tertiary level facilities and specialized transmission from parents to children by their
institutes providing specific treatments. genetic inheritance but all do not cause visual
Utilizing the field staffs, eye patients may be impairment [1]. A genetic or inherited disease
screened at the community and referred to the may pass on from parent to their children through
health facilities for further diagnosis and treat- the coded information in the genes, which deter-
ment. Sociodemographic data may reveal the mines the type of genetic disorder. When involved
consanguineous marriage history and will genes are located on the chromosomes numbered
help diagnosing genetic eye diseases. Thus 1 to 22, it is called autosomal, and when resided
proper utilization of health system will not on the sex chromosomes, it is called X-linked or
only ensure eye care of patients but also to sex-linked. The characteristics of X-linked inher-
create opportunities of research with data gen- itance are that males are affected whereas females
eration providing magnitude of eye disease are usually unaffected carriers.
with a focus to genetic eye disorder in this Genetic factors are responsible for over 60%
large population. of childhood blindness (congenital glaucoma,
ocular malformations, atrophy of the optic nerve,
retinitis pigmentosa, etc.), also associated with
A. H. M. Enayet Hussain (*) · K. Islam
Directorate General of Health Services, serious eye diseases, including glaucoma and
Mohakhali, Dhaka, Bangladesh macular degeneration in adults. Some common

https://doi.org/10.1007/978-981-13-0884-0_13
132 A. H. M. Enayet Hussain and K. Islam
ocular genetic diseases that are found here are as Later, Bangladesh National Council for Blindness
follows [2]: (BNSB) was formed in 1978 to implement
National Eye Care Plan. The government demon-
• Corneal dystrophies. strated its commitment to prevent blindness and
• Anterior segment dysgenesis. ratified vision 2020 – the Right to Sight in 2003
• Aniridia. [4]. The Ministry of Health and Family Welfare
• Uveal melanoma. (MOH&FW) following the global eye health
• Glaucoma. action plan 2014–2019 (2013) emphasized better
• Cataract. integration of eye health into national health
• Norrie disease. plans [5]. Vision 2020 committees were formed
• Retinoschisis. in all districts, which enhanced eye care services
• Retinitis pigmentosa. through GO, NGO, and private partnership [6].
• Choroideremia. Moreover, an intervention package which was
• Albinism. piloted in 2016 demonstrated that existing health
• Color vision deficiencies. system may be utilized to detect children with
• Inherited optic neuropathies. avoidable childhood blindness at community and
• Anophthalmia. manage through referral at the higher health
• Keratoconus. facilities having ophthalmic unit [7]. Though the
• Marfan syndrome. health system is challenged to ensure ophthalmic
• Stargardt disease. personnel, equipment, in handling existing cata-
• Pseudoexfoliation syndrome. ract and emerging eye problems like diabetic
retinopathy, ocular trauma, retinopathy of prema-
turity, etc., MOH&FW need to know prevailing
13.2 Epidemiology types of eye diseases with magnitude and identify
the gaps in health system to ensure universal eye
Global burden of visual impairment from genetic care.
causes is not known though it seems that in
industrialized countries significant percentage of
blindness is due to genetic cause [1]. No litera- 13.4 E
ye Care and Research
ture is found yet on genetic eye disease in Opportunity for Genetic Eye
Bangladesh, though blindness is a major public Disorder
health problem in the country and majority of
which is preventable. National blindness and low Bangladesh has unique health system and infra-
vision survey of Bangladesh (2003) revealed that structure in public sector starting from commu-
prevalence of bilateral blindness was 1.52% with nity clinic delivering primary healthcare (PHC)
an estimated 650,000 blind adults aged 30 years mostly preventive and promotive up to tertiary
and older in population. The reasons were unop- level facilities and national institutes offering
erated cataract (73.3%), refractive error (18.8%), specialized treatments to the patients. Utilizing
macular degeneration (1.8%), uncorrected apha- this health system, the country has made progress
kia (1.1%), etc. The study recommended national in public health with a remarkable reduction of
eye plan for effective eye care services [3]. under 5 mortality between 1993 (<5 MR 133)
and 2014 (<5 MR 46) and achieved the
Millennium Development Goal (MDG) 4 well
13.3 Bangladesh Initiatives ahead of time; the target was 48 deaths per 1000
live births by 2015.
Ispahani Islamia Eye Institute and Hospital The success story is indebted to the public
was the first structured initiative to address health impact of several programs like expanded
the eye healthcare in the country in 1960. program of immunization (EPI) where all basic
13 Genetic Disease in Ophthalmology: Healthcare and Research Opportunity in Bangladesh 133
vaccine coverage by age 12 months between units with ophthalmologists to confirm diagnosis
2004 and 2014 increased from 68% to 78%, and provide specific treatment and contributing
nutrition program where children under age 5 substantially to the management of referred eye
showed reduction in stunting between 2004 and patients from all over the country.
2014 from 51% to 36%, maternal health program Suspected case identification by frontline
which decreased total fertility rate (TFR) from health workers will provide an idea about case
6.3 (1975) to 2.3 (2014) children born per woman load at community. Confirming diagnosis and
[8], and also reduced maternal mortality since treating the patient at ophthalmologic unit will
1990 to 2010 with an estimated reduction of 66% provide case-based proportion of eye disease
[9]. Utilizing the same health system eye care can including genetic eye disorder attending the facil-
be ensured along with identification and manage- ity. Sociodemographic and family history will
ment of genetic eye disorder and creating research explore genetic eye disorder which is related to
opportunity in the field. families and prevalent in offspring of consan-
Currently the primary level health infrastruc- guineous marriage. This is prevalent in the South
ture is the community clinic (CC), operated by Asian community in Great Britain and some
Community Health Care Provider (CHCP), situ- other parts of world where they migrate.
ated within half an hour walking distance of the Consanguineous marriage is also common in
people and mandated to serve 6000 catchment Bangladesh, and the reason behind is strong cul-
population. There are more than 13,000 opera- tural issues like cultural possessiveness, property
tional CCs each of which is managed by inheritance, religion, and ethnicity.
Community Group and supported by 3 commu- Reddy et al. in their study identified that in
nity support groups. In 4th Health, Population, East London in the 1980s, over 50% of marriages
Nutrition (HPN) sector program, five multipur- in the Pakistani community were consanguineous
pose community health volunteers (CHVs) are compared to 1% in general population and the
being recruited for each CC constituting a total children of consanguineous unions have increased
number of 65,000 CHVs for the whole country. postneonatal mortality and childhood morbidity
They will be working as bridge between commu- compared with other ethnic groups. The predom-
nity and health facility in addition to providing inant ethnic community in East London is
preventive and promotive eye care [6, 10, 11]. Bangladeshi having tradition of consanguineous
At the Upazila (subdistrict) health complex marriages. The study identified 13% of children
(UHC), there are field staffs attached to the CC, (45/342) with an ocular genetic disorder or pos-
along with CHCP and multipurpose CHVs. They sible ocular genetic disorder; of them 22%
would be trained to screen eye patients while (10/45) had a history of consanguinity with an
doing their routine domiciliary visits and refer- inheritance pattern of 30% autosomal recessive
ring patients to the UHC for diagnosis and man- (3/10), 20% autosomal dominant, and 50%
agement. The doctors, nurses, and paramedics of X-linked/unknown/isolated cases. In the remain-
UHC would be trained on ophthalmic manage- ing non-consanguineous families (35/45), 22%
ment, and the complicated cases would be were autosomal recessive, 17% autosomal domi-
referred to district or tertiary hospitals or at nant, and 60% X-linked/unknown/isolated cases.
national level where there are National Institute The vast majority of cases (9/10) with a history of
of Ophthalmology and Hospital, Ispahani Islamia consanguineous marriage had South Asian ances-
Eye Institute and Hospital (IIEI&H), specialized try. The predominant inheritance pattern of ocu-
eye hospitals, and medical colleges. These lar genetic disorders in patients with
national level institutes are from government, consanguineous parents is expected to be autoso-
NGO, and private sectors having ophthalmologic mal recessive [12].
134 A. H. M. Enayet Hussain and K. Islam
Two-thirds of childhood blindness in the getting rid of social stigma, psychosocial trauma,
Middle East, with a prevalence ranging from and financial difficulties but will also help the
47% in Tunisia to 86% in Kuwait, is due to health system not to be overburdened with genetic
genetic diseases (autosomal recessive disorders), eye disorders. Proper database will provide infor-
attributed to high rates of consanguineous mar- mation regarding healthcare seeking pattern and
riage. Genetic eye disease is also common in success rate of treatment. Moreover sustainable
Egypt (40%) and assumed to cause at least half of strengthening of health system with mid-level eye
all cases of childhood blindness. Ahmed Gomaa personnel and basic eye equipment will also
assessed availability and use of genetic counseling ensure universal health coverage of patients with
services in Egypt, evaluated parents’ attitudes eye disorder. However awareness creation of com-
and satisfaction with these services, and also munity, health literacy, strong community involve-
assessed ethno-cultural beliefs about the causes ment, and community support are needed to
of genetic disorders. He found that parents’ per- maximize the efficiency of such programs.
ceptions of genetic disease varied; many attrib-
uted the condition to will of Allah, though Compliance with Ethical Requirements The authors
majority understood it as a condition inherited A.H.M. Enayet Hussain and Khaleda Islam declares that
they have no conflict of interest.
within family. They were compliant with doc-
tors’ advice and wanted to discover risk of having
another affected child, without understanding
level of risk. Abortion is prohibited in Islam, and
References
mothers were often blamed for their child’s blind- 1. http://www.who.int/blindness/causes/priority/en/
ness with consequences like divorce, husband index9.html
taking another wife, social stigma, having no 2. Mathebula SD. A review of ocular genetics and inher-
more children, and financial difficulties. The ited eye diseases. S Afr Optom. 2012;71(4):179–89.
3. Dineen BP, Bourne RRA, Ali SM, Huq DMN,
study found that main barriers to service uptake Johnson GJ. Prevalence and causes of blindness and
were lack of motivation, cost, long waiting lists, visual impairment in Bangladeshi adults: results
distance, lack of awareness among doctors, and of the National Blindness and low vision survey of
insufficient services [13]. While ensuring eye Bangladesh. Br J Ophthalmol. 2003;87(7):820–8.
4. Ministry of Health and Family Welfare (MoH&FW)
care, these types of studies can be conducted in Bangladesh. National Eye Care Plan for implementa-
Bangladesh through existing health system. tion of Vision 2020 in Bangladesh.
5. Universal eye health: a global action plan 2014–2019.
World Health Organization; 2013. ISBN 978 92 4
150656 4 (NLM classification: WW 140). http://
13.5 Summary www.who.int/blindness/actionplan/en/
6. Ministry of Health and Family Welfare (MoH&FW)
Proper utilization of existing health system will Bangladesh. 4th Health, Population and Nutrition
not only ensure screening of eye disorder at the Sector Programme (4th HPNSP); 2017.
7. Preventing avoidable childhood blindness in
community, early diagnosis, and timely treatment Bangladesh: a pilot intervention (2016). http://www.
but will also provide information related to eye searo.who.int/bangladesh/cblindness/en/
disorder with a focus on genetic causes. Genetic 8. National Institute of Population Research and
counseling service may be established for consan- Training (NIPORT), Mitra and Associates, and ICF
International. Bangladesh Demographic and Health
guineous marriage, which may help the parents to Survey 2014. Dhaka, Bangladesh, and Rockville,
understand the risk of having children with genetic Maryland, USA: NIPORT, Mitra and Associates, and
eye disease and deciding to have no more children. ICF International; 2016.
The program will not only help parents from
13 Genetic Disease in Ophthalmology: Healthcare and Research Opportunity in Bangladesh 135
9. Chowdhury S, Banu LA, Chowdhury TA, Rubayet 12. Reddy MA, Purbrick R, Petrou P. The prevalence
S, Khatoon S. Achieving millennium development of patients with ocular genetic disorders attending a
goals 4 and 5 in Bangladesh. BJOG. 2011;118(Suppl. general paediatric ophthalmology clinic in the east
2):36–46. end of London. Eye. 2009;23:1111–4. https://doi.
10. Directorate General of Health Services (DGHS),
org/10.1038/eye.2008.212. published online 11 July
Ministry of Health and Family Welfare (MoH&FW). 2008 & Macmillan Publishers Limited2009
Community Based Health Care (CBHC). http://www. 13. Genetic eye diseases and genetic counselling services
communityclinic.gov.bd/ in Egypt. Ahmed Gomaa Staff Ophthalmologist,
11. World Health Organization (WHO); Community clinics Flying Eye Hospital, ORBIS International.
in Bangladesh: Bringing health care to the doorstep of Community Eye Health Journal, vol 20(61), March
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events/community-clinics-bangladesh-story/en/
Update on the Japan Eye Genetics
Consortium (JEGC) 14
Takeshi Iwata
Abstract Keywords
Japan Eye Genetics Consortium (JEGC) was Japanese · Hereditary retinal disease · Optic
launched in 2011 to identify gene mutations neuropathy · Glaucoma · Gene mutation ·
responsible for 37 hereditary retinal diseases Whole genome/exome sequence · Knock-in
including hereditary optic neuropathy and mouse · patient iPS cells
hereditary glaucoma in Japanese population.
More than 2,300 DNA samples have now been
collected from 30 university ophthalmology 14.1 Launch of JEGC
departments in Japan for whole genome/
exome analysis. Our study shows that approx- JEGC was established by six ophthalmology
imately 80% of families with inherited retinal departments, RIKEN (Yokohama), National
disease carry novel gene mutations. Number Institute of Genetics (Mishima), and Tokyo
of new genes were identified and expected for Medical Center funded by the Japanese Ministry
dozens more. This high heterogynous genetic of Health, Labour and Welfare and later by the
background of Japanese patients with novel Japanese Agency for Medical Research and
gene mutations is a challenge to our consor- Development (AMED). In 2014, JEGC expanded
tium to identify all disease-causing mutations to current size of 30 university ophthalmology
within the time frame of research funding. departments when all board member of the
JEGC is also responsible to identify molecular Japanese Society for Clinical Electrophysiology
mechanism of disease onset for each mutation of Vision (President, Prof. Masayuki Horiguchi,
and apply these seed information for therapeu- MD, PhD, Fujita Health University) joined the
tic development. consortium (Table 14.1). Every 3 years the
research grant has been renewed with additional
objective for this consortium. The current
research objective is to contenuously accumu-
T. Iwata (*) late genotype-phenotype information of Japanese
National Institute of Sensory Organs, Tokyo Medical patients and to identify molecular mechanism for
Center, National Hospital Organization, Tokyo, Japan
e-mail: [email protected];
each mutant to develop potential therapeutic
http://iwatalab.org (Fig. 14.1).

https://doi.org/10.1007/978-981-13-0884-0_14
138 T. Iwata
Table 14.1 List of JEGC member Table 14.1 (continued)

Name Institution Name Institution
Takehi Iwata National Hosipital Organization KazuoTsubota Keio University
Tokyo Medical Center Akiko Maeda Case Western Reserve
Toshihide St. Marianna University University Department of
Nishimura Ophthalmology and Visual
Yoshihide RIKEN (Institute of Physical Sciences
Hayashizaki and Chemical Research) Kosuke Noda Hokkaido University
Kazusshige National Hospital Organization AtsuhiroTanigawa Fujita Health University
Tsunoda Tokyo Medical Center Syuji Yamamoto Jin Eye Clinic
Mineo Kondo Mie University Hiroyuki Jin Eye Clinic
Miroko Tera saki Nagoya University Yamamoto
Kei Shinoda Saitama Medical University Makoto Araie Kanto Central Hospital of the
Takaaki Hayashi The Jikei University of Medicine Mutual Aid Association of
Kazuki Kuniyoshi Kindai University Public School Teachers
Shuhei Kameya Nippon Medical School Makoto Aaihara Tokyo University
Kaoru Fujinami National Hosipital Organization Toru Nakazawa Tohoku University
Tokyo Medical Center Tetsuju Sekiryu Fukushima Medical University
Nobuhiro Tsukuba Primate Research Kenji Kashiwagi University of Yamanashi
Shimozawa Center, National Institute of Kenjiro Kozaki Keio University
Biomedical Innovation Caminci Piero RIKEN (Institute of Physical
Shinji Ueno Nagoya University and Chemical Research)
Masayuki Fujita Health University Takeo Fukuchi Niigata University
Hohguchi Atsuhshi Hayashi University of Toyama
SyuichiYamamoto Chiba University Katsuhiro Hosono Hamamatsu University School
Manami Kuze Matsusaka Central General of Medicine
Hospital Keisuke Mori International University of
AtsushiMizota Teikyo University Health and Welfare
Nobuhisa Naoi Miyazaki University Kazutoshi Tokyo University
Shigeki MachkJa Dokkyo Medical University, Yoshitake
Koshigaya Hospital Yuriko Minegishi National Hospital Organization
YoshiakiShimada Fujita Health University, Tokyo Medical Center
Banbuntane Hotokukai Hospital Daisuke lejima National Hospital Organization
Makoto Nakamura Kobe University Tokyo Medical Center
Hisashi Fujikado Osaka University Aakiko Suga National Hospital Organization
Yoshihiro Hotta Hamamatsu University School Tokyo Medical Center
of Medicine Brian National Hospital Organization
MasayoTakahashi RIKEN (Institute of Physical P. Rossmiller Tokyo Medical Center
and Chemical Research) Mao Nakayama National Hospital Organization
Kiyofumi Gifu University Tokyo Medical Center
Motiduki Yu Teruyama National Hospital Organization
Akira Murakami Juntendo University Tokyo Medical Center
Hiroyuki Kondo University of Occupational and Eiko Sanbe National Hospital Organization
Environmental Health Tokyo Medical Center
Susumu Ishida Hokkaido University Natsuko Teikyo University
Mitsuru Nakazawa Hirosaki University Nakamura
Teruhisa Hatase Niigata University Go Mawatah Miyazaki University
Tatsuo Matsunaga National Hospital Organization Kentaro Kurata Hamamatsu University School
Tokyo Medical Center of Medicine
Yozo Mryake Aichi Mecical University Norihiro Yamada Saitama Medical University
(continued)
14 Update on the Japan Eye Genetics Consortium (JEGC) 139
Fig. 14.1 Scheme of JEGC objective to collect family samples for clinical trials. Both functional study and collection
of phenotypic information are time-consuming process requiring dedicated clinicians and scientists
blue cone monochromatism, 19. gyrate atrophy,

14.2 JEGC Targeted Retinal 20. bradyopsia, 21. retinoschisis, 22. familial
Diseases drusen, 23. familial AMD, 24. bestrophinopathy,
25. Wnt signaling retinopathy, 26. Stickler syn-
JEGC covers 37 retinal diseases (1. Leber con- drome, 27. Wagner syndrome, 28. dominant optic
genital amaurosis, 2. retinitis pigmentosa, 3. atrophy (DOA), 29. mitochondrial retinopathy,
enhanced S-cone syndrome, 4. Usher syndrome, 30. Leber’s hereditary optic neuropathy (LHON),
5. Stargardt disease (STGD), 6. macular dystro- 31. ocular albinism, 32. oculocutaneous albi-
phy (non-STGD) or cone (cone-rod) dystrophy, nism, 33. albinism with systemic abnormalities,
7. occult macular dystrophy, 8. cone (cone-rod) 34. angioid streaks, 35. retinoblastoma, 36.
dystrophy with normal fundus appearance, 9. hereditary optic neuropathy, 37. hereditary glau-
North Carolina macular dystrophy, 10. foveal coma) for collection of genotypic and phenotypic
hypoplasia, 11. microphthalmus/nanophthalmus, information of patient and family (Fig. 14.2).
12. congenital stationary night blindness (CSNB), Phenotypic characterization is examined by fun-
13. Oguchi’s disease, 14. flecked retina syn- dus photograph, optical coherence tomography,
drome, 15. Bietti crystalline corneoretinal dystro- electroretinography, fluorescein angiography,
phy, 16. choroideremia, 17. achromatopsia, 18. autofluorescence, and other diagnostic methods.
140 T. Iwata
Fig. 14.2 Retinal
diseases collected by
JEGC. Over 1,300
pedigrees are currently
collected aiming for
5,000, which is
estimated to cover 10%
of total Japanese patients
14.3 JEGC Diagnostic of Japanese menus to English for future use as

and Genotype-Phenotype Global Eye Genetics Consortium (GEGC)
Database for Deep Learning Genotype-Phenotype Database (Fig. 14.3).
Currently, genotype-phenotype information of
All 37 eye diseases (Fig. 14.2) were selected by 2,300 individual are now entered into the system.
JEGC to collect information on patient’s family The system is constantly reprogrammed for eas-
phenotype information and blood/saliva samples ier input and quicker output of genotype-
for DNA extraction. The consortium came to an phenotype data. In 2018, artificial intelligence
agreement that the phenotypic information is (AI) server (IBM Japan, AI Vision) was con-
critical for grouping of patients with similar phe- nected to the database for deep learning of col-
notype, for natural history study, and patient lected genotype and phenotype. The aim of this
recruitment to clinical trials. To maintain high AI connection is to create a diagnostic support
diagnostic quality, “disease leader” was appointed system for rare retinal disease and to improve
to ophthalmologist with experience for selected genetic analysis to extract most likely disease-
retinal disease. Disease leaders gather twice a causing gene mutation from the whole genome/
year at Tokyo Medical Center to exchange infor- whole exome analysis. Linking AI drug discov-
mation and to improve protocol for data collec- ery system is also in consideration. Higher qual-
tion. Online JEGC Genotype-Phenotype ity of genotypic and phenotypic data is critical
Database was modified in 2017 with replacement for the deep learning process.
Fig. 14.3 JEGC Genotype-Phenotype Database was developed to collect data and to it transparent to JEGC members.
The database is now linked with artificial intelligence server for deep learning of these rare retinal diseases
142 T. Iwata
14.4 I dentification of Novel Gene localized to the connecting cilia between outer
Mutations for Retinal and inner photoreceptor [4]. Whole exome analy-
Diseases sis was performed on 147 families with retinal
degenerations, and novel mutations in C21orf2
14.4.1 Identification of Novel Gene were found in Japanese patients with autosomal
CCT2 Responsible for Leber recessive retinitis pigmentosa (arRP) with skele-
Congenital Amaurosis tal defects and with autosomal recessive cone-
rod dystrophy (arCRD) [5]. The patients in each
Leber congenital amaurosis (LCA) is a heredi- family carried compound heterozygous muta-
tary early-onset retinal dystrophy with severe tions p.V111 M and p.Y107H or homozygous
macular degeneration. In collaboration with Prof. mutation p.Y107C, respectively. Bother muta-
Xunlun Sheng, Director of Ningxia Eye Hospital tions are localized in the leucine-rich repeat
in China, we identified novel compound hetero- C-terminal domain (LRRC) required for protein
zygous mutations in chaperonin-containing stabilization. The effect of the mutations was
TCP-1, subunit 2 (CCT2) gene, which encodes examined by in vitro assays. In vitro expression
the chaperone protein CCTβ [1]. This CCTβ proof mutant C21orf2 cDNA showed reduced pro-
tein is a component of eight subunits (CCT α-θ) tein levels and abnormal cytoplasmic localization
forming a two-ring structure of molecular chap- compared to the wild type. Since C21orf2 is
erone. Previous study has shown zebrafish required for ciliogenesis, the data suggested that
mutants of CCTβ exhibit abnormal eye pheno- reduced levels of functional C21orf2 can induce
type, while its mutation and association with photoreceptor degradation through abnormal
human disease have not been reported [2]. The cilia formation, leading to arRP or arCRD.
identified novel CCTβ mutants T400P and
R516H are biochemically instable, and affinity
for adjacent subunit CCTγ was significantly 14.4.3 Novel Gene Mutations
affected in both mutants. The patient-derived in Known Genes
induced pluripotent stem cells (iPSCs) carrying
these CCTβ mutants were less proliferative than Our data shows approximately 20% of family
the control iPSCs. Decreased proliferation under collected with retinal disease carries known gene
Cct2 knockdown in 661 W cells was significantly mutation and approximately 20% with novel
rescued by wild-type CCTβ expression. However, mutations in known disease genes [6–13].
the expression of T400P and R516H didn’t Phenotypical characteristic in Japanese popula-
exhibit the significant effect. In mouse retina, tion with different genetics background are being
both CCTβ and CCTγ are expressed in the retinal studied in these families.
ganglion cells and connecting cilium of photore-
ceptor cells. The Cct2 knockdown decreased its
major client protein, transducing β1 (Gβ1). 14.5 Effective Therapeutic
Knock-in zebrafish [3] and mouse is now being Development Using Patient
developed for further pathological study. iPS Cells and Knock-In
Mouse Model
14.4.2 Identification of Novel Gene JEGC objective is to identify molecular mecha-

C21orf2 Responsible nisms for each disease-causing gene mutation,
for Retinitis Pigmentosa which will serve as seeds information to develop
and Cone-Rod Dystrophy new drugs or to alternatively use drugs currently
available for diseases other than eye. A number
C21orf2 encodes a ciliary protein expressed of tools are now available to mimic patient’s con-
through developing and mature stage of the retina dition in vitro and in vivo. These techniques
include use of patient iPS cells, which can be identify interacting proteins by proteomic analy-
transformed to retinal pigment epithelial (RPE) sis. This analysis resulted with mutant protein
cells, retinal ganglion cells (RGC), photorecep- interaction with TANK-binding kinase 1 (TBK1).
tors, and other retinal cell types. In these differ- The inhibitor for TBK1, amlexanox, a FDA- and
entiated cells, mutant protein localization and PMDA-approved drug for allergic rhinitis and
behavior can be directly observed and compared asthma, was later used to successfully disassoci-
with cells derived from normal individual. In ate mutant OPTN-TBK1 interaction [16]. To test
JEGC, iPS cells are generated from patients with if this mutant OPTN-TBK1 disassociation can
novel mutation or unique phenotype and stored in lead to improvement of the disease, OPTN E50K
biobank for use among consortium members for knock-in mice was generated by CRISPR/Cas9
functional studies. [3]. We administered amlexanox by oral adminis-
The CRISPR/Cas9 system to generate knock- tration 5 days a week for 1 year, which showed
in animal has become important technique to
significant neural protection compared to non-
confirm abnormal behavior of the mutant protein treated mice (Fig. 14.4). Amlexanox is currently
in vivo. This technique is currently applied to prepared for clinical trial funded by the National
zebrafish, mice, and cynomolgus macaque mon- Hospital Organization of Japan for NTG patients
keys to develop knock-in models. This technique with OPTN mutations.
is effective especially when amino acid sequence We are working on another successful transi-
of mutant protein is highly homologous between tion of basic to translational research on another
human and targeted animal. Knock-in mouse can gene mutation in JEGC [17].
be generated in a month and begin observation
for abnormal phenotype within a year. A number
of knock-in mice for novel genes are being devel- 14.6 Discussion and Future
oped in JEGC and periodically examined by Prospects
fundus photo, optical coherence tomography

(OCT), and electroretinogram (ERG) for any JEGC has now added proteomic and transcrip-
abnormal change in retinal structure and tomic analysis to the genome analysis in 2017 to
function. study molecular mechanism of each disease-
Optineurin (OPTN), a gene responsible for causing mutation. The Genotype-Phenotype
hereditary normal tension glaucoma (NTG), is Database plays major role to accumulate basic
well studied with myocilin, another gene respon- research and clinical information in one place,
sible for hereditary glaucoma with high intraocu- and to use the same database with patient’s natu-
lar pressure (IOP) [14]. A Japanese family with ral history for patient recruitment for future clini-
OPTN E50K mutation was identified by Dr. cal trials. Artificial intelligence (AI) server is now
Kazuhide Kawase at Gifu University, and patient connected to this database for deep learning of
iPS cells were generated to differentiate neural genotype-phenotype correlation. We hope this
cells. We soon noticed that mutant OPTN protein process will help improve clinical diagnostic
was accumulating in the endoplasmic reticulum and improve identification of disease-
of the patient-derived neural cells [15]. causing gene mutation for selection of
Immunoprecipitation experiment of mutant and future treatments.
normal OPTN was performed independently to
144 T. Iwata
Fig. 14.4 Fundus and OCT image of OPTN E50K mutant and control treated or non-treated with amlexanox at 1, 6,
and 12 months of age. Neuroprotection of amlexanox-treated NTG mice at 12 months (red asterisk). DW: distilled
water, A: amlexanox
Acknowledgments I would like to thank all the patients

and researchers participated in this study. This work was References
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Genetics and Susceptibility
of Retinal Eye Diseases in India 15
Sunita Mohan, Uthra Satagopan,
Soumittra Nagasamy, Sundaram Natarajan,
and Govindasamy Kumaramanickavel

As per the World Health Organization, genetic
eye disorders are one of the top ten major Genetic eye disorders are one of the top ten
causes of global ocular health burden. AMD causes of global ocular health burden (www.who.
and DR take major share of the adult eye dis- int/). It is increasingly evident that ophthalmic
eases component that particularly affects the clinical practice by the physician would be defi-
neurovascular retina. More than 100 genes are cient, if dealt without genetic knowledge and
known to cause Mendelian types of retinal information [148]. Retinal diseases are one of the
degenerations including syndromic and non- leading causes of childhood blindness [44], and
syndromic RP, and it is presumed that this inherited retinal disease has a prevalence of 1 in
constitutes only 60% of all the genes known 4000 that is one of the common causes of blind-
so far, and the remaining are yet to be identi- ness in children and working adults. Mendelian
fied. The burden of genetic disorders in India diseases such as retinitis pigmentosa (RP), con-
is significant, and very many significant genes genital stationary night blindness (CSNB), and
like RPE65 have been identified with consan- Stargardt disease have been reported to be caused
guineous autosomal recessive pedigrees by pathogenic genetic mutations, while adult
obtained from this region. onset retinal diseases such as age-related macular
degeneration (AMD) and diabetic retinopathy
Keywords (DR) have been shown to be hereditary through
Leber congenital amaurosis · family-based studies and have been associated
Phototransduction pathway · Retinitis with variations in genes involved in the diseases’
pigmentosa · Stargardt disease · Visual cycle pathways. This review would provide overall
information on the clinical and genetic aspects of
retinal eye diseases, focusing on status of research
in India in the field.
S. Mohan (*) · S. Natarajan · G. Kumaramanickavel AMD and DR take major share of the adult
Aditya Jyot Foundation for Twinkling Little Eyes,
Mumbai, India eye diseases component that particularly affects
e-mail: [email protected] the neurovascular retina. Over 19 genes/genomic
U. Satagopan regions cause susceptibility to AMD, out of
Center for Medical Genetics, Chennai, India which 2 genes complement factor H (CFH) and
S. Nagasamy age-related maculopathy susceptibility 2
MedGenome, Bangalore, India (ARMS2) have been widely reported, whereas the

https://doi.org/10.1007/978-981-13-0884-0_15
148 S. Mohan et al.
rest are minor common low effect alleles. 15.2 Epidemiology

However, environment factors like aging, diet, and Inheritance
smoking, and hypertension contribute signifi-
cantly to the disease onset and morbidity [55]. A Global prevalence of AMD is 8% (45–85 years)
number of single-locus genetic association stud- with a rising estimation of 196 million in 2020
ies and genome-wide association studies (GWAS) to 288 million by 2040 and with a higher preva-
have helped in identifying few genes that may lence among Europeans compared to Asians
have a role in DR pathogenesis, yet the multifac- [150]. DR affects 93 million people globally
torial involvement in DR pathology makes gene and is also expected to rise to alarming propor-
mapping quite challenging [74]. tions in the next few decades [157]. In India the
About 124 genes are known to cause DR prevalence is around 15–18% among the
Mendelian retinal degenerations including syn- diabetic population over 40 years of age [114,
dromic and non-syndromic RP, and it is presumed 139].
that this constitutes only 60% of all the genes RP affects 1 in 3000–7000 people in a conser-
known so far, and the remaining are yet to be vative estimate [18]. The prevalence in North
identified [4, 37]. About 17 genes have been China was found to be thrice higher when com-
reported to be responsible for congenital station- pared to rural Beijing (≥40 years [57, 159]).
ary night blindness (CSNB) out of which some However in India it appears to be more frequent.
are unique to the disease, whereas some are com- A prevalence of about 1 in 750 in rural central
mon to RP as well, affecting both phototransduc- India (≥30 years, [101]), 1 in 930 in urban South
tion pathway and visual cycle [163]. Vision India, and 1 in 372 in rural India has been esti-
sciences particularly genetics attracted intense mated for RP (≥40 years, [127]); the reason for
studies, and hence so far 4000 mutations have such high prevalence is not clear but could be due
been identified in the 124 genes (Table 15.1); to consanguineous marital practice that is widely
genes like ABCA4, BEST1, CDH23, CEP290, prevalent in southern India. Consanguinity adds
CRB1, EYS, MYO7A, OFD1, RPE65, RPGR, to the proportion of autosomal recessive eye dis-
RHO, PRPH2, and USH2A each have 100 muta- eases in the Indian population [73, 104].
tions described with ABCA4 alone having 700 Consanguineous marriages not only in South
mutations [27]. There is a high degree of clinical Asia and North Africa but also in London, due to
and genetic heterogeneity with overlapping phe- the South Asian and North African immigrant
notypes in retinal degeneration leaving a huge populations, are causing different counseling
challenge in diagnostics, genetic counseling, and approaches in pediatric clinic for patients with
gene therapies [103]. Syndromic RP alone has 40 ocular genetic disorders [116].
genes which are ciliopathies – Alstrom syn-
drome, Bardet-Biedl syndrome, Jeune syndrome
or asphyxiating thoracic dystrophy, Joubert syn- 15.3 Phototransduction Pathway
drome, and Meckel-Gruber syndrome. Zellweger and Visual Cycle
is a spectrum of peroxisomal disorders caused by
a dozen genes including Refsum disease. Kearns- Electromagnetic photo-energy is bioprocessed to
Sayre syndrome and neuropathy-ataxia-RP a neuroelectrical impulse in the retina to be per-
(NARP) are caused by mitochondrial mutations ceived in the occipital lobe as vision. The physi-
[153]. The National Eye Institute has created a ology and biochemistry of vision are broadly
gene screening center and repository for patients divided into phototransduction (PT) in the neuro-
with inherited ocular genetic disorders to facili- retina and visual cycle (VC) in the retinal pig-
tate and network scientists and clinicians to fur- ment epithelium (RPE). In PT the final outcome
ther research [45]. is a neuroelectrical impulse, whereas in VC it is
15 Genetics and Susceptibility of Retinal Eye Diseases in India 149
Table 15.1 The list of retinal degenerative diseases genes classified based on the physiology and biochemistry of the
neuroretina and retinal pigment epithelium, including syndromes
No Function Genes
1 Phototransduction cascade RHO (rhodopsin (G-protein coupled photon receptor)), PDE6A (rod cGMP-
phosphodiesterase α-subunit (G-protein effector enzyme)), PDE6B (rod
cGMP-phosphodiesterase β-subunit (G-protein effector enzyme)), CNGA1 (rod
cGMP-gated cation channel α-subunit), CNGB1 (rod cGMP-gated cation
channel β-subunit), SAG (arrestin (rhodopsin deactivation)), GUCA1B
(guanylate cyclase activator 1B), RDH12 (retinol dehydrogenase 12), PDE6G
(phosphodiesterase 6G, CGMP-specific, rod, gamma)
2 Vitamin A metabolism ABCA4 (ATP-binding cassette protein A4 [photoreceptor disc membrane
flippase for vitamin A]), RLBP1 (retinaldehyde-binding protein [11-cis-
retinaldehyde carrier]), RPE65 (retinal pigment epithelium 65, vitamin A
trans-cis isomerase), LRAT (lecithin retinol acetyltransferase (synthesizes
vitamin A esters)), RGR (RPE-vitamin A G-protein coupled receptor (photon
receptor in RPE), RBP3 (retinol-binding protein 3, interstitial), MVK
(mevalonate kinase)
3 Structural or cytoskeletal RDS (peripherin (outer disc segment membrane protein)), ROM1 (rod outer
elements segment protein), FSCN2 (fascin [actin-bundling protein]), TULP1 (tubby-like
protein), CRB1 (crumbs homologue (transmembrane protein, adherent
junctions)), RP1 (microtubule-associated protein [microtubule formation and
stabilization])
4 Signaling, cell-cell NRL (neural retina leucine zipper), FAM161A (family with sequence similarity
interaction, synaptic 161, member A), SEMA4A (semaphorin B, transmembrane immune system
interaction, tissue protein), CDH23 (cadherin 23 (adhesion receptor)), PCDH15 (protocadherin
development and 15 [adhesion receptor]), USH1C (Usher syndrome type IC ([integrating scaffold
maintenance protein harmonin])), USH2A (Usher syndrome type IIA [Usher network
protein]), MASS1 (monogenic audiogenic seizure susceptibility 1 [Usher
network protein]), USH3A (Usher syndrome type IIIA [transmembrane protein
clarin 1]), RP2, plasma membrane-associated protein
5 RNA intron-splicing PRPF31 (precursor mRNA-processing factor 31 [spliceosome component]),
factors PRPF8 (precursor mRNA-processing factor 8 [spliceosome component]),
PRPF3 (precursor mRNA-processing factor 3 [spliceosome component]), RP9
(PIM1-associated protein [RNA splicing factor]), PRPF4 (pre-MRNA
processing factor 4), PRPF6 (pre-MRNA processing factor 6), SNRNP200
(Small Nuclear Ribonucleoprotein 200kDa (U5)), DHX38 (DEAH (Asp-Glu-
Ala-His) box polypeptide 38))
6 Trafficking of intracellular MYO7A (myosin 7A [melanosome motility protein]), USH1G scaffold
proteins protein-containing ankyrin repeats and SAM domain [Usher’s type I protein
traffic regulator]
7 Maintenance of cilia/ BBS1, BBS2, BBS4, BBS5, BBS7 (Bardet-Biedl syndrome), ARL6 (ADP-
ciliated cells (possible role ribosylation factor-like), MKKS, McKusick-Kaufman syndrome (BBS6
in intracellular trafficking) protein), TTC8 (tetratricopeptide repeat domain 8), PTHB1 (parathyroid
hormone-responsive B1), RPGR (ciliary protein, cargo of PDE6D for ciliary
trafficking) (viii) pH regulation (choriocapillaris) – CA4 (carbonic anhydrase IV
(carbon dioxide/bicarbonate balance)
8 Phagocytosis MERTK, mer tyrosine kinase proto-oncogene [RPE receptor involved in outer
segment phagocytosis]
9 Transcription factors NR2E3 (nuclear receptor subfamily 2, group E, member 3), CRX (cone-rod
homeobox), ZNF513 (zinc finger protein 513)
10 Photoreceptor ARL2BP (ADP-ribosylation factor-like 2 binding protein)
maintenance and function
11 Regulator of cell growth IMPDH1 (inosine-5’-monophosphate dehydrogenase type I [guanine
nucleotide synthesis])
12 Cellular structure IMPG2 (interphotoreceptor matrix proteoglycan 2), MAK (male germ
cell-associated kinase), PROM1 (prominin-like protein 10)
(continued)
150 S. Mohan et al.
Table 15.1 (continued)
No Function Genes
13 Photoreceptor structure CLRN1 (clarin 1), DHDDS (dehydrodolichyl diphosphate synthase)
14 Anion channel BEST1 (bestrophin 1)
15 Ubiquitin related KLHL7 (ubiquitin-proteasome protein degradation), TOPORS (ubiquitin-
protein ligase)
16 Cell signaling EYS (eyes shut homologue (Drosophila)), CERKL (ceramide kinase-like
(ceramide converting enzyme))
17 Cell division NEK2 (NIMA (never in mitosis gene A)-related kinase 2)
18 Others BBS10 (vertebrate-specific chaperonin-like protein), C2orf71 (chromosome 2
open reading frame 71), C8orf37 (chromosome 8 open reading frame 37),
PRCD (progressive rod-cone degeneration), SPATA7 (spermatogenesis
associated 7), EMC1 (ER membrane protein complex subunit 1), GPR125
(G-protein coupled receptor 25), KIAA1549 (UPF0606 family, protein coding
gene), SLC7A14 (solute carrier family 7, member 14)
Modified as per Refs. [39, 51, 103]
the recycling of vitamin A. Several genes func- 15.4 Genetics Overview
tion critically in both the components to
effectively optimize both PT and VC, to perceive The burden of genetic disorders in India is sig-
vision. The electrical impulse is generated in the nificant [111, 142]. Considerable amount of eye
photoreceptors (rods and cones) and transmitted genetic studies has been performed in the Middle
through neurotransmitters to the bipolar cells and East [63]. Countries like Egypt are providing eye
then to the occipital lobe through the visual genetic counseling services [46]. Twenty-nine
pathway. different mutations causing various forms of reti-
In dark, the photoreceptors are in a constant nal degenerative disorders, with similar human
state of depolarization, and glutamate neuro- clinical phenotypes, are found in canines starting
signals pass from them to the bipolar cells. At this from stationary night blindness to cone-rod
state the PRs have their calcium-gated channels degenerations, which are ideal to be used as mod-
open; hence, ions like calcium and sodium pass els for preclinical trials [22].
into them freely, whereas irrespective of this
mechanism potassium constantly keeps passing
out of them freely. However in light, these chan- 15.4.1 Retinitis Pigmentosa
nels close and prevent Ca++/Na++ from entering
the PR, but the K++ continues to be expelled; this RP starts majority of times with night blindness,
causes hyperpolarization of PR and initiates to as an early symptom, and later leads on to legal
release the neurotransmitters to send signal to the or total blindness. The fundus image is classical
bipolar cells. PT is triggered by photon-striking with mid-periphery pepper-salt appearance pig-
retinal in the rhodopsin molecule to convert to the mentation and attenuated arteries. The pigmenta-
activated form, which allows the binding of trans- tion appears due to the tilting of balance of outer
ducin, to rhodopsin. α-Subunit of transducin is segment disc shedding and RPE scavenging; in
released from its β- and γ-subunits of transducin RP the shedding is overwhelming, leaving them
which then binds to phosphodiesterase; this step to be seen as pigments in the fundus.
converts cyclic GMP to GMP and makes the RP is inherited in all Mendelian forms, includ-
opened cGMP-gated channels to close causing ing syndromic and mitochondrial inheritances.
hyperpolarization. Autosomal dominant (adRP – with a global aver-
age proportion of 30–40%) is more common in genes region or genome-wide SNP genotyping
North America, autosomal recessive (arRP, arrays (Illumina Infinium arrays) on consanguin-
50–60%) in southern India, and X-linked eous and non-consanguineous families have reit-
recessive (xlRP, 5–15%) in Britain. aR and xl erated the efficiency of identifying the disease
onset is quite early compared to aD, since the causative gene/variation in autosomal recessive
normal gene compensates for some time. RP disease using this methodology. These studies
genes have been classified in multiple ways, reported identification of homozygous regions
since there are 124 causative genes; however, the common between the affected sibs in 5/10 fami-
most simple way to classify is according to the lies; homozygosity in 10/34 families followed by
physiology and biochemistry, like genes pertain- detection of mutations in TULP1, RLBP1,
ing to phototransduction cascade, vitamin A ABCA4, RPE65, and RP1 in 5 of these 10 fami-
metabolism, structural and cytoskeletal elements, lies; and mutations in TULP1, NR2E3, MRFP,
signaling, RNA intron-splicing factors, traffick- and SPATA7 in 4/26 arRP families, respectively
ing of intracellular proteins, maintenance of cilia, [58–60, 75, 129].
phagocytosis, transcription factors, photorecep- Homozygosity mapping followed by whole
tor maintenance and function, regulator of cell exome sequencing (WES) or WES alone has
growth, cellular structure, photoreceptor struc- been employed, and the causative genes were
ture, anion channel, ubiquitin related, cell signal- identified as TTC8 and CRB1 in each one and
ing, cell division, and others (Table 15.1. EYS in two arRP families, respectively. Analyzing
Modified as per [51, 103]). 100 sporadic RP cases by WES has identified
mutations in CRB1 and EYS in 2 and 8 cases,
15.4.1.1 RP Genetic Studies in India respectively [32, 48, 158]. RHO gene, though is a
There are few reports on autosomal recessive and candidate for ADRP, a homozygous missense
autosomal dominant non-syndromic RP from mutation, has been reported in the same in an
India. Homozygosity mapping has been exten- Indian arRP family [72]. Mutations in codon 345
sively employed to map either known or novel (V>M/L) and 347 (P>S/A/R/Q/L/T) in RHO
candidate loci/genes in autosomal recessive dis- gene are most frequent cause of ADRP, as
eases in both consanguineous and non- reported from studies on various ethnicities
consanguineous families. The first report from worldwide. However, screening 100 Indian RP
India on homozygosity mapping on arRP identi- patients from 76 families revealed V345M muta-
fied RP22 locus in two families [38]. However, tion in an AD family with 3 affected and in 1 spo-
the causative gene in this locus is yet to be identi- radic case [33]. A complete gene screening of
fied. A second new locus, RP28 on chromosome ADRP genes, RHO, PRPF31, RP1, and IMPDH1
2p11–2p15, was identified in an Indian family in 48 isolated and 53 ADRP families, revealed a
with multiple consanguineous marriages again missense mutation (p.Gly106Arg) and a deletion
using homozygosity mapping [49]. The linkage c.358_359delAA leading to a frameshift and pre-
to this 16cM region was further fine mapped to a dicted protein truncation p.(Lys120GlufsX122)
minimum critical region of 1.06cM between the in RHO gene in each one of an isolated case and
D2S2225 and D2S296 markers in a second Indian a splice-site mutation IVS6+1G>A in PRPF31
family [69]. FAM161A was identified as the can- gene in an ADRP family. The splice mutation
didate gene in the RP28 locus in 2010, using observed in the ADRP family presented with
ChIP and parallel sequencing [76]. Further muta- incomplete penetrance as one of the clinically
tions in FAM161A were reported in two more normal individual too harbored the variation [43].
consanguineous arRP families and one out of Similarly, in a report by Saini et al., a deletion
hundred sporadic Indian RP patients using whole mutation c.59_65del7 (p.Gly20AlafsX43) in
exome sequencing [166]. Also, few earlier reports PRPF31 was seen in a large ADRP family with
on homozygosity mapping using either microsat- ten affected members and one unaffected consis-
ellite markers flanking the known candidate tent with high incomplete penetrance of the gene.
152 S. Mohan et al.
This deletion was absent in other unaffected to the interplay of three prime factors that vary
members and ethnic-matched controls [122]. among the patients: (i) severity of their ABCA4
Linkage mapping in an ADRP family of 34 genotype, (ii) relative sensitivity of the foveal
members with 14 affected mapped a novel locus cones to the genotype, and (iii) the relative sensi-
on 6q23 with a disease co-segregating region of tivity of the RPE to the genotype [125].
about 25 Mb and a maximum two-point LOD Early manifestation may only consist of some
score of 3.8 [58–60]. There are no reports yet on yellowish flecks and a macula with a snail’s slime
xlRP from India. aspect. In the later stages, the macula may show
bull’s eye pattern with RPE atrophy or a beaten-
bronze atrophy aspect. The functional changes
15.4.2 Stargardt Disease remain usually restricted to the posterior pole of
the eye, but they sometimes also affect the periph-
Stargardt disease (STGD) is an autosomal reces- eral retina. Fluorescein angiography plays a key
sive form of juvenile hereditary macular degen- role in the diagnosis of Stargardt’s, as it evidences
eration characterized by bilateral discrete dark choroid (silence choroidien), a characteris-
yellowish round or pisiform flecks around the tic sign of the disease that probably results from
macula at the level of RPE [54]. It is one of the the accumulation of lipofuscin in the RPE [5].
commonly hereditary forms of juvenile macular Also relative sparing of the peripapillary RPE is a
degeneration with an estimated prevalence of 1 in fairly reliable diagnostic sign of ABCA4-
8000 to 10,000 individuals. In more than 95% of associated retinal disease more evident of fluo-
cases, STGD is caused by mutations in ABCA4 rescein angiography.
gene, and the remaining 5% is caused by muta-
tions in the ELOVL4, PRPH2, BEST1, or PROM1 15.4.2.1 Stargardt Disease Genetic
gene. ABCA4 encodes a transmembrane trans- Studies in India
porter protein that is responsible for clearance of The only report of ABCA4 and Stargardt disease
a retinoid intermediate of the visual cycle from from India is on screening 5 STGD patients using
the intradiscal lumen of the outer segments of the targeted NGS with 184 retinal genes panel.
rods and the cones. The three-step pathophysiol- Mutations were identified in all five: four com-
ogy in a mutated ABCA4 gene includes a defec- pound heterozygous and one homozygous that
tive rim protein, encoded by mutated ABCA4 includes two novel mutations [9].
gene causing an accumulation of toxic by-product
A2E in the RPE cells leading to the death of pho-
toreceptors [132, 165]. 15.4.3 Vitelliform Macular Dystrophy
Fundus flavimaculatus (FFM) is an allelic
subtype of Stargardt disease that has been associ- Vitelliform macular dystrophy (VMD) is a slowly
ated with mutation in the ABCA4 gene and the progressive macular dystrophy. Two forms of
PRPH2 gene. FFM has a later age of onset. If loss VMD with similar features have been described.
of visual acuity begins in the first two decades, The early-onset form (known as Best disease/Best
the designation STGD is preferred; if it begins macular dystrophy) is inherited in an autosomal
later in life and has a more progressive course, dominant fashion and appears in childhood, while
the term FFM is preferred [147]. No racial predi- the adult-onset form with an uncertain inheritance
lection has been observed. Men and women are pattern begins later in the mid-adulthood and tends
equally affected. Both eyes are usually equally to cause vision loss that worsens slowly over time
and symmetrically affected. STGD patients typi- [109, 89]. Best macular dystrophy (BMD) is one
cally complain of decreased visual acuity, gradu- of the most common Mendelian macular dystro-
ally diminishing to 20/200. Clinical presentation phies, occurring in about 1 in 10000 individuals;
in STGD varies greatly in the age of onset, pre- the prevalence of adult-onset form is not known.
senting symptoms and the fundus appearance. Retinal findings are not generally present at
The variations in the clinical presentation are due birth and typically do not manifest until ages
5–10 years. Affected individuals have the charac- unknown. Two distinct phenotypes are recog-
teristic lesion, a typical yellow yolk-like macular nized based on full-field ERGs, complete CSNB
lesion on fundus examination. Over time, a (CSNB1A-45%) and incomplete CSNB
pseudo-hypopyon appears due to gravitation of (CSNB2A-55%) [96, 97]. The complete and
the yellow material inferiorly in the subretinal incomplete CSNBs are model disorders of bipo-
space, followed by deep and irregular pigmenta- lar cell dysfunction caused by dysfunction of on
tion giving it a scrambled egg appearance. and off bipolar cells. Complete CSNB can be
Lesions are usually bilateral but can be unilateral. inherited in an X-linked recessive or autosomal
Visual acuity is often 20/20 or better in eyes with recessive manner [96, 128]. The incomplete form
undisturbed vitelliform lesions. Peripheral vision appears to be more common in people of Dutch-
and dark adaptation remain normal. The progno- German Mennonite descent [16]; however, it has
sis is good till the fifth decade after which visual been reported in families with many different
acuity declines in one or both eyes due to CNV, ethnic backgrounds.
scarring, or geographic atrophy. Mutations in the NYX and CACNA1F genes
The diagnosis of BMD is based on character- cause complete and incomplete forms of
istic fundus appearance, a light peak/dark trough XL-CSNB, respectively. NYX and CACNA1F
ratio of less than 1.5 on EOG with a normal ERG genes encode proteins that are specifically
and a family history of BMD. A normal EOG, expressed in the retina: nyctalopin and voltage-
however, does not exclude the possibility of dependent calcium channel for complete and
BMD [17, 77, 88, 93, 149]. incomplete CSNB, respectively [10, 135].
VMD is caused by mutations in the BEST1 Mutations in these genes affect the synaptic
and PRPH2 genes. BEST1 mutations are respon- transmission from photoreceptors (rods and
sible for BMD. The BEST1 gene encodes for cones) to the bipolar cells. In the complete form
bestrophin protein that localizes to the basolat- of XL-CSNB, the function of rods is severely dis-
eral membrane of RPE and appears to function as rupted and that of cones is only mildly affected.
a calcium-sensitive chloride channel In the incomplete form, incomplete defect of syn-
(Marmorstein AD et al). Mutations in the BEST1 apses in the ON and OFF bipolar cells in both the
gene lead to impaired uptake of calcium across rod and cone pathways occurs, but they do pre-
RPE leading to alterations in the adhesiveness serve the ability to detect the light [12, 98].
between interphotoreceptor matrix and the RPE XL-CSNB is characterized by nonprogressive
or diminution of outer segment phagocytosis, retinal findings with visual acuity ranging from
both of which are sensitive to the levels of cal- 20/30 to 20/200 associated with refractive error,
cium [87; 50]. In most of the cases of adult onset, most typically myopia, but occasionally hypero-
the mutation is unknown, and less than a quarter pia, nystagmus, strabismus, normal color vision,
have mutations traced to the PRPH2 and BEST1 and normal fundus examination [3, 16].
gene. The PRPH2 gene encodes peripherin 2, a Individuals with CSNB1A generally report
protein essential for the normal functioning of severe night blindness, while individuals with
photoreceptors, mutation of which causes vision CSNB2A do not uniformly report severe night
loss by disrupting structures that contain light- blindness. Other findings include defective dark
sensing pigments [15, 36]. adaptation; carriers of an NYX or CACNA1F
mutation usually do not develop any of the visual
problems related with XL-CSNB. However, car-
15.4.4 Congenital Stationary Night riers may have retinal changes detected with an
Blindness ERG. Diagnosis is based on clinical findings,
characteristic findings on ERG, family history,
Congenital stationary night blindness (CSNB) is and molecular genetic testing of NYX and
a nonprogressive retinal condition of the scotopic CACNA1F, the only two genes in which mutation
vision. The prevalence of this condition is is known to cause XL-CSNB.
154 S. Mohan et al.
15.4.4.1 Congenital Stationary Night abnormalities. Visual acuity is severely reduced

Blindness Genetic Studies to counting fingers or worse in the majority of
in India cases, and perimetry shows an enlarging dense
A pilot study of eight complete CSNB cases from central or centrocecal scotoma. Post-acute epi-
India identified mutations in all and in the candi- sode, the atrophic phase ensues in which the optic
date genes TRPM1, GRM6, and GPR179 [85]. discs become atrophic within 6 weeks of onset.
Further, using NGS-based targeted re-sequencing, Significant improvements in visual acuity are
a homozygous and a compound heterozygous rare, and in most individuals, vision remains
mutations were identified in SLC24A1 gene in severely impaired (Kirkman et al [67]). The hall-
two cases, confirming the inheritance pattern and mark of LHON is the selective degeneration of
genotype-phenotype correlation of autosomal the retinal ganglion cell layer and optic nerve.
recessive Rigg’s type CSNB to this gene [106]. The diagnosis of LHON is based on characteris-
tic fundus findings in optic disc and vascular
changes in the acute phase.
15.4.5 Leber Hereditary Optic LHON is caused by mutations in the MT-ND1,
Neuropathy MT-ND4, MT-ND4L, or MT-ND6 genes. These
genes are found in the mtDNA and encode sub-
Leber hereditary optic neuropathy (LHON) is a units of NADH dehydrogenase. Mutations in any
rare form of vision loss associated with mater- of these genes disrupt this process and lead to
nally inherited mitochondrial DNA mutations. increased mitochondrial reactive oxygen species
The prevalence of LHON in most populations is production that trigger retinal ganglion cell death
unknown, and the relative frequency of the differ- via an apoptotic mechanism [11, 29, 162].
ent LHON-causing mtDNA variants varies However, the selective vulnerability of retinal
throughout the world. Overall, the m.11778G>A ganglion cells in LHON remains unexplained. A
variant is the most prevalent, accounting for 70% significant percentage of people with a mutation
of cases among northern European [83] and associated with LHON do not develop any fea-
approximately 90% of cases in Asian populations tures of the disorder. Specifically, more than 50%
[56, 90]. It affects 1 in 8500 to 1 in 50,000 people of males with a mutation and more than 85% of
in northeast England and Finland, respectively females with a mutation never experienced vision
[86, 112, 133]. loss or related health problems.
LHON is characterized acute or subacute,
severe painless unilateral loss of central vision 15.4.5.1 Leber Hereditary Optic
during the young adult life. The fellow eye Neuropathy Genetic Studies
becomes subsequently affected within weeks or in India
months of the first. Males are about four to five So far there are seven reports on LHON from
times more likely to be affected than females India, screening either the primary mutations
[160]. In the acute phase, the affected individuals alone or the entire mitochondrial genome. The
are usually entirely asymptomatic until they first report, a case study on two families identi-
develop visual blurring affecting the central fied MT:G3640A and MT:G11778A primary
visual field in one eye; similar symptoms appear mutations, respectively [143]. Analyses of whole
in the other eye at an average of 2–3 months later. mitochondrial genome in a north Indian cohort of
In about 25% of cases, visual loss is bilateral at 30 cases and 20 controls revealed 6 pathogenic
onset. The ocular fundus may have a characteris- mutations including the primary mutations in 12
tic appearance that includes disc swelling, edema cases. The percentage of all variations, synony-
of the peripapillary nerve fiber layer, retinal tel- mous and non-synonymous, was higher in cases
angiectasia, and increased vascular tortuosity. as compared to controls [70, 71]. A similar obser-
These changes can be subtle, and approximately vation was seen in a study on 75 LHON cases and
20% of the affected individuals show no fundal 40 controls from South India, wherein primary
mutations were identified in 27 cases and 4 other into the photochromophore [14]. The inheritance
LHON-associated mutations as well. The above pattern for families with mutations in PRPH2 is
study also reports association of haplogroup M consistent with autosomal dominant inheritance,
with LHON in the cohort [121]. In a study on 187 while mutations in RDH5 result in an autosomal
LHON families, 8 were positive for the primary recessive pattern. Mutations in RLBP1
mutation, MT:T14484C. A haplogroup analysis (retinaldehyde-binding protein 1) have also been
revealed that they belonged to diverse haplogroup found in some families.
contrary to the observed association of hap-
logroup J to MT:T14484C in the western popula-
tion [64, 65]. A case report of two families with 15.4.7 Oguchi Disease
primary mutation, MT:G11778A along with
MT:A1555G (usually associated with Oguchi disease is a CSNB characterized by the
aminoglycoside-induced non-syndromic hearing Mizuo-Nakamura phenomenon [95], a unique
loss), observed varying severity in the two cases morphological and functional abnormality in
but with no involvement of the auditory sense, which retina appears normal after prolonged dark
thus suggesting role of nuclear modifying genes adaptation, but on exposure to light, the retina
and environmental factors in the disease severity displays a golden sheen with an unusually dark
[64, 65]. Overall the frequency of LHON primary macula [20]. It was originally discovered in Japan
mutations observed in Indian cohorts is ~34%, where the prevalence is the highest but has been
whereas it contributes to >95% of the cases in subsequently reported in European, American,
western population [70, 71, 121, 138]. Pakistani, and Indian patients [20, 21, 92, 145].
Individuals with Oguchi disease have a non-
progressive night blindness since young child-
15.4.6 Fundus Albipunctatus hood with normal day vision, but they often claim
improvement of light sensitivities when they
Fundus albipunctatus (FA), a flecked retinal dis- remain for long time in a dark environment. The
order, is a subgroup of CSNB, characterized by visual functions, including visual acuity, visual
nonprogressive night blindness and delayed dark field, and color vision, are usually normal. On
adaptation, usually presenting in early childhood. clinical examination, in addition to eliciting
The fundi show numerous small, yellow dots in Mizuo-Nakamura phenomenon, they have a dark
the retinal pigment epithelium scattered through- adaptation curve with a cone component but no
out the fundus, which may or may not involve the rod-cone break and exhibit gradual recovery of
macula [100]. The dark adaptation curve of full rod sensitivity after prolonged dark adapta-
affected individuals shows prolonged recovery of tion of 1–2 h. The clinical diagnosis is confirmed
cone and rod sensitivity. The ERG cone and rod by genetic testing.
amplitudes are markedly reduced after 30–40 min Oguchi disease is caused by mutations in the
of dark adaptation; however, they may come to SAG (S-antigen, retina, and pineal gland) or
normal or near-normal levels after many hours of GRK1 (G-protein coupled receptor kinase 1)
adaptation. ([40]; Yamamoto et al. [155] Hayashi et al. [53]).
FA is a genetically heterogeneous disorder The SAG gene codes for arrestin, a member of the
with mutations in two genes, PRPH2 (also known rod phototransduction pathway (Oguchi type 1),
as RDS) and RDH5 [47, 105, 156]. The PRPH2 while the GRK1 gene codes for the rhodopsin
gene encodes peripherin 2, a protein involved in kinase that works with arrestin in shutting off
the formation and stability of the photoreceptors. rhodopsin after it has been activated by a photon
The RDH5 gene encodes enzyme 11-cis-retinol (Oguchi type 2). Some mutations in the SAG
dehydrogenase that catalyzes the final step in the gene are associated with Oguchi disease and RP
biosynthesis of 11-cis-retinaldehyde and trans- in the same family suggesting that mutations in
ports it to the photoreceptors for incorporation SAG lead to RP.
156 S. Mohan et al.
15.4.7.1 Oguchi Disease Genetic retinal phenotypes can be observed: (a) preserved
Studies in India para-arteriole retinal pigment epithelium
Till date there are two publications on genetics of (PPRPE) in individuals with CRB1 pathogenic
Oguchi disease from India. In a study on consan- variants; (b) “translucent RPE,” white dots, and a
guineous family with three affected sisters, map- peculiar star-shaped maculopathy in individuals
ping with markers on chromosome 2q revealed with RPE65 pathogenic variants; and (c) a pro-
linkage to a region between D2S172 and D2S345 gressive macular atrophic lesion presenting in
[91]. In another report two affected sibs were infancy or later in some individuals. Because of
identified with a nonsense mutation in codon 193 its sharply defined borders, this lesion has been at
of the SAG gene [92]. times called a macular coloboma [23]. The diag-
nosis of LCA is established by clinical findings.
15.4.8 Leber Congenital Amaurosis 15.4.8.1 Leber Congenital Amaurosis

Genetic Studies in India
Leber congenital amaurosis (LCA) is an early- There are very few reports on genetics of LCA
onset childhood severe rod-cone dystrophy with from India. These studies have been either
a very poor prognosis. LCA occurs in 2–3 per screening one or two candidate genes in a small
100,000 newborns being the commonest genetic cohort or participation in multicenter studies
cause of inherited blindness in childhood consti- again with very few Indian subjects or identify-
tuting more than 5% of all retinal dystrophies. It ing the candidate gene using homozygosity map-
appears to be more prevalent where consanguin- ping. The screening of RPE65 gene in cohort of
ity is common [130]. 60 and 20 cases by Gandra et al. [81] and Ramana
Visual function is usually poor, accompanied et al. [115] has identified a mutations in 1 and 3
by nystagmus, sluggish or near-absent pupillary cases, respectively. A study by Sundaresan et al.
responses, photophobia, high hyperopia, and ker- [137] to know the frequency of 104 mutations in
atoconus [26]. A characteristic finding is 8 genes that contribute to 30% of LCA in north-
Franceschetti’s oculo-digital sign, comprising ern American population showed that only 1 of
eye poking, pressing, and rubbing [35]. The fun- the 38 case studies harbored a reported mutation.
dus appearance is extremely variable; the retina In another study on 30 LCA cases, wherein
may initially appear normal, followed by pig- direct sequencing of RPE65 gene was followed
mentary retinopathy reminiscent of retinitis pig- by screening of reported pathogenic mutations
mentosa observed later in childhood. The ERG is using APEX technology, mutations were identi-
characteristically “non-detectable” or severely fied in 36% of the cases [144]. These reports
subnormal. indicate the genetic heterogeneity of the disease
LCA has an autosomal recessive pattern of in our population and also involvement of novel
inheritance with more than half cases resulting variations in disease causation as reported in
from mutations in at least 17 different genes. other ethnicities. Homozygosity mapping that
These genes play a variety of roles in the normal has shown to map the candidate gene with >90%
development of the photoreceptors, phototrans- frequency was employed in a cohort of consan-
duction, and the functioning of cilia. Mutations guineous LCA families, and in 11 of 12, the can-
in any of these genes disrupt the development and didate gene and the causative variation were
function of the retina, resulting in early vision identified [134, 136, 137].
loss. Mutations in CEP290, CRB1, GUCY2D,
and RPE65 genes are the most common causes of 15.4.8.2 Therapies Under Investigation
the disorder, mutations in other genes account for in LCA
a smaller percentage of cases, and in about 30% In a naturally occurring Briard dog model of
of all people with LCA, the cause of the disorder LCA resulting from mutation of RPE65, gene
is unknown [31]. Of note, three more specific therapy utilizing AAV-mediated RPE65 has been
shown to restore visual function, an effect that testing. RS1 is the only gene known to be associ-
has been documented to last for more than 5 years ated with XLJR. More than 196 pathogenic vari-
[1]. The results of three simultaneous Phase I ants in RS1 gene have been associated with XLJR
clinical treatment trials of AAV-mediated RPE65 [66]. RS1 gene mutations result in a decrease or
gene therapy in humans were recently reported complete loss of functional retinoschisin, an
[8, 24, 52, 84]. Initial results demonstrated safety extracellular protein that exists as a novel
and showed slight improvement in vision in both disulfide-linked octamer and is expected to play a
bright and dim light. Clinical and laboratory crucial role in cellular organization of the retina
studies suggest that persons with CEP290-related [152]. Some individuals with X-linked juvenile
LCA may also be good candidates for gene ther- retinoschisis do not have a mutation in the RS1
apy. [24] studied the retinal architecture of gene. In these individuals, the cause of the disor-
CEP290-mutant mice and humans. In the mouse der is unknown.
retina, dramatic retinal remodeling was evident
by age 4–6 weeks. Cross-sectional imaging of 15.4.9.1 Therapies Under Investigation
affected human retinas performed using OCT for X-linked Juvenile
indicated preservation of foveal cones. The rela- Retinoschisis
tive sparing of foveal cone cells, despite severe A mouse model of human X-linked juvenile reti-
visual dysfunction, suggests an opportunity for noschisis was studied to determine whether sup-
cell rescue. plementation with functional normal retinoschisin
protein can produce improvement in ERG func-
tion and retina morphology [94, 164]. Subsequent
15.4.9 X-linked Juvenile evaluation of the mouse model confirmed that it
Retinoschisis appropriately mimics structural features of
human X-linked juvenile retinoschisis.
X-linked juvenile retinoschisis (XLJR) is a bilat- Replacements of the deficient protein through the
eral maculopathy with onset in the first decade of use of a neomycin resistance cassette or through
life. XLJR is inherited in X-linked recessive pat- the use of an AAV vector were both successful,
tern. The prevalence of XLJR is estimated to be suggesting that, with additional study, gene ther-
1 in 5000 to 25,000 men worldwide [140]. apy could become a viable strategy for therapeu-
Presentation is usually between the ages of 5 and tic intervention [68].
10 years; affected males generally present with Other researchers showed that older mice had
reading difficulties. Visual acuity deteriorates significantly reduced benefit from AAV Rs1h
during the first two decades and may remain sta- cDNA (the mouse ortholog of human RS1) gene
ble until the fifth–sixth decades when further transfer compared to younger mice that had res-
deterioration occurs due to progressive maculop- cue of retinal structure and function. These ben-
athy [6]. Clinically, areas of schisis (splitting of efits along with retinoschisin expression persisted
the nerve fiber layer) in the macula giving the for over 15 months. Other studies showed that
impression of a spoke wheel pattern may be vis- intravitreal injection of a rAAV8 vector contain-
ible. Schisis of the peripheral retina, predomi- ing the mouse Rs1h cDNA under the control of a
nantly inferotemporally, in approximately 50% human retinoschisin promoter in Rs1h knockout
of individuals has also been described [34]. mice yielded strong retinoschisin expression and
XLJR progresses to retinal detachment in an structural and functional improvements. Wild-
estimated 5–22% of affected individuals. Retinal type retinoschisin delivered and expressed in
detachment can occur in infants with severe reti- human retinal culture cells has been shown to
noschisis. About 4–40% of individuals with undergo protein folding, subunit assembly, and
XLJR develop vitreous hemorrhage. The diagno- secretion mostly independent of endogenously
sis of XLJR is based on fundus findings, electro- expressed, defective retinoschisin protein. This
physiologic testing, and molecular genetic suggests that gene therapy could be possible for
158 S. Mohan et al.
affected individuals who have residual, patho- inal description of Norrie disease were from
genic RS1 protein expression [99]. Scandinavia [118].
Byrne et al. [19] explained that cell targeting The NDP gene encodes protein Norrin of the
and appropriate vector choice are very important Wnt cascade which is essential for the specializa-
to the success of retinal gene therapy. This group tion of retinal cells for their unique sensory capa-
demonstrated that different cell types were able bilities. It is also involved in the establishment of
to secrete retinoschisin, transporting the protein blood supply to the tissues of retina and the inner
across the retina. Photoreceptor cell secretion of ear and the development of other body systems.
this protein produced the best long-term rescue. In order to initiate the Wnt cascade, norrin must
Another therapeutic approach involves in vivo- bind to another protein called frizzled-4.
directed evolution of AAV variants to deliver the Alteration in the norrin protein interferes with its
wild-type gene to the outer retina after injection ability to bind to frizzled-4, resulting in the signs
to the vitreous humor of the eye. The authors sug- and symptoms of Norrie disease [25, 79, 154].
gested that this has the potential to be a broadly Clinically ND is characterized by bilateral
applicable gene delivery method for inherited grayish yellow elevated mass (pseudogliomas)
retinal diseases [28]. secondary to peripheral avascular retina, neovas-
Possible drawbacks of using viral vectors cular membranes, and tractional retinal detach-
(e.g., the risks of oncogenicity, immunogenicity, ments [146]. A child with ND unanimously
and the possible persistence of such vectors in the presents with congenital blindness, and majority
brain after intravitreal injection) have triggered of them develop sensorineural hearing loss [108].
an interest in non-viral systems. A combination Almost half of the males with ND have develop-
of solid lipid nanoparticles, dextran, and prot- mental delay/intellectual disability or behavioral
amine as well as EGFP and RS1 plasmids has abnormalities. Though the general health is nor-
been used to develop non-viral vectors for mal, the life span may be shortened secondary to
X-linked juvenile retinoschisis treatment. risks associated with intellectual disability, blind-
Researchers studied the in vitro transfection ness, and/or hearing loss, such as increased risk
capacity, cellular uptake, and intracellular traf- of trauma, aspiration pneumonia, and complica-
ficking of these vectors in ARPE-19 cells. In vivo tions of seizure disorder [146]. The diagnosis of
intravitreal, subretinal, and topical forms of vec- ND is based on the combination of clinical find-
tor administration in Wistar rat eyes were also ings and molecular genetic testing of NDP.
evaluated. EGFP expression in various cell types
depended on the administration route. This work
suggests that these non-viral vectors may be use- 15.4.11 Refsum Disease
ful in treating X-linked juvenile retinoschisis,
other degenerative retinal diseases, and ocular Refsum disease is an inborn error of lipid metab-
surface diseases as well [30]. olism characterized by a tetrad of retinitis pig-
mentosa, peripheral neuropathy, cerebellar
ataxia, and elevated protein levels in the cerebro-
15.4.10 Norrie Disease spinal fluid (CSF) without an increase in the
number of cells. It is inherited in an autosomal
Norrie disease (ND) is an XL recessive neuro- recessive pattern. The prevalence of Refsum dis-
logical syndrome in which affected males are ease is unknown, although the condition is
blind at birth or early infancy. It is caused by thought to be uncommon.
mutations in the NDP gene. ND is a rare disorder; Refsum disease is characterized by anosmia
its exact incidence is unknown [151]. It has been and early-onset retinitis pigmentosa, both being
reported in all ethnic groups; although no ethnic universal findings with variable combinations of
group appears to predominate, most of the indi- neuropathy, deafness, ataxia, and ichthyosis
viduals reported in the first decades after the orig- [131]. Onset of symptoms ranges from age
7 months to over 50 years. However, due to 15.5 Syndromic RP

insidious onset, it is difficult to know exactly
when the symptoms first started [131]. In gen- Sometimes RP occurs as part of syndromes in
eral, individuals with retinitis pigmentosa due to which other organs are affected. These conditions
Refsum disease keep some visual function until are called syndromic RP. Some of the syndromic
late in life although with severely concentrically RP are Usher syndrome, Bardet-Biedl syndrome
constricted visual fields [119]. Early-onset dis- (BBS), Refsum syndrome, Alstrom syndrome,
ease is not essentially associated with a poor etc.
prognosis for life span. Cardiac arrhythmia and
heart failure secondary to cardiomyopathy are
severe health problems developing later in life. 15.5.1 Usher Syndrome
It should be noted that the full assemblage of
signs and symptoms is rarely seen in an affected Usher syndrome is an inherited condition that is
individual, and most of these features develop characterized by hearing loss and progressive
with age. loss of vision due to retinitis pigmentosa and, in
The diagnosis of Refsum disease requires some cases, due to cataract. Majority of patients
analysis of phytanic acid concentration in retain some central vision throughout their life-
plasma or serum followed by either molecular time [126]. Three types of Usher syndrome have
genetic testing or enzyme analysis. When pres- been identified depending upon the severity of
ent, normal plasma phytanic acid levels essen- the disease and based on age of onset [62]. Type
tially rule out the Refsum disease. More than I, which is further subdivided into seven sub-
90% of all cases of Refsum disease result from types, is defined by complete deafness at birth or
mutations in the PHYH gene that causes defi- by the first year of life. Progressive vision loss
ciency of phytanoyl-CoA hydroxylase; the rest and delayed sitting and walking due to problems
are caused by mutations in a PEX7 gene that with inner ear and thereby with balance are rec-
lead to deficiency of the PTS2 receptor. Milder ognizable in childhood. While type II is identi-
phenotypes caused by pathogenic variants in fied through hearing loss from birth and visual
PEX7 have also been reported [141]. Mutations impairment in adolescence or adulthood, type III
in either the PHYH or PEX7 gene disrupt the is characterized by onset of clinical symptoms in
usual functions of peroxisomes, including the adulthood.
breakdown of phytanic acid, accumulation of About 3–6% of all childhood deafness and
which is toxic to cells, although it is unclear 50% of adult deaf-blindness are caused by Usher
how an excess of this substance affects vision syndrome [126]. The estimated incidence of type
and smell and causes other specific features of I is about 4 in 100,000, while type II, rate of inci-
Refsum disease. dence unknown, is the most predominant form of
the disorder. Type III accounts to only a small
15.4.11.1 Therapies percentage of Usher syndrome except Finnish
Under Investigation population [110]. Usher syndrome is an autoso-
At present, the potential of enzyme replacement mal recessive disorder. Genes responsible for
therapy (ERT) similar to that for lysosomal stor- causing the disorder have been identified through
age diseases (e.g., Hurler syndrome, Fabry dis- linkage studies on families with affected individ-
ease, and Gaucher disease) is under investigation. uals. Nearly 16 chromosomal locations and 13
This may eventually replace dietary restrictions genes have been recognized to cause Usher
and plasma- or lipapheresis. In the long run, gene syndrome.
therapy may be the treatment of choice, but many It is understood that Usher syndrome resem-
issues need to be resolved before it can be bles cytoskeletal abnormalities, having a pheno-
applied. type that occurs due to defective organization of
microtubules that affects the stability and f unction
160 S. Mohan et al.
of photoreceptors and sensory cells in the inner At least two gene therapy trials, aimed at
ear. Mutations in MYO7A, CDH23, PCDH15, restoring vision in patients, are underway: one on
USH1C, USH1G, and CIB2 genes are known to patients suffering from Usher syndrome type IB
cause Usher syndrome type I (Keats), out of carrying MYO7A mutation and the other on those
MYO7A gene has been reported predominantly in suffering from type II carrying a mutation in
Usher syndrome type IB. Several homozygous USH2A gene. While hearing loss can be treated
missense and few deletion mutations have been to an extent by cochlear implant, the restoration
identified in this gene. Experiments in mice mod- of vision largely depends on the successful out-
els have shown the interaction of harmonin b, come of the trials that would inspire trials involv-
CDH23, and MYO7A genes which bring about ing other gene mutations as well.
proper shaping of hair bundles and cohesion of
stereocilia [13]. About 35–39% of mutations
causing Usher syndrome are observed in MYO7A 15.5.2 Bardet-Biedl syndrome (BBS)
and CDH23 genes, and the R666X and IVS27-
1G-C mutations constitute about 38% of muta- BBS is a pleiotropic disorder characterized by
tions reported in that locus [107]. Riazuddin et al. clinical symptoms such as progressive RP, poly-
[117] reported about 17 homozygous mutations dactyly, obesity, hypogonadism, genital anoma-
in 23 Pakistani families. lies, cognitive disabilities, and renal conditions
Studies suggest that Usher syndrome types I [82]. Other minor features include delayed
and II follow similar pathogenic mechanism, in speech and development and dental anomalies.
terms of the organization of hair bundles and Genetically heterogeneous in nature, Bardet-
their adhesion. A scaffold protein harmonin, Biedl Syndrome (BBS) was first described by
USH2A, VLGR1, and NBC3 genes are found to Lawrence and Moon when they reported a family
express in both photoreceptor and inner ear hair with four siblings who presented with retinal
cells, and it is believed that the pathogenesis of dystrophy, obesity, spastic paraparesis, and men-
type II is brought about by the interaction tal retardation. Bardet and Biedl later described
between these molecules. Mutations in GPR98 an additional phenotype associated with the syn-
and USH2A genes have been associated with drome, namely, polydactyly, and the term
Usher syndrome type II. More than 200 muta- Lawrence Moon Bardet-Biedl syndrome came
tions have been identified in Usher syndrome into existence. Several reports that followed sug-
type IIA. However a few mutations are known to gested overlapping of the phenotypes, and cur-
cause only retinitis pigmentosa and not hearing rently it is known by the standard term BBS. It is
loss, reason for which remains to be determined. a disorder characterized by ciliopathy in which
Majority of the mutations detected are thought to eight proteins that assemble to form a BBSome
result in shorter or no functional usherin protein; complex involved in signal trafficking in cilia are
the mechanism underlying the pathogenesis is implicated. BBS is an autosomal recessive disor-
still unknown [2, 123]. Type III is the least com- der, and about 22 genes have been reported in
mon of the types of the syndrome recognized so BBS till now, namely, BBS1, BBS2, BBS4, BBS5,
far, except for Finnish population in which it BBS7, BBS9, BBS10, BBS12, ARL6, BBIP1,
accounts for about 40% of all cases of Usher syn- CEP290, IFT172, IFT27, INPP5E, KCNJ13,
drome. The clinical symptoms start in late child- LZTFL1, MKKS, MKS1, NPHP1, SDCCAG8,
hood and with progression leading to profound TRIM32, and TTC8. While BBS genes accounts
impairment by middle age. Mutations in CLRN1 for about 70–80% of the mutations reported in
gene have been implicated in type III, among BBS so far, BBS1 gene is considered to contrib-
which the Tyr176Ter and Met120Lys mutations ute predominantly with about 40% of all the
are common. The mechanism by the dysfunc- mutations reported.
tional clarin 1 protein, which is thought to be the Being an autosomal recessive disorder, homo-
result of mutated gene, is not yet understood. zygous mutations are commonly found. However
rarely triallelism is observed in BBS. Katsanis Chlamydomonas reinhardtii, Volvox carteri, and
et al. [61] reported a BBS family in which two Natronomonas pharaonis, respectively, are used
unaffected siblings carried a homozygous BBS1 to stimulate the retina, paving way for preclinical
gene mutation, while affected sibling carried an studies soon [41]. Gene therapy is a good option
additional heterozygous mutation in BBS1 or in recessive diseases, whereas in dominant disor-
BBS6 gene. Epistatic modifier effects have also ders, it may not work; hence, alternatively siRNA
been observed in BBS. Patients who carried het- and microRNA pathways are explored, including
erozygous mutations in BBS2 and BBS6 genes in using neurotrophic factors to stabilize the photo-
addition to BBS1 homozygous mutation have receptor degeneration [42].
been observed to present severe and early-onset
phenotypes compared to those who had only 15.5.2.1 Bardet-Biedl Syndrome
BBS1 homozygous mutations [7]. Ciliopathy Genetic Studies in India
genes such as MKS1, MKS3, and CEP290 genes There is only one report on molecular genetics of
have also been reported to exert epistatic effects Bardet-Biedl syndrome from India. A study on
on BBS gene mutations [161]. Although BBS1 30 families identified disease causative mutations
gene mutations predominate the overall muta- in 24 (80%) of them, 22 in BBS candidate genes,
tions reported in BBS, they are also associated and 2 in other ciliopathy gene, ALMS1. Of the
with milder phenotypes when compared to those identified variations, mutation in BBS3 gene
reported in other BBS genes. Among the BBS including the novel recurrent mutation (p.I91T)
genes, BBS1, BBS2, BBS3, and BBS4 are fre- accounted for 18% [124].
quently associated with ocular and digital pheno-
types seen in BBS. Advanced technologies such
as homozygosity mapping, exome sequencing, 15.6 Conclusions
and next-generation sequencing have helped in
the molecular analysis of mutations in the clini- Gene mapping and characterization of genes
cally and genetically highly heterogenic BBS causing inherited or familial ocular genetic disor-
phenotype. Although the impact of molecular ders has been a priority and focus area in fighting
diagnosis on therapy needs to be explored yet, blindness initiative. Extensive studies have been
their role in preventive and prenatal diagnosis is carried out in the western countries like the USA,
of importance. Canada, the UK and Europe; however, there is
To understand hereditary retinal dystrophies, need for more work to be carried out in countries
extensive research has been attempted in animal in South Asia including India. Even in the whole
sciences to characterize clinical, functional, struc- of Asia, including Hong Kong, Singapore, Japan,
tural, and molecular genetic studies in feline to China, and South Asia, all put together cannot
create interventional strategies for genes like CRX match with the quantity and quality of work the
and CEP290 [102]. Interventional strategies in West has done in ocular genetic studies, even
inherited retinal degenerative diseases have though two-thirds of the population live in Asia
moved from huge barriers to prospective experi- with these diseases. There is a growing need for
mental successes through gene therapy, alterna- Asia and the West to network and perform more
tive pharmacological approaches, neuroprotection, studies to identify all the genes that cause various
optogenetics, and cellular therapy [120]. ocular disorders which are genetic in nature both
Therapeutic animal models for RP have shown Mendelian and complex disorders.
that rhodopsin gene therapy-based augmentation
and supplementation plus suppression are needed Compliance with Ethical Requirements The paper is
for the survival of the rods [78]. Interestingly, in not a original work but a compilation of latest updates in
the concerned field. Therefore, does not need ethical
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Unique Patient Populations in Asia
for Genetic Eye Research 16
Himshikha Bhutani, Neel Kamal Sharma,
and Akshay Anand
Abstract unique patient data set and needs elaborate

DNA level similarities within the human race and well divided study. Area wise description
may have far-reaching consequences, but of patient population in Asia for genetic eye
environment is a major factor determining the research has been provided in the following
final outcome of genetics. It is therefore chapter.
mostly agreed that individual population study
should be an integral part of any major Keywords
research programme. Asia is the most popu- Population · Genetics · Asia · Eye diseases ·
lated continent on Earth, and with the immense Polymorphism
diversity that it holds, it is essential to identify
unique patient populations for efficient genetic
eye research. Consanguineous marriages are
common in many Asian countries, and their 16.1 Introduction
consequences hold prevalence in the field of
medical genetics. Novel ocular pathways have It is mostly agreed that modern humans consti-
been suggested by ocular genetic conditions tute a population which is quite young in its age,
thus far unique to the region. Population- but the ‘origin’ of the modern human being
specific genetic intervention is important for remains a debatable issue. The scientific commu-
better diagnosis and management of eye dis- nity is divided between supporters of the ‘multi-
eases. Each population is accompanied by a regional’ hypotheses on one side and those
supporting the ‘Garden of Eden’ hypotheses on
the other side [1]. With the advent of modern
H. Bhutani genetic approaches, attention has been diverted
Alumna, Neuroscience Research Laboratory, towards extensive DNA level similarities among
Department of Neurology, Postgraduate Institute of
Medical Education and Research, Chandigarh, India the human race, but major distinguishing features
among groups like Asians, Australian aborigines
N. K. Sharma
Armed Forces Radiobiology Research Institute, and Europeans point towards uniqueness and
Uniformed Services University of the Health may be brought about by environmental factors,
Sciences, Bethesda, MD, USA which clearly demands individual population
A. Anand (*) study [2].
Neuroscience Research Laboratory, Department of Studies in ocular genetics opened the path to
Neurology, Postgraduate Institute of Medical gene discovery on human chromosomes [3, 4].
Education and Research, Chandigarh, India

https://doi.org/10.1007/978-981-13-0884-0_16
170 H. Bhutani et al.
Currently, the burden of genetic diseases is on a both Mendelian and complex traits [11]. In the
rise, and it has been pointed out that routine oph- case of eye diseases, Mendelian inheritance pat-
thalmology clinics may not be sufficient to deal tern is normally followed by ocular conditions
with this increasing burden of patients in need of that have an early onset, whereas conditions
simultaneous counselling and treatment [5]. appearing in adult life exhibit complex inheri-
Advances in molecular genetics have made diag- tance patterns. Data generated by an increasing
nosis and prevention of certain eye diseases an number of studies is promoting genetic counsel-
accomplished reality [6]. Since heredity is finally ling prior to arranged marriages and prenatal test-
a product of interaction between genetic and ing of disease causing mutations. At the same
environmental factors, so the differences encoun- time, as ocular genetics advances, ethical issues
tered at regional levels may stem from social associated with such predictive testing are also
practices pertaining to specific populations with cropping up [12].
each affecting evolution in its own way. Patient Researchers have highlighted the need to
populations are thus structured and distributed in study the evolutionary history of genetically
their own unique manner. diverse populations like those in Asian countries
before designing further investigations for health
and disease conditions [13]. The vision 2020 pro-
16.2 T
he Genetic Diversity gramme has managed to achieve impressive tar-
of Asian Population gets in combating major eye diseases that result
in blindness [14], but such initiatives could reach
Asia is the largest continent on Earth and almost their maximum potential if supported by
equally diverse. Cultural differences among population-specific genetic intervention for bet-
Asians play a vital role in influencing gene flow ter diagnosis and management of eye diseases.
which may be due to caste restrictions on mar-
riages. An example of this is a study conducted in
Southern India, wherein the researchers studied 16.3 G
enetic Eye Research
maternal and paternal lineages using the mito- and Asian Populations
chondrial genome (mtDNA) and Y-chromosomal
DNA, respectively, and confirmed that females in Genetic eye research in complex eye diseases has
this population have a greater intercaste mobility recognized several susceptible genes, but result
as compared to men. It was found that the reproducibility becomes dependent on the popu-
Y-chromosomal data did not correspond to social lation at hand. It is thus important to identify
rank differences between castes, whereas mtDNA unique populations especially because of ances-
distances did [7]. The control region of mtDNA try which also plays a major role in determining
has also been studied in the Sindh region of risk factors associated with diseases [15]. The
Pakistan so as to generate data for population importance of risk factors in eye diseases may be
genetic epidemiological investigations [8]. The judged from an example of a study conducted on
non-recombining feature of mtDNA and data pooled from across the major continents of
Y-chromosomal DNA has helped to establish the world, so as to assess the prevalence of retinal
sound evidence in support of a north-south divide vein occlusion [RVO]. Hypertension is a risk fac-
within the East Asian population [9]. tor associated with RVO, and since Asian and
Founder events pertaining to specific popula- Hispanic populations have higher recorded cases
tions play complex roles during the course of of hypertension, consequently these two popula-
evolution and may result in higher rates of reces- tions also reported a higher prevalence of branch
sive diseases in these populations, for example, retinal vein occlusion (BRVO) [16].
the ‘Finnish founder mutations’ [10]. Such events Starting from the colour of the eye, genes such
may also create population isolates which usually as OCA2 (oculocutaneous albinism II gene) and
turn out to be excellent candidates for mapping HERC2 (HECT and RLD domain containing E3
16 Unique Patient Populations in Asia for Genetic Eye Research 171
ubiquitin protein ligase 2) have been studied with disease shows a 100% penetrance in Turkish and
respect to country of origin to reveal that these Slovakia populations (possibly due to certain
genes face multiple selection pressures [17]. founder effects), whereas this penetrance rate is
Ulivi et al. conducted a study to identify the lesser for Saudi Arabian population [26].
genetics of eye colour across the Silk Road and Defining the age and racial categorization of a
they indicated towards a probability of genetic population is of utmost importance before dis-
polymorphisms contributing to the phenotype in cussing any results of such studies. As pointed
a manner specific to each population, by interact- out by Al-Mansouri et al., their study yielded a
ing with OCA2 and HERC2 genes [18]. Even if percentage prevalence of glaucoma at 1.73% in a
one considers the morphology of the eye, then Qatar population aged 40 years and above [27].
comparisons within the broader Asian population This figure is low compared to an Oman-based
are further followed by interethnic differences so population study where the authors observed a
much so that direct transferability of results is prevalence of 4.75%. It is to be noted here that
limited and clinical procedures have to be planned the age of population in the latter case was
accordingly [19]. 30 years and above. Also, Oman hosts people of
Wong et al. have pointed out the lack of pre- African origin, which might account for the
cise and standardized Asian data required for a higher prevalence [28].
meta-analysis to be conducted in the area of eye Achromatopsia is an autosomal recessive
diseases [20]. A lot of data, linking DNA disorder, and it mostly occurs due to mutations in
sequence variation to diseases, is available from any of these three genes: CNGA3 (cyclic
European studies, but it may lose its validity if nucleotide-gated channel, alpha-3), CNGB3
applied in a parallel manner to Asian populations (cyclic nucleotide-gated channel, beta-3) and
without further investigations [21, 22]. GNAT2 (guanine nucleotide-binding protein,
alpha-transducing activity polypeptide 2). In
CNGA3 gene, two mutations (Arg283Trp and
16.4 Population Division Gly397Val) were found to be associated with this
disease in two United Arab Emirates-based
16.4.1 West Asians families. Large family sizes and endogamy are
important factors taken into consideration for
Buphthalmos or primary congenital glaucoma genetic eye research in these populations [29].
(PCG) is highly prevalent in Saudi Arabia, and
since it follows an autosomal recessive pattern of
inheritance, so a high frequency of occurrence is 16.4.2 East Asians
usually reported where consanguineous relation-
ships are common. Mutations in genes coding for East Asia is currently facing an epidemic with
cytochrome P450, family 1, subfamily B, poly- ever increasing cases of short-sightedness or
peptide 1 (CYP1B1) and latent-transforming myopia [30]. Studies examining the effect of
growth factor beta-binding protein 2 (LTBP2) are genes and environmental factors on the develop-
most extensively studied in relation to this dis- ment of refractive error are extensively available.
ease [23]. In the year 1997, Stoilov et al. first A single nucleotide polymorphism (SNP) in the
reported three different mutations in the CYP1B1 CTNND2 (catenin delta 2) gene has been found
gene linked to PCG in a Turkish population to be associated with high myopia in Chinese
(transcontinental Eurasia) study [24]. The dis- and Japanese cohorts in Singapore [31].
tribution of high variable CYP1B1 mutations Hammond et al., in a study conducted on a British
stands at 90–100% in Saudi Arabia and Gypsy female twin population, have underlined the
population of Slovakia (Europe). In Saudi Arabia importance of interpreting the results of such
alone, 96% cases are linked to mutations in the studies in a population-specific manner. So even
CYP1B1 gene [23, 25]. It may be noted that the though their results depict an 85% heritability of
spherical equivalence with genetic effects Many loci have been identified to be playing a
accounting for maximum population variance in role in placing specific ethnic groups at a higher
a manner parallel to Finnish and Chinese twin risk to develop the disease. A meta-analysis by
studies nevertheless, environmental changes in a Fan et al. utilized the previously identified 34
specific population hold strong at all times [32]. AMD loci [from the International AMD
One such change could be urbanization of popu- Genomics Consortium] to narrow down to 8 loci
lations. For example, a study conducted by Pan specifically associated with PCV. The authors
et al. on a multi-ethnic Asian population within also found a very high genetic correlation
Singapore indicated a higher prevalence of myo- between PCV and typical neovascular AMD
pia in the younger generation as compared to (tAMD), and certain risk alleles showed up more
adults among all the three cohorts studied. This frequently in Asians as compared to Europeans
may be due to extreme changes in socio- [37].
environmental variables which accompany Primary angle-closure glaucoma (PAC) is
urbanization [33]. Similarly, a study conducted quite prevalent among East Asians. Anterior
on a Japanese adult population from Tajimi city chamber depth (ACD) is a risk factor, but a single
revealed a lower prevalence of blindness and low risk factor can never justify this high prevalence
vision as compared to data from other parts of the [38]. For primary congenital glaucoma (PCG),
world. The authors reasoned that this may be due corresponding to data outlined above for West
to increasing public awareness coupled with a Asians, the distribution of high variable CYP1B1
sound medical cover available to citizens irre- mutations stands at 15–20% among Japanese and
spective of their economic background [34]. The Chinese populations [25].
impact of environment is so strong that it has The OCA2 gene, mentioned in a previous sec-
been suggested that the prevalence of myopia tion in relation with eye pigmentation, was stud-
among children depends less on their genetic lin- ied by Murray et al. in East Asian populations,
eage and more on the environment they grow up and their work revealed that out of the two known
in [35]. Environment being a very broad concept OCA2 polymorphisms, the rs74653330 allele
needs geographical considerations so as to nar- showed up almost exclusively in northern parts of
row down certain parameters, and two studies East Asia, whereas the rs1800414 allele showed
that deserve special mention within East Asia are up frequently throughout the broader region of
the SiMES (Singapore Malay Eye Study) and the East Asia [39]. Overall, there is a need to estab-
TPS (Tanjong Pagar Survey). Both these studies lish a database that stores ethnic-specific genetic
were based on data collected from subjects aged information for eye diseases because disease-
40–79 years of age within Singapore [20]. While associated mutations and even the phenotypic
on one hand, the SiMES studied the Malay popu- expressions of such genes may show ethnic varia-
lation of Singapore, on the other hand, the TPS tions [40].
studied the Chinese population of Singapore. The
foundation behind conducting separate studies
within the same geographical region highlights 16.4.3 South Asians
the importance of ethnic differences while study-
ing eye diseases [36]. Ethnicity regulates the The untapped potential of gene mapping for
socio-environmental backdrop, which in turn health benefits in South Asian populations has
decides the outcome of such studies. been highlighted in a study by Nakatsuka et al.
Age-related macular degeneration (AMD) This study moves beyond the usual domain of
is a heritable disorder, and the elderly population consanguineous marriages of close relatives
is very likely to be affected by it. Polypoidal resulting in recessive diseases and instead focuses
choroidal vasculopathy (PCV) is a subtype of on founder events in a way parallel to founder
exudative (Wet) AMD, and East Asians show a groups recognized in European studies. In this
high predisposition towards this eye disease. way even non-consanguineous marriages get
included in the screening process for recessive dominant in deciding the course of AMD as com-
disease-associated mutations resulting from pared to environmental factors [44].
recent shared ancestors [41]. A study of CNGA3 and CNGB3 genes men-
The Indian Genome Variation Consortium tioned above for West Asians with respect to
sought to address, among many other questions, achromatopsia, revealed two unique mutations in
the issue of association between HapMap popula- two separate Pakistani families. These two
tions and Indian populations so as to better identify mutations were firstly, a missense mutation in
populations ‘at-risk’ of certain diseases. Though CNGA3 gene which leads to replacement of a
the study acknowledged a correspondence between serine residue for an arginine residue (p.R274S)
these two populations, but it also documented vari- in the final protein product and secondly, a frame
ous levels of genetic combinations based on ethnic- shift mutation in CNGB3 resulting in premature
ity. The rs1056827 allele of CYP1B1 gene, termination of the protein (p.V609WfsX9) [45].
discussed above for West and East Asian popula-
tions in relation with PCG, was identified as one of
the 12 base SNPs valuable for categorizing popula- 16.5 Summary
tions of unidentified ethnicity as either large popu-
lations that are primarily caste based or tribal Even as the controversy surrounding the exact ori-
isolated populations with 100% precision. Within gin of modern human beings continues, the need
the entire geographical boundary of India, this to study individual human populations keeps get-
allele showed an observed frequency ranging from ting stronger. Genetic eye research has been piv-
0.06 to 0.64 with central and east regions account- otal in leading towards breakthrough discoveries
ing for lowest frequency and western and south- in the field of genomics. These discoveries can
eastern borders showing a very high frequency positively impact patient populations if each of
[42]. This gradual build-up of allele frequency dif- the data set is matched to ethnic and ancestral his-
ferences is important in structuring populations. It tory of specific populations. Asia is multi-cultural
has been noted that most alleles are pervasive in and multi-ethnic in its societal background, and
nature; therefore, genetic variation encountered in most of the people conform to a much conserved
human populations are actually a result of varying system of marriages. With such practices and also
degrees of frequencies of these alleles and not an a high degree of recent shared ancestors, the
isolated manifestation [43]. genetic makeup of an average Asian may predis-
A study conducted by Gemmy Cheung et al. pose the individual to various eye diseases. In
to compare a central rural Indian population order to tackle with the oncoming burden of such
with an urban Indian population residing in diseases, ophthalmology clinics need genetic
Singapore revealed an interesting outcome in counselling units equipped with sound knowledge
AMD patients. Considering the vast difference of the unique patient populations at hand. We
between the environments encountered by these have thus attempted to describe patient population
two populations, a corresponding difference in area wise, keeping genetic eye research at the
disease prevalence was not obtained. It was thus forefront. Table 16.1 summarizes the information
concluded that genetic factors may be more pre- with regard to major populations discussed.
Table 16.1 Summarized representation of major populations discussed

Region Eye disease Associated gene Population studied
West Asia PCG CYP1B1 Saudi Arabian
Achromatopsia CNGA3 and CNGB3 UAE
East Asia Myopia CTNND2 Chinese and Japanese [in Singapore]
South Asia Achromatopsia CNGA3 and CNGB3 Pakistani
PCG CYP1B1 Indian
Compliance with Ethical Requirements Himshikha lence of retinal vein occlusion: pooled data from pop-
Bhutani, Neel Kamal Sharma and Akshay Anand declare ulation studies from the United States, Europe, Asia,
that they have no conflict of interest. and Australia. Ophthalmology. 2010;117(2):313–9.
No human or animal studies were performed by the 17. Sturm RA, Duffy DL. Human pigmentation genes
authors for this book chapter. under environmental selection. Genome Biol.
2012;13(9):248.
18. Ulivi S, Mezzavilla M, Gasparini P. Genetics of eye
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Retina Genes in Chinese
17
Jingna He, Wai Kit Chu, Li Ma, Calvin C. P. Pang,
and Guy L. J. Chen
Abstract ing age-related macular degeneration, polypoi-

Retina is a multilayered structure containing dal choroidal vasculopathy diabetic retinopathy,
several different cell types crucial for visual retinitis pigmentosa, Best vitelliform macular
functions. Light signals are converted into dystrophy, and Stargardt disease.
electrochemical signals on the retina and trans-
ferred to the brain through the optic nerve. A Keywords
dysfunctional retina leads to various retinal Retina genes · Chinese · Retinal diseases
diseases that can result in vision impairment
and even irreversible blindness. Genetic fac-
tors play important roles in retina structure and
functions. In recent decades, there are signifi- 17.1 Introduction
cant advancements in mapping retina genes
associated with retinal diseases, some are Retina is a multilayered structure containing sev-
monogenic and some multifactorial in etiol- eral different cell types. For the light to reach the
ogy. Some retinal diseases overlap in clinical rod and cone photoreceptors at the back of the
courses and even genetic constitutions, with retina, it has to pass through the ganglion cell layer
both similarities and differences in presenta- and the layers of bipolar, Müller, and horizontal
tions among ethnic populations. We have cells [1]. The outer segments of these photorecep-
investigated CFH, HTRA1, BEST1, CETP, tors contain pigments that absorb various wave-
ABCA4, RHO, RP1, CYP4V2, and other genes lengths of the light and convert the light signal into
in Chinese patients with retinal diseases includ- electrochemical signals. These electrochemical
signals are then transferred to the ganglion cells
and eventually to the brain through the optic nerve
J. He · W. K. Chu · L. Ma · C. C. P. Pang [2]. Retina plays essential roles in vision. A dys-
Department of Ophthalmology and Visual Sciences,
The Chinese University of Hong Kong, functional retinal would lead to various retinal dis-
Hong Kong, China eases, which could result in vision loss.
G. L. J. Chen (*) In recent decades, advent in high-throughput
Department of Ophthalmology and Visual Sciences, genomic technologies for large number of patient
The Chinese University of Hong Kong, samples and vigorous studies in family pedigrees
Hong Kong, China has led to identification of many retina genes that
Department of Ophthalmology and Visual Sciences, contribute directly or interactively with other fac-
Prince of Wales Hospital, Hong Kong, China tors to various retina genes. There has been sig-

https://doi.org/10.1007/978-981-13-0884-0_17
178 J. He et al.
Fig. 17.1 Mapped and identified genes in major retinal diseases
nificant advancement in understanding the vision initially in adolescence, followed by los-

contribution of both genetic and environmental ing side vision in young adulthood and central
factors (Fig. 17.1). While properties of some ret- vision at older ages [3]. Usually RP is a disease
ina genes have helped to understand disease that is confined to the eyes. But about 20–30%
mechanism and thus aid in development of effec- RP patients are associated with complex diseases
tive treatment modes, more understanding is still such as Usher syndrome and Bardet-Biedl syn-
needed to delineate pathophysiology of many drome (BBS), which associate with hearing
potentially blinding retinal diseases, especially impairment and ciliopathy, respectively [3]. The
those with complex etiology. In this chapter, the prevalence of RP is around 1 in 4000 worldwide.
genetic and environmental factors of retinal dis- More than one million people are affected by RP
eases that occur in the Chinese population will be [3]. Inheritance patterns of RP are mainly autoso-
reviewed. Some of them are monogenic diseases, mal dominant (30–40% of total cases), autosomal
and some are multifactorial in etiology. recessive (50–60%), and X-linked (5–15%) RP
[3]. In China, the prevalence of RP is very similar
to the global data, which was about 0.03% (1 in
17.2 Monogenic Retina Diseases 3784) in Eastern, mid-Southern, North-Western,
and Northern China, based on a mass screening
17.2.1 Retinitis Pigmentosa (RP) of 196,777 people [4]. In 2006, a population-
based, cross-sectional cohort study was con-
Retinitis pigmentosa (RP) is a group of heredi- ducted for 4439 study subjects aged older than
tary retinal diseases that feature degeneration of 40 years. The prevalence of RP showing
rod and cone photoreceptors [3]. In most RP pathological fundus appearances and visual func-
patients, rod cells would degenerate before the tion losses was about 1 out of 1000 elderly
cones. These patients usually lose their night Chinese in Northern China, which could be
17 Retina Genes in Chinese 179
extrapolated to around 1.3 million patients based c.768_770delCAT, p.Ile256del; c.1040C>T, p.

on the total population in that area [5]. Pro347Leu; c.310G>A, p.Val104Ile; and
RP is essentially a monogenic disorder inher- c.895G>T, p.Ala299Ser [8]. In Table 17.1, we
ited as Mendelian trait. At least 89 RP-associating summarize RHO variations in RP of Chinese
genes have been mapped (https://sph.uth.edu/ret- population-based studies.
net/sum-dis.htm#B-diseases). Rhodopsin (RHO) To date 61 gene loci have been mapped in
was identified in RP in 1990 [6]. Most RP genes autosomal recessive RP, and about 20% of reces-
are only involved in only one form of Mendelian sive RP are caused by mutations in USH2A [3].
inheritance (autosomal dominant or recessive), In the Chinese population, many mutations in
while a few genes such as NRL, RP1, and RHO USH2A have been determined (Table 17.2). They
could be inherited in both forms. Including all the would be useful for the molecular diagnosis and
mutations found to cause non-syndromic RP, disease management of Usher syndrome.
about 3100 disease-causing mutations have been Comparing with autosomal dominant and reces-
reported in the Human Gene Mutation Database sive RP, less genes have been mapped for
(HGMD). Another 1200 mutations have been X-linked RP. There are six of them: OFD1, RP2,
reported to cause Usher syndrome and BBS [7]. RPGR, RP6, RP24, and RP34. RPGR accounts
In autosomal dominant RP, 28 loci have been for 70% of X-linked RP [3]. In the Chinese popu-
identified. Mutations in most of these genes lation, several RPGR mutations have been found
account for a small proportion of RP cases except (Table 17.3).
the RHO on 3q22.1, which causes around 25% of
autosomal dominant RP [3]. A sequencing study
on all coding regions and adjacent intronic 17.2.2 Leber Congenital Amaurosis
regions of RHO in 248 Chinese probands with (LCA)
non-X-linked RP detected eight heterozygous
nucleotide changes in RHO, c.628G>T, p. Theodor Leber, a German ophthalmologist, first
Val210Phe; c.945C>G, p.Asn315Lys; c.527C>T, described Leber congenital amaurosis (LCA) in
p.Ser176Phe; c.568G>T, p.Asp190Tyr; 1896, which is now used to describe a group of
Table 17.1 Sequence variations detected in RHO of RP patients in Chinese

DNA sequence variations Amino acid changes References
c.527C > T p.Ser176Phe [8]
c.568G > T p.Asp190Tyr
c.628G > T p.Val210Phe
c.768_770delCAT p.Ile256del
c.945C > G p.Asn315Lys
c.1040C > T p.Pro347Leu
c.310G > A p.Val104Ile
c.895G > T p.Ala299Ser
c.-300_-302delTTT No amino acid change [9]
c.-201C > T No amino acid change
c.625G > A p.Val209Met
c.891C > T p.Ser297Ser
c.155C > T p.Arg21Cys [10]
c.423G > C p.Cys110Ser
c.639G > T p.Gly182Val
c.653 T > G p.Cys187Gly
c.369C > T p.Thr92Ile
c.627A > G p.Tyr178Cys
c.409_426delGTGGTGGTGTGTAAGCCC No amino acid change
180 J. He et al.
Table 17.2 Sequence variations detected in USH2A of RP patients in Chinese

c.4384delA p.Thr1462Leufs*2 [11]
IVS47 + 1G > A Splice site
c.13156A > T p.Ile4386Phe
c.8559-2A > G No amino acid change [12]
c.8272G > T p.Glu2758X [13]
c.12376_12378ACT > TAA p.Thr4126X
c.6875_6876insG p.Arg2292ArgfsX39 [14]
c.8284C > G p.Pro2762Ala [15]
c.9958G > T p.Gly3320Cys
c.14287G > C p.Gly4763Arg
c.8559-2 T > C No amino acid change
c.11235C > G p.Tyr3745X
c.2802 T > G p.Cys934Trp [16]
c.8232G > C p.Trp2744Cys
c.1876C > T p.Arg626X
c.6249delT p.Ile2084fs
c.3788G > A p.Trp1263X
c.9492_9498delTGATGAT p.Asp3165fs
c.7123delG p.Asp3165fs
c.14403C > G p.Asp3165fs
c.5200G > C p.Gly1734Arg [17]
IVS32 + 1G > A No amino acid change
Table 17.3 Sequence variations detected in RPGR of RP patients in Chinese

c.1059 + 1 G > T No amino acid change [18]
c.2002dupC p.His668PfsX4
c.2236_2237delCT p.Glu746RfsX22
c.2899delG p.Phe967LfsX121
c.2417_2418insG p.Glu806fs [19]
c.2233_34delAG p.Glu746RfsX22 [20]
c.2236_2237delGA p.Glu746RfsX22
c.2403_2404delAG p.Glu802GfsX31
c.851C > G No amino acid change
c.2260G > T No amino acid change
g.ORF15 + 556delA p.Lys184fs [21]
g.ORF15 + 483_484delGA p.Glu746ArgfsX768 [22]
g.ORF15 + 652_653delAG p.Flu802GlyfsX833
g.ORF15 + 650_653delAGAG p.Thr801ThrfsX813
retinal dystrophy causing blindness before the inherited retinal dystrophies and 20% of children
age of 1 year. LCA is a congenital disease associ- with visual impairment around the world [24]. In
ating with poor pupil responses, nystagmus, and the Chinese population, the prevalence of LCA is
a severely subnormal or undetectable full-field not known. LCA is genetically heterogeneous. To
electroretinogram (ERG) [23]. It affects 1 in date, mutations in more than 25 genes have been
33,000 newborns worldwide and 1 in 81,000 in identified in LAC patients. Only one gene, CRX,
North America. LCA represents around 5% of all involves in both autosomal dominant and reces-
sive forms. There are 3 genes inherited in autoso- juvenile-onset macular degeneration. In the sub-
mal dominant trait and 23 genes autosomal retinal and sub-RPE spaces, there are large
recessive. The three frequently mutated LCA deposits of yellow pigmented materials in BVMD
genes are CEP290, GUCY2D, and CRB1 [25]. patients, giving a typical yolk-like macular lesion
In a Han Chinese cohort, screening of variants on fundus photo. The appearance of the lesion
in 15 genes in 87 unrelated LCA patients identi- looks like egg yolk, and then it would develop
fied 35 pathogenic mutations, which 66% of all into a pseudohypopyon stage resulting in detach-
detected variants. In these Chinese patients, vari- ment of the neural retina from the RPE layer. The
ants in GUCY2D are the most common causes of deposits would become disorganized with pig-
LCA (16.1% of study patients), followed by ment accumulation, causing an increased thick-
CRB1 (11.5%), RPGRIP1 (8%), RPE65 (5.7%), ness of RPE/choroid layer. A thinner retinal layer
SPATA7 (4.6%), CEP290 (4.6%), CRX (3.4%), is also observed following the photoreceptor
LCA5 (2.3%), MERTK (2.3%), AIPL1 (1.1%), degeneration and central vision deterioration
and RDH12 (1.1%). The prevalence is different [30]. BVMD is a rare disease. Prevalence was
from other populations. Three mutations in 2 in 10,000 in Sweden, 1.5 in 100,000 in
GUCY2D gene have been detected in this study, Denmark, and between 1 in 16,500 and 1 in
c.164C>T, c.935C>T, and c.2302C>T [26]. 21,000 in a US study [31].
Variations in the GUCY2D gene causes 10–20% Mutations in the BEST1 gene, also called
of recessive LCA and up to 40% of dominant VMD2, were initially found associated with
corn dystrophy or corn-rod dystrophy [27]. BVMD. It is located on chromosome 11q13 and
GUCY2D codes for the retinal-specific guanylate encodes the BEST1 protein, mainly expressed in
cyclase 1, which localizes in the outer segment of RPE, but also in the testis, placenta, and brain.
the photoreceptors. It is involved in the photo- There are 585 amino acids in human BEST1, the
transduction recovery process for cGMP first 350 amino acids conserved in different spe-
resynthesis. cies. BEST1 works as an anion channel and a
Whole exome sequencing (WES) of 41 regulator of intracellular calcium signalling in
Chinese LCA families found 17 variants in 19 RPE [31]. To date, five retinal degeneration dis-
genes [28]. GUCY2D, CRB1, RPGRIP1, eases have reported associations with BEST1
CEP290, and CRX were the five most frequently mutations. They are Best vitelliform macular
mutated genes, consistent with results of an ear- dystrophy, autosomal recessive bestrophinopa-
lier study [26]. In another study on Chinese LCA thy, adult-onset vitelliform macular dystrophy,
families, direct Sanger sequencing of 7 genes autosomal dominant vitreoretinochoroidopathy,
(AIPL1, CRB1, CRX, GUCY2D, LRAT, RDH12, and retinitis pigmentosa. To date, 269 mutations
RPE65) in 117 LCA families identified homozy- have been reported in the BEST1 gene across dif-
gous or compound heterozygous mutations in ferent ethnicities (http://www.huge.uniregens-
107 families. Heterozygous autosomal dominant burg.de/BEST1_database/variants.
mutations were identified in three families and an php?action=search_unique&select_db=BEST1).
X-linked mutation in one family. In total, 136 In the Chinese population, we have directly
mutations were found [29]. sequenced of BEST1 in 27 subjects from 7
Chinese BVMD families and 100 unrelated
healthy Chinese subjects. Each BVMD family
17.2.3 Best Vitelliform Macular was found to have a unique mutation. We identi-
Dystrophy fied seven missense mutations (p.Thr2Asn, p.
Leu75Phe, p.Ser144Asn, p.Arg255Trp, p.
Best vitelliform macular dystrophy (BVMD) was Pro297Thr, p.Asp301Gly, p.Arg218Cys). Two of
first described by a physician Friedrich Best in these six novel mutations are located within the
1905. BVMD is commonly inherited in autoso- four common mutation clusters within the BEST1
mal dominant form. It is a progressive and gene [32].
182 J. He et al.
Five other studies screened BEST1 mutations the photoreceptor outer segment. Mutations in
in Chinese. In 13 patients of 12 unrelated Chinese ABCA4 also contribute to other retinal diseases
families affected by BVMD, 5 mutations (p.T4I, including retinitis pigmentosa and age-related
p.A291V, p.R218C, p.Q293H, and p.D301G) macular degeneration (AMD) [38].
were identified [33]. In another study direct We also identified mutations in ABCA4 gene
sequencing of BEST1 in a 10-year-old patient, his in our Hong Kong Chinese cohort. We genotyped
family members and 200 unrelated subjects 140 AMD, 18 STGD patients, and 95 normal
revealed heterozygous mutations c.292G>A (p. control subjects for 15 ABCA4 exons. In STGD,
Glu98Lys) in exon 4 and c.1608C>T (p. p. Arg2040Stop was detected in two patients but
Thr536Thr) in exon 10. These two mutations not in controls. Thus it is likely a disease-causing
were not found in any of the patient’s unaffected mutation. Another sequence alteration p.
family members or in the normal controls [34]. Thr1428Met was found in 2 STGD patients, 18
Liu et al. identified six mutations (p.Ser16Phe, AMD patients, and 15 normal people, implying
p.Ser144Asn, p.Glu292Lys, p.Glu300Lys, p. Thr1428Met could be a polymorphism [39].
Thr307Asp, p.Arg47His) from 13 BVMD indi-
viduals from 6 unrelated families [35]. Recently,
Tian et al. identified 36 disease-associating vari- 17.3 Multifactorial Retina
ants in BEST1, including 28 (77.8%) missense, 3 Diseases
(8.3%) nonsense, 4 (11.1%) splicing, and 1
(2.8%) frameshift mutations from 17 BVMD 17.3.1 Age-Related Macular
patients and 20 autosomal recessive bestrophi- Degeneration
nopathy (ARB) patients. Results of these studies
showed the spectrum of BEST1 mutations in Age-related macular degeneration (AMD) is a
Chinese patients is different from that of progressive chronic retinal disease and a leading
Caucasian patients [36]. cause of vision loss in the elderly population
worldwide [40]. There are two principal forms of
AMD, exudative AMD, also called wet AMD,
17.2.4 Stargardt Disease and geographic atrophy (GA), also referred to as
dry AMD. In exudative AMD, fibrous scarring
Stargardt disease (STGD) was first described by often happens after leaking of fluid, blood, and
Karl Stargardt in 1909, representing a set of juve- lipid from the retinal blood vessels as choroidal
nile macular degeneration diseases. STGD is neovascularization penetrates into the neural ret-
typically inherited in an autosomal recessive ina. AMD was considered untreatable until pho-
trait. The prevalence of STGD is 1 in 10,000 [37] todynamic therapy was available [40]. In 2006 a
and is characterized with yellowish flecks around landmark clinical trial showed that monthly intra-
the macula and degeneration of RPE and the neu- vitreal injections of ranibizumab, an antibody
ral retina layer. Patients with STGD are affected inhibiting a protein vascular endothelial growth
bilaterally, with gradual decline in vision between factor (VEGF), could prevent vision loss in
6 and 20 years old. These patients usually have nearly 95% of AMD patients [41, 42]. A similar
no prior history of abnormal visual acuity [37]. antibody, bevacizumab was also developed and
All recessively inherited cases of STGD are could be supplied at a lower cost with compara-
due to mutations in the photoreceptor-specific ble efficacy to ranibizumab [43]. There have been
ATP-binding cassette transporter gene ABCA4 many epidemiological studies on AMD over the
(formerly known as ABCR), which encodes the years worldwide. It is notable that the proportion
ABCR protein. ABCA4 acts as a flippase for of blindness caused by AMD in East Asian popu-
N-retinylidene-phosphatidylethanolamine (PE) lations has increased from 5% in 1990 to 6.9% in
to facilitate the transport of all-trans retinalde- 2010. It is now the third leading cause of blind-
hyde from intradiscal space into the cytoplasm of ness in East Asia [44]. By 2040, Asia is predicted
to contribute to the highest population of AMD were found, and especially CEPT p.Asp442Gly
patients in the world [45]. (rs2303790) is specific to the East Asian popula-
Strong evidence of genetic influences on the tion which is strongly associated with exudative
development of AMD has been given by familial AMD with an odd ratio of 1.70 [54].
aggregation, segregation, linkage, and twin stud- AMD is a multifactorial disease with complex
ies. Since 2005, genetic loci have been reported etiology involving genetic and environmental
to be associated with AMD as a result of GWAS. factors. In Western populations, risk factors for
The complement factor H (CFH) gene on 1q32 AMD older age are the major risk for AMD, with
was reported for dry AMD and the ARMS2/HTRA1 more than 10% of people older than 80 years hav-
locus on the 10q26 gene cluster for wet AMD ing late AMD [55]. Dark iris pigmentation, previ-
with Hong Kong Chinese as the primary study ous cataract surgery, and hyperopic refraction are
cohort [46–49]. In Chinese study subjects, we ocular risk factors for AMD [56–58]. Systemic
have found higher HTRA1 protein expression in risk factors including cigarette smoking, obesity,
human vitreous with HTRA1 rs2672598 C alleles. sunlight exposure, and cardiovascular diseases
But rs11200638 was not correlated with HTRA1 have been found in AMD. Smoking is a strong
level in human vitreous [50]. Sequencing of and most consistent risk factor for AMD [59]. In
HTRA1 revealed higher prevalence of the Asian AMD, age is also the major risk factor for
c.34delCinsTCCT allele in controls (8.0%) than Asian AMD and cigarette smoking the most con-
in nAMD patients (1.9%) [50]. In cultured retina sistent systemic risk factor [60–62]. In studies on
pigment epithelial cells (ARPE-19), the recombi- Asian populations, other demographic and sys-
nant HTRA1 c.34delCinsTCCT variant protein temic risk factors have been reported, including
was more localized in the endoplasmic reticulum male gender, hypertension, hyperlipidemia, high
of RPE cells compared with the wild-type pro- levels of high-density lipoprotein (HDL), chronic
tein, and its secretion was delayed. ARPE-19 kidney disease (CKD), hepatitis V surface anti-
cells expressing HTRA1 c.34delCinsTCCT vari- gen, liver cancer, coronary heart disease, lower
ant had higher cell viability, lower cell apoptosis, education levels, and increased serum white
and less response to anoikis, supporting its pro- blood cell counts [63]. Both genetic susceptibil-
tective role [51]. ity and environmental factors are important deter-
Associations of AMD with C2, CFB, C3, and minants to the onset and progression of AMD.
CFI in the complement pathway have also been
reported [46, 52, 53]. Several genes in the HDL
cholesterol pathway, LIPC, CETP, ABCA1, and 17.3.2 Polypoidal Choroidal
LPL, have been reported to associate with Vasculopathy (PCV)
AMD. Apolipoprotein E (ApoE) has been sug-
gested to be associated with AMD in the Dutch PCV is a vascular disease in the choroid. It occurs
population, but our study in Chinese did not show more commonly in Asian than Western popula-
association. Other genes like COL10A1, tions [64–68]. In GA, the retinal pigment epithe-
COL8A1, TIMP3, and VEGFA have also been lium (RPE), choriocapillaris, and photoreceptors
associated with AMD [40]. show cumulative atrophy, which lead to retinal
Genome-wide association study (GWAS) and degeneration. Unlike AMD, there is less epide-
exome-wide association study (EWAS) showed 9 miological data about PCV. It is difficult to obtain
out of the 21 loci in East Asia that are strongly accurate estimation of PCV prevalence based on
associated with AMD in European populations. a population-based screening with only fundus
These loci are ARMS2/HTRA1 rs10490924, CFH photos. Fluorescein angiography and indocya-
rs10737680, CETP rs3764261, ADAMTS9 nine green (ICG) staining are required to diag-
rs6795735, C2-CFB rs429608, CFI rs4698775, nose PCV [69]. Hospital- or clinic-based
TGFBR1 rs334353, APOE rs4420638, and cross-sectional studies have been conducted to
VEGFA rs943080. In addition, four other foci estimate prevalence of PCV. In Asia, 22.3%–
184 J. He et al.
61.6% prevalence has been reported, compared SNP rs10490924 and HTRA1 SNP rs11200638
to 8%–13% prevalence in Caucasian [70–73]. conferred greater risks to AMD than to PCV [81].
A systematic review on the genetic associa- In the high-density lipoprotein (HDL) metabolic
tions of PCV with 56 polymorphisms in 19 genes/ pathway, CETP is a susceptibility gene for neo-
loci [83] revealed polymorphisms in 5 genes/loci vascular AMD and PCV independent of CFH and
that are involved in the complement pathway HTRA1 [82]. According to genotype studies in
(CFH p.Try402His, rs1061170; CFH p.Ile62Val, our Chinese cohort with neovascular AMD and
rs800292; C2, rs547154; CFB, rs4151657; PCV, there are consistent associations of the
RDBP, rs3880457; and SKIV2L, rs2075702 and CFH, ARMS2/HTRA1, and CETP genes, indicat-
rs429608). Two genes/loci are related to the ing sharing of partially similar molecular mecha-
inflammatory pathway (TNFRSF10A- nisms. However, the different genetic effects
LOC389641, rs13278062 and BEST-C4orf14- from the complement pathways also show exis-
POLR2B-IGFBP7, rs1713985). Extracellular tence of additional genetic and environmental
matrix/basement membrane regulatory pathway factors affecting them to different extents [83].
(ARMS2 p.A69S rs10490924 and HTRA1 pro-
moter rs11200638) and lipid metabolism path-
way (CETP rs3764261) were also reported to be 17.3.3 Diabetic Retinopathy
associated with PCV [63].
In the Hong Kong Chinese population, we Diabetes mellitus (DM) is one of the fastest
identified specific genetic associations in AMD growing chronic diseases globally. Various com-
and PCV in Chinese. We found angiopoietin 2 plications in DM can be grouped into macrovas-
(ANGPT2) a susceptibility gene for PCV and cular and microvascular complications. Diabetic
nAMD [74]. Two haplotype-tagging SNPs retinopathy (DR) belongs to the microvascular
rs4455855 and rs13269021 in ANGPT2 associ- complication of DM and is a leading cause of
ated with PCV and nAMD in Chinese and vision loss in middle-aged working population
Japanese [74]. Angiopoietin 2 is elevated in aque- [84]. Thirty percent of patients with DM would
ous of patients with nAMD [75]. We reported the result in DR, and 5–10% of them may develop
first association of placental growth factor (PGF) into proliferative DR. Severe DR affect patients
rs2268615 and rs2268614 with exudative AMD physically and emotionally, costing heavy health-
[76]. The first exome sequencing study on PCV care-related resources [84].
utilized Chinese patients as primary cohort and There are proliferative and nonproliferative
identified a rare c.986A>G (p.Lys329Arg) vari- forms of DR according to the growth of the new
ant in the FGD6 gene that conferred risks to PCV, retinal blood vessels. In patients having mild and
but not to AMD [77]. This is the first gene identi- nonproliferative DR, if left untreated, the reti-
fied for PCV. nopathy could progress into moderate or severe
We conducted a series of genotyping studies nonproliferative DR or even to proliferative
on PCV and neovascularized AMD in the Chinese DR. In the proliferative stage, abnormal new reti-
population. Our data showed SKIV2L is a suscep- nal blood vessels would appear. In some DR
tibility gene for neovascular AMD, independent patients, diabetic macular edema (DME) may
of CFH and HTRA1, but not for PCV [78]. For also happen [85].
genes in the complement pathway, C3, SNP Recent clinical trials showed that the progres-
rs17030, is not associated with neovascular AMD sion of DR can be reduced by controlling blood
but with PCV, though only in males but not glucose level, timely laser retinal photocoagula-
females [79]. Serpin peptidase inhibitor, clade G, tion treatment, and intraocular administration of
member 1 (SERPING1) gene associated with anti-vascular endothelial growth factor (anti-
AMD or PCV in Caucasians but not in Chinese or VEGF) agents. According to World Health
Japanese, indicating ethnic diversity in the Organization (WHO), DR accounts for 4.8%
genetic etiology [80]. We have found ARMS2 (37 million people) of blindness worldwide [86].
There are approximately 93 million people with DR in association with AR -106C/T variants in
DR worldwide, 17 million with proliferative DR, Chinese Han population [97].
21 million with diabetic macular edema, and IL-6 genotype of rs1800795 GC and rs1800796
28 million with vision-threatening DR [87]. In GG might affect risk for type 2 DM patients suf-
Chinese, DR prevalence among the type 1 DM fering from proliferative DR in a Chinese study
was reported to be 14% [88]. In rural areas of [98]. Association of rs507392, rs1617640, and
China, the overall prevalence of type 2 DR has rs551238 minor alleles of erythropoietin with
been reported to be 43.1% and prevalence of pro- increased DR risk has been reported [99]. Liu
liferative DR, macular edema, and vision- et al. reported rs10946398 of CDKAL1 was inde-
threatening retinopathy 1.6%, 5.2%, and 6.3%, pendently associated with DR in Chinese Han
respectively [89]. A study in urban areas of China population [100]. Complement 5 (C5) rs2269067
found the prevalence of DR among adult diabetic GG genotype and -2518 GG genotype, G allele
Chinese was about 27.9% [90], which was much of MCP-1, and rs1927914 of TLR4 have con-
lower than that in the rural area. ferred risks for proliferative DR of type 2 DM
DR is complex in clinical manifestations and patients in Chinese Han population [101–103].
in genetics. Twin studies, family studies, candi- Long duration of diabetes, hyperglycemia,
date gene studies, linkage studies, and GWAS and hypertension are the most consistent risk fac-
had reported VEGF, receptor for advanced glyca- tors for the progression of DR. From 50 to 90%
tion end products (RAGE), endothelial nitric of patients develop DR after more than 20 years
oxide synthase (eNOS), and aldose reductase of suffering diabetes. About 1% reduction in gly-
(AR) to be involved in the pathogenesis of DM or cated hemoglobin (HbA1c) serum level is associ-
DR. ated with an approximate 35% reduction in risk
In the VEGF gene, several polymorphisms of DR development, 15–25% reduction in DR
have been reported: rs833061 (−460T/C), progression, 25% reduction in vision-threatening
rs699947 (−2578C/A), rs2010963 [(405G/C) DR, and 15% reduction in blindness. A 10 mm
and (634G/C)], and rs3025039 (+936C/T). In the Hg reduction in systolic blood pressure was
Chinese population, -460C/T was also correlated reportedly associated with an approximate
with nonproliferative DR [91]. Significant asso- 40–50% reduction in DR progression [84].
ciations of DR were reported for SNP rs699947, Cataract surgery, pregnancy, puberty, and
rs833061, rs13207351, and rs2146323 [92]. Yang nephropathy are other common risk factors for
et al. suggested that polymorphisms in the pro- the progression of DR.
moter region of the VEGF would increase the
risk of DR in Chinese patients with type 2 diabe-
tes mellitus (T2DM) [93]. 17.4 Conclusive Remarks
eNOS, RAGE, and AR are not well reported in
the Chinese population. The function of eNOS Retina plays essential roles in vision. A dysfunc-
protein is to regulate vascular tone by inhibiting tional retina leads to various retinal diseases,
smooth muscle contraction and platelet aggrega- which could result in vision loss or even irrevers-
tion. There was no significant association ible blindness. Recent advancements in genetic
between eNOS-4b/a polymorphism and DR in studies have identified retina genes. There are
patients with type 2 diabetes mellitus [94, 95]. similarities and differences in the effects of
RAGE protein regulates oxidative stress and genetic factors among different ethnic popula-
endothelial function in T2DM. The p.G82S poly- tions. In future, intensive studies in gene effects
morphism in RAGE was associated with DR in and environmental influences should be con-
one reported Chinese study [96]. AR converts ducted for understanding the mechanisms and
glucose to sorbitol in the polyol pathway. A meta- devising new treatment modes for various retinal
analysis showed significantly increased risks for diseases.
186 J. He et al.
Compliance with Ethical Requirements Jingna He, 15. Chen X, Sheng X, Liu X, Li H, Liu Y, Rong W, Ha
Wai Kit Chu, Li Ma, Calvin C.P. Pang, and Guy L.J. Chen S, Liu W, Kang X, Zhao K, Zhao C. Targeted next-
declare that they have no conflict of interest. generation sequencing reveals novel USH2A muta-
No human or animal studies were performed by the tions associated with diverse disease phenotypes:
authors for this article. implications for clinical and molecular diagnosis.
PLoS One. 2014;9(8):e105439.
16. Xu W, Dai H, Lu T, Zhang X, Dong B, Li Y. Seven
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Leber Congenital Amaurosis
in Asia 18
Sharola Dharmaraj, Anshuman Verma,
P. Sundaresan, and Chitra Kannabiran
Abstract in some Asian populations has been noted,

Leber congenital amaurosis (LCA) is a het- and gene identification using homozygosity
erogeneous infantile retinal dystrophy pre- mapping and testing for common mutations
senting with severe visual loss, nystagmus, is possible, but the prevalence of mutations is
sluggish pupillary responses and an extin- not always identical to cohorts in the Western
guished electroretinogram (ERG). LCA world. The advent of next-generation, whole
accounts for 5% of inherited retinal degener- genome and exome sequencing in addition to
ative disorders worldwide. To date at least 30 gene chip technology have revolutionised
genes are known to either cause or be associ- genetic and molecular diagnosis. Phenotype-
ated with this condition. The genes perform a genotype correlation of this disorder in some
structural or functional role in the visual instances has made the choice of laboratory
pathway. Mutations in several of these genes diagnosis rapid and easier. An accurate
causing LCA have been identified in Asian genetic diagnosis has become mandatory to
populations (AIPL1, ALMS1, CABP4, CCT2, access upcoming treatment options. Gene
CEP290, CLUAP1, CRB1, CNGA3, CRX, therapy for LCA has been encouraging
CTNNA1, CYP4V2, GDF6, GUCY2D, recently as shown in the clinical trials involv-
IFT140, IMPDH1, IQCB1, KCNJ13, LCA5, ing RPE65-related LCA both in canines and
LRAT, MERTK, MYO7A, NMNAT1, OTX2, humans.
PEX1, PNPLA6, POC1, PRPH2, RD3,
RDH12, RPE65, RPGRIP1, SPATA7 and Keywords
TULP1). An increased rate of consanguinity LCA · RPE65 · Clinical trials · Gene therapy ·
Consanguinity · Mutations · Asian ·
Heterogeneity · Genotype · Phenotype ·
Treatment · Sequencing · AIPL1 · ALMS1 ·
CABP4 · CCT2 · CEP290 · CLUAP1 · CRB1 ·
S. Dharmaraj (*)
Moorfields Eye Hospital, London, UK CNGA3 · CRX · CTNNA1 · CYP4V2 · GDF6 ·
GUCY2D · IFT140 · IMPDH1 · IQCB1 ·
A. Verma · P. Sundaresan
Aravind Medical Research Foundation, KCNJ13 · LCA5 · LRAT · MERTK · MYO7A ·
Aravind Eye Hospital, Madurai, India NMNAT1 · OTX2 · PEX1 · PNPLA6 · POC1 ·
C. Kannabiran PRPH2 · RD3 · RDH12 · RPE65 · RPGRIP1 ·
LV Prasad Eye Institute, Hyderabad, India SPATA7 · TULP1

https://doi.org/10.1007/978-981-13-0884-0_18
192 S. Dharmaraj et al.
18.1 Introduction The incidence of RP, the more common retinal

dystrophy, is 1 in 3500 live births [6]. However,
Leber congenital amaurosis is one of the most RP appears to be more common in regions with a
severe forms of retinal dystrophy presenting in high degree of consanguineous unions, for exam-
infancy or early childhood with the absence of ple, Saudi Arabia and South India [7]. Recessive
photoreceptor function [1]. LCA is a genetically gene defects may be higher in consanguineous
and clinically heterogenous disorder causing pro- populations. For example, in the south of India
found visual loss, nystagmus, poorly reactive where maternal uncle marriages and first-cousin
pupils and a markedly diminished electroretino- marriages still continue to the present day
gram (ERG) [1, 2]. Theodor Leber in 1869 first because of cultural, social or economic reasons,
described the condition, but confirmation of the the occurrence of autosomal recessive disease is
diagnosis was made easier by Adolphe increased depending on the closeness of the
Franceschetti, a Swiss ophthalmologist who genetic relationship. In inbred populations where
noted the attenuated electroretinographic individuals are related in more than one way, the
responses in patients with LCA in 1954. Theodor risk of recessive genetic disease is further
Leber even in those early days had noted its clini- increased.
cal heterogeneity. The heterogeneity of LCA had Retinal abnormalities, one of the major causes
been well documented prior to the era of molecu- of childhood blindness, account for 22% of child-
lar diagnosis [3]. However, genetic heterogeneity hood blindness in India [4, 8].
was established only in the latter part of the last The clinical history has been well documented
century. Visual acuity of <6/60 in the better eye is over the last century, and LCA accounts for a
defined as blindness [4]. Currently at least 30 proportion of blind school admissions each year
genes have been implicated in LCA, and they are in several countries in Asia. The incidence of reti-
involved in preserving photoreceptor and retinal nal blindness in a referral hospital in Bengal was
pigment epithelial structure and function. The 9.5% [9]. Most cases of LCA are transmitted in
same genes implicated in LCA in some instances an autosomal recessive fashion, but dominant
also cause other retinal disorders like retinitis forms of LCA have been described [10–12]. LCA
pigmentosa, cone-rod dystrophy and early-onset like RP is also observed in conjunction with other
retinal dystrophy. LCA is at the severe end of the systemic and syndromic disorders, like cerebellar
genetic continuum of photoreceptor dysfunction disease (Joubert syndrome), epiphyseal disease
and loss, while cone-rod dystrophy is at the (Saldino-Mainzer syndrome) and renal disease
milder end of this spectrum. In addition LCA (Senior-Løken syndrome). Decreased visual acu-
may also be associated with non-retinal condi- ity as a result of heritable retinal disease occurs in
tions. Mutations in genes causing LCA are linked 1 in 3000 people [13]. More recently the geno-
with cerebellar disease (Joubert syndrome), type of many of these syndromic forms has been
epiphyseal disease (Saldino-Mainzer syndrome), identified. Currently more than 261 retinal dis-
renal disease (Senior-Løken syndrome) and neu- ease genes have been identified (RetNet URL
ral tube defects (Meckel-Gruber syndrome). https://sph.uth.edu/retnet/).
18.2 Epidemiology 18.3 Clinical Diagnosis
Hereditary retinal disease accounts for approxi- The diagnosis of heritable retinal disease has
mately 5% of blindness worldwide, and Leber been based on the clinical features. However,
congenital amaurosis accounts for 5% of all with the knowledge of molecular information
inherited retinal dystrophies [5]. LCA accounts and greater use of imaging and electroretinogra-
for 1–3 in 100,000 live births per year [5]. phy, a more accurate diagnosis is possible.
18 Leber Congenital Amaurosis in Asia 193
Molecular genetics has brought better under- peripheral vision, motion detection and decreased
standing to the inheritance of retinal dystrophies, vision in scotopic conditions (nyctalopia). Night
the role of functional proteins and their biochem- blindness and loss of peripheral visual fields are
ical pathways. noted. Loss of cone photoreceptor cells results in
The diagnosis of heritable retinal disease is loss of central vision (acuity loss), trichromatic
possible following the elucidation of a detailed colour discrimination (dyschromatopsia), vision
family history and thorough clinical investiga- loss in photopic conditions (hemeralopia) and
tions. Examination includes not only the affected ocular discomfort under photopic conditions
individuals but also potential carriers and other (photoaversion or photophobia). In many retinal
family members who may have subtle clinical degenerations, apoptosis of the photoreceptor
findings. Family history and examination will cells leads to decreased oxygen need. This altered
elucidate the inheritance pattern. The clinical oxygen tension stimulates autoregulation in the
examination consists of assessing best corrected retinal vasculature causing attenuation of the
visual acuity, optical assessment (under cyclople- blood vessels as a secondary effect noted oph-
gia in children) with slit-lamp biomicroscopy and thalmoscopically along with optic disc pallor.
dilated fundoscopy. Other relevant investigations Patients with LCA present in early infancy
include electrophysiological (ERG, dark adapta- with wandering or roving eyes, photophobia and
tion curves and EOG) and psychophysical testing an inability to focus. Often there is a history of
(static or kinetic visual field tests) and imaging photophobia or photo attraction. Eye poking
and molecular studies. Imaging includes fundus (digito-ocular phenomena of Franceschetti) is
photography, optical coherence tomography, often noted in a large number of patients, often
scanning laser ophthalmoscopy, fundus fluores- resulting in enophthalmos due to atrophy of
cein angiography and retinal autofluorescence. It orbital fat. The entoptic phenomena induced by
is also important to perform a thorough medical eye poking may be a habitual behaviour observed
and genetic examination in an effort to identify in some patients. Digito-ocular signs noted in
other clinical features suggestive of a syndromic about 25% of patients include recurrent eye rub-
form of retinal degeneration. bing, eye pressing and eye poking and are not
LCA includes both peripheral and central reti- pathognomonic of LCA [18–20]. The presence of
nal disease leading to profound loss of vision enophthalmos or eye poking did not show an
early in life. In several inherited retinal disorders, adequate correlation with the molecular diagno-
photoreceptor cell death occurs as a result of sis. Sequelae of this habitual visual behaviour
apoptosis [14]. RP, the commonest retinal dystro- may include enophthalmos, keratoconus and
phy, is characterised by progressive cell death of ocular infections [21]. However, it is often very
rods and cones. Cone cell loss occurs when rod difficult to break this habit in children.
outer segment degenerates to more than 75% as Decreased pupillary responses to light are
observed by electroretinography [15]. Cone loss observed in most patients. The retinae on oph-
correlates spatially and temporally with rod loss thalmoscopy are either essentially normal in their
and this has been noted in pathological examina- appearance or show varying degrees of pigmen-
tion too [16, 17]. Retinal dystrophies could pres- tary changes [22, 23]. Additional ocular features
ent in early infancy, whereby the arrested may include cataracts, keratoconus and kerato-
development of the photoreceptors is followed by globus [23–25]. Keratoconus, observed in some
degeneration. Early-onset severe retinal dystro- patients, may be explained by genetic suscepti-
phy has been defined as a subset of the LCA bility or the secondary effect of the digito-ocular
spectrum. When dystrophies present in later life, phenomena [23, 26, 27].
the degeneration occurs after the photoreceptors The overall phenotype in LCA is influenced
have developed, but their survival is not sus- by pathological changes in non-retinal tissues.
tained. Loss of rod photoreceptors causes loss of The presence of cataracts, keratoconus and high
grades of refractive errors further increases the patients prolong survival. Constant monitoring of
severity of the phenotype. High hypermetropia renal function is mandatory throughout child-
and myopia have also been reported [28]. hood in these patients. Hand X-rays showing
Strabismus, too, has been a common finding and cone-shaped epiphysis may be useful in making
is often related to nystagmus and poor oculomo- the diagnosis of the Saldino-Mainzer disease. An
tor control. To be able to minimise altered head MRI study of the brain is essential to detect
position and nystagmus in some patients, surgical abnormalities of the cerebellar vermis associated
correction is undertaken. with Joubert syndrome and also to screen for
Visual acuity in this disease ranges from light other associated structural anomalies. A consul-
perception (LP) to vision 20/400, although rarely tation by a neurogenetics team and a metabolic
better visual acuity of 20/80 has been observed workup are important. The history of develop-
[23]. Relatively better vision has been noted in mental milestones, visual behaviour, psychomo-
patients with CRB1 and RPE65 mutations [29]. tor delay and social behaviour are important in
Progression of the disorder is characterised by understanding development, as LCA has been
worsening visual function, increased retinal pig- associated with both autism and psychomotor
mentary and atrophic changes, attenuation of delay. Psychomotor development is delayed in
retinal vasculature and optic nerve atrophy. This the setting of blindness due to the lack of ade-
was observed in one third of LCA patients for quate visual input which is essential for early
which the underlying gene defects were not iden- development and neurobiological processing [18,
tified [20]. Worsening is also noted in patients 30]. Neuro- and psychomotor developmental
with other ocular findings like keratoconus and delay was noted in 8.1% of patients with LCA
cataract, which add to the decreased clarity of the [31]. Clinically, it is also important to elicit a
media [23, 26]. detailed family history, draw a complete pedigree
The electroretinogram (ERG) is non-and chart ethnicity and genealogical origins. In
detectable, extinguished or markedly reduced as the light of understanding the phenotype of LCA
a result of both rod and cone photoreceptor dys- better, it is relevant to elicit the history of photo-
function. ERGs obtained in early infancy quite phobia, photo attraction and night blindness. It is
often need to be recorded again in 1 year for also important to ascertain milestones, regression
reliability. and visual behaviour in terms of stability, pro-
Since an LCA-like phenotype may co-exist gression or deterioration.
with a significant systemic disease, a measure of The differential diagnosis of LCA includes
clinical suspicion must accompany each workup peroxisomal disorders (Zellweger disease, adre-
of an infant with LCA. Relevant investigations noleukodystrophy and infantile Refsum disease),
include ERG, serum very-long-chain fatty acid neuronal ceroid lipofuscinosis (NCL) and vari-
(VLCFA) assays, abdominal ultrasonography, ous forms of chorioretinal dystrophy (i.e. con-
MRI (magnetic resonance imaging) and develop- genital stationary night blindness, achromatopsia,
mental assessment. VLCFAs are raised in peroxi- juvenile RP, albinism and high myopia). Albinism
somal disease, which may mimic the retinal is the most common misdiagnosis [32]. Cortical
phenotype of LCA along with liver, spleen and blindness, delay in visual maturation and optic
central nervous system involvement. The pres- nerve hypoplasia may also be mistakenly diag-
ence of cystic changes in the kidneys or liver on nosed as LCA since all these disorders are associ-
ultrasonography is indicative of Senior-Løken or ated with severe visual loss. In peroxisomal
Arima syndrome, respectively. When renal dis- disease, retinal dysfunction is characterised by
ease is identified in Senior-Løken syndrome, markedly attenuated photopic and scotopic ERGs
morbidity increases; hence it is important to and is associated with changes in the central ner-
screen patients ultrasonographically and bio- vous system. Peroxisomes are ubiquitous cell
chemically. Early renal transplants in these organelles involved in lipid metabolism. Peroxins
encoded by the PEX family of genes are respon- Renal dysplasia, pigmentary retinal dystro-
sible for various aspects of assembly and biogen- phy, cerebellar ataxia and skeletal dysplasia con-
esis of peroxisomes. Peroxisomes contain stitute syndrome Saldino-Mainzer or conorenal
vitamin A storing lipid droplets that are distrib- syndrome, named because of the cone-shaped
uted along the basal and lateral surfaces of the epiphyses of the hands. The Saldino-Mainzer
retinal pigment epithelium where receptors for (conorenal) syndrome includes renal histopatho-
retinal binding protein (RBP) are located. Műller logic changes consistent with a primarily glomer-
cells also contain a high number of peroxisomes. ular disorder and short distal phalanges with
The exact mechanism of loss of photoreceptor cone-shaped epiphysis. Interstitial fibrosis and
function is not completely understood. However, tubular cysts lead to renal insufficiency [37].
the phenotypic appearance of the retina along The cerebello-oculo-renal (CORS) syndromes
with the electroretinographic abnormalities is include Joubert and other syndromes that exhibit
similar to LCA. The presence of photoreceptor a “molar tooth sign” on magnetic resonance
dysfunction has been attributed to mutations in imaging which consist of cerebellar aplasia or
the PEX genes. hypoplasia, thickened superior cerebellar pedun-
The presence of LCA with nephronophthisis in cles and a large interpeduncular fossa [38].
Senior-Løken syndrome; nephronophthisis, cone- Pathologically this represents significant brain-
shaped epiphyses of the hand and cerebellar stem malformation.
ataxia in Saldino-Mainzer (conorenal) syndrome; The LCA observed in patients with Joubert
and vermis hypoplasia, oculomotor anomalies syndrome is stable, although symptoms associ-
and neonatal respiratory apnoea in Joubert syn- ated with renal or hepatic involvement present in
drome has been documented [33, 34]. Joubert childhood or early adolescence, necessitating
syndrome has been associated with LCA and careful observation of blood pressure, blood urea
multicystic kidneys [35]. Alström syndrome pres- nitrogen, urinalysis, liver function tests and
ents with LCA, cardiomyopathy, short stature, abdominal ultrasonography to detect early renal
deafness and diabetes mellitus. Although cardio- and hepatic pathology. Visual impairment pres-
myopathy is noted at infancy, diabetes mellitus ents in early infancy. The presence of tachypnoea
occurs in the second or third decade [36]. The admixed with periods of central apnoea, seizures
Arima syndrome, cerebro-oculo-hepatorenal syn- and brainstem abnormalities help make the diag-
drome, is a rare though well-defined entity that nosis of Joubert syndrome. High-quality mag-
includes hepatic involvement. The clinical hetero- netic resonance imaging (MRI), polysomnograms
geneity of this renal-retinal syndrome is indicated and ophthalmic evaluation to assess visual func-
by the variable age of onset of the renal and retinal tion are indicated.
abnormalities. The link between the renal and Mutations in the gene encoding the ciliary/
retinal systems in Senior- Løken (renal-retinal) centrosomal protein CEP290 (NPHP6) or neph-
syndrome is the cilium. Homozygous mutations rocystin-6 lead to Joubert syndrome, and due to
in NPHP1 on chromosome 2q13, encoding neph- interactions with ATF4, a transcription factor
rocystin and NPHP4 (nephroretinin) on chromo- involved in cAMP-dependent cyst formation, it is
some 1p36 are one cause of the clinical syndrome now possible to understand the phenotype of reti-
of Senior-Løken. The interaction of nephrocystin, nal degeneration and renal failure. CEP290 is
nephroretinin and the tubulin of cilia forms a located in renal epithelial centrosomes and in the
complex in the primary cilia and co-localises in connecting cilia of photoreceptors [39].
the renal epithelial cells. In the retina, the con- Mutations in CEP290 were found to be the most
necting cilium in photoreceptor cells plays a key frequent cause of LCA in North American and
role in the transport of molecules from the inner to West European populations [40].
the outer segment.
18.4 Pathogenesis H82Y mutation in AIPL1, histopathological

examination showed almost total absence of pho-
Few pathological studies on retinal degenerative toreceptor cells, decreased ganglion cells,
disease have been reported in which the genetic increased vacuolisation of the nerve fibre layer
defect was known. Similarly, few studies on the with retinal gliosis and a characteristic alteration
morphological appearance (light and electron of arterioles with venular dilation. Increased
microscopy) of the retina in patients with LCA areas of retinal atrophy, sclerosis and attenuation
have been undertaken. Studies of prenatal LCA of the blood vessels and pigment accumulation
gene expression and regulation in the foetus will were also observed. Both the RPE and the
provide greater depth in understanding the cellu- Bruch’s membrane were intact with a normal
lar basis of this disorder. Case studies of CRX, optic nerve [44]. This appearance correlates well
RPE65 and GUCY2D –related LCA have been with the phenotypic appearance observed in
reported. AIPL1-associated LCA [26]. Human retinal
CRX expression in the foetus is known to degeneration in both GUCY2D- and RPE65-
occur at 10–12 weeks in the foveal region when associated LCA appears to occur prenatally, lead-
the rods are generated in the retina, and this is ing to profound photoreceptor dysfunction [45].
followed by further CRX expression that pro- Interestingly, although the retinal appearance on
ceeds towards the peripheral retina [41]. ophthalmoscopy may appear normal, histopatho-
An important comparative pathological study logical studies may show different changes as
examined retinal tissue from a foetus with RPE65 observed in the foetal appearance of both
mutations and compared the histopathology and GUCY2D- and RPE65-associated LCA retinas
immunohistochemistry with an age-matched nor- with decreased photoreceptor cells and disorgan-
mal foetal retina [42]. Compared to normal foetal ised outer segments. This may have important
retina, the affected retina displayed cell loss and implications in the considerations for clinical tri-
thinning of the outer nuclear (photoreceptor) als in the prenatal and postnatal stages and the
layer and decreased immunoreactivity for key extent of photoreceptor cell rescue possible. As
phototransduction proteins and aberrant synaptic more human foetal retinal tissue is studied,
and inner retinal organisation. The homozygous greater understanding of gene function in early
RPE65 mutation C330Y abolished detectable development may be possible.
expression of RPE65 within the RPE of affected
eyes, and ultrastructural examination revealed
the presence of lipid and vesicular inclusions not 18.5 LCA Genetics
seen in normal RPE. The mutant eye demon-
strated thickening, detachment and collagen fibril The main goal of research into the genetics of
disorganisation in the underlying Bruch’s mem- LCA is to identify the molecular basis of the dis-
brane, and the choroid was distended and abnor- ease and to develop therapies. LCA is largely
mally vascularised, in comparison with the inherited in an autosomal recessive pattern; auto-
control eye [42]. These human foetal findings somal dominant inheritance has been described
contrast with the late-onset ocular changes infrequently. Being inherited in an autosomal
observed in animal models, indicating the prob- recessive disorder, homozygosity (identity-by-
lems of inferring human retinal pathophysiology descent (IBD) mapping has been a useful tool for
from animal models [43]. This work also under- identifying genes causing LCA, especially in
lined the early prenatal pathogenic insult and families with consanguineous unions. However,
congenital nature of human LCA in contrast to more recently in the last decade, targeted whole
human RP, which is a disease of later onset. exome sequencing and DNA microarrays have
In retinal tissue obtained from a 22-year-old ushered in greater identification of novel genes
patient with LCA harbouring a novel heterozygous and mutations. Almost a decade ago, mutations
Table 18.1 Causative LCA and LCA-associated genes
LCA Gene No. of Chromosomal
type symbol Protein exons locus Visual function Disorders
LCA1 GUCY2D Retinal guanylate cyclase 2D 20 7p13.1 Synthesis of cGMP during visual cycle ARLCA
CORD
LCA2 RPE65 Retinoid isomerohydrolase 14 1p31 11-cis-retinol regeneration from AR LCA
all-trans-retinyl ester in retinoid cycle ESORD
process
LCA3 SPATA7 Spermatogenesis-associated 7 15 14q31.3 Not clear LCA, Juvenile RP
LCA4 AIPL1 Aryl-hydrocarbon-interacting 6 17p13.1 Protein trafficking, folding and LCA, RP
protein-like stabilization in phototransduction process
LCA5 LCA5 Lebercilin 9 6q14.1 Protein transport across the AR LCA
photoreceptor connecting cilium
18 Leber Congenital Amaurosis in Asia
LCA6 RPGRIP1 Retinitis pigmentosa GTPase 25 14q11 Protein transport processes into the AR LCA
regulator-interacting protein 1 photoreceptor outer segment AR LCA and neurodevelopmental delay
LCA7 CRX Cone-rod homeobox 4 19q13.1 Transcriptional factor regulating AD LCA
photoreceptor cell-specific gene AD cone-rod dystrophy, RP
transcription
LCA8 CRB1 Crumbs 1 cell polarity 18 1q31-q32 Photoreceptor morphogenesis AR LCA
component RP
LCA9 NMNAT1 Nicotinamide nucleotide 9 1p36 NAD biosynthesis AR LCA
adenylyltransferase1 ESORD
AR SLS and LCA
LCA10 CEP290 Centrosomal protein 290 60 12q21.32 Localization of ciliary and AR LCA
phototransduction proteins in retinal AR SLS
photoreceptor cells AR Joubert syndrome
AR Meckel syndrome
LCA11 IMPDH1 Inosine monophosphate 18 7q31.3-q332 Retinal guanine synthesis AD LCA
dehydrogenase 1 AD RP
LCA12 RD3 Retinal degeneration 3 3 1q32.3 Outer segment ciliary transport of AR LCA
guanylate cyclase
LCA13 RDH12 Retinal dehydrogenase12 9 14q24.1 Reduction of cis and trans retinal in the AR LCA
retinoid cycle ARRP
(continued)
197
198

LCA14 LRAT Lecithin retinol acyltransferase 5 4q31 Chemical transformations of vitamin A LCA, early-onset ERSOD
(all-trans-retinol) into 11-cis retinal, in
retinoid cycle
LCA15 TULP1 Tubby-like protein 1 15 6p21.3 Protein transport across the AR LCA
photoreceptor connecting cilium AR ESORD
LCA16 KCNJ13 Potassium voltage-gated 3 2q37.1 Regulating potassium channel across cell Snowflake degeneration
channel subfamily J member 13 membrane
LCA17 GDF6 Growth differentiation factor 6 2 8q22.1 Development of the eye AD microphthalmia, Klippel-Feil
syndrome
Chorioretinal coloboma
(LCA-like)
LCA18 PRPH2 Peripherin 2 5 6p21.1 Stabilization of the outer segment of the Digenic RP, ADRP, progressive macular
photoreceptors degeneration, macular dystrophy
LCA
… OTX2 Orthodenticle homeobox 2 5 14q22.3 Differentiation of photoreceptors Microphthalmia (MCOPS5) and combined
pituitary hormone deficiency (CPHD6)
LCA-like
… IFT140 Intraflagellar transport140 40 16p13.3 Ciliary transport between the inner and AR Saldino-Mainzer syndrome
outer segment of photoreceptors ARRP
ARLCA
… PNPLA6 Patatin-like phospholipase 37 19p13.2 Membrane lipid homeostasis Oliver-MacFarlane syndrome,
domain-containing protein 6 trichomegaly-retina pigmentary
degeneration and dwarfism
… CABP4 Calcium-binding protein 4 8 11q13.1 Maintenance of synaptic function Stationary night blindness type 2B
Cone-rod synaptic disorder
POC1 POC1 centriolar protein B 13 12q21.33 Ciliogenesis basal body and centrosome AR JS, PKD and LCA
integrity AR CORD
… MERTK MER tyrosine kinase 24 2q13 Outer segment phagocytosis by RPE ARRP ARRCD
proto-oncogene EOSORD
ARLCA
S. Dharmaraj et al.
… IQCB1 IQ motif-containing protein B1 16 3q13.33 Photoreceptor ciliary function AR SLS
(nephronophthisis 5) LCA
… ALMS1 Centrosome and basal body- 23 2p13.1 Photoreceptor ciliogenesis ARLCA
associated protein Centriole structure and function Alström disease
… CNGA3 Cyclic nucleotide-gated channel 9 2q11.2 Visual and olfactory signal transduction AR achromatopsia, ARCORD
alpha 3
… MYO7A Myosin 7A 55 11q13.5 Photoreceptor organization Usher syndrome (1B), deafness AR,
decreased vestibular function, retinal
degeneration,
USH3-like
CTNNA1 Catenin alpha 1 27 5q31.2 Cell adhesion Butterfly-shaped pigment dystrophy
ADMD
LCA
18 Leber Congenital Amaurosis in Asia
CCT2 Chaperone-containing TCP-1 17 Chaperone stability AR LCA

DTHD1 Death domain containing 1 12 4p14 Signalling and apoptosis Myopathy and LCA
CLAUP1 Claustrin-associated protein 1 15 16p13.3 Essential for ciliogenesis and LCA
photoreceptor maintenance
PEX1 Peroxisomal biogenesis factor 1 7q21.2 Biogenesis of peroxisomes Infantile Refsum, Zellweger, neonatal
adrenoleukodystrophy
(LCA-like retinal appearance)
CYP4V2 ARLCA
Bietti’s corneoretinal dystrophy
RPE Retinal pigment epithelium
199
in all LCA- associated genes accounted for 70% leading to loss of function. CEP290, GUCY2D,
of the clinically diagnosed LCA in Caucasian CRB1, RPE65 and CRX account for a major por-
populations [46]. LCA occurs due to mutations in tion of LCA. The individual mutational fre-
the genes involved directly or indirectly in the quency of these genes may vary from one ethnic
visual pathways. There are at least 30 such genes population to another [46, 48, 49]. CEP290
identified, and new genes are constantly being mutation is relatively frequent in European and
discovered (Table 18.1). With discovery of genes American patients with LCA while remarkably
causing LCA comes the understanding of protein low or absent in other regions, including India.
function. Photoreceptor structure and morpho- [50]. A particular CEP290 mutation,
genesis involve CRB1, CRX, GDF6 and OTX2, c.2991 + 1655A > G that causes truncated protein
while phototransduction involves AIPL1, formation due to aberrant splicing, accounts for
GUCY2D and RD3. LRAT, RDH12 and RPE65 20% of LCA in Europe [40]. Recently, genes
are involved in the retinoid cycle. Interestingly (ALMS1, CNGA3, IQCB1 and MYO7A) related to
the detection of genes involved in the ciliome has syndromic or non-syndromic retinal disorders
increased remarkably in the last decade, and have been reported to be genetically linked with
these include ALMS1, CEP290, CLUAP1, LCA [51]. Interestingly, genes causing LCA, like
IFT140, LCA5, RPGRIP1 and TULP1. Mutations CRX, LRAT, MERTK, RPE65, CEP290 and
in these genes cause ciliopathies. TULP1, have also been implicated in other early-
These known genes account for disease in onset retinal degeneration like retinitis pigmen-
70–80% or more of patients with LCA currently, tosa (RP) and cone-rod dystrophy (CORD). Both
suggesting that new undiscovered genes account RP and CORD are less severe than LCA, and the
for the remaining 20–30% cases. A recent study onset of disease is not as early as LCA.
from India using a 20-LCA gene panel involving For their study of LCA, the ocular genetic
92 patients with LCA detected mutations in 61% laboratories in LV Prasad employed direct
[47]. LCA is a rare disease, and many studies sequencing of candidate genes for mutations that
undertaken worldwide have not always men- are disease-associated. The patients recruited
tioned the origin of the patients, and LCA cohorts were from different regions of India. Testing of a
reported in the West include DNA from the wider few selected candidate genes for LCA –
diaspora of Asian patients from the East, too. The GUCY2D, CRB1, RD3, RPE6 and NMNAT1 –
number of LCA-causing mutations detected has showed a combined frequency for all these genes
increased over the past two decades, and most of approximately 12% in a total of 100 probands
studies have not always included all the most tested. Among these, pathogenic changes were
recently identified genes in their sequencing, and detected in GUCY2D (2%), CRB1 (2%), RPE65
as a result the relative percentages of the muta- (4%), RD3 (<1%) and NMNAT1 (4%). Mutations
tional load are quite variable as are the popula- in the RD3 gene are associated with <1% of cases
tions studied. Nevertheless due to enormous with LCA from different parts of India and in dif-
international collaborative efforts, significant ferent populations across the world reported in
understanding of both the clinical phenotype and the literature with a similar low frequency of
the genotype has been possible. A large number mutations [52, 53].
(~800) of mutations are known to cause LCA The frequency of mutations in different popu-
(mutation database), but many of these are pri- lations is remarkable. While in Chinese and
vate mutations found only in 1 family. The muta- Indian families the commonest gene mutations
tions have been missenses, deletions, insertions are in GUCY2D, in Saudi Arabia TULP1 muta-
and frameshift and splice site mutations. tions are most common. In Belgian populations
Missense mutations account for the greatest CEP290 was most commonly mutated, while in
number of mutations in any cohort. Several muta- Indonesian, Italian and Danish cohorts, RPE65
tions cause null alleles and truncated proteins mutations were the commonest [47, 48, 54–57].
The above tables have listed many genes associ- mutations in both AIPL1 copies, the survival of
ated with LCA; however, several of them cause an adult rods is more dependent on AIPL1 function
LCA-like fundus appearance which could be nor- than cones. However, the absence of AIPL1 in
mal or show varying degrees of vascular attenua- early retinal development leads to both rod and
tion or pigmentary disturbance. Genes like GDF6, cone dysfunction as AIPL1 is required for the
CABP4, PEX and ALMS1 cause other disorders. normal development of both rod and cone photo-
MYO7A PNPLA6, CYP4AV2, CLAUP1, receptors [79]. The phenotype is severe, present-
DTHDI, CCT2, CTNNA1 and CNGA3 need more ing early with loss of vision and varying areas of
research to validate them as genes causative of atrophy and hyperpigmentary changes in the ret-
LCA, and there are likely others that require ina. Keratoconus has been described in some pro-
more molecular studies to validate them as genes bands [26, 47]. The outer retina in young patients
causing LCA. with AIPL1 mutations is preserved, and it has
Using homozygosity mapping and linkage been observed on OCT, providing an opportunity
analysis, several genes causing LCA were identi- for gene therapy [80]. Mutations in this gene
fied. Whole exome sequencing and candidate causing LCA have been observed in Indian,
analysis contributed vastly to the identification of Pakistani, Indonesian, Arab and Chinese popula-
more causative genes for LCA. The genes associ- tions (Table 18.2).
ated with transduction include GUCY2D, RD3
and AIPL1, while those involved with morpho-
genesis are CRB1, CRX and OTX2. Retinoid 18.5.2 ALMS1
recycling in the RPE are dependent on RDH12,
LRAT and RPE65. The ciliome is important for ALMS1, a centrosome and basal body associated
maintaining structure and function in the photo- protein, is involved in photoreceptor ciliogenesis.
receptor cell and is involved in transport of In infancy the presence of markedly decreased
metabolites and impulses between the inner and visual function, marked by severely extinguished
outer segment of rods and cones. ALMS1, rod and cone function coupled with mutations in
CEP290, CLAUP1, IFT140, IQCB1, LCA5, ALMS1, identified the phenotype in these patients
RPGRIP1, SPATA7 and TULP1 play a pivotal as Alström disease [81]. Alström disease is asso-
role in the ciliome. ciated with obesity, loss of visual function,
hyperopia, non-recordable ERGs and hearing
loss. Cardiomyopathy, hyperinsulinemia, hyper-
18.5.1 AIPL1 tension and hypertriglyceridemia may be associ-
ated, too. Mutations causing LCA were identified
The aryl-hydrocarbon-interacting protein-like in Saudi Arabian patients [82].
(AIPL1) gene located on chromosome 17p13.1
has six exons and is expressed in the inner seg-
ment, nucleus, perinuclear region, synaptic ter- 18.5.3 CCT2
minals and cytoplasm of the photoreceptor cells.
It is expressed in the developing retina and is CCT2 is a chaperone-containing T-complex poly-
essential for the normal development of the rods peptide, and mutations involved chaperone insta-
and cones [75–77]. Mutations in AIPL1 cause bility. Patients with LCA have a severe ocular
AD CORD, juvenile ARRP and ARLCA [76]. In phenotype with attenuation of retinal vasculature,
LCA, W278X, the commonest mutation in AIPL1, disc pallor and thin disorganised retina. Eye pok-
alters the structure, stability and transport of the ing was observed [83]. Two novel compound het-
protein [78]. In keeping with greater loss of rod erozygous mutations in a Chinese family have
function both in heterozygote carriers of AIPL1- been reported c.1198A > G and c.1547G > A.
associated LCA and in patients with LCA with
Table 18.2 Mutation and Asian LCA

% Reported in
worldwide
No. Gene OMIM Gene populations Asian LCA
1 600179 GUCY2D 6–21 Japanese (Hosona et al.)
Saudi Arabian (Safieh et al., Li et al.)
Bedouin Saudi Arabian (Gradstein et al.)
Indian (Verma et al., Srilekha et al., Srikrupa et al.)
Chinese (Chen Y et al., Li Y et al.)
2 180069 RPE65 3–16 Japanese (Wada et al., Katagiri et al.)
Saudi Arabian (Li Y et al.)
Indian (Ramprasad et al., Verma et al., Srilekha et al.,
Srikrupa et al.)
Arab (Beryozkin et al.)
Indonesia (Sitorus et al.)
Chinese (Li Y et al.)
3 609868 SPATA7 – Indian (Srilekha et al., Srikrupa et al.)
4 604392 AIPL1 5–10 Indian (Verma et al., Srilekha et al., Srikrupa et al.)
Indonesia (Sitorus et al.)
Pakistani (Khaliq et al., Damji et al.)
5 611408 LCA5 1–2 Indian (Ramprasad et al., Mackay et al., Srikrupa et al.)
Pakistani (Ahamad et al., Mackay et al.)
Afghan (Mackay et al.)
Iraqi (Mackay et al.)
Taiwanese (Mackay et al.)
Korean (Seong et al.)
Chinese (Li Y et al., Mackay et al.)
6 605446 RPGRIP1 4–6 Pakistan (McKubbin et al.)
Saudi Arabian (McKubbin et al., Khan et al.)
Egyptian (Abouzeid et al.)
Chinese (Li Y et al., Huang et al., Chen Y et al.)
Indian (Srikrupa et al.)
Japanese (Suzuki T et al.)
Thai (Jinda et al.)
Iranian (Imani et al.)
7 602225 CRX 1–3 Chinese (Chen Y et al., Li Y et al.)
Indian (Verma et al., Srikrupa et al.)
Japanese (Nakamura et al.)
(continued)
% Reported in
worldwide
8 604210 CRB1 9–13 Palestinian (Abouzeid et al., Beryozkina et al.)
Israeli (Beryozkina et al.)
Indian (Srilekha et al., Srikrupa et al.)
Saudi Arabian (Li et al.)
Iranian (Ghofrani et al.)
Chinese (Chen Y et al., Yang et al., Li Y et al.)
Pakistan (Khaliq et al.)
Japanese (Kuniyoshi et al.)
9 608700 NMNAT1 – Chinese (Jn X et al.)
Han Chinese (Tong H et al.)
Pakistan (Koenekoop et al.)
Japanese (Coppeiters et al.)
Thai (Jinda et al.)
10 610142 CEP290 20 Arab (Aboussair et al.)
Chinese (Chen et al.)
Chinese (Li et al.)
Thai (Jinda et al.)
11 146690 IMPDH1 8 Chinese (Chen et al.)
Japanese (Wang et al.)
12 180040 RD3 <1 Indian (Srikrupa et al.)
13 608830 RDH12 4–5 Indian (Sunderamurthy et al., Srilekha et al., Srikrupa
et al.)
Chinese (Li et al.)
Japanese (Kuniyoshi et al.)
14 604863 LRAT <1 (Srikrupa et al.)
15 602280 TULP1 1 Arab Israeli (Abbesi et al.)
Saudi Arabian (Li et al.)
16 603208 KCNJ13 – Middle Eastern (Sergouniotis et al.)
Jordian (Pattnaik et al.)
17 601147 GDF6 –
18 60813 PRPH2 –
19 610125 OTX2 –
20 614260 IFT140 – Chinese (Xu et al.)
21 603197 PNPLA6 –
22 608965 CABP4 –
23 614784 POC1 – Iraqi (Beck et al.)
24 604705 MERTK 2 Chinese (Li et al.)
(continued)
% Reported in
worldwide
25 609237 IQCB1 3 Turkish (Otto)
Indian (Verma et al., Srilekha et al., Srikrupa et al.)
Saudi Arabian (Wang et al.)
Thai (Jinda et al.)
26 606844 ALMS1 – Chinese (Li et al.)
Saudi Arabian (Safieh et al.)
Thai (Jinda et al.)
27 600053 CNGA3 –
28 276900 MYO7A –
29 608970 CTNNA1 – Thai (Jinda et al.)
30 605139 CCT2 – Chinese (Minegish et al.)
31 616979 DTHD1
32 616787 CLAUP1
33 602859 PEX1
34 608614 CYP4V2 Thai (Jinda et al.)
Table references: Hosono et al. [58], Verma et al.[59], Srilekha et al. [60], Chen et al. [61], Gradstein et al. [62], Li et al.
[55, 63], Katagiri et al. [64], Wada and Tamai [65], McKay et al. [66] McKibbin et al. [67], Suzuki et al. [68], Nakamura
et al. [69], Beryozkin et al. [70] Kuniyoshi et al. [71], Imani et al. [72], Huang et al. [73], Li et al. [74]
OMIM Online Mendelian inheritance in man, AD Autosomal dominant, AR Autosomal recessive, CORD Cone-rod
dystrophy, LCA Leber congenital amaurosis, RP Retinitis pigmentosa, SLS Senior-Løken syndrome, JS Joubert
syndrome
18.5.4 CEP290 18.5.5 CLUAP1
CEP290 is a centrosomal protein (290 kDa) that CLUAP1 (clusterin-associated protein 1) is associ-

controls ciliary transport between the inner and ated with ciliogenesis and is critical in photorecep-
outer segments of photoreceptors. CEP290 was tor function. Hypomorphic mutations were
the first gene noted to cause syndromic LCA identified in CLUAP1 (hypomorphic mutations
[40]. The commonest intronic mutation result in partial function). Compound heterozy-
(c.2991 + 1655A > G) creates a strong splice gous mutations have been detected in Joubert syn-
donor site and inserts a cryptic exon in the drome and orofacial with digital overlap syndrome,
CEP290 mRNA (Jacobson SG IOVS 2017 [40, too [89]. Hypomorphic mutations associated with
84]. OCT and full-field sensitivity testing showed LCA have been identified in this gene [90].
dissociation of structure and function of the pho-
toreceptors. CEP290 mutations are the common-
est mutations in Western populations and are a 18.5.6 CRB1
frequent cause of non-syndromic LCA [85].
Mutations cause diffuse retinal degeneration. The crumbs homolog 1 (CRB1) gene was iso-
Foveal cones are preserved as noted on OCT lated by subtractive hybridisation. It is expressed
[86]. Clinical features include renal failure, in the apical membranes of retinal photoreceptors
ataxia, hypotonia and Joubert syndrome [85]. [91]. CRB1 maps to 1q31–32.1 and consists of 12
Mutations were reported in Meckel-Gruber syn- exons. It is expressed in the adult and foetal brain.
drome [87]. CEP 290 mutations were noted in CRB1 is involved in cell-cell interaction and may
patients with anosmia [88]. be responsible for the maintenance of retinal cell
polarity. CRB1 is essential for the integrity of the Interestingly CRX interacts with NRL and mis-
external limiting membrane and photoreceptor sense mutations in NRL cause ADRP and not
cell morphogenesis [92]. It is needed to maintain LCA, while recessive mutations cause thickening
an organised layer of photoreceptor cells during of the retina and an unusual pigmentary retinopa-
light exposure, and in its absence adhesion thy [106, 107]. Patients have nonprogressive
between the photoreceptor cells and the Mϋller markedly decreased visual acuity with diffuse
cells is lost resulting in both structural and func- pigmentary retinal changes and ateliotic maculas
tional changes. Light exposure results in an [108, 109].
increase in focal retinal lesions [93]. Mutations in
this gene are associated with a severe form of RP,
RP12, and with LCA [94–97]. Mutations in 18.5.8 CNGA3
CRB1 were detected in patients with RP and
Coats-like vasculopathy and in patients with RP Cyclic nucleotide-gated cation channel (CNGA3)
and preserved para-arteriolar retinal pigment epi- encodes one of the alpha subunits of the ion chan-
thelium [98]. The retinal phenotype is variable nels necessary for phototransduction. Mutations
and nummular pigment clumping and white dots have been detected in patients with AR achroma-
have been observed in LCA. Slow progression of topsia. Mutations associated with LCA were
the disease has been noted [91, 95, 97]. Mutations detected in a Saudi Arabian pedigree [51].
also result in RP without para-arteriolar RPE
preservation. High hyperopia was noted with a
compound heterozygous mutation in a consan- 18.5.9 CTNNA1
guineous family of Palestinian descent [99].
Characteristically an unlaminated thickened ret- CTNNA1, catenin alpha 1, plays a role in cell
ina is noted on OCT [100]. adhesion and connects cadherins on the plasma
membrane to the actin filaments inside the cell.
Mutations cause AD butterfly-shaped macular
18.5.7 CRX dystrophy [110]. Mutations in a Thai patient with
LCA were reported [111].
The cone-rod homeobox gene (CRX) on chromo-
some 19q13 encodes a transcription factor impor-
tant in embryonic photoreceptor development. 18.5.10 CYP4V2
CRX belongs to the otx/otd homeobox family of
genes and is similar to the human OTX1 and Cytochrome P450 family 4 subfamily V is a gene
OTX2 homeodomain proteins [101]. It is that encodes a member of the cytochrome P450
expressed in the pineal gland and in developing protein involved in oxidizing substrates in the
and mature retinal photoreceptor cells. CRX is metabolism of fatty acid precursors. Mutations
also expressed in the inner nuclear layer [41]. It is have been noted in Bietti corneoretinal crystalline
necessary for the maintenance of normal cone dystrophy and more recently in LCA [111, 112].
and rod function [102]. This cone-rod otx-like
photoreceptor homeobox transcription factor is
capable of trans-activating several photoreceptor 18.5.11 DTHDI
cell-specific genes and plays a crucial role in dif-
ferentiation. Mutations in CRX are known to DTHDI (death domain containing 1) is involved
cause AD LCA, autosomal dominant cone-rod in cell signalling and apoptosis, and mutations
dystrophy and autosomal dominant retinitis pig- have been reported in a patient with LCA and
mentosa [11, 12, 103, 104]. Some AD mutations myopathy [113].
in CRX are associated with LCA [10, 11, 105].
18.5.12 GDF6 inner and outer segments of photoreceptors.

Mutations cause a ciliopathy involving the skel-
Growth differentiation 6 encodes a secreted eton and eye. IFT140-associated LCA causes
ligand of the TGF-beta superfamily of proteins. poor vision, nystagmus, peripheral RPE mottling
This protein is required for bones and joints in with depigmented spots in the retina and non-
the axial skeleton. Mutations cause Klippel-Feil recordable ERGs. The phenotype consists of reti-
syndrome, dominant microphthalmia, chorioretinal pigment epithelial atrophy [119].
nal coloboma and a phenotype not similar to
LCA, although an LCA reference has been
assigned to this gene. 18.5.15 IMPDH1
Inosine monophosphate dehydrogenase 1

18.5.13 GUCY2D (IMPDH1) on chromosome 7q31.3–q32 is
involved in cyclic nucleoside metabolism within
Retinal guanylate cyclase 2D, the first gene photoreceptors. Mutations in IMPDH1 cause
linked to LCA on chromosome 17p13.1, encodes ADRP (RP10) and ADLCA [120, 121].
a retinal protein that is involved in phototrans-
duction. GUCY2D synthesises the intracellular
messenger of photoreceptor excitation cGMP 18.5.16 IQCB1
and is regulated by the intracellular calcium-
sensitive proteins GCAPs. RETGC1 is a protein The IQ calmodulin-binding motif-containing B1
involved in photorecovery in the phototransduc- protein encodes nephrocystin 5 that localises to the
tion cascade, and mutations in the gene hinder primary cilia of renal epithelium and connecting
the restoration of cGMP in the rods and cones to cilium of photoreceptors. The IQCB1 protein inter-
the basal state, resulting in permanent closure of acts with RPGR and calmodulin in the photorecep-
the cGMP-gated channels [114]. Mutations in tor connecting cilia. The LCA phenotype includes
GUCY2D were first identified in patients with distinct retinal blood vessel straightening and
LCA [114]. However, mutations in patients with altered pigmentation around the vascular arcades
CORD have been detected [115]. Nevertheless [122]. Senior-Løken syndrome involves cystic kid-
more mutations causing LCA in cohorts world- ney disease (nephronophthisis) and retinitis pig-
wide have been noted [47, 116, 117]. It is one of mentosa or Leber congenital amaurosis;
the major causes of LCA, and of the 140 muta- homozygous mutations were detected in several
tions reported, 88% cause AR LCA. patients with LCA patients, some of whom later
The fundus in infants appears normal initially, developed kidney disease [123]. In addition to
but in later years varying degrees of peripheral LCA, psychomotor delay and midface hypoplasia
pigmentary changes are noted. A greyish tapetal were observed in an Arab proband who had a muta-
reflex has been noted in many of these patients. tion in the IQCB1 gene. Sequence variants were
However, the ERG has been vastly diminished, noted in the ACAT1 and FGFR2 genes, too [51].
and the OCT shows perifoveal thinning [117,
118].
18.5.17 KCNJ13
18.5.14 IFT140 KCNJ13 (potassium channel) that controls

inward potassium flux in RPE is expressed in the
IFT140 encodes an intraflagellar transport com- apical microvilli. A novel mutation resulting in
plex A (IFTA) that pays a role in the maintenance loss of channel function was identified in a
of transport in the connecting cilia between the Jordanian pedigree. Early progressive retinal
degeneration involving both rods and cones was scent macula and a hyperfluoroscent fovea signi-
noted [124]. Retinal degeneration and thinning, fying continued metabolic activity of the foveal
with nummular pigment distribution, have been cones. Most mutations have been null mutations
observed [125]. Mutations cause autosomal dom- and have been identified in several Asian pro-
inant snowflake vitreoretinal degeneration [126]. bands [67]. Homozygous mutation was noted in a
Vitreoretinal dystrophy and cataracts were noted Pakistani family who presented with LCA and
in Saudi Arabian families [127]. cataract [134]. LCA5 mutations were identified in
several Asian populations [135].
18.5.18 LCA5
18.5.19 LRAT
LCA5 (protein product, lebercilin) maps to chro-
mosome 6q14.1, a region rich with retinal genes. Lecithin retinol acyltransferase (LRAT) maps to
The gene LCA5 was identified as it was part of 4q31 and encodes a polypeptide of 230 amino
the ciliome, and mutations in the LCA5 gene acids. LRAT is synthesised in the retinal pigment
account for about 2% of LCA [128]. The affected epithelium [136]. LRAT catalyses the initial
individuals of a family (Old Order River Brethren) series of reactions in the conversion of all trans-
of German-Swiss descent mapping to chromo- retinol to all trans-retinyl ester in the RPE and
some 6q11–q16, presented with visual acuities in plays a vital role in the regeneration of the visual
the order of 20/100–20/400, high hyperopia and chromophore. Patients with mutations in LRAT
normal fundi in early infancy and childhood and causing LCA present with decreased visual func-
mutations, were identified in lebercilin. tion, night blindness and photophilia, and severe
Interestingly one of the patients showed a hetero- retinal atrophy and fibrosis have been noted on
zygous change in GUCY2D (C2174T) which did fundoscopy. OCT confirmed photoreceptor loss
not cosegregate with the disease and may exert a and disrupted retinal lamination was noted [137].
modifying influence on the phenotype causing an Mutations in LRAT lead to early-onset severe
increase in retinal degeneration [118, 129]. retinal dystrophy [138].
However, the phenotype of another family from
Pakistan mapping to the same locus was quite
different. Progressive, severe maculopathy con- 18.5.20 MERTK
sisting of pigmentary disturbance and retinal pig-
mentary atrophy was noted [130]. Atrophic The MER tyrosine kinase proto-oncogene is
retinal maculopathy was a distinct feature in the expressed in RPE and is involved in phagocyto-
Pakistani kindred; however, despite a similar sis. Mutations are known to cause ARRP,
ophthalmoscopic appearance, the LCA5 locus childhood- onset rod-cone dystrophy and
was excluded in a Turkish pedigree [131]. LCA. Mutations have been noted in Chinese fam-
Mutations in LCA5 also cause cone dystrophy ilies with LCA [63].
wherein the phenotype showed no remarkable
peripheral retinal changes, and novel biallelic
mutations were detected in a Chinese sibship 18.5.21 MYO7A
[132]. The LCA phenotype is severe, presenting
with nyctalopia, hyperopia and markedly MYO7A Myosin 7A encodes a myosin, a mecha-
decreased visual function. Hypopigmented white nochemical protein when mutated causes Usher
spots have been noted on fundoscopy [133]. syndrome IB characterised by deafness, abnor-
Retinal pigment atrophy and loss of photorecep- mal gait and retinal degeneration [139]. LCA was
tors in the outer retinal layer are noted on OCT, noted in a Saudi Arabian pedigree [51].
and fundus autofluorescence shows a hypofluoro-
18.5.22 NMNAT1 diminished ERGs [146]. Patients with Oliver-

McFarlane syndrome (OMS) have a complex
Nicotinamide nucleotide adenylyltransferase 1 phenotype characterised by blindness due to
on chromosome 1p36 is involved in nicotinamide severe photoreceptor degeneration, dwarfism due
adenine dinucleotide biosynthesis and localises to pituitary growth hormone deficiency, tricho-
to the cell nucleus. NMNAT1 mutations reduce megaly and progressive alopecia [147].
the enzymatic activity of NMNAT1 in NAD bio- Occasionally, OMS is associated with mental
synthesis which affects protein binding and neu- retardation, distal muscle weakness/wasting and
roprotection. The locus was linked initially to a ataxia, due to axonal peripheral neuropathy.
Pakistani pedigree and mutations were identified Mutations have been identified in patients with
later [140]. Visual acuity was the perception of spastic paraplegia [148]. Human
light in all affected individuals. Posterior subcap- organophosphorous-induced delayed neuropathy
sular lens opacities, macular staphylomas, vary- (OPIDN) occurs when PNPLA6 becomes
ing degrees of retinal white spots and pigmentary phosphorylated by these organophosphates, fol-
retinopathy with optic atrophy were noted [141]. lowed by dealkylation of the phosphoryl enzyme,
The LCA phenotype is one with severe macu- inhibiting the catalytic domain.
lar and optic atrophy with attenuation of retinal
vasculature and marked visual loss. The E257K
mutation was the commonest and was observed 18.5.25 POC1
in a great majority of patients in several studies
[142–144]. Centriolar protein chlamydomonas homolog 1b
(POC1) localises to the centriole close to the cil-
ium of photoreceptors and plays a role in mainte-
18.5.23 OTX2 nance and duplication of the centriole in the basal
body of cells. It also localises to the synapsis in
Orthodenticle homeobox 2 (OTX2) is a transcrip- the outer plexiform layer of the retina. Mutations
tion factor involved in the differentiation of pho- were first detected in patients with CORD [149].
toreceptors and interacts with CRX, NRL and Mutations causing CORD were reported in a
TR-beta 2. Mutations in addition to early-onset Turkish cohort [150]. LCA with polycystic kid-
retinal dystrophy also cause short stature as a ney disease and Joubert syndrome was reported
result of abnormal pituitary function. Fundoscopy in an Iraqi family of consanguineous origin [151].
shows fine hyperpigmentation of the RPE and the
peripapillary zone [145].
18.5.26 PRPH2
18.5.24 PNPLA6 Peripherin 2 retinal degeneration slow (PRPH2)

helps stabilise the outer segment of photorecep-
Patatin-like phospholipase domain containing tors. Mutations are known to cause ADRP, auto-
protein 6 (PNPLA6) is involved in the deacety- somal dominant macular dystrophy, retinitis
lation of membrane lipids in the central nervous punctata albescens, digenic RP and AR LCA. The
system. This protein is expressed in the inner seg- children stare at lights. Asymptomatic parents
ment of photoreceptors. In one case report of a (heterozygous carriers) show peripheral retinal
compound heterozygous mutation in PNPLA6, pigmentary changes [152]. Recessive PRPH2
the patient had a reduction of vision with nystag- mutations cause prominent maculopathy in
mus. It causes severe retinal degeneration and adulthood.
18.5.27 RD3 18.5.30 RPGRIP1
Retinal degeneration 3 (RD3) is an accessory The retinitis pigmentosa guanosine triphospha-

chaperone protein for transport of GUCY2D and tase (GTPase) regulator-interacting protein 1
GUCY2F from photoreceptors. Mutations cause gene (RPGRIP1) on chromosome 14q11 encodes
macular and retinal degeneration [52, 53]. a protein that is involved in disc morphogenesis
Mutations cause early-onset severe retinal dystro- through regulating actin cytoskeleton dynamics
phy. Phenotypically they present with disorgan- [165]. RPGRIP1 consists of 24 exons, and the
isation of the retina with thinning of the inner predicted protein consists of 1259 amino acids.
nuclear, ganglion cell and nerve fibre layers [153]. RPGRIP1 was identified as an interactor of
RPGR (retinitis pigmentosa GTPase regulator);
both localise to the connecting cilium. RPGRIP1
18.5.28 RDH12 anchors RPGR to the connecting cilium.
Mutations in RPGR cause X-Linked RP (RP3)
RDH12 (LCA13) localises to the inner segment [166]. Mutations in RPGRIP1 lead to LCA and
of the photoreceptors and is involved in the trans- AR CORD [167–169]. Increased frequency of
formation of 11-cis retinol to 11-cis retinal of the mutations were noted in patients from Pakistan
phototransduction cascade. The phenotype pres- and Saudi Arabia. [67].
ents with marked retinal degeneration and macu- In an Egyptian proband, neurodevelopmental
lar atrophy [154]. delay and LCA were noted with a severe ocular
phenotype and marked decrease on VEP testing
[170]. The phenotype is severe and presents with
18.5.29 RPE65 poor vision and drusen-like deposits [163]. Bone
spicule-like pigmentary changes have been noted
The retinal pigment epithelium 65 (RPE65) gene in the retina [47].
on chromosome 1p31 encodes a protein which
plays a pivotal role in retinoid metabolism. RPE65
is a non-glycosylated 65 kD microsomal protein 18.5.31 RDH12
expressed in retinal pigment epithelium and is
conserved in mammals, reptiles and birds. It is The retinol dehydrogenase 12 (RDH12) gene on
necessary for the production of 11-cis vitamin chromosome 14q23.3–q24.1 has been implicated
A. Mutations in RPE65 disrupt the synthesis of in early-onset severe retinal dystrophy and LCA
the opsin chromophore ligand 11-cis retinal and [171, 172]. It encodes a photoreceptor protein
are partial to total loss of isomerization [155]. The that is important in retinoid metabolism. The
deactivation of sensory transduction by opsins gene consists of 7 exons and encodes a protein of
causes light-independent retinal degeneration in 316 amino acids. RDH12 belongs to the super-
LCA. RPE65 defects result in progressive photo- family of short-chain alcohol dehydrogenases
receptor cell death. Both LCA and severe ARRP and reductases. RDH12 may be involved in the
have been reported with mutations in RPE65 [56, conversion of 11-cis retinol to 11-cis retinal dur-
116, 156–162]. The most number of RPE-65 ing the regeneration of cone visual pigments.
mutations have been detected in Denmark [54]. RDH12 is expressed predominantly in the neuro-
Chorioretinal atrophy and pigmentary retinopathy retina in photoreceptors [173]. Phenotype
have been noted in patients [163]. Relatively pre- includes atrophic macular lesions and disorgan-
served central vision and retinal structure was ised retinal architecture [47, 154].
noted in Chinese patients [164].
18.5.32 SPATA7 Although some genes have been associated with

LCA, it has not always been very appropriate.
LCA3/SPATA7 spermatogenesis-associated pro- Better phenotyping and genotyping with more
tein 7 localises to the cilium of the photorecep- extensive laboratory work could eliminate this
tors and is involved in protein trafficking and problem. Capb4 is a gene mutated in cone-rod
recruiting RPGRIP to the cilium. Mutations synaptic disorder and not LCA as reported. Genes
cause peripheral retinal degeneration and juve- associated with colobomas and photoreceptor
nile RP. Phenotype comprises severe progressive dysfunction in addition to central nervous system
rod-cone degeneration and relatively preserved abnormalities are not generally causative of
foveal structure [174]. Patients presenting with LCA.
this subset of LCA had enophthalmos, nystagmus
in early life, moderate hyperopia, variable stra-
bismus, profound visual loss, retinal pigment 18.6 Molecular Testing
mottling and clumping. Older individuals of the
Saudi Arabian kindred had perception of light, Clinicians managing patients with LCA now can
nuclear or cortical cataracts, optic atrophy, send a simple cheek swab or blood to genetic
clumping of retinal pigment, atrophic maculas labs. High-throughput screening methods such as
and vascular attenuation [47, 175]. LCA mutation chips and next-generation
sequencing-based exome-sequencing could be
undertaken to arrive at a genotype. DNA
18.5.33 TULP1 microarray-based chip (Asper chip, www.asper-
bio.com) offers a rapid, cost-effective and accu-
Tubby-like protein (TULP1) gene is analogous to rate genotyping method for detection of all
the tub gene in mice that is responsible for known disease-associated variations in LCA
maturity-onset obesity, insulin resistance, retinal [180]. It contains a collection of specific probes
degeneration and neurosensory hearing loss. Tub which target specific variations. This chip pro-
is known to cause early progressive retinal degen- vides a cost-effective option for mutation detec-
eration in mice. Human TULP1 maps to chromo- tion and may be used for molecular diagnosis to
some 6p21.3, consists of 15 exons and is study LCA cohorts. Causal genes in LCA have
expressed exclusively in the retina. Mutations in been identified using linkage and homozygosity
this gene are known to cause early retinal dystro- mapping, candidate gene analysis, whole genome
phy [176]. The human tubby TU and tubby-like and whole exome sequencing.
proteins TULP1, TULP2 and TULP3 belong to a
highly evolutionarily conserved gene family and
play an important role in obesity, sensorineuronal 18.7 Genotype-Phenotype
degeneration and development [177]. The TULPs Correlation
are expressed in the brain, retina and cochlea.
TULP1 localises to the perinucleolar cap region LCA constitutes the most severe form of retinal
in photoreceptor cells and is involved in protein dystrophy since retinal function is profoundly
trafficking. Tulp1 labelling is noted in the inner decreased, as evidenced by extinguished ERGs in
segments of rods and cones in foetal retinas at infancy. Although progress in the understanding
~8 weeks [178]. TULP1 is a transcription factor of the molecular mechanisms underlying LCA
involved in the control of downstream genes in has steadily grown, genotype-phenotype correla-
photoreceptors and is involved in retinal phago- tions are not straightforward. However, the clini-
cytosis. Mutation screening and direct sequenc- cal heterogeneity of this disorder spares some
ing revealed a novel splice site mutation in an individuals from extreme severity, allowing for
Arab Israeli family [179]. Mutations causing some residual retinal function [116, 118, 161,
LCA were noted in an Indian cohort [47]. 181–183].
The diagnosis and distinction between early- analysed by [191]. Mutations common in Western
onset retinitis pigmentosa and LCA come with populations include CEP290 CRB1, GUCY2D
the extinguished or markedly attenuated ERG RDH12 and RPE65, while common mutations in
obtained in patients with LCA during early Asian populations include TULP1 and GUCY2D.
infancy. Leber in his early description of the dis- Mutations in the known LCA-associated
ease included the early-onset severe retinal dys- genes may show overlapping phenotypes [29,
trophy. The phenotype in LCA is not only 116, 117, 191, 192]. CRB1 mutations have been
determined by the primary defect but is also detected in RP with PPRPE and also in ARRP
influenced by the genetic background and the without PPRPE, besides causing LCA [193]. As
environment. The dysfunctional biochemical, the number of patients in whom both genotypic
structural and physiological pathways involved and phenotypic data become available, it may be
in vision in LCA have an impact on the pheno- possible to generate a clearer picture regarding
type. All the gene products involved in visual significant genotype-phenotype correlations.
function are diverse in their subcellular locations All LCA-related genes show a high degree of
and functions. The timing of the insult, too, influ- allelic heterogeneity and this contributes to the
ences the phenotypic appearance. The natural increased phenotypic variability [194]. The phe-
passage of time further alters the retinal appear- notypic variability seen in patients from diverse
ance in LCA. Mutations in the known genes can populations with mutations in the same gene fur-
be detected in about 70% of patients with LCA ther proves the diversity of this disease.
[184]. Early severe onset retinal dystrophy is RDH12-associated LCA presents as progres-
noted more commonly in patients with mutations sive rod-cone degeneration perhaps due to the
in LRAT, RDH12 and RPE65. A severe LCA phe- longer survival of cones in these retinas as is also
notype with markedly decreased visual function observed in RPE65-associated LCA. Interestingly,
is noted in patients with mutations in AIPL1 and both these genes are involved in retinoid metabo-
GUCYD. Both LCA and ESORD share remark- lism. With the increasing inv olvement of rods in
able clinical and genetic overlap. LCA and the longer survival of cones, photopic
The natural history of LCA has been reported function may be preserved longer, as noted in
to be stable; however, slow deterioration, or in a RDH12, AIPL1 and RPE65-associated
few cases some transient improvement, too, has LCA. Early rod dysfunction is also noted in the
been noted [23, 116, 183, 185–187]. Summarising carriers of AIPL1-associated LCA [26].
several longitudinal studies, stability of clinical Peripapillary RPE sparing was noted in patients
course and visual function was observed in 75% with RDH12 mutations. Retinal thinning was
of patients with LCA, deterioration in 15% and noted on spectral-domain OCT. Generalised fun-
an improvement in visual function in 10% of dus autofluorescence was diminished [195].
patients [185, 186, 189]. Comparing the pheno- RPE65 and TULP1-associated LCA present
types in patients with a molecular diagnosis, as severe early-onset retinal dystrophies. Absence
patients with GUCY2D mutations have a stable of autofluorescence is observed in the RPE65-
clinical course, while those with RPE65 muta- associated phenotype [182]. Photoreceptor func-
tions show progressive deterioration [162, 187, tion that can be rescued as delineated by the
190]. The collation of several studies with known presence of normal autofluorescence in some
genotypes reveals that more than half of the the patients with LCA in the second decade of life
majority of patients with LCA run a stable visual has been reported [196]. Increasingly poor vision
course, while approximately a fourth of them at night is a common complaint in patients with
improve and less than a fourth deteriorate. mutations in genes affecting the retinoid metabo-
Maculopathy has been noted in patients with lism as noted in RPE65 mutations [116, 187].
mutations in TULP1 CRB1 AIPL1 NMNAT1. Patients with mutations in AIPL1 and RPE65
Twenty-five genes and their phenotypes were have a prominent pigmentary maculopathy [26].
AIPL1-associated LCA shows early macular severe LCA phenotype [26]. Though most LCA
involvement in a significant proportion of variants are rare, some are recurrent, and hence, it
patients. is worthwhile to use a microarray disease chip as
Patients with CRB1 mutations in several a first-pass screening tool in every new case of
instances show a Coats-like phenotype and a LCA. The use of the LCA disease chip in any
thickening of the retinal layers by OCT, unlike given patient with the clinical diagnosis of LCA
that seen in other patients. Retinal thinning is has more than 50% likelihood of detecting one or
easier to understand as it occurs due to thinning two mutations [184].
of the RPE and the photoreceptor layer. Both the The frequency of mutations in each subtype of
presences of a Coats-like phenotype and RP with LCA is dependent on the populations screened.
PPRPE due to CRB1 mutations hitherto lack a In an Indonesian LCA cohort, RPE65 mutations
sufficient explanation. GUCY2D-associated were predominant (9.5%), followed by AIPL1
LCA is a severe but stable phenotype with fre- mutations (4.8%), while no mutations in
quent complaints of photophobia, due perhaps to GUCY2D were detected [56]. On the other hand,
the impaired production of cGMP with the perin a largely North African population, GUCY2D
sistent closure of the cGMP-gated cation chan- mutations were more common [117, 200]. A pre-
nels [114]. CRX- and IMPDH1-associated LCA dominance of CRB1 mutations (~13%) was
is the only subtype of LCA with a dominant observed in the Netherlands and Germany, while
mode of transmission, wherein affected parents CRB1 mutations accounted for 11% and 10% of
have affected offspring and several de novo muta- patients from the United States and North Africa/
tions have been identified [11, 12, 121, 186]. France, respectively [97, 98, 117]. A predomi-
Mutations in rare recessive diseases such as nance of GUCY2D mutations has been detected
LCA may have a founder basis. Therefore, the in patients from Mediterranean countries [117,
presence of founder mutations was explored. A 200]. Patients of Asian descent have mutations in
common Finnish founder mutation in GUCY2D GUCY2D, AIPL1 or RPE65 [26, 47, 157].
causing LCA (2943delG) has been detected even Modifier genes studied so far play a small role in
in an outbred population. F565S appears to be a disease. In LCA the role of modifiers has been
common mutation in GUCY2D occurring in noted in disease progression. Ateliotic maculas
families of North African descent [197]. Another (failure to achieve perfection) have been noted
common founder mutation detected in RPE65 wherein the macula failed to develop or function
(Y368H) accounts for disease in an isolated as normal in CRX-related LCA [11, 12, 201,
Dutch population [162]). A founder mutation 202]. Severe ateliosis of the macula has been
(c.95-2A > T; IVS2-2A > T) in RPE65, in a North noted in AIPL1- and RDH12-related LCA, while
African Jewish community, has made it possible foveal aplasia is noted in NMNAT1-related LCA.
to enrol these patients for gene therapy [198]. To Ocular coherence tomography has been a
date, few recurrent mutations have been found in helpful aid in understanding the relative preser-
the LCA genes. The intronic CEP290 mutation vation of the retina and could potentially influ-
(c.2991 + 1655A > G) which creates a cryptic ence the nature of therapeutic intervention.
splice donor site resulting in the insertion of an Characteristically an unlaminated thickened ret-
aberrant pseudoexon into more than 50% of the ina is noted on OCT in CRB1-related LCA [100].
CEP290 transcripts and the W278X mutation in Normal foveal thickness with a central island of
AIPL1 are common. The latter has been detected outer nuclear layer has been noted in CEP290,
homozygously in consanguineous families as LCA5, RPGIP1 and TULP1-related LCA on
well as in combination with other mutations in OCT [86]. GUCY2D-related LCA shows normal
patients born of non-consanguineous union, and retinal thickness and a subnormal macular thick-
it shows a high incidence in populations of South ness. CEP290 severe phenotype, some foveal
East Asian descent [26, 199]. The presence of cones were preserved as noted on OCT [86].
this mutation in most instances leads to a very Peripapillary RPE sparing was noted in patients
with RDH12 mutations, and retinal thinning was reported in patients from Korea [204]. Studies
noted on spectral-domain OCT. from the Middle East, Korea, Japan, Indonesia,
Pakistan and India have helped define mutations
in LCA. RPGRIP1 mutations are more common
18.8 Carriers of LCA Mutations in the reported LCA cohort in Northern Pakistan
[67]. TULP1 mutations were more common in
It has been noticed that several phenotypically Saudi Arabian pedigrees [55]. The detection of
normal carrier parents harbouring heterozygous novel genes in Asian populations may be easier
mutations in the LCA-associated genes have with homozygosity mapping especially in regions
abnormalities in rod or/and cone ERGs, sugges- where consanguinity is high. In Asia, charting the
tive of mild rod or/and cone dysfunction [26, 31, genetic patterns and identifying the genotypes
203]. Drusen-like deposits were noted in carriers will also not only help identify phenotypes in the
of mutations in RPE65, AIPL1, CRB1 and Asian populations but also in the Asian diaspora.
RPGRIP1, while mild peripheral chorioretinal It is also important to identify the genotypes and
atrophy was detected in carriers of AIPL1 and phenotypes of patients with LCA to prepare for
RPE65 mutations [31]. Retinal changes in the therapy. Treatable forms of LCA in Asian popu-
form of inferior retinal atrophy have been lations may be more rapidly identified as both
detected in carriers of CRB1 mutations. These genetic and clinical diagnoses become more
findings indicate that carriers of LCA-associated streamlined. Greater collaboration, reliable core
mutations can now be identified in several LCA sequencing facilities, sharing of clinical and
families based on ERGs showing mild rod or genetic information and easy access to public
cone dysfunction. Biallelic or triallelic mutations databases have made it easier to collate data and
have been detected, and they may modify the understand the significance of various genetic
phenotype [47]. changes. Genetic testing, as part of the clinical
workup of ophthalmic patients, is more common
now in several centres, and liaising with labs in
18.9 Asian Perspective different parts of the world to process samples of
DNA and provide genotyping results will become
Asian populations are greater than the popula- more valuable to patients where complete facili-
tions in Western countries; however, published ties for all genetic diagnosis are not yet available.
data from Asia regarding LCA is small. This dis- It has also become cost-effective to share facili-
order which affects 1–3 in a 100,000 worldwide ties and use genetic laboratory services far from
is likely underreported in some parts of Asia. In home to obtain useful information for patients
regions where consanguinity is high, the preva- and clinicians.
lence may be higher. In some regions consan-
guinity is the norm and not an exception, making
autosomal recessive disease more prevalent. 18.10 Management
Sequencing Han Chinese and Korean genomes
enabled identification of SNPs, variants and The diagnosis for most LCA patients has meant a
mutations in these ethnic groups. More Asian lifetime of coping with debilitating visual loss as
genomes may be sequenced in future which will there have been no treatment options. In the last
aid in the better understanding of SNPs, variants few decades, greater molecular understanding of
and mutations in Eastern populations. the disease has been possible, paving the way for
Just as some causal genes are greater in some a new look at treatment options. In a continuing
Western populations (CEP290 mutations), some effort to understand the evolution and natural his-
may be greater in the diverse Eastern popula- tory of the disease, it is important to examine the
tions. Mutations in CEP290 have not been patients at regular periodic intervals. Serial fundus
photography, optical coherence tomography 18.11 Gene Therapy

(OCT) and electroretinography (ERG) document
any structural and functional changes. As apoptosis may be the final common pathway
For AR LCA, there is a one in four risks for of cell death in retinal degeneration, modifying
offspring being affected when parents are carri- the process could delay degeneration. Gene ther-
ers. Recurrence risk for the progeny of affected apy using trophic growth factors enhances the
patients is low in the absence of consanguinity, as survival both ultrastructurally and functionally of
noted in a previous study [117]. In some parts of photoreceptors in retinal degeneration.
Asia, patients with this largely recessive disease Great progress has been made in gene therapy
may have a strong family history because of con- studies and the use of animal models in retinal
sanguinity. When considering AD LCA in genetic disease [207]. The retina is considered to be a
counselling, the recurrence risk for children of region of immune privilege, thus making it a via-
affected patients is 50%. The stigma of this blind- ble area for gene therapy [208]. Genetically
ing disease is worse outside the close confines of based therapy could involve either replacement
kinsmanship in some rural populations. or suppression of dominant-negative mutations.
The present practical management for LCA Adenovirus vectors are non-enveloped, double-
patients is visual rehabilitation and training with stranded DNA viruses capable of gene transfer in
visual aids. Supportive management has included retinal cells. Adeno-associated and recombinant
the correction of refractive errors, cataract extrac- adenovirus vectors have been used successfully
tion, intraocular lens implantation and strabismus for gene transfer in the retina [209, 210]. Adeno-
surgery. On the other hand, novel treatment for associated virus (AAV) and lentivirus have been
LCA is becoming a real option now, although used to transfect RPE cells with therapeutic genes
considerable preparation is necessary in terms of to delay photoreceptor death in rodents [207,
genotyping and phenotypic analysis. Gene ther- 211]. The size of genetic material to be packaged
apy trials for RPE65-associated LCA have been in viral DNA for effective transfection has led to
underway in four centres. Gene therapy involves the use of lentiviruses in animal models.
a normal copy of a curative gene being intro- Intraocular gene transfer via subretinal injec-
duced into target cells to restore its function. The tions, notwithstanding uveitis, has shown remark-
success story of gene therapy for RPE65- able recovery of photoreceptor function in Briard
associated LCA provides an insight into potential dogs with RPE65-associated LCA. Swedish
cures for other types of inherited childhood Briard dogs with a naturally occurring 4 bp dele-
blindness. tion in RPE65 showed improvement of electro-
Navigational vision and an ability to appreci- retinographic function following injections with
ate contrast are vital for patients. Rods, being recombinant adeno-associated virus (AAV) car-
necessary for cone survival, become essential to rying normal RPE65 complementary DNA
replace degenerating rods to salvage the cones (cDNA) [212]. An added milestone in the treat-
and thus preserve vision. On the other hand, it ment of congenital blindness as observed in LCA
may also be beneficial to protect the cones from is the use of successful in utero gene therapy. The
rod-mediated cell loss. Either trophic factors or in utero delivery of human RPE65 cDNA to the
pharmacologically derived nano-molecules mim- RPE cells using AAV2/AAV1 capsid in RPE65
icking the same function could be used to prevent knockout mice results in efficient transduction
the degeneration of cones which are so vital for with measurable levels of rhodopsin and restora-
vision [17, 205, 206]. Gene therapy to correct the tion of visual function [213].
underlying molecular defect, pharmacotherapy, Gene replacement therapy for RPE-based
retinal cell transplantation and the use of visual retinal dystrophies rescues photoreceptor function
prosthesis are the way forward. as noted in the RCS rats with wild-type mertk
therapy and in Briard dogs with RPE65 gene ther- the Goldmann visual field examination and
apy [214]. Interestingly, out of 55 blind dogs used mobility testing to assess differences in the abil-
in the study, 90% began to recover vision which ity of the patients to navigate a standardised
demonstrated that inherited blindness could be obstacle course before and after the therapy.
improved. This led to three contemporaneous These tests results have shown improvement in
human clinical trials in three different centres, visual acuity, mobility, visual behaviour and reti-
including Moorfields Eye Hospital, UK; Children’s nal functions after treatment [222].
Hospital of Philadelphia, USA; and Scheie Eye No serious adverse effects or systemic toxicity
Institute of the University of Pennsylvania with has been reported. However, some adverse out-
Shands Children’s Hospital of the University of comes that were observed in few cases include
Florida. Published results from these trials were retinal detachment, choroidal effusions, transient
ground-breaking [215, 216]. All the patients had a ocular hypotony and ocular hypertension related
modest improvement in measures of retinal func- with the administration of topical steroids. The
tion and on subjective tests of visual acuity. These outcome of a recent study for a long-term post-
studies have provided the basis for further gene treatment follow-up in LCA patients as well as in
therapy trials in patients with LCA. Since then, canine models showed that gene therapy improves
several LCA gene therapy clinical trials accessing vision but photoreceptor degeneration progresses
safety and efficacy have been performed success- unabated [220]. The early effect of therapy up to
fully [217–219]. The results of clinical trials using 6–12 months sustains visual improvement, but
RPE65 gene therapy in adults initially and chil- over a period of time, there was no consistent
dren later have proved the safety and efficacy of improvement in visual acuity, and there was no
this form of therapy albeit with a non-persistence improvement in the ERG along with the decrease
of visual restoration. in retinal thickness measured by OCT [223]. The
A crucial step in gene therapy for LCA lack of long-term effects of gene augmentation
involves the delivery of the vector carrying the therapy suggests a need for a combinatorial strat-
RPE65 gene that enables it to transfect the target egy for effective outcome. Besides gene replace-
cells to induce subsequent in vivo generation of ment therapy, more recently genetic editing has
the RPE65 protein. The subretinal space has been become an option for patients with the common
used by many researchers to inject the vector as it Cep290 splice site mutation (c.2991 + 1655A > G)
acts as a relatively immune privileged site and wherein antisense oligonucleotide (ASO) treat-
also easily transfects the target cells – the retinal ment restores pre-mRNA splicing thereby
pigment epithelium and photoreceptors. In post- increasing CEP290 protein so necessary for effi-
treatment follow-up, it was observed that safety cient ciliary transport in photoreceptors [224,
and efficacy of gene transfer with rAAV2-RPE65 225]. Clinical trials are now underway using
vector in LCA patients extended to at least 1 year ASO for Cep290-related LCA.
[220]. In the 3-year follow-up study, it was shown The RPE65 gene replacement trials by
that there was no evidence of systemic dissemi- Bainbridge et al. noted that the effect of the gene
nation of vector sequences and no evidence of function decreases over time and the natural
humoral immune response or cell-mediated course of retinal degeneration continues.
immune responses to AAV2 capsid or RPE65 McGuire and Testa noted a sustained stable
protein [221]. improvement of acuity nystagmus and visual
Posttrial objective measures included evalua- filed over 3 years [215, 222, 226]. This gene
tion of the pupillary light reflex, nystagmus test- replacement trial could also be applicable in LCA
ing and optical coherence tomography. Subjective caused by mutations in AIPL1 CEP290 GUCY2D
measures included standard tests of visual acuity, LCA5 and RPGRIP [227].
18.12 Pharmacotherapy tent stem cells (iPSC) and fulfilling the need for
functional RPE and photoreceptor cells are a step
Human ciliary neurotrophic factor (CNTF) seques- towards ameliorating immunologic adverse
tered in semipermeable capsules and implanted in events noted in allogenic transplants [234]. iPSC
the vitreous cavity maintain cone survival [228, cells have been used to evaluate novel mutation
229]. Since both rod and cone dysfunction occurs pathogenicity and gene-based rescue.
in LCA, the use of trophic factors in this disorder Ciliogenesis has been restored using CEP290
could be explored. The protective effect of neuro- gene transfer in cells proving another avenue for
trophic factors like ciliary neurotrophic factor has gene- and cell-based therapy [235].
been exploited in gene transfer, and a delay in reti-
nal degeneration has been noted [230].
Oral treatment using multiple doses of 18.14 Retinal Prosthesis
QLT091001 (QLT) which is a stable synthetic
compound converted in the body to 9-cis retinal Using the intact inner retina to carry impulses to
that combines with opsin to form isorhodopsin the visual cortex has been possible using retinal
and is able to activate the phototransduction cas- prosthesis. The use of a visual prosthesis whereby
cade on exposure to light improves visual func- electrical stimulation bypasses damaged nerve
tion in RPE65 and LRAT-related LCA [231]. endings has been used in the treatment of retinal
This drug bypasses the block in the retinoid cycle degenerative disorders [236]. Both the crude
and concentrates in the retina improving visual appreciation of the sensation of light and the abil-
fields, acuity and functional MRI values. ity to detect motion have been possible using
these prostheses [237]. The Argus system works
by direct stimulation of the inner retina via epi-
18.13 Cell Transplantation retinal microelectrodes. Visual information gath-
ered by a video camera mounted on external
Replacement stem cells provide mutation- glasses is converted to pixelated images by an
independent regenerative therapy. Since LCA is a external processor, and this is transmitted to the
heterogeneous disease implicating genes involved microarray electrode array at the macula which
in several different pathways, the use of cell in turn transmits it to the occipital cortex via the
transplantation circumvents individual gene- optic nerve [238, 239]. The occipital cortex is not
related mechanisms of overcoming retinal degen- totally dysfunctional in the absence of light-
eration. Transplantation serves to replace sensitive input, but activation of the visual cortex
damaged cells or provide growth and survival is observed by positron emission tomography
factors to cells to prevent further cell death. The (PET) scanning and during functional magnetic
role of stem or precursor cell transplants in reti- resonance imaging while Braille reading. The
nal degenerative disease provides a potential period of cross-modal plasticity extends into the
route to photoreceptor cell renewal. Photoreceptor second decade of life in blind patients [240].
survival following stem cell transplants occurs Transretinal electrical stimulation using multi-
due to the release of trophic factors both close to channel arrays have shown results [241].
the transplant and away from it [17, 206]. Rod- However, electronic photoreceptor prosthesis are
dependent cone viability factors play a role in the limited by their biocompatibility, longevity, sta-
rescue process [232]. Human embryonic-derived bility and image quality [242]. Neuroprosthesis
stem cell RPE and pluripotent stem cell-derived have been used to help with visual rehabilitation
RPE have been used in LCA to recover visual [243]. Promising results with persistent VEPs
function [233]. Converting patient-specific were observed in canine models with an Okayama
somatic cells to be reprogrammed into pluripo- University-type retinal prosthesis (OURePTM),
a photoelectric dye-coupled polyethylene film a comprehensive resource on human genetic vari-

that generates electric potential in nearby neu- ation. The data from the 1000 Genomes Project
rons in response to light [244]. was made available to the worldwide scientific
Implantation of a cortical stimulation unit community through freely accessible public
attached to a camera and a laptop computer has databases.
been able to increase visual potential [245]. As Later the 100,000 Genomes Project, a UK
both tactile and auditory pathways are well devel- Government initiative, was launched to sequence
oped in subjects with profound loss of vision, whole genomes from patients using the National
voice and tactile technology are being made use Health Service. The focus is on rare diseases,
of to simulate vision [246]. Using optogenetics to cancers and infectious diseases. The genomic and
restore degenerated retina relying on the intact clinical data are linked and made available to
inner neural pathway to convey impulses has patients, clinicians and researchers making it
offered some treatment possibility [247]. possible to understand individuals and their dis-
The genetic introduction of light-sensitive eases better when planning treatment options.
opsins to drive the visual pathway has been sug- LCA in most instances results in the profound
gested [248]. loss of photoreceptor function, and gene therapy
directed at this disease involves long-term stable
expression of transgenes for several decades.
18.15 Future Perspectives Although the pace of translational research in
retinal degenerative diseases has been fairly fast,
Animal models of LCA have increased our com- several difficulties still need to be overcome. The
prehension of the disease and have provided an duration of gene expression over a lifetime to
avenue for clinical trials, although they may not confer therapeutic efficacy, indelibility of transla-
truly represent human disease. Molecular diag- tional control of transgene expression and the
nosis in LCA will permit better prediction of improved safety of therapy continue to be impor-
visual prognosis. tant issues.
Gene therapy, cell transplantation, New patient cohorts in Asia, in addition to
pharmacogenomics- based therapy and retinal characterised European/American cohorts, could
prosthesis are all future considerations in the be instrumental in finding new candidate genes
treatment of this disease. Pioneering treatment LCA. In India established ocular research centres
for LCA involves issues of consent especially in and medical research foundations are involved
the paediatric age group, ethical approval, safety with research in patients with LCA focusing on
and the efficacy of new clinical trials. With involved genes, mutations, phenotypes and
greater collaboration within the scientific com- molecular mechanisms in the pathogenesis of the
munity, the pharmaceutical industry and the reg- disease. Similar centres in other parts of Asia
ulatory bodies will propel the therapeutic options have been instrumental in identifying mutations
for this rare congenital retinal dystrophy. in patients with LCA in order to stratify patients
Providing support to patients and their families is for treatment. Autozygosity-guided sequencing
furthered by support groups who have also helped approach may be used as a powerful tool in
accelerate research and involvement in clinical detecting novel genes.
trials. Finding novel genes expressed in the RPE and
The aim of the 1000 Genomes Project was to the photoreceptors using the Affymetrix
find genetic variants with frequencies of at least GeneChip technology will provide valuable
1% in the populations studied. The 1000 Genomes information regarding candidate genes, and this
Project took advantage of cost-effective sequenc- will be of great value in gene identification.
ing and was the first project to sequence the Variability in the human genome in people of
genomes of a large number of people, to provide Asian origin is now being studied, and the
100,000 genome project (publically funded UK Most mutations detected are in coding regions
National Health Service project) will shed further of genes, but there is mounting evidence for
light on the significance of variants in different hidden non-coding changes or structural variants
ethnic groups and their impact on disease. altering protein function. The databases of eye
The first Asian genome of the Han Chinese diseases, exome variant server and exome
and the later sequencing of the Korean genome sequencing data in addition of sharing of pheno-
have showed several differences in minor allele typic information will increase the knowledge of
frequency, novel single-nucleotide polymor- the disease and provide valuable insight into
phisms (SNPs) and copy number variants (CNVs) treatment options that may be more
which could potentially affect the expression of gene-specific.
several genes. These insights have led to the Autozygosity mapping being more time-
importance of sequencing several other ethnic consuming and less cost-effective in comparison
genomes in the 1000 genome project with exome sequencing yields better rates of
TGP. Cataloguing human variation in different detection for pathogenic changes.
populations will also be helpful in understanding Providing replacement for the mutant factors,
minor allele frequencies, disease variants and inhibiting apoptotic pathways or stimulating anti-
traits in other Asian populations [249]. apoptotic pathways, downregulating mutant gene
Despite targeted next-generation sequencing expression, providing gene replacement and spe-
and analysis of copy number variants (CNV), cifically targeting gene correction will help pave
causative mutations have not always been identi- the way for treating this hitherto untreatable
fied because they occur in non-coding regions disease.
and 5’ UTR in disease genes. Including these
hidden mutations in gene chips and panels will
increase detection rates. 18.16 Summary
Understanding modifiers and their role in dis-
ease will shed light on the molecular mechanisms During the past two decades, several populations
involved in LCA. Gene therapy in animal models of patients have been tested genetically using
of AIPL1, GUCY2D, RPGRIP1 and CEP290 has gene panels; the numbers of genes incorporated
also been promising, paving the way for human in these panels increased year on year as more
trials. The establishment of better diagnostics and were discovered. The percentage of gene muta-
treatment options for LCA would herald a new tions identified has not always reflected the true
phase of genomic medicine and hope for families. frequency of gene mutations in any given popula-
Next-generation sequencing allows unpar- tion. However, certain mutations like the W278X
ralled sequencing at an affordable cost in a timely in AIPL1 are consistently higher in Asian popula-
fashion. Exome sequencing of the known LCA- tions. The frequency of the intronic mutation
related genes is a reliable first-pass test when the (c.2991 + 1655A > G) in CEP290 is higher in
clinical diagnosis has been confirmed but the outbred Western populations of America and
genotype is yet to be identified. The detection of Europe unlike in Asia. CEP290 mutations have
founder mutations will simplify mutation detec- not yet been identified in Arabian populations.
tion in inbred populations. Outbred populations With the identification of more mutations in
will validate the results obtained from inbred Asian cohorts, the LCA microarray mutation
ones. chip could be updated, and its use as a first-pass
WES is a powerful tool for rapid analysis of genetic screening tool could potentially yield
known disease genes in large patient cohorts. greater results.
In the 1990s linkage analysis and candidate Leber’s congenital amaurosis and fine mapping of
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The Genetics of Inherited Retinal
Diseases in the Israeli 19
and Palestinian Populations:
A Lesson from Populations
with High Rates of Consanguinity
Mor Hanany and Dror Sharon
Abstract large variability of retinal phenotypes among

Inherited retinal diseases (IRDs) are disorders patients, while mutations in the same gene can
that cause visual loss mainly due to photore- result either in the same phenotype or variable
ceptor degeneration. The prevalence of IRDs phenotypes that are usually mutation-
in the Israeli and Palestinian populations was dependent. There is currently no cure for the
reported to be higher compared to other stud- vast majority of IRD types; however recent
ied populations. The structures of the Israeli advances bring new hope for curing or at least
and Palestinian populations are unique mainly delaying the degeneration process in the near
because of the large number of ethnic groups. future.
In addition, high rates of consanguinity and
intra-community marriages resulted in a high Keywords
proportion of families with autosomal reces- Consanguinity · Gene · Genotype-phenotype
sive inheritance patterns. The study of Israeli correlation · Heterogeneous disease ·
and Palestinian IRD families resulted so far in Homozygosity mapping · Mutation ·
the identification of mutations in 74 IRD Population · Retinitis pigmentosa
genes, including 23 novel genes that were
identified mainly using the homozygosity
mapping and whole exome sequencing tech-
niques. The history and tradition of these pop- 19.1 Introduction
ulations led to common founder mutations
that are usually subpopulation-specific. Such Inherited retinal diseases (IRDs) are heteroge-
mutations allow a more efficient genetic anal- neous groups of disorders causing visual loss
ysis in searching for the causative gene. mainly due to degeneration of photoreceptors.
However, some founder mutations are shared IRDs include rod-dominated diseases such as
among different ethnicities and are likely to be retinitis pigmentosa (RP) in which rod photore-
the result of a common origin of these ethnic ceptor loss precedes cone involvement, cone-
groups, which may have an estimated diver- dominated diseases such as cone-rod degeneration
gence time of a few thousand years. There is a (CRD) in which cone photoreceptor loss pre-
cedes rod involvement, and regional degenera-
M. Hanany · D. Sharon (*) tions such as Stargardt disease in which a specific
Department of Ophthalmology, Hadassah-Hebrew retinal region is degenerated. The total number of
University Medical Center, Jerusalem, Israel genes responsible for these heterogeneous dis-

https://doi.org/10.1007/978-981-13-0884-0_19
234 M. Hanany and D. Sharon
eases is still unknown but is estimated to be over autosomal dominant (AD, 30–40%), and
300 (RETNET database at https://sph.uth.edu/ X-linked (XL, 5–15%) [84]. Due to the relatively
retnet/). Heterogeneity is an important feature of high rates of consanguinity and intra-community
IRDs at both the genetic and clinical aspects, and marriages in the Israeli and Palestinian popula-
IRDs are considered the most heterogeneous tions, the AR inheritance pattern is much more
group of inherited diseases in humans. common reaching 80% in the vicinity of
Heterogeneity complicates genetic counseling Jerusalem and even over 90% in the Arab-Muslim
and testing since the mode of inheritance and population [31]. A summary of all reported inher-
potential causative genes are difficult for inter- itance patterns is depicted in Fig. 19.1b clearly
pretation in many cases. The genetic cause of emphasizing the high prevalence of the AR inher-
various retinal phenotypes in the Israeli and itance pattern (92%).
Palestinian populations has been reported so far
(Table 19.1 and Fig. 19.1a), with RP, Usher syn-
drome (USH), achromatopsia (ACHM), and 19.4 Prevalence of IRDs
Leber congenital amaurosis (LCA), being the
most common phenotypes. The prevalence of IRDs in the Israeli and
Palestinian populations has been published for
nonsyndromic RP [31] and achromatopsia [30]
19.2 T
he Structure of the Israeli and is available for congenital stationary night
and Palestinian Populations blindness (CSNB- Sharon, unpublished data). In
all cases, disease prevalence in the Israeli and
The Israeli and Palestinian populations have Palestinian populations was found to be higher
unique structures and include different ethnic than reported in other populations that are known
groups including Jews of various origins, Arab to have lower rates of consanguineous marriages.
Muslims, Bedouins, Arab Christians, and Druze. The prevalence of nonsyndromic RP in the vicin-
Consanguinity and intra-community marriages ity of Jerusalem was found to be 2.5–3 times
have been reported to be high in different ethnic higher compared to the European and American
groups for many generations, stemming from his- populations. Similarly, the prevalence of achro-
toric, ethnic, religious, and cultural causes that matopsia among Arab Muslims who reside in the
shaped the genetic landscape of the populations vicinity of Jerusalem was found to be about
and contributed dramatically to the relatively 1:5000 individuals, which is much higher than
high prevalence of autosomal recessive (AR) dis- the prevalence (1:30,000–1:50,000 individuals)
eases, including ocular diseases, in these popula- reported in various populations. Only one popu-
tions. [30, 31, 83] lation, the Pingelap Island population, was
reported to have a higher achromatopsia preva-
lence of 4–10% [85] due to a single founder
19.3 Inheritance Patterns of IRDs mutation in the CNGB3 gene. The interesting
story of this population and the effect of the dis-
IRDs can be inherited in different patterns, ease on their daily life are well described in The
depending mainly on the population history and Island of the Colorblind book by Oliver Sacks.
structure. About 50% of families include a single
affected individual, and therefore it is not possi-
ble to determine the inheritance pattern prior to 19.5 Genetic Analyses of IRDs
gene identification, although most cases are
likely to be AR. In the USA and Europe, the most Various methods have been used along the last
common inheritance patterns in families with at 30 years to aid in the identification of the genetic
least two affected individuals are AR (50–60%), cause of IRDs, including the traditional posi-
Table 19.1 A list of IRD genes reported to cause retinal diseases in the Israeli and Palestinian populations
Novel identified genes
Inheritance Number of Relevant Novel as Novel as nonsyndromic
Gene name pattern Disease families publication IRD gene IRD gene Genetic analysis method
ABCA4 AR STGD 1 [1] Linkage and HM
6 [2] Gene screening
ACO2 AR Infantile cerebellar-retinal 2 [3] + HM and WES
degeneration
ADAM9 AR CRD 2 [4] + HM
RP 2 [5] WES
AIPL1 AR LCA 5 [6] Gene screening
1 [5] WES
ALMS1 AR CRD 1 [7] WES
ARL2BP AR RP 1 [8] + HM and WES
ARL6 AR BBS 1 [9] + Positional-candidate
approach
ARMC9 AR JS 1 [10] + MIPs and NGS
BBS1 AR RP 1 [5] WES
BBS2 AR RP 4 [11] + WES
BBS4 AR BBS 1 [12] Gene screening
BBS7 AR BBS 1 [12] Gene screening
BEST1 AR Best 1 [13] Gene screening
CDHR1 AR RD 1 [14] WES
1 [15] WES
1 [5] WES
RP 1 [5] WES
CRD 1 [16] HM
CDH3 AR HJMD 4 [17] + HM
19 The Genetics of Inherited Retinal Diseases in the Israeli and Palestinian Populations: A Lesson…

EEM 1 [23] Gene screening
RP 1 [5] WES
235
(continued)
236

CEP78 AR CRD + SNHL 5 [24] + HM and WES
CEP250 AR USH 1 [25] + HM and WES
CEP290 AR LCA 1 [26] HM
CERKL AR RD 7 [27] HM
CHM XL CHM 1 [28] WES
CNGA1 AR RP 2 [5] WES
CNGA3 AR ACHM 10 [29] Gene screening
CNGB1 AR RP 1 [5] WES
CNGB3 AR ACHM 8 [30] Gene screening
CNNM4 AR Jalili syndrome 2 [32] + Positional-candidate
approach
1 [5] WES
CRB1 AR LCA 1 [33] HM
8 [34] Gene screening and HM
Early RP 1 [35] Linkage analysis
5 [34] Gene screening and HM
RP 5 [34] Gene screening and HM
CYP4V2 AR RP 1 [5] WES
C2ORF71 AR RP 1 [36] + HM
1 [25] WES
1 [5] WES
C8ORF37 AR RP 2 [37] + HM and NGS
1 [15] WES
DHDDS AR RP 9 [38] + HM
7 [31] Founder screening
M. Hanany and D. Sharon
EYS AR RP 10 [39] HM
1 [26] HM
1 [28] WES
FAM161A AR RP 20 [40] + HM
1 [41] HM
1 [5] WES
GUCY2D AR LCA 4 [6] Gene screening
1 [42] HM
AD CRD 6 [15] WES
HGSNAT AR RP 2 [43] + WES
IDH3A AR RP 1 [44] + WES
IMPG2 AR RP 2 [45] + HM
KCNV2 AR CDSRR 5 [46] Gene screening
LCA5 AR LCA 1 [26] HM
MAK AR RP 1 [47] + HM and WES
1 [5] WES
MFRP AR RP 1 [26] HM
MTTP AR Abetalipoproteinemia 6 [48] Gene screening
MYO7A AR USH1 15 [49] Gene screening
2 [50] HM
1 [51] HM and WES
2 [28] WES
NPHP4 AR SLS 1 [28] WES
NRL AR RP 1 [5] WES
ESCS 2 [52] Gene screening
NR2E3 AR ESCS/GFS/RP 10 [53] Gene screening
2 [26] HM
1 [5] WES
PAX6 AD Aniridia 1 [54] Gene screening

PCDH15 AR USH1 8 [56] Linkage
237
(continued)
238

PDE6A AR RP 1 [5] WES
PDE6B AR RP 1 [51] HM and WES
PDE6G AR Early RP 1 [58] + HM
PEX6 AR Zellweger syndrome 1 [59] Gene screening
Heimler syndrome 1 [60] Gene screening
PLA2G5 AR Late-onset RP 1 [5] WES
PRCD AR RP 9 [61] HM
PROM1 AR CRD 1 [62] HM
1 [26] HM
1 [63] HM
PRPF3 AD RP 1 [5] WES
RAB28 AR CRD 1 [64] + HM and WES
RDH5 AR FAP 14 [65] Gene screening
1 [5] WES
RDH12 AR Early RP 1 [35] HM
7 [26] HM
4 [5] WES
RHO AD RP 9 [66] Gene screening
OPN1LW/ XL BCM 6 [67] Gene screening
OPN1SW
RPE65 AR LCA 10 [6] Gene screening
1 [5] WES
RP 2 [5] Gene screening
RPGR XL RP 1 [68] Gene screening
2 [28] WES
RP1 AR RP 1 [5] WES
RP2 XL RP 1 [31] Gene screening
SCAPER AR RP 1 [69] + WES
SLC38A8 AR Foveal hypoplasia 3 [70] HM and WES
M. Hanany and D. Sharon
SPATA7 AR LCA 1 [5] WES
Early RP 1 [71] HM
TRIM32 AR BBS 1 [72] + Positional-candidate
approach
TSPAN12 AR FEVR 1 [5] WES
1 [73] WES
TULP1 AR LCA 1 [74] Linkage
2 [26] HM
1 [5] WES
USH1C AR RP 9 [75] + WES
USH2A AR USH2 3 [76] Gene screening
USH2, RP 1 [77] Gene screening
RP 2 [5] WES
USH3A AR USH3 5 [80] Linkage and gene
screening
USH1G AR USH1 1 [50] WES
ACHM achromatopsia, BBS Bardet-Biedl syndrome, BCM blue cone monochromatism, CDSRR cone dystrophy with supernormal rod response, CHM choroideremia, CRD cone-
rod degeneration, EEM ectodermal dysplasia, ectrodactyly, and macular dystrophy, ESCS enhanced S-cone syndrome, FAP fundus albipunctata, FEVR familial exudative vitreo-
retinal disease, GFS Goldmann-Favre syndrome, HJMD congenital hypotrichosis associated with juvenile macular dystrophy, HM homozygosity mapping, JS Joubert syndrome,
LCA Leber congenital amaurosis, MIPs molecular inversion probes, RD retinal degeneration, RP retinitis pigmentosa, SLS Senior-Loken syndrome, SNHL sensorineural hearing
loss, STGD Stargardt disease, USH Usher syndrome, WES whole exome sequencing
239
tional cloning approach that is based on genetic genes have been reported during the last 7 years
linkage, the gene candidate approach, homozy- (Fig. 19.2- green bars), mainly using
gosity mapping, and whole exome sequencing next-generation sequencing techniques, such as
(WES). The approach to be used is determined WES. Although most of the novel genes were
by multiple factors and mainly the population identified by analyzing a small number of
structure, clinical phenotype, and inheritance Israeli and Palestinian families, some were
pattern. In many Asian populations, consanguin- found to be major IRD genes in these popula-
ity rates are relatively high, and therefore the tions, including CDH3 (16 families), FAM161A
homozygosity mapping approach was a major (25 families), and DHDDS (16 families), and
tool for gene identification. In this method, the all three have been identified using the homo-
genome of a patient is analyzed by studying the zygosity mapping approach. FAM161A, for
genotype of a large number of polymorphisms example, has been identified by performing
allowing one to determine which genomic homozygosity mapping on a large number of
regions are at a homozygous state. Such regions consanguineous families with RP leading to the
are highly likely to contain the causative muta- identification of a large homozygous region on
tion not only in consanguineous families but also chromosome 2 [40], overlapping with a previ-
in families of intra- community marriages. ously reported RP locus (RP28). Screening all
Disease-causing mutations have been reported genes within the interval led to the identifica-
thus far in 74 genes in the Israeli and Palestinian tion of three null mutations in FAM161A, which
populations (Table 19.1), with CNGA3 and was not considered as a good candidate prior to
FAM161A being the causative genes in the largest mutation screening. Similarly, DHDDS has
number of reported families (Fig. 19.1c). Most of been identified by homozygosity mapping in
the studies have been reported since 2007 small families of Ashkenazi Jewish origin and
(Fig. 19.2- red graph). Homozygosity mapping gene prioritization [38].
was indeed a valuable tool for gene identification The identification of PDE6G as a novel cause
(Table 19.1) and was used as the major mapping of early-onset RP [58] is unique and tangled with
technique or in conjunction with other methods the history of the Arab-Muslim population in
such as WES, to more accurately locate the caus- North Israel, who live in small towns, which
ative gene. were originally settled by a small number of
related individuals. Therefore, most of the genetic
diseases frequent among Israeli Arabs are due to
19.6 I dentification of Novel IRD founder effects. Using genome-wide homozy-
Genes gosity mapping, Dvir and colleagues [58] identi-
fied a homozygous 4.7-Mb region on chromosome
Due to the large number of genes in which muta- 17q25.3 in patients who reside in the same town
tions can cause IRDs, each studied population and suffer from early-onset RP. The authors then
shows a different proportion of contributing applied the candidate approach and identified
genes. Therefore, newly studied populations have an excellent candidate gene within the linked
a high probability to aid in the identification of region: PDE6G which encodes for the inhibitory
novel disease-causing genes. γ subunit of rod photoreceptor cyclic GMP phos-
Studying Israeli and Palestinian IRD fami- phodiesterase. A homozygous canonical splice-
lies resulted so far in the identification of 23 site mutation was then identified using Sanger
novel genes: 20 that were not previously asso- sequencing of all coding exons. Interestingly,
ciated with a retinal disease and 3 that were ini- although the mutation was missing in 256 Arab-
tially associated with syndromic IRD and later Muslim controls, a relatively high percentage of
were found to cause a nonsyndromic IRD phe- controls who reside in the same village (7 out of
notype, usually in a genotype-phenotype corre- 84, carrier frequency of 8.3%) were found to be
lation (Table 19.1). The vast majority of novel heterozygous for this mutation.
19 The Genetics of Inherited Retinal Diseases in the Israeli and Palestinian Populations: A Lesson… 241
Fig. 19.1 Pie chart analyses of IRD characteristics in the which mutations were reported in the Israeli and
Israeli and Palestinian populations. (a) Different IRD phe- Palestinian populations. (See also Table 19.1)
notypes. (b) IRD Inheritance patterns. (c) IRD genes in
19.7 F
ounder Mutations and Their tively frequent existence of an AR disease in an
Importance isolated population usually suggests a founder
effect. The Arab-Muslim and Jewish populations
Founder mutations are pathogenic variants in Israel and the Palestinian territories are specu-
observed in a group of individuals that is or was lated, mainly by historical records, to originate
geographically or culturally isolated, in which at from a single founder population, with an esti-
least one of the ancestors carried them. The rela- mated divergence time of a few thousand years.
Table 19.2 IRD-causing founder mutations in the Israeli and Palestinian populations
Gene Mutation name Number of families Subpopulation References
CNGA3 p.Val529Met 9 AMJ [29, 30]
4 OJ [29]
p.Ile314del 12 AMJ [30]
DHDDS p.Lys42Glu 16 ASH [31, 38]
EYS p.Thr135Leufs*26 13 NAJ [26, 31]
FAM161A p.Arg523* 5 Mainly NAJ [40]
p.Thr452Serfs*3 19 [5, 31, 40]
MYO7A p.Ala826Thr and IVS4+2T>G 7 NAJ [49]
PCDH15 p.Arg245* 10 ASH [56, 57]
PRCD p.Arg22* 9 AMO [61]
RPE65 IVS2-2A > T 10 NAJ [6]
USH1C p.Gly407Glufs*58 9 YJ [75]
USH3A p.Asn48Lys 18 ASH [80–82]
AMJ Arab Muslims (vicinity of Jerusalem), AMO Arab Muslims (other areas), ASH Ashkenazi Jews, NAJ North African
Jews, OJ Oriental Jews, YJ Yemenite Jews
Fig. 19.2 IRD-related publications and novel genes and 2017. The most common novel genes identified by
reported by analyzing Israeli and Palestinian families. The studying the Israeli and Palestinian populations are
graph shows the number of publications (red line) and marked by arrows
novel genes (green bars) reported between the years 1997
Founder mutations identified in the Israeli has its own unique set of founder mutations. The
Jewish population, and mainly in Ashkenazi Ashkenazi Jewish population segregates a variety
Jews, were also reported to be prevalent among of genetic conditions caused by prevalent founder
non-Israeli Jews residing in North America [38, mutations, including Tay-Sachs disease, cystic
86]. At least 40 Mendelian disorders have been fibrosis, and Usher syndrome [87]. Founder
described in various Jewish subpopulations [87]; mutations in the Israeli population are usually
for some disorders, each of these subpopulations restricted to a specific ethnic group, e.g., the
USH3A, p.Asn48Lys mutation in Ashkenazi Jews 19.8 Genotype-Phenotype

[81], or the CERKL, c.238+1G>A mutation Correlations
among patients of Jewish Yemenite origin [27].
On the other hand, some mutations have been Different mutations within the same gene usually
identified in multiple Jewish ethnic groups, result in the same phenotype. Out of the 74 genes
including two founder mutations in FAM161A listed in Table 19.1, mutations in 63 of the genes
that were identified in Jews from three different (85%) have been described to cause a single phe-
origins (Ashkenazi Jews, Oriental Jews, and notype in the studied populations. On the other
North African Jews) [40]. These mutations were hand, a few unique cases show variable pheno-
not reported so far in other populations. types that can be mutation-dependent.
Interestingly, a few disease-causing mutations An example of a clear genotype-phenotype
are shared among the Arab-Muslim and the correlation has been reported in the USH1C gene
Jewish populations, most of which are pan-ethnic [75]. Dozens of different mutations in USH1C
and appear in almost every studied population are known to cause Usher syndrome type 1 (con-
(e.g., the p.Gly1961Glu variant in ABCA4). genital sensorineural deafness, vestibular dys-
However, one CNGA3 mutation tells a highly function, and RP). However, using a combined
interesting story [29]. The c.1585G>A (p. approach of homozygosity mapping and WES, a
Val529Met) mutation in this gene has been homozygous frameshift mutation has been iden-
reported in Arab Muslims from the vicinity of tified in 16 patients of a Yemenite Jewish origin
Jerusalem, Oriental Jews, as well as a few with RP and either normal hearing or late-onset
European families. Haplotype analysis demon- (after the age of 40 years) mild-to-severe hearing
strated that the European alleles have at least two loss. What is special about this particular muta-
different origins that are clearly different from tion that does not result in severe early-onset
the one that is shared by the Arab Muslims and deafness? Similar to other genes in the human
Oriental Jewish patients who carried this allele. genome, USH1C can produce at least four differ-
This shared haplotype was relatively large, ent proteins through the alternative splicing
spanning 21.5 cM (~11 Mbp) with an estimated mechanism. The identified mutation (c.1220del;
age of about 5000 years, indicating a common p.Gly407Glufs*58) is located in an alternative
origin of these two ethnic groups. exon (#15) that is mainly expressed in the retina,
A classical founder mutation in the Arab- and therefore loss of this isoform leads mainly to
Muslim population is the one reported in the retinal degeneration.
PRCD gene [61]. Prior to this study, only a single Mutations in some genes, however, can lead to
patient (residing in Bangladesh) has been reported variable phenotypes, with no clear genotype-
to suffer from RP due to a PRCD mutation. By phenotype correlation. The best example is prob-
analyzing a large set of patients (18 individuals ably the c.238+1G>A (IVS1+1G>A) mutation in
who belong to nine families) residing in the same the CERKL gene [27]. This founder mutation is
Arab-Muslim village, the authors identified a relatively common among patients of Yemenite
homozygous nonsense mutation in PRCD with a Jewish origin with a carrier frequency of 4.4%.
high carrier frequency (10%) in normal controls Interestingly, among the 24 patients who shared
who reside in the same village. These results the same CERKL genotype (homozygous for
therefore provide a strong and important confir- c.238+1G>A), some were diagnosed with RP,
mation for the role of PRCD in the etiology of RP while others were diagnosed with cone-rod
in humans. degeneration (CRD). Electroretinography (ERG)
The identification and characterization of such analysis of the patients did not help in clarifying
common mutations in a specific ethnic popula- the clinical dilemma, as often a similar degree of
tion allows sensitive and specific use of genetic rod and cone dysfunction was present rather than
testing for carrier screening, genetic counseling, preferential involvement of one photoreceptor type
and diagnostic purposes. or the other [27]. In addition, marked variability
in ERG responses has been noted among patients, photoreceptor degeneration [89]. Aiming to study
similar to the variability reported among patients the effect of 9-cis β-carotene on progression of
who carry mutations in the same gene. There is retinal degeneration, 29 patients with RP of
currently no clear explanation for such variabil- unknown etiology were treated with 9-cis
ity, but environmental factors as well as modifier β-carotene in a randomized crossover trial [90].
genes might contribute to this phenomenon. Although no improvement was evident in visual
acuity, treated patients had better retinal function
based on ERG testing.
19.9 Therapeutic Modalities As new treatment modalities are constantly
in IRDs being developed in Israeli research laboratories,
including stem-cell therapy, retinal implants,
There is currently no cure for the vast majority nanotechnology, and artificial vision, this field is
of IRD types; however recent parallel advances likely to progress rapidly during the coming
in various fields bring new hope for curing or at 5–10 years.
least delaying the degeneration process in the
near future. One of these modalities is gene aug-
mentation therapy in which an adeno-associated 19.10 Future Perspective
virus (AAV) containing a normal copy of the
gene (in which mutations are the cause of dis- Identification of the vast majority or even all
ease) is injected into the subretinal space. Three genes that cause IRDs in the Israeli and
Israeli patients who are homozygous for a Palestinian populations is a matter of time. As
founder RPE65 splicing mutation underwent this DNA analysis dramatically improves and inter-
treatment [6], leading to an increase in vision in national collaborations become the prominent
the treated area as early as 15 days after the way of collaboration, the route for identifying all
intervention. An interesting story in this regard is genes and mutations is relatively short. It is
the CNGA3 gene. While in Europe and in the likely to assume that all major causative genes
USA, mutations in CNGB3 are the major cause have been identified in populations that were
of achromatopsia, in the Israeli and Palestinian studied during the last 10 years and the remain-
populations, two major founder mutations in ing genes are likely to affect a relatively small
CNGA3 make this gene the major cause of the number of families worldwide, making interna-
disease (84% of solved achromatopsia families) tional collaborations across countries and conti-
[30]. A careful clinical assessment of CNGA3 nents crucial to prove the pathogenicity of
patients showed that in over 50% of patients, mutations identified in novel genes. Consortia
rods are also involved and that under dark- and like the European Retinal Disease Consortium
light-adapted conditions, patients use rod-medi- (ERDC, http://www.erdc.info) and the Asian
ated pathways. This study determined the effi- Eye Genetics Consortium (AEGC, http://www.
cacy outcome measures for gene augmentation asianeyegenetics.org) are likely to enhance this
therapy of CNGA3 that will include chromatic process dramatically. This progress will lead to
light-adapted psychophysics, with attention to more focused efforts in developing therapeutic
the photoreceptor basis of the response, and modalities for IRDs, including gene- and even
quantitation of photoaversion. In parallel to this mutation- specific therapies (such as gene aug-
study, a homozygous CNGA3 mutation was also mentation therapy and translational read-through
found to cause achromatopsia in Israeli sheep therapy) as well as general therapeutic modali-
flock that was successfully used for gene aug- ties, such as stem-cell therapy. It is clear that not
mentation therapy [88]. a single therapy will cure or improve vision in all
Supplementary of vitamin A and its deriva- patients, but different modalities will be needed
tives have been studied for many years as poten- to be tailored to different stages of diseases and
tial treatments for slowing down the process of different etiologies.
As the carrier frequency for IRD mutations is autosomal-recessive retinitis pigmentosa. Am J Hum
Genet. 2013;93:321–9.
predicted to be relatively high (1 out of 5–6 indi- 9. Chiang AP. mfl. Comparative genomic analysis identi-
viduals), new offsprings will be born with genetic fies an ADP-ribosylation factor-like gene as the cause
mutations that will cause disease, unless a treat- of Bardet-Biedl syndrome (BBS3). Am J Hum Genet.
ment is applied. Although it is difficult to predict 2004;75:475–84.
10. Van De Weghe JC. mfl. Mutations in ARMC9, which
the time needed to develop therapy for most encodes a basal body protein, cause Joubert syndrome
IRD types, both the large number of different in humans and ciliopathy phenotypes in zebrafish.
treatments that are being developed and the large Am J Hum Genet. 2017. https://doi.org/10.1016/j.
number of teams involved in this important effort ajhg.2017.05.010.
11. Shevach E. mfl. Association between missense muta-
are likely to yield results within a decade or tions in the BBS2 gene and nonsyndromic retinitis
two. Additionally, with proper genetic counsel- Pigmentosa. JAMA Ophthalmol. 2015;133:312.
ing for families with IRD-causing mutations, it is 12. Lindstrand A. mfl. Copy-number variation contributes
likely that the prevalence of IRDs will decline to the mutational load of Bardet-Biedl syndrome. Am
J Hum Genet. 2016;99:318–36.
within 1–2 generations. 13. Bitner H. mfl. A homozygous frameshift mutation in
BEST1 causes the classical form of Best disease in an
Compliance with Ethical Requirements Mor Hanany autosomal recessive mode. Invest Ophthalmol Vis Sci.
and Dror Sharon declare that they have no conflict of 2011;52:5332–8.
interest. 14. Duncan JL. mfl. Identification of a novel mutation in
No human or animal studies were performed by the the CDHR1 gene in a family with recessive retinal
authors for this article. degeneration. Arch Ophthalmol. 2012;130:1301.
15. Lazar CH. mfl. Whole exome sequencing reveals
GUCY2D as a major gene associated with cone and
cone-rod dystrophy in Israel. Invest Ophthalmol
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Occult Macular Dystrophy
(Miyake’s Disease) 20
Kazushige Tsunoda
Abstract icrostructure of the photoreceptors have

m
Occult macular dystrophy (OMD; OMIM been detected by spectral-domain optical
613587), first described by Miyake et al. in coherence tomography (SD-OCT) in eyes
1989, is an inherited macular dystrophy with Miyake’s disease (Tsunoda K, Usui T,
characterized by a progressive decrease in Hatase T, et al, Retina J Retinal Vitreous Dis
the visual acuity in eyes with a normal 32:1135–47, 2012, Fujinami K, Kameya S,
appearing fundus and normal fluorescein Kikuchi S, et al, Invest Ophthalmol Vis Sci,
angiograms (Miyake Y, Ichikawa K, Shiose 57:4837–46, 2016). The most common
Y, Kawase Y, Am J Ophthalmol,108:292–9, mutation is the c.133C > T, p.Arg45Trp
1989). The full- field electroretinograms mutation in exon 2, and there is another hot
(ERGs) are usually normal; however the spot between amino acid numbers 1194 and
focal macular ERGs, multifocal ERGs, and 1201 in exon 4 which is downstream of the
pattern ERGs are abnormal. Heterozygous doublecortin domain (Fujinami K, Kameya
mutations in the retinitis pigmentosa 1-like 1 S, Kikuchi S, et al, Invest Ophthalmol Vis
(RP1L1) gene (OMIM 608581) cause this Sci, 57:4837–46, 2016, Kabuto T, Takahashi
ocular condition, (Akahori M, Tsunoda K, H, Goto-Fukuura Y, et al, Mol Vis, 18:1031–
Miyake Y, et al, Am J Hum Genet, 87:424–9, 9, 2012, Davidson AE, Sergouniotis PI,
2010, Tsunoda K, Usui T, Hatase T, et al, Mackay DS, et al, Hum Mutat, 34:506–14,
Retina J Retinal Vitreous Dis 32:1135–47, 2013). In addition to the typical phenotype
2012, Miyake Y, Tsunoda K, Jpn J of OMD, extensive retinal dysfunction such
Ophthalmol, 59:71–80, 2015) and OMD as generalized cone dysfunction and gener-
with the RP1L1 mutations has been spe alized rod-cone dysfunction has been docu-
cifically designated as Miyake’s disease mented in patients with biallelic mutations
(Fujinami K, Kameya S, Kikuchi S, et al, in the RP1L1 gene (Davidson AE,
Invest Ophthalmol Vis Sci, 57:4837–46, Sergouniotis PI, Mackay DS, et al, Hum
2016). Characteristic changes in the Mutat, 34:506–14, 2013, Kikuchi S, Kameya
S, Gocho K, et al, Biomed Res Int,
2015:545243, 2015).
K. Tsunoda (*) Keywords

Division of Vision Research, National Institute of Occult macular dystrophy · Miyake’s disease ·
Sensory Organs, Tokyo, Japan RP1L1 gene · Progressive occult maculopathy

https://doi.org/10.1007/978-981-13-0884-0_20
250 K. Tsunoda
20.1 Introduction can range from 10 to 30 years [3]. In particular

cases with central foveal sparing, patients remain
Occult macular dystrophy (OMD; OMIM asymptomatic although they have dysfunction of
613587) is an inherited macular dystrophy char- photoreceptors at the parafoveal regions [13].
acterized by a progressive decrease in the visual There are only a few cases where the decimal
acuity in eyes with an essentially normal appear- visual acuity decreases to less than 0.1 even in
ing fundus and normal fluorescein angiograms senile patients with a long duration of OMD. The
[1]. The important signs of OMD are normal full- localized macular dysfunction can be confirmed
field electroretinograms (ERGs) but abnormal either by Goldmann perimetry or automated
focal macular ERGs (FMERGs), multifocal static perimetry as a relative central scotoma. In
ERGs (mfERGs), and pattern ERGs. These find- patients examined shortly after the onset, a rela-
ings indicate that the retinal dysfunction was con- tive central scotoma is not detected by Goldmann
fined to the macula. Since the initial report by perimetry but can be detected by static perimetry.
Miyake et al. in 1989, there have been a number Due to the normal fundus appearances and nor-
of reports describing the phenotype of this disor- mal full-field ERGs, OMD is often misdiagnosed
der, but the clinical characteristics varied in dif- as optic neuropathy of unknown origin, amblyo-
ferent reports [9–12]. In 2010, our laboratory pia, or nonorganic visual loss. There are also
found that dominant mutations in the RP1L1 cases with senile cataract, which can be later
gene were responsible for OMD, [2–4] and a diagnosed as OMD due to unexpectedly low
number of mutations in the RP1L1 gene have visual acuity following cataract surgery.
been recently reported in patients of various eth-
nicities [5–7]. The genotypic and phenotypic
investigations have confirmed that patients with 20.4 Retinal Appearances
OMD harboring RP1L1 mutations, Miyake’s dis-
ease, have homogeneous clinical characteristics The ophthalmoscopic appearances, fluorescein
and should be strictly segregated from macular angiography (FA), indocyanine green angiogra-
dysfunction without genetic cause [3–5]. In this phy, and fundus autofluorescence (AF) are nor-
review, the clinical and genetic characteristics of mal in OMD [3, 4] (Fig. 20.1). In some cases
Miyake’s disease will be presented. with long duration, round-shaped very weak
staining at the fovea may be present in FA.
The AF images are generally normal in the
20.2 Clinical Features entire posterior pole region. However, some cases
(~50%) may demonstrate a round-shaped areas
of increased AF signals at the fovea [14]. The
round-shaped areas of increased AF are very faint
20.3 Clinical Course of OMD in some cases and more apparent in others. The
Caused by RP1L1 Gene relationship between duration of the disease and
Mutation intensity of the increased AF signal has not been
determined.
Patients with OMD initially complain of blurred
vision, color vision abnormalities, and photopho-
bia. Most of the patients with RP1L1 mutations 20.5 Optical Coherence
have a family history of dominant inheritance. Tomography (OCT)
The age of onset varies from 6 to 60 years, and
the onset of the visual disorder may be different Spectral-domain OCT is a very important method
in the two eyes in some cases. Patients are gener- in the diagnosis of OMD [3, 13, 14]. In patients
ally affected in both eyes, and the visual acuity with the RP1L1 mutation, the most prominent
gradually decreases for a long duration which features on OCT are the abnormalities of the two
20 Occult Macular Dystrophy (Miyake’s Disease) 251
Fig. 20.1 Fundus photographs, multifocal electroretino- eye with OMD (left) and normal eye (right). The responses
grams (mfERGs), and optical coherence tomographic in the central loci are extinguished in the eye with OMD.
(OCT) images of a patient with a RP1L1 mutation (p. (c); OCT images horizontally profiled along the foveola in
Arg45Trp, heterozygous). (a); Fundus photographs (left), OMD (top) and normal eye (bottom). The ellipsoid zone
fluorescein angiograms (center), and fundus autofluores- (EZ) appears blurred, and the interdigitation zone (IZ) is
cence (right), showing no abnormal findings. (b); Trace absent in the macular region in the eye with OMD
arrays of mfERGs tested with 61 hexagonal stimuli in an
highly reflective lines in the OCT images at the have normal visual function, these outer retinal
macular region. These lines correspond to the microstructures appear normal. In longer dura-
ellipsoid zone (EZ) and interdigitation zone (IZ) tion cases, e.g., >30 years, both the photorecep-
in the photoreceptor layer (Fig. 20.1). The EZ at tors and outer nuclear layers are thinnest at the
the fovea appears thickened and blurred in the fovea; however, the retinal pigment epithelium
early to middle stages of OMD and disrupted or remains unchanged.
absent in the later stages. The IZ cannot be clearly It is notable that there are asymptomatic fam-
observed in the macular area even at the early ily members who have RP1L1 mutations in some
stage of OMD. In the peri-macular regions which families with dominant OMD [13]. The OCT
252 K. Tsunoda
images of these asymptomatic cases demon- d ownstream of the doublecortin domain [5–7].
strated photoreceptor abnormalities only in the The genetic background leading to the OMD
parafoveal regions, viz., absence of the IZ and may be a variant; however, other genetic causes
blurring of the EZ. However, the microstructures contributing to this disease have not been deter-
of the photoreceptors are preserved at the foveal mined. In addition to the typical phenotype of
center. The sparing of the photoreceptor layer at OMD, extensive retinal dysfunction such as gen-
the central foveal accounts for the well-preserved eralized cone dysfunction and generalized rod-
visual acuity in the asymptomatic patients. It has cone dysfunction has been documented in
not been determined whether the sparing repre- patients with biallelic mutations of the RP1L1
sents an initial phase of typical OMD or a sub- gene [7, 8].
type of macular lesion associated with OMD. The RP1L1 gene was originally cloned as a
gene derived from common ancestors with the
retinitis pigmentosa 1 (RP1) gene, which is
20.6 Electrophysiology responsible for 5–10% of all autosomal dominant
retinitis pigmentosa (RP) worldwide. It is located
Electrophysiological tests are the key for the on chromosome 8 [16, 22–25]. An immunohisto-
diagnosis of OMD. Both the scotopic (rod) and chemical study on cynomolgus monkeys showed
photopic (cone) responses of the full-field ERGs that RP1L1 was expressed in rod and cone photo-
are normal; however the focal macular ERGs, receptors, and it is believed to play important
multifocal ERGs, and pattern ERGs are abnormal roles in the morphogenesis of the photoreceptors
even at the very early stage of the macular dys- [16, 17]. Heterozygous RP1L1 knockout mice
function in OMD patients[1, 4] (Fig. 20.1). The were reported to have normal retinal morphol-
amplitudes of the cone-induced responses of the ogy, while homozygous knockout mice devel-
full-field ERGs may be borderline or slightly oped subtle retinal degeneration [17]. However,
reduced in some cases with the RP1L1 mutation, the RP1L1 protein has a very low degree of over-
[15] where the region of dysfunctional retina all sequence identity (39%) between humans and
expands over the macula toward the periphery. mice compared to the average values of sequence
These cases may be better referred to as central similarities observed between human and mice
cone dystrophy rather than macular dystrophy proteins. The cellular mechanisms that explain
from the viewpoint of electrophysiology. why only the macular region is impaired in
human OMD patients have not been determined.
20.7 Genetics
20.8 M
iyake’s Disease and Occult
Heterozygous mutations in the retinitis pigmen- Maculopathy
tosa 1-like 1 (RP1L1) gene (OMIM 608581)
cause this ocular condition, and OMD with the Patients with OMD having the RP1L1 mutation
RP1L1 mutations has been specifically desig- have a similar clinical course and share the com-
nated as Miyake’s disease [2, 3, 5]. The RP1L1 mon OCT findings: blurred EZ and absence of IZ
protein was suggested to be involved in the main- of the photoreceptors in the macular region. We
tenance of the morphological and functional categorized these patients as OMD with classical
characteristics of the photoreceptors, [16, 17] and phenotype and those lacking at least one of these
a number of mutations in the RP1L1 gene have two features as nonclassical phenotype [5]. In a
been reported [2, 3, 6, 7, 15, 18–21]. The most cohort throughout Japan, a significant association
common mutation is the c.133C > T, p.Arg45Trp was found between the OCT phenotypes and
mutation in exon 2, [2, 3, 7, 15, 18, 20, 21] and molecular genotypes; OMDs with RP1L1 muta-
there is another hot spot between amino acid tions have classical phenotype, and those without
numbers 1194 and 1201 in exon 4 which is RP1L1 mutations have nonclassical phenotypes.
20 Occult Macular Dystrophy (Miyake’s Disease) 253
Fig. 20.2 Classification
of occult macular
dysfunction syndrome
Occult macular
dysfunction syndrome
includes three
subcategories: (a)
RP1L1-associated occult
macular dystrophy
(Miyake’s disease), (b)
other hereditary occult
macular dystrophy
caused by other gene
abnormalities, and (c)
non-hereditary occult
macular dystrophy-like
syndrome (progressive
occult maculopathy)
There are two types of pathophysiology asso- 2. Akahori M, Tsunoda K, Miyake Y, et al. Dominant
ciated with bilateral progressive central cone mutations in RP1L1 are responsible for occult macu-
lar dystrophy. Am J Hum Genet. 2010;87:424–9.
dysfunction with normal fundus [5] (Fig. 20.1). 3. Tsunoda K, Usui T, Hatase T, et al. Clinical charac-
One is a Mendelian hereditary occult macular teristics of occult macular dystrophy in family with
dystrophy caused by genetic abnormalities such mutation of Rp1l1 gene. Retina J Retina Vitr Dis.
as the RP1L1 mutations (Miyake’s disease) and 2012;32:1135–47.
4. Miyake Y, Tsunoda K. Occult macular dystrophy. Jpn
possibly other unknown gene mutations, i.e., J Ophthalmol. 2015;59:71–80.
other occult macular dystrophies. The other type 5. Fujinami K, Kameya S, Kikuchi S, et al. Novel RP1L1
is a retinopathy with clinical signs of the occult variants and genotype-photoreceptor microstructural
macular dysfunction syndrome, i.e., clinical and phenotype associations in cohort of Japanese patients
with occult macular dystrophy. Invest Ophthalmol Vis
ERG findings similar to hereditary occult macu- Sci. 2016;57:4837–46.
lar dystrophy but not related to the Mendelian 6. Kabuto T, Takahashi H, Goto-Fukuura Y, et al. A new
genetic abnormality. We refer to this non-Men- mutation in the RP1L1 gene in a patient with occult
delian form as progressive occult maculopathy macular dystrophy associated with a depolarizing pat-
tern of focal macular electroretinograms. Mol Vis.
(Fig. 20.2). 2012;18:1031–9.
7. Davidson AE, Sergouniotis PI, Mackay DS, et al. RP1L1
Compliance with Ethical Requirements The proce- variants are associated with a spectrum of inherited reti-
dures used adhered to the tenets of the Declaration of nal diseases including retinitis pigmentosa and occult
Helsinki, and approval to perform this study was obtained macular dystrophy. Hum Mutat. 2013;34:506–14.
from the Review Board/Ethics Committee of the National 8. Kikuchi S, Kameya S, Gocho K, et al. Cone dystrophy
Institute of Sensory Organs, National Hospital in patient with homozygous RP1L1 mutation. Biomed
Organization, Tokyo Medical Center. Res Int. 2015;2015:545243.
9. Miyake Y, Horiguchi M, Tomita N, et al. Occult mac-
ular dystrophy. Am J Ophthalmol. 1996;122:644–53.
10. Fujii S, Escano MF, Ishibashi K, Matsuo H, Yamamoto
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occult macular dystrophy (OMD). Klin Monatsbl serous retinal detachment associated with a novel
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photoreceptor abnormalities in asymptomatic patients 20. Ahn SJ, Cho SI, Ahn J, Park SS, Park KH, Woo
with RP1L1 mutations in families with occult macular SJ. Clinical and genetic characteristics of Korean
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autosomal dominant occult macular dystrophy. Arch Multimodal approach to monitoring and investigating
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Bowne SJ, Daiger SP, Malone KA, et al.
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Optom. 2012;89:684–91. paralog of the retinitis pigmentosa 1 (RP1) gene. Mol
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pigmentosa 1-like1 gene (RP1L1): a novel candidate EL, Dryja TP. Mutations in a gene encoding a new
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Clinical Genetics of Vitelliform
Macular Dystrophy: An Asian 21
Perspective
Sung Wook Park, Chang Ki Yoon, Dae Joong Ma,

Un Chul Park, and Hyeong Gon Yu
Abstract because the clinical features of VMD are simi-

Vitelliform macular dystrophy (VMD) is a lar to those of exudative age-related macular
group of macular dystrophy characterized by degeneration (AMD), choroidal neovascular-
the subretinal accumulation of yellow yolk- ization (CNV), or central serous chorioreti-
like materials which predominantly affect the nopathy (CSC). Here, in addition to describing
macula. Best vitelliform macular dystrophy is the clinical characteristics of VMD, this chap-
among the most common autosomal dominant ter focuses on the clinical genetics of BEST1
(AD) retinal dystrophy, caused by mutations gene in VMD.
in the BEST1 gene. Since first identification of
BEST1 gene in 1998, molecular biology and Keywords
pathophysiology of BEST1 gene and vitelli- Vitelliform macular dystrophy · Bestrophin-1
form macular dystrophy were studied. Recent · Best vitelliform macular dystrophy ·
advances in genetic analysis have described Adult-onset vitelliform macular dystrophy ·
over 200 different human BEST1 mutations to BEST1 gene mutation · Genome editing
date, associated with a broad spectrum of
ocular diseases, called bestrophinopathy.

However, the genotype-phenotype correlation
in VMD is largely unexplored. Genetic test is 21.1 Introduction
clinically important in the diagnosis of VMD
Macular dystrophy is a group of heritable disor-
S. W. Park · D. J. Ma · U. C. Park · H. G. Yu (*) ders that cause ophthalmoscopically visible mac-
Department of Ophthalmology, Seoul National ular abnormalities. Vitelliform macular dystrophy
University College of Medicine, Seoul, (VMD) is a group of macular dystrophy charac-
Republic of Korea terized by the subretinal accumulation of yellow
Department of Ophthalmology, Seoul National yolk-like materials which predominantly affects
University Hospital, Seoul, Republic of Korea the macula. Best vitelliform macular dystrophy
Retinal Degeneration Research Laboratory, (BVMD) is named after Friedrich Best who
Biomedical Research Institute, Seoul National described a family with a history of early-onset
University Hospital, Seoul, Republic of Korea
e-mail: [email protected] macular degeneration in 1905 [1]. BVMD is
among the most common autosomal dominant
C. K. Yoon
Department of Ophthalmology, Inje University (AD) retinal dystrophy, caused by mutations in
College of Medicine, Busan, Republic of Korea the BEST1 gene. Since the first identification of

https://doi.org/10.1007/978-981-13-0884-0_21
256 S. W. Park et al.
BEST1 gene in 1998 [2], molecular biology and 21.3 Molecular Biology
pathophysiology of BEST1 gene in VMD have
been studied. Recent advances in genetic analysis The BEST1 gene consists of 11 exons that encode
have described over 200 different human BEST1 the bestrophin-1 protein (585 amino acids).
mutations to date, associated with a broad spec- Bestrophin-1 is a retinal pigment epithelium
trum of ocular diseases, called a bestrophinopa- (RPE) protein hypothesized to function as a Ca2+-
thy [3, 4]. Bestrophinopathy includes five activated Cl- channel (CaCC), or a regulator of
clinically distinct categories BVMD, adult-onset ion transport [14]. Bestrophin-1 is predominantly
vitelliform macular dystrophy (AVMD), autoso- expressed in the basolateral membrane of the
mal recessive bestrophinopathy (ARB), autoso- RPE [15]. X-ray structure of chicken BEST1-Fab
mal dominant vitreoretinochoroidopathy complexes indicates that Bestrophin-1 forms a
(ADVIRC), and retinitis pigmentosa. AVMD was homo-pentamer and functions as a CaCC [16].
first described by Gass in 1974 who initially Disease-causing mutations are prevalent within
termed it peculiar foveomacular dystrophy [5]. the gating apparatus. In addition, Bestrophin-1
AVMD is one of the most common forms of mac- functions as a regulator of intracellular calcium
ular dystrophy as well [6]. Many investigators signaling and influences transepithelial electrical
suggested that AVMD is a mild form of BVMD properties [17]. Recently, patient’s stem cell-
within the same spectrum because the clinical derived RPE is used for the function of bestro-
features of AVMD were similar to those of early- phin-1 and reveals that bestrophin-1 assembles
stage BVMD and the age of onset were highly into a key calcium-sensing chloride channel in
variable [7–9]. Clinically, BVMD is distin- human RPE [18]. Further study using RPE cells
guished from AVMD by earlier age of onset, from patient-derived induced pluripotent stem
larger lesion size, and an abnormal electrooculo- cells (iPSc) harboring BEST1 mutations is
gram (EOG). Clinical features of VMD are simi- required to elucidate the exact functional role of
lar to those of exudative age-related macular bestrophin-1.
degeneration (AMD), choroidal neovasculariza-
tion (CNV), or central serous chorioretinopathy
(CSC). Thus, genetic test is clinically important 21.4 Clinical Features
in the diagnosis of VMD. Here, in addition to
describing the clinical characteristics of VMD, 21.4.1 BVMD
this chapter focuses on the clinical genetics of
BEST1 gene in VMD (BVMD and AVMD). BVMD is an early-onset autosomal dominant
disorder showing extremely variable penetrance
and expressivity. The diagnosis of BVMD shows
21.2 Epidemiology and Asian a bimodal age distribution; the first maximum
Perspective peak was made during the childhood, but the sec-
ond peak was made following puberty and
VMD is an autosomal dominant macular dystro- extending into the sixth decade of life [19].
phy with an estimated prevalence of 1 in 10,000 Before the era of genetic analysis, the diagnosis
in the USA [10], 2/10,000 in Sweden [11], of BVMD was based on typical fundus findings,
1.5/100,000 in Denmark [12], and 1 in 16,500 to family history, and a decreased Arden ratio (light
1 in 21,000 in Olmsted County, Minnesota, USA peak/dark trough) of EOG with a normal electro-
[13]. Males are more affected than females (3:1) retinogram (ERG), which may contribute to vari-
[11, 12]. Despite the update of novel mutations of ability of penetrance, expressivity, and onset age.
BEST1 in Asian VMD patients, there was no report BVMD is caused by dysfunction of
of the prevalence of VMD in Asian countries. Bestrophin-1 protein, a CaCC protein located on
Thus, a study of the prevalence of VMD with a the basolateral membrane of RPE, which causes
genetic analysis in Asian countries is necessary. abnormal fluid and ion exchange that decreases
21 Clinical Genetics of Vitelliform Macular Dystrophy: An Asian Perspective 257
pumping of fluid from the subretinal space, and ible on OCT below the neurosensory retina,
results in swelling of RPE and subretinal lipofus- located between the EZ and the RPE. The disrup-
cin accumulation [20]. Histopathologically, auto- tion of outer retinal layers and neurosensory reti-
fluorescent material was accumulated in the outer nal detachment with subretinal fluid occur in
retina and the subretinal space in BVMD, which is many cases [28, 29]. The yellowish subretinal
considered as indigestible components of photore- material is intensely hyperautofluorescent in FAF
ceptor outer segments that accumulate due to the imaging. FA shows marked hypofluorescence in
lack of direct apposition of the outer segments and the zone covered by lesion by blockage of fluo-
the RPE [21]. Eventual phagocytosis of these older rescence. Metamorphopsia, blurred vision, and a
materials over time would load the RPE cells and decrease of central vision can occur.
may account for excessive accumulation of abnor- In the third pseudohypopyon stage, the vitelli-
mal lipofuscin in RPE cells across the entire fun- form material accumulates inferiorly and devel-
dus [22]. These findings coincide with the ops a fluid level. On OCT, the upper part of the
decreased Arden ratio of EOG, less than 1.5, seen lesion is observed as hyporeflective area located
in BVMD, which suggest generalized dysfunction between RPE and EZ, with clumping of hyperre-
of the RPE. Even otherwise asymptomatic carriers flective material on the posterior retinal surface.
of BEST1 mutations will exhibit an altered EOG The lower part of the lesion, where the vitelliform
[23]. Full-field ERG is generally normal, but the material is still accumulated, shows a highly
multifocal ERG amplitudes of the central and peri- reflective area located in the subretinal space. FA
central responses were significantly reduced in the shows hypofluorescence in the lower part resulted
majority of patients [24]. However, the photore- from the blockage by the vitelline material. The
ceptor structure evaluated cellular imaging with superior part shows hyperfluorescent due to trans-
adaptive optics scanning light ophthalmoscopy mission defects linked to RPE and chorioretinal
was retained within active BVMD lesions, even in atrophy in the early phase. FAF shows loss of
apparently advanced disease [25, 26]. autofluorescence, particularly in the upper part.
Five progressive stages can be defined based The fourth vitelliruptive stage is characterized
on fundus examination [20, 27]. However, these by the partial reabsorption of the vitelliform
stages are not observed in all patients, nor do they material. This vitelliform material becomes less
occur consecutively. The first previtelliform stage homogeneous to develop a “scrambled-egg”
is characterized by the absence of symptoms and appearance. OCT shows an optically empty
subtle RPE changes such as RPE mottling and a lesion between EZ and RPE, with clumping of
small yellow spot. On optical coherence tomog- hyperreflective material on the posterior retinal
raphy (OCT), RPE and ellipsoid zone (EZ) dis- surface like the upper part of the pseudohypo-
ruption was detectable in a small fraction of eyes pyon. The areas of focal RPE hypertrophy can be
[28, 29]. A slight thickening of the interdigitation observed as hyperreflective mottling on the RPE
zone was also observed [30]. EOG is abnormal layer on some parts. FAF shows decreased of
and fluorescein angiogram (FA) shows window autofluorescence centrally but increased autoflu-
defects. Visual acuity remains intact in most orescence at the outer border of the lesion.
patients. The previtelliform lesions are character- In the last atrophic/fibrotic stage, RPE atro-
ized by absence or only slight autofluorescence phy and loss of central vision occur after rupture
on fundus autofluorescence (FAF) imaging. and reabsorption of the cystic lesion. FA shows
The second vitelliform shows a well- hyperfluorescence without leakage. OCT reveals
circumscribed, circular, homogeneous, yellow- thinning of all the retinal layers and diffuse dis-
opaque, 0.5–3 disc diameter sized, yolk-like appearance of outer retinal layers within the
macular lesions. The remaining part of fundus macular area, with highly hyperreflective thick-
usually has normal appearance, but multifocal ening at the RPE level [29, 31]. Atrophic lesions
lesions also can be seen. The accumulation of are characterized by decreased autofluorescence
hyperreflective vitelliform material is clearly vison FAF.
Choroidal neovascularization (CNV) may reduced Arden ratio, which is obviously abnor-
develop and can lead to form a disciform scar. mal in BVMD. The macular lesion appears as
Patients usually underwent sudden visual distur- hyperautofluorescent in FAF. The vitelliform
bance with central scotoma and/or metamor- deposit usually appears as initially hypofluores-
phopsia, showing a macular hemorrhage on cent but gradually becomes hyperfluorescent on
fundus examination. In that case, FA shows the edges by staining of the dye in FA [39] and
hyperfluorescence because of CNV and leakage. hypofluorescent on indocyanine green angiogra-
Intravitreal injection of anti-vascular endothelial phy (ICGA). OCT reveals a dome-shaped hyper-
growth factor (VEGF) agent was effective in reflective lesion located between the retina and
treating CNV complicated with BVMD and safe RPE [40]. The foveal thinning and EZ disruption
even in children [32–34]. are also observed and probably explain the pro-
Patients with BVMD undergo a progressive gressive visual loss [41, 42].
decrease of vision over time. In a study that eval- AVMD progression is characterized by frag-
uated the course of visual decline of 53 patients mentation and reabsorption of the vitelliform
in BVMD with BEST1 mutation [35], the median material [6]. Macular atrophy progressively
age of onset of visual symptoms was 33 years. replaces the vitelliform deposits at the advanced
Twenty-five percent of patients retain visual acu- stages of the disease in most cases [42], but most
ity of 20/40 or better at the age of 66 years. Other patients retain reading vision throughout life [43,
study evaluated 47 patient with BVMD; 74% of 44]. CNV may be complicated in few cases; 6 out
patient older than 30 years had 20/100 or worse of 51 patients developed CNV after a 6-year fol-
visual acuity at least one eye [36]. low-up [45]. Anti-VEGF therapies have shown to
be effective in the treatment of CNV associated
with AVMD [46].
21.4.2 AVMD
Gass reported a three-generation family and six 21.5 Genetic Aspects

sporadic patients characterized by one-third disc
diameter sized bilateral subfoveal vitelliform 21.5.1 BVMD
lesions with onset between the ages of 30 and
50 years accompanied by slowly progressive Currently, only genetic test for mutation analysis
visual loss as “peculiar foveomacular dystrophy.” of the BEST1 gene leads to confirmation of a
They also showed occasional paracentral drusen, clinical diagnosis of BVMD. Note that individu-
normal to slightly subnormal response on EOG als with clinical findings of BVMD occasionally
but normal ERG and color vision [5]. AVMD have a normal EOG, turning out to have a patho-
shows a variable genetic inheritance, although genic variant of BEST1 [47]. In case of atypical
most cases are sporadic [37]. Patients with BVMD [3], genetic test for confirmation should
AVMD may be asymptomatic but become symp- be performed. Over 200 BEST1 mutations with
tomatic in the fourth or fifth decade of life with significant clinical heterogeneity require a thor-
blurred vision, metamorphopsia, or scotoma and ough genetic analysis and clinical examinations
typically have slow progression of vision loss to better understanding of genotype-phenotype
[38]. Patients with AVMD typically present a correlations in BVMD. Most mutations of BEST1
round, yellowish subretinal deposit in one-third gene in BVMD and AVMD are missense muta-
to one disc diameter size within the macular area, tions. Table 21.1 shows a list of missense muta-
similar fundus finding to the vitelliform stage of tions of BEST1 gene in BVMD and AVMD.
BVMD. Most genetic studies were performed in
The initial yellow lesion may present in only Western countries including the USA, England,
one eye and appear as small yellow flecks in the Sweden, Denmark, Germany, the Netherlands,
paracentral area. EOG shows a normal or slightly Italy, and France. BEST1 mutations are extremely
Table 21.1 BEST1 missense mutations in BVMD and AVMD

Mutations a.a Mutation nucleotide Associated isease Inheritance Ethnicity References
1 Thr2Ala c.4A > G BVMD AD Japanese [48, 49]
AVMD AD Iowa, USA
2 Thr2Asn c.5C > A BVMD AD Chinese [50]
3 Thr2Ile c.5C > T AVMD AD Iowa, USA [49]
4 Ile3Asn c.8 T > A Atypical BVMD AD USA [51]
5 Ile3Thr c.8 T > C BVMD AD Dutch [52]
6 Thr4Ala c.10A > G BVMD AD French [53]
7 Thr4Ile c.11C > T AVMD AD Chinese [54, 49]
Iowa, USA
8 Tyr5His c.13 T > C AVMD AD Iowa, USA [49]
9 Tyr5Term c.15C > A Multifocal AD French [55]
BVMD
10 Thr6Ala c.16A > G BVMD De novo USA [56]
11 Thr6Arg c.17C > G BVMD AD Iowa, USA [57, 58]
AD USA or
Swiss
12 Thr6Lys c.17C > A AVMD AD Iowa, USA 49,
13 Thr6Pro c.16A > C BVMD AD Dutch [59, 52, 60–62, 2]
BVMD AD Dutch
Multifocal AD Dutch
BVMD
AVMD AD German
BVMD AD Dutch
BVMD AD Dutch
14 Ser7Asn c.20G > A BVMD AD Japanese [48]
15 Val9Ala c.26 T > C AVMD AD French [53, 2]
Swedish
16 Val9Glu c.26 T > A BVMD Unknown Portuguese [63]
17 Val9Leu c.25G > C BVMD AD USA [64, 49]
AVMD AD Iowa, USA
18 Val9Met c.25G > A BVMD AD German [65, 61, 66]
BVMD AD German
BVMD AD German
19 Ala10Thr c.28G > A BVMD AD German [61, 66]
BVMD AD German
20 Ala10Val c.29C > T BVMD AD Dutch [59, 62]
BVMD AD Dutch
21 Asn11Ile c.32A > T BVMD AD German [67]
22 Arg13Cys c.37C > T AVMD AD Iowa, USA [49]
23 Arg13His c.38G > A BVMD AD Chinese [54, 68]
BVMD AD USA
24 Arg13Pro c.38G > C AVMD AD Iowa, USA [49]
25 Gly15Arg c.43G > C BVMD AD Slovenian [69]
26 Gly15Asp c.44G > A BVMD AD Italian [53]
27 Ser16Phe c.47C > T BVMD AD Chinese [70, 71]
BVMD AD French
28 Ser16Tyr c.48C > A BVMD AD Dutch [59, 60]
Multifocal AD Dutch
BVMD
(continued)
29 Phe17Cys c.50 T > G BVMD AD French [71, 58]
AD USA or
Swiss
30 Phe17Ser c.50 T > C AVMD AD Iowa, USA [49]
31 Arg19Leu c.56G > T AVMD AD Iowa, USA [49]
32 Leu20Val c.58C > G BVMD AD Danish [12]
33 Leu21Val c.61C > G BVMD AD German [61, 72]
BVMD AD English,
Canadian
34 Trp24Cys c.72G > T BVMD AD USA or [58, 66]
Swiss
BVMD AD German
35 Arg25Gln c.74G > A BVMD AD German [66]
36 Arg25Trp c.73C > T BVMD AD Japanese [48, 53, 73, 58,
BVMD AD French 61]
BVMD AD Italian
BVMD AD USA or
Swiss
BVMD AD German
37 Gly26Arg c.76G > C BVMD AD German [67]
38 Ser27Arg c.81C > G BVMD AD German [61]
39 Tyr29His c.85 T > C BVMD AD German [67]
40 Lys30Arg c.89A > G BVMD AD USA or [58]
Swiss
41 Lys30Asn c.90G > C AVMD AD Iowa, USA [49]
42 Glu35Lys c.103G > A BVMD Unknown Portuguese [63]
43 Leu41Pro c.122 T > C BVMD AD German [67]
44 Arg47Cys c.139C > T AVMD AR Iowa, USA [49]
45 Arg47His c.728C > T BVMD AD Chinese [70, 61]
AVMD AD German
46 Gln58Leu c.173A > T BVMD AD German [65, 61]
BVMD AD German
47 Tyr72Asp c.214 T > G AVMD AD Iowa, USA [49]
48 Ile73Asn c.218 T > A BVMD AD French [71]
49 Ile73Phe c.217A > T BVMD AD USA [64]
50 Leu75Phe c.223C > T BVMD AD Chinese [50]
51 Ile76Asn c.227 T > A AVMD AD Iowa, USA [49]
52 Ile76Val c.226A > G BVMD AD Iowa, USA [49]
53 Phe80Leu c.240C > A BVMD AD Japanese [48, 58]
BVMD AD USA or
Swiss
54 Phe80Val c.238 T > G BVMD AD USA [64]
55 Val81Met c.241G > A BVMD AD Japanese [48, 49]
BVMD AD Iowa, USA
56 Leu82Val c.244C > G BVMD AD Danish [12, 74, 52, 62]
BVMD AD German
BVMD AD Dutch
BVMD AD Danish
57 Phe84Val c.250 T > G AVMD AD Iowa, USA [49]
(continued)
58 Tyr85His c.253 T > C BVMD AD Danish [12, 74, 2]
BVMD AD Danish
BVMD AD Swedish
59 Val89Ala c.266 T > C BVMD AD Swedish [75]
60 Thr91Ile c.272C > T BVMD AD French [53, 58]
AD USA or
Swiss
61 Arg92Cys c.274C > T BVMD AD Italian, [53, 62]
French
BVMD AD Swedish
62 Arg92Gly c.274C > G AVMD AD Italian [53]
63 Arg92His c.275G > A BVMD AD Danish [12, 74, 71]
BVMD AD Danish
BVMD AD French
64 Arg92Ser c.274C > A BVMD AD German [65, 61]
BVMD AD German
65 Trp93Arg c.277 T > C AVMD AD Iowa, USA [49]
66 Trp93Cys c.279G > C BVMD AD Swedish [2]
67 Gln96Arg c.287A > G BVMD AD Danish [12]
68 Gln96Glu c.286C > G AVMD AD Iowa, USA [49]
69 Gln96His c.288G > C BVMD AD Dutch [59, 62]
BVMD AD Dutch
70 Asn99Lys c.297C > A BVMD AD German [61]
71 Asn99Tyr c.295A > T BVMD AD Iowa, USA [49]
72 Leu100Arg c.299 T > G BVMD AD German [67, 61]
BVMD AD German
73 Pro101Leu c.302C > T AVMD AD Iowa, USA [49]
74 Pro101Thr c.301C > A BVMD AD USA or [58]
Swiss
75 Tryp102Arg c.304 T > C BVMD AD German [67]
76 Asp104Glu c.312C > A BVMD AD Swedish [2]
77 Asp104His c.301G > C BVMD AD German [67]
78 Arg105Gly c.313G > C BVMD AD Slovenian [69]
79 Phe113Leu c.339C > G BVMD AD Chinese [76]
80 Arg130Ser c.388C > A BVMD AD USA [64]
81 Asn133Lys c.399C > G BVMD AD USA or [58]
Swiss
82 Leu134Val c.400C > G BVMD AD Dutch [59, 77, 60]
BVMD AD French
Multifocal BVMD AD Dutch
83 Gly135Ser c.403G > A BVMD AD USA or [58, 62]
Swiss
BVMD AD Swedish
84 Leu140Arg c.419 T > G BVMD AD USA or [58]
Swiss
85 Arg141His c.422G > A BVMD AD USA or [58, 61]
Swiss
AD German
86 Arg141Ser c.421C > A BVMD AR Iowa, USA [49]
(continued)
Val143Phe c.427G > T AVMD AD Iowa, USA [49]
87 Ser144Asn c.431G > A BVMD AD Chinese [70, 50]
BVMD AD Chinese
88 Ser144Gly c.430A > G Multifocal AD French [55]
BVMD
89 Ala195Val c.584C > T BVMD AD Japanese [48, 59, 60, 67,
BVMD AD Dutch 58]
Multifocal AD Dutch
BVMD AD German
BVMD AD USA or
Swiss
90 Ile201Thr c.602 T > C BVMD AD USA or [58]
Swiss
91 Ser209Asn c.626G > A BVMD AD English, [61]
Canadian
92 Leu211Thr c.632 T > C BVMD AD USA or [58]
Swiss
93 Arg218Cys c.652C > T BVMD AD Chinese [54, 12, 67, 71,
BVMD AD Danish 50, 58, 62, 68]
BVMD AD German
BVMD AD French
BVMD AD Chinese
BVMD AD USA or
Swiss
BVMD AD Dutch
BVMD AD USA
94 Arg218Gly c.652C > G BVMD AD Italian [73]
95 Arg218His c.653G > A BVMD AD Japanese [48, 59, 71, 58]
BVMD AD Dutch
BVMD AD French
BVMD AD USA or
Swiss
96 Arg218Ser c.652C > A BVMD AD German [67, 62]
BVMD AD Swedish
97 Arg218Gln c.654 T > G BVMD AD Dutch [66]
98 Gln220Pro c.659A > C AVMD AD Iowa, USA [49]
99 Cys221Phe c.662G > T BVMD AD Iowa, USA [49]
100 Cys221Trp c.663 T > G BVMD De novo Italy [78]
101 Gly222Glu c.665G > A BVMD AD Japanese [48]
102 Gly222Val c.665G > T BVMD AD USA or [58]
Swiss
103 Leu224Met c.670C > A BVMD AD German [61]
104 Leu224Pro c.671 T > C BVMD AD USA or [58]
Swiss
105 Tyr227Asn c.679 T > A BVMD AD Dutch [59, 58, 66, 2]
BVMD AD USA or
Swiss
BVMD AD Dutch
BVMD AD Dutch
106 Tyr227Cys c.680A > G BVMD AD USA or [58, 66]
Swiss
BVMD AD Dutch
(continued)
107 Tyr227Phe c.680A > T BVMD AD German [79]
108 Trp229Gly c.685 T > G BVMD AD Chinese [80]
109 Ile230Asn c.689 T > A AVMD AD Iowa, USA [49]
110 Ile230Trh c.689 T > C BVMD AD French [53]
111 Ser231Arg c.693 T > G BVMD AD German [61]
112 Ser231Thr c.692G > C BVMD AD French [77]
113 Ile232Asn c.695 T > A BVMD AD German [79]
114 Pro233Ala c.697C > G BVMD AD Swedish [81]
115 Pro233Gln c.698C > A BVMD AD French [77]
116 Pro233Leu c.698C > A AVMD AD Iowa, USA [49]
117 Leu234Pro c.698C > T BVMD Unknown USA [18]
118 Val235Leu c.703G > C BVMD AD French [71]
119 Val235Met c.703G > A BVMD AD Dutch [66]
120 Thr237Arg c.710C > G BVMD AD German [67, 61]
BVMD AD German
121 Thr237Ser c.709A > T BVMD AD German [79]
122 Thr241Asn c.722C > A BVMD AD German [67]
123 Val242Met c.724G > A BVMD AD Japanese [48]
124 Ala243Thr c.727G > A BVMD AD Danish [12, 61, 58]
BVMD AD German
BVMD AD USA or
Swiss
125 Ala243Val c.728C > T BVMD AD Italian [53, 67, 61]
BVMD AD German
AVMD AD German
126 Arg255Trp c.763C > T BVMD AD Chinese [50]
127 Pro274Arg c.821C > G AVMD AR Iowa, USA [49]
128 Phe276Leu c.828C > G BVMD AD USA or [58]
Swiss
129 Tyr284Cys c.851A > G BVMD AD Iowa, USA [49]
130 Arg291Val c.872C > T BVMD AD Chinese [54]
131 Glu292Lys c.874G > A BVMD AD Chinese [70, 82]
BVMD AD USA
132 Gln293His c.879G > C BVMD AD Chinese [54], 77]
BVMD AD French
133 Gln293Lys c.877C > A BVMD AD Dutch [59, 62]
BVMD AD Dutch
134 Leu294Val c.880C > G BVMD AD German [67]
135 Ile295Thr c.884 T > C BVMD AD German [67, 83]
BVMD AD Japanese
136 Ile295Val c.883A > G BVMD AD Iowa, USA [49]
137 Asn296His c.886A > C BVMD AD USA or [58]
Swiss
138 Asn296Lys c.891C > A Multifocal AD Dutch [60]
BVMD
139 Asn296Ser c.887A > G BVMD AD Danish [12, 71]
BVMD AD French
140 Pro297Ala c.889C > G BVMD AD USA or [58, 66]
Swiss
BVMD AD Dutch
141 Pro297Ser c.889C > T BVMD AD Iowa, USA [49]
(continued)
142 Pro297Thr c.889C > T BVMD AD Chinese [50]
143 Phe298Cys c.893 T > G BVMD AD USA [64]
144 Phe298Ser c.893 T > C BVMD AD Dutch [59, 60, 67]
Multifocal AD Dutch
BVMD
BVMD AD German
145 Phe298Val c.892 T > G BVMD Unknown English [84]
146 Gly299Ala c.896G > C BVMD AD Dutch [59, 52]
BVMD AD Dutch
147 Gly299Arg c.895G > A BVMD AD French [77]
148 Gly299Glu c.896G > A BVMD AD Swedish [2]
149 Glu300Asp c.900G > C BVMD AD Iowa, USA [49, 58, 68]
BVMD AD USA or
Swiss
BVMD AD USA
150 Glu300Lys c.898G > A BVMD AD Chinese [70, 61, 58]
BVMD AD German
BVMD AD USA or
Swiss
151 Asp301Asn c.901G > A BVMD AD German [61]
152 Asp301Glu c.903 T > G BVMD AD German [65, 67, 61, 68]
BVMD AD German
BVMD AD German
BVMD AD USA
153 Asp301Gly c.902A > G BVMD AD Chinese [50, 54]
BVMD AD Chinese
154 Asp302Ala c.905A > C BVMD AD Danish [12, 64, 59]
BVMD AD USA
BVMD AD Dutch
155 Asp302Asn c.904G > A BVMD AD Danish [12]
156 Asp302Gly c.905A > G BVMD AD USA or [58]
Swiss
157 Asp302His c.904G > C BVMD AD French [85]
158 Asp302Val c.905A > T BVMD AD USA or [58]
Swiss
159 Asp303Asn c.907G > A BVMD AD Italian [86]
160 Asp303Glu c.909 T > A BVMD AD French [85]
161 Asp303Gly c.908A > G AVMD AD Iowa, USA [49]
162 Asp304Asn c.910G > A AVMD AD Iowa, USA [49]
163 Asp304Gly c.911A > G BVMD AD Italian [86]
164 Asp304Val c.911A > T BVMD Unknown Portuguese [63]
165 Phe305Leu c.915 T > A BVMD AD Italian [47]
166 Phe305Ser c.914 T > C BVMD AD Dutch [66]
167 Phe305Tyr c.914 T > A AVMD AD Iowa, USA [49]
168 Glu306Asp c.918G > C BVMD AD Japanese [48, 58]
BVMD AD USA or
Swiss
169 Glu306Gly c.917A > G BVMD AD USA or [58]
Swiss
170 Thr307Asp c.920C > A BVMD AD Chinese [70]
171 Thr307Ala c.919A > G BVMD AD USA or [58]
Swiss
(continued)
172 Thr307Ile c.902C > T BVMD AD USA or [58, 68]
Swiss
AD USA
173 Asn308Ser c.923A > G BVMD AD French [85]
174 Trp309Arg c.925 T > C AVMD AD Iowa, USA [49]
175 Ile310Thr c.929 T > C BVMD AD Germany [61]
176 Val311Gly c.932 T > G BVMD AD Germany [61]
177 Asp312Asn c.934G > A AVMD AD Germany [61]
178 Asp312Glu c.936C > A BVMD AD Danish [12, 74]
BVMD AD Danish
179 Gln316His c.948G > T AVMD AR Iowa, USA [49]
180 Gln316Pro c.947A > C AVMD AD Iowa, USA [49]
181 Pro346His c.1037C > A BVMD AD Japanese [48]
182 Val492Ile c.1474G > A AVMD AD Iowa, USA [49]
183 Glu557Lys c.1669G > A AVMD AD Iowa, USA [49]
heterogenous, but several mutations have been sis identified 12 BEST1 variants in 13 probands
frequently found (Thr6Pro, Arg25Trp, (81%). Of these, ten variants (Tyr2Arg, Arg25Trp,
Arg218Cys, Tyr227Asn, Arg243Val, Ile295del, Phe80Leu, Val81Met, Ala195Val, Arg218His,
Gle300Asp, Asp301Glu, and Asp302Asn). Gly222Glu, Val242Met, Asp304del, and
Interestingly, these frequent mutations are ethnic Glu306Asp) have been previously reported in
specific (44.4% of Asp302Asn in Danish [12] BVMD, while two variants (Ser7Asn and
and 36.8% of Arg25Trp in Italian [86]). Pro346His) were novel disease-causing
Currently, only limited reports are available in mutations.
Asian genetic studies of BEST1 from Chinese In Korea, we report a BVMD patient (Fig. 21.1)
[50, 54, 70, 76, 80, 87–89], Japanese [48, 83], carrying Asn296Lys mutation which is a caus-
and Korean [9]. The mutation spectrum of the ative mutation of multifocal BVMD in German
BEST1 gene in Asian patients of BVMD is dif- patient [60]. Arg218Leu is a novel disease-
fered from those in Western patients [88]. Six causing mutation in BVMD (Fig. 21.2). These
novel missense mutations (Thr2Asn, Leu75Phe, findings expand the spectrum of BEST1 genetic
Ser144Asn, Arg255Trp, Pro297Thr, and variation in Asian and will be valuable for genetic
Asp301Gly) and one previously reported muta- counseling for patients with BVMD [88].
tion (Arg218Cys) were identified [50]. Three BVMD shows variable expressivity and
novel mutations Tyr4Ile [54], Ala291Val [54], incomplete penetrance at the clinical level.
and Phe113Leu [76] in BVMD were reported. Disease-causing effect of BEST1 mutations
Lin [80] reported two novel heterozygous muta- seems to be cumulative over time [79]. In
tions 304delAsp and Trp229Gly in Chinese genotype-phenotype relationship of Dutch study
BVMD patients. Liu [70] reported four previ- [59], median age of onset of visual symptoms
ously reported mutations (Ser16Phe, Ser144Asn, was 33 years (range, 2–78). The cumulative risk
Glu292Lys, and Glu300Lys) and two novel of VA below 0.5 (20/40) was 50% at 55 years and
disease-causing mutations (Thr307Asp, 75% at 66 years. The cumulative risk of VA
Arg47His) in Chinese patients with BVMD. decline less than 0.3 (20/63) was 50% by age
In Japanese study [48], 22 patients including 66 years and 75% by age 74 years. Most patients
16 probands from 16 families with BVMD were (96%) had missense mutations; the Thr6Pro,
analyzed. All 16 probands exhibited characteris- Ala10Val, and Tyr227Asn mutations were most
tic BVMD fundus appearances, abnormal EOG, common. Visual decline was significantly faster
and normal ERG responses with the exception of in patients with an Ala10Val mutation than either
one diabetic retinopathy proband. Genetic analy- the Thr6Pro or the Tyr227Asn mutation.
Fig. 21.1 Best vitelliform macular dystrophy (BVMD). tive vitelliform materials at RPE in both eyes. (c, h)
A 32-year-old man carrying p.Asn296Lys mutation in the Fluorescein angiography (FA) shows late pooling of fluo-
BEST1 gene was incidentally found on routine fundus rescein dye at the egg lesion. (d, i) Fundus autofluores-
examination for a pilot license. The visual acuities (VA) cence (FAF) image of the vitelliruptive lesion shows
were 20/20 in both eyes. (a, f) Bilateral BVMD of vitel- increased autofluorescence at inferior part of ruptured
liruptive stage shows scattered yellow-white vitelliform vitelliform lesions and at the border of the serous retinal
deposits. (b, g) Vertical optical coherent tomography detachment. (e, j) Indocyanine green angiography (ICGA)
(OCT) shows serous retinal detachment and hyperreflec- shows active leakage spot in the left eye
Fig. 21.2 Best vitelliform macular dystrophy (BVMD). OCT reveals marked RPE loss at the fovea (f). Six months
A 39-year-old man carrying Arg218Leu mutation in the later, FAF shows dispersed materials with hyperautofluo-
BEST1 gene had multiple injections of anti-VEGF agents rescent (c), and OCT reveals the disappearance of subfo-
(ten for right eye and five for left eye) in both eyes. At veal pillar with a progression to vitelliruptive stage (d) in
initial visiting in our institute, vitelliform stage of right the right eye. FAF shows central hypoautofluorescent and
eye (a) reveals highly reflective subfoveal pillar without surrounding hyperfluorescent lesions. OCT reveals that
surrounding SRF (b). Small round vitelliform lesion with hypoautofluorescent lesion corresponds to the enlarged
central cicatricial change was found in the left eye (e). RPE loss (h)
In the recent Chinese study, despite typical 21.6 Future Perspectives

macular appearance of BVMD, no clear for Therapy
genotype-phenotype correlation was observed
[88]. In Asian BVMD cohort, genetic tests should The development of gene and cell therapies is
be performed for the diagnosis with thorough promising in various retinal diseases. Indeed, the
clinical examinations to elucidate a genotype- results of clinical trials using iPSC-derived RPE
phenotype correlation. cells in wet age-related macular degeneration
[94] or AAV/RPE65 vectors in Leber’s congeni-
tal amaurosis [95] were already reported.
21.5.2 AVMD Therapeutic intervention of inherited retinal dys-
trophy should be primarily aimed at the restora-
In AVMD, several mutations in BEST1 gene have tion of normal gene (i.e., BEST1 gene in BVMD
been identified including p.Ala146Lys [90], p. and AVMD). However, until decade ago, this
Thr6Pro, p.Arg47His, P.Ala243Val, p.Asp312Asn therapeutic goal was ideal but unachievable due
[61], and p.Ile38Ser [9]. Table 21.1 includes the to the lack of a proper biotechnology. Recent
list of missense mutations in AVMD. In addition, advances in genome editing technology using
AVMD is associated with mutations in PRPH2 CRISPR system and gene delivery system are
[91], IMPG1 [92], IMPG2 [93]. promising and harness the CRISPR-based
Age of onset is a major criterion to distinguish genome editing for the therapeutic applications.
BVMD from AVMD [64]. Thus, systematic Since its first therapeutic applications in retinal
screening of BEST1 and PRPH2 has been sug- disease using wet AMD animal models [96, 97],
gested in BVMD and AVMD. BEST1 screening in vivo genome editing using CRISPR-Cas9
should be recommended to patients with an age enlarged its therapeutic applications both in
of onset less than 40 years, and PRPH2 screening genetic diseases harboring mutations [98, 99] and
should be recommended to patients with an age nongenetic degenerative diseases [96, 97, 100].
of onset more than 40 years. For an onset between Conventional concept of gene therapy to
30 and 40 years, PRPH2 can be screened if no deliver normal copy of BEST1 gene into RPE
mutation has been detected in BEST1. In this would be effective in the treatment of VMD of
screening approach, we found PRPH2 mutation haploinsufficiency phenotype, which is caused
of p.Pro219_Pro221delinsPro in a 39-year-old by BEST1 mutations that exclusively result in a
female without BEST1 mutation (Fig. 21.3). loss of sufficient wild-type protein. In addition,
Fig. 21.3 Adult-onset vitelliform macular dystrophy Pro221delinsPro in PRPH2 gene suffered from dysmor-
(AVMD). A 39-year-old woman carrying Pro219_ phopsia of the right eye. OCT reveals subfoveal vitelliform
lesion in the right eye (a) and left eye (b)
simple destruction of mutant proteins at the DNA Compliance with Ethical Requirements Sung Wook
Park, Chang ki Yoon, Dae Joong Ma, Un Chul Park, and
level is achievable by genome editing of mutant Hyeong Gon Yu declare that they have no conflict of
BEST1 allele using CRISPR-Cas9. interest.
Currently, many BEST1 mutations cause All procedures followed were in accordance with the
VMD through dominant negative effect. In addi- ethical standards of the responsible committee on institu-
tional review board and with the Helsinki Declaration of
tion, over 200 mutations of BEST1 gene, large 1975, as revised in 2000. Informed consent was obtained
amounts of BEST1 mutations are missense muta- from all patients for being included in the study.
tions; thus, a precise base-editing using base-
editors enables a literally complete recovery of
normal gene [101, 102]. According to the recent References
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Adeno-Associated Virus (AAV)-
Mediated Gene Therapy for Leber 22
Hereditary Optic Neuropathy
Kunpeng Xie, Shuai Ming, Mingzhu Yang,

Xuemin Jin, and Bo Lei
Abstract Keywords
Leber hereditary optic neuropathy (LHON) is Leber hereditary optic neuropathy · Allotopic
the first clinically characterized maternally expression · AAV2-ND4 · Gene therapy ·
inherited mitochondrial disorder. Up to now, Retinal ganglion cell
more than 30 pathogenic point mutations in
mitochondrial DNA (mtDNA) coding for the
respiratory chain subunits of complex I genes, 22.1 Introduction
which are highly susceptible to disrupted ATP
production and oxidative stress, have been Recently, gene therapy for monogenic inherited
identified to cause LHON. The fundamental eye diseases has become a hot field. Promising
cell type affected in LHON is the retinal gan- results from different clinical trials have been
glion cells. Many researches facilitated the reported for treating Leber hereditary optic neu-
progress of animal models in vivo and cell ropathy (LHON). LHON is a maternally inher-
culture in vitro that have been used to deter- ited disease caused by point mutations in
mine the effects of the genetic mutations upon mitochondrial DNA (mtDNA), and it is consid-
the clinical phenotype and to explore potential ered as the most common mitochondrial disorder
novel therapies. More recently, clinical studies [1]. Three mtDNA mutations including
applying gene therapy have shown promising m.3460G > A in MT-ND1, m.11778G > A in
results in treating LHON. This article reviewed MT-ND4, and m.14484 T > C in MT-ND6 genes
the efficacy and safety of recombinant adeno- are found in almost 95% LHON patients [2].
associated virus 2 carrying ND4 (rAAV2- They are considered as the primary mutations for
ND4) in clinical trials and its allotopic
causing LHON, and each mutation represents a
expression in the LHON patients with the significant risk of severe vision loss [3]. The
G11778A mutation, which accounts for the prevalence of vision loss due to the three primary
majority of this vision-threatening disorder. mutations in LHON is one in 31,000 in the
Northern UK [4]. Other epidemiological studies
reported a prevalence of one in 39,000 and one in
50,000 in the Netherlands and Finland, respec-
Author’s Kunpeng Xie and Shuai Ming have been contrib- tively [5, 6]. LHON is characterized by incom-
uted equally to this chapter.
plete penetrance and is far more common in men.
K. Xie · S. Ming · M. Yang · X. Jin · B. Lei (*) Usually, up to 50% of men and only 10% of
Henan Eye Institute, Henan Eye Hospital, Henan women with the gene mutations present the
Provincial People’s Hospital, Zhengzhou, China

https://doi.org/10.1007/978-981-13-0884-0_22
274 K. Xie et al.
phenotype with typical symptoms of acute or for missing protein product. It is well known that
subacute pain-free vision loss in one eye. Similar mitochondrial structure includes two layers of
symptoms may appear in the other eye 8 weeks membrane, which is the physical barrier for sus-
later averagely. The onset occurs typically in tained gene expression following gene delivery
early adulthood, with little or no propensity to into the mitochondrial matrix compartment [13].
recover [7, 8]. Affected individuals are usually Under these conditions, allotopic gene expres-
asymptomatic until they develop central vision sion is employed to treat disease caused by
loss in one eye. mtDNA mutations. Adeno-associated viral vector
Although great effort has been made, there is serotype 2 (AAV2) is one of the most commonly
still no effective treatment for this devastating used vectors for allotropic gene expression. As a
disease. Idebenone (a short-chain derivative of therapeutic strategy, it has been developed to
coenzyme Q10), EPI-743 (parabenzoquinone deliver the MT-ND4 gene construction to com-
analogue of coenzyme Q10 and idebenone), and pensate for the m.11778G > A mutation [14].
vitamin B12 are reported to be effective in some AAV is a small, non-enveloped, icosahedral
LHON patients, while the medication did not virus that contains a linear single-stranded DNA
work in other patients [9]. Therefore, to find new genome of 5 kb [15]. The AAV genome includes
regime for LHON, gene therapy has been two frames: rep is required for DNA replication,
explored in several studies due to the unique and cap encodes all the three structural proteins
characteristics of the eye, including immune which form the icosahedral capsid and an essen-
privilege, easy implementing of surgical inter- tial protein for capsid assembled within the
vention, and symmetrical disease progression nucleolus [16]. The AAV genome is flanked by
[10, 11]. The present article reviewed the efficacy two 145-bp-long palindromic inverted terminal
and safety of recombinant adeno-associated virus repeats (ITRs) which form hairpin-loop second-
2 (AAV2) carrying ND4 (rAAV2-ND4) in clini- ary structures at the strand termini. Rep and cap
cal trials and its allotopic expression in the LHON are replaced by the exogenous DNA, which is
patients with G11778A mutation, which accounts provided together with the adenoviral helper
for the majority of this vision-threatening regarding the AAV productive infection in the
disorder. packaging cells [17]. AAV vectors are capable to
infect quiescent cells and mediate long-term
expression of transgenes. Various serotypes
22.2 mtDNA Mutations exhibit tropisms for different subsets of retinal
and Construction cells. Among all of the AAV vectors, AAV2 is
of AAV-ND4 Vector the most commonly used AAV serotype and has
been applied to several genetic and degenerative
Point mutations in the ND1 (G3460A), ND4 eye diseases including LHON. In a series of lab-
(G11778A), and ND6 (T14484C) genes account oratory experiments and clinic trials, ND4 sub-
for 95% of the LHON patients. Among them unit of complex I is packaged into AAV and then
ND4 mutation (G11778A) identified in 70% injected into the vitreous of rodent, nonhuman
patients impacts the most important complex I primate, and ex vivo human eye. AAV2-ND4 is
subunits of the mitochondrial respiratory chain modified with mitochondrial targeting signals
and leads to reduction of ATP production and and 3′ UTRs from nuclear genes whose mRNA
increase of reactive oxygen species (ROS) [3]. has been found to localize to the mitochondrial
Complex I defect is the main cause of retinal gan- surface, which results in targeting of the hybrid
glion cell (RGC) loss and optic atrophy [12]. mRNA to the mitochondrial surface and in the
Although gene therapy has gained great progress increase in the amount of protein fully imported
in the treatment for hereditary optic eye disease in mitochondria [18].
including LHON caused by mtDNA mutations, In a study by Guy and colleagues, the ND4
classical gene therapy is not applied to LHON yet subunit of complex I is recoded and imported
22 Adeno-Associated Virus (AAV)-Mediated Gene Therapy for Leber Hereditary Optic Neuropathy 275
into the mitochondria from the cytoplasm by the study of Li’s group [21, 23]. The discrepancy
adding a targeting sequence derived from the P1 of occurrence of transient uveitis between the
isoform of the subunit c of ATP synthase [19]. two studies remains unknown. One possible
Afterward, the nuclei encode ND4 with the interpretation may be that one study used predni-
appended subunit c of ATP synthase targeting sone while the other did not. However, involve-
sequences as P1ND4v2, which is inserted into a ment of neutralizing antibodies (NAbs) against
self-complementary adeno-associated virus vec- AAV2 can’t be excluded, because one of the
tor (scAAV2) (Y444, 500, 730F) and named patients in Feuer’s study showed elevation of
scAAV2-P1ND4v2 [20]. In another study by NAbs [20]. NAbs to AAV2 in serum and aqueous
Yang et al., an AAV2-ND4 vector is constructed humor were evaluated in the study by Guy et al.
[21]. The DNA sequence is synthesized as two Elevated serum NAbs are found in 3 of all 14
parts: the mitochondrial positioning signal cases. Though four patients with the most
(MTS) of the COX10 plus the coding sequence improvement in visual acuity after treatment had
of ND4, with 5′ end carrying a KpnI site and 3′ the highest levels of serum NAbs, they had simi-
end carrying a SalI site, and COX10 3′ UTR, lar low levels of aqueous humor NAbs, which
with 5′ end carrying a SalI site and 3′ end carry- suggested high level of serum NAbs was not
ing a BamHI site [14, 22]. Then the two associated with prognosis of visual acuity.
sequences are inserted into the plasmid pAAV-
2neo carrier successively [23].
22.4 AAV-ND4-Mediated Gene
Therapy Improved Patients’
22.3 T
he Safety of AAV-ND4- Visual Acuity
Mediated Gene Therapy
for LHON In all clinical trials reported by Yang and col-
leagues, the change in best-corrected visual acu-
Safety is the most important issue to be addressed ity (BCVA) was the primary endpoint for LHON
in gene therapy. In the study by Wan et al., gene therapy [21]. Improvement of visual acuity
patients with negative anti-AAV2 antibody were was defined as an improvement of 0.3 or more
recruited for clinical trials. Oral prednisone was with logMAR. This definition minimized the
administered 1 week before and for 2 months influence of subjective factors and changes in
thereafter to avoid a potentially severe immune vision greater than or equal to 0.3 logMAR dur-
response induced by gene therapy [23]. During ing the 36-month follow-up period. Five patients
the follow-up period of 36 months, there was no achieved vision improvement in the injected eyes
adverse event such as cataracts, retinal detach- between 3 and 6 months after gene therapy.
ment, and endophthalmitis, or other ocular tissue Interestingly, vision improvement was also
damage in any of the subjects. Also, systemic observed between 3 and 12 months in the contra-
examinations did not reveal any abnormal lateral un-injected eyes. Thus vision improve-
changes. None of the patients have remarkable ment in the injected eyes had a time window,
change in the concentrations of serum AAV2, while vision recovery observed in the fellow eyes
ND4, and IFN-γ. In the clinical trials carried out happened randomly. There were four patients
by Guy and colleagues, there were no serious with no significant improvement in visual acuity
safety concerns associated with allotopic gene 36 months after gene therapy [21].
therapy in 14 participants treated with low- and There was no difference in vision improve-
medium-dose vector. The primary adverse event ment between patients with a disease duration
was anterior uveitis related to the transgene. less than 2 years and those more than 2 years.
Fortunately, the inflammation response was mild, Though there was no difference with regard to
asymptomatic, and transient and required no the primary endpoint, gene therapy in the injected
treatment. Anterior uveitis was not reported in eye displayed an overall protection against the
276 K. Xie et al.
further vision loss. No patient demonstrated a injected eyes, and it occurred within 6 months
more serious decline in visual acuity from the after injection, suggesting that the vision recov-
baseline. ery of the contralateral eyes may be a result from
In the study reported by Guy and colleagues, gene therapy.
vision improvement was defined as three lines or The mechanism by which gene therapy
more [19]. One of the six patients with duration affected the contralateral eye was investigated
of vision loss longer than 12 months had visual [25]. In animal experiments, a fluorescent gold
improvement, while four of six patients with tracer was used to explore the link between the
duration of vision loss within 1 year had visual two eyes. The results demonstrated a physical
improvement. Furthermore, the results indicated communication between the two eyes via the
that vision recovery may be associated with the optic chiasm. Studies by Luo et al. [26] showed
treatment dosages and duration of disease. In that a few regenerating nerve axons crossed the
those participants with chronic vision loss more optic chiasm into the contralateral optic nerve
than 12 months and marked loss of RNFL, low and grew toward the contralateral retina. These
dose of gene transfer presented no effect on data suggested the possibility of direct communi-
visual acuity. However, when given a medium cation between the optic nerves, which may con-
dose, one of the participants with marked loss of tribute to the visual acuity improvement of the
the RNFL showed an increase in visual acuity contralateral eyes.
equivalent to three lines on the ETDRS chart. In
those participants with acute visual loss who also
had normal RNFL, an injection of low dose 22.5 AAV-ND4-Mediated Gene
increased the acuity by three lines on the ETDRS Therapy Prevented the Loss
chart. Vision improvements in the treated eyes of the RNFL
were usually evident within 7–30 days after
injection, as quickly as that reported in the Previous studies have shown that RNFL thick-
Chinese cohort. Vision of these patients may ness continuously declines in LHON patients
improve further with longer follow-up period. [27, 28]. To evaluate the effect of gene therapy on
The rapid and persistent improvement may result RNFL thickness, RNFL thickness was examined
from the rapid and persistent expression of the in the Chinese subjects. As expected, the RNFL
transgene in the eyes, which was demonstrated in thickness in the un-injected eyes decreased even
rodent studies [24]. Animal studies may provide after the gene therapy. However, RNFL thickness
important information; however, care must be in the injected eyes did not change, suggesting
taken in extrapolating the results in rodent to that loss of RNFL terminated. These results sug-
humans, particularly under pathological gested that gene therapy provided RNFL protec-
conditions. tion for the injected eyes, but did not have
It should be noted that there are two unex- protective effect on the un-injected eyes
pected phenomena observed in the 3-year LHON 24 months after gene therapy. Studies reported by
gene therapy follow-up. Firstly, though visual Guy et al. [19] showed that the temporal RNFL
acuity improvement of patients was above the of treated patients followed up for 12 months was
baseline after treatment, some patients presented not damaged by allotopic ND4 gene therapy.
BCVA fluctuations during the 3-year follow-up. One-year posttreatment values were similar to
Visual acuity fluctuations may relate to stability those obtained before treatment. In contrast, the
of protein expression after ND4 transfection, thickness of the temporal RNFL of the fellow
other persistent disease factors, and/or patient- eyes continued to decrease. OCT data from both
specific factors. Secondly, visual acuity of the groups showed that allotopic ND4 was not harm-
contralateral eye appeared to be improved in ful to visual function and did not damage the
some patients. Improvements of the un-injected temporal RNFL subserving macular fibers tar-
eyes never occur before the improvements of the geted by an intravitreal AAV2 injection. These
22 Adeno-Associated Virus (AAV)-Mediated Gene Therapy for Leber Hereditary Optic Neuropathy 277
results might pave the way for injection of high- This yeast gene would treat all mutations associ-
dose cohorts in the following trials. ated with complex I disease by replacing the
entire complex I, not just the affected ND4 sub-
unit [29, 30]. All of these developments bring
22.6 AAV-ND4 Decreased much optimism about the future of LHON treat-
the PERG Amplitudes ment. However, the efficacy, adverse effects, and
duration of treatment benefit have yet to be deter-
In the study by Wan et al., VEP results showed mined by long-term follow-up.
that the latency period of the P100 wave of the
injected eyes in some patients was shortened (a Acknowledgments This study is supported by the
P100 wave <105 ms is normal) [23]. At 6 months National Natural Science Foundation of China grants
(81470621, 81770949), National Key Clinical Specialties
after intravitreal injection, the P100 amplitudes Construction Program of China, Henan Science and
of the injected eyes were increased, but VEP Technology Bureau (182102310145), Henan Provincial
results were unstable. In the study by Guy et al., Clinical Research Center, and Henan Key Laboratory of
results suggested that PERG amplitudes wors- Ophthalmology and Visual Science. The authors alone are
responsible for the content and writing of the paper.
ened more in the treated eyes than the fellow eyes
(P¼ 0.009 exchangeable; P ¼ 0.011 autoregres- Compliance with Ethical Requirements The authors
sive) by approximately 0.05 mV [19]. Besides, in have no conflict of interest to declare.
Guy’s study, they inferred that the drop in PERG
amplitudes seemed to be related to the injection,
because the gene expression from the vector
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Stargardt Disease in Asian
Population 23
Xiao Liu, Yu (Yokokawa) Fujinami, Lizhu Yang,
Gavin Arno, Kaoru Fujinami
Abstract Keywords
Stargardt disease 1 (STGD1; MIM 248200) is Stargardt disease · ABCA4 · Macular
the most prevalent inherited macular dystro- dystrophy · Cone–rod dystrophy · Retinitis
phy, which is an autosomal recessive condi- pigmentosa · Asian · Genetics
tion caused by pathogenic variants in the
ABCA4 gene (ATP-binding cassette subfam-
ily A member 4; MIM 601691). Clinical and
molecular genetic investigations of STGD1/
ABCA4 have been intensively performed over
the last 10 years, and understanding the under- L. Yang
lying pathophysiology promotes ongoing and Laboratory of Visual Physiology, Division of Vision
planned human clinical therapeutic trials. We Research, National Institute of Sensory Organs,
herein describe the phenotypic and genotypic National Institute of Sensory Organs, Tokyo Medical
Centre, Tokyo, Japan
characteristics of the disease, pathogenesis,
therapeutic approaches, and recent findings in Keio University, Tokyo, Japan
Asian population. Peking Union Medical College Hospital,
Beijing, China
G. Arno
East Asian Inherited Retinal Disease Society (EAIRDs) Laboratory of Visual Physiology, Division of Vision
Research, National Institute of Sensory Organs,
X. Liu National Institute of Sensory Organs, Tokyo Medical
Laboratory of Visual Physiology, Division of Vision Centre, Tokyo, Japan
National Institute of Sensory Organs, Tokyo Medical UCL Institute of Ophthalmology, London, UK
Centre, Tokyo, Japan Moorfields Eye Hospital, London, UK
Keio University, Tokyo, Japan K. Fujinami (*)
Third Military Medical University, Chongqing, China Laboratory of Visual Physiology, Division of Vision
Y. (Yokokawa) Fujinami National Institute of Sensory Organs, Tokyo Medical
Laboratory of Visual Physiology, Division of Vision Centre, Tokyo, Japan
National Institute of Sensory Organs, Tokyo Medical Keio University, Tokyo, Japan
Centre, Tokyo, Japan UCL Institute of Ophthalmology, London, UK
Keio University, Tokyo, Japan Moorfields Eye Hospital, London, UK
Yokokawa Clinic, Osaka, Japan e-mail: [email protected]

https://doi.org/10.1007/978-981-13-0884-0_23
280 X. Liu et al.
23.1 Introduction onset, progression, psychophysical and electro-

physiological findings, and variable prognosis [4,
Stargardt disease or Stargardt macular dystrophy 5, 7–17].
(STGD1: Online Mendelian Inheritance in Man In 1997, causative mutations in the ABCA4
identifier; 248200), first described by Karl (ATP-binding cassette subfamily A member 4:
Stargardt in 1909, is one of the most common Online Mendelian Inheritance in Man identifier;
macular dystrophy [1–5]. The prevalence of 601691) gene was firstly reported in patients with
STGD1 has been estimated to be 10–12.5 per autosomal recessive Stargardt macular dystrophy
100,000 [2, 6]. Most cases present with central [18]. The carrier frequency for a mutation in
visual loss, which often begins within the first/ ABCA4 may be as high as 1:20, and the true prev-
second decades of life, and there is typically alence of retinopathy caused by disease-causing
macular atrophy with yellow-white flecks at the ABCA4 variants is likely much higher than that of
level of the retinal pigment epithelium (RPE) of STGD1 [6, 19]. The vast allelic heterogeneity of
the posterior pole on ophthalmoscopy (Fig. 23.1). ABCA4 is also clearly demonstrated by the num-
However, there are various manifestations result- ber of reported sequence variations (>1000) in
ing in a large spectrum of clinical presentations, the ABCA4 gene [6, 8, 19–26].
Fig. 23.1 Typical findings of Stargardt disease (STGD1) and multiple foci of abnormal AF (b). Spectral-domain
Fundus photography shows macular atrophy with yellow- optical coherent tomography (SD-OCT) demonstrated the
white flecks at the level of the retinal pigment epithelium thinned sensory retina and RPE at the macula with
(RPE) at the posterior pole (a). Autofluorescence (AF) multiple hyper refractive lesions corresponding to the
imaging reveals the area of low density at the macula flecks (c)
23 Stargardt Disease in Asian Population 281
Clinical and molecular genetic investigations 23.3 Disease Mechanism

of STGD1/ABCA4 have been intensively per-
formed over the last 10 years, and understanding ABCA4, formerly described as ABCR, is a mem-
the underlying pathophysiology promotes ongo- ber of the ABC transporter gene superfamily,
ing and planned human clinical therapeutic trials encoding the retinal specific transmembrane pro-
[2, 27–29]. However, most of these researches tein, a member of the ATP-binding cassette trans-
are focusing on the Caucasian patients, and char- porter superfamily [38, 39]. ABCA4 is localised
acteristics of Asian or African patients are still to the rim of the rod–cone outer segment discs
unknown [30–34]. We herein describe the pheno- and involved in the active transport of retinoids
typic and genotypic characteristics of the disease,from photoreceptor to RPE in the retinoid cycle
pathogenesis, therapeutic approaches, and recent [38–41]. There are two transmembrane domains
findings in Asian population. (TMD), two glycosylated extracellular domains
(ECD), and two nucleotide-binding domains
(NBD) (Fig. 23.2) [39].
23.2 Molecular Genetics The retinoid cycle consists of enzyme-
catalysed reactions converting all-trans retinal,
The ABCA4 gene (cytogenetic location, 1p22.1; generated with photobleaching of rhodopsin/
genomic coordinates (GRCh38), 1:93,992,836- cone opsin, back to 11-cis retinal [38–40, 42,
94,121,148) is a large and highly polymorphic 43]. All-trans retinal is released from the light-
gene. The estimated size of ABCA4 is 6,819 bp activated rhodopsin/cone opsin into the rod–
encoding a 2,273-amino acid protein, including cone outer segments to form a complex with
50 exons [18, 35]. Over 1000 variants in the phosphatidylethanolamine (PE) resulting in
ABCA4 gene are associated with macular dystro- N-retinylidene-phosphatidylethanolamine
phy, cone dystrophy, cone–rod and ‘rod–cone’ (N-ret-PE); then this complex is actively trans-
dystrophy [4, 5, 7, 9–12, 14–17, 20]. Recently, ported to the disc surface by ABCA4
the phenotype caused by ABCA4 gene aberration (Fig. 23.2).
is described as ‘ABCA4-associated retinal disor- Failure of transport due to ABCA4 dysfunc-
der.’ [6, 14] This allelic heterogeneity makes it tion/mislocalisation leads to inefficient removal
challenging to establish genotype–phenotype of N-ret-PE from photoreceptor outer segments
correlations [11]. Generally, null/deleterious and results in an accumulation of bisretinoid
variants are associated with earlier-onset disease compounds in outer segment discs, and ulti-
with more severe phenotype and missense vari- mately in toxic levels of bisretinoid A2PE in pho-
ants with later-onset disease with milder pheno- toreceptor membranes [23, 39, 41, 44]. A2PE is
type; meanwhile, certain missense variants can hydrolysed to form the highly toxic metabolite
have severe functional effects similar to nulls N-retinylidene-N-retinyl-ethanolamine (A2E),
(e.g. p.Leu541Pro/p.Ala1038Val (complex), p. which accumulates as a major component of
Glu1022Lys, p.Cys1490Tyr, p.Glu1087Lys, lipofuscin in RPE cells and ultimately causes
p.Thr1526Met, p.Arg1640Trp, and p. RPE dysfunction and death with subsequent pho-
Cys2150Tyr) [11, 17, 20, 36]. The interaction toreceptor dysfunction/loss (Fig. 23.2) [42, 45].
between the variants (including disease-causing The previous studies of STGD1 mouse model
and benign variants) may also affect the func- (ABCA4 knockout) support the aforementioned
tional effects [37]. Certain missense variants pathogenesis, although there are limitations such
including p.Arg2030Gln are commonly observed as lack of a macula in mice and the mild pheno-
in the mildest ABCA4-associated phenotype, type in mouse model showing a later-onset dis-
foveal-sparing Stargardt disease (FS-STGD1) ease with slower degeneration than that of typical
[12, 16]. human patients with STGD1 [40, 46].
Fig. 23.2 A schematic of ABCA4 protein structure, retinoid activated rhodopsin/cone opsin into the rod–cone outer seg-
cycle, transport of all-trans retinal, and disease mechanism ments to form a complex with phosphatidylethanolamine
The ABCA4 gene transcribes a large retina-specific ABCA4 (PE) resulting in N-retinylidene-phosphatidylethanolamine
protein with two transmembrane domains (TMD), two gly- (N-ret-PE); then this complex is actively transported to the
cosylated extracellular domains (ECD), and two nucleotide- disc surface by ABCA4 (c). Failure of this transport results in
binding domains (NBD) (a). The retinoid cycle consists of accelerated deposition of a major lipofuscin fluorophore,
enzyme-catalysed reactions converting all-trans retinal, gen- N-retinylidene-N-retinylethanolamine (A2E), in the RPE,
erated with photobleaching of rhodopsin/cone opsin, back to which causes RPE dysfunction and cell death, with subse-
11-cis retinal (b). All-trans retinal is released from the light- quent photoreceptor cell loss overtime
23.4 Clinical Characteristics of FFERGs (Fig. 23.6) [8]. A longitudinal study

demonstrated prognostic implications and that
Patients with STGD1 commonly present with they do not reflect stages of disease: Group 1, the
progressive bilateral central vision loss. The best prognosis; Group 2, intermediate variable
onset is often the first/second decades of life but prognosis; and Group 3, the worst prognosis [4].
variable [4, 5, 12, 14, 47]. The onset relates to the All patients with initial rod ERG involvement
disease severity; an earlier-onset disease is asso- demonstrated clinically significant electrophysi-
ciated with more deleterious variants compared ological deterioration; only 20% of patients with
with adult-onset disease (more frequently, mis- normal FFERGs showed clinically significant
sense variants) [14]. progression [4]. Such data are supported by asso-
Comprehensive investigations are crucial for ciation with genotype grouping and are also rel-
clinical diagnoses, including fundus photogra- evant in the design, patient selection, and
phy, AF imaging, spectral-domain optical coher- monitoring of potential therapeutic interventions
ence tomography (SD-OCT), and [4].
electrophysiological findings (Figs. 23.3, 23.4, STGD1 with a later age of onset has been
23.4, 23.5, and 23.6, Tables 23.1, 23.2, and 23.3). increasingly been recognised recently. Patients
Clinical classifications are useful to assess the with late-onset STGD1 often have foveal-sparing
disease severity and associate with genotype phenotype (FS-STGD1) (Fig. 23.7) [10, 12, 49,
group classification (Table 23.4). 50]. Patients with FS-STGD1 frequently have
At an early stage, ophthalmoscopy can reveal preserved visual acuity and relatively maintained
a normal or minimal retinal abnormalities, foveal structure and function [12]. Interestingly,
including foveal reflex abnormality and RPE dis- SD-OCT often demonstrates the outer retinal
turbance, with or without vision loss [14]. Retinal tabulation at the edge of atrophy, which suggests
imaging with AF, SD-OCT, and electrophysio- the primal damage of this phenotype is RPE/cho-
logical assessment (including pattern, full-field, roid. On the other hand, patients with foveal atro-
and multifocal electroretinograms; PERG, phy show the sensory retinal atrophy at the fovea
FFERG, mfERG) is useful for the diagnosis [2, at the early stage. Therefore, the presence of two
14, 15, 48]. It is of note that paediatric patients distinct phenotypes (non-FS-STGD1 and
with STGD1 may not have retinal flecks on fun- FS-STGD1) suggests that there may be more
doscopy or AF at the early stage and develop than one disease mechanism in ABCA4-
them associated with increasing macular atrophy associated retinal disorder [12]. The fact that the
overtime (Fig. 23.4). In very early childhood- distribution of disease-causing variants between
onset disease with relatively preserved vision, the two phenotypes is different can support this
macular atrophy involves the parafovea and hypothesis [12].
spares the foveola, and these changes are pre-
dated by symmetrical yellowish-white fine dots
at the central macula in some cases [14, 15, 48]. 23.5 Asian Population
Electrophysiological assessment is helpful in
confirming the diagnosis of STGD1 and in pro- The number of studies focusing on Asian
viding better-informed advice on prognosis. A population is relatively small; however several
classification of three functional phenotypes studies have been performed in Asian patients
based on electrophysiological findings is well- with STGD1/ABCA4-associated retinal disorder.
established: Group 1, severe PERG abnormality The prevalent variants of recent reports from
(macular dysfunction) with normal FFERGs; Chinese, Japanese, Korean, Indian, and South
Group 2, severe PERG abnormality with addi- Asian have been summarised in Table 23.6.
tional generalised cone dysfunction of FFERGs; Jing et al. reported the disease-causing vari-
and Group 3, severe PERG abnormality with ants in a Chinese cohort of 161 unrelated patients
additional generalised cone and rod dysfunction with STGD1 (96 patients) and cone–rod dystro-
284 X. Liu et al.
Fig. 23.3 Classification for fundus appearance in Table 23.1. (Fujinami et al. Clinical and Molecular
STGD1. Fundus classification is performed based on the Characteristics of Childhood-Onset Stargardt Disease.
presence of macular atrophy, flecks, foveal sparing, and Ophthalmology 2015)
peripheral atrophy. Detailed descriptions are presented in
Fig. 23.4 Development of macular atrophy and flecks. atrophy and macular and/or peripheral flecks are devel-
At baseline, fundus photography reveals normal or mini- oped, which are recognisable both by fundus photography
mal macular abnormalities with evidence of macular and AF imaging
abnormality detected by AF. Overtime, marked macular
phy (65 patients) [51]. The prevalent variants Fukui et al. reported the disease-causing vari-
were p.Tyr808Ter (15 alleles), p.Phe2188Ser (12 ants in a Japanese cohort with Stargardt disease
alleles), p.Ser34-Leu35del (10 alleles), and p. and retinitis pigmentosa [52]. The prevalent vari-
Asn965Ser (10 alleles). All these variants are not ants were c.1760+2T>G (five alleles), p.
described as prevalent variants in Caucasian Leu1894Leu (four alleles), p.Leu1938Leu (four
population. alleles), c.5838-11G>A (four alleles), and p.
Pro1948Pro (four alleles). These variants are dif-
286 X. Liu et al.
Fig. 23.5 Autofluorescence patterns and progression. AF tions are presented in Table 23.2. (Fujinami et al. A longi-
classification is performed based on the size of the area tudinal study of Stargardt disease: quantitative assessment
with low signal and background features (heterogeneous/ of fundus autofluorescence, progression, and genotype
homogeneous). A severe type shows rapid progression correlations. Invest Ophthalmol Vis Sci. 2013)
associated with null ABCA4 variants. Detailed descrip-
ferent from those of Chinese population, although reports of FS-STGD1 in Japanese population,
clinical presentation of the high proportion was which could be relatively common in Japanese
retinitis pigmentosa. Interestingly, these are [10, 53].
Fig. 23.6 Functional phenotype and electrophysiological All patients with initial rod ERG involvement demon-
progression Full-field electroretinogram (FFERG) and pat- strated clinically significant electrophysiological deterio-
tern ERG (ERG) of three representative cases from each ration overtime; only 20% of patients with normal FFERGs
ERG group and a normal subject are shown. ERG group showed clinically significant progression. (Lois et al.
classification is performed based on the presence of macu- Phenotypic subtypes of Stargardt macular dystrophy-fun-
lar dysfunction (abnormal PERG or macular ERG), gener- dus flavimaculatus. Arch Ophthalmol 2001; Fujinami et al.
alised cone dysfunction (abnormal light-adapted FFERG), A longitudinal study of Stargardt disease: clinical and elec-
and generalised rod dysfunction (abnormal dark-adapted trophysiological assessment, progression, and genotype
FFERG). Detailed descriptions are presented in Table 23.3. correlations. Am J Ophthalmol 2013)
288 X. Liu et al.
Table 23.1 Classification for fundus appearance in Table 23.4 Classification for genotype classification in
Stargardt disease ABCA4 retinopathy
Grade Normal fundus Genotype Two or more likely deleterious variants
1 A
Grade Macular and/or peripheral flecks without Genotype One deleterious variant and one or more
2 central atrophy B missense or in frame insertion/deletion
Grade Central atrophy without flecks variant(s)
3a Genotype Two or more missense or in frame
Grade Central atrophy with macular and /or C insertion/deletion variants
3b peripheral flecks Fujinami et al. Clinical and Molecular Characteristics of
Grade Paracentral atrophy with macular and/or Childhood-Onset Stargardt Disease. Ophthalmology 2015
3c peripheral flecks, without a central atrophy
Grade Multiple extensive atrophic changes of the
4 RPE, extending beyond the vascular arcades Song et al. reported safety and potential effi-
Fujinami et al. Clinical and Molecular Characteristics of cacy of subretinal transplantation of human
Childhood-Onset Stargardt Disease. Ophthalmology 2015 embryonic stem cell (hESC)-derived RPE cells in
four Asian patients: two with dry age-related
macular degeneration and two with Stargardt
Table 23.2 Classification for autofluorescence pattern in macular dystrophy [54]. This is the first report of
Stargardt disease
therapeutic trials for STGD1 focusing on Asian
Pattern Localised low AF signal at the fovea population. Otherwise, there are no reports
1 surrounded by a homogeneous background
with/without perifoveal foci of high or low
describing disease-causing ABCA4 variants in a
signal large cohort of Korean population, which
Pattern Localised low AF signal at the macula appeared on PubMed (https://www.ncbi.nlm.nih.
2 surrounded by a heterogeneous background gov/pubmed).
and widespread foci of high or low AF Battu et al. reported three recurrent disease-
signal extending anterior to the vascular
arcades causing variants in five unrelated patients with
Pattern Multiple areas of low AF signal at posterior STGD1: p.Gly1961Glu (two alleles), p.
3 pole with a heterogeneous background and/ Thr971Asn (two alleles), and p.Arg2149Ter (two
or foci of high or low signal alleles) [55]. Lee et al. described the genotypic
Fujinami et al. A longitudinal study of Stargardt disease: spectrum of ABCA4-associated disease in 38
quantitative assessment of fundus autofluorescence, pro-
patients of South Asian descent and reported two
gression, and genotype correlations. IOVS 2013
major prevalent variants: p.Gly1961Glu (17
alleles) and c.859-9T>C (7 alleles) [33].
Table 23.3 Classification for electrophysiologic find- Interestingly, p.Gly1961Glu is the most prevalent
ings in Stargardt disease variant in Caucasian population and also fre-
Group Macular dysfunction (normal full-field quently found in individuals of Somalian ances-
1 ERG) try [56]; thereby the genetic route of this variant
Group Macular dysfunction with generalised cone could be shared between Asian and Caucasian
2 dysfunction
[11, 16, 17, 57].
Group Macular dysfunction with generalised cone
3 and rod dysfunction Overall, large cohort studies in Asian popula-
ERG electroretinogram
tion are still lacking, and international collabora-
Lois et al. Phenotypic subtypes of Stargardt macular tive studies by Asian Eye Genetics Consortium
dystrophy-fundus flavimaculatus. Arch Ophthalmol 2001; (AEGC; http://asianeyegenetics.org/) and East
Fujinami et al. A longitudinal study of Stargardt disease: Asia Inherited Retinal Disease Consortium
clinical and electrophysiologic assessment, progression,
and genotype correlations. Am J Ophthalmol 2013
(EAIRDc; https://www.fujinamik.com/east-asia-
inherited-retinal-disease) are needed, aiming to
Fig. 23.7 Fundus imaging and spectral-domain optical mal damage of this phenotype is RPE/choroid. Detailed
coherent tomography of foveal sparing phenotype Patients descriptions for the pattern classification are presented in
with foveal-sparing STGD1 (FS-STGD1) have preserved Table 23.5 (Fujinami K et al. Clinical and molecular anal-
visual acuity and relatively maintained foveal structure ysis of Stargardt disease with preserved foveal structure
and function. The outer retinal tabulation (arrowed) at the and function. Am J Ophthalmol. 2013)
edge of atrophy is often observed, which suggests the pri-
290 X. Liu et al.
Table 23.5 Fundus pattern for Stargardt disease with A Phase I/II stem cell therapy trial with sub-
foveal-sparing phenotype
retinal transplantation of hESC-derived RPE
Foveal-sparing Patchy parafoveal atrophy cells has been ongoing in patients with severe
pattern 1 surrounded by numerous yellow-
white flecks
advanced STGD1 (ClinicalTrials.gov Identifier:
Foveal-sparing Numerous yellow-white flecks at the NCT01469832), given RPE cell dysfunction/loss
pattern 2 posterior pole without atrophy is believed to precede photoreceptor cell dys-
Foveal-sparing Mottled RPE changes and/or function/loss in STGD1 [29]. There have been no
pattern 3 localised parafoveal yellow-white safety concerns to date.
flecks
Several pharmacological agents have been
Foveal-sparing Multiple patchy atrophic lesions,
pattern 4 extending beyond the arcades specifically developed that target different aspects
Fujinami et al. Clinical and Molecular Analysis of
of the retinoid cycle and are potentially beneficial
Stargardt Disease with Preserved Foveal Structure and in slowing or preventing progression in STGD1
Function. Am J Ophthalmol 2013より [61]. The aims of these agents are either [1]
reducing the formation of toxic products of the
develop/apply therapeutic approaches in Asian retinoid cycle, by reducing delivery of vitamin A
population. or inhibition of various enzymes participating in
the cycle, or [2] directly targeting toxic metabo-
lites such as A2E. Visual cycle modulators are
23.6 Therapeutic Approaches candidates for the former treatment [62–64]. A
Phase II clinical trial with a chemically modified
Several treatment approaches have been devel- vitamin A, which does not dimerise and stops
oped for STGD1, and clinical trials for gene N-ret-PE and A2E formation, has been ongoing
replacement, stem cell therapy, pharmacological (ClinicalTrials.gov Identifier NCT02402660)
agents, and retinal prosthesis have been ongoing [65–67].
[2, 27, 28].
Gene replacement therapy has been increas-
ingly applied to photoreceptor diseases, aiming 23.7 Conclusion
to slow or prevent further retinal degeneration.
Adeno-associated virus (AAV) vectors have been STGD1 is one of the most common causes of
the major choice for gene transfer system of inherited retinal disease associated with ABCA4
human gene therapy; however there is a limita- disease-causing variants. STGD1 is highly het-
tion for the size, that is, the ABCA4 gene is larger erogeneous both phenotypically and genetically,
than the current AAV vector capacity [58]. and investigations have been performed to under-
Considering the larger cargo capacity of lentivi- stand the underlying disease mechanisms, which
ruses, subretinal injection of a lentivirus vector allows to conduct multiple clinical trials.
delivering ABCA4 has been developed and is in However, these trials have been performed
an ongoing Phase I/II clinical trial (ClinicalTrials. mostly in Caucasian population, and further
gov Identifier: NCT01367444). There have been robust longitudinal studies, probing genotype–
no safety concerns in the first three cohorts of phenotype and structure–function associations in
subjects with relatively advanced disease and the Asian population, are crucial in order to provide
final cohort with less severe disease now being improved prognostication and genetic counsel-
recruited [28, 59, 60]. ling, as well as to initiate therapeutic trials.
Table 23.6 Prevalent ABCA4 variants in Asian population
Ethnicity Number Recruitment Prevalent variants
of criteria Variant 2 Variant 3
affected
Reports subjects Variant 1 Variant 4
Jiang et el. Screening of Chinese 161 161 unrelated c.2424C>G, c.6563 T>G, p. c.101_106delCTTTAT, c.2894A>G,
ABCA4 Gene in a patients with p.Tyr808Ter (15 Phe2188Ser (12 p.Ser34-Leu35del (10 alleles) p.Asn965Ser (10
Chinese Cohort With STGD1 (96 alleles) alleles) alleles)
Stargardt Disease or patients) and
Cone-Rod Dystrophy CRD (65
With a Report on 85 patients)
Novel Mutations.
Investigative
Ophthalmology & Visual
23 Stargardt Disease in Asian Population
Science January 2016

Fukui T, et al. "ABCA4 Japanese 110 110 unrelated c.1760+2T>G, c.5682G>C, c.6285T>C, p.Asp2095Asp (3 c. 5644A>G, p.
gene mutations in patients with splice site p.Leu1894Leu (4 alleles) Met1882Val (2
Japanese patients with STGD1 (10 alteration (5 alleles) c.5814A>G, alleles)
Stargardt disease and patients), alleles) p.Leu1938Leu (4
retinitis pigmentosa. " arRP(96 alleles) c.5838-
Investigative patients), and 11G>A, splice site
Ophthalmology & Visual CRD (4 alteration (4 alleles)
Science 2002. patients) c.5844A>G,
p.Pro1948Pro (4
alleles)
Song, et al. Treatment of Korean 2 2 unrelated c.983A>T, p.Glu328Val (1 allele) c.1933G>A, p.Asp645Asn (1 allele) c.3106G>A, p.Glu1036Lys (1
Macular Degeneration patients with allele) c.2894A>G, p.Asn965Ser (1 allele) c.4972A>C, p.Ser1658Arg (1 allele)
Using Embryonic Stem STGD1
Cell-Derived Retinal
Pigment Epithelium:
Preliminary Results in
Asian Patients. Stem Cell
Reports 2015.
(continued)
291
292
Ethnicity Number Recruitment Prevalent variants

of criteria Variant 2 Variant 3
affected
Reports subjects Variant 1 Variant 4
Battu, R, et al. Indian 5 5 unrelated c.5882G>A, c.2912C>A, c.6445C>T, p.Arg2149Ter (2 c.4918C>T,
Identification of Novel patients p.Gly1961Glu (2 p.Thr971Asn (2 alleles) p.Arg1640T (1
Mutations in ABCA4 withSTGD1 alleles) alleles) allele)rpc.514G>A,
Gene: Clinical and p.Gly172Ser (1
Genetic Analysis of allele) c.570+1G>A,
Indian Patients with Splice site alteration
Stargardt Disease. (1 allele)
Biomed Research c.2616C>G,
International 2015. p.Tyr872Ter (1
allele)
Lee W et al. Genotypic South 38 Not described c.5882G>A, c.859-9T>C, splice c.93G>A, p.Trp31Ter (2 alleles), c.91T>C, p.
spectrum and phenotype Asian p.Gly1961Glu (17 site alteration Trp31Arg (2 alleles), c.1531C>T, p.Arg511Cys (2
correlations of ABCA4- alleles) (7alleles) alleles)c.2894A>, p.Asn965Ser (2 alleles),
associated disease in c.2966T>C, p.Val 989Ala (2 alleles), c.5917del, p.
patients of south Asian Val1973Ter (2 alleles)c.6191C>T, p.Ala2064Val (2
descent. Eur J Hum alleles), c.6729+5_6729+19del, splice site alteration
Genet. 2017. (2allele)c.179C>T, p.Ala60Val/c.3830C>T,p.
Thr1277Met (complex; 2 alleles)
CRD cone–rod dystrophy, STGD1 Stargardt disease, arRP autosomal recessive retinitis pigmentosa
X. Liu et al.
Acknowledgement We are grateful to Prof Michel 3. Ueber Familiaere KS. progressive degeneration in
Michaelides, Prof Yozo Miyake, Dr Kazushige Tsunoda, der Makulagegend des Auges. Graefes Arch Clin Exp
Prof Takeshi Iwata, Prof Graham E. Holder, Prof Anthony Ophthalmol. 1909;71:534–49.
T. Moore, Prof Andrew R. Webster, Prof Anthony 4. Fujinami K, Lois N, Davidson AE, et al. A longitudinal
G. Robson, Prof Andrew Webster, Dr Rupert W Strauss, study of Stargardt disease: clinical and electrophysi-
Dr Preena Tanna, Dr Kamron N. Khan, Dr Ana Fakin, ologic assessment, progression, and genotype correla-
Prof Hendrik P. N. Scholl, Prof Shiying Li, Prof Ruifang tions. Am J Ophthalmol. 2013;155(6):1075–88.
Sui, Prof Se Joon Woo, Kwangsic Joo, Dr Panagiotis 5. Fujinami K, Lois N, Mukherjee R, et al. A longitu-
Sergouniotis, Dr Eva Lenassi, Dr Toshihide Kurihara, and dinal study of Stargardt disease: quantitative assess-
Prof Kazuo Tsubota for the data collection and their ment of Fundus autofluorescence, progression, and
insightful comments. genotype correlations. Invest Ophthalmol Vis Sci.
2013;54(13):8181–90.
Funding Kaoru Fujinami is supported by Grant-in-Aid 6. Burke TR, Tsang SH. Allelic and phenotypic het-
for Young Scientists (A) and Fund for the Promotion of erogeneity in ABCA4 mutations. Ophthalmic Genet.
Joint International Research, Fostering Joint International 2011;32(3):165–74.
Research, the Ministry of Education, Culture, Sports, 7. Fishman GA, Stone EM, Grover S, et al. Variation of
Science and Technology (Japan); the Japan Agency for clinical expression in patients with Stargardt dystro-
Medical Research and Development (Japan); the Specified phy and sequence variations in the ABCR gene. Arch
Disease Research Program on Intractable Diseases, the Ophthalmol. 1999;117(4):504–10.
Ministry of Health Labour and Welfare (Japan); National 8. Lois N, Holder GE, Bunce C, et al. Phenotypic sub-
Hospital Organization Network Research Fund (Japan); types of Stargardt macular dystrophy-fundus flavi-
Foundation Fighting Blindness Career Development maculatus. Arch Ophthalmol. 2001;119(3):359–69.
Award Clinical Research Fellowship Program (USA); and 9. Yatsenko AN, Shroyer NF, Lewis RA, Lupski
the Great Britain Sasakawa Foundation, Butterfield JR. Late-onset Stargardt disease is associated with
Awards for UK–Japan collaboration in medical research missense mutations that map outside known func-
and public health practice (UK). tional regions of ABCR (ABCA4). Hum Genet.
2001;108(4):346–55.
10. Fujinami K, Akahori M, Fukui M, et al. Stargardt dis-
The sponsor or funding organisation had no role in the
ease with preserved central vision: identification of a
design or conduct of this research.
putative novel mutation in ATP-binding cassette trans-
porter gene. Acta Ophthalmol. 2011;89(3):E297–E8.
Conflict of Interest Liu X, Fujinami YY, Lizhu Y, and 11. Fujinami K, Sergouniotis PI, Davidson AE, et al. The
Arno G declare that they have no conflict of interest. clinical effect of homozygous ABCA4 Alleles in 18
patients. Ophthalmology. 2013;120(11):2324–31.
Financial Disclosures Fujinami K has received research 12. Fujinami K, Sergouniotis PI, Davidson AE, et al.

grants from Astellas Pharma Inc., and Foundation Fighting Clinical and molecular analysis of Stargardt disease
Blindness. Fujinami K has received a speaker honorarium with preserved foveal structure and function. Am
from Santen Pharmaceutical Co., Ltd., Astellas Pharma J Ophthalmol. 2013;156(3):487–501.
Inc., Japanese Ophthalmological Society, and Japanese 13. Singh R, Fujinami K, Chen LL, et al. Longitudinal fol-
Retinitis Pigmentosa Society. low-up of siblings with a discordant Stargardt disease
phenotype. Acta Ophthalmol. 2014;92(4):e331–e2.
14. Fujinami K, Zernant J, Chana RK, et al. Clinical and
molecular characteristics of childhood-onset Stargardt
disease. Ophthalmology. 2015;122(2):326–34.
15. Fujinami K, Singh R, Carroll J, et al. Fine central mac-
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Retinoblastoma Genes in Chinese
Studies 24
Bi Ning Zhang, Yuning Jiang, Wai Kit Chu,
Winnie W. Y. Lau, Simon T. C. Ko, Kwong Wai Choy,
Calvin C. P. Pang, Guy L. J. Chen,
and Jason C. S. Yam
Abstract in Chinese. Contrasts with other ethnic groups

Retinoblastoma is the commonest pediatric will be made. These studies help us to under-
intraocular malignancy across most ethnic stand the mechanisms of inactivating RB1, its
populations. Its genetic basis follows a two-hit functional consequences, and maintaining
model, in which two events are needed to genome stability. Evidences have also been
inactivate both alleles of the disease-causative obtained on epigenetic contribution to retino-
gene, RB1. Loss of heterozygosity (LOH) is a blastoma, especially by DNA repair genes. On
major driving force to inactivate the whole the basis of the genetic and epigenetic find-
RB1 gene. Epigenetic modifications such as ings, development of potential alternative
DNA methylation involving different genes therapy for retinoblastoma will be discussed,
and microRNA (miRNA) expressions also together with key issues in attempts of aware-
play important roles in retinoblastoma tumori- ness campaign for early detection and genetic
genesis. Functionally, as RB1 is important in counselling.
maintaining chromosomal stability and cell
cycle progression, loss of its function would Keywords
underlie aberrations in other chromosomal Retinoblastoma · RB1 · LOH · Methylation ·
regions. In addition, genes other than RB1 Chromosomal instability
have been identified as direct or indirect
causes of retinoblastoma tumorigenesis. In
this chapter, we review the major genetic stud-
ies of retinoblastoma that have been conducted
B. N. Zhang · Y. Jiang · W. K. Chu · C. C. P. Pang

J. C. S. Yam (*) K. W. Choy
Department of Ophthalmology and Visual Sciences, Department of Obstetrics and Gynaecology,
The Chinese University of Hong Kong, The Chinese University of Hong Kong,
Kowloon/Hong Kong, China Hong Kong, China
G. L. J. Chen
W. W. Y. Lau Department of Ophthalmology and Visual Sciences,
Hong Kong Eye Hospital, Hong Kong, China The Chinese University of Hong Kong,
Kowloon/Hong Kong, China
S. T. C. Ko
Department of Ophthalmology, Tung Wah East Department of Ophthalmology and Visual Sciences,
Hospital, Hong Kong, China Prince of Wales Hospital, Hong Kong, China

https://doi.org/10.1007/978-981-13-0884-0_24
298 B. N. Zhang et al.
24.1 Retinoblastoma live births during 1979–2003 [4] and 1 in 17,373
Epidemiology live births during 1998–2011 [5]. Incidence of
1 in 18,000 live births had been reported in Hong
Retinoblastoma is the most common childhood Kong [6]. According to hospital records from
intraocular cancer affecting all populations. It 2009 to 2014, there were 35 eyes diagnosed as
accounts for 3% of all cancers occurring in chil- retinoblastoma in Hong Kong, approximately 3–9
dren younger than 15 years old [1]. Its incidence new cases per year (unpublished data). There is no
is approximately 40–60 per million live births strong geographic difference in incidences of reti-
worldwide, which corresponds to 1 per 15,000– noblastoma by gender or laterality. However,
25,000 live births. In the United States and studies from Brazil and Mexico have reported an
Europe, the age-adjusted incidence is 2–5 cases inverse association between the incidence of reti-
per 1 million children (approximately 1 in noblastoma and socioeconomic level index [7, 8].
14,000–18,000 live births) [2]. The disease has no In the United States, an increased incidence has
validated geographic or populational differences been correlated with low levels of maternal edu-
in incidence. The greatest burden is recorded in cation and poverty [9]. Table 24.1 summarizes the
large populations that have high birth rates, such age-standardized incidence rates (ASIR) of reti-
as Africa, with reportedly approximately six to noblastoma in Asia. Some countries including
ten cases per one million children [2]. Population- China and Japan employed survival rate or the
based studies have been conducted in Asia. total incidence number for statistical analysis of
Singapore has reported an incidence of 1 in retinoblastoma, and these data are therefore not
15,789 live births [3]. In Taiwan it was 1 in 21,691 included.
Table 24.1 Age-standardized incidence rates (ASIR) of retinoblastoma in Asia

Region Race/ethnicity Case no ASIR/million Study period References
United Statesa White 346 2.8 2000–2010 [104]
Hispanic 274 3.6
Black 119 3.5
APIb 75 3.1
AI/ANb 10 2.1
Total 824 –
Singapore Asian 46 2.4 (<9 years) 1968–1995 [3]
11.1 (<5 years)
India Asian 82 7.6 (boys) 1990–2001 [14]
7.9 (girls)
Korea Asian 411 4.6 1999–2011 [105]
Taiwan Asian 380 8.58 (0–4) 1979–2003 [4]
0.28 (5–9)
0.05 (10–14)
Thailand Asian (Khon Kaen) 31 4.4 1990–2009 [106]
Asian (Chiang Mai) 20 4.0
Asian (Songkhla) 24 4.6
Jordan Asian 40 9.32 2006–2010 [107]
Saudi Arabia Asian 597 7.7 (<5 years) 1983–2012 [108]
3.5 (<15 years)
Vietnam Asian 13 9.1 (boys) 1995–1997 [109]
4.1 (girls)
The Asian population in this area is counted
a
API, Native Hawaiian/Asian-Pacific Islander; AI/AN, American Indian/Alaska native

b
24 Retinoblastoma Genes in Chinese Studies 299
24.2 Clinical Presentation those of Europe, the United States, and Japan,
and Complications deaths of children due to retinoblastoma could be
substantially reduced worldwide [18]. The prog-
Retinoblastoma is a cancer of very young chil- nosis of retinoblastoma also depends on the stage
dren. In our Hong Kong Chinese cohort, approxi- of presentation at diagnosis. According to our
mately 60% were diagnosed before 2 years and hospital record in Hong Kong from 2009 to 2014,
95% before 5 years [unpublished data]. About 34% of cases were at late stage at presentation.
68% of cases present with leukocoria, which is Early diagnosis is essential to improve prognosis,
the white reflex in the pupil. This manifestation is especially for children with a positive family his-
often firstly noticed by parents under dim illumi- tory. For these children, screening by fundus
nation or flash photography. It is the most com- examination should be performed under anesthe-
mon presentation of retinoblastoma in Hong sia at regular intervals. For surviving children that
Kong [6]. The second most frequent sign is stra- grow up and become parents, their infants should
bismus, which accounts for 10–20% of cases and have a dilated eye examination in the first month
usually correlates with macular involvement. of life. Genetic testing of RB1 should be con-
Some children may also present with pain, glau- ducted. Infants with a positive genetic test should
coma, poor vision, orbital cellulitis, anterior uve- be examined on a monthly basis. For infants who
itis, and buphthalmos. As the tumor progresses, do not develop retinoblastoma but with a positive
some patients may develop orbital or metastatic genetic test, monthly exams should be conducted
systemic extra-central nervous system (CNS) continually throughout the first year. Siblings of
disease. Patients with bilateral disease usually patients should also be examined continually until
present at a younger age, in the first 12 months age 3–5 years, and it is important to check the
after birth. The median age of presentation is RB1 mutation status [18].
18 months in our Hong Kong cohort.
Retinoblastoma growth is underneath the ret-
ina layer and toward the vitreous. With its pro- 24.3 Introduction of the RB1 Gene
gression, the choroidal or scleral layer and optic
nerve may be entangled. Progression through the Before the retinoblastoma-causative gene RB1
ocular coats results in invasion of the tumor into was identified, a two-hit hypothesis was proposed
orbit, choroid, anterior chamber, and even to sys- to describe the genetic basis of retinoblastoma by
temic circulation. Tumor progression through the Knudson in 1971 based on the observation of 48
optic nerve leads to systemic and central nervous retinoblastoma cases [19]. Two mutation events
system metastasis. Saving patients’ life is the pri- are needed to develop retinoblastoma. The
ority of managing retinoblastoma. Compared genomic origin of the first mutation event also
with the developed world where most cases are affects the heredity [19]. For inherited retinoblas-
detected early, retinoblastoma is often detected toma, a germline mutation is required to predis-
late resulting in an enlarged eye with locally inva- pose risk in the offspring. In combination with a
sive disease in developing countries. In Hong second spontaneous mutation, the complete inac-
Kong, the survival rate is 95.1% during 1980– tivation of the disease-causing gene would lead to
2009, with 5 deaths among 104 patients, whereas development of retinoblastoma in the offspring.
the survival rate in the United States approaches For the non-hereditary form, the two spontaneous
100%. However the survival rate is lower in many mutations can occur in somatic cells of the off-
other parts of the world: 80–89% in South spring. In the early 1980s, several studies found
America [10], 83% in Iran [11], 81% in China close association of retinoblastoma with abnor-
[12, 13], 48% in India [14], and 20–46% in Africa mities on chromosome 13, where the RB1 locus
[15, 16]. Consequently 3000–4000 annual deaths was identified [20–22]. The clear relationship
were estimated globally [17]. Notably, if survival between RB1 locus with retinoblastoma and other
rates of these relatively lower areas approaching malignancies indicated the existence of
tumor-suppressor genes [23, 24]. In 1986, a DNA promote pRB intramolecular interactions and
segment containing RB1 locus at 13q14 was iso- conformation change [35]. The protein structure
lated, and deletions in the region were found in (Fig. 24.1a) underlies the function and the post-
retinoblastoma [25]. In 1987, two studies reported translational modification of pRB. Deciphering
the transcripts of RB1 gene with abnormal mRNA the protein structural information has greatly
expression in some cases of retinoblastoma [26, facilitated our understanding of the multifunc-
27]. The RB amino acid sequence was subse- tionality of this tumor-suppressor protein [36].
quently predicted [26] and deletion hotspots
identified [27]. These studies provided molecular
evidence for Knudson’s two-hit hypothesis and 24.5 RB1 Inactivation
suggested RB1 gene as a causative gene for reti-
noblastoma. In an experimental model, an exog- Germline RB1 mutations often give rise to bilat-
enous normal RB1 gene was introduced into eral retinoblastoma, while individual retinal cells
RB-inactivated tumor cells by retrovirus to res- acquiring somatic changes on both alleles result
cue the tumorigenesis phenotype in cell lines and in unilateral tumor. There are several possibilities
in nude mice [28]. Thus, RB1 has been validated causing inactivation of the RB1 alleles. In most
as a bona fide tumor-suppressor gene. circumstances, single nucleotide mutations or
small deletions account for the inactivation of the
first RB1 allele [37]. When the first mutation hap-
24.4 RB1: The Key Player pens in the constitutional germline cell, all cells
in the body are inherited with one mutated RB1
The RB1 gene (NM_000321) spans around allele. Nature of the second mutation can be vari-
200 kb. The protein-coding transcript (RB1–201) able, including another mutation, which can be
contains 27 exons with some large introns [29]. de novo, epigenetic modifications, or loss of het-
The exon 13–17 region is frequently mutated in erozygosity (LOH). In diploid organisms, there
multiple tumor types and is denoted as the poten- are several ways to gain LOH. For short genomic
tial “hotspot” [27, 29]. Human RB protein (pRB) fragments less than 2 kb, recombination via gene
consists of 928 amino acids and is a member of conversion or double crossovers between chro-
“pocket protein” family [30]. pRB can be divided mosomes is the main source of LOH [38]. Long-
into three major domains: central pocket domain range LOH is often related to repairing DNA
(aa 379–791), pRB C-terminus domain (RbC; aa breaks which are deleterious [38]. To repair DNA
792–928), and pRB N-terminal domain (RbN; aa breaks, the required DNA strand integrates into
1–378). The central pocket domain is essential to the homologous chromosome and initiates the
its function [31] and is composed of A and B complementary DNA sequence to repair the bro-
cyclin-like subdomains [31]. This central pocket ken region, the phenomenon of break-induced
is the target to be inactivated by viral oncopro- replication. The integrated strand could replicate
teins, such as human papilloma virus E7 [31] and DNA to the end of the chromosome, causing a
SV40 large T antigen [32]. The pRB C-terminus long-track LOH between the DNA breakage site
domain contains five cyclin-dependent kinase and the end of the chromosome. DNA double-
(CDKs) phosphorylation sites [33], which are strand break (DSB) repair between homologs at
important for the recruitment of and modification the RB1 locus could lead to more than 100-fold
by cyclins and CDKs. Such activity is involved in induction of LOH upon DSB induction [39].
regulation of cell cycle progression. Two cyclin- Our study in a Hong Kong Chinese cohort of
like folds form a globular entity at the pRB 42 sporadic retinoblastoma patients identified
N-terminal domain [34]. This domain interacts 15 RB1 mutations in 16 patients, 8 of them
with the central pocket domain to regulate the germline mutations. There were nine stop
conformation of pRB [34]. Phosphorylation at codons and four splice site variations, the for-
the sites T373, S608/S612, and T821/T826 could mer leading to truncations of pRB affecting its
Fig. 24.1 (a) Retinoblastoma protein (pRB) structure. causing pRB conformation change and dissociation of the
(b) In G1 phase, highly expressed cyclin D activates binding transcription factor E2F. Released E2F induces
CDK4 and CDK6. Activated CDKs phosphorylate pRB, cell cycle-related gene expression and cell enters S phase
central large pocket domain [40]. No methyla- mutations and late-onset of the disease were
tion across the CpG dinucleotides of the RB1 mostly found in bilateral retinoblastoma patients,
promoter among all 42 patients was found. while children with nonsense, frameshift, or mis-
Among 15 patients who had both normal and sense mutations had relatively early onset. While
retinoblastoma tissues available for analysis of ethnic segregation of mutations was observed
3 microsatellite markers (D13S153, D13S156, [47], similar relations were found in a study on
and D13S128), 9 (60%) of them showed LOH at 85 Chinese patients, with null mutations giving a
13q14, and they did not express nuclear RB. The higher percentage of bilateral (96.8%) and early
results thus showed inactivation of the RB1 gene diagnosed (10.7 months) retinoblastoma than in-
is mainly caused by loss-of-function mutations frame mutations (66.7% and 13.5 months,
and LOH, but not epigenetic effect of promoter respectively) [48]. Among 1173 Dutch retino-
methylation [40]. In a Chinese retinoblastoma blastoma patients, RB1 mutations occurred in
cohort in Beijing, in the north of China, 10 of 92% bilateral or familial patients, and about 10%
the 47 patients had RB1 exon mutations, while 4 unilateral sporadic patients [49]. About 80% of
had CpG island hypermethylation in the RB1 RB1 mutations are nonsense or frameshift muta-
promoter. LOH was also detected in 60% of 30 tions causing truncated pRB [50, 51]. A small
cases, at loci D13S153, D13S262, and D13S284 proportion of retinoblastoma patients, 2–5%, do
[41]. In another cohort in Chongqing in Western not carry a RB1 mutation, and some possess
China, screening of LOH with 14 microsatellite amplified copy numbers of other oncogene [49,
markers in 16 sporadic retinoblastoma patients 52]. There are also RB1 mutation carriers that do
showed 12 (75%) had LOH at 13q14 [42]. not develop retinoblastoma, but offspring from
Similar studies had been conducted in other eth- such unaffected parents may inherit a potential
nic populations. In Russia, a retinoblastoma disease-causing mutation. There are a small pro-
molecular screening study involving 45 families portion of low-penetrance families. For clinical
identified 23 RB1 mutations, and 9 (20%) exhib- assessments and genetic counselling, detailed
ited abnormal methylation status in the RB1 information of the RB1 sequence and inheritance
promoter and 70% showed LOH [43]. In an patterns have to be available [51].
Indian cohort, 72.9% of 54 retinoblastoma
patients showed LOH of RB1 [44]. These stud-
ies show that LOH plays an important role in 24.7 T
he Classical Cell Cycle
tumorigenesis of retinoblastoma. Regulatory Pathway
A rare cause of RB1 inactivation is chro-
mothripsis, which is a phenomenon of tens to The lack of functional pRB can predispose
hundreds of rearrangements of genome sequence patients to retinoblastoma development. pRB reg-
happening at one time [45]. Focal chromothripsis ulates the cell cycle progression through control-
was found on chromosome 13 in three retinoblas- ling the transcription process [53] (Fig. 24.1b). In
toma cases [46]. More studies are needed to the four phases of a cell cycle, G1, S, G2, and M
investigate the impacts of chromothripsis in phase, the first three phases are interphase, during
retinoblastoma. which the cell is prepared for cell division mainly
by doubling the genomic materials and undergo-
ing physiological metabolisms. The M phase is
24.6 Mutations in RB1 the mitosis phase, during which chromosomes
and cytoplasm divide into two daughter cells.
A database (RBGMdb) was constructed form Other proteins are also involved to regulate pro-
meta-analysis of 932 RB1 mutations, showing gression of the cell cycle, including cyclins and
40% of mutations located in 16 putative mutation the cyclin-dependent kinases (CDKs). Cyclins are
hot spots from exon 12 to exon 23, while others expressed at specific stages of the cell cycle [54].
scattered throughout the gene [47]. Splicing For example, in G1 phase, cyclin D is expressed
in a higher level and activates CDK4 and CDK6, ligated directly to each other [67]. As NHEJ path-
which are able to phosphorylate pRB. Highly way does not require the terminal sequence
phosphorylated pRB was found in rapid prolifer- homology, inaccurate repair and even DNA
ating cells, while dephosphorylation of pRB sequence loss could happen at the breakage sites.
induced cell growth arrest [55, 56]. The phosphor- Thus, NHEJ is considered to be “error-prone.”
ylated pRB formed a complex with the transcrip- On the contrary, HR is considered to be “error-
tion factor E2F [57, 58], while the uncomplexed free” as the homologous sequences are highly
form of E2F1 could drive the quiescent cells to similar to the original broken DNA. Since mam-
enter S phase [59]. Based on these observations, a malian cells prefer to use NHEJ for DSB repair
model has been proposed in that when there is a [68], the chance of chromosomal instability
loss of pRB activity, E2F is able to activate the raised tremendously in RB1-deficient cells.
transcription of other genes necessary for DNA Therefore, RB1 mutations can be considered as
synthesis and cell cycle progression [60]. the primary driving force of tumorigenesis. The
RB1-mutated cells could then accumulate addi-
tional mutations that could lead to other features
24.8 R
B1 and Chromosomal of cancer development.
Instability
Inactivated RB1 initiates the onset of retinoblas- 24.9 Recurrent Chromosomal

toma by dysregulating cell cycle progression. Aberrations
Cancer has several hallmark features: replicative in Retinoblastoma
immortality, invasion and metastasis, angiogenesis,
cell death avoidance, and sustainable proliferation In the human genome, there are regions known as
[61, 62]. Therefore, there is a need to understand fragile sites that are difficult to be replicated dur-
the relationships of pRB inactivation and these ing S phase. These fragile sites are prone to form
phenomena. Other than regulation of the cell cycle, gaps and breakages, which could be observed
pRB is also involved in maintaining genomic sta- during mitosis, particularly when under replica-
bility. For sporadic cancers, one cause of genomic tion stresses [69]. In one study constitutional
instability is DNA replication stress [63]. As men- chromosomal instability investigation at fragile
tioned above, in RB1-inactivated cells, the RB-free sites was conducted in 36 retinoblastoma patients,
E2F can promote the expression of cell cycle pro- 2 of them hereditary form and the rest sporadic.
gression genes and lead to premature entering into High prevalence of chromosome breakages at
S phase. In these cells, the existing nucleotide pool 3p14, 6p23, 13q14, 13q22, and 16q22-23 loci
may not be sufficient in availability for the required was identified. Tumor karyotype analysis found
high-level DNA replication [64], which could that 50% of this cohort showed structural aberra-
result in accumulating DNA replication stress. tions on chromosomes 13, 1, 6, and 16 [70]. Our
Nucleotide deficiency during S phase would lead to study on 15 microdissected retinoblastoma sam-
inadequate replication of the genome, leaving some ples showed 73% having chromosomal changes
unreplicated DNA regions as single-strand breaks at one or more loci on chromosomes 19, 20, 21,
(SSBs). These SSBs could be further converted 22, and X [71]. Single allele loss was more com-
into double-strand breaks (DSBs) by a nearby mon on chromosome 19 (33%) and 20 (27%).
DNA replication fork [65]. The most frequent recurrent allelic loss happened
There are essentially two cellular strategies to on 19q13 between D19S902 and D19S571, sug-
repair these DSBs: homologous recombination gesting this locus may be associated with tumori-
(HR) and nonhomologous end joining (NHEJ). genesis [71]. Comparative genomic hybridization
In the HR pathway, the ends of DSBs integrated (CGH) studies on retinoblastoma karyotype have
into the homologous sequence to repair the DSBs been performed and recurrent changes docu-
[66]. In the NHEJ pathway, the ends of DSBs are mented [72, 73].
24.9.1 Chromosome 1q oncogenes in this area were DEK, a DNA-binding

protein, and E2F3, a pRB-regulated target tran-
In 179 retinoblastoma samples, 95 (53%) were scription factor [81].
found to have gained an additional copy of chro-
mosome arm 1q (+1q) [72]. Although occurrence
of +1q is common in other cancers, its presenta- 24.9.4 Chromosome 13q
tion in retinoblastoma is different. Specifically,
the +1q was not highly expressed, and the addi- Gain and loss of chromosome 13q have been
tional regions were often distributed along the found in CGH studies. The minimal common
whole chromosome arm. LRRN5 (1q32.1) was region of gain (MRG) was enriched in 13q32-34
found overexpressed in half of tumor samples by region [82]. However, no candidate gene other
Southern blotting [74]. The protein LRRN5 than RB1 has been identified in this region.
belongs to the leucine-rich repeat superfamily
and possesses cell adhesion or signal transduc-
tion functions. KIF14 (1q32.1) got a 100- to 24.9.5 Chromosome 16q
1000-fold overexpression in 90% retinoblastoma
retinas than normal fetal retinas [75]. KIF14 is a By the combination of LOHand QM-PCR, and
kinesin family member and works as a microtu- comparison between tumors and normal retinas,
bule motor protein. Other genes in this region the CDH11 gene was found mutated in more than
were expressed differentially in various studies 40% of retinoblastoma samples in two separate
including CENPF, ENSA, HTCD37, SYT11, studies [83, 84]. This gene encodes an integral
DAR, SNX27, JTB, LASS2, SYT11, MDM4, and membrane protein, which functions in cell-cell
ZNF281 [76]. adhesion. It may be involved in the infiltration of
retinoblastoma cells into the optic nerve [85].
Another gene RBL2 (16q12.2) has been found
24.9.2 Chromosome 2p lost in some retinoblastoma patients [86]. When
the retinoblastoma cell line WERI was treated
The transcription factor MYCN (2p24.1), in chro- with 5-Aza-dC, a DNA methylation inhibitor, the
mosome 2p, has been reported as a strong prog- expression of RBL2 was restored [86]. This result
nostic indicator of neuroblastoma, and its indicated that RBL2 expression was epigeneti-
expression has been found elevated in various cally suppressed in retinoblastoma.
cancers [77]. About 10–200 copies of MYCN
amplification have been found in retinoblastoma
samples and the Y79 retinoblastoma cell line. 24.10 Epigenetics
MYCN expression was highly elevated in all ten in Retinoblastoma
retinoblastoma samples examined in a study [78].
The RB1 gene is subjected to numerous epigene-
tic modifications. Stable genome has been
24.9.3 Chromosome 6p observed in some patients, while epigenetic dys-
regulations have also been reported. Epigenetic
Isochromosome 6p [i(6p)], with chromosome modification clusters are summarized in
arm 6q lost and replaced by duplicating the 6p Fig. 24.2.
arm, is unique in retinoblastoma. Unbalanced
structural abnormality and i(6p) were found in
60% retinoblastomas [79]. Despite i(6p), 6p22 24.10.1 Methylation
gain was seen in more than 50% retinoblastoma
by CGH studies [80]. By using quantitative mul- An early study on methylation status of RB1 pro-
tiplex PCR, the most commonly gained bona fide moter in a cohort of 21 patients found association
24 Retinoblastoma Genes in Chinese Studies
Fig. 24.2 Summary of RB1 information on Ensembl transcription factor binding sites H3K27Ac mark [112], and dbSNP 150 [115] using the University of California at
[110–112], ENCODE methylation data [113], CpG island tracks [114], ENCODE Santa Cruz (UCSC) database (http://genome.ucsc.edu/) [116]
305
of hypermethylation of the CpG island with resulting in translational repression [93]. It plays
tumor development [87]. Later studies confirmed regulatory roles in the initiation and progression
the correlation between RB1 hypermethylation of human cancers [94]. Dysregulation by miRNA
and decreased RB1 expression level [88]. Besides in retinoblastoma has been screened in microar-
RB1, promoter methylation of other genes also ray analyses [95–97]. The let-7 family members
affects retinoblastoma development. Our previ- were involved in repressing oncogenes such as
ous work found MGMT (10q26) promoter hyper- the RAS family members and MYC [95]. Another
methylation would lead to MGMT inactivation miRNA cluster miR17–92 regulates cell prolif-
and to impairment of the functions of this enzyme eration, differentiation, and angiogenesis. It was
in retinoblastoma [89, 90]. In 8 of 23 retinoblas- overexpressed in retinoblastoma tissues [96].
toma samples, MGMT promoter hypermethyl- Furthermore, it was under direct regulation of
ation was detected [89]. Similar observation was E2F transcription factor. Therefore, in pRB-
obtained in another cohort of 10 out of 68 sam- deficient cells, the activated E2F could further
ples [90]. O-6-methylguanine-DNA methyltrans- induce the expression of the miR17–92 cluster.
ferase (MGMT) is an important enzyme in Alternatively, the miR17–92 cluster could regu-
preventing DNA mismatch mutation. Under nor- late the expression of E2F. Thus there is a regula-
mal conditions, deoxycytidine (dC) is incorpo- tory feedback loop between the miR17–92 cluster
rated into the newly synthesized DNA strand and E2F [98, 99].
opposite to deoxyguanosine (dG) during DNA
replication. When there is alkylation on an oxy-
gen atom (O6) of dG, incorrect incorporation of 24.11 Genetic Counselling,
thymidine (dT) would happen opposite to the Awareness Campaign,
O-6-methylguanine, which gives rise to DNA and Development of Novel
mismatch mutation and further compromises the Treatment
genome integrity in retinoblastoma. MGMT con-
verts O-6-methylguanine into dG to prevent this Retinoblastoma is a malignancy of early life, so
mismatch mutation. Another DNA mismatch early diagnosis and treatment are extremely
repair protein MLH1 (3q21.3) also showed pro- important. Genetic counselling is an integral part
moter hypermethylation, which was associated of the management of retinoblastoma patients
with null MLH1 protein expression in retinoblas- and their families. It is needed to assist parents in
toma [91]. MLH1 coordinates the detection of predicting the genetic features of retinoblastoma,
the distorted DNA structure caused by the mis- which could be used to estimate the risk of hav-
match and removal of the mismatch DNA ing retinoblastoma in other family members. In
sequence [92]. Another example of promoter Hong Kong, all children are screened for retino-
hypermethylated gene is RASSF1A (3q21.3). blastoma at birth and at 5 years old, and the mean
RASSF1A is a tumor-suppressor gene, which age of retinoblastoma presentation is 10 months
belongs to the RAS-association domain family. after birth for bilateral cases and 26 months for
Its promoter was found hypermethylated in more unilateral cases. The advocate of early recogni-
than 50% of retinoblastoma samples, in 10 of 17 tion of leukocoria with a smartphone flash pho-
samples in a US cohort [92] and 56 of 68 samples tography by parents could effectively enhance
in our Hong Kong Chinese cohort [86]. the early identification of retinoblastoma.
Recently we have launched a Hong Kong
Retinoblastoma Awareness Campaign to promote
24.10.2 MicroRNA (miRNA) early parental recognition of signs of retinoblas-
toma by linking with the citywide vaccination
MicroRNAs are small noncoding RNAs of 20–22 program for all newborn babies.
nucleotides that work as gene regulatory mole- Current treatments of retinoblastoma mainly
cules by targeting mRNAs for degradation, involve the application of chemotherapy, focal
treatment (laser or cryotherapy), or plaque radio- retinoblastoma is pan-ethnic, and research con-
therapy [100, 101]. For advanced retinoblastoma, ducted in different parts of the world has attrib-
enucleation is unavoidable. It causes total loss of uted to advancements of detection, treatment,
vision and imposes on the affected children psy- and counselling of the disease, and more con-
chological and mental trauma. The management certed international efforts are in view.
of retinoblastoma aims to save the patients’ life,
reducing the risk of long-term secondary tumors, Compliance with Ethical Requirements Bi Ning
preserve globe, and maximize vision. Recent Zhang, Yuning Jiang, Wai Kit Chu, Winnie W.Y. Lau,
Simon T.C. Ko, Kwong Wai Choy, Calvin C.P. Pang, Guy
study found retinoblastoma affected mainly the L.J. Chen, and Jason C.S. Yam declare that they have no
cone precursor cells in the retina [102]. In the conflict of interest.
cone precursor cells, high levels of MDM2 were No human or animal studies were performed by the
detected. MDM2 suppresses p53-mediated apop- authors for this article.
tosis. As RB1 inactivation accelerates cell cycle,
triggering apoptosis could stop the formation of
cancer from these RB1-deficient cells. However, References
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Genetics of Retinoblastoma: Basic
Research and Clinical Applications 25
Usha Kim, K. Thirumalairaj, Aloysius Abraham,
Shanthi Radhakrishnan, B. Devarajan,
V. R. Muthukkaruppan, and A. Vanniarajan
Abstract faster turnaround time. The newly adopted

Retinoblastoma is the first genetic cancer of sequential genetic analysis method had con-
the eye, where the inheritance of the disease is siderably reduced the cost and provided an
directly demonstrated. This prototype cancer affordable method of genetic screening.
has served as a model for studying multiple
other cancers. Retinoblastoma affects the Keywords
vision, eye, and even life of young children up Retinoblastoma · Genetic testing · Genetic
to the age of 5 years. Developing countries counseling · Clinical applications · Gene
like India harbor the large number of retino- panel · Pediatric cancer · Translational
blastoma patients, and most of the patients research · Genetics · Mutations · RB1
present at advanced stage of the disease that
makes the treatment difficult. It is hence pro-
posed that early detection is necessary for the
appropriate treatment and management of the 25.1 Introduction
disease. Basic research had largely helped the
clinic to identify the patients with genetic pre- Retinoblastoma (RB) (OMIM: 180200), an
disposition to develop retinoblastoma. embryonal tumor of the developing retina, can
Effective use of genetic methods including affect early childhood from developing fetuses to
next-generation sequencing had largely 5-year-old children. It is the most common tumor
improved the patient care through high- of the eye contributing about 90% of ocular
throughput, large gene panel analysis and tumors and 4% of all tumors in young children
U. Kim B. Devarajan
Department of Orbit, Oculoplasty and Ocular Department of Bioinformatics, Aravind Medical
Oncology, Aravind Eye Hospital, Madurai, India Research Foundation, Madurai, India
K. Thirumalairaj · A. Abraham · A. Vanniarajan (*) V. R. Muthukkaruppan
Department of Molecular Genetics, Aravind Medical Advisor-Research, Aravind Medical Research
Research Foundation, Madurai, India Foundation, Madurai, India
R. Shanthi
Department of Pathology, Aravind Eye Hospital,
Madurai, India

https://doi.org/10.1007/978-981-13-0884-0_25
314 U. Kim et al.
(NCRP-ICMR 1999–2000). There is an esti- 25.2 Knudson’s Hypothesis

mated incidence of 5000 new cases per year and Clinical Presentation
worldwide. The incidence of RB is even much of Retinoblastoma
higher with 1500–1800 new cases in developing
countries like India compared to 280 new cases Retinoblastoma is the first genetic cancer of the
in the USA every year [1, 12]. This is primarily eye, where the inheritance of the disease is
attributed to the increased population size and directly demonstrated and further research has
delayed presentation of the disease in the devel- helped to understand RB in detail (Table 25.1).
oping countries [1]. RB may occur either as unilateral or bilateral
The delayed presentation is largely due to the
socioeconomic factors such as lack of access to
healthcare facilities, ignorance about early signs, Table 25.1 From bed to bench and back to bed: a
research path to RB1 genetic testing
illiteracy, poverty, lack of trained personnel, and
1886: Retinoblastoma inheritance (Albert 1987)
inadequate infrastructure [2]. The globe salvage
First evidence of susceptibility to cancer inherited
becomes difficult at the advanced stages, but the from a parent to a child by Brazilian ophthalmologist
cure rate is higher with the surgical removal of Hilário de Gouvêa. Two out of seven children born to
the globe filled with tumor. If the tumor is a father who was successfully treated for childhood
retinoblastoma also developed the disease.
detected early even before the gross appearance,
1971: Two-hit hypothesis (Knudson 1971)
it is possible to treat it completely, and vision can
Alfred Knudson showed that two mutations are
also be preserved. Genetic methods have proven required for the occurrence of retinoblastoma by the
to be useful in identifying the predisposed RB at analysis of 48 cases of retinoblastoma with laterality
least in patients having a positive family history. and the presence of a family history. In familial cases,
one hit was inherited and the other one was acquired
The various genetic studies in Indian RB patients
later; in sporadic tumors, both changes were somatic
and prospects of implementing them in the clinic and are acquired during development.
are well documented in a recent review [1]. 1973: Tumor suppressor gene (Comings 1973)
The outcome of the research has been wit- David Comings articulated a general framework for a
nessed in the recent years through the incorpora- role of tumor suppressor genes. He explained that
germline mutation in regulatory genes that suppressed
tion of the heritability factor for RB, and it is the
tumorigenesis followed by the somatic loss of the
first cancer where it has been widely accepted in homologous allele caused the inherited tumors
clinical practice [3]. Earlier, the treatment of RB including retinoblastoma.
was only dependent on the clinical diagnosis. 1983: Localization of retinoblastoma gene (Cavenee
However, with the explosion of the molecular et al. 1983)
Webster Cavanee and colleagues localized the
methods, the ophthalmologist is now armed with
retinoblastoma gene (RB1) to chromosome 13; Loss of
better ways of diagnosis and management. In heterozygosity was also demonstrated by the analysis
many academic centers, the research labs are of the inherited and sporadic cancers.
located in close vicinity to the clinics for 1986–1987: Isolation and cloning of RB1 (Friend et al.
improved communication between the clinician 1986; Lee et al. 1987; Fung et al. 1987)
and geneticist. This also facilitates the patient to Stephen Friend and colleagues isolated a human
cDNA that mapped to the RB region in 1986. The next
undergo the necessary tests for clinical and year, RB1 was cloned by chromosome walking by two
molecular diagnosis in a single visit. The clinical independent groups — Wen-Hwa Lee and Yuen-Kai
and genetic data are shared between the depart- Fung.
ments in order to provide the appropriate coun- 1989: Genetic testing of RB1 (Yandell et al. 1989)
seling to the patient and the family. This chapter David Yandell and his peers had used DNA
sequencing to identify point mutations in RB1 gene
will focus on the current updates in basic research and reported that somatic mutations caused non-
in genetics and its usefulness in the diagnosis and hereditary retinoblastoma, and germline mutations
management of RB. caused hereditary retinoblastoma.
25 Genetics of Retinoblastoma: Basic Research and Clinical Applications 315
depending upon the inheritance pattern. Bilateral 25.3 Cost-Effective RB1 Genetic
RB is usually heritable caused either due to Testing
inherited or de novo germline mutations, and
unilateral RB is usually nonheritable caused by RB1 is a large gene having 27 exons spread across
somatic mutations. This difference in the inheri- 180 kb intervened by introns as long as 60kb. The
tance pattern was explained by Knudson promoter, exon 1 and several other exons have
46 years ago even before the RB1 gene was high GC content and repeat regions. Earlier,
described [4]. According to Knudson’s hypoth- karyotyping and Southern blotting was used to
esis, mutation in both alleles of RB1 gene was detect whole RB1 gene deletions [6, 7]. Point
necessary for initiation of RB. The two-hit mutations were detected by single-strand confor-
hypothesis of Knudson was further demon- mation polymorphism and denaturing high per-
strated in other pediatric tumors such as Wilms’ formance liquid chromatography [8, 9], but with
tumor, neuroblastoma, and adult tumors includ- a low resolution. Later, multi-technique
ing hemangioblastoma and renal cell carcinoma approaches were employed utilizing allele spe-
[5]. cific PCR, quantitative multiplex polymerase
In case of bilateral RB, one mutation is chain reaction (QM PCR) and Sanger sequencing
already inherited, and hence the time taken for to capture the complete spectrum of mutations
the occurrence of second mutation is less, and [10, 11]. These multi-technique approaches
hence the patients with bilateral RB present remain time consuming, labour-intensive, and
early. In case of unilateral RB, both mutations expensive.
occur in the retinal cells during the development, In order to overcome all these bottlenecks, a
and RB may present late during the 2–3 years or new method with four simple steps based on the
after. Apart from those individuals who get RB, order of mutations as observed in the database
there were unaffected carriers who can transmit and literature was reported [12]. Analysis of the
the mutant gene (without presenting with the database revealed that mutations are predomi-
disease). In these carriers, the second random nantly present in 8 exons having fragile codons
mutation did not occur, and hence they did not leading to truncation. Based on the frequency of
get the disease [4]. the pathogenic mutations (Fig. 25.1), a new strat-
Knudson’s hypothesis, which was based on egy was suggested for RB1 genetic screening.
the statistical analysis of the laterality, focality, The first step involves Sanger sequencing of the
and family history of RB patients, was revisited selected exons, multiplex ligation dependent
recently in the Indian context with the genetic probe amplification (MLPA) in the step 2 fol-
data. lowed by Sanger sequencing of other set of exons
In the study of 73 RB patients, 31 were bilat- in step 3 and 4. The outcome of this study clearly
eral, and 42 were unilateral. Multiple tumors matched with the published data in the order of
were seen more common in bilateral group than nonsense mutations, deletions/duplications,
unilateral group. The mean age at diagnosis for splice variants and novel mutations [12].
bilateral and unilateral cases was 9.82 ± 11.52 A similar sequential mutation detection strat-
and 24.02 ± 15.11, respectively, and the mean egy was also reported in Tunisian RB patients in
difference in two groups was found to be statisti- order to lower analytical efforts and reduce the
cally significant. In bilateral group, 21 (68%) cost of genetic testing. Out of 20 unrelated
cases presented at an age less than 1 year, while patients with familial and/or de novo bilateral
in unilateral group, most of the patients (18.43%) RB, 19 patients had oncogenic mutations.
presented during the third year. Thus bilateral Mutational mosaicism was found in one unilater-
tumors presented earlier as compared to unilat- ally affected father of a bilateral proband and
eral tumors. This study has reemphasized incomplete penetrance was observed in two
Knudson’s hypothesis and correlated it with the mothers. RNA analysis in a family with the same
age of the presentation [38]. mutation showed an in-frame loss of exon 9. This
316 U. Kim et al.
Filled boxes represent the exons Each open box corresponds to 5-20 mutations
Fig. 25.1 Frequency of pathogenic mutation across the RB1 gene
study emphasized the need for genetic testing to also included in a single panel, and whole assay
reveal or exclude incomplete penetrance specifi- is performed, and reports are generated in 3 days
cally in parents of patients with sporadic disease [14]. In another study of 50 unrelated patients
[13]. with RB using the TruSight Cancer panel and run
on a MiSeq platform, germline pathogenic muta-
tions that included missense, nonsense, splice
25.4 Current Prospects of Next- site, indel, and structural variants were identified
Generation Sequencing in 66% (33/50) of the cases [15].
Another important application of NGS is the
Since the spectrum of mutations identified in detection of low-level mosaicism. Sporadic RB is
RB1 gene varies widely, multiple methods are sometimes caused by de novo mutations at a
required for detecting different types of muta- threshold of 15–20%, which cannot be detected
tions. Sanger sequencing has been the choice for by Sanger sequencing. Through an ion-torrent-
detection of point mutations and indels. Copy based deep sequencing, the frequency of point
number changes including loss or gain of few mutations in lymphocyte DNA was found to be
exons to whole RB1 gene can be detected by increased from 96% to 97% for bilateral RB and
methods such as QMPCR, multiplex ligation- from 13% to 18% for unilateral RB in a referral
dependent probe amplification (MLPA), quanti- lab in the USA [16]. NGS could detect as little as
tative real-time PCR, and array comparative 1% mutant through an artificial mosaicism cre-
genomic hybridization (array CGH). With the ated by serial dilution of mutants [17]. When a
technical advances, next-generation sequencing series of 30 patients with sporadic RB with no
(NGS) provides the opportunity to find point RB1 mutations were retested, 3 had low-level
mutations, indels, and copy number changes as mosaic variants, varying in frequency between 8
well in a single run and make a direct impact on and 24% [18].
clinical management of RB. Targeted NGS had been shown to be a power-
NGS-based approaches provide a better way ful tool in the molecular diagnosis of RB through
of genetic testing through enhanced sensitivity the analysis of the RB patients from India. The
and faster turnaround and are cost-effective com- algorithms used could detect both SNVs and
pared to the conventional methods. In a recent CNVs [19]. Although NGS provides an advantage
study of RB patients, in addition to the detection over the Sanger sequencing, still the algorithms
of other variants, amplification of MYCN was used for detecting deletions and duplications are
evolving. Hence a combination of targeted NGS GATA5, TP53, VHL, and GSTP1 along with the
with array comparative genomic hybridization recurrently methylated MGMT, RB1, and
was suggested through an analysis of 65 RB CDKN2 were found to be hypermethylated in
patients and found a mutation detection rate of RB tumors [26].
96.5% in bilateral and 22% in unilateral [17]. MicroRNAs (miRNAs) are single-stranded
Moreover, only the patient samples were ana- noncoding RNA molecules that contribute to
lyzed in many studies, and interpretation was posttranscriptional downregulation of gene
made based on the variants identified in the patient expression. It is noted that microRNA-21 pos-
samples. However, in some cases, the variants sesses the oncogenic potential to target several
identified might be benign without being cause of tumor suppressor genes, including PDCD4, and
the disease. It is important to include the family thereby downregulates RB1 expression and regu-
members along with patients in order to confirm lates tumor progression and metastasis [27].
the variants as pathogenic through co-segregation Inactivation of miR-17-92 suppresses RB forma-
analysis and classify them as inherited or de novo tion in mice, and co-silencing of miR-17/20a and
[19]. The enhanced ability of mutation detection p53 cooperatively decreases the viability of
by NGS has significant implications for improved human RB cells [28]. In a miRNome landscape
clinical diagnosis, genetic counseling, surveil- analysis, core cluster of 30 miRNAs were found
lance, and management of RB. to be highly expressed in RB tumors [29]. Recent
studies focus on studying the levels of miRNAs
in body fluids which may serve as a biomarker
25.5 C
urrent Research Focused for RB.
toward Clinical Applications
It is now understood that the biallelic inactivation 25.6 Implications of Genetic

of RB1 is important for the development of RB Testing
tumor. The loss of two RB1 alleles in turn will
trigger the other changes leading to genomic Genetic testing of RB1 gene has made a signifi-
instabilities leading to copy number gains in cant impact in the clinical management by (i)
genes, such as MDM4, KIF14, MYCN, DEK, and predicting the risk, (ii) assessing the carrier sta-
E2F, and loss of CDH11 [20]. It has been reported tus, (iii) confirming the diagnosis, and (iv) prena-
that a small proportion of RB can be caused by tal or preimplantation diagnosis. Based on the
the copy number gains of MYCN alone even mutation and its inheritance pattern, classifica-
without the inactivation of the RB1 gene [21]. tion of retinoblastoma into heritable and nonher-
Customized NGS panels are now under develop- itable is possible (Table 25.2).
ment to target the genes that are involved in the
tumor progression. Table 25.2 Clinical implications of heritable and non-
Differential gene regulation of various can- heritable retinoblastoma
cer pathways and DNA damage pathways had Nonheritable
been found in RB [22, 23]. It is proposed that Heritable retinoblastoma retinoblastoma
many of these genes are regulated by epigenetic Generally detected early Generally detected late
rather than genetic mechanisms as described in (0–2 years) (3–5 years)
syk. Further assays with inhibitors of these Mostly bilateral Mostly unilateral
Mostly multifocal Mostly unifocal
genes showed promising results for tumor
Either inherited or de Not inherited
reduction and could be the potential drugs for novo
RB patients. Further reduction of the tumor Increased risk of No risk of secondary
through inhibitors suggests the newer therapeu- secondary tumors tumors
tic drugs for RB patients [24, 25]. In another Higher risk of RB in Low risk of RB in sibling
study, genes such as MSH6, CD44, PAX5, sibling and offspring and offspring
318 U. Kim et al.
25.6.1 Predicting the Risk 25.6.3 Confirmation of Diagnosis
Genetic testing of RB1 will facilitate the risk pre- Genetic testing of RB1 may also serve as a tool
diction in the siblings and offspring of the for confirming the clinical diagnosis. The pre-
affected individuals [30]. (a) Close surveillance dominant sign of the RB such as leucocoria often
of the sibling/offspring is required if the inherited mimics other eye conditions including but not
germline mutation is detected as the risk is limited to persistent hyperplastic primary vitre-
estimated to be 50%. (c) If the mutation is found ous (PHPV), Coats’ disease, ocular toxocariasis,
to be de novo germline, the risk will reduce to 5% and retinopathy of prematurity. The eye will be
in the sibling possibly due to gonadal mosaicism painful in most of these cases similar to RB and
of one of the parents and 50% risk in the off- end up in enucleation. Genetic testing of RB1 in
spring. (d) If the mutation is found to be somatic, most of these tissues obtained from enucleated
the risk of RB reduces to less than 1% in sibling globes will help in confirming the diagnosis of
and offspring which is equal to the general popu- RB along with the histopathological analysis,
lation risk. thereby excluding the other etiologies.
If the mutation is identified in a family having This is important for further treatment proto-
one of the parents and a child affected with RB, cols as chemotherapeutic drugs were adminis-
then the next sibling is at a higher chance of tered even after enucleation if the extraocular
developing RB. In this case, early screening of spread was noticed by histological analysis.
the sibling soon after birth will help in surveil- Some patients present with unilateral RB without
lance of the disease in the child. Early diagnosis any family history but with a germline mutation
through the genetic testing will help in providing detected in constitutional cells. These patients
the appropriate treatment to save the eye and may turn to bilateral within few months, and
vision. If the heritable mutation in the family is hence a close surveillance will help in saving the
not identified in the child examined after birth, other eye and vision which otherwise may
further surveillance may not be necessary and become worsen.
could avoid unnecessary examinations under
anesthesia. The patients with RB1 mutation will
have an increased risk of developing pinealoblas- 25.6.4 Prenatal or Preimplantation
toma and lifelong risk of developing secondary Genetic Diagnosis
tumors, and hence periodical surveillance is
necessary. The survival of the RB patients has considered
increased with improved treatment procedures.
Many adult patients who wanted to plan their
25.6.2 Assessing the Carriers family were anxious to know whether they would
of the RB1 Mutations inherit the disease and like to know the possible
ways of avoiding the children with RB. In case of
Rarely, there may be carriers of the RB1 muta- patients having germline mutations, there would
tions without any disease phenotype, either due be an increased risk of inheriting RB to the child,
to the nonoccurrence of the second mutation in and hence a prenatal genetic testing would help
the retinal cells or spontaneous regression of the in identifying the risk of RB in the growing fetus.
tumor. However, they can potentially pass on the If mutation was detected in fetal DNA, the
mutation to the next generation. It is hence pregnancy could be terminated to avoid the child
important to screen the complete family when a with RB or continued further. If the pregnancy
mutation is identified in the patient for the accu- was continued, the screening of the child imme-
rate risk prediction for the next sibling. diately after birth is required to check for RB and
further treatment. If the mutation was not detected 3. Soliman SE, Racher H, Zhang C, MacDonald H,
Gallie BL. Genetics and molecular diagnostics in
in the fetal DNA, a chance of the inheritance retinoblastoma--an update. Asia Pac J Ophthalmol
would be reduced to less than <1, and a normal (Phila). 2017;6(2):197–207.
child was expected. Preimplantation genetic test- 4. Knudson AG. Mutation & Cancer: statistical study
ing might also help to avoid the embryos carrying of retinoblastoma. Proc Natl Acad Sci U S A.
1971;68:820–3.
mutations that can potentially cause RB. After 5. Maher ER, Yates JRW, MA F-S. Statistical anal-
testing the embryos, the transfer of embryos ysis of the two stage mutation model in von
without the mutation would produce an offspring Hippel-Lindau disease, and in sporadic cerebellar
free of RB. haemangioblastoma and renal cell carcinoma. J Med
Genet. 1989;27:311–4.
6. Harbour JW. Overview of RB gene mutations in
patients with retinoblastoma. Implications for clinical
25.7 Summary genetic screening. Ophthalmology. 1998;105:1442–7.
7. Harini R, Ata-ur-Rasheed M, Shanmugam MP, Amali
J, Das D, Kumaramanickavel G. Genetic profile of
It is now clear that basic research in retinoblas- 81 retinoblastoma patients from a referral hospital in
toma genetics has paved a way for the transla- southern India. Indian J Ophthalmol. 2001;49:37–42.
tional research and supported the clinical care 8. Sugano K, Yoshida T, Izumi H, Umezawa S, Ushiama
through early detection and intervention of reti- M, Ichikawa A, Hidaka A, Murakami Y, Kodama T,
Suzuki S, Kaneko A. Outpatient clinic for genetic
noblastoma. The enhanced sensitivity of muta- counseling and gene testing of retinoblastoma. Int
tion detection by NGS has significant implications J Clin Oncol. 2004;9(1):25–30.
for improved clinical diagnosis, genetic counsel- 9. Houdayer C, Gauthier-Villars M, Laugé A, Pagès-
ing, surveillance, and management of RB. The Berhouet S, Dehainault C, Caux-Moncoutier V,
Karczynski P, Tosi M, Doz F, Desjardins L, Couturier
cost of the analysis has been considerably J, Stoppa-Lyonnet D. Comprehensive screening
reduced by adapting newer strategies of genetic for constitutional RB1 mutations by DHPLC and
testing. In the developing countries like India, it QMPSF. Hum Mutat. 2004;23(2):193–202.
is important to utilize the genetic methods for 10. Parsam VL, Kannabiran C, Honavar S, Vemuganti
GK, Ali MJ. A comprehensive, sensitive and economi-
early detection of the disease which would reduce cal approach for the detection of mutations in the RB1
the economic burden and social stress due to the gene in retinoblastoma. J Genet. 2009;88:517–27.
loss of vision and the eye. 11. Price EA, Price K, Kolkiewicz K, Hack S, Reddy MA,
Hungerford JL, Kingston JE, Onadim Z. Spectrum
of RB1 mutations identified in 403 retinoblastoma
Acknowledgment This work was supported by Aravind
patients. J Med Genet. 2014;51:208–14.
Eye Foundation, USA; the Indian Council of Medical
12. Thirumalairaj K, Abraham A, Devarajan B, Gaikwad
Research, New Delhi; and Aravind Medical Research
N, Kim U, Muthukkaruppan V, Vanniarajan A. A step-
Foundation, Madurai.
wise strategy for rapid and cost-effective RB1 screen-
ing in Indian retinoblastoma patients. J Hum Genet.
Compliance with Ethical Requirements Conflict of 2015;60(9):547–52.
interest: The authors Usha Kim, K. Thirumalairaj, 13. Jeridi AH, Bouguila H, Ansperger-Rescher B, Baroudi
Aloysius Abraham, R. Shanthi, B. Devarajan, O, Mdimegh I, Omran I, Charradi K, Bouzayene H,
VR. Muthukkaruppan, and A. Vanniarajan declare that Benammar-Elgaaïed A, Lohmann DR. Genetic test-
they have no conflict of interest. ing in Tunisian families with heritable retinoblastoma
using a low cost approach permits accurate risk pre-
diction in relatives and reveals incomplete penetrance
in adults. Exp Eye Res. 2014;124:48–55.
14. Li WL, Buckley J, Sanchez-Lara PA, Maglinte DT,
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Genotype-Phenotype Correlation
in Retinal Degenerations 26
Sripriya Srivatsan, Mathavan Sinnakaruppan,
Vikas Khetan, Sundaram Natarajan,
Sangeetha Srinivasan, and Rajiv Raman

Vitreoretinal degenerations affect a significant
proportion of people world-wide, which, in Monogenic diseases of the retina and vitreous
the most severe stages, can result in legal affect approximately 1 in 2000 individuals or
blindness. Investigations on molecular, genetic more than 2 million people worldwide [1].
and environmental basis of these degenera- Clinical features can range from legal blind-
tions have contributed to additional knowl- ness in the most severe forms of retinal degen-
edge with respect to differential diagnosis erations (Leber congenital amaurosis, LCA) to
according to the genotype and gene therapy. less severe or rather mild retinal dysfunctions
This chapter summarizes the phenotype and (night blindness, achromatopsia). In the past
genotype of the vitreoretinal degenerations two decades, the knowledge about the molecu-
under four major groups: (1) rod-dominated lar basis of retinal diseases has tremendously
diseases, (2) cone-dominated diseases, (3) progressed, and evidence for the contribution
generalised retinal degenerations (affecting of genetic factors but also environmental cir-
both photoreceptor cell types, rods and cones) cumstances is continuously accumulating.
and finally (iv) exudative as well as erosive However, with the available techniques, a reli-
vitreoretinopathies. able molecular diagnosis is possible for only
half of the affected individuals or families with
Keywords monogenic forms of retinal diseases [2]. In
Genotype · Phenotype · Retina · Degeneration addition, the predictive value of a mutation or
risk allele for multifactorial disorders is prob-
lematic since the phenotypic and/or symptom-
atic consequences are highly variable.
Nevertheless, this knowledge has greatly
S. Srivatsan · M. Sinnakaruppan improved the understanding of molecular aeti-
SNONGC Department of Genetics and Molecular ology to some extent. The diseases discussed
biology, Vision Research Foundation, Chennai, India in this article are categorised in four major
V. Khetan · S. Srinivasan · R. Raman (*) groups: (1) rod-dominated diseases, (2) cone-
Shri Bhagwan Mahavir Vitreoretinal Services, dominated diseases, (3) generalised retinal
Sankara Nethralaya, Chennai, India degenerations (affecting both photoreceptor
cell types, rods and cones) and finally (iv) exu-
S. Natarajan dative as well as erosive vitreoretinopathies.
Aditya Jyot Eye Hospital, Mumbai, India

https://doi.org/10.1007/978-981-13-0884-0_26
324 S. Srivatsan et al.
Each disorder is further described as genotype autosomal recessive CSNB is associated with
and phenotype to understand the genotype- mutations in CABP4, CACNA2D4, GNAT1 and
phenotype correlation to some extent. SLC24A1 [3].
Oguchi Disease
26.2 Non-syndromic Retinal Phenotype Oguchi disease is an autosomal
and Vitreoretinal Diseases recessive stationary rod disease. It is differenti-
ated from CSNB by the characteristic fundus
26.2.1 Disease of Rod Photoreceptor change where golden yellow fundus reflex was
Cells (Stationary observed which returns to its normal appearance
and Progressive) after long dark adaptation.
26.2.1.1 Stationary Rod Diseases Genotype Genetically also it does not overlap
with CSNB where rod phototransduction genes
Congenital Stationary Night Blindness like arrestin gene (s-antigen, SAG) and rhodopsin
Phenotype As the name implies, congenital sta- kinase gene (G protein-coupled receptor kinase
tionary night blindness (CSNB) is a nonprogres- 1, GRK1) are shown to be causative in many
sive visual impairment present at birth. It is a rod patients.
degenerative disease leading to characteristic fea-
tures of night vision impairment and decreased Fundus Albipunctatus
visual acuity. CSNB is classified according to Phenotype Fundus albipunctatus is an autoso-
ERG findings, where in Schubert-Bornschein mal recessive congenital night blindness differ-
type, the b wave is smaller than the a wave and entiated from CSNB due to fundus involvement.
Riggs type is defined by proportionally reduced a Also it is differentially diagnosed from Oguchi
and b waves [3]. by phenotype difference, i.e. presence of white to
white-yellow dots in the perimacular or periph-
Genotype Genetically it shows autosomal dom- ery of the retina.
inant, recessive and X-linked recessive pattern of
inheritance. Genotype The genes so far associated with this
disease are RDH5, RLBP1 and RPE65 [4].
Riggs–Type CSNB Autosomal dominant genes
involved in Riggs-type CSNB are RHO, GNAT 26.2.1.2 Progressive Rod Diseases
and PDE6B, while only one gene SLC24A1 is
inherited recessively. Retinitis Pigmentosa (RP)
Phenotype RP is a heterogeneously inherited
Schubert-Bornschein–Type CSNB It is subdi- progressive retinal degeneration with charac-
vided as complete and incomplete according to teristic symptoms of night blindness, tunnel
ERG. The complete form has ON-bipolar cell vision and progressive decrease in central
dysfunction and hence characterised by reduced vision. Retinal manifestations include narrow-
rod b-wave response. The autosomal recessive ing of arterioles, pigmentation within the ret-
genes in complete CSNB are GRM6, GNB3, ina combined with loss of pigmentation and
TRPM1, GPR179 and LRIT3. NYX is inherited as retinal pigment epithelial cells resulting in a
X-linked recessive form. bone-spicule presentation. More advanced
stages present with cataract, pale optic disc and
Incomplete CSNB involves both ON- and severe narrowing of the arterioles. The condi-
OFF-bipolar cell dysfunction caused due to genes tion is generally bilateral but can present with
involved presynaptically and in glutamate no detectable fundus findings but with degen-
release. The X-linked incomplete CSNB is asso- eration of the outer retinal layers (also called
ciated with mutation in CACNA1F, while the RP sine pigmento) to sectoral or diffuse retinal
26 Genotype-Phenotype Correlation in Retinal Degenerations 325
involvement. To assess the disease status and Only three genes have been associated
progression, electroretinographic measure- with X-linked RP, OFD1, RP2 and RPGR. Of
ments provide a sensitive method. In contrast this, RPGR is more frequently associated,
to CSNB, which predominantly affects the b and it is also shown to be involved in syn-
wave, RP is characterised by reduced or absent dromic/atypical RP like in primary ciliary
a and b waves in the ERG. dyskinesias. The loci associated so far are
Xp21.3-p21.2 (RP6), Xq26-q27 (RP24) and
J uvenile RP or Early–Onset RP Xq28 (RP34). Y-linked inheritance is reported
Phenotype Juvenile RP is always confused with in one Chinese family [5].
Leber congenital amaurosis as it affects rod and Pericentral RP is defined as one which affects
cone photoreceptors simultaneously. While LCA only the near-central retina and spared the periph-
is defined as blindness earlier to 1 year of age, ery. Mutation studies in these patients have
EORP is considered in those kids with progres- shown mutations in RHO, USH2A, PDE6B and
sive blindness later than 1 year. HGSNAT genes.
Many syndromes have associated RP –
Genotype The autosomal recessive form has Alstrom syndrome include abetalipoproteinae-
mutation in SPATA7, LRAT and TULP1, and mia, Refsum syndrome, Bardet-Biedl syndrome,
autosomal dominant form has mutation in AIPL1 Usher syndrome, etc.
gene. RHO is among the most prominent
RP-associated genes. RHO constitutes a seven-
Late–Onset RP transmembrane receptor protein which initiates
Genotype The onset is from the second decade the phototransduction cascade upon absorption
onwards and is inherited as autosomal dominant/ of light by its chromophore 11-cis retinal. The
recessive, X-linked and also y-linked. Autosomal vast majority of mutations show a classical auto-
recessive inheritance is more frequently reported somal dominant inheritance leading to RP, the
with mutations in ABCA4, AGBL5, ARHGEF18, mechanism of which is fairly well understood as
ARL6, ARL2BP, BBS1, BBS2, BEST1, C2orf71, being either a gain-of-function or a dominant
C8orf37, CERKL, CLRN1, CNGA1, CNGB1, negative effect of the mutated protein.
CRB1, CYP4V2, DHDDS, DHX38, EMC1, EYS,
FAM161A, GPR125, HGSNAT, IDH3B, IFT140,
IFT172, IMPG2, KIAA1549, KIZ, LRAT, MAK, 26.2.2 Cone and Cone-Rod Diseases
MERTK, MVK, NEK2, NEUROD1, NR2E3, (Stationary and Progressive)
NRL, PDE6A, PDE6B, PDE6G, POMGNT1,
PRCD, PROM1, RBP3, REEP6, RGR, RHO, 26.2.2.1 Stationary Cone
RLBP1, RP1, RP1L1, RPE65, SAG, SAMD11, Dysfunction/Colour Vision
TRNT1, TTC8, TULP1, USH2A, ZNF408 and Defect
ZNF513.The recessive loci mapped so far are The stationary cone dysfunction group of dis-
16p12 (RP22), 4q32-q34 (RP29) and 1p21.1- eases are characterised by colour vision abnor-
p13.3 (RP32). malities, photophobia and electro- or
psychophysical evidence of abnormal function-
The autosomal dominant RP account to about ing of cone. They are clinically and genetically
30% of cases and are caused by ADIPOR1, ARL3, heterogeneous. The stationary cone dystrophies
BEST1, CA4, CRX, FSCN2, GUCA1B, HK1, are mostly congenital, and hence they usually
IMPDH1, KLHL7, NR2E3,NRL, PRPF3, PRPF4, display a normal rod response.
PRPF6, PRPF8, PRPF31, PRPH2, RDH12,
RHO, ROM1, RP1, RP9,RPE65, SEMA4A, Achromatopsia Achromatopsia are autosomal
SNRNP200, SPP2 and TOPORS. The dominant recessive cone diseases that are classified as i) a
locus associated is 6q23 (RP63). more severe form called complete achromatopsia
where all three types of cones are affected and ii) CORDs feel intense photophobia and variable
a less severe form called incomplete achromatop- degrees of colour vision abnormalities. Central
sia where patients have high visual acuity and scotomas can be also present upon visual field
mild cone response in ERG. There are five genes testing. The fundus examination frequently
associated with the disease – CNGA3, CNGB3, reveals pigment deposits and retinal atrophy in the
GNAT2, PDE6H, PDE6C and ATF6 [6]. All these macular region. To discriminate CORDs from
genes are shown to be a part of phototransduction CODs and macular degeneration, additional oph-
pathway in cone cells. Of these, incomplete thalmologic examinations are needed (fluorescein
achromatopsia involves mutation in CNGA3 and angiography, fundus autofluorescence, ERG). In
PDE6H only [7, 8]. contrast to CODs, CORDs show a peripheral reti-
nal involvement, and the ERG is characterised by
Oligoconetrichromacy Oligoconetrichromacy is a decrease in both cone and rod responses.
a very rare cone disease which projects reduced However, cone responses are more severely
visual acuity and cone response in ERG while a affected than the rod-specific ERG components.
normal colour vision and retinal phenotype. It In pure CODs or early CORDs, scotopic ERG is
has a genetic overlap with genes of achromatop- normal. In later disease stages, night blindness
sia with association of recessive mutation in occurs, and the loss of the peripheral visual field
CNGA3, CNGB3, GNAT2 and PDE6C. is progressing. This is different in rod-dominated
retinal diseases, where night blindness is one of
Blue Cone Monochromatism (BCM) It is also a the first symptoms and the disease progresses
rare disease of cone dysfunction characterised by from the periphery to the centre.
severe impairment in colour discrimination, pho-
tophobia and reduced visual acuity. It is also Genotype It is inherited in autosomal dominant,
sometimes referred as a form of incomplete or recessive and X-linked recessive manner. The
atypical achromatopsia. But the distinguishing genes involve in autosomal dominant CORDs are
factor is the loss of function mutation in OPN1LW AIPL1, CRX, GUCA1A, GUCY2D, PITPNM3,
and OPN1MW genes encoding ‘L’ (red) and ‘M’ PROM1, PRPH2, RIMS1, SEMA4A and
(green) cone pigments. These genes are present UNC119.The loci involved are 17q and 10q26.
in X chromosomes, and hence these diseases are
X-linked recessive. The autosomal recessive CORD genes include
ABCA4, ADAM9, RPGRIP1, CDHR1, C8orf37,
26.2.2.2 Cone and Cone-Rod RAB28, TTLL5, POC1B, ATF6, C21orf2,
Dystrophies (COD and CORD) CACNA2D4, CERKL, CNNM4, IFT81, KCNV2,
This involves cone degeneration in childhood or RAX2 and RDH5.The unidentified gene locus
early adult stage and subsequent degeneration of involved is 1q12-q24. The X-linked recessive
rods later. Genetically it is highly heterogeneous progressive CORD is associated with mutation in
with overlapping genotype to many retinal degen- RPGR, CACNA1F genes and Xq27 loci.
erative diseases like RP, LCA, macular degenera-
tion, rod dystrophies, etc.
26.2.3 Macular Degeneration
Phenotype The progressive cone diseases are
generally more severe than the progressive rod- 26.2.3.1 Monogenic
dominated phenotypes (e.g. RP). Although some Best Disease Best disease, also called as Best
peripheral vision is preserved, it can lead to legal vitelliform dystrophy, is an early-onset macular
blindness earlier than RP. Affected individuals dystrophy caused by mutation in BEST1 (VMD2)
experience first symptoms of decreased visual gene. This gene encodes bestrophin-1 that func-
acuity at school during the first decade of life. In tions as a calcium chloride channel. It is inherited
addition, patients with progressive CODs and as an autosomal dominant disease. Incomplete
penetrance is also noted in few cases [9]. There is recessive manner, while ELOVL4 [18] is found to
also a separate class of bestrophinopathy called be mutated in autosomal dominant families.
the autosomal recessive bestrophinopathy (ARB)
caused by recessive mutation in BEST1 gene Fundus flavimaculatus (FFM) is an allelic dis-
[10]. order of Stargardt’s disease with mutations in
ABCA4 and PRPH2 [19]. It is characterised as a
Sorsby’s Fundus Dystrophy Sorsby’s fundus late-onset form of Stargardt’s disease.
dystrophy is an age-related autosomal dominant
macular dystrophy occurring in the fourth decade Age–Related Macular Degeneration
of life [11]. Mutation in tissue inhibitor of metal-(AMD) ARMD is a multifactorial disorder with
loproteinase 3 (TIMP3) gene is shown to be asso- associated genetic and environmental factors.
ciated with the disease [9]. ARMD is classified as dry and wet AMD depend-
ing on the presence or absence of drusen. It is
Malattia Leventinese and Doyne Honeycomb autosomal dominant and is also genetically het-
Retinal Dystrophy Malattia leventinese (ML) erogeneous with association of high-risk poly-
and Doyne honeycomb retinal dystrophy morphism in genes like FBLN6, CFH, VEGF,
(DHRD) are autosomal dominant diseases that LRP6, HTRA1, HLA, MMP9, ARMS2, C3 and
have a very strong phenotypic similarity to age- TLR4 and risk variants in ABCR, FBLN5, ERCC6,
related macular degeneration (AMD). Unlike ELOVL4, APOE, ACE, SOD2, ABCA4, PON1,
AMD, they are monogenic where mutations in RAX2, CST3, CX3CR1, CFI, C9, C2 and CFB.
EFEMP1 (EGF-containing fibrillin-like extracel- AMD is also associated with mitochondrial gene
lular matrix protein 1) gene are shown to be asso- mutation in MTTL1 [20]. About 50% of the dis-
ciated with it [12]. ease risk is attributed to CFH polymorphism
Y402H [21, 22]. There are also reports that show
X-Linked Juvenile Retinoschisis Retinoschisis is that the combination of environmental factors
an X-linked recessive disorder caused by mutation like smoking and body mass index along with
in RS1 gene encoding retinoschisin [13]. There is Y402H variation increases the risk of AMD [23].
also a study of single three-generation family with Reduced risk of AMD has been associated with
eight affected members showing typical autoso- common deletion that encompasses both the
mal dominant pattern of the disease [14]. CFHR1 and CFHR3 genes [24].
26.2.3.2 Multigenic
Adult-Onset Vitelliform Macular dystrophy 26.2.4 Generalised Photoreceptor
(AVMD) Vitelliform macular dystrophies Diseases
(VMDs) are characterised by round yellow depos-
its at the centre of the macula and contain lipofus- 26.2.4.1 Leber Congenital Amaurosis
cin deposits in the fundus autofluorescence. Phenotype Leber congenital amaurosis is a
severe form of retinal degenerative disease diag-
AVMD are late-onset autosomal dominant nosed in children earlier than 1 year of age. This
genetically heterogeneous macular dystrophies disease was described initially by Theodore
caused by mutation in PRPH2 (VMD3), IMPG1 Leber in 1869 as a congenital form of retinitis
(VMD4) and IMPG2 (VMD5) [15]. pigmentosa [25].The clinically distinguishing
features of LCA include severe visual impair-
Stargardt’s Disease (STGD) It is the common ment present at birth or shortly thereafter, extin-
juvenile macular degeneration with clinical and guished or non-recordable ERG, pendular or
genetic heterogeneity. So far three genes are searching nystagmus, photophobia and digito-
associated with it – ABCA4 [16], PROM1 and ocular sign (Franceschetti-Leber phenomenon),
ELOV4. PROM1 [17] is inherited in autosomal with progressive retinal degeneration [26].
Genotype LCA is both clinically and geneti- retinal pigment epithelial cells leading to severe
cally heterogeneous. So far, 29 candidate genes blindness [33]. In contrast to mutations that affect
have been identified. They are GUCY2D (LCA1), only the REP1 locus leading to non-syndromic
RPE65 (LCA2), SPATA7(LCA3), AIPL1(LCA4), CHM, large gene deletions are known to cause
LCA5(LCA5), RPGRIP1(LCA6), CRX (LCA7), syndromic phenotypes [34].
CRB1(LCA8), NMNAT1(LCA9), CEP290
(LCA10), IMPDH1(LCA11), RD3 (LCA12), The initial phase of an ongoing multicentric
RDH12 (LCA13), LRAT(LCA14), TULP1 clinical trial reported promising results. Six
(LCA15), KCNJ13 (LCA16), GDF6 (LCA17), patients were administered a subfoveal injection
PRPH2 (LCA18), CNGA3, CLUAP1, DTHD1, of adeno-associated viral (AAV) vector-encoding
IQCB1, , MERTK, MYO7A, OTX2, ALMS1, REP1. There was a significant improvement in
CABP4 and CCT2. Most of them are inherited in visual acuity and in rod and cone functions [35].
an autosomal recessive manner except CRX,
IMPDH1 and OTX2 which are associated with 26.2.4.3 Gyrate Atrophy
autosomal dominant inheritance pattern [27, 28]. of the Choroid and Retina
Phenotype Gyrate atrophy of the choroid and
One of the genes which is associated with retina is a progressive condition associated with
LCA was designated RPE65 [29]. It encodes a significantly increased plasma ornithine levels,
protein consisting of 533 amino acid residues chorioretinal degeneration, myopia and early
with high abundance in the retinal pigment epi- cataracts. There might be a mild skeletal muscle
thelium. The protein is an isomerase and involved weakness and mental retardation, but these
in the conversion of all-trans retinol to 11-cis reti- extraocular symptoms are present only
nal. Gene transfer of the RPE65 gene in the eyes occasionally.
of patients provides the first example for a suc-
cessful gene therapy in human patient with this
severe form of retinal dystrophy [30–32]. LCA Genotype It is inherited as an autosomal reces-
like ocular phenotype is also observed in few sive disease and is shown to be caused by
syndromes like Alstrom syndrome (ALMS1 gene homozygous or compound heterozygous muta-
mutation), infantile neuronal ceroid lipofuscino- tion in the ornithine aminotransferase (OAT)
ses, Senior-Loken syndrome (IQCB1 gene muta- gene [36].
tion), Joubert syndrome (CEP290 gene mutation)
and thiamine-responsive megaloblastic anaemia
(SLC19A2 gene mutation). 26.2.5 Vitreoretinopathies
26.2.4.2 Choroideremia 26.2.5.1 Erosive Vitreoretinopathies

Phenotype Choroideremia is an X-linked domi- (ERVR)
nant chorioretinal dystrophy. Symptoms begin Phenotype Erosive vitreoretinopathy belongs to
with night blindness during teenage years, and a group of disorders called the hereditary vitreo-
the disease progressively affects degeneration of retinopathies [37]. Apart from erosive vitreoreti-
photoreceptor cells, retinal pigment epithelial nopathy, Stickler syndrome, Wagner’s disease
cells, choriocapillaris and choroid, leading to and Goldmann-Favre syndrome also belong to
complete blindness. hereditary vitreoretinopathies and are character-
ised by marked vitreous syneresis. The differen-
Genotype Choroideremia is caused by mutation tial diagnosis for ERVR phenotypically involves
in CHM gene. CHM encodes Rab escort protein marked visual field defect, poor night vision,
1 (REP1), a family of GTP-binding proteins that abnormal electroretinographic findings and asso-
regulate vesicular traffic. Absence of REP1 leads ciated retinal pigment epithelial changes [38].
to degeneration of choroid, photoreceptors and This along with absence of COL2A1 gene muta-
tion helps in differential diagnosis of ERVR and Genotype It is genetically heterogeneous with
Stickler syndrome. six candidate genes identified so far. The autoso-
mal dominant STL includes mutations in COL2A1
In most of the patients, the first clinical sign gene (STL type I) [44], COL11A1 gene (STL type
becomes obvious during the late teens. The II) [45] and COL11A2 gene (STL type III) [46].
effected vitreous is frequently described as Among these STL type III is a nonocular Stickler
‘empty’ with veils and strands. Except for auto- syndrome. Incomplete penetrance was also
somal dominant vitreoretinochoroidopathy, reti- observed in a family with COL2A1 dominant
nal detachment is a common feature in all. mutation resulting in variable age of onset [47].
Wagner’s and erosive vitreoretinopathy present
with tractional retinal detachment/snowflake The autosomal recessive STL includes muta-
degeneration, while Stickler syndrome presents tions in COL9A1 (STL type IV) [44] and COL9A2
with rhegmatogenous retinal detachment. genes (STL type V) [48, 49]. Also, autozygome
and exome analysis in a family identified a novel
Genotype Genotypically ERVR is an autoso- missense variant in LOXL3 as the likely candi-
mal dominant disease and is considered to be date cause [50]. Mutation in these genes affects
an allelic disorder of Wagner’s disease. Brown the fibrillar type II/XI collagen molecules
et al. [39] observed a significant linkage of expressed in vitreous interfering in the proper
both ERVR and Wagner’s disease to regions on formation of secondary vitreous.
chromosome 5q13–14 [39]. On screening the
critical region using 13 microsatellite markers, nhanced S–Cone Syndrome (ESCS)
E
a CSPG2/Versican splice site variant was ESCS patients have an unusual gain of function
observed in families of ERVR and Wagner’s of photoreceptors with varying degree of severity
disease. CSPG2/Versican functions to maintain of retinal degeneration. The more severe type is
the integrity of vitreous by keeping the colla- the Goldmann-Favre syndrome (GFS). GFS is
gen molecules apart. The splice variant results characterised by a liquefied vitreous body with
in the balance shifts of the CSPG2/Versican preretinal band-shaped structures, cataract, mac-
isoforms [40], thus leading to disease ular retinoschisis, pigment loss and severely
phenotype. affected ERG. The patients have severely reduced
number of rods and L and M types of cones but
Selective Phenotypes of Few ERVRs increased S cones.
Stickler Syndrome (STL) Phenotype The association of nummular pig-

Phenotype Stickler syndrome was initially des- mentary deposits with white-yellow dots at the
ignated as hereditary progressive arthro- level of the RPE along the vascular arcades, focal
ophthalmopathy, a connective tissue disorder hyperpigmentation within the arcades and foveal
involving skeletal, orofacial, ocular and auditory or peripheral schisis is clinically suggestive of
abnormalities [41]. Along with the characteristic ESCS. The ERG features are the rod-specific
vitreous syneresis observed in all hereditary vit- undetectable ERG; the ERG to a standard flash is
reoretinopathies, various other abnormalities simplified and delayed, with a similar waveform
reported with this syndrome includes mild spon- under photopic and scotopic conditions; and, also
dyloepiphyseal dysplasia or arthritis, cleft pal- of importance, the 30-Hz flicker is delayed and of
ate, both conductive and sensorineural hearing lower amplitude than the single-flash photopic
loss and ocular findings such as retinal detach- ERG a wave. Further, abnormally large, delayed,
ment, high myopia and cataract [42]. The preva- simplified waveform S-cone ERG responses (rel-
lence of Stickler syndrome is 1 in 10,000 ative to the size of the conventional ERGs) are
newborns [43]. present in majority of patients.
Genotype The molecular cause is related to 26.3 Syndromic Retinal Diseases

mutations of nuclear receptor gene (NR2E3).
26.3.1 Usher Syndrome (USH)
26.2.5.2 Exudative Vitreoretinopathy
(EVR) Phenotype Clinically, Usher syndrome is clas-
Exudative vitreoretinopathy is characterised by sified in three subtypes, depending on the sever-
heterogeneous retinal vascular changes that vary ity and age of onset of disease manifestations in
from being avascular to developing retinal neo- the retina and ear. The most severe form is Usher
vascularisation, vitreoretinal traction, subretinal syndrome type I. Patients with this subtype have
exudation and retinal detachment. profound and congenital deafness and vestibular
dysfunction, leading to delayed motor develop-
Phenotype EVR is characterised by an incom- ment and adolescent-onset RP. Usher type II is
plete blood vessel development in the retinal less severe with normal vestibular function; mild
periphery and retinal folds or retinal detachment. to severe sensorineural hearing loss, which is
The clinical diagnosis is usually made in the first nonprogressive in most cases; and a later onset of
years of life. Upon fluorescein angiography, the RP in adolescence or adulthood. Patients with
retinal periphery appears avascular. A fibrovascu- Usher III also have a milder but progressive form
lar mass may develop that is associated with reti- of deafness, and approximately 50% also mani-
nal exudates. This mass may extend to the ciliary fest vestibular problems.
body and peripheral lens capsule. The clinical
manifestations are highly variable, and some Genotype Genes associated with USH type I
patients do not experience marked visual are MYO7A (USH1B), Harmonin (USH1C),
impairment. CDH23 (USH1D), PCDH15 (USH1F), SANS
(USH1G) and CIB2 (USH1J). Three loci associ-
Genotype In most cases, it is considered ated with USH1 are 21q21 (USH1E), 15q22-q23
familial and is shown to inherit in autosomal (USH1H), 10p11.21-q21.1(USH1K). USH type
dominant, autosomal recessive and X-linked II genes include Usherin (USH2A), GPR98
recessive form. Genes that are associated with (USH2C) and DFNB31 (USH2D), and USH type
dominant form of the disease are Wnt signal- III genes are CLRN1 and HARS (USH3A).
ling pathway proteins such as FZD4 [51] on
11q14 encoding the putative Wnt receptor friz- Digenic inheritance was also reported in few
zled-4, LRP5 gene on 11q13.4 encoding low- cases – atypical Ushers was reported with the
density lipoprotein receptor-related protein and combination of mutant homozygous CEP250 and
cadherin-associated protein, beta1 (CTNNB1) heterozygous C2orf71 [57], in USH3 patient
[52] on chromosome 3p22. Other genes include with homozygous CLRN1 mutation and hetero-
tetraspanin family of protein encoded by zygous MYO7A mutation [58]. With ubiquitous
TSPAN12 [53] on 7q31 that functions with the gene expression, the USH genes mostly function
norrin receptor complex and increases norrin/ as cell adhesion proteins and scaffolds, in actin-
beta-catenin signalling, ZNF408 [54], a tran- based intracellular trafficking and Ca2+-mediated
scription factor that functions in vascular devel- signalling [59].
opment. While LRP5 gene mutation was also
associated with autosomal recessive EVR [55],
X-linked recessive EVR is caused by mutation 26.3.2 Bardet-Biedl Syndrome (BBS)
in NDP gene on chromosome Xp11 encoding
norrin protein which is involved in a pathway Phenotype Bardet-Biedl syndrome is a
that regulates neural cell differentiation and ciliopathy disorder caused by disruption in cili-
proliferation [56]. ary biogenesis and trafficking. It is clinically
h eterogeneous and characterised by primary clin- 26.3.4 Refsum Syndrome (Batten

ical features such as rod-cone dystrophy, obesity, Disease)
renal dysfunction, reduced intelligence, polydac-
tyly, male hypogonadism and secondary features Refsum disease belongs to the peroxisome bio-
such as hepatic fibrosis, diabetes mellitus, hyper- genesis disorders (PBD), which also include
cholesterolaemia, reproductive abnormalities, Zellweger syndrome, neonatal adrenoleukodys-
short stature, speech defects and developmental trophy and rhizomelic chondrodysplasia
delay. There is a progressive loss in visual acuity punctata.
eventually leading to legal blindness in about 3/4
of the patients, usually by the second decade of Phenotype Refsum disease is an inborn error of
life. lipid metabolism caused by deficiency of fatty
acid alpha-oxidation. Clinically it is character-
Genotype It is also genetically heterogeneous ised by retinitis pigmentosa, anosmia, and cere-
with 26 candidate genes identified so far: bellar ataxia [62], and plasma phytanic acid
BBS1, BBS2, ARL6 (BBS3), BBS4, BBS5, levels are abnormal. Other systemic conditions
MKKS (BBS6), BBS7, TTC8 (BBS8), BBS9, include neuropathy, ichthyosis, cardiomyopathy,
BBS10, TRIM32 (BBS11), BBS12, MKS1 arrhythmia and eventually heart failure later in
(BBS13), CEP290 (BBS14), BBS15, life.
SDCCAG8 (BBS16), LZTFL1 (BBS17), BBIP1
(BBS18), IFT27 (BBS19), BBS20, C8orf37 Genotype RS is an autosomal recessive disease.
(BBS21), ADIPOR1, IFT172, INPP5E, Genetically, two genes were associated with the
KCNJ13 and NPHP1. Oligogenic inheritance diseases – phytanoyl-CoA 2-hydroxylase gene
was also observed in BBS [60], and the pres- (PHYH) where more than 90% of patients were
ence of pathogenic mutations in different genes shown to have mutation in this gene and PEX7
has resulted in severe disease phenotype in gene where 10% of patients have shown muta-
patients [61]. tions in this gene [63]. Three more genes were
found to be associated with the disease, namely,
PEX1, PEX26 and PXMP3.
26.3.3 Senior–Loken Syndrome
(SLSN)
26.3.5 Joubert Syndrome
Phenotype Senior-Loken syndrome is a renal-
retinal syndrome which is a congenital, cilio- Phenotype Joubert syndrome is a ciliopathic,
pathic autosomal recessive disease that is neurodevelopmental disorder with heterogeneous
characterised by nephronophthisis and Leber clinical manifestations. Classic findings include
congenital amaurosis. the ‘molar tooth sign’ which comprises of mal-
formation of the cerebellum and the brain stem
Genotype Genetically it is heterogeneous with and hypotonia and developmental delay.
seven genes and one locus associated with the Ophthalmic manifestations include retinal dys-
disease. SLSN1 is caused by NPHP1 (nephrocys- trophy, abnormal eye movements and difficulty
tin 1) gene, SLSN3 is associated with locus on in smooth pursuits and gaze and tracking. It clini-
chromosome 3q22, and SLSN4 is caused by cally overlaps with Bardet-Biedl syndrome and
mutation in NPHP4 (nephroretinin) gene, SLSN5 Meckel-Gruber syndrome.
by NPHP5 (IQCB1) gene, SLSN6 by NPHP6
(CEP290) gene, SLSN7 by SDCCAG8 gene, Genotype JS is an inherited autosomal reces-
SLSN8 by WDR19 gene and SLSN9 by sive disorder. Mutations in 33 genes have
TRAF3IP1 gene. shown to be associated with Joubert syndrome.
JBTS1 is caused by INPP5E gene, JBTS2 by artery hypoplasia with cardiac abnormalities and
mutation in the TMEM216 gene, JBTS3 by a butterfly-like vertebral arch.
AHI1 gene, JBTS4 by NPHP1 gene, JBTS5 by
CEP290 gene, JBTS6 by TMEM67 gene, Genotype So far two genes belonging to
JBTS7 by mutation in the RPGRIP1L, JBTS8 NOTCH signalling pathway are shown to be
by ARL13B, JBTS9 by mutation in CC2D2A, associated with Alagille syndrome – JAG1 [72]
JBTS10 by CXORF5(OFD1) gene, JBTS11 by and NOTCH2 [73]. The majority of cases can be
TTC21B gene, JBTS12 by mutation in the explained by sequence alterations in JAG1. JAG1
KIF7 gene, JBTS13 by TCTN1 gene, JBTS14 is a transmembrane protein that constitutes a
by TMEM237 gene, JBTS15 by CEP41 gene, ligand in the notch pathway.
JBTS16 by mutation in the TMEM138 gene,
JBTS17 by C5ORF42 gene mutation, JBTS18 Since the notch signalling is an important
by mutation in the TCTN3 gene, JBTS19 by developmental pathway, relevant for several
ZNF423 gene, JBTS20 by TMEM231 gene, tissues, it is not surprising that ALGS1 includes a
JBTS21 by mutation in the CSPP1 gene, wide range of clinical manifestations (hepatic,
JBTS22 caused by mutation in the PDE6D cardiac, vascular, skeletal, ocular, facial, renal,
gene, JBTS23 by KIAA0586 gene, JBTS24 pancreatic and neuronal) [74]. Interestingly, the
caused by mutation in the TCTN2 gene, majority of the JAG1 mutations are nonsense
JBTS25 by CEP104 gene, JBTS26 by mutation mutations or small deletions leading to premature
in the KIAA0556 gene, JBTS27 by B9D1 gene, termination codons. Furthermore, some deletions
JBTS28 by mutation in the MKS1 gene, affect the entire coding region of JAG1. This sup-
JBTS29 by TMEM107 gene and JBTS30 by ports the hypothesis that a reduced gene dosage
mutation in the ARMC9 gene [64]. Others (haploinsufficiency) may be the basis of the
genes associated recently are C2CD3 [65], disease.
CEP120 [66] and POC1B [67]. Only JBTS10
is X-linked recessive [68], and the rest all are
inherited in autosomal recessive pattern. Early 26.3.7 Alstrom Syndrome
genetic diagnosis also helps in clinical man-
agement in Joubert syndrome as few systemic Phenotype Alstrom syndrome is a rare cilio-
complications have a late onset like JBTS1 and pathic disease with characteristic features of type
3 are restricted to central nervous system II diabetes mellitus, retinal degeneration (cone-
(CNS) defects, while JBTS2 is shown to have rod dystrophy or Leber congenital amaurosis),
clinical impact on the kidney, retina and liver obesity and sensorineural deafness [75]. Other
apart from CNS abnormalities [69]. systemic associations include dilated or restric-
tive cardiomyopathy, insulin resistance syn-
drome, liver involvement with cirrhosis and
26.3.6 Alagille Syndrome multiple organ failure. Despite its similarities to
Bardet-Biedl syndrome, mental retardation,
Phenotype Alagille syndrome is an autosomal polydactyly and hypergonadism do not belong to
dominant disease with reduced penetrance, the repertoire of features in Alstrom syndrome.
involving liver, heart, skeletal, ocular and facial
abnormalities. Ophthalmic manifestations
include posterior embryotoxon, iris abnormali- Genotype Until now, mutations in ALMS1 gene,
ties, retinal hypopigmentation with RPE speck- located on chromosome 2, have only been associ-
ling [70], angulated retinopathy [71] and optic ated with this disease in an autosomal recessive
nerve head abnormalities in about a third of the pattern. ALMS1 is localised in the centrosomes,
individuals. The ocular findings do not have and they play an important role in centriole struc-
major significance. Systemic manifestations ture and function [76]. Accumulation of intracel-
include malformation of bile ducts, pulmonary lular vesicles in the inner segments and
mislocalisation of rhodopsin to the outer nuclear called oto-sino-pulmonary disease as it shows

layer indicate that ALMS1 may play a role in characteristic chronic abnormalities of the ear,
intracellular trafficking . sinus and lower airways. Approximately half of
the patients have situs inversus (mirror orienta-
tion of thoraco-abdominal organs). Also, male
26.3.8 Neuronal Ceroid infertility is common, and complex congenital
Lipofuscinosis (NCN) heart disease can occur. The combination of situs
inversus totalis, chronic sinusitis and bronchiec-
Phenotype Neuronal ceroid lipofuscinosis is a tasis is also known as the Kartagener triad.
neurodegenerative disease caused due to impair-
ment in lysosomal storage. They are character- Almost all men with PCD are infertile from
ised by the accumulation of autofluorescent sperm immotility. Additional symptoms or clini-
material in different cell types including neurons. cal manifestations may include hydrocephalus,
Clinical manifestations comprise epileptic sei- retinitis pigmentosa, polycystic kidney disease,
zures, progressive psychomotor retardation, liver cysts and biliary atresia. All those features
visual loss and premature death. By the age of are rare in PCD.
onset and clinical features presented, these are
classified as congenital, infantile, late infantile, Genotype Genetically it is reported as an auto-
juvenile, adult and Northern epilepsy (epilepsy somal recessive disease in most cases involving
with mental retardation) [77]. 28 genes and 2 loci. Ciliary dyskinesia 1(CILD1)
is caused by recessive mutation in DNAI1 gene,
Genotype It is also genetically heterogeneous CILD2 by DNAAF3 gene, CILD3 by DNAH5
with eight genes identified so far. The congenital gene, CILD5 by HYDIN gene, CILD6 by
NCN form is caused by CTSD (CLN10) gene. TXNDC3 gene, CILD7 by DNAH11 gene, CILD9
Infantile NCL is caused by PPT1 (CLN1) and by DNAI2 gene, CILD10 by KTU gene, CILD11
KCTD7 (CLN1) gene. Late infantile form is by RSPH4A gene, CILD12 by RSPH9 gene,
associated with PPT1 (CLN1), TPP1 (CLN2), CILD13 by DNAAF1 gene, CILD14 by CCDC39
CLN5, CLN6, MFSD8 (CLN7), CLN8 and CTSD gene, CILD15 by CCDC40 gene, CILD16 byD-
genes (CLN10). Juvenile form is associated with NAL1 gene, CILD17 byCCDC103 gene, CILD18
PPT1 (CLN1), TPP1 (CLN2), CLN3, CLN9 and by HEATR2 gene, CILD19 by LRRC6 gene,
ATP13A2 (CLN12) genes. The Northern epilepsy CILD20 by CCDC114 gene, CILD21 by DRC1
variant is associated with CLN8. Adult NCL also gene, CILD22 by ZMYND10 gene, CILD23 by
known as Kufs disease is due to mutation in ARMC4 gene, CILD24 by RSPH1 gene, CILD25
CTSD (CLN10), PPT1 (CLN1), CLN3, CLN5, by DYX1C1 gene, CILD26 by C21ORF59 gene,
CLN6 [78], CTSF (CLN13) and GRN (CLN11) CILD27 by CCDC65 gene, CILD28 by SPAG1
genes. The above forms are autosomal recessive, gene, CILD29 by CCNO gene, CILD30
while the autosomal dominant adult NCL, also byCCDC151 gene, CILD32 by RSPH3 gene,
known as Parry disease, is caused by DNAJC5 CILD33 by GAS8 gene, CILD34 by DNAJB13
(CLN4B) gene [79].The most prevalent NCLs gene, CILD35 by TTC25 gene and CILD37 by
are juvenile-onset CLN3 and late infantile-onset DNAH1 gene [80]. Apart from these the ciliary
CLN2 diseases. biogenesis proteins of MCIDAS [81] and DNAH8
[82] are also associated with ciliary dyskinesias.
The loci identified so far are CILD4 on 15q13
26.3.9 Primary Ciliary Dyskinesias and CILD8 on 15q24-q25.
(PCD)
Apart from these X-linked recessive PCD is
Phenotype Primary ciliary dyskinesias are a cil- also reported along with other conditions such as
iopathic disorder involving motile cilia. It is also mutation in RPGR in patients with X-linked reti-
nitis pigmentosa [83] and PCD and mutation in ing copy number variations in 552 achromatopsia
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CYP1B1 Gene Mutation in Primary
Congenital Glaucoma 27
Rita S. Sitorus
Abstract potential new molecular targets to treat this

Primary congenital glaucoma (PCG) is the threatening blindness disease in children will
most common childhood glaucoma affecting be developed in the near future.
children from birth to age 3 years and is a
major cause of blindness in this young Keywords
population. CYP1B1 · Gene · Mutation · Children ·
Mutations in the CYP1B1 gene, associated Primary congenital glaucoma
with GLC3 locus, have been found to cause
PCG in children worldwide and are the domi-
nant genetic cause for pediatric glaucoma in
the Middle East and Central Europe. Our
27.1 Introduction
study in a small number of Indonesian and
European PCG families supports the previous
Primary congenital glaucoma (PCG) is a rare
studies that reported mutations of the CYP1B1
form of glaucoma and is usually inherited in an
gene being responsible for the PCG pheno-
autosomal recessive mode with incomplete pen-
type. A different pattern of CYP1B1 disease-
etrance [1]. PCG is the most common childhood
causing mutations and benign variants appears
glaucoma and one of the most important causes
to exist in Indonesian patients when compared
of childhood blindness. In a separate school for
to patients from other ethnic backgrounds.
the blind study in Indonesia, we reported pediat-
Other genes such as LTBP2 and PXDN
ric glaucoma, mostly the primary congenital
gene have been reported recently by several
form, accounted for 8.2% of the treatable causes
studies as being associated with pediatric
of blindness in children [2].
glaucoma.
PCG is caused by unknown developmental
The exact mechanisms of how these gene
defect(s) of the trabecular meshwork and anterior
abnormalities actually cause primary congeni-
chamber angle. It manifests clinically during the
tal glaucoma remain unclear. However, with
neonatal or infantile period. The disease is char-
the promising research progress so far, it is
acterized by high intraocular pressure (IOP),
hoped that we will understand more about the
buphthalmos with corneal enlargement, and
pathogenesis of this disease and eventually
breaks in Descemet’s membrane.1 In Saudi
Arabia, the incidence is estimated to be 1:2500,
R. S. Sitorus (*) whereas in western countries, the incidence is
Department of Ophthalmology, Faculty of Medicine estimated to be less than 1:30000 [3, 4].
Universitas Indonesia, Cipto Mangunkusumo
Hospital, Jakarta, Indonesia

https://doi.org/10.1007/978-981-13-0884-0_27
338 R. S. Sitorus
27.2 Clinical Features tive open reading frame starts in the second exon
and is 1629 bp in length [7–9]. More than 150
Primary congenital glaucoma often manifests in mutations including missense, nonsense, regula-
the first years of life, especially during neonatal tory, and insertions and/or deletions in CYP1B1
or infantile period. The later the onset, the less have been associated with PCG [5] and are the
severe the defect, hence often leading to better main known cause of PCG. In ethnically mixed
prognosis. Also, the later the onset, the less clini-populations, mutations were found in 20–30% of
cal manifestation develops. Typically, it presents patients with PCG,11 whereas in consanguineous
as triad of epiphora, blepharospasm, and photo- populations, the prevalence increases to 85% [3,
phobia. During the first year of life, glaucoma is 8, 10].
often suspected whenever changes in scleral and
corneal architecture are found, such as buphthal-
mos with corneal enlargement (Fig. 27.1). 27.4 Genetic Analysis of CYP1B1
Diagnosis is usually made based upon clinical in Indonesian and European
findings. A comprehensive ophthalmologic Patients
assessment is mandatory due to pending diagno-
sis, and treatment may cause irreversible end- We screened the coding region of the CYP1B1
stage eye disease and permanent visual loss. gene in 21 patients clinically diagnosed as con-
genital glaucoma [11]. Twelve of them were of
Indonesian descent and nine of European descent.
27.3 Genetic Aspects of PCG Blood samples were obtained from subjects and
their relatives. We use oligonucleotides primers
PCG is a genetically heterogeneous disorder in reported by Bejjani et al. to amplify the coding
terms of disease-associated loci, penetrance, and exons of the CYP1B1 gene by performing PCR
expressivity. To date, GLC3 loci have been deter- analysis [3]. Subsequently, PCR products were
mined with four specific loci identified: GLC3A analyzed using single-strand conformation poly-
(MIM 231300) on chromosome 2p22-p21, morphism analysis (SSCP).
GLC3B on chromosome 1p36.2-36.1, GLC3C Our study identified CYP1B1 mutations in
on chromosome 14q24.3, and GLC3D on chro- 33.3% and 22.2% of subjects screened from
mosome 14q24.2-24.3 [5]. The GLC3A locus co- Indonesian and European patients, respectively
localizes to the CYP1B1 gene (MIM 601771) on [11]. Five distinct disease-causing mutations
chromosome 2p21. were identified in 6 of the 21 index cases:
CYP1B1 is a member of the cytochrome P450 V364 M, S215I, E281X, 1407del12bp, and
superfamily enzyme, which is the largest known 1410del13bp.
enzyme of the human cytochrome P450 pathway In the Indonesian patients, the missense muta-
that is primarily expressed in the trabecular tions V364 M (3/12 patients) and S215I (1/12
meshwork, iris, retina, and ciliary body. This patients) were identified in Indonesian patients.
enzyme contributes to the development of normal Mutations were found exclusively in Indonesian-
trabecular meshwork microarchitecture by Sundanese patients, which suggest that V364 M
metabolizing essential molecules that are perhaps is the most common mutation in this ethnic
used in a signaling pathway, possibly a steroid group. We ruled out V364 M as recent founder
[6]. mutation in this ethnic group by testing six short
The human CYP1B1 gene consists of three sequence repeat markers at 2p24–12. Alleles var-
exons of which the first is noncoding. The puta- ied among all tested patients for the informative
27 CYP1B1 Gene Mutation in Primary Congenital Glaucoma 339
Fig. 27.1 A 9-month

infant with Primary
Congenital
Glaucoma, showing
buphthalmos, corneal
haze, and increased
intraocular pressure
Fig. 27.2 Direct sequencing of the 5′ amplimer of exon tide in codon 215 in exon 2 leads to the substitution of a
2 in case 7. (a) Direct sequencing representing a novel Ile residue for a Ser residue. (b) FokI restriction endonu-
mutation S215I . A G → T transition at the second nucleo- clease digest (lanes 1 and 3, controls; lane 2, case 7)
markers. The V364 M mutation has been reported patient and is a novel mutation (Fig. 27.2). It was
in a Japanese study in the compound heterozy- singly heterozygous (only one mutant allele was
gous state in 1 of 11 families [17]. This mutation, detected). No genomic DNA from the unaffected
however, has never been reported in white popu- parents was provided for segregation analysis;
lations, and we also did not find this mutation in however, this missense mutation was not present
our European patients. The possibility exists that in 50 chromosomes from randomly selected nor-
the mutation originated from an ancient founder mal subjects.
and then spread throughout Asia, since even In a Saudi Arabian PCG study, other distinct
cases 1 and 2 (sibs) shared only one allele for missense mutations G61E, R468W, and D374N
some markers, despite being the product of a con- were reported as the most common mutations,
sanguineous marriage. The V364 M mutation accounting for 72%, 12%, and 7% of the tested
might also have occurred independently owing to alleles, respectively. In Brazilian patients, a sin-
a mutational hot spot caused by the surrounding gle truncating mutation g.4340delG (20.2%) was
sequence (GTC GTG GGG). the most frequent mutation and was different
S215I mutation, a serine to isoleucine transi- from Turkish PCG families who showed an equal
tion affecting the second nucleotide of codon frequency of truncating and missense mutations
215 in exon 2, was identified in an Indonesian in the tested samples [6, 12].
340 R. S. Sitorus
In contrast to the Indonesian PCG families, Many studies have demonstrated CYP1B1 as a
the truncating mutations account for the majority critical gene responsible for PCG [3–6, 8, 10–12,
of the CYP1B1 mutations identified in the 15, 17, 19–28], which is directly related to the
European PCG families (Table 27.1). All of the integrity of trabecular meshwork. CYP1B1 par-
mutations identified probably truncate the open ticipates in the normal development and function
reading frame. They include one nonsense muta- of the eye by metabolizing essential molecules
tion (E281X), one frameshift mutation that are probably used in a signaling pathway [6].
(c.1410del13bp), and one in-frame mutation The detailed mechanism of CYP1B1 alteration
(c.1407del12bp). These mutations are expected which may contribute to the pathogenesis of PCG
to eliminate amino acids 79–377 from the has, however, remained unclear [28].
carboxy- terminus of the CYP1B1 polypeptide.
Thus, if a stable protein is synthesized, every
mutant molecule would lack at least the heme- 27.5 C
YP1B1 Mutation Reported
binding region, which is essential for the function in Various Population
of the cytochrome P450 molecule. Therefore, it is with PCG
expected that these mutations will result in func-
tional null alleles. Various disease-causing mutations in CY1B1
Peters’ anomaly has been reported as being gene recently reported from different populations
correlated with mutation in CYP1B1 [13, 14]. In were shown in Table 27.2.
our study, we identified E281X/del355RVGA CYP1B1 mutations occur in 87% of familial
mutation (case 5). It appears that this entity does and 27% of sporadic cases of PCG worldwide
not result from a single specific mutation or type [6]. Five major groups of mutations in CYP1B1
of mutation since in our case, the combination of have been found in PCG: missense mutations,
a stop mutation and a deletion shows a similar frameshift or truncating mutations, mutations
phenotype to a stop mutation (W57X) and a mis- triggering the nonsense-mediated mRNA decay
sense change (M1T), which by themselves do not of CYP1B1 [6, 28], and mutations in the pro-
predict the phenotype of Peters’ anomaly. moter or control regions of the gene.
Different pattern of single nucleotide poly-
morphisms (SNPs) appears to exist in the
Indonesian population compared to European 27.6 O
ther Genes Associated
(white) patients. Benign sequence variants such with PCG
as A453S and L432 V have been identified in
European patients only, while the allele fre- Several studies reported LTBP2 and PXDN genes
quency of the noncoding variant IVS1-12 t/c was to be associated with pediatric glaucoma. A new
much higher in European than in Indonesian locus (GLC3D) harboring the LTBP2 gene has
patients (60% compared to 15.8%). been characterized in developmental glaucoma,
However, as in other reported studies [12, 16], but its role in classical cases of PCG is yet to be
we also found that R48G and A119S were always understood.
co-inherited, indicating that these two SNPs are Ali M et al. [20] reported null mutations in
linked [16]. We could not confirm the hypothesis LTBP2 cause PCG in four consanguineous fami-
that the combination of four well-known poly- lies from Pakistan and in patients of Gypsy eth-
morphisms, R48G, A119S, L432 V, and N453S, nicity. LTBP2 maps to chromosome 14q24.3 but
establishes a pathogenic allele when co-inherited, is around 1.3 Mb proximal to the documented
as previously reported [17]. GLC3C locus. It remains to be determined
Table 27.1 CYP1B1 gene: genotype-phenotype correlations on mutations reported in our study [11]
Sequence change Ethnic Age at Therapy, IOP at Visual acuity
Case Gender allele1/allele2 Predicted effect Exon background Consanguinity diagnosis Age at onset started at age diagnosis RE/LE
1 F c.1436G-A V364 M 3 Indonesian- Yes 19 y <3 y M, 19 y High/30– HM/P*
homozygous homozygous Sundanese 40
2 M c.1436G-A V364 M 3 Indonesian- Yes 15 y <3 y M, 15 y High/30– LP/LP*
3 M c.1436G-A V364 M 3 Indonesian- Yes 15 y <3 y M, 15 y High/30– LP/LP*
4 M c.1436G-A/ND V364 M 3 Indonesian- No 16 y <3 y M, 16 y High/30– LP/LP*
homozygous Sundanese 40
27 CYP1B1 Gene Mutation in Primary Congenital Glaucoma
5 F c.1189G- E281X/ 2/3 Turkish No 3 mth Congenital TET, 3 mth 25/25 1,0/1,0
T/1407del12bp del355RVGA CPC, 11
mth
CPC, 3 y
6 F c.1410del13bp/ND Frameshift/ND 3 Italian No 2 mth 4d T, 9 mth 17/20 1,6/2,4 cyc/
degree
7 M c.992G-T/ND S215I/ND 2 Indonesian- No 24 y <3 y M, 24 y High/30– LP/LP*
Sundanese 40
*HM hand movement, LP light perception. †By PL + TAC procedure; M, pilocarpine + acetazolamide, CPC: sequence data refer to GenBank entry HSU56438 and the
sequence given by Sutter et al. [18] and Stoilov et al. [19]
341
342 R. S. Sitorus
Table 27.2 CYP1B1 disease-causing mutation recently reported in patients with PCG in various population
No. Mutations Population References
1 p.F231I, p.P437A, p.G61E, c.535delG Tunisia Bouyacoub
[20]
2 p.Arg355*/p.Arg355, p.Arg390Cys/c.1209_1210insTCATGCCACC, p. United Lim [21]
Glu387Lys/c.1209_1210insTCATGCCACC, States
p.Trp57*/c.1209_1210insTCATGCCACC,
p.Arg355*/c.1209_1210insTCATGCCACC,
p.Glu387Lys/c.1064_1076delGAGTGCAGGCAGA, p.Trp57*/p.Ala106Asp
3 p.Arg355fsX69, p.Thr404fsX30, p.Arg469Trp, p.Thr404fsX30 Spanish Milla [22]
162-kb del(2p21.1), p.Asp449fsX6, p.Arg368His, p.Trp57Stop, p.
Ser464PhefsX12, p.Gly61Glu, p.Leu277Stop
4 c.970_971dupAT; p.T325SfsX104, p.G329S, p.V419Gfs11X Korean Kim [23]
5 g.4339delG (predominant), p.R163C, p.C470Y, g.4330-4331delTG, p.R163C, Moroccan Hilal [24]
p.E173K, g.4330-4331delTG, p.E229K, p.R390S, p.R368H, p.R469W, p.
C470Y, g.7901-7913del13bp
6 A106D, E173X, F261 L, E262X, W341X, P513_K514del Spanish Campos-
P52L, G61E, Y81N, E229K, P400S Mollo [25]
7 g.3972delC, g.4168_4169insGACCGGCCGGCCTTCGCC, g.8209_8213delA Chinese Chen [26]
GCAGinsTTGTTGAAAAA, p.I60M, p.V95A, p.L107 V, p.P118S, p.F134S,
p.N203S, p.A287S, p.D291G, p.N319S, p.V363D, p.R368L
8 E173K. N498D, G61E Egyptian El-Ashry
and Saudi [27]
Arabian
Fig. 27.3 Mutation detection and confirmation of the Direct sequencing of the wild-type amplimer indicating
V364 M mutation. NlaIII restriction endonuclease digest. the amino acids Arg 355, Val 356, Gln 357, and Ala 358
Lane 1, case 1; lane 2, case 2; lane 3, case 4; lane 4, case abolished by the deletion
3; lanes 5 and 6, controls; lane M1, marker 25 bp ladder;
lane M2, marker 100 bp ladder
whether LTBP2 is the GLC3C gene or whether a related glaucoma; however the glaucoma may be
second adjacent gene is also implicated in PCG. secondary (lens-related) rather than primary.
LTBP2 mutations were also identified in auto- LTBP2 DNA sequencing of individuals with pri-
somal recessive congenital/infantile glaucoma mary congenital glaucoma identified 14 sequence
with the clinical spectrum of primary megalocor- variants comprising 7 transitions and 7 transver-
nea, spherophakia with ectopic lens, and lens- sions. These variants were inherited in a hetero-
27 CYP1B1 Gene Mutation in Primary Congenital Glaucoma 343
zygous manner and included 3 coding References

nonsynonymous and 11 coding synonymous
SNVs [21]. PXDN gene is also reported to cause 1. Cascella R, Strafella C, Germani C, Novelli G, Ricci
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nant cause of primary congenital glaucoma in Saudi
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research progress, a better understanding of the coma (PCG) maps to chromosome 14q24.3. Fort
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Acknowledgments Table 27.1, Figs. 27.2 and 27.3, and
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Diabetic Retinopathy: Clinical,
Genetic, and Health Economics 28
(An Asian Perspective)
Siddhita Nare, Sunita Mohan, Uthra Satagopan,

Sundaram Natarajan,
and Govindasamy Kumaramanickavel

Diabetes mellitus is the fastest growing dis-
ease in the world that is estimated to reach 28.1.1 Prevalence of Diabetes
nearly half a billion in 2045, and a third of Mellitus (DM) and Diabetic
them would have microvascular complication Retinopathy (DR)
like diabetic retinopathy (DR). Hyperglycemia,
hypertension, and dyslipidemia are some of Diabetes mellitus (DM), a noncommunicable
the controllable risk factors. DR is classified complexly origin disease, is considered as one of
into nonproliferative, proliferative, and macu- the most challenging health problems rising at a
lar edema types. Many molecular factors like tremendous pace globally. DM is estimated to
VEGF, ALR2, eNOS, MTHFR, ACE, IGF, and rise to 629 million by 2045 from 425 million in
RAGE and its associated single nucleotide 2017 [1]. According to IDF 2017 report, 159 mil-
polymorphisms play a critical role in the pro- lion in Western Pacific and 82 million in Southeast
cess of neovascularization. Some of the drug Asian adults are living with DM [1]. China, India,
discovery and newer treatment regimens are Indonesia, and Pakistan represent 48% of the
based on these molecular factors. More global burden [1]. With the incidence of DM
research by the clinicians, epidemiologists, increasing at an alarming rate, the number of
and vision scientists is necessary to reduce the people with diabetic retinopathy (DR), a micro-
visual morbidity and disease burden of DR in vascular complication, is expected to surge from
the community. 126.6 million in 2010 to 191.0 million by 2030
[2]. DR ranks the fifth most common cause of
Keywords global blindness (moderate and severe vision
Diabetic retinopathy · Health economics · impairment) [3] accounting for 4.8% of the cases
Genetic susceptibility · Type 2 diabetes of blindness throughout the world [4]. It is one of
mellitus · Prevalence the leading causes of visual impairment and
blindness in the working-age population (24–
70 years) in both developing and developed
S. Nare (*) · S. Mohan (*) · S. Natarajan countries [5].
G. Kumaramanickavel A pooled analysis of 22,896 people with DM
Aditya Jyot Foundation for Twinkling Little Eyes, from 35 population-based studies in the USA,
Mumbai, Maharashtra, India
Australia, Europe, and Asia (between 1980
U. Satagopan and 2008) showed that the overall prevalence of
Centre for Medical Genetics, Chennai, India

https://doi.org/10.1007/978-981-13-0884-0_28
346 S. Nare et al.
any DR in T1DM (type 1 diabetes mellitus) and rural areas [12]. Patients with DM having retinal
T2DM (type 2 diabetes mellitus)) was 34.6% complication spent approximately INR 13922
(95%CI 34.5–34.8), with 10.2% having vision- (USD 214.38) per month [12]. For a chronic dis-
threatening diabetic retinopathy (VTDR) [6]. ease like DM and hence DR, there is an unmet
The prevalence of DR appears to be higher in urgency for development of a cost-effective ther-
patients with T1DM than in those with T2DM apy that is dependent on a basic understanding of
[3]. Nearly all persons suffering from T1DM the pathophysiological progression of DR.
develop retinopathy, while more than 77% of per-
sons with T2DM may develop retinopathy after
20 years’ duration of diabetes [5], and approxi- 28.2 Patho-mechanisms
mately 25% of persons with DR may develop and Biology of Retinopathy
macular edema [7]. In India, the overall crude
prevalence of DM is 4.7% in urban and 1.9% in Retinopathy is a slow-progressing disease,
rural areas [8], and 7.3–26.2% of these develop mainly characterized by damage of the microvas-
DR [9]. In a population-based door-to-door sur- culature of the retina. The onset and progression
vey in the urban slums of Western India, we of retinopathy are triggered by numerous factors
reported the DR prevalence of 1.41% in general including extended duration of diabetes, poor
population and 15.4% in type 2 DM [10]. The control of blood glucose, elevated blood pres-
prevalence of severe retinopathy in patients with sure, and dyslipidemia [13–16].
T1DM has diminished over the past 35 years due Histological studies, using postmortem retinas
to improved medical care [11], but the recent epi- of diabetic patients, have revealed several cellular
demic of T2DM requires a new understanding of changes: selective endothelial and mural cell loss
the biology of DM. The growing number of dia- (including pericytes), presence of mural cell
betic patients and the longer life span in aging ghosts, endothelial clusters, acellularity and
population imply an increase in patients suffering microaneurysms [17, 18], basement membrane
from DR, which not only affects the quality of thickening, presence of hemorrhage in the inner
life of the individuals and their families but also nuclear layer (INL) and outer plexiform layer
increases the medical and economic burden of (OPL), as well as eosinophilic exudates in the
the society. OPL [18] .
Further, immunological and immunohisto-
chemical studies have shown hypertrophy of
28.1.2 Social Burden and Health Müller cells throughout the inner and outer dia-
Economics betic retina and increased apoptosis [19]; expres-
sion of pro- and antiapoptotic molecules in
DM and its complications like DR impose a huge ganglion and glial cells, respectively [20]; and
economic burden on the global healthcare. elevated levels of vascular endothelial growth
According to the International Diabetes factor (VEGF) in retinal blood vessels of diabetic
Federation (IDF) estimation, globally USD patients with pre-proliferative or no retinopathy
727 billion is spent for DM (20–79 years age stages [21]. Alternation in several other factors,
group), whereas USD 850 billion was spent (19– including somatostatin [22], cortistatin [23], 𝛼A-
99 years age group) in 2017. The expenditure on and 𝛼B-crystallins, advanced glycation end prod-
diabetes is projected to reach USD 776 billion ucts (AGEs), and receptor for AGE (RAGE) [24],
(20–79 years age group) and USD 958 billion as well as apolipoprotein A1 (ApoA1) [25], was
(18–99 years age group) by 2045 [1]. A recently also observed in the postmortem tissues.
published study from India estimated that the Advanced molecular studies revealed abnor-
total annual expenditure on DM care was, on an mal levels of expression of mRNA and proteins
average, INR (Indian rupee) 10,000 (USD 154) of various chemokines [26, 27], cytokines [26–
in urban areas and INR 6260 (USD 96.42) in 29], inflammatory markers [29–31], and
28 Diabetic Retinopathy: Clinical, Genetic, and Health Economics (An Asian Perspective) 347
angiogenic factors [27, 29, 30, 32] in aqueous lipoproteins leaking from retinal capillaries into
humor, serum, or urine from diabetic patients. the extracellular space of the retina [41] is also a
Although these morphological and molecular major cause of vision loss and can occur at any
studies provide a better picture of the pathogen- stage of DR.
esis of DR at a cell and molecular level, they IDF estimates globally 64% of people are liv-
failed to provide mechanistic biological pathway. ing with DME, while 58% with DR face difficul-
However, based on these observations, several ties in performing daily activities [1]. Vision loss
mechanisms and interlinked biological pathways results from retinal detachment if patients are left
such as hyperglycemia and oxidative stress- untreated. Several emerging automated technolo-
mediated AGE products and inflammation, endo- gies have demonstrated promise in assisting with
plasmic stress (ES)-mediated unfolded protein the diagnosis of sight-threatening DR leading to
response (UPR) and apoptosis, hypoxia- and prompt referral for the timely management of the
ischemia-mediated angiogenesis, activation of treatable conditions.
protein kinase C, polyol production, and hexos-
amine pathways have been postulated to be
responsible for retinopathy complications in DM 28.4 Genetics and Epigenetics
[33–36]. As these mechanisms and pathways are of DR
interlinked [37], the strategies to prevent the
development/progression of this complication DR is a complex disease, strongly influenced by
become complicated. both genetics and environment. Single nucleo-
tide polymorphisms in genes encoding for the
molecules and other enzymes, cytokines, and
28.3 Clinical Diagnosis growth factors involved in DR pathophysiology
and Classification of DR have been associated with risk for DR in various
populations. Among the candidate genes studied
From a clinical standpoint, it is clear that the pri- for variations and association with DR, VEGF,
mary driving factors in DR pathogenesis are aldose reductase (ALR2), endothelial nitric
uncontrolled hyperglycemia, hypertension, and oxide synthase (eNOS), methylene tetrahydro-
dyslipidemia. However, recent evidences have folate reductase (MTHFR), and RAGE have
also suggested neurodegeneration as an early been widely studied in various populations
event in the pathogenesis of DR [38]. (Table 28.1).
The diagnosis of DR essentially remains clini- Elevated serum and vitreous VEGF levels
cal in nature, the gold standard being dilated eye have been associated with PDR suggesting pos-
exam and serial fundus images. DR is classified sible increase in VEGF gene expression in
as either nonproliferative diabetic retinopathy DR. The C(−634)G promoter VEGF polymor-
(NPDR) or proliferative diabetic retinopathy phism has been associated with risk for DR [67]
(PDR) based on the presence of neovasculariza- and DME in Japanese cohorts [68], similarly
tion that typifies the proliferative form [13]. G(−1154)A and C(− 7)T, and T(−1498)C poly-
Nonproliferative features of retinopathy, include morphisms are reported to be associated with risk
microaneurysms, intraretinal hemorrhage, hard for DR in Caucasian [69] and South Indian popu-
exudates, venous beading, and intraretinal micro- lations [70], respectively. VEGF-460C variation
vascular anomalies (IRMAs), and the prolifera- might accelerate the pathogenesis of retinal neo-
tive form features the neovascularization (NV) vascularization in T2DM patients as suggested
that bleeds easily resulting in vitreous hemor- by association studies in Indian population [59].
rhage, subsequent fibrosis, and tractional retinal In Chinese patients with T2DM, SNPs rs699947,
detachment [39, 40]. DME characterized by rs833061, and rs13207351 at the promoter region
increased vascular permeability and deposition of the VEGF gene might have association with
of hard exudate at the central retina secondary to predisposition DR [71].
Table 28.1 Genetic association studies for DR, PDR, and DME in various population
348
Gene Chromosome location Variation Disease Type Association Population References

AR 7q35 C [-106] T, DR T2DM Significant association—risk Iranian, Japanese, [42, 43]
CC genotype Egyptian
DR T2DM No significant association Chinese [44]
C [-106] T, C allele DR T1DM Significant association—risk Asia, South [45]
America, Europe,
and Australia
AKR1B1 -106C>T, [rs759853], DR T2DM Significant association—risk North Indian [46]
TT genotype population
Z-2 Microsatellite DR T2DM High risk Asian Indian [47]
DR T1DM/T2DM Confer risk White Ancestry [48]
DR T2DM Involved in the development Japanese [49]
of DR population
DR T2DM early onset of DR Chinese population [50]
in Hong Kong
DR T2DM Risk Caucasian [51]
C[-12]G DR T2DM Risk China [52]
Z-4 DR T1DM Risk Japanese [53]
Z+2 DR T1DM Confer protection White Ancestry [48]
against DR
ALAR2 Z+2 DR T2DM associated with South Indian [54]
susceptibility to DR Cohort
eNOS 7q36 VNTR 4b/a, a allele PDR T2DM Significant association—risk Slovenian [45]
T-786C & C774T DR T1DM Risk for early onset French [55]
severe DR
Intron4 VNTR DR T2DM Risk Chinese, German, [56]
Japanese
DME T2DM Risk Japanese [56]
RAGE 6p21.3 Gly82Ser, DR T2DM Significant association—risk North Indian, [45]
Ser82genotype Chinese
-374A T/A Sight T1DM Risk associated Scandinavian [57]
threatening DR origin
1704T allele DR DM Risk associated East Asian [58]
S. Nare et al.
VEGF 6p21 [-460 C/T], C allele PDR, DR T2DM Significant association—risk Indian, Caucasian [45, 59]
[+936C/T] DR T2DM Positive association—risk Asian [45]
[-2578C/A] DR T2DM Significant association—risk Chinese [45]
rs13207351 DR T2DM Positive association—risk Chinese [45]
rs699947[-2578 A/C] DR T2DM Positive association—risk Asians and [45]
Europeans
rs699947[-2578C/A] DR T2DM Positive association—risk Asians and [45]
Caucasians
rs3025039[+936 C/T] PDR T2DM Positive association—risk Bengali Hindu [45]
rs2010963 [+405 G/C] PDR T2DM Positive association—risk Bengali Hindu [45]
rs2010963[-634G/C], DR T2DM Positive association—risk Caucasian, [45]
C allele Japanese
rs2010963[-634G/C], DME T2DM Risk Japanese [45]
C allele
+405CC genotype DR T2DM Risk associated Japanese [60]
CG genotype DR T2DM Risk associated Indian [60]
[-1154]C DR T2DM Risk Caucasian [56]
C[-634]G DR T2DM Increase the risk for DR South Indian [61]
in patients with Cohort
microalbuminuria
ACE 17q23 Deletion polymorphism Advanced DR T2DM Risk Japanese [56]
Insertion/ Deletion DR/NPDR T2DM Risk Pakistani [62]
PPARγ 3p25 Gly482Ser DR T2DM Risk Slovenia [56]
Vit D receptor 12q13 Fok1 polymorphism DR T1DM Risk French [56]
Taq 1 Polymorphism DR T1DM Tt- Risk in poor glycemic French [56]
Adiponectin 3q27 G276T DR T2DM Risk Japanese [56]
MTHFR 1q36 C677T DR T2DM Risk Chinese [56]
Glycoprotein 5q23 807T Advanced T2DM Risk Sweden [56]
28 Diabetic Retinopathy: Clinical, Genetic, and Health Economics (An Asian Perspective)
stages of DR
TGFβ 9q13 R25P PDR T2DM Risk Caucasian [56]
Neuro-peptide Y 7p15 L7P DR T2DM Risk Finnish [56]
Alpha2/beta1 17q21 Intron 7 polymorphism DR T2DM Risk Japanese [56]
integrin
HLA 6p21 DR9 & DQA1 DR T1DM Risk Senegal [56]
(continued)
349
350

MCP-I 17q11.2 rs1024611 [-2518 A/G] PDR T2DM Positive association—risk Korean [45]
AA genotype
rs1024611 [-2518 A/G] PDR & NPDR T2DM Positive association—risk Han Chinese [45]
G allele
rs1024611 [-2518 A/G] DR T2DM Increased onset Japanese [45]
G allele
MnSOD 6q25.3 A16V[C47T] AV DR DM Positive association—risk North Iranian [45]
genotype
iNOS 17q11.2 13-repeat genotype DR T2DM associated with South Indian [54]
susceptibility to DR Cohort
TNF 6p21.3 15-repeat genotype DR T2DM associated with South Indian [54]
[β gene] susceptibility to DR Cohort
NcoI PDR T2DM β2 allele is genetic factor Caucasian – Slovak [63]
for incidence of PDR
[GT]n microsatellite DR, PDR T2DM Allele 4 [103 bp] is a low Asian Indian [64]
risk for developing
retinopathy, Allele 8 [111
bp] is associated with PDR
PEDF gene 17p13.1 T130T DR T2DM Moderate protective South Indian [65]
polymorphism association Cohort
IGF-1 12q23.2 promoter [CA] 18 DR T2DM for more high risk for developing Southern Indian [66]
repeat genotype than 15 years DR and PDR sample cohort
S. Nare et al.
Among Chinese Han individuals with T2DM, Lindholam (2006) reported an association
polymorphism -634G/C of the VEGF gene was between RAGE(−374 T/A) polymorphism and
not correlated with NPDR or PDR; however, type 1 diabetes [57].
polymorphism-460C/T of the VEGF gene was Saleem et al. (2015) observed a significant
correlated with NPDR, and C allele was associ- association between insertion deletion polymor-
ated with lower NPDR risk than T allele [72]. phism rs4646994 in intron 16 and DR and NPDR,
The gene ALR2 that codes for aldose reduc- but not with PDR in Pakistani cohort [62].
tase, the rate-limiting enzyme of the polyol path- Matsumoto reported significant association in
way, has a particular Z-2 promoter microsatellite Japanese population between the presence of the
repeat which has not only been associated with D allele polymorphism in the ACE gene and
genetic susceptibility to DR in Caucasian [48, advanced diabetic retinopathy (ADR) in Japanese
73] and Asian Indian populations [47] with subjects with T2DM [81].
T2DM but also has been shown through func- An 18-repeat polymorphism in the promoter
tional studies to enhance gene expression in of IGF-1 gene is a susceptibility genotype for
response to hyperglycemia. Also an association DR, and its clinical severity in a Southern Indian
was observed between DR and the C-106 T, CC cohort is found by Uthra et al. [66] who also
genotype in the T2DM patients in Iranian [42] reported lack of association of PRKCB1 gene
and Japanese population [74] but not in Chinese promoter polymorphisms and moderate protec-
population [44]. Kaur et al. reported significant tive association of PEDF gene polymorphism
association of AKR1B1 -106C > T polymorphism with DR in the same cohort [65].
(homozygous recessive TT genotype) with reti- Monocyte chemoattractant protein-1 (MCP-1)
nopathy in North Indian patients [75]. Studies is a chemokine specific for monocytes and baso-
from Asia, South America, Europe, and Australia phils. MCP-1 rs1024611 (−2518 A/G) AA geno-
showed an association between C(−106)T poly- type was significantly associated with PDR in
morphism and the risk of DR in T1DM but not T2DM in Korean patients. On the other hand, the
type 2 DM [45]. The Z-4 allele was significantly G allele of the same polymorphism in Han
associated with patients with proliferative reti- Chinese patients was significantly associated
nopathy in Japanese population. While the same with high-risk PDR in T2DM. A study in
study reported an association of Z + 2 allele with Japanese patients with T2DM also reported that
patients without retinopathy [53], a South Indian the G allele was significantly associated with DR
study reported an association of Z + 2 allele with [45].
risk for DR [54]. Results on Z + 2 allele from Single gene association studies have not been
T1DM white ancestors were comparable to comprehensively informative about the role of
Japanese study [48]. the DNA polymorphisms in disease pathogene-
Increased production of nitric oxide by down- sis, and hence approaches like haplotype analy-
regulation of eNOS has been shown to result in sis, linkage disequilibrium, and functional studies
angiogenesis in animal models [76]. The intron4 are expected to throw more light in this area.
27-bp (VNTR) has been consistently associated Analysis of a handful of genetic variations is
with risk for DR in Japanese [77], German, and often insufficient to understand the genetic etio-
Caucasian [78] populations. pathology of polygenic disease as DR. Hence,
Ser82 allele in the RAGE gene is a low-risk researchers now rely on robust technologies such
allele for developing DR in Asian Indian patients as genome-wide association studies. Imperatore
with T2DM [54, 79], whereas Vanita V. showed et al. (1998), in their study on Pima Indians with
significant association of p.Gly82Ser polymor- T2DM, showed some evidence of linkage to
phism in RAGE with DR in T2DM patients [80]. chromosomes 3 (LOD = 1.36) and 9 (LOD = 1.46)
However, studies from Malaysia, the USA, for diabetic retinopathy, although the evidence
Europe, and Asia reported no associations was insufficient for genome-aide studies [82].
between RAGE polymorphisms and DR [45]. Looker et al. (2007) performed a genome-linkage
352 S. Nare et al.
analysis for DR and found evidence of linkage to [88], significant genotype-specific personalized
chromosome 1p (LOD = 3.1 by single-point anal- management strategies for patients could evolve.
ysis and 2.58 by multipoint analysis) [83]. Gene therapy experiments targeting renin angio-
Similarly, another study on Mexican Americans tensin system (RAS) pathway and antioxidant
with T2DM revealed suggestive linkage on chro- enzyme activities are also underway owing to the
mosome 3 (LOD = 3.41) and chromosome 12 limitations of the current inhibitor-based treat-
(LOD = 2.47) [84]. However, fine mapping of ments, which collectively hold promise in the
critical genomic regions, which harbor possible effective management of diabetic microvascular
susceptibility genes, has not yet been reported. complications in the retina [89].
Genome-wide meta-analysis performed by
Grassi et al. (2011) identified an intragenic SNP
on chromosome 6 rs227455, located more than 28.6 Future Trends
200 kb from two undesignated genes LOC728275
and LOC728316, and rs10521145 in strong link- Research in DR has to be a concerted effort
age disequilibrium with copy number variation between the ophthalmologist, the epidemiologist,
CNVR6685.1 on chromosome 16 to have strong and the vision scientist. Such efforts have led to
association with risk for sight-threatening DR the current knowledge that we have in the field.
[85]. A genome-wide association study on However, further research is needed to iden-
Taiwanese population reported a risk association tify molecular genetics and biological factors that
of SNPs located in five novel chromosomal could be applied as early genetic or biomarkers to
regions in and around MYSM1, PLXDC2, identify the target population to reduce the bur-
HS6ST3, and ARHGAP22 genes; the latter two den of visual impairment or blindness in a com-
found to have significant role in endothelial cell munity. Besides, such pathway analysis would
angiogenesis and increased capillary permeabil- also lead to the discovery of newer drugs that
ity [86]. A three-stage genome-wide association would reduce the morbidity or disease burden.
study carried out in a Japanese cohort revealed a Granting agencies should focus on these areas of
borderline significance of association of an research as DM is one of the rapidly rising dis-
intronic SNP in long intragenic noncoding RNA eases with epidemic proportions.
RP1-90 L14 adjacent to KIAA1009/QN1/CEP162
gene, suggesting a possible role of ciliary- Conflict of Interest None of the authors have any propri-
associated genes in the pathogenesis of DR due etary interests or conflicts of interest related to this
submission.
to the involvement of CEP162 gene [87].
However, identification of genes and genetic
variations conferring risk for the development of
DR and recognition of pre-symptomatic individ- References
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Glaucoma Genes in East Asian
Studies 29
Shi Yao Lu, Clement C. Y. Tham, Pancy O. S. Tam,
Shisong Rong, Calvin C. P. Pang, Guy L. J. Chen,
and Wai Kit Chu
Abstract types of glaucoma including primary open-

Glaucoma is a leading cause of irreversible angle glaucoma (POAG), primary angle-
blindness worldwide. Genetic studies in glau- closure glaucoma (PACG), and exfoliation
coma provide evidences of genes and loci glaucoma (XFG). While many GWAS utilized
related to the disease development and insights East Asian cohorts as primary cohorts, the
of the pathogenesis in glaucoma. Gene muta- effects of the variants contributing to glau-
tions with strong associations to the disease coma susceptibility vary across ethnic groups
were found in family linkage studies in which due to differential genetic background. In this
the glaucoma patients usually had early-onset chapter, we focus on the genetics of different
and severe disease features. From these stud- glaucoma forms in East Asian populations.
ies, several genes, such as MYOC, OPTN,
TBK1, and TIE2, have been found to be related Keywords
to glaucoma. On the other hand, sequence Glaucoma · Genome-wide association study ·
variants linked to common and late-onset East Asians
forms of glaucoma were mainly discovered
from genome-wide association studies
(GWAS). Up to date, 15 GWAS have identi-
fied common variants for the risk of different 29.1 Introduction
S. Y. Lu · C. C. Y. Tham · P. O. S. Tam · C. C. P. Pang Glaucoma, a leading cause of irreversible blind-

W. K. Chu (*) ness worldwide, is a group of optic neuropathies
Department of Ophthalmology and Visual Sciences, with characteristic structural optic nerve fiber
The Chinese University of Hong Kong,
Hong Kong, China loss and functionally visual field defects.
e-mail: [email protected] Glaucoma is more common and could progress
S. Rong more rapidly in the aging population [1]. Recent
Department of Ophthalmology, Massachusetts Eye epidemiological evidences reported the preva-
and Ear, Harvard Medical School, Boston, MA, USA lence of glaucoma was 3.54% for people from 40
G. L. J. Chen to 80 years old worldwide. This prevalence is at a
Department of Ophthalmology and Visual Sciences, similar rate of 3.4% in Asia [2, 3]. Glaucoma is a
The Chinese University of Hong Kong,
heterogeneous disease including subtypes of dif-
Hong Kong, China
ferent etiology, prevalence, genetic susceptibility,
clinical manifestations, and treatment strategies.
Prince of Wales Hospital, Hong Kong, China

https://doi.org/10.1007/978-981-13-0884-0_29
358 S. Y. Lu et al.
Based upon the anatomical structure of the ocular tion of glaucoma genetics including family link-
anterior segment, it can be divided into two major age analysis, re-sequencing study, genome-wide
subgroups, open-angle (OAG) and closed-angle association study (GWAS), and next-generation
(ACG). Structural variations in the anterior sequencing (NGS) [8–13]. Most glaucoma
chamber angle could affect the drainage rate of patients are non-symptomatic at the early stage.
aqueous humor, fluid secreted from the ciliary Therefore, early detection is a challenge for dis-
body and removed through the trabecular mesh- ease management. One important purpose of
work and Schlemm’s canal, leading to changes in genetic studies is to identify individuals at risk by
the intraocular pressure (IOP) [4]. Elevated IOP developing precise genetic screenings of high
is an important risk factor for most of the glau- disease susceptibility. These genetic screenings
coma types. Normal tension glaucoma (NTG), a will help the decisions on early intervention to
form of open-angle glaucoma, however, can prevent or slow down the disease progression.
occur with no IOP elevation [5]. In addition, Understanding of genetic factors and biological
glaucoma can develop at young age. There is also mechanisms of different glaucomas provides
congenital glaucoma that occurs in infancy essen- information to devise new therapeutics to
tially following the Mendelian inheritance. improve treatments and to develop gene-based
Congenital glaucoma is a rare disease, triggered therapies [14].
by rare mutations with strong genetic effects,
while later-onset glaucoma is influenced usually
by common variants with weaker genetic effects 29.2 Pathophysiology
[6]. Furthermore, genetic variations can not only of Glaucoma
influence the risks of primary glaucoma but also
some cases of secondary glaucoma such as exfo- The pathophysiology of glaucoma is complex
liation glaucoma (XFG) [7]. and still not fully understood. Glaucomatous
Genetic factors contribute to a significant pro- optic neuropathies are associated with progres-
portion of risk in glaucoma (Fig. 29.1). Different sive neuroretinal rim narrowing and effects on
methodologies have been applied for investiga- the optic cups. Retinal nerve fiber layer (RNFL)
Fig. 29.1 Overlaps between the genes associated with one or more glaucoma subtypes in East Asians
29 Glaucoma Genes in East Asian Studies 359
thinning and arcuate visual field loss are common [28]. High occurrence in the former populations
features of most glaucoma patients, indicating is likely associated with consanguinity. PCG has
damages in the optic nerve head [15]. Optic nerve been found to be associated with myocilin
injury is generally considered as the dysfunction (MYOC) mutations, with Glu230Lys,
or loss of neurons such as retinal ganglion cells Arg272Stop, and Ser313Phe reported in a
(RGCs). One of the putative causes for RGC Chinese study and p.Leu228Ser and p.Glu240Gly
death is axon injury by the displacement of lam- identified in a South Korean study [29, 30]. The
ina cribrosa, which is a sieve-like membrane for most important gene for PCG is CYP1B1 (cyto-
RGC axons to exit to the posterior part of the chrome P450 family 1 subfamily B member (1),
sclera [16]. Elevated IOP raises the pressure gra- which accounts for most congenital glaucoma
dient through the lamina cribrosa. Deformation patients. CYP1B1 mutations which specifically
of this tissue causes damages to the optic nerve occur in East Asians have been reported. In a
head and RGC axons [17]. In angle-closure glau- Japanese study, four CYP1B1 mutations,
coma, structural disruptions affect positioning of Asp192Val, Ala330Phe, Val364Met, and
the peripheral iris with respect to the trabecular Arg444Gln, were identified in PCG [31]. A
meshwork and peripheral cornea, leading to Chinese study reported 11 novel CYP1B1 muta-
obstruction of aqueous outflow and consequently tions in PCG including g.3985C>G (p.Ile60Met),
elevation in IOP [4]. In open-angle glaucoma, g.4089T>C (p.Val95Ala), g.4124C>G (p.
although the anterior chamber angle is open, Leu107Val), g.4157C>T (p.Pro118Ser),
there are metabolic or structural derangements g.4206T>C (p.Phe134Ser), g.4413A>G (p.
affecting drainage of aqueous humor, such as Asn203Ser), g.4664G>A (p.Ala287Ser),
defects of mitochondrial function, reduction of g.4677A>G (p.Asp291Gly), g.4761A>G (p.
endothelial cells, and abnormal accumulation of Asn319Ser), g.7925T>A (p.Val363Asp), and
extracellular matrix components in the trabecular g.7940G>T (p.Arg368Leu) [29]. Subsequently,
meshwork [18–20]. Elevated IOP could induce Kim et al. reported 11 mutations from 22 of 85
glaucoma through ischemia, oxidative stress, Korean PCG patients. In particular, two of these
autophagy, excitotoxic signaling, and immune mutations, p.sGly329Ser and p.
response [21–24]. However, the IOP of NTG Val419Glyfs11Stop, were novel [30]. CYP1B1
patients is within the normal range which is usu- has been shown to form digenic interaction with
ally defined as 10–21 mm Hg. Reduced ocular MYOC, resulting in higher risk of glaucoma [32].
perfusion pressure, the net gradient between sys-
temic blood and intraocular pressure, is also a
risk factor for optic nerve damage and glaucoma 29.4 Familial Normal Tension
[25, 26]. Perfusion of vasculature and resultant Glaucoma (NTG)
oxidative stress in the optic nerve might be asso-
ciated with NTG pathophysiology [27]. Familial NTG is a rare and early-onset type of
POAG, which has been reported to associate with
mutations in optineurin (OPTN) and TANK-
29.3 Primary Congenital binding kinase 1 (TBK1). OPTN Glu50Lys is the
Glaucoma (PCG) most frequent mutation identified in familial
NTG in East Asians; several studies have identi-
PCG is a serious childhood glaucoma usually fied OPTN mutations in early-onset NTG indi-
developed before 3 years old, compared to a vidual patients instead of NTG families [33]. In a
later-onset time in juvenile-onset primary open- Korean study, homozygous Met98Lys, homozy-
angle glaucoma (JOAG). Prevalence of PCG var- gous Thr34Stop, heterozygous Arg271Cys, and
ies widely across different ethnic populations, combined heterozygous Thr34Stop with
reportedly from 1/1250 in Middle East popula- Arg545Gln mutations in OPTN were found in
tions and South Asians to 1/70,000 in Caucasians early-onset NTG patients. Moreover, a combined
360 S. Y. Lu et al.
heterozygous mutation Thr123Stop and linked to JOAG and late-onset POAG in East
Ile288Stop in MYOC in a female NTG patient Asians (Table 29.1). A Japanese study in 1997
was found [34]. Arg158Gln in MYOC was found detected a new mutation Pro370Leu in MYOC
from an early-onset NTG patient in Japan [35]. from a JOAG family. In this family, the father
Susceptible loci in TBK1 in familial NTG were and the daughter were diagnosed of having glau-
first identified from three NTG pedigrees in the coma at 26 and 16 years old, respectively.
United States: GGO-441 from an African- Gly367Arg was identified in a female diagnosed
American family and GGA-416 and GGA-1159 with POAG at 45 years old from another Japanese
from two Caucasian families. In the African- family [41]. A Korean family study in 1999
American family, three copy number variations reported two mutations Arg46Stop and Thr353Ile
(CNVs) within the GLC1P locus, located on in MYOC in JOAG [42]. However, our study in
chromosome 12q14, co-inherited with the southern Chinese in Hong Kong suggested that
patients. Interestingly, TBK1 is located in the both the Arg46stop and Thr353Ile in MYOC
GLC1P locus and was expressed in ganglion could be found in both POAG patients and nor-
cells, nerve fiber layer, and microvasculature of mal controls. Moreover, our study found a novel
the human retina [36]. A subsequent study dis- mutation, Arg91Stop in MYOC, in a late-onset
covered a novel TBK1 mutation, in locus GGJ- POAG [9]. We also reported a Arg46stop trunca-
414, from a Japanese NTG pedigree [37]. As both tion in MYOC in one JOAG patient, four late-
OPTN and TBK1 are reported to involve in onset POAG, and nine normal controls [43]. The
autophagy and NF-ΚB signaling, these pathways mutation Ala363Thr was first reported in a muta-
may play important roles in NTG [38]. tion screening study in Japanese JOAG patients
[35] and another missense MYOC mutation,
Cys245Tyr, from a Chinese JOAG family in
29.5 Juvenile-Onset Primary Hong Kong by our group [8]. The variant
Open-Angle Glaucoma Arg46Stop was also reported by a Chinese JOAG
(JOAG) study in Taiwan together with Val56Ala,
c.604 + 228A > T in the intron, and
Primary open-angle glaucoma (POAG), char- c.1515 + 73G > C in the 3′-untranslated region
acterized by normal anatomic configuration of of MYOC [44]. A nonsynonymous substitution
the aqueous humor outflow pathway, is the Asp384Asn in MYOC was first identified from a
most common form of glaucoma in the United northern Chinese JOAG family [45] and
States and Western Europe. Globally, the high- Pro254Arg from a JOAG family in Sichuan of
est prevalence of POAG is in Africa, 4.20%, Western China. Patients in this family suffered
while PACG is in Asia (1.09%) [3]. JOAG is an severe visual field loss and elevated IOP at
early-onset form of POAG before the age of 31 mm Hg in both eyes, with disease onset
40 years old. The first glaucoma gene locus, younger than 40 years of age [46]. Two com-
GLC1A on chromosome 1q24.3, was identified bined heterozygous mutations (c.-83G > A and
in a large Caucasian family [39]. The respon- c.764 T > C, coding for Leu255Pro, and
sible gene in this locus for POAG was found to c.369C > T and c.864C > T, coding for
be trabecular meshwork-inducible glucocorti- Thr123 = and Ile288=, respectively) were found
coid response (TIGR), which is also called in two Korean JOAG patients [34]. Screening for
myocilin (MYOC) [40]. sequence variations in the MYOC promoter did
not show mutations in both HK Chinese and
Korean POAG patients [47, 48]. These results
29.5.1 MYOC imply that the modulation of MYOC expression
might not affect glaucoma development. In addi-
A number of studies have been conducted to tion, MYOC mutations were also identified in
investigate mutations in MYOC and other loci late-onset POAG (Glu300Lys, Tyr471Cys,
Table 29.1 Novel mutations/loci discovered from JOAG patients in East Asian
Gene (HGNC locus designation) Chromosome Location Sequence alteration Ethnic group References
MYOC 1q24.3 Exon Pro370Arg Japanese [41]
(GLC1A) Exon Gly367Arg Japanese [41]
Exon Arg46X Korean [42]
Exon Thr353Ile Korean [42]
Exon Ala363Thr Japanese [35]
Exon Cys245Tyr Southern [8]
Chinese
Exon Val56Ala Taiwan Chinese [44]
Intron c.604 + 228A > T Taiwan Chinese [44]
3′ UTR c.1515 + 73G > C Taiwan Chinese [44]
Exon Asp384Asn Northern [50]
Chinese
Exon Pro254Arg Southern [46]
Chinese
Exon Leu255Pro Korean [34]
Exon Thr123= Korean [34]
Exon Ile288= Korean [34]
Unknown gene 15q22-q24 No No reported Southern [66]
(GLC1N) reported Chinese
EFEMP1 2p15-p16 No No reported Southern [67]
(GLC1H) reported Chinese
Ile360Asn, Gly451Asn, Ala363The, Phe369Leu, reticulum stress could be a potential therapeutic

and Thr448Pro) as well as familial NTG patients strategy for glaucoma patients with MYOC
(Arg158Gln, and a combined mutation of mutations [61].
Thr123= and Ile288=) from Hong Kong, Japan
and Korea [9, 34, 35, 43, 49, 50, 51]. These
results indicate that MYOC mutations also 29.5.2 Other Gene Loci
appeared in the late-onset POAG patients.
Overall, MYOC mutations account for 2–4% of MYOC mutations account for 8–36% of JOAG in
late-onset POAG [6]. different reported studies. It is also induced by
MYOC encodes a secreted glycoprotein mod- mutations in other genes and environmental fac-
ulating different signaling pathways to regulate tors [6]. Several POAG related loci, GLC1H,
cell-cell junctions, cell-matrix adhesion, cyto- GLC1J, GLC1K, GLC1M, and GLC1N, were
skeletal network, and cell migration in adjacent identified in JOAG patients. GLC1J (9q22) and
cells [52–54]. MYOC mutations associating GLC1K (20p12) are the only two JOAG associat-
with glaucoma and increased IOP are consid- ing loci reported in Caucasian studies [62]. Our
ered to mainly affect the physiology in trabecu- previous work found GLC1M, 5q22.1-q32, was
lar meshwork and the aqueous humor outflow associated with autosomal dominant JOAG in a
pathway [55, 56]. However, changes in MYOC Philippine family [63]. NRG2 and SPARC from
expressions were not associated with ocular the GLC1M locus were excluded to be the causal
hypertension and glaucoma development [57– genes of JOAG [64, 65]. GLC1N, 15q22-q24,
59]. The misfolded mutant MYOC could trigger was identified in a Chinese JOAG family [66].
endoplasmic reticulum stress in trabecular Genes in both GLC1M and GLC1N linked to
meshwork cells to induce resistance in aqueous JOAG are still unknown. GLC1H, 2p16.3-p15,
humor outflow and IOP elevation [60]. An ani- was also reported to associate with JOAG in a
mal study showed that reducing endoplasmic Chinese family [67]. A mutation in EFEMP1
362 S. Y. Lu et al.
(c.418C > T, Arg140Trp) in this locus was identi- rs1042522 in tumor protein p53 (TP53) signifi-
fied by exome sequencing in an African-American cantly associated with NTG in Hong Kong
family with adult-onset POAG [67, 68]. In addi- Chinese [73]. Both the TNF and TP53 genes
tion, we found significant association of POAG that play key roles in apoptosis and t are related
with interactions between two mutations, MYOC to tumorigenesis. The neurotrophin-4 (NTF4)
(Thr353Ile) and OPTN (IVS15 + 10G > A), indi- gene stimulated the receptor tropomyosin
cating polygenic etiology of glaucoma [69]. receptor kinase B on retinal ganglion cells and
suppressed cell death [74–76]. Mutations of
NTF4 have been identified in POAG patients
29.6 Adult-Onset Primary Open- from Europe and Asia. The nonsynonymous
Angle Glaucoma (POAG) variants, Cya7Tyr, Glu84Lys, Ala88Val,
Arg90His, Arg206Trp, Arg206Gln, and
29.6.1 MYOC, OPTN, and WDR36 Arg209Gly, were reported in a European study,
while three disease-causing mutations,
To identify mutations and common variants asso- Leu113Ser, Gly157Ala, and Ala182Val, were
ciated with POAG development, target genes or reported in two Chinese studies [77–79]. The
loci were explored in case-control studies by CYP1B1 gene was first reported for primary
direct sequencing and genotyping. Some com- congenital glaucoma. However, Pro93Ser,
mon variants and mutations associating with the Arg259Cys, Ala295Thr, and Leu475Pro of
early-onset POAG, such as MYOC and OPTN, CYP1B1 were identified from POAG patients
contributed to the risks of developing adult-onset in a northern Chinese study [80]. Oxidative
POAG in East Asians [9, 34, 35, 43, 49–51]. stress is proposed to cause cellular damage and
Candidate gene-based investigations of late-onset optic neuropathy. Enzymes involving in anti-
POAG have reported loci with differential allelic oxidation have been investigated in POAG. A
frequencies between POAG patients and healthy study in Sichuan, Western China, genotyped
controls. The WD repeat domain 36 gene the catalase (CAT) gene, which encodes an
(WDR36), located in the GLC1G locus on chro- antioxidant enzyme secreted into the aqueous
mosome 5q22.1, is related to POAG especially humor, and found SNP rs769217 in CAT asso-
for high tension glaucoma (HTG). A study in ciated with POAG [81]. Studies on loci identi-
southern Chinese identified one disease-fied from GWAS related to glaucoma
predisposing mutation, Ile713Val in WDR36 with endophenotype found that SNPs in atonal
a frequency of 3.7% in HTG patients. In addition, homolog 7 (ATOH7) and raftlin lipid raft linker
three SNPs (rs13153937, rs10038177, and 1 (RFTN1) were associated with increased
rs11241095) were significantly associated with risks of POAG in Hong Kong Chinese [82].
HTG, while no WDR36 SNPs was associated NTG is affected by genetic factors other than
with NTG or JOAG [70]. A genome-wide linkage those for HTG. Apolipoprotein E (APOE),
scanning for IOP-related loci in Mongolian located in 19q13.2, is related to Alzheimer’s dis-
patients found an association between the WDR36 ease [83, 84]. APOE is involved in lipid transport
locus and IOP regulation [71]. in the metabolism of neuronal cell membrane.
Several SNPs in APOE were associated with
POAG in Caucasians [85, 86]. Our two studies in
29.6.2 Other Associated Genes Hong Kong Chinese confirmed the association
between the APOE epsilon 4 allele and NTG and
Tumor necrosis factor (TNF), coding for a also indicated the interaction of APOE and glau-
monocyte- derived cytotoxin that causes cell coma genes MYOC and OPTN [69, 87]. NCK2,
death, was first associated with POAG in a encoding the NCK adaptor protein 2, was linked
Chinese study [72]. SNP rs1800629 in TNF to NTG in a Japanese cohort [88]. Another
had significant association with HTG and SNP Japanese study found three SNPs in Toll-like
receptor 4 (TLR4), rs10759930, rs1927914, and fied from GWAS have provided insights of bio-
rs7037117, significantly affected risks for NTG logical pathways that might be involved in
[89]. TLR4 participates in the activation of innate pathophysiology of POAG. For instance, identifi-
and adaptive immune responses. However, the cations of CAV1/CAV2 and ABCA1 suggest the
association could not be replicated in a Korean roles of lipid metabolism in POAG, while the dis-
study. Furthermore, only SNP rs7037117 in covery of PMM2 implies that the fructose and
TLR4 was associated with NTG in a recessive mannose metabolism is related to POAG
model in southern Chinese [90, 91]. [103–107].
29.6.3 GWAS for POAG 29.7 Primary Angle-Closure

Glaucoma (PACG)
Apart from the abovementioned genetic factors,
there are also sequence alterations in other genes PACG is characterized as a condition of acutely
reported in POAG. However, many of them only or chronically raised IOP due to an abnormal
had weak association and could not be replicated configuration of the anterior chamber angle. The
in later studies, probably due to small sample closed angle acts as a physical barrier to the aque-
sizes and ethnic differences between the study ous humor outflow. Extremely high IOP is a fea-
populations. GWAS is a powerful genomic tech- ture of PACG to trigger optic nerve degeneration.
nology to discover disease-related loci. So far, 9 Asians have increased predisposition to PACG
GWAS studies in POAG have been reported, and compared to Europeans and Africans. More than
SNPs in 15 genes/loci reached genome-wide sig- 80% of PACG cases are in Asia [108]. GWAS has
nificant level (P ≤ 5 × 10−8). These loci are near identified most of the known loci for PACG, two
or located in CAV1/CAV2, TMCO1, GWAS in PACG and one in anterior chamber
CDKN2B-AS1, SIX1/SIX6, 8q22, ABCA1, depth (ACD). These studies reported that there
AFAP1, GMDS, ARHGEF12, PMM2, TGFBR3, was one SNP associated with both PACG and
TXNRD2, ATXN2, FOXC1, and GAS7 [92–100]. ACD and nine genes/loci were identified, includ-
Among these nine GWAS, four of them included ing PLEKHA7, COL11A1, PCMTD1/ST18,
East Asian cohorts (Table 29.2) [92, 95, 97, 98]. ABCC5, EPDR1, GLIS3, CHAT, FERMT2, and
Variants in CDKN2B-AS1 were consistently DPM2/FAM102A [11, 109, 110]. In addition,
detected in GWAS in different populations with genotype analysis of POAG-associated SNPs in
top-tier statistical significances. Moreover, it case and control cohorts suggested that only two
showed stronger relationship to NTG compared SNPs, rs2276035 at ARHGEF12 and rs12150284
to other types of POAG [94, 100]. Human and at GAS7, were significantly associated with
animal studies have shown expressions of PACG [110]. The first PACG GWAS and ACD
CDKN2B-AS1 in the inner retinal nuclear layer, GWAS discovered genotypic associations in
ganglion cell layer, and trabecular meshwork. Asian, while the latter PACG GWAS involved
Variants in CDKN2B-AS1 were shown to increase cohorts from 24 countries across five continents.
RGC death [101, 102]. Another GWAS showed Most genes reported from these GWAS were
that FNDC3B rs4894796 was in association with found to be expressed in human ocular tissues
POAG in Asians (OR = 0.89, P = 7.93 × 10−8) but except PCMTD1 and ST18. In particular, mRNA
not in Caucasians (OR = 0.99, P = 0.71) [98]. of EPDR1, GLIS3, FERMT2, DPM2, and
FNDC3B is expressed in trabecular meshwork, FAM102A were found in human iris, ciliary body,
optic disc, and optic nerve. Another FNDC3B and trabecular meshwork [4]. Expressions of
SNP rs6445055 was significantly associated with these genes in the ocular anterior segment tissues
IOP in a GWAS [12]. These evidences implied indicated possible effects on the configuration of
that the FNDC3B gene is involved in regulating the anterior chamber angle and the drainage path-
aqueous humor outflow and IOP. Genes identi- way of the aqueous humor. Additionally,
364
Table 29.2 POAG loci discovered by GWAS involving East Asian

SNP Discovery cohort Replication cohort
Gene Country Ethnic Size (case vs control) Country Ethnic Size (case vs control) References
CAV1/ rs4236601 Iceland Caucasian 1263 vs 34,877 Sweden, UK, Australia, Caucasian; Asian 2474 vs 2644 [92]
CAV2 China
CDKN2B rs1063192 Japan Japanese 1394 vs 6599 Japan Japanese 1802 vs 7212 [95]
SIX1/SIX6 rs10483727
ABCA1 rs2487032 Southern China Chinese 1007 vs 1009 China and Singapore Chinese 1899 vs 4965 [97]
PMM2 rs3785176
TGFBR3 rs1192415 Multiple Multiethnic 3504 vs 9746 Multiple Multiethnic 9173 vs 26,780 [98]
FNDC3B rs4894796
S. Y. Lu et al.
PLEKHA7, EPDR1, and FERMT2 played roles tion glaucoma (XFG), which is the most common
in cell adhesion and junction. Abnormal function identifiable type of open-angle glaucoma world-
of these gene products might influence the tight- wide [120–122]. XFG individuals have higher
ness of cell adhesion in anterior segment tissues, mean IOP, more aggressive visual field loss, and
leading to changes in the anatomic structure in worse prognosis with resistance to IOP-lowering
the chamber angle [109, 110]. SNPs in ABCC5 medications compared with POAG patients. The
did not show genome-wide association with mechanism underlying abnormal aggregation of
PACG but ACD. Genotype-phenotype analyses extracellular materials that trigger XFS is still not
showed that SNPs in ABCC5 were significantly fully understood, but likely involved both genetic
associated with PACG in a Chinese study [11, and environmental factors [123].
111]. Other candidate genes reported by PACG Unlike POAG, there is no candidate gene or
GWAS included COL11A1. It is involved in locus that has been identified for XFS/XFG from
fibrillogenesis and extracellular matrix binding familial studies. The first gene contributing sig-
that may have potential to alter the tissue struc- nificant risk for XFS was reported by a GWAS in
ture [112]. CHAT participates in neurotransmitter which the primary cohort contained 75 cases and
biosynthetic processes and DPM2 in biosynthesis 14,470 healthy controls from Iceland. The gene
of dolichol-phosphate mannose [113, 114]. LOXL1, encoding lysyl oxidase homolog 1, and
Mutations of GLIS3 have been reported in con- SNPs rs3825942, rs1048661, and rs2165241 in
genital diabetes and common GLIS3 SNPs also this region were genome-wide associated with
related to diabetes [115–117]. Finally, a meta- XFS [124]. The associations have been replicated
analysis revealed seven polymorphisms in five in other Caucasian populations and different eth-
genes/loci associated with PACG: rs17427817 nic groups including Chinese, Japanese, and
and rs5745718 at HGF, rs1043618 at HSP70, Indian [7, 125–129]. In addition, there was no
rs2510143 and rs3814762 at MFRP, rs3918249 association between LOXL1 locus and POAG in
at MMP9, and rs7830 at NOS3 [118]. In sum- all reported populations [130–132]. However,
mary, the candidate genes identified from both there is some evidence showing allelic reversal of
GWAS and meta-analysis could suggest new tar- rs3825942 on XFS in South Africans [133]. This
get genes for further investigation in biological allelic reversal was further supported by a subse-
functions and exploration of underlying mecha- quent GWAS, in which the primary Japanese
nism of PACG pathophysiology. cohort showed the A allele of rs3825942 with
OR = 9.87 (P = 2.13 × 10−217), while the OR of
this allele in non-Japanese (Caucasian, African,
29.8 E
xfoliation Syndrome (XFS) Indian, and Chinese) is 0.49 (P = 2.35 × 10−31).
and Glaucoma Another locus, rs4926244, in CACNA1A was also
reported with significant association with XFS by
XFS is an age-related disease characterized by this GWAS. These findings implicated a complex
the deposition of abnormal amyloid-like fibers in mechanism of allelic effects at the LOXL1 locus
the extracellular matrix in different tissues pri- and other gene factors in the pathogenesis of XFS
marily in the eyes. Aggregates of pigments and [134]. Furthermore, a recent GWAS with a larger
fibrillar materials in the anterior segment tissues sample size including 13,838 cases and 110,275
such as ciliary body, trabecular meshwork inner controls from 24 countries across 6 continents
surface, and Schlemm’s canal would induce confirmed the previous associations and found 5
blockage of aqueous outflow and lead to increased more XFS loci (POMP, TMEM136, AGPAT1,
IOP, resulting in the development of secondary RBMS3, and near SEMA6A) [135]. These studies
glaucoma [119]. Epidemiology studies showed expanded the spectrum of gene candidates
that about one fourth of XFS patients had ele- involved in the biological pathways and pro-
vated IOP and 30% of them developed exfolia- cesses that contribute to XFS/XFG development.
366 S. Y. Lu et al.
29.9 Conclusive Remarks 8. Fan BJ, Leung DY, Wang DY, Gobeil S, Raymond V,
Tam PO, Lam DS, Pang CP. Novel myocilin mutation
in a Chinese family with juvenile-onset open-angle
In summary, a number of genes and loci were glaucoma. Arch Ophthalmol. 2006;124(1):102–6.
identified by genetic studies of glaucoma patients 9. Lam DS, Leung YF, Chua JK, Baum L, Fan DS,
in East Asia. Most of them are specific to only Choy KW, Pang CP. Truncations in the TIGR
gene in individuals with and without primary
one type of glaucoma, while several genes play open-angle glaucoma. Invest Ophthalmol Vis Sci.
roles in multiple disease forms, such as MYOC in 2000;41(6):1386–91.
POAG and ARHGEF12 and GAS7 in both late- 10. Leung YF, Baum L, Lam DS, Fan DS, Chua JK,
onset POAG and PACG (Fig. 29.1). This infor- Pang CP. Absence of trabecular meshwork-inducible
stretch response (TISR)/oculomedin gene and proxi-
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biological pathways to account for pathogenic coma patients. Hum Genet. 2000;107(4):404–5.
mechanisms in glaucoma. Better understanding 11. Nongpiur ME, Khor CC, Jia H, Cornes BK, Chen
of genetic and biological architecture of glau- LJ, et al. ABCC5, a gene that influences the anterior
chamber depth, is associated with primary angle clo-
coma is required for the development of early sure glaucoma. PLoS Genet. 2014;10(3):e1004089.
diagnosis and potential novel treatments for 12. Hysi PG, Cheng CY, Springelkamp H, Macgregor S,
glaucoma. Bailey JNC, Wojciechowski R, et al. Genome-wide
analysis of multi-ancestry cohorts identifies new loci
influencing intraocular pressure and susceptibility to
Compliance with Ethical Requirements Shiyao Lu,
glaucoma. Nat Genet. 2014;46(10):1126–30.
Clement C.Y. Tham, Pancy O.S. Tam, Shisong Rong,
13. Jeoung JW, Seong MW, Park SS, Kim DM, Kim
Calvin C.P. Pang, Guy L.J. Chen, and Wai Kit Chu declare
SH, Park KH. Mitochondrial DNA variant discov-
that they have no conflict of interest. No human or animal
ery in normal-tension glaucoma patients by next-
studies were performed by the authors for this article.
generation sequencing. Invest Ophthalmol Vis Sci.
2014;55(2):986–92.
14. Fan BJ, Tam PO, Choy KW, Wang DY, Lam DS,
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Quantitative Trait for Glaucoma
30
Sarangapani Sripriya, Ferdina Sharmila,
Suganya Kandeepan, and Ronnie George
Abstract Keywords
Dissecting the genetic architecture of complex Quantitative traits · Glaucoma ·
or multifactorial disorders always demanded Endophenotypes
different strategies in addition to the classical
gene mapping methods, due to the high level
of clinical and genetic heterogeneity. An endo- 30.1 Quantitative Traits or
phenotype or intermediate phenotype is a Endophenotypes
measurable trait with a strong genetic compo-
nent (determined by heritability and twin stud- An endophenotype, also known as intermediate
ies) which associates with a disease. phenotype or a quantitative trait, is a quantifiable
Endophenotypes for POAG include intraocu- biological trait that is heritable and influences the
lar pressure (IOP), central corneal thickness course of primary disease. These traits are con-
(CCT), cup area (CA), vertical cup-disc ratio sidered important in understanding the genetics
(VCDR) and disc area (DA). The loci fo of the primary disease as they contribute to the
these genetic determinants are called as quan- varying disease severity observed for a disease in
titative trait loci (QTLs). Identifying the QTLs question and thus could potentially explain the
for the clinical risk factors is considered as an heterogeneity in the disease presentation [1]. In
effective strategy for mapping the genes con- single gene disorders, whether they are dominant
tributing to the overall disease pathology. We or recessive, the phenotype is discrete and quali-
review the various ocular QTs and the insights tative, whereas, in complex diseases, the pheno-
gained into the pathogenesis of glaucoma type is heterogeneous which is accounted due to
through QTL mapping. shift in the quantifiable risk predisposing clinical
features. Many human quantitative trait loci
(QTL) have been shown to exhibit additive or
recessive inheritance patterns, indicating the con-
S. Sripriya · F. Sharmila · S. Kandeepan tribution from multiple genes (or alleles) for phe-
SNONGC Department of Genetics and Molecular notypic expression.
Biology, Vision Research Foundation, Chennai, India
Co-segregation at a single genetic locus or at
R. George (*) numerous interacting loci could exhibit a cumu-
Smt. Jadhavbai Nathamal Singhvee Glaucoma
lative effect and result in a clinical presentation.
Services, Medical Research Foundation,
Chennai, India Such additive effects on susceptibility to com-
e-mail: [email protected] plex traits are over-represented in communities

https://doi.org/10.1007/978-981-13-0884-0_30
374 S. Sripriya et al.
with higher frequency of consanguineous mar- of the disease. The search for glaucoma genes
riage because siblings in consanguineous fami- have been through linkage studies which focussed
lies would inherit two copies of the same allele or
on families affected by Mendelian forms of the
set of alleles, contributing to the trait. This could
disease. The loci mapped to POAG from GLCA
manifest as a greater variation in the magnitude to GLCP did not explain a significant proportion
of a quantitative trait because of the additive of the genetic basis for POAG. Out of the regions
effects of the allele(s). mapped, the MYOC, OPTN and WDR36 genes
These endophenotypes however individually were widely studied across various populations
exert only small effects but collectively result in
in family-based studies. MYOC-associated glau-
complex diseases. An overt expression of the coma accounted for 3–5% of POAG cases world-
combined effects of these factors which crosses awide with more than 100 reported mutations
hypothetical threshold manifests as a disease worldwide, some of which were population spe-
such as in schizophrenia, bipolar disorder, cific (Gln368Stop, Arg46Stop, Gln48His).
depression, etc. For example, deficits in working OPTN (GLC1E, 10p13), the other gene stud-
memory are known to increase the risk for schizo-ied more frequently across various populations,
phrenia and are considered as an endophenotype has a mutation frequency of 16.7% in hereditary
for the disease. At the molecular level, specificforms of normal tension glaucoma (NTG) and
alleles associated with the risk of dopaminergic 1–2% of POAG cases. Specific mutations like
dysregulation in the prefrontal cortex modulate E50K have been frequently observed in POAG
effect on working memory which subsequently patients and are also correlated with various clin-
increases the risk for schizophrenia. Thus identi-
ical features. POAG patients harbouring the
fication of quantitative traits and their geneticE50K mutation have an earlier age of onset, a
loci (called quantitative trait loci) is considered
more advanced optic nerve cupping and a more
as an alternative to traditional disease association
frequent need for surgical intervention [3]. In the
studies, especially in complex diseases. With Indian context, this mutation was not observed,
respect to neurological disorders, as listed above,
and we have reported the possible role of SNPs
endophenotypes are selected for further genetic rather than mutations in POAG patients. The
analysis when they exhibit specific properties [2].
other gene observed in <1% of normal tension
Some of the requisites are that the endopheno- POAG cases is TBK1 (GLC1P, 12q14) that codes
types should be heritable (heritability defines the
for serine/threonine kinase with a major role in
proportion of the phenotype variance due to the regulation of inflammatory responses [4].
genetic components) and play a causal role in theTBK1-OPTN protein is shown to mediate the
disorder, the effect size of a particular gene interaction between mitophagy and immune
should be greater in relation to the endopheno- response [5]. This has been shown to have a
type than to the clinical syndrome and they potential therapeutic implication for neurodegen-
should vary continuously in the general popula- erative diseases like glaucoma. Yet, these details
tion. Grouping patients based on the endopheno- were not sufficed to explain the pathology of
types is considered to be effective for mapping glaucoma.
the linked genetic loci and the genes involved. The WD repeat domain 36 (WDR36: GLC1G,
5q22) gene codes for a nucleolar protein impli-
cated in the maturation of 18 s rRNA. The
30.2 From Endophenotypes observed frequency of mutations ranges from
to Disease 1.6% to 17% of POAG patients [6] that was how-
ever not replicated in subsequent studies. Similar
Genetic predisposition has been one of the major to the observation of OPTN gene variants in the
risk factors for glaucoma, and the discovery of Indian population, the WDR36 variants rather
genes predisposing to glaucoma through family- than mutations were suggested to be associated
based studies has been the initial approach to with the risk of POAG. Mutations in NTF4 gene
understand the molecular events in the pathology (GLC1O, 19q13.33) contributed to 1.7% of
30 Quantitative Trait for Glaucoma 375
POAG patients of European origin [7] that has pressure are some of the quantitative traits of the
not been replicated in other populations (India, eye of which the optic disc parameters, CCT and
China). The other gene that was mapped in IOP are well studied for their association with
family-based studies and later replicated in case- POAG.
control studies is ASB10 (GLC1F, 7q36).
Besides, copy number variations in TBK1, Axial Length (AL) Axial length (AL) of the eye is
genomic duplications (in CDKN2B-AS1, near an important refractive parameter which repre-
GAS7 and TMCO1) and deletions (in SIX6 and sents the length between the anterior and posterior
ATOH7) have been reported in POAG cases. poles of the eye. It is determined by the combina-
As discussed above, the genes identified tion of anterior chamber depth, lens thickness and
through family-based studies and then replicated posterior chamber depth. AL increases most rap-
through case-control studies accounted to a fre- idly in infancy with growth slowing by adoles-
quency of 1–5% of POAG in the general popula- cence and remains constant in adulthood. A study
tion. The limitations in family based studies such on 148 normal eyes of 79 patients ranging from
as the unavailability of multiple members affected premature newborns to 36-year-old adults showed
large families, sample size and technological lim- that the mean AL measured 16.8 mm in a full-
itations restricted the understanding of the molec- term newborn baby and 23.6 mm in an adult [8].
ular pathology of POAG in earlier phase of This increase with age causes a shift in refraction
glaucoma research. Subsequent technological in the eye from hyperopia towards myopia. The
innovations, clinical phenotype standardisation final refractive state of the eye is a result of the
and recognising the need for a consortium-based combined changes in lens and corneal curvature.
approach gave a new dimension to the genetic A 1 mm elongation of AL without these compen-
studies in POAG. The utility of classical genetics sations is equivalent to a myopic shift
that shifted to genome-wide association studies (AL > 28 mm) of −2 or −2.5 dpt, thereby estab-
(GWAS) through case-control study designs lishing the fact that the components of the visual
combined with a consortium-based approach system are in close interaction with one another
proved effective in better understanding the during the entire maturation process. Heritability
genetics of POAG. In addition to disease associa- of axial length ranges from 0.89 to 0.94 [9].
tions, studies on the possible association of the
genetic variants with the various clinically mea-
surable risk factors for glaucoma (QTs or quanti- Intraocular Pressure (IOP) Aqueous humour
tative traits) are being studied across various formed by the ciliary body is drained primarily
population towards understanding of the glau- through the trabecular outflow pathways, which
coma genetics. Identification of genetic factors includes the trabecular meshwork, the juxtacan-
for QTs that are highly heritable with significant alicular connective tissue, the endothelial lining
variations amidst the general population (e.g. of Schlemm’s canal and the collecting channels
IOP, CCT, optic nerve parameters) is useful in and the aqueous veins [10]. IOP depends on the
mapping the genes contributing to the overall dis- rate at which the aqueous humour is drained out
ease pathology. This strategy based on endophe- and in normal cases the rate of production equals
notypes to map glaucoma genes is based on the rate of removal. When the rate of removal is
ranking individuals based on the variable risk less than the production, it starts to accumulate,
traits rather than as patients and controls. leading to increase in IOP. Thus elevated IOP is
the major risk factor of glaucoma which affects
the retinal ganglion cells leading to axonal
30.3 Ocular Quantitative Traits degeneration, abnormal optic nerve head appear-
ance and finally blindness. IOP shows circadian
Refraction parameters, anterior chamber depth, variations and fluctuation and remains as the
axial length, optic disc parameters, central cor- only modifiable and hence treatable risk factor
neal thickness, corneal curvature and intraocular for glaucoma. In the general population, elevated
IOP (over the upper normative limit of 21 mmHg) of the ratio was estimated as 0.48 according to
is not always accompanied by glaucomatous the parent-child correlation and 0.56 based on the
damage. While those with elevated IOP are more data from siblings of the population-based
likely to have glaucoma, glaucomatous changes Salisbury Eye Evaluation [19]. In a large twin
can occur with IOP in the physiological range study conducted in 1114 twins, inclusive of both
too (normal tension glaucoma). The heritability mono and dizygotic twins revealed an intraclass
estimates for IOP varies from 0.29 to 0.50. correlation coefficients of 0.79 for disc area, 0.83
Short- and long-term fluctuations in IOP, non for cup area and 0.80 for cup-disc area ratio in
genetic factors probably accounted for its lower MZ pairs and 0.30, 0.37 and 0.35, respectively, in
heritability [11]. DZ pairs. An additive genetic effect was also
observed for disc area, cup area and cup-disc area
Charlesworth et al. mapped the 10q22 as the ratio (77.3%, 82.7% and 78.6%, respectively)
locus for maximum IOP [12]. Interestingly the and thus concluded that 80% of these phenotype
same region has been associated with systemic variances are genetically determined.
hypertension in a Japanese population.
Expression QT analyses in UK twins identified a The loci associated with these parameters by
new locus FAM125B – a membrane complex GWAS-based studies included ATOH7, CDC7/
involved in vesicular trafficking process [13]. TGFBR3 and SALL1, CARD10 for the optic disc
Another GWAS in Australian cohort and later area, and CDKN2B, SIX1, SCYL1/LTBP3, SIX6,
replicated in a UK cohort showed evidence of CHEK2 and DCLK1, in addition to ATOH7, for
association with 7p21 near GLCCI1 and ICA1 the VCDR [20–24]. Of the genes listed above,
[14]. A study by van Koolwijk et al. revealed an ATOH7, CDKN2B and SIX1 are replicated for the
association with GAS7 and TMCO1 on chromo- association with POAG in other cohorts like the
somes 17p13.1 and 1q24.1, respectively [15]. Rotterdam Eye Study with the Twin UK study
GWAS in subjects of European ancestry also [25] with borderline associations for
identified significant associations with IOP at CDC7/TGFBR3 and SALL4 (both p = 0.04).
TMCO1 (rs7518099-G, p = 8.0 × 10−8) and with CARD10 was not found to be associated with
other common variants in multiple genomic African-Caribbean POAG cases [14], whereas
regions (CDKN2B-AS1, GAS7, CAV1/CAV2 CHEK2 was reported to be associated with
and SIX1/SIX6) in regulating IOP [16]. Other VCDR and high tension glaucoma among the
loci found to be associated with IOP so far Japanese [26] but not in Europeans [27]. Multiple
include FNDC3B, ABCA1, ABO, 11p11.2 and studies have provided strong evidence of associa-
ARHGEF12 [17, 18]. tion of ATOH7, CDKN2B(-AS1) and SIX1/SIX6
with POAG [14, 25–30].
Optic Disc Parameters Characteristic changes
on the optic nerve head are an important feature ATOH7, Atonal Homolog of Drosophila Gene
of glaucoma. Optic disc area and vertical cup- (10q21.3) ATOH is the atonal homolog of dro-
disc ratio (VCDR) are important quantitative sophila, named for its chordotonal stretch recep-
traits that are altered in glaucoma. VCDR have tor mutant phenotype [31]. It is a basic proneural
been a longstanding method of documenting disc helix-loop-helix (bHLH) transcription factor
change in glaucoma and can be influenced by the expressed in the early retinal progenitors. This has
optic disc size/area. In addition there exist racial been shown to be essential for the initiation of
variations in optic disc size. Familial aggregation neurogenesis and production of retinal ganglion
for cup-disc ratio was initially reported through cells (RGCs) [32]. Defects in the ATOH7 gene
extended family studies by Armaly et al., in 2016. affect the RGC number, fate and differentiation of
The correlation coefficient of vertical CDR was the other retinal cells [33, 34]. GWAS in European,
0.25 in siblings and 0.24 in parents and children, Australian, Caucasians and Indian populations
as reported by Beaver Dam Eye study. Heritability have shown association of rs1900004, rs3858145,
rs17231602 and rs4746741 with ODA [22–24, CARD10 Gene (22q 13.1) This protein is
30, 35]. Kamron et al. have documented that involved in the regulation of caspase activation
homozygous mutations (p.E49V and p. and apoptosis. It activates NF-kappaB, a well-
P18RfsX69) in this gene play a role in the devel- characterised transcription factor involved in sig-
opment of both the anterior and the posterior nalling pathway [24, 35]. Variations in this gene
chamber of the eye and are important in retinal have been proposed to influence the
vascular development and hyaloid regression ODA. Associations between CARD10 and ODA
[36]. [24] have been reported in Asian and Singaporean
cohort.
SIX1/SIX6 Gene (14q23) These homeoprotein

members belong to the SIX/sine oculis family Central Corneal Thickness Corneal biomechan-
of homeobox transcription factors. The ical properties are shown to influence intraocular
SIX1/SIX6 gene complex has been significantly pressure (IOP) measurements that include central
associated with POAG in several GWAS. It was corneal thickness (CCT) and corneal hysteresis
observed that SIX6 missense variants deregu- (CH), corneal resistance factor (CRF) and
lated its expression and affected its normal corneal-compensated intraocular pressure
function in controlling retinal progenitor cell (IOPcc). Studies like the Ocular Hypertension
proliferation during eye development, hence Treatment Study (OHTS) and European
influencing susceptibility to POAG [37]. Glaucoma Prevention Study have revealed that
Functionally this protein is involved in the regu- CCT is an important risk factor associated with
lation of retinal progenitor cell proliferation glaucoma. Familial correlation studies (Eskimos),
during eye development. Animal models have heritability study based on twin studies, have
shown retinal hypoplasia, reduction in eye size demonstrated strong heritability estimates for
and reduction in eye volume of the optic nerve CCT ranging from 0.6 to 0.7 till 0.95 among dif-
[38–40]. ferent ethnic groups [43]. Glaucoma progression
was shown to be twofold higher in the eyes with
thinner central corneas. In certain studies, CH
CDC7/TGFBR3 Gene (1p22) Transforming has been shown to be associated with the diagno-
growth factor (TGF) is a multifunctional cytosis and risk of progression of POAG. CCT could
kine which modulates the development of many be an independent risk factor for POAG apart
tissue and repair processes by binding to its from other factors; hence it has been proposed as
receptor TGFBR3 [37]. This gene has been impli- an intermediate trait or endophenotype for POAG
cated in POAG development by its association [44]. A recent study from the International
with VCDR (rs1063192) in GWAS. Functionally Glaucoma Genetics Consortium (IGGC) identi-
it is known to interact with CDKN2A, another fied 16 loci significantly associated with CCT
gene known to be associated with VCDR [45]. Pathway analyses suggested that collagen
[14, 40, 41]. and extracellular matrix pathways are important
regulators of CCT. Genes associated include
ZNF469, FOXO1 and FNDC3B [46], COL5A and
CDNK2B Gene (9p21) This gene is a member COL8A2 [47, 48] and AKAP13 [49] in both
of the family of cyclin-dependent kinase (CDK) Caucasian (GWAS by Gutenberg and Rotterdam
inhibitors which plays a role in cell cycle regula- study) and Asian cohorts [50]. Quantitative anal-
tion, influencing the proliferation/differentiation ysis of CCT and a subsequent analysis of POAG
balance. GWAS has shown association with revealed SNPs in two cell adhesion molecules,
VCDR in various populations [30, 42]. The pro- NTM and CNTNAP4, that may increase POAG
tein is involved in the pathogenesis of optic nerve susceptibility in a subset of cases [51]. Analysis
degeneration and POAG. of genes controlling CCT in multi-ethnic Indians
shows strong evidence of association at four ysis has limited value given that the disease may
novel loci: IBTK on chromosome 6q14.1, CHSY1 not be manifested in younger individuals.
on chromosome 15q26.3 and intergenic regions Identifying genetic biomarkers for disease-
on chromosomes 7q11.2 and 9p23 [49]. A study associated endophenotypes addresses these
in Caucasians identified a missense mutation in limitations.
COL8A1 associated with thin CCT [52].
Segmental chromosomal duplications or dele- Compliance with Ethical Requirements This article
tions encompassing FOXC1 were associated does not contain any studies with human participants per-
with increased CCT in families with early-onset formed by any of the authors. This article does not contain
any studies with animals performed by any of the authors.
glaucoma phenotypes [53]. Sarangapani Sripriya, Ferdina Sharmila, Suganya
Kandeepan and Ronnie George declare that they have no
conflict of interest.
Zinc Finger Nuclease (ZNF469) Gene
(16q24) The protein functions either as a nuclear
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Genetics of Exfoliation Syndrome
in Asians 31
Prakadeeswari Gopalakrishnan, Aravind Haripriya,
Banushree Ratukondla,
Abstract Keywords
Exfoliation syndrome (XFS) is common, age- XFS · SNP · Genetics · GWAS
related, and systemic microfibrillopathy. XFS
is also a major recognizable cause of second-
ary open-angle glaucoma and blindness Abbreviations
worldwide. It is considered to be a connective
tissue disorder of extracellular matrix (ECM) BIRC6 Baculoviral IAP repeat-
that targets the tissues of the anterior eye seg- containing 6
ment through abnormal production and exces- CACNA1A Calcium voltage-gated channel
sive accumulation of the fibrillary-flaky subunit alpha 1 A
white-grayish material. The etiopathogenesis CAD Coronary artery disease
of XFS is still obscure, ten decades of research CLU Clusterin
with XFS from the period of its first descrip- CNTNAP2 Contactin-associated protein-
tion. On the other hand, many explanations like 2
were made with both genetic and nongenetic ECM Extracellular matrix
factors, which are believed to be one among GST Glutathione S-transferase
the causal mechanisms for XFS. This chapter GWAS Genome-wide association study
provides evidence regarding the well-studied HLA Human leukocyte antigens
genetic, nongenetic, and other associated IncRNA Long noncoding RNA
risk factor-based argument which enriches IOP Intraocular pressure
our understanding of this complex inherited LOXL1 Lysyl oxidase-like 1
disorder for future research. LOXL1-AS1 LOXL1 antisense RNA
MMPs Matrix metalloproteinases
PEXS Pseudoexfoliation syndrome
P. Gopalakrishnan · P. Sundaresan (*)
Department of Genetics, Aravind Medical Research POAG Primary open-angle glaucoma
Foundation, Dr G. Venkataswamy Eye Research SNPs Single nucleotide polymor-
Institute, Madurai, India phisms
e-mail: [email protected] TM Trabecular meshwork
A. Haripriya TNF-α Tumor necrosis factor-α
Intraocular Lens and Cataract Clinic, Aravind Eye XFG Exfoliation glaucoma
Hospital, Madurai, India
XFM Exfoliation material
B. Ratukondla XFS Exfoliation syndrome
Aravind Eye Hospital, Madurai, India

https://doi.org/10.1007/978-981-13-0884-0_31
382 P. Gopalakrishnan et al.
31.1 Introduction rare disorder in which anterior layer of lens cap-

sule delaminates, appears to be thin fluttering
Exfoliation syndrome (XFS) is a late-onset and membrane in the anterior eye segment in glass-
conformational disorder of fibrillin, causing blowers exposed to intensely hot and open fires
progressive elastotic degeneration and perhaps [8, 9]. A Swiss ophthalmologist, Alfred Vogt,
associated systemically with cerebrovascular and called this exfoliation of lens capsule as “glau-
cardiovascular diseases [1]. It is a well-recognized coma capsulare (capsular glaucoma)” [10].
risk factor for secondary glaucoma and known as Further, Georgiana Dvorak-Theobald an ocular
a major reason for the irreversible blindness pathologist noticed the precipitates and accre-
worldwide. Ritch et al. have estimated that tions of those unknown molecules around the
around 60–70 million people worldwide get anterior eye segment and proposed the term
affected with XFS [2]. It is clinically character- called “pseudoexfoliation.” She distinguished it
ized by the deposition of the fibrillary-flaky from “true exfoliation” of the lens capsule by
white-grayish small dandruff-like material over pointing out their differences [11].
the different ocular tissues of the anterior
segment including anterior lens capsule, lens
zonules, iris, trabecular meshwork (TM), cornea, 31.2 Worldwide Epidemiology
ciliary body, and the lamina cribrosa of the optic of the Exfoliation Syndrome
nerve [3, 4]. This deposition of the exfoliation and Asian Perspective
material is not just limited to the eye which also
occurs in non-ocular tissues like blood vessels The prevalence of the XFS is more predomi-
and elastic connective tissues of the lung, kidney, nantly observed in elderly people after their
liver, gallbladder, and cerebral meninges [5]. fourth decade. However, the distribution of XFS
varies between the different ethnic populations
[12]. This wide-ranging frequency of the XFS is
31.1.1 Discovery of Exfoliation evidenced by many studies including India
Syndrome and Origin (South India), 3.8% [13]; India (Tamil Nadu),
of the Term 6.0% [14]; India (Central India), 0.95% [15];
“Pseudoexfoliation” India (Western India), 30% [16]; India (Andhra
Pradesh), 3.01% [17], 0.6% [18], and 10.1%
Over a hundred years ago, John G. Lindberg, [19]; Nepal (Gurungs), 8.2%; Nepal (Tamangs),
Finnish ophthalmologist, was first to describe the 0.3% [20]; Myanmar, 3.4% [21]; Kashmir,
clinical condition which is now known as exfolia- 26.32% [22]; Japan, 3.4% [23]; Republic of
tion syndrome (XFS) or pseudoexfoliation syn- China, 2.38% [24]; Hong Kong (Chinese), 0.4%
drome (PEXS). He observed and described it as [25]; Singapore (Chinese), 0.2% [26]; Singapore
grayish flakes and fringes at the pupillary border (Malay), 0.46% [27]; Singapore (Chinese), 2.1%;
and noted the strange material formed a mem- Singapore (Malay), 4.5%; and Singapore
brane on the anterior lens surface in senile cata- (Indian), 9.7% [28]; Australia (Blue Mountains),
ract patients which was published as a thesis at 2.3% [29]; Australia (Victoria), 0.98% [30];
the University of Helsinki in 1917 and translated Australia (Central Australia), 16.3% [31];
in English later [6]. He also elucidated that this Finland, 22.1% [32]; France, 5.5% [33]; Greece
new phenomenon was shown to be common in (Epirus), 24.3% [34]; Greece (Crete), 16.1%
both cataract patients and non-cataractous indi- [35]; Greenland (Eskimos), 4.5% [36]; Iceland,
viduals above 55 years, and it was observed in 10.7% [37]; Belgrade, 17.5% [38]; Jordan, 9.1%
50% of the individuals with glaucoma. XFS was [39]; Northern Jordan, 10.3% [40]; Iran, 13.1%
more prevalent with advancing age, which is an [41]; Pakistan, 6.45% [42]; Saudi Arabia, 3.5%
important decisive factor [7]. In 1922, Elschnig [43]; Nigeria, 2.7% [44]; Norway, 16.9% [45];
described “true exfoliation of the lens capsule,” a Urban South Africa, 1.4% (white population) and
31 Genetics of Exfoliation Syndrome in Asians 383
20% (black population) [46]; Turkey, 7.2% [47] nents of the elastic system and on ultrastructural
and 10.1% [48]; USA (Framingham), 1.8% [49]; indications from the developing XFM by degen-
and USA (Southeastern), 3.2% [50]. Overall, erating the elastic microfibrils [56]. Currently, the
these reports suggest that genetic and environ- various elements representing as the precursor
mental factors might have a major influence on molecule of the dysregulated cellular mechanism
the phenotypic expression of XFS. involved in XFS are poorly understood.
31.3 Etiology and Proposed 31.4 Pathology and Clinical

Pathophysiological Features of Exfoliation
Mechanism of Exfoliation Syndrome
Syndrome
Busacca et al. made the first histological observa-
The foremost pathological mechanism involved tion in XFM which suggested that the flaky mate-
in exfoliation syndrome is the deposition of the rials arose from the aqueous humor and may not
flaky grayish-white amyloid-like material on the be from lens capsules [57]. In contrast, electron
ocular tissues such as lens, pupillary borders, iris, microscopic analysis clearly suggested the
trabecular meshwork, ciliary body, cornea, and involvement of the lens capsule in XFS by the
lamina cribrosa toward the anterior segment of presence of XFM upon the lens capsule, anterior
the eye that bathed by aqueous humor [3, 5]. The equatorial regions, and inner half of the lens
gradual deposition of the exfoliation material known as central disk but not in lens epithelium
(XFM) impedes the aqueous humor flow by [58]. Other electron microscopic analysis showed
blocking the sieve natured layers of trabecular the XFM deposition on the posterior surface of
meshwork, Schlemm’s canal, ciliary muscles, the iris and ciliary body along with their internal
and uveal meshwork that drains out the aqueous limiting membranes. XFM deposition was also
humor through both conventional and noncon- observed deeply into the space between the epi-
ventional pathways [4, 51, 52]. This blocked or thelial cells of the iris, whereas their epithelial
misdirected aqueous humor outflow may lead to cells were normal with intact nuclei and cyto-
increased intraocular pressure (IOP) of the eye, plasm. In the iris, unusual pigmented granules
which subsequently ends with irreversible blind- were noticed outside the epithelial cells, while
ness leading to a clinical condition known as few cells got ruptured, and those ruptured cells
exfoliation glaucoma. contained the filaments of the XFM, which were
To understand the nature of the exfoliation also observed in stroma and surrounding blood
material, three different theories [4] were pro- vessels [59]. Lectin staining with XFS lens cap-
posed by using biochemical and immunohisto- sule, zonules, and iris suggested the complex car-
chemical approaches, including (1) amyloid bohydrate composition in XFM with O-linked
theory, even though XFM showed positive label- sialomucin-type and N-linked oligosaccharide
ling with a crude anti-amyloid A antiserum [53] chains [60]. Electron microscopic immunohisto-
and few observed XFS cases associated with pri- chemistry observations revealed that XFM mate-
mary amyloidosis [54] but lacked any conclusive rial from lenses, trabecular meshwork, bulbar
evidence. (2) Basement membrane theory was conjunctivas, and lid skin almost had similar
proposed due to the fact that production of XFM ultrastructure and identical immunoreactivity to
may disrupt the basement membrane metabolism the antibodies including vitronectin, fibronectin,
which was also supported by frequent association laminin, and elastin suggesting XFS to be a sys-
of XFM and defective basement membrane of temic disorder [61].
various cell types [55]. (3) Elastic microfibril the- The preoperative corneal endothelial cell den-
ory was proposed based on the frequent structural sity was reduced in XFS patients compared with
association of the exfoliation fibers with compo- age-matched controls, whereas the cataract sur-
gery in XFS patient also showed a similar induced scan of XFS in Finnish family had demonstrated
endothelial cell changes without increase in an autosomal dominant mode of inheritance with
endothelial cell loss during postoperative period incomplete penetrance [71]. In a population-
[62]. In the early 2000s, Inoue et al. also reported based Finnish Twin Cohort Study for determin-
the significant reduction of corneal endothelial ing the inheritance of XFS, 34 XFS patients were
cell density and thinner central cornea in XFS analyzed, but the heritability of XFS was not pos-
eyes than in non-XFS eyes [63]. The corneal sible to assess by the classical twin method due to
subbasal nerve plexus variables, endothelial cell smaller number of twin pairs [72]. XFS perhaps
densities, and mean anterior and posterior is a mitochondrial disorder because of the mater-
stromal keratocyte were significantly lower in nal transmission tendency, 99.9% of the mito-
XFS subjects, while their basal epithelial cell chondria inherited from the mother [73]. Reduced
density was normal [64]. mitochondrial numbers was revealed in the iris
The other clinical manifestations related to sphincter muscles of XFS patients through elec-
XFS are (a) iris depigmentation which leads to tron microscopic analysis [74]. But it demands
pupillary transillumination defects, (b) mild further studies to provide definitive proof for
hyperpigmentation of the trabecular meshwork, mitochondrial inheritance.
and (c) zonular dehiscence which causes lens
subluxation and loss of the zonular support which
makes cataract surgeries more challenging with 31.6 Clinical and Systemic
vitreous loss and lens dislocation [65]. Complications Associated
with Exfoliation Syndrome
31.5 G
enetic Inheritance Pattern The subjects with XFS have been suggested to be
of Exfoliation Syndrome strongly susceptible to cardiovascular risk [75]
and cerebrovascular complications [76], and it
Being a late-onset disorder, determining the had always been a matter of controversy. XFS
inheritance pattern is a challenging task as patients are suggested to be screened for coronary
genetic analysis with more than two generations artery disease (CAD), irrespective of the age and
is barely possible due to mortality. Despite of sex [77]. A meta-analysis study has elucidated
these complications, studies with human leuko- that XFS has been associated with an increased
cyte antigens (HLA) in XFS support the genetic risk of vascular disease [78]. Although, additional
predisposition for the development of XFS. A data should be acquired for the key correlations,
study from Irish subjects showed the association an increased incidence of cardiovascular disor-
of the 14 HLA antigens (HLA-Al, A33, B8, B47, ders in XFS patients and several other common
B51, B53, B57, B62, DR3, DR12, and DR13) features in the pathogenesis suggests that XFS
with exfoliation syndrome [66]. A study from may be an independent risk factor for cardiovas-
Swedish XFS patients reported a significant asso- cular disease or it may occur as a part of systemic
ciation with HLA Bw35 compared to primary disorders with cardiovascular implications [79]. It
open-angle glaucoma (POAG) patients [67]. In is difficult to obtain the accurate causality infor-
contrast, Norwegian patients did not show asso- mation about XFS, glaucoma, and CAD, which of
ciation with HLA-A and HLA-B compared to the three occurred first [80]. Recently, a meta-
POAG subjects [68]. XFS was known to be a analysis by Siordia et al. has observed the associa-
familial condition, and the transmission to the tion between XFS and ischemic heart disease,
second generation was through an affected while there was no correlation found with myo-
mother in Icelandic families [69]. XFS was cardial infarction, chronic ischemic heart disease,
observed to be transmitted as a late-onset autoso- angina, and hypertension with XFS [81]. However,
mal dominant trait without a maternal transmis- other studies did not show a clear association
sion in three Gozo families [70]. Genome-wide between XFS and ischemic heart disease, arterial
hypertension and diabetes mellitus [82, 83] and (SNPs) in the candidate genes such as lysyl
aortic aneurysm, and peripheral artery disease oxidase-like 1 (LOXL1), LOXL1 antisense RNA
[84]. A study has reported that the prevalence of (LOXL1-AS1), calcium voltage-gated channel
XFS in diabetes patients was shown to be less subunit alpha1 A (CACNA1A), and contactin-
than nondiabetic patients [85]. Further, studies associated protein-like 2 (CNTNAP2). Genes of
also showed that XFS or XFG (exfoliation glau- cellular pathways implicated in XFS are homo-
coma) is not correlated to mortality when either of cysteine metabolism genes, adenosine receptor
them represented with cardiovascular and cere- A3, matrix metalloproteinases (MMPs), glutathi-
brovascular complications [86, 87]. Overall, one S-transferase (GST), clusterin (CLU), and
reports are still contradictory regarding the sig- tumor necrosis factor (TNF)-α. The nongenetic
nificant correlation with XFS and systemic disor- factors include sun exposure [88, 89], geographi-
ders. In our study, the prevalence of hypertension, cal history [90], consumption of caffeine, and
coronary artery heart disease, and diabetes melli- low folate intake [91].
tus was statistically higher in XFS patients when
compared to the control group (Table 31.1). This
significant difference in the prevalence of the sys- 31.7.1 Lysyl Oxidase–Like 1 (LOXL1)
temic complications suggests the strong associa-
tion with XFS. However, further studies are Thorleifsson et al. performed the first genome-
necessary to explore and understand the underly- wide association study (GWAS) with 274 XFG
ing mechanism. cases/14,672 controls and identified three major
single nucleotide polymorphisms (SNPs) in
LOXL1 gene on chromosome 15, which was
31.7 Genetic and Nongenetic strongly associated with XFS and XFG [92]. The
Factors of Exfoliation two coding SNPs rs1048661 (G/T; Arg141Leu)
Syndrome and rs3825942 (A/G; Gly153Asp) were present
in the exon 1 of the LOXL1, while the other SNP
Both genetic and nongenetic factors play a role in rs2165241 (C/T) was located near the intron 1 of
the development of XFS. The genetic factors LOXL1. These two coding variants were observed
include the single nucleotide polymorphisms in 98% of the XFS cases but were also observed
Table 31.1 Systemic complications associated with XFS patients and cataract controls (CC)
Systemic complications XFS CC P-value Odds ratio (95% CI)
Electrocardiogram n (%) n (%) 0.145
Normal ECG 691(86.9) 378(89.8) 1.32 (0.91–1.93)
Abnormal ECG 104(13.1) 43(10.2)
Total 795(100.0) 421(100.0)
Hypertension n (%) n (%) 0.013
HT 215(25.2) 84(19.1) 1.43 (1.08–1.90)
Non-HT 637(74.8) 357(80.9)
Total 852(100.0) 441(100.0)
CAHD* n (%) n (%) 0.008
CAHD 42(4.9) 8(1.8) 2.81 (1.31–6.04)
Non-CAHD 809(95.1) 433(98.2)
Total 851(100.0) 441(100.0)
Diabetes mellitus n (%) n (%) 0.033
DM 158(18.5) 61(13.8) 1.42 (1.03–1.95)
Non-DM 694(81.5) 380(86.2)
Total 852(100.0) 441(100.0)
XFS exfoliation syndrome, CC cataract control, *CAHD coronary artery heart disease
in 85% of the unaffected individuals. The LOXL1 31.7.2 LOXL1 Antisense RNA
is a member of the lysyl oxidase family of pro- (LOXL1–AS1)
teins with five members LOX, LOXL1, LOXL2,
LOXL3, and LOXL4. This LOXL1 is a copper- Hauser et al. showed many risk variants at LOXL1
dependent monoamine that is actively involved in exon 1/intron 1 which are strongly associated
the covalent cross-linking of the elastin and col- with XFS in four different ethnic populations;
lagen polymers during extracellular matrix for- some were reversed in one or more populations.
mation and remodeling [93]. LOXL1 knockout The intron 1 region comprises of a promoter for
mice showed a distinctive phenotype such as the long noncoding RNA (lncRNA) LOXL1-AS1,
reduced elastic fiber formation, impaired connec- which is on the opposite strand of LOXL1. The
tive tissue repair, pelvic organ prolapses, skin expression of LOXL1-AS1 was affected due to
laxity, enlarged lung airspaces, impaired blood- XFS-related cell stressors, including oxidative
aqueous barrier, and lens abnormalities with cat- and mechanical stress. Remarkably, a haplotype
aract formation [94–98]. of risk alleles for three intronic SNPs is shown to
The association of the two coding variants be reducing the promoter activity of LOXL1-AS1
rs1048661 (Arg141Leu) and rs3825942 by 43% in lens epithelial [103]. As lncRNAs
(Gly153Asp) was globally replicated in all the transcription is sufficient to regulate gene expres-
populations worldwide [99]. The risk allele for sion either positively or negatively at both neigh-
these coding SNPs showed a reversal risk allelic boring and distant loci [104], now the altered
association in one or more populations [100]. LOXL1-AS1 expression may affect expression of
The variant rs1048661 (Arg141) showed multiple other XFS-related genes located
decreased risk in Asian cohorts, while the throughout the genome. Thus, it suggests an
increased risk was observed in all other popula- interesting possibility that dysregulated expres-
tions. Likewise, the variant rs3825942 (Gly153) sion of the LOXL1-AS1 lncRNA may play a role
showed decreased risk in South Africans, whereas in the pathogenesis of XFS and XFG.
increased risk was seen in all other populations
studied. The functional role of these variants in
the pathogenesis of the disease is still obscure, 31.7.3 Calcium Voltage–Gated
but these variants are expected to be in linkage Channel Subunit Alpha1
disequilibrium with any unknown functional A (CACNA1A)
variant, which should be answered by further
genetic analysis. A multicentered GWAS reported the association
Recently, epigenetic regulation of gene between the CACNA1A locus and XFS suscepti-
expression in the LOXL1 locus has been explored bility with 7000 XFS cases and 20,000 controls
in Uygur population which identified hypermeth- from 17 countries [105]. The associated variant
ylation of CpG islands in the LOXL1 promoter rs4926244 that lies in an intronic region near the
region in anterior lens capsule specimens from 3′ end of the CACNA1A was first observed in
XFS patients compared to controls. LOXL1 Japanese population then replicated as multi-
mRNA levels were reduced in the XFS lens cap- centered GWAS analysis. The risk allele for
sules, suggesting that hypermethylation down- rs4926244 was shown to be consistent across all
regulates gene expression at this locus [101]. major populations, with an allele frequency
Additionally, immunohistochemical analysis ranges between 10 and 40%. The in silico analy-
demonstrated the significant expression of sis suggests that the risk allele for rs4926244 is
LOXL1 and fibrillin-1 (FBN-1) toward outer sur- associated with the reduced CACNA1A mRNA
face of the lens capsule. Significant expression of expression levels in peripheral blood cells [105].
LOXL1 in lens epithelial cell nuclei was In accordance with this, another study has dem-
observed, whereas the cytoplasmic expression of onstrated a moderate mRNA expression of
LOXL1 was lower in XFS patients [102]. CACNA1A in the trabecular meshwork, lens cap-
sule, and retina, as well as a low expression in the an essential role in the functional decay of tra-
cornea, iris, ciliary body, and choroid by compar- becular meshwork [119], while another study did
ing 20 XFS eyes with 20 control eye; however, no not show any association with the mitochondrial
significant difference could be established [106]. DNA [120]. Recently, Want et al. reported that
CACNA1A encodes the α1A subunit of the the Tenon fibroblast cells of XFS display features
type P/Q voltage-dependent calcium channel on associated with autophagy and mitochondrial
chromosome 19. Calcium channels are said to be dysfunction [121].
responsible for the transport of calcium ions
across cell membranes and play a vital role in a
cell’s ability to generate and transmit electrical 31.8 Summary
signals. An electron microscopy studies on
human XFS eyes showed the presence of high Molecular genetic approaches stand to be a
calcium concentrations in direct association with powerful tool to unravel the genetic basis of
aggregating XFS fibrils [107]. Thus, it can be any disease. Candidate gene studies are neces-
postulated that the dysregulated function of a cal- sary to unlock the heritable locks involved in
cium channel could lead to alterations in calcium the pathogenesis of the complex genetic disor-
concentrations, which may result in the forma- ders including exfoliation syndrome. Numerous
tion of XFS aggregates [105]. research efforts like multicentered genome-
Recently, the expanded GWAS on XFS from wide association studies have been carried out
24 countries followed by the replication in 18 to understand the complicated mechanism
countries identified the genetic association of a involved in the exfoliation syndrome. The
rare protective allele at LOXL1 (rs201011613: future research should be focused to underpin
A/T; p.Phe407) and 5 new genome-wide signifi- the major functional component which is acting
cant susceptibility loci (i) rs7329408 (A/G): at the background of the all well-studied high-
POMP:13q12, (ii) rs11827818 (G/A): TMEM136: risk candidate genes associated with exfoliation
11q23.3, (iii) rs3130283 (A/C): AGPAT1: 6p21, syndrome.
(iv) rs12490863 (A/G): RBMS3: 3p24, and (v)
rs10072088 (G/A): SEMA6A:5q23 as an addi- Compliance with Ethical Requirements Prakadeeswari
tional clarification for the genetic basis of XFS Gopalakrishnan, Aravind Haripriya, Banushree
Ratukondla, and Periasamy Sundaresan declare that they
[108]. have no conflict of interest. No human or animal studies
were performed by the authors for this article.
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Proteomics of Neurodegenerative
Disorders of the Eye 32
Kim Ramasamy, Krishnadas Ramasamy,
Dharmalingam Kuppamuthu,
and Jeya Maheshwari Jayapal
Abstract Keywords
Neurodegenerative eye diseases refer to those Neurodegeneration · Age-related macular
ocular conditions where the retina is affected degeneration · Diabetic retinopathy ·
and include pathologies such as diabetic reti- Glaucoma · Proteomics
nopathy, age-related macular degeneration,
and glaucoma. All these three diseases are the
leading causes of irreversible blindness. 32.1 Introduction
Although they affect different parts of the ret-
ina, the death of retinal cells that is responsi- Diabetic retinopathy, age-related macular degen-
ble for vision loss is the common unifying eration, and glaucoma are the three major degen-
factor of the three conditions. These diseases erative diseases of the retina that causes
are generally multifactorial, relatively asymp- irreversible blindness. All the three diseases have
tomatic, and progressive. Proteomics of the been included in the list of priority eye diseases
tissues and fluids of the posterior compart- of the World Health Organization as they have
ment of the eye has advanced our understand- emerged as potential threats, particularly for the
ing on the pathological mechanisms middle-aged and the ageing population of many
underlying all three conditions. These studies middle-income and industrialized countries.
have also contributed to the identification of Age-related macular degeneration (AMD) ranks
candidate biomarkers that upon validation third among the global causes of visual impairment
would be helpful in the early diagnosis of and is the primary cause of legal blindness in many
these diseases. countries. It is a chronic and progressive disease of
the central retina of the elderly population. There
are two subgroups of AMD, atrophic (dry form) and
K. Ramasamy exudative (wet form). In most patients, the AMD
Retina-Vitreous Services, Aravind Eye Hospital, onsets as the dry form, which can advance either to
Madurai, India the late dry AMD (geographic atrophy) or linked to
K. Ramasamy choroidal neovascularization (CNV) resulting in
Glaucoma Services, Aravind Eye Hospital, wet AMD. Geographic atrophy leads to progres-
Madurai, India
sive vision loss, with a decrease in dark-adapted
D. Kuppamuthu · J. M. Jayapal (*) function and a significant drop in acuity. On the
Department of Proteomics, Aravind Medical
Research Foundation, Madurai, India other hand, neovascular AMD causes sudden and
e-mail: [email protected] severe vision loss.

https://doi.org/10.1007/978-981-13-0884-0_32
394 K. Ramasamy et al.
The increase in diabetes among many popu- Table 32.1 Comparison of the prevalence of AMD, DR,
and glaucoma worldwide and in Asian population
lation groups has caused diabetic retinopathy
(DR) to be added to the WHO’s priority eye dis- Prevalence (%)
eases. DR is a microvascular complication that Global Asian
Age-related macular degeneration [63]
develops in both type 1 and type 2 diabetics.
Early AMD 8.01 6.8
Beyond 20 years of diabetes, nearly all type 1
Late AMD 0.37 0.37
and 50–80% of type 2 diabetic patients have Any AMD 8.69 7.38
some form of DR [34]. The early stage of DR is Glaucoma [6]
non-proliferative (NPDR), which progresses POAG 3.05 2.31
from mild to moderate to severe form. A subset PACG 0.5 0.73
of NPDR patients progress further to the Any glaucoma 3.54 3.54
advanced stage characterized by neovascular- Diabetic Retinopathy [38, 54]
ization called proliferative diabetic retinopathy Among type 1 diabetics 77.3 5.3–15.1
(PDR). At any stage of DR, patients may also Among type 2 diabetics 25.2 12.1–23.0
develop macular edema, which is exudation and
accumulation of fluids in the macular region.
Both DME and PDR are associated with poor 32.3 Etiology
visual outcomes.
Glaucoma has remained a public health con- The exact cause(s) for any of these three neurode-
cern for long due to difficulties in its early diag- generative diseases have not been clearly defined,
nosis combined with a need for lifelong but many studies over the years have implied
treatment. Glaucoma is asymptomatic in the various factors as probable causes. AMD, DR,
early stages, and the disease advances with sub- and glaucoma are all multifactorial diseases, and
stantial neural damage. When symptoms do many factors (such as genetic, environmental) do
occur, there is already a significant reduction in have a role in the onset of the disease.
the visual field that greatly affects the quality of The hallmark of AMD is the appearance of
life. Glaucomas can be classified as open-angle drusen, the extracellular deposits that accumulate
glaucoma or angle-closure glaucoma. Both between the basement membrane of the retinal
these types of glaucomas can be primary dis- pigment epithelium (RPE) and Bruch’s mem-
eases with characteristic optic neuropathy and a brane. Basal linear deposits between the RPE
normal or elevated IOP with no other specific basement membrane and the inner collagenous
pathological cause. Secondary glaucoma refers zone of Bruch’s membrane is a specific marker
to an increased IOP with an identifiable patho- for early AMD [51]. These deposits lead to pro-
logical cause, such as trauma, certain medica- gressive defects in macular functions.
tions like corticosteroids, inflammation, tumor, Accumulation of RPE lipofuscin that appears as
pigment dispersion, or pseudoexfoliation. As diffuse and irregular patches of autofluorescence
primary open-angle glaucoma (POAG) and pri- is the earliest sign of AMD. Confluent areas of
mary angle-closure glaucoma (PACG) account RPE cell death with a concomitant atrophy of the
for the majority of glaucomas, these two types overlying photoreceptors are characteristic of
will be discussed here. late stage of dry AMD, also called geographic
atrophy. In wet AMD, hypoxic-driven VEGF
secretion induces choroidal neovascularization
32.2 Epidemiology: Global that can lead to subretinal or sub-RPE
and Asian hemorrhage.
Important risk factors in the development of
The prevalence of the three ocular diseases in the DR are long duration of diabetes, poorly con-
global scenario as well as with specific reference trolled hyperglycemia, and hypertension. Chronic
to the Asian population is shown in Table 32.1. hyperglycemia induces changes in both the
32 Proteomics of Neurodegenerative Disorders of the Eye 395
n eural retina and retinal vasculature. Apoptosis presence of macrophages in areas where the
of RGCs and activation of glial cells result in a Bruch’s membrane is thin or breached [30]. The
local inflammatory response in the neural retina. growth of choroidal blood vessels through the
Concomitantly, changes in the vascular endothe- Bruch’s membrane into the RPE or subretinal
lium result in increased leukostasis, an early space can lead to hemorrhage resulting in severe
event that leads to occlusions in blood vessels. vision loss.
Many factors, particularly the dysregulation of The early stage of DR is characterized by reti-
insulin signaling and formation of advanced gly- nal vascular changes such as thickening of the
cation end products, have a role in the initiation basement membrane and loss of pericytes.
of changes in the retina. Hyperglycemia-induced changes in the retinal
Glaucoma has been conventionally thought to cells include activation of glial cells, which along
be due to high intraocular pressure (IOP). Although with RPE have important roles in early microvas-
many individuals with high IOP may not develop cular impairment. Progression of NPDR is char-
glaucoma, increase in IOP is considered as a risk acterized by severe endothelial damage, which
factor. In open-angle glaucoma patients, there is an together with vasoconstriction and capillary
increase in resistance to aqueous humor outflow occlusion results in pathological angiogenesis. In
through the trabecular meshwork (TM), while in the proliferative stage, severe hypoxia leads to an
angle-closure patients, the access to the drainage increase in the pro-angiogenic factors resulting in
through TM is obstructed by the iris. PACG is a neovascularization. These new vessels can grow
more aggressive form of glaucoma and is the dom- into the vitreous body, and since they are fragile,
inant form among Asian population [17]. Disorders they have a tendency to rupture and leak blood
of the iris, the lens, and retrolenticular structures into the vitreous humor.
can cause PACG. Non-pupil block mechanisms Glaucoma is a heterogeneous group of dis-
have been shown to be responsible for angle clo- eases, and the pathophysiology of glaucoma is
sure in Asian patients [50]. believed to be multifactorial. Many theories of
glaucoma progression have been proposed and
are reviewed by Davis et al. [8]. According to the
32.4 Pathology mechanical theory, increased IOP might lead to
the compression of the nerve fiber bundle causing
A number of basic and clinical research studies in a discontinuity in the axonal transport of neuro-
the last few decades have identified multiple tropic factors resulting in apoptotic death of
pathological events underlying the neurodegen- RGCs. Yet another ischemic theory of glaucoma
erative eye diseases. Specific hallmarks charac- progression suggests that hemodynamic altera-
terize the stages of the disease as they progress tions independently or in conjugation with raised
and are discussed here briefly. IOP result in optic nerve head damage. And,
AMD affects primarily the RPE, photorecep- mitochondrial dysfunction and RGC axonal
tors, and Bruch’s membrane. In the nonexudative transport dysregulation activate a series of events
form, the early stage is characterized by the culminating in RGC cell death. Loss of RGCs by
appearance of basal laminar deposits (BLamD) apoptotic cell death leads to a decrease in the
that increase with AMD progression, and the late, function of these cells that is considered to be
amorphous form of BLamD correlates with the responsible for loss of visual field.
severity of RPE degeneration [51]. Analysis of
the drusen components revealed the presence of a
large number of molecular constituents including 32.5 Clinical Features
proteins such as immunoglobulins, complement
components, and other proteins implying the role AMD, DR, and glaucoma are progressive diseases
of immunological and inflammatory process in that are asymptomatic in the early stages. Their
AMD pathology. This is further supported by the diagnosis is dependent on the clinical features.
Patients in the early stages of AMD clinically areas. The retina is the ocular tissue that is pri-
exhibit focal thickening of Bruch’s membrane marily affected in all these three diseases.
along with areas of hypo- and hyperpigmentation Comparative analysis of the retina or vitreous
in the RPE. Patients who progress to geographic humor (VH) from the patients and healthy donors
atrophy exhibit a sharp demarcated area of depig- allows the identification of disease-specific pro-
mentation indicative of RPE atrophy. And, those teome alterations. Proteins altered in the early
who develop neovascular AMD have RPE atro- stages might represent the causative mechanisms,
phy with subretinal or intraretinal hemorrhage. while those identified in the late stages could pos-
Neovascular AMD patients develop rapid vision sibly be due to the accumulated effect or second-
loss, while it is slower over a period of many ary consequences.
years in geographic atrophy. Proteome analysis of RPE, Bruch’s mem-
The earliest detectable clinical features of DR brane, and choroid-RPE complex has identified a
are the presence of microaneurysms and dot number of factors involved in AMD pathology.
intraretinal hemorrhages, both of which increase Many studies have shown the involvement of
in number as the disease progresses along with complement cascade, inflammation, and oxida-
the additional appearance of cotton wool spots. tive stress in AMD [13, 44, 67]. Dysregulation of
Advancement to PDR stage is clinically pre- the complement system activation is an estab-
sented with the formation of new blood vessels lished pathological component of AMD. Genetic
that upon rupture can leak blood into the vitre- variations in the complement genes, namely, the
ous. Additionally, blood vessels can grow into the complement factor H [12], the central comple-
vitreous body resulting in the detachment of the ment component C3, and factors B and C2 [20],
retina. Both vitreous hemorrhage and retinal have been associated with the AMD susceptibil-
detachment are sight-threatening conditions in ity. An increase in the level of the complement
DR. pathway proteins has been reported in the AH
Distinct and characteristic changes appear in [66], VH [36], RPE cells, and choroid-RPE com-
the optic nerve head and retinal nerve fiber due to plex [55] of AMD patients. Further, complement
RGC cell death and loss of optic nerve fiber. proteins are detected in the drusen deposits and
These changes can be identified during ophthal- Bruch’s membrane of AMD patients [67]. The
moscopic examination of the optic nerve head. In complement C3 and complement regulator, vitro-
glaucoma patients, there is a progressive deterio- nectin, was reported to be elevated in wet AMD
ration of visual fields, with an initial loss in the tissues but not in the advanced dry AMD tissues
peripheral vision that progresses in a centripetal [67].
manner until only the central vision remains. AMD affects both the macula and the periph-
ery of the retina. Evidence for this comes from
the study by Ethen and group [13] who
32.6 Proteomics demonstrated that 60% of the altered proteins are
of Neurodegenerative Eye specific to either the macula or periphery.
Diseases Inflammation-related proteins are elevated in the
foveal or macular region, while bestrophin 1,
32.6.1 Understanding rhodopsin, ras homolog family member A, and
the Pathological Mechanisms ras homolog family member C are the proteins
found in high levels in the periphery.
A large number of studies in the last two decades Proteomics have been helpful in identifying
have immensely contributed to our understanding the stage-specific changes in the retina during
of the molecular events underlying these dis- AMD progression. Yuan et al. [67] established a
eases, and proteomic studies have a major contri- database of 901 proteins in macular Bruch’s
bution. Here, we discuss primarily how proteomic membrane/choroid complex of AMD and normal
analysis have advanced our knowledge in these donors. Complement proteins, DAMPs, and
other immune response and host defense proteins stages has been valuable to understand the early
were found to be increased in AMD suggesting changes leading to neurodegeneration of the ret-
the involvement of inflammatory processes in ina. A 2D-based proteomic study on diabetic
both initiation as well as progression of the dis- human donor RPE identified the molecular
ease. RPE cells perform a range of complex func- changes associated with diabetes prior to the
tions that are important for proper visual function. onset of DR [9]. Majority of the proteins altered
Loss of function of RPE cells are the initial events in diabetic RPE were involved in metabolism,
in atrophic AMD ultimately leading to death of chaperone function, protein degradation, synthe-
these cells. Nordgaard and group [44] identified sis and transport, oxidoreductases, cytoskeletal
the changes in the RPE proteome of human donor structure, and retinoid metabolism. Many of
eyes across four progressive stages of these proteins have been reported to be altered
AMD. Impaired stress response and mitochon- during diabetes in non-ocular tissues, and this is
drial biogenesis were observed in the early stages the first report on their alteration in the retina.
of AMD. Proteins involved in apoptotic signaling Proteomics of the human retina from DR donor
pathways were altered as the disease progressed eyes is quite limited. Most of the studies on reti-
and are of significant concern as RPE cells have nal proteome changes have been carried out using
limited ability to renew. αA and αB crystallin lev- animal models. Rat is a widely accepted model as
els were found to be increased indicating a cel- they exhibit histological changes in the early
lular response to oxidative stress [13]. As four stages of diabetes similar to that observed in
retinoid processing proteins were uniquely ele- humans. Quin and group [48] established a 2D
vated in early- or mid-stage AMD, retinoids and proteome map of the rat retina to study the
lipofuscin might be presumed to play a role in proteome- wide changes induced by early dia-
AMD initiation. Proteins involved in cellular betic retinopathy. Proteins were differentially
growth and proliferation, hematological disease, expressed as well as posttranslationally modified.
and cell morphology were found to be particu- Some proteins such as HSPs 70.1A and 8, and
larly altered in the early or mid-stage of the dis- platelet-activating factor, were uniquely
ease [67]. Late-stage changes were observed in expressed in diabetic retina, while beta-catenin,
proteins that regulate retinoic acid and regenera- phosducin, aldehyde reductase, succinyl-CoA
tion of the rhodopsin chromophore. Distinct sets ligase, and dihydropyrimidase-related protein
of proteins are altered in advanced dry AMD and were differentially regulated. Ya-Dong and group
neovascular AMD implying the differences in the [64] also published a similar proteome-wide
mechanisms of progression. Galectin-3, the study on rat retina. This study identified numer-
advanced glycation end product receptor 3, is the ous proteins including αA-crystallin,
most significantly elevated in more than 80% of glyceraldehyde-3-phosphate dehydrogenase, and
advanced dry AMD samples, while neutrophil glutamine synthetase to be differentially
α-defensins 1–3 are the most abundant in neovas- expressed in the rat neural retina suggesting that
cular AMD. Both forms of advanced AMD sam- diabetes induces complex changes in the retina.
ples showed increases in metalloproteinase Quin’s group induced DM in rats using strepto-
inhibitor 3 and S100-A9 level. Cellular compro- zotocin that creates an insulin deficiency, and
mise, drug metabolism, and molecular transport thus, the retinal proteome reflects the changes as
proteins decreased in geographic atrophy, in type 1 diabetes. On the other hand, Wang’s
whereas cell cycle, tissue development, and cel- group generated a type 2 diabetes model by feed-
lular development-related proteins were at lower ing rats with high-fat diet followed by a low-dose
levels in neovascular AMD [67]. injection of streptozotocin that creates only insu-
In diabetic retinopathy, changes in the neural lin resistance, and not deficiency. Altered expres-
retina occur even before the onset of clinical sion of crystallins was also reported in diabetic
symptoms in the retinal vasculature. Examining rat retina by Fort et al. [14]. They showed
the retinal proteome in the diabetic and early DR increased expression of the isoforms of α-, β-,
and γ-crystallin, and their 2D-DIGE approach levels (an indicator of cell damage), implying a
was helpful in identifying that these crystallins role for IRBP in neurodegeneration. Two studies
undergo multiple posttranslational modifications specifically examined changes in the DR vitreous
during diabetes. The authors also demonstrated with special reference to diabetic macular edema,
that γ-crystallins and β-crystallins were upregu- as this is a risk factor for vision loss in DR
lated in the ganglion cells and inner retinal neu- patients. These studies show increase in hemo-
rons, respectively. As the upregulation of these pexin, PEDF, ApoA4, ApoH, Trip-II, PRBP, and
crystallins coincided with the onset of DR symp- VDBP and decrease in clusterin, transthyretin,
toms, these proteins might have a role in vascular and crystallin-S, and these changes might be
remodeling in diabetes. A little later, VanGuilder DME specific [23, 45].
et al. [61] used a multimodal proteomic approach Proteome analysis of the retina from glauco-
to achieve an in-depth coverage of the retinal pro- matous human donor eyes as well as animal mod-
teome that also validated the protein alterations els provides an insight into glaucoma-induced
reported earlier. However, the authors did not alterations. Tezel and group published a series of
find any difference in VEGF expression, which is finding, all based on the proteomics of glaucoma-
contradictory to the already reported increase in tous retina. Initially, through gel-based analysis,
VEGF expression. Ly and group [43] studied they identified the proteins that were altered as
retinal membrane proteome of 10-week-old dia- well as posttranslationally modified in glaucoma-
betic db/db mice treated with antihyperglycemic tous rat retina [57, 65]. These studies demon-
drug. Proteome alterations include a decreased strated the involvement of 14-3-3 proteins in
expression of proteins related to synaptic trans- cellular signaling in glaucomatous neurodegener-
mission (e.g., vesicular glutamate transporter 1) ation. Later, through a quantitative gel-free
and cell signaling in diabetic mice. This study 2D-LC-MS/MS, Tezel and group [58] identified a
also shows that antihyperglycemic agents can large number of proteins that were differentially
only partially ameliorate diabetes-induced regulated in both human and rat retina during
changes in the membrane-associated signaling glaucoma. A twofold upregulation of hemoglobin
proteins. expression was detected in the retina as well as
Additional insights on the pathogenic mecha- the optic nerve head macroglia. Additional in vitro
nisms in DR come from the proteome analysis of experiments indicated that glial expression of Hb
VH, AH, and serum/plasma. Using in-depth pro- was induced by hypoxia through EPO signaling.
filing methods, the changes in the vitreous pro- The same group also suggested that the comple-
teome have been analyzed in both NPDR and ment system [59] and TLR signaling [42] are acti-
PDR stages. Proteins involved in complement, vated in the human retina during glaucoma.
coagulation, and kallikrein-kinin system have Activation of complement through classical and
been consistently found to be altered across many lectin pathway was also accompanied by a
studies [2, 16, 40]. In addition, apolipoproteins, decrease in the levels of complement factor H
immunoglobulins, and cellular adhesion mole- [59]. Stowell and group [56] used nonhuman pri-
cules are also altered [40]. Many studies demon- mate model of glaucoma, and using a label-free
strate the involvement of factors other than VEGF quantitative MS methods showed that cytoskele-
in vascular changes. Dyer et al. [11] showed that tal remodeling may play a role in the early retinal
carbonic anhydrase mediates an increase in vas- response to chronic IOP elevation. Tezel and
cular permeability as observed in DME and group [59] compared the human retinal proteome
PDR. Beta-2 integrin pathway is yet another fac- of ocular hypertensive donor with that of glauco-
tor leading to VEGF-induced changes during DR matous and normal donor to understand how high
[27]. Garcia-Ramirez [19] studies on the retinal IOP leads to neurodegeneration [59]. This study
proteome detected lower mRNA and protein lev- identified an initial period of intrinsic stress and
els of interphotoreceptor retinoid-binding protein defense response during ocular hypertension, and
(IRBP) with a concomitant increase in the GFAP these responses along with an ongoing cell death
process characterized glaucoma. A recent study disease pathology. Hence, the differential pres-
on the human retinal proteome of glaucoma ence or alteration in the level of proteins can be
patients suggest that an impairment of energy used as a marker. Many such markers have been
metabolism and stress response occurs during the identified in the retina, VH, AH, and serum/
process of retinal neurodegeneration [15]. These plasma for AMD, DR, and glaucoma. Those
changes were also reflected in the aqueous humor markers identified through proteome analysis are
of glaucoma patients [25]. Bhattacharya and considered in this review, and some of these have
group [3] examined the optic nerve to understand already been discussed in the previous section.
the molecular pathology of POAG. Through pro- Additional markers are listed here.
teomic approach, they identified PAD2 protein to Early diagnosis and treatment of AMD are
be present exclusively in glaucomatous optic critical for preventing AMD-related blindness.
nerve and also provide evidence for the transla- Phospholipid transfer protein (PLTP) and
tional modulation of PAD2 expression suggesting mannan-binding lectin serine protease (MASP-1)
that this enzyme might be involved in optic nerve have been suggested as plasma biomarkers for
damage in POAG. Some studies have examined early AMD. The discriminatory power of these
the proteome of cultured trabecular meshwork biomarkers is enhanced when combined with the
(TM) to identify changes in response to treatment risk genotypes, age-related maculopathy suscep-
with dexamethasone [5] or TGF-β2 [69]. These tibility 2, and complement factor H genes [31].
studies suggest that both treatments induce Vinculin is another potential plasma biomarker
changes in the ECM and secreted proteins in TM, for AMD [32]. Proteome analysis of the exo-
similar to changes observed in glaucoma. Other somal proteins in AH enabled the identification
studies have implicated cochlin, a deafness disor- of molecular chaperone proteins, and proteins
der protein [4], and copine1, a calcium-dependent related to the autophagy-lysosomal pathway have
membrane-binding protein [68] to be increased in been identified as potential biomarkers and thera-
glaucomatous TM. Grus et al. [22] identified peutic target proteins for AMD [29].
transthyretin to be present at high levels in the AH Pharmacological intervention either to target Src
of glaucomatous patients and proposed that this kinase with the aim of preventing cytoskeletal
protein might play a role in the onset of glaucoma rearrangements in the retinal pigment epithelium
through the formation of amyloid deposits. Duan (RPE) and neuronal retina or to help rebuild dam-
and group [10] showed significant alteration in aged IPM may provide fresh avenues of treat-
AH proteome of POAG patients, and a study by ment for patients suffering from AMD [37].
Kaeslin and group [28] suggested the proteome Proteome studies on DR have identified many
changes in AH reflect an imbalanced metabolism, candidate protein markers such as a polipoproteins
lack of ROS detoxification, and low-grade and (ApoA-1, ApoH), complement system proteins
chronic inflammatory processes that occur during (C3, FI, C4b, C9, CFB), coagulation cascade pro-
glaucoma. Proteomic studies have mostly been teins (prothrombin, antithrombin III, factor XII,
carried out for POAG, and surprisingly, pro- fibrinogen A), and other proteins like alpha-1
teomics of PACG is a completely unexplored antitrypsin, peroxiredoxin, zinc-alpha glycopro-
area. tein, and angiotensinogen [2, 16, 18, 52] that
were increased in the vitreous humor. On the
other hand, proteins that were present at lower
32.6.2 Disease–Specific Candidate levels in DR vitreous include PEDF, IRBP,
Protein Markers ITIH3, calsyntein-1, interphotoreceptor retinoid-
binding protein, neuroserpin, and extracellular
Quantitative proteomic studies identify proteins SOD [16, 18]. Wang and group [62] through a
that are significantly altered in a pathological 2D-based MS approach reported for the first time
condition. These proteins may or may not be a alterations in DDAH1, tubulin α-1B chain,
part of specific pathways but are involved in the γ-enolase, cytosolic acyl-CoA thioester hydro-
lase, malate dehydrogenase, and phosphatidyl- as ELAM 1, apolipoprotein B and E, phospholi-

ethanolamine- binding protein 1. Many groups pase C, muscle cell differentiation and function
have examined the serum or plasma profile to proteins such as myotrophin, and heat shock pro-
identify candidate biomarkers for DR. Proteins, teins (Hsp60, Hsp90) also show differential
namely, hemoglobin, CD160 antigen, β2-GPI, expression in the AH of POAG patients [49].
AHSG, α1-AGP, and apo-A1, were elevated, Grus et al. [22] reported transthyretin as one of
while afamin, protein arginine the most abundant proteins in the AH of glauco-
N-methyltransferase, vitamin binding protein, matous patients. On the other hand, autotaxin is
gelsolin, RBP1, NUD10, and neuroglobin were an abundant protein in normal AH, but its lyso-
detected at lower levels in the serum of DR phospholipase D activity was significantly ele-
patients [21, 39, 41]. Kim and group [33] adopted vated in glaucoma [24]. Pieragostino and group
a comprehensive approach where they identified [46] profiled the proteome of tears of newly diag-
proteins to be altered in the VH of DR patients nosed and untreated glaucomatous patients
and combined it with those reported in literature. (naïve) with that of patients undergoing therapy
They subsequently validated these markers in with prostanoid analogues. Proteins, namely,
NPDR patient serum and suggested 28 candidate lipocalin-1, lysozyme C, lactotransferrin, proline-
proteins that can distinguish moderate NPDR rich-protein 4, prolactin-inducible protein, zinc-
group from DM patients without DR. Further, alpha-2-glycoprotein, polymeric immunoglobulin
they proposed a more specific multi-biomarker receptor, cystatin S, Ig kappa chain C region, Ig
panel for the early-stage DR comprising of apo- alpha-2 chain C region, immunoglobulin J chain,
4, complement C7, clusterin, and inter-alpha- and Ig alpha-1 chain C region, were all upregu-
trypsin inhibitor heavy chain H2 [26, 33]. Two lated in naïve POAG patients. Some of these pro-
groups identified circulating antibodies to be DR teins have also been found to be altered in the AH
specific. Sinha et al. [53] propose anti-MPO anti- of glaucomatous patients [47]. Biomarkers for
body as a marker for the progression from NPDR POAG from different tissues (TM, optic nerve)
to PDR as the levels of this antibody correlated and fluids (AH, serum/plasma) with reference to
with the severity of DR. Ahn et al. [1] examined the specific functional category are discussed in
specifically for anti-retinal autoantibodies by detail by Knepper et al. [35].
screening the retinal proteins using human sera
from DM and DR patients and found anti-
aldolase antibody to be elevated in DR patients. 32.7 Limitations
Ting and group [60] have reviewed the biomark-
ers for diabetic retinopathy from all other studies Proteome analysis of the retinal cells and ocular
and have provided a comprehensive list of vitre- fluids has been very helpful in identifying the
ous and serum biomarkers. factors and events that contribute to the pathol-
Crabb and group [7] quantified proteomic ogy of these three neurodegenerative eye dis-
change in aqueous humor (AH) from human eases. Many of these studies have also identified
POAG donors. Among the differentially candidate biomarkers. Upon validation in a larger
expressed proteins in AH, carbonic anhydrase 1, dataset, these proteins will be useful in predicting
Cu-Zn superoxide dismutase, and insulin-like the high-risk subgroups or for an early diagnosis.
growth factor-binding protein 7 were upregu- However, the limitation lies in the source of the
lated, while C-Jun-amino-terminal kinase-tissue or fluid for assaying these markers. Many
interacting protein 4, glutathione synthetase, of these markers have been identified in the ret-
synaptotagmin 5, and ten different crystallins ina, VH, or AH of patients, and these tissue or
were downregulated. Nearly 44% of the AH pro- fluids cannot be collected from normal individu-
teins were detected in the patient plasma suggest- als for ethical reasons. Hence, it is important to
ing that glaucoma-induced changes can be identify markers for these retinal diseases in
monitored in the blood. Additional proteins such sources such as tears or blood that is easily acces-
sible as well as those that are disease specific. Yet 4. Bhattacharya SK, Rockwood EJ, Smith SD, Bonilha
VL, Crabb JS, Kuchtey RW, Robertson NG,
another limitation lies in the amount of tissues/ Peachey NS, Morton CC, Crabb JW. J Biol Chem.
fluids available for proteomic analysis, and in 2005;280:6080–6084.
many cases, the starting material is insufficient 5. Bollinger KE, Crabb JS, Yuan X, Putliwala T, Clark
for multiple analysis. Proteomic technology has AF, Crabb JW. Mol Vis. 2012;18:2001–2011.
6. Chan EW, Li X, Tham YC, Liao J, Wong TY, Aung T,
advanced exponentially, and the newer platforms Cheng CY. Br J Ophthalmol. 2016;100:78–85.
generate a huge amount of data. Also, a parallel 7. Crabb JW, Jang GF, Zhang L, Crabb JS, Willard
development in the analysis software is lacking, B, Stowell C, Eisengart J, Burgoyne C, Rockwood
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Genomic Approaches to Eye
Diseases: An Asian Perspective 33
Bharanidharan Devarajan, Ayyasamy Vanniarajan,
Abstract identifying causative variants in various eye

Recent advances in genomic technologies, diseases with emphasis on Asian data and dis-
particularly next-generation sequencing cuss its implementation in the clinical
(NGS) methods, have brought a paradigm settings.
shift in discovering eye disease-associated
genetic variants from linkage and genome- Keywords
wide association studies to NGS-based Genome sequencing · Exome sequencing ·
genome/exome studies. Whole-genome Variant filtering · Eye diseases · Molecular
sequencing (WGS) remains prohibitively diagnosis · Asian population
expensive for most applications and requires
concurrent development of bioinformatic
approaches to expeditiously analyze the large
data sets; whole-exome sequencing (WES) is 33.1 Introduction
now made as a viable approach to uncover
unknown etiology with a limited number of The latest WHO figures state about 180 million
probands with eye disease. WES focuses on people are visually impaired; 22 million are blind
only the protein-coding sequence of human in Asia alone. Over 526 ocular clinical pheno-
genome, has become a powerful tool with types have been shown with inheritance in Online
many advantages in the research setting, and Mendelian Inheritance in Man database (https://
moreover is now being implemented into the omim.org/), which may lead to low vision and
clinical diagnostic arena. Here, we review the blindness. The inheritance of eye diseases, attrib-
current literature on technical approaches and uted to the deleterious genetic alterations, relies
to provide recommendations for bioinformatic on a large population or aggregation of a disease
analysis focusing WES and WGS methods. in the generations of family. The identification of
We highlight its successful applications for deleterious genetic alterations contributing to
Mendelian and complex eye diseases is the first
B. Devarajan (*) step toward the development of genetic screening
Department of Bioinformatics, Aravind Medical tests and novel therapeutic interventions. The
Research Foundation, Madurai, India high-throughput genome-wide association stud-
e-mail: [email protected] ies (GWAS) have been used extensively to iden-
A. Vanniarajan · P. Sundaresan tify genetic risk factors of inherited eye diseases.
Department of Molecular Genetics, Aravind Medical However, it has had several complications due to
Research Foundation, Madurai, India

https://doi.org/10.1007/978-981-13-0884-0_33
404 B. Devarajan et al.
incomplete penetrance and linkage disequilib- discovery in eye diseases are highlighted and
rium, and it can find only the common risk vari- concluding its use and challenges in the clinical
ants [1]. The novel and rare deleterious variants, settings.
which require to account for much of the inheri-
tance, remain uncovered in GWAS [2]. This
could be circumvented by conventional Sanger 33.2 Whole–Genome and Whole–
DNA-sequencing method. Despite steady Exome Sequencing–
improvement in the Sanger method, it remains Technical Design
costly, time-consuming, and low throughput. The
advent of next-generation sequencing (NGS) has Although the NGS platforms employ different
emerged as a high-throughput genomic approach sequencing approaches, they share a common
to overcome all the limitations. feature that is massively parallel sequencing of
Next-generation massively parallel sequenc- clonally amplified or single DNA molecules that
ing technology enables the comprehensive are spatially separated in a flow cell. It generates
genetic sequencing of all or part of the human hundreds of megabases to gigabases of DNA
genome, and its improvements in chemistries and sequence from each run based on the platforms.
workflows have paved the way into the clinical In recent past, it has improved immensely since
diagnostic arena. Its efficiency enables the avail- their advent in 2015 that the current Illumina
ability of high-speed and low-cost genomic NovaSeq 6000 platform is capable of producing
sequencing of multiple samples. The whole- six TB of sequencing data per run. The new NGS
genome sequencing (WGS), i.e., sequencing the systems called single-molecule sequencers from
entire genome, and whole-exome sequencing Pacific Biosciences [5] and Nanopore [6] can
(WES), i.e., sequencing the protein-coding provide high read lengths and resolution of DNA
regions alone, advance the discovery of novel modifications. Since most of published exome
variant/gene and therapeutic interventions. WES and genome studies have used Illumina technol-
has been the successful cost-effective tool for the ogy, the following sections will focus on Illumina
discovery of casual variants associated with sim- sequencing.
ple Mendelian as well as complex inheritance WES only covers 1–1.5% [7] of the human
patterns. WES has become available as a diag- genome and operationally defined by the consen-
nostic test performed in Clinical Laboratory sus coding sequence (CCDS) database (http://
Improvement Amendments-certified and College www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi),
of American Pathologists-accredited clinical lab- yet this portion of the genome houses approxi-
oratories [3, 4]. WGS is now technically feasible mately 85% of the known disease-causing vari-
and cost-effective; however, it is still expensive ants [8]. To achieve WES as technically possible,
as the cost of data analysis and storage has been complete coverage of all exons of known disease-
much higher than initially expected. Nevertheless, associated gene is needed. This is still a challeng-
recent developments in NGS platforms with ing process that certain regions in the genome are
faster turnaround times are accelerating the NGS difficult to sequence including GC-rich regions,
implementation into the clinical domain. Yet, repeat expansions (interspersed or tandem
NGS labs have been challenged with new chem- repeat), and regions of high sequence homology
istries and instrumentation as well as complexity (from pseudogenes or gene families) and difficult
in the data analysis. to analyze bioinformatically as well. Further,
In this review, first, the current technical large insertions and deletions (indels), copy num-
aspects of exome and genome sequencing, fol- ber variants, and other structural variants are very
lowed by the bioinformatic considerations and difficult, which can be achieved using WGS as it
challenges, are discussed. Subsequently, the covers complete genome.
applications of NGS technology to analyze whole Basically, the routine sequencing protocol
exomes and genomes for the causative variants includes genomic DNA fragmentation, library
33 Genomic Approaches to Eye Diseases: An Asian Perspective 405
construction, cluster generation, and sequencing. neously by multiplexing. The libraries with adapt-

The initial preparatory steps are the same for ers containing distinct index sequences, are used
genome and exome libraries, wherein the frag- to identify the samples, can be pooled together in
mented genomic DNA is converted into an oligo- each run. Resulting reads made available in
nucleotide adapter-tagged library. Multiple FASTQ format files are aligned to the human ref-
methods of genomic DNA fragmentation such as erence genome that are further run through com-
nebulization, sonication, restriction enzyme putational pipelines for variant calling and
digestion, chemical methods, or sonication by subsequent downstream analysis.
adaptive focused acoustics result in DNA frag- Approximately 623 genes are known to be
ments about 200–600 base pair size range. All associated with eye disease (https://omim.org/)
DNA fragments are then end repaired and ligated out of approximately 22,000 genes in the human
to platform-specific adapter oligonucleotides, genome. For WES, an enrichment approach
which is capable of hybridizing to the oligonucle- needs to be strategically designed to provide
otides on the sequencer’s flow cell surface, a complete coverage of known disease-associated
thick glass fluidic device (Illumina Inc., San genes as technically feasible for the coding
Diego, California). Before hybridizing, in the regions and flanking intron-exon boundaries
case of exome library enrichment, the libraries (e.g., 10–50 base pairs). Additionally, by careful
are hybridized with exome/target-specific cap- characterization of capture efficiency and cover-
ture probes. The captured libraries are further age using bioinformatic algorithms, specific deep
purified and enriched with exome kits and, in intronic and untranslated regions associated with
some protocols, amplified by limited cycles of diseases can be included in the capture enrich-
polymerase chain reaction. Several exome cap- ment. Subsequently, the Sanger sequencing can
ture/enrichment kits from different vendors are be used to cover the low-coverage regions likely
available. Though they differ in the targeted cap- to be associated with the patient’s clinical pheno-
ture regions, capture probe length, probe compo- type. In contrast to WES, without having capture
sition, and performance characteristics, the most probe enrichment, WGS results in less-biased
commonly used capture approach involves sequence coverage that allows inspection of
hybridization with Agilent SureSelect probe deeper intronic and regulatory regions. Further,
technology (Agilent Technologies, Santa Clara, the all structural variations are detected with spe-
California), followed by Roche Nimblegen Inc. cific bioinformatic algorithms. However, WGS
(Madison, Wisconsin) and Illumina Inc. (San still holds increase in sequencing cost and com-
Diego, California). putational resources.
The enriched libraries hybridized on the flow
cell are then bridge amplified to generate clusters
of DNA clones. Sequencing of clonal clusters 33.3 Bioinformatic Analysis
proceeds in a cyclic manner with reversible dye- for WES and WGS
terminator chemistry, allowing only the first
nucleotide base to be incorporated, that is, fluo- Several open-source bioinformatic algorithms
rescently labeled and reversibly terminated and commercial software exist to analyze WES
nucleotides. The high-sensitivity imaging optics and WGS large data files and process into variant
are used to capture the fluorescent output of clus- call files (VCF) with high-quality variants, which
ters on the flow cell after each base post incorpo- require substantial computational resource and
ration. This process is repeated to yield a strand bioinformatic expertise. Each lab has unique
(read) whose length is dependent on the number pipeline made up of open-source, in-house devel-
of sequencing cycles. In paired-end sequencing, oped, and/or commercial software; the WES
the same cluster is also sequenced from the oppo- pipeline we use is shown in Fig. 33.1. The bioin-
site end, generating two reads per library. The formatic analysis, although there are variations in
sequencing of many samples can be run simulta- the protocol, is a common multistep process for
Raw Reads
(FastQ)
Clinical
Read Mapping Phenotype
In-house Scripts:
(BWA and Novaalign) >20X Coverage and
satisfactory Phred Score
Discard Duplicates Mode of MAF (Study Dependent)
Processing of Alignment
Local InDel Realignment Inheritance Functional variants
(picard, GATK)
Base quality recalibration
SNV and InDel Calling Known candidate

(GATK HC and Samtools) genes /variant In silio pathogenicity
pathogenicity prediction, structural
bioinformatics and
Combined Raw VCF Visual analysis
Expression and
Function of gene
Annotation of interest
(Annovar and inhouse tools) Pathogenic Variants
and
Annotated VCF Variant Filtering and possibly pathogenic
Prioritization variants
Fig. 33.1 A clinical exome sequencing analysis workflow for variant identification
WES and WGS. The primary analysis starts with identify them in only the patient or patient with
the processes of initial alignment and mapping of biological parents (trio) if they are available. The
sequence reads to the human genome reference trio sequencing has been demonstrated as a pre-
in order to generate a binary alignment and map- ferred diagnostic approach, which allows for a
ping file after the quality control analysis of NGS more detailed analysis of variant inheritance pat-
data (Fig. 33.1). The secondary analysis is the terns. A common first step in trio studies is to
variant preparation, involving variant calling and apply filters based on suspected disease inheri-
annotations, for the downstream clinical interpre- tance patterns and disease frequency. In a pre-
tation (tertiary analysis). The tertiary analysis sumed rare inherited disorder or general exome
includes variant incidence, prioritization, classifi- analysis workflow (Fig. 33.1), a common starting
cation, and integration with the clinical features. point is to filter common variants that are in the
Several reviews are referred here [9–11] for the population assuming that rare variants are caus-
primary and secondary analyses of WES and ative. This filtering step may differ based on the
WGS data and have emphasized the sensitivity individual patients and laboratories. In common,
and accuracy of different algorithms in identifica- variants with high minor allele frequency (MAF)
tion of variants. The tertiary analysis, a complex equal to or greater than 1% in public databases
process, requires customized data-mining pro- will be removed. Several public databases
cess for the identification of a disease-causing or include the 1000 Genomes Project (http://
disease-associated variant, which is depending www.1000genomes.org/), ESP6500 (http://evs.
on the phenotype. gs.washington.edu/EVS/), dbSNP (http://www.
ncbi.nlm.nih.gov/SNP/), ExAC (http://exac.
broadinstitute.org/), and GenomeAD (http://gno-
33.3.1 Variant Filtering in Mendelian mad.broadinstitute.org), which now serve as a
Diseases valuable resource for MAF estimations. MAF
cutoff around 1% and 0.5% or less may be used
WES/WGS have dramatically altered the tradi- for rare recessive disorders and a dominant or
tional linkage-analysis landscape of finding rare X-linked disorder, respectively. Next, the remain-
variant in Mendelian disease, and we can now ing variants are filtered using the functional
impact of the variant. The truncating variants is still challenging to determine the causative
(stop gain/loss, start loss, or frameshift), mis- SVs. For example, causative CNVs may not be
sense variants, and canonical splice-site variants identical [17], and different SVs may be obtained
are considered first as protein function-altering with different technologies and bioinformatic
variants, followed by silent and in-frame indels approaches for a given sample. Regardless of
affecting protein-coding regions. Missense vari- these challenges, detection of SVs is important as
ants are further prioritized by their functional their involvement in human eye diseases is
impact on the protein using in silico tools such as increasingly being recognized.
SIFT (http://sift.jcvi.org/) and Polyphen2 (http:// Finally, a careful examination is required for
genetics.bwh.harvard.edu/pph2/) and others [12]. the filtered variants prior to confirm their signifi-
This prioritized set is further stratified whether cance in the clinical practice, since more number
those are present in the same gene, those are pre- of variants could be prioritized as potential caus-
dicted to be the most deleterious, and those are ative variants through the above-described filter-
residing in the genes that are implicated in the ing methods. For example, the genome of healthy
patient’s phenotype. Subsequent variant analysis individual carries large number (at least 100 per
is differed in trio sequencing compared to patient genome) of loss-of-function variants, and yet
sequencing alone, wherein mode of inheritance is they include rare disease-causing (causal) vari-
applied. For example, homozygous or compound ants [18]. To distinguishing causal variants from
heterozygous variants will be retained for reces- the many potentially rare and functional variants
sive mode of inheritance whereas heterozygous that are commonly present, substantial time and
variants for dominant mode of inheritance. For de effort must be taken to suggest causality. In pre-
novo variants, trio sequencing is highly recom- sumed monogenic-disease cases, first one must
mended. On an average one to two de novo vari- evaluate genes and variant previously implicated
ants appear in a typical exome, which tend to be in disease phenotypes before exploring novel.
more deleterious than inherited variants, and are Second, the new gene can be implicated only
difficult to deduce from trio sequencing [13]. when variants in the same gene and similar clini-
The detection of structural variations (SVs) cal features have been implicated in multiple
including copy number variations (CNVs) may individuals, along with unaffected controls [19],
be treated separately, since the process of their which requires considerable expertise and collab-
detection differs substantially from the pipelines orative input from physician and genetician.
used to identify and small InDels and single Further genetic and functional studies are impor-
nucleotide variants (SNVs). Detecting SVs has tant to validate experimentally the predicted
several challenges, and a number of approaches causal variants. Despite the current limitations of
and commonly used methods include read depth, variant interpretation capabilities, the rapid pace
allele frequency, paired-end mapping, split-read of discovery in variant calling methods and new
mapping, and de novo assembly and have been diseases-associated genes may help WES/WGS
reviewed elsewhere [14–16]. SVs, like other vari- methods to achieve its clinical translation.
ants, can be common (polymorphisms with no
clinical significance) or rare and potentially dam-
aging, which often causes several clinical pheno- 33.3.2 Variant Filtering in Complex
types. Using mode of inheritance, total rare SV Diseases
burden, size, and number of dosage-sensitive
genes in the SV region, causative SVs can be pri- Prioritizing variants of WGS/WES data in a com-
oritized. In trio sequencing, SVs may present in plex disease is very difficult and requires a differ-
patient alone compared to unaffected individuals, ent approach. GWAS has been used mostly for
which helps to identify causative SVs. In addi- investigating the genetic architecture of complex
tion, a number of public databases are available diseases by following the common variant
to annotate the potential clinical SVs. However, it [MAF > 0.05]/common disease hypothesis [20].
In GWAS, several millions of single nucleotide of currently available algorithms are reviewed
polymorphisms (SNPs) are assayed in thousands elsewhere [26, 27].
of individuals with a phenotype and unrelated
controls. However, challenges are still existing
that most of the variants identified so far confer a 33.4 Whole–Exome Sequencing
small increase in risk and explain only a small in Eye Diseases
proportion of familial clustering [20]. This miss-
ing heritability can be explained by the alterna- WES, primarily very successful in Mendelian
tive hypothesis that rare [MAF < 0.01] and disorders, has also offers in identifying causal
low-frequency [MAF 0.01–0.05] variants are variants in patients and families with complex
likely to be strong effect and heritable [21]. diseases. In case of Mendelian disorders, screen-
WGS/WES are mostly employed to deduce rare ing the long list of known genes using targeted
and low-frequency variants that are likely to be NGS does not reveal causal variants; for exam-
causal in complex phenotypes. Thus far, the ple, in most of the RD cases as described below,
interpretations of causal variants can be challeng- WES is proved to be efficient to discover novel
ing that the allelic architecture of complex dis- causal variants and genes. In case of complex eye
eases can be underpinned by a combination of diseases such as cataract, glaucoma, diabetic reti-
spectrum of frequency and rare variants. For nopathy, age-related macular degeneration, and
example, both common and rare variants have other degenerative eye disorders, it is difficult to
been identified in AMD patients using GWAS identify causative variants due to the fact that it
and NGS approaches [22, 23]. Currently, WGS is may be caused by combination of many genetic
the preferred approach to exhaustively study the factors, and the involvement of environmental
full allelic spectrum of variations in complex factors may affect the identification. Despite the
traits. The possible explanation is that rare vari- fact, WES is still the preferred choice of method
ants in or near gene targets display larger average to identify causative variants. The major exome
effects on phenotype, and both regulatory vari- sequencing studies presented here were in
ants of comparable allele frequencies and com- the context of Asia-Pacific and the data is illus-
mon genetic variants are indeed causal [21, 24]. trated in Fig. 33.2.
Thus, one must focus on the functional genomic
approaches that include transcriptome and epig-
enome and along with association signal from 33.4.1 Retinal Dystrophies
GWAS that would help to understand their
involvement in the complex phenotype. Even rig- Retinal dystrophies (RD) contribute about 40% of
orous evaluation of each variant with functional all inherited eye diseases, and there are about 30
consequences is required because the single vari- genes earlier reported to be associated with
ant displays small effect in complex disease. RD. Exome studies had been found to be power-
Several statistical methods have been developed ful in identifying the molecular cause where the
to evaluate the effect of multiple variants that known genes did not show any mutation. In an
have functional consequences, most of them Indian Muslim family, three out of eight members
using variant aggregation approaches to address were affected with autosomal recessive cone dys-
this issue. These methods can be classified into trophy. WES showed a homozygous frameshift
two main types as burden and variance compo- mutation leading to truncation in the affected
nent tests or a mixture of both [25]. Burden tests members, and the same is found to be heterozy-
collapse the number of variants in a certain region gous in the unaffected family members in the
or gene between cases and controls, while vari- CNGB3 gene [28]. In a consanguineous Israeli
ance component tests distinguish variants with family with early-onset cone-rod dystrophy
effect compared to variant with no effect in a (CRD) and muscular dystrophy, exome sequenc-
single gene [25]. The overview and performance ing and homozygosity mapping identified variants
Cataract Anopthalmia/Micropthalmia
ALDH1A3, FOXE3, VSX2
High Myopia GJA8
Congenital [41] Usher [46]
ZNF644
Cataract Syndrome Congenital Glaucoma
[42]
CRYGD BPES USH2A PXDN, LTBP2
BBS [39] FOXL2 [44]
BBS4
[47] Alstrom
CRYGC [52]
[45] ARS syndrome
CRYAA
PRMD5 ALMS1
[40]
[43] [51]
2011 2012 2013 2014 2015 2016 2017
RP CD
RDH12, CNGB1, arRP CRD
LCA GUVY2D, CDHR1, CNGB3
CERKL, USH2A, EYS, SAG,
BBS4
USH2A, TULP1, C8orf37 arRP [28]
EYS, PDE6B,
[38] [33] EYE
DFNB31 C2orf71, CNGA1
[34] [35]
[49]
RD CRB1
RD [36]
ALMS1, DYSF
EMC, GPR125,
[29]
K1AA1549, C21orf2,
RPE65, LCA5,
ACBD5, DTHD1
USH2A, CNGB1,
[31]
ALMS, FAM161A,
CRD GUCY2D
CNGB3, PDE6C, [30]
ABCA4,
RPGRIP1, RPGR,
CACNA1F
[32]
CSNB
LRIT3
[37]
Fig. 33.2 Timeline of WES and WGS-based studies of eye diseases in Asian populations
in ALMS1 and DYSF that are linked genetically candidates, ACBD5 and DTHD1 were observed
and physically on chromosome 2. Although both for the first time in the syndromic RD cases [31].
the genes were reported in Alstrom syndrome, the The power of exome sequencing was best wit-
affected individuals did not have any symptoms nessed in a study of the 47 Chinese families
other than mild muscular dystrophy [29]. through the identification of potential pathogenic
In a study of 26 Pakistani families with inher- mutations in 10 families (21.28%) in the list of 25
ited retinal degenerations, 9 causal mutations known genes of retinal dystrophies. The earlier
along with 6 novel variants in RPE65, LCA5, study from the same group had identified muta-
USH2A, CNGB1, FAM161A, CERKL, and tions in only 7 out of 130 families (5.38%) using
GUCY2D were identified in 13 families. In addi- the Sanger sequencing of complete exons in 5
tion to the causal variants, a total of 200 variants genes and reported mutations in 17 genes [32]. In
were also identified that are unique to Pakistani another study with cone-dominated retinopathy,
population as they were not reported in any of the WES followed by segregation analysis in six
genomic databases [30]. Genomic approaches Israeli families revealed mutations in known reti-
including autozygome-guided mutation analysis nopathy genes: GUCY2D gene in three families
and exome sequencing were performed in a large and one each in CDHR1 and C8orf37. Further
cohort of 150 families with retinal dystrophies. targeted screening of additional cone-dominated
Six novel candidate disease genes (C21orf2, families led to identification of GUCY2D muta-
EMC1, KIAA1549, GPR125, ACBD5, and tions in four other families [33].
DTHD1) were identified in addition to causative Retinitis pigmentosa (RP) is a heterogeneous
genetic lesions in the known genes. Of the novel group of inherited disorders characterized by
progressive degeneration of the retinal photore- gested for this condition, and no mutations were
ceptor cells. In a large cohort study of Japanese identified by screening these known genes. Hence
RP patients, exome sequencing of 30 RP patients whole-exome sequencing with linkage analysis
identified disease-causing gene mutations of was used to identify the causative mutation in a
CNGA1, EYS, and SAG along with potential Chinese family. Through linkage, 11 candidate
disease-causing gene variants of USH2A, EYS, mutations were selected, and finally CRYGD was
TULP1, and C2orf71. Further screening of most found to be as the gene responsible for the cata-
frequent CNGA1 gene mutation in 69 patients ract phenotype [39]. In another study of two
revealed 1 patient with a homozygous mutation Korean families with autosomal dominant con-
[34]. Similarly, in a large cohort of 14 Indian genital cataract (ADCC), a recurrent CRYAA
autosomal recessive retinitis pigmentosa (arRP) missense mutation in family A and a novel
families and 100 sporadic RP cases, a spectrum CRYGC frameshift mutation in family B were
of novel EYS mutations including a frameshift identified [40]. Another exome study of proband
mutation, 2 were stop-gain mutations, 1 was a with ADCC has found a recurrent missense muta-
splicing mutation, and the others were missense tion in GJA8 gene. This mutation was confirmed
mutations that were identified in 2 families and 8 by Sanger sequencing and found to be co-
sporadic patients [35]. In another study of 2 segregating with the affected family members but
Chinese families with 1 family having 3 affected was not detected in unrelated unaffected controls.
members and 100 Indian sporadic families, This study demonstrates the utility of the exome
exome sequencing revealed 4 novel mutations sequencing in a clinical setup for identification of
and 1 reported mutation in CRB1 gene, which known and unknown disease-causing variants
has been known to cause severe retinal dystro- [41]. Exome analysis was also carried out in two
phies [36]. affected individuals from a Han Chinese family
Congenital stationary night blindness (CSNB) with high myopia and identified a mutation in
is a genetically heterogeneous retinal disorder ZNF644 that was related to the phenotype.
inherited as either X-linked or autosomal reces- Further Sanger sequencing analysis of ZNF644
sive. Whole-exome sequencing in one simplex gene in 300 sporadic cases of high myopia
complete CSNB case which was negative for the detected 5 additional mutations in 11 different
known genes NYX, GRM6, TRPM1, and patients that were absent in 600 normal controls
GPR179 led to the identification of a missense [42].
and a nonsense mutation in LRIT3 gene.
Subsequent Sanger sequencing of 89 individuals
showed a nonsense and a frameshift mutation in 33.4.3 Ocular Syndromes
the same gene in another individual with com-
plete CSNB [37]. WES was also employed in a Axenfeld–Rieger syndrome (ARS; OMIM
consanguineous Saudi Arabian family with Leber 180500) is a rare developmental disorder inher-
congenital amaurosis (LCA) and identified a ited in an autosomal dominant manner affecting
novel missense mutation in BBS4, which segre- the cornea, iris, lens, and angle, which is primar-
gated with the disease. Further analysis in zebraf- ily caused by mutations in CYP1b1, PITX2, and
ish also indicated that the mutation is pathogenic FOXC1. A novel heterozygous missense variant
[38]. in PRDM5 gene was identified in a proband with
ARS, which co-segregates with the disease and
found to be absent in matched controls and
33.4.2 Cataract and Refractive Errors genomic databases [43]. In another study WES
was performed in patients with Usher syndrome
Congenital cataract causing blindness in children (USH), which is a multisensory degenerative dis-
is primarily inherited in an autosomal dominant order with deafness and blindness and geneti-
manner. There are about 30 candidate genes sug- cally heterogeneous. A novel and two known
mutations in USH2A were detected in two difficult. Thus far, only very few studies have
affected patients that were absent in an unaf- been published to identify candidate variants in
fected relative and further confirmed by direct eye diseases. In the literature, we found only
sequencing and co-segregation analysis [44]. three studies of ocular diseases using WGS in
WES also revealed a novel homozygous splice Asian ethnic population. The first study pub-
BBS1 mutation in four patients from two consan- lished by Nishiguchi and colleagues performed
guineous Pakistani families with Bardet-Biedl WGS on 16 unrelated patients from North
syndrome, which is genetically heterogeneous America or Japan with autosomal recessive reti-
disorder characterized by rod-cone dystrophy nitis pigmentosa (arRP) in search of pathogenic
with other non-ocular features [45]. variants [49]. Homozygous or compound hetero-
zygous mutations in seven genes (EYS, PDE6B,
USH2A, CNGB1, CERKL, RDH12, and
33.4.4 Other Eye Diseases DFNB31) were detected. Of these, there was a
2.3 kb deletion in USH2A and an inverted dupli-
Anophthalmia and microphthalmia (A/M) are cation of ∼446 kb in EYS, which would not be
genetically heterogeneous disease caused by detected through WES. Moreover, a homozygous
mutations in at least 20 genes that show different frameshift variant (p.L206 fs) in the ciliary gene
modes of inheritance. Exome sequencing of eight NEK2 was identified as a new arRP gene [49]. In
samples with anophthalmia and microphthalmia another study, whole-genome low-coverage
from Pakistani and Indian families identified sequencing (WGLCS) was used to characterize
three novel mutations including two mutations in the breakpoints of blepharophimosis–ptosis–epi-
ALDH1A3 gene and a missense mutation in canthus inversus syndrome (BPES, OMIM
FOXE3 gene. Additionally, two previously 110100) and affects eyelid formation and ovarian
reported mutations were identified in FOXE3 and function, in Han Chinese families. Four break-
VSX2 [46]. In another study involving patients points were identified in the FOXL2 locus and
with primary congenital glaucoma (PCG), WES show that disruption of FOXL2 gene can be accu-
was performed in four individuals belonging to rately and rapidly detected using WGLCS [50].
three different CYP1B1-negative Pakistani fami- Recently, WGS was performed on a Chinese
lies and identified two mutations in the LTBP2 quartet family with two siblings predominantly
gene and one in the PXDN gene [47]. affected by cone-rod dystrophy and short stature
to identify the full spectrum of the two siblings’
genetic variations. Two compound heterozygous
33.5 Whole-Genome Sequencing mutations (p.S1301X; p.R2146X) shared
in Eye Diseases between two siblings in ALMS1 gene were iden-
tified [51]. Taken together, WGS substantially
The limitations of WES, include only 2% cover- improves the detection of full spectrum of patho-
age of the human genome, missing pathogenic genic variants in the eye diseases. With the costs
intronic variants that may affect transcripts, about of whole-genome sequencing continuously
3 to 5% of missed exome targets, may not com- decreasing, WGS is now likely the preferred
pletely characterize copy number variations and choice in the clinical settings.
breakpoints for other structural variants [25, 48],
leads to unsolved cases. For example, approxi-
mately 20% of unsolved RP cases were detected 33.6 Use of WES/WGS
as described previously [31]. In contrast, WGS in the Clinical Settings
shows evident in avoiding these limitations and
offers complete exomic coverage and identifica- Compared to whole-genome sequencing, exome
tion of noncoding pathogenic variations [48]. sequencing has become a widely used method for
However, the data analysis of WGS is still very identifying the molecular basis of genetic eye
disorders, specifically to detect rare causal vari- family and need for genetic counseling. The
ants for Mendelian disorders. In recent years, American College of Medical Genetics and
WES has been successfully employed in the clin- Genomics (ACMG) has proposed recommenda-
ical settings, with the diagnostic rate of ~25% tions on informed consent process, reporting of
[52]. To test diagnostic rate in retinal dystrophies, incidental findings and clinical laboratory stan-
WES and autozygosity-guided mutation analysis dards for next-generation sequencing [53, 54].
were performed in a large set of simplex and mul- However, individual laboratories may have their
tiplex families with different RD phenotypes. own policies for incidental findings, including
The study showed WES is superior and also which type of variant to be disclosed (e.g., patho-
found six novel candidate genes. Molecular diag- genic and possibly pathogenic). But then, their
nostic rate of WES was 80% and 74% in simplex assay design should follow an adequate read
and multiplex cases, respectively, compared to depth and coverage, and accuracy in identifying
42% and 52% in simplex and multiplex cases by variants includes confirmation of other orthogo-
autozygome-guided sequencing [31]. However, nal methods for CNVs and other structural
recommendations of WES use in the clinical lab- variants.
oratories are still evolving with many challenges
that delay the common use of WES in the molec-
ular diagnosis. Mainly, the incomplete coverage 33.7 Conclusions and Future
of exonic regions due to sequence architecture Prospects
(e.g., high G + C content) and evolving various
variant detection and interpretation methods The presented literature here reveals that NGS-
(e.g., copy number variants detection) limit the based genomic approaches to most of the eye dis-
diagnostic rate. The evolution of genome infor- eases have so far been widely used and showed
mation (e.g., http://gnomad.broadinstitute.org) its utility in the clinical settings. However, WES-
may change the understanding of variant inter- based methods showed its maximum utility in
pretation. Moreover, additional information from identifying novel causal variants and genes in eye
family studies and input from clinicians about diseases, especially in retinal degenerations.
patients may help to increase the diagnostic rate. Regardless of its success, evolution of variant
More recently, the use of WGS in clinical set- interpretation knowledge is required, and trans-
tings has been enabled. Although WGS is becom- lating the findings into the clinical settings is a
ing more and more affordable and providing challenge in itself. Though the large amounts of
comprehensive view of genetic alterations, the data from both clinic and sequencing research
bigger challenge of computational analysis of projects are available, creation of dedicated loci-
high volume of data exists. More importantly, specific databases for eye diseases is on major
individuals with high bioinformatic skills are need to enhance the process of variant interpreta-
needed for the analysis of data set since the con- tion. Indeed, new analysis methods to deal with
stant change of bioinformatic analysis and algo- various rare variants, discovered in millions of
rithms along with the modifications of sequencing variants through WGS and WES approaches, into
chemistries. Regardless, the use of NGS in the particular causal variants can have important
clinical settings provides a specific treatment or implications in the disease management. In the
medical management that can have an impact on near future, collaborative effort, combination of
the clinical outcome. This poses unique chal- WGS and transcriptomic approaches, and the
lenges that include informed consent process, large cohort study may be of high priority to pri-
possibility of proband versus trio sequencing, oritize candidate variants for follow-up.
cost and turnaround time, and how to interpret
and communicate the results, including inciden- Conflict of Interest The authors declare no conflict of
interest.
tal findings and data sharing, to the patient and
Compliance with Ethical Requirements No animal or review of informatic approaches. Cancer Genet.
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Myopia Genes in Asians
34
Shumin Tang, Yu Meng Wang, Aziz K. W. Kam,
Tommy C. Y. Chan, Calvin C. P. Pang,
Jason C. S. Yam, and Guy L. J. Chen
Abstract Keywords
Myopia is the most common ocular disorder Myopia · Refractive errors · Genetics · Asians
causing visual impairment worldwide. It is a
public health issue in many parts of the world.
Compared with Caucasian or other ethnicities,
its prevalence in Asians, especially Japanese, 34.1 Introduction
Koreans, and Chinese, is much higher.
Environmental and genetic factors play impor- Myopia is the most common ophthalmic disorder
tant roles in myopia development. Myopia is a posing risk to visual impairment worldwide [1,
multifactorial disease. Time spent outdoors, 2]. Its occurrence and severity are much more
amount of near work, and educational level serious in East Asians than other ethnic popula-
influence myopia onset and progression. Recent tions, affecting as many as 90% of high school
advances in modern technology and molecular students in East Asia [1]. It is a public health
biology including linkage analyses, candidate problem. The longer axial length of myopic eyes
gene analysis, genome-wide association stud- is mismatched with the optical power of cornea
ies (GWAS), whole-exome sequencing (WES), and lens, focusing images in front of the retina,
and next-generation sequencing (NGS) have leading to blurred distant vision [3]. Although
led to mapping and identifying many myopia- myopic refraction can be corrected by spectacles,
associated gene loci and variants. Understanding contact lens, and/or refractive surgery, highly
the genetic basis may help in myopia prediction myopic eyes with elongated axial length and
and prevention. This review is to summarize thinned sclera are at increased risk for vision-
recent major findings in myopia genetics with a threatening complications, such as glaucoma, ret-
focus in Asian populations. inal detachment, choroidal neovascularization,
macular hole, and myopic foveoschisis [4–7].
Therefore, the rapidly rising prevalence of myo-
S. Tang · Y. M. Wang · T. C. Y. Chan · C. C. P. Pang · pia poses socioeconomic burden to individual
J. C. S. Yam · G. L. J. Chen (*)
Department of Ophthalmology and Visual Sciences, and the society.
The Chinese University of Hong Kong, Both environmental and genetic factors play
Hong Kong, China important roles in myopia development.
e-mail: [email protected] Increased time spent outdoor reduces the inci-
A. K. W. Kam dence and progression of myopia in school chil-
Department of Ophthalmology and Visual Sciences, dren [8, 9]. Other factors include near work and
Prince of Wales Hospital, Hong Kong, China

https://doi.org/10.1007/978-981-13-0884-0_34
418 S. Tang et al.
education. In addition, more than 20 chromo- studies [31, 32]. In addition, children with myo-
somal loci and 100 genes have been identified to pic parents are more likely to be myopic [33–35].
be associated with myopia or refractive errors via In 1994, Zadnik et al. revealed a longer eyeball
linkage analyses, candidate gene, genome-wide length in those pre-myopic children with family
association studies (GWAS), next-generation history of myopia, when comparing to those
sequencing, and whole-exome sequencing without family history [36]. In 2008, our group
(WES) [10–23]. However, some of the gene asso- further demonstrated that parental history of
ciations are inconsistent among reported studies. myopia would increase the growth rate of eyeball
So far no individual gene has been proven to [37]. Children with two myopic parents had the
solely account for the etiology of myopia. The greatest annual axial length (AL) growth of
mechanism of myopia development is complex 0.37 mm, followed by children with one myopic
and not fully explained. This review is to sum- parent (AL growth = 0.26 mm), and children with
marize the major findings in myopia genetics no myopic parents (AL growth = 0.20 mm) [37].
with a focus in Asian populations. Both myopia onset and progression are affected
by genetic factors.
34.2 E
thnic Differences in Myopia
Prevalence 34.4 The Myopia Loci
Prevalence of myopia varies among different eth- Many myopia loci have been discovered by link-
nicities with predominance in Asia. The age analysis. Linkage region of the entire genome
Refractive Error Study in Children (RESC), was first identified, and that region was further
employing standardized sampling strategies and analyzed by allelic association analysis. Till now,
measurement methods, determined the preva- 25 myopia loci have been reported in the Online
lence of refractive error in children across differ- Mendelian Inheritance in Man (OMIM). Among
ent ethnicities and cultures worldwide. Among them, 23 were located at the autosomal chromo-
children aged 5–15 years, the myopia prevalence some, and the other two were on the X-linked
was 21.6% in northern China [24] and higher at chromosome (Table 34.1).
38.1% in southern China [25]. In contrast, the
prevalence was much lower at 7% in Chile and
4% in South Africa [26, 27]. In Australia, the 34.4.1 MYP1
Sydney Myopia Study (SMS) investigated chil-
dren from grade 1 (6-year-old) to grade 7 The first locus identified for high myopia,
(12-year-old), and the prevalence were 1.43% MYP1, was reported in 1990 and located at
and 11.9%, respectively [28, 29]. Across the Xq28, inherited in X-linked recessive manner in
same age and using the same protocol as SMS in a syndromic family with X-linked myopia,
the Anyang County in northern China, the preva- astigmatism, impaired vision, and optic nerve
lence was 3.9% and 67.3%, respectively [30]. head hypoplasia [38]. In 2010, the X-linked
These population-based studies consistently sug- recessive inheritance of MYP1 locus was con-
gested a higher prevalence of myopia in Asians. firmed in a large non-syndromic family with
high myopia [39]. However, causative mutation
was not identified in the coding and adjacent
34.3 Inheritability of Myopia intronic regions of GPR50, PRRG3, CNGA2,
and BGN. In 2011, Ratnamala et al. refined
Family aggregation studies and twin studies indi- the MYP1 locus on Xq28 in the non-syndromic
cated inheritability in myopia and supported the high myopia family and excluded 13 positional
role of genetics in myopia development. Myopia candidate genes as the high myopia causative
heritability was reported up to 90% from twin genes in this region [40].
Table 34.1 Summary of reported myopia loci
Myopia loci Related gene Location Phenotype Ethnicity Participants MIM # References
MYP1 Xq28 X-linked Caucasian 1 family 310,460 [38]
MYP2 18p11.31 Autosomal dominant Northern European and Chinese 8 families 160,700 [41, 42]
34 Myopia Genes in Asians
MYP3 LUM, TGF1 12q21-q23 Autosomal dominant Italian/German 1 family 603,221 [45]
MYP5 COL1A1, CHAD 17q21-q22 Autosomal dominant English/Canadian 1 family 608,474 [48]
MYP6 SCO2 22q12 Autosomal dominant Jewish American 44 families 608,908 [49, 50]
MYP7 PAX6 11p13 Multifactorial Caucasian 221 pairs of dizygotic twins 609,256 [51]
MYP8 3q26 Multifactorial 609,257
MYP9 4q12 Multifactorial 609,258
MYP10 8p23 Multifactorial 609,259
MYP11 RRH 4q22-q27 Autosomal dominant Chinese 1 family 609,994 [52]
MYP12 SAG, DGKD 2q37.1 Autosomal dominant Northern European 1 family 609,995 [53]
MYP13 Xq23-q27.2 X-linked Chinese 300,613 [54]
MYP14 1p36 NA Ashkenazi Jewish 49 families 610,320 [55]
MYP15 PCDH15, ZWINT 10q21.1 Autosomal dominant Caucasian 1 family 612,717 [56]
MYP16 5p15.33-p15.2 Autosomal dominant Chinese 3 families 612,554 [57]
MYP17, MYP4 7p36; 7p15 Autosomal dominant French and Algerian; 23 families; 96 families 608,367 [58–60]
African-American
MYP18 14q22.1-q24.2 Autosomal recessive Chinese 1 family 255,500 [61]
MYP19 5p15.1-p13.3 Autosomal dominant Chinese 1 family 613,969 [62]
MYP20 13q12.12 Autosomal dominant Chinese 419 high myopia and 669 controls 614,166 [20]
MYP21 ZNF644 1p22.2 Autosomal dominant Chinese 1 family 614,159 [63]
MYP22 CCDC111 4q35.1 Autosomal dominant Chinese 1 family 615,420 [64]
MYP23 LRPAP1 4p16.3 Autosomal recessive Saudi Arabian 3 families 615,431 [65, 66]
MYP24 SLC39A5 12q13.3 Autosomal dominant Chinese 1 family 615,946 [67]
MYP25 P2HA2 5q31.3 Autosomal dominant Chinese A three-generation family 617,238 [68]
419
420 S. Tang et al.
34.4.2 MYP2 pia. The extracellular matrix protein COL1A1

and chondroadherin (CHAD) on 17q were pro-
The MYP2 locus was identified on 18p by Young posed as potential candidate genes for high myo-
et al. in 1998 through a genome-wide scan for pia [48].
myopia loci in eight multigenerational high myo- The MYP6 locus on 22q12 region was discov-
pia families with autosomal dominant pattern; ered among 44 Ashkenazi Jewish families with
one of the pedigrees was ethnic Chinese [41, 42]. myopia less than −1.0D by performing the
In 2003, our group investigated the coding exons genome-wide linkage scan [49]. SCO2 gene on
of the transforming growth factor-beta-induced 22q12, which codes for a copper homeostasis
factor (TGIF) on the MYP2 locus by screening protein in the mitochondrial cytochrome c oxi-
the DNA sequence among 71 high myopia sub- dase activity, has been identified for high myopia
jects and 105 control subjects, all Hong Kong in an 11-member family with the autosomal
Chinese. Six single-nucleotide polymorphisms dominant inheritance [50].
(SNPs) showed a significant difference between The MYP7, MYP8, MYP9, and MYP10 loci
patients and controls, and four of them caused have been identified by Hammond et al. who per-
codon changes, suggesting TGIF as a susceptibil- formed a genome-wide scan in 221 dizygotic
ity gene for high myopia [43]. In 2005, Scavello twin pairs with different refractions. Totally four
et al. studied nine candidate genes on the MYP2 loci were observed at chromosomes 11p13
locus in a Caucasian cohort but did not identify (MYP7), 3q26 (MYP8), 4q12 (MYP9), and 8p23
any alteration on these genes in high myopia sub- (MYP10). As the PAX6 gene was located on the
jects [44]. MYP7 locus, the tagging SNPs showed strong
evidence of linkage, suggesting a role of PAX6 in
myopia [51].
34.4.3 MYP3 MYP11 on chromosome 4q22-q27 was identi-
fied in an autosomal dominant Chinese family of
The MYP3 locus at 12q21-q23 was identified in a 12 members affected with high myopia. Retinal
large German/Italian family of high myopia with pigment epithelium-derived rhodopsin homolog
autosomal dominant inheritance [48]. Decorin on gene (RRH), located on the MYP11 locus, was
12q23 and lumican (LUM) on 12q21.3-q22 were excluded as a causative gene, as subse-
suggested as candidate genes, in view of their quent sequence analysis did not identify any
roles in coding the proteoglycan proteins, an causative mutations [52].
important component of the extracellular matrix MYP12 on chromosome 2q37.1 was identified
of sclera [45]. However, the lumican gene could in a large US family originated from Northern
not be identified on the MYP3 locus in their sub- Europe with high myopia in an autosomal domi-
sequent study [46]. In 2010, Lin et al. detected nant pattern. However, sequencing analyses of the
the haplotype of four SNPs at the promoter region potential candidate genes in the region, S-antigen
of LUM gene, which was significantly different (SAG) and diacylglycerol kinase-delta (DGKD),
between high myopia subjects and controls in a did not find any causative mutations [53].
Taiwan Chinese population. A novel SNP of MYP13 was identified in a four-generation
LUM (c.1567 C/T) associated with high myopia Chinese family of high myopia with X-linked
was identified [47]. inheritance, mapping to Xq23-q25. In addition to
high myopia, the affected individuals had reduced
permanent visual acuity and typical high myopic
34.4.4 Other Myopia Loci fundal changes [54].
MYP14 on chromosome 1p36 was detected
The MYP5 locus on 17q21-q22 was identified in from 49 multigenerational Ashkenazi Jewish
a multigenerational English/Canadian family families by conducting quantitative trait locus
with autosomal dominant pattern of severe myo- linkage analysis [55].
34 Myopia Genes in Asians 421
MYP15 was mapped to 10q21.1 among a large MYP22 was identified in a four-generation
Hutterite family from South Dakota segregating Chinese family with autosomal dominant high
high myopia [56]. Protocadherin 15 (CDH15) myopia. Exome sequencing identified a missense
and ZWIO interactor (ZWINT), within the link- mutation in the CCDC111 gene that was segre-
age region, were screened, but no causative muta- gated with disease in the family while absent in
tion was found. 270 Chinese controls [64].
MYP16 was identified by our group when we MYP23 was identified in three consanguine-
completed one of the first genome-wide scans in ous Saudi Arabian families of non-syndromic
Asians, on the chromosome 5p15.33-p15.2, in extreme myopia by exome sequencing, which
three Hong Kong Chinese pedigrees with autoso- revealed two homozygous truncating mutations
mal dominant high myopia [57]. in the LRPAP1 gene on chromosome 4p16.3.
Naiglin et al. found MYP4 locus on chromo- Linkage analysis showed one peak on chromo-
some 7q36 in 21 French families and 2 some 4 with a LOD score of 7 [65]. Subsequently,
Algerian families with autosomal dominant high LRPAP1 gene mutations were identified in 298
myopia in 2002 [58]. Subsequently in 2008, Chinese families with early-onset high myopia
Paget et al. studied 26 families and demonstrated [66].
significant linkage to chromosome 7p15 named MYP24 was found in a three-generation
MYP17 in a nonparametric model [59]. Ciner Chinese family segregating autosomal dominant
et al. reported results of a QTL linkage analysis high myopia by whole-genome linkage analysis
for ocular refraction in 96 African-American and exome sequencing, with SLC39A5 mutations
families with myopic probands to chromosome in high myopia individuals [67].
7p15 [60]. MYP25 was identified in a three-generation
MYP18 on chromosome 14q22.1-q24.2 was Chinese family segregating with autosomal dom-
identified from a consanguineous Chinese family inant high myopia by whole-exome sequencing.
with autosomal recessive high myopia vis A missense mutation in the P4HA2 gene on chro-
genome-wide linkage analysis [61]. mosome 5 was found in 5 high myopic members
MYP19 was identified on chromosome of the family, but not in 626 controls [68].
5p15.1-p13.3 in a linkage analysis in a four- It is notable that starting from MYP16 reported
generation Chinese family segregating autosomal in 2008, eight myopia loci were identified in
dominant high myopia [62]. Chinese pedigrees.
MYP20, unlike the previously reported loci,
was identified in a genome-wide association
study of 493,947 SNPs in 419 Han Chinese 34.5 M
yopia Genes Identified
individuals with high myopia and 669 unrelated from GenomeWide
controls. The SNP rs9318086 at 13q12.12 Association Studies (GWAS)
showed significant association with high myo-
pia (P = 1.91E-16). The genes MIPEP, Since 2009, a total of 13 GWAS have been
C1QTNF9B-AS1, and C1QTNF9B were at the reported for myopia, refractive errors, and axial
locus, and two of them (MIPEP and C1QTNF9B) length (AL) (Table 34.2). Seven of them investi-
were expressed in the retina and retinal pigment gated genetic variants for high myopia or myo-
epithelium [20]. pia, of which six were conducted in Asians and
MYP21 on chromosome 1p22.2 was identified one in Caucasians. In 2009, Nakanishi et al.
by exome sequencing and segregation analysis in reported the first GWAS for myopia. SNP
a five-generation Han Chinese family with auto- rs577948, within 200 kb DNA encompassed by
somal dominant inheritance of high myopia, with BLID and LOC399959, was associated with high
a missense mutation in the ZNF644 gene, which myopia in Japanese [18]. In 2011, rs10034228 in
was expressed in human retinal and retinal pig- MYP11 was associated with high myopia in a
ment epithelium [63]. Chinese cohort [17]. Shi and our group reported
Table 34.2 Genes identified for myopia, refractive errors, and axial length from genome-wide association studies
422
Sample size P value

Authors Years Case Control Disease/phenotype Ethnicity SNP Chromosome Related gene OR (pooled) References
Nakanishi 2009 839 1914 High myopia Japanese rs577948 11 BLID, LOC399959 1.37(1.21–1.54) 2.22E–07 [18]
et al.
Li et al. 2011 2891 10,071 High myopia Chinese rs10034228 4 MYP11 0.81(0.76–0.86) 7.70E–13 [17]
Shi et al. 2011 3222 6311 High myopia Chinese rs9318086 13 MIPEP 1.64(1.46–1.85) 1.91E–16 [20]
Fan et al. 2012 4944 AL/high myopia Chinese, rs4373767 1 ZC3H11B 0.75(0.68–0.84) 2.69E–10 [11]
Malay
Khor et al. 2013 1603 3427 High myopia Singapore rs13382811 2 ZFHX1B 1.33 7.44E10–9 [13]
Chinese, rs6469937 8 SNTB1 0.75 6.08E10–8
Japanese
Shi et al. 2013 2758 4605 High myopia Han rs2730260 7 VIPR2 1.77(1.47–2.14) 8.95E–14 [19]
2741 4599 Chinese rs4455882 8 SNTB1 0.59(0.47–0.73) 2.13E–11
Kiefer 2013 25,999 19,772 Myopia European rs12193446 6 LAMA2 0.79(0.76–0.81) 1.4E10–45 [14]
et al.
rs1381566 11 LRRC4C 1.15(1.12–1.18) 3E10–26
rs17648524 16 RBFOX1 1.1(1.08–1.12) 1.3E10–22
rs7744813 6 KCNQ5 0.91(0.89–0.93) 6.6E10–22
rs3138142 12 RDH5 0.89(0.87–0.91) 1.8E10–20
chr8:60178580 8 TOX/CA8 0.91(0.90–0.93) 3.5E10–19
rs524952 15 GOLGA8B/GJD2 1.09(1.07–1.11) 5.6E10–19
rs2137277 8 SFRP1 0.9(0.88–0.92) 4.7E10–16
rs1550094 2 PRSS56 1.09(1.07–1.11) 1.3E10–15
rs2908972 17 SHISA6 1.07(1.06–1.09) 4.5E10–13
rs17412774 2 PABPCP2 0.93(0.92–0.95) 1.1E10–12
rs11145746 9 TJP2 1.09(1.06–1.11) 2.3E10–11
rs28412916 15 RASGRF1 1.07(1.05–1.09) 3.5E10–11
rs5022942 4 BMP3 1.08(1.05–1.10) 1.4E10–10
rs745480 10 RGR 1.06(1.04–1.08) 2.5E10–10
rs2155413 11 DLG2 1.06(1.04–1.08) 4.7E10–10
rs13091182 3 ZBTB38 0.94(0.92–0.96) 9E10–10
rs17400325 2 PDE11A 1.14(1.10–1.19) 1.9E10–9
rs17428076 2 DLX1 0.94(0.92–0.96) 2.8E10–9
rs6480859 10 KCNMA1 1.06(1.04–1.08) 1.2E10–8
chr14:54413001 14 BMP4 0.95(0.93–0.96) 1.7E10–8
S. Tang et al.
Sample size P value
rs4291789 13 ZIC2 1.07(1.05–1.09) 2.1E10–8
Beta (for
quantitative trait)
Cheng 2013 20,747 AL European, rs4074961 1 Intron 4 of RSPO1 0.07 4.00E–13 [10]
et al. Asian
rs994767 1 7 kb upstream of −0.07 9.60E–12
ZC3H11B
rs9811920 3 Intron 1 of C3orf26 0.08 4.90E–11
rs12193446 6 Intron 58 of 0.12 1.20E–08
34 Myopia Genes in Asians
LAMA2
rs11073058 15 57 kb upstream of 0.07 4.30E–11
GJD2
rs12321 22 3' UTR of ZNRF3 −0.05 4.10E–08
Hysi et al. 2010 17,684 Refractive errors Caucasian rs939658 15 RASGRF1 −0.15 1.85E–09 [12]
Solouki. 2010 15,608 Refractive errors Caucasian rs634990 15 GJD2 and ACTC1 −0.23 2.21E–14 [21]
et al.
Verhoeven 2013 45,758 Refractive errors European, rs1652333 1 CD55 −0.112 3.05E10–12 [23]
et al. Asian
rs1656404 2 PRSS56 −0.153 7.86E10–11
rs1881492 2 CHRNG −0.139 5.15E10–11
rs14165 3 CACNA1D 0.10 2.14E10–8
rs1960445 4 BMP3 −0.114 1.25E10–6
rs12205363 6 LAMA2 0.24 1.79E10–12
rs4237036 8 CHD7 0.09 1.82E10–8
rs7837791 8 TOX 0.11 3.99E10–12
rs7829127 8 ZMAT4 0.12 3.69E10–10
rs7042950 9 RORB −0.096 4.15E10–8
rs10882165 10 CYP26A1 −0.107 1.03E10–11
rs7084402 10 BICC1 −0.108 2.06E10–13
rs11601239 11 GRIA4 −0.095 5.92E10–9
rs3138144 12 RDH5 0.12 4.44E10–12
rs2184971 13 PCCA 0.09 2.11E10–8
rs8000973 13 ZIC2 0.08 5.1E10–8
rs524952 15 GJD2a −0.158 1.44E10–15
(continued)
423
424
Sample size P value

rs4778879 15 RASGRF1a −0.102 4.25E10–11
rs17183295 17 MYO1D −0.131 9.66E10–11
rs4793501 17 KCNJ2 0.08 2.79E10–8
rs12971120 18 CNDP2 0.10 1.85E10–7
Stambolian 2013 26,953 Refractive errors European rs10500355 16 RBFOX1 −0.11 3.90E–09 [22]
et al. ancestry
Fan et al. 2016 50,356 Refractive errors European, rs60843830 2 FAM150B-ACP1 −0.10 1.27E10–9 [69]
Asian
rs10946507 6 LINC00340 −0.08 2.24E10–8
rs8023401 15 FBN1 −0.13 2.85E10–9
rs16949788 15 DIS3L-MAP2K1 −0.13 2.19E10–8
rs10880855 12 ARID2-SNAT1 −0.09 4.38E10–8
rs10853531 18 SLC14A2 −0.11 2.54E10–8
S. Tang et al.
a high myopia-associated SNP rs9318086 in a 34.6 M

yopia Candidate Genes
combined Chinese cohort. The locus contains in Asians
three genes, MIPEP, C1QTNF9B-AS1, and
C1QTNF9B [20]. A GWAS in Singapore includ- A number of myopia candidate genes have been
ing Chinese and Malays revealed the protective suggested from various association studies. We
effect of the minor C allele of rs4373767 on herein categorize these genes into sclera-related
ZC3H11B against high myopia and longer genes, growth factor-related genes, master con-
AL. The neighboring genes SLC30A10 and trol genes, muscarinic-related genes, and other
LYPLAL1 were expressed in the retinal pigment genes (Table 34.3).
epithelium and sclera [11]. A genome-wide meta-
analysis on high myopia and 286,031 SNPs in a
combined Han Chinese cohort of 665 cases and 34.6.1 Sclera-Related Genes: Matrix
960 controls identified two myopia-associated Metallopeptidase Genes,
genes, VIPR2 and SNTB1 [19]. Khor et al. found Collagen-Related Genes,
association of SNP rs13382811 in ZFHX1B with and Proteoglycan-Related
severe myopia in Chinese and Japanese [13]. In Gene
20 genetic regions associated with the age of
myopia onset, 16 of them were near to genes In myopes, the axial length is elongated, and
involved in eye development in Caucasian [14]. sclera is stretched resulting in its thinning and
In addition to high myopia, the myopia-related remodeling. Sclera is comprised of extracellular
quantitative traits including refractive errors and matrix, matrix-secreting fibroblasts, collagen,
AL have been investigated in six GWAS or and proteoglycans. Scleral remodeling is believed
genome-wide meta-analyses. SNP rs8027411 on to be a critical determinant in the development of
15q25 was associated with refractive errors in myopia and is mediated by increasing collagen
Caucasians [12]. Another Caucasian study identi- degradation and decreasing collagen and proteo-
fied a myopia locus at chromosome 15q14, where glycans production to reduce extracellular matrix
the GJD2 and ACTC1 genes were found to express production [70]. The vitreal concentration of
in the retina and sclera in animal models [21]. A matrix metallopeptidase 2 (MMP2) was found to
genome-wide meta-analysis among multi-ances- be higher in high myopic eyes than non-myopic
try cohorts including 37,382 European and 8376 eyes [71]. Animal studies showed that the scleral
Asians reported 18 refractive error-associated MMP expression was increased in form-
SNPs [23]. Meta-analysis of the GWAS data of deprivation myopic tree shrews and in guinea
five European cohorts identified association of pigs [72–75]. MMP1 and MMP2 genes were
RBFOX1 rs10500355 with refractive error [22]. found to be associated with refractive error in
RBFOX1 was involved in neuronal development Amish families [76] but not in Asians. Scleral tis-
and maturation. In a study involving both sue contains about 90% of collagen by weight
European and Asian study subjects, eight loci [77]. Collagen, type I, alpha 1 (COL1A1) gene
were associated with axial length, and four of was shown to be associated with high myopia in
them also associated with refractive errors [22]. Chinese and Japanese populations [78, 79].
Recently, Fan et al. conducted a meta-analysis of Proteoglycans are other major components of the
gene-environment-wide association scans for scleral extracellular matrix to regulate collagen
refractive errors in European and Asian myopia fibril assembly and interaction [80]. The LUM
subjects, including the SNP- education interac- gene is a member of small leucine-rich proteo-
tion. Six loci for refractive error have been identi- glycan (SLRP) gene family [80]. Proteoglycans
fied in the European ancestry individuals and are major components of the scleral extracellular
three in the Asians. Notably, the Asian loci showed matrix, which plays an important role in
significant interaction with education level, but regulating collagen fibril assembly and interac-
this effect was less evident in Europeans [69]. tion, and are intensely related to the structure and
426 S. Tang et al.
Table 34.3 Candidate genes for myopia in Asians

Gene symbol Gene name Location Ethnicity Phenotype References
Collagen-related genes
COL1A1 Collagen type I alpha 1 17q21.33 Chinese, High myopia (≤-6D) [78, 79]
Japanese
Proteoglycan-related genes
LUM Lumican 12q21.33 Chinese High myopia (≤-10D) [47, 81, 82]
Growth factor-related genes
TGFB1 Transforming growth 19q13.2 Chinese High myopia (≤-6D) [85, 86]
factor-beta 1
TGFB2 Transforming growth 1q41 Chinese High myopia (≤-6.5D) [87]
factor-beta 2
TGIF Transforming growth 18p11.31 Chinese High myopia (≤-6D) [43]
factor-beta-induced factor
HGF Hepatocyte growth factor 7q21.11 Chinese High myopia (≤-10D) [89]
MET Hepatocyte growth factor 7q31.2 Chinese High myopia (≤-6D) [90]
receptor
IGF1 Insulin-like growth factor 1 12q23.2 Chinese High myopia (≤-9D) [92]
Master control genes
PAX6 Paired box 6 11p13 Chinese, High myopia (≤-6D) [100–104]
Japanese
Muscarinic-related genes
CHRM1 Cholinergic receptor 11q12.3 Chinese High myopia (≤-6.5D) [107]
muscarinic 1
CHRM2 Cholinergic receptor 7q33 Chinese High myopia (≤-6D) [108]
muscarinic 2
CHRM3 Cholinergic receptor 1q43 Chinese
muscarinic 3
CHRM4 Cholinergic receptor 11p11.2 Chinese
muscarinic 4
Others
LAMA1 Laminin, alpha 1 18p11.31-p11.23 Chinese High myopia (≤-6D) [109]
UMODL1 Uromodulin-like 1 21q22.3 Japanese High myopia (≤-9.25D) [110]
CRYBA4 Crystallin beta A4 22q12.1 Chinese High myopia (≤-8D) [111]
BMP2K BMP2-inducible kinase 4q21.21 Chinese High myopia (≤-6D) [112]
MYOC Myocilin 1q24.3 Chinese High myopia (≤-6D) [113]
function of the sclera [80]. LUM polymorphisms in a decrease in collagen synthesis of sclera [84].
were found to be associated with high myopia in TGFB1 has been reported as a susceptibility gene
Chinese populations [47, 81, 82]. for high myopia in Chinese populations [85, 86].
Another scleral structure-related gene, TGFB2
gene, was also associated with the development of
34.6.2 Growth Factor-Related Genes high myopia in Chinese [87]. The aqueous con-
centration of TGFB2 was higher in Chinese sub-
Ocular elongation involves active scleral growth jects with longer axial length [88]. In addition,
[83]. Growth factors, such as transforming growth our group identified the transforming growth fac-
factor-beta 1 (TGFB1), are expressed in ocular tor-beta-induced factor (TGIF) gene as a candi-
tissues to regulate fibroblast proliferation and col- date gene for high myopia in Chinese [43].
lagen production of sclera. Animal studies showed Hepatocyte growth factor (HGF) is an
that isoform-specific changes in TGFB1 resulted important multifunctional cytokine for cellular
scattering and proliferation. Han et al. found the myopia development [105, 106]. In Chinese,
association of HGF gene with high myopia in a high myopia is associated with the muscarinic
Han Chinese population [89]. Furthermore, the acetylcholine receptor genes CHRM1, CHRM2,
hepatocyte growth factor receptor (MET) gene CHRM3, and CHRM4 [107, 108]. Other
was also associated with high myopia in Chinese myopia- associated genes identified in Asian
[90]. Animal studies have demonstrated the role populations include laminin alpha 1 (LAMA1)
of insulin and insulin-like growth factor 1 (IGF1) [109], uromodulin- like 1 (UMODL1) [110],
in retinal development and ocular growth [91]. crystallin beta A4 (CRYBA4) [111], BMP2-
The IGF1 gene was associated with high myopia inducible kinase (BMP2K) [112], and myocilin
in Chinese adults [92]. This association was rep- (MYOC) [113].
licated in the Egyptians [93] but not in other
Asian populations [94–96].
34.7 Rare Variants in Asians
34.6.3 PAX6 The advent of advanced and high-throughput

genomic technologies, specifically genome-
The paired box 6 (PAX6) gene is a member of the wide association studies (GWAS), whole-exome
paired-domain Pax family. It regulates tissue- sequencing (WES), and next-generation
specific expression of diverse molecules, includ- sequencing, provide important tools to discover
ing transcription factors, cell adhesion molecules, gene variants for myopia or refractive errors. In
hormones, and structural proteins [97]. The 2011, Shi et al. first identified causative muta-
PAX6 protein is a transcriptional factor, regulat- tions in ZNF644 among 11 Chinese high myo-
ing the eyeball development [98, 99]. In 2004, pic patients by exome sequencing [63]. In 2013
Hammond et al. first revealed PAX6 as a suscep- a novel missense variant of the CCDC111 gene
tibility gene for myopia [51]. Later, two Chinese was discovered for high myopia in Chinese
family studies showed the presence of PAX6 vari- patients [64]. SLC39A5 mutations regulating the
ants in extreme refractive error [100]. Our group BMP/TGF-β pathway were identified in a
found that the AC and AG dinucleotide repeats in Chinese high myopia family and a sporadic
the PAX6 P1 promoter affected its transcription patient [67]. A mutation of the LEPREL1 gene
activities and was associated with high myopia was identified in an autosomal-recessive high
[101]. SNP rs662702 at the 3′ untranslated region myopia family with co-existing early-onset cat-
(UTR) of PAX6 was reported as a risk marker for aract. LEPREL1 is involved in the collagen
extreme myopia in a Taiwan Chinese study [102]. modifications in the eye development [114].
The PAX6 haplotypes GTAA, AGTG, and AGTA, Another causative gene for high myopia,
defined by rs2071754, rs3026393, rs1506, and P4HA2, was also identified by the same group
rs12421026, were associated with high myopia [68]. Jiang et al. identified a h omozygous frame-
[103]. Meta-analysis of the published data con- shift mutation in LRPAP1 gene in a Chinese
firmed association of PAX6 rs644242 with consanguineous family with high myopia [66].
extreme and high myopia [104]. Frameshift mutations in LOXL3 were identified
in two of the 298 probands with early-onset high
myopia [115]. In 2017, Jin et al. pioneered a
34.6.4 Muscarinic Related and Other whole-exome study for high myopia in 18 unre-
Genes lated Chinese trios. All probands had early-
onset high myopia before the age of 6 with both
Animal and clinical studies have showed the parents non-myopic. The results revealed de
effect of atropine, a muscarinic acetylcholine novo mutations in the BSG gene in early-onset
antagonist, in retarding axial elongation and high myopia [116].
428 S. Tang et al.
34.8 Gene-Environment variants. However, currently none of the reported

Interaction genes has been shown to account for even a mod-
est fraction of risk of myopia. Furthermore,
Both genetic and environmental factors contrib- inconsistency of data exists among various stud-
ute to myopia development in all studied popula- ies. The intrinsic genetic constitutions and exter-
tions over the world. Independent effect of each nal environmental factors that lead to the
factor is not possible to be separated from each strikingly high prevalence and severity of myopia
other. Interactions are involved. For example, in East Asians as compared with other ethnic
children with myopic parents are more likely to populations have to be explored and understood
be raised in myopiogenic environment than those through vigorous and large-scale research with
with non-myopic parents. Gene-environment multicenter efforts. Replication and aggregation
interaction studies have been reported recently cohort studies to confirm the true association are
[69, 117]. Joint meta-analysis approach on SNP necessary. Functional studies on identified genes
main effects and SNP-environment interactions should be conducted to throw light on the mecha-
was conducted [118]. Fan et al. investigated the nism of myopia. The role of environmental fac-
interaction effect of education with those SNPs tors to genetic influences should be further
identified from previous GWAS on refractive explored to explain the heterogeneity of myopia
error in Chinese, Malay, and Indian. Three development.
genetic loci SHISA6-DNAH9, GJD2, and
ZMAT4-SFRP1 exhibited a strong association Compliance with Ethical Requirements Shumin Tang,
with myopic refractive error in individuals with Yu Meng Wang, Aziz K. W. Kam, Tommy C. Y. Chan,
Calvin C. P. Pang, Jason C. S. Yam, and Guy L. J. Chen
higher secondary or university education. Low declare that they have no conflict of interest. No human or
level of education may attenuate the effect of risk animal studies were performed by the authors for this
alleles on myopia [117]. The Consortium for article.
Refractive Error and Myopia (CREAM) also per-
formed a joint meta-analysis to test gene-
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Keratoconus Genes in Chinese
35
Yu Meng Wang, Ka Wai Kam, Tommy C. Y. Chan,
Alvin L. Young, Vishal Jhanji, Guy L. J. Chen,
and Calvin C. P. Pang
Abstract no complete cure. Keratoconus is a multifac-

Keratoconus is a corneal disease characterized torial disease resulting from the interaction of
by conical protrusion and progressive thinning environmental, behavioral, and genetic fac-
of the cornea, resulting in various degrees of tors. Its progression has been associated with
visual impairment. Keratoconus affects all structural, biochemical, cellular, and molecu-
ethnic groups, but the prevalence is higher lar alternations in corneal collagen lamellae,
among the Asian populations. The onset of higher systemic oxidative stress, and modifi-
keratoconus is insidious and often occurs dur- cations in corneal proteins. However, the eti-
ing late childhood. Early diagnosis is difficult. ology of keratoconus remains unclear and the
If untreated, the disease often progresses irre- exact regulatory mechanism still elusive.
versibly and can lead to blindness. Nowadays, There is evidence of familial aggregation,
corneal collagen cross-linking has shown monozygotic twin concordance, association
some promising results in retarding or halting with other genetic diseases, and the ethnic dif-
keratoconus progression, but currently there is ference in prevalence and incidences. This
chapter attempts to summarize the current
knowledge and research of keratoconus epide-
miology, pathology, and genetics, with a par-
Y. M. Wang · T. C. Y. Chan · C. C. P. Pang (*) ticular focus for studies in the Chinese
Department of Ophthalmology and Visual Sciences, population.
The Chinese University of Hong Kong, Hong Kong
Eye Hospital, Kowloon, Hong Kong
e-mail: [email protected] Keywords
Keratoconus · Genetics · Epidemiology ·
K. W. Kam · A. L. Young · G. L. J. Chen
Department of Ophthalmology and Visual Sciences, Pathology · Chinese
Prince of Wales Hospital, Shatin, Hong Kong 35.1 Introduction
V. Jhanji
Department of Ophthalmology and Visual Sciences, Keratoconus is characterized by progressive cor-
neal thinning, irregular astigmatism, and loss of
vision [1]. About 20% of the patients require
UPMC Eye Center, University of Pittsburgh School
keratoplasty for visual rehabilitation worldwide
of Medicine, Pittsburgh, PA, USA

https://doi.org/10.1007/978-981-13-0884-0_35
436 Y. M. Wang et al.
[1, 2]. Keratoconus exists in all ethnic groups k eratoconus. In a genome-wide association study
with reported prevalence ranging from 54.5 to (GWAS) that involves our Hong Kong Chinese
230 per 100,000 [2–5]. Previous studies indicated samples, 26 loci associated with central corneal
that Asians have a high incidence of keratoconus thickness (CCT) have been identified. Among
[6–8]. Onset of the disease typically starts at them, FOXO1 and FNDC3B conferred a higher
puberty or early adulthood followed by variable risk of keratoconus [18]. An exome sequencing
progression [1, 4]. The diagnosis of keratoconus analysis also found a WNT10A variant that is
is made by a combination of clinical history, slit associated with corneal thickness and an
lamp examination, and corneal tomographic find- increased risk of keratoconus [19]. However,
ings [9]. Despite rapid advancements of ophthal- most of these associations were inconsistent
mic investigative technologies and imaging across different study cohorts. Roles of the genes
techniques in recent years, early diagnosis of the and loci remain inconclusive. In this chapter, we
disease remains challenging. Furthermore, the attempt to summarize the current knowledge of
rate of disease progression is variable. Despite its keratoconus epidemiology, pathology, and genet-
progressive nature, a few large-scale observa- ics, especially in the Chinese population.
tional cohort studies have examined the natural
history and rate of progression. Factors associ-
ated with a higher likelihood of progression 35.2 Epidemiology
included young age of disease onset, corneal
scaring, and worse visual acuity at disease onset During the past decade, a large number of kerato-
[10, 11]. Corneal cross-linking is able to stabilize conus studies have been conducted in European,
the corneal curvature and visual function. Studies American, Asian, African, and Australian popu-
of our Hong Kong Chinese cohort of 45 keratoco- lations that contribute important information that
nus patients have shown cross-linking halts the help detection and treatment (Table 35.1).
progression [12]. The etiology of keratoconus is Globally, the prevalence of keratoconus ranged
multifactorial, with both genetic and environ- from 54.5 to 230 per 100,000 [2–5]. Such wide
mental risk factors, such as ultraviolet light expo- range may be partly due to the nonuniform diag-
sure, eye rubbing, and consanguinity, contributing nostic criteria employed by different studies and
to the disease pathology [13]. Previous studies partly caused by genetic variations. Hence rigor-
have suggested the inheritance of keratoconus ous, multiethnic, population-based epidemiologi-
development, including twin studies [14], famil- cal studies for keratoconus are needed. While
ial aggregation studies [15], and linkage analyses occurrence of keratoconus is polyethnic, Asians
[16, 17]. Genome-wide association studies have higher incidence, younger age of onset, and
(GWAS) and candidate gene association studies faster progression compared to other ethnicities
have identified over 150 gene polymorphisms in [6–8]. Among Asians, Indians [20], Pakistanis
more than 60 genes/loci related to the risk of [21, 22], Saudi Arabians [23], and Iranians (22.3–
Table 35.1 Reported incidence of keratoconus worldwide

Region Coverage Study period Incidence Source
American Olmsted County, 1935–1982 54.5 per 100,000 Kennedy RH et al. 1986 [2]
Minnesota
Saudi Arabia Asir Province 2001–2002 20 per 100,000 Assiri AA et al. 2005 [23]
Iranian Yazd province 2008–2009 22.3–24.9 per 100,000 Ziaei H et al. 2012 [24]
Japanese Tokyo 2000 12 per 100,000 (males) 5.6 per Ota R et al. 2002 [25]
100,000 (females)
Chinese Beijing 2011 700–1100 per 100,000 Xu L et al. 2012 [26]
35 Keratoconus Genes in Chinese 437
24.9 per 100,000) [24] have higher prevalence able to discriminate normal and keratoconus cor-
and incidence. The incidence is relatively lower neas [28–30]. Although the diagnosis of advanced
(12 in 100,000 males and 5.6 in 100,000 females) keratoconus can still be made based on the pres-
in the Japanese [25]. In Chinese, a population- ence of abnormal corneal tomographic indices,
based study calculated the prevalence of steep differentiation of forme fruste keratoconus from
cornea/keratoconus of Chinese to be 0.9 ± 0.2% normal corneas can be difficult [31]. Albeit an
in the Beijing Eye Study [26]. The low preva- abundance of indices, there is currently no con-
lence may not represent the prevalence for the sensus on the optimal cutoff values or a single
whole of China. More population-based epide- best parameter in diagnosing or monitoring kera-
miological studies in different parts of China are toconus. Keratoconus corneas also have changes
required. According to currently reported epide- in biomechanical properties. Ocular Response
miologic data, there are indications that ultravio- Analyzer (ORA, Reicherts®) [32] and Corvis ST
let irradiation exposure, geographic locations, (Oculus Optikgeräte GmbH; Wetzlar, Germany)
and environment-related ocular conditions, such [33, 34] have been used to perform biomechani-
as atopy, eye rubbing, and consanguinity, may be cal assessment and help differentiating keratoco-
the underlying causes for the higher prevalence. nus from normal eyes. Combining tomographic
and biomechanical data collected by the
Pentacam and Corvis ST devices, we found com-
35.3 Symptoms and Diagnosis parable diagnostic abilities to differentiate kera-
toconus from normal subjects [35, 36]. The
Clinical features of keratoconus vary according Amsler-Krumeich classification is a commonly
to the stage of disease. There is a spectrum in used diagnostic tool. It combines information
physical signs: from keratoconus suspect to from biomicroscopy, keratometry, refraction, and
forme fruste keratoconus, to progressive pachymetry for keratoconus staging [37, 38]. The
advanced keratoconus. It is therefore difficult to ultimate goal of clinicians and researchers is to
determine the exact onset of disease. As the dis- improve the sensitivity of screening methods in
ease progresses, slit lamp should detect order to accurately and reliably identify early-
Fleischer’s ring, Vogt’s striae, and central or para- stage keratoconus, perform accurate classifica-
central stromal thinning. In advanced stages, cor- tion, initiate intervention, and prevent iatrogenic
neal scars can be evident [10]. But the patients keratectasia following elective laser refractive
may present with the complication of acute surgery.
hydrops. In earlier stages of the disease, the cor-
nea may appear normal on a cursory slit lamp
examination, but ophthalmic investigations by 35.4 Disease Etiology
retinoscopy, keratometry, and corneal topogra- and Pathology
phy may yield additional evidence to aid diagno-
sis. The increasing utilization of tomographic Keratoconus corneas tend to have reduced num-
techniques and corneal imaging greatly assist the ber and altered orientation of collagen lamellae,
detection and monitor its progression. Major top- causing a reduction in biomechanical stability
ographic patterns found in keratoconus include [39]. In vivo confocal images show abnormal
asymmetric bowtie, skewed radial axis, and infe- features in all layers of the cornea, including
rior steepening [27]. Scheimpflug tomography, abnormal epithelial and stromal keratocytes [40].
especially Pentacam® (Oculus Optikgeräte Reduction in keratocyte density has also been
GmbH; Wetzlar, Germany), is widely applied for documented in keratoconic corneas [41]. Higher
diagnosis of keratoconus. Various diagnostic systemic oxidative stress was associated with
indices derived from tomography, such as keratoconus progression [42]. Matrix-degrading
Keratoconus Severity Index (KSI) and Belin- enzymes (MMPs), which were responsible for
Ambrosio Enhanced Ectasia Display (BAD), are the degradation of the main components of
extracellular matrix and corneal membranes, studies in Asia, including Hong Kong [60],
were present in keratoconic corneas and involved Malaysia [61], and Korea [62].
in the pathogenesis [43, 44]. Previous studies
also identified inflammatory cytokines that were
associated to keratoconus [45, 46]. Proteome 35.6 Family Aggregation
analysis demonstrated degenerative process in and Linkage Studies
keratoconus with abnormal mitochondrial func-
tions, increased cell death, and lipid metabolism A study from the United States reported two pairs
[47]. However, no conclusive molecular mecha- of discordant monozygotic twins for keratoco-
nism has been identified, while the etiology nus. Some family members demonstrated corneal
should be complex, affected by interactive topographical abnormality, suggesting genetic
genetic and environmental factors [13]. abnormality [14]. A profile study in New Zealand
Keratoconus has been linked to systemic disor- that included familial aggregation analysis has
ders including Down syndrome, Ehlers-Danlos identified that the keratoconus familial rate is
syndrome, and osteogenesis imperfect [6, 48]. 23.5% [15]. Linkage analysis studies have identi-
However, there is no proven direct cause-and- fied chromosomal loci in isolated, i.e., sporadic,
effect relationship. Allergic diseases have been keratoconus patients, including 2p24 [63], 3p14-
suggested to be associated with developmental q13 [64], 5q14.3-q21.1 [17], 13q32 [65],
keratoconus [49, 50]. Reports based on clinical 16q22.3-q23.1 [66], and 20q12 [16]. However,
observation have implicated eye rubbing as a risk among all these loci, no disease-causing mutation
factor for keratoconus [48]. Contact lens use has has been identified. Family history of keratoco-
been implicated in the development of keratoconus has been reported in about 14% of the cases
nus but still with little evidence [51, 52]. The eti- [10], but the definitive role of inheritance patterns
ology and pathology of keratoconus are has not been determined. There are no obvious
complex. clinical differences between familial and spo-
radic keratoconus. As a genetic disease, kerato-
conus has shown weak penetrance and
35.5 Inherited Disease- demonstrated significant variability of expres-
Associated Risk Factors sion. While family aggregation and linkage stud-
ies for keratoconus showed that genetic factors
Keratoconus is reportedly associated with vari- were associated with familial inheritance, the
ous inherited diseases. It had been proposed to be majority of the reported keratoconus patients are
part of generalized heritable disorders, such as sporadic.
Ehlers-Danlos syndrome [53]. Osteogenesis
imperfecta (OI) is a connective tissue inherited
disorder involving genes encoding the synthesis 35.7 Keratoconus Candidate
of type 1 collagen. The ocular features in an Genes
Italian family indicated the association of OI with
keratoconus [54]. Leber congenital amaurosis 35.7.1 Visual System Homeobox 1
(LCA) is a retinal dystrophic disease which can (VSX1)
lead to retinopathy and severe visual impairment.
Keratoconus associated with LCA has been The visual system homeobox 1 (VSX1) gene is a
reported in patients from Pakistan [55, 56], Israel candidate keratoconus gene with elusive patho-
[57], and Australia [58]. One Chinese study genetic mechanism [67, 68]. The VSX1 Q175H
reported that 0.86% of the 233 investigated kera- mutation was shown to play a causative role in an
toconus patients had Down syndrome [59]. Indian familial segregation study [69]. The shared
However, no such associations were noted in haplotype (p.Leu268His) that was identified in
pediatric Down syndrome populations in other five Indian keratoconus patients from two
u nrelated families suggested the possibility of a results. They found two of them, rs1324183
founder effect which required elucidation [70]. (MPDZ-NF1B, chr9:13557491; P = 0.001;
However, VSX1 mutations were absent in iso- OR = 1.68) and rs9938149 (BANP-ZNF49,
lated keratoconus patients [71, 72]. The associa- chr16:88331640; P = 0.010, OR = 1.47), con-
tions were not consistent in two case-control ferred significant association with keratoconus.
studies conducted in South Korea [73, 74]. A sig- An association analysis between CCT-associated
nificant association between keratoconus and variants and keratoconus in a Saudi Arabian pop-
VSX1 genetic alterations (p.R166W and p. ulation attempted to validate eight CCT-
H244R) was identified in Iranian patients [75]. associated SNPs. But none of the association
reached statistical significance [48]. However,
the result might not be conclusive due to limita-
35.7.2 Interleukin 1 Beta (IL1B) tion in sample size and population variation. In a
Han Chinese population in northern China, ten
Keratoconus has been considered to be a nonin- MPDZ-NF1B SNPs were assessed. rs1324183
flammatory corneal disease. However, genetic was associated with increased risk of keratoco-
evidence proved that chronic inflammation may nus (OR = 3.1) [82].
exist in the pathology. Interleukin 1 beta (IL1B) is
a mediator of keratocyte apoptosis [76, 77]. In a
Japanese population, polymorphisms in interleu- 35.7.5 COL4A3, COL4A4, and COL5A1
kin 1 beta (IL1B) promoter region were associ-
ated with keratoconus [78]. Screening for IL1 COL4A3, COL4A4, and COL5A1 are related to
gene cluster mutations in a Korean cohort identi- corneal collagen structure and development dur-
fied −31*C and − 511*T linkages, associated ing embryonic development. Genetic association
with an increased risk for keratoconus (P = 0.012, of variants in these three genes in keratoconus
OR = 2.38, 95% CI = 1.116–5.046) [79]. patients were identified in European and
American cohorts [83, 84]. Another study in an
Iranian keratoconus cohort evaluated the possible
35.7.3 Lysyl Oxidase (LOX) relationship between COL4A4 gene polymor-
phisms and keratoconus and revealed rs2229813
Lysyl oxidase (LOX) is related to copper- as a risk mutation [85].
dependent amine oxidase influencing the devel-
opment of lysine-derived cross-links in
extracellular matrix proteins, such as collagen 35.7.6 Other Genes
and elastin, which play an important role in the
pathogenesis of keratoconus [80]. An association The substitution c.214 + 242C > T in IL1RN and
study in an Iranian population identified that a novel deletion c.2558 + 149_2558 + 203del54 in
lysyl oxidase (LOX) rs1800449 risk allele A con- SLC4A11 were related with keratoconus in an
ferred risk for keratoconus [80]. Ecuadorian family [86]. c.2262A > C (p.
Gln754His) mutation in DOCK9, which is
expressed in corneal epithelium as activator of
35.7.4 MPDZ-NF1B small G-proteins involved in intracellular signal-
ing networks, was shown to contribute to familial
MPDZ (multiple PDZ domain crumbs cell polar- keratoconus [87]. A genomic deletion within
ity complex component) and nuclear factor I B intron 2 close to the 5′ splice junction of the
(NF1B or NFIB) were suggested as a genetic risk superoxide dismutase 1 (SOD1) gene was identi-
of keratoconus. Sahebjada et al. [81] investigated fied in familial keratoconus, suggesting SOD1 to
the association between keratoconus in Australia be a candidate keratoconus gene. SOD1 is
and six CCT-associated SNPs based on GWAS involved in the superoxide radical metabolism
and oxygen toxicity defense [88]. Keratoconus, than 4000 controls identified variations at the
epithelial basement membrane corneal dystrophy, HGF locus with keratoconus susceptibility in
and Fuchs’ endothelial corneal dystrophy were Caucasians [93]. The risk factor allele
associated with zinc finger E-box-binding homeo- (rs3735520), located near the HGF, has strong
box 1 (ZEB1) gene mutation. ZEB1 is involved in association with keratoconus in European origin
epithelial mesenchymal transition [89]. In 89 [96]. HGF was also significantly associated with
patients from a French cohort, 38 had a history of keratoconus in an Australian cohort [97]. HGF
atopic dermatitis or ichthyosis vulgaris, and 5 of regulates epithelial cell motility and growth dur-
them were carriers of filaggrin (FLG) mutants ing wound healing. In 2012, another study ana-
[90]. Linkage analysis and genetic association lyzed association results of the Lysyl Oxidase
indicate involvement of the calpastatin (CAST) gene (LOX) polymorphisms from a GWAS inves-
gene in genetic susceptibility to keratoconus in tigation in two independent cohorts of keratoco-
262 patients in 40 white keratoconus families nus patients, involving 222 Caucasian patients,
[91]. CAST is involved in many aspects of cell 687 African Americans, 3324 Caucasian con-
physiology, including proliferation, apoptosis, trols, and 307 individuals from 70 keratoconus
and migration, and therefore has been proposed to families. The results strongly presented genetic
be having potential involvement in the mecha- evidence that LOX variants lead to increased sus-
nisms of keratoconus development [91]. ceptibility to keratoconus, with meta P values of
2.5 x 10−7 and 4.0 x 10−5 for LOX SNPs rs2956540
and rs10519694, respectively [94]. Our previous
35.8 Keratoconus Genome-Wide GWAS study has identified 26 loci associated
Association Studies with central corneal thickness. Among them,
FOXO1 SNP rs2721051 P = 2.7 x 10−10 and
Genome-wide association studies (GWAS), FNDC3B SNP rs4894535 P = 4.9 x 10−9 con-
together with candidate gene association studies, ferred a higher risk of keratoconus [18].
have identified over 150 polymorphisms in more A comprehensive GWAS with 222 keratoco-
than 60 genes/loci related to keratoconus [92]. nus Caucasian patients and 3324 suggested a
GWAS studies on keratoconus are summarized in novel potential keratoconus locus SNP rs4954218
Table 35.2. Several genes/loci were validated, at 2q21.3, containing a candidate gene
such as HGF [93], LOX [94], FOXO1 and RAB3GAP1 [95]. RAB3GAP1 expressed a cata-
FNDC3B [18], and RAB3GAP1 [95]. A GWAS lytic subunit of GTPase-activating protein spe-
study with 933 keratoconus patients and more cific for the Rab3 subfamily, which participated
Table 35.2 Allelic associations of gene variations with keratoconus using cohorts from both GWAS and subsequent
replication studies
Sample size Test for
No. Studies Study design Country Ethnicity Gene and locus Case Control HWE
1 Burdon KP GWAS + Australia, Whites HGF and 12 loci 933 4164 n.r.
et al. 2011 validation America
2 Li X et al. 2012 GWAS + America Whites 3p26,2q21.3,19q13.3 222 3324 In
validation and 12 loci HWE
3 Bykhovskaya Y GWAS + America Whites LOX 222 3324 In
et al. 2012 validation HWE
4 Lu Y et al. 2013 GWAS + Australia, Whites FOXO1 and FNDC3B 874 6085 n.r.
validation Northern
Ireland and
America
5 Cuellar-Partida Exome Australia Whites WNT10A 621 1680 n.r.
G et al. 2015 sequencing
GWAS genome-wide association study, n.r. not reported
in normal eye development [98]. Another exome MPDZ-NF1B was associated with an increased
sequencing analysis in Australian patients risk of keratoconus (OR = 3.1) [82].
reported a WNT10A variant associated with cor- Our previous GWAS identified 26 loci associ-
neal thickness and an increased risk of keratoco- ated with central corneal thickness, among them,
nus [19]. However, as the results across different FOXO1 and FNDC3B conferred a higher risk of
study cohorts were inconsistent, the roles of these keratoconus [18]. The candidate genes VSX1 and
genes/loci are inconclusive. IL1A showed genetic variations and mutations in
Han Chinese keratoconus population [102]. An
association analysis in Asians identified four
35.9 Keratoconus Meta–Analysis novel loci associated with central cornea thick-
ness in chromosomal regions 6q14.1, 7q11.21,
We have conducted a meta-analysis on 53 single- 9p23, and 15q26.3. Rs1538138 located on chro-
nucleotide polymorphisms (SNPs) in 28 genes/ mosome 6q14.1 near the IBTK gene was the most
loci to find out their genetic associations with significant, meta P = 2.10 x 10−11 [103].
keratoconus. Among them, eight single-Independent genome-wide association studies of
nucleotide polymorphisms (SNPs) in six genes/ corneal curvature across 10,008 Asian samples of
loci are associated with keratoconus in the white Chinese, Malay, and Indian ancestries in
population. Five genes/loci were originally from Singapore identified two loci that were associ-
genome-wide association studies, including ated with corneal curvature variation: FRAP1 on
FOXO1 (rs2721051), RXRA-COL5A1 chromosome 1p36.2 and PDGFRA on chromo-
(rs1536482), FNDC3B (rs4894535), IMMP2L some 4q12 [104]. Meanwhile, mitochondrial
(rs757219; rs214884), and BANP-ZNF469 DNA (mtDNA) is involved in mitochondrial
(rs9938149). The COL4A4 gene (rs2229813; function and affects the generation of reactive
rs2228557) was identified in previous candidate oxygen species. One study in China demon-
gene studies [92]. strated that decreased integrity, content, and
increased transcript level of mtDNA are associ-
ated with keratoconus [105]. Though mitochon-
35.10 Keratoconus Genetic Studies drial haplogroups H and R were identified in
in Chinese Saudi Arabian keratoconus patients [106],
another study in Han Chinese suggested that
Reported studies on keratoconus genetics in mtDNA copy number, but not haplogroup, is
Chinese are summarized in Table 35.3. related to keratoconus [107]. In the future, large-
Polymorphisms in IL1 gene have association scale multicenter genomic studies should be con-
with risk of keratoconus in the Han Chinese pop- ducted in Chinese cohorts to establish the genetic
ulation. The altered levels of IL1 gene could profile of keratoconus.
induce keratocyte apoptosis preceding keratoco-
nus [99]. Besides, polymorphisms of a candidate
keratoconus gene TGFBI have been confirmed to 35.11 Conclusive Remarks
be associated with keratoconus in Han Chinese
[100]. TGFBI plays a role in tissue injury and Keratoconus is a global visual threat with com-
repair that interacts with extracellular matrix pro- plicated environmental and genetic causative fac-
tein, which may also be involved in the pathogen- tors. The progression of keratoconus can be
esis of keratoconus [101]. In a northern Han imperceptible, and the diagnosis of keratoconus
Chinese cohort of keratoconus, 10 SNPs were in the early stage is not easy. The pathogenesis of
studied: rs4894535 (FNDC3B), rs3735520 keratoconus is heterogeneous and complex.
(HGF), rs1324183 (MPDZ-NF1B), rs1536482 Epidemiological studies showed higher preva-
(RXRA-COL5A1), rs7044529 (COL5A1), and lence, earlier onset, and greater progression in
rs9938149 (BANP-ZNF49). SNP rs1324183 in Asians. Both environmental and genetic factors
442
Table 35.3 Allelic associations of gene variations with keratoconus in Chinese

Associated Sample size Outcome
allele vs.
No. Gene/locus SNP Study design Phenotype reference allele Case Control P OR (95% CI) References
1 IL1A rs2071376 CG KCN A vs. C 115 101 0.017 1.968 Wang Y [99]
(1.313– et al.
3.425) 2016
rs2071376 CG KCN A vs. C 97 101 0.0487 1.51 Wang Y [102]
(1.00–2.26) et al.
2013
2 IL1B rs1143627 CG KCN C vs. T 115 101 <0.0001 2.846 Wang Y [99]
(1.631– et al.
4.968) 2016
rs16944 CG KCN A vs. G 115 101 0.002 2.401 Wang Y [99]
(1.396– et al.
4.161) 2016
3 MPDZ-NF1B rs1324183 CG KCN A vs. C 210 191 0.005 3.108 Hao XD [82]
(1.366– et al.
7.072) 2015
rs1324183 GWAS + CCT A vs. C 2681 2.92 × 10–4 β: −3.83 Cornes [103]
validation SE:1.06 BK et al.
2012
4 LOX rs2956540 CG KCN G vs. C 210 191 0.042 0.664 Hao XD [82]
(0.447– et al.
0.986) 2015
5 FOXO1 rs2721051 GWAS + CCT T vs. C 874 6085 2.7 × 10–10 1.62 Lu Y [18]
validationa (1.4–1.88) et al.
2013
6 FNDC3B rs4894535 GWAS + CCT T vs. C 874 6085 4.9 × 10–9 1.47 Lu Y [18]
validationa (1.29–1.68) et al.
2013
Y. M. Wang et al.
7 VSX1 rs56157240 CG KCN T vs. A 97 101 0.0499 6.42 Wang Y [102]
(0.77–53.78) et al.
2013
rs12480307 CG KCN C vs. T 97 101 0.0499 6.42 Wang Y [102]
(0.77–53.78) et al.
2013
rs6050307 CG KCN T vs. G 97 101 1.22 × 10–7 0.05 Wang Y [102]
(0.01–0.23) et al.
2013
8 FRAP1 rs17036350# GWAS + CC T vs. C 4289 4.06 × 10–13 n.r. Han S [104]
validation et al.
2011
35 Keratoconus Genes in Chinese
9 PDGFRA rs2114039# GWAS + CC C vs. T 4289 1.33 × 10–9 n.r. Han S [104]

validation et al.
2011
10 6q14.1 near rs1538138 GWAS + CCT T vs. C 2681 9.49 × 10–5 β: −3.87 Cornes [103]
IBTK validation SE:0.99 BK et al.
2012
11 15q26.3 rs4965359 GWAS + CCT A vs. G 2681 1.24 × 10–4 β: −3.52 Cornes [103]
2012
12 7q11.21 rs4718428 GWAS + CCT G vs. T 2681 0.0180 β: −2.31 Cornes [103]
2012
CG candidate gene association study, GWAS genome-wide association study, KCN keratoconus, CCT central corneal thickness, CC corneal curvature. n.r not reported
a
GWAS involve Chinese, replication in Caucasians #Lead SNP
443
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Granular Corneal Dystrophy Type 2:
Prevalence in South Korea, 36
Molecular Pathogenesis,
and Therapeutic Approaches
Hun Lee, Seung-il Choi, Kyung Eun Han,

Tae-im Kim, and Eung Kweon Kim
Abstract growth factor-β-induced gene protein

Recent advances in the genetics of corneal (TGFBIp) in the corneal stroma in the form of
dystrophies have facilitated more precise clas- hyaline and amyloid, which interfere with cor-
sification of the disease and elucidation of the neal transparency. The heterozygous form of
molecular mechanisms involved in the patho- GCD2 is generally mild when the patients are
genesis of the different types of corneal dys- young, accompanied by only a few visually
trophies. Granular corneal dystrophy type 2 insignificant corneal opacities. However, as
(GCD2) is an autosomal dominant disorder patients age, increased diffuse haze will
associated with the arginine-to-histidine sub- appear, resulting in decreased vision owing to
stitution at codon 124 (R124H) in the trans- reduced passage of light rays through the
forming growth factor-β-induced gene visual axis. In contrast, patients with the
(TGFBI) on chromosome 5q31. The hall- homozygous form of GCD2 have severe
marks of GCD2 include the age-dependent visual impairment beginning early in child-
progressive accumulation of transforming hood. Here, we discuss the current state of
knowledge on GCD2, including the epidemi-
ology, clinical manifestations, molecular
This work was supported by a grant of the KoreaHealth
Technology R&D Project through the Korea Health pathogenesis, and treatment modalities.
Industry Development Institute (KHIDI), funded by the
Ministry of Health & Welfare, Republic of Korea (grant
number : HI16C1009). T.-i. Kim
Department of Ophthalmology, Vision Research
H. Lee
Institute, Severance Hospital, Yonsei University
Department of Ophthalmology, International St.
College of Medicine, Seoul, South Korea
Mary’s Hospital, Catholic Kwandong University
College of Medicine, Incheon, South Korea Corneal Dystrophy Research Institute, Yonsei
University College of Medicine, Seoul, South Korea
Department of Ophthalmology, Vision Research
Institute, Severance Hospital, Yonsei University E. K. Kim (*)
College of Medicine, Seoul, South Korea Department of Ophthalmology, Vision Research
Institute, Severance Hospital, Yonsei University
S.-i. Choi
College of Medicine, Seoul, South Korea
Corneal Dystrophy Research Institute,
Yonsei University College of Medicine, Corneal Dystrophy Research Institute, Yonsei
Seoul, South Korea University College of Medicine, Seoul, South Korea
K. E. Han Severance Biomedical Science Institute, Yonsei
Department of Ophthalmology, Ewha Womans University College of Medicine, Seoul, South Korea
University, Seoul, South Korea e-mail: [email protected]

https://doi.org/10.1007/978-981-13-0884-0_36
450 H. Lee et al.
Keywords 36.2 Epidemiology of GCD2

Granular corneal dystrophy type 2 · Prevalence
· Molecular mechanism · Genetics · South Direct measurement of a population-based sam-
Korea ple to determine the prevalence of GCD2 would
be prohibitively expensive owing to the relatively
low prevalence of GCD2 in most countries. Our
group investigated the prevalence of GCD2 in the
36.1 Introduction Korean population by identifying the number of
patients with the homozygous form of GCD2 as a
Recent advances in the genetics of corneal dys- measure of the frequency of heterozygous GCD2
trophies have facilitated more precise classifi- [4]. We found 16 families, including 21 children,
cation of the disease and elucidation of the who were homozygous for the R124H mutation.
molecular mechanisms involved in the patho- All parents of homozygous patients available for
genesis of the different types of corneal dystro- examination in the study were themselves hetero-
phies. The International Committee for zygous for the R124H mutation. Thus, with the
Classification of Corneal Dystrophies (IC3D) assumption of the Hardy-Weinberg equilibrium,
provides updated data to ophthalmologists by we used the variable q for the prevalence of the
incorporating traditional definitions of corneal GCD2 allele (A’) in the Korean population and
dystrophies with new genetic, clinical, and the variable p for the prevalence of the normal
pathologic information. Granular corneal allele (A). The prevalence of the normal allele
dystrophy type 2 (GCD2) is an autosomal (A) is (1 − q), and for convenience, we let the

dominant disorder associated with the argi- variable p = (1 − q). The Hardy-Weinberg equa-
nine-to-histidine substitution at codon 124 tion says that the genotype frequencies for the
(R124H) in the transforming growth factor-β- normal allele (AA), heterozygous allele (AA’),
induced gene (TGFBI) on chromosome 5q31 and homozygous allele (A’A’) are given by p2,
[1]. The hallmarks of GCD2 include the age- 2pq, and q2, respectively [4], and the sum of the
dependent progressive accumulation of prevalence for normal, heterozygous, and homo-
transforming growth factor-β-induced gene zygous alleles must be exactly one

protein (TGFBIp) in the corneal stroma in the (p2 + 2pq + q2 = 1). Using the Korean registration-
form of hyaline and amyloid, which interfere based census of 47,041,434 individuals, the esti-
with corneal transparency [2, 3]. The heterozy- mated overall prevalence (combining
gous form of GCD2 is generally mild when the heterozygotes and homozygotes) of GCD2 is
patients are young, accompanied by only a few 11.5 per 10,000 persons (q = 0.000574) based
visually insignificant corneal opacities. upon the Hardy-Weinberg principle. In another
However, as patients age, increased diffuse study conducted in South Korea, GCD2 was the
haze will appear, resulting in decreased vision most frequent mutation among 268 patients with
owing to reduced passage of light rays through TGFBI corneal dystrophies [5].
the visual axis. In contrast, patients with the
homozygous form of GCD2 have severe visual
impairment beginning early in childhood. 36.3 Asian Perspective
However, the exact molecular mechanisms
mediating the pathogenesis of GCD2 are not According to the results of several studies, substi-
fully understood. tution of arginine for histidine at codon 124
In this chapter, we discuss the current state of (R124H) is considered to be the most frequently
knowledge on GCD2, including the clinical man- observed mutation in the Asian population. In
ifestations, epidemiology, molecular mecha- China, granular corneal dystrophy type 1 (GCD1)
nisms, and treatment modalities. is the most popular mutation, followed by lattice
36 Granular Corneal Dystrophy Type 2: Prevalence in South Korea, Molecular Pathogenesis… 451
corneal dystrophy type 1 (LCD1) and GCD2 [6]. d ystrophies, i.e., the granular form (GCD types 1
According to Japanese studies, R124H mutation and 2), lattice form (LCD types 1, 3, and 4), and
is the most common mutation, up to 72% of diffuse Bowman’s layer deposits (Reis-Bucklers
patients with corneal dystrophies [7, 8]. The sec- and Thiel-Behnke corneal dystrophies) [2].
ond most frequently observed mutation is either GCD2 exhibits the clinical and histological
LCD1 or GCD1. In Western countries, LCD1 is characteristics of both granular and lattice dys-
more common. Notably, the prevalence of TGFBI trophies. Clinically, small and faint superficial
corneal dystrophies is likely to be underestimated stromal deposits observed in the first or second
as these data were collected only from published decade of life with a slit lamp are the earliest
studies. manifestations (Fig. 36.1) [1, 10]. The heterozy-
gous form of GCD2 is generally mild, and visu-
ally insignificant corneal opacities appear at a
36.4 Etiology and Clinical young age. These granular deposits accumulate
Features with age. In particular, lattice-like amyloid
lesions appear in some patients in the deeper
Substitution of arginine for histidine at codon stroma with superficial granular lesions, and a
124 (R124H) in the transforming growth factor- superficial diffuse anterior stromal haze appears
β-induced gene (TGFBI) on chromosome 5q31 is in some older patients (Fig. 36.2). Coalescence of
always found in GCD2 [1]. Age-dependent pro- the opacities may impair visual acuity in elderly
gressive accumulation of hyaline and amyloid patients with the heterozygous form of the dis-
and the production of TGFBIp, an extracellular ease. However, the homozygous form of GCD2
matrix protein, in the corneal epithelia and stroma has earlier onset with faster progression showing
are also generally observed in GCD2 [2, 3]. Most many gray-white, discrete deposits in the superfi-
TGFBIp (also known as keratoepithelin) cial cornea compared with the heterozygous form
expressed under normal conditions is thought to of GCD2 [11–13]. Specifically, these deposits
be produced by the corneal epithelium. During can be observed as early as 3 years of age in
wound healing in the normal human cornea, patients with the homozygous form of GCD2 and
TGFBIp is found in the epithelium and fibroblasts increase in size with age (Fig. 36.3). Thus,
near the wound, which suggests that keratocytes patients with the homozygous form of GCD2
can also produce TGFBIp [9]. TGFBIp can be have severe visual impairment from early in
deposited in corneal tissue in the three major childhood, eventually requiring surgery. Our
forms of dominant TGFBI-linked corneal group reported that some granular corneal
Fig. 36.1 Slit-lamp
photograph of a 23-year-
old female patient with
heterozygous granular
corneal dystrophy type 2.
Two small granular
deposits are present in the
anterior stroma
452 H. Lee et al.
Fig. 36.2 Slit-lamp
Multiple crumb-shaped
granular deposits are
present in the anterior
stroma, and deep
lattice-like deposits are
present in the deep stroma.
A diffuse haze is present
between granules
Fig. 36.3 Slit-lamp
photograph of a 6-year-old
male patient with
homozygous granular
Multiple crumb-shaped
granular deposits are
present in the anterior
stroma
d eposits disappear partially after painful corneal form of GCD2 in Korea is 1 in 870, more than
erosion attack [14]. The dropping out of the cen- 300 patients have experienced poor vision after
ter of deposits would result in the formation of undergoing LASIK or LASEK procedures in
ring-shaped opacities [14]. Histologically, granu- Korea.
lar deposits in the anterior stroma stain with
Masson’s trichrome, while the lattice-like lesions
in the deep stroma stain with Congo red. Electron 36.5 Molecular Pathogenesis
microscopy has revealed the presence of rod- of GCD2
shaped or trapezoidal electron-dense areas sur-
rounded by tubular microfibrils [15]. The pathogenesis of GCD2 has been shown to be
Notably, in patients with the heterozygous associated with altered morphological character-
form of the disease, manifestation of the damage istics of corneal fibroblasts, vulnerability of cor-
to the cornea is exacerbated when LASIK or neal fibroblasts to oxidative stress, mitochondrial
LASEK procedures are applied (Figs. 36.4 and dysfunction, defective autophagy, and delayed
36.5). Because the prevalence of the heterozygous mutant-TGFBIp secretion.
Fig. 36.4 Slit-lamp
old male patient with
corneal dystrophy type 2 at
6 years after
LASIK. Newly formed
small granules (arrows) are
present along the LASIK
flap interface with multiple
crumb-shaped granular
deposits (arrow heads) in
the anterior stroma
Fig. 36.5 Slit-lamp
corneal dystrophy type 2
after LASEK. Newly
formed small granules
(arrows) are present with a
crumb-shaped granular
deposit (arrow head) in the
anterior stroma
36.5.1 Morphological Properties mutant-TGFBIp and cathepsin D (an enzyme

of GCD2 Corneal Fibroblasts found in lysosome) than wild-type cells [17].
Transmission electron microscopy analyses have
Keratocytes (often called corneal fibroblasts revealed that GCD2 corneal fibroblasts contain
when cultured) are the predominant cellular com- fragmented or elongated mitochondria, whereas
ponent of the corneal stroma and are mainly normal corneal fibroblasts contain small, round
involved in maintaining corneal transparency and mitochondria [19, 20]. Disorganized and dilated
the extracellular matrix environment [16]. In eyes mitochondria are often observed, particularly in
with GCD2, corneal fibroblasts harbor multiple GCD2 homozygous corneal fibroblasts at late
vesicles of different sizes containing amorphous passages. Corneal fibroblasts from patients with
material showing a senescence-like morphology the homozygous form of GCD2 do not survive
with increases in size [17, 18]. Additionally, cul- for more than eight passages in vitro, whereas
tured GCD2 corneal fibroblasts exhibit a much those from normal donors can survive for more
more extensive pattern of colocalization for than 24 passages.
454 H. Lee et al.
36.5.2 Oxidative Stress in GCD2 of electrons through electron transport chain

Corneal Fibroblasts redox reactions coupled with proton transfer
across the inner mitochondrial membrane in the
In mammalian cells, aerobic metabolic pro- mitochondria. Superoxide (O2•−), hydroxyl radi-
cesses lead to the production of reactive oxygen cals (•OH), and hydrogen peroxide (H2O2) are
species (ROS) in mitochondria and peroxi- formed as a side effect of this process [23].
somes. Excessive ROS can cause oxidative Excessive production of O2− and H2O2 results in
damage to proteins, lipids, and DNA. Because oxidative injury to the tissue. To maintain a
the cornea should be transparent in order to healthy status, many types of mammalian cells
allow light transmission, it should be avascular. have developed both enzymatic and nonenzy-
The cornea is also constantly exposed to a wide matic ROS scavenging mechanisms. When the
spectrum of light, including ultraviolet (UV) redox-active species metabolic system is
light, which causes tissue stress and ROS gen- unbalanced, more oxidizing agents are produced,
eration [21]. These factors make the cornea and oxidative stress occurs. Indeed, increased
more vulnerable to oxidative stress than other amounts of malondialdehyde (MDA),
tissues. Accordingly, the cornea is rich in anti- 4-hydroxy-2-nonenal (HNE), protein carbonyl
oxidant enzyme systems that aid in the removal groups, and 8-hydroxy-2′-deoxyguanosine
of ROS generated by UV light absorption [22]. (8-OHdG) are frequently associated with oxida-
Oxygen is reduced to water (H2O) by the p assage tive damage (Fig. 36.6) [24–29].
Fig. 36.6 Oxidative stress, antioxidant defenses system, and release of 8-OHdG, protein carbonyl group, malondi-
and keratocyte damage. A primary source of reactive oxy- aldehyde (MDA), and 4-hydroxynonenal (HNE) compo-
gen species (ROS) production is mitochondrial NADPH/ nents. Cells have protective antioxidant systems, including
NADH oxidase. Hydrogen peroxide (H2O2) is converted superoxide dismutase (SOD), catalase, glutathione per-
to the hydroxyl radical (•OH) in the presence of transition oxidase (GPx), glutathione reductase (GR), and glutathi-
metals, such as iron (+Fe). The hydroxyl radical induces one (GSH). Upon oxidation, GSH forms glutathione
DNA, protein, and lipid damage, leading to cell damage disulfide (GSSG)
Our group examined the mitochondrial features extracellular protein aggregates, which eventu-
of heterozygous GCD2 corneas using electron ally result in cellular death. To control the quality
microscopy and demonstrated that GCD2 corneas of intracellular proteins and organelles, cells have
have many mitochondria that are dilated and special surveillance systems. Two major systems,
degenerative and/or contain vesicles with amor- the ubiquitin/proteasome system (UPS) and the
phous material [30]. MitoTracker and cytochrome autophagy system, are responsible for the prote-
c staining showed increased mitochondrial activity olysis and removal of abnormal proteins [32].
in mutated cells in early passages in homozygous Several studies have shown that the accumulation
GCD2 corneal fibroblasts cultured for four to eight of disease-related proteins is highly dependent on
passages. In late-passage mutant cells, decreases the UPS and/or the autophagy system [33, 34].
in depolarized mitochondria, cellular prolifera- Degradation of proteins by the UPS is also known
tion, and complexes I–V expression were observed. to be a regulated, specific process [35].
Treatment of the cultured cells with butylated Degradation of a target protein by the UPS occurs
hydroxyanisole, an antioxidant, was shown to in two successive steps: (1) several ubiquitin
result in decreased intracellular ROS and reduced molecules are conjugated to the target protein, a
mitochondrial oxidative damage [30]. process that is catalyzed by ubiquitin-activating
Our group also demonstrated that oxidative enzyme (E1), ubiquitin-conjugating enzyme
damage is involved in GCD2 pathogenesis by (E2), and a substrate-specific ubiquitin-protein
showing that levels of 4-HNE, MDA, and protein ligase (E3); and (2) the tagged protein is degraded
carbonyl groups were significantly elevated in into small peptides by the 26S proteasome com-
cultured GCD2 corneal fibroblasts compared plex, which recognizes only ubiquitin-conjugated
with those in normal corneal fibroblasts [31]. We proteins [36, 37].
also examined MDA distribution in the corneal Autophagy occurs by sequestering molecules
tissues from patients with GCD2 by immunohis- and organelles for degradation by lysosomes.
tochemical staining and demonstrated that MDA Autophagy can be classified into the following
expression was significantly elevated in the cor- three types: microautophagy, chaperone-
neal stroma and epithelium from patients with mediated autophagy, and macroautophagy [38].
GCD2; notably, however, MDA immunoreactiv- Hereafter, we refer to the process of macroau-
ity was also detectable in corneal epithelial cells tophagy as autophagy. In the autophagy path-
from age-matched normal controls [31]. way, bulky cytoplasmic materials and organelles
Moreover, the expression levels of Cu/ are packaged into double-membrane-bound ves-
Zn-superoxide dismutase (SOD), Mn-SOD, glu- icles called autophagosomes. These autophago-
tathione peroxidase (GPx), and glutathione somes then fuse with lysosomes, and damaged
reductase (GR) proteins in GCD2 corneal fibro- material is degraded within these fused vesicles
blasts are significantly elevated compared with through the functions of lysosomal enzymes
those in normal corneal fibroblasts. Intracellular (Fig. 36.7) [39].
ROS and H2O2 levels are significantly elevated in In the context of CD2, mutant TGFBIp has
GCD2 corneal fibroblasts compared with those in been shown to accumulate in the autophagolyso-
wild-type corneal fibroblasts [31]. somal compartment of corneal fibroblasts [17,
18]. In GCD2 corneal fibroblasts, mutant-
TGFBIp colocalizes extensively with
36.5.3 Accumulation microtubule-associated protein 1 light chain 3b
and Degradation of TGFBIp (LC3)-enriched cytosolic vesicles, cathepsin D,
via Autophagy Pathway and lysosomal enzymes, indicating that TGFBIp
in GCD2 Corneal Fibroblasts is degraded by autophagy [17, 18]. Furthermore,
when GCD2 corneal fibroblasts are treated with
Many degenerative disorders are characterized bafilomycin A1, an inhibitor of the fusion of
by the accumulation of intracellular or autophagosomes to lysosomes, caspase-3 and
456 H. Lee et al.
Fig. 36.7 The autophagy process. In the presence of an vesicle, the autophagosome. The outer membrane of the
autophagy inducer, cytosolic proteins and organelles are autophagosome docks and directly fuses with a lysosome
sequestered by a pre-autophagosomal membrane structure to form an autolysosome. The sequestered material is
called the phagophore. The phagophore membrane then degraded inside the autolysosome and recycled later
expands and encloses its cargo to form a double-membrane
poly (ADP-ribose) polymerase 1 (PARP1) are of autophagy via a mammalian target of

activated [17]. Based on these results, defective rapamycin (mTOR)-dependent pathway and con-
autophagy is thought to contribute to GCD2 cor- sequently eliminates mutant-TGFBIp from
neal fibroblast dysfunction. GCD2 corneal fibroblasts (Table 36.1) [41].
Hence, induction of autophagy may be an When melatonin and rapamycin are applied
option for GCD2 treatment even though autoph- together, they have greater effects on mutant-
agy itself is not sufficient for the removal of all TGFBIp clearance than either drug alone.
mutant-TGFBIp from GCD2 corneal fibroblasts Our group also showed that mitomycin C
[18]. When rapamycin, an autophagy inducer, is (MMC) application during photorefractive kera-
applied to cultured GCD2 corneal fibroblasts, tectomy (PRK) to GCD2 corneas did not inhibit
mutant-TGFBIp levels are reduced. However, the exacerbation of GCD2 after laser ablation
normal TGFBIp levels in wild-type corneal fibro- [42]. When MMC was applied to cultured fibro-
blasts did not change [17]. This suggests that blasts harvested from normal, heterozygous
rapamycin or its related analogs may be suitable GCD2 and homozygous GCD2 cornea, MMC
agents for the treatment of patients with GCD2. caused apoptosis in GCD2 corneal fibroblasts,
Because rapamycin is currently used in the clini- with homozygous cells being most vulnerable.
cal setting to treat several diseases, application of Because TGFBIp can be absorbed from outside
rapamycin in patients with GCD2 may be a of corneal fibroblasts into the cell and can be
promising strategy [17, 40]. Our group also dem- degraded in lysosomes, application of MMC to
onstrated that melatonin functions as an inducer GCD2 corneas to inhibit the formation of deposits
Table 36.1 Effects of melatonin on TGFBIp, autophagy, is not desirable [43]. Further studies are needed
and mTOR signaling pathway
to fully elucidate the roles of keratocytes in the
Wild Granular corneal pathogenesis of GCD2.
Parameters type dystrophy type 2
LC3 I + +
LC3 II + +
Autophagosome + n.t. 36.5.4 TGFBIp Regulation via TGF-β
ATG5 + n.t. Signaling Pathway:
mTOR n.c. n.t. Therapeutic Applications
p-mTOR (2481) n.c. n.t.
p-mTOR (2448) + + The transforming growth factor-β (TGF-β) is a
GβL n.c. n.t. multifunctional cytokine that regulates diverse
Raptor n.c. n.t. cellular and physiological processes, including
Rictor n.c. n.t. proliferation, differentiation, and extracellular
TGFBIp − −
matrix homeostasis [44]. This pathway is initiated
-, decrease; +, increase; n.t., not tested; n.c., not changed by TGF-β binding to type I (TβRI) and type II
significantly; LC3, microtubule-associated protein 1 light
chain 3; ATG5, autophagy-related gene 5; mTOR, mam- (TβRII) TGF-β receptors, both of which can acti-
malian target of rapamycin; GβL, G-protein-β-subunit- vate multiple downstream signaling pathways to
like protein; Raptor, regulatory-associated protein of alter gene transcription (Fig. 36.8). The activated
mTOR; Rictor, rapamycin-insensitive companion of receptor complex phosphorylates the downstream
mTOR; TGFBIp, transforming growth factor-β-induced
protein transcription factors Smad2 and Smad3, promot-
Fig. 36.8 Major transforming growth factor-β signaling binds to this receptor complex, intracellular Smad2 and
pathways. Latent TGF-β complexes are activated. Smad3 are recruited and phosphorylated at their C-termini
Activated TGF-β then assembles the productive signaling by TβRI. Phosphorylated Smad2 and Smad3 subsequently
heteromeric receptor complex. In the signaling receptor bind to Smad4 and translocate to the nucleus, where they
complex, the type II receptor (TβRII) activates its intracel- modulate the transcription of target genes in collaboration
lular serine/threonine kinase function, resulting in multi- with cofactors and other transcription factors (TFs)
ple intracellular signaling cascades. Thus, once TGF-β1
458 H. Lee et al.
Table 36.2 Effects of lithium on TGFBIp, autophagy, 36.6.2 Keratoplasty

cellular proliferation, and TGF-β signaling
Wild Granular corneal dystrophy Anterior lamellar keratoplasty (ALKP) and PKP
Parameters type type 2
are traditionally used to treat deep deposits of
TGFBIp − −
stromal corneal dystrophies. ALKP may be con-
LC3 I n.c. n.t.
LC3 II + n.t. sidered for primary cases of GCD2 or cases of
Smad3 − n.t. recurrence following PTK for preservation of the
p-Smad3 n.c. n.t. deep corneal stroma. Visual acuity after ALKP,
(S423/425) however, is often less than that after DALK due
GSK3α/β n.c. n.t. to irregularities and/or scarring on the donor-to-
p-GSK3α/β + n.t. recipient interface [48, 49]. Several research
(S21/9)
groups have performed homologous penetrating
Cell proliferation n.c. n.c.
central limbokeratoplasty; however, the success
-, decrease; +, increase; n.t., not tested; n.c., not changed
significantly; LC3, microtubule-associated protein 1 light
rate of this procedure has not been shown to be
chain 3; GSK3, glycogen synthase kinase 3 satisfactory because donor epithelial cells are
eventually replaced by host epithelial cells
[50–53].
ing their association with Smad4. The Smad com-
plex then translocates to the nucleus to regulate
the transcription of target genes (Fig. 36.8) [45]. 36.7 Conclusion
Because TGFBIp expression is induced by TGF-
β, reducing intracellular TGFBIp by controlling In summary, we presented recent literature
TGF-β signaling may be a useful approach for describing the genetics of GCD2 in Asia and
drug development in TGFBI-linked corneal dys- the pathophysiology of GCD2. Lithium and
trophies [46]. Our group reported that lithium melatonin have been proposed as possible ther-
treatment of corneal fibroblasts reduces the phos- apeutic agents for the management of GCD2.
phorylation of Smad3 (S423/425) and the expres- Until now, surgical treatments, including PTK,
sion of normal and mutant TGFBIp, suggesting ALKP, DLKP, and PKP, have been used to
that lithium may be a potential useful compound maintain adequate vision in patients with
in the treatment of TGFBI-linked corneal dystro- GCD2. More studies of the prevalence of
phies (Table 36.2) [46]. GCD2, both in Asia and around the world, as
well as continuous development of therapeutic
agents are required.
36.6 Treatment Modalities
Conflict of Interest Eung Kweon Kim is in the medical
36.6.1 Laser Ablation advisory board member of Avellino Lab USA. Hun Lee,
Seung-il Choi, Kyung Eun Han, and Tae-im Kim declare
that they have no conflict of interest.
Phototherapeutic keratectomy (PTK) has been
used for the removal of shallow corneal depos-
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About the Editors
Dr. Gyan Prakash is the founding President of Dr. Prakash created and established over a
Asian Eye Genetics Consortium (AEGC), now dozen innovative and fully operational product
renamed as Global Eye Genetics Consortium development programs in the pharmaceutical and
(GEGC). He has a deep-rooted dedication and biotechnology industries. He served as the
passion for global health programs, international Project Scientist for Pfizer’s first therapeutic bio-
research collaborations, and more than 30 years logic (first therapeutic monoclonal Antibody –
of experience in international health research, E5), and then for fluconazole, the number one
teaching, and mentorship. Dr. Prakash has selling antifungal drug in the world. Dr. Prakash
worked across many biomedical disciplines earned his first international program manage-
including infectious diseases, drug development, ment experience working at the Pfizer world
eye diseases, substance and drug abuse, oncol- headquarters in New York in many leadership
ogy, neurology, and biomedical technology. roles over several years. At Pfizer, he adminis-
Dr. Prakash has served as Director of the Office tered thirty-four international clinical programs
of International Program Activities (OIPA) at across the globe for the largest selling antifungal
National Eye Institute in the USA since 2012, and drugs in the world, fluconazole and voriconazole,
worked across interdisciplinary and geographic which have been used by millions of HIV/AIDS
boundaries to lead strategic programs and opportu- and cancer patients and saved millions of lives
nities for advancing scientific research, training, around the world in the immunosuppressed and
developing next generation of biomedical work- immunocompromised patient populations. While
force, and promoting global engagement of at Pfizer and then at NIH-NIAID, he worked with
researchers at all career stages. Earlier in his career, the clinical investigators and scientists in low and
he worked with the industry leaders to create three middle income countries on various programs
new companies (Mesa Diagnostics in Albuquerque, related to infectious and neglected tropical dis-
New Mexico, Metastatin Pharmaceuticals in eases such as TB, malaria, influenza, and visceral
Bethesda, Maryland, and AMAR International in leishmaniasis. His experience in non-
Reston, Virginia), led teams at established compa- communicable diseases spans from eye diseases,
nies (Pfizer International in New York, Johnson cancer, and drug and substance abuse. Before
and Johnson/Ortho Diagnostics in California, and joining the National Institutes of Health, he
PRA International in Virginia), and mentored the served as the Chief Operating Officer and Senior
next generation of global health scientists and R & Fellow at AMAR International, Inc., a life sci-
D managers at NIH-Global Health Interest Group ence program management company in Reston,
(NIH-GHIG), George Mason University, Virginia, USA, where he led biomedical program
University of Maryland, and Johns Hopkins management teams for the US Government for
University. large programs. Previously, Dr. Prakash served as

https://doi.org/10.1007/978-981-13-0884-0
462 About the Editors
the President and CEO of Metastatin India, China, Brazil, Australia, the UK, Spain,
Pharmaceuticals, Inc., a cancer biologics- France, Germany, and many other countries. Dr.
development stage company in Bethesda, Prakash has been affiliated with several leading
Maryland, USA. universities in the USA and abroad (Johns
Dr. Prakash has served in the capacity of Hopkins, University of Maryland, Georgetown
senior scientific advisor for several public and University, George Mason University, Cambridge
private pharmaceutical/biotech companies, as University in the UK, and Sun-yat Sen University
Director for the programs of American Society in China) as Adjunct and visiting Professor in
for Microbiology, and a member on national teaching role and serving on various committees.
committee for a major medical and professional He played a key role in founding of a new trans-
association. Dr. Prakash held an Adjunct NIH Global Health Interest Group (NIH-GHIG)
Professorship at Robert H. Smith School of in 2012, providing infrastructure and mentorship
Management, University of Maryland and served to post-doctoral fellows and next generation of
on the advisory board of New Market Growth scientists across NIH interested in global health.
Fund of Dingman Center for Entrepreneurship. Dr. Prakash has been the founder of a nationally
In 2001, he was appointed an Adjunct Professor recognized program “Science for the Future” that
at School of Management at George Mason won the Blue Ribbon Award from The White
University in Virginia where he established the House. The program provided guidance in devel-
first graduate program on bioscience manage- oping hands-on science as part of the early learn-
ment in the USA. Dr. Prakash has published sev- ing in elementary schools and was featured on
eral original papers in peer-reviewed journals and “The Frontline” at the Public Broad-casting
has a major biotechnology book, Nucleic Acid Service (PBS).
and Monoclonal Antibody Probes, to his credit
that he published with Marcel Decker of Dr. Takeshi Iwata received his Ph.D. from the
New York. He has also coauthored a manual of Department of Agriculture at Meijo University in
business of biotechnology and has presented Japan and moved to the National Eye Institute
numerous invited lectures around the world. (NEI)/National Institute of Health (NIH), USA,
Dr. Prakash earned his graduate degrees, M.S., as postdoctoral fellow in a retina genetic labora-
and Ph.D. in Medical Microbiology (University tory, headed by Dr. George Inana. The laboratory
of Illinois at Urbana-Champaign, USA) and an had just identified the first gene responsible for
MBA in Pharmaceutical Management/Marketing genetic eye disease called gyrate atrophy caused
(St. Joseph’s University, USA). He received pro- by a mutation in ornithine aminotransferase
fessional training at the UCLA School of (OAT) gene. Dr. Iwata’s first job was to use this
Management and Centers for Disease Control OAT cDNA probe to hybridize OAT pseudogenes
(CDC) in Atlanta. Prior to immigrating to the in X-chromosome and perform linkage analysis
USA in 1978, Dr. Prakash earned a B.Sc. for X-linked retinitis pigmentosa families. He
(Biology and Chemistry) and a M.Sc. moved with Dr. Inana to Bascom Palmer Eye
(Biochemistry) at University of Allahabad in Institute, University of Miami School of Medicine
India. He received the University Gold Medal in in Miami, Florida, to further work on other
the University for securing the highest rank in the hereditary retinal diseases. After 2 years, he
undergraduate program. Dr. Prakash has received returned to the NEI laboratory headed by Dr.
a number of national and international awards Deborah Carper to work on another major retinal
and recognitions in his career including a disease, the diabetic retinopathy. His work was to
UNESCO Fellowship, AAAS award, the White identify the mechanism of transcriptional regu-
House Blue Ribbon Award, Dr. Brahma Prakash lation for two genes, aldose reductase and sorbi-
Memorial Oration Award, and the Aditya Jyot tol dehydrogenase, in polyol pathway associated
Foundation Award. He has served as invited with the disease. These retinal and gene related
speaker in many parts of the world, such as Japan, work in the USA motivated him to significantly
About the Editors 463
expand the research when he returned to Tokyo, Genetics Consortium (JEGC) for Hereditary
Japan, to head the first laboratory at the National Retinal Diseases. A diagnostic system and a data-
Institute of Sensory Organs (NISO), Tokyo base developed and described in Chap. 2 has
Medical Center, National Hospital Organization inspired him to help establish the Asian Eye
(NHO). The NHO is a group of 143 National Genetics (AEGC, http://asianeyegenetics.org)
Hospitals with 52,000 beds and more than 59,000 using the same system. He now serves as the
total employees. Ten Research Centers in Japan president of AEGC.
are affiliated to the NHO and located in major Dr. Iwata has received awards from
cities with each center targeting specific medical Cooperative Cataract Research Group, National
research area. The NISO was developed as the Foundation for Longevity Science, Foundation
first sensory organs research institution in Japan Preventing Blindness, Japanese Association for
to focus on vision, hearing, and vocalization Complement Research, Japan Retinitis
research. Pigmentosa Society, International Society for
Dr. Iwata is currently running three main proj- Eye Research (ISER) Special Recognition
ects related to age-related macular degeneration Award, and has given keynote lectures around the
(AMD), normal tension glaucoma (NTG), and globe including major universities in the USA,
hereditary retinal diseases as the Director of the UK, China, India, and recently at RD 2016 meet-
Molecular and Cellular Biology Division. His ing in Kyoto, Japan. Dr. Iwata has served as a
research goes beyond identification of disease- committee member in number of local and inter-
causing gene to explore the mutant biological national organizations including the Association
behavior in vitro and in vivo. His laboratory for Research in Vision and Ophthalmology
recently identified the abnormal transcriptional (ARVO) and the International Society for Eye
regulation of HTRA1 gene at the most highly Research (ISER). He has served as a committee
associated genome region on chromosome 10 member of the ARVO Foundation Award
(Iejima et al., J Biol Chem 2014). This hypothesis Committee, ARVO Global Research Training
was confirmed when HTRA1 was overexpressed Committee, and ARVO Executive Committee
in mouse which led to the development of choroi- Member for the Advocacy Pillar. He has served
dal neovascularization (Nakayama et al., Invest two terms as a counselor for ISER and recently
Ophthalmol Vis Sci 2015). His research for nor- served as the vice president (Asia & Pacific) and
mal tension glaucoma focuses on optineurin program chair for the XXII Biennial Meeting of
(OPTN) gene responsible for hereditary ISER 2016 in Tokyo, Japan. Dr. Iwata has over
NTG. His laboratory identified the OPTN E50K 100 publications in scientific journals, reviews,
mutant protein that interacts with TANK-binding and book chapters. He currently serves on the
protein 1 (TBK1), which leads to the precipita- editorial board for Journal of Ocular Biology,
tion of OPTN in the endoplasmic reticulum Diseases, and Informatics and Eye and Brain.
(Minegishi et al., Hum Mol Genet 2013). When Dr. Iwata has supported international collabo-
inhibitor chemical for TBK1 was applied, this rations between researchers, laboratories, insti-
precipitation was significantly reduced. The work tutions, and consortia. Recently, he played a
has now expanded to the identification of FDA major role in establishing several Collaborative
approved TBK1 inhibitor drug for worldwide Research Agreements including the ones
clinical trial (Minegishi, Nakayama, Iejima et al., between NISO and NEI=NIH in the USA,
Prog Retin Eye Res 2016). In 2011, Dr. Iwata Buffalo Niagara Medical Center Campus in the
started with six Japanese ophthalmologists from USA, Aditya Jyot Eye Hospital, Mumbai in
different universities specialized in retinal elec- India, and Moorfield Eye Hospital-University
trophysiology to systematically analyze the College, London in the UK. These collabora-
genetics of family with hereditary retinal dis- tions have brought fruitful results to NISO-
eases. This small group has now expanded to 26 Tokyo Medical Center. As the President of
universities and institutions as the Japan Eye AEGC (now GEGC), Dr. Iwata is actively
464 About the Editors
involved in identifying the key leaders around the Dystrophy started operation in September, 2016,
world to build effective plans for future genetic initiated by Dr. Kaoru Fujinami. These AEGC
eye research. He led the third annual meeting of (now GEGC) database should accelerate thera-
AEGC (now GEGC) during ARVO 2016 in peutic developers to decide which gene to focus
Seattle with over 45 attendants representing each and who to contact for patient access. Dr. Iwata
region of Asia. The meeting participants agreed has worked with Dr. Gyan Prakash since 2013 to
to share eye genetic information by constructing build eye genetic research programs in Asia and
a common AEGC (now GEGC) database con- other parts of the world. This book is not only a
taining useful genotype-phenotype information landmark achievement for GEGC, but it serves as
along with natural history of patients for each a guiding document for all future collabora-
mutation recorded. AEGC (now GEGC) database tive research programs for the Global Eye
for Stargardt’s Disease and Occult Macular Genetics Consortium.
Index
A Autosomal recessive (AR), 42–44, 48, 50, 53, 72, 94,

ABCA4, 44, 51, 94, 122, 123, 126, 148, 149, 152, 182, 114–115, 126, 128, 133, 134, 142, 148, 151–153,
235, 243, 280–283, 286, 288, 290, 325–327 155, 156, 158–160, 171, 178, 179, 181, 182, 192,
ADAMTSL4, 43, 53 196, 204, 213, 234, 256, 280, 324–333, 337, 342,
Adeno-associated virus 2 carrying ND4 (AAV2-ND4), 348, 408, 410, 411, 419, 421, 427
274, 275, 277
Aditya Jyot Foundation for Twinkling Little
Eyes (AJFTLE), 6–10 B
Aditya Jyot Research in Vison and Ophthalmology Bardet biedl syndrome (BBS), 88, 94, 98, 148, 149,
(AJRVO), 6–9 159–161, 178, 179, 325, 330–332, 411
Adult-onset vitelliform macular dystrophy (AVMD), BEST1 gene mutation, 94, 108, 109, 148, 152, 153, 181,
181, 256, 258–265, 267, 268, 327 182, 255–268, 325, 326
Age related macular degeneration (ARMD), 3, 6, 8, 19, Best vitelliform macular dystrophy (BVMD), 94, 181,
58, 63, 64, 72, 95, 147, 172, 173, 182–184, 256, 182, 255, 266, 267
267, 268, 288, 327, 393–396, 399, 408 Bestrophin-1, 109, 150, 256, 268, 326, 396
AIPL1, 122, 181, 196, 200, 201, 211–213, 215, Bibliometrics, 13, 15, 16, 18
218, 235, 326, 328 Biobank, 29, 32, 90, 143
Alagille syndrome, 332 Biospecimens, 38
Allotopic expression, 274
ALMS1, 161, 199–201, 204, 235, 328, 332, 409, 411
Alstrom syndrome, 148, 159, 195, 325, 328, C
332–333, 409 CABP4, 198, 201, 324, 328
Asia, 2, 3, 9, 14, 23, 85, 90, 113–115, 148, 161, 173, Capacity building, 24–25
182, 183, 197, 298, 345, 348, 351, 357, 360, 362, Cataract, 3, 8, 14, 23–25, 58, 63, 71, 76–81, 86, 87, 94,
363, 366, 403, 408, 417, 418, 428, 438, 458 102, 108, 109, 132, 159, 183, 185, 193, 194, 207,
Asian Eye Genetics Consortium (AEGC), 210, 214, 250, 275, 324, 328, 329, 343, 382, 383,
1, 13, 15, 16, 18 385, 386, 408, 410, 427
Asian population, 2, 9, 73, 82, 90, 154, 170–173, 182, CCT2, 94, 99, 100, 142, 199, 201, 204, 328
183, 207, 211, 213, 218, 240, 280–290, CEP290, 148, 156, 157, 160, 161, 181, 195, 197, 200,
394, 395, 409, 418, 427, 450 201, 203, 204, 211–213, 215, 218, 328, 331, 332
Asians, 1, 14, 15, 18, 64, 73, 77, 81, 86, 87, 89, 90, Children, 25, 26, 34, 45, 64, 86, 88, 98, 131, 133, 134,
113, 121, 133, 148, 154, 169–173, 182, 183, 147, 172, 180, 193, 208, 214, 215, 258, 298, 299,
200, 202–204, 207, 211–213, 217, 218, 240, 302, 306, 307, 313, 318, 327, 337, 376, 410, 417,
244, 255–268, 280–290, 345–352, 357–366, 378, 418, 428, 450
382–387, 403–412, 417–428, 436, 441, 450–451 Chinese, 14, 58, 77, 171, 178, 200, 276, 283, 299, 325,
Asia-Pacific Academy of Ophthalmology (APAO), 3–6 359, 382, 420, 436–439, 441–444
Autosomal dominant (AD), 44, 45, 49, 52, 59, 72, 87, Choroideremia (CHM), 94, 121, 123–125, 127, 132, 139,
94, 95, 97, 102, 104, 123, 126, 133, 150–152, 239, 328, 334
155, 178–181, 196, 201, 204, 205, 207, 208, 214, Chromosomal Instability, 303
234, 252, 255, 256, 268, 324–330, 332, 333, 361, Clinical data collection, 6, 30, 36, 37
384, 410, 419–421, 450 Clinical phenotype, 64, 79, 150, 200, 240, 375, 403, 407

https://doi.org/10.1007/978-981-13-0884-0
466 Index
Clinical trials, 29, 121, 128, 139, 141, 143, 182, 184, G
196, 215, 217, 267, 273–275, 277, 290, 328 GDF6, 198, 200, 201, 203, 206, 328
CLUAP1, 200, 204, 328 Gene, 3, 30, 58, 72, 87, 94, 113, 121, 141, 148, 169, 180,
CNGA3, 171, 173, 199–201, 204, 205, 236, 240, 192, 234, 250, 255, 273, 280, 299, 324, 338, 347,
242–244, 326, 328 358, 373, 385, 404, 418, 436, 450
Common data elements (CDEs), 37, 38 Gene mutation, 3, 64, 87, 94, 100, 141–143, 157, 160,
Cone rod diseases, 325–326 161, 200, 218, 250, 273, 327–330, 332,
Cone-rod dystrophy (CRD/CORD), 36, 44, 52–53, 121, 337, 421, 440
126–129, 139, 142, 200, 201, 204, 206, 208, 209, Gene therapy, 73, 116, 118, 121, 128, 156–161, 201,
325–326, 408, 411 212, 214, 215, 217–219, 267, 273–277, 290,
Congenital stationary night blindness (CSNB), 48, 51, 328, 352
94, 139, 147, 148, 153–155, 194, Genetic, 2, 14, 26, 29, 41, 57, 71, 77, 86, 94, 113, 121,
234, 324, 325, 410 131, 137, 147, 169, 178, 192, 234, 250, 256, 274,
Consanguineous marriage, 94, 133, 134, 148, 281, 314, 326, 338, 347, 373, 383, 394, 403, 417,
151, 172, 339, 374 436, 450
Consanguinity, 45, 94, 133, 148, 156, 213, 214, Genetic disease, 30, 86, 89, 131, 170, 192, 240,
233–245, 341, 359, 436, 437 267, 438
CRB1, 45, 94, 122, 148, 149, 151, 156, 181, 194, 197, Genetic susceptibility, 64, 77, 78, 87, 183, 193,
200, 201, 203–205, 211–213, 236, 325, 328, 410 351, 357, 440
CRX, 45, 52, 53, 122, 149, 161, 180, 181, 196, 197, Genetic testing, 6, 30, 34, 36, 44, 48, 51, 53, 89, 153,
200–202, 205, 208, 212, 325, 326, 328 155, 157–159, 213, 243, 299, 315–319, 334
CTNNA1, 199, 201, 204, 205 Genome editing, 267, 268
CYP1B1, 43, 72, 171–173, 337, 359, 362, 410, 411 Genome sequencing, 129, 404, 411
CYP4V2, 199, 204, 205, 236, 325 Genome wide association study (GWAS), 3, 42, 62–64,
72, 90, 148, 183, 185, 351, 352, 358, 362–365,
376–378, 385–387, 403, 407, 408, 418, 421–425,
D 427, 428, 436, 439–441, 443, 444
Data sharing, 2, 6, 29, 32 Genotype, 2, 3, 5, 6, 29–31, 38, 44, 51, 58, 64, 95, 98,
Database, 2–6, 9–11, 14, 17, 20, 30, 33, 35, 39, 42, 44, 101, 104, 107, 109, 137, 141, 143, 152, 154,
45, 51–53, 90, 101, 126, 134, 141, 143, 172, 200, 182, 184, 185, 192, 200, 210, 211, 213, 218,
213, 217, 218, 396, 401, 403, 404, 406, 407, 219, 240, 243, 258, 265, 267, 268, 281,
409, 410, 412 283, 286, 287, 290, 323, 341, 348–350,
Diabetic retinopathy (DR), 8, 64, 72, 86, 147, 184–185, 362, 363, 365, 399, 450
265, 345–352, 393, 394, 397, 400, 408 Genotype-phenotype correlation, 38, 143, 154, 210–213,
243–244, 258, 267, 268, 323
Glaucoma, 3, 14, 25, 43, 58, 71, 86, 131, 139, 171, 299,
E 337, 357, 374, 382, 393
East Asians, 64, 170–173, 182, 183, 212, 348, Global Eye Genetics Consortium (GEGC), 2, 9, 141
357–366, 417, 428 Granular corneal dystrophy type 2, 450–454, 456–458
Endophenotypes, 61, 63, 362, 373–375, 377, 378 GUCY2D, 45, 95, 97, 122, 156, 181, 196, 200–202,
Enhances S-cone syndrome (ESCS), 329–330 206, 207, 209, 211, 212, 215, 218,
Epidemiology, 72–73, 89, 90, 122, 132, 148, 192, 256, 237, 326, 328, 409
298–299, 365, 382–383, 394, 436–437, 450
Erosive vitreoretinopathies (ERVR), 323, 328–330
Exfoliation syndrome (XFS), 365, 382–387 H
Exome sequencing, 8, 94, 107, 122, 123, 126, 151, 161, Health economics, 345–352
184, 196, 201, 210, 218, 219, 362, 404–406, Health system, 23, 44, 132–134
408–411, 421, 427, 436, 441 Hereditary retinal disease, 3, 94, 178, 192
Exudative vitreoretinopathy (EVR), 73, 330 Heritability, 61, 63, 77, 79–81, 171, 314, 374–377,
Eye diseases, 2, 8, 9, 14, 15, 29–31, 39, 42, 72, 86, 384, 408
88–90, 94, 108, 118, 131, 141, 147–161, Heterogeneity, 33, 36, 37, 58, 78, 101–102,
170–173, 218, 273, 274, 393–401, 403–412 148, 156, 192, 195, 210, 211, 234, 258,
EyeGene, 9, 29, 32 280, 281,
327, 334, 373, 428
Heterogeneous disease, 97, 216, 233–234, 357, 411
F Homozygosity mapping (HM), 151, 156, 161, 201, 210,
Fundus albipunctatus, 155, 324 213, 239, 240, 243, 408
Index 467
I Myopia, 6, 8, 86, 153, 171, 172, 194, 328, 375,

IFT140, 198, 200, 201, 203, 206, 325, 328 410, 417–428
IMPDH1, 45, 49, 122, 149, 151, 197, 203,
206, 212, 325, 328
Indian Eye Research Group (IERG), 7, 8 N
Inheritability, 418 National Eye Institute (NEI), 1, 2, 9, 17, 19–21, 29–31,
Inherited retinal disease (IRD), 88, 113, 115, 147, 158, 33, 37, 38, 117, 119, 148
233–245, 290 National Institute of Sensory Organs (NISO), 1, 6
IQCB1, 199–201, 204, 206, 328, 331 Nepal, 13, 15, 76–82, 115, 382
Neurodegeneration, 347, 397–399
Neuronal ceroid lipofuscinosis (NCN), 194, 328, 333
J Next-generation sequencing (NSG), 33, 44, 45, 48,
Japanese, 3, 9, 15, 19, 20, 52, 137, 140–143, 171, 172, 51–53, 121–123, 152, 154, 161, 210, 218, 219,
184, 202, 203, 285, 293, 339, 347–352, 359–362, 235, 236, 240, 316–317, 319, 358, 378,
364, 365, 376, 386, 410, 421, 425, 439, 451 404, 406, 408, 412, 418, 427, 428
Joubert syndrome (JS), 148, 192, 194, 195, 197, 204, Ningxia, 93–109, 142
208, 239, 328, 331–332 NMNAT1, 197, 200, 203, 208, 211, 212, 328
K O
KCNJ13, 160, 198, 203, 206–207, 328, 331 Occult macular dystrophy (OMD), 52, 53, 94,
Keratoconus, 42, 51, 53, 132, 156, 193, 194, 139, 250–253
201, 435–444 Ocular diseases, 30, 44, 57, 71, 80, 234, 256, 394, 411
Knock-in mouse, 142, 143 Ocular genetics, 3, 44, 57, 89, 118, 119, 132, 133, 148,
161, 169, 170, 200
Oguchi disease, 118, 155–156, 324
L Ophthalmic genetics, 8, 14, 15, 18, 20, 30, 31, 116
LCA5, 45, 181, 197, 200–202, 207, 212, 215, 237, 409 Ophthalmology, 2, 13, 25, 43, 71, 82, 86,
Leber congenital amaurosis (LCA), 44, 45, 48, 52, 94, 115, 133, 137, 170, 352
97–100, 139, 142, 156–157, 179–181, 202, Optic neuropathy, 3, 14, 58, 61, 132, 139, 250,
234–239, 323, 325–328, 332, 410, 438 357, 358, 362, 394
Leber hereditary optic neuropathy (LHON), 94, 139, OTX2, 198, 200, 201, 203, 208, 328
154–155, 273–277
Linkage analysis, 87, 114, 117, 201, 219, 236, 358, 406,
410, 418, 420, 421, 428, 436, 438, 440 P
Loss of heterozygosity (LOH), 300, 302, 304 Pacific peoples, 41–47, 50, 52–54
LRAT, 122, 149, 181, 198, 200, 201, 203, 207, 211, Pathology, 95, 97, 113, 131, 148, 195, 351, 358, 374,
325, 328 375, 378, 383–384, 395, 396, 399, 400, 436–439
Patient iPS cells, 142, 143
PDE6B, 44, 48, 50, 53, 122, 149, 238, 324, 325, 411
M Pedigree, 44, 48, 51, 59, 77–81, 94, 98, 104, 107,
Macular dystrophy, 73, 139, 152, 205, 208, 250, 252, 114–117, 124, 127, 140, 177, 194, 205–208, 213,
255, 256, 281, 326, 327 360, 378, 420, 421
Malaysia, 3, 15, 19, 57, 351, 438 Persistent Fetal Vasculature (PFV), 8
Māori, 44 PEX1, 199, 204, 331
MERTK, 122, 149, 181, 198, 200, 207, 325, 328 Phenotypes, 10, 29–31, 36, 37, 44, 48, 51–53, 58, 61, 64,
Methylation, 302, 304–306 77, 79, 87, 94, 95, 97, 100, 101, 103, 108, 109,
Miyake’s disease, 250–253 123, 127, 128, 137, 141–143, 148, 153, 156,
Molecular diagnosis, 87, 123, 128, 161, 179, 192, 193, 159–161, 171, 193–195, 200, 201, 205–213, 217,
210, 211, 217, 314, 316, 323, 412 234, 240, 241, 243, 250, 252, 267, 274, 281, 283,
Molecular mechanisms, 9, 10, 97, 137, 142, 143, 184, 287, 289, 300, 318, 323, 340, 343, 373, 374, 376,
210, 217, 218, 438, 450 378, 386, 403, 405–408, 410, 419, 422, 426, 442
Molecular research, 57, 58, 71 Philippines, 15, 85–90, 361
Moorfields, 5, 9, 17, 18, 118, 215 Phototransduction pathway, 148–150, 155
Mutations, 3, 43, 59, 72, 94, 116, 121, 141, 148, 170, PNPLA6, 198, 201, 203, 208
179, 192, 234, 250, 255, 273, 280, 299, 315, 324, POC1, 198, 203, 208
338, 358, 374, 409, 420, 439 Polymorphism, 3, 6, 60–62, 64, 65, 114, 171, 172, 182,
MYO7A, 94, 122, 148, 149, 160, 199–201, 204, 207, 184, 185, 218, 240, 327, 338, 340, 347, 349–351,
237, 242, 328, 330 365, 385, 407, 408, 420, 426, 436, 439–441, 444
468 Index
Population, 2, 13, 24, 31, 42, 58, 76, 85, 93, 122, 132, 280, 288, 318, 323, 332, 345–352, 393, 394, 397,
142, 148, 169, 178, 192, 234, 277, 298, 314, 338, 400, 408, 409, 438, 462
345, 357, 374, 382, 393, 403, 417, 436, 450 RP1L1 gene, 250, 252
Population research, 23 RPE65, 45, 48, 116, 118, 122, 148, 149, 156, 157, 181,
Prevalence, 3, 23–25, 33, 42, 53, 64, 76, 77, 81, 82, 194, 196, 197, 200–202, 209, 211–216, 238, 242,
86–88, 101, 104, 122, 132, 134, 147, 148, 244, 267, 324, 325, 328, 409
152–155, 157, 158, 170–173, 178, 180–185, 213, RPGRIP1, 122, 126, 181, 197, 200–202, 209, 213, 218,
234, 245, 256, 268, 273, 280, 303, 329, 338, 345, 326, 328
346, 357, 359, 360, 382, 385, 394, 417, 418, 428,
436, 437, 441, 450–454, 456–458
Primary ciliary dyskinesias (PCD), 325, 333–334 S
Primary congenital glaucoma (PCG), 171–173, 337, 359, Senior-loken syndrome (SLSN), 204, 206, 239, 328, 331
362, 411 Sequencing, 6, 10, 33, 44, 77, 94, 100, 101, 103, 105,
Progressive occult maculopathy, 253 107–109, 119, 121, 143, 151, 179, 181–184, 196,
Proteomics, 8, 9, 89, 143, 393–401 200, 210, 213, 217, 218, 240, 316, 339, 342, 358,
PRPH2, 52, 95, 97, 122, 148, 152, 153, 155, 198, 203, 362, 378, 404–412, 418, 420, 421, 427, 428, 441
208, 267, 268, 325–328 Single nucleotide polymorphism (SNP), 61–64, 77, 81,
Public health, 25, 30, 44, 78, 81, 82, 89, 90, 132, 293, 151, 171, 184, 185, 347, 352, 362–364, 385,
394, 417 420–422, 425, 427, 428, 440, 441
South Korea, 3, 15, 19, 439, 450–454, 456–458
SPATA7, 44, 45, 122, 150, 181, 197, 201, 202, 210, 239,
Q 325, 328
Quantitative trait, 61, 63, 79, 420, 425 Stargardt disease (STGD), 31, 36, 121, 123, 139, 152,
182, 233, 239, 280–290, 327
Stargardt-like macular dystrophy, 123–126
R Stickler Syndrome (STL), 62, 139, 329
RB1, 64, 88, 299–304, 307, 315–318
RD3, 44, 45, 197, 200, 201, 203, 209, 328
RDH12, 122, 149, 181, 197, 200, 201, 203, 209, 211, T
213, 238, 325, 328, 411 Targeted sequencing, 125, 128
Refractive error, 87, 132, 153, 171, 194, 214, 410, 418, Tokyo Medical Center (TMC), 1, 2, 137, 138, 141, 253
421–425, 427, 428 Training, 2, 5, 24, 25, 58, 86, 89, 90, 106, 214
Refsum symdrome (batten disease), 159, 325, 331 Translational research, 143, 217, 319
Research collaboration, 1, 2 Treatment, 10, 11, 29, 30, 38, 39, 60, 71, 73, 87, 88, 90,
Research funding, 4–6, 293 101, 118, 121, 132–134, 143, 157–159, 170, 178,
Retina, 48, 52, 53, 63, 64, 71–73, 76, 94, 95, 97, 99–101, 184, 185, 213–219, 244, 245, 258, 267, 268,
104, 105, 108, 115, 123, 139, 142, 147, 148, 152, 274–277, 290, 306–307, 314, 318, 319, 338, 343,
153, 155–158, 161, 177–185, 195, 196, 198, 201, 352, 357, 358, 366, 394, 399, 412, 436, 450, 455,
205, 208–210, 212, 214, 216, 217, 243, 252, 257, 456, 458
258, 276, 280, 282, 299, 307, 313, 323–325, 328, TULP1, 45, 122, 126, 149, 198, 200, 201, 203, 210, 211,
330, 332, 338, 346, 347, 352, 360, 387, 393, 213, 239, 325, 328, 410
395–401, 417, 421, 425 Type 2 diabetes mellitus (T2DM), 77, 185, 332, 346–352
Retina genes, 177–185
Retinal, 139–143, 346, 347, 399
Retinal degenerations (RD), 38, 102, 124, 142, 148, 181, U
183, 193, 195–197, 199, 200, 204, 207–210, Usher syndrome (USH), 19, 88, 94, 101, 139, 159–160,
214–216, 239, 243, 244, 252, 290, 323, 409, 412 178, 179, 199, 234, 236, 239, 243, 325, 410
Retinal diseases, 3, 9, 10, 36, 44, 72, 87, 88, 94, 113,
115, 139–142, 147, 152, 158, 177, 178, 182,
185, 192, 193, 214, 233–245, 267, 290, 323, V
326, 330, 400 Variant filtering, 406–408
Retinal ganglion cells (RGC), 63, 100, 142, 143, 154, Vision 2020, 23, 71, 132, 170
274, 359, 362, 363, 375, 376, 395, 396 Visual cycle (VC), 97, 118, 148–150, 152, 197, 290
Retinitis pigmentosa (RP), 3, 31, 36, 72, 73, 86, 88, 94, Vitelliform macular dystrophy (VMD), 45, 52, 152–153,
101–104, 113, 114, 121–123, 131, 132, 139, 140, 255–268, 327
142, 147, 150–152, 156, 158–160, 178–179, 181,
182, 192, 200, 204–206, 209, 211, 233, 252, 256,
293, 324–325, 327, 331, 333, 334, 409 W
Retinoblastoma, 43–44, 58, 64, 88, 139, 298–307, 313 Whole genome/exome sequence (WGS/WES), 2, 6, 33,
Retinopathies, 8, 58, 64, 65, 72, 73, 86, 95, 97, 98, 132, 100, 107, 129, 141, 151, 181, 210, 218, 219,
139, 156, 184, 185, 205, 208, 209, 253, 256, 265, 235–240, 243, 404–412, 418, 427

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Essentials in Ophthalmology

Series Editor: Arun D. Singh

More information about this series at http://www.springer.com/series/5332

ISSN 1612-3212 ISSN 2196-890X (electronic)

Library of Congress Control Number: 2017937303

© Springer Nature Singapore Pte Ltd. 2019

The Asian Eye Genetics Consortium (AEGC) was established in 2014 to

Cleveland Clinic Foundation Arun D. Singh

had established world-known research centers, the dispersion of genetic

International Council of Ophthalmology (ICO)

L. V. Prasad Eye Institute Gullapalli N. Rao

Watanabe, Toshiro Mikami, and Selvakumar Rajendran from Springer pro-

Bethesda, MD, USA Gyan Prakash

1 Asian Eye Genetics Consortium (AEGC):

10 Hereditary Eye Disease in Ningxia Hui

23 Stargardt Disease in Asian Population.......................................... 279

34 Myopia Genes in Asians................................................................. 417

About the Editors.................................................................................... 461

Abstract including Australia, Bangladesh, China, India,

© Springer Nature Singapore Pte Ltd. 2019 1

Currently, two countries – Japan and India –

Prof. Govindasamy Kumaramanickavel, tion in genetic research. He delivered an oration

AJFTLE participated in the poster and presen-

1.5  he First AEGC-Dedicated

In the year 2016, the AJFTLE in India established

To facilitate the best design of a database, a

1.6.1 AEGC Infrastructure be shipped to a site that could undertake the

1.6.6 Future Work we will begin to expand this concept to other

Abstract research, but Journal Impact Factors™ and num-

© Springer Nature Singapore Pte Ltd. 2019 13

internal (620, 2.509%), and endocrinology/

Abstract 3.1 Community Eye Health

© Springer Nature Singapore Pte Ltd. 2019 23

R. S. Parrish, K. E. Goetz, and S. J. Tumminia

Abstract 4.1 Introduction

© Springer Nature Singapore Pte Ltd. 2019 29

4.8 Community Access GENE® Research Resource. A full list of studies

© Springer Nature Singapore Pte Ltd. 2019 41

VSX1, and ZEB1 genes, we have not identified

5.1.3 Anterior Segment Dysgenesis

A NZ Māori woman had a historical diagnosis of

The NZ IRD database includes 48 families with 5.2.2 A

5.3 X-Linked Disease 5.3.2 X-Linked Retinoschisis

5.3.1 Choroideremia Of seven probands with X-linked retinoschisis

5.3.3 X-Linked Congenital and an ORF15 deletion c.2630delA, which segre-

5.4.3 CRX-associated CORD 5.4.5 BBS9 Cord

(Leu69Ile), and c.1014_1015delinsTT p. 5.5 Conclusions

Abstract tion, while SNP of 2245G/A was found to be

© Springer Nature Singapore Pte Ltd. 2019 57

resulting in many ­laboratories conducting sim- 6.2 Glaucoma and Genetics

66. IDF. International Diabetes Federation Diabetes

© Springer Nature Singapore Pte Ltd. 2019 71

7.2 Genetics associated with PLEKHA7 and COL11A1 genes.

References 5. Fahim AT, Daiger SP, Weleber RG. Nonsyndromic

Abstract cal mechanisms underlying the development

© Springer Nature Singapore Pte Ltd. 2019 75

8.2 Age-Related Cataract:

Cataract is a complex multifactorial disease extended pedigrees provide optimal opportuni-

“affected” status and those falling below the 8.7.3 C

Cleveland Clinic Foundation Arun D. Singh

L. V. Prasad Eye Institute Gullapalli N. Rao

Bethesda, MD, USA Gyan Prakash

1 Asian Eye Genetics Consortium (AEGC):

10 Hereditary Eye Disease in Ningxia Hui

23 Stargardt Disease in Asian Population.......................................... 279

34 Myopia Genes in Asians................................................................. 417

About the Editors.................................................................................... 461

1.5 he First AEGC-Dedicated

1.6.1 AEGC Infrastructure be shipped to a site that could undertake the

1.6.6 Future Work we will begin to expand this concept to other

Abstract 3.1 Community Eye Health

Abstract 4.1 Introduction

4.8 Community Access GENE® Research Resource. A full list of studies

5.1.3 Anterior Segment Dysgenesis

5.3 X-Linked Disease 5.3.2 X-Linked Retinoschisis

5.3.1 Choroideremia Of seven probands with X-linked retinoschisis

5.3.3 X-Linked Congenital and an ORF15 deletion c.2630delA, which segre-

5.4.3 CRX-associated CORD 5.4.5 BBS9 Cord

(Leu69Ile), and c.1014_1015delinsTT p. 5.5 Conclusions

resulting in many laboratories conducting sim- 6.2 Glaucoma and Genetics

7.2 Genetics associated with PLEKHA7 and COL11A1 genes.

8.2 Age-Related Cataract:

9.3.5 Molecular Profiling 9.4 Genetic Services

10.4 Usher Syndrome 10.4.3 Genetic Research

10.4.1 Introduction The homozygous variant that affects the canoni-

10.6.2 Clinical Features line phosphatase (ALP) level, and reduced

10.6.3 Genetic Research mutant fibrillin-1 (residues 584–950) was con-

11.2 Beginnings: The Prince

Abstract 12.1 Introduction

12.2 Epidemiology of RP in Korea MAK, TULP1, GUCA1A, GUCA1B, PRPH2,

14.3 JEGC Diagnostic of Japanese menus to English for future use as

14.4.2 Identification of Novel Gene JEGC objective is to identify molecular mecha-

Abstract 15.1 Introduction

rest are minor common low effect alleles. 15.2 Epidemiology

15.4.4.1 Congenital Stationary Night abnormalities. Visual acuity is severely reduced

15.4.8 Leber Congenital Amaurosis 15.4.8.1 Leber Congenital Amaurosis

7 months to over 50 years. However, due to 15.5 Syndromic RP

18.1 Introduction The incidence of RP, the more common retinal

18.2 Epidemiology 18.3 Clinical Diagnosis

18.4 Pathogenesis H82Y mutation in AIPL1, histopathological

18.5.4 CEP290 18.5.5 CLUAP1

18.5.12 GDF6 inner and outer segments of photoreceptors.

18.5.14 IFT140 KCNJ13 (potassium channel) that controls

18.5.22 NMNAT1 diminished ERGs [146]. Patients with Oliver-

18.5.24 PNPLA6 Peripherin 2 retinal degeneration slow (PRPH2)