PRENATAL TESTING FOR

GENETIC MUTATION
CARRIER PARENTS
Dr Ritika Bajaj
MBBS, MS (OBGYN), Fellowship in Maternal Fetal Medicine (AIIMS, New Delhi)
Consultant Fetal Medicine
Jindal IVF & Sant Memorial Hospital, Chandigarh
 IMPACT OF GENETIC DISEASE

SPONTANEOUS MISCARRIAGES NEWBORN INFANTS CHILDHOOD

• 40-50% of all recognized 1st • 2%-3% of neonates have at • Genetic disorders account
trimester pregnancy loss have a least one major congenital for-
chromosomal abnormality anomaly • 50% of childhood blindness
• 50% of these are caused • 50% of childhood deafness
exclusively or partially by • 50% of all cases of severe
genetic factors learning difficulties
• Incidence of chromosomal
abnormalities in neonates-
1 in 200
• Incidence of single gene
disorders in neonates- 1 in
100

As infectious causes of perinatal mortality decline, the relative contribution of genetic causes in this subgroup has increased
 PRESENTING COMPLAINTS IN CLINICAL
PRACTISE

• Infertility- Karyotype
• Recurrent pregnancy loss- Karyotype
• Abnormal ultrasound findings in fetus during pregnancy
• History of stillbirth/ unexplained death of previous child during infancy
• History of intellectual disability/ developmental delay in previous child/ close relative
• History of genetic disorder in family (mental handicap/ physical handicap/ malformation/
lethality)
 COMMON QUESTIONS THAT BRING COUPLES TO
FETAL MEDICINE SPECIALIST/ CLINICAL GENETICIST

• Why did it happen? • Genetic diagnosis

• Can it happen again? • Recurrence risk

• What can be done in next pregnancy? • Prenatal testing

 TYPES OF GENETIC DISORDERS
CLASSIC CHROMOSOME MICRODELETIONS/ MENDELIAN DISORDERS
DISORDERS MICRODUPLICATIONS/
COPY NUMBER VARIANTS • Mutation at a single
• Full aneuploidies (Trisomy/ genetic locus
Monosomy) • Very small imbalances • Autosomal Dominant/
• Partial aneuploidies • Not detected on Standard Autosomal Recessive
(Deletions/ Duplications of Karyotype • X-linked dominant/ X-
sufficient size that they can • May not always lead to linked Recessive/ Y-linked
be seen on Microscope) clinical abnormality
• Phenotype practically always
abnormal
APPROACH TO A COUPLE WITH HISTORY OF GENETIC DISEASE

• 3 generation family history (PEDIGREE), Ascertain the pattern of inheritance

• Try to make a clinical diagnosis (Role of clinical geneticist)
• GENETIC TESTING OF THE INDEX CASE BASED ON PRESUMPTIVE CLINICAL DIAGNOSIS
• Battery of genetic tests available, different tests give different information
• Which genetic test to use- Depends upon the presumptive clinical diagnosis
• NO SINGLE GENETIC TEST TO DIAGNOSE ALL GENETIC CONDITIONS
• Many genetic tests have a Turn Around Time of 4-6 weeks
• Ideally Molecular Genetic testing should be done in the preconception period or at first ANC visit itself

• Prenatal testing possible if exact molecular diagnosis is available

• Majority of genetic disorders do not have a cure
• Current focus-
 Prevention of genetic disorder (PGT)
 Timely prenatal diagnosis
TOOLS FOR PRENATAL DIAGNOSIS

INVASIVE TESTING

• CVS (Chorionic Villus Sampling)

• AMNIOCENTESIS
 AMNIOCENTESIS

Process of withdrawing amniotic fluid from uterine cavity

for diagnostic/ therapeutic purposes

Optimal gestation – 16-17wks

Early amniocentesis (<15wks) – Increased fetal loss,

complication rates, culture failure

COMPLICATIONS-

DRY TAP –
Fetal membranes tent over needle tip. More common in
early amniocentesis (Incomplete fusion of amnion, chorion
& decidua parietalis)

FETAL LOSS –
1 in 300 to 1 in 500. Can occur upto 4 wks after procedure

BLOODY TAP –
< 1% cases. Blood almost always of maternal origin. Usually
does not affect amniotic cell growth
Amniocentesis done at 19 wks

Patient had history of bleeding PV off & on

USG- Subchorionic hematoma

RBCs in pellet after centrifugation

 CVS

OPTIMAL GESTATION – After 11 weeks (Once NT, NB

Scan is available)

Advantage of CVS over amniocentesis –

• DNA analysis can usually be carried out directly
on villi obviating the need and delay of a cell
culture as required after amniocentesis 
• Yield of cells and DNA from CVS much greater
than 20ml of amniotic fluid
• Provides a shift towards earlier diagnosis and
option of termination at an earlier gestation

COMPLICATIONS –

Fetal loss – Comparable to amniocentesis in

experienced hands

LIMITATIONS-
Mosaicism – 1-2% samples
CASE NO. 1
CHROMOSOMAL ABNORMALITY
Balanced Chromosomal Translocation In Either Parent

Karyotype- 46,XX,t(8;10)(p21;q22)

Balanced reciprocal translocation involving

breakage at specific breakpoints on ch- 8 & 10 and
exchange of fragments
 REPRODUCTIVE IMPLICATIONS OF BALANCED RECIPROCAL
TRANSLOCATION IN EITHER PARENT

Behavior of Balanced reciprocal

translocation at meiosis-

• Problems arise at meiosis because

chromosomes involved in translocation
cannot pair normally to form Bivalents
• They form a cluster known as
Pachytene Quadrivalent
• Each chromosome aligns with
homologous material in quadrivalent
Pattern of Segregation Segregating Chromosome Constitution
Chromosomes in Gamete
2:2
Alternate A+D Normal
B+C Balanced translocation
Adjacent-1 (non- A+C
homologous centromeres or Unbalanced (combination
segregate together B+D of partial monosomy and
Adjacent-2 (homologous A+B partial trisomy in zygote)
centromeres segregate Or
together) C+D
3:1
3 chromosomes A+B+C Unbalanced, leading to
A+B+D trisomy in zygote
A+C+D
B+C+D
1 chromosome A Unbalanced, leading to
B Monosomy in zygote
C
D
 RISKS IN RECIPROCAL TRANSLOCATION

• When counseling a carrier of balanced

translocation, risk of birth of an abnormal
baby depends upon the particular
rearrangement

• This risk is usually somewhere between 1%

and 10%

Karyotype of child- 46,XX,der(8)t(8;10)(p21;g22)mat

Additional material on short arm of ch- 8 due to maternal

balanced translocation
CASE NO. 2

Achieved normal milestones & was attending

regular school till 13 yrs of age
At 13 yrs of age-
• Eating difficulty with mild drooling
• Gait disturbances
• Tremors
• Speech difficulty
• Dystonia

• Ceruloplasmin levels, 24hr urinary copper

excretion, Slit lamp examination of eyes, MRI
findings s/o of Wilson’s disease

• When couple presented to us at 12 wks POG,

eldest child was already on treatment for
Wilson/s disease
• However, NO Genetic evaluation had been
done
 DILEMMAS IN THIS
CASE

• Couple was from a remote area and had not brought the affected child
• Already 12 wks of gestation & no genetic diagnosis available
• In view of clinical diagnosis of Wilson’s disease, 25% recurrence risk in each pregnancy

• Role of Clinical geneticist very important

• Sample of affected child collected from home in this case as complete evaluation
and clinical diagnosis was available and logistic issues

• Couple counselled that prenatal diagnosis can be provided only if genetic testing of
previous child reveals pathogenic variation in ATP7B gene

• Coordination with lab in view of urgency of genetic diagnosis in prenatal testing

Amniocentesis done at 16+4 weeks of gestation after pathogenic mutation identified in ATP7B gene

FETUS UNAFFECTED
CASE NO. 3

Clinical suspicion of Osteopetrosis

Baby died at 7.5 months of age

Genetic testing of baby could not be done

30
DNA of baby not available

ROLE OF TARGETED CARRIER SCREENING

OF PARENTS
• Because clinical evaluation of previous child had
been done, clinical diagnosis (Osteopetrosis)
made by Geneticist was available

• Based on clinical diagnosis, Targeted Carrier

screening was able to identify mutation in
parents

• Both parents heterozygous for c.674G>A

mutation in exon 3 of TCIRG1 gene

• 60% cases of infantile osteopetrosis are due to

mutations in TCIRG1 gene

• Because pathogenic mutation was already

identified when couple presented to us, CVS was
done for prenatal diagnosis
CVS- FETUS UNAFFECTED
CASE NO. 4 Both parents Beta Thalassemia Minor

WIFE-
Hb- 11gm%, HbA2- 4.9%
HUSBAND
Hb- 13.4gm%, HbA2- 4.8%

Late presentation- 18+6 wks

Counselled regarding 25% risk of Thalassemia major

Amniocentesis done

Trio sequencing done to save time

Pathogenic variant identified in parents and fetal DNA

evaluated for the same mutation
 • On an average, 10% of Indians are carriers for Beta
Thalassemia gene mutation

• HPLC/ Hb electrophoresis should be offered to all

women who come for pre-conceptional
counselling or at the First ANC visit

• Hb should not be used as a criteria for offering Hb

electrophoresis/ HPLC as B Thalassemia carriers
can have Hb in the range of 10-11 gm%

• In patients who present late for prenatal diagnosis-

We counsel the parents and take a written consent
explaining the need for legal route if report
becomes available after legal limit for MTP and
fetus is found to be affected

FETUS- BETA THALASSEMIA MAJOR

CASE NO. 5

FTLSCS, B Wt.- 3.0 kg

Age-appropriate milestones till 6 months of age
H/O Recurrent LRTI
Abdominal distension, Hepatomegaly,
jaundice at 6 months of age
Hepatorenal failure
Died at 7 months of age

H/0 5 abortions at 3 months POG

Cause for abortions- Not identified

Died at 7 months
Age d/t Hepatic
failure
• Genetic evaluation of child had been done
• Variant c.82G>T identified in SLC25A20 gene
• Variant was classified as VOUS
(VOUS- Variant Of Uncertain Significance)

• SLC25A20 gene is associated with Carnitine-

acylcarnitine translocase deficiency

• Carnitine-acylcarnitine translocase deficiency is AR

metabolic disorder of long-chain fatty acid oxidation

• Clinical features- Neurologic abnormalities,

hypotonia, Cardiomyopathy, skeletal muscle
damage, LIVER DYSFUNCTION
 VOUS (VARIANTS OF UNCERTAIN SIGNIFICANCE)

ACMG recommends a 5-tier system of classification of Variants

(1) Pathogenic (2) Likely pathogenic (3) Uncertain significance (4) Likely benign (5) Benign

Counseling for VOUS should preferably be undertaken by Clinical geneticists

VARIANT REANALYSIS
• Knowledge of variants is continuously evolving so an unreported variant or a VOUS at the time of
initial Exome sequencing may be labelled pathogenic in future
• Testing additional family members may result in re-classification of variants

Testing should be ordered only by specialists who are comfortable with the interpretation and
explanation of results

Information should include options for reproductive decision making, pregnancy and perinatal
management
Evaluation by clinical geneticist

Both parents underwent testing for the variant

identified in SLC25A20 gene
Sanger sequencing confirmed the presence of
variant in SLC25A20 gene in both parents

As parents were Heterozygous carriers for

SLC25A20 gene, VOUS variant was reclassified as
Likely pathogenic

Genetic counselling done regarding 25% risk of

recurrence

Parents opted for prenatal testing by CVS

FETUS – HETEROZYGOUS (UNAFFECTED)

 CASE NO. 6
Husband- Myotonic Dystrophy Type I
Mild DM1 (Mild myotonia)
People with mild DM1 may have fully active
lives & a normal or minimally shortened
lifespan

Parents counselled

• Myotonic Dystrophy is Autosomal Dominant

Myotonic Dystrophy Triplet CTG repeat disorder with 50% risk of
Type I
transmission to children
• ANTICIPATION –
• Fetus may inherit repeat lengths
considerably longer than transmitting parent
• May lead to increasing disease severity &
decreasing age of onset in successive
generations
Molecular genetic diagnosis was available in father

Couple opted for prenatal testing

CVS done at 12 weeks

FETUS UNAFFECTED
CASE NO. 7
• 5th pregnancy- Unexplained IUD @ 28 wks

• Genetic testing of fetus done

• Fetus Heterozygous for mutation in CFTR & BTD

gene

• Genetic counselling done

• Need to rule out carrier status of parents as both

diseases (Cystic Fibrosis & Biotinidase deficiency)
cause significant morbidity

• Both parents found carriers for mutation in BTD

POG
12 wks
gene (Biotinidase deficiency)
 In Present pregnancy- CVS done for both-
• Fragile X syndrome
• Biotinidase defeciency

TAKEAWAY-

Whenever we find a genetic variant, we

need to evaluate fully (even with very rare
conditions)

What is rare in literature or in western

world may not be very rare in our settings

FETUS AFFECTED (HOMOZYGOUS FOR BTD GENE MUTATION)

CASE NO. 8
10 yrs old Male child with DMD (Duchenne Muscular
Dystrophy)
GENTIC ANALYSIS-
Deletion of exon 48-50 in Dystrophin gene

Molecular Genetic testing of Mother-

No Deletion or Duplication in 79 Exons of Dystrophin gene

DMD
DMD pathogenic variant identified in affected Male is not detectable in Maternal leukocyte DNA
• It could be a ‘de novo’ pathogenic variant
• It could be because of Maternal germline mosaicism

What is Germline Mosaicism?

Some of the mother’s egg cells carry the Dystrophin gene mutation while other egg cells donot

Likelihood of maternal germline mosaicism in DMD is 15% to 20%

Even when the mother is not a carrier, it is possible that she has Germline mosaicism for the
mutation and is at risk of having a second son with DMD

Prenatal testing should be offered to mother in all subsequent pregnancies

CASE NO. 9

Present pregnancy-

NT/ NB Scan-
CRL- 54mm
NT- 3.5mm (>99th centile)
Unossified NB
FETUS- TRISOMY 18
Prenatal testing in genetic mutation carrier
parents is much more than CVS or Amniocentesis

In fact, that might be the easiest part

Knowledge of Clinical Genetics is the backbone of

prenatal testing in Single Gene Disorders

THANK YOU
 PRACTICAL CONSIDERATIONS-

• R/O Down syndrome if either

parent carries Robertsonian
translocation involving ch- 21

Source of Robertsonian Risk of having

translocation baby with
Trisomy 21
Mother carries 13q21q or 14q21q 10%
translocation
Father carries 13q21q or 14q21q 1% to 3%
translocation
Either parent carries 21q21q 100%
translocation
CASE NO. 2 ROBERTSONIAN TRANSLOCATION IN EITHER PARENT

• Robertsonian translocation- Breakage of two

acrocentric chromosomes (13, 14, 15, 21 and 22) at
or close to their centromeres with subsequent
fusion of their long arms

• Short arms of each chromosome are lost (no clinical

importance as they contain genes only for
ribosomal RNA for which there are multiple copies
on other acrocentric chromosomes)

• Total chromosome number is reduced to 45

• This is a functionally balanced rearrangement