Lab Diagnosis

GENOMIC IMPRINTING
LAB DIAGNOSIS OF GENETIC DISORDERS
2024
Genomic
Imprinting
• It is a genetic phenomenon by which certain
genes are expressed in a PARENT-OF-ORIGIN
specific manner.
• Selective inactivation of either maternal or

paternal allele before fertilization.
• Transmitted to all somatic cells.

• 1) Prader-Willi syndrome
del of band q12 on chromo15 (paternal)
Mental retardation, short stature, hypotonia, obesity,
small hands and feet, hypogonadism.
• 2) Angleman syndrome
del of same chromo region from mother
Mental retardation, ataxic gait, seizures,
inappropriate laughter –Happy puppets
The genetic basis of these two imprinting disorders is
• Prader-Willi syndrome series of genes are involved -

SNORP family of genes that encode small nucleolar
RNAs.
• These RNAs are noncoding molecules that are involved

in posttranscriptional modifications of ribosomal RNAs
and other small nuclear RNAs.
• Loss of SNORP functions is believed to contribute to

Prader-Willi syndrome, but the precise mechanisms are
unclear.
 Angelman syndrome, the affected gene is a
ubiquitin ligase that is involved in catalyzing the
transfer of activated ubiquitin to target protein
substrates.
Expressed in specific regions of the brain.
Absence of UBE3A inhibits synapse formation

and synaptic plasticity.
Indication of prenatal chromosome analysis
• Advanced maternal age >34yrs
• A previous child with a chromosomal abnormality
• A carrier parent of X-linked genetic disorder/ translocation or inversion
• Fetal anomalies observed on ultrasound
• Routine maternal blood screening indicating an increased

risk of Down syndrome (trisomy 21) or another trisomy.
At present usually performed cells obtained by amniocentesis,

chorionic villus biopsy , umbilical cord blood.
Prenatal diagnosis
• Amniocentesis
◦ most common modality
◦ performed at 15-17 weeks of gestation
• Chorionic Villus Sampling (CVS)

◦ second most common
◦ performed at 10-12 weeks of gestation
• Percutaneous umbilical blood sampling (PUBS)

◦ performed in second and third trimesters
◦ usually prompted by ultrasound abnormalities of fetus
Indication of postnatal chromosome analysis
• Multiple congenital abnormalities
• Unexplained mental/developmental retardation
• Suspected aneuploidy (down’s)
• Suspected unbalanced autosome (imprinting)
• Suspected sex chromosome abnormality
• Suspected fragile X syndrome

In older patients
• Inherited cancer syndrome
• Atypically mid monogenic disease
• Neurodegenerative disorders
( Familial Alzheimer disease, Huntington disease)
• Infertility (to rule out sex chromo abn)
• Multiple spontaneous abortions

• Diagnosis & Management of infectious disease:
• Detection of micro-organism specific genetic material
[ human immunodeficiency virus [HIV],
mycobacteria, human papillomavirus, herpesvirus in central
nervous system)
• Identification of specific genetic alteration in the
microbes- drug resistance
• Determination of treatment efficacy (e.g., assessment of
viral loads in HIV, Epstein-Barr virus, and hepatitis C
virus infection)
Diagnosis & management of
cancer:
• Detection of tumor specific mutations

• Determination of clonality
• Identification of specific genetic alterations
• Determination of treatment efficacy
• Detection of drug resistant secondary malignancy
Conventional karyotyping
• For diagnosis of cytogenetic disorders
• Ex: down syndrome
DNA based assays

• Southern blot – Huntington disease
• Over a period of time

• Sanger DNA sequencing in 1977
• PCR in 1983
• These 2 methods allowed routine sequencing of any known
segment of DNA
Diagnosis of genetic diseases
• Cytogenetic analysis: Karyotyping
• Molecular methods
Karyotyping
• Arrest of mitosis in metaphase by colchicine
followed by staining of chromosomes.
• In interphase cells, the genetic material is

dispersed and chromosomes are not visible.
• Identification of each chromosome on pattern of

distinctive light and dark bands.
• Giemsa stain – G banding

Karyotyping
• Described as: 47,XY, +21 (Downs)
• Total no of chromo, sex chromo, abnormality
• Short arm – p / Long arm – q
• A,B,C,D,E,F,G depends on chromosome
length
• A longest / G shortest
• ARM-REGION-BAND-Sub-BAND,
• numbering from the centromere progressing
distally
Banded karyotype of X chromosome
notation
Xp21.2
refers to a
chromosomal
segment
located on the
short arm
of the X
chromosome,
in region 2,
band 1, and
sub-band 2.
FISH
• Fluorescent in situ hybridization: (FISH)
A technique used to identify the presence of

specific chromosomes or chromosomal regions
through hybridization (attachment) of
fluorescently-labelled DNA probes to denatured
chromosomal DNA.
FISH
Uses fluorescent
labelled DNA
fragments, ~10,000
base pairs, to bind
(or not bind) to its
complement
• Step 1. Preparation of probe. A probe is a fluorescently-
labelled segment of DNA complementary to a chromosomal
region of interest.
• Step 2. Hybridization. Denatured chromosomes fixed on a

microscope slide are exposed to the fluorescently-labelled
probe. Hybridization (attachment) occurs between the probe
and complementary (i.e., matching)chromosomal DNA.
• Step 3. Visualization. Following hybridization, the slide is
examined under a microscope using fluorescent microscopy.
Fluorescent signals indicate the presence of complementary
chromosomal DNA; absence of fluorescent signals indicate
absence of complementary chromosomal DNA
Advantage of FISH
FISH detects small (submicroscopic)
chromosome - Is applicable to interphase cells
 Deletions
 Duplications
Translocations
• PCR & Detection of DNA sequence alterations:
 Sanger sequencing
 Pyro sequencing
 Single based primer extensions
 Restriction fragment length analysis
 Amplicon length analysis
 Real time PCR
 NGS
Single base primer extension
• Useful for identifying mutation at a specific nucleotide
position.
• An interrogating sequencing primer is added to the PCR

product.
• Binds only one base upstream of the target.
• Differentially colored terminator fluorescent nucleotides also

used.
• Very sensitive to detect 1-2% of mutated alleles.
• Disadvantage is only producing one base pair of sequencing

data.
Restriction fragment length analysis
• Advantage of digestion of DNA with endonucleases
that recognize and cut DNA at specific sequences.
• Amplified PCR product may be digested and

normal and mutated PCR products yield different
size fragments.
• These different bands are identified by

electrophoresis
Amplicon length analysis
• Here 2 primers that flank the region containing
trinucleotide repeats at 5’end.
• Amplification done.
• Large difference in the number of repeats from
the normal individual and mutated individual
• This is easily distinguished by gel

electrophoresis
PCR
Real time PCR
• Use of fluorophore indicators can detect and quantify the presence

of particular nucleic acid sequences in real time
• Used to monitor the frequency of cancer cells bearing characteristic

genetic lesion
• Also used to detect the somatic point mutation in oncogenes
• Advantage is avoiding the need for post PCR analysis

MULTIPLEX LIGATION DEPENDENT PROBE AMPLIFICATION
• It is a variation of the multiplex polymerase chain

reaction that permits amplification of multiple
targets with only a single primer pair
• Used to detect deletions & duplications of any size
• Including anomalies that too large detected by PCR

& too small detected by FISH
Southern Blotting
• Detection of a specific DNA sequence in DNA samples
• Hybridization of radiolabeled sequence specific probes to genomic

DNA
• This has been digested by restriction enzyme
• Separated by gel electrophoresis
• Its rarely used but remains useful in detection of large
trinucleotide expansion disease.

Sanger sequencing
• Amplified DNA is mixed with DNA polymerase , DNA primer,

Nucleotides , 4 dead end nucleotides labelled with different
fluorescent tags.
• Reaction produce series of DNA molecules of possible length
• The size are separated by capillary electrophoresis
• The exact sequence can be read & compared with normal
sequence to detect the presence of the mutations.
• Particularly used when analysis of large genes or multiple genes is
required.
Pyrosequencing
• When one or more nucleotides are incorporated into
the growing strands the pyrophosphate released and
participated in a secondary reaction that produce light
& measured by photo detector.
• More sensitive than sanger sequencing for detection of

as little as 5% mutated alleles present in the
background of normal alleles.
Next generation sequencing
• The term used to describe several newer DNA sequencing

technologies that are capable of producing large amount of
sequence data in a massively parallel manner.
• Differ from sanger sequencing by its input sample

requirements
• In sanger – Single simple homogenous template DNA needed
• In NGS – No need of such material
• Any DNA from almost any source can be used.

Diagnosis of single gene disorders
by recombinant DNA
• Direct gene diagnosis

• Indirect DNA diagnosis: linkage analysis
Linkage analysis
• When information about gene sequencing is
lacking in a disease
• To differentiate abnormal chromosome from
normal based on polymorphisms (variations)
in DNA sequences
• Site polymorphism
• Length polymorphism
Others
• Southern blotting: change in structure of specific

loci can be detected.
• SNP(single nucleotide polymorphism)array
• GWAS(genomic wide association studies)
• Epigenetic alterations(study of heritable chemical
modification of DNA or chromatin that does not
alter DNA sequence itself)

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GENOMIC IMPRINTING

LAB DIAGNOSIS OF GENETIC DISORDERS

• Selective inactivation of either maternal or

• Transmitted to all somatic cells.

• Prader-Willi syndrome series of genes are involved -

• These RNAs are noncoding molecules that are involved

• Loss of SNORP functions is believed to contribute to

Expressed in specific regions of the brain.

Absence of UBE3A inhibits synapse formation

• Advanced maternal age >34yrs

• A previous child with a chromosomal abnormality

• A carrier parent of X-linked genetic disorder/ translocation or inversion

• Fetal anomalies observed on ultrasound

• Routine maternal blood screening indicating an increased

At present usually performed cells obtained by amniocentesis,

• Chorionic Villus Sampling (CVS)

• Percutaneous umbilical blood sampling (PUBS)

• Multiple congenital abnormalities

• Unexplained mental/developmental retardation

• Suspected aneuploidy (down’s)

• Suspected unbalanced autosome (imprinting)

• Suspected sex chromosome abnormality

• Suspected fragile X syndrome

• Atypically mid monogenic disease

• Infertility (to rule out sex chromo abn)

• Multiple spontaneous abortions

• Detection of tumor specific mutations

DNA based assays

• Over a period of time

• In interphase cells, the genetic material is

• Identification of each chromosome on pattern of

• Giemsa stain – G banding

A technique used to identify the presence of

• Step 2. Hybridization. Denatured chromosomes fixed on a

• An interrogating sequencing primer is added to the PCR

• Binds only one base upstream of the target.

• Differentially colored terminator fluorescent nucleotides also

• Very sensitive to detect 1-2% of mutated alleles.

• Disadvantage is only producing one base pair of sequencing

• Amplified PCR product may be digested and

• These different bands are identified by

• Large difference in the number of repeats from

the normal individual and mutated individual

• This is easily distinguished by gel

• Use of fluorophore indicators can detect and quantify the presence

• Used to monitor the frequency of cancer cells bearing characteristic

• Also used to detect the somatic point mutation in oncogenes

• Advantage is avoiding the need for post PCR analysis

• It is a variation of the multiplex polymerase chain

• Used to detect deletions & duplications of any size

• Including anomalies that too large detected by PCR

• Hybridization of radiolabeled sequence specific probes to genomic

• This has been digested by restriction enzyme

• Separated by gel electrophoresis

• Its rarely used but remains useful in detection of large

trinucleotide expansion disease.

• Amplified DNA is mixed with DNA polymerase , DNA primer,

• More sensitive than sanger sequencing for detection of

• The term used to describe several newer DNA sequencing