following presentation covers different diagnostic methods utilised for diagnosis of genetic defects in brief.

1. DIAGNOSIS OF GENETIC
DEFECTS
DR. AISHWARYA SINHA

2. There are a dizzying number of techniques and indications for performing
molecular genetic diagnostic tests on patient.
The burden of choice is often problematic, both for molecular pathologists who
design tests and for clinicians who need to choose the optimal test for their
patients.

3. LABORATORY CONSIDERATION
Pathologists developing tests focus on
To choose the most appropriate diagnostic technique, one must have a thorough
knowledge of the spectrum of genetic anomalies that are responsible for the
disease in the patient population under study.
SENSITIVITY
REPRODUCIBILITY
SPECIFICITY
ACCURACY

4. INDICATION FOR ANALYSIS OF INHERITED
GENETIC ALTERATIONS
Advanced maternal age
• A parent known to carry a balanced chromosomal rearrangement, which
greatly increases the frequency of abnormal chromosome segregation during
meiosis and the risk of aneuploidy in the fertilized ovum
• Fetal anomalies observed on ultrasound
• Routine maternal blood screening indicating an increased risk of Down
syndrome (trisomy 21) or another trisomy

5. Prenatal testing may also be considered for fetuses at known risk for Mendelian
disorders (e.g., cystic fibrosis, spinal muscular atrophy) based on family history.
At present it is usually performed on cells obtained by
Following birth, testing is ideally done as soon as the possibility of constitutional
genetic disease arises, done on peripheral blood.
AMINOCENTESIS
CHORIONIC VILLI BIOPSY
UMBILICAL CORD
BLOOD

6. In newborns or children, indications are as follows:
• Multiple congenital anomalies
• Suspicion of a metabolic syndrome
• Unexplained intellectual disability, and/or developmental delay
• Suspected aneuploidy (e.g., features of Down syndrome) or other syndromic
chromosomal abnormality (e.g., deletions, inversions)
• Suspected monogenic disease, whether previously described in the family or
new.

7. In older patients, testing logically becomes more focused toward genetic
diseases that manifest at later stages of life.
the more common indications include the following:
• Inherited cancer syndromes (triggered by either family history or an unusual
cancer presentation, such as multiple cancer types or unusually young age at
diagnosis)
• Atypically mild monogenic disease (e.g., attenuated cystic fibrosis)
• Neurodegenerative disorders (e.g., familial Alzheimer disease, Huntington
disease

8. KARYOTYPING
Karyotyping is the process of pairing and ordering all the chromosomes of
an organism, thus providing a genome-wide snapshot of an individual's
chromosomes.
Karyotypes are prepared using standardized staining procedures that reveal
characteristic structural features for each chromosome.
Can reveal conditions like - aneuploidy
deletions
duplication
translocation

9. Karyotypes are prepared from mitotic cells that have been arrested in
the metaphase or prometaphase portion of the cell cycle.
Source of these cells- For prenatal diagnosis, amniotic fluid or chorionic villus
specimens are used as the source of cells.
dividing cells are arrested in metaphase by addition of colchicine, which poisons
the mitotic spindle.
stains that bind with DNA and generate characteristic banding patterns for
different chromosomes, Q- banding, G-banding.

10. Q-banding R-banding
C-banding

11. Organizing chromosome in karyograms for review- chromosomes are aligned
along a horizontal axis shared by their centromeres. Centromere placement can
also be used to identify the gross morphology, or shape, of chromosomes.
Using karyogram to detect chromosomal abnormalities- G-banded karyograms
are routinely used to diagnose a wide range of chromosomal abnormalities in
individuals.

12. Karyogram of Down syndrome

13. PCR and DETECTION OF DNA SEQUENCE
ALTERATIONS
PCR analysis, which involves synthesis of relatively short DNA fragments from a
DNA template, has been a mainstay of molecular diagnostics for the last few
decades.
By using appropriate heat-stable DNA polymerases and thermal cycling, the
target DNA lying between designed primer sites is exponentially amplified from
as little as one original copy, greatly simplifying secondary sequence analysis.

14. SANGER SEQUENCING

15. NEXT GENERATION SEQUENCING

16. the genetic material (DNA or
RNA) is fragmented
oligonucleotides of known
sequences are attached
fragments interact with the
chosen sequencing system
The bases of each fragment
are then identified by their
emitted signals.
The main difference between
Sanger sequencing and 2G NGS
stems from sequencing volume,
with NGS allowing the processing of
millions of reactions in parallel,
resulting in high-output, higher
sensitivity, speed and reduced cost.

18. SINGLE BASE PRIMER EXTENSION
an oligonucleotide primer hybridizes to a
complementary region along the nucleic acid to
form a duplex
Using a DNA polymerase, the oligonucleotide
primer is enzymatically extended by a single base
in the presence of all four nucleotide terminators
The presence of all four terminators suppresses
misincorporation of non-complementary
nucleotides.
identity the incorporated terminator using various
method,.

19. RESTRICTION FRAGMENT LENGTH
ANALYSIS
is a difference in homologous DNA
sequences that can be detected by the
presence of fragments of different lengths
after digestion of the DNA samples in
question with specific restriction
endonucleases.
An RFLP probe is a labeled DNA sequence
that hybridizes with one or more
fragments of the digested DNA sample
after they were separated by gel
electrophoresis, thus revealing a unique
blotting pattern characteristic to a specific
genotype at a specific locus.

20. REAL TIME PCR

21. The principle of real-time PCR relies on
the use of fluorescent dye. In general,
the principle of the present method is
stated below,
“The amount of the nucleic acid
present into the sample is quantified
using the fluorescent dye or using the
fluorescent-labeled oligos.” When a
dye or probe binds with the target
template, it releases a fluorochrome
which resultantly emits fluorescence
for the detector to detect. The
detector captures a signal as a positive
template amplification.

22. MOLECULAR ANALYSIS OF GENOMIC
ALTERATIONS
A significant number of genetic lesions involve large deletions, duplications, or
more complex rearrangements that are not readily amenable to detection using
PCR or DNA sequencing approaches. Such genomic-scale alterations can be
studied using a variety of hybridization-based techniques.
Fluorescence in Situ Hybridization
(FISH)

23. FLOURESCENCE IN SITU HYBRIDISATION
(FISH)

24. Performed on Used in
Pre natal sample
Peripheral blood cells
Cancer biopsies
Archvial tissue section
aneuploidy
Subtle microdeletion
Complex translocation
Gene amplification