Topic 5: technologies in genetics

Question 1

What conditions are associated with chromosome abnormalities?

Answer 1

Numerous medical conditions, such as spontaneous abortions, congenital anomalies, birth defects, intellectual disabilities, infertility, cancer, and other pathologies

Question 2

What does the human karyotype consist of?

Answer 2

46 chromosomes with 22 homologous pairs of autosomes and 1 pair of sex chromosomes (XX for female and XY for male).

Question 3

What are the two major types of chromosomal abnormalities?

Answer 3

Constitutional abnormality and acquired abnormality.

Question 4

What are the categories within numerical chromosomal abnormalities?

Answer 4

Monosomy and trisomy.

Question 5

What are the categories within structural chromosomal abnormalities?

Answer 5

Balanced or unbalanced.

Question 6

How can constitutional chromosomal abnormalities affect normal development?

Answer 6

Through dosage effect, direct damaging effect, discrepant parental origin, and position effect.

Question 7

What kinds of chromosomal abnormalities are reported in tumor cells?

Answer 7

Deletions, duplications, and translocations.

Question 8

What are unbalanced structural rearrangements?

Answer 8

Chromosomal abnormalities with excess or lack of DNA in the genome, affecting dosage-sensitive genes.

Question 9

What are balanced structural rearrangements?

Answer 9

Not losing or gaining material, just moving it around, including reciprocal translocation, inversion, and Robertsonian translocation.

Question 10

What is the role of cytogenetic analysis in hematology and oncology?

Answer 10

Identification of abnormalities related to cancer initiation and progression. Important for diagnosis, prognosis, and therapeutic decisions.

Question 11

What are unbalanced structural rearrangements especially problematic for?

Answer 11

Dosage-sensitive genes, such as haploinsufficient and triplosensitive genes.

Question 12

What are examples of unbalanced structural rearrangements?

Answer 12

Deletion (terminal or interstitial), duplications (often in tandem), ring and supernumerary marker.

Question 13

How are chromosomal abnormalities categorized?

Answer 13

Numerical (e.g., monosomy or trisomy) and structural (balanced or unbalanced).

Question 14

What morphological groups can chromosomes be classified into?

Answer 14

Metacentric, submetacentric, and acrocentric.

Question 15

What is a dosage effect in terms of chromosomal abnormalities?

Answer 15

A loss or gain of chromosomal material, whether for a whole chromosome or a part of a chromosome, that affects normal development.

Question 16

What is a direct damaging effect in chromosomal abnormalities?

Answer 16

Disruption of a gene at the breakpoint of a rearrangement, such as a double-stranded break.

Question 17

What is an effect due to the discrepant parental origin of a chromosome or chromosomal segment?

Answer 17

Known as genomic imprinting, it can lead to conditions such as uniparental disomy, where both copies of a chromosome are inherited from one parent.

Question 18

What is a position effect in chromosomal abnormalities?

Answer 18

A gene in a new chromosomal environment functions inappropriately, such as when a gene comes close to a strong enhancer.

Question 19

What is constitutional mosaicism?

Answer 19

A condition where an additional cell line with a different chromosomal complement arises in embryonic or pre-embryonic life and becomes part of the organism.

Question 20

What is a chimera in terms of chromosomal abnormalities?

Answer 20

An organism derived from two different zygotes, resulting in different cell lines within the same individual.

Question 21

What is G-banding in chromosome analysis?

Answer 21

G-banding involves trypsin treatment followed by Giemsa staining to produce distinctive and reproducible patterns of transverse light and dark bands along the chromosomes.

Question 22

What are the benefits and limitations of G-banding?

Answer 22

Benefits: Viewing entire genome, visualizing individual cells and chromosomes, detecting balanced and unbalanced rearrangements.
Limitations: Resolution limit around 5-10 Mb, requires actively growing cells.

Question 23

What is Fluorescence In Situ Hybridization (FISH)?

Answer 23

A DNA probe labeled with a fluorescent dye hybridizes to the metaphase or interphase chromosome, allowing the study of alterations at the submicroscopic level.

Question 24

What are the benefits and limitations of FISH?

Answer 24

Benefits: Fast, higher resolution than G-banding, can study non-dividing tissues, shorter TAT for results.
Limitations: Limited to the region of the genome complementary to the probe.

Question 25

What is Quantitative Fluorescence (QF)-PCR used for?

Answer 25

QF-PCR is used to study multiple DNA polymorphic microsatellite markers on chromosomes 13, 18, 21, X, and Y for rapid detection of common trisomies and monosomy X.

Question 26

What are the steps in QF-PCR?

Answer 26

DNA extraction, microsatellite marker PCR amplification using fluorescent labeled primer, capillary electrophoresis of fluorescent DNA fragments, analysis of microsatellite loci size and height.

Question 27

What are the benefits and limitations of QF-PCR?

Answer 27

Benefits: Fast TAT, automated, cost-effective. Limitations: Targeted approach, will not detect anomalies affecting other chromosomes.

Question 28

What is Chromosomal Microarray (CMA)?

Answer 28

CMA involves mixing patient DNA and control DNA to hybridize to their complementary sequences on the array, identifying copy number gains and losses across the genome.

Question 29

What are the benefits and limitations of CMA?

Answer 29

Benefits: Whole genome approach, less reliance on clinician's suspicion, higher resolution. Limitations: Cannot identify balanced chromosome rearrangements, cannot establish the location or orientation of chromosome segment duplications.

Question 30

What factors are considered in the interpretation of CMA data?

Answer 30

It is imperative to distinguish between pathogenic, Variation of Unclear Significance (VUS) and benign CNVs by incorporating family studies, information about the gene content of the region in question, and data from CNV databases and current literature

Question 31

How is chromosome compaction achieved for chromosome analysis?

Answer 31

By the addition of various proteins (histones, condensins) and arrangement of the chromatin into loops. From interphase to metaphase chromosome, the compaction level goes from 1 to 10,000.

Question 32

How are metaphase chromosomes obtained for analysis?

Answer 32

Blood is sampled, phytohemagglutinin is added to stimulate lymphocyte division, and mitotic spindle is disabled (e.g., with methotrexate) to stop cell division at mitosis stage.

Question 33

What are the main steps of the FISH technique?

Answer 33

Denaturation of DNA, hybridization (overnight), post-hybridization washes, and visualization of signals under the fluorescent microscope.

Question 34

What types of probes are used in FISH?

Answer 34

Centromere-specific and locus-specific probes

Question 35

What is the role of capillary electrophoresis in QF-PCR? (i.e how do we interpret the data?)

Answer 35

During capillary electrophoresis, amplified fluorescent microsatellite fragments of different lengths migrate at different speeds, and the fluorescence is measured.

Question 36

How does CMA identify copy number gains and losses?

Answer 36

Red and green intensities are measured, indicating equivalent dosage between the two genomes (yellow) or a relative gain (green) or loss (red) in the patient sample

Question 37

What are some of the clinical applications of CMA?

Answer 37

First-line test for fetuses with major malformations and patients with unexplained developmental delay, intellectual disability, autism, multiple congenital anomalies, or dysmorphic features.

Question 38

What are morbid and non-morbid genes in the context of CNVs?

Answer 38

Morbid genes are associated with diseases, while non-morbid genes are not.

Question 39

what is a microsatellite?

Answer 39

- A microsatellite is a tract of repetitive DNA. - Microsatellites are present across the genome and the length of the tracts vary between chromosomes and between individuals.