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Flashcards in Genetic Testing Deck (18)

CHROMOSOMAL ANALYSIS: general indications for use.

Suspected abnormality of chromosome number or structure
(deletion, insertion, rearrangements). Frequently obtained from pregnant women > 35 years
(amniocentesis or chorionic villus sampling), from patients with congenital abnormalities, from families with
multiple miscarriages and/or fertility problems, and directly from certain cancer biopsies.


Chromosomal analysis can diagnose:

aneuploidies (abnormal chromosome number), chromosome deletions, duplications, and insertions of moderate to large size (>3,000-5,000 kb / 3-5 Mb), and


Chromosomal analysis cannot diagnose:

single gene deletions, point mutations, small deletions, duplications, and
insertions, methylation defects, trinucleotide repeat abnormalities.



Used to diagnose deletions, some translocations, and
abnormalities of copy number. Often used to detect cytogenetic changes that are at or beyond the
limits of resolution obtained by high-resolution chromosomal analysis. FISH for duplications works
better on cells in interphase than metaphase (metaphase the chromatin is very compact)


FISH Can Diagnose:

recognized microdeletion syndromes, recognized chromosomal rearrangements
(in cancers), and gene copy numbers (cancers). Also useful in diagnosing anueploidies (e.g.
trisomy 13, 18, 21) in the prenatal setting.


FISH Cannot Diagnose:

Cannot Diagnose: deletions, rearrangements that are not specifically tested for (i.e. FISH probes
are specifically designed for each condition). FISH is not always able to detect duplications of
gene regions. Point mutations and small deletions cannot be diagnosed with this approach.


Examples of microdeletion syndromes diagnosible with FISH:

PW, Cri-du-chat, angelman, DiGeorge, Williams,



Chromosomal analysis, expression array


What differentiates CMA from expression array?

Expression Arrays: Used to test the RNA expression
of genes (i.e. which genes are turned ‘on’ or ‘off’).
These are semi-quantitative and test the activity of
genes (see figure) rather than just the presence or
absence of a gene or genetic variant (expression
arrays). These have a small, but likely growing role, in

Chromosomal Microarray Analysis (CMA): These
have a big role in clincal genetics currently. These
look for chromosomal DNA losses and gains (so called ‘deletion/duplication’ studies). Sometimes
this is also called array comparative genomic hybridization (aCGH) analysis.


What is array comparative genomic hybridization? (aCGH)

Another term for chromosomal microarray analysis (CMA)


General Uses and Indications of CMA:

CMA has become fairly standard for looking for small
genomic deletions/insertions. You can think of this as a superior method to looking for
chromosomal gains than losses than traditional chromosomal analysis because the resolution of
the CMA is vastly superior to chromosomal analysis. The probe size used these days is between
100-200 Kb so they can pick up smaller changes than can be appreciated by chromosome
analysis. Currently, some labs use >~200 Kb for deletions and >~400 Kb for duplications.


CMA can diagnose:

aneuploidies, unbalanced chromosomal rearrangements, chromosome deletions
and duplications > 200 Kb and 400 Kb, respectively.


CMA cannot diagnose? What is the smallest resolution of CMA

Deletions/Duplications below the resolution of CMA, nucleotide mutations,
balanced chromosomal rearrangements; (~200-400kb)


General Uses and Indications of DNA sequencing (4 requirements)

1. You must know or suspect a specific genetic diagnosis
2. The gene must have been identified
3. The mutation must be detectable by sequencing (deletions, insertions, rearrangements are not always found by sequencing)
4. The mutation must be located in a region of the gene that is actually sequenced (promoter and deep-intronic mutations often missed by commercial tests)


DNA sequencing Can Diagnose:

Mutations in known genes (mutation can be previously reported or can be novel),
polymorphic variants, small (1 to ~100 nucleotide) deletion/insertions. Ideal for looking at the
sequence of a known disease gene


DNA sequencing Cannot Diagnose:

The technique is very specific, assaying only the region of the gene(s) for which the test has been designed. Frequently, many clinical genetic tests do NOT routinely
sequence all parts of a gene (e.g. promoters, introns). This means that although the approach is often very specific, clinical sensitivity is frequently below 100% (this is an important concept to understand). This technique cannot easily detect larger deletions/insertions, rearrangements, and most chromosomal abnormalities.


NextGen DNA Methodology:

Uses massively-parallel sequencing of individual DNA molecules and is likely to replace PCR based DNA sequencing within a few years (and is actually already in clinical use as of early 2012).


What is the distinction between allelic and genetic heterogeneity?

Allelic heterogeneity means that several mutations in the same gene may cause the same phenotype. Genetic heterogeneity means that multiple genes are associated with the same phenotype.

Example: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease caused by mutations in at least 10 different genes. HCM shows both allelic and genetic heterogeneity