Genetic Testing

Question 1

Chromosomal Analysis

Answer 1

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 (dysmorphisms, structural organ defects, mental and/or growth retardation), from families with multiple miscarriages and/or fertility problems, and directly from certain cancer biopsies.

Question 2

Chromosomal Analysis can diagnose

Answer 2

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

Question 3

Chromosomal Analysis cannot diagnose

Answer 3

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

Question 4

FISH

Answer 4

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)

Question 5

FISH can diagnose

Answer 5

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.

Question 6

FISH cannot diagnose

Answer 6

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.

Question 7

Examples of Microdeletion Syndromes

Answer 7

Cri-du-chat, Smith-Magenis, DiGeorge (22qdel), Williams syndrome, Wolf-Hirschhorn, Prader-Willi syndrome, Angelman syndrome.

Question 8

Expression Arrays

Answer 8

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 oncology

Question 9

Chromosomal Microarray Analysis (CMA)

Answer 9

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.

Question 10

General Uses and Indications of CMA

Answer 10

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. •

Question 11

CMA can diagnose

Answer 11

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

Question 12

CMA cannot Diagnose

Answer 12

Deletions/Duplications below the resolution of CMA, nucleotide mutations, balanced chromosomal rearrangements

Question 13

DNA sequencing

Answer 13

Widely used method of mutation detection based on Sanger-Gilbert method. Able to identify the genetic mutation causing many disease conditions

Question 14

DNA sequencing general uses and indications

Answer 14

Used to identify sequence changes (mutations) in specific genes. In general you need the following:
o You must know or suspect a specific genetic diagnosis
o The gene must have been identified
o The mutation must be detectable by sequencing (deletions, insertions, rearrangements are not always found by sequencing)
o 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)

Question 15

DNA sequencing can diagnose

Answer 15

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 •

Question 16

DNA sequencing cannot diagnose

Answer 16

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.

Question 17

NextGen DNA Methodology

Answer 17

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).

Question 18

Genetic testing is used for several purposes:

Answer 18

diagnosis, risk estimation, pregnancy planning, population screening

Question 19

A genetic test which gives a ‘normal’ (or ‘wild-type’) genetic sequence is often referred to a ‘_______’ by clinicians

Answer 19

negative result

Question 20

A genetic test which gives an ‘abnormal’ (or ‘mutant’ genetic sequence for example) is often referred to as being ‘‘_______” or ‘‘_______’ by clinicians (again, be wary of patient confusion)

Answer 20

positive

mutant

Question 21

polymorphism is defined as

Answer 21

a genetic mutation which is present in 1% of the population studied.

Question 22

informative genetic test result is

Answer 22

one where the information from a genetic test definitively diagnoses or excludes the disease in question. Put another way, an informative genetic test result is one that is reliably either a true positive or true negative result (ruling-in or ruling-out disease/risk, respectively)

Question 23

non-informative genetic test result usually refers to

Answer 23

a situation where the genetic test result is normal, but it is not possible to definitively exclude disease/disease risk. A non-informative genetic test result leaves open the possibility that an underlying pathogenic mutation exists, but was missed/not detected by the test.

Question 24

Allelic Heterogeneity refers to the fact that

Answer 24

multiple mutations in a particular gene (or at a particular loci) can cause disease.

Question 25

Example of allelic heterogeneity

Answer 25

Cystic fibrosis is an autosomal recessive disease caused my mutations in one gene, CFTR. Over 1,000 different mutations have been reported. Cystic fibrosis shows allelic heterogeneity but is genetically homogenous (e.g. NO Genetic Heterogeneity).

Question 26

Genetic Heterogeneity:

Answer 26

multiple genes (when mutated) associated with the same phenotype

Question 27

Example of genetic heterogeneity

Answer 27

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