Genetic Testing Flashcards
Describe Genotyping:
This method is used to determine presence/absence of known genetic variant.
It is mostly 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, and the disorder should exhibit *little or no allelic heterogeneity. Genotyping is cost - effective when there are few variants; as the number of candidate variants, grows, sequencing quickly becomes cheaper.
What can genotyping diagnose?
Previously- described mutations in known genes, polymorphic variants.
Whar CANNOT Diagnose?
The technique is very specific, assaying only the specific mutation (s) for which the test has been designed. The mutation(s) identified should represent the majority of causative mutations in the gene of interest, otherwise a negative result is uninformative.
Describe Fragment Analysis:
This method refers to sizing of PCR products by capillary electrophoresis or, historically, gel electrophoresis.
General Uses and Indications
: Used to identify mutations that are expected to differ in PCR amplicon size (e.g., insertions / deletions). 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 expected mutation must be of a type expected to result in a larger or smaller amplicon than wild-type, and there must be no other size polymorphisms within the amplicon.
- Can Diagnose: Small - medium (1 to ~2000 nucleotide) deletion/insertions, repeat expansions .
- Cannot Diagnose:
This technique will not detect sequence changes other than insertions/deletions.
Sanger Sequencing
General Uses and Indications: 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 ident ified
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)
• 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
• 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 l_arger deletions/insertions, rearrangements, and most chromosomal abnormalities._
MASSIVELY -PARALLEL SEQUENCING / NEXT - GENERATION SEQUENCING :
Uses massively - parallel sequencing of individual DNA molecules and is likely to replace majority of Sanger DNA sequencing within a fe w years . Has been in clinical use since 2012. Powerful tool for identifying genetic etiology in difficult cases.
What is most commonly used for?
Used to identify sequence changes in a number of circumstances, but usually most powerful when there is significant genetic or allelic heterogeneity, or when the clinical diagnosis is uncertain. Used in limited by expanding fashion to identify copy - number changes and large deletions.
o You do not necessarily have to suspect a specific genetic diagnosis !! (although it’s helpful)
o The gene must have been identified
-The mutation must be detectable by analysis algorithm (sequence variants and small- moderate insertions/deletions are easy to detect, cytogenetic abnormalities less so)
o The mutation must be located in a region of the genome that is captured (promoter and deep-intronic regions are ommited).
- Can Diagnose: Mutations in known genes (mutation can be previously reported or can be novel), polymorphic variants, small (1 to ~100 n ucleotide) deletion/insertions. Ideal for looking at the sequence of a known disease gene s in highly heterogeneic diseases, or for clinical cases for which the diagnosis is uncertain.
- Cannot Diagnose: The technique is very powerful, but typically does not detect large repeat expansions (e.g., Fragile X, HD). This technique cannot easily detect large deletions/insertions, rearrangements, and most chromosomal abnormalities, although the technologies and algorithms are constantly improving .
What is allelic heterogeneity?
Allelic Heterogeneity refers to the fact that multiple mutations in a particular gene (or at a particular loci) can cause disease. (Allelic heterogeneity in the research setting can also refer to the present of multiple non-pathogenic polymorphisms within a gene)
o Example: Cystic fibrosis is an autosomal recessive disease caused by mutations in one gene, CFTR . Over 1,5 00 different mutations have been reported. Cystic fibrosis shows allelic heterogeneity but is genetically homogenous (e.g. NO Genetic Heterogeneity).
What is genetic heterogeneity?
Genetic Heterogeneity refers to mutations in multiple genes associated with the same phenotype.
Example: Hypertrophic card iomyopathy (HCM) is an autosomal dominant disease caused by mutations in at least 10 different genes. HCM shows both allelic and genetic heterogeneity.
What is pleiotrophy?
Pleiotropy refers to mutations in a single gene can cause multiple disorders
