week 2: genetic testing Flashcards
- Define what constitutes a “genetic test.”
Analyzing an individual’s genetic material to determine predisposition to a particular health condition or to confirm a diagnosis of genetic disease
Examining a sample of blood, or other body fluid or tissue for biochemical, or genetic markers that indicate presence or absence of genetic disease
- Describe the basic approaches, advantages, limitations, and interpretations of different types of genetic tests.
Examining a sample of blood, or other body fluid or tissue (again looking for biochemical, genetic or chromosomal markers)
Diagnostic testing:
-symptomatic patient
-confirm/assign diagnosis from test
Ex: Karyotype analysis in new infant born with trisomy 21
Predictive Testing:
-asymptomatic patient, generally with fam hist of genetic disease
-can predict disease or help assign risk
Ex: HD testing in an asymptomatic adult child of a parent with confirmed HD (I think heart disease)
BRCAI testing in an asymptomatic adult child of a parent with confirmed BRCA1 related BC (BC= breast cancer, also related to ovarian cancer)
Prognostic Testing:
determine likely disease course/severity/response to treatment
most common in cancer setting
ex: EGFR somatic mutation analysis in NSCLC to determine likelihood of response to tyrosine kinase inhibitors (TKIs)
Carrier Testing:
- asymptomatic patient
- determine risk of affected offspring
ex: cystic fibrosis carrier screening
Actual tests (rather than reasoning):
Genotyping analysis:
-if little genetic heterozygosity, it is fast, cost-effective and informative
-genotype at 1 or a few loci examined
can diagnose: previously described mutations in known genes, polymorphic variants
can’t diagnose: very specific,
old methods: gel electrophoresis, restriction digest, sequencing
newer methods: Taqman, FRET, Invader assay
PCR (Polymerase Chain Reaction):
-start with small amt of DNA
amplify the DNA using primers specific for the targeted area
-double the amt of DNA with every cycle
from there 2 electrophoresis types:
Capillary Electrophoresis
-flourescently-labelled PCR products are injected electrokinetically into polymerfilled capillaries. Voltage applied, (-) DNA migrate towards (+) anode
Gel Electrophoresis
-PCR products + dye loaded into gel
-apply voltage, (-) DNA migrate to (+) anode
size standard in adjacent well allows PCR fragment size estimation
Sanger sequencing:
Used to identify sequence specific changes
need: -know/suspect a specific genetic diagnosis
-gene must have been identified
-mutation must be detectable by sequencing (deletions, insertions, rearrangements not always found in sequencing)
-mutation must be in sequenced region
can diagnose: mutations in known genes,ideal for looking at sequence of a known disease gene
cannot diagnose: clinical sensitivity is below 100% even though frequencing is very specific, again limitations with the insertions/deletions etc
Next generation sequencing aka massively parallel sequencing:
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.
does well: small variants and deletions or duplications
limited: long string of repeats (fragile X), large deletions/duplications/rearrangements, pseudogenes
acquired disease: good at detecting low level mutations (like treatment resistance mutations like MRD)
copy number variants and structural rearrangemnets are current frontier of NGS
- Interpret genetic testing results and distinguish between “informative” and “non-informative” results.
informative: genetic test result results in a definitive diagnosis or excludes the disease in question (true positive or true negative result is given)
non-informative: genetic test refers to a situation where the genetic test result is normal, but not possible to definitively eliminate a disease/risk
Let’s talk interpretation and shit:
Coding sequence change: c.XXXXX
-position “c.1” is the “A” in the “ATG” initation codon
Predicted protein change: p.XXXX
-positon “p.1” is the methionine initation codon (AUG)
Reading mutations
Missense mutation:
FGFR3 c.1138G>A, p.Gly380Arg
Basically the individual nucleotide 1138 on gene FGFR3 was changed from a G to an A, which resulted in the protein at 380 getting changed from a Gly to Arg
Nonsense mutation:
FGFR3 c.1137 C>G, pTyr379*
Prematurally stops at 379 because Tyr got changed
Frameshift mutation:
FGFR3 c.1564_1565insA, p.Leu522Hisfs4
(insertion)
FGFR c.1564_1565delCT, p. Leu522Glyfs3
location of where insertion/deletion occcurred, how it affected things down hte road
- Explain how allelic heterogeneity and genetic heterogeneity can affect the performance of genetic tests.
genetic heterogeneity: a single phenotype/genetic disorder may be caused by mutations at multiple genes/loci
The issue with this is that you can perform a test for the most likely mutation location to result in a disease, but it is difficult to test every possible mutation location that will result in a disease
ex of a disease: Cancer predisposition syndrome
Allelic heterogeneity:
a single phenotype/genetic disorder caused by multiple mutations within the same gene
-true of most genetic disorders
>= 1500 cystic-fibrosis-associated variants in CFTR
The issue with this is that even though some are present, it maybe misleading because they dont have all the necessary mutations for a disease to manifest. Can also make it difficult when determining what will happen with offspring; perhaps it takes the summation of genetic mutations between the two parents to manifest the genetic disorder
- Discuss the ethical and legal issues related to the confidentiality of genetic test results.
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