Direct Testing Flashcards
Why does the common test for cystic fibrosis have only an 85% accuracy rate?
Allelic heterogeneity; this tests for the common mutations that account for 85% of cases
What is the goal of direct testing?
Determine the genotype for 1 or more specific mutations or susceptibility alleles
“Direct testing has high analytic validity for”…
the specific alleles being assayed
How does direct testing work?
Oligonucleotides specific for the mutation are used to determine patients’ genotype:
–Only perfect hybrids are detected (due to the mutation, the abnormal DNA and the normal oligonucleotide will not pair
What are 3 types of direct testing explained in class?
- Single gene mutation (example-factor V leiden)
- Mutation panel–screen for the various genotypic mutations associated with a phenotype (such as the 23 common cystic fibrosis mutations)
- Gene panel–screen for multiple genes associated with a phenotype (such as screening for factor V leiden and prothrombin2010 in patients with hypercoagulability)
T/F: Direct testing is often not very specific for the alleles that are tested.
F–It is
T/F: Direct testing is unable to determine if a person is homozygous or heterozygous for the allele.
F–It can (PCR fluorescence testing) (specific primers for specific alleles)
Direct testing (with PCR) depends on _____ _____ of the oligonucleotide and patient DNA.
Perfect complementarity
T/F: Allele specific primers can detect differences in one base pair.
T: they won’t anneal correctly (she calls it “single base discretion”)
A trinucleotide repeat disorder is direct tested based on:
allele sizing
PCR product will vary in size based on repeat copy number
Describe PCR testing for a trinucleotide repeat disorder.
- Gene-specific primers anneal to areas flanking the repeat regions
- PCR amplification occurs across the repeat region
- PCR products are separated based on size (thus based on number of repeats)
What disease is associated with the trinucleotide repeat sequence “CGG”?
Fragile X
What disease is associated with the trinucleotide repeat sequence “CAG”?
Huntington
What may be observed in families, which appears to be related to the size of a trinucleotide repeat?
Anticipation
When using PCR to analyze a pedigree, what feature of the Huntington allele is being studied?
The degree of expansion of trinucleotide repeat region
What is notable about the interpretation of results in genetic test reports?
They are interpreted in standardized nomenclature
Primary uses of direct testing (2):
- Limited allelic heterogeneity
2. Defined mutation panel
Advantages of direct testing: (2)
- Very accurate
2. Reasonable cost
Limitations of direct testing:
Information limited to specific alleles tested
(This is an implication of allelic heterogeneity–in other words: an obligate carrier of CF with a negative test result will STILL be an obligate carrier)
Disorders in which a gene contains tandem repeats of 3 bp.
Trinucleotide repeat expansion disorders
Most common form of inherited mental retardation
Fragile X
Disorder in which a 3bp expansion in the 5’ untranslated region of FMR1 gene interferes with its expression
Fragile X
What shuts down gene expression in Fragile X?
Methylation associated with expansion
2 minor phenotypic abnormalities that are often present in premutation carriers of Fragile X?
- premature ovarian failure
2. fraX associated tremor/ataxia syndrome
