Direct Testing

Question 1

Why does the common test for cystic fibrosis have only an 85% accuracy rate?

Answer 1

Allelic heterogeneity; this tests for the common mutations that account for 85% of cases

Question 2

What is the goal of direct testing?

Answer 2

Determine the genotype for 1 or more specific mutations or susceptibility alleles

Question 3

“Direct testing has high analytic validity for”…

Answer 3

the specific alleles being assayed

Question 4

How does direct testing work?

Answer 4

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

Question 5

What are 3 types of direct testing explained in class?

Answer 5

1. Single gene mutation (example-factor V leiden)
2. Mutation panel–screen for the various genotypic mutations associated with a phenotype (such as the 23 common cystic fibrosis mutations)
3. Gene panel–screen for multiple genes associated with a phenotype (such as screening for factor V leiden and prothrombin2010 in patients with hypercoagulability)

Question 6

T/F: Direct testing is often not very specific for the alleles that are tested.

Answer 6

F–It is

Question 7

T/F: Direct testing is unable to determine if a person is homozygous or heterozygous for the allele.

Answer 7

F–It can (PCR fluorescence testing) (specific primers for specific alleles)

Question 8

Direct testing (with PCR) depends on _____ _____ of the oligonucleotide and patient DNA.

Answer 8

Perfect complementarity

Question 9

T/F: Allele specific primers can detect differences in one base pair.

Answer 9

T: they won’t anneal correctly (she calls it “single base discretion”)

Question 10

A trinucleotide repeat disorder is direct tested based on:

Answer 10

allele sizing

PCR product will vary in size based on repeat copy number

Question 11

Describe PCR testing for a trinucleotide repeat disorder.

Answer 11

1. Gene-specific primers anneal to areas flanking the repeat regions
2. PCR amplification occurs across the repeat region
3. PCR products are separated based on size (thus based on number of repeats)

Question 12

What disease is associated with the trinucleotide repeat sequence “CGG”?

Answer 12

Fragile X

Question 13

What disease is associated with the trinucleotide repeat sequence “CAG”?

Answer 13

Huntington

Question 14

What may be observed in families, which appears to be related to the size of a trinucleotide repeat?

Answer 14

Anticipation

Question 15

When using PCR to analyze a pedigree, what feature of the Huntington allele is being studied?

Answer 15

The degree of expansion of trinucleotide repeat region

Question 16

What is notable about the interpretation of results in genetic test reports?

Answer 16

They are interpreted in standardized nomenclature

Question 17

Primary uses of direct testing (2):

Answer 17

1. Limited allelic heterogeneity

2. Defined mutation panel

Question 18

Advantages of direct testing: (2)

Answer 18

1. Very accurate

2. Reasonable cost

Question 19

Limitations of direct testing:

Answer 19

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)

Question 20

Disorders in which a gene contains tandem repeats of 3 bp.

Answer 20

Trinucleotide repeat expansion disorders

Question 21

Most common form of inherited mental retardation

Answer 21

Fragile X

Question 22

Disorder in which a 3bp expansion in the 5’ untranslated region of FMR1 gene interferes with its expression

Answer 22

Fragile X

Question 23

What shuts down gene expression in Fragile X?

Answer 23

Methylation associated with expansion

Question 24

2 minor phenotypic abnormalities that are often present in premutation carriers of Fragile X?

Answer 24

1. premature ovarian failure

2. fraX associated tremor/ataxia syndrome

Question 25

2 Types of trinucleotide repeat disorders?

Answer 25

1. expansions in noncoding regions that interfere with normal gene expression
2. expansions of polyglutamine tracts in coding sequences

Question 26

What category of trinucleotide repeat disorder is associated with a loss of function?

Answer 26

expansions in noncoding regions that interfere with normal gene expression

Question 27

What category of trinucleotide repeat disorder is associated with a gain of function? What is this symptom?

Answer 27

• expansions of polyglutamine tracts in coding sequences

- neuronal degeneration

Question 28

Disorder caused by an expansion in the 3’ UTR in DMPK gene which disrupts normal splicing of other genes

Answer 28

Myotonic dystrophy

Question 29

Disorder in which expansion of polyglutamine tract produces proteins which form toxic aggregates

Answer 29

Huntington’s disease

Question 30

Autosomal dominant adult onset neurodegenerative disorder.

Answer 30

Huntington’s disease

Question 31

What is the result of meiotic instability?

Answer 31

Continued expansion of TNR’s

Question 32

What is associated with increasing trinucleotide repeats numbers?

Answer 32

Earlier age of onset (anticipation)

Question 33

What is anticipation?

Answer 33

progressively earlier age of onset with increased severity in successive generations

Question 34

What causes anticipation?

Answer 34

successive increase in copy number

Question 35

T/f: In premutation carriers, there is calculable a risk of expansion.

Answer 35

T (apparently; it’s shown in %)

Question 36

What are 2 likely mechanisms for trinucleotide repeat expansion?

Answer 36

1. strand slippage

2. altered DNA conformation during replication

Question 37

Huntington’s is more unstable in (male/female) meiosis.

Answer 37

Male

Question 38

Fragile X is more unstable in (male/female) meiosis.

Answer 38

Female

Question 39

What is a possible result of minor instability in trinucleotide repeat expansions during MITOSIS?

Answer 39

somatic mosaicism