Clinical Applications of Molecular Biology I

Question 1

What are the types of variants?

Answer 1

1. Single nucleotide variants/polymorphisms
2. Indels
3. Repeat expansion mutations
4. Copy Number Variants/Polymorphisms
5. Structural variants
6. Uniparental disomy

Question 2

What are the molecular diagnostic assay types? When is it used?

Answer 2

1. Single locus or targeted assays
   → patient has a characteristic phenotype suggesting a specific disorder
   → suspected disorder is linked to one of a small number of genes
   → confirmation of a suspected or familial condition
2. Genome wide assays
   → patient has complex phenotype associated w/ many potential genes

Question 3

How is DNA labelled?

Answer 3

Accomplished with Nucleotide Analogues
→ End-labeled
→ Incorporated label

Question 4

What are repeat expansion disorders?

Answer 4

A wide variety of conditions that are caused by expansions of simple repeats
→ repeats may be in coding sequence or intronic sequence

Question 5

What is an example of a repeat expansion disorder?

Answer 5

Huntington’s Disease
→ progressive adult-onset, inherited, neurodegenerative disorder
→ Expansions of CAG repeat sequence in HTT gene resulting in polyglutamine sequence in protein

Myotonic Dystrophy Type 2 (DM2)
→ adult onset
→ progressive proximal muscle weakness & myotonia
→ 10000 CCTG repeats

Question 6

How to diagnose for HD?

Answer 6

Sizing a PCR product containing the CAG repeat expansion
→ <35 repeats = no disease
→ 36-39 repeats = incomplete penetrance
→ 40+ repeats = full penetrance

Question 7

How to diagnose for DM2?

Answer 7

TP-PCR / RP-PCR
→ triplet primed or repeat primed

Amplification will creat products at all possible multiples of 1 repeat unit

When PCR products are run on capillary electrophoresis machine products can be observed with a peak at each repeat unit expanding past the normal range for DM2 alleles

Question 8

In what disease is the fusion gene, BCR-ABL, found? What does it do and what is it targeted by?

Answer 8

Chronic myelogenous leukemia (CML)

Drives cell proliferation

Targetd by tyrosine kinase inhibitors

Question 9

What does karyotyping detect?

Answer 9

1. Large structural changes (translocation)
2. Large deletions/insertions (5 to 10+ Mbp)

Question 10

What is the purpose of microarray technology?

Answer 10

Multiple simultaneous measurements by hybridization of labeled probe to immobilized target on solid substrate

Question 11

What are the applications of microarray technology?

Answer 11

1. Genotyping of single nucleotide variants
2. Copy number variation detction
3. Detecting mRNA expression differences

Question 12

How does SNP genotyping by microarray work?

Answer 12

Each SNP has 2 probes
1. A
2. B

After normalization, examine intensity of signal for A and B across individuals

Result should be 3 clusters of signal
1. AA
2. AB
3. BB

Question 13

What are the two major classes of array that can be used to detect CNVs?

Answer 13

SNP arrays
→ B allele frequency (BAF)
B/(A+B) x AB = 0.5

CGH arrays
→ array of long oligonucleotides (45-80 bp) or large clones
→ competitive hybridization