Molecular Testing

Question 1

Germline mutations

Answer 1

inherited, present in every cell

Question 2

Somatic mutations

Answer 2

acquired, present in diseased tissue

Question 3

Molecular diagnostic tests for germline mutation

Answer 3

confrim diagnosis when suspicious clinical features shown
screen ‘at risk’ mutation carriers
prenatal diagnosis
screen pop

Question 4

Molecular diagnostic tests for somatic mutation

Answer 4

usually in tumours
diagnosis and classification
prognosis
prediction of tumour response to chemo

Question 5

Challenges of molecular testing

Answer 5

lost of different mutations - diff. tests needed
hotspots
different mutations, same gene - different phenotype
locus heterogeneity

Question 6

Hotspots

Answer 6

region of genes with high frequency of mutation

usually in functionally important areas, allowing testing

Question 7

Example of hotspots

Answer 7

Duchenne’s and also Becker’s muscular dystrophy
deletions in Dystrophin gene
D: frameshift - total loss of function
B: in-frame deletions - partial

Question 8

Locus heterogeneity

Answer 8

mutations in different genes in a pathway may give same syndrome, usually due to malfunction of physiological pathways
some functions depend on multimeric complexes - loss of one may lead to disruption of whole complex
increases no. of tests to screen syndrome

Question 9

Imprinting

Answer 9

eg. Angelman syndrome

caused by deletion of similar region - silencing duoble copy in epigenetic modification

Question 10

Expansion mutation

Answer 10

expansion of triplet repeat sequences (coding)

eg. alter protein processing - myotonic dystrophy

Question 11

Polymerase chain reaction

Answer 11

used to amplify specific region of interest
in-vitro DNA replication
requires small no. starting material, produces HUGE amounts of targeted product
product analysed

Question 12

Post PCR analysis

Answer 12

size
presence/absence of product
mutation scanning
sequencing

Question 13

Size

Answer 13

electrophoresis
allows ID of expansion mutations
polymorphic microsatellite markers to be followed for linkage analysis
using size markers: assess of threshold crossed
polymorphic microsatellite can be used to trak mutation if near mutant allele

Question 14

Presence/absence of product

Answer 14

absence - ID deletion of exons/genes/sequence

multiplex PCR uses several primers to amplify several regions which can then be analysed

Question 15

Example of multiplex PCR

Answer 15

MPLA, ARMS
sensitivity of PCR to primer-binding site base changes, use as tool to detect known mutations
no mutation: primer doesn’t bind, no PCR product
several mutations detected in same assay

Question 16

Mutation scanning

Answer 16

for allelic heterogeneity, a lot of regions may need to be scanned, so methods used:
Heteroduplex formation - altered migration on electrophoresis and binding on HPLC columns
Single strand conformation - altered by base changes, altered migration

Question 17

Sequencing

Answer 17

eg. Sanger sequencing

validate mutations - not always easy, and is expensive, therefore scanning typically done first

Question 18

Methylation specific PCR

Answer 18

used to detect imprinted/epigenetically silenced alleles
DNA modified by bisulphite reaction, conerts non-methylated cytosines to thymine
methylation specific primers then used to test for presence of PCR product
maternal/paternal specific primers