Molecular Testing

Question 1

Germline Mutation

Answer 1

Inhereted mutations present in every cell

Question 2

Somatic Mutation

Answer 2

Acquired mutations present in diseased tissue

Question 3

Clinical Utility of molecular diagnostic tests for germline mutations

Answer 3

Confirms diagnosis

Screen at risk mutation carriers

Prenatal diagnosis

Screening populations

Pharmacogenetic testing (how genes dictate response to drugs)

Question 4

Clinical Utility of molecular diagnostic tests for somatic mutations

Answer 4

Identification of tumours and prediction of tumour response to chemotherapy

Prognosis

Question 5

Challenge facing molecular tests

Answer 5

Huge variety of mutations that require various different tests

Question 6

Mutation hotspots example

Answer 6

Mutations in Hungtington’s disease occur in polyQ region of HTT gene

In CF, 80% mutations are in ΔF508

They are usually in functionally important areas

Question 7

Duchenne’s and Becker’s Muscular Dystrophy

Answer 7

X-linked recessive Deletions in dystrophin gene

Different mutations in same gene cause different phenotype

DMD due to frameshift mutations cuasing truncated protein and total loss of function

BMD due to in-frame deletions causing partial loss of function

60% of mutations in both occur in two hot spots

Question 8

How does the dystrophin protein work

Answer 8

A huge membrane bound protein that helps with calcium release and contraction of actin fibres

Question 9

Describe symptom development of DMD and BMD

Answer 9

DMD - symptoms appear 2-5 yo; loss of ambulation by 12. Death by cardiac/respiratory complications in 3rd decade

BMD - May survive to old age

Question 10

How does locus Heterogeneity affect molecular testing

Answer 10

Increases the amount of testing needed as one symptom may arise from the dysfunction of many different proteins (e.g. one protein in a complex)

Question 11

Imprinting

Answer 11

Only one allele is expressed from birth; silencing done by epigenetic modification

Deletion of a gene whether on maternal or paternal gene affects syndrome

Question 12

Where is DNA used in genetic tests derived from

Answer 12

Lymphocytes in blood mainly

Sometimes mouthwash cell/buccal scrapes from babies

Chorionic villi/amniocentesis

Question 13

Why is PCR such a game changer

Answer 13

Requires tiny quantities of starting material to produce huge amounts of target product which can then be analysed using various assays

Question 14

Expansion mutation

Answer 14

Form of mutation involving expansion of triplet repeat sequences in coding or non-coding region

Question 15

Anticipation characteristic of expansion mutations

Answer 15

Age of onset is lower and/or worse and/or more common in successive generations as progressive repeats makes mutation worse

Different diseases have different thresholds as this leaves out any residual function

(Huntington’s has >40)

Question 16

How can polymorphic mutations ease post PCR analysis

Answer 16

Can be used as markers even for when mutations are unknown (generally near locus of mutated gene)

Question 17

Multiplex PCR

Answer 17

Using several primers to amplify several regions simultaneously to then be analysed

Question 18

Figure out how to identify this and what this tells you… It’s not that hard you did it in school

Question 19

figure out Multiplex Ligation Probe

Question 20

Methylation specific PCR

Answer 20

When DNA is methylated; they are modified by bisulphite reaction where methylated cytosines are resistant to bisulfites but non-methylated get converted to cytosine

Methylation specific primers can be used to test for presence/absence of PCR product

We know which allele on which parental chromosome should be methylated in what way, so the results from analysis can indicate where mutation is and what the syndrome is (where revelant)

Question 21

Mutation Scanning

Answer 21

If we have multiple PCR products but know the mutation is only in one place, we can narrow down the analysis by using the physical characteristics of the strand like heteroduplex formation or other methods

Question 22

For analysis of PCR products, what is the most definitive answer

Answer 22

Sequencing

Question 23

Methods of sequencing

Answer 23

Dideoxy/Sanger sequencing

Pyroseqencing

Question 24

Compare pyrosequencing and sanger sequencing

Answer 24

Pyrosequencing is more sensitive but cannot sequence as much length of DNA as sanger

Question 25

Next Generation Sequencing

Answer 25

Modern form of sequencing where full genome can be sequenced relatively cheaply and quickly

Relies on PCR

Will have huge impact on medicine