Molecular Testing

Question 1

Mutations and disease

Answer 1

Gene mutations make a significant contribution to human disease;
Inherited mutations (germline mutations) are present in every cell;
Acquired mutations are somatic and present in the diseased tissue;
Detection of both germline and somatic mutations is important in clinical management;

Question 2

Clinical utility of molecular diagnostic tests (Germline)

Answer 2

Confirm diagnosis where clinical features arouse suspicion;
Screen ‘at risk’ mutation carriers;
Prenatal diagnosis;
Pharmacogenetic testing;
Screen populations;

Question 3

If Germline mutation is identified in one family member…

Answer 3

Others should be tested

Example: HNPCC - autosomal dominant condition;

Question 4

Clinical utility of molecular diagnostic tests (somatic)

Answer 4

Usually done n tumours;
Diagnosis and classification - some tumours identified by mutation;
Prognosis;
Prediction of tumour response to chemotherapy;

Question 5

Challenges facing molecular tests

Answer 5

Lots of different types of mutations ranging from single base mutations up to changes in large chromosomal fragments;
Different types of mutations require different types of test;
If different types of mutation in same gene give the same effect, multiple tests may be required;
Some genes have regions that show high freq of mutation (hotspots);
In others, multiple different mutations may give the same phenotype (allele heterogeneity);
Different mutations within the same gene may give different phenotype;
Mutations in different genes in a pathway may give same syndrome (locus heterogeneity);

Question 6

Types of mutations

Answer 6

Point mutations - single base changes;
Small insertions or deletions;
Large deletions or duplications;
Structural rearrangements - translocations, inversions;
Small expansions: Huntington’s disease;

Question 7

Duchenne’s and Becker’s muscular dystrophy

Answer 7

Deletions occur in dystrophin gene;
How different mutations in the same gene cause different phenotype;
DMD due to frameshift mutations causing truncated protein and total loss of function;
BMD due to in-frame deletions causing partial loss of function;
60% mutations = deletions of whole exons in 2 hotspots;

Question 8

Locus heterogeneity

Answer 8

Syndromes often result from malfunction of physiological pathways eg. Many genes responsible for hearing loss;

Some functions depend on multimeric comples. Loss of nay member may disrupt the whole complex; eg. Mismatch repair;

It increases the number of tests required to screeen for syndrome

Question 9

Imprinting

Answer 9

All genes ar represented twice; in small minority of genes expressed from only one parental chromosome;
Silencing by epigenetic modification (methylation of cytosines);
Prader-willi syndrome and angelman syndrome;
Similar events occur in tumours at tumour suppressor loci;

Question 10

What are sources of DNA;

Post PCR analysis

Answer 10

PCR;
Post PCR analysis:
Size analysis to detect expansion mutations;
For X-linked deletions in males;
Detect common point mutations - ARMS;

Question 11

Genetic tests are performed on DNA derived from

Answer 11

Lymphocytes from blood;
Mouthwash cells;
Chorionic villi (placenta)/amniocentesis;

Question 12

PCR

Answer 12

Used to amplify a specific region of interest (eg.hotspot in gene);
In-vitro DNA replication;
Requires tiny quantities of starting material and produces huge amounts of target product;
PCR product can be analysed using a variety of assays;

Question 13

Post PCR analysis: Size

Answer 13

PCR product can be analysed for size by electrophoresis;
This will allow identification of expansion mutations such as Huntington’s disease;
It allows polymorphic microsatellite markers to be followed for linkage analysis;

Question 14

Expansion mutation

Answer 14

Special form of mutation involving expansion of triplet repeat sequences;
Expansions may be within coding or non-coding;
May exert effect by turning off gene (fragile X syndrome), altered protein processing (myotonic dystrophy) or producing non-functional protein (Huntington’s chorea);
May show anticipation- age of onset lower or severity worse or increased incidence of affected individuals in successive generations;
Due to progressive increase in size of repeats;
There are thresholds above which allele cause disease;

Question 15

Use of polymorphic marker in post PCR analysis - size

Answer 15

If there is a polymorphic marker near to a mutant alley it can be used to track mutation;
Rather than for the sequence for the mutation every time, the presence of the polymorphic marker is used to indicate presence of mutant allele;
If the specific mutation is unknown, there may be no choice other than to track the polymorphic marker;

Question 16

Post PCR analysis - presence or absence of product

Answer 16

Absence used to identify deletion of exons/genes/sequence;

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

Question 17

Amplification refractory multiplex system (ARMS)

Answer 17

Sensitivity of PCR to primer-binding site base changes can be used as a tool to detect known mutations;

Useful when there are very few mutations since primers are mutation specific;
If the mutation is not thee the prime will not bind and thee will be no PCR product;

Several mutations can be detected in same assay;

Question 18

Post PCR analysis - mutation scanning

Answer 18

In cases of allergic heterogeneity , a lot of regions may need to be tested;
Testing each one by sequencing would be expensive;
Strategies used to allow PCR product to be tested for sequence change before definitive sequencing

Question 19

Post PCR analysis: scanning

Answer 19

Dideoxy sequencing (Sanger sequencing) is the most commonly used method;
Newer methods include pyrosequencing;
It’s is not always easy and is expensive;
Better to use scanning methods before deciding when to sequence;

Question 20

Pyrosequencing compared to Sanger sequencing

Answer 20

Pyro - new and slightly more sensitive but has some shortcomings;

Question 21

Methylation specific PCR

Answer 21

Can be used to detect imprinted or epigenetically silenced alleles;
DNA is modified by bisulphate reaction which converts non-methylated cytosines to thymine;
Methylation specific primers can then be used to test for the presence or absence of a PCR product;