Molecular tests

Question 1

Inherited mutations are called ….

Answer 1

Germline

Question 2

Acquired mutations are called ….

Answer 2

Somatic

Question 3

Clinical utility of molecular diagnostic tests in somatic mutations

Answer 3

Usually carried out on tumours ; diagnosis/classifications/prognosis

Question 4

Types of mutation

Answer 4

Point mutations (single base change) 
Small/large insertions or deletions 
Gene amplification (more copies of gene than needed)

Question 5

What is allele heterogeneity

Answer 5

When multiple different mutations within the same gene may give the same phenotype

Question 6

What are genetic hotspots

Answer 6

Regions within a gene which show a high freq of mutation

Question 7

What is locus heterogeneity

Answer 7

Mutations in different genes in a pathways may give the same syndrome

Question 8

DNA for use in molecular tests are derived from

Answer 8

Lymphocytes in blood
Mouth cells
Chorionic villi / amniocentesis

Question 9

Post PCR analysis : size

Answer 9

PCR product can be evaluated for size by electrophoresis
Allows identification of expansion mutations e.g huntingtons disease

Question 10

Expansion mutations

Answer 10

when the number of triplets present in a mutated gene is greater than the number found in a normal gene
May be in coding or non-coding regions
Possible effects : turning off gene , altering protein processing, producing non-functional gene
May show anticipation

Question 11

What is anticipation (in terms of expansion mutations) ?

Answer 11

When age of onset lowers/severity of disease increases/increased incidence in successive generations
Due to progressive increase in number of repeats

Question 12

What is DNA polymorphism

Answer 12

The different DNA sequences among individuals e.g variation is single base pairs/many base pairs or repeated sequences

Question 13

Post PCR analysis : presence or absence of product

Answer 13

Identifies deletions of exons/gene sequences

Question 14

Methylation specific PCR

Answer 14

Can be used to detect imprinted or epigenetically silenced alleles
DNA is modified by bisulphate react which converts non-methylated cytosines to thymines
Methylation specific primers can be used to tes for presence or absence of methylation

Question 15

What is genomic imprinting

Answer 15

In a small minority of genes, only one allele from one parental chromosome is expressed

silencing is achieved by epigenetic modification

Question 16

Post PCR analysis : mutation scanning

Answer 16

In cases of alleles heterogeneity a lot of regions need to be tested
Several strategies may need used to test the PCR product for sequence change before definitive sequencing

Question 17

Post PCR analysis : sequencing

Answer 17

Dideoxy (Sanger) sequencing is most commonly used
Pyrosequencing is a newer method
All mutations should be validated by sequencing but its in not always easy/cheap

Question 18

What are misense substitution mutations

Answer 18

Change leads to incorrect amino acid

Question 19

What are nonsense substitution mutations

Answer 19

Change leads to stop codon

Question 20

What is an in-frame insertion mutation

Answer 20

When number of bases added is a multiple of 3

Question 21

Sequence change due to misense mutations ?

Answer 21

Change in AA sequence or aberrant splicing

Question 22

Common effect of frameshift mutations

Answer 22

Truncated protein as stop codons often generated

Question 23

In-frame mutations may lead to

Answer 23

Inappropriate expansion of sequence/deletion of crucial sequences

Question 24

Large scale mutations - name examples

Answer 24

Numerical changes (amplifications/deletions) change the number of gene copies 
Or structural changes (translocation ) which place a gene at another site 
May create new protein or impact regulation of gene

Question 25

Promoter changes - how do they occur and what is the effect ?

Answer 25

Promotors may be activated by mutation or viral insertion giving increased transcription
Promoters may be inactivated via imprinting at birth or epigenetic hypermethylation during lifetime

Question 26

Identify the types of mutation responsible for each change in protein function

Answer 26

Question 27

If there are mutiple sites of mutation, ______ ________ will be necessary

Answer 27

Mutation scanning

Question 28

What causes Duchenne and beckers muscle dystrophy

Answer 28

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

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

both are x-linked recessive progressive muscle wasting diseases

Question 29

What is heteroduplex analysis

Answer 29

Heteroduplex are DNA molecules with one or more mismatched pairs

homoduplexes are DNA molecules with no mismatched pairs

mutant allele form heteroduplexes with the primer in PCR reactions which have bulges and can be verified by electron miscroscope