Overview Of Genome Technologies In Genetic Diagnostics

Question 1

Why do you need to do PCR for diagnostics?

Answer 1

Amplify enough DNA molecules so we have sufficient material for downstream applications

Question 2

What is fragment analysis used for?

Answer 2

Detect repeat expansions or other small size changes

Question 3

What is fragment analysis?

Answer 3

PCR followed by capillary electrophoresis

Question 4

What is an example of a repeat expansion disease?

Answer 4

Huntingtons

Question 5

What is the mutation in huntingtons?

Answer 5

CAC repeat expansion in the huntingtin HTT gene

Question 6

Why is the huntingtons mutation bad?

Answer 6

The resulting protein is toxic and accumulates in neurons causing cell death

Question 7

How is huntingtons diagnosed?

Answer 7

Fragment analysis

Question 8

What mutation causes cutaneous vasculitis?

Answer 8

R1042G mutation in gene C3

Question 9

What does FISH stand for?

Answer 9

Fluorescent in situ hybridisation

Question 10

What is FISH used for?

Answer 10

Detect large chromosomal abnormalities

Question 11

What is the method for FISH?

Answer 11

Design fluorescent probe to chromosomal region of interest
Denature probe and target
Mix probe and target DNA

Question 12

What does CGH stand for in array-CGH?

Answer 12

Comparative genome hybridisation

Question 13

What colour is the patient DNA labelled in array-CGH?

Answer 13

Green

Question 14

What colour is the control DNA labelled in array-CGH?

Answer 14

Red

Question 15

In array-CGH, what does an increase in green signal indicate?

Answer 15

A gene in the patient not present in control

Question 16

What does MLPA stand for?

Answer 16

Multiplex ligand dependent probe amplification

Question 17

What is MLPA?

Answer 17

Variation of PCR that permits amplification of multiple targets

Question 18

What does a MPLA probe consist of and what do they do?

Answer 18

Two oligonucleotides that recognise adjacent target sites on the DNA

Question 19

What is MLPA used for?

Answer 19

To detect abnormal copy numbers at specific chromosomal locations

Question 20

What do the two probes in MLPA detect?

Answer 20

One contains the sequence recognised by the forward primer

One contains the sequence recognised by the reverse primer

Question 21

What has to happen before amplification can take place in MLPA?

Answer 21

Both probes hybridised and ligated into a complete probe

Question 22

What happens after amplification in MLPA?

Answer 22

Fragment analysis of the product

Question 23

What is next generation sequencing used for?

Answer 23

Enriching to sequence the only known disease genes relevant to the phenotype

Question 24

What is the next generation sequencing method?

Answer 24

Target enrichment
Capture regions of interest with baits
Carry out hybridisation and column purification

Question 25

What are the ethical considerations needed for exome and genome sequencing?

Answer 25

Inspect relevant genes first when analysing
Long patient consent process
Need some form of strategy for reporting incidental findings

Question 26

What does the 100,000 genome project sequence?

Answer 26

Rare diseases- index cases and families

Cancer- germline and tumour samples

Question 27

What does the 100,000 genome project do?

Answer 27

Brings direct benefit of whole genome sequencing and genetics to patients

Question 28

What are tier 1 variants in the 100,000 genome project?

Answer 28

Known pathogenic,

protein truncating

Question 29

What are tier 2 variants in the 100,000 genome project?

Answer 29

Protein altering (missense)
Intronic (splice site)

Question 30

What are tier 3 variants in the 100,000 genome project?

Answer 30

Loss- of-function variants in genes not on the disease gene panel

Question 31

Where is most of the genetic testing in the UK done?

Answer 31

NHS diagnostic laboratory

Question 32

What is the main role of the NHS diagnostic laboratory?

Answer 32

Help consultants reach a genetic diagnosis for patients to help guide their treatment and clinical management

Question 33

What is clinical validity?

Answer 33

How well a test predicts the phenotype

Question 34

What is clinical utility?

Answer 34

How well a test adds to the management of the patient

Question 35

What are the options for the outcome of a diagnostic test?

Answer 35

Pathogenic mutation
Normal variation
Novel variant

Question 36

How do you establish if a mutation is pathogenic?

Answer 36

Mode of inheritance
Genetic databases of published and unpublished data
Different types of mutagens
Missense/intronic mutation