Genetic Testing ✅

Question 1

How is testing for single gene disorders usually performed?

Answer 1

DNA sequencing techniques

Question 2

How do DNA sequencing techniques detect single gene disorders?

Answer 2

They detect single nucleotide and other small-scale sequence alterations

Question 3

What is DNA sequencing often complemented by?

Answer 3

The use of copy number analysis techniques

Question 4

What do copy number analysis techniques detect?

Answer 4

Deletions or duplications

Question 5

How was most DNA sequencing carried our until recently?

Answer 5

Sanger sequencing

Question 6

What happens in Sanger sequencing?

Answer 6

The target gene is amplified in small fragments using the polymer chain reaction (PCR), which are each sequenced separately

Question 7

What does mutation testing typically require for genes affected by loss-of-function mutations?

Answer 7

Sequencing of all exons of the gene

Question 8

What is the limitation of Sanger sequencing?

Answer 8

It does not reliably detect larger scale copy number alterations

Question 9

What is the result of Sanger sequencing not reliably detecting larger scale copy number alterations?

Answer 9

Comprehensive testing of such genes also requires a targeted copy number analysis technique

Question 10

Give an example of a targeted copy number analysis technique?

Answer 10

MLPA (multiplex ligation-dependent probe amplification)

Question 11

Why is targeted copy number analysis usually preferable to genome-wide copy number analysis?

Answer 11

It currently has greater sensitivity for small, s ingle gene, or intragenic copy number abnormalities

Question 12

What is the implication of gain of function mutations often clustering in hot-spots on testing?

Answer 12

Mutation testing to detect these can often be performed by targeted sequencing of selected exons or regions of the gene

Question 13

How are triplet repeat mutations detected?

Answer 13

Targeted PCR or Southern blotting based tests

Question 14

What triplet repeat mutations be detected by DNA sequencing?

Answer 14

Not usually

Question 15

What do targeted PCR or Southern blotting techniques assess in triplet repeat mutations?

Answer 15

The size of a gene’s triplet repeat tract

Question 16

What is the advantage of ‘next generation’ sequencing techniques?

Answer 16

They allow ‘massively parallel’ sequencing of millions DNA fragments simultaneously, permitting larger scale genetic testing at lower cost per gene than conventional Sanger sequencing

Question 17

Give 4 examples of current uses of next generation sequencing

Answer 17

• Sequencing of a single gene in large numbers of samples
• Sequencing of panels of genes with overlapping phenotypes
• Sequencing the coding sequence of all 20,000 known genes (exome sequencing)
• Whole genome sequencing

Question 18

Give an example of where sequencing of a single gene in a large number of samples may be useful?

Answer 18

Cystic fibrosis (CFTR)

Question 19

Give an example of a situation where sequencing panels of genes with overlapping phenotypes might be helpful?

Answer 19

Sequencing the 13 genes known to cause Fanconi’s anaemia

Question 20

Give an example of when sequencing the coding sequence of all 20,000 known genes may be useful?

Answer 20

Currently predominantly a research technique used when a single gene disorder is considered likely, but where targeted gene testing fails to identify a cause

Question 21

Give an example of where whole genome sequencing may be useful?

Answer 21

Predominantly a research technique used where more targeted approaches have failed

Question 22

What is the problem with exome sequencing and whole genome sequencing?

Answer 22

They generate very large quantities of data

Question 23

What is the problem with the large amount of data produced by exome and whole genome sequencing?

Answer 23

• Challenge and cost of managing the data
• Hard to find single disease causing mutation
• Difficult to differentiate disease causing mutations from innocent, non-disease causing variants
• Ethical considerations

Question 24

What are the ethical considerations with exome and whole genome sequencing?

Answer 24

Possibility of identifying an incidental gene mutation of medical consequence, e.g. mutation causing untreatable adult onset disease

Question 25

What is the implication of the issues associated with exome and whole genome sequencing?

Answer 25

Currently favour more targeted approaches when testing for single gene disorders when they are available

Question 26

Is next generation sequencing capable of detecting copy number alterations?

Answer 26

Yes, but technically difficult

Question 27

What is the result of it being difficult to detect copy number variations using next generation sequencing?

Answer 27

Currently, conventional copy number testing techniques are used where a copy number abnormality is considered likely

Question 28

Is next generation sequencing suitable for detection of triplet repeat mutations?

Answer 28

No