Genetic Testing ✅ Flashcards
How is testing for single gene disorders usually performed?
DNA sequencing techniques
How do DNA sequencing techniques detect single gene disorders?
They detect single nucleotide and other small-scale sequence alterations
What is DNA sequencing often complemented by?
The use of copy number analysis techniques
What do copy number analysis techniques detect?
Deletions or duplications
How was most DNA sequencing carried our until recently?
Sanger sequencing
What happens in Sanger sequencing?
The target gene is amplified in small fragments using the polymer chain reaction (PCR), which are each sequenced separately
What does mutation testing typically require for genes affected by loss-of-function mutations?
Sequencing of all exons of the gene
What is the limitation of Sanger sequencing?
It does not reliably detect larger scale copy number alterations
What is the result of Sanger sequencing not reliably detecting larger scale copy number alterations?
Comprehensive testing of such genes also requires a targeted copy number analysis technique
Give an example of a targeted copy number analysis technique?
MLPA (multiplex ligation-dependent probe amplification)
Why is targeted copy number analysis usually preferable to genome-wide copy number analysis?
It currently has greater sensitivity for small, s ingle gene, or intragenic copy number abnormalities
What is the implication of gain of function mutations often clustering in hot-spots on testing?
Mutation testing to detect these can often be performed by targeted sequencing of selected exons or regions of the gene
How are triplet repeat mutations detected?
Targeted PCR or Southern blotting based tests
What triplet repeat mutations be detected by DNA sequencing?
Not usually
What do targeted PCR or Southern blotting techniques assess in triplet repeat mutations?
The size of a gene’s triplet repeat tract
What is the advantage of ‘next generation’ sequencing techniques?
They allow ‘massively parallel’ sequencing of millions DNA fragments simultaneously, permitting larger scale genetic testing at lower cost per gene than conventional Sanger sequencing
Give 4 examples of current uses of next generation sequencing
- 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
Give an example of where sequencing of a single gene in a large number of samples may be useful?
Cystic fibrosis (CFTR)
Give an example of a situation where sequencing panels of genes with overlapping phenotypes might be helpful?
Sequencing the 13 genes known to cause Fanconi’s anaemia
Give an example of when sequencing the coding sequence of all 20,000 known genes may be useful?
Currently predominantly a research technique used when a single gene disorder is considered likely, but where targeted gene testing fails to identify a cause
Give an example of where whole genome sequencing may be useful?
Predominantly a research technique used where more targeted approaches have failed
What is the problem with exome sequencing and whole genome sequencing?
They generate very large quantities of data
What is the problem with the large amount of data produced by exome and whole genome sequencing?
- 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
What are the ethical considerations with exome and whole genome sequencing?
Possibility of identifying an incidental gene mutation of medical consequence, e.g. mutation causing untreatable adult onset disease
What is the implication of the issues associated with exome and whole genome sequencing?
Currently favour more targeted approaches when testing for single gene disorders when they are available
Is next generation sequencing capable of detecting copy number alterations?
Yes, but technically difficult
What is the result of it being difficult to detect copy number variations using next generation sequencing?
Currently, conventional copy number testing techniques are used where a copy number abnormality is considered likely
Is next generation sequencing suitable for detection of triplet repeat mutations?
No
