Genetic Testing for Mitochondrial Disorders Part 1 Flashcards
Where is genetic diagnosis of mtDNA disorders undertaken in the UK?
- In the UK, genetic testing for common mitochondrial DNA mutations is undertaken by several diagnostic genetics labs within UKGTN.
- A specialist service for rare mutation analysis and/or further characterisation of mutations is provided as part of the NHS Highly Specialised Services funded Rare Mitochondrial Disorders Service for Adults and Children (centres in Oxford, London and Newcastle).
Why do mtDNA heteroplasmy levels often vary between tissues in an individual?
- At mitosis, mtDNA molecules segregate randomly to the daughter cells.
- Pathogenic mtDNA mutations can be selected against in certain rapidly proliferating tissues, such as haematopoietic stem cells (leukocyte precursors), leading to reduced heteroplasmy levels with increasing age for certain mutations in blood DNA (such as m.2343A>G).
- Pathogenic mtDNA mutations can also accumulate in post-mitotic tissues, as a consequence of mitochondrial proliferation triggered by the cell in an attempt to improve mitochondrial function.
Why is the sample type for genetic testing for heteroplasmic mtDNA mutations an important consideration in genetic testing?
- mtDNA heteroplasmy levels often vary between tissues in an individual.
- In view of the variation in heteroplasmy levels betwen tissues for some mtDNA mutations, the ideal samples are from post-mitotic, clinically affected tissues such as the muscle or liver. Post-mitotic tissues are the ones that have the highest levels of heteroplasmy in general.
- However, muscle and liver samples are invasive meaning they are costly to sample, unpleasant for the patient and not always safe to sample.
- Therefore, in practice, alternative samples are often taken such as blood, urine, mouthwash /saliva / buccal.
What are the considerations that need to be taken into account when deciding what tests to use for primary mtDNA diseases?
Considerations:
1) . Sensitivity - ability to detect low levels of heteroplasmy.
2) . Specificity - absence of false positives.
3) . Quantitative - to determine the level / percentage heteroplasmy.
4) . Robust
What are the main technologies available for the detection of mtDNA heteroplasmy?
1) . To test for known common mutations:
- Pyrosequencing (lower limit of heteroplasmy approximately 1-10%).
- Real time PCR (lower limit of heteroplasmy approximately 1-5%).
- Digital PCR, including droplet digital PCR (lower limit of heteroplasmy approximately 1%).
- Fluorescent restriction digest PCR (lower limit of heteroplasmy approximately 1-10%).
2) . To test for large-scale mtDNA rearrangements (mainly deletions):
- Long range PCR (lower limit of heteroplasmy approximately 1-5%).
- Southern blotting (lower limit of heteroplasmy approximately 1-10%).
- Real time PCR.
3) . To test for mutations in the whole mtDNA:
- Sanger sequencing (lower limit of heteroplasmy approximately 30%).
- NGS (lower limit of heteroplasmy dependant on read depth, can be <1%).
What are the main technologies available to test for mtDNA heteroplasmy common mutations?
To test for known common mutations:
- Pyrosequencing (lower limit of heteroplasmy approximately 1-10%).
- Real time PCR (lower limit of heteroplasmy approximately 1-5%).
- Digital PCR, including droplet digital PCR (lower limit of heteroplasmy approximately 1%).
- Fluorescent restriction digest PCR (lower limit of heteroplasmy approximately 1-10%).
What are the main technologies available to test for large-scale mtDNA rearrangements (mainly deletions)?
To test for large-scale mtDNA rearrangements (mainly deletions):
- Long range PCR (lower limit of heteroplasmy approximately 1-5%).
- Southern blotting (lower limit of heteroplasmy approximately 1-10%).
- Real time PCR.
What are the main technologies available to test for mutations in the whole mtDNA?
To test for mutations in the whole mtDNA:
- Sanger sequencing (lower limit of heteroplasmy approximately 30%).
- NGS (lower limit of heteroplasmy dependant on read depth, can be <1%).
Give an overview of pyrosequencing technology.
- Can be used to detect mtDNA common mutations.
- Carry out a standard PCR except generally you need to obtain a single stranded amplicon. Usually what is done is one of the primers for the PCR is biotinylated and then you can purify just the single strand.
1) . Hybridisation of a primer to single stranded PCR amplicon and incubation with enzymes.
2) . Addition of dGTP, dATP*, dTTP or dCTP, which is incorporated if complementary, and an equimolar quantity of pyrophosphate (PPi) is released.
3) . PPi drives the production of a proportional amount of light via sulfurylase, luciferin and luciferase.
4) . The light is detected and quantified by a CCD camera.
5) . Apyrase degrades unincorporated nucleotides.
6) . Further dNTPs are added sequentially. - Software automatically determines the percentage heteroplasmy on the basis of the peak heights.
Give an overview of fluorescent restriction digest PCR technology.
- Can be used to detect mtDNA common mutations.
- This approach makes use of creation or loss of a restriction site in the presence of the mutation to be tested.
- Therefore, this is not possible for all mutations but is often possible by using partially mismatched primer to artificially create a restriction site.
OVERVIEW:
- PCR followed by addition of a 5’ fluorescent labelled primer to the last cycle.
- Restriction enzyme digest.
- Capillary electrophoresis.
- E.g. Normal gene will have one restriction site, mutant will have two and they will produce different sized products on digestion and capillary electrophoresis. The ratio of peaks enables you to determine the proportion of heteroplasmy.
What are the most commonly used techniques for mtDNA rearrangement analysis? Outline these techniques.
1) . Long range PCR.
- Long range PCR across the region of mtDNA where almost all deletions occur (major ark, 10.8 kb region between origins of replication). If there is a deletion then the products and bands will be smaller.
- Not quantitative - will be skewed due to preferential amplification of the smaller band.
- Is sensitive and will detect low levels of deletion.
2) . Southern blotting.
- Typically carried out using a restriction enzyme that linearises the mtDNA such as PvuII and SnaBI.
- PvuII has a restriction site away from the commonly deleted region.
- SnaBI has a restriction site within the deleted region.
- Not as skewed as long rage PCR - is quantitative but difficult to accurately quantify with normal tech.
What is the best technique to screen for mutations throughout the whole mitochondrial genome?
NGS:
Many possible strategies and methodologies. For example:
- Amplify whole mtDNA in 2 overlapping long range PCR amplicons.
- Fragmentation and library preparation for NGS.
- Sequence (e.g. using Illumina MiSeq or Life Technologies Ion Torrent) to read depth of >1000 reads.
Dependant on read depth can detect low level heteroplasmy and accurately quantitate based on the number of mutant versus normal reads.
