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Mitochondrial DNA (mtDNA) testing is a technique used in the investigation of suspected primary mitochondrial conditions.

Clinical applications

Primary mitochondrial conditions can present at any age with a wide range of symptoms, and can be caused by variants in mtDNA or nuclear DNA. At present, identification of mtDNA variants, including heteroplasmic variants, requires a dedicated sequencing approach. Heteroplasmic variants can be present at varying levels in different tissues, so an mtDNA variant may be absent from blood DNA but detectable in another tissue. For this reason, mtDNA testing may be performed in more than one tissue if the suspicion of an underlying primary mitochondrial condition is particularly strong.

How does it work?

Step one

DNA is extracted from blood. Targeted testing for common mtDNA variants is performed, with the specific variants tested dependent on the clinical presentation. For Leber hereditary optic neuropathy, for example, the three most common variants are tested for (m.3460G>A, m.11778G>A, m.14484T>C). For broader presentations of suspected mitochondrial disease, m.3243A>G, m.8344A>G and m.8993T>G/C are typically tested for, being the common causes of mitochondrial encephalopathy lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy with ragged red fibres (MERRF), neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) and maternally inherited diabetes and deafness (MIDD).

Modified restriction fragment length polymorphism analysis is carried out to test for m.3243A>G, m.8344A>G and m.8993T>G/C and involves fluorescent polymerase chain reaction (PCR) and digestion with the appropriate enzyme, followed by capillary electrophoresis, which allows for the detection and accurate quantification of the level of heteroplasmy in one test. Other variants are typically tested for using targeted Sanger sequencing.

Step two

If a diagnosis is not identified via targeted testing, the next step is to undertake sequencing of the entire mitochondrial genome, which is roughly 16.6Kb in length. MtDNA is targeted by performing a long-range PCR, which can amplify the entire mtDNA molecule, followed by massively parallel sequencing (sometimes called next-generation sequencing). As many mtDNA molecules are sequenced, even low levels of heteroplasmic variants can be identified.

Step three

If no variants are identified from steps one or two but mitochondrial disease is still suspected, it may be worth repeating mtDNA testing using DNA from another tissue. This is most commonly performed in DNA extracted from muscle tissue; however, in some cases it may be possible to sequence DNA from uroepithelial cells, liver tissue or cardiac tissue. In particular, targeted testing for the m.3243A>G variant is commonly performed using DNA from uroepithelial cells.

What are the advantages?

Targeted testing of mtDNA offers faster turnaround times for common causes of primary mitochondrial conditions. Full mtDNA testing is very sensitive to low levels of heteroplasmy.

What are the limitations?

Because a heteroplasmic variant may be absent from certain tissues, absence of a mitochondrial variant in a specific tissue such as blood does not rule out a mitochondrial condition. This is particularly the case for large-scale rearrangements, which may only be detected in muscle-derived DNA.

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  • Last reviewed: 19/09/2023
  • Next review due: 19/09/2025
  • Authors: Dr William L Macken
  • Reviewers: Dr Lianne Gompertz, Dr Mary O’Driscoll