Mitochondrial DNA Testing

Mitochondrial DNA (mtDNA) is a small circular genome of 16,569 base pairs that encodes 37 genes needed for oxidative phosphorylation. It is inherited only from the mother. Each cell holds hundreds to thousands of copies, and the proportion of mutated copies (heteroplasmy) determines whether a person is affected. Disease usually appears when the mutated fraction passes a threshold, often 60 to 90%, which depends on the variant and the tissue.

mtDNA disease causes a wide range of problems that involve high energy organs: muscle, brain, heart, eye, ear, liver, kidney and pancreas. Syndromes include MELAS (m.3243A>G), MERRF (m.8344A>G), NARP and Leigh syndrome (m.8993T>G and T>C), Leber hereditary optic neuropathy (m.11778G>A, m.3460G>A, m.14484T>C), and maternally inherited diabetes and deafness (m.3243A>G).

Single large deletions cause Kearns-Sayre syndrome, chronic progressive external ophthalmoplegia and Pearson syndrome. Aminoglycoside induced deafness is linked to m.1555A>G.

Most mitochondrial disease, particularly in children, is caused by variants in nuclear genes that serve the mitochondria (for example, POLG, SURF1, and many others), and these are tested by gene panels or exome sequencing.

Testing can be targeted (a specific common variant), or full sequencing of the whole mtDNA by next generation sequencing, with detection and measurement of heteroplasmy. Deletions are found by long range PCR, sequencing or quantitative methods. DNA can come from blood, urine sediment, buccal cells or muscle. The result takes 2 to 6 weeks.

Mitochondrial disease is estimated to affect about 1 in 4000 to 1 in 5000 people, and about 1 in 200 people carry a pathogenic mtDNA variant at low levels without disease.

Indications.

• Multisystem disease with features suggesting a mitochondrial disorder: stroke like episodes, seizures, myopathy, ophthalmoplegia, diabetes with deafness, cardiomyopathy, lactic acidosis, and a family history of maternal inheritance.
• Painless subacute visual loss in a young adult (Leber hereditary optic neuropathy).
• Maternally inherited deafness or diabetes.
• Leigh syndrome and other severe infantile encephalopathy, where mtDNA and nuclear genes are both tested.
• Testing of maternal relatives of a proband, and at risk women asking about reproductive options.
• Aminoglycoside exposure risk, in families with maternally inherited deafness.
• Investigation of muscle biopsy findings (ragged red fibres, cytochrome oxidase deficiency).

Not indicated as a first test in an infant with a suspected mitochondrial disease where nuclear genes are more likely, unless the family history suggests maternal inheritance.

Specimen requirements.

• EDTA blood (3 to 5 mL) for common point variants. For variants that fall in blood over time (for example, m.3243A>G), urine sediment, buccal cells or muscle give better heteroplasmy estimates.
• Skeletal muscle biopsy for deletions and for tissue specific variants.
• A clinical summary with lactate, imaging and biochemical findings, and the maternal family tree.
• Consent after counselling about maternal inheritance and its implications for relatives.

Normal appearances.

The mtDNA sequence matches the reference sequence apart from common population variants (haplogroup defining polymorphisms), which are not disease causing. No heteroplasmic pathogenic variant is found over the detection limit.

Interpretation.

• Pathogenic variant with a high heteroplasmy level: consistent with disease, and the level helps to explain severity. Symptoms can occur at levels as low as 20 to 30% for some variants in muscle.
• A pathogenic variant at low heteroplasmy in blood: possible in a person with symptoms, since levels in blood fall with age for some variants. A test on urine or muscle gives a better estimate.
• Homoplasmic variants (all copies mutated) in Leber hereditary optic neuropathy: disease penetrance is incomplete (about 50% of men and 10% of women develop visual loss).
• Single large scale deletions: arise de novo in most patients and are rarely passed on, so recurrence risk is low (about 1 in 24).
• Maternal transmission: a mother with a heteroplasmic variant can pass on a different proportion to each child. Recurrence risks are hard to give, and range widely.
• Variants of uncertain significance: require supporting evidence from muscle biochemistry, histology, heteroplasmy in different tissues, and segregation in maternal relatives.
• Results are interpreted together with the clinical picture, lactate, imaging, muscle biopsy and nuclear gene testing.

Critical values and findings needing immediate action.

• A pathogenic m.1555A>G or related variant: avoid aminoglycoside antibiotics, and counsel maternal relatives.
• A pathogenic POLG or mtDNA variant in a patient with seizures: avoid sodium valproate because of the risk of fatal liver failure.
• Stroke like episodes, severe lactic acidosis, or a metabolic crisis in a known carrier: urgent specialist care.
• Features of cardiac involvement (cardiomyopathy, conduction defects) in a carrier: cardiac assessment and monitoring.
• Maternal relatives of a proband planning pregnancy: urgent genetic counselling.

Limitations and artefacts.

• Heteroplasmy differs between tissues, and blood may under represent the burden for some variants, giving false negative results.
• Sanger sequencing detects heteroplasmy at levels of only about 15 to 20%, so next generation sequencing is better for low level variants.
• Nuclear copies of mtDNA sequences (numts) can cause false positive calls, and careful laboratory methods are needed.
• Many variants have uncertain significance, and a single variant does not prove disease.
• Normal mtDNA tests do not exclude mitochondrial disease, since most cases arise from nuclear genes.
• Deletions need special methods, and their detection in blood is poor.
• The level of heteroplasmy in an oocyte cannot be predicted from the maternal level, and prenatal testing has limits.
• A mitochondrial disease picture can result from other metabolic and acquired causes.

Special groups.

• Children: nuclear genes are tested with mtDNA, and early diagnosis affects management.
• Young adults with optic neuropathy: urgent testing for Leber hereditary optic neuropathy, and counselling about smoking, alcohol and toxins as triggers.
• Women who carry variants: reproductive options include prenatal diagnosis, preimplantation genetic testing, egg donation, and, where permitted, mitochondrial donation.
• Maternal relatives: testing is offered after counselling, and they are advised about drugs to avoid.
• Patients with diabetes and deafness: testing for m.3243A>G, with screening for cardiac, renal and neurological disease.
• Regions: mtDNA testing is available in few centres, and diagnosis often rests on clinical features, lactate, muscle biopsy and exclusion of other causes. Aminoglycoside deafness is a preventable disorder, so antibiotic choice and family counselling are important.

Follow up tests.

• Next generation sequencing of the whole mtDNA genome, with heteroplasmy quantification.
• Nuclear gene panel or exome sequencing.
• Muscle biopsy with histochemistry and respiratory chain enzyme assays.
• Blood and cerebrospinal fluid lactate and pyruvate, amino acids and organic acids.
• Brain MRI and magnetic resonance spectroscopy, electrocardiogram and echocardiogram, audiometry, ophthalmological examination, and glucose testing.
• Testing of maternal relatives.
• Genetic counselling.