Mitochondrial DNA Variant with Low Heteroplasmy in Blood
A 28-year-old man presents with bilateral optic atrophy, ataxia, and peripheral neuropathy. His mother has mild hearing loss and diabetes, and his maternal uncle had a stroke-like episode at age 35. The clinical team suspects a mitochondrial disorder and orders mitochondrial DNA (mtDNA) sequencing from blood. The laboratory uses next-generation sequencing of the entire mtDNA genome with a sensitivity of 5% variant allele fraction (heteroplasmy). The result is negative. The clinician requests repeat testing on a different tissue. The laboratory offers testing on a saliva sample and a urine sediment sample. The patient's blood sample was collected 2 years ago and stored at -80°C. The laboratory's policy is to perform mtDNA sequencing on blood first, and if negative, on urine sediment.
1. Explain why the blood mtDNA sequencing might be negative despite a likely mitochondrial disorder, and which tissue would be most appropriate for repeat testing.
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Mitochondrial DNA variants can be heteroplasmic, meaning the mutant load can vary among tissues. Blood often has lower heteroplasmy for certain mtDNA variants, especially those affecting oxidative phosphorylation, because hematopoietic cells may select against high mutant loads. Therefore, a negative blood test does not rule out a mitochondrial disorder. Urine sediment is enriched for renal tubular cells and often shows higher heteroplasmy for mtDNA variants than blood, especially for variants causing mitochondrial encephalomyopathy. Saliva may also be useful but can be contaminated with leukocytes. The most appropriate tissue for repeat testing is urine sediment, as it is non-invasive and has higher sensitivity for mtDNA variants. The laboratory should also consider testing a affected tissue such as muscle if the urine sediment is negative, but that is invasive.
2. What are the quality control and reporting considerations for mtDNA heteroplasmy testing?
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Quality control for mtDNA heteroplasmy testing includes ensuring the limit of detection is validated (e.g., 5% VAF), using appropriate positive and negative controls, and avoiding contamination with nuclear mitochondrial pseudogenes (NUMTs). The laboratory should report the heteroplasmy level and the tissue tested, and note that heteroplasmy can vary among tissues. The report should include a statement about the limitations of blood testing and the possibility of false negatives due to low heteroplasmy. If a variant is found, the report should specify the gene, the nucleotide change, and the heteroplasmy percentage. The laboratory should also follow ACMG/AMP guidelines for mtDNA variant classification, which consider the variant's frequency in population databases, conservation, and functional studies. Additionally, the report should recommend genetic counseling because mtDNA variants are maternally inherited.