Detection of unrecognized low-level mtDNA heteroplasmy may explain the variable phenotypic expressivity of apparently homoplasmic mtDNA mutations

Mitochondrial DNA (mtDNA) mutations are an important cause of human disease. Most mtDNA mutations are found in heteroplasmy, in which the proportion of mutant vs. wild-type species is believed to explain some of the observed high phenotypic heterogeneity. However, homoplasmic mutations also observe phenotypic heterogeneity, which may be in part due to undetected low levels of heteroplasmy. In the present report, we have developed two assays, using DHPLC and Pyrosequencing (Biotage AB, Uppsala, Sweden), for reliably and accurately detecting low-level mtDNA heteroplasmy. Using these assays we have identified a three-generation family segregating two mtDNA mutations in heteroplasmy: the deafness-related m.1555A>G mutation in the 12S rRNA gene (MTRNR1) and a new variant (m.15287T>C) in the cytochrome b gene (MTCYB). Both heteroplasmic mtDNA mutations are transmitted through generations in a random manner, thus showing differences in mutation load between siblings within the family. In addition, the developed assays were also used to screen a group of deaf subjects of unknown etiology for the presence of heteroplasmy for both mtDNA variants. Two additional heteroplasmic m.1555A>G samples, previously considered as homoplasmic, and two deaf subjects carrying m.15287T>C variant were identified, thus confirming the high specificity and reliability of the approach. The development of assays for reliably detecting low-level heteroplasmy, together with the study of heteroplasmic mtDNA transmission, are essential steps for a better knowledge and clinical management of mtDNA diseases.