Development of a New Technique for Quantifying Mitochondrial DNA in Single Muscle Fibers

Mitochondrial diseases (MDs) are the most common metabolic disorders. Due to their great clinical and genetic heterogeneity, their diagnosis relies exclusively on the identification of pathogenic variants in nuclear genes or in mitochondrial DNA (mtDNA). However, to date, 50% of affected patients remain without a definitive diagnosis. The advent of next-generation sequencing (NGS) has improved diagnostic yield, but many identified variants remain of uncertain significance (VUS), preventing a definitive diagnosis. The clinical interpretation of these newly identified rare variants therefore represents a major challenge. In the context of MDs, one of the major criteria for mtDNA variant pathogenicity is a good correlation between heteroplasmy level and tissue or cellular involvement. Heteroplasmy refers to the coexistence, within the same cell or tissue, of mutated and non-mutated mtDNA molecules. Pathogenic mtDNA variants are most often heteroplasmic, with the most affected tissues harboring a higher proportion of mutated mtDNA. In muscle biopsies from patients with MDs, muscle fibers may show a cytochrome c oxidase (COX) enzymatic deficiency (COX-negative fibers), reflecting dysfunction of the mitochondrial respiratory chain (MRC). Single-fiber analysis makes it possible to isolate muscle fibers by LASER microdissection and to quantify the heteroplasmy level of a variant within them. The presence of a high heteroplasmy level in COX-negative fibers, in contrast to fibers without deficiency (COX-positive fibers), is strong evidence supporting the pathogenicity of the variant. In previous projects, we tested two variant quantification techniques (PCR-RFLP and NGS), but these methods remain too labor-intensive or costly and are therefore difficult to maintain in routine practice. This pilot study aims to develop a new method for quantifying heteroplasmy levels using digital PCR. Faster and less expensive, this approach could simplify technical implementation and reduc