Exploring Heteroplasmic Variants in mtDNA: Insights from Single-Cell Transcriptomics

Mitochondrial DNA (mtDNA) heteroplasmy, which is the coexistence of wild-type and mutant mtDNA variants within the same cell, plays a critical role in modulating cellular phenotypes, disease severity, and penetrance. Bulk RNA sequencing cannot detect cell-to-cell heteroplasmy variability, limiting our understanding of the pathological mechanisms of mtDNA variants. In this study, we leveraged single-cell RNA sequencing (scRNA-seq) combined with a robust bioinformatics pipeline to characterize mtDNA heteroplasmy. We employed four fibroblast lines from patients harboring heteroplasmic mtDNA pathogenic variants in genes encoding respiratory complex I subunits. While RNA heteroplasmy corresponded to DNA-based measurements at the bulk level, single-cell analysis uncovered a diverged distribution in three out of four lines: most cells had near-homoplasmic (wild-type or mutant) mtDNA, with few cells showing intermediate levels. Furthermore, we found that high mutation levels correlate with transcriptional profile changes, although these responses were highly sample-specific, suggesting that the nuclear background and cellular context critically influence mitochondrial dysfunction and compensatory mechanisms. Our findings highlight the potential of single-cell technologies to better understand the complex link between mtDNA genetic diversity and mitochondrial phenotypic variability and to study crucial aspects of mitochondrial biology and pathology, such as clonal dynamics, at single-cell resolution.