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

Abstract

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 phenotype as well as disease severity and penetrance. Bulk RNA sequencing is not able to detect cell-to-cell variability in heteroplasmy, limiting our understanding of mitochondrial pathological mechanisms. In this study, we leverage 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 corresponds to DNA-based measurements at the bulk-level, single-cell analysis uncovers a diverged distribution: most cells have near-homoplasmic (wild-type or mutant) mtDNA, with few cells showing intermediate levels. Furthermore, we find that high mutation levels correlate with transcriptional profile changes, though these responses are highly sample-specific, suggesting that nuclear background and cellular context critically influence mitochondrial dysfunction and compensatory mechanisms. Our findings highlight the power of single-cell technologies to better understand the complex link between mtDNA genetic diversity and mitochondrial phenotypic variability, and to study crucial aspects in mitochondrial biology and pathology, such as clonal dynamics, at single-cell resolution.