Single-cell multi-omics resolved analysis of mitochondrial genome-wide mutational burden, constraint, and mosaicism

Mitochondria contain their own circular, multi-copy genome, encoding essential components of the mitochondrial respiratory chain vital for cellular metabolism. Mitochondrial DNA (mtDNA) mutations occur more frequently than nuclear mutations and are associated with various diseases. While single-cell sequencing has allowed analysis of mtDNA variant heteroplasmy, a holistic view of mtDNA mutational landscapes in individual cells has remained limited, prohibiting the investigation of fundamental questions in mitochondrial genetics. Here, we leverage mitochondrial single-cell ATAC-seq (mtscATAC-seq) and mtDNA-hypermutated POLGD274A knock-in HEK293 cell lines to introduce two metrics—single-cell mtDNA mutations per million base pairs (scmtMPM) and heteroplasmy-weighted mitochondrial local constraint scores (scwMSS)—to capture cellular mutational loads and somatic mosaicism. We demonstrate that individual POLGD274A cells are characterized by complex mutational landscapes, with pathogenic mutations and truncating variants only present at subthreshold levels, indicative of their negative selection. In human healthy donors and mitochondriopathy patients, we identify constrained mutations in complex I, highlighting previously unrecognized mtDNA mutational landscape heterogeneity present on the single-cell level. Overall, scmtMPM and scwMSS provide a novel framework to investigate fundamental properties of mitochondrial genetics, disease, and somatic mosaicism in human tissues.