Fig. 1: Study flowchart.

Whole-genome sequencing was conducted to identify mitochondrial DNA (mtDNA) heteroplasmy in 10,986 individuals across seven epidemiological cohorts. Cohort- and ancestry-specific association analyses were performed to investigate the relationship between mtDNA heteroplasmy and nuclear DNA (nDNA) methylation levels at CpG sites. Random-effects meta-analyses were subsequently conducted in both pooled and race-stratified datasets. CpG sites demonstrating statistically significant associations (false discovery rate [FDR] < 0.05) were subjected to downstream bioinformatic analyses, including gene ontology enrichment and transcriptomic integration. Experimental validation using targeted genome editing in cell lines, followed by pyrosequencing, provided functional evidence supporting a causal role of mtDNA heteroplasmic variation in modulating nuclear epigenetic states. Integrative analyses further linked heteroplasmy-associated CpGs to complex traits through methylation quantitative trait loci (mQTL) mapping, interrogation of the Epigenome-Wide Association Studies (EWAS) Catalog and Genome-Wide Association Studies (GWAS) Catalog, and Mendelian randomization. Finally, composite methylation scores derived from heteroplasmy-associated CpG sites were evaluated for association with all-cause mortality and cardiovascular disease risk.