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

Area of Science:

• Mitochondrial Biology
• Genetics
• Cellular Phenotyping

Background:

• Mitochondrial DNA (mtDNA) heteroplasmy, the coexistence of wild-type and mutant mtDNA, influences cellular phenotypes and disease.
• Bulk RNA sequencing misses cell-to-cell variability in mtDNA heteroplasmy, hindering understanding of pathological mechanisms.
• Investigating mtDNA variant impact requires methods that capture cellular heterogeneity.

Purpose of the Study:

• To characterize mtDNA heteroplasmy using single-cell RNA sequencing (scRNA-seq).
• To analyze cell-to-cell variability in mtDNA heteroplasmy distribution.
• To correlate mtDNA mutation levels with transcriptional changes at the single-cell level.

Main Methods:

• Employed scRNA-seq and a bioinformatics pipeline on fibroblast lines with pathogenic mtDNA variants.
• Analyzed four patient-derived fibroblast lines harboring heteroplasmic mtDNA variants in respiratory complex I genes.
• Compared single-cell RNA heteroplasmy with bulk DNA measurements.

Main Results:

• Single-cell analysis revealed a diverged mtDNA heteroplasmy distribution in three of four lines, with most cells being near-homoplasmic.
• RNA heteroplasmy at the single-cell level differed from bulk DNA measurements.
• High mtDNA mutation levels correlated with sample-specific transcriptional profile changes, influenced by nuclear background and cellular context.

Conclusions:

• Single-cell technologies offer a powerful tool to dissect mtDNA genetic diversity and mitochondrial phenotypic variability.
• scRNA-seq can resolve clonal dynamics and cellular heterogeneity in mtDNA heteroplasmy.
• Cellular context critically influences mitochondrial dysfunction and compensatory responses to mtDNA variants.