# Quantifying variability of mitochondrial markers in m3243A > G myopathy

## TL;DR

This study measures how mitochondrial dysfunction varies within muscles of patients with a specific mitochondrial disease, providing thresholds to better track disease progression and treatment effects.

## Contribution

The study quantifies spatial variability of mitochondrial markers in m.3243A > G myopathy and establishes thresholds for meaningful change.

## Key findings

- OXPHOS-deficient fiber variability increases with anatomical distance within the same muscle.
- mtDNA copy number variability modestly increases with distance, while m.3243A > G heteroplasmy remains stable.
- Thresholds for meaningful change in OXPHOS deficiency are 13.8% for NDUFB8 and 9.8% for MT-CO1.

## Abstract

Myopathy is a prevalent and disabling feature of mitochondrial disease, in which skeletal muscle accumulates fibres with mitochondrial dysfunction in a variable mosaic pattern. This intra-individual spatial heterogeneity, a key consideration in longitudinal assessments, remains largely uncharacterised, hindering mechanistic studies and clinical trials by obscuring or confounding findings. We quantified this variability in m.3243 A > G-related myopathy, a leading cause of adult mitochondrial disease. Post-mortem biopsies from quadriceps femoris and tibialis anterior muscles of four patients were analysed for single-fibre deficiency in oxidative phosphorylation (OXPHOS) complex I and IV, while homogenate mitochondrial DNA (mtDNA) copy number and m.3243 A > G heteroplasmy were respectively determined by quantitative PCR and pyrosequencing. Bootstrapped combinatorial analyses established thresholds for minimum meaningful change above the 97.5th percentile, while accounting for anatomical biopsy distancing. Spatial variability in the proportion of OXPHOS-deficient fibres increased with distancing; within the same muscle, this threshold was 13.8% for NDUFB8 and 9.8% for MT-CO1. Variability in mtDNA copy number modestly increased with distance, while m.3243 A > G heteroplasmy remained largely stable, with within-muscle thresholds of 1,136 copies per nucleus and 8.2%, respectively. These findings provide assay-specific thresholds and offer mechanistic and translational insights for trial design, patient monitoring, and reliable detection of disease progression or therapeutic response.

The online version contains supplementary material available at 10.1038/s41598-025-33106-3.