Challenging mtDNA tRNA Variant Guidelines: Emphasizing Single-Cell Analysis through Four Novel Variants

The broad clinical and genetic heterogeneity of mitochondrial diseases makes diagnosis challenging. Accurate characterization of novel variants is crucial to reduce diagnostic uncertainty, guide treatment, and enable reliable genetic counseling. In this study, we validated a single-cell NGS-based analysis approach by comparison with conventional PCR-RFLP and applied it to five mtDNA VUS identified in patients evaluated at our national reference center for mitochondrial disorders (CALISSON). Variant interpretation was assessed using multiple frameworks, including Yarhams scoring, Wongs specifications, and the ClinGen guidelines, highlighting differences between these scoring systems and the limitations of current recommendations in fully integrating functional evidence and tissue segregation data. We implemented a classification approach that incorporates these aspects to achieve a more clinically meaningful interpretation. This analysis enabled the reclassification of four novel variants (m.9998T>C in MT-TG, m.7530A>G in MT-TD, and m.4271G>C and m.4305A>G in MT-TI), providing a definitive molecular diagnosis. Further validation on a larger set of variants will be required, as well as the establishment of standardized criteria for single-fiber analyses, including minimum fiber numbers, thresholds for COX-negative fibers, and statistical significance. Overall, this study underscores the critical importance of integrating robust functional evidence into mtDNA variant interpretation and provides insights for refining existing guidelines to improve diagnostic accuracy and support clinical decision-making.