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 variants identified in patients evaluated at our national reference center for mitochondrial disorders (CALISSON). Variant interpretation was assessed using multiple frameworks, including Yarham's scoring, Wong's specifications, and the ClinGen guidelines. We report four novel variants (m.9998 T > C in MT-TG, m.7530A > G in MT-TD, and m.4271G > C and m.4305A > G in MT-TI), including three for which single-fiber analysis provided strong evidence supporting pathogenicity. However, these functional results alone were not sufficient to enable reclassification under the current ClinGen framework. These findings highlight differences between the various scoring systems and illustrate the limitations of current recommendations in fully integrating functional evidence and tissue segregation data. We therefore suggest that adjusted weighting of existing criteria may improve variant classification. Nevertheless, classification frameworks must preserve reproducibility across laboratories, and the criteria proposed here should be considered preliminary points for reflection requiring further validation in larger cohorts, as well as the establishment of standardized criteria for functional studies, including single-fiber analyses.