Challenging mtDNA tRNA variant guidelines: Emphasizing single-cell analysis through four novel variants.

Area of Science:

• Genetics
• Molecular Biology
• Biochemistry

Background:

• Mitochondrial diseases present significant diagnostic challenges due to their genetic and clinical heterogeneity.
• Accurate identification and classification of novel variants are essential for reducing diagnostic uncertainty, guiding patient treatment, and providing genetic counseling.

Purpose of the Study:

• To validate a single-cell next-generation sequencing (NGS) approach for characterizing mitochondrial DNA (mtDNA) variants.
• To assess the utility of current variant interpretation frameworks, including ClinGen guidelines, for classifying novel mtDNA variants.

Main Methods:

• A single-cell NGS-based analysis was validated against conventional PCR-RFLP.
• The approach was applied to five mtDNA variants from patients at a national reference center (CALISSON).
• Variant interpretation utilized Yarham's scoring, Wong's specifications, and ClinGen guidelines.

Main Results:

• Four novel mtDNA variants were identified, with three showing strong pathogenic evidence via single-fiber analysis.
• Current ClinGen framework limitations were highlighted, as functional data alone was insufficient for reclassification.
• Discrepancies among scoring systems and limitations in integrating functional evidence were observed.

Conclusions:

• Current variant classification frameworks may require adjustments to better incorporate functional evidence and tissue segregation data.
• Standardization of functional studies, including single-fiber analyses, is crucial for improving reproducibility and reliability.
• Further validation in larger cohorts is necessary to refine classification criteria for mitochondrial DNA variants.