In Silico Evidence Curation and Pathogenicity Reclassification of MYOD1 Variants of Uncertain Significance in Rhabdomyosarcoma

In pediatric oncogenes, missense Variants of Uncertain Significance (VUS) pose a major challenge in clinical genetics and cancer predisposition screening. MYOD1 encodes a master basic helix-loop-helix (bHLH) transcription factor vital for myogenic differentiation, and specific alterations are key drivers in embryonal and sclerosing/spindle cell Rhabdomyosarcoma (RMS). For this study, 54 germline missense VUS entries for MYOD1 were extracted from NCBI ClinVar and batch annotated using Ensembl VEP. Cross-referencing allele frequencies against gnomAD exomes and in silico pathogenicity meta-predictors (REVEL, CADD PHRED, and AlphaMissense) were integrated according to the ACMG/AMP variant interpretation framework. 57.4% of variants demonstrated benign-leaning predictions (BP4, PM2), whereas 13.0% exhibited strong, concordant in silico pathogenicity (PP3, PM2). 29.6% remained ambiguous or exhibited intermediate scores. This systematic in silico evidence curation effectively stratifies MYOD1 VUS entries, prioritizing specific deleterious candidates for downstream wet-lab validation in rhabdomyosarcoma models.