Computational protein stability analysis of SCN1A missense variants reveals domain-dependent stability patterns.

objectiveTo determine whether computational protein-stability predictions discriminate pathogenic from benign SCN1A missense variants, and to characterize the structural distribution of predicted destabilization among pathogenic variants.

methodsOn an AlphaFold3-predicted Nav1.1 structure, FoldX, and Rosetta Cartesian ΔΔG were computed for a single ClinVar snapshot of pathogenic/likely-pathogenic (P/LP) and benign/likely-benign (B/LB) missense variants and its extension to ClinVar variants of uncertain significance (VUS) and gnomAD v4.1 variants; membrane-aware RosettaMP was applied to the patch-clamp subgroup. Pathogenic variants were stratified by functional domain.

resultsPathogenic variants were more destabilizing than benign (FoldX 2.61 vs. 0.31 kcal/mol, p = 1.27 × 10 SIGNIFICANCE: Computational stability analysis thus adds a mechanistic layer complementary to the conventional gating-dysfunction view, distinguishing a destabilized pore-region subset-for which proteostasis impairment is a candidate, though unproven, mechanism-from a structurally tolerated S4 subset whose pathogenicity is stability-independent. As a hypothesis-generating rather than mechanism-defining approach, this stratification prioritizes candidate variants-including the 18.5% of VUS that are strongly destabilizing-for direct functional and surface-expression validation in SCN1A-related epilepsies.  PLAIN LANGUAGE SUMMARY: We used computational modeling to predict how thousands of SCN1A genetic variants influence the stability of the Nav1.1 sodium channel protein. Disease-causing variants tended to destabilize the protein more than benign variants, and variants in the pore region-where ions flow through the channel-were predominantly destabilizing. This is consistent with loss-of-function arising from misfolding and degradation of the channel protein in this subset of variants. By contrast, variants in the voltage-sensing region were often structurally tolerated, indicating that their disease-causing effects likely arise through a different mechanism that requires direct functional measurement to define. Accordingly, the analysis nominates a candidate pore-region subset potentially affected by proteostasis impairment and a complementary stability-neutral subset warranting functional evaluation.