Fig. 5: Proposed strategy for characterizing pathogenicity of variants.
Missense variants of interest are initially evaluated using computational analysis and the solubility/misfolding assay followed by further functional analysis as needed. Computational analysis is performed to determine the minor allele frequency of the variant in the general population (e.g., gnomAD), to predict if the variant is destabilizing (e.g., FoldX), and to determine if the variant is evolutionarily conserved (e.g., ConSurf). The rapid solubility assay is used for functional assessment. In the case of a sequence variant that is absent or extremely rare in the population (PM2) and is also insoluble (PS3), then per American College of Medical Genetics and Genomics (ACMG) guidelines1 this variant would be categorized as likely pathogenic and hence clinically actionable. Computational models could provide further evidence of pathogenicity (PP3). In cases where variants have wild type (WT)-like solubilities, further functional studies (e.g., heterologous expression, patient-specific induced pluripotent stem cells [iPSCs]) are needed to exclude other mechanisms of pathogenesis than domain misfolding. If a variant is soluble, functional studies are negative (BS3) and computational models are negative (BP4), then it will be classified as likely benign.
