Next-generation genome sequencing has revolutionized genetic testing, identifying numerous rare disease-associated gene variants. However, to impute pathogenicity, computational approaches remain inadequate and functional testing of gene variant is required to provide the highest level of evidence. The emergence of AlphaFold2 has transformed the field of protein structure determination, and here we outline a strategy that leverages predicted protein structure to enhance genetic variant classification. We used the gene IRF6 as a case study due to its clinical relevance, its critical role in cleft lip/palate malformation, and the availability of experimental data on the pathogenicity of IRF6 gene variants through phenotype rescue experiments in irf6-/- zebrafish. We compared results from over 30 pathogenicity prediction tools on 37 IRF6 missense variants. IRF6 lacks an experimentally derived structure, so we used predicted structures to explore associations between mutational clustering and pathogenicity. We found that among these variants, 19 of 37 were unanimously predicted as deleterious by computational tools. Comparing in silico predictions with experimental findings, 12 variants predicted as pathogenic were experimentally determined as benign. Even with the recently published AlphaMissense model, 15/18 (83%) of the predicted pathogenic variants were experimentally determined as benign. In comparison, mapping variants to the protein revealed deleterious mutation clusters around the protein binding domain, whereas N-terminal variants tend to be benign, suggesting the importance of structural information in determining pathogenicity of mutations in this gene. In conclusion, incorporating gene-specific structural features of known pathogenic/benign mutations may provide meaningful insights into pathogenicity predictions in a gene-specific manner and facilitate the interpretation of variant pathogenicity.
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