ACMG/AMP interpretation of BRCA1 missense variants: Structure-informed scores add evidence strength granularity to the PP3/BP4 computational evidence

Classification of missense variants is challenging. Lacking compelling clinical and/or functional data, ACMG/AMP lines of evidence are restricted to PM2 (rarity code applied at supporting level) and PP3/BP4 (computational evidence based mostly on multiple-sequence-alignment conservation tools). Currently, the ClinGen ENIGMA BRCA1/2 Variant Curation Expert Panel uses BayesDel to apply PP3/BP4 to missense variants located in the BRCA1 RING/BRCT domains. The ACMG/AMP framework does not refer explicitly to protein structure as a putative source of pathogenic/benign evidence. Here, we tested the value of incorporating structure-based evidence such as relative solvent accessibility (RSA), folding stability (ΔΔG), and/or AlphaMissense pathogenicity to the classification of BRCA1 missense variants. We used MAVE functional scores as proxies for pathogenicity/benignity. We computed RSA and FoldX5.0 ΔΔG predictions using as alternative input templates for either PDB files or AlphaFold2 models, and we retrieved pre-computed AlphaMissense and BayesDel scores. We calculated likelihood ratios toward pathogenicity/benignity provided by the tools (individually or combined). We performed a clinical validation of major findings using the large-scale BRIDGES case-control dataset. AlphaMissense outperforms ΔΔG and BayesDel, providing similar PP3/BP4 evidence strengths with lower rate of variants in the uninformative score range. AlphaMissense combined with ΔΔG increases evidence strength granularity. AlphaFold2 models perform well as input templates for ΔΔG predictions. Regardless of the tool, BP4 (but not PP3) is highly dependent on RSA, with benignity evidence provided only to variants targeting buried or partially buried residues (RSA ≤ 60%). Stratification by functional domain did not reveal major differences. In brief, structure-based analysis improves PP3/BP4 assessment, uncovering a relevant role for RSA.