VarMeter2: An enhanced structure-based method for predicting pathogenic missense variants through Mahalanobis distance.

Various computational methods have been developed to predict the pathogenicity of missense variants, which is crucial for diagnosing rare diseases. Recently, we introduced VarMeter, a diagnostic tool for predicting variant pathogenicity based on normalized solvent-accessible surface area (nSASA) and mutation energy calculated from AlphaFold 3D models, and validated it on arylsulfatase L. To evaluate the broader applicability of VarMeter and enhance its predictive accuracy, here we analyzed 296 pathogenic and 240 benign variants extracted from the ClinVar database. By comparing structural features including nSASA, mutation energy, and predicted local distance difference test (pLDDT) score, we identified distinct characteristics between pathogenic and benign variants. These features were used to develop VarMeter2, which classifies variants based on Mahalanobis distance. VarMeter2 achieved a prediction accuracy of 82 % for the ClinVar dataset, a marked improvement over the original VarMeter (74 %), and 84 % for published missense variants of N-sulphoglucosamine sulphohydrolase (SGSH), an enzyme associated with Sanfillippo syndrome A. Application of VarMeter 2 to SGSH variants in our clinical database identified a novel SGSH variant, Q365P, as pathogenic. The recombinant Q365P protein lacked enzymatic activity as compared with wild-type SGSH. Furthermore, it was largely retained in the endoplasmic reticulum and failed to reach the Golgi, probably due to misfolding. Protein stability assays confirmed reduced stability of the variant, further explaining its loss of function. Consistently, the patient homozygous for this variant was diagnosed with Sanfilippo syndrome A. These results underscore the predictive power and versatility of VarMeter2 in assessing the pathogenicity of missense variants.