Accurate identification and mechanistic evaluation of pathogenic missense variants with <i>Rhapsody-2</i>

Understanding the effects of missense mutations or single amino acid variants (SAVs) on protein function is crucial for elucidating the molecular basis of diseases/disorders and designing rational therapies. We introduce here <i>Rhapsody-2</i>, a machine learning tool for discriminating pathogenic and neutral SAVs, significantly expanding on a precursor limited by the availability of structural data. With the advent of AlphaFold2 as a powerful tool for structure prediction, <i>Rhapsody-2</i> is trained on a significantly expanded dataset of 117,525 SAVs corresponding to 12,094 human proteins reported in the ClinVar database. Adopting a broad set of descriptors composed of sequence evolutionary, structural, dynamic, and energetics features in the training algorithm, <i>Rhapsody-2</i> achieved an AUROC of 0.94 in 10-fold cross-validation when all SAVs of a particular test protein (mutant) were excluded from the training set. Benchmarking against a variety of testing datasets demonstrated the high performance of <i>Rhapsody-2</i>. While sequence evolutionary descriptors play a dominant role in pathogenicity prediction, those based on structural dynamics provide a mechanistic interpretation. Notably, residues involved in allosteric communication and those distinguished by pronounced fluctuations in the high-frequency modes of motion or subject to spatial constraints in soft modes usually give rise to pathogenicity when mutated. Overall, <i>Rhapsody-2</i> provides an efficient and transparent tool for accurately predicting the pathogenicity of SAVs and unraveling the mechanistic basis of the observed behavior, thus advancing our understanding of genotype-to-phenotype relations.