There will always be variants of uncertain significance. Analysis of VUSs

The ACMG/AMP guidelines include five categories of which variants of uncertain significance (VUSs) have received increasing attention. Recently, Fowler and Rehm claimed that all or most VUSs could be reclassified as pathogenic or benign within few years. To test this claim, we collected validated benign, pathogenic, VUS and conflicting variants from ClinVar and LOVD and investigated differences at gene, protein, structure, and variant levels. The gene and protein features included inheritance patterns, actionability, functional categories for housekeeping, essential, complete knockout, lethality and haploinsufficient proteins, Gene Ontology annotations, and protein network properties. Structural properties included the location at secondary structural elements, intrinsically disordered regions, transmembrane regions, repeats, conservation, and accessibility. Gene features were distributions of nucleotides, their groupings, codons, and location to CpG islands. The distributions of amino acids and their groups were investigated. VUSs did not markedly differ from other variants. The only major differences were the accessibility and conservation of pathogenic variants, and reduced ratio of repeat-locating variants in VUSs. Thus, all VUSs cannot be distinguished from other types of variants. They display one form of natural biological heterogeneity. Instead of concentrating on eradicating VUSs, the community would benefit from investigating and understanding factors that contribute to phenotypic heterogeneity.