Complementary computational and experimental evaluation of missense variants in the ROMK potassium channel

The renal outer medullary potassium (ROMK) channel is essential for potassium transport in the kidney, and its dysfunction is associated with a salt-wasting disorder known as Bartter syndrome. Despite its physiological significance, we lack a mechanistic understanding of the molecular defects in ROMK underlying most Bartter syndrome-associated mutations. To this end, we employed a ROMK-dependent yeast growth assay and tested single amino acid variants selected by a series of computational tools representative of different approaches to predict each variants' pathogenicity. In one approach, we used in silico saturation mutagenesis, i.e. the scanning of all possible single amino acid substitutions at all sequence positions to estimate their impact on function, and then employed a new machine learning classifier known as Rhapsody. We also used two additional tools, EVmutation and Polyphen-2, which permitted us to make consensus predictions on the pathogenicity of single amino acid variants in ROMK. Experimental tests performed for selected mutants in different classes validated the vast majority of our predictions and provided insights into variants implicated in ROMK dysfunction. On a broader scope, our analysis suggests that consolidation of data from complementary computational approaches provides an improved and facile method to predict the severity of an amino acid substitution and may help accelerate the identification of disease-causing mutations in any protein.