Evidence for G6PD variant classification from multiplexed functional assays.

G6PD deficiency is one of the most common enzyme deficiencies worldwide, and increases the likelihood of adverse reactions to certain drugs and foods. As availability of routine sequencing and biobanking increases, more variants in G6PD have been identified but not connected to phenotypes, since most G6PD-deficient individuals are asymptomatic until they encounter a trigger. This is further complicated since over 60% of 1,559 known genetic variants in G6PD are variants of uncertain significance and thus cannot guide drug prescribing and dosing. To resolve which variants are clinically meaningful and avoid harm from adverse drug reactions, we conducted two high-throughput functional assays: one for G6PD activity, and one for abundance. We measured the function of 9,504 missense, nonsense, and synonymous G6PD variants. The patterns of variant effect on activity and abundance confirmed the importance of structural NADP+ for G6PD activity and abundance, and G6PD dimerization for G6PD activity. Based on the ability of our functional assay scores to accurately classify G6PD variants of known clinical effect, we generated evidence that 4,773 missense variants contribute to G6PD deficiency and 2,295 are unlikely to contribute to G6PD deficiency. Our data can be used to deepen our understanding of G6PD as a protein, and to close the gap in classification for variants of uncertain significance to improve implementation of genetic medicine for G6PD deficiency.