Multiplexed Assay of Variant Effect for human G6PD activity in Saccharomyces cerevisiae

G6PD deficiency is the most common enzyme deficiency worldwide, and increases the likelihood of adverse reactions to certain drugs and foods. Identifying people at risk is challenging, since most are asymptomatic until they encounter a trigger. This is further complicated since over 60% of 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 a high-throughput functional assay for G6PD activity, by ability of human G6PD to rescue growth of Saccharomyces cerevisiae lacking their G6PD homolog, ZWF1. We created a barcoded G6PD variant library, associated barcodes with variants using PacBio sequencing, and transformed zwf1D yeast with the library. We sequenced barcodes at ten time points over 72 hours of growth to determine variant function. In total we measured the function of 9,144 missense, nonsense, and synonymous G6PD variants. 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.