Abstract Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in the world, affecting over 400 million people. G6PD is of particular importance in red blood cells, since it is the sole source of NADPH needed for detoxification of reactive oxygen species. Individuals with G6PD deficiency have variants of the enzyme with decreased activity, which can lead to hemolysis after infections or exposure to oxidants, including antibiotics, antimalarials, and certain foods and household items. The frequency of G6PD variants is higher in areas where malaria is historically endemic, as some variants offer protection against severe malarial infections; identification of individuals with G6PD deficiency is critical since the antimalarial primaquine is one of the drugs that can lead to hemolysis. However, diagnosis of G6PD deficiency by activity- based assays can be challenging or even misleading during a hemolytic crisis, with frequent false negatives, leading to an interest in improving genetic tests. Furthermore, prospective genetic testing can determine fetal risk and allow triggers to be avoided during pregnancy. Accurate interpretation of G6PD genetic variation requires association between genetic sequence and variant function, which is known for less than half of identified G6PD variants. There is a need for a system that can robustly characterize the function of a large number of G6PD variants in order to improve variant interpretation. Previous studies and our own preliminary data have shown that human G6PD functionally complements Zwf1, the homolog in S. cerevisiae, baker’s yeast. We have established a system to measure the function of G6PD by its ability to rescue the growth of zwf1Δ yeast in conditions of oxidative stress. I hypothesize that there is a great diversity of function in G6PD variants and combinations of variants that have not yet been classified or identified in the human population. I will use our yeast system to (1) classify nearly all possible G6PD variants by their ability to rescue growth of zwf1Δ S. cerevisiae, increasing our knowledge of the relative activity of variants both previously observed in humans and those yet to be discovered. Since most G6PD variants have only been studied on the most common haplotype background, I will also study the effect of diverse genetic backgrounds by (2) introducing the most common background haplotypes into our G6PD variant library to study the effect of genetic background on variant function. This is of utmost importance due to the high prevalence of G6PD loss-of- function mutations in people of African, Asian, and Mediterranean descent. Finally, I will investigate a specific mechanism by which genetic variants alter G6PD function by (3) measuring acetylation and activity of G6PD variants, and their relationship to deacetylase and cancer risk factor SIRT2. This study will increase our understanding of the relationship between G6PD sequence and function, as well as...