Functional interpretation of human G6PD variants using multiplexed analyses in S. cerevisiae

摘要 葡萄糖-6-磷酸脱氢酶（G6 PD）缺乏症是世界上最常见的酶病， 影响了超过4亿人G6 PD在红细胞中特别重要，因为它是红细胞的唯一来源。 的NADPH需要解毒的活性氧。患有G6 PD缺乏症的人 活性降低的酶变体，可导致感染或暴露于 氧化剂，包括抗生素、抗疟药以及某些食品和家居用品。G6 PD的频率 在历史上疟疾流行的地区， 严重的疟疾感染;由于抗疟疾药物的使用， 伯氨喹是可导致溶血的药物之一。然而，通过活性诊断G6 PD缺乏症- 在溶血危象期间，基于的测定可能具有挑战性甚至是误导性的，具有频繁的假阴性， 这引起了人们对改进基因测试的兴趣。此外，前瞻性基因检测可以确定胎儿 风险，并允许在怀孕期间避免触发。准确解读G6 PD遗传变异 需要基因序列和变异功能之间的关联，这是已知的不到一半的 G6 PD变体。需要一种能够鲁棒地表征大的生物体的功能的系统。 G6 PD变体的数量，以改善变体解释。以前的研究和我们自己的初步研究 数据显示，人G6 PD在功能上补充了Zwf 1，即S.酿酒厂 酵母我们已经建立了一个系统来测量G6 PD的功能，通过其拯救生长的能力， zwf 1 Δ酵母在氧化应激条件下的生长。我假设G6 PD的功能有很大的多样性， 在人类群体中尚未分类或鉴定的变体和变体的组合。我 我们将使用我们的酵母系统（1）根据其拯救生长的能力对几乎所有可能的G6 PD变体进行分类。 zwf1Δ S.酿酒酵母，增加了我们的知识的相对活性的变体都以前观察到的， 人类和那些尚未被发现的。由于大多数G6 PD变体仅在大多数情况下进行了研究， 共同的单倍型背景，我也将研究不同的遗传背景的影响，通过（2）引入 最常见的背景单倍型到我们的G6 PD变体文库中，以研究遗传效应。 变量函数的背景。这是非常重要的，因为高患病率的G6 PD损失- 非洲人、亚洲人和地中海人后裔的基因突变。最后，我将研究一个具体的 遗传变异改变G6 PD功能的机制，通过（3）测量G6 PD的乙酰化和活性 变异，以及它们与脱乙酰酶和癌症风险因子SIRT 2的关系。这项研究将增加我们的 了解G6 PD序列与功能之间的关系，并告知关键的临床 通过改进对可能致病的变异的识别来做出决策。

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 inform critical clinical decisions by improving identification of variants that are likely to be pathogenic.