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

摘要 葡萄糖-6-磷酸脱氢酶(G6PD)缺乏症是世界上最常见的酶病， 影响了超过4亿人。G6PD在红细胞中特别重要，因为它是红细胞的唯一来源 活性氧物种解毒所需的NADPH。G6PD缺乏症患者有 酶的变种，活性降低，在感染或接触后可导致溶血 氧化剂，包括抗生素、抗疟疾药物以及某些食品和家居用品。G6PD的频率 在疟疾历史上流行的地区，变种的比例更高，因为一些变种提供了预防疟疾的保护 严重的疟疾感染；确认G6PD缺乏的个体至关重要，因为抗疟疾 伯喹是一种可能导致溶血的药物。然而，通过活动来诊断G6PD缺乏症- 在溶血危机期间，基于基础的分析可能具有挑战性，甚至具有误导性，经常出现假阴性， 这导致了人们对改进基因测试的兴趣。此外，预期的基因测试可以确定胎儿 风险，并允许在怀孕期间避免触发因素。G6PD基因变异的准确解释 需要遗传序列和变异功能之间的联系，这一点只有不到一半的人知道 确定了G6PD变异体。需要一种系统，该系统能够强有力地表征大型 G6PD变种的数量，以改进变种解释。以前的研究和我们自己的初步研究 数据表明，人类G6PD在功能上与酿酒酵母中的同源物Zwf1互补。 酵母。我们已经建立了一套系统来衡量G6PD的功能，它是通过它的挽救生长的能力来衡量的 氧化应激条件下的ZWF1Δ酵母。我推测G6PD的功能有很大的多样性 在人类群体中尚未被分类或鉴定的变种和变种组合。我 将使用我们的酵母系统来(1)根据G6PD变体拯救生长的能力对几乎所有可能的G6PD变体进行分类 Zwf1ΔS.cerevisiae，增加了我们对之前在 人类和那些尚未被发现的人。由于大多数G6PD变异体只被研究了最多 共同的单倍型背景，我还将通过(2)介绍不同的遗传背景来研究不同遗传背景的影响 将最常见的背景单倍型纳入我们的G6PD变异体文库，研究遗传效应 不同功能的背景知识。由于G6PD缺失率很高，这一点至关重要。 非洲人、亚洲人和地中海人后裔的功能突变。最后，我将调查一个具体的 基因变异通过(3)测定G6PD乙酰化和活性改变G6PD功能的机制 变异，以及它们与脱乙酰酶和癌症风险因子SIRT2的关系。这项研究将增加我们的 了解G6PD序列与功能的关系，并为危重临床提供信息 通过改进对可能致病的变异体的识别来做出决定。

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.