Identification and Functional Validation of Human Infertility Alleles

摘要 在美国，大约10%的人患有不孕症，其中约一半人被认为患有不孕症 遗传基础。然而，在绝大多数患者中，潜在的原因仍然不确定。 传统的识别遗传病基因座的方法，如GWAs，已经被 不孕不育表型的异质性和参与生殖的大量基因。不过， 很可能有许多“不育”等位基因在种群中分离，影响着不同的过程。 配子发育的各个阶段。我们的目标是识别这些等位基因，它们的性质，以及它们在体内对 繁殖。在以前的资助下，我们使用了一种截然不同的方法来解决涉及到的问题 生化和小鼠体内人类编码变体的预测和建模。在这里，我们建议使用 识别和表征不育变异(特别是SNPs)的创新策略 群体中的次要等位基因。拥有高通量基因组学、生殖基因组学专业知识的多学科团队 遗传学、蛋白质组学、计算生物学和转录调控已经被组合在一起来鉴定两者 影响“生育”基因的蛋白质编码和调控变异。具体目标是：1)使用 人类非同义SNPs的计算方法和高通量体外检测 可能扰乱蛋白质功能的生殖基因。这些等位基因将在小鼠身上精确建模 使用CRISPR/Cas9基因组编辑，并进行彻底表型以告知患者诊断。2)利用 人类变异的亚生育小鼠模型，表现出交叉减少，以了解 卵巢功能不全(POI)和复发性妊娠丢失。3)鉴定人类生殖细胞的调控 通过使用CHRO-SEQ技术鉴定活性(Erna转录)增强子，随后 小鼠转基因检测。我们还将通过利用GTEx来鉴定位于配子发生启动子中的eQTL， 高通量载体构建技术和表达分析。 该项目的成功实施将构成有史以来对 识别和验证人类群体中的编码和非编码遗传变异 男性和女性都有不孕症。由于变种是由数百万人共同携带的，所以这个项目可以有一个 精确基因组学时代对生殖遗传学领域的重大而持久的影响。

Abstract Approximately 10% of people in the U.S. suffer from infertility, about half of whom are thought to have a genetic basis. However, the underlying causes remain undetermined in the great majority of patients. Traditional methods for identifying inherited disease loci, such as GWAS, have been confounded by heterogeneity of infertility phenotypes and the large numbers of genes involved in reproduction. Nevertheless, there are probably numerous “infertility” alleles segregating in populations, affecting diverse processes at all stages of gamete development. Our goal is to identify these alleles, their nature, and their in vivo impacts to reproduction. Under previous funding, we used a radically different approach to the problem that involved prediction and modeling of human coding variants biochemically and in mice. Here, we propose to employ innovative strategies for identifying and characterizing infertility variants (particularly SNPs) that segregate as minor alleles in populations. A multidisciplinary team with expertise in high-throughput genomics, reproductive genetics, proteomics, computational biology, and transcriptional regulation has been assembled to identify both protein-coding and regulatory variants affecting “fertility” genes. The Specific Aims are to: 1) Use computational approaches and high-throughput in vitro assays to identify nonsynonymous SNPs in human reproduction genes that are likely to disrupt protein function. These alleles will be precisely modeled in mice using CRISPR/Cas9 genome editing, and thoroughly phenotyped to inform patient diagnosis. 2) Exploit subfertile mouse models of human variants, exhibiting decreased chiasmata, to understand mechanisms of premature ovarian insufficiency (POI) and recurrent pregnancy loss. 3) Identify human germ cell regulatory variants via indentification of active (eRNA-transcribing) enhancers using ChRO-seq technology, followed by mouse transgenic assays. We will also identify eQTL residing in gametogenesis promoters by exploiting GTEx, high-throughput vector-building technology, and expression assays. Successful execution of this project would constitute the most comprehensive study ever conducted to identify and validate both coding and non-coding genetic variants in human populations that contribute to infertility in both sexes. Since the variants are carried by millions of people collectively, this project can have a major and lasting impact on the field of reproductive genetics in the precision genomics era.