GENOMICS OF SPERMATOGENIC IMPAIRMENT

描述(申请人提供)：严重生精功能衰竭(SF)影响近1%的男性，约占所有男性不育病例的20%，表现为射精液中精子稀少(少精子症)或无精子(无精子症)。医生在为SF患者提供有用指导方面的能力受到严重限制，这些指导涉及使用辅助生殖技术(ART)成功治疗的机会及其对ART衍生后代的潜在负面后果，因为目前超过一半的SF病例没有可识别的原因。尽管如此，我们小组的临床成员在近十年来一直在男性不育的治疗中常规使用基因检测，因为众所周知，几个Y染色体微缺失可以诊断生精失败，并对手术取精成功具有预后意义。哺乳动物精子发生需要1000多个基因，200多个小鼠基因敲除表现为SF；我们假设超过20%的特发性SF病例是由单基因或寡基因缺陷引起的。我们最近发表了一项基于阵列的初步研究，表明SF的众多遗传原因仍有待发现，并且DMRT1基因的反复缺失是SF的一种常见的、新的遗传原因。在这个提案中，我们描述了将在这些发现的基础上极大地扩展以确定SF的新的遗传原因的实验。我们将对1000名特发性无精子症男性和1000名精子密度正常的对照组男性进行外显子全基因组测序。有了这些数据，我们将制作一个完整的遗传变异图谱，使用特殊的计算和实验试剂来加强我们对富含重复序列的Y染色体的分析。我们将使用非靶向变异和基于基因的测试进行全基因组病例对照分析，并通过将变异聚合到通路或基因组注释中来测试许多生物学假说。我们建议使用有效的两阶段设计来复制前1%的关联变体和前5%的关联基因。我们设计了对3种不同形式的SF突变具有最大敏感性的第一阶段研究。在第一阶段(A)，我们重新使用作为目标1的一部分产生的对照数据，通过精子计数的定量特征分析来复制中等效应的常见风险因素。在第一阶段(B)，我们使用极端表型抽样策略，从顶端和底端10%的精子数分布中抽取新患者样本，最大限度地发挥我们的能力来检测极其罕见的大效应单基因/孟德尔变异。在第一阶段(C)中，我们使用来自患有原发性卵巢功能不全(POI)的女性的现有遗传数据来测试调节女性和男性性腺功能的基因/遗传变异。然后，在i(a，b，c)中至少一种中复制的感兴趣的变体将在3000例无精子症病例和3000例对照的中国队列中进行验证。在短期内，我们的发现将提高临床医生的能力 使用基因数据为患者提供咨询并改善患者护理。从长远来看，对生精障碍的遗传基础的表征可以为无精子症男性使用基因治疗策略铺平道路。

DESCRIPTION (provided by applicant): Severe spermatogenic failure (SF) affects nearly 1% of all men, accounting for approximately 20% of all cases of male infertility, and manifests as either scarcity (oligozoospermia) or absence (azoospermia) of sperm in ejaculate. Physicians are severely limited in their ability to offer useful direction for SF patients pertaining to the chances for successful treatment using assisted reproductive techniques (ART) and potential negative consequences in their ART-derived offspring because more than half of SF cases currently have no identifiable cause. Nonetheless, clinical members of our group have used genetic testing routinely in the management of male infertility for nearly a decade, as several Y chromosome microdeletions are known to be diagnostic of spermatogenic failure, and are prognostic for the success of surgical sperm retrieval. Over 1,000 genes are required for spermatogenesis in mammals, and over 200 mouse knockouts manifest SF; we hypothesize that over 20% of idiopathic SF cases are due to single-gene or oligogenic defects. We have recently published an array-based pilot study demonstrating that numerous genetic causes of SF remain to be discovered, and that recurrent deletions of the gene DMRT1 are a frequent, novel genetic cause of SF. In this proposal we describe experiments that will dramatically expand upon these findings to identify novel genetic causes of SF. We will perform whole exome sequencing of 1000 idiopathic azoospermic men, and 1000 control men with normal sperm concentrations. With these data we will produce an integrated map of genetic variants, using special computational and experimental reagents to enhance our analysis of the repeat-rich Y chromosome. We will perform a genomewide case-control analysis using untargeted variant and gene-based testing, as well as testing numerous biological hypotheses by aggregating variants into pathways or genomic annotations. We propose to use an efficient two-stage design to replicate the top 1% of associated variants and top 5% of associated genes. We have designed the stage I studies for maximum sensitivity to 3 distinct forms of SF mutations. In stage I(a) we re-use the control data generated as part of aim 1 to replicate common risk factors of moderate effect using a quantitative-trait analysis of sperm count. In stage I(b) we use an extreme phenotype sampling strategy to sample new patients from the top and bottom 10% of the sperm count distribution, maximizing our power to detect extremely rare monogenic/Mendelian variants of large effect. In stage I(c) we use existing genetic data from women with primary ovarian insufficiency (POI) to test for genes/genetic variants that modulate gonadal function in women as well as men. Variants of interest that are replicated in at least one of I(a,b,c) will then be taken forward for validation in a Chinese cohort of 3,000 azoospermic cases and 3,000 controls. In the short term, our findings will improve the ability of clinicians to use genetic data to counsel patients and improve patient care. In the long term, characterization of the genetic basis of spermatogenic failure could pave the way for the use of gene therapy strategies in azoospermic men.