STUDY QUESTION Can a targeted whole exome sequencing (WES) on a cohort of women showing a primary ovarian insufficiency (POI) phenotype at a young age, combined with a study of copy number variations, identify variants in candidate genes confirming their deleterious effect on ovarian function? SUMMARY ANSWER This integrated approach has proved effective in identifying novel candidate genes unveiling mechanisms involved in POI pathogenesis. WHAT IS KNOWN ALREADY POI, a condition occurring in 1% of women under 40 years of age, affects women’s fertility leading to a premature loss of ovarian reserve. The genetic causes of POI are highly heterogeneous and several determinants contributing to its prominent oligogenic inheritance pattern still need to be elucidated. STUDY DESIGN, SIZE, DURATION WES screening for pathogenic variants of 41 Italian women with non-syndromic primary and early secondary amenorrhoea occurring before age 25 was replicated on another 60 POI patients, including 35 French and 25 American women, to reveal statistically significant shared variants. PARTICIPANTS/MATERIALS, SETTING, METHODS The Italian POI patients’ DNA were processed by targeted WES including 542 RefSeq genes expressed or functioning during distinct reproductive or ovarian processes (e.g. DNA repair, meiosis, oocyte maturation, folliculogenesis and menopause). Extremely rare variants were filtered and selected by means of a Fisher Exact test using several publicly available datasets. A case-control Burden test was applied to highlight the most significant genes using two ad-hoc control female cohorts. To support the obtained data, the identified genes were screened on a novel cohort of 60 Caucasian POI patients and the same case-control analysis was carried out. Comparative analysis of the human identified genes was performed on mouse and Drosophila melanogaster by analysing the orthologous genes in their ovarian phenotype, and two of the selected genes were fruit fly modelled to explore their role in fertility. MAIN RESULTS AND THE ROLE OF CHANCE The filtering steps applied to search for extremely rare pathogenic variants in the Italian cohort revealed 64 validated single-nucleotide variants/Indels in 59 genes in 30 out of 41 screened women. Burden test analysis highlighted 13 ovarian genes as being the most enriched and significant. To validate these findings, filtering steps and Burden analysis on the second cohort of Caucasian patients yielded 11 significantly enriched genes. Among them, AFP, DMRT3, MOV10, FYN and MYC were significant in both patient cohorts and hence were considered strong candidates for POI. Mouse and Drosophila comparative analysis evaluated a conserved role through the evolution of several candidates, and functional studies using a Drosophila model, when applicable, supported the conserved role of the MOV10 armitage and DMRT3 dmrt93B orthologues in female fertility. LARGE SCALE DATA The datasets for the Italian cohort generated during the current study are publicly available at ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/): accession numbers SCV001364312 to SCV001364375. LIMITATIONS, REASONS FOR CAUTION This is a targeted WES analysis hunting variants in candidate genes previously identified by different genomic approaches. For most of the investigated sporadic cases, we could not track the parental inheritance, due to unavailability of the parents’ DNA samples; in addition, we might have overlooked additional rare variants in novel candidate POI genes extracted from the exome data. On the contrary, we might have considered some inherited variants whose clinical significance is uncertain and might not be causative for the patients’ phenotype. Additionally, as regards the Drosophila model, it will be extremely important in the future to have more mutants or RNAi strains available for each candidate gene in order to validate their role in POI pathogenesis. WIDER IMPLICATIONS OF THE FINDINGS The genomic, statistical, comparative and functional approaches integrated in our study convincingly support the extremely heterogeneous oligogenic nature of POI, and confirm the maintenance across the evolution of some key genes safeguarding fertility and successful reproduction. Two principal classes of genes were identified: (i) genes primarily involved in meiosis, namely in synaptonemal complex formation, asymmetric division and oocyte maturation and (ii) genes safeguarding cell maintenance (piRNA and DNA repair pathways). STUDY FUNDING/COMPETING INTEREST(S) This work was supported by Italian Ministry of Health grants ‘Ricerca Corrente’ (08C621_2016 and 08C924_2019) provided to IRCCS Istituto Auxologico Italiano, and by ‘Piano Sostegno alla Ricerca’ (PSR2020_FINELLI_LINEA_B) provided by the University of Milan; M.P.B. was supported by Telethon-Italy (grant number GG14181). There are no conflicts of interest.

中文翻译：

---

靶向全外显子组测序和果蝇模型揭示原发性卵巢功能不全的分子基础

研究问题 是否可以对一组年轻时表现出原发性卵巢功能不全 (POI) 表型的女性进行靶向全外显子组测序 (WES)，结合拷贝数变异研究，确定候选基因中的变异，确认它们对卵巢的有害影响功能？总结答案 已证明这种综合方法可有效识别新的候选基因，揭示与 POI 发病机制有关的机制。已知情况 POI 是一种发生在 40 岁以下女性中的 1% 的疾病，会影响女性的生育能力，导致卵巢储备过早丧失。POI 的遗传原因是高度异质的，仍然需要阐明导致其突出的寡基因遗传模式的几个决定因素。研究设计，尺寸，持续时间 WES 筛查 41 名在 25 岁之前发生非综合征性原发性和早期继发性闭经的意大利女性的致病性变异，并在另外 60 名 POI 患者（包括 35 名法国和 25 名美国女性）中重复进行，以揭示具有统计学意义的共有变异。参与者/材料、设置、方法 意大利 POI 患者的 DNA 由靶向 WES 处理，包括 542 个在不同生殖或卵巢过程（例如 DNA 修复、减数分裂、卵母细胞成熟、卵泡发生和更年期）中表达或发挥作用的 RefSeq 基因。使用几个公开可用的数据集通过 Fisher Exact 测试过滤和选择极其罕见的变体。应用病例对照负担测试来突出使用两个特别对照女性队列的最重要的基因。为了支持获得的数据，鉴定的基因在 60 名高加索 POI 患者的新队列中进行了筛选，并进行了相同的病例对照分析。通过分析其卵巢表型中的直系同源基因，对小鼠和黑腹果蝇进行了人类鉴定基因的比较分析，并对选定的两个基因进行了果蝇模拟，以探索它们在生育中的作用。主要结果和机会的作用 用于在意大利队列中搜索极其罕见的致病性变异的过滤步骤显示，在 41 名接受筛查的女性中，有 30 名在 59 个基因中存在 64 个经过验证的单核苷酸变异/插入缺失。负担测试分析强调了 13 个卵巢基因是最丰富和最重要的。为了验证这些发现，对第二批白种人患者的过滤步骤和负担分析产生了 11 个显着富集的基因。其中，AFP、DMRT3、MOV10、FYN 和 MYC 在两个患者队列中均显着，因此被认为是 POI 的有力候选者。小鼠和果蝇比较分析通过几个候选者的进化评估了保守作用，并且在适用时使用果蝇模型进行的功能研究支持了 MOV10 armitage 和 DMRT3 dmrt93B 直系同源物在女性生育力中的保守作用。大规模数据当前研究期间生成的意大利队列数据集可在 ClinVar 数据库 (http://www.ncbi.nlm.nih.gov/clinvar/) 上公开获得：登录号 SCV001364312 至 SCV001364375。限制，谨慎的原因 这是一项有针对性的 WES 分析，可在先前通过不同基因组方法鉴定的候选基因中寻找变异。对于大多数调查的散发病例，由于无法获得父母的DNA样本，我们无法追踪父母的遗传；此外，我们可能忽略了从外显子组数据中提取的新候选 POI 基因中的其他罕见变体。相反，我们可能考虑了一些临床意义不确定的遗传变异，并且可能不是患者表型的原因。此外，关于果蝇模型，在未来为每个候选基因提供更多的突变体或 RNAi 菌株以验证它们在 POI 发病机制中的作用将非常重要。研究结果的更广泛意义 我们研究中整合的比较和功能方法令人信服地支持 POI 的极其异质的寡基因性质，并证实了一些保护生育力和成功繁殖的关键基因在进化过程中的维持。确定了两类主要基因：(i) 主要参与减数分裂的基因，即联会复合体形成、不对称分裂和卵母细胞成熟；(ii) 保护细胞维持的基因（piRNA 和 DNA 修复途径）。研究资助/竞争兴趣这项工作得到了意大利卫生部向 IRCCS Istituto Auxologico Italiano 提供的“Ricerca Corrente”（08C621_2016 和 08C924_2019）以及由大学提供的“Piano Sostegno alla Ricerca”（PSR2020_FINELLI_LINEA_B）的支持米兰; 公安部 得到了 Telethon-Italy 的支持（授权号 GG14181）。没有利益上的冲突。