Targeted whole exome sequencing and Drosophila modelling to unveil the molecular basis of primary ovarian insufficiency.
Targeted whole exome sequencing and Drosophila modelling to unveil the molecular basis of primary ovarian insufficiency.
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靶向全外显子组测序和果蝇建模揭示原发性卵巢功能不全的分子基础。
DOI：
10.1093/humrep/deab192
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发表时间：
2021-10-18
期刊：
影响因子：
--
通讯作者：
Finelli P
中科院分区：
文献类型：
--
作者：
Bestetti I;Barbieri C;Sironi A;Specchia V;Yatsenko SA;De Donno MD;Caslini C;Gentilini D;Crippa M;Larizza L;Marozzi A;Rajkovic A;Toniolo D;Bozzetti MP;Finelli P
对年轻时表现出原发性卵巢功能不全（POI）表型的女性队列进行靶向全外显子组测序（WES），结合拷贝数变异研究，是否可以识别候选基因中的变异，确认其对卵巢功能的有害影响？这种整合的方法已被证明是有效的，在确定新的候选基因揭示参与POI发病机制。POI是一种发生在1%的40岁以下女性中的疾病，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性的，其突出的寡基因遗传模式的几个决定因素仍然需要阐明。对41名25岁以前发生的非综合征性原发性和早期继发性闭经的意大利妇女进行WES致病性变异筛查，并对另外60名POI患者（包括35名法国妇女和25名美国妇女）进行重复，以揭示统计学上显著的共同变异。意大利POI患者的DNA由靶向WES处理，包括在不同的生殖或卵巢过程（例如DNA修复、减数分裂、卵母细胞成熟、卵泡发生和绝经）期间表达或起作用的542个RefSeq基因。使用几个公开可用的数据集，通过Fisher精确检验过滤和选择极其罕见的变体。使用两个特别对照雌性队列，应用病例对照负担检验来突出最显著的基因。为了支持所获得的数据，在60名白人POI患者的新队列中筛选鉴定的基因，并进行相同的病例对照分析。通过分析小鼠和黑腹果蝇卵巢表型中的orthopathy基因，对人类鉴定的基因进行比较分析，并对其中两个选定的基因进行果蝇建模，以探索其在生育中的作用。在意大利队列中用于搜索极其罕见的致病性变异的过滤步骤显示，在41名筛查女性中的30名中，59个基因中有64个经验证的单核苷酸变异/插入缺失。负荷测试分析突出了13个卵巢基因是最丰富和最重要的。为了验证这些发现，对第二组高加索患者进行过滤步骤和负担分析，得到11个显著富集的基因。其中，AFP、DMRT 3、MOV10、FYN和MYC在两个患者队列中均具有显著性，因此被认为是POI的强候选者。小鼠和果蝇比较分析通过几种候选物的进化评估了保守作用，并且在适用时使用果蝇模型的功能研究支持MOV10 armitage和DMRT3 dmrt93 B直系同源物在雌性生育力中的保守作用。本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/）中公开获得：登录号SCV001364312至SCV001364375。这是一个有针对性的WES分析，在先前通过不同的基因组方法鉴定的候选基因中寻找变体。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异。相反，我们可能会考虑一些遗传变异，其临床意义是不确定的，可能不是患者表型的原因。此外，关于果蝇模型，在未来具有可用于每个候选基因的更多突变体或RNAi菌株以验证它们在POI发病机制中的作用将是极其重要的。在我们的研究中整合的基因组，统计，比较和功能的方法令人信服地支持POI的极端异质性寡基因的性质，并确认维护整个进化的一些关键基因的生育力和成功的繁殖。鉴定了两类主要的基因：（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，以及（ii）保护细胞维持的基因（piRNA和DNA修复途径）。这项工作得到了意大利卫生部资助的“Ricerca Corrente”（08C621_2016和08C924_2019）提供给IRCCS Istituto Estiologico Italiano，以及米兰大学提供的“Piano Sostegno alla Ricerca”（PSR2020_FINELLI_LINEA_B）的支持; M.P.B.由Telethon-Italy（资助号GG14181）支持。没有利益冲突。
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? This integrated approach has proved effective in identifying novel candidate genes unveiling mechanisms involved in POI pathogenesis. 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. 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. 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. 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. 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. 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. 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). 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.
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影响因子：
16.2
作者：
Bultje, Ronald S.;Castaneda-Castellanos, David R.;Jan, Lily Yen;Jan, Yuh-Nung;Kriegstein, Arnold R.;Shi, Song-Hai
通讯作者：
Shi, Song-Hai
影响因子：
4
作者：
Bozzetti, Maria Pia;Specchia, Valeria;Giangrande, Angela
通讯作者：
Giangrande, Angela
DOI：
10.1056/nejmoa1309635
发表时间：
2014-03-06
期刊：
The New England journal of medicine
影响因子：
--
作者：
Caburet S;Arboleda VA;Llano E;Overbeek PA;Barbero JL;Oka K;Harrison W;Vaiman D;Ben-Neriah Z;García-Tuñón I;Fellous M;Pendás AM;Veitia RA;Vilain E
通讯作者：
Vilain E
影响因子：
3.3
作者：
Durdevic, Zeljko;Ephrussi, Anne
通讯作者：
Ephrussi, Anne
影响因子：
6.1
作者：
Bramble, Matthew S.;Goldstein, Ellen H.;Vilain, Eric
通讯作者：
Vilain, Eric