应用人类iPS细胞模型研究Xq26-q28区关键基因缺失导致原发性卵巢功能不全的分子机制

Primary ovarian insufficiency (POI) is a type of common disorder which leads to serious deterioation to the reproductive health of the females. Heterozygous deletion of Xq26-q28 is one of the important causes, but the molecular mechanism is still unclear. We have evidence to believe that the decreased expression level of genes escaping X inactivation might play a role in the POI etiology. In our previous studies, we found that induced pluripotent stem cells (iPSC) could be used as a model for POI mechanism research and the ability of primordial germ cells differentiation (PGC specialization) in iPSCs from POI patients with Xq26-q28 deletion was decreased compared to those from females with a normal karyotype. In this study, we propose to investigate the mechanisms of POI in three aspects using the established iPSC model: first, we will compare the biological characters of the induced PGCs between the normal and POI derived iPSCs; second, we will compare the gene expression profile of the induced PGCs between the normal and POI derived iPSCs using single cell RNA transcriptome technique and screen for the critical genes which escape the X inactivation; third, by combining the results of the first two components, we will select one or two critical genes and study whether the ability of PGC specification is contributed by these genes using RNAi and over expression techniques, and confirm whether the deletion of these genes is present in the idiopathic POI patients. This study will overcome the bottleneck of lacking an experimental model in studying the molecular mechanism of of POI and provide the useful data for treating POI via recovering ovarian function in POI patients using targeted gene therapy.

原发性卵巢功能不全（POI）是一类常见的严重损害女性生殖健康的疾病，Xq26-q28杂合性缺失（缺失型）是重要病因之一，但分子机制不清楚，一批逃逸X失活基因的表达下降可能起重要作用。前期的研究发现，诱导性多能干细胞（iPSC）可作为POI疾病模型，缺失型POI患者iPSC诱导成原始生殖细胞（PGC特化）能力下降。本研究拟以构建的iPSC为模型进一步研究：比较正常和缺失型POI的iPSC诱导的PGC的分化、增殖及凋亡等生物学特征；应用单细胞转录组学技术比较正常和POI的iPSC诱导的PGC的表达谱，筛选逃逸X失活的关键基因；结合上述两方面的结果，挑选1-2个关键基因，应用RNAi和过表达技术来探讨所筛选基因与POI的关系，并在特发性POI患者中验证所筛选基因是否为致病基因。本研究将突破缺乏有效模型研究缺失型POI发生分子机制的瓶颈，为最终实现通过靶向基因治疗恢复这些患者的卵巢功能提供基础。

项目的背景.女性原发性卵巢功能不全（POI）是不孕症的重要原因，但具体机制不清楚。以X染色体部分缺失患者的皮肤成纤维细胞诱导多能干细胞为模型，有助于寻找致病基因及致病机制。参与POI的基因众多，而POI与非梗阻性无精子症(NOA)，都属于配子发生障碍，有共同通路，因此，研究NOA可能帮助了解POI的发病机制。.主要研究内容.用Xq26-q28部分缺失POI患者和正常女性皮肤成纤维细胞，构建了POI疾病的iPSCs（POI1-iPS和POI2-iPS）和正常对照iPSCs（hEF-iPS），分析POI的相关基因。进而收集大样本特发性POI，验证iPS分析的结果。同时，考虑到NOA与POI均属配子发生障碍，可能有共同病因，也收集部分NOA患者用于验证。对收集的POI患者和NOA患者进行全外显子测序，寻找配子发生障碍的候选基因，通过构建小鼠突变模型复制相应的表型明确致病基因，对基因敲除小鼠进一步通过RNA测序、蛋白质组学、免疫荧光等，研究候选基因致病机制。.重要结果以及关键数据.1).POI1-iPS和POI2-iPS和正常hEF-iPS，有相同的细胞学特点及自我更新能力，均能诱导分化为原始生殖细胞，分析发现FAM122C, IKBKG,RBMX与POI相关（Human Reproduction，2015）。.2).发现STAG3基因突变为家族性POI的病因(Clinical Genetics,2017)；.3).首次发现减数分裂基因DMC1和XRCC2同时导致女性POI及男性NOA，证实人POI和NOA有共同的路径(Journal of Medical Genetics,2018; Clinical Genetics,2018);.4).发现POI相关FOXL2基因新突变，细胞模型证实了突变的致病性及其分子机制(Molecular Genetics & Genomic Medicine, 2017);.5).在2个NOA家系中，发现TDRD7是NOA的致病新基因（Genetics in Medicine,2017）。.6).累计发表文章24篇，其中SCI论文19篇。.科学意义.筛选POI的致病基因，明确POI与NOA有共同的致病机制，加深了我们对于配子发生障碍病因学的了解，为最终实现通过靶向基因，恢复这些患者的生殖功能提供了依据。