TBX6基因拷贝数变异导致卵巢储备功能降低的剂量机理研究

Female carriers with TBX6/16p11.2 copy number gain or loss were reported to manifest defects in ovarian reserve. However, there is still a lack of in-depth study in the genotype-phenotype correlation, let alone the underlined mechanisms. Previously, the applicant constructed mouse models with Tbx6 up-regulated or down-regulated, mimicing the human TBX6 gene copy number gain or loss, both of which manifested decreases follicles and subfertility. Based on the phenotypes of mouse models in our research and human carriers from the reports, the applicant proposed a theoretical model of "TBX6 copy number variations leading to defects of ovarian reserve by dosage effect". For verification, the ovarian function will be analyzed by fertility and hormone detection. Classical histological section method and advanced tissue-clearing and three-dimensional reconstruction technology will be used to analyze the numbers and construction of follicles at different developmental stage. The oocytes’ quality will also be revealed by 1st and 2nd polar body extrusion and postzygote development study. This work will shed light on the molecular mechanisms beside the dosage effect from primordial follicle activation to follicular atresia induced by granulosa cells apoptosis by using conventional biochemistry, immunochemistry, molecular biology technology, transcriptomics and bioinformatics technologies. This project will reveal the important role of TBX6 gene dosage in ovarian reserve and help the fertility preservation and genetic counseling for female carriers with TBX6/16p11.2 copy number variation.

TBX6/16p11.2的拷贝数重复和缺失携带者均有报道出现卵巢储备功能降低相关表型，然而基因型-表型的关联及其机制仍然缺乏深入研究。申请人前期构建了模拟拷贝数变异的Tbx6剂量上升和下降的小鼠模型，观察到卵泡减少、生育力低下等卵巢储备相关表型。基于初步结果和文献支持，申请人提出“TBX6拷贝数变异通过剂量效应导致卵巢储备功能降低”的理论模型，拟通过生育力及激素水平检测卵巢功能，并结合经典的组织切片技术和前沿的组织透明化与三维重建技术对每个发育阶段的卵泡进行定性和定量来分析卵泡数量及构成；从极体排出和体外受精后合子发育情况来分析卵子质量，以此建立不同剂量与表型之间的关联；利用生化、免疫组化、分子生物学、转录组学及生物信息学技术从原始卵泡过度激活和颗粒细胞凋亡引发卵泡闭锁两条途径分析其中的致病机制。本项目将揭示TBX6基因与卵巢储备的剂量效应关系，促进相关突变携带者的生育力维护和遗传咨询。

卵巢早衰困扰了近1%的育龄期妇女，对女性生育力造成严重的损害；除此之外，卵巢功能下降引发的健康问题也给女性身心带来严重的影响。遗传因素是导致卵巢早衰的重要原因之一，早期分子诊断给生育力保护和健康管理奠定了基础。申请人前期发现TBX6基因剂量改变导致不同的出生缺陷遗传病，该研究还发现TBX6基因剂量上升可以导致卵巢早衰这类新表型。该研究建立了卵巢早衰的新型表型鉴定方法，即利用组织透明化技术结合whole-mount免疫荧光标记技术进行卵巢3D重构，实现单细胞水平的卵巢储备统计，相比传统的组织切片方法更准确。同时，该研究还发现BMP4/SMAD6通路的过度激活是TBX6剂量上升导致始基卵泡过度活化及卵子加速丢失的关键调控通路。由于TBX6位于人类基因组16p11.2区段，该区段拷贝数增加是导致颈椎发育畸形的致病原因。因此，该研究不仅揭示了TBX6基因剂量升高与卵巢早衰的紧密联系，还提示携带TBX6/16p11.2变异的患者进行卵巢功能监测的必要性，为这类患者的生育力保护、早期分子诊断和遗传咨询提供理论依据。