TUBB8基因在卵子成熟障碍患者群体中的遗传变异筛查及CRISPR/Cas9介导的TUBB8突变小鼠基因治疗研究

Oocyte maturation failure is one of the severe infertile diseases. The key clinical feature is oocyte maturation arrest in repeated cycles of controlled ovarian hyperstimulation，which eventually leads to the failure of pregnancy. At present, there is no way to solve the problem by assisted reproductive technologies and oocyte donation is the only solution. Over the years, the pathogenic mechanism of oocyte maturation failure has been unclear. We first found that TUBB8 mutations can lead to oocyte maturation failure. However, it is not clear that whether there are many types of genetic variations of TUBB8 gene in a large-scale of patients with oocyte maturation failure as well as the related clinical phenotype and pathogenic mechanism. The project, based on our previous work, will search for new genetic variation of TUBB8 gene by gene targeted sequencing in a large-scale of samples collected from patients with oocyte maturation failure and further reveal the clinical phenotype and pathogenic mechanism, which could provide a reliable experimental basis for the TUBB8 gene as a molecular marker for diagnosis of oocyte maturation failure. Furthermore, this project will be the first to establish TUBB8 mutation transgenic mouse model of oocyte maturation failure and perform gene therapy in TUBB8 gene mutant mice by CRISPR/Cas9 gene editing technology, providing new ideas and research methods for the clinical treatment of patients with oocyte maturation failure.

卵子成熟障碍是一种严重的不孕不育疾病，临床表现为反复促排卵治疗始终无法获得成熟卵子，最终导致助孕失败。目前现有的辅助生殖技术无法治疗该疾病，卵子捐赠是唯一的解决手段。多年来，卵子成熟障碍的致病机制研究一直处于空白。我们首次发现人TUBB8基因突变会导致卵子成熟障碍。但是，TUBB8基因在大样本的卵子成熟障碍患者群体中是否存在多种遗传变异类型以及临床表型和致病机理目前尚不清楚。本项目研究将在前期工作基础上，利用搜集完成的卵子成熟障碍患者样本，通过TUBB8基因靶向测序寻找新的遗传变异，深入和全面的揭示其临床表型和致病机制，为将TUBB8基因作为卵子成熟障碍的分子诊断标记物提供可靠的实验依据。此外，本项目研究将首次建立TUBB8基因突变的卵子成熟障碍小鼠疾病模型，并利用CRISPR/Cas9基因编辑技术对TUBB8基因突变小鼠进行基因治疗，为卵子成熟障碍患者的临床治疗提供新的思路和研究方向。

在不孕女性的辅助生殖治疗中获得健康的成熟卵子是治疗成功的必备条件。但是，有0.1～1%的患者经过反复促排卵治疗后始终无法获得成熟的卵子，被称之为卵子成熟障碍，直接导致助孕失败。现有的辅助生殖技术对卵子成熟障碍患者的临床治疗无能为力，卵子捐赠是唯一的解决办法。因此，对卵子成熟障碍的诊断和治疗是目前急需解决的问题。多年来，卵子成熟障碍的致病机制研究一直处于空白。我们首次发现人TUBB8基因突变会导致卵子成熟障碍。但是，TUBB8基因在大样本的卵子成熟障碍患者群体中是否存在多种遗传变异类型和临床表型以及是否有可行的治疗手段都尚不清楚。本项目研究将在前期工作的基础上，从临床和基础研究两个方面开展：（1）构建卵子成熟障碍患者生物样本库，通过TUBB8基因靶向测序在卵子成熟障碍患者群体中寻找到多种新的遗传变异和多样的临床表现，深入和全面的揭示其临床表型和致病机制，为卵子成熟障碍患者的分子诊断提供可靠的实验依据。（2）建立TUBB8基因突变的卵子成熟障碍小鼠模型，填补了国际上缺乏卵子成熟障碍动物模型的空白，为卵子成熟障碍的机制研究提供了理想的动物模型。（3）探索性利用CRISPR/Cas9基因编辑技术对TUBB8基因突变小鼠进行基因治疗，为卵子成熟障碍患者的临床治疗提供新的思路和研究手段。