人类弱畸形精子症候选新基因DNAH10突变通过破坏精子鞭毛组装导致男性不育的分子机制研究

精子鞭毛多发形态异常（MMAF）是一类主要由遗传缺陷导致的弱畸形精子症，是男性不育的常见原因。本项目前期在643个MMAF患者中筛查到5例患者携带未报道的内动力蛋白臂组分DNAH10双等位基因突变，Dnah10敲除雄鼠和模拟患者点突变的敲入雄鼠均重现MMAF表型，表现为严重的鞭毛组装异常，提示DNAH10是MMAF的候选新基因，但其致病机理不清楚。结合文献调研与前期研究，我们推测该基因缺陷引起精子鞭毛内运输和精子领运输障碍是导致鞭毛组装异常的关键机制。为此，我们拟以患者精液和模式小鼠为材料，结合辅助生殖技术、蛋白质组学、Co-IP-MS质谱、电镜、RNAi等技术，阐明DNAH10的生物学特征及其突变后所致疾病的生殖干预策略；探究DNAH10参与精子鞭毛组装的分子机制。本研究将通过对DNAH10功能的解读，加深对精子鞭毛组装机制的理解，为MMAF患者的分子诊断、遗传咨询和个体化治疗提供参考。

Asthenoteratozoospermia is one common cause of male infertility, wherein multiple morphological abnormalities of the flagella (MMAF) is one specific type mainly caused by genetic defects. Our preliminary study identified bi-allelic DNAH10 mutations in five patients from 643 Chinese MMAF-affected men. DNAH10 encodes a component of inner dynein arm, and this gene has never been reported to be related to MMAF/human infertility before. Importantly, one Dnah10-knockout mouse model and one Dnah10-knockin mouse model carrying a homozygous variant equivalent to that in one patient were conducted, and both mutant adult males were sterile and presented with typical MMAF phenotype, including complete immobility of sperm and abnormal flagellar morphology. These findings indicate that DNAH10 is a new MMAF candidate, however, the underlying mechanism by which DNAH10 defect results in MMAF is unknown. Based on our preliminary findings and an extensive literature research, we propose that disturbances in intraflagellar transport and/or intramanchette transport could be the key mechanism to explain the abnormal flagellum assembly caused by DNAH10 deficiency. To prove the hypothesis, this project will take advantage of the semen of patients and Dnah10 mutant mouse models, and combine with assisted reproductive technology, proteomics, Co-IP-MS mass spectrometry and electron microscopy, to reveal the biological characteristics of DNAH10 and the reproductive intervention strategies for MMAF-affected individuals with bi-allelic DNAH10 variants, and to explore the molecular mechanism of DNAH10 involved in the flagellum assembly. By uncovering of the function of DNAH10, this study will deepen our understanding of the assembly mechanism for sperm flagella, and provide MMAF patients with reference for molecular diagnosis, genetic counseling and individualized therapy.