精子鞭毛形成相关基因CFAP58在男性不育中的作用及分子机制研究

精子鞭毛多发形态异常（MMAF）是一种严重的男性不育症，因其遗传病因高度异质，仍有近40%患者病因不明。申请人前期对90例MMAF行全外显子测序，发现新致病基因CFAP58功能缺失突变，患者精子表现短尾或卷尾，鞭毛中段缩短，轴丝中央二连管缺失及微管排列紊乱。通过CRISPR/Cas9获得Cfap58基因敲除小鼠，发现Cfap58-/-小鼠完全不育，附睾精子表现为短尾和卷尾，与MMAF患者精子表型高度一致，提示CFAP58是MMAF的致病基因，但其确切致病机制尚不清楚。本课题拟：1）采用基因敲除小鼠模型进一步验证Cfap58的致病性，探索其在精子发生和鞭毛形成过程中的作用；2）利用患者睾丸组织和Cfap58KO小鼠模型，通过免疫共沉淀和蛋白质组测序寻找CFAP58结合蛋白及调控基因，揭示其调控机制；3）进一步探索CFAP58突变在MMAF中的大概频率，为临床男性不育症诊疗提供重要理论依据。

The multiple morphological abnormalities of the flagella (MMAF) phenotype is among the most severe forms of sperm defects responsible for male infertility. Recent studies have demonstrated that MMAF is genetically heterogeneous and pathogenic mechanisms in the genetically unexplained MMAF account for about 40%. Recently, in a large cohort of 90 infertile male individuals with MMAF, we have now identified five unrelated individuals harboring mutations in CFAP58, which caused coiled and short flagella presenting with shortened mitochondrial sheath, disorganization in fibrous sheaths and the absence of central microtubules in most flagella. Using CRISPR/Cas9 technology, we generated Cfap58 knockout model and Cfap58-deficient male mice presented with completely infertile. The morphology of Cfap58 deficient sperm was entirely abnormal, mainly with short and coiled flagella, which was in accordance with CFAP58-mutated men. These results indicated that the MMAF phenotypes in CFAP58-mutated subjects are likely to be explained by the bi-allelic LoF variants in CFAP58, but the exact molecular mechanism needs to be further explored. Thus, our project is aimed to: 1) assess the impact of Cfap58 absence on mouse spermatogenesis and sperm flagellation by Cfap58-KO model; 2) elucidate the mechanisms of Cfap58 in spermatogenesis and sperm flagellar formation by performing proteomic analysis on the testes of CFAP58-mutated men and Cfap58-/-mice, combining co-immunoprecipitation and mass spectrometry; 3) analyze the pathogenicity of CFAP58 in MMAF patients, providing new clues to personalized diagnosis and treatment for male infertility.