新发现的人β防御素126基因突变与精子功能异常的相关性研究

A glycosylated polypeptide, β-defensin 126 (DEFB126), derived from the epididymis and adsorbed onto the sperm surface, has been implicated in efficient movement of sperm in mucosal fluids of the female reproductive tract. A recent study shows that ?20% of men carry a dysfunctional polymorphism in the open reading frame of DEFB126 gene. The density and activity of sperms in men with the homozygous variant genotype appear to be normal, but these sperms have a reduced ability to penetrate cervical mucus, and this polymorphism in men is associated with reduced fertility. Our recent study found a novel frame-shift mutation of human DEFB126 gene, which may result in an inactive protein and impair sperm function. In addition, we found that the frequency of DEFB126 mutation is region-dependent in China, varied from 1.4% to 39%. Given the significant impairment of reproductive function by DEFB126 gene mutation, we will further investigate the effect of sequence variation in DEFB126 on reproductivity in a cohort study of the adult males of the Shui Nationality in Guizhou province, who carry high mutation rate of DEFB126 gene. In this research plan, we will analyze the answer sheet of questionnaire survey, genotype allele frequency of DEFB126 gene using real-time PCR from adult males of the Shui Nationality, and use multiple-factor Statistics to analyze the correlation between DEFB126 gene mutation and reproductivity in the population. The results will bring us the relevance of sperm fertility and the mutation of DEFB126 gene, especially for the novel DEFB126 mutant. Moreover, to further study the mutations of DEFB126 on sperm function, we will generate human DEFB126-specific antibody, measure the cervical mucus penetration ability of sperm carrying the novel mutation of DEFB126 gene, test sperm fertility coated by anti-DEFB126 antibody, and identify sarccharide component of DEFB126 using lectin chip assays. The obtained epidemiological data and experimental results of sperm parameters will provide us the relevance of the novel mutation of DEFB126 and sperm infertility, and help us develop a novel diagnostic criteria used in clinical evaluation of Assistant Reproductive Technology.

DEFB126是由附睾分泌并包裹到精子表面的一种β防御素，最近文献报道男性DEFB126基因突变率高达20%，纯合子突变精子密度和活力均正常，但穿透宫颈粘液能力降低，导致男性生育力下降。我们新近也发现一个DEFB126基因中新的移码突变，可能导致该蛋白无活性。此外，我们发现DEFB126突变在我国不同地区存在显著差异，变化区间为1.4%-39%。鉴于DEFB126基因突变对男性生育影响显著，我们拟以突变率高的贵州水族育龄男性为研究对象，通过问卷调查和精液检查获得生育信息，荧光定量PCR检查DEFB126基因突变，以多因素统计法获取该人群DEFB126基因突变（特别是新突变）与生育的相关性；自制DEFB126特异性抗体用于抗体封闭实验；通过宫颈粘液穿透试验和凝集素芯片实验等，分析DEFB126基因新突变对精子功能的影响。项目研究所获结果可为辅助生殖临床男性不育病因诊断提供理论依据和方法技术。

DEFB126是覆盖在精子表面高度糖基化的蛋白，在人群中具有较高的2D/D突变率。通过本项目研究，我们发现了一个新的移码突变，4D/D突变。另外，经大规模人群样本分析发现2D/D突变和精子活力不相关，这与前面报道一致；而我们新发现的4D/D突变与男性不育显著相关，并且突变会导致精液中圆细胞数显著增加，经westerning blotting检测，发生2D/D突变精子上的DEFB126蛋白表达不稳定， 而发生4D/D突变的精子，则没有DEFB126蛋白表达，说明4D/D突变会导致DEFB126缺失表达，并具有更严重的不育特征。此外，为了研究DEFB126突变导致精子糖被的变化，我们首次把凝集素芯片应用到精子上，建立了目前最全的人精子凝集素结合谱；通过比较DEFB126不同基因型的凝集素结合谱，发现6个凝集素与2D/D突变精子结合显著降低，进一步大规模样本验证和检测发现ABA和MPL可以作为临床检测由于DEFB126突变引起的低生育或不育人群的潜在标志物，为不明原因不育男性致病因素开拓新思路。