ZP1突变在卵透明带发生中的“隐性负性作用”研究

The human zona pellucida is composed of four glycoproteins (ZP1, ZP2, ZP3, and ZP4) and has an important role in reproduction. In our preview study, we identified a caustive mutation in ZP1 (GenBank accession number KJ489454), leading to the eggs that lack a zona pellucida. We proposed the hypothesis of the pathogenic mechanism that the mutant ZP1 promotes interaction with and sequestration of the other ZP proteins. The intracellular sequestration of these proteins could impede their trafficking to the extracellular milieu, thus precluding the formation of a zona pellucida and leading to infertility (The New England Journal of Medicine 2014; 370 (13):1220-6). The hypothesis is similar to dominant negative that the single copy of mutant protein without its own function could affect other normal proteins, but the dominant negative is different for the normal phenotype in the Heterozygote in our found. So, we further infer there is a recessive-negative effect that the two copy of mutant proteins without its own function but affecting other normal proteins result in the disease. Our project plans to research on the mouse model of the novel homozygous & heterozygous mutation, using COIP, RT-PCR and Western Blot technologies to quantify the expression of the mutant ZP1 and normal ZPs, to verify the interaction between the mutant ZP1 and normal ZP2/3/4 and to elucidate the recessive-negative effect.

人卵透明带由四种蛋白组成(ZP1-4)，在生殖过程起关键作用。前期我们发现zp1上的致病突变（GenBank: KJ489454），导致透明带缺失，提出“突变ZP1与其它ZPs在卵细胞内相互作用，阻断透明带蛋白向细胞外转运而致透明带缺失”的机制假说(the New England Journal of Medicine，2014)。该假说类似“显性负效应”致病机理：杂合子的突变蛋白自身无功能还影响其他正常蛋白的生理功能。但不同的是我们研究中杂合子均表型正常，进一步推测可能存在一种“隐性负性作用”，指一对等位基因都突变丧失自身功能且影响其它蛋白功能才致病。本项目拟在前期定性实验结果支持基础上，以新发现突变zp1杂合/纯合疾病模式小鼠为模型，采用CO-IP、RT-PCR及Western Blot定量检测突变ZP1及正常ZPs表达，验证突变ZP1和正常ZPs间的聚合作用，诠释“隐性负性作用"。

人卵透明带由四种糖蛋白组成（ZP1-4），在生殖过程发挥重要作用。前期我们首次发现由于卵子透明带缺失导致家族性不孕的新疾病（OMIM:615774），筛查出符合常染色体隐性遗传模式的新致病基因ZP1（GenBank KJ489454)，提出“一定数量的变异ZP1在卵子内发生负性作用以低聚其他透明带蛋白，阻止透明带蛋白向细胞外转运导致透明带缺失，致使患者不孕”的致病机制假说(NEJM)。本课题将前期构建成功的携带相同zp1突变的疾病模式小鼠进一步的功能研究，验证突变的致病性。本实验在突变小鼠上获得部分卵子丢失透明带的情况，因此导致其不能在体内受精发育成早期胚胎，从而导致不孕。在体外细胞实验中验证了突变ZP1蛋白的负性效应。本研究进一步在模式大鼠上得到了更完美的展现。已经发表论文一篇，在修两篇及撰写高质量论文一篇，本课题的研究结论可以初步阐明一种新的致病机制“隐性负性作用”。