ZP基因变异与卵细胞异常的相关性研究

发布时间：2018-06-16 11:37

本文选题：透明带基因 + 卵细胞　；参考：《山东大学》2017年硕士论文

【摘要】：研究背景成熟的卵细胞是保证正常受精,胚胎发育以及着床的关键,是妊娠维持及胎儿生长的重要影响及决定因素。ZP(zonapellucida,透明带)基因属于卵细胞特异性表达基因,编码ZP蛋白,人类的ZP蛋白包括ZP1,ZP2,ZP3和ZP4;小鼠的ZP仅包括ZP1,ZP2和ZP3,与人的高度同源,小鼠的ZP结构也与人的相似。ZP最早在初级卵泡中合成,在卵细胞成熟过程中,ZPmRNAs的拷贝数显著增加,ZP厚度随着卵细胞的生长增加。敲除雌鼠的任何一个ZP基因,均可导致其生育障碍和/或卵细胞形态异常,说明ZP基因在卵细胞成熟和受精过程中起着至关重要的作用。在辅助生殖实验室中,我们发现有部分较为罕见的原发性不孕患者,在不同的促排卵周期中均表现为卵细胞成熟障碍或形态异常,无法正常受精。因此我们推测:ZP基因突变是否与这部分患者卵细胞异常有关。研究目的分析ZP(ZP1,ZP2,ZP3和ZP4)基因突变是否参与卵细胞成熟阻滞或者形态异常的发病。研究方法选择多周期重复表现为卵细胞异常的辅助生殖助孕患者作为研究对象,自2011年至2015年共收集92例(属较为罕见病例),其中卵细胞成熟阻滞(组Ⅰ)49例,表现为卵细胞成熟阻滞(GV期和/或MI期阻滞);卵细胞形态异常(组Ⅱ)43例,表现为卵细胞胞质异常或透明带等异常。纳入的研究对象同时满足:(1)21岁≤年龄≤40岁;(2)染色体核型均为正常(46,XX);(3)多个(不少于两个)IVF/ICSI周期中卵细胞异常(成熟阻滞或形态异常)表现一致。对照组选择行辅助生殖助孕的,卵细胞形态无异常且正常受精,并生育过至少一个正常孩子的女性,共373例。提取所有受试者的外周血DNA,对其ZP1-ZP4四个透明带编码基因的外显子及其侧翼序列进行基因突变筛查,分析ZP1-ZP4基因的外显子及其侧翼序列是否存在单个碱基的改变(点突变)、缺失、插入、重复或移码突变。结果在组Ⅰ的49例卵细胞成熟阻滞患者中,除了已知的单核苷酸多态性(SNP)位点,未发现ZP1-ZP4有新的变异。在组Ⅱ的43例卵细胞形态异常患者中,发现了四处变异,其中两处位于ZP1基因:c.247TC(p.W83R)和c.1413GA(P.W471X);两处位于 ZP2 基因:c.1599GT(p.R533S)和 c.1696TC(p.C566R)。这四处位点共在四个不同的病例中检测到,在373例对照组中未检测到,同时在常用数据库(dbSNP、ExAC、1000 Genomes Project Database、NHLBI GO ESP)中进行筛查亦未见报道。这四处变异位点在各个物种之间高度保守;利用生物信息学软件预测,发现四处位点均为有害突变。含有ZP基因变异的病例大约占组Ⅱ的9%(4/43),均为原发性不孕患者。其中患者#224同时携带有c.247TC(ZP1)和c.1413GA(ZP1)两处变异,该患者表现为两个周期卵全部退变、碎裂。患者#006携带有c.1413GA(ZP1),该变异为无义突变,ZP1翻译提前终止,透明带结构域(ZPD)部分缺失,该患者三个促排卵周期所获卵全部退变。患者#189携带c.1599GT(ZP2),在三个促排卵周期中,所获卵多数退变、碎裂。患者#198携带c.1696TC(ZP2),在三个不同方案的促排卵周期中,所获卵多数退变、ZP异常。结论ZP基因变异可能与卵细胞退变或透明带等异常导致的原发性不孕有关,这些变异位点所造成的功能影响需要进一步实验证实。
[Abstract]:The mature egg cell is the key to ensure normal fertilization, embryo development and implantation. It is an important influence and determinant of pregnancy maintenance and fetal growth..ZP (Zonapellucida, zona pellucida) gene belongs to the specific expression gene of egg cell, encoding ZP protein, and human ZP protein including ZP1, ZP2, ZP3 and ZP4; ZP only includes ZP1, ZP in mice. 2 and ZP3, highly homologous to human, the ZP structure of mice is also similar to that of human.ZP in the primary follicle. In the process of egg maturation, the number of copies of ZPmRNAs increases significantly, and the thickness of ZP increases with the growth of the egg cells. The knockout of any one of the ZP genes of the female rats can lead to their fertility disorder and / or the abnormal morphology of the egg cells, indicating ZP Genes play a vital role in the maturation and fertilization of egg cells. In the auxiliary reproductive laboratory, we have found that some of the relatively rare patients with primary infertility have an ooocyte maturation disorder or abnormal morphology during the different cycle of ovulation, and can not be fertilized normally. Therefore, we speculate whether the ZP gene mutation is associated with this The purpose of this study is to analyze whether ZP (ZP1, ZP2, ZP3, and ZP4) gene mutations participate in the pathogenesis of oocyte maturation block or morphological abnormalities. The research method selected 92 cases from 2011 to 2015. There were 49 cases of oocyte maturation block (group I), which showed mature oocyte block (GV phase and / or MI phase block); abnormal oocyte morphology (Group II), 43 cases of abnormal oocyte cytoplasm or zona pellucida. The subjects included (1) 21 years of age < < < < 40 years'; (2) chromosome karyotype were normal (46, XX); (3) multiple (no less than two) of the IVF/ICSI cycle of abnormal oocyte (mature or morphologically abnormal). The control group chose to assist reproductive pregnancy with no abnormal and normal fertilization, and had 373 women who had had at least one normal child. A total of 373 cases were extracted from all the subjects' peripheral blood, and four of their ZP1-ZP4 zona pellucida were encoded. Gene mutation screening for gene exons and their flanking sequences to analyze whether there is a single base change (point mutation), deletion, insertion, repetition or code mutation in the exons and flanking sequences of the ZP1-ZP4 gene. Results in 49 cases of egg cell maturity block in group I, except the known single nucleotide polymorphisms (SNP) sites, have not been found. There are new variations in ZP1-ZP4. In 43 patients with abnormal oocyte morphologic abnormalities in group II, two of them are located in the ZP1 gene: c.247TC (p.W83R) and c.1413GA (P.W471X); two loci are located in the ZP2 gene: c.1599GT (p.R533S) and c.1696TC (p.C566R). These loci are detected in four different cases and in 373 control groups. It was not detected, and the screening was not reported in the common database (dbSNP, ExAC, 1000 Genomes Project Database, NHLBI GO ESP). The four heterotopic sites were highly conserved among the species. Using bioinformatics software, the sites were found to be catastrophic mutations. The cases containing the ZP gene mutation accounted for about 9% of the group II. 4/43), all of which were primary infertility. The patient #224 also carried two variations of c.247TC (ZP1) and c.1413GA (ZP1). The patient showed all two cycles of degeneration and fragmentation. The patient #006 carried c.1413GA (ZP1), the mutation was a nonsense mutation, the ZP1 translation was terminated, the zona pellucida domain (ZPD) partial deletion, and three ovulation in the patient. All the eggs obtained from the cycle were degenerated. The patient #189 carried c.1599GT (ZP2), and in the three ovulation cycle, most of the eggs were degenerated and fractured. The patient #198 carried c.1696TC (ZP2), and during the ovulation cycle of the three different schemes, the majority of the eggs were degenerated and ZP was abnormal. Conclusion the genetic variation of ZP may be caused by abnormal changes in the oocyte or the clear zone of the oocyte. Infertility is related to the functional effects of these mutation sites. Further experiments are needed to confirm this.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.8

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