卵子成熟障碍与MARF1基因变异相关性的研究

发布时间：2018-02-16 13:17

本文关键词： MARF1 卵子成熟障碍突变单核苷酸多态性　出处：《山东大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景卵子成熟障碍是引起女性不孕的原因之一。主要包括细胞核成熟和细胞质成熟两方面。卵子核成熟主要指卵子从减数分裂阻滞中恢复,并再次停滞于减数第二次分裂等待受精。目前生殖临床对于卵子核成熟与否的判断主要采用形态学观察的方法判断减数分裂进程。若卵细胞质内见生发泡(Gv),无极体,则为Gv期卵；若无生发泡也无极体,为第一次减数分裂中期(MI)卵。2012年,Youqiang Su et al报道乙基亚硝基脲(ENU)诱导小鼠Marf1基因突变,引起卵子减数分裂停滞于Gv期,导致小鼠不孕。人类MARF1基因位于16号染色体1区3带,包括27个外显子,编码1742个氨基酸,与小鼠Marf1编码的蛋白具有86%同源性,构成包含3个RNA识别结构域(RRMs)的蛋白。目前尚未见人类卵母细胞减数分裂停滞的MARF1基因突变相关报道,因此,本课题将对卵子成熟障碍患者MARF1基因的关键外显子进行测序,探讨人类卵子成熟障碍是否与MARF1基因变异相关。研究方法研究对象选自2008-2013年就诊于山东大学附属生殖医院的患者。研究对象满足以下条件：1)年龄40岁以下；2)染色体核型正常,均为46,XX；3)两个或两个以上IVF/ICSI周期中70%以上卵子出现成熟障碍。对照选择无流产史且生育过正常孩子的妇女200人。提取其外周血全基因组DNA,应用聚合酶链反应(PCR)扩增其外显子及相应侧翼序列,对PCR产物进行直接测序,与NCBI提供的正常序列比较,分析MARF1基因8、11、12及16-27外显子及侧翼序列是否存在异常碱基改变、插入或缺失。结果对91例卵子减数分裂障碍患者MARF1基因的第8、11、12及16-27号外显子及侧翼序列测序发现了一处位于26号外显子的新发同义突变(c.4852GA),两处新发单核苷酸多态性位点,分别为位于25号外显子的c.4715CG和位于21号外显子的c.3979AT(此处为中国人新发单核苷酸多肽位点),两处新发SNP位点基因型频率与对照组相比未见统计学差异。此外还发现五个已知的SNP位点,分别是rs28670396、rs7185421、rs28509427、rs58075088、rs202242803。结论本研究未发现人类卵子成熟障碍与MRAF1基因突变相关的直接证据,但新发现同义突变的功能仍需进一步研究和验证。
[Abstract]:Background the obstacle of egg maturation is one of the causes of female infertility. It mainly includes nuclear maturation and cytoplasmic maturation. Egg nuclear maturation mainly refers to the recovery of egg from meiosis block. At present, the judgement of egg nucleus maturation or not in reproductive clinic mainly uses morphological observation method to judge the meiosis process. In 2012, Youqiang Su et al reported that ENU-induced mutation of Marf1 gene in mice, resulting in meiosis in GV stage. The human MARF1 gene, located in region 3 of chromosome 16, contains 27 exons and encodes 1742 amino acids, having 86% homology with the protein encoded by mouse Marf1. There are no reports on the mutation of MARF1 gene in human oocytes with meiosis stagnation. Therefore, the key exons of MARF1 gene in patients with oocyte maturation disorder will be sequenced in this study. To investigate whether the disorder of human egg maturation is related to the variation of MARF1 gene. Methods the subjects were selected from patients who were admitted to the affiliated Reproductive Hospital of Shandong University from 2008 to 2013. The subjects met the following conditions: 1) the age below 40 years old and 2)) the karyotype was normal. In two or more IVF/ICSI cycles, more than 70% oocytes developed maturation disorders. In contrast, 200 women with no history of abortion and who had given birth to normal children were selected. The genomic DNA of peripheral blood was extracted and amplified by polymerase chain reaction (PCR). Their exons and their corresponding flanking sequences, The PCR products were directly sequenced and compared with the normal sequences provided by NCBI. The results showed that there were abnormal base changes, insertions or deletions in the exons 8111112 and 16-27 of MARF1 gene and their flanking sequences. Results in 91 patients with meiosis, a new synonymous mutation, c. 4852 GAA, and two new single nucleotide polymorphisms were found in exons 8111112 and 16-27 of MARF1 gene and flanking sequences. C. 4715CG at exon 25 and c. 3979AT at exon 21, respectively. The genotypes of the two new SNP loci were not significantly different from those of the control group. Five known SNP sites, They are rs28670396rs7185421rs28509427rs58075088andrs20242803.Conclusion there is no direct evidence of the relationship between MRAF1 gene mutation and human oocyte maturation disorder, but the function of the newly discovered synonymous mutation still needs to be further studied and verified.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.8

【共引文献】