ESR2基因多态性与河南汉族人群男性不育的相关性分析

发布时间：2018-07-06 09:18

本文选题：Y染色体微缺失 + ESR2基因　；参考：《郑州大学》2014年硕士论文

【摘要】：研究背景和目的由世界卫生组织(WHO)规定,夫妇生活1年以上并且没有采用任何避孕措施,仍然无法生育的称为不孕不育,由男方因素造成的称为不育症。近年来,不孕不育的比例在已婚的适龄夫妇中越来越高,并且,不孕不育给社会带来了很多负面影响。男性不育症中,病因很多并且相当复杂,比如外伤、睾丸发育不良、内分泌或免疫性疾病、梗塞、服用激素药物或肿瘤药物、环境毒物、不良生活习惯以及遗传因素等等。还有少数男性不育不能给出基于分子水平的合理解释,被称为特发性男性不育,这其中约有50%是由遗传因素造成的。近年来,大量研究发现,染色体异常、Y染色体微缺失及相关基因的突变都与男性生殖能力的改变有关。越来越多的学者验证了Y染色体长臂远端AZF区域微缺失与精子发生障碍有重要关系。基于这一结论,要想研究相关基因多态性与男性生殖能力的相关关系,必须先排除Y染色体AZF微缺失的影响。 1996年,Mosselman等人首次从睾丸组织中克隆出了雌激素受体的另一种亚型,雌激素受体β (Estrogen receptor β)后来被称为ERβ即ESR2。研究发现,ESR2基因rs4986938G/A位点位于ESR2基因的第8外显子的3’非编码区第1730号核苷酸处。而rs1256049G/A位点则位于ESR2基因的第5外显子的配体结合区的第1082号核苷酸处。2010年,Mohammad Reza Safarinejad等人对伊朗人群ESR2基因这两个位点多态性与男性不育的相关性研究发现,单倍型与男性不育有关系。2011年台湾学者Wen Lee等人的研究发现,ESR2基因的rs4986938、rs1256049位点的多态性与精液浓度有关,rs1256049位点的多态性还与精子运动能力有关。2012年日本学者Tsutomu Ogata等对ESR2基因的多个SNPs位点多态性与男性不育的相关性研究中显示,单倍型“TGTAGA"可能是男性不育的风险单倍型,但其中rs1256049位点的等位基因G/A在病例组和对照组中的频率分布没有显著差异。目前,ESR2基因多态性与男性不育的相关性在不同研究报道中的结果不尽相同,并且国内没有这方面研究的相关报道,故本研究选取ESR2基因的rs4986938、rs1256049位点进行研究,拟分析ESR2基因多态性与河南汉族人群男性不育是否相关。本研究首先用多重聚合酶链式反应(Mutiplex Polymerase chain reaction Mutiplex PCR)对研究对象的病例组进行了Y染色体AZF区域微缺失的检测,排除AZF微缺失之后,对病例组剩下的研究对象再利用聚合酶链式反应-限制性片段多态性(Polymerase chain reaction restriction-fragment length polymorphism, PCR-RFLP)技术对河南汉族人群ESR2基因的rs4986938位点和rs1256049位点多态性进行分析,拟研究这两个位点多态性在河南汉族人群中的分布情况及其与男性生殖能力的相关性,从而为男性不育的临床检测与治疗提供相关理论依据。研究对象：病例组收集2012年11月到2013年3月来郑州大学第一附属医院门诊检验科就诊的397例不育症患者的精液标本,患者年龄22-42岁(平均年龄29.4±6.1岁)。患者均根据世界卫生组织(WHO)标准进行常规精液分析,并且连续三次检查确诊为无精子症、少精子症(精子密度20×106/m1)。患者均排除生殖器手术、睾丸炎、精索静脉曲张、内分泌疾病、肝肾类疾病、染色体异常、生殖器异常等情况。对照组随机选取通过正常手段生育至少一个健康孩子的健康男性347例作为对照组,年龄22-42岁(平均年龄31.7±2.6岁)。所有病例组和对照组的研究对象均来自河南地区,饮食结构大致相同,并且都知情同意。研究方法：抽取精液用经典的酚-氯仿方法提取基因组DNA,PCR扩增目的片段,再经过Rsa Ⅰ和Alu Ⅰ限制性内切酶酶切,最后用2.5%的琼脂糖凝胶电泳对扩增片段和酶切片段进行基因型检测,紫外分析凝胶成像系统观察记录。统计学处理：所有得到的数据都采用SHEsis在线软件和SPSS17.0统计软件进行分析,检验水准为：a=0.05。病例组和对照组均使用SHEsis在线软件进行Hardy-Weinberg(HWE)检测,以估计所选资料的人群代表性。使用SPSS17.0计算OR值以及95%置信区间,进行基因突变对男性不育发生的风险性估计。用在线软件SHEsis进行单倍型分析。结果： 1.检测出397例病例组中有50例Y染色体微缺失。 2.河南汉族人群中,ESR2基因的rs4986938以及rs1256049位点基因型频率都符合Hardy-Weinberg平衡,表明其具有良好的人群代表性。 3.本研究中,rs4986938位点在患者组和对照组对比中,基因型和等位基因频率差异具有统计学意义(P0.05)。 4.对rs4986938位点进行的密度分组分析中,GG基因型在重度少精和轻度少精组间差异显著(P0.05)。 5.在本研究中,对于rs1256049位点两组之间的对比显示：基因型和等位基因频率差异具有统计学意义(P0.05)。 6.对rs1256049位点进行的密度分组分析,GG基因型在重度少精和极重、无精组间差异显著(P0.05)。 7.在河南汉族人群中,对rs4986938G/A和rs1256049G/A两个位点在男性不育组与正常对照组间进行单倍型分析,发现A-G、G-A、G-G单倍型在两组间的差异有统计学意义(P0.05)。结论： 1.ESR2基因rs4986938位点的A等位基因与rs1256049位点的A等位基因可能均是河南汉族人群男性不育的遗传危险因子。 2.ESR2基因rs4986938和rs1256049位点的A-G/G-A单倍型可能是河南汉族人群男性不育的风险单倍型,G-G单倍型是男性不育的保护单倍型。 3.ESR2基因多态性可能与河南汉族男性不育的发生相关。
[Abstract]:Background and purpose of research
The WHO (WHO) stipulates that couples who have lived for more than 1 years and have not adopted any contraceptive measures, are still infertile, called infertility, and are called infertility caused by the male factors. In recent years, the proportion of infertility is getting higher and higher among married couples, and infertility has brought a lot of negative effects to the society. In male infertility, there are many and quite complex causes, such as trauma, dysplasia of the testicles, endocrine or immune diseases, infarction, taking hormone drugs or tumor drugs, environmental poison, bad habits and hereditary factors. Sexual infertility, about 50% of which are caused by genetic factors. In recent years, a large number of studies have found that chromosomal abnormalities, Y chromosome microdeletion and mutations in related genes have been associated with changes in male reproductive capacity.
More and more scholars have verified that the microdeletion of the distal AZF region of the long arm of the Y chromosome has an important relationship with the disturbance of spermatogenesis. Based on this conclusion, to study the correlation between genetic polymorphism and male reproductive ability, the effect of AZF microdeletion on the Y chromosome must be eliminated first.
In 1996, Mosselman and others first cloned another subtype of estrogen receptor from the testicular tissue. The estrogen receptor beta (Estrogen receptor beta) was later known as ER beta, or ESR2. study found that the ESR2 gene rs4986938G/A loci was located at the 3 'non coding region 1730th nucleotides of the eighth exon of the ESR2 gene. The relationship between the two loci polymorphism of the ESR2 gene in Iran population and the relationship between the two loci polymorphism of the ESR2 gene in Iran population and the male infertility in the number 1082nd nucleotides of the ligand binding region of the fifth exon of the ESR2 gene found that the haplotype and male infertility were related to the study of the ESR2 gene of the ESR2 gene of the Taiwan scholar Wen Lee, which was found by the Taiwan scholar in.2011. Rs4986938, the polymorphism of the rs1256049 locus is related to the concentration of semen. The polymorphism of the rs1256049 locus is also related to the motility of the sperm. The correlation between the polymorphisms of the multiple SNPs loci and male infertility of the ESR2 gene, such as Tsutomu Ogata of the Japanese scholar,.2012 years, shows that the haplotype "TGTAGA" may be the risk haplotype of male infertility. There is no significant difference in the frequency distribution of the allele G/A of the rs1256049 locus in the case group and the control group. At present, the correlation between the ESR2 gene polymorphism and male infertility is not the same in the different research reports, and there is no related report in this field at home. Therefore, this study selects the rs4986938, rs1256049 of the ESR2 gene. We studied the association between ESR2 gene polymorphism and male infertility in Henan Han population.
In this study, the Mutiplex Polymerase chain reaction Mutiplex PCR was used to detect the microdeletion of the Y chromosome AZF region in the case group of the subjects. After eliminating the AZF microdeletion, the remaining subjects of the case group were reused by the polymerase chain reaction restriction fragment polymorphism (Polymerase chain). Reaction restriction-fragment length polymorphism, PCR-RFLP) technique was used to analyze the rs4986938 and rs1256049 loci polymorphism of the ESR2 gene in Henan Han population. The distribution of the two loci polymorphism in the Han population in Henan and the correlation with male reproductive ability were studied, which was the clinical study of male infertility. The relevant theoretical basis is provided for the detection and treatment.
Research object:
Case group
The semen specimens of 397 infertile patients, aged 22-42 years (average age 29.4 + 6.1 years), were collected from November 2012 to March 2013 at the First Affiliated Hospital of Zhengzhou University. The patients were performed routine semen analysis according to the WHO (WHO) standard and were diagnosed as azoospermia and oligozoospermia in three consecutive cases. The disease (sperm density is 20 x 106/m1). Patients are excluded from genital surgery, orchitis, varicocele, endocrine diseases, liver and kidney diseases, chromosomal abnormality, and genital abnormalities.
control group
A total of 347 healthy men with normal health at least one healthy child were randomly selected as the control group, aged 22-42 years (mean age 31.7 + 2.6 years).
All cases and controls were from Henan, with similar dietary patterns and informed consent.
Research methods:
The extract semen was extracted by classical phenol chloroform method to extract genomic DNA, PCR to amplify the target fragment, and then Rsa I and Alu I restriction endonuclease was cut. Finally, the amplified fragment and enzyme cut fragment were detected by 2.5% agarose gel electrophoresis, and the UV analysis gel imaging system was observed.
Statistical treatment:
All the data obtained were analyzed using SHEsis online software and SPSS17.0 statistical software. The test level was that the a=0.05. case group and the control group all used SHEsis online software for Hardy-Weinberg (HWE) detection to estimate the representative of the selected data. The OR value and the 95% confidence interval were calculated by SPSS17.0, and the gene mutation was carried out. Risk assessment of male infertility. Haplotype analysis was performed using online software SHEsis.
Result:
1. out of 397 cases, 50 cases of Y chromosome microdeletions were detected.
2. of the Han population in Henan, the genotype frequencies of the rs4986938 and rs1256049 loci of the ESR2 gene all conformed to the Hardy-Weinberg balance, indicating that they have a good representation of the population.
3. in this study, there was a statistically significant difference in genotype and allele frequencies between the rs4986938 locus and the control group (P0.05).
4. in the density grouping analysis of rs4986938 locus, GG genotype was significantly different between severe oligozoospermia group and mild oligozoospermia group (P0.05).
5. in this study, the comparison between the two loci of rs1256049 locus showed that there was a statistically significant difference in genotype and allele frequency (P0.05).
6. according to the density grouping analysis of rs1256049 locus, GG genotype was significantly different between severe oligozoospermia and heavy inseminal group (P0.05).
7. in Henan Han population, the two loci of rs4986938G/A and rs1256049G/A were analyzed by haplotype between male infertility group and normal control group. It was found that the difference of A-G, G-A and G-G haplotypes between the two groups was statistically significant (P0.05).
Conclusion:
The A alleles at the rs4986938 loci of the 1.ESR2 gene and the A alleles at the rs1256049 locus may be the genetic risk factors for male infertility in Henan Han population.
The A-G/G-A haplotypes of the 2.ESR2 gene rs4986938 and rs1256049 loci may be the risk haplotypes of male infertility in Henan Han population, and the haplotype of G-G is the protective haplotype of male infertility.
3.ESR2 gene polymorphism may be associated with male infertility in Henan Han population.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R698.2

【参考文献】