【作者】 刘相东；

【导师】 赵跃然；

【作者基本信息】 山东大学 ， 临床检验诊断学， 2009， 博士

【摘要】 研究背景多囊卵巢综合征(PCOS)是育龄女性最常见的一种高度异质性的内分泌代谢紊乱性疾病,临床表现多种多样,典型的表现为不同程度的月经异常、卵巢多囊性改变、高雄激素血症、不孕、多毛、痤疮、肥胖等,并常伴有随年龄增长而日益明显的胰岛素抵抗／高胰岛素血症和高脂血症,常合并2型糖尿病和心血管疾病。PCOS的确切发病原因至今尚未阐明,可能与遗传和环境因素有关。PCOS的家族聚集性提示遗传因素在其发病机制中起重要作用,PCOS复杂的生化特征可能各自存在着遗传易感性,或者他们之间存在着某种共同的遗传学联系,多方面的研究结果显示,该病可能是由多个基因异常和多种环境因素如营养、精神、心理等因素共同作用的结果。PCOS的发病可能与下丘脑、垂体、卵巢、肾上腺、胰腺等的功能异常,环境因素和遗传因素有关。PCOS存在明显的家族聚集性特点。早年有病例报道双胞胎姐妹同时患有PCOS。而迄今为止最大规模的双胎PCOS研究也发现单卵双胎同时患PCOS的几率是双卵双胎的两倍,研究者认为遗传因素在PCOS的发病中起到决定性作用。PCOS与2型糖尿病有共同的病因学,胰岛素抵抗及高胰岛素血症是2型糖尿病和PCOS发生、发展的共同的病理生理基础。胰岛素抵抗及高胰岛素血症将这两种复杂疾病联系起来,对于PCOS患者来说,胰岛素抵抗及高胰岛素血症不仅是PCOS患者糖代谢异常的基本特征,胰岛素抵抗还可以通过诱发高雄激素血症、影响卵泡发育和子宫内膜生长等,从而引发PCOS患者的生殖功能障碍,近年来的研究逐步认为,胰岛素抵抗是PCOS病理生理过程的中心环节,并且与人种背景无关。研究资料表明,在患有PCOS的女性中,其2型糖尿病的发病率较不患有PCOS的女性显著升高。2型糖尿病同PCOS一样,也是一种多基因病,具有很强的遗传易感性,在不同人种／种族之间,其患病率有很大差异,其遗传学背景具有多基因性、微效性和高度复杂性的特点。PCOS与2型糖尿病不仅仅具有相同的病因学,还在临床表现、疾病发生特点、群体遗传学背景和流行病学分布特征上具有众多的相似性,鉴于这两种疾病的相似性,特别是在遗传学背景上的相似性,研究者们常常将2型糖尿病的疾病相关基因作为PCOS的疾病相关基因研究的候选基因。最近,多项研究在2型糖尿病患者中发现了两个与2型糖尿病的发病密切相关的单核苷酸多态位点,即转录因子7类似物2(transcription factor7-like2, TCF7L2)基因中的单核苷酸多态·(single nuclear polymorphisms,SNP)位点rs290487(C)和rs11196218(G)。此外,一项大规模的全基因族研究研究在欧洲2型糖尿病人群中发现了另一个2型糖尿病的风险位点,即周期素依赖的激酶5调节亚单位相关蛋白1类似物1 (cyclin-dependent kinase 5 regulatory subunit associated protein 1-like 1, CDKAL1)基因中的SNP位点rs7756992(G)。依据PCOS与2型糖尿病的相似性以及候选基因原理,我们假设,TCF7L2基因中的单核苷酸多态位点rs290487(C)和rs11196218(G),以及CDKAL1基因中的rs7756992(G)位点,不仅是2型糖尿病的风险位点,同时也是PCOS的疾病相关位点。目的①通过分析PCOS患者的基本资料和内分泌及代谢特征表型资料,确定PCOS病人胰岛素抵抗的相关因素。②通过分析比较TCF7L2基因中的rs290487(C)和rs11196218(G),以及CDKAL1基因中的rs7756992(G)多态位点的基因型频率和等位基因频率在PCOS病例组与对照组中分布的差异,比较不同基因型的PCOS病人的内分泌及代谢特征表型的差别,研究TCF7L2基因中的单核苷酸多态位点rs290487(C)和rs11196218(G),以及CDKAL1基因中的rs7756992(G)位点的不同基因型与PCOS不同临床表型的相关性,探讨TCF7L2基因和CDKAL1基因在PCOS的发病及病理生理的变化过程中的相关作用,从而验证我们的假设是否正确。研究方法研究对象：本研究依据2003年鹿特丹标准选择在山东省立医院生殖医学中心门诊就诊的PCOS患者826例,并选择620名健康的非糖尿病女性作为对照。两组年龄具有可比性,所有研究对象均为山东汉族人且无亲缘关系。分别测定PCOS患者的激素、生化指标、OGTT,测量身高、体重、腰围和臀围,计算体重指数(BMI)、腰臀比例及HOMA-IR指数。自肘静脉抽取外周血,抗凝处理后,提取全血基因组DNA。基因分型：本研究采用TaqMan-MGB探针技术进行基因分型。根据方法原理设计一对等位基因特异性的MGB探针和一对扩增引物。PCR反应结束后,立即使用实时荧光PCR仪分析进行基因分型。PCR反应及基因型分析在ABI 7500实时荧光PCR反应仪上进行。基因分型完成后,将所有被分型的标本中的2%进行测序,以确定该基因分型方法的准确性。统计学分析：病例组PCOS患者的基本资料和内分泌代谢特征表型资料的比较采用独立样本t检验的方法进行分析。计算病例组与对照组中三个多态位点各基因型的频率及等位基因频率,Hardy-Weinberg遗传平衡定律检验使用x~2检验来计算,病例组与对照组之间基因型频率与等位基因频率的比较采用x~2检验的方法。PCOS组中患者的内分泌及代谢特征表型与基因型的关联性分析采用协方差分析(ANCOVA)的方法,并将BMI作为协变量引入。结果1、胰岛素抵抗组和胰岛素非抵抗组患者的血清T、FSH、LH和PRL水平之间没有差异,而胰岛素抵抗组的PCOS患者的BMI和各个时点的血糖及血浆胰岛素水平均高于胰岛素非抵抗组的PCOS患者。2、肥胖组和非肥胖组的PCOS患者的血清T、FSH、LH和PRL水平之间没有差异,除服糖后180分钟的血糖水平在两组患者中没有差异外,肥胖组PCOS患者的HOMA-IR和各个时点的血糖及血浆胰岛素水平均高于非肥胖组的PCOS患者。3、PCOS患者的HOMA-IR与BMI呈正相关。4、在PCOS病例组与对照组中,三个多态位点的基因型频率和等位基因频率的分布没有差异。5、在PCOS病例组中,三个多态位点的基因型频率和等位基因频率在胰岛素抵抗组和非胰岛素抵抗组患者中的分布没有差异。6、在PCOS病例组中,三个多态位点的基因型频率和等位基因频率在肥胖组和非肥胖组患者中的分布没有差异。7、在PCOS病例组中,PCOS患者的各种内分泌及代谢特征与这三个多态位点的基因型分布之间没有相关性。结论TCF7L2基因中的单核苷酸多态位点rs290487(C)和rs11196218(G),以及CDKAL1基因中的rs7756992(G)位点不是PCOS的疾病相关基因位点,未发现这三个位点的基因型分布与PCOS患者的内分泌及代谢表型特征表型之间存在相关性。更多还原

【Abstract】 Background Polycystic ovary syndrome (PCOS) is a highly heterogeneous endocrine disorder of women that always results in infertility. It is characterized by oligomenorrhea or amenorrhea, hyperandrogenism, insulin resistance, infertility, acne, obesity and multiple small subcapsular cystic follicles in the ovary on ultrasonography. It affects about 5%-10%of women of reproductive age. Recently, it was demonstrated that PCOS was also a metabolic disorder. The etiopathogenisis of PCOS is still unknown. PCOS displays evident familial aggregation. The interaction between genetic susceptibility and environmental factors plays a possible role in the pathogenesis of PCOS.PCOS and type 2 diabetes mellitus are both diseases of multifactorial inheritance. The genetic backgrounds of these two disorders are complex. They share many common features in both clinical metabolism manifestations and epidemiology characters. Insulin resistance and hyperinsulinemia is the link between PCOS and type 2 diabetes. The risk of developing type 2 diabetes is significantly higher in women with PCOS than in women without PCOS. Therefore, the known susceptibility genes for type 2 diabetes are reasonable candidates for investigation of the genetic basis of PCOS.A number of studies had shown the powerful association between the common variants, rs7903146 (T) and rs 12255372 (T), in the gene encoding transcription factor 7-like 2 (TCF7L2) and type 2 diabetes in European ancestry populations. However, rs7903146 and rs12255372 were not associated with type 2 diabetes in Han Chinese population. Since the risk alleles of the two loci were rare and the statistical power was inadequate in Chinese. Two studies identified that another two SNPs (rs290487 [C] and rs11196218 [G]) in gene TCF7L2 were associated with type 2 diabetes in Han Chinese populations.Recently, a genome-wide association study identified a novel risk locus, single nucleotide polymorphism (SNP) rs7756992 (G) in the cyclin-dependent kinase 5 (CDK5) regulatory subunit associated protein 1-like 1 (CDKALI) gene (with an allelic specific odds ratio [OR] of 1.20,95%confidence interval [95%CI] of 1.13-1.27), for type 2 diabetes in several case-control cohorts of European ancestry. The association between rs7756992 and type 2 diabetes in individuals from Hong Kong Han Chinese was also identified (OR [95%CI] =1.25[1.11-1.40]).We hypothesize that these three novel risk loci for type 2 diabetes, rs7756992 in gene CDKAL1, rs290487 and rs11196218 in gene TCF7L2, may also contribute to the risk for PCOS in Han Chinese women. In this study, we performed a case-control study to test our hypothesis.Objective To test whether the SNPs rs290487 and rs11196218 in gene TCF7L2 and rs7756992 in gene CDKAL1 associated with PCOS. To analyze the associations between the genotype distribution of these three SNPs and the clinical features of PCOS patients.MethodsSubjects A total of 826 women with PCOS were recruited from the outpatient clinic of Shandong Provincial Hospital. Recruitment was based on the revised Rotterdam diagnostic criteria. We recruited 620 healthy nondiabetic women with regular menstrual cycles as a control group. None of the controls had hyperandrogenism or other endocrine disorders related to PCOS. All patients and controls were Han Chinese women and were recruited from the same area. After undergoing a physical examination, including measurement of abdominal and hip circumferences and the measurement of hormone, an oral glucose tolerance test (OGTT) was performed in PCOS patients. Body mass index (BMI) was used to evaluate obesity. Insulin sensitivity in the fasting state was estimated by the homeostasis model assessment of insulin resistance (HOMA-IR).Genotyping Peripheral blood from participants was collected and the genomic DNA was isolated. Genotypes were identified by TaqMan genotyping technologies. Both the PCR reaction and the analysis were performed on the ABI 7500 real-time PCR system.Statistical analysis The Hardy-Weinberg equilibrium test was performed using the chi-square test.Genotype and allele frequencies for the case and control groups were compared using the chi-square test. Analysis of covariance (ANCOVA) was used to analyze the associations between the genotype of these three SNPs and the clinical features of PCOS patients in PCOS group, introducing BMI as the covariate. P<0.05 was considered statistically significant.Results1. There are no significant differences of the T, FSH, LH and PRL between the PCOS patient with or without insulin resistance. The serum glucose and plasma insulin level of patients with insulin resistance in OGTT are significant higher than that of patients without insulin resistance.2. There are no significant differences of the T, FSH, LH and PRL between the PCOS patient with or without obesity. The serum glucose and plasma insulin level of patients with obesity in OGTT are significant higher than that of patients without obesity, except the serum glucose level at the time point of 180 min.3. There is a positive correlation between the HOMA-IR and BMI of PCOS patient.4. No significant differences in genotypes of these three SNPs and allele frequencies were found between PCOS patients and healthy controls. 5. No significant differences in genotype and allele frequencies of these three SNPs were found between PCOS patients with or without insulin resistance.6. No significant differences in genotype and allele frequencies of these three SNPs were found between PCOS patients with or without obesity.7. No associations were observed between genotype of these three SNPs and the quantitative clinical features of PCOS patients in PCOS group after adjustment for BMI.Conclusions The risk allele of the SNPs rs290487 and rs11196218 in gene TCF7L2 and rs7756992 in gene CDKALI have no associations with PCOS or PCOS-related clinical features.更多还原