INSR基因rs2252673位点单核苷酸多态性与多囊卵巢综合征发病的关系

Association between single nucleotide polymorphism of rs2252673 of INSR gene and polycystic ovarian syndrome

目的 研究胰岛素受体（INSR）基因rs2252673位点单核苷酸多态性（SNP）与多囊卵巢综合征（PCOS）发病易感性的相关性,进一步确证INSR基因在PCOS发病中的风险.方法 选择2007年7月-2013年4月在山东大学附属生殖医院就诊的224例汉族PCOS患者（PCOS组）和192例因输卵管原因或男方原因的不孕患者（对照组）;根据体质指数（BMI）对PCOS患者进行再分组,分为肥胖组（BMI≥25 kg/m2） 88例、非肥胖组（BMI＜25 kg/m2） 136例.同时以PCOS患者为先证者,采用由其父亲、母亲以及患者本人组成的224个满足经典传递不平衡检验（TDT）分析的核心家系为观察对象.提取基因组DNA,采用PCR技术和直接测序法分析INSR基因rs2252673位点SNP基因型,比较PCOS组与对照组患者、肥胖组与非肥胖组PCOS患者间基因型与等位基因频率的差异;并比较不同基因型的PCOS患者间临床、生化指标的差异,临床、生化指标包括年龄、BMI、睾酮、雌二醇、LH、FSH、空腹血糖（FBG）、空腹胰岛素（FINS）、总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白（HDL）、低密度脂蛋白（LDL）.采用Hardy-Weinberg平衡检验分析INSR基因rs2252673位点是否符合Hardy-Weinberg平衡定律,TDT分析法验证INSR基因rs2252673位点在PCOS核心家系中是否存在危险等位基因的过度传递.结果 PCR产物的测序结果显示,rs2252673位点存在CC、CG和GG 3种基因型.（1）PCOS组CC、CG和GG基因型频率分别为8.0％、38.8％和53.1％,对照组CC、CG和GG基因型频率分别为14.6％、42.2％和43.2％;PCOS组C、G等位基因频率分别为27.5％、72.5％,对照组分别为35.7％、64.3％.PCOS组与对照组患者间基因型和等位基因频率分别比较,差异均有统计学意义（P＜0.05）.（2）不同基因型（包括CC、CG和GG基因型）的PCOS患者间各临床、生化指标比较,差异均无统计学意义（P＞0.05）.将基因型CG和GG患者合并后,CG＋GG基因型的PCOS患者的TC水平明显高于CC基因型患者（t=2.072,P=0.048）,HDL水平明显低于CC基因型患者（t=2.274,P=0.026）;CG＋GG基因型患者的FBG、FINS水平也有升高的趋势,但分别与CC基因型患者比较,差异均无统计学意义（P＞0.05）.（3）肥胖组GG、CG和CC基因型频率分别为52.3％、39.8％和8.0％,非肥胖组分别为53.7％、38.2％和8.1％;肥胖组G和C等位基因频率分别为72.2％和27.8％,非肥胖组分别为72.8％和27.2％.肥胖组与非肥胖组PCOS患者间基因型、等位基因频率分别比较,差异均无统计学意义（x2=0.054,P=0.974; x2=0.022,P=0.883）.（4） Hardy-Weinberg平衡检验显示,INSR基因rs2252673位点基因型分布符合Hardy-Weinberg平衡定律（P＞0.05）;TDT分析显示,INSR基因rs2252673位点存在传递不平衡,且危险等位基因G在患病子代中存在过度传递现象,差异有统计学意义（传递基因∶未传递基因=120∶88;x2=4.923,P=0.027）.结论 INSR基因rs2252673位点SNP与PCOS的发病易感性相关,且这种相关性不受BMI的影响.携带INSR基因rs2252673位点G等位基因的女性可能有较高的PCOS发病风险.

Objective This study is designed to determine whether an association exists between single nucleotide polymorphism （SNP） variant rs2252673 of insulin receptor（INSR） gene and polycystic ovarian syndrome （PCOS） in Han Chinese in order to identify INSR as a genetic susceptibility factor for PCOS.Methods A total of 224 women with PCOS,192 controls and 672 participants consisting of 224 trios （mother,father and offspring with PCOS） were recruited from the Hospital for Reproductive Medicine Affiliated to Shandong University,from July 2007 to April 2013.Genomic DNA was extracted according to the manufacturer＇ s protocol.SNP rs2252673 of INSR gene was amplified by PCR and then sequenced on an automated sequencer.Moreover,clinical and metabolic features of the patients with PCOS were compared according to the genotypes.The subjects were divided into twot groups according to body mass index （BMI）,and then the results were compared between two groups.And the transmitted disequilibrium test （TDT） was applied for data analysis.Results （1） There were three kinds of genotype of CC,CG and GG.Genotype frequencies of rs2252673 were 8.0％,38.8％,53.1％ and 14.6％,42.2％,43.2％ in the PCOS group and the control group,respectively.The allele frequencies of C and G were 27.5％,72.5％ and 35.7％,64.3％ in the PCOS group and the control group,respectively.There were statistical differences in genotype frequencies and allele frequencies between two groups （all P＜0.05）.（2）No significant differences were observed in the different genotype according to clinical and metabolic characteristics of women with PCOS （P＞0.05）.But when merging the genotype CG and GG,carriers of the CG and GG genotypes in women with PCOS were slightly associated with total cholesterol （TC） levels （t=2.072,P=0.048） and lower high-density lipoprotein （HDL） levels （t=2.274,P=0.026）.Although statistical significance was not achieved,there was an increased tendency in fasting blood glucose （FBG） and fasting insulin （FINS） levels in CG and GG genotypes in PCOS cases.（3）Between the obesity and the non-obesity with PCOS,there was no statistical significance in the genotype and allele frequencies （x2=0.054,P=0.974; x2=0.022,P=0.883）.（4）The results of families based analysis shown that genotype distribution of the SNP rs2252673 was in Hardy-Weinberg equilibrium （P＞0.05）.After the TDT,the G allele in SNP rs2252673 was over transmitted in families （transmitted∶ non-transmitted=120∶ 88; x2=4.923,P=0.027）.There was a transmitted disequilibrium in rs2252673,which implies the association of INSR and PCOS were independent of population stratification.Conclusions There were a association between the SNP variant rs2252673 of INSR gene and the susceptibility to PCOS in Han Chinese women,which was independently of body mass index.The carrier of G allele frequency of rs2252673 may have higher risk of PCOS.