IgA肾病患者Cosmc基因启动子甲基化状态的检测

Detection of the Methylation State of Cosmc Gene Promoter from Patients With IgA Nephropathy

目的检测IgA肾病(IgA nephropathy,IgAN)患者β1,3半乳糖基转移酶(β1,3galactosyltransferase,β1,3GT)分子伴侣Cosmc(core1β3-Gal-T specific molecular chaperone)基因启动子的甲基化状态。方法选择2006年5月至2007年2月在中山大学附属第一医院确诊为IgA肾病的23例患者,纳入IgAN组(n=23);病理学分级分别为Ⅰ级2例,Ⅱ级4例[Ⅰ级+Ⅱ级组(n=6)],Ⅲ级6例[Ⅲ级组(n=6)],Ⅳ级8例,Ⅴ级3例[Ⅳ级+Ⅴ级组(n=11)]。选择同期在中山大学附属第一医院就诊的非IgA肾病患者纳入非IgAN组(n=10),健康体检者纳入正常对照组(n=23)。三组受试者年龄、性别构成比较,差异无显著意义(P>0.05)(本研究遵循的程序符合中山大学附属第一医院人体试验委员会制定的伦理学标准,得到该委员会批准,分组征得受试对象监护人的知情同意,并与其签署临床研究知情同意书)。IgAN诊断标准:①以IgA为主的免疫球蛋白在肾小球系膜区和或血管袢沉积;②排除狼疮性肾炎、紫癜性肾炎及乙肝病毒相关性肾炎等继发性肾脏疾病。IgAN病理学分级参照Lee分级标准,分为Ⅰ～Ⅴ级。分离外周血B淋巴细胞,提取DNA,对DNA进行亚硫酸氢钠修饰。采用巢式甲基化特异性聚合酶链反应(nested methylation specific Polymerase Chain Reaction,nMSP)检测外周血B淋巴细胞Cosmc基因启动子CpG岛甲基化状态。扩增产物经克隆、测序验证。结果 IgAN组、非IgAN组及正常对照组外周血B淋巴细胞Cosmc基因启动子CpG岛均呈部分甲基化。IgA肾病不同病理学分级组外周血B淋巴细胞Cosmc基因启动子CpG岛均呈部分甲基化。结论尚未发现IgA肾病患者外周血B淋巴细胞Cosmc基因启动子检测位点范围内CpG岛存在异常甲基化。

Objective To investigate the methylation state of CpG island of the Cosmc gene promoter of peripheral B lymphocyte from patients with IgA nephropathy （IgAN）.Methods From May 2006 to February 2007,23 biopsy-proven IgAN patients in First Affiliated Hospital of Sun Yat-sen University were included in IgAN group.They were divided into three groups according to Lee pathologic grade：grade Ⅰ ＋ grade Ⅱ group（n=6）,grade Ⅲ group （n=6）,grade Ⅳ ＋ grade Ⅴgroup （n=11）.At the same time,another 10 biopsy-proven non-IgAN patients were included in non-IgAN group.And 23 health people were included in normal control group.There were no significant differences in age and gender among IgAN group,non-IgAN group and normal control group（P〉0.05）.The study protocol was approved by the Ethical Review Board of Investigation in Human Being of First Affiliated Hospital of Sun Yat-sen University.Informed consent was obtained from all participants.Diagnosis criterion of IgAN was based on the manifestation of generalized glomerular mesangial proliferation with presence of IgA as the sole or predominant immunoglobulin deposition in the mesangial area of glomeruli.Patients with systemic disease such as Schonlei-Henoch purpura,systemic lupus erythematosus,or hepatitis B virus-associated glomerulonephritis were excluded.The severity of renal lesions was graded according to Lee′s classifications.Peripheral B lymphocytes were isolated.DNA were extracted from peripheral B lymphocytes and treated by natrium bisulfurosum.The methylation state of CpG island of the Cosmc gene promoter was detected by means of nested methylation specific Polymerase Chain Reaction （nMSP）,then cloning and sequencing.Results CpG islands of the Cosmc gene promoters from normal control group,IgAN group and non-IgAN group were partially methylated.CpG islands of the Cosmc gene promoters from different pathologic grade groups of IgAN were partially methylated.Conclusion The methylation state of detected cites of CpG island of Cosmc gene promoter of peripheral B lymphocyte from IgAN patients might not be abnormal.