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目的研究先天性心脏病患儿CITED2基因启动子区CpG岛的甲基化情况,并探讨其在该病发病中所起的作用。方法收集43例先天性心脏病患儿及2例意外死亡儿童右心耳组织,对CITED2基因进行突变筛查,同时利用生物信息学筛选,分别用亚硫酸氢盐修饰结合测序(BSP)及甲基化特异性PCR(MSP)检测CITED2基因启动子区CpG岛的甲基化情况,并运用实时荧光定量PCR检测CITED2基因mRNA的表达。结果先天性心脏病组启动子区存在4例杂合子突变(均为-341 T>G),此突变对CITED2基因mRNA的表达无影响。其中BSP法成功检测到先天性心脏病组甲基化阳性率为83.3%(10/12),MSP法分析发现甲基化阳性率为84.2%(16/19),同时CITED2基因异常甲基化使其mRNA的表达明显减低(P<0.05)。结论先天性心脏病中存在CITED2基因CpG岛的异常甲基化,此甲基化下调了CITED2基因mRNA的表达。
Objective To investigate the methylation status of CITED2 promoter CpG island in children with congenital heart disease and to explore its role in the pathogenesis of the disease. Methods Forty-three children with congenital heart disease and two children with unintentional death were enrolled in this study. The CITED2 gene was screened by mutation and bioinformatics screening was performed. The methylation status of CITED2 promoter in CpG island was detected by MSP and the expression of CITED2 mRNA was detected by real-time fluorescence quantitative PCR. Results There were 4 heterozygous mutations (all -341 T> G) in the promoter region of congenital heart disease group, which had no effect on the expression of CITED2 mRNA. The positive rate of methylation in the congenital heart disease group was 83.3% (10/12) by BSP method. The positive rate of methylation was 84.2% (16/19) by MSP method, while the abnormal methylation of CITED2 gene MRNA expression was significantly reduced (P <0.05). Conclusion Abnormal methylation of CITED2 gene CpG island exists in congenital heart disease, and this methylation down-regulates the expression of CITED2 gene mRNA.