法洛四联症患儿心肌中VANGL2基因的启动子甲基化与表达

Promoter methylation and expression of the VANGL2 gene in the myocardium of pediatric patients with tetralogy of fallot.

作者信息

Yuan Yuan, Gao Yan, Wang Huijun, Ma Xiaojing, Ma Duan, Huang Guoying

机构信息

Children's Hospital of Fudan University, Shanghai, China.

出版信息

Birth Defects Res A Clin Mol Teratol. 2014 Dec;100(12):973-84. doi: 10.1002/bdra.23291. Epub 2014 Sep 8.

DOI:10.1002/bdra.23291

PMID:25200836

Abstract

BACKGROUND

Tetralogy of Fallot (ToF) is the most common form of cyanotic congenital heart disease and is a major cause of significant morbidity and mortality. VANGL2 is a critical gene in the planar cell polarity pathway that plays an important role in the development of the heart. This study investigates the methylation status of the promoter region of VANGL2 and the expression pattern of VANGL2 in cardiac tissue.

METHODS

The promoter region of VANGL2 was sequenced in 200 ToF patients and 400 control subjects. Methylation levels were measured in four regions of the VANGL2 promoter (B1-1: -282 bp ∼ -117 bp, B1-2: -117 bp ∼ 41 bp, B2: 8 bp ∼ 157 bp, B3: 132 bp ∼ 401 bp) by bisulfite sequencing PCR in the right ventricular outflow tract of the myocardium. Quantitative real-time PCR and immunohistochemistry were used to detect the mRNA and protein expression levels, respectively.

RESULTS

No mutations were found in the promoter region, but two SNPs (rs11582932 T>G, rs11265385 T>G) were found in ToF patients and controls with similar frequencies (p>0.05). The overall methylation status of the VANGL2 promoter was significantly higher in ToF patients than in controls (p=0.0234). Specifically, the methylation levels of regions B1-1 and B3 were significantly higher in ToF patients (p=0.0042, p=0.0418). Both the VANGL2 mRNA and protein levels were significantly lower in ToF patients than in controls (p<0.05).

CONCLUSION

The aberrant VANGL2 promoter methylation and the decreased gene expression in ToF patients may provide important clues for the development of ToF.

摘要

背景

法洛四联症（ToF）是最常见的青紫型先天性心脏病，是导致严重发病和死亡的主要原因。VANGL2是平面细胞极性通路中的关键基因，在心脏发育中起重要作用。本研究调查了VANGL2启动子区域的甲基化状态以及VANGL2在心脏组织中的表达模式。

方法

对200例ToF患者和400例对照者的VANGL2启动子区域进行测序。通过亚硫酸氢盐测序PCR在心肌右心室流出道中测量VANGL2启动子四个区域（B1-1：-282 bp ∼ -117 bp，B1-2：-117 bp ∼ 41 bp，B2：8 bp ∼ 157 bp，B3：132 bp ∼ 401 bp）的甲基化水平。分别使用定量实时PCR和免疫组织化学检测mRNA和蛋白表达水平。

结果

启动子区域未发现突变，但在ToF患者和对照者中发现了两个单核苷酸多态性（rs11582932 T>G，rs11265385 T>G），频率相似（p>0.05）。ToF患者中VANGL2启动子的总体甲基化状态显著高于对照者（p=0.0234）。具体而言，ToF患者中B1-1和B3区域的甲基化水平显著更高（p=0.0042，p=0.0418）。ToF患者中VANGL2的mRNA和蛋白水平均显著低于对照者（p<0.05）。