法洛四联症患者心肌组织基因甲基化变异及机制研究
发布时间：2018-03-06 00:33
本文选题：先天性心脏病 切入点：圆锥动脉干畸形 出处：《复旦大学》2012年博士论文 论文类型：学位论文
【摘要】：先天性心脏病(congenital heart disease),简称先心病,是指胚胎发育过程中心脏和血管结构发生异常的一类疾病。近年来出生缺陷调查资料显示,先心病已跃居我国出生缺陷畸形首位,发病率占新生活产婴儿的6‰～10‰。圆锥动脉干畸形(conotruncal defects,CTD)是先心病中最为严重的一种类型,约占先心病的15～20%,临床上表现为法洛四联症、肺动脉闭锁、大动脉转位、右室双出口、永存动脉干等多种复杂心血管畸形,是造成我国婴儿和新生儿死亡最主要的原因之一。法洛四联症(tetralogy of Fallot, TOF)是圆锥动脉干畸形中最常见的类型,约占整个先心病的10%,它具有四个明显的特征：室间隔缺损、主动脉骑跨、肺动脉狭窄和右心室肥厚。尽管目前的手术矫正效果良好,仍然有大约0.5%到6%的疾病患者在术后死于各种并发症等,确切病因尚不明确。研究证实,环境因素和遗传因素均可能参与先心病的发生。环境因素对疾病发生的影响,主要表现在表观遗传学层面。但到目前为止,表观遗传学与先心病的关系鲜有报道。 本课题以法洛四联症患者右室流出道梗阻组织样本为研究对象,以法医尸检非心源性死亡心脏相应部位组织为对照,研究患者心肌组织DNA甲基化的变化情况,评估DNA甲基化状态与疾病发生的相关性,建立法洛四联症DNA甲基化特征谱,探讨法洛四联症患者的表观遗传学改变,为该疾病的病因研究和发病机制探讨提供新的思路和线索。 本课题研究内容分为三个部分： 第一部分,对法洛四联症患者心肌组织整体甲基化水平进行研究,检测患者基因组整体甲基化状态,分析整体甲基化变化与疾病发生风险的相关性。LINE-1基因约占人整体基因组的17～25%,该基因的甲基化水平在一定程度上反映了整体基因组甲基化状态。本研究通过MassArray EpiTYPER甲基化分析平台研究了LINE-1基因在32例病例组织和15例正常对照心肌组织中的甲基化水平,通过软件对结果进行分析。 第二部分,研究法洛四联症患者心脏组织中MBD2(methyl-CpG binding protein2)和三种DNA甲基化转移酶(DNA methyltransferases, DNMT s)的表达,分析这些基因表达之间的关系以及对整体甲基化水平的影响。首先从48例病例心脏组织和16例正常对照组织中提取总RNA,验证RNA的完整性；其次,以QRT-PCR方法对MBD2和三种DNA甲基化转移酶mRNA表达水平进行检测；最后,通过软件对实验结果进行分析。 第三部分,整理出已报道的与先心病发生密切相关的71个基因,通过对基因启动子区进行分析(http://genome.ucsc.edu/),设计了113个扩增子,这些扩增子分布在基因启动子区的CpG岛,CpG岛岸和非CpG岛的转录起始区(http://www.epidesigner.com/)。利用MassArray EpiTYPER平台对113个扩增子甲基化状态进行检测,通过软件对结果进行分析,获得具有明显甲基化差异的基因,然后在更大样本量中对这些基因的甲基化状态进行进一步的验证,建立法洛四联症患者心肌组织特异性的甲基化谱；以QRT-PCR方法检测有明显甲基化差异基因的mRNA水平,分析基因的甲基化状态对其mRNA表达水平的影响。 研究结果 第一部分：通过对LINE-1甲基化状态分析,发现法洛四联症患者右室流出道梗阻组织整体甲基化水平显著低于正常对照组(59.70%vs57.95%,p=0.0021)。该疾病的发生风险随着甲基化程度的降低而显著增高(OR=14.7,95%CI:1.8-117.7,p=0.014)。被检者工作特征曲线分析(ROC)证实了以基因组总体甲基化水平预测TOE的准确性(AUC=0.78,95%CI:0.65-0.91;p=0.002).通过对年龄和性别与LINE-1甲基化状态的相关性分析,发现整体基因组甲基化水平与年龄和性别没有相关性。 第二部分：DNA甲基转移酶和MBD2基因的mRNA水平在法洛四联症患者心肌组织中的表达水平均显著低于正常对照组(p0.001),尤其是DNMT1和DNMT3B降低最明显。两两相关性分析结果显示,在正常组中仅有DNMT1与DNMT3A表现出正相关(r=0.718,p=0.002),而整体基因组甲基化状态与DNA甲基转移酶和MBD2基因表达不相关；在TOF病例组中,各DNA甲基转移酶和MBD2基因表达之间彼此均表现出明显的正相关(p0.05),整体基因组甲基化水平与MBD2之间表现为负相关(r=-0.579,p=0.005)。 第三部分：通过MassArray EpiTYPER技术平台,在预实验中检测了10例患者心脏组织和6例正常对照组织中71个基因(113个扩增子)的甲基化状态。结果表明,在113个扩增子中有26个扩增子(涉及到26个基因)表现出显著的差异性。其中17个基因(CFC1B, DVL2, EGFR, EDNRA, EVC2, GJA5, HAND1, HAS2, HSPG2, MED13L, NFATC1, NKX2-5, NFATC2, PAX3, TBX5, TEK, ZFPM2)的甲基化表现为上调,9个基因(EDN1, HOXA3, MYH6, NR2F2, NRG1, NRP1, PDGFRA, SLC2A10, TBX20)的甲基化为下调。这26个基因中,所检测的甲基化区域位于CpG岛的基因包括：DVL2, EDN1, EGFR, EVC2, HAND1, HAS2, HSPG2, NKX2-5, NRG1, NFATC2, PAX3, PDGFRA, SLC2A10, TBX20;位于CpG岛岸的基因包括：EDNRA, MED13L, NFATC1, NR2F2, NRP1, TBX5, ZFPM2;位于转录起始位点区域(无CpG岛)的基因包括：CFC1B, GJA5, HOXA3, MYH6,TEK。综合考虑上述基因在心脏发育中的作用,以及其甲基化差异的程度,我们最后明确了11个候选基因(CFC1B, EGFR, EVC2, GJA5, HAND1, NKX2-5, NR2F2, NFATC2, TBX5, TBX20, ZFPM2),其中包括9个甲基化上调的基因和2个下调的基因。通过扩大样本量(42病例组织和22例正常对照组织),对上述结果进行了验证,结果与预实验相符。同时,我们检测了这11个候选基因的mRNA水平,结果表明在病例组织中,除HAND1基因表达上调外,其它基因的表达均显著低于正常组p0.05)。 总之,在本研究中,我们发现法洛四联症患者右心室流出道梗阻部位心肌组织的基因组整体甲基化水平显著低于正常组,同时,该疾病的发生风险随着甲基化程度的降低而显著增加；甲基化转移酶DNMT1和DNMT3B表达水平的显著降低可能与疾病的发生有一定关系；明确了法洛四联症候选基因DNA甲基化特征谱,为法洛四联症的病因和发病机制研究提供了新的表观遗传学思路。
[Abstract]:Congenital heart disease (congenital heart disease), fCHD, refers to a kind of disease with abnormal embryonic development of heart and vascular structure in the process. In recent years, the data of birth defect survey, congenital heart disease has become our birth defects first, the incidence rate of 6 per thousand births to new life accounted for 10 per thousand. Conotruncal defects (conotruncal, defects, CTD) in congenital heart disease is the most serious type, about 15 to 20% hand heart disease, clinical manifestations of tetralogy of Fallot, pulmonary atresia, transposition of the great arteries, double outlet of right ventricle, truncus artery and other complex cardiovascular malformations, is one of the reasons China's infant and neonatal death. The main tetralogy of Fallot (tetralogy of Fallot, TOF) is the most common type of conotruncal malformation, congenital heart disease accounts for about 10%, it has four obvious features: ventricular septal defect, the main Riding the artery, pulmonary artery stenosis and right ventricular hypertrophy. Although the surgery effect is good, there are still about 0.5% to 6% of the patients died of various complications after operation, the exact cause is not clear. The research confirmed that both environmental and genetic factors may occur in congenital heart disease. The effects of environmental factors on the disease the main performance at the epigenetic level. But so far, table relationships rarely reported epigenetic and congenital heart disease.
This topic in tetralogy of Fallot patients with right ventricular outflow tract obstruction tissue samples as the research object, the forensic autopsy of non cardiogenic cardiac death corresponding parts of tissue as a control, the change of myocardial tissue in patients with DNA methylation, DNA methylation related assessment and diseases, the establishment of tetralogy of Fallot DNA methylation profiles patients with tetralogy of Fallot, epigenetic changes, and provide new clue for the study on etiology and pathogenesis of the disease is discussed.
The research content of this topic is divided into three parts:
The first part of the overall level of methylation in myocardial tissue of patients with tetralogy of Fallot, detection of patients with whole genome methylation status, analysis of the relationship between.LINE-1 gene methylation changes and overall risk of disease accounted for about 17 of the whole human genome to 25%, methylation of this gene reflects the whole genome methylation status in a certain degree the study by MassArray EpiTYPER. The methylation of LINE-1 gene methylation level in 32 cases and 15 cases of normal tissue in myocardial tissue analysis platform, based on the results of the software analysis.
The second part, the MBD2 of patients with tetralogy of Fallot in heart tissue (methyl-CpG binding protein2) and three DNA methyltransferase (DNA methyltransferases, DNMT s) expression, analysis of the relationship between the expression of these genes and their impact on the overall level of methylation. Total RNA was extracted from the first 48 cases and 16 cases of heart tissue the normal control group, verify the integrity of RNA; secondly, the method of QRT-PCR in MBD2 and three DNA were detected the expression level of methyl transferase mRNA; finally, the experiment results are analyzed.
The third part, sort out has been reported with CHD is closely related to 71 genes, through the analysis of the promoter region of the gene (http://genome.ucsc.edu/), designed 113 amplicons, these amplicons located in the promoter region of CpG Island, CpG island and the transcription initiation site of non CpG Island (http://www.epidesigner.com/ of the 113 amplicons). The methylation status was detected by MassArray EpiTYPER platform, based on the results of analysis software, with obvious differences in methylation of the gene, and then based on these methylation status because of further validation in a larger sample, establish the methylation profile of myocardial tissue in patients with tetralogy of Fallot. Specific to QRT-PCR; detection methods have significant differences in methylation levels of mRNA gene, the effect of methylation analysis of gene expression of mRNA.
Research results
The first part: through the analysis of the methylation status of LINE-1, found in patients with tetralogy of Fallot right ventricular outflow tract obstruction in the overall level of methylation was significantly lower than the normal control group (59.70%vs57.95%, p=0.0021). The risk of the disease with the methylation level decreased significantly increased (OR=14.7,95%CI:1.8-117.7, p=0.014). By analysis the work characteristics the curve (ROC) confirmed the prediction accuracy of TOE in whole genome methylation level (AUC=0.78,95%CI:0.65-0.91; p=0.002). The correlation of age and gender and LINE-1 methylation analysis, found that the overall methylation level was not correlated with age and sex.
The second part: the expression of DNA methyltransferase and MBD2 gene mRNA level in myocardial tissue of patients with tetralogy of Fallot were significantly lower than that in normal control group (p0.001), especially DNMT1 and DNMT3B were the lowest. 22 correlation analysis showed that in the normal group, only DNMT1 and DNMT3A showed a positive correlation (r=0.718. P=0.002), and whole genome methylation status and DNA methyltransferase and MBD2 gene expression are not related; in the case of TOF group, the DNA methyltransferase and MBD2 gene expression between each other showed a significant positive correlation (P0.05), the whole genome methylation level and MBD2 showed negative correlation (r=-0.579. P=0.005).
绗笁閮ㄥ垎锛氶，
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