本文選題：先天性心臟病　切入點(diǎn)：圓錐動(dòng)脈干畸形　出處：《復(fù)旦大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：先天性心臟病(congenital heart disease),簡(jiǎn)稱先心病,是指胚胎發(fā)育過(guò)程中心臟和血管結(jié)構(gòu)發(fā)生異常的一類疾病。近年來(lái)出生缺陷調(diào)查資料顯示,先心病已躍居我國(guó)出生缺陷畸形首位,發(fā)病率占新生活產(chǎn)嬰兒的6‰～10‰。圓錐動(dòng)脈干畸形(conotruncal defects,CTD)是先心病中最為嚴(yán)重的一種類型,約占先心病的15～20%,臨床上表現(xiàn)為法洛四聯(lián)癥、肺動(dòng)脈閉鎖、大動(dòng)脈轉(zhuǎn)位、右室雙出口、永存動(dòng)脈干等多種復(fù)雜心血管畸形,是造成我國(guó)嬰兒和新生兒死亡最主要的原因之一。法洛四聯(lián)癥(tetralogy of Fallot, TOF)是圓錐動(dòng)脈干畸形中最常見(jiàn)的類型,約占整個(gè)先心病的10%,它具有四個(gè)明顯的特征：室間隔缺損、主動(dòng)脈騎跨、肺動(dòng)脈狹窄和右心室肥厚。盡管目前的手術(shù)矯正效果良好,仍然有大約0.5%到6%的疾病患者在術(shù)后死于各種并發(fā)癥等,確切病因尚不明確。研究證實(shí),環(huán)境因素和遺傳因素均可能參與先心病的發(fā)生。環(huán)境因素對(duì)疾病發(fā)生的影響,主要表現(xiàn)在表觀遺傳學(xué)層面。但到目前為止,表觀遺傳學(xué)與先心病的關(guān)系鮮有報(bào)道。 本課題以法洛四聯(lián)癥患者右室流出道梗阻組織樣本為研究對(duì)象,以法醫(yī)尸檢非心源性死亡心臟相應(yīng)部位組織為對(duì)照,研究患者心肌組織DNA甲基化的變化情況,評(píng)估DNA甲基化狀態(tài)與疾病發(fā)生的相關(guān)性,建立法洛四聯(lián)癥DNA甲基化特征譜,探討法洛四聯(lián)癥患者的表觀遺傳學(xué)改變,為該疾病的病因研究和發(fā)病機(jī)制探討提供新的思路和線索。 本課題研究?jī)?nèi)容分為三個(gè)部分： 第一部分,對(duì)法洛四聯(lián)癥患者心肌組織整體甲基化水平進(jìn)行研究,檢測(cè)患者基因組整體甲基化狀態(tài),分析整體甲基化變化與疾病發(fā)生風(fēng)險(xiǎn)的相關(guān)性。LINE-1基因約占人整體基因組的17～25%,該基因的甲基化水平在一定程度上反映了整體基因組甲基化狀態(tài)。本研究通過(guò)MassArray EpiTYPER甲基化分析平臺(tái)研究了LINE-1基因在32例病例組織和15例正常對(duì)照心肌組織中的甲基化水平,通過(guò)軟件對(duì)結(jié)果進(jìn)行分析。 第二部分,研究法洛四聯(lián)癥患者心臟組織中MBD2(methyl-CpG binding protein2)和三種DNA甲基化轉(zhuǎn)移酶(DNA methyltransferases, DNMT s)的表達(dá),分析這些基因表達(dá)之間的關(guān)系以及對(duì)整體甲基化水平的影響。首先從48例病例心臟組織和16例正常對(duì)照組織中提取總RNA,驗(yàn)證RNA的完整性；其次,以QRT-PCR方法對(duì)MBD2和三種DNA甲基化轉(zhuǎn)移酶mRNA表達(dá)水平進(jìn)行檢測(cè)；最后,通過(guò)軟件對(duì)實(shí)驗(yàn)結(jié)果進(jìn)行分析。 第三部分,整理出已報(bào)道的與先心病發(fā)生密切相關(guān)的71個(gè)基因,通過(guò)對(duì)基因啟動(dòng)子區(qū)進(jìn)行分析(http://genome.ucsc.edu/),設(shè)計(jì)了113個(gè)擴(kuò)增子,這些擴(kuò)增子分布在基因啟動(dòng)子區(qū)的CpG島,CpG島岸和非CpG島的轉(zhuǎn)錄起始區(qū)(http://www.epidesigner.com/)。利用MassArray EpiTYPER平臺(tái)對(duì)113個(gè)擴(kuò)增子甲基化狀態(tài)進(jìn)行檢測(cè),通過(guò)軟件對(duì)結(jié)果進(jìn)行分析,獲得具有明顯甲基化差異的基因,然后在更大樣本量中對(duì)這些基因的甲基化狀態(tài)進(jìn)行進(jìn)一步的驗(yàn)證,建立法洛四聯(lián)癥患者心肌組織特異性的甲基化譜；以QRT-PCR方法檢測(cè)有明顯甲基化差異基因的mRNA水平,分析基因的甲基化狀態(tài)對(duì)其mRNA表達(dá)水平的影響。 研究結(jié)果 第一部分：通過(guò)對(duì)LINE-1甲基化狀態(tài)分析,發(fā)現(xiàn)法洛四聯(lián)癥患者右室流出道梗阻組織整體甲基化水平顯著低于正常對(duì)照組(59.70%vs57.95%,p=0.0021)。該疾病的發(fā)生風(fēng)險(xiǎn)隨著甲基化程度的降低而顯著增高(OR=14.7,95%CI:1.8-117.7,p=0.014)。被檢者工作特征曲線分析(ROC)證實(shí)了以基因組總體甲基化水平預(yù)測(cè)TOE的準(zhǔn)確性(AUC=0.78,95%CI:0.65-0.91;p=0.002).通過(guò)對(duì)年齡和性別與LINE-1甲基化狀態(tài)的相關(guān)性分析,發(fā)現(xiàn)整體基因組甲基化水平與年齡和性別沒(méi)有相關(guān)性。 第二部分：DNA甲基轉(zhuǎn)移酶和MBD2基因的mRNA水平在法洛四聯(lián)癥患者心肌組織中的表達(dá)水平均顯著低于正常對(duì)照組(p0.001),尤其是DNMT1和DNMT3B降低最明顯。兩兩相關(guān)性分析結(jié)果顯示,在正常組中僅有DNMT1與DNMT3A表現(xiàn)出正相關(guān)(r=0.718,p=0.002),而整體基因組甲基化狀態(tài)與DNA甲基轉(zhuǎn)移酶和MBD2基因表達(dá)不相關(guān)；在TOF病例組中,各DNA甲基轉(zhuǎn)移酶和MBD2基因表達(dá)之間彼此均表現(xiàn)出明顯的正相關(guān)(p0.05),整體基因組甲基化水平與MBD2之間表現(xiàn)為負(fù)相關(guān)(r=-0.579,p=0.005)。 第三部分：通過(guò)MassArray EpiTYPER技術(shù)平臺(tái),在預(yù)實(shí)驗(yàn)中檢測(cè)了10例患者心臟組織和6例正常對(duì)照組織中71個(gè)基因(113個(gè)擴(kuò)增子)的甲基化狀態(tài)。結(jié)果表明,在113個(gè)擴(kuò)增子中有26個(gè)擴(kuò)增子(涉及到26個(gè)基因)表現(xiàn)出顯著的差異性。其中17個(gè)基因(CFC1B, DVL2, EGFR, EDNRA, EVC2, GJA5, HAND1, HAS2, HSPG2, MED13L, NFATC1, NKX2-5, NFATC2, PAX3, TBX5, TEK, ZFPM2)的甲基化表現(xiàn)為上調(diào),9個(gè)基因(EDN1, HOXA3, MYH6, NR2F2, NRG1, NRP1, PDGFRA, SLC2A10, TBX20)的甲基化為下調(diào)。這26個(gè)基因中,所檢測(cè)的甲基化區(qū)域位于CpG島的基因包括：DVL2, EDN1, EGFR, EVC2, HAND1, HAS2, HSPG2, NKX2-5, NRG1, NFATC2, PAX3, PDGFRA, SLC2A10, TBX20;位于CpG島岸的基因包括：EDNRA, MED13L, NFATC1, NR2F2, NRP1, TBX5, ZFPM2;位于轉(zhuǎn)錄起始位點(diǎn)區(qū)域(無(wú)CpG島)的基因包括：CFC1B, GJA5, HOXA3, MYH6,TEK。綜合考慮上述基因在心臟發(fā)育中的作用,以及其甲基化差異的程度,我們最后明確了11個(gè)候選基因(CFC1B, EGFR, EVC2, GJA5, HAND1, NKX2-5, NR2F2, NFATC2, TBX5, TBX20, ZFPM2),其中包括9個(gè)甲基化上調(diào)的基因和2個(gè)下調(diào)的基因。通過(guò)擴(kuò)大樣本量(42病例組織和22例正常對(duì)照組織),對(duì)上述結(jié)果進(jìn)行了驗(yàn)證,結(jié)果與預(yù)實(shí)驗(yàn)相符。同時(shí),我們檢測(cè)了這11個(gè)候選基因的mRNA水平,結(jié)果表明在病例組織中,除HAND1基因表達(dá)上調(diào)外,其它基因的表達(dá)均顯著低于正常組p0.05)。 總之,在本研究中,我們發(fā)現(xiàn)法洛四聯(lián)癥患者右心室流出道梗阻部位心肌組織的基因組整體甲基化水平顯著低于正常組,同時(shí),該疾病的發(fā)生風(fēng)險(xiǎn)隨著甲基化程度的降低而顯著增加；甲基化轉(zhuǎn)移酶DNMT1和DNMT3B表達(dá)水平的顯著降低可能與疾病的發(fā)生有一定關(guān)系；明確了法洛四聯(lián)癥候選基因DNA甲基化特征譜,為法洛四聯(lián)癥的病因和發(fā)病機(jī)制研究提供了新的表觀遺傳學(xué)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).
絎笁閮ㄥ垎錛氶，

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