本文關(guān)鍵詞： 右心發(fā)育不良綜合征 第二生心區(qū) 轉(zhuǎn)錄因子 先天性心臟病 單核苷酸變異　出處：《北京協(xié)和醫(yī)學(xué)院》2013年博士論文　論文類型：學(xué)位論文

【摘要】：目的：右心發(fā)育不良綜合征(Hypoplastic Right Heart Syndrome, HRHS)是以不同程度的右心室發(fā)育不良及三尖瓣和(或)肺動(dòng)脈瓣的發(fā)育不良(閉鎖或狹窄)為特征的一組先天性心臟病(Congenital Heart Disease, CHD);第二生心區(qū)(Second Heart Field, SHF)做為右室發(fā)育的細(xì)胞來源,其相關(guān)基因在胚胎期調(diào)控右室及流出道發(fā)育中發(fā)揮重要作用�；蚬こ虅�(dòng)物實(shí)驗(yàn)顯示,敲除SHF轉(zhuǎn)錄因子,存活動(dòng)物表型與HRHS一致。故提出假設(shè),SHF發(fā)育相關(guān)基因發(fā)生突變可能導(dǎo)致HRHS的發(fā)生；本實(shí)驗(yàn)計(jì)劃通過候選基因二代測(cè)序的方法探尋SHF發(fā)育相關(guān)基因變異與HRHS發(fā)生的關(guān)系。 方法：本實(shí)驗(yàn)收集在我院接受手術(shù)治療的31例HRHS患兒做為病例組,及383例單純冠心病病人(心臟超聲排除結(jié)構(gòu)性心臟病)做為對(duì)照。外周血提取DNA。取第一批16個(gè)HRHS患者DNA對(duì)17個(gè)基因Hand2, Smydl, Mef2c, Tbx20, Foxhl, Isletl, Gata4, Gata5, Gata6, Nkx2-5, Foxcl, Foxc2, Foxa2, Tbxl, Shh, Fgf8,Fgf10全長(zhǎng)進(jìn)行PCR擴(kuò)增,擴(kuò)增產(chǎn)物進(jìn)行Ion Torrent二代測(cè)序,將測(cè)序資料與千人基因組中1092個(gè)正常人的信息比對(duì)并生物信息學(xué)分析；選取基因功能區(qū)的變異位點(diǎn)進(jìn)行sanger測(cè)序驗(yàn)證,將驗(yàn)證位點(diǎn)在31例病例及383例對(duì)照中進(jìn)行Sequenom MassARRAY(?)SNP檢測(cè)。 結(jié)果：將17個(gè)基因的exon, promotor, enhancer binding cite,3'UTR及5'UTR等功能區(qū)做為一個(gè)總體進(jìn)行分析發(fā)現(xiàn),Foxa2, Nkx2-5, Hand2, Tbxl, Fgf8, Isl-1, Foxc2, Gata6, Mef2c, Tbx20等10個(gè)基因低頻變異(MAF≤5%)出現(xiàn)頻率病例組明顯高于千人基因組(p0.05)；其中Foxa2(p=3.16×10-16), Nkx2-5(p=2.91×10-8), Hand2(p=2.48×10-6), Tbx1(p=2.48×10-6), Fgf8(p=1.96×10-4), Isl-1(p=1.96×10-4), Foxc2(p=0.014), Gata6(p=0.014)這8個(gè)基因功能區(qū)的低頻變異在1000Genomes中為0,而在HRHS組卻有不同程度的低頻變異。Mef2c的功能區(qū)低頻變異較無功能區(qū)低頻變異能夠使HRHS的發(fā)病風(fēng)險(xiǎn)增加22倍(OR值為23.49,95%CI為8.27-66.69,p=7.8×10-15),而Tbx20的低頻變異增加HRHS發(fā)病風(fēng)險(xiǎn)15倍(OR值為16.54,95%CI為4.27-64.11,p=8.3×10-6)。選取19個(gè)變異位點(diǎn)并進(jìn)行Sanger測(cè)序驗(yàn)證,位點(diǎn)均存在,進(jìn)一步質(zhì)譜分析發(fā)現(xiàn)常見變異Gata5:rs6061550CT攜帶TT純合突變者發(fā)生HRHS的風(fēng)險(xiǎn)是攜帶CC+CT的5.51倍(OR=5.51,95‰CI=2.55-11.89,p=2.02×10-6)。而Foxc2rs34221221AG若以野生純合子AA做為參照,則突變純合子GG可以增加HRHS的發(fā)病風(fēng)險(xiǎn)2.6倍(OR=3.6,95%CI=1.20-10.81,p=0.043)。 結(jié)論：第二生心區(qū)發(fā)育相關(guān)基因低頻變異和常見變異都在HRHS發(fā)生中發(fā)揮重要作用。Foxa2, Nkx2-5, Hand2, Tbxl, Fgf8, Isl-1, Foxc2, Gata6, Mef2c, Tbx20等10個(gè)基因功能區(qū)的低頻變異能夠顯著增加HRHS的發(fā)病風(fēng)險(xiǎn)；而Gata5及Foxc2.常見變異也能顯著增加HRHS風(fēng)險(xiǎn)。據(jù)此,我們推測(cè)HRHS的發(fā)生存在三種可能性,其一是SHF相關(guān)基因易感SNP在特定環(huán)境作用下導(dǎo)致了HRHS的發(fā)生；其二是SHF基因低頻變異引起的信號(hào)通路障礙導(dǎo)致了HRHS的發(fā)生；或者是鑲嵌模型,即常見變異增加了個(gè)體易感性的同時(shí),低頻變異導(dǎo)致了HRHS的發(fā)生。
[Abstract]:Objective: Hypoplastic Right Heart Syndrome (HRHS) is a group of congenital heart diseases characterized by different degrees of right ventricular dysplasia and tricuspid and / or pulmonary valve dysplasia (atresia or stenosis). The second Heart field (SHF) was used as a cell source for the development of the right ventricle. Its related genes play an important role in regulating the development of right ventricle and outflow tract at embryonic stage. Genetic engineering animal experiments show that knockout of SHF transcription factors, The phenotypes of surviving animals were consistent with those of HRHS. It was suggested that mutation of SHF development-related genes might lead to the occurrence of HRHS, and the relationship between mutation of SHF development-related genes and occurrence of HRHS was explored by the method of candidate gene second-generation sequencing. Methods: 31 patients with HRHS received surgical treatment in our hospital were selected as the case group. And 383 patients with coronary heart disease (heart ultrasound excluded structural heart disease) were used as controls. The first group of 16 patients with HRHS were selected for PCR amplification of 17 genes (Hand2, Smydl, Mef2c, Tbx20, Foxhl, Isletl, Gata4, Gata5, Gata6, Nkx2-5, Foxcll, Foxc2, Foxa2, Tbxl, Shh, Fgf8Fgf10). The amplified products were sequenced in the second generation of Ion Torrent. The sequence data were compared with the information of 1 092 normal persons in the human genome and the bioinformatics analysis was carried out. The mutation sites in the functional region of the gene were selected for sanger sequencing. Sequenom MassARRAYA was performed in 31 cases and 383 controls. SNP detection. Results: the functional regions of 17 genes, such as exon, promoter, enhancer binding citean3UTR and 5UUTR, were analyzed as a whole. It was found that the frequency of 10 genes, such as Foxa2, Nkx2-5, Hand2, Tbxl, Fgf8, Isl-1, Foxc2, Gata6, Mef2c, Tbx20 and so on, was significantly higher in the case group than in the case group. Among them, Foxa2(p=3.16 脳 10-16, Nkx2-5(p=2.91 脳 10-8, Hand2(p=2.48 脳 10-6, Tbx1(p=2.48 脳 10-6, Fgf8(p=1.96 脳 10-4, Isl-1(p=1.96 脳 10-4, Foxc2p0.014, Gata6P0. 014) the low frequency variation of the functional regions of Foxa2(p=3.16 脳 10-16, Nkx2-5(p=2.91 脳 10-8, Hand2(p=2.48 脳 10-6, Tbx1(p=2.48 脳 10-6, Fgf8(p=1.96 脳 10-4, Foxc2p0.014, Gata6P0. 014) was 0 in 1000 genomes, but in HRHS group, the low frequency variation of the functional region of Mef2c was higher than that of the non-functional region. The odds ratio of risk increase was 23.49 / 95 CI = 8.27-66.69 脳 10 ~ (-15), while the low frequency variation of Tbx20 increased the risk of HRHS by 15 times / r = 16.54.11 / 95 / CI = 4.27-64.11p ~ (8.3) 脳 10 ~ (-6). 19 mutation sites were selected and Sanger sequencing was performed. Further mass spectrometry analysis showed that the risk of developing HRHS in common variant Gata5:rs6061550CT carriers with TT homozygous mutant was 5.51 times of CC CT, and the risk of HRHS was 5.51 times higher than that of CC CT, and 2.02 脳 10 ~ (-6) Foxc2rs34221221AG with wild homozygote AA. The mutation homozygote GG could increase the risk of HRHS by 2.6 times. Conclusion: the low-frequency variation and common variation of the genes related to the development of the second heart region play an important role in the pathogenesis of HRHS. The low-frequency variation of 10 gene functional regions, I. E. Foxa2, Nkx2-5, Hand2, Tbxl, Fgf8, Isl-1, Foxc2, Gata6, Mef2cand Tbx20, can significantly increase the risk of HRHS. On the other hand, Gata5 and Foxc2.Common variation can also increase the risk of HRHS. Based on this, we speculate that there are three possibilities in the occurrence of HRHS. One is that the susceptible SNP of SHF related gene leads to the occurrence of HRHS under the specific environment. The other is that the signal pathway obstacle caused by low frequency variation of SHF gene leads to the occurrence of HRHS, or mosaic model, that is, common variation increases individual susceptibility, and low frequency variation leads to the occurrence of HRHS.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R725.4

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本文編號(hào)：1508272