第二生心区发育相关基因变异在右心发育不良综合征发生中的作用

发布时间：2018-02-13 13:29

本文关键词： 右心发育不良综合征第二生心区转录因子先天性心脏病单核苷酸变异　出处：《北京协和医学院》2013年博士论文　论文类型：学位论文

【摘要】：目的：右心发育不良综合征(Hypoplastic Right Heart Syndrome, HRHS)是以不同程度的右心室发育不良及三尖瓣和(或)肺动脉瓣的发育不良(闭锁或狭窄)为特征的一组先天性心脏病(Congenital Heart Disease, CHD);第二生心区(Second Heart Field, SHF)做为右室发育的细胞来源,其相关基因在胚胎期调控右室及流出道发育中发挥重要作用。基因工程动物实验显示,敲除SHF转录因子,存活动物表型与HRHS一致。故提出假设,SHF发育相关基因发生突变可能导致HRHS的发生；本实验计划通过候选基因二代测序的方法探寻SHF发育相关基因变异与HRHS发生的关系。方法：本实验收集在我院接受手术治疗的31例HRHS患儿做为病例组,及383例单纯冠心病病人(心脏超声排除结构性心脏病)做为对照。外周血提取DNA。取第一批16个HRHS患者DNA对17个基因Hand2, Smydl, Mef2c, Tbx20, Foxhl, Isletl, Gata4, Gata5, Gata6, Nkx2-5, Foxcl, Foxc2, Foxa2, Tbxl, Shh, Fgf8,Fgf10全长进行PCR扩增,扩增产物进行Ion Torrent二代测序,将测序资料与千人基因组中1092个正常人的信息比对并生物信息学分析；选取基因功能区的变异位点进行sanger测序验证,将验证位点在31例病例及383例对照中进行Sequenom MassARRAY(?)SNP检测。结果：将17个基因的exon, promotor, enhancer binding cite,3'UTR及5'UTR等功能区做为一个总体进行分析发现,Foxa2, Nkx2-5, Hand2, Tbxl, Fgf8, Isl-1, Foxc2, Gata6, Mef2c, Tbx20等10个基因低频变异(MAF≤5%)出现频率病例组明显高于千人基因组(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)这8个基因功能区的低频变异在1000Genomes中为0,而在HRHS组却有不同程度的低频变异。Mef2c的功能区低频变异较无功能区低频变异能够使HRHS的发病风险增加22倍(OR值为23.49,95%CI为8.27-66.69,p=7.8×10-15),而Tbx20的低频变异增加HRHS发病风险15倍(OR值为16.54,95%CI为4.27-64.11,p=8.3×10-6)。选取19个变异位点并进行Sanger测序验证,位点均存在,进一步质谱分析发现常见变异Gata5:rs6061550CT携带TT纯合突变者发生HRHS的风险是携带CC+CT的5.51倍(OR=5.51,95‰CI=2.55-11.89,p=2.02×10-6)。而Foxc2rs34221221AG若以野生纯合子AA做为参照,则突变纯合子GG可以增加HRHS的发病风险2.6倍(OR=3.6,95%CI=1.20-10.81,p=0.043)。结论：第二生心区发育相关基因低频变异和常见变异都在HRHS发生中发挥重要作用。Foxa2, Nkx2-5, Hand2, Tbxl, Fgf8, Isl-1, Foxc2, Gata6, Mef2c, Tbx20等10个基因功能区的低频变异能够显著增加HRHS的发病风险；而Gata5及Foxc2.常见变异也能显著增加HRHS风险。据此,我们推测HRHS的发生存在三种可能性,其一是SHF相关基因易感SNP在特定环境作用下导致了HRHS的发生；其二是SHF基因低频变异引起的信号通路障碍导致了HRHS的发生；或者是镶嵌模型,即常见变异增加了个体易感性的同时,低频变异导致了HRHS的发生。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R725.4

【共引文献】