HIRA基因在法洛四联症发病中作用机制初步研究

发布时间：2018-07-06 15:53

本文选题：先天性心脏病 + 圆锥动脉干畸形　；参考：《复旦大学》2014年硕士论文

【摘要】：先天性心脏病(简称先心病)是胎儿期心脏及大血管发育异常所致的先天性畸形,发病率在活产婴儿中占6%0-10%0。我国每年约出生15万先心病患儿,造成社会家庭严重的精神经济负担。青紫型先心病是先心病中最严重的类型,畸形复杂,可致顽固性低氧血症,未经治疗,早期死亡率高达80%。圆锥动脉干畸形占青紫型先心的60%-70%。法洛四联症是圆锥动脉干畸形最常见的类型,约占所有先心病的12%,基本病理改变为右室流出道梗阻、室间隔缺损、主动脉骑跨及右心室肥厚。研究其发病机制有利于预防、产前诊断及早期干预,对于减轻社会家庭精神、经济负担有重要意义。目前普遍接受的观点为先心病是内在遗传易感因素和外界环境致畸因素共同作用的结果。先心病候选基因的研究是目前先心病发病机制研究的热点。圆锥动脉干畸形是遗传综合症DiGeroge综合症(DGS)的主要心脏表现,研究发现,80%-100%DGS患者伴染色体22q11.2的单倍体微缺失。目前该区域已分离出20-30个候选基因,HIRA基因位于该区域,作为DGS的候选基因之一,受到广泛关注。在动物实验中,下调鸡胚HIRA在神经嵴细胞中表达,90%鸡胚发生永存动脉干畸形；下调小鼠HIRA基因表达,小鼠心脏袢化失败。法洛氏四联症作为最常见的圆锥动脉干畸形,也是22q11.2微缺失的常见表型。据此推测,HIRA基因可能是TOF的候选基因。但是人类TOF与HIRA的相关研究非常有限。本课题组前期通过TOF患儿右室流出道心肌HIRA基因表达研究发现HIRA基因在TOF患儿表达较正常降低。其表达降低的原因有待于进一步探讨。本课题拟对法四患儿HIRA基因进行编码区序列分析,探讨HIRA蛋白结构变化与TOF发生的关系：同时分析HIRA基因3’UTR区序列,探寻可能与3’UTR区结合的micro RNAs,找出TOF患儿右室流出道心肌HIRA表达降低的原因。初步解释HIRA基因在TOF发病中的作用机制。第一部分HIRA基因编码区序列分析目的：对中国TOF患儿中HIRA基因编码区进行序列分析,探讨HIRA基因编码区变异与TOF的关系。方法：收集TOF患儿外周血278例及年龄和性别与病例组尽量匹配的健康儿童外周血500例,提取基因组DNA,对HIRA基因全部编码区进行测序,突变位点病例组扩大到500例,对氨基酸的改变进行生物信息学预测,将野生型HIRA基因mRNA与突变后mRNA注入斑马鱼单细胞时期后观察斑马鱼表型进行突变功能验证。结果：1.在12外显子发现1个已报导的SNP c.G1339GA (rs150603624),其发生频率与NCBI dbSNP数据库报导的频率无统计学差异。2.第17外显子发现1个突变c.C2301CA,频率为1/500,用生物信息学网站预测发现其氨基酸改变对其蛋白质功能影响不大,在物种间保守性不高,推测该突变可能为良性突变。3.第23外显子发现1个突变c.C3026CT,频率为2/500,生物信息学网站预测发现其氨基酸改变对其蛋白质功能影响大,突变位点在不同特种间高度保守,推测该突变可能为致病性突变,突变mRNA注射后斑马鱼的心脏畸形发生率明显高于野生型。小结：HIRA基因23外显子突变c.C3026CT可能为致病性突变,可能导致先心病的发生。第二部分HIRA基因3'UTR序列分析及相关micro RNAs研究目的：对中国TOF患儿HIRA基因3'UTR进行序列分析,探讨HIRA基因3'UTR序列变异对调控其micro RNAs的影响。方法：收集TOF患儿外周血278例及年龄和性别与病例组尽量匹配的健康儿童外周血500例,提取基因组DNA,对HIRA3'UTR进行测序,用生物信息学网站预测SNP位点周围的micro RNAs,用双荧光素酶报告基因检测系统及real-timePCR检测micro RNA与HIRA 3'UTR的相互作用。结果：1.HIRA基因3'UTR SNP(rs:117447448)位点的分布在病例组和对照组中存在统计学差异(P=0.001)。2.在线预测SNP(rs:117447448)位点上游8 bp存在microRNA 328的结合位点。3.共同转染p-silencer+miR328质粒和pgl3-promoter+HIRA3'UTR质粒293细胞荧光素酶的活性(0.0125±0.0006)低于单独转染pgl3-promoter+HIRA3'UTR质粒的活性(0.01963±0.0038),其差异有统计学意义(P=0.0327)。转染p-silencer+miR328质粒的293T细胞HIRA基因的表达(1.03961±0.0772)较转染p-silencer空载质粒的表达(1.60865±0.2749)降低,差异有统计学意义(P=-0.037)。4.用SNP(rs:117447448)位点不同基因型的质粒与miR328共同转染293细胞,不同基因型的荧光素酶活性无统计学差异(P=0.38)小结HIRA基因3'UTR区rs:117447448SNP位点可能与TOF的发生相关：HIRA基因是micro RNA328的靶基因。rs:117447448SNP位点不是micro RNA328作用于HIRA基因的主要靶位。
[Abstract]:Congenital heart disease (congenital heart disease) is a congenital malformation caused by abnormal development of the heart and large blood vessels in the fetal stage. The incidence of congenital heart disease is 6%0-10%0. in live born infants, which is about 150 thousand children born with congenital heart disease every year in China, causing serious mental and economic burdens in social families. Intractable hypoxemia, untreated, the early death rate up to 80%. conical artery stem deformities 60%-70%. Fallot is the most common type of conical artery stem deformity, accounting for about 12% of all congenital heart disease. The basic pathological changes are right ventricular outflow tract obstruction, ventricular septal defect, aortic riding span and right ventricular hypertrophy. Its pathogenesis is beneficial to prevention, prenatal diagnosis and early intervention are of great significance for reducing social family spirit and economic burden. At present, it is widely accepted that congenital heart disease is the result of inherent genetic susceptibility factors and external environment teratogenic factors. The study of candidate genes of congenital heart disease is the study of the pathogenesis of congenital heart disease. Conical artery stem deformity is the main cardiac manifestation of genetic syndrome DiGeroge syndrome (DGS). The study found that 80%-100%DGS patients with chromosome 22q11.2 haploid microdeletion. 20-30 candidate genes have been isolated in this region. The HIRA gene is located in the region and is one of the candidate genes for DGS. In the experiment, the down regulated chicken embryo HIRA was expressed in the neural crest cells, and the 90% chicken embryo had persistent arteriosk deformity; the HIRA gene was downregulated and the heart loop of the mice failed. The Fallot tetralogy was the most common conical artery stem deformity and the common phenotype of 22q11.2 microdeletion. According to this, the HIRA gene may be a candidate gene of TOF. The related research of human TOF and HIRA is very limited. In the earlier period, we found that the expression of HIRA gene in the right ventricular outflow tract of children with TOF was lower than normal in the right ventricular outflow tract of children with the HIRA gene expression. The reason for the decrease of the expression of the HIRA gene is to be further discussed. This subject is intended to analyze the sequence of the coding region of the HIRA gene in four children of France and explore HIRA. The relationship between the changes of protein structure and the occurrence of TOF: simultaneous analysis of the 3 'UTR region of the HIRA gene and the micro RNAs that may be combined with the 3' UTR region to find out the decrease of HIRA expression in the right ventricular outflow tract in children with TOF. The preliminary interpretation of the mechanism of the HIRA gene in the pathogenesis of TOF. The sequence analysis of the coding region of HIRA gene in children with TOF was carried out to investigate the relationship between the mutation of the HIRA gene coding region and the TOF. Methods: 278 cases of peripheral blood of children with TOF and 500 cases of healthy children's peripheral blood which were matched with age and sex and case group were collected, the genomic DNA was extracted, the whole coding region of the HIRA gene was sequenced, and the case group of the mutation site was enlarged. In 500 cases, the changes of amino acids were predicted by bioinformatics. The mutant function of the wild type HIRA gene mRNA and the mutant mRNA was observed after the single cell period of zebrafish. Results: 1. the 1 reported SNP c.G1339GA (rs150603624) was found in exon 12, and the frequency of its occurrence and the NCBI dbSNP Database Report The frequency of.2. seventeenth exon found 1 mutations c.C2301CA, the frequency was 1/500, and the bioinformatics website predicted that the amino acid changes had little influence on its protein function, and the conservatism between species was not high. It was presumed that the mutation may be a benign mutation of.3. twenty-third exon 1 mutation c.C3026CT, the frequency of 2/500, birth rate. The prediction of the information science website found that the amino acid changes have great influence on its protein function, and the mutation site is highly conserved in different special rooms. It is suggested that the mutation may be a pathogenic mutation. The incidence of heart malformation of zebrafish after mRNA injection is obviously higher than that of the wild type. The mutation of HIRA gene 23 exon mutation c.C3026CT may be pathogenicity. Mutation, may lead to the occurrence of congenital heart disease. Second part HIRA gene 3'UTR sequence analysis and related micro RNAs research purposes: sequence analysis of HIRA gene 3'UTR in children with TOF in Chinese children, to explore the effect of the 3'UTR sequence variation of HIRA gene on the regulation of micro RNAs. Methods: 278 cases of peripheral blood and age, sex and case group were collected. 500 cases of peripheral blood of healthy children were matched, genomic DNA was extracted, HIRA3'UTR was sequenced, micro RNAs around SNP site was predicted by bioinformatics website, and the interaction between micro RNA and HIRA 3'UTR was detected by double luciferase reporter gene detection system and real-timePCR. The distribution in the case group and the control group was statistically different (P=0.001).2. on-line prediction of the SNP (rs:117447448) site upstream 8 bp existence microRNA 328 binding site.3. co transfection p-silencer+miR328 plasmids and pgl3-promoter+HIRA3'UTR plasmid 293 cell luciferase activity (0.0125 + 0.0006) lower than the single transfection of pgl3-promoter+HIRA. The activity of 3'UTR plasmid was (0.01963 + 0.0038), and the difference was statistically significant (P=0.0327). The expression of HIRA gene in 293T cells transfected with p-silencer+miR328 plasmid (1.03961 + 0.0772) was lower than that of transfected p-silencer empty plasmid (1.60865 + 0.2749), and the difference was statistically significant (P=-0.037).4. with the different genotype of SNP (rs:117447448) loci. The plasmids and miR328 co transfected 293 cells, and there was no significant difference in the luciferase activity of different genotypes (P=0.38). The rs:117447448SNP locus in 3'UTR region of HIRA gene may be related to the occurrence of TOF. The HIRA gene is the target gene of micro RNA328, which is not the main target of micro RNA328.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R725.4

【共引文献】