云南省漾濞县先心病流行病学调查及散发房间隔缺损易感基因分析和突变研究

发布时间：2018-06-16 01:21

本文选题：先天性心脏病 + 房间隔缺损　；参考：《昆明医科大学》2016年博士论文

【摘要】：[研究背景和目的]CHD是一种最常见的先天性出生缺陷。既往研究表明：CHD的发病率存在区域和民族间差异。自2001年开始,昆明医科大学附属延安医院科研团队对云南省各地区进行CHD流行病学调研。经过15年的工作,我们调研的地区已经覆盖云南省全境,超过20万中小学在校学生纳入筛查范围。第一部分漾濞县流行病学调查属于整个系统流调的一个部分,旨在调查云南省大理漾濞彝族自治县3-16岁人群CHD患病率,并对相关因素进行分析,以了解该地区人群CHD发生现状和分布特点。结合国内外文献报道及我们第一部分的流行病学调查提示：ASD的发病率已经超过VSD和PDA成为CHD最常见的类型。到目前为止,大部分ASD的发生遗传学机制尚不明确。鉴于此,我们的第二部分拟采用全外显子测序技术结合个体化的生物信息学分析方法,从整个基因组层面探索ASD患者的遗传学背景,以寻找ASD新的易感基因和突变位点。[方法]第一部分：采用横断面流行病学调查方法,于2015年1月对云南省大理漾濞彝族自治县3-16岁在校学生共计9945人进行分层整群抽样分析。以问卷调查结合体格检查为初筛,对初筛可疑阳性的病例进行心脏彩色超声多普勒检查以明确诊断。第二部分：采集昆明医科大学附属延安医院心脏大血管外科行ASD修补术患者术中修剪下废弃房间隔组织5例,同时采集患者外周血作为自身对照。分别提取心肌来源和外周血来源的基因组DNA。将心肌来源的DNA作为WES样本,HiSeq2500二代测序仪进行WES。测序所得原始数据经转换和ANNOVAR软件注释得到所有外显子区域的SNV,对这些SNV进行数据库过滤(dbSNP、 1000GEMO、NHLBI)和个体化生物信息学分析后,优先筛选出部分稀有突变位点作为候选基因位点。使用Sanger测序法排除其中的假阳性位点后,剩余位点作为拟验证对象,Mass-Array技术将5‘例ASD样本外周血来源的DNA与同一患者心肌组织来源的DNA进行比对。将比对一致的位点使用Mass-Array在421例ASD患者和507例健康志愿者中进行验证。最后,使用siRNA基因干扰技术沉默H9C2心肌细胞HYDIN基因,采用qRT-PCR方法检测HYDIN siRNA对HYDIN基因的干扰效率。对成功沉默HYDIN的心肌细胞进行细胞迁移能力(划痕实验)、细胞凋亡情况和细胞周期分布情况的检测(]ACS检测),以明确HYDIN基因在H9C2心肌细胞中的作用。[结果]第一部分：通过体格检查,检出疑似病例共计505例,经心脏彩超确诊CHD患者共计79例,漾濞县CHD总患病率为7.94‰。其中,男性：7.17‰,女性：8.69‰;女性患病率高于男性,差异有统计学意义(P0.05)。漾濞县彝族患病率8.259‰,高于该县总体水平,差异有统计学意义(P0.05)。不同年龄段患病率：3-6岁：4.63‰,7-12岁6.45‰,13-16岁：11.11%o,组间比较差异有统计学意义(P0.05)。对漾濞县9个乡镇进行分别统计后发现：各乡镇患病率有差异,CHD患病率和当地GDP水平负相关；亚型分析后发现：ASD是漾濞县CHD的最常见类型。第二部分：5例ASD患者WES共得原始数据20.7GB,经转换和ANNOVAR软件注释得到外显子区域单核苷酸变异共计181762个。通过dbSNP、1000GEMO和NHLBI数据库过滤得到稀有突变713个,个体化生物信息学分析后优先选择25个候选基因的33个稀有突变位点进行Sanger测序法验证,排除假阳性位点1个。对剩余32个位点进行外周血和心肌组织来源的DNA的同一比对,结果显示：心肌组织来源的DNA和外周血来源的DNA 32个位点突变类型一致,未发现体细胞突变现象。使用Mass-Array对这32个位点进行临床样本验证,结果显示：TTN、HYDIN、ZFPM1、XPO1、FOXL2基因可能是新的ASD易感基因。MYH6基因23862952、XPO1基因61708404位点可能与部分ASD发生相关,FOXL2基因138665410位点(NM_023067_c.C155G:p.A52G)在房间隔缺损组突变频率高达25.9%,对照组未检测到该位点突变,统计学分析差异有统计学意义(P0.05)该突变可能与ASD发生高度相关；最后,通过siRNA干扰技术,我们沉默了H9C2心肌细胞中的HYDIN基因,对沉默HYDIN基因的H9C2细胞进行检测：划痕实验发现细胞迁移能力减弱；流式细胞仪检测细胞凋亡和细胞周期发现：沉默HYDIN基因的H9C2细胞停留在G1期的增加,S期减少,细胞凋亡增加。[结论]1.漾濞县CHD总患病率高于云南省平均水平,彝族CHD患病率高于其它民族；2.漾濞县男女CHD患病率有差异,女性多于男性；3.漾濞县各乡镇患病率有差异,患病率与当地经济水平负相关；4.漾濞县CHD发病率在近15年有逐年下降的趋势；5.ASD是漾濞县CHD的最常见类型；6.应用WES结合个体化后期生物信息学分析的方法寻找散发ASD易感基因是可行的；7.散发ASD遗(?)异质性强,基因突变谱范围广,大规模临床样本验证是必要的;8.体细胞突变可能不是ASD的发生原因；9.TTN、HYDIN、ZFPM1、XPO1、FOXL2可能是新发现的ASD易感基因；10.MYH6基因23862952、XPO1基因61708404位点的稀有突变可能与部分ASD发生相关；11.FOXL2基因138665410位点可能与ASD发生高度相关,该突变引起ASD的具体机制值得进一步研究。
[Abstract]:[background and purpose]CHD is one of the most common congenital birth defects. Previous studies have shown that the incidence of CHD has regional and ethnic differences. Since 2001, the Yanan hospital research team affiliated to the Kunming Medical University has conducted CHD epidemiological investigations in various regions of Yunnan province. After 15 years of work, our research area has already been investigated. Covering the entire territory of Yunnan Province, more than 200 thousand primary and secondary school students were included in the screening range. The first part of the epidemiological survey in Yangbi county belongs to a part of the whole system flow. The purpose is to investigate the prevalence of CHD in the Yangbi Yi Autonomous County, Dali, Yunnan Province, and to analyze the related factors in order to understand the status of CHD in the population in this area and to understand the status of the population in this area. Distribution characteristics. Combined with domestic and foreign literature and our epidemiological survey in the first part, the incidence of ASD has exceeded VSD and PDA as the most common type of CHD. So far, most of the genetic mechanisms of ASD are not yet clear. In view of this, our second part is intended to be combined with exons sequencing and individualization. The bioinformatics analysis method was used to explore the genetic background of ASD patients from the whole genome level in order to find new susceptibility genes and mutation sites of ASD. [method] Part 1: a total of 9945 students at 3-16 years old in Yangbi Yi Autonomous County of Dali, Yunnan Province, were stratified by cross-sectional epidemiological survey method in January 2015. Group sampling analysis. With a questionnaire survey combined with physical examination as the initial screening, color ultrasound Doppler examination for suspicious positive cases was made to make a clear diagnosis. Second part: collect 5 cases of discarded atrial septum under surgical trimming of ASD repair in Yanan Hospital of Yanan hospital, affiliated to Kunming Medical University, and collect the patients at the same time. The genomic DNA. extracted from the source of cardiac muscle and the source of peripheral blood from the peripheral blood was used as the WES sample, and the DNA of the HiSeq2500 two generation sequencer was transformed by WES. sequencing and the ANNOVAR software was annotated to get all the exons SNV, and the SNV was filtered (dbSNP, 1000GEMO,). After NHLBI) and individualized bioinformatics analysis, some rare mutation sites were selected as candidate loci. Sanger sequencing was used to exclude the false positives and the remaining sites were used as the target. The Mass-Array technique was 5 'DNA of the peripheral blood from ASD samples from the DNA of the same patient's myocardial tissue origin. Mass-Array was used in 421 patients with ASD and 507 healthy volunteers. Finally, the siRNA gene interference technique was used to silence the HYDIN gene of H9C2 cardiomyocytes and the qRT-PCR method was used to detect the interference efficiency of HYDIN siRNA to the HYDIN gene. Force (scratch test), cell apoptosis and cell cycle distribution detection (]ACS detection) to identify the role of HYDIN gene in H9C2 cardiac myocytes. [results] Part 1: 505 cases of suspected cases were detected by physical examination, 79 cases were diagnosed by cardiac color Doppler, and the total prevalence rate of CHD in Yangbi County was 7.94 per thousand. Sex: 7.17 per thousand, female: 8.69 per thousand; the prevalence rate of female was higher than that of male, the difference was statistically significant (P0.05). The prevalence rate of Yi nationality in Yangbi county was 8.259 per thousand, higher than that of the county, the difference was statistically significant (P0.05). The prevalence rate of different age groups: 3-6 years, 4.63 per thousand, 7-12 years old and 6.45 per thousand, 13-16 years of age: 11.11%o, there was statistically significant difference between groups (P0. 05) 9 villages and towns in Yangbi county were statistically analyzed: the prevalence rate of each township was different, the incidence of CHD was negatively correlated with the local GDP level. After subtype analysis, it was found that ASD was the most common type of CHD in Yangbi county. The second part: 5 cases of ASD patients received the original data 20.7GB, and the exons area single was obtained by transfer and ANNOVAR software annotation. A total of 181762 nucleotide variations were obtained. 713 rare mutations were obtained through dbSNP, 1000GEMO and NHLBI databases. After the individualized bioinformatics analysis, 33 rare mutation sites of 25 candidate genes were selected for Sanger sequencing, and 1 false positives were excluded. The remaining 32 loci were derived from the peripheral blood and myocardial tissue sources. The same comparison of DNA showed that the 32 sites of DNA and DNA in the source of cardiac tissue were the same, and no somatic mutation was found. The 32 loci were verified by clinical samples using Mass-Array. The results showed that TTN, HYDIN, ZFPM1, XPO1, and FOXL2 genes may be the new.MYH6 gene 238629 of the ASD susceptibility gene. 52, the 61708404 site of XPO1 gene may be associated with partial ASD, and the mutation frequency of the 138665410 site of FOXL2 gene (NM_023067_c.C155G:p.A52G) in the atrial septal defect group is up to 25.9%, and the control group has not detected the mutation. The statistical difference is statistically significant (P0.05) the mutation may be highly correlated with ASD; finally, through siRNA Interference technique, we silenced the HYDIN gene in the H9C2 cardiomyocytes, and detected the H9C2 cells of the silent HYDIN gene. The scratch test found that the cell migration ability was weakened; the flow cytometry detected the apoptosis and the cell cycle: the H9C2 cells with the silent HYDIN gene remained in the G1 phase, the S phase decreased, and the apoptosis increased. Conclusion the total prevalence rate of CHD in]1. Yangbi county is higher than that of Yunnan Province, and the prevalence rate of CHD in Yi nationality is higher than that of other ethnic groups. The prevalence rate of CHD in 2. Yangbi counties is different, that of women is more than that of men. The prevalence rate of each Township in Yangbi county is different, and the prevalence rate is negatively correlated with the local economic level; 4. the incidence of CHD in Yangbi county has a trend of declining year by year in the last 15 years. 5.ASD is the most common type of CHD in Yangbi County; 6. it is feasible to find out ASD susceptible genes by using WES combined with individual later bioinformatics analysis; 7. sporadic ASD remains (?) heterogeneity is strong, gene mutation spectrum is wide, large scale clinical sample verification is necessary; 8. body cell mutation may not be the cause of ASD; 9.TTN, HYDIN, ZFPM1, XPO1, FOXL2 may be a newly found ASD susceptibility gene; 10.MYH6 gene 23862952, the rare mutation at the 61708404 site of the XPO1 gene may be associated with some ASD; the 138665410 locus of the 11.FOXL2 gene may be highly related to ASD. This mutation causes the specific mechanism of ASD to be further studied.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.1

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