本文選題：先天性心臟病 + 房間隔缺損　； 參考：《昆明醫(yī)科大學(xué)》2016年博士論文

【摘要】：[研究背景和目的]CHD是一種最常見的先天性出生缺陷。既往研究表明：CHD的發(fā)病率存在區(qū)域和民族間差異。自2001年開始,昆明醫(yī)科大學(xué)附屬延安醫(yī)院科研團(tuán)隊對云南省各地區(qū)進(jìn)行CHD流行病學(xué)調(diào)研。經(jīng)過15年的工作,我們調(diào)研的地區(qū)已經(jīng)覆蓋云南省全境,超過20萬中小學(xué)在校學(xué)生納入篩查范圍。第一部分漾濞縣流行病學(xué)調(diào)查屬于整個系統(tǒng)流調(diào)的一個部分,旨在調(diào)查云南省大理漾濞彝族自治縣3-16歲人群CHD患病率,并對相關(guān)因素進(jìn)行分析,以了解該地區(qū)人群CHD發(fā)生現(xiàn)狀和分布特點(diǎn)。結(jié)合國內(nèi)外文獻(xiàn)報道及我們第一部分的流行病學(xué)調(diào)查提示：ASD的發(fā)病率已經(jīng)超過VSD和PDA成為CHD最常見的類型。到目前為止,大部分ASD的發(fā)生遺傳學(xué)機(jī)制尚不明確。鑒于此,我們的第二部分?jǐn)M采用全外顯子測序技術(shù)結(jié)合個體化的生物信息學(xué)分析方法,從整個基因組層面探索ASD患者的遺傳學(xué)背景,以尋找ASD新的易感基因和突變位點(diǎn)。[方法]第一部分：采用橫斷面流行病學(xué)調(diào)查方法,于2015年1月對云南省大理漾濞彝族自治縣3-16歲在校學(xué)生共計9945人進(jìn)行分層整群抽樣分析。以問卷調(diào)查結(jié)合體格檢查為初篩,對初篩可疑陽性的病例進(jìn)行心臟彩色超聲多普勒檢查以明確診斷。第二部分：采集昆明醫(yī)科大學(xué)附屬延安醫(yī)院心臟大血管外科行ASD修補(bǔ)術(shù)患者術(shù)中修剪下廢棄房間隔組織5例,同時采集患者外周血作為自身對照。分別提取心肌來源和外周血來源的基因組DNA。將心肌來源的DNA作為WES樣本,HiSeq2500二代測序儀進(jìn)行WES。測序所得原始數(shù)據(jù)經(jīng)轉(zhuǎn)換和ANNOVAR軟件注釋得到所有外顯子區(qū)域的SNV,對這些SNV進(jìn)行數(shù)據(jù)庫過濾(dbSNP、 1000GEMO、NHLBI)和個體化生物信息學(xué)分析后,優(yōu)先篩選出部分稀有突變位點(diǎn)作為候選基因位點(diǎn)。使用Sanger測序法排除其中的假陽性位點(diǎn)后,剩余位點(diǎn)作為擬驗(yàn)證對象,Mass-Array技術(shù)將5‘例ASD樣本外周血來源的DNA與同一患者心肌組織來源的DNA進(jìn)行比對。將比對一致的位點(diǎn)使用Mass-Array在421例ASD患者和507例健康志愿者中進(jìn)行驗(yàn)證。最后,使用siRNA基因干擾技術(shù)沉默H9C2心肌細(xì)胞HYDIN基因,采用qRT-PCR方法檢測HYDIN siRNA對HYDIN基因的干擾效率。對成功沉默HYDIN的心肌細(xì)胞進(jìn)行細(xì)胞遷移能力(劃痕實(shí)驗(yàn))、細(xì)胞凋亡情況和細(xì)胞周期分布情況的檢測(]ACS檢測),以明確HYDIN基因在H9C2心肌細(xì)胞中的作用。[結(jié)果]第一部分：通過體格檢查,檢出疑似病例共計505例,經(jīng)心臟彩超確診CHD患者共計79例,漾濞縣CHD總患病率為7.94‰。其中,男性：7.17‰,女性：8.69‰;女性患病率高于男性,差異有統(tǒng)計學(xué)意義(P0.05)。漾濞縣彝族患病率8.259‰,高于該縣總體水平,差異有統(tǒng)計學(xué)意義(P0.05)。不同年齡段患病率：3-6歲：4.63‰,7-12歲6.45‰,13-16歲：11.11%o,組間比較差異有統(tǒng)計學(xué)意義(P0.05)。對漾濞縣9個鄉(xiāng)鎮(zhèn)進(jìn)行分別統(tǒng)計后發(fā)現(xiàn)：各鄉(xiāng)鎮(zhèn)患病率有差異,CHD患病率和當(dāng)?shù)谿DP水平負(fù)相關(guān)；亞型分析后發(fā)現(xiàn)：ASD是漾濞縣CHD的最常見類型。第二部分：5例ASD患者WES共得原始數(shù)據(jù)20.7GB,經(jīng)轉(zhuǎn)換和ANNOVAR軟件注釋得到外顯子區(qū)域單核苷酸變異共計181762個。通過dbSNP、1000GEMO和NHLBI數(shù)據(jù)庫過濾得到稀有突變713個,個體化生物信息學(xué)分析后優(yōu)先選擇25個候選基因的33個稀有突變位點(diǎn)進(jìn)行Sanger測序法驗(yàn)證,排除假陽性位點(diǎn)1個。對剩余32個位點(diǎn)進(jìn)行外周血和心肌組織來源的DNA的同一比對,結(jié)果顯示：心肌組織來源的DNA和外周血來源的DNA 32個位點(diǎn)突變類型一致,未發(fā)現(xiàn)體細(xì)胞突變現(xiàn)象。使用Mass-Array對這32個位點(diǎn)進(jìn)行臨床樣本驗(yàn)證,結(jié)果顯示：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位點(diǎn)可能與部分ASD發(fā)生相關(guān),FOXL2基因138665410位點(diǎn)(NM_023067_c.C155G:p.A52G)在房間隔缺損組突變頻率高達(dá)25.9%,對照組未檢測到該位點(diǎn)突變,統(tǒng)計學(xué)分析差異有統(tǒng)計學(xué)意義(P0.05)該突變可能與ASD發(fā)生高度相關(guān)；最后,通過siRNA干擾技術(shù),我們沉默了H9C2心肌細(xì)胞中的HYDIN基因,對沉默HYDIN基因的H9C2細(xì)胞進(jìn)行檢測：劃痕實(shí)驗(yàn)發(fā)現(xiàn)細(xì)胞遷移能力減弱；流式細(xì)胞儀檢測細(xì)胞凋亡和細(xì)胞周期發(fā)現(xiàn)：沉默HYDIN基因的H9C2細(xì)胞停留在G1期的增加,S期減少,細(xì)胞凋亡增加。[結(jié)論]1.漾濞縣CHD總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.漾濞縣男女CHD患病率有差異,女性多于男性；3.漾濞縣各鄉(xiāng)鎮(zhèn)患病率有差異,患病率與當(dāng)?shù)亟?jīng)濟(jì)水平負(fù)相關(guān)；4.漾濞縣CHD發(fā)病率在近15年有逐年下降的趨勢；5.ASD是漾濞縣CHD的最常見類型；6.應(yīng)用WES結(jié)合個體化后期生物信息學(xué)分析的方法尋找散發(fā)ASD易感基因是可行的；7.散發(fā)ASD遺(?)異質(zhì)性強(qiáng),基因突變譜范圍廣,大規(guī)模臨床樣本驗(yàn)證是必要的;8.體細(xì)胞突變可能不是ASD的發(fā)生原因；9.TTN、HYDIN、ZFPM1、XPO1、FOXL2可能是新發(fā)現(xiàn)的ASD易感基因；10.MYH6基因23862952、XPO1基因61708404位點(diǎn)的稀有突變可能與部分ASD發(fā)生相關(guān)；11.FOXL2基因138665410位點(diǎn)可能與ASD發(fā)生高度相關(guān),該突變引起ASD的具體機(jī)制值得進(jìn)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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R541.1

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