【作者】 林彬；

【导师】 孔祥银；

【作者基本信息】 上海交通大学 ， 遗传学， 2012， 博士

【摘要】 先天性心脏病(简称先心病)是一种由于胎儿心脏发育异常而导致心血管结构畸形的复杂性疾病,在我国有较高的发病率,给患者和社会造成了沉重的负担。为了研究中国散发性先心病的遗传因素,我们综合了多种先进的遗传学手段和方法(包括基因芯片,第二代测序技术等)对中国散发性先心病人群的稀有变异进行了检测。我们将66名不同临床亚型的先心病患者分为4组,将每组患者的基因组DNA等量混合后,分别进行外显子组测序。对测序数据进行分析及验证后,我们一共发现了843个变异,我们又在800名正常人中检测是否存在上述变异,而只保留正常人群中不存在的变异,结果我们在散发性先心病人群中识别了387个稀有变异,其中包括351个错义突变,14个剪接位点突变以及22个无义突变。在此基础上我们挑选了一系列的候选基因,在217名散发性先心病患者中对它们的编码区进行测序,并排除在正常人群也存在的变异,最终我们在PKD1L1基因中发现了19个稀有变异(P=9.434e-14),在DLC1基因中发现了11个稀有变异(P=1.208e-8),在FAM71A基因中发现了6个稀有变异(P=8.932e-5)。我们还分析了400名正常人的外显子组测序数据,在PKD1L1基因中只发现了4个稀有变异,这说明相对于正常人群,PKD1L1基因的稀有变异显著地富集在散发性先心病人群中(P=2.736e-6)。此外,SIFT软件的预测结果表明,PKD1L1基因的19个稀有变异在平均水平上会对蛋白的结构或功能造成影响(P<0.001)。另一方面,我们利用基因芯片技术对58名先心病患者进行了全基因组扫描,发现了一些稀有的拷贝数变异。我们首次在中国散发性先心病人群中进行了外显子组测序,识别了多个先心病人群特异的稀有变异,建立了中国散发性先心病人群的稀有变异图谱,更为重要的是,我们发现PKD1L1基因的稀有变异与先心病的发生有密切关系,这些为对中国散发性先心病遗传因素的后续研究打下了坚实的基础。更多还原

【Abstract】 Congenital heart disease(CHD) is a complex disease that the structures of the heart or great vessels are abnormal when the fetus was born. In China, the incidence of CHD is now close to 1%, which makes thousands of neonates born with heart defects. To identify the risk factors of sporadic CHD, kinds of powerful tools were used to detect the rare variations in a sporadic CHD cohort. We performed 4 pooled exome sequencing on 66 CHD patients according to the clinical subtypes of CHD. After data analysis and validation, we genotyped the SNV(single nucleotide variation) sites in 800 normal samples to rule out the common variations. Here we present a rare variations spectrum which contains 387 non-synonymous mutations in a sporadic CHD cohort, including 351 missense mutations, 14 splicing site mutations and 22 nonsense mutations. On that basis, several candidate genes were selected to sequence the coding regions in an expanded CHD population(n=217). Finally we identified 19 rare variations in PKD1L1(P=9.434e-14), 11 rare variations in DLC1(P=1.208e-8) and 6 rare variations in FAM71A(P=8.932e-5). Compared with the rare variations frequency of PKD1L1 in 400 normal samples, our data demonstrate that the rare variations of PKD1L1 are significantly recruited in the CHD cohort(P=2.736e-6). In addition, the damage evaluation of these rare variants using SIFT scores indicates that they are more inclined to disrupt the protein(P<0.001). In the other hand, we used Affymetrix 500 K array to scan the genomes of 58 CHD patients and a few copy number variations were also identified. For the first time, our study identifies a series of rare variations which were only found in the Chinese sporadic CHD cohort, and more importantly, we provide strong evidence that the rare variations of PKD1L1 are associated with the sporadic congenital heart disease. All of these efforts are essential for the continued studies to explore the genetic factors of CHD.更多还原