【作者】 邱广蓉；

【导师】 孙开来；

【作者基本信息】 中国医科大学 ， 遗传学， 2004， 博士

【摘要】 目的 先天性心脏病(Congenital Heart Disease，CHD)是一类严重危害婴幼儿健康的先天畸形，发病率占活产婴儿的4‰～50‰。其中，约有80％的CHD仅表现为心脏畸形，而不伴有其他系统的先天异常，称之为单纯性先天性心脏病(simple congenital heart disease)。目前认为，CHD主要是胚胎心脏发育异常所致，遗传因素在CHD的发病过程中发挥重要作用，遗传率(度)为55％～65％，但其遗传方式和外显率等均不清楚，故CHD致病基因研究进展缓慢。研究CHD的分子遗传学机制，不仅为阐明CHD的发生机理和遗传干预提供理论依据，同时对丰富人类基因组学的理论和实践应用，具有十分重要的意义，也为其他先天畸形的研究提供可借鉴的科学方法。 心脏的发育是一个极其复杂的过程。它涉及胚胎发育过程中不同时间、不同空间的若干个基因先后表达，也涉及细胞的迁移、分化、增生及精确的相互作用。国外学者应用荧光原位杂交、免疫组化、基因剔除等方法，通过对果蝇、琥珀鱼、鸡胚、鼠等心脏发育的研究，发现了一些与心脏发育相关的基因，如Nkx2-5、Pitx2、erbB2、MEF2C、dHAND、Endothelin、Irx4、Ufdl、NF-ATc、Sox-6。这些基因在进化过程中高度保守的特征为我们寻求CHD的相关基因提供了有利的线索。 目前国内外关于人类CHD的研究主要集中于孕早期环境因素和一些伴有心脏畸形的综合征，发现了引起心脏畸形的候选区域，如22q11、10p13、4p16，以及CHD相关基因如TBX5、Jagged-1、TFAP2B等。但在单纯性CHD的研究方面，仅Nkx2-5／Csx基因被证实与之有关。因此，国内外对人类单纯性CHD发病机理的研究尚待深入。 本课题组前期研究工作发现位于12q13区域内的D12S1056位点与单纯性CHD显著相关(P<0.01)，提示在此位点附近可能存在单纯性CHD遗传易感基因。为进一步证实，本研究在D12S1056位点附近选择10个微卫星多态遗传标记:D12S1056、D12S1293、D12S83、D1251655、D1251662、D125334、D125137、D125102、D1251702和D12S1691，对62个单纯性CHD核心家系186位成员进行基因型分析和传递不平衡检验(Transmission Dise-叫lib五uml’est，TDT)，首次将单纯性eHD遗传易感基因定位至12q13区域内3.4cM，井采用候选克隆策略，首次证明该候选区域内Gli基因与单纯性CHD相关。方法 标本:62个单纯性CHD核心家系共186名成员，其中VSD37人、ASD8人、PDA功人、凡7人。62个单纯性CHD核心家系均由中国医科大学附属第一临床学院、第二临床医院和沈阳军区陆军总院心脏外科提供。所有患者均具有典型的临床表现，经心脏超声和手术确诊。取所有CHD患者及其家系成员静脉血2而，构椽酸钠抗凝后一20“C冻存，备用。 1.微卫星多态位点的选择 根据各个微卫星多态位点的等位基因数目、杂合度及多态信息含量(Polymo印hi‘:Inf’ormation Content，pIC)值，选择染色体12q13区域内10个微卫星多态位点进行TDT检验。其中D1251056、D12S1293来自人类合作连锁中心(The Cooperative Human Link鳃e Center，CHLC)，DzZss3、D12S1655、D12S一662、D125334、D125137、D12S102、D1251702、D12S一691选自基因组数据库(Genome DataBase，GDB)。 2.基因型分析 常规饱和氯化钠盐析法提取外周血DNA。荧光标记PCR技术扩增微卫星多态位点。酚/氯仿抽提法纯化PCR产物。经95“C变性3而n后，在ABI PlllSMT“310型遗传分析仪上进行毛细管电泳。电泳介质为POP一4胶，电泳条件为15Kv电泳24而n。Genescan Analysis 2.02软件自动分析基因型。 3.TDT检验和遗传多态性分析 对62个核心家系成员进行TDT检验。对有意义的微卫星多态位点在中国北方汉族人群中进行遗传多态性分析。采用直接计数法计算各微卫星多态位点等位基因频率、基因型频率和杂合度。依据Hardy一Weinberg定律计算各微卫星多态位点基因型频率和杂合度预期值，采用x，检验进行Hardy一Weinbe铭平衡吻合度检验。 4.Gli基因与单纯性CHD相关性研究 在Gii基因编码区选择3个可引起氨基酸改变的cSNP:EI looQ、G933D和G1012V。常规PCR扩增后，采用PCR一RFLP和DHPLC两种方法对62个单纯性CHD核心家系进行基因型分析。其中，EI looQ和G933D两个cSNP分别具有Ec0RI和Tfil酶切位点。应用wAvE⑧系统温度预测软件对扩增片段序列进行分析，确定3个。SNP的熔解温度(Tm):G933 D Tm为60.3OC，Ell00Q Tni为58.5”C，G1012VTm为61.3”C。PCR产物经缓慢变性后进行DHPLC检测。3个csNP的不同基因型经测序证实。统计学分析包括:应用在线Hardy一Weinberg检测程序分析基因型的Hardy一Weinberg平衡吻合度;采用ETDT软件分别对Gli基因3个cSNP进行单位点关联分析;应用ZLD软件进行配对连锁不平衡检验(连锁不平衡程度以D‘表示);应用TRANSMrr version 2.5软件进行单倍型分析。结果 1.毛细管电泳结果 Rllo经荧光扫描为蓝色，R6G经荧光扫描为绿色。单峰为纯合子，对称双峰为杂合子。 2.基因型分析结果 62个核心家系在10个微卫星多态位点基?更多还原

【Abstract】 ObjectiveCongenital heart disease ( CHD) is a kind of congenital malformation that does severe harm to inborn’ health and the morbidity is from 4%c to 50%c. About 80% percent of congenital heart diseases only manifest malformations in cardiovascular system without any abnormality of other systems, which is called simple congenital heart disease. It is considered that congenital heart disease results from the abnormal development of the embryonic cardiovascular system and the genetic factors play an important role in the pathogenesis of CHD. The heritabil-ity of CHD is from 55% to 65% , but it is not clear about the hereditary’ patterns and penetrance, and little advances have been made in the researches on the susceptibility genes of CHD. Therefore, it is of great significance that the molecular genetic mechanisms of CHD should be discussed, which can provide the theorical bases for the genetic interfere of CHD, enrich the theory and practical application of human genomics and offer an useful method from which researches on other congenital malformations can draw lessons.Cardiac development is a complicated progress that involves not only spatial and temporal expression of many genes but also the migration, differentiation, proliferation of cells and accurate interaction between cells. With the studies on the cardiac Development of Drosophila, zebrailsh, chick embryo and mouse, some cardiac development - related genes have been identified by the means of fluorescence in situ hybridization, immumohistochemistry or gene knockout, such as Nkx2 -5, Pitx2, erbB2, MEF2C, dHAND, Endothelin, Irx4, Ufd1, NF - ATc, Sox - 6 and so on. The highly conservative characteristics of thesegenes provide an useful clue for us to look for cardiac development - related genes.At present, researches on human CHD mainly focus on the environmental factors at early stage of pregnancy and many syndromes with cardiac malformations. Candidate regions for cardiac malformations such as 22qll, 10pl3, 4pl6 and CHD related genes such as TBX5, Jagged - 1, TFAP2B, are identified. However, only Nkx2 - 5/Csx is confirmed with regard to the pathogenesis of simple CHD. Therefore, little has been known about the pathogenesis of human simple CHD and further researches are urgent.Previous researches in our group showed that D12S1056 locus in the region of 12ql3 was remarkably associated with simple CHD ( P < 0. 01) , suggesting there might be simple CHD susceptibility genes around D12S1056. In order to confirm it, ten microsatellite DNA markers were chosen around D12S1056, that is, D12S1056, D12S1293, D12S83, D12S1655, D12S1662, D12S334, D12S137, D12S102, D12S1702 and D12S1691. Genotyping and transmission disequilibrium test were done in 186 members from 62 core pedigrees of simple CHD. For the first time, the simple CHD susceptibility gene was narrowed to 3.4cM in 12ql3. At the same time, Gli gene in the candidate region was firstly confirmed associated with simple CHD using candidate cloning stragety.MethodsSamples186 members from 62 core pedigrees include 37 patients with ventricular septal defect, 8 patients with atrial septal defect, 10 patients with Patent Ductus Arterisus, and 7 patients with Fallot s Tetralogy. All patients had typical manifestation and were confirmed by cardiac ultrasonics and surgical operation. Vein-ous blood of 186 members from 62 core pedigrees were kept at -20C for use after anti - coagulation.Choose of microsatllite DNA markersAccording to the number of alleles, heterozygosity and polymorphic information content, 10 microsatellite DNA markers in 12q13 were chosen for TDT.Among these microsatellite DNA markers, D12S1056 and D12S1293 were from the Cooperative Human Linkage Center (CHLC) , while D12S83, D12S1655, D12S1662, D12S334, D12S137, D12S102, D12S1702 and D12S1691 were from Genome DataBase (GDB).GenotypingDNA was extracted from veinous blood routinely and all microsatellite DNA markers were amplified by fluorencent - labeling polymerase chain reaction. PCR products were pu更多还原