人类单纯性先天性心脏病遗传易感基因的定位与鉴定
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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、ASD
8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?
Objective
Congenital 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 resear
ches 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 these
genes 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.
Methods
Samples
186 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 markers
According 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).
Genotyping
DNA was extracted from veinous blood routinely and all microsatellite DNA markers were amplified by fluorencent - labeling polymerase chain reaction. PCR products were pu
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、ASD
8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?
Objective
Congenital 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 resear
ches 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 these
genes 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.
Methods
Samples
186 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 markers
According 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).
Genotyping
DNA was extracted from veinous blood routinely and all microsatellite DNA markers were amplified by fluorencent - labeling polymerase chain reaction. PCR products were pu
引文
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20．钟秋安，仇小强．先天性心脏病的病因研究进展．中国儿童保健杂志，2003；11(4)：259-261
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27. Schott JJ, Benson DW, Basson CT, et al. Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science, 1998;28:108-111
28．邱广蓉，孙桂凤，孙开来，等．Hox基因与人类室间隔缺损的相关性研究．中华医学杂志，2001；81(3)：174-175
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35. Krantz ID, Smith R, Colliton RP, et al. Jagged1 mutations in patients ascertained with isolated congenital heart defects. Am J Hum Genet, 1999; 84:56-60
36. Goldmuntz E. The epidemiology and genetics of congenital heart disease. Clin Perinatol, 2001 ;28 (1): 1-10
37. Giglio S, Graw SL, Gimelli G, et al. Deletion of a 5-cM region at chromo-
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51．孙红霞，杜玮南，方福德．人类疾病基因定位常用的统计学分析方法．中国医学科学院学报，2001；23(1)：86-88
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64．贺林．解码生命．第1版．北京：科学出版社．2000，78-80
65．张奎星，朱鼎良，黄薇．多基因遗传病基因研究的策略和方法．生理科学进展，2001；32(3)：215-219
66．黄文，朱佩芳，王正国．人类疾病相关基因定位研究的策略和方法．2003；25(4)：349-351
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69. Lander ES. The new genomics: global views of biology. Science, 1996; 274: 536-539
70. Kruglyak L. Prospects for whole-genome linkage disequilibrium mapping of common disease genes. Nat Genet, 1999 ;22:139-144
71. Wang DG, Fan JB, Siao CJ, et al. Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. Science, 1998 ;280: 1077-1082
72．崔静，倪鹏生，沈福民．单核苷酸多态．国外医学遗传学分册，2000；23(2)：80-82
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18．王晓明，张国成，韩美玉．B19病毒对先天性心脏病先天感染的探证及电镜观察．第四军医大学学报，2001；22(4)：340-343
19. Loffredo CA, Silbergeld EK, Ferencz C, et al. Association of transposition of the great arteries infants with maternal exposures to herbicides and rodenticides. Am J Epidemiol, 2001; 153 (6) :529-536
20．钟秋安，仇小强．先天性心脏病的病因研究进展．中国儿童保健杂志，2003；11(4)：259-261
21. Srivastava D and Olson EN. A genetic blueprint for cardiac development. Nature, 2000;407: 221-226
22. Lindsay EA, Botta A, Jurecic V, et al. Congenital heart disease in mice deficient for DiGeorge syndrome region. Nature, 1999 ;401:379-383
23. Gelb BD. Molecular genetics of congenital heart disease. Curr Opin Cardiol, 1997;12(3):321-328
24. Basson CT, Huang T, Lin RC, et al. Different TBX5 interactions in heart and limb defined by Holt-Oram syndrome mutations. Proc Nalt Acad Sci USA, 1999;96:2919-2924
25. Satoda M, Zhao F, Diaz GA, et al. Mutations in TFAP2B cause Char syndrome a familial form of patent ductus arteriosus. Nature Genet, 2000;25:42-46
26. Eldadah ZA, Hamosh A, Biery NJ, et al. Familial Tetralogy of Fallot caused by mutation in the jaggedl gene. Hum Mol Genet, 2001;10 (2): 163-169
27. Schott JJ, Benson DW, Basson CT, et al. Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science, 1998;28:108-111
28．邱广蓉，孙桂凤，孙开来，等．Hox基因与人类室间隔缺损的相关性研究．中华医学杂志，2001；81(3)：174-175
29. Lazzeroni LC and Lange KA. A conditional inference framework for extending the transmission/disequilibrium test. Hum Hered, 1998 ;48(2):67-81
30. Lazzeroni L and Lange K. Permutation tests in genetic epidemiology. Wellcome Sammer School, Oxford, U. K. 1997
31. Olser EN and Srivastava D. Molecular pathways controlling heart development. Science, 1996, 272:671-675
32. Eisenberg LM and Markwald RR. Molecular regulation of atrioventricular valvuloseptal morphogenesis. Circ Res, 1995, 77 (1): 1-6
33. Green EK, Priestley MD, Waters J, et al. Detailed mapping of a congenital heart disease gene in chromosome 3p25. J Med Genet, 2000 ;37 (8):581-587
34. Vaughan CJ and Basson CT. Moiecular-determinants of atrial and ventricular septal defects and patent ductus arteriosus. Am J Med Genet, 2000;97(4): 304-309
35. Krantz ID, Smith R, Colliton RP, et al. Jagged1 mutations in patients ascertained with isolated congenital heart defects. Am J Hum Genet, 1999; 84:56-60
36. Goldmuntz E. The epidemiology and genetics of congenital heart disease. Clin Perinatol, 2001 ;28 (1): 1-10
37. Giglio S, Graw SL, Gimelli G, et al. Deletion of a 5-cM region at chromo-
some 8p23 is associated with a spectrum of congenital heart defects. Circulation, 2000; 102 (4):432-437
38. Christiansen J, Dyck JD, Elyas BG, et al. Chromosome 1q21.1 contiguous gene deletion is associated with congenital heart disease. Cire Res, 2004, Apr 29 [Epub ahead of print]
39. Howard TD, Guttmacher AE, McKinnon W, et al. Autosomal dominant postaxial polydactyly, nail dystrophy, and dental abnormalities map to chromosome 4p16, in the region containing the Ellis-van Creveld syndrome locus. Am J Hum Genet, 1997;61(6):1405-1412
40. Barlow GM, Chen XN, Shi ZY, et al. Down syndrome congenital heart disease: a narrowed region and a candidate gene. Genet Med, 2001 ;3(2) :91-101
41. Innis JW. Role of Hox gene on human development. Curr Opin Pediatr, 1997; 9 (6) : 617-622
42. Nielsen PR, Nietlispach D, Mott HR, et al. Structure of the HP1 chromodomain bound to histone H3 methylated at lysine 9. Nature, 2002;416 (6876): 103-107
43. Freitag M, Hickey PC, Khlafallah TK, et al. HP1 is essential for DNA methylation in neurospora. Mol Cell, 2004;13 (3):427-434
44. Piacentini L, Fanti L, Berloco M, et al. Heterochromatin protein 1 (HP1) is associated with induced gene expression in Drosophila euchromatin. J Cell Biol, 2003 ;161 (4) :707-714
45. Fuks F, Hurd PJ, Deplus R, et al. The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase. Nucleic Acids Res, 2003 ;31 (9) :2305-2312
46. Lin CY, Li CC, Huang PH, et al. A developmentally regulated ARF-like 5 protein (ARL5), localized to nuclei and nucleoli, interacts with heterochromatin protein 1. J Cell Sci, 2002; 115 (Pt 23): 4433-4445
47. Mullor JL, Dahmane N, Sun T, et al. Wnt signals are targets and mediators of Gli function. Curr Biol, 2001;11 (10) :769-773
48. Koyabu Y, Nakata K, Mizugishi K, et al. Physical and functional interactions between Zic and Gli proteins. J Biol Chem, 2001;276(10):6889-
6892
49. Altmuller J, Palmer LJ, Fischer G, et al. Genomewide scans of complex human diseases: true linkage is hard to find. Am J Hum Genet, 2001 ;69:936-950
50. Lander ES and Schork NJ. Genetic dissection of complex traits. Science, 1994 ;2;65:2037-2047
51．孙红霞，杜玮南，方福德．人类疾病基因定位常用的统计学分析方法．中国医学科学院学报，2001；23(1)：86-88
52. Weeks DE and Hardy LD. The affected-pedigree-member method: power to detect linkage. Hum Hered, 1995 ;45:13-24
53. Shaid DJ and Sommer SS. Comparison of statistics for candidate gene association studies using cases and parents. Am J Genet, 1994;44(2):502
54. Ghosh S. Probability and complex disease gene. Nat Genet, 1995;9 (3): 223
55. Chu S, Zhu D, Wang G, et al. Linkage analysis of cytokine and cytokine-related receptor gene loci and essential hypertension in Chinese. Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 2002;19(3):221-224
56. Chu S, Zhu D and Xiong M. Linkage analysis of candidate genes for glucose and lipid metabolism with essential hypertension. Zhonghua Yi Xue Za Zhi, 2001; 81 (1): 20-22
57. Nakagawa Y, Kawaguchi Y, Twells RC, et al. Fine mapping of the diabetes-susceptibility locus, IDDM4, on chromosome 11 q13. Am J Hum Genet, 1998 ;63(2):547-556
58. Marron MP, Zeidler A, Raffel LJ, et al. Genetic and physical mapping of a type 1 diabetes susceptibility gene (IDDM12) to a 100-kb phagemid artificial chromosome clone containing D2S72-CTLA4-D2S105 on chromosome 2q33. Diabetes, 2000 ;49 (3) :492-499
59. Li WD, L D, Wang S, et al. Linkage and linkage disequilibrium mapping of genes influencing human obesity in chromosome region 7q22.1-7q35. Diabetes, 2003 ;52(6) :1557-1561
60. Liang SG, Sadovnick AD, Remick RA, et al. A linkage disequilibrium study of bipolar disorder and microsatellite markers on 22q13. Psychiatr
Genet, 2002;12(4):231-235
61. Freedman R, Leonard S, Gault JM, et al. Linkage disequilibrium for schiz-ophrenia at the chromosome 15q13-14 locus of the alpha7-nicotinic acetylcholine receptor subunit gene (CHRNA7). Am J Med Genet, 2001; 105 (1):20-22
62. Mujaheed M, Corbex M, Lichtenberg P, et al. Evidence for linkage by transmission disequilibrium test analysis of a chromosome 22 microsatellite marker D22S278 and bipolar disorder in a Palestinian Arab population. Am J Med Genet, 2000;96(6):836-838
63. Wright P, Dawson E, Donaldson PT, et al. A transmission/disequilibrium study of the DRB1*04 gene locus on chromosome 6p21.3 with schizophrenia. Schizophr Res, 1998 ;32(2):75-80
64．贺林．解码生命．第1版．北京：科学出版社．2000，78-80
65．张奎星，朱鼎良，黄薇．多基因遗传病基因研究的策略和方法．生理科学进展，2001；32(3)：215-219
66．黄文，朱佩芳，王正国．人类疾病相关基因定位研究的策略和方法．2003；25(4)：349-351
67. Collins FS, Guyer MS and Charkravarti A. Variations on a theme: cataloging human DNA sequence variation. Science, 1997 ;278:1580-1581
68. Risch N and Merikangas K. The future of genetic studies of complex human disease. Science, 1996 ;273:1516-1517
69. Lander ES. The new genomics: global views of biology. Science, 1996; 274: 536-539
70. Kruglyak L. Prospects for whole-genome linkage disequilibrium mapping of common disease genes. Nat Genet, 1999 ;22:139-144
71. Wang DG, Fan JB, Siao CJ, et al. Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. Science, 1998 ;280: 1077-1082
72．崔静，倪鹏生，沈福民．单核苷酸多态．国外医学遗传学分册，2000；23(2)：80-82
73. Kinzler KW, Ruppert JM, Bigner SH, et al. The Gli gene is a member of the Kruppel family of zinc finger proteins. Nature, 1988; 332 (6162): 371
-374
74. Ruppert JM, Kinzler KW, Wong AJ, et al. The Gli-Kruppel family of human genes. Mol Cell Biol, 1988 ;8(8):3104-3113
75. Capdevila J, Vogan KJ, Tabin CJ, et al. Mechanisms of left-right determination in vertebrates. Cell, 2000;101 :9-21
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