心臟是胚胎發育過程中最早發育的器官之一，一般認為先天性心臟病的發生與中樞神經系統之心臟神經脊 (cardiac neural crest) 發育得正常與否有密切的關係。小兒先天性心臟病 (congenital heart diseases) 每一年在台灣所造成的死亡率為兒童癌症的兩倍。探討造成先天性心臟病的致病基因與心臟發育的相關性在優生學和治療策略的發展中，將可提供早期預防、診斷或開發藥物的有效途徑。先天性心臟病發生的時間與胚胎發育早期、心臟形成有密切的關係。雖然藥物和母體環境皆可能造成先天性心臟病的發生，但家族中若有罹患先天性心臟病，在發生的比例較一般無先天性心臟並家族要高，顯示先天性心臟病與個體基因型 (genotypes) 有關。本研究利用來自高雄榮總小兒科共 239 個小兒先天性心臟病家庭總計 245 位病人。其中有 83 位病人被診斷是心室中膈缺損，為 34.7% ，所佔的比例最高。進行病人與親代血液採樣及基因組DNA之萃取，進一步利用位於第 22 對染色體十個涵蓋DiGeorge syndrome 之微衛星型 (microstallite) 遺傳標記，進行台灣小兒先天性心臟病與心臟發育相關基因型的探討。進一步進行染色體細部基因定位 (fine mapping) 與局部候選基因分析方式 (local candidate analysis) ，希望藉此研究將導致台灣小兒先天性心臟病的基因定位並縮小範圍到第 22 對染色體內較小之區域。目前結果顯示有二十五個心室中膈缺損的家族顯示在 22q11 的位置上有 loss of heterozygosity 的現象，分別在 D22S264, D22S303, D22S420, D22S427, D22S941, D22S944, D22S1638, D22S1648 發生。將進一步研究該區域與心臟發育相關的候選基因 TBX1, DGCR6, UFD1L 之基因是否有所突變。另外，利用單股結構多樣性的實驗 (single strand conformation polymorphisms) 分析其他三個位於不同染色體上但與心臟發育相關的基因 CSX, JAG1, TBX5 是否有所突變而造成心室中膈缺損。目前研究結果顯示TBX5的第四個、第五個、第九個及第十個的表現子，在245位病人與其親代間並沒有發現突變，所以我們即證明這四個表現子對於這245位小兒先天性心臟病病人的心臟發育無直接關係。但我們仍會繼續研究這三個基因其他表現子的序列是否在這些病人中是否有發生突變的情形。

Objective. Congenital heart disease (CHDs) in Taiwan cause twice as many children die each year comparing with childhood cancers. Prevalent CHDs are ventricular septal defects (VSDs) which accounted for ~40% Taiwanese population averagely. Studies on heart development-related genes on the human genome will provide valuable information for early diagnosis/prevention in eugenics and the development of therapeutic strategies.
Methods. A total of 239 CHD families from Kaohsiung Veteran General Hospital, including 713 individuals with 245 affected, participated in this study. Among these CHDs families, 83 were diagnosed as VSDs, accounted for 34.7% of all CHDs. We initiated using a semi-quantitative fluorescent PCR method applying ten highly polymorphic markers that located within 22q11, genotyping analysis for deletion or loss of heterozygosity. In those cases that are identified as chromosome 22q11-independent VSDs, cardiac development-related candidate genes TBX5, CSX and JAG1 analyses were performed by Single-Strand Conformation Polymorphisms (SSCPs) and Temporal Temperature Gradient Gel Electrophoresis (TTGE) analyses to identify whether any genomic mutation/deletion exists.
Results. So far, there are twenty-five VSD affected individuals have been identified as loss of heterozygosity (LOH) at loci D22S264, D22S303, D22S420, D22S427,D22S941, D22S944, D22S1638 and D22S1648. Candidate gene approaches will therefore be carried out within chromosome 22q11 subregion in these individuals.
Conclusions. The frequency of CHD necessitating intervention in patients referred for cardiovascular evaluation after diagnosis of a chromosome 22q11 deletion. Routine screening for CHDs, including VSD and other imaging studied to identify the any microdeletion(s) or LOH.

中文摘要......................................................Ⅰ
英文摘要......................................................Ⅲ
英文縮寫表....................................................Ⅳ
前言...........................................................1
材料與方法....................................................24
結果..........................................................31
討論..........................................................49
結論..........................................................55
參考文獻......................................................56

1. Nora J.J., Nora A.H., Update on counseling the family with a first-degree relative with congenital heart defect. Am. J. Med. Genet. 1988; 29:137-142
2. Cprrea V.A., McCarter R., Downing J., et al. White-black differences in cardiovascular malformations in infancy and socioeconomic factors. Am. J. Epidemiol. 1991; 134:4:393-402
3. Bristow J., The search for genetic mechanisms of congenital heart disease. Cell. Mol. Biol. Res. 1995; 41:307-319
4. Hoffman J.I., Incidence of congenital heart disease. Ⅱ. Prenatal incidence. Pediatr. Cardiol. 1995; 16:155-165
5. Yi R.S., Kai S.H., Jer Y.W., et al. Genetic analysis of Chromosome 22q11.2 Markers in Congenital Heart Disease. J. Clin. Lab. Analysis 2003; 17:28-35
6. Payne R.M., Johnson M.C., Grant J.W., et al. Toward a molecular understanding of congenital heart disease. Circulation 1995; 95:479-504
7. Hoffman, J. and Christianson, R., Congenital heart disease in a cohort of 19,502 births with long-term follow-up. Am. J. Cardiol.1978; 42:641
8. Boughman J., Berg K., and Astemborski J., Familial risks of congenital heart disease in a population based epidemiologic study. Am. J. Med. Genet. 1987; 26:839-49
9. Morrow B., Goldberg R., Carlson C., et al. Molecular definition of 22q11 deletions in 151 velo-cardio-facial syndrome patients. Am. J. Hum. Genet. 1995; 56:1391-1403
10. Driscoll D.A., Salvin J., Sellinger B., et al. Prevalence of 22q11 microdeletions in DiGeorge and velo-cardio-facial syndromes: implications of genetic counseling and prenatal diagnosis. J. Med. Genet. 1993; 30:813-817
11. Carlson C., Sirotkin H., Pandita C., et al. Molecular definition of 22q11 deletions in 151 velo-cardio-facial syndrome patients. Am. J. Hum. Genet. 1997; 61:620-629
12. Lupski J.R., Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits. Trends. Genet. 1998; 14:417–422
13. Driscoll D.A., Spinner N.B., Budarf M.L., et al. Deletions and microdeletions 22q11.2 in velocardiofacial syndrome. Am. J. Med. Genet. 1992; 44:261-268
14. Heather E.M. and Bernice E.M., REVIEW ARTICLE: Genomic Disorders on 22q11. Am. J. Hum. Genet. 2002; 70:1077-1088
15. Ryan A.K., Goodship J.A., Wilson D.I., et al. Spectrum of clinical features associated with interstitial chromosome 22q11 deletions: a European collaborative study. J. Med. Genet. 1997; 34:798–804
16. McDonald-McGinn D.M., Kirschner R., Goldmuntz E., et al. The Philadelphia story: the 22q11.2 deletion: report on 250 patients. Genet. Couns. 1999; 10:11–24
17. Vitelli F., Morishima M., Taddei I., et al. Tbx1 mutation causes multiple cardiovascular defects and disrupts neural crest and cranial nerve migratory pathways. Hum. Mol. Genet. 2002; 11:915–922
18. Lindsay E.A., Botta A., Jurecic V., et al. Congenital heart disease in mice deficient for the DiGeorge syndrome region. Nature 1999; 401:379–383
19. Kimber W.L., Hsieh P., Hirotsune S., et al. Deletion of 150 kb in the minimal DiGeorge/velocardiofacial syndrome critical region in mouse. Hum. Mol. Genet. 1999; 8:2229–2237
20. Lee M.L., Chaou W.T., Wang Y.M., et al. A new embryonic linkage between chromosome 22q11 deletion and a right ductus from a right aortic arch in a neonate with DiGeorge syndrome. Int. J. Cardiol. 2001; 79:315–316
21. Munoz S., Garay F., Flores I., et al. Heterogeneidad de la presentacion clinica del sindrome de microdelecion del cromosoma 22, region q11. Revista. Medica. de Chile. 2001; 129:515–521
22. Cohen E., Chow E.W.C., Weksberg R., et al. Phenotype of Adults With the 22q11 Deletion Syndrome. Am. J. Med. Genet. 1999; 86:359-365
23. Chow E.W.C., Zipursky R.B., Mikulis D.J., et al. Structural Brain Abnormalities in Patients with Schizophrenia and 22q11 Deletion Syndrome. Bio. Psy. 2001; in press.
24. Scutt L.E., Chow E.W.C., Weksberg R., et al. Patterns of Dysmorphic Features in Schizophrenia. Am. J. Med. Genet. 2001; 105:713-723
25. Gelb B.D., Recent advances in the understanding of genetic causes of congenital heart defects. Frontiers in Bioscience 2001; 5:d321-333
26. Srivastava D., Genetic assembly of the heart: implications for congenital heart disease. Trends. Vardiovasc. Med. 2001; 9:11-18
27. Kelley R.I., Zackai E.H., Emanuel B.S., et al. The association of the Digeorge anomalad with partial monosomy of chromosome 22. J. Pediatr. 1982; 101:197-200
28. Antonio B., Digeorge syndrome: the use of model organisms to dissect complex genetics. Hum. Mol. Genet. 2002; 11:2363-2369
29. Scambler P.J., Carey A.H., Wyse R.K., et al. Microdeletions within 22q11 associated with sporadic and familial Digeorge syndrome. Genomics 1991; 10:201-206
30. Discoll D.A., Spinner N.B., Budarf M.L., et al. Deletions and microdeletions of 22q11.2 in velo-cardio-facial syndrome. Am. J. Med. Genet. 1992; 44:261-268
31. Burn J. and Goodship J., Congenital heart disease. Emery and Rimoin’s Principles and Practice of Medical Genetics. Churchill Livingstone, New York, 1996; 767-803
32. Goldmuntz E., and Emanuel B.S., Genetic disorders of cardiac morphogenesis. The Digeorge and velocardiofacial syndromes. Circ. Res. 1997; 80:497-443
33. Ryan A.K., Goodship J.A., Wilson D.I., et al. Spectrum of clinical features associated with interstitial chromosome 22q11 deletions: a European collaborative study. J. Med. Genet. 1997; 34:798-804
34. Baldini A., DiGeorge syndrome: the use of model organisms to dissect complex genetics. Hum. Mol. Genet. 2002; 11:2363-2369
35. Lindsay E.A., Chromosomal microdeletions: dissecting del22q11 syndrome. Nat. Rev. Genet. 2001; 2:858-868
36. Basson C.T., Bachinsky D.R., Lin R.C., et al. Mutations in human TBX5 cause limb and cardiac malformation in Holt-Oram syndrome. Nat. Genet. 1997; 15:30-35
37. Li Q.Y., Newbury-Ecob R.A., Terrett J.A., et al. Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family. Nat. Genet. 1997; 15:21–29
38. Newbury-Ecob R.A., Leanage R., Raeburn J.A., et al. Holt-Oram syndrome: a clinical genetic study. J. Med. Genet. 1996; 33:300-307
39. Basson C.T., Huang T., Lin R.C., et al. Different TBX5 interactions in heart and limb defined by Holt-Oram syndrome mutations. Proc. Natl. Acad. Sci. USA 1999; 96:1919-1924
40. Yelon D., Ticho B., Halpern M.E., et al. Parallel roles for the bHLH transcription factor HAND2 in zebrafish and pectoral fin development. Development 2000; 127:2573-2582
42. Basson, C.T., Cowley, G.S., Solomon, S.D., et al. The clinical and genetic spectrum of the Holt-Oram syndrome. N. Engl. J. Med. 1994; 330:885–891
43. Bruneau, B.G., Logan, M., Davis, N., et al. Chamber-specific cardiac expression of Tbx5 and heart defects in Holt- Oram syndrome. Dev. Biol. 1999; 211:100–108
44. Newbury-Ecob, R.A., Leanage, R., Raeburn, J.A., et al. Holt-Oram syndrome: a clinical genetic study. J. Med. Genet. 1996; 33:300–307
45. Papaioannou V.E., and Silver L.M., The T-box gene family. Bioessays 1998; 20:9–19
46. Weintraub R.G., and Menahem S., Growth and congenital heart disease. J. Paediatr. Child. Health. 1993; 29:95-8
47. Rebecca B., Saenz M.D., Diane K., et al. Caring for Infants with Congenital Heart Disease and Their Families. American Family Physician 1999; 59:1857-1866
48. Ingeborg S., Diether L., Frederik D.S., et al. VEGF: A modifier of the del22q11 (DiGeorge) syndrome? Nat. Med. 2003; 9:173-182
49. Yasuhiko S., Caramai N.K., Hironori N., et al. Ventricular septal defect and cardiomyopathy in mice lacking the transcription factor CHF1/Hey2. PNAS 2002; 99:16197-16202
50. Gehring W. J., Affolter M., and Burglin T. Homeodomain proteins. Annu. Rev. Biochem. 1994; 63:487-526
51. Harvey R. P. NK-2 homeobox genes and heart development. Dev. Biol. 1996; 178:203-206
52. Gruschus J.M., Tsao D.H., Wang L.H., et al. Interactions of the vnd/NK-2 homeodomain with DNA by nuclear magnetic resonance spectrosocopy: basis of binding specificity. Biochemistry 1997; 36:5372-5380
53. Weidong Z., Ichiro S., Yukio H., et al. Functional Analyses of Three Csx/Nkx-2.5 Mutations That Cause Human Congenital Heart Disease. J. Bio. Chem. 2000; 275:35291-35296
54. Hideko K., Bora L., Jean J.S., et al. Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease. J. Clin. Invest. 2000; 106:299-308
55. Schott J.J., Benson D.W., Basson C.T., et al. Congenital Heart Disease Caused by Mutations in the Transcription Factor NKX2-5. Science. 1998; 281:108-111
56. Benson D.W., Silberbach G.M., Kavanaugh-McHugh A., et al. Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways. J. Clin. Invest. 1999; 104:1567-1573
57. Komuro I., and Izumo S., Csx: A Murine Homeobox-Containing Gene Specifically Expressed in the Developing Heart. Proc. Natl. Acad. Sci. U.S.A. 1993; 90:8145–8149
58. Lints T.J., Parsons L.M., Hartley L., et al. Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 1993; 119:419–431
59. Bodmer R., The gene tinman is required for specification of the heart and visceral muscles in Drosophila. Development 1993; 118:719–729
60. Lyons I., Parsons L.M., Hartley L., et al. Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5. Genes Dev.1995; 9:1654–1666
611. Li L., Krantz I.D., Deng Y., et al. Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1. Nat. Genet. 1997; 16:243-251.
62. Yuan S.R., Kohsaka T., Ikegaya T., Mutational analysis of the Jagged1 gene in Alagille syndrome families. Hum. Mol. Genet. 1998; 7:1363-1369.
63. Crosnier C., Driancourt C., Raynaud N., et al. Mutations in JAGGED1 gene are predominately sporadic in Alagille syndrome. Gastroenterology 1999; 116:1141-1148
64. Lewis J., Notch signalling and the control of cell fate choices in vertebrates. Semin. Cell. Dev. Biol. 1998; 9:583-589
65. Bollag R.J., Siegfriend Z., Cebra-Thomas J.A., et al. An ancient family of embryonically expressed mouse genes sharing a conserved protein motif with the T locus. Nat. Genet. 1994; 7:383-389
66. Papaioannou V.E. and Silver L.M., The T-box gene family. Bioessays 1998; 20:9-19
67. Smith J., T-box gene: What they do and how they do it. Trends Genet. 1999; 15:154-158
68. Chapman D.L., Garvey N., Hancock S., et al. Expression of the T-box family genes, Tbx1-bx5, during early mouse development. Dev. Dyn. 1996; 206:379-390
69. Garg V., Yamagishi C., Hu T., et al. Tbx1, a Digeorge syndrome candidate gene, is regulated by Sonic hedgehog during pharyngeal arch development. Dev. Biol. 2001; 235:62-73
70. Abu-Issa R., Smyth G., Smoak I., et al. Fgf8 is required for pharyngeal arch and cardiovascular development in the mouse. Development 2002; 129:4613-4625
71. Frank D.U., Fotheringham L.K., Brewer J.A., et al. An Fgf8 mouse mutant phenocopies human 22q11 deletion syndrome. Development 2002; 129:4591-4603
72. Vitelli F., Taddei I., Morishima M., et al. A genetic link between Tbx1 and fibroblast groeth factor signaling. Development 2002; 129:4605-4611
74. Kispert A. and Hermann B.G. The Brachyury gene encodes a novel DNA binding protein. EMBO J. 1993; 12:4898-4899
75. Kispert A. Koschorz B. and Herrmann B.G. The T protein encoded by Brachyury is a tissue-specific transcription factor. EMBO J. 1995; 14:4769-4772
76. Yi C.H., Terrett J.A., Li Q.Y., et al. Identification, mapping, and phylogenomic analysis of four new human members of the T-box gene family: EOMES, TBX6, TBX18, and TBX19. Genomics 1999; 15:10-20
77. Bollag R. J., Siegfried Z., Cebra-Thoman J.A., et al. An ancient family of embryonically expressed mouse henes sharing a conserved protein motif with the T locus. Nat. Genet. 1994; 7:383-389
78. Wattler S., Russ A., Evans M., et al. A combined analysis of genomic and primary protein structure defines the phylogenetic relationship of new members if the T-box family. Genomics 1998; 48:24-33
79. Agulnik S.I., Ruvinsky I. and Silver L.M., Three novel T-box genes in Caenorhabditis elegans. Genome 1997; 40:458-464
80. Tushar K. Ghosh, Elizabeth A. Packham, Andrew J. Bonser, et al. Characterization of the TBX5 binding site and analysis of mutations that cause Holt-Oram syndrome. Hum. Mol. Genet. 2001; 18:1983-1994
81. Benoit G. Bruneau, Georges Nemer, Joachim P. Schmitt, et al. A Murine Model of Holt-Oram Syndrome Defines Roles of the T-Box Transcription Factor Tbx5 in Cardiogenesis and Disease. Cell 2001; 106:709-721
82. Papaioannou, V.E., and Silver, L.M. The T-box gene family. Bioessays 1998; 20:9–19
83. Bruneau, B.G., Logan, M., Davis, N., Levi, T., Chamber-specific cardiac expression of Tbx5 and heart defects in Holt- Oram syndrome. Dev. Biol. 1999; 211:100–108
84. Chapman, D. L., Garvey, N., Hancock, S., et al. Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development. Dev. Dyn. 1996; 206:379–390
85. Bamshad M., Lin R.C., Law D.J., et al. Mutations in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome. Nat. Genet. 1997; 16:311–315
86. Merscher S., Funke B., Epstein J.A., et al. TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell 2001; 104:619–629
87. Kispert A., and Hermann B.G., The Brachyury gene encodes a novel DNA binding protein. EMBO 1993; 12:4898–4899
88. Borresen, A.L., et al. Constant Denaturant Gel Electrophoresis as a Rapid Screening Technique for p53 Mutations. Proc. Natl. Acad. Sci. 1991; 88:8405-8409
89. Jerome L.A., Papaioannou V.E., Nature Genet. 2001; 27:286-291
90. Bonnet D., Cormier-Daire V., Kachaner J., et al. Microstallite DNA markers detects 95％ of chromosome 22q11 deletions. Am. J. Med. Genet. 1997; 68:182-184
91. Driscoll D.A., Emanuel B.S., Mitchell L.E., et al. PCR assay for screening patients at risk for 22q11.2 deletion. Genet. Test. 1997; 1:109-113
92. Francesca V., Masae M., Ilaria T., et al. Tbx1 mutation causes multiple cardiovascular defects and discrupts neural crest and cranial nerve migratory pathways. Hum. Mol. Genet. 2002; 11:915-922
93. Driscoll D.A., Budarf M.L., and Emanuel B.S., Antenatal diagnosis of Digeorge syndrome. Lancet. 1991; 338:1390-1391
94. Puder K.S., Humes R.A., Gold R.L., et al. The genetic implication for preceding generations of the prenatal diagnosis of interrupted aortic arch in association with unsuspected DiGeorge anomaly. Am. J. Obstet. Gynecol. 1995; 173:239-41
95. Davidson A., Khandelwal M., and Punnett H.H., Prenatal diagnosis of the 22q11 deletion syndrome. Prenat. Diagn. 1997; 17:380-3