**6. Abbreviations**

ASD= atrial septal defect; VSD= ventricular septal defect; CSD= cardiac septal defect; SRF= serum response factor (c-fos serum response element-binding transcription factor); FGF=fibroblast growth factor; BMP= bone morphogenetic protein; Nodal= nodal homolog; Wnt= wingless-type MMTV integration site family; Shh= sonic hedgehog homolog; Ihh= Indian hedgehog homolog; VEGF= vascular endothelial growth factor; NFATc1= nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1; ß-catenin= catenin (cadherin-associated protein), beta 1; TGF-ß= transforming growth factor, beta 1; EGF= epidermal growth factor (beta-urogastrone) ; erbB= v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog; NF1= neurofibromin 1;MEF2= myocyte enhancing factor 2; Hand= transcription factor protein; RUNX2= runtrelated transcription factor 2; NOTCH= Notch homolog; Hesr1/Hey1= hairy/enhancer-ofsplit related with YRPW motif 1; BAF60C= a subunit of chromatin-remodelling complex BAF; SMYD1= histone methyltransferase.

#### **7. References**

134 Congenital Heart Disease – Selected Aspects

the chromatin architecture of extensive regions of DNA. Non-coding RNA, in the context of this review, refers to transcripts expressed and processed in the nucleus much like any protein coding gene, but lacking an open reading frame and often transcribed antisense to bona fide protein coding genes. Dysregulation of miRNAs might result in congenital heart disease in humans. Further studies of miRNAs in CHD are required. **Epigenetics** There is increasing evidence that epigenetic modifications, arising primarily through DNA methylation and histone modifications may have as important a role as genetics in certain diseases, such as cancer, birth defects, developmental disorders, and psychiatric disorders. **Bioinformatics** Unprecedented growth in the interdisciplinary domain of biomedical informatics reflects the recent advancements in genomic sequence availability, high-content biotechnology screening systems, as well as the expectations of computational biology to command a leading role in drug discovery and disease characterization. These forces have moved much of life sciences research almost completely into the computational domain. Human genome project has succeeded, and postgenome era is following. Human genome comprises 30, 000-40, 000 genes, but their functions, relation, interaction, and regulation remain unknown. Bioinformatics is a powerful and indispensable tool in exploring the

Congenital heart disease (CHD) is the most common type of birth defect. Despite of the many advances in our understanding of cardiac development and many genes related to cardiac development identified, the fundamental etiology for the majority of cases of congenital heart disease remains unknown. CHD is a multifactorial complex disease, with environmental and genetic factors playing important roles. A number of causative genes of selected congenital heart defects and genetic syndromes have been found. The molecular mechanisms of CHD may include mutations in components of the cardiac gene network, altered haemodynamics, regulatory pathway of cardiac genes , microRNA dysfunction , epignetics, adult congenital heart diseases and so on. The molecular basis of CHD is an exciting and rapidly evolving field. The continuing advances in the understanding of the molecular mechanisms of CHD will hopefully result in improved genetic counseling and

This study was supported by the Ruikang Hospital Natural Science Foundation (code:

SRF= serum response factor (c-fos serum response element-binding transcription factor); FGF=fibroblast growth factor; BMP= bone morphogenetic protein; Nodal= nodal homolog; Wnt= wingless-type MMTV integration site family; Shh= sonic hedgehog homolog; Ihh= Indian hedgehog homolog; VEGF= vascular endothelial growth factor; NFATc1= nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1; ß-catenin= catenin

ASD= atrial septal defect; VSD= ventricular septal defect; CSD= cardiac septal defect;

molecular mechanisms of CHD [90, 91].

care of affected individuals and their families.

**5. Acknowledgements** 

ZKZ201001).

**6. Abbreviations** 

**4. Conclusions** 


Molecular Mechanisms of Congenital Heart Disease 137

[35] Muncke N, Jung C, Rüdiger H, et al. Missense mutations and gene interruption in

[37] Heritage ML, MacMillan JC, Anderson GJ. DHPLC mutation analysis of Jagged1 (JAG1)

[38] Robinson SW, Morris CD, Goldmuntz E, et al. Missense mutations in CRELD1 are

[39] Tomita-Mitchell A, Maslen CL, Morris CD, et al. GATA4 sequence variants in patients

[40] Zhu L, Zhou G, Poole S, et al. Characterization of the interactions of human ZIC3

[41] Harrison CA, Gray PC, Fischer WH, et al. An activin mutant with disrupted ALK4 binding blocks signaling via type II receptors. *J Biol Chem.* 2004;279(27):28036-44. [42] Besser D. Expression of nodal, lefty-a, and lefty-B in undifferentiated human

[43] Ferland RJ, Gaitanis JN, Apse K, et al. Periventricular nodular heterotopia and Williams

[44] Fan C, Liu M, Wang Q. Functional analysis of TBX5 missense mutations associated with

[45] Zhao F, Weismann CG, Satoda M, et al. Novel TFAP2B mutations that cause Char

[46] Ko JM, Kim JM, Kim GH, et al. PTPN11, SOS1, KRAS, and RAF1 gene analysis, and

[47] Roberts AE, Araki T, Swanson KD, et al. Germline gain-of-function mutations in SOS1

[48] Wincent J, Holmberg E, Strömland K, et al. CHD7 mutation spectrum in 28 Swedish patients diagnosed with CHARGE syndrome. *Clin Genet.* 2008;74(1):31-8. [49] Temtamy SA, Aglan MS, Valencia M, et al. CHD7 mutation spectrum in 28 Swedish patients diagnosed with CHARGE syndrome. *Hum Mutat.* 2008;29(7):931-8. [50] Ulucan H, Gül D, Sapp JC, et al. Extending the spectrum of Ellis van Creveld

[51] Li D, Yu J, Gu F, et al. The roles of two novel FBN1 gene mutations in the genotype-

with congenital heart disease. *Eur J Med Genet.* 2008;51(6):527-35.

(transposition of the great arteries). *Circulation.* 2003;108(23):2843-50. [36] Finelli P, Pincelli AI, Russo S, et al. Disruption of friend of GATA 2 gene (FOG-2) by a

gonadal dysgenesis. *Clin Genet.* 2007;71(3):195-204..

mutants with GLI3. *Hum Mutat.* 2008;29(1):99-105.

syndrome. *Am J Med Genet A.* 2006;140(12):1305-11.

Holt-Oram syndrome. *J Biol Chem*.2003;278(10):8780-5.

cause Noonan syndrome. *Nat Genet.* 2007;39(1):70-4.

marfanoid habitus. *Genet Test.* 2008;12(2):325-30.

2002;20(6):481.

2003;72(4):1047-52

2004;279(43):45076-84.

2001;69(4):695-703.

2008;9:92

*Genet.* 2008;53(11-12):999-1006.

PROSIT240, a novel TRAP240-like gene, in patients with congenital heart defect

de novo t(8;10) chromosomal translocation is associated with heart defects and

reveals six novel mutations in Australian alagille syndrome patients. *Hum Mutat.*

associated with cardiac atrioventricular septal defects. *Am J Hum Genet.*

embryonic stem cells requires activation of Smad2/3. *J Biol Chem.* 

syndrome provide a genotype-phenotype correlation. *Am J Hum Genet.*

genotype-phenotype correlation in Korean patients with Noonan syndrome. *J Hum* 

syndrome: a large family with a mild mutation in the EVC gene. *BMC Med Genet.*

phenotype correlations of Marfan syndrome and ectopia lentis patients with


[17] Xiao H, Zhang YY. Understanding the role of transforming growth factor-beta

[18] Moon JI, Birren SJ. Target-dependent inhibition of sympathetic neuron growth via modulation of a BMP signaling pathway. *Dev Biol.* 2008; 315(2):404-17. [19] Mjaatvedt CH, Nakaoka T, Moreno-Rodriguez R, et al. The outflow tract of the heart is recruited from a novel heart-forming field. *Dev. Biol.* 2001; 238 (1): 97–109. [20] Bruneau BG. The developmental genetics of congenital heart disease.*Nature*. 2008;

[21] High FA, Epstein JA. The multifaceted role of Notch in cardiac development and

[22] Armstrong EJ, Bischoff J. Heart valve development: endothelial cell signaling and

[23] Yutzey KE, Colbert M, Robbins J. Ras-related signaling pathways in valve

[24] Joziasse IC, van de Smagt JJ, Smith K, et al. Genes in congenital heart disease: atrioventricular valve formation. *Basic Res Cardiol*. 2008;103(3):216-27. [25] Pierpont ME, Basson CT, Benson DW, et al. Genetic basis for congenital heart defects:

[26] Ikeda Y, Hiroi Y, Hosoda T, et al. Novel point mutation in the cardiac transcription

[27] McElhinney DB, Geiger E, Blinder J, et al. NKX2.5 mutations in patients with congenital

[28] Elliott DA, Kirk EP, Yeoh T, et al. Cardiac homeobox gene NKX2–5 mutations and

[29] Schubbert S, Zenker M, Rowe SL, et al. Germline KRAS mutations cause Noonan

[30] Tartaglia M, Kalidas K, Shaw A, et al. PTPN11 mutations in Noonan syndrome:

[31] Toko H, Zhu W, Takimoto E, et al. Csx/Nkx2-5 Is Required for Homeostasis and

[32] Akazawa H, Komuro I. Cardiac transcription factor Csx/Nkx2-5: Its role in cardiac

[33] Goldmuntz E, Bamford R, Karkera JD, et al. CFC1 mutations in patients with

[34] Davit-Spraul A, Baussan C, Hermeziu B, et al. CFC1 gene involvement in biliary atresia with polysplenia syndrome. *J Pediatr Gastroenterol Nutr.* 2008;46(1):111-2.

development and diseases. *Pharmacol Ther.*2005;107(2):252-68.

current knowledge: a scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics. *Circulation.* 

factor CSX/NKX2.5 associated with congenital heart disease. *Circ J.* 2002;66:561–

congenital heart disease: associations with atrial septal defect and hypoplastic left

molecular spectrum, genotype-phenotype correlation, and phenotypic

Survival of Cardiac Myocytes in the Adult Heart. *J Biol Chem.* 2002;277(27):24735-43.

transposition of the great arteries and double-outlet right ventricle. *Am J Hum* 

development:ebb and flow. *Physiology* (Bethesda). 2005 ;20:390-7.

*Pharmacol Physiol.*2008;35(3):335-41.

disease. *Nat Rev Genet.* 2008;9(1):49-61.

differentiation. *Circ Res.* 2004;3;95(5):459-70.

heart disease. *J Am Coll Cardiol.* 2003;42:1650 –1655.

heart syndrome. *J Am Coll Cardiol.* 2003;41:2072–2076.

heterogeneity. *Am J Hum Genet.* 2002;70:1555–1563.

syndrome. *Nat Genet.* 2006;38:331–336.

*Genet.* 2002;70(3):776-80.

451(7181):943-8.

2007;115:3015–3038.

563.

signalling in the heart: overview of studies using genetic mouse models. *Clin Exp* 


Molecular Mechanisms of Congenital Heart Disease 139

[70] Kwon C, Arnold J, Hsiao EC, et al. Canonical Wnt signaling is a positive regulator of mammalian cardiac progenitors. *Proc Natl Acad Sci U S A*.2007;104(26):10894-9. [71] Miller CT, Swartz ME, Khuu PA, et al. mef2ca is required in cranial neural crest to effect

[72] Ren X, Li Y, Ma X, et al. Activation of p38/MEF2C pathway by all-trans retinoic acid in

[73] Ueno S, Weidinger G, Osugi T, et al. Biphasic role for Wnt/beta-catenin signaling in

[74] Groenendijk BC, Stekelenburg-de Vos S, Vennemann P, et al. The endothelin-1 pathway

[75] Couzin J. Breakthrough of The Year: Small RNAs make big splash, *Science.* 2002;298

[76] Liu CG, Calin GA, Meloon B, et al. An oligonucleotide microchip for genome-wide

[77] Lee CT, Risom T, Strauss WM. MicroRNAs in mammalian development. *Birth Defects* 

[78] Zhang B, Wang Q, Pan X. MicroRNAs and their regulatory roles in animals and plants,

[79] Zhao Y. Ransom JF, Li A, et al. Dysregulation of cardiogenesis, cardiac conduction, and

[80] Zhao Y, Samal E, Srivastava D. Serum sponse factor regulates a muscle-specific

[81] Kwon C, Han Z, Olson EN, et al. MicroRNA1 influences cardiac differentiation in

[82] Wong AH, Gottesman II, Petronis A. Phenotypic differences in genetically identical

[83] Armstrong L, Lako M, Dean W, et al. Epigenetic modification is central to genome reprogramming in somatic cell nuclear transfer. *Stem Cells*.2006; 24(4):805-814. [84] Jacinto FV, Esteller M, Mutator pathways unleashed by epigenetic silencing in human

[85] Warnes CA. The adult with congenital heart disease: Born to be bad? *J Am Coll Cardiol.* 

[86] Cambien F, Tiret L, Genetics of cardiovascular diseases: from single mutations to the

[87] Gatzoulis MA. Adult congenital heart disease: a cardiovascular area of growth in

[88] Cohen JC. Genetic approaches to coronary heart disease. *J Am Coll Cardiol*. 2006;48:A10–

urgent need of additional resource allocation. *Int. J. Cardiol*. 2004; 97 (Suppl 1): 1–

microRNA that targets Hand2 during cardiogenesis, *Nature*.2005;436 (7048):214-

Drosophila and regulates Notch signaling, *Proc Natl Acad Sci USA.* 2005;102

organisms: the epigenetic perspective. *Human Molecular Genetics,* 2005, 14(1):R11–

cell cycle in mice lacking miRNA-1-2. *Cell.* 2007;129 (2):303–317.

cardiac specification in zebrafish and embryonic stem cells. *Proc Natl Acad Sci U S* 

and the development of cardiovascular defects in the haemodynamically

microRNA profiling in human and mouse tissues. *Proc Natl Acad Sci U S A.* 

Endothelin1 signaling in zebrafish. *Dev Biol.* 2007;308(1):144-57.

challenged chicken embryo. *J Vasc Res*. 2008;45(1):54-68.

cardiac myoblasts. *Life Sci.* 2007;81(2):89-96.

*Res C Embryo Today.* 2006;78 (2):129-139.

*J Cell Physiol* .2007;210 (2): 279-289.

cancer. *Mutagenesis.* 2007;22(4):247-53.

whole genome. *Circulation*,2007;116;1714-1724 .

*A.* 2007;104(23):9685-90.

(5602):2296-2297.

2004;101(26):9740-4.

(52):18986–18991.

220.

R18.

2.

A14.

2005;46:1–8.


[52] Singh KK, Rommel K, Mishra A, et al. TGFBR1 and TGFBR2 mutations in patients with

[53] Niihori T, Aoki Y, Narumi Y, et al. Germline KRAS and BRAF mutations in cardio-

[54] Roberts A, Allanson J, Jadico SK, et al. The cardiofaciocutaneous syndrome.*J Med Genet.*

[55] Nava C, Hanna N, Michot C, et al. Cardio-facio-cutaneous and Noonan syndromes

[56] Quezada E, Gripp KW. Costello syndrome and related disorders.*Curr Opin Pediatr.*

[57] Clark EB. Mechanisms in the pathogenesis of congenital heart disease. In: Pierpont ME,

[58] Clark EB. Pathogenetic mechanisms of congenital cardiovascular malformations

[59] Benoit G. Bruneau. The developmental genetics of congenital heart disease. *Nature*.

[60] Hyun C, Lavulo L. Congenital heart diseases in small animals: Part I. Genetic pathways and potential candidate genes. *The Veterinary Journal*; 2006; 171 (2): 245–255. [61] Hiroi Y, Kudoh S, Monzen K, et al. Tbx5 associates with Nkx2-5 and synergistically promotes cardiomyocyte differentiation. *Nat. Genet.* 2001;28 (3): 276–280. [62] Goldmuntz E, Geiger E, Benson DW. NKX2.5 mutations in patients with tetralogy of

[63] Stennard FA, Costa MW, Elliott DA, et al. Cardiac T-box factor Tbx20 directly interacts

[64] Pashmforoush M, Lu JT, Chen H, et al. Nkx2-5 pathways and congenital heart disease:

[66] Moskowitz IP, Kim JB, Moore ML, et al. A molecular pathway including Id2, Tbx5, and

[67] Ching YH, Ghosh TK, Cross SJ, et al. Mutation in myosin heavy chain 6 causes atrial

[68] Varadkar P, Kraman M, Despres D, et al. Notch2 is required for the proliferation of cardiac neural crest-derived smooth muscle cells. *Dev Dyn.* 2008;237(4):1144-52. [69] Del Monte G, Grego-Bessa J, González-Rajal A, et al. Monitoring Notch1 activity in

cardiomyopathy and complete heart block. *Cell .*2004;117 (3): 373–386. [65] Pu WT, Ishiwata T, Juraszek AL, et al. GATA4 is a dosage-sensitive regulator of cardiac

with Nkx2-5, GATA4, and GATA5 in regulation of gene expression in the

loss of ventricular myocyte lineage specification leads to progressive

Nkx2-5 required for cardiac conduction system development. *Cell*. 2007;129(7):1365-

development: evidence for a feedback regulatory loop. *Dev Dyn*. 2007;236(9):2594-

facio-cutaneous syndrome. *Nat Genet.* 2006;38(3):294-6.

2006;27(8):770-7.

2007;19(6):636-44.

Nijoff; 1986. p. 3 –11.

2008;451(7181):943-8.

revisited. *Semin Perinatol* 1996;20:465– 72.

fallot. *Circulation*. 2001;104 (21): 2565–2568.

developing heart. *Dev Biol*. 2003 ;262(2):206-24.

morphogenesis. *Dev. Biol.* 2004;275 (1): 235–244.

septal defect. *Nat. Genet*. 2005;37 (4): 423–428.

71.

76.

614.

2006 Nov;43(11):833-42.

features of Marfan syndrome and Loeys-Dietz syndrome. *Hum Mutat.* 

due to mutations in the RAS/MAPK signalling pathway: genotype-phenotype relationships and overlap with Costello syndrome. *J Med Genet.* 2007;44(12):763-

Moller J, editors. The genetics of cardiovascular disease. Boston, MA' Martinus-


**6** 

*1USA 2Japan* 

*Drosophila* **Model of Congenital Heart Diseases** 

Congenital heart defects (CHD) are the most common birth defects, occurring in about 0.7% of all newborn infants. There are multiple lines of evidence that genetic components are involved in developing CHD pathogenesis. An important aspect in understanding disease mechanisms is that in addition to contributions from a single disease-causing gene (usually seen in many familial cases of CHD), a multitude of other genetically interacting loci can also influence the severity or progression of the disease, often diagnosed as idiopathic CHD. It is likely that such genetic interactions underlie a large proportion of cases of idiopathic CHD, where a direct link to known cardiogenic genes yet to be identified. Recent advances in stem cell research and in the growing field of systems biology provide a tremendous amount of new data leading to new hypotheses and to new heart disease gene candidates that may also have potential roles during heart formation and establishment of cardiac function. Usually, these hypotheses are tested in cell-based assays and eventually in the mouse model, however both systems have their own particular set of limitations. In this article we review recent advancements in using *Drosophila melanogaster* as a model organism

to study basic mechanisms of heart development, cardiac function and disease.

**2. Comparison between** *Drosophila* **and vertebrate cardiogenesis** 

The early development of the *Drosophila* heart shows remarkable similarities with its vertebrate counterparts, both morphologically and genetically (for review, see Bodmer, 1995; Bier and Bodmer, 2004). Our understanding of the regulation of cardiac development by a core cardiac transcription factor network (Venkatesh et al., 2000; Cripps and Olson, 2002; Olson, 2006; Bodmer and Frasch, 2010) began with the identification of the *Drosophila Nkx2.5* homologue *tinman* twenty years ago (Bodmer et al., 1990; Azpiazu and Frasch, 1993; Bodmer, 1993). One decade later, the completion of the sequencing of the *Drosophila*, mouse and human genomes has led to the identification of fly homologues of most cardiac transcription factors. The *Drosophila* model allowed the extensive genetic screening and functional analysis of Tinman (NKX2.5 Yin et al., 1997; Akasaka et al., 2006; Zaffran et al., 2006; Qian et al., 2011; Ryu et al., 2011), Hand (dHAND, eHAND, Han and Olson, 2005; Han

**1. Introduction** 

Georg Vogler1, Rolf Bodmer1 and Takeshi Akasaka1,2

*Sanford-Burnham Medical Research Institute, La Jolla* 

*2Cardiology and Catheterization Laboratories, Shonan Kamakura General Hospital, Kanagawa* 

*1Development and Aging Program,* 

