ELN, Elastin

This gene encodes a protein that is one of the two components of elastic fibers. Deletions and mutations in this gene are associated with supravalvular aortic stenosis (SVAS) and autosomal dominant cutis laxa [43].

#### TBX5, T-box 5

This gene is a member of a phylogenetically conserved family of genes that share a common DNA-binding domain, the T-box. The encoded protein may play a role in heart development and specification of limb identity. Mutations in this gene have been associated with Holt-Oram syndrome [44].

TFAP2B, transcription factor AP-2 beta

This gene encodes a member of the AP-2 family of transcription factors. This protein functions as both a transcriptional activator and repressor. Mutations in this gene result in autosomal dominant Char syndrome, suggesting that this gene functions in the differentiation of neural crest cell derivatives [45].

PTPN11, protein tyrosine phosphatase, non-receptor type 11

The protein encoded by this gene is a member of the protein tyrosine phosphatase (PTP) family. PTPs are known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. Mutations in this gene are a cause of Noonan syndrome as well as acute myeloid leukemia [46].

SOS1, son of sevenless homolog 1

This gene encodes a protein that is a guanine nucleotide exchange factor for RAS proteins, membrane proteins that bind guanine nucleotides and participate in signal transduction pathways. Mutations in this gene are associated with gingival fibromatosis 1 and Noonan syndrome type 4 [47].

CHD7, chromodomain helicase DNA binding protein 7

This gene encodes a protein that contains several helicase family domains. Mutations in this gene have been found in some patients with the CHARGE syndrome [48, 49].

#### EVC, Ellis van Creveld syndrome

This gene encodes a protein containing a leucine zipper and a transmembrane domain. This gene has been implicated in both Ellis-van Creveld syndrome (EvC) and Weyers acrodental dysostosis [50].

#### FBN1, fibrillin 1

This gene encodes a member of the fibrillin family. Mutations in this gene are associated with Marfan syndrome, isolated ectopia lentis, autosomal dominant Weill-Marchesani syndrome, MASS syndrome, and Shprintzen-Goldberg craniosynostosis syndrome [51].

This gene encodes a member of the TGF-beta family of proteins. The encoded protein is secreted and plays a role in left-right asymmetry determination of organ systems during development.Mutations in this gene have been associated with left-right axis malformations,

This gene encodes a protein that is one of the two components of elastic fibers. Deletions and mutations in this gene are associated with supravalvular aortic stenosis (SVAS) and

This gene is a member of a phylogenetically conserved family of genes that share a common DNA-binding domain, the T-box. The encoded protein may play a role in heart development and specification of limb identity. Mutations in this gene have been associated

This gene encodes a member of the AP-2 family of transcription factors. This protein functions as both a transcriptional activator and repressor. Mutations in this gene result in autosomal dominant Char syndrome, suggesting that this gene functions in the

The protein encoded by this gene is a member of the protein tyrosine phosphatase (PTP) family. PTPs are known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. Mutations

This gene encodes a protein that is a guanine nucleotide exchange factor for RAS proteins, membrane proteins that bind guanine nucleotides and participate in signal transduction pathways. Mutations in this gene are associated with gingival fibromatosis 1 and Noonan

This gene encodes a protein that contains several helicase family domains. Mutations in this

This gene encodes a protein containing a leucine zipper and a transmembrane domain. This gene has been implicated in both Ellis-van Creveld syndrome (EvC) and Weyers acrodental

This gene encodes a member of the fibrillin family. Mutations in this gene are associated with Marfan syndrome, isolated ectopia lentis, autosomal dominant Weill-Marchesani syndrome, MASS syndrome, and Shprintzen-Goldberg craniosynostosis syndrome [51].

in this gene are a cause of Noonan syndrome as well as acute myeloid leukemia [46].

gene have been found in some patients with the CHARGE syndrome [48, 49].

LEFTYA, left-right determination factor 2

particularly in the heart and lungs [42].

autosomal dominant cutis laxa [43].

with Holt-Oram syndrome [44].

SOS1, son of sevenless homolog 1

EVC, Ellis van Creveld syndrome

syndrome type 4 [47].

dysostosis [50]. FBN1, fibrillin 1

TFAP2B, transcription factor AP-2 beta

differentiation of neural crest cell derivatives [45].

PTPN11, protein tyrosine phosphatase, non-receptor type 11

CHD7, chromodomain helicase DNA binding protein 7

ELN, Elastin

TBX5, T-box 5

TGFBR2, transforming growth factor receptor 2

This gene encodes a member of the Ser/Thr protein kinase family and the TGFB receptor subfamily. Mutations in this gene have been associated with Marfan Syndrome, Loeys-Deitz Aortic Aneurysm Syndrome, and the development of various types of tumors [52].

KRAS, v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog

This gene, encodes a protein that is a member of the small GTPase superfamily. The transforming protein that results is implicated in various malignancies, including lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas and colorectal carcinoma [53].

BRAF, v-raf murine sarcoma viral oncogene homolog B1

This gene encodes a protein belonging to the raf/mil family of serine/threonine protein kinases. This protein plays a role in regulating the MAP kinase/ERKs signaling pathway, which affects cell division, differentiation, and secretion. Mutations in this gene are associated with cardiofaciocutaneous syndrome [53].

MEK1, MAP2K1, mitogen-activated protein kinase 1

The protein encoded by this gene is a member of the dual specificity protein kinase family, which acts as a mitogen-activated protein (MAP) kinase kinase. This kinase is involved in many cellular processes such as proliferation, differentiation, transcription regulation and development [54].

MEK2, MAP2K2*,* mitogen-activated protein kinase 2

The protein encoded by this gene is a dual specificity protein kinase that belongs to the MAP kinase kinase family. This kinase is known to play a critical role in mitogen growth factor signal transduction. Mutations in this gene cause cardiofaciocutaneous syndrome (CFC syndrome) [55].

HRAS, v-Ha-ras Harvey rat sarcoma viral oncogene homolog

This gene belongs to the Ras oncogene family. The products encoded by these genes function in signal transduction pathways. Mutations in this gene cause Costello syndrome. Defects in this gene are implicated in a variety of cancers [56].

### **2.2 Pathogenic mechanisms of congenital heart disease**

Phenotypes of CHD vary from small ASD and VSD, which may go undetected throughout life, to large ASD and VSD, which are significantly symptomatic. Clinically significant anomalies range from persistence of fetal circulation (eg, patent ductus arteriosus) to complex defects such as transposition of the great vessels, single ventricle anomaly, hypoplastic left heart syndrome, and complex variants of heterotaxy. The etiological factors of many genetic syndromes and familial CHD have been identified, but the genetic basis of majority of "sporadic" CHD remains unknown. It is hypothesized that susceptibility resulted from single nucleotide polymorphisms or key gene(s), with the interaction of environmental factors, which disturb normal cardiac development, result in cardiac defects. There are six causative mechanisms according to pathogenetic classification of congenital cardiovascular malformations: ectomesenchymal tissue migration abnormalities (causing conotruncal malformations and aortic arch anomalies); intracardiac blood flow defects

Molecular Mechanisms of Congenital Heart Disease 129

Hairless (Su(H)). Su(H) binds to the regulatory sequences of the Enhancer-of-Split locus, upregulating the expression of basic helix-loop-helix proteins, which in turn regulate the expression of downstream target genes. Upon ligand binding, a signal is transmitted intracellularly by a process involving the proteolytic cleavage of the receptor and the subsequent nuclear translocation of the Notch intracellular domain (NICD)(Figure 3). Alagille syndrome is an autosomal dominant disorder characterized by developmental abnormalities of the liver, heart, eye, skeleton and, at lower penetrance, several other organs. Most cases of Alagille syndrome are caused by *JAG1* mutations, although a small number of Alagille syndrome patients with *NOTCH2* mutations have been identified. The cardiac defects associated with Alagille syndrome include pulmonary artery stenosis and hypoplasia, pulmonic valve stenosis, and tetralogy of Fallot. These defects are likely to be due to a requirement for Notch signaling-mediated differentiation of cardiac neural crest cells into smooth muscle cells, which has been demonstrated in a mouse model. Bicuspid aortic valve affects 1-2% of the population, making it the most common congenital cardiac malformation. Bicuspid aortic valve predisposes one to aortic valve calcification. Aortic valve calcification was linked to Notch regulation of the transcription factor RUNX2.Heterozygous mutations in the *NOTCH1* gene were found in two families with autosomal-dominant aortic valve disease. *NOTCH1* mutations are also found in 4% of sporadic bicuspid aortic valve patients. The formation of bicuspid aortic valve might reflect the role of Notch signaling in regulating the epithelial-mesenchymal transition required for the generation of the heart valves [20, 69, 70]. Recently, mutations in Notch1 in humans have been shown to cause aortic valve defects and activation of Notch1 in mouse leads to abnormal cardiogenesis characterized by deformities of the ventricles and atrioventricular canal. Additionally, mutations in various Notch signaling pathway genes, including Jagged1, mind bomb 1, Hesr1/Hey1, and Hesr2/Hey2, result in cardiac defects, such as pericardial edema, atrial and ventricular septal defects, cardiac

cushion, and valve defects [71-74].

Fig. 2. Interaction of NKX2.5,TBX5 and GATA4

(causing septal defects and left or right heart obstructive malformations); cell death abnormalities (causing septal defects and valve abnormalities); extra cellular matrix abnormalities (causing atrioventricular canal defects); abnormal targeted growth (causing partial or total anomalous pulmonary venous return and cor triatriatum); and abnormal situs and looping (causing left–right positioning problems) [57, 58].

### **2.2.1 Mutations in components of the cardiac gene network cause CHD**

Heart development is controlled by a highly conserved network of transcription factors that connect signaling pathways with genes of muscle growth, patterning, and contractility. The core transcription factor network consists of *NKX2, MEF2, GATA, TBX,*  and *Hand.* Dozens of other transcription factors contribute to cardiogenesis, in many cases by serving as accessory factors for these core regulators. Autoregulatory and cross regulatory of the cardiac gene network maintain the cardiac phenotype once the network has been activated by upstream inductive signals. Mutations in components of the cardiac gene Network cause CHD [59, 60].

Mutations in *NKX2.5* cause a spectrum of congenital heart defects, including atrial-septal defects (ASDs), ventricular-septal defects (VSDs), and cardiac conduction abnormalities. Mutations in TBX5 cause the congenital disease Holt–Oram syndrome, which is characterized by truncations of the upper limbs and heart malformations [61, 62]. Mutations in *GATA4*, some of which disrupt its interaction with *TBX5*, cause ASDs and VSDs. In mouse models, haploinsufficiency for Nkx2-5 or Tbx5 resulted in an increased incidence of structural heart disease, confirming that normal heart development is sensitive to small changes in expression levels of Nkx2-5 and Tbx5. GATA4 also is an essential, dosagedependent regulator of cardiac morphogenesis. The missense mutation in Gata4 specifically disrupted the Gata4-Tbx5 interaction while maintaining its ability to interact with Nkx2.5. In previous studies, Tbx5 had been shown to interact with Nkx2.5, demonstrating that all three transcription factors could physically interact *in vitro*. In summary, a mutation in any of these three genes can result in human CSD and suggests that these three genes may work to direct common molecular pathways critical for cardiac septum formation. [63, 64]. Consistent with this, mutations in *MYH6*, a downstream transcriptional target of GATA4 and TBX5, was implicated as a cause of human atrial septal defects .TBX5, GATA4 and NKX2-5 function together only to activate genes. The overlapping expression patterns and complex interactions of these transcription factors allow fine regulation of cardiac gene expression and morphogenesis [20, 65-67] (Figure 2).

#### **2.2.2 Regulatory pathway of cardiac genes**

Several types of congenital heart disease involve valve defects of varying severity. Notch signaling is an ancient intercellular signaling mechanism that plays an important role during valve development. Mutations affecting signaling proteins and downstream pathways can lead to valve disease. In mammals, four Notch family receptors have been described: NOTCH1 through to NOTCH4 [20, 68]. The Notch ligands are encoded by the Jagged (*JAG1*  and *JAG2*) and Delta-like (*DLL1*, *DLL3* and *DLL4*) gene families. The Notch signaling pathway is an evolutionarily conserved mechanism used by metazoans to control cell fate decisions through local cell interactions. The notch gene encodes a single-pass transmembrane protein receptor that interacts with its ligands, Delta and Serrate/Jagged. Upon binding of the ligand, the intracellular domain of Notch (NIC) undergoes proteolytic cleavage, and is translocated to the nucleus. In the nucleus, NIC binds to its major downstream effector, Suppressor-of-

(causing septal defects and left or right heart obstructive malformations); cell death abnormalities (causing septal defects and valve abnormalities); extra cellular matrix abnormalities (causing atrioventricular canal defects); abnormal targeted growth (causing partial or total anomalous pulmonary venous return and cor triatriatum); and abnormal

Heart development is controlled by a highly conserved network of transcription factors that connect signaling pathways with genes of muscle growth, patterning, and contractility. The core transcription factor network consists of *NKX2, MEF2, GATA, TBX,*  and *Hand.* Dozens of other transcription factors contribute to cardiogenesis, in many cases by serving as accessory factors for these core regulators. Autoregulatory and cross regulatory of the cardiac gene network maintain the cardiac phenotype once the network has been activated by upstream inductive signals. Mutations in components of the cardiac

Mutations in *NKX2.5* cause a spectrum of congenital heart defects, including atrial-septal defects (ASDs), ventricular-septal defects (VSDs), and cardiac conduction abnormalities. Mutations in TBX5 cause the congenital disease Holt–Oram syndrome, which is characterized by truncations of the upper limbs and heart malformations [61, 62]. Mutations in *GATA4*, some of which disrupt its interaction with *TBX5*, cause ASDs and VSDs. In mouse models, haploinsufficiency for Nkx2-5 or Tbx5 resulted in an increased incidence of structural heart disease, confirming that normal heart development is sensitive to small changes in expression levels of Nkx2-5 and Tbx5. GATA4 also is an essential, dosagedependent regulator of cardiac morphogenesis. The missense mutation in Gata4 specifically disrupted the Gata4-Tbx5 interaction while maintaining its ability to interact with Nkx2.5. In previous studies, Tbx5 had been shown to interact with Nkx2.5, demonstrating that all three transcription factors could physically interact *in vitro*. In summary, a mutation in any of these three genes can result in human CSD and suggests that these three genes may work to direct common molecular pathways critical for cardiac septum formation. [63, 64]. Consistent with this, mutations in *MYH6*, a downstream transcriptional target of GATA4 and TBX5, was implicated as a cause of human atrial septal defects .TBX5, GATA4 and NKX2-5 function together only to activate genes. The overlapping expression patterns and complex interactions of these transcription factors allow fine regulation of cardiac gene

Several types of congenital heart disease involve valve defects of varying severity. Notch signaling is an ancient intercellular signaling mechanism that plays an important role during valve development. Mutations affecting signaling proteins and downstream pathways can lead to valve disease. In mammals, four Notch family receptors have been described: NOTCH1 through to NOTCH4 [20, 68]. The Notch ligands are encoded by the Jagged (*JAG1*  and *JAG2*) and Delta-like (*DLL1*, *DLL3* and *DLL4*) gene families. The Notch signaling pathway is an evolutionarily conserved mechanism used by metazoans to control cell fate decisions through local cell interactions. The notch gene encodes a single-pass transmembrane protein receptor that interacts with its ligands, Delta and Serrate/Jagged. Upon binding of the ligand, the intracellular domain of Notch (NIC) undergoes proteolytic cleavage, and is translocated to the nucleus. In the nucleus, NIC binds to its major downstream effector, Suppressor-of-

situs and looping (causing left–right positioning problems) [57, 58].

gene Network cause CHD [59, 60].

expression and morphogenesis [20, 65-67] (Figure 2).

**2.2.2 Regulatory pathway of cardiac genes** 

**2.2.1 Mutations in components of the cardiac gene network cause CHD** 

Hairless (Su(H)). Su(H) binds to the regulatory sequences of the Enhancer-of-Split locus, upregulating the expression of basic helix-loop-helix proteins, which in turn regulate the expression of downstream target genes. Upon ligand binding, a signal is transmitted intracellularly by a process involving the proteolytic cleavage of the receptor and the subsequent nuclear translocation of the Notch intracellular domain (NICD)(Figure 3). Alagille syndrome is an autosomal dominant disorder characterized by developmental abnormalities of the liver, heart, eye, skeleton and, at lower penetrance, several other organs. Most cases of Alagille syndrome are caused by *JAG1* mutations, although a small number of Alagille syndrome patients with *NOTCH2* mutations have been identified. The cardiac defects associated with Alagille syndrome include pulmonary artery stenosis and hypoplasia, pulmonic valve stenosis, and tetralogy of Fallot. These defects are likely to be due to a requirement for Notch signaling-mediated differentiation of cardiac neural crest cells into smooth muscle cells, which has been demonstrated in a mouse model. Bicuspid aortic valve affects 1-2% of the population, making it the most common congenital cardiac malformation. Bicuspid aortic valve predisposes one to aortic valve calcification. Aortic valve calcification was linked to Notch regulation of the transcription factor RUNX2.Heterozygous mutations in the *NOTCH1* gene were found in two families with autosomal-dominant aortic valve disease. *NOTCH1* mutations are also found in 4% of sporadic bicuspid aortic valve patients. The formation of bicuspid aortic valve might reflect the role of Notch signaling in regulating the epithelial-mesenchymal transition required for the generation of the heart valves [20, 69, 70]. Recently, mutations in Notch1 in humans have been shown to cause aortic valve defects and activation of Notch1 in mouse leads to abnormal cardiogenesis characterized by deformities of the ventricles and atrioventricular canal. Additionally, mutations in various Notch signaling pathway genes, including Jagged1, mind bomb 1, Hesr1/Hey1, and Hesr2/Hey2, result in cardiac defects, such as pericardial edema, atrial and ventricular septal defects, cardiac cushion, and valve defects [71-74].

Fig. 2. Interaction of NKX2.5,TBX5 and GATA4

Molecular Mechanisms of Congenital Heart Disease 131

MicroRNAs are natural, single-stranded, non–protein-coding small RNA molecules (~22 nucleotides) that regulate gene expression by binding to target mRNAs and suppress its translation or initiate its degradation. Mature miRNAs are processed from -70 nucleotides long precursor miRNA (pre-miRNAs) that form hairpin secondary structures and that are often evolutionarily conserved. Pre-miRNAs are transcribed from miRNA genes. Although the specific biological roles of most miRNAs are still unknown, functional characterizations of a few of them suggest that these small RNA molecules are involved in many processes of animal development and physiology[75-77] .For example, miR-1 and miR-133 control cardiac and skeletal muscle development [78, 79]. Both genes are under the control of serum response factor, indicating that they are part of a developmental programme regulated by cardiac transcription factors. It has been shown that miR-1 targets the cardiac transcription factor HAND2. Deletion of *miR-1-2* results in heart defects that include VSDs; surviving mice have conduction system defects and increased cardiomyocyte proliferation. Dysregulation of miRNAs might result in congenital heart

Epigenetics refers to DNA and chromatin modifications that play a critical role in regulation of various genomic functions, and it was then redefined as the study of heritable traits that are not dependent on the primary sequence of DNA. Although the genotype of most cells of a given organism is the same (with the exception of gametes and the cells of the immune system), cellular phenotypes and functions differ radically, and this can be (at least to some extent) controlled by differential epigenetic regulation that is set up during cell differentiation and embryonic morphogenesis [82,83]. Once the cellular phenotype is established, genomes of somatic cells are 'locked' in tissue-specific patterns of gene expression, generation after generation. This heritability of epigenetic information

Fig. 4. Altered haemodynamics leads to CHD

**2.2.4 MicroRNA dysfunction** 

disease in human [80, 81](Figure 5).

Fig. 5. MicroRNA dysfunction results in CHD

**2.2.5 Epignetics** 

Fig. 3. Notch pathway

#### **2.2.3 Altered haemodynamics**

Haemodynamic forces have been demonstrated to play an important role in cardiac development. When these forces are impaired or when genes involved in growth and differentiation are not functioning correctly, malformations may arise. Shear stress is one of those haemodynamic forces, and the expression of many genes, including those of the endothelin pathway, changes in response to alterations in shear stress. For example, ligating the right lateral vitelline vein of chicken embryos results in cardiovascular malformations similar to those observed in knockout mice studies of components of the endothelin-1/endothelin-converting enzyme-1/ endothelin-A receptor pathway. In zebrafish, altering haemodynamics mechanically or genetically has profound consequences on heart morphology. In mice, a recent study pinpointed altered haemodynamics as a key intermediate between altered outflow tract morphogenesis and signaling events in branchial-arch artery remodeling. In human, complex congenital heart diseases with an outflow tract defect, such as tetralogy of Fallot, can be accompanied by 'accessory' congenital heart diseases, such as persistent right-sided aortic arch. Because the heart functions during its morphogenesis, haemodynamic forces might participate in cardiac morphogenesis [20, 71, 74] (Figure 4).

Fig. 4. Altered haemodynamics leads to CHD
