**2.1.1 Causative genes of CHD**

The etiological factors of many genetic syndromes and familial CHD have been identified, but the genetic basis of majority of "sporadic" CHD remains unknown. With the progress in molecular genetics and developmental biology, many genes associated heart development have been identified. When searching computer databases such as NCBI Gene Bank for "cardiac or heart", we can indentify 1154 loci in human. Search for"(heart or cardiac)and development", limited to human, 630 genes were found. A number of selected congenital heart defects and genetic syndromes have been found to be associated with mutations in a variety of single genes. The mutations were found only in affected individuals, were not present in control samples, and were demonstrated to change protein structure or function.

plate-specific expressed genes NKX2.5, SRF, GATA4, TBX5, and HAND2, compose the core regulatory network of cardiac morphogenesis, controlling heart looping, left-right symmetry and chambers formation. SRF regulates the differentiation of coronary vascular smooth muscle cells [17, 18]. Genes that involved in epicardial development include FOG-2, vascular cell adhesion molecule 1, integrins, erythropoietin, and erythropoietin receptor. Specific genes such as the NOTCH receptor, Jagged (JAG), WNT, transforming growth factor beta 2 (TGF ß2) and bone morphogenic proteins have been implicated in cardiac neural crest development in the mouse[12, 19-21]. Retinoic acid signal pathway is involved in the regulation of cardiac looping. Complex signal pathways are implicated in the crosstalk between endocardium and myocardium to form endocardial cushion and heart valves, including VEGF, NFATc1, Notch, Wnt/ß-catenin, BMP/TGF-ß, EGF, erbB, NF1

signal pathways [10, 22-24].

Fig. 1. Eteology of CHD

**2.1.1 Causative genes of CHD** 

**2. Molecular mechanisms of congenital heart disease** 

The etiological factors of many genetic syndromes and familial CHD have been identified, but the genetic basis of majority of "sporadic" CHD remains unknown. With the progress in molecular genetics and developmental biology, many genes associated heart development have been identified. When searching computer databases such as NCBI Gene Bank for "cardiac or heart", we can indentify 1154 loci in human. Search for"(heart or cardiac)and development", limited to human, 630 genes were found. A number of selected congenital heart defects and genetic syndromes have been found to be associated with mutations in a variety of single genes. The mutations were found only in affected individuals, were not present in control samples, and were demonstrated to change protein structure or function.


Disease genes of CHD identified to date are summarized in Table 1, and the functions of these causative genes are summarized as following [25].

Table 1. Causative genes of CHD

For example, NKX2-5, Homeobox-containing genes play critical roles in regulating tissuespecific gene expression essential for tissue differentiation, as well as determining the temporal and spatial patterns of development. It has been demonstrated that a Drosophila

Molecular Mechanisms of Congenital Heart Disease 125

Homeobox-containing genes play critical roles in regulating tissue-specific gene expression essential for tissue differentiation, as well as determining the temporal and spatial patterns of development. Mutations in NKX2-5 result in loss of heart formation in the embryo,

This gene encodes a member of the epidermal growth factor (EGF)- Cripto, Frl-1, and Cryptic (CFC) family. These proteins play key roles in intercellular signaling pathways during vertebrate embryogenesis. Mutations in this gene can cause autosomal visceral heterotaxy. This protein is involved in left-right asymmetric morphogenesis during organ

Also known as THRAP2, The evolutionarily conserved THRAP genes encode a family of proteins that regulate embryonic development. THRAP2 is involved in early development

The zinc finger protein encoded by this gene is a widely expressed member of the FOG family of transcription factors. The family members modulate the activity of GATA family proteins, which are important regulators of hematopoiesis and cardiogenesis in mammals

The jagged 1 protein encoded by JAG1 is the human homolog of the Drosophilia jagged protein. Human jagged 1 is the ligand for the receptor notch 1. Mutations that alter the

Epidermal growth factor-like repeats are a class of cysteine-rich domains that mediate interactions between proteins of diverse function. CRELD1 is the founding member of a

This gene encodes a member of the GATA family of zinc-finger transcription factors. This protein is thought to regulate genes involved in embryogenesis and in myocardial differentiation and function. Mutations in this gene have been associated with cardiac septal

This gene encodes a member of the ZIC family of C2H2-type zinc finger proteins. Mutations

Activins are dimeric growth and differentiation factors which belong to the transforming growth factor-beta superfamily of structurally related signaling proteins. These receptors

suggesting that NKX2-5 is essential for heart formation [31, 32].

PROSIT240, MED13L, mediator complex subunit 13-like

CFC1, cripto, FRL-1, cryptic family 1

development [33, 34].

of the heart and brain [35].

[36].

defects [39].

ZFPM2, zinc finger protein, multitype 2

Jagged 1, jagged 1 (Alagille syndrome)

family of matricellular proteins [38]. GATA4, GATA binding protein 4

ZIC3, Zic family member 3 heterotaxy 1

ACVR2B, activin A receptor, type 2, beta

are all transmembrane proteins [41].

in this gene cause X-linked visceral heterotaxy [40].

jagged 1 protein cause Alagille syndrome [37]. CRELD1, cysteine-rich with EGF-like domains 1

homeobox-containing gene called 'tinman' is expressed in the developing dorsal vessel and in the equivalent of the vertebrate heart. Mutations in tinman result in loss of heart formation in the embryo, suggesting that tinman is essential for Drosophila heart formation. Furthermore, abundant expression of Csx, the presumptive mouse homolog of tinman, is observed only in the heart from the time of cardiac differentiation. CSX, the human homolog of murine Csx, has a homeodomain sequence identical to that of Csx and is expressed only in the heart, again suggesting that CSX plays an important role in human heart formation. Studies have recently shown that nonsyndromic CHD can result from single-gene defects. Schott et al identified mutations in *NKX2.5* in 4 kindreds with atrial septal defects and atrioventricular conduction delay without other apparent syndromic features. The mutations were found only in affected individuals, were not present in control samples, and were demonstrated to change protein structure or function [26-28].

Noonan Syndrome is a genetic multiple malformation disorder that includes short stature, typical facial dysmorphism, webbed neck, chest deformity, and cardiovascular abnormalities. The cardiac involvement is observed in 80% to 90% of affected individuals, with valvar pulmonic stenosis and hypertrophic cardiomyopathy being the most common. Other congenital heart defects observed in Noonan Syndrome are secundum atrial septal defect, atrioventricular septal defect, mitral valve abnormalities, aortic coarctation, and tetralogy of Fallot. Noonan Syndrome is genetically heterogeneous, which means that there are at least 3 Noonan Syndrome disease genes, *PTPN11*, *SOS1*, and *KRAS* [29]. It is *PTPN11*, which encodes a protein tyrosine phosphatase called SHP-2. SHP-2 plays an important role in signal transduction for a wide variety of biological processes, including the formation of the semilunar valves. Mutations in the *PTPN11* gene are observed in 40% to 50% of Noonan Syndrome patients [25, 30].


#### **2.1.2 Functions of the causative genes of CHD**

Table2 shows the functions of the causative genes of CHD.

NKX2-5, NK2 transcription factor related, locus 5

Table 2. Inborn Errors Causing CHD

Homeobox-containing genes play critical roles in regulating tissue-specific gene expression essential for tissue differentiation, as well as determining the temporal and spatial patterns of development. Mutations in NKX2-5 result in loss of heart formation in the embryo, suggesting that NKX2-5 is essential for heart formation [31, 32].

CFC1, cripto, FRL-1, cryptic family 1

124 Congenital Heart Disease – Selected Aspects

homeobox-containing gene called 'tinman' is expressed in the developing dorsal vessel and in the equivalent of the vertebrate heart. Mutations in tinman result in loss of heart formation in the embryo, suggesting that tinman is essential for Drosophila heart formation. Furthermore, abundant expression of Csx, the presumptive mouse homolog of tinman, is observed only in the heart from the time of cardiac differentiation. CSX, the human homolog of murine Csx, has a homeodomain sequence identical to that of Csx and is expressed only in the heart, again suggesting that CSX plays an important role in human heart formation. Studies have recently shown that nonsyndromic CHD can result from single-gene defects. Schott et al identified mutations in *NKX2.5* in 4 kindreds with atrial septal defects and atrioventricular conduction delay without other apparent syndromic features. The mutations were found only in affected individuals, were not present in control samples, and

Noonan Syndrome is a genetic multiple malformation disorder that includes short stature, typical facial dysmorphism, webbed neck, chest deformity, and cardiovascular abnormalities. The cardiac involvement is observed in 80% to 90% of affected individuals, with valvar pulmonic stenosis and hypertrophic cardiomyopathy being the most common. Other congenital heart defects observed in Noonan Syndrome are secundum atrial septal defect, atrioventricular septal defect, mitral valve abnormalities, aortic coarctation, and tetralogy of Fallot. Noonan Syndrome is genetically heterogeneous, which means that there are at least 3 Noonan Syndrome disease genes, *PTPN11*, *SOS1*, and *KRAS* [29]. It is *PTPN11*, which encodes a protein tyrosine phosphatase called SHP-2. SHP-2 plays an important role in signal transduction for a wide variety of biological processes, including the formation of the semilunar valves. Mutations in the *PTPN11* gene are observed in 40% to 50% of Noonan

Transcription factors Signaling proteins Vascular extracellular matrix

FBN-1 Elastin

were demonstrated to change protein structure or function [26-28].

Syndrome patients [25, 30].

Genes affected

GATA4 TBX1 TBX5 NKX2.5 dHAND TFAP2 ZFPM2

**2.1.2 Functions of the causative genes of CHD** 

NKX2-5, NK2 transcription factor related, locus 5

Table 2. Inborn Errors Causing CHD

Table2 shows the functions of the causative genes of CHD.

PTPN11 Jagged 1 DMPK CFC1 SOS1 TGFBR2 KRAS BRAF MEK1 MEK2 HRAS ACVR2B CRELD1 LEFTYA

This gene encodes a member of the epidermal growth factor (EGF)- Cripto, Frl-1, and Cryptic (CFC) family. These proteins play key roles in intercellular signaling pathways during vertebrate embryogenesis. Mutations in this gene can cause autosomal visceral heterotaxy. This protein is involved in left-right asymmetric morphogenesis during organ development [33, 34].

PROSIT240, MED13L, mediator complex subunit 13-like

Also known as THRAP2, The evolutionarily conserved THRAP genes encode a family of proteins that regulate embryonic development. THRAP2 is involved in early development of the heart and brain [35].

ZFPM2, zinc finger protein, multitype 2

The zinc finger protein encoded by this gene is a widely expressed member of the FOG family of transcription factors. The family members modulate the activity of GATA family proteins, which are important regulators of hematopoiesis and cardiogenesis in mammals [36].

Jagged 1, jagged 1 (Alagille syndrome)

The jagged 1 protein encoded by JAG1 is the human homolog of the Drosophilia jagged protein. Human jagged 1 is the ligand for the receptor notch 1. Mutations that alter the jagged 1 protein cause Alagille syndrome [37].

CRELD1, cysteine-rich with EGF-like domains 1

Epidermal growth factor-like repeats are a class of cysteine-rich domains that mediate interactions between proteins of diverse function. CRELD1 is the founding member of a family of matricellular proteins [38].

GATA4, GATA binding protein 4

This gene encodes a member of the GATA family of zinc-finger transcription factors. This protein is thought to regulate genes involved in embryogenesis and in myocardial differentiation and function. Mutations in this gene have been associated with cardiac septal defects [39].

ZIC3, Zic family member 3 heterotaxy 1

This gene encodes a member of the ZIC family of C2H2-type zinc finger proteins. Mutations in this gene cause X-linked visceral heterotaxy [40].

ACVR2B, activin A receptor, type 2, beta

Activins are dimeric growth and differentiation factors which belong to the transforming growth factor-beta superfamily of structurally related signaling proteins. These receptors are all transmembrane proteins [41].

Molecular Mechanisms of Congenital Heart Disease 127

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

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

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

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

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

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.

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

Aortic Aneurysm Syndrome, and the development of various types of tumors [52].

TGFBR2, transforming growth factor receptor 2

carcinoma [53].

development [54].

(CFC syndrome) [55].

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

BRAF, v-raf murine sarcoma viral oncogene homolog B1

associated with cardiofaciocutaneous syndrome [53]. MEK1, MAP2K1, mitogen-activated protein kinase 1

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

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

Defects in this gene are implicated in a variety of cancers [56].

**2.2 Pathogenic mechanisms of congenital heart disease** 
