management of CHD has reduced morbidity and mortality for many CHDs, increasing the

1. INTRODUCTION

Congenital heart disease (CHD) defines a large set of structural and functional abnormalities that arise during cardiac embryogenesis. It accounts for nearly one-third of all major congenital anomalies. With a worldwide annual birth rate around 150 million births, this corresponds to 1.35 million live births with CHD every year, representing a major public health issue (van der Linde et al., 2011). CHD is also identified in 10% of stillbirths and is presumed to be a substantive cause of early fetal death (Jorgensen et al., 2014). Numerous studies have shown that the overall incidence of CHD is relatively comparable across populations (after accounting for the ascertainment bias associated with improved imaging over the past decades) (

et al., 2004; Gelb and Chung, 2015). The distribution of heart lesions is broadly similar with a few notable examples such as the relatively greater incidence of right-sided over left-sided defects among Asians (Pradat et al., 2003; Wu et al., 2010).

Recent improvements in pre- and neonatal diagnosis as well as medical and surgical

management of CHD has reduced morbidity and mortality for many CHDs, increasing the

prevalence of affected children who survive the first year of life. More than 80% of patients

with complex inborn malformation are now reaching adulthood (Kaemmerer and Hess,

2005; Marelli et al., 2007). It has been estimated that adult patients with CHD, the so-

called GUCHs, were 1 million in US in the year 2000, and will be 1.4 million in 2020

(Andreassi and Picano, 2014). Numbers are similar in EU (Baumgartner et al, 2010). The

management of this population represent a social concern and a major scientific challenge

since GUCHs are a heterogeneous group of patients which need for routinely hospital

admissions and are predisposed to age-related cardiac diseases. Additionally, a GUCH

patient who reaches reproductive age demands a better understanding of genetic

transmission, disease penetrance and risk of CHD recurrence.

Thus, the knowledge of molecular mechanisms underlying CHD pathogenesis, is a major challenge in order to establish new preventive strategies and new treatments.

Genetic abnormalities appear to be the primary cause of CHD, but identifying precise defects has proven challenging, principally because CHD is a complex genetic trait.

Several studies showed that chromosomal defects and single-gene mutations can cause CHD, often in the context of a syndrmic disease (Pierpont et al., 2007). For Tetralogy of Fallot (TOF) and/or atrioventricular canal defects, examples include trisomy 21 and TBX5 point mutations underlying Down and Holt-Oram syndromes, respectively (Basson et al., 1997; Mori and Bruneau, 2004). The discovery of 22q11 microdeletions, usually arising de novo, added significant improvement in our understanding of conotruncal forms of CHD as

these account for substantial percentages of some cardiac lesions especially in patients with CATCH syndorme (e.g., 34% of truncus arteriosus and 16% of TOF) (Peyvandi et al., 2013).

However, the most likely pathogenetic scenario in the majority of CHD cases involves

multiple factors, as different genetic loci, somatic mutations, epigenetic factors as well as

the combination of these factors (Figure 1).

Figure 1. Genetic and epigenetic mechanisms involved in the CHD pathogenesis.

TF= transcription factor; UTR= untraslated region; AC= acetylation

1.1 Genetic causes of isolated CHD

CHD is believed to be a multifactorial disorder, due to the interaction between genetics and environmental factors. Although during the last decades remarkable progresses have been made, the genetic mechanisms underlying non-syndromic, sporadic CHD, which account for at least 80% of all the CHDs, are poorly understood.

1.1-1 Transcription factors and CHD

The list of gene point mutations that cause isolated, non-syndromic CHD is rapidly

expanding. A specific class of genes involved in transcriptional controls, the so called

transcription factors (TFs), has been identified as the major player in the cardiac development (Olson, 2006; Fahed et al., 2013).

The availability of experimental models in which specific genes are inactivated or over expressed has allowed the identification of several TFs, highly conserved in organisms ranging from insects to higher vertebrates, and their involvement in heart development (Wessel and Willems, 2010) (Figure 2).

Figure 2. Transcription factors implicated in cardiac morphogenesis and in the CHD pathogenesis.

Cardiac TFs play fundamental roles in regulating the expression of genes responsible for

the cardiac-lineage determination, valvulogenesis, conduction-system and heart chamber

formation, in a mutually reinforcing transcriptional network in which each factor regulates

the expression of the others in order to stabilize and reinforce the cardiac gene program

(Bruneau, 2008; Andersen et al., 2014). Thus, for a correct heart development, a spatiotemporal and quantitative regulation of cardiac TFs must occur.

The critical importance of the transcriptional regulation of gene expression for cardiogenesis is highlighted by the constant discovery of mutations in genes encoding for those TFs (Kodo K et al., 2012), although the frequency of these mutations in sporadic, non syndromic cases of CHD is very low, ranging from 0% to among 3% (Posch et al., 2008; Blue et al., 2012).

In particular, GATA4 and NKX2.5 are the most studied and well characterized cardiac transcription factors critical for normal heart development. Both factors synergistically activate cardiac transcription, suggesting functional convergence of two critical cardiac pathways (Figure 3) (Durocher et al., 1997).

Figure 3. Involvement of GATA4 and NKX2.5 during cardiogenesis.

GATA binding protein 4 (GATA4)

GATA4 belongs to the zinc finger superfamily of TFs characterized by a conserved zinc finger DNA-binding domain, which recognizes the GATA motif (5’-A/TGATAA/G-3’) of target gene promoters. GATA4 is one of the earliest transcription factors expressed in developing cardiac cells (at E7.0–E7.5), specifically in the proepicardium, endocardial cushions and cardiomyocytes, and continues to be expressed in cardiac myocytes throughout the adult life (Watt et al., 2004).

During the fetal life, it controls cardiogenesis, promoting cardiomyocytes differentiation and reprogramming fibroblasts into cardiomyocytes. Animal studies showed that embryos of mice knockout for GATA4 gene lacked a ventral pericardial cavity and heart tube fusion between E8.5 and E10.5 (Yang et al., 2014; Molkentin et al.1997; Kuo et al., 1997).

Moreover, mice with hypomorphic Gata4 alleles show a reduced viability, promoting the development of congenital heart diseases (Pu et al., 2004). Complementary, gain-of- function studies reveled that an over expression of GATA4 enhances cardiogenesis and stimulates formation of terminally differentiated cardiomyocytes (Grépin et al., 1997). Thus, controlled levels of GATA4 are fundamental and mutations altering these levels can result in the most common type of cardiac malformation, such as septal defects, double outlet right ventricle, TOF and hypoplasia of the ventricular myocardium (Yang et al., 2012, 2013;

Wang et al., 2013; Yang et al., 2014; Warburton et al.,2014).

GATA4 has been shown to cooperate with a number of other TFs and co-activators expressed in the heart, like TBX5, dHAND and MEF2 (Morin et al., 2000; Dai et al., 2002;

Dodou et al., 2004; Moskowitz et al., 2011; Yamak et al., 2014; Carter et al., 2014; Misra

et al., 2014). Specifically, the C-terminal zinc finger domain of GATA4 is required for the

binding with the cardiac homeodomain NKX2.5, causing a conformational change that

exposes the NKX2.5 activation domains and promoting the cardiac sarcomeric protein gene expression (Durocher et al., 1997). GATA4 and NKX2.5 are mutual co-factors since only acting together could initiate the cardiogenesis (Durocher et al., 1997; Charron and Nemer,1999).

NK2 transcription factor related, locus 5 (NKX2.5)

The homeodomain protein NKX2.5, mapped to chromosome 5q31.1, belongs to the NK-2 family of transcription factors and contains three highly conserved regions (the homeodomain, the tinman and the NK2 domain), which are responsible to bind specific DNA sequences and to regulate protein expression.

NKX2.5 controls many aspects of cardiac development, from specification and proliferation of cardiac precursors to later events, such as formation and maintenance of the conduction system (Harvey et al., 2002; Reamon-Buettner and Borlak, 2010). Notably, NKX2.5 is also expressed in the second heart field (SHF), supporting a central role in the transcriptional network that controls the development of right ventricle and outflow tract (Black, 2007).

Absence of Nkx2.5 in mice results in embryonic lethality with a failure of cardiac looping and chamber formation, deficient myocardial differentiation and also defects of the conduction system in perinatal mice (Lyons et al., 1995; Tanaka et al., 1999).

NKX2.5 physically interacts with other cardiac TFs, collaborating with them to promote

cardiomyocytes differentiation and chamber identity (Yamagishi et al., 2001; Sepulveda et

al., 2002; Moskowitz et al., 2007; Vincentz et al., 2008;). In humans, mutations in NKX2.5

have been found in patients with variable clinical phenotype of CHD, including

atrioventricular septal defects, TOF, ventricular septal defects, hypoplasic left heart

syndrome and L-transposition of the great arteries (Benson, 2010; Reamon-Buettner and Borlak, 2010).

After the discovery of a critical role of TFs in cardiogenesis, several studies have been performed in order to identify genetic mutations potentially involved in human heart defects.

Actually, only few genes robustly associated with nonsyndromic CHDs and often related with a wide range of CHD phenotypes have been identified. Thus, the genetics behind the onset of cardiac defects is complicated by the variable (often low) penetrance and low frequency (ranging from 0 to 3%) of those mutations. The high-throughput sequencing approach is likely the only tool to investigate these variants so far (Pulignani, 2013; Vecoli., 2014).

1.1-2 Somatic mutations

During the last decade, a major focus in the genetics of CHD has been the investigation of the potential role of somatic mutations.

Somatic cells are defined as any cells of the body excluding sperm and egg cells. Somatic mutations, arising early during embryogenesis, may affect the structure, function and survival of tissue derived from a subset of cells, leading to the so called “mosaicism”.

Mosaicism refers to a mixture of cells of different genetic composition in one individual and may result in a milder disease phenotype or can unmask the expression of a mutation that would otherwise be lethal to the embryo (Erickson, 2010).

In 2004, a series of evidences proposed the ‘somatic hypothesis’ as a potential

pathogenetic mechanism of isolated CHD (Reamon-Buettner and Borlak, 2004a and

2004b). In particular, sequence analyses revealed mutations in GATA4, NKX2.5, TBX5

and HAND1 genes extracted from diseased cardiac tissues. These genetic variants

resulted totally absent in the DNA from unaffected cardiac tissues of the same malformed hearts. These observations have suggested an involvement of somatic mutations in the development of CHDs, stimulating much research and discussion regarding the “somatic hypothesis” of CHD (Reamon-Buettner and Borlak, 2006; Reamon-Buettner, 2007; Draus et al., 2009; Wang et al, 2011; Salazar et al., 2011; Esposito et al.,2011).

1.1-3 Novel genetic mutations

The sporadic nature of most of CHD could be due to rare, novel, highly penetrant gene mutations.

A smaller subset of CNVs, often large and novel, have been reported in left-sided defects (White et al., 2014), TOF (Silversides et al., 2012; Soemedi et al., 2012) and other isolated cases of CHDs (Payne et al., 2012; Warburton et al., 2014). The Pediatric Cardiac Genomics Consortium, recently, completed an elegant study of exome sequencing performed in parent-offspring trios, revealing a marched excess of de novo point/insertion- deletion mutations in several genes highly expressed in the heart during cardiogenesis, collectively contribute to at least 10% of severe CHDs (Zaidi et al., 2013).

Exploration of genes with novel mutation, in particular those leading to haploinsuffiency, may reveal further inside into the complex etiology and pathogenesis of congenital heart diseases.

1.2 Epigenetics and CHD

Epigenetic factors play a critical role in modulating cardiac lineage specification and cardiogenesis as well as in maintaining transcriptional programs, acting in concert with signaling pathways and TFs.

Epigenetics has been defined as ‘the study of mitotically and meiotically heritable alterations in gene expression that are not caused by changes in DNA sequence’

(Waddington et al., 1942). These changes are mediated through complex

environment/genome interactions. Accumulating evidences strongly suggest that environmental exposure “in utero” can influence the so-called “epigenome”, resulting in cardiac defects or diseases developing later in life (van Driel et al., 2008; Obermann-Borst et al., 2011).

Cytosine methylation, post-translational modification of histone proteins and remodeling of chromatin as well as RNA-based mechanisms are included in this category of epigenetic regulators, which influence gene expression by modulating the availability of regulatory DNA regions to DNA-binding proteins, such as transcription factors.

Cytosine methylation at 5’-CpG-3 dinucleotide is the most studied epigenetic base modification in the eukaryotic genome. Generally, CpG islands in the gene promoter are protected from DNA methylation, whereas CpG sites in gene-coding or non-coding region are commonly methylated. However, the methylation pattern could be disrupted in diseases and methylated CpG islands can be silenced or downregulated.

DNA methylation has been proposed as a potential mechanism involved in CHDs that can influence the transcription regulation of genes essential for the cardiogenesis (Sheng et al., 2014; Yuan et al., 2014; Xu et al., 2014). Notably, a recent study of Sheng W. and colleagues associated a lower LINE-1 methylation level with the TOF risk (Sheng et al., 2012). The same tendency was observed by the same group in NKX2 and HAND1 genes in a TOF population, correlating the difference in the methylation status to the TOF risk (Sheng W et al., 2014).

The function of TFs is intimately associated with the status of the chromatin at particular targets. Chromatin remodeling and histone modifications can affect the charge on histone residues, thereby strengthening or neutralizing the affinity of DNA to the histone, rendering the DNA more or less accessible to bind TFs and co-regulatory proteins.

The major modulators of chromatin structure are chemical modifications of histones

(mainly methylation, acetylation and phosphorylation) and the action of ATP-dependent

chromatin remodeling complexes. Loss of function mutations in these factors lead to severe CHDs, highlighting the importance of a proper chromatin regulation during cardiogenesis (Kaynak, 2003; Ho et al., 2009; Takeuchi and Bruneau, 2009).

Interestingly, the presence of a feedback loop between these complexes and specific TFs, such as GATA4 and NKX2.5, is necessary to regulate genes that are involved in cardiac differentiation, cell proliferation and hypertrophy. The absence of this interaction has been related to atrial and ventricular septal defects in some syndromes associated with CHDs (Prall et al., 2007; Nimura et al., 2009; Wang et al., 2009). Therefore, interactions between chromatin remodeling factors and transcription factors provide an additional level of regulation of gene expression.

1.2-1 microRNAs

The identification of a small non-coding RNAs, such as microRNAs (miRNAs), has added

an additional layer of post-transcriptional regulatory complexity to the gene networks. The

major role of miRNAs in both physiologic and pathophysiologic processes is rapidly

emerging. Indeed, identification of specific miRNAs in definite cardiac cell types has led to

the discovery of important regulatory roles for these molecules during cardiomyocytes

differentiation, cell cycle and conduction as well as during stages of cardiac hypertrophy in

adults, indicating that miRNAs may be as important as transcription factors in controlling

cardiac gene expression (Wang et al., 2008). miRNAs are a class of evolutionarily

conserved small (20–26 nucleotides in length) non-protein-coding RNAs that regulate

gene expression by affecting mRNA stability and/or translation. The miRNA interaction

with the target transcript is mediated by Watson–Crick base pairing with sequences that

are generally located in the 3’untranslated region (UTR) of target mRNAs (Lytle et

al.,2007). The critical region for that binding is the so-called ‘seed’ region, defined as 7/8

nucleotides at the 5’end of the mature miRNA.

Several expression studies characterized the cellular miRNA’s profile involved in the development of CHDs, through the investigation of their interaction with gene patterns important for cardiac development (Kuhn et al., 2008; O’Brien et al., 2012; Yu et al., 2012).

MiR-1 was the first miRNA shown to regulate fundamental aspects of heart development (Zhao et al., 2005). Overexpression of miR-1 in the embryonic heart, decreasing the level of the TF HAND2, inhibits cardiomyocytes proliferation and prevents expansion of the ventricular myocardium, causing lethality due to deficiency of cardiomyocytes and insufficient muscle mass (Zhao et al., 2005). Additionally, deletion of miR1-2 has profound consequences on the conduction system and cardiomyocytes proliferation, resulting in heart defects like ventricular septal defects (Zhao et al., 2007). MiR-133a-1/133a-2 double knockout in mice results in inappropriate expression of serum response factor and cyclin D2, leading to late embryonic and neonatal lethality due to ventricular septal defects and chamber dilatation (Liu et al., 2008). Disruption of miR-138 function led to an abnormal ventricular cardiomyocytes morphology and cardiac function (Morton et al., 2008). Deletion of either miR-106b–25 or miR- 106a–363 combined with the miR-17 to miR-92 null allele can result in embryonic lethality, accompanied by severe ventricular and atrial septal defects (Ventura et al., 2008).

The efficiency of miRNA–mRNA pairing, and consequently gene regulation, can be

influenced by a number of factors, such as the presence of variants in miRNA-binding sites

of mRNA 3’UTRs. Since miRNAs can inhibit translation and/or promote mRNA

degradation depending on its degree of complementarity with the target, nucleotide

variants in the 3’UTR could impact the miRNA-dependent gene regulation either by

weakening or by reinforcing the binding sites (Landi et al., 2011).