Two novel and functional DNA sequence variants within an upstream enhancer of the human NKX2-5 gene in ventricular septal defects

doi:10.1016/j.gene.2013.04.043

Gene

Volume 524, Issue 2, 25 July 2013, Pages 152-155

https://doi.org/10.1016/j.gene.2013.04.043 Get rights and content

Highlights

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An upstream enhancer of the human NKX2-5 gene was analyzed in VSD patients.
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Two novel heterozygous DNA sequence variants were identified in VSD patients.
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The variants significantly decreased transcriptional activities of the enhancer in vitro.
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The DNA sequence variants may contribute to a small group of VSD.

Abstract

Mortality in patients with congenital heart disease (CHD) is significantly increased even with successful surgeries. The main causes are late cardiac complications, such as heart failure and arrhythmia, probably due to genetic defects. To date, genetic causes for CHD remain largely unknown. NKX2-5 gene encodes a highly conserved homeobox transcription factor, which is essential to the heart development in embryos and cardiac function in adults. Mutations in NKX2-5 gene have been implicated in diverse types of CHD, including ventricular septal defect (VSD). As NKX2-5 is a dosage-sensitive regulator, we have speculated that changed NKX2-5 levels may mediate CHD development by influencing cardiac gene regulatory network. In previous studies, we have analyzed the NKX2-5 gene promoter and a proximal enhancer in VSD patients. In the present study, we further genetically and functionally analyzed an upstream enhancer of the NKX2-5 gene in large cohorts of VSD patients (n = 340) and controls (n = 347). Two novel heterozygous DNA sequence variants (DSVs), g.17483576C>G and g.17483564C>T, were identified in three VSD patients, but none in controls. Functionally, these two DSVs significantly decreased the activity of the enhancer (P < 0.01). Another novel heterozygous DSV, g.17483557Ins, was found in both VSD patients and controls with similar frequencies (P > 0.05). Taken together, our data suggested that the DSVs within the upstream enhancer of the NKX2-5 gene may contribute to a small number of VSD. Therefore, genetic studies of CHD may provide insight into designing novel therapies for adult CHD patients.

Introduction

Congenital heart disease (CHD) is the most common birth defect in humans, which affects 4–10 per 1000 live births in different populations, which is the main cause of death in infants (Go et al., 2013). Mutations in more than 40 genes, including GATA transcription factor 4 (GATA4), T-box transcription factor 5 (TBX5), NK2 transcription factor related, locus 2 (NKX2-5), have been implicated in a small portion of CHD patients (Bruneau, 2008, McCulley and Black, 2012, Wessels and Willems, 2010). To date, genetic causes and underlying molecular mechanisms for CHD remain largely unknown.

NKX2-5 gene is a highly conserved homeobox protein gene and expressed in the developing heart, as well as in adult heart. In the embryonic heart, NKX2-5 plays essential roles in cardiac progenitor determination, cardiomyocyte differentiation, cardiac morphogenesis and conduction system development (Habets et al., 2002, Jamali et al., 2001, Jay et al., 2004, Lyons et al., 1995, Moskowitz et al., 2007, Pashmforoush et al., 2004, Prall et al., 2007, Stanley et al., 2002, Tanaka et al., 1999a). In adult heart, NKX2-5 is required for cardiomyocyte homeostasis and postnatal formation of ventricular conduction system (Meysen, 2007, Toko et al., 2002). More than 30 mutations in NKX2-5 gene have been identified in diverse types of CHD, including atrial septal defects, ventricular septal defects (VSD) and tetralogy of Fallot (Benson et al., 1999, Elliott et al., 2003, Gioli-Pereira et al., 2010, McElhinney et al., 2003, Rauch et al., 2010, Reamon-Buettner and Borlak, 2010, Schott et al., 1998, Stallmeyer et al., 2010). Several lines of evidence from genetic studies in model animals have confirmed the association of NKX2-5 gene mutations with cardiac phenotypes (Biben, 2000, Jay et al., 2004, Kasahara, 2001, Lyons et al., 1995, Tanaka et al., 1999a, Terada et al., 2011).

NKX2-5 gene expression is controlled by a complex module of regulatory regions, including promoter, proximal and upstream independent enhancers (Liberatore et al., 2002, Lien et al., 1999, Lien et al., 2002, Reecy et al., 1999, Schwartz and Olson, 1999, Searcy et al., 1998, Tanaka et al., 1999b). NKX2-5, like GATA4 and TBX-5, is a dosage-sensitive regulator in the embryonic development (Meysen, 2007, Mori et al., 2006, Pu et al., 2004). Dosage-sensitive interdependence between cardiac transcription factors and chromatin remodeling complexes has been reported in the developing heart (Takeuchi et al., 2011). Thus, we have hypothesized that DNA sequence variants (DSVs) within the regulatory regions of the NKX2-5 gene may mediate CHD development. In previous studies, we have analyzed the promoter and a proximal enhancer of the NKX2-5 gene and identified a few DSVs in VSD patients (Pang et al., 2012, Qin et al., 2012).

An upstream enhancer region (− 9435 bp to − 8922 bp) of the NKX2-5 gene has been demonstrated to direct endogenous NKX2-5 gene expression in the cardiac crescent and early heart tube in mice (Lien et al., 1999). Cross-species alignment indicated that part of the upstream enhancer of the mouse NKX2-5 gene, − 9432 bp to − 9171 bp, was conserved in genomic sequence of the human NKX2-5 gene. To further understand the roles of mutations and variants within the NKX2-5 gene and its regulatory regions in CHD, we genetically and functionally analyzed the upstream enhancer region of the human NKX2-5 gene in large cohorts of VSD patients and healthy controls in this study.

Section snippets

Patients and controls

All the VSD patients (n = 340, male 131, female 209, age range from 3 month to 35 years, median age 8.03 years) were recruited from Jining Medical University Affiliated Hospital, Jining Medical University, Jining, Shandong, China. The VSD patients were diagnosed according to medical records, physical examination, electrocardiogram, three-dimensional echocardiography and cardiac surgery. The ethnic-matched healthy controls (n = 347, male 303, female 44, age range from 6 month to 13 years, median age 3.19

Results

The upstream enhancer of the mouse NKX2-5 gene (− 9435 bp to − 8922 bp) was aligned with genomic sequence of the human NKX2-5 gene. A part of the mouse enhancer (− 9432 bp to − 9171 bp) was conserved at similar location in genomic sequence of the human NKX2-5 gene (− 10,489 bp to − 10,228 bp), which contains three GATA factor binding sites. DNA region covering the enhancer region (− 10,495 bp to − 9930 bp) was genetically analyzed by PCR-based bi-directional sequencing. The distributions of the DSVs identified

Discussion

We have previously analyzed a proximal enhancer and promoter regions of the NKX2-5 gene and identified a few DSVs in CHD patients (Pang et al., 2012, Qin et al., 2012). In the present study, we further genetically and functionally analyzed an upstream cardiac enhancer of the NKX2-5 gene in VSD patients and controls. Within the enhancer, two novel heterozygous DSVs (g.17483564C>T and g.17483576C>G) were found in VSD patients, but none in controls. Functional analysis showed that these two DSVs

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 81070173) and the Shandong Provincial Natural Science Foundation (ZR2010HL030 and ZR2010HM111).

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Genetic variations of NKX2-5 in sporadic atrial septal defect and ventricular septal defect in Chinese Yunnan population
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Citation Excerpt :
NKX2-5 is a dosage-sensitive regulator during embryonic development. Huang et al. (Huang et al., 2013) hypothesize that, the expression levels of NKX2-5 gene rather than the mutant protein may play important roles in CHD. Two sequence variants locating in the promoter region of NKX2-5 gene can significantly enhance the transcriptional activities of NKX2-5 gene promoter, suggesting that they may contribute to the VSD etiology by altering NKX2-5 gene expression levels.
Congenital heart disease (CHD) is the most common birth abnormality, and more than 40% CHD subtypes are sporadic atrial septal defect (ASD) and ventricular septal defect (VSD). The etiology of ASD and VSD remains largely unknown. NKX2-5 gene is a highly conserved homeobox protein gene and expressed in the developing heart. Its mutations can cause sporadic ASD and VSD. This study aimed to investigate the genetic variations of NKX2-5 in ASD and VSD in Chinese Yunnan population. The whole 2 coding exon and partial flanking intron sequences of NKX2-5 gene were screened using DNA sequencing in 107 ASD patients and 391 VSD patients as well as 487 healthy individuals (control) who had parental origin (three generations) from the Yunnan province in China. Results found that, 4 reported single nucleotide polymorphisms (SNPs) (rs2277923, rs3729753, rs703752 and rs202071628) were detected. A novel heterozygous DNA sequence variant (DSV) (1500G > C) in the 3′UTR region of NKX2-5 gene were identified in 2 VSD patients, but none in ASD and controls. One single nucleotide polymorphism (rs2277923), the frequency of which was significantly higher in ASD group, and the allele and genotype were associated with the occurrence of ASD. Besides, a weak statistical association existed between rs703752 and VSD (uncorrected P = 0.028). The novel DSV (1500G > C) of NKX2-5 gene may contribute to a small number of VSD, and rs2277923 SNP may contribute to the risk of sporadic ASD in Chinese Yunnan population.
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Citation Excerpt :
In vertebrates, cardiac morphogenesis is a complex dynamic process that requires the accurate spatial and temporal cooperation of cardiac cell commitment, differentiation, proliferation and migration, and both environmental and genetic risk factors may disturb this biological process of cardiogenesis, resulting in a wide spectrum of CHDs [Fahed et al., 2013]. Recently, aggregating evidence underscores the genetic origin of CHD, and in addition to chromosomal deletions and copy number variants [Digilio et al., 2013; Fagerberg et al., 2013; Fahed et al., 2013; Liu et al., 2014; Rahikkala et al., 2013; Sanchez-Castro et al., 2013; Soemedi et al., 2012; van Trier et al., 2013], a great number of mutations, including autosomal dominant, autosomal recessive, X-linked and somatic mutations, in more than 60 genes have been associated with CHD [Ackerman et al., 2012; Al Turki et al., 2014; Aoki et al., 2013; Arrington et al., 2012; Breckpot et al., 2012; Chang et al., 2013; Cowan et al., 2014; Fahed et al., 2013; Huang et al., 2013, 2014; Jiang et al., 2013; Lahm et al., 2013; Lalani and Belmont, 2014; Shi et al., 2014; Tan et al., 2012; Wang et al., 2012, 2013, 2014; Wei et al., 2014, 2013; Yang et al., 2013; Yuan et al., 2013; Zaidi et al., 2013; Zhao et al., 2014]. Among these well established CHD-associated genes, most encode cardiac transcription factors, and the cardiac transcription factor genes Nkx2.5 and GATA4 are the most frequently linked to non-syndromic CHD [Beffagna et al., 2013; Costa et al., 2013; Fahed et al., 2013; Granados-Riveron et al., 2012; Huang et al., 2013; Lalani and Belmont, 2014; McCulley and Black, 2012; Qin et al., 2012; Reamon-Buettner et al., 2013; Wang et al., 2013; Xiang et al., 2014; Yang et al., 2012, 2013a, 2013b].
Congenital heart disease (CHD) is the most common form of birth defect and is the most prevalent non-infectious cause of infant death. A growing body of evidence documents that genetic defects are involved in the pathogenesis of CHD. However, CHD is a genetically heterogeneous disease and the genetic basis underpinning CHD in an overwhelming majority of patients remain unclear. In this study, the coding exons and flanking introns of the Nkx2.6 gene, which codes for a homeodomain-containing transcription factor important for normal cardiovascular development, were sequenced in 320 unrelated patients with CHD, and two novel heterozygous Nkx2.6 mutations, p.V176M and p.K177X, were identified in two unrelated patients with CHD, respectively, including a patient with tetralogy of Fallot and a patient with double outlet of right ventricle and ventricular septal defect. The mutations were absent in 400 control chromosomes and the altered amino acids were completely conserved evolutionarily across species. Due to unknown transcriptional targets of Nkx2.6, the functional consequences of the identified mutations at transcriptional activity were evaluated by using Nkx2.5 as a surrogate. Alignment between human Nkx2.6 and Nkx2.5 proteins showed that V176M-mutant Nkx2.6 was equivalent to V182M-mutant Nkx2.5 and K177X-mutant Nkx2.6 was equal to K183X-mutant Nkx2.5, and introduction of V182M or K183X into Nkx2.5 significantly diminished its transcriptional activating function when compared with its wild-type counterpart. To our knowledge, this is the first report on the association of Nkx2.6 loss-of-function mutation with increased susceptibility to tetralogy of Fallot or double outlet of right ventricle and ventricular septal defect, providing novel insight into the molecular mechanism of CHD.
Prevalence and spectrum of GATA4 mutations associated with sporadic dilated cardiomyopathy
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In mice, targeted deletion of NKX2-5 resulted in impaired cardiac growth and chamber formation, deranged gene regulatory network, and early embryonic death, while cardiac-specific knockout of NKX2-5 gave rise to progressive cardiomyopathy and complete heart block (Lyons et al., 1995; Pashmforoush et al., 2004; Prall et al., 2007). In humans, mutations in the NKX2-5 gene have been involved in different kinds of congenital heart diseases, including cardiac septal defects, valvular deformations, tetralogy of Fallot, transposition of the great arteries and hypoplastic left heart, and in left ventricular contractile dysfunction and adult-onset familial DCM (Costa et al., 2013; Huang et al., 2013; Qin et al., 2012; Reamon-Buettner and Borlak, 2010; Schott et al., 1998). These results justify evaluating the prevalence and spectrum of GATA4 mutations associated with sporadic DCM.
Dilated cardiomyopathy (DCM) is the most frequent type of primary myocardial disorder responsible for substantial morbidity and mortality. DCM is the third most common cause of heart failure and the most common reason for heart transplantation. A recent study has implicated GATA4 mutation in the pathogenesis of familial DCM. However, the prevalence and spectrum of GATA4 mutations associated with sporadic DCM remain unclear. In this study, the coding exons and exon–intron boundaries of the GATA4 gene, which encodes a cardiac transcription factor crucial for normal cardiogenesis, were sequenced in 220 unrelated patients with sporadic DCM. A total of 200 unrelated ethnically-matched healthy individuals used as controls were genotyped. The functional characteristics of the mutant GATA4 were assayed in contrast to its wild-type counterpart using a luciferase reporter assay system. As a result, 3 novel heterozygous GATA4 mutations, p.V39L, p.P226Q and p.T279S, were identified in 3 unrelated patients with sporadic DCM, with a mutational prevalence of approximately 1.36%. The missense mutations were absent in 400 control chromosomes and the altered amino acids were completely conserved evolutionarily across species. Functional analysis showed that the GATA4 mutants were consistently associated with significantly decreased transcriptional activity and markedly reduced the synergistic activation between GATA4 and NKX2-5. This study firstly links GATA4 mutations to increased susceptibility to sporadic DCM and provides novel insight into the molecular etiology underlying DCM, suggesting the potential implications for the early prophylaxis and allele-specific treatment of this common form of cardiomyopathy.
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¹: These authors contributed equally to the work.

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Two novel and functional DNA sequence variants within an upstream enhancer of the human NKX2-5 gene in ventricular septal defects

Highlights

Abstract

Introduction

Section snippets

Patients and controls

Results

Discussion

Conflict of interest

Acknowledgments

Semin. Cell Dev. Biol.

J. Mol. Cell. Cardiol.

J. Am. Coll. Cardiol.

Int. J. Cardiol.

J. Biol. Chem.

J. Mol. Cell. Cardiol.

Dev. Biol.

Dev. Biol.

Dev. Cell

Curr. Top. Dev. Biol.

J. Am. Coll. Cardiol.

Dev. Biol.

Dev. Biol.

Cell

Cell

Cell

Dev. Biol.

Gene

Gene

J. Biol. Chem.

Transl. Res.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Dev. Biol.

J. Biol. Chem.

Transl. Res.

Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways

J. Clin. Invest.

Cardiac septal and valvular dysmorphogenesis in mice heterozygous for mutations in the homeobox gene Nkx2-5

Circ. Res.

The developmental genetics of congenital heart disease

Nature

Epigenetics and cardiovascular development

Annu. Rev. Physiol.