TCF21 rs12190287 Polymorphisms Are Associated with Ventricular Septal Defects in a Chinese Population

Abstract

Aims: TCF21 knockout mice display cardiac defects, including ventricular septal defects (VSDs). Functional rs12190287 polymorphisms located within the 3′ untranslated region (3′-UTR) of TCF21 were associated with a risk of coronary heart disease in the European and Eastern populations. However, whether rs12190287 polymorphisms in the TCF21-3′UTR confer predisposition to congenital heart disease (CHD) is unclear.

Methods: A case–control study was designed consisting of 781 nonsyndromic VSD patients and 867 non-CHD control subjects. The genotype frequency of rs12190287 polymorphisms was determined by real-time polymerase chain reaction.

Results: There were significant differences in the genotype and allele frequencies of rs12190287 between the cases and controls in a Chinese population. Allele G of rs12190287 was significantly associated with an increased risk of VSD in a Chinese population.

Conclusions: Our results demonstrate that rs12190287 polymorphisms confer predisposition to VSDs in the Chinese population studied here.

Introduction

Congenital heart disease (CHD) is the most common type of birth defect, accounting for one-third of all major congenital disorder anomalies (van der Linde et al., 2011). CHD may occur as part of chromosomal and Mendelian syndromes. However, most instances occur sporadically as isolated nonsyndromic defects and may result from interactions between genetic and environmental factors (Blue et al., 2012). Ventricular septal defects (VSDs) are the most common form of CHD, occurring in more than 1 in 300 live births, as well as a frequent component of more complex lesions, such as DiGeorge syndrome (DGS) (Hoffman, 1995; Hoffman and Kaplan, 2002). Some human syndromic and sporadic cases of VSD have been associated with mutations in the transcription factors NKX2.5, TBX5, and GATA4 (Gruber and Epstein, 2004).

TCF21 is a member of the basic helix-loop-helix (bHLH) transcription factor family, which plays crucial roles in cell fate specification and differentiation during organ development (Massari and Murre, 2000). Recent studies have found that TCF21 knockout mouse embryos display Tetralogy of Fallot (TOF), including a VSD, overriding aorta, and pulmonic stenosis (Harel et al., 2012). Genome-wide association studies found that the single nucleotide polymorphism (SNP) rs12190287 located in the 3′ untranslated region (3′-UTR) of TCF21 is associated with coronary heart disease in both Caucasian and East Asian populations (Schunkert et al., 2011; Nyegaard et al., 2012). We hypothesized that rs12190287 polymorphisms may play a role in the pathogenesis of CHD. In this study, we investigated the association of rs12190287 with patients with VSDs in a Chinese population. Our data showed that allele G at variant rs12190287 is a contributing risk factor for VSDs.

Materials and Methods

We enrolled 781 isolated patients with VSDs with a mean age of 8.57 ± 11.71 years (Table 1) and 867 control subjects from the Fujian Medical University. Cases that had clinical features of structural malformations involving another organ system or known chromosomal abnormalities were excluded. Exclusion criteria also included a positive family history of CHD in a first-degree relative (parent, sibling, or child). Informed consent was obtained from the patients or their guardians. The patients underwent routine clinical examination and ultrasonic echocardiogram, which were further confirmed by open heart surgery.

Table 1.

Characteristics of Ventricular Septal Defect Cases

Characteristic	Cases (n = 781)
Age, years (mean)	8.57 ± 11.71
Sex
Males	447
Females	334
VSD
Conoventricular defect	587
Conal defect	162
Muscular defect	6
Atrioventricular canal defect	26

VSD, ventricular septal defects.

The control subjects were non-CHD adult outpatients in the same geographic region. Given that neither age nor sex has been shown to have associations with VSD, we did not match the age or sex between cases and controls. This study was approved by the Ethics Committee of Fujian Medical University and adhered to the tenets of the Declaration of Helsinki. For each participant, ∼2 mL whole blood was obtained to extract genomic DNA for genotyping analysis.

A genotype analysis of rs12190287 in the cases and the control subjects was performed using TaqMan-based real-time polymerase chain reaction (PCR) (forward primer sequence: 5′-CAGCAATAGTGACCTCATTA-3′; reverse primer for allele G: 5′-CAAATAGACAGGTGGATGAAC-3′; reverse primer for allele C: 5′-AAATAGACAGGTGGAT GAAG-3′). The TaqMan probe, TGGCGACCAC ATTACC AAGC, was labeled at the 5′ end with 6-carboxyfluoroscein (FAM) and at the 3′ end with Black Hole Quencher 1 (BHQ-1). PCR amplification was performed using an ABI Prism 7,500 Sequence Detection System (Applied Biosystems) under the following conditions: one cycle 95°C for 3 min, followed by 40 cycles of 95°C for 5 s and 61°C for 30 s. The ΔCt value was used to differentiate alleles G and C. Quality control of real-time PCR for genotyping was validated by DNA sequencing. Ten percent of the samples were randomly selected for direct sequencing.

The comparisons of genotype and allele frequencies were evaluated using the χ² test. The association of the SNP genotype with CHD risk was estimated by computing odds ratios (ORs) and 95% confidence intervals (CIs) from logistic regression analysis. The Hardy–Weinberg equilibrium was evaluated using the χ² test in the control subjects. All the statistical analyses were performed with the computer software Statistical Package for Social Sciences (SPSS) for Windows (version 18.0).

Results

The frequency distributions of the different genotypes for rs12190287 (C/G) in patients with VSDs and those in the control group are shown in Table 2. The observed genotype frequencies of the controls were in the Hardy–Weinberg equilibrium (p = 0.21), suggesting that there was no population stratification and no sampling bias. To evaluate the risk of VSDs associated with the rs12190287 genotype, logistic regression analyses were conducted. As shown in Table 1, there were significant differences in genotype and allele frequency of rs12190287 between the VSD cases and controls. Individuals carrying allele G of rs12190287 showed a 21.0% (OR = 1.21; 95% CI = 1.057–1.391; p = 0.0061) increase in VSD risk. Using the CC genotype as the reference genotype, the GG genotype increased the risk of VSD by 1.446-fold (OR = 1.446; 95% CI = 1.101–1.899; p = 0.0079), but the GC genotype did not increase VSD risk (OR = 1.206; 95% CI = 0.964–1.508; p = 0.101). We also observed that the rs12190287 variant homozygote GG was associated with a significant increase in VSD risk in both the recessive genetic model (OR = 1.598; 95% CI = 1.248–2.047; p = 0.000205) and the dominant genetic model (OR = 1.351; 95% CI = 1.095–1.668; p = 0.005).

Table 2.

Distribution of rs12190287 in Ventricular Septal Defect Cases and Controls and its Effect on Ventricular Septal Defect Risk

Genotypes	Cases (N = 781), N (%)	Controls (N = 867), N (%)	OR (95% CI)	p
CC	224 (28.68)	294 (33.91)	1.00 (reference)
GC	373 (47.76)	406 (46.83)	1.206 (0.964–1.508)	0.101
GG	184 (23.56)	167 (19.26)	1.446 (1.101–1.899)	0.0079
CC/CG	597 (76.44)	700 (80.74)	1.00 (reference)
GG	184 (23.56)	167 (19.26)	1.598 (1.248–2.047)	0.000205
CC	224 (28.68)	294 (33.91)	1.00 (reference)
CG/GG	557 (71.32)	573 (66.09)	1.351 (1.095–1.668)	0.005
C allele	821 (52.56)	994 (57.32)	1.00 (reference)
G allele	741 (47.44)	740 (42.68)	1.21 (1.057–1.391)	0.0061

OR, odds ratio; CI, confidence interval.

Discussion

In the present study, we provide evidence of a significant association between the genotype and allele frequencies of rs12190287 C/G polymorphism in patients with VSDs but not in those of the control group.

TCF21 rs12190287 was associated with coronary heart disease in both Caucasians and Asians (Schunkert et al., 2011; Nyegaard et al., 2012; Bastami et al., 2016). Wang et al. (2014) showed that rs12190287 polymorphisms are significantly associated with myocardial infarction in a Chinese population. Fujimaki et al. (2015) reported that rs12190287 polymorphisms are significantly associated with hypertension in Japanese populations. In the current study, we found that rs12190287 polymorphisms are associated with VSDs in a Chinese population. It is noteworthy that allele C represented a risk factor for disease in previous studies, while our data showed that allele G increases the risk of VSD in our study population. Together, these data suggest that rs12190287 is a disease-related susceptibility locus implicated in cardiovascular disease that has different polymorphic effects on different tissues and in different stages of development. TCF21 knockout mouse embryos displayed the TOF phenotype, a type of severe outflow tract (OFT) development and alignment defect, including a VSD, overriding aorta, and pulmonic stenosis (Harel et al., 2012). This raises the possibility that rs12190287 polymorphisms may confer susceptibility to TOF.

Pharyngeal mesoderm cells are a subset of the head mesoderm contributing to broad regions of the heart and head musculature (Tzahor and Evans, 2011). Recent studies found that TCF21, TBX1, and LHX2 are genetically linked in the pharyngeal mesoderm transcriptional network, which controls cardiac and head muscle morphogenesis (Harel et al., 2012). It has been reported that the mesoderm-specific deletion of TBX1 causes severe pharyngeal patterning and cardiovascular defects, including VSDs and cardiac OFT defects (Zhang et al., 2006). TBX1 haploinsufficiency is probably a major contributor to human DGS and to murine models of the syndrome (Lindsay et al., 2001; Merscher et al., 2001). Mutations in TBX1 have been found in some patients showing a DGS-like phenotype (Yagi et al., 2003). Approximately 80% of DGS patients are born with CHD, including VSDs and OFT malformations (McDonald-McGinn and Sullivan, 2011). Mesodermal expression of MOZ, a member of the MYST family of histone acetyltransferases, is essential for the normal activation of the TBX1 gene and is necessary for cardiac septum development (Vanyai et al., 2015). The mesoderm-specific deletion of MOZ results in a high penetrance of VSDs and overriding aorta (Vanyai et al., 2015). Miller et al. (2013) identified a cis-acting mechanism by which the TCF21 G allele at variant rs12190287 disrupts an atypical activator protein 1-like enhancer element to decrease the transcriptional control of TCF21 gene expression. Further study showed that the rs12190287 allele G results in a mismatch that disrupts miR-224 binding and accessibility of this region of the TCF21 3′-UTR. The minor allele leads to dysregulated TCF21 gene expression (Miller et al., 2014). This implies that rs12190287 polymorphisms may influence the mesodermal expression of TCF21, which disrupts cardiac septum and OFT development.

Recent studies have shown that TCF21 is expressed in the epicardium and the proepicardial organ of embryonic zebrafish, chick, and mouse hearts (Hidai et al., 1998; Serluca, 2008; Braitsch et al., 2012). Loss of TCF21 in mice leads to epicardial blistering, increased smooth muscle differentiation on the surface of the heart, and a paucity of interstitial fibroblasts, with perinatal lethality (Braitsch et al., 2012). The epicardium is a mesothelial cell layer essential for vertebrate heart development and relevant for cardiac repair postinjury in adults (Limana et al., 2010; von Gise and Pu, 2012). Tandon et al. (2013) reported that TCF21 associates with cofactors involved in transcriptional regression, such as Pbx1 and Ctbp2, in the epicardium. Stankunas et al. (2008) reported that embryos with different combinations of Pbx mutations display a spectrum of cardiac malformations that include persistent truncus arteriosus (PTA), TOF, overriding aorta with a VSD, and bicuspid aortic valves. Each of the observed cardiac defects represents developmental abnormalities affecting distinct stages of cardiac OFT development, correlating with Pbx gene dosage (Stankunas et al., 2008). Arrington et al. (2012) showed that nonsynonymous variants in Pbx genes are associated with CHD, including OFT malformations. Ctbp2-null embryos exhibit defects in heart morphogenesis characterized by a dilated pericardium and failure of cardiac looping (Chinnadurai, 2003). Glessner et al. (2014) reported that Ctbp2 may be a pathogenic gene in CHD cases with 10q subtelomeric deletions. This implies that rs12190287 polymorphisms may disrupt cardiac development by influencing the expression of TCF21 and its interaction with other proteins in the epicardium.

In summary, our study demonstrates that rs12190287 polymorphisms confer genetic susceptibility to VSDs within the study population. However, the exclusion criteria for the cases did not include environmental factors, such as maternal diabetes mellitus and maternal teratogen exposures, which may result in selection bias and a false-positive correlation. In addition, our study represents a relatively small sample size; therefore, a larger population-based study should be conducted in the future.

Acknowledgments

The authors thank all the subjects who participated in this study. The authors appreciate critical reading of the manuscript by Dr. Gregory Roderiquez (National Institutes of Health, Maryland). This work was supported by the National Key Research and Development Program (2016YFC1000501), the National Natural Science Fund (No. 81470525), Important National Science & Technology Specific Projects (2014ZX09304307-002), and the National Nonprofit Institute Research Grant of NRIFP (2016GJM01).

Author Disclosure Statement

No competing financial interests exist.