Background
A congenital heart defect (CHD) is an abnormality in the structure and great vessels of the heart, which is present at birth. About 19–75 people per 1000 are born with CHD each year, depending on which types of defects are included[
1] and the incidence is higher if stillbirths were included[
2]. Tetralogy of Fallot (TOF) is the most common complex congenital heart disease[
3], accounting for 10% of all congenital heart diseases[
4]. The four distinct anatomical features that characterize TOF are pulmonary outflow tract obstruction, ventricular septal defect, overriding aortic root and right ventricular hypertrophy[
5]. TOF is considered to be a neural crest cell and/or second heart field related conotruncal heart defects that occur during embryonic development[
6]. Although advances in surgical techniques, cardiopulmonary bypass and postoperative care in the last few decades have brought us today to where survival after complete repair is greater than 98% in large congenital heart surgery programs[
7] and have improved the life quality of TOF patients to an excellent condition, late sudden cardiac death remains a persistent risk, with an estimated incidence of 0.5% to 6%[
8], however, the exact etiology of TOF remains unclear. Some studies have reported that some of these defects related to TOF may be associated with chromosomal aneuploidy, including trisomies 13, 18 and 21[
9]. In addition to chromosomal rearrangements, mutations in a number of genes have also been found to be associated with TOF. A variety of gene insertion/deletion has been detected in patients with TOF. These genes include
JAG1[
10,
11],
NKX2-5 and
GATA4[
12,
13] etc. However, a single gene mutation or deletion can be identified only in a small percentage of cases[
14]. Thus, we hypothesize that aberrant epigenetic regulation may be another important factor associated with TOF. Epigenetics refers to the heritable changes in genome function that occur without a change in the primary DNA sequence. These are characterized by covalent modifications of cytosine bases and histones, and changes in the positioning of nucleosomes[
15]. Currently, the most widely studied epigenetic modification in humans is DNA methylation. DNA methylation can control the transcriptional activity of genes by various mechanisms, which occurs almost exclusively in the context of CpG dinucleotides[
16]. The CpG dinucleotides tend to cluster in regions called CpG islands. Studies have proven that over 60% of human gene promoters are overlapped with CpG islands and are usually unmethylated in normal cells[
17]. However, recent work on colon cancer has demonstrated that DNA methylation not only occurs at CpG islands, but also takes place in regions of lower CpG density that lie in close proximity (~2 kb) to the CpG islands, which is known as the CpG island shore[
18]. The CpG island shores are the most enriched with functional CpG sites and have variable methylation, which are closely associated with transcriptional regulation. Most of the tissue-specific DNA methylation seems to occur, not at CpG islands, but at CpG island shores[
19,
20].
Methylation within gene promoters and CpG-dense sequences (CpG islands) have the highest functional relevance to gene expression control and the aberrant methylation changes contribute to many diseases[
15]. The methylation patterns of multiple genes can provide different types of useful information about cancer cells[
21]. The CpG island methylator phenotype, referring to the concurrent methylation of multiple genes, is a useful marker for tumor progression and has been reported in hepatocellular carcinoma[
22]. However, although a lower methylation level of global genome has been demonstrated in TOF patients[
23], little is known about gene-specific DNA methylation changes in patients with TOF.
In this study, we selected 71 CHD candidate genes, based on the evidences of methylation microarray performed previously[
24], transcriptional studies, mouse models and their close association with the heart development, to explore their promoter DNA methylation changes and their association with TOF development.
Discussion
DNA methylation constitutes an important epigenetic regulation mechanism in many eukaryotes[
27]. In previous study, we have demonstrated the hypomethylation of LINE-1, which can serve as an indicator of global DNA methylation[
28], in the myocardial tissue of TOF patients[
23]. In the present study, quantitative methylation analysis was performed on the promoter region of 71 CHD candidate genes in the myocardial tissue of TOF cases and normal controls using the Sequenom MassARRAY platform. This system combines base-specific enzymatic cleavage with MALDI-TOF mass spectrometry and creates a highly accurate, sensitive, and high-throughput method for the quantitative analysis of DNA methylation at CpG units[
29]. The robustness of this approach for quantifying methylated and unmethylated DNA has been confirmed by the Sequenom group[
30]. We found that 26 of the 71 candidate genes showed significant differences in the methylation levels when comparing TOF cases to controls (
p < 0.05). In which, 17 genes were up regulation and 9 genes were down regulation. DNA methylation analysis for multiple genes in TOF cases enables us to reveal the complex etiology of CHD from a novel aspect and provides potential development of new treatments for TOF disease.
The methylation statuses at different regions of the gene promoter may have different effects on the gene’s activities. The 26 genes showing significant differences in the methylation levels can be grouped to three categories according to the location of amplicons in the gene promoter. Fourteen amplicons (14 genes) were located in the CpG islands, 7 amplicons (7 genes) were located in the CpG island shore, and 5 amplicons (5 genes) were covering the region near the TSS.
Seven CHD candidate genes, including
EGFR, EVC2, NFATC2, NR2F2, TBX5, CFC1B and
GJA5, were chosen for further validation in a larger number of TOF cases because of their nominally significant differences in methylation levels and the important roles they play in the development of the heart.
EGFR, a receptor tyrosine kinase in the
ErbB family, activates several signaling cascades that convert extra-cellular cues into appropriate cellular responses and has been demonstrated to be associated with the congenital left ventricular outflow tract obstruction[
31].
EVC2 plays a critical role in bone formation and skeletal development and mutations in
EVC2 are associated with Ellis van Creveld syndrome in which 50-60% of congenital heart defects occur[
32].
NFATC2 is a DNA binding protein with a REL-homology region (RHR) and an NFAT-homology region (NHR). It plays a central role in inducing gene transcription during the immune response. Bourajjaj M. et all, 2008, have shown that
NFATC2 is a necessary mediator of calcineurin-dependent cardiac hypertrophy and heart failure[
33].
NR2F2 is a ligand inducible transcription factor that is involved in the regulation of many different genes and the deletion of
NR2F2 was considered to possibly contribute to congenital heart defects[
34].
TBX5 is a member of the T-box transcription factor family and plays an important role in heart development and the specification of limb identity, which is very well known associated with Holt Oram syndrome, a developmental disorder affecting the heart and upper limbs[
35]. The regulatory variation in the TBX5 enhancer can lead to the same phenotype as a mutation in the gene.
CFC1B is an important factor in embryo development. Mutations in this gene have been reported in Chinese children with congenital heart disease[
36]. The
GJA5 gene encodes for the cardiac gap junction protein connexin 40. A variant in the carboxyl-terminus of connexin 40 alters GAP junctions and increases the risk for TOF[
37].
Aberrant DNA methylation of CpG islands has been widely observed in human tumors and is associated with gene silencing when it occurs in promoter areas[
38]. Multiple genes showing increased or decreased methylation simultaneously have been found in colorectal cancer[
39] and in duodenal adenocarcinomas[
40]. In the present study, we found significantly higher methylation levels in the promoter CpG islands of
EGFR, EVC2 and
NFATC2 in TOF cases compared with controls. The simultaneous higher methylation of
EGFR, EVC2 and
NFATC2 may represented a CpG island methylator phenotype in TOF development and provide useful clues as to the development of an epigenetic classification of the disease with prognostic and therapeutic potential. Moreover,
EGFR and
EVC2 were found to have significant negative correlations between methylation values and respective mRNA expression levels (Figure
3A,B ,
p < 0.05), indicating that methylation changes at the CpG island region of the two genes may have influences on gene expression, though the exact control mechanism requires further study. Although the mRNA level of
NFATC 2 was significantly lower in TOF cases compared to the controls in our study (
p < 0.05), no significant correlation between methylation level and mRNA expression was observed (Figure
3C,
p > 0.05). We concluded that the analyzed methylation region at the promoter CpG island of
NFATC2 might not be involved in regulating the gene transcription.
A recent genome-wide analysis of DNA methylation showed that 76% of differential tissue methylation regions were not located in CpG islands, but in CpG island shore[
18]. In this study, we found a decreased methylation level at the CpG island shore for
NR2F2 and an increased methylation level for
TBX5. We cannot ascertain the factor that contributed to the lower methylation level of
NR2F2. However, the methylation status of
NR2F2 were found not to be associated with its mRNA level, suggesting that methylation changes at the CpG island shore of
NR2F2 might not influence transcriptional activity. Further studies are needed to explore whether the methylation changes in the other region of
NR2F2 promoter influences its mRNA level. Interestingly, the methylation status of the
TBX5 gene was significantly negatively correlated with its mRNA level, indicating that increased methylation level at the CpG island shore of
TBX5 may inhibit this gene transcriptional activity.
DNA methylation can directly inhibit transcription by precluding the recruitment of DNA binding proteins from their target sites[
41]. Cao et al. found that CpG site-specific methylation can alter binding affinities of specific transcription factors, which can differentially activate or repress transcription[
42]. Consistent with this, Tihomira D et al. have reported that, although methylation of all CpG sites resulted in the silencing of
EphA5 promoter activity, lower levels of methylation resulted in differential activation or repression of
EphA5 promoter activity, depending on the sites methylated[
43]. In the current study, the methylation levels at the promoter region near the TSS of
CFC1B and
GJA5 were significantly higher in TOF cases compared to normal controls. In addition, other than
GJA5, one significant negative correlation was observed between the methylation status and mRNA level of
CFC1B, indicating that the methylation change at the promoter region near the TSS of
CFC1B may play an important role in regulating the gene transcriptional activity. How the altered methylation statuses of
CFC1B influence mRNA level requires further study.
However, in the present study, we have not examined the protein levels of these genes in the myocardial tissue because of the limited samples. The potential alterations of the protein levels and their associations with the transcription level would be explored in the further study.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SW participated in study concept and design and coordination of the study, helped with the statistical analysis and drafted the manuscript. QY, WY, WH and MX participated in TOF sample acquisition and helped to draft the manuscript. ZP participated in normal control sample acquisition and helped to draft the manuscript. CL, MD and HG conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors have read and approved the final manuscript.