Main

Tetralogy of fallot (TOF) is a combination of four birth defects, including pulmonary stenosis, ventricular septal defect, overriding aortic roots, and hypertrophy of the right ventricle (1). TOF occurs in 3.6 out of every 10,000 live births and accounts for about 10% of all congenital heart defects (2). The mortality rate in untreated TOF patients reaches 50% by age 6 y; however, when receiving the cardiac surgery, children with simple forms of TOF enjoy good long-term survival with an excellent quality of life (3). Although treatment of TOF disease has advanced dramatically over the past few decades, the exact etiology remains unknown (4). Human genetic studies have identified numerous genes that are responsible for inherited and sporadic congenital heart diseases. Most of these genes, such as ZFPM2, encode transcription factors that regulate specific events in heart development, such as ventricular septum or outflow tract morphogenesis (5). ZFPM2 gene encodes a zinc finger protein and can physically interact with the N-terminal zinc finger of GATA4 to modulate its activity and thereby influence the transcription of target genes. This interaction is required for normal morphogenesis of heart during organogenesis (6). ZFPM2-/- mice embryo showed grossly normal in appearance at E12.5, however, at E13.5–15, the heart of ZFPM2-/- mice exhibited the defects seen in human tetralogy of fallot and died at midgestation (7). The mutations in ZFPM2 have also been observed in the human TOF cases. De Luca et al. (8) have identified two missense variants in the ZFPM2 in the 1 of 178 patients with TOF. Although the whole exons of ZFPM2 were sequenced and several new mutations were observed in the TOF patients, the mutation percentage is very lower (8,9,10). For the nongenetic mutation-caused TOF patients, the epigenetic mechanisms, such as DNA methylation, should be considered in the development of TOF (11,12).

Currently, the DNA methylation is the most widely studied epigenetic modification in humans, which occurs almost exclusively in the context of CpG dinucleotides and regulates the transcriptional activity of genes by various mechanisms (13). The clusters of CpG dinucleotides consist of the CpG island regions and about 60% of gene promoters in human are overlapped with CpG islands and usually unmethylated in normal cells (14). The regions that lie in close proximity (~2 kb) to the CpG islands are known as the CpG island shores (15). Although the CpG island shores have lower CpG site density, they frequently have functional CpG sites and show variable methylation levels and responsible for gene transcriptional activity. Most of the tissue-specific gene DNA methylation seems to occur, not at CpG islands, but at CpG island shores (16,17).

Methylation within gene promoters has the closely functional relevance to gene expression regulation, and the aberrant methylation changes may contribute to many diseases (18). Although the mutations in ZFPM2 gene have been reported in patients with TOF, little is known about its DNA methylation changes in the TOF patients without gene mutations.

The goal of the present study was to explore the DNA methylation changes of ZFPM2 and its epigenetic regulation in TOF patients. The potential results may provide important clues to assist the development of new treatments for TOF as well as give a deeper understanding of the etiology of this disease.

Results

Methylation Status Analyses for ZFPM2 Gene in TOF Patients and Controls

The methylation status of ZFPM2 gene was analyzed using the Sequenom MassARRAY platform (Sequenom). We focused on the promoter region of ZFPM2 gene from −2,000 bp to +200 bp (relative to transcription start site). Based on the information of the ZFPM2 promoter region analyzed by the UCSC Genome Browser (Santa Cruz, San Francisco, CA), we designed the amplicons and polymerase chain reaction (PCR) primers using the EpiDesigner software (Sequenom) and found only one amplicon in the CpG island shore region (ZFPM2_ R1: −1,752 bp ~ −1,323 bp, Figure 1a ), which is 430 base pairs in length and contains 6 CpG units. Primer sequences used are listed in Table 1 .

Figure 1
figure 1

The methylation status for ZFPM2_R1 and ZFPM2_R2. (a) The schematic represents the distribution of ZFPM2_R1 and ZFPM2_R2 in the promoter region. (b) Median methylation levels for ZFPM2_R1 between normal and tetralogy of fallot subjects. (c) BSP sequencing results of ZFPM2_R2. All the values represent median with interquartile range. TSS, transcription start sites; red thick bars, CpG islands; R1, MassARRAY amplicon; R2, BSP sequencing region. *P < 0.05 (Mann-Whitney test). BSP, bisulfite sequencing polymerase chain reaction.

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Table 1 Primer sets for MassArray methylation analysis and BSP
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The methylation level of ZFPM2_ R1 was measured in specimens from 10 TOF patients and 6 age-matched controls for the first screening. The results indicated that the methylation values for ZFPM2_R1 was significantly higher in the TOF patients, with a median of 83.00% (interquartile range (IQR): 78.50–85.42%, N = 9, one male, aged 19 mo was excluded), as compared to 59.63% in control subjects (IQR: 44.79–73.83%; P = 0.0256, N = 6) ( Figure 1b ).

To explore the methylation status of promoter CpG island of ZFPM2 gene, which can not be detected by the Sequenom MassARRAY system (Sequenom), BSP was performed to measure the methylation level of ZFPM2_ R2 (−950 bp ~ −534 bp, Figure 1a ) located in the promoter CpG island of ZFPM2 gene in two TOF patients and in two controls, which is 416 base pairs in length and contains 17 CpG units. The BSP primers used in the study are listed in Table 1 . As shown in Figure 1c , the methylation levels of the ZFPM2_ R2 were very low and showed no significant difference between the TOF patients and controls.

Validation of the Methylation Level of ZFPM2_ R1 in TOF Patients and Controls

According to the date of the first screening, the different methylation levels for ZFPM2_ R1 were further validated by the Sequenom MassARRAY system in a larger cohort of 33 TOF patients and six age-matched controls. As shown in the Figure 2a , the methylation value of ZFPM2_ R1 was significantly higher in patients with TOF, with a median value of 79.67% (IQR: 68.33–86.30%, N = 33), while the control subjects are 59.63% (IQR: 44.79–73.83%; P = 0.0279, N = 6).

Figure 2
figure 2

Median methylation levels for ZFPM2_R1 between normal controls and tetralogy of fallot patients. (a) In independent validated cohorts. (b) In combined group of screening and independent validated cohorts. *P < 0.05 (Mann-Whitney test).

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Furthermore, when the ZFPM2_ R1 methylation levels in the 33 TOF patients and the initial 9 TOF subjects were combined and compared to that of the control subjects, a significantly higher ZFPM2_ R1 methylation level was found in TOF patients, with a median value of 80.32% (IQR: 73.54–85.75%, N = 42) as compared to 59.63% (IQR: 44.79–73.83%; P = 0.0186, N = 6, Figure 2b ) in controls.

Considering the difference of age in the individual subjects in the TOF patients and normal controls, which may influence the methylation values, a correlation of ZFPM2 methylation level with age was analyzed and no significant association was found between the ZFPM2 methylation values and age in the normal controls (r = −0.174, P = 0.714; N = 6, Figure 3a ) or in TOF patients (r = 0.115, P = 0.467; N = 42, Figure 3b ).

Figure 3
figure 3

Correlation of ZFPM2 methylation level and age. (a) In the control group (N = 6). (b) In the tetralogy of fallot patients (N = 42).

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Expression Levels of ZFPM2 mRNA in Patients With TOF and Controls

The mRNA expression level of the ZFPM2 gene was determined by quantitative real-time polymerase chain reaction in 42 TOF patients and 6 controls. The primers used in this study are listed in Table 2 .

Table 2 Primer sequences and product length for PT-PCR analysis
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The normalized Ct mean (▵Ct), ▵▵Ct, and relative quantification (2−▵▵Ct) values for ZFPM2 gene in each samples are listed in Table 3 . The relative quantification value (2−▵▵Ct) of control 1 was set as 1 and used for normalizations for all samples. As shown in Table 4 , ZFPM2 mRNA relative expression levels (relative quantification values) showed significantly lower in patients with TOF compared with controls (P < 0.05).

Table 3 The normalized Ct mean (▵Ct), ▵▵Ct, and RQ (2−▵▵Ct) values for ZFPM2 gene in each individual samples
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Table 4 Relative mRNA expression levels of ZFPM2 in TOF patients and controls
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Association Between the Methylation Statuses and mRNA Level of ZFPM2 Gene

A correlation analysis was performed to identify any relationships between ZFPM2 methylation statuses and its respective mRNA levels in TOF patients.

As shown in Figure 4a , a significant association between the methylation statuses of ZFPM2_R1 and its mRNA levels was observed for the nine TOF patients in the first screening (r = −0.75, P = 0.026, N = 9). In a larger cohort of 33 TOF patients, there was a strong association between the methylation statuses and mRNA level for the ZFPM2 gene (r = −0.42, P = 0.014, N = 33, Figure 4b ). Interestingly, a higher significant correlation was found between the ZFPM2_R1 methylation statuses and mRNA levels (r = −0.40, P = 0.008, N = 42, Figure 4c ) in the combination cohort of the initial 9 and independent 33 TOF patients.

Figure 4
figure 4

Association of ZFPM2 methylation status with its mRNA levels in tetralogy of fallot patients. (a) In the first screening cohort; (b) in independent validated cohorts; (c) in combined group of first screening and independent validated cohorts. *P < 0.05, **P < 0.01.

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To further explore whether DNA methylation regulates gene expression of ZFPM2 in vitro, we used human cardiac myocyte (HCM) cell lines (ScienCell, San Diego, CA) in this study due to lack of well-established TOF disease cell lines. The HCM cell lines were treated with a DNA methyltransferase inhibitor (5-aza-2’-deoxyctidie) (Sigma, St.Louis, MO), which can inhibit the activity of DNA methyltransferase and reduce the DNA methylation level of genome. We then analyzed the methylation status and mRNA levels of ZFPM2 gene before and after treatment with 5-aza-2’-deoxyctidie in this cell. As shown in Figure 5a , b , the methylation values of the ZFPM2_ R1 were very low and showed a significant difference before and after treatment with 5-aza-2’-deoxyctidie. The BSP primers for ZFPM2_ R1 used in the study are listed in Table 1 . Moreover, we found a significantly increased expression of the ZFPM2 gene in the cell treated with 5-aza-2’-deoxyctidine Figure 5c . These data indicated that the promoter methylation of ZFPM2 gene maybe associated with its gene expression.

Figure 5
figure 5

BSP sequencing results of ZFPM2_R1 and mRNA expression levels of ZFPM2. (a) BSP sequencing results of ZFPM2_R1 in HCM cells; (b) BSP sequencing results of ZFPM2_R1 in HCM cells treated with 5-aza-2’-deoxycytidine at 5 μmol for 48 h; (c) mRNA expression levels of ZFPM2 in the HCM cell before and after treated with 5-aza-2’-deoxycytidine at 1, 3, and 5 μmol, respectively, for 48 h.

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Discussion

Epigenetic refers to the study of heritable changes in gene regulation occurring without changes in the DNA sequence, including DNA methylation, histone modification, chromatin remodeling, and long noncoding RNAs. Epigenetic plays a key role in the regulation of tissue homeostasis and disease development (19). The development of TOF disease may be associated with the abnormality of epigenetic regulation. In our previous studies, we have observed that LINE-1 methylation levels, which may serve as a potential indicator of global DNA methylation status, were lower in the cardiac tissue of TOF patients and increased the risk of TOF development (20). The decreased LINE-1 methylation levels may be caused by the lower expression of DNMT1 and DNMT3B (21). In the present study, we focus on the DNA methylation changes within the promoter region of the ZFPM2 gene. We used the Sequenom MassARRAY platform (Sequenom) to analyze the methylation status of ZFPM2 gene. The accuracy of this approach for quantifying methylated and unmethylated DNA has been validated by the Sequenom group (22) and our previous study (23). The methylation levels for the promoter region of ZFPM2 gene were initially explored in the cardiac tissues of 10 TOF patients and 6 age-matched controls, and a significant difference in the methylation levels of CpG island shore of ZFPM2 gene (ZFPM2_R1) was observed (P = 0.0256). Moreover, BSP was used to explore the methylation change in the promoter CpG island of ZFPM2 gene (ZFPM2_R2) and no significant difference was observed ( Figure 1c ).

ZFPM2 is a multi-zinc-finger protein that is coexpressed with Gata4 in the developing heart beginning at E8.5. Svensson et al. (24) used targeted mutagenesis to explore the role of Zfpm2 in normal cardiac development and found Zfpm2-deficient mice died of congestive heart failure at E13 with a syndrome of tricuspid atresia that includes an elongated left ventricular outflow tract, rightward displacement of the aortic valve, and pulmonic stenosis. Moreover, miR-130a has also been reported to regulate the ZFPM2 gene expression and plays an important role in the regulation of cardiac development (25). Promoter DNA methylation of a gene is tightly associated with its transcription activity. DNA hyper-methylation of CpG islands in the gene promoter region has been widely reported to associate with the gene silencing (26). In the current study, we examined the methylation status of CpG islands in the promoter area and mRNA levels of ZFPM2 gene. Although the ZFPM2 mRNA levels were significantly lower in patients with TOF disease compared with controls (P < 0.05), the methylation status of promoter CpG island was very low and there was no significant difference between the TOF patients and controls. These findings suggested that the methylation levels of CpG islands in the ZFPM2 promoter may not influence its gene expression.

The CpG island shore refers to the region located in close proximity (~2 kb) to the CpG islands. Recent study has proved that 76% of differential tissue methylation regions were not located in CpG islands, but in the CpG island shores (27). Rao et al. (28) have reported that, within the promoter of Cav1 gene, the CpG island shore methylation, not the CpG islands, regulates the gene expression in the breast cancer. Although there is no significant difference in the CpG island methylation of ZFPM2 gene in the initial study, the CpG island shore methylation in the ZFPM2 promoter showed significant increased in the TOF patients ( Figure 1b ). The differential methylation levels of the CpG island shore were then validated and confirmed in an independent cohort ( Figure 2a ). When combining the data of the two independent cohorts, interestingly, the methylation levels of the CpG island shore were significant differently even more between the TOF patients and controls ( Figure 2b ).

Considering the age difference in the individual subjects, which have been reported to influence DNA methylation patterns (29), we analyzed the association of ZFPM2 CpG island shore methylation with age and found no significant association in the normal controls ( Figure 3a ) or in TOF patients ( Figure 3b ), suggesting that the age may not influence the ZFPM2 methylation levels.

Moreover, we also analyzed the association of methylation level of ZFPM2 gene with gene expression in the patients with TOF disease. A negative association between methylation status and mRNA level for ZFPM2 gene was found in the TOF cases ( Figure 4 ). To further explore whether the DNA methylation regulates the gene expression of ZFPM2 in vitro, we used HCM cell lines in this study due to lack of well-established TOF disease cell lines. The HCM cell lines were treated with a DNA methyltransferase inhibitor (5-aza-2’-deoxyctidie), which can inhibit the activity of DNA methyltransferase and reduce the DNA methylation level of genome. Interestingly, we observed a reduced DNA methylation value and increased expression for ZFPM2 gene in cells after treatment with 5-aza-2’-deoxyctidie. These findings indicated that promoter methylation of the ZFPM2 gene may influence the gene expression and contribute to the development of TOF disease.

As it is difficult to collect cardiac tissue samples from healthy control and TOF patients, we were unable to obtain enough complete matched samples. Further studies with larger samples or in cell level with more methods are warranted to confirm our findings and reveal how aberrant methylation change contributes to TOF development.

Conclusion

The present study has explored the methylation status of ZFPM2 and found significant changes of methylation levels at the CpG island shore in the ZFPM2 promoter region. Moreover, aberrant methylation status of ZFPM2 genes showed significant negative correlations with its corresponding mRNA expressions, indicating that the DNA methylation changes may contribute to their transcription regulation in the TOF patients.

Methods

Patients and Controls

All surgical specimens of TOF subjects were obtained from the Children’s Hospital of the Fudan University, Shanghai, China. Cardiovascular diagnosis was performed by echocardiography and confirmed by surgery. The surgical procedure is same for all despite different ages. Moreover, we mainly focused on the methylation changes in the TOF subjects, while the patients with other problem and defects were excluded in this study, which included heart failure, trisomy 21, 22q11 deletion, gene mutation, other chromosomal anomalies, or extracardiac major- or minor-associated anomalies.

A total of 43 patients with TOF were recruited in this study. Ten patients with TOF in first screening cohort included 7 (70%) males and 3 (30%) females, ranging in age from 5.0 to 48.0 mo (8.0 (6.0–19.0), median (interquartile range)). Thirty-three patients with TOF in the independent validated cohort included 20 (60.6%) males and 13 (39.4%) females, ranging in age from 3.0 to 48.0 mo (12.0 (7.5–24.0)).

The control group was taken from autopsy specimens from healthy subjects. The control subjects were collected at the forensic medicine department of the Fudan University, Shanghai, China. The postmortem interval for the control subjects was as shortly as possible, not more than 24 h. Six age-matched control subjects were collected for the study, including four (66.7%) males and two (33.3%) females, ranging in age from 6.0 to 54.0 mo (15.0 (10.5–30.0)). Anagraphical characteristics of the study subjects were summarized in Table 5 .

Table 5 Anagraphical characteristics of TOF patients and control subjects
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All the tissue samples were obtained from right ventricular outflow tract and saved in RNA later solution (Ambion, Austin, TX) immediately after surgical resection or autopsy and stored until use. So, the cellular composition or tissue quantity of controls is comparable to that of TOF patients, excluding any tissue heterogeneity that may affect methylation results.

Written informed consents were obtained from the parents or relatives of all the study subjects. This study was approved by the local ethics committee of the Fudan University.

DNA Extraction and Sodium Bisulfite Conversion

A QIA amp DNA Mini Kit (Qiagen, Hilden, Germany) was used to extract genomic DNA from the heart tissue samples of TOF and normal control subjects according to manufacturer’s instructions. The concentration and purity of genomic DNA were determined by absorbance at 260 and 280 nm by NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington). EZ DNA Methylation Kit (Zymo Research, Orange, CA) was used to perform sodium bisulfite modification for the genomic DNA strictly according to manufacturer’s instructions. The bisulfite converted genomic DNA was resuspended in 10 μl elution buffer and stored at −80 °C until the samples were ready for analysis.

Quantitative MassARRAY Analysis of Gene Methylation Status

Quantitative methylation analysis for ZFPM2 gene was performed using the EpiTyper by MassArray (Sequenom) based upon base-specific cleavage and MALDI-TOF mass spectrometry, as recommended by the manufacturer. The robustness of this approach for quantifying methylated and unmethylated DNA has been demonstrated by the Sequenom groups (22). The promoter regions of the ZFPM2 gene were analyzed by the UCSC Genome Browser (Santa Cruz). The amplicons and PCR primers used in this system were designed with the EpiDesigner software (Sequenom). For each reverse primer, an additional T7 promoter tag for in vivo transcription was added, as was a 10-mer tag on the forward primer to adjust for the melting temperature differences. All experiments were performed as we described before (20). The methylation status for ZFPM2 CpG island was detected by bisulfite sequencing PCR (BSP) (30).

RNA Extraction and Quantitative real-time PCR

Total RNA was extracted from all tested heart tissue samples using Trizol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The extracted RNA quality and integrity were validated before use. RNA was reverse-transcribed using PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Biotechnology, Dalian, China) and integrity of synthesized cDNA was confirmed using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as endogenous control. Quantitative real-time PCR was performed using SYBR Premix Ex Taq GC (Perfect Real Time, TaKaRa). The reaction volume includes 5 μl SYBR Premix Ex Taq GC, 0.2 μmol/l of each primer, 0.2 μl ROX1 Reference Dye and 100 ng cDNA. Reactions were performed in triplicate and analyzed using an ABI 7900 Sequence Detection System (Applied Biosystems, Foster, CA). The standard 2−▵▵Ct method was used to calculate the relative expression levels of mRNA. The β-2 microglobulin (B2M) and the GAPDH gene were used as the endogenous controls for normalization in this study.

Cell Culture and In Vitro Methylation Status and mRNA Levels of ZFPM2 Gene

HCM cell lines were purchased from ScienCell Research Laboratories (ScienCell) and used in this study due to lack of well-established TOF disease cell lines. Cells were cultured in Dulbecco’s Minimum Essential Medium supplemented with 10% fetal bovine serum (Gibco, NY) and penicillin-streptomycin at the condition of 37 °C in 5% CO2. The HCM cells were treated with 5-aza-2’-deoxycytidine (Sigma) at 1, 3, and 5 μmol respectively for 48 h. Medium was changed daily with fresh drugs. The genomic DNA and RNA of the cell before and after treatment with 5-aza-2’-deoxyctidie (Sigma) were extracted for the analysis of methylation status and mRNA levels. The methylation status of ZFPM2_R1 was determined by BSP and mRNA level was measured by real-time RT-PCR assay as described above.

Statistical Analysis

Data were analyzed using GraphPad Prism (version 5.0; GraphPad Software, San Diego, CA). Mann-Whitney U-test or unpaired t test was performed to evaluate the significance of any differences between TOF and control groups. Spearman correlation analysis was performed to evaluate the correlations between the methylation status of ZFPM2 gene and its mRNA levels. All statistical analysis was two-sided and P < 0.05 was considered statistically significant.

Statement of Financial Support

This work was supported by grants from the National Natural Sciences Foundation of China (81570282, 81570283, and 81370198), Shanghai Municipal Commission of Science and Technology Research Project (11JC1401400).

Disclosure

The authors declare that they have no competing interests.