Epigenetic control mechanisms play key roles in the regulation of tissue homeostasis and disease development. This includes DNA methylation, histone modification, the regulation of mRNA stability, translation by non-coding RNAs and differential RNA splicing . DNA methylation constitutes an important epigenetic regulation mechanism in many eukaryotes and has been extensively studied . In a previous study, decreased LINE-1 methylation levels were found in the cardiac tissue of TOF patients. The lower LINE-1 methylation level may be associated with increased TOF risk  and could serve as an indicator of global DNA methylation . In the current study, we initially performed quantitative methylation analysis of NKX2-5, GATA4 and HAND1 genes in the right ventricular myocardium tissues of 10 TOF patients and 6 age matched control subjects using the Sequenom MassARRAY platform and found a significant difference in the methylation levels of NKX2-5_M3 and HAND1_M1 in TOF patients compared with controls (p < 0.05). The methylation values for NKX2-5_M3 and HAND1_M1 were then validated in a larger cohort TOF subjects. The difference in methylation levels for NKX2-5_M3 and HAND1_M1 were compared, between TOF patients and controls, in two independent cohorts. A significant difference was observed in the methylation status of NKX2-5_M3 when comparing the 20 TOF patients to the 6 controls (p = 0.0383). The methylation levels for the promoter region of the HAND1 gene (HAND1_M1) were also significantly different between the 20 TOF patients and the 6 controls (p = 0.0137). Interestingly, the significant difference in the methylation levels for NKX2-5_M3 and HAND1_M1 increased when comparing the 30 TOF cases to the 6 controls (p = 0.0074, p = 0.0054, respectively).
The MassARRAY EpiTYPER® for quantitative analysis of DNA methylation combines base-specific enzymatic cleavage with MALDI-TOF mass spectrometry. This combination provides a highly accurate, sensitive and high-throughput method for the quantitative analysis of DNA methylation. The EpiTYPER software provides convenient solutions for data analysis and export, however,the Sequenom EpiTYPER has a 5% technical error rate . Although the robustness of this approach for confirming the accuracy of Sequenom methylation analysis results has been demonstrated, we used two alternate methods,pyrosequence and BSP,to test the methylation analysis results of NKX2-5 and GATA4. No apparent differences were detected among these methods. This data supports the accuracy of the Sequenom MassARRAY methylation analysis results in our study.
Increasing evidence suggests that single gene mutations are present in a broad spectrum of genes involved in cardiac structure and function. Factors, such as NKX2-5, GATA4 and HAND1, are among the earliest transcription factors expressed in the developing heart and are crucial in the activation of cardiac-specific genes . Wang et al. sequenced all exons and their boundaries in the NKX2-5, GATA4 and HAND1 genes from non-syndromic TOF children and controls and found no evidence that somatic mutations in NKX2-5, GATA4 and HAND1 play a role in the pathogenesis of TOF . Although mutations in NKX2-5, GATA4 and HAND1 have been observed in patients with TOF, they are present only in a small percentage of TOF cases and cannot be considered to be the main cause of TOF. Based on these findings, we hypothesized that the aberrant DNA methylation modifies these genes and likely contributes to the development of TOF. In the present study, no significant difference in GATA4 methylation levels were observed between TOF patients and controls, thus the methylation status of GATA4 likely has no influence on its gene expression. Furthermore, we found that mRNA levels of the NKX2-5 and HAND1 genes were significantly lower in TOF patients compared to controls (p < 0.05). We also found significant negative correlations between methylation status and mRNA level for NKX2-5 and HAND1 in the 30 TOF cases (Figure 7A and B). Interestingly, the associations between methylation status and mRNA level for NKX2-5 and HAND1 were confirmed in the 20 TOF cases without including the initial 10 TOF subjects (Figure 7C and D). This demonstrates a consistent, significant association via two independent comparisons. These findings indicate that changes in the methylation levels for NKX2-5 and HAND1 may influence gene expression and contribute to the development of TOF.
One limitation of this study was that we were unable to obtain enough complete age-matched samples from TOF patients and healthy controls due to the difficulty of obtaining cardiac tissue samples. Further studies with larger number of subjects or samples are warranted in order to confirm our findings that aberrant methylation contributes to TOF development. Moreover, based only on the current results from clinical samples, we cannot ascertain whether the noted methylation changes occurred after the heart was already formed or after the heart defect already existed. Thus, we cannot determine whether these changes are reflective of disease physiology or related to disease etiology. Related research will be conducted using cell lines or animal models in future studies.