Tetralogy of fallot (TOF) is a congenital defect caused by the improper development of the right side of the heart . TOF accounts for 10% of all congenital heart defects, with an incidence of 3.6 per 10,000 live births . It is characterized by four distinct anatomical features: pulmonary outflow tract obstruction, ventricular septal defects (VSD), overriding aortic roots, and right ventricular hypertrophy . TOF malformations can be lethal. Although treatment has advanced dramatically over the past few decades, 0.5% to 6% of TOF patients who survive after treatment suffer sudden cardiac death . Research into congenital heart disease has come a long way since the first description and classification of such conditions. Improvements in utero diagnosis and surgical techniques have considerably brightened the prospects of infants born with congenital heart diseases, but true biological insights into this set of developmental diseases have been gained only recently, and their exact etiology remains unknown . Heredity is likely to play an important role in the development of TOF . In recent years, some studies have proved the existence of a correlation between TOF and gene mutations . NKX2-5 and HAND1 are known to act as regulatory genes during cardiac development. They are evolutionarily inflexible in their regulation of the differentiation of cardiac muscle cells and morphogenesis of the heart [7, 8]. Mutations in NKX2-5 and HAND1 have been identified in patients with TOF . TBX20, a member of the T-box transcription factor family, interacts directly with NKX2-5, GATA4, and GATA5 in the regulation of gene expression in the developing heart . Mutations and over-expression of these genes have been identified in patients with TOF . Some studies have reported chromosomal abnormalities in infants and fetuses with conotruncal cardiac malformations . TOF also has a strong association with San Luis Valley Recombinant Chromosome 8 syndrome and trisomy 21 . The causes of TOF are complex. In addition to disorders in the DNA sequence, epigenetic regulation has been proven to be associated with CHD . Despite advances in uncovering the molecular basis of these epigenetic mechanisms, their roles in cardiovascular development, tissue homeostasis, and cardiovascular disease are largely unknown . Alterations of DNA methylation patterns have been found in many types of cancers, such as lung cancer, brain tumors, and hepatocellular carcinoma (HCC) . The cancer genome is frequently characterized by hypermethylation of specific genes concurrently with an overall decrease in the level of 5′ methyl cytosine. This hypomethylation of the global genome promotes chromosomal instability, translocation, gene disruption, and reactivation of endoparasitic sequences .
Long interspersed nucleotide element-1 (LINE-1) is a repetitive element. It constitutes 17–25% of the human genome . LINE-1 elements are moderately CpG rich, and most heavily methylated CpGs are located in the 5′-UTR, where they serve as internal promoters . Because LINE-1 sequences are frequently repeated and widely interspersed human retrotransposons, their methylation level can serve as a surrogate marker of global genomic DNA methylation . Hypomethylation in the promoter region of LINE-1 causes transcriptional activation of LINE-1 element, which causes transposition of the retroelement and chromosomal alteration . One recent report has shown that global LINE-1 hypomethylation can repress genome-wide gene expression . Alterations of LINE-1 methylation status have been observed frequently in some diseases, such as colon cancer , neural tube defects , and systemic lupus erythematosus . This has been shown to be a good prognostic marker in certain cancers . Maternal LINE-1 DNA hypomethylation has been found to be associated with increased occurrence of non-syndromic CHDs . LINE-1 showing higher methylation levels was also observed in Alzheimer’s disease (AD) . These findings suggest that changes in LINE-1 methylation may not be restricted to cancer but may instead be present in other diseases and show hypo- or hype-methylation status under different conditions.
However, it remains unclear whether changes in LINE-1 methylation are correlated with TOF. Although mutations in NKX2-5, HAND1 and TBX20 have been found in patients with TOF, together they only account for a very small percentage of patients . In addition, little is known about whether changes in methylation are present in these specific genes.
In the present study, to determine whether alterations in LINE-1 methylation exist in the TOF tissue sample and are associated with the risk of TOF, we measured the methylation level of LINE-1 elements in the TOF patients and controls and evaluated the association between LINE-1 methylation status and TOF. The promoter methylation status of NKX2-5, HAND1 and TBX20 and their possible association with LINE-1 methylation were also investigated.