Changes And Mechanisms Of Gene Methylation In The Children With Tetralogy Of Fallot

Congenital heart defect (CHD) is a structure defect of the heart and great vessels during embryonic development, which account for6%o～10%o in newborn infants, ranking top one birth defects according to the recent survey data. Conotruncal defects (CTD) is a kind of severe cardiovascular abnormality, including tetralogy of fallot, pulmonary atresia, transposition of the great arteries, double outlet of the right ventricle and persistent truncus arteriosus, which represent15-20%of all congenital heart defects and is the majorcause of death of infant and newborn in China. Tetralogy of fallot (TOF) is a congenital defect caused by the improper development of the right side of the heart. TOF accounts for10%of all CHD and is the most common complex conotruncal defects. It is characterized by four distinct anatomic features: pulmonary outflow tract obstruction, ventricular septal defect, overriding aortic root and right ventricular hypertrophy. The TOF malformations can be lethal. Although treatment has advanced dramatically in the last few decades, there are0.5%to6%of the TOF patients who survive after the surgery die of different kinds of complication. But the exact etiology remains unclear. Studies have proven that both environmental factors and genetic factors may lead to the development of TOF concurrently. But, epigenetic, especially the role of DNA methylation in the occurrence of the TOF disease is rarely reported.In this study, we used the cardiac tissue samples from the right ventricular outflow tract of patients with TOF as subject, the cardiac tissue samples from the same place of non-cardiac caused death from forensic autopsy as normal control to study the DNA methylation status in the subjects. By analyzing the change of DNA methylation level, assessing the relationships between the risk of TOF development and genome-wide DNA methylation level and establishing the specific DNA methylation pattern of TOF disease, we provide important scientific clues to explore new strategies for etiology and mechanism of pathogensis of TOF from the epigenetic point of view.Study contents:Section1:We studied the genome-wide DNA methylation level in the cardiac tissue from the patients with TOF and analyzed the change of DNA methylation status and its association with TOF in Chinese infants. Long interspersed nucleotide element-1(LINE-1) is one of the repetitive elements and constitutes17%-25%of the human genome. Because LFNE-1sequences are highly repeated and widely interspersed human retrotransposons, their methylation status can serve as a surrogate marker for the level of global genomic DNA methylation. In this study, we measured and analyzed the methylation change of LINE-1element in32patients with TOF and15normal controls by the MassArray EpiTYPER platform.Section2:We detected the transcription levels of DNMT1, DNMT3A, DNMT3B and MBD2and analyzed the association among them and with global DNA methylation status. We extracted the total RNA from48patients with TOF and16normal controls and verified the total RNA integrity. Then, we used the QRT-PCR method to detect the mRNA expression changes and analyzed the experimental datas by software.Section3:Based on data from related reference, we collected71candidate genes associated tightly with the development of heart or heart malformation. The promoter regions of these candidate genes were analyzed from the website http://genome.ucsc.edu/.113amplicons and PCR primers were designed with Methprimer (http://epidesigner.com) in this study. These amplicons distribute in72CpG island (64%),24CpG island shore (21%) and17amplicon without CpG island (15%). In small samples trials, we detected the methylation levels of the promoter of these genes in10patients with TOF and6controls using the Sequenom MassARRAY platform. Then, we verified the experiment results in larger scale of samples and established the specific DNA methylation pattern of TOF disease. Finally, we measured the mRNA level of11genes in which the DNA methylation level show significant different, and analyzed their association with corresponding methylation status.Study results:Section1:The methylation level of LINE-1was statistically significant lower in patients with TOF compared to the controls (59.70%vs57.95%, p=0.0021). The risk of the TOF development was significantly increased with the lower degree of methylation levels (OR=14.7,95%CI:1.8-117.7, p=0.014). An ROC curve analysis showed a relatively high accuracy of using the LINE-1methylation level in predicting the presence of TOF (AUC=0.78,95%CI:0.65-0.91;p=0.002). Neither age nor gender was associated with the LINE-1methylation level in patients and controls.Section2:The mRNA levels of DNMTs and MBD2showed statistically significant decreases in TOF patients (p<0.001). Especially, the expression of DNMT1and DNMT3B reduced significantly compared to normal control (p<0.0001). Pairwise correlation analysis showed that, in control samples, only DNMT1showed significant positive correlation with DNMT3A mRNA level (r=0.718, p=0.002). There had no relationship of the genome-wide DNA methylation status and the expression levels of MBD2and three DNA methyltransferases. But in TOF patients, the expression levels of the DNMT1, DNMT3A, DNMT3B and MBD2had correlated positively relationship with each other and showed significant (p<0.05). There had a significantly negative correlation of the global DNA methylation with the expression of MBD2(r=-0.579,p=0.005) in patients with TOF.Section3:We detected the methylation levels of the promoter regions of71candidate gene (including113amplicons totally) in10TOF patients and6normal controls by the Sequenom MassARRAY platform in the small size trials. The results indicated that the median methylation values of26amplicon for, referring to26genes, show significant different between the patients and controls (p<0.05). Seventeen genes, including CFC1B, DVL2, EGFR, EDNRA, EVC2, GJA5, HAND1, HAS2, HSPG2, MED13L, NFATC1, NKX2-5, NFATC2, PAX3, TBX5, TEK, ZFPM2, show up-regulated methylation levels. Nine genes, including EDN1, HOXA3, MYH6, NR2F2, NRG1, NRP1, PDGFRA, SLC2A10, TBX20, shows down-regulation. Moreover, there are14amplicons, referring to14genes (DVL2, EDN1, EGFR, EVC2, HAND1, HAS2, HSPG2, NKX2-5, NRG1, NFATC2, PAX3, PDGFRA, SLC2A10, TBX20), which located in the CpG island,7amplicons, referring to7genes (EDNRA, MED13L, NFATC1, NR2F2, NRP1, TBX5, ZFPM2), locating in the CpG island shore, and5amplicons, referring to5genes(CFClB, GJA5, HOXA3, MYH6,TEK), covering the region near transcription start site (TSS). On the basis of the data analyzed above, the methylation status for eleven candidate genes, including CFC1B, EGFR, EVC2, GJA5, HAND1, NKX2-5, NR2F2, NFATC2, TBX5, TBX20, ZFPM2, were further confirmed in the larger scales of samples, including42patients with TOF and22normal controls. Moreover, the mRNA levels for the eleven candidate genes were detected, showing significant reduced in the patients with TOF (p<0.05) in addition to HAND1which showed significant increased compared to normal control. In summary, we first confirmed that the global DNA methylation levels in the outflow tract tissue of TOF patients had significantly lower compared to normal controls. The risk of TOF increased with the reduction of the degree of global DNA methylation level. Lower expression of DNMT1and DNMT3B may play an important role in the pathogenesis of TOF. We established the specific DNA methylation pattern of candidate genes of TOF disease. These findings may provide important clues in the heart development and the pathogensis study of conotruncal defect.