Study On Cx43Gene Sequence Analysis, Expression And Its Regulation Mechanism In Patients With Tetralogy Of Fallot

Congenital heart disease (CHD),one of the most common birth defect, affects approximately6%o-9%o of children and is the leading cause of death in infants younger than one year of age. The data from WHO shows that there are1.5million children born with CHD each year in the world, while in our country the data is150,000to200,000. Conotruncal defects (CTD), caused by abnormal development of conotruncus, accounts for30%of CHD. Tetralogy of Fallot (TOF), accounting for10%of CHD, is the most common type of cyanosis CHD. TOF malformations can be lethal, without surgery patients will die before adult. Even after surgery,0.5%to6%of patients will die of sudden cardiac death due to ventricular tachycardia, which causes a heavy burden on the family and society. Therefore, it is important to study the pathogenesis of TOF. As the most prevalent connexin (Cx) in mammal heart, animal experiments have confirmed that Cx43(connexin43) gene is an important candidate gene for TOF. However, it is still obscure whether Cx43is also important in TOF patients. This study aimd to clarify the relationship between TOF patients and Cx43and the role Cx43played in the pathogenesis of TOF by detecting its coding and promoter sequence variation, expression and regulation in TOF patients. This study will not only partly clarify the role of Cx43in the pathogenesis of human TOF, but also provide a new way for the research of other birth defects.PartⅠ Sequence analysis of Cx43gene in TOF patientsObjective:Detect the sequence variation of the coding and promoter region of Cx43in TOF patients and analyze its role in the pathogenesis of TOF.Methods:A cohort of200TOF patients were recruited in the study,200normal children were used as controls. PCR and DNA sequencing were performed for the detections of DNA variation of Cx43gene in TOF patients.Results:1.Two novel synonymous mutations including c.G455A, c.G716A were identified in the coding region of Cx43in1TOF patient, respectively.2.Four heterozygous mutations in the promoter region, including T-153C,T-157C, T-266C and A-1462G were detected in TOF patients. T-266C was detected in2TOF patients, while C-153T, T-157C, A-1462G were each detected in1TOF patient. We also identified seven known single nucleotide polymorphisms (SNPs), including C-284T(rs9597379),A-416C(rs2071166),G-535A(rs2071165),G-925C(rs1925223),A-1408G(rs75153918),G-1431GA(rs73532207),T-1958C(rs37631741) and four novel SNPs (T-1032C,G-1415A,T-1478C,and C-1569T), which we found in the Chinese population but did not find in the NCBI dbSNP or1000Genome Project database. The chi-square tests for these SNPs showed that the allele distribution of G/A at the site-1431(OR=0.698,95%CI=0.500261-0.975258, p=0.034) and genotype distribution of TT/TC/CC at the site-1958(p=0.047) were significantly different between the TOF patients and controls. All potential pathogenic mutations and SNPs were located in the5’-UTR of Cx43.It is predicted that there is an Nkx2-5gene binding site65bp downstream of the-1431site.Conclusions:1. Gene mutations in the Cx43coding region are rare in TOF patients.2. Mutations and SNPs in the promoter region of Cx43may influence the binding of some transcription factors such as Nkx2-5and play important role in human TOF.Part ⅡCx43gene expression in the right ventricular outflow tract myocardium of TOF patientsObjective:Detect the expression of Cx43mRNA and protein in the right ventricular outflow tract (RVOT) myocardium of TOF patients and illustrate the role of its abnormal expression in the pathogenesis of TOF.Methods:Myocardial samples from the right RVOT were collected from30patients undergoing cardiac surgery in our hospital.10normal RVOT myocardial samples, from victims of traffic accidents, were collected as controls, whose age and gender were matched as closely as possible to those of the TOF patients. Real-time PCR and immunohistochemistry were used to detect the mRNA and protein expression of Cx43gene, respectively.Results:1. Real-time PCR analysis revealed that the mRNA expression of Cx43in the RVOT myocardium was significantly increased in TOF patients compared to the controls (p=0.0006).2. Immunohistochemical staining showed that Cx43gene was irregularly distributed in the cytoplasm and cell membranes of the RVOT myocardial cells in the TOF patients but that it was regularly distributed in the intercalated disc in the controls. Cx43protein expression was increased in the TOF patients.Conclusions:Abnormal transcriptional and post-transcriptional regulation may exist, resulting in the increased expression of Cx43mRNA and protein in TOF patients. Abnormal distribution and over-expression of Cx43may affect the migration of neural crest cell, as well as the signal transduction of some important signaling pathways and transcription factors in heart development, leading to the abnormalities of RVOT obstruction.Part ⅢPreliminary study of regulation mechanism of abnormal Cx43expression in the RVOT of TOF patientsObjective:Detect the methylation status of Cx43gene promoter and enhancer, the expression of Cx43-related microRNAs (miRNAs) and Cx43-related transcriptional factors, clarify their role in Cx43abnormal expression and pathogenesis of TOF.Methods:Myocardial samples from the right RVOT were collected from30patients undergoing cardiac surgery in our hospital.8normal RVOT myocardial samples, from victims of traffic accidents, were collected as controls, whose age and gender were matched as closely as possible to those of the TOF patients. MassARRAY EpiTyper platform was used to detect the methylation status of Cx43gene promoter and enhancer, while luciferase assays were used to verify the function of abnormal CpG sites; Real-time PCR was used to detect the expression of ten Cx43-related miRNAs, while DNA sequencing was used to detect the sequence variation of miRNAs binding sites in the3’UTR of Cx43; Real-time PCR was also used to detect the expression of Cx43-related transcriptional factors.Results:1. None of the eight CpG sites in the promoter region of Cx43was found to be differentially methylated in the TOF patients compared to the controls (p>0.05). The average methylation of3CpG sites in one of the enhancer regions in the first intron of Cx43was lower in TOF patients as compared to the controls (p=0.007).In vitro experiments showed that after transfecting the plasmid containing the enhancer sequence into HEK293T cells, the luciferase activity of the cells significantly increased (3.36-fold) compared to the controls. Then, we methylated the sequence in vitro using M.SssI. After transfecting the methylated plasmid into HEK293cells, the luciferase activity of the cells significantly decreased (60%reduction).2. The expression of miR-1and miR-206was significantly decreased in TOF patients as compared with the controls (p=0.0001, p=0.0112, respectively). No obvious differences were observed in the expression of miR-130a, miR-19a, miR-30a, miR-30c, miR-30d and miR-130b between TOF patients and the controls. MiR-30e and miR-144expression were not detected in the RVOT myocardium. Sequence analysis of miR-1and miR-206putative binding sites in the3’UTR of Cx43identified no mutations and differentially distributed SNPs.3. No obvious differences were observed in the expression of Nkx2-5and p53between TOF patients and controls.Conclusions:1. Hypomethylation was existed in one enhancer of Cx43gene in TOF patients, which may influence the binding of some transcriptional factors and lead to over-expression of Cx43mRNA in transcriptional level.2. MiR-1and miR-206is down-regulated in TOF patients, which may cause an up-regulation of Cx43protein in post-transcriptional level.3. Over-expression of Cx43might not be associated with the expression of Nkx2-5and p53.