Preliminary Study On The Mechanism Of RXRA Gene In Pathogenesis Of Tetralogy Of Fallot

Conotruncal defects (CTD), induced by abnormal development of the outflow tract (OFT), account for almost30%of congenital heart disease (CHD) and can be categorized as TGA, DORV, tetralogy of Fallot, and PTA. The genetic causes for CTD remain largely unknown. Tetralogy of Fallot (TOF) is is the most common type of cyanosis CHD. TOF malformations can be lethal; patients will die before adult without surgery. Therefore, it is important to study the pathogenesis of TOF. Retinoid X receptor alpha (RXRA), a ligand-dependent transcript factor, plays a critical role in multiple aspects of cardiogenesis including the development of outflow tract (OFT). Taken together, RXRA may be a potential candidate gene of TOF. To date, RXRA gene has not been analyzed and reported in TOF patients. As the most prevalent connexin (Cx) in mammal heart, animal experiments have confirmed that Cx43(connexin43) gene is an important candidate gene for TOF. What’s more, the expression level of Cx43in the RVOT myocardium was significantly increased in TOF patients compared to the controls according to our previous research. 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. The aim of the study was to clarify the relationship between TOF patients and RXRA and the role RXRA played in the pathogenesis of TOF by investigating its coding and promoter sequence variation, expression and regulation in TOF patients and the possible mechnism of RXRA and Cx43in pathogenesis of TOF.Part Ⅰ Sequence analysis of RXRA gene in TOF patientsObjective:To detect the sequence variation of the coding and promoter region of RXRA in TOF patients and analyze its role in the pathogenesis of TOF.Methods:A cohort of213TOF patients were recruited in the study,500normal children were used as controls. Genomic DNA was extracted from peripheral blood. PCR was used to amplify the coding and promoter region of RXRA from genomic DNA. PCR products were sequenced by ABI Prism Bigdye system. Results:1. Two novel synonymous mutations including c.237G/A and c.288A/G were identified in the coding region of RXRA in TOF patient. There were no statistically significant change in the genotype and allele frequencies of the SNPs between TOF patients and control group (p>0.05).2. One novel heterozygous mutation,-1191A/G (according to the transcription start site), was found in one TOF patients, but in none of controls. One novel heterozygous mutation,-1287C/T, was found in one TOF patients, and in one of controls. Two novel single-nucleotide polymorphisms,-800C/A and-760C/T were found in both TOF patients and controls. There were no statistically significant change in the genotype and allele frequencies of the two SNPs between TOF patients and control group (p>0.05).3. Several transcription factor binding sites within the region containing these single-nucleotide variations were found by searching the website: http://www.cbrc.jp/research/db/TFSEARCH.html.Conclusions:1. Gene mutations in the RXRA coding region are rare in TOF patients.2. Mutation and SNPs in the promoter region of RXRA may influence the binding of some transcription factors and play important role in human TOF.Part ⅡRXRA gene expression in the right ventricular outflow tract myocardium of TOF patientsObjective:To investigate the expression of RXRA mRNA 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. Real-time PCR and immunohistochemistry were used to detect the mRNA and protein expression of RXRA gene, respectively.Results:1. Real-time PCR analysis revealed that the mRNA expression of RXRA in the RVOT myocardium was significantly decreased in TOF patients compared to the controls.2. Immunohistochemical staining showed that the expression level of RXRA protein was decreased in the TOF patients.Conclusions:Abnormal transcriptional and post-transcriptional regulation may exist, resulting in the decreased expression of RXRA mRNA and protein in TOF patients.Part ⅢPreliminary study of regulation mechanism of abnormal Cx43expression in the RVOT of TOF patientsObjective:To preliminarily clarify the possible regulation mechanism of RXRA in TOF patients by detecting the methylation status of RXRA gene promoterMethods:Myocardial samples from the right RVOT were collected from26patients undergoing cardiac surgery in our hospital.6normal 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. Methylation status of the RXRA promoter region in TOF patients and controls were detected by BSP cloning-based sequencing. Dual-luciferase assay combined with methylation assay in vitro were performed to determine the transcription activity of unmethylated and methylated CpG region in RXRA promoter.Results:1. The methylation status of CpG region containing CpG sites1-23(-1000～-1453) in the RXRA promoter region was statistically higher in TOF patients than the controls.2. By searching the web site http://www.cbrc.jp/research/db/TFSEARCH.html we found that this CpG region contained several transcription factor sites including SP1.3. Dual-luciferase assays combined with methylation assay in vitro showed that this CpG region had transcriptional regulate activity and methylation of this region can depress its transcriptional regulate activity.Conclusions:The aberrant methylation at RXRA promoter may be responsible for the aberrant gene expression in RVOT myocardium of TOF patients. These findings suggest that TOF may also be an epigenetic disease.Part IVPreliminary study on mechanism of RXRA protein and Cx43protein in pathogenesis of TOFObjective:To preliminarily clarify the possible mechanism of RXRA protein and Cx43protein in pathogenesis of TOF.Methods:HEK293T cells were transiently cotransfected with expression vectors of RXRA and Cx43, western blot and immunofluorescence assays were used to detect the expression level of Cx43protein while real-time PCR was used to measure the transcpition level of Cx43mRNA. Western blot was used to test the change of Cx43protein after9-cis RA treatment and RNA interference of RXRA respectively.Results:1. Both Western blot and immunofluorescence assays revealed that the expression level of Cx43protein was signifficantly decreaed in HEK293T cells transiently cotransfected with expression vector of RXRA and Cx43compared with cells cotransfected with RXRA-Control and Cx43.2. Real-time PCR indicated that the transcription level of Cx43mRNA was signifficantly decreaed in HEK293T cells transiently cotransfected with N4flag-RXRA and Cx43-3myc compared with cells cotransfected with N4flag-Control and Cx43-3myc.3. Western blot revealed that the expression level of Cx43protein was signifficantly decread after9-cis RA treatment and increaed after RNA interference of RXRA.Conclusions:RXRA may inhibit the expression of Cx43at transcription level; the decrseaed expression level of RXRA in TOF patients resulted in aberrant overexpression of Cx43and subsequently took part in the abnormalities of RVOT obstruction.