Studies On DNA Sequences, Expression And Methylation Of VANGL2,TGFβ2, PAX3Genes In Patients With Tetralogy Of Fallot

Congenital heart disease （CHD） is one of the most commonly seen congenital malformations in children, which can severely influence mortality and life quality of children. Conotruncal defect （CTD） is a kind of complex congenital heart disease, it can cause hypoxemia and irreversible acidosis during neonatal period, thus, leads to early death. The pathogenesis of CTD has already been more highlighted. Reciprocity of multi-gene and environment are accepted as the cause of CHD. Tetralogy of Fallot （TOF） is the common conotruncal defects. VANGL2, TGFβ2and PAX3gene knockout animals have appeared in the phenotype of conotruncal defects. Thus, these genes were considered as candidate genes for conotruncal development. Our studies were to investigate the DNA sequence changes and examine the methylation and the expression level of VANGL2, TGFβ2and PAX3genes in patients with TOF. The results will help to illustrate the pathogenesis of conotruncal defects.Part â…  Sequence changes of VANGL2, TGFβ2, PAX3genes in patients with Tetralogy of FallotObjective:To examine the DNA sequence changes of VANGL2, TGFβ2and PAX3genes with Tetralogy of Fallot, and to observe whether these genes are the susceptibility genes for TOF.Methods:A cohort of100pediatric patients with Tetralogy of Fallot was recruited in the study,200normal children were used as control. PCR and genotyping were performed for the detections of DNA changes of VANGL2, TGFβ2and PAX3genes.Results:1. Sequence changes of VANGL2Among all TOF patients, only3single nucleotide changes including33543A>AG in exon7,34641G>GA in exon8and9010T>TG in5’-UTR were found, however all of these3heterozygous changes do not alternate the amino acid of the VANGL2protein. There were no significant differences in allelic frequencies and genotypes frequencies of position33543and34641in coding region between the TOF group and the Control. There were significant differences in allelic frequencies （X2=6.865, P=0.032） of position9010in5’-UTR between the TOF group and the Control.2. Sequence changes of TGFβ2No mutations were identified in coding region in all TOF patients. However,3single nucleotide changes including9126A>AC,9353A>AG and9040₉043del CTTC in5’-UTR were found. There were no significant differences in allelic frequencies and genotypes frequencies of position9126and9353between the TOF group and the Control. There were significant differences in allelic frequencies （X2=17.469, P<0.001） of position9040₉043in5’-UTR between the TOF group and the Control.3. Sequence changes of PAX3A single nucleotide change AA>GG in the position95457in exon2was detected in all examined people. Two single nucleotide changes including97596G>GC and9763297655del GTGTGTGTGTGTGTGTGTGTGTGT in5’-UTR were found in TOF patients. There were significant differences in allelic frequencies and genotypes frequencies （X2=8.711, P=0.013; X2=4.367, P=0.037） of position97596between the TOF group and the Control. There were significant differences in allelic frequencies （X2=72.0, P<0.001） of position97632₉7655in5’-UTR between the TOF group and the Control.There were5patients with97654₉7655del GT and1patients with98064G>GA in5’-UTR in all100TOF patient, which were thought as two pathogenic mutations in5’-UTR in TOF patients.Conclusions:1. SNPs at position9010T>TG in5’-UTR of VANGL2is associated with the susceptibility of TOF.2. SNPs at position9040₉043del CTTC in5’-UTR of TGFβ2is associated with the susceptibility of TOF. CTTC allelic may be susceptible allelic to TOF.3. SNPs at position97596G>GC and97632₉7655del GTGTGTGTGTGTGTGTGTGTGTGT in5’-UTR of PAX3is associated with the susceptibility of TOF. GTGTGTGTGTGTGTGTGTGTGTGT allelic in97632₉7655may be susceptible allelic to TOF.4. Two pathogenic mutations in5’-UTR of PAX3were identified in Chinese TOF patients （97654₉7655del GT and98064G>GA）.Part IIMethylation at5’-UTR and expression of VANGL2, TGFβ2, PAX3genes in right ventricular outflow tract myocardium in TOF patientsObjective:To examine the CpG island methylation and expression of VANGL2, TGFβ2, and PAX3genes in the myocardium of patients with TOF, and to illustrate the roles of these genes in the pathogenesis of TOF.Methods:Five pediatric patients with Tetralogy of Fallot was recruited in the study. The CpG island methylation status was detected at5’-UTR of VANGL2, TGFβ2and PAX3genes using COMPARE-MS. The mRNA expression of VANGL2, TGFβ2and PAX3was detected in right ventricular outflow tract myocardium using real-time PCR. The protein expression of VANGL2, TGFβ2and PAX3genes was detected using Immunohistochemistry.Results:1. The methylation levels of VANGL2, TGFβ2and PAX3at their5’-UTR CpG island in the TOF group were lower than the control.2. The mRNA expression of TGFβ2and PAX3in myocardium was significantly higher in the TOF group than that in the control, while the mRNA expression of VANGL2was significantly lower in the TOF group than that in the control.3. The protein expression of PAX3in myocardium was significantly higher in the TOF group than that in the control. The protein expression of TGFβ2was not changed significantly in the TOF group as compared with the control. However, it was expressed obviously in the mesenchymal tissue surrounding the myocardial cells. The protein expression of VANGL2was significantly lower in the TOF group than that in the control.Conclusions:1. The increased expression of TGFβ2, PAX3might be related to the development of TOF, which may be regulated by the lower methylation level of TGFβ2, PAX3genes.2. The decreased expression of VANGL2might also be related to the development of TOF, which may be related to lower methylation level of the gene, interacting with other pathways.