The Expression Of HIRA Gene And Its Regulation Mechanism In Patients With Tetralogy Of Fallot

Congenital heart disease (CHD) is one of the most commonly seen congenital malformations in children, which can severely influences mortality, morbidity and life quality of children, and is one of the main reasons for early death of the newborns and infants. Conotruncal defect (CTD) is a kind of complex congenital heart disease, which can cause hypoxemia and irreversible acidosis during neonatal period, thus, leading to early death. The pathogenesis of CTD has already been more highlighted. Reports showed that the major cardiac abnormality was conotruncal defects in patients with22q11microdeletion syndrome, while some patients with conotruncal defects had22q11microdeletion. Deletion of genes such as HIRA, located on the segment of22q11microdeletion, may be relevant to conotruncal defects in animal models. However, although conotruncal defect is the cardiac phenotype in22q11microdeletion syndrome, it is still controversial for the association of non-syndromic conotruncal defects and22q11microdeletion. On the other hand, the role of the genes in22q11in human being is still unclear. Our studies were to investigate the DNA promoter sequence changes and examine the expression and methylation level of HIRA gene in patients with TOF. The results will help to illustrate the pathogenesis of conotruncal defects.PartⅠSequence changes at promoter of HIRA gene in patients with Tetralogy of FallotObjective:To examine DNA sequence changes of the promoter of HIRA gene in patients with Tetralogy of Fallot.Methods:A cohort of100pediatric patients with TOF were recruited in the study,200normal children were used as control. PCR and genotyping were performed for the detection of promoter DNA sequence changes of HIRA gene.Results:We examined the promoter DNA sequence (1000bp upstream of transcriptional start site) changes of HIRA gene, four heterozygous mutations including-574G>GA, -552G>GC,-305A>AG and-137C>CT were identified in the promoter region of four TOF childrens respectively, which were not present in200controls. Among them,-137site can be combined with transcription factor CP2.Another five SNP including-890A>AG (rs1128399),-736C>CA (rs4585115),-632T>TG (rs2277837),-630C>CA and-458T>TC (rs111802956) were found in the promoter. Among them,-630C>CA was a novel SNP, which was not found in the UCSC database. There were no significant differences in allelic frequencies and genotypes frequencies of these five single nucleotide polymorphisms between TOF group and the Control. However, the-458site can be combined with three transcription factors including GATA-1, GATA-2and GATA-3.Conclusions:1. Four heterozygous mutations (including-574G>GA>-552G>GC.-305A>AG and-137C>CT) and five SNPs (including-890A>AG.-736C>CA.-632T>TG、-630C>CA and-458T>TC) were found in the promoter of HIRA gene of TOF patients..2. The heterozygous mutation on-137site (C>CT) and the SNP on-458site (T>TC) in the promoter of HIRA gene may affect the transcription factors:CP2(on-137site) and GATA-1、GATA-2、GATA-3(on-458site), thus leading to the change of mRNA expression of this gene.Part IIExpression of HIRA gene in right ventricular outflow tract myocardium in TOF patientsObjective:To investigate the expression of HIRA gene in the myocardium of patients with TOF.Methods:The mRNA expression in right ventricular outflow tract myocardium of HIRA gene was detected using real-time PCR in39patients with TOF. The protein expression of HIRA gene was detected using Immunohistochemistry in12patients with TOF.Results:1. The mRNA expression of HIRA gene in myocardium was significantly lower in the TOF group than that in the control. 2. The protein expression of HIRA gene in myocardium was significantly lower in the TOF group than that in the control as well.Conclusions:1. The abnormal low expression of HIRA gene in myocardium of the TOF group may participate in the pathogenesis of TOF.2. The low expression of HIRA gene in myocardium of the TOF group prompted the presence of abnormal regulation at the transcriptional level.Part ⅢMethylation at promoter of HIRA gene in right ventricular outflow tract myocardium in TOF patientsObjective:To examine the methylation status at promoter region of HIRA gene in the myocardium of patients with TOF, and to illustrate its association with low expression of HIRA gene.Methods:The promoter methylation status of HIRA gene was detected by using bisulfite-PCR (BSP) in12patients with TOF.Results:The overall methylation level of HIRA gene at its promoter region is low in both TOF group and the control (percentage of methylation status<5%). There is no significant difference of the overall and each CpG site methylation status between the TOF group and the control. And the methylation status of each sample has no relationship with its mRNA、protein expression respectively.Conclusions:The low expression of HIRA gene in the TOF patients may not be associated with its promoter methylation status.