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

Congenital heart disease is a birth defect due to abnormal heart and big vessel development during embryogenesis, which affects 6%o to 10‰ live births. About 150 thousand children with congenital heart disease are born in China every year, causing great burden to society and families. Cyanosis congenital heart disease (CCDH)is the most serious type among CHD. The mortality rate of CCDH is about 80% without early treatment. Conotruncal defects accounts for 60% to 70% of CCHD.TOF is the most common type of conotrncal defects which is characterized as pulmonary infundibular stenosis, overidding aorta, ventricular septum defect and right ventricular hypertrophy. Therefore, it is important to study the pathogenesis of TOF. It is generally accepted that both genetic and environmental factors play important roles in the pathogenesis of congenital heart disease. The study of candidate genes of CHD has become a hotspot.Conotruncal defect is the main cardiac manifestation of DiGeroge Syndrome(DGS).80% to 100% of DGS has microdeletion of chromosome 22q11.2.HIRA is located in this region. HIRA gene Knockout chicken and mice develop similar heart defects. So we suspect that HIRA is the candidate gene for TOF. But the study of the relationship between HIRA and human TOF is limited. Our research group has proved previously that expression of HIIRA gene is declined compared to normal controls. The reason is to be identified.The aim of the study was to clarify the relationship between the pathogenesis of TOF and HIRA by investigating its coding and 3’UTR region, the alteration of which is related to the structure of HIRA protein and micro RNAs.Part ⅠSequence analysis of HIRA gene in TOF patientsObjective:To detect the sequence variation of the coding region in TOF patients and analyze its role in the pathogenesis of TOF.Methods:A cohort of 278 TOF patients were recruited in the study,500 normal children were used as controls. Genomic DNA was extracted from peripheral blood. All coding regions were sequenced. The case number was increased to 500 is a mutation is detected. The mutation was analyzed with biological network or software. Zebrafish was used as animal model to testify the function of the mutation.Results:1. A reported SNP (rs150603624) was found in exon 12. The frequency was not statistically from the database of NCBI dbSNP.2.A mutation (c.C2301CA) was found in exon 17 with a frequency of 1/500. Structural analysis shows that the effect of the mutation on protein of HIRA is benign.3.A mutation (c.C3026CT) was found in exon 23,with a frequency of 2/500. Structural analysis showed that the effect of the mutation on protein HIRA is damaging. The mutation site is highly conserved in different species. Heart malformation rate was higher after the injection of mutated HIRA mRNA than WT.Conclusions:The mutation in exon 23(c.C3026CT) may play an important role in the pathogenesis of congenital heart disease.part ⅡSequence analysis of the HIRA gene 3’UTR region and the study of the related micro RNAObjective:To detect the variation of 3’UTR of HIRA gene in TOF patients in China and the effect on the related micro RNAs.Methods:We screened for mutations and SNPs in 278 unrelated probands with isolated TOF and 500 controls. We used Target Scan to predict micro RNAs that are possibly combined with 3’UTR region of HIRA gene. Dual-luciferase assay and real-time PCR were performed to detect the inhibition activity of micro RNAs on target genes.Results:1.There was statistically significant change in the allele frequencies of the existing SNPs (rs:117447448) between TOF patients and control group (P=0.001).2.The combining site of miR328 was predicted to be 8 bp up the SNP site.3.Dual-luciferase assay showed decreased standard luciferase level after co-transfected with miR328 (0.01963 ±0.0038 vs 0.0125±0.0006,P=0.0327). So is the expression of HIRA(1.60865±0.2749 vs 1.03961±0.0772, P=0.037).4.the luciferase level was not affected by the SNP (rs:l 17447448) (P=0.38)Conclusions:The SNP (rs:117447448) of 3’UTR region of HIRA gene is related to tetralogy of Fallot. HIRA is the target gene of miR328.Although the study cannot prove that the SNP (rs:117447448) affect micro RNA328 combining with the target gene HIRA, it provides an important clue to in-depth study of the 3’UTR region of HIRA gene in the pathogenesis of TOF.