An EGLN1 Mutation May Regulate Hypoxic Response In Cyanotic Congenital Heart Disease Through The PHD2/HIF-1A Pathway

Background:Congenital heart diseases（CHD）is the most common birth defect.CHD are found in 7–10 of every 1000 live births.Taking into account the severe clinical symptoms and high medical expense of CHD,It has been one of the cardiovascular diseases relating to national well-being and people’s livelihood^[1-3].Cyanotic congenital heart disease（CCHD）,a term describing the most severe congenital heart diseases characterized by the anatomic malformation of a right to left shunt,includes tetralogy of Fallot（TOF）,transposition of the great arteries（TGA）,double outlet of right ventricle（DORV）,pulmonary artery（PA）defects,single ventricle and so on.Although only approximate 0.08–0.12%of live births are affected by CCHD,far less than the incidence of CHD,patients with CCHD always present severe clinical features such as hypoxia,dyspnea,and heart failure.Many patients present cyanosis before they could benefit from surgical treatment^[4].Based on the specific anatomic malformation,patients with CCHD usually develop chronic hypoxia,which induces hypoxemia that significantly contributes to poor prognosis in CCHD,with dysplasia,convulsions,and sudden death,even after an urgent surgical intervention^[5-7].Consequently,the ability of hypoxic response determines the prognosis of CCHD patients.Currentstudieshavedemonstratedthatthe PHD2（prolyl-4-hydroxylase2）/HIF-1A（hypoxia-inducible factor-1A）is a key regulator of hypoxic adaptation.Under normoxia,PHD2,encoded by EGLN1 gene,triggers the oxygen-dependent hydroxylation of ODD within HIF-1A.This hydroxylation produces a binding site for the von Hippel–Lindau（VHL）protein and then results in the proteasomal degradation of HIF-1A.During hypoxia,however,such hydroxylation is blocked.As a result,HIF-1A is released from VHL and form a heterodimer with the HIF-1B subunit,resulting in HIF-1A accumulation,angiogenesis,erythropoiesis,and glycolysis.It was reported by many studies that angiogenesis and erythropoiesis enhance hypoxic response through the PHD2/HIF-1A pathway,improvingcell survivalunderhypoxic conditions^[8].Many gene mutations and abnormal embryo may contribute to CCHD,such as NKX2-5,GATA4,TFAP2B and so on^[9].PHD2/HIF-1A,as the most important hypoxic pathway,plays a crucial role in hypoxic adaptation in CCHD.But the mechnism of hypoxic adaptation in CCHD carried EGLN1mutation is unknown.Objective:To assess the associations of single polymorphisms（SNPs）of the EGLN1 gene and hypoxic response in CCHD and research its gene mechanism.To better understand the CCHD at the genic level and provide better individual suggestions for improving the hypoxia in CCHD.To provide new targets and ideas for drug research in CCHD.Methods:EGLN1 was selected for next-generation sequencing in 126CCHD patients in Children’s Hospital of Chongqing Medical University.DNA was extracted from peripheral blood with MagPure Blood DNA Mini KF Kit（Magen,NO:MD5111-01）.The obtained DNA was submitted to high-throughput sequencing of the EGLN1 gene,including the 5’UTR and3’UTR,exons,and the 2 kb region upstream the promoter.Then,the gene variants were identified by Sanger sequencing,with the DNA-Star software used for data analysis.Then,those CCHD were divided into mutation group and wild-type group respectively.The incidence of hypoxia and collateral vessel formation between the two groups were analyzed.According to the results,we obtain the CDS of EGLN1 by PCR and insert it into the GV230 vector,generating different genotype recombined plasmids of EGLN1 such as mutation genotype,wild-type genotype.Otherwise,these recombined plasmids were verified by sanger sequencing.Then,The effects of different EGLN1 genotypes on its target genes,including HIF-1A,VEGF,EPO and HK1,were detected in HEK293 cells transfected with recombinant plasmids by Western Blot and real-time PCR.SPSS 20.0 was used for statistical analyses.Data are mean±standard error（SE）.Enumeration data were assessed the Chi-square test.Differences among groups were analyzed by one way analysis of variance（ANOVA）.P<0.05 was considered statistically significant.Results:A missense variant of EGLN1 c.380G>C（rs1209790）was found in 46 patients（46/126）,resulting in the replacement of cysteine by the serine.Those results of sanger sequencing were coincident with the next-generating sequencing.Furthermore,the hypoxia incidence of carriers is 36.96%（17/46）and that of wild-type is 65%（52/80）.The rate of collateral vessel formation of carriers is 50%（23/46）and that of wild-type is27.5%（22/80）.Those mutation carriers has lower hypoxia incidence and higher rate of collateral vessel formation,compared with the wild type（P<0.05）.During hypoxia,EGLN1 mutation reduced PHD2 expression compared with the wild type,with higher HIF-1A,VEGF and EPO expression levels in the mutant.No difference in HK1 expression was observed between the mutant and wild type.No differences among those target genes were observed during normoxia.Conclusion:These carriers had a lower rate of hypoxia and higher rate of collateral vessel formation compared with the wild-type group.During hypoxia,EGLN1 mutation reduced EGLN1 expression compared with the wild type,with higher HIF-1A,VEGF and EPO expression levels in the mutant,resulting in the angiogenesis,erythropoiesis which can improve the hypoxic adaptation.So,we consider that the EGLN1 c.380G>C mutation does good to the CCHD and improves hypoxic adaptation through the PHD2/HIF-1A pathway,which may provide a molecular mechanism for hypoxic adaptation in CCHD.The effects of the EGLN1 c.380G>C mutation on CCHD prognosis deserve further investigation.