The Correlation Between HIF-1Α Gene Polymorphism And Hypoxic Tolerance In Cyanotic Congenital Heart Disease

BackgroundCongenital heart disease(CHD)is the most common pediatric cardiovascular disease.CHD has been the most common birth defects in our country since 2009,and the leading cause of infant death.At present,the incidence of CHD in live births is from 8‰ to 12‰ worldwide.Cyanotic congenital heart disease(CCHD)is the most serious type of CHD,which represents 10–20% of all CHD cases.All CCHD is represented by cyanosis because that abnormal cardiac structure leads hypoxia venous blood directly into systemic circulation.Hypoxemia is the common symptom of CCHD and the main cause of death.Hypoxemia in CCHD is more severe than that in acyanotic congenital heart disease(ACHD),and chronic hypoxemia affects the growth and development of children seriously.Hypoxia tolerance is the basis of CCHD survival.We can often observe that individuals with the same structure and hypoxic condition have different clinical manifestations and prognosis,which indicate that different individuals have different hypoxia tolerance abilities.Hypoxia tolerance abilities of CCHD are affected by the individual genetic background to some extent.Hypoxia inducible factor-1 alpha(HIF-1α)is a DNA binding protein induced by hypoxia which promotes cell survival under hypoxia condition.It suggests that HIF-1α gene may be associated with CCHD hypoxia tolerance.Some studies have confirmed that chronic hypoxia can increase the expression of HIF-1α protein in cardiac tissue of CCHD,which may be involved in CCHD hypoxia tolerance.A large number of studies have confirmed that the progression and prognosis of many diseases are closely related to the specific gene polymorphisms,but the correlation between HIF-1α gene polymorphism and CCHD hypoxia tolerance is unclear.ObjectiveTo explore the relationship between HIF-1α gene polymorphism and CCHD hypoxia tolerance,to reveal the role of CCHD genetic background in hypoxia tolerance.To provide theoretical basis for clinical prognosis and individualized treatment.MethodsHIF-1α was selected sequenced in 97 CCHD cases and 108 healthy controls,the next sequencing covered all exons,3’UTR,5’UTR and ‐2kb regions of 5’ upstream in HIF-1α gene.HIF-1α gene polymorphisms were screened by bioinformatics analysis.Distribution of each polymorphic allele and genotype in CCHD and ACHD group was analyzed by chi square test.The DNA fragment including related gene polymorphism was cloned and sequenced by Sanger.According to the above results,HIF-1α promoter region containing-1496 loci was cloned and inserted into p GL3-Basic luciferase reporter vector.Recombinant plasmid identified by enzyme digestion and sequencing.Finally two kinds of luciferase expression vector were constructed(p GL3-Basic-HIF-1α-AA and p GL3-Basic-HIF-1α-DD).Recombinant plasmids were transfected into HEK293 T and H9C2 cells by liposome.Luciferase assay was performed with the dual luciferase assay reagent after lysis the transfected cells with the passive lysis buffer.The final activity ratios reflect the activity of mutant human HIF-1α promoter.ResultsAfter the Next Generation Sequencing,33 high frequency polymorphisms were screened(MAF > 10%).Only the allele frequency of polymorphism site-1496 A > D(D represents a single base deletion mutation),rs138141447,in HIF-1α gene promoter region was observed to be significantly different between CCHD and ACHD cases(P < 0.05).Sanger sequencing results of rs138141447 were 93.2% consistent with the next generation sequencing.Sanger sequencing result is the gold standard.For HIF-1α gene promoter-1496 loci,allele frequencies of A and D in CCHD were 77.8% and 22.2%,in ACHD were 89.4% and 10.6%(χ2 = 10.04,P﹤0.01).AA,AD,DD genotype frequencies in CCHD were 58.76%,38.14% and 3.1%,in ACHD group were 78.7%,21.3% and 0%(χ2 = 12.42,P﹤0.01).Different genotype HIF-1α gene promoters encompassing-1496 were successfully subcloned into p GL3-Basic vector.In HEK293 T and H9C2 cells,transcriptional activity of DD genotype promoter was significantly higher than that of AA genotype by comparing the relative luciferase activity of recombinant plasmid.Different genotype recombinant plasmid under the same oxygen concentration had significant difference relative luciferase activity(P<0.05).Same genotype recombinant plasmid under different oxygen concentration had similar relative luciferase activity(P > 0.05).There was no interaction effect between different genotypes and different oxygen concentrations(P > 0.05).Oxygen concentration had no effect on relative luciferase activity of different genotype recombinant plasmid.ConclusionsThe sequencing results showed that HIF-1α promoter rs138141447,the allele frequencies and genotype frequencies are statistically significant difference between CCHD and ACHD group.D allele frequency in CCHD group is significantly higher than that in ACHD group.This polymorphism may be associated with CCHD hypoxia tolerance.Luciferase assay showed that transcriptional activity of DD genotype increased significantly,which confirmed that rs138141447 is a functional polymorphism and D allele is a protective allele.It also showed that the individual carrying rs138141447 DD genotype have better hypoxia tolerance ability.