Relationship Between The Polymorphisms Of The Hypoxia-response Associated Genes And Congenital Heart Disease Susceptibility In Chinese Population At Different Altitudes

Objective: This study was conducted to explore the relationship between the polymorphisms of hypoxia-response associated genes SESN2,CCN1 and the susceptibility to congenital heart disease(CHD)in case-control populations at different altitudes.The genotype-phenotype molecular mechanism related to hypoxia was preliminarily explored and analyzed.Methods:(1)658 CHD patients and 681 healthy control Han populations were recruited at different altitudes to analyze the correlation between SESN2 gene polymorphism and CHD susceptibility.SESN2 polymorphism and SESN2 gene expression in myocardial tissue were used to evaluate e QTL effect.Chromatin immunoprecipitation sequencing data of cardiac tissue or cell in the ENCODE database and Roadmap database annotated epigenetic markers in the up-stream and down-stream regions of SESN2 gene.Culture of HEK293 T and H9c2 cells and extraction of nuclear proteins.The binding of DNA sequence harboring rs492554 and rs12406992 to nuclear protein was detected by electrophoretic mobility shift assay.Protein mass spectrometry and transcription factor binding motif analysis were used to identify candidate transcription factors.The luciferase reporter gene assay evaluated the effect of candidate transcription factors and different allelic sites of rs492554 and rs12406992 on the transcription level of down-stream genes.Gene set enrichment analysis was performed to explore the relationship between SESN2 and the hypoxia gene set in cardiac tissue expression profiles of the GEO database.Recombinant lentivirus and specific small interfering RNA were used to construct H9c2 cells with Sesn2 overexpression and knockdown,respectively.Hypoxia-mimetic cobalt chloride was used to construct cell hypoxia conditions,and the levels of cell viability,oxidative stress,cell proliferation and apoptosis of each experimental group were detected.Western blotting was used to detect the levels of Sesn2,Hif-1α,p-p38,p38,Ccnd1,Bax and Bcl2 in each group.(2)To explore the relationship between CCN1 polymorphism and CHD susceptibility in 395 patients with CHD and 486 healthy controls from different altitudes in Gansu.Haplotype analysis and haplotype network analysis were performed with SHEsis software and Pop ART software.Multiple linear regression analysis was used to evaluate the relationship between CCN1 gene polymorphism and erythrocyte parameter levels in different populations.Results:(1)The rs492554 T allele of SESN2 gene was associated with a reduced risk of CHD in high altitude areas(OR=0.73,95%CI=0.59-0.92,P=0.008);But there was no statistical correlation with the risk of CHD in low altitude areas(OR=0.79,95%CI=0.45-1.40,P=0.48).Rs492554 protective T allele was associated with higher SESN2 expression.Gel electrophoretic mobility shift assay combined with protein mass spectrometry showed that the DNA probe containing T allele of rs492554 site had high binding affinity to POU2F1.Through the annotation of epigenetic markers,it was found that rs492554 was located in the potential enhancer region of SESN2 gene.Subsequently,it was found that the rs12406992 site located in the potential promoter region had a strong linkage disequilibrium with rs492554,and the sequence around rs12406992 also conformed to the binding motif of POU2F1.Luciferase reporter gene analysis showed that the T-C haplotype of rs492554-rs12406992 could significantly increase the expression of downstream genes.Embryonic rat-heart-derived H9c2 cells treatment under cobalt chloride-imitated hypoxia showed that Sesn2 protects H9c2 cells from oxidative stress,cell cycle G1 arrest and apoptosis caused by cobalt chloride mimicking hypoxia by affecting the levels of p-p38,p38,Ccnd1,Bax and Bcl2.(2)The C allele at rs3753793 of CCN1 gene(OR=0.59,95%CI=0.42-0.81,P=0.001),the A allele at rs2297141(OR=0.66,95%CI=0.49-0.90,P=0.008)and the CA haplotype of rs3753793-rs2297141(OR=0.58,95%CI=0.42-0.82,P=0.002)were associated with reducing the risk of atrial septal defect(ASD).Further subgroup analysis by different altitudes and ethnics were performed,the results showed that the rs2297141 is related to the risk of ASD in the Han population in the plateau area of2500～4287 meters under the recessive inheritance mode(OR=0.32,95%CI=0.13-0.78,P=0.011).The proportion of C-A haplotype of rs3753793-rs2297141 in high-altitude population was higher than that in low-altitude population.The C allele of rs3753793 and the A allele of rs2297141 were also associated with the difference of red blood cell count and hemoglobin concentration in populations at different altitudes.Conclusion:(1)rs492554 associated with the risk of CHD at high altitude,the protective T allele of rs492554 show a preferential binding affinity to POU2F1;POU2F1 may mediate the interaction between the rs492554 enhancer region and the rs12406992 promoter region and increase the expression of downstream gene SESN2,thereby reducing the risk of CHD.The interaction between SESN2 gene rs492554 polymorphism and high-altitude hypoxic environment may be one of the reasons affecting the occurrence of CHD in high-altitude areas.(2)The C allele at rs3753793 and the A allele at rs2297141 of the CCN1 gene were associated with a reduced risk of ASD in the Chinese Gansu Han population.The frequency of the C allele of rs3753793 and the A allele of rs2297141 was higher in the high-altitude population.The CCN1 gene polymorphism may affect the hypoxia response and associate with the adaptability of high-altitude environments,which may also be the reason for its association with ASD risk at high altitude.