Association Study Of Alternative Splicing Associated SNPs With Congenital Heart Disease And Clopidogrel Resistance

Background:Congenital heart disease（CHD）is a congenital defect disease with high incidence and mortality.Many studies have found that genetic polymorphisms of genes related to heart development are related to the occurrence of susceptibility to CHD.In recent years,studies have found that alternative splicing（AS）of genes is an important factor affecting heart development.The present work aims to study the relationship between the single nucleotide polymorphism（SNP）involved in AS of TBX5,BMP2,PABPC1 and NKX2-5 genes and the susceptibility of sporadic CHD in the Chinese population.Methods:In this study,blood genomes of 314 patients with sporadic CHD and391 healthy controls were collected.The 18 SNPs of TBX5,BMP2,PABPC1 and NKX2-5 genes were screened out by using databases（1000-genomes,db SNP,Hap Map）and AS analysis software（Human Splicing Finder and Alamut Visual）for correlation the study.Massarray typing technology was used to genotype 705 samples.Analysis of genotype,haplotype,linkage disequilibrium and CHD susceptibility by using SPSS23.0and SHEsis software.Results:The rs12425471 of the TBX5 gene is significantly related to the susceptibility of atrial septal defect（ASD）,ventricular septal defect（VSD）and tetralogy of Fallot（TOF）.The analysis of different inheritance patterns shows that the rs12425471 G>A of the TBX5 gene is in the dominant model（AA+GA/GG,P=0.036,OR=0.401,95%CI=0.223-0.722）,under the heterozygous model（GA/GG,P=0.004,OR=0.267,95%CI=0.130-0.548）and under the additive model（AA+GG/GA,P=0.004,OR=3.843,95%CI=1.873-7.888）significantly affect the risk of ASD.The rs12425471G>A of the TBX5 gene is in the allele model（A/G,P=0.023,OR=0.372,95%CI=0.217-0.640）,and the dominant model（AA+GA/GG,P=0.024,OR=0.371,95%CI=0.210-0.656）significantly affect the risk of VSD.The rs12425471 G>A of the TBX5 gene is under the allele model(A/G,P=5.4×10^-5,OR=3.032,95%CI=1.886-4.875),under the dominant model(AA+GA/GG,P=1.80×10^-7,OR=6.56,95%CI=3.263-13.192),under the heterozygous model(GA/GG,P=3.09×10^-8,OR=7.268,95%CI=3.605-14.653),the additive model(AA+GG/GA,P=2.28×10^-8,OR=0.132,95%CI=0.066-0.267)significantly affects the risk of TOF.The rs1786321 G>C of PABPC1 gene is under the allele model（C/G,P=0.022,OR=0.536,95%CI=0.373-0.770）,and the recessive model（CC/CG+GG,P=0.003,OR=0.092,95%CI=0.022-0.383）and homozygous model（CC/GG,P=0.002,OR=0.086,95%CI=0.020-0.369）significantly affect the risk of ASD.In addition,rs10850327,rs11067074,rs16944153,rs2555030,rs3825215,and rs7304774 loci of the TBX5 gene,rs1049007,rs235767,rs235768,and rs3819804 loci of the BMP2 gene,rs1039153,rs2226712,rs3133548,rs3133-5 loci of the PABPC1gene and rs3729753 and rs703752 of NKX2-5 gene show no significant difference between the control group and CHD group or CHD subgroup.According to haplotype analysis,CTATGGT(P=4.11×10^-15,OR=0.158,95%CI=0.095-0.264),CTGTGCC（P=0.022,OR=1.742,95%CI=1.077-2.820）,CTGTGGT(P=4.33×10^-15,OR=8.058,95%CI=4.605-14.10)and TCGTGCC（P=0.024,OR=0.764,95%CI=0.605-0.966）four haplotypes on the TBX5 gene are related to the susceptibility of CHD.The TGTTC（P=0.002,OR=3.146,95%CI=1.443-6.859）haplotype on the PABPC1 gene is related to the susceptibility of CHD.Conclusion:The rs12425471 of TBX5 gene and rs1786321 of PABPC1 gene are associated with the incidence of CHD in the Chinese population,especially rs12425471affects the risk of ASD,VSD and TOF,and rs1786321 affects the ASD risk.There is no significant correlation between rs10850327,rs11067074,rs16944153,rs2555030,rs3825215,rs7304774,rs1049007,rs235767,rs235768,rs3819804,rs1039153,rs2226712,rs3133548,rs3133580,rs3729753,rs703752 and susceptibility to CHD and different subtypes of CHD.The rs12425471 and rs1786321 might affect the occurrence of CHD by changing the AS process of genes.The results of this study provide new and important clues to the genetic mechanism of variable gene splicing affecting the pathogenesis of CHD.Background: Coronary heart disease（CAD）is a common cardiovascular disease.Thrombolytic therapy is very important.Clopidogrel is one of the commonly used antithrombotic drugs,but some patients will cause clopidogrel resistance（CR）to occur after taking it.More and more studies have confirmed the relationship between ABCB1 gene polymorphism and clopidogrel resistance,but the molecular mechanism is still unclear.This article aims to study the relationship between the ABCB1 gene polymorphism in patients with coronary heart disease and clopidogrel resistance in the Chinese population,and to further analyze whether the ABCB1 gene single nucleotide polymorphism（SNP）affects the alternative splicing of the gene.Methods: A total of 741 blood samples were collected in our experiment（including 316 CR samples and 425 NCR controls）.Finally,five SNPs of ABCB1 gene were selected for research by consulting literature and databases.741 samples were genotyped by Massarray typing technology.Analysis of genotype,haplotype,linkage disequilibrium and CR susceptibility by using SPSS23.0 and SHEsis softwares.Results: The rs1045642 of the ABCB1 gene,in the allele model,the frequencies of the G allele and A allele in the control group are 61.6% and 38.4%,and the frequencies of the G allele and A allele in the CR group are 53.5% and 46.5%,compared with the G allele,the A allele has differences between groups（P=0.020,OR=1.398,95% CI=1.135-1.723）;in the homozygous model,the frequency of GG and AA genotype in the control group are 72.2% and 27.8%,and the genotype frequency of GG and AA genotype in the clopidogrel resistant group are 56.6% and 43.4%,the distribution of AA genotype and GG genotype is different between the two groups（P=0.020,OR=1.989,95%CI=1.301-3.040）.Patients with CAD who carry the A allele at the rs1045642 of the ABCB1 gene are more likely to develop CR.The alleles and genotypes of rs4148727,rs2032582,rs3789243 and rs1858923 of ABCB1 gene show no significant difference between the CR group and NCR group.In haplotype analysis,it is found that TGAGG haplotype can significantly reduce the risk of CR（P=0.040,OR=0.663,95%CI=0.447-0.983）.Using bioinformatics to predict that the rs1045642 polymorphic site of ABCB1 gene produces a new exon splicing enhancer sequence and its ability to bind to the SR protein has changed.Conclusion: The rs1045642 locus of the ABCB1 gene is significantly related to the risk of CR in the Chinese population.Patients with CAD who carry the A allele on the rs1045642 locus of the ABCB1 gene are more likely to develop CR.There is no significant correlation between rs4148727,rs2032582,rs3789243 and rs1858923 of ABCB1 gene and the risk of CR in Chinese population.The TGAGG haplotype can significantly reduce the risk of CR.The rs1045642 polymorphism might affect the occurrence of CR by changing the AS process of ABCB1 gene.