Association Of +138I/D And Lys198Asn Polymorphisms In Endothelin-1 Gene With Early Onset Of Coronary Artery Disease Among Jiangxi Han-ethnic Population And The Possible Mechanisms

Part ⅠAssociation of +138I/D and Lys198 Asn Polymorphisms in Endothelin-1 Gene with Early Onset of Coronary Artery Disease among Jiangxi Han-Ethnic PopulationBackground: Human Endothelin-1(ET-1)gene polymorphism is closely associated with coronary artery disease(CAD).This study aimed to investigate the association of two SNPs(+138 I/D and Lys198Asn)of ET-1 gene with early-onset of CAD in Jiangxi Han-ethnic population,in order to provide some new suggestions for the local people to be given an early diagnosis of CAD as well as preventing measurements to keep away from it.Methods: Genotyping of the two SNPs(+138 I/D and Lys198Asn)was performed in88 early-onset CAD patients(≤55 years for males;≤60 years for females)and 52 volunteer control subjects using polymerase chain reaction-direct sequencing method.Blood samples were obtained after a 12-h overnight fast to gain assessments of blood sugar,lipid as well as liver and kidney function.Some clinical data such as blood pressure,height,body of weight,were collected in the same time.In addition,some surveys of life habits were also evaluated.The data were performed with SPSS(SPSS17.0)software.It has statistical significance for P<0.05.Results:(1)The distributions of sex and lifestyle factors,as well as various quantitative variables(age,BMI,TC,LDL-C,FBS,SBP,DBP)were evaluated in all subjects.Most variables were significantly higher in cases than in controls(P<0.05),except for the distributions of both sex and age.(2)Polymorphisms of both +138 I/D and Lys198 Asn had been tested in Hardy-Weinberg equilibrium(p>0.05).(3)The genotype frequencies of Lys198 Asn in patients were76.1%(G/G),2.3%(T/T),21.6%(G/T),respectively,and T allele frequency was 12.1 %.On the contrary,in controls,the G/G,G/T,and T/T genotype frequencies were 92.3%,7.7%,and 0% respectively with the T allele frequency of 3.9%.(4)Fisher’s exact test showed that T allele(OR =3.38,P = 0.02)and GT/TT genotype(OR = 3.76,P = 0.02)of ET-1 gene Lys198 Asn were associated with increasing risk of early-onset CAD,while the other SNP had no significant effect.(5)No associations in patients were observed between most of high risk factors of CAD and the polymorphisms of the two loci.But the proportion of carrying T allele genotype during the smoking patients was significantly higher than that of carrying G/G homozygote.Multivariate logistic regression analysis showed that smoking was the single independent variable related to early-onset CAD(P<0.05).Conclusions: Asn allele frequency in Lys198 Asn loci was significantly correlated with the incidence of EOCAD in Jiangxi Han-ethnic population.Smoking can greatly increase the risk of EOCAD during the local inhabitants carring with Asn allele.There was no correlation of the allele frequency in the +138 I/D locus with the incidence of EOCAD during Jiangxi Han-ethnic population.Part ⅡPossible Mechanisms of the Asn Allele at Lys198 Asn Locus Significantly Increasing the Risk of Early-Onset Coronary Artery Disease among Jiangxi Han-Ethnic PopulationBackground: Asn allele frequency in Lys198 Asn loci might be associated with the pathogenesis of early-onset CAD in Jiangxi Han-ethnic population.To further investigate the possible mechanisms,we had overexpressed the different alleles,by means of constructing expression vector in vitro,to demonstrate the effect of this polymorphism on ET-1 protein expression.Methods: According to the experimental results of the first part of this study,the DNA fragments containing the Lys or Asn alleles were extracted and then cloned between Mlu I and BgⅢ sites in the firefly luciferase reporter vector p GL3.0 Basic with a tagged-flag using standard molecular biology techniques.Recombinant plasmids were transfected into HEK293 T cells by Lipofectamine for 24 hours,and empty plasmid was transfected simultaneously to serve as control group.Cells were incubated for an additional 24 hours together with each of TNF-α,IL-6,or LPS,the concentration of which was either 10 ng/m L or 20 ng/m L.Western blot was used to measure the protein expression of ET-1 in cells.Data were processed by SPSS 17.0and it is considered to be significant for P<0.05.Results: Higher levels of heterologous ET-1 protein expression were detected in the cells transfected with either Lys198 or Asn198 plasmids as compared to the cells without ET-1 plasmid transfected vector.No difference of the heterologous protein expressive levels was observed between Lys198 plasmid and Asn198 plasmid transfected cells in the unstimulated state.The levels of the protein expression was higher in cells transfected with Asn198 plasmid compared to that of Lys198 plasmid(with a P value less than 0.05),upon stimulation by inflammatory factors such as TNF-α or IL-6,of different concentrations(either 10 ng/m L or 20 ng/m L).But those differences between the two groups were not observed upon stimulation by LPS of either concentration.Conclusion: The Asn allele at the rs5370 locus in the ET-1 gene could significantly upregulate the protein expression of ET-1 compared to the Lys allele,upon stimulation by both TNF-α and IL-6.The polymorphism at the rs5370 locus in ET-1gene could not affect the protein expression of ET-1 upon stimulation by LPS.The alleles at rs5370 locus could show unlike impacts on protein expression of ET-1under stimulated by some specific inflammatory factors,which might lead to an apparent difference to be vulnerable to EOCAD during Jiangxi Han-ethnic population.