| Objective To investigate the association between the single nucleotide polymorphisms of rs10754558 locus in NLRP3 gene and rs2043211 locus in CARD8 gene and the susceptibility to lower extremity arteriosclerosis obliterans(ASO)in Qingdao,Shangdong han male population by bioinformatics method and molecular genetics technology.Methods A total of 758 Han male patients with ASO in Qingdao and 793 age matched healthy Han male controls were included in this case-control study.The rs10754558 locus in the NLRP3 gene and rs2043211 locus in the CARD8 gene were genotyped by using TaqMan SNP Genotyping Assays.The concentration of IL-1β in serum of all subjects was determined by ELISA kit.The concentrations of IL-1β among different genotypes were compared by ANOVA.Quanto 1.2.4 software was used to evaluate the effectiveness of statistics.The experimental data were statistically analyzed with SPSS 20.0 statistical software package.The Hardy-Weinberg balance was used to test the genetic balance of each group to evaluate whether the subject in each group was representative in the population.The clinical data and blood biochemical index in ASO group and control group were compared by two independent samples t test;Since the genotype distribution and allele frequency were recorded in enumeration data,χ2 test was used for statistical analysis of enumeration data.Analysis of variance was used to analyze the disease susceptible genotype-phenotype correlation of ASO.The relative risk degree was expressed with the odds ratio(OR value)and its 95%confidence interval(95%CI),and the gene-gene.interaction was analyzed by Logistic Regression.Take P<0.05 as a statistically significant difference.Results(1)The genotype frequencies of NLRP3 rs10754558 locus and CARD8 rs2043211 locus in the ASO group and control group were all in line with the genetic balance(P>0.05),with the group representativeness,according to the Hardy-Weinberg balance pinciple.(2)The differences of the genotype frequencies of CC,CG,GG in NLRP3 rs10754558 locus in ASO group(32.32%,50.00%and 17.68%)and control group(38.84%,47.67%and 13.49%)were significant(P<0.05).The differences of the allele frequencies of C,G in ASO group(57.32%,42.68%)and control group(62.67%,37.33%)were also significant(P<0.05).Those carrying genotype GG had a higher risk for ASO than those carrying genotype CC(OR=1.574,95%CI 1.161-2.135,P=0.003).Those carrying allele G had a higher risk for ASO than those carrying allele C(OR=1.250,95%CI 1.083-1.444,P=0.002).After the adjustment of smoking index,total cholesterol,low density lipoprotein,fasting blood glucose,systolic blood pressure and body mass index,we found that the ASO risk of those carrying genotype GG was still high,which was 1.448 times for individuals carrying genotype CC(OR=1.448,95%CI 1.046-1.461,P = 0.004).Individuals carrying allele G were still at higher risk for ASO,which was 1.193 times for carrying allele C(OR = 1.193,95%CI 1.032-1.358,P=0.003).(3)The differences of the genotype frequencies of AA,AT,TT in CARD8 rs2043211 locus in ASO group(25.20%,48.94%and 25.86%)and control group(31.40%,47.04%and 21.56%)were significant(P<0.05).The differences of the allele frequencies of A,T in ASO group(49.67%,50.33%)and control group(54.92%,45.08%)were also significant(P<0.05).Those carrying genotype TT had a higher risk for ASO than those carrying genotype AA(OR=1.494,95%CI 1.131-1.974,P=0.005).Those carrying allele T had a higher risk for ASO than those carrying allele A(OR=1.234,95%CI 1.072-1.421,P=0.003).After the adjustment of smoking index,total cholesterol,low density lipoprotein,fasting blood glucose,systolic blood pressure and body mass index,we found that the ASO risk of those carrying genotype TT was more higher,which was 1.525 times for individuals carrying genotype AA(OR=1.525,95%CI 1.158-2.009,P=0.003).Individuals carrying allele T were at even higher risk for ASO,which was 1.252 times for carrying allele A(OR=1.252,95%CI 1.088-1.443,P=0.002).(4)Using variance analysis to analyze the genotype-phenotype correlation of the NLRP3 rs1075558 locus with the susceptibility to ASO.The results are shown in table 2.4.The triglyceride(P=0.019),cholesterol(P=0.031)and IL-1β(P=0.011)were significantly different among genotypies CC,CG and GG in the NLRP3 rs10754558 locus.There was no significant difference in other biochemical indicators and the rates of past complications(hyperlipidemia,diabetes,hypertension and ischemic heart disease)and smoking index among different genotypes.(5)Using variance analysis to analyze the genotype-phenotype correlation of the CARD8 rs2043211 locus with the susceptibility to ASO.The results are shown in table 2.5.The low density lipoprotein(P=0.037)and IL-1β(P=0.014)were significantly different among genotypies AA,AT and TT in the CARD8 rs2043211 locus.However,there was no significant difference in other biochemical indicators and the rates of the past complications(hyperlipidemia,diabetes,hypertension and ischemic heart disease)and smoking index among different genotypes.(6)In order to make it clear what is the pathological mechanism about NLRP3 rs10754558 locus and CARD8 rs2043211 locus polymorphisms affecting ASO pathogenesis,the concentration of serum IL-1β was determined by ELISA method for all the research objects in the case group and control group.The results showed that the concentration of IL-1β in ASO group was significantly higher than that in the control group(8.21 ± 1.23 ng/L vs.4.97 ±1.07 ng/L,P=0.009,FIG.2.7).In NLRP3 rs10754558 locus,in the control group,IL-1β concentration of genotype GG(n=107)was significantly higher than that of genotype CC(n=308)(5.21±1.03 ng/L vs.3.57 ±1.11 ng/L,P=0.004,FIG.2.8);in ASO group,IL-1β concentration of genotype GG(n=134)was significantly higher than that of genotype CC(n=245)(8.41 ±1.01 ng/L vs.6.97±1.06 ng/L,P=0.003,FIG.2.9).In CARD8 rs2043211 locun,in the control group,the IL-1β concentration of genotype TT(n=171)was higher than that of genotype AA(n=249)(5.08 ± 1.43 ng/L vs.4.42 ±1.35 ng/L,P=0.004,FIG.2.10);in ASO group,the IL-1β concentration of genotype TT(n=196)was significantly higher than that of the genotype AA(n=191)(8.75±1.06 ng/L vs.7.94±0.93 ng/L,P=0.019,FIG.2.11).In ASO group,the IL-1β concentration of GG/TT genotype combination(n=38)was ignficantly higher than that of CC/AA genotype combination(n=65)(9.24±1.24 ng/L vs.6.65±1.19 ng/L,P=0.009,FIG.2.12).(7)Logistic Regression analysis was done to analyze whether there were biological interactions between the genotypes of the two polymorphism loci in the genetic susceptibility to ASO.After correcting the body mass index,systolic blood pressure,fasting blood glucose,low density lipoprotein,total cholesterol and smoking index,the results showed that there were interactions between NLRP3 rs10754558 gene and CARD8 rs2043211 gene(Pinteraction=0.038).But when the concentration of IL-1β was taken in as a correction factor,we got χ2 = 3.812,Pinteraction=0.114,which suggested that there were interactions between the genotypes of the two polymorphism loci in the genetic susceptibility to ASO,the interactions were not affected by body mass index,systolic blood pressure,fasting blood glucose,low density lipoprotein,total cholesterol and smoking index,but were consistent with the concentration of IL-1β.Conclusion The polymorphisms of NLRP3 rs10754558 gene and CARD8 rs2043211 gene were closely related to lower extremity arteriosclerosis obliterans in the Han male population in Qingdao area,Shandong.In this study population,the genotype GG of NLRP3 rs10754558 locus and the genotype TT of CARD8 rs2043211 locus were related to the genetic susceptibility to ASO.These two gene loci could interact with each other,which could lead to the development of ASO by affecting the production and activation of IL-Iβ.This study provided new insights into the pathogenesis of ASO and provided new potential molecular targets for the treatment and prevention of ASO. |
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