| Objectives To investigate the association of 9 polymorphisms of MTOR,AKT1 and ULK1 gene and the susceptibility to anti-neutrophil cytoplasmic autoantibody(ANCA)-associated vasculitis(AAV)in Guangxi population.Methods A total of 214 patients with AAV at the inpatient and outpatient department at the second affiliated hospital of Guangxi Medical University(the former west hospital of the first affiliated hospital of Guangxi Medical University)from January 2005 to June 2020 were enrolled in this case-control study,as well as 211 healthy controls from the Medical Center of this hospital during the same period.Multiplex polymerase chain reaction(PCR)combined with high-throughput sequencing technology was performed to genotype the candidate SNPs of the DNA samples of all subjects.The frequencies of the alleles and genotypes were obtained by direct calculation.SPSS 23.0 statistical software was used to conduct the Hardy-Weinberg Equilibriu(HWE)test of the control group,and the chi-square test or the exact probability method was performed to compare the distributions of allele and genotype frequencies between the case group and the control group.Binary unconditional logistic regression analysis was applicated to calculate odds ratio(OR)and 95%confidence interval(confidence interval,CI)to evaluate the impact of the genotypes and alleles of each SNP on the AAV risk.Haploview 4.2 and SNPstats software were used for genetic model,linkage disequilibrium and haplotype analyses.Multifactor dimensionality reduction(MDR)was used to evaluate the impact of SNP-SNP interaction on the risk of AAV.Regulome DB and Haplo Reg 4.1 online software were used to annotate each SNP,and the Genotype-Tissue Expression(GTEx)database was used to analyze the Expression Quantitative Trait Loci(e QTL).P-value less than 0.05(two-side)was thought to be statistically significant.Results 1.There was no statistically significant difference in the distribution of gender between the AAV group and control group(P=0.829),while the average age and age stratification(<60/≥60)between the two groups were statistically different(P<0.01).2.All SNPs in this study were according with HWE.The distribution of alleles and genotypes of AKT1 rs2498786 and rs5811155 and the genotypes distribution of ULK1 rs4964879 were significantly different between the AAV group and control group(P<0.05).The G allele of AKT1 rs2498786 and the ins allele of rs5811155 were significantly associated with a decreased risk of AAV(G: OR=0.533,95%CI=0.388-0.789,P=0.001;ins:OR=0.609,95%CI=0.448-0.827,P=0.001).The CG genotype of AKT1rs2498786 showed a lower risk of AAV(OR=0.515,95%CI=0.339-0.782,P=0.002).Compared with those carrying the del/del genotype of AKT1rs5811155,the del/ins genotype or ins/ins genotype carriers had a lower risk of AAV(del/ins: OR=0.549,95%CI=0.365-0.825,P=0.004;ins/ins: OR=0.460,95%CI=0.219-0.963,P=0.039).Compared with the AA genotype of ULK1rs4964879,the GA genotype was liked to an increased risk of AAV(OR=1.846,95%CI=1.213-2.811,P=0.004).After adjusting for age and gender,the polymorphisms still had an effect on the risk of AAV in Guangxi population except for the ins/ins genotype of AKT1 rs5811155.However,no significant association was detected between the remaining SNPs and the risk of AAV in Guangxi population(P>0.05).3.In the AAV subgroup analysis,the CG genotype of AKT1 rs2498786 was associated with a reduced risk of AAV with MPO-ANCA positive(OR=0.488,95%CI=0.313-0.761,P=0.002).This association was still remained after adjusting for age and gender(P<0.05).The G allele of AKT1 rs2498786 reduced the risk of AAV with MPO-ANCA positive(OR=0.520,95%CI=0.355-0.762,P=0.001).In AKT1 rs5811155,the del/ins genotype or ins/ins genotype was associated with a decreased susceptibility of AAV with MPO-ANCA positive(P<0.05).After adjusting for age and gender,both two genotypes were still associated with the risk of MPO-AAV(P<0.05).The ins allele was associated with the lower susceptibility of AAV with MPO-ANCA positive(OR=0.572,95%CI=0.412-0.794,P=0.001).In ULK1rs4964879,the GA genotype increased the risk of AAV with MPO-ANCA positive by 1.651 times(OR=1.651,95%CI= 1.062-2.567,P=0.021).After adjusting for age and gender,the GA genotype was still associated with an increased risk of AAV with MPO-ANCA positive(P<0.05).However,the allele of ULK1 rs4964879 was not associated with the risk of AAV with MPO-ANCA positive(P>0.05).In addition,there was no significant associations of the alleles and genotypes of the remaining SNPs and the susceptibility of AAV with MPO-ANCA positive(P>0.05).4.In the stratified analysis of ethnicity,gender,and age,the polymorphisms of MTOR rs2536 and rs1034528 were associated with increasing the risk of AAV in the Guangxi Zhuang population,and rs1057079 could reduce the susceptibility of AAV in the Guangxi female population(P<0.05).AKT1 rs2494737 was associated with the risk of AAV in Guangxi Zhuang population and female,and the polymorphism of rs2498786 was associated with the risk of AAV in Guangxi Han population,Zhuang population,female,young and middle-aged people(<60 years old),and elderly people(≥60 years old)(P<0.05).The polymorphism of rs5811155 was related to the incidence of AAV in Guangxi Han population,Zhuang population and female(P<0.05).The polymorphism of ULK1 rs78172903 was associated with an increase risk of AAV in female,and the polymorphism of rs4964879 could increase the susceptibility of AAV in Guangxi Han population,female,and young and middle-aged people(P<0.05).There was no statistical correlation between rs12303764 and the risk of AAV in the stratified analysis(P>0.05).5.In the genetic model analysis,under the co-dominant model,those carring CG genotype of AKT1 rs2498786 had a significantly lower risk of AAV(OR=0.51,95%CI=0.34-0.78,P=0.0027).The CG and GG genotypes carriers had a lower risk of AAV under dominant model(OR=0.50,95%CI=0.33-0.75,P<0.001).In the co-dominant model and the dominant model,compared with the del/del genotype,the del/ins genotype and del/ins+ins/ins genotype AKT1 rs5811155 were correlated with a reduced risk of AAV(P<0.05).In ULK1 rs4964879,the GA genotype was associated with an increased risk of AAV under codominance model(OR=1.85,95%CI=1.21-2.81,P=0.014).Under dominant model,the GA and GG genotypes increased the risk of AAV(OR=1.68,95%CI=1.13-2.49,P=0.01).After adjusting for age and gender,AKT1 rs2498786,rs5811155,and ULK1 rs4964879 were related to the risk of AAV in the co-dominant model and the dominant model.However,it was found that the remaining SNPs were not associated with the risk of AAV in the analysis of each genetic model(P>0.05).6.In the linkage disequilibrium analysis,there was strong linkage disequilibrium(LD)among rs2536,rs1057079 and rs1034528 in MTOR gene.Besides,strong LD was also found between rs2498786 and rs5811155 in AKT1 gene,as well as rs12303764 and rs4964879 in ULK1 gene.In the haplotype analysis,the AKT1rs2498786G-rs5811155 ins haplotype reduced the risk of AAV by 55%(OR=0.55,95%CI=0.37-0.81,P=0.0024).The remaining haplotypes were not associated with the susceptibility of AAV(P>0.05).7.In the MDR analysis,there was no interaction among these SNPs in this study on the risk of AAV in Guangxi population.The function annotation results showed that all SNPs had potential molecular biological functions.Conclusions 1.ULK1 rs4964879 was a risk factor to increase the susceptibility of AAV and AAV with MPO-ANCA positive in Guangxi population,while AKT1 rs2498786 and rs5811155 were the protective factors to reduce the risk of AAV and AAV with MPO-ANCA positive in Guangxi population.2.MTOR rs2536,rs1034528 and rs1057079,AKT1 rs2494737rs2498786 and rs5811155,ULK1 rs78172903 and rs4964879 were associated with the susceptibility to AAV in Guangxi population in different ethnic groups(Han/Zhuang),different gender and different age stratification(<60/≥60).3.Gene polymorphisms related to m TOR signaling pathway might be associated to the risk of AAV in Guangxi population. |
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