Association Of TLR4and NLRP3Polymorphisms With Susceptibility To Primary Gouty Arthritis In A Chinese Han Population

Background and ObjectiveHyperuricemia is the biochemical basic of gout development. The studies have demonstrated that genetic polymorphisms of SLC2A9. URAT1are key regulators of urate homeostasis, the inheritance of one predisposing variant of SLC2A9or URAT1increases the risk for an individual to develop gout. Why only about10%certain hyperuricemie individuals are predisposed to gout?There may be other genes unrelated to the urate metabolism also contribute to the disease susceptibility. Interleukin1β (IL1β) is a key inflammatory factor for gouty arthritis, toll-like receptor (TLR)4and Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing (NLRP)3inflammasome signaling pathways are involved in the inflammatory mechanism of gout, but the concrete mechanism is still unclear. We will investigate the association of TLR4and NLRP3polymorphisms with susceptibility to primary gouty arthritis in a Chinese Han population. Peripheral blood mononuclear cells (PBMCs) TLR4, NLRP3, and IL1β mRNA and serum IL11β were measured in different genotype carriers, and correlations between TLR4gene SNP and TLR4mRNA, IL11β, between NLRP3SNP and NLRP3, IL11β mRNA, serum IL11β were investigated.Materials and Methods1) Populations:Five hundred and eighty-three consecutive patients with primary gouty arthritis (GA) attending the Affiliated Hospital of North Sichuan Medical College between January2007and February2013were included in the study. A total of459age and gender-matched men without gout who underwent regular physical examination at the Affiliated Hospital of North Sichuan Medical College between June2008and February2013were included as controls subjects in this study. Clinical data were carefully recorded, and a medical history was obtained from all of the subjects. All the participants are from the Chinese Han population. Blood samples were obtained from all of the participants.2) All the genotyping assays of TLR4and NLRP3genes polymorphisms loci (rs4986790Asp299Gly, rs4986791Thr399Ile, rs2149356T>G, rs4612666T>C, rs1539019A>C and rs10754558G>C) were measured using TaqMan probes that specifically target the alternate alleles.3) Peripheral blood mononuclear cells (PBMCs) TLR4, NLRP3, and IL1β mRNA levels were measured using RT-qPCR, and serum IL1β production was detected using ELISA, in different genotypes carriers.4) Statistical analysis:Statistical analysis was performed using SPSS (Statistical Package for the Social Sciences)16.00. The genotype distribution was analyzed for deviations from the Hardy-Weinberg equilibrium (HWE) using χ2analyses. Associations between genotypes and GA were estimated by computing the odds ratios (OR) and their95%confidence intervals (CI) from mullivariate logistic regression analysis with the adjustment for confound factors. The statistical power was calculated using PS software.Results1) There were no significant deviations from HWE both in GA and controls groups in the six SNPs (P>0.05, respectively).2) All the subjects were found to be homozygous for the wild-type TLR4alleles (Asp/Asp, Thr/Thr) on TLR4Asp299Gly and Thr399Ile genotyping. Significant differences were observed between the GA and HC groups with respect to genotype and allele frequencies of TLR4gene rs2149356(χ2=16.23,17.08; P=0.0003,3.58X10-5), frequencies of TT genotype and T allele were much higher in GA group than those in HC group (P<0.05, respectively), and TT genotype is the risk factor for gout (adjusted OR=2.09).3) The frequencies of CC, CT and TT genotypes of NLRP3gene rs4612666in GA and HC groups were0.299/0.504/0.197and0.303/0.536/0.161, respectively. The frequencies of C/T allele in GA and HC groups were0.551/0.449,0.571/0.429. No significant differences were observed between GA and HC groups with respect to genotype and allele frequencies (P>0.05, respectively).4) The frequencies of CC, CA and AA genotypes of NLRP3gene rs1539019in GA and HC groups were0.166/0.508/0.326,0.153/0.479/0.368, respectively. The frequencies of C/A allele in GA and HC groups were0.42/0.58,0.392/0.608. No significant differences were observed between GA and HC groups with respect to genotype and allele frequencies (P>0.05, respectively).5) The frequencies of CC, CG and GG genotypes of NLRP3gene rs10754558in GA and HC groups were0.295/0.506/0.199,0.379/0.466/0.155, respectively. The frequencies of C/G allele in GA and HC groups were0.548/0.452,0.612/0.388. Significant differences were observed between the GA and HC groups with respect to allele and genotype frequencies (x2=8.66,9.103; P=0.003,0.011). Multivariate logistic regression analysis revealed that significantly increased risk of GA was associated with the GG genotype (adjusted OR=2.66), compared with the GG genotype.6) Three of the NLRP3SNP loci (rs4612666, rs10754558and rs1539019) were in LD (D’>0.8, r2>0.3). The frequency of CCA haplotype was significantly decreased in GA cases compared with healthy controls (P<0.05), while the frequency of CGA haplotype was significantly increased (P<0.05). The risk for developing GA was significantly increased in individuals carrying CGA haplotype (P=0.02, OR=1.683,95%CI=1.081-2.619; Figure2B), while decreased in individuals with CCA haplotype (P=0.0007, OR=0.622,95%CI-0.472-0.820).7) PBMCs TLR4. IL1β mRNA and serum IL1β levels were much higher in GA group than those in HC group (P<0.05, respectively), while NLRP3mRNA in PBMCs was much lower in GA group than that in HC group (P<0.05).8) In acute GA patients, PBMCs TLR4mRNA expression and serum IL1β in TT homozygotes carriers of TLR4gene rs2149356were significantly higher than those in GG homozygotes carriers (P<0.05, respectively); while in non-acute GA patients, PBMCs TLR4mRNA expression in the TT genotype carriers was significantly decreased compared with the GG genotype carriers (P<0.05).9) In acute GA patients, PBMCs NLRP3, IL1βmRNA expression and serum IL1β level in the GG genotype carriers of NLRP3gene rs10754558were significantly increased compared with the CC genotype carriers (P<0.05, respectively); in non-acute GA patients, NLRP3mRNA expression in the GG genotype carriers was significantly reduced compared with the CC genotype carriers (P<0.05), the trend of IL1βmRNA expression and serum IL1β level was accordance with the acute GA patients.10) In GA patients, significant differences were observed among the GG, GT and TT three genotypes of TLR4gene rs2149356with respect to HDL, apoA1, LDL and GLOB (P<0.05; respectively); significant differences were also observed among the CC, CG and GG three genotypes of NLRP3gene rs10754558with respect to lymphocyte counts and apolipoprotein B100(P<0.05; respectively).Conclusions1) Serum uric acid, alcohol consumption, BMI, purine-rich foods intake, triglycerides, hypertension and smoking are the independent risk factors for developing gout in Chinese Han population.2) There is no evidence for involvement of the TLR4gene Asp299Gly and Thr399Ile, NLRP3gene rs4612666T>C and rs1539019A>C polymorphisms in susceptibility to primary gouty arthritis in the Chinese Han population. 3) The TLR4gene rs2149356SNP might be associated with GA susceptibility, and TT genotype might be the risk factor for developing gout.4) The NLRP3gene rs10754558SNP might be associated with GA susceptibility. GG genotype and CGA haplotype might be the risk factor for developing gout.5) The TLR4gene rs2149356and NLRP3gene rs10754558SNPs might be involved in regulating TLR4-IL1β and NLRP3inflammasome signaling pathways in GA patients.6) The TLR4gene rs2149356and NLRP3gene rs10754558SNPs might also be involved in regulating lipid metabolism in GA patients.