Gene Identification And Function Study Of Primary Gout

BackgroundGout is a common form of arthritis caused by the deposition of monosodium urate crystals in joints,and it affects 1%-2%of adults in developed countries.The prevalence and incidence of gout have risen rapidly in recent decades in China because of diet and lifestyle changes.Although the pathogenesis of gout remains unclear,genetic susceptibility is thought to be an important factor.Indeed,the biological processes underlying regulation of this disorder are assumed to involve complex interplay among genetic,environmental and lifestyle factors.Therefore,identification of a major gout susceptibility gene would be an important step toward successful molecular diagnostics and would potentially facilitate the development of targeted therapies for patients with gout.Despite rencent years progress,it is likely that many other unidentified genes contribute to the formation of monosodium urate(MSU)crystals and clinical presentation of acute gout arthritis and chronic tophaceous disease.Genome-wide linkage studies of large families with follow-up fine mapping are considered particularly effective for identifying variants with large effects.Our family-based linkage analysis indicated the maximum Logof Odds(LOD)score reached 1.50 at marker D4S1572(at recombination fraction 0 = 0.00),which is adjacent to the 4q25 region.Genome-wide linkage studies of large families with follow-up fine mapping are considered particularly effective for identifying variants with large effects.For instance,genome-wide linkage analysis was performed on 21 multiplex pedigrees with gout from Taiwan,and chromosome 4q25(logarithm of odds = 4.29)was found to be strongly associated with gout.Therefore,we speculate that a certain gene in 4q25 region may play a role in gout susceptibility.Several studies have demonstrated that the inflammatory/immune response and glucose/lipid metabolism play important roles in the pathogenesis of gout and that variants in the associated genes are related to this disease.Within the chromosome 4q25 region,8 genes of 95 genes have been shown to participate in regulating inflammatory/immune pathways or glucose/lipid metabolism.ObjectiveIn this study,we focused mainly on candidate genes in the 4q25 region and revealed the role of epidermal growth factor(EGF)in the pathogenesis of gout inflammation.Materials and MethodsPart ⅠStudy cohort.We recruited a total of 480 male gout patients and 480 male gout-free controls for the first stage of our analysis and an additional 1017 male gout patients and 1897 healthy(non-gout)male controls for the fine mapping stage.All patients and controls were unrelated individuals of self-reported Han Chinese ethnicity.The diagnosis of gout was based on the preliminary Gout Classification Criteria published by the American College of Rheumatology(ACR)in 1977 for use in either the clinical setting or in population-based epidemiologic studies.This study was approved by the Ethics Committee of the Affiliated Hospital,Qingdao University and conducted in accordance with the ethical guidelines of the 1975 Declaration of Helsinki.All subjects gave written informed consent.Genotyping.A 2-mL peripheral blood sample was collected from each study participant into an Ethylene Diamine Tetraacetic Acid(EDTA)tube.Genomic DNA was extracted using a whole-blood DNA isolation kit.SNPs(Single nucleotide polymorphisms)were selected from candidate genes in the 4q25 genomic region(the cytoBand table in the UCSC Table Browser defines 4q25 as the genomic sequence in chr4:107700000-114100000，hg19).Samples were genotyped in the first stage of analysis using Illumina GoldenGate assay and Illumina BeadStudio software according to the manufacturer’s instructions.The original raw genotype dataset contained genotyping information for 960 samples and 96 SNPs.Following automatic clustering,the SNPs were ranked according to their GenTrain scores(from 0 to 1).Those with GenTrain scores of 0.5 were manually checked and adjusted according to the Illumina guidelines.Samples and SNPs with call rates below 90%were excluded.In the second stage of analysis,93 tag SNPs in EGF and Elovl6 were selected for genotyping using a MassArray system.All procedures were performed according to the manufacturer’s instructions.Approximately 15 ng of genomic DNA was amplified by multiplex polymerase chain reaction,and then the products were used in locus-specific single-base extension reactions.The resulting products were desalted and transferred to a 384-element SpectroCHIP array.Allele detection was accomplished using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,and mass spectrograms were analyzed using MassArray TYPER software.For quality control,analysis was repeated for 10%of the samples,which were randomly selected.Statistical analysis.All SNPs were tested for Hardy-Weinberg equilibrium(HWE)in the control populations for both stages.Any SNPs that deviated from HWE(P<0.001)were excluded from subsequent analyses.The genotype and allele frequencies for the cases and controls were assessed using the x2 test based on 2 × 3 and 2×2 contingency tables.Additionally,the Cochran-Armitage trend test was used for categorical data analysis.Because the incidence of gout is related to both age and body mass index(BMI),we used age and BMI as covariates.The associations of candidate gene SNPs with gout were evaluated using the PLINK toolset.In inferential analysis,odds ratio(ORs),95%confidence interval(CIs),and the corresponding P values were used to detect associations between the different genotypes and the presence of disease.All significant associations were corrected for multiple testing using the max(T)permutation procedure for both stages(100,000 permutations).Haplotype reconstruction and haplotype-based association tests were performed using the Haploview program.Individual haplotypes and their estimated population frequencies were inferred using the default parameters,and P values adjusted for multiple testing using the permutation procedure(100,000 permutations)were also assessed.One-way analysis of variance was performed to assess the impacts of the genotypes on the various clinical indices at presentation(alternatively,the Kruskal-Wallis test was used when indicated).The threshold P value was set to 0.05,and a P<0.05 was considered significant.The LD plot was generated using the Haploview program,and the blocks were defined using a custom settings:CI minima for strong LD(lower,0.7;upper,0.95),upper CI maximum for strong recombination(0.9),and fraction of strong LD in informative comparisons(≥ 0.7).Markers with a frequency<0.05 were excluded.Power analysis.We estimated the statistical power of this study using the Genetic Power Calculator program(http://pngu.mgh.harvard.edu/～purcell/gpc/qtlassoc.html)and the following genetic model:a 22%risk allele frequency(similar to the minor allele frequency of rs2298999 in our study);and a 1.14%prevalence of gout in the Chinese population.Part ⅡSamples of peripheral blood were obtained from patients with acute gouty arthritis(n=30),patients with chronic gouty arthritis(n = 30)and healthy control subjects(n =41).The diagnosis of acute gouty arthritis was made according to the European League Against Rheumatism/American College of Rheumatology(EULAR/ACR).The diagnosis of chronic gout based on the clinical manifestations and symptoms of gout.5ml peripheral venous blood samples were collected and serum EGF levels were determined with ELISA kit.THP-1 cells were stimulated with different concentrations of MSU crystals for 4h,12h,24h,48h,the culture supernatants were collected for EGF detection by ELISA kit,and the cells were extracted for detection of EGF RNA level by Reverse Transcription-Polymerase Chain Reaction(RT-PCR).THP-1 cells were stimulated by 100ug/ml MSU crystals for 24h,IL-1β and pro-IL-1β were been detected.Adding different concentrations of EGF for 2 hours,levels of IL-1β in culture supernatants of THP-1 cells were measured by ELISA kit and levels of pro-IL-1β in cells were detected by Western blot analysis.Statistical analysisAll statistical analyses were performed using SPSS software,and graphics were generated using Prism software.Multiple group comparisons were analyzed by one-way analysis of variance.Differences between two groups were analyzed by Student’s t test.All data are shown as mean ± SEM.P<0.05 was considered statistically significant.ResultsPart ⅠCandidate SNP selection.We selected eight candidate genes,three of which encode proteins that play roles in inflammatory/immune pathways:epidermal growth factor(EGF);lymphoid enhancer-binding factor 1(LEF1);and oligosaccharyltransferase complex subunit(OSTC).The other five are involved in the regulation of glucose and lipid metabolism:cytochrome P450,family 2,subfamily U,polypeptide 1(CYP2U1);elongation of very-long-chain-fatty-acid-like family member 6(ELOVL6);phospholipase A2,group 12(PLA2G12A);sphingomyelin synthase 2(SGMS2);and hydroxyacyl-CoA dehydrogenase(HADH).Genotyping.In the first stage of analysis,96 tag SNPs in the eight candidate genes in the 4q25 region were genotyped in 480 male gout patients and 480 matched controls.SNPs with call rates below 90%and PHWE<0.001 were excluded from subsequent analysis.A total of 91 SNPs and 960 subjects passed this initial quality control stage.After correction for multiple testing,the SNP rs12504538 in Elovl6 was found to be significantly associated with gout(Padjusted = 0.00595).In the second stage,we attempted to select genes upstream and downstream of Elovl6 as the target region for fine mapping analysis.However,because the gene downstream of Elovl6 is a pseudogene,the upstream genes EGF and Elovl6 were chosen as candidate genes for further analysis.MassArray system was used to genotype 93 tag SNPs in EGF and Elovl6 in an additional independent population of 1017 male gout patients and 1897 matched controls.The 91 SNPs that met the criteria(call rate>90%,MAF>0.05 and PHWE 0.001)were included in subsequent analysis.Power analysis revealed a power of over 80%for detecting an association at a relative risk of 1.4-1.6(for heterozygotes and homozygotes)using an additive model.We identified a significant association between the T allele(minor)of EGF rs2298999 and gout in our Chinese Han population(odds ratio(OR)= 0.77,95%confidence interval(Cl)= 0.67-0.88,Padjusted = 6.42x10-3).Statistical analysis using the Cochran-Armitage trend test showed similar patterns.No significant differences in allele frequencies between the gout patients and controls were detected for any of the other SNPs investigated.Linkage disequilibrium(LD)analysis.LD analysis was used to investigate whether any EGF haplotype was correlated with gout.rs2298999 was located near seven other SNPs(rs11569057,rs2237054,rs11569090,rs10857004,rs10010695,rs6533485 and rs11569126)within a block.These eight SNPs form six common haplotypes(H1-H6),with frequencies of 0.324,0.157,0.133,0.125,0.109,and 0.044,respectively,among the gout patients,and 0.281,0.157,0.157,0.12,0.114,and 0.057,respectively,among the controls.The results of the haplotype association analysis were consistent with the single SNP analysis.The risk haplotype H1 carried the risk C allele of rs2298999,and was found to be the most frequent haplotype in our analysis(32.4%in gouts,28.1%in controls).While,the three other haplotypes(H2,H4 and H5),also carrying the C risk allele,were observed with similar frequencies between gout and control groups.Taken together,the H1 was the haplotype that showed the most significant association with gout(Padjusted = 0.027).Bioinformatics analysis.Furthermore,ENCODE data from the UCSC database indicated that SNP rs2298999 is located in a region with 11 other SNPs with histone H3 acetylation at lysine 27(H3K27ac),which is a marker of active enhancers.This suggests that rs2298999 is located within a functional region.No significant expression Quantitative Trait Loci(eQTLs)were found for SNP rs2298999 in all eQTL tissues in GTex database29,and we considered that further work is needed to determine the effect of rs2298999 on the expression of EGF gene.Correlation analysis of genotypes and gout clinical features.We also investigated the potential associations between specific genotypes and different clinical indices of gout.An increase in the mean(±SD)disease duration was found to be correlated with increased mutation severity;the duration was 4.72(±4.79)years in the T/T group compared with 5.12(±5.42)years and 6.38(±6.91)years in the C/T and C/C groups,respectively.This increased disease duration was found to be significant using the Kruskal-Wallis test(P= 0.023).Part ⅡSerum EGF levels in patients with acute gouty arthritis were significantly]ower than those in controls(20.99±1.80 vs.30.60±11.16，P=0.032).We used RT-PCR and Western blot to test the expression levels of EGF.The results showed that the expression levels of EGF were significantly increased in 100ug/ml MSU-induced THP-1 cells for 24h compared with that of CON(P<0.05).Moreover,we found that 10 ng/ml EGF could suppress MSU-induced IL-1β(P<0.05)and pro-IL-1β protein expression in THP-1 cells.ConclusionIn summary,to the best of our knowledge,this study is the first to provide evidence of an association between EGF and gout in a Chinese Han population.EGF serum levels might be associated with the risk of acute gouty arthritis.EGF plays an anti-inflammatory role by inhibit IL-1β and pro-IL-1β protein expression in MSU-induced gouty cell model.These findings increase the current understanding of the genetic architecture of gout and suggest that EGF could serve as potential therapeutic targets in the treatment of gouty arthritis.