| Part ?Association of polymorphisms in TNF and GRN genes with ankylosing spondylitis in a Chinese Han populationBackgroundSpondyloarthritis(SpA)is a group of chronic inflammatory diseases with common clinical and genetic characteristics.The most common manifestation of SpA is ankylosing spondylitis(AS).One distinct feature of AS is familial clustering.The gene which has the strongest association with AS is human leukocyte antigen(HLA)-B27.In recent years,it has been found that non-HLA-B27 genes play an important role in the pathogenesis of AS.Therefore,it is of great significance to study the role of non HLA-B27 genes in the pathogenesis of AS.Single Nucleotide Polymorphisms(SNPs)refer to DNA sequence polymorphisms caused by mutations in single nucleotides at the genome level.SNPs,comprising the most abundant type of genetic variation and easy to be genetyped,are now the most important study object in the study of genetic variation and complex traits.The tumor necrosis factor(TNF)gene,also known as TNFA,TNF-alpha,is located at 6p21.33,and is a non-HLA gene.The gene encodes a kind of pro-inflammatory cytokine belonging to the tumor necrosis factor superfamily.This cytokine is mainly secreted by macrophages.It can bind to its receptors TNFRSF1A/TNFR 1 and TNFRSF1B/TNFBR to function.Recent studies have shown that TNF plays an important role in the pathogenesis of AS.The strongest evidence that TNF is a key cytokine for spinal arthritis is that the use of TNF inhibitors in clinical practice can effectively improve the symptoms and signs of patients with spinal arthritis including ankylosing spondylitis.At the tissue level,TNF inhibitors can cause a range of histological improvements,including reducing inflammatory infiltration of neutrophils and macrophages and reducing new bone formation.There have been many studies on the relationship between SNPs and TNF gene in AS,but it is controversial in different studies.Therefore,it is necessary to further study the relationship between the SNPs of TNF gene and AS.The granulin precursor gene is located at 17q21.31 and can encode granule protein precursor.Cleavage of this signal peptide produces mature granule which can be further cleaved into various active peptides.These peptides and intact granulin proteins regulate cell growth.However,different members of the granulin family can act as inhibitors,agonists or have dual actions on cell growth.Granulin has been considered to be involved in many autoimmune diseases in recent years,.It has been indicated that the precursor protein of granule protein(PGRN)is a key regulator in inflammation,and the effect may be mediated by blocking the binding of TNF to its receptors.It has been found in other studies that the patients with psoriatic arthritis(PSA)with positive progranulin antibodies(PGRN Ab)are associated with a higher frequency of developing tendinitis and dactylitis.Both AS and PsA belong to seronegative spondylitis(SpA).Therefore,it is significant to research whether the polymorphisms of TNF and GRN are associated with AS.Objective:To investigate the association of the polymorphisms of TNF and GRN in ankylosing spondylitis(AS)in a Chinese population in Shandong,China.Methods:1.Study SubjectsA total of 861 AS patients were enrolled as the case group,all of whom were from the Han nationality in Shandong,China.The patients were diagnosed in our hospital from January 2014 to December 2015.The diagnosis of all patients complied with the New York Standard revised in 1984,and the clinical symptoms,signs and family history of AS patients were recorded in detail.HLA-B27 was detected by flow cytometry,and all patients underwent sacroiliac joint X-ray examinations.According to the international unified sacroiliac joint grading standard,the sacroiliac joint imaging examination of the patients were determined by the imaging experts.Other autoimmune diseases were excluded in all patients.The control group of 864 cases belonging to the Chinese Han healthy population in Shandong were from the Medical Center of Shandong Provincial Hospital Affiliated to Shandong University.There were no autoimmune diseases,infectious diseases,tumors or other diseases in the group,whose age and gender were matched with the AS group.The informed consent from all the participants were received before they entered into study.2.Experimental Methods2.1 Specimen Collection 2ml of fasting peripheral venous blood was collected from every AS patient and the control group,placed in EDTA anticoagulation tubes,and stored in-80? refrigerator for testing.2.2 Genomic DNA Extraction The DNA of the case and control group were extracted by using genomic DNA extraction kits following the instructions.2.3 The Measurement of Concentration and Purity of DNA 2mL of the DNA sample was transferred out and was diluted to 1/25 concentration by added into 48ul of triple distilled water.Then the concentration and purity of DNA were measured by the UV spectrophotometer.2.4 The Selection of SNP Loci According to the dbSNP database,the data of SNPs of TNF and GRN genes were obtained.The distribution frequency of each SNP loci was determined by HaploView,and the TagSNP was screened by the Tagger function.The initial screening needs to meet the minor allele frequency>5%and the linkage disequilibrium coefficient r2>0.8.Based on the screening results,11 tagSNPs were finally determined.2.5 Genotyping Taqman probe real-time PCR method was used to perform genotyping according to the steps.2.6 Analysis of Test Results Data of FAM and VIC fluorescence signals amplified by using TaqMan-PCR collection and processing were performed by using SDS software(Applied Biosystem,version 2.0).3.Statistical Analysis3.1 Hardy-Weinberg Equilibrium Test Hardy-Weinberg equilibrium test was performed by using SHEsis software.3.2 Statistical Analysis of Genotype and Allele Frequencies of the Case Group and Control Group Alleles,genotypes and haplotypes were analyzed by using the chi-square test with SPSS24.0 software.The SHEsis program was used to calculate the haplotype frequency,odds ratio,and 95%confidence interval.3.3 Association Analysis between Patient Clinical/Laboratory Indicators and SNPs SPSS 24.0 statistical software was used for statistical analysis of the collected data.3.4 Analysis of Linkage Disequilibrium and Haplotype HaploView 2.0 and SHEsis statistical software were used to statistically analyze the linkage disequilibrium and haplotype between all loci.4.Biological information function annotation of SNPs GTEx,Haploreg and Regulome DB databases were used to annotate the function of rs1799964,rs1800629,rs1800630 and rs769178.Results1.Basic InformationA total of 861 AS patients and 864 healthy controls were genotyped.Among AS patients,there were 634 male patients and 227 female patients,aged 15-71,with an average age of 30.56±10.98.The control group included 619 males and 245 females,aged 14-73,with an average age of 31.97 ±11.23.All the subjects were from the Han ethnic group in Shandong.There was no significant difference in age and gender between the case group and the control group(p>0.05).2.Hardy-Weinberg Equilibrium TestThe observed and expected values of alleles and genotypes of all SNP loci in this experiment are in good agreement with all p-values>0.05,which are in line with Hardy-Weinberg equilibrium law.3.Distribution of TNF Genotype Frequency and Allele Frequency in AS and Normal Population3.1 Sites with Statistical Differences For rs1799964,the difference of genotype distribution between the AS group and the control group was statistically significant(p<0.0001),and the difference of the allele distribution between the two groups was also statistically significant(p<0.0001,OR=0.60,95%Cl 0.50-0.71),which suggesting that the C allele is a protective gene for AS.For the rs1800629,the difference of genotype distribution between the AS group and the control group was statistically significant(p=0.0004),and the difference of the allele distribution between the two groups was also statistically significant(p=0.0001;OR=0.60,95%CI 0.39-0.74),which suggesting that the A allele is a protective gene for AS.For rs 1800630,the difference of genotype distribution between the AS group and the control group was statistically significant(p<0.0001),and the difference of the allele distribution between the two groups was also statistically significant(p<0.0001,95%CI 0.48-0.72),which suggesting that the A allele is a protective gene for AS.For rs769178,the difference of genotype distribution between the AS group and the control group was statistically significant(p<0.0001),and the difference of the allele distribution between the two groups was also statistically significant(p<0.0001,95%CI 2.18-3.09),which suggesting that the T allele is a risk factor for AS.3.2 Sites without Statistical Differences For rs361525,there was no statistically significant difference in genotype and allele frequency between the AS group and the control group.4.GRN Genotype Frequencies and Allele Frequencies Distribution in AS and Normal PopulationForrs850713,rs3785817,rs3760365,rs4792939,rs25646,and rs5848,there were no statistically significant differences in genotype and allele frequencies between the AS group and the control group.So the polymorphisms in GRN are not related to AS susceptibility in our Chinese Han population.5.Analysis of the Genotype Genetic Pattern of Loci with Significant Differences in TNF Allele Frequency DistributionFor rs 1799964,the carriers of the C/C and C/T homozygote showed a significantly lower risk of AS under the dominant model,and the carriers of the C/C homozygote showed a significantly lower risk of AS under the recessive model.For rsl 800629,the carrers of the A/A and A/G homozygote showed a significantly lower risk of AS under the dominant model,and the carriers of the A/A homozygote showed a significantly lower risk of AS under the recessive model.For rs1800630,the carriers of the A/A and A/C homoztgote showed a significantly lower risk of AS under the dominant model,and the carriers of the A/A homozygote showed a significantly lower risk of AS under the recessive model.For rs769178,the carriers of the T/T and G/T homozygote showed a significantly higher risk of AS under the dominant model,and the carriers of the T/T homozygote showed a significantly higher risk of AS under the recessive model.6.Association Analysis of TNF Gene Polymorphisms with Clinical/Laboratory Indicators in Patients with ASRs1799964,rs1800629,rs1800630,and rs769178 were not significantly associated with gender,age,course,family history,HLA-B27 positive,peripheral joint involvement,and uveitis in AS patients.7.Haplotype AnalysisStatistical results show that there is a weak linkage disequilibrium between the research loci of TNF and GRN genes.The CGAGG,CGCAG,TACGG,TGCGG,TGCGT haplotypes of the five research loci of TNF gene and the CTGACA haplotypes of the six research loci of GRN gene were statistically different between the AS group and thecontrol group.8.Bioinformatics Analysis of TNFThe eQTL analysis of rsl 800629?rsl 800630?rs769178 and rs1799964 in the GTEx database showed that the minor alleles of the aforementioned SNPs could significantly change the expression of several genes,suggesting that rs1800629,rs100630,rs769178 and rs1799964 might play a biological role in the development of ankylosing spondylitis by regulating the expression of other genes.Conclusion1.It was confirmed for the first time in our study that the polymorphisms of rs769178 was associated with AS susceptibility,and the allele frequency and genotype of rs18006299 rs1800630,rs769178,rs1799964 in AS group were significantly different from those of control group.The polymorphisms in TNF were related to AS in the Chinese Han population in Shandong,and eQTL analysis shows that it may affect AS susceptibility by affecting the expression of certain genes.But it was not found that the clinical manifestations of AS were related to the SNPs of TNF.2.The polymorphisms in GRN were not related to AS susceptibility in our Chinese Han population in Shandong.Considering that the distribution of SNPs is significantly different in different regions and ethnic groups,it is necessary to perform further studies of larger samples and different population to confirm whether there is an association between the GRN gene and AS.Part ?Associations of Polymorphisms in ERAP1 with Ankylosing Spondylitis:A Meta-analysisBackgroundAnkylosing spondylitis(AS)is a chronic,systemic,inflammatory disease characterized by involvement of the central axis and peripheral joints,ligaments,and tendon attachment points.AS is a highly heritable disorder and the human leukocyte antigen(HLA)-B27 gene is the gene most strongly associated with AS.However,recent studies have found that non-major histocompatibility complex(MHC)genes also play an important role in the pathogenesis of AS.It is important to study the role of non-MHC genes in the pathogenesis of AS to further clarify the etiology and pathogenesis of AS.It has been found that endoplasmic reticulum aminopeptidase(ERAP)1 belonging to non-MHC genes plays an important role in the risk of AS in recent studies.It has been found in many case-control studies that the polymorphisms of EARP1 are associated with AS,but the conclusions are not consistent,The differences among ethnicity may partly explain the inconsistent results.Meta-analysis is an effective standard analysis method that summarizes the results of many studies.There have been many related meta-analysis related to ERAP1 gene in the past,but the database and research population selected by the previous studies are not consistent,and there have been many new articles on the relationship between ERAP1 gene polymorphism and AS in the past two years.Therefore,in this study,we performed a meta-analysis study combining genome wide association studies(GWAS)and case-control studies to explore whether ERP1 is associated with AS susceptibility to the overall population and subgroups.ObjectiveThe meta-analysis was used to determine the relationship between the single nucleotide polymorphisms of ERAP1 gene and the susceptibility of AS in the general population and different ethnic groups.Methods1.General DataWe used "ankylosing spondylitis","ERAP1" and "polymorphism" as the key words.The Pubmed,Embase,and Cochrane databases were searched,and related free words were also searched.English was the selected language.2.Screening MethodsDocuments were screened according to the criteria for inclusion and exclusion that had been developed in advance.The information extracted from each study included:first author,year of publication,country and ethnicity of the study population,number of case groups and control groups,and number of alleles for the ERAP1 polymorphism.The quality of the included studies was evaluated by using the Newcastle-Ottawa scale(NOS)Literature Quality Evaluation Scale.3.Statistical AnalysisReview Manager 5.3 was used for statistical analysis.The OR value and 95%confidence interval were used as the effect index,and the overall population and the subgroup analysis by ethnicity were analyzed.I2 was used for quantitative analysis of heterogeneity.For data with obvious heterogeneity,sensitivity analysis was performed.Funnel plot was used to analyze publication bias.Results1.Literature Search ResultsA total of 105 studies including 92 from Pubmed databases,21 from Embase databases,and 2 from Cochrane databases were obstained by the preliminary literature search.After carefully reading the title and abstract of the literature,we finally selected 31 articles according to the inclusion and exclusion criteria,and a total of 26,291 AS patients and 45,779 healthy controls were included.2.Meta Analysis ResultsIncluding rs27044,rs17482078,rs10050860,rs30187,rs2287987,rs27980,rs27037,rs27434,rs26653,rs1065407,rs27529,rs27582,rs469876,rs7711564,rs27038,rs3734016,rs11209032,rs26618,rs27895,rs17481856,rs13167972,a total of 21 loci were analyze and followed to perform subgroup analysis.The results showed that the A allele of rs27434 was a risk factor for AS in the overall population,the Caucasian population,the Middle East population,and the East Asian population.There was no significant association at the loci of rs27980,rs27582,rs469876,rs27038,rs11209032,rs26618,rs27895,rs17481856 between AS and control groups in the overall population and subgroups.There were different associations in the other 12 loci with AS in the overall population and subgroups.In the overall population,the SNPs of rs27434,rs17482078,rs10050860,rs30187,rs2287987,rs27037,rs26653,rs7711564,rs3734016 and rs13167972 are associated with susceptibility of AS.In the Caucasian population,the SNPs of rs27044,rs17482078,rs10050860,rs30187,rs2287987,rs27037,rs27434,rs26653,rs1065407,rs13167972 are associated with susceptibility of AS.In the Middle Eastern populations,the SNPs of rs27044,rs27434,and rs26653 are associated with AS.In the East Asian populations,the SNPs of rs30187,rs27037,rs27434,rs27529,and rs7711564 are associated with AS,ConclusionsOur meta-analysis indicated that the SNPs of the ERAP1 gene are related to AS susceptibility in the overall population,the Caucasian population,the Middle Eastern population,and the East Asian population.The effect of each locus on AS susceptibility varies among different races.Functional annotation of rs27434 suggests that it may affect AS susceptibility by influencing the expression of ERAP1 and indirectly affecting the role of TNF.Further race-specific association studies are needed to be performed to determine the genetic relationship between SNPs of ERAP1 and AS susceptibility in different populations. |
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