Relationship Between HLA-B27Polymorphism, Non-MHC Gene Polymorphism And Ankylosing Spondylitis

Background Ankylosing spondylitis （AS） is characterized by inflammation anddestruction of sacroiliac joint and stiffness of spine. The patient can be resulted in Jointdeformities and Disability, finally loss the ability of work. It affect the patient’s qualityof life seriously. It is a highly heritable arthropathy, and heritability>90%. AS isaffecting approximately0.3%of Han Chinese in20-30age. The number of male of ASare10times than female. The pathogenesis of which is poorly understood. So there isno effective treatment measures at present. In recent years, the genome-wide associationstudy （GWAS） in AS has a rapid progress. The research showed that not only MHCgene, but also the non-MHC gene, with the genes IL23R、ERAP1、IL1R2、ANTXR2,associated with AS, and leaded to the study of susceptibility in AS having a greatprogress. However, the current studies mainly focused on loci associated with AS indifferent population, and there was little study that researched the fuction of gene in thedevelopment of AS. Therefore it is necessary to find the genetic variation section withAS susceptibility, and study the fuction of gene associated with AS.Objective Our research is to study the association between HLA-B27and non-MHCincluding ANTXR2, TNFAIP3, ERAP1genetic polymorphism and AS in north ChineseHan, and Investigate their function in AS, and analysis the interaction betweenHLA-B27and ERAP1, and depth show the relationship between HLA-B27polymorphism，non-MHC gene polymorphism and AS, and their fuction in Geneticpathogenesis of AS. Methods1.602AS patients and598health controls from Beijing Han Chinese wereselected for case-control study to research the association between B27geneticpolymorphism and AS. All patients met the modified New York criteria for AS.Sequence-specific primers PCR （PCR-SSP） was manipulated to analyze HLA-B27polymorphism. Chi-square test was used to compared the B27subtype fequencebetween cases and controls, and the distribution between different regions. The Fisherexact test was used when the excepted count less than5.2.602AS patients and619health controls from Beijing Han Chinese were selected forcase-control study to study the association between non-MHC genetic polymorphismand AS. Selected19tagSNPs of ANTXR2,6tagSNPs of TNFAIP3and4tagSNPs ofERAP1, genotyping the samples was performed using the Sequenom iPlex and ligasedetection reaction platforms. Flow cytometry was used to test the effect of geneticvariants on expression of the capillary morphogenesis protein-2（CMP2）. TheHaploview program was appled to test the genotypic distributions of SNPs forHardy-Weinberg equilibrium, to estimate linkage disequilibrium, and to define thehaplotype blocks. Unphased program was used to analysis the allelic, genotypic andhaplotypic association. The Bonferroni correction was applied to correct a p value fromindividual haplotype association. Study power were estimated by Quantol software.Comparison of a difference in CMP-2protein expression between two groups wasanalyzed by paired T test.3.According to the HLA-B27subtypes disease group were grouped, and analysed theassociate of ERAP1, to explore the interaction between the HLA-B27and ERAP1geneassociated with AS.Results1.There were82.4%and7.5%B27-positive individuals in cases and controls,respectively. Associated analyses confirmed the strong positive correlation（OR=58,p=6.5x10-163） between B27and AS. We detected five B27geneticpolymorphisms, B*2705, B*2704, B*2702, B*2707and B*2724. among which B*2704, B*2705and B*2702were all in correlation with AS and the most relevantpolymorphism was B*2704（p=1.4x10-68）. However B*2707and B*2724has nocorrelation with AS. In addition, the distribution of B27genetic polymorphism betweenNorthern Han, Shanghai, Guangdong and Taiwan AS patients were in large differences（the most significant different B27polymorphism was B*2705, p=6.28x10-52）, and therewere markedly differences with the distribution of B27genetic polymorphisms andodds ratio for AS occurrence in the above four regional populations.2. We first found that3SNPs in ANTXR2（rs4690127, p=0.013; rs6823031,p=0.029; rs2867698, p=0.044） were significantly associated with AS, using genotypeanalysis. And2SNPs in ANTXR2（rs4690127, p=0.004; rs6823031, p=0.011） weresignificantly associated with AS, using allele analysis. We also confirmed the SNPrs4333130was associated with AS （p=0.013）. Results of the2-marker and3-markersliding window omnibus test showed many haplotypes were significantly associatedwith AS, the most significant of which was the haplotypers4690127-rs6823031-rs4333130(p=2.5×10^-4). Although rs12647691（Ala357Pro）wasno associated with AS, the haplotype rs4690127-rs12647691-rs4333130was stronglyassociated with AS (p=2.7×10^-4). The cis-acting interactions between rs6534639andrs4333130was associated with AS （p=0.027）.3. No associated was observed between TNFAIP3and AS.4. rs27434in ERAP1were significantly associated with AS, p=0.049ingenotype analysis, p=0.036in allele analysis. We first found the haplotypers27980-rs27582and rs27037-rs27980-rs27582were strongly associated with AS(p=3.8×10^-7and p=1.6×10^-6).5. rs27434was significantly associate with the HLA-B*2702subtype （p=0.01）,and rs27582was associate with the HLA-B*2704subtype （p=0.047）. Whereas we sawno association with ERAP1in HLA-B27-negative cases.6. Result of Flow cytometry analysis showed that expression of CMP2in peripheral blood mononuclear cells （PBMCs） was significantly lower inLPS-stimulation group than in the control group （p=0.018）, and that in subjects carryingthe rs6534639-CC,rs6534654-GG,rs4690127-CC, rs6823031-TT,rs12647691-GG andrs4333130-TT genotype, expression of CMP2was significantly lower in theLPS-stimulation group than in the control group.ConclusionThis study was first obtained some new discovery about the associated betweenHLA-B27subtype, ANTXR2, TNFAIP3, ERAP1genes and AS, and explored thefunction of ANTXR2gene polymorphism in AS on the protein expression level. Wealso study the interaction between the HLA-B27and ERAP1gene in AS. Generallyspeaking, the conclusion of this study can be briefly summarized as the followingpoints:1.This study confirms the high degree correlation between B27and AS, and B*2704isthe most AS relevant polymorphism for North Han Chinese.2. The study results demonstrate for the first time that genetic polymorphisms inANTXR2are associated with AS in Han Chinese.3. The study results also demonstrate TNFAIP3associated with RA and SLE is notassociated with AS in Han Chinese.4. Our results suggest that the ERAP1gene variants together with HLA-B*2702andHLA-B*2704strongly contribute to disease susceptibility in patients with AS in northHan Chinese.5. The effect of ANTXR2genetic variants on expression of the ANTXR2gene maycontribute to the etiology of AS.The above study of AS provide a reliable genetic data, and propose new researchdirections.