The Study Of Susceptible Gene Relevant To Ossification In Ankylosing Spondylitis

Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease thatprimarily affects the axial skeleton and characterized by chronic inflammation of thesacroiliac joints and the vertebral column, which ultimately leads to new boneformation and ankylosis. Strong genetic factors have been implicated in the etiologyof AS and HLA-B27confers the greatest susceptibility. By combination of thegenome-search meta-analysis(GSMA) method and the previous AS genome scans,studies confirmed that HLA loci is the greatest susceptible region to AS, non-HLAregions also contain AS susceptibility genes. By meta-analysis of genomewide scansfor AS, we found that6p22.3-p21.1is the greatest susceptible region to AS,6pter-p22.3、3p22.1-p14.1and16q23.1-qter are hot regions,16q12.2-q23.1、6p21.1-q15、10q23.33-q26.13、17pter-p12、2p12-q22.1、2q34-q35、5q34-qter、2p23.2-p16.2、11pter-p15.1、9q31.1-q33.3also associated with AS. These regionsor regions near by may provide targets for further fine mapping and candidate geneloci.At present, most of AS susceptibility genes are inflammation-related, only feware associated with calcification and/or ectopic ossification. New bone formation is amain process in AS and AS patients show notable ectopic ossification in the spine.Several signaling pathways have been described in the mechanisms controlling newbone formation, and ectopic ossification is mainly regulated by Wnt and bonemorphogenetic protein (BMP) pathway. Therefore, members of WNT and BMPpathway are potential ossification-related candidates for AS susceptibility. Ourprevious study also identified a novel single nucleotide polymorphism (SNP) site(c.4488+74G> A) of low-density lipoprotein receptor-related protein5(LRP5) genewas associated with AS in Chinese Han population. On the early basis, weinvestigated the association of ossification-related candidate genes polymorphismswith AS in Chinese Han population. The research content mainly divided into two parts: The first part,investigate the association of three genes MSX2，WNT5A andRHOA polymorphisms with AS in Chinese Han population by TaqMan Assay method.The second part, we assessed the association of9previously identified SNPs in theTNAP and ANKH genes in Chinese AS patients by Multiplex Snapshot.OBJECTIVE：In this study, we investigated the association of AS with MSX2,WNT5A, RHOA, TNAP and ANKH genes to determine whether tag SNPs coveringthe three candidate genes influence the susceptibility of AS in a Chinese Hanpopulation.METHODS：This study adopts the method of "case-control", In the TaqManAssay method study,336patients with AS and336unrelated healthy individuals ascontrols were selected. Multiplex Snapshot method involving278AS patients and286unrelated healthy controls. Hardy–Weinberg equilibrium (HWE) and the frequenciesof genotype, haplotypes were analyzed for all SNPs by SHEsis-online. Chi-squaretests were applied in case-control analyses and the strength of association wereestimated by the odds radio (OR) and95%confidence intervals (95%CI). P<0.05wasconsidered statistically significant difference. Multiple testing corrections wereconducted by SHEsis, and the number of permutations was10,000. Multivariatelogistic regression was carried out through SPSS16.0and Haploview4.0was used forLinkage analysis.RESULTS:1) Multiple Snapshot method resultsThere were significant differences in genotype (permutated p=0.00481) andallele (permutated p=0.0126) frequencies of the rs26307ANKH SNP between ASpatients and controls, and rs27356showed a marginal statistic significance level(genotype permutated p=0.055, allele permutated p=0.0596). The T allele ofrs26307significantly lower the risk of developing AS (OR=0.634,95%CI=0.475-0.847). Haplotype analysis identified the ANKH haplotype rs26307(C)/rs27356(T) as apredisposing factor for AS (OR=1.53,95%CI=1.165-2.071, p=0.0026,permutation10000corrected p=0.0103); the minor haplotype combination ofrs26307(T) and rs27356(C) defined significantly a protective common haplotypes(OR=0.659,95%CI=0.487-0.890, p=0.0064, permutation10000corrected p=0.0264).2) TaqMan Assay method resultsOnly WNT5A rs472631did we find significant differences in allele and genotypefrequencies between two groups (genotype permutated p=0.029, allele permutated p=0.016). The C allele was speculated to restrain susceptibility to AS (OR=0.75) andC> T mutation increased the risk of AS. Haplotype analysis identified the ANKHhaplotype rs472631(C)/rs11918967(C) as a protective factor for AS(OR=0.722,95%CI=0.567~0.918, permutation10000corrected p=0.008); haplotype rs472631(T)/rs11918967(G) as a predisposing factor for AS (OR=1.304,95%CI=1.001-1.700, permutation10000corrected p=0.049).CONCLUSION: Our findings showed that WNT5A and ANKH polymorphismsare associated with susceptibility to AS in Chinese Han population.WNT5A andANKH may play important roles in the susceptibility of AS in the Chinese Hanpopulation.