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Nat Genet. Author manuscript; available in PMC Nov 28, 2011.
Published in final edited form as:
Published online Jan 10, 2010. doi:  10.1038/ng.513
PMCID: PMC3224997

Genome-wide association study of ankylosing spondylitis identifies non-MHC susceptibility loci


To identify susceptibility loci for ankylosing spondylitis, we undertook a genome-wide association study in 2,053 unrelated ankylosing spondylitis cases among people of European descent and 5,140 ethnically matched controls, with replication in an independent cohort of 898 ankylosing spondylitis cases and 1,518 controls. Cases were genotyped with Illumina HumHap370 genotyping chips. In addition to strong association with the major histocompatibility complex (MHC; P < 10−800), we found association with SNPs in two gene deserts at 2p15 (rs10865331; combined P = 1.9 × 10−19) and 21q22 (rs2242944; P = 8.3 × 10−20), as well as in the genes ANTXR2 (rs4333130; P = 9.3 × 10−8) and IL1R2 (rs2310173; P = 4.8 × 10−7). We also replicated previously reported associations at IL23R (rs11209026; P = 9.1 × 10−14) and ERAP1 (rs27434; P = 5.3 × 10−12). This study reports four genetic loci associated with ankylosing spondylitis risk and identifies a major role for the interleukin (IL)-23 and IL-1 cytokine pathways in disease susceptibility.

Ankylosing spondylitis is a common cause of inflammatory arthritis, with a prevalence of ~5 per 1,000 in European populations1. It is characterized by inflammation of the spine and sacroiliac joints causing pain and stiffness and ultimately new bone formation and progressive joint ankylosis. Hip and peripheral joint arthritis is common, and inflammation may also involve extra-articular sites such as the uveal tract, tendon insertions, proximal aorta and, rarely, the lungs and kidneys. The disease is strongly associated with the gene HLA-B27; however, only 1%–5% of HLA-B27-positive individuals develop ankylosing spondylitis, and there is increasing evidence to suggest that other genes must also be involved25. Association has previously been confirmed between ankylosing spondylitis and SNPs in IL23R at chromosome 1p23 and ERAP1 (previously known as ARTS-1) at chromosome 5p15 (ref. 6), and linkage has been demonstrated at genome-wide significance to chromosome 6p (where HLA-B is encoded) and chromosome 16q (lod score 4.7)7. We report here the first genome-wide association study (GWAS) for ankylosing spondylitis.

To identify ankylosing spondylitis susceptibility genes, we performed a GWAS in a sample of ankylosing spondylitis cases among Australian, British and North American individuals of European descent (n = 2,053 in the final data set), using data from previously genotyped, ethnically matched British and North American individuals as controls (n = 5,140). Cases were genotyped with Illumina HumHap370 genotyping chips; 288,662 SNPs were available for study that were common to case and all control data sets after quality-control filtering (see Online Methods). After data cleaning, a modest overall inflation of test statistics remained, with a genomic inflation factor (λ) of 1.06 (ref. 8), excluding SNPs in the MHC (Supplementary Fig. 1). We then genotyped a total of 163 SNPs in a replication cohort of 898 British ankylosing spondylitis cases and 1,518 unselected British controls. The SNPs genotyped included 49 ancestry-informative SNPs and 114 SNPs in 105 chromosomal regions selected from the discovery sample on the basis of their strength of association in that sample and because of close proximity to genes of biologically plausible involvement in ankylosing spondylitis (Supplementary Table 1). Of the confirmation SNPs, 102 markers from 95 regions passed quality control filters and are reported here.

As expected, SNPs in the MHC on chromosome 6p were strongly associated with ankylosing spondylitis (rs7743761 P = 5.0 × 10−304). Association was evident across a very broad region surrounding the MHC, including five SNPs lying in a 153-kb region at 26.0–26.1 Mb from the p-telomere (5.4 Mb from HLA-B), which achieved P < 10−5. The most associated SNP in this region was rs3734523 (P = 1.6 × 10−6). However, conditional logistic regression analysis suggested that this was unlikely to represent a separate independent association because conditioning on five of the most significant SNPs from the MHC (rs7743761, rs2596501, rs3915971, rs2516509, rs1265112) caused the association to disappear (P = 0.27).

Excluding the MHC and surrounding regions, 25 SNPs from six independent loci were significantly associated with ankylosing spondylitis, including the known ankylosing spondylitis–associated genes ERAP1 and IL23R, and two new loci, chromosomes 2p15 and 21q22 (Table 1 and Supplementary Fig. 2). We also observed strong association within two more genes, ANTXR2 and IL1R2, with support in both the discovery and confirmation data sets.

Table 1
Genome-wide significant loci typed in both discovery cohort and replication study

Both non-MHC genes previously associated with ankylosing spondylitis, ERAP1 and IL23R, were significantly associated in this data set. The most strongly associated SNPs were rs30187 (P = 2.6 × 10−11) and rs11209026 (P = 9.1 × 10−14), confirming the strong association observed for these SNPs in the initial discovery set6.

We used SNP imputation to investigate association strength at untyped markers of the six non-MHC loci associated with ankylosing spondylitis. Considering IL23R, only marginally stronger association was observed with one imputed SNP (rs11465817, P = 1.2 × 10−10) than with the strongest associated genotyped SNP, rs11209026 (P = 2.3 × 10−9) (Fig. 1a). IL23R has ten exons, with marker rs11209026 encoding a Q381R substitution in exon 9, and rs11465817 falling in intron 9, suggesting that this is the critical region involved in the association of IL23R with ankylosing spondylitis.

Figure 1
SNP association plots for ankylosing spondylitis–associated regions. Discovery cohort association significance is plotted against the left hand y axis as −log10 (P-value). Genetic coordinates are as per NCBI dbSNP genome build 128 (October ...

In ERAP1, the imputed data revealed a block of SNPs lying in a 4.6-kb region between rs27529 (in exon 9) and rs469758 (in intron 12) achieving P < 10−11, more than 50 times more significant than any other imputed SNP (Fig. 1b). In this region, only marker rs30187 is coding (R528K). It has previously been demonstrated that rs30187 causes a significant reduction in aminopeptidase activity toward a synthetic peptide substrate as well as alterations in substrate affinity9. Molecular modeling of the ERAP1 protein suggests that Arg528 lies at the mouth of the putative enzyme substrate pocket, perhaps explaining the lower aminopeptidase activity of this genetic variant. ERAP1 variants also correlate significantly with expression. Strong cis-regulation of ERAP1 expression in lymphoblastoid cell lines was seen from SNPs close to and within ERAP1, including the marker rs30187 (C allele reduced expression, P = 0.00015)10. In our study, we saw no difference in ERAP1 expression in peripheral blood mononuclear cells (PBMCs) from ankylosing spondylitis cases compared with controls (Supplementary Table 2), suggesting that this is a less likely explanation of the mechanism of association of ERAP1 with ankylosing spondylitis.

Three SNPs at the 2p15 locus achieved genome-wide significance in the discovery set: rs10865331 (P = 6.1 × 10−15), rs10865332 (P = 3.5 × 10−10) and rs4672503 (P = 9.3 × 10−10). No imputed SNP was more significantly associated than rs10865331. In the replication study we genotyped two SNPs in this locus, both of them confirming the discovery set findings: rs4672495 (P = 8.4 × 10−4) and rs10865331 (P = 5.5 × 10−6). The combined level of association of these SNPs was highly significant: rs4672495 (P = 3.2 × 10−9) and rs10865331 (P = 1.9 × 10−19). Combining the imputed and genotyped data, there is a block of SNPs lying between marker rs10865331 and rs4672507 in tight linkage disequilibrium (LD) (r2 > 0.8) with >1,000 times stronger significance than any other SNP at this locus, encompassing a 23-kb region likely to contain the causative variant(s) responsible for the association observed (Fig. 1c). No genes are encoded within this region, the nearest gene to the most strongly associated marker rs10865331 being 100 kb distant (B3GNT2). We are not aware of this region being associated previously with any known disease. B3GNT2 encodes UDP-GlcNAc: betaGal beta-1,3-N-acetylglucosaminyltransferase 2, a protein not as yet known to have any immunological function.

At chromosome 21q22, three SNPs across an 11-kb region achieved genome-wide significance in the discovery cohort: rs2242944 (P = 2.7 × 10−14), rs2836878 (P = 4.9 × 10−12) and rs378108 (P = 6.1 × 10−11) (Fig. 1d). SNP rs2242944 also showed strong association in the confirmation cohort (P = 5.6 × 10−7) and in the combined analysis (P = 8.3 × 10−20). The nearest gene to the most strongly associated SNP, rs2242944, is 82 kb distant (PSMG1, proteasome assembly chaperone 1). This region has recently been associated with pediatric-onset inflammatory bowel disease (IBD), in which the most strongly associated SNP was rs2836878; positive association was seen with over-representation of the minor allele, as was the case in our ankylosing spondylitis data set (P = 4.1 × 10−10). This SNP is in strong LD with the strongest ankylosing spondylitis–associated marker, rs2242944 (r2 = 0.6, D = 1)11. Increased expression of PSMG1 was observed in colonic biopsies from IBD cases, and it was suggested that this may be the gene involved at this locus. Ankylosing spondylitis and IBD are closely related conditions, with ~70% of those with ankylosing spondylitis having microscopic terminal ileitis resembling Crohn’s disease12 and ~10% of those with IBD having ankylosing spondylitis. Crohn’s disease and ankylosing spondylitis are each associated with IL23R SNPs, and it is likely that further shared genetic susceptibility factors exist. We saw strong association even among those cases with no clinical IBD (n = 1,159 cases, rs2242944, P = 1.3 × 10−9), indicating that the association was present even in cases of primary ankylosing spondylitis in the absence of clinically manifest IBD.

PSMG1 was not differentially expressed in PBMCs from cases with active ankylosing spondylitis compared with healthy controls (Supplementary Fig. 3), nor in relationship to ankylosing spondylitis–associated chromosome 21q22 SNPs. A large recombination hotspot lying between PSMG1 and the ankylosing spondylitis–associated SNPs makes it unlikely that the association signal observed is due to effects from SNPs located in or close to PSMG1. We feel that its remoteness to the associated locus, absence of differential expression with disease, and lack of evidence of a relevant biological function make it an unlikely candidate to be directly involved in ankylosing spondylitis susceptibility. Rather, we hypothesize that the chromosome 2p15 and 21q22 regions harbor either noncoding RNA species or hitherto unreported protein-coding genes that are likely to be involved in susceptibility to ankylosing spondylitis. To investigate this further, we performed a transcriptome-wide profiling study of expressed sequence tags and small RNAs derived from PBMCs from four active ankylosing spondylitis cases and three healthy controls using Illumina’s deep sequencing approach. No small regulatory RNAs such as microRNAs were seen within the regions of highly associated SNPs at either locus, although, consistent with recent findings13, these were identified in association with transcription start sites of flanking genes outside the disease-associated region (Supplementary Fig. 4). At both loci, we identified sequence tags derived from long RNAs. These either represent long mRNA-like noncoding RNA species or, alternatively, previously undescribed mRNA isoforms originating from distal promoters of adjacent protein-coding genes.

Fourteen SNPs in a 61-kb region encompassing IL1R2 achieved nominal significance, with the strongest association observed with genotyped markers at rs2310173 (P = 8.3 × 10−6) and with imputed markers at rs10185424 (P = 5.4 × 10−6) (Supplementary Fig. 3a). Marker rs2310173 was also associated with ankylosing spondylitis in the replication study (P = 0.018) and showed a high level of significance in the combined analysis (P = 4.8 × 10−7). IL-1R2 is cleaved from cell membranes, possibly by ERAP1 (ref. 14) and acts as a decoy receptor, interfering with the binding of IL-1 to IL-1RI. One possible explanation for the associations of ERAP1 and IL1R2 with ankylosing spondylitis is that the disease-associated genetic variants affect cleavage of IL-1R2 from the cell surface. In this respect, we note that several SNPs in TNFRSF1A achieved moderate levels of association in the discovery set (strongest associated SNP, rs1800693, P = 6.9 × 10−5). TNFRSF1A encodes tumor necrosis factor receptor 1, which may also be cleaved from the cell surface by ERAP1 (ref. 15). No support for this association was seen in our replication study, but SNPs in TNFRSF1A have been associated with both ulcerative colitis and Crohn’s disease previously16,17, providing some support for this association with ankylosing spondylitis. Tumor necrosis factor overexpression in mice leads to inflammatory bowel disease and to sacroiliitis resembling ankylosing spondylitis, and is dependent on expression of TNFRSF1A (ref. 18).

ANTXR2, recessive mutations of which cause juvenile hyaline fibromatosis (MIM228600) and infantile systemic hyalinosis (MIM236490), encodes capillary morphogenesis protein-2 (CMP2). The SNP rs4333130 was associated with ankylosing spondylitis in both the discovery cohort (P = 7.5 × 10−7) and replication cohort (P = 0.029) as well as overall (P = 9.3 × 10−8). In the imputed data set, no markers were more strongly associated (Supplementary Fig. 3b). A functional explanation for this association with ankylosing spondylitis is not clear.

The power of this study to detect small to moderate genetic effects was modest. We calculate that the discovery phase of the study has 2%–21% power to identify SNPs conferring an additive allelic odds ratio of 1.2 with minor allele frequencies of 0.1–0.5 at α = 5 × 10−7, assuming D′ = 0.9 and the marker and disease-associated allele frequencies are equal. Further GWAS with larger sample sizes will therefore be useful and likely to identify more genes associated with ankylosing spondylitis. The identification of four genetic loci newly associated with ankylosing spondylitis extends our understanding of the genetic etiology of this disorder and provides an important foundation for future hypothesis-driven research into the pathogenesis of this common and debilitating condition.


Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturegenetics/.

Supplementary Material

Supplementary Text and Figures


We would like to thank all participants, with and without ankylosing spondylitis, who provided the case and control DNA and clinical information necessary for this study. We are also very grateful for the invaluable support received from the National Ankylosing Spondylitis Society (UK) and Spondyloarthritis Association of America in subject recruitment. We are extremely grateful to all the families who took part in this study, the midwives for their help in recruiting them and the whole Avon Longitudinal Study of Parents And Children (ALSPAC) team, which includes interviewers, computer and laboratory technicians, clerical workers, research scientists, volunteers, managers, receptionists and nurses. This study was funded by US National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) grants P01-052915 and R01-AR046208. Funding was also received from the University of Texas at Houston Clinical and Translational Science Award grant UL1RR024188, Cedars-Sinai General Clinical Research Center grant MO1-RR00425, Intramural Research Program of NIAMS/National Institutes of Health, and Rebecca Cooper Foundation (Australia). This study was funded in part by the Arthritis Research Campaign (UK), by the Wellcome Trust (grant number 076113) and by the Oxford Radcliffe Hospital Biomedical Research Centre ankylosing spondylitis chronic disease cohort (theme code A91202). M.A.B. is funded by the National Health and Medical Research Council (Australia). The UK Medical Research Council, the Wellcome Trust and the University of Bristol provide core support for ALSPAC.


COMPETING INTERESTS STATEMENT The authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturegenetics/.

Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/.

Note: Supplementary information is available on the Nature Genetics website.


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