Jennifer J. Pointon

Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility

Ankylosing spondylitis is a common form of inflammatory arthritis predominantly affecting the spine and pelvis that occurs in approximately 5 out of 1,000 adults of European descent. Here we report the identification of three variants in the RUNX3, LTBR-TNFRSF1A and IL12B regions convincingly associated with ankylosing spondylitis (P < 5 × 10−8 in the combined discovery and replication datasets) and a further four loci at PTGER4, TBKBP1, ANTXR2 and CARD9 that show strong association across all our datasets (P < 5 × 10−6 overall, with support in each of the three datasets studied). We also show that polymorphisms of ERAP1, which encodes an endoplasmic reticulum aminopeptidase involved in peptide trimming before HLA class I presentation, only affect ankylosing spondylitis risk in HLA-B27–positive individuals. These findings provide strong evidence that HLA-B27 operates in ankylosing spondylitis through a mechanism involving aberrant processing of antigenic peptides.

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.

Association between the interleukin 23 receptor and ankylosing spondylitis is confirmed by a new UK case–control study and meta-analysis of published series

OBJECTIVESnIt has been shown previously that IL-23R variants are associated with AS. We conducted an extended analysis in the UK population and a meta-analysis with the previously published studies, in order to refine these IL-23R associations with AS.nnnMETHODSnThe UK case-control study included 730 new cases and 1331 healthy controls. In the extended study, the 730 cases were combined with 1088 published cases. Allelic associations were analysed using contingency tables. In the meta-analysis, 3482 cases and 3150 controls from four different published studies and the new UK cases were combined. DerSimonian-Laird test was used to calculate random effects pooled odds ratios (ORs).nnnRESULTSnIn the UK case-control study with new cases, four of the eight SNPs showed significant associations, whereas in the extended UK study, seven of the eight IL-23R SNPs showed significant associations (P < 0.05) with AS, maximal with rs11209032 (P < 10(-5), OR 1.3), when cases with IBD and/or psoriasis were excluded. The meta-analysis showed significant associations with all eight SNPs; the strongest associations were again seen not only with rs11209032 (P = 4.06 x 10(-9), OR approximately 1.2) but also with rs11209026 (P < 10(-10), OR approximately 0.6).nnnCONCLUSIONSnIL-23R polymorphisms are clearly associated with AS, but the primary causal association(s) is(are) still not established. These polymorphisms could contribute either increased or decreased susceptibility to AS; functional studies will be required for their full evaluation. Additionally, observed stronger associations with SNPs rs11209026 and rs11465804 upon exclusion of IBD and/or psoriasis cases may represent an independent association with AS.

Genetic Dissection of Acute Anterior Uveitis Reveals Similarities and Differences in Associations observed with Ankylosing Spondylitis

To use high‐density genotyping to investigate the genetic associations of acute anterior uveitis (AAU) in patients with and those without ankylosing spondylitis (AS).

The chromosome 16q region associated with ankylosing spondylitis includes the candidate gene tumour necrosis factor receptor type 1-associated death domain (TRADD)

Objective To replicate and refine the reported association of ankylosing spondylitis (AS) with two non-synonymous single nucleotide polymorphisms (nsSNPs) on chromosome 16q22.1. Methods Firstly, 730 independent UK patients with AS were genotyped for rs9939768 and rs6979 and allele frequencies were compared with 2879 previously typed historic disease controls. Secondly, the two data sets were combined in meta-analyses. Finally, 5 tagging SNPs, located between rs9939768 and rs6979, were analysed in 1604 cases and 1020 controls. Results The association of rs6979 with AS was replicated, p=0.03, OR=1.14 (95% CI 1.01 to 1.28), and a trend for association with rs9939768 detected, p=0.06, OR=1.25 (95% CI 0.99 to 1.57). Meta-analyses revealed association of both SNPs with AS, p=0.0008, OR=1.31 (95% CI 1.12 to 1.54) and p=0.0009, OR=1.15 (95% CI 1.06 to 1.23) for rs9939768 and rs6979, respectively. New associations with rs9033 and rs868213 (p=0.00002, OR=1.23 (95% CI 1.12 to 1.36) and p=0.00002 OR=1.45 (95% CI 1.22 to 1.72), respectively, were identified. Conclusions The region on chromosome 16 that has been replicated in the present work is interesting as the highly plausible candidate gene, tumour necrosis factor receptor type 1 (TNFR1)-associated death domain (TRADD), is located between rs9033 and rs868213. It will require additional work to identify the primary genetic association(s) with AS.

ERAP1 and ankylosing spondylitis.

The strong genetic association of ERAP1 (endoplasmic reticulum aminopeptidase 1) with ankylosing spondylitis (AS), which is restricted to HLA-B27 positive cases, has profound pathogenetic implications. ERAP1 is involved in trimming peptides to optimal length for binding to HLA class 1 molecules, thereby not only affecting the stability and processing of HLA-B27 but also influencing the peptide repertoire presented to the immune system. This could have secondary effects on specific adaptive or autoimmune responses in AS. However, it appears increasingly likely that the pathogenic effect of ERAP1 may be mediated through effects on innate immunity, such as altering the interaction between HLA-B27 and immune receptors such as the killer immunoglobulin-like receptors (KIR) found on a range of innate immune cells or via the endoplasmic reticulum unfolded protein response. ERAP1 variants associated with reduced endopeptidase activity appear to be protective against AS, raising the possibility that ERAP1 inhibition could represent a future treatment strategy.

Ankylosing spondylitis is associated with the anthrax toxin receptor 2 gene (ANTXR2)

Objectives ANTXR2 variants have been associated with ankylosing spondylitis (AS) in two previous genome-wide association studies (GWAS) (p∼9×10−8). However, a genome-wide significant association (p<5×10−8) was not observed. We conducted a more comprehensive analysis of ANTXR2 in an independent UK sample to confirm and refine this association. Methods A replication study was carried out with 2978 cases and 8365 controls. Then, these were combined with non-overlapping samples from the two previous GWAS in a meta-analysis. Human leukocyte antigen (HLA)-B27 stratification was also performed to test for ANTXR2-HLA-B27 interaction. Results Out of nine single nucleotide polymorphisms (SNP) in the study, five SNPs were nominally associated (p<0.05) with AS in the replication dataset. In the meta-analysis, eight SNPs showed evidence of association, the strongest being with rs12504282 (OR=0.88, p=6.7×10−9). Seven of these SNPs showed evidence for association in the HLA-B27-positive subgroup, but none was associated with HLA-B27-negative AS. However, no statistically significant interaction was detected between HLA-B27 and ANTXR2 variants. Conclusions ANTXR2 variants are clearly associated with AS. The top SNPs from two previous GWAS (rs4333130 and rs4389526) and this study (rs12504282) are in strong linkage disequilibrium (r2≥0.76). All are located near a putative regulatory region. Further studies are required to clarify the role played by these ANTXR2 variants in AS.

A common functional variant of endoplasmic reticulum aminopeptidase 2 (ERAP2) that reduces major histocompatibility complex class I expression is not associated with ankylosing spondylitis

Sir, The strong genetic association between AS and HLA-B27 has defied explanation for nearly 40 years. However, the additional discovery of a strong association between AS and ERAP1 (endoplasmic reticulum aminopeptidase 1), a gene that almost certainly operates in the trimming of peptides for optimal binding to MHC class I molecules, has rekindled hopes of rapid advances in this field [1]. It has been suggested that another aminopeptidase, endoplasmic reticulum aminopeptidase 2 (ERAP2), may act in concert with ERAP1, trimming residues inefficiently removed by ERAP1 [2]. The association described recently between ERAP2 and Crohn’s disease, which shares many clinical and genetic overlaps with AS [3], suggests that ERAP2 is worthy of further study in AS. An experiment of nature allows us to do this relatively simply. n nERAP2 has evolved under balancing selection, similar to the MHC, and includes a high-frequency variant (∼50%) that influences antigen presentation [4]. A single nucleotide polymorphism (SNP), rs2248374 (A to G), located within the 5′ canonical splice site of exon 10, results in an alternatively spliced ERAP2 mRNA that is degraded by nonsense-mediated decay (NMD). Homozygosity for the minor G allele (carried by ∼25% of the population) results in failure to express ERAP2 protein; in turn this genotype is also associated with reduced surface MHC Class I expression on human B cells [4]. Such a dramatic phenotypic effect provides a great opportunity for studying the potential role of ERAP2 in AS, analogous to using a knockout model. n nWe therefore specifically tested for differences in frequency of rs2248374 in 470 sporadic AS cases and 420 healthy, ethnically matched blood donors to determine whether this functionally important variant is implicated in AS. All patients were Caucasians, of UK origin and fulfilled the modified New York criteria for AS. Eighty-four per cent of the AS patients were HLA-B27 positive. All patients gave informed consent and ethical approval was obtained (Multicentre Research Ethics Committee 98/5/23). Genotyping involved restriction fragment length polymorphism (RFLP) analysis of PCR products performed under standard conditions. Primers (forward 5′-GCATCCATGGCTAATGTGCR and reverse 5′-GTTGTGGGAAAGCCGAACTA) amplified a 370-bp product that, in the presence of the G allele, was digested to 214 and 156u2009bp products by HphI. Genotype and allele frequencies in AS cases and controls were compared using the Cochrane–Armitage test of trend and the chi-squared test, respectively. This study had 80% power under a log-additive model to detect an odds ratio (OR) of 1.3 with a population risk of AS of 0.04% and a risk allele frequency of 46% (frequency in our controls) at a significance level of 0.05. There were no significant differences between allele or genotype frequencies in AS cases and controls at rs2248374 (Table 1). Additionally, due to the potential functional significance of homozygosity at the minor G allele (i.e. no functional ERAP2 protein), we also compared the frequency of GG homozygotes in AS cases and controls, but these proved almost identical again (Table 1). n n n nTable 1 n nGenotypes, minor allele frequency (MAF), OR, 95% CI and P-value for the ERAP2 SNP rs2248374 n n n nThe association between ERAP1 and AS is the second only to HLA-B27, but formal testing for additional association with the neighbouring gene, ERAP2, had not been previously undertaken. A study of familial AS that typed one marker within ERAP2 reported that an ERAP1/ERAP2 haplotype was over-represented in affected family members [5]. However, such studies are frequently confounded by linkage disequilibrium effects. If genetic variation at ERAP2 were to play a role in susceptibility to AS, then a dramatic loss-of-function variant, such as rs2248374, would appear to be a prime candidate for the ‘causal variant’ at this locus. However, we have now excluded any significant effect arising from this important functional ERAP2 variant. Nonetheless, the possibility of other associations between ERAP2 and AS has not been excluded by this study. This could be done only by systematic mapping of the gene with tagging SNPs in a very large sample after controlling for the significant linkage disequilibrium between ERAP1 and ERAP2 would this be achievable. Our study was insufficiently powered to do this.

Exome-wide study of ankylosing spondylitis demonstrates additional shared genetic background with inflammatory bowel disease

Ankylosing spondylitis (AS) is a common chronic immune-mediated arthropathy affecting primarily the spine and pelvis. The condition is strongly associated with HLA-B*27 as well as other human leukocyte antigen variants and at least 47 individual non-MHC-associated variants. However, substantial additional heritability remains as yet unexplained. To identify further genetic variants associated with the disease, we undertook an association study of AS in 5,040 patients and 21,133 healthy controls using the Illumina Exomechip microarray. A novel association achieving genome-wide significance was noted at CDKAL1. Suggestive associations were demonstrated with common variants in FAM118A, C7orf72 and FAM114A1 and with a low-frequency variant in PNPLA1. Two of the variants have been previously associated with inflammatory bowel disease (IBD; CDKAL1 and C7orf72). These findings further increase the evidence for the marked similarity of genetic risk factors for IBD and AS, consistent with the two diseases having similar aetiopathogenesis.