B P Wordsworth

Replication of association of IL1 gene complex members with ankylosing spondylitis in Taiwanese Chinese

Objective: To test the association of interleukin 1 (IL1) gene family members with ankylosing spondylitis (AS), previously reported in Europid subjects, in an ethnically remote population. Methods: 200 Taiwanese Chinese AS patients and 200 ethnically matched healthy controls were genotyped for five single nucleotide polymorphisms (SNPs) and the IL1RN.VNTR, markers previously associated with AS. Allele, genotype, and haplotype frequencies were compared between cases and controls. Results: Association of alleles and genotypes of the markers IL1F10.3, IL1RN.4, and IL1RN.VNTR was observed with AS (p<0.05). Haplotypes of pairs of these markers and of the markers IL1RN.6/1 and IL1RN.6/2 were also significantly associated with AS. The strongest associations observed were with the marker IL1RN.4, and with the two-marker haplotype IL1RN.4–IL1RN.VNTR (both pu200a=u200a0.004). Strong linkage disequilibrium was observed between all marker pairs except those involving IL1B-511 (D′ 0.4 to 0.9, p<0.01). Conclusions: The IL1 gene cluster is associated with AS in Taiwanese Chinese. This finding provides strong statistical support that the previously observed association of this gene cluster with AS is a true positive finding. These authors contributed equally to the study.

Interleukin-1 promoter region polymorphism role in rheumatoid arthritis: a meta-analysis of IL-1B-511A/G variant reveals association with rheumatoid arthritis

OBJECTIVESnIL-1 has a central role mediating inflammation and joint destruction in RA. Single nucleotide polymorphisms (SNPs) and haplotype structure in the promoter region can modulate IL-1 function. This study examined the effects of four common promoter SNPs in the IL-1 region on susceptibility and clinical characteristics of RA in British Caucasian patients and assessed the risk of RA by meta-analysis of published studies.nnnMETHODSnUsing PCR-based methods, 756 RA patients and 625 healthy controls (HCs) were genotyped for IL-1A (-889 C/A, rs17561), IL-1B (-511 A/G, rs16944), IL-1B (-1464 C/G, rs1143623) and IL-1B (-3737 G/A, rs4848306) SNPs. Further meta-analysis was performed for IL-1B (-511 A/G) incorporating 3712 RA patients and 2331 HC from six association studies.nnnRESULTSnThe IL-1B (-1464 C/G) G allele was found to be less common in the RA group [P = 0.01; odds ratio (OR) 1.24; 95% CI 1.04, 1.48]. There was no association between IL-1 SNPs and the presence of HLA-DRB1 shared epitope, RF or clinical characteristics. Meta-analysis revealed statistically significant association between IL-1B (-511 A/G) and RA (P = 0.02; pooled OR 1.13; 95% CI 1.02, 1.26).nnnCONCLUSIONSnThere may be a protective effect in RA from the IL-1B (-1464 C/G) G variant. No direct association between the polymorphisms studied and clinical severity characteristics were observed. Further meta-analysis revealed IL-1B (-511 A/G) to be associated with increased susceptibility to RA.

Analysis of killer immunoglobulin-like receptor genes in ankylosing spondylitis

Objectives: To assess the possible association of killer immunoglobulin-like receptor (KIR) genes, specifically KIR3DL1, KIR3DS1 and KIR3DL2, with ankylosing spondylitis (AS). Methods: 14 KIR genes were genotyped in 200 UK patients with AS and 405 healthy controls using multiplex polymerase chain reaction. Sequence-specific oligonucleotide probes were used to subtype 368 cases with AS and 366 controls for 12 KIR3DL2 alleles. Differences in KIR genotypes and KIR3DL2 allele frequencies were assessed using the χ2 test. Results: KIR3DL1 and KIR3DS1 gene frequencies were very similar in cases with AS and controls (odds ratiou200a=u200a1.5, 95% confidence interval 0.8 to 3.0, and odds ratiou200a=u200a1.02, 95% confidence interval 0.2 to 5.3, respectively). KIR3DL2 allele frequencies were not significantly different between cases with AS and controls. Conclusions: Neither the KIR gene content of particular KIR haplotypes nor KIR3DL2 polymorphisms contribute to 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.

No association of KIR3DL1 or KIR3DS1 or their alleles with ankylosing spondylitis.

We determined the alleles of KIR3DL1 and KIR3DS1 in a cohort of British Caucasian ankylosing spondylitis (AS) patients and HLA-B27-positive controls. We found no association in frequencies of the alleles of these genes in AS. In addition, no differences were found when the patients and controls were differentiated by gender.

The genetic association of RUNX3 with ankylosing spondylitis can be explained by allele-specific effects on IRF4 recruitment that alter gene expression

Objectives To identify the functional basis for the genetic association of single nucleotide polymorphisms (SNP), upstream of the RUNX3 promoter, with ankylosing spondylitis (AS). Methods We performed conditional analysis of genetic association data and used ENCODE data on chromatin remodelling and transcription factor (TF) binding sites to identify the primary AS-associated regulatory SNP in the RUNX3 region. The functional effects of this SNP were tested in luciferase reporter assays. Its effects on TF binding were investigated by electrophoretic mobility gel shift assays and chromatin immunoprecipitation. RUNX3 mRNA levels were compared in primary CD8+ T cells of AS risk and protective genotypes by real-time PCR. Results The association of the RUNX3 SNP rs4648889 with AS (p<7.6×10−14) was robust to conditioning on all other SNPs in this region. We identified a 2u2005kb putative regulatory element, upstream of RUNX3, containing rs4648889. In reporter gene constructs, the protective rs4648889 ‘G’ allele increased luciferase activity ninefold but significantly less activity (4.3-fold) was seen with the AS risk ‘A’ allele (p≤0.01). The binding of Jurkat or CD8+ T-cell nuclear extracts to the risk allele was decreased and IRF4 recruitment was reduced. The AS-risk allele also affected H3K4Me1 histone methylation and associated with an allele-specific reduction in RUNX3 mRNA (p<0.05). Conclusion We identified a regulatory region upstream of RUNX3 that is modulated by rs4648889. The risk allele decreases TF binding (including IRF4) and reduces reporter activity and RUNX3 expression. These findings may have important implications for understanding the role of T cells and other immune cells in AS.

An ankylosing spondylitis-associated genetic variant in the IL23R-IL12RB2 intergenic region modulates enhancer activity and is associated with increased Th1-cell differentiation.

Objectives To explore the functional basis for the association between ankylosing spondylitis (AS) and single-nucleotide polymorphisms (SNPs) in the IL23R-IL12RB2 intergenic region. Methods We performed conditional analysis on genetic association data and used epigenetic data on chromatin remodelling and transcription factor (TF) binding to identify the primary AS-associated IL23R-IL12RB2 intergenic SNP. Functional effects were tested in luciferase reporter assays in HEK293T cells and allele-specific TF binding was investigated by electrophoretic mobility gel shift assays. IL23R and IL12RB2 mRNA levels in CD4+ T cells were compared between cases homozygous for the AS-risk ‘A’ allele and the protective ‘G’ allele. The proportions of interleukin (IL)-17A+ and interferon (IFN)-γ+ CD4+ T-cells were measured by fluorescence-activated cell sorting and compared between these AS-risk and protective genotypes. Results Conditional analysis identified rs11209032 as the probable causal SNP within a 1.14u2005kb putative enhancer between IL23R and IL12RB2. Reduced luciferase activity was seen for the risk allele (p<0.001) and reduced H3K4me1 methylation observed in CD4+ T-cells from ‘A/A’ homozygotes (p=0.02). The binding of nuclear extract to the risk allele was decreased ∼3.5-fold compared with the protective allele (p<0.001). The proportion of IFN-γ+ CD4+ T-cells was increased in ‘A/A’ homozygotes (p=0.004), but neither IL23R nor IL12RB2 mRNA was affected. Conclusions The rs11209032 SNP downstream of IL23R forms part of an enhancer, allelic variation of which may influence Th1-cell numbers. Homozygosity for the risk ‘A’ allele is associated with more IFN-γ-secreting (Th1) cells. Further work is necessary to explain the mechanisms for these important observations.

Investigation of a possible extended risk haplotype in the IL23R region associated with ankylosing spondylitis

The IL23R region on chromosome 1 exhibits complex associations with ankylosing spondylitis (AS). We used publicly available epigenomic information and historical genetic association data to identify a putative regulatory element (PRE) in the intergenic region between IL23R and IL12RB2, which includes two single-nucleotide polymorphisms (SNPs) independently associated with AS—rs924080 (P=2 × 10−3) and rs11578380 (P=2 × 10−4). In luciferase reporter assays, this PRE showed silencer activity (P<0.001). Haplotype and conditional analysis of 4230 historical AS cases and 9700 controls revealed a possible AS-associated extended haplotype, including the PRE and risk variants at three SNPs (rs11209026, rs11209032 and rs924080), but excluding the rs11578380 risk variant. However, the rs924080 association was absent after conditioning on the primary association with rs11209032, which, in contrast, was robust to conditioning on all other AS-associated SNPs in this region (P<2 × 10−8). The role of this putative silencer on some IL23R extended haplotypes therefore remains unclear.

Evidence for a second ankylosing spondylitis-associated RUNX3 regulatory polymorphism

Objectives To explore the functions of RUNX3 single nucleotide polymorphisms (SNPs) associated with ankylosing spondylitis (AS). Methods Individual SNP associations were evaluated in 4230 UK cases. Their effects on transcription factor (TF) binding, transcription regulation, chromatin modifications, gene expression and gene interactions were tested by database interrogation, luciferase reporter assays, electrophoretic mobility gel shifts, chromatin immunoprecipitation and real-time PCR. Results We confirmed the independent association of AS with rs4265380, which was robust (P=4.7×10−6) to conditioning on another nearby AS-associated RUNX3 SNP (rs4648889). A RUNX3 haplotype incorporating both SNPs was strongly associated with AS (OR 6.2; 95%u2009CI 3.1 to 13.2, P=1.4×10−8). In a large UK cohort, rs4265380 is associated with leucocyte counts (including monocytes). RUNX3 expression is lower in AS peripheral blood mononuclear cells than healthy controls (P<0.002), independent of rs4265380 genotype. Enhancer function for this RUNX3 region was suggested by increased luciferase activity (approximately tenfold; P=0.005) for reporter constructs containing rs4265380. In monocytes, there was differential allelic binding of nuclear protein extracts to a 50u2009bp DNA probe containing rs4265380 that was strongly augmented by lipopolysaccharide activation. TF binding also included the histone modifier p300. There was enrichment for histone modifications associated with active enhancer elements (H3K27Ac and H3K79Me2) that may be allele dependent. Hi-C database interrogation showed chromosome interactions of RUNX3 bait with the nearby RP4-799D16.1 lincRNA. Conclusions The association of AS with this RUNX3 regulatory region involves at least two SNPs apparently operating in different cell types. Monocytes may be potential therapeutic targets in AS.

THU0364 Rare Erap1 Allotype Combinations Do Not Explain The ERAP1 Association with Ankylosing Spondylitis

Background Single nucleotide polymorphisms (SNPs) in ERAP1 are strongly associated with ankylosing spondylitis (AS) (1,2). ERAP1 is an aminopeptidase involved in the generation of optimal peptide epitopes for presentation by T-cells in the context of major histocompatibility complex (MHC) class I molecules such as HLA-B27. One small study (17 AS cases and 19 controls) suggested that the frequencies of functionally different ERAP1 allotypes differ between patients and controls in AS (3,4). In this study 13 ERAP1 alleles were identified, many of which were rare and previously unreported; allotype *001 was reported in 44% of cases and 8% controls; allotype *002 in 3% of cases and 45% of controls; allotype *005 in 11% of cases and 29% of controls. The authors also reported that the frequencies of particular allotype combinations differed greatly between AS and controls. For example, the allotype combination *001+*005 was found in 53% of cases, but no controls. This allotype combination (and several other less common combinations) were reported exclusively in AS cases, and were shown in parallel in vitro experiments to be associated with poor processing of peptides and altered HLA class I expression. The results of these unexpected ERAP1 associations clearly required verification in a larger population sample in view of their potentially dramatic implications for understanding one of the most enduring mysteries of the pathogenesis of AS, namely its association with HLA-B27. Objectives Our study was performed in a much larger sample to assess the veracity of previous suggestions that rare ERAP1 alleles and allotype combinations are associated with AS. Methods We genotyped 213 AS families and 46 control families using KASP™ technology for 5 SNPs (M349V, K528R, D575N, R725Q, Q730E) to infer ERAP1 allotypes. Allotype and genotype frequencies were compared. Results Several of the reported rare variants were not observed in our sample. We did not confirm the previously reported ERAP1 associations with rare allotype combinations; the *001 allotype was seen in 17.1% of cases and 23.9% controls. Allotype *002 was seen in 34.7% of cases and 25% controls. The *005 allotype, which was common in the previous report among cases, was not seen in either of our AS cases or controls. The *001+*005 allotype combination, which was observed in 53% of cases in the previous small study was completely absent from both our larger sample of 213 cases and 46 controls. Conclusions We were unable to confirm the suggested association of AS with rare ERAP1 allotype combinations in this much larger population study. We were also unable to confirm the diversity of ERAP1 haplotypes previously suggested. Further investigation will be required to clarify the correlation of ERAP1 allotype combinations with both functional effects and their potential association with AS. References Evans DM, et al. Nat Genet 2011;43:761–7. Cortes A, et al. Nat Genet 2013;45:730–40. Reeves E, et al. PNAS 2014;111:17594–9. Reeves E, et al. J Immunol 2013;191:35–43. Disclosure of Interest None declared