Amity R. Roberts

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 2 kb 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.

ERAP1 association with ankylosing spondylitis is attributable to common genotypes rather than rare haplotype combinations.

Significance Ankylosing spondylitis (AS) is a common inflammatory arthritis of the spine. It is associated with two genes involved in antigen processing and presentation to the immune system, HLA-B*27 and ERAP1 (endoplasmic reticulum aminopeptidase 1), which act synergistically in AS. Previous reports have suggested that rare ERAP1 variants associated with dramatically altered antigen processing function are responsible. In contrast, we show here conclusively that it is common variants of ERAP1 that are mainly responsible for protection/susceptibility in AS rather than rare ERAP1 variants and/or unusual combinations of ERAP1 variants. This has important potential implications for future studies addressing the development of ERAP1 inhibitors as new treatments not only for AS but also in other diseases genetically associated with ERAP1. We investigated the proposal that ankylosing spondylitis (AS) is associated with unusual ERAP1 genotypes. ERAP1 haplotypes were constructed for 213 AS cases and 46 rheumatoid arthritis controls using family data. Haplotypes were generated from five common ERAP1 single nucleotide polymorphisms (SNPs)—rs2287987 (M349V), rs30187 (K528R), rs10050860 (D575N), rs17482078 (R725Q), and rs27044 (Q730E). Haplotype frequencies were compared using Fisher’s exact test. ERAP1 haplotypes imputed from the International Genetics of AS Consortium (IGAS) Immunochip study were also studied. In the family study, we identified only four common ERAP1 haplotypes (“VRNQE,” “MKDRQ,” “MRDRE,” and “MKDRE”) in both AS cases and controls apart from two rare (<0.5%) previously unreported haplotypes. There were no examples of the unusual ERAP1 haplotype combination (“*001/*005”) previously reported by others in 53% of AS cases. As expected, K528-bearing haplotypes were increased in the AS family study (AS 43% vs. control 35%), due particularly to an increase in the MKDRQ haplotype (AS 35% vs. control 25%, P = 0.01). This trend was replicated in the imputed Immunochip data for the two K528-bearing haplotypes MKDRQ (AS 33% vs. controls 27%, P = 1.2 × 10–24) and MKDRE (AS 8% vs. controls 7%, P = 0.004). The ERAP1 association with AS is therefore predominantly attributable to common ERAP1 haplotypes and haplotype combinations.

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 1.14 kb 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% CI 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 50 bp 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.

The severity of ankylosing spondylitis and responses to anti-tumour necrosis factor biologics are not influenced by the tumour necrosis factor receptor polymorphism incriminated in multiple sclerosis.

Genetic polymorphism (rs1800693) of TNFRSF1A (type 1 tumour necrosis factor receptor) encodes a potentially anti-inflammatory soluble truncated form of the p55 receptor, which is associated with predisposition to multiple sclerosis but protection against ankylosing spondylitis (AS). We analysed 2917 UK Caucasian cases by linear and logistic regression for associations of rs1800693 with disease severity assessed by the Bath Ankylosing Spondylitis measures of disease activity and function (BASDAI, BAS-G and BASFI) and/or responses to anti-TNF therapy. In contrast to predictions, rs1800693 GG homozygotes actually had significantly worse BASDAI (mean 4.2, 95% CI: 4–4.5) than AA homozygotes (mean 3.8, 95% CI: 3.7–4) in both the unadjusted (difference = 0.4, p = 0.006) and adjusted analyses (difference = 0.2–0.5, p = 0.002–0.04 depending on the adjustment model). We found no evidence that rs1900693 predicted functional status (BASFI) or global disease scores (BAS-G), and it exerted no influence on either the intention to treat with or efficacy of anti-TNF treatment.

Reply to Reeves et al.: No evidence for rare ERAP1 haplotypes and haplotype combinations in ankylosing spondylitis.

The comments on our recent paper (1) by Reeves et al. (2) concentrate on the functional effects of novel ERAP1 (endoplasmic reticulum aminopeptidase 1) haplotypes and haplotype combinations. The very nice in vitro functional experiments, which Reeves et al. have previously published (3) and highlight in their letter (2), clearly show that various combinations of different amino acid substitutions in synthetic ERAP1 molecules can have profound functional effects. This is unsurprising, but the relevance of many of the “polymorphisms” that these authors have described and their contribution to “real life” haplotypic combinations and their involvement in the pathophysiology of ankylosing spondylitis (AS) is doubtful. Most of the “new” rare variants described by Reeves et al. in patients and healthy controls have not been confirmed by others and are not recorded in standard DNA databases (1, 4). We suggest that repeat sequencing of the relevant exons of genomic DNA from the original 19 AS cases and 17 controls (rather than from cDNA) might be the best way to resolve this issue. Reeves et al.’s elegant functional studies (2, 3) compare many ERAP1 combinations that are either exceptionally rare or that we do not recognize at all in the general population or in people with AS in much larger studies (1, 4). For example, there is no corroborating evidence to suggest that the “*003,” “*004,” “*006,” “*007,” “*009,” “*012,” or “*013” ERAP1 haplotypes actually exist. Our data also suggest that “*005” is exceptionally rare (allele frequency ∼0.001) (1), in contrast to the previous suggestion by Reeves et al. that it has an allele frequency of 0.29 in AS cases (3). Many of their comparisons in the in vitro functional studies are between artificial haplotype combinations that include these spurious variants. However, the functional data also include valuable comparisons of variants that are known to exist, such as *001 and *002. It is quite clear that when ERAP1deficient cells are reconstituted with the combination of *001 + *002 (common in the general population and also in AS), there is restoration of peptide trimming activity and HLA class I cell surface expression. In contrast, reconstitution with the *001 + *001 combination has little effect. However, this is most likely explained simply by the presence of the hypofunctional R528 in *001 compared with the AS-predisposing normally functioning 528K in *002. One does not have to invoke an influence from other residues necessarily to explain this. We note that this reconstituting effect is apparent for all six haplotype combinations studied by Reeves et al. (3) containing *002, except *002 + *006 (which we have never actually observed in more than 28,000 individuals). The relevance of functional effects arising from artificial ERAP1 haplotypes or combinations of haplotypes seems highly doubtful. We believe that the onus is now on Reeves et al. to provide additional genomic sequencing data to support the concept that these exceptionally rare unusual ERAP1 haplotypes and haplotype combinations actually exist.

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

OP0299 Susceptibility To Ankylosing Spondylitis Is Influenced Independently by Two Closely Adjacent Runx3 Snps, That Show Strong Functional Effect in Different Cell Types

Background More than 60 genes are associated with ankylosing spondylitis (AS), many of which (RUNX3, EOMES, TBX21, ZMIZ1, IL23R, IL6R, ERAP-1, IL7 and IL7R) are involved in diverse immunological processes.1 To understand the functional basis for these genetic associations is one of the greatest scientific challenges in complex polygenic diseases. For example, the association between AS and the single nucleotide polymorphism (SNP) rs4648889 located in a 2kb regulatory locus upstream the promoter of RUNX3 can be explained by allele-specific effects on transcription factor (TF) recruitment (including IRF4) that alter gene expression, specifically in CD8+ T-cells.2 Objectives 22 SNPs in the RUNX3 locus were initially found to be associated with AS. Here we focus on an independently associated SNP adjacent to rs4648889 which affects gene regulation in CD14+ monocytes. Methods We used the Encyclopedia of DNA Elements (ENCODE) data to dissect the epigenetic and transcriptional landscape of the RUNX3 locus; we performed in silico analysis and in vitro functional studies to characterize the effects of this particular genetic variant, providing critical functional evidence for its role in AS. Results (1) Conditional analysis on 4230 AS cases and 9700 matched controls established the primacy of rs4648889 association (1.3×10–14) with AS in this region. However, there was also an independent second signal (1.7×10–7) with rs4265380, adjacent to rs4648889, highlighting the importance of this locus and a likely functional role for rs4265380. (2) ENCODE data on CD14+ monocytes, revealed a robust peak for DNA accessibility (DNase1 Hypersensitivity) and TFs ChIP-seq overlapping rs4265380. There was also a strong overlying peak for histone modification H3K4Me1, normally associated with active regulatory region. (3) The effect of the rs4265380 dimorphism on TF binding was evaluated by electrophoretic mobility shift assays (EMSA) using nuclear extract from U937 and THP-1 (monocyte-derived cell lines) focusing on specific TFs involved in monocyte regulation, inflammation and chromatin modifiers (C-Fos, p300 i.e.). (4) Analysis of the expression of inflammasome-related genes revealed some striking differences (i.e CARD6 and IL-6, upregulated of 5 and 177 fold, respectively) influenced by the rs4265380 genotype. Conclusions We have identified functional differences in the transcriptional regulation of RUNX3 associated with AS-associated SNPs at this locus. Two individual adjacent SNPs exert their independent effects in two separate cell types (CD8+ T-cells and monocytes). These observations are critically important not only in identifying cell types that play a pathogenic role in AS but also in defining potential therapeutic drug targets. References IGAS et al. Nat Genet. 2013 Jul;45(7):730–8 Vecellio M. et al, Ann Rheum Dis. 2015 Oct 9 pii: annrheumdis-2015–207490 Disclosure of Interest None declared

OP0231 Differential Transcription Factor Binding Could Explain The Genetic Association of Ankylosing Spondylitis with Polymorphisms in The IL23R-IL12RB2 Intergenic Region

Background Genome-wide association studies have revealed the polygenic nature of ankylosing spondylitis (AS). More than 60 genetic influences have been identified but most of these are non-coding sequences (1–3). Protection against AS is afforded by a loss of function mutation in the cytoplasmic tail of the IL23 receptor (IL23R), but there is also a second independent association in this region (2). Objectives This study explores the functional basis for the second independent association between AS and single nucleotide polymorphisms (SNPs) in the IL23R-IL12RB2 intergenic region. Methods We performed conditional analysis on genetic association data at IL23R and used epigenetic data on chromatin remodelling and transcription factor (TF) binding to identify the primary AS-associated 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. The involvement of candidate TFs in DNA binding was investigated by antibodies in these experiments. We measured mRNA expression levels of nearby genes in CD4+ T-cells and compared these between cases homozygous for the risk “A” allele and the protective “G” allele. The proportions of IL-17A+ and IFN-γ+ CD4+ T-cells were measured by FACS and also correlated to patient genotype. Results Conditional analysis identified rs11209032 as the primary causal SNP within a putative enhancer between IL23R and IL12RB2. Reduced luciferase activity was seen for the risk “A” allele (p<0.001), and reduced H3K4me1 methylation observed in CD4+ T-cells from “A/A” homozygotes (p=0.02). Nuclear extract binding to the risk “A” allele was decreased ∼3.5-fold compared to the protective allele (p<0.001). Reduced nuclear factor binding was observed when antibody to TWIST1 was included. 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. Homozygotes for the risk “A” allele have more Interferon gamma-secreting (Th1) cells compared to those with the protective “G” allele. Further work is necessary to explain how TWIST1 contributes to these important observations. References Evans DM, Spencer CCA, Pointon JJ, et al. Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility. Nat Genet 2011;43:761–7. Cortes A, Hadler J, Pointon JP, et al. Identification of multiple risk variants for ankylosing spondylitis through high-density genotyping of immune-related loci. Nat Genet 2013;45:730–40. Reveille JD, Sims A-M, Danoy P, et al. Genome-wide association study of ankylosing spondylitis identifies non-MHC susceptibility loci. Nat Genet 2010;42:123–7. Acknowledgement This work was supported by the Arthritis Research UK (grant 20402, 20235, 19536, 18797, and 20796); and the NIHR comprehensive Biomedical Research Centre (immunity and inflammation theme A93081); NIHR Oxford Musculoskeletal Research Unit. Disclosure of Interest None declared