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To explore the functional basis for the association between ankylosing spondylitis (AS) and single-nucleotide polymorphisms (SNPs) in the IL23R-IL12RB2 intergenic region.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.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.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.

Original publication

DOI

10.1136/annrheumdis-2015-208640

Type

Journal article

Journal

Annals of the rheumatic diseases

Publication Date

12/2016

Volume

75

Pages

2150 - 2156

Addresses

Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, UK.

Keywords

Th1 Cells, Humans, Spondylitis, Ankylosing, Receptors, Interleukin, DNA, Intergenic, Flow Cytometry, Cell Differentiation, Genotype, Polymorphism, Single Nucleotide, Alleles, Adult, Female, Male, Receptors, Interleukin-12, Genetic Variation, Genetic Association Studies, HEK293 Cells