title: An ankylosing spondylitis-associated genetic variant in the IL23R-IL12RB2 intergenic region modulates enhancer activity and is associated with increased Th1-cell differentiation.
abstract: OBJECTIVE
  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.
authors.affiliation: Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, UK.
authors.forename: Amity R
authors.initials: AR
authors.lastname: Roberts
authors.affiliation: Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, UK.
authors.forename: Matteo
authors.initials: M
authors.lastname: Vecellio
authors.affiliation: Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, UK.
authors.forename: Liye
authors.initials: L
authors.lastname: Chen
authors.affiliation: Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, UK.
authors.forename: Anna
authors.initials: A
authors.lastname: Ridley
authors.affiliation: Division of Clinical Neurology, Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford, UK.|Wellcome Trust Centre for Human Genetics, Roosevelt Drive, University of Oxford, Oxford, UK.
authors.forename: Adrian
authors.initials: A
authors.lastname: Cortes
authors.affiliation: Wellcome Trust Centre for Human Genetics, Roosevelt Drive, University of Oxford, Oxford, UK.
authors.forename: Julian C
authors.initials: JC
authors.lastname: Knight
authors.affiliation: Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Bowness
authors.affiliation: Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, UK.
authors.forename: Carla J
authors.initials: CJ
authors.lastname: Cohen
authors.affiliation: Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University of Oxford, Oxford, UK.
authors.forename: B Paul
authors.initials: BP
authors.lastname: Wordsworth
mesh_terms: D000328:Adult; D000483:Alleles; D002454:Cell Differentiation / Q000235:genetics*; D021901:DNA, Intergenic; D005260:Female; D005434:Flow Cytometry; D056726:Genetic Association Studies; D014644:Genetic Variation; D005838:Genotype; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D020641:Polymorphism, Single Nucleotide; D018123:Receptors, Interleukin / Q000235:genetics*; D053707:Receptors, Interleukin-12 / Q000235:genetics*; D013167:Spondylitis, Ankylosing / Q000235:genetics*; D018417:Th1 Cells / Q000502:physiology*
chemical_list: D021901:DNA, Intergenic; C508439:IL12RB2 protein, human; C515500:IL23R protein, human; D018123:Receptors, Interleukin; D053707:Receptors, Interleukin-12
keywords: Ankylosing Spondylitis; Gene Polymorphism; T Cells