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Evidence for a second ankylosing spondylitis-associated RUNX3 regulatory polymorphism

Abstract

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.

  • ankylosing spondylitis
  • gene polymorphism
  • spondyloarthritis
  • epidemiology

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Footnotes

  • Contributors MV, AC, ARR, JE, MAB and BPW conceived and designed the experiments. MV, ARR, JE and AC performed the experiments. MV, AC, ARR, JE, CJC, MAB, PB and BPW analysed the data. MV, AC, ARR, CJC, JCK, MAB, PB and BPW wrote the manuscript.

  • Funding MV was funded by NIHR Oxford Comprehensive Biomedical Research Centre (immunity and inflammation theme A93081) and arthritis research UK (grant 21428). ARR was funded by arthritis research UK (grant 20402). JCK and PB by the European Research Council under the European union’s seventh framework programme (FP7/2007-2013)/ERC grant agreement no. 281824, arthritis research UK (grant 20773) and the NIHR Oxford Comprehensive Biomedical Research Centre. MAB is funded by the National Health and Medical Research Council (Australia) senior principal research fellowship. Additional funding was provided by arthritis research UK (18797, 19536, 20402, 20796), the NIHR Thames Valley Collaborative Research Network and National Ankylosing Spondylitis Society (UK).

  • Competing interests None declared.

  • Patient consent Detail has been removed from this case description/these case descriptions to ensure anonymity. The editors and reviewers have seen the detailed information available and are satisfied that the information backs up the case the authors are making.

  • Ethics approval Central Oxford research ethics committee COREC 06/Q1606/139 and OXREC B 07/Q1605/35).

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data sharing statement Access to unpublished raw data relating to this experiments is available on application to the lead author MV.

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