Background/Purpose: The JIA risk haplotypes, like those of other autoimmune diseases, are highly enriched for H3K4me1/H3K27ac histone marks, epigenetic features typically associated with functional enhancers. However, enhancer function cannot be assumed based on chromatin features alone, but must be demonstrated experimentally. Furthermore, the presence of a functional enhancer on a risk haplotype is not prima facieevidence that the enhancer plays a role in genetic risk. We therefore used in vitro reporter assays to query 2 JIA risk haplotypes, IL2RAand IL6R, to confirm the presence of functional enhancers in select H3K4me1/H3K27ac marked regions and determine the effects of JIA-associated genetic variants on enhancer function.

Methods: We used the pGL4 reporter system to query enhancer effects across a 1,000 bp region within the first intron of the IL2RAgene and a 2,483 bp intergenic region upstream of the IL6R transcription start site.  SNPs within both of these regions are in strong linkage disequilibrium (LD) with the tag SNPs that identify these JIA risk loci.  The pGL4 vector contains a minimal SV40 promoter that is, by itself, inefficient at driving luciferase function. We tiled three ~500 bp constructs across the regions of interest and cloned these constructs into pGL4 vectors, which were then transfected into Jurkat T cells.  Cells were incubated for 24 hr, after which we assessed luciferase production.  We repeated these same experiments using constructs containing genetic variants within theIL2RAintronic enhancer (rs117119468 C- >T and rs12722502 C- >T) that we identified in children with JIA using whole genome sequencing. We also tested the effects of 4 variants on the IL6Rhaplotype: rs540546266 (C >T), rs532200576 (T >C), rs9651053 (G >A), and rs540215820 (C >T).

Results: Within the H3K4me1/H3K27ac-marked regions that we queried, within the first we observed enhancer activity across a 657 bp region (chr10:6092661-chr10:6093317) within the first intron of IL2RAand in a 1,474 bp intergenic region upstream of the IL6Rgene region in Jurkat T cells. The IL2RA enhancer increased luciferase activity by 5-8 fold (compared with a control vector that contained only the minimal SV40 promoter and the luciferase gene) and the IL6R enhancer increased activity by 3-5 fold. A construct carrying a sequence within the first intron of the IL2RAgene that was not marked by the H3K4me1/H23K27ac histone signature showed no enhancement of luciferase expression compared to background. Constructs containing the JIA-associated rs117119468 C- >T allele abolished the enhancer activity within the IL2RAlocus, while the construct containing the rs12722502 C- >T allele reduced luciferase activity by 30% (p< 0.05).  In contrast, all 4 SNPs within the IL6Rlocus increased enhancer activity (p< 0.03) by 25-30%.

Conclusion: The JIA-associated risk loci, IL2RA and IL6R , contain enhancers that is are active in lymphoid cells. JIA-associated genetic variants identified by WGS and/or conventional genotyping alter enhancer activity at these loci. These findings represent conclusive evidence for the presence of non-coding, functional elements within JIA risk loci and demonstrate that JIA-associated genetic variants alter these functions.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-juvenile-idiopathic-arthritis-associated-il2ra-and-il6r-haplotypes-contain-enhancers-whose-functions-are-altered-by-jia-associated-genetic-variants/