Abstract
BackgroundPolymorphisms in the interferon regulatory factor 5 (IRF5) gene are associated with susceptibility to systemic lupus erythematosus, rheumatoid arthritis and other diseases through independent risk and protective haplotypes. Several functional polymorphisms are already known, but they do not account for the protective haplotypes that are tagged by the minor allele of rs729302.MethodsPolymorphisms in linkage disequilibrium (LD) with rs729302 or particularly associated with IRF5 expression were selected for functional screening, which involved electrophoretic mobility shift assays (EMSAs) and reporter gene assays.ResultsA total of 54 single-nucleotide polymorphisms in the 5' region of IRF5 were genotyped. Twenty-four of them were selected for functional screening because of their high LD with rs729302 or protective haplotypes. In addition, two polymorphisms were selected for their prominent association with IRF5 expression. Seven of these twenty-six polymorphisms showed reproducible allele differences in EMSA. The seven were subsequently analyzed in gene reporter assays, and three of them showed significant differences between their two alleles: rs729302, rs13245639 and rs11269962. Haplotypes including the cis-regulatory polymorphisms correlated very well with IRF5 mRNA expression in an analysis based on previous data.ConclusionWe have found that three polymorphisms in LD with the protective haplotypes of IRF5 have differential allele effects in EMSA and in reporter gene assays. Identification of these cis-regulatory polymorphisms will allow more accurate analysis of transcriptional regulation of IRF5 expression, more powerful genetic association studies and deeper insight into the role of IRF5 in disease susceptibility.
Highlights
The interferon regulatory factor 5 (IRF5) gene occupies a prominent place among the genetic factors involved in susceptibility to rheumatic and autoimmune diseases [1]
The selected single-nucleotide polymorphism (SNP) were evaluated for their effect in binding nuclear proteins as an indication of their possible role in cis-regulation of IRF5 expression by means of electrophoretic mobility shift assay (EMSA)
To identify SNPs strongly correlated with rs729302 or with the protective haplotypes, we genotyped 54 polymorphisms located predominantly within 21.5 kb of the 5’ region of IRF5 (Additional file 2: Table S1) in 95 Spanish controls
Summary
The interferon regulatory factor 5 (IRF5) gene occupies a prominent place among the genetic factors involved in susceptibility to rheumatic and autoimmune diseases [1] It is associated with a large series of diseases, either definitively, as with systemic lupus erythematosus (SLE) [2,3,4,5,6,7], Sjögren’s syndrome, systemic sclerosis, primary biliary cirrhosis and rheumatoid arthritis; or more tentatively, as with granulomatosis with vasculitis, multiple sclerosis, inflammatory bowel disease and atherosclerosis [1]. Rs10954213, creates an early polyadenylation site that leads to shorter IRF5 mRNA isoforms with longer halflives [4] This single-nucleotide polymorphism (SNP) has the strongest evidence for a role in cis-regulatory element of IRF5 [12,13]. Several functional polymorphisms are already known, but they do not account for the protective haplotypes that are tagged by the minor allele of rs729302
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