Background Systemic lupus erythematosus is a multisystemic autoimmune disease characterised by the production of autoantibodies to nuclear antigens. We have identified a variant in intron 1 of complement receptor 2 (CR2/CD21) that is associated with decreased risk of lupus (rs1876453; Pmeta = 4.2 × 10–4, OR = 0.85). Its effect is strongest in subjects with anti-dsDNA antibodies (case-control Pmeta = 7.6 × 10–7, OR = 0.71; case-only Pmeta 1.9 × 10–4, OR = 0.75), suggesting a preferential association with this endophenotype. rs1876453, located 97 nucleotides from the 5’ end of CR2 intron 1, alters the binding of multiple protein complexes, including one containing CTCF, and is associated with increased B cell-specific expression of the adjacent gene, complement receptor 1 (CR1/CD35). The transcriptional mechanism connecting these observations remains unclear, and we hypothesised that long noncoding RNA (lncRNA) play a role.
Materials and methods cDNA was generated by reverse transcription from RNA purified from the Raji B cell line as well as from human tonsil and spleen, peripheral blood mononuclear cells, and purified primary B cells. PCR was performed using 5’ and 3’ primers that targeted spliced exons from known lncRNA sequences in the intergenic region 5’ of CR2, in the CR2 gene, and in CR1 intron 1. Quantitative PCR of primary B cell transcripts was performed using cDNA transcribed using random primers and MultiScribe reverse transcriptase (Applied Biosystems), customised lncRNA primers and probe that targeted spliced exons, Taqman assays for U6 snRNA and b-actin mRNA, and the Applied Biosystems 7500 Real-Time PCR System. Relative expression levels of lncRNA, normalised to either U6 snRNA (A) or b-actin (B), were calculated using the comparative CT method. P values were determined using a two-tailed Student t test and a p value of <0.05 was considered significant.
Results We confirmed the presence of annotated lncRNAs in the CR2-CR1 genomic region in various cell types. One annotated lncRNA located downstream of rs1876453 in CR2 intron 1 was readily detected in B cells. We determined the allele-specific expression of this lncRNA by quantitative RT-PCR and found that it was ∼3-fold increase in individuals with the minor protective allele at rs1876453 (p = 0.0025 normalised to U6 snRNA and p = 0.0054 normalised to beta-actin).
Conclusions Our data suggest that the generation of pathogenic autoantibodies associated with early, active, and severe lupus is modified by expression of a CR2 lncRNA that appears to have long-range effects. Examination of its mechanism and effects may therefore reveal a novel target for the treatment of lupus.
Acknowledgements This study was approved by the Colorado Multiple Institutional Review Board (Protocol 06–0501).
Funding for this work was provided by:
NIH R01 AI070983
NIH K24 AI078004
NIH T32 AR007534
University of Colorado School of Medicine
University of Colorado Department of Medicine
Charley J. Smyth Endowed Chair for Rheumatology Research
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