Arthritis & Rheumatism, Volume 60,
October 2009 Abstract Supplement
The 2009 ACR/ARHP Annual Scientific Meeting
Philadelphia October 16-21, 2009.
A Functional RANKL Promoter SNP Associated with Younger Age at Onset of Rheumatoid Arthritis
Tan1, W., Wu1, H., Derber2, L. A., Lee3, D. M., Shadick3, N. A., Smith4, E. A., Gersuk5, V. H.
UCLA, LA, CA
UAB, Birmingham, AL
David Geffen School of Medicine at UCLA, LA, CA
U Colorado Denver, Aurora, CO
Brigham and Women's Hosp, Boston, MA
MUSC, Charleston, SC
Benaroya Rsch Ini., Seattle, WA
U Pittsburgh, Pittsburgh, PA
Children's Hosp, Cincinnati, OH
UNC-Chapel Hill, Chapel Hill, NC
U Colo Denver, Aurora, CO
We previously reported the association of co-occurrence of HLA-DRB1 shared epitope (SE) and RANKL SNPs with younger at onset of RA in 182 RF+ European American (EA) early RA patients, which prompted us to fine map RANKL causal SNPs and study additional cohorts: 298 African-Americans (AA) RA (RF+= 211, CCP+= 172), 501 EA RA (RF+= 317, CCP+= 344) and 80 RF+ EA JIA.
SNPs were genotyped using TaqMan or pyrosequencing. RANKL expression levels in plasma, PBMC and isolated T cells were quantified using ELISA and RT-PCR. In vitro site-directed mutagenesis of rs7984870 within the 2kb RANKL promoter was performed to drive the luciferase reporter gene in transfected cells.
Fine-mapping a 64-kb RANKL region using 19 SNPs in an extended 210 EA RF+ early RA patients showed association of minor allele homozygotes of 4 RANKL SNPs (rs5803141, rs7984870 and rs9525641 in the promoter region, and rs1054016 in the 3'UTR) with 5 yrs earlier mean age at RA onset (p < 0.05). The minor allele homozygote frequency for those 4 SNPs had increasing proportions in EA normals (1011%), RF+ RA (1820%), and RF+ EA polyarticular JIA (2326%) patients (p = 0.02-0.009). Among those, rs7984870 CC genotype exhibited the strongest statistical significance (p = 0.009). This association was confirmed in 172 anti-CCP+ AA patients (7 and 5 yrs earlier for rs5803141 and rs7984870, respectively, p < 0.05), and replicated in an independent 344 anti-CCP+ EA early RA patients (4 yrs earlier for rs7984870, p < 0.05). Co-occurrence of rs7984870 CC genotype and HLA-DRB1*04 showed a strong younger age (mean 9.2 yrs earlier) at onset in RF+ and anti-CCP+ subgroups of both EA and AA patients (p = 0.01-0.00003). Plasma levels of RANKL were 2-fold higher in RF+ RA patients carrying rs7984870 CC vs. GG (10 vs. 13, p < 0.001) but not in normals (5 vs. 19). Levels of RANKL mRNA in normal PBMCs (17 vs. 21) or T cells (4 vs. 4) were not different in those carrying either homozygous rs7984870 genotypes. However, after 48h incubation with IL-2, significantly elevated RANKL mRNAs were observed in normal control T cells carrying the rs798470 CC genotype compared to those carrying the GG genotype (4 vs. 4, p < 0.05). Transfection of the rs7984870 CC RANKL promoter displayed higher transcription activity than the GG genotype in both stromal and osteoblast cell lines only after stimulation with b-FGF or TNFa (p < 0.05).
Association of CC genotype of rs7984870 with younger RA onset has been replicated in several independent cohorts, and confers an elevated promoter activity after stimulation. Elevated inducible RANKL mRNA and protein levels may predispose to earlier development of seropositive (RF+ or anti-CCP+) RA in EA and AA patients.
To cite this abstract, please use the following information:
Tan, W., Wu, H., Derber, L. A., Lee, D. M., Shadick, N. A., Smith, E. A., et al; A Functional RANKL Promoter SNP Associated with Younger Age at Onset of Rheumatoid Arthritis [abstract]. Arthritis Rheum 2009;60 Suppl 10 :380