A functional RANKL polymorphism associated with younger age at onset of rheumatoid arthritis.
OBJECTIVE
We previously observed the association of the co-occurrence of the HLA-DRB1 shared epitope (SE) and RANKL single-nucleotide polymorphisms (SNPs) with younger age at the onset of rheumatoid arthritis (RA) in 182 rheumatoid factor (RF)-positive European American patients with early-onset RA. The aim of this study was to fine-map the 48-kb RANKL region in the extended cohort of 210 European American RF-positive patients with early RA, to seek replication of RA-associated SNPs in additional RA cohorts of 501 European Americans and 298 African Americans, and to explore the functional consequences of RA-associated SNPs.


METHODS
SNP genotyping was conducted using pyrosequencing or TaqMan polymerase chain reaction (PCR) assays. Associations of rs7984870 with RANKL expression in plasma, peripheral blood mononuclear cells, and isolated T cells were quantified using enzyme-linked immunosorbent assay and reverse transcription-PCR. Site-directed mutagenesis of rs7984870 within the 2-kb RANKL promoter was performed to drive the luciferase reporter gene in osteoblast and stromal cell lines. Interaction of DNA and protein was determined by electrophoretic mobility shift assay.


RESULTS
A single promoter SNP, rs7984870, was consistently significantly associated with earlier age at the onset of RA in 3 independent seropositive (RF or anti-cyclic citrullinated peptide antibody) RA cohorts but not in seronegative RA patients. The C risk allele of rs7984870 conferred 2-fold higher plasma RANKL levels in RF-positive patients with RA, significantly elevated RANKL messenger RNA expression in activated normal T cells, and increased promoter activity after stimulation in vitro via differential binding to the transcription factor SOX5.


CONCLUSIONS
The RANKL promoter allele that increased transcription levels upon stimulation might promote interaction between activated T cells and dendritic cells, predisposing to a younger age at the onset of RA in seropositive European American and African American patients.
Tan
Wenfeng
W
David Geffen School of Medicine at the University of California, Los Angeles, USA.
Wu
Hui
H
Zhao
Jian
J
Derber
Lezlie A
LA
Lee
David M
DM
Shadick
Nancy A
NA
Conn
Doyt L
DL
Smith
Edwin A
EA
Gersuk
Vivian H
VH
Nepom
Gerald T
GT
Moreland
Larry W
LW
Furst
Daniel E
DE
Thompson
Susan D
SD
Jonas
Beth L
BL
Holers
V Michael
VM
Glass
David N
DN
Chen
Pojen P
PP
Bridges
S Louis
SL
Weinblatt
Michael E
ME
Paulus
Harold E
HE
Tsao
Betty P
BP
D000328:Adult; D001741:Black or African American / Q000235:genetics; D017668:Age of Onset; D001172:Arthritis, Rheumatoid / Q000208:ethnology / Q000235:genetics*; D005260:Female; D020022:Genetic Predisposition to Disease / Q000208:ethnology / Q000235:genetics; D005838:Genotype; D006801:Humans; D008297:Male; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic / Q000235:genetics*; D053245:RANK Ligand / Q000097:blood / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D055755:SOXD Transcription Factors / Q000502:physiology; D044465:White People / Q000235:genetics
D053245:RANK Ligand; D012333:RNA, Messenger; C490301:SOX5 protein, human; D055755:SOXD Transcription Factors; C505688:TNFSF11 protein, human