Although the role of RANKL
in RA has been well established, little is known about the contribution of its genetic variations to RA onset. We previously observed a strong association between the combined presence of HLA-DRB1*04
polymorphisms with a younger age of RA onset in 182 RF+ EA early RA WCPR patients (21
). In this extended study of 210 RF+ RA WCPR patients, we fine mapped the RANKL
locus and observed association of the minor allele of 4 SNPs (rs5803141, rs7984870, rs9525641 and rs1054016) with younger age at RA onset. Subsequent replication studies of these 4 SNPs showed association of a single SNP in the promoter, the minor C allele of rs7984870, with younger age of RA onset in two independent cohorts (ACPA+ EA BRASS and AA CLEAR). These two independent RA cohorts were early RA (<24 months after symptom onset) long-term observational cohorts, and have an accurately documented date of RA onset similar to WCPR cohort.
In both the previous (21
) and extended WCPR cohort, the association between rs9525641 and younger age of RA onset was present; however, the formerly observed RA-associated rs922996 was not confirmed. Recently, a RANKL
intron 1 rs2277438 has been associated with RA in European population (534 cases and 516 controls) (35
). This SNP was also tested in our WCPR EA cohort, but had weak LD with promoter SNP rs7984870 (r2
= 0.17) and did not show the association with age of disease onset. Possible explanations for the inability to replicate RANKL
SNPs (rs5803141, rs9525641, rs922996, rs1054016 and rs2277438) in some of the studied cohorts include genetic, ethnic, and clinical heterogeneity across study populations as well as the probability that these SNPs may be linked to the actual RANKL
causal allele. Several genome-wide association studies (GWAS) in RA have identified and replicated more than 20 risk loci (2
), but have not included the RA-associated RANKL
SNP described here. Given that the association with younger age of RA onset was only observed in CC homozygotes, we used a recessive genetic model to estimate the power of GWAS to capture this risk allele under the assumption of relative risk of 1.3 (36
). A sample size of 10,000 cases and 10,000 controls could provide 65% power to reach a p value of 10−7
, which might explain why this SNP was not identified in previous GWAS in RA (2
). However, the consistent presence of the observed genetic association in 3 independent panels and the functional properties of the rs7984870 allele in transcription regulation of RANKL
in our study make this promoter polymorphism an excellent candidate for a causal variant contributing to younger age of RA onset.
SE is a recognized genetic risk predisposing to RA onset and serves as a binding site for arthritogenic peptides, allowing their presentation to CD4+ T cells. Similar to our previous report, the co-occurrence of rs7984870 risk allele and SE displayed the earliest age of RA onset in RF+ or ACPA+ RA patients. One possible hypothesis is that co-occurrence of RANKL polymorphisms and SE contributed to younger RA onset in ACPA+/RF+ patients by causing abnormally stimulated RANKL expression, which dysregulates dendritic/T cell communication and breaks the immune tolerance to self-components in the joint, thereby enhancing T-cell reactivity towards arthritogenic antigens.
A growing number of genetic variants have been reported to contribute to ACPA+ RA (6
). In our replication studies performed in 298 AA and 501 additional EA patients, the significant genetic association of rs7984870 with earlier RA onset was only shown in the ACPA+ patients but not in ACPA− RA, suggesting that the RANKL
allele might be a novel disease predisposing variant in ACPA+ RA subset. Alternatively, the lack of a significant association might be attributed to the modest sample size of seronegative RA patients used in these studies.
Our study provides evidence that risk C allele of rs7984870 conferred a 2-fold higher plasma soluble RANKL level in RF+ RA patients and significantly elevated RANKL
isoform 1 mRNAs expression by activated control T cells, as well as increased promoter activity after cytokine stimulation in vitro
. Over expression of RANKL
results in functional alteration of epidermal DCs (38
) and enhances survival of DCs (13
). Transfer of RANKL-stimulated MRL/lpr DCs pulsed with collagen into MRL/lpr mice have been shown acceleration autoreactivity of T and B cells and enhance the productions of pathogenic cytokines in the recipients (13
). The minor C allele of rs7984870 contributed to higher RANKL
expression and protein level upon activation and thus, might affect initiation of immune responses between DCs and T cells. In addition, immunoreceptor tyrosine-based activation motif (ITAM) associated receptor networks have been implicated in providing co-stimulation for RANKL signaling (39
); thus, another possible explanation is that genetic variants causing differences in RANK
L expression could therefore result in abnormal cross-talk in signaling networks and alteration of cellular responses to various extracellular stimuli.
An important issue is how this RANKL
variant affects the expression of RANKL
. Our data suggests that the minor C allele of rs7984870 creates a binding site for transcription factor SOX5, a member of the SOX family, which might explain observed alterations in RANKL
transcription. SOX family have been primarily characterized as important transcriptional regulators involved in determination of cell fate and tissue specification during a number of developmental processes (40
). Elevated SOX5
transcripts have been related to invasive growth and reduced apoptosis in tumors and have been associated with progression of certain cancers (41
). Recently, SOX6
, another member of the SOX family, which shares 91% amino acid homology in the leucine-zipper motif with SOX5, was identified as a low bone mineral density locus in a large-scale meta-analysis of GWAS studies (44
). Taken together, the differential binding of the minor C allele of rs7984870 to SOX5
transcriptional regulator might influence bone metabolism, and facilitate invasive growth and resistance to apoptosis in the synovium, leading to RA manifestations.
In summary, the association of CC genotype of rs7984870 with younger RA onset was identified and replicated in several independent cohorts in this study. This novel genetic risk factor, RANKL rs7984870 CC genotype, conferred elevated promoter activity after stimulation by cytokines, potentially via binding to transcription factor SOX5. Elevated inducible RANKL mRNA and protein levels might result in enhanced interaction between activated T cells presenting arthritogenic peptides to DCs, predisposing to earlier development of seropositive (RF+ or ACPA+) RA in EA and AA patients. Our evidence suggests that the combination of CC genotype of rs7984870 RANKL polymorphism, SE, and abnormal humoral immune responses interacts to permit an earlier onset of RA.