Recent progress in genetic studies of rheumatoid arthritis (RA) has revealed several new loci as risk factors for disease development (1–7). However, all newly found variations outside the HLA locus confer only limited, although statistically significant, increased risk of RA. The strongest association with anti–citrullinated protein antibody (ACPA)–positive RA was repeatedly reported for the HLA–DRB1 gene, and it is evident that this genetic locus plays a central role in susceptibility to disease in different Caucasian populations. RA is a complex disease with many different factors, and it is rational to discern which of the combinations of these factors results in the most aggressive form of the disease. Our own and other previous studies demonstrated an unexpected high increase in risk associated with exposure to smoking in the presence of shared epitope (SE) alleles of the HLA–DRB1 gene, with regard to susceptibility to ACPA-positive and/or rheumatoid factor–positive RA, which we considered strong evidence for an interaction (8–16).
According to the current state of knowledge, the association between the HLA–DRB1 variations and susceptibility to ACPA-positive RA is related to more than 1 allele (*0101, *0401, *0404, *0405, *0408, *1001, and *1402). These alleles share a common amino acid sequence (70QRRAA74, 70RRRAA74, or 70QKRAA74) in the third hypervariable region of the DRB1 molecule and have therefore been denoted SE alleles (17–20). The SE residues constitute a part of the antigen-binding site forming the fourth anchoring pocket in the HLA groove. The epitope motif hypothetically serves as a binding site for arthritogenic peptides, allowing presentation to CD4+ T cells and generation of T cell autoimmune responses, and may possibly induce certain B cells to differentiate into plasma cells, leading to the production of ACPAs (15).
ACPAs occur in ∼60% of RA patients and in 2% of healthy populations and are rather rare in patients with other inflammatory diseases (15, 21). The occurrence of ACPAs is observed several years before onset of disease (22) and is closely linked to the presence of SE alleles. More specifically, the association of RA with the SE, which is the strongest genetic risk factor for the disease, is observed exclusively within the ACPA-positive patient subset (8, 9, 15).
Several environmental factors that appear to predispose to or protect against development of RA have been described, but findings have been ambiguous (16, 23–27). However, the main environmental risk factor for RA detected to date is smoking (8, 13). A strong gene–environment interaction between tobacco exposure and the SE in the ACPA-positive subset of patients has been repeatedly demonstrated in several studies in Europe (8, 10–13), whereas neither smoking nor the SE confers an increased risk of ACPA-negative RA. However, when replication of the demonstrated gene–environment interaction was assessed in 3 North American cohorts by Lee et al (28), evidence of a gene–environment interaction between smoking and SE alleles for ACPA formation could be observed in only 1 of those cohorts. This discrepancy could possibly be explained by different procedures for recruiting controls and patients, diverse methodologies for evaluation of smoking, and the existence of different sorts of environmental exposure. In a recent study, van der Helm-van Mil et al (8) performed gene–environment analyses stratifying for the *01, *04, and *10 groups in an investigation of 421 patients, with ACPA-negative patients as controls. Interestingly, through use of a multiplicative model, they observed an interaction between tobacco exposure and DRB1*01 and *10 in ACPA-positive RA, but no interaction was evident for the *04 alleles.
We undertook a large population-based, case–control study to scrutinize the gene–environment interaction between smoking and SE alleles in RA. The goal of our investigation was to ascertain whether all HLA–DRB1 SE alleles (HLA–DRB1*01, HLA–DRB1*04, HLA–DRB1*10) present a similar interaction effect or whether the interaction is restricted to a particular DRB1 SE group. In addition, we assessed the relevance of studying the different subtypes of the *04 group. More specifically, we focused on *0401, *0404, *0405, and *0408 (i.e., the “true” SE alleles in the *04 group). Hence, from this population-based study, we report findings from analyses of the *01, *10, and *04 groups separately, as well as subtypes of the *04 group, in the context of smoking and ACPA status in RA.
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Our data illustrate that regardless of the fine specificity of the SE alleles of DRB1, there is an interaction between these genetic risk factors and smoking. When comparing the RR between ever smokers and carriers of any SE allele (i.e., single or double SE allele) for the DRB1*01/*10 group (RR 4.9 [95% CI 3.0–7.8]) and the DRB1*04 group (RR 8.7 [95% CI 5.7–13.1]), the latter group has a higher relative risk, although this difference is not statistically significant.
This study was designed to address whether an interaction between smoking and SE alleles, as previously observed, occurs with all HLA–DRB1 SE alleles (HLA–DRB1*01, HLA–DRB1*04, HLA–DRB1*10) or whether it is restricted to any particular DRB1 group (35–37). In a first step we separately analyzed 2 groups of SE alleles, DRB1*01/*10 and DRB1*04 (instead of only 1 combined group, which previously also included a few individuals with unidentified DRB1*04 non-SE alleles [*0402 and *0403]). We subsequently specifically focused on *0401 and *0404 alleles, which are the most common alleles within the DRB1*04 group.
The observed independent effects of smoking and SE alleles in our study are concordant with previously reported results indicating that smoking and SE are primarily associated with ACPA-positive RA (10–13). However, conclusions from another study in which ACPA-positive RA patients were compared with ACPA-negative RA patients (without healthy controls) were somewhat different, and interaction of DRB1*04 alleles with smoking was not demonstrated (8). This discrepancy may be due to differences in study design and in the ways of assessing interaction. Our study is based on a case–control cohort of relatively large size, and we believe that it might represent a better estimate of independent as well as combined influences from genetic and environmental risk factors.
As an attempt to measure the interaction between smoking and SE alleles, we used the attributable proportion due to interaction and demonstrated significant gene–environment interactions for both single and double SE alleles in ACPA-positive disease. Interestingly, we observed an increased relative risk with any of the SE allele groups, DRB1*01/*10 and DRB1*04. However, the relative risk was highest for carriers of double DRB1*0401 and/or *0404 alleles (RR 39.6 [95% CI 18.6–84.4]; attributable proportion due to interaction 0.8 [95% CI 0.6–0.98]). It was previously reported that the DRB1*01 allelic group confers less risk of developing ACPA-positive RA in comparison with the DRB1*04 allelic group (8), as also observed in our study (Tables 2–4 and Figure 1). Although the different SE alleles are associated with different magnitudes of increased risk of ACPA-positive RA, their interaction with smoking seems to be similar, as indicated by the magnitude of the values for attributable proportion due to interaction (Tables 2–4).
The molecular mechanisms underlying the observed risk and interaction concerning smoking and SE alleles are still unknown, but some speculations have been published. One hypothesis is that long-term exposure to cigarette smoke may induce mechanisms that accelerate deimination of arginine to citrulline in autoantigens present in the lungs. Since citrullination increases the binding of modified peptides to antigens containing the SE motif and thereby enhances the immunogenicity of the proteins, a break of tolerance toward citrullinated proteins might be induced in those individuals carrying the SE alleles (15, 38). Another possibility concerns the presence of substances in smoke which might act as adjuvants, triggering the innate immune system to contribute to development of arthritis, similar to what has been reported in animal models of adjuvant-induced arthritis (39, 40). However, the possibility still remains that the HLA–DRB1 gene involvement in the gene–environment interaction is not primary, but rather is dependent on another genetic factor in linkage disequilibrium in this locus, such as variations in HLA–DQ (41, 42). Finally, we cannot exclude the possibility of a pure genetic interaction between the HLA–DRB1 gene and the putative gene involved in the behavioral trait, which includes smoking.
Taking advantage of the availability of data from a large population-based, case–control study of RA, we also reinvestigated the possibility of an independent effect of SE alleles or of an interaction between SE alleles and smoking in the development of ACPA-negative RA. We conclude that the SE alleles do not seem to confer an increased risk of ACPA-negative RA, either on their own or in combination with smoking. In conclusion, we have demonstrated that regardless of fine specificity, all SE DRB1 alleles strongly interact with smoking in the development of ACPA-positive RA.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Ms Lundström had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Lundström, Alfredsson, Klareskog, Padyukov.
Acquisition of data. Lundström, Alfredsson, Padyukov.
Analysis and interpretation of data. Lundström, Källberg, Alfredsson, Klareskog, Padyukov.