SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

An interaction effect for developing rheumatoid arthritis (RA) was previously observed between HLA–DRB1 shared epitope (SE) alleles and smoking. We aimed to further investigate this interaction between distinct SE alleles and smoking regarding the risk of developing RA with and without anti–citrullinated protein antibodies (ACPAs).

Methods

We used data regarding smoking habits and HLA–DRB1 genotypes from 1,319 patients and 943 controls from the Epidemiological Investigation of Rheumatoid Arthritis, in which 972 patients and 488 controls were SE positive. Subsequently, 759 patients and 328 controls were subtyped for specific alleles within the DRB1*04 group. Odds ratios with 95% confidence intervals (95% CIs) were calculated by means of logistic regression. Interaction was evaluated by calculating attributable proportion due to interaction, with 95% CIs.

Results

A strong interaction between smoking and SE alleles in the development of ACPA-positive RA was observed for all DRB1*04 SE alleles taken as a group (relative risk [RR] 8.7 [95% CI 5.7–13.1]) and for the *0401 and *0404 alleles (RR 8.9 [95% CI 5.8–13.5]) and the *01 and *10 alleles (RR 4.9 [95% CI 3.0–7.8]) as specific, separate groups, with similar strength of interaction for the different groups (attributable proportion due to interaction 0.4 [95% CI 0.2–0.6], 0.5 [95% CI 0.3–0.7], and 0.6 [95% CI 0.4–0.8], respectively).

Conclusion

There is a statistically significant interaction between distinct DRB1 SE alleles and smoking in the development of ACPA-positive RA. Interaction occurs with the *04 group as well as the *01/*10 group, demonstrating that regardless of fine specificity, all SE alleles strongly interact with smoking in conferring an increased risk of ACPA-positive RA.

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.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Study population.

This work is derived from a population-based, case–control study titled the Epidemiological Investigation of Rheumatoid Arthritis (EIRA). Individuals for whom information was available regarding smoking habits, ACPA status, and SE status were included in the study (1,319 patients and 943 controls), and all subjects were Caucasian. Of these 1,319 patients and 943 controls, 972 patients and 488 controls were SE positive. Subsequently, 759 patients and 328 controls were subtyped for specific alleles within the DRB1*04 group. The current report is based on incident cases of RA from different parts of Sweden recruited between May 1996 and December 2005. A person who fulfilled the 1987 revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (29) and who had never had a previous RA diagnosis was defined as a case. For each potential case, a control subject was randomly selected from the Swedish national population registry, taking into consideration the subject's age, sex, and residential area. The EIRA study design, including identification of patients and controls, data collection, and definition of smoking habits, has been described elsewhere (30). The Ethics Committee of Karolinska Institutet approved the study. Distribution of age, sex, and ACPA status is depicted in Table 1.

Table 1. Baseline characteristics of the cases and controls*
 Controls (n = 943)Cases (n = 1,319)
  • *

    ACPA = anti–citrullinated protein antibody.

Age, mean ± SD years52.1 ± 11.851.1 ± 12.4
Women, %72.471.5
ACPA-positive, %1.960.7

Definition of smoking status.

Cases and controls were classified as “ever smokers” or “never smokers” according to their reported smoking habits. Briefly, subjects who reported that they regularly smoked cigarettes during or before the year they were included in the EIRA study were defined as ever smokers, and those who reported that they had never smoked tobacco before or during the inclusion year were defined as never smokers.

Genetic analysis.

Two-digit and 4-digit HLA–DRB1 typing was conducted using sequence-specific primer–polymerase chain reaction (PCR) (DR low-resolution kit [2-digit], DRB1*04 subtyping kit [4-digit]; Olerup SSP, Saltsjöbaden, Sweden), and the PCR products were loaded onto 2% agarose gels. An interpretation table was used to determine the specific genotype according to the manufacturer's instructions.

Statistical analysis.

Using unconditional logistic regression models, we calculated odds ratios (ORs) together with 95% confidence intervals (95% CIs) for RA associated with combinations of different SE alleles and smoking. As a first step we analyzed the DRB1*04 SE alleles (i.e., the combination of any of the subtypes *0401, *0404, *0405, and *0408 as a risk factor). In the following analyses we excluded the more rare *0405 and *0408 subtypes and only considered the *0401 allele and/or the *0404 allele as risk factors. Finally, we studied the interaction between HLA–DRB1*01 and/or *10 and smoking, since there were too few carriers of HLA–DRB1*10 to perform analyses with sufficient power. When the effect of different HLA–DRB1 SE alleles was estimated, individuals without HLA–DRB1 SE alleles of any type were used as the reference group.

ORs were interpreted as relative risks (RRs) due to the recruitment procedure. Interaction, defined by departure from additivity of effects as described by Rothman and others (termed “biologic interaction” by many investigators) (31–33), between different HLA–DRB1 SE alleles and smoking was evaluated by calculating the attributable proportion due to interaction together with a 95% CI (34). All analyses were performed using the software package SAS 9.1.3 (SAS Institute, Cary, NC).

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Interaction between smoking and HLA–DRB1*04 alleles in ACPA-positive RA.

Due to considerable difference in the frequency of different SE alleles, even some analyses for interaction in a relatively large study will lack power. For this reason, we first tested for interaction in individuals having any of the SE alleles from the DRB1*04 group. As shown in Table 2, we observed a strong interaction between smoking and the SE allele group *0401, *0404, *0405, or *0408 in individuals with at least 1 of these alleles, with regard to the risk of developing ACPA-positive RA. The interaction between HLA–DRB1*04 and smoking in ACPA-positive disease remained when the analysis was limited to only the *0401 and *0404 alleles (Table 3). The relative risk of disease associated with combinations of *0401/*0404 alleles and smoking was very high (for ever smokers with a single SE allele, RR 6.6 [95% CI 4.3–10.2]; for ever smokers with 2 SE alleles, RR 39.6 [95% CI 18.6–84.4]), as illustrated in Figure 1. The attributable proportion due to interaction between smoking and a single *0401 or *0404 allele was 0.4 (95% CI 0.2–0.6), and that between smoking and double alleles was 0.8 (95% CI 0.6–0.98), demonstrating a significant gene–environment interaction with involvement of the DRB1*04 group of alleles.

Table 2. Interaction between smoking and DRB1*04 alleles (*0401, *0404, *0405, *0408)*
SE allele status, smoking statusACPA-positive RAACPA-negative RA
No. of cases/no. of controlsRR (95% CI)No. of cases/no. of controlsRR (95% CI)
  • *

    Relative risk (RR) was adjusted for age, sex, and residential area.

  • The single *04 group was defined by the presence of a single HLA–DRB1*0401, *0404, *0405, or *0408 shared epitope (SE) allele. The double *04 group was defined by the presence of two *0401, *0404, *0405, and/or *0408 SE alleles. The any *04 group was defined by the presence of either one or two *0401, *0404, *0405, and/or *0408 SE alleles.

  • For those with anti–citrullinated protein antibody (ACPA)–positive rheumatoid arthritis (RA), the attributable proportion due to interaction was 0.37 (95% confidence interval [95% CI] 0.1–0.6) in the single *04 group, 0.72 (95% CI 0.5–0.95) in the double *04 group, and 0.4 (95% CI 0.2–0.6) in the any *04 group.

  • §

    Never smokers without HLA–DRB1*01, *04, or *10 SE alleles.

None    
 Never smokers38/154Referent§90/154Referent§
 Ever smokers82/2991.1 (0.7–1.7)137/2990.8 (0.6–1.1)
Single*04    
 Never smokers79/833.9 (2.4–6.3)49/831.0 (0.6–1.5)
 Ever smokers214/1456.4 (4.2–9.7)82/1451.0 (0.7–1.4)
Double*04    
 Never smokers34/158.7 (4.2–17.7)8/151.0 (0.4–2.4)
 Ever smokers102/1431.1 (15.8–61.0)10/141.4 (0.6–3.2)
Any*04    
 Never smokers115/984.8 (3.0–7.5)58/981.0 (0.7–1.5)
 Ever smokers322/1628.7 (5.7–13.1)94/1621.0 (0.7–1.5)
Table 3. Interaction between smoking and DRB1*04 alleles (*0401, *0404) in ACPA-positive RA*
SE allele status, smoking statusNo. of cases/no. of controlsRR (95% CI)
  • *

    RR was adjusted for age, sex, and residential area. See Table 2 for definitions.

  • The single *0401/*0404 group was defined by the presence of a single HLA–DRB1*0401 or *0404 SE allele. The double *0401/*0404 group was defined by the presence of two *0401 and/or *0404 SE alleles. The any *0401/*0404 group was defined by the presence of either one or two *0401 and/or *0404 SE alleles. The attributable proportion due to interaction was 0.4 (95% CI 0.2–0.6) in the single *0401/*0404 group, 0.8 (95% CI 0.6–0.98) in the double *0401/*0404 group, and 0.5 (95% CI 0.3–0.7) in the any *0401/*0404 group.

  • Never smokers without HLA–DRB1*01, *04, or *10 SE alleles.

None  
 Never smokers38/154Referent
 Ever smokers82/2991.1 (0.7–1.7)
Single*0401/*0404  
 Never smokers77/833.8 (2.4–6.2)
 Ever smokers218/1446.6 (4.3–10.2)
Double*0401/*0404  
 Never smokers30/139.0 (4.2–19.2)
 Ever smokers92/1039.6 (18.6–84.4)
Any*0401/*0404  
 Never smokers107/964.6 (2.9–7.2)
 Ever smokers309/1548.9 (5.8–13.5)
thumbnail image

Figure 1. Risk of anti–citrullinated protein antibody–positive rheumatoid arthritis in smokers and nonsmokers conferred by having 0, 1, or 2 copies of the shared epitope alleles HLA–DRB1*0401 and *0404. Values are relative risks (RRs) and 95% confidence intervals (95% CIs). The attributable proportion due to interaction between smoking and a single *0401 or *0404 allele was 0.4 (95% CI 0.2–0.6), and that between smoking and double alleles was 0.8 (95% CI 0.6–0.98).

Download figure to PowerPoint

Interaction between smoking and HLA–DRB1*01 and *10 alleles in ACPA-positive RA.

To test for interaction between smoking and non-DRB1*04 SE alleles, we performed additional analyses using nonsmoking, non–SE-positive individuals as the reference group. In addition, due to the design of the HLA typing assay, we were able to perform discrimination of the DRB1*0103 allele, which was considered to be a non-SE allele, within the *01 group. As is apparent from our data, the SE alleles HLA–DRB1*01 and *10 exhibited interaction with smoking in ACPA-positive RA (Table 4).

Table 4. Interaction between smoking and DRB1*01 and *10*
ACPA status, smoking statusNo SE alleleAny*01/*10 SE allele
No. of cases/no. of controlsRR (95% CI)No. of cases/no. of controlsRR (95% CI)
  • *

    RR was adjusted for age, sex, and residential area. See Table 2 for definitions.

  • Presence of either one or two HLA–DRB1*01 and/or *1001 SE alleles. For those with ACPA-positive RA, the attributable proportion due to interaction was 0.6 (95% CI 0.4–0.8).

  • Never smokers without HLA–DRB1*01, *04, or *10 SE alleles.

Positive    
 Never smokers38/154Referent27/671.8 (1.0–3.2)
 Ever smokers82/2991.2 (0.8–1.9)99/914.9 (3.0–7.8)
Negative    
 Never smokers90/154Referent37/670.9 (0.5–1.5)
 Ever smokers137/2990.8 (0.6–1.2)69/911.4 (0.9–2.2)

Interaction between smoking and HLA–DRB1 SE alleles in ACPA-negative RA.

Our data confirmed previous findings of no increased risk for smokers of developing ACPA-negative RA (Tables 2 and 4). No single group of the SE alleles (DRB1*01, *04, or *10) either conferred independent risk or showed interaction with smoking regarding the risk of developing ACPA-negative RA.

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We thank Associate Professor R. A. Harris for linguistic advice and Eva Jemseby and Gull-Britt Almgren for invaluable technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES