Exposure to certain environmental factors within genetically predisposed individuals is thought to be an underlying cause of the development of rheumatoid arthritis (RA), a complex autoimmune disease affecting ∼1% of the adult population (1). Epidemiologic research has suggested that cigarette smoking is a strong environmental risk factor for RA (2–5). Evidence of dose effects for both smoking and the gene–environment effects of smoking have been demonstrated within the Nurses' Health Study (NHS) (2, 6). Genetic variants associated with an increased risk of RA within the HLA complex have been known for decades (7, 8), and numerous studies have shown strong gene–environment interactions between HLA and smoking (3, 6, 9–12).
Glutathione S-transferase (GST) genes are a widely expressed supergene family encoding biotransforming enzymes that catalyze the conjugation of glutathione and are involved in the detoxification of cytotoxic carcinogens and metabolites. GST substrates found in cigarette smoke include α- and β-unsaturated carbonyls, polycyclic aromatic hydrocarbons, and reactive oxygen species (ROS), which may lead to cellular damage through oxidative stress. Variations in these genes reduce glutathione conjugation, and therefore may increase susceptibility to the harmful effects of carcinogen exposure and oxidative stress (13–19).
Polymorphisms within the Mu (GSTM1), Theta (GSTT1), and Pi (GSTP1) classes of GST have been identified. Individuals with homozygous deletions at the M1 and T1 loci of GST (GSTM1-null and GSTT1-null, respectively) have no functional enzymatic activity (13–19). In GSTP1, an A-to-G single-nucleotide polymorphism (SNP) may result in reduced enzymatic activity for the AG and GG genotypes when compared with the AA genotype (18, 20, 21).
Numerous studies have examined the possible associations between GST polymorphisms and disease risk or disease-phenotype determination. It has been hypothesized that GST variations are associated with susceptibility to various cancers (22, 23), most notably lung cancer (17–19). Previous studies have shown relationships between the GSTT1-null or GSTM1 polymorphisms and RA risk and severity, but results have been inconsistent with regard to possible interactions between the GST variants and smoking (3, 24–27). Significant GST–smoking interactions have been reported (3, 16, 17, 22), suggesting that underlying environmental exposures play an important role in the effect of GST genotypes.
Heme oxygenase 1 (HO-1), the inducible form of heme oxygenase, catabolizes heme groups into biliverdin, free iron, and carbon monoxide. Heme oxygenase has been shown to have antioxidant, antiinflammatory, and cytoprotective properties and to be up-regulated under the presence of nicotine (28–32). An A-to-T SNP that may affect the extent of the HO-1 production response has been identified; subjects with the TA or TT genotypes have lower HO-1 expression when compared with those with the AA genotype (31). Previous studies have examined the associations between HMOX1 and several diseases (31, 32), including lung cancer (33). Recent studies have identified a role for HMOX1 enzymes in RA biology (34) and have shown associations between HMOX1 polymorphisms and RA susceptibility and severity (35, 36).
Our present study focuses on 3 genetic polymorphisms within the GSTM1-null, GSTT1-null, and GSTP1 (rs1695) classes and 1 polymorphism within the HMOX1 gene promoter (rs2071716). We hypothesized that significant gene–environment interactions exist between these genetic variations and heavy smoking. We studied the effect of these interactions on the risk of all RA and the risk of RA serologic phenotypes. We further hypothesized that the interactions would be stronger in relation to the risk of seropositive RA.
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Interest in the associations of GST and HMOX1 genes with RA risk stems from the role of the genes in the detoxification of carcinogens in cigarette smoke and in the protection against oxidative stress caused by ROS. This nested case–control study demonstrated gene–environment interactions for both GSTT1-null and HMOX1 risk alleles with heavy smoking (>10 pack-years) in the NHS cohort, although only the interactions between GSTT1-null and heavy smoking remained significant after correction for multiple comparisons. These interactions were strongest in the risk of seropositive RA. The effect of interactions between GSTT1-null and heavy smoking on the risk of seropositive RA was replicated in an independent Swedish case–control sample. In both cohorts, no significant interactions were observed between the GSTM1-null or GSTP1 alleles and heavy smoking. Moreover, we observed no significant relationships with the risk of seronegative RA.
To the best of our knowledge, this is the first study to examine and identify a convincing interaction between the GSTT1-null polymorphism and heavy smoking in the risk of seropositive RA, with replication in an independent cohort. A previous study by Bohanec Grabar et al, in which only RA cases were assessed, identified a significant interaction between GSTT1-null and smoking in relation to disease activity as the RA phenotype, observing an OR for high disease activity of 8.64 (95% CI 2.00 to 37.43) in smokers with GSTT1-null compared with smokers with GSTT1-present (24). We observed that the interaction between GSTT1-null and heavy smoking led to an increased risk for developing RA, with stronger effects on seropositive RA. We did not study the phenotype of RA disease activity. Our study showed that among subjects with the GSTT1-null polymorphism, heavy smokers were at a 3.1-times increased risk of developing RA compared with never or light smokers. This increase in risk was even greater for seropositive RA, in which we observed a 4.3-times increased risk among the subjects with GSTT1-null.
A similar association was seen in our replication analysis, in which we observed a 3.5-times increased risk of ACPA-positive RA among individuals with GSTT1-null who were heavy smokers. When we examined the effect of GSTT1-null among groups defined by smoking levels of ≤10, 10–20, and >20 pack-years, to determine whether this effect varies for even higher levels of smoking, we obtained similar results for those with a history of 10–20 pack-years and those with a history of >20 pack-years of smoking (results not shown). Our observation of modest interactions between GSTT1-null and ever smoking in the NHS cohort but no significant interaction in the EIRA cohort supports the importance of considering the dose effect of smoking in analyses of interactions (2, 6).
The effect of interaction between GSTT1-null and heavy smoking on RA risk may be due to the lack of enzymatic activity associated with the GSTT1-null polymorphism, which may decrease the detoxification of certain cytotoxic carcinogens and metabolites found in cigarette smoke and subsequently increase the harmful effects of heavy smoking, a proven risk factor for RA (2–5). These results establish the importance of considering genetic background when studying the effect of environmental risk factors.
Despite the similar functions between GST genes, we did not observe significant interactions between GSTM1-null or GSTP1 and heavy smoking in the risk of RA, as we had observed with GSTT1-null. These differing results may be due to the differences in catalytic activity between the GST classes. The GST-Theta class has a lower affinity toward glutathione conjugates, leading to less product inhibition and thus higher catalytic efficiency, and has an ∼10 times higher enzymatic activity rate when compared with the GST-Mu and GST-Pi classes (13, 19, 47). There are also established differences in specific substrates for GSTT1, GSTM1, and GSTP1 and their roles in detoxification and toxification (13–16).
Results of previous studies on interactions between GSTM1 polymorphisms and smoking in RA have been inconsistent (3, 24, 26). Mattey et al observed that ever smokers with the GSTM1-null polymorphism were at higher risk for a more severe disease outcome, but Bohanec Grabar et al did not observe a significant interaction between GSTM1 and ever smoking in relation to RA disease activity (24, 26). Criswell et al observed a significant interaction between GSTM1 and exposure to tobacco smoke in the risk of RA (OR 2.10, 95% CI 1.13 to 3.89), which suggests that smoking is a stronger risk factor among individuals with GSTM1 present (3). After correction for multiple comparisons, we observed no significant associations with GSTM1 in the risk of RA.
This is also the first study to examine the effect of possible interactions between HMOX1 and heavy smoking on RA risk. We observed interactions between HMOX1 and heavy smoking for both the risk of all RA and the risk of seropositive RA in the NHS sample. Our results suggest that among individuals carrying at least 1 HMOX1 risk allele, heavy smoking was associated with a 1.9-times increased risk of all RA and a 2.3-times increased risk of seropositive RA, compared with never or light smokers. However, this result was not significant after correction for multiple comparisons and we did not observe significant associations in the replication analyses in the EIRA cohort. More research is needed to determine the validity of this association.
A number of studies have shown gene–environment interactions between HLA and smoking in the risk of seropositive RA (3, 6, 9–12). Thus, it is important to consider the impact that HLA may have on our results. We examined possible gene–gene interactions between HLA and our polymorphisms of interest and found no significant associations (results not shown).
Limitations of this study include the inability to determine heterozygous deletions for GSTM1 and GSTT1 polymorphisms, and the lack of information on the anti-CCP status in the NHS cohort. The fact that data on the anti-CCP status were lacking is due to the absence of plasma samples in almost half of our patients with RA, and the fact that many of the cases were diagnosed prior to the widespread use of the anti-CCP test. However, RF status was available from medical record reviews, and other gene–environment interactions are similar for RF- and anti-CCP–seropositive phenotypes in RA (9, 10). Our rates of seropositive RA in this study (59% anti-CCP+ and/or RF+) are similar to those reported in a large US registry study that recruited patients from rheumatology practices across the US (48). Furthermore, we only had data on incident RA, and therefore could not study disease severity phenotypes.
The NHS cohorts are comprised primarily of middle-to-older–age Caucasian women with high education levels. This lack of diversity in the population may raise concerns about the generalizability of our results, and these interactions should be studied in other cohorts. However, the restriction of our genetic analyses to Caucasian women limits the potential for population stratification, and thus may also be viewed as a strength. While limiting our analyses to self-reported Caucasian ancestry does not remove all concerns about population stratification, prior research has examined the potential for this bias extensively within the NHS and found no evidence of significant population stratification (49, 50).
Other strengths of this study include the prospective nature of the information collected, including detailed information on smoking status, and the access to a large, independent case–control sample for replication. Our sample size makes this one of the largest studies examining the effects of GST and HMOX1 genes on RA risk. Despite this large sample, the power to detect a significant interaction was still limited. Based on the observed ORs for the association of RA risk with specific genes (OR 1.1) and heavy smoking (OR 1.8), we had, at most, 24% power and 40% power to detect a significant gene × environment interaction (OR of 1.5) in the NHS and EIRA study, respectively. This should be considered when interpreting negative results.
In summary, we observed significant multiplicative and strong additive interactions between the GSTT1-null polymorphism and heavy smoking, with the strongest associations seen in the risk of seropositive RA. In replication analyses, we found that the interaction between GSTT1-null and heavy smoking that was observed in relation to the risk of seropositive RA could be replicated in a study assessing the risk of ACPA-positive RA. This suggests that we have identified a truly novel association with the risk of seropositive RA. Although we observed significant interactions between the HMOX1 risk alleles and heavy smoking in the risk of RA in the NHS, these results could not be replicated. No significant interactions were seen in relation to the risk of seronegative RA, supporting the hypothesis that different risk factors and different pathways may exist between the seropositive and seronegative RA phenotypes.
Our results add new evidence to the hypothesis that gene–environment interactions play a significant role in the complex etiology of RA. Additional research is needed to examine the validity of these interactions and to study the potential biologic pathways involved. Future studies should focus on how to incorporate these findings, in addition to the findings on other risk factors previously identified, into larger models that can be used for RA prediction.
- Top of page
- 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. Mr. Keenan 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. Keenan, Chibnik, Bengtsson, Klareskog, Alfredsson, Karlson.
Acquisition of data. Keenan, Chibnik, Cui, Ding, Padyukov, Kallberg, Bengtsson, Alfredsson, Karlson.
Analysis and interpretation of data. Keenan, Chibnik, Cui, Ding, Padyukov, Kallberg, Klareskog, Alfredsson, Karlson.