Rheumatoid arthritis (RA) is a systemic inflammatory disorder that is associated with an increased incidence of cardiovascular events (1–3) and increased mortality from cardiovascular disease (4), compared with that in the general population. Comorbidity and mortality in RA are associated with disease severity, and survival is particularly poor among patients with severe extraarticular disease manifestations (5, 6). It is therefore of major importance to identify specific prognostic factors that could influence the risk of developing systemic complications, and to elucidate the pathogenic mechanisms underlying the extraarticular manifestations of RA (ExRA).
Suggested predictors of ExRA include clinical, serologic, and genetic factors. The reported association between disease progression and HLA–DRB1*04 subtypes (7–9) and the occurrence of ExRA may be due to effects of these genes on the T cell repertoire (10–12). Studies of T cells in patients with ExRA have demonstrated clonal expansion of CD4+,CD28null T cells with unusual characteristics, including the expression of killer immunoglobulin-like receptors (KIRs) (13, 14). KIR molecules interact with class I major histocompatibility complex (MHC) molecules, which suggests a role for class I MHC gene products in the regulation of abnormal T cells in RA (15). Investigators in a case–control study of a limited sample reported that HLA–C*03 and *05 alleles were associated with vasculitis in patients with RA (15).
There is very limited information about the role of HLA–C in RA. A meta-analysis of studies of the Native American Pima and Yakima tribes, in which more than 90% of the population have the RA-associated shared epitope (SE) of HLA–DRB1 but fewer than 5% are clinically affected by RA, revealed that homozygosity for the HLA–C7 and C8 alleles was significantly more frequent in subjects in whom RA developed (16). In a recent study of a well-characterized early RA cohort, HLA–C7 was associated with extensive inflammation and a need for more aggressive treatment (17). Taken together, these results suggest that HLA–C alleles may influence disease susceptibility and/or disease outcome.
Smoking is a risk factor for RA (18) and has also been associated with a more severe disease phenotype (19, 20). It is well established that rheumatoid nodules are more likely to develop in smokers with RA (21), and results of several studies have suggested that smoking is an important predictor of severe ExRA (22, 23). It is unknown whether the relationship between smoking and ExRA is influenced by genetic background, including genes specifically associated with ExRA.
The purpose of the present study was to investigate associations between HLA–C alleles, smoking, and severe ExRA in a multicenter case–control study of well-characterized patients, using predefined criteria for ExRA (5, 24). To our knowledge, this is the largest sample of patients with severe ExRA ever reported.
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- PATIENTS AND METHODS
A total of 159 patients were identified as having severe ExRA in accordance with the predefined criteria (5, 23). Forty-six patients had vasculitis, defined as biopsy-proven vasculitis (n = 28) or major cutaneous vasculitis diagnosed by a dermatologist (n = 18). Additional subgroups analyzed were those with neuropathy (mononeuropathy or polyneuropathy [n = 41]), ILD [n = 27], Felty's syndrome [n = 21], and pericarditis [n = 27]). Neuropathy was defined by clinical diagnosis and supported by electroneurography; only 8 patients (19%) underwent a nerve biopsy. Eighteen patients with neuropathy had motor neuron symptoms, of whom 7 also had evidence of vasculitis.
Patients with ExRA were compared with 178 non-ExRA controls. Disease duration and age at RA onset were similar between patients and controls (mean 11.3 years versus 12.5 years for duration of RA, and mean 50.1 years versus 50.4 years for age) (Table 1). There was a trend toward a relative predominance of male patients in the ExRA group (P = 0.06). This comparison is, however, skewed because of the matching of cases and controls by sex in 2 of the subsamples. Among the patients included at the Mayo Clinic, 165 of 170 (97.1%) were of Northern European ancestry.
Table 1. Demographic data and clinical predictors of ExRA*
| ||ExRA patients||Non-ExRA controls||P|
|No. of patients||159||178|| |
|Age at RA diagnosis, mean ± SD years||50.1 ± 14.4||50.4 ± 14.8||0.87|
|Disease duration, mean ± SD years||11.3 ± 11.2||12.5 ± 11.3||0.34|
|No. male/no. female||75/84||66/112||0.06|
|Smokers at RA diagnosis, %†||48.5||30.0||0.001|
|RF positive, %‡||87.2||58.3||<0.0001|
|ANA positive, %§||60.8||33.8||<0.0001|
A positive result for RF or ANAs at any time was significantly associated with ExRA (both P < 0.0001). Patients with ExRA were also significantly more likely than controls to have been smokers at the time of RA diagnosis (48.5% versus 30.0%; P = 0.001). Among the patients in whom vasculitis developed, 54% had been smokers at the time of RA onset. Smoking was associated with an increased risk of vasculitis (OR 2.04, 95% CI 1.04–3.97). The highest proportion of smokers was found in the mononeuropathy group (70%), indicating that smoking is most strongly associated with the most severe extraarticular manifestations of RA.
The global distribution of HLA–C alleles significantly differed between patients with ExRA and non-ExRA controls (P = 0.031) (Table 2). The most frequent HLA–C allele in patients with ExRA was HLA–C3 (allele frequency 0.266), whereas the most frequent genotype in non-ExRA controls was HLA–C7 (allele frequency 0.243). The difference in the distribution of HLA–C alleles between the 2 groups was mainly due to a trend toward a higher frequency of HLA–C3 in the ExRA group (P = 0.054) and lower frequencies of HLA–C2 (P = 0.034) and HLA–C8 (P = 0.053) in patients with ExRA compared with non-ExRA controls. The association with ExRA overall was not overwhelmingly strong for any single allele (Table 2).
Table 2. HLA–C allele frequencies in patients with ExRA compared with non-ExRA control patients*
|Allele||Non-ExRA controls||Patients with ExRA|
The patients with RA-associated vasculitis (19 men and 27 women, mean age at RA diagnosis 48.8 years, mean duration of RA 10.1 years, 91% RF positive, 62% ANA positive, 54% smokers) had a high frequency of other severe ExRA (neuropathy [n = 15], ILD [n = 7], Felty's syndrome [n = 3], pericarditis [n = 3]). The global distribution of HLA–C alleles was significantly different in patients with vasculitis compared with non-ExRA controls (P = 0.014) (Table 2). This was mainly due to an association of the HLA–C3 allele with vasculitis (allele frequency 0.411 in vasculitis patients versus 0.199 in non-ExRA controls; P < 0.001) and a decreased frequency of HLA–C7 (allele frequency 0.122 and 0.243, respectively; P = 0.018). The OR for vasculitis in patients with HLA–C3 was 4.15 (95% CI 2.14–8.08). There was also a significantly increased frequency of HLA–C3 in patients with neuropathy (allele frequency 0.329; P = 0.016 versus non-ExRA controls), and a similar trend was observed in patients with ILD (HLA–C3 allele frequency 0.315; P = 0.089 versus non-ExRA controls), but not in patients with pericarditis or Felty's syndrome (Figure 2).
Figure 2. Variation of the frequency of HLA–C3 by disease phenotype in rheumatoid arthritis (RA). HLA–C3 was more frequent in patients with extraarticular RA (ExRA)–associated vasculitis than in non-ExRA controls (P < 0.001) with a similar pattern for vasculitis-related neuropathy (Neuro) and interstitial lung disease (ILD) (P = 0.016 and P = 0.08, respectively, versus non-ExRA controls).
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The global test for homozygosity for HLA–C alleles in patients with vasculitis compared with non-ExRA controls did not indicate a significant difference (P = 0.11), although homozygosity for HLA–C3 was more frequent among patients with vasculitis (16%) than among non-ExRA controls (8%) (P = 0.35), and homozygosity for HLA–C7 was negatively associated with vasculitis (2% versus 12% of non-ExRA controls; P = 0.03). Among all patients with ExRA, 10% were homozygous for HLA–C3, and 7% were homozygous for HLA–C7.
The impact of HLA–C3 and smoking habits at RA diagnosis on the risk of vasculitis was assessed by logistic regression. In a model including both factors, both HLA–C3 (adjusted OR 3.70, 95% CI 1.83–7.48) and smoking (adjusted OR 2.02, 95% CI 1.01–4.02) remained significantly associated with vasculitis. The interaction term for HLA–C3 and smoking was not significantly associated with vasculitis (P = 0.28). When RF positivity and presence of ANAs were added to the model, HLA–C3 (P < 0.001), but not smoking, remained significantly associated with vasculitis.
Haplotype analysis did not demonstrate any significant difference in the distribution of HLA–DRB1;DQ;C haplotypes between ExRA patients and non-ExRA controls (P = 0.12), suggesting that the difference in the distribution of HLA–C alleles could not be attributed to linkage disequilibrium with DRB1;DQ haplotypes. The most frequent haplotype in both groups was DRB1*0401;DQ8;C3 (haplotype frequency 0.069 in ExRA patients versus 0.065 in non-ExRA controls; P = 0.73). The DRB1*0401;DQ7;C7 haplotype was negatively associated with ExRA (haplotype frequency 0.003 in ExRA patients versus 0.037 in non-ExRA controls; P = 0.01), whereas there was a trend toward a positive association with the DRB1*0401;DQ7;C3 haplotype (haplotype frequency 0.022 in ExRA patients versus 0.010 in non-ExRA controls; P = 0.20).
HLA–DRB1*04 SE alleles were present in 57% of the patients with ExRA. As previously reported (34), the presence of the DRB1*04 SE was more frequent in patients with RA-associated vasculitis compared with non-ExRA controls (OR 2.1, 95% CI 1.0–4.2). HLA–C3 was associated with a significantly increased risk of vasculitis both in patients with DRB1*04 SE alleles (OR 2.7, 95% CI 1.2–6.1) and in those without DRB1*04 SE alleles (OR 8.3, 95% CI 2.0–34.2).
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- PATIENTS AND METHODS
In this large sample of patients with severe ExRA, we found an association between HLA–C3 and rheumatoid vasculitis. Patients with ExRA overall were more likely than RA control patients without extraarticular disease to be RF positive and ANA positive, and also were more likely to be smokers at the time of RA onset. Smoking and HLA–C3 were independent predictors of vasculitis. The allele frequencies of HLA–C3 also tended to be increased in ExRA patients with neuropathy and ILD, but not in those with pericarditis and Felty's syndrome. HLA–C7 was negatively associated with vasculitis.
The association between vasculitis and HLA–C3 confirms earlier observations from a smaller study (15). Of note, HLA–C3 was strongly associated with vasculitis, and also with neuropathy, but not with other extraarticular manifestations, suggesting that class I MHC genes have a specific importance in vasculitis manifestations. The increase in HLA–C3 in the subgroup with neuropathy is in accordance with data showing that neuropathy in patients with RA often is due to underlying vasculitis (35). The overlap between vasculitis and neuropathy in our sample may be more extensive than is evident, since neural biopsy was not routinely performed. Similarly, vascular mechanisms dependent on HLA–C3 may be important in some patients with ILD. In contrast, HLA–C alleles do not seem to contribute to the risk of pericarditis and Felty's syndrome. These findings underline the heterogeneity of RA and of ExRA, although our failure to detect an effect could be due to problems of sample size or patient selection.
In a recent study of patients with early RA, HLA–C7 was found to be a predictor of the need for more aggressive disease treatment (17). The biologic basis for this is currently not understood, and the negative association between HLA–C7 and vasculitis in our study is intriguing. One possible explanation is that it is simply a reflection of the increase in HLA–C3 in patients with vasculitis. Alternatively, it could be that early active disease associated with HLA–C7 led to more intensive early antirheumatic treatment, and that this treatment protected patients carrying HLA–C7 from developing vasculitis.
HLA–C gene products and other class I MHC molecules interact with T cell receptors on CD8+ T cells, and with KIR molecules on natural killer cells and a subset of T cells (36). CD4+,CD28null T cells, which have been associated with ExRA (13) and coronary artery disease (37), express the stimulatory receptor KIR2DS2 more frequently than other stimulatory KIR molecules (38). KIR2DS2 can function as a costimulatory molecule or as an independent stimulatory receptor on T cells (39). Yen et al, who found an association between vasculitis in patients with RA and HLA–C*03 and *05, also reported an association between the KIR2DS2 gene and vasculitis (15). Based on this and our current findings, it may be hypothesized that the interaction between KIR2DS2 or other KIR molecules and HLA–C molecules is important in the pathogenesis of RA-associated vasculitis. However, a recent study found no evidence of binding of HLA–Cw3 to KIR2DS2 tetramers in vitro (40). Studies of the role of class I MHC interactions with ligands under inflammatory conditions are needed to understand their biologic relevance.
Smoking has previously been found to be a predictor of severe ExRA, including vasculitis (22, 23). Smoking is also associated with an increased risk of developing RA in the general population (18, 41) and is a known risk factor for atherosclerotic vascular disease (42). Since the effect of smoking and HLA–C3 on the risk of vasculitis in patients with RA is not synergistic, the mechanisms involved are likely to be different. Smoking may be associated with vascular damage and antigen modification, whereas HLA–C3 may be more specifically involved in T cell activation.
The increased number of RF-positive and ANA-positive patients in the ExRA group is in accordance with previous findings (29, 43). This supports a role for immune complexes in the pathogenesis of vasculitis (44) and other extraarticular manifestations (45), and suggests that not only T cells, but also B cell abnormalities are important in the development of ExRA. Given the emergence of new treatment strategies directed specifically against B cells (46) and against T cell costimulation (47) in RA, such therapies could be of particular benefit in patients with severe ExRA. Further studies of the pathogenesis of ExRA and of potential therapeutic targets are needed to understand the role of these therapies in the management of ExRA and for development of new treatment approaches.
The patients included in this study were recruited from 4 different centers. The background RA population from which they originated is not fully characterized, at least for the patients seen at Lund University Hospital and at the Mayo Clinic (that is, they were not derived from, for example, population-based cohorts). Nevertheless, these patients were recruited during a period when there was a particular interest in patients with severe ExRA at each of the centers, suggesting that they should reflect the majority of patients with ExRA seen and be representative of the ExRA population as a whole.
The ethnic heterogeneity of the studied patient samples needs to be considered in multicenter studies of genetic markers. However, the majority of the patients included at the Mayo Clinic were white subjects of North European origin, similar to the patients from southern Sweden. Thus, our result could be generalized to RA patients with a similar ethnic background, but not to other populations.
HLA–C3 was a strong predictor of vasculitis in patients lacking HLA–DRB1*04 SE alleles, suggesting that HLA–C and HLA–DR genes influence the RA disease process through different pathways. Furthermore, haplotype studies showed that the association between vasculitis and HLA–C3 was not due to linkage disequilibrium with HLA–DRB1 and DQB1, but we cannot exclude the possibility that linkage with other genes within the MHC might explain our results. The suggested biologic role of class I MHC gene products in T cell regulation (15) supports the possibility of a role for HLA–C, but further studies are needed to clarify this.
The presence of HLA–C3 and smoking are independent predictors of vasculitis in patients with RA. Smoking, RF, and ANAs are all associated with severe extraarticular disease manifestations. Based on our findings, we suggest that HLA–C genotypes influence the disease process in RA and contribute to the heterogeneity of the disease, including the development of systemic extraarticular manifestations.