Rheumatoid arthritis (RA) is a complex genetic disorder, with an estimated heritability of 60% (1). HLA class II molecules are the most powerful of the recognized genetic factors and contribute to at least 30% of the total genetic effect (2). Extensive evidence exists showing the association between certain frequently occurring HLA–DRB1 alleles (*0101, *0102, *0401, *0404, *0405, *0408, *0410, *1001, and *1402) and susceptibility to and the severity of RA (3–5). The indicated alleles share a conserved amino acid sequence (QKRAA, QRRAA, or RRRAA; also called the shared epitope [SE]) at positions 70–74 in the third hypervariable region of the DRβ1 chain. These residues are part of an α-helical domain forming one side of the antigen-presenting binding site. According to the SE hypothesis, the SE motif itself is directly involved in the pathogenesis of RA by allowing the presentation of a peptide to arthritogenic T cells.
Although the predisposing effects of the SE-encoding HLA–DRB1 alleles are generally accepted, controversy exists regarding the possible protective effects of certain HLA–DRB1 alleles. These alleles contain, instead of the SE, another common anchor region consisting of the amino acids DERAA. HLA–DRB1 alleles that express this DERAA sequence (DRB1*0103, *0402, *1102, *1103, *1301, *1302, and *1304) may protect against RA (6–8). Some evidence suggests that disease is less erosive in patients carrying the DERAA sequence. However, few studies have addressed the effect of DERAA on disease severity, and interpretation of the results of these studies is hampered either by a retrospective design with variable disease duration (9, 10) or by small numbers of patients carrying the DERAA sequence. Wagner et al (11) performed a prospective study, but only 7 DERAA-positive patients were followed up for 4 years. Moreover, it is not clear whether the effect of DERAA-encoding HLA–DRB1 alleles is truly protective or whether a protective effect is attributable to the concomitant absence of predisposition SE–encoding HLA–DRB1 alleles.
Several of the initial reports on the protective effects of the DERAA haplotype are based on the experience at the Leiden Early Arthritis Clinic (6, 12). This cohort study began in 1993, and since then, the cohort has expanded considerably; presently, >1,800 patients are included. Using this expanded cohort, we assessed the association of DERAA-encoding HLA–DRB1 alleles with susceptibility to RA and with RA severity, taking advantage of the fact that, at present, a substantial number of patients are being followed up prospectively. This large cohort allows for a determination of the possible protective effects of DERAA-encoding HLA–DRB1 alleles in the presence of an equal number of SE-encoding HLA–DRB1 alleles, thereby permitting differentiation between protection and nonpredisposition. Furthermore, the available clinical data allowed us to determine whether patients with RA who exhibit an extreme of the phenotypic spectrum by achieving clinical remission harbor a different distribution of HLA alleles compared with patients in whom disease is persistent. The present data show that HLA–DRB1 alleles encoding the DERAA sequence were associated with less severe disease at all time points during 4 years of followup and conferred a lower risk of developing RA.
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- PATIENTS AND METHODS
In this study, we investigated the associations between HLA class II alleles and RA and described the protective effects of DERAA-encoding HLA–DRB1 alleles on RA severity and susceptibility. The question of whether the effect of DERAA is truly protective or is merely the result of the absence of predisposing SE-encoding HLA–DRB1 alleles has been surrounded by some controversy. In the current study, the comparison of subgroups allowed differentiation of the effects of protection and nonpredisposition. This study shows that DERAA-encoding HLA–DRB1 alleles independently reduce the risk of developing RA.
More importantly, however, our study in a large prospective cohort shows that at all time points during 4 years of followup, DERAA-encoding alleles were associated with less severe radiographic destruction in patients who were predisposed to the development of severe RA because of the presence of SE alleles. The protective effect of DERAA remained after stratification for anti-CCP antibodies. Stratification for smoking, another risk factor for severe disease, showed that DERAA confers protection, particularly in patients who are predisposed to more severe disease because of smoking. Taken together, these data indicate that the protective influence of DERAA can be detected in patients who are predisposed to the development of severe disease because of the presence of SE alleles, anti-CCP antibodies, or smoking. For patients with RA in whom the rate of joint destruction is low, such as those who are SE negative and anti-CCP negative, the current data set is not sufficiently powered to answer the question of whether a protective effect of DERAA is present. Intriguingly, the differences in Sharp/van der Heijde scores between DERAA-positive and DERAA-negative patients (in the presence of an SE allele) were as large as the differences in Sharp/van der Heijde scores between SE-positive and SE-negative patients (data not shown). Thus, the protective effect of DERAA-encoding alleles on radiographic joint destruction seems to be of a magnitude similar to that for the predisposing effect of SE alleles.
The chance of achieving clinical remission is lower for patients carrying a predisposition allele but is not higher in patients carrying protection alleles. Although at present, we cannot explain these observations, our findings suggest that the disease-promoting mechanisms that are associated with SE alleles are distinct from the mechanisms involved in tempering disease progression. In this respect, it is tempting to speculate that the protective pathways associated with the expression of DERAA-encoding HLA alleles are able to dampen the effector pathways underlying bone and cartilage breakdown, but that they do not affect the principal pathway that drives chronicity.
Although in the current study, the number of patients with 4 years of followup is higher than that in previous studies on the protective effect of DERAA on RA severity, the present study lacked sufficient power to address the question of a dose effect of DERAA. This is attributable to the finding that homozygosity for DERAA in RA patients is rare (2% of the RA patients in this cohort). Of these 8 patients, 5 had undergone 2 years of followup at the time of analysis, 1 had undergone followup for 3 years, and only 2 had undergone followup for 4 years. Remarkably, the total mean ± SEM Sharp/van der Heijde scores for these patients were 1.0 ± 1 at inclusion, 1.6 ± 1 at the 2-year followup, and 0 ± 0 at the 4-year followup, indicating that RA patients with 2 copies of DERAA seem to have a nondestructive disease course. Because the radiographic scores of the patients homozygous for DERAA were lower than those of patients heterozygous for DERAA, a gene-dose effect is possible. However, the number of homozygous patients is too low to allow definite conclusions to be reached.
Although few data are currently available on the association between protective HLA class II alleles and RA severity, results of well-designed studies on the association between protective HLA alleles and disease susceptibility are available (8, 22, 23). However, the definition of protective alleles differs in these studies. De Vries et al (22) considered alleles with amino acid D at position 70 as being protective. Thus, more alleles than those encoding for D70ERAA were classified as protective (e.g., HLA–DRB1*07, *1201, and *1501). Reviron et al (23) reached a different conclusion: that alleles with a neutral or negative electric charge in their P4 pocket protect against the development of RA. Such alleles contain not only the DERAA-encoding HLA–DRB1 alleles, but also other HLA alleles, including HLA–DRB1*08. Our results confirm and extend these observations by focusing on the DERAA-encoding HLA alleles and by analyzing the effects of these alleles on disease severity. The observed effects of the presence of DERAA might be the direct result of the DERAA-encoding alleles or might be the result of HLA haplotypes that contain the DERAA-encoding HLA–DRB1 alleles.
The known predisposing effects of the SE alleles on RA susceptibility and severity was confirmed in this study. Previously, our group hypothesized that predisposition to RA is not only controlled by SE alleles but is also conferred by HLA–DQ alleles (24). Support for a role of HLA–DQ came from studies on collagen-induced arthritis in HLA–DQ–transgenic mice (25). The so-called RA-protection hypothesis further implied that DERAA is protective only in the presence of certain DQ3 or DQ5 heterodimers (24). The data from the current study were analyzed using both HLA–DRB1 and HLA–DR/DQ genotypes, and similar results were obtained. The predisposing HLA–DQ and DRB1 alleles are strongly associated in our population; therefore, differentiation of the individual effects of HLA–DR and HLA–DQ was not feasible. Because results of the present study demonstrate that DERAA not only protects against RA in patients with predisposing HLA–DR alleles or HLA–DR/DQ genotypes but also confers a lower risk of developing RA in patients without these predisposition genotypes, the previously published RA-protection hypothesis should be amended.
It has been demonstrated that peptides carrying the DERAA motif are naturally processed by human antigen-presenting cells, and it has been suggested that the protective effect of DERAA is mediated by a specific protective T cell response (26). Although our results clearly show that the presence of a predisposing haplotype is not required to observe the protective effect associated with DERAA, it is conceivable that the DERAA sequence itself is presented toward T cells with protective activities. Interestingly, alleles carrying the DERAA sequence, particularly DRB1*13 alleles, not only protect against (severe) RA but have also been associated with a milder outcome in other diseases, such as a reduced progression to active chronic hepatitis C and B (27, 28), a lower incidence of cervical carcinoma (29), and a reduced incidence of rejection of renal transplants (30). These findings are intriguing and point to the importance of elucidating the biologic pathways underlying these associations, because they might unveil new insights about immune regulation in relation to the HLA system.