The association of the DRB1 shared epitope (SE) genotypes with rheumatoid arthritis (RA) has been extensively reported (1–16). However, it is less certain whether they predict severity or susceptibility, whether they are associated with seronegative RA, whether the associations differ between sexes, and whether there are additional unrecognized associations between genotype and RA. The SE is a sequence in the third hypervariable region (amino acids 67–71; Leu-x-x-Glu-Arg/Lys) of the DRB1 locus common to several different DRB1 alleles. A continuing controversy is whether different DRB1 alleles that contain the SE do, in fact, have similar effects on RA susceptibility and/or severity and whether the SE “dose” (1 versus 2 copies) affects susceptibility or severity. A number of investigators have found an association between the SE and disease severity (1–7), while others have not (8–16), even though the populations investigated were all or mostly white, in both the studies showing and those not showing an association. These issues are important to our understanding of genetic and environmental interactions in RA, since susceptibility might imply an abnormal response to a particular pathogen or pathogens, while severity might imply a more or less intensive immunologic response to a perturbation. If different genotypes with the SE epitope are associated with different effects, then it is clear that alleles, rather than any single sequence motif, determine the extent of risk.
Unfortunately, the interaction between treatment and disease severity has not been studied (1–16), yet differences in aggressiveness of treatment between genetic groups could easily confound severity/genetic associations, and the effects of this bias could vary greatly depending on the treatment philosophy implemented for the cohort. Currently used disease-modifying antirheumatic drugs (DMARDs) have major effects on the usual dependent variables, including disease activity, function, and radiographic score (17–22). Thus, the potential bias is that more severe disease is associated with one genetic group versus another. The treating physician, of course unaware of the patient's genetic group, sees the aggressive disease and treats vigorously. After the treatment response, the genetically more aggressive disease now appears to be less severe than the other.
Study of these issues has proven difficult for a number of reasons. A study group obtained by selecting consecutive patients in a clinic can be biased toward patients with more severe disease since sicker patients attend clinic more frequently. Most studies have been cross-sectional; only a few have been longitudinal. As described above, more severely ill patients may be treated with stronger agents earlier in the course of their disease. Distinctions between seropositive and seronegative patients also may be confounded, since some patients with early RA are seronegative although they later will become seropositive. It is likely, particularly in inception cohorts, that some seronegative patients do not have RA at all and that their diagnoses may change over time. The numbers of patients in most earlier studies have been small (often not more than 100), control groups needed for susceptibility testing may have had even fewer subjects, and controls may not have been ethnically matched. Finally, a large number of possible associations have been examined simultaneously.
We sought to clarify these issues by assembling a large inception cohort of white RA patients initially seen during the first 6 months of disease. This cohort was first studied cross-sectionally, as described in this report, and is being followed up longitudinally. We have extensive clinical and treatment data collected prospectively, with latex fixation (rheumatoid factor [RF]) and C-reactive protein (CRP) testing performed at a central laboratory. We were able to examine 21 separate genotypic groups both alone and aggregated, to use a large control group, to match ethnically by including only white patients and controls, to investigate for associations with rare alleles, and to examine confounding by extraneous variables such as treatment indication bias.
We sought to address the following questions: 1) Are all genotypes containing the SE associated with greater susceptibility, or do different DRB1 alleles containing the SE motif have different effects on disease risk? 2) Are there additional previously unrecognized associations between RA and DRB1 alleles? 3) Are there differences in disease severity (disability, pain, patient global assessment, RF status, erythrocyte sedimentation rate [ESR], CRP) associated with different alleles containing the SE or with combinations of these alleles? 4) Are there differences in use of DMARDs and prednisone associated with specific genotype groups and if so, could this bias account for reduction in disease severity in certain genotype groups (treatment indication bias)? 5) Are there differences in associations between seropositive and seronegative patients and/or between men and women? 6) Are there increased risks with 2 epitope copies versus 1, and if so, are these similar for all alleles or unique to certain homozygous or heterozygous combinations?
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- PATIENTS AND METHODS
This large data set from an inception cohort of white RA patients contains extensive HLA–DRB1 genotyping, demographic, clinical, and treatment data. In examining data obtained within the first 6 months of study and the first year of disease, we have been able to confirm the general findings of DRB1 associations described previously. In addition, with the large number of patients (n = 793) included in the present study, we have been able to postulate some new associations, demonstrate differences among DRB1 alleles containing the shared epitope, and develop insights into a treatment bias that may have contributed to confusion in the earlier literature.
One issue that has remained controversial has been the association, or lack thereof, between the SE genotypes and seronegative RA. Clinically, it is obvious that any group of RA patients labeled “seronegative” is very likely to include some who will later become seropositive. Some patients become seropositive later in the disease course, and some are inconsistently seropositive. As a consequence, any genotype associations that are found only in seropositive patients are likely to be present also in “seronegative” patients but to appear with reduced frequency, creating false-negative results. We found only minimal examples of this “contamination” of seronegative groups by seropositive patients in our cohort, perhaps because we used a high-quality central laboratory for RF testing, also used rheumatologist-confirmed diagnoses, and had 2 serum samples analyzed for seropositivity for most patients. Our data strongly suggest that the SE associations in whites are limited to RA patients who are seropositive, which raises again the issue as to whether seropositive and seronegative RA are the same disease.
Genotypes containing DRB1*0401, unlike other SE-containing DRB1 alleles, increase susceptibility or penetrance even if only 1 copy of the SE is present. We note that DRB1*0401 encodes Lys in amino acid position .71, while all other DRB1 alleles containing the SE encode Arg. In contrast, DRB1*0404- and *10-containing genotypes appear to require 2 copies of the SE to increase susceptibility. DRB1*0401 in combination with either itself or *0404 further increases the association with susceptibility. All combinations of 2 copies of the epitope are significantly associated with susceptibility. Clearly there is much heterogeneity of risk among DRB1 alleles containing the SE, and clinical associations appear to be dependent as much on the specific genotype as on the presence and number of the SE.
The association of DRB1*0401 with heightened susceptibility is also supported by its association with younger age at disease onset and male sex, if one assumes that younger onset implies greater patient susceptibility as does male sex; such attributes might imply lessened resistance to a perturbation in individuals who would otherwise be more resistant to RA. These sex difference findings are robust, and the younger age and greater proportion of males among patients with DRB1*0401/0404 have been reported previously by others (1, 5). Again, the message is that of heterogeneity in susceptibility among DRB1 alleles containing the SE. Additional genetic factors, such as tumor necrosis factor microsatellite markers (32), HLA–A, HLA–DQ, or others, may be involved, or environmental factors such as smoking or infection (33, 34) could interact differently with different genotypes.
We found an increase in frequency of genotypes containing the *10 allele in combination with another SE DRB1 allele among seropositive RA patients, an observation that has not been described previously. The numbers remain rather small, however, and this association should be considered hypothetical at present. Of potential clinical significance, this subgroup is the only group examined in which there was an association with disease severity and increased DMARD treatment.
Our cohort is large, with well-established diagnoses and careful protocol-based outcome observations, and with extensive treatment data. The patient sample is likely to be broadly representative of rheumatologist-treated RA patients in North America. Yet, we did not find severity effects as assessed with disability, pain, patient global score, or laboratory variables. We did not study radiologic outcomes, although recent work has suggested that there is little association between epitope status and radiologic outcomes after up to 5 years (8, 13). We did not include data on extraarticular features, since we were examining patients during the first year of disease, in which nodules are rare and other extraarticular features are generally not seen.
Previous studies have not investigated the potential biases introduced by treatment status in relation to the question of whether genotype can predict severity of illness. It is clear that these biases can be of major importance. Consider 2 patients first seen by a rheumatologist after 5 months of illness and enrolled in an inception cohort. One has severe disease and his primary physician prescribed MTX only 3 months after disease onset. The other has less severe disease and is still DMARD-naive when enrolled in the cohort. Since MTX treatment is known to result in decreases in HAQ disability scores of ∼0.30–0.40 units within a few months (17–19, 31), the biologically more severely affected patient may well have lesser apparent disease severity, since any apparent differences in severity between genotypes are much smaller than the effects of the MTX treatment. Thus, a genotype associated with disease severity might lead to more vigorous treatment and an ensuing response, which could more than account for the differences in severity at the time of study.
Radiographs were not available for many of the patients at this point in the study, but a treatment bias could also invalidate severity data involving radiographic erosions; the rate of erosion development is substantially reduced by a number of contemporary treatments. Longitudinal study may help elucidate some of these issues but will likely not conclusively answer the questions since treatment bias will be more and more complex with greater disease duration and multiple treatments. Only if clinical data are available for all patients in a cohort prior to institution of DMARD therapy can the bias be avoided; we hope that by observing our patients who meet this criterion we will be able to provide more definitive answers. In general, however, we believe that any associations of disease severity with specific genotypes or with number of epitope copies must be small and not of clinical significance, at least in early disease.
Nevertheless, we believe there may be hidden associations of disease severity with certain genotypes which cannot yet be identified with certainty. We found, for example, that the patients with DRB1*10 carrying an additional allele with the SE were more likely to have been treated with MTX. Since treating physicians were blinded to genotype and our analysis in this instance was limited to seropositive patients (some clinicians might use seropositivity itself as an indication for more aggressive treatment), it seems possible that the treating physician may have made the more aggressive treatment choice on the basis of greater perceived severity.
It is likely that the magnitude of treatment indication bias is not the same across different published studies. It will be largest when the philosophy of treatment for the cohort is most aggressive. Future studies must consider the effects of treatment upon the apparent disease severity associated with specific genotype groups.