Juvenile arthritis, whether classified as juvenile rheumatoid arthritis (JRA) according to the American College of Rheumatology (ACR) criteria (1) or as juvenile idiopathic arthritis (JIA) according to the International League of Associations for Rheumatology (formerly, the International League Against Rheumatism) criteria (2), is the most common chronic autoimmune rheumatic disease of childhood. It is characterized by the presence of chronic arthritis before the age of 16 years that lasts for at least 6 weeks, and represents a heterogeneous group of patients with different disease subtypes. Each subtype exhibits its own clinical, immunologic, and genetic characteristics. Using the ACR criteria, JRA comprises 3 different types of onset and 3 different types of disease course, which makes JRA largely different from adult-onset RA. The exception is that ∼5% of patients with JRA experience disease onset at or after puberty and are positive for both IgM rheumatoid factor (RF) and HLA–DR4, representing the childhood equivalent of adult-onset RA. In contrast, in some younger patients with JRA, especially those with pauciarticular-onset disease, HLA–DR4 haplotypes have a protective effect (3–5).
Recently, antibodies to citrullinated proteins have received a great deal of attention in terms of their use as a novel and more specific test that can aid in the diagnosis of adult RA and in the determination of disease prognosis (6–8). This autoantibody system includes antiperinuclear factor, antikeratin antibodies, antifilaggrin antibodies, anticitrullinated fibrin antibodies, and anti-Sa (citrullinated vimentin) antibodies (9–14). Based on the development of a synthetic cyclic citrullinated peptide (CCP) by Schellekens et al, for which serum reactivity can be assessed by enzyme-linked immunosorbent assay (ELISA) (15), the antibodies are now commonly referred to as anti-CCP antibodies. Anti-CCP antibody ELISA testing has shown a specificity of 98% in sera from adults with established RA and 96% in sera from adults with early RA (15). The sensitivity of this anti-CCP antibody ELISA was 68% and 48% in sera from patients with established RA and early RA, respectively (15). Using the second-generation anti-CCP assay (CCP-2) in patients with established RA, the sensitivity of the test improved to 82%, with a specificity of 98.5% (16).
Anti-CCP antibodies have been detected in the serum of children with chronic arthritis, particularly those with polyarticular-onset, RF-positive disease (17, 18). In adults, the presence of anti-CCP antibodies has been associated with specific HLA alleles constituting the shared epitope (SE) (19, 20). This association of anti-CCP antibodies with the SE has not been evaluated in JRA.
In this study, we addressed 2 questions, as follows. First, are there any associations between HLA haplotypes (specifically HLA–DR) and the presence of anti-CCP antibodies? Second, do anti-CCP antibodies in a JRA cohort including simplex and multiplex families have any associations with clinical phenotypes? To address the latter question, we tested for associations between anti-CCP antibodies and several clinical manifestations and laboratory findings of the disease, including the presence of erosions, antinuclear antibodies (ANA), and iritis. We further assessed the relationship between anti-CCP antibodies and both onset type and disease course and evaluated multiplex families for the degree of concordance of anti-CCP antibodies.
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- PATIENTS AND METHODS
The results of our studies show that anti-CCP antibodies are significantly associated with the presence of HLA–DR4 alleles in patients with JRA. Anti-CCP antibodies are also associated with polyarticular onset, a polyarticular course, and erosive disease. Similar associations were observed with RF, and those patients who had polyarticular-onset JRA and were RF positive were much more likely to have anti-CCP antibodies.
JRA is distinct from RA, except for a particular subset of patients. It is well known that in patients with late onset of polyarticular disease (usually RF-positive female patients), a disease very similar to RA develops. Not surprisingly, this is the group of patients in whom anti-CCP antibodies have been shown to be more prevalent, although in a smaller proportion of patients than that reported in RA (18).
Anti-CCP antibodies have been shown to be present in the serum of patients with JRA. Two recent European studies showed prevalence rates of 2% and 15% among all JIA subtypes (17, 18). In one study, only 1 patient with polyarticular-onset, RF-positive disease was included, and that patient was negative for anti-CCP antibodies. The other study showed that among 11 patients with polyarticular-onset, RF-positive disease the prevalence of anti-CCP antibodies was 73%. This value is similar to the prevalence of this autoantibody in adults with RA. We observed a prevalence of anti-CCP antibodies of 5.6% when all JRA subtypes in our cohort were included. When only patients with polyarticular disease were included, anti-CCP antibodies were observed in 13%, and further limiting this cohort to patients with polyarticular-onset, RF-positive disease resulted in a prevalence of 57%. These observations suggest that the presence of anti-CCP antibodies is an immunologic feature in this particular subset of patients, who have the childhood equivalent of adult-onset RA.
Our results also established an association between the presence of anti-CCP antibodies and a more severe polyarticular disease course characterized by the presence of erosions. Our study was not prospective, and the time course for detection of anti-CCP antibodies and development of erosions cannot be determined. In addition, we had radiographic data for only 76% of our subjects, leading to a concern that our analyses might not have been representative of the entire cohort. However, there was little difference in mean disease duration (9.8 years versus 9.2 years) and onset type (for pauciarticular onset, 57% versus 70%; for polyarticular onset, 36% versus 25%; and for systemic onset, 6% versus 5%) between those subjects who had radiographs and those who did not, respectively, suggesting that the missing data most likely led only to a reduction in power, not to bias. Although prospective studies are needed to determine the prognostic significance of anti-CCP antibodies in erosive disease, it is possible that the presence of these antibodies at an early stage in the subset of patients with polyarticular-onset JRA might be useful in identifying patients who are at a higher risk for the development of more aggressive disease.
Associations between different HLA alleles and JRA subtypes have been described. The HLA–DR4 allele, which has been strongly associated with adult-onset RA, confers risk for the development of polyarticular-onset JRA but has a protective effect against the development of pauciarticular-onset JRA in younger children (3–5). Other JRA-associated HLA–DR alleles (i.e., HLA–DR1, DR8, and DR11) were not associated with anti-CCP antibodies. Although HLA–DR4 is observed in the subset of patients with an older age at disease onset, HLA–DR11 and DR8 are found in the group with a younger age at disease onset, which is consistent with the associated subtype distribution (23).
Even though HLA–DR1 can be present in both patients with younger age at the time of disease onset and in those with older age at onset and is part of the SE, we did not find any association between the presence of this allele and anti-CCP antibodies. HLA–DR subtyping was not performed, and it is possible that patients in the study who possessed HLA–DR1 alleles did not possess the SE, which is found only in association with the HLA–DRB1*0101 subtype. Another possibility is that the power of the study was inadequate to detect a weaker association between HLA–DR1 and anti-CCP antibodies.
The presence of HLA–DR4 was associated with the presence of anti-CCP antibodies in the JRA population; these findings are similar to those in RA. It has been proposed that the interaction between citrullinated self peptides and the SE carried by HLA–DR alleles such as DRB1*0404 and *0401 may result in the induction of anti-CCP antibodies in patients with adult-onset RA (25). In fact, citrullinated peptides fit better in the HLA–DR4 antigen binding groove than do similar arginine-containing peptides, linking anti-CCP antibodies to the SE hypothesis of RA (25). Our data demonstrate that a similar response may occur in the subset of patients with RF-positive, polyarticular-onset JRA.
An association of HLA–DR4 and anti-CCP antibodies was also observed in healthy children enrolled in a prospective study of diabetes (DAISY) (24). Anti-CCP antibodies were observed in <1% of these children, but all children with anti-CCP antibodies carried the HLA–DR4 allele. This association with HLA–DR4 was not demonstrated for RF. This finding provides further support for the theory that citrullination of peptides allows for a better fit in the HLA–DR4 antigen binding groove, leading to production of anti-CCP antibodies.
In multiplex families with JRA, concordance of clinical phenotype for this cohort has been reported previously (22). We evaluated concordance of anti-CCP antibodies in multiplex families with JRA and found that the majority were concordant negative for anti-CCP antibodies, 2% were concordant positive, and 3% were discordant. The concordance of autoantibodies was statistically significant, but the high rate of concordant-negative values, along with the low frequency of anti-CCP antibody positivity, may limit the clinical significance and temper interpretation of the kappa statistic. The most common type of JRA onset in the multiplex population is pauciarticular, and this onset type is not associated with anti-CCP antibodies. This likely explains the low frequency of anti-CCP antibodies and the difficulty with interpreting concordance rates in this population. In previous studies of the same population, a high concordance rate for the presence or absence of ANA and RF was observed, suggesting that multiplex families have similar serologic features, and the current findings may add to this conclusion (22).
The association between HLA–DR4 and anti-CCP antibodies was also observed in the multiplex population. It is noteworthy that both sibpairs concordant for anti-CCP antibodies were also concordant for the subtype of JRA with a polyarticular course. This finding reinforces the notion that anti-CCP antibodies are associated not only with onset type but also, and possibly more clinically relevant, with disease course. As discussed above, this was not a prospective study, and a predictive value of anti-CCP antibodies for the development of a polyarticular course of JRA cannot be assigned.
In this study, an existing registry was used to test for autoantibodies that were not included as part of the original study design. There were some limitations of the study design, including the fact that some of the data in the registry were based on chart review, and some data were missing. The chart review method could specifically affect the ability to detect iritis, because some cases of iritis might be missed when assessment was performed in this way. Although some data were missing, we believe that for the majority of patients clinical information was complete enough to allow valid statistical analysis. Specifically, in 76–91% of subjects data on radiographic erosions, iritis, ANA, and disease course were available.
The controls used in this study were children enrolled in a prospective study of the development of type 1 diabetes. These children were healthy but were at higher risk for the development of autoimmune diabetes, and thus the prevalence of anti-CCP and RF autoantibodies may have been slightly higher than that in the general population. Therefore, if anything, this may have resulted in an underestimation of the difference in antibody prevalence between certain types of JRA and the general population of children. The healthy controls in this study were younger than the patients with JRA, and it is possible that the rate of anti-CCP antibody positivity would be higher in older healthy children. However, the rate of anti-CCP antibody positivity in healthy adults is also low, and we would not expect the age difference to significantly affect the results.
Another limitation of the study is that in the patients with JRA, serum samples were obtained several years after the onset of disease. Therefore, we cannot comment on the likelihood of anti-CCP antibodies being observed early in JRA. In addition, tests for anti-CCP antibodies were conducted at only 1 time point, making it impossible to determine the time course for development of these antibodies. We have made the assumption that anti-CCP antibodies remain present over time, without significant seroconversion from either negative to positive or positive to negative. This assumption is based in part on observations by Mikuls et al, showing that anti-CCP antibodies do not convert from positive to negative following the initiation of disease-modifying antirheumatic drug therapy (26). If there were a high rate of conversion from negative to positive, anti-CCP antibodies would not be as useful in predicting the prognosis in patients with early-onset JRA. Conversely, if a high rate of conversion from positive to negative was associated with control of disease, the results of this study might underestimate the prevalence of anti-CCP antibodies in JRA. Analogous to RA, in which anti-CCP antibodies have been observed both prior to the diagnosis of RA and in early undifferentiated arthritis, we would expect anti-CCP antibodies to be present early in JRA, but prospective studies are needed to determine whether this is true. In addition, we would expect the presence or absence of anti-CCP antibodies to remain relatively constant over time, but this could be proven only by prospective studies.
Because JRA comprises many disease types, we thought it was necessary to analyze our data within disease-type subgroups, even though this resulted in small numbers and large confidence intervals for some of the analyses. We would not have been able to detect many of these clinically relevant associations without this type of subgroup analysis.
The association of the presence of anti-CCP antibodies and a polyarticular disease course should be further investigated. Future studies will prospectively determine the prevalence of this antibody prior to or at the onset of disease, as well as further delineate its value in the prediction of outcome, especially in the subset of older patients with polyarticular-onset JRA.