he current study was undertaken to explore heterogeneity within the subgroup of JIA patients currently classified as having persistent oligoarticular disease and ended up also identifying commonalities between polyarticular and oligoarticular subtypes. Since some clinical observations and genetic studies (9
) suggest that the age at which disease begins may have biologic implications, we investigated whether the age at onset is reflected by the molecular phenotype of the disease. The rationale for choosing age 6 years as the cutoff was based on genetic studies showing that some major histocompatibility complex (MHC) genes operative in JIA appear to have a “window-of-effect” during which time they may contribute to risk of disease, but that they may be neutral or even protective at other times (9
). For many of the MHC genes, this window is limited to the first 6 years of life.
The comparison of PBMC gene expression profiles obtained from oligoarticular JIA patients with age at onset <6 years versus those with age at onset ≥6 years identified 832 probe sets representing differentially expressed genes. Hierarchical clustering of these 832 probe sets revealed 2 main clusters distinguishing the 2 groups of patients. One cluster (cluster a) comprised genes with higher levels of expression in patients with late-onset oligoarticular JIA and was enriched for the genes related to cellular immunity and myeloid cell lineage (). This cluster overlaps with the monocyte signature we previously reported in a subset of older patients with polyarticular JIA (7
The other cluster (cluster b) was highly enriched for genes related to humoral immunity encoding for immunoglobulins, other B lymphocyte–related cell surface markers, and B cell–specific proteins including several transcription factors (). Additional evidence that cluster b is derived from B lymphocytes comes from a comparison of the list of probe sets in this cluster with B cell signatures generated by other groups. Of 324 probe sets in cluster b (), 97 overlapped with a list of probe sets representing differentially expressed genes in purified B cells (19
). In contrast, there was no overlap with the signatures of other purified cell types, including T cells and granulocytes (19
A separate study by Hystad et al (20
) used microarray analyses to identify gene expression signatures associated with various stages of B cell differentiation (i.e., hematopoietic stem cells, early B cells, pro–B cells, pre–B cells, and immature B cells). Of the 324 probe sets in cluster b, many overlap with genes that are expressed predominantly in immature B cells, including several transcription factors (TCF3, EBF, EIF5B). The overlap also includes many immunoglobulin-related genes as well as genes encoding cell surface markers CD19 and CD79A. Combined with the absence of transcripts from the gene encoding CD27, a marker of memory B cells, it appears that cluster b is derived from relatively early stage transitional and perhaps mature naive B cells. This is consistent with a recent report describing detectable expansion of transitional B cells in peripheral blood of patients with oligoarticular JIA (21
Martini previously suggested that 1 goal of the ILAR classification, to define homogeneous subgroups, might be better served by combining patients with oligoarticular, RF-negative polyarticular, or psoriatic arthritis and exploring the value of variables such as age at onset, symmetry of arthritis, and ANA status to define subsets (22
). This concept was supported by a subsequent study showing that ANA-positive patients were similar in terms of age at onset, sex ratio, frequency of symmetric arthritis, and frequency of iridocyclitis (14
). In addition, it was shown that lymphoid neogenesis and plasma cell infiltration of the synovium were more frequent in ANA-positive JIA patients rather than being related to disease activity or severity (23
). The results in the current study are supportive of patient regrouping, since we identified a B cell signature and a high preponderance of ANA-positive patients in early-onset oligoarticular JIA. Further, the B cell signature was present in patients with early-onset RF-negative polyarticular JIA. In the same manner, the myeloid signature was present in patients with late-onset oligoarticular and those with late-onset RF-negative polyarticular disease. Taken together, the signatures identified in this study suggest that early-onset JIA and late-onset JIA have similarities that cross the boundaries proscribed by the JIA classification, the derivation of which was based mainly on the number of active joints.
In our study, we enrolled patients as early in the disease course as possible, and therefore age at onset and age at sampling were similar. In general, healthy younger children have a slightly higher proportion of B lymphocytes in their peripheral circulation (24
), and the first 6 years of life are characterized by a particularly steep increase in the levels of serum immunoglobulins. Therefore, we considered age-related normal physiologic changes as a possible explanation for the observed differences between patients with early-onset disease and those with late-onset disease. To address this possibility, samples from healthy controls were analyzed to assess the contribution of age. Using the same statistical methods and similar sample sizes, we identified only 22 probe sets detecting differentially expressed genes between samples from control subjects age <6 years and samples from control subjects age ≥6 years. Of these, 11 overlapped with the 832 probe sets from samples from patients with persistent oligoarticular disease. One caveat, however, is that fewer subjects in the control group than in the patient group were age <2 years. Nevertheless, additional evidence that the observed gene expression differences are a feature of disease rather than simply a reflection of age is the ability of the patterns from the 832 probe sets to separate samples from younger and older patients with oligoarticular disease, but not from younger and older control subjects, by either hierarchical clustering or support vector machine analyses.
The study was expanded to assess whether differences related to age at onset could be identified in other subtypes of JIA. Patterns similar to those in oligoarticular JIA were found in patients with RF-negative polyarticular JIA but not in patients with systemic arthritis, indicating disease subtype specificity. It was also apparent using PCA that the age at onset was a more important characteristic than the JIA subtype, which is defined to a large extent by the number of affected joints.
In our study, the B cell signature was present in patients with disease onset at age <6 years. The fact that healthy children in this age group have a physiologic increase in the activity of B cells is intriguing. One might speculate that this early “physiologic hyperactivity” of B cells during development contributes, in the context of additional genetic and environmental factors, to susceptibility to JIA. Interestingly, a similar age-dependent effect seems to play a role in the development of B cell autoimmunity to a myelin surface antigen in pediatric multiple sclerosis (25
Overall, the differential gene expression patterns that distinguish patients with early- or late-onset oligoarticular or polyarticular JIA suggest that different pathologic mechanisms may be active depending on the age at disease onset. Moreover, the expression differences related to age at onset appear to be more robust than the number of joints involved at classifying samples from patients with oligoarticular and polyarticular JIA subtypes. Considering other genetic and antibody repertoire differences, we propose that age at onset should be considered in future efforts to refine JIA classification.