Blood leukocyte microarrays to diagnose systemic onset juvenile idiopathic arthritis and follow the response to IL-1 blockade

Abstract

Systemic onset juvenile idiopathic arthritis (SoJIA) represents up to 20% of juvenile idiopathic arthritis. We recently reported that interleukin (IL) 1 is an important mediator of this disease and that IL-1 blockade induces clinical remission. However, lack of specificity of the initial systemic manifestations leads to delays in diagnosis and initiation of therapy. To develop a specific diagnostic test, we analyzed leukocyte gene expression profiles of 44 pediatric SoJIA patients, 94 pediatric patients with acute viral and bacterial infections, 38 pediatric patients with systemic lupus erythematosus (SLE), 6 patients with PAPA syndrome, and 39 healthy children. Statistical group comparison and class prediction identified genes differentially expressed in SoJIA patients compared with healthy children. These genes, however, were also changed in patients with acute infections and SLE. An analysis of significance across all diagnostic groups identified 88 SoJIA-specific genes, 12 of which accurately classified an independent set of SoJIA patients with systemic disease. Transcripts that changed significantly in patients undergoing IL-1 blockade were also identified. Thus, leukocyte transcriptional signatures can be used to distinguish SoJIA from other febrile illnesses and to assess response to therapy. Availability of early diagnostic markers may allow prompt initiation of therapy and prevention of disabilities.

Figures

Figure 1.

Differential gene expression in PBMCs isolated from SoJIA patients and healthy controls. 17,454 genes passing the control criteria were tested. Genes expressed at statistically different levels between the two groups (P < 0.01; Wilcoxon-Mann-Whitney test, Bonferroni correction) were rearranged by hierarchical clustering to reveal differential expression. Expression values are normalized per gene to the healthy group. Transformed expression levels are indicated by color scale, with red representing relative high expression and blue indicating relative low expression. A list of the genes shown in this figure is available in Table S2.

Figure 2.

Class prediction. (A) (Left) Eight healthy and eight SoJIA samples obtained from our initial study group were used as a training set to generate a list of classifier genes displaying the best ability to discriminate patients from healthy controls. In our training set, 100% of healthy and 88% of SoJIA patients were classified accurately. (Right) Those classifier genes were then tested on a test set (eight Healthy and eight SoJIA), and 100% of patients were classified accurately. Expression values were normalized per gene to the healthy group. Samples and genes were arranged by hierarchical clustering. Transformed expression levels are indicated by color scale, with red representing relative high expression and blue indicating relative low expression. The list of the genes from this figure is shown in Table I. (B) Specificity of the SoJIA signature. The 50 best classifier genes from A were used to classify a test set of 39 healthy controls, 16 SoJIA, 31 S. aureus, 15 S. pneumoniae, 30 E. coli, 18 influenza A, 38 SLE patients, and 6 PAPA syndrome patients. The number of samples within each disease group predicted as SoJIA is represented on top of the figure. Genes were arranged by hierarchical clustering. Transformed expression levels are indicated by color scale, with red representing relative high expression and blue relative low expression.

Figure 3.

SoJIA-specific signature. (A) Genes expressed at statistically different levels in the SoJIA patient group compared with healthy volunteers (P < 0.01; Wilcoxon-Mann-Whitney test) were selected (4,311 probe sets). p-values were similarly obtained from patients suffering from S. aureus, S. pneumoniae, E. coli, influenza A, and SLE. Each of these cohorts was compared with the corresponding control group. p-values are represented according to color scale: turquoise, low p-value; pink, high p-value. (B) 88/4,311 transcripts were found expressed at statistically different levels in the SoJIA patient group compared with healthy controls (P < 0.01; Wilcoxon-Mann-Whitney test), but not in all of the other groups compared with their healthy controls (P > 0.5; Wilcoxon-Mann-Whitney test). (C) The 88 genes from B were hierarchically clustered in the 107 samples used in the meta-analysis. Expression values of those 88 genes were normalized per gene to the healthy group. (D) The 12 most significant genes (P < 0.0001 in SoJIA group) were used as predictors for an independent test set of 10 healthy patients, 9 patients with febrile syndromes to rule out the diagnosis of SoJIA (2 of these patients were later on found to suffer from other diseases), 15 S. aureus, 5 S. pneumoniae, 20 E. coli, 8 influenza A, 22 SLE, and 6 PAPA syndrome patients. Expression values of those 12 genes were normalized per gene to the healthy group. Genes were arranged by hierarchical clustering. Transformed expression levels are indicated by color scale, with red representing relative high expression and blue indicating relative low expression. The list of the 88 and 12 genes shown in B–D is displayed in Table II. (E) The 12 gene signature displayed in D is shown in 16 healthy controls, 23 samples from 19 patients with established diagnosis of SoJIA while presenting systemic symptoms, and 11 patients with established diagnosis of SoJIA in whom the systemic symptoms had subsided but arthritis persisted. Only one patient with confirmed clinical diagnosis of SoJIA did not display the signature while having systemic symptoms (*).

Figure 4.

Transcription of CLIC-2 in blood cell subsets. (A) Total RNA was extracted from the PBMCs of 16 healthy donors and 16 SoJIA patients; B cells, T cells, and monocytes from 3 healthy donors and 4 SoJIA patients, and neutrophils from 6 healthy donors and 7 SoJIA patients. Amplified cRNA was hybridized on Affymetrix HG-U133 chips. Raw intensity values from each chip were first pre-scaled to the 500 target intensity value in Affymetrix Microarray suite before being imported and analyzed in GeneSpring 6.1. (B) Total RNA was extracted from the PBMCs of 16 healthy donors and 16 SoJIA patients, mDCs from 9 healthy donors, and pDCs from 6 healthy donors. Amplified cRNA was hybridized to Affymetrix HG-U133 chips. Raw intensity values from each chip were pre-scaled to the 500 target intensity value in Affymetrix Microarray suite before being imported and analyzed in GeneSpring 6.1.

Figure 5.

Anakinra effect on the specific SoJIA signature. Transcription levels (raw values) corresponding to the genes that best differentiate SoJIA patients from healthy controls (from Fig. 1 and Table S2) were analyzed before and after the initiation of Anakinra in eight patients. The 10 genes with most significant p-values are displayed (p-values were calculated using a paired, two-tailed t test).