Peripheral blood gene expression as a novel genomic biomarker in complicated sarcoidosis

Abstract

Sarcoidosis, a systemic granulomatous syndrome invariably affecting the lung, typically spontaneously remits but in ~20% of cases progresses with severe lung dysfunction or cardiac and neurologic involvement (complicated sarcoidosis). Unfortunately, current biomarkers fail to distinguish patients with remitting (uncomplicated) sarcoidosis from other fibrotic lung disorders, and fail to identify individuals at risk for complicated sarcoidosis. We utilized genome-wide peripheral blood gene expression analysis to identify a 20-gene sarcoidosis biomarker signature distinguishing sarcoidosis (n = 39) from healthy controls (n = 35, 86% classification accuracy) and which served as a molecular signature for complicated sarcoidosis (n = 17). As aberrancies in T cell receptor (TCR) signaling, JAK-STAT (JS) signaling, and cytokine-cytokine receptor (CCR) signaling are implicated in sarcoidosis pathogenesis, a 31-gene signature comprised of T cell signaling pathway genes associated with sarcoidosis (TCR/JS/CCR) was compared to the unbiased 20-gene biomarker signature but proved inferior in prediction accuracy in distinguishing complicated from uncomplicated sarcoidosis. Additional validation strategies included significant association of single nucleotide polymorphisms (SNPs) in signature genes with sarcoidosis susceptibility and severity (unbiased signature genes - CX3CR1, FKBP1A, NOG, RBM12B, SENS3, TSHZ2; T cell/JAK-STAT pathway genes such as AKT3, CBLB, DLG1, IFNG, IL2RA, IL7R, ITK, JUN, MALT1, NFATC2, PLCG1, SPRED1). In summary, this validated peripheral blood molecular gene signature appears to be a valuable biomarker in identifying cases with sarcoidoisis and predicting risk for complicated sarcoidosis.

Figure 1. Identifying gene signatures in sarcoidosis. Panel A. Enriched pathways among complicated sarcoidosis-associated genes.

The top ranking KEGG pathways are listed for each population. The red line indicates the cutoff of significance (adjusted p-value<0.05). The number of genes in each pathway is shown beside the pathway name. Panel B. Heatmap of patients with complicated sarcoidosis and healthy controls. Red represents increased gene expression; Blue represents down-regulation. “++”: patients with complicated sarcoidosis; “−”: healthy controls. Panel C. Principal component analysis on expression values of the 20-gene signature. X-axis: principal component 1 with eigenvalue; Y-axis: principal component 2 with eigenvalue. Left panel: patients with complicated sarcoidosis and healthy controls; middle panel: patients with complicated sarcoidosis, uncomplicated sarcoidosis and healthy controls; and right panel: patients with complicated sarcoidosis and uncomplicated sarcoidosis. HC: healthy controls; US: patients with uncomplicated sarcoidosis; and CS: patients with complicated sarcoidosis. Panel D. Comparison between the 20-gene signature and the TCR/JS/CCR signaling pathway gene signature. The distribution of prediction accuracy is based on 1,000 times of five-fold cross-validation. The dashed lines indicate the average classification accuracy for the 20-gene signature or the TCR/JS/CCR signaling pathway gene signature. Left panel: all sarcoidosis patients versus healthy controls; and right panel: patients with complicated sarcoidosis versus patients with uncomplicated sarcoidosis.

Figure 2. Validation in independent datasets.

The upper panels show the comparison between the 20-gene signature and the TCR/JS/CCR signaling pathway gene signature. The distribution of prediction accuracy is based on 1,000 times of five-fold cross-validation. The dashed lines indicate the average classification accuracy for the 20-gene signature or the TCR/JS/CCR signaling pathway gene signature. The lower panels show the results of principal component analysis on expression values of the 20-gene signature. X-axis: principal component 1 with eigenvalue; Y-axis: principal component 2 with eigenvalue.

Figure 3. Boxplot of expression of the 20 signature genes.

The dark grey points and lines indicate the geometric mean of expression in each category. HC: healthy controls; US: patients with uncomplicated sarcoidosis; and CS: patients with complicated sarcoidosis. Y-axis: log₂-transformed expression values.