Urinary Proteomics Identifying Novel Biomarkers for the Diagnosis of Adult-Onset Still's Disease

Abstract

Adult-onset Still's disease (AOSD) is a systemic, multigenic autoinflammatory disease, and the diagnosis of AOSD must rule out neoplasms, infections, and other autoimmune diseases. Development of a rapid and efficient but non-invasive diagnosis method is urgently needed for improving AOSD therapy. In this study, we first performed a urinary proteomic study using isobaric tags for relative and absolute quantification (iTRAQ) labeling combined with liquid chromatography-tandem mass spectrometry analysis in patients with AOSD and healthy control (HC) subjects. The urinary proteins were enriched in pathways of the innate immune system and neutrophil degranulation, and we identified that the α-1-acid glycoprotein 1 (LRG1), orosomucoid 1 (ORM1), and ORM2 proteins were highly expressed in patients with AOSD. The elevated urine levels of LRG1, ORM1, and ORM2 were further validated by enzyme-linked immunosorbent assay in active patients with AOSD, disease controls, and HC subjects. Receiver operating characteristic curves showed that the areas under the curve of LRG1, ORM1, and ORM2 were 0.700, 0.837, and 0.736, respectively (all p < 0.05). Furthermore, we found that the urine levels of LRG1, ORM1, and ORM2 were positively correlated with the systemic score and erythrocyte sedimentation rate and that the urine levels of LRG1 were positively correlated with interleukin 1β (IL-1β), IL-6, and IL-18 levels, whereas the urine levels of ORM1 were positively correlated with the IL-1β level. Together, our study identified novel urinary markers for non-invasive and simple screening of AOSD.

Keywords: adult-onset Still’s disease; biomarker; orosomucoid; urinary proteomics; α-1-acid glycoprotein 1.

FIGURE 1

Urinary proteomic analysis in patients with AOSD and HC subjects. (A) Heatmap showing the differentially expressed proteins between patients with AOSD (B group) and HC subjects (A group). (B) A volcano plot displaying that LRG1, ORM1, and ORM2 were differentially expressed in patients with AOSD. GO analysis (C) and Reactome pathway analysis (D) showing the enriched pathways in urine samples of AOSD patients.

FIGURE 2

The urinary levels of LRG1, ORM1, and ORM2 were elevated in patients with active AOSD. The levels of LRG1 (A), ORM1 (B), and ORM2 (C) in patients with active AOSD (○; n = 70), RA (∇; n = 24), neoplasm (□; n = 27), infection (▲; n = 14), and HC subjects (🌑; n = 50) were determined by ELISA. (D) ROC curves for LRG1 (blue line), ORM1 (red line), and ORM2 (black line) levels to distinguish AOSD from non-AOSD subjects. The correlation between the levels of LRG1 (E), ORM1 (F), and ORM2 (G) and the systemic score of AOSD. *p < 0.05; **p < 0.01; and ***p < 0.001.

FIGURE 3

Correlation of urinary LRG1, ORM1, and ORM2 levels with laboratory values in AOSD patients. *p < 0.05; **p < 0.01; and ***p < 0.001.