Fatigue in Sjögren's Syndrome: A Search for Biomarkers and Treatment Targets

Abstract

Background: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease, where patients often suffer from fatigue. Biological pathways underlying fatigue are unknown. In this study aptamer-based SOMAscan technology is used to identify potential biomarkers and treatment targets for fatigue in pSS. Methods: SOMAscan® Assay 1.3k was performed on serum samples of healthy controls (HCs) and pSS patients characterized for interferon upregulation and fatigue. Differentially expressed proteins (DEPs) between pSS patients and HC or fatigued and non-fatigued pSS patients were validated and discriminatory capacity of markers was tested using independent technology. Results: Serum concentrations of over 1,300 proteins were compared between 63 pSS patients and 20 HCs resulting in 58 upregulated and 46 downregulated proteins. Additionally, serum concentrations of 30 interferon positive (IFNpos) and 30 interferon negative (IFNneg) pSS patients were compared resulting in 25 upregulated and 13 downregulated proteins. ELISAs were performed for several DEPs between pSS patients and HCs or IFNpos and IFNneg all showing a good correlation between protein levels measured by ELISA and relative fluorescence units (RFU) measured by the SOMAscan. Comparing 22 fatigued and 23 non-fatigued pSS patients, 16 serum proteins were differentially expressed, of which 14 were upregulated and 2 were downregulated. Top upregulated DEPs included neuroactive synaptosomal-associated protein 25 (SNAP-25), alpha-enolase (ENO1) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1). Furthermore, the proinflammatory mediator IL36a and several complement factors were upregulated in fatigued compared to non-fatigued pSS patients. ROC analysis indicated that DEPs showed good capacity to discriminate fatigued and non-fatigued pSS patients. Conclusion: In this study we validated the use of aptamer-based proteomics and identified a novel set of proteins which were able to distinguish fatigued from non-fatigued pSS patients and identified a so-called "fatigue signature."

Keywords: SOMAscan; Sjögren's syndrome; fatigue; interferon; proteomics.

Figure 1

Differential protein expression in serum of pSS and healthy controls. Heatmap of differentially expressed proteins measured by SOMAscan technology in serum samples of pSS patients (pSS) (n = 63) and healthy controls (CON) (n = 20) clustered unsupervised within the groups (A) and volcano plot (B) visualizing the same DEPs. The correlation between RFU determined by SOMAscan and protein levels determined by ELISA is shown in (C).

Figure 2

Differential protein expression in serum of fatigued pSS patients. Heatmap showing the unsupervised clustering of differentially expressed proteins between fatigued (n = 22) and non-fatigued (n = 23) pSS patients (A) and volcano plot (B) visualizing the same DEPs. (C) Correlation between RFU determined by SOMAscan and protein levels determined by ELISA (for ENO1 and EGF) and Immage nephelometer (for C3 and C4). (D) Comparison of complement levels, IFN and ESSDAI score between fatigued and non-fatigued pSS patients.

Figure 3

Discriminatory capacity of markers for fatigue in pSS. (A) ROC curves of positively and negatively predictive proteins (2LogFC>1) for fatigue in pSS. (B) Boxplots of differentially expressed proteins between fatigued (n = 22) and non-fatigued (n = 23) pSS patients.