Preclinical validation of salivary biomarkers for primary Sjögren's syndrome

Abstract

Objective: Sjögren’s syndrome (SS) is a systemic autoimmune disease with a variety of presenting symptoms that may delay its diagnosis. We previously discovered a number of candidate salivary biomarkers for primary SS using both mass spectrometry and expression microarray analysis. In the current study, we aimed to verify these candidate biomarkers in independent patient populations and to evaluate their predictive values for primary SS detection.

Methods: In total, 34 patients with primary SS, 34 patients with systemic lupus erythematosus (SLE), and 34 healthy individuals were enrolled for the validation studies. Salivary protein biomarkers were measured using either Western blotting or enzyme-linked immunosorbent assay, and the messenger RNA (mRNA) biomarkers were measured using quantitative polymerase chain reaction. Statistical analysis was performed using R software, version 2.9.

Results: Three protein biomarkers (cathepsin D [CPD], α-enolase, and ß₂-microglobulin [ß₂m]) and 3 mRNA biomarkers (myeloid cell nuclear differentiation antigen [MNDA], guanylate binding protein 2 [GBP-2], and low-affinity IIIb receptor for the Fc fragment of IgG) were significantly elevated in patients with primary SS compared with both SLE patients and healthy controls. The combination of 3 protein biomarkers, CPD, α-enolase, and ß₂m, yielded a receiver operating characteristic (ROC) value of 0.99 in distinguishing primary SS from healthy controls. The combination of protein biomarkers ß₂m and 2 mRNA biomarkers, MNDA and GBP-2, reached an ROC of 0.95 in discriminating primary SS from SLE.

Conclusion: We have successfully verified a panel of protein and mRNA biomarkers that can discriminate primary SS from both SLE and healthy controls. If further validated in patients with primary SS and those with sicca symptoms but no autoimmune disease, these biomarkers may lead to a simple yet highly discriminatory clinical tool for diagnosis of primary SS.

Figure 1

Validation of protein biomarkers, cathepsin D (A), alpha-enolase (B) and B2M (C), in independent patient (pSS) and control (SLE and healthy control) populations (n=34 for each group). B2M was validated by ELISA and mean±SEM is plotted. Cathepsin D and alpha-enolase were validated by western blotting, and the bar figures indicate the normalized levels of cathepsin D and alpha-enolase among three groups (mean±SEM).

Figure 1

Validation of protein biomarkers, cathepsin D (A), alpha-enolase (B) and B2M (C), in independent patient (pSS) and control (SLE and healthy control) populations (n=34 for each group). B2M was validated by ELISA and mean±SEM is plotted. Cathepsin D and alpha-enolase were validated by western blotting, and the bar figures indicate the normalized levels of cathepsin D and alpha-enolase among three groups (mean±SEM).

Figure 2

Validation of mRNA biomarkers, MNDA, FCGR3B, and GIP2, in pSS (n=25), SLE (n=26) and healthy control (n=26) samples. The bar figures indicate the normalized levels of mRNA biomarkers among three groups (mean±SEM). Note that the lower the Ct values, the higher the levels of the mRNA.