Background The identification of new complementary diagnostic markers to RF and anti-CCP is a clinical imperative since a substantial proportion of patients are seronegative, especially in early disease when access to prompt, effective therapy can significantly improve patient outcomes. We have reported that one such marker, serum 14-3-3eta, increases the diagnostic sensitivity of RF and anti-CCP by 20-30%. Since 14-3-3eta is normally intracellular and is externalized through mechanisms related to the pathophysiology of RA, this may lead to a specific 14-3-3eta auto-antibody response.
Objectives We examined 1) whether 14-3-3eta auto-antibodies have diagnostic utility for RA 2) if 14-3-3eta represents a novel citrullination target and 3) whether auto-antibodies to the native (anti-14-3-3eta) and citrullinated (anti-cit-14-3-3eta) forms, alone or in combination, identify anti-CCP -ve RA patients.
Methods In silico modeling and epitope mapping were performed to identify putative 14-3-3eta auto-antibody immunogenic and citrullination sites. To develop a specific auto-antibody assay to native 14-3-3eta, 11 sites were used for screening in 30 healthy and 58 RA patients with a rheumatologist-confirmed diagnosis (28 anti-CCP +ve and 30 -ve). A composite score was generated for complementary peptides with specificity set at 100% for RA with sensitivities ranging from 34-62%. To develop a specific anti-cit 14-3-3eta assay, full-length recombinant human 14-3-3eta was enzymatically citrullinated (cit) with purified peptidylarginine deiminase. Reactivity to both cit and non-cit-14-3-3eta was measured in 30 anti-CCP -ve RA patients and 30 anti-CCP -ve healthy controls to evaluate their differential expression. Mean and median fluorescence intensity [1 MFI = 1 unit (U)] was determined and corresponding t-tests and Mann-Whitney U-tests were used to examine differences within and between groups. Diagnostic utility was assessed by area under the ROC curve (AUC) and likelihood ratios (LR) were determined for optimal cut-offs of anti-14-3-3eta and anti-cit-14-3-3eta. The expression of these two markers alone and together in anti-CCP -ve RA patients was assessed.
Results Median anti-14-3-3eta and anti-cit-14-3-3eta autoantibody expression was higher in RA patients (681U and 306U) versus healthy controls (371U and 100U), respectively; p<0.0001. Means were also significantly higher (p<0.001). ROC analysis for anti-14-3-3eta and anti-cit-14-3-3eta versus healthy controls gave an AUC of 0.93 (95% CI 0.88-0.98; p<0.0001) and 0.79 (95% CI 0.68-0.91; p<0.0001), respectively. For anti-14-3-3eta, the best cut-off of 476U had specificity and sensitivity of 90% and 80%, respectively, with a LR+ of 7.9 and LR- of 0.22. For anti-cit-14-3-3eta, the optimal cut-off of 320U had specificity and sensitivity of 90% and 50%, respectively, with a LR+ of 5 and LR- of 0.56. At these cut-offs, anti-14-3-3eta and anti-cit-14-3-3eta together identify 83% of anti-CCP -ve RA patient.
Conclusions Auto-antibodies to 14-3-3eta are differentially expressed in RA patients versus healthy controls. 14-3-3eta is also a novel citrullination target and auto-antibodies to its native and citrullinated forms combine to capture a substantial portion of anti-CCP negative RA patients.
Disclosure of Interest W. Maksymowych: None Declared, V. Bykerk: None Declared, D. van der Heijde: None Declared, R. Landewé: None Declared, M. Murphy: None Declared, A. Marotta Employee of: Augurex Life Sciences Corp
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