Article Text

PDF
SAT0036 Auto-Antibodies to Pan and Citrullinated 14-3-3 ETA are Expressed in Anti-Ccp Negative RA
  1. W. Maksymowych1,
  2. V. Bykerk2,
  3. D. van der Heijde3,
  4. R. Landewé4,
  5. M. Murphy5,
  6. A. Marotta5
  1. 1University of Alberta, Edmonton, Canada
  2. 2Hospital for Special Surgery, New York, United States
  3. 3Leiden University, Leiden
  4. 4Academic Medical Center, Amsterdam, Netherlands
  5. 5Augurex Life Sciences Corp, North Vancouver, Canada

Abstract

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

Statistics from Altmetric.com

Request permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.