Background/Purpose Citrullination is a post-translational modification whereby arginine (Arg) is deiminated by PAD enzymes to form citrulline (Cit). In RA, current ACPA assays measure the ACPA titer without determining whether the observed reactivity is specific for the citrullinated or arginilated form of the peptide. Brink showed that autoantibodies (AAb) directed to uncitrullinated peptides may occur prior to the development of cit specific antibodies. Consequently, important information related to citrullination and its relationship to clinical outcomes might be overlooked. 14-3-3η is normally an intracellular protein whose extracellular expression in RA leads to the development of specific pan-reactive AAb to the native protein that inform joint damage prognosis, and also cit-14-3-3η-reactive AAbs. In this study we examined 14-3-3η cit:arg reactivity and its complementarity to ACPA in diagnosing RA.

Methods A total of 623 subjects were evaluated; 190 DMARD-naïve early RA patients (READE n=139, Sherbrooke n=40, iCOBRA n=11), 115 with classified established RA (n=65 from the RAPPORT cohort; 50 from the year 3 time point of the BeSt study). 318 controls constituted 106 healthy and 212 disease controls with connective tissue disease, OA, AS and autoimmune conditions. AAb levels were measured using the MSD ECL platform. Seven (7) cit/arg sites on 14-3-3η were examined for differential AAb reactivity between RA and controls using the Kruskal-Wallis test and ROC curve analysis.  The paired t-test was used to determine differences in cit:arg reactivity. Anti-cit-14-3-3η positivity at an individual cit site was defined by a 50% increase in reactivity to the cit site over the corresponding arg site, with overall anti-cit positivity defined as positive at a least one cit site.

Results The significantly higher reactivity of anti-cit 14-3-3η in RA versus controls, based on ROC analysis and assessment of cit:arg fold reactivity, resulted in the prioritization of 4 cit sites (1, 2, 4, 7) which reside on the protein surface. Reactivity was not confined to the cit form of the peptide since significantly higher reactivity to the arg peptide was also apparent in RA versus controls. Importantly, 50% of early and 54% of established RA patients were anti-cit-14-3-3η positive. While the Fisher Exact test revealed a significant association between anti-cit-14-3-3η and ACPA positivity (LR = 19.1 p<0.0001), in ACPA negatives, 25% of early and 41% of established RA patients were positive for anti-cit-14-3-3η. Combining ACPA and anti-cit-14-3-3η reactivity improved the early RA capture rate (table).

Conclusion In RA, AAbs exist against 14-3-3η native and citrullinated protein. Anti cit-14-3-3η AAbs are expressed in early and established RA and complement ACPA as a diagnostic aid.  Relative cit:arg AAb reactivity should be accounted for in citrullination assays to properly assess the burden of citrullination in RA.

Ref. Brink M et al. ARD. 2014 Jul;73(7):e46.