Background/Purpose: Anti-Cyclic Citrullinated Peptide (anti-CCP) antibodies are one of the strongest risk factors for the development of rheumatoid arthritis (RA). Anti-CCP antibodies are generated against citrullinated proteins, which arise when peptidylarginine deiminases (PADs) make post-translational modifications to proteins by the deamination of arginine residues to citrulline. PAD2 and PAD4 isomer expression is associated with RA and serum anti-PAD4 antibodies are strongly associated with anti-CCP antibody positivity and aggressive disease phenotype. However, the specific role of serum PAD4 in anti-CCP antibody positive (CCP+) at-risk individuals who do not have synovitis, and its relationship with arthritis development, is unclear.Our objectives were to (i) investigate baseline serum PAD2/4 levels in CCP+ at-risk individuals with musculoskeletal symptoms but no synovitis, compared with CCP+ early RA patients and healthy controls and (ii) to investigate the relationship between serum PAD2/4 levels and future arthritis development.

Methods: Stored serum samples were selected from the Leeds CCP study. We selected: (i) CCP+ at-risk individuals with high anti-CCP antibody levels ( >100 U/ml) and Early Morning Stiffness (EMS) lasting ≥30 minutes (n=79) (ii) early, treatment-naïve RA patients (n=34) and (iii) asymptomatic healthy controls (n=44). Follow-up data was collated for all CCP+ at-risk individuals to determine future arthritis progression status. PAD2/4 levels were assayed via Enzyme-linked Immunosorbent Assay (ELISA), performed in triplicate, following manufacturer’s instructions, Cayman Chemicals PAD2 (Item No. 501450) and PAD4 (Item No. 501460). All results were analysed using R (version 4.3.1).

Results: All groups were balanced for age, sex and smoking status (Table 1). Anti-CCP antibody levels and duration of EMS were similar for CCP+ at-risk individuals and early RA patients (Table 1). PAD4 but not PAD2 levels were higher in early RA patients compared with healthy controls (p=0.00922). There was no statistically significant difference in PAD4 levels between CCP+ at-risk individuals and healthy controls (p=0.084). However, when CCP+ at-risk individuals were separated according to future arthritis progression status, a significant increase in PAD4 levels in future progressors (n=50) compared to both future non-progressors (n=29) (p=0.022) and healthy controls (p=0.01) was observed (Figure 1). PAD4 levels were similar in healthy controls and future non-progressors (p=0.888) as well as in future progressors and early RA patients (p=0.975). There was no statistically significant difference in PAD2 levels between any of the groups.

Conclusion: These data suggest serum PAD4 levels are increased in CCP+ at-risk individuals who progress to RA compared with CCP+ future non-progressors and healthy controls. Serum PAD4 levels are also similar in Pre-RA and RA. PAD4 levels may therefore have utility in predicting arthritis development and PAD4 may also be a potential therapeutic target for arthritis prevention in those at high risk.

Table 1 – Comparison of demographic, clinical and biomarker data between the three patient groups.

aANOVA test b Fisher’s Exact Test c t-test d Wilcoxon rank sum test. Definitions: EMS: Early morning stiffness; CCP

titre: 2nd generation anti-Cyclic Citrullinated Peptide antibody titre; PAD2: peptidylarginine deiminase 2; PAD4:

peptidylarginine deiminase 4; F: Female; M: Male.

Figure 1 – PAD2 and PAD4 levels according to patient group. Plot completed via Wilcoxon rank sum test with

no additional covariate adjustments. Definitions: ns: no statistical significance; * statistical significance at

p < 0.05; **: statistical significance at p < 0.01; ***: statistical significance at p < 0.001.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/elevated-serum-peptidylarginine-deiminase-4-pad4-levels-in-anti-ccp-antibody-positive-at-risk-individuals-with-arthralgia-who-progress-to-rheumatoid-arthritis/