Background:The presence of anti-citrullinated protein antibodies (ACPAs) is a hallmark of rheumatoid arthritis (RA) that precede the development of the disease by years and is used for its clinical diagnosis. However, there are RA subjects that test negative for ACPA and thus the early diagnosis on these patients may be delayed. Furthermore, the presence or absence of ACPA in RA supports the hypothesis that on these two subsets of patients underlie different pathogenesis and clinical outcomes.Objectives:In this work, we searched for serum autoantibodies useful to assist the early diagnosis of ACPA-seronegative RA and its management.Methods:We profiled the serum autoantibody repertoire of 80 ACPA-seronegative and 80 ACPA-seropositive RA subjects from the Swedish population-based Epidemiological Investigation of RA (EIRA) cohort. A suspension bead array platform built on protein fragments within Human Protein Atlas and selected from an initial untargeted screening using arrays containing 2660 total antigens was employed to identify IgG and IgA serum autoantibodies. A validation phase on antigen suspension bead arrays was carried out on another set of samples from EIRA containing 386 ACPA-seropositive, 358 ACPA-seronegative and 372 randomly selected control subjects of the same age and sex. A sample-specific threshold based on 20 times the median absolute deviation plus the median of all signals was selected to determine the reactivity of samples. The Wilcoxon rank sum test and Fisher’s test were applied for the comparison of autoantibody levels and reactivity frequencies between the groups.Results:Our data revealed four antigens associated with the ACPA status (Table 1). Testis-specific Y-encoded-like protein 4 (TSPYL4) showed significantly higher IgG reactivity frequency in ACPA-seronegative subjects compared to ACPA-seropositive (8% vs. 3%; P<0.05). Significant differences at IgG autoantibody levels (P<0.05) were also observed between ACPA-seronegative subjects and controls for this specific antigen. Significantly higher IgG autoantibody levels (P<0.05) towards another antigen, dual specificity mitogen-activated protein kinase kinase 6 (MAP2K6), were also observed in ACPA-seronegative subjects compared to ACPA-seropositive and controls. In contrast, we found significantly higher IgG autoantibody levels (P<0.05) in ACPA-seropositive individuals compared to ACPA-seronegative and controls towards two antigens, anosmin-1 (ANOS-1) and muscle related coiled-coil protein (MURC). ANOS-1 shows also significantly higher IgG reactivity frequency in ACPA-seropositive individuals compared to ACPA-seronegative and controls (22%, 9% and 6% respectively; P<0.05). Interestingly, three out of the four antigens discovered to be associated with the ACPA status in early RA are highly expressed in lungs and heart, two of the main extraarticular sites affected in RA. No significant differences were observed at IgA levels for any of the antigens analyzed.Table 1.Scheme of the different phases of the study, the features within each phase and the results. The reactivity to four antigens allows to distinguish ACPA-seronegative (ACPA-), ACPA seropositive (ACPA+) and controls.PhasesUntargeteddiscoveryTargeteddiscoveryTargetedvalidationNumber of samples80 ACPA-80 ACPA-358 ACPA-372 Controls80 ACPA+80 ACPA+386 ACPA+Antigen arrayplatformPlanararraysSuspensionbead array 1Suspensionbead array 2Number of antigens26606227Number of candidatebiomarkers6227 4 (TSPYL4,MAP2K6,ANOS1,MURC)Conclusion:Upon further validation in other early RA sample cohorts, our data suggest the measurement of these four autoantibodies may be useful for the early diagnosis of ACPA-seronegative RA and give insight into the pathogenesis of the different RA subsets.Characters from table content including title and footnotes:Disclosure of Interests:None declared
Genetic and environmental contributions to the association between ADHD and affective problems in early childhood-A Swedish population-based twin study
