University of Brescia/MGH Monza, Monza and Brianza, Italy
Disclosure(s): No financial relationships with ineligible companies to disclose
Background/Purpose: IgG4-related disease (IgG4-RD) is a chronic immune-mediated disease typified by mass-forming lesions. Self-antigens driving the oligoclonal expansion of plasmablasts have previously been reported. However, broad screening of the IgG repertoire from IgG4-RD patients for reactivity across the human proteome has yet to be done. The goal of this study was to identify novel, potentially causal, disease-specific autoantigens by broadly screening the human proteome. Methods: The HuProt microarray, displaying 21,216 proteins in total, including 15,889 unique human proteins, was used to screen plasma samples from IgG4-RD patients and controls across discovery and validation batches. The discovery batch included IgG4-RD (N=30) and systemic sclerosis (SSc, N=30). The validation batch included IgG4-RD (N=42, 28 of which were not included in the initial batch), idiopathic pulmonary fibrosis (N=20), sarcoidosis (N=20), and healthy donors (N=20). Microarray data was normalized and analyzed for autoantigens selectively bound by IgG4-RD samples after adjusting for false discovery rate. Autoantibodies with frequencies >15% among IgG4-RD were selected for unsupervised hierarchical clustering. A separate analysis focused on the highest titer autoantibodies across cohorts was also conducted. Selected autoantigens were validated using a luciferase immunoprecipitation system (LIPS) assay. Results: FAM84A and LIMS1 were the two autoantigens that most accurately distinguished IgG4-RD from SSc in the discovery batch, while expected autoantigens distinguished SSc including those against centromeric proteins, RNA polymerase III subunits, and topoisomerase (Figure 1). Anti-FAM84A was present in 9/30 (30%) and anti-LIMS1 in 6/30 (20%) of the IgG4-RD cohort versus 0% in SSc and 1/30 (3%), respectively. The validation batch confirmed the presence of FAM84A and LIMS1 autoantibodies and identified four additional proteins to form a cassette of 6 autoantigens distinguishing IgG4-RD from HD. The additional four autoantigens included CCDC97, MAGEE1, ISM2 and SCG2. The nine anti-FAM84A samples from the discovery batch were reproducibly reactive to FAM84A in the validation batch. After excluding all overlapping samples between batches, frequencies of these autoantibodies were 7/28 (25%), 7/28 (25%), 14/28 (50%), 12/28 (43%), 7/28 (25%) and 5/28 (18%), respectively (Figure 2). Reactivity to at least one of these 6 autoantigens was observed in 35 of the 42 (83%) IgG4-RD patients and the IgG4-RD cohort additionally clustered into three distinct groups based on antigen specificity (Figure 3). Analysis focused on the highest titer autoantibodies identified ANXA11, as has been previously reported, and FAM84A. ANXA11 and FAM84A were validated by LIPS, with anti-FAM84A responses observed in 12% (23 of 192) of IgG4-RD patients and anti-ANXA11 in 12.5% (24 of 192). Anti-FAM84A patients showed enrichment for Mikulicz phenotype while anti-ANXA11 was linked to pancreatobiliary involvement. Conclusion: The novel cassette of 6 autoantigens including FAM84A, LIMS1, CCDC97, MAGEE1, ISM2, and SCG2 distinguished IgG4-RD from controls. Further efforts to validate these findings are needed.
