Identification of anti-DOK2 antibodies in patients with autoimmune hepatitis via a human protein microarray

Background: Autoantibodies such as antinuclear antibodies (ANAs) and anti-smooth muscle antibodies (ASMAs), which are detected in many cases of autoimmune hepatitis (AIH), are not disease-specific; thus, they have limited value in disease diagnosis. In this study, we aimed to identify disease-specific autoantibodies related to AIH diagnosis or pathogenesis. Methods: We analyzed human protein microarrays (containing more than 16,000 human proteins, including membrane, cytoplasmic, and nuclear proteins, synthesized in a wheat germ cell-free system using cDNA). To identify AIH-specific autoantibodies, we performed a cross-sectional study of AIH patients (n=67) and matched healthy controls (HCs) (n=25) via ELISA. An independent cohort of 123 AIH patients and 75 HCs was analyzed for validation. All patient data were collected before corticosteroid therapy. We also evaluated the associations between candidate protein antigen expression and clinical findings in AIH patients. Candidate protein antigen expression in liver tissues was confirmed by immunohistochemistry. Results: According to comprehensive human protein microarray analysis, docking protein 2 (DOK2) was selected from 6 antigens upregulated in AIH patients. The mean anti-DOK2 level was significantly greater in AIH patients (36.1–37.4 AU) than in those with other liver diseases (2.6–9.2) or HCs (1.0–4.5 AU) in the exploration and validation cohorts ( p value/AUROC, vs. HCs, <0.0001/0.87–0.90). Serum anti-DOK2 levels were positively correlated with serum IgG levels and liver histological activity. The serum IgG level was an independent predictor of increased anti-DOK2 antibody levels in patients with AIH. Immunostaining revealed that DOK2-positive cells were present in the hepatic lobules and portal tracts of patients with AIH. Conclusions: We provide the first evidence that serum anti-DOK2 antibody levels are associated with the diagnosis and disease activity of AIH patients.