Screening Of Serum Markers Of Behcet 's Disease And IgG4 - Related Diseases By High - Throughput Protein Chip Technique

Behcet’s disease (BD) is a recurrent common systemic autoimmune disease of unknown etiology. The clinical manifestations of BD is complex, characterized by oral ulcers, genital ulcers, vasculitis, ophthalmia, thereby causing serious consequences of infection, blindness, aneurysm, which causing great pain to the patient and affecting the life quality of patients seriously. Studies have shown that the incidence of Behcet’s disease in China was significantly higher than in other regions, about 14 times of in Americas region.Patients with BD benefit from earlier clinical diagnosis and treatment. However, as there is no reliable serum markers, the diagnosis of BD depends mainly on non-specific clinical manifestations and need to discriminate BD from a variety of autoimmune diseases, which result in retard, misdiagnosis and mistreatment of patients with BD. We used a human proteome microarray with-21,065 unique full-length human proteins to investigate autoantibodies associated with BD. Seventy-five sera specimens from forty BD, fifteen autoimmune diseases controls and twenty normal control were screened for BD-associated autoantibodies with proteome microarrays.36 BD-associated autoantibodies target antigens were identified. To confirm the sensitivity, specificity and clinical value of these BD-associated autoantibodies, a focused autoantigen microarray with 36 BD-associated autoantibodies target antigens was applied to profile a larger cohort of sera, including 130 BD,103 autoimmune diseases controls(forty TA, forty aw and twenty-there SS), and 110 normal control. The positive rate of anti-CTDP1 (RNA polymerase Ⅱ subunit A C-terminal domain phosphatase) antibodies in BD patients positive rate is 23.85%, which is significantly higher than in autoimmune diseases control 4.85%(X2=15.87, P=6.79×10-5) and normal controls 6.4%(X2=13.67, P= 0.000218).There is no significant difference of positive rate of the other 35 BD-associated autoantibodies target antigens identified from high-throughput protein chip between the BD group, autoimmune disease control and healthy control group. Western blot results confirmed that anti-CTDP1 antibodies verified by the focused autoantigen microarray of candidate BD autoantigens are BD-specific autoantibodies present in the serum of patients with BD.IgG4-related disease (IgG4-RD) is a newly recognized multi-organ involvement autoimmune disease, characterized by tissue swelling, IgG4+ lymphoplasmacytic cells infiltration, and elevated serum IgG4 concentrations。However, the elevated IgG4 in serum is not a specific marker of IgG4-RD. Recently, too much emphasis on the role of elevated serum IgG4 concentrations and infiltration of IgG4-positive cells in tissues in the diagnosis of IgG4-related diseases, resulting in increasing numbers of misdiagnosis of patients with IgG4-related diseases.We used a human proteome microarray with-21,065 unique full-length human proteins to investigate autoantibodies associated with IgG4-RD. Thirty sera specimens from IgG4-RD and thirty from normal control were screened for IgG4-RD associated autoantibodies with proteome microarrays. The average positive protein number in IgG4-RD was 68.8±72.3,which is lower than that in normal control(234.4±372.4). In order to ensure the clinical specificity of markers, we using a positive rate of lower than 6.67%(2/30) within normal control as a cutoff, we found the positive rate of all the selected IgG4-RD associated autoantibodies are ≤20% in IgG4-RD. As the limited clinical value of all the selected IgG4-RD associated autoantibodies, we no longer construct a IgG4-RD focused autoantigen microarray to profile a larger cohort of sera.Results of this study indicated that the elevated serum IgG4 in patient with IgG4-RD showed low reactivity to the 21,065 unique full-length human proteins,which presented a brand new research topic on IgG4-RD,whether the autoimmunity of IgG4-RD is abnormal in vivo? We will study on protein levels whether the IgG4 in patients with IgG4-RD is oligoclonal immunoglobulin by analysis the amino acid sequence of IgG4, and confirm on nucleic acid level whether the IgG4+ plasma blasts is oligoclonal cells by single cell sequencing.our research showed that screening small numbers of samples with high-throughput protein chip combined with profiling a larger cohort of sera with disease focused autoantigen microarray and validating by immunoblotting techniques is a effective research strategy to identify serum markers of autoimmune disease.