Study On Blood Proteomics, Immunoglobulin Glycosylation And Autoantibodies In Patients With Behcet's Syndrom

Beh?et’s syndrome(BS)is a recurrent systemic vascular autoimmune/inflammatory disease.It is highly heterogeneous and has multiorgan involvement,including oral ulcers,genital ulcers,uveitis,and skin lesions,which affect the quality of life of patients.It can be fatal owing to ruptured vascular aneurysms or severe neurological complications during the disease development.However,despite many efforts to study BS,few specific biomarkers have been found for diagnosis of BS,and the pathogenesis remains uncompletely unknown.Focusing on plasma proteins,immunoglobulin glycosylation and autoantibodies,this study sought to identify BS specific biomarkers showing better performance,and explore the relationship between specific molecules and the pathogenesis of BS.The main content is divided into the following three chapters:Chapter Ⅰ Blood proteomics in Beh?et’s syndromePlasma is rich in proteins,which are important molecules performing human physiological functions.It is an ideal sample source for disease biomarker screening.Nowadays,the high-throughput omics platform enables us to obtain a large amount of human physiological and pathological information In this study,we aimed to elucidate the pathogenesis and heterogeneity associated with different organs and identify the biomarkers for clinical assessment and treatment of patients with BS.Using our in-depth proteomic platform with a data-independent acquisition mass spectrometer and antibody microarray,the expression levels of 759 proteins in plasma samples from 98 patients with BS and 31 healthy controls(HC)were measured.The BS group had 220 differentially expressed proteins,which well discriminate 88.6%of the patients and 95.5%of the HCs.The bioinformatic analyses revealed that these differentially expressed proteins participated in different biological processes associated with BS pathogenies.including complement activation,wound healing,angiogenesis,leukocyte-mediated immunity,and so on.Furthermore,the first proteomic landscape of the organ-resolved BS was constructed,providing the proteomic features of BS associated with different organs and protein targets for the development of therapeutic treatment.Three proteins(HABP2,TNC,and SERPINA3)that indicate the disease severity and the subset with vascular events were validated in an independent cohort of 108 patients with BS and 29 HCs by using enzyme-linked immunosorbent assay,and their increased levels aid in the clinical assessment of vascular BS and targeted therapy.Chapter Ⅱ Glycosylation of immunoglobulin in Beh?et’s syndromeThe activation of B cells and the abnormal increase of immunoglobulin are related to the phenotype of BS.Recently,studies have focused on the change of immunoglobulin glycosylation in autoimmune diseases,which largely affect the function of immunoglobulins.Our study aimed to explore the significantly changed glycosylation structure of immunoglobulin in patients with BS,and then identify glycosylation markers for diagnosis.Using chip technology,we first quantified the concentrations of immunoglobulin(IgG,IgG1-4,IgA,IgAl-2)in plasma of 97 patients with BS,30 healthy controls and 15 disease controls(Takayasu arteritis,ANCA associated vasculitis and Sjogren’s syndrome).After the plasma screening of lectin array,we identified the changes of 47 lectin binding glycans in immunoglobulins and their subtypes.After adjusting the individual plasma immunoglobulin concentration,the levels of glycosylation markers among the groups were compared using statistical analysis.Compared with healthy and disease controls,the level of 33 immunoglobulin specific glycans binding to different lectins was significantly correlated with BS,Glycan changes in IgG were the most common,accounting for 87.9%(29/33)of all changes.The glycan level of GaIβ4G1cNAc(binding ECL)decreased significantly in BS,while the others increased significantly in BS.Among them,the fold change of the expression level of 13 IgG glycans and 1 IgA glycan in BS compared with controls groups was greater than 1.2.Our study revealed the changes of plasma immunoglobulin levels and glycosylation in patients with BS for the first time,and the candidate markers will be verified in a larger cohort.The results provide the insight in the pathogenesis and help indentify new biomarkers of BS.Chapter Ⅲ Novel autoantibodies in Beh?et’s syndromeAutoantibodies are antibodies against one or more proteins produced by the immune system.Their production is of significance for the occurrence and development of various autoimmune diseases.The detection of autoantibodies helps in the diagnosis of autoimmune diseases.Several BS related specific autoantibodies have been reported.However,their application in clinic is limited due to their poor sensitivity or specificity and the lack of validation in larger cohort.In this study,a new proteomic technique.Nucleic Acid Programmable Protein Array(NAPPA)was used to screen the plasma from 40 patients with BS and 20 healthy controls in order to identify the BS specific autoantibodies with better specificity and sensitivity.The candidate markers were determined by scoring manully the positive signal dots.Several potential target antigens were identifid with positive BS plasma reaction,in which the reactivity of IgG was significantly higher than that of IgA.Five proteins were identified as potential target antigen molecules of BS related autoantibodies:CCDC134-IgG/IgA,GOT2-IgG/IgA,SGK1-IgG/IgA,SIX3-IgG,NDUFB8-IgG,GPR62-IgG/IgA.Next,we used ELISA or Western blot to verify in an independent cohort comprised of 98 BS patients and 41 healthy controls.The results of ELISA showed that there was no significant difference in the levels of anti SIX3 antibody,anti CCDC134 antibody,anti GOT2 antibody and anti SGK1 antibody between BS group and healthy control group,or the trend was opposite to that of results from discovery cohort;The results of wsetern blot showed that the plasma pool of BS and HC had no reactivity to SGK1 and CCDC134,but had reactivity to got2,which however,shows no difference between the groups.Therefore,these protein molecules were excluded as BS related target antigens.For candidate proteins,GPR62,NDUFB8 and SIX3,they will be further verified in an independent cohort in the future.