Publications

Abstract

OBJECTIVE: Rheumatoid factors (RFs) are associated with systemic disease in primary Sjogren's syndrome (SS) and may be pathogenic as mixed cryoglobulins. Current detection methods cannot resolve RFs at a molecular level. Here, we perform the first proteomic and transcriptomic analysis of secreted and membrane-bound RF IgM in primary SS and identify unique heavy (H)-chain peptide signatures for RF clonotype tracking. METHODS: Purified H-chains of serum RFs (IgH-RF) from 15 primary SS patients were subjected to de novo mass spectrometric sequencing. The circulating B-cell immunoglobulin (Ig) repertoire was determined by massively parallel sequencing of IGH RNA from matched PBMCs (n=7). RF-specific H-chain complementarity determining region 3 (HCDR3) peptides were identified by searching IgH-RF peptide sequences against the corresponding IGH RNA sequence libraries. HCDR3 peptides were used as biomarkers to track serum RF clonotypes using quantitative multiple reaction monitoring (MRM). RESULTS: Serum RFs were clonally restricted and composed of shared sets of IGHV1-69, 3-15, 3-7 and 3-74 subfamilies. Cryoprecipitable RFs from patients with mixed cryoglobulinemia (MC) were distinguishable from nonprecipitating RFs by a higher frequency of amino acid substitutions and identification of stereotypic HCDR3 transcripts. Potentially pathogenic RF clonotypes were detected in serum by MRM years before presentation with MC. Levels of an IGHV4-34 RF IgM decreased following immunosuppression and remission of MC. CONCLUSION: Cryoprecipitable RF clonotypes linked to vasculitis in primary SS have different molecular profiles to nonprecipitating RFs, suggesting different underlying mechanisms of production. The combined omics workflow herein provides molecular biomarkers for tracking and removal of pathogenic RF clones. This article is protected by copyright. All rights reserved.