Flinders University and SA Pathology, Bedford Park, South Australia, Australia.
Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia.
Arthritis Rheumatol. 2018 Oct;70(10):1617-1625. doi: 10.1002/art.40539. Epub 2018 Aug 3.
Rheumatoid factors (RFs) are associated with systemic disease in primary Sjögren's syndrome (SS) and may be pathogenic as mixed cryoglobulins. Current detection methods cannot resolve RFs at a molecular level. This study was undertaken to perform the first proteomic and transcriptomic analysis of secreted and membrane-bound IgM-RF in primary SS and identify unique heavy-chain peptide signatures for RF clonotype tracking.
Purified heavy chains of serum RFs from 15 patients with primary SS were subjected to de novo mass spectrometric sequencing. The circulating B cell Ig repertoire was determined by massively parallel sequencing of IGH RNA from matched peripheral blood mononuclear cells (n = 7). RF-specific heavy-chain third complementarity-determining region (CDR3) peptides were identified by searching RF heavy-chain peptide sequences against the corresponding IGH RNA sequence libraries. Heavy-chain CDR3 peptides were used as biomarkers to track serum RF clonotypes using quantitative multiple reaction monitoring.
Serum RFs were clonally restricted and composed of shared sets of IgM heavy-chain variable region (Ig V ) 1-69, 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 heavy-chain CDR3 transcripts. Potentially pathogenic RF clonotypes were detected in serum by multiple reaction monitoring years before patients presented with MC. Levels of Ig V 4-34 IgM-RF decreased following immunosuppression and remission of MC.
Cryoprecipitable RF clonotypes linked to vasculitis in primary SS have different molecular profiles than nonprecipitating RFs, suggesting different underlying mechanisms of production. The combined omics workflow presented herein provides molecular biomarkers for tracking and removal of pathogenic RF clones.
类风湿因子（RFs）与原发性干燥综合征（SS）的系统性疾病有关，并且可能作为混合冷球蛋白存在致病性。目前的检测方法无法在分子水平上解析 RF。本研究旨在对原发性 SS 中分泌型和膜结合 IgM-RF 进行首次蛋白质组学和转录组学分析，并确定用于 RF 克隆型跟踪的独特重链肽特征。
从 15 例原发性 SS 患者的血清 RF 中纯化重链，进行从头质谱测序。通过对匹配的外周血单核细胞（n=7）中的 IGH RNA 进行大规模平行测序来确定循环 B 细胞 Ig 库。通过将 RF 重链肽序列与相应的 IGH RNA 序列文库进行搜索，鉴定 RF 特异性重链第三互补决定区（CDR3）肽。使用定量多重反应监测，使用重链 CDR3 肽作为生物标志物跟踪血清 RF 克隆型。
血清 RF 是克隆受限的，由共享的 IgM 重链可变区（Ig V）1-69、3-15、3-7 和 3-74 亚家族组成。混合冷球蛋白血症（MC）患者的可沉淀 RF 与非沉淀 RF 不同，其氨基酸替换频率更高，并且鉴定出了刻板的重链 CDR3 转录本。通过多重反应监测，在患者出现 MC 之前的多年前就在血清中检测到了潜在致病性 RF 克隆型。免疫抑制和 MC 缓解后，Ig V 4-34 IgM-RF 水平降低。
与原发性 SS 血管炎相关的可沉淀 RF 克隆型具有与非沉淀 RF 不同的分子谱，提示其产生的潜在机制不同。本文提出的组合组学工作流程为跟踪和清除致病性 RF 克隆提供了分子生物标志物。
