Abstract
Crohn's Disease (CD) is a relapsing inflammation of the gastrointestinal tract that affects a young working age population and is increasing in developing countries. Half of all sufferers will experience stricturing or fistulizing intestinal complications that require extensive surgical interventions and neither genes nor clinical risk factors can predict this debilitating natural history. We applied discovery and verification phase studies as part of an NCI-FDA modeled biomarker pipeline to identify differences in the low-mass (<25kDa) blood-serum proteome between CD behavioral phenotypes. A significant enrichment of epithelial component proteins was identified in CD patients with intestinal complications using quantitative proteomic profiling with label-free Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). DAVID 6.7 (NIH) was used for functional annotation analysis of detected proteins and immunoblotting and multiple reaction monitoring (MRM) to verify a priori findings in a secondary independent cohort of complicated CD (CCD), uncomplicated inflammatory CD (ICD), Th1/17 pathway inflammation controls (rheumatoid arthritis), inflammatory bowel disease controls (ulcerative colitis), and healthy controls. Seventy-six high-confidence serum proteins were modulated in CCD versus ICD by LC-MS/MS (p < 0.05, FDR q<0.01), annotating to pathways of epithelial barrier homeostasis (p < 0.01). In verification phase, a putative serology panel developed from discovery proteomics data consisting of desmoglein-1, desmoplakin, and fatty acid-binding protein 5 (FABP5) distinguished CCD from all other groups (p = 0.041) and discriminated complication in CD (70% sensitivity and 72.5% specificity at score ≥1.907, AUC = 0.777, p = 0.007). An MRM assay secondarily confirmed increased FABP5 levels in CCD (p < 0.001). In a longitudinal subanalysis-cohort, FABP5 levels were stable over a two-month period with no behavioral changes (p = 0.099). These studies along the biomarker development pipeline provide substantial proof-of-principle that a blood test can be developed specific to transmural intestinal injury. Data are available via the PRIDE proteomics data repository under identifier PXD001821 and PeptideAtlas with identifier PASS00661.
Highlights
Crohn’s disease (CD)1 is a progressive Inflammatory Bowel Disease (IBD) in which more than half of all patients will experience a stricturing (SCD) or fistulizing (FCD) complication within 10 years from diagnosis [1, 2]
We examine the low-mass (Ͻ25kDa) serum protein fraction between CD behavioral phenotypes in discovery and qualification phase proteomic studies of the National Cancer Institute-Food and Drug Administration (NCI-FDA) developed clinical biomarker pipeline [13]
Desmosomal and Anti-Microbial Proteins Selected for Qualification Phase Proteomics—DSG1, DSK and fatty acid-binding protein 5 (FABP5) proteins were selected for immunoblot qualification based on significant differential regulation (p Ͻ 0.01) by label-free LC-MS/MS (Fig. 2A–2C) and inclusion in the significantly enriched clusters and Gene Ontology (GO) terms by functional annotation analysis (BHcorrected EASE score p Ͻ 0.01) (Fig. 1C; Table II)
Summary
Study Population and Sample Collection—Serum samples were obtained from subjects recruited from Concord Repatriation General Hospital and Bankstown-Lidcombe Hospital, Sydney Australia. Label-Free LC-MS/MS—Low-mass fraction serum samples were prepared with C18 stage tips (Thermo Scientific, IL, USA) according to manufacturer recommendations except for an 80% Acetonitrile, 0.1% formic acid elution buffer. Statistical comparisons between groups were performed by one-way ANOVA with multiple testing correction by FDR q value significance set at Ͻ 0.01(18). Immunoblot Assay—Dot blots for desmoglein-1 (DSG1), desmoplakin (DSK), and fatty acid-binding protein 5 (FABP5) proteins were performed in serum samples following standard procedures [21]. Discriminant function analysis was used to determine discriminability of DSG1, DSK, and FABP5 for CCD and the subsequent discriminant function coefficients were used to weight each protein value contribution to a combined biomarker panel score— hereafter referred to as the Serum Epithelial Components (SEC) score.
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