High-Throughput Characterization of Blood Serum Proteomics of IBD Patients with Respect to Aging and Genetic Factors

Abstract

To date, no large scale, systematic description of the blood serum proteome has been performed in inflammatory bowel disease (IBD) patients. By using microarray technology, a more complete description of the blood proteome of IBD patients is feasible. It may help to achieve a better understanding of the disease. We analyzed blood serum profiles of 1128 proteins in IBD patients of European descent (84 Crohn's Disease (CD) subjects and 88 Ulcerative Colitis (UC) subjects) as well as 15 healthy control subjects, and linked protein variability to patient age (all cohorts) and genetic components (genotype data generated from CD patients). We discovered new, previously unreported aging-associated proteomic traits (such as serum Albumin level), confirmed previously reported results from different tissues (i.e., upregulation of APOE with aging), and found loss of regulation of MMP7 in CD patients. In carrying out a genome wide genotype-protein association study (proteomic Quantitative Trait Loci, pQTL) within the CD patients, we identified 41 distinct proteomic traits influenced by cis pQTLs (underlying SNPs are referred to as pSNPs). Significant overlaps between pQTLs and cis eQTLs corresponding to the same gene were observed and in some cases the QTL were related to inflammatory disease susceptibility. Importantly, we discovered that serum protein levels of MST1 (Macrophage Stimulating 1) were regulated by SNP rs3197999 (p = 5.96E-10, FDR<5%), an accepted GWAS locus for IBD. Filling the knowledge gap of molecular mechanisms between GWAS hits and disease susceptibility requires systematically dissecting the impact of the locus at the cell, mRNA expression, and protein levels. The technology and analysis tools that are now available for large-scale molecular studies can elucidate how alterations in the proteome driven by genetic polymorphisms cause or provide protection against disease. Herein, we demonstrated this directly by integrating proteomic and pQTLs with existing GWAS, mRNA expression, and eQTL datasets to provide insights into the biological processes underlying IBD and pinpoint causal genetic variants along with their downstream molecular consequences.

Trial registration: ClinicalTrials.gov NCT00771667.

Conflict of interest statement

We have read the journal's policy and the authors of this manuscript have the following competing interests. The following authors: SET, CB, KL and RD are paid employees of Janssen R&D LLC. The work is partially supported by Janssen R&D LLC.

Figures

Fig 1. Heatmap of top significant age-related proteins.

Included in the heatmap are all microarray probes with pvalue ≤ 1E-4 in at least one cohort. The UC, CD and NC cells are color-coded according to the Wald t-test of the age coefficient of the protein levels in each cohort, with the t test further reported in each cell. The last 2 columns report previously known association of gene mRNA levels with age within different tissues. Shades of green indicate increase of protein or mRNA level with aging; shades of red indicate decrease of protein or mRNA level with aging; the white color indicates lack of evidence in either direction.

Fig 2. Increase of Albumin levels with aging in CD and UC.

Scatterplot of the Albumin protein level vs patients age, separately for UC patients (left panel) and CD patients (baseline data only is displayed, right panel). Age in years on the horizontal axis; mean-centered and adjusted log2-protein expression on the vertical axis (adjusted for sex and plateID).

Fig 3. Variation of APOE levels with aging in CD, UC and NC subjects.

Forest plot of the estimated log2-FC of APOE protein levels (probe SL000276) per 10 years increase in age, with 95% confidence intervals, as obtained from the differential expression analysis performed separately in Crohn’s Disease (CD), Ulcerative Colitis (UC) and Normal Controls (NC) subjects. Estimated log2-FCs and 95% Confidence Intervals are further reported on the right.

Fig 4. Enrichment for GWAS signals in blood protein-QTLs of CD patients.

Expected uniform -log10(relative rank) of the protein-SNPs (nominal pvalue ≤ 1E-5) within the full GWAS SNPs list on the horizontal axis; observed–log10(relative rank) on the vertical axis. CD: Crohn’s Disease; UC: Ulcerative Colitis; BMI: Body Mass Index; SCZ: Schizofrenia; Stroke: Ischemic Stroke; T2D: Type-2 Diabetes. References for all the studies are reported in the Methods section.

Fig 5. Locuszoom plot of MST1, CD and UC association pvalues around the MST1 gene.

It is worth noting that our proteomics platform has 4 probes in this chromosomal region, targeting 4 different proteins: IMPDH2 (probe SL010928), MST1 (probe SL005202), MST1R (probe SL004637) and MAPKAPK3 (probe SL004765). Of these, MST1 is most significantly associated with the IBD GWAS SNP in this locus (S3 Fig), and the association pattern was highly consistent with the CD and UC GWAS peaks (Fig 5).