Genetic and Transcriptomic Bases of Intestinal Epithelial Barrier Dysfunction in Inflammatory Bowel Disease

Abstract

Background: Intestinal barrier defects are common in patients with inflammatory bowel disease (IBD). To identify which components could underlie these changes, we performed an in-depth analysis of epithelial barrier genes in IBD.

Methods: A set of 128 intestinal barrier genes was selected. Polygenic risk scores were generated based on selected barrier gene variants that were associated with Crohn's disease (CD) or ulcerative colitis (UC) in our study. Gene expression was analyzed using microarray and quantitative reverse transcription polymerase chain reaction. Influence of barrier gene variants on expression was studied by cis-expression quantitative trait loci mapping and comparing patients with low- and high-risk scores.

Results: Barrier risk scores were significantly higher in patients with IBD than controls. At single-gene level, the associated barrier single-nucleotide polymorphisms were most significantly enriched in PTGER4 for CD and HNF4A for UC. As a group, the regulating proteins were most enriched for CD and UC. Expression analysis showed that many epithelial barrier genes were significantly dysregulated in active CD and UC, with overrepresentation of mucus layer genes. In uninflamed CD ileum and IBD colon, most barrier gene levels restored to normal, except for MUC1 and MUC4 that remained persistently increased compared with controls. Expression levels did not depend on cis-regulatory variants nor combined genetic risk.

Conclusions: We found genetic and transcriptomic dysregulations of key epithelial barrier genes and components in IBD. Of these, we believe that mucus genes, in particular MUC1 and MUC4, play an essential role in the pathogenesis of IBD and could represent interesting targets for treatment.

Figure 1. Quartile analysis of the barrier risk scores in patients and controls

The percentage of individuals in the quartiles (Q1-Q4) of the CD barrier risk scores (A) and UC barrier risk scores (B). Comparisons of the number of individuals in Q1 and Q4 was done with Chi-squared testing. *Statistically significant (p<2.2x10^-16 for A and B). CD, Crohn’s disease; UC, ulcerative colitis; Q, quartile.

Figure 2. Enrichment analysis of associated barrier genes (≥1 SNP) per component

Bar plots representing the percentage of significant genes in each of the barrier components for CD (left) and UC (right). Only the set of regulating proteins was significantly enriched for UC in associated barrier genes compared to the other barrier components using Fisher Exact testing (*p<0.05). CD, Crohn’s disease; UC, ulcerative colitis.

Figure 3. Relative expression levels of eight barrier genes with quantitative reverse transcription PCR

Bar plots representing the relative expression levels of eight barrier genes (A-H) measured by qRT-PCR in colon (white bars) from controls (n=11), active UC (n=72), inactive UC (n=22), active CD patients (n=8) and inactive CD patients (n=26); and ileum (black bars) from controls (n=11), active CD (n=51) and inactive CD patients (n=14). The expression levels are normalised to beta-actin. Data are expressed as medians with interquartile range. Comparisons between the subgroups were performed with Mann-Whitney U testing. Significant differences as described in the main text are indicated (*p<0.05, **p<0.01). UC, ulcerative colitis; CDc, colon of Crohn’s disease patients; CDi, ileum of CD patients.

Figure 4. Schematic overview of the main results

The genetic and transcriptomic approaches in this study identified the potential role of particular epithelial barrier genes and components in the context of IBD. We found that disease-associated variants were significantly enriched in MUC19 (secreted mucin), MUC22 (membrane-bound mucin), TFF1 (stabilizing mucus layer protein) and PTGER4 (regulating protein) for CD, and MUC21 (membrane-bound mucin), MUC22 (membrane-bound mucin), GNA12 (regulating protein) and HNF4A (regulating protein) for UC. The most enriched barrier component was the set of regulating proteins for both CD and UC. At mRNA level, persistent changes in ileal and colonic MUC1 (membrane-bound mucin) and MUC4 (membrane-bound mucin) expression were found during inactive disease for CD and UC, pinpointing to a possible central role of these genes in IBD onset/relapse. In uninflamed CD ileum, also CLDN8 expression remained strongly dysregulated. Genetic predispositions and expression changes may together induce barrier dysfunction of the intestinal epithelium in IBD patients, and result in an enhanced uptake of harmful luminal antigens and initiation of inflammation. Symbols within the mucus layer are explained below the figure. Structures within the epithelial cells are defined by the category names. Arrows indicate persistent up- or downregulation of the genes during inactive disease. ^asignificant enrichment of variants associated with CD; ^bsignificant enrichment of variants associated with UC.