Molecular classification of Crohn's disease reveals two clinically relevant subtypes

Abstract

Objective: The clinical presentation and course of Crohn's disease (CD) is highly variable. We sought to better understand the cellular and molecular mechanisms that guide this heterogeneity, and characterise the cellular processes associated with disease phenotypes.

Design: We examined both gene expression and gene regulation (chromatin accessibility) in non-inflamed colon tissue from a cohort of adult patients with CD and control patients. To support the generality of our findings, we analysed previously published expression data from a large cohort of treatment-naïve paediatric CD and control ileum.

Results: We found that adult patients with CD clearly segregated into two classes based on colon tissue gene expression-one that largely resembled the normal colon and one where certain genes showed expression patterns normally specific to the ileum. These classes were supported by changes in gene regulatory profiles observed at the level of chromatin accessibility, reflective of a fundamental shift in underlying molecular phenotypes. Furthermore, gene expression from the ilea of a treatment-naïve cohort of paediatric patients with CD could be similarly subdivided into colon-like and ileum-like classes. Finally, expression patterns within these CD subclasses highlight large-scale differences in the immune response and aspects of cellular metabolism, and were associated with multiple clinical phenotypes describing disease behaviour, including rectal disease and need for colectomy.

Conclusions: Our results strongly suggest that these molecular signatures define two clinically relevant forms of CD irrespective of tissue sampling location, patient age or treatment status.

Keywords: CROHN'S DISEASE; ENERGY METABOLISM; GENE REGULATION; IBD CLINICAL; RNA EXPRESSION.

Figure 1. Two distinct molecular subtypes in adult CD

(A) PCA analysis of RNA-seq data from colon tissue from 21 CD and 11 non-IBD patients shows two distinct clusters. (B) 849 genes are differentially expressed between the two CD subclasses (adjusted p<0.05, DEseq), defined as colon-like and ileum-like. Known markers of colon and ileum are highlighted. (C) Genes upregulated in colon-like (top) and ileum-like (bottom) CD subclasses overlap previously defined colon-specific and ileum-specific genes, respectively. (D) Differentially accessible regions (DARs) identified using FAIRE-seq (p<0.05, two-sided t-test, normalized read counts, 300-bp windows) show distinct profiles in colon-like and ileum-like CD samples. (E) Colon-like-associated DARs are enriched and ileum-like-associated DARs are depleted near genes upregulated in colon-like samples (p≤0.01, permutation test). (F) Ileum-like-associated DARs are enriched and colon-like-associated DARs are depleted near genes upregulated in ileum-like samples (p<0.001, permutation test).

Figure 2. Treatment-naïve pediatric CD samples show similar molecular subtypes

(A) PCA analysis of combined RNA-seq data from adult colon tissue and pediatric ileum tissue from CD and non-IBD patients shows separation of tissue types (PC1) and replicates ileum-like and colon-like clusters (PC2). Expression of APOA1 (blue-pink, low-high) in pediatric samples aligns well with subclasses. (B) Hierarchical clustering of RNA-seq data using 500 colon- and ileum-specific genes show clusters of genes associated with ileum-like and colon-like samples across both the adult colon and pediatric ileum cohorts, as well as genes associated with tissue of origin.

Figure 3. Pathways enriched for differentially expressed genes associated with CD phenotypes

Pathway enrichments were determined using GSAA (FDR<0.1, permutation test) for all pairwise comparisons between all CD, colon-like CD, ileum-like CD, and non-IBD samples. Separate analyses in (A) adult colon and (B) pediatric ileum show similar and unique pathways associated with CD phenotypes. Each circle represents Reactome-defined pathway with the size reflecting the number of genes in the pathway. Pathways were grouped based on similarity in gene membership, and labels describing multi-pathway clusters are shown. See Supplementary Figures 3–4 for CD vs non-IBD comparisons and full list of pathways.