Analysis of Systemic Epigenetic Alterations in Inflammatory Bowel Disease: Defining Geographical, Genetic and Immune-Inflammatory influences on the Circulating Methylome

Abstract

Background: Epigenetic alterations may provide valuable insights into gene-environment interactions in the pathogenesis of inflammatory bowel disease [IBD].

Methods: Genome-wide methylation was measured from peripheral blood using the Illumina 450k platform in a case-control study in an inception cohort (295 controls, 154 Crohn's disease [CD], 161 ulcerative colitis [UC], 28 IBD unclassified [IBD-U)] with covariates of age, sex and cell counts, deconvoluted by the Houseman method. Genotyping was performed using Illumina HumanOmniExpressExome-8 BeadChips and gene expression using the Ion AmpliSeq Human Gene Expression Core Panel. Treatment escalation was characterized by the need for biological agents or surgery after initial disease remission.

Results: A total of 137 differentially methylated positions [DMPs] were identified in IBD, including VMP1/MIR21 [p = 9.11 × 10-15] and RPS6KA2 [6.43 × 10-13], with consistency seen across Scandinavia and the UK. Dysregulated loci demonstrate strong genetic influence, notably VMP1 [p = 1.53 × 10-15]. Age acceleration is seen in IBD [coefficient 0.94, p < 2.2 × 10-16]. Several immuno-active genes demonstrated highly significant correlations between methylation and gene expression in IBD, in particular OSM: IBD r = -0.32, p = 3.64 × 10-7 vs non-IBD r = -0.14, p = 0.77]. Multi-omic integration of the methylome, genome and transcriptome also implicated specific pathways that associate with immune activation, response and regulation at disease inception. At follow-up, a signature of three DMPs [TAP1, TESPA1, RPTOR] were associated with treatment escalation to biological agents or surgery (hazard ratio of 5.19 [CI: 2.14-12.56], logrank p = 9.70 × 10-4).

Conclusion: These data demonstrate consistent epigenetic alterations at diagnosis in European patients with IBD, providing insights into the pathogenetic importance and translational potential of epigenetic mapping in complex disease.

Keywords: DNA methylation; Mendelian randomization; epigenetic clock; gene expression; genetics; inflammatory bowel diseases [IBD]; methylation; prognosis; quantitative trait loci.

Figure 1.

[A] Manhattan plot of top differentially methylated positions [DMPs] in IBD vs controls in Scandinavia. Yellow dots represent hits that replicate in the UK cohort and purple dots represent hits that replicate in Spain. [B] Volcano plot of the top DMPs across Scandinavia. Horizontal dashed line represents Holm significance. The y-axis for both plots represents −log₁₀[p-values] with the horizontal line representing statistical significance after Holm testing.

Figure 2.

Correlation between DNA methylation probes and gene expression in IBD [Holm p < 0.05] where no significant correlation was seen in non-IBD for these probes [Holm p > 0.05]. The y-axis represents gene names. Bar colours represent CpG probe location within the gene. TSS200 and TSS1500, 200 or 1500 nucleotides upstream of the transcriptional start sites. Within regulatory regions of a gene (5ʹ untranslated region [5ʹUTR], 3ʹ untranslated region [3ʹUTR]) or body of the gene [Body].

Figure 3.

Multi-omic factor analysis [MOFA] integrating IBD genome-wide association analysis [GWAS] polymorphisms, mRNA expression of IBD-related genes and differentially IBD-related methylated positions [DMPs]. [A] MOFA heatmap demonstrating the variance explained by each factor. [B–E] Analyses demonstrating the correlation of the MOFA factors with inflammatory markers such as hsCRP, IBD, age and smoking. [F] Boxplots for Factors 1 and 4 in IBD and controls. [G] Boxplot showing the association of Factor 1 with disease extent in UC. [H, I] Correlation plots of the key methylation and mRNA expression genes that define Factor 1 and Factor 4. [J] Gene ontology analysis of the top MOFA Factor 1 and Factor 4.

Figure 4.

A three-probe methylation panel predicts treatment escalation in inflammatory bowel disease [IBD]. Kaplan–Meier survival curve demonstrating the proportion of IBD patients who did not require a treatment escalation at diagnosis. High [red] and low [blue] risk represents patient groups defined as high or low risk of treatment escalation based on the three-probe panel.