Two-stage genome-wide methylation profiling in childhood-onset Crohn's Disease implicates epigenetic alterations at the VMP1/MIR21 and HLA loci

Abstract

Background: As a result of technological and analytical advances, genome-wide characterization of key epigenetic alterations is now feasible in complex diseases. We hypothesized that this may provide important insights into gene-environmental interactions in Crohn's disease (CD) and is especially pertinent to early onset disease.

Methods: The Illumina 450K platform was applied to assess epigenome-wide methylation profiles in circulating leukocyte DNA in discovery and replication pediatric CD cohorts and controls. Data were corrected for differential leukocyte proportions. Targeted replication was performed in adults using pyrosequencing. Methylation changes were correlated with gene expression in blood and intestinal mucosa.

Results: We identified 65 individual CpG sites with methylation alterations achieving epigenome-wide significance after Bonferroni correction (P < 1.1 × 10(-7)), and 19 differently methylated regions displaying unidirectional methylation change. There was a highly significant enrichment of methylation changes around GWAS single nucleotide polymorphisms (P = 3.7 × 10(-7)), notably the HLA region and MIR21. Two-locus discriminant analysis in the discovery cohort predicted disease in the pediatric replication cohort with high accuracy (area under the curve, 0.98). The findings strongly implicate the transcriptional start site of MIR21 as a region of extended epigenetic alteration, containing the most significant individual probes (P = 1.97 × 10(-15)) within a GWAS risk locus. In extension studies, we confirmed hypomethylation of MIR21 in adults (P = 6.6 × 10(-5), n = 172) and show increased mRNA expression in leukocytes (P < 0.005, n = 66) and in the inflamed intestine (P = 1.4 × 10(-6), n = 99).

Conclusions: We demonstrate highly significant and replicable differences in DNA methylation in CD, defining the disease-associated epigenome. The data strongly implicate known GWAS loci, with compelling evidence implicating MIR21 and the HLA region.

FIGURE 1

A, Schematic representation of study design, n = ratio of CD samples to control samples. B, Log fold-change (log₂ mean methylation in CD/mean methylation in controls) for all probes with nominally significant (uncorrected P < 0.05, n = 3620) methylation changes in both pediatric cohorts. Data are binned; colors of shorter wavelength indicate higher frequency. C, A 50 kb of genomic regions are more likely to contain GWAS SNPs for IBD or CD if they also contain more significant disease-associated methylation changes (P = 3.66 × 10⁻⁷). D, Manhattan plot of disease-associated methylation changes; horizontal line corresponds to significance after Bonferroni correction.

FIGURE 2

A, Separation by diagnosis is achieved by plotting beta values for combinations of 2 Illumina 450k probes with significant disease-associated methylation changes in the discovery cohort. Beta values for both discovery (open) and replication (filled) cohorts are shown. Area under the curve for the model based on each probe combination shown in the top right of each panel. B, Replication of disease-associated methylation changes in 7 significant probes by pyrosequencing in 40 adults. HC, healthy controls.

FIGURE 3

Schema for the selection of targets for further study, showing examples and total numbers for each set. GWAS risk loci correspond to all CD and IBD results from recent GWAS meta-analysis, inclusion within epigenome-wide significance and differentially methylated regions sets based on individual probe significance surviving Bonferroni correction, and being identified as a DMR by the modified ChAMP algorithm, respectively (Methods). VMP1/MIR21 shown at the intersection of all 3 sets.

FIGURE 4

A, The significance of disease-associated methylation changes in all Illumina 450k probes in VMP1. A schematic representation of the gene is overlaid (bars represent exons, lines represent introns), the height of which corresponds to a Bonferroni corrected P < 0.05. B, Expanded view of the 3′ end of VMP1 in (A), with MIR21 primary transcript (line) and mature MIR21 (bar) plotted below. C, Beta values for each sample, colored by diagnosis, at all Illumina 450k probes across VMP1 (excluding the unmethylated 5′ CpG island). Background shading highlights probes contained in the DMR.

FIGURE 5

A, Replication of VMP1/MIR21 hypomethylation at cg16936953 by pyrosequencing of the same region in 172 adults. B, Increased leukocyte MIR21 primary transcript in CD measured by qPCR (n = 66). C, Microarray data showing significantly increased pri-miR-21 mRNA in response to inflammation in CD and UC. VMP1 increased in CD, but not UC or control.