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.
Article first published online 20 August 2014.
*Gastrointestinal Unit, Centre for Genetics and Experimental Medicine, Institute of Genetics and Molecular Medicine, Western General Hospital, Edinburgh, United Kingdom;
†Gastrointestinal Research Group, Division of Applied Medicine, University of Aberdeen, Aberdeen, United Kingdom;
‡Department of Paediatric Gastroenterology, Royal Hospital for Sick Children, Glasgow, United Kingdom; and
§Paediatric Gastroenterology and Nutrition, Child Life and Health, University of Edinburgh, Royal Hospital for Sick Children, Edinburgh, United Kingdom.
Reprints: Jack Satsangi, DPhil, Gastrointestinal Unit, Centre for Genetics and Experimental Medicine, Institute of Genetics and Molecular Medicine, Western General Hospital, Edinburgh, EH4 2XU, United Kingdom (e-mail: firstname.lastname@example.org).
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The authors have no conflicts of interest to disclose.
A.T. Adams, N.A. Kennedy, and R. Hansen are co-first authors. G.L. Hold and J. Satsangi as co-senior authors.
Received July 08, 2014
Accepted July 08, 2014