Epigenetic alterations including DNA methylation and microRNAs may provide important
insights into gene-environment interaction in complex immune diseases such as
inflammatory bowel disease (IBD). An integrative genome-wide approach was used to analyse
whole blood genetic, DNA methylation and gene expression data in 240 newly diagnosed IBD
patients and 190 controls. Using the Illumina 450k array, differences in whole blood DNA
methylation were observed in IBD cases versus controls including 439 differentially
methylated positions (DMPs) and 5 differentially methylated regions (DMRs). The top DMP
(RPS6KA2, discovery Holm adjusted p=1.22×10-16, replication p=1×10-9) and DMRs (VMP1,
ITGB2, TXK) were replicated in an independent cohort using pyrosequencing. Paired genetic
and epigenetic data allowed the identification of methylation quantitative trait loci (meQTL);
two of the five DMRs (VMP1, ITGB2) demonstrated significant association with genetic
polymorphisms. Methylation in the VMP1/microRNA-21 region was significantly associated
with two single nucleotide polymorphisms (cg18942579 -rs10853015 [meQTL FDR adjusted
p=9.4 × 10-5], cg16936953 - rs8078424 [meQTL FDR adjusted p=8.8 × 10-5]), both of which
are in linkage disequilibrium with a known IBD susceptibility variant (rs1292053). Separated
leukocyte methylation data highlight the cell type of origin of epigenetic signals seen in whole
blood. IBD-associated hypermethylation within the TXK gene transcription start-site
negatively correlated with gene expression in whole blood and CD8+ T-cells, but not other cell
types, highlighting that cell-specificity and gene location-specificity of DNA methylation
change is critical when associating methylation and gene expression. These data offer
significant translational potential as diagnostic biomarkers. Least absolute shrinkage and
selection operator (lasso) modelling identified 30 methylation probes can be used to
accurately discriminate IBD cases from controls (Area under receiver operating characteristic
curve = 0.898, sensitivity = 90.6%, specificity = 84.7%).
MicroRNAs (miRNA) are small non-coding nucleic acids that have the capacity to modulate
gene expression. MiRNAs have been increasingly implicated in many of the important IBD
pathogenic pathways including autophagy, intestinal epithelial barrier integrity and the Th17
pathway. In common with all epigenetic mechanisms, miRNA expression is dynamic and cell-specific.
Small RNA sequencing (RNA-seq) was performed on RNA extracted from CD14+,
CD4+ and CD8+ cells isolated from 8 newly diagnosed cases of ileal or ileocolonic CD and 8
age and sex matched controls. There was a median of 2.4 million reads per sample (range
132,800-12.8 million reads per sample). One microRNA was differentially expressed in CD
compared with controls (hsa-miR-503-5p log fold change = 0.7, FDR adjusted p = 9.1 × 10-5)
in CD4+ lymphocytes, however this finding did not remain significant when alternative
normalisation methods were used. The small number of cases used in microRNA analyses
raises the possibility of both type I and II error, and limits the ability to draw firm conclusion
from this series of experiments.
Site-specific differences in DNA methylation in IBD relate to underlying genotype and
associate with cell-specific alteration in gene expression. This is the most detailed
characterisation of the epigenome carried out in IBD to date. The findings strongly validate
this approach in complex disease, are replicable, and provide clear translational opportunities
