Genome-Wide Methylation Profiling in 229 Patients With Crohn's Disease Requiring Intestinal Resection: Epigenetic Analysis of the Trial of Prevention of Post-operative Crohn's Disease (TOPPIC)

Abstract

Background & aims: DNA methylation alterations may provide important insights into gene-environment interaction in cancer, aging, and complex diseases, such as inflammatory bowel disease (IBD). We aim first to determine whether the circulating DNA methylome in patients requiring surgery may predict Crohn's disease (CD) recurrence following intestinal resection; and second to compare the circulating methylome seen in patients with established CD with that we had reported in a series of inception cohorts.

Methods: TOPPIC was a placebo-controlled, randomized controlled trial of 6-mercaptopurine at 29 UK centers in patients with CD undergoing ileocolic resection between 2008 and 2012. Genomic DNA was extracted from whole blood samples from 229 of the 240 patients taken before intestinal surgery and analyzed using 450KHumanMethylation and Infinium Omni Express Exome arrays (Illumina, San Diego, CA). Coprimary objectives were to determine whether methylation alterations may predict clinical disease recurrence; and to assess whether the epigenetic alterations previously reported in newly diagnosed IBD were present in the patients with CD recruited into the TOPPIC study. Differential methylation and variance analysis was performed comparing patients with and without clinical evidence of recurrence. Secondary analyses included investigation of methylation associations with smoking, genotype (MeQTLs), and chronologic age. Validation of our previously published case-control observation of the methylome was performed using historical control data (CD, n = 123; Control, n = 198).

Results: CD recurrence in patients following surgery is associated with 5 differentially methylated positions (Holm P < .05), including probes mapping to WHSC1 (P = 4.1 × 10^-9, Holm P = .002) and EFNA3 (P = 4.9 × 10^-8, Holm P = .02). Five differentially variable positions are demonstrated in the group of patients with evidence of disease recurrence including a probe mapping to MAD1L1 (P = 6.4 × 10^-5). DNA methylation clock analyses demonstrated significant age acceleration in CD compared with control subjects (GrimAge + 2 years; 95% confidence interval, 1.2-2.7 years), with some evidence for accelerated aging in patients with CD with disease recurrence following surgery (GrimAge +1.04 years; 95% confidence interval, -0.04 to 2.22). Significant methylation differences between CD cases and control subjects were seen by comparing this cohort in conjunction with previously published control data, including validation of our previously described differentially methylated positions (RPS6KA2 P = 1.2 × 10^-19, SBNO2 = 1.2 × 10^-11) and regions (TXK [false discovery rate, P = 3.6 × 10^-14], WRAP73 [false discovery rate, P = 1.9 × 10^-9], VMP1 [false discovery rate, P = 1.7 × 10^-7], and ITGB2 [false discovery rate, P = 1.4 × 10^-7]).

Conclusions: We demonstrate differential methylation and differentially variable methylation in patients developing clinical recurrence within 3 years of surgery. Moreover, we report replication of the CD-associated methylome, previously characterized only in adult and pediatric inception cohorts, in patients with medically refractory disease needing surgery.

Keywords: Aging; Crohn's disease; DNA methylation; Epigenetics; Inflammatory bowel disease; Surgery.

Graphical abstract

Figure 1

Flowchart of cohorts and analyses.

Figure 2

ShinyMethyl output for quality control for TOPPIC methylation data. (A) Average negative control probe intensities. (B) Median intensity of M channel against median intensity of the u channel. (C, D) M-value intensities before and after functional normalization. (E–G) MDS during processing steps. (E) Raw data. (F) Following quantile normalization. (G) Following filtering of SNPs and sex chromosomes. (H) ShinyMethyl sex-prediction plot. No samples were mismatched for sex.

Figure 3

Batch correction for TOPPIC-only methylation cohort. (A, C, E) QQ plots and Lambda values for the (A) TOPPIC cohort following BMIQ and quantile normalization, (C) Combat correction for Chip (21 batches), and (E) Combat correction for position on array (12 batches). (B, D, F) Multidimensional scaling plots showing the first 2 principal components (B) TOPPIC cohort following BMIQ and quantile normalization, (D) Combat correction for Chip (21 batches), and (F) Combat correction for position on array (12 batches). Inner color, between array batch; outer color, intra-array batch; triangles, technical replicates.

Figure 4

QQ plots and Lambda values for the originally combined TOPPIC and BIOM datasets following BMIQ and quantile normalization (A), followed by Combat correction for methylation chip (B) and location within each chip (C). MDS scaling plot of the first 2 principal components for the originally combined of TOPPIC and BIOM datasets following BMIQ and quantile normalization (D), followed by Combat correction for methylation chip (E), and location within each chip (F). Colors (blue, red) denote different experimental batches. Green labelled points denote technical replicates included across chips, plates, runs, and batches.

Figure 5

Principal component plot of the first 2 components (PC1, PC2) using 1000 most variable probes of the combined TOPPIC and BIOM cohorts.Colors correspond to technical replicates. Shapes refer to 450K scan date. (A) All replicates. (B) Replicates 1 and 2 removed.

Figure 6

(A) Violin and box plots of DMPs associated with disease recurrence (clinical end point) following surgery for Crohn's disease. (B) Violin and box plots of DVPs associated with disease recurrence (clinical end point) following surgery for Crohn's disease (defined as increase in Crohn’s disease activity index of more than 150 and an increase of 100 points from baseline measurement and institution of immunosuppressive treatment or further surgery).

Figure 9

cis meQTLs of DVP/DMP probes. Top SNP shown. Age, sex, smoking status used as covariates. MAF of <10% filtered. Cis distance 1 × 10⁶, P value threshold <2 × 10^-6.

Figure 10

Cis meQTLs of DVP or DMP probes associated with Crohn’s disease recurrence following surgery. Top SNP shown. Age, sex, smoking status used as covariates. MAF of <10% filtered. Cis distance 1 × 10⁶, P value threshold <2 × 10^-6.

Figure 7

(A) Aryl hydrocarbon receptor repressor ARHH/cg05575921 methylation in smokers and nonsmokers and exsmokers in the entire cohort (combined CD and control subjects in both cohorts). (B) Correlation plot between smoking and exsmoker/nonsmoker log fold change beta value in Gao et al meta-analysis and in the present study (DMPs, Holm P < .05, entire cohort CD and control subjects combined). (C) Venn diagram of overlapping probes in Gao et al meta-analysis, smokers versus nonsmokers (DMPs, Holm P < .05) in total cohort (CD and control subjects combined), Crohn’s disease patients only, and control subjects only. The 9 smoking-associated CpGs seen only in the Crohn’s cohort are listed in the box. (D) Venn diagram of Crohn’s disease versus control DMPs in the entire cohort without using smoking as a covariate, entire cohort using smoking as a covariate, and in the smoking-associated DMPs in the Crohn’s only cohort. There are 4 CpGs that overlap that are both associated with Crohn’s (vs control, DMPs) and smoking (smoking vs exsmoker/never smoker, DMPs) that are listed in the boxes.

Figure 8

Epigenetic age analysis using methods by (from right to left) Horvath (DNAmAge),Hannum,tissue specific (skin and blood clock),phenoAge,and GRIMage clocks. (A) Correlation plot of methylation age (y-axis) and biologic age (x-axis) using methods above, inset, density plot of methylation age). Cor, Pearsons R Correlation estimate. (B) Boxplots of age acceleration using methods above in patients with Crohn’s disease requiring surgery (CD_TOPPIC), newly diagnosed Crohn’s disease patients (CD_BIOM), and control subjects. (C) Boxplots of age acceleration in patients included in the TOPPIC trial who went on to develop recurrence or no recurrence following surgery. (C) Box plot for each methylation clock age acceleration and smoking status, current, exsmoking (recorded in the BIOM cohort), exsmoker/never smoker (grouped together as part of the TOPPIC cohort), and never smoked (recorded in the BIOM cohort). Ns = P > .05, ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001 (Wilcox test).