HumanMethylation450 array

The HumanMethylation450 array (Illumina, San Diego, USA) was used to obtain genome-wide methylation levels. This array comprises 485,000 CpG sites throughout the genome and covers 99% of RefSeq genes and 96% of CpG islands. In order to reduce technical bias, patients and controls were randomly divided over the analysis. DNA was bisulfite converted using the EZ DNA methylation kit of ZYMO® and subsequently submitted for analysis on the HumanMethylation450 array, outsourced to GenomeScan, Leiden, The Netherlands (ISO/IEC 17025 approved). The quality of raw methylation data was assessed using the MethylAid script in R (GenomeScan’s Guidelines for Successful Methylation Experiments Using the Illumina Infinium® HumanMethylation BeadChip). All the samples met the quality criteria. To analyze our data, we used a statistical method for single-sample analysis described by Rezwan et al. [9] adapted to a MINFI package in R (based on the Crawford-Howell t test). Prior to this analysis, all probes known to involve polymorphic sites (minor allele frequency, MAF > 0.01), cross-hybridization probes, and probes located on sex chromosomes were removed from the dataset. The workflow and QC details are depicted in the Additional file 1. The 450 k data was normalized with the function preprocessFunnorm of MINFI [10].

Differentially methylated positions (DMP) with assigned adjusted P value for M-values smaller than 0.05 were considered as significant (adjusted according to the false discovery rate; FDR).