DNA was isolated from 200 µL of whole blood using a MagNA Pure LC DNA Isolation Kit and MagNA Pure LC 2.0 Instrument (Roche, Basel, Switzerland), according to the manufacturer’s protocol. DNA was quantified using a broad range Quant-iT™ dsDNA Broad Range Assay Kit (Invitrogen™, Carlsbad, CA, USA). All the DNA samples underwent quality control using a PCR reaction for sex determination. For all of the samples for which the DNA concentration was above 40 ng/uL, and for which the sex determined by PCR was consistent with that described in the questionnaire were enrolled to the further procedures. A total of 117 DNA samples met the criteria for the bisulfide conversion step and further microarray analysis. A quantity of 500 ng DNA from each sample was independently treated with sodium bisulfite using an EZ DNA Methylation Kit™ (Zymo Research, Irvine, CA, USA). A genome-wide DNA methylation analysis was performed using a recently developed MethylationEPIC BeadChip (Illumina, San Diego, CA, USA), which covers more than 850,000 CpG sites. All procedures were conducted according to the manufacturers’ instructions.
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