This research used combined bioinformatic methods to identify differentially methylated regions (DMRs) in newly diagnosed patients with Graves' disease (GD). Peripheral blood from six GD patients and controls was collected and methyl-DNA immunoprecipitation (MeDIP), and NimbleGen Human DNA Methylation 3 x 720 K promoter plus CpG island microarrays were further analyzed. DMRs were categorized into low-methylated genes and high-methylated genes, which were mapped into a protein-protein interaction (PPI) network constructed by a dataset. Then, six candidate genes were validated in an expanded population with 32 GD patients and 30 controls using bisulfite amplicon sequencing. Top 10 hub genes revealed by PPI analysis were CRHR1, CAMK2A, SERPINA1, RANBP9, ICAM1, ADRB2, KRTAP13-1, PTPRA, S100A2, and KPRP. Five CpG sites of CDKN2C (51436061), SERPINA1 (94856657), B3GNT2 (62422532 and 62422689), and IRS4 (107979477) were validated, having significantly different methylation levels between GD patients and controls. Based on gender stratification, nine significant CpG sites of CDKN2C (51436061), SERPINA1 (94855831), and B3GNT2 (62422301, 62422327, 62422356, 62422365, 62422374, 62422532, and 62422689) were detected between female GD patients and controls. The methylation level of 62422532 of B3GNT2 was significantly associated with levels of serum TGAb and TRAb. In addition, the methylation level of 62422689 of B3GNT2 showed significant correlation with the age of GD patients. In the analysis of prediction of transcription factor binding at specific CpG sites in B3GNT2 promoter region, paired box protein 5 (Pax-5) and CCAAT/enhancer-binding protein (C/EBP beta) might be under the influence of methylation at CpG sites 62422365 and 62422532, respectively. CDKN2C, SERPINA1, IRS4, and especially B3GNT2 were potential aberrantly methylated genes related to GD. These findings might supply the latest information of DNA methylation in the GD disease.
