Building on previous EWAS in AD, which have primarily focused on single CpG analysis, we applied our recently developed pipeline that aims to identify DMRs harboring multiple statistically significant CpGs exhibiting concordant disease-associated changes in methylation [ 32].
Many genes contained multiple significant sites, with an average of 4.7 CpGs per gene and a maximum of 114 CpGs.
Several of the significant CpGs overlapped with or were immediately adjacent to transcription factor binding sites (TFBSs) - including two of the FDR-significant CpG units in promoter 5.
Of these CpGs, four CpGs and two genes containing significant CpGs were replicated in an independent study of DNAm and EOC risk.
The DM analysis using a moderated t-test identified a total of 64,049 significant CpG sites after the Benjamini-Hochberg (BH) multiple hypothesis testing correction with a threshold of P <0.05.
Two CpG units had nominally significant case control differences in methylation, CpG 14 (Z = −2.37, p = 0.018) and 9,10 (Z = −2.017, p = 0.044); at each of the significant CpG units, cases had higher MMFs than controls.
This was also evident at the individual CpG level of DNA methylation analysis (below), where the majority of significant CpG units were in region D3.
We noted that several of these significant CpG units were at or in the immediate vicinity of TFBSs.
Table 2 lists the 10 most significant CpG sites in the phase I study.
109 of the 110 significant CpG sites were located on autosomes.
