External and internal validation of the observed changes. (A) Correlation of the DMRs identified in 26 samples using the HELP method and an external dataset containing 87 human kidney samples analyzed using Illumina Infinium 450K arrays. We found concordant regulation of 1,061 transcripts (98%) from the 1,092 mapped genes using this validation dataset. Of transcripts that showed both differential methylation and expression, 404 (97%) were confirmed. (B) An example of a DMR identified by the HELP assay and confirmed in the validation dataset. This DMR is localized in the intronic region of COLIVA1. (C) Methylation status and (D) gene expression of COLIVA1 in the original HELP dataset in control and CKD samples. (E) MassArray confirmation of methylation status of the COLIVA1 locus (blue is mean ± standard deviation of control samples, red is mean ± standard deviation of CKD samples). (F) Methylation status of the COLIVA1 locus in the validation dataset (Infinium 450K arrays). The data represent the mean differences in absolute methylation levels of individual cytosines at the COLIVA1 locus. (G) Immunohistochemistry of COLIVA1 expression in control (CTL), diabetic (DM), DKD and CKD kidneys.