Fig. 4

Identification of candidate differentially methylated genes in gastric cardiac IM. a Unsupervised hierarchical cluster heatmap of selected DMPs in promoter CpG islands. A total of 54 DMPs showed extremely high (mean β > 0.7 in IM and mean β < 0.7 in normal) or extremely low (mean β < 0.3 in IM and mean β > 0.3 in normal) levels of DNA methylation. Of these, 43 CpG sites (79.6%) were hypermethylated, and 11 (20.4%) were hypomethylated. Note that IM and normal control are distinctly divided by unsupervised hierarchical clustering. Each row represents a CpG probe, and each column indicates an independent sample. The cells are colored according to methylation level (blue = unmethylated, red = fully methylated) at each CpG in each sample, as described by the color key. For each sample (column), the methylation levels are plotted (bottom), and the corresponding histology and age is shown in the bars directly above the heatmap. b Diagram showing the number of DMPs identified in each step by using the extra data filtering strategy. Three genes, HOXA5, DLEU7, and PXYLP1, harboring multiple DMPs (i.e., ≥ 3 DMPs identified in a single gene) were selected for validation and are indicated in the red dotted rectangle. c Differentially methylated levels in neighboring CpGs of HOXA5. Each data point represents the mean β value of the group, and error bars show SDs. The y-axis shows the absolute methylated fraction (β value) of each CpG site; the x-axis shows the CpG ID coordinates. Error bars represent means and standard deviations. d Relative mRNA expression of IM (n = 11) and normal (n = 11) gastric cardiac tissue (n = 11) measured by quantitative real-time PCR. Gene expression in IM was normalized against that in normal tissue. The mean value (± SD) is shown. P value was calculated by Mann–Whitney U test. Error bars represent standard deviations