We next examined the possibility that the CBS hotspots were associated with changes in expression of nearby genes. We restricted the analysis to the four CBS hotspots that had at least three mutated samples with gene expression data in the TCGA cohort (N = 35 samples). We validated the results using expression data from the SG cohort (N = 14 samples). Since the chromatin structure is generally cell-type invariant41,42 and there is no published Hi–C data from gastric tissue, we used the Hi–C data from IMR90 cells published by Dixon et al.41 to examine the 3D chromatin structure around each hotspot (Supplementary Figs. 8–10). We identified the flanking TAD boundary nearest to each hotspot, and tested the association between the mutation status of each hotspot and the expression of genes within the two adjacent TADs. We found genes with nominally altered expression for three of the four hotspots (Fig. 5), two of them remain significant after correcting for multiple testing in each region.Fig. 5Association of CBS hotspot mutations and cis-gene expression. a, d, g Association between mutation status of the CBS hotspot and expression levels of neighboring genes (two-sided Wilcoxon rank-sum test). Upregulated genes are shown above the x-axis, and downregulated genes are shown below the x-axis. Non-expressed genes are shown with empty circles on the x-axis (normalized count < 10 in all samples). b, e, h The reference sequence and mutated alleles at the three CBS hotspots. The mutations in tumors with expression data are underlined. c, f, i The gene expression of CENPQ (c), KCNQ5 (f), and SPG20 (i) in normal gastric tissue, and tumors with and without mutations at the corresponding CBS hotspot. P-values were adjusted using the Benjamini–Hochberg method