The next hotspot we tested is located on chromosome 6 with 9 mutated samples. Tumors with mutations at this hotspot had significantly lower expression of the KCNQ5 gene (Wilcoxon P = 0.0059, adjusted P = 0.047), located ~200 kb downstream of the hotspot (Fig. 5d–f). A similar trend in KCNQ5 expression was observed using the expression data from the SG cohort (Supplementary Fig. 11b). A recent study by Umer et al. found the same mutation hotspot by analyzing motif-breaking mutations40. Using an electrophoretic mobility shift assay, Umer et al. confirmed that the chr6:73,122,103 A>G mutation disrupts CTCF binding. In addition, it has been reported that CTCF is involved in the spatial organization of the KCNQ5 locus, and knockdown of CTCF downregulates KCNQ5 expression46.