H3^K27Mand H3^K27Imutations occur in human AML with RUNX1 alterations. (A) Discovery of histone H3 mutations in RNA sequencing (RNAseq) of primary AML. Variant allele frequencies (VAFs) and RNAseq coverage (variant/total reads) are provided for sequenced AML samples with H3^K27 mutations (2 of 415 investigated specimens). (B) Validation of H3 mutation status. Genomic DNA from AML cells (top) and buccal swaps (bottom) was amplified using gene-specific primers and subjected to Sanger sequencing. Mutated nucleotides are boxed in red. (C) H3 variant expression varies based on the mutated H3 gene family member. Gene expression values of H3.1 and H3.3 genes in the 2 H3^K27-mutant AML specimens are plotted. In patient 04H138 (top panels), the HIST1H3H^K27M variant is present on the highest expressed H3 (27.6% of all H3.1 genes, 0.31% of all H3.1 and H3.3 genes; highlighted in bold). In patient 12H183 (bottom panels), expression of the HIST1H3F gene constitutes 4.4% of total canonical H3 and 0.10% of total H3 gene expression. (D) Summary of mutational contexts of H3^K27-mutant AML samples. Note that all specimens with H3^K27 mutations (in blue) have aberrations in the RUNX1 gene (highlighted in red). (E) Statistical analysis (Fisher’s exact test) of coassociations between H3K27 mutations and RUNX1 anomalies in the Leucegene or combined Leucegene and The Cancer Genome Atlas (TCGA) cohorts. Inf, infinite; na, not applicable; n.s., not significant; RPKM, reads per kilobase million.