Figure 1

(A) Genomic organization of the EZH2 locus, alternative exons and protein domain structure. The location of the mutation affecting Y641 in exon 15 of the EZH2 gene and protein is indicated with a red asterisk. (B) Illustration of sequencing results. Three of the five distinct mutations and amino acid replacements in codon 641 from different lymphoma samples as detected by capillary sequencing (left) or Illumina WTSS (right). (C) A multiple alignment of EZH2, EZH1 (its paralog), the Drosophila ortholog E(Z) and six other human SET domain proteins demonstrates the intra and inter-species sequence conservation of SET domains. Conservation codes reported by ClustalX are shown above²⁴. The predominant mutation in EZH2 affects a key tyrosine in the catalytic site of the SET domain (orange) conserved in the Drosophila ortholog E(Z). With one exception, all EZH2 mutations in FL and DLBCL alter this amino acid. The exception was a double mutant (FL), with a second somatic mutation affecting N635 (blue). All mutants comprise 5 of the 8 possible non-synonymous variants of this codon (lower right, in red). Notably, the five observed amino acid changes were not found at equal frequencies. We detected a slight enrichment for Y641F (49%) followed by Y641S (21%), Y641N (15%) and Y641H (13%) and only a single example of Y641C (2%)(Supplementary Table 5). Of the unobserved variants (D, blue), two would result in a truncated protein and the third would introduce an aspartate residue. The pattern and nature of these changes (A->G, A->T, T->G, T->A), indicated to us that these mutations do not likely arise from AID-induced somatic hypermutation at this locus²⁵.