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NOTCH2 mutations in SMZL. (A, top) The 34 exons of NOTCH2 are shown as gray boxes flanked by the 5′- and 3′-untranslated (UTR) regions of exons 1 and 34, respectively, above the protein domain structure of NOTCH2 including 36 epidermal growth factor-like repeats (EGFR, blue; mediates ligand binding), three LNR domains (purple; prevents ligand-independent activation), the HD (pink; prevents ligand-independent activation), a single-pass transmembrane region (TM, light blue), RBP-Jκ–associated module (RAM, black; required for NOTCH signaling), six ankyrin repeats (AR, red; bind the CSL transcription factor), the transactivation domain (TAD, white), and the proline-, glutamate-, serine- and threonine-rich domain (PEST, magenta). (A, Middle) Three mutations in the TAD and the PEST domain downstream of the AR region were identified in the SMZL discovery cohort. (A, Lower) Targeted Sanger sequencing of the SMZL validation cohort uncovered the same as well as additional missense (blue triangles), nonsense and frameshift (red circles) mutations in the HD, TAD and PEST domains. Constitutional tissue from a total of 19 patients confirmed somatic acquisition of these mutations in all samples (solid symbols). Sanger sequencing confirmation was performed in at least two independent replicates. (B) The locations of mutations in hematolymphoid malignancies in the NOTCH2 (7 total; top) and NOTCH1 (>800 total; bottom) genes in the COSMIC database are displayed adjacent to NOTCH2 and NOTCH1 amino acid sequence alignment. Mutations within both genes cluster in the HD and TAD/PEST domains. Specific amino acid residues and the predicted consequence of all NOTCH2 mutations in COSMIC are also displayed. (C) Mutations in HCS are confined to the C-terminal of the NOTCH2 protein and cluster within the TAD and PEST domains. The p.R2400X mutation seen in 9 cases of SMZL is also present in at least one case of HCS as an inherited mutation (open symbol). De novo mutations are displayed as solid symbols.