Exome sequencing identifies GRIN2A as frequently mutated in melanoma

Abstract

The incidence of melanoma is increasing more than any other cancer, and knowledge of its genetic alterations is limited. To systematically analyze such alterations, we performed whole-exome sequencing of 14 matched normal and metastatic tumor DNAs. Using stringent criteria, we identified 68 genes that appeared to be somatically mutated at elevated frequency, many of which are not known to be genetically altered in tumors. Most importantly, we discovered that TRRAP harbored a recurrent mutation that clustered in one position (p. Ser722Phe) in 6 out of 167 affected individuals (∼4%), as well as a previously unidentified gene, GRIN2A, which was mutated in 33% of melanoma samples. The nature, pattern and functional evaluation of the TRRAP recurrent mutation suggest that TRRAP functions as an oncogene. Our study provides, to our knowledge, the most comprehensive map of genetic alterations in melanoma to date and suggests that the glutamate signaling pathway is involved in this disease.

Figure 1

Distribution of new non-synonymous recurrent mutations. Seven new non-synonymous recurrent mutations identified in this study are presented on relevant protein schematics. Black arrows indicate locations of recurrent mutations and conserved protein functional domains are indicated as colored boxes (1, immunoglobulin I-set domain; 2, fibronectin type III domain; 3, neogenin C terminus; 4, phosphoinositide-specific phospholipase C, efhand-like; 5, phosphatidylinositol-specific phospholipase C, X domain; 6, phosphatidylinositol-specific phospholipase C, Y domain; 7, C2 domain; 8, PDZ domain; 9, nitric oxide synthase, oxygenase domain; 10, flavodoxin; 11, FAD binding domain; 12, oxidoreductase NAD-binding domain; 13, LRRNT, leucine rich repeat N-terminal domain; 14, leucine rich repeat; 15, immunoglobulin I-set domain; 16, fibronectin type III domain; 17, major facilitator superfamily).

Figure 2

Effect of mutant TRRAP on colony formation and apoptosis. (a) Mutant TRRAP induces cell transformation. Foci formation of NIH 3T3 cells transfected with the indicated constructs or empty vector control. Ras p.Gly12Val was included as a positive control for cell transformation. (b) Detection of TRRAP and KRas protein expression in lysates of transiently transfected NIH 3T3 cells by immunoblot analysis. (c) Immunoblot of cell lysates from HEK 293T cells transiently transfected with either control vector or shRNAs that target TRRAP. For normalization, lysates were analyzed in parallel by anti–α-tubulin immunoblotting. NS, non specific. (d) Immunoblot of melanoma cells transduced with shRNA targeting TRRAP and immunoblotted with anti-TRRAP. (e–h) TRRAP mutation confers resistance to apoptosis. Apoptosis quantification of melanoma cell lines transduced with shRNA control or shRNAs targeting TRRAP by Hoechst 33258-staining. Cells were grown in growth medium containing 2.5% serum for the indicated times. Error bars, s.d. (i,j) Immunoblot analysis of representative melanoma lines presented in e–h using the indicated antibodies to assess PARP cleavage. WT, wild type.

Figure 3

Location of somatic mutations in GRIN2A. A schematic of human GRIN2A is presented, with conserved functional domains indicated as colored blocks. Somatic mutations are indicated with arrows and amino acid changes. Recurrent mutations and nonsense mutations are indicated as orange arrows and black boxes, respectively. Conserved domains: SP, signal peptide; PBP1_iGluR_NMDA_NR2, N-terminal leucine/isoleucine/valine-binding protein LIVBP-like domain of the NR2 subunit of NMDA receptor family; PBPb, bacterial periplasmic substrate-binding protein; Lig_chan, ligand-gated ion channel; NMDAR2_C, N-methyl D-aspartate receptor 2B3 C terminus.