Exome sequencing identifies recurrent somatic mutations in EIF1AX and SF3B1 in uveal melanoma with disomy 3

Abstract

Gene expression profiles and chromosome 3 copy number divide uveal melanomas into two distinct classes correlating with prognosis. Using exome sequencing, we identified recurrent somatic mutations in EIF1AX and SF3B1, specifically occurring in uveal melanomas with disomy 3, which rarely metastasize. Targeted resequencing showed that 24 of 31 tumors with disomy 3 (77%) had mutations in either EIF1AX (15; 48%) or SF3B1 (9; 29%). Mutations were infrequent (2/35; 5.7%) in uveal melanomas with monosomy 3, which are associated with poor prognosis. Resequencing of 13 uveal melanomas with partial monosomy 3 identified 8 tumors with a mutation in either SF3B1 (7; 54%) or EIF1AX (1; 8%). All EIF1AX mutations caused in-frame changes affecting the N terminus of the protein, whereas 17 of 19 SF3B1 mutations encoded an alteration of Arg625. Resequencing of ten uveal melanomas with disomy 3 that developed metastases identified SF3B1 mutations in three tumors, none of which targeted Arg625.

Figure 1

Exome sequencing of uveal melanomas. Sequencing of 22 uveal melanomas (E-1 to E-22) that were randomly chosen from a cohort of 374 uveal melanomas identified 6 recurrently mutated genes. Tumors are grouped according to their chromosome 3 status. All mutations identified in either GNAQ or GNA11 were missense mutations affecting Gln209. All mutations were verified by Sanger sequencing and are listed in Supplementary Table 3.

Figure 2

Results of targeted resequencing. Resequencing of EIF1AX (exons 1 and 2) and SF3B1 (exons 12–16) in 89 uveal melanomas with monosomy 3 (n = 35), disomy 3 (n = 31), partial monosomy 3 (n = 13) and disomy 3 with metastasis (n = 10). All mutations identified in tumor DNA were also analyzed in matched blood DNA samples, but no germline mutations were detected. (a) Sixty-six uveal melanomas randomly chosen from our cohort of 374 subjects, excluding tumors with partial monosomy 3 and disomy 3 with metastasis. (b) Results of EIF1AX and SF3B1 resequencing of all tumors with partial monosomy 3 and disomy 3 with metastasis in this cohort from which DNA was available. BAP1 mutation screening was performed in all tumors with partial monosomy 3 and disomy 3 with metastasis from which DNA was available. Exons 4 and 5 of GNAQ and GNA11 were sequenced in all tumors to detect recurrent mutations affecting Arg183 and Gln209. All mutations identified in either GNAQ or GNA11 were missense mutations affecting Gln209. (c) Distribution of SF3B1 mutations in uveal melanoma. Arg625 in the HD5 repeat was altered in 18 uveal melanomas. All non–codon 625 mutations in SF3B1 were found in only one tumor. Distinct SF3B1 mutations outside of codon 625 in tumors from individuals who died because of uveal melanoma metastases are colored in blue, yellow and red.

Figure 3

Alignment of EIF1AX amino acid sequences from 19 uveal melanomas with an EIF1AX mutation. The first 15 N-terminal residues are shown. Amino acids affected by mutations are restricted to positions 2–15 in the unstructured NTT. Codon 6 (Gly6) is split by intron 1. All EIF1AX mutations lead to amino acid substitutions (red) or short deletions of one or two amino acids.