SF3B1 mutations are associated with alternative splicing in uveal melanoma

Abstract

Uveal melanoma, the most common eye malignancy, causes severe visual morbidity and is fatal in approximately 50% of patients. Primary uveal melanoma can be cured by surgery or radiotherapy, but the metastatic disease is treatment refractory. To understand comprehensively uveal melanoma genetics, we conducted single-nucleotide polymorphism arrays and whole-genome sequencing on 12 primary uveal melanomas. We observed only approximately 2,000 predicted somatic single-nucleotide variants per tumor and low levels of aneuploidy. We did not observe an ultraviolet radiation DNA damage signature, but identified SF3B1 mutations in three samples and a further 15 mutations in an extension cohort of 105 samples. SF3B1 mutations were associated with good prognosis and were rarely coincident with BAP1 mutations. SF3B1 encodes a component of the spliceosome, and RNA sequencing revealed that SF3B1 mutations were associated with differential alternative splicing of protein coding genes, including ABCC5 and UQCC, and of the long noncoding RNA CRNDE.

Significance: Our data show that despite its dismal prognosis, uveal melanoma is a relatively simple genetic disease characterized by recurrent chromosomal losses and gains and a low mutational burden. We show that SF3B1 is recurrently mutated in uveal melanoma, and the mutations are associated with aberrant alternative splicing.

Figure 1. Somatic mutations in uveal melanoma.

(A) Comparison of predicted somatic structural variation in uveal, cutaneous, acral and mucosal melanoma subtypes. (B) Comparison of non-synonymous point mutation rates identified from whole genome and exome sequencing studies in various solid tumors (Details and references in Supplementary Methods). (C) Proportion of predicted somatic single nucleotide variants in uveal melanoma genomes by class of mutation. (D) Frequency of bases ±1 bp of C>T/G>A mutations in the uveal melanoma genomes.

Figure 2. Alternative splicing in SF3B1 mutant uveal melanoma.

(A - C) Plots showing normalized RNA-seq reads for UQCC (A), CRNDE (B) and ABCC5 (C) in SF3B1 wildtype (orange) and SF3B1 mutant (mauve) tumors. Above the graphs we show representations of the splicing events. Exons are represented as boxes, with major splicing events indicated by the solid lines/solid boxes and minor splicing by the dotted lines/hashed boxes. Te: terminal exon; e3, e4, e5, e6: exons 3, 4, 5, 6; i5: intron 5. (D) Heat map of the 8 differentially spliced genes validated by RT-QPCR in a cohort of 74 independent uveal melanoma samples (16 SF3B1 mutant, 58 SF3B1 wild-type). The brackets present the splicing form that was measured, together with exons number involved in each case (e: exon, i: intron, ae: alternative exon). Primer sequences used are presented in Supplementary Table 12 and p-values (Mann-Whitney U test) are indicated without adjustment for the 8 tests. Primary data is shown in Supplementary Fig. S6. Genes that are alternatively spliced are shown in blue; non-spliced genes are shown in red; unexpressed genes are shown in yellow. For each sample, the status of the SF3B1 gene is indicated.