Spliceosome mutations exhibit specific associations with epigenetic modifiers and proto-oncogenes mutated in myelodysplastic syndrome

Abstract

The recent identification of acquired mutations in key components of the spliceosome machinery strongly implicates abnormalities of mRNA splicing in the pathogenesis of myelodysplastic syndromes. However, questions remain as to how these aberrations functionally combine with the growing list of mutations in genes involved in epigenetic modification and cell signaling/transcription regulation identified in these diseases. In this study, amplicon sequencing was used to perform a mutation screen in 154 myelodysplastic syndrome patients using a 22-gene panel, including commonly mutated spliceosome components (SF3B1, SRSF2, U2AF1, ZRSR2), and a further 18 genes known to be mutated in myeloid cancers. Sequencing of the 22-gene panel revealed that 76% (n=117) of the patients had mutations in at least one of the genes, with 38% (n=59) having splicing gene mutations and 49% (n=75) patients harboring more than one gene mutation. Interestingly, single and specific epigenetic modifier mutations tended to coexist with SF3B1 and SRSF2 mutations (P<0.03). Furthermore, mutations in SF3B1 and SRSF2 were mutually exclusive to TP53 mutations both at diagnosis and at the time of disease transformation. Moreover, mutations in FLT3, NRAS, RUNX1, CCBL and C-KIT were more likely to co-occur with splicing factor mutations generally (P<0.02), and SRSF2 mutants in particular (P<0.003) and were significantly associated with disease transformation (P<0.02). SF3B1 and TP53 mutations had varying impacts on overall survival with hazard ratios of 0.2 (P<0.03, 95% CI, 0.1-0.8) and 2.1 (P<0.04, 95% CI, 1.1-4.4), respectively. Moreover, patients with splicing factor mutations alone had a better overall survival than those with epigenetic modifier mutations, or cell signaling/transcription regulator mutations with and without coexisting mutations of splicing factor genes, with worsening prognosis (P<0.001). These findings suggest that splicing factor mutations are maintained throughout disease evolution with emerging oncogenic mutations adversely affecting patients' outcome, implicating spliceosome mutations as founder mutations in myelodysplastic syndromes.

Figure 1.

Distribution of all mutations detected in our cohort of patients. The top row represents 117 mutated MDS cases where the shade of the bar indicates the cytogenetic risk groups according to the inset key. The rows beneath represent individual gene mutations denoted by colored bars and specified on the left hand side. Bars with black stripes indicate nonsense mutations, including splice-site mutations, while bars without stripes represent missense mutations. ● Indicates mutations with <10% mutant allele burden.

Figure 2.

Clonal evolution and disease progression in a patient with RARS. Sequencing analysis of sequential samples in patient A (UPN RC060337) with an SF3B1 mutation. The three horizontal rows represent samples collected at different time points: diagnosis, transformation to AML and disease progression after a short-lasting remission. Accumulation of the oncogenic mutations and changes in mutant allele burden levels are seen through disease progression along with an increase in the blast count. Sanger sequencing was used to confirm/determine the mutation status throughout the experiment. 454 sequencing confirmed the mutation level differences at start and end points of the experiment. WT-wild type.

Figure 3.

Overall survival for patients with SF3B1 mutations (n=24) compared to those with wild type SF3B1 (n=130) (A) and overall survival for patients with spliceosome mutations (n=59) compared to patients without splicing factor mutations (n=99) (B).

Figure 4.

Overall survival (A) and progression-free survival (B) for patients with spliceosome mutations (n=59), stratified according to patients with splicing factor mutations (S) co-existing with mutations of genes involved in cell signaling/transcription regulation (CS/TR) or TP53 mutations versus splicing factor mutations co-existing with epigenetic modifier (EM) mutations versus patients with splicing factor mutations alone.