Different Mutant Splicing Factors Cause Distinct Missplicing Events and Give Rise to Different Clinical Phenotypes in Myelodysplastic Syndromes

Introduction

Splicing factor (SF) mutations represent a novel class of driver mutations in myelodysplastic syndromes (MDS), where SF3B1 and SRSF2 are most frequently affected. Although abnormal RNA splicing is thought to play a central role in the pathogenic mechanism of mutated SFs, little is known about exact gene targets whose abnormal splicing is responsible for the pathogenesis of MDS.

Methods

We enrolled a total of 480 patients with MDS, for whom complete clinical and pathological data were available. RNA sequencing was performed for bone marrow mononuclear cells (BM/MNCs) and/or CD34+ cells from 215 MDS patients. Observed splicing junctions were compared between samples with and without each SF mutation. In SF-mutated cases, NMD could cause severe degradation of abnormal transcripts and obscure the effect of SF-mutants. To sensitively detect abnormal transcripts otherwise degraded by nonsense-mediated RNA decay (NMD), analysis was also performed on BM/MNCs from 7 patients and CD34+ bone marrow cells from 3 patients with or without inhibition of NMD by cycloheximide (CHX). Common mutations and copy number variations were also investigated using targeted sequencing.

Results

SF3B1 and SRSF2 mutations were associated with distinct clinical phenotypes and outcomes. SF3B1-mutated cases typically showed isolated erythroid dysplasia and high proportion of ring sideroblasts, whereas SRSF2 mutations correlated with a significantly higher incidence of myeloid and megakaryocyte dysplasia (P<.001), higher proportion of bone marrow blasts (P=.02) and lower degree of erythroid dysplasia and proportion of ring sideroblasts (P<.001). SF3B1- and SRSF2-mutated myeloid neoplasms were also associated with a significantly different outcome, SRSF2-mutated neoplasms having a significantly shorter survival (HR=5.35, P<.001).

To explore the molecular basis of these distinct features in terms of splicing defects, RNA sequencing data from SF3B1-mutated (n = 68) and SRSF2-mutated (n = 39) BM/MNCs and CD34+ cells were compared with those without known SF mutations (n = 91) to detect splicing events significantly enriched in SF-mutated cells. In total 748 and 589 splicing events were enriched in SF3B1- and SRSF2-mutated samples. Among these, 203 (27%) of SF3B1-specific events were observed almost exclusively in SF3B1-mutated samples;193 (95%) were caused by misrecognition of 3' splice sites of which ~50% resulted in frameshift. In contrast, in SRSF2-mutated cases, the predominant defects were alternative exon usage, which accounted for for 80% of the abnormalities. However, the effect of mutant-SRSF2 on abnormal splicing was generally small, with 89% showing only <3× more abnormal transcripts in SRSF2-mutated. Similar results were obtained for BM/MNCs for both mutations.

Splicing defects of both SF-mutations involved substantially different set of genes. Aberrant splicing enriched in SF3B1- and SRSF2-mutated samples involved 12 and 7 cancer-related genes defined by the Cancer Gene Census with no genes overlapped in between. Of special interest among these was EZH2, which showed 2 SRSF2-associated alternative exons with a premature termination codon. EZH2 transcripts having these exons are expected to be susceptible to NMD-mediated degradation. A similar defect was also detected in another component of the polycomb repressive complex 2 (PRC2), implicated in compromised function of PRC2 in SRSF2-mutated cases. On the other hand, 2 genes involved in mitochondrial heme synthesis were significantly affected in SF3B1-mutated samples. In addition, an additional gene engaged in heme synthesis, ABCB7, was identified from experiments using NMD inhibition to detect 'masked splicing'. ABCB7 is one of the causative genes for congenital sideroblastic anemia and uniformly showed reduced expression in SF3B1-mutated samples, most likely due to abnormal splicing.

Conclusion

SF3B1 and SRSF2 mutations have distinct impacts on clinical phenotypes and outcomes together with RNA splicing. SF3B1 mutations caused misrecognition of 3' splice sites, frequently resulting in truncated gene products and/or decreased expression due to NMD. SRSF2 mutations were characterized by modest but more widespread alterations in exon usage of genes including multiple components of PRC2. Our results provide insights into the pathogenesis of SF-mutated MDS.