Components Of RNA Splicing Machinery Research Articles

Distinct and convergent consequences of splice factor mutations in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are characterized by recurrent somatic alterations often affecting components of RNA splicing machinery. Mutations of splice factors SF3B1, SRSF2, ZRSR2 and U2AF1 occur in >50% of MDS. To assess the impact of spliceosome mutations on splicing and to identify common pathways/genes affected by distinct mutations, we performed RNA-sequencing of MDS bone marrow samples harboring spliceosome mutations (including hotspot alterations of SF3B1, SRSF2 and U2AF1; small deletions of SRSF2 and truncating mutations of ZRSR2), and devoid of other common co-occurring mutations. We uncover the landscape of splicing alterations in each splice factor mutant MDS and demonstrate that small deletions in SRSF2 cause highest number of splicing alterations compared with other spliceosome mutations. Although the mis-spliced events observed in different splice factor mutations were largely non-overlapping, a subset of genes, including EZH2, were aberrantly spliced in multiple mutant groups. We also verified aberrant splicing of key genes USP9X, USP24 (deubiquitinating enzymes), LUC7L2 (splice factor) and EED (PRC2 component) in MDS harboring small deletions of SRSF2. Pathway analysis revealed that mis-spliced genes in different mutant groups were enriched in RNA splicing and transport as well as several signaling cascades, suggesting converging biological consequences downstream of distinct spliceosome mutations. Our study reveals splicing signatures of each splice factor mutation and identifies shared and distinct sets of mis-spliced genes and affected biological processes in different spliceosome mutant MDS.

The Frequency of SF3B1 Mutations in Thai Patients with Myelodysplastic Syndrome

Genetic mutations in genes encoding critical component of RNA splicing machinery including SF3B1 are frequently identified and recognized as the pathogenesis in the development of myelodysplatic syndrome (MDS). In this study, PCR sequencings specific for SF3B1 exon 13, 14, 15, and 16 were performed to analyse genomic DNA isolated from bone marrow samples of 72 newly diagnosed MDS patients. We found that 10 of 72 (14%) patients harbor SF3B1 missense mutations including E622D (1/72), R625C/G (2/72), H662Q (1/72), K666T (1/72), K700E (4/72) and G740E (1/72), respectively. Mutations were predominantly located on exon 14 and 15 of SF3B1 coding sequence. Interestingly, patients with SF3B1 mutations exhibited higher platelet counts (195×109/L VS. 140×109/L, p-value = 0.025) as well as lower hemoglobin levels (81 g/L VS. 92 g/L, p-value = 0.009) and associated with ring sideroblast phenotype (p-value < 0.001) when compared with patients without the SF3B1 mutation. In summary, we reported the frequency of SF3B1 mutations in Thai patients with different subtypes of MDS. SF3B1 mutations were predominantly occurred in MDS-RS and considered as favourable prognosis value. This study further highlighted the clinical important of SF3B1 mutations analysis for the classification of MDS.

Response to Treatment Among SF3B1 Mutated Myelodysplastic Syndromes (MDS): A Case-Control Study from the MDS Clinical Research Consortium (MDS CRC)

IntroductionSomatic mutations in SF3B1 ,a gene encoding a core component of RNA splicing machinery, have been identified in patients (pts) with myelodysplastic syndrome (MDS). The SF3B1 mutation (MT) is more commonly detected in pts with ring sideroblasts (RS) morphology and is associated with favorable outcome. The pattern of response among SF3B1 mutated MDS pts to available treatment options, including erythropoiesis stimulating agents (ESA), hypomethylating agents (HMA) and lenalidomide is not known. The distinct underlying disease biology among such pts may alter response to treatment.MethodsPts treated at MDS CRC institutions with MT vs wild-type SF3B1 (WT) controls were matched 1:2. Matching criteria were age at diagnosis, year of diagnosis and International Prognostic Scoring System (IPSS) category at diagnosis. IPSS category was split into two groups (Low or Int-1 vs. Int-2 or High). Matching was performed using the R package by calculating a propensity score, which was then used to determine the two most similar WT SF3B1 patients for each SF3B1-mutated pt, without replacement. Additionally, to be included in the population, pts also had to have been treated with one of the following: ESAs, HMA, or lenalidomide. Response to treatment was evaluated by international Working Group criteria (IWG 2006) and classified as response if hematological improvement or better was achieved (HI+). Survival was calculated from date of treatment until date of death or last known follow-up, unless otherwise noted.Results:We identified 48 Pts with MT and 96 matched controls. Table 1 summarizes baseline characteristics comparing MT vs WT SF3B1 cohorts. SF3B1 MT was detected more often in association with RS, as expected. The majority of pts had lower-risk disease by IPSS and revised IPSS (IPSS-R). Pts with MT had higher platelets than controls. The most common concomitant somatic mutations observed were TET2 (30%), DNMT3A (21%), and ASXL1 (7%). Median follow-up time from diagnosis was 35 months (mo). Median overall survival (OS) from diagnosis was significantly longer for patients with SF3B1 MT (108.5 mo (68.8, NA)) than wild-type controls (28.3 mo (22.3, 36.4); p < 0.001). Patients with an SF3B1 MT had a decreased hazard of death (hazard ratio [HR]: 0.49 (95% confidence limits [95% CL]: 0.29, 0.84); p = 0.009)ESA was the first line therapy for 43 pts (88%) with MT and 55 WT Pts (56%). For ESA treated pts, 14 out 40 MT Pts responded (35%) compared to 9/56 among WT Pts (16%), p 0.032. Among those treated with HMA therapy, 5 out 21 (24%) MT pts responded compared to 11/46 (24%) WT Pts (p 0.99). Finally, for Pts treated with lenalidomide 4/16 (25%) and 4/21 (19%) responded among SF3B1 MT and WT Pts respectively, p 0.7.ConclusionsSF3B1 somatic mutation in MDS is commonly associated with RS, lower risk disease, and better OS. Pts with SF3B1 mutation had higher response to ESA compared WT SF3B1. No difference in response to HMA or lenalidomide was observed compared to WT patients. Response rates to lenalidomide and HMA were low in both MT patients and controls. Biologically rational therapies are needed that target this molecular disease subset.Table 1Baseline characteristicsSF3B1 MT (n=48)SF3B1 WT (n=96)P valueAgemedian65670.6Gendermale29 (60%)64(67%)0.5RaceWhite44/45 (98%)83/90 (92%)0.34WHO classificationRA RARS RCMD RARS-T Del5 q RAEB-I RAEB-II MDS-U MDS/MPN CMML3 24 8 4 1 3 3 2 0 06 9 17 2 6 10 9 3 11 9IPSSLow Int-1 Int-2 High29 (60%) 16 (33%) 3 (6%) 021 (22%) 69 (72%) 4 (4%) 2 (2%)< 0.001IPSS-RVery low Low Intermediate High Very High15 (31%) 26 (54%) 5 (10%) 2 (4%) 011 (11%) 37 (39%) 26 (27%) 18 (19%) 4 (4%)<0.001Lab values (mean)Hgb Platelets ANC myeloblasts9.7 274 2.63 19.6 108 1.92 20.46 <0.001 0.04 0.05 DisclosuresKomrokji:Novartis: Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding; Incyte: Consultancy; Pharmacylics: Speakers Bureau. Padron:Novartis: Speakers Bureau; Incyte: Research Funding. List:Celgene Corporation: Honoraria, Research Funding. Steensma:Incyte: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Onconova: Consultancy. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Clinical significance of SF3B1 mutations in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms

AbstractIn a previous study, we identified somatic mutations of SF3B1, a gene encoding a core component of RNA splicing machinery, in patients with myelodysplastic syndrome (MDS). Here, we define the clinical significance of these mutations in MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). The coding exons of SF3B1 were screened using massively parallel pyrosequencing in patients with MDS, MDS/MPN, or acute myeloid leukemia (AML) evolving from MDS. Somatic mutations of SF3B1 were found in 150 of 533 (28.1%) patients with MDS, 16 of 83 (19.3%) with MDS/MPN, and 2 of 38 (5.3%) with AML. There was a significant association of SF3B1 mutations with the presence of ring sideroblasts (P < .001) and of mutant allele burden with their proportion (P = .002). The mutant gene had a positive predictive value for ring sideroblasts of 97.7% (95% confidence interval, 93.5%-99.5%). In multivariate analysis including established risk factors, SF3B1 mutations were found to be independently associated with better overall survival (hazard ratio = 0.15, P = .025) and lower risk of evolution into AML (hazard ratio = 0.33, P = .049). The close association between SF3B1 mutations and disease phenotype with ring sideroblasts across MDS and MDS/MPN is consistent with a causal relationship. Furthermore, SF3B1 mutations are independent predictors of favorable clinical outcome, and their incorporation into stratification systems might improve risk assessment in MDS.

Somatic Mutation of SF3B1, a Gene Encoding a Core Component of RNA Splicing Machinery, in Myelodysplasia with Ring Sideroblasts

Abstract 3Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by dysplasia in one or more cell lines, ineffective hematopoiesis, and variable risk of progression to acute myeloid leukemia (AML). As any other neoplasm, MDS is expected to be driven by mutation, and its clonal evolution is likely a multistep process in which several genetic events occur. Somatic mutations of TET2 have been found in about 25% of MDS patients, while additional mutant genes (including ASXL1, ETV6, EZH2, IDH1, IDH2, RUNX1, and TP53) have been detected in smaller proportions of patients, particularly in those with poor prognosis.Refractory anemia with ring sideroblasts (RARS) is a phenotypically well-defined subtype of MDS, characterized by 15% or more ring sideroblasts (RS, erythroblasts with perinuclear iron-loaded mitochondria) in the bone marrow. We reasoned that the identification of recurrently mutated genes in RARS could provide novel insights into molecular pathogenesis of MDS, and used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 8 patients with RARS.We identified 62 point mutations across the 8 patients, and the mutation spectrum showed a predominance of transitions, especially C>T/G>A mutations. Within 5/8 patients studied, the observed proportion of reads reporting a mutant allele showed significantly greater variability than expected by chance, indicating that the population of malignant cells was genetically heterogeneous. In 6/8 RARS patients, we identified recurrent somatic mutations (found in granulocytes but not in T-lymphocytes) in a gene that encodes a core component of the RNA splicing machinery, SF3B1. Based on the proportion of reads reporting the mutant allele, the mutations all appeared to be heterozygous and present in the dominant clone of cells.To characterize the spectrum and frequency of SF3B1 mutations in greater detail, both in myeloid malignancies and other cancers, we undertook targeted resequencing of the gene. In particular, we studied patients with MDS, myelodysplastic/myeloproliferative neoplasm (MDS/MPN) or AML evolving from MDS. Somatic mutations of SF3B1 were found in 150/533 (28.1%) patients with MDS, 16/83 (19.3%) patients with MDS/MPN, and 2/38 (5.3%) patients with AML. The gene was also mutated in 1–5% of diverse other tumor types. All mutations appeared to be heterozygous substitutions, and we observed no frameshift indels, splice site mutations or nonsense substitutions. The mutations clustered in exons 12–15 of the gene, and K700E accounted for 97/168 (57.7%) of the variants observed. SF3B1 mutations were less deleterious than expected by chance, implying that the mutated protein retains structural integrity with altered function. Gene expression profiling revealed SF3B1 mutations are associated with down-regulation of key gene networks, including core mitochondrial pathways.Close relationships were found between mutant SF3B1 and presence of RS (P<.001), and between mutant allele burden and percentage of RS (P=.002). Overall, 83/105 (79%) of patients with RARS, 30/54 (57.7%) of those with refractory cytopenia with multilineage dysplasia and RS, and 12/18 (66.7%) of those with RARS associated with marked thrombocytosis (RARS-T) carried a somatic mutation of SF3B1. On the other hand, 97% of patients carrying a mutant SF3B1 had RS, and the mutant gene had a positive predictive value for RS of 97.7% (95% CI, 93.5–99.5%). We then studied the prognostic significance of the genetic lesion. In multivariable analysis including established risk factors, SF3B1 mutations were independently associated with better overall survival (HR=0.18, P=.028) and lower risk of progression to AML (HR=0.32, P=.048).In conclusion, mutations in SF3B1 implicate abnormalities of mRNA splicing, a pathway not previously known as a target for mutation, in the pathogenesis of MDS. The close relationship between this molecular lesion and RS is consistent with a causal relationship, and makes SF3B1 the first gene to be strongly associated with a specific morphological feature in MDS. Finally, SF3B1 mutations are independent predictors of favorable clinical outcome, and their detection may improve risk assessment in MDS.The first two authors equally contributed to this paper, which is on behalf of the International Cancer Genome Consortium Chronic Myeloid Disorders Working Group. Disclosures:No relevant conflicts of interest to declare.

Identification of Novel Somatic Mutations in SF3B1, a Gene Encoding a Core Component of RNA Splicing Machinery, in Myelodysplasia with Ring Sideroblasts and Other Common Cancers

Identification of Novel Somatic Mutations in SF3B1, a Gene Encoding a Core Component of RNA Splicing Machinery, in Myelodysplasia with Ring Sideroblasts and Other Common Cancers

Zebrafish usp39 Mutation Leads to rb1 mRNA Splicing Defect and Pituitary Lineage Expansion

Loss of retinoblastoma (Rb) tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39) mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130), and cdkn1a (p21) expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.