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

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 139-139 ◽  
Author(s):  
Yusuke Shiozawa ◽  
Luca Malcovati ◽  
Aiko Sato-Otsubo ◽  
Anna Gallì ◽  
Kenichi Yoshida ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rna Sequencing ◽  
Myelodysplastic Syndromes ◽  
Rna Splicing ◽  
Splice Sites ◽  
Clinical Phenotypes ◽  
Cd34 Cells ◽  
Ring Sideroblasts ◽  
Exon Usage ◽  
Splicing Defects

Abstract 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. Disclosures No relevant conflicts of interest to declare.

Comprehensive Analysis of Aberrant RNA Splicing in Myelodysplastic Syndromes

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 826-826 ◽  
Author(s):  
Yusuke Shiozawa ◽  
Sato Sato-Otsubo ◽  
Anna Gallì ◽  
Kenichi Yoshida ◽  
Tetsuichi Yoshizato ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rna Sequencing ◽  
Myelodysplastic Syndromes ◽  
Rna Splicing ◽  
Comprehensive Analysis ◽  
Erythroid Cells ◽  
Splice Sites ◽  
Cd34 Cells

Abstract Introduction Splicing factor (SF) mutations represent a novel class of driver mutations highly prevalent in myelodysplastic syndromes (MDS), where four genes, including SF3B1, SRSF2, U2AF1, and ZRSR2, are most frequently affected. SF3B1 and SRSF2 mutations show prominent specificity to RARS/RCMD-RS and CMML subtypes, respectively. 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 implicated in the pathogenesis of MDS or about the molecular mechanism that explains the unique subtype specificity of SF mutations, especially to those subtypes characterized by increased ring sideroblasts. Methods To address these issues, comprehensive analysis of abnormal RNA splicing was performed for a total of 336 MDS patients with different SF mutations. High-quality RNA was extracted from bone marrow mononuclear cells (BM/MNCs) and/or CD34+ cells and subjected to high-throughput sequencing, followed by exhaustive detection of splicing junctions for all relevant reads. Aberrant splicing events associated with different SF mutations were explored by comparing observed splicing junctions between samples with and without mutations. To specifically determine the role of SF3B1 mutations in ring sideroblast formation, CD34+ bone marrow cells from 13 patients with or without SF3B1 mutations were differentiated in vitro into erythroid cells. RNA sequencing was performed on cells recovered on day 7 and day 14 and differentially spliced genes in erythroid cells between SF3B1-mutated and unmutated samples were investigated. Results SF3B1, SRSF2, U2AF1, and ZRSR2 were mutated in 28%, 18%, 5%, and 7% of the patients, respectively. First, we compared SF3B1-mutated samples with those without known SF mutations. RNA sequencing of CD34+ cells revealed 230 splicing events significantly enriched in SF3B1-mutated cases, of which 90% (n = 206) were caused by misrecognition of 3' splice sites. A similar result was obtained in the experiment for BM/MNCs, where 177 (83%) out of 206 splicing events significantly enriched in SF3B1-mutated samples were caused by misrecognition of 3' splice sites. These observations were in accordance with the known function of SF3B1 in branch point recognition in the U2 snRNP complex. In both BM/MNCs and CD34+ cells, approximately 70% of the unusual 3' splice sites were located from 5 to 25 bases downstream from the authentic junctions. The bases immediately upstream to these 3' splice sites were more often pyrimidines, which was not accordance with the general rule: the bases next to 3' splice sites are purines, especially guanines. About 50% of these altered 3' splice sites resulted in frameshift, indicating that SF3B1 mutations cause deleterious effects in many genes simultaneously. Next, to explore the genes whose abnormal splicing is responsible for increased ring sideroblast formation, RNA sequencing was carried out for erythroid progenitor cells differentiated in vitro from CD34+ cells from MDS patients with or without SF3B1 mutations. We found that a total of 146 altered 3' splice sites were significantly associated with SF3B1 mutations, of which 87 were overlapped to the aberrant splice sites shown to be enriched in SF3B1 mutated primary MDS specimens. These splice sites were found in genes involved in heme biosynthesis, cell cycle progression, and DNA repair and their consequence was mostly deleterious due to aberrant frameshifts. Abnormal splicing events associated with other SF mutations were also identified. Among these, the most common abnormalities associated with mutated SRSF2 and U2AF1 were alternative exon usage. Misrecognition of 3' splice sites was also common in U2AF1-mutated cases. ZRSR2 mutations were associated with retentions of U12 introns, which is consistent with the known role of ZRSR2 as an essential component of the minor (U12-type) spliceosome. Conclusion SF mutations were associated with characteristic abnormal splicing changes in primary MDS samples as well as in vitro cultured cells. Our results provide insights into the pathogenic role of SF mutations in MDS. Disclosures No relevant conflicts of interest to declare.

Definition of RAEB-I and RAEB-Ii According to the Who Classification of Myelodysplastic Syndromes (MDS): Problems Emerging from a Large Multicenter Registry.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1432-1432
Author(s):  
Alessandro Levis ◽  
L. Godio ◽  
M. Girotto ◽  
M. Bonferroni ◽  
T. Callegari ◽  
...  
Keyword(s):  
Internal Medicine ◽  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Bone Marrow Cells ◽  
Clinical Information ◽  
Bone Marrow Trephine Biopsy ◽  
A Value ◽  
Cd34 Cells ◽  
Definition Of

Abstract BACKGROUND. TheWHO classification of myelodysplastic syndromes (MDS) is based on the evaluation of bone marrow morphology. The two categories of REAB-I and RAEB-II are apparently easy to differentiate on the basis of bone marrow blast percent. However there are no so far data about the differences among cytology, histology and immunophenotypic evaluation of blasts in order to discrimante non-RAEB from RAEB-I and RAEB-II categories. PATIENTS AND METHODS. The Piemonte MDS Registry was born in 1999 thanks to the cooperation of both Haematology and Internal Medicine departments of our region, with the following aims: a) to follow homogeneous guidelines in diagnosis and treatment of MDS; b) to collect epidemiological and clinical information on a large group of patients; c) to cryopreserve bone marrow cells for molecular biology studies. When obtaining an informed consent, data of patients were prospectively centrally recorded through our web site. A retrospective analysis on differences in diagnosing RAEB, comparing conventional cytology on bone marrow smears (CBM), histochemical evaluation of CD34+ cells on bone marrow trephine biopsy (HBM), and cytofluorimetric count of CD34+ and CD117+ cells (IBM) has been done. RESULTS. From June 1999 to December 2003, 633 MDS patients were registered from 37 different institutions: 364 (57%) from haematology and/or academic institutions and 269 (43%) from internal medicine departments of community hospitals. Mean age was 72 (range 23–69). The actual diagnostic distribution of cases according to the WHO criteria based on only morphology evaluation of bone marrow smears was: non-RAEB 429 (68%), RAEB-I 134 (21%), and RAEB-II 70 (11%). Information about the quantification of blasts with both CBM and HBM techniques was avilable in 243 cases. An IBM evaluation was also available in 89 out of this 243 cases. A disagreement between CBM and HBM was evident in 65/243 cases (27%), with HBM over-evaluating and under-evaluating WHO class on the basis of blasts count in 54/243 (22%) and 11/243 cases respectively. When comparing CBM and IBM the disagreement was even higher in 29/89 cases (33%), with IBM over-evaluating blast percent in 9 (10%) and under-evaluating it in 20 cases (23%). The disagreement betwen HBM and IBM was maximum with a value of 39%. The role of CBM in predicting a different prognosis of non-RAEB, RAEB-I and RAEB-II was confirmed. However, when comparing the prognostic value of the three different methods of computing bone marrow blasts, IBM was the best in order to define the good prognostic non-RAEB group. CONCLUSIONS. The distinction among non-RAEB, RAEB-I and RAEB-II is far from beeing highly accurate and reproducible. Important differences are present among CBM, HBM and IBM. While CBM remain the conventional standard system, IBM could offer a tool better and more reproducible than CBM in order to define MDS categories on the basis of blast percentage. A large multicenter study could be useful in order to clarify this point.

Aberration Of SF3B1 Results In Deregulated Splicing Of Key Genes and Pathways In Myelodysplastic Syndromes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2747-2747
Author(s):  
Hamid Dolatshad ◽  
Marta Fernandez-Mercado ◽  
Bon Ham Yip ◽  
Chris J Smith ◽  
Martin Attwood ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Lines ◽  
Rna Processing ◽  
Exon Skipping ◽  
Erythroid Differentiation ◽  
Aberrant Splicing ◽  
Cd34 Cells ◽  
Ring Sideroblasts ◽  
Exon Usage

Abstract The recent discovery of a variety of somatic splicesomal mutations in the myelodysplastic syndromes (MDS) has revealed a new leukaemogenic pathway involving spliceosomal dysfunction. Pre-mRNA splicing proceeds by way of two phosphoester transfer reactions and is catalyzed by the spliceosome, which consists of the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs) and numerous non-snRNP proteins. The snRNPs are involved in recognising short conserved sequences of the pre-mRNA, including the 5′ and 3′ splice sites and the branch site, and in positioning the reactive nucleotides for catalysis. The spliceosome is a dynamic molecular machine, undergoing several major structural rearrangements during its functional cycle. Mutation of the Splicing Factor 3B, subunit 1 (SF3B1) gene is common in MDS, occurring in over 70% of patients whose disease is characterised by ring sideroblasts (RARS). The close association between SF3B1 mutation and ring sideroblasts is consistent with a causal relationship, and makes this the first gene to be strongly associated with a specific feature of MDS. Sf3b1 heterozygous knockout mice show the presence of ringed sideroblasts. In order to investigate the role of SF3B1 haploinsufficiency in MDS we have silenced SF3B1 using siRNA in the myeloid cell lines K562, TF-1, SKM1, HeL and OCIM2. Cell growth was impaired in all the cell lines with SF3B1 knockdown. Using Flow Cytometry, cell cycle analysis showed a significant increase in cells in the sub-G0 phase as well as G2/M arrest in the cell lines. We also observed impaired erythroid differentiation in hemin treated K562 and TF-1 cell lines with SF3B1 knockdown. Gene expression profiling (GEP) was performed in two cell lines with SF3B1 knockdown (K562 and TF1). Deregulated pathways and gene ontology categories included cell cycle regulation and alternative splicing using Ingenuity Pathway Analysis. We next performed Gene Set Enrichment Analysis (GSEA). The GSEA showed a significant enrichment of nonsense-mediated mRNA decay (NMD) genes that were up-regulated in cells with SF3B1 knockdown, suggesting NMD activation following SF3B1 silencing. We used Human Exon-Junction arrays (Affymetrix) to evaluate global transcript exon usage in the K562 and TF1 cell lines with SF3B1 knockdown. We observed significant differential exon usage in genes involved in RNA degradation, spliceosome, cell cycle and apoptosis. We further observed aberrant splicing of the candidate gene ABCB7 showing exon skipping and TP53 gene showing exon skipping as well as intron retention. We have investigated the changes in the transcriptome in CD34+ cells from MDS patients with SF3B1 mutation by RNA sequencing and found many genes showing significant differential exon usage including CCND1, EIF3B, FKBP1A, BCL2 and RB1. Using Ingenuity Pathway Analysis we identified alternative splicing pattern of genes involved in cell cycle, RNA processing, mTOR signalling and P53 signalling pathways. We have studied CD34+ cells from MDS patients with SF3B1 mutation in vitro and observed impairment in cell growth compared to CD34+ cells from healthy controls or from MDS patients without splicing mutations. In colony forming assays we observed a decrease in the number of erythroid or myeloid colonies derived from CD34+ cells of patients with SF3B1 mutation compared to patient CD34+ cells without splicing factor mutation. The identification of SF3B1 downstream targets in SF3B1 mutant and wild-type erythroid and myeloid colonies from MDS patients is in progress using RNA sequencing. Our results show that knockdown of SF3B1 in haematopoietic cell lines results in impaired cell growth, deregulated global gene expression and aberrant splicing. Studies of the haematopoietic progenitor CD34+ cells of patients with SF3B1 mutation show impaired cell growth and erythroid differentiation as well as deregulation of many pathways including the cell cycle and RNA processing. The identification of the key target genes affected by the common splicing mutations in MDS is critical to our understanding of how the mutations contribute to the pathogenesis of this disorder. Disclosures: Maciejewski: NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Triple Negative (JAK2 exon 12 /14 and MPL wild type) Myelofibrosis Have Higher Expression Of CDC25A and Greater Sensitivity To CDC25A Inhibition Compared To JAK2 Mutant Cases

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5259-5259
Author(s):  
Ali Tabarroki ◽  
Valeria Visconte ◽  
Heesun J. Rogers ◽  
Juraj Bodo ◽  
Li Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rna Sequencing ◽  
Western Blotting ◽  
Rna Splicing ◽  
Conflicts Of Interest ◽  
Direct Sequencing ◽  
Wild Type ◽  
Illumina Platform ◽  
Sequencing Technologies ◽  
Stat Signaling

Abstract Pharmacologic therapies that target the JAK-STAT pathway are clinically used to alleviate splenomegaly and disease-related constitutional symptoms in MF. However, it is clear that some patients develop intolerance or resistant to this therapy. Furthermore, there are MF related complications especially cytopenias that are not alleviated by these therapies. Therefore, alternative and complementarytherapies are warranted in the management of MF. We hypothesized that other pathways downstream of the JAK-STAT signaling pathway can play a role in the pathophysiology of MF. We used whole exome (WES) and RNA sequencing technologies to interrogate new molecular markers and pathways which can serve as novel targets for this disease. In 4 MF patients [JAK2 mutant (MUT) =2, and wild type (WT) =2], WES was performed using the Illumina platform. All of the variants were filtered based on PHRED score (>=30) with coverage was set at 30X. Analysis of data in JAK2/MPL WT patients demonstrated the presence of 263 candidate genes. After clarifying the status of tumor nucleotide variants in each gene compared to germline (CD3+) fraction, 7 genes (RBL1, ADSS, ZNF717,MUC4, TUBB4Q and CDC25A) were selected for further somatic confirmation by direct sequencing. Among these genes, only alteration in CDC25A, a regulator of cyclinE/cdk2 (cyclin-dependent kinase-2) and cyclinA/cdk2 kinase, was confirmed to be somatic. This genetic change was previously reported as somatic by WES in lung cancer although not confirmed by direct sequencing (Bartkova et al, Nature, 2005, Apr 14; 434 (7035):864-70). Based on these observations and since CDC25A acts as a downstream effector of JAK-STAT signaling, we hypothesized that, CDC25A phosphatase, may be a driver in MF pathogenesis. The transcriptome of two patients, one MUT and one WT for JAK2 was then analyzed. RNA was isolated from bone marrow (BM) cells of healthy individuals (HI) (N=3). cDNA was made from 1.5-3 ug of RNA and fragmented for library preparation. RNA-sequencing was performed on 20 million sequence reads. Paired-end 90 base pair reads were generated on an Illumina HiSeq2000 sequencer and aligned to the human genome 19. RNA-splicing patterns were analyzed by a bioinformatics algorithm and gene expression analysis was carried out using GSEA (Visconte V; Blood. 2012). By using FDR<0.2, 11,460 genes were expressed. Further analysis demonstrated, CDC25A was over-expressed in both cases compared to HI but interestingly more highly expressed in JAK2 WT cases [fold change (FC): 0.39].This finding was validated by performing Western blotting and immunohistochemistry (IHC). To evaluate the protein expression of CDC25A in 10 MF (JAK2 MUT=5, JAK WT=5) patients and 5 HI, western blotting was performed; and higher expression in WT and MUT compared to HI were observed. Furthermore, its expression was also higher in WT compared to MUT cases. This is in contrast to a previous report by Gautier EF et al (Blood, 2012 Feb 2;119 (5):1190-9) where CDC25A expression was less in JAK2 WT cases compared to MUT cases. IHC was performed to confirm the difference of expression level of CDC25A in JAK2 MUT and WT bone marrow samples (N=8). IHC showed that JAK2 WT samples had many positive megakaryocytes stained with CDC25A antibody (>80%) while JAK2 MUT samples had only a few positive megakaryocytes (<20%). To test the feasibility of targeting this pathway in patients with MF and to assess for differential response between JAK2 MUT and WT cases, a potent cell permeable 7-substituted quinolinedione derivedCDC25 phosphatase inhibitor (NSC663284) was tested in JAK2 MUT (N=2) vs WT (N=1). Cell proliferation was determined by Trypan Blue and MTT assay after cell exposure to different concentrations of the inhibitor [3, 5, 7, 10 and 30uM] in 24 hours observation. NSC663284 induced higher dose-dependent cell growth inhibition in JAK2 WT compared to MUT cases (% of viable cells in WT vs MUT using previously mentioned concentrations, 3 uM= 98% vs 86%, 5 uM= 93% VS 77%, 7 uM= 88% vs 65%, 10 uM= 71% vs 43% and 30 uM= 25% vs 61%; p=0.01).In conclusion, CDC25A is more highly expressed in patients with wild type JAK2 compared to the mutant counterpart and primary cells from WT JAK2 patients demonstrate higher sensitivity to CDC25A inhibition, warranting further clinical testing of this therapeutic strategy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Prognostic interaction between bone marrow morphology and SF3B1 and ASXL1 mutations in myelodysplastic syndromes with ring sideroblasts

2018 ◽  
Vol 8 (2) ◽  
Author(s):  
Abhishek A. Mangaonkar ◽  
Terra L. Lasho ◽  
Christy M. Finke ◽  
Naseema Gangat ◽  
Aref Al-Kali ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Bone Marrow Morphology ◽  
Ring Sideroblasts

scholarly journals Next Generation RNA Sequencing of Acute Promyelocytic Leukemia (APL) Identifies Novel Long Non Coding RNAs Including New Variants of MIR181A1HG That Are Differentially Expressed during Myeloid Differentiation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1031-1031
Author(s):  
Frederic Lammer ◽  
Marion Klaumuenzer ◽  
Maximilian Mossner ◽  
Johann Christoph Jann ◽  
Anna Hecht ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rna Sequencing ◽  
Expression Analysis ◽  
Myeloid Differentiation ◽  
In Vitro Differentiation ◽  
Hematopoietic Stem ◽  
Genomic Deletion ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Introduction: Recently we identified a recurrent acquired genomic deletion on chromosome 1q as a potential new marker in approximately 14% of APL patients predicting a significantly increased risk of relapse (Nowak D et al., Genes Chromosomes and Cancer 2012). The deleted region contains the coding sequences for the microRNAs hsa-mir-181a1 and hsa-mir-181b1, which have been implicated as prognostic factors in Acute Myeloid Leukemia (AML) and a corresponding host gene (MIR181A1HG). To elucidate biologic mechanisms associated with the described genomic deletion we performed targeted sequencing of the affected region and RNA sequencing of APL samples carrying the deletion versus samples not carrying the deletion with subsequent validation of novel variants of MIR181A1HG. Methods: Explorative sequencing of genomic DNA in the chromosomal subband 1q31.3, pos. 197073900-197196158 (hg18) was performed using the amplicon sequencing workflow of the Roche 454 platform sequencing 5000 bp fragments tiling a region of approximately 120 kb on n=3 APL samples. Corresponding patient samples from molecular remission were used as germline controls. Whole transcriptome sequencing of poly-A enriched RNA was performed on n=6 samples of bone marrow blasts of APL patients either carrying a deletion of the mir181a1/b1 coding region (n=3) or not carrying a deletion (n=3). RNA Sequencing was performed using the HiSeq2000 platform. Data analysis was carried out using Bowtie vers. 2.2.30, TopHat vers. 2.0.12 for alignment and mapping and the Cufflinks package vers. 2.2.1 for transcriptome assembly and expression analysis all using default settings and hg19 as reference genome. Validation of newly identified variants and differential expression of MIR181A1HG was carried out by RACE PCR and qRT-PCR on cDNA from primary leukemic blasts of APL patients (n=45), CD34+ cells from healthy donors (n=29). In vitro differentiation assays with concomitant gene expression analysis of MIR181A1HG variants were performed with CD34+ cells from healthy donors. Results: Genomic sequencing of the recurrently deleted region revealed no somatically acquired mutations in the analyzed APL samples. Differential gene expression analysis using FPKM values (Fragments Per Kilobase Of Exon Per Million Fragments Mapped) inferred from RNA sequencing data of APL samples carrying a genomic deletion of 1q31.3 versus non-deleted samples identified n=58 genes significantly downregulated in deleted samples and n=31 upregulated genes. Interestingly, among the differentially regulated genes, BAALC, a factor recently shown to be prognostically relevant in APL was significantly upregulated 13 fold in the unfavourable group of samples with 1q31.3 deletions. Furthermore, RNA sequencing revealed numerous new isoforms of known transcripts as well as novel long non-conding RNA (lncRNA) sequences. Among these were a total of 6 new transcript variants of the MIR181A1HG gene in the recurrently deleted region on chromosome 1q31.3. One novel 5600bp lncRNA covering the coding regions for the hsa-mir-181a1/b1 was 24 fold overexpressed in samples carrying the recurrent 1q31.3 deletions. Expression analysis of MIR181A1HG in blasts of APL patients, CD34+ cells, unselected bone marrow cells and granulocytes of healthy donors revealed significantly elevated levels of MIR181A1HG in APL cells as compared to healthy CD34+ cells and almost absent expression in unselected bone marrow and granulocytes. This indicated a possible role for MIR181A1HG in APL blasts and hematopoietic stem cells. Subsequent in vitro differentiation experiments of primary healthy CD34+ cells showed that MIR181A1HG is downregulated 7 fold within 14 days of cytokine induced myeloid differentiation. Furthermore, MIR181A1HG was downregulated 5 fold during ATRA induced differentiation of NB4 cells. Conclusion: RNA sequencing of APL cells demonstrated numerous novel uncharacterized lncRNAs whose expression is associated with clinical risk and which merit further investigation. Identification of novel isoforms of MIR181A1HG, which are highly expressed in APL blasts and purified CD34+ cells suggest a potential role for this lncRNA in hematopoietic stem cells and response to ATRA induced differentiation of APL cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Genomic Landscape of Pediatric Myelodysplastic Syndromes

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 956-956 ◽  
Author(s):  
Jason R. Schwartz ◽  
Michael P. Walsh ◽  
Jing Ma ◽  
Tamara Lamprecht ◽  
Shuoguo Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Rna Splicing ◽  
Copy Number ◽  
Mapk Pathway ◽  
Chromosome 7 ◽  
Refractory Anemia ◽  
Juvenile Myelomonocytic Leukemia ◽  
Germline Variants ◽  
Genomic Landscape

Abstract Myelodysplastic syndromes are uncommon in children (incidence of ~2 cases/million) and have a poor prognosis. Despite the wealth of knowledge about the genomic landscape of adult MDS, much less is understood about pediatric MDS, and many recurrent mutations found in adults are not common in children (Hirabayashi, Blood 2012). Furthermore, the clinical presentation, bone marrow morphology, and cytogenetic abnormalities are also different when comparing adult and pediatric MDS, suggesting disparate underlying mechanisms. Here we describe the somatic and germline genomic landscape of pediatric MDS using whole exome sequencing (WES) and RNA-sequencing. We evaluated 88 diagnostic bone marrow samples obtained from the St. Jude Children's Research Hospital Tissue Bank from patients diagnosed between 1988 and 2015. This cohort contains 34 primary MDS, including Refractory Cytopenia of Childhood/RCC (n=19) and Refractory Anemia with Excess Blasts/RAEB (n=15). For comparison, we also included 32 treatment-related (tMDS), 14 MDS/MPN (including 10 Juvenile Myelomonocytic Leukemia/JMML), and 8 cases of AML with Myelodysplasia-Related Changes/AML-MRC (including 5 cases that previously would have been classified as RAEB in transformation/RAEB-T). WES was completed for 87 tumor/normal pairs (tumor only, n=1) using the Nextera Rapid Capture Expanded Exome (Illumina). Normal comparator gDNA was obtained from flow-sorted lymphocytes and variants were classified as germline if present at greater than 30% variant allele frequency (VAF) in the lymphocyte sample; thus, bone marrow mosaicism cannot be excluded. Mean sequencing coverage for the tumor and normal samples were 102x and 105x, respectively. An average of 7.9 variants were observed per patient in the primary MDS cohort (RCC=6.3, RAEB=10.2), compared to 25.5/patient in the tMDS cohort (p=0.001). Copy number information, obtained from WES data, determined that deletions involving chromosome 7 were frequent (n=28, 32%). Approximately 50% of RCC cases had deletions involving chromosome 7 (9 of 19), compared to only 20% of RAEB cases (3 of 15). Amplification of chromosomes 8 (n=7, 8%) and 21 (n=6, 7%), and deletions of 17 (n=5, 6%) were present at low frequency. In total, we detected 43 additional copy number abnormalities (including 9 cryptic chromosome 7 abnormalities) with WES compared to standard conventional karyotyping. RAS/MAPK pathway mutations were present in 42% of the patients (49 total mutations in 37 cases, including 4 germline variants). Fourteen of the 34 primary MDS cases (41%) had at least one RAS/MAPK mutation, including 13 somatic and 2 germline variants. Mutations in RNA splicing genes (germline, n=0; somatic, n=7; 8% of cohort) were less common, unlike what is observed in adult MDS. As expected, 2 patients with JMML harbored germline variants in PTPN11 and NF1. Surprisingly, presumed germline variants were detected in RRAS and NF1 in patients with primary MDS. Germline variants in transcription factors seen in familial MDS/AML (e.g., RUNX1, CEBPA, ETV6, GATA2) were uncommon, although a germline GATA2 variant was found in a single AML-MRC case. RNA-seq using the TruSeq (Illumina) Stranded RNA protocol was performed on 70 samples with suitable RNA. Fusion transcripts were uncommon in primary MDS, while fusions involving KMT2A, NUP98, RUNX1, MECOM, and ETV6were detected in the tMDS and AML-MRC cohorts. Although many of the mutations affecting the RAS/MAPK pathway were in common genes (NRAS, PTPN11, NF1, or CBL), many other mutations were in genes less frequently reported to be mutated in myeloid neoplasms, such as BRAF, SOS1, RIT1 and RRAS. We demonstrated that the mutations in BRAF (G469A, D594N, N581I) and SOS1 (E433K, G328R, S548R) found in our cohort activate the RAS/MAPK pathway to variable levels, as measured by ERK phosphorylation. In addition, expression of the BRAFvariants conferred IL3-independence in Ba/F3 cells. In conclusion, we show that the genomic landscapes of pediatric and adult MDS are different, namely in patterns of RAS/MAPK pathway and RNA splicing gene mutations, thus supporting the notion that MDS in adults and children comprise unique biological entities. The enrichment of RAS/MAPK mutations in pediatric MDS suggests biologic overlap with JMML and may provide direction for future therapeutic options. Disclosures No relevant conflicts of interest to declare.

Immunophenotypic Diagnostic Score for MDS Taking into Account Various Cell Populations.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1445-1445
Author(s):  
Arnaud Millaret ◽  
Carlos Galmarini ◽  
Danielle Treille Ritouet ◽  
Luc M. Gerland ◽  
Charles M. Dumontet
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Wilcoxon Test ◽  
Cell Populations ◽  
Acute Leukemias ◽  
Diagnostic Score ◽  
Normal Bone ◽  
Morphological Studies ◽  
Cd34 Cells ◽  
Lower Expression

Abstract Immunophenotyping has proven to be an interesting approach in patients with myelodysplastic syndromes (MDS)(Maynadie et al., Blood. 2002 Oct 1;100(7):2349–56). Most studies in these diseases have focused on the analysis of blast cells. We chose to determine the potential interest of markers expressed on other bone marrow compartments, namely granulocytes, monocytes and the CD34 compartment. Fourteen marrow samples from patients with MDS and 10 normal bone marrow were analyzed. The median age in the MDS group and the control group was 64.4 and 59.8 years, respectively. Flow cytometry analysis of bone marrow samples was performed on a Facs Calibur, using four-color panels. The following antigens were analyzed: CD7, CD11c, CD14, CD15, CD16, CD33, CD34, CD45, CD36, CD38, CD56, CD71, CD117 and HLA-DR. The various compartments analyzed were defined using CD45/SSC gating, combined with SSC/CD34 gating for CD34+ cells. The median percentage of blasts in the MDS marrows was < 5%. Seven patients had a hemoglobin value < 100 g/dl. Five patients had a leukocyte count < 3.109/l. Six patients had a platelet count < 100.1012/L. Threshold values were determined by ROC analysis. Comparison of the expression levels of markers between MDS and normal bone marrows was performed using a Wilcoxon test. Analysis was performed on bone marrow granulocytes, monocytes and CD34 cells. Granulocytes were found to express higher levels of CD33 in MDS than in normal marrows (p=0.007). CD34 + cells in MDS marrows expressed significantly higher levels of CD11c (p=0.007), CD117 (p=0.023) and CD33 (p=0.013) than their normal counterparts, with a trend towards lower expression of CD71 (p=0.053). Monocytes in MDS marrows expressed significantly lower levels of CD16 (p=0.006), CD36 (p=0.004) and CD117 (p=0.007) than their normal counterparts, with a trend towards lower expression of CD11c (p=0.059). We did not observe any differences in the antigen expression of MDS erythroblasts in comparison to their normal counterparts. A diagnostic score (0–7) was constructed taking into account the following seven parameters: expression of CD33 on granulocytes; CD11c, CD33 and CD117 on CD34+ cells; CD16, CD36 and CD117 on monocytes. This score distinguished all cases of MDS samples from controls. These results suggest that analysis of cell populations other than blasts could be useful in patients with MDS and that an immunophenotypic panel composed of markers commonly used in the diagnosis of acute leukemias could constitute a useful tool, in complement to morphological studies, for the diagnosis of myelodysplastic syndromes.

Expression Profiling of CD34+ Cells in Patients with Myelodysplastic Syndromes with and without a del(5q).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2363-2363
Author(s):  
Andrea Pellagatti ◽  
Aristotle Giagounidis ◽  
Sally Killick ◽  
Lisa J. Campbell ◽  
Vicki Wraight ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Multiple Testing ◽  
Suppressor Gene ◽  
T Test ◽  
Metastasis Suppressor ◽  
Multiple Testing Correction ◽  
Hematopoietic Stem ◽  
Putative Tumor Suppressor Gene ◽  
Cd34 Cells

Abstract The myelodysplastic syndromes (MDS) are a heterogeneous group of haematopoietic malignancies, characterized by blood cytopenias, ineffective hematopoiesis and hypercellular bone marrow. MDS are believed to arise from the malignant transformation of a hematopoietic stem cell. We have used Affymetrix microarray technology to determine gene expression profiles in bone marrow CD34+ cells of 17 MDS patients and 6 healthy controls. Nine patients with RA and 8 patients with RAEB were included in the study. Eleven of 17 patients had a del(5q). CD34+ cells were isolated from bone marrow samples of MDS patients and controls using MACS magnetic cell separation columns (purity greater than 85%). Extracted total RNA was amplified using the Two-Cycle Target Labelling and Control Reagent package (Affymetrix). Biotin-labelled fragmented cRNA was hybridized to GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix), covering over 47,000 transcripts representing 39,000 human genes. Cell intensity calculation and scaling was performed using GeneChip Operating Software (GCOS) and data analysis using GeneSpring 6.2. Genes up-regulated by >2 fold in the majority of MDS patients include IFITM1, an interferon-inducible protein implicated in the control of cell growth, and KANGAI1, a metastasis suppressor gene. IFITM1 was up-regulated in 15 of 17 MDS patients. Up-regulation of DLK1, previously reported in MDS, was confirmed in 8 of 17 MDS patients. Genes down-regulated by >2 fold in the majority of MDS patients include FOSB (a transcription factor which regulates cell proliferation and differentiation), COX2 (anti-apoptotic, promotes cell survival), Gravin/AKAP12 (a putative tumor suppressor gene), CD24 and MME. FOSB was down-regulated in 16 of 17 MDS patients. The results for several genes have been confirmed by real-time quantitative PCR (TaqMan). MDS patients with a del(5q) could be discriminated from those without a del(5q), using a set of 12 genes obtained applying a parametric t-test with multiple testing correction (P<0.01). Five of the 12 genes (DCP2, HTGN29, ANKHD1, CSNK1A1 and RBM22) mapped to chromosome 5q and their expression levels were lower in MDS patients with del(5q) than those without del(5q). Using a parametric t-test with multiple testing correction (P<0.05), 221 genes were found to be significantly different between MDS patients and controls. These 221 genes were ranked by their power to discriminate the two classes. The eight genes with the highest prediction strength were used for leave-one-out cross-validation. Twenty-one of 23 samples (91%) were correctly classified as MDS or control and one was not assigned. This set of genes is being tested on an independent test set to perform class prediction. This study has identified genes differentially expressed in the CD34+ cells of MDS patients that may be important in the molecular pathogenesis of this disorder.

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