scholarly journals Aberrant splicing of genes involved in haemoglobin synthesis and impaired terminal erythroid maturation in SF 3B1 mutated refractory anaemia with ring sideroblasts

2015 ◽  
Vol 171 (4) ◽  
pp. 478-490 ◽  
Author(s):  
Simona Conte ◽  
Shintaro Katayama ◽  
Liselotte Vesterlund ◽  
Mohsen Karimi ◽  
Marios Dimitriou ◽  
...  
Keyword(s):  
Refractory Anaemia ◽  
Aberrant Splicing ◽  
Ring Sideroblasts ◽  
Erythroid Maturation ◽  
Haemoglobin Synthesis

scholarly journals Haematological and molecular responses in refractory anaemia with ring sideroblasts and thrombocytosis treated with lenalidomide

2013 ◽  
Vol 92 (2) ◽  
pp. 179-180 ◽  
Author(s):  
Jo Caers ◽  
Kaoutar Hafraoui ◽  
Aurore Keutgens ◽  
Jean-Hubert Caberg ◽  
Frederic Lambert ◽  
...  
Keyword(s):  
Refractory Anaemia ◽  
Molecular Responses ◽  
Ring Sideroblasts

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.

Iron Overload, Oxidative Damage and Ineffective Erythropoiesis in Myelodysplastic Syndromes

2010 ◽  
Vol 00 (04) ◽  
pp. 34 ◽  
Author(s):  
Rosangela Invernizzi ◽  
Keyword(s):  
Oxidative Damage ◽  
Iron Overload ◽  
Myelodysplastic Syndromes ◽  
Iron Homeostasis ◽  
Current Knowledge ◽  
Early Stage ◽  
Refractory Anaemia ◽  
Ineffective Erythropoiesis ◽  
Ring Sideroblasts ◽  
High Level

A high level of apoptosis may be responsible for the ineffective haematopoiesis in myelodysplastic syndromes (MDS). Recently, it has been demonstrated that the erythroid apoptosis of low-risk MDS is initiated at a very early stage in stem cells and is associated with mitochondrial dysfunction. However, the underlying pathogenetic mechanisms causing malfunctioning of mitochondria and initiation of the intrinsic apoptotic cascade are not completely clear. Recent studies suggest a close relationship between impaired iron metabolism and pathogenesis of myelodysplasia. In fact, iron overload, which is apparent in refractory anaemia with and without ring sideroblasts, may lead to the generation of intracellular free radicals, thereby causing oxidative damage and inducing apoptosis in haematopoietic progenitors. This review summarises current knowledge supporting the role of iron-related oxidative damage in the pathogenesis of MDS. The relationship between mitochondrial iron homeostasis impairment and ineffective erythropoiesis in refractory anaemia with ring sideroblasts as well as the various functions of the cytosolic and mitochondrial ferritins are also discussed.

Haemoglobin Synthesis during Erythroid Maturation in β-Thalassaemia

1972 ◽  
Vol 240 (101) ◽  
pp. 190-192 ◽  
Author(s):  
J. B. CLEGG ◽  
D. J. WEATHERALL
Keyword(s):  
Erythroid Maturation ◽  
Haemoglobin Synthesis

Erythropoiesis In SF3B1 Mutated RARS Is Disrupted During Terminal Erythroid Maturation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2408-2408
Author(s):  
Simona Conte ◽  
Liselotte Vesterlund ◽  
Shintaro Katayama ◽  
Mohsen Karimi ◽  
Elli Papaemmanuil ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Culture System ◽  
Refractory Anemia ◽  
Erythroid Progenitors ◽  
Allele Burden ◽  
Recurrent Mutations ◽  
Ring Sideroblasts ◽  
Erythroid Maturation ◽  
Allelic Burden

Abstract Refractory anemia with ring sideroblasts (RARS) is characterized by severe anemia, erythroid apoptosis and aberrant mitochondrial ferritin accumulation in erythroblasts. Dominant mutations in SF3B1, a core component of the splicing machinery are detected in more than 75% of patients. Of the recurrent mutations, SF3B1 alone is associated with the RARS phenotype. We recently showed that SF3B1 suppresses the expression of the mitochondrial transporter protein ABCB7, which in turn mediates erythroid failure in RARS. The exact mechanisms remain unclear, hence potential targets for treatment have not been identified. Fifty-six patients with RARS or RCMD-RS were subjected to targeted sequencing of which 52 (93%) had SF3B1 mutations and 43 also other recurrent mutations mostly involving epigenetic regulators. To explore the mechanisms behind the disrupted RARS erythropoiesis molecular and biological features of erythroid progenitors from SF3B1 mutated RARS and normal bone marrow (NBM) were monitored in vitro throughout a well-established 14-days liquid culture system. Normal and RARS transcriptomes (RNAseq) were analyzed at two time points (CD34+ cells at day 0 and early erythroblasts at day 4) during differentiation, followed by validation in an extended cohort of 11 RARS and 4 NBM using Taqman Low Density Array (TLDA). CD34+ RARS progenitors were characterized by activation of genes involved in the oxidative stress pathway and in particular genes involved in defense against oxidative stress, such as SEPP-1. This activation dropped dramatically at day 4. By contrast, early differentiation in RARS displayed a marked failure to up-regulate genes in the autophagy pathway, which is essential for removal of organelles and terminal maturation to erythrocytes. We then followed the clone size of SF3B1 in 8 erythroid cultures, two of which also underwent myeloid differentiation. 4 cultures used RARS patients with stable untreated anemia (Hb 107 g/L ± 15), a high percentage of ring sideroblasts and a relatively normal reticulocyte count. SF3B1 allelic burden was assessed by pyrosequencing at day 0, 4, 7 and 14. To exclude the possibility that the culture system itself promoted survival of mutated progenitors we also analyzed isolated GPA+ progenitors from uncultured mononuclear BM, and finally we analyzed blood reticulocytes from the 4 untreated patients. We observed a normal expansion of both erythroid and myeloid progenitors during culture, with a stable allele burden (± 10%) in all patients. In fact, cultured and non-cultured erythroid progenitors showed a higher allelic burden than the corresponding myeloid cells. However, the allele burden in reticulocytes was only 60% of that found in the freshly isolated GPA+ fraction, indicating that reticulocytes to a greater extent derived from unmutated cells (Figure 1). In summary, we show that genes involved in autophagy are markedly down-regulated during early erythroid differentiation in RARS. While SF3B1 mutations do not confer a growth disadvantage to differentiating erythroid bone marrow progenitors, the final step of erythroid maturation to reticulocytes is clearly affected. Hence, we demonstrate a novel interference of the SF3B1 mutation with terminal erythroid maturation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Low rate of calreticulin mutations in refractory anaemia with ring sideroblasts and marked thrombocytosis

Leukemia ◽  
2014 ◽  
Vol 28 (6) ◽  
pp. 1374-1376 ◽  
Author(s):  
J Broséus ◽  
E Lippert ◽  
A S Harutyunyan ◽  
S Jeromin ◽  
E Zipperer ◽  
...  
Keyword(s):  
Refractory Anaemia ◽  
Ring Sideroblasts ◽  
Low Rate

Refractory anaemia with ring sideroblasts (RAS): Caracterization and prognostic factors

1991 ◽  
Vol 15 ◽  
pp. 18
Author(s):  
T. Vallespí ◽  
F. López ◽  
D. Irriguible ◽  
G. Sanz ◽  
A. Juliá ◽  
...  
Keyword(s):  
Prognostic Factors ◽  
Refractory Anaemia ◽  
Ring Sideroblasts

scholarly journals Mutant Splicing Factor 3b Subunit 1 (SF3B1) Causes Dysregulated Erythropoiesis and a Stem Cell Disadvantage

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 828-828 ◽  
Author(s):  
Esther A. Obeng ◽  
Marie E. McConkey ◽  
Dean Campagna ◽  
Rebekka K. Schneider ◽  
Michelle C. Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Macrocytic Anemia ◽  
Splicing Factor ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Erythroid Precursors ◽  
Ring Sideroblasts ◽  
Erythroid Maturation ◽  
Mutant Cells

Abstract Recurrent, heterozygous, somatic mutations in components of the mRNA spliceosome complex were recently identified in over 60% of myelodysplastic syndrome (MDS) patients. Splicing factor mutations are thought to be founding mutations in MDS based on their allele fraction at diagnosis. Splicing factor 3b Subunit 1 (SF3B1) is the most frequently mutated splicing factor in MDS. SF3B1 mutations are highly associated (70 – 85% of cases) with refractory anemia with ring sideroblasts (RARS), a morphologic subtype of MDS characterized by the presence of erythroid precursors with perinuclear iron-laden mitochondria in the bone marrow. The pathophysiological role of SF3B1 mutations in MDS has yet to be elucidated. To explore the biology of SF3B1 mutations, we generated a heterozygous conditional knock-in mouse model of the most common SF3B1 mutation, K700E. Heterozygous conditional knock-in of Sf3b1K700E leads to a progressive macrocytic anemia, with normal absolute neutrophil and platelet counts. Over the course of 15 months, mutant mice developed a statistically significant macrocytic anemia (hemoglobin of 11.4 g/dL vs. 14 g/dL, p = 0.004; MCV of 63.1 fL vs. 58.4 fL, p = 0.008) associated with elevated plasma erythropoietin levels (257.5 pg/mL vs. 101 pg/mL, p = 0.0016). Analysis of hematopoietic stem and progenitor cells at 12 and 65 weeks after induction showed a similar percentage of stem (LT-HSC, ST-HSC, MPP, LSK) and progenitor (LK, CMP, GMP, MEP, pre CFU-E) cells in Sf3b1K700E and wild-type animals. Histopathologic analysis revealed no significant difference in spleen weights, but increased erythroid islands in the red pulp of mutant animal spleens; suggestive of ineffective erythroid maturation. Sf3b1K700E animals have a normocellular bone marrow with rare ring sideroblasts. No ring sideroblasts were identified in wild-type controls. No overt hematological malignancies were identified during the observation period, however two mutant animals succumbed to significant anemia (2 of 11, 18%) compared to zero deaths in the wild-type controls. To further characterize the erythroid-specific phenotype observed in Sf3b1K700E mice, mutant and wild-type animals were treated with phenylhydrazine, a drug that induces intravascular hemolysis. Sf3b1K700E mice had a more rapid onset of anemia and a higher reticulocytosis during count recovery compared to wild-type controls. Analysis of the bone marrow and spleens was notable for a higher percentage of immature erythroid precursors (R2/basophilic erythroblasts) and a lower percentage of more mature erythroid precursors (R4/orthochromatophilic erythroblasts) in mutant animals, consistent with impaired erythroid maturation. An in vitro erythroid differentiation assay using purified ckit+ progenitor cells from Sf3b1K700E mice yielded significantly fewer erythroblasts (p = 0.0226) when compared to cells from wild type mice due to a statistically significant increase in the percentage of mutant cells in G0 (p=0.018). Similarly, noncompetitive transplantation assays highlighted the cell intrinsic nature of these erythroid-specific findings, as mutant cells did not show a defect in repopulating recipients, however Sf3b1K700E recipients developed a progressive macrocytic anemia. Competitive transplantation assays demonstrated a competitive disadvantage in Sf3b1K700E hematopoietic stem cells. Engraftment was lower in Sf3b1K700E compared to wild-type recipients 4 weeks (33.9% vs. 54.4%, p = 0.002) and 16 weeks (29% vs. 62.4%, p = 0.0013) after transplantation. These findings are consistent with the fact that RARS patients have a lower risk of progression to acute myeloid leukemia compared with other MDS subtypes. Taken together, our results demonstrate that heterozygous mutations in Sf3b1 lead to aberrant erythroid maturation and ineffective hematopoiesis in mice. These findings are consistent with the clinical picture seen in RARS patients. The results from the competitive transplantation studies may be consistent with the more favorable prognosis seen in patients with RARS, as our data suggest that additional genetic or epigenetic alterations must be acquired in SF3B1K700E cells to facilitate the development of clonal dominance. Disclosures No relevant conflicts of interest to declare.

SIGNIFICANCE OF RING SIDEROBLASTS IN REFRACTORY ANAEMIA WITH EXCESS OF BLASTS

1987 ◽  
Vol 65 (1) ◽  
pp. 119-120 ◽  
Author(s):  
Yataro Yoshida ◽  
Shigeru Oguma ◽  
Haruto Uchino ◽  
Tadashi Maekawa
Keyword(s):  
Refractory Anaemia ◽  
Ring Sideroblasts
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