scholarly journals Congenital Disorders of Ribosome Biogenesis and Bone Marrow Failure

2010 ◽  
Vol 16 (1) ◽  
pp. S12-S17 ◽  
Author(s):  
Kathleen M. Sakamoto ◽  
Akiko Shimamura ◽  
Stella M. Davies
Keyword(s):  
Bone Marrow ◽  
Ribosome Biogenesis ◽  
Bone Marrow Failure ◽  
Congenital Disorders

A Novel Radiosensitivity Phenotype in Shwachman-Diamond Syndrome Is Mediated By ER Stress Response

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3618-3618
Author(s):  
Nimrat Chatterjee ◽  
Christopher Lee Williams ◽  
Saleh Bhar ◽  
Alison A Bertuch
Keyword(s):  
Bone Marrow ◽  
Dna Repair ◽  
Stress Response ◽  
Er Stress ◽  
Ribosome Biogenesis ◽  
Bone Marrow Failure ◽  
Hek293 Cells ◽  
80S Ribosome ◽  
Er Stress Response ◽  
Shwachman Diamond Syndrome

Abstract Shwachman-Diamond syndrome (SDS), an autosomal recessive disorder, is characterized by bone marrow dysfunction, exocrine pancreatic insufficiency, congenital abnormalities, and leukemia predisposition (Myers et al., 2012). Most patients with SDS harbor biallelic mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SBDS is known to play a role in ribosome biogenesis by enabling eviction of the ribosome anti-association factor eIF6 from the 60S ribosomal subunit, to allow formation of the 80S ribosome (Wong et al., 2011). SBDS-depleted cells are, therefore, defective in ribosome assembly. In addition, absence of SBDS sensitizes cells to ultraviolet irradiation, translation inhibitors, and endoplasmic reticulum (ER) stressors, such as tunicamycin (Ball et al., 2009). A recent report indicated that lymphoblastoid cell lines (LCLs) derived from two SDS patients accumulated more DNA damage after being exposed to ionizing radiation (IR) (Morini et al., 2015). A deficiency in DNA repair was alluded to as a possible cause, however, the mechanism underlying this previously unreported phenotype was not determined. In this study, we investigated LCLs derived from five SDS patients with biallelic SBDS mutations and found all to be hypersensitive to IR in a colony survival assay. In this assay, increasing doses of IR resulted in a significantly lower survival fraction in SDS-compared to control-LCLs. We found SBDS expression to increase in control-cells when stressed with IR, suggesting that SBDS is a stress response protein and its absence in SDS-LCLs induces hypersensitivity to IR. Because knockdown of SBDS in HEK293 cells induces an ER stress response (Ball et al., 2009), we examined the expression of the ER stress response factor phospho-eIF2α in untreated and IR exposed SDS-LCLs and found phospho-eIF2α expression to be markedly increased compared to controls. This result indicated that SDS-LCLs may have an activated ER stress response, as was further confirmed by exposing these cells to additional ER stressors, tunicamycin and H2O2, and observing a similar upregulation of phospho-eIF2α. Because ER stress is known to suppress DNA double strand break (DSBR) (Yamamori et al., 2013), we examined the expression of Rad51 and Ku70, which are required for the homology-directed and nonhomologous end-joining pathways of DSBR, respectively. Surprisingly, we found Rad51 and Ku70 protein levels to be repressed in SDS-LCLs compared to controls, both with and without exposure to IR. Collectively, these data support the hypothesis that, in addition to its role in ribosome biogenesis, SBDS is a stress response protein that plays an important role in regulating the ER stress response. In SDS-cells, where SBDS is lacking, activated ER stress represses DNA repair proteins rendering cells hypersensitive to IR and other stresses. This novel pathway to ER stress induction may contribute to the bone marrow failure and cancer predisposition seen in SDS patients. Disclosures No relevant conflicts of interest to declare.

Premature Senescence in Hematopoietic Stem and Progenitor Cells in Ribosomopathy-Associated Bone Marrow Failure Syndromes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 298-298
Author(s):  
Hengjun Chao ◽  
Johnson M. Liu
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Ribosome Biogenesis ◽  
Bone Marrow Failure ◽  
Premature Senescence ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Bone Marrow Failure Syndromes ◽  
P53 Activation ◽  
Adult Bone

Abstract Introduction: Aged hematopoietic stem cells (HSCs) are known to functionally decline and are prone to development of myeloid malignancies. Recent work has highlighted the twin roles of replication stress and decreased ribosome biogenesis as drivers for the accumulation of DNA damage and senescence. Certain bone marrow failure syndromes, including Shwachman-Diamond syndrome (SDS), Diamond-Blackfan anemia (DBA), and the acquired 5q- syndrome, are characterized by defects in ribosome biogenesis. Furthermore, recent work has suggested a role for p53 activation, through the 5S ribonucleoprotein particle (RNP), in driving cells to senescence following perturbation of ribosome biogenesis. Methods and Results: Here, we have used multiplexing flow cytometry protocols to define, enumerate, and characterize hematopoietic cells of distinct differentiation stages and lineages in 2 DBA cord bloods and 4 adult bone marrows (2 SDS, 1 DBA, and 1 patient with a diminutive somatic deletion of 5q: ages 27, 32, 40, and 30, respectively), as compared with 4 normal cord bloods and 6 normal adult bone marrows. We included a patient with bona fide MDS (diminutive somatic deletion of 5q including RPS14 in a young adult) to compare with the SDS and DBA patients, who do not meet criteria for MDS. Our preliminary results revealed significant defects in the primitive HSC and multipotent progenitor (MPP) compartments in both DBA and SDS. Specifically, we found in DBA and SDS bone marrow and cord blood samples (compared to normal controls): significantly decreased numbers of primitive HSCs (Lin-CD34+CD133+CD38-CD45RA-CD49f+CD90+) and MPPs (Lin-CD34+CD133+CD38-CD45RA-CD49f-CD90-); increased levels of apoptosis and dysregulated proliferation; and G0-1/S cell cycle arrest. We also found significant increases in senescence-associated β-galactosidase staining and G0-1/S cell cycle arrest in Lin-CD34+ and Lin-CD34+CD38-CD133+ subpopulations in all 4 adult patient bone marrows, as compared with normal adult bone marrows processed in identical fashion [see Fig. 1 for representative data from Lin-CD34+CD133+ hematopoietic progenitor cells (HPCs) from one SDS patient]. Foci of the phosphorylated form of the variant histone H2AX (γH2AX) mark DNA damage, and γH2AX staining was similarly increased in comparison to controls (Fig. 1). The mechanism whereby disturbed ribosome biogenesis induces senescence has been suggested as involving 5S RNP-mediated p53 activation. However, our experiments did not demonstrate increased levels of p53 in the SDS patient marrows, as assessed by intracellular staining. Levels of p16, a well known marker of senescence, were markedly increased in the SDS patient samples, when compared to controls. Finally, in the 2 DBA cord bloods analyzed, there was increased senescence-associated β-galactosidase staining but to a lesser degree than in the adult bone marrow samples (as might be expected with temporal progression). Discussion: Taken together, our data suggest that ribosomopathies (which often present in childhood) are disorders of premature senescence. Consequent DNA damage accumulation and decreased repair and compensation may account for the development of MDS and acute myeloid leukemia, disorders seen in young ribosomopathy patients that ordinarily are rare in the general pediatric and young adult population. Disclosures No relevant conflicts of interest to declare.

scholarly journals Characterizing Dyskeratosis Congenita Caused By Parn Mutations in the Zebrafish

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3744-3744
Author(s):  
Anna Cordeiro ◽  
Adam P Deveau ◽  
Santhosh Dhanraj ◽  
Yigal Dror ◽  
Jason Berman
Keyword(s):  
Bone Marrow ◽  
Ribosome Biogenesis ◽  
Catalytic Domain ◽  
Conflicts Of Interest ◽  
Stop Codon ◽  
Bone Marrow Failure ◽  
Premature Stop Codon ◽  
Telomere Maintenance ◽  
Specific Gene ◽  
Hematopoietic Stem

Inherited bone marrow failure syndromes (IBMFs) are a group of rare genetic disorders characterized by deficient hematopoiesis and extra-hematologic traits. Most known entities are related to a specific gene or group of genes, but others still remain unclassified. In many cases, the involved proteins are required for critical processes involved in cell survival, such as ribosome biogenesis, maintenance of telomere length, and DNA repair. Importantly, patients with IBMFs have higher risks of developing a variety of cancers from leukemia to solid tumours of the head and neck. In 2015, we published four patients from three different families with mutations in the Poly(A)-specific ribonuclease (PARN) gene. This gene encodes a ribonuclease which is involved in degradation of the poly(A) tails, which regulate mRNA turnover, and thus gene expression. Three of the patients presented with several degrees of mental illness and/or developmental delay. The fourth patient, harbored both a monoallelic deletion and a point mutation at the catalytic domain of the protein, and presented with bone marrow failure and hypomyelination, similar to a severe form of dyskeratosis congenital (DC) known as Hoyeraal-Hreidarsson syndrome. In the last two decades, zebrafish (Danio rerio) has emerged as an excellent animal model for human disease, and is especially relevant in hematology, since many of the transcription factors and cell types are highly conserved. Zebrafish have a single PARN ortholog,with 64% sequence identity to the human gene. Using CRISPR-Cas9 genome editing and a combination of six sgRNAs, we generated a 1.2 kb deletion in the zebrafish parn ortholog extending from exon 5 to 13, causing a premature stop codon. Homozygous fish were generated by incrossing to replicate the complete loss-of-function observed in the patient with the DC-like phenotype. Using whole-mount in situ hybridization (WISH) at 48 hours post-fertilization (hpf), we observed a decrease in the number of several mature myeloid cell lineages including neutrophils (labeled with mpx; p<0.0001), macrophages (lcp1; p=0.0005) and mast cells (cpa5; p=0.0005). We also observed a decrease in the amount of hemoglobin (o-dianisidine staining; p<0.0001). However, the number of hematopoietic stem cells (HSCs) was unchanged in parn mutants. This data parallels similar findings using parn directed splice site and translation start site morpholinos. PARN is described as a protein involved in RNA processing, but has also been associated with telomere maintenance. This latter process is crucial for cell senescence and genome integrity, and is a known cause of several IBMFSs. The telomerase ribonucleoprotein complex is highly active in hematopoietic stem and progenitor cells (HSPCs) and plays a role in cell differentiation. This complex is composed of a reverse transcriptase (TERT), RNA template (TERC), and the dyskerin protein complex (DKC1), mutations of which represent a common cause of DC. qPCR analysis in zebrafish parn mutants revealed a 2.98-fold reduction in tert expression compared with the wild type fish. Combined, these findings suggest that PARN plays an important role in HSC differentiation into myeloid and erythroid lineages, resulting in a bone marrow failure phenotype. Our model provides a unique in vivo platform to study the role of PARN in hematopoiesis and for identifying compounds that restore normal blood cell ratios, which may have the potential to prevent future leukemic transformation. Disclosures No relevant conflicts of interest to declare.

The Effects of Pathogenic Dkc1 Mutations on Telomerase and Ribosome Biogenesis.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1163-1163
Author(s):  
Baiwei Gu ◽  
Jian-meng Fan ◽  
Rachel Idol ◽  
Monica Bessler ◽  
Philip J Mason
Keyword(s):  
Bone Marrow ◽  
Mammalian Cells ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Bone Marrow Failure ◽  
Mutant Cell ◽  
3D Structure ◽  
Tamoxifen Treatment ◽  
Variable Expression ◽  
Mutant Cells

Abstract Abstract 1163 Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome (BMF) associated with a predisposition to cancer. It is diagnosed by the triad of abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia. X-linked DC, accounting for around one third of DC patients, is due to the mutations in the DKC1 gene which encodes the protein dyskerin. Dyskerin is a component of small nucleolar RNA particles (H/ACA snoRNP) active in the pseudouridylation of specific residues in nascent ribosomal RNA (rRNA). It also plays an important role in maintaining the telomere repeats at the ends of chromosomes by forming and stabilizing the telomerase complex. Patients with Dkc1 mutations show a wide variation of severity emphasizing the variable expression of the disease and the influence of other genetic and environmental factors. Most mutations are clustered, in the 3D structure, in a region important for RNA binding and few mutations are present in the pseudouridine synthesis activity domain. The relationship between the severity of DC and the position of the mutation in dyskerin is still unclear. It will be interesting to compare the pathogenic effects of Dkc1 mutations in different positions of dyskerin protein. Since the production of mutant lines of mouse is time consuming, expensive, and may be difficult, here we established a suitable in vitro system to express mutant dyskerin in MEF cells. We began with MEF cells containing a Dkc1 gene that is deleted in the presence of Cre. Mutated dyskerin introduced by a retroviral vector is expressed and wild type (WT) dyskerin is deleted by retrovirally introduced Cre-ERT2 and subsequent tamoxifen treatment, which activates the Cre-ERT2 protein. MEF cells are then single cell cloned and selected cells that grew well and contained for the mutant dyskerin gene but not the WT gene. We obtained cell lines with several mutations including I38T, H68Q, S121G and A353V as well as WT control. All of these mutations are recurrent ones and cause severe bone marrow failure in patients. Interestingly, we also obtained a D125A mutant cell line. This mutation has not been found in human patients but is an alteration of a crucial catalytic aspartic acid residue that is essential for pseudouridylation activity. All of these Flag-tagged dyskerin proteins showed a normal nucleolar sub-cellular distribution pattern. We chose the cells expressing similar levels of flag-tagged dyskerin to WT dyskerin to do further experiments. The exception to this was the D125A mutation because all the clones express tiny amounts of flag-tagged dyskerin. First, we measured the Dkc1 mRNA level of these mutant cells and found the expression of Dkc1 mRNA in these mutant cells is 3–8 fold higher than in WT cells (25 fold higher for D125A). This result indicates that the mutant dyskerin proteins in these cells are very unstable, so more mRNA has to be produced to maintain growth. Then, we studied the growth curves of these cells and found that all mutant cells showed more or less slower growth rate than WT. Notably, the D125A mutant cells showed extremely slow growth. We find that the expression of Terc is lower than WT in all mutant cells (10∼50% compared to WT) while telomerase activity was not significantly decreased except for the D125A mutant. By Q-FISH, we showed the telomere length of all mutant cells is shorter than WT control cells and the number of telomere free ends is increased in these mutant cells. These findings indicate that all Dkc1 mutations affect the telomerase function and cause rapidly shortening telomeres. In ribosome biogenesis we found that, although I38T, H68Q, S121G and A353V mutant cells did not show significant changes in the ribosomal profile and ribosomal processing, D125A mutant cell showed significantly delayed maturation of rRNA as well as dramatically decreased amounts of mature 80S ribosomes. Further investigation revealed that the D125A mutation abolished in vivo pseudouridylation of rRNA. Together with our previous results this study further indicates that compromised telomerase function is the major effect of the pathogenic Dkc1 mutations. We also show, surprisingly, that mammalian cells can grow and survive with no pseudouridine in their rRNA. Disclosures: Bessler: Alexion Pharmaceutical Inc: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Taligen: Consultancy.

scholarly journals Nonsense Suppression Therapy: New Hypothesis for the Treatment of Inherited Bone Marrow Failure Syndromes

2020 ◽  
Vol 21 (13) ◽  
pp. 4672 ◽  
Author(s):  
Valentino Bezzerri ◽  
Martina Api ◽  
Marisole Allegri ◽  
Benedetta Fabrizzi ◽  
Seth J. Corey ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Solid Tumors ◽  
Ribosome Biogenesis ◽  
Ex Vivo ◽  
Bone Marrow Failure ◽  
Nonsense Suppression ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes ◽  
Telomere Biology ◽  
Nonsense Suppressor

Inherited bone marrow failure syndromes (IBMFS) are a group of cancer-prone genetic diseases characterized by hypocellular bone marrow with impairment in one or more hematopoietic lineages. The pathogenesis of IBMFS involves mutations in several genes which encode for proteins involved in DNA repair, telomere biology and ribosome biogenesis. The classical IBMFS include Shwachman–Diamond syndrome (SDS), Diamond–Blackfan anemia (DBA), Fanconi anemia (FA), dyskeratosis congenita (DC), and severe congenital neutropenia (SCN). IBMFS are associated with high risk of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and solid tumors. Unfortunately, no specific pharmacological therapies have been highly effective for IBMFS. Hematopoietic stem cell transplantation provides a cure for aplastic or myeloid neoplastic complications. However, it does not affect the risk of solid tumors. Since approximately 28% of FA, 24% of SCN, 21% of DBA, 20% of SDS, and 17% of DC patients harbor nonsense mutations in the respective IBMFS-related genes, we discuss the use of the nonsense suppression therapy in these diseases. We recently described the beneficial effect of ataluren, a nonsense suppressor drug, in SDS bone marrow hematopoietic cells ex vivo. A similar approach could be therefore designed for treating other IBMFS. In this review we explain in detail the new generation of nonsense suppressor molecules and their mechanistic roles. Furthermore, we will discuss strengths and limitations of these molecules which are emerging from preclinical and clinical studies. Finally we discuss the state-of-the-art of preclinical and clinical therapeutic studies carried out for IBMFS.

Haemopoietic stem cell transplantation

2010 ◽  
pp. 4571-4580
Author(s):  
E.C. Gordon-Smith ◽  
Emma Morris
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Failure ◽  
Severe Combined Immunodeficiency ◽  
Congenital Disorders ◽  
Combined Immunodeficiency ◽  
Haemopoietic Stem Cell ◽  
Bone Marrow Failure Syndromes ◽  
Lysosomal Diseases ◽  
Haematological Disorders ◽  
Haemopoietic Stem Cell Transplantation

Haemopoietic stem cells (HSC) give rise to the blood cell lineages and the cells of the immune system, and their transplantation may be an appropriate part of the management of conditions including (1) malignant haematological disorders e.g. leukaemia, lymphoma, myeloma (2) bone marrow failure syndromes—e.g. aplastic anaemia; (3) congenital disorders—(a) haematological—e.g. Fanconi’s anaemia; (b) immunological—severe combined immunodeficiency; (c) metabolic—e.g. lysosomal diseases....

scholarly journals The human Shwachman-Diamond syndrome protein, SBDS, associates with ribosomal RNA

Blood ◽  
2007 ◽  
Vol 110 (5) ◽  
pp. 1458-1465 ◽  
Author(s):  
Karthik A. Ganapathi ◽  
Karyn M. Austin ◽  
Chung-Sheng Lee ◽  
Anusha Dias ◽  
Maggie M. Malsch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Ribosomal Rna ◽  
Ribosome Biogenesis ◽  
Actinomycin D ◽  
Bone Marrow Failure ◽  
Ribosomal Subunit ◽  
Autosomal Recessive Disorder ◽  
Multifunctional Protein ◽  
Rrna Transcription ◽  
Shwachman Diamond Syndrome

Abstract Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic dysfunction, and leukemia predisposition. Mutations in the SBDS gene are identified in most patients with SDS. SBDS encodes a highly conserved protein of unknown function. Data from SBDS orthologs suggest that SBDS may play a role in ribosome biogenesis or RNA processing. Human SBDS is enriched in the nucleolus, the major cellular site of ribosome biogenesis. Here we report that SBDS nucleolar localization is dependent on active rRNA transcription. Cells from patients with SDS or Diamond-Blackfan anemia are hypersensitive to low doses of actinomycin D, an inhibitor of rRNA transcription. The addition of wild-type SBDS complements the actinomycin D hypersensitivity of SDS patient cells. SBDS migrates together with the 60S large ribosomal subunit in sucrose gradients and coprecipitates with 28S ribosomal RNA (rRNA). Loss of SBDS is not associated with a discrete block in rRNA maturation or with decreased levels of the 60S ribosomal subunit. SBDS forms a protein complex with nucleophosmin, a multifunctional protein implicated in ribosome biogenesis and leukemogenesis. Our studies support the addition of SDS to the growing list of human bone marrow failure syndromes involving the ribosome.

scholarly journals Mechanisms of somatic transformation in inherited bone marrow failure syndromes

Hematology ◽  
2021 ◽  
Vol 2021 (1) ◽  
pp. 390-398
Author(s):  
Haruna Batzorig Choijilsuren ◽  
Yeji Park ◽  
Moonjung Jung
Keyword(s):  
Bone Marrow ◽  
Ribosome Biogenesis ◽  
Bone Marrow Failure ◽  
Clonal Evolution ◽  
Germline Mutations ◽  
Partial Recovery ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes ◽  
Somatic Transformation ◽  
Genomic Studies

Abstract Inherited bone marrow failure syndromes (IBMFS) cause hematopoietic stem progenitor cell (HSPC) failure due to germline mutations. Germline mutations influence the number and fitness of HSPC by various mechanisms, for example, abnormal ribosome biogenesis in Shwachman-Diamond syndrome and Diamond-Blackfan anemia, unresolved DNA cross-links in Fanconi anemia, neutrophil maturation arrest in severe congenital neutropenia, and telomere shortening in short telomere syndrome. To compensate for HSPC attrition, HSPCs are under increased replication stress to meet the need for mature blood cells. Somatic alterations that provide full or partial recovery of functional deficit implicated in IBMFS can confer a growth advantage. This review discusses results of recent genomic studies and illustrates our new understanding of mechanisms of clonal evolution in IBMFS.

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