scholarly journals Pluripotent stem cell model of Shwachman–Diamond syndrome reveals apoptotic predisposition of hemoangiogenic progenitors

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Takayuki Hamabata ◽  
Katsutsugu Umeda ◽  
Kagehiro Kouzuki ◽  
Takayuki Tanaka ◽  
Tomoo Daifu ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Cell Model ◽  
Bone Marrow Failure ◽  
Pancreatic Insufficiency ◽  
Autosomal Recessive Disorder ◽  
Exocrine Pancreatic Insufficiency ◽  
Stem Cell Model ◽  
Shwachman Diamond Syndrome ◽  
Induced Pluripotent

Abstract Shwachman–Diamond syndrome (SDS), an autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, and skeletal abnormalities, is caused by mutations in the Shwachman–Bodian–Diamond syndrome (SBDS) gene, which plays a role in ribosome biogenesis. Although the causative genes of congenital disorders frequently involve regulation of embryogenesis, the role of the SBDS gene in early hematopoiesis remains unclear, primarily due to the lack of a suitable experimental model for this syndrome. In this study, we established induced pluripotent stem cells (iPSCs) from patients with SDS (SDS-iPSCs) and analyzed their in vitro hematopoietic and endothelial differentiation potentials. SDS-iPSCs generated hematopoietic and endothelial cells less efficiently than iPSCs derived from healthy donors, principally due to the apoptotic predisposition of KDR+CD34+ common hemoangiogenic progenitors. By contrast, forced expression of SBDS gene in SDS-iPSCs or treatment with a caspase inhibitor reversed the deficiency in hematopoietic and endothelial development, and decreased apoptosis of their progenitors, mainly via p53-independent mechanisms. Patient-derived iPSCs exhibited the hematological abnormalities associated with SDS even at the earliest hematopoietic stages. These findings will enable us to dissect the pathogenesis of multiple disorders associated with ribosomal dysfunction.

SBDS Protein Plays a Role in Ribosome Biogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 185-185
Author(s):  
Karthik A. Ganapathi ◽  
Karyn M. Austin ◽  
Maggie Malsch ◽  
Akiko Shimamura
Keyword(s):  
Ribosome Biogenesis ◽  
Actinomycin D ◽  
Bone Marrow Failure ◽  
Pancreatic Insufficiency ◽  
Protein Translation ◽  
Exocrine Pancreatic Insufficiency ◽  
Nucleolar Localization ◽  
28S Rrna ◽  
Wild Type ◽  
Shwachman Diamond Syndrome

Abstract Shwachman-Diamond syndrome is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency, bone marrow failure, and leukemia predisposition. The majority of patients with Shwachman-Diamond syndrome harbor mutations in the SBDS gene. SBDS is a novel gene of unknown function and is highly conserved throughout evolution. Studies of the yeast orthologue, YLR022c/SDO1, suggest that SBDS may play a role in ribosome biogenesis. In support of this hypothesis, we have found that the SBDS protein shuttles in and out of the nucleolus. Previously we have shown that SBDS nucleolar localization is regulated in a cell cycle-dependant manner. We now find that SBDS nucleolar localization is also lost following exposure to actinomycin D, suggesting that SBDS nucleolar localization is dependent on active ribosomal RNA (rRNA) transcription. In cell survival assays, SBDS−/− patient-derived cells are sensitive to actinomycin D treatment relative to normal control cells. Introduction of the wild-type SBDS cDNA into SBDS−/− cells corrects their actinomycin D sensitivity, confirming that the observed sensitivity is SBDS-dependent. In contrast, SBDS−/− cells do not exhibit increased sensitivity to cyclohexamide, a protein translation inhibitor. Consistent with this result, SBDS protein co-localizes with ribosomal precursor subunits but not with mature polysomes upon sucrose gradient sedimentation. No differences in polysome profiles are observed between SBDS−/− cells and wild type control cells. Gel filtration studies suggest that SBDS associates into a complex with other proteins. SBDS co-immunoprecipitates with other nucleolar proteins involved in rRNA biogenesis. RNA immunoprecipitation studies reveal that SBDS also associates with the 28S rRNA but not the 18S rRNA. These findings support the hypothesis that SBDS plays a role in ribosome biogenesis

scholarly journals Hematologic abnormalities in Shwachman Diamond syndrome: lack of genotype-phenotype relationship

Blood ◽  
2005 ◽  
Vol 106 (1) ◽  
pp. 356-361 ◽  
Author(s):  
Taco W. Kuijpers ◽  
Mariel Alders ◽  
Anton T. J. Tool ◽  
Clemens Mellink ◽  
Dirk Roos ◽  
...  
Keyword(s):  
Sequence Data ◽  
Pancreatic Insufficiency ◽  
Allogeneic Bone Marrow Transplantation ◽  
Fetal Hemoglobin ◽  
Autosomal Recessive Disorder ◽  
Exocrine Pancreatic Insufficiency ◽  
Allogeneic Bone ◽  
Cytogenetic Aberrations ◽  
Shwachman Diamond Syndrome

Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by short stature, exocrine pancreatic insufficiency, and hematologic defects. The causative SBDS gene was sequenced in 20 of 23 unrelated patients with clinical SDS. Mutations in the SBDS gene were found in 75%, being identical in 11 patients. Hematologic parameters for all 3 lineages were determined over time such as absolute neutrophil counts (ANCs), granulocyte functions, and erythroid and myeloid colony formation (erythroid burst-forming unit [BFU-E] and granulocyte-monocyte colony-forming unit [CFU-GM]) from hematopoietic progenitor cells, percentage of fetal hemoglobin (HbF), and platelet counts. Persistent neutropenia was present in 43% in the absence of apoptosis and unrelated to chemotaxis defects (in 65%) or infection rate. Irrespective of the ANC in vivo, abnormal CFU-GM was observed in all patients with SDS tested (14 of 14), whereas BFU-E was less often affected (9 of 14). Cytogenetic aberrations occurred in 5 of 19 patients in the absence of myelodysplasia. One child died during allogeneic bone marrow transplantation. In conclusion, neutropenia and defective chemotaxis did not result in severe clinical infection in SDS. CFU-GMs were impaired in all patients tested. From the SBDS sequence data, we conclude that in patients with genetically proven SDS a genotype-phenotype relationship in SDS does not exist in clinical and hematologic terms.

scholarly journals The Shwachman-Diamond SBDS protein localizes to the nucleolus

Blood ◽  
2005 ◽  
Vol 106 (4) ◽  
pp. 1253-1258 ◽  
Author(s):  
Karyn M. Austin ◽  
Rebecca J. Leary ◽  
Akiko Shimamura
Keyword(s):  
Bone Marrow Failure ◽  
Pancreatic Insufficiency ◽  
Intracellular Localization ◽  
Gene Mutations ◽  
Exocrine Pancreatic Insufficiency ◽  
Nucleolar Localization ◽  
Shwachman Diamond Syndrome ◽  
Low Levels ◽  
Cycle Dependent ◽  
Novel Protein

AbstractShwachman-Diamond syndrome (SDS) is an autosomal recessively inherited disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The gene for this syndrome, SBDS, encodes a highly conserved novel protein. We characterized Shwachman-Bodian-Diamond syndrome (SBDS) protein expression and intracellular localization in 7 patients with SDS and healthy controls. As predicted by gene mutation, 4 patients with SDS exhibited no detectable full-length SBDS protein. Patient DF277, who was homozygous for the IVS2 + 2 T>C splice donor mutation, expressed scant levels of SBDS protein. Patient SD101 expressed low levels of SBDS protein harboring an R169C missense mutation. Patient DF269, who carried no detectable gene mutations, expressed wild-type levels of SBDS protein to add further support to the growing body of evidence for additional gene(s) that might contribute to the pathogenesis of the disease phenotype. The SBDS protein was detected in both the nucleus and the cytoplasm of normal control fibroblasts, but was particularly concentrated within the nucleolus. SBDS localization was cell-cycle dependent, with nucleolar localization during G1 and G2 and diffuse nuclear localization during S phase. SBDS nucleolar localization was intact in SD101 and DF269. The intranucleolar localization of SBDS provides further supportive evidence for its postulated role in rRNA processing.

Dilated cardiomyopathy in a case of Shwachman–Diamond syndrome

2011 ◽  
Vol 21 (5) ◽  
pp. 588-590 ◽  
Author(s):  
Liliane Kopel ◽  
Paulo S. Gutierrez ◽  
Silvia G. Lage
Keyword(s):  
Dilated Cardiomyopathy ◽  
Cardiac Failure ◽  
Bone Marrow Failure ◽  
Pancreatic Insufficiency ◽  
Exocrine Pancreatic Insufficiency ◽  
Bone Marrow Failure Syndrome ◽  
Organ Systems ◽  
History Of ◽  
Shwachman Diamond Syndrome

AbstractThe Shwachman–Diamond syndrome is an autosomal recessive bone marrow failure syndrome with exocrine pancreatic insufficiency. Additional organ systems, such as the liver, heart and bone, may also be affected. We report a patient with a long history of cardiac failure and diagnosis of dilated cardiomyopathy with intermittent neutropenia. Periodic follow-up revealed progressive cardiac failure and pulmonary hypertension. A diagnosis of Shwachman–Diamond syndrome was made at the autopsy.

scholarly journals Enhanced p53 Levels Are Involved in the Reduced Mineralization Capacity of Osteoblasts Derived from Shwachman–Diamond Syndrome Subjects

2021 ◽  
Vol 22 (24) ◽  
pp. 13331
Author(s):  
Annalisa Frattini ◽  
Simona Bolamperti ◽  
Roberto Valli ◽  
Marco Cipolli ◽  
Rita Maria Pinto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Ribosome Biogenesis ◽  
Collagen Type ◽  
Bone Marrow Failure ◽  
Pancreatic Insufficiency ◽  
Collagen Type I ◽  
Type I ◽  
Protein Levels ◽  
Shwachman Diamond Syndrome

Shwachman–Diamond syndrome (SDS) is a rare autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, and skeletal abnormalities, caused by loss-of-function mutations in the SBDS gene, a factor involved in ribosome biogenesis. By analyzing osteoblasts from SDS patients (SDS-OBs), we show that SDS-OBs displayed reduced SBDS gene expression and reduced/undetectable SBDS protein compared to osteoblasts from healthy subjects (H-OBs). SDS-OBs cultured in an osteogenic medium displayed a lower mineralization capacity compared to H-OBs. Whole transcriptome analysis showed significant differences in the gene expression of SDS-OBs vs. H-OBs, particularly in the ossification pathway. SDS-OBs expressed lower levels of the main genes responsible for osteoblastogenesis. Of all downregulated genes, Western blot analyses confirmed lower levels of alkaline phosphatase and collagen type I in SDS-OBs than in H-OBs. Interestingly, SDS-OBs showed higher protein levels of p53, an inhibitor of osteogenesis, compared to H-OBs. Silencing of Tp53 was associated with higher collagen type I and alkaline phosphatase protein levels and an increase in SDS-OB mineralization capacity. In conclusion, our results show that the reduced capacity of SDS-OBs to mineralize is mediated, at least in part, by the high levels of p53 and highlight an important role of SBDS in osteoblast functions.

Mouse Model for Shwachman-Diamond Syndrome with the R126T Disease Mutation Leads to Severe Growth and Developmental Deficiencies with Impairment of Hematopoiesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1283-1283
Author(s):  
Siyi Zhang ◽  
Jian Zhong ◽  
Olga I. Gan ◽  
John E. Dick ◽  
Johanna M. Rommens
Keyword(s):  
Ribosome Biogenesis ◽  
Bone Marrow Failure ◽  
Pancreatic Insufficiency ◽  
Mutant Mice ◽  
Null Alleles ◽  
Wild Type ◽  
Disease Mutation ◽  
Disease Phenotypes ◽  
Growth Difference ◽  
Shwachman Diamond Syndrome

Abstract Shwachman-Diamond syndrome (SDS) is a bone marrow failure syndrome characterized by exocrine pancreatic insufficiency and skeletal abnormalities, as well as hematological dysfunction. SDS is caused by mutations in SBDS, a highly conserved gene that has been suggested to be involved in RNA metabolism and/or ribosome biogenesis. It is essential, based on our mouse knock-out model studies and the absence of observed patients with the combination of two null alleles. We have generated an allele with the disease mutation, R126T, in the murine ortholog in order to develop new models of SDS. This mutation has been interpreted to be hypomorphic in nature, as it occurs in combination with the common null disease allele in two patients. Interestingly, these patients also have severe hematological disease phenotypes. SbdsR126T/+mice develop normally and show no disease phenotypes, in accordance with the recessive inheritance of SDS. However, the SbdsR126T/R126T and SbdsR126T/- mice did not survive birth, and exhibited marked size reduction. The growth difference became apparent in embryos during the mid-fetal period, at E12.5–14.5; there were also noted disturbances of major organs including the skeleton, brain and lung. Comparable deficiencies were noted overall, but the SbdsR126T/- embryos were consistently more severely affected than were SbdsR126T/R126T embryos. Examination of hematopoietic progenitors from the fetal livers of SbdsR126T/R126T and SbdsR126T/- mutant mice also showed marked reductions in BFU-E, CFU-G, CFU-M, CFU-GM and CFU-GEMM numbers, as revealed by standard colony formation assays, when compared to wild type and heterozygote littermates. In additional studies, primary fibroblast cultures from E16.5 day mutant embryos were found to exhibit slower growth and an extended G1 phase of the cell cycle compared to fibroblasts from wild type embryos. Further, total protein synthesis was measured by incorporation of radio-labeled amino acids and found to be notably reduced in the mutant fibroblasts. The small size, organ deficiencies and early death of the mutant mice together with the cellular deficiencies, which indicate severe autologous growth problems, emphasize the severe consequences of loss of Sbds. These animal disease models provide new avenues for investigation to elucidate basic functions of SBDS and the pathobiology of SDS.

scholarly journals Defective ribosome assembly in Shwachman-Diamond syndrome

Blood ◽  
2011 ◽  
Vol 118 (16) ◽  
pp. 4305-4312 ◽  
Author(s):  
Chi C. Wong ◽  
David Traynor ◽  
Nicolas Basse ◽  
Robert R. Kay ◽  
Alan J. Warren
Keyword(s):  
Ribosome Biogenesis ◽  
Bone Marrow Failure ◽  
Pancreatic Insufficiency ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Ribosome Assembly ◽  
Restrictive Temperature ◽  
Poor Growth ◽  
Shwachman Diamond Syndrome ◽  
Subunit Joining

AbstractShwachman-Diamond syndrome (SDS), a recessive leukemia predisposition disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, skeletal abnormalities and poor growth, is caused by mutations in the highly conserved SBDS gene. Here, we test the hypothesis that defective ribosome biogenesis underlies the pathogenesis of SDS. We create conditional mutants in the essential SBDS ortholog of the ancient eukaryote Dictyostelium discoideum using temperature-sensitive, self-splicing inteins, showing that mutant cells fail to grow at the restrictive temperature because ribosomal subunit joining is markedly impaired. Remarkably, wild type human SBDS complements the growth and ribosome assembly defects in mutant Dictyostelium cells, but disease-associated human SBDS variants are defective. SBDS directly interacts with the GTPase elongation factor-like 1 (EFL1) on nascent 60S subunits in vivo and together they catalyze eviction of the ribosome antiassociation factor eukaryotic initiation factor 6 (eIF6), a prerequisite for the translational activation of ribosomes. Importantly, lymphoblasts from SDS patients harbor a striking defect in ribosomal subunit joining whose magnitude is inversely proportional to the level of SBDS protein. These findings in Dictyostelium and SDS patient cells provide compelling support for the hypothesis that SDS is a ribosomopathy caused by corruption of an essential cytoplasmic step in 60S subunit maturation.

scholarly journals Mutation Screening of Elongation Factor 2 in Shwachman-Diamond Syndrome Patients Lacking Mutations in the SBDS Gene

ISRN Genetics ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-4
Author(s):  
Elena Nicolis ◽  
Marco Cipolli
Keyword(s):  
Autosomal Recessive ◽  
Bone Marrow Failure ◽  
Pancreatic Insufficiency ◽  
Mutation Screening ◽  
Elongation Factor ◽  
Autosomal Recessive Disorder ◽  
Deleterious Mutations ◽  
Italian Experience ◽  
Elongation Factor 2 ◽  
Shwachman Diamond Syndrome

Shwachman-Diamond syndrome is an autosomal recessive disorder characterized by bone marrow failure, pancreatic insufficiency, and skeletal abnormalities. Mutations in SBDS gene explain, by literature, 90% of SDS cases. The Italian experience shows that only the 5% of individuals diagnosed as affected by SDS on clinical and hematological grounds lack mutations in the SBDS gene. It is well established that SBDS protein is essential for the assembly of mature ribosomes. The yeast SBDS ortholog functions within a pathway containing elongation factor-like 1, homologous to human GTPase elongation factor-2, to promote the release and recycling of the nucleolar shuttling factor Tif6 from cytoplasmic pre-60S subunits in a cascade targeted to form the active ribosome. We considered that mutations of genes that disrupt pathways shared by SBDS may result in disease with comparable clinical features. EEF2 was evaluated as a candidate gene by mutation screening in clinically defined SDS which lack mutations in the SBDS gene. To date, no deleterious mutations were found in EEF2 in four Italian patients without SBDS mutations, but with a clinical diagnosis of SDS.

Suppression of the Hematopoietic Defect in TF-1 Cells Depleted of Shwachman-Diamond Syndrome Protein: Correlation with Decreased eIF6 Levels

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1270-1270
Author(s):  
Sharon Singh ◽  
Lionel Blanc ◽  
Adrianna Henson ◽  
Gulay Sezgin ◽  
Steven R Ellis ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Pancreatic Insufficiency ◽  
Exocrine Pancreatic Insufficiency ◽  
Yeast Cells ◽  
Missense Mutations ◽  
Bone Marrow Failure Syndrome ◽  
Increased Risk ◽  
Shwachman Diamond Syndrome ◽  
Biallelic Mutations

Abstract Abstract 1270 Shwachman Diamond syndrome (SDS) is a rare autosomal recessive bone marrow failure syndrome mainly characterized by neutropenia, exocrine pancreatic insufficiency and an increased risk of myelodysplastic syndrome and leukemia. The phenotype in patients is variable for unclear reasons, but approximately 90% of patients have biallelic mutations in the SBDS gene. At least one action of the SBDS protein is to couple with the GTPase ELF1 to facilitate release of the eIF6 protein from the 60S ribosome subunit, thus enabling joining of the 60S and 40S ribosome subunits, a function that has prompted many to consider SDS a “ribosomopathy”. We created a cellular model of SDS using TF-1 erythroleukemia cells transduced with lentiviral vectors containing two different shRNAs against SBDS or a scrambled sequence. Clones were grown under puromycin selection and a clone from each shRNA was selected. In each clone, knockdown of SBDS was verified at the protein level by western blot, and expression levels of SBDS were less than 1%. Both clones underwent differentiation to either myeloid or erythroid colonies by culturing in GM-CSF or erythropoietin, respectively. The 2–12 clone had a significant decrease in the number and size of both myeloid and erythroid colonies (see Table) when compared with the scrambled shRNA control. In contrast, the 1–7 clone had the same number of myeloid and erythroid colonies as the control. Previous work by other investigators in SDS yeast models revealed that missense mutations in the anti-association factor, Tif6 suppress the slow growth phenotype of SDS-mutant yeast cells. In exploring the molecular basis for the difference in phenotype observed in our TF-1 cells, we therefore focused on eIF6, the human ortholog of Tif6. The 2–12 clone had similar expression of the eIF6 protein when compared to the scrambled control. However, the 1–7 clone had a significantly decreased amount of eIF6 protein compared to the control. DNA sequencing did not reveal any mutations in the eIF6 gene, and quantitative RT-PCR showed similar levels of eIF6 mRNA transcripts, suggesting that the differences in eIF6 protein levels may be due to post-translational modifications. Pressato and colleagues (Br J Haematol 157:503, 2012) have recently speculated that the relatively benign course of SDS patients with a deletion of chromosome 20q may be due to loss of the eIF6 protein (whose gene is located on 20q). Our findings add to the hypothesis that antagonizing eIF6 may modify or rescue the SDS phenotype, possibly by reducing the requirement of SBDS in giving rise to 60S subunits lacking eIF6. Scramble colonies +/− SE 2–12 colonies +/−SE 1–7 colonies +/− SE Myeloid 131+/−4.4 112+/−3.5 p<0.01 135+/−6.8 p=0.64 Erythroid 89+/−8.4 48+/−4 p<0.001 89+/− 4.7 p=0.94 Disclosures: No relevant conflicts of interest to declare.

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