The Shwachman-Diamond SBDS protein localizes to the nucleolus

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.

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SBDS Protein Plays a Role in Ribosome Biogenesis.

Blood ◽

10.1182/blood.v108.11.185.185 ◽

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

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Dilated cardiomyopathy in a case of Shwachman–Diamond syndrome

Cardiology in the Young ◽

10.1017/s1047951111000308 ◽

2011 ◽

Vol 21 (5) ◽

pp. 588-590 ◽

Cited By ~ 5

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.

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

Blood ◽

10.1182/blood.v120.21.1270.1270 ◽

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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Loss of the Mouse Ortholog of the Shwachman-Diamond Syndrome Gene (Sbds) Results in Early Embryonic Lethality

Molecular and Cellular Biology ◽

10.1128/mcb.00091-06 ◽

2006 ◽

Vol 26 (17) ◽

pp. 6656-6663 ◽

Cited By ~ 72

Author(s):

Siyi Zhang ◽

Mingjun Shi ◽

Chi-chung Hui ◽

Johanna M. Rommens

Keyword(s):

Expression Pattern ◽

Essential Gene ◽

Bone Marrow Failure ◽

Pancreatic Insufficiency ◽

Critical Role ◽

Exocrine Pancreatic Insufficiency ◽

In Vivo Model ◽

Mammalian Development ◽

Shwachman Diamond Syndrome

ABSTRACT Mutations in SBDS are responsible for Shwachman-Diamond syndrome (SDS), a disorder with clinical features of exocrine pancreatic insufficiency, bone marrow failure, and skeletal abnormalities. SBDS is a highly conserved protein whose function remains largely unknown. We identified and investigated the expression pattern of the murine ortholog. Variation in levels was observed, but Sbds was found to be expressed in all embryonic stages and most adult tissues. Higher expression levels were associated with rapid proliferation. A targeted disruption of Sbds was generated in order to understand the consequences of its loss in an in vivo model. Consistent with recessive disease inheritance for SDS, Sbds +/− mice have normal phenotypes, indistinguishable from those of their wild-type littermates. However, the development of Sbds −/− embryos arrests prior to embryonic day 6.5, with muted epiblast formation leading to early lethality. This finding is consistent with the absence of patients who are homozygous for early truncating mutations. Sbds is an essential gene for early mammalian development, with an expression pattern consistent with a critical role in cell proliferation.

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Normative growth charts for Shwachman-Diamond syndrome from Italian cohort of 0–8 years old

BMJ Open ◽

10.1136/bmjopen-2018-022617 ◽

2019 ◽

Vol 9 (1) ◽

pp. e022617 ◽

Cited By ~ 3

Author(s):

Marco Cipolli ◽

Gloria Tridello ◽

Alessio Micheletto ◽

Sandra Perobelli ◽

Emily Pintani ◽

...

Keyword(s):

Bone Marrow Failure ◽

Pancreatic Insufficiency ◽

National Registry ◽

Exocrine Pancreatic Insufficiency ◽

Birth Date ◽

Growth Charts ◽

Healthy Population ◽

Average Birth Weight ◽

Shwachman Diamond Syndrome ◽

Reference Tool

ObjectivesShwachman-Diamond syndrome (SDS) is a rare autosomal recessive disorder. Its predominant manifestations include exocrine pancreatic insufficiency, bone marrow failure and skeletal abnormalities. Patients frequently present failure to thrive and susceptibility to short stature. Average birth weight is at the 25th percentile; by the first birthday, >50% of patients drop below the third percentile for height and weight.The study aims at estimating the growth charts for patients affected by SDS in order to give a reference tool helpful for medical care and growth surveillance through the first 8 years of patient’s life.Setting and participantsThis retrospective observational study includes 106 patients (64 M) with available information from birth to 8 years, selected among the 122 patients included in the Italian National Registry of SDS and born between 1975 and 2016. Gender, birth date and auxological parameters at repeated assessment times were collected. The General Additive Model for Location Scale and Shape method was applied to build the growth charts. A set of different distributions was used, and the more appropriate were selected in accordance with the smallest Akaike information criterion.ResultsA total of 408 measurements was collected and analysed. The median number of observations per patient amounted to 3, range 1–11. In accordance with the methods described, specific SDS growth charts were built for weight, height and body mass index (BMI), separately for boys and girls.The 50th and 3rd percentiles of weight and height of the healthy population (WHO standard references) respectively correspond to the 97th and 50th percentiles of the SDS population (SDS specific growth charts), while the difference is less evident for the BMI.ConclusionsSpecific SDS growth charts obtained through our analysis enable a more appropriate classification of patients based on auxological parameters, representing a useful reference tool for evaluating their growth during childhood.

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

Scientific Reports ◽

10.1038/s41598-020-71844-8 ◽

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.

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Mutations of the SBDS gene are present in most patients with Shwachman-Diamond syndrome

Blood ◽

10.1182/blood-2004-04-1516 ◽

2004 ◽

Vol 104 (12) ◽

pp. 3588-3590 ◽

Cited By ~ 69

Author(s):

Jill R. Woloszynek ◽

Robert J. Rothbaum ◽

Amy S. Rawls ◽

Patrick J. Minx ◽

Richard K. Wilson ◽

...

Keyword(s):

Bone Marrow ◽

Natural History ◽

Pancreatic Insufficiency ◽

Exocrine Pancreatic Insufficiency ◽

Compound Heterozygous ◽

Loss Of Function ◽

Multisystem Disorder ◽

Clinical Criteria ◽

Function Mechanism ◽

Shwachman Diamond Syndrome

Shwachman-Diamond Syndrome (SDS) is a rare multisystem disorder characterized by exocrine pancreatic insufficiency, bone marrow dysfunction, and metaphyseal chondrodysplasia. Recent studies show that mutations of SBDS, a gene of unknown function, are present in the majority of patients with SDS. In the present study, we show that most, but not all, patients classified based on rigorous clinical criteria as having SDS had compound heterozygous mutations of SBDS. Full-length SBDS protein was not detected in leukocytes of SDS patients with the most common SBDS mutations, consistent with a loss-of-function mechanism. In contrast, SBDS protein was expressed at normal levels in SDS patients without SBDS mutations. These data confirm the absence of SBDS mutations in this subgroup of patients and suggest that SDS is a genetically heterogeneous disorder. The presence (or absence) of SBDS mutations may define subgroups of patients with SDS who share distinct clinical features or natural history.

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The SBDS Gene Regulates Viability of Myeloid Precursor Cells and Is Associated with High ROS Production and Telomere Shortening in the SBDS Downregulated Cells.

Blood ◽

10.1182/blood.v108.11.1639.1639 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1639-1639 ◽

Cited By ~ 2

Author(s):

Masafumi Yamaguchi ◽

Kingo Fujimura ◽

Hanae Toga ◽

Asim Khwaja ◽

Naoki Okamura ◽

...

Keyword(s):

Telomere Length ◽

Fas Ligand ◽

Bone Marrow Failure ◽

Pancreatic Insufficiency ◽

Exocrine Pancreatic Insufficiency ◽

Serum Starvation ◽

Ros Production ◽

High Concentration ◽

Maximum Cell Density ◽

Telomere Shortening

Abstract Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11 and disease-associated mutations were reported in the Shwachman-Bodian-Diamond syndrome (SBDS) gene resulting in a truncated and inactive protein. SBDS is a member of a highly conserved protein family in diverse species including archaea and eukaryotes. It is widely expressed in many tissues and its function is still unknown. In order to investigate the function of the protein, we have established two stably infected 32Dcl3 cell lines, expressing two different shRNAi against SBDS. The downregulation of SBDS was confirmed by Western blotting with anti human SBDS antibody. The growth of 32Dcl3 cells was significantly decreased in cells with down regulation of SBDS cells when compared to that of cells stable transfected with a control shRNAi cells. The maximum cell density of mIL-3 dependent cell growth was about 50% in SBDS downregulated cells (2.9x105 cells/ml vs 1.3x105 cells/ml, respectively). The SBDS downregulated cells were sensitive to serum starvation. The Annexin V binding was same as control in the presence of 10% FCS. However, after exposure to 1 % FCS, apoptotic cells were much increased in SBDS downregulated cells(16.45±4.2% in control cells vs 25.19±0.2% in SBDS downregulated cells, p=0.036). Fas and Fas ligand was not detected in SBDS downregulated cells, and both Bcl2 and BclxL expression was same between the cells. To further characterize the potential mechanism for cell kill as a consequence of SBDS knockdown, we examined the telomere length by Flow-FISH. The telomere length of SBDS downregulated cells was significantly shorter than control cells after 2 weeks infection (13.3±2.85 kb in SBDS knockdown cells vs 15.6±3.19 kb in control cells, p=0.013. Cells were passaged 6 times after infection). In addition, reactive oxygen species (ROS) production of SBDS downregulated cells was much higher than control infected cells. This high ROS production correlated with the rapid telomere shortening, and predisposition of the cells to apoptosis. We also characterized the consequence of SBDS protein knockdown on neutrophil differentiation. Knockdown cells showed normal differentiation as defined by morphology and the neutrophil maturation markers, Mac1, CD11b but proliferation was decreased by 90%. In summary, we have an established SDS model cell line and have demonstrated that SBDS is not required for neutrophil maturation. After loss of SBDS, ROS production was increased, and high concentration of ROS resulted in increased sensitivity to apoptosis stage and abnormal telomere shortening. These data give insights into the pathways affected by loss of SBDS function, together with mechanistic hypotheses that can be tested in primary samples form patients with SDS.

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Mutations of the SBDS Gene Result in Nuclear Mislocalization.

Blood ◽

10.1182/blood.v112.11.2034.2034 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2034-2034

Author(s):

Masafumi Yamaguchi ◽

Kingo Fujimura ◽

Hanae Toga-Yamaguchi ◽

Valentina Svetic ◽

Naoki Okamura ◽

...

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Bone Marrow Failure ◽

Pancreatic Insufficiency ◽

Exocrine Pancreatic Insufficiency ◽

Expression Level ◽

Wild Type ◽

Nuclear Localization Signals ◽

Localization Signal ◽

Domain I

Abstract Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11. We have previously reported that Shwachman-Bodian- Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells were sensitive to apoptotic stimuli, indicating that SBDS acts to maintain survival of granulocyte precursor cells. (Exp Hematol35; 579, 2007). A wide variety of mutations in SBDS gene has been identified, and almost of all patients show truncated immature proteins, p.K62X (c.183_184TA>CT) or p.C84fsX3 (c.258+2T>C). However, it is not yet clear how these truncated proteins affect cellular processes that result in the SDS phenotype. The SBDS protein is localized to the nucleoli but does not have the canonical nuclear localization signal. In order to clarify the molecular basis of pathogenicity of mutated SBDS proteins, we explored the subcellular distribution of normal and mutant SBDS proteins in Hela and 32Dcl3 cells. Using various N-terminal and C-terminal deletion constructs, we found N-terminal region, domain I (1-87 amino acid residue) in particular, was necessary to localize to the nucleus. The disease related mutations (C31W, K33E, N34I, L71P) and the mutations which are conserved among the species in the domain I (E44K, K62E, D70N, E82K) were generated. C31W and N34I mutants failed to localize SBDS to the nuclei. The SV40 derived nuclear localization signal was fused to these mutated SBDS protein, and these proteins were clearly localized to the nuclei. In addition to the mislocalization, the protein expression level of these mutants showed a dramatic decrease compared to the wild type. We also established SBDS wild type and domain I overexpressed 32Dcl3 cell. SBDS wild type overexpressed cells could differentiate to normal neutrophils in the presence of mG-CSF, however domain I overexpressed cells did not differentiate. Almost of all cells showed apoptosis in this domain I overexpressed cells in the presence of mG-CSF, and this was very similar like SBDS RNAi knockdown cells. The localization of endogenous SBDS protein was also analyzed in this domain I overexpressed cells. The domain I was concentrated to nuclei, however endogenous SBDS protein was diffused to cytosol. Conclusions: The present findings enable us to document the nuclear localization signals in SBDS domain I, and that the shuttling protein would promote SBDS to nuclei. These results also showed that mislocalization and/or low expression level of mutated SBDS protein would cause SDS.

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