Impaired Ribosome Maturation In Human Cells Depleted of Shwachman-Diamond Syndrome Protein SBDS

Abstract Abstract 2242 Background: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine dysfunction, neurocognitive and skeletal abnormalities, and bone marrow failure. Mutations in SBDS have been shown to cause SDS. From experiments on its yeast ortholog (Haematologica 2010. 95:57-64), SBDS has been implicated in maturation and function of the 60S ribosomal subunit. In particular, subunit maturation in the SDS yeast model was associated with delayed export and accumulation of 60S-like particles in the nucleoplasm. Methods and Results: To clarify its role in human cells, erythroleukemia TF-1 cells were transduced with lentiviral vectors expressing short hairpin RNA (shRNA) against SBDS. Immunoblot assays confirmed approximately 60% knockdown in individual TF-1 cell clones expressing different shRNAs. The growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to cells stably transduced with shRNA against a scrambled SBDS sequence. Using Hoechst 33342/Pyronin Y staining and flow cytometry, we also found an increased percentage of knockdown cells retained at the G0/G1 cell cycle phase. To address whether near-complete knockdown of SBDS affected ribosome synthesis as it does in yeast cells, we silenced SBDS in A549 cells. Our data revealed a reduction in polysomes but in contrast to what was observed in yeast, there was no evidence of half-mer polysomes indicative of decreased 60S subunits participating in translation. The absence of half-mers is not unusual in mammalian systems, so to better analyze the effect of SBDS on 60S subunit maturation subunit localization was assessed by co-transfection with a vector expressing a fusion between human RPL29 and enhanced GFP. Preliminary studies indicated a higher percentage of SBDS-depleted cells with nuclear localization of 60S subunits, when compared with normal controls (Fig. 1). Conclusions: Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Depletion of the Shwachman-Diamond Syndrome Protein in Hematopoietic Progenitor Cells Impairs Growth, Colony Formation, and Ribosome Function.

Blood ◽

10.1182/blood.v112.11.2046.2046 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2046-2046

Author(s):

Gulay Sezgin ◽

Abdallah Nihrane ◽

Steven Ellis ◽

Johnson M. Liu

Keyword(s):

Cell Cycle ◽

Fluorescent Protein ◽

Bone Marrow Failure ◽

Gradient Centrifugation ◽

Flow Cytometric Analysis ◽

Ribosomal Subunit ◽

Autosomal Recessive Disorder ◽

Cycle Phase ◽

Cell Extracts ◽

Shwachman Diamond Syndrome

Abstract Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine dysfunction, skeletal abnormalities, and bone marrow failure, which can evolve to leukemia. Mutations in SBDS have been shown to cause SDS. We and other investigators have suggested that SBDS orthologs in yeast play a role in biogenesis and function of the 60S ribosomal subunit. To clarify the unknown function of SBDS in hematopoiesis, human erythroleukemia TF-1 cells were transduced with lentiviral vectors expressing the green fluorescent protein (GFP), neomycin phosphotransferase, and small interfering RNA (siRNA) against SBDS. After transduction, cells were selected for neomycin resistance and then sorted by flow cytometry. To probe for SBDS, an antibody against the carboxyl-terminus of human SBDS was generated, and individual TF-1 cell clones expressing different siRNAs were confirmed to knock down SBDS expression by Western blot analysis. Our experiments were aimed at analyzing the cellular effects of SBDS knockdown. The growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to cells stably transduced with siRNA against a scrambled SBDS sequence. Using propidium iodide staining and flow cytometric analysis, we found an increased percentage of knockdown cells retained at the G0/G1 cell cycle phase. To address whether TF-1 cells expressing siRNA against SBDS have a selective deficiency of 60S ribosomal subunits, cell extracts were prepared and polysome profiles examined after sucrose gradient centrifugation. In preliminary experiments, TF-1 cells expressing siRNA against SBDS appeared to show a reduction in free 60S subunits and 80S subunits with a shift toward smaller polysomes, compared to cells expressing the scrambled sequence siRNA. We conclude that depletion of SBDS results in a significant growth and clonogenic defect in TF-1 hematopoietic cells. Our preliminary results also suggest defects in ribosome function and cell cycle transit, which may provide an integrated molecular explanation for the hematopoietic defect in SDS since nucleolar stress has been linked to cell cycle arrest and p53 stabilization.

Download Full-text

Shwachman-Diamond Syndrome and the Quality Control of Ribosome Assembly

Blood ◽

10.1182/blood.v128.22.sci-42.sci-42 ◽

2016 ◽

Vol 128 (22) ◽

pp. SCI-42-SCI-42

Author(s):

Alan J. Warren

Keyword(s):

Quality Control ◽

Ribosomal Proteins ◽

Active Sites ◽

Structural Integrity ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Ribosomal Subunit ◽

Ribosome Assembly ◽

Final Maturation ◽

Shwachman Diamond Syndrome

Abstract The synthesis of new ribosomes is a fundamental conserved process in all cells. Ribosomes are pre-assembled in the nucleus and subsequently exported to the cytoplasm where they acquire functionality through a series of final maturation steps that include formation of the catalytic center, recruitment of the last remaining ribosomal proteins and the removal of inhibitory assembly factors. Surprisingly, a number of key factors (SBDS, DNAJC21, RPL10 (uL16)) involved in late cytoplasmic maturation of the large (60S) ribosomal subunit are mutated in both inherited and sporadic forms of leukemia. In particular, biallelic mutations in the SBDS gene cause Shwachman-Diamond syndrome (SDS), a recessive bone marrow failure disorder with significant predisposition to acute myeloid leukemia. By using the latest advances in single-particle cryo-electron microscopy to elucidate the function of the SBDS protein, we have uncovered an elegant mechanism that couples final maturation of the 60S subunit to a quality control assessment of the structural integrity of the active sites of the ribosome. Further molecular dissection of this pathway may inform novel therapeutic strategies for SDS and leukemia more generally. References: 1. Weis F, Giudice E, Churcher M,et al. Mechanism of eIF6 release from the nascent 60S ribosomal subunit. Nat Struct Mol Biol, (2015) Nov;22(11):914-9. 2. Wong CC, Traynor D, Basse N, et al. Defective ribosome assembly in Shwachman-Diamond syndrome. Plenary Paper, Blood. 2011 Oct 20;118(16):4305-12. 3. Finch AJ, Hilcenko C, Basse N, et al. Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome. Genes Dev (2011) 25: 917-929. 4. Menne TM, Goyenechea B, Sánchez-Puig N, et al. The Shwachman-Bodian-Diamond syndrome protein mediates translational activation of ribosomes in yeast. Nature Genetics (2007) 39: 486-95. Disclosures No relevant conflicts of interest to declare.

Download Full-text

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.

Download Full-text

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

Blood ◽

10.1182/blood-2007-02-075184 ◽

2007 ◽

Vol 110 (5) ◽

pp. 1458-1465 ◽

Cited By ~ 93

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.

Download Full-text

Expression of Human SBDSR126T in Sbds Null Background Shows Eif6 Dysregulation: An Adult Zebrafish Model for Shwachman-Diamond Syndrome

Blood ◽

10.1182/blood-2019-129014 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3737-3737

Author(s):

Usua Oyarbide Cuervas-Mons ◽

Matthew Snyderman ◽

Jacek Topczewski ◽

Seth J. Corey

Keyword(s):

Transgenic Line ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Ribosomal Subunit ◽

Zebrafish Model ◽

80S Ribosome ◽

Early Juvenile Stage ◽

Increased Risk ◽

Skeletal Defects ◽

Shwachman Diamond Syndrome

Background. Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic insufficiency, skeletal defects, neutropenia, and an increased risk of myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). SDS occurs in 1/75,000 births, and biallelic mutations in the SBDS gene account for ~90% of patients. The SBDS protein is highly conserved. SBDS interacts physically with EFL1 to release EIF6 from the cytoplasmic pre-60S ribosomal subunit and promote the assembly of the mature 80S ribosome. The SBDS R126T allele is found in combination with the common K62X mutation in some SDS patient. A recent study showed that the SBDSR126T is not able to activate the GTPase activity of the EFL1, affecting the release of EIF6 from the 60S surface. Methods. We created a zebrafish knockout line that phenocopies the SDS with neutropenia, pancreas atrophy, small size (Figure 1A), and decreased 80S ribosomes. To rescue those fish from early mortality, we generated a new transgenic line Tg(ubi:SBDSR126T:pA) expressing the missense variant R126T, a disease-associated allele. Results. The sbds knockout fish die after 21 days post fertilization (dpf), corresponding to an early juvenile stage. However, the SBDSR126T transgenic line in the background of the sbds knockout can live for at least 12 months. This is in strong contrast to the mouse SbdsR126T/R126T line that do not survive to birth. Transgenically-rescued fish displayed a small size phenotype resembling SDS (Figure 1B). Levels of ribosomal proteins Rpl5 and Rpl11 were lower in the sbds knockout at 21 dpf but they were normal in the transgenic line at 6 months. We also observed a concordant regulation of Sbds and Eif6 expression (Figure 1C,D). sbds null fish showed a significant upregulation of cdkn1a, while in their transgenic siblings levels were normal (Figure 1E). Moreover, mpx was upregulated in the transgenic line with the null background (Figure 1F). Analysis of neutrophil and monocyte counts are being performed and will be reported. Conclusions. Our novel SBDSR126T zebrafish model survives until adulthood, which will allow us to carry out a number of informative assays such as stress response, gene expression, and polysome profiles in different organs. Rpl5 and Rpl11 levels are affected in sbds mutants but not in the transgenic line. Activation of cdkn1a (p21) in sbds mutants might lead to apoptosis and death. The normal levels of cdkn1a in the transgenic line might be non-deleterious, as loss of Tp53 activation can rescue some models of bone marrow failure. In addition, loss of sbds or expression of SBDSR126T affect Eif6 levels in zebrafish. Importantly, some patients with SBDS deficiency acquire interstitial deletions of chromosome 20, resulting in the loss of the EIF6 gene. This might be a potential mechanism to suppress the defect in ribosome biogenesis by reducing the copy number of the EIF6 gene and has been related to a lower risk of MDS/AML comparing to other SDS patients. Our adult model of Shwachman-Diamond Syndrome can provide new insights into the pathogenesis of SDS and its progression to malignancy, which can be used to identify novel targets for AML/MDS therapy. Figure 1 Disclosures No relevant conflicts of interest to declare.

Download Full-text

New Bone Marrow Failure Genes: DNAJC21 and ERCC6L2

Blood ◽

10.1182/blood.v130.suppl_1.sci-22.sci-22 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. SCI-22-SCI-22

Author(s):

Inderjeet Dokal

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Nuclear Export ◽

Conflicts Of Interest ◽

Excision Repair ◽

Bone Marrow Failure ◽

Retinal Dystrophy ◽

Ribosomal Subunit ◽

Genotoxic Stress ◽

Biallelic Mutations

A significant number of cases with bone marrow failure present with one or more extra-hematopoietic abnormality. This suggests a constitutional or genetic basis, and yet many of them remain uncharacterized. Through exome sequencing, we have recently identified two sub groups of these cases, one defined by germline biallelic mutations in DNAJC21 (DNAJ homolog subfamily C member 21) and the other in ERCC6L2 (excision repair cross complementing 6 like-2). Patients with DNAJC21 mutations are characterized by global bone marrow failure in early childhood. They can also have a variable number of extra-hematopoietic abnormalities such as short stature and retinal dystrophy. The encoded protein associates with ribosomal RNA (rRNA) and plays a highly conserved role in the maturation of the 60S ribosomal subunit. Lymphoblastoid patient cells exhibit increased sensitivity to the transcriptional inhibitor actinomycin D and reduced levels of rRNA. Characterisation of mutations has revealed impairment in interactions with cofactors (PA2G4, HSPA8 and ZNF622) involved in 60S maturation. DNAJC21 deficiency results in cytoplasmic accumulation of the 60S nuclear export factor PA2G4, aberrant ribosome profiles and increased cell death. Collectively these findings demonstrate that biallelic mutations in DNAJC21 cause disease due to defects in early nuclear rRNA biogenesis and late cytoplasmic maturation of the 60S subunit. Patients harbouring biallelic ERCC6L 2 mutations are characterized by bone marrow failure (in childhood or early adulthood) and one or more extra-hematopoietic abnormality such as microcephaly. Knockdown of ERCC6L2 in human cells significantly reduces their viability upon exposure to the DNA damaging agent irofulven but not etoposide and camptothecin suggesting a role in nucleotide excision repair. ERCC6L2 knockdown cells and patient cells harbouring biallelic ERCC6L2 mutations also display H2AX phosphorylation that significantly increases upon genotoxic stress, suggesting an early DNA damage response. ERCC6L2 is seen to translocate to mitochondria as well as the nucleus in response to DNA damage and its knockdown induces intracellular reactive oxygen species (ROS). Treatment with the ROS scavenger, N-acetyl-cysteine, attenuates the irofulven-induced cytotoxicity in ERCC6L2 knockdown cells and abolishes its traffic to mitochondria and nucleus in response to this DNA damaging agent. Collectively, these observations suggest that ERCC6L2has a pivotal rolein DNA repair and mitochondrial function. In conclusion, ERCC6L2 and DNAJC21 have an important role in maintaining genomic stability and ribosome biogenesis, respectively. They bring into focus new biological connections/pathways whose constitutional disruption is associated with defective hematopoiesis since patients harbouring germline biallelic mutations in these genes uniformly have bone marrow failure. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Both p53-Dependent and -Independent Pathways Contribute to Erythroid Dysplasia in a Zebrafish Model for Diamond Blackfan Anemia.

Blood ◽

10.1182/blood.v114.22.177.177 ◽

2009 ◽

Vol 114 (22) ◽

pp. 177-177 ◽

Cited By ~ 1

Author(s):

Elspeth Payne ◽

Hong Sun ◽

Barry H. Paw ◽

A. Thomas Look ◽

Arati Khanna-Gupta

Keyword(s):

Bone Marrow ◽

Ribosomal Protein ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Ribosomal Subunit ◽

Specific Effect ◽

Independent Component ◽

Diamond Blackfan Anemia ◽

Increased Risk ◽

Erythroid Maturation

Abstract Abstract 177 Diamond Blackfan Anemia (DBA) is a congenital autosomal dominant bone marrow failure syndrome of childhood manifested as profound anemia. The disease is characterized by enhanced sensitivity of hematopoietic progenitors to apoptosis with evidence of stressed erythropoiesis. In addition to bone marrow defects, DBA patients often have craniofacial, genitourinary, cardiac and limb abnormalities and have an increased risk of developing hematopoietic malignancies and osteosarcoma. Twenty-five percent of patients with DBA have heterozygous mutations in the ribosomal protein S19 (RPS19) gene, which encodes a component of the 40S ribosomal subunit. Additionally, a growing percentage of DBA patients lacking a mutation in the RPS19 gene have been shown to have mutations in other ribosomal protein genes. These observations support the hypothesis that DBA is a disease of altered ribosome assembly and function. It is unclear how defects in ribosomal proteins have such a specific effect on erythroid maturation and cause increased apoptosis in the erythroid compartment. An attempt to model DBA by homozygous deletion of the Rps19 gene in mice proved to be embryonic lethal, and heterozygous mice appeared to fully compensate for the loss of one Rps19 allele, in contrast to the disease observed in humans. However, two groups have successfully modeled DBA in zebrafish using an antisense morpholino (MO) approach. These studies demonstrated that similar to the human disease, rps19 deficiency leads to defective erythropoiesis, increased apoptosis and to developmental abnormalities. A central role for the tumor suppressor p53 was suggested in one of these studies. It has previously been shown that any MO injection into zebrafish embryos can lead to the activation of the p53 pathway. Therefore, in order to clarify whether p53-independent effects also contributed to the DBA phenotype in zebrafish, we utilized the p53e7/e7 line that harbors a mutation within the p53 DNA-binding domain. Splice site and validated 5'UTR MOs targeting zebrafish rps19 were injected into one-cell stage embyros that were wildtype (WT) for p53 (AB) or mutated p53e7/e7. Staining for hemoglobin at 48 hours post fertilization showed a profound reduction in circulating blood in both p53 wild-type and p53 mutant embryos. Although p53 mutants injected with rps19 MO show a similar reduction in hemoglobin expression to WT morphants, they have a marked improvement in their developmental defects. A 20% decrease in expression of the transcription factor GATA-1 was observed in the rps19 morphants in the p53 mutant background compared to control MO injection. The implications of this finding are being further investigated and extended to include a panel of additional erythroid-specific factors. We have observed no increase in the levels of cell death, as measured by acridine orange (AO) staining or expression of the p53-regulated apoptosis associated gene PUMA, in the p53 mutant background. Taken together, our observations indicate that the phenotype observed in DBA has both a p53-dependent and a p53-independent component. We hypothesize that the p53-dependent component of DBA is likely responsible for the increased apoptosis associated with DBA while the erythroid maturation defect is associated, in large part, with a p53-independent component. Our studies are currently focused on identifying the players in the latter pathway. These investigations should shed light on thus far undefined pathways that will likely open new avenues for drug design and development for DBA. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Knockdown of Shwachman-Diamond Syndrome Gene, SBDS, Induces Increased Expression of Galectin-1 and Impaired Cell Growth.

Blood ◽

10.1182/blood.v120.21.2360.2360 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2360-2360

Author(s):

Masafumi Yamaguchi ◽

Kingo Fujimura ◽

Hirokazu Kanegane ◽

Hanae Toga-Yamaguchi ◽

Naoki Okamura

Keyword(s):

Cell Culture ◽

Cell Growth ◽

Growth Inhibition ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Growth Failure ◽

Dependent Manner ◽

Growth Inhibitors ◽

C Cells ◽

Shwachman Diamond Syndrome

Abstract Abstract 2360 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 the Shwachman-Bodian-Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells which established by SBDS shRNAi were sensitive to apoptotic stimuli, and led to growth inhibition, indicating that SBDS acts to maintain survival of granulocyte precursor cells (Exp Hematol 35;579,2007). The precise mechanism by which the loss of SBDS inhibits growth of cells remains elusive. In order to clarify the impaired cell growth of SBDS knockdown cells, we analyzed two SDS patients (c.183_184TA>CT and c.258+2T>C) derived EB virus transformed lymphoblast cells (LCL). The growth of both LCL-SDS cell lines was considerably lower than control donor cells (LCL-C) which occurred in within 3 days of culture (1.4×106 cells/ml in LCL-C vs 5×105 cells/lm in LCL-SDS). LCL-C cells divided until 5 days, however, growth of LCL-SDS cells was saturated in 3days. When LCL-SDS cells were seeded to the fresh medium, LCL-SDS cells proliferated again. Conditional medium from 5 days SDS-LCL cell culture was then added to the culture of both LCL-C and LCL-SDS cell. This LCL-SDS conditional medium inhibited both LCL-C and LCL-SDS cell growth (50% and 60%, respectively), suggesting that growth inhibitors were secreted from LCL-SDS cells. In order to find growth inhibitors, we performed differential display. By annealing control primer based GeneFishing PCR screening, we found galectin-1 mRNA level was increased in LCL-SDS cells. We also confirmed that Galectin-1(Gal1) protein expression was markedly increased in LCL-SDS cells by western blot and conforcal microscopy. Gal1 was found to membrane bound, and it is plausible that Gal1 was secreted to the medium. In order to isolate Gal1 protein from medium, medium was passed through lactose agarose. Gal1 protein was purified from LCL-SDS cell culture medium, not from LCL-C cells. The inhibitory effect of Gal1 was confirmed using recombinant human Galectin-1 (rhGal1), which had similar dynamics to that of conditional medium from LCL-SDS cells. rhGal1 the proliferation of both LCL-C and LCL-SDS cells in a dose dependent manner. After exposure to rhGal1, Annexin V positive cells were increased in LCL-C cells (13.78±2.09% in control vs 16.83±2.81% in rhGal1, p=0.02). However, there was no difference in Gal1 induced apoptosis between LCL-C and LCL-SDS cells. In order to rescue growth failure of LCL-SDS cells, lactose, which modulates the binding between galectin and its substrate, was supplemented to the medium. Though lactose showed the growth inhibition of LCL cells, the viability of LCL-SDS cells was much higher than LCL-C cells. LCL-SDS cells were easily aggregated, however, the colony of LCL-SDS cell was much smaller in the presence of lactose. We also confirmed that Gal1 protein was overexpressed in SBDS knockdown 32Dcl3 cells, which were established by SBDS shRNAi. Conclusion: Overexpressed Gal1 was found from SDS patient's derived LCL cells and SBDS shRNAi knockdown 32Dcl3 cells. Gal1 was also found in the conditional medium of LCL-SBDS cells, and secreted Gal1 inhibited the cell proliferation. These results indicated that Galectin-1 partially involved in growth failure of SBDS deficient cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Genotype-Phenotype Associations in Patients with Fanconi Anemia: National Cancer Institute Cohort

Blood ◽

10.1182/blood-2020-137200 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 4-5

Author(s):

Burak Altintas ◽

Neelam Giri ◽

Lisa J. McReynolds ◽

Blanche P. Alter

Keyword(s):

Bone Marrow ◽

Fanconi Anemia ◽

Upper Limb ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Skin Pigmentation ◽

Autosomal Recessive Disorder ◽

Gene Variants ◽

Pathway Gene ◽

Significant Difference

Fanconi anemia (FA) is a predominantly autosomal recessive disorder resulting from mutations in one of >22 genes involved in the FA/BRCA DNA repair pathway. FA is characterized by multiple congenital abnormalities, progressive bone marrow failure (BMF) and cancer predisposition. Genetic heterogeneity and diverse clinical presentations challenge early diagnosis and optimal management. We previously reviewed the genotype-phenotype associations in FA from literature cases (Fiesco-Roa MO et al. Blood Rev. 2019). We now report the results from the NCI cohort. We studied 147 patients with FA in the NCI inherited bone marrow failure syndromes Cohort Study (ClinicalTrials.gov, NCT00027274) to explore genotype phenotype associations by genes, location in the FA/BRCA pathway (upstream, ID complex, downstream), and compare information on the clinic cohort (CC) and field cohort (FC) patients. 57 patients (CC) were evaluated at the NIH Clinical Center between 2002 and 2020. Details on 90 patients in the FC were obtained from the review of medical records. The sex ratio (M:F) was similar (0.6:1 and 0.8:1). Patients in the FC were younger than in the CC (p=0.004) with median ages 27 (3-68) years for the CC and 19 (0-57) for the FC. The main genotypes in the CC were 59% FANCA, 17% FANCC, 6% FANCI and in the FC were 60% FANCA, 13% FANCC and 8% FANCG. At least one FA type physical abnormality was present in all CC patients and 73/79 (92%) FC patients (phenotype data not reported on 11 FC patients). >3/8 VACTERL-H features (Vertebral, Anal, Cardiac, Tracheo-esophageal fistula (TEF), Esophageal or duodenal atresia, Renal, upper Limb (radial ray) and Hydrocephalus) were present in 32% of CC patients and 16% of FC (p=0.04). At least 4/6 PHENOS features (skin Pigmentation, small Head, small Eyes, other central Nervous system (CNS) anomalies, Otology and Short stature) were present in 54% of CC patients and 34% FC (p=0.02). The types and frequencies of phenotypic abnormalities are shown in figure 1. 17 patients in the CC (30%) and 10 in the FC (13%) had both VACTERL-H and PHENOS (p=0.01). We excluded patients with unknown genotype or phenotype from further analysis. In the CC, cardiac abnormalities were more common in patients with FANCI or ID complex gene variants than in all others (p=0.02 and 0.001, respectively) as were VACTERL-H and structural CNS abnormalities in patients with ID complex variants (p=0.03 and 0.006, respectively). In the FC, VACTERL-H, imperforate anus and hydrocephalus were more common in patients with FANCD1 genotype (p=0.03, 0.009 and 0.004, respectively) and downstream pathway gene variants (p=0.004, <0.001 and 0.03, respectively). PHENOS, renal and neurodevelopmental abnormalities were less common in patients with upstream genes variants (p=0.001, 0.009 and <0.001, respectively). Upper limb abnormalities were less common in patients with FANCC genotype (p=0.007). BMF was present in 121/147 (88%) patients; 33% had been transfusion-dependent and 26% received androgen therapy. Clonal cytogenetic abnormalities were seen in 30%; 17% developed myelodysplastic syndrome at a median age of 17 (1.4-44) years and 6 patients developed acute myeloid leukemia at a median age of 19 (12-29) years. 72 (49%) patients underwent bone marrow transplant at a median age of 9.5 (1.5-44) years for BMF, MDS or leukemia. There was no significant difference between the FC and CC. The median survival age of our cohort is 38 (95% CI 34-43) years and at least 80% of our patients are >18 years of age. Kaplan-Meier survival estimates are presented in figure 2. Solid tumors developed in 30/135 (22%) patients with available data; median age at first cancer was 30 (2-44) years. The most common tumor was head and neck squamous cell carcinoma (n=15 patients), followed by skin (n=8) and anogenital cancers (n=6); many patients developed multiple cancers. Detailed hematologic, cancer, endocrine outcomes and survival analyses are ongoing. Overall, renal and upper limb abnormalities were reported in most of the patients in both CC and FC, as shown previously (Alter BP et al. Mol Syndromol. 2013). Data from the CC were more complete than from the review of charts from the FC highlighting that the clinical in person evaluation of patients provides detailed characterization of FA phenotypes and more accurate assessment of genotype-phenotype associations. This will facilitate timely diagnosis, surveillance and clinical management of patients with FA. Disclosures No relevant conflicts of interest to declare.

Download Full-text

SBDS and eIF6 Modulate Ribosome Subunit Joining in Shwachman Diamond Syndrome,

Blood ◽

10.1182/blood.v118.21.3438.3438 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3438-3438

Author(s):

Nicholas Burwick ◽

Scott Coats ◽

Akiko Shimamura

Keyword(s):

Stromal Cells ◽

Ribosome Biogenesis ◽

Conflicts Of Interest ◽

Ribosomal Subunit ◽

Marrow Stromal Cells ◽

Vitro Assay ◽

Amino Terminal ◽

Shwachman Diamond Syndrome ◽

Subunit Joining

Abstract Abstract 3438 Shwachman Diamond syndrome (SDS) is an autosomal recessive marrow failure syndrome with a predisposition to leukemia. Over 90% of SDS patients harbor biallelic mutations in the SBDS gene. SBDS has been implicated in several cellular functions including ribosome biogenesis and mitotic spindle stabilization. Deletion of SBDS orthologues in yeast results in a severe slow growth phenotype and depressed polysomes. Homozygous deletion of Sbds in murine models results in early embryonic lethality, while conditional deletion of Sbds in mouse liver demonstrates accumulation of 40S and 60S subunits and halfmer formation consistent with impaired ribosome joining. SBDS facilitates the release of eIF6, a factor that prevents ribosome joining. The dramatic phenotypic and polysome changes noted in these experimental models were not observed in cells derived from SDS patients. SDS patient cells have only a mildly reduced growth rate compared to heatlhy controls, and polysome profiles do not demonstrate depressed polysomes or halfmer formation. Since complete abrogation of SBDS expression is lethal and biallelic null mutations in SBDS have not been reported, we examined the role of SBDS and eIF6 in SDS patients and human cell models. We first investigated whether ribosome subunit homeostasis is impaired in SDS patient cells. We find that the 60S:40S ribosomal subunit ratio is consistently reduced in bone marrow stromal cells from SDS patients of different genotypes (n=4). This impairment in 60S:40S ratio is demonstrated in both SDS patient stromal cells and patient lymphoblasts. Stable lentiviral knockdown of SDS in normal marrow stromal cells recapitulates the reduction in 60S:40S ratio. SBDS and eIF6 co-sediment in polysome gradients of human SDS cells. This co-sedimentation is specific for the 60S ribosomal subunit. Since eIF6 has a role as an anti-joining factor, we next developed an in vitro assay to test for ribosome subunit joining in human cells. In this assay, we validate that over-expression of eIF6 results in reduced ribosome joining, and eIF6 knockdown promotes ribosome joining. Moreover, we find that SDS patient stromal cells and patient lymphoblasts both demonstrate impaired ribosome subunit joining, compared with healthy controls. Importantly, the addition of wild type SBDS or depletion of eIF6 improve ribosome joining in SDS patient cells. We demonstrate that the amino terminal sequences of SBDS are necessary but not sufficient for the association of SBDS with the 60S ribosomal subunit. Insertion of a patient-derived N-terminal SBDS point mutation also results in decreased association of SBDS with the 60S ribosomal subunit. These structure-function studies may help to inform genotype:phenotype correlations in SDS. The role of defective ribosome joining in promoting the SDS hematopoietic phenotype is of particular interest. Ongoing studies are interrogating the role of eIF6 modulation on the hematopoietic phenotype in SBDS- depleted cells. Insights garnered from these experiments will help inform the development of novel agents to improve the hematopoetic defect in human SDS. Disclosures: No relevant conflicts of interest to declare.

Download Full-text