Mutation of ribosomal protein RPS24 in Diamond-Blackfan anemia results in a ribosome biogenesis disorder

Abstract Variants in ribosomal protein (RP) genes drive Diamond-Blackfan anemia (DBA), a bone marrow failure syndrome that can also predispose individuals to cancer. Inherited and sporadic RP gene variants are also linked to a variety of phenotypes, including malignancy, in individuals with no anemia. Here we report an individual diagnosed with DBA carrying a variant in the 5′UTR of RPL9 (uL6). Additionally, we report two individuals from a family with multiple cancer incidences carrying a RPL9 missense variant. Analysis of cells from these individuals reveals that despite the variants both driving pre-rRNA processing defects and 80S monosome reduction, the downstream effects are remarkably different. Cells carrying the 5′UTR variant stabilize TP53 and impair the growth and differentiation of erythroid cells. In contrast, ribosomes incorporating the missense variant erroneously read through UAG and UGA stop codons of mRNAs. Metabolic profiles of cells carrying the 5′UTR variant reveal an increased metabolism of amino acids and a switch from glycolysis to gluconeogenesis while those of cells carrying the missense variant reveal a depletion of nucleotide pools. These findings indicate that variants in the same RP gene can drive similar ribosome biogenesis defects yet still have markedly different downstream consequences and clinical impacts.

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Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond Blackfan anemia patients

Blood ◽

10.1182/blood-2013-01-478321 ◽

2013 ◽

Vol 122 (6) ◽

pp. 912-921 ◽

Cited By ~ 57

Author(s):

Loïc Garçon ◽

Jingping Ge ◽

Shwetha H. Manjunath ◽

Jason A. Mills ◽

Marisa Apicella ◽

...

Keyword(s):

Stem Cells ◽

Ribosomal Protein ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Ribosome Biogenesis ◽

Ribosomal Protein Genes ◽

Diamond Blackfan Anemia ◽

Key Points ◽

Induced Pluripotent

Key PointsRibosome biogenesis and hematopoiesis are impaired in iPSCs from DBA patients. The abnormalities of DBA iPSCs are ameliorated by genetic restoration of the defective ribosomal protein genes.

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Diamond Blackfan Anemia at the Crossroad between Ribosome Biogenesis and Heme Metabolism

Advances in Hematology ◽

10.1155/2010/790632 ◽

2010 ◽

Vol 2010 ◽

pp. 1-8 ◽

Cited By ~ 18

Author(s):

Deborah Chiabrando ◽

Emanuela Tolosano

Keyword(s):

Ribosomal Protein ◽

Ribosome Biogenesis ◽

Leukemia Virus ◽

Ribosomal Subunit ◽

Pure Red Cell Aplasia ◽

Feline Leukemia Virus ◽

Congenital Defects ◽

Diamond Blackfan Anemia ◽

Heme Metabolism ◽

Gene Coding

Diamond-Blackfan anemia (DBA) is a rare, pure red-cell aplasia that presents during infancy. Approximately 40% of cases are associated with other congenital defects, particularly malformations of the upper limb or craniofacial region. Mutations in the gene coding for the ribosomal protein RPS19 have been identified in 25% of patients with DBA, with resulting impairment of 18S rRNA processing and 40S ribosomal subunit formation. Moreover, mutations in other ribosomal protein coding genes account for about 25% of other DBA cases. Recently, the analysis of mice from which the gene coding for the heme exporter Feline Leukemia Virus subgroup C Receptor (FLVCR1) is deleted suggested that this gene may be involved in the pathogenesis of DBA. FLVCR1-null mice show a phenotype resembling that of DBA patients, including erythroid failure and malformations. Interestingly, some DBA patients have disease linkage to chromosome 1q31, where FLVCR1 is mapped. Moreover, it has been reported that cells from DBA patients express alternatively spliced isoforms of FLVCR1 which encode non-functional proteins. Herein, we review the known roles of RPS19 and FLVCR1 in ribosome function and heme metabolism respectively, and discuss how the deficiency of a ribosomal protein or of a heme exporter may result in the same phenotype.

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Characterization of the Hematologic Phenotype of rpS6 Heterozygous Null Mice as a Model of the Erythroid Failure In Diamond-Blackfan Anemia

Blood ◽

10.1182/blood.v116.21.2033.2033 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2033-2033

Author(s):

Sioban B. Keel ◽

Janis L. Abkowitz

Keyword(s):

Ribosomal Protein ◽

Ribosome Biogenesis ◽

Conflicts Of Interest ◽

Macrocytic Anemia ◽

Ribosomal Subunit ◽

T Test ◽

Ribosomal Protein Genes ◽

Diamond Blackfan Anemia ◽

Student’S T ◽

Student’S T Test

Abstract Abstract 2033 Diamond-Blackfan Anemia (DBA) is a congenital form of pure red cell aplasia characterized by a hypoproliferative, macrocytic anemia, congenital anomalies, and a predisposition to cancer. DBA, along with a growing number of human diseases, is linked to defects in ribosome biogenesis. Mutations in at least 10 ribosomal protein genes of both the 40S and 60S ribosomal subunits have now been identified in over 50% of patients with DBA (Narla A, et al. Blood 2010; 115) resulting in ribosomal protein haploinsufficency and in turn a defect in ribosome biogenesis. It remains, however, unknown how these events culminate in erythroid marrow failure. The study of this pathophysiology has been hindered by a lack of animal models. We became aware of the Rps6-deleted mouse as a potential murine model of DBA (Volarevic S, et al. Science 2000; 288). RPS6 is another 40S ribosomal subunit protein required for ribosomal subunit assembly. Haploinsufficiency of RPS6 causes a phenotype reminiscent of DBA during embryogenesis (Panic L, et al. Mol Cell Biol 2006; 26), however, the erythropoietic phenotype of the conditionally-deleted Rps6 heterozygous mouse was unknown. The purpose of these studies is to fully characterize the erythroid phenotype of this mouse as a model of DBA. We demonstrate that deletion of one Rps6 allele in mice results in a macrocytic anemia and leukopenia (an absolute neutropenia and lymphocytopenia, Table 1). Though this finding is not typical, neutropenia has been described in DBA. Like DBA, the anemia is hypoproliferative (corrected reticulocyte counts were equivalent in rpS6 heterozygous and control mice: 3.3% ± 0.21, n= 3 vs. 3.6 ± 0.33, n=3; two-tailed Student's t-test, p= 0.08, which is an inappropriately low value given the deleted animals’ anemia). Flow cytometric analyses of bone marrow and spleen double-stained for Ter119 and transferrin receptor (CD71) demonstrate impaired early erythroid differentiation, evidenced by a relative expansion in the proerythroblast and basophilic erythroblast populations. Hematopoietic colony assays confirm this early defect. These data suggest that haploinsufficiency of rpS6 impacts both erythropoiesis and granulopoiesis, and since the mice are not thrombocytopenic, the effect appears lineage specific, rather than occurring in a common progenitor cell. Polysome profiles to confirm a defect in ribosome biogenesis are pending. Since heterozygous mice recapitulate the erythroid phenotype of DBA, we treated the mice with standard and potential DBA therapies. Specifically, mice received 2 mg/kg/day of prednisone for 12 weeks. There was no improvement in the hemoglobin or MCV in treated animals. As DBA and 5q- syndrome myelodysplastic syndrome (MDS) share an erythroid phenotype and both result from a haploinsufficiency of a ribosomal protein, we also tested whether the macrocytic anemia in rpS6 heterozygous mice responds to lenalidomide (Revlimid®, gift from Celgene Corporation, San Diego, CA). Mice received 3 mg/kg/day of lenalidomide by oral gavage for 12 weeks. The hemoglobin increased in control mice and markedly increased in rpS6 heterozygous mice after 12 weeks of therapy (13.5 ± 0.4 to 14.9 ± 0.2, p= 0.0 and 7.9 g/dL ± 0.9 to 10.3 ± 0.8, p= 0.01, respectively; mean ± SEM, Student's t-test, paired). Additionally, the MCV decreased with therapy in both groups (49.1 fL ± 1.4 to 41.1 ± 0.2, p=0.005 and 57.4 ± 1.1 to 53.77 ± 1.4, p=0.08). With the caveat that we did not monitor drug levels achieved in vivo, these data suggest that lenalidomide improves hemoglobinization and deserves further study in DBA. Disclosures: No relevant conflicts of interest to declare.

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The Frequency and Characteristics of the Ribosomal Protein L5 (RPL5) and Ribosomal Protein L11 (RPL11) Gene Mutations in Patients with Diamond-Blackfan Anemia in the Czech National Registry

Blood ◽

10.1182/blood.v112.11.3103.3103 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3103-3103

Author(s):

Dagmar Pospisilova ◽

Radek Cmejla ◽

Jana Cmejlova ◽

Helena Handrkova ◽

Jan Stary ◽

...

Keyword(s):

Ribosomal Protein ◽

Ribosome Biogenesis ◽

Stop Codon ◽

Point Mutations ◽

Ribosomal Subunit ◽

Premature Stop Codon ◽

National Registry ◽

Red Cell ◽

Large Ribosomal Subunit ◽

Diamond Blackfan Anemia

Abstract Introduction: Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia that is usually diagnosed during early infancy. Apart from defects in red cell maturation, the disorder is also associated with various physical anomalies in 40% of patients. Mutations in the ribosomal protein (RP) S19 were found in 25% of patients, while mutations in other proteins of a small ribosomal subunit (RPS17 and RPS24) were published only in a small fraction of patients. Recently, mutations in RPL5, RPL11 and RPL35a of a large ribosomal subunit were also disclosed in several DBA patients. Results: The Czech DBA registry currently comprises 31 patients. Mutations in RPL5 were identified in 8/31 patients (26%), and mutations in RPL11 in 2/31 patients (6.5%), implying that mutations in RPL5 account for more Czech DBA cases than mutations in RPS19 (22.6%). As for the classification of mutations, all types were identified, including a nonsense mutation (in RPL11), point mutations (in RPL11 and RPL5), a supposed splicing defect and a small insertion and deletions (all in RPL5). Except for point mutations, all other changes were predicted to cause frameshift with premature stop codon. Since identified alterations were found neither in dbSNP nor in 52 healthy controls, and in two families mutations segregate with the disease, we conclude that they represent true DBA-causative mutations. Although the Czech DBA Registry is rather small, we performed a direct comparison of the group of patients with RPS19 mutations (n=7) with the group of patients with RPL5 mutations (n=8). No differences were found in sex ratio, steroid responsiveness, severity or course of the disease or the treatment outcome. However, patients with RPL5 mutations were generally born small for gestational age (SGA) compared with patients from the RPS19-mutated group. Only one patient (12.5%) with an RPL5 mutation was born with normal birth weight compared to four patients (57.1%) with RPS19 mutations. The second difference was even more striking: all patients with RPL5 mutations had flat thenar and some also an additional thumb anomaly, while no thumb anomalies were observed in patients with RPS19 mutations. It is questionable whether normal RPL5 function is in some way more important for proper thumb development than RPS19. Discussion: The identification of mutations in the genes in DBA patients is also interesting from another point of view. Both proteins RPL5 and RPL11 have been reported to be implicated in the activation of p53 through the interaction with the MDM2 protein, suppressing its E3 ubiquitin ligase function that otherwise directs p53 to a rapid degradation. It is noteworthy that yet another RP of a large ribosomal subunit was described, having exactly the same function – RPL23. Because no RPL23 mutations in our DBA patients were found, the primary function of RPL5 and RPL11 in ribosome biogenesis and/or translation underlies DBA phenotype rather than the conjoint role of RPL5, RPL11 and RPL23 in the p53 regulation. Conclusions: We identified 6 and 2 different mutations in the RPL5 and RPL11 genes, respectively, expanding the repertoire of known DBA-associated mutations. No mutations in the RPL23 were identified, suggesting that aberrant p53 activation due to mutations in RPL5 and RPL11 seems unlikely to be the primary cause of DBA. Patients with RPL5 mutations are more commonly born SGA and have higer frequency of thumb anomalies.

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Mice with ribosomal protein S19 deficiency develop bone marrow failure and symptoms like patients with Diamond-Blackfan anemia

Blood ◽

10.1182/blood-2011-08-371963 ◽

2011 ◽

Vol 118 (23) ◽

pp. 6087-6096 ◽

Cited By ~ 82

Author(s):

Pekka Jaako ◽

Johan Flygare ◽

Karin Olsson ◽

Ronan Quere ◽

Mats Ehinger ◽

...

Keyword(s):

Bone Marrow ◽

Rna Interference ◽

Ribosomal Protein ◽

Mouse Models ◽

Bone Marrow Failure ◽

Macrocytic Anemia ◽

Down Regulation ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia ◽

Ribosomal Protein S19

Abstract Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 down-regulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNA interference that allows an inducible and graded down-regulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene.

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Ribosomal Protein S6 Gene Haploinsufficiency Is Associated with Activation of a p53-Dependent Checkpoint during Gastrulation

Molecular and Cellular Biology ◽

10.1128/mcb.00751-06 ◽

2006 ◽

Vol 26 (23) ◽

pp. 8880-8891 ◽

Cited By ~ 89

Author(s):

Linda Panić ◽

Sanda Tamarut ◽

Melanie Sticker-Jantscheff ◽

Martina Barkić ◽

Davor Solter ◽

...

Keyword(s):

Cell Cycle ◽

Embryonic Development ◽

Ribosomal Protein ◽

Ribosome Biogenesis ◽

Fetal Liver ◽

Genetic Inactivation ◽

Developmental Program ◽

Ribosomal Protein S6 ◽

M Phase ◽

Insight Into

ABSTRACT Nascent ribosome biogenesis is required during cell growth. To gain insight into the importance of this process during mouse oogenesis and embryonic development, we deleted one allele of the ribosomal protein S6 gene in growing oocytes and generated S6-heterozygous embryos. Oogenesis and embryonic development until embryonic day 5.5 (E5.5) were normal. However, inhibition of entry into M phase of the cell cycle and apoptosis became evident post-E5.5 and led to perigastrulation lethality. Genetic inactivation of p53 bypassed this checkpoint and prolonged development until E12.5, when the embryos died, showing decreased expression of D-type cyclins, diminished fetal liver erythropoiesis, and placental defects. Thus, a p53-dependent checkpoint is activated during gastrulation in response to ribosome insufficiency to prevent improper execution of the developmental program.

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Eukaryotic protein uS19: a component of the decoding site of ribosomes and a player in human diseases

Biochemical Journal ◽

10.1042/bcj20200950 ◽

2021 ◽

Vol 478 (5) ◽

pp. 997-1008

Author(s):

Dmitri Graifer ◽

Galina Karpova

Keyword(s):

Ribosome Biogenesis ◽

Lymphocytic Leukemia ◽

Human Diseases ◽

Tumor Suppressor P53 ◽

Translational Fidelity ◽

Gene Encoding ◽

Ribosomal Subunits ◽

Translational Machinery ◽

Diamond Blackfan Anemia ◽

Ribosomal Function

Proteins belonging to the universal ribosomal protein (rp) uS19 family are constituents of small ribosomal subunits, and their conserved globular parts are involved in the formation of the head of these subunits. The eukaryotic rp uS19 (previously known as S15) comprises a C-terminal extension that has no homology in the bacterial counterparts. This extension is directly implicated in the formation of the ribosomal decoding site and thereby affects translational fidelity in a manner that has no analogy in bacterial ribosomes. Another eukaryote-specific feature of rp uS19 is its essential participance in the 40S subunit maturation due to the interactions with the subunit assembly factors required for the nuclear exit of pre-40S particles. Beyond properties related to the translation machinery, eukaryotic rp uS19 has an extra-ribosomal function concerned with its direct involvement in the regulation of the activity of an important tumor suppressor p53 in the Mdm2/Mdmx-p53 pathway. Mutations in the RPS15 gene encoding rp uS19 are linked to diseases (Diamond Blackfan anemia, chronic lymphocytic leukemia and Parkinson's disease) caused either by defects in the ribosome biogenesis or disturbances in the functioning of ribosomes containing mutant rp uS19, likely due to the changed translational fidelity. Here, we review currently available data on the involvement of rp uS19 in the operation of the translational machinery and in the maturation of 40S subunits, on its extra-ribosomal function, and on relationships between mutations in the RPS15 gene and certain human diseases.

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5′UTR Variants of Ribosomal Protein S19 Transcript Determine Translational Efficiency: Implications for Diamond-Blackfan Anemia and Tissue Variability

PLoS ONE ◽

10.1371/journal.pone.0017672 ◽

2011 ◽

Vol 6 (3) ◽

pp. e17672 ◽

Cited By ~ 11

Author(s):

Jitendra Badhai ◽

Jens Schuster ◽

Olof Gidlöf ◽

Niklas Dahl

Keyword(s):

Ribosomal Protein ◽

Translational Efficiency ◽

Diamond Blackfan Anemia ◽

Ribosomal Protein S19

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Molecular Analysis of Diamond Blackfan Anemia and Genotype-Phenotype Correlation: Experience from the Canadian Inherited Marrow Failure Registry

Blood ◽

10.1182/blood.v126.23.3621.3621 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3621-3621

Author(s):

Omri Avraham Arbiv ◽

Bozana Zlateska ◽

Robert J. Klaassen ◽

Conrad Fernandez ◽

Rochelle Yanofsky ◽

...

Keyword(s):

Ribosomal Protein ◽

Research Funding ◽

Gene Panel ◽

Ribosomal Protein Genes ◽

Severe Phenotype ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia ◽

Genetic Groups ◽

Number Of Patients ◽

The Impact

Abstract Background/Objectives: Diamond Blackfan anemia (DBA) is an inherited disorder characterized by chronic hypoproductive anemia, physical malformations, and an increased risk of malignancies. At least 12 DBA genes have been identified, which include various ribosomal protein genes and the transcription factor GATA1. The aims of our study were (1) to identify the mutation spectrum of DBA patients, utilizing a cohort of patients enrolled on the Canadian Inherited Marrow Failure Registry (CIMFR) and (2) to determine whether specific hematological abnormalities, malformations, and outcomes are associated with specific mutations. Methods: Patients were enrolled on the CIMFR, which is a multicenter cohort study of inherited bone marrow failure syndromes (IBMFS). Genetic testing was performed using one or more of the following tests: Sanger sequencing, next generation sequencing (NGS) DBA gene panel, a comprehensive NGS IBMFS gene panel developed in our laboratory, or comparative genetic hybridization (CGH). Severity of the hematological disease was dichotomized according to a patient's requirement for chronic treatment: those who were maintained on corticosteroids, blood transfusions, or received a hematopoietic stem cell transplantation were considered to have a more severe phenotype than those who did not require hematological treatment. Chi-square tests with a Fisher's exact test correction were used to compare genetic groups with at least 5 patients on observed phenotypes. Results: 71 patients with DBA have been enrolled in our registry. A causal mutation has been identified in 36 of these patients, with the following rates: RPS19 (n=11), RPL11 (n=7), RPL5 (n=6), RPS26 (n=5), RPL35a (n=2), RPS24 (n=2), and one of each RPS7, RPS29, RPS17. Remarkably, a substantial number of patients in our population-based cohort (19.4%) had mild hematological phenotype requiring no therapy. Patients with RPL11 mutations tended to have a less severe DBA phenotype, while patients with RPS19 mutations tended to have a more severe phenotype (p=0.04). In terms of non-hematological malformations, we found no differences in cardiac, stature and craniofacial malformations across the groups compared (all p>0.1). However, patients with RPL5 mutations had significantly more hand malformations (p=0.02), and patients with RPS26 mutations had more genitourinary malformations (p=0.04). To control for the impact of mutation severity on the observed phenotype, we compared the prevalence of mutations that are predicted to result in truncated or lack of protein from the respective allele (large copy-number variation, nonsense, or indel frameshift) to mutations that are predicted to be hypomorphic or affect function (splicing, indel/inframe and, missense) between mutation categories. There were no differences among genetic groups in the severity of their mutations (p=0.58). Conclusions: Mutations in a wide spectrum of ribosomal protein genes underlie DBA cases in Canada, which approximate those observed by other registries in Western countries. Patients with DBA caused by RPL11 mutations tended to have a milder hematological phenotype, while patients with RPS19 mutation tended to have a more severe phenotype. Mutations in RPS26 and RPL5 are associated with genitourinary and hand malformations, respectively. Our findings may help improve counseling of DBA patients and their family. Future studies are needed to replicate our results and determine whether these findings can help personalize care. Disclosures Lipton: Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

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