Erythrocyte Adenosine Deaminase Levels Are Elevated in Classic Diamond Blackfan Anemia but Not in the 5q- Syndrome

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1501-1501
Author(s):  
Anupama Narla ◽  
Natalie Louise Davis ◽  
Corinne LaVasseur ◽  
Carolyn Wong ◽  
Bertil Glader
Keyword(s):  
Bone Marrow ◽  
Ribosomal Protein ◽  
Adenosine Deaminase ◽  
Statistical Power ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Phenotypic Variability ◽  
Reciprocal Relationship ◽  
Diamond Blackfan Anemia ◽  
Chromosome 5Q

Abstract Diamond Blackfan anemia (DBA) is congenital bone marrow failure syndrome characterized by red cell aplasia. It was the first disease linked to ribosomal dysfunction with 70% of patients having haploinsufficiency of a ribosomal protein (RP) gene. More recently, several patients with DBA have been shown to have mutations in GATA-1. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), is also characterized by a severe anemia that is caused by heterozygous loss of RPS14 on chromosome 5q. The reciprocal relationship between these two diseases, which are now collectively known as the ribosomopathies, has spurred interest in how we may be able to better understand the pathophysiology of these disorders to broaden therapeutic options, and to improve diagnosis. Erythrocyte adenosine deaminase (eADA) levels have been used for the last three decades in the diagnosis of DBA based on the finding in 1983 that eADA enzyme levels are significantly elevated in patients with DBA. It is now known that more than 75% of DBA patients have elevated eADA levels. The goal of the present study is to measure the levels of eADA in patients with the 5q- syndrome and determine whether they are elevated implying that eADA may be specific to ribosomal haploinsufficiency in general or normal suggesting that elevated eADA may be specific to DBA. Under a Stanford Univeristy approved IRB protocol, we measured eADA levels in patients with the 5q- syndrome who were undergoing routine blood draws for clinical purposes. The patients with Diamond Blackfan anemia are either followed at our center in Stanford University or were previously referred to us for eADA testing with accompanying clinical information. Analysis was performed using SAS 9.3 (SAS Institute, Carey, NC). Continuous variables were presented as means and standard deviations (SD) and analyzed using 2-tailed T-Test. Complete results by mutation type are summarized in Tables 1 and 2. In a disorder with the marked genotypic and phenotypic variability such as Diamond Blackfan anemia (DBA), it is critical to have sensitive tests to aid with the diagnosis. Erythrocyte adenosine deaminase (eADA) levels have long been used as supportive diagnostic criteria. The test has been especially useful in distinguishing DBA from other causes of anemia and other inherited bone marrow failure syndromes. However, the reason for the elevation of eADA in DBA remains unknown. Our goal was to study eADA levels in the 5q- syndrome where there is an acquired ribosomal haploinsufficiency (from loss of RPS14 on chromosome 5q), which is in contrast to the congenital haploinsufficiency seen in DBA. Interestingly, we found that the eADA levels were normal in the 5q- syndrome suggesting that eADA is specific for DBA. We then studied eADA levels in patients with DBA that is not the results of a ribosomal protein (RP) mutation but instead due to mutations in GATA-1. Once again, we found that eADA levels were normal in this cohort suggesting that elevated eADA is specific feature of DBA due to RP mutations. It is interesting to note that patients with RPS19 mutations, who tend to have a milder clinical phenotype, and patients without an identified mutation had lower (but still elevated) levels of eADA when compared to other RP mutations, in particular RPL11. A larger number of patient samples in future studies would improve the statistical power to compare eADA levels between individual RP mutations. This is the first report to examine the value of eADA in non-classical DBA and in another disorder of ribosomal haploinsufficiency, namely del(5q) MDS. In summary, we found that an elevated eADA strongly suggests the diagnosis of classic DBA although a normal eADA does not exclude the diagnosis, particularly in the setting of GATA-1 mutations. There is no utility for using eADA in the diagnosis of the 5q- syndrome despite the connection of the disease with impaired ribosome function. The reason for elevated eADA in classic DBA remains to be defined. Disclosures No relevant conflicts of interest to declare.

scholarly journals Understanding the Role of Erythrocyte Adenosine Deaminase in the Ribosomopathies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5152-5152
Author(s):  
Anupama Narla ◽  
Bertil Glader ◽  
Carolyn Wong
Keyword(s):  
Bone Marrow ◽  
Adenosine Deaminase ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Reciprocal Relationship ◽  
Bone Marrow Failure Syndrome ◽  
Predictive Values ◽  
Diamond Blackfan Anemia ◽  
Chromosome 5Q ◽  
Bone Marrow Failure Syndromes

Abstract Diamond Blackfan anemia (DBA) is congenital bone marrow failure syndrome characterized by red cell aplasia and a predisposition to cancer. It was the first disease linked to ribosomal dysfunction with 70% of patients having haploinsufficiency of a ribosomal protein (RP) gene with RPS19 being the most frequently mutated. The 5q- syndrome is a subtype of myelodysplastic syndrome (MDS) also characterized by a severe anemia that is caused by heterozygous loss of RPS14 on chromosome 5q. These patients also have a predisposition to cancer. The reciprocal relationship between these two diseases, which are now collectively known as the ribosomopathies, has spurred interest in how we may be able to better understand the pathophysiology of these ribosomopathies to broaden therapeutic options, and to improve diagnosis. Erythrocyte adenosine deaminase (eADA) levels have been used for last three decades in the diagnosis of Diamond Blackfan anemia based on the finding in 1983 that eADA enzyme levels are significantly elevated in patients with DBA. It is now known the eADA levels are elevated in >75% of patients with DBA. Furthermore, it has a sensitivity of 84%, specificity of 95% and positive and negative predictive values of 91% for the diagnosis of DBA compared with other inherited bone marrow failure syndromes. Our goal was to study the levels of eADA in patients with the 5q- syndrome to determine whether they were elevated (suggesting that eADA may be specific to ribosomal haploinsufficiency in general) or normal (suggesting that eADA may be specific to DBA). Under a Stanford approved IRB, we are testing adult patients with the 5q syndrome who have not been transfused in the previous 3 months (which would affect the results of the test) and who have a confirmed deletion of the 5q- region by cytogenetics. To date, all of the 4 MDS samples, sent to us from collaborating hematologists, have had a normal eADA level. These findings suggest that elevation of eADA may be specific to DBA and not a general feature of ribosomal haploinsufficiency although our sample size is small and we plan to test additional samples. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 177-177 ◽  
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.

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

Blood ◽  
2011 ◽  
Vol 118 (23) ◽  
pp. 6087-6096 ◽  
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.

Ribosomal Protein Genes S10 and S26 Are Commonly Mutated in Diamond-Blackfan Anemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 175-175 ◽  
Author(s):  
Hanna T. Gazda ◽  
Mee Rie Sheen ◽  
Leana Doherty ◽  
Adrianna Vlachos ◽  
Valerie Choesmel ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Large Scale ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Ribosomal Protein Genes ◽  
Variants Of Unknown Significance ◽  
Diamond Blackfan Anemia ◽  
Increased Risk ◽  
Unknown Significance ◽  
Risk Of Cancer

Abstract Abstract 175 Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by anemia usually presenting during infancy or in early childhood, birth defects, and increased risk of cancer. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, in particular craniofacial, upper limb, heart and urinary system defects, that are present in ∼30–50% of patients. To date, DBA has been associated with mutations in seven ribosomal protein (RP) genes, S19, S24, S17, L35A, L5, L11, and S7 in about ∼43% of patients. To complete our large scale screen of RP genes in a DBA population, we sequenced 49 ribosomal protein genes in our DBA patient cohort of 117 probands. Together with our previous efforts of screening 29 RP genes, this completes the analysis of all known RP genes (excluding RPS4Y on the Y chromosome). Here we report probable mutations in four more RP genes, RPS10, RPS26, RPL19, and RPL26, and variants of unknown significance in six more, RPL9, RPL14, RPL23A, RPL7, RPL35, and RPL3. RPS10 and RPS26 are now confirmed DBA genes as we identified five mutations in RPS10 in five probands and nine mutations in RPS26 in 12 probands. We found single small frameshifting deletions in RPL19 and RPL26 in single DBA families. Pre-rRNA analysis in lymphoblastoid cells from patients bearing mutations in RPS10 and RPS26 showed elevated levels of 18S-E pre-rRNA. This accumulation is consistent with the phenotype observed in HeLa cells after knock-down of RPS10 or RPS26 expression with siRNAs, which indicate that mutations in the RPS10 and RPS26 genes in DBA patients affect the function of the proteins. This brings to 11, the total number of RP genes mutated in ∼54% of patients with DBA, with nine more genes harboring variants of unknown significance requiring further study. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Acquired ribosomopathies in leukemia and solid tumors

Hematology ◽  
2017 ◽  
Vol 2017 (1) ◽  
pp. 716-719 ◽  
Author(s):  
Adrianna Vlachos
Keyword(s):  
Bone Marrow ◽  
Ribosomal Protein ◽  
Solid Tumors ◽  
Ribosomal Proteins ◽  
Bone Marrow Failure ◽  
Ribosomal Protein Gene ◽  
Small Subunit ◽  
Gene Encoding ◽  
Bone Marrow Failure Syndrome ◽  
Diamond Blackfan Anemia

AbstractA mutation in the gene encoding the small subunit-associated ribosomal protein RPS19, leading to RPS19 haploinsufficiency, is one of the ribosomal protein gene defects responsible for the rare inherited bone marrow failure syndrome Diamond Blackfan anemia (DBA). Additional inherited and acquired defects in ribosomal proteins (RPs) continue to be identified and are the basis for a new class of diseases called the ribosomopathies. Acquired RPS14 haploinsufficiency has been found to be causative of the bone marrow failure found in 5q– myelodysplastic syndromes. Both under- and overexpression of RPs have also been implicated in several malignancies. This review will describe the somatic ribosomopathies that have been found to be associated with a variety of solid tumors as well as leukemia and will review cancers in which over- or underexpression of these proteins seem to be associated with outcome.

Functional Consequences Of RPS29 Germline Mutations In Diamond-Blackfan Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2465-2465
Author(s):  
Lisa Mirabello ◽  
Elizabeth R Macari ◽  
Lea Jessop ◽  
Timothy Myers ◽  
Neelam Giri ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Blood Cell ◽  
Ribosomal Protein ◽  
Board Of Directors ◽  
Bone Marrow Failure ◽  
Germline Mutations ◽  
Zebrafish Embryos ◽  
Advisory Committees ◽  
Diamond Blackfan Anemia ◽  
Equity Ownership

Abstract Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome (IBMFS) characterized by red blood cell aplasia, variable physical anomalies, and increased risks of cancer. Approximately half of DBA cases have a germline mutation or deletion in a ribosomal protein gene (i.e., RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, or RPS26), or in GATA1, an X-linked hematopoietic transcription factor. The inheritance of mutated ribosomal protein genes is autosomal dominant (AD), but de novo germline mutations may also occur. We previously reported a novel nonsynonymous ribosomal protein S29 (RPS29) mutation, I31F, which segregated with DBA in a large multi-case family (NCI-193). We now present functional data that link this RPS29 mutation to the DBA phenotype and report an additional large multi-case DBA family (NCI-38) with another novel RPS29 mutation. Individuals in this study are participants in the IRB-approved longitudinal cohort IBMFS study at the NCI. We have identified a second large family with DBA, NCI-38, in which the male proband had steroid-responsive anemia as a child, elevated erythrocyte adenosine deaminase (eADA = 1.26 IU/g Hb, normal <0.96), and has been in remission for approximately 20 years. Two unaffected male half-siblings have no symptoms of DBA, and 1 female half-sibling has only a borderline elevated eADA (1.00 IU/g Hb). The proband’s mother is an asymptomatic obligate carrier because her full brother and two half-siblings have DBA or an unspecified bone marrow failure; her eADA is normal (0.8 IU/g Hb). We performed whole-exome sequencing using Nimblegen v2 capture arrays and paired-end sequencing on the Illumina HiSeqTM. After applying quality control filters, removing variants present in publicly available databases (1000Genomes, ESP, Kaviar, and dbSNP), and applying an AD inheritance model, we identified an I50T mutation in RPS29 that tracked with AD inheritance in this family (NCI-38). The sibling with elevated eADA and the mother also carried this RPS29 mutation while the two asymptomatic healthy siblings were mutation negative. I50T, like I31F, occurs in a very highly evolutionarily conserved region of the RPS29 protein and in silico prediction programs (i.e., SIFT, Polyphen 2, Condel, MutationTaster) predict this variant to be deleterious. The original DBA family, NCI-193, has AD DBA due to the heterozygous I31F mutation in RPS29. We used quantitative rtPCR to determine RPS29 expression in the proband with the I31F mutation compared with a healthy individual with wildtype RPS29. The proband demonstrated haploinsufficiency of RPS29 mRNA, with levels consistent with approximately 50% expression compared with wildtype RPS29 values. Zebrafish are ideal for modeling ribosomal protein knockdown because hematopoietic regulation is conserved with mammals, they are amenable to high throughput in vivo genetics, and ribosomal proteins can be knocked down in the embryo with morpholino technology. The rps29-/- mutant zebrafish displays many aspects of the DBA phenotype including significant defects in red blood cell development, shown by a decrease in hemoglobin levels. We used this as a model to determine if the RPS29 mutation identified in our DBA cases could rescue the hemoglobin defect. The rps29-/- zebrafish embryos were injected with 50pg of either wildtype or the mutated (I31F) human RPS29 RNA. The wildtype human RPS29 RNA rescued the hemoglobin phenotype (increased hemoglobin levels) of the rps29-/- zebrafish embryos, whereas the mutated human RPS29 RNA could not (no increase in hemoglobin levels; P <0.01). In summary, germline mutations in RPS29 can cause AD DBA. RPS29 is an essential protein in the 40s small subunit of the ribosome, important for ribosomal RNA processing and ribosome biogenesis. Using functional assays, we show for the first time that the mutant RPS29 in our DBA cases results in haploinsufficiency of RPS29. The patient-associated I31F RPS29 mutation failed to rescue the defective hematopoiesis in the rps29-/- mutant zebrafish DBA model and provides further evidence that RPS29 is a DBA-associated gene. Disclosures: Zon: FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

scholarly journals Rpl22 Deficiency Predisposes Hematopoietic Stem and Progenitor Cells to Leukemogenesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 899-899 ◽  
Author(s):  
Bryan Harris ◽  
Jaqueline Perrigoue ◽  
Rachel M. Kessel ◽  
Shawn Fahl ◽  
Stephen Matthew Sykes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Stem Cell ◽  
Ribosomal Protein ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Ectopic Expression ◽  
Hematopoietic Stem ◽  
Increased Risk

Abstract Mutations and deletions in ribosomal proteins are associated with a group of diseases termed ribosomopathies. Collectively, these diseases are characterized by ineffective hematopoiesis, bone marrow failure, and an increased risk of developing myelodysplastic syndrome (MDS) and subsequently acute myeloid leukemia (AML). This observation highlights the role of dysregulation of this class of proteins in the development and progression of myeloid neoplasms. Analysis of gene expression in CD34+ hematopoietic stem cells (HSC) from 183 MDS patients demonstrated that ribosomal protein L22 (Rpl22) was the most significantly reduced ribosomal protein gene in MDS. Interestingly, we observed that AML patients with lower expression of Rpl22 had a significant reduction in their survival (TCGA cohort, N=200, Log Rank P value <0.05). To assess the mechanism of reduced expression, we developed a FISH probe complementary to the RPL22 locus and assessed for deletion of this locus in an independent set of 104 MDS/AML bone marrow samples. Strikingly, we found that RPL22 deletion was enriched in high-risk MDS and secondary AML cases. We, therefore, sought to investigate whether reduced Rpl22 expression played a causal in leukemogenesis. Using Rpl22-/- mice, we found that Rpl22-deficiency resulted in a constellation of phenotypes resembling MDS. Indeed, Rpl22-deficiency causes a macrocytic reduction in red blood cells, dysplasia in the bone marrow, and an expansion of the early hematopoietic stem and progenitor compartment (HSPC). Since MDS has been described as a disease originating from the stem cell compartment, we next sought to determine if the hematopoietic defects were cell autonomous and resident in Rpl22-/- HSC. Competitive transplantation revealed that Rpl22-/- HSC exhibited pre-leukemic characteristics including effective engraftment, but a failure to give rise to downstream mature blood cell lineages. Importantly, there was a strong myeloid bias in those downstream progeny derived form Rpl22-/- HSC. Because human MDS frequently progresses to AML, we examined the potential for Rpl22-deficient HSC to be transformed upon ectopic expression of the MLL-AF9 oncogenic fusion. Indeed, Rpl22-deficient HSPC exhibited an increased predisposition to transformation both in vitro and in vivo, in MLL-AF9 knockin mice. To determine how Rpl22-deficiency increased the transformation potential of HSC, we performed whole transcriptome analysis on Rpl22-/- HSC. Interestingly, four expression signatures were observed that were consistent with the altered behavior exhibited by Rpl22-/- HSC. Rpl22-deficient HSC exhibited increased expression of: 1) genes associated with stem cell function, consistent with the basal expansion and effective engraftment of Rpl22-/- HSC upon adoptive transfer; 2) markers of the myeloid lineage, providing a potential explanation for the myeloid bias exhibited by Rpl22-/- HSC; 3) cell cycle regulators, consistent with the increased proliferation exhibited by Rpl22-/- HSC; and 4) components of the mitochondrial respiratory chain, a metabolic program on which leukemic stem cell function depends. Together, these data suggest that Rpl22 controls a program of gene expression that regulates the predisposition of HSPC to myeloid transformation. Disclosures No relevant conflicts of interest to declare.

Diamond-Blackfan Anemia: Erythropoiesis Lost in Ribosome Biosynthesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-2-SCI-2
Author(s):  
Stefan Karlsson ◽  
Johan Flygare ◽  
Pekka Jaako ◽  
David Bryder
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Macrocytic Anemia ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia

Abstract Abstract SCI-2 Diamond-Blackfan anemia (DBA) is a rare congenital erythroid hypoplasia that presents early in infancy. The classic hematologic profile of DBA consists of macrocytic anemia with selective absence of erythroid precursors in a normocellular bone marrow, normal or slightly decreased neutrophil, and variable platelet count. During the course of the disease some patients show decreased bone marrow cellularity that often correlates with neutropenia and thrombocytopenia. DBA is a developmental disease since almost 50% of the patients show a broad spectrum of physical abnormalities. All known DBA disease genes encode for ribosomal proteins that collectively explain the genetic basis for approximately 55% of DBA cases. Twenty-five percent of the patients have mutations in a gene encoding for ribosomal protein S19 (RPS19). All patients are heterozygous with respect to RPS19 mutations suggesting a functional haploinsufficiency of RPS19 as basis for disease pathology. Despite the recent advances in DBA genetics, the pathophysiology of the disease remains elusive. Cellular studies on patients together with successful marrow transplantation have demonstrated the intrinsic nature of the hematopoietic defect. DBA patients have a variable deficit in burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors. The frequency of immature hematopoietic progenitors in DBA patients is normal but their proliferation is impaired in vitro. Generation of animal models for RPS19-deficient DBA is pivotal to understand the disease mechanisms and to evaluate novel therapies. Several DBA models have been generated in mice or zebrafish. Although these models have provided important insights on DBA, they are limited in a sense that the hematopoietic phenotype and molecular mechanisms are likely to be influenced by the level of RPS19 downregulation. We have generated mouse models for RPS19-deficient DBA by taking advantage of transgenic RNAi. These models are engineered to contain a doxycycline-regulatable RPS19-targeting shRNA, allowing a reversible and dose-dependent downregulation of RPS19 expression. We demonstrate that the RPS19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count and the severity of the phenotype depends on the level of RPS19 downregulation. We show further that a chronic RPS19 deficiency leads to irreversible exhaustion of hematopoietic stem cells and subsequent bone marrow failure. Overexpression of RPS19 following gene transfer rescues the proliferative and apoptotic phenotype of RPS19-deficient hematopoietic progenitors in vitro, demonstrating that the phenotype is specifically caused by the RPS19 deficiency. Expression analysis of RPS19-deficient hematopoietic progenitors reveals an activation of the p53 pathway. By intercrossing the DBA mice with p53 null mice we demonstrate that inactivation of p53 in vivo results in a variable rescue of the hematopoietic phenotype depending on the level of RPS19 downregulation. Therefore, we conclude that increased activity of p53 plays a major role in causing the DBA phenotype but that other hitherto unidentified pathways also play a role, specifically in patients that have low levels of functional RPS19. Disclosures: No relevant conflicts of interest to declare.

Array Comparative Genomic Hybridization of Ribosomal Protein Genes In Diamond-Blackfan Anemia Patients; Evidence for Three New DBA Genes, RPS8, RPS14 and RPL15, with Large Deletion or Duplication

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1007-1007 ◽  
Author(s):  
Hanna Gazda ◽  
Michael Landowski ◽  
Christopher Buros ◽  
Adrianna Vlachos ◽  
Colin A. Sieff ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Comparative Genomic Hybridization ◽  
Array Comparative Genomic Hybridization ◽  
Large Scale ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Comparative Genomic ◽  
Genomic Hybridization ◽  
Exon 2 ◽  
Diamond Blackfan Anemia

Abstract Abstract 1007 Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, usually presenting during infancy or in early childhood. Although anemia is the most prominent feature of DBA, the disease is also characterized by cancer predisposition, growth retardation and congenital malformations, in particular craniofacial, upper limb, heart and urinary system defects, which are present in ∼30-50% of patients. We completed our large scale sequencing of 80 ribosomal protein (RP) genes and found eight of them mutated in DBA. In total, together with three RP genes identified by others, there are 11 genes mutated in ∼54% of DBA patients; RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, RPS26, RPL19 and RPL26. To search for moderate and large RP gene deletions and duplications we performed high resolution array comparative genomic hybridization on 80 DNA samples from DBA patients who did not have mutations in the 11 known RP genes. We found a deletion of exon 2 and 3 (4800 bp), deletion of the coding region, and duplication of exons 2 and 3 (488 bp) in RPS19 gene in three probands; three deletions of exons 1, 2 and 3 in RPS17 in three probands (2920 bp, 2886 bp and 3018 bp); and deletion of exons 1, 2 and 3 of the RPS26 gene. We also identified two deletions and a duplication in three RP genes previously not found mutated in DBA; RPS8 duplication of exon 3 (764 bp), RPS14 deletion of exons 2, 3, 4 and 5 (2568 bp) and RPS15 deletion of exon 4 (1995 bp). The deletions and duplications are being confirmed by multiplex PCRs. Interestingly, RPS14 was previously identified as a 5q- syndrome gene demonstrating that abnormality of this protein can cause both DBA and 5q- syndrome. These data bring to 14 the total number of RP genes mutated in DBA. Disclosures: No relevant conflicts of interest to declare.

Gene transfer improves erythroid development in ribosomal protein S19–deficient Diamond-Blackfan anemia

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2724-2731 ◽  
Author(s):  
Isao Hamaguchi ◽  
Andreas Ooka ◽  
Ann Brun ◽  
Johan Richter ◽  
Niklas Dahl ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Ribosomal Protein ◽  
Viral Vectors ◽  
Bone Marrow Failure ◽  
Erythroid Progenitors ◽  
Bone Marrow Failure Syndrome ◽  
Expression Levels ◽  
Diamond Blackfan Anemia ◽  
Ribosomal Protein S19

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by a specific deficiency in erythroid progenitors. Forty percent of the patients are blood transfusion–dependent. Recent reports show that the ribosomal protein S19 (RPS19) gene is mutated in 25% of all patients with DBA. We constructed oncoretroviral vectors containing theRPS19 gene to develop gene therapy for RPS19-deficient DBA. These vectors were used to introduce the RPS19 gene into CD34+ bone marrow (BM) cells from 4 patients with DBA withRPS19 gene mutations. Overexpression of theRPS19 transgene increased the number of erythroid colonies by almost 3-fold. High expression levels of the RPS19transgene improved erythroid colony-forming ability substantially whereas low expression levels had no effect. Overexpression of RPS19 had no detrimental effect on granulocyte-macrophage colony formation. Therefore, these findings suggest that gene therapy for RPS19-deficient patients with DBA using viral vectors that express the RPS19gene is feasible.

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