Diamond Blackfan Anemia is a rare congenital disease that is primarily characterized by reduced erythroid progenitors. DBA pathogenesis has been associated with genes encoding ribosomal proteins (RPs) which are important in translation. However, this fails to explain why erythropoiesis is specifically disrupted. Our lab previously found that aberrant splicing of the human transcript encoding heme exporter, FLVCR1, is involved in DBA pathogenesis; and that RPS19 implicated in 25% of DBA patients, regulates FLVCR1 transcript splicing. This thesis investigated the role of another DBA associated gene encoding RPS17, in the regulation of FLVCR1 splicing and disrupted erythropoiesis in DBA. My findings further support the role of FLVCR1 aberrant splicing in DBA and provide evidence suggesting that RPS17 may not be a candidate DBA gene. Furthermore, my study implicates a potential role for RPS19 transcript levels in defective erythroid differentiation observed in DBA.

Diamond Blackfan Anemia is a rare congenital disease that is primarily characterized by reduced erythroid progenitors. DBA pathogenesis has been associated with genes encoding ribosomal proteins (RPs) which are important in translation. However, this fails to explain why erythropoiesis is specifically disrupted. Our lab previously found that aberrant splicing of the human transcript encoding heme exporter, FLVCR1, is involved in DBA pathogenesis; and that RPS19 implicated in 25% of DBA patients, regulates FLVCR1 transcript splicing. This thesis investigated the role of another DBA associated gene encoding RPS17, in the regulation of FLVCR1 splicing and disrupted erythropoiesis in DBA. My findings further support the role of FLVCR1 aberrant splicing in DBA and provide evidence suggesting that RPS17 may not be a candidate DBA gene. Furthermore, my study implicates a potential role for RPS19 transcript levels in defective erythroid differentiation observed in DBA.

The focus of this thesis is the role of ribosomal proteins in hematopoiesis and development. Ribosomal proteins are mutated in patients with Diamond Blackfan anemia (DBA). These mutations primarily affect blood tissues, as DBA patients have a macrocytic anemia. We have identified hematopoietic defects in zebrafish with a mutation in ribosomal protein S29 (rps29). \(Rps29^{-/-}\) embryos have defects in hematopoietic stem cell formation, aorta specification, and hemoglobinization. Embryos also have increased numbers of apoptotic cells, and microarray analysis reveals up-regulation of a p53 gene signature. All of the hematopoietic phenotypes are rescued by p53 mutation, demonstrating that p53 activation induced by ribosomal protein knockdown is mediating the \(rps29^{-/-}\) mutant phenotype. In addition, polysome profiles of mutant embryos identify a decrease in 80s monosome and polysome fractions. Preliminary RNA sequencing analysis of the polysome fractions suggested a shift in genes being translated in the mutant. We performed a chemical screen on rps29 embryos. Using embryo morphology and vascular expression patterns as read-outs, 600 compounds of known bioactivity were screened. One compound, A-3, improves embryo morphology, and a structurally related compound, W-7, rescues the vasculature defect. These compounds are calmodulin inhibitors, and A-3 can also rescue the hemoglobin defect in \(rps29^{-/-}\) embryos. To elucidate the compounds’ mechanism of action, A549 and \(CD34^+\) cells with RPS19 knocked down by shRNA were treated with chemical hits. In these cells, calmodulin inhibitors cause a decrease of p21 even with p53 induction. These data support a model where calmodulin inhibition can inhibit the p53 pathway upon ribosomal protein knockdown. In parallel to our zebrafish studies, we generated induced pluripotent stem (iPS) cells from DBA patient fibroblasts as a part of a large-scale collaboration. Three iPS lines are validated, and a total of 27 lines will be generated from patients with mutations in RPS19, RPL5, and RPL11. Testing for defects in blood differentiation and determining the role of p53 in these lines will enable validation of this system as a model of DBA. The iPS lines can subsequently be used for chemical and genetic screening to identify novel DBA pathways and potential therapies.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

L’anémie de Blackfan-Diamond (ABD) est une maladie hématologique rare qui touche 4 à 7 individus/ million de naissances. Cette maladie se manifeste par une érythroblastopénie congénitale sévère (≤ 5% de précurseurs érythroïdes dans la moelle osseuse). L’anémie est arégénérative et souvent macrocytaire et associée à des malformations osseuses dans 40% des cas. 70% des patients sont porteurs d’une mutation hétérozygote pour un gène de protéine ribosomique impliquée dans la traduction cellulaire. Les gènes les plus fréquemment mutés sont les gènes RPS19 (25%), RPL11 (5%) et RPL5 (7%). La maladie est hétérogène et évolutive. Les liens entre la traduction cellulaire et l’érythropoïèse ne sont pas bien élucidés. Les objectifs de cette thèse ont été d’étudier les métabolismes de l’hème et du fer ainsi que l’expression des globines dans des cellules de patients atteints d'ABD et dans un modèle shARN ciblant l'expression de ces trois gènes afin de comprendre les causes du tropisme érythroïde de la maladie. Ce travail de recherche a permis de mettre en évidence un défaut majeur de synthèse des globines ayant pour conséquence une augmentation de la quantité d’hème libre et une production de formes réactives de l'oxygène toxiques dans les cellules des patients qui pourraient expliquer en partie l’apoptose cellulaire et le déficit de globules rouges. Alors que le métabolisme du fer ne semblait pas altéré dans l'ABD, l’étude de l’expression de différentes protéines importantes pour l’érythropoïèse au cours de la différenciation érythroïde in vitro dans des conditions contrôles et chez des patients a permis de confirmer et de caractériser le retard de différenciation cellulaire en cas de mutation des gènes RPL5 et RPL11. Ce travail montre que le retard de différenciation et le défaut d'hémoglobinisation mis en évidence s'expliquent par un déficit du facteur de transcription GATA-1 qui est primordial au cours de l'érythropoïèse. Ce déficit de GATA-1 dans des cellules déficitaires en RPL11 est dû à une dégradation de sa protéine chaperonne HSP70. La restauration de HSP70, permet d'augmenter l'expression de GATA-1 et d'améliorer la différenciation érythroïde et l'hémoglobinisation cellulaire pour le gène RPL11. Ces résultats permettent de mieux comprendre le tropisme érythroïde de l'ABD et de proposer HSP70 comme une cible thérapeutique prometteuse dans son traitement. / Diamond-Blackfan anemia (DBA) is a rare hematologic disease that affects 4 to 7 individuals / million births. This disease is characterized by a severe congenital erythroblastopenia (less than 5% erythroid precursors in the bone marrow). Anemia is agerenative, often macrocytic and associated with bone malformations in 40% of cases. 70% of patients carry a heterozygous mutation for a ribosomal protein gene involved in cell translation. The most frequently mutated genes are RPS19 (25%), RPL11 (5%) and RPL5 (7%) genes. The disease is heterogeneous and can evolve. The link between cell translation and erythropoiesis is not well understood. The objectives of this thesis were to study haem and iron metabolisms as well as the expression of globins in DBA patients cells and CD34+ cells transduced with shRNA targeting the expression of these three genes in order to understand the causes of the erythroid tropism of the disease. This research has highlighted a major defect of globin synthesis resulting in an increase in the amount of free heme and a production of toxic ROS in patients' cells that could explain in part cell apoptosis and red blood cell deficiency. While iron metabolism did not appear to be altered in DBA, the study of the expression of various important proteins for erythropoiesis in normal CD34+ or DBA cells during erythroid differentiation in vitro confirmed a strong cell differentiation delay for RPL5 and RPL11 mutations. This work shows that the delay of differentiation and the lack of hemoglobinization can be explained by a deficiency of the transcription factor GATA-1, which is essential during erythropoiesis. This deficiency of GATA-1 in shRPL11 cells is due to a degradation of its chaperone protein HSP70. The restoration of HSP70 increases the expression of GATA-1 and improves erythroid differentiation and cellular hemoglobinization for the shRPL11 condition. These results provide a better understanding of the erythroid tropism of ABD and suggest a role for HSP70 as a promising therapeutic target in its treatment.

<p>Ribosomal proteins are components of the ribosome, the protein synthesis machinery. The ribosomal protein S19 gene (<i>RPS19</i>) is mutated in Diamond-Blackfan anemia, DBA, which is a rare congenital anemia with absence or reduction of erythroid precursors in bone marrow. In this thesis, the role of RPS19 in erythropoiesis is investigated.</p><p>A genetic analysis of <i>RPS19</i> in 24 DBA cases was performed. Four novel <i>RPS19</i> mutations were identified with evidence of wide clinical expression of the disease.</p><p>Due to the clinical overlap in Transient Erythroblastopenia of Childhood, TEC, and DBA, the two diseases may be caused by a common genetic factor. In a study of seven TEC families, all affected shared at least one parental haplotype in the <i>RPS19</i> gene region. Coding exons of <i>RPS19</i> were normal for all affected, although mutations in intronic and regulatory sequences are not excluded. This indicates a genetic factor behind TEC and a possible association between <i>RPS19</i> and TEC. </p><p>To investigate the role of RPS19 in erythropoiesis in a mammal, we created a mouse model for the targeted disruption of the homologue <i>Rps19</i> on the C57BL/6J genetic background. Null mutants are embryonic lethal prior to implantation. The <i>Rps19</i>^+/- mice, however, are viable with normal development including the hematopoietic system. The <i>Rps19</i> transcript level in <i>Rps19</i>^+/- mice is normal. Accordingly, RPS19 protein levels are similar in <i>Rps19</i>^+/- and <i>Rps19</i>^+/+ mice. This argues for a transcriptional up-regulation to compensate for the loss of one <i>Rps19</i> allele. </p><p>Peripheral blood is normal in <i>Rps19</i>^+/- mice also on the FVB/NJ strain which argues against strain-specific effects of the <i>Rps19</i> disruption. Preliminary results indicate a reduced erythroid proliferation in response to erythropoietin in <i>Rps19</i>^+/- mice, suggesting the requirement of both <i>Rps19</i> alleles for normal erythroid proliferation under stress. This would support a mechanism by which haplo-insufficiency for RPS19 causes DBA.</p>

Ribosomal proteins are components of the ribosome, the protein synthesis machinery. The ribosomal protein S19 gene (RPS19) is mutated in Diamond-Blackfan anemia, DBA, which is a rare congenital anemia with absence or reduction of erythroid precursors in bone marrow. In this thesis, the role of RPS19 in erythropoiesis is investigated. A genetic analysis of RPS19 in 24 DBA cases was performed. Four novel RPS19 mutations were identified with evidence of wide clinical expression of the disease. Due to the clinical overlap in Transient Erythroblastopenia of Childhood, TEC, and DBA, the two diseases may be caused by a common genetic factor. In a study of seven TEC families, all affected shared at least one parental haplotype in the RPS19 gene region. Coding exons of RPS19 were normal for all affected, although mutations in intronic and regulatory sequences are not excluded. This indicates a genetic factor behind TEC and a possible association between RPS19 and TEC. To investigate the role of RPS19 in erythropoiesis in a mammal, we created a mouse model for the targeted disruption of the homologue Rps19 on the C57BL/6J genetic background. Null mutants are embryonic lethal prior to implantation. The Rps19+/- mice, however, are viable with normal development including the hematopoietic system. The Rps19 transcript level in Rps19+/- mice is normal. Accordingly, RPS19 protein levels are similar in Rps19+/- and Rps19+/+ mice. This argues for a transcriptional up-regulation to compensate for the loss of one Rps19 allele. Peripheral blood is normal in Rps19+/- mice also on the FVB/NJ strain which argues against strain-specific effects of the Rps19 disruption. Preliminary results indicate a reduced erythroid proliferation in response to erythropoietin in Rps19+/- mice, suggesting the requirement of both Rps19 alleles for normal erythroid proliferation under stress. This would support a mechanism by which haplo-insufficiency for RPS19 causes DBA.

Diamond-Blackfan anemia (DBA) is a severe congenital anemia characterized by a defect in red blood cell production. The disease is associated with growth retardation, malformations, a predisposition for malignant disease and heterozygous mutations in either of the ribosomal protein (RP) genes RPS7, RPS17, RPS19, RPS24, RPL5, RPL11 and RPL35a. In a cellular model for DBA, siRNA knock-down of RPS19 results in a relative decrease of other ribosomal (r) proteins belonging to the small subunit (RPS20, RPS21, RPS24) when compared to r-proteins from the large ribosomal subunit (RPL3, RPL9, RPL30, RPL38). RPS19 mutant cells from DBA patients show a similar and coordinated down-regulation of small subunit proteins. The mRNA levels of the small subunit r-proteins remain relatively unchanged. We also show that RPS19 has an extensive number of transcriptional start sites resulting in mRNAs of variable 5’UTR length. The short variants are translated more efficiently. Structural sequence variations in the 5’UTR of RPS19 found in DBA patients show a 20%-30% reduced translational activity when compared to normal transcripts. Primary fibroblast from DBA patients with truncating mutations in RPS19 or RPS24 showed specific cell cycle defects. RPS19 mutant fibroblasts accumulate in the G1 phase whereas the RPS24 mutant cells show a defect in G2/M phase. The G1 phase arrest is associated with a reduced level of phosphorylated retinoblastoma (Rb) protein, cyclin E and cdk2 whereas the G2/M phase defect is associated with increased levels of p21, cyclin E, cdk4 and cdk6. RPS19 interacts with PIM-1 kinase. We investigated the effects of targeted disruptions of both Rps19 and Pim-1 in mice. Double mutant (Rps19+/-, Pim-1-/-) mice have increased peripheral white- and red blood cell counts when compared to the wild-type mice (Rps19+/+, Pim-1+/+). Bone marrow cells in Rps19+/-, Pim-1-/- mice showed up-regulated levels of c-Myc and the anti-apoptotic factors Bcl2, Bcl-xl and Mcl-1 and reduced levels of the apoptotic factors Bak and Caspase 3 as well as the cell cycle regulator p21. In summary, this thesis clarifies several mechanisms in the pathogenesis of DBA. Mutations in RPS19 results in coordinated down-regulation of several small subunit r-proteins causing haploinsufficiency for the small ribosomal subunit. RPS19 have multiple transcriptional start sites and mutations in the RPS19 5’UTR found in DBA patients result in reduced translational activity. At the cellular level, mutations in RPS19 and RPS24 cause distinct cell cycle defects and reduced cell proliferation. Finally, PIM-1 kinase and RPS19 cooperates in the proliferation of myeloid cells.

Diamond Blackfan Anemia (DBA) is a congenital disorder characterized by a specific reduction in erythroid progenitor cells. Approximately 55% of patients have heterozygous mutations in ribosomal protein with 25% of these mutations in RPS19. However, it is unclear how a defect in ribosomal proteins specifically disrupts erythroid development. FLVCR1, a heme exporter, has been implicated as a potential DBA factor. FLVCR1 is essential for erythropoiesis as its disruption leads to apoptosis and disrupted erythroid differentiation. Though no FLVCR1 mutations have been found in DBA patients, our lab has shown that it is aberrantly spliced in DBA erythroid cells. Using RPS19 reduced K562 erythroid cells, I found that disruption of RPS19 leads to aberrant FLVCR1 splicing, disrupted erythropoiesis and reduced Tra2-β, ASF2 and SRp30c protein expression. This was specific to DBA as I did not find these features in a cell culture model of Shwachmann Diamond Syndrome, another ribosomal disorder.