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.

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.

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.

Diamond-Blackfan anemia (DBA) is a rare congenital syndrome that presents with ane- mia and selective deficiency of erythroid precursors, while other blood lineages are usu- ally unaffected. Approximately half of the patients display additional somatic anoma- lies and growth retardation. The therapy is mostly symptomatic and is dominated by corticosteroids, other modalities include regular blood transfusions or hematopoietic stem cell transplantation. At the beginning of this work, only two DBA causal genes were known, RPS19 and RPS24, being mutated in approximately 1/4 of all DBA patients. The goals of this work were to study the consequences of the known DBA causal mutations on cellular level and to find novel DBA causal genes. To date, over a half of DBA patients have been reported to carry a mutation in one of nine known DBA causal genes, including RPS17, RPL11 and RPL5, that are reported in this dissertation. All confirmed DBA causal genes encode for ribosomal proteins (RPs) that were essential for ribosome assembly. We further hypothesized a non- ribosomal protein participating in this process might be involved in DBA pathogenesis, too. In one DBA patient, we identified a rare sequence variant in one such candidate, a protein arginine methyltransferase 3 (PRMT3). We reported that the patient PRMT3...

Diamond-Blackfan anemia (DBA) is a rare congenital syndrome that presents with ane- mia and selective deficiency of erythroid precursors, while other blood lineages are usu- ally unaffected. Approximately half of the patients display additional somatic anoma- lies and growth retardation. The therapy is mostly symptomatic and is dominated by corticosteroids, other modalities include regular blood transfusions or hematopoietic stem cell transplantation. At the beginning of this work, only two DBA causal genes were known, RPS19 and RPS24, being mutated in approximately 1/4 of all DBA patients. The goals of this work were to study the consequences of the known DBA causal mutations on cellular level and to find novel DBA causal genes. To date, over a half of DBA patients have been reported to carry a mutation in one of nine known DBA causal genes, including RPS17, RPL11 and RPL5, that are reported in this dissertation. All confirmed DBA causal genes encode for ribosomal proteins (RPs) that were essential for ribosome assembly. We further hypothesized a non- ribosomal protein participating in this process might be involved in DBA pathogenesis, too. In one DBA patient, we identified a rare sequence variant in one such candidate, a protein arginine methyltransferase 3 (PRMT3). We reported that the patient PRMT3...

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The aim of this thesis is to investigate the effect of genetic mutations on the pathophysiology of two human disorders: Diamond-Blackfan Anemia (DBA) and isolated congenital nail dysplasia. The first part of this thesis (Paper I-III) investigates the mechanism associated with DBA. DBA is a rare bone marrow failure syndrome characterized by the absence or decrease of erythroid precursor cells. The disease is further associated with growth retardation, malformations, predisposition to malignant disease and heterozygous mutations in ribosomal protein (RP) genes. The second part of this thesis (Paper IV) investigates the genetic basis of isolated autosomal recessive nail dysplasia characterized by pachyonychia and onycholysis of both finger- and toenails. It further dissects the molecular mechanisms regulating nail development. In the first study, we investigated the previously reported RPS19/PIM-1 interaction by generating a combined Rps19/Pim-1 knockout mouse model. We found that allelic Rps19 insufficiency and Pim-1 deficiency have a cooperative effect on murine hematopoiesis resulting in increased myeloid cellularity associated with cell cycle alterations and reduced apoptosis. In the second study, we analyzed primary fibroblasts from DBA patients with truncating mutations in RPS19 or RPS24 and observed a marked delay in cellular growth associated with specific cell cycle defects. In the third study, we discovered that recombinant RPS19 binds its own mRNA and that the binding is altered when two DBA-associated RPS19 mutations are introduced. In the fourth study, we identified mutations in the WNT signaling receptor Frizzled 6 (FZD6). We observed that the nonsense mutant fails to interact with the first downstream effector Dishevelled. Fzd6 mutant mice displayed claw malformations and we detected a transient Fzd6 expression in the distal digits at the embryonic time point for nail development. In summary, this thesis elucidates several mechanisms in the etiology of DBA and congenital nail dysplasia and mechanisms regulating nail development.