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Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. Current therapies for DBA have undesirable side effects, including iron overload from repeated transfusions or infections from immunosuppressive drugs and stem cell transplantation. Nemo-like Kinase (NLK) is hyperactivated in erythroid progenitors in murine and human models as well as DBA patients. In an RPS19-insufficient human model, genetic silencing of NLK increased erythroid expansion by 2.2 fold, indicating that aberrant NLK activation contributes to disease pathogenesis. A high-throughput inhibitor screen identified a compound that inhibits NLK (IC50:440nM) and increased erythroid expansion in murine (5.4 fold) and human (6.3 fold) models of DBA with no effect on wild type erythropoiesis (EC50: 0.7 µM). Virtually identical results were observed in CD34+ progenitors from 3 DBA patient bone marrow aspirates with 2.3, 1.9 and 2.1 fold increases in CD235+ erythroblast generation. In erythroid progenitors, RPS19 insufficiency increased phosphorylation of the mTORC1 component Raptor, reducing mTOR activity by 82%. This was restored to basal levels upon inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTOR activity with leucine has been proposed to increase translational efficiency in DBA patients. Probably due to NLK phosphorylation of raptor, DBA patients did not respond as anticipated. While leucine treatment mildly increased mTOR activity in both control and RPS19-insufficiency, combining leucine with NLK inhibition increased mTOR activity to 142% of control and significantly improved erythroid expansion. Identification of aberrantly activated enzymes, such as NLK, offer therapeutic promise used alone, or in combination with existing therapies, as druggable targets in the clinical management of DBA.

Pearson marrow pancreas syndrome (PS) is a multisystem disorder caused by mitochondrial DNA (mtDNA) deletions. Diamond-Blackfan anemia (DBA) is a congenital hypoproliferative anemia in which mutations in ribosomal protein genes and GATA1 have been implicated. Both syndromes share several features including early onset of severe anemia, variable nonhematologic manifestations, sporadic genetic occurrence, and occasional spontaneous hematologic improvement. Because of the overlapping features and relative rarity of PS, we hypothesized that some patients in whom the leading clinical diagnosis is DBA actually have PS. Here, we evaluated patient DNA samples submitted for DBA genetic studies and found that 8 (4.6%) of 173 genetically uncharacterized patients contained large mtDNA deletions. Only 2 (25%) of the patients had been diagnosed with PS on clinical grounds subsequent to sample submission. We conclude that PS can be overlooked, and that mtDNA deletion testing should be performed in the diagnostic evaluation of patients with congenital anemia.

Classical 5q- syndrome is an acquired macrocytic anemia of the elderly. Similar to Diamond Blackfan anemia (DBA), an inherited red cell aplasia, the bone marrow is characterized by a paucity of erythroid precursors. RPS14 deletions in combination with other deletions in the region have been implicated as causative of the 5q- syndrome phenotype. We asked whether smaller, less easily detectable deletions could account for a syndrome with a modified phenotype. We employed single-nucleotide polymorphism array genotyping to identify small deletions in patients diagnosed with DBA and other anemias lacking molecular diagnoses. Diminutive mosaic deletions involving RPS14 were identified in a 5-year-old patient with nonclassical DBA and in a 17-year-old patient with myelodysplastic syndrome. Patients with nonclassical DBA and other hypoproliferative anemias may have somatically acquired 5q deletions with RPS14 haploinsufficiency not identified by fluorescence in situ hybridization or cytogenetic testing, thus refining the spectrum of disorders with 5q- deletions.

In a zebrafish mutagenesis screen to identify genes essential for myelopoiesis, we identified an insertional allele hi1727, which disrupts the gene encoding RNA helicase dead-box 18 (Ddx18). Homozygous Ddx18 mutant embryos exhibit a profound loss of myeloid and erythroid cells along with cardiovascular abnormalities and reduced size. These mutants also display prominent apoptosis and a G1 cell-cycle arrest. Loss of p53, but not Bcl-xl overexpression, rescues myeloid cells to normal levels, suggesting that the hematopoietic defect is because of p53-dependent G1 cell-cycle arrest. We then sequenced primary samples from 262 patients with myeloid malignancies because genes essential for myelopoiesis are often mutated in human leukemias. We identified 4 nonsynonymous sequence variants (NSVs) of DDX18 in acute myeloid leukemia (AML) patient samples. RNA encoding wild-type DDX18 and 3 NSVs rescued the hematopoietic defect, indicating normal DDX18 activity. RNA encoding one mutation, DDX18-E76del, was unable to rescue hematopoiesis, and resulted in reduced myeloid cell numbers in ddx18hi1727/+ embryos, indicating this NSV likely functions as a dominant-negative allele. These studies demonstrate the use of the zebrafish as a robust in vivo system for assessing the function of genes mutated in AML, which will become increasingly important as more sequence variants are identified by next-generation resequencing technologies.

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. The disease typically presents within the first year of life. Approximately 70% of DBA patients possess a mutations in one of at least 12 ribosomal proteins, with RPS19 and RPL11 being the most prevalent, accounting for over 25% and 5% of cases respectively. Current therapies for DBA have undesirable side effects, including iron overload from repeated transfusions or infections from immunosuppressive drugs and stem cell transplantation. Nemo-like Kinase (NLK) is chronically hyper-activated in RPS19- and RPL11-haploinsufficient murine and human models of DBA, as well as erythroid progenitors from DBA patients. In an RPS19-insufficient human model, genetic silencing of NLK (shRNA) increased erythroid expansion by 2.2 fold, indicating aberrant NLK activation contributes to the pathogenesis of the disease. In an independent, high-throughput kinase inhibitor screen examining progenitor expansion in RPS19-insufficient Kit+ murine cells, a number of compounds were identified that increased progenitor expansion. SB431542 and SD208 are recognized TGFβ inhibitor compounds, but were the only TGFβ inhibitors of a panel of 11 that increased progenitor expansion. Both active compounds robustly inhibited NLK activity in vitro and in vivo while the remaining 9 inhibitors had no significant impact on NLK. Both compounds increased erythroid expansion in murine (3.1 and 5.4 fold) and human (3.2 and 6.3 fold) models of DBA with no effect on wild type erythropoiesis (EC50 5 µM and 0.7 µM). No further increase in erythroid expansion was observed when NLK expression was silenced by shRNA. Virtually identical results were observed in CD34+ progenitors from 3 DBA patient bone marrow aspirates with 2.3, 1.9 and 2.1 fold increases in CD235+ erythroblast generation compared to untreated. NLK hyperactivation was limited to differentiating committed erythroid progenitors and was not detected in megakaryocytic, other myeloid progenitors or lymphoblastoid cells lines from DBA patients. During differentiation, non-erythroid lineages upregulate miR181, which results in NLK transcript degradation and loss of NLK expression. The absence of NLK in non-erythroid progenitors prevents NLK activation during ribosomal insufficiency. CRISPR/Cas9 mutation of the miR181 binding site in the NLK 3'UTR in RPS19-insufficient CD34+ HSPCs prevented NLK downregulation, increased NLK activity, and sensitized megakaryocyte and other myeloid lineages (80.5% and 76% reduction relative to controls). This is comparable to the erythroid expansion defect in RPS19-insufficiency (80.7% reduction). In differentiating erythroid progenitors, RPS19 insufficiency increased phosphorylation of the mTORC1 component Raptor (5.3 fold), reducing mTOR activity by 82%. This was restored to basal levels upon pharmacological or genetic inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTORC1 activity with leucine has been proposed to increase translational efficiency in DBA patients. Probably due to NLK phosphorylation of raptor, DBA patients did not respond as anticipated. While leucine treatment did increase mTOR activity in both control (100% to 188%) and RPS19-insufficiency (27 % to 42% of control), the combined treatment of leucine with NLK inhibition resulted in increased mTOR activity to 142% of control and significantly improved erythroid expansion. Identification of aberrantly activated enzymes, such as NLK, that are specifically expressed in erythroid progenitors, offer therapeutic promise as potential druggable targets in the clinical management of DBA that can be used in combination with existing therapies. Disclosures Glader: Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Porteus:CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Flygare:LU Holding: Patents & Royalties: Patent.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, congenital abnormalities, and cancer predisposition. Small ribosomal subunit genes RPS19, RPS24, and RPS17 are mutated in approximately one-third of patients. We used a candidate gene strategy combining high-resolution genomic mapping and gene expression microarray in the analysis of 2 DBA patients with chromosome 3q deletions to identify RPL35A as a potential DBA gene. Sequence analysis of a cohort of DBA probands confirmed involvement RPL35A in DBA. shRNA inhibition shows that Rpl35a is essential for maturation of 28S and 5.8S rRNAs, 60S subunit biogenesis, normal proliferation, and cell survival. Analysis of pre-rRNA processing in primary DBA lymphoblastoid cell lines demonstrated similar alterations of large ribosomal subunit rRNA in both RPL35A-mutated and some RPL35A wild-type patients, suggesting additional large ribosomal subunit gene defects are likely present in some cases of DBA. These data demonstrate that alterations of large ribosomal subunit proteins cause DBA and support the hypothesis that DBA is primarily the result of altered ribosomal function. The results also establish that haploinsufficiency of large ribosomal subunit proteins contributes to bone marrow failure and potentially cancer predisposition.

Diamond-Blackfan anaemia (DBA) is a congenital anaemia and broad developmental disease that develops soon after birth. The anaemia is due to failure of erythropoiesis, with normal platelet and myeloid lineages, and it can be managed with steroids, blood transfusions, or stem cell transplantation. Normal erythropoiesis after transplantation shows that the defect is intrinsic to an erythroid precursor. DBA is inherited in about 10-20% of cases, and genetic studies have identified mutations in a ribosomal protein gene, RPS19, in 25% of cases; there is evidence for involvement of at least two other genes. In yeast, RPS19 deletion leads to a block in ribosomal RNA biogenesis. The critical question is how mutations in RPS19 lead to the failure of proliferation and differentiation of erythroid progenitors. While this question has not yet been answered, understanding the biology of DBA may provide insight not only into the defect in erythropoisis, but also into the other developmental abnormalities that are present in about 40% of patients, and into the cancer predisposition that is inherent to DBA.

Diamond-Blackfan anemia (DBA) is a rare congenital pure red cell aplasia. Previous studies indicate that mutations of a gene on chromosome 19q13.2, which encodes a ribosomal protein, are responsible for 25% of cases. Recent investigations suggest both the presence of a second candidate region on chromosome 8p and non-19q, non-8p disease. In linkage analysis studies of 28 multiplex DBA families, we identified 8 families with disease linkage to chromosome 1q31. In 4 families, the disease linked exclusively to 1q31. Here, we report that the FLVCR gene on 1q31, which encodes a cytoplasmic heme exporter associated with red cell aplasia in cats, is not involved in DBA in these families.

Summary The genetic basis of Diamond–Blackfan anaemia (DBA), a congenital erythroid hypoplasia that shows marked clinical heterogeneity, remains obscure. However, the fact that nearly one-quarter of patients harbour a variety of mutations in RPS19, a ribosomal protein gene, provides an opportunity to examine whether haplo-insufficiency of RPS19 protein can be demonstrated in certain cases. To that end, we identified 19 of 81 DBA index cases, both familial and sporadic, with RPS19 mutations. We found 14 distinct insertions, deletions, missense, nonsense and splice site mutations in the 19 probands, and studied mutations in 10 patients at the RNA level and in three patients at the protein level. Characterization of the mutations in 10 probands, including six with novel insertions, nonsense and splice site mutations, showed that the abnormal transcript was detectable in nine cases. The RPS19 mRNA and protein in CD34+ bone marrow cells identified haplo-insufficiency in three cases predicted to have one functional allele. Our data support the notion that, in addition to rare DBA patients with the deletion of one allele, the disease in certain other RPS19 mutant patients is because of RPS19 protein haplo-insufficiency.

Diamond-Blackfan anemia (DBA) is a rare congenital hypoplastic anemia that usually presents early in infancy and is inherited in 10% to 20% of cases. Linkage analysis has shown that DBA in many of both dominant and recessive DBA families mapped to chromosome 19q13.2 leading to the cloning of a gene on chromosome 19q13.2 that encodes a ribosomal protein, RPS19. However, subsequently, mutations of the RPS19 gene have only been identified in 25% of all patients with DBA. This study analyzed 14 multiplex DBA families, 9 of which had 19q13.2 haplotypes inconsistent with 19q linkage. A genome-wide search for linked loci suggested the presence of a second DBA locus in a 26.4-centimorgan (cM) interval on human chromosome 8p. Subsequently, 24 additional DBA families were ascertained and all 38 families were analyzed with additional polymorphic markers on chromosome 8p. In total, 18 of 38 families were consistent with linkage to chromosome 8p with a maximal LOD score with heterogeneity of 3.55 at D8S277 assuming 90% penetrance. The results indicate the existence of a second DBA gene in the 26.4-cM telomeric region of human chromosome 8p23.3-p22, most likely within an 8.1-cM interval flanked by D8S518 and D8S1825. Seven families were inconsistent with linkage to 8p or 19q and did not reveal mutations in the RPS19 gene, suggesting further genetic heterogeneity. (Blood. 2001;97:2145-2150)

Congenital hypoplastic or Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder characterized by a paucity of red blood cells and their precursors in the bone marrow. The majority of cases of DBA are due to haploinsufficient mutations in ribosomal protein genes and in rare cases result from GATA1 mutations. However, nearly half of the DBA cases do not have an identified genetic etiology. While analyzing whole exome sequencing data from a cohort of over 450 patients with a clinical diagnosis of DBA, we encountered the case of a male child of a first cousin consanguineous union who was diagnosed with DBA as an infant and remained transfusion dependent. The patient responded to corticosteroid therapy for a year as a toddler, but this treatment was discontinued due to side effects. The patient subsequently remained transfusion dependent and at 6 years of age an allogeneic bone marrow transplant from a matched maternal aunt was performed. Surprisingly, despite achievement of robust donor chimerism, the patient remained transfusion dependent. Unfortunately the patient developed severe graft-versus-host disease and died of resultant complications. Both the potential recessive nature of the mutation, given parental consanguinity, and the lack of anemia correction following transplant made this case extremely unusual. Thus we evaluated this patient's whole exome sequencing data. We identified a homozygous recessive mutation in the erythropoietin gene (EPO), which resulted in an R150Q substitution in the mature EPO protein. This mutation was absent from a cohort of 60,706 individuals depleted for Mendelian disease and fit the model of complete penetrance in the family. The R150Q mutation was expected to disrupt the high-affinity binding site to the EPO receptor (EPOR). However, we found by producing recombinant proteins that the EPO R150Q mutation only reduced the EPOR binding affinity by 3-fold. Surprisingly, the patient had an over 100-fold elevation in their serum EPO levels, suggesting that this mutation did not cause disease through altered affinity. Rather we observed altered EPOR binding kinetics by this mutant ligand. There was a slightly increased on-rate with a much faster dissociation rate (t1/2 of 10 seconds for the mutant vs. 6 minutes for the wild type). Using human erythroid cells and primary hematopoietic stem and progenitor cells, we could show that this mutant ligand never reached the same efficacy as the wild type (WT) EPO in promoting erythroid differentiation and proliferation. To better characterize this abnormal activity, we examined downstream signaling responses. We found identical phosphorylation of STAT5 at maximally potent concentrations of the WT (1 nM) and R150Q mutant (100 nM) EPO. By surveying a broad array of >120 phosphorylation events using intracellular flow cytometry, we demonstrated that maximal levels of STAT3 and STAT1 phosphorylation were reduced by 30% and 25%, respectively, with the R150Q (100 nM) compared to WT (1 nM) EPO. To determine the mechanistic basis for variation in downstream effector activation by the R150Q mutant ligand, we used inhibitors of both the JAK2 kinase and the SHP1/2 phosphatases that are respectively up- and downstream of STAT phosphorylation. While SHP1/2 inhibition did not alter STAT phosphorylation, JAK2 inhibition by ruxolitinib more potently inhibited STAT1/3 phosphorylation compared to STAT5. Interestingly, treatment with a low dose of ruxolitinib (40 nM) reduced erythroid proliferation to the same extent at maximally potent concentrations of the WT or R150Q EPO, demonstrating that the impairment in signaling by the R150Q EPO was primarily due to reduced JAK2 activity. Finally, we utilized single molecule fluorescent imaging of EPOR dimerization at the intact cell surface to directly show that the kinetically-biased R150Q EPO has a reduced ability to promote productive dimerization as compared to the WT EPO, even at maximally potent concentrations. Collectively, our results demonstrate how the R150Q mutant EPO - the first pathogenic mutation in EPO identified in humans - results in biased agonism of EPOR signaling through reduced receptor dimerization and consequently impaired JAK2 activation. More broadly our findings reveal how variation of cytokine-receptor binding kinetics can be used to tune downstream responses, which has broad implications for modulating the activity of numerous hematopoietic cytokines. Disclosures No relevant conflicts of interest to declare.

Diamond Blackfan anemia is a rare congenital hypoplastic anemia that usually presents early in infancy. Congenital anomalies, in particular of the head and upper limbs, are present in about 25% of reported patients. The disease is characterized by a moderate to severe macrocytic anemia, occasional neutropenia or thrombocytosis, a normocellular bone marrow with erythroid hypoplasia, and an increased risk of developing leukemia. Recent genetic studies have led to the identification of mutations in the ribosomal protein RPS19 in approximately 25% of sporadic and familial cases, a second gene on chromosome 8p, and evidence for an additional locus (or loci). The pathogenesis is unknown. The majority of patients respond to prednisone, and often erythropoiesis can be maintained with low doses of the drug. Both remissions and increased resistance to steroid treatment can occur. Patients who do not respond to treatment are usually transfusion dependent, although responses to high dose steroid, androgen, and interleukin-3 have been observed. Bone marrow transplantation can be curative.

Chronic constitutional aregenerative anemia with absent or decreased red cell precursors in bone marrow. Macrocytosis elevated fetal hemoglobin and increased eADA. Physical abnormalities in about 40% of DBA cases including craniofacial and thumb abnormalities, atrial or ventrucular septal defects, short stature, mild retardation, etc. Hematologic malignancy: in 2.5% of all reported cases of DBA; primarily ANLL with no FAB preference but also ALL, Hodgkin's disease. Solid tumors include carcinoma of liver, stomach, osteogenic sarcoma. Age of malignancy onset from 2 to 43 years. Disease-related and treatment-related factors, i.e., allosensitization and iron overload, contribute to malignancy.

Mutations in the RPS19 gene have been identified in 25% of individuals affected by Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia characterized by an aregenerative anemia and a variety of malformations. More than 60 mutations in the five coding exons of RPS19 have been described to date. We previously reported a mutation (c.-1 + 26G>T) and an insertion at -631 upstream of ATG (c.-147_-146insGCCA) in the noncoding region. Because DBA phenotype is extremely heterogeneous from silent to severe and because haploinsufficiency seems to play a role in this process, it is likely that genetic variations in the noncoding regions affecting translation of RPS19 can modulate the phenotypic expression of DBA. However, to date, very few studies have addressed this question comprehensively. In this study, we performed detailed sequence analysis of the RPS19 gene in 239 patients with DBA and 110 of their relatives. We found that 6.2% of the patients with DBA carried allelic variations upstream of ATG: 3.3% with c.-1 + 26G>T; 2.5% with c.-147_-146insGCCA; and 0.4% with c.-174G>A. Interestingly, the c.-147_-146insGCCA, which has been found in a black American and French Caribbean control population, was not found in 500 Caucasian control chromosomes we studied. However, it was found in association with the same haplotype distribution of four intronic polymorphisms in our patients with DBA. Although a polymorphism, the frequency of this variant in the patients with DBA and its association with the same haplotype raises the possibility that this polymorphism and the other genetic variations in the noncoding region could play a role in DBA pathogenesis.

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome linked to mutations in ribosomal protein (RP) genes that result in the impaired proliferation of hematopoietic progenitor cells. The etiology of DBA is not completely understood; however, the ribosomal nature of the genes involved has led to speculation that these mutations may alter the landscape of messenger RNA (mRNA) translation. Here, we performed comparative microarray analysis of polysomal mRNA transcripts isolated from lymphoblastoid cell lines derived from DBA patients carrying various haploinsufficient mutations in either RPS19 or RPL11. Different spectrums of changes were observed depending on the mutant gene, with large differences found in RPS19 cells and very few in RPL11 cells. However, we find that the small number of altered transcripts in RPL11 overlap for the most part with those altered in RPS19 cells. We show specifically that levels of branched-chain aminotransferase-1 (BCAT1) transcripts are significantly decreased on the polysomes of both RPS19 and RPL11 cells and that translation of BCAT1 protein is especially impaired in cells with small RP gene mutations, and we provide evidence that this effect may be due in part to the unusually long 5'UTR of the BCAT1 transcript. The BCAT1 enzyme carries out the final step in the biosynthesis and the first step of degradation of the branched-chain amino acids leucine, isoleucine, and valine. Interestingly, several animal models of DBA have reported that leucine ameliorates the anemia phenotypes generated by RPS19 loss. Our study suggests that RP mutations affect the synthesis of specific proteins involved in regulating amino acid levels that are important for maintaining the normal proliferative capacity of hematopoietic cells.

Diamond-Blackfan anemia (DBA) is a congenital red blood cell aplasia inherited in an autosomal dominant pattern caused by mutations in ribosomal protein (RP) genes and in an X-linked recessive pattern by GATA1 mutations. Heterozygous mutations and large deletions in 11 RP genes, RPS19, RPS24, RPS17, RPL5, RPL11, RPL35A, RPS7, RPS10, RPS26, RPL26, and RPL15, are present in ∼65% of DBA patients. DBA is associated with congenital abnormalities in ∼50% of patients and with increased risk of malignancy. To investigate the molecular pathogenesis of RPL5 and RPS24 gene mutations, we generated two murine lines of heterozygous mice, Rpl5 and Rps24, by knocking-out exons 1-8 in the Rpl5 gene and exons 2-3 in the Rps24 gene in C57BL/6 mice. Knock-out of both alleles of Rpl5 and Rps24 genes are embryonic lethal. In contrast, heterozygous mice exhibited normal hematological phenotype, as well as normal Rpl5 and Rps24 RNA and protein levels in their tissues, suggesting that the presence of one allele was sufficient to support the normal function of ribosomal proteins L5 and S24 in mice. To evaluate the risk of cancer development in Rpl5 +/- and Rps24 +/- mice, we monitored these mice and wild type mice until late age. Out of 21 Rpl5 +/- mice (between the ages of 14 and 26 months), two mice developed tumors at 22 months of age and two mice were euthanized due to severe dermatitis at the same age. Similarly, we have been monitoring 23 Rps24 +/- mice between 15 and 26 months of age. One of these mice developed a tumor at 17 months of age, five mice were euthanized due to severe dermatitis between the ages of 17 and 19 months, and two mice were euthanized due to injuries at ages 15 and 29 months. We also monitored 20 control wild-type mice ranging from 13 to 26 months of age. To this date, no tumors have been detected in wild-type mice, although nine of these mice developed severe dermatitis and were euthanized. Histological and immunohistochemical studies were performed to determine the nature of tumors in Rpl5 +/- and Rps24 +/- mice. Comparison of tumor tissues with normal skin from wild-type or Rpl5 +/- and Rps24 +/- with no detected tumors showed that all tissues had normal epidermis and underlying dermis, but connective tissues from tumor sections consisted of a densely cellular neoplasms composed of predominantly atypical spindle shaped cells arranged in intersecting fascicles. The tumor cells had strong cytoplasmic reactivity for vimentin and negative staining for S100, CD45, and pan-keratin, consistent with a high-grade spindle cell sarcoma. Recent studies conducted by the DBA Registry of North America revealed that out of 608 DBA patients, 18 with median age of 41 years developed various types of cancer including sarcomas, colon cancer, and acute myeloid leukemia. The relative risk of cancer in DBA was increased 5.4 fold compared to general population (Vlachos, et al., 2012). Our studies also suggest the correlation between ribosomal protein gene mutations and cancer. However, further studies are required to better understand the underlying molecular mechanism. Disclosures: No relevant conflicts of interest to declare.

Pearson marrow pancreas syndrome (PS) is a congenital multisystem disorder characterized by sideroblastic anemia, pancreatic insufficiency, metabolic acidosis, and other defects, and is caused by mitochondrial DNA (mtDNA) deletions. Diamond Blackfan anemia (DBA) is a congenital hypoproliferative anemia with associated physical malformations, and in which mutations in ribosomal protein (RP) genes and GATA1 have been implicated. The clinical presentation of both of these bone marrow failure (BMF) syndromes shares several features including early onset of severe anemia, sporadic genetic inheritance, variable penetrance and manifestations, and episodes of spontaneous hematologic improvement. PS is less frequently occurring than DBA, with estimated incidences of < 1/1,000,000 versus 1/100,000 respectively, and therefore less often encountered by hematologists. We hypothesized that some patients in whom the leading clinical diagnosis is DBA actually have PS. To test this hypothesis, we retrospectively evaluated DNA samples from a cohort of patients that were submitted to a research study for DBA genetic testing. The study cohort consists of clinical samples and/or data from 362 patients, with a primary inclusion criterion of known or suspected congenital anemia. Prior genetic studies from this cohort have yielded the novel identification or confirmation of mutations and deletions in several genes implicated in DBA (e.g. RP genes, GATA1), which are to date identifiable in 175/362 samples (48%), a proportion consistent with that found in independent DBA registries. We screened peripheral blood DNA samples available from 173 genetically uncharacterized patients using a long PCR strategy, and found that 8 samples (4.6%) contained large mtDNA deletions. Deletion mapping and Southern blot analysis on DNA from these 8 patients confirmed the presence of a single deletion event within each patient, ranging in size from 2.3 - 7.0 kb of the 16.6 kb mitochondrial genome, existing as monomer or multimer mtDNA species, and in a proportion ranging from 55-80% of total mtDNA, all of which are consistent with the molecular diagnosis of PS. Follow-up with referring providers in the 1 month to 8 year time span since sample submission revealed that 2 of the 8 patients (25%) were subsequently diagnosed with PS. Of the remaining 6 undiagnosed patients, 2 had died from complications of bone marrow transplantation, performed for worsening cytopenias and concern for myelodysplasia; one patient died from bacterial sepsis; and 3 were alive with the provisional diagnosis of DBA. One of the 3 patients had become transfusion-independent. Review of bone marrow examinations revealed that the pathological hallmarks of ringed sideroblasts and/or vacuolization of precursors described in PS were inconsistently present or reported in the diagnostic evaluation. We conclude that PS is frequently overlooked in the diagnostic evaluation of children with congenital anemia. Establishing the diagnosis of PS, as distinct from DBA and other BMF disorders, holds important implications for patient management and family counseling. mtDNA deletion testing should be performed in the initial genetic evaluation of all patients with congenital anemia. Disclosures: Szczepanski: Octapharma AG: Investigator Other.

Background: DBA is a rare genetic disorder characterized by faulty ribosome biogenesis, leading to pro-apoptotic erythropoiesis and red cell failure. The Diamond Blackfan Anemia Registry (DBAR) was established in 1993 to provide a robust database for investigating the biology and epidemiology of DBA. The DBAR defines remission in DBA as independence from red cell transfusion or corticosteroid therapy for greater than 6 months. An understanding of factors influencing DBA remission may provide insights into the pathophysiology of DBA and ultimately can lead to improved treatment options. Method: Patients were enrolled in the DBAR with informed consent. Patients who met the remission criteria for this study were evaluated. Patients who reported a remission were contacted and completed a “remission questionnaire”; further information was obtained from the patients’ physicians and verified, where possible, from medical records. Results: Of 555 patients enrolled in the DBAR, 67 patients have experienced a remission (actuarial likelihood approximately 20%). The male: female ratio for remission patients vs. the total DBAR population is 1:0.97 vs. 1:1.03, respectively. Sixty-four percent of patients have congenital anomalies, compared to 47% in the total population; the difference is not statistically significant. All categories of congenital anomalies (e.g. orofacial, cardiac, renal, skeletal, etc.) are represented in the remission population. The median age at diagnosis is 2.9 months (range, 0 to 14.9 years). The majority of patients were started on 2mg/kg/day of prednisone or methylprednisolone at a median age of 3.6 months (range, 4 days to 15.2 years). Sixty-six percent entered remission while on steroid therapy. The median total duration on steroids was 36 months (range, 1 month to 37.6 years) and the median duration from start of steroids to beginning of taper was 3.6 months (range, 1 month to 4.6 years). Seventeen percent of patients went into remission with less than one year of steroids and 55% were in remission within 5 years of start of steroids. Fifteen percent remitted while receiving chronic transfusion therapy. Six percent never received steroids prior to remission and 7.5% of remitters initially responded to steroids but became steroid refractory prior to remission. Median duration of treatment to remission and duration of remission were 16 months (range, 6 months to 38.2 years) and 14 years (range, 1 to 46.8 years) respectively. The median age of remission was 6.4 years (range, 0.8 to 39 years); males 6.5 years (range, 0.8 to 39 years) and females 6.3 years (range, 1.1 to 26 years). Of note, remissions were observed in DBA patients or in affected family members of probands with mutations in 4 of the 6 genes (RPS 19, RPS 24, RPL35a, and RPS11) known to be mutated in DBA as well as in those with no known mutation, suggesting that remission is not restricted to a particular genotype. Additional patients are being genotyped. Conclusion: Remission in patients with DBA is not an uncommon event. Steroid responsiveness is not a prerequisite for remission. There is no obvious phenotypic or genotypic difference between remission and non-remission patients. The expression of a remission phenotype within multiplex families is quite variable. We conclude that remission is not restricted to a particular phenotype or genotype and that the likelihood of remission is influenced by unknown modifier genes and/or epigenetic factors.

Diamond-Blackfan anemia (DBA), a form of congenital red cell aplasia with marked clinical heterogeneity and increased risk of malignancy, has been associated with mutations in ribosomal protein (RP) gene RPS19 in 25% of probands and in RPS24 or RPS17 in ∼2% of patients. Thus, DBA appears to be a disorder of ribosome synthesis. To test the hypothesis that mutations in other RP genes may also cause DBA, we carried out direct sequencing of candidate RP genes. Genomic DNA samples from 96 unrelated DBA probands (14 familial and 82 sporadic cases) without RPS19 or RPS24 mutations were screened for mutations in RPS3a, RPS13, and RPS16 (previous studies revealed that RPs S19, S24, S3a, S13, and S16 are involved in binding of eIF-2 to the 40S subunit); RP genes L18, L13A, L36, L28, L18A, L40, S5, S9, S11, and S28 (located on chromosome 19); and RP genes, L5, L11, L22, S8, and S27 (on chromosome 1). PCR primers were designed to amplify the coding exons and intron/exon boundaries. We found multiple mutations in two RP genes, L5 and L11. Subsequently we sequenced these two genes in 42 additional DNA samples from DBA probands. In total, we screened 5′UTR, promoter and coding regions, and exon/intron boundaries of RPL5 and RPL11 in 138 DBA unrelated probands. We identified 14 mutations in RPL5 in 138 probands (∼10%), 13 of which are nonsense mutations, deletions or insertions of 1–5 nucleotides causing frameshift and premature termination. One missense mutation, 418G>A, results in a G140S substitution. We found nine mutations in RPL11 in138 DBA probands (6.5%), including five acceptor or donor splice site mutations (introns 1–4) and four deletions or insertions of 1–4 nucleotides causing frameshifts (codons 32-120). None of these sequence changes were found on the NCBI (http://www.ncbi.nlm.nih.gov/SNP/) or the HapMap (http://www.hapmap.org/) SNP lists. Both genes, as well as RPL23 have recently been demonstrated by others to activate the p53 tumor suppressor protein by inhibiting MDM2-mediated p53 ubiquitination and degradation. Moreover, knockdown of any of these genes by siRNAs markedly reduced p53 induction by the ribosomal biogenesis stressor, actinomycin-D. These findings suggest that DBA patients with mutated L5 and L11 proteins may have inadequate p53 pathway activation and (consistent with clinical observations) be at increased risk for neoplasia. We are currently investigating the role of RPL5 and RPL11 mutations in ribosomal biogenesis and in the p53-mediated cell cycle arrest and apoptosis in DBA patients.

DBA is an inherited bone marrow failure syndrome characterized by hypoproliferative anemia, congenital abnormalities and cancer predisposition. Ribosomal genes RPS19 and 24 are mutated in 25% and 3% of DBA patients respectively. To identify additional genetic abnormalities in DBA, we evaluated 2 unrelated children with DBA and sub-telomeric deletions of chromosome 3q by comparative genomic hybridization. The larger deletion spanned 11 Mb from 3q28 to the telomeric region and included 72 gene candidates. The second deletion involved 4 Mb from 3q29 to the telomeric end and included 52 known or hypothetical genes. The overlapping deletion region contained a previously reported 1.5 Mb microdeletion-associated syndrome that did not involve hematologic abnormalities, leaving 24 candidate genes. Gene expression microarray analysis from patient-derived EBV cell lines demonstrated down regulation of 7 of these candidate genes, one of which was RPL35a, a component of the large ribosomal subunit. We screened for mutations of RPL35a by direct sequencing of PCR-amplified genomic DNA from 149 DBA probands (125 sporadic, 24 familial) and 180 normal control subjects. We identified three probands with sequence changes in the RPL35a coding region: 1) an in-frame deletion in exon 3 (82-84CTT), causing a deletion of leucine at codon 28, 2) a nonsense mutation in exon 4 (298C>T), leading to an Arg102Stop and a 9 amino acid C-terminal truncation and 3) a missense mutation in exon 3 (97G>A) leading to a Val33Ile change. In the patient derived EBV cell line, the latter sequence change also resulted in an aberrant exon 3 splice site leading to a frame shift following codon 32. All of the probands with RPL35a mutations were sporadic cases. These sequence variations were not observed in the control subjects. Four lentiviral-based siRNA constructs targeting RPL35a were used to test the functional consequences of reduced RPL35a expression. Hematopoietic cell lines (TF-1 and UT-7/epo) transduced with the RPL35a directed siRNA constructs demonstrated decreased growth and viability compared to control siRNAs. Northern blot analysis demonstrated abnormal processing of large ribosomal subunit RNA with decreased mature 5.8S and 28S as well as decreased precursor 12S and 32S rRNA. Orthophosphate labeling confirmed a kinetic defect in large subunit rRNA processing, characterized by increased amounts of 45S and 41S rRNA with decreases of the precursors to and the mature 28S and 5.8S rRNAs. Mature 18S rRNA levels were unaffected, suggesting a defect in rRNA processing within the first internal transcribed sequence (ITS1). These data demonstrate that DBA can be caused by alterations in large as well as small ribosomal subunit proteins. These observations further support the hypothesis that altered ribosome homeostasis and function, rather than extra-ribosomal gene functions, is the central mechanism leading to DBA.

Diamond-Blackfan anemia (DBA) is a broad developmental disease, characterized by anemia, bone marrow erythroblastopenia, physical anomalies, and an increased incidence of malignancy. Ribosomal protein S19 gene (RPS19) is mutated in approximately 25% of DBA probands. However, its role in the pathogenesis of DBA remains to be determined. Using global gene expression analysis (Affymetrix HG-U133A chips, >22,000 probe sets) we compared highly purified multipotential (P) (CD34+CD71−CD45RA−) erythroid (E) (CD34+CD71hiCD45RA), and myeloid (M) (CD34+CD71lowCD45RA+) bone marrow progenitors from three RPS19 mutated and six control samples. For statistical analysis we applied Geometric Fold Change Analysis and Significance Analysis of Microarrays. As we have previously shown the highest number, 545, of significantly changed genes (≥2 fold up- or down-regulated), was identified in diseased vs control E progenitors, while only 106 and 72 genes were dysregulated in P and M progenitors, respectively. In addition to 10 ribosomal protein genes down-regulated in DBA samples, we found several genes involved in translation, including EIF5B, EIF2C2, EEF1D and EEF1E1 significantly under-expressed in diseased erythroid progenitors. Quantitative real-time PCR revealed the expression of 18S rRNA 3.5 to 7-fold up-regulated in the DBA P populations, 1.5–4-fold in the E populations, and unchanged in the M populations. We also found transcriptional control genes TAF9L, TAF12, TCF3, NFYA, ELYS, NFYB and CNOT8, greatly down-regulated mostly in the DBA E populations. In addition we found the erythroid transcription factor, c-Myb, 6-fold down-regulated in the DBA E populations. Importantly, we identified 29 cancer-related genes, oncogenes and tumor suppressor genes, including RAB2, RABL4, RAB20, RAB21, RB1 and PHB significantly dysregulated in the P, E or M DBA populations. We also studied the relationship between P/E, P/M and E/M populations separately in the diseased and control samples. This analysis revealed 3,846 genes ≥2-fold changed between diseased E and P populations (485 in control P/E) while the number of dysregulated genes between diseased P/M and E/M were 1,660 and 1,042, respectively, (controls 330 and 378, respectively). Our data show that at the molecular level, erythroid progenitors are the most affected in DBA. Identification of expression changes for multiple cancer-related genes suggests a molecular basis for the increased risk for malignancy in these patients. The results suggest that RPS19 mutation and RPS19 protein insufficiency in DBA patients leads to impairment of ribosomal biogenesis by dysregulated stoichiometry of ribosomal components and subsequent reduction of protein translation. However, it is also possible that specific targets such as c-MYB are affected through an extra-ribosomal role of RPS19. Since disruption of c-Myb is characterized by complete failure of fetal erythropoiesis, our data suggest a link between RPS19 mutations and reduced erythropoiesis in DBA.

Diamond-Blackfan anemia is usually characterized by anemia, absence or insufficiency of erythroid precursors in bone marrow, growth retardation and diverse congenital anomalies that are present in approximately half of patients, indicating that DBA is a broad disorder of development. Mutations of RPS19 are found in approximately 25% of DBA patients. There is good evidence for a second DBA gene, located on chromosome 8, and further genetic heterogeneity of the disease is likely. The aim of this study is to determine the most disturbed molecular pathways in DBA patients, based on gene expression changes in bone marrow cells. Knowing these pathways will possibly enable us to decipher the pathogenic mechanisms of DBA and find other genes involved in the disease. Bone marrow cells from 6 normal individuals and 3 DBA patients with RPS19 mutations, currently in remission, were FACS separated into 3 populations: primitive (P), erythroid (E) and myeloid (M) containing CD34+CD71-CD45RA-, CD34+CD71hiCD45RA- and CD34+CD71lowCD45RA+ cells, respectively. The purity of each sorted population was >97%. As a control for cell sorting accuracy, methylcellulose assay demonstrated that the P populations were highly enriched in primitive BFU-E and CFU-GEMM colonies, the E populations gave rise to BFU-E and CFU-E colonies in more than 90% of the CFCs, while more than 99% colonies from M populations were CFU-G, CFU-M and CFU-GM. RNA targets from these three FACS sorted cellular subsets was hybridized to Affymetrix HG-U133A chips (>22,000 probe sets). The data from all 27 samples were analyzed by hierarchical clustering and Principal Component Analysis, and each cell population was also studied separately. All pairwise comparisons among 27 datasets showed correlations with r=0.86–0.99. Hierarchical clustering identified three major specimen clusters, perfectly overlapping with the three different cell populations under study. Principal Component 1 and 2 separated the three studied subgroups P, E, and M. In each cell population analysis, 3 patient samples were compared to 6 control samples using 1)Significance Analysis of Microarrays with fold change 2 or greater and false discovery rate 1%, 2)Geometric Fold Change analysis and 3)Filter on Fold Change GeneSpring application (arithmetic analysis). All fold change analyses revealed the most significantly changed transcripts in patients vs. control individuals in E (45 upregulated and 184 downregulated) and P populations. The most changed genes in E subgroup were apoptosis related genes, namely TNFRSF10B and TNFRSF6 (CD95/Fas), upregulated in patients 10 and 3 fold, respectively. Other most changed genes were cancer related and genes involved in developmental processes and nucleic acid binding. Additionally, several ribosomal protein genes, namely RPL10L, RPL28, RPL36, RPL13, RPL27a and RPL37a were significantly underexpressed in P and E populations of DBA patients. All three analyses showed that RPL10L, RPL28 and RPL36 are underexpressed in the M population. This finding indicates that ribosomal protein genes are closely co-regulated and that RPS19 protein abnormalities result in downregulation of the additional ribosomal protein genes in both erythroid and nonerythroid cells in DBA patients.

Diamond-Blackfan Anemia (DBA) is a rare congenital hypoplastic anemia that usually presents early in infancy and is inherited in 10–20% of cases. Recently, identification of a patient with a translocation t(X;19)(p21;q13) led to the cloning of a gene on chromosome 19q13.2 that encodes a ribosomal protein, RPS19. However, mutations of RPS19 have only been identified in 25% of 19q-linked and sporadic cases, suggesting a greater degree of genetic heterogeneity in DBA we carried out linkage analysis and searched for RPS19 mutations in 38 multiplex families. Although approximately 40% of families were consistent for linkage to the 19q13.2 region, mutation analysis revealed mutations in only 3 families. Three different missense mutations were identified; 2 of them, in exon 4 (G185A;R62Q) and in exon 5 (T395G;L131R), are novel mutations, while the third, in exon 3 (G171A;R56Q) has been described. We excluded these kindreds from further linkage analysis. A genome-wide search for loci linked to the DBA phenotype suggested the presence of a second DBA locus on chromosome 8p. To define the critical interval more precisely we used additional polymorphic markers on chromosome 8p and applied GENEHUNTER program for calculation of parametric two-and multipoint LOD scores with heterogeneity (HLOD). A significant HLOD score (higher than 3.3 with 49% families linked to the locus) was found for the 8.1cM 8p telomeric region flanked by D8S518 and D8S1825. Six of 35 families were not consistent for linkage either to 19q or 8p, which strongly suggested further genetic heterogeneity. In conclusion, our results show evidence of a second DBA gene on 8p 23.3-22, most likely within an 8.1cM interval flanked by D8S1825 and D8S518, and also provides further support for the existence of at least one additional gene locus.

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia with marked clinical heterogeneity, an increased risk of malignancy and mutations in ribosomal protein (RP) S19 in 25% of probands. To identify other gene(s) mutated in DBA and investigate their expression and function, we performed a genome-wide screen using a 10,000 single nucleotide polymorphism mapping set (Affymetrix) on a large family comprising 10 informative meioses. We found linkage of the DBA phenotype to regions on chromosome 8q, 10 and 6. The RP gene RPS24, is located in the linked region on chromosome 10; we sequenced exons, intron-exon boundaries and the promoter regions in this family, and found a nonsense mutation (316C>T) in exon 4 of RPS24 in five affected individuals, and a wild type sequence in five unaffected family members. This mutation causes the change of Gln106STOP and is predicted to result in formation of a truncated RPS24 protein. Subsequently, we sequenced DNA from 215 unrelated DBA probands, 30 with RPS19 mutations and 185 without. We found another nonsense mutation in exon 2 in a sporadic case, and a splice site deletion resulting in skipped exon 2 in another proband and in his father; over 200 control individuals did not have any of the above sequence changes, indicating that they are pathogenic mutations. To explore the normal role of RPS24 and consider how its dysfunction might result in DBA we performed real time RT-PCR (rt-PCR) and western blotting experiments on 20 normal human tissues and on lymphoblastoid cell lines from diseased and control individuals. Interestingly, rt-PCR of total human RPS24 and RPS19 mRNA revealed a tissue-specific variation in expression level. We found co-ordinate expression of both genes in the majority of studied tissues. Lymphoblastoid cell lines from both probands with nonsense mutations showed a reduced level of RPS24 mRNA, suggesting degradation of mutated transcripts due to nonsense mediated decay, while the RPS19 mRNA level in these patients was normal or elevated. Western blot experiments revealed a reduction of RPS24 protein in lymphoblastoid cell lines from all three mutated probands compared to control samples. Interestingly, co-ordinate expression of RPS24 and RPS19 protein was found in these patients as well as in other patients with RPS19 mutations or without any mutations, suggesting co-regulation of RP expression. To determine whether recruitment of mRNA to polysomes was impaired in DBA patients, we separated lymphoblast cell line lysates from nine diseased and four control individuals on sucrose gradients. We did not detect any significant difference in the RNA ratio of polysome-bound/free ribosomal subunits between diseased and control samples (p In summary, our results suggest that in addition to RPS24 and RPS19, which are mutated in ~ 27% of index cases, other DBA genes are also RP genes or genes involved in ribosome biogenesis or translation, and reinforce the notion that DBA is a ribosomal disease. This study opens new avenues for studying and understanding the pathogenesis of DBA.

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.

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.

Mutations in ribosomal protein S19 (RPS19) gene have been found in 25% of patients affected with Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. We have previously shown that several RPS19 mutant proteins (V15F, InsAG36, W33stop, Y48stop, R56stop, M75stop, R94stop, 274del31, InsG238, G127Q and L131P) exhibit decreased levels of protein expression and do not localize to the nucleolus like the wild type protein in transfected Cos-7 cells. In contrast, other mutants (W52C, T55M, R56Q, R62W, 24Del18, G120S) exhibit normal levels of protein expression and normal nucleolar localization. We hypothesized that decreased levels of expression of mutant proteins such as V15F, G127Q, and L131P may be due to proteosomal degradation. In order to validate our hypothesis, we analyzed the effects of two proteasome inhibitors (MG132 and lactacystin) on mutant RPS19 protein expression levels and their subcellular localization. Following treatment with proteosome inhibitors, the mutant proteins with missense mutations (V15F, G127Q and L131P) were expressed at levels similar to that of wild type protein and localized in the nucleolus. Similarly, proteasome inhibitors also restored the expression levels and normal subcellular localization to RPS19 with non-sense mutations (InsG238, R94stop, and 274del31) that resulted in the translation of RPS19 protein with at least 80 aminoacids. In marked contrast, proteosome inhibitors failed to restore the expression levels of RPS19 with the non-sense mutants that led to synthesis of shortened proteins (InsAG36, W33stop, Y48stop, R56stop, M75stop). Even in the presence of proteosome inhibitors we noted a dramatic decrease in the levels of expression of these mutant proteins and proteins expressed were localized in the cytoplasm. Our findings imply an important role for proteosomal degradation pathway in regulating the expression levels of RPS19. They further suggest that proteasome inhibitors could be considered as a potential treatment for some steroid resistant DBA affected individuals with specific RPS19 mutations.