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Studies of allelic variation underlying genetic blood disorders have provided important insights into human hematopoiesis. Most often, the identified pathogenic mutations result in loss-of-function or missense changes. However, assessing the pathogenicity of noncoding variants can be challenging. Here, we characterize two unrelated patients with a distinct presentation of dyserythropoietic anemia and other impairments in hematopoiesis associated with an intronic mutation in GATA1 that is 24 nucleotides upstream of the canonical splice acceptor site. Functional studies demonstrate that this single-nucleotide alteration leads to reduced canonical splicing and increased use of an alternative splice acceptor site that causes a partial intron retention event. The resultant altered GATA1 contains a five–amino acid insertion at the C-terminus of the C-terminal zinc finger and has no observable activity. Collectively, our results demonstrate how altered splicing of GATA1, which reduces levels of the normal form of this master transcription factor, can result in distinct changes in human hematopoiesis.

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 1 in 100,000 to 200,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this genetically heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole exome sequencing (WES). Overall, we identified rare and predicted damaging mutations in likely causal genes for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and in one of 19 previously reported genes encoding for a diverse set of ribosomal proteins (RPs). Using WES exon coverage estimates, we were able to identify and validate 31 deletions in DBA associated genes. We also observed an enrichment for extended splice site mutations and validated the diverse effects of these mutations using RNA sequencing in patientderived cell lines. Leveraging the size of our cohort, we observed several robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. In addition to comprehensively identifying mutations in known genes, we further identified rare mutations in 7 previously unreported RP genes that may cause DBA. We also identified several distinct disorders that appear to phenocopy DBA, including 9 individuals with biallelicCECR1mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain > 5% of DBA cases. Overall, this comprehensive report should not only inform clinical practice for DBA patients, but also the design and analysis of future rare variant studies for heterogeneous Mendelian disorders.

Abdulhay et al. report that an intronic genetic variant alters GATA1 splicing and presents as a distinct form of dyserythropoietic anemia in two unrelated patients. Functional studies demonstrate that the novel GATA1 isoform lacks observable activity and leads to a decrease in wild-type GATA1 levels in affected individuals., Studies of allelic variation underlying genetic blood disorders have provided important insights into human hematopoiesis. Most often, the identified pathogenic mutations result in loss-of-function or missense changes. However, assessing the pathogenicity of noncoding variants can be challenging. Here, we characterize two unrelated patients with a distinct presentation of dyserythropoietic anemia and other impairments in hematopoiesis associated with an intronic mutation in GATA1 that is 24 nucleotides upstream of the canonical splice acceptor site. Functional studies demonstrate that this single-nucleotide alteration leads to reduced canonical splicing and increased use of an alternative splice acceptor site that causes a partial intron retention event. The resultant altered GATA1 contains a five–amino acid insertion at the C-terminus of the C-terminal zinc finger and has no observable activity. Collectively, our results demonstrate how altered splicing of GATA1, which reduces levels of the normal form of this master transcription factor, can result in distinct changes in human hematopoiesis.

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation. A global reduction in ribosome levels in Diamond-Blackfan anemia profoundly alters translation of a select subset of transcripts, thereby impeding erythroid lineage commitment. Keyword: hematopoiesis; lineage commitment; GATA1; genetics; ribosome; translation; Diamond-Blackman anemia; erythropoiesis, National Institutes of Health (U.S.) (Grants R01 DK103794), National Institutes of Health (U.S.) (Grant R33 HL120791)

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.

Defects in ribosome biogenesis are associated with a group of diseases called the ribosomopathies, of which Diamond-Blackfan anemia (DBA) is the most studied. Ribosomes are composed of ribosomal proteins (RPs) and ribosomal RNA (rRNA). RPs and multiple other factors are necessary for the processing of pre-rRNA, the assembly of ribosomal subunits, their export to the cytoplasm and for the final assembly of subunits into a ribosome. Haploinsufficiency of certain RPs causes DBA, whereas mutations in other factors cause various other ribosomopathies. Despite the general nature of their underlying defects, the clinical manifestations of ribosomopathies differ. In DBA, for example, red blood cell pathology is especially evident. In addition, individuals with DBA often have malformations of limbs, the face and various organs, and also have an increased risk of cancer. Common features shared among human DBA and animal models have emerged, such as small body size, eye defects, duplication or overgrowth of ectoderm-derived structures, and hematopoietic defects. Phenotypes of ribosomopathies are mediated both by p53-dependent and -independent pathways. The current challenge is to identify differences in response to ribosomal stress that lead to specific tissue defects in various ribosomopathies. Here, we review recent findings in this field, with a particular focus on animal models, and discuss how, in some cases, the different phenotypes of ribosomopathies might arise from differences in the spatiotemporal expression of the affected genes., Summary: This paper reviews recent data on Diamond Blackfan anemia and discusses them in connection with other ribosomopathies.

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.

Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA)[superscript 1, 2], congenital asplenia[superscript 3] and T cell leukemia[superscript 4]. Yet, how mutations in genes encoding ubiquitously expressed proteins such as these result in cell-type– and tissue-specific defects remains unknown[superscript 5]. Here, we identify mutations in GATA1, encoding the critical hematopoietic transcription factor GATA-binding protein-1, that reduce levels of full-length GATA1 protein and cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can lead to decreased GATA1 mRNA translation, possibly resulting from a higher threshold for initiation of translation of this mRNA in comparison with other mRNAs. In primary hematopoietic cells from patients with mutations in RPS19, encoding ribosomal protein S19, the amplitude of a transcriptional signature of GATA1 target genes was globally and specifically reduced, indicating that the activity, but not the mRNA level, of GATA1 is decreased in patients with DBA associated with mutations affecting ribosomal proteins. Moreover, the defective hematopoiesis observed in patients with DBA associated with ribosomal protein haploinsufficiency could be partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations affecting ubiquitous ribosomal proteins can result in human disease., National Institutes of Health (U.S.) (Grant P01 HL32262), National Institutes of Health (U.S.) (Grant U54 HG003067-09)

Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow disorder with mutations in ribosomal protein genes. Several animal models have been developed to study the pathological mechanism of DBA. Previously, we reported that the complete knock-out of both Rpl5 and Rps24 alleles were lethal, while heterozygous Rpl5+/- and Rps24+/- mice showed normal phenotype. To establish a more efficient mouse model for mimicking DBA symptoms, we have taken advantage of RNAi technology to generate an inducible mouse model utilizing tetracycline-induced down-regulation of Rpl5. After two weeks of treatment with doxycycline in drinking water, a subset of treated shRNA Rpl5+/- adult mice developed mild anemia while control mice had normal complete blood counts. Similarly, treated shRNA Rpl5+/- mice developed reticulocytopenia and bone marrow erythroblastopenia. Detection of DBA symptoms in these mice make them a valuable DBA model for studying the pathological mechanism underlying DBA and for further assessment of the disease and drug testing for novel therapies.

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.

Diamond-Blackfan anemia (DBA) is an inherited red blood cell aplasia that usually presents during the first year of life. The main features of the disease are normochromic and macrocyctic anemia, reticulocytopenia, and nearly absent erythroid progenitors in the bone marrow. The patients also present with growth retardation and craniofacial, upper limb, heart and urinary system congenital malformations in ~30–50% of cases. The disease has been associated with point mutations and large deletions in ten ribosomal protein (RP) genes RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, RPS26 and RPL26 and GATA1 in about 60–65% of patients. Here we report a novel large deletion in RPL15, a gene not previously implicated to be causative in DBA. Like RPL26, RPL15 presents the distinctive feature of being required both for 60S subunit formation and for efficient cleavage of the internal transcribed spacer 1 (ITS1). In addition, we detected five deletions in RP genes in which mutations have been previously shown to cause DBA: one each in RPS19, RPS24, and RPS26, and two in RPS17. Pre-ribosomal RNA processing was affected in cells established from the patients bearing these deletions, suggesting a possible molecular basis for their pathological effect. These data identify RPL15 as a new gene involved in DBA and further support the presence of large deletions in RP genes in DBA patients.

Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.

Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. Identifying therapeutics for DBA requires circumventing the paucity of primary patient blood stem and progenitor cells. To this end, we adopted a reprogramming strategy to generate expandable hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs) from DBA patients. Reprogrammed DBA progenitors recapitulate defects in erythroid differentiation, which were rescued by gene complementation. Unbiased chemical screens identified SMER28, a small-molecule inducer of autophagy, which enhanced erythropoiesis in a range of in vitro and in vivo models of DBA. SMER28 acted through autophagy factor ATG5 to stimulate erythropoiesis and up-regulate expression of globin genes. These findings present an unbiased drug screen for hematological disease using iPSCs and identify autophagy as a therapeutic pathway in DBA., National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (Grant R24-DK092760), National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (Grant R24-DK49216), National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (Grant U54DK110805), National Heart, Lung, and Blood Institute (Grant UO1-HL100001), National Heart, Lung, and Blood Institute (Grant U01HL134812), National Heart, Lung, and Blood Institute (Grant R01HL04880), National Institutes of Health (U.S.) (Grant R24OD017870-01)

International audience; Diamond-Blackfan anemia (DBA) is an inherited form of pure red cell aplasia that usually presents in infancy or early childhood and is associated with congenital malformations in ∼30-50% of patients. DBA has been associated with mutations in nine ribosomal protein (RP) genes in about 53% of patients. We completed a large scale screen of 79 RP genes by sequencing 16 RP genes (RPL3, RPL7, RPL8, RPL10, RPL14, RPL17, RPL19, RPL23A, RPL26, RPL27, RPL35, RPL36A, RPL39, RPS4X, RPS4Y1, and RPS21) in 96 DBA probands. We identified a de novo two-nucleotide deletion in RPL26 in one proband associated with multiple severe physical abnormalities. This mutation gives rise to a remarkable ribosome biogenesis defect that affects maturation of both the small and the large subunits. We also found a deletion in RPL19 and missense mutations in RPL3 and RPL23A, which may be variants of unknown significance. Together with RPL5, RPL11, and RPS7, RPL26 is the fourth ribosomal protein regulating p53 activity that is linked to DBA.

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.

International audience; Diamond-Blackfan anemia (DBA), an inherited bone marrow failure syndrome characterized by anemia that usually presents before the first birthday or in early childhood, is associated with birth defects and an 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 approximately 30%-50% of patients. DBA has been associated with mutations in seven ribosomal protein (RP) genes, RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, and RPS7, in about 43% of patients. To continue our large-scale screen of RP genes in a DBA population, we sequenced 35 ribosomal protein genes, RPL15, RPL24, RPL29, RPL32, RPL34, RPL9, RPL37, RPS14, RPS23, RPL10A, RPS10, RPS12, RPS18, RPL30, RPS20, RPL12, RPL7A, RPS6, RPL27A, RPLP2, RPS25, RPS3, RPL41, RPL6, RPLP0, RPS26, RPL21, RPL36AL, RPS29, RPL4, RPLP1, RPL13, RPS15A, RPS2, and RPL38, in our DBA patient cohort of 117 probands. We identified three distinct mutations of RPS10 in five probands and nine distinct mutations of RPS26 in 12 probands. 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 knockdown of RPS10 or RPS26 expression with siRNAs, which indicates that mutations in the RPS10 and RPS26 genes in DBA patients affect the function of the proteins in rRNA processing.

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 anemia (DBA) is a rare congenital disease affecting erythroid precursor differentiation. DBA is emerging as a paradigm for a new class of pathologies potentially linked to disorders in ribosome biogenesis. Three genes encoding ribosomal proteins have been associated to DBA: after RPS19, mutations in genes RPS24 and RPS17 were recently identified in a fraction of the patients. Here, we show that cells from patients carrying mutations in RPS24 have defective pre-rRNA maturation, as in the case of RPS19 mutations. However, in contrast to RPS19 involvement in the maturation of the internal transcribed spacer 1, RPS24 is required for processing of the 5' external transcribed spacer. Remarkably, epistasis experiments with small interfering RNAs indicate that the functions of RPS19 and RPS24 in pre-rRNA processing are connected. Resolution of the crystal structure of RPS24e from the archeon Pyroccocus abyssi reveals domains of RPS24 potentially involved in interactions with pre-ribosomes. Based on these data, we discuss the impact of RPS24 mutations and speculate that RPS19 and RPS24 cooperate at a particular stage of ribosome biogenesis connected to a cell cycle checkpoint, thus affecting differentiation of erythroid precursors as well as developmental processes.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome associated with ribosomal protein (RP) gene mutations. Recent studies have also demonstrated an increased risk of cancer predisposition among DBA patients. In this study, we report the formation of soft tissue sarcoma in the Rpl5 and Rps24 heterozygous mice. Our observation suggests that even though one wild-type allele of the Rpl5 or Rps24 gene prevents anemia in these mice, it still predisposes them to cancer development.

Diamond-Blackfan anemia (DBA) is a rare congenital red-cell aplasia characterized by anemia, bone-marrow erythroblastopenia, and congenital anomalies and is associated with heterozygous mutations in the ribosomal protein (RP) S19 gene (RPS19) in approximately 25% of probands. We report identification of de novo nonsense and splice-site mutations in another RP, RPS24 (encoded by RPS24 [10q22-q23]) in approximately 2% of RPS19 mutation-negative probands. This finding strongly suggests that DBA is a disorder of ribosome synthesis and that mutations in other RP or associated genes that lead to disrupted ribosomal biogenesis and/or function may also cause DBA.

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.

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.

The nemaline myopathies (NMs) are a clinically and genetically heterogeneous group of disorders characterized by nemaline rods and skeletal muscle weakness. Mutations in five sarcomeric thin filament genes have been identified. However, the molecular consequences of these mutations are unknown. Using Affymetrix oligonucleotide microarrays, we have analyzed the expression patterns of >21,000 genes and expressed sequence tags in skeletal muscles of 12 NM patients and 21 controls. Multiple complementary approaches were used for data analysis, including geometric fold analysis, two-tailed unequal variance t test, hierarchical clustering, relevance network, and nearest-neighbor analysis. We report the identification of high satellite cell populations in NM and the significant down-regulation of transcripts for key enzymes of glucose and glycogen metabolism as well as a possible regulator of fatty acid metabolism, UCP3. Interestingly, transcript level changes of multiple genes suggest possible changes in Ca 2+ homeostasis. The increased expression of multiple structural proteins was consistent with increased fibrosis. This comprehensive study of downstream molecular consequences of NM gene mutations provides insights in the cellular events leading to the NM phenotype.

Mutations affecting the ribosome lead to several diseases known as ribosomopathies, with phenotypes that include growth defects, cytopenia, and bone marrow failure. Diamond-Blackfan anemia (DBA), for example, is a pure red cell aplasia linked to the mutation of ribosomal protein (RP) genes. Here we show the knock-down of the DBA-linked RPS19 gene induces the cellular self-digestion process of autophagy, a pathway critical for proper hematopoiesis. We also observe an increase of autophagy in cells derived from DBA patients, in CD34+ erythrocyte progenitor cells with RPS19 knock down, in the red blood cells of zebrafish embryos with RP-deficiency, and in cells from patients with Shwachman-Diamond syndrome (SDS). The loss of RPs in all these models results in a marked increase in S6 kinase phosphorylation that we find is triggered by an increase in reactive oxygen species (ROS). We show that this increase in S6 kinase phosphorylation inhibits the insulin pathway and AKT phosphorylation activity through a mechanism reminiscent of insulin resistance. While stimulating RP-deficient cells with insulin reduces autophagy, antioxidant treatment reduces S6 kinase phosphorylation, autophagy, and stabilization of the p53 tumor suppressor. Our data suggest that RP loss promotes the aberrant activation of both S6 kinase and p53 by increasing intracellular ROS levels. The deregulation of these signaling pathways is likely playing a major role in the pathophysiology of ribosomopathies., Author Summary Diseases linked to mutations affecting the ribosome, ribosomopathies, have an exceptionally wide range of phenotypes. However, many ribosomopathies have some features in common including cytopenia and growth defects. Our study aims to clarify the mechanisms behind these common phenotypes. We find that mutations in ribosomal protein genes result in a series of aberrant signaling events that cause cells to start recycling and consuming their own intracellular contents. This basic mechanism of catabolism is activated when cells are starving for nutrients, and also during the tightly regulated process of blood cell maturation. The deregulation of this mechanism provides an explanation as to why blood cells are so acutely affected by mutations in genes that impair the ribosome. Moreover, we find that the signals activating this catabolism are coupled to impairment of the highly conserved insulin-signaling pathway that is essential for growth. Taken together, our in-depth description of the pathways involved as the result of mutations affecting the ribosome increases our understanding about the etiology of these diseases and opens up previously unknown avenues of potential treatment.

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 (DBA) is a hypoplastic anemia characterized by impaired production of red blood cells, with approximately half of all cases attributed to ribosomal protein gene mutations. We performed exome sequencing on two siblings who had no known pathogenic mutations for DBA and identified a mutation in the gene encoding the hematopoietic transcription factor GATA1. This mutation, which occurred at a splice site of the GATA1 gene, impaired production of the full-length form of the protein. We further identified an additional patient carrying a distinct mutation at the same splice site of the GATA1 gene. These findings provide insight into the pathogenesis of DBA, showing that the reduction in erythropoiesis associated with the disease can arise from causes other than defects in ribosomal protein genes. These results also illustrate the multifactorial role of GATA1 in human hematopoiesis.

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.

International audience; Diamond-Blackfan Anemia (DBA) is characterized by a defect in erythroid progenitor differentation and, clinically, by anemia and malformations. DBA exhibits an autosomal dominant pattern of inheritance with incomplete penetrance. Currently nine genes, all encoding ribosomal proteins (RP), have been found mutated in approximately 50% of patients. Experimental evidence supports the hypothesis that DBA is primarily the result of defective ribosome synthesis. By means of a large collaboration among six centers, we report here a mutation update that includes nine genes and 220 distinct mutations, 56 of which are new. The DBA Mutation Database now includes data from 355 patients. Of those where inheritance has been examined, 125 patients carry a de novo mutation and 72 an inherited mutation. Mutagenesis may be ascribed to slippage in 65.5% of indels, whereas CpG dinucleotides are involved in 23% of transitions. Using bioinformatic tools we show that gene conversion mechanism is not common in RP genes mutagenesis, notwithstanding the abundance of RP pseudogenes. Genotype-phenotype analysis reveals that malformations are more frequently associated with mutations in RPL5 and RPL11 than in the other genes. All currently reported DBA mutations together with their functional and clinical data are included in the DBA Mutation Database.

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), a congenital bone-marrow-failure syndrome, is characterized by red blood cell aplasia, macrocytic anemia, clinical heterogeneity, and increased risk of malignancy. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital anomalies that are present in approximately 30%-50% of patients. The disease has been associated with mutations in four ribosomal protein (RP) genes, RPS19, RPS24, RPS17, and RPL35A, in about 30% of patients. However, the genetic basis of the remaining 70% of cases is still unknown. Here, we report the second known mutation in RPS17 and probable pathogenic mutations in three more RP genes, RPL5, RPL11, and RPS7. In addition, we identified rare variants of unknown significance in three other genes, RPL36, RPS15, and RPS27A. Remarkably, careful review of the clinical data showed that mutations in RPL5 are associated with multiple physical abnormalities, including craniofacial, thumb, and heart anomalies, whereas isolated thumb malformations are predominantly present in patients carrying mutations in RPL11. We also demonstrate that mutations of RPL5, RPL11, or RPS7 in DBA cells is associated with diverse defects in the maturation of ribosomal RNAs in the large or the small ribosomal subunit production pathway, expanding the repertoire of ribosomal RNA processing defects associated with DBA.