Congenital Pure Red Cell Aplasia Research Articles

High expression of NADH Ubiquinone Oxidoreductase Subunit B11 induces catheter-associated venous thrombosis on continuous blood purification.

Venous thromboembolism (VTE) is a common vascular disease of venous return disorders, including deep venous thrombosis and pulmonary embolism (PE), with high morbidity and high mortality. However, the relationship between oxidative phosphorylation and NDUFB11 and venous thromboembolism is still unclear. The venous thromboembolism datasets GSE48000 and GSE19151 were downloaded, and the differentially expressed Genes (DEGs) were screened. The protein-protein interaction (PPI) network was constructed. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for functional enrichment analysis. The comparative toxicogenomics database (CTD) was used to identify the diseases most associated with the core genes. TargetScan was used to screen miRNA regulating central DEGs. Western blotting (WB) experiment and real-time quantitative PCR (RT-qPCR) experiment were performed. A total of 500 DEGs were identified. GO analysis showed that the DEGs were mainly enriched in ATP synthesis coupled electron transport, respiratory electron transport chain, cytoplasm, enzyme binding, nonalcoholic fatty liver disease, oxidative phosphorylation, and Alzheimer disease. Enrichment items were similar to GO and KEGG enrichment items of DEGs. The result of CTD showed that 12 genes (RPS24, FAU, RPLP0, RPS15A, RPS29, RPL9, RPL31, RPL27, NDUFB11, RPL34, COX7B, RPS27L) were associated with chemical and drug-induced liver injury, inflammation, kidney disease, and congenital pure red cell aplasia. WB and RT-qPCR results showed that the expression levels of 12 genes in venous thromboembolism were higher than normal whole blood tissue samples. NDUFB11 is highly expressed in catheter-related venous thromboembolism during continuous blood purification, which may lead to the formation of venous thrombosis through oxidative phosphorylation pathway.

Pure Red Cell Aplasia Encountered in a Tertiary Care Hematology Laboratory: A Series of Nine Distinctive Cases.

Pure red cell aplasia (PRCA) is characterized by severe anemia with reticulocytopenia and bone marrow erythroblastopenia. The early erythroblasts are markedly decreased; however, in rare instances, they may be normal or raised in number. There are varied etiologies, namely congenital or acquired and primary or secondary. The congenital PRCA is known as "Diamond-Blackfan anemia." Thymomas, autoimmune disease, lymphomas, infections, and drugs also may be familiar associates. However, the etiologies of PRCA are numerous, and many diseases/infections can be associated with PRCA. The diagnosis rests on clinical suspicion and appropriate laboratory workup. We evaluated nine cases of red cell aplasia, having severe anemia with reticulocytopenia. Nearly half of the cases showed adequate erythroid (> 5% of the differential count) but with a maturation arrest. The adequacy of the erythroid could confuse the hematologist and may even delay the diagnosis. Hence, it is empirical that PRCA could be considered a differential in every case of severe anemia with reticulocytopenia, even in the presence of adequate erythroid precursors in the bone marrow.

Research Progress on Pathogenesis of Congenital Pure Red Cell Aplasia---Review

Congenital pure red cell aplasia, also known as Diamond-Blackfan anemia (DBA), is a hereditary disease characterized by pure red cell aplasia and congenital malformation. Its main clinical features are anemia, dysplasia, and tumor susceptibility. Ribosomal protein (RP) gene mutation is the main pathogenesis of DBA. The most common type of gene mutation is RPS19 gene mutation. Heterozygous mutations in as many as 19 RP genes and other non-RP genes mutations have been identified in DBA. This review summarized briedfly the latest research advances in the pathogenesis of DBA.

Whole Exome Sequencing of a Patient with Atypical Congenital Pure Red Cell Aplasia Has Enabled the Identification of a Novel Key Actor of Erythropoiesis That May be Involved in CDAI Physiopathology

Background: The development of next generation sequencing techniques has brought important insights into the molecular mechanisms of erythropoiesis and how these processes can be perturbed in human diseases. This strategy may be valuable in some hereditary erythroid disorders where a subset of patients does not carry any mutations in the supposed causal gene and for which transgenic mouse models do not recapitulate the phenotype, suggesting that additional genetic events may be involved in pathogenesis. Here, we report the case of an adult patient presenting with atypical pure red cell aplasia associated with facial dysmorphy and chronic leg ulcers. Whole exome sequencing revealed a heterozygous missense mutation (R725W) in the CDAN1 gene, which has been previously reported in congenital dyserythropoietic anemia type I (CDAI). However, this mutation was also detected in her healthy brother, suggesting that this event alone was not sufficient to explain her phenotype. According to this hypothesis, we found an additional germline heterozygous nonsense mutation (Q732X) in the MMS22L gene, which was not shared by her unaffected relatives. MMS22L is a protein involved in homologous recombination-dependent repair of stalled or collapsed replication forks. Additionally, MMS22L is able to bind newly synthesized soluble histones H3 and H4 and exhibits a histone chaperone activity. MMS22L loading onto ssDNA during homologous recombination is promoted by the histone chaperone ASF1. Interestingly, CDAN1 acts as a negative regulator of ASF1 by mediating its sequestration in the cytoplasm, which results in the blocking of histone delivery. Aims: As MMS22L has never been reported in erythropoiesis before, we aimed to investigate the role of MMS22L in human erythropoiesis. Based on the data summarized above, the purpose of this study was also to determine the effect of combined inactivation of MMS22L and CDAN1 on in vivo erythropoiesis, while exploring the functional cooperation between both proteins. Results: To decipher the role of MMS22L in human erythropoiesis, we assessed the consequences of complete MMS22L inactivation in human cord blood CD34+ progenitors as well as in CD36+ immature erythroblasts using shRNA lentiviruses. This resulted in a severe decrease of cell proliferation and differentiation due to G1 cell cycle arrest, with a slight increase of apoptosis. Interestingly, this phenotype was not observed when MMS22L was inactivated in the granulo-monocytic lineage, in which differentiation was maintained, suggesting that erythroid cells, that are highly proliferative, are more sensitive to MMS22L inactivation. To better understand the effect of combined CDAN1 and MMS22L haploinsufficiency observed in the proband, we used zebrafish as an in vivo model. Mms22l and cdan1 expression were simultaneously or separately downregulated by about 50% using antisens morpholino oligomers. 48 hours later, zebrafish embryos were stained with o-dianisidine to detect hemoglobin-containing cells. We found that combined knock-down of mms22l and cdan1 resulted in severe anemia, while knock-down of mms22l or cdan1 alone did not lead to any erythroid disorder. This experiment provides a proof-of-concept, indicating that the phenotype of the proband is indeed caused by the combination of both MMS22L and CDAN1 mutations. Finally, in order to decipher the cooperation between MMS22L and CDAN1 we used the human erythroid UT-7 cell line. We found that CDAN1 inactivation resulted in a severe decrease in MMS22L expression within the nucleus, suggesting that CDAN1 may regulate MMS22L expression or localization. We therefore wanted to confirm these results by assessing MMS22L expression in B-EBV cell lines established from two CDAI patients with CDAN1 compound heterozygous mutations. We found a great decrease in MMS22L expression within the nucleus of the CDAI patients' cells when compared to three control B-EBV cell lines. Based on these results, we suggest that impairment of MMS22L trafficking to the nucleus could be involved in CDA1 physiopathology. Conclusion: Through comprehensive genetic analysis of a single case with atypical congenital anemia, we demonstrated for the first time that MMS22L, a cell cycle regulator, is essential for the process of erythropoiesis. The crosstalk between MMS22L and CDAN1 is currently under investigation and could bring important new insights into the physiopathology of CDAI. Disclosures Hermine: Novartis: Research Funding; Alexion: Research Funding; AB Science: Consultancy, Current equity holder in publicly-traded company, Honoraria, Patents & Royalties, Research Funding; Celgene BMS: Consultancy, Research Funding; Roche: Consultancy.

Incidence and Clinico - Hematological pattern of Diamond Blackfan Anemia: 10 years experience in a single center

The study aimed to determine the incidence and clinico-hematological pattern of Diamond Blackfan Anemia (DBA), a congenital form of pure red cell aplasia (PRCA). A retrospective observational descriptive cohort study was conducted in the Department of Pediatric Hematology and Transfusion Medicine, The Children’s Hospital and The Institute of Child Health, Lahore, for a period of 10 years (January 2008 to December 2017.) Data was analyzed using SPSS software. Among 19,475 cases reviewed, 74 patients (0.4%) were diagnosed as congenital PRCA with a male to female ratio of 4.2:1 and a mean age of 5months. In this cohort, 23% of patients had a positive family history. Thirty-four percent of patients presented with congenital anomalies involving cleft lip and palate (23.5%), thumb and upper limb defects (20.5%), craniofacial anomalies (14.7%), and cardiac and urogenital malformations in 8.8 and 5.8% patients, respectively. Red blood cell (RBC) counts and hemoglobin (Hb) concentration levels were low with a range of 0.5–3.0 × 1012/μl and 2.5–6.5 g/dl, respectively. Fetal hemoglobin (HbF) levels remained within the range of 2–20%. Bone marrow aspirate showed <5% erythroblasts in all patients. The specific clinico-hematological pattern as described in our study not only helps in early diagnosis and management of the disease and also paves way to establish a data registry.

Favorable outcomes of hematopoietic stem cell transplantation in children and adolescents with Diamond-Blackfan anemia

AbstractDiamond-Blackfan anemia (DBA) is a congenital pure red cell aplasia associated with congenital abnormalities and cancer predisposition. Allogeneic hematopoietic stem cell transplantation (HSCT) can correct the hematological phenotype and is indicated in transfusion-dependent patients. In 70 children reported to the German DBA and French HSCT registries, HSCT was performed from 1985 to 2017. Median age at HSCT was 5.5 years (range, 0.9-17.3 years). Two-thirds of patients (64%) were transplanted from a matched sibling donor (MSD), and most procedures were performed after the year 1999 (73%). Primary engraftment was achieved in all patients. One patient developed secondary graft failure. Cumulative incidence of acute graft-versus-host disease (GVHD) was 24% for °II-IV (95% confidence interval [CI], 16% to 37%) and 7% for °III-IV (95% CI, 3% to 17%); cumulative incidence of chronic GVHD was 11% (95% CI, 5% to 22%). The probability of chronic GVHD-free survival (cGFS) was 87% (95% CI, 79% to 95%) and significantly improved over time (<2000: 68% [95% CI, 47% to 89%] vs ≥2000: 94% [95% CI, 87% to 100%], P < .01). cGFS was comparable following HSCT from a MSD and an unrelated donor (UD). Of note, no severe chronic GVHD or deaths were reported following MSD-HSCT after 1999. The difference of cGFS in children transplanted <10 years of age compared with older patients did not reach statistical significance (<10 years: 90% [95% CI, 81% to 99%] vs 10-18 years 78% [95% CI, 58% to 98%]). In summary, these data indicate that HSCT is efficient and safe in young DBA patients and should be considered if a MSD or matched UD is available. HSCT for transfusion dependency only must be critically discussed in older patients.

Pure red cell aplasia, a disease of a great diversity

Pure red cell aplasia (PRCA) is a type of normocytic or sometimes macrocytic anemia characterized by reticulocytopenia. The cause of hypo erythropoiesis is varied. It could be of a congenital or an acquired type. A congenital PRCA is different than anemia caused by congenital dyserythropoiesis. The classification of acquired PRCA can be primary when no cause is identified or secondary due to underling or associated pathology. The primary PRCA is a disease of exclusion. There are many disorders such as autoimmune disorders, hematological and nonhematological malignancies, some infections, and medications that cause secondary PRCA. Immune-mediated mechanism plays an important role in both primary and secondary types. Therefore, immunomodulating agents are important in the treatment of primary type and many secondary types. Other therapy modalities include surgery, plasmapheresis, and hemopoietic stem cell transplantation. The assessment of hemoglobin level and absolute reticulocyte count with the frequency of blood transfusion are important to monitor therapy response achievement. Achievement of complete response is important to eliminate demands for blood transfusion and to avoid blood-transfusion risks. Herein, we review this disorder which has broad diversity, its causes, approach to its cause, therapy modalities.

Stem Cell Transplantation for Diamond-Blackfan Anemia. a Retrospective Study on Behalf of Severe Aplastic Anemia Working Party of the European Blood and Marrow Transplantation Group (EBMT)

Bader: Riemser, Neovii: Research Funding; Medac: Patents & Royalties, Research Funding; Amgen (Brasil), Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy. Risitano:Alexion: Honoraria, Research Funding, Speakers Bureau; Achillion: Research Funding; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Biocryst: Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Research Funding, Speakers Bureau; Amyndas: Consultancy; Samsung: Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amyndas: Consultancy; Biocryst: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Research Funding; Ra Pharma: Research Funding; Alnylam: Research Funding; Alnylam: Research Funding; Achillion: Research Funding. Peffault de Latour:Pfizer: Consultancy, Honoraria, Research Funding; Amgen: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria, Research Funding.

Identification of a novel RPS26 nonsense mutation in a Chinese Diamond-Blackfan Anemia patient

BackgroundDiamond-Blackfan anemia (DBA), a congenital pure red cell aplasia (PRCA), is characterized by normochromic macrocytic anemia, reticulocytopenia, and nearly absent erythroid progenitors in the bone marrow. DBA10, a subset of DBA, is an autosomal dominant disease caused by a mutation in RPS26. So far, there are 30 disease-causing variants in RPS26 being reported, however, only three of them are small insert mutations.Case presentationHere we report a three-month Chinese boy who presents with anemia from postnatal day 2. He was suspected to have Diamond-Blackfan anemia, according to the clinical result. Thus, whole-exome sequencing was performed for precise diagnosis.ConclusionHere, a novel insert mutation c.96dupG in RPS26 was identified by whole-exome sequencing, which caused neonatal DBA in a Chinese boy. This is the first case report of a Chinese DBA10 patient who carries a small insertion in the RPS26 gene. These findings expand the mutation diversity of RPS26 and demonstrate the clinical presentations of the Chinese DBA10 patient.

Diamond-Blackfan Anemia Phenotype Caused By Deficiency of Adenosine Deaminase 2

Diamond-Blackfan anemia (DBA) is a prototypic ribosomopathy and remains the most common cause of congenital pure red cell aplasia (PRCA). In 2/3 of patients, ribosomal protein haploinsufficiency is disease-causing, while in remaining 1/3 the genetic etiology is unknown. Recently, deficiency of ADA2 (DADA2) due to biallelic CECR1 -mutations was reported in patients with systemic autoinflammatory disease presenting with early onset vasculopathy, strokes, antibody deficiency, and in some cases variable cytopenias. Based on the clinical findings in an ADA2-deficient patient with PRCA resembling DBA, we aimed to define the prevalence and clinical picture of DADA2 within DBA patient cohorts. Patients enrolled in the national observational DBA registry in Germany were evaluated for the presence of mutations in CECR1 gene; additional nonconsecutive patients from the French and Turkish registries within the European DBA (EuroDBA) consortium were part of this study. Functional studies included profiling of polysomes and pre-rRNAs in patient-derived EBV-cell lines, CECR1 RT-PCR, measurements of autophagy and apoptosis, and analysis of erythropoiesis in zebrafish embryos. Systematic mutational and copy number analysis had identified typical ribosomal haploinsufficiency in 169/242 patients (70%). Out of 73 remaining patients, full CECR1 -sequencing was accomplished in 68 cases, of which 4 (6%) carried biallelic CECR1 -mutations. Additional 3 patients with biallelic CECR1 -mutations and DBA phenotype were referred from Germany (the index PRCA case), France and Turkey. In contrast to typical autoinflammatory DADA2 (caused by missense biallelic CECR1 -mutations) all patients studied here had at least one CECR1 -allele affected by truncating/stop-gain/deletion mutation leading to mRNA degradation in patient cells. Low or missing ADA2 enzyme activity in plasma confirmed DADA2, while erythrocyte ADA (eADA) levels and MCV were normal. Transfusion-dependent hypoproliferative anemia developed at a median age of 5 weeks (birth-14 years), while hypogammaglobulinemia developed in all cases either initially or during disease course. Notably, a transient hematologic response to steroids was achieved in 5/7 patients, but no improvement was observed in 2 patients treated with TNF-inhibitor; all patients at one point became heavily transfusion-dependent. Systemic vasculitis or cerebral complications were not observed in our cohort. At the last follow-up, 6/7 patients were alive; 3 had successfully undergone hematopoietic stem cell transplantation (HSCT) with myeloablative conditioning and 1 patient had died due to septic shock. Next, we addressed the question if ribosome biogenesis is affected in ADA2-deficient patient cells. Using pre-rRNA maturation assays, polysome profiling and Western blots we established that ribosome biogenesis is normal in DADA2-related PRCA and there is no increase of TP53 stabilization over basal levels in patient LCLs. Analysis of CECR1 -morpholino zebrafish embryos revealed early anemia with lethal phenotype. Although there was no evidence for extrinsic (e.g. immune-mediated) pathomechanisms in our patients, it remains to be answered if CECR1 loss directly affects erythroid development. Finally, the association between elevated levels of eADA (=ADA1) specific to classical DBA and decreased ADA2 enzyme levels in DADA2-related PRCA remains obscure. In summary, DADA2 can phenotypically mimic DBA and thus extends the spectrum of congenital PRCA. Ribosome synthesis seems not to be affected by CECR1 mutations. DADA2 should be considered in patients with DBA-like phenotype but with normal eADA/MCV and hypogammaglobulinemia, allowing for early stratification aimed at HSCT in affected individuals. DisclosuresGrosse:Addmedica: Membership on an entity's Board of Directors or advisory committees.

Alteration of heme metabolism in a cellular model of Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital pure red cell aplasia often associated with skeletal malformations. Mutations in ribosomal protein coding genes, mainly in RPS19, account for the majority of DBA cases. The molecular mechanisms underlying DBA pathogenesis are still not completely understood. Alternative spliced isoforms of FLVCR1 (feline leukemia virus subgroup C receptor 1) transcript coding for non-functional proteins have been reported in some DBA patients. Consistently, a phenotype very close to DBA has been described in animal models of FLVCR1 deficiency. FLVCR1 gene codes for two proteins: the plasma membrane heme exporter FLVCR1a and the mitochondrial heme exporter FLVCR1b. The coordinated expression of both FLVCR1 isoforms regulates an intracellular heme pool, necessary for proper expansion and differentiation of erythroid precursors. Here, we investigate the role of FLVCR1 isoforms in a cellular model of DBA. RPS19-downregulated TF1 cells show reduced FLVCR1a and FLVCR1b mRNA levels associated with heme overload. The downregulation of FLVCR1 isoforms affects cell cycle progression and apoptosis in differentiating K562 cells, a phenotype similar to DBA. Taken together, these data suggest that alteration of heme metabolism could play a role in the pathogenesis of DBA.

English

Anemia in children may arise from a wide variety of pathogenetic mechanisms that include congenital and acquired disorders. Often the diagnostic considerations include disorders that are not seen commonly in adults and lifelong disorders that arise in children and persist throughout life. Consideration of diverse causes of anemia such as red cell membrane disorders, red cell enzymopathies, congenital dyserythropoietic anemias, congenital sideroblastic anemias, and hereditary pure red cell aplasia (Diamond-Blackfan anemia), as well as infectious causes such as parvovirus B19 infection, often is required when diagnosing anemia in an infant or young child. Knowledge of these entities that are important causes of anemia in the pediatric population, including clinical manifestations and laboratory workup, will aid in recognition of the specific disease entities and effective workup of pediatric red cell disorders. Inherited and acquired red cell disorders in children include a diverse group of disorders that include red cell enzyme disorders, red cell membrane disorders, congenital dyserythropoietic anemia, sideroblastic anemia, hemoglobinopathies, and red cell aplasias. Most of these disorders are manifested in the child by anemia with hypoproliferative or hemolytic mechanisms underpinning the development of anemia. Hypoproliferative anemia arises secondary to decreased overall RBC precursors or ineffective erythropoiesis, suggesting an abnormality in bone marrow production or erythroid precursors. Conversely, the anemia may be hemolytic in nature, reflecting abnormalities in RBC structure or biologic function that affect RBC life span and are associated with normal to increased erythropoiesis. Clinical manifestations, RBC characteristics, specific laboratory testing, and bone marrow findings are all useful in trying to classify the cause of the anemia and define a specific disease process. INTRODUCTION: Pure red cell aplasia (PRCA) is an uncommon disorder in which maturation arrest occurs in the formation of erythrocytes. Erythroblasts are virtually absent in bone marrow; however, WBC and platelet production is normal. The anemia due to PRCA is usually normocytic but can be macrocytic. In 1922, Kaznelson recognized that this condition was a different entity from aplastic anaemia, which presents with pancytopenia. The characteristics of PRCA include a severe anemia, a reticulocyte count of less than 1%, and the presence of less than 0.5% mature erythroblasts in the bone marrow. The bone marrow is usually normocellular. The etiology of PRCA is heterogeneous. A congenital form of PRCA was initially described by Joseph in 1936 and by Diamond and Blackfan in 1938. Congenital PRCA is a lifelong disorder and is associated with physical abnormalities. PRCA can be transient and reversible. Transient erythroblastopenia of childhood (TEC) can occur after viral infections. PRCA due to medications is also often reversible when these medications are discontinued. PRCA due to infections is often reversible. In adults, most cases of chronic PRCA are idiopathic, and a cause cannot be established. Secondary PRCA occurs in patients with conditions such as autoimmune disorders, thymomas, hematologic malignancies,

Molecular mechanisms of pathology and treatment in Diamond Blackfan Anaemia

Diamond Blackfan Anaemia (DBA) is a rare congenital pure red cell aplasia that may be associated with facio-skeletal developmental defects. The disease is caused by mutations in one of at least ten ribosomal proteins, which results in haploinsufficiency and an imbalance between the synthesis of rRNA and ribosomal proteins during ribosome biogenesis. Such imbalance results in stabilization and activation of the tumour suppressor gene TP53. The loss of ribosomes also results in reduced mRNA translation capacity, and may affect translation of specific erythroid transcripts more than average. The contribution of these two mechanisms to impaired erythropoiesis is discussed. The most effective and relatively safe therapy is treatment with glucocorticoid hormone, but responsiveness differs between patients. The molecular and cellular mechanisms involved in treatment are discussed in the context of DBA.

High Frequency of Large Gene Deletions Detected by Multiplex Ligation-Dependent Probe Amplification in Diamond Blackfan Anemia,

Abstract 3428 Background:Diamond-Blackfan anemia (DBA,#MIM105650) is a rare congenital pure red cell aplasia characterized by normochromic macrocytic anemia, reticulocytopenia, and normocellular bone marrow with a selective deficiency of erythroid precursors.Although mutations of eleven ribosomal protein (RP) genes have been detected in more than 50% of DBA patients the remaining patients appear to have intact ribosomal protein genes using stadard sequencing methods (Boria et al. Hum Mut 2010).We previously described the detection of three large RPS19 deletions using the MLPA (Multiplex Ligation-dependent Probe Amplification) technique (Quarello et al. Haematologica 2008). As MLPA is an efficient and rapid technique that detects gene dosage alterations, we thus decided to apply this approach also to other RP genes. Aim:To search for unidentified RP large deletions we applied the MLPA technique in Italian DBA patients who have been found mutation-negative by sequencing. Methods:Italian DBA patients without RP genes mutations (73/156, 47%) were included in this study. The analysis was performed using a homemade MLPA kit following the recommendations provided by MRC Holland (Amsterdam, The Netherlands, www.mlpa.com). The probes were designed to detect deletions of six RP genes (RPS17, RPS19, RPS26, RPL5, RPL11, RPL35A). Deletions of probe recognition sequences were apparent by a 35–50% reduced relative peak area of the amplification product of that probe. Results:The results of the MLPA assay revealed that 13 out of the 73 probands (18%) had a multi-exonic deletion in one of the six DBA genes analyzed. We identified four deletions of the RPS17 gene, three of the RPS26 gene, three of the RPL35A, two of the RPL11 gene and one of the RPL5 gene. No additional RPS19 deletions were found. DBA patients with deletions showed a severe phenotype with a very high percentage of transfusion dependence (85%). Somatic malformations were observed only in two patients. Conclusion:We detected a high percentage of deletions of known DBA genes in a cohort of patients in whom no mutations were found by RP sequencing.Mutation screening of the RP genes with a combination of sequencing and MLPA reached an overall detection rate of 61.5% (96/156). In our cohort, large genomic deletions represent up to 18% of all mutations detected.In conclusion, we stress the high percentage of identified RP genes deletions in DBA patients. We also highlight that a gene-dosage technique, such as MLPA, should complement sequencing in a clinical environment since only a combined approach of this kind permits the comprehensive detection of all mutations in the DBA RP genes. Disclosures:No relevant conflicts of interest to declare.

Disorders of sex development and Diamond-Blackfan anemia: is there an association?

Diamond-Blackfan anemia (DBA) is a rare disorder characterized by congenital pure red cell aplasia. Mutations in ribosomal protein S19 (RPS19) have been identified in 25% of DBA patients. More recently, mutations in other ribosomal protein genes, namely RPS7, RPS15, RPS24, RPS17, RPS27A, RPL35a, RPL36, RPL11, and RPL5, have also been found in patients with DBA. Approximately 30-40% of affected patients have various associated physical anomalies, mostly craniofacial and at the extremities, but also cardiac or urogenital malformations. Anomalies of the urogenital tract in DBA patients comprise changes in the kidney (dysplasia, agenesis, duplication, horseshoe kidney) and genitalia (hypospadias). To date, disorders of sex development (DSD) have only been described once in association with DBA. We report here four DBA patients who exhibited DSD.

Crosstalk between Erythropoiesis and Iron Metabolism

Oxygen delivery is essential to sustain all vital functions. Vertebrate organisms have, therefore, evolved sophisticated and tightly regulated mechanisms to ensure continuous red cell synthesis. Chronic or acute blood losses trigger multiple feedback mechanisms aimed at increasing erythropoiesis and minimizing the damage associated with profound anemia and hypoxia. Iron is an element of paramount importance for erythropoiesis, being an essential part of the hemoglobin metalloprotein that ensures oxygen absorption, transport, and delivery. Therefore, it is not surprising that iron absorption and transport are closely linked to the demands of erythropoiesis. However, the mechanisms underlying this interconnected relationship have only recently started being understood. The focus of this special issue is on iron metabolism, erythropoiesis, and their close association, with a special emphasis on proteins regulating iron metabolism and how erythropoiesis affects their expression. The mechanistic view will be integrated with the clinical observations in disease such as myelodysplastic syndromes, sickle cell anemia, Diamond-Blackfan anemia, and anemia of inflammation. And finally, in light of these new discoveries, new potential therapies for well-known disorders such as beta-thalassemia will be discussed. In the last decade, new discoveries have fueled the field of iron metabolism. The papers by H. Li and Y. Z. Ginzburg[1],E. Nemeth [2], L. Cianetti et al. [3], and T. Tanno and J. L. Miller [4] provide a comprehensive and updated overview of the proteins involved in regulating dietary iron absorption, plasma iron concentrations, tissue iron distribution, and erythropoiesis. The review by H. Li and Y. Z. Ginzburg [1] introduces the mechanisms of crosstalk between iron and erythropoiesis and their function in different diseases. E. Nemeth [2] describes the role of the peptide hormone hepcidin in iron physiology and in many iron-related disorders and highlights the potential of hepcidin agonists and antagonist as future therapeutic tools for iron disorders. The paper by L. Cianetti et al. [3] describes the hepcidin receptor and iron transporter ferroportin and its role during normal and pathological erythroid differentiation. The paper by T. Tanno and J. L. Miller [4] describes the relationship between iron regulation and ineffective erythropoiesis, focusing on potential mechanisms for pathological iron overloading. Improved knowledge on proteins that interconnect iron metabolism and erythropoiesis is opening new prospective to develop new therapies, such as in beta-thalassemia. The review by L. Melchiori et al. [5] provides important evidence that use of Jak2 inhibitors or hepcidin agonists may ameliorate the abnormal erythropoiesis and iron overload in β-thalassemia. The interdependence of erythropoiesis and iron regulation extends beyond normal metabolism to include several pathological conditions. Diseases that are primarily associated with erythroid defects often lead to iron overload, maldistribution, or deficiency. The reviews by E. Messa et al. [6] and by M. A. Badawi et al. [7] summarize the historical progress and recent clinical developments using iron chelation in patients with myelodysplastic syndromes. Similarly, the paper by R. Raghupathy et al. [8] provides an overview of transfusional iron overload patients with sickle cell disease, highlighting how to prevent iron overload in this disorder. Diamond-Blackfan anemia is a rare congenital pure red-cell aplasia that presents during infancy. D. Chiabrando and E. Tolosano's [9] review focused upon heme and ribosomal biogenesis in this disease and why deficiency of the heme exporter Feline Leukemia Virus subgroup C Receptor (FLVCR1) may cause a related phenotype. Finally, the review by E. A. Price and S. L. Schrier [10] provides a comprehensive and updated overview of anemia of inflammation, focusing on many of key clinical issues that remain to be clarified, such as understanding of mechanisms of anemia of inflammation in “noninflammatory” diseases, optimal methods for distinguishing anemia of inflammation from iron deficiency anemia, and understanding the contributory role of various pathologic mechanisms in individual human diseases that lead to anemia of inflammation. We hope that this special issue will stimulate curiosity, interest, and new research directions in exploring the field of iron and erythropoiesis, with the ultimate goal to develop new drugs and improve the management of disorders associated with altered iron metabolism, abnormal erythropoiesis, and anemia of inflammation. Stefano Rivella Elizabeta Nemeth Jeffery Lynn Miller

Differential Down-Regulation of RPL35a in Human Bone Marrow Progenitors Demonstrates a p53 Independent Mechanism Mediating the Diamond Blackfan Anemia Phenotype.

Abstract 1096Poster Board I-118We have previously reported the first known mutations in a gene that encodes a large ribosomal subunit protein, Rpl35a that are linked to Diamond Blackfan Anemia (DBA), a congenital pure red cell aplasia. We have demonstrated that down-regulation of RPL35a results in decreased cellular proliferation, increased apoptosis, impaired ribosomal RNA processing and defective ribosome biogenesis. In order to examine the mechanisms by which defects in RPL35a lead to the development of DBA, we have developed a lentiviral system containing shRNA to down-regulate RPL35a and RPS19, a gene encoding a small ribosomal protein known to be defective in about 25% of DBA cases. Defects in these cellular processes have been linked to disruption of cell cycle progression through activation of nucleolar associated regulatory mechanisms. Cell cycle analysis of the normal human bone marrow progenitors transduced with the specific shRNA demonstrates a G1 arrest when compared to cells transduced with a luciferase shRNA control construct. Similar findings were made in the p53-mutant cell line, TF-1. In contrast, cell cycle analysis of the p53-mutant UT-7/Epo cell line demonstrates a primarily G2/M arrest. To further investigate the mechanism of the cell cycle arrest in bone marrow progenitors, p53 expression was analyzed by Western Blot. Only a nearly complete knockdown resulted in increased p53 expression; shRNA affecting a haploinsufficient state had no evidence of increased p53 expression. However, clonogenic studies of human bone marrow progenitors transduced with shRNA constructs that approximate haploinsufficiency demonstrate a selective absence of erythroid colonies compared to myeloid colonies, thus mimicking the phenotype of DBA. In contrast, bone marrow progenitors transduced with shRNA that causes near complete absence of expression of RPL35a have neither myeloid nor erythroid clonogenic potential consistent with a stem cell catastrophe. These data may suggest a p53 independent pathway can mediate the effects of ribosomal protein deficiency in DBA as well as demonstrate a model for further understanding abnormalities in ribosomal proteins that result in an in vitro phenocopy of altered DBA hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Ribosomal Deficiencies Cause Translational Deregulation of Genes Crucial for Erythropoiesis.

Abstract 179Diamond Blackfan anemia (DBA) is a rare congenital pure red cell aplasia in which mutations in distinct ribosomal proteins (RPs) have been identified in 50% of patients. Mutated genes include Ribosomal Protein Small 19 (RPS19), RPS24 and RPS17, components of the 40S ribosomal subunit, and Ribosomal Protein Large 11 (RPL11), RPL5 and RPL35A, components of the 60S subunit. It is not known why mutations in such ubiquitously expressed proteins cause such a strong erythroid phenotype. Previously, we showed that selective translation of transcripts with a complex RNA structure in the 5'untranslated region (5'UTR) is crucial in Stem Cell Factor (SCF) dependent expansion of the early erythroid compartment. Transcripts such as Immunoglobulin binding protein 1 (Igbp1) are actively recruited into translating polyribosomes upon SCF signaling which activates the PI3K-mTOR pathway leading to release of eukaryote initiation factor 4E (eIF4E), a limiting factor for assembly of the scanning complex. The scanning complex also involves the ribosomal subunits. We therefore investigated if polysome recruitment of specific mRNAs is affected in erythroblasts deficient for RPs. Mouse primary erythroblasts derived from p53 deficient and wild-type (wt) fetal livers were cultured in presence of erythropoietin, SCF and dexamethasone under serum free conditions. Downregulation of either Rps19 or Rpl11 by lentivirus-delivered shRNA resulted in severely reduced proliferation and inhibition of differentiation in comparison to nontransduced cells or cells expressing a scrambled control shRNA. Analysis of subpolysomal and polysome-bound RNA by sucrose gradient centrifugation showed a specific reduction of the 40S and 60S ribosomal subunit upon knock down of Rps19 and Rpl11, respectively. Subpolysomal and polysome-bound RNA fractions from 3 independent experiments were used for expression profiling. Surprisingly, the polysome recruitment of transcripts that require SCF and increased eIF4E availability to be translated was not affected by RP deficiency. The ratio of polysome association was calculated per gene for each experiment and datasets from wt, scrambled treated, Rps19 and Rpl11 deficient conditions were compared using F-test with random variance model (p<0.0005). This revealed a distinct set of mRNAs, including Fxc1 (fractured callus expressed-1), Siva1 (apoptosis inducing gene), Csde1 (cold shock domain containing E1) and Cdc25B (cell dividion cycle homolog B) as being down regulated from translating polyribosomes upon RP deficiency. This downregulation was independent from the presence of p53, or glucocorticoids and also occurred in cells deficient for other ribosomal proteins (Rps14, Rpl8). Currently we are analyzing selective translation of these genes in erythroblasts of DBA patients. Erythroblasts could be cultured from peripheral blood of DBA patients although expansion potential of the cultures was decreased compared to control cultures. Protein expression of several identified target genes (CSDE1, CDC25B) was down regulated in erythroblasts derived from DBA patients as compared to controls, whereas their transcript level on total mRNA remained unchanged, indicating a specific translation defect. Further investigation showed that loss of Csde1 perturbed cell cycle progression and erythroid differentiation. In conclusion, we identified transcripts whose translation is selectively affected in RP deficient erythroblasts cultured from mouse fetal livers or DBA patients. We propose that the erythroid phenotype in DBA patients is caused by defective translation of specific set of mRNAs, which are essential for erythroid development. Disclosures:Pospisilova:Grant NS9935-3 from Ministry of Health, Czech Republic: Research Funding.

Acute cholecystitis caused by Haemophilus influenzae in a child

A 6-year old girl was admitted to our hospital with high fever and right upper quadrant abdominal pain. At 5 years of age she had undergone allogeneic bone marrow transplantation because of intractable congenital pure red cell aplasia, after which she had asymptomatic cholelithiasis. Imaging studies and laboratory findings on admission suggested acute cholecystitis with a gallstone. The patient recovered completely after laparoscopic cholecystectomy was performed and was discharged on day 7 after the operation. Haemophilus influenzae grew on the aspirated purulent bile. It should be noted that H. influenzae can cause cholecystitis in children.

Multiplex ligation-dependent probe amplification enhances molecular diagnosis of Diamond-Blackfan anemia due to RPS19 deficiency

Diamond-Blackfan anemia (DBA,#MIM105650) is a rare congenital pure red cell aplasia characterized by nor-mochromic macrocytic anemia, reticulocytopenia, and normocellular bone marrow with a selective deficiency of erythroid precursors. Defects in the RPS19 gene on chromosome 19q13.2 are the main