Dyserythropoietic Anemia Research Articles

Disparate Phenotypes Of SEC23B Deficiency In Humans and Mice

Congenital dyserythropoietic anemia type-II (CDAII) is an autosomal recessive disease characterized by moderate anemia and increased bone marrow (BM) bi/multi-nucleated erythroblasts. CDAII results from mutations in SEC23B, one of two closely related mammalian SEC23 paralogs. SEC23 is a core component of COPII coated vesicles, which transport secretory proteins from the Endoplasmic Reticulum (ER) to the Golgi apparatus. Bone marrow transplantation cures CDAII, suggesting that the pathologic defect in this disease is restricted to the hematopoietic compartment. However, the mechanism by which SEC23B-deficiency results in CDAII remains unknown. We previously reported that mice homozygous deficient for SEC23B (Sec23bgt/gt) exhibit massive pancreatic degeneration. The latter resulted in perinatal mortality precluding evaluation of the adult hematopoietic compartment. To examine the impact of SEC23B-deficiency on adult murine hematopoiesis, fetal liver cells (FLC) were harvested from Sec23bgt/gt or wildtype (WT) control E17.5 embryos and transplanted into lethally irradiated C57BL/6J mice. Recipients of Sec23bgt/gt FLC had normal peripheral blood counts and were indistinguishable from recipients of WT FLC. To test for a more subtle hematopoietic defect, Sec23bgt/gt FLCs were tested directly against WT FLC for their ability to reconstitute hematopoiesis in a competitive repopulation assay. SEC23B deficient FLC exhibited no competitive disadvantage at reconstituting erythropoiesis relative to WT FLC over 18 weeks of follow-up. Transplant of marrow from these chimeric animals into secondary recipients demonstrated continued equivalence of Sec23bgt/gt and WT hematopoietic stem cells. We also generated a second, conditional Sec23b allele, in which exons 5 and 6 are flanked by loxP sites (Sec23bfl). Deletion of exons 5 and 6 with Cre-recombinase results in a frame shift leading to a stop codon in exon 7. Mice with erythroid-specific SEC23B deficiency were generated by crossing the Sec23bfl allele to an EpoR-Cre transgene. Sec23bfl/-/EpoR-CreTg+ mice maintained normal erythropoiesis indistinguishable from their WT littermates. Pancreas-specific knock-out generated by crossing the Sec23bfl allele to p48-Cre or Pdx1-Cre transgenes generated phenotypes indistinguishable from complete SEC23B deficiency, demonstrating that loss of pancreatic Sec23b expression alone is sufficient to explain the perinatal lethality observed in Sec23bgt/gt and Sec23b-/- mice. Our results conclusively demonstrate that in contrast to humans, SEC23B-deficiency results in massive pancreatic degeneration in mice, but no CDAII in these animals.To investigate the cause of the disparate human and mouse SEC23B-deficient phenotypes, SEC23B/SEC23A expression ratios were examined in endogenous tissues from both species. This ratio (normalized to the ratio in liver mRNA as 1.0) was higher in mouse pancreas (12.7) compared to BM (2.6), with the reverse pattern observed in human BM (7.8) relative to pancreas (5.5). These data, taken together with the high degree of identity between SEC23A and SEC23B (∼ 85% amino acid identity), suggest that the tissue-specific functions of SEC23A and SEC23B may have shifted during evolution between humans and mice. To test the role of SEC23A, we generated a mouse with a conditional Sec23a allele, in which exon 3 is flanked by loxP sites (Sec23afl). Cre-recombinase mediated deletion of exon 3 results in a frame shift leading to a stop codon in exon 7 (Sec23a-). Mice with erythroid-specific SEC23A-deficiency (Sec23afl/-/EpoR-CreTg+) maintained normal red blood cell counts indistinguishable from their WT littermates.In summary, we have shown that SEC23B-deficient humans and mice exhibit disparate phenotypes. We have also demonstrated variations in the gene expression programs for SEC23A and SEC23B potentially explaining the pancreatic phenotype of SEC23B-deficiency in mice and the erythroid phenotype in humans. These results suggest that the two SEC23 paralogs have overlapping functions and that therapeutic strategies that increase the expression of either SEC23A or SEC23B in erythroid cells might be effective in CDAII. Further studies of the overlapping functions of SEC23A and SEC23B and their relevant protein cargos should provide new insight into the pathogenesis of CDAII. Disclosures:No relevant conflicts of interest to declare.

Development Of a Comprehensive Rapid Next-Generation Sequencing Assay For The Diagnosis Of Inherited Hemolytic Anemia

Hereditary hemolytic anemia is caused by defects in hemoglobin, in the red blood cell (RBC) cytoskeleton proteins, or by deficiencies in RBC enzymes. RBC cytoskeletal disorders include hereditary spherocytosis (HS), elliptocytosis (HE), pyropoikilocytosis (HPP), and stomatocytosis (HSt), which are typically inherited as autosomal dominant disorders but can also present as recessive forms, frequently severe. The RBC enzymopathies, such as glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK) deficiency, are X-linked or recessively inherited disorders causing hemolytic anemia. Although rare, congenital dyserythropoietic anemias (CDA) are inherited red cell lineage disorders which occasionally, especially CDA-II, can be misdiagnosed as HS. Diagnosis of an inherited hemolytic anemia not due to a hemoglobin disorder is based upon morphology of the RBCs, functional analysis of the RBCs through ektacytometry or osmotic fragility, and enzymatic assays. The diagnosis of severely affected patients is complicated by transfusion dependence. Diagnosis based on genetic mutations is attractive, especially in transfused patients, and provides additional insight into the mechanisms of disease. Despite these advantages, genetic diagnoses have been limited by expense and long turn-around times for clinical results. To address these issues, we have developed a rapid comprehensive clinical next-generation sequencing-based assay that evaluates 27 genes with published disease-causing mutations for RBC cytoskeletal disorders, enzymopathies, and CDAs. The protein-coding exons plus 25 bases of exon/intron junction as well as promoter sequences with known relationship to clinical phenotypes were included in the design. Genomic DNA was digested with a panel of 8 restriction enzymes and oligonucleotide probes were used to enrich the target regions. Enriched samples were then sequenced on an Illumina MiSeq benchtop sequencer with 150 base pair, paired-end reads. Enrichment and sequencing were completed within 48 hours. Sequencing reads were aligned to the human genome reference sequence and analysis of coverage and variants was completed using NextGENe software.Initial validation included 5 affected probands, 1 affected sibling of a proband, 4 parental samples, and 2 unrelated control individuals with no history of hemolytic anemia. Overall, > 99% of all nucleotides in the regions of interest had at least 20X sequencing coverage. Our assay confirmed a previously identified maternally inherited nonsense mutation, Y1089X, and a paternally inherited A970D missense variant in SPTA1 in a patient (SPCA) with severe HS. The A970D SPTA1 missense change was also seen in two unaffected parents and an unaffected control, further validating that this missense variant alone does not cause dominant HS. The common SPTA1 allele, αLELY was seen in the clinically unaffected mother of SPCA, but was not found in the affected child, suggesting that this allele must be inherited in trans with a pathologic SPTA1 mutation to have clinical effect. Analysis of additional probands revealed both dominant and recessive forms of HS due to mutations in ANK1. Patient AAHS2 presented with typical dominant HS, and was found to have a novel ANK1 frameshift mutation, c.3464delG. Patient HEEM presented with severe transfusion-dependent anemia and was found to have one reported ANK1 missense mutation, T1075I (ankyrin Tubarao) in the spectrin-binding domain, and one novel nonsense mutation, Y735X. In addition, we identified novel amino acid changes in the newly identified dehydrated HSt gene PIEZO1. HEGR, a patient with hemolytic anemia, splenomegaly and portal vein thrombosis in infancy was found to have a novel 6 base pair insertion at R1462 in PIEZO1. Thal1 was previously diagnosed with dominant β-thalassemia, however had a more severe presentation than expected. We found a PIEZO1missense mutation R2302H in Thal1 that likely explains the severe phenotype.The initial validation of this comprehensive sequencing assay has demonstrated that this is a robust and rapid diagnostic tool for patients with severe hereditary hemolytic anemia. Simultaneous investigation of the key protein-coding genes involved in proper RBC function and survival, provides new insight into the variable phenotypes of patients with hemolytic anemia and ultimately will improve the management of patients with severe disease. Disclosures:No relevant conflicts of interest to declare.

Stage Selective Requirement For The N-Terminus Of GATA1 During Erythropoiesis

The X-linked blood transcription factor GATA1 is required for the survival and maturation of erythroid cells. Loss of Gata1 causes profound anemia and related mid-gestation lethality in mouse models and human GATA1 mutations are associated with congenital dyserythropoietic anemia, congenital thrombocytopenia, porphyria, Diamond-Blackfan anemia (DBA) and acute megakaryoblastic leukemia in children with Down syndrome (DS-AMKL). In DBA, DS-AMKL, and a subset of congenital anemia, GATA1 mutations cause the exclusive production of a shorter GATA1 isoform, termed GATA1s. The GATA1s isoform retains both zinc fingers and binds DNA and cofactors accordingly, but lacks the N-terminal 83 residues of full-length GATA1. Intriguingly, exclusive production of GATA1s promotes AMKL in the context of Down syndrome (trisomy 21), while disomic individuals harboring the same type of mutations suffer from anemia and severe red cell defects. A mutation causing exclusive production of Gata1s in mice does not appear to affect adult hematopoiesis, but has been shown to cause a marked expansion of megakaryocytes in the fetal liver. The effects of the replacement of Gata1 with Gata1s during fetal liver erythropoiesis, however, remain uncharacterized.To investigate the effects of exclusive Gata1s production during fetal hematopoiesis, we performed comprehensive phenotypic and mechanistic studies using Gata1s knock-in embryos. We found significant changes in myelo-erythroid differentiation beyond the known expansion of megakaryocytes. Flow cytometric analysis revealed altered erythroid differentiation at embryonic days 12.5 and 14.5, evidenced by decreased Ter119 and increased CD71 expression, characteristic of delayed erythroid maturation. Changes in expression of myelo-erythroid progenitor commitment markers were also discovered. Specifically, we observed marked decreases in pre-CFU-E and CFU-E committed progenitors and an increase in pre-GM and pre-MegE populations. Our findings are consistent with a bias towards megakaryopoiesis at the expense of erythroid commitment caused by the expression of Gata1s in place of full-length Gata1. A shift in differentiation was also observed in the embryonic granulocyte/ macrophage lineage, with an increased generation of macrophages with fewer developing granulocytes. Mechanistically, we found that expression of erythroid -specific Gata1 target genes such as Alas2, Slc4a1 and Klf1 are markedly reduced in the Gata1 knock-in erythroid cells, indicating that Gata1s is a less effective transcriptional activator than full-length Gata1. In particular, given that Klf1 functions to promote erythroid specification downstream of the megakaryocyte-erythrocyte progenitor, our results suggest that Gata1s may promote megakaryocyte differentiation at the expense of erythroid differentiation, in part, by failing to activate Klf1.Taken together, our studies demonstrate a stage-specific requirement for full-length Gata1 during embryonic erythropoiesis. Furthermore, erythroid defects associated with exclusive production of Gata1s in humans may result from an incomplete activation of the erythroid transcriptional program. Disclosures:Crispino:Sanofi: Research Funding.

The Patient With Transfusion-Dependent Anemia: Diagnosis and Directed Management With Targeted Next Generation Sequencing and Copy Number Analysis

Management of the patient with transfusion-dependent anemia (TDA) is complex. Diagnosis is frequently difficult as numerous disorders may lead to TDA, including bone marrow failure syndromes, congenital dyserythropoietic anemias, or inherited hemolytic anemias. Assigning the diagnosis may be problematic as transfused blood or reticulocytosis confound diagnostic testing, or, mutant erythrocytes are so unstable, they are rapidly destroyed. Complications of chronic transfusion include iron overload, infection risk, alloimmunization, cost, and inconvenience. TDA is an excellent candidate for targeted next generation sequencing. There is significant genotypic variability and many of the associated genetic loci are very large, making traditional sequencing strategies cumbersome. We studied 21 patients with TDA using genome-wide targeted exon capture followed by high-throughput next generation DNA sequencing (whole-exome sequencing, WES) using a NimbleGen SeqCap EZ Exome v2.0 solution-based capture system followed by next-generation sequencing on a HiSeq 2000 with paired-end sequencing at 75bp read length. The male:female ratio was 13:8. Age at referral ranged from 2 months to 14 years. All patients were transfusion dependent by 6 months of age. Working diagnoses included possible marrow failure syndrome, congenital dyserythropoietic anemia, and possible enzyme or membrane defect. Variant analyses were performed using the GATK pipeline. Targeted filtering and annotation of protein changing variants in 154 erythrocyte disease genes were performed using the ANNOVAR algorithm. Variants were assessed by mutation prediction and conservation programs including PolyPhen2, Sift, LRT, and Mutation Taster. Variants were also assessed for occurrence and frequency Thousand Genomes, Exome Sequencing Project, dbSNP, on line and local mutation databases, and PubMed. Copy number variants were assessed by ExomeCount and visual inspection. Potential disease-associated variants were validated by Sanger sequencing of DNA from the proband and parents. Interpretation was made using historical, clinical, laboratory and genetic data. The most common diagnosis was hereditary spherocytosis due to alpha spectrin gene (SPTA1) mutations, found in 7 patients. Two patients had deleterious mutations in both SPTA1 alleles; one with nonsense mutations in trans died of liver failure associated with iron overload, the other with nonsense and splicing mutations in trans remains transfusion dependent. One patient homozygous for an SPTA1 missense mutation in a highly conserved, functionally important amino acid had a sibling homozygous for the same mutation die in the perinatal period due to complications of anemia. Finally, one patient with SPTA1 nonsense and missense mutations in trans became transfusion independent post splenectomy. Ten patients had defects in erythrocyte metabolism. Mutations in the pyruvate kinase gene PKLR were found in 6 patients; two of these patients had deletions in the PKLR gene locus suggested by WES and confirmed by Gap PCR and MLPA. Three patients had bi-allelic mutations in the glucose phosphate isomerase gene and one had bi-allelic mutations in the hexokinase gene. Homozygosity was found in 4 of 10 patients with metabolic gene mutations. A single patient had beta thalassemia major with homozygous beta-globin gene mutations. Confirmatory functional studies are underway in three patients. Two TD patients had bone marrow findings suggestive of hypoplastic anemia; one had a missense mutation in a highly conserved residue of RPS7, recently associated with Diamond Blackfan anemia; the other had a deleterious mutation in FANCI predicted to function as a dominant negative. Functional studies are underway in a third patient with likely deleterious, membrane-linked variants. Application of targeted WES to TDA allows precise diagnosis to guide appropriate therapy, e.g. splenectomy or transplant; it allows genetic counseling of associated family members, and permits diagnosis and expectant management of future pregnancies. Targeted WES is an excellent tool for application to monogenic hematologic diseases where genotypic variability, i.e. mutations in numerous genes, leads to the same clinical phenotype. Examples include bone marrow failure syndromes, hemolytic anemia, congenital neutropenia, and immunodeficiency syndromes. Disclosures:No relevant conflicts of interest to declare.

Congenital dyserythropoietic anemia in China: a case report from two families and a review

Congenital dyserythropoietic anemias (CDAs) are a group of hereditary disorders characterized by ineffective erythropoiesis and distinct morphological abnormalities of erythroblasts in the bone marrow. Most cases of CDA, caused by a wide spectrum of mutations, have been reported from Europe and Mediterranean countries, while a few cases have been described in China. Here, we present three cases of CDA, one from one family and two from a second unrelated family, with typical morphologic features and clinical presentations. Sequence analysis of CDA-related genes revealed that the proband with CDA Ι in the first family was a compound heterozygote of CDAN1 with mutation IVS-12+2T>C and c. 3389C>T, while both probands with CDA ΙΙ in the second family were a homozygote of the SEC23B gene with mutation c.938G>A (R313H). This study suggests that more patients with CDA, sharing a phenotype and genetic background like those of European and Mediterranean origin, remain to be diagnosed and reported in China.

John W.Stirling, AlanCurry and BrianEydenDiagnostic Electron Microscopy – A Practical Guide to Interpretation and Technique (RMS – Royal Microscopical Society)John Wiley & Sons, West Sussex, UK, 2013. 468 pages. Price £71.25 (Hardback, Amazon). ISBN 978‐1‐119‐97399‐7

I first came across a pre-publication copy of this book at a conference on a trade stand and instantly knew that I wanted to read it from just having a quick flick through. This new and exciting book covers a wide range of the most common areas in diagnostic pathology at the ultrastructural level, including chapters on muscle, nerve and CADASIL. There are also extremely informative and helpful chapters about the practicalities of processing, cutting and viewing different types of specimen. Each chapter is written by specialists in that particular field. The book itself has a very pleasing lay out and text which is clear, flowing and easily accessible. The electron micrographs are numerous and high in quality, with clear and concise figure legends which are well referenced within the main body of the text. It is easy to dip in and out of and find the particular area of interest. The editors of this book state that they have not attempted to reproduce previous encyclopaedic texts of TEM in diagnostic pathology. However for a relatively small book a lot of ground is covered. Chapters cover the ultrastructural pathology of renal disease, transplant renal biopsies, skeletal muscle, nerve, tumours, microbial ultrastructure, ciliary disorders and sperm centriolar abnormalities, lysosomal storage diseases, CADASIL, platelet disorders, congenital dyserythropoietic anaemia types I and II, Ehlers-Danlos Syndrome, and occupational and environmental lung disease. I like the fact that where appropriate many of the chapters start by describing and illustrating ‘normal’ tissue, something you never normally see in diagnostic electron microscopy. The book also covers the practical aspects of electron microscopy, including how to cut up and process samples in order to gain the maximum amount of information from them. Detailed protocols are included and, having had to do TEM on a histological section on a slide for the first time recently, I can speak from experience and say that the protocols are clear, easy to follow and really do work. There is also a section on trouble shooting problems with sectioning of blocks which is useful, and a discussion of hot topics in modern diagnostic electron microscopy. This includes chapters covering digital imaging in diagnostic EM, the uncertainly of measurement and the impact of microwave technology and telemicroscopy. From a neuropathological point of view the CADASIL chapter provides a logical and practical approach for how to screen the sample for the presence of the classical GOM deposits that confirm a positive diagnosis of CADASIL. The electron micrographs in this chapter show clear examples of the GOM deposits and also show that they should not be confused with other non-specific electron dense deposits often see in samples that are submitted as possible CADASIL. The chapters on skeletal muscle and nerve cover 64 pages in total and give a good but brief overview of these tissues. The chapters cover practical aspects of how to handle and process these tissues, the ultrastructure of normal tissue, possible artefacts, and pathological changes. The chapter on lysosomal storage diseases is particularly useful for those rare occasions when you see them in clinical practice. The chapter provides a good overview of these diseases, the majority of which have CNS involvement. The chapter contains an array of electron micrographs demonstrating the ultrastructural findings of some of these diseases in skin biopsies, which are the most cost effective first line diagnostic tool in these cases. Overall I feel that this book is a valuable and useful addition to the bookshelf of anyone involved in electron microscopy who works in a diagnostic setting, be they an electron microscopist or a pathologist. It covers a lot of ground in just 468 pages. Priced at £71.25 (http://www.amazon.co.uk), it offers excellent value for money. This book is also available as a kindle edition with a price of £49.88 (http://www.amazon.co.uk). The clear explanations, electron micrographs and practical advice (that really works) make this a good all round diagnostic EM reference book. I would highly recommend it.

Successful treatment of an infant with CDA type II by intrauterine transfusions and postnatal stem cell transplantation

Congenital dyserythropoietic anemias are rare hematological disorders leading to ineffective erythropoiesis with chronic anemia, complicated by iron overload. Here we present a remarkable clinical course of an infant with CDA type II who first presented as a severe fetal hydrops, requiring serial intrauterine red cell transfusions. While postnatal transfusion dependency persisted, the patient was successfully transplanted with a myeloablative conditioning regimen and peripheral blood stem cells of a matched donor. We believe that allogeneic HSCT is a reasonable therapeutic approach for patients with very severe CDA, even if only a matched unrelated donor is available.

Tissue iron evaluation in chronically transfused children shows significant levels of iron loading at a very young age

Chronic blood transfusions start at a very young age in subjects with transfusion-dependent anemias, the majority of whom have hereditary anemias. To understand how rapidly iron overload develops, we retrospectively reviewed 308 MRIs for evaluation of liver, pancreatic, or cardiac iron in 125 subjects less than 10 years old. Median age at first MRI evaluation was 6.0 years. Median liver iron concentrations in patients less than 3.5 years old were 14 and 13 mg/g dry weight in thalassemia major (TM) and Diamond-Blackfan anemia (DBA) patients, respectively. At time of first MRI, pancreatic iron was markedly elevated (> 100 Hz) in DBA patients, and cardiac iron ( R₂* >50 Hz) was present in 5/112 subjects (4.5%), including a 2.5 years old subject with DBA. Five of 14 patients (38%) with congenital dyserythropoietic anemia (CDA) developed excess cardiac iron before their 10th birthday. Thus, clinically significant hepatic and cardiac iron accumulation occurs at an early age in patients on chronic transfusions, particularly in those with ineffective or absent erythropoiesis, such as DBA, CDA, and TM, who are at higher risk for iron cardiomyopathy. Performing MRI for iron evaluation in the liver, heart, and pancreas as early as feasible, particularly in those conditions in which there is suppressed bone marrow activity is very important in the management of iron loaded children in order to prescribe appropriate chelation to prevent long-term sequelae. .

Characteristic phenotypes associated with congenital dyserythropoietic anemia (type II) manifest at different stages of erythropoiesis

Congenital dyserythropoietic anemia type II is an autosomally recessive form of hereditary anemia caused by SEC23B gene mutations. Patients exhibit characteristic phenotypes including multinucleate erythroblasts, erythrocytes with hypoglycosylated membrane proteins and an apparent double plasma membrane. Despite ubiquitous expression of SEC23B, the effects of mutations in this gene are confined to the erythroid lineage and the basis of this erythroid specificity remains to be defined. In addition, little is known regarding the stage at which the disparate phenotypes of this disease manifest during erythropoiesis. We employ an in vitro culture system to monitor the appearance of the defining phenotypes associated with congenital dyserythropoietic anemia type II during terminal differentiation of erythroblasts derived from small volumes of patient peripheral blood. Membrane protein hypoglycosylation was detected by the basophilic stage, preceding the onset of multinuclearity in orthochromatic erythroblasts that occurs coincident with the loss of secretory pathway proteins including SEC23A during erythropoiesis. Endoplasmic reticulum remnants were observed in nascent reticulocytes of both diseased and healthy donor cultures but were lost upon further maturation of normal reticulocytes, implicating a defect of ER clearance during reticulocyte maturation in congenital dyserythropoietic anemia type II. We also demonstrate distinct isoform and species-specific expression profiles of SEC23 during terminal erythroid differentiation and identify a prolonged expression of SEC23A in murine erythropoiesis compared to humans. We propose that SEC23A is able to compensate for the absence of SEC23B in mouse erythroblasts, providing a basis for the absence of phenotype within the erythroid lineage of a recently described SEC23B knockout mouse.

Congenital dyserythropoietic anemia type II with novel mutations in SEC23B and HFE2 genes: a Chinese family survey

To report novel mutations SEC23B gene in congenital dyserythropoietic anemia (CDA). By direct sequencing method, we sequenced CDAN1 and SEC23B genes in a Chinese CDA II patient, presented with chronic fatigue and dark urine, as well as his family members. Serum hepcidin was assayed by mass spectrometry. We found a c.71G>A mutation and a c.74C> A mutation in the patient. In addition, a heterozygous c.55A>G mutation of HFE2 gene was found in some family members. The level of serum hepcidin of the patient was below the detection limit (<1 nmol/L). Contrary with what have been reported previously in the Europe, especially in the Italy, the gene mutations identified in this case was different and novel. The two novel mutations contribute to the diagnosis of CDAII and are the first report in East Asian CDAII patients.

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,

Congenital dyserythropoietic anemia type III (CDA III) is caused by a mutation in kinesin family member, KIF23

AbstractHaplotype analysis and targeted next-generation resequencing allowed us to identify a mutation in the KIF23 gene and to show its association with an autosomal dominant form of congenital dyserythropoietic anemia type III (CDA III). The region at 15q23 where CDA III was mapped in a large Swedish family was targeted by array-based sequence capture in a female diagnosed with CDA III and her healthy sister. Prioritization of all detected sequence changes revealed 10 variants unique for the CDA III patient. Among those variants, a novel mutation c.2747C>G (p.P916R) was found in KIF23, which encodes mitotic kinesin-like protein 1 (MKLP1). This variant segregates with CDA III in the Swedish and American families but was not found in 356 control individuals. RNA expression of the 2 known splice isoforms of KIF23 as well as a novel one lacking the exons 17 and 18 was detected in a broad range of human tissues. RNA interference–based knock-down and rescue experiments demonstrated that the p.P916R mutation causes cytokinesis failure in HeLa cells, consistent with appearance of large multinucleated erythroblasts in CDA III patients. We conclude that CDA III is caused by a mutation in KIF23/MKLP1, a conserved mitotic kinesin crucial for cytokinesis.

SP0076 Emerging genetics and molecular aspects of autoinflammatory bone disease

Autoinflammatory disorders of the bone are innate immune system disorders that result in chronic sterile inflammation in the bone. Chronic recurrent multifocal osteomyelitis (CRMO) is the most common autoinflammatory bone...

Nonimmune hydrops fetalis caused by G6PD deficiency hemolytic crisis and congenital dyserythropoietic anemia

We present a case of a female neonate who had a nonimmune hydrops fetalis and severe hemolytic anemia due to a rare combination of glucose-6-phosphate dehydrogenase (G6PD) deficiency and congenital dyserythropoietic anemia. We conclude that in severe cases with persistent anemia one should search after delivery for a second reason other than G6PD deficiency alone.

Homozygous mutations in a predicted endonuclease are a novel cause of congenital dyserythropoietic anemia type I.

The congenital dyserythropoietic anemias are a heterogeneous group of rare disorders primarily affecting erythropoiesis with characteristic morphological abnormalities and a block in erythroid maturation. Mutations in the CDAN1 gene, which encodes Codanin-1, underlie the majority of congenital dyserythropoietic anemia type I cases. However, no likely pathogenic CDAN1 mutation has been detected in approximately 20% of cases, suggesting the presence of at least one other locus. We used whole genome sequencing and segregation analysis to identify a homozygous T to A transversion (c.533T>A), predicted to lead to a p.L178Q missense substitution in C15ORF41, a gene of unknown function, in a consanguineous pedigree of Middle-Eastern origin. Sequencing C15ORF41 in other CDAN1 mutation-negative congenital dyserythropoietic anemia type I pedigrees identified a homozygous transition (c.281A>G), predicted to lead to a p.Y94C substitution, in two further pedigrees of SouthEast Asian origin. The haplotype surrounding the c.281A>G change suggests a founder effect for this mutation in Pakistan. Detailed sequence similarity searches indicate that C15ORF41 encodes a novel restriction endonuclease that is a member of the Holliday junction resolvase family of proteins.

脾摘により輸血依存から脱した congenital dyserythropoietic anemia（CDA）II 型の1 例

脾摘により輸血依存から脱した congenital dyserythropoietic anemia（CDA）II 型の1 例

Congenital dyserythropoietic anemia type 1 with a novel mutation in the CDAN1 gene previously diagnosed as congenital hemolytic anemia

The congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of genetic disorders of red cell production. They are characterized by ineffective erythropoiesis and dyserythropoiesis. Here, we present the clinical description and mutation analysis of a Japanese female with CDA type 1. She has long been diagnosed with unclassified congenital hemolytic anemia from the neonatal period. However, bone marrow morphology and genetic testing of the CDAN1 gene at the age of 12years confirmed the afore-mentioned diagnosis. Thus, we should be aware of the possibility of CDA if the etiology of congenital anemia or jaundice cannot be clearly elucidated.

Micromapping of Very Rare Anemias: The Model of CDA

The congenital dyserythropoietic anemias, first described in 1966 and classified into four distinct types in 1968, are still very rare. However, many cases were described in recent years, mainly in European Countries. The detection of mutations of the CDAN1-gene (Tamary et al. from Israel) in all cases of CDA I and of the SEC23B-gene in almost all cases of CDA II (Schwarz, Iolascon, Heimpel et al. from Germany and Italy, Zanella, Bianchi et al. from Italy) stimulated greater awarness of the CDAs and resulted in many recent reports from all continents. Period prevalance of the the last 50 years in the European countries collated in the German Registry on CDAs were calculated in 2009 and published in 2010. The cumulated incidence of both types combined variedwidely between European regions, with minimal values of 0.08 cases⁄million in Scandinavia and 2.60 cases⁄million in Italy. CDA II is more frequent than CDA I, with an overall ratio of approximately 3.2, but the ratio also varied between different regions. The most likely explanations for the differences are both differences in the availability of advanced diagnostic procedures and different levels of the awarenessfor the diagnosis of the CDAs. The estimations reported here are most probably below the true incidence rates, because of failure to make the correct diagnosis and to underreporting. Limited data did not suggest differing levels of risk in identified ethnic groups. Here, we report the first results of an update of these data and decribe a project to extent epidemiological research to all regions of the world. Based on the experience with the previous attempt to measure, performed in the framework of ENERCA, we discuss the limitations and methodological problems and present the hypothesis, that the CDAs are not dependent from environmental conditions such as malaria or other contagious diseases, but the apparent prevalence data depend rather on factors of the medical system and the rates of consanguinity in different cultures. Since the prognosis of the CDAs depend on appropriate management, consultation by specialized centers of rare diseases, today available in Europe, could improve life expectancy and quality of life of individuals affected by this very rare diseases.

Erythroid transcription factor EKLF/KLF1 mutation causing congenital dyserythropoietic anemia type IV in a patient of Taiwanese origin: Review of all reported cases and development of a clinical diagnostic paradigm

KLF1 is an erythroid specific transcription factor that is involved in erythroid lineage commitment, globin switching and terminal red blood cell maturation. Various mutations of KLF1 have been identified in humans, which have led to both benign and pathological phenotypes. The E325K mutation, within the second zinc finger of the KLF1 gene, has been shown to cause a new form of congenital dyserythropoietic anemia (CDA) now labeled as CDA type IV. We report the fourth documented case of this mutation, and propose a clinical diagnostic model to better identify this disease in other patients. Our patient is a Taiwanese child who presented to us at 8years of age with severe hemolytic anemia, splenomegaly, elevated fetal hemoglobin (HbF), iron overload, and dyserythropoiesis in the bone marrow. KLF1 sequence analysis revealed a G-to-A transition in one allele of exon 3, which resulted in the substitution of a glutamate 325 by a lysine. Flow cytometry analysis revealed decreased protein expression of CD44 on the red blood cells, and decreased red blood cell deformability as measured using an ektacytometer. Blood typing revealed his red blood cells to be Co(a-b-), In(b-), LW(ab-) and Lu(b+), even though DNA testing predicted that he would be Co(a+b-) and LW(a+b-). This newly discovered CDA combines features of a hemoglobinopathy, RBC membrane defect and hereditary persistence of HbF (HPFH) which are not seen in the previous types of CDA. Increased awareness of this phenotype may improve the more prompt and accurate diagnosis of these patients.

Hypomorphic mutations of SEC23B gene account for mild phenotypes of congenital dyserythropoietic anemia type II

Congenital dyserythropoietic anemia type II, a recessive disorder of erythroid differentiation, is due to mutations in SEC23B, a component of the core trafficking machinery COPII. In no case homozygosity or compound heterozygosity for nonsense mutation(s) was found. This study represents the first description of molecular mechanisms underlying SEC23B hypomorphic genotypes by the analysis of five novel mutations. Our findings suggest that reduction of SEC23B gene expression is not associated with CDA II severe clinical presentation; conversely, the combination of a hypomorphic allele with one functionally altered results in more severe phenotypes. We propose a mechanism of compensation SEC23A-mediated which justifies these observations.