New Insights in Congenital Dyserythropoietic Anemias

Red cell disease New insights in congenital dyserythropoietic anemias

A. Iolascon1,2 ABSTRACT M.R.Esposito1 Dyserythropoiesis appears to be a qualitative and a quantitative defect of erythropoiesis and occurs in a wide range of diseases embracing a number of conditions that primarily affect the nucleus or the 1CEINGE Biotecnologie Avanzate, Napoli, Italy cytoplasm of erythroblasts or the environment in which erythropoiesis takes place. This could be an 2Department of Biochemistry acquired or an inherited defect; it may include dyserythropoiesis as the principal (congenital dysery- and Medical Biotechnologies, thropoietic anemias, CDA) or a secondary characteristic (thalassemia syndromes; unstable hemoglo- University Federico II of Naples, bins or thiamine- responsive anemias). One of the main problems of the CDAs is their heterogeneity, Italy both at clinical and genetic levels. In general, the features of dyserythropoiesis, in terms of ineffective erythropoiesis, should be demonstrated by several criteria: bone marrow evaluation by light microscopy, ultrastructural features, assessment of red cell production and destruction, and studies of Hematology Education: iron metabolism. Certainly the main assessment method, and one which is easy to perform, is light the education program for the microscopy. Biochemical approach (such as in CDA II) or a molecular approach (such as in CDA I or II) annual congress of the European are also indicated. Mapping and cloning showed that these conditions have different molecular mech- Hematology Association anisms that induce disturbances of cell maturation and of cell division during erythropoiesis. Cloning of codanin 1 and Sec23b genes allowed for the molecular characterization of these two 2011;5:316-322 conditions. Moreover, cases with clinical and morphological features of CDA , both type I and II, and without mutations in these genes clearly demonstrated genetic heterogeneity. The molecular basis of several cases was recently obtained by characterization of forms due to eKLF-1 and GATA-1 defects.

he congenital dyserythropoietic ane- increased hemoglobin turnover, such as mild mias (CDAs) comprise a group of rare jaundice, and low or absent haptoglobin, as Thereditary disorders that are charac- in hemolytic anemia, with a reticulocytosis terized by ineffective erythropoiesis as the that does not correspond to the degree of predominant mechanism of anemia and by anemia. The bone marrow is always hyper- distinct morphological abnormalities of ery- cellular, exclusively due to a pronounced throblasts in the bone marrow. Diagnosis increase of erythroblasts, with increased ery- has to exclude other congenital anemias thropoietic/granulopoietic ratio (normal ref- such as the thalassemia syndromes, certain erence values 0.3 – 1.0). All types of CDA types of pathological hemoglobins or hered- share a high incidence of cholelithiasis and itary sideroblastic anemias, megaloblastic distinct iron loading. As in other forms of anemia due to vitamin deficiencies or anemia with ineffective erythropoiesis, this myelodysplastic syndromes.1,2 is due to up-regulation of iron resorption, The term “dyserythropoiesis” was first mediated by hepcidine19. Extramedullary used by Crookston et al.3 (for cases later clas- hematopoiesis presenting as paravertebral sified as CDA II) and by Wendt and bulks may be observed in all types of CDA. Heimpel4 (for cases later classified as CDA I). CDA type III was reported in 1962 in a The working classification (CDA I, II and III) Swedish family accounting for 34 patients. mainly due to Heimpel studies is still used in This observation allowed for the mapping of clinical practice. There are, however, fami- the gene on chromosome 15. Very few cases lies that fulfill the general definition of the are present in literature and this impeded CDAs, but do not conform to any of the further molecular insights. This review will three classical types (Table 1). These classify- deal with new insights on the two most fre- ing criteria could explain part of the genetic quent forms of CDA: type I and II. heterogeneity of these conditions, since dyserythropoiesis appears to be a morpho- logic criteria common to several (inherited CDA type I and acquired) conditions. Recent identifica- tion of several causative genes could help in Until August 2009, 169 cases from 143 reclassifying these disorders (Table 1). families with CDA I were recorded in the lit- Ineffective erythropoiesis in combination erature, and hence this condition appears with moderately shortened red cell lifespan, approximately 3 times less frequently than particularly in type II, are common features. CDA II. Most families presenting were A dyserythropoietic anemia could be sus- Western European and Arab, but single cases pected in presence of symptoms and signs of were also reported from the USA, India,

| 316 | Hematology Education: the education programme for the annual congress of the European Hematology Association | 2011; 5(1) London, United Kingdom, June 9-12, 2011

Table 1. Characteristic features of different types of congenital dyserythropoietic anemias.

CDA type I II III familial III sporadic Variants

Inheritance Autosomal-recessive Autosomal-recessive Dominant Recessive/X-linked Autosomal-recessive

Cases reported ~150 ~370 3 families <10 >20

Morphology Abnormal chromatin Binuclearity of mature Giant multinucleated Giant multinucleated CDA I-like structure, chromatin erythroblasts erythroblasts erythroblasts CDA II-like bridges others

Gene codanin SEC23B 15q (21-25) eKLF1 / GATA1 unknown 15q (15.1.3) 20p (11.2)

Associated Skeleton, others Variable, rare B-Cells variable CNS dysmorphology Retina others

Japan, and Australia.5 mutations were detected.9,10 Approximately 90% of The clinical picture includes anemia, sometimes with patients with a bone marrow suggesting CDA I have neonatal appearance, jaundice, splenomegaly, codanin gene mutations. Interestingly, families with hepatomegaly, frequent and diverse dysmorphisms, definite phenotype of CDA I in which no mutation of predominantly affecting the digits (particularly syn- the CDAN1-gene could be found suggested either a pro- dactyly in hands or feet, absence of nails or supernumer- moter defect or a mutation in another gene11 (genetic ary toes)1-6 and a progressive build up of iron overload. heterogeneity). These observations, associated with the The degree of anemia is variable not only between fam- exclusion of linkage with chromosome 15 in several ilies, but for unexplained reasons (modifier genes) may families, suggested the presence of a second disease also be different among siblings.6 Most patients have locus.11 life-long anemia with hemoglobin concentration The vast majority of patients with confirmed diagno- between 7 and 11 g/dL. Occasionally, there are severe sis of CDA I showed mutations of at least one allele cases requiring transfusion in-utero7 or immediately from exons 6 to 28 within CDAN1, and more than 30 after birth, and regular blood transfusions during child- unique mutations have been identified so far (Table 2). hood and adolescence.6 On the other side there are Interestingly, no homozygotes or compound heterozy- patients with only low-borderline hemoglobin but dis- gotes for null-type mutations have been identified, sup- tinct macrocytosis. The anemia is usually macrocytic porting an earlier notion that codanin-1 may have a with MCV between 100 and 120 fl, but may be normo- unique function and may be essential during develop- cytic in childhood. ment. Light microscopy of bone marrow demonstrates 30 to The mutated gene (CDAN1) encodes a ubiquitously 60% of early and late polychromatic erythroblasts expressed protein (codanin-1) of unknown function.9 showing characteristic and partially bizarre abnormali- This protein is still not well characterized, but it seems ties of nuclear shape and size and of chromatin struc- to be related to chromosome structure and it must be ture, but proerythroblasts and immature basophilic ery- involved in mitotic process. Tamary et al. have previous- throblasts usually appear normal. There are large poly- ly shown that codanin-1 is a direct transcriptional target ploid cells, and a minority of cells are bi- or multinucle- of the E2F1 transcription factor and that the levels of ated; in contrast to CDA II, nuclei are of different size codanin-1 increase during S-phase and decrease during and stain. The hallmark of CDA I is incompletely divid- mitosis.12 Furthermore, they conducted a yeast two- ed cells with thin chromatin bridges between pairs of hybrid screen using a human bone marrow library and erythroblasts, which may also be seen between two found that codanin-1 binds to Asf1a. Asf1 (anti silencing nuclei in a single cell. Electron microscopy studies function) is a H3/H4 histone chaperone involved in the demonstrated that heterochromatin is denser than nor- chromatin structure dynamics by its role in nucleosome mal and forms sharply delineated clumps with small assembly and disassembly. Coimmunoprecipitation translucent vacuoles, giving rise to the description of experiments confirmed that histone chaperone Asf1a is “Swiss cheese appearance”, and cytoplasm may pene- a direct binding partner of codanin-1. They suggested trate through widened pores of the nuclear envelope.8 that binding of codanin-1 inhibits dissociation of his- The gene responsible for CDA I (CDAN1 gene) was tones from Asf1a, which therefore cannot be deposited mapped to the long arm of chromosome 15 between onto DNA.13 15q15.1q15.3 by homozygosity mapping in four Very recently, another contribution attempts to define Bedouin families with a high degree of consanguinity. the role of codanin-1 in pathophysiology of CDA type Similar results were reported in patients from Europe I. Renella et al. investigated localization, distribution and and the Near East. The CDAN1 gene was successively interactions of codanin-1 in CDA I patients and generat- cloned with 28 exons spanning 15 Kb encoding a pro- ed a murine knock-out model for CDAN1. They found tein named codanin-1. In nine unrelated patients of no gross differences between normal and patient sam- European, Bedouin and Asian origin, different point ples both in the amounts of histone proteins or various

Hematology Education: the education programme for the annual congress of the European Hematology Association | 2011; 5(1) | 317 | 16th Congress of the European Hematology Association

Table 2. Mutational spectrum of CDAN1/CDAN2

Gene Exon Nucleotide change°/* Legacy namea/b Protein domain affected Ref. for published mutations

CDAN1 (53 cases) missense 2 c.156 C>G F52L Tamary, 2005 Br J Haematol 6 c.1078 T>C F360L Heimpel, 2006 Blood 12 c.1796 A>G N599S Dgany, 2002 Am J Hum Genet 14 c.2015 C>T P672L Dgany, 2002 Am J Hum Genet 14 c.2062 C>T R688W Tamary, 2005 Br J Haematol 14 c.2069 T>C V690A Ru, 2008 Ann Hematol 14 c.2092 G>A E698K Dgany, 2002 Am J Hum Genet 14 c.2140 C>T R714W Dgany, 2002 Am J Hum Genet 14 c.2173 C>T R725W Heimpel, 2006 Blood 15 c.2248 G>T G750C Heimpel, 2006 Blood 19 c.2602 T>A F868I Dgany, 2002 Am J Hum Genet 19 c.2605 G>A V869M Dgany, 2002 Am J Hum Genet 20 c.2650 A>G T884A Tamary, 2005 Br J Haematol 24 c.3107 C>A S1036F Dgany, 2002 Am J Hum Genet 24 c.3124 C>T R1042W Dgany, 2002 Am J Hum Genet 24 c.3128 A>T D1043V Dgany, 2002 Am J Hum Genet 24 c.3194 G>A R1065Q Heimpel, 2006 Blood 26 c.3389 C>T P1130L Dgany, 2002 Am J Hum Genet nonsense 14 c.2044 C>T R682X Dgany, 2002 Am J Hum Genet 18 c.2539 C>T Q847X Tamary, 2005 Br J Haematol 23 c.2992 C>T R998X Dgany, 2002 Am J Hum Genet 27 c.3547 C>T Q1183X Heimpel, 2006 Blood splicing 8 c.1367+1 G>A Tamary, 2005 Br J Haematol 12 c.1860+5 G>A Tamary, 2005 Br J Haematol 21 c.2868+2 insCCG Heimpel, 2006 Blood 28 c.3558 del -10 to +31 Heimpel, 2006 Blood frameshift 6 c.1117_19 delGTT V373del Ahmed, 2006 Blood 12 c.1789_1791 delGAG E597del Heimpel, 2006 Blood 23 c.3024 insTT G1008Gfs23X Heimpel, 2006 Blood 24 c.3145 insT S1049Ffs25X Heimpel, 2006 Blood CDAN2 (129 cases) missense 2 c.40 C>T R14W Segment 1 Schwarz, 2009 Nat Genet. 2 c. 53 G>A R18H Schwarz, 2009 Nat Genet. 2 c.197 G>A C66Y Zink finger Fermo, 2010 Blood Cells Mol Dis 4 c.325 G>A E109K Schwarz, 2009 Nat Genet. 7 c.716 A>G D239G Trunk Schwarz, 2009 Nat Genet. 8 c.938 G>A R313H Schwarz, 2009 Nat Genet. 8 c.953T>C I318T Schwarz, 2009 Nat Genet. 9 c.1043 A>C D348A Bianchi, 2009 Hum Mutat. 10 c.1157 A>G Q386R Schwarz, 2009 Nat Genet. 11 c.1254 T>G I418M β-sheet Russo, 2010 Am J Haematol 11 c.1307 C>T S436L Russo, 2010 Am J Haematol 12 c.1385 A>G Y462C Schwarz, 2009 Nat Genet. 13 c.1489 C>T R497C Schwarz, 2009 Nat Genet. 13 c.1508 G>A R503Q Iolascon, 2010 Haematologica 14 c.1571 C>T A524V Helical Schwarz, 2009 Nat Genet. 14 c.1588 C>T R530W Schwarz, 2009 Nat Genet. 14 c.1589 G>A R530Q Iolascon, 2010 Haematologica 14 c.1654 C>T L552F Russo, 2010 Am J Haematol 15 c.1685 A>G Y562C Iolascon, 2010 Haematologica 15 c.1733 T>C L578P Russo, 2010 Am J Haematol 15 c.1735 T>A Y579N Russo, 2010 Am J Haematol 16 c.1808 C>T S603L Bianchi, 2009 Hum Mutat. 16 c.1832 G>C R611P Russo, 2010 Am J Haematol 16 c.1858 A>G M620V Russo, 2010 Am J Haematol 17 c.1968 T>G F656L Gelsolin Iolascon, 2010 Haematologica 18 c.2101 C>T R701C Bianchi, 2009 Hum Mutat. 18 c.2129 C>T T710M Amir, Acta Haematologica in press 19 c.2166 A>C K723Q Segment 6 Iolascon, 2010 Haematologica 20 c.2270 A>C H757P Russo, 2010 Am J Haematol

| 318 | Hematology Education: the education programme for the annual congress of the European Hematology Association | 2011; 5(1) London, United Kingdom, June 9-12, 2011

Table 2. Mutational spectrum of CDAN1/CDAN2

Gene Exon Nucleotide change°/* Legacy namea/b Protein domain affected Ref. for published mutations

nonsense 3 c.235 C>T R79X Zink finger Schwarz, 2009 Nat Genet. 5 c.367 C>T R123X Segment 2 Russo, 2010 Am J Haematol 5 c.568 C>T R190X Trunk Bianchi, 2009 Hum Mutat. 6 c.649 C>T R217X Schwarz, 2009 Nat Genet. 7 c.790 C>T R264X Schwarz, 2009 Nat Genet. 8 c.970 C>T R324X Schwarz, 2009 Nat Genet. 9 c.1015 C>T R339X Russo, 2010 Am J Haematol 10 c.1201C>T R401X Segment 3 Schwarz, 2009 Nat Genet. 14 c.1603 C>T R535X Helical Russo, 2010 Am J Haematol 14 c.1648 C>T R550X Iolascon, 2010 Haematologica 14 c.1660 C>T R554X Bianchi, 2009 Hum Mutat. splicing 2 c.221+31 A>G Zink finger Russo, 2010 Am J Haematol 3 c.279+3 A>G Russo, 2010 Am J Haematol 6 c.689+1 G>A Trunk Schwarz, 2009 Nat Genet. 9 c.1109+1 G>A Russo, 2010 Am J Haematol 9 c.1109+5 G>A Russo, 2010 Am J Haematol 19 c.2149-2 A>G Gelsolin Russo, 2010 Am J Haematol frameshift 3 c.222-817_366+4242del Q75EfsX7 Zink finger Schwarz, 2009 Nat Genet. 5 c.387(del G) L129LfsX26 Trunk Russo, 2010 Am J Haematol 5 c.428_428delAinsCG D143AfsX35 Bianchi, 2009 Hum Mutat. 9 c.1063delG D355IfsX8 Schwarz, 2009 Nat Genet. 16 c.1821delT H608IfsX7 Helical Bianchi, 2009 Hum Mutat. 16 c.1857_1859delCAT I619del Russo, 2010 Am J Haematol 17 c.1962-64 (delT) T654TfsX13 Gelsolin Iolascon, 2010 Haematologica 19 c.2150 (delC) A717VfsX7 Russo, 2010 Am J Haematol

°CDAN1: The nucelotides are numbered from the A of the ATG initiation codon (ENST00000356231). All mutations have been revised according to the rules of nomenclature of HGVS database (http://www.hgvs.org/mutnomen/). *CDAN2: The nucelotides are numbered from the A of the ATG initiation codon (ENST00000377475). a CODANIN: Accession number: Q8IWY9 (UniProtKB/Swiss-Prot). b SEC23B: Accession number: Q15437 (UniProtKB/Swiss-Prot). epigenetic marks of histone tails, suggesting that his- gotes die in-utero before the onset of primitive erythro- tone signatures involved in maintenance of chromatin poiesis, suggesting that Cdan1 has other critical roles structure and epigenetic regulation, are globally main- during embryogenesis. tained in CDA I.14 Another unsolved problem in CDA type I was the Using a hybridoma technique, they demonstrated bone-marrow response of interferon administration. codanin-1 distribution in both nucleus and cytoplasm of Literature data demonstrate that interferon remains an cells in normal primary human erythroblasts. This local- effective treatment of anaemia of CDA I for a long peri- ization pattern was unchanged in CDA I erythroblasts. od, without any noticeable side-effects.15 Furthermore, No differences were found in the distribution patterns its effects on iron status parameters raise the question of of RNA-polymerase-II, PML, nucleophosmin, HP1β and its use even in patients moderately anemic but with a HP1g between patients and control erythroblasts. marked iron overload. Increased expression of GDF-15, However, the localization of HP1βα, a key component which represents a negative regulator of hepcidin, of heterochromatin, was found to be markedly per- seems to be characteristic of this type of anemia and turbed. HP1α accumulates in the Golgi apparatus of related to increased apoptosis on bone marrow ery- CDA I but not normal erythroblasts. They confirmed throblasts.16 that the abnormal localization of HP1α in CDA I patients is confined to the intermediate erythroblast maturation stage, where the characteristic ultrastructur- CDA type II al chromatin pattern of CDA I is observed. Furthermore, they suggested that an abnormality in codanin-1 could CDA type II is an autosomal recessive disorder also be responsible for the aberrant localization of HP1α. known as hereditary erythroblastic multinuclearity Interestingly, by confocal immunofluorescence, they with a positive acidified serum lysis test (HEMPAS).4 also found codanin-1 co-localizes partially with The severity of anemia varies from mild to severe. SEC23B, the protein mutated in CDA II, suggesting a Diagnosis of CDA II is usually made later in life com- molecular link between the two major types of CDA. pared with CDA I, because symptoms can be mild.17,18 As expected by the molecular studies in human Patients may come to attention because of the anemia patients, the total absence of codanin-1 is lethal. combined with jaundice (90% of cases), splenomegaly Renella et al. generated the first murine Cdan1 gene- (70%), or hepatomegaly (45%). CDA II patients with trap model demonstrating its widespread expression Gilbert syndrome had a greater tendency to form gall- during embryonic development. Cdan1gt/gt homozy- stones than those without.19 The evolution of CDA II is

Hematology Education: the education programme for the annual congress of the European Hematology Association | 2011; 5(1) | 319 | 16th Congress of the European Hematology Association doomed, once again, by iron overload and biliary com- secretory pathway in eukaryotic cells is critical for plications, and in about 20% of patients by liver cirrho- membrane homeostasis, localization of proteins within sis secondary to iron overload. In certain cases, the pres- cells and secretion of extracellular factors. During a bud- ence of posterior mediastinal or paravertebral masses ding reaction, cytoplasmic coat proteins (COPs) are consisting of extramedullary hemopoietic tissue could assembled on a membrane surface, capture cargo mole- be present.20-26 The anemia of CDA II is normocytic, cules and polymerize into a cage sculpting different- hemoglobin levels are somewhat lower in children than sized cargo vesicles.38-39 So far, severe mutations in genes in later life, ranging to 8 and 11 g/dl with a normal MCV, encoding COP II components have been assigned to and the peripheral blood shows basically aniso-poikilo- human genetic disorders. SAR1B defects cause chylomi- cytosis without specific types of poikilocytes, with cron retention disease, Anderson disease and basophilic stippling of cells and few, occasionally binu- Marinesco-Sjogren syndrome40, and SEC23A is mutated cleated, mature erythroblasts. Relative reticulocyte in cranio-lenticulo-sutural dysplasia.41 counts are normal or moderately increased. The bone A recent study on 42 patients with CDA type II marrow is hypercellular with erythroid hyperplasia but, showed a correlation between the mutations and vari- in contrast to CDA I, is of normoblastic appearance ous biological parameters. In this study, patients were with a large number of binucleate (10%-35%) and divided into two groups: (1) patients with two missense rarely multinucleate late polychromatic erythroblasts.27 mutations and (2) patients with one nonsense and one On electron microscopic examination, vesicles of endo- missense mutation. Compound heterozygosity for a plasmic reticulum (ER) containing proteins normally missense and nonsense mutation tended to produce found in this organelle, calreticulin, glucose regulated more severe clinical presentations than homozygosity protein (GRP78) and protein disulfide isomerise,28-31 or compound heterozygosity for two missense muta- appear to be running beneath the plasma membrane in tions. Homozygosity or compound heterozygosity for this disorder. two nonsense mutations were never found, and it was Erythrocytes of CDA II patients lyse in acidified supposed to be lethal.42 serum (Ham test) because of an IgM class antibody that In a recent study, we characterized the allelic distribu- recognizes an antigen present on CDA II cells but absent tion of SEC23B gene mutations found in CDA II on normal cells. The test checks whether red blood cells patients from Italy compared to those found in interna- become more fragile when they are placed in mild acid. tional cases. Until now, 53 different causative mutations Usually, this test is done to diagnose paroxysmal noctur- have been described (Table 2). We substantially con- nal hemoglobinuria (PNH), although it is increasingly firmed our previous reports42-43 as the most representa- being replaced by a flow cytometry. The technical diffi- tive mutations in both cohorts were R14W and E109K. culty of this test, and the fact that cross-testing of more Nevertheless, we also showed that the high recurrence than 30 normal sera is needed to obtain a reliable result, of SEC23B-R14W mutation among the Italian CDA II has undermined its usefulness.1 patients is due to a founder effect, while the most fre- In addition to the tendency to lyse in acidified sera, quent variant E109K showed a common genetic back- red blood cells of CDA II patients agglutinate in sera ground among European patients (Russo R. et al., in sub- containing anti-i antigens. This test is sensitive but not mission 122-11- EJHG). specific, and results cannot be compared among differ- Very recently Sec23b deficient mice (Sec23b gt/gt) from ES ent institutions because of the different anti-i sera used. cells with a genetrap cassette inserted into the last intron of The diagnostic hallmark of CDA II is the analysis of Sec23b was generated. Sec23b gt/gt mice die at birth with erythrocyte membrane proteins by sodium dodecyl sul- destruction of the exocrine pancreas. Interestingly, 8 to 12- phate polyacrylamide gel electrophoresis (SDS-PAGE) week-old lethally irradiated C57BL/6J recipients trans- by identifying the narrower band size and faster migra- planted with fetal liver cells from either wildtype or Sec23b tion of band-3 and band-4.5 proteins.32-35 Exceptional gt/gt showed no haematological findings.44 These data cases that do not show the characteristic SDSPAGE pat- demonstrate that Sec23b deficient humans and mice exhib- tern have been reported, but it is recommended that it disparate phenotypes, apparently restricted to CDA II in these should be considered CDA II-like conditions. humans and a prominent neonatal pancreatic insufficiency Research on the abnormalities found in CDA II red in mice. blood cells has yielded several additional tests such as western blotting analysis of endoplasmic reticulum pro- teins of red blood cells (GRP-78, calreticulin, PDI). Others congenital dyserythropoietic anemias Genome-wide linkage analysis localized the disease gene (CDAN2) to a 5-cM region of chromosome In the past, several genes have been already described 20q11.236 but only recently has the causative gene for associated to congenital dyserythropoietic anemias. CDA II been identified.37 The study was epoch-making One of these is the transcriptional factor GATA-1. because it not only identified the responsible gene, but Mutations in GATA-1 have been found to have impor- also showed that the gene defects directly led to produc- tant clinical significance, and are directly linked to tion of binucleated cells, which is a hallmark of CDA deregulated formation of certain blood cell lineages. type II. It has been demonstrated that short hairpin Five are rare syndromes caused by defects in GATA-1 RNA (shRNA)-mediated suppression of SEC23B expres- gene expression or a malformed protein product. These sion recapitulates the cytokinesis defect. Knockdown of disorders are: X-linked thrombocytopenia (XLT), X- zebrafish Sec23b also leads to aberrant erythrocyte linked thrombocytopenia with thalassemia (XLTT), development. The gene encoding the secretory COPII congenital erythropoietic porphyria (CEP), transient component SEC23B was mutated CDA type II. The myeloproliferative disorder (TMD) and acute

| 320 | Hematology Education: the education programme for the annual congress of the European Hematology Association | 2011; 5(1) London, United Kingdom, June 9-12, 2011 megarakaryoblastic leukemia (AMKL) associated with (HIDS) to mevalonic aciduria (MA), a severe metabolic trisomy 21, and anemia associated with the production disease. Genomic sequencing of the mevalonate kinase of GATA-1s.45 GATA-1 has two zinc fingers essential for gene revealed compound heterozygosity for a missense normal function. The C-terminal finger is necessary for mutation previously described in MA (V310M) and a DNA binding. The N-terminal finger mediates interac- novel missense mutation (Y116H). In contrast, sequenc- tion with FOG-1, a cofactor for GATA-1. ing of SEC23B gene revealed no mutations, suggesting A family with X-linked dyserythropoietic anaemia that the bone marrow abnormalities were causally relat- and thrombocytopenia due to a substitution of methio- ed to the MKD.56 nine for valine at amino acid 205 of GATA-1 was described.46 This highly conserved valine is necessary for interaction of the amino-terminal zinc finger of GATA-1 References with its essential cofactor, FOG-1 (for friend of GATA-1). 1. Renella R, Wood WG. The congenital dyserythropoietic ane- They showed that the V205M mutation abrogates the mias. Hematol Oncol Clin North Am. 2009; 23:283-306. interaction between Gata-1 and Fog-1, inhibiting the 2. Iolascon A, Russo R and Delaunay J. Congenital dyserythrpoi- ability of GATA-1 to rescue erythroid differentiation in etic anemias. Curr. Op. Hematol. 2011; (May), in press. an erythroid cell line deficient for GATA-1 (G1E). Their 3. Heimpel H, Wendt F. Congenital dyserythropoietic anemia with karyorhexis and multinuclearity of erythroblasts. Helv findings underscore the importance of FOG-1:GATA-1 Med Acta. 1968;34:103-112. associations in both megakaryocyte and erythroid devel- 4. Crookston JH, Crookston MC, Burnie KL, Francombe WH, opment, and suggest that other X-linked anemias or Dacie JV, Lewis SM. Hereditary erythroblastic multinuclearity associated with a positive acidified-serum test: a type of congen- thrombocytopenias may be caused by defects in ital dyserythropoietic anaemia. Br J Haematol. 1969;17:11-23. GATA1. This mutation most closely resembles those of 5. Heimpel H, Matuschek A, Ahmed M, Bader-Meunier B, Colita mice with the ‘knockdown’ Gata1 mutation. A, Delaunay J et al. Frequency of congenital dyserythropoietic anemias in Europe. Eur J Haematol. 2010;85:20-5. Another group described a family with a novel single 6. Heimpel H, Schwarz K, Ebnöther M, Goede JS, Heydrich D, base mutation that results in an amino acid substitution Kamp T et al. Congenital dyserythropoietic anemia type I (Gly208Arg) within the highly conserved portion of the (CDA I): molecular genetics, clinical appearance, and progno- GATA-1 N-terminal finger domain, leading to dysery- sis based on long-term observation. Blood. 2006;107:334-40. 47 7. Iolascon A, Delaunay J. Close to unraveling the secrets of con- thropoietic anemia and macrothrombocytopenia. genital dyserythropoietic anemia types I and II. Haematologica. In the 90s a 10-year-old female Danish case was 2009;94:599-602. extensively described.48-51 She showed severe anemia at 8. Heimpel H, Kellermann K, Neuschwander N, Högel J, Schwarz K. The morphological diagnosis of congenital birth and required repeated transfusion during child- dyserythropoietic anemia: results of a quantitative analysis of hood. The clinical characteristic exhibited by the patient peripheral blood and bone marrow cells. Haematologica. 2010; was persistent expression of ε and ζ embryonic globin, 95:1034-6. 9. Dgany O, Avidan N, Delaunay J, Krasnov T, Shalmon L, an HbF level of 40%, novel intra-erythroblastic and Shalev H et al. Congenital dyserythropoietic anemia type I intra-erythrocytic inclusions and deficiency of erythroid (CDAN1) is caused by mutations in codanin-1. Am. J. Hum. proteins CD44 and Aquaporin 1. The marrow aspirate Genet. 2000;71:1467-74. studies revealed active erythropoiesis with some 10. Tamary H, Dgany O, Proust A, Krasnov T, Avidan N, Eidelitz- Markus T et al. Clinical and molecular variability in congenital dyserythropoietic features. Blood grouping tests further dyserythropoietic anemia type I. Br J Haematol. 2005;130:628-34. showed that the child has the very rare Co(a-b-) blood 11. Ahmed MR, Chehal A, Zahed L, Taher A, Haidar J, group phenotype and is negative for the high incidence Shamseddine A et al. Linkage and mutational analysis of the 50 CDAN1 gene reveals genetic heterogeneity in congenital antigen AnWj. dyserythropoietic anemia type I. Blood. 2006;107:4968-9. Only recently an erythroid transcriptional factor alter- 12. Noy-Lotan S, Dgany O, Lahmi R, Marcoux N, Krasnov T, ation has been elucidated. It was the case of two Yissachar N et al. Codanin-1, the protein encoded by the gene mutated in congenital dyserythropoietic anemia type I patients with a hitherto unclassified CDA in whom a (CDAN1), is cell cycle-regulated. Haematol. 2009;94:629-37. missense mutation in KLF1 has been identified. One of 13. Tamary H, Marcoux N, Noy-Lotan S, Yaniv I and Dgany O. these was a Danish patient previously described. The Codanin 1, the product of the gene mutated in congenital first patient described showed a similar clinical pheno- dyserythropoietic anemia type I (CDAN1), binds to histone chaperone Asf1a and inhibits its nucleasome assembly activ- type of Danish. KLF1 is an erythroid transcription factor, ity. Blood. ASH abstract book 2010 n°1004. and extensive studies in mouse models have shown that 14. Renella R, Roberts N, Brown JM, De Gobbi M, Bird L, it plays a critical role in the expression of globin genes, Hassanali T et al. Codanin-1 mutations in congenital dysery- thropoietic anemia type I affect HP1-alpha localization in ery- but also in the expression of a wide spectrum of genes throblasts. Blood. 2011 March (Epub ahead of print). potentially essential for erythropoiesis.52-54 The unique 15. Goede JS, Benz R, Fehr J, Schwarz K, Heimpel H. Congenital features of this CDA confirm the key role of KLF1 dur- dyserythropoietic anemia type I with bone abnormalities, mutations of the CDAN 1 gene and significant responsiveness ing human erythroid differentiation. Sequencing of to alpha-interferon therapy. Ann Hematol. 2006;85:591-5. KLF1 in both patients revealed the presence of de novo 16. Tamary H, Shalev H, Perez-Avraham G, Zoldan M, Levi I, c.973G>A (p.E325K) mutation that had never been Swinkels DW et al. Elevated growth differentiation factor 15 expression in patients with congenital dyserythropoietic ane- reported and was not present in 96 regular blood, sug- mia type I. Blood. 2008;112:5241-4. gesting it was the disease-causing mutation. The 17. Heimpel H, Anselstetter V, Chrobak L, Denecke J, Einsiedler B, authors suggested that mutations in KLF1 cause a hith- Gallmeier K et al. Congenital dyserythropoietic anemia type erto unclassified CDA.55 II: epidemiology, clinical appearance, and prognosis based on long-term observation. Blood. 2003;102:4576-5581. Finally, in recent months the case of a patient with 18. Iolascon A, D’Agostaro G, Perrotta S, Izzo P, Tavano R, mevalonate kinase deficiency (MKD) and congenital Miraglia del Giudice B. Congenital dyserythropoietic anemia dyserythropoietic anemia has been described. The clin- type II: molecular basis and clinical aspects. Haematologica. 1996;81:543-59. ical phenotype was variable, ranging from the hyperim- 19. Perrotta S, Del Giudice EM, Carbone R, Servedio V, Schettini munoglobulinemia D and periodic fever syndrome F Jr, Nobili B et al. Gilbert’s syndrome accounts for the pheno-

Hematology Education: the education programme for the annual congress of the European Hematology Association | 2011; 5(1) | 321 | 16th Congress of the European Hematology Association

typic variability of congenital dyserythropoietic anemia type 39. Fromme JC, Orci L, Schekman R. Coordination of COPII vesi- II (CDA II). J Pediatr. 2000;136:556-9. cle trafficking by Sec23. Trends Cell Biol. 2008; 18:330-336. 20. Bird AR, Jacobs P, Moores P. Congenital dyserythropoietic 40. Jones B, Jones EL, Bonney SA, Patel HN, Mensenkamp AR, anaemia (type II) presenting with haemosiderosis. Acta Eichenbaum-Voline S et al. Mutations in a Sar1 GTPase of Haematol. 1987;78:33-6. COPII vesicles are associated with lipid absorption disorders. 21. Lugassy G, Michaeli J, Harats N, Libson E, Rachmilewitz EA. Nat Genet. 2003;34:29-31. Paravertebral extramedullary hematopoiesis associated with 41. Boyadjiev SA, Fromme JC, Ben J, Chong SS, Nauta C, Hur DJ improvement of anemia in congenital dyserythropoietic ane- et al. Cranio-lenticulo-sutural dysplasia is caused by a SEC23A mia type II. Am J Hematol. 1986;22:295-300. mutation leading to abnormal endoplasmic-reticulum-to- 22. Hines GL. Paravertebral extramedullary hematopoiesis (as a pos- Golgi trafficking. Nat Genet. 2006;38:1192-7. terior mediastinal tumor) associated with congenital dyserythro- 42. Iolascon A, Russo R, Esposito MR, Asci R, Piscopo C, Perrotta poietic anemia. J Thorac Cardiovasc Surg. 1993;106:760-1. S et al. Molecular analysis of 42 patients with congenital 23. Imran A, Mawhinney R, Swirsky D, Hall C. Paravertebral dyserythropoietic anemia type II: new mutations in the SEC23B extramedullary haemopoiesis occurring in a case of congenital gene and a search for a genotype-phenotype relationship. dyserythropoietic anaemia type II. Br J Haematol. 2008;140:1. Haematologica. 2010;95:708-15. 24. Steiner W, Denzlinger C, Weiss M. Paravertebral extramedullary 43. Russo R, Esposito MR, Asci R, Gambale A, Perrotta S, hematopoiesis in congenital dyserythropoietic anemia type II Ramenghi U et al. Mutational spectrum in congenital dysery- (CDA II). Rontgenpraxis. 1995;48:110-2. thropoietic anemia type II: identification of 19 novel variants 25. Halpern Z, Rahmani R, Levo Y. Severe hemochromatosis: the in SEC23B gene. Am J Hematol. 2010; 85:915-20. predominant clinical manifestation of congenital dyserythro- 44. Vasievich M, Zhang B, Rinehart J, Jones M, Maillard I, poietic anemia type 2. Acta Haematol. 1985;74:178-80. Ginsburg D et al. Sec23b deficiency in mice results in pancre- 26. Tamura H, Matsumoto G, Itakura Y, Terai H, Ikebuchi K, atic desctruction and defective long term hemopoietic stem Mitarai T et al. A case of congenital dyserythropoietic anemia cell function. Blood. ASH abstract blook 2010 n° 2038. type II associated with hemochromatosis. Intern Med. 1992; 45. Ciovacco WA, Raskind WH, Kacena MA. Human phenotypes 31:380-4. associated with GATA-1 mutations. Gene. 2008;427:1-6. Epub 27. Verwilghen RL, Lewis SM, Dacie JV, Crookston JH, Crookston 2008 Sep 30. MC. HEMPAS: congenital dyserythropoietic anaemia (type II). 46. Nichols KE, Crispino JD, Poncz M, White JG, Orkin SH, Maris Q J Med. 1973;42:257-78. JM et al. Familial dyserythropoietic anaemia and thrombocy- 28. Breton-Gorius J, Daniel MT, Clauvel JP, Dreyfus B. topenia due to an inherited mutation in GATA1. Nat Genet. [Ultrastructural abnormalities of erythroblasts and erythro- 2000;24:266-70. cytes in 6 cases of congenital dyserythropoietic anemia]. Nouv 47. Del Vecchio GC, Giordani L, De Santis A, De Mattia D. Rev Fr Hematol. 1973;13:23-49. Dyserythropoietic anemia and thrombocytopenia due to a 29. Verwilghen RL, Tan P, De Wolf-Peeters C, Broeckaert-van novel mutation in GATA-1. Acta Haematol. 2005;114:113-6. Orshoven, Louwagie AC. Cell membrane anomaly impeding 48. Wickramasinghe SN, Illum N, Wimberley PD. Congenital cell division. Experientia. 1971;27:1467-8. 30. Wong KY, Hug G, Lampkin BC. Congenital dyserythropoietic dyserythropoietic anaemia with novel intra-erythroblastic and anemia type II: ultrastructural and radioautographic studies of intra-erythrocytic inclusions. Br J Haematol. 1991;79:322-30. blood and bone marrow. Blood. 1972;39:23-30. 49. Tang W, Cai SP, Eng B, Poon MC, Waye JS, Illum N et al. 31. Alloisio N, Texier P, Denoroy L, Berger C, Miraglia del Giudice Expression of embryonic zeta-globin and epsilon-globin E, Perrotta S et al. The cisternae decorating the red blood cell chains in a 10-year-old girl with congenital anemia. Blood. membrane in congenital dyserythropoietic anemia (type II) 1993;81:1636-40. originate from the endoplasmic reticulum. Blood. 1996;87: 50. Parsons SF, Jones J, Anstee DJ, Judson PA, Gardner B, Wiener 4433-9. E et al. A novel form of congenital dyserythropoietic anemia 32. Mawby WJ, Tanner MJ, Anstee DJ, Clamp JR. Incomplete gly- associated with deficiency of erythroid CD44 and a unique cosylation of erythrocyte membrane proteins in congenital blood group phenotype [In(a-b-), Co(a-b-)]. Blood. 1994; dyserythropoietic anaemia type II (CDA II). Br J Haematol. 83:860-8. 1983;55:357-68. 51. Agre P, Smith BL, Baumgarten R, Preston GM, Pressman E, 33. Baines AJ, Banga JP, Gratzer WB, Linch DC, Huehns ER. Red cell Wilson P et al. Human red cell Aquaporin CHIP. II. Expression membrane protein anomalies in congenital dyserythropoietic during normal fetal development and in a novel form of congen- anaemia, type II (HEMPAS). Br J Haematol. 1982; 50:563-74. ital dyserythropoietic anemia. J Clin Invest. 1994;94:1050-8. 34. Scartezzini P, Forni GL, Baldi M, Izzo C, Sansone G. 52. Nuez B, Michalovich D, Bygrave A, Ploemacher R, Grosveld F. Decreased glycosylation of band 3 and band 4.5 glycoproteins Defective haematopoiesis in fetal liver resulting from inactiva- of erythrocyte membrane in congenital dyserythropoietic tion of the EKLF gene. Nature. 1995;375:316-8. anaemia type II. Br J Haematol. 1982;51:569-76. 53. Perkins AC, Sharpe AH, Orkin SH. Lethal beta-thalassaemia in 35. Tomita A, Parker CJ. Aberrant regulation of complement by mice lacking the erythroid CACCC-transcription factor EKLF. the erythrocytes of hereditary erythroblastic multinuclearity Nature. 1995;375:318-22. with a positive acidified serum lysis test (HEMPAS). Blood. 54. Drissen R, von Lindern M, Kolbus A, Driegen S, Steinlein P, 1994;83:250-9. Beug H. The erythroid phenotype of EKLF-null mice: defects 36. Gasparini P, Miraglia del Giudice E, Delaunay J, Totaro A, in hemoglobin metabolism and membrane stability. Mol Cell Granatiero M, Melchionda S et al. Localization of the congen- Biol. 2005;25:5205-14. ital dyserythropoietic anemia II locus to chromosome 20q11.2 55. Arnaud L, Saison C, Helias V, Lucien N, Steschenko D, by genome wide search. Am J Hum Genet. 1997;61:1112-6. Giarratana MC et al. A dominant mutation in the gene encod- 37. Schwarz K, Iolascon A, Verissimo F, Trede NS, Horsley W, ing the erythroid transcription factor KLF1 causes a congenital Chen W et al. Mutations affecting the secretory COPII coat dyserythropoietic anemia. Am J Hum Genet. 2010;87:721-7. component SEC23B cause congenital dyserythropoietic ane- Epub 2010 Nov 4. mia type II. Nat Genet. 2009;41:936-40. 56. Samkari A, Borzutzky A, Fermo E, Treaba DO, Dedeoglu F, 38. Lee MC, Miller EA, Goldberg J, Orci L, Schekman R. Bi-direc- Altura RA. A novel missense mutation in MVK associated tional protein transport between the ER and Golgi. Annu. Rev. with MK deficiency and dyserythropoietic anemia. Pediatrics. Cell Dev. Biol. 2004; 20:87-123. 2010;125:e964-8. Epub 2010 Mar 1.

| 322 | Hematology Education: the education programme for the annual congress of the European Hematology Association | 2011; 5(1)