The congenital dyserythropoietic anaemias

BaillieÁre's Clinical Haematology Vol. 12, No. 4, pp. 691±705, 1999

5 The congenital dyserythropoietic anaemias J. Delaunay

MD, PhD

Professor of Genetics INSERM U 473, Service d'HeÂmatologie, d'Immunologie et de CytogeÂneÂtique, Service de Biologie SpeÂcialiseÂe, HoÃpital de BiceÃtre, Assistance Publique-HoÃpitaux de Paris, Faculte de MeÂdecine Paris-Sud, 78 rue du GeÂneÂral-Leclerc, 94275, Le Kremlin-BiceÃtre, France

A. Iolascon

MD, PhD

Professor of Paediatrics Dipartimento di Biomedicina dell'EtaÁ Evolutiva, UniversitaÁ degli Studi di Bari, Centro Interuniversitario per lo Studio delle Malattie Ereditarie dell'EtaÁ Evolutiva, UniversitaÁ di Bari, Bari, Italy

Congenital dyserythropoietic anaemias (CDA) are a category of rare genetic diseases that a€ect erythropoiesis. Dyserythropoiesis is associated with abnormal erythroblasts and leads to altered red cells, the amount of which is insucient. There are three main, well-de®ned CDAs, CDA I, II and III. Their characterization is based on a careful examination of the bone marrow under light and electron microscopes. In addition, a number of rare or unique forms of dyserythropoiesis have been reported. At least with respect to CDA I to III, the clinical evaluation is reaching an ever increasing re®nement: age of discovery, determinants of iron overload and/or biliary complications. Over the past few years, a more promising breakthrough has been the localization of the genes responsible for CDA I, II and III, that is, 15q15.1-q15.3, 20q11.2 and 15q21-q25, respectively. Epidemiological studies have now become possible. The identi®cation of the genes is pending. Key words: haematology; genetics; bone marrow; erythroblasts; nucleus; iron overload; dysmorphology; linkage analysis.

INTRODUCTION The term `Kongenitale dyserythropoetische AnaÈmie', or congenital dyserythropoietic anaemia (CDA), was coined by Wendt and Heimpel in 1967.1 A year later, these authors established the prescient classi®cation of CDA I, II and III.2 Indeed, this classi®cation has proved to be essentially correct ever since. For example, in 1969, Crookston et al3 also used the term congenital dyserythropoietic anaemia in order to designate CDA II. Altogether, CDA designates a group of inherited disorders in which erythropoiesis is quantitively and qualitatively altered. The number of erythroblasts reaching maturity is All correspondence to: Jean Delaunay, Service d'HeÂmatologie, d'Immunologie et de CytogeÂneÂtique, HoÃpital de BiceÃtre, 78 rue du GeÂneÂral-Leclerc, 94275 Le Kremlin-BiceÃtre, France. Tel: ‡‡ (33) 1 45 21 20 16. Fax: ‡‡ (33) 1 45 21 28 47. E-mail: [email protected] 0950±3536/99/040691+15 $12.00/0

c 1999 Harcourt Publishers Ltd *

692 J. Delaunay and A. Iolascon

decreased because they are destroyed in the bone marrow. Erythroblasts display characteristic dysplastic features involving the nucleus in the ®rst place, and these have been the basis of the current classi®cation. Descendent erythrocytes are themselves abnormal and show a reduced life span. Thus CDA combines to various extents a central defect in red cell production and a more or less pronounced peripheral increase in red cell destruction. Hence the frequent haemolysis that accompanies anaemia. A number of other CDAs have been described subsequently, but as a rule they remain incompletely documented. Light and electron microscopy of the bone marrow have always been and remain central to the diagnosis of CDA. Erythrokinetic studies have delineated the central and peripheral components of anaemia. Recently, knowledge about CDA has taken a decisive genetic turn through the localization of the genes responsible for the three main CDAs. The characterization of the genes is pending. The three main CDAs and the others will be considered here. However, dyserythropoietic features that accompany a variety of congenital or acquired disorders will only be enumerated. We will place the emphasis on the genetic trend that is now emerging. Several reviews on CDA have appeared in recent years4±7, some with an exhaustive survey of their morphological aspects.6,7 CONGENITAL DYSERYTHROPOIETIC ANAEMIA TYPE I Mode of inheritance and incidence Congenital dyserythropoietic anaemia type I, or CDA I, is an autosomal recessive condition. Its incidence is usually very low, although no accurate data are available. Alter8 stated that 60 cases (in how many kindreds?) had been registered. Heimpel collated 98 patients from 77 families from his own unpublished observations and the literature.9 Cases originated mostly from European countries and, more rarely, from the USA, Japan and Russia. In France, we have recognized fewer than 10 a€ected families over the last 20 years. There may be isolates in which the disease frequency dramatically rises. Tamary et al10 studied 20 patients in seven families among Israeli Bedouins. In practice, consanguinity should always be searched for. Clinical and laboratory ®ndings A continuous spectrum exists between moderate and severe forms of CDA I. The age of diagnosis is variable. In a retrospective study, Shalev et al11 showed that 17 out of 31 CDA I patients had ®rst been seen in the neonatal period with signi®cant anaemia. The more severe the presentation, the earlier the discovery. The common haematological form The common haematological form of moderate intensity is discovered in childhood or adolescence, sometimes later. Anaemia is mild and associated with intermittent jaundice, splenomegaly and, sometimes, hepatomegaly. Haemoglobin ¯uctuates around 9 g/100 ml, usually staying above the transfusional threshold. The reticulocyte count is normal or low. A strong tendency to macrocytosis is observed. Mature red cells show morphological abnormalities: anisocytosis with prevalent elliptocytosis, and poikilocytosis with dacryocytosis. Punctate basophilia is common and Cabot's rings may be seen. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) shows

Congenital dyserythropoietic anaemias 693

no gross protein alteration. A slight decrease of protein 4.1, thought to be a secondary phenomenon, is constantly present (12, J. Delaunay et al, unpubl. results). It would account, presumably, for the pronounced elliptocytic component. The concentration of serum bilirubin is increased and that of haptoglobin is decreased. Bearing witness to the destruction of erythroblasts in the bone marrow, the activity of serum thymidine kinase is increased.13 The evolution of CDA I is doomed by iron overload (liver cirrhosis, skin pigmentation and endocrine dysfunction) and biliary complications (bile duct obstruction, pancreatitis, perforation with bile peritonitis and life-threatening sepsis). Using light microscopy, the bone marrow shows erythroid hyperplasia and dysplastic features in the erythroblasts. There may be cytoplasmic stippling. Some nuclei are irregular or karyorrhectic. One notes an increased number of binucleate erythroblasts, the two nuclei being often unequal. More rarely, erythroblasts are trior tetranucleated. Some nuclei remain connected in some cells. Very typical of CDA I are the long chromatin strands that may connect the nuclei of two separate cells (Figure 1A). Electron microscopy shows a spongy condensation of chromatin, often depicted as the `Swiss cheese abnormality'. In the intercellular bridges, chromatin is surrounded by longitudinal bundles of microtubules. The nuclear membrane displays invaginations bringing cytoplasm and sometimes organelles (iron-laden mitochondria, autophagic vacuoles) into the nuclear region (for a review, see Wickramasinghe7). The percentage of haemaglobin A2 has been found to be increased10, as well as the a/non-a globin chain synthesis ratio10,12,14, thus mimicking a b-thalassaemia trait. They presumably account for a secondary phenomenon. Red cell enzymes, osmotic fragility, serum vitamin B12 and folate should be found to be normal. Severe forms Severe forms of CDA I occur at, or even before, birth. Haemoglobin may drop to 3 gm/100 ml. Bone marrow examination using both the light and the electron microscope displays the usual features. Prior to birth, intra-uterine transfusions may be necessary. Following birth, a heavy regimen of transfusions must be started. However, it is unusual for such demand to be lifelong, with the attendant risk of iron overload. In one patient, the number of bone marrow burst-forming units erythroid (BFU-E) was found to be increased fourfold compared with normals.15 Tamary et al10 pointed out that the peripheral condensation of chromatin in CDA I was similar to that seen in cells undergoing apoptosis. However, they failed to observe DNA ladders in the bone marrow. The dysmorphology side of CDA I Whatever the extent of the haematological presentation, it is important to assess the presence of morphological body abnormalities. These are sometimes the presenting features and infants or children are often referred to Dysmorphology Units in the ®rst place. They include the following, in various combinations and degree of severity: short stature, syndactyly, hypoplasia or loss of one or several nails (®ngers and/or toes), hypoplasia or loss of one or several distal phalanges (®ngers and/or toes), additional metatarsal, clubfeet, sacral dimple, very blue almond-shaped eyes, hypertelorism, wide nasal bridge, micrognatism, large mouth with thick lower lips, large ears and blond hair.16,17 There is much to speculate on regarding dysmorphic changes in CDA I. They remind one of Blackfan±Diamond anaemia, Fanconi anaemia or thrombocytopenia with absent radii.

694 J. Delaunay and A. Iolascon

Figure 1. Morphological abnormalities in the bone marrow in the major CDAs. A. CDA I, in a French patient. A chromatin bridge between two cells is visible in the centre of the picture. B. Bone marrow in CDA II, in an Italian patient. Several binucleated erythroblasts are visible. C. Bone marrow in CDA III, in the Swedish VaÈsterbotten family (Courtesy of Dr Herbert SandstroÈm, and with permission from the Annales de PeÂdiatrie. Delaunay J, 1999; 46: 64±72). A spectacular gigantoblast occupies the centre of the picture.

Congenital dyserythropoietic anaemias 695

A unique association In one sibship, three members combined CDA I, deafness and azoospermia.18 The most likely hypothesis is the occurrence of a deletion removing the CDA I gene along with other genes. No deletion was visible on the basis of cytogenetics. a-Interferon as a treatment of CDA I Apart from non-speci®c treatments, dealt with in the section on Treatment, below, we will report here on tentative speci®c treatments for CDA I. Recombinant human erythropoietin appeared to be of no use19, but a-interferon proved to be of value. It was a chance discovery, made in a 28-year-old female with transfusion-dependent CDA I and post-transfusion chronic viral hepatitis C.20 a-Interferon had been used against hepatitis and turned out to cure the anaemia. Haemoglobin reached normal values after 24 weeks of treatment, but dropped again upon cessation of the latter. The improvement brought about by a-interferon was underlain by a reduction of the ine€ective erythropoiesis as shown by erythrokinetic studies, and of the cellular abnormalities as revealed by electron microscopy. Similar results were reported by Wickramasinghe21: improvement of the abnormalities visible using electron microscopy was real, but partial. a-Interferon was also used successfully in an infant presenting with a severe form of CDA I (22, G. Tchernia et al, unpubl. results). Again the need for transfusions disappeared, and electron microscopy stigmata resolved in part. How a-interferon operates on CDA I is unknown. a-Interferon eciency may be connected in some way with the fact that Epstein±Barr virus (EBV)-transformed lymphoblastoid cells from CDA I patients release less a-interferon into the medium than normal cells.23 Genetics and epidemiology Tamary et al24 mapped the gene responsible for CDA I, or the CDAN1 gene, to 15q15.1q15.3 based on homozygosity mapping analysis in the above-mentioned Israeli Bedouins. Twenty-®ve individuals from four large consanguineous families were investigated using 14 markers that encompassed a 12 cM interval. Linkage disequilibrium was found for marker D15S779. Cross-over events narrowed down the region of interest to a chromosomal segment stretching between markers D15S779 and D15S778, and assigned the CDAN1 gene to a 0.5 cM interval. The CDAN1 gene maps close to the EPB42 gene (15q15-q21)25,26, which is the only erythroid-related gene in the CDAN1 gene interval and encodes protein 4.2. (The total absence of protein 4.2 causes a rare and atypical variety of hereditary sperocytosis.) More remote from, and unlinked to, the CDAN1 gene stands the CDAN3 gene, responsible for CDA III.27 The CDA I Bedouin patients harboured eight di€erent haplotypes. It was estimated that the involved mutation had occurred about 400 years ago. Using four markers, Hodges et al28 haplotyped six unrelated English and two related Lebanese patients. Each of the English patients had di€erent haplotypes while the Lebanese had the same haplotype. Only one English patient was homozygous for marker D15S779. It seems likely that the Lebanese patients were homozygous for the responsible mutation in the CDAN1 gene, and that the English patients were heterozygotes, but this will not be ascertained until the mutations are de®nitely elucidated. The possibility exists that the same mutation has occurred in a recurrent manner, at a hot spot, within the framework of distinct haplotypes. Hodges et al28 could not tell how the Lebanese haplotype related to the haplotypes reported in the Israeli

696 J. Delaunay and A. Iolascon

Bedouins24, because of the use of a di€erent nomenclature. At this point, it is reasonable to assume that CDA I, like most rare genetic disorders, has a multicentric origin and may be caused by a variety of mutations in one gene. (For the time being, there is no hint of a second locus involved in CDA I.) Concerning the individual expression and, in particular, the presence or absence of morphological abnormalities, it has not been established whether they would stem mostly from the nature of the mutation involved, and therefore remain essentially the same within a given kindred, or whether they would also be in¯uenced by other genetic factors and thereby give any a€ected family member a unique phenotype. al-Fawaz and al-Mashhadani29 reported a girl who displayed anaemia, jaundice and hepatosplenomegaly in the neonatal period, and who required four blood transfusions in the ®rst 7 months of life; her brother had never been transfused when anaemia and jaundice were discovered at the age of two. This particular example would support the second hypothesis, yet the issue requires further documentation. CONGENITAL DYSERYTHROPOIETIC ANAEMIA TYPE II Congenital dyserythropoietic anaemia type II, or CDA II, was recognized by Heimpel and Wendt on morphological criteria in 1967.2 Later, Crookston et al described two distinctive laboratory features: the positivity of the acidi®ed serum haemolysis and the anti-i agglutination.3 These authors called the condition HEMPAS (Hereditary Erythroblastic Multinuclearity with Positive Acidi®ed Serum). Mode of inheritance and incidence The mode of inheritance of CDA II is autosomal recessive. It is the most common form of CDA. An International Registry for CDA II, begun some years ago by A. Iolascon et al (Dipartimento di Biomedicina dell'EtaÁ Evolutiva, UniversitaÁ degli Studi di Bari, Bari, Italy), systematically established the ancestry of patients and the follow-up of the disease: date of diagnosis, evolution of the clinical and laboratory data since then, determination of genetic markers in the patients and healthy family members. The overall enrolled population amounts to 81 patients from 63 families, comprising 56 Italian patients from 43 families and 25 non-Italian patients from 20 families (April, 1999). Clinical ®ndings Diagnosis of CDA II is usually made at birth, infancy, childhood or early adulthood. The main clinical ®ndings are anaemia, jaundice and splenomegaly or hepato-splenomegaly. The severity of anaemia is variable. Quite rarely one encounters other manifestations: frontal and parietal bossing (pronounced erythroid hyperplasia), mental retardation. Posterior mediastinal tumours (extramedullar erythropoiesis)30 and parvovirus infections31 are much rarer than in hereditary spherocytosis (HS) and other hereditary haemolytic anaemias. The evolution of CDA II is doomed, once again, by iron overload and biliary complications. CDA II patients with Gilbert syndrome had a 4.75-fold greater tendency to form gallstones than CDA II patients without the latter syndrome.32 Interestingly, the risk of Gilbert syndrome-related gallstone formation increased in a similar fashion (4.66-fold) in patients with hereditary spherocytosis.33 There may be more severe forms of CDA II. They often turn out to be the result of association with other conditions. Analysis of ®ve transfusion-dependent CDA II cases

Congenital dyserythropoietic anaemias 697

showed that three of them also carried a b-thalassaemic trait. Co-inheritance of CDA II and other red cell defects, mild though the latter may be, can lead to transfusiondependence. Recently, we had the opportunity of following two pregnancies (one was gemellar) in two sisters with CDA II: neither required transfusions (J. Delaunay et al, unpubl. results). Laboratory data Red cell counts show a haemoglobin value that usually remains above the transfusion threshold, yet it may go below on the occasion of acute infection. The reticulocyte count is normal or low. The blood ®lm shows basically aniso-poikilocytosis and basophilic stippling. The concentration of serum bilirubin and the activity of serum lactate dehydrogenase (LDH) are increased. The concentration of haptoglobin is decreased. The Ham test is positive in about 30% of fresh ABO-compatible sera and the i antigen is increased. Using light microscopy, bone marrow exhibits erythroid hyperplasia (about 5±10 times more erythroblasts than normal). Early erythroblasts are relatively normal, but about 10±40% of more mature erythroblasts (late polychromatophilic and oxyphilic erythroblasts) are bi- or multinucleated (Figure 1). In binucleated cells, the nuclei are roughly equivalent. There are pseudo-Gaucher cells. Using electron microscopy, the most dramatic feature, which is very speci®c, is the presence of elongated vesicles that run parallel and beneath the plasma membrane34,35 (for a review see Wickramasinghe7). Using immunogold electron microscopy, an antibody directed against protein disulphide isomerase (an endoplasmic reticulum protein) showed binding to these vesicles, strongly suggesting that these were cisternae of the endoplasmic reticulum. Their persistence can be shown even in circulating red cells.36 SDS-polyacrylamide gel electrophoresis revealed numerous changes. Band 3 repeatedly appeared to have a narrower aspect and a faster migration (for a review, see Iolascon et al4). The slightly decreased molecular weight of band 3 is consistent with its reduced glycosylation (for a review, see Fukuda37). In contrast, glycolipids were overglycosylated.38,39 Using Western blotting of circulating red cell membrane proteins, Alloisio et al36 showed the presence of three minor proteins with apparent molecular weights of 74, 59 and 58 kDa. They correspond to the glucose-regulated protein (GRP78), the (aforementioned) protein disulphide isomerase, and calreticulin, a high anity Ca2‡ binding protein, respectively. All these proteins are constitutive proteins of the endoplasmic reticulum. Their presence further supports the hypothesis that the cisternae running beneath the plasma membrane belong to a fraction of the endoplasmic reticulum that failed to be cleared away as a consequence of dyserythropoiesis. The glycosylation defect is not an exclusive attribute of erythroid band 3. Some serum glycoproteins show incompletely processed N-glycans.40 CDA II may be viewed as a disease stemming from a glycosylation and/or a deglycosylation defect. Mice lacking a functional a-mannosidase II develop a dyserythropoietic anaemia concurrent with the loss of erythrocyte complex N-glycans.41 However, we assume that the glycosylation alterations in CDA II are secondary, and that the ultimate cause of the condition lies in one of the many genes that control erythropoiesis. The glycosylation abnormalities may correlate with the retarded processing in the endoplasmic reticulum. The e€ects of the reduced glycosylation of band 3 on its function have been studied.42 Analysis of the anion transport (inhibition of sulphate ¯ux by di-

698 J. Delaunay and A. Iolascon

isothiocyanodihydrostilbene disulphenate, or H2-DIDS) demonstrated that the transport activity was decreased. An increased number of band 3 aggregates was observed. These aggregates could bind against band 3 naturally-occurring antibodies, which are able to mediate the phagocytic removal of red blood cells by the cells of the reticuloendothelial system. Both the phagocytic index (red blood cell/macrophage) and the amount of membrane bound IgG was increased in CDA II erythrocytes. These results suggest that the mild haemolysis exhibited by CDA II patients may be ascribed to the clustering of band 3. The latter leads to IgG binding and phagocytosis, but not to a secondary modi®cation of the red blood cell membrane skeleton. These data are consistent with the fact that red cells are mostly destroyed in the spleen, as shown by erythrokinetic studies, and would support a possible bene®cial e€ect of splenectomy.

Genetics and epidemiology Reduction in the activities of some enzymes involved in glycan synthesis and/or the amount of the corresponding messenger RNA were reported for N-acetylglucosaminyltransferase II, galactosyltransferase and a-mannosidase II.43±45 However, the genes encoding a-mannosidase II and N-acetylglucosaminyltransferase II, and the gene encoding yet another enzyme, a-mannosidase IIx, are not linked to CDA II.46 They map to chromosomes 14, 15 and 5, respectively (the gene encoding galactosyltransferase was not investigated). Indeed, a genome-wide search was necessary to localize the gene responsible for CDA II, the CDAN2 gene. Following the analysis of 145 markers, which excluded about 50% of the genome, evidence for linkage was obtained using markers D20S195 and D20S107. The highest lod score obtained was with marker D20S863. Several cross-over events helped de®ne the limits for the CDAN2 gene location. The region of interest spans from marker D20S890 to D20S908 and corresponds to a 5 cM interval, approximately. The CDAN2 gene maps to 20q11.2.47 The above-mentioned Registry also demonstrated that approximately 10% of the CDA II cases were not linked to chromosome 20 and that at least one other CDAN2 locus must exist.48 Such cases are usually severe and transfusion-dependent. CDA II alleles were indirectly traced in families using microsatellite haplotyping. The highest linkage disequilibrium was with markers D20S863 and D20S841. Many Italian patients, residing in or originating from southern Italy, carried the same alleles of these markers. This suggests a founder e€ect and a genetic isolate developed in southern Italy and `leaked' because of northward migrations. In non-Italian patients, the linkage of CDA II to other alleles of these markers was also observed. This hints at a multicentric origin for CDA II and thereby at the possibility of distinct subjacent mutations (A. Iolascon et al, unpubl. results). The tools of molecular genetics sometimes produce unexpected ®ndings. In a sibship of seven, three had overt CDA II, associated with well-de®ned microsatellite haplotypes. A fourth member had the same haplotypes, yet she had no CDA II at all (save, perhaps, a light abnormality of band 3). We assumed that some genetic event occurred in a precursor of one of the germ cells from which she was descended, which caused suppression of CDA II expression. It could have been (i) a double recombination event that took place between the known markers closest to the CDAN2 gene, and replaced the mutated allele with the wild one without altering the microsatellite assortment, or (ii) a de novo mutation that restored most, if not all, of the function of the mutated protein.49

Congenital dyserythropoietic anaemias 699

CONGENITAL DYSERYTHROPOIETIC ANAEMIA TYPE III Congenital dyserythropoietic anaemia type III, or CDA III, is the rarest of the three wellcharacterized CDAs. Indeed, its initial description took place before that of CDA I and II.50 A large fraction of the patients investigated belongs to one large Swedish family, also known as the VaÈsterbotten family.51 Other familial cases have been reported, one in the USA50 and another in Argentina.52 Yet, most of our knowledge about CDA III comes from the Swedish family and does not necessarily apply to other kindreds. The inheritance pattern is dominant. Sporadic cases have also been reported in various populations, possibly stemming from de novo mutations. It is nevertheless questionable as to whether these sporadic cases could be the outcome of recessive mutations present in the homozygous (or in the compound heterozygous) state. There is no certainty as to the involvement of the same gene as that in the familial form of CDA III. Familial CDA III is clinically well-tolerated. It is associated with some fatigue and mild icterus. Splenomegaly is inconstant. Haemoglobin is decreased, although it does not make transfusion necessary. The reticulocyte count is normal or low. Serum haptoglobin concentration is low. Serum bilirubin concentration and LDH activity are increased. There is no iron overload. Blood smears show anisocytosis and basophilic stippling. There is no macrocytosis. Using light microscopy (Figure 1C), the bone marrow exhibits erythroid hyperplasia, with sometimes spectacular, giant multinucleate erythroblasts (with up to 12 nuclei). Using electron microscopy, the salient features are accounted for by the massive multinuclearity, clefts within heterochromatin, autophagic vacuoles and ironladen mitochondria (for a review, see Wickramasinghe7). The Swedish family was large enough to allow the emergence, to a signi®cant frequency, of some remarkable features. These would make CDA III part of a broader, hitherto unreported syndrome. They include (i) increase of the serum thymidine kinase activity53, (ii) visual abnormalities with subjacent macular degeneration and angioid streaks54, and (iii) monoclonal gammapathies and myeloma.55 Sporadic CDA III may comprise occasional distinctive features: mongoloid face, `hair on end' appearance of the skull (using X-rays), mental retardation, iron overload, malignant condition (Hodgkin's disease, T-cell lymphoma). Based on linkage analysis within the Swedish family, the gene responsible for familial CDA III, or the CDAN3 gene, has been mapped to 15q21-q25 over a 4.5 cM interval.27 OTHER CONGENITAL DYSERYTHROPOIETIC ANAEMIAS As well as the three main, well-characterized CDAs, a variety of conditions with dyserythropoietic features have been reported. A tentative classi®cation has been proposed by Wickramasinghe7,56 and is summarized in Table 1. Generally speaking, the mode of inheritance is not always documented, the question of genetic homogeneity within each class is not settled, and nothing is known regarding the genes involved. (i) CDA IV is a severe, transfusion-dependent condition.57 Hydrops fetalis was present in one case. Its mode of inheritance would be recessive. (ii) CDA V has been reported very rarely, once in a 19 year old Mauritian male.58 As well as episodic nausea, abdominal discomfort and jaundice, the patients displayed unconjugated hyperbilirubinaemia (primary shunt hyperbilirubinaemia), erythroid hyperplasia without dyserythropoiesis and possible slight anaemia.

700 J. Delaunay and A. Iolascon Table 1. Distinctive features of CDA IV to VII. CDA IV

Severe, transfusion-dependent anaemia Erythroid hyperplasia with a slight increase of the erythroblasts Absence of precipitated protein within erythroblasts

CDA V

Normal or near normal haemoglobin Normal or slightly elevated mean cell volume Hyperbilirubinaemia (unconjugated in the main) Slightly or pronounced normoblastic erythroid hyperplasia without dysplasia

CDA VI

Normal or near-normal haemoglobin Macrocytosis Megaloblastic hyperplasia

CDA VII

Severe, transfusion-dependent anaemia Erythroid hyperplasia with abnormalities of erythroblast nuclei Intra-erythroblastic inclusions (a- and b-globin chains)

From S. N. Wickramasinghe7,56 (With permission).

(iii) CDA VI is characterized by little or no anaemia, a pronounced microcytosis, an erythroid hyperplasia with megaloblastic erythropoiesis (unrelated to vitamin B12 and folate de®ciency) and non-speci®c dysplastic changes in erythroblasts.59 The mode of inheritance seems to be recessive. (iv) CDA VII is associated with severe, transfusion-dependent anaemia.60 There was a strong erythroid hyperplasia with dyserythropoiesis:basophilic stippling and nuclear shape abnormalities in polychromatophilic erythroblasts. The most peculiar feature was the presence of inclusions in polychromatophilic erythroblasts and also in some erythrocytes. Immuno-electron microscopy showed that these inclusions were neither a- nor b-globin chains. This Danish patient had a persistence of embryonic and fetal haemoglobins. CD44 was absent in the red cells (but not in the leukocytes). The In (a-b-) phenotype correlated with the absence of CD44. The high incidence red blood cell antigen AnWj, which is also associated with CD44, was missing as well.61 Remarkably, the channel-forming integral protein (CHIP)-associated Colton antigens were diminished, and the CHIP itself was decreased by 90%. Yet no mutation was found in the gene encoding the CHIP.62 Other types of dyserythropoiesis have been reported very rarely, sometimes only from one case. In a transfusion-dependent Gipsy female, blood smears showed red cells with all shapes and sizes, including dacryocytes and gigantocytes.63 The erythrocyte membrane proteins exhibited four additional, uncharacterized bands. Dyserythropoiesis was recognized on the basis of erythrokinetics and the morphological appearance of the erythroid precursors. Because the patient was born from consanguineous parents, who were healthy, it was supposed that she was homozygous for some mutation. To our knowledge, no other similar case has ever been reported. We have no clue about its pathophysiology. Taken together, these rare or unique conditions need further characterization, especially with regard to their genetics. HOW TO CHARACTERIZE A CDA IN PRACTICE? In many instances, the diagnosis may be quite straightforward, based on tests summarized in Table 2. The bone marrow examination is central. The red cell membrane

Congenital dyserythropoietic anaemias 701

protein pattern in CDA II (using SDS-polyacrylamide gel electrophoresis) is absolutely speci®c and also seems to be absolutely constant. If added to clinical, routine haematological and genetic data, the bone marrow examination will be con®rmatory. Such tests as the Ham test, or the assessment of i antigens, may become optional. Erythrokinetic studies using 59Fe and 51Cr, for the evaluation of ine€ective erythropoiesis and peripheral haemolysis, are no longer necessary in common cases. Analyses for transmission of genetic markers can be carried out for the three main CDA types, and this has allowed preliminary epidemiological studies, as we have seen. One may hope that in the near future the search for mutations will be feasible. In contrast, rare or unique varieties of CDA require exhaustive investigations, but even then a satisfactory explanation for the underlying cause of the disease may not be obtained. It is important to screen the mutation responsible for the Gilbert syndrome, that is 0 the addition of one TA ((TA)7 versus (TA)6) in the 5 -region of the UDP-glucuronosyltransferase 1 gene.64 Its e€ect on biliary complications, which has been proved in CDA II32, might apply to some other CDAs, in particular to CDA I. It is also important to de®ne the iron status, especially in CDA I and CDA II which are iron-loading conditions, using an assay of serum iron and serum ferritin concentrations, and by assessment of the saturation of serum transferrin. The onset and evolution of iron overload varies from one patient to another. It remains to be seen whether mutations associated with genetic haemochromatosis (H63Y and C282Y) in the HLA-H gene65 would have a negative impact on iron overload in CDA (mostly CDA I and CDA II). Haemosiderosis is the most important long term complication, except in those patients protected by ongoing iron loss through menstruation, pregnancy or haemosiderinuria. Iron accumulation does occur, either from transfusions or from increased intestinal absorption even in untransfused patients. Death may occur from intractable heart failure. Excessive iron deposition can also result in diabetes mellitus, hypogonadotropic hypogonadism and liver cirrhosis. For these reasons, blood transfusions should be minimized and iron therapy contraindicated. Finally it should be noted that dyserythropoiesis may accompany a number of other diseases: thalassaemia66, haemoglobin C67 and E68, hereditary sideroblastic anaemia69,

Table 2. Exhaustive elements for the diagnosis of CDA. Basic characterization . Clinical assessment (search for CDA-associated non-haematological manifestations) . Complete genealogical tree (inheritance pattern) . Red cell indices . Reticulocyte count . Blood smears . SDS-PAGE of red cell membrane proteins, and Western blots . Bone-marrow (light and electron microscopy) . Serum bilirubin (unconjugated hyperbilirubinaemia); serum haptoglobin; serum LDH . Serum free transferrin receptor; serum ferritin . Folic acid and vitamin B12 assessment (secondary de®ciency is frequent) Optional tests . Ferrokinetic studies . Serum thymidine kinase . Globin chain synthesis . Ham-test and anti-i agglutination

702 J. Delaunay and A. Iolascon

aplastic anaemia70, myelodysplastic syndromes71, preleukaemic or leukaemic states72,73, bone marrow transplantation74, AIDS75, malaria76,77, or kala-azar.78 TREATMENT There is no speci®c treatment for CDA, except for a-interferon in CDA I, as we have seen. Otherwise, treatment will be largely supportive. Transfusions should be avoided due to the major risk of iron overload, especially in CDA I and CDA II. The usefulness of splenectomy is dubious in the vast majority of cases; however this issue has not been documented in depth. However, cholecystectomy is often indicated. Morbidity and mortality following cholecystectomy are expected to be lower in the paediatric age group. Iron therapy should be avoided, especially in CDA I and CDA II. We are not ready to discuss genetic counselling at this time. More clinical data and molecular genetic data are required. Nevertheless, it seems that CDAs are usually mild enough to be out of the scope of genetic counselling. Only severe cases, which are the least understood and may derive from intermingled diseases, might be considered to be within the scope of genetic counselling. CONCLUSIONS While rare CDAs remain a problem, because thorough understanding cannot grow from cases seen from time to time, CDA I, II and III have now produced a considerable body of knowledge. Much has still to be added up, however, through careful clinical studies, long term follow-ups and the appraisal of susceptibility factors. The elucidation of the responsible genes, which encode proteins that we hardly suspect, will open startling perspectives to further fundamental knowledge. Acknowledgements We thank Professor G. Tchernia for constant stimulating discussions, Professor S. N. Wickramasinghe, Professor H. SandstroÈm and Professor Y. Yawata for their valuable help, Dr F. MieÂlot and T. Cynober for their advice in cytology, and Dr L. Roda for providing us with cases from French Polynesia. This work was supported by the Institut National de la Sante et de la Recherche MeÂdicale (Unite 473), the DeÂleÂgation aÁ la Recherche Clinique de l'Assistance PubliqueHoÃpitaux de Paris (CRC96082), the Fondation pour la Recherche MeÂdicale, the Faculte de MeÂdecine Paris-Sud; the `Telethon Projects E-645', the `MURST' Progetti Co®nanziamento and by the University of Bari (Italy). REFERENCES 1. Wendt F & Heimpel H. Kongenitale dyserythropoietische AnaÈmie bei einem zweieiigen Zwillingspaar. Medizinische Klinik 1967; 62: 172±177. * 2. Heimpel H & Wendt F. Congenital dyserythropoietic anaemia with karyorrhexis and multinuclearity of erythroblasts. Helvetica Medica Acta 1968; 34: 103±115. 3. Crookston JH, Crookston MC, Burnie KL et al. Hereditary erythroblastic multinuclearity associated with a positive acidi®ed-serum test: a type of congenital dyserythropoietic anaemia. British Journal of Haematology 1969; 17: 11±26.

Congenital dyserythropoietic anaemias 703 4. Iolascon A, D'Agostaro G, Perrotta S et al. Congenital dyserythropoietic anemia type II: molecular basis and clinical aspects. Haematologica 1996; 81: 542±558. 5. Marks WM & Mitus J. Congenital dyserythropoietic anemias. American Journal of Hematology 96; 51: 55±63. 6. Wickramasinghe SN. Dyserythropoiesis and congenital dyserythropoietic anaemias. British Journal of Haematology 1997; 98: 785±797. * 7. Wickramasinghe SN. Congenital dyserythropoietic anaemias: clinical features, haematological morphology and new biochemical data. Blood Reviews 1998; 12: 178±200. 8. Alter BP. Inherited bone marrow failure syndromes. In Handin RI, Stossel TP & Lux SE (eds) Principles and Practice of Hematology, pp 227±291. Philadelphia: JB Lippincott Company, 1995. 9. Heimpel H. CDA I: epidemiology and clinical presentation. In Proceedings of the Workshop on Congenital Dyserythropoietic Anaemias. Villa Vigoni, Loveno di Menagio, Como, Italy, May 17±18, 1999. *10. Tamary H, Shalev H, Luria D et al. Clinical features and studies of erythropoiesis in Israeli Bedouins with congenital dyserythropoietic anemia type I. Blood 1996; 87: 1763±1770. 11. Shalev H, Tamary H, Shaft D et al. Neonatal manifestations of congenital dyserythropietic anemia type I. Journal of Pediatrics 1997; 131: 95±97. 12. Alloisio N, Jaccoud P, DorleÂac E et al. Alterations of globin chain synthesis and of red cell membrane protein in congenital dyserythropoietic anemia I and II. Pediatric Research 1982; 16: 1016±1021. 13. Wickramasinghe SN, Hasan R, Menike D et al. Serum thymidine kinase in congenital dyserythropoietic anaemia type I and homozygous beta-thalassaemia. European Journal of Haematology 1997; 59: 333±334. 14. Wickramasinghe SN & Pippard MJ. Studies of erythroblast function in congenital dyserythropoietic anaemia, type I: evidence of impaired DNA, RNA, and protein synthesis and unbalanced globin chain synthesis in ultrastructurally abnormal cells. Journal of Clinical Pathology 1986; 39: 881±890. 15. Florensa L, Woessner S, Almarcha S et al. Erythroid colonies derived from BFU-E from the bone marrow in a patient with type I congenital dyserythropoietic anemia. Sangre 1990; 35: 219±221. 16. Brichard B, Vermylen C, Schei€ JM et al. Two cases of congenital dyserythropoietic anaemia type I associated with unusual skeletal abnormalities of the limb. British Journal of Haematology 1994; 86: 201±202. 17. Le Merrer M, Girot R, Parent P et al. Acral dysostosis dyserythropoiesis syndrome. European Journal of Pediatrics 1995; 154: 384±388. 18. Tamary H, Shalmon L, Shalev H et al. Approach to CDA type 1 gene [Abstract]. Haematologica 1999; 84: 112. 19. Tamary H, Shalev H, Pinsk V et al. No response to recombinant human erythropoietin therapy in patients with congenital dyserythropoietic anemia type I. Pediatric Hematology and Oncology 1999; 16: 165±168. *20. Lavabre-Bertrand T, Blanc P, Navarro R et al. Alpha-interferon therapy for congenital dyserythropoiesis type I. British Journal of Haematology 1995; 89: 929±932. 21. Wickramasinghe SN. Response of CDA type I to alpha-interferon. European Journal of Haematology 1997; 58: 121±123. 22. Tchernia G, Dommergues JP, Zupan V et al. Severe congenital dyserythropoietic anaemia type I. Antenatal management, transfusion support, and a-interferon therapy. In Proceedings of the Workshop on Congenital Dyserythropoietic Anaemias. Villa Vigoni, Loveno di Menagio, Como, Italy, May 17±18, 1999. 23. Wickramasinghe SN, Hasan R & Smythe J. Reduced interferon-alpha production by Epstein±Barr virus transformed B-lymphoblastoid cell lines and lectin-stimulated lymphocytes in congenital dyserythropoietic anaemia type I. British Journal of Haematology 1997; 98: 295±298. *24. Tamary M, Shalmon L, Shalev H et al. Localisation of the gene for congenital dyserythropoietic anemia to chromosome 15q15.1-15.3. American Journal of Human Genetics 1998; 62: 1062±1069. 25. Najfeld V, Ballard SG, Menninger J et al. The gene for human erythrocyte protein 4.2 maps to chromosome 15q15. American Journal of Human Genetics 1992; 50: 71±75. 26. Sung LA, Chien S, Fan YS et al. Human erythrocyte protein 4.2: isoform expression, di€erential splicing, and chromosomal assignment. Blood 1992; 79: 2763±2770. *27. Lind L, SandstroÈm H, Wahlin A et al. Localization of the gene for congenital dyserythropoietic anemia type III, CDAN3, to chromosome 15q21-q25. Human Molecular Genetics 1995; 4: 109±112. 28. Hodges VM, Molloy GY & Wickramasinghe SN. Genetic heterogeneity of congenital dyserythropoietic anemia type I. Blood 1999; 94: 1139±1140. 29. al-Fawaz IM & al-Mashhadani SA. Congenital dyserythropoietic anaemia type I. Report of two siblings from Saudi Arabia. Acta Haematologica 1995; 93: 50±53. 30. Lugassy G, Michaeli J, Harats N et al. Paravertebral extramedullary hematopoiesis associated with improvement of anemia in congenital dyserythropoietic anemia type II. American Journal of Hematology 1986; 22: 295±300.

704 J. Delaunay and A. Iolascon 31. West NC, Meigh RE, Mackie M & Anderson MJ. Parvovirus infection associated with aplastic crisis in a patient with HEMPAS. Journal of Clinical Pathology 1986; 39: 1019±1020. 32. Perrotta S, Carbone R, Servedio V et al. Gilbert syndrome accounts for the phenotypic variability of congenital dyserythropoietic anemia Type II (CDA II). Journal of Pediatrics (in press). 33. Miraglia del Giudice E, Perrotta S, Nobili B et al. Coinheritance of Gilbert syndrome increases the risk of developing gallstones in patients with hereditary spherocytosis. Blood 1999; 94: 2259±2262. 34. Wong KY, Hug G & Lampkin BC. Congenital dyserythropoietic anemia type II: ultrastructural and radioautographic studies of blood and bone marrow. Blood 1972; 39: 23±30. 35. Breton-Gorius J, Daniel MT, Clauvel JP & Dreyfus B. Anomalies structurales des eÂrythroblastes et des eÂrythrocytes dans six cas de dyseÂrythropoieÁse congeÂnitale. Nouvelle Revue FrancËaise d'HeÂmatologie 1973; 13: 23±49. *36. Alloisio N, Texier P, Denoroy L et al. The cisternae decorating the red blood cell membrane in congenital dyserythropoietic anemia (type II) originate from the endoplasmic reticulum. Blood 1996; 87: 4433±4439. 37. Fukuda MN. Congenital dyserythropoietic anaemia type II (HEMPAS) and its molecular basis [Review]. BaillieÁre's Clinical Haematology 1993; 6: 493±511. 38. Bouhours JF, Bouhours D & Delaunay J. Abnormal fatty acid composition of erythrocyte glycosphingolipids in congenital dyserythropoietic anaemia type II. Journal of Lipid Research 1985; 26: 435±441. 39. Zdebska E, Anselstetter V, Pacuszka T et al. Glycolipids and glycopeptides of red cell membranes in congenital dyserythropoietic anaemia type II (CDAII). British Journal of Haematology 1987; 3: 385±391. 40. Fukuda MN, Gaetani GF, Izzo P et al. Incompletely processed N-glycans of serum glycoproteins in congenital dyserythropoietic anaemia type II (HEMPAS). British Journal of Haematology 1992; 82: 745±752. 41. Chui D, Oh-Eda M, Liao YF et al. Alpha-mannosidase-II de®ciency results in dyserythropoiesis and unveils an alternate pathway in oligosaccharide biosynthesis. Cell 1997; 90: 157±167. 42. De Franceschi L, Turrini F, Miraglia del Giudice E et al. Decreased band 3 anion transport activity and band 3 clusterization in congenital dyserythropoietic anemia type II. Experimental Hematology 1998; 26: 869±873. 43. Fukuda MN, Dell A & Scartezzini P. Primary defect of congenital dyserythropoietic anaemia type II. Failure in glycosylation of erythrocyte lactosaminoglycan proteins caused by lowered N-acetylglucosaminyltransferase II. Journal of Biological Chemistry 1987; 262: 7195±7206. 44. Fukuda MN, Masri KA, Dell A et al. Defective glycosylation of erythrocyte membrane glycoconjugates in a variant of congenital dyserythropoietic anemia type II: association of low level of membrane-bound form of galactosyltransferase. Blood 1989; 73: 1331±1339. 45. Fukuda MN, Masri KA, Dell A et al. Incomplete synthesis of N-glycans in congenital dyserythropoietic anemia type II caused by a defect in the gene encoding a-mannosidase II. Proceedings of the National Academy of Sciences of the USA 1990; 87: 7443±7447. 46. Iolascon A, Miraglia del Giudice E, Perrotta S et al. Exclusion of three candidate genes as determinants of congenital dyserythropoietic anemia type II (CDA-II). Blood 1997; 90: 4197±4200. *47. Gasparini P, Miraglia del Giudice E, Delaunay J et al. Localization of congenital dyserythropoietic anemia II (CDA II) locus to chromosome 20 (20q11.2) by genomewide search. American Journal of Human Genetics 1997; 61: 1112±1116. 48. Iolascon A, De Mattia D, Perrotta S et al. Genetic heterogeneity of congenital dyserythropoiesis anemia II (CDA II). Blood 1998; 9: 2593±2594. 49. Beauchamp-Nicoud A, Schischmano€ PO, Alloisio N et al. Suppression of CDA II expression in a homozygote. British Journal of Haematology 1999; 106: 948±953. 50. Wol€ JA & Von Hofe FH. Familial erythroid multinuclearity. Blood 1951; 6: 1274±1283. 51. BergstroÈm I & Jacobsson L. Hereditary erythroreticulosis. Blood 1962; 19: 296±303. 52. Accame EA & de Tezanos Pinto M. Diseritropoyesis congeÂnita con poliploidia eritroblaÂstica. A propoÂsito de una variedad hallada en la Mesopotamia argentina. Sangre 1981; 26: 545±555. 53. SandstroÈm H, Wahlin H, Eriksson M & BergstroÈm I. Serum thymidine kinase in congenital dyserythropoietic anemia type III. British Journal of Haematology 1994; 87: 653±654. 54. SandstroÈm H, Wahlin H, Eriksson M et al. Angioid streaks are part of a familial syndrome in dyserythropoietic anemia (CDA III). British Journal of Haematology 1997; 98: 845±849. 55. SandstroÈm H, Wahlin H, Eriksson M et al. Intravascular haemolysis and increased prevalence of myeloma and monoclonal gammapathy in congenital dyserythropoietic anemia, type III. European Journal of Haematology 1994; 52: 42±46. 56. Wickramasinghe SN. Congenital dyserythropoiesis anaemias other than types I to III. In Proceedings of the Workshop on Congenital Dyserythropoietic Anaemias. Villa Vigoni, Loveno di Menagio, Como, Italy, May 17±18, 1999. 57. Carter C, Darbyshire PJ & Wickramasinghe SN. A congenital dyserythropoietic anaemia presenting as hydrops fetalis. British Journal of Haematology 1989; 72: 289±290.

Congenital dyserythropoietic anaemias 705 58. Bird AR, Knottenbelt E, Jacobs P & Maigrot J. Primary shunt hyperbilirubinaemia: a variant of the congenital dyserythropoietic anaemias. Postgraduate Medical Journal 1991; 67: 396±398. 59. Wickramasinghe SN, Andrews VE & O'Hea AM. Congenital dyserythropoiesis characterized by marked macrocytosis, vitamin B12- and folate-independent megaloblastic change and absence of the de®ning features of congenital dyserythropoietic anaemia types I or III. British Journal of Haematology 1996; 95: 73±76. 60. Wickramasinghe SN, Illum N & Wimberley PD. Congenital dyserythropoietic anaemia with novel intraerythroblastic and intra-erythrocytic inclusions. British Journal of Haematology 1991; 79: 322±330. 61. Parsons SF, Jones J, Anstee DJ et al. A novel form of congenital dyserythropoietic anemia associated with de®ciency of erythroid CD44 and a unique blood group phenotype [In(a-b-), Co(a-b-)]. Blood 1994; 83: 860±868. 62. Agre P, Smith BL, Baumgarten R et al. Human red cell aquaporin CHIP. II. Expression during normal fetal development and in a novel form of congenital dyserythropoietic anemia. Journal of Clinical Investigation 1994; 94: 1050±1058. 63. Pothier B, Morle L, Alloisio N et al. Aberrant pattern of red cell membrane and cytosolic proteins in a case of congenital dyserythropoietic anaemia. British Journal of Haematology 1987; 66: 393±400. 64. Bosma PJ, Chowdhury JR, Bakker C et al. The genetic basis of the reduced expression of bilirubin UDPglucuronosyltransferase 1 in Gilbert syndrome. New England Journal of Medicine 1995; 333: 1171±1175. 65. Feder JN, Gnirke A, Thomas W et al. A novel MHC I-like gene is mutated in patients with hereditary haemochromatosis. Nature Genetics 1996; 13: 399±408. 66. Thein SL, Hesketh C, Taylor P et al. Molecular basis for dominantly inherited inclusion body betathalassaemia. Proceedings of the National Academy of Sciences of the USA 1990; 87: 3924±3928. 67. Wickramasinghe SN, Akinyanju OO & Hughes M. Dyserythropoiesis in homozygous haemoglobin C disease. Clinical Laboratory Haematology 1982; 4: 373±381. 68. Wickramasinghe SN, Hughes M, Wasi P et al. Ultrastructure and cell cycle distribution of erythropoietic cells in heterozygotes and homozygotes for haemoglobin E. British Journal of Haematology 1984; 57: 685±694. 69. Cazzola M, Barosi G, Gobbi PG et al. Natural history of idiopathic refractory sideroblastic anemia. Blood 1988; 71: 305±312. 70. Frisch B, Lewis SM & Sherman D. The ultrastructure of dyserythropoiesis in aplastic anemia. British Journal of Haematology 1975; 29: 545±552. 71. Head DR, Kopecky K, Bennet JM et al. Pathogenic implication of internuclear bridging in myelodysplastic syndrome: An Eastern Cooperative Oncology Group/Southwest Oncology Group Cooperative Study. Cancer 1989; 64: 2199±2202. 72. Gahn B, Haase D, Unterhalt M et al. De novo AML with dysplastic hematopoiesis: cytogenetic and prognostic signi®cance. Leukaemia 1996; 10: 946±951. 73. Berkowitz LR, Ross DW & Orringer EP. Hairy cell leukemia with acquired dyserythropoiesis. Archives of Internal Medicine 1980; 140: 554±555. 74. Macon WR, Tham KT, Greer JP & Wol€ SN. Ringed sideroblasts: a frequent observation after bone marrow transplanatation. Modern Pathology 1995; 8: 782±785. 75. Wickramasinghe SN, Beatty C, Shiels S et al. Ultrastructure of the bone marrow in HIV infection: evidence of dyserythropoiesis and stromal cell damage. Clinical and Laboratory Haematology 1992; 14: 213±229. 76. Abdalla S, Weatherall DJ, Wickramasinghe SN & Hughes M. The anemia of P. falciparum malaria. British Journal of Haematology 1980; 46: 171±183. 77. Wickramasinghe SN, Looareesuwan S, Nagachinta B & White NJ. Dyserythropoiesis and ine€ective erythropoiesis in Plasmodium vivax malaria. British Journal of Haematology 1989; 72: 91±99. 78. Wickramasinghe SN, Abdalla SH & Kasili EG. Ultrastructure of bone marrow in patients with visceral leishmaniasis. Journal of Clinical Pathology 1987; 40: 267±275.