Congenital dyserythropoietic anemia type III

Congenital dyserythropoietic anemia type III HERBERT SANDSTRÖM,* ANDERS WAHLIN° *Family Medicine and °Hematology, Medicine, Department of Public Health and Clinical Medicine, Umeå University,

in the congenital dyserythropoietic anemias (CDA) resemble those seen in vitamin B12 or folate deficiency, and disorders of hemoglobin synthesis including thalassemia and hereditary sideroblastic anemia. 1 The term dyserythropoiesis includes abnormalities in both the morphologic and kinetic aspects of erythropoiesis and implies a disturbance during the development of the erythrocyte irrespective of the primary cause.This leads to the production of defective erythroblasts and erythrocytes and an increased rate of phagocytosis of these cells by bone marrow macrophages. 2he CDAs are rare inherited disorders with clinical manifestations of ineffective erythropoiesis and a variable degree of anemia.On the basis of the morphologic alterations of the bone marrow and on serologic grounds, in 1968 Heimpel and Wendt classified CDA into three different types. 3][6][7] The clinical severity of these anemias is variable.Some patients are completely asymptomatic and others may require blood transfusions.The diagnosis and classification of the CDAs have so far been based on clinical characteristics, morphologic features of the bone marrow and a few serologic tests.Since clinical and laboratory findings may be subtle, it is reasonable to assume that CDA may be misdiagnosed and confused with other congenital or acquired disorders such as myelodysplastic syndrome, atypical thalassemia, or Gilbert's syndrome. 8The conspicuous bone marrow findings in CDA-III may also be misdiagnosed as acute erythroleukemia. 9n CDA-II the erythrocyte membrane glycoprotein band 3 shows a marked reduction of polylactosamines and migrates slightly faster than normal in SDS polyacrylamide gel electrophoresis.Analysis of N-glycans from CDA-II erythrocyte membranes has revealed incompletely processed N-glycan structures indicating defective glycosylation at the N-acetylglucosaminyltransferase II (GnT-II) and/or α-mannosidase II (MII) steps, 10 but besides this observation very little is known about the pathologic mechanisms underlying the CDAs.
In 1995, our group located the genetic defect in a Swedish family with CDA-III to chromosome 15q21-25. 11In an Israeli Bedouin family with CDA-I the genetic lesion was subsequently also located to the long arm of chromosome 15 but in a different region, 15q15.1-15.3. 12In a study of a large number of Italian families with CDA-II the disease gene has been located to 20q11.2. 13 D yserythropoiesis is often the result of an acquired defect in a stem cell and its progeny, e.g.myelodysplastic syndrome or leukemia with abnormal development of cell lines, or abnormal environmental conditions within the bone marrow, for instance the toxic effect of chemotherapy or deficiency of vitamin B12, folate or iron inhibiting normal cell proliferation.Some of the morphologic changes Congenital dyserythropoietic anemia type III CDA-III is the rarest form of dyserythropoietic anemia.In a family with 37 affected cases living in the North Swedish county Västerbotten, the disorder is inherited as an autosomal dominant trait with full penetrance of the bone marrow changes. 9,14The majority of known CDA-III patients belong to the Västerbotten family and two other families, an American family, and an Argentinian family.The mode of inheritance is probably also autosomal dominant in the American family.This family has four affected members, who formed the basis of the original description of CDA-III reported by Wolff and Von Hofe in 1951. 15The mode of inheritance has not been clarified in eight Argentinian cases but, again, an autosomal dominant mode of inheritance has been suggested. 6,16Only sporadic cases have been reported from other countries and, overall, fewer than 60 cases are reported in the literature.[19] In 1951 Wolff and van Hofe reported the cases of a woman and her three children with mild anemia and remarkable bone marrow findings with abundant multinucleated erythroblasts.The condition was named familial erythroid multinuclearity and was later classified as CDA-III.These were the first cases of CDA-III reported in the literature. 15In 1954 a 35-year old man was investigated at the Department of Medicine in Umeå in northern Sweden because of anemia.The investigations revealed bone marrow changes similar to those occurring in erythroleukemia without there being clinical enlargement of the liver, spleen or lymph nodes.After five years of follow-up there were no signs of progression of the disease.Based on this case Bergström and Jacobsson published a study of the Västerbotten family descending from a couple born in the late 19 th century.They identified 15 subjects in the family with similar findings and named the disorder hereditary benign erythroreticulosis. 9They showed that the disorder is inherited as an autosomal dominant trait with full penetrance of the bone marrow changes but with minor variations in the clinical picture.[22]

Clinical findings
In an investigation of twenty members of the Västerbotten family with CDA-III, 35% regularly suffered weakness, fatigue, or headache.The patients reported more intense symptoms during infections, following trauma, and during pregnancy.Five patients (20%) had received blood transfusions, four of them in late pregnancy or after delivery.Twenty percent reported episodes of abdominal pain interpreted as biliary symptoms and two (10 %) had had a cholecystectomy.However, in spite of these symptoms most of the patients regarded themselves as healthy.Six patients (30%) had mild jaundice, but the physical examination was otherwise normal.No patient had splenomegaly. 20though the Swedish and American families are not related to each other the degree of anemia as well as other clinical and laboratory abnormalities in the four patients in the the American CDA-III family discussed above were similar to those found in the Västerbotten family.No information is available concerning urinary hemosiderinuria in the American family, and eye lesions and gammopathy have only been reported in the Västerbotten family.Preliminary results of coupling analyses of a few samples from the American family are compatible with, but do not prove, the occurrence of a common genetic basis of the disorder in the two families (unpublished data).
Despite some similarities there are considerable differences in the clinical picture between CDA-III cases reported in the literature.This may indicate a variable phenotypic expression of the same genetic alteration or different genetic abnormalities leading to a similar phenotype.For example, none of the patients described by Wolf and von Hofe nor any of the patients in the Västerbotten family had an enlarged liver or spleen, but splenomegaly was present in eight patients and hepatomegaly in four Argentinian cases. 168][19] In the sporadic cases of CDA-III there are some reports of rare clinical findings such as mongoloid facies and mental retardation, 18 malignant Tcell lymphoma, 23 Hodgkin's disease, 24 development of severe iron overload and cirrhosis 25 and extramedullary hematopoiesis. 26

Laboratory findings
Sixty-five percent of patients belonging to the Västerbotten CDA-III family have mild or moderate anemia.Their median hemoglobin concentration is 118 g/L (range 80-143).The blood film shows moderate or marked anisocytosis and poikilocytosis, basophilic stippling of the red cells and large erythrocytes (macrocytes).The MCV is normal or slightly elevated.The morphology and numbers of leukocytes and platelets are normal.The relative number of reticulocytes is normal or slightly low.Haptoglobin is always low or absent and lactate dehydrogenase elevated.Most patients have a slight increase in bilirubin and aminotransferase levels.There are no significant changes in serum iron, transferrin or ferritin concentrations.Iron staining of the urinary sediment regularly shows hemosiderinuria. 14Serum thymidine kinase is markedly increased in all patients in the Västerbotten family. 20he anemia seems to be the result of both ineffective erythropoiesis and hemolysis.The hemolysis is intravascular, as demonstrated by the urinary hemosiderin, explaining why patients with the Västerbotten type of CDA-III do not have any problem with iron overload unlike patients with some other types of CDA. 14 Agglutination and hemolysis of erythrocytes with anti-i and anti-I antibody was normal in two cases, 22 and increased in two others. 18,25The acidified serum lysis test (Ham's test) is negative. 14Studies of the erythrocyte membrane shows only minor changes in membrane proteins.A small difference in relative molecular mass (Mr) has been observed for band 3 in SDS polyacrylamide-electrophoresis in patients with CDA-III. 22This type of discrepancy has also been observed Congenital dyserythropoietic anemia type III in CDA-II, although to a greater extent. 10The relevance of this finding is unclear but may suggest that glycosylation of band 3 could also be affected in CDA-III.

Bone marrow
Light microscopy of bone marrow aspirates shows erythroid hyperplasia with bi-or multinucleated erythroblasts and some giant mononucleated polychromatic erythroblasts. 6Giant multinucleated erythroblasts, with up to twelve nuclei of variable size, are typical of CDA III (Figure 1 and 2).Anisocytosis, macrocytosis, and poikilocytosis are regularly seen (Figure 3).Myelopoiesis and thrombopoiesis are normal.In the Västerbotten family the bone marrow mor-phology was stable over 10-25 years in 17 re-examined patients.Various non-specific dysplastic features can be seen by electron microscopy, e.g.abnormally long intranuclear clefts, abnormally large autophagic vacuoles, iron-laden mitochondria and intracytoplasmic myelin figures.Other findings are duplication or extensive myelinization of large parts of the nuclear membrane and lobulation of the nuclear outline. 22,27tudies of DNA content of erythroblasts and DNA synthesis show that some of the erythroblasts seem to become arrested during the cell cycle after a period in S-phase.Both mononucleated and multinucleated erythroblasts contain an increased amount of DNA, up to 28c and 48c, respectively, compared to normal (2c).When the separate nuclei within a multinucleated cell are unequal in size, the nuclei usually have different DNA contents and when this difference is marked, the DNA content may differ between <1c and 8c. 18,19,22

Gammopathy and myeloma
Serum electrophoresis was performed in 25 of the Västerbotten patients.In five cases an M-component was present, in all cases of IgG-kappa type.One patient had myeloma, the others had monoclonal gammopathy.In one of the five patients the M-component was also present in the urine.The median age of these patients was 53 years (range 35-80).The patient with myeloma had an M-component for 15 years and smoldering myeloma for 10 years with >30% plasma cells in the bone marrow but without bone disease or any other signs of progress.Another deceased patient with CDA-III in this family had had myeloma with an IgG-λ M-component. 14,21

Eye changes
A few patients belonging to the Västerbotten CDA-III family have developed visual impairment in later life.Ten patients, with and without visual impair-   © F e r r a t a S t o r t i F o u n d a t i o n ment, were investigated with eye fundus photography and fluorescein angiography.Five healthy relatives were also investigated in the same way.The investigation showed angioid streaks in eight of the investigated patients.(Dr O. Sandgren, personal communication). 21Skin biopsies taken from five patients to exclude pseudoxanthoma elasticum were negative.Angioid streaks are a condition usually associated with systemic disorders such as pseudoxanthoma elasticum, Paget's disease of the bone, sickle cell anemia, and β-thalassemia. 28The characteristic ocular sign is a peripapillary ring with irregularly radiating subretinal linear breaks in Bruch's membrane extending in all directions (Figure 4).The changes are usually bilateral and of varying width from barely visible with an ophthalmoscope to three to four times as wide as the retinal vessels and thus detectable in routine eye fundus investigation.Fluorescein angiography can be used to detect early angioid streaks. 29

Localization of the disease gene (CDAN3)
Fifty-six members from the Västerbotten family were studied.Using linkage analyses and recombination data the genetic defect in CDA-III (CDAN3) was located to chromosome 15 (15q21-25) within a distance of 11 cM. 11Additional investigations have limited the distance to 4.5 cM in 15q22 (Dr.L. Lind, personal communication).

Diagnosis
Despite the fact that congenital dyserythropoietic anemias are very rare the diagnosis should be considered in neonates, children or adults with anemia and normal to slightly elevated MCV, especially when anisocytosis and poikilocytosis are present and the anemia is familial.Laboratory findings of hemolysis and elevated bilirubin strengthen the suspicion. 8The investigation should include a blood-count, MCV and red cell volume histogram, reticulocyte count and blood smear.A bone marrow examination is usually diagnostic and should be performed early.A thor-ough family history focusing on anemia, jaundice and visual impairment is essential.Conditions that may be confused with CDA are MDS, erythroleukemia, congenital and acquired hemolytic disorders, thalassemias, PNH and certain infections such as AIDS, malaria and kala azar. 30In order to exclude megaloblastic anemia serum B12 and folate or homocysteine levels should be checked.Hemoglobin electrophoresis should be done in order to demonstrate a possible hemoglobinopathy.Serum thymidine kinase is elevated in CDA-III but has recently been shown to be high also in CDA-I. 31Measurement of serum thymidine kinase is useful in family investigations when bone marrow aspirate is not available, for instance in small children. 20

Management
The anemia in CDA III, if present, is usually mild and does not require any intervention.Transfusion is needed only in exceptional cases with severe anemia, for instance in pregnancy.Iron substitution should not be given if iron deficiency is not present.Increased erythropoiesis may cause increased requirement of folate, and treatment with 5 mg folate per week has been suggested although serum folate is usually normal or only slightly lowered. 30Our studies show that patients with the Västerbotten type of CDA-III have an increased risk of developing monoclonal gammopathy, myeloma and eye changes (angioid streaks). 21Patients with M-proteins should be examined annually with routine laboratory investigations including electrophoresis.Eye fundus examination by an ophthalmologist is recommended at the time of diagnosis.Thereafter, fundus photography is recommended every second to third year in patients over 50 years of age.Photocoagulation of early neovascular changes in the retina seems to prevent visual impairment in patients with angioid streaks associated with other disorders. 32Although the benefit of such procedures has not been proven in angioid streaks associated with CDA-III they may be useful also in this condition.Current data do not indicate any benefit of regular follow-up in younger patients without gammopathy and eye lesions.

Perspectives
Although the genetic lesion causing CDA-III has been mapped to chromosome 15q22 in the Västerbotten family the function of the gene is completely unknown.The occurrence of multinucleated erythroblasts containing nuclei of different degrees of maturation may indicate that the gene product causes a disturbance during mitosis in erythropoietic cells.However, the gene operating in CDA-III obviously also causes abnormal functions in other cell lines, as demonstrated by the occurrence of monoclonal gammopathy and angioid streaks among the same members of the family who carry the CDA-III trait.The American CDA-III family, originally described by Wolff and von Hofe, seems to have the same phenotype as the Västerbotten family, although monoclonal gammopathy and angioid streaks have not been reported in this family.It is possible that the genetic lesion in the American and Västerbotten families is identical, but the different phenotypes report-

Figure 1 .
Figure 1.Typical bone marrow smear from a patient with CDA-III and myeloma.To the upper left there is a large multinucleate erythroblast with six nuclei, adjacent to a binucleate plasma cell.May-Grünwald-Giemsa staining (Figure by courtesy of Dr. J. Hultdin).

Figure 4 .
Figure 4. Angioid streaks in a 69-year-old female with CDA-III.Fluorescein angiogram of the left eye.(Photo by courtesy of Dr. O. Sandgren).© F e r r a t a S t o r t i F o u n d a t i o n