New Sporadic Case of Congenital Dyserythropoietic Anemia Type III in an Aged Woman: Detailed Description of Ultrastructural Findings

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1 American Journal of Hematology 70:72±76 (2002) New Sporadic Case of Congenital Dyserythropoietic Anemia Type III in an Aged Woman: Detailed Description of Ultrastructural Findings Erica Sigler, 1,2 Dina Shaft, 3 Mordechai Shtalrid, 1 Lev Shvidel, 1 Alain Berrebi, 1,3 and Peretz Resnitzky 3 * 1 Department of Hematology, Kaplan Medical Center, Rehovot, Israel, Af liated with the Medical School of the Hebrew University, Jerusalem, Israel 2 Blood Bank, Kaplan Medical Center, Rehovot, Israel, Affiliated with the Medical School of the Hebrew University, Jerusalem, Israel 3 Efrati Research Institute for Blood Cells and Cytology, Kaplan Medical Center, Rehovot, Israel, Af liated with the Medical School of the Hebrew University, Jerusalem, Israel We describe a new case of congenital dyserythropoietic anemia (CDA) type III. This least common type of CDA was diagnosed at the age of 59 in a 70-year-old woman who suffered from a young age from mild macrocytic anemia,while the long follow up since diagnosis documented a benign clinical course. No family history of blood diseases was obtained and no anemia was documented in the medical records of any of her four children. The bone marrow (BM) examination on light microscopy revealed a severe erythroid hyperplasia with the presence of giant multinucleated erythroblasts. Ultrastructural examination of the BM disclosed the presence of many large multinucleated erythroblasts bearing a variety of ultrastructural ndings: nuclear clefts,autophagic vacuoles,iron-loaded mitochondria,and intracytoplasmic myelin gures. In addition, extensive hyperlobulation of the nucleus and partial loss of nuclear membrane with ``spilling'' of nuclear material to the adjacent cytoplasm was also noted in some of the erythroblasts. These last two ndings have not been previously described in CDA III. Am. J. Hematol. 70:72±76,2002. ã 2002 Wiley-Liss,Inc. Key words: anemia; dyserythropoiesis; CDA III; ultrastructure INTRODUCTION The congenital dyserythropoietic anemias (CDA) are a heterogeneous group of rare hereditary diseases characterized by ine ective erythropoiesis and dysplastic changes in the erythroid precursors. Three major types of CDA (I, II, and III) as well as a number of variants have been described [1,2]. CDA III is the least common of the three classical types. The disorder was inherited as an autosomal recessive character in three large families, and in one of those, linkage analysis studies have localized the responsible gene to chromosome 15q21-q25 [3]. In addition, the disease may appear as sporadic with an inherited autosomal recessive character or may arise de novo as a spontaneous dominant mutation [4,5]. The main laboratory nding is a mild macrocytic anemia, and the disease runs a relatively benign course, which makes it possible for most patients to reach longevity. Bone marrow ã 2002 Wiley-Liss,Inc. (BM) examination by light microscopy reveals multinucleated large erythroid precursors and other dyserythropoietic features. The reported ultrastructural studies demonstrate various dysplastic features in the erythroblasts, such as multinuclearity, intranuclear clefts, iron-loaded mitochondria, and intracytoplasmic autophagic vacuoles and myelin gures [6 8]. We present here a detailed electron microscopic (EM) analysis of the erythroblasts in a new case of CDA III of the sporadic form in an elderly woman. *Correspondence to: Dr. P. Resnitzky, The Efrati Research Institute for Blood Cells and Cytology, Kaplan Medical Center, 76100, Israel. PeretzResnitzky@wis .weizmann.ac.il Received for publication 2 July 2001; Accepted 15 November 2001 Published online in Wiley InterScience ( com). DOI: /ajh.10086

2 Case Report: EM Description in CDA III 73 CASE REPORT A 59-year-old female patient who had emigrated from Egypt was seen in 1988 for the rst time in our Hematological Outpatient Clinic because of anemia. The family history was irrelevant and the patient was unaware of any case of blood disease among family members. Her four children were healthy, and no anemia was documented in their medical records. The past history revealed a mild macrocytic anemia for many years, which did not require special treatment though she occasionally received blood transfusions during pregnancies. After the delivery of her fourth child, at the age of 32, a diagnosis of Sheehan syndrome was made and she was put on replacement therapy with cortisone and a thyroid preparation. Cholecystectomy was performed at the age of 35 because of symptomatic cholelythiasis. Physical examination revealed no special abnormalities. The patient did not complain of visual disturbances, and the ophthalmoscopic examination was normal. The laboratory data showed: Hb 9.7 g/dl, Ht 30%, MCV 103 fl, reticulocytes 1.2%, WBC and platelets in the normal range. The blood lm revealed marked anisopoikilocytosis, hypochromia, and basophilic stippling of red blood cells. The BM examination on light microscopy revealed a severe erythroid hyperplasia with the presence of giant multinucleated erythroblasts. The granulocytic and megakaryocytic lineages were normal, and the iron stores were normal, although in subsequent examinations (in December 1996 and January 1999) an increased amount of iron in macrophages and a few ring sideroblasts were noted. The bilirubin, the liver enzymes, total protein, protein electrophoresis, iron and transferin, ferritin, folic acid, and vitamin B 12 levels were all in the normal range. The urine sediment negatively stained for hemosiderin. Haptoglobin was low (5 mg/dl), and the LDH was slightly increased (450 IU/mL). Hemoglobin electrophoresis, Coombs' test, Ham's test, sucrose lysis test, osmotic fragility, and G6PD were normal. The red cells were not agglutinated by anti-i serum. The erythropoietin level was mildly elevated (181 miu/ml, normal range miu/ml). During the following 9 years, the patient required occasional blood transfusions while being hospitalized for infectious conditions complicated by addisonian crises. During the last 2 years the patient developed chronic atrial brillation with signs of congestive heart failure, and the laboratory tests revealed a drop in Hb to 8 g/dl and a rise in LDH to 1,400 IU/mL and in ferritin to 1,500 ng/ml. The spleen and liver remain within normal limits, and she requires a blood transfusion every 3 4 months. Fig. 1. Multinuclearity in abnormal erythroblasts. (A) Large multinucleated late erythroblast containing ve nuclei of different sizes,while an already extruded one can be seen nearby (arrow). The cytoplasm contains many iron-loaded mitochondria (arrowhead) and a cluster of ferritin (F) (original magni cation 39,300). (B) Binucleated erythroblast with an intermediate degree of maturity containing two nuclei of different shapes and sizes. A nuclear cleft is evident in the larger indented nucleus (arrow) and the cytoplasm contains many iron-loaded mitochondria (arrowhead) (original magni cation 39,527).

3 74 Case Report: Sigler et al. EM Studies Ultrastructural examination of the BM, which was performed on two occasions (in 1996 and in 1999), disclosed the presence of many large multinucleated erythroblasts bearing a variety of ultrastructural ndings (Figs. 1 4). In some multinucleated cells the shape and size of the di erent nuclei di er considerably, and occasionally one of them displays a ``spongy'' appearance of the heterochromatin, while the other(s) do not. Asynchrony in the process of nuclear expelling was noted between the di erent nuclei in a given multinucleated cell. Intranuclear clefts were evident in many cells; in part of the cells the extensive clefting caused the formation of lobules. Partial loss of nuclear membrane with the ``spilling'' of nuclear material to the adjacent cytoplasm was evident in some cells. The cytoplasm of many erythroblasts contained iron-loaded mitochondriae, clusters of ferritin, autophagic vacuoles, and myelin gures. DISCUSSION We report a patient with mild to moderate chronic macrocytic anemia, laboratory ndings of ine ective erythropoiesis, and BM morphological features that t the criteria for diagnosis of CDA III. The diagnosis was made at an advanced age after other causes of dyserythropoiesis had been ruled out, and the clinical and morphological changes remained stable for almost 10 years. Because no family history was obtained, this case seems to be one of a sporadic mutation, similar to those previously described [4,5,9]. In spite of the similarity in BM morphology among reported cases of CDA III, the clinical manifestations in the di erent patients are variable and it seems that they are not related to the particular type of inheritance. Iron overload secondary to ine ective erythropoiesis and increased absorption from the gastrointestinal tract was observed in some patients with the sporadic form of the disease [4,9] but not in others [5], nor in patients su ering from the familial type of the disease [10]. In cases in which iron overload was absent, hemosiderinuria due to intravascular hemolysis was noted. This pattern was observed in both familial and sporadic cases of CDA III [5,10,11]. The same discrepancy was already emphasised about the presence or absence of splenomegaly in familial or sporadic cases [2]. In our patient, though the liver and spleen remained in the normal range and no overt clinical signs of hemochromatosis appeared, the laboratory tests indicated that iron overload progressed with time. Fig. 2. ``Hyperlobulation'' in abnormal erythroblasts. (A) Large late erythroblast showing multiple nuclear clefts (arrows),myelin gures (M),clusters of ferritin (F),and ironloaded mitochondria (arrowhead) (original magni cation 35,570). (B) Late erythroblast showing multiple nuclear clefts (arrow) and lobulation of the nucleus (original magni cation 35,080). (C) Detail of the ``hyperlobulated'' region of B showing nuclear pockets (arrow) (original magni cation 311,600). The ultrastructural ndings in the erythroblasts of patients with CDA III include multinuclearity, nuclear clefts, autophagic vacuoles, iron-loaded

4 Case Report: EM Description in CDA III 75 Fig. 4. Abnormal late erythroblast showing a lobulated nucleus with many intranuclear clefts,partial loss of nuclear membrane and ``spilling'' of nuclear material to the cytoplasm. A part of the nucleus at the right shows a ``spongy'' appearance. Iron-loaded mitochondria and two large autolytic areas are present in the cytoplasm (original magni cation 36,640). Fig. 3. ``Spilling'' of nuclear material to cytoplasm of abnormal erythroblasts. (A) Erythroblast showing disappearance of parts of nuclear membrane and nuclear material outside to the nucleus in the adjacent cytoplasm which is largely vacuolated (original magni cation 314,800). (B) Part of an abnormal early erythroblast showing intranuclear cleft (arrow),loss of nuclear membrane (arrowhead) and extensive ``spilling'' of nuclear material to the cytoplasm (original magni cation 321,700). mitochondria, and intracytoplasmic myelin gures [6 8]. The EM picture as a whole in the present case ts the above descriptions. However, in our patient two additional features were evident in a substantial number of BM erythroblasts. The rst one is the striking nuclear hyperlobulation (Fig. 2) which seems to be the result of a particular extensive clefting tendency of the nucleus. Though lobulation of the nuclear membrane has been mentioned in CDA III [5], this phenomenon was not described in detail and not emphasised as a EM pattern in CDA III in the previously reported cases. In the present case the nuclear clefting in many erythroblasts was so striking that the entire nucleus or a part of it almost became hyperlobulated (Figs. 2 and 4). The second observed feature in our case was the disappearance of segments of nuclear membrane with the concomitant ``spilling'' of nuclear material to the surrounding cytoplasm (Figs. 3 and 4). This nding has not been described in the previously reported cases of CDA III or in other types of CDA or as an unspeci ed sign in severe dyserythropoiesis of di erent etiologies. Interestingly, in CDA I, a wide dilatation of nuclear pores with the concomitant invagination of cytoplasm into the nuclear area is a very frequent EM feature. However ``spilling'' of nuclear material to the cytoplasm has not been described. We did not nd that feature in 20 cases of CDA Iwe recently reported

5 76 Case Report: Sigler et al. [12], even in cases with marked dyserythropoietic changes. As the number of ultrastructural studies already reported in CDA III is relatively small, it could be that the two above previously undescribed ndings are actually present in other cases as well. The pathogenesis of the above-described erythroblastic morphological changes present in CDA III is not clear. However, the striking tendency to nuclear clefting, hyperlobulation, and ``spilling'' of nuclear material into cytoplasm would suggest an underlying structural membrane defect. EM studies would be an important tool in the diagnosis of CDAs particularly during investigation of anemia of obscure etiology in aged persons in which that diagnosis would at rst seem to be improbable. In addition, analysis of ultrastructural ndings in new cases of CDA III may be of help for the understanding of pathogenesis and for its further classi cation. REFERENCES 1. Heimpel H, Wendt F. Congenital dyserythropoietic anaemia with karyorrhexis and multinuclearity of erythroblasts. Helv Med Acta 1968;34: Wickramasinghe SN. Dyserythropoiesis and congenital dyserythropoietic anaemias. Br J Haematol 1997;98: Lind L, SandstroÈ m H, Wahlin A, Eriksson M, Nilsson-Sojka B, SikstroÈ m C, Holmgren G. Localization of the gene for congenital dyserythropoietic anaemia type III, CDAN3 to chromosome 15q21-q25. Hum Mol Genet 1995;4: Goudsmit R, Beckers D, de Bruijne JI, Engelfriet CP, James J, Morselt AFW, Reynierse E. Congenital dyserythropoietic anaemia, type of III. Br J Haematol 1972;23: Wickramasinghe SN, Parry TE, Williams C, Bond AN, Hughes M, Crook S. A new case of congenital dyserythropoietic anaemia, type III: studies of the cell cycle distribution and ultrastructure of erythroblasts and of nucleic acid synthesis in marrow cells. J Clin Pathol;35: BjoÈ rksten B, Holmgren G, Roos G, Stenling R. Congenital dyserythropoietic anemia type III: an electron microscopic study. Br J Haematol 1978;38: Wickramasinghe SN, Goudsmit R. Some aspects of the biology of multinucleate and giant mononucleate erythroblasts in a patient with CDA, type III. Br J Haematol 1979;41: Wickramasinghe SN, Wahlin A, Anstee D, Parsons SF, Stopps G, BergstroÈ m I, Eriksson M, SandstroÈ m H, Shiels S. Observations on two members of the Swedish family with congenital dyserythropoietic anaemia, type III. Eur J Haematol 1993;50: Clauvel JP, Cosson A, Breton-Gorius J, Flandrin G, Faille A, Bonnet-Gajdos M, Turpin F, Bernard J. Dyse rythropoieá se congeâ nitale. Nouv Rev Fr Hematol 1972;12: SandstroÈ m H, Wahlin A, Eriksson M, BergstroÈ m I, Wickramasinghe SN. Intravascular haemolysis and increased prevalence of myeloma and monoclonal gammopathy in congenital dyserythropoietic anaemia, type III. Eur J Haematol 1994;52: BergstroÈ m I, Jacobsson L. Hereditary benign erythroreticulosis. Blood 1962;19: Tamary H, Shalev H, Luria D, Shaft D, Zoldan M, Shalmon L, Gruinspan A, Stark B, Chaison M, Shinar E, Resnitzky P, Zaizov R. Clinical features and studies of erythropoiesis in Israeli Bedouins with congenital dyserythropoietic anemia Type I. Blood 1996;87: