Congenital dyserythropoietic anaemias (CDA) are a rare group of diseases characterized by an ineffective erythropoiesis and a large spectrum of cytological nuclear anomalies of erythroid precursors, which are the mainstay of classification. In spite of recent genetic advances in the identification of the genes involved in several CDA subtypes, including CDAN1, SEC23B and KLF1 genes, in CDA type I (CDAI) (Dgany et al, 2002; Renella et al, 2011), CDA type II (CDAII) (Schwarz et al, 2009) and unclassified CDA (Arnaud et al, 2010) respectively, these diseases remain extremely heterogeneous. The up-regulation of iron absorption is mediated by low hepcidin levels consecutive to ineffective erythropoiesis, and the concept of a ‘physiological erythroid regulator’ was suggested almost 20 years ago (Finch, 1994) but its exact nature remains unknown. We report a new variant of CDA with major and impressive unclassified cytological anomalies and contrasting with both a very slight impairment of haematopoiesis and a very severe liver iron overload.
This 33-year-old man born from non-consanguineous Caucasian parents presented a past history of very slight non-regenerative anaemia (Hb > 105 g/l) with hyperbilirubinaemia since birth. He never received any red blood cell transfusions. He also presented hypospadias, mental retardation and hypogonadism. His follow up stopped when aged 17 years. His mother and his sister had a normal haemogram and no iron overload. His father could not be assessed. The patient was referred again at the age of 33 years for asthenia. His spleen was slightly enlarged (18 × 16 × 9·5 cm) and a haemogram demonstrated a moderate normocytic, normochromic anaemia (Hb, 108 g/l; haematocrit, 32%; mean corpuscular volume, 91 fl; mean corpuscular haemoglobin, 31 pg;mean corpuscular haemoglobin concentration, 340 g/l and 2% (60 × 109/l) reticulocytes. Microscopic blood film examination showed dyserythropoietic red cells: basophilic stippling, tear-drop cells and cabot rings. Karyorrhexis was found among the few acidophilic erythroblasts observed. Total bilirubin was 27·4 μmol/l (normal range [N] < 17 μmol/l) while haptoglobin and lactate dehydrogenase levels were normal. The serum erythropoietin level was at 97 iu/l (N: 5–30 iu/l). Folate and vitamin B12 levels, haemoglobin electrophoresis, enzyme glucose-6-phosphate dehydrogenase, pyruvate kinase, 5′nucleotidase enzymes, erythrocyte membrane protein electrophoresis and ektacytometry enabled the exclusion of other causes of dyserythropoiesis. Karyotypes of both bone marrow cells and constitutional cells were normal. Bone marrow aspirate showed a major hyperplasia of the erythroid cell lineage, which represented 58% of bone marrow cells. Dyserythropoietic changes affected 85% of the erythroid precursors with predominant nuclear fragmentation of the vast majority of late erythroblasts (Fig 1A–A’). There were a few binuclear erythroblasts (Fig 1A) but no ‘double membrane’ features like those depicted in CDAII. Electron microscopy identified some erythroblasts with an abnormal electron-dense heterochromatin (Fig 1B) but there was no internuclear chromatin bridge, like in CDAI, and no double membrane beneath the cytoplasmic membrane, as in CDAII. Erythroblasts with karyorrhexis were frequent (30%) (Fig 1B’), but the bone marrow did not contain giant multinucleate erythroblasts, as found in CDAIII. Perls colouration and electron microscopy did not show any iron-loaded mitochondria. Sanger sequencing of the entire coding open reading frame and the flanking intronic sequences of the CDAN1, SEC23B and KLF1 genes failed to identify any mutation. Markers of iron status showed heavy iron overload with a serum ferritin concentration at 3480 μg/l (N: 20–300 μg/l) and a 100% transferrin saturation. Liver iron concentration was 559 mol/g liver dw (N < 36 mol/g) determined on a liver biopsy (tissue iron score was 42). Hepatic iron overload was associated to severe fibrosis (F3, Metavir score). Soluble transferrin receptor dosage was normal. There was no HFE mutation (C282Y, H63D, S65C) and SLC40A1 (Ferroportin 1 gene) sequence was normal. Serum hepcidin (enzyme-linked immunosorbent assay, Intrinsic LifeSciences, La Jolla, CA) was decreased, AT 19·7 μg/l (N in men: 110–245 μg/l); serum growth differentiation factor 15 (GDF15) was 2477 pg/ml (control: 325–555 pg/ml). Erythroid proliferation and differentiation were studied in liquid culture performed as previously described (Claessens et al, 2002). Cytological analysis demonstrated that the onset of terminal erythroid differentiation was delayed compared to normal bone marrow. Mature polychromatophilic and acidophilic erythroblasts appeared from day 15 instead of day 12 and cytologically abnormal erythroblasts represented 35% of the cells at day 20 demonstrating that dyserythropoiesis was preserved by ex vivo expansion. Cumulative cell number for the patient was a third lower than controls (Fig 2A) corresponding to a defect in proliferative capacities as shown by an arrest after only one cell division during the examined period of 3 days (Fig 2B) without excess of apoptosis (Fig 2C–D).
This reported case of very mild anaemia clearly presents the hallmarks of CDA complicated by massive hepatic iron overload. However, it does not match with any previously described case of CDA according to morphological classification, recent genetic analyses (Dgany et al, 2002; Schwarz et al, 2009; Arnaud et al, 2010) or any form of genetic haemochromatosis. This case illustrates that, despite the recent advances in genetics and mouse models (Renella et al, 2011), CDA subtypes remain extremely heterogeneous and are probably multiple unrelated genetic conditions. In vitro analysis of erythropoiesis showed evidence of a defect of terminal erythroid maturation without apoptosis excess, in contrast to some CDAII (Schwarz et al, 2009; Cazzola & Invernizzi, 2010). The universal mechanism by which ineffective erythropoiesis suppresses hepcidin is not yet fully understood, and the involvement of different members of the transforming growth factor-β super family of cytokines, has been reported (Tanno et al, 2007; Truksa et al, 2009). Although GDF15 was found to be elevated in our patient, much higher levels ranging from 5000 to 15 000 pg/ml were reported in CDAI cases, with more discrete iron overload (Tamary et al, 2008). This new case of CDA is interesting because the mechanism involved in iron overload is slightly or not dependent on the degree of anaemia (very mild in our case), but more directly related to the blockade in erythroid differentiation occurring at the late erythroblast stage. This case could be considered as a typical pure genetic disease of the erythroid regulator, but that one is still to be discovered.