Pathogenesis of iron overload in congenital dyserythropoietic anemia type II (CDAII)

Iron homeostasis plays a crucial role in normal erythropoiesis as well as in iron loading anemias, such as β-thalassemia intermedia and CDAs (congenital dyserythropoietic anemias), which represent interesting human models to study abnormal erythropoiesis. CDAs are genetic disorders which result in intramedullary destruction of developing erythroid cells (ineffective erythropoiesis). The disorder of erythropoiesis results in the paradoxical association of a bone marrow showing marked erythroid hyperplasia and anemia with a suboptimal reticulocyte response. Three forms of CDAs are well known: the type II is the most frequent, whose causative gene, SEC23B, is involved in ER-Golgi network. Cirrhosis and hemochromatosis, diabetes and gallstones are common complications due to abnormal iron accumulation. The mechanisms responsible for such complications mainly due to iron overload remain to be elucidated. In this work we investigated the role of SEC23B gene in iron overload through studies in vivo e in vitro. In in vivo studies we evaluated growth differentiation factor 15 (GDF15), soluble transferrin receptor (sTfR), soluble hemojuvenil (sHJV), ferritin and hepcidin levels in serum or plasma samples of CDAII patients as iron overload markers. Serum or plasma levels of these molecules were correlated with iron biochemical and hematological parameters to identify prognostic markers to predict the onset of iron overload in CDAII. So these measurements in infancy or childhood may be explored as a disease severity marker for development of preventative treatment strategies for CDAII children or others with ineffective erythropoiesis. In in vitro studies we performed a characterization of CDAII iron overload in human hepatocarcinoma (Huh7) cell line and in human erythromyeloblastoid leukemia (K562) cell line silenced for SEC23B to analyse gene expression of iron metabolism genes. We also characterized the new erythroid regulator erythroferrone (Erfe) both in vivo in CDAII patients and also in vitro in cell lines silenced for SEC23B gene expression. The levels sTfR are indicative of the number of red blood cell precursors and their demand for iron. In our study, sTfR was increased in CDAII patients and negatively correlated with hepcidin and serum ferritin, despite the predicted role of sTfR as an inhibitor of hepcidin. Together with previous findings, sTfR failed to affect hepcidin concentrations, suggesting that sTfR may not be a major suppressor of hepcidin in CDAII patients. In order to investigate whether sHJV down-regulates hepcidin contributing to iron loading pathology, for the first time its levels was analysed in CDAII. We reported high levels of sHJV in CDAII patients compared to healthy controls and its correlation with the severity of hematological phenotype and also iron overload in CDAII. Erythropoietin (EPO) is the main signalling molecules which mediates erythropoiesis and helps the maturation and development of erythroblasts in the later developmental stages. EPO levels in CDAII patients are increased. This data confirms that anemia in CDAII could be due to a deficiency of hematopoietic function of bone marrow, because the level of EPO is increased in anemic patient who has an insufficient number of new red blood cells. Our results reveals that EPO is involved in iron overload decreasing hepcidin expression. Also GDF15 is involved in the iron accumulation in CDAII patients. Its concentrations correlate with the severity of anaemia and serum iron availability in patients, so it reflects the magnitude of the ineffective erythropoiesis. Our data support a role for GDF15 in blunting hepcidin expression in response to iron overload and increasing iron absorption under erythropoietic stress conditions. So, it may be possible to combine information about GDF15 concentrations with other indicators to predict disease severity in CDAII patients.