Diamond-Blackfan anemia (DBA) is a rare congenital red cell aplasia characterized by macrocytic anemia, reticulocytopenia and reduced number of erythroblasts in the bone marrow. Etiology and symptoms of DBA are closely associated with mutations of genes coding for 12 ribosomal proteins. Approximately 40% of patients are dependent on regular transfusions. In chronically transfused DBA patients, severe iron overload with tissue hemosiderosis with the need for iron chelation develops and has a substantial impact on morbidity and mortality of DBA patients. Liver is the most affected organ, but extra-hepatic iron accumulation, although less well documented, appears to occur early and at high frequency. Despite these facts a detailed analysis of iron metabolism in DBA is missing.

We therefore aimed to determine selected markers of erythropoid activity and iron metabolism including the levels of the key regulator of iron homeostasis, hepcidin, in 14 DBA patients from the Czech National DBA Registry. Ten patients receive regular transfusions, two are treated with steroids and two are in the remission. None of the patients had signs of an inflammatory process at the time of the examination. Following markers of iron metabolism were analysed: iron, total iron binding capacity, transferrin saturation, ferritin and soluble transferrin receptor (TfR). Serum hepcidin was measured by proteomic analysis. Levels of rowth differentiation factor 15 (GDF 15), aputative marker of erythroid activity, were measured by ELISA. Pearls’ staining was used for the liver iron detection. In all transfusion dependent patients, low levels of soluble TfR and reduced number of erythroblasts in the bone marrow confirm severally suppressed erythropoiesis which corresponded with dramatically elevated serum erythropoietin (EPO) levels. Analysis of iron parameters showed increased transferrin saturation and high ferritin levels. The two patients who are currently in the remission had near normal blood counts and iron parameters. Liver biopsy on 5 selected patients revealed excessive iron accumulation in both liver hepatocytes and macrophages which can be attributed to increased iron uptake and non-effective erythrocytes-derived iron recycling, respectively. Measurement of hepcidin levels showed significant elevation in DBA cohort in comparison with healthy controls (p>0.0005). The highest hepcidin was detected for transfusion dependent patients, the lowest (near normal) for patients in remission. As erythropoiesis is known to produce a signal for hepcidin suppression, patients in remission with restored erythropoietic activity are likely able to attenuate hepcidin expression and increase the pool of iron available for improved erythropoiesis. On the other hand repeated blood transfusions contribute to the suppression of erythropoiesis thus likely relieving hepcidin synthesis and worsening iron recycling. We also found that elevated hepcidin positively correlated with ferritin levels. No correlation was detected with EPO or GDF15, the putative negative regulator of hepcidin production. These results indicate that EPO is an indirect suppressor of hepcidin synthesis and that GDF15 possibly plays a hepcidin-regulatory role in the disease states associated with ineffective erythropoiesis but not in DBA.

In conclusion we propose that in DBA patients iron overload develops as a consequence of the combination of repeated transfusions and absent or reduced erythropoiesis in the bone marrow. At the systemic level the increased hepcidin promotes iron retention in macrophages and thus is responsible for impaired iron recycling.

Acknowledgments

Grant support: Ministry of Health, Czech Republic, grant No. NT11059 to DP and NT/13587 to VD, Czech Grant Agency, grant No. P305/11/1745, and Internal Grant of Palacky University Olomouc (LF_2013_010). We thank dr. Thomas Ganz for validation of our HPLC-MS-based hepcidin measurements.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.

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