Hepcidin mutation in a β-thalassemia major patient with persistent severe iron overload despite chelation therapy

Abstract

Hepcidin, a 25 amino acid peptide expressed in the liver, has been shown to have a major role in iron homeostasis controlling iron release from reticuloendothelial and intestinal epithelial cells. Human hepcidin is encoded as a prohormone called pre-prohepcidin (84 amino acid) containing a consensus furin cleavage site preceding the C-terminal 25 amino acid bioactive peptide. It is induced by inflammation and iron and decreased by hypoxia and anemia [1]. b-Thalassemia major (TM) is a hereditary hemolytic anemia resulting from the reduction or complete absence of b-globin chain synthesis. Patients with TM and other refractory anemias receiving regular blood transfusions become iron overloaded at the rate of approximately 0.3– 0.5 mg/kg body weight per day. Indeed, iron overload results from transfusional iron and increased intestinal absorption due to inefficient erythropoiesis. Hepcidin mRNA expression was found to be decreased in the liver of C57Bl/6 Hbb (a murine model of thalassemia major) and in patients with thalassemia major urinary hepcidin was disproportionately suppressed in regard to iron burden [2]. In thalassemia major, low hepcidin levels result in increased iron absorption and greater release of stored iron into the circulation and contribute to iron accumulation in liver, endocrine glands and heart. Iron chelator deferoxamine (DFO) reduces iron burden in TM, but limitations of its infusion have stimulated the design of oral chelators which result in better compliance. Thus, almost all TM patients achieve an acceptable iron status. We report a TM case in which mutations in hepcidin could have interfered with distribution and removal of body iron by chelators. The patient was a 23-year-old woman, homozygous for b39 thalassemia major, regularly transfused since 1 year of age, receiving an average of 4 units of packed red cells monthly (corresponding to 7.5 g/year of iron) and chelated by subcutaneous infusion of DFO (40 mg/kg day, 6 days/ week) since early infancy. In her record, a persistent high iron burden was documented despite chelation treatment. The patient developed hypothyroidism and hypogonadotropic hypogonadism and was on hormonal replacement therapy. The echocardiography evaluation showed a slight cardiac left ventricular hypertrophy and an ejection fraction of 66%. Liver iron concentration was estimated at 20 years by Superconducting Quantum Interference Device biomagnetic liver susceptometry method (SQUID-BLS). Despite chelation treatment, the iron concentration evidenced by SQUID was at very high levels (5,230 lg/g liver dw). At the time of the study, non-transferrin-bound iron (NTBI), detected by high-performance liquid chromatography (HPLC) [3], was 3.66 lM (normal value -0.70 ± 0.50); serum ferritin levels and transferrin saturation, measured by standards method, were 4,323 ng/mL and 110%, respectively (normal values 150 ± 100, 35 ± 15). Genomic DNA was isolated from leukocytes by standard method. Exons 1–3 of hepcidin gene were amplified with different pairs of primers: the first pair amplified exon 1, including the promoter and 50-UTR region, the second L. Duca P. Delbini I. Nava M. D. Cappellini Department of Internal Medicine, Maggiore Policlinico, Mangiagalli and Regina Elena Foundation IRCCS, University of Milan, Milan, Italy