Identification of a New Mutation in the 5′-UTR of Hepcidin Gene in beta-Thalassemia Major (TM) Patients.

Abstract Hepcidin is a peptide hormone produced in the liver that acts as negative regulator of iron absorption from the enterocytes and of iron release from macrophages. Iron overload and inflammation up-regulate hepcidin synthesis, while anaemia and hypoxia suppress hepcidin expression. Thalassaemia Major (TM) is a hereditary haemolytic anaemia requiring long-life blood transfusions treatment with consequent iron overload. In β-thalassemias is a disorder in which hepcidin is regulated by opposing influences of ineffective erythropoiesis and concomitant iron overload. In order to get further insights on iron regulation in thalassemias, we screened hepcidin and HFE genes in fourty-three TM regularly transfused patients and sixty control subjects. Blood from TM was taken at least 48 hours after chelation therapy and just before blood transfusion. DNA was prepared from peripheral blood, according to standard protocols. Hepcidin and HFE sequences were amplified with PCR using specific primers and PCR products were sequenced, after purification, in a automatic sequencer. Moreover in all patients serum pro-hepcidin was evaluated by ELISA competitive binding assay (DRG,Germany); iron status was evaluated by serum ferritin (SF), percentage of transferrin saturation (TS) by standard procedures and non transferrin bound iron (NTBI) in serum by HPLC after nitrilotriacetic acid (NTA) chelation. Serum IL-6 as inflammation marker was measured by lateral flow immunoassay (Milenia QuickLine, Germany). Molecular analysis detected an undescribed G→T mutation at position +23 of the 5′-untranslated region in two unrelated TM patients; no mutations were found in control subjects. The probands have been regularly transfused since the age of 1 year, receiving 2–3 units of packed red cells and treated with Deferoxamine 40 mg/Kg/day 6 days/week. The first patient, wild type for HFE mutation, was a 29-years-old compound heterozygous IVS II-745/IVS I-110 man. The SF was 1052 ng/ml, TS 94% and NTBI 1.77 μM. Serum pro-hepcidin value was in normal range (213 ng/ml). The proband’s father was heterozygous for the same hepcidin mutation and showed signs of mild iron overload (SF 491 ng/ml and NTBI 0.50 μM). The second patient was a 26-years-old homozygous β039 man with high levels of SF (4346 ng/ml), TS (169%) and NTBI (2.10 μM), while serum pro-hepcidin was 269 ng/ml. HFE analysis revealed a homozygous genotype for H63D mutation. The patient’s mother was heterozygous for hepcidin and H63D mutation and showed mild iron overload (SF 500 ng/ml; NTBI 0.22 μM) whereas the father, heterozygous only for H63D, had normal iron status. According to recent findings (Bridle et al, 2003) our results indicate that hepcidin mutation in association with H63D synergizes the effect on iron homeostasis and it could be responsible for the development of marked iron overload poorly responsive to chelation therapy in β-thalassemia patients.