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Mutations in the only known mammalian iron exporter ferroportin cause a rare iron overload disorder termed ferroportin disease. Two distinct clinical phenotypes are caused by different disease mechanisms: mutations in ferroportin either cause loss of iron export function or gain of function due to resistance to hepcidin, the peptide hormone that normally downregulates ferroportin. The aim of the present study was to examine the disease mechanisms of the thus far unclassified A69T and D181V ferroportin mutations. We overexpressed wild-type and mutant ferroportin fused to green fluorescent protein in human embryonic kidney cells and used a (59)Fe-assay, intracellular ferritin concentrations, confocal microscopy and flow cytometry to study iron export function, subcellular localization and the responsiveness to hepcidin. While the A69T ferroportin mutation seems not to affect the iron export function it causes dose-dependent hepcidin resistance. We further found that D181V mutated ferroportin is iron export defective and hepcidin resistant, similar to the loss of function mutations A77D and C367X. This indicates that intact iron export might be necessary for hepcidin-induced downregulation of ferroportin. This hypothesis was investigated by studying the hepcidin response under modulation of iron availability. Incubation of wild-type ferroportin overexpressing cells with holo-transferrin increases the hepcidin effect whereas chelating extracellular ferrous iron causes hepcidin resistance. In this study we present data that postulates to classify the D181V ferroportin mutation as loss of function and the A69T mutation as dose-dependent hepcidin resistant and outline a possible causal link between iron export function and the hepcidin effect., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Recent identification of genetic variants in iron storage disease has changed the classification system and diagnostic algorithms for hemochromatosis. Clinical diagnosis of the disease requires phenotypic evidence of iron overload because the commonly disease-associated HFE genotypes have an incomplete penetrance. Furthermore, approximately 20% of patients with a clinical diagnosis of hemochromatosis have no disease-associated genotype, which underlines the importance of clear phenotypic criteria of hemochromatosis. A diagnosis of hemochromatosis cannot be made even in patients with liver cirrhosis simply on the basis of genetic testing that indicates that iron overload is the cause of the disease and not its consequence. Proper diagnosis requires integration of clinical presentation, family history, and the results of biochemical and histopathologic tests. Here we propose a rational diagnostic algorithm for hepatic iron overload syndromes and illustrate potential pitfalls by presenting a family study in a pedigree with rare HFE variants (H63D and E168Q), in cis on the same chromosome. Although the clinical suspicion of hemochromatosis was confirmed by histology, chemical analysis of liver tissue revealed a normal hepatic iron concentration, which is compatible with the genetic finding of 1 normal and 1 doubly mutated allele. In conclusion, clinical suspicion of hemochromatosis and elevated serum iron parameters should prompt HFE genotyping for C282Y and H63D. Should they be uninformative, further genetic tests should be recommended only if iron overload in liver tissue has been confirmed chemically.

Abstract: The hormone hepcidin is produced mainly in the liver in response to iron loading and inflammation and secreted into the circulation as a 25-amino acid peptide. The 84-amino acid prohormone undergoes limited proteolytic cleavage at a conserved proprotein convertase (PC) recognition site. In addition to the 25-amino acid hepcidin, N-terminally truncated isoforms of lower biological activity are found in plasma and urine. Here we show that a redundant system of proprotein convertases cleaves prohepcidin at the predicted site releasing active hepcidin-25 from the proprotein. In addition to furin mediated cleavage of prohepcidin, we found prohepcidin peptidase activity of proprotein convertases PC5/6, PC7/LPC, PC1/3 and PC2 which was specific for the release of hepcidin-25 from prohepcidin as shown by mass spectrometry. In native tissue extracts, a calcium-dependent prohepcidin peptidase activity is present specifically releasing the 25-mer hepcidin isoform from the recombinant prohormone. In contrast, the 20-mer isoform of hepcidin is generated by a calcium-independent tissue activity which cleaves the 25-mer peptide but has no activity on the entire prohormone. This finding demonstrates the presence of an additional peptidase in this inactivation mechanism for hepcidin. An inhibitor of prohepcidin cleavage was designed and synthesized from d-amino acids (QRRRRR). Biochemical studies indicated that this is a potent and generic inhibitor of prohepcidin cleavage. Biochemical and inhibitor studies of endogenous tissue peptidase activities support the implication of proprotein convertases in the activation of hepcidin. Inactivation of the peptide hormone by N-terminal truncation is mediated by other distinct peptidases, which appear to act sequentially to initial release of hepcidin-25 from the proprotein. [Copyright &y& Elsevier]

Background & Aims: Classical ferroportin disease is characterized by hyperferritinemia, normal transferrin saturation, and iron overload in macrophages. A non-classical form is characterized by additional hepatocellular iron deposits and a high transferrin saturation. Both forms demonstrate autosomal dominant transmission and are associated with ferroportin gene (SLC40A1) mutations. SLC40A1 encodes a cellular iron exporter expressed in macrophages, enterocytes, and hepatocytes. The aim of the analysis is to determine the penetrance of SLC40A1 mutations and to evaluate in silico tools to predict the functional impairment of ferroportin mutations as an alternative to in vitro studies. Methods: We conducted a systematic review of the literature and meta-analysis of the biochemical presentation, genetics, and pathology of ferroportin disease. Results: Of the 176 individuals reported with SLC40A1 mutations, 80 were classified as classical phenotype with hyperferritinemia and normal transferrin saturation. The non-classical phenotype with hyperferritinemia and elevated transferrin saturation was present in 53 patients. The remaining patients had normal serum ferritin or the data were reported incompletely. Despite an increased hepatic iron concentration in all biopsied patients, significant fibrosis or cirrhosis was present in only 11%. Hyperferritinemia was present in 86% of individuals with ferroportin mutations. Bio-informatic analysis of ferroportin mutations showed that the PolyPhen score has a sensitivity of 99% and a specificity of 67% for the discrimination between ferroportin mutations and polymorphisms. Conclusions: In contrast to HFE hemochromatosis, ferroportin disease has a high penetrance, is genetically heterogeneous and is rarely associated with fibrosis. Non-classical ferroportin disease is associated with a higher risk of fibrosis and a more severe overload of hepatic iron. [ABSTRACT FROM AUTHOR]