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    2019-08-11


    Authors' contributions
    Competing interests
    Transparency document
    Introduction In vertebrates, ferroportin (Fpn) is the only macromolecule known to be able to release elemental iron from cells. This polytopic transmembrane protein belongs to the Major Facilitator Superfamily of transporters and it is expressed in duodenal enterocytes, liver Kupffer cells, splenic red-pulp macrophages, periportal hepatocytes and placenta [1]. Its activity controls the total iron content in the organism by transferring dietary iron from enterocytes to plasma. At the same time, Fpn exports into the circulation recycled and stored iron from macrophages, hepatocytes and other cells types. In addition, the protein drives out iron from placental cells pouring it in the fetal circulation. Fpn is modulated post-translationally by hepcidin, a 25 amino acids long hormone that binds the protein and induces its internalization and degradation [2]. High plasma concentrations of hepcidin inhibit iron export from cells causing depletion of cell-surface Fpn. On the contrary, when hepcidin levels are low, Fpn remains highly expressed and localized on the cell membrane and retains its activity [3]. Heterozygous missense mutations or single amino Cell Counting Kit-8 (CCK-8) deletions in SLC40A1, the gene coding for Fpn, have been demonstrated to cause type 4 hemochromatosis [4], [5] an autosomal dominant iron overload condition with phenotypic manifestations that can vary greatly from one patient to another. This mainly depends on the causative mutations that can be generally distinguished in two subclasses. The majority of pathogenic variants, defined as “loss of function” since they lead to decreased iron export from cells, give rise to type 4A hemochromatosis, an atypical form of primary iron overload, better known as “ferroportin disease” [6]. Ferroportin disease is characterized by early elevated plasma ferritin levels, normal transferrin saturation percentage (TS%) and by iron overload localized mainly in Kupffer cells and other macrophages, that may result in spleen enlargement. On the other hand, more unusual SLC40A1 “gain of function” mutations, conferring partial or complete resistance to hepcidin-mediated Fpn degradation and consequent Fpn hyperactivity, are responsible for type 4B hemochromatosis. The manifestations of this rarer form of the disease do not seem to significantly diverge from those observed in autosomal recessive hemochromatosis related to HFE, TFR2, HJV and HAMP genes, in which iron overload is prevalently localized in hepatocytes and in other parenchymal cells and patients display, as the first biochemical serum abnormality, an elevated TS% followed by progressive increase of ferritin levels [7]. Indeed, the distinction between the two forms of type 4 hemochromatosis is not always so sharp due to the fact that SLC40A1 heterozygous mutations can result in a spectrum of intermediate phenotypes. Furthermore, the effect of different mutations on Fpn function and trafficking can be predicted only on the basis of putative models of the protein. In fact, an experimentally determined three-dimensional structure of human Fpn is currently unavailable and the mechanism by which Fpn transports iron is not yet completely understood [1], [8]. With the intent to provide additional information about the mechanisms underlying the wide phenotypic heterogeneity existing in type 4 hemochromatosis, here we discuss molecular and clinical findings in a group of patients in whom a SLC40A1 single copy missense variant was identified. Three of the detected SLC40A1 mutations were novel and their effect was functionally analyzed and correlated to a three-dimensional model of Fpn recently proposed by some of us [8], [9].
    Materials and methods
    Results and discussion
    Conclusions The results here presented allow to further confirm the gain of function nature of the p.A69T SLC40A1 mutation and to assert that Fpn p.D181N and p.R296Q mutants can be classified as full and partial loss of function, respectively. Replacement of G204 with arginine appears to cause a more complex defect with impact both on iron export function and hepcidin sensitivity. This finding confirms once again the difficulty of predicting the effect of a mutation on the molecular properties of Fpn and of providing exhaustive explanations to the wide variability of the phenotype in type 4 hereditary hemochromatosis.