Ferroportin Disease Research Articles

Twenty Years of Ferroportin Disease: A Review or An Update of Published Clinical, Biochemical, Molecular, and Functional Features.

Iron overloading disorders linked to mutations in ferroportin have diverse phenotypes in vivo, and the effects of mutations on ferroportin in vitro range from loss of function (LOF) to gain of function (GOF) with hepcidin resistance. We reviewed 359 patients with 60 ferroportin variants. Overall, macrophage iron overload and low/normal transferrin saturation (TSAT) segregated with mutations that caused LOF, while GOF mutations were linked to high TSAT and parenchymal iron accumulation. However, the pathogenicity of individual variants is difficult to establish due to the lack of sufficiently reported data, large inter-assay variability of functional studies, and the uncertainty associated with the performance of available in silico prediction models. Since the phenotypes of hepcidin-resistant GOF variants are indistinguishable from the other types of hereditary hemochromatosis (HH), these variants may be categorized as ferroportin-associated HH, while the entity ferroportin disease may be confined to patients with LOF variants. To further improve the management of ferroportin disease, we advocate for a global registry, with standardized clinical analysis and validation of the functional tests preferably performed in human-derived enterocytic and macrophagic cell lines. Moreover, studies are warranted to unravel the definite structure of ferroportin and the indispensable residues that are essential for functionality.

Ferroportin Disease Caused by a Heterozygous Variant p.Cys326Phe in the SLC40A1 Gene and the Efficacy of Therapeutic Phlebotomy in Children.

Therapeutic phlebotomy is recommended for treating hereditary hemochromatosis. However, the procedure and its efficacy for children remain unclear. We describe a young female patient with ferroportin disease, which was confirmed from excess iron deposition within hepatocytes and by identifying a heterozygous variant p.Cys326Phe in SLC40A1. She had been followed without phlebotomy. Liver histology at age 13 years revealed iron deposition progression. Phlebotomy was initiated and her iron markers and imaging findings improved without severe adverse effects. Therapeutic phlebotomy for children is effective and well-tolerated and should be considered as early as possible after a hemochromatosis diagnosis.

Influence of Ferroportin Disease Mutations on Manganese Accumulation and Toxicity (P24-060-19)

ObjectivesHemochromatosis is a frequent genetic disorder characterized by the accumulation of excess iron across tissues. Mutations in the FPN1 gene, encoding a cell-surface iron exporter ferroportin (Fpn), are responsible for hemochromatosis type 4, also known as ferroportin disease. Recently, Fpn has been implicated in the regulation of manganese (Mn), another essential nutrient required for numerous cellular enzymes. However, the roles of Fpn in Mn regulation remain ill defined, and the impact of disease mutations on cellular Mn levels is unknown. Thus, this study aimed to define the role of Fpn in Mn regulation and determine the functional consequences of ferroportin disease mutations in cellular Mn levels. MethodsThus far, over 50 mutations in Fpn have been identified in hemochromatosis type 4/ferroportin disease. To test whether these mutations alter cellular Mn metabolism, we constructed an expression vector encoding human Fpn with a C-terminal HA epitope tag and introduced nine clinically relevant mutations by site-directed mutagenesis. Based on previously reported in vitro functional results, we selected five ferroportin disease mutations from each of the two groups: five loss-of-function (LOF) mutations (G80S, R88G, D157G, D157Y, and V162Δ) and four gain-of-function (GOF) mutations (N144H, N144T, C326S, and and S338R). ResultsHere, we provide evidence that Fpn can export Mn from cells into extracellular space. Fpn appears to play protective roles in Mn-induced cellular toxicity and oxidative stress. Finally, disease mutations interfere with Fpn’s role in controlling Mn levels as well as the stability of Fpn. ConclusionsThese results define the function of Fpn as an exporter of both iron and Mn and highlight the potential involvement of Mn dysregulation in ferroportin disease. Funding SourcesNational Institutes of Health (NIH) to Y.A.S. (K99/R00 ES024340).

Functional properties of mouse ferroportin, a cellular iron‐export protein

Iron homeostasis is a topic of great public‐health importance because of the high prevalence of iron‐deficiency anemia and of hereditary disorders that cause iron overload. Ferroportin (Fpn)—the only known mammalian cellular iron exporter—is expressed on the plasma membrane of macrophages, enterocytes, and hepatocytes. Fpn, under the control of the iron‐regulatory hormone hepcidin, serves a critical role in the regulation of systemic iron. Whereas we have previously characterized the functional properties of human Fpn, a great deal of research in iron homeostasis utilizes mouse models, highlighting the need to study mouse Fpn in vitro. For example, the flatiron mouse model of classical ferroportin disease bears the mutation H32R in mFpn and is characterized by systemic iron deficiency and macrophage iron loading. The flatiron mouse also appears to exhibit a manganese phenotype [Seo YA and Wessling‐Resnick M (2015) FASEB J 28, 2726–2733], raising the possibility that mouse Fpn (mFpn) serves a role in Mn metabolism, although Mn is not a substrate of human Fpn. We therefore tested the hypothesis that mouse but not human Fpn can transport Mn. We characterized the functional properties of mFpn by using radiotracer assays in RNA‐injected Xenopus oocytes expressing mouse or human Fpn. We found that mFpn can export several transition metals, with selectivity Fe > Co > Ni, but not Cd. Whereas the H32R mutant abolished mFpn‐mediated Fe efflux relative to wildtype, neither wildtype nor mutant mFpn could transport Mn. mFpn‐mediated Fe efflux was pH‐dependent with estimated optimal pH of 8.2 ± (SEM) 0.2 when [Ca2+]o = 2 mM. mFpn‐mediated Fe efflux was activated by extracellular Ca2+, proceeded independently of extracellular Na+, and was inhibited 51% ± 1% by 10 μM hepcidin (30‐min preincubation). Our data (i) reveal that human and mouse Fpn share similar functional properties and (ii) do not support the notion that Fpn can transport Mn.Support or Funding InformationNIH grants R01 DK107309, P30 DK078392, and R25 HL115473, and the American Physiological SocietyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel SLC40A1 p.Y333H mutation with gain of function of ferroportin: A recurrent cause of haemochromatosis in China.

Haemochromatosis type 4, also known as ferroportin disease, is an autosomal dominant genetic disorder caused by pathogenic mutations in the SLC40A1 gene, which encodes ferroportin 1 (FPN1). We have identified a novel SLC40A1 p.Y333H mutation in our previous study. In the present study, we tried to investigate the frequency and pathogenicity of the SLC40A1 p.Y333H mutation in haemochromatosis in China. Patients were analysed for SLC40A1 p.Y333H as well as mutations in the other classic haemochromatosis-related genes by Sanger sequencing. To analyse iron export capacity of the SLC40A1 p.Y333H mutant, the 293T cells were transfected with the SLC40A1 p.Y333H construct and then treated with hepcidin after exposure to ferric ammonium citrate. Cellular localization of mutant FPN1, expression of FPN1 and intracellular ferritin were analysed by immunofluorescence and Western blotting. Of 22 unrelated cases with primary iron overload, three cases (3/22, 13.6%) harboured the SLC40A1 p.Y333H, with no missense mutations identified in any other classical haemochromatosis-related genes including HFE, HJV, HAMP and TFR2. Pedigree analysis showed that three probands and the son of one proband had haemochromatosis of stage 3, while the son of another proband with age of 16 showed elevated transferrin saturation but normal serum ferritin level. In vitro studies showed the mutant p.Y333H ferroportin was resistant to hepcidin, affecting the subsequent internalization and degradation of FPN1, and was associated with ferroportin gain of function. The SLC40A1 p.Y333H mutation is associated with gain of function of ferroportin, representing one of the major aetiological factors of haemochromatosis in China.

Inherited Disorders of Iron Overload.

Dietary iron absorption and systemic iron traffic are tightly controlled by hepcidin, a liver-derived peptide hormone. Hepcidin inhibits iron entry into plasma by binding to and inactivating the iron exporter ferroportin in target cells, such as duodenal enterocytes and tissue macrophages. Hepcidin is induced in response to increased body iron stores to inhibit further iron absorption and prevent iron overload. The mechanism involves the BMP/SMAD signaling pathway, which triggers transcriptional hepcidin induction. Inactivating mutations in components of this pathway cause hepcidin deficiency, which allows inappropriately increased iron absorption and efflux into the bloodstream. This leads to hereditary hemochromatosis (HH), a genetically heterogenous autosomal recessive disorder of iron metabolism characterized by gradual buildup of unshielded non-transferrin bound iron (NTBI) in plasma and excessive iron deposition in tissue parenchymal cells. The predominant HH form is linked to mutations in the HFE gene and constitutes the most frequent genetic disorder in Caucasians. Other, more severe and rare variants are caused by inactivating mutations in HJV (hemojuvelin), HAMP (hepcidin) or TFR2 (transferrin receptor 2). Mutations in SLC40A1 (ferroportin) that cause hepcidin resistance recapitulate the biochemical phenotype of HH. However, ferroportin-related hemochromatosis is transmitted in an autosomal dominant manner. Loss-of-function ferroportin mutations lead to ferroportin disease, characterized by iron overload in macrophages and low transferrin saturation. Aceruloplasminemia and atransferrinemia are further inherited disorders of iron overload caused by deficiency in ceruloplasmin or transferrin, the plasma ferroxidase and iron carrier, respectively.

Ferroportin disease mutations influence manganese accumulation and cytotoxicity.

Hemochromatosis is a frequent genetic disorder, characterized by the accumulation of excess iron across tissues. Mutations in the FPN1 gene, encoding a cell surface iron exporter [ferroportin (Fpn)], are responsible for hemochromatosis type 4, also known as ferroportin disease. Recently, Fpn has been implicated in the regulation of manganese (Mn), another essential nutrient required for numerous cellular enzymes. However, the roles of Fpn in Mn regulation remain ill-defined, and the impact of disease mutations on cellular Mn levels is unknown. Here, we provide evidence that Fpn can export Mn from cells into extracellular space. Fpn seems to play protective roles in Mn-induced cellular toxicity and oxidative stress. Finally, disease mutations interfere with the role of Fpn in controlling Mn levels as well as the stability of Fpn. These results define the function of Fpn as an exporter of both iron and Mn and highlight the potential involvement of Mn dysregulation in ferroportin disease.-Choi, E.-K., Nguyen, T.-T., Iwase, S., Seo, Y. A. Ferroportin disease mutations influence manganese accumulation and cytotoxicity.

The interactions between iron and copper in genetic iron overload syndromes and primary copper toxicoses in Japan.

Iron and copper are trace elements essential for health, and iron metabolism is tightly regulated by cuproproteins. Clarification of the interactions between iron and copper may provide a better understanding of the pathophysiology and treatment strategy for hemochromatosis, Wilson disease, and related disorders. The hepcidin/ferroportin system was used to classify genetic iron overload syndromes in Japan, and ceruloplasmin and ATP7B were introduced for subtyping Wilson disease into the severe hepatic and classical forms. Interactions between iron and copper were reviewed in these genetic diseases. Iron overload syndromes were classified into pre-hepatic iron loading anemia and aceruloplasminemia, hepatic hemochromatosis, and post-hepatic ferroportin disease. The ATP7B-classical form with hypoceruloplasminemia has primary hepatopathy and late extra-hepatic complications, while the severe hepatic form is free from ATP7B mutation and hypoceruloplasminemia, and silently progresses to liver failure. A large amount of iron and trace copper co-exist in hepatocellular dense bodies of all iron overload syndromes. Cuproprotein induction to stabilize excess iron should be differentiated from copper retention in Wilson disease. The classical form of Wilson disease associated with suppressed hepacidin25 secretion may be double-loaded with copper and iron, and transformed to an iron disease after long-term copper chelation. Iron disease may not be complicated with the severe hepatic form with normal ferroxidase activity. Hepatocellular dense bodies of iron overload syndromes may be loaded with a large amount of iron and trace copper, while the classical Wilson disease may be double-loaded with copper and iron.

Oligomerization of ferroportin and the mechanism of autosomal dominance in ferroportin disease

Ferroportin (Fpn)—the only known cellular iron‐export protein—plays a critical role in iron homeostasis by mediating iron export from macrophages and enterocytes. Rare, loss‐of‐function mutations in human Fpn (e.g. V160del) cause classical ferroportin disease, characterized by macrophage iron loading and iron‐restricted anemia. Ferroportin disease displays an autosomal–dominant inheritance pattern. Because all of the disease‐associated mutations are missense, the dominant–negative effect is most readily explained as resulting from oligomerization of Fpn protein. As a first step in testing this hypothesis, we have examined the functional activity of wildtype (wt) and mutant human Fpn co‐expressed in RNA‐injected Xenopus oocytes. We measured 55Fe efflux from control oocytes, or oocytes expressing wtFpn or V160del GFP proteins with or without co‐expression of a wtFpn–mCherry fusion protein. The RNA dose eliciting half‐maximal iron‐export activity was 12.1 ± (SEM) 1.6 ng for wtFpn–GFP (P = 0.005) and 11.7 ± 2.2 ng for wtFpn–mCherry (P = 0.013), so we injected 12 ng RNA of each construct in subsequent co‐expression experiments. 55Fe efflux activity resulting from co‐expression of wtFpn–GFP and wtFpn–mCherry constructs was additive (Fig 1). Whereas expression of V160del–GFP alone did not stimulate 55Fe efflux, co‐expression of the mutant with wtFpn–mCherry stimulated 55Fe efflux to roughly the same degree as either of the wildtype constructs alone. Our results therefore provide no evidence of a dominant–negative effect of V160del. Our next step will be to test co‐expression of wtFpn with other loss‐of‐function mutants. We will examine the oligomeric structure of wildtype and mutant Fpn by using freeze‐fracture electron microscopy to examine the interior (p) face of the inner lamella of the plasma membrane, as previously described for several membrane‐transport proteins [Eskandari S et al (1998) Proc Natl Acad Sci USA 95, 11235–11240]. We anticipate that our study will further elucidate the structure–function of Fpn and help to explain the autosomal dominance of ferroportin disease.Support or Funding InformationNIH–NIDDK grant R01 DK107309This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structure-function analysis of ferroportin defines the binding site and an alternative mechanism of action of hepcidin

AbstractNonclassical ferroportin disease (FD) is a form of hereditary hemochromatosis caused by mutations in the iron transporter ferroportin (Fpn), resulting in parenchymal iron overload. Fpn is regulated by the hormone hepcidin, which induces Fpn endocytosis and cellular iron retention. We characterized 11 clinically relevant and 5 nonclinical Fpn mutations using stably transfected, inducible isogenic cell lines. All clinical mutants were functionally resistant to hepcidin as a consequence of either impaired hepcidin binding or impaired hepcidin-dependent ubiquitination despite intact hepcidin binding. Mapping the residues onto 2 computational models of the human Fpn structure indicated that (1) mutations that caused ubiquitination-resistance were positioned at helix-helix interfaces, likely preventing the hepcidin-induced conformational change, (2) hepcidin binding occurred within the central cavity of Fpn, (3) hepcidin interacted with up to 4 helices, and (4) hepcidin binding should occlude Fpn and interfere with iron export independently of endocytosis. We experimentally confirmed hepcidin-mediated occlusion of Fpn in the absence of endocytosis in multiple cellular systems: HEK293 cells expressing an endocytosis-defective Fpn mutant (K8R), Xenopus oocytes expressing wild-type or K8R Fpn, and mature human red blood cells. We conclude that nonclassical FD is caused by Fpn mutations that decrease hepcidin binding or hinder conformational changes required for ubiquitination and endocytosis of Fpn. The newly documented ability of hepcidin and its agonists to occlude iron transport may facilitate the development of broadly effective treatments for hereditary iron overload disorders.

Genetic hemochromatosis: Pathophysiology, diagnostic and therapeutic management

The term hemochromatosis (HC) corresponds to several diseases characterized by systemic iron overload of genetic origin and affecting both the quality of life and life expectancy. Major improvement in the knowledge of iron metabolism permits to divide these diseases into two main pathophysiological categories. For most HC forms (types 1, 2, 3 and 4B HC) iron overload is related to cellular hepcidin deprivation which causes an increase of plasma iron concentration and the appearance of plasma non-transferrin bound iron. In contrast, iron excess in type 4A ferroportin disease is related to decreased cellular iron export. Whatever the HC type, the diagnosis rests on a non-invasive strategy, combining clinical, biological and imaging data. The mainstay of the treatment remains venesection therapy with the perspective of hepcidin supplementation for hepcidin deprivation-related HC. Prevention of HC is critical at the family level and, for type 1 HC, remains a major goal, although still debated, at the population level.

Characterization of three novel pathogenic SLC40A1 mutations and genotype/phenotype correlations in 7 Italian families with type 4 hereditary hemochromatosis

Mutations of SLC40A1 encoding ferroportin (Fpn), the unique cellular iron exporter, severely affect iron homeostasis causing type 4 hereditary hemochromatosis, an autosomal dominant iron overload condition with variable phenotypic manifestations. This disease can be classified as type 4A, better known as “ferroportin disease”, which is due to “loss of function” mutations that lead to decreased iron export from cells, or as type 4B hemochromatosis, which is caused by “gain of function” mutations, conferring partial or complete resistance to hepcidin-mediated Fpn degradation.In this work, we discuss clinical and molecular findings on a group of patients in whom a SLC40A1 single copy missense variant was identified. Three novel variants, p.D181N, p.G204R and p.R296Q were functionally characterized. Fpn D181N and R296Q mutants can be classified as full or 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 the difficulty of predicting the effect of a mutation on the molecular properties of Fpn in order to provide an exhaustive explanation to the wide variability of the phenotype in type 4 hereditary hemochromatosis.

Ferroportin disease: pathogenesis, diagnosis and treatment.

Ferroportin Disease (FD) is an autosomal dominant hereditary iron loading disorder associated with heterozygote mutations of the ferroportin-1 (FPN) gene. It represents one of the commonest causes of genetic hyperferritinemia, regardless of ethnicity. FPN1 transfers iron from the intestine, macrophages and placenta into the bloodstream. In FD, loss-of-function mutations of FPN1 limit but do not impair iron export in enterocytes, but they do severely affect iron transfer in macrophages. This leads to progressive and preferential iron trapping in tissue macrophages, reduced iron release to serum transferrin (i.e. inappropriately low transferrin saturation) and a tendency towards anemia at menarche or after intense bloodletting. The hallmark of FD is marked iron accumulation in hepatic Kupffer cells. Numerous FD-associated mutations have been reported worldwide, with a few occurring in different populations and some more commonly reported (e.g. Val192del, A77D, and G80S). FPN1 polymorphisms also represent the gene variants most commonly responsible for hyperferritinemia in Africans. Differential diagnosis includes mainly hereditary hemochromatosis, the syndrome commonly due to either HFE or TfR2, HJV, HAMP, and, in rare instances, FPN1 itself. Here, unlike FD, hyperferritinemia associates with high transferrin saturation, iron-spared macrophages, and progressive parenchymal cell iron load. Abdominal magnetic resonance imaging (MRI), the key non-invasive diagnostic tool for the diagnosis of FD, shows the characteristic iron loading SSL triad (spleen, spine and liver). A non-aggressive phlebotomy regimen is recommended, with careful monitoring of transferrin saturation and hemoglobin due to the risk of anemia. Family screening is mandatory since siblings and offspring have a 50% chance of carrying the pathogenic mutation.

The dynamics of hepcidin-ferroportin internalization and consequences of a novel ferroportin disease mutation.

The hepcidin-ferroportin axis underlies the pathophysiology of many iron-associated disorders and is a key target for the development of therapeutics for treating iron-associated disorders. The aims of this study were to investigate the dynamics of hepcidin-mediated ferroportin internalization and the consequences of a novel disease-causing mutation on ferroportin function. Specific reagents for ferroportin are limited; we developed and characterized antibodies against the largest extracellular loop of ferroportin and developed a novel cell-based assay for studying hepcidin-ferroportin function. We show that hepcidin-mediated ferroportin internalization is a rapid process and could be induced using low concentrations of hepcidin. Targeted next-generation sequencing utilizing an iron metabolism gene panel developed in our group identified a novel ferroportin p.D84E variant in a patient with iron overload. Wild-type and mutant ferroportin constructs were generated, transfected into HEK293 cells and analysed using an all-in-one flow-cytometry-based assay to study the effects on hepcidin-mediated internalization and iron transport. Consistent with the classical phenotype of ferroportin disease, the p.D84E mutation results in an inability to transport iron and hepcidin insensitivity. These results validate a recently proposed 3D-structural model of ferroportin and highlight the significance of this variant in the structure and function of ferroportin. Our novel ferroportin antibody and assay will be valuable tools for investigating the regulation of hepcidin/ferroportin function and the development of novel approaches for the therapeutic modulation of iron homeostasis.

Expanding the clinical and molecular spectrum of ferroportin disease

Ferroportin disease is an autosomal dominant hepatic iron overload syndrome where two distinct clinical and biochemical phenotypes have been reported. Classical ferroportin disease is due to a loss of iron export function and characterized by low to normal transferrin saturation with macrophage iron storage. Non-classical ferroportin disease is due to a resistance of ferroportin to hepcidin mediated internalization and degradation of the protein. The aim of this study was to characterize the functional consequences of the ferroportin gene (SLC40A1) mutation p.Arg178Gln.

Ferroportin Deficiency Decreases Manganese Accumulation in the brain after Olfactory Manganese Exposure

Manganese (Mn) is both essential, yet toxic when present in excessive amounts. In particular, accumulation of Mn in the brain produces neurotoxic effects, disrupting motor function and behavior. A significant route of entry is uptake of airborne Mn, since this metal is efficiently transported into the brain through the nasal epithelium. Ferroportin (Fpn; SLC40A1) can function in the transport of Mn in addition to iron (Fe). Our previous studies have shown that Fpn deficiency impaired Mn metabolism in flatiron (ffe/+) mice, a genetic model of human “ferroportin disease”. Our recent studies have further shown that Fpn deficiency alters body Mn levels, and promotes brain Mn accumulation in flatiron mice, suggesting that toxicity related to Mn exposure could be modified by deficiency of Fpn export function. Therefore, we characterized the impact of Fpn deficiency on Mn accumulation in the brain after olfactory Mn exposure. Ffe/+ mice and wild‐type control (+/+) mice were intranasally instilled with MnCl2 (5 mg/kg) daily for 3 weeks. Mn and Fe levels in the blood, liver, and microdissected brain tissues were determined using inductively coupled plasma mass spectrometry (ICP‐MS). Flatiron mice had reduced blood Fe levels (532.3 vs. 475.5 mg/kg; P=0.0107) and higher Fe levels in liver (44.54 vs. 56.91 mg/kg; P=0.0481) compared to +/+ mice. These data confirm the Fe deficiency and loading phenotype of flatiron mouse associated with ferroportin disease. Blood Mn levels were significantly reduced in flatiron mice compared to +/+ mice (0.0315 vs. 0.0353 mg/kg, P=0.0332). These data agree with our previous findings of the Mn deficiency phenotype of flatiron mouse. Mn instillation increased Mn levels in the liver, which were not affected by Fpn deficiency (Mn X FFE interaction effect; P=0.0369). Flatiron mice showed higher Mn levels in most brain regions, including the olfactory bulbs (0.224 vs. 0.001 mg/kg, P < 0.0001), prefrontal cortex (0.273 vs. 0.163 mg/kg), hippocampus (0.230 vs. 0.180 mg/kg), cerebellum (0.196 vs. 0.172 mg/kg), and brain stem (0.253 vs. 0.174 mg/kg) compared to +/+ mice. Mn instillation increased Mn levels in brain regions, including olfactory bulbs (Mn effect, P=0.0013), cortex (Mn effect, P=0.0092), and midbrain (Mn effect, P=0.0033) compared with vehicle control. Interestingly, Mn‐instilled flatiron mice had significantly reduced Mn levels in the olfactory bulbs (FFE effect, P=0.0013), and most brain regions including the cortex, midbrain, hippocampus, and cerebellum compared to Mn‐instilled +/+ mice. Furthermore, Mn‐instilled flatiron mice displayed reduced Fe levels in the olfactory bulbs (FFE effect, P=0.0013) and elevated Fe levels in prefrontal cortex (FFE effect, P=0.0078) compared to Mn‐instilled +/+ mice. Taken together, these results indicate that olfactory Mn exposure decreases brain Mn accumulation in flatiron mice. These findings further suggest that Fpn deficiency reduces susceptibility to neurotoxicity induced by inhaled Mn.

Human macrophage ferroportin biology and the basis for the ferroportin disease.

Ferroportin (FPN1) is the sole iron exporter in mammals, but its cell‐specific function and regulation are still elusive. This study examined FPN1 expression in human macrophages, the cells that are primarily responsible on a daily basis for plasma iron turnover and are central in the pathogenesis of ferroportin disease (FD), the disease attributed to lack‐of‐function FPN1 mutations. We characterized FPN1 protein expression and traffic by confocal microscopy, western blotting, gel filtration, and immunoprecipitation studies in macrophages from control blood donors (donor) and patients with either FPN1 p.A77D, p.G80S, and p.Val162del lack‐of‐function or p.A69T gain‐of‐function mutations. We found that in normal macrophages, FPN1 cycles in the early endocytic compartment does not multimerize and is promptly degraded by hepcidin (Hepc), its physiological inhibitor, within 3‐6 hours. In FD macrophages, endogenous FPN1 showed a similar localization, except for greater accumulation in lysosomes. However, in contrast with previous studies using overexpressed mutant protein in cell lines, FPN1 could still reach the cell surface and be normally internalized and degraded upon exposure to Hepc. However, when FD macrophages were exposed to large amounts of heme iron, in contrast to donor and p.A69T macrophages, FPN1 could no longer reach the cell surface, leading to intracellular iron retention. Conclusion: FPN1 cycles as a monomer within the endocytic/plasma membrane compartment and responds to its physiological inhibitor, Hepc, in both control and FD cells. However, in FD, FPN1 fails to reach the cell surface when cells undergo high iron turnover. Our findings provide a basis for the FD characterized by a preserved iron transfer in the enterocytes (i.e., cells with low iron turnover) and iron retention in cells exposed to high iron flux, such as liver and spleen macrophages. (Hepatology 2017;65:1512‐1525)

肝細胞癌を合併したフェロポルチン病B型の1例

症例は50歳代男性．肝機能障害と肝腫瘤の精査目的で入院となった．血液検査では，血清肝酵素の軽度上昇，高度の高フェリチン血症，トランスフェリン飽和度高値を認めた．腹部CT・MRIで，びまん性鉄沈着が疑われる肝実質内に径15 mmの単発の腫瘤を認め，肝細胞癌を疑い施行した腫瘍生検でdysplastic noduleと診断された．また，非腫瘤部の肝細胞およびKupffer細胞には強い鉄沈着を認め，線維化はF2であった．鉄過剰症に対して瀉血療法を開始した．

Detection of a rare mutation in the ferroportin gene through targeted next generation sequencing.

Hereditary haemochromatosis (HH) is a disorder characterised by increase of serum iron parameters and gradual iron accumulation in parenchymal organs. Since the discovery of the genetic defects of the most common form of hereditary haemochromatosis (type 1 haemochromatosis [HFE]), many observations have shown that not only rare/familial mutations in HFE can be present but also that mutations in other genes (transferrin receptor 2 [TFR2], hepcidin [HAMP], hemojuvelin [HJV], and ferroportin [SLC40A1]) can lead to rarer genetic forms of iron overload, referred as non-HFE haemochromatosis1. The genetic heterogeneity is particularly evident in the Italian population where only two-thirds of haemochromatosis patients are HFE C282Y homozygotes2, requiring expensive and time-consuming gene specific genotyping to define the molecular diagnosis. Therefore, “second level” genetic tests should be developed for a rapid and simultaneous study of the haemochromatosis genes. The most common non-HFE haemochromatosis is the autosomal dominant haemochromatosis type 4 (HH4), caused by mutations in the SLC40A1 gene, encoding for cellular iron exporter ferroportin. Ferroportin acts as receptor of hepcidin, the key regulator of iron metabolism. The hepcidin/ferroportin interaction induces the internalisation of the complex, causing a decrease in iron export and its retention in the cell3. HH4 can be phenotypically classified in two groups: patients may have hyperferritinaemia with normal/low transferrin saturation (type-A HH4, ferroportin disease) or both serum iron parameters increased (type-B HH4, non-classical ferroportin disease)4. This depends on ferroportin impairment that could be classified as “loss-of-function” or “gain-of-function”. In the first case (type-A), the mutated protein is not expressed on the membrane and it is not able to exert its exporting function. In the second case (type-B), the mutated iron exporter becomes resistant to the activity of hepcidin, causing increased iron absorption. Several mutations have been characterised in HH4 patients, spreading along the entire gene sequence4. The position of the correspondent amino acidic change is important to define the HH4 clinical phenotype5. Among the different ferroportin mutations, the p.A69T variant, despite the causal nucleotide change not being annotated in dbSNP, has been described in a 52-year old Italian woman with diabetes and type-B HH4, on the basis of hepatocyte iron overload6, but no iron parameters or clinical history of the patient were described. The pathogenic role of this mutation has been subsequently confirmed through in vitro experiments in which mutant cDNA has been over-expressed7. These studies demonstrated that p.A69T mutated ferroportin has a partial resistance to hepcidin. Here we report an Italian patient with a severe iron overload phenotype in whom a careful clinical characterisation led to a diagnose of p.A69T non-classical ferroportin disease through the combination of capture of technology with a next generation sequencing (NGS) platform.

Hémochromatoses : un monde en pleine mutation

Due to major advances in the understanding of iron metabolism as well as in the bioche- iuical, imaging, and genetic domains: i) The nosologicalframework of hemochromatosis (HC) encompasses not only HFE-HC, by far the most frequent HC form, but also non-HFE HC diseases which comprise essentially juvenile HC and the ferroportin disease. ii) The diagnostic approach has become totally non invasive, based on clinical, imaging and biological data. iii) The treatment remains, for most forms, based on venesections, but the innovative emerging therapeutic approach is represented by hepcidin supplementation.