1972 haematologica | 2017; 102(12)

Received: July 10, 2017.

Accepted: September 25, 2017.

Pre-published: November 3, 2017.

©2017 Ferrata Storti FoundationMaterial published in Haematologica is covered by copyright.All rights are reserved to the Ferrata Storti Foundation. Use ofpublished material is allowed under the following terms andconditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode. Copies of published material are allowed for personal or inter-nal use. Sharing published material for non-commercial pur-poses is subject to the following conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode,sect. 3. Reproducing and sharing published material for com-mercial purposes is not allowed without permission in writingfrom the publisher.

Correspondence: antonello.pietrangelo@unimore.it

Ferrata StortiFoundation

EUROPEANHEMATOLOGYASSOCIATION

Haematologica 2017Volume 102(12):1972-1984

REVIEW ARTICLE

doi:10.3324/haematol.2017.170720

Check the online version for the most updatedinformation on this article, online supplements,and information on authorship & disclosures:www.haematologica.org/content/102/12/1972

Introduction

The name Ferroportin Disease (FD) refers to a clinical entity that differs from allother known forms of hereditary iron overload, including hemochromatosis (HC)[synonymous for hereditary hemochromatosis (HH)], i.e. the syndrome due toeither HFE or non-HFE hemochromatosis gene mutations.1 In humans, a numberof genetic disorders associate with systemic iron overload (Table 1) while othersare caused by iron misdistribution and are associated with the regional accumula-tion of iron in subcellular compartments (e.g. mitochondria in Friedreich ataxia) orcertain cell types and organs (e.g. basal ganglia in neuroferritinopathy) (Table 1). Instrict terms, the latter disorders may not all qualify as true iron-overload states, asthe total body iron content may not be increased. FD, which today accounts forone of the commonest forms of hereditary iron overload disorder besides HFE-hemochromatosis, is characterized by a unique pathogenic basis and clinical pres-entation and, unlike HC, has been reported worldwide, regardless of ethnicity. Ferroportin Disease (phenotype MIM number 606069, gene/Locus; MIM num-

ber 604653; https://www.omim.org/entry/606069?search=ferroportin%20dis-

Ferroportin Disease (FD) is an autosomal dominant hereditary ironloading disorder associated with heterozygote mutations of the fer-roportin-1 (FPN) gene. It represents one of the commonest causes of

genetic hyperferritinemia, regardless of ethnicity. FPN1 transfers ironfrom the intestine, macrophages and placenta into the bloodstream. InFD, loss-of-function mutations of FPN1 limit but do not impair ironexport in enterocytes, but they do severely affect iron transfer inmacrophages. This leads to progressive and preferential iron trapping intissue macrophages, reduced iron release to serum transferrin (i.e. inap-propriately low transferrin saturation) and a tendency towards anemia atmenarche or after intense bloodletting. The hallmark of FD is markediron accumulation in hepatic Kupffer cells. Numerous FD-associatedmutations have been reported worldwide, with a few occurring in differ-ent populations and some more commonly reported (e.g. Val192del,A77D, and G80S). FPN1 polymorphisms also represent the gene variantsmost commonly responsible for hyperferritinemia in Africans.Differential diagnosis includes mainly hereditary hemochromatosis, thesyndrome commonly due to either HFE or TfR2, HJV, HAMP, and, in rareinstances, FPN1 itself. Here, unlike FD, hyperferritinemia associates withhigh transferrin saturation, iron-spared macrophages, and progressiveparenchymal 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). Anon-aggressive phlebotomy regimen is recommended, with careful mon-itoring of transferrin saturation and hemoglobin due to the risk of ane-mia. Family screening is mandatory since siblings and offspring have a50% chance of carrying the pathogenic mutation.

Ferroportin disease: pathogenesis, diagnosisand treatmentAntonello Pietrangelo

Center for Hemochromatosis, Department of Internal Medicine II, University of Modenaand Reggio Emilia Policlinico, Modena, Italy

ABSTRACT

ease&highlight=ferroportin%20disease) is due to patho-genic (usually missense) mutations of the ferroportin1gene (FPN1; SLC40A1) which encodes the only ironexporter so far identified in mammals;2-4 lack-of-functionmutations impair the iron-export capability of FPN1, par-ticularly in cells with high iron turnover, such as tissuemacrophages. Unlike the mutations causing FD, other raremutations of FPN1 (such as N144H, C326Y, C326S andC326F),5-8 do not impair protein expression at the cellmembrane or its iron export capability, but make FPN1resistant to the inhibitory effect of hepcidin, the physio-logical FPN1 inhibitor (see below under Pathogenesis).This causes unchecked iron export activity of FPN1; theresulting clinical disorder is different from FD and indistin-guishable from other forms of hereditary HC (Tables 1and 2).

Definition and classificationThe OMIM database classifies the two forms of heredi-

tary iron overload due to FPN1 mutations within the sametaxonomic category as “hemochromatosis type 4”(https://www.omim.org/entry/606069?search=ferro-portin%20disease&highlight=ferroportin%20disease).Similar terminology has then been adopted by Orphanetwith the inclusion of two subcategories: hemochromato-sis type 4A (referring to classic FD due to lack-of-functionFPN1 mutations) and hemochromatosis type 4B (referringto FD due to gain-of-function FPN1 mutations)( h t t p : / / w w w . o r p h a . n e t / c o n s o r / c g i -bin/OC_Exp.php?Lng=EN&Expert=139491). These classi-fications have been incorporated into recent publications,with some variants.9,10 Disease naming and classification(taxonomy) can vary depending on different criteria, suchas pathogenic genes, mechanisms, clinical manifestations,etc. Ideally, disease taxonomy (and names) should alsohelp clinicians to recognize, diagnose, and cure diseases.In this context, the taxonomy adopted by OMIM andOrphanet, by embracing two pathogenically and clinicallydifferent disorders caused by mutations in the same geneunder the term “hemochromatosis”, may fail to reachthose objectives. Over the past decades, the termhemochromatosis has been inconsistently used in the lit-erature and in clinical practice to imprecisely refer to: i)any form of body iron overload; ii) tissue iron overloadcausing organ damage and disease; iii) genetically deter-mined iron overload; and, recently, iv) HFE-related iron

overload.11 Recent discoveries in the field have shownthat, regardless of the underlying genetic defect, a numberof hereditary iron loading disorders (i.e. those due to loss-of-function mutations of HFE, TfR2, HJV, HAMP and gain-of-function mutations of FPN1) belong to the same syn-dromic entity as they share the pathogenic basis (lack ofhepcidin function-activity), biochemical expressivity (hightransferrin saturation and high serum ferritin), liverpathology features (iron accumulation in parenchymalcells with iron-spared Kupffer cells until late stage), dam-age and disease of distinct target organs (liver, heart,endocrine glands, joints), and the therapeutic approachwith optimal response to phlebotomy.11 As discussed inthe following sections, each individual feature reportedabove is different in classic FD.1 Therefore, using the term“hemochromatosis” for the classic FD or the term“Ferroportin Disease” for FPN1-associated HC, is mislead-ing, particularly for clinicians, since clinical suspicion,diagnostic strategy and management differ profoundly.Based on these considerations, and on our present under-standing of the pathogenesis and clinical manifestationsof these disorders, it is proposed that the disorder due tolack-of-function mutations of FPN1 is termed “FerroportinDisease”, as originally described,1 and the disorder due togain-of-function FPN1 mutations is termed “FPN-1 associ-ated hemochromatosis” (Table 1). Instead, in analogy withother protein-related classifications (e.g. ferritinopathies;hemoglobinopathies), both disorders due to lack- andgain-of-function mutations of FPN1 may well be includedin a broader taxonomic category named “ferro-portinopathies”.

Historical aspectsIn 1996, the HFE hemochromatosis gene, whose C282Y

homozygote mutation is responsible for most cases of HHin Caucasians, was identified.12 Soon after, it becameapparent that not all hereditary iron overload disorderscould be explained by HFE mutations, particularly inSouthern Europe, where an active search for other geneslinked to genetic iron overload flourished. From 2000 to2004, all known non-HFE genes associated so far withHC, namely transferrin receptor 2 (TfR2),13 FPN1,5 hep-cidin (HAMP),14 and hemojuvelin (HJV)15 were identified. A few years earlier, in 1999, a distinct and somehow

unusual phenotype had been reported in a large familywith hereditary iron overload from Italy. Selective iron

Ferroportin Disease

haematologica | 2017; 102(12) 1973

Table 1. Human hereditary disorders associated with iron overload and iron mis-distribution. Iron overload Iron mis-distribution Disorder/cause Pattern of iron accumulation Disorder/cause Pattern of iron accumulation

Hereditary hemochromatosis (HFE-TfR2-, Systemic (iron accumulation in X-linked sideroblastic anemias Systemic (mitochondria)HJV-, HAMP-, FPN1-associated) parenchymal cells) Ferroportin Disease Systemic (preferential iron accumulation Friedreich ataxia Systemic (mitochondria) in macrophages) Aceruloplasminemia Systemic Neuroferritinopathy Regional (brain) Atransferrinemia Systemic DMT-1 deficiency Regional (mainly liver) H-ferritin-related iron overload Systemic Hereditary iron-loading anemias with inefficient Systemic (early iron erythropoiesis accumulation in hepatocytes due to increased iron absorption)

loading of liver macrophages, hyperferritinemia co-exist-ing with normal-low transferrin saturation, and tendencyto anemia after intense phlebotomy were the hallmarks ofthe disorder.16 In 2001, all affected family members werereported to be heterozygous for a c. 230 C→A substitu-tion resulting in the replacement of alanine 77 with aspar-tate in FPN1.17 This entity was subsequently named FD.1On the other hand, FPN1-related HC, due to a gain-of-

function mutation of FPN1 (p.N144H), was first reportedby Njajou et al. in 2001.5 Yet, it is worth mentioning thatthe first clinical description of an “autosomal dominant”form of classic HC had been already reported by Eason etal. in a Melanesian kindred in 1990.18 In this same popu-lation, Arden et al.19 have later linked the HC phenotypeto the NI44T gain-of-function mutation of FPN1.

Ferroportin biology and physiology and FDFPN1, the product of the FPN1 (SLC40A1) gene, trans-

fers iron from the external milieu (i.e. maternal blood orintestinal lumen) and from internal sites of iron storageand recycles it into the bloodstream. In fact, it is highlyexpressed in liver and spleen macrophages, the luminalsite of enterocytes and placental syncytiotrophoblasts.2-4FPN1 is regulated at different levels by a number of fac-

tors, including transcriptionally by heme,20 translationallyby the iron-regulatory proteins (IRPs),21 and posttransla-tionally mainly by hepcidin, the iron hormone. Hepcidin

is produced by the liver in response to iron, inflammation,and a variety of stressors.22-25 Hepcidin binds to the extra-cellular loop of FPN1 and triggers its ubiquitinylation onlysine residues located in the intracellular domain leadingto internalization and degradation in lysosomes.26-28 Thismechanism allows a finely-tuned control of iron effluxfrom enterocytes and macrophages toward the blood-stream when more iron is needed during active erythro-poiesis (in this case, hepcidin synthesis is inhibited by ery-throid signals), or blood iron must be controlled due topathogen proliferation/growth or incipient iron overload(here hepcidin synthesis is induced by inflammatory oriron mediators, respectively) (reviewed by Drakesmith,Nemeth and Ganz29).FPN1 topology and membrane organization have long

been addressed with controversial results concerninglocalization of the N- and C-terminal extremities andnumber of transmembrane segments.30-39 Recently, aninward-open conformation of the transporter has beenpredicted,34,37 with a cluster of residues lying in the centralcore of the protein important for iron traffic and consistentwith an iron-binding site37 and residues involved in hep-cidin binding fully accessible in the outward-openmodel.37 The inward-open form may represent the restingstate of the protein, and the outward-open state as a con-formation attainable only in the presence of intracellulariron, i.e. when FPN1 shuttles between the two conforma-

Table 2. Main features of Ferroportin Disease and other hereditary iron overload disorders in humans.Disorder Affected gene Known or Epidemiology Genetics Mechanism for Clinical Main Clinical (symbol / location) postulated gene increased cellular onset clinical course product function iron deposits (decade) manifestation

I. Ferroportin Disease Solute carrier Iron exporter Affects Caucasians Autosomal Iron retention Any • Liver disease Mild family 40 (iron-regulated (SLC40A1 / 2q32) and non Caucasians dominant due to decreased • Marginal anemia transporter), member 1 from cells iron export including macrophages, enterocytes, syncityotrophoblasts II. Hemochromatosis Hemochromatosis Hepcidin regulator Affects Caucasians 4th-5th

gene (HFE / 6p21.3) of North European descent Autosomal Transferrin-receptor 2 Hepcidin Affects recessive Mild- (TfR2 / 7q22) regulator Caucasians and 3rd-4th severe non Caucasians Increased iron Solute carrier Iron exporter Affects Caucasians accumulation family 40 (iron- from cells including and non Caucasians in parenchymal •Liver Disease regulated transporter), macrophages, Autosomal cells due to 4th-5th • Arthropathy

member 1 enterocytes, dominant increased • Cardiomyopathy (SLC40A1 / 2q32) syncityotrophoblasts transferrin and • Endocrinopathy Hepcidin antimicrobial Degradation Affects Caucasians non-transferrin peptide of ferroportin and non Caucasians bound iron (HAMP /19q13.1) and downregulation Autosomal import 2nd-3rd • Hypogonadism and of iron efflux recessive cardiomyopathy Severe from cells • Liver disease Hemojuvelin Hepcidin Affects Caucasians (HJV/ 1p21) regulator and non Caucasians III. Aceruloplasminemia Ceruloplasmin Iron efflux Affects Caucasians Autosomal Decreased 2nd-3rd • Neurological Severe (CP / 3q23-q25) from cells and non Caucasians recessive iron efflux manifestations • Anemia IV. A (hypo)transferrinemiaTransferrin • Iron transport in the Affects Caucasians Autosomal Increased iron 1st-2nd Anemia Severe (Tf / 3q21) bloodstream and non Caucasians recessive influx

A. Pietrangelo

1974 haematologica | 2017; 102(12)

tions (Figure 1A). The selective binding of hepcidin to theouter-facing conformation would, therefore, guaranteethat FPN1 degradation can occur only when intracellulariron is abundant37 and actively pumped through the chan-nel38 (Figure 1B). Recently, the crystal structures of a bac-terial homolog of FPN1, BbFPN, has been resolved in boththe outward- and inward-facing states, and a homologymodel with human FPN1 has been developed.39According to the Au, FPN1 has 12 TM helices, as previous-ly predicted,32 and is divided into two halves, one formingthe N lobe, the other the C lobe connected by a longcytosolic loop, with a central cavity between the lobesthat is open towards the extracellular side and not accessi-ble from the intracellular side (Figure 1A). FPN1 undergoesan intra-domain conformational rearrangement during thetransport cycle. When hepcidin enters the central cavity

between the N and C lobes, and interacts with the hep-cidin-binding site located on the C lobe, it elicits twoeffects: a) it increases the accessibility of the intracellularloops that harbor the ubiquitination sites to the ubiquitinligases; and b) it arrests the conformational transition ofFPN1 from the outward-facing state to the inward-facingstate, inhibiting the access of iron from the cytoplasm tothe substrate-binding site within the intracellular gate(Figure 1B).39

Molecular pathogenesisThe general pathophysiological basis of FD is well

defined and relies on the impaired iron export from theiron storage/recycling site (particularly macrophages)towards the bloodstream. Figure 2 shows the basic irontransport defect of the FD as opposed to FPN1-associated

Ferroportin Disease

haematologica | 2017; 102(12) 1975

Figure 1. Biology of ferroportin and postulated pathobiology of Ferroportin Disease (FD). (A) Structure-function relationship of iron-export ferroportin activity.39 (B)Putative mechanisms of hepcidin binding to FPN and its degradation.39 (C) Postulated basis for FD. (Upper panel) In cells undergoing relatively low iron flux, such asenterocytes, the product of the FPN wild-type allele is able to reach the plasma membrane and export iron. For clarity, mutated FPN1 was not depicted at the cellsurface: based on previous in vitro work, it has been postulated that some mutant FPN1 can still reach the cell surface and preserve some iron-transport compe-tence, but this is still controversial. (Lower panel) In cells undergoing high iron turnover, such as macrophages, increased requests for iron export impose highdemands on FPN traffic leading to a 'traffic jam' within the endocytic/plasmamembrane and degradation compartments and inappropriately low wild-type allele prod-uct targeting to the cell membrane.54 (D) Postulated effect of FPN mutations that affect formation of the intracellular gate and access to the iron binding site.39

HC. In the latter cases, as discussed above, mutations thataffect the hepcidin binding site and or FPN1 ubiquitinationresult in reduced FPN 'sensitivity' to hepcidin, leading tothe FPN1-related HC phenotype. This has been nicelyexemplified by an informative murine model correspon-ding at the mutation of the hepcidin binding site.40In spite of these advances, the molecular pathogenesis

of FD has long been elusive. A number of in vitro studies,mostly using over-expressed exogenous wild-type andmutant FPN1 in a variety of cell lines, have investigatedFPN1 biology and function and the effect of differentFPN1 mutants on protein traffic and iron-transfer capabil-ity, although with conflicting results, depending on thecell line or methodology used.30-32,34,35,38,41-47In this context, it has been actively debated whether

FPN1 haplo-insufficiency would explain FD or whetherthe disease results from a dominant-negative effect. It hasbeen argued that if haplo-insufficiency was the explana-tion for FD, then nonsense mutations should also result inthe disorder; however, so far, the vast majority of reportedmutations in FD are missense mutations.48 In addition, atargeted gene deletion in the murine Fpn1 gene has littleeffect in heterozygous animals,49 whereas the flatiron (ffe)mouse with a missense mutation in Fpn1 that affects itslocalization and iron export activity when over-expressedin vitro, present a phenotype similar to human patients.50 Instudies using exogenous tagged protein in vitro, Fpn1 formsmultimers and mutant Fpn1 prevents cell membrane local-ization of wild-type Fpn1.33,42,50 A multimeric protein,through a dominant-negative effect, would better explainthe autosomal dominant trait of FD. However, other stud-ies from different groups have provided experimental evi-dence to support the opposite conclusion and showed thatFpn1 is a monomer in cultured cells35,51,52 and in vivo.53More recently, Sabelli et al.,54 using for the first-time cul-tured macrophages from FD patients, found that endoge-nous FPN1 shows a similar localization to that in donormacrophages, except for greater accumulation in lyso-somes, suggesting a higher degradation rate of mutantFPN1. Unexpectedly, and contrary to previous studiesusing over-expressed mutant protein in cell lines, FPN1 inFD macrophage circulates in the early endocytic compart-ment, does not multimerize, it reaches the plasma mem-brane, is iron-transport competent (although to a lesserextent than normal macrophages), and is promptly inter-nalized and degraded upon exposure to hepcidin.However, when FD macrophages are exposed to largeamounts of heme iron, in contrast to donor macrophages,FPN1 can no longer reach the cell surface, leading tomarked intracellular iron retention. Based on these obser-vations, a model of FD has been proposed in which FPN1monomers, in spite of the fact that half proteins are mutat-ed, can still reach the cell surface and export iron in cellsthat are exposed in vivo to a relatively low flux of iron,such as enterocytes (Figure 1C).54 On the contrary, in cellsundergoing high iron turnover in vivo, such as tissuemacrophages, sufficient FPN1 is prevented from reachingthe plasma membrane, possibly due to a 'traffic jam' in thedegradation and/or endocytic cycling pathways. Thismodel is consistent with the clinical manifestation of FDcharacterized by early iron accumulation in hepaticKupffer cells and normal transferrin saturation, indicatingthat mutant FPN1 activity does not limit intestinal irontransfer; the latter becomes critically low in young femalesat menarche or after aggressive phlebotomy, when high

iron demands for erythropoiesis likely impose increasedFPN1 traffic/cycling within tissue macrophages.1,16,17 (Seebelow under Clinical Manifestations and Diagnosis andTreatment). This study did not address the question as towhether mutant or only wild-type FPN1 reaches the plas-ma membrane and whether mutant-FPN1 is transportcompetent. Previous studies have not been conclusive.Exogenously expressed p.A77D and p.Val162del FPN1mutants have been found to be iron-transport incompe-tent in all studies, but able to reach the cell membrane insome,34,35,38,41,43 and not in others.30,31 The p.G80S FPN1mutant has been localized at the cell surface in two pub-lished studies,43,55 and found iron transport competent inone55 and incompetent in the other.43According to Taniguchi et al.,39 the mutation sites asso-

ciated with FD are mainly mapped onto the inter-lobeinterface, mostly on the intracellular side, and form theintracellular gate. These mutations would, therefore,destabilize the inter-lobe interactions, thereby affectingthe stable formation of the intracellular gate and reducingthe iron transport activity of FPN1 (Figure 1D). It is possi-ble that different mutants differently affect iron transportcapability of FPN1; while this may be better overcome bythe normal allele product in cells with low iron turnoversuch as enterocytes or hepatocytes, it may be further ham-pered in cells like macrophages where the additional 'traf-fic jam' in the endocytic-plasmamembrane compartmentwill aggravate the basic defect (see above).

Genetics and epidemiologyA list of published mutations associated with FD and

FPN1-related HC is reported in Table 3.5-8,17,43,56-102Numerous mutations of the FPN1 gene have been identi-fied so far in probands with the classic FD phenotype ofFrench-Canadian, Melanesian, Thai, Japanese andEuropean heritages.A few common FPN1 mutations have been reported in

independent pedigrees, in different countries (e.g.Val192del;56,60,72-79,92 A77D,17,59,60 G80S.43,55,56,61-63 It is nowbelieved that the most frequently reported FPN1 muta-tions, such as the p.Val162del, are more frequently iden-tified than other SLC40A1 mutations because they haveoccurred multiple times in isolated populations ratherthan occurring once and spreading to different popula-tions, as indicated by the identification of a de novop.Val162del variant in an isolated case of FD.79FPN1 variants are highly prevalent in African popula-

tions. The first prevalent FPN1 variant reported in Africansand Black Americans was the common Q248H polymor-phism (p.Gln248His).82,83,100-102 Interestingly, global analysisof variants in the SLC40A1 gene (which includes muta-tions associated with both the FD and FPN1-associatedHH) revealed an allele frequency of 0.0364%, giving a pre-dicted pathogenic genotype carrier rate of 1 in 1373, a fig-ure that approaches the frequency of HFE-HC.103 This waslargely due to the relatively high allele frequencies for twoSLC40A1 variants (p.Asp270Val84,85 and p.Arg371Trp56) inthe African populations; the predicted SLC40A1 patho-genic genotype carrier rate of these two variants is 1 in 197among the African population.103 The Q248H,101,102 thep.Asp270Val and the p.Arg371Trp and other FPN1 poly-morphic variants84 may also predispose to iron overload;but no clear evidence for this has been provided (e.g. lackof functional studies), while the possibility remains that,because of the small sample size, these observations could

A. Pietrangelo

1976 haematologica | 2017; 102(12)

Ferroportin Disease

haematologica | 2017; 102(12) 1977

Table 3. Disease-associated mutations of the FPN1 gene. Gene SLC40A1 (RefSeq NM_014585.5, NP_055400.1)A. Ferroportin disease Nucleotide change Amino acid change Type of variation* Phenotype Reference

1. c.134C>A p.Ala45Glu Missense ferroportin diseasea 562. c.205G>A p.Ala69Thr Missense ferroportin disease 573. c.206C>T p.Ala69Val Missense ferroportin diseasea 564. c.212C>T p.Ser71Phe Missense ferroportin diseasea 565. c.214G>T p.Val72Phe Missense ferroportin disease 586. c.230C>A p.Ala77Asp Missense ferroportin disease 17,59,607. c.238G>A p.Gly80Ser Missense ferroportin disease 43,55,56,61-638. c.239G>T p.Gly80Val Missense ferroportin disease 649. c.252C>G p.Asp84Glu Missense ferroportin disease 9110. c.262A>G p.Arg88Gly Missense ferroportin disease 56, 6511. c.263G>C p.Arg88Thr Missense ferroportin disease 6612. c.386T>C p.Leu129Pro Missense ferroportin disease 6713. c.454A>T p.Ile152Phe Missense ferroportin disease 6814. c.469G>A p.Asp157Asn Missense ferroportin disease 5815. c.469G>T p.Asp157Tyr Missense ferroportin disease 6916. c.470A>C p.Asp157Ala Missense ferroportin disease 7017. c.470A>G p.Asp157Gly Missense ferroportin disease 56,69,7118. c.473G>T p.Trp158Leu Missense ferroportin disease 5619. c.474G>T p.Trp158Cys Missense ferroportin disease 4720. c.484_486del p.Val162del Deletion ferroportin disease 56,60,72-79,9221. c.521A>T p.Asn174Ile Missense ferroportin disease 6122. c.532C>G p.Arg178Gly Missense ferroportin disease 7723. c.533G>A p.Arg178Gln Missense ferroportin disease 56,6524. c.539T>C p.Ile180Thr Missense ferroportin disease 6625. c.542A>T p.Asp181Val Missense ferroportin disease 56,64,6926. c.546G>T p.Gln182His Missense ferroportin disease 56, 7127. c.553A>G p.Asn185Asp Missense ferroportin disease 56, 8028. c.554A>C p.Asn185Thr Missense ferroportin diseasea 5629. c.610G>A p.Gly204Ser Missense ferroportin diseasea 5630. c.689C>A p.Thr230Asn Missense ferroportin diseasea 6931. c.695C>A p.Ala232Asp Missense ferroportin disease 8132. c.698T>C p.Leu233Pro Missense ferroportin disease 68,6933. c.744G>T p.Gln248His Missense ferroportin diseasea 82,83,100-10234. c.797T>C p.Met266Thr Missense ferroportin diseasea 6935. c.800G>A p.Gly267Asp Missense ferroportin disease 6436. c.809A>T p.Asp270Val Missense ferroportin disease 84,8537. c.968G>T p.Gly323Val Missense ferroportin disease 7138. c.1035G>C p.Leu345Phe Missense ferroportin diseasea 6939. c.1051A>G p.Ile351Val Missense ferroportin diseasea 6940. c.1111C>T p.Arg371Trp Missense ferroportin diseasea 5641. c.1112G>A p.Arg371Gln Missense ferroportin diseasea 5642. c.1328C>T p.Pro443Leu Missense ferroportin diseasea 6943. c.1402G>A p.Gly468Ser Missense ferroportin diseasea 8644. c.1466G>A p.Arg489Lys Missense ferroportin disease 4645. c.1467A>C p.Arg489Ser Missense ferroportin disease 8746. c.1468G>A p.Gly490Ser Missense ferroportin disease 56,65,6947. c.1469G>A p.Gly490Asp Missense ferroportin disease 88, 6948. c.1520A>G p.His507Arg Missense ferroportin diseasea 8949. c.1681A>G p.Arg561Gly Missense ferroportin diseasea 90

continued on the next page

be attributable to chance or that the polymorphisms iden-tified may be in linkage disequilibrium with other disease-causing loci. Yet, taken together, the collected data makeFPN1 the gene most frequently associated with hereditaryhyperferritinemia in Africans.

Clinical manifestations and diagnosisAs discussed, FD is caused by loss-of-function muta-

tions in FPN1. These mutations impair iron export, partic-ularly from reticuloendothelial macrophages. The result isiron accumulation in macrophages of the spleen, liver, andbone (reflected by high levels of SF) (Figure 3). At liver his-tology, parenchymal cells of these organs are largelyspared (Figure 3), but discrete hepatocytic iron depositsare also appreciable, due to defective FPN1 activity inhepatocytes, even at early stages.16 Clinical presentationappears heterogeneous, but overall expressivity is milderthan classic HC, and the associated liver disease is usuallynot as severe (Table 2 and Figure 3).1,16,17,56 As occurs in clas-sic forms of HFE HC, also in the FD host factors (menses,blood loss, etc.), co-inheritance of mutations of other iron-genes or variants in genes associated to antioxidantdefense and organ fibrosis, and associated pathologicalconditions (metabolic syndrome, viral hepatitis, etc.) mayall affect the phenotype. Hypochromic anemia is notuncommon in young menstruating females. Owing to the mild clinical expressivity reported in the

literature, doubts have been raised on the penetrance ofthe genetic defect and the rationale for iron-removal ther-apy. However, there is limited and usually not detailedclinical information in the published reports; this, and thelack of prospective studies, still hamper our understandingof the actual clinical impact of the disorder. In the onlyreport published so far, 6 members of the pedigree inwhich FD was first described16 were followed for 11-24years.104 The proband, aged 83, who had carried an occultHBV infection since the age of 56, developed a liver cancerin a non-cirrhotic liver after discontinuation of a 20-yearlong phlebotomy program; 2 siblings, who had also inter-rupted treatment, showed a fibrosis progression. These

clinical data, while of interest, do not allow definite con-clusions to be drawn as to a pathogenic link between ironaccumulation in FD and liver damage and disease. The hallmark of classic FD is marked iron accumulation

in Kupffer cells (Figure 3). Kupffer cells are vital to the pro-duction of fibrogenic mediators, to immunological tumorsurveillance, and disposal of transformed hepatocytes.105Selective and massive iron overload may impair theseactivities and favor fibrogenesis and carcinogenesis.Moreover, as discussed above, hepatocytic iron accumula-tion also takes place in FD, although to a much lesserextent than in HFE- and non-HFE HC, and the establishedpro-oxidant damaging activity of iron in parenchymal cellsmay also contribute to disease progression. Unlike HFE-HC, the pattern of inheritance of FD is

autosomal dominant. Therefore, either parent carries thepathogenic mutation of FPN1 and presents with unex-plained hyperferritinemia. In addition, the proband carriesa 50% risk of having an affected child. The disease mustbe suspected in any individual with unexplained hyperfer-ritinemia and low-normal transferrin saturation (TS), ornon-parenchymal cell siderosis at liver biopsy or liver andspleen iron accumulation at MRI (Table 4). Hyperferritinemia in FD appears very early in life, and

unexplained hyperferritinemia with normal TS in a childshould prompt MRI evaluation to evaluate iron accumu-lation in liver, spleen and bone marrow106 (see below).Figure 4 shows a proposed algorithm for the diagnosis of

FD. If hyperferritinemia associates with high TS (as con-firmed in at least two sequential determinations) but in theabsence of anemia, a typical picture of HFE- and non HFE-HC (including FPN1-associated HC due to gain-of-functionFPN1 mutations) is ruled out a priori. If hyperferritinemiaassociates with high TS and anemia, a typical picture ofhereditary hemoglobinopathies and red cell defects oratransferrinemia, FD is again ruled out (Figure 4).On the contrary, in subjects with increased serum fer-

ritin and low or normal TS, the workup should focus oncommon causes of secondary hyperferritinemia and otherrare causes of hereditary hyperferritinemia to confirm the

A. Pietrangelo

1978 haematologica | 2017; 102(12)

B. Ferroportin1-associated hemochromatosis

1. c.-59_-45del Deletion hemochromatosis 65 (heterozygous state)2. c.190T>A p.Tyr64Asn Missense (heterozygous state) hemochromatosisb 933. c.190T>C p.Tyr64His Missense (heterozygous state) hemochromatosis 944. c.430A>C p.Asn144His Missense (heterozygous state) hemochromatosisb 55. c.430A>G p.Asn144Asp Missense (heterozygous state) hemochromatosisb 66. c.431A>C p.Asn144Thr Missense (heterozygous state) hemochromatosisb 197. c.718A>G p.Lys240Glu Missense (heterozygous state) hemochromatosis 958. c.977G>A p.Cys326Tyr Missense (heterozygous state) hemochromatosis 96, 979. c.977G>C p.Cys326Ser Missense (heterozygous state) hemochromatosisb 710. c.977G>T p.Cys326Phe Missense (heterozygous state) hemochromatosisb 811. c.1014T>G p.Ser338Arg Missense (heterozygous state) hemochromatosisb 9812. c.1502A>G p.Tyr501Cys Missense (heterozygous state) hemochromatosis 9913. c.1510G>A p.Asp504Asn Missense (heterozygous state) hemochromatosis 6914. c.1520A>G p.His507Arg Missense (heterozygous state) hemochromatosisb 47*All reported ferroportin mutations are at the heterozygous state (autosomal dominant trait). aReported data do not allow a classic Ferroportin Disease phenotype to beassigned. bPatients carrying a ferroportin missense mutation in whom a classic hemochromatosis phenotype is confirmed by a liver biopsy.

continued from the previous page

diagnosis of FD (Figure 4). First, common causes of hyper-ferritinemia, such as metabolic disorders, inflammation,cancer, etc., should be considered. If they are not found, orif the hyperferritinemia persists after their treatment, thenext step depends on whether or not anemia is present. Inthe absence of overt anemia, if liver and spleen iron con-tent are increased at MRI or liver biopsy shows prominentKupffer cell iron load, FD disease should be consideredand genetic testing performed for confirmation of diagno-sis (Figure 4). Another common cause of hereditary hyper-ferritinemia with normal TS associated with iron accumu-lation and anemia is Gaucher disease, usually associatedwith hepatosplenomegaly, cytopenia, abnormal coagula-tion, bone disease, and neuropathic manifestations.107 In the absence of body iron accumulation, but in the

presence of elevated SF levels and normal TS, autosomaldominant hyperferritinemia with cataract (due to muta-tions of the iron responsive element in the 5' untranslatedregion of the L ferritin mRNA108) or without cataract,109should be considered. If overt anemia is present, but TS isnormal/low, aceruloplasminemia should be suspected, arare autosomal recessive disease due to loss of function

mutations in ceruloplasmin (CP) and resulting in iron over-load in the liver and pancreas, and progressive neurode-generation, diabetes and retinal degeneration.110Brain MRI with typical iron accumulation in basal gan-

glia and thalamus may help confirm the diagnosis. Asmentioned above, another rare genetic disease presentingwith hyperferritinemia and anemia is atransferrinemia/hypotransferrinemia111 which, however, is characterizedby increased transferrin saturation due to extremely lowserum transferrin levels. Differential diagnosis mainly includes the classic (HFE)

and non-classic (TfR2, HAMP, HJV and FPN1) forms ofHH, all characterized by early and progressive increase ofTS followed by elevation of serum ferritin as iron accumu-lation increases in parenchymal cells of the liver, pancreas,heart and other organs (Table 2). As discussed, unlike HH,in FD clinical expressivity is milder. Abdominal MRI is a useful non-invasive tool to catego-

rize and diagnose the disorder, as it can differentiatepatients with FD, characterized by the SSL triad (spleen,spine, liver) iron retention (Figure 5B), from all other formsof HH, including FPN1-HC, associated with liver iron

Ferroportin Disease

haematologica | 2017; 102(12) 1979

Figure 2. The basis for abnormal iron transferinto the bloodstream in Ferroportin Disease asopposed to FPN-associated hereditaryhemochromatosis.

overload but normal spleen and bone marrow iron content(Figure 5D).112

TreatmentVenesection is the cornerstone of therapy also in FD, but

it may not be tolerated equally in all patients, and low TSwith anemia may be rapidly established despite SF stillbeing elevated.1 Macrophage iron overload is very resist-ant to iron withdrawal in this disorder, even in patientswho are apparently well-treated (Figure 5C). Therefore,unlike HH, not only serum ferritin, but especially TSshould be carefully monitored during therapy. In addition,therapy should not aim at reaching the usual HH targetsfor iron depletion (TS below 20%, SF 50 ng/L or slightanemia) but be more conservative. There are no studieson the optimal phlebotomy schedule in FD. In practical

terms, a monthly/bi-monthly phlebotomy session for 1-2years, depending on the underlying mutation, allows anacceptable state of iron depletion to be reached, whilemaintenance therapy (usually a phlebotomy session every4-6 months) should be continued for life. A reasonable tar-get for therapy is an SF level of 100-200 ng/mL. In certaincases, such Ft values may still reflect some iron loading oftissue macrophages (Figure 5C), but the associated clinicalrisk is negligible. Ideally, the optimal target is the lowestacceptable ferritin level for TS and hemoglobin levels notbelow the lower limit of normal. The (controversial)dietary restrictions sometimes recommended for patientswith HH (avoiding vitamin C or iron-rich or enrichedfoods) do not apply to FD due to the different pathogenicbasis as compared to HH: normal/sufficient enterocyteiron absorption and normal/marginally increased iron

A. Pietrangelo

1980 haematologica | 2017; 102(12)

Table 4. Suspecting and diagnosing Ferroportin Disease. When to suspect Essential for diagnosisSex Ethnicity Age, y Signs

Either Any 10-80 • Unexplained hyperferritinemia and normal Heterozygosity for FPNmutation and or inappropriately low transferrin saturation hyperferritinemia with normal or inappropriately • Isolated hyperferritinemia in father or mother low transferrin saturation and Kupffer cell iron • Sinusoidal (Kupffer cell) iron overload at liver overload at liver biopsy biopsy or spleen (and liver), iron accumulation at MRI in patients with unexplained hyperferritinemia and normal or inappropriately low transferrin saturation y: years; MRI: magnetic resonance imaging.

Figure 3. The different stages and outcomes of “iron retention” in Ferroportin Disease versus “iron accumulation” in FPN1-associated hemochromatosis (HC).Liver histology pictures are reproduced with the permission of Sabelli et al.54

accumulation in parenchymal cells in the FD versusincreased iron absorption and marked iron accumulationin parenchymal cells in HH. Iron chelation may be an option in selected cases.79Siblings of patients with FD, like their offspring, must

undergo screening since they have a 50% chance of beingsusceptible.

Conclusions

FPN1 is a multipass membrane iron-exporter that hasevolved in mammals to assure sufficient iron deliveryfrom the external milieu and internal sites of iron storageand recycling to the bloodstream, mainly to support theerythron activity. Overall, the FPN1/SLC40A1 gene isessential for humans and total loss (homozygote muta-

tion) of its product is incompatible with life.49 Loss-of-function of one FPN1 allele in humans results in FD, char-acterized by a preserved intestinal iron export activity butcompromised iron export from tissue macrophages. Thisleads to progressive iron retention in liver, spleen and bonemarrow macrophages, resulting in inappropriately lowiron delivery to circulating transferrin and marginal iron-restricted erythropoiesis that may result in overt anemiawhen bone marrow demands are increased (e.g. menar-che, aggressive phlebotomy regimen). Gain-of-functionmutations of FPN1 preclude the inhibitory activity of hep-cidin, thereby leading to unrestricted iron transfer to thebloodstream and causing a rare form of HH.The pathogenic, biochemical and clinical signatures of

FD are symmetrical and opposite to HFE and non HFE-HH: normal/sufficient enterocyte iron absorption,marked iron accumulation in non parenchymal cells in

Ferroportin Disease

haematologica | 2017; 102(12) 1981

Figure 4.Diagnostic algorithm for Ferroportin Disease and hereditary hyperferritinemia. ACD/AI: anemia of chronic disease/anemia of inflammation. *Gaucher dis-ease may present with or without siderosis depending on the disease stage. **Advanced ACD/AI may also present with siderosis at MRI (usually spleen and bonemarrow).

the FD versus increased iron absorption and marked ironaccumulation in parenchymal cells in HH; hyperferritine-mia with normal/low transferrin saturation in FD versushyperferritinemia and high transferrin saturation in HH;intolerance to aggressive phlebotomy regimens in FDversus optimal response to intense phlebotomy in HH;mild and benign clinical course in FD versus potentiallysevere clinical expressivity in HH; vertical hereditarytransmission and presentation at each generation of FDversus recessive transmission of most forms of HH(except FPN1-HH).

While the molecular pathogenesis of FD is becomingmore and more defined, the long-term effect of massiveiron retention in tissue macrophages in the setting ofchronic inflammatory/infectious or degenerative disor-ders is still unclear. Today, isolated or unexplained hyperferritinemia rep-

resents one of the commonest reasons for referral.Knowing that FD is one of the most frequent geneticcauses of hyperferritinemia, regardless of ethnicity, it isimportant to maintain a high diagnostic suspicion forthis disorder.

A. Pietrangelo

1982 haematologica | 2017; 102(12)

References

1. Pietrangelo A. The ferroportin disease.Blood Cells Mol Dis. 2004;32(1):131-138.

2. Abboud S, Haile DJ. A novel mammalianiron-regulated protein involved in intracellu-lar iron metabolism. J Biol Chem.2000;275(26):19906-19912.

3. Donovan A, Brownlie A, Zhou Y, et al.Positional cloning of zebrafish ferroportin1identifies a conserved vertebrate ironexporter. Nature. 2000;403(6771):776-781.

4. McKie AT, Marciani P, Rolfs A, et al. A novelduodenal iron-regulated transporter, IREG1,implicated in the basolateral transfer of ironto the circulation. Mol Cell. 2000;5(2):299-309.

5. Njajou OT, Vaessen N, Joosse M, et al. Amutation in SLC11A3 is associated withautosomal dominant hemochromatosis. NatGenet. 2001;28(3):213-214.

6. Wallace DF, Clark RM, Harley HA,Subramaniam VN. Autosomal dominantiron overload due to a novel mutation of fer-roportin1 associated with parenchymal ironloading and cirrhosis. J Hepatol.2004;40(4):710-713.

7. Sham RL, Phatak PD, West C, Lee P,

Andrews C, Beutler E. Autosomal dominanthereditary hemochromatosis associatedwith a novel ferroportin mutation andunique clinical features. Blood Cells MolDis. 2005;34(2):157-161.

8. Chen SR, Yang LQ, Chong YT, et al. Novelgain of function mutation in the SLC40A1gene associated with hereditary haemochro-matosis type 4. Intern Med J.2015;45(6):672-676.

9. Brissot P, Loreal O. Iron metabolism andrelated genetic diseases: A cleared land,keeping mysteries. J Hepatol. 2016;64(2):505-515.

10. Hollerer I, Bachmann A, Muckenthaler MU.Pathophysiological consequences and bene-fits of HFE mutations: 20 years of research.Haematologica. 2017;102(5):809-817.

11. Pietrangelo A. Hemochromatosis: anendocrine liver disease. Hepatology.2007;46(4):1291-1301.

12. Feder JN, Gnirke A, Thomas W, et al. Anovel MHC class I-like gene is mutated inpatients with hereditary haemochromatosis.Nat Genet. 1996;13(4):399-408.

13. Camaschella C, Roetto A, Cali A, et al. Thegene TFR2 is mutated in a new type ofhaemochromatosis mapping to 7q22. NatGenet. 2000;25(1):14-15.

14. Rivard SR, Lanzara C, Grimard D, et al.Juvenile hemochromatosis locus maps tochromosome 1q in a French Canadian pop-ulation. Eur J Hum Genet. 2003;11(8):585-589.

15. Papanikolaou G, Samuels ME, Ludwig EH,et al. Mutations in HFE2 cause iron overloadin chromosome 1q-linked juvenilehemochromatosis. Nat Genet. 2004;36(1):77-82.

16. Pietrangelo A, Montosi G, Totaro A, et al.Hereditary hemochromatosis in adults with-out pathogenic mutations in the hemochro-matosis gene [see comments]. N Engl J Med.1999;341(10):725-732.

17. Montosi G, Donovan A, Totaro A, et al.Autosomal-dominant hemochromatosis isassociated with a mutation in the ferro-portin (SLC11A3) gene. J Clin Invest.2001;108(4):619-623.

18. Eason RJ, Adams PC, Aston CE, Searle J.Familial iron overload with possible autoso-mal dominant inheritance. Aust NZ J Med.1990;20:226-230.

19. Arden KE, Wallace DF, Dixon JL, et al. Anovel mutation in ferroportin1 is associatedwith haemochromatosis in a SolomonIslands patient. Gut. 2003;52(8):1215-1217.

20. Marro S, Chiabrando D, Messana E, et al.

Figure 5. Abdominal magnetic resonance imag-ing (MRI) pattern of Ferroportin Disease (FD). MRIscans. T2*-weighted gradient-echo sequenceswere used to detect iron accumulation. (A) Normalsubject. (B) FD. (C) FD after completion of phle-botomy program (note that excess iron is stilldetectable in the liver and spine in spite of normalserum ferritin and transferrin saturation levels).(D) Ferroportin-associated hereditary hemochro-matosis: iron accumulation involves only the liverand spares the spleen and spine (arrows).

A B

C D

Heme controls ferroportin1 (FPN1) tran-scription involving Bach1, Nrf2 and aMARE/ARE sequence motif at position -7007 of the FPN1 promoter. Haematologica.2010;95(8):1261-1268.

21. Lymboussaki A, Pignatti E, Montosi G, et al.The role of the iron responsive element inthe control of ferroportin1/IREG1/MTP1gene expression. J Hepatol. 2003;39(5):710-715.

22. Krause A, Neitz S, Magert HJ, et al. LEAP-1,a novel highly disulfide-bonded human pep-tide, exhibits antimicrobial activity. FEBSLett. 2000;480(2-3):147-150.

23. Pigeon C, Ilyin G, Courselaud B, et al. A newmouse liver-specific gene, encoding a pro-tein homologous to human antimicrobialpeptide hepcidin, is overexpressed duringiron overload. J Biol Chem. 2001;276(11):7811-7819.

24. Park CH, Valore EV, Waring AJ, Ganz T.Hepcidin, a urinary antimicrobial peptidesynthesized in the liver. J Biol Chem.2001;276(11):7806-7810.

25. Vecchi C, Montosi G, Zhang K, et al. ERstress controls iron metabolism throughinduction of hepcidin. Science.2009;325(5942):877-880.

26. Nemeth E, Tuttle MS, Powelson J, et al.Hepcidin regulates cellular iron efflux bybinding to ferroportin and inducing its inter-nalization. Science. 2004;306(5704):2090-2093.

27. Qiao B, Sugianto P, Fung E, et al. Hepcidin-induced endocytosis of ferroportin isdependent on ferroportin ubiquitination.Cell Metab. 2012;15(6):918-924.

28. Ross SL, Tran L, Winters A, et al. MolecularMechanism of Hepcidin-MediatedFerroportin Internalization RequiresFerroportin Lysines, Not Tyrosines or JAK-STAT. Cell Metab. 2012;15(6):905-917.

29. Drakesmith H, Nemeth E, Ganz T. Ironingout Ferroportin. Cell Metab. 2015;22(5):777-787.

30. Schimanski LM, Drakesmith H,Merryweather-Clarke AT, et al. In vitrofunctional analysis of human ferroportin(FPN) and hemochromatosis-associated FPNmutations. Blood. 2005;105(10):4096-4102.

31. Drakesmith H, Schimanski LM, Ormerod E,et al. Resistance to hepcidin is conferred byhemochromatosis-associated mutations offerroportin. Blood. 2005;106(3):1092-1097.

32. Liu XB, Yang F, Haile DJ. Functional conse-quences of ferroportin 1 mutations. BloodCells Mol Dis. 2005;35(1):33-46.

33. De Domenico I, Ward DM, Musci G, KaplanJ. Evidence for the multimeric structure offerroportin. Blood. 2007;109(5):2205-2209.

34. Wallace DF, Harris JM, Subramaniam VN.Functional analysis and theoretical modelingof ferroportin reveals clustering of mutationsaccording to phenotype. Am J Physiol CellPhysiol. 2010;298(1):C75-84.

35. Rice AE, Mendez MJ, Hokanson CA, ReesDC, Bjorkman PJ. Investigation of the bio-physical and cell biological properties of fer-roportin, a multipass integral membraneprotein iron exporter. J Mol Biol.2009;386(3):717-732.

36. Le Gac G, Ka C, Joubrel R, et al. Structure-function analysis of the human ferroportiniron exporter (SLC40A1): effect ofhemochromatosis type 4 disease mutationsand identification of critical residues. HumMutat. 2013;34(10):1371-1380.

37. Bonaccorsi di Patti MC, Polticelli F, Cece G,et al. A structural model of human ferro-portin and of its iron binding site. FEBS J.2014;281(12):2851-2860.

38. Praschberger R, Schranz M, Griffiths WJ, etal. Impact of D181V and A69T on the func-tion of ferroportin as an iron export pumpand hepcidin receptor. Biochim BiophysActa. 2014;1842(9):1406-1412.

39. Taniguchi R, Kato HE, Font J, et al.Outward- and inward-facing structures of aputative bacterial transition-metal trans-porter with homology to ferroportin. NatCommun. 2015;6:8545.

40. Altamura S, Kessler R, Grone HJ, et al.Resistance of ferroportin to hepcidin bindingcauses exocrine pancreatic failure and fataliron overload. Cell Metab. 2014;20(2):359-367.

41. McGregor JA, Shayeghi M, Vulpe CD, et al.Impaired iron transport activity of ferro-portin 1 in hereditary iron overload. JMembr Biol. 2005;206(1):3-7.

42. De Domenico I, Ward DM, Nemeth E, et al.The molecular basis of ferroportin-linkedhemochromatosis. Proc Natl Acad Sci USA.2005;102(25):8955-8960.

43. De Domenico I, McVey Ward D, Nemeth E,et al. Molecular and clinical correlates in ironoverload associated with mutations in ferro-portin. Haematologica. 2006;91(8):1092-1095.

44. De Domenico I, Ward DM, Langelier C, etal. The molecular mechanism of hepcidin-mediated ferroportin down-regulation. MolBiol Cell. 2007;18(7):2569-2578.

45. Mayr R, Janecke AR, Schranz M, et al.Ferroportin disease: A systematic meta-analysis of clinical and molecular findings. JHepatol. 2010;53(5):941-949.

46. Griffiths WJ, Mayr R, McFarlane I, et al.Clinical presentation and molecular patho-physiology of autosomal dominanthemochromatosis caused by a novel ferro-portin mutation. Hepatology. 2010;51(3):788-795.

47. Mayr R, Griffiths WJ, Hermann M, et al.Identification of Mutations in SLC40A1That Affect Ferroportin Function andPhenotype of Human Ferroportin IronOverload. Gastroenterology. 2011;140(7):2056-2063 e2051.

48. Pietrangelo A, Caleffi A, Corradini E. Non-HFE hepatic iron overload. Sem Liv Dis.2011;31(3):302-318.

49. Donovan A, Lima CA, Pinkus JL, et al. Theiron exporter ferroportin/Slc40a1 is essentialfor iron homeostasis. Cell Metab.2005;1(3):191-200.

50. Zohn IE, De Domenico I, Pollock A, et al.The flatiron mutation in mouse ferroportinacts as a dominant negative to cause ferro-portin disease. Blood. 2007;109(10):4174-4180.

51. Goncalves AS, Muzeau F, Blaybel R, et al.Wild-type and mutant ferroportins do notform oligomers in transfected cells. BiochemJ. 2006;396(2):265-275.

52. Schimanski LM, Drakesmith H, Talbott C,et al. Ferroportin: lack of evidence for multi-mers. Blood Cells Mol Dis. 2008;40(3):360-369.

53. Pignatti E, Mascheroni L, Sabelli M, Barelli S,Biffo S, Pietrangelo A. Ferroportin is amonomer in vivo in mice. Blood Cells MolDis. 2006;36(1):26-32.

54. Sabelli M, Montosi G, Garuti C, et al.Human macrophage ferroportin biology andthe basis for the ferroportin disease.Hepatology. 2017;65(5):1512-1525.

55. McDonald CJ, Wallace DF, Ostini L, Bell SJ,Demediuk B, Subramaniam VN. G80S-linked ferroportin disease: classical ferro-portin disease in an Asian family and reclas-sification of the mutant as iron transport

defective. J Hepatol. 2011;54(3):538-544.56. Le Lan C, Mosser A, Ropert M, et al. Sex and

acquired cofactors determine phenotypes offerroportin disease. Gastroenterology.2011;140(4):1199-1207.e1-2.

57. Ferbo L, Manzini PM, Badar S, et al.Detection of a rare mutation in the ferro-portin gene through targeted next genera-tion sequencing. Blood Transfus.2016;14(6):531-534.

58. Pelucchi S, Mariani R, Salvioni A, et al.Novel mutations of the ferroportin gene(SLC40A1): analysis of 56 consecutivepatients with unexplained iron overload.Clin Genet. 2008;73(2):171-178.

59. Subramaniam VN, Wallace DF, Dixon JL,Fletcher LM, Crawford DH. Ferroportin dis-ease due to the A77D mutation in Australia.Gut. 2005;54(7):1048-1049.

60. Lim FL, Dooley JS, Roques AW, Grellier L,Dhillon AP, Walker AP. Hepatic iron concen-tration, fibrosis and response to venesectionassociated with the A77D and V162del "lossof function" mutations in ferroportin dis-ease. Blood Cells Mol Dis. 2008;40(3):328-333.

61. Corradini E, Montosi G, Ferrara F, et al. Lackof enterocyte iron accumulation in the ferro-portin disease. Blood Cells Mol Dis.2005;35(3):315-318.

62. Mougiou A, Pietrangelo A, Caleffi A,Kourakli A, Karakantza M, Zoumbos N.G80S-linked ferroportin disease: the firstclinical description in a Greek family. BloodCells Mol Dis. 2008;41(1):138-139.

63. Wolff F, Bailly B, Gulbis B, Cotton F.Monitoring of hepcidin levels in a patientwith G80S-linked ferroportin disease under-going iron depletion by phlebotomy. ClinChim Acta. 2014;430:20-21.

64. Cremonesi L, Forni GL, Soriani N, et al.Genetic and clinical heterogeneity of ferro-portin disease. Br J Haematol. 2005;131(5):663-670.

65. Cunat S, Giansily-Blaizot M, Bismuth M, etal. Global sequencing approach for charac-terizing the molecular background of hered-itary iron disorders. Clin Chem.2007;53(12):2060-2069.

66. Bach V, Remacha A, Altes A, Barcelo MJ,Molina MA, Baiget M. Autosomal dominanthereditary hemochromatosis associatedwith two novel Ferroportin 1 mutations inSpain. Blood Cells Mol Dis. 2006;36(1):41-45.

67. Moreno-Carralero MI, Munoz-Munoz JA,Cuadrado-Grande N, et al. A novel mutationin the SLC40A1 gene associated withreduced iron export in vitro. Am J Hematol.2014;89(7):689-694.

68. Girelli D, De Domenico I, Bozzini C, et al.Clinical, pathological, and molecular corre-lates in ferroportin disease: A study of twonovel mutations. J Hepatol. 2008;49(4):664-671.

69. Callebaut I, Joubrel R, Pissard S, et al.Comprehensive functional annotation of 18missense mutations found in suspectedhemochromatosis type 4 patients. Hum MolGenet. 2014;23(17):4479-4490.

70. Saja K, Bignell P, Robson K, Provan D. Anovel missense mutation c.470 A>C(p.D157A) in the SLC40A1 gene as a causeof ferroportin disease in a family withhyperferritinaemia. Br J Haematol.2010;149(6):914-916.

71. Hetet G, Devaux I, Soufir N, Grandchamp B,Beaumont C. Molecular analyses of patientswith hyperferritinemia and normal serumiron values reveal both L ferritin IRE and 3new ferroportin (slc11A3) mutations. Blood.

Ferroportin Disease

haematologica | 2017; 102(12) 1983

2003;102(5):1904-1910.72. Devalia V, Carter K, Walker AP, et al.

Autosomal dominant reticuloendothelialiron overload associated with a 3-base pairdeletion in the ferroportin 1 gene(SLC11A3). Blood. 2002;100(2):695-697.

73. Wallace DF, Pedersen P, Dixon JL, et al.Novel mutation in ferroportin1 is associatedwith autosomal dominant hemochromato-sis. Blood. 2002;100(2):692-694.

74. Cazzola M, Cremonesi L, Papaioannou M,et al. Genetic hyperferritinaemia and reticu-loendothelial iron overload associated witha three base pair deletion in the codingregion of the ferroportin gene (SLC11A3). BrJ Haematol. 2002;119(2):539-546.

75. Melis MA, Cau M, Congiu R, et al. A muta-tion in the TMPRSS6 gene, encoding a trans-membrane serine protease that suppresseshepcidin production, in familial iron defi-ciency anemia refractory to oral iron.Haematologica. 2008;93(10):1473-1479.

76. Roetto A, Merryweather-Clarke AT, DaraioF, et al. A valine deletion of ferroportin 1: acommon mutation in hemochromastosistype 4. Blood. 2002;100(2):733-734.

77. Speletas M, Kioumi A, Loules G, et al.Analysis of SLC40A1 gene at the mRNAlevel reveals rapidly the causative mutationsin patients with hereditary hemochromato-sis type IV. Blood Cells Mol Dis.2008;40(3):353-359.

78. Zoller H, McFarlane I, Theurl I, et al. Primaryiron overload with inappropriate hepcidinexpression in V162del ferroportin disease.Hepatology. 2005;42(2):466-472.

79. Unal S, Piperno A, Gumruk F. Iron chelationwith deferasirox in a patient with de-novoferroportin mutation. J Trace Elem Med Biol.2015;30:1-3.

80. Morris TJ, Litvinova MM, Ralston D,Mattman A, Holmes D, Lockitch G. A novelferroportin mutation in a Canadian familywith autosomal dominant hemochromato-sis. Blood Cells Mol Dis. 2005;35(3):309-314.

81. Relvas L, Claro MT, Bento MC, Ribeiro ML.Novel human pathological mutations. Genesymbol: SLC40A1. Disease: haemochro-matosis, type 4. Hum Genet. 2009;125(3):338.

82. Gordeuk VR, Caleffi A, Corradini E, et al.Iron overload in Africans and African-Americans and a common mutation in theSCL40A1 (ferroportin 1) gene small star,filled. Blood Cells Mol Dis. 2003;31(3):299-304.

83. Beutler E, Barton JC, Felitti VJ, et al.Ferroportin 1 (SCL40A1) variant associatedwith iron overload in African-Americans.Blood Cells Mol Dis. 2003;31(3):305-309.

84. Zaahl MG, Merryweather-Clarke AT, KotzeMJ, van der Merwe S, Warnich L, RobsonKJ. Analysis of genes implicated in iron reg-ulation in individuals presenting with pri-mary iron overload. Hum Genet.2004;115(5):409-417.

85. Lee PL, Gaasterland T, Barton JC. Mild ironoverload in an African American man withSLC40A1 D270V. Acta Haematol.

2012;128(1):28-32.86. Lee PL, Gelbart T, West C, Barton JC.

SLC40A1 c.1402G-->a results in aberrantsplicing, ferroportin truncation after glycine330, and an autosomal dominant hemochro-matosis phenotype. Acta Haematol.2007;118(4):237-241.

87. Koyama C, Wakusawa S, Hayashi H, et al. AJapanese family with ferroportin diseasecaused by a novel mutation of SLC40A1gene: hyperferritinemia associated with arelatively low transferrin saturation of iron.Intern Med. 2005;44(9):990-993.

88. Jouanolle AM, Douabin-Gicquel V, HalimiC, et al. Novel mutation in ferroportin 1gene is associated with autosomal dominantiron overload. J Hepatol. 2003;39(2):286-289.

89. Yamakawa N, Oe K, Yukawa N, et al. ANovel Phenotype of a HereditaryHemochromatosis Type 4 with Ferroportin-1 Mutation, Presenting with JuvenileCataracts. Intern Med. 2016;55(18):2697-2701.

90. Sussman NL, Lee PL, Dries AM, SchwartzMR, Barton JC. Multi-organ iron overload inan African-American man with ALAS2R452S and SLC40A1 R561G. ActaHaematol. 2008;120(3):168-173.

91. Wallace DF, McDonald CJ, Ostini L, Iser D,Tuckfield A, Subramaniam VN. The dynam-ics of hepcidin-ferroportin internalizationand consequences of a novel ferroportin dis-ease mutation. Am J Hematol.2017;92(10):1052-1061.

92. Wallace DF, Browett P, Wong P, Kua H,Ameratunga R, Subramaniam VN.Identification of ferroportin disease in theIndian subcontinent. Gut. 2005;54(4):567-568.

93. Rivard SR, Lanzara C, Grimard D, et al.Autosomal dominant reticuloendothelialiron overload (HFE type 4) due to a new mis-sense mutation in the FERROPORTIN 1gene (SLC11A3) in a large French-Canadianfamily. Haematologica. 2003;88(7):824-826.

94. Raszeja-Wyszomirska J, Caleffi A,Milkiewicz P, Pietrangelo A. Ferroportin-related haemochromatosis associated withnovel Y64H mutation of the SCL40A1 gene.Prz Gastroenterol. 2014;9(5):307-309.

95. Del-Castillo-Rueda A, Moreno-CarraleroMI, Alvarez-Sala-Walther LA, et al. Twonovel mutations in the SLC40A1 and HFEgenes implicated in iron overload in aSpanish man. Eur J Haematol. 2011;86(3):260-264.

96. Viprakasit V, Merryweather-Clarke AT,Chinthammitr Y, et al. Molecular Diagnosisof the First Ferroportin Mutation (C326Y) inthe Far East Causing a Dominant Form ofInherited Iron Overload. Blood. 2004;104(11):3204.

97. Lok CY, Merryweather-Clarke AT,Viprakasit V, et al. Iron overload in the Asiancommunity. Blood. 2009;114(1):20-25.

98. Wallace DF, Dixon JL, Ramm GA, AndersonGJ, Powell LW, Subramaniam VN. A novelmutation in ferroportin implicated in ironoverload. J Hepatol. 2007;46(5):921-926.

99. Letocart E, Le Gac G, Majore S, et al. A novelmissense mutation in SLC40A1 results inresistance to hepcidin and confirms the exis-tence of two ferroportin-associated ironoverload diseases. Br J Haematol. 2009;147(3):379-385.

100.Barton JC, Acton RT, Rivers CA, et al.Genotypic and phenotypic heterogeneity ofAfrican Americans with primary iron over-load. Blood Cells Mol Dis. 2003;31(3):310-319.

101.Gordeuk VR, Diaz SF, Onojobi GO, et al.Ferroportin Q248H, dietary iron, and serumferritin in community African-Americanswith low to high alcohol consumption.Alcohol Clin Exp Res. 2008;32(11):1947-1953.

102.Barton JC, Acton RT, Lee PL, West C.SLC40A1 Q248H allele frequencies andQ248H-associated risk of non-HFE ironoverload in persons of sub-Saharan Africandescent. Blood Cells Mol Dis. 2007;39(2):206-211.

103.Wallace DF, Subramaniam VN. The globalprevalence of HFE and non-HFE hemochro-matosis estimated from analysis of next-generation sequencing data. Genet Med.2016;18(6):618-626.

104.Corradini E, Ferrara F, Pollicino T, et al.Disease progression and liver cancer in theferroportin disease. Gut. 2007;56(7):1030-1032.

105.Manifold IH, Triger DR, Underwood JC.Kupffer-cell depletion in chronic liver dis-ease: implications for hepatic carcinogene-sis. Lancet. 1983;2(8347):431-433.

106.Galicia-Poblet G, Cid-Paris E, Lopez-AndresN, et al. A Pediatric Case Report ofFerroportin Disease. J Pediatr GastroenterolNutr. 2015;63(6):e205-e207.

107.Mistry PK, Lopez G, Schiffmann R, BartonNW, Weinreb NJ, Sidransky E. Gaucher dis-ease: Progress and ongoing challenges. MolGenet Metab. 2017;120(1-2):8-21.

108.Beaumont C, Leneuve P, Devaux I, et al.Mutation in the iron responsive element ofthe L ferritin mRNA in a family with domi-nant hyperferritinaemia and cataract. NatGenet. 1995;11(4):444-446.

109.Kannengiesser C, Jouanolle AM, Hetet G, etal. A new missense mutation in the L ferritincoding sequence associated with elevatedlevels of glycosylated ferritin in serum andabsence of iron overload. Haematologica.2009;94(3):335-339.

110.Miyajima H, Nishimura Y, Mizoguchi K,Sakamoto M, Shimizu T, Honda N. Familialapoceruloplasmin deficiency associatedwith blepharospasm and retinal degenera-tion. Neurology. 1987;37(5):761-767.

111.Heilmeyer L, Keller W, Vivell O, Betke K,Woehler F, Keiderling W. [Congenital atrans-ferrinemia]. Schweiz Med Wochenschr.1961;91:1203.

112.Pietrangelo A, Corradini E, Ferrara F, et al.Magnetic resonance imaging to identify clas-sic and nonclassic forms of ferroportin dis-ease. Blood Cells Mol Dis. 2006;37(3):192-196.

A. Pietrangelo

1984 haematologica | 2017; 102(12)