Comprehensive functional annotation of 18 missense mutations found in suspected hemochromatosis type...

Comprehensive functional annotationof 18 missense mutations found in suspectedhemochromatosis type 4 patients

Isabelle Callebaut1, Rozenn Joubrel2, Serge Pissard3, Caroline Kannengiesser4,

Victoria Gerolami5, Cecile Ged6, Estelle Cadet7, Francois Cartault8, Chandran Ka2,

Isabelle Gourlaouen2, Lenaick Gourhant9,{, Claire Oudin4, Michel Goossens3,

Bernard Grandchamp4, Hubert De Verneuil6, Jacques Rochette7, Claude Ferec2

and Gerald Le Gac2,9,{,∗

1IMPMC, Sorbonne Universites – UMR CNRS 7590, UPMC Univ Paris 06, Museum d’Histoire Naturelle, IRD UMR 206,

Paris, France, 2Laboratoire de Genetique Moleculaire et d’Histocompatibilite, Inserm U1078, Universite de Brest, SFR

SnInBioS, CHRU de Brest, Etablissement Francais du Sang – Bretagne, Brest, France, 3Laboratoire de Genetique,

UPEC (Universite Paris Est Creteil), GHU Henri Mondor, Creteil, France, 4Hopital Bichat, Departement de Genetique,

Inserm U1149 – Center for Research on Inflammation, Universite Paris Diderot, AP-HP, Paris, France, 5Biologie

Moleculaire, CHU de Marseille, Hopital Conception, 6Inserm U1035, Biotherapies des Maladies Genetiques et Cancers,

Universite de Bordeaux, CHU de Bordeaux, Pole de Biologie et Pathologie, Bordeaux, France, 7Laboratoire de Genetique

Moleculaire, UPJV EA4666, CHU d’Amiens, Amiens, France, 8Service de Genetique, CHU de la Reunion, Saint-Denis,

France and 9CHRU de Brest, Inserm CIC0502, Brest, France

Received February 20, 2014; Revised and Accepted April 2, 2014

Hemochromatosis type 4 is a rare form of primary iron overload transmitted as an autosomal dominant traitcaused by mutations in the gene encoding the iron transport protein ferroportin 1 (SLC40A1). SLC40A1 muta-tions fall into two functional categories (loss- versus gain-of-function) underlying two distinct clinical entities(hemochromatosis type 4A versus type 4B). However, the vast majority of SLC40A1 mutations are rare missensevariations, with only a few showing strong evidence of causality. The present study reports the results of an inte-grated approach collecting genetic and phenotypic data from 44 suspected hemochromatosis type 4 patients,with comprehensive structural and functional annotations. Causality was demonstrated for 10 missense var-iants, showing a clear dichotomy between the two hemochromatosis type 4 subtypes. Two subgroups ofloss-of-function mutations were distinguished: one impairing cell-surface expression and one altering onlyiron egress. Additionally, a new gain-of-function mutation was identified, and the degradation of ferroportinon hepcidin binding was shown to probably depend on the integrity of a large extracellular loop outside of thehepcidin-binding domain. Eight further missense variations, on the other hand, were shown to have no discern-ible effects at either protein or RNA level; these were found in apparently isolated patients and were associatedwith a less severe phenotype. The present findings illustrate the importance of combining in silico and biochem-ical approaches to fully distinguish pathogenic SLC40A1 mutations from benign variants. This has profoundimplications for patient management.

†On behalf of the French National Network for the Molecular Diagnosis of Inherited Iron-Overload Disorders and Clinical Investigators (Jean-BaptisteNousbaum, Caroline de Kerguenec, Pascale Poullin, Dominique Capron, Georges Barjonet, Samir Berber, Olivier Rosmorduc, Nicolas Ferry, DenisVital-Durand).

∗To whom correspondence should be addressed at: Centre Hospitalier Regional Universitaire de Brest, Hopital Morvan, Bat 5bis, Laboratoire de GenetiqueMoleculaire et d’Histocompatibilite, 2 Avenue Foch, 29200 Brest, France. Tel: +33 298445064; Fax: +33 298430555; Email: [email protected]

# The Author 2014. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

Human Molecular Genetics, 2014, Vol. 23, No. 17 4479–4490doi:10.1093/hmg/ddu160Advance Access published on April 8, 2014

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INTRODUCTION

Hemochromatosis type 4 (OMIM #606069), also called ferro-portin disease, is an inborn error in iron metabolism transmittedthrough autosomal dominant inheritance and associated withmutations in the solute-carrier family 40 member 1 (SLC40A1)gene. The disease is characterized by wide clinical hetero-geneity, showing marked differences in type of target celland iron deposition, sub-phenotypes and degree of penetrance(1–3). Although rare, hemochromatosis type 4 is the secondmost common cause of hereditary iron overload, after HFE-related hemochromatosis (4).

SLC40A1, also known as ferroportin 1 or FPN1 (Uni-Prot#Q9NP59), is the sole iron export protein reported in mammalsand is expressed in all types of cell that handle major iron flow, in-cluding iron-recycling macrophages, iron-storing hepatocytesand absorptive enterocytes (5). SLC40A1 is predominantlyregulated by the liver-derived peptide hepcidin, which bindsSLC40A1 on the cell surface and induces internalization and deg-radation (6). Hepcidin-SLC40A1 interaction is critical in bothnormal iron homeostasis and common iron metabolism patholo-gies, including not only inherited disorders and iron overload butalso non-genetic diseases and anemia (7–9).

SLC40A1 gene mutations fall into two functional categories,underlying two distinct clinical entities (hemochromatosistype 4A or classical ferroportin disease, versus type 4B or non-classical ferroportin disease) (3, 9, 10). Loss-of-functionmutations, which are more frequent, are associated with hemo-chromatosis type 4A, characterized by relative plasma iron defi-ciency (manifesting in normal-to-low transferrin saturation)and preferential iron retention in reticuloendothelial cells (man-ifesting in high serum ferritin concentrations). As the diseaseprogresses, iron is deposited in hepatocytes, normally accom-panied by a rise in transferrin saturation. Aggressive phlebotomyregimens can be a problem in the early stages of the disease,when patients often display borderline anemia. Gain-of-functionmutations, on the other hand, result in higher plasma ironconcentrations (manifesting in elevated transferrin saturationlevels), due to abnormal iron release from iron-recycling macro-phages and enterocytes. Hyperferritinemia is secondary to theplasma iron burden and is mostly associated with iron depositsin liver parenchymal cells (hepatocytes). These biochemicaland histological features mimic the natural history of typicalHFE-related hemochromatosis. Hence, patients with ferroportingain-of-function mutations usually respond well to phlebotomy.

Thus far, a total of 50 SLC40A1 variations have been shown tobe associated with iron overload phenotypes (11–13): 46 mis-sense mutations, three deletions (c.259-45del, p.Val162deland p.Gly330del) and one splicing mutation (c.1402 G . A).To fully understand the allelic heterogeneity found at theSLC40A1 locus, it is noteworthy that few variations have beenidentified in multiple pedigrees worldwide (1, 12, 13), indicatingthat only private or population-specific variants contribute todisease . Additionally, a lack of genotype–phenotype correl-ation has been reported between unrelated patients and withinfamilies (1, 2, 13). Finally, it must be borne in mind that onlya small number of variations have been investigated in vitroand that functional studies have produced conflicting results(14–19). Hence, despite identification in patients, not all 50

SLC40A1 variations can be firmly established as disease-causingmutations.

Establishing thepathogenicityofSLC40A1variations isofmajordiagnostic importance, with implications for other family membersand for therapeutic approaches targeting iron withdrawal. It canalso aid in identifying individuals at high risk of liver fibro-genesis and carcinogenesis (20, 21). The present study providesevidence that an integrated strategy combining structural andfunctional annotations with clinical findings can reduce therisk of misdiagnosis.

RESULTS

Molecular analysis of 44 hyperferritinemia patients andfrequency of SLC40A1 variations in a Caucasian controlpopulation

Eighteen heterozygous SLC40A1 variations (1 single amino aciddeletion and 17 missense mutations) were identified in 44 indi-viduals (22 female, 22 male; Table 1). All patients were negativefor genotypes known to cause hemochromatosis types 1–3(in the HFE, HJV, HAMP and TFR2 genes) and for mutationsin the FTL gene 5′ untranslated region (hyperferritinemia-cataract syndrome; OMIM#600886) and exon 1 (hyperferritine-mia without iron overload and cataract; OMIM#134790).

To our knowledge, the p.Asp157Tyr, p.Thr230Asn,p.Met266Thr, p.Leu345Phe, p.Ile351Val and p.Asp504AsnSLC40A1 variations have not been previously reported; theothers were previously observed either in pedigrees or insingle patients (11–13).

A total of 734 DNA samples from healthy subjects, exclusive-ly from north-western France (Brittany), were investigated tocontrol the frequency of the 18 SLC40A1 variations. None ofthe 18 mutated alleles was detected. Allele frequencies in thecontrol population were estimated at 0.00034 on Wald’smethod .

Identification of nine loss-of-function mutations: distinctionbetween SLC40A1 mislocation and intrinsic inabilityto export iron

The iron-exporting function of the 18 SLC40A1 variants wasassessed by first assaying the cytosolic iron storage protein fer-ritin, the expression of which is tightly regulated by the intracel-lular iron pool. HEK293T cells were transiently transfected withplasmids encoding SLC40A1-V5 fusion proteins in the presenceof holotransferrin (to increase background ferritin levels). Thep.Ala77Asp missense mutation, which significantly damagesferroportin structure and is known to prevent cell-surface local-ization (15), was used as a negative control. Figure 1A shows thatcells expressing the p.Ile180Thr, p.Thr230Asn, p.Glu248His,p.Met266Thr, p.Leu345Phe, p.Ileu351Val, p.Pro443Leu,p.Asp504Asn or p.Arg561Gly variant were iron-depleted,similar to those expressing wild-type (WT) SLC40A1, indi-cating that these nine variants did not alter the iron-exportingfunction of the cells. In contrast, cells expressing the p.Gly80Ser,p.Arg88Gly, p.Val162del, p.Asp157Gly, p.Asp157Tyr, p.Asp181Val, p.Leu233Pro, p.Gly490Asp or p.Gly490Ser variantwere iron-loaded, similar to those expressing the p.Ala77Asp

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Table 1. Description of the study sample: SLC40A1 variations, family relationships, biological and clinical data

Variation Familyrelationships

Patient Gender Agea

(years)Serumferritin(mg/l)

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AST(UI/l)

ALT(UI/l)

GGT(UI/l)

CRP(mg/l)

RBC(Tera/l)

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Alcoholconsumptionb

Clinicalobservations

p.Gly80Ser Index case FRA-05-005 F 49 702 24 22 29 5 12.1 91.0 AbstinentDaughter FRA-05-010 F 29 519 47 17 15 ,0.3 4.6 14.9 96.0 AbstinentSon FRA-05-004 M 27 1200 130 Abstinent Fatigue

p.Arg88Gly Index case FRA-03-002 F 41 4129 54 27 34 19 4.4 13.5 93.0 AbstinentBrother FRA-06-004 M 38 2840 52 24 4.5 13.9 88.4 Abstinent Fatigue

p.Asp157Gly Index case FRA-06-007 M 64 4459 35 32 38 25 3.3 5.3 15.7 90.0 Moderate ArthralgiaDaughter FRA-06-002 F 42 1590 20 17 28 19 0.6 4.4 13.0 91.0 Moderate Fatigue

p.Val162del Index case FRA-02-009 F 31 2578 35 19 22 15 ,2 3.7 11.4 95.7 ModerateBrother FRA-02-008 M 21 71.2 32 16 19 18 ,2 5.1 15.2 89.8 Moderate Fatigue,

arthralgiap.Val162del Index case FRA-02-014 F 45 977 14 25 23 17 2 4.6 13.1 91.0 Abstinent Fatigue,

arthralgiaDaughter FRA-02-012 F 18 1572 16 19 8 4 4.5 12.7 88.4 Abstinent

p.Val162del Index case FRA-05-008 M 19 2268 25 16.6 ModerateSister FRA-05-018 F 19 2198 18 Abstinent

p.Asp181Val Index case FRA-01-010 F 37 1679 24 14 13 16 3 5.0 12.8 90.3 Moderate FatigueSister FRA-01-007 F 25 1173 28 19 15 13 26.6 4.5 13.6 87.7 Moderate FatigueUncle FRA-01-009 M 57 8515 49 17 21 51 3 4.6 15.1 96.1 Moderate Fatigue,

hepatomegalyp.Leu233Pro Index case FRA-06-001 F 52 4293 76 46 91 35 14.4 96.0 .300 Moderate

Son FRA-06-005 M 23 1552 40p.Gly490Asp Index case FRA-06-006 F 54 3890 1.1 12.3 80.0 160

Daughter FRA-06-008 F 25 910 32p.Asp504Asn Index case FRA-05-002 M 60 1514 89 37 68 86 4.6 16.5 108.0 320 Moderate Hepatomegaly

Brother FRA-05-011 M 52 1605 102 36 113 4.9 15.7 93.0Unrelated

patientsp.Gly80Ser FRA-02-013 M 46 3109 35 4.8 14.1 94.0 Negligiblep.Arg88Gly FRA-06-003 F 77 4910 39 27 35 10 7 3.7 11.4 93.0 150 Abstinent Arthralgia,

hepatomegaly,type 2 diabetes

p.Asp157Gly FRA-02-007 F 66 .6000 53 Abstinent Fatiguep.Asp157Tyr FRA-02-016 M 64 2213 35 34 36 30 5.2 11.7 73.0 Moderatep.Val162del FRA-01-001 M 52 12178 37 29 28 23 5 6.5 17.0 77.6 Abstinent Fatigue,

hepatomegalyp.Ile180Thr FRA-05-009 M 37 604 30 4.5 13.9 90.5p.Asp181Val FRA-01-006 F 36 1973 29 22 20 27 6 4.4 13.2 89.5 Moderate Fatiguep.Thr230Asn FRA-02-003 M 64 1015 30 31 52 33 ,1 5.2 15.4 91.0 Moderate Arthralgiap.Gln248His FRA-03-001 M 35 1305 38 40 20 110 5 5.2 86.0 Moderate Fatiguep.Met266Thr FRA-05-012 M 40 1910 44 69 48 582 4.7 16.0 101.0 Heavyp.Leu345Phe FRA-04-009 F 53 620 41p.Ile351Val FRA-01-005 F 60 1293 32 15 14 17 3.6 3.8 11.8 91.7 Abstinent Fatiguep.Pro443Leu FRA-04-003 M 585 17 28 20 2 4.8 14.4 86.0 Abstinent Arthralgiap.Gly490Asp FRA-02-005 M 53 4377 22 32 34 51 3 4.5 14.5 94.0 Moderate Fatiguep.Gly490Asp FRA-05-006 F 32 1853 33 21 22 14.8 Moderate Fatigue,

arthralgiap.Gly490Asp FRA-05-013 F 49 2151 38 14 15 14 3.9 10.9 89.2 Abstinent Fatigue,

arthralgia,hepatomegaly

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loss-of-function mutation. Comparable ferritin levels werefound in cells transfected with the commercial pcDNA3.1-V5-His vector (no-SLC40A1).

Secondly, the cellular location of the nine iron-export defect-ive variants was examined. Cultured HEK293T cells were tran-siently cotransfected with plasmids encoding either full-lengthhuman ferroportin or human leukocyte antigen (HLA)-A, bothproteins being fused to a V5 epitope tag. Twenty-four hoursafter transfection, proteins were biotinylated, purified and ana-lyzed by western blot and densitometry. HLA-A was used ascontrol and standard for normalization, being a cell-surfaceprotein with no known role in iron metabolism. As expected,WT SLC40A1 expression resulted in cell-surface localiza-tion, whereas the p.Ala77Asp mutant showed markedlyreduced localization (Fig. 1B). Eight of the nine tested vari-ants (p.Val162del, p.Gly80Ser, p.Asp157Gly, p.Asp157Tyr,p.Asp181Val, p.Leu233Pro, p.Gly490Asp and p.Gly490Ser)were also found to cause ferroportin mislocalization; that is,60% or less of the protein was detected at the cell surface as com-pared with WT SLC40A1. Only the p.Arg88Gly variant properlyreached the plasma membrane (101%).

The impact of the ferroportin p.Arg88Gly variant on theiron-export function was specifically re-investigated usingradioactively labeled iron, as previously reported (15). Kineticsexperiments revealed that the p.Arg88Gly variant was not ableto export 55Fe in amounts comparable with WT ferroportin(Fig. 1C) but was more active than the p.Ala77Asp control;Student’s t-test highlighted significant differences betweenboth variants and WT ferroportin (Fig. 1D; P , 0.01 and0.001, respectively) and between the two variants (P , 0.001).

Identification of a new gain-of-function mutation:p.Asp540Asn amino acid substitution

To investigate whether the p.Ile180Thr, p.Thr230Asn, p.Glu248His,p.Met266Thr, p.Leu345Phe, p.Ile351Val, p.Pro443Leu,p.Asp504Asn and p.Arg561Gly variants could modify responseto hepcidin, transiently transfected HEK293T cells were initiallytreated with the full-length 25 amino acid mature hepcidinpeptide for 16 h, and cell-surface ferroportin levels wereassessed by western blot and densitometry. The p.Cys326Tyrmutant was used as positive control, completely ablating hepci-din binding and thus preventing ferroportin/hepcidin complexinternalization (22). Adding hepcidin to cells expressing WTSLC40A1 resulted in the disappearance of ferroportin from theplasma membrane, while the p.Cys326Tyr mutant inducedno change (Figs. 2A and B). The western blot pattern of thep.Asp504Asn variant, unlike the other eight tested variants,was similar to that of the p.Cys326Tyr mutant.

To confirm these results, radiolabeled hepcidin was added toferroportin-overexpressing cells. As expected, after 3 h incuba-tion with [125I]-hepcidin, the WT SLC40A1-V5 fusion proteinwas efficiently internalized, showing the highest level of intra-cellular radioactivity (Fig. 2C) whereas that of cells expressingthe p.Cys326Tyr mutant was indistinguishable from the levelin cells transfected with the commercial pcDNA3.1-V5-Hisvector (no-SLC40A1). Consistent with our previous findings, amarked decrease in [125I]-hepcidin accumulation was alsoobserved in cells expressing the p.Asp504Asn variant (P ,0.001), whereas the p.Ile180Thr, p.Thr230Asn, p.Glu248His,T

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p.Met266Thr, p.Leu345Phe, p.Ile351Val, p.Pro443Leu andp.Arg561Gly variants were found to increase intracellular[125I]-hepcidin in amounts similar to WT SLC40A1.

Structure–function analysis

We recently built a 3D model of ferroportin based on homologywith the crystal structure of a bacterial protein (EmrD) of themajor facilitator superfamily (MFS) (15). Combined with theresults presented in Figures 1 and 2, this 3D model provided anovel opportunity for structure–function analysis.

As illustrated in Figure 3 and Supplementary Material,Figure S1 and discussed in detail below, a clear distributioninto separate functional subclasses was observed:

† Loss-of-function mutations (Fig. 3A). Most loss-of-function mutations were located at the cytoplasmic endof the membrane-spanning domains.

– Mutations affecting intracellular trafficking and cell-surface localization. The effects of the p.Val162del,p.Asp157Tyr and p.Gly490Asp mutants (red in Fig. 3A)on the structure of ferroportin were previously predicted(15). However, a novel experimental MFS 3D structure(YajR), in an outward-facing conformation (23), recentlyprovided important new information on the probable roleof p.Asp157, which could not be anticipated from previous-ly observed conformations: p.Asp157, located in helix H4and highly conserved in the MSF family, is predictedto be involved in a charge-relay system with the othertwo highly conserved amino acids (p.Asp84 and p.Arg88)of the MSF-specific or conserved A motif (betweenhelices H2 and H3). This system, together with inter-domain charge-helix dipole interaction, probably plays acritical role in the stability of the outward-facing con-formation and in regulating conformational transition.The p.Asp157Tyr substitution might thus play a role inprotein stability, as demonstrated for YajR and otherMSF proteins (23). p.Gly80 is located at the very end ofhelix H2 (orange in Fig. 3A), at a position that is strictly con-served not only across ferroportin proteins from differentspecies but also across the various structures of MFS trans-porters (Supplementary Material, Figure S1). p.Gly80bends helix H2, which, like the irregularities observed inother cavity-lining helices, is probably important to thestructural flexibility of the protein and/or the tight helix–

helix packing observed in the outward-facing conformationof MSF transporters (23); pGly80Ser substitution maytherefore destabilize this local conformation. p.Leu233 isincluded in the long intracellular segment IS4 linkinghelices H6 and H7 (orange in Fig. 3A, not modeled). Itshould be noted that p.Leu233 constitutes part of a smallcluster including other hydrophobic amino acids, which istypical of an extended structure and may form a shortb-sheet with another predicted extended structure encom-passing p.Val160-p.Val161-p.Val162. This local structure,which is potentially disturbed by Leu233Pro substitution,may therefore contribute to a specific pore-entry architec-ture. Finally, p.Asp181 is located within helix H5, at a pos-ition that is highly conserved across ferroportin proteins(orange in Fig. 3A). The Asp side-chain is oriented nottoward the pore but toward the lipid bilayer, but is able toform a stabilizing salt bridge with the preceding p.Arg178(top inset in Fig. 3A), which is also highly conserved andmay form a hydrogen bond with p.Asn174; p.Asp181Valsubstitution may thus disturb the insertion of helix H5 inthe lipid bilayer.– Mutations preventing iron egress. p.Arg88, which is theonly amino acid for which substitution resulting in aloss-of-function mutation does not impair cellular localiza-tion of the protein, is located at the extreme N-terminus ofhelix H3, at the end of the IS2 loop-linking helices H2 andH3 (pink in Fig. 3A). Following our model, p.Arg88 isinvolved in a tightly linked network of interactions stabiliz-ing not only the IS2 loop between helices H2 and H3 (inter-action with H2 p.Trp82) but also the IS4 loop betweenhelices H6 and H7 (interactions with p.Met216 andp.Tyr220) (bottom inset in Fig. 3A). Thus, it is likely thatsubstitution of p.Arg88 by a glycine residue has no struc-tural impact, allowing this variant to be correctly foldedand addressed to the plasma membrane (Fig. 1B). Rather,the arginine residue may indirectly play a functional roleby maintaining the local conformation of loops on the intra-cellular side. Notably, p.Arg88 is located in the immediatevicinity of p.Ile152 and p.Asn174, at the intracellular end ofthe transmembrane domain, highlighting the importance ofthis region for iron egress (bottom inset Fig. 3A). Followingour model, the side-chain of p.Ile152, at the intracellularend of helix H4, is oriented toward the pore, whereas thatof p.Asn174, at the intracellular end of helix H5, pointstowards the lipid bilayer but may, like p.Asp181, form ahydrogen bond with p.Arg178.

Figure 1. Effect of ferroportin variants on intracellular iron accumulation, cell-surface expression and iron export. (A) HEK293T cells were transiently transfectedwith CMV-regulated plasmids containing no ferroportin sequence (no-SLC40A1), full-length WT human ferroportin cDNA or a mutated sequence in the presence of1 mg/ml holotransferrin. After 48 h, intracellular ferritin levels were determined by ELISA and normalized to the total protein concentration. Error bars represent theSD of three independent experiments (performed in triplicate). (B) HEK293T cells were transiently co-transfected with plasmids encoding either a V5-tagged ferro-portin protein (WT or variant) or a V5-tagged HLA-A protein. HLA-A was used as a control and as a standard for normalization, being a cell-surface protein with noknown role in iron metabolism. At 24 h after transfection, cell-surface proteins were selectively purified and analyzed by western blotting using a peroxidase-conjugated mouse anti-V5 antibody. Densitometric scans of SLC40A1 levels (normalized to HLA-A) are shown in the lower part of the figure. The error bars representthe standard deviation of three independent experiments. (C) HEK293T cells were grown in 20 mg/ml 55Fe-transferrin for 24 h before being washed and transientlytransfected with WT or mutated SLC40A1-V5 expression plasmids. After 15 h, cells were washed and then serum starved for up to 36 h. The 55Fe exported into thesupernatant was collected at various time points. Data are presented as percentage cellular radioactivity at time zero. Each point represents the mean SD; n ¼ 3 in eachgroup. The data are representative of three separate experiments (see below). (D) The graph shows the percentage of 55Fe collected from the supernatant of SLC40A1-transfected and no-SLC40A1-transfected cells at 36 h. The error bars represent the SD of three independent experiments (performed in triplicate; n ¼ 9). P-valueswere calculated using Student’s t-test. ∗P , 0.01 and ∗∗P , 0.001, compared with WT SLC40A1.




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† The newly identified p.Asp504Asn gain-of-function muta-

tion. p.Asp504 is located within a large loop (ES6) thatwas not modeled due to its divergence from the MFS

template structure (light green in Fig. 3A). However, itshould be noted that, as previously shown for p.His507(15), p.Asp504 is located at the entrance to the iron

Figure 2. Effect of ferroportin variants on hepcidin-induced internalization. (A and B) HEK293T cells were transiently co-transfected with CMV-regulated plasmidsexpressing HLA(A)-V5 and either WT SLC40A1-V5 or SLC40A1-V5 variants. At 16 h posttransfection, the cells were incubated in the presence or absence of hep-cidin (0.5 mM; Biochem) for 3 h. Plasma membrane proteins were purified and analyzed by western blotting and densitometry. The data are expressed as percentageferroportin in cells not treated by hepcidin, according to the formula 100 × (SLC40A1 – hepcidin/SLC40A1 + hepcidin). Error bars are the SD of three independentexperiments. (C) HEK293T cells expressing WT SLC40A1-V5 or variant SLC40A1-V5 were treated with [125I]-hepcidin and cell-associated radioactivity was mea-sured. Each bar represents the average of three independent experiments (performed in triplicate). Data were normalized to the amount of radioactivity bound to WTSLC40A1-V5-expressing cells, and the amount bound to untransfected cells was subtracted as background for each point. P-values were calculated using Student’st-test. ∗∗P , 0.001 and ∗∗∗P , 0.0001, compared with WT SLC40A1.




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Figure 3. Structural analysis of variants. The variants were localized within a ribbon representation (lateral view) of the previously reported 3D structural model ofhuman ferroportin (15). Non-modeled loops are indicated by dotted lines. (A) Loss-of-function (red, orange and pink) and gain-of-function (green) mutations. Pinkindicates amino acid positions at which variants did not impair membrane localization, in contrast to red and orange positions. The two insets focus on the environmentof p.Asp181/pAsn174 (top) and p.Arg88 (bottom). (B) Positions of amino acids showing neutral variations. The inset provides an orthogonal view of the 3D model,viewed from the extracellular side.




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transport channel, not far from the critical p.Cys326 residue(22, 24), which is consistent with the hepcidin resistancereported for both the p.Asp504Asn (Fig. 2) andp.His507Arg mutations (25).

† Neutral substitutions. None of the 8 SLC40A1 substitutionsthat apparently do not alter the ferroportin iron exportfunction or down-regulation by hepcidin are predictedto cause structural disturbance. As shown in Figure 3Band Supplementary Material, Figure S1, the p.Thr230,p.Glu248, p.Met266, p.Pro443 and p.Arg561 residues areincluded in loops linking the transmembrane helices or inthe C-terminal tail (p.Arg561) at some distance from theferroportin pore (light blue in Fig. 3A). These positionsare therefore likely not critical for the folding or ironexport function of ferroportin. The other three residues(p.Ile180, p.Leu345 and p.Ile351) are located on the hydro-phobic side of transmembrane helices, oriented toward thelipid region (blue on Fig. 3B). The hydrophobic characteris, however, conserved at these three positions for the ob-served mutations, which should therefore not be structurallydamaging.

Combined use of in silico and in vitro splicing assays forinterpretation of the 8 SLC40A1 variations withno impact at protein level

Analysis was extended by assessing the effects of thep.Ile180Thr, p.Thr230Asn, p.Glu248His, p.Met266Thr,p.Leu345Phe, p.Ile351Val, p.Pro443Leu and p.Arg561Gly var-iations on pre-mRNA splicing. Different algorithms andSLC40A1 minigenes were used; no abnormalities were found(see Supplementary Material, Results and Fig. S2). Results of

in vitro investigations for the 18 ferroportin variants are summar-ized in Table 2.

Genotype–phenotype correlations

Familial recurrence of iron overload is an alternative meansof assessing the clinical relevance of SLC40A1 variations. Var-iations detected in at least two relatives with hyperferritinemia(defined as serum ferritin .300 mg/l in males and .200 mg/lin females) were investigated: i.e. 8 of the 10 SLC40A1 variationswith clear functional impact; exceptions were the p.Asp157Tyrand p.Gly490Ser loss-of-function mutations (Table 1). Nofamily discordances were detected, apart from the well-knownp.Val162del mutation, which was associated with distinct pheno-types in one sib pair: the index case (FRA-02-009) displayed sig-nificant hyperferritinemia (2578 mg/l), while her younger brother(FRA-02-008) showed normal iron values.

The p.Asp504Asn gain-of-function mutation was identifiedin two brothers with hyperferritinemia (.1500 mg/l) and wasfound to increase transferrin saturation levels (.85%). Abdom-inal MRI was available for the index case (SupplementaryMaterial, Figure S3) and showed a marked reduction in signal in-tensity in the liver, consistent with iron overload (HIC estimatedat 320 mmol/g). No signal reduction was observed in spleen orbone marrow.

In total, 34 patients harboring a loss-of-function mutationwere identified: 14 males and 20 females. In most of thesepatients, hyperferritinemia contrasted with normal or low trans-ferrin saturation levels. Serum ferritin concentrations correlatedsignificantly and positively with age in both males and females(Supplementary Material, Fig. S4). Only five patients (two malesand three females) displayed .45% transferrin saturation. It isnoteworthy that four of these patients presented particularly highserum ferritin concentrations (hyperferritinemia .4000 mg/l),and that these four patients were among the oldest: the twomales were diagnosed after 42 years of age, which was themedian age in the group of 14 males carrying a loss-of-functionmutation, and the two females after 52 years of age, which wasthe 75th age percentile in the group of 20 females carrying aloss-of-function mutation. MRI, performed in the 52-year-oldfemale positive for the p.Leu233Pro mutation, found mixediron overload with evidence of iron deposition in liver (HIC.300 mmol/g), spleen and bone marrow.

All of the neutral SLC40A1 variations came from a singlepatient. All of these patients presented moderate-to-high serumferritin concentrations (620–1910 mg/l) and normal transferrinsaturation levels. Whether hyperferritinemia was accompaniedby iron overload was unknown, as none of the patients underwentliver biopsy or MRI, and no information was available ontherapeutic phlebotomy. One 40-year-old man, positive forthe new SLC40A1 p.Met266Thr substitution, declared heavyalcohol consumption and at diagnosis displayed a mean cor-puscular volume of 101 fl, a gamma-glutamyl transpeptidaselevel of 582 IU/l and a ratio of serum aspartate aminotransferaseto serum alanine aminotransferase of 1.4. No other obviouscause of acquired hyperferritinemia was found in patients withpolymorphic SLC40A1 variants. Interestingly, males who har-bored a neutral variant displayed significantly lower ferritinvalues than those who harbored a loss-of-function mutation(P ¼ 0.0054; Fig. 4).

Table 2. Classification of the 18 ferroportin substitutions into loss-of-functionversus gain-of-function mutations and neutral variants

Variation Functional categoryLoss of function Gain of function Neutral

Abnormalcell-surfacerouting

Reducedironexport

Total resistance tohepcidininhibition

p.Gly80Ser x xp.Arg88Gly xp.Asp157Gly x xp.Asp157Tyr x xp.Val162del x xp.Ile180Thr xp.Asp181Val x xp.Thr230Asn xp.Leu233Pro x xp.Gln248His xp.Met266Thr xp.Leu345Phe xp.Ile351Val xp.Pro443Leu xp.Gly490Asp x xp.Gly490Ser x xp.Asp504Asn xp.Arg561Gly x




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DISCUSSION

Lack of penetrance and the influence of gender and co-morbidfactors have been postulated to explain part of the clinical hetero-geneity attributed to SLC40A1 variations (1, 2), in addition to thewell-recognized dichotomy between loss-of-function andgain-of-function mutations (1, 2). Although it seems reasonablethat SLC40A1 mutations may not always be phenotype determi-nants and that other factors modulate the severity of iron over-load, it is important to bear in mind that evidence for thecausality of many non-synonymous changes and small deletionsis at best circumstantial. Regardless of the conditions underlyingphenotypic heterogeneity, there is an unmet need for more nar-rowly defined phenotypes and more effective functional charac-terization strategies.

The present study classified 9 SLC40A1 variants as loss-of-function mutations (Fig. 1). With few exceptions, the diseaseassociated with these mutations involved isolated hyperferriti-nemia (Table 1). In spite of markedly elevated ferritin levels inthe majority of patients (hyperferritinemia.10 001 000 mg/lin 28 of 34 patients), the clinical manifestations were rathermoderate. They included: fatigue (n ¼ 13), joint pain (n ¼ 8),hepatomegaly (n ¼ 1) and type 2 diabetes (n ¼ 1). Hepaticiron overload was found on MRI in five patients, with nosign of liver fibrosis. Serum ferritin concentrations increasedwith age, independently of gender (Supplementary Material,Figure S4). Interestingly, the patients showing elevated trans-ferrin saturation were among the oldest. MRI demonstrated amixed pattern of iron accumulation in parenchymal and non-parenchymal cells in a 52-year-old woman with serum ferritin.4000 mg/l and 76% transferrin saturation. Overall, these find-ings support the idea that hemochromatosis type 4A consists inprogressive iron overload with biochemical and histologicalhallmarks that are clearly different from those of classical HFE-hemochromatosis (4, 13).

Particular attention was paid to the p.Arg88Gly amino acidsubstitution: of the 10 loss-of-function mutations (includingthe p.Ala77Asp control), only the p.Arg88Gly ferroportinmutant was defective for iron egress while being normallyaddressed to the cell surface (Fig. 1B–D). In the literature,such a functional defect has been reported only for thep.Ile152Phe and p.Asn174Ile clinical mutations (15, 26, 27).

Accordingly, the present structural predictions suggest thatp.Arg88 is located in the immediate vicinity of the p.Ile152 andp.Asn174 residues in a region that might be directly involvedin iron egress (Fig. 3B). Deciphering the precise function ofthe p.Arg88, p.Ile152, p.Asp174 and neighboring residues mayimprove understanding of the ferroportin iron export mechanism.

Apart from the three cases in the present study (Table 1), thep.Arg88Gly mutation was also reported in four French patients:three family members and one apparently unrelated individual(1, 28). It was systematically associated with classical ferropor-tin disease (hemochromatosis type 4A), although there were po-tential confounding factors (metabolic syndrome, excessivealcohol intake) in two patients (11). p.Arg88Gly substitutionshould thus be considered recurrent in the French population.Another mutation, causing replacement of glycine by the acidicpolar residue aspartic acid at position 490 (p.Gly490Asp),appears to be particularly frequent in patients originatingfrom the Reunion Island (nine patients in the present study;Table 1). It would be interesting to perform a population geneticsstudy on this French island located in the Indian Ocean, which islikely to represent a genetic isolate.

Furthermore, a typical hemochromatosis phenotype wasfound in two brothers carrying the new p.Asp504Asn gain-of-function mutation (Table 1; Supplementary Material, Fig. S3).In vitro assessment suggested total resistance to hepcidin inhib-ition (Fig. 2), as previously demonstrated for the p.Cys326Tyrand p.Cys326Ser disease-causing mutations (22, 29). Accordingto the present 3D model of ferroportin, aspartic acid 504 is part ofan extracellular loop (ES6) at the entrance to the iron exportchannel (Fig. 3A). We previously reported that this extracellularloop could not be accurately modeled due to its size and relativedivergence from the MFS structure template (15). Consequently,the distance between aspartic acid 504 and cysteine 326, whichhas proved critical for the docking of hepcidin with ferroportin(22, 24), is difficult to estimate. Similar clinical and functionalobservations were, however, made by Mayr et al. reportingp.His507Arg substitution in a third-generation Caucasian pedi-gree (25); testing hepcidin activity in presence of synthetic ferro-portin peptides, they argued that the strongly conserved histidine507 residue was directly involved in the interaction of hepcidinwith ferroportin. A recent study on small peptides mimickinghepcidin activity showed that the hepcidin-binding domain(HBD) of ferroportin is larger than previously thought (24),extending to amino acids adjacent to the region betweenglycine 323 and serine 343 (30); the question of whether it alsoextends to residues outside the region between helix 7 andhelix 8 and, particularly, to the extracellular loop containingthe p.Arg504 and p.His507 residues (ES6), clearly remains open.

Complementary in vitro and in silico studies demonstratedthat eight missense variations had no noticeable effects on ferro-portin function and its interaction with hepcidin (Figs. 1–3) oron SLC40A1 pre-mRNA splicing (Supplementary Material,Figure S2). These non-synonymous ferroportin variants, identi-fied in eight single patients showing moderate increases in serumferritin and normal transferrin saturation (Table 1; Fig. 4) mostlikely corresponded to neutral polymorphisms. It should beborne in mind in this regard that elevated serum ferritin isfrequent in clinical practice and can be caused by a variety ofconditions that do not necessarily result in excess iron. Further-more, in many patients, hyperferritinemia remains unexplained

Figure 4. Serum ferritin levels in male carriers of a loss-of-function mutation or anon-functional polymorphism at the SLC40A1 locus. Box and whisker plotsshowing median and interquartile range in the box (50th, 25th and 75th percen-tiles) and minimum to maximum range (whiskers). P-value was calculated usingMood’s median test – Pearson’s x2-test.




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after assessment (31). The present study excluded mutations inthe other four hemochromatosis genes (HFE, HJV, HAMP andTFR2) and in the L-ferritin-encoding gene, which has recentlybeen associated with elevated levels of glycosylated serum fer-ritin without iron overload (32). On the other hand, alcoholabuse emerged as the most probable cause of hyperferritinemiain the patient carrying the new p.Met266Thr substitution.C-reactive protein levels were found to be normal in five otherpatients. There was not enough information to ascertain meta-bolic abnormalities or other obvious underlying causes of hyper-ferritinemia.

Another possible explanation, however, is that hypomorphicmutations were overlooked under the present experimental con-ditions. A supporting argument might be that the eight missensemutations are infrequent in the French population, so that thelikelihood of any of them contributing to false diagnosis ofhyperferritinemia can be expected to be low. On the otherhand, the present control population was representative only ofpatients originating from the north-western part of France,likely underestimating the frequency of SLC40A1 variants inpatients with different genetic histories; e.g. p.Glu248His sub-stitution has a minor allele frequency of 5.7–6.6% in subjectsof African-American ancestry (Supplementary Material, Table S1)and is thought to be even more frequent in native Africans (33).

It should be noted that the common p.Glu248His substitutionis associated with a tendency for elevated serum ferritin invarious African-American and native African groups (34–37),whereas the risk of iron overload associated with p.Glu248Hishas not been established (33, 36). Consistently with both thepresent observations and previous in vitro findings (18, 29, 38,39), it has been suggested that the p.Glu248His polymorphismcauses iron overload only when associated with other geneticand/or environmental factors (33); the p.Glu248His substitutionis thereby supposed to have slight functional impact and not to bea major genetic determinant of iron excess. Whether or not thisapplies to the apparently less frequent variants identified in thepresent study, classified as neutral, will be hard to determine.

In summary, the present findings show that the functionalconsequences of missense mutations in the gene-encoding ferro-portin are crucial for interpreting the observed clinical hetero-geneity, further highlighting the need to clarify the variouscausal hypotheses found in the literature. The present studyalso demonstrates that interpreting missense mutations at theSLC40A1 locus will increase knowledge of structure–functionrelationships in ferroportin.

MATERIALS AND METHODS

Patients

The present study involved six laboratories in the Frenchnetwork for the molecular diagnosis of inherited iron overloaddisorders (University Hospitals of Amiens, Paris-Bichat,Bordeaux, Brest, Creteil and Marseille). Biological, clinical,socio-demographic, lifestyle and molecular genetic data werecollected for all consenting patients with suggested moleculardiagnosis of ferroportin disease or hemochromatosis type 4. Phe-notypes were assessed retrospectively, based on a questionnairecompleted by the physicians who had initially ordered moleculartesting. When available, familial and abdominal MRI data were

also collected. All 44 patients with complete molecular geneticdata (i.e. Sanger sequencing of the HFE, HJV, HAMP, TFR2,SLC40A1 and FTL genes) were recruited over the period fromJuly 2011 to July 2013. Informed consent for molecularstudies was obtained from all patients, in accordance with theDeclaration of Helsinki, and, in line with French ethical guide-lines, the Clinical Research Ethics Committee of the UniversityHospital of Brest approved the study on October 25, 2010.

In vitro experiments and molecular modeling

In vitro investigations and structural predictions were performedas previously described (36–39) and are presented in detail in theSupplementary Material, Documents.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

Conflict of Interest statement. None declared.

FUNDING

This work was supported by the Programme Hospitalier de Re-cherche Clinique French Hospital Clinical Research Program(PHRC National 2009; Brest University Hospital – UF0857).The authors thank Dr Virginie Scotet for statistical support,and Dr Jian-Min Chen and Pr. Cedric Le Marechal for criticalreading.

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