HFE Protein Research Articles

Expression of hereditary hemochromatosis C282Y HFE protein in HEK293 cells activates specific endoplasmic reticulum stress responses.

BackgroundHereditary Hemochromatosis (HH) is a genetic disease associated with iron overload, in which individuals homozygous for the mutant C282Y HFE associated allele are at risk for the development of a range of disorders particularly liver disease. Conformational diseases are a class of disorders associated with the expression of misfolded protein. HFE C282Y is a mutant protein that does not fold correctly and consequently is retained in the Endoplasmic Reticulum (ER). In this context, we sought to identify ER stress signals associated with mutant C282Y HFE protein expression, which may have a role in the molecular pathogenesis of HH.ResultsVector constructs of Wild type HFE and Mutant C282Y HFE were made and transfected into HEK293 cell lines. We have shown that expression of C282Y HFE protein triggers both an unfolded protein response (UPR), as revealed by the increased GRP78, ATF6 and CHOP expression, and an ER overload response (EOR), as indicated by NF-κB activation. Furthermore, C282Y HFE protein induced apoptotic responses associated with activation of ER stress. Inhibition studies demonstrated that tauroursodeoxycholic acid, an endogenous bile acid, downregulates these events. Finally, we found that the co-existence of both C282Y HFE and Z alpha 1-antitrypsin protein (the protein associated with the liver disease of Z alpha 1-antitrypsin deficiency) expression on ER stress responses acted as potential disease modifiers with respect to each other.ConclusionOur novel observations suggest that both the ER overload response (EOR) and the unfolded protein response (UPR) are activated by mutant C282Y HFE protein.

The Hereditary Hemochromatosis protein HFE and its chaperone β2-microglobulin localise predominantly to the endosomal-recycling compartment

Hereditary Hemochromatosis is an iron overload disease most frequently associated with mutations in the HFE gene. While clinical studies of the disease have received extensive attention by various groups, the localisation, trafficking and function of the HFE protein, and its chaperone β2-microglobulin (β2M), require further investigation. In this study, we present data on the cellular localisation of HFE and its clinically relevant mutants in HuTu 80 cells. We find by confocal microscopy that HFE localises to the endosomal-recycling compartment (ERC), with minimal localisation to sorting or late endosomes. Interestingly, we also demonstrate that β2M localises to the ERC where it co-localises with HFE. We find that exogenous expression of HFE results in enhanced β2M cellular levels and that β2M is necessary for cell surface expression of HFE. Finally, we have analysed the functional effects of exogenous expression of HFE and β2M on transferrin binding to the cell surface. In summary, our study sheds light on the localisation and functional effects of the HFE and its chaperone protein β2M.

A novel homozygous frameshift deletion c.471del of HFE associated with hemochromatosis

A 47-year-old white male patient who manifested biochemical evidence of iron overload was found not to be a carrier of the three most common mutations, C282Y, H63D and S65C, of the HFE gene. Sequencing of the patient's entire HFE-coding region revealed a presence of a previously undescribed frameshift deletion c.471del in exon 3 resulting in a premature termination of a nonsense HFE protein. Interestingly, the patient was a homozygous carrier of this novel mutation and his hemochromatosis phenotype can be explained by the fact that he has no intact HFE protein. To the best of our knowledge, this is the first description of a complete loss of function of the HFE gene because of a homozygous mutation. The patient's son was found to be a heterozygous carrier of the mutation and has so far exhibited no indications of iron overload. Similarly, A*02-B*40/A*02-B*40 homozygous human leukocyte antigen (HLA) genotype was determined in the patient and heterozygous A*02-B*40/A*03-B*35 HLA genotype in his son. Thus, the novel HFE frameshift deletion c.471del was linked to the HLA-A*02-B*40 haplotype.

Transcriptional repression of the HFE gene promoter by IRF‐1

Hereditary hemochromatosis (HH) due to HFE protein deficiency is an autosomal recessive disorder, relatively common among Caucasians, that causes excessive iron accumulation in several tissues leading to their damage. Characterization of the HFE gene promoter is important for comprehending iron metabolism and homeostasis. To this end, we cloned the 1.7 kb promoter fragment into pGL3 vector (HG1, −1680) and constructed a panel of deletion mutants: HG2 (−875), HG3 (−580) and HG4 (−210). TESS analysis implicated the presence of sites for Sp1 at −631 and −442, AP-1 at −68 and IRF-1 at −580 from the transcription start site. In comparison to pGL3 alone (1-fold), expression of luciferase reporter in HG1 and HG2 in HEK293T cells exhibited a strong reduction (0.002-fold), while HG4 was activated (13-fold). Despite both HG1 and HG2 being unaffected by co-expressed MEKK-1 and cJun/Fos, HG4 showed significant activation (65-fold), indicating that the AP-1 site is functional, though silenced in HG1 and HG2. The Sp1 site(s) in HG2 appeared to be responsive (0.16-fold) to exogenous Sp1, but were also repressed. Absence of the IRF-1 site in HG3 restores a 4-fold activation. EMSA analysis of the IRF-1 site confirms the interaction of nuclear proteins with this sequence. These results provide evidence that the IRF-1 site functions as a negative element and strongly down-regulates the HFE gene promoter. NIH funds support this study.

Iron-independent specific protein expression pattern in the liver of HFE-deficient mice

Hereditary hemochromatosis type I is an autosomal-recessive iron overload disease associated with a mutation in HFE gene. The most common mutation, C282Y, disrupts the disulfide bond necessary for the association of HFE with beta-2-microglobulin and abrogates cell surface HFE expression. HFE-deficient mice develop iron overload indicating a central role of the protein in the pathogenesis of hereditary hemochromatosis type I. However, despite significant effort, the role of the HFE protein in iron metabolism is still unknown. To shed a light on the molecular mechanism of HFE-related hemochromatosis we studied protein expression changes elicited by HFE-deficiency in the liver which is the organ critical for the regulation of iron metabolism. We undertook a proteomic study comparing protein expression in the liver of HFE deficient mice with control animals. We compared HFE-deficient animals with control animals with identical iron levels obtained by dietary treatment to identify changes specific to HFE deficiency rather than iron loading. We found 11 proteins that were differentially expressed in the HFE-deficient liver using two-dimensional electrophoresis and mass spectrometry identification. Of particular interest were urinary proteins 1, 2 and 6, glutathione- S-transferase P1, selenium binding protein 2, sarcosine dehydrogenase and thioredoxin-like protein 2. Our data suggest possible involvement of lipocalins, TNF-alpha signaling and PPAR alpha regulatory pathway in the pathogenesis of hereditary hemochromatosis and suggest future targeted research addressing the roles of the identified candidate genes in the molecular mechanism of hereditary hemochromatosis.

Hemochromatosis

This review acknowledges the recent and dramatic advancement in the field of hemochromatosis and highlights the surprising analogies with a prototypic endocrine disease, diabetes. The term hemochromatosis should refer to a unique clinicopathologic subset of iron-overload syndromes that currently includes the disorder related to the C282Y homozygote mutation of the hemochromatosis protein HFE (by far the most common form of hemochromatosis) and the rare disorders more recently attributed to the loss of transferrin receptor 2, HAMP (hepcidin antimicrobial peptide), or hemojuvelin or to certain ferroportin mutations. The defining characteristic of this subset is failure to prevent unneeded iron from entering the circulatory pool as a result of genetic changes compromising the synthesis or activity of hepcidin, the iron hormone. Like diabetes, hemochromatosis results from the complex, nonlinear interaction between genetic and acquired factors. Depending on the underlying mutation, the coinheritance of modifier genes, the presence of nongenetic hepcidin inhibitors, and other host-related factors, the clinical manifestation may vary from simple biochemical abnormalities to severe multiorgan disease. The recognition of the endocrine nature of hemochromatosis suggests intriguing possibilities for new and more effective approaches to diagnosis and treatment.

Consequences of expressing mutants of the hemochromatosis gene ( HFE ) into a human neuronal cell line lacking endogenous HFE

HFE mutations have traditionally been associated with the iron overload disorder known as hemochromatosis. Recently, it has become clear that the two most common mutations in the HFE gene, H63D and C282Y, may be genetic modifiers for risk of neurodegenerative disorders and cancer, respectively. We developed human neuroblastoma stable cell lines that express either wild-type (WT) or mutant HFE to determine the cellular consequences of the mutant forms of HFE. The presence of the C282Y mutation was associated with relatively higher labile iron pool and iron regulatory protein activity than WT or H63D HFE. Targeted gene arrays revealed that the signal transduction pathway was up-regulated in the C282Y cells. H63D cells had higher levels of lipid peroxidation, protein oxidation, and lower mitochondrial membrane potential, suggesting higher baseline stress. This cell line was also more vulnerable to exposure to oxidative stress agents and more responsive to iron chelation than the C282Y cells. These data demonstrate that the different mutations in the HFE gene have unique effects on the cells and provide insights into how the different mutations may have different clinical consequences. The results also raise multiple novel questions for future study about the function of the HFE protein.

Genetic Study of Variation in Normal Mouse Iron Homeostasis Reveals Ceruloplasmin as an HFE-Hemochromatosis Modifier Gene

Genetic hemochromatosis is one of the most common genetic disorders, with progressive tissue iron overload leading to severe clinical complications. In Northern European populations, genetic hemochromatosis is usually caused by homozygosity for the C282Y mutation in the HFE protein. However, penetrance of this mutation is incomplete, suggesting that other genetic and environmental factors contribute to its differential biologic or clinical expression. To identify genes modifying iron homeostasis, we screened the 27 recombinant congenic strains of the C3H/DiSnA-C57BL/10ScSnA/Dem series for tissue and serum iron indices and genotyped 18 microsatellite markers in (C3H/DiSnA x HcB-2) F2 hybrid mice. We identified 1 locus encompassing the Ceruloplasmin (Cp) gene with a strong linkage with liver iron, serum iron, and transferrin levels but not with spleen iron. Sequencing of Cp showed an R435X nonsense mutation in exon 7 in C3H/DiSnA mice. To evaluate whether Cp might act as a modifier gene of genetic hemochromatosis, we intercrossed C3H Hfe(-/-) and C3HDiSnA Cp(R435X/R435X) mice. As expected, we found that double-mutant mice deposited more iron in the liver than mice defective for either one or both genes. In contrast, Hfe(-/-) x Cp(R435/R435X) or Cp(R435X/R435X) x Hfe(+/-) showed 30% decrease in liver iron when compared with single mutant mice. This study highlights the existence of complex interactions between Cp and HFE and represents the first example of a modifier gene with a protective effect, in which heterozygosity reduces the iron load in the context of HFE deficiency.

Expression and Polarized Localization of the Hemochromatosis Gene Product HFE in Retinal Pigment Epithelium

Hereditary hemochromatosis is an autosomal recessive disorder of iron overload leading to oxidative stress. Mutations in HFE are responsible for approximately 90% of cases of this disease. HFE is the principal regulator of iron homeostasis, and the process involves interaction with transferrin receptor (TfR)-1, transferrin receptor (TfR)-2, and beta2-microglobulin (beta2M). Expression of HFE has not been investigated in the retina. In the present study, the expression of HFE and the HFE-interacting proteins TfR1, TfR2, and beta2M were analyzed in mouse retina. RT-PCR was used to detect the expression of HFE mRNA in neural retina and the RPE-eyecup. Expression of HFE in intact retina was investigated by in situ hybridization, immunofluorescence, and immunogold electron microscopy. Expression of HFE-interacting proteins was also analyzed using similar techniques. RT-PCR showed predominant expression of HFE mRNA in the RPE-eyecup. In situ hybridization in intact retina revealed that HFE mRNA is expressed almost exclusively in RPE Immunofluorescence and immunogold electron microscopy showed that HFE protein was specifically associated with the basolateral membrane of RPE. Expression of the HFE-interacting proteins TfR1, TfR2, and beta2M was also evident in the retina. This is the first report on the expression of HFE in the retina. The specific localization of HFE and its interacting proteins, TfR1 and TfR2, at the basolateral membrane of RPE is relevant to the regulation of iron homeostasis in this cell. Patients with hemochromatosis may have impairment of iron homeostasis in RPE, potentially contributing to age-related RPE dysfunction and retinal degeneration.

Hereditary Hemochromatosis Protein, HFE, Interaction with Transferrin Receptor 2 Suggests a Molecular Mechanism for Mammalian Iron Sensing

HFE and transferrin receptor 2 (TFR2) are membrane proteins integral to mammalian iron homeostasis and associated with human hereditary hemochromatosis. Here we demonstrate that HFE and TFR2 interact in cells, that this interaction is not abrogated by disease-associated mutations of HFE and TFR2, and that TFR2 competes with TFR1 for binding to HFE. We propose a new model for the mechanism of iron status sensing that results in the regulation of iron homeostasis.

Expression of the hemochromatosis gene modulates the cytotoxicity of doxorubicin in breast cancer cells

The antineoplastic agent doxorubicin inhibits cell growth through mechanisms that include an interaction with iron, resulting in the generation of cytotoxic reactive oxygen species (ROS). Prior studies have shown that the wild-type hemochromatosis gene (wt HFE) may downregulate iron uptake and alter iron homeostasis in cells. We therefore tested the hypothesis that expression of wt HFE would affect the cytotoxicity of doxorubicin. Human breast cancer MCF-7 cells were transfected with an expression plasmid for a FLAG-tagged wt HFE gene [fwtHFE(+) cells], to examine the impact of wt HFE expression on doxorubicin-induced apoptosis. Our results show that, in MCF-7 cells, fwtHFE expression resulted in a reduction in cellular iron uptake and a decrease in the growth inhibitory effects of doxorubicin. Two micromolar doxorubicin inhibited the growth of fwtHFE(+) and fwtHFE(-) MCF-7 cells by 34% and 61%, respectively. In parallel, doxorubicin induced caspase-3-like activity in fwtHFE(-) cells, but not in fwtHFE(+) cells. On analysis with a DCF fluorescence assay, ROS could be detected in fwtHFE(-) cells but not in fwtHFE(+) cells exposed to doxorubicin. Western blot analysis of breast biopsy samples from patients revealed immunoreactive HFE and transferrin receptor proteins in both normal and malignant breast tissues. Our studies suggest that HFE expression and its consequent effect on cellular iron homeostasis may modulate doxorubicin-induced oxidative stress and apoptosis in breast cancer cells. Further investigation is warranted to determine whether HFE expression in tumor cells impacts on the clinical efficacy of doxorubicin.

Identification of the Segments of the Mouse Transferrin Receptor 1 Required for Mouse Mammary Tumor Virus Infection

Most enveloped viruses enter cells through binding of virion surface envelope proteins to receptors found on the plasma membrane of the cell. The beta retrovirus mouse mammary tumor virus (MMTV) uses transferrin receptor 1 (TfR1) to enter cells in a pH-dependent mechanism, probably co-trafficking with TfR1 to an acidic compartment where virus entry occurs. We have shown here that, although mouse and rat TfR1 function as entry receptors, cat, dog, hamster, or human TfR1s do not support MMTV infection. We also demonstrated that MMTV entry is independent of transferrin, iron, and the TfR1 cofactor hereditary hematochromatosis HFE protein. Using chimeric mouse/human hybrid TfR1 constructs, we determined the site of interaction with MMTV and found that it maps to two segments physically disparate from the TfR and HFE binding sites. Thus, MMTV has apparently evolved to enter cells independently of the iron status of the host.

Characterization of Hfe mutations in brain tumor cells

Mutants of Hfe, a gene that regulates cellular iron uptake, have been associated with an increase in hepatocarcinoma and neurodegenerative diseases. In this study we characterize the expression and effect of the Hfe mutations, H63D and C282Y, in brain tumor cells and tissues to understand the role of the Hfe in brain tumor carcinogenesis. We determined the expression level and genotype of Hfe in brain tumor tissue and found that the majority of high-grade tumors expressed Hfe protein. The mutation rate for Hfe, specifically the C282Y mutation, is higher in brain tumor patients compared to the general population. We also generated stable human neuroblastoma cells carrying the wild type (WT) and mutant Hfe genes to determine the response of Hfe mutant cells to chemotherapeutic agents and gamma irradiation. The cells that carry the C282Y mutation proliferate at a much higher rate than cells carrying the H63D or WT Hfe. The C282Y cells fail to differentiate when exposed to retinoic acid or cAMP. Temodar is not cytotoxic to these cells, but they are vulnerable to BCNU. However, when H63D or WT Hfe cells are treated with these agents normal differentiation and susceptibility is observed. In addition, the C282Y cells are resistant to gamma irradiation while the WT or H63D cells are sensitive to gamma irradiation. Targeted gene arrays reveal that several signaling pathway and drug metabolism genes were changed in chemically treated C282Y cells. In conclusion, Hfe protein is strongly expressed in brain tumor cells, and the cellular data indicate that the C282Y mutation significantly alters gene expression, proliferation, differentiation, and response to anti-tumor drugs.

Release of the Soluble Transferrin Receptor Is Directly Regulated by Binding of Its Ligand Ferritransferrin

The human transferrin receptor (TfR) is shed by an integral metalloprotease releasing a soluble form (sTfR) into serum. The sTfR reflects the iron demand of the body and is postulated as a regulator of iron homeostasis via binding to the hereditary hemochromatosis protein HFE. To study the role of transferrin in this process, we investigated TfR shedding in HL60 cells and TfR-deficient Chinese hamster ovary cells transfected with human TfR. Independent of TfR expression, sTfR release decreases with increasing ferritransferrin concentrations, whereas apo-transferrin exhibits no inhibitory effect. To investigate the underlying mechanism, we generated several TfR mutants with different binding affinities for transferrin. Shedding of TfR mutants in transfected cells correlates exactly with their binding affinity, implying that the effect of ferritransferrin on TfR shedding is mediated by a direct molecular interaction. Analysis of sTfR release from purified microsomal membranes revealed that the regulation is independent from intracellular trafficking or cellular signaling events. Our results clearly demonstrated that sTfR does not only reflect the iron demand of the cells but also the iron availability in the bloodstream, mirrored by iron saturation of transferrin, corroborating the important potential function of sTfR as a regulator of iron homeostasis.

Structural Allostery and Binding of the Transferrin·Receptor Complex

The structural allostery and binding interface for the human serum transferrin (Tf)*transferrin receptor (TfR) complex were identified using radiolytic footprinting and mass spectrometry. We have determined previously that the transferrin C-lobe binds to the receptor helical domain. In this study we examined the binding interactions of full-length transferrin with receptor and compared these data with a model of the complex derived from cryoelectron microscopy (cryo-EM) reconstructions (Cheng, Y., Zak, O., Aisen, P., Harrison, S. C. & Walz, T. (2004) Structure of the human transferrin receptor.transferrin complex. Cell 116, 565-576). The footprinting results provide the following novel conclusions. First, we report characteristic oxidations of acidic residues in the C-lobe of native Tf and basic residues in the helical domain of TfR that were suppressed as a function of complex formation; this confirms ionic interactions between these protein segments as predicted by cryo-EM data and demonstrates a novel method for detecting ion pair interactions in the formation of macromolecular complexes. Second, the specific side-chain interactions between the C-lobe and N-lobe of transferrin and the corresponding interactions sites on the transferrin receptor predicted from cryo-EM were confirmed in solution. Last, the footprinting data revealed allosteric movements of the iron binding C- and N-lobes of Tf that sequester iron as a function of complex formation; these structural changes promote tighter binding of the metal ion and facilitate efficient ion transport during endocytosis.

Intron 2 [IVS2, T-C +4] HFE gene mutation associated with S65C causes alternative RNA splicing and is responsible for iron overload

A patient with congenital liver fibrosis revealed a high transferrin saturation index and iron overload on liver biopsy. He did not carry the most frequent HFE mutations: C282Y or H63D. Heterozygosity was detected for S65C. Unknown HFE mutations were also sought using a combined denaturing high performance liquid chromatography (DHPLC)/direct sequence approach and another point mutation, a transition T-C (nt 4910), at the fourth base of the donor splice site of intron 2 [HFE, intervening sequence (IVS) 2, T-C +4] was found. Family screening revealed that a daughter carried both S65C and [IVS2, T-C +4]. : The existence in our proband of a partly-altered HFE protein in the region encoded by exon 2 might be responsible for the histologically-demonstrated iron overload.

HIV-1 Nef down-regulates the hemochromatosis protein HFE, manipulating cellular iron homeostasis

The multifunctional Nef protein of HIV-1 is important for the progression to AIDS. One action of Nef is to down-regulate surface MHC I molecules, helping infected cells to evade immunity. We found that Nef also down-regulates the macrophage-expressed MHC 1b protein HFE, which regulates iron homeostasis and is mutated in the iron-overloading disorder hemochromatosis. In model cell lines, Nef reroutes HFE to a perinuclear structure that overlaps the trans-Golgi network, causing a 90% reduction of surface HFE. This activity requires a Src-kinase-binding proline-rich domain of Nef and a conserved tyrosine-based motif in the cytoplasmic tail of HFE. HIV-1 infection of ex vivo macrophages similarly down-regulates naturally expressed surface HFE in a Nef-dependent manner. The effect of Nef expression on cellular iron was explored; iron and ferritin accumulation were increased in HIV-1-infected ex vivo macrophages expressing wild-type HFE, but this effect was lost with Nef-deleted HIV-1 or when infecting macrophages from hemochromatosis patients expressing mutated HFE. The iron accumulation in HIV-1-infected HFE-expressing macrophages was paralleled by an increase in cellular HIV-1-gag expression. We conclude that, through Nef and HFE, HIV-1 directly regulates cellular iron metabolism, possibly benefiting viral growth.

Systemic Regulation of Intestinal Iron Absorption

The intestinal absorption of the essential trace element iron and its mobilization from storage sites in the body are controlled by systemic signals that reflect tissue iron requirements. Recent advances have indicated that the liver-derived peptide hepcidin plays a central role in this process by repressing iron release from intestinal enterocytes, macrophages and other body cells. When iron requirements are increased, hepcidin levels decline and more iron enters the plasma. It has been proposed that the level of circulating diferric transferrin, which reflects tissue iron levels, acts as a signal to alter hepcidin expression. In the liver, the proteins HFE, transferrin receptor 2 and hemojuvelin may be involved in mediating this signal as disruption of each of these molecules decreases hepcidin expression. Patients carrying mutations in these molecules or in hepcidin itself develop systemic iron loading (or hemochromatosis) due to their inability to down regulate iron absorption. Hepcidin is also responsible for the decreased plasma iron or hypoferremia that accompanies inflammation and various chronic diseases as its expression is stimulated by pro-inflammatory cytokines such as interleukin 6. The mechanisms underlying the regulation of hepcidin expression and how it acts on cells to control iron release are key areas of ongoing research.

The Q283P amino-acid change in HFE leads to structural and functional consequences similar to those described for the mutated 282Y HFE protein

In Caucasians, 4-35% of hemochromatosis patients carry at least one chromosome without a common HFE mutation (i.e. C282Y, H63D and S65C). Several studies have now shown that iron overload phenotypes in such patients can be associated with uncommon HFE mutations. We previously supported implication of the C282Y/Q283P compound heterozygous genotype in hemochromatosis phenotypes and, based on molecular dynamics simulations, proposed that the Q283P substitution prevents normal folding of the HFE alpha3-domain. In the current work, we have used HeLa cells carrying wild-type or Q283P-mutant HFE cDNA under the control of a tetracycline-sensitive promoter to functionally characterise the Q283P mutation. Experiments using cells over-expressing wild-type HFE confirm the existence of beta2microglobulin(beta2m)/HFE and HFE/transferrin receptor 1 (TfR1) interactions, as well as the capacity of HFE to reduce transferrin-mediated iron uptake. In contrast, neither beta2m/HFE nor HFE/TfR1 complex formation was detected in cells over-expressing the mutated form of HFE. Moreover, the 283P HFE protein was found to have a very limited effect on the major cellular iron uptake pathway. Combined, our results indicate that the Q283P mutation leads to structural and functional consequences similar to those described for the main hereditary hemochromatosis mutation. As a consequence, our study has implications for the screening of hemochromatosis patients that have one or two copies of HFE which lack the main mutations. It also highlights that protein structure prediction methods could be more generally used to better interpret relationships between rare genotypes and molecular diagnosis of a human inherited disorder.

Hémochromatose HFE : approche pathogénique et diagnostique

HFE hemochromatosis is the most frequent genetic iron overload disease. It is linked to the C282Y mutation of the HFE protein, protein encoded by the HFE gene, which is located on chromosome 6. The mechanisms accounting for iron excess are not only digestive hyperabsorption of iron but also excessive recycling of macrophagic iron coming from erythrophagocytosis and secreted into the blood. Both mechanisms are linked to an HFE-related hepatic failure in producing hepcidin, a key hormone of body iron regulation. The marked phenotypic variability of C282Y homozygosity expression is likely related to both genetic and environmental factors. The HFE gene discovery has rendered non invasive the positive diagnostic of HFE hemochromatosis, which is now based first on an increased level of plasma transferrin saturation leading to the request of the HFE mutation. Then, hepatic MRI is a reliable method to quantify iron overload. The HFE gene discovery has also paved the road of an enlarged field of differential diagnoses corresponding to novel entities of non-HFE related genetic iron overload syndromes.