Modulate Hepcidin Expression Research Articles

Hepcidin in Human Iron Disorders: Diagnostic Implications

The peptide hormone hepcidin plays a central role in regulating dietary iron absorption and body iron distribution. Many human diseases are associated with alterations in hepcidin concentrations. The measurement of hepcidin in biological fluids is therefore a promising tool in the diagnosis and management of medical conditions in which iron metabolism is affected. We describe hepcidin structure, kinetics, function, and regulation. We moreover explore the therapeutic potential for modulating hepcidin expression and the diagnostic potential for hepcidin measurements in clinical practice. Cell-culture, animal, and human studies have shown that hepcidin is predominantly synthesized by hepatocytes, where its expression is regulated by body iron status, erythropoietic activity, oxygen tension, and inflammatory cytokines. Hepcidin lowers serum iron concentrations by counteracting the function of ferroportin, a major cellular iron exporter present in the membrane of macrophages, hepatocytes, and the basolateral site of enterocytes. Hepcidin is detected in biologic fluids as a 25 amino acid isoform, hepcidin-25, and 2 smaller forms, i.e., hepcidin-22 and -20; however, only hepcidin-25 has been shown to participate in the regulation of iron metabolism. Reliable assays to measure hepcidin in blood and urine by use of immunochemical and mass spectrometry methods have been developed. Results of proof-of-principle studies have highlighted hepcidin as a promising diagnostic tool and therapeutic target for iron disorders. However, before hepcidin measurements can be used in routine clinical practice, efforts will be required to assess the relevance of hepcidin isoform measurements, to harmonize the different assays, to define clinical decision limits, and to increase assay availability for clinical laboratories.

Inactivation of prolyl hydroxylase domain (PHD) protein by epigallocatechin (EGCG) stabilizes hypoxia-inducible factor (HIF-1α) and induces hepcidin (Hamp) in rat kidney

HIF-1α plays a key role in iron uptake and transport in the liver, whose activity is tightly linked to the repression of hepcidin (Hamp). Hamp prevents intestinal iron uptake and cellular efflux by negatively modulating ferroportin. Hamp is also expressed in the kidneys, where transcriptional control by HIF-1α remains poorly understood. We show that the administration of epigallocatechin gallate (EGCG) results in a considerable Hamp expression in rat kidneys. We also provide evidence to show that EGCG inhibited prolyl hydroxylase (PHD) activity, essential for HIF-1α degradation in vivo and in vitro. Rats that were dosed with EGCG (60mg/kg, intraperitoneal) over a 7day time course stabilized HIF-1α protein in kidney tissues. Interestingly, Hamp gene expression was induced, even after subjecting rats to a 4h hypoxia treatment (8% oxygen). Using Hep3B cells, we determined that EGCG conferred its inhibitory action by complexing with PHD, altering its catalytic iron center and thus preventing HIF-1α hydroxylation. These data demonstrate EGCG’s therapeutic potential in modulating hepcidin expression in diseases associated with altered iron metabolism.

A tincture of hepcidin cures all: the potential for hepcidin therapeutics

Iron overload as a result of blood transfusions and excessive intestinal iron absorption can be a complication of chronic anemias such as β-thalassemia. Inappropriately low levels of hepcidin, a negative regulator of iron absorption and recycling, underlie the pathophysiology of the intestinal hyperabsorption. In this issue of the JCI, Gardenghi et al. demonstrate that increasing hepcidin expression to induce iron deficiency in murine β-thalassemia not only mitigates the iron overload, but also the severity of the anemia. These data illustrate the therapeutic potential of modulating hepcidin expression in diseases associated with altered iron metabolism.

ß-Thalassemic Mice Require Functional Hfe to Modulate Hepcidin Expression In Response to Iron Overload

AbstractAbstract 2063ß-Thalassemia is a genetic disorder characterized by decreased or absent production of ß-globin chains, leading to ineffective erythropoiesis, anemia and iron overload. Hepcidin, the hormone that controls iron homeostasis, is regulated by several mechanisms, including erythropoiesis, iron overload, inflammation and hypoxia. In the absence of transfusion therapy, patients with ß-thalassemia major exhibit a severe ineffective erythropoiesis that suppresses hepcidin expression. However, in patients or animal affected by ß-thalassemia intermedia (th3/+), iron overload is associated with a milder form of ineffective erythropoiesis. In this study we investigated whether th3/+ mice retain the ability to modulate hepcidin expression in response to iron load, despite their increased erythropoietic activity. We analyzed some of the genes involved in the regulation of hepcidin, in particular, genes that are upregulated by iron overload in wt mice. These included Bmp6, a strong modulator of Hamp in response to iron, and Id1, Atoh8 and Smad7, other targets of the Bmp/Smad pathway. Analysis of the phosphorylation of the Smad protein complex is in progress. In addition, we generated mice affected by ß-thalassemia intermedia lacking the Hfe gene (Hfe-th3/+), in an attempt to determine whether or not this gene is involved in hepcidin regulation in this disorder. We analyzed th3/+ mice at 2, 5 and 12 months of age. In 2-month-old th3/+ mice hepcidin expression was significantly low compared to wt mice. As th3/+ mice age and their iron overload worsens, hepcidin expression increases showing similar and elevated levels in th3/+ compared to wt animals, respectively at 5 and 12 months. At 2 months, hepcidin expression normalized to liver iron concentration exhibited even lower levels in th3/+ mice compared to wt animals. This ratio did not change in aging th3/+ animals, despite the fact that their liver iron concentration increased over time (0.66, 1.24, and 1.45 ug/mg of dry weight at 2, 5 and 12 months, respectively). The expression levels of Bmp6, Id1, Atoh8 and Smad7 followed a similar pattern, being generally downregulated at 2 months compared to wt mice. However, as iron overload progressed, th3/+ mice exhibited increased expression of these genes compared to wt mice. Similar to what was observed with hepcidin, their expression was low in th3/+ mice at all ages when normalized to liver iron concentration. These observations indicate that hepcidin expression in ß-thalassemia increases over time and is regulated by the relative levels of ineffective erythropoiesis and iron overload. We also investigated the relationship between Hfe and hepcidin in response to iron in ß-thalassemia. We transplanted the ß-thalassemic phenotype into lethally irradiated wt or Hfe-KO mice, generating th3/+ and Hfe-th3/+ animals, respectively. Compared to th3/+ mice, we observed that Hfe-th3/+ animals had increased hepatic iron (3.09 vs 1.29 ug/mg of dry weight, p≤0.05) and serum iron (232 vs 162 ug/dL, p≤0.05), with no significant changes in splenic iron concentration. The Hfe-th3/+ mice also exhibited increased hemoglobin levels (9.4 vs 7.8 g/dL, p≤0.001) due to an increase in both red cell counts (8.9 vs 8.0 ×106 cells/uL, p≤0.01) and mean corpuscular hemoglobin levels (10.6 vs 9.7 pg, **p≤0.05). However, this did not reduce splenomegaly or ineffective erythropoiesis. We also analyzed the levels of hepcidin, Bmp6, Id1, Smad7 and Atoh8 in 5-month-old mice. At his time point expression of most of these genes was similar between wt, th3/+ and Hfe-th3/+ mice. Only expression of Bmp6 was elevated in the two thalassemic groups compared to wt mice. When the levels of hepcidin, Bmp6, Id1, Smad7 and Atoh8 expression were normalized to liver iron content, we observed significant reductions in Hfe-th3/+ mice compared to th3/+ animals. Taken together, these observations indicate that iron overload can partially counteract the repressive effect of ineffective erythropoiesis on hepcidin expression in th3/+ mice. Moreover, lack of Hfe further impairs the ability of hepcidin and other iron regulated genes to respond to iron overload, aggravating this feature in thalassemic mice. Overall, this indicates that Hfe plays a positive role in the regulation of hepcidin in ß-thalassemia. Disclosures:No relevant conflicts of interest to declare.

Hydroxyurea Induces Hepcidin Expression In Monocytes In Sickle Cell Anemia Patients

AbstractAbstract 2653The antimicrobial peptide hormone hepcidin is a key regulator of iron metabolism. Although mainly produced in the liver, hepcidin is also known to be synthesized by monocytic-macrophagic cells. We have previously shown that hepcidin is overexpressed in mononuclear cells in patients with sickle cell anemia (SCA), but whether monocyte-derived hepcidin production is related to iron metabolism remains unknown.To gather further insight into the role of hepcidin in monocytes, we collected peripheral blood samples from adult SCA patients (n=54) and normal age- and race-matched controls (CTRL, n=13) for assessment of hematological parameters, biochemical markers of iron status (serum iron and transferrin saturation - TfSat), hemolysis (lactate dehydrogenase-LDH) and inflammation (C reactive protein - CRP), and separation of peripheral blood monocytes by Ficoll-Hypaque and Percoll gradients for gene expression analyses of genes involved in iron metabolism signaling (HAMP encoding hepcidin, and three genes belonging to different pathways known to influence hepcidin expression, STAT3, SMAD4 and TLR4). Plasma GDF-15 levels were also measured, as this protein has been shown to be a potent downregulator of hepcidin. SCA patients were further stratified according to the number of previous blood transfusions and to treatment with hydroxyurea (HU). All patients were in steady-state, had no history of iron chelation treatment and were not enrolled in a regular transfusion program. 18 patients were receiving HU and 15 from the non-HU group had received over 20 blood transfusions during their lifetime.As expected, SCA patients had elevated LDH levels, but no differences were found between control and SCA groups regarding serum iron, TfSat or CRP levels. Except for a higher red cell mean corpuscular volume, patients on HU did not differ significantly from patients not using HU. Plasma GDF-15 levels were higher in SCA patients (2146±506.4pg/mL) than in control individuals (228.5±21.0pg/mL, p<0.0001). Among the genes studied, HAMP expression was significantly increased in the SCA group as a whole compared to the CTRL group, but SCA patients on HU had higher levels of monocytic hepcidin expression when compared to the remaining individuals (CTRL 0.043±0.030, SCA on HU 1.240±0.426, remaining SCA 0.332±0.093, p=0.0196). There were no significant correlations between monocytic hepcidin expression and hemoglobin levels, hematocrit, leucocyte or reticulocyte counts, serum iron, LDH or CRP levels, TfSat or transfusion history. STAT3, SMAD4 and TLR4 gene expressions did not differ significantly, suggesting that a possible alternative cause for hepcidin upregulation unrelated to known mechanisms by IL-6, BMP6, LPS or GDF-15 could be an unexpected effect of hydroxyurea. To further investigate if HU was able to modulate hepcidin expression, we performed experiments with THP-1, a human monocytic lineage, since in vitro analysis would allow us to exclude the influence of circulating cytokines elevated in SCA patients. THP-1 cells were cultured in RPMI medium enriched with 10% fetal bovine serum at 37°C and 5% CO2 atmosphere, and were submitted to treatment with water as control, or HU dissolved in water in increasing concentrations (100μM, 400μM and 1600μM) for 6 hours (n≥4). Cell viability was not affected by treatment (>90% viable cells at all experiments), and HAMP gene expression was increased up to four times in the cell cultures exposed to HU (p=0.03), while STAT3 and SMAD4 expressions remained unchanged.We have shown that hepcidin expression is upregulated in monocytes in SCA patients, particularly in those receiving HU, and that HU is capable of inducing this expression in an in vitro model, independently from inflammatory cytokine-mediated stimulation. Our data suggest that, although liver-derived hepcidin has been shown to have a major role in iron metabolism, its monocyte-derived counterpart does not seem to be directly influenced by iron status and may have other functions. Some studies have demonstrated that hepcidin in other species has anti-inflammatory effects in vitro, and that patients with SCA on HU shift to a lower inflammatory status. Thus, monocytic hepcidin overexpression might be a response against the chronic inflammatory state in SCA, and HU treatment may enhance this response. This is the first description of monocyte-derived hepcidin in SCA and the influence of HU on its expression. Disclosures:No relevant conflicts of interest to declare.

PP-010 Serum hepcidin: a predictor of early graft dysfunction following orthotopic liver transplantation?

<h3>Introduction</h3> The hepatic peptide hepcidin is a critical regulator of iron metabolism. Serum levels of hepcidin correlate with liver function in humans and rodent studies have shown that liver injury modulate hepcidin expression. This is the first study of hepcidin levels in patients undergoing liver transplantation. We show that post-transplant serum hepcidin levels identify patients with severe preservation/reperfusion injury and hence predict graft dysfunction following liver transplantation. <h3>Methods</h3> Serial serum hepcidin levels were measured by SELDI-TOF mass spectrometry in 11 patients undergoing liver transplantation. Liver biopsies (T0) were taken during transplantation in 9/11 patients. Clinicopathological, biochemical and haematological data were compared. <h3>Results</h3> 5/11 patients had poor graft function after transplantation (determined by graft cholestasis, elevated PT and AST levels). Patients with poor graft function were more likely to have evidence of moderate/severe preservation injury at T0 biopsy than patients with normal graft function and were more likely to have received a liver from a non-heart beating (NHB) than a heart-beating donor (3/5 vs 1/7, respectively). While there was no difference in hepcidin levels between the two groups at the time of transplantation (90 vs 76 ng/ml p=0.68), the five patients with poor graft function had significantly higher hepcidin levels on day 3 post-transplantation compared to the six patients with mild preservation injury (Mean 135 (91–179) vs 39 (17–61) ng/ml, respectively, p&lt; 0.01 ANOVA). Mean serum hepcidin values rose by 393% between day 1 and day 3 post transplant in the group with poor graft function whereas there was a 49% drop in serum hepcidin level in the group with normal function. The rise in hepcidin values at day 3 anticipated the onset of graft cholestasis in all of the patients with severe preservation injury. <h3>Conclusion</h3> This is the first study of serum hepcidin levels in patients undergoing liver transplantation. We show that hepcidin values rapidly fall in patients with normal graft function following transplantation, but remain elevated in patients with early graft dysfunction. Elevated serum hepcidin levels accurately predicted the onset of preservation injury and anticipated graft dysfunction. Moreover we show that hepcidin levels are a better determinant of future graft dysfunction than AST or CRP. Early identification of graft dysfunction is important as it allows for modification of immunosuppression. We show that day 3 hepcidin levels correlate well with the severity of graft injury and can be used as a predictor of early graft dysfunction.

Gender-related variations in iron metabolism and liver diseases

The regulation of iron metabolism involves multiple organs including the duodenum, liver and bone marrow. The recent discoveries of novel iron-regulatory proteins have brought the liver to the forefront of iron homeostasis. The iron overload disorder, genetic hemochromatosis, is one of the most prevalent genetic diseases in individuals of Caucasian origin. Furthermore, patients with non-hemochromatotic liver diseases, such as alcoholic liver disease, chronic hepatitis C or nonalcoholic steatohepatitis, often exhibit elevated serum iron indices (ferritin, transferrin saturation) and mild to moderate hepatic iron overload. Clinical data indicate significant differences between men and women regarding liver injury in patients with alcoholic liver disease, chronic hepatitis C or nonalcoholic steatohepatitis. The penetrance of genetic hemochromatosis also varies between men and women. Hepcidin has been suggested to act as a modifier gene in genetic hemochromatosis. Hepcidin is a circulatory antimicrobial peptide synthesized by the liver. It plays a pivotal role in the regulation of iron homeostasis. Hepcidin has been shown to be regulated by iron, inflammation, oxidative stress, hypoxia, alcohol, hepatitis C and obesity. Sex and genetic background have also been shown to modulate hepcidin expression in mice. The role of gender in the regulation of human hepcidin gene expression in the liver is unknown. However, hepcidin may play a role in gender-based differences in iron metabolism and liver diseases. Better understanding of the mechanisms associated with gender-related differences in iron metabolism and chronic liver diseases may enable the development of new treatment strategies.

Hepcidin Regulation by the BMP Pathway.

Abstract SCI-25Systemic iron balance is regulated by the key iron regulatory hormone hepcidin. Secreted by the liver, hepcidin inhibits iron absorption from the diet and iron mobilization from body stores by decreasing cell surface expression of the iron export protein ferroportin. Iron administration increases hepcidin expression, thereby providing a feedback mechanism to limit further iron absorption, while anemia and hypoxia inhibit hepcidin expression, thereby increasing iron availability for erythropoiesis. Hepcidin excess is thought to have a role in the anemia of inflammation, while hepcidin deficiency is thought to be the common pathogenic mechanism underlying the iron overload disorder hereditary hemochromatosis, due to mutations in the genes encoding hepcidin itself (HAMP), HFE, transferrin receptor 2 (TFR2), or hemojuvelin (HFE2). Notably the precise molecular mechanisms by which iron levels are “sensed” and how this iron “signal” is transduced to modulate hepcidin expression have remained elusive. We have recently discovered that hemojuvelin is a co-receptor for the bone morphogenetic protein (BMP) signaling pathway, and that hemojuvelin-mediated BMP signals increase hepcidin expression at the transcriptional level. In addition to patients with HFE2 mutations and Hfe2 knockout mice, other genetic mouse models associated with impaired hepatic BMP signaling through a global knockout of the ligand Bmp6, or selective hepatic knockout of an intracellular mediator of BMP signaling, Smad4, also cause inappropriately low hepcidin expression and iron overload. Exogenous BMP6 administration in mice increases hepatic hepcidin expression and reduces serum iron, while BMP6 antagonists inhibit hepatic hepcidin expression, mobilize reticuloendothelial cell iron stores and increase serum iron. Not only does the BMP6-hemojuvelin-SMAD pathway regulate hepcidin expression and thereby systemic iron homeostasis, but also the BMP6-SMAD pathway itself is regulated by iron. Acute iron administration in mice increases phosphorylation of Smad proteins in the liver, and chronic changes in dietary iron modulate hepatic Bmp6 mRNA expression and phosphorylation of Smad proteins concordantly with Hamp mRNA expression. Together, these data support the paramount role of the BMP6-hemojuvelin-SMAD signaling pathway in the iron-mediated regulation of hepcidin expression and systemic iron homeostasis, and suggest that modulators of this pathway may be an alternative therapeutic strategy for treating disorders of iron homeostasis. Recent work elucidating the role of the BMP signaling pathway in hepcidin regulation and systemic iron homeostasis will be presented. DisclosuresBabitt:Ferrumax Pharmaceuticals, Inc.: Equity Ownership.

Hepatocyte growth factor (HGF) suppresses hepcidin expression in hepatocytes by interfering with BMP‐mediated hepcidin induction

Hepcidin, a 25‐amino‐acid peptide synthesized in the liver, is the iron regulatory hormone. Suppression of hepcidin in the setting of anemia appears to depend on a soluble factor expressed in the bone marrow during erythropoiesis. Here we describe that hepatocyte growth factor (HGF) suppresses hepcidin mRNA in a dose‐response fashion, in the concentration range detected in human serum. A similar response is observed in primary cells or cell lines transfected with a hepcidin promoter‐luciferase reporter. HGF is a ligand for the receptor tyrosine kinase (RTK), Met. In HepG2 cells transfected with the hepcidin‐luciferase reporter, the luminescence is specifically suppressed by HGF and not other ligands of RTKs, PDGF and EGF. BMPs 2, 4, 6 and 9 are strong inducers of hepcidin, and the BMP2/4 pathway also mediates hepcidin induction by iron‐transferrin. We report that HGF prevents a BMP2‐mediated increase in hepcidin mRNA and the activity of the luciferase reporter. HGF also prevented the induction by BMP2 of an unrelated BMP‐sensitive luciferase reporter. In primary hepatocytes, the BMP‐sensitive transcript ID1 is also downregulated by HGF. Thus, HGF may be a novel suppressor of hepcidin, modulating hepcidin expression by interfering with BMP signaling. We speculate that HGF could mediate the suppression of hepcidin in chronic liver diseases.This research is supported by National Institutes of Health grant #1F30DK082151.

Role of Iron in Inducing Oxidative Stress in Thalassemia: Can It Be Prevented by Inhibition of Absorption and by Antioxidants?

The pathophysiology of thalassemia is, to a certain extent, associated with the generation of labile iron in the pathological red blood cell (RBC). The appearance of such forms of iron at the inner and outer cell surfaces exposes the cell to conditions whereby the labile metal promotes the formation of reactive oxygen species (ROS) leading to cumulative cell damage. Another source of iron accumulation results from increased absorption due to decreased expression of hepcidin. The presence of labile plasma iron (LPI) was carried out using fluorescent probes in the FACS. RNA expression of hepcidin was measured in two models of thalassemic mice. Hepcidin expression was also measured in human hepatoma HepG2 cells following incubation with thalassemic sera. LPI was identified and could be quantitatively measured and correlated with other parameters of iron overload. Hepcidin expression was downregulated in the livers of thalassemic mice, in major more than in intermedia. Thalassemic sera down regulated hepcidin expression in HepG2 liver cells. A possible way to decrease iron absorption could be by modulating hepcidin expression pharmacologically, by gene therapy or by its administration. Treatment with combination of antioxidants such as N-acetylcysteine for proteins and vitamin E for lipids in addition to iron chelators could neutralize the deleterious effects of ROS and monitored by quantitation of LPI.

A role for tumour necrosis factor α in human small bowel iron transport

Cytokines are integral to the development of anaemia of chronic inflammation. Cytokines modulate hepcidin expression and iron sequestration by the reticuloendothelial system but their direct effects on small bowel iron transport are not well characterized. The aim of the present study was to examine the local effects of TNFalpha (tumour necrosis factor alpha) on small bowel iron transport and on iron transporter expression in the absence of hepcidin. The effects of TNFalpha on iron transport were determined using radiolabelled iron in an established Caco-2 cell model. The effect of TNFalpha on the expression and localization of the enterocyte iron transporters DMT-1 (divalent metal transporter 1), IREG-1 (iron-regulated transporter 1) and ferritin was determined utilizing Caco-2 cells and in a human ex vivo small bowel culture system. TNFalpha mediated an early induction in both iron import and iron export, which were associated with increased DMT-1 and IREG-1 mRNA and protein expression (P<0.05). However, by 24 h, both iron import and iron export were significantly inhibited, coinciding with an induction of ferritin heavy chain (P<0.05) and a decrease in DMT-1 and IREG-1 to baseline levels. In addition, there was a relocalization of IREG-1 away from the basolateral cell border and increased iron deposition in villous enterocytes. In conclusion, TNFalpha has a direct effect on small bowel iron transporter expression and function, leading to an inhibition of iron transport.

Ischemia‐reperfusion of rat liver modulates hepcidin in vivo expression

The recently identified acute-phase response antimicrobial peptide hepcidin has been postulated to maintain iron homeostasis by modulating iron absorption at both the intestinal and macrophage levels. Hepcidin has also been reported to be responsible for anemia associated with chronic inflammatory diseases, and in anemia in patients with hepatic adenomas. Since Kupffer cells are known to be the primary contributor to early-phase ischemia-reperfusion injury in the liver and iron is known to modulate Kupffer cell production of proinflammatory cytokine and reactive oxygen species, we investigated hepcidin in vivo expression in the well-established rat partial-liver ischemia-reperfusion model. We found that both liver ischemia alone and liver ischemia-reperfusion significantly induced serum and liver hepcidin levels. Furthermore, currently proposed mediators of in vivo hepcidin expression, such as interleukin-6, signal transducers and activators of transcription-family transducers, and CCAAT/enhancing binding protein-alpha do not appear to modulate hepcidin expression in the liver ischemia-reperfusion acute inflammatory model. In this study we report the first in vivo evidence of liver ischemia and liver ischemia-reperfusion modulation of hepcidin expression. In conclusion, in the well-characterized liver ischemia-reperfusion model of acute inflammation, mechanism(s) other than interleukin-6 signal transduction via signal transducers and activators of transcription-3 may be responsible for hepcidin induction.

Hepcidin in Iron Metabolism

Hepcidin, which has been recently identified both by biochemical and genomic approaches, is a 25 amino acid polypeptide synthesized mainly by hepatocytes and secreted into the plasma. Besides its potential activity in antimicrobial defense, hepcidin plays a major role in iron metabolism. It controls two key steps of iron bioavailability, likely through a hormonal action: digestive iron absorption by enterocytes and iron recycling by macrophages. In humans, this could explain that low levels of hepcidin found during juvenile haemochromatosis and HFE-1 genetic haemochromatosis are associated with an iron overload phenotype. Conversely, an increase of hepcidin expression is suspected to play a major role in the development of anemia of chronic inflammatory diseases. However, the regulatory mechanisms of hepcidin expression are multiple, including iron-related parameters, anemia, hypoxia, inflammation and hepatocyte function. Therefore, many physiological and pathological situations may modulate hepcidin expression and subsequently iron metabolism. A better knowledge of the biological effects of hepcidin and of its expression regulatory mechanisms will clarify the place of hepcidin in the diagnosis and treatment of iron-related diseases.

Mutations in HFE2 cause iron overload in chromosome 1q-linked juvenile hemochromatosis.

Juvenile hemochromatosis is an early-onset autosomal recessive disorder of iron overload resulting in cardiomyopathy, diabetes and hypogonadism that presents in the teens and early 20s (refs. 1,2). Juvenile hemochromatosis has previously been linked to the centromeric region of chromosome 1q (refs. 3-6), a region that is incomplete in the human genome assembly. Here we report the positional cloning of the locus associated with juvenile hemochromatosis and the identification of a new gene crucial to iron metabolism. We finely mapped the recombinant interval in families of Greek descent and identified multiple deleterious mutations in a transcription unit of previously unknown function (LOC148738), now called HFE2, whose protein product we call hemojuvelin. Analysis of Greek, Canadian and French families indicated that one mutation, the amino acid substitution G320V, was observed in all three populations and accounted for two-thirds of the mutations found. HFE2 transcript expression was restricted to liver, heart and skeletal muscle, similar to that of hepcidin, a key protein implicated in iron metabolism. Urinary hepcidin levels were depressed in individuals with juvenile hemochromatosis, suggesting that hemojuvelin is probably not the hepcidin receptor. Rather, HFE2 seems to modulate hepcidin expression.