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1.  Deletion of Hemojuvelin, an Iron-Regulatory Protein, in Mice Results in Abnormal Angiogenesis and Vasculogenesis in Retina Along With Reactive Gliosis 
Purpose.
Loss-of-function mutations in hemojuvelin (HJV) cause juvenile hemochromatosis, an iron-overload disease. Deletion of Hjv in mice results in excessive iron accumulation and morphologic changes in the retina. Here, we studied the retinal vasculature in Hjv−/− mice.
Methods.
Age-matched wild-type and Hjv−/− mice were used for fluorescein angiography and preparation of retinal cryosections, flat-mounts, and trypsin-digested blood vessels. Retinal angiogenesis was monitored by immunofluorescent detection of isolectin-B4, endoglin, and VEGF. Retinal vasculogenesis was monitored by immunofluorescent detection of collagen IV. Reactive gliosis was assessed based on the expression of glial fibrillary acidic protein and vimentin and CD11b/c as markers for Müller cells and microglia.
Results.
Between 18 and 24 months of age, retinas of Hjv−/− mice displayed marked disruptions in angiogenesis and vasculogenesis. Blood vessels in Hjv−/− mice were tortuous and dilated, with a decrease in the tight-junction protein occludin. There was also evidence of neovascularization in Hjv−/− mice with blood vessels appearing in the vitreous, which were leaky. There was reactive gliosis in these mice involving both Müller cells and microglia. Such changes were not detected at 2 weeks of age. Even at the age of 4 months, retinas of Hjv−/− mice were almost normal with changes just beginning to appear. Thus, the vascular changes in Hjv−/− mouse retinas represent an age-dependent phenomenon.
Conclusions.
Deletion of Hjv in mice leads to abnormal retinal angiogenesis/vasculogenesis, with proliferation of new, leaky blood vessels in the vitreous. These changes are accompanied with reactive gliosis involving Müller cells and microglia.
Deletion of Hemojuvelin (HJV) in mice is associated with excessive iron accumulation and morphologic changes in the retina. In this study, we find that HJV knockout mice have abnormal retinal angiogenesis and vasculogenesis along with robust reactive gliosis involving Müller cells and microglia.
doi:10.1167/iovs.13-13677
PMCID: PMC4053073  PMID: 24812553
iron; angiogenesis; retinal vasculature; gliosis
2.  Iron-mediated retinal degeneration in hemojuvelin knockout mice 
The Biochemical journal  2012;441(2):599-608.
Summary
Hemochromatosis is a genetic disorder of iron overload resulting from loss-of-function mutations in genes coding for the iron-regulatory proteins HFE, transferrin receptor 2, ferroportin, hepcidin, and hemojuvelin (HJV). Recent studies have established the expression of all the five genes in retina, indicating their importance in retinal iron homeostasis. We previously demonstrated that Hjv is expressed in retinal pigment epithelium (RPE), outer and inner nuclear layers, and ganglion cell layer. Here we report on the consequences of Hjv deletion on the retina in mice. Hjv-null mice at ≥ 18 months of age had increased iron accumulation in retina with marked morphological damage compared to age-matched controls; these changes were not found in younger mice. The retinal phenotype in Hjv-null mice included hyperplasia of RPE. We isolated RPE cells from wild type and Hjv-null mice and examined their growth patterns. Hjv-null RPE cells were less senescent and exhibited a hyperproliferative phenotype. Hjv-null RPE cells also showed upregulation of Slc7a11 that codes for the ‘transporter proper’ subunit xCT in the heterodimeric amino acid transporter cystine/glutamate exchanger (xCT/4F2hc). Bone morphogenic protein (BMP) 6 could not induce hepcidin expression in Hjv-null RPE cells, confirming that retinal cells require Hjv for induction of hepcidin via BMP6 signaling. Hjv is a glycosylphosphatidylinositol-anchored protein, and the membrane-associated Hjv is necessary for BMP6-mediated activation of hepcidin promoter in RPE cells. Taken together, these studies confirm the biological importance of Hjv in the regulation of iron homeostasis in the retina and in RPE.
doi:10.1042/BJ20111148
PMCID: PMC3710445  PMID: 21943374
hemojuvelin; hemochromatosis; retinal pigment epithelium; hepcidin; bone morphogenic protein; knockout mouse
3.  Molecular Diagnostic and Pathogenesis of Hereditary Hemochromatosis 
Hereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by enhanced intestinal absorption of dietary iron. Without therapeutic intervention, iron overload leads to multiple organ damage such as liver cirrhosis, cardiomyopathy, diabetes, arthritis, hypogonadism and skin pigmentation. Most HH patients carry HFE mutant genotypes: homozygosity for p.Cys282Tyr or p.Cys282Tyr/p.His63Asp compound heterozygosity. In addition to HFE gene, mutations in the genes that encode hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1) have been associated with regulation of iron homeostasis and development of HH. The aim of this review was to identify the main gene mutations involved in the pathogenesis of type 1, 2, 3 and 4 HH and their genetic testing indication. HFE testing for the two main mutations (p.Cys282Tyr and p.His63Asp) should be performed in all patients with primary iron overload and unexplained increased transferrin saturation and/or serum ferritin values. The evaluation of the HJV p.Gly320Val mutation must be the molecular test of choice in suspected patients with juvenile hemochromatosis with less than 30 years and cardiac or endocrine manifestations. In conclusion, HH is an example that genetic testing can, in addition to performing the differential diagnostic with secondary iron overload, lead to more adequate and faster treatment.
doi:10.3390/ijms13021497
PMCID: PMC3291973  PMID: 22408404
hemochromatosis; primary iron overload; HFE; high-resolution melting; HJV; molecular diagnostic
4.  Tumour necrosis factor alpha downregulates human hemojuvelin expression via a novel response element within its promoter 
Background
Iron homeostasis is chiefly regulated by hepcidin whose expression is tightly controlled by inflammation, iron stores, and hypoxia. Hemojuvelin (HJV) is a bone morphogenetic protein co-receptor that has been identified as a main upstream regulator of hepcidin expression; HJV mutations are associated with a severe form of iron overload (Juvenile haemochromatosis). Currently however, there is no information on how HJV is regulated by inflammation.
Methods
To study the regulation of Hjv expression by inflammation and whether Hfe has a role in that regulation, control and LPS-injected wild type and Hfe KO mice were used. Moreover, human hepatoma cells (HuH7) were used to study the effect of IL-6 and TNF-α on HJV mRNA expression.
Results
Here we show that LPS repressed hepatic Hjv and BMPs, while it induced hepcidin 1 expression in wild-type and Hfe KO mice with no effect on hepatic pSMAD 1, 5, 8 protein levels. In addition, exogenous TNF-α (20 ng/mL) decreased HJV mRNA and protein expression to 40% of control with no effect on hepcidin mRNA expression in 24 hours. On the other hand, IL-6 induced hepcidin mRNA and protein expression with no effect on HJV mRNA expression levels. Moreover, using the HJV promoter-luciferase reporter fusion construct (HJVP1.2-luc), we showed that the basal luciferase activity of HJVP1.2-luc was inhibited by 33% following TNF-α treatment of HuH7 transfected cells suggesting that the TNF-α down-regulation is exerted at the transcriptional level. Additionally, mutation of a canonical TNF- alpha responsive element (TNFRE) within HJVP1.2-luc abolished TNF-α response suggesting that this TNFRE is functional.
Conclusions
From these results, we conclude that TNF-α suppresses HJV transcription possibly via a novel TNFRE within the HJV promoter. In addition, the results suggest that the proposed link between inflammation and BMP-SMAD signalling is independent of HJV and BMP ligands.
doi:10.1186/1423-0127-19-83
PMCID: PMC3500654  PMID: 22998440
Inflammation; Hemojuvelin; TNF-α
5.  Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload 
Journal of Clinical Investigation  2005;115(8):2180-2186.
Iron homeostasis plays a critical role in many physiological processes, notably synthesis of heme proteins. Dietary iron sensing and inflammation converge in the control of iron absorption and retention by regulating the expression of hepcidin, a regulator of the iron exporter ferroportin. Human mutations in the glycosylphosphatidylinositol-anchored protein hemojuvelin (HJV; also known as RGMc and HFE2) cause juvenile hemochromatosis, a severe iron overload disease, but the way in which HJV intersects with the iron regulatory network has been unclear. Here we show that, within the liver, mouse Hjv is selectively expressed by periportal hepatocytes and also that Hjv-mutant mice exhibit iron overload as well as a dramatic decrease in hepcidin expression. Our findings define a key role for Hjv in dietary iron sensing and also reveal that cytokine-induced inflammation regulates hepcidin expression through an Hjv-independent pathway.
doi:10.1172/JCI25683
PMCID: PMC1180556  PMID: 16075058
6.  A mouse model of juvenile hemochromatosis 
Journal of Clinical Investigation  2005;115(8):2187-2191.
Hereditary hemochromatosis is an iron-overload disorder resulting from mutations in proteins presumed to be involved in the maintenance of iron homeostasis. Mutations in hemojuvelin (HJV) cause severe, early-onset juvenile hemochromatosis. The normal function of HJV is unknown. Juvenile hemochromatosis patients have decreased urinary levels of hepcidin, a peptide hormone that binds to the cellular iron exporter ferroportin, causing its internalization and degradation. We have disrupted the murine Hjv gene and shown that Hjv–/– mice have markedly increased iron deposition in liver, pancreas, and heart but decreased iron levels in tissue macrophages. Hepcidin mRNA expression was decreased in Hjv–/– mice. Accordingly, ferroportin expression detected by immunohistochemistry was markedly increased in both intestinal epithelial cells and macrophages. We propose that excess, unregulated ferroportin activity in these cell types leads to the increased intestinal iron absorption and plasma iron levels characteristic of the juvenile hemochromatosis phenotype.
doi:10.1172/JCI25049
PMCID: PMC1180543  PMID: 16075059
7.  BMP-6 is a key endogenous regulator of hepcidin expression and iron metabolism 
Nature genetics  2009;41(4):482-487.
Juvenile hemochromatosis is an iron overload disorder caused by mutations in the genes encoding the major iron regulatory hormone hepcidin (HAMP)1 and hemojuvelin (HFE2)2. We have previously shown that hemojuvelin is a bone morphogenetic protein (BMP) co-receptor and that BMP signals regulate hepcidin expression and iron metabolism3,4. However, the endogenous BMP regulator(s) of hepcidin in vivo is unknown. Here, we show that in vitro, compared with soluble hemojuvelin (HJV.Fc), the homologous DRAGON.Fc more potently inhibits hepcidin induction by BMP-2 or BMP-4, but less potently inhibits BMP-6. In vivo, HJV.Fc or a neutralizing BMP-6 antibody inhibits hepcidin expression and increases serum iron, while DRAGON.Fc has no effect. Notably, Bmp6 null mice have a phenotype resembling hereditary hemochromatosis with reduced hepcidin expression and tissue iron overload. Finally, we demonstrate a physical interaction between HJV.Fc and BMP-6, and we show that BMP-6 increases hepcidin expression and reduces serum iron in mice. These data support a key role for BMP-6 as a ligand for HJV and an endogenous regulator of hepcidin expression and iron metabolism in vivo.
doi:10.1038/ng.335
PMCID: PMC2810136  PMID: 19252486
8.  Loss of Hfe Leads to Progression of Tumor Phenotype in Primary Retinal Pigment Epithelial Cells 
Purpose.
Hemochromatosis is a disorder of iron overload arising mostly from mutations in HFE. HFE is expressed in retinal pigment epithelium (RPE), and Hfe−/− mice develop age-related iron accumulation and retinal degeneration associated with RPE hyperproliferation. Here, the mechanism underlying the hyperproliferative phenotype in RPE was investigated.
Methods.
Cellular senescence was monitored by β-galactosidase activity. Gene expression was monitored by real-time PCR. Survivin was analyzed by Western blot and immunofluorescence. Migration and invasion were monitored using appropriate kits. Glucose transporters (GLUTs) were monitored by 3-O-methyl-D-glucose uptake. Histone deacetylases (HDACs) were studied by monitoring catalytic activity and acetylation status of histones H3/H4.
Results.
Hfe−/− RPE cells exhibited slower senescence rate and higher survivin expression than wild type cells. Hfe−/− cells migrated faster and showed greater glucose uptake and increased expression of GLUTs. The expression of HDACs and DNA methyltransferase (DNMTs) also was increased. Similarly, RPE cells from hemojuvelin (Hjv)-knockout mice, another model of hemochromatosis, also had increased expression of GLUTs, HDACs, and DNMTs. The expression of Slc5a8 was decreased in Hfe−/− RPE cells, but treatment with a DNA methylation inhibitor restored the transporter expression, indicating involvement of DNA methylation in the silencing of Slc5a8 in Hfe−/− cells.
Conclusions.
RPE cells from iron-overloaded mice exhibit several features of tumor cells: decreased senescence, enhanced migration, increased glucose uptake, and elevated levels of HDACs and DNMTs. These features are seen in Hfe−/− RPE cells as well as in Hjv−/− RPE cells, providing a molecular basis for the hyperproliferative phenotype of Hfe−/− and Hjv−/− RPE cells.
Hfe−/− and Hjv−/− retinal pigment epithelial (RPE) cells exhibit several features of tumor cells: decreased senescence, enhanced migration, increased glucose uptake, and elevated levels of Histone deacetylases and DNA methyltransferase. These biochemical parameters underlie the hyperproliferative phenotype of Hfe-/- and Hjv-/- RPE cells.
doi:10.1167/iovs.12-10312
PMCID: PMC3544423  PMID: 23169885
9.  Proteomic Analysis and Molecular Modeling Characterize the Iron-Regulatory Protein, Hemojuvelin/Repulsive Guidance Molecule c 
The Biochemical journal  2013;452(1):87-95.
Hemojuvelin (HJV) plays a key role in iron metabolism in mammals by regulating expression of the liver-derived hormone, hepcidin, which controls systemic iron uptake and release. Mutations in HJV cause juvenile hemochromatosis, a rapidly progressing iron overload disorder in humans. HJV, also known as repulsive guidance molecule c (RGMc), is a member of the three-protein RGM family. RGMs are GPI-linked glycoproteins that share ~50% amino acid identity and several structural motifs, including the presence of 14 cysteines in analogous locations. Unlike RGMa and RGMb, HJV/RGMc is composed of both single-chain and two-chain isoforms. To date there is no structural information for any member of the RGM family. Here we have mapped the disulfide bonds in mouse HJV/RGMc using a proteomics strategy combining sequential mass spectrometry (MS) steps composed of electron transfer dissociation (ETD) and collision-induced dissociation (CID), in which ETD induces cleavage of disulfide linkages, and CID establishes disulfide bond assignments between liberated peptides. Our results identify an HJV/RGMc molecular species containing 4 disulfide linkages. We predict using ab initio modeling that this molecule is a single-chain HJV/RGMc isoform. Our observations outline a general approach using tandem MS and ab initio molecular modeling to define unknown structural features in proteins.
doi:10.1042/BJ20121845
PMCID: PMC3890427  PMID: 23464809
hemojuvelin; repulsive guidance molecule; iron metabolism; mass spectrometry; molecular modeling; disulfide bonds; protein structure
10.  Hepcidin regulation: ironing out the details 
Journal of Clinical Investigation  2007;117(7):1755-1758.
Hepcidin is a peptide hormone secreted by the liver that plays a central role in the regulation of iron homeostasis. Increased hepcidin levels result in anemia while decreased expression is the causative feature in most primary iron overload diseases. Mutations in hemochromatosis type 2 (HFE2), which encodes the protein hemojuvelin (HJV), result in the absence of hepcidin and an early-onset form of iron overload disease. HJV is a bone morphogenetic protein (BMP) coreceptor and HJV mutants have impaired BMP signaling. In this issue of the JCI, Babitt and colleagues show that BMPs are autocrine hormones that induce hepcidin expression (see the related article beginning on page 1933). Administration of a recombinant, soluble form of HJV decreased hepcidin expression and increased serum iron levels by mobilizing iron from splenic stores. These results demonstrate that recombinant HJV may be a useful therapeutic agent for treatment of the anemia of chronic disease, a disorder resulting from high levels of hepcidin expression.
doi:10.1172/JCI32701
PMCID: PMC1904333  PMID: 17607352
11.  Hemojuvelin and bone morphogenetic protein (BMP) signaling in iron homeostasis 
Mutations in hemojuvelin (HJV) are the most common cause of the juvenile-onset form of the iron overload disorder hereditary hemochromatosis. The discovery that HJV functions as a co-receptor for the bone morphogenetic protein (BMP) family of signaling molecules helped to identify this signaling pathway as a central regulator of the key iron hormone hepcidin in the control of systemic iron homeostasis. This review highlights recent work uncovering the mechanism of action of HJV and the BMP-SMAD signaling pathway in regulating hepcidin expression in the liver, as well as additional studies investigating possible extra-hepatic functions of HJV. This review also explores the interaction between HJV, the BMP-SMAD signaling pathway and other regulators of hepcidin expression in systemic iron balance.
doi:10.3389/fphar.2014.00104
PMCID: PMC4026703  PMID: 24860505
hemojuvelin; bone morphogenetic protein; hepcidin; iron; hemochromatosis; repulsive guidance molecule
12.  Evidence for distinct pathways of hepcidin regulation by acute and chronic iron loading 
Hepatology (Baltimore, Md.)  2011;53(4):1333-1341.
In response to iron loading, hepcidin synthesis is homeostatically increased to limit further absorption of dietary iron and its release from stores. Mutations in HFE, transferrin receptor 2 (Tfr2), hemojuvelin (HJV) or bone morphogenetic protein 6 (BMP6) prevent appropriate hepcidin response to iron, allowing increased absorption of dietary iron, and eventually iron overload. To understand the role each of these proteins plays in hepcidin regulation by iron, we analyzed hepcidin mRNA responsiveness to short and long-term iron challenge in iron-depleted Hfe, Tfr2, Hjv and Bmp6 mutant mice. After 1-day (acute) iron challenge, Hfe−/− showed a smaller hepcidin increase than their wild-type strain-matched controls, Bmp6−/− nearly no increase, and Tfr2 and Hjv mutants no increase in hepcidin expression, indicating that all four proteins participate in hepcidin regulation by acute iron changes. After a 21-day (chronic) iron challenge, Hfe and Tfr2 mutants increased hepcidin expression to nearly wild-type levels but a blunted increase of hepcidin was seen in Bmp6−/− and Hjv−/− mice. BMP6, whose expression is also regulated by iron, may mediate hepcidin regulation by iron stores. None of the mutant strains (excepting Bmp6−/− mice) had impaired BMP6 mRNA response to chronic iron loading. Conclusion: TfR2, HJV and BMP6 and, to a lesser extent, HFE, are required for the hepcidin response to acute iron loading, but are partially redundant for hepcidin regulation during chronic iron loading, and are not involved in the regulation of BMP6 expression. Our findings support a model in which acute increases in holotransferrin concentrations transmitted through HFE, TfR2 and HJV augment BMP receptor sensitivity to BMPs. A distinct regulatory mechanism that senses hepatic iron may modulate hepcidin response to chronic iron loading.
doi:10.1002/hep.24178
PMCID: PMC3074982  PMID: 21480335
Hereditary hemochromatosis; bone morphogenetic protein 6; hemojuvelin; HFE; transferrin receptor 2
13.  Effect of Iron Overload and Iron Deficiency on Liver Hemojuvelin Protein 
PLoS ONE  2012;7(5):e37391.
Introduction
Hemojuvelin (Hjv) is a key component of the signaling cascade that regulates liver hepcidin (Hamp) expression. The purpose of this study was to determine Hjv protein levels in mice and rats subjected to iron overload and iron deficiency.
Methods
C57BL/6 mice were injected with iron (200 mg/kg); iron deficiency was induced by feeding of an iron-deficient diet, or by repeated phlebotomies. Erythropoietin (EPO)-treated mice were administered recombinant EPO at 50 U/mouse. Wistar rats were injected with iron (1200 mg/kg), or fed an iron-deficient diet. Hjv protein was determined by immunoblotting, liver samples from Hjv−/− mice were used as negative controls. Mouse plasma Hjv content was determined by a commercial ELISA kit.
Results
Liver crude membrane fraction from both mice and rats displayed a major Hjv-specific band at 35 kDa, and a weaker band of 20 kDa. In mice, the intensity of these bands was not changed following iron injection, repeated bleeding, low iron diet or EPO administration. No change in liver crude membrane Hjv protein was observed in iron-treated or iron-deficient rats. ELISA assay for mouse plasma Hjv did not show significant difference between Hjv+/+ and Hjv−/− mice. Liver Hamp mRNA, Bmp6 mRNA and Id1 mRNA displayed the expected response to iron overload and iron deficiency. EPO treatment decreased Id1 mRNA, suggesting possible participation of the bone morphogenetic protein pathway in EPO-mediated downregulation of Hamp mRNA.
Discussion
Since no differences between Hjv protein levels were found following various experimental manipulations of body iron status, the results indicate that, in vivo, substantial changes in Hamp mRNA can occur without noticeable changes of membrane hemojuvelin content. Therefore, modulation of hemojuvelin protein content apparently does not represent the limiting step in the control of Hamp gene expression.
doi:10.1371/journal.pone.0037391
PMCID: PMC3356351  PMID: 22629388
14.  Characteristics of participants with self-reported hemochromatosis or iron overload at HEIRS Study initial screening 
American journal of hematology  2008;83(2):126-132.
There are few descriptions of young adults with self-reported hemochromatosis or iron overload (H/IO). We analyzed initial screening data in 7,343 HEmochromatosis and IRon Overload Screening (HEIRS) Study participants ages 25–29 years, including race/ethnicity and health information; transferrin saturation (TS) and ferritin (SF) measurements; and HFE C282Y and H63D genotypes. We used denaturing high-pressure liquid chromatography and sequencing to detect mutations in HJV, TFR2, HAMP, SLC40A1, and FTL. Fifty-one participants reported previous H/IO; 23 (45%) reported medical conditions associated with H/IO. Prevalences of reports of arthritis, diabetes, liver disease or liver cancer, heart failure, fertility problems or impotence, and blood relatives with H/IO were significantly greater in participants with previous H/IO reports than in those without. Only 7.8% of the 51 participants with previous H/IO reports had elevated TS; 13.7% had elevated SF. Only one participant had C282Y homozygosity. Three participants aged 25–29 years were heterozygous for potentially deleterious mutations in HFE2, TFR2, and HAMP promoter, respectively. Prevalences of self-reported conditions, screening iron phenotypes, and C282Y homozygosity were similar in 1,165 participants aged 30 years or greater who reported previous H/IO. We conclude that persons who report previous H/IO diagnoses in screening programs are unlikely to have H/IO phenotypes or genotypes. Previous H/IO reports in some participants could be explained by treatment that induced iron depletion before initial screening, misdiagnosis, or participant misunderstanding of their physician or the initial screening questionnaire.
doi:10.1002/ajh.21053
PMCID: PMC3773364  PMID: 17726683
15.  Severe Iron Overload with a Novel Aminolevulinate Synthase Mutation and Hepatitis C Infection. A Case Report 
A 55 year old man with a history of chronic hepatic C infection was found to have severe hemochromatosis: hepatic cirrhosis, cardiomyopathy, arrhythmia, hypogonadism, diabetes and bronzed skin color. After 50 phlebotomies, he underwent a combined heart and liver transplant. Genetic analyses identified a novel mutation in the iron responsive element of the ALAS2 gene. No mutations were found in other genes associated with adult or juvenile hemochromatosis including HFE, transferrin receptor-2 (TfR2), ferroportin (SLC40A1), hepcidin (HAMP) and hemojuvelin (HJV). We suggest that the ALAS2 mutation together with chronic hepatitis C infection may have caused the severe iron overload phenotype.
doi:10.1016/j.bcmd.2008.08.001
PMCID: PMC2696479  PMID: 18823803
ALAS2; iron responsive element; iron overload; hepatitis C; liver transplant; hemochromatosis
16.  Altered hepatic BMP signaling pathway in human HFE hemochromatosis 
Blood cells, molecules & diseases  2010;45(4):308-312.
Human hemochromatosis (HC) has been associated with the common C282Y polymorphism of HFE or rare pathogenic mutations of TfR2, HJV, FPN and HAMP. All forms of human HC seem to be caused by low or inadequate levels of hepcidin, the iron hormone. We and others have recently shown that Hfe−/−mice exhibit an impairment in the bone morphogenetic protein (BMP) signaling pathway controlling hepcidin. However, all data indicating the central role of BMPs in hepcidin regulation and an impaired BMP/SMAD signaling in HC have been collected in mice. In this study we investigated whether also in humans the expression of BMP signaling targets, SMAD7 and Id1, are associated with liver iron concentration (LIC) and whether such regulation is disrupted in HFE-HC. We correlated the mRNA expression, assessed by RT-PCR, of HAMP, SMAD7 and Id1 with LIC in liver biopsies from patients with normal iron status, HFE-HC or non-HC hepatic iron overload. We found that in human liver, not only HAMP, but also SMAD7 and Id1 mRNA significantly correlate with the extent of hepatic iron burden. However, this correlation is lost in patients with HFE-HC, but maintained in subjects with non-hemochromatotic iron overload. These data indicate that in human HFE-HC a disrupted BMP/SMAD signaling in the liver is key in the pathogenesis of the disease.
doi:10.1016/j.bcmd.2010.08.010
PMCID: PMC3295241  PMID: 20863724
iron overload; bone morphogenetic proteins; hepcidin; SMAD proteins
17.  Effects of iron loading on muscle: genome-wide mRNA expression profiling in the mouse 
BMC Genomics  2007;8:379.
Background
Hereditary hemochromatosis (HH) encompasses genetic disorders of iron overload characterized by deficient expression or function of the iron-regulatory hormone hepcidin. Mutations in 5 genes have been linked to this disease: HFE, TFR2 (encoding transferrin receptor 2), HAMP (encoding hepcidin), SLC40A1 (encoding ferroportin) and HJV (encoding hemojuvelin). Hepcidin inhibits iron export from cells into plasma. Hemojuvelin, an upstream regulator of hepcidin expression, is expressed in mice mainly in the heart and skeletal muscle. It has been suggested that soluble hemojuvelin shed by the muscle might reach the liver to influence hepcidin expression. Heart muscle is one of the target tissues affected by iron overload, with resultant cardiomyopathy in some HH patients. Therefore, we investigated the effect of iron overload on gene expression in skeletal muscle and heart using Illumina™ arrays containing over 47,000 probes. The most apparent changes in gene expression were confirmed using real-time RT-PCR.
Results
Genes with up-regulated expression after iron overload in both skeletal and heart muscle included angiopoietin-like 4, pyruvate dehydrogenase kinase 4 and calgranulin A and B. The expression of transferrin receptor, heat shock protein 1B and DnaJ homolog B1 were down-regulated by iron in both muscle types. Two potential hepcidin regulatory genes, hemojuvelin and neogenin, showed no clear change in expression after iron overload.
Conclusion
Microarray analysis revealed iron-induced changes in the expression of several genes involved in the regulation of glucose and lipid metabolism, transcription and cellular stress responses. These may represent novel connections between iron overload and pathological manifestations of HH such as cardiomyopathy and diabetes.
doi:10.1186/1471-2164-8-379
PMCID: PMC2151772  PMID: 17949489
18.  Of mice and men: the iron age 
Journal of Clinical Investigation  2005;115(8):2079-2082.
Recently, mutations causing juvenile hemochromatosis have been identified in a novel gene, hemojuvelin (HJV), located on chromosome 1. Mouse models of this disease have now been developed by 2 groups, Huang et al. and Niederkofler et al., through targeted disruption of the Hjv gene (see the related articles beginning on pages 2180 and 2187). These mutant mice will allow further investigation into the role of HJV in the regulation of iron homeostasis, a role that to date remains elusive.
doi:10.1172/JCI25642
PMCID: PMC1180554  PMID: 16075054
19.  Accelerated CCl4-Induced Liver Fibrosis in Hjv-/- Mice, Associated with an Oxidative Burst and Precocious Profibrogenic Gene Expression 
PLoS ONE  2011;6(9):e25138.
Hereditary hemochromatosis is commonly associated with liver fibrosis. Likewise, hepatic iron overload secondary to chronic liver diseases aggravates liver injury. To uncover underlying molecular mechanisms, hemochromatotic hemojuvelin knockout (Hjv-/-) mice and wild type (wt) controls were intoxicated with CCl4. Hjv-/- mice developed earlier (by 2-4 weeks) and more acute liver damage, reflected in dramatic levels of serum transaminases and ferritin and the development of severe coagulative necrosis and fibrosis. These responses were associated with an oxidative burst and early upregulation of mRNAs encoding α1-(I)-collagen, the profibrogenic cytokines TGF-β1, endothelin-1 and PDGF and, notably, the iron-regulatory hormone hepcidin. Hence, CCl4-induced liver fibrogenesis was exacerbated and progressed precociously in Hjv−/− animals. Even though livers of naïve Hjv−/− mice were devoid of apparent pathology, they exhibited oxidative stress and immunoreactivity towards α-SMA antibodies, a marker of hepatic stellate cells activation. Furthermore, they expressed significantly higher (2–3 fold vs. wt, p<0.05) levels of α1-(I)-collagen, TGF-β1, endothelin-1 and PDGF mRNAs, indicative of early fibrogenesis. Our data suggest that hepatic iron overload in parenchymal cells promotes oxidative stress and triggers premature profibrogenic gene expression, contributing to accelerated onset and precipitous progression of liver fibrogenesis.
doi:10.1371/journal.pone.0025138
PMCID: PMC3178612  PMID: 21966437
20.  Iron-Dependent Regulation of Hepcidin in Hjv−/− Mice: Evidence That Hemojuvelin Is Dispensable for Sensing Body Iron Levels 
PLoS ONE  2014;9(1):e85530.
Hemojuvelin (Hjv) is a bone morphogenetic protein (BMP) co-receptor involved in the control of systemic iron homeostasis. Functional inactivation of Hjv leads to severe iron overload in humans and mice due to marked suppression of the iron-regulatory hormone hepcidin. To investigate the role of Hjv in body iron sensing, Hjv−/− mice and isogenic wild type controls were placed on a moderately low, a standard or a high iron diet for four weeks. Hjv−/− mice developed systemic iron overload under all regimens. Transferrin (Tf) was highly saturated regardless of the dietary iron content, while liver iron deposition was proportional to it. Hepcidin mRNA expression responded to fluctuations in dietary iron intake, despite the absence of Hjv. Nevertheless, iron-dependent upregulation of hepcidin was more than an order of magnitude lower compared to that seen in wild type controls. Likewise, iron signaling via the BMP/Smad pathway was preserved but substantially attenuated. These findings suggest that Hjv is not required for sensing of body iron levels and merely functions as an enhancer for iron signaling to hepcidin.
doi:10.1371/journal.pone.0085530
PMCID: PMC3883712  PMID: 24409331
21.  HFE gene mutation, C282Y causing hereditary hemochromatosis in Caucasian is extremely rare in Korean population. 
Journal of Korean Medical Science  2000;15(2):179-182.
Hereditary hemochromatosis (HFE), which affects 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals in Western population, results in multiple organ damage caused by iron deposition, and is treatable if detected early. C282Y mutation in HFE gene has been known to be responsible for the most hereditary hemochromatosis cases and 5-10% of white subjects are heterozygous for this mutation. However, the prevalence of hemochromatosis in the Asian population was reported to be very low and ethnic heterogeneity has been suspected. The aim of our study was to determine the prevalence of heterozygosity and homozygosity for the C282Y HFE gene mutations in 502 unrelated Koreans. Results revealed that none of them had the mutant gene, suggesting a significant ethnic difference when compared with Caucasians. Our study excluded underlying possibility of hereditary hemochromatosis in Korean which could mimic the findings of alcoholic liver disease with iron overload or liver cirrhosis with chronic hepatitis C.
PMCID: PMC3054623  PMID: 10803694
22.  SLC40A1 Q248H allele frequencies and Q248H-associated risk of non-HFE iron overload in persons of sub-Saharan African descent 
Blood cells, molecules & diseases  2007;39(2):206-211.
The ferroportin polymorphism SLC40A1 Q248H (exon 6, cDNA 744G→T; Gln248His) occurs in persons of sub-Saharan African descent with and without iron overload, and is associated with elevated serum ferritin concentrations (SF). However, the risk of iron overload associated with Q248H has not been defined. We tabulated previously reported Q248H allele frequency estimates in African Americans and Native Africans, and computed the risk of iron overload associated with Q248H in subjects who lacked HFE C282Y. The aggregate Q248H allele frequency in 1,038 African Americans in two cohorts from Alabama and one cohort each from Washington, D.C. and California was 0.0525 (95% CI: 0.0451, 0.0652); there was no significant difference in frequencies across these cohorts. The aggregate frequency in 259 Natives from southeast Africa in two cohorts was 0.0946 (95% CI: 0.0694, 0.1198); the difference between the frequencies of these cohorts was not significant. The aggregate Q248H frequencies in African Americans and Native Africans differed significantly (0.0525 vs. 0.0946, respectively; p = 0.0021). There were reports of 24 unrelated African Americans and 15 unrelated Native Africans without HFE C282Y who had iron overload. In African Americans, the odds ratio (OR) of Q248H-associated risk of iron overload using 610 C282Y-negative control subjects unselected for SF was 1.57 (95% CI: 0.52, 4.72; p = 0.29). In Native Africans, the OR using 208 control subjects unselected for SF was 1.05 (95% CI: 0.28, 3.90; p = 0.58). We conclude that the frequency of SLC40A1 Q248H is significantly lower in African Americans than in Native Africans. Although OR estimates of iron overload in African Americans and Native Africans with Q248H were greater than unity, the increased OR were not statistically significant.
doi:10.1016/j.bcmd.2007.03.008
PMCID: PMC1986732  PMID: 17490902
ferroportin; genetics; hemojuvelin A310G; mutation
23.  Recent advances in understanding haemochromatosis: a transition state 
Journal of Medical Genetics  2004;41(10):721-730.
Mutations in the hepcidin gene HAMP and the hemojuvelin gene HJV have recently been shown to result in juvenile haemochromatosis (JH). Hepcidin is an antimicrobial peptide that plays a key role in regulating intestinal iron absorption. Hepcidin levels are reduced in patients with haemochromatosis due to mutations in the HFE and HJV genes. Digenic inheritance of mutations in HFE and HAMP can result in either JH or hereditary haemochromatosis (HH) depending upon the severity of the mutation in HAMP. Here we review these findings and discuss how understanding the different types of haemochromatosis and our increasing knowledge of iron metabolism may help to elucidate the host's response to infection.
doi:10.1136/jmg.2004.020644
PMCID: PMC1735598  PMID: 15466004
24.  Non-HFE hemochromatosis 
Hereditary hemochromatosis (HH) is an autosomal recessive disorder classically related to HFE mutations. However, since 1996, it is known that HFE mutations explain about 80% of HH cases, with the remaining around 20% denominated non-HFE hemochromatosis. Nowadays, four main genes are implicated in the pathophysiology of clinical syndromes classified as non-HFE hemochromatosis: hemojuvelin (HJV, type 2Ajuvenile HH), hepcidin (HAMP, type 2B juvenile HH), transferrin receptor 2 (TFR2, type 3 HH) and ferroportin (SLC40A1, type 4 HH). The aim of this review is to explore molecular, clinical and management aspects of non-HFE hemochromatosis.
doi:10.5581/1516-8484.20120079
PMCID: PMC3460409  PMID: 23049448
Hemochromatosis; Iron overload; Iron metabolism disorders
25.  Juvenile Hemochromatosis, Genetic Study and Long-term Follow up after Therapy 
BACKGROUND
Hereditary hemochromatosis (HH) is a very rare disease in Iran and reported cases are all negative for HFE mutation. We report a family affected by severe juvenile hemochromatosis (JH) with a detailed molecular study of the family members.
METHODS
We studied a pedigree with siblings affected by juvenile HH and followed them for 3 years. Microsatellite and gene sequencing analysis was performed for all family members.
RESULTS
Two siblings (the proband and his sister, aged 26 and 30 years, respectively) were found to have clinical findings of JH. The proband’s brother, who presented with hyperpigmentation, died of probable JH at the age of 24 years. Gene sequencing analysis showed that the proband has a homozygote c.265T>C (p.C89R) HJV mutation + a heterozygote c.884T>C (p.V295A) mutation of HFE. The affected proband’s sister presented with the same HJV c.265T>C (p.C89R) homozygote mutation. In addition, we found the HJV c.98-6C>G polymorphic variant in both the sister and proband (homozygote). Sequencing of hepcidin (HAMP), TfR2, and FPN revealed no mutation.
CONCLUSION
We have shown that molecular analysis of the HH related gene is a powerful tool for reliable diagnosis of JH and, in conjunction with magnetic resonance imaging (MRI) and noninvasive liver stiffness measurement by elastography, is adequate tool for management and follow up of HH.
PMCID: PMC4034669  PMID: 24872867
Juvenile Hemochromatosis; Hemojuvelin Mutation; Genetic Study; Iran

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