Pathogenesis Of Hereditary Hemochromatosis Research Articles

DENND3 p.L708V activating variant is involved in the pathogenesis of hereditary hemochromatosis via the RAB12/TFR2 signaling pathway.

Pathogenic variants in HFE and non-HFE genes have been identified in hereditary hemochromatosis (HH) in different patient populations, but there are still a considerable proportion of patients with unexplained primary iron overload. We recently identified in Chinese patients with unexplained primary iron overload a recurrent p.L708V variant in the differentially expressed in normal and neoplastic cells domain 3 (DENND3) gene, functioning as a guanine nucleotide exchange factor for small GTpase Rab12 which down-regulates TfR expression in mice. We aim to investigate the pathogenicity and the underlying mechanism of the DENND3 p.L708V variant in HH patients. Patients with primary iron overload were analyzed for DENND3 p.L708V. TFR2 and hepcidin expression in livers were examined in HH patients harboring DENND3 p.L708V. The effects of DENND3 p.L708V on RAB12/TFR2 and downstream iron metabolic pathways were investigated in vitro and in vivo. Six of 31 patients with HH (19.35%) harbored the DENND3 p.L708V variant. The expression of TFR2 and hepcidin was decreased in the liver of HH patients with DENND3 p.L708V. Cells transfected with the DENND3 p.L708V vector showed up-regulation of RAB12 expression and TFR2 degradation in lysosomes, and down-regulation of the pSMAD1/5 and hepcidin. Mice models infected with adeno-associated virus expressing DENND3 p.L708V variant showed higher total serum iron concentrations and decreased HAMP level, increased amount of iron accumulation and the down-regulated of TFR2 expression in the liver. The DENND3 p.L708V activating variant down-regulates hepcidin expression through the DENND3/RAB12/TFR2 axis, which may represent a potential novel pathogenic factor of HH.

HJV and HFE Play Distinct Roles in Regulating Hepcidin.

Hereditary hemochromatosis (HH) is an iron overload disease that is caused by mutations in HFE, HJV, and several other genes. However, whether HFE-HH and HJV-HH share a common pathway via hepcidin regulation is currently unclear. Recently, some HH patients have been reported to carry concurrent mutations in both the HFE and HJV genes. To dissect the roles and molecular mechanisms of HFE and/or HJV in the pathogenesis of HH, we studied Hfe(-/-), Hjv(-/-), and Hfe(-/-)Hjv(-/-) double-knockout mouse models. Hfe(-/-)Hjv(-/-) mice developed iron overload in multiple organs at levels comparable to Hjv(-/-) mice. After an acute delivery of iron, the expression of hepcidin (i.e., Hamp1 mRNA) was increased in the livers of wild-type and Hfe(-/-) mice, but not in either Hjv(-/-) or Hfe(-/-)Hjv(-/-) mice. Furthermore, iron-induced phosphorylation of Smad1/5/8 was not detected in the livers of Hjv(-/-) or Hfe(-/-)Hjv(-/-) mice. We generated and phenotypically characterized Hfe(-/-)Hjv(-/-) double-knockout mice. In addition, because they faithfully phenocopy clinical HH patients, these mouse models are an invaluable tool for mechanistically dissecting how HFE and HJV regulate hepcidin expression. Based on our results, we conclude that HFE may depend on HJV for transferrin-dependent hepcidin regulation. The presence of residual hepcidin in the absence of HFE suggests either the presence of an unknown regulator (e.g., TFR2) that is synergistic with HJV or that HJV is sufficient to maintain basal levels of hepcidin.

A Novel Rat Model of Hereditary Hemochromatosis Due to a Mutation in Transferrin Receptor 2

AbstractAbstract 612Sporadic iron overload has been reported previously in rats but the underlying cause has not been ascertained. In this study, phenotypic analysis of a subpopulation of Wistar rats designated Hsd:HHCL revealed a low incidence of histologically detected liver iron overload. One rat out of 132 screened animals exhibited liver iron accumulation in a predominantly periportal, hepatocellular distribution; this male rat expressed low RNA levels of the iron regulatory hormone hepcidin and low protein levels of transferrin receptor 2, a membrane protein essential for hepcidin expression in humans and mice and mutated in forms of hereditary hemochromatosis, a disease of excessive intestinal iron absorption and progressive tissue iron overload. Sequencing of the transferrin receptor 2 gene in the iron-overloaded rat revealed a novel Ala679Gly polymorphism affecting a highly conserved residue. Quantitative trait locus mapping revealed that a transferrin receptor 2 polymorphism correlated strongly with serum iron and transferrin saturations in male rats. Transfection of Tfr2 expression constructs into tissue culture cell lines revealed that the Gly679 Tfr2 variant is expressed at a lower level than the Ala679 variant. Selective breeding of rats carrying this polymorphism and characterization of iron metabolism in the resulting progeny indicated that homozygosity for the Ala679Gly allele leads to a hemochromatosis phenotype. The Hsd:HHCL rat is the first genetic rat model of hereditary hemochromatosis and may prove useful for understanding the molecular mechanisms underlying the regulation of iron metabolism and the pathogenesis of hereditary hemochromatosis. Disclosures:No relevant conflicts of interest to declare.

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.

HFE gene mutations in patients with primary iron overload: Is there a significant improvement in molecular diagnosis yield with HFE sequencing?

Rare HFE variants have been shown to be associated with hereditary hemochromatosis (HH), an iron overload disease. The low frequency of the HFE p.C282Y mutation in HH-affected Brazilian patients may suggest that other HFE-related mutations may also be implicated in the pathogenesis of HH in this population. The main aim was to screen for new HFE mutations in Brazilian individuals with primary iron overload and to investigate their relationship with HH. Fifty Brazilian patients with primary iron overload (transferrin saturation > 50% in females and 60% in males) were selected. Subsequent bidirectional sequencing for each HFE exon was performed. The effect of HFE mutations on protein structure were analyzed by molecular dynamics simulation and free binding energy calculations. p.C282Y in homozygosis or in heterozygosis with p.H63D were the most frequent genotypic combinations associated with HH in our sample population (present in 17 individuals, 34%). Thirty-six (72.0%) out of the 50 individuals presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation ( n = 11, 22.0%). One novel mutation (p.V256I) was indentified in heterozygosis with the p.H63D mutation. In silico modeling analysis of protein behavior indicated that the p.V256I mutation does not reduce the binding affinity between HFE and β2-microglobulin (β2M) in the same way the p.C282Y mutation does compared with the native HFE protein. In conclusion, screening of HFE through direct sequencing, as compared to p.C282Y/p.H63D genotyping, was not able to increase the molecular diagnosis yield of HH. The novel p.V256I mutation could not be implicated in the molecular basis of the HH phenotype, although its role cannot be completely excluded in HH-phenotype development. Our molecular modeling analysis can help in the analysis of novel, previously undescribed, HFE mutations.

Analysis of HFE And Non-HFE Gene Mutations in Brazilian Patients with Hemochromatosis

BACKGROUNDApproximately one-half of Brazilian patients with hereditary hemochromatosis (HH) are neither homozygous for the C282Y mutation nor compound heterozygous for the H63D and C282Y mutations that are associated with HH in Caucasians. Other mutations have been described in the HFE gene as well as in genes involved in iron metabolism, such as transferrin receptor 2 (TfR2) and ferroportin 1 (SCL40A1).AIMSTo evaluate the role of HFE, TfR2 and SCL40A1 mutations in Brazilian subjects with HH.PATIENTS AND METHODSNineteen male subjects (median age 42 [range: 20–72] years) with HH were evaluated using the Haemochromatosis StripAssay A®. This assay is capable of detecting twelve HFE mutations, which are V53M, V59M, H63D, H63H, S65C, Q127H, P160delC, E168Q, E168X, W169X, C282Y and Q283, four TfR2 mutations, which are E60X, M172K, Y250X, AVAQ594-597del, and two SCL40A1 mutations, which are N144H and V162del.RESULTSIn our cohort, nine (47%) patients were homozygous for the C282Y mutation, two (11%) were heterozygous for the H63D mutation, and one each (5%) was either heterozygous for C282Y or compound heterozygous for C282Y and H63D. No other mutations in the HFE, TfR2 or SCL40A1 genes were observed in the studied patients.CONCLUSIONSOne-third of Brazilian subjects with the classical phenotype of HH do not carry HFE or other mutations that are currently associated with the disease in Caucasians. This observation suggests a role for other yet unknown mutations in the aforementioned genes or in other genes involved in iron homeostasis in the pathogenesis of HH in Brazil.

SELDI-TOF MS detection of urinary hepcidin

Hepcidin is a 25-residue hepatic peptide that regulates iron absorption from the diet and tissue iron distribution. Inappropriately low Hepcidin expression is implicated in the pathogenesis of hereditary hemochromatosis and iron-loading anemias, like the thalassemias. Increased hepcidin expression mediates iron retention in the anemias of inflammation and plays a pathogenic role in iron-refractory iron-deficiency anemia (IRIDA). Because of its clinical importance, Hepcidin is expected to be a useful biomarker for diagnosis and management of iron-related disorders. So far an ELISA for human hepcidin and SELDI-TOF-MS based approaches have been applied to monitor urinary and/or serum hepcidin levels. Here we report a modified protocol for SELDI-TOF based detection of human, urinary hepcidin. We show that CM10 Proteinchips are superior to NP20 Proteinchips commonly used in previously reported protocols to sensitively and accurately detect urinary hepcidin. Application of this modified hepcidin assay accurately detects increased hepcidin levels in the urine of sepsis patients.

UGT1A1 gene polymorphism as a potential factor inducing iron overload in the pathogenesis of type 1 hereditary hemochromatosis

Aim Hereditary hemochromatosis is a common genetic disorder characterized by iron overload and subsequent organ damage. It is caused in most cases by HFE gene mutations which penetrance can be affected by many factors. The aim of this study was to establish the role of UGT1A1 gene polymorphism and serum bilirubin concentration in the pathogenesis of hereditary hemochromatosis. Methods Biochemical, histopathological and genetic data indicating iron excess and serum total bilirubin concentration were determined in 32 patients with the type 1 hereditary hemochromatosis. Fluorescent molecular probes assays were used for genotyping of UGT1A1*28 and UGT1A1*60 mutations in these individuals. Results High incidence and a significant correlation of UGT1A1 gene mutations with increased serum bilirubin level and lower grades of liver tissue inflammatory activity were observed in study participants. UGT1A1*28 and UGT1A1*60 mutations were strongly linked together. Two of the subjects presented very rare genotypes of UGT1A1 gene: (TA)(5/7) and c.-64G>C heterozygotes. Conclusions UGT1A1 gene polymorphism and as its consequence of high serum bilirubin level may promote iron accumulation in hemochromatosis patients by reducing the activity of inflammation. We proposed a possible mechanism of this interaction.

Hfe Acts in Hepatocytes to Prevent Hemochromatosis

Hereditary hemochromatosis (HH) is a prevalent, potentially fatal disorder of iron metabolism hallmarked by intestinal hyperabsorption of iron, hyperferremia, and hepatic iron overload. In both humans and mice, type I HH is associated with mutations in the broadly expressed HFE/Hfe gene. To identify where Hfe acts to prevent HH, we generated mice with tissue-specific Hfe ablations. This work demonstrates that local Hfe expression in hepatocytes serves to maintain physiological iron homeostasis, answering a long-standing question in medicine and explaining earlier clinical observations.

Physiologic systemic iron metabolism in mice deficient for duodenal Hfe

AbstractMutations in the Hfe gene result in hereditary hemochromatosis (HH), a disorder characterized by increased duodenal iron absorption and tissue iron overload. Identification of a direct interaction between Hfe and transferrin receptor 1 in duodenal cells led to the hypothesis that the lack of functional Hfe in the duodenum affects TfR1-mediated serosal uptake of iron and misprogramming of the iron absorptive cells. Contrasting this view, Hfe deficiency causes inappropriately low expression of the hepatic iron hormone hepcidin, which causes increased duodenal iron absorption. We specifically ablated Hfe expression in mouse enterocytes using Cre/LoxP technology. Mice with efficient deletion of Hfe in crypt- and villi-enterocytes maintain physiologic iron metabolism with wild-type unsaturated iron binding capacity, hepatic iron levels, and hepcidin mRNA expression. Furthermore, the expression of genes encoding the major intestinal iron transporters is unchanged in duodenal Hfe-deficient mice. Our data demonstrate that intestinal Hfe is dispensable for the physiologic control of systemic iron homeostasis under steady state conditions. These findings exclude a primary role for duodenal Hfe in the pathogenesis of HH and support the model according to which Hfe is required for appropriate expression of the “iron hormone” hepcidin which then controls intestinal iron absorption.

The Regulation of Cellular Iron Metabolism

While iron is an essential trace element required by nearly all living organisms, deficiencies or excesses can lead to pathological conditions such as iron deficiency anemia or hemochromatosis, respectively. A decade has passed since the discovery of the hemochromatosis gene, HFE, and our understanding of hereditary hemochromatosis (HH) and iron metabolism in health and a variety of diseases has progressed considerably. Although HFE-related hemochromatosis is the most widespread, other forms of HH have subsequently been identified. These forms are not attributed to mutations in the HFE gene but rather to mutations in genes involved in the transport, storage, and regulation of iron. This review is an overview of cellular iron metabolism and regulation, describing the function of key proteins involved in these processes, with particular emphasis on the liver's role in iron homeostasis, as it is the main target of iron deposition in pathological iron overload. Current knowledge on their roles in maintaining iron homeostasis and how their dysregulation leads to the pathogenesis of HH are discussed.

Tratamiento de la hemocromatosis hereditaria tipo 1 con magnesio oral

An essential step in the pathogenesis of hereditary hemochromatosis seems to be the increased expression of a duodenal divalent cation transporter (DMT1) responsible for absorption of non-heminic iron2+. The objective of the present study was to ascertain whether the competitive blockade of DMT1 by the administration of high doses of oral Mg2+ reduces iron absorption in patients homozygous for the C282Y mutation.Iron absorption was evaluated by a low dose iron absorption test in 15 patients before and after treatment with oral magnesium (809.6 mg every 8 hours) for two weeks.We did not observe secondary effects or significant differences in iron absorption before or after magnesium treatment (14.7 micromol/L; 95% confidence interval [CI], 9.8-19.6 vs 14.9 micromol/L; 95% CI, 8.5-21.4, P = 0.7).Treatment with oral magnesium does not reduce iron absorption in homozygous C282Y patients. This treatment can not be used in these subjects.

Transferrin receptor 1

With the discovery that transferrin serves as the iron source for hemoglobin-synthesizing immature red blood cells came the demonstration that a cell surface receptor, now known as transferrin receptor 1, is required for iron delivery from transferrin to cells. (A recently described second transferrin receptor, with as yet poorly understood function, will not be discussed in this brief review.) In succeeding years transferrin receptor 1 was established as a gatekeeper for regulating iron uptake by most cells, and the transferrin-to-cell endocytic pathway characterized in detail. HFE, the protein incriminated in the pathogenesis of hereditary hemochromatosis, a disorder of progressive and toxic iron overload, competes with transferrin for binding to receptor, thereby impeding the uptake of iron from transferrin. Mutation of HFE destroys this competition, thus facilitating access of transferrin and its iron to cells. Availability of the crystal structure of transferrin receptor 1, along with those of transferrin and HFE, opened research on molecular mapping of the transferrin-HFE- transferrin receptor interfaces by correlated synchrotron-generated hydroxyl radical footprinting and cryo-electron microscopy. The emerging challenge is to relate structure to the functional effects of receptor binding on the iron-binding and iron-releasing properties of transferrin within the iron-dependent cell.

What's new in hemochromatosis.

Hereditary hemochromatosis (HHC) is an inherited disorder of iron metabolism affecting approximately 1 in 200-300 individuals of Northern European descent. Over time, the continued deposition of iron in parenchymal cells of many organs can eventually lead to diabetes mellitus, cardiomyopathy, and hepatic cirrhosis, the last of which is frequently followed by hepatocellular carcinoma. Although the complications of HHC can be devastating, its clinical management is simple and effective if the disease is identified early in its progression. The recent elucidation of the HFE gene has provided insight into the pathogenesis of HHC and provided a means for the early identification of individuals in whom HHC may develop. Two mutations have been implicated in HHC: C282Y and H63D. The former occurs in a homozygous state seen in 75-100% of patients with HHC. The high correlation of HFE to HHC has caused it to be considered as a candidate gene for population-based genetic testing for diagnosis and detection of predisposition to HHC. In addition, mechanisms of iron transport and metabolism are unfolding and are providing clues to the enigma of iron homeostasis and the pathophysiology of iron overload.

Genes that modify the hemochromatosis phenotype in mice.

Hereditary hemochromatosis (HH) is a prevalent human disease caused by a mutation in HFE, which encodes an atypical HLA class I protein involved in regulation of intestinal iron absorption. To gain insight into the pathogenesis of hemochromatosis, we have bred Hfe knockout mice to strains carrying other mutations that impair normal iron metabolism. Compound mutant mice lacking both Hfe and its interacting protein, beta-2 microglobulin (B2m), deposit more tissue iron than mice lacking Hfe only, suggesting that another B2m-interacting protein may be involved in iron regulation. Hfe knockout mice carrying mutations in the iron transporter DMT1 fail to load iron, indicating that hemochromatosis involves iron flux through DMT1. Similarly, compound mutants deficient in both Hfe and hephaestin (Heph) show less iron loading than do Hfe knockout mice, indicating that iron absorption in hemochromatosis involves the function of Heph as well. Finally, compound mutants lacking Hfe and the transferrin receptor accumulate more tissue iron than do mice lacking Hfe alone, consistent with the idea that interaction between these two proteins contributes to the control of normal iron absorption. In addition to providing insight into the pathogenesis of HH, our results suggest that each of these genes might be a candidate modifier of the human hemochromatosis phenotype.

The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding.

We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis called HFE. The gene product, a member of the major histocompatibility complex class I-like family, was found to have a mutation, Cys-282 --> Tyr (C282Y), in 85% of patient chromosomes. This mutation eliminates the ability of HFE to associate with beta2-microglobulin (beta2m) and prevents cell-surface expression. A second mutation that has no effect on beta2m association, H63D, was found in eight out of nine patients heterozygous for the C282Y mutant. In this report, we demonstrate in cultured 293 cells overexpressing wild-type or mutant HFE proteins that both the wild-type and H63D HFE proteins form stable complexes with the transferrin receptor (TfR). The C282Y mutation nearly completely prevents the association of the mutant HFE protein with the TfR. Studies on cell-associated transferrin at 37 degrees C suggest that the overexpressed wild-type HFE protein decreases the affinity of the TfR for transferrin. The overexpressed H63D protein does not have this effect, providing the first direct evidence for a functional consequence of the H63D mutation. Addition of soluble wild-type HFE/beta2m heterodimers to cultured cells also decreased the apparent affinity of the TfR for its ligand under steady-state conditions, both in 293 cells and in HeLa cells. Furthermore, at 4 degrees C, the added soluble complex of HFE/beta2m inhibited binding of transferrin to HeLa cell TfR in a concentration-dependent manner. Scatchard plots of these data indicate that the added heterodimer substantially reduced the affinity of TfR for transferrin. These results establish a molecular link between HFE and a key protein involved in iron transport, the TfR, and raise the possibility that alterations in this regulatory mechanism may play a role in the pathogenesis of hereditary hemochromatosis.