Transferrin Receptor 2 Mutations Research Articles

Inherited Disorders of Iron Overload.

Dietary iron absorption and systemic iron traffic are tightly controlled by hepcidin, a liver-derived peptide hormone. Hepcidin inhibits iron entry into plasma by binding to and inactivating the iron exporter ferroportin in target cells, such as duodenal enterocytes and tissue macrophages. Hepcidin is induced in response to increased body iron stores to inhibit further iron absorption and prevent iron overload. The mechanism involves the BMP/SMAD signaling pathway, which triggers transcriptional hepcidin induction. Inactivating mutations in components of this pathway cause hepcidin deficiency, which allows inappropriately increased iron absorption and efflux into the bloodstream. This leads to hereditary hemochromatosis (HH), a genetically heterogenous autosomal recessive disorder of iron metabolism characterized by gradual buildup of unshielded non-transferrin bound iron (NTBI) in plasma and excessive iron deposition in tissue parenchymal cells. The predominant HH form is linked to mutations in the HFE gene and constitutes the most frequent genetic disorder in Caucasians. Other, more severe and rare variants are caused by inactivating mutations in HJV (hemojuvelin), HAMP (hepcidin) or TFR2 (transferrin receptor 2). Mutations in SLC40A1 (ferroportin) that cause hepcidin resistance recapitulate the biochemical phenotype of HH. However, ferroportin-related hemochromatosis is transmitted in an autosomal dominant manner. Loss-of-function ferroportin mutations lead to ferroportin disease, characterized by iron overload in macrophages and low transferrin saturation. Aceruloplasminemia and atransferrinemia are further inherited disorders of iron overload caused by deficiency in ceruloplasmin or transferrin, the plasma ferroxidase and iron carrier, respectively.

Transferrin Receptors TfR1 and TfR2 Bind Transferrin through Differing Mechanisms.

Hereditary hemochromatosis (HH), a disease marked by chronic iron overload from insufficient expression of the hormone hepcidin, is one of the most common genetic diseases. One form of HH (type III) results from mutations in transferrin receptor-2 (TfR2). TfR2 is postulated to be a part of signaling system that is capable of modulating hepcidin expression. However, the molecular details of TfR2's role in this system remain unclear. TfR2 is predicted to bind the iron carrier transferrin (Tf) when the iron saturation of Tf is high. To better understand the nature of these TfR-Tf interactions, a binding study with the full-length receptors was conducted. In agreement with previous studies with truncated forms of these receptors, holo-Tf binds to the TfR1 homologue significantly stronger than to TfR2. However, the binding constant for Tf-TfR2 is still far above that of physiological holo-Tf levels, inconsistent with the hypothetical model, suggesting that other factors mediate the interaction. One possible factor, apo-Tf, only weakly binds TfR2 at serum pH and thus will not be able to effectively compete with holo-Tf. Tf binding to a TfR2 chimera containing the TfR1 helical domain indicates that the differences in the helical domain account for differences in the on rate of Tf, and nonconserved inter-receptor interactions are necessary for the stabilization of the complex. Conserved residues at one possible site of stabilization, the apical arm junction, are not important for TfR1-Tf binding but are critical for the TfR2-Tf interaction. Our results highlight the differences in Tf interactions with the two TfRs.

Hematopoietic deletion of transferrin receptor 2 in mice leads to a block in erythroid differentiation during iron-deficient anemia.

Iron metabolism and erythropoiesis are inherently interlinked physiological processes. Regulation of iron metabolism is mediated by the iron-regulatory hormone hepcidin. Hepcidin limits the amount of iron released into the blood by binding to and causing the internalization of the iron exporter, ferroportin. A number of molecules and physiological stimuli, including erythropoiesis, are known to regulate hepcidin. An increase in erythropoietic demand decreases hepcidin, resulting in increased bioavailable iron in the blood. Transferrin receptor 2 (TFR2) is involved in the systemic regulation of iron metabolism. Patients and mice with mutations in TFR2 develop hemochromatosis due to inappropriate hepcidin levels relative to body iron. Recent studies from our laboratory and others have suggested an additional role for TFR2 in response to iron-restricted erythropoiesis. These studies used mouse models with perturbed systemic iron metabolism: anemic mice lacking matriptase-2 and Tfr2, or bone marrow transplants from iron-loaded Tfr2 null mice. We developed a novel transgenic mouse model which lacks Tfr2 in the hematopoietic compartment, enabling the delineation of the role of Tfr2 in erythroid development without interfering with its role in systemic iron metabolism. We show that in the absence of hematopoietic Tfr2 immature polychromatic erythroblasts accumulate with a concordant reduction in the percentage of mature erythroid cells in the spleen and bone marrow of anemic mice. These results demonstrate that erythroid Tfr2 is essential for an appropriate erythropoietic response in iron-deficient anemia. These findings may be of relevance in clinical situations in which an immediate and efficient erythropoietic response is required. Am. J. Hematol. 91:812-818, 2016. © 2016 Wiley Periodicals, Inc.

Functional consequences of transferrin receptor-2 mutations causing hereditary hemochromatosis type 3.

Hereditary hemochromatosis (HH) type 3 is an autosomal recessive disorder of iron metabolism characterized by excessive iron deposition in the liver and caused by mutations in the transferrin receptor 2 (TFR2) gene. Here, we describe three new HH type 3 Spanish families with four TFR2 mutations (p.Gly792Arg, c.1606-8A>G, Gln306*, and Gln672*). The missense variation p.Gly792Arg was found in homozygosity in two adult patients of the same family, and in compound heterozygosity in an adult proband that also carries a novel intronic change (c.1606-8A>G). Two new nonsense TFR2 mutations (Gln306* and Gln672*) were detected in a pediatric case. We examine the functional consequences of two TFR2 variants (p.Gly792Arg and c.1606-8A>G) using molecular and computational methods. Cellular protein localization studies using immunofluorescence demonstrated that the plasma membrane localization of p.Gly792Arg TFR2 is impaired. Splicing studies in vitro and in vivo reveal that the c.1606-8A>G mutation leads to the creation of a new acceptor splice site and an aberrant TFR2 mRNA. The reported mutations caused HH type 3 by protein truncation, altering TFR2 membrane localization or by mRNA splicing defect, producing a nonfunctional TFR2 protein and a defective signaling transduction for hepcidin regulation. TFR2 genotyping should be considered in adult but also in pediatric cases with early-onset of iron overload.

Clinical Testing of Five Hereditary Hemochromatosis-Related Genes: Preliminary Evidence for the Benefit of Next Generation Sequencing

▪IntroductionHereditary hemochromatosis (HH) is a genetic form of iron overload. In cases of excessive iron deposition, serious clinical manifestations may occur, such as liver damage, cardiomyopathy, diabetes, and arthritis. First described in 1996, the HFE gene leads to autosomal recessive HH with reduced penetrance. In other words, two mutations in the HFE gene need to be present in a patient in order to develop symptoms of HFE-related HH, but not all patients with two mutations are affected. In the last 15 years, 4 additional genes were discovered that cause HH: HAMP (hepcidin), HFE2 (hemojuvelin), SLC40A1 (ferroportin), and TFR2 (transferrin receptor 2). HAMP, HFE2 and TFR2 mutations are inherited in a recessive pattern, whereas SLC40A1 mutations are inherited in a dominant pattern. HAMP and HFE2 mutations cause a severe, early-onset form of HH. There is some evidence that sequence changes in HAMP, HFE2 and TFR2 may interact with homozygous HFE mutations, causing a more severe phenotype. Current HH testing guidelines only exist for the most common HFE mutations (C282Y and H63D), with no specific recommendations regarding full gene sequencing for any of the HH genes. Recent research suggests that sequential sequencing may be beneficial in patients who test negative for the most common HFE mutations, exhibit a more severe or early-onset phenotype compared to what is normally seen in HFE-related HH, and/or are of non-Caucasian ethnicity. Next Generation Sequencing (NGS) is a new high-throughput sequencing technology that allows testing of multiple genes concurrently and can detect rare and novel HH-causing mutations that are not typically assayed using targeted methods. However, sequencing can also identify sequence changes known as variants of uncertain significance (VUS) - changes that have not yet been characterized as disease-causing or benign. This abstract summarizes the results of clinical NGS for the five HH-related genes, and shows preliminary evidence as to its’ increased diagnostic yield for HH diagnosis.MethodsPatients were referred for clinical full gene sequencing of HFE, HAMP, HFE2, SLC40A1, and/or TFR2 using Next Generation Sequencing (Illumina MiSeq). Results from patients with a clinical indication of iron overload or HH who were tested from 9/2013 - 7/2014 were reviewed. The diagnostic yield of sequencing for all five HH genes was determined. Patients who only had sequencing for a subset of the five genes were analyzed separately. Patients who had testing for a familial mutation were excluded from the review.ResultsIn total, 56 patients underwent HH-related NGS. Thirty-five (62.5%) were males and 21 (37.5%) were females. Ages ranged from 3-77yrs (avg. 40.9yrs). Fifty-one percent were Caucasian, 9% Hispanic, 4% African American, 16% Asian, and 20% not specified.Forty-one patients were tested for all five genes. HH-causing mutations were found in 9 patients: 5 (12.2%) were c.187C>G (p.H63D) HFE homozygous, 1 (2.4%) was c.845G>A (p.C282Y) HFE homozygous, and 3 (7.3%) had mutations in non-HFE genes: SLC40A1 c.430A>T (p.N144Y) heterozygous, SLC40A1 c.533G>A (p.R178Q) heterozygous, and HFE2 c.959G>T (p.R178Q) homozygous. Nine patients (22%) were heterozygous carriers of an HFE mutation. One or more VUSes were found in 5 patients (12.2%). In 18 patients (43.9%), no pathogenic mutations or VUSes were found. There were 15 additional patients who only had sequencing of 1-3 of the available genes. Results for those patients consisted of 1 H63D HFE homozygote, 3 HFE heterozygotes (2 H63D and 1 C282Y) and 1 VUS.ConclusionsThe new sequencing technology of NGS makes it possible to test multiple genes at the same time. In this sample, sequencing of HFE, HAMP, HFE2, SLC40A1, and TFR2 genes resulted in an additional diagnostic yield compared to HFE C282Y and H63D testing alone. In patients who have a genetic explanation for their HH, management can be personalized based on genotype-phenotype correlation (e.g. N144Y SLC40A1 mutations may lead to reduced phlebotomy tolerance) and at-risk family members can be screened. In addition, all patients in this sample with non-HFE positive results were reportedly Caucasian, highlighting the benefit of sequencing regardless of ethnic background. This preliminary study is an important step toward gaining a better understanding of the genetics of HH. Ultimately, NGS data may make it possible to update current clinical guidelines for HH. DisclosuresRabideau:Invitae: Employment, Equity Ownership.

An Essential Role For Transferrin Receptor 2 In Erythropoiesis During Iron Restriction

Iron deficiency and iron overload are common clinical conditions that impact on the health and wellbeing of up to 30% of the world's population. Understanding mechanisms regulating iron homeostasis will provide improved strategies for treating these disorders. The liver-expressed proteins matriptase-2 (encoded by TMPRSS6), HFE and transferrin receptor 2 (TFR2) play important and opposing roles in systemic iron homeostasis by regulating expression of the iron regulatory hormone hepcidin. Mutations in TMPRSS6 lead to iron refractory iron deficiency anemia, whereas mutations in HFE and TFR2 lead to the iron overload disorder hereditary hemochromatosis. To elucidate the competing roles of these hepcidin regulators, we created mice lacking matriptase-2, Hfe and Tfr2.Tmprss6-/-/Hfe-/-/Tfr2-/- mice had iron deficiency anemia resulting from hepatic hepcidin over-expression and activation of Smad1/5/8, indicating that matriptase-2 predominates over Hfe and Tfr2 in hepcidin regulation. Surprisingly, this anemia was more severe than in the Tmprss6-/- mice, demonstrated by more extensive alopecia, lower hematocrit and significant extramedullary erythropoiesis in the spleen. There was increased expression of erythroid-specific genes in the spleens of Tmprss6-/-/Hfe-/-/Tfr2-/- mice, consistent with the extramedullary erythropoiesis. Expression of Tfr2 but not Hfe in the spleen was increased in the Tmprss6-/- mice compared to wild type and correlated with the expression of erythroid genes, suggesting that Tfr2 is expressed in erythroid cells. Further analysis of gene expression in the bone marrow suggests that the loss of Tfr2 in the erythroid cells of Tmprss6-/-/Hfe-/-/Tfr2-/- mice causes a delay in the differentiation process leading to a more severe phenotype.In conclusion, our results indicate that Hfe and Tfr2 act upstream of matriptase-2 in hepcidin regulation or in a way that is overridden when matriptase-2 is deleted. These results indicate that inhibition of matriptase-2 would be useful in the treatment of iron overload conditions such as hereditary hemochromatosis. We have also identified a novel role for Tfr2 in erythroid differentiation that is separate from its canonical role as a regulator of iron homeostasis in the liver. This important role of Tfr2 in erythropoiesis only becomes apparent during conditions of iron restriction. Our results provide novel insights into mechanisms regulating and linking iron homeostasis and erythropoiesis. Disclosures:No relevant conflicts of interest to declare.

N-Linked Glycosylation Is Required for Transferrin-Induced Stabilization of Transferrin Receptor 2, but Not for Transferrin Binding or Trafficking to the Cell Surface

Transferrin receptor 2 (TfR2) is a member of the transferrin receptor-like family of proteins. Mutations in TfR2 can lead to a rare form of the iron overload disease, hereditary hemochromatosis. TfR2 is proposed to sense body iron levels and increase the level of expression of the iron regulatory hormone, hepcidin. Human TfR2 (hTfR2) contains four potential Asn-linked (N-linked) glycosylation sites on its ectodomain. The importance of glycosylation in TfR2 function has not been elucidated. In this study, by employing site-directed mutagenesis to remove glycosylation sites of hTfR2 individually or in combination, we found that hTfR2 was glycosylated at Asn 240, 339, and 754, while the consensus sequence for N-linked glycosylation at Asn 540 was not utilized. Cell surface protein biotinylation and biotin-labeled Tf indicated that in the absence of N-linked oligosaccharides, hTfR2 still moved to the plasma membrane and bound its ligand, holo-Tf. However, without N-linked glycosylation, hTfR2 did not form the intersubunit disulfide bonds as efficiently as the wild type (WT). Moreover, the unglycosylated form of hTfR2 could not be stabilized by holo-Tf. We further provide evidence that the unglycosylated hTfR2 behaved in manner different from that of the WT in response to holo-Tf treatment. Thus, the putative iron-sensing function of TfR2 could not be achieved in the absence of N-linked oligosaccharides. On the basis of our analyses, we conclude that unlike TfR1, N-linked glycosylation is dispensable for the cell surface expression and holo-Tf binding, but it is required for efficient intersubunit disulfide bond formation and holo-Tf-induced stabilization of TfR2.

Control of iron metabolism – Lessons from neonatal hemochromatosis

When hepatologists begin their investigations at the scene of liver damage, iron is certainly on the list of ‘‘usual suspects’’ to be rounded up. Hemochromatosis, a well-known cause of chronic liver disease, is the most prevalent genetic disorder in adults [1]. Serum iron parameters are therefore included in most guidelines on how to investigate patients with elevated transaminases or chronic liver disease [2]. The hemochromatosis (HFE) genotype is an important piece of evidence in patients with liver disease and elevated transferrin saturation with hyperferritinemia, because homozygosity for the C282Y allele of HFE is considered sufficient to diagnose hemochromatosis under these circumstances [1]. Compound heterozygosity for C282Y and H63D has been almost removed from the watch-list and is currently considered a mere risk factor for liver diseases – sufficient to cause hemochromatosis or even cirrhosis only in combination with other health burdens. Hence, a partner in crime for HFE mutation must be found in patients with liver disease and C282Y/H63D compound heterozygosity [3]. If there is evidence for iron overload in the absence of hemochromatosis-associated HFE genotypes and the hepatopathologist finds iron confined to hepatocytes, sharp investigators are well advised to search for the culprit in the genes encoding transferrin receptor 2 (TFR2) and ferroportin (SLC40A1) or even to hunt for combined mutations in these genes if a patient presents with severe iron overload or early-onset disease [4]. If the patient is adolescent or before the 3rd decade of life and presents with heart failure or hypogonadism in addition to – often overlooked – liver disease, other genes will be committed in custody. Mutations in HJV and HAMP, which respectively encode hemojuvelin and hepcidin, will be sufficient to identify juvenile hemochromatosis [5]. Through the power of genetics, iron-related diseases are now subject to close scrutiny. In addition, genetic studies have greatly advanced our understanding of hemochromatosis. This progress is reflected in the proposed re-classification of human iron

Global Sequencing for the Molecular Background of Hereditary Hemochomatosis In Brazilian Patients

AbstractAbstract 5146 INTRODUTION:Most hereditary hemochromatosis (HH) patients are homozygous for the p.C282Y mutation in the HFE gene. But rare HFE variants have been shown to be associated with HH. In addition, four main types of non-HFE HH are caused by mutations in the hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1) genes. The main aim of this study was to screen for HFE, HJV, HAMP, TFR2 and SLC40A1 mutations and to investigate their relationship with HH. MATERIAL E METHODS:Fifty-one Brazilian patients with primary iron overload (transferrin saturation > 50% in females and 60% in males) were eligible. Subsequent bidirectional sequencing for each exon of HFE, HJV, HAMP, TFR2 and SLC40A1 genes was performed. The effect of HFE p.V256I novel mutations on protein structure was analyzed by in silico molecular dynamic and free energy calculations. RESULTS:Thirty-seven (72.5%) out of the 51 patients presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n = 11, 21.6%). In addition, the p.S65C mutation was found in heterozygosis with p.H63D in two patients and the homozygous genotype for the p.H63D was found in two patients. One novel mutation (p.V256I) was indentified in heterozygosis with the p.H63D mutation. In silico modeling analysis of protein behavior suggested 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. Sequencing HJV revealed one patient presenting Juvenile hemochromatosis (JH) (homozygous genotype for the HJV p.G320V mutation); two patients carrying heterozygous genotype for the p.E302K mutation; and one patient with heterozygosis p.A310G polymorphism. Sequencing HAMP revealed one patient carrying p.P48G novel mutation in the heterozygous form. Three and five non-pathogenic polymorphisms were observed in the TFR2 and SLC40A1 genes, respectively. Sequencing SLC40A1 also identified one patient with homozygous genotype for the p.R561G described mutation; and one patient with homozygous genotype for the p.G204S novel mutation. CONCLUSION:The HFE p.C282Y in homozygosis or in heterozygosis with p.H63D was the most frequent mutation associated with HH in our sample population. The novel HFE p.V256I mutation could not be implicated in the molecular basis of the HH phenotype. Two described mutations, HJV p.E302K and SLC40A1 p.R561G, could have functional consequences according to previously studies contributing to HH phenotype. Two novel mutations, HAMP p.R48G and SLC40A1 p.G204S, may be implicated with iron overload in these patients, but further studies are need to explain their impact on proteins. Disclosures:No relevant conflicts of interest to declare.

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.

Defective trafficking and localization of mutated transferrin receptor 2: implications for type 3 hereditary hemochromatosis

Transferrin receptor 2 (TfR2), a homologue of transferrin receptor 1 (TfR1), is a key molecule involved in the regulation of iron homeostasis. Mutations in TfR2 result in iron overload with similar features to HFE-associated hereditary hemochromatosis. The precise role of TfR2 in iron metabolism and the functional consequences of disease-causing mutations have not been fully determined. We have expressed wild-type and various mutant forms of TfR2 that are associated with human disease in a mouse liver cell line. Intracellular and surface analysis shows that all the TfR2 mutations analyzed cause the intracellular retention of the protein in the endoplasmic reticulum, whereas the wild-type protein is expressed in endocytic structures and at the cell surface. Our results indicate that the majority of mutations that cause type 3 hereditary hemochromatosis are a consequence of the defective localization of the protein.

HFE Modulates Transferrin Receptor 2 Levels in Hepatoma Cells via Interactions That Differ from Transferrin Receptor 1-HFE Interactions

Mutations in the transmembrane glycoproteins transferrin receptor 2 (TfR2) and HFE are associated with hereditary hemochromatosis. Interactions between HFE and transferrin receptor 1 (TfR1) have been mapped to the alpha1- and alpha2-helices in HFE and to the helical domain of TfR1. Recently, TfR2 was also reported to interact with HFE in transfected mammalian cells. To test whether similar HFE residues are important for both TfR1 and TfR2 binding, a mutant form of HFE (W81AHFE) that has an approximately 5,000-fold lower affinity for TfR1 than HFE was employed. As expected, W81AHFE does not interact with TfR1. However, we found that the same mutation in HFE does not affect the TfR2/HFE interaction. This finding indicates that the TfR2/HFE and TfR1/HFE interactions are distinct. We further observed that, unlike TfR1/HFE, Tf does not compete with HFE for binding to TfR2 and that binding is independent of pH (pH 6-7.5). TfR2-TfR1 and HFE-HLA-B7 chimeras were generated to map the domains of the TfR2/HFE interaction. TfR1 and HLA-B7 were chosen because of their similar overall structures with TfR2 and HFE, respectively. We mapped the interacting domains to the putative stalk and protease-like domains of TfR2 located between residues 104 and 250 and to the alpha3 domain of HFE, both of which differ from the TfR1/HFE interacting domains. Furthermore, we found that HFE increases TfR2 levels in hepatic cells independent of holo-Tf.

Relative contribution of iron genes, dysmetabolism and hepatitis C virus (HCV) in the pathogenesis of altered iron regulation in HCV chronic hepatitis

Hepatitis C virus (HCV) chronic hepatitis predisposes to iron overload, which negatively influences the prognosis of this infection. Since the underlying mechanisms of this iron overload are undefined, we analyzed the prevalence of altered iron parameters, and the relative contribution of viral, metabolic, and genetic factors in Italian patients. We studied the metabolic and biochemical characteristics of 143 previously untreated, biopsied patients with HCV who were not alcohol abusers. Hepatic iron was determined according to Deugnier, HFE genotype by restriction analysis, hepcidin, hemojuvelin, ferroportin-1, and transferrin receptor-2 mutations by denaturing high performance liquid chromatography and sequencing. Increased transferrin saturation was observed in 20%, hyperferritinemia in 22%, and histological iron deposition in 32% of patients. Ferritin was independently correlated with iron stores and host metabolic parameters, whereas hepatic iron deposition was correlated with ferritin and histological severity of hepatitis. Sinusoidal iron deposition was associated with metabolic alterations, including body mass index, insulin resistance, and LDL cholesterol. Conversely, the prevalence of HFE mutations and serum ferritin values increased with the severity of steatosis. The prevalence of HFE and beta-globin mutations was not different from that of controls (31% and 2%, respectively). No tranferrin receptor-2, hemojuvelin, or ferroportin-1 mutations were detected, but two patients carried the -72C>T hepcidin promoter mutation. The C282Y HFE mutation, hepcidin and beta-globin mutations influenced iron stores. Both carriers of the -72C>T Hepcidin mutation had beta-thalassemia trait, moderate iron overload, and liver cirrhosis. Iron genes influence iron overload and steatosis development, but the major burden is related to HCV itself and host metabolic factors.

Hepcidin Expression Is Regulated by a Complex of Hemochromatosis-Associated Proteins.

Hemochromatosis is a common genetic disease resulting from increased dietary iron absorption and tissue iron deposition. Mutations in five unrelated genes are known to cause hemochromatosis in humans and mice. These encode the classic hemochromatosis protein (HFE), transferrin receptor 2 (TFR2), the iron exporter ferroportin (FPN), hemojuvelin (HJV), and the circulating anti-microbial peptide hepcidin (HAMP). Hepcidin binds to FPN, causing its internalization and degradation, thus decreasing cellular iron release. A basic understanding of the pathophysiology of FPN and hepcidin mutations has recently been elucidated; however, it was still unclear how mutations in HFE, TFR2, and HJV cause hemochromatosis. All are associated with decreased hepcidin production and inappropriately high levels of ferroportin activity. HFE, TFR2 and HJV are normally expressed in the hepatic cells that produce hepcidin. With collaborators, we showed that HJV acts as a bone morphogenetic protein (BMP) co-receptor. HJV binds to the BMP ligands and forms a complex with Type I BMP receptors, resulting in signaling through a SMAD pathway and induction of hepcidin expression. Disease causing mutations in HJV abrogate BMP co-receptor activity, and hepatocytes from Hjv−/ − mice have a blunted response to BMP2. HFE was known to form a complex with the classical transferrin receptor, TFR1. Several models have been proposed implicating this complex in the regulation of normal iron homeostasis, but they have not taken the role of hepcidin into account. To examine the HFE/TFR1 interaction in vivo, we developed mice expressing a mutant form of TFR1 that should constitutively interact with HFE. We found that these transgenic animals have a phenotype similar to Hfe−/ − mice, suggesting that TFR1 serves to sequester HFE to silence its activity. We next asked whether HFE might also participate in BMP signaling. We found that forced expression of HFE in a hepatoma cell line induces transcription of a reporter gene linked to the hepcidin promoter. It also induces transcription from a heterologous promoter containing BMP-responsive elements, suggesting that HFE works through the BMP pathway. In contrast, forced expression of TFR2 did not amplify expression of either reporter, but it prevented cellular release of a soluble cleavage product of HJV. Furthermore, we showed that both HFE and TFR2 are associated with HJV in a stable protein complex that can be isolated by co-immunoprecipitation or Ni-affinity chromatography. TFR2 appears to aid in the recruitment of HFE to this complex. We conclude that HFE and TFR2 thus serve to amplify BMP signaling through an HJV/BMP receptor pathway. Our findings provide a compelling explanation for the similar clinical hemochromatosis phenotypes resulting from mutations in these genes.

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

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

Inactivation of the murine Transferrin Receptor 2 gene using the Cre recombinase: loxP system.

Transferrin Receptor 2 (TfR2) is a key molecule involved in the regulation of iron homeostasis. Mutations in TfR2 lead to type 3 hemochromatosis in humans. We have developed mice with a targeted deletion of TfR2. The Cre-recombinase:loxP system used to create the mice allows both full deletion and tissue-specific deletion of TfR2. The development of these mice will provide new models for type 3 hemochromatosis and assist in determining the role of TfR2 in iron metabolism.

Hepcidin in HFE-associated hemochromatosis: Another piece of the “iron” puzzle

Hepcidin in HFE-associated hemochromatosis: Another piece of the “iron” puzzle

Hemochromatosis Due to Mutations in Transferrin Receptor 2

ABSTRACT A rare recessive disorder which leads to iron overload and severe clinical complications similar to those reported in HFE-related hemochromatosis has been delineated and sometimes called hemochromatosis type 3. The gene responsible is Transferrin Receptor 2 (TFR2), which maps to chromosome 7q22. The TFR2 gene presents a significative homology to transferrin receptor (TFRC) gene, encodes for a transmembrane protein with a large extracellular domain, is able to bind transferrin, even if with lower affinity than TFRC. The TFR2 function is still unclear. The transcript does not contain IRE elements and is not modified by the cellular iron status. At variance with TFRC, interactions between TFR2 and HFE do not occur, at least in their soluble forms. TFR2 is spliced in two alternative forms, alfa and beta. The alfa form is strongly expressed in the liver. The beta form, codified from a start site in exon 4 of the alpha, has a low and ubiquitous expression. Using anti-TFR2 monoclonal antibodies we have confirmed expression of the protein in the liver but also in duodenal epithelial cells, and studied the protein functional behaviour in cell lines, in response to iron addition, iron deprivation and olo-transferrin exposure. Our results suggest a regulatory role of TFR2 in iron metabolism. Five TFR2 homozygous mutations have been documented in HFE3 patients: a nonsense mutation (Y250X); a C insertion that causes a frameshift and a premature stop codon (E60X); a missense mutation (M172K); a 12 basepair deletion in exon 16, that causes 4 aminoacid loss (AVAQ 594-597del) in the extracellular domain of TFR2; a missense mutation in exon 17 (Q690P). The mutation analysis supports the hypothesis that all are private mutations. The pathogenetic role of TFR2 in hemochromatosis has been recently further demonstrated through the targeted expression of the Y250X human mutation in mice, which develop sings of iron overload identical to the human disease. Although the rarity of TFR2 mutations limits their usefulness in diagnostic/screening programs, their study can contribute to a better understanding of the protein function.

Transferrin Receptor-2 Gene and Non-C282Y Homozygous Patients with Hemochromatosis

ABSTRACTMore than 80% of the patients affected by hereditary hemochromatosis, a common inherited iron disorder, are homozygotes for the 845G → A (C282Y) mutation of the HFE gene. However, depending on the population, 10–20% of hereditary hemochromatosis can be linked either to other HFE genotypes, particularly the compound heterozygous state for C282Y and the 187 C → G (H63D) mutation, or to mutations of new other genes. Recently, Camaschella et al. (Nat. Genet. 25, 14–15, 2000) identified a stop mutation (exon 6 nt 750 C → T, Y250X) on the transferrin receptor-2 (TFR2) gene in two unrelated Sicilian families with hereditary hemochromatosis. The TFR2 gene is a transferrin receptor gene homologue that seems to be involved in iron metabolism. Moreover, one of the patients described by Camaschella et al. was a H63D homozygote. H63D homozygosity can be associated with various phenotypes from asymptomatic subjects to patients with a typical form of hereditary hemochromatosis. Thus, the Y250X mutation could be the molecular defect responsible for hereditary hemochromatosis in subjects with atypical HFE genotypes. We have searched for the Y250X mutation in 63 unrelated French subjects. Forty-three had a diagnosis of hereditary hemochromatosis based on classical criteria. This group included 12 H63D homozygotes, 3 C282Y heterozygotes, and 3 patients with none of the two most prevalent HFE mutants. These 18 patients had no other HFE sequence change and were subsequently subjected to DNA sequencing of the 15 last exons and flanking sequences of the TFR2 gene. The 25 remaining hereditary hemochromatosis patients who were tested for the Y250X mutant were compound heterozygotes for the C282Y and H63D mutations. Finally, we also tested for this TFR2 mutation 20 H63D homozygotes with milder manifestations of iron overload and no acquired cause of iron overload. None of the 63 tested subjects had the Y250X mutation. Concurrently, none of the 18 hereditary hemochromatosis patients who had their TFR2 gene sequenced had any deleterious mutation. Thus, TFR2 mutations are not responsible for hemochromatosis in non-C282Y homozygous patients of our area.