Hereditary hemochromatosis (HH) is a common autosomal recessive disease characterized by increased iron absorption and progressive iron storage that results in damage to major organs in the body. Recently, a candidate gene for HH called HFE encoding a major histocompatibility complex class I-like protein was identified by positional cloning. Nearly 90% of Caucasian HH patients have been found to be homozygous for the same mutation (C282Y) in the HFE gene. To test the hypothesis that the HFE gene is involved in regulation of iron homeostasis, we studied the effects of a targeted disruption of the murine homologue of the HFE gene. The HFE-deficient mice showed profound differences in parameters of iron homeostasis. Even on a standard diet, by 10 weeks of age, fasting transferrin saturation was significantly elevated compared with normal littermates (96 ؎ 5% vs. 77 ؎ 3%, P < 0.007), and hepatic iron concentration was 8-fold higher than that of wild-type littermates (2,071 ؎ 450 vs. 255 ؎ 23 g͞g dry wt, P < 0.002). Stainable hepatic iron in the HFE mutant mice was predominantly in hepatocytes in a periportal distribution. Iron concentrations in spleen, heart, and kidney were not significantly different. Erythroid parameters were normal, indicating that the anemia did not contribute to the increased iron storage. This study shows that the HFE protein is involved in the regulation of iron homeostasis and that mutations in this gene are responsible for HH. The knockout mouse model of HH will facilitate investigation into the pathogenesis of increased iron accumulation in HH and provide opportunities to evaluate therapeutic strategies for prevention or correction of iron overload.

Hereditary hemochromatosis (HH) is the most common autosomal recessive disorder known in humans. A candidate gene for HH called HFE has recently been cloned that encodes a novel member of the major histocompatibility complex class I family. Most HH patients are homozygous for a Cys-2823Tyr (C282Y) mutation in HFE gene, which has been shown to disrupt interaction with ␤ 2 -microglobulin; a second mutation, His-633Asp (H63D), is enriched in HH patients who are heterozygous for C282Y mutation. The aims of this study were to determine the effects of the C282Y and H63D mutations on the cellular trafficking and degradation of the HFE protein in transfected COS-7 cells. The results indicate that, while the wild-type and H63D HFE proteins associate with ␤ 2 -microglobulin and are expressed on the cell surface of COS-7 cells, these capabilities are lost by the C282Y HFE protein. We present biochemical and immunof luorescence data that indicate that the C282Y mutant protein: (i) is retained in the endoplasmic reticulum and middle Golgi compartment, (ii) fails to undergo late Golgi processing, and (iii) is subject to accelerated degradation. The block in intracellular transport, accelerated turnover, and failure of the C282Y protein to be presented normally on the cell surface provide a possible basis for impaired function of this mutant protein in HH.Hereditary hemochromatosis (HH) is a common autosomal recessive disorder characterized by iron overload of parenchymal cells in many organs including the liver, pancreas, heart, joints, and endocrine organs due to increased iron absorption in the gastrointestinal tract (1-4). Clinical consequences of iron accumulation in these organs include cirrhosis of the liver, hepatocellular carcinoma, diabetes, heart failure, arthritis, and hypogonadism. Within the Caucasian population, 1 in 300-400 individuals is homozygous and 1 in 8-10 individuals is heterozygous for HH (3,5). Recently, Feder et al. (6) reported that 83% of 178 American HH patients were homozygous for the same missense mutation (C282Y) in a novel major histocompatibility complex (MHC) class I-like gene, originally called HLA-H, but now designated HFE (7). [Although Feder et al. (6) originally designated the HH candidate gene HLA-H, this designation had already been assigned to a pseudogene and the HH locus had already been assigned the name HFE by the nomenclature committee (7).] Eight of nine patients with HH who were heterozygous for this mutation were found to have a different missense mutation (H63D) on the other HFE allele, although 17% of the normal population also carried one H63D allele. These findings were confirmed by Beutler et al. (8), who found that 82% of 147 HH patients were homozygous for the C282Y mutation and 10 were compound heterozygotes for C282Y and H63D alleles. Subsequent studies reported that 72-91% of French patients (9, 10), 64% of Italian patients (11), and 100% of Australian patients (12) were homozygous for the C282Y mutation. Independent support for HFE as the HH gene comes ...

Hereditary hemochromatosis (HH) is a common genetic disorder characterized by excess absorption of dietary iron and progressive iron deposition in several tissues, particularly liver. The vast majority of individuals with HH are homozygous for mutations in the HFE gene. Recently a second transferrin receptor (TFR2) was discovered, and a previously uncharacterized type of hemochromatosis (HH type 3) was identified in humans carrying mutations in the TFR2 gene. To characterize the role for TFR2 in iron homeostasis, we generated mice in which a premature stop codon (Y245X) was introduced by targeted mutagenesis in the murine Tfr2 coding sequence. This mutation is orthologous to the Y250X mutation identified in some patients with HH type 3. The homozygous Tfr2 Y245X mutant mice showed profound abnormalities in parameters of iron homeostasis. Even on a standard diet, hepatic iron concentration was several-fold higher in the homozygous Tfr2 Y245X mutant mice than in wild-type littermates by 4 weeks of age. The iron deposition in the mutant mice was predominantly hepatocellular and periportal. The mean splenic iron concentration in the homozygous Tfr2 Y245X mutant mice was significantly less than that observed in the wild-type mice. The homozygous Tfr2 Y245X mutant mice also demonstrated elevated transferrin saturations. There were no significant differences in parameters of erythrocyte production including hemoglobin levels, hematocrits, erythrocyte indices, and reticulocyte counts. Heterozygous Tfr2 Y245X mice did not differ in any measured parameter from wild-type mice. This study confirms the important role for TFR2 in iron homeostasis and provides a tool for investigating the excess iron absorption and abnormal iron distribution in iron-overload disorders.iron ͉ liver ͉ gene targeting T ransferrin receptor (TFR)2 is a recently identified type II membrane protein with significant homology to the classical transferrin receptor (TFR1). Both TFR1 and TFR2 are capable of transporting transferrin-bound iron into the cell (1) and supporting cell growth (2). However, their properties differ in several critical ways. TFR2 has a lower affinity for holotransferrin than does TFR1 (1, 3). The expression pattern of TFR2 mRNA is quite distinct from TFR1 as well (4, 5). In particular, the TFR2 gene is expressed at much higher levels in liver compared with other tissues, whereas the TFR1 gene demonstrates little hepatic expression. TFR1 and TFR2 differ also in their response to changes in cellular iron status. The TFR1 transcript contains multiple iron-responsive elements in the 3Ј-untranslated region. These elements stabilize the TFR1 transcript under conditions of low cellular iron. The TFR2 transcript does not contain these elements, and TFR2 message and TFR2 protein levels vary little with changes in iron status (4, 5). The observation that hepatic expression of Tfr2 persists despite iron overload in a murine Hfe gene knockout model of hereditary hemochromatosis (HH) led us to suggest that Tfr2-mediated iron uptake contributes...

Hereditary hemochromatosis (HH) is a common autosomal recessive disease associated with loss of regulation of dietary iron absorption and excessive iron deposition in major organs of the body. Recently, a candidate gene for HH (also called HFE) was identified that encodes a novel MHC class I-like protein. Most patients with HH are homozygous for the same mutation in the HFE gene, resulting in a C282Y change in the HFE protein. Studies in cultured cells show that the C282Y mutation abrogates the binding of the recombinant HFE protein to ␤ 2 -microglobulin (␤ 2 M) and disrupts its transport to the cell surface. The HFE protein was shown by immunohistochemistry to be expressed in certain epithelial cells throughout the human alimentary tract and to have a unique localization in the cryptal cells of small intestine, where signals to regulate iron absorption are received from the body. In the studies presented here, we demonstrate by immunohistochemistry that the HFE protein is expressed in human placenta in the apical plasma membrane of the syncytiotrophoblasts, where the transferrinbound iron is normally transported to the fetus via receptormediated endocytosis. Western blot analyses show that the HFE protein is associated with ␤ 2 M in placental membranes. Unexpectedly, the transferrin receptor was also found to be associated with the HFE protein͞␤ 2 M complex. These studies place the normal HFE protein at the site of contact with the maternal circulation where its association with transferrin receptor raises the possibility that the HFE protein plays some role in determining maternal͞fetal iron homeostasis. These findings also raise the question of whether mutations in the HFE gene can disrupt this association and thereby contribute to some forms of neonatal iron overload. 1) originally designated the HH candidate gene HLA-H, this designation had already been assigned to a pseudogene and the HH locus had already been assigned the name HFE by the nomenclature committee (27).] They found 83% of 178 HH patients to be homozygous for the same missense mutation (C282Y) in the HFE gene. Eight of nine HH patients who were heterozygous for this mutation were found to have a different missense mutation (H63D) on the other HFE allele (1). On the basis of these findings, they proposed that a mutation in the HFE gene is the molecular basis for most cases of HH. The high frequency of the C282Y mutation in HH patients has been confirmed by at least five other studies (2-6).The human HFE protein predicted from the cDNA sequence is composed of 343 amino acids. It is most homologous to major histocompatibility complex (MHC) class I molecules that are integral membrane proteins with three extracellular loops (␣1, ␣2, and ␣3), a transmembrane region, and a short cytoplasmic tail. The C282Y mutation was predicted to disrupt a critical disulfide bond in the ␣3 loop of the HFE protein and abrogate binding of the mutant HFE protein to ␤ 2 -microglobulin (␤ 2 M) and its transport to and presentation on the cell surface. Feder et ...