Progressive Iron Overload Research Articles

HFE Genotyping by Amplification Refractory Mutation System–Denaturing HPLC

Hereditary hemochromatosis [(HC); MIM 235200] is one of the most frequent genetic diseases in Caucasian populations [prevalence up to 1 in 300 in northern Europe, with an estimated carrier frequency of 1 in 10 (1)]. Caused by a progressive iron overload, it is characterized by severe complications and a potentially lethal progression that could be entirely prevented with regular iron removal by means of bleeding (2). The availability of effective preventive treatment highlights the value of early detection. The discovery of the HFE gene by Feder et al. (3) has yielded a genetic test for disease risk. Two mutations, C282Y and H63D, located respectively in exons 4 and 2, impair HFE function (4) and are frequently tested for, along with a third variant located on exon 2 (S65C). Two genotypes (C282Y homozygote and compound heterozygote C282Y/H63D) are associated with risk of HC (5). Because many could benefit from testing, numerous methods have been described in addition to conventional PCR restriction assays (3). Described methods include real-time PCR with fluorescent probes (6)(7)(8)(9) or SYBR green® melting curve (10), conventional PCR restriction assay with the use of HPLC (11) or capillary electrophoresis to resolve restriction fragments, allele-specific PCR products resolved on slab gels (12)(13) or by capillary electrophoresis (14)(15), and PCR followed by a single-nucleotide extension step (11), electrochemiluminescence detection (16), or reverse hybridization assay (17)(18). Denaturing HPLC (DHPLC) is a new method for single- nucleotide polymorphism detection, which has been effective in terms of cost per analysis, sensitivity, and specificity (19). However, one limitation of DHPLC is that in most cases it hardly differentiates homozygous mutants from homozygous wild-type genotypes, and therefore, it is not recommended for diseases characterized by a few highly prevalent mutations. …

Autosomal-dominant hemochrom-atosis is associated with a mutation in the ferroportin (SLC11A3) gene

Hemochromatosis is a progressive iron overload disorder that is prevalent among individuals of European descent. It is usually inherited in an autosomal-recessive pattern and associated with missense mutations in HFE, an atypical major histocompatibility class I gene. Recently, we described a large family with autosomal-dominant hemochromatosis not linked to HFE and distinguished by early iron accumulation in reticuloendothelial cells. Through analysis of a large pedigree, we have determined that this disease maps to 2q32. The gene encoding ferroportin (SLC11A3), a transmembrane iron export protein, lies within a candidate interval defined by highly significant lod scores. We show that the iron-loading phenotype in autosomal-dominant hemochromatosis is associated with a nonconservative missense mutation in the ferroportin gene. This missense mutation, converting alanine to aspartic acid at residue 77 (A77D), was not seen in samples from 100 unaffected control individuals. We propose that partial loss of ferroportin function leads to an imbalance in iron distribution and a consequent increase in tissue iron accumulation.

Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice.

We previously reported the disruption of the murine gene encoding the transcription factor USF2 and its consequences on glucose-dependent gene regulation in the liver. We report here a peculiar phenotype of Usf2(-/-) mice that progressively develop multivisceral iron overload; plasma iron overcomes transferrin binding capacity, and nontransferrin-bound iron accumulates in various tissues including pancreas and heart. In contrast, the splenic iron content is strikingly lower in knockout animals than in controls. To identify genes that may account for the abnormalities of iron homeostasis in Usf2(-/-) mice, we used suppressive subtractive hybridization between livers from Usf2(-/-) and wild-type mice. We isolated a cDNA encoding a peptide, hepcidin (also referred to as LEAP-1, for liver-expressed antimicrobial peptide), that was very recently purified from human blood ultrafiltrate and from urine as a disulfide-bonded peptide exhibiting antimicrobial activity. Accumulation of iron in the liver has been recently reported to up-regulate hepcidin expression, whereas our data clearly show that a complete defect in hepcidin expression is responsible for progressive tissue iron overload. The striking similarity of the alterations in iron metabolism between HFE knockout mice, a murine model of hereditary hemochromatosis, and the Usf2(-/-) hepcidin-deficient mice suggests that hepcidin may function in the same regulatory pathway as HFE. We propose that hepcidin acts as a signaling molecule that is required in conjunction with HFE to regulate both intestinal iron absorption and iron storage in macrophages.

The gene TFR2 is mutated in a new type of haemochromatosis mapping to 7q22.

Haemochromatosis is a common recessive disorder characterized by progressive iron overload, which may lead to severe clinical complications. Most patients are homozygous for the C282Y mutation in HFE on 6p (refs 1-5). A locus for juvenile haemochromatosis (HFE2) maps to 1q (ref. 7). Here we report a new locus (HFE3) on 7q22 and show that a homozygous nonsense mutation in the gene encoding transferrin receptor-2 (TFR2) is found in people with haemochromatosis that maps to HFE3.

Genetics of hemochromatosis.

Hereditary hemochromatosis (HHC) is a common autosomal recessive disorder of iron metabolism that results in progressive iron overload and can be fatal if untreated. The hemochromatosis gene (HFE) was identified by positional cloning in 1996. Two missense mutations have been described in HFE. The majority of HHC patients are homozygous for a cysteine-to-tyrosine substitution (C282Y); however, a small number are homozygous for a histidine-to-aspartic-acid substitution (H63D) or are heterozygous for both of these mutations. Mechanisms by which C282Y and H63D may disrupt the normal functioning of HFE have been suggested, but the role of HFE in the process of normal iron metabolism has yet to be clearly defined.

Polymorphisms in the HFE Gene

Hereditary hemochromatosis is an autosomal recessive disease characterized by progressive iron overload. Recently, a candidate gene named HFE was isolated on the short arm of the chromosome 6 within which two mutations were identified: C282Y and H63D. To date, only homozygosity for the C282Y mutation is considered as a diagnostic criterion of hemochromatosis. 7.6% of the patients studied in our laboratory did not carry two copies of the C282Y mutation. On the other hand, a dysmetabolic iron overload syndrome has recently been described and the search for the C282Y and H63D mutations revealed that none of the patients was homozygous for C282Y while 67% exhibited one of the mutations. The possibility of a new mutation in the HFE gene has been raised to explain the disease in the remaining patients, as well as, in the few hemochromatotic patients without two copies of the C282Y mutation. The aim of this study was to search for new mutations in the HFE gene in 16 such patients. Direct sequencing of exons and 3 introns did not reveal any new mutation but identified a few polymorphisms.

Non-C282Y familial iron overload: evidence for locus heterogeneity in haemochromatosis.

Haemochromatosis (HC) is an autosomal recessive disease with progressive iron overload leading to midlife onset of clinical complications. The causal gene (HFE) maps to 6p, in close linkage with the...

The Hemochromatosis 845 G→A and 187 C→G Mutations: Prevalence in Non-Caucasian Populations

Hemochromatosis, the inherited disorder of iron metabolism, leads, if untreated, to progressive iron overload and premature death. The hemochromatosis gene, HFE, recently has been identified, and characterization of this gene has shown that it contains two mutations that result in amino acid substitutions-cDNA nucleotides 845 G-->A (C282Y) and 187 C-->G (H63D). Although hemochromatosis is common in Caucasians, affecting >=1/300 individuals of northern European origin, it has not been recognized in other populations. The present study used PCR and restriction-enzyme digestion to analyze the frequency of the 845 G-->A and 187 C-->G mutations in HLA-typed samples from non-Caucasian populations, comprising Australian Aboriginal, Chinese, and Pacific Islanders. Results showed that the 845 G-->A mutation was present in these populations (allele frequency 0.32%), and, furthermore, it was always seen in conjunction with HLA haplotypes common in Caucasians, suggesting that 845 G-->A may have been introduced into these populations by Caucasian admixture. 187 C-->G was present at an allele frequency of 2.68% in the two populations analyzed (Australian Aboriginal and Chinese). In the Australian Aboriginal samples, 187 C-->G was found to be associated with HLA haplotypes common in Caucasians, suggesting that it was introduced by recent admixture. In the Chinese samples analyzed, 187 C-->G was present in association with a wide variety of HLA haplotypes, showing this mutation to be widespread and likely to predate the more genetically restricted 845 G-->A mutation.

Global prevalence of putative haemochromatosis mutations.

Haemochromatosis is a genetic disease associated with progressive iron overload, and is common among populations of northern European origin. HLA-H is a recently reported candidate gene for this condition. Two...

Hereditary hemochromatosis: A prevalent disorder of iron metabolism with an elusive etiology

Hereditary hemochromatosis is a prevalent inherited disorder with an estimated frequency of homozygosity of 0.2 to 0.45% in Caucasians. The disease is characterized by progressive iron overload until a massive accumulation of body iron occurs. Undetected, the disorder eventually can produce either cirrhosis, diabetes mellitus, cardiac disease, arthritis, or hepatocellular carcinoma or a combination of these manifestations. Early diagnosis and treatment prevents organ damage and normalizes life expectancy. Screening studies to detect hemochromatosis are most effectively accomplished by measurement of the serum iron and total iron binding capacity. Treatment is most effectively performed by frequent phlebotomy until body stores are empty and then 3 to 4 times yearly for life. The basic defect of hemochromatosis appears to increase iron absorption, decrease iron excretion, and produce preferential deposit of iron in hepatic parenchymal cells rather than Kupffer cells. The genetic abnormality of hemochromatosis is located on chromosome 6 in close association with the gene for HLA antigens. Recent speculation postulates that tumor necrosis factor may be involved in the etiology of this disease because of its location on chromosome 6 and its effect upon iron transport.

Where does the gene for hemochromatosis lie in relation to HLA-A?

Hereditary Hemochromatosis (HFE) is one of the most common inherited disorders with an estimated frequency of homozygous patients of 0.002‐0.0045. The disease is characterized by increased intestinal iron absorption and progressive iron overload. Affected subjects show clinical symptoms of parenchymal organ damage after the third‐fourth decade of life and have a 200‐fold increased risk of developing hepatocellular carcinoma. Early diagnosis and treatment prevent complications and may normalize life expectancy of patients. The biochemical and genetic defects leading to progressive iron accumulation are still unknown, but the HFE gene is tightly linked to HLA complex on the short arm of chromosome 6. Utilizing HLA serotypes and the study of several polymorphic markers of 6p21, a linkage analysis of the disease locus was performed in a series of Italian hemochromatosis families. The data obtained by linkage analysis and the study of a family with a double recombinant allowed us to better define the HFE gene location with respect to HLA‐class I A and F loci.

Iron overload in β2-microglobulin-deficient mice

The present paper describes the results of a comparative histological and quantitative analysis of iron distribution in tissues of β 2m−/− and β 2m+/− mice of different ages. Progressive hepatic iron overload, indistinguishable from that observed in human hemochromatosis, was found only in mice homozygous for the mutated β 2m gene. Total iron measurements done by flame atomic absorption showed statistically significant differences between liver samples from 5 β 2m+/− heterozygotes (468 ± 174 μg/g of dry weight) and 9 mice homozygous for the mutated β 2m gene with average total hepatic iron levels of 1583 ± 424 μg/g of dry weight.

Hunting the hemochromatosis gene: progress and problems.

Hereditary hemochromatosis (HFE) is an inherited recessive disorder which causes progressive iron overload. Homozygotes for the affected gene develop symptoms of parenchymal organ damage and especially liver cirrhosis in midlife. Early diagnosis is important in order to prevent symptoms. The protein responsible for the increased iron absorption is unknown. The tight association of the disease gene with HLA-A has been known for nearly 20 years, but its precise localization remains uncertain. Linkage and linkage disequilibrium analyses in different populations have focussed on two possible locations of the gene either very close to HLA-A, or at the telomeric site of 6p in the vicinity of the D6S105 marker.

Linkage analysis of 6p21 polymorphic markers and the hereditary hemochromatosis: localization of the gene centromeric to HLA-F.

Hereditary Hemochromatosis (HFE) is one of the most common inherited disorders with an estimated frequency of homozygous patients of 0.002-0.0045. The disease is characterized by increased intestinal iron absorption and progressive iron overload. Affected subjects show clinical symptoms of parenchymal organ damage after the third-fourth decade of life and have a 200 fold increased risk of developing hepatocellular carcinoma. Early diagnosis and treatment prevent complications and may normalize life expectancy of patients. The biochemical and genetic defects leading to progressive iron accumulation are still unknown, but the HFE gene is tightly linked to HLA complex on the short arm of chromosome 6. Utilizing HLA serotypes and the study of several polymorphic markers of 6p21, a linkage analysis of the disease locus was performed in a series of Italian hemochromatosis families. The data obtained by linkage analysis and the study of a family with a double recombinant allowed us to better define the HFE gene location with respect to HLA-class I A and F loci.

Metabolism of iron from (3,5,5-trimethylhexanoyl) ferrocene in rats: A dietary model for severe iron overload

The feeding of diets enriched with (3,5,5-trimethylhexanoyl)ferrocene (TMH-ferrocene) has been shown recently to produce a severe experimental iron overload in rats and has been considered as an adequate animal model for hereditary haemochromatosis in humans. We synthesized three 59Fe-labelled ferrocene compounds with different lipophilic characters ferrocene, TMH-ferrocene, and 1,1'-bis (3,5,5-trimethylhexanoyl)ferrocene [(TMH) 2-ferrocene] and studied the metabolism of iron from these compounds in comparison with the hydrophilic ferrous sulphate in rats with iron deficiency, and normal and increased iron stores. The bioavailability of iron from TMH-ferrocene (whole body retention, 48% from a 5 mg Fe dose) was twice as high as from ferrocene and six times higher than from (TMH) 2-ferrocene and ferrous sulphate. In contrast to the well-known iron salts (ferrous sulphate), the intestinal absorption of TMH-ferrocene iron was independent from the dose (1 or 5 mg Fe) and similar in iron-deficient and iron-loaded rats, indicating that the intestinal absorption of the TMH-ferrocene is not regulated by the body iron stores. After intestinal absorption, TMH-ferrocene iron in the portal blood is transported to the liver independently from transferrin. In contrast to absorbed ferrocene, iron from TMH-ferrocene is almost completely released from the hydrocarbon moiety within the liver. Depending on the body iron stores, TMH-ferrocene iron is then incorporated preferentially into haemoglobin (iron-deficient rats) or added to the iron stores in the liver (iron-loaded rats). A transient storage of the 59Fe-label in fat tissue was observed only from oral ferrocene but not from TMH-ferrocene. Due to the outstandingly high bioavailability of TMH-ferrocene, the chronic feeding of this compound resulted in a fast and progressive iron overload in rats (liver iron: 16.9 mg Fe g wet weight after 10 weeks of feeding a diet containing 0.5% TMH-ferrocene), and can be regarded as the best characterized and most useful animal model for severe hepatocellular iron overload in humans.

Hepatic Ferritin Uptake and Hepatic Iron

The effect of hepatic iron on the uptake of ferritin was studied by perfusing livers from normal, iron-deficient and iron-loaded rats with 125I-labeled ferritin. Unlabeled ferritin with tracer doses of labeled ferritin in concentrations of 0.02 to 2,700 nmol/L were studied. Rats were made iron deficient by feeding an established iron-deficient diet for 3 wk. Rats were iron loaded by injection of iron dextran (50 mg/wk) for 3 wk. The mean percentage of uptake of ferritin was similar for doses ranging from 0.22 to 22.2 nmol/L of 125I-labeled ferritin. Uptake of ferritin in the normal animal was saturable, with an apparent maximal velocity of uptake of approximately 9.1 pmol/gm/min and a Michaelis-Menten constant of approximately 5 nmol/L at 37 degrees C. Uptake was minimal at 4 degrees C. The mean uptake of ferritin was 78% +/- 10% in the iron-deficient rats (mean hepatic iron = 1.5 mumol/gm), 79% +/- 10% in the normal animals (mean hepatic iron = 9.2 mumol/gm) and 78% +/- 8% in the iron-loaded animals (mean hepatic iron = 192 mumol/gm). In this experimental system, modulation of hepatic iron did not affect uptake of ferritin, suggesting that regulation of the hepatic ferritin receptor may not depend on hepatic iron content. The rapid uptake of ferritin by the liver despite iron overload is consistent with other observations of the nonregulation of non-transferrin-bound iron by hepatic iron and may play a role in the progressive iron overload seen in hemochromatosis.

HLA typing in idiopathic hemochromatosis: distinction between homozygotes and heterozygotes with biochemical expression.

In a study of 20 families with idiopathic hemochromatosis, relatives of probands were classified as either homozygous, heterozygous, or normal according to their HLA phenotype. An abnormality in the serum iron concentration, total iron-binding capacity, or serum ferritin concentration was present in all homozygotes and in 25% of heterozygotes. In heterozygotes, the mean total iron-binding capacity was significantly decreased, and the mean hepatic iron concentration was significantly increased compared to normals. However, in contrast to homozygotes, clinical evidence of iron overload was not observed in heterozygotes, and there was no biochemical or histological evidence of liver disease resulting from excessive iron stores. Progressive iron overload did not develop in 44 heterozygotes who were studied for up to 16 yr.