Search

Hepcidin, a 25-amino-acid antimicrobial peptide, is the central regulator of iron homeostasis. Hepcidin transcription is upregulated by inflammatory cytokines, iron, and bone morphogenetic proteins and is downregulated by iron deficiency, ineffective erythropoiesis, and hypoxia. The iron transporter ferroportin is the cognate receptor of hepcidin and is destroyed as a result of interaction with the peptide. Except for inherited defects of ferroportin and hepcidin itself, all forms of iron-storage disease appear to arise from hepcidin dysregulation. Studies using multiple approaches have begun to delineate the molecular mechanisms that regulate hepcidin expression, particularly at the transcriptional level. Knowledge of the regulation of hepcidin by inflammation, iron, erythropoiesis, and hypoxia will lead to an understanding of the pathogenesis of primary hemochromatosis, secondary iron overload, and anemia of inflammatory disease.

The response of hepcidin transcription to iron has been repeatedly documented in living mice, but it is difficult to demonstrate the response in ex vivo systems. We have hydrodynamically transfected mice with plasmid constructs composed of a murine hepcidin 1 promoter and fragments of the promoter fused to a firefly luciferase reporter. This method enabled us to quantitate the response of the hepcidin promoter to short-term feeding of a high-iron diet to mice that have been maintained on an iron-deficient diet. We show that the region of the promoter between 1.6 Kb and 1.8 Kb upstream from the start of translation is essential for the response to iron. The promoter region between -260 bp and -1.6 Kb is not essential for the iron responsiveness of hepcidin promoter. The iron-responsive region that we have mapped is the same region required for the in vitro response of HepG2 cells to stimulation with bone morphogenetic proteins and differs from the LPS/IL-6 responsive area.

Hepcidin is an acute-phase response antimicrobial peptide that has emerged as a central regulator of iron absorption. Circulating hepcidin levels have been shown to affect iron uptake, release and storage. Hepcidin is mainly liver-derived and regulated, at least in part, transcriptionally. Hypoxia, erythroid demand, iron content and inflammation each have been shown to influence hepcidin mRNA expression in intact animals. In vitro, regulation of hepcidin by cytokines and by hypoxia is readily demonstrated in primary hepatocytes or in hepatocyte lines, but incubating the same cell lines with iron does not increase transcription of hepcidin. Thus, how iron excess stimulates hepcidin production in hepatocytes remains unknown. In addition, there is no current technique available that can investigate how iron induces hepcidin expression. To provide a better understanding of hepcidin gene expression in response to these regulatory stimuli, we have established a whole animal in vivo bioluminescence imaging assay to measure the activity of hepcidin promoter constructs in the animals' liver after hydrodynamic transfection of hepcidin promoter/luciferase constructs into mice. Transfected hepcidin promoter constructs were shown to respond to both inflammatory and iron stimuli in vivo. This work highlights the ability of this new imaging technique to investigate the key regions of the hepcidin promoter involved in iron induction of hepcidin expression.

Background: Alcoholic liver disease is associated with increased hepatic iron accumulation. The liver-derived peptide hepcidin is the central regulator of iron homeostasis and recent animal studies have demonstrated that exposure to alcohol reduces hepcidin expression. This down-regulation of hepcidin in vivo implies that disturbed iron sensing may contribute to the hepatosiderosis seen in alcoholic liver disease. Alcohol intake is also a major factor in expression of the hemochromatosis phenotype in patients homozygous for the C282Y mutation of the HFE gene., Methods: To assess the effect of alcohol in mice with iron overload, alcohol was administered to mice with disrupted Hfe and IL-6 genes and Tfr2 mutant mice and their respective 129x1/SvJ, C57BL/6J, and AKR/J wild-type congenic strains. Iron absorption, serum iron levels, and hepcidin expression levels were then measured in these mice compared with water-treated control mice., Results: Alcohol was shown to have a strain-specific effect in 129x1/SvJ mice, with treated 129x1/SvJ mice showing a significant increase in iron absorption, serum iron levels, and a corresponding decrease in hepcidin expression. C57BL/6J and AKR/J strain mice showed no effect from alcohol treatment. 129x1/SvJ mice heterozygous or homozygous for the Hfe knockout had a diminished response to alcohol. All 3 strains were shown to have high blood alcohol levels., Conclusions: The effect of alcohol on iron homeostasis is dependent on the genetic background in mice. In an alcohol-susceptible strain, mutation of the Hfe gene diminished the response of the measured iron indices to alcohol treatment. This indicates that either maximal suppression of hepcidin levels had already occurred as a result of the Hfe mutation or that Hfe was a component of the pathway utilized by EtOH in suppressing hepcidin production and increasing iron absorption.

Soluble transferrin receptor-1 (sTfR1) concentrations are increased in the plasma under two conditions that are associated with increased iron absorption, i.e. iron deficiency and increased erythropoiesis. To determine the possible role of sTfR1 as a signaling mechanism for iron absorption, a hydrodynamic gene transfer technique was established to express transfected plasmid constructs of human sTfR1 (hsTfR1) and murine sTfR1 (msTfR1) from the livers of C57BL/6 mice. Iron absorption, serum iron levels and hepcidin expression were then measured. The hydrodynamic gene transfer technique proved to be an effective approach to achieving sustained expression of sTfR1 in mice. Although expression of high levels of sTfR1 significantly increased serum iron levels, repeated experiments showed that neither hsTfR1 nor msTfR1 had any effect on iron absorption or hepcidin mRNA expression levels. Thus, despite its attractiveness as a potential modifier of iron absorption, sTfR1 levels do not exert a regulatory effect on iron absorption., (Copyright 2006 S. Karger AG, Basel.)

The goal of this study was to determine whether statistical modeling of population data for a phenotypic marker could reflect a major locus gene defect. Identifying mutations in the HFE gene makes it possible to assess the association between transferrin saturation (TS) subpopulations and HFE mutations. Data were analyzed from 27 895 white patients who attended a health appraisal clinic and who had TS and common mutations of HFE determined. Mixture distribution modeling of TS was performed, and the proportion of HFE mutations in TS subpopulations was assessed on a probability basis. Three subpopulations of TS were identified, consistent with Hardy-Weinberg conditions for major locus effects. For men, 72% of the subpopulation with the highest mean TS had HFE gene mutations; they were primarily homozygotes or compound heterozygotes. Seventy-three percent of the subpopulation with moderate mean TS also had HFE gene mutations; they were predominantly simple heterozygotes. Sixty-seven percent of the subpopulation with the lowest mean TS were wild-type homozygotes. Similar results were observed for women. These results suggest that statistical modeling of population clinical laboratory test data can reveal the influence of a major locus gene defect and perhaps can be applied to other aspects of body metabolism than iron.

Background: There is a marked difference in the degree of expression of the homozygous C282Y HFE genotype that is associated with hereditary hemochromatosis. It has been reported that individuals with the haptoglobin 2-2 type manifest increased iron concentrations, including serum iron, transferrin saturation, and ferritin., Methods: We studied 232 patients, 115 homozygous for the c.845G-->A (C282Y) mutation and 117 matched controls with the wild-type HFE genotype, for haptoglobin phenotypes. Haptoglobin types were determined by electrophoresis of the denatured protein. The HFE genotype was determined by allele-specific oligonucleotide hybridization. Ferritin and transferrin saturation were measured by standard methods., Results: There was no relationship between haptoglobin type and ferritin concentration or transferrin saturation., Conclusions: The effect of haptoglobin type on iron homeostasis cannot account for the marked phenotypic variation that is seen in patients homozygous for the HFE C282Y mutation.

Hereditary hemochromatosis is characterized by marked variation of expression of the defect: very few homozygotes with the C282Y/C282Y HFE genotype have full-blown clinical disease, a larger number show biochemical stigmata of iron overload, and some seem normal biochemically. The following candidate genes have been examined in detail to determine whether polymorphisms in them may be responsible for this variation: transferrin, transferrin receptor 1, transferrin receptor 2, ferritin-L, ferritin-H, IRP1, IRP2, HFE, beta(2) microglobulin, mobilferrin/calreticulin, ceruloplasmin, ferroportin, NRAMP1, NRAMP2 (DMT1), haptoglobin, heme oxygenase-1, heme oxygenase-2, hepcidin, USF2, ZIRTL, duodenal cytochrome b ferric reductase (DCYTB), TNFalpha, keratin 8, and keratin 18. The coding sequence, exon-intron junctions, and promoters of each of these genes was sequenced in DNA from 20 subjects: 5 HFE C282Y/C282Y with clinical disease, 5 HFE C282Y/C282Y with normal/low ferritin levels and no disease, 5 wt/wt with high ferritin and transferrin saturation, and 5 wt/wt normal controls. When coding or promoter polymorphisms were encountered, DNA from large numbers of ethnically defined subjects was examined for these polymorphisms and a relationship between their existence and abnormalities of iron homeostasis was sought. Only in the case of one transferrin mutation did we find a strong relationship between the polymorphism and iron deficiency anemia. The putative genes that affect the expression of HFE mutations remain elusive.

None of the methods for assessing total body iron burden in patients with hemochromatosis is satisfactory. Although it is commonly believed that a relationship exists between serum ferritin levels and total iron burden, the extent of this relationship has not previously been documented. In the present investigation we measured the total body iron burden of 88 patients with putative hemochromatosis, 54 of whom were homozygotes for the 845G-->A (C282Y) mutation. The total body iron stores were estimated from the volume of red cells removed during therapeutic phlebotomy corrected for an estimated 2 mg/day dietary iron absorbed during the phlebotomy period; the amount of storage iron was compared to the serum ferritin, serum iron, unsaturated iron binding capacity, and transferrin saturation before the beginning of phlebotomy. The serum ferritin proved to be the best predictor of body iron stores. The correlation between all of the analytes and the body iron burden was greater in patients homozygous for the C282Y mutation than in those who were not, including the compound heterozygotes for C282Y and H63D. The body iron burden tended to be greater in patients homozygous for the C282Y mutation than the other patients at any other given ferritin level. We conclude that the serum ferritin level does provide some information regarding total iron burden but even in the case of C282Y homozygotes, the correlation is not very strong., (Copyright 2002 S. Karger AG, Basel)

Iron, an essential element for central nervous system (CNS) function, has frequently been found to accumulate in brain regions that undergo degeneration in neurological diseases such as Alzheimer disease, Parkinson disease, Friedreich ataxia and other disorders. However, the precise role of iron in the cause of many neurodegenerative diseases is unclear. To assist in understanding the potential importance of iron in CNS disease, this review summarizes the present knowledge in the areas of CNS iron metabolism, homeostasis and disregulation of iron balance caused by mutations in genes encoding proteins involved in iron transport, storage and metabolism. This review encompasses neurodegenerative disorders associated with both iron overload and deficiency to highlight areas where iron misregulation is likely to be important in the pathophysiology of several human brain diseases., (Copyright 2002 S. Karger AG, Basel)

The effect of five different transferrin variants (TFv1, TFv2, TFv3, TFv4, and TFv5) on the hemoglobin level, mean corpuscular volume (MCV), ferritin level, percent transferrin saturation (%TS), and the unsaturated iron binding capacity (UIBC) was investigated in subjects with defined HFE haplotypes, 919 persons undergoing health screening and 113 patients with clinical hemochromatosis. The most common variant is TFv4; the population distribution of this variant was also studied. None of the variants were found to have an effect on any of the parameters of iron metabolism that were investigated. Moreover, the frequency of these variants in patients with clinically significant hemochromatosis was no different from that in the general population. We conclude that these polymorphisms in transferrin do not play a role in the expression of hemochromatosis, nor do they produce any other significant changes in iron metabolism., (Copyright 1999 Academic Press.)

Erythrocytosis, increased whole blood oxygen affinity, and iron overload were found in a 37-yr-old man. Electrophoretic techniques to demonstrate a hemoglobin variant showed no abnormality. Structural studies of the hemoglobin from this patient revealed an abnormal hemoglobin previously described as Hemoglobin Olympia, a high-affinity variant. Study of three generations in this family showed increased hepatic iron or iron absorption in some members of all three generations studied. The findings in this family are consistent with an increase in iron absorption due to the consequences of Hemoglobin Olympia and the heterozygous state for hemochromatosis (allele for hemochromatosis associated with HLA-A3, B7) or the presence in the family pedigree of three different alleles for hemochromatosis (alleles for hemochromatosis associated with HLA-A3. B7, HLA-A3, B15, and HLA-A9, B44) with the heterozygous state being manifest with increased iron absorption.

Barton JC, West C, Lee PL, Beutler E. A previously undescribed frameshift deletion mutation of HFE (c.del277; G93fs) associated with hemochromatosis and iron overload in a C282Y heterozygote. A 62-year-old white man with a hemochromatosis phenotype was found to be heterozygous for the C282Y mutation of the HFE gene. The H63D and S65C mutations of HFE were not present. As most C282Y heterozygotes do not develop a hemochromatosis phenotype, the coding region of the patient's HFE gene was sequenced and a previously undescribed frameshift mutation was identified in exon 2 (c.del277; G93fs) that resulted in a premature stop-codon. There were no coding region mutations of the ferroportin gene ( FPN1). We performed human leukocyte antigen (HLA) typing of the patient and his brother who was heterozygous for the C282Y HFE mutation unassociated with a hemochromatosis phenotype. They shared only C282Y and the HLA haplotype A*03, B*14; hence, the c.del277 mutation was linked to the HLA haplotype A*02, B*44 and therefore not on the same chromosome as the C282Y mutation. Thus, the present patient's only intact HFE protein is C282Y, and this may explain his hemochromatosis phenotype. [ABSTRACT FROM AUTHOR]

A patient with clinically manifest hereditary hemochromatosis was found to be heterozygous for the c.845 A→G (C282Y) mutation. As simple heterozygotes for this mutation do not develop the hemochromatosis phenotype, the coding region of the patient's HFE gene was sequenced and a previously undescribed nonsense mutation was identified at c.211 C→T (R74X). The patient's brother who also had the hemochromatosis phenotype shared his HFE genotype. To determine how common such mutations might be, the coding and 5′ region of the HFE genes of 11 subjects who had been found in a large population survey to be heterozygous for the C282Y mutation and had elevated ferritin levels were sequenced. No mutations were found. Sequencing of the HFE gene also revealed two polymorphisms that had not previously been noted, - 467 C→G and - 970 T→G. Neither of these mutations appear to cause an abnormality in iron metabolism. [ABSTRACT FROM AUTHOR]