Dysregulated hepcidin response to dietary iron in male mice with reduced Gnpat expression.

Exome sequencing has identified the glyceronephosphate O-acyltransferase (GNPAT) gene as a genetic modifier of iron overload in hereditary hemochromatosis (HH). Subjects with HFE (Homeostatic Iron Regulator) p.C282Y mutations and the GNPAT p.D519G variant had more iron loading compared with subjects without the GNPAT variant. In response to an oral iron challenge, women with GNPAT polymorphisms loaded more iron as compared with women without polymorphisms, reinforcing a role for GNPAT in iron homeostasis. The aim of the present study was to develop and characterize an animal model of disease to further our understanding of genetic modifiers, and in particular the role of GNPAT in iron homeostasis. We generated an Hfe/Gnpat mouse model reminiscent of the patients previously studied and studied these mice for up to 26 weeks. We also examined the effect of dietary iron loading on mice with reduced Gnpat expression. Gnpat heterozygosity in Hfe knockout mice does not play a role in systemic iron homeostasis; Gnpat+/- mice fed a high-iron diet, however, had lower hepatic hepcidin (HAMP) mRNA expression, whereas they have significantly higher serum iron levels and transferrin saturation compared with wildtype (WT) littermates on a similar diet. These results reinforce an independent role of GNPAT in systemic iron homeostasis, reproducing in an animal model, the observations in women with GNPAT polymorphisms subjected to an iron tolerance test.

Evaluation of a bone morphogenetic protein 6 variant as a cause of iron loading.

BACKGROUND: Atypical iron overload without variation in the five clinically associated hereditary hemochromatosis genes is now recognized; however, their etiology remains unknown. Since the identification of iron overload in the bone morphogenetic protein 6 (Bmp6) knockout mouse, the search has been on for clinically pathogenic variants in the BMP6 gene. A recent report proposes that variants in the pro-peptide region of BMP6 are the underlying cause of several cases of iron overload. We performed targeted next-generation sequencing on three cases of atypical iron overload with Asian ethnicity and identified a p.Q118dup (aka p.E112indelEQ, p.Q115dup, p.Q118_L119insQ) variant in BMP6. The purpose of this study was to characterize the molecular function of the identified BMP6 variant. Molecular characterization by immunofluorescence microscopy and Western blotting of transfected cells, bioinformatics, and population analyses was performed. RESULTS: In contrast to reports for other BMP6 pro-peptide variants in this region, our data indicates that this variant does not affect the function of the mature BMP6 protein. CONCLUSIONS: Our data suggest that assignment of disease causation in clinical cases of iron overload to pro-peptide variants in BMP6 should thus be treated with caution and requires biological characterization.

The dynamics of hepcidin-ferroportin internalization and consequences of a novel ferroportin disease mutation.

The hepcidin-ferroportin axis underlies the pathophysiology of many iron-associated disorders and is a key target for the development of therapeutics for treating iron-associated disorders. The aims of this study were to investigate the dynamics of hepcidin-mediated ferroportin internalization and the consequences of a novel disease-causing mutation on ferroportin function. Specific reagents for ferroportin are limited; we developed and characterized antibodies against the largest extracellular loop of ferroportin and developed a novel cell-based assay for studying hepcidin-ferroportin function. We show that hepcidin-mediated ferroportin internalization is a rapid process and could be induced using low concentrations of hepcidin. Targeted next-generation sequencing utilizing an iron metabolism gene panel developed in our group identified a novel ferroportin p.D84E variant in a patient with iron overload. Wild-type and mutant ferroportin constructs were generated, transfected into HEK293 cells and analysed using an all-in-one flow-cytometry-based assay to study the effects on hepcidin-mediated internalization and iron transport. Consistent with the classical phenotype of ferroportin disease, the p.D84E mutation results in an inability to transport iron and hepcidin insensitivity. These results validate a recently proposed 3D-structural model of ferroportin and highlight the significance of this variant in the structure and function of ferroportin. Our novel ferroportin antibody and assay will be valuable tools for investigating the regulation of hepcidin/ferroportin function and the development of novel approaches for the therapeutic modulation of iron homeostasis.

Next-generation sequencing: Application of a novel platform to analyze atypical iron disorders.

The development of targeted next-generation sequencing (NGS) applications now promises to be a clinically viable option for the diagnosis of rare disorders. This approach is proving to have significant utility where standardized testing has failed to identify the underlying molecular basis of disease. We have developed a unique targeted NGS panel for the systematic sequence-based analysis of atypical iron disorders. We report the analysis of 39 genes associated with iron regulation in eight cases of atypical iron dysregulation, in which five cases we identified the definitive causative mutation, and a possible causative mutation in a sixth. We further provide a molecular and cellular characterization study of one of these mutations (TFR2, p.I529N) in a familial case as proof of principle. Cellular analysis of the mutant protein indicates that this amino acid substitution affects the localization of the protein, which results in its retention in the endoplasmic reticulum and thus failure to function at the cell surface. Our unique NGS panel presents a rapid and cost-efficient approach to identify the underlying genetic cause in cases of atypical iron homeostasis disorders.

Functional analysis of matriptase-2 mutations and domains: insights into the molecular basis of iron-refractory iron deficiency anemia.

Mutations in the TMPRSS6 gene are associated with severe iron-refractory iron deficiency anemia resulting from an overexpression of hepcidin, the key regulator of iron homeostasis. The matriptase (MT)-2 protein (encoded by the TMPRSS6 gene) regulates hepcidin expression by cleaving hemojuvelin [HJV/hemochromatosis type 2 (HFE2)], a bone morphogenetic protein (BMP) coreceptor in the hepcidin regulatory pathway. We investigated the functional consequences of five clinically associated TMPRSS6 variants and the role of MT-2 protein domains by generating epitope-tagged mutant and domain-swapped MT-2-MT-1 (encoded by the ST14 gene) chimeric constructs and expressing them in HepG2/C3A cells. We developed a novel cell culture immunofluorescence assay to assess the effect of MT-2 on cell surface HJV expression levels, compatible with HJV cleavage. The TMPRSS6 variants Y141C, I212T, G442R, and C510S were retained intracellularly and were unable to inhibit BMP6 induction of hepcidin. The R271Q variant, although it has been associated with iron-refractory iron deficiency anemia, appears to remain functional. Analysis of the chimeric constructs showed that replacement of sperm protein, enterokinase, and agrin (SEA), low-density-lipoprotein receptor class A (LDLRA), and protease (PROT) domains from MT-2 with those from MT-1 resulted in limited cell surface localization, while the complement C1r/C1s, Uegf, Bmp1 (CUB) domain chimera retained localization at the cell surface. The SEA domain chimera was able to reduce cell surface HJV expression, while the CUB, LDLRA, and PROT domain chimeras were not. These studies suggest that the SEA and LDLRA domains of MT-2 are important for trafficking to the cell surface and that the CUB, LDLRA, and PROT domains are required for cleavage of HJV.

A critical role for murine transferrin receptor 2 in erythropoiesis during iron restriction.

Effective erythropoiesis requires an appropriate supply of iron and mechanisms regulating iron homeostasis and erythropoiesis are intrinsically linked. Iron dysregulation, typified by iron-deficiency anaemia and iron overload, is common in many clinical conditions and impacts the health of up to 30% of the world's population. The proteins transmembrane protease, serine 6 (TMPRSS6; also termed matriptase-2), HFE and transferrin receptor 2 (TFR2) play important and opposing roles in systemic iron homeostasis, by regulating expression of the iron regulatory hormone hepcidin. We have performed a systematic analysis of mice deficient in these three proteins and show that TMPRSS6 predominates over HFE and TFR2 in hepcidin regulation. The phenotype of mice lacking TMPRSS6 and TFR2 is characterized by severe anaemia and extramedullary haematopoiesis in the spleen. Stress erythropoiesis in these mice results in increased expression of the newly identified erythroid iron regulator erythroferrone, which does not appear to overcome the hepcidin overproduction mediated by loss of TMPRSS6. Extended analysis reveals that TFR2 plays an important role in erythroid cells, where it is involved in terminal erythroblast differentiation and the regulation of erythropoietin. In conclusion, we have identified an essential role for TFR2 in erythropoiesis that may provide new targets for the treatment of anaemia.

Parenteral vs. oral iron: influence on hepcidin signaling pathways through analysis of Hfe/Tfr2-null mice.

Treatment for iron deficiency anemia can involve iron supplementation via dietary or parenteral routes that result in different cellular iron distributions. The effect of the administered iron on the iron regulatory system and hepcidin in the liver has not been well studied. Hepcidin, the liver-expressed central iron-regulatory peptide, is itself regulated through the bone morphogenetic protein (BMP)/SMAD signaling pathway. Specifically, Bmp6 expression is upregulated in response to iron and induces hepcidin through phosphorylation of Smad1/5/8. The hemochromatosis-associated proteins Hfe and transferrin receptor 2 (Tfr2) are known upstream regulators of hepcidin, although their precise roles are still unclear. To investigate the mechanisms of this regulation and the roles of the Hfe and Tfr2, we subjected wild-type, Hfe(-/-), Tfr2(-/-), and Hfe(-/-)/Tfr2(-/-) mice to iron loading via dietary or parenteral routes. Systematic analysis demonstrated that Tfr2 is required for effective upregulation of Bmp6 in response to hepatocyte iron, but not nonparenchymal iron. Hfe is not required for Bmp6 upregulation, regardless of iron localization, but rather, is required for efficient downstream transmission of the regulatory signal. Our results demonstrate that Hfe and Tfr2 play separate roles in the regulatory responses to iron compartmentalized in different cell types and further elucidates the regulatory mechanisms controlling iron homeostasis.

Hepatic iron deposition does not predict extrahepatic iron loading in mouse models of hereditary hemochromatosis.

Hereditary hemochromatosis is characterized by tissue iron loading and associated organ damage. However, the phenotype can be highly variable. The relationship between iron loading of different organs and the temporal nature of its deposition is still not well understood. We examined the progression of tissue iron loading in three mouse models to advance our understanding of the natural history of iron deposition in hereditary hemochromatosis. Wild-type, Hfe(-/-), Tfr2(-/-), and Hfe(-/-)/Tfr2(-/-) mice were analyzed at 3, 5, 10, 26, and 52 weeks, respectively. Hepatic, splenic, cardiac, and pancreatic iron concentrations were determined. Expression of both iron-regulatory and fibrosis genes was determined by quantitative real-time PCR in livers and hearts of 52-week-old mice. In all models, hepatic iron increased rapidly, plateauing before 10 weeks at different levels, depending on the genotype. Iron deposition in the pancreas and heart occurred after maximal iron loading of the liver was reached and was most marked in the Hfe(-/-)/Tfr2(-/-) mice. Although a significant positive correlation was identified between pancreatic and cardiac iron in all models at 52 weeks, there was no correlation between hepatic and either pancreatic or cardiac iron. There is variability in the timing and extent of tissue iron loading within a genotype, suggesting that hepatic iron levels in hereditary hemochromatosis may not accurately predict the iron content of other organs.

Iron loading and oxidative stress in the Atm-/- mouse liver.

Ataxia-Telangiectasia (A-T) is an autosomal recessive disorder resulting in a myriad of abnormalities, including progressive neurodegeneration and cancer predisposition. At the cellular level, A-T is a disease of chronic oxidative stress (OS) causing damage to proteins, lipids, and DNA. OS is contributed to by pro-oxidative transition metals such as iron that catalyze the conversion of weakly reactive oxygen species to highly reactive hydroxyl radicals. Iron-associated OS has been linked to neurodegeneration in Alzheimer's and Parkinson's diseases and development of lymphoid tumors (which afflict ∼30% of A-T patients). To investigate iron regulation in A-T, iron indexes, regulatory genes, and OS markers were studied in livers of wild-type and Ataxia telangiectasia mutated (Atm) null mice on control or high-iron diets. Atm(-/-) mice had increased serum iron, hepatic iron, and ferritin and significantly higher Hepcidin compared with wild-type mice. When challenged with the high-iron diet, Bmp6 and Hfe expression was significantly increased. Atm(-/-) mice had increased protein tyrosine nitration and significantly higher Heme Oxygenase (decycling) 1 levels that were substantially increased by a high-iron diet. Ferroportin gene expression was significantly increased; however, protein levels were unchanged. We demonstrate that Atm(-/-) mice have a propensity to accumulate iron that is associated with a significant increase in hepatic OS. The iron-induced increase in hepcidin peptide in turn suppresses ferroportin protein levels, thus nullifying the upregulation of mRNA expression in response to increased OS. Our results suggest that increased iron status may contribute to the chronic OS seen in A-T patients and development of disease pathology.

Blunted hepcidin response to inflammation in the absence of Hfe and transferrin receptor 2.

The induction of the iron-regulatory peptide hepcidin by proinflammatory cytokines is thought to result in the withholding of iron from invading pathogens. Hfe and transferrin receptor 2 (Tfr2) are involved in the homeostatic regulation of hepcidin and their disruption causes hereditary hemochromatosis (HH). To determine whether either Hfe or Tfr2 is involved in the inflammatory pathway regulating hepcidin, we analyzed the effect of inflammation in 3 mouse models of HH. The inflammatory response and indicators of iron homeostasis were measured in wild-type, Hfe(-/-), Tfr2(-/-), and Hfe(-/-)/Tfr2(-/-) mice injected with lipopolysaccharide (LPS). The administration of LPS significantly reduced serum iron in wild-type and Hfe(-/-) mice, with smaller reductions in Tfr2(-/-) and Hfe(-/-)/Tfr2(-/-) mice. Low basal levels of hepcidin in the Hfe(-/-)/Tfr2(-/-) mice were increased in response to LPS, but remained significantly lower than in the other strains of mice. These results suggest that despite the absence of Hfe and Tfr2, hepcidin is responsive to inflammation; however, the low basal expression and subsequent low levels of circulating hepcidin are insufficient to reduce serum iron effectively. This suggests that in HH, the iron-withholding response to invading pathogens may be inadequate, and this is especially the case in the absence of both Hfe and Tfr2.

G80S-linked ferroportin disease: classical ferroportin disease in an Asian family and reclassification of the mutant as iron transport defective.

BACKGROUND & AIMS: Hereditary iron overload associated with mutations in the ferroportin gene produces a dichotomy of phenotypes resulting from either increase or decrease in iron efflux capacity. In this study, we examined the molecular basis of iron overload in a family of Vietnamese origin, characterized the molecular and cellular defect, and correlated it with the clinical and pathological phenotype. METHODS: We analyzed the ferroportin gene by DNA sequencing. The molecular characterization was performed by immunofluorescence microscopy analysis of transfected cells. We analyzed ferritin levels, in cells expressing wild-type and mutant ferroportin, to define the nature of the molecular defect in iron transport. RESULTS: We identified a G to A nucleotide change at position 238 in the ferroportin gene leading to the G80S substitution. Cellular analysis of the mutant protein indicates that this amino acid change does not affect the localization of the protein but does affect its ability to transport iron. CONCLUSIONS: The G80S mutation results in a mutated ferroportin associated with iron overload and is predicted to be defective in iron export.