Ferroportin diseases: functional studies, a link between genetic and clinical phenotype.

Ferroportin (FPN) mediates iron export from cells and this function is modulated by serum hepcidin. Mutations in the FPN gene (SLC40A1) lead to autosomal dominant iron overload diseases related either to loss or to gain of function, and usually characterized by normal or low transferrin saturation versus elevated transferrin saturation, respectively. However, for the same mutation, the phenotypic expression may vary from one patient to another. Using in vitro overexpression of wild-type or mutant FPN proteins, we characterized the functional impact of five recently identified FPN gene mutations regarding FPN localization, cell iron status, and hepcidin sensitivity. Our aim was to integrate functional results and biological findings in probands and relatives. We show that while the p.Arg371Gln (R371Q) mutation had no impact on studied parameters, the p.Trp158Leu (W158L), p.Arg88Gly (R88G), and p.Asn185Asp (N185D) mutations caused an iron export defect and were classified as loss-of-function mutations. The p.Gly204Ser (G204S) mutation induced a gain of FPN function. Functional studies are useful to determine whether or not a FPN gene mutation found in an iron overloaded patient is deleterious and to characterize its biological impact, especially when family studies are not fully informative and/or additional confounding factors may affect bio-clinical expression.

A novel N491S mutation in the human SLC11A2 gene impairs protein trafficking and in association with the G212V mutation leads to microcytic anemia and liver iron overload.

BACKGROUND: DMT1 is a transmembrane iron transporter involved in iron duodenal absorption and cellular iron uptake. Mutations in the human SLC11A2 gene coding DMT1 lead to microcytic anemia and hepatic iron overload, with unexpectedly low levels of plasma ferritin in the presence of iron stores. DESIGN AND METHODS: We report a patient with a similar phenotype due to two mutations in the SLC11A2 gene, the known p.Gly212Val (G212V) mutation and a novel one, p.Asn491Ser (N491S). To assess the expression of DMT1 in human liver, we studied the expression of the four DMT1 mRNA isoforms by real-time quantitative PCR in control human liver samples. We also studied the effect of G212V and N491S DMT1 mutations on RNA splicing in blood leukocytes and cellular trafficking of dsRed2-tagged-DMT1 protein in the human hepatic cell line HuH7. RESULTS: Our results showed that i) only the isoforms 1B-IRE and 1B-nonIRE were significantly expressed in human liver; ii) the G212V mutation did not seem to affect mRNA splicing and the N491S mutation induced a splicing alteration leading to a truncated protein, which seemed quantitatively of low relevance; and iii) the N491S mutation, in contrast to the G212V mutation, led to abnormal protein trafficking. CONCLUSIONS: Our data confirm the major role of DMT1 in the maintenance of iron homeostasis in humans and demonstrate that the N491S mutation, through its deleterious effect on protein trafficking, contributes together with the G212V mutation to the development of anemia and hepatic iron overload.

Sex and acquired cofactors determine phenotypes of ferroportin disease.

BACKGROUND & AIMS: Ferroportin disease is characterized by iron overload. It has an autosomal-dominant pattern of inheritance and has been associated with mutations in the SLC40A1 gene, which encodes the cellular iron exporter ferroportin. Since the first description in 2001, about 30 mutations have been reported; the heterogeneity of ferroportin disease phenotypes has led to the hypothesis that the nature of the mutation affects the function of the protein in different ways. We studied genotypes and phenotypes of a large cohort of patients with ferroportin disease. METHODS: We studied clinical, biochemical, imaging, histologic, and genetic data from 70 affected subjects from 33 families with 19 mutations. RESULTS: We found that ferroportin disease, at the time of diagnosis, has limited consequences in the absence of cofactors. Data indicated that transferrin saturation, which correlated with fibrosis and levels of alanine aminotransferase, might be a marker of disease severity. Although the study was performed in a large number of families, we observed incomplete penetrance and no correlation between genotypes and phenotypes. CONCLUSIONS: Members of families with ferroportin disease should be screened for biochemical parameters of iron metabolism as well as genotype to detect silent mutations that might cause disease with acquired or genetic cofactors. Patients should be followed up long term to identify potential complications of the disease.

Molecular diagnosis of genetic iron-overload disorders.

Genetic iron overload has long been confined to the picture of classical hemochromatosis related to the HFE C282Y mutation (type 1 hemochromatosis). C282Y homozygosity affects approximately three people out of 1000 of the Caucasian population, representing one of the most frequent genetic predispositions. It has, however, rapidly become clear that the HFE C282Y mutation is not the sole culprit in genetic iron overload. Several novel mutations in HFE and other genes have been discovered and related to various entities, which are now known as types 2, 3 and 4 hemochromatosis. These diseases are far less frequent than the classical type 1 hemochromatosis but, by contrast, are not limited to the Caucasian population. Molecular diagnosis obviously plays a key role in the diagnostic strategy. In the future, it will undoubtedly enable not only identification of new diagnostic markers, but also provide potential molecular targets for pathophysiologically based innovative therapeutic approaches.

A new mutation in the hepcidin promoter impairs its BMP response and contributes to a severe phenotype in HFE related hemochromatosis.

Low levels of hepcidin are responsible for the development of iron overload in p.Cys282Tyr HFE related hemochromatosis. Every genetic factor lowering the hepcidin gene expression could contribute to a more severe phenotype in HFE hemochromatosis. Based on this hypothesis, we identified a heterozygous nc.-153 C>T mutation in the hepcidin gene promoter sequence in a patient homozygous for the p.Cys282Tyr HFE mutation who presented massive iron overload, resisting to well conducted iron depletive treatment. Our results demonstrate that the nc.-153 C>T mutation, located within a BMP-RE (Bone Morphogenetic Protein-Responsive Element): i) decreases the transcriptional activity of the hepcidin promoter, ii) alters its IL-6 (Interleukin-6) total responsiveness, and iii) prevents the binding of the SMAD protein complex (1/5/8 and 4) to the BPM-RE. In conclusion, our results suggest that a mutation in the BMP-RE of hepcidin promoter may impact on human iron metabolism.

A new missense mutation in the L ferritin coding sequence associated with elevated levels of glycosylated ferritin in serum and absence of iron overload.

BACKGROUND: Elevated serum ferritin levels are frequently encountered in clinical situations and once iron overload or inflammation has been ruled out, many cases remain unexplained. Genetic causes of hyperferritinemia associated to early cataract include mutations in the iron responsive element in the 5' untranslated region of the L ferritin mRNA, responsible for the hereditary hyperferritinemia cataract syndrome. DESIGN AND METHODS: We studied 91 probands with hyperferritinemia comprising 25 family cases belonging to families with at least two cases of unexplained hyperferritinemia, and 66 isolated cases. In the families, we also analyzed 30 relatives. Hyperferritinemia was considered as unexplained when transferrin saturation was below 45% and/or serum iron below 25 mumol/L and/or no tissue iron excess was detected, when inflammation had been ruled out and when iron responsive element mutation was absent. We carried out sequencing analysis of the FTL gene coding the L ferritin. RESULTS: A novel heterozygous p.Thr30Ile mutation in the NH2 terminus of L ferritin subunit was identified in 17 probands out of the cohort. The mutation was shown to cosegregate with hyperferritinemia in all the 10 families studied. No obvious clinical symptom was found associated with the presence of the mutation. This unique mutation is associated with an unusually high percentage of ferritin glycosylation. CONCLUSIONS: This missense mutation of FTL represents a new cause of genetic hyperferritinemia without iron overload. We hypothesized that the mutation increases the efficacy of L ferritin secretion by increasing the hydrophobicity of the N terminal "A" alpha helix.

Common variants in the BMP2, BMP4, and HJV genes of the hepcidin regulation pathway modulate HFE hemochromatosis penetrance.

Most cases of genetic hemochromatosis (GH) are associated with the HFE C282Y/C282Y (p.Cys282Tyr/p.Cys282Tyr) genotype in white populations. The symptoms expressed by C282Y homozygotes are extremely variable. Only a few suffer from an overt disease. Several studies have suggested that, in addition to environmental factors, a genetic component could explain a substantial part of this phenotypic variation, although very few genetic factors have been identified so far. In the present study, we tested the association between common variants in candidate genes and hemochromatosis penetrance, in a large sample of C282Y homozygotes, using pretherapeutic serum ferritin level as marker of hemochromatosis penetrance. We focused on two biologically relevant gene categories: genes involved in non-HFE GH (TFR2, HAMP, and SLC40A1) and genes involved in the regulation of hepcidin expression, including genes from the bone morphogenetic protein (BMP) regulatory pathway (BMP2, BMP4, HJV, SMAD1, SMAD4, and SMAD5) and the IL6 gene from the inflammation-mediated regulation pathway. A significant association was detected between serum ferritin level and rs235756, a common single-nucleotide polymorphism (SNP) in the BMP2 genic region (P=4.42x10-5). Mean ferritin level, adjusted for age and sex, is 655 ng/ml among TT genotypes, 516 ng/ml in TC genotypes, and 349 ng/ml in CC genotypes. Our results further suggest an interactive effect on serum ferritin level of rs235756 in BMP2 and a SNP in HJV, with a small additive effect of a SNP in BMP4. This first reported association between common variants in the BMP pathway and iron burden suggests that full expression of HFE hemochromatosis is linked to abnormal liver expression of hepcidin, not only through impairment in the HFE function but also through functional modulation in the BMP pathway. Our results also highlight the BMP regulation pathway as a good candidate for identification of new modifier genes.

[Pathophysiology and genetics of classic HFE (type 1) hemochromatosis]. / Physiopathologie et génétique de l'hémochromatose HFE de type 1.

Hereditary type 1 HFE hemochromatosis is associated with homozygosity for the p.Cys282Tyr mutation of the HFE gene (C282Y mutation). The p.Cys282Tyr mutation of the HFE gene leads to an abnormal reduction in hepatic expression of hepcidin, a protein that appears to control the release of iron from enterocytes and macrophages towards plasma. Abnormally low hepcidin levels promote an increase in the bioavailability of plasma iron, characterized by elevated transferrin saturation and the appearance of non transferrin bound iron. This nontransferrin-bound iron is avidly taken up by the liver, heart, and pancreas, the principal target organs for systemic iron overload. The variable penetrance of this disease is related to environmental and genetic factors. Among the genetic factors, mutations of some newly identified genes may aggravate the phenotype of iron overload associated with homozygosity for the p.Cys282Tyr mutation of the HFE gene; these new genes include those of hemojuvelin (HJV), transferrin receptor 2 (TfR2), and hepcidin (HAMP).