Iron supplementation in mouse expands cellular innate defences in spleen and defers lethal malaria infection.

The co-endemicity of malnutrition, erythrocytopathies, transmissible diseases and iron-deficiency contribute to the prevalence of chronic anaemia in many populations of the developing world. Although iron dietary supplementation is applied or recommended in at risk populations, its use is controversial due to undesirable outcomes, particularly regarding the response to infections, including highly prevalent malaria. We hypothesized that a boosted oxidative stress due to iron supplementation have a similar impact on malaria to that of hereditary anaemias, enhancing innate response and conditioning tissues to prevent damage during infection. Thus, we have analysed antioxidant and innate responses against lethal Plasmodium yoelii during the first five days of infection in an iron-supplemented mouse. This murine model showed high iron concentration in plasma with upregulated expression of hemoxygenase-1. The sustained homeostasis after this extrinsic iron conditioning, delayed parasitemia growth that, once installed, developed without anaemia. This protection was not conferred by the intrinsic iron overload of hereditary hemochromatosis. Upon iron-supplementation, a large increase of the macrophages/dendritic cells ratio and the antigen presenting cells was observed in the mouse spleen, independently of malaria infection. Complementary, malaria promoted the splenic B and T CD4 cells activation. Our results show that the iron supplementation in mice prepares host tissues for oxidative-stress and induces unspecific cellular immune responses, which could be seen as an advantage to promote early defences against malaria infection.

Microcytic anemia in a pregnant woman: beyond iron deficiency.

Sideroblastic anemias are a heterogeneous group of disorders characterized by anemia of varying severity and the presence of ringed sideroblasts in bone marrow. The most common form of inherited sideroblastic anemia is X-linked sideroblastic anemia (XLSA). In many XLSA patients, anemia responds variably to supplementation with pyridoxine (vitamin B6). We describe the case of a pregnant female with XLSA who had a novel mutation on the ALAS2 gene (c.1218G > T, p.Leu406Phe). Oral chelation therapy was contraindicated and high-dose vitamin B6 would have possible side effects in pregnancy. Serum hepcidin level was very low, indicating increased absorption of iron secondary to ineffective erythropoiesis. Therapy was begun with a low dose of pyridoxine that was increased post-partum. The patient's liver showed moderate iron deposits. During a subsequent 3-month period of pyridoxine supplementation, serum ferritin level and transferrin saturation decreased, hemoglobin content and serum hepcidin level normalized, and morphologic red cell abnormalities improved markedly. The patient responded well to treatment, showing the pyridoxine responsiveness of this novel ALAS2 mutation. The baby girl had the same mutation heterozygously, and although she was neither anemic nor showed abnormalities in a peripheral blood smear, she had a mild increment in RDW and her condition is now being followed.

A novel mutation in the SLC40A1 gene associated with reduced iron export in vitro.

Ferroportin disease is an inherited disorder of iron metabolism and is caused by mutations in the ferroportin gene (SLC40A1). We present a patient with hyperferritinemia, iron overload in the liver with reticuloendothelial distribution and also in the spleen, and under treatment with erythropheresis. A molecular study of the genes involved in iron metabolism (HFE, HJV, HAMP, TFR2, SLC40A1) was undertaken. In vitro functional studies of the novel mutation found in the SLC40A1 gene was performed. The patient was heterozygous for a novel mutation, c.386T>C (p.L129P) in the SLC40A1 gene; some of his relatives were also heterozygous for this mutation. In vitro functional studies of the L129P mutation on ferroportin showed it impairs its capacity to export iron from cells but does not alter its sensitivity to hepcidin. These findings and the iron overload phenotype of the patient suggest that the novel mutation c.386T>C (p.L129P) in the SLC40A1 gene has incomplete penetrance and causes the classical form of ferroportin disease.

Familial porphyria cutanea tarda in Spain: characterization of eight novel mutations in the UROD gene and haplotype analysis of the common p.G281E mutation.

Porphyria cutanea tarda (PCT) results from decreased activity of uroporphyrinogen decarboxylase (UROD) in the liver. Deficiency in this enzyme results in accumulation of highly carboxylated porphyrins responsible for the disease. PCT usually occurs in adulthood and is characterized by cutaneous photosensitivity, hyperpigmentation, skin fragility and hypertrichosis. Familial PCT (F-PCT) occurs in 20-30% of patients in whom UROD gene mutations in heterozygosity decrease the enzymatic activity to about 50% in all tissues. The rare homozygous form of F-PCT (hepatoerythropoietic porphyria) has more severe clinical features and onset in childhood. In Spain, F-PCT is molecularly heterogeneous and the most frequent UROD mutation is p.G281E. In the present study, we searched for the molecular defect causing F-PCT in a group of Spanish patients and investigated whether the p.G281E mutation in the Spanish population came from a single or various origins. Among seventeen F-PCT patients, sixteen UROD mutations were identified, including eight novel ones: six missense (p.A23V, p.L78P, p.W180G, p.T196I, p.E278G and p.V279M), one frameshift (c.233delT) and one splice site mutation (c.774G>C). Prokaryotic expression studies showed the detrimental effect for each missense mutation, whereas reverse transcription-PCR and sequencing demonstrated that the novel splice site mutation caused exon 7 skipping. Moreover, haplotype analysis performed in Spanish families with the p.G281E mutation indicated that this lesion is associated with at least five haplotype backgrounds. These results extend knowledge on the molecular heterogeneity of F-PCT and suggest multiple origins of the p.G281E mutation.

Mutations in the HFE, TFR2, and SLC40A1 genes in patients with hemochromatosis.

Hereditary hemochromatosis causes iron overload and is associated with a variety of genetic and phenotypic conditions. Early diagnosis is important so that effective treatment can be administered and the risk of tissue damage avoided. Most patients are homozygous for the c.845G>A (p.C282Y) mutation in the HFE gene; however, rare forms of genetic iron overload must be diagnosed using a specific genetic analysis. We studied the genotype of 5 patients who had hyperferritinemia and an iron overload phenotype, but not classic mutations in the HFE gene. Two patients were undergoing phlebotomy and had no iron overload, 1 with metabolic syndrome and no phlebotomy had mild iron overload, and 2 patients had severe iron overload despite phlebotomy. The patients' first-degree relatives also underwent the analysis. We found 5 not previously published mutations: c.-408_-406delCAA in HFE, c.1118G>A (p.G373D), c.1473G>A (p.E491E) and c.2085G>C (p.S695S) in TFR2; and c.-428_-427GG>TT in SLC40A1. Moreover, we found 3 previously published mutations: c.221C>T (p.R71X) in HFE; c.1127C>A (p.A376D) in TFR2; and c.539T>C (p.I180T) in SLC40A1. Four patients were double heterozygous or compound heterozygous for the mutations mentioned above, and the patient with metabolic syndrome was heterozygous for a mutation in the TFR2 gene. Our findings show that hereditary hemochromatosis is clinically and genetically heterogeneous and that acquired factors may modify or determine the phenotype.

Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination.

Ferroportin exports iron into plasma from absorptive enterocytes, erythrophagocytosing macrophages, and hepatic stores. The hormone hepcidin controls cellular iron export and plasma iron concentrations by binding to ferroportin and causing its internalization and degradation. We explored the mechanism of hepcidin-induced endocytosis of ferroportin, the key molecular event in systemic iron homeostasis. Hepcidin binding caused rapid ubiquitination of ferroportin in cell lines overexpressing ferroportin and in murine bone marrow-derived macrophages. No hepcidin-dependent ubiquitination was observed in C326S ferroportin mutant which does not bind hepcidin. Substitutions of lysines between residues 229 and 269 in the third cytoplasmic loop of ferroportin prevented hepcidin-dependent ubiquitination and endocytosis of ferroportin, and promoted cellular iron export even in the presence of hepcidin. The human ferroportin mutation K240E, previously associated with clinical iron overload, caused hepcidin resistance in vitro by interfering with ferroportin ubiquitination. Our study demonstrates that ubiquitination is the functionally relevant signal for hepcidin-induced ferroportin endocytosis.

Molecular analysis of the UROD gene in 17 Argentinean patients with familial porphyria cutanea tarda: characterization of four novel mutations.

Porphyria cutanea tarda (PCT) is caused by decreased activity of uroporphyrinogen decarboxylase (UROD) in the liver. The disease usually occurs in adulthood and is characterized by cutaneous photosensitivity, hyperpigmentation, skin fragility and hypertrichosis, due to the accumulation of porphyrins produced by oxidation of uroporphyrinogen and other highly carboxylated porphyrinogens overproduced as a result of the enzyme deficiency. PCT is generally sporadic, but about 20-30% of patients have familial-PCT (F-PCT) which is associated with heterozygosity of mutations in the UROD gene. In the present study we have found the molecular defect in seventeen unrelated Argentinean patients with F-PCT, identifying a total of eleven UROD gene mutations: four novel and seven previously described. The novel mutations were: a guanine insertion at the 5' splice junction of intron 2, a three nucleotide deletion causing the lost of valine 90, a deletion of 22 bp in exon 6 and a deletion of part of the polyadenylation signal. Prokaryotic expression studies showed that the novel amino acid deletion resulted in an inactive protein. Mutations c.10insA and p.M165R, previously found in Argentinean patients, were recurrent in this study; they are the most frequent in Argentina accounting for 40% of the mutant alleles characterized to date.

Mutaciones en los genes HFE y TFR2 en un paciente español con hemocromatosis / Mutations in HFE and TFR2 genes in a Spanish patient with hemochromatosis

La enfermedad por sobrecarga de hierro está originada por diversas anomalías genéticas. El estudio genético de esta enfermedad confirma su carácter hereditario y nos permite ofrecer consejo genético a los familiares en primer grado. Hemos realizado resonancia magnética y biopsia de hígado en un paciente asintomático con más de 1.000 mg/l de ferritina en suero, y hemos analizado los genes implicados en el metabolismo del hierro. El fenotipo de sobrecarga de hierro se confirmó por la presencia de un patrón de depósito de hierro en el hígado con predominio periportal que sugiere la existencia de una enfermedad genética. En el caso que presentamos, el estudio genético reveló que el paciente es doble heterocigoto para las mutaciones c.187C>G (p.H63D) y c.840C>G (p.F280L) en los genes HFE y receptor 2 de transferrina (TFR2), respectivamente(AU)

Iron overload disease has a wide variety of genotypes. The genetic study of this disease confirms its hereditary nature and enables us to provide genetic counseling for first-degree relatives. We performed magnetic resonance imaging and liver biopsy in an asymptomatic patient with more than 1,000 mg/L of serum ferritin and studied the genes involved in this condition. The phenotype of iron overload is confirmed by a predominantly periportal pattern of iron deposits in the liver suggestive of genetic disease. In the case we present the molecular study revealed a double heterozygosity for the mutations c.187C>G (p.H63D) and c.840C>G (p.F280L) in the HFE and transferrin receptor 2 (TFR2) genes, respectively(AU)

Hiperferritinemia, ferropenia y síndrome metabólico en un paciente con una nueva mutación en el gen TFR2 y otra en el gen FTL. Estudio familiar / Hyperferritinemia, ferropenia and metabolic syndrome in a patient with a new mutation of gene TFR2 and another in gene FTL. A family study

Fundamento y objetivo: La hiperferritinemia es un hallazgo común en la práctica clínica diaria que puede ser congénita o adquirida y no siempre se asocia con sobrecarga férrica. La hiperferritinemia genética puede acompañarse de sobrecarga, como en la hemocromatosis hereditaria, o cursar con cataratas sin sobrecarga, en ese caso es el síndrome hereditario de hiperferritinemia y cataratas. Por otra parte, el síndrome metabólico puede cursar con hiperferritinemia y sobrecarga leve-moderada pero sin aumento de saturación de transferrina. Presentamos una familia con hiperferritinemia.Pacientes y método: Estudio de una familia con hiperferritinemia dual, congénita y adquirida, con análisis de los genes implicados en el metabolismo del hierro. Resultados:Los pacientes con síndrome hereditario de hiperferritinemia y cataratas tienen la mutación c.-167C>T en heterocigosis en el gen FTL. El paciente con síndrome metabólico presenta, además, una nueva mutación en heterocigosis en el gen TFR2 (c.1259G>A, p.Arg420His). Conclusiones: La hiperferritinemia, habitualmente casual, supone para el clínico un reto diagnóstico por sus diversidades fenotípicas y genotípicas, siendo necesario aunar esfuerzos en investigación básica y clínica para la asistencia de los pacientes (AU)

Background and objetives: Hyperferritinemia is a common finding in clinical practice. This condition can be congenital or acquired, although it is not always associated with iron overload. Genetic hyperferritinemia is associated with iron overload, hereditary hemochromatosis, or cataracts that progress without iron overload (hereditary hyperferritinemia-cataract syndrome). Metabolic syndrome is associated with hyperferritinemia and mild iron overload, with no increase in transferrin saturation. We report a family with hyperferritinemia. Patients and methods: We present the study of a family with dual hyperferritinemia (congenital and acquired) and an analysis of the genes involved in iron metabolism. Results: Patients with hereditary hyperferritinemia-cataract syndrome have the mutation c.-167C>T in the FTL gene; patients with metabolic syndrome present a new mutation in the TFR2 gene (c.1259G>A, p.Arg420His).Conclusions: The phenotypic and genotypic diversity of hyperferritinemia makes it a diagnostic challenge for clinicians. Basic research and clinical research should be combined to ensure better patient care (AU)

[Hyperferritinemia, ferropenia and metabolic syndrome in a patient with a new mutation of gene TFR2 and another in gene FTL. A family study]. / Hiperferritinemia, ferropenia y síndrome metabólico en un paciente con una nueva mutación en el gen TFR2 y otra en el gen FTL. Estudio familiar.

BACKGROUND AND OBJECTIVES: Hyperferritinemia is a common finding in clinical practice. This condition can be congenital or acquired, although it is not always associated with iron overload. Genetic hyperferritinemia is associated with iron overload, hereditary hemochromatosis, or cataracts that progress without iron overload (hereditary hyperferritinemia-cataract syndrome). Metabolic syndrome is associated with hyperferritinemia and mild iron overload, with no increase in transferrin saturation. We report a family with hyperferritinemia. PATIENTS AND METHODS: We present the study of a family with dual hyperferritinemia (congenital and acquired) and an analysis of the genes involved in iron metabolism. RESULTS: Patients with hereditary hyperferritinemia-cataract syndrome have the mutation c.-167C>T in the FTL gene; patients with metabolic syndrome present a new mutation in the TFR2 gene (c.1259G>A, p.Arg420His). CONCLUSIONS: The phenotypic and genotypic diversity of hyperferritinemia makes it a diagnostic challenge for clinicians. Basic research and clinical research should be combined to ensure better patient care.

Two novel mutations in the SLC40A1 and HFE genes implicated in iron overload in a Spanish man.

The most common form of hemochromatosis is caused by mutations in the HFE gene. Rare forms of the disease are caused by mutations in other genes. We present a patient with hyperferritinemia and iron overload, and facial flushing. Magnetic resonance imaging was performed to measure hepatic iron overload, and a molecular study of the genes involved in iron metabolism was undertaken. The iron overload was similar to that observed in HFE hemochromatosis, and the patient was double heterozygous for two novel mutations, c.-20G>A and c.718A>G (p.K240E), in the HFE and ferroportin (FPN1 or SLC40A1) genes, respectively. Hyperferritinemia and facial flushing improved after phlebotomy. Two of the patient's children were also studied, and the daughter was heterozygous for the mutation in the SLC40A1 gene, although she did not have hyperferritinemia. The patient presented a mild iron overload phenotype probably because of the two novel mutations in the HFE and SLC40A1 genes.

The hereditary hyperferritinemia-cataract syndrome: a family study.

Ferritin is an acute-phase reactant that is elevated in the course of infectious, inflammatory, autoimmune, and oncological diseases and the hemophagocytic syndrome. In asymptomatic patients, isolated hyperferritinemia may be due to different causes depending on whether or not it is accompanied by iron overload. Hyperferritinemia values above 300 ng/ml and an excess of body iron levels may be indicative of hemochromatosis. However, if such values develop in the absence of iron overload, they may be secondary to hemochromatosis type 4a (ferroportin disease) or more often to hereditary hyperferritinemia-cataract syndrome (HHCS; Aguilar-Martinez et al., Am J Gastroenterol 100:1185-1194, 2005; Ferrante et al., Eur J Gastroenterol Hepatol 17:1247-1253, 2005). HHCS results from different mutations in the L-ferritin gene (FTL) on chromosome 19 (19q13.1), causing autosomal dominant transmission (Bertola et al., Curr Drug Targets Immune Endocr Metabol Disord 4:93-105, 2004). We present a child with HHCS due to the allelic variant c.-167C>T (C33T) in the iron-responsive element region of the FTL gene. When pediatricians encounter an asymptomatic patient with isolated hyperferritinemia in the absence of iron overload, they should consider the possibility of HHCS, especially if other members of the family have developed cataracts from a young age.

Hepcidin treatment in Hfe-/- mice diminishes plasma iron without affecting erythropoiesis.

BACKGROUND: Iron is essential for mammalian metabolism and its cellular concentration is controlled by regulating its acquisition and storage. Haemochromatosis is a condition involving iron overload that is characterised by increased duodenal iron absorption and a progressive accumulation of iron in vital organs. Hepcidin is the main hormone that regulates iron homoestasis and it is secreted by the liver. MATERIALS AND METHODS: We have studied how extended hepcidin administration affects the iron load status, plasma and tissue iron concentration, erythropoiesis and the expression of proteins involved on iron homeostasis in haemochromatotic (Hfe(-/-)) and wild-type mice. RESULTS: Hepcidin reverted the high plasma iron concentrations in Hfe(-/-) mice to normal values. The high concentration of hepatic iron was not altered in the liver of these Hfe(-/-) mice. Hepcidin administration did not disturb erythropoiesis in either Hfe(-/-) or wild-type mice and likewise, hepcidin did not modify the expression of any protein analysed in the liver, duodenum or spleen of Hfe(-/-) and wild-type mice. These data confirm that hepcidin administration diminishes plasma iron concentrations. CONCLUSION: Treatment with sustained doses of hepcidin diminishes plasma iron concentrations in Hfe(-/-) mice.

Smoking but not homozygosity for CYP1A2 g-163A allelic variant leads to earlier disease onset in patients with sporadic porphyria cutanea tarda.

Porphyria cutanea tarda (PCT) results from decreased activity of hepatic uroporphyrinogen decarboxylase (UROD). Both sporadic and familial forms are characterised by typical cutaneous lesions triggered by genetic/environmental factors. Studies in rodents showed that cytochrome P4501A2 (CYP1A2) plays a central role in the synthesis of a competitive inhibitor of hepatic UROD, but there is little evidence in humans. The impact of smoking and CYP1A2 g-163C > A allelic variant upon first appearance of clinical signs was investigated in 102 patients (80 sporadic-PCT) and 150 healthy donors from Spain. We found an increase in the frequency of CYP1A2 g-163A allele in patients with PCT when compared with controls, although the more inducible A/A genotype had no effect on the onset age. In sporadic-PCT, smoking leads to earlier onset of clinically overt disease in moderate-to-heavy smokers (>or=10 cigarettes/day). In conclusion, this study provides evidence that smoking hastens the onset of cutaneous symptoms in sporadic-PCT patients.

Bone marrow-derived cells promote liver regeneration in mice with erythropoietic protoporphyria.

BACKGROUND: Bone marrow transplantation can reverse hepatic protoporphyrin accumulation and prevent the hepatobiliary complications characteristic of erythropoietic protoporphyria. The aim of this study was to assess the recruitment capacity of bone marrow cells in the damaged liver and their possible contribution to the improved or recovered hepatic function in a murine model of erythropoietic protoporphyria (EPP). METHODS: Lethally irradiated female EPP mice were transplanted with bone marrow cells from healthy male mice and were monitored during 12 or 36 weeks. Two groups of animals killed 12 weeks after transplant were also treated with granulocyte colony-stimulating factor. RESULTS: Cell transplantation decreased porphyrin contents in erythrocytes and liver. Improved hepatic structure and function and reduced hepatic fibrosis were observed, especially 36 weeks after transplant. Bone marrow-derived cells (22%-35%) were identified in the liver of recipient mice by means of fluorescence in situ hybridization (chrY-FISH) or green fluorescent protein staining and were characterized by immunofluorescence staining. The livers of recipients contained 20% to 30% myofibroblasts (alpha-smooth muscle actin-positive cells), 40% CK19-positive cells, and 10% to 28% hepatocytes (albumin-positive cells) derived from the donor bone marrow. CONCLUSIONS: Bone marrow-derived cells play a significant role in restoring and regenerating hepatic tissue in EPP mice. Hepatic repair was associated with fibrogenesis, enhanced by granulocyte colony-stimulating factor treatment, and almost normal liver structure and function was observed in the long term (36 weeks posttransplant).

Bone marrow transplantation into hemochromatotic mice decreases hepatic and duodenal iron overload.

Human hereditary hemochromatosis is a disorder of iron homeostasis characterized by increased absorption of iron and its deposition in parenchymal organs. The maintenance of iron homeostasis is regulated by molecules involved in the absorption, transport, storage and redox of iron. The potential of hematopoietic stem cell therapy for liver diseases has been studied in some experimental animal models. Our objective was to evaluate the effect of bone marrow transplantation from wild type mice on the status of iron overload in Hfe knockout hemochromatotic mice (Hfe(-/-)). The transplanted cells were detected in the liver (11% of the total cells) and characterized as hepatocytes and myofibroblasts. They were also detected in the duodenum and characterized as myofibroblasts. The iron content in the Hfe(-/-) mice descended 2.9-fold in the liver and 2.4-fold in the duodenum 6 months after transplantation. Non-significant changes of relative mRNA abundance of genes of iron metabolism were observed in the liver and duodenum of Hfe(-/-) transplanted mice. At 6 months after transplantation the proportion of Hfe mRNA in Hfe(-/-) mice reached 3.8% of the levels in wild type mice in the liver and 1.6% in the duodenum. In conclusion, adult stem cells from bone marrow transplant were able to differentiate into hepatocytes and myofibroblasts in hemochromatotic mice. Bone marrow transplantation assisted in reducing the iron overload in this murine model of hemochromatosis. These findings might contribute to the knowledge of pathways involved in the regulatory system of iron homeostasis.