RESUMO
Clinical severity is heterogeneous among patients suffering from congenital erythropoietic porphyria (CEP) suggesting a modulation of the disease (UROS deficiency) by environmental factors and modifier genes. A KI model of CEP due to a missense mutation of UROS gene present in human has been developed on 3 congenic mouse strains (BALB/c, C57BL/6, and 129/Sv) in order to study the impact of genetic background on disease severity. To detect putative modifiers of disease expression in congenic mice, hematologic data, iron parameters, porphyrin content and tissue samples were collected. Regenerative hemolytic anemia, a consequence of porphyrin excess in RBCs, had various expressions: 129/Sv mice were more hemolytic, BALB/c had more regenerative response to anemia, C57BL/6 were less affected. Iron status and hemolysis level were directly related: C57BL/6 and BALB/c had moderate hemolysis and active erythropoiesis able to reduce iron overload in the liver, while, 129/Sv showed an imbalance between iron release due to hemolysis and erythroid use. The negative control of hepcidin on the ferroportin iron exporter appeared strain specific in the CEP mice models tested. Full repression of hepcidin was observed in BALB/c and 129/Sv mice, favoring parenchymal iron overload in the liver. Unchanged hepcidin levels in C57BL/6 resulted in retention of iron predominantly in reticuloendothelial tissues. These findings open the field for potential therapeutic applications in the human disease, of hepcidin agonists and iron depletion in chronic hemolytic anemia.
Assuntos
Hepcidinas/metabolismo , Ferro/metabolismo , Porfiria Eritropoética/genética , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Modelos Animais de Doenças , Feminino , Hemólise , Hepcidinas/genética , Sobrecarga de Ferro/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Porfiria Eritropoética/etiologia , Porfiria Eritropoética/metabolismo , Porfirinas/metabolismo , Uroporfirinogênio III Sintetase/genéticaRESUMO
CKD occurs in most patients with acute intermittent porphyria (AIP). During AIP, δ-aminolevulinic acid (ALA) accumulates and promotes tubular cell death and tubulointerstitial damage. The human peptide transporter 2 (PEPT2) expressed by proximal tubular cells mediates the reabsorption of ALA, and variants of PEPT2 have different affinities for ALA. We tested the hypothesis that PEPT2 genotypes affect the severity and prognosis of porphyria-associated kidney disease. We analyzed data from 122 individuals with AIP who were followed from 2003 to 2013 and genotyped for PEPT2 At last follow-up, carriers of the PEPT2*1*1 genotype (higher affinity variant) exhibited worse renal function than carriers of the lower affinity variants PEPT2*1/*2 and PEPT2*2/*2 (mean±SD eGFR: 54.4±19.1, 66.6±23.8, and 78.1±19.9 ml/min per 1.73 m2, respectively). Change in eGFR (mean±SD) over the 10-year period was -11.0±3.3, -2.4±1.9, and 3.4±2.6 ml/min per 1.73 m2 for PEPT2*1/*1, PEPT2*1*2, and PEPT*2*2*2 carriers, respectively. At the end of follow-up, 68% of PEPT2*1*1 carriers had an eGFR<60 ml/min per 1.73 m2, compared with 37% of PEPT2*1*2 carriers and 15% of PEPT2*2*2 carriers. Multiple regression models including all confounders indicated that the PEPT2*1*1 genotype independently associated with an eGFR<60 ml/min per 1.73 m2 (odds ratio, 6.85; 95% confidence interval, 1.34 to 46.20) and an annual decrease in eGFR of >1 ml/min per 1.73 m2 (odds ratio, 3.64; 95% confidence interval, 1.37 to 9.91). Thus, a gene variant is predictive of the severity of a chronic complication of AIP. The therapeutic value of PEPT2 inhibitors in preventing porphyria-associated kidney disease warrants investigation.
Assuntos
Porfirias/complicações , Porfirias/genética , Insuficiência Renal Crônica/genética , Simportadores/genética , Doença Aguda , Idoso , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Índice de Gravidade de DoençaRESUMO
In 90% of people with erythropoietic protoporphyria (EPP), the disease results from the inheritance of a common hypomorphic FECH allele, encoding ferrochelatase, in trans to a private deleterious FECH mutation. The activity of the resulting FECH enzyme falls below the critical threshold of 35%, leading to the accumulation of free protoporphyrin IX (PPIX) in bone marrow erythroblasts and in red cells. The mechanism of low expression involves a biallelic polymorphism (c.315-48T>C) localized in intron 3. The 315-48C allele increases usage of the 3' cryptic splice site between exons 3 and 4, resulting in the transcription of an unstable mRNA with a premature stop codon, reducing the abundance of wild-type FECH mRNA, and finally reducing FECH activity. Through a candidate-sequence approach and an antisense-oligonucleotide-tiling method, we identified a sequence that, when targeted by an antisense oligonucleotide (ASO-V1), prevented usage of the cryptic splice site. In lymphoblastoid cell lines derived from symptomatic EPP subjects, transfection of ASO-V1 reduced the usage of the cryptic splice site and efficiently redirected the splicing of intron 3 toward the physiological acceptor site, thereby increasing the amount of functional FECH mRNA. Moreover, the administration of ASO-V1 into developing human erythroblasts from an overtly EPP subject markedly increased the production of WT FECH mRNA and reduced the accumulation of PPIX to a level similar to that measured in asymptomatic EPP subjects. Thus, EPP is a paradigmatic Mendelian disease in which the in vivo correction of a common single splicing defect would improve the condition of most affected individuals.
Assuntos
Ferroquelatase/genética , Oligonucleotídeos Antissenso/uso terapêutico , Protoporfiria Eritropoética/terapia , Linhagem Celular , Feminino , Humanos , Masculino , Linhagem , Polimorfismo Genético , Protoporfirinas/metabolismo , Splicing de RNA , RNA Mensageiro/genéticaRESUMO
Hemolysis occurring in hematologic diseases is often associated with an iron loading anemia. This iron overload is the result of a massive outflow of hemoglobin into the bloodstream, but the mechanism of hemoglobin handling has not been fully elucidated. Here, in a congenital erythropoietic porphyria mouse model, we evaluate the impact of hemolysis and regenerative anemia on hepcidin synthesis and iron metabolism. Hemolysis was confirmed by a complete drop in haptoglobin, hemopexin and increased plasma lactate dehydrogenase, an increased red blood cell distribution width and osmotic fragility, a reduced half-life of red blood cells, and increased expression of heme oxygenase 1. The erythropoiesis-induced Fam132b was increased, hepcidin mRNA repressed, and transepithelial iron transport in isolated duodenal loops increased. Iron was mostly accumulated in liver and spleen macrophages but transferrin saturation remained within the normal range. The expression levels of hemoglobin-haptoglobin receptor CD163 and hemopexin receptor CD91 were drastically reduced in both liver and spleen, resulting in heme- and hemoglobin-derived iron elimination in urine. In the kidney, the megalin/cubilin endocytic complex, heme oxygenase 1 and the iron exporter ferroportin were induced, which is reminiscent of significant renal handling of hemoglobin-derived iron. Our results highlight ironbound hemoglobin urinary clearance mechanism and strongly suggest that, in addition to the sequestration of iron in macrophages, kidney may play a major role in protecting hepatocytes from iron overload in chronic hemolysis.
Assuntos
Anemia Hemolítica/metabolismo , Hepatócitos/metabolismo , Hepcidinas/metabolismo , Ferro/metabolismo , Anemia Hemolítica/sangue , Anemia Hemolítica/complicações , Anemia Hemolítica/genética , Animais , Apoptose , Transporte Biológico , Biomarcadores , Modelos Animais de Doenças , Eritrócitos/metabolismo , Eritropoese , Expressão Gênica , Heme/metabolismo , Hepcidinas/sangue , Hepcidinas/genética , Humanos , Ferro/urina , Sobrecarga de Ferro/etiologia , Sobrecarga de Ferro/metabolismo , Macrófagos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Baço/fisiologia , Estresse FisiológicoRESUMO
The iron-regulatory peptide hepcidin exhibits antimicrobial activity. Having previously shown hepcidin expression in the kidney, we addressed its role in urinary tract infection (UTI), which remains largely unknown. Experimental UTI was induced in wild-type (WT) and hepcidin-knockout (Hepc-/-) mice using the uropathogenic Escherichia coli CFT073 strain. Compared with infected WT mice, infected Hepc-/- mice showed a dramatic increase in renal bacterial load. Moreover, bacterial invasion was significantly dampened by the pretreatment of WT mice with hepcidin. Infected Hepc-/- mice exhibited decreased iron accumulation in the renal medulla and significant attenuation of the renal inflammatory response. Notably, we demonstrated in vitro bacteriostatic activity of hepcidin against CFT073. Furthermore, CFT073 repressed renal hepcidin, both in vivo and in cultured renal cells, and reduced phosphorylation of SMAD kinase in vivo, suggesting a bacterial strategy to escape the antimicrobial activities of hepcidin. In conclusion, we provide new mechanisms by which hepcidin contributes to renal host defense and suggest that targeting hepcidin offers a strategy to prevent bacterial invasion.
Assuntos
Anti-Infecciosos/farmacologia , Infecções por Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Hepcidinas/metabolismo , Hepcidinas/farmacologia , Infecções Urinárias/metabolismo , Animais , Anti-Infecciosos/metabolismo , Carga Bacteriana/genética , Células Cultivadas , Contagem de Colônia Microbiana , Citocinas/metabolismo , Infecções por Escherichia coli/microbiologia , Feminino , Hepcidinas/genética , Ferro/metabolismo , Medula Renal/citologia , Medula Renal/metabolismo , Medula Renal/microbiologia , Camundongos , Camundongos Endogâmicos CBA , Camundongos Knockout , Nefrite/metabolismo , Nefrite/microbiologia , Nefrite/patologia , Neutrófilos , Fosforilação , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Infecções Urinárias/microbiologiaRESUMO
Disorders of iron metabolism are among the most common acquired and constitutive diseases. Hemochromatosis has a solid genetic basis and in Northern European populations it is usually associated with homozygosity for the C282Y mutation in the HFE protein. However, the penetrance of this mutation is incomplete and the clinical presentation is highly variable. The rare and common variants identified so far as genetic modifiers of HFE-related hemochromatosis are unable to account for the phenotypic heterogeneity of this disorder. There are wide variations in the basal iron status of common inbred mouse strains, and this diversity may reflect the genetic background of the phenotypic diversity under pathological conditions. We therefore examined the genetic basis of iron homeostasis using quantitative trait loci mapping applied to the HcB-15 recombinant congenic strains for tissue and serum iron indices. Two highly significant QTL containing either the N374S Mon1a mutation or the Ferroportin locus were found to be major determinants in spleen and liver iron loading. Interestingly, when considering possible epistatic interactions, the effects of Mon1a on macrophage iron export are conditioned by the genotype at the Slc40a1 locus. Only mice that are C57BL/10ScSnA homozygous at both loci display a lower spleen iron burden. Furthermore, the liver-iron lowering effect of the N374S Mon1a mutation is observed only in mice that display a nonsense mutation in the Ceruloplasmin (Cp) gene. This study highlights the existence of genetic interactions between Cp, Mon1a, and the Slc40a1 locus in iron metabolism, suggesting that epistasis may be a crucial determinant of the variable biological and clinical presentations in iron disorders.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte de Cátions/genética , Ceruloplasmina/genética , Epistasia Genética , Hemocromatose/veterinária , Ferro/metabolismo , Camundongos/genética , Doenças dos Roedores/genética , Animais , Feminino , Hemocromatose/genética , Hemocromatose/metabolismo , Fígado/metabolismo , Masculino , Camundongos/metabolismo , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Locos de Características Quantitativas , Doenças dos Roedores/metabolismo , Baço/metabolismoRESUMO
Mutations in the uroporphyrinogen III synthase (UROS) gene cause congenital erythropoietic porphyria (CEP), an autosomal-recessive inborn error of erythroid heme biosynthesis. Clinical features of CEP include dermatologic and hematologic abnormalities of variable severity. The discovery of a new type of erythroid porphyria, X-linked dominant protoporphyria (XLDPP), which results from increased activity of 5-aminolevulinate synthase 2 (ALAS2), the rate-controlling enzyme of erythroid heme synthesis, led us to hypothesize that the CEP phenotype may be modulated by sequence variations in the ALAS2 gene. We genotyped ALAS2 in 4 unrelated CEP patients exhibiting the same C73R/P248Q UROS genotype. The most severe of the CEP patients, a young girl, proved to be heterozygous for a novel ALAS2 mutation: c.1757 A > T in exon 11. This mutation is predicted to affect the highly conserved and penultimate C-terminal amino acid of ALAS2 (Y586). The rate of 5-aminolevulinate release from Y586F was significantly increased over that of wild-type ALAS2. The contribution of the ALAS2 gain-of-function mutation to the CEP phenotype underscores the importance of modifier genes underlying CEP. We propose that ALAS2 gene mutations should be considered not only as causative of X-linked sideroblastic anemia (XLSA) and XLDPP but may also modulate gene function in other erythropoietic disorders.
Assuntos
5-Aminolevulinato Sintetase/genética , Mutação de Sentido Incorreto , Porfiria Eritropoética/genética , Uroporfirinogênio III Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Sequência de Aminoácidos , Anemia Sideroblástica/genética , Anemia Sideroblástica/metabolismo , Anemia Sideroblástica/patologia , Sequência de Bases , Pré-Escolar , Eletroforese em Gel de Poliacrilamida , Saúde da Família , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Genótipo , Humanos , Lactente , Cinética , Masculino , Dados de Sequência Molecular , Linhagem , Porfiria Eritropoética/metabolismo , Porfiria Eritropoética/patologia , Protoporfiria Eritropoética/genética , Protoporfiria Eritropoética/metabolismo , Homologia de Sequência de Aminoácidos , Índice de Gravidade de Doença , Espectrofotometria , Uroporfirinogênio III Sintetase/metabolismo , Uroporfirinogênios/metabolismoRESUMO
BACKGROUND & AIMS: Chronic, progressive hepatobiliary disease is the most severe complication of erythropoietic protoporphyria (EPP) and can require liver transplantation, although the mechanisms that lead to liver failure are unknown. We characterized protoporphyrin-IX (PPIX)-linked hepatobiliary disease in BALB/c and C57BL/6 (Fechm1Pas) mice with mutations in ferrochelatase as models for EPP. METHODS: Fechm1Pas and wild-type (control) mice were studied at 12-14 weeks of age. PPIX was quantified; its distribution in the liver, serum levels of lipoprotein-X, liver histology, contents of bile salt and cholesterol phospholipids, and expression of genes were compared in mice of the BALB/c and C57BL/6 backgrounds. The in vitro binding affinity of PPIX for bile components was determined. RESULTS: Compared with mice of the C57BL/6 background, BALB/c Fechm1Pas mice had a more severe pattern of cholestasis, fibrosis with portoportal bridging, bile acid regurgitation, sclerosing cholangitis, and hepatolithiasis. In C57BL/6 Fechm1Pas mice, PPIX was sequestrated mainly in the cytosol of hepatocytes and Kupffer cells, whereas, in BALB/c Fechm1Pas mice, PPIX was localized within enlarged bile canaliculi. Livers of C57BL/6 Fechm1Pas mice were protected through a combination of lower efflux of PPIX and reduced synthesis and export of bile acid. CONCLUSIONS: PPIX binds to bile components and disrupts the physiologic equilibrium of phospholipids, bile acids, and cholesterol in bile. This process might be involved in pathogenesis of sclerosing cholangitis from EPP; a better understanding might improve diagnosis and development of reagents to treat or prevent liver failure in patients with EPP.
Assuntos
Colangite Esclerosante/prevenção & controle , Hepatócitos/metabolismo , Células de Kupffer/metabolismo , Porfiria Eritropoética/metabolismo , Protoporfirinas/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Colangite Esclerosante/genética , Colangite Esclerosante/metabolismo , Colangite Esclerosante/patologia , Colesterol/metabolismo , Modelos Animais de Doenças , Ferroquelatase/genética , Ferroquelatase/metabolismo , Regulação da Expressão Gênica , Genótipo , Hepatócitos/patologia , Células de Kupffer/patologia , Lipoproteína-X/sangue , Cirrose Hepática/metabolismo , Cirrose Hepática/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fenótipo , Fosfolipídeos/metabolismo , Mutação Puntual , Porfiria Eritropoética/complicações , Porfiria Eritropoética/genética , Porfiria Eritropoética/patologia , Índice de Gravidade de DoençaRESUMO
Mouse bone marrow erythropoiesis is homeostatic, whereas after acute anemia, bone morphogenetic protein 4 (BMP4)-dependent stress erythropoiesis develops in the spleen. The aim of this work was to compare spleen stress erythropoiesis and bone marrow erythropoiesis in a mouse model of zymosan-induced generalized inflammation, which induces long-lasting anemia and to evaluate the ability of erythropoietin (Epo) injections to correct anemia in this setting. The effects of zymosan and/or Epo injections on erythroid precursor maturation and apoptosis, serum interferon-γ levels, hematologic parameters, and spleen BMP4 expression were analyzed, as well as the effect of zymosan on red blood cell half-life. We found that bone marrow erythropoiesis is suppressed by inflammation and does not respond to Epo administration, despite repression of erythroblast apoptosis. On the contrary, a robust erythropoietic response takes place in the spleen after Epo injections in both control and zymosan-induced generalized inflammation mice. This specific response implies Epo-mediated induction of BMP4 expression by F4/80(+) spleen macrophages, proliferation of stress burst-forming units-erythroid, and increased number of spleen erythroblasts. It allows only partial recovery of anemia, probably because of peripheral destruction of mature red cells. It is not clear whether similar BMP4-dependent stress erythropoiesis can occur in human bone marrow after Epo injections.
Assuntos
Anemia/prevenção & controle , Proteína Morfogenética Óssea 4/metabolismo , Modelos Animais de Doenças , Eritropoese/fisiologia , Eritropoetina/uso terapêutico , Inflamação/prevenção & controle , Anemia/induzido quimicamente , Animais , Apoptose , Western Blotting , Medula Óssea/metabolismo , Células Precursoras Eritroides/metabolismo , Humanos , Inflamação/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Receptores da Eritropoetina/metabolismo , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Baço/citologia , Baço/metabolismo , Zimosan/toxicidadeRESUMO
The hereditary porphyrias comprise a group of eight metabolic disorders of the heme biosynthesis pathway. Each porphyria is caused by abnormal function at a separate enzymatic step resulting in a specific accumulation of heme precursors. Porphyrias are classified as hepatic or erythropoietic, based on the organ system in which heme precursors (δ-aminolevulinic acid [ALA], porphobilinogen and porphyrins) are overproduced. Clinically, porphyrias are characterized by acute neurovisceral symptoms, skin lesions or both. However, most if not all the porphyrias impair hepatic or gastrointestinal function. Acute hepatic porphyrias present with severe abdominal pain, nausea, constipation, confusion and seizure, which may be life threatening, and patients are at risk of hepatocellular carcinoma without cirrhosis. Porphyria Cutanea presents with skin fragility and blisters, and patients are at risk of hepatocellular carcinoma with liver iron overload. Erythropoietic protoporphyria and X-linked protoporphyria present with acute painful photosensitivity, and patients are at risk of acute liver failure. Altogether, porphyrias are still underdiagnosed, but once they are suspected, early diagnosis based on measurement of biochemical metabolites that accumulate in the blood, urine, or feces is essential so specific treatment can be started as soon as possible and long-term liver complications are prevented. Screening families to identify presymptomatic carriers is also crucial to prevent overt disease and chronic hepatic complications.
Assuntos
Porfirias/diagnóstico , Porfirias/terapia , Humanos , Porfirias/classificação , Porfirias/genética , Fatores de RiscoRESUMO
Mitochondria supply cells with ATP, heme, and iron sulfur clusters (ISC), and mitochondrial energy metabolism involves both heme- and ISC-dependent enzymes. Here, we show that mitochondrial iron supply and function require iron regulatory proteins (IRP), cytosolic RNA-binding proteins that control mRNA translation and stability. Mice lacking both IRP1 and IRP2 in their hepatocytes suffer from mitochondrial iron deficiency and dysfunction associated with alterations of the ISC and heme biosynthetic pathways, leading to liver failure and death. These results uncover a major role of the IRPs in cell biology: to ensure adequate iron supply to the mitochondrion for proper function of this critical organelle.
Assuntos
Proteínas Reguladoras de Ferro/metabolismo , Ferro/metabolismo , Mitocôndrias/metabolismo , Animais , Metabolismo Energético , Heme/biossíntese , Proteína 1 Reguladora do Ferro/deficiência , Proteína 1 Reguladora do Ferro/genética , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/deficiência , Proteína 2 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/metabolismo , Proteínas Reguladoras de Ferro/deficiência , Proteínas Reguladoras de Ferro/genética , Falência Hepática/etiologia , Falência Hepática/metabolismo , Falência Hepática/patologia , Camundongos , Camundongos TransgênicosRESUMO
The main porphyrin in rodent Harderian glands (HGs) is the heme precursor protoporphyrin IX (PPIX). Rhythmic variations in PPIX levels have yet to be studied in rodent HGs. Moreover, the mode of regulation of heme biosynthesis in this organ is poorly documented in the rat. The aim of this study was to determine day-night PPIX levels as well as day-night activity and mode of expression of the porphyrinogenic enzymes delta-aminolevulinate synthase (ALA-S) and ferrochelatase (Fech) in the rat HG. The mRNA expression of ABCG2/Bcrp1 was also investigated. Male Wistar rats acclimatized to 12 h light (L): 12 h dark (D) cycles were sacrificed in the middle of both the L and D spans, and HG and liver tissues were collected. We report here that the HG contains an extremely high level of PPIX, 630- to 670-fold higher than in the liver, without a day-night difference, which is the consequence of both low Fech gene expression (5- to 7-fold lower than in the liver) and ALA-S over-expression (4- to 7-fold higher in the HG than liver). Fech and PPIX transporter ABCG2/Bcrp1 do not exhibit day-night variation, whereas HG ALA-S levels are significantly higher during the scotophase. Interestingly, when melatonin (10 mg/kg) is administered in the middle of the light phase, it increases ALA-S mRNA levels in the HG to the ones observed during the middle of the D span. Continuous light exposure abolishes the day-night ALA-S variation in the HG that is observed under standard 12 L:12 D conditions. Our results suggest that melatonin and environmental lighting regulate ALA-S gene expression in the rat HG.
Assuntos
Glândula de Harder/efeitos dos fármacos , Melatonina/metabolismo , Porfirinas/metabolismo , 5-Aminolevulinato Sintetase/metabolismo , Animais , Ferroquelatase/metabolismo , Fluorometria/métodos , Luz , Fígado/metabolismo , Masculino , Protoporfirinas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
BACKGROUND & AIMS: Genetic hemochromatosis is one of the most common genetic disorders, with progressive tissue iron overload leading to severe clinical complications. In Northern European populations, genetic hemochromatosis is usually caused by homozygosity for the C282Y mutation in the HFE protein. However, penetrance of this mutation is incomplete, suggesting that other genetic and environmental factors contribute to its differential biologic or clinical expression. METHODS: To identify genes modifying iron homeostasis, we screened the 27 recombinant congenic strains of the C3H/DiSnA-C57BL/10ScSnA/Dem series for tissue and serum iron indices and genotyped 18 microsatellite markers in (C3H/DiSnA x HcB-2) F2 hybrid mice. RESULTS: We identified 1 locus encompassing the Ceruloplasmin (Cp) gene with a strong linkage with liver iron, serum iron, and transferrin levels but not with spleen iron. Sequencing of Cp showed an R435X nonsense mutation in exon 7 in C3H/DiSnA mice. To evaluate whether Cp might act as a modifier gene of genetic hemochromatosis, we intercrossed C3H Hfe(-/-) and C3HDiSnA Cp(R435X/R435X) mice. As expected, we found that double-mutant mice deposited more iron in the liver than mice defective for either one or both genes. In contrast, Hfe(-/-) x Cp(R435/R435X) or Cp(R435X/R435X) x Hfe(+/-) showed 30% decrease in liver iron when compared with single mutant mice. CONCLUSIONS: This study highlights the existence of complex interactions between Cp and HFE and represents the first example of a modifier gene with a protective effect, in which heterozygosity reduces the iron load in the context of HFE deficiency.
Assuntos
Ceruloplasmina/genética , Ligação Genética , Hemocromatose/genética , Hemocromatose/metabolismo , Ferro/metabolismo , Animais , Códon sem Sentido , Cruzamentos Genéticos , Modelos Animais de Doenças , Eritrócitos/metabolismo , Genótipo , Hemocromatose/sangue , Hemocromatose/fisiopatologia , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/genética , Homeostase/genética , Ferro/sangue , Fígado/metabolismo , Escore Lod , Macrófagos/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Penetrância , Fagocitose , Fenótipo , Locos de Características Quantitativas , Baço/metabolismo , Transferrina/metabolismoRESUMO
Patients with deficiency in ferrochelatase (FECH), the last enzyme of the heme biosynthetic pathway, experience a painful type of skin photosensitivity called erythropoietic protoporphyria (EPP), which is caused by the excessive production of protoporphyrin IX (PPIX) by erythrocytes. Controversial results have been reported regarding hematologic status and iron status of patients with EPP. We thoroughly explored these parameters in Fechm1Pas mutant mice of 3 different genetic backgrounds. FECH deficiency induced microcytic hypochromic anemia without ringed sideroblasts, little or no hemolysis, and no erythroid hyperplasia. Serum iron, ferritin, hepcidin mRNA, and Dcytb levels were normal. The homozygous Fechm1Pas mutant involved no tissue iron deficiency but showed a clear-cut redistribution of iron stores from peripheral tissues to the spleen, with a concomitant 2- to 3-fold increase in transferrin expression at the mRNA and the protein levels. Erythrocyte PPIX levels strongly correlated with serum transferrin levels. At all stages of differentiation in our study, transferrin receptor expression in bone marrow erythroid cells in Fech(m1Pas) was normal in mutant mice but not in patients with iron-deficiency anemia. Based on these observations, we suggest that oral iron therapy is not the therapy of choice for patients with EPP and that the PPIX-liver transferrin pathway plays a role in the orchestration of iron distribution between peripheral iron stores, the spleen, and the bone marrow.
Assuntos
Anemia Hipocrômica/metabolismo , Eritrócitos/metabolismo , Ferroquelatase/metabolismo , Ferro/metabolismo , Transferrina/metabolismo , Anemia Hipocrômica/sangue , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Células Cultivadas , Ferroquelatase/genética , Hepcidinas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Mutantes , Especificidade de Órgãos , RNA Mensageiro/metabolismo , Distribuição Tecidual , Transferrina/análiseRESUMO
Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis that results from a partial deficiency of ferrochelatase (FECH). Recently, we have shown that the inheritance of the common hypomorphic IVS3-48C allele trans to a deleterious mutation reduces FECH activity to below a critical threshold and accounts for the photosensitivity seen in patients. Rare cases of autosomal recessive inheritance have been reported. We studied a cohort of 173 white French EPP families and a group of 360 unrelated healthy subjects from four ethnic groups. The prevalences of the recessive and dominant autosomal forms of EPP are 4% (95% confidence interval 1-8) and 95% (95% confidence interval 91-99), respectively. In 97.9% of dominant cases, an IVS3-48C allele is co-inherited with the deleterious mutation. The frequency of the IVS3-48C allele differs widely in the Japanese (43%), southeast Asian (31%), white French (11%), North African (2.7%), and black West African (<1%) populations. These differences can be related to the prevalence of EPP in these populations and could account for the absence of EPP in black subjects. The phylogenic origin of the IVS3-48C haplotypes strongly suggests that the IVS3-48C allele arose from a single recent mutational event. Estimation of the age of the IVS3-48C allele from haplotype data in white and Asian populations yields an estimated age three to four times younger in the Japanese than in the white population, and this difference may be attributable either to differing demographic histories or to positive selection for the IVS3-48C allele in the Asian population. Finally, by calculating the KA/KS ratio in humans and chimpanzees, we show that the FECH protein sequence is subject to strong negative pressure. Overall, EPP looks like a Mendelian disorder, in which the prevalence of overt disease depends mainly on the frequency of a single common single-nucleotide polymorphism resulting from a unique mutational event that occurred 60,000 years ago.
Assuntos
Ferroquelatase/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único/genética , Protoporfiria Eritropoética/epidemiologia , Protoporfiria Eritropoética/genética , Sequência de Bases , Análise Mutacional de DNA , Etnicidade/genética , França/epidemiologia , Componentes do Gene , Genética Populacional , Haplótipos/genética , Humanos , Padrões de Herança/genética , Dados de Sequência Molecular , Filogenia , Prevalência , Seleção Genética , Análise de Sequência de DNA , Estatísticas não Paramétricas , População Branca/genéticaRESUMO
Hereditary coproporphyria (HCP), an autosomal dominant acute hepatic porphyria, results from mutations in the gene that encodes coproporphyrinogen III oxidase (CPO). HCP (heterozygous or rarely homozygous) patients present with an acute neurovisceral crisis, sometimes associated with skin lesions. Four patients (two families) have been reported with a clinically distinct variant form of HCP. In such patients, the presence of a specific mutation (K404E) on both alleles or associated with a null allele, produces a unifying syndrome in which hematological disorders predominate: 'harderoporphyria'. Here, we report the fifth case (from a third family) with harderoporphyria. In addition, we show that harderoporphyric patients exhibit iron overload secondary to dyserythropoiesis. To investigate the molecular basis of this peculiar phenotype, we first studied the secondary structure of the human CPO by a predictive method, the hydrophobic cluster analysis (HCA) which allowed us to focus on a region of the enzyme. We then expressed mutant enzymes for each amino acid of the region of interest, as well as all missense mutations reported so far in HCP patients and evaluated the amount of harderoporphyrin in each mutant. Our results strongly suggest that only a few missense mutations, restricted to five amino acids encoded by exon 6, may accumulate significant amounts of harderoporphyrin: D400-K404. Moreover, all other type of mutations or missense mutations mapped elsewhere throughout the CPO gene, lead to coproporphyrin accumulation and subsequently typical HCP. Our findings, reinforced by recent crystallographic results of yeast CPO, shed new light on the genetic predisposition to HCP. It represents a first monogenic metabolic disorder where clinical expression of overt disease is dependent upon the location and type of mutation, resulting either in acute hepatic or in erythropoietic porphyria.
Assuntos
Coproporfiria Hereditária/genética , Coproporfiria Hereditária/patologia , Coproporfirinogênio Oxidase/genética , Mutação/genética , Porfirias Hepáticas/genética , Porfirias Hepáticas/patologia , Sequência de Aminoácidos , Coproporfiria Hereditária/enzimologia , Coproporfirinogênio Oxidase/química , Coproporfirinogênio Oxidase/metabolismo , Éxons/genética , Expressão Gênica , Heme/biossíntese , Humanos , Sobrecarga de Ferro/metabolismo , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Linhagem , Porfirias Hepáticas/enzimologia , Estrutura Secundária de Proteína , Homologia de SequênciaRESUMO
We have recently demonstrated that in an autosomal dominant porphyria, erythropoietic protoporphyria (EPP), the coinheritance of a ferrochelatase (FECH) gene defect and of a wild-type low-expressed FECH allele is generally involved in the clinical expression of EPP. This mechanism may provide a model for phenotype modulation by minor variations in the expression of the wild-type allele in the other three autosomal dominant porphyrias that exhibit incomplete penetrance: acute intermittent porphyria (AIP), variegata porphyria (VP) and hereditary coproporphyria (HC), which are caused by partial deficiencies of hydroxy-methyl bilane synthase (HMBS), protoporphyrinogen oxidase (PPOX) and coproporphyrinogen oxidase (CPO), respectively. Given the dominant mode of inheritance of EPP, VP, AIP and HC, we first confirmed that the 200 overtly porphyric subjects (55 EPP, 58 AIP, 56 VP; 31 HC) presented a single mutation restricted to one allele (20 novel mutations and 162 known mutations). We then analysed the available single-nucleotide polymorphisms (SNPs) present at high frequencies in the general population and spreading throughout the FECH, HMBS, PPOX and the CPO genes in four case-control association studies. Finally, we explored the functional consequences of polymorphisms on the abundance of wild-type RNA, and used relative allelic mRNA determinations to find out whether low-expressed HMBS, PPOX and the CPO alleles occur in the general population. We confirm that the wild-type low-expressed allele phenomenon is usually operative in the mechanism of variable penetrance in EPP, but conclude that this is not the case in AIP and VP. For HC, the CPO mRNA determinations strongly suggest that normal CPO alleles with low-expression are present, but whether this low-expression of the wild-type allele could modulate the penetrance of a CPO gene defect in HC families remains to be ascertained.