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Duodenal Cytochrome B and Hephaestin Expression is Regulated by the Soluble HFE Isoform

2013-04-26T14:19:11Z

Title: Duodenal Cytochrome B and Hephaestin Expression is Regulated by the Soluble HFE Isoform Authors: Silva, Bruno; Martins, Rute; Proença, Daniela; Faustino, Paula Abstract: INTRODUCTION: Hereditary Hemochromatosis is an autosomal recessive disorder characterized by excessive intestinal iron absorption and iron deposition in organs such as liver, heart and pancreas, potentially leading to cirrhosis, hepatocelular carcinoma, diabetes, cardiac failure and arthritis. This disorder is mainly due to mutations in HFE gene. HFE protein associates with beta-2 microglobulin (B2M) for trafficking to the cell surface. However, the HFE’s role on iron homeostasis is not completely cleared. It may regulate hepcidin expression in the liver and iron trafficking in the duodenum. Several HFE alternative splicing transcripts have been reported, but their structural and functional characterization have been poorly studied. MATERIALS AND METHODS: Aiming to investigate the putative biological role of an alternative HFE transcript originated by the intron 4 inclusion, we measured its expression level in several human tissues by quantitative Real-Time PCR. Also, we produced the corresponding GFP-tagged HFE variant. HepG2 cells were transfected with this construct and protein cellular location analyzed by immunofluorescence, using B2M, TfR1 and calnexin antibodies. In parallel, immunoprecipitation was performed. Finally the intron 4 inclusion variant was over-expressed in a human duodenum adenocarcinoma cell line (Hutu-80) under normal and iron overload conditions and the expression of several iron metabolism genes (TFR1, DMT1, DCYTB, SLC40A1 and HEPH) evaluated by quantitative Real-Time PCR. RESULTS: We have found that the intron 4 inclusion transcript has an ubiquitous expression in the analyzed tissues, being its relative expression higher in duodenum and lower in the liver. Also, we found that this variant gives rise to a truncated protein (sHFE) that is secreted by the cells and is able to maintain its interaction with B2M. Its overexpression in HuTu-80 cells showed that sHFE down-regulates the duodenal cytochrome b (CYBRD1) expression in about 20% independently of cellular iron status, as it happens with the HFE_full length protein. Also, sHFE seems to be involved in the down-regulation of hephaestin (HEPH) expression, being its effect higher in the presence of iron overload (reduction of ~40 and ~50%, respectively). CONCLUSIONS: Through this study we might have unveiled the contribution of the HFE’s intron 4 inclusion splice variant to the maintenance of iron homeostasis. sHFE may be secreted into the bloodstream and act in remote tissues such as the duodenum, down-regulating the expression of some of the iron metabolism related genes, as CYBRD1 and HEPH, and consequently reducing dietary iron absorption. Also we are currently exploring the hypothesis of a possible effect of sHFE in the expression of other iron metabolism related genes in hepatic cells and macrophages.

Clinical impact of HFE Mutations in portuguese patients with Chronic Hepatitis C

2013-04-26T14:14:50Z

Title: Clinical impact of HFE Mutations in portuguese patients with Chronic Hepatitis C Authors: Ferreira, Joana; Baldaia, Cilénia; Inácio, Ângela; Bicho, Manuel; Velosa, José; Faustino, Paula; Serejo, Fátima Abstract: INTRODUCTION: Chronic hepatitis C (CHC) is often associated with alterations in iron and lipid metabolisms, which may affect the long-term prognosis and the response to antiviral treatment. Some studies suggested that the occurrence of HFE mutations may contribute to modulate these metabolisms in CHC. Here, the prevalence of two HFE mutations (C282Y and H63D) was determined in a group of Portuguese CHC patients and the findings were correlated with clinical, histological and virulogical features. MATERIALS AND METHODS: 183 CHC patients (118 males and 65 females), mean age 45.84±11.46 years and IMC 25.45±3.96 Kg/m2. Eighty two (44.8%) were treated with standard antiviral therapy and divided into 3 groups: non response (NR)-25.6%, relapse (RR)-9.8% and sustained response (SR)-64.6%. HCV-RNA and genotype were determined by PCR. Liver steatosis, fibrosis stage and degree of necroinflammation (grading) were assessed by liver biopsy (Peter Scheuer score) and clinical parameters were measured by standard techniques: AST, ALT, GGT, lipid profile (LDL, HDL, total cholesterol and triglycerides), iron metabolism (iron, ferritin, transferrin and transferrin saturation), haptoglobin, ceruloplasmin, insulin, glucose, peptide-C and HOMA. Antioxidant potential (tGSH/GSSG ratio) was evaluated by spectrofluorimetry. HFE_H63D and C282Y polymorphisms were analyzed by PCR-RFLP and statistical analysis was performed with SPSS 16.0 (level of significance of p<0.05). Patients’ exclusion criteria: other chronic liver diseases, alcohol ingestion >40g/day, HIV infection, metabolic and autoimmune diseases. RESULTS: Sixty two patients (33.9%) carried one or two mutant H63D allele (HD+DD), being 4.4% homozygous (DD). C282Y polymorphism was present in 5.5% of the patients; all were heterozygous. No difference was found comparing HFE_H63D and _C282Y polymorphisms with the type of antiviral response. Regarding H63D, we observe a decrease in the degree of necroinflammation (grading) and in tGSH/GSSG ratio and an increase in total cholesterol for carriers of the mutant allele (HD+DD) comparing to HH individuals (p=0.004; p=0.006; p=0.042, respectively). For C282Y, our study revealed that heterozygous CY had higher serum iron and transferrin saturation levels (p=0.038; p=0.006, respectively) and lower total cholesterol (p<0.0001). In the total studied population, this last clinical parameter was found to be increased in patients with less necroinflammation and steatosis (p=0.023; p=0.046) and patients with higher fibrosis stages (moderate and intense) showed higher serum iron levels. CONCLUSIONS: These data suggest a relevant role of HFE_H63D and C282Y polymorphisms in some clinical and histological features of chronic hepatitis C.

"Double trouble” or digenic disorder in Complex I deficiency

2013-02-12T16:38:52Z

Title: "Double trouble” or digenic disorder in Complex I deficiency Authors: Almeida, L.S.; Ferreira, M.; Nogueira, C.; Furtado, F.; Evangelista, T.; Santorelli, F.M.; Vilarinho, L. Abstract: Complex I (CI) deficiency is a defect of OXPHOS caused by mutations in the mitochondrial or nuclear genomes. To date disease-causing mutations have been reported in all mitochondrial-encoded subunits and 22 nuclear genes. In about 50% of the patients no mutations are found, suggesting that undiscovered factors are an important cause of disease. In this study we report a consanguineous family from Southern Portugal with three affected children presenting with CI deficiency and 3-methylglutaconic aciduria type IV.

Plasma and red blood cell proteome in sickle-cell disease

2013-02-12T12:53:30Z

Title: Plasma and red blood cell proteome in sickle-cell disease Authors: Charro, Nuno; Vaz, Fatima; Morais, Anabela; Lavinha, João; Penque, Deborah Abstract: Sickle-cell disease (SCD) is a clinically heterogeneous autosomal recessive monogenic chronic anaemia characterized by recurrent episodes of severe vaso-occlusion, haemolysis and infection. Painful crises are the major SCD clinical manifestation probably due to significant increase in dense red blood cells (RBC) and reduction of their ability to pass through capillaries. Using proteomic strategies, we aim to discover novel and better SCD prognosis biomarkers as early predictors of the transition from steady-state to crisis namely vaso-occlusive episodes, thus, allowing a prompt and specific therapeutic intervention