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

2013-04-01T00:00:00Z

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

2013-04-01T00:00:00Z

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.

Efficient IDUA Gene Mutation Detection with Combined Use of dHPLC and Dried Blood Samples

2013-04-03T00:00:00Z

Title: Efficient IDUA Gene Mutation Detection with Combined Use of dHPLC and Dried Blood Samples Authors: Ribeiro, Diogo; Cardoso, Ana; Duarte, Ana Joana; Vieira, Luís; Amaral, Olga Abstract: Development of a simple mutation directed method in order to allow lowering the cost of mutation testing using an easily obtainable biological material. Assessment of the feasibility of such method was tested using a GC-rich amplicon. A method of denaturing high-performance liquid chromatography (dHPLC) was improved and implemented as a technique for the detection of variants in exon 9 of the IDUA gene. The optimized method was tested in 500 genomic DNA samples obtained from dried blood spots (DBS). With this dHPLC approach it was possible to detect different variants, including the common p.Trp402Ter mutation in the IDUA gene. The high GC content did not interfere with the resolution and reliability of this technique, and discrimination of G-C transversions was also achieved. This PCR-based dHPLC method proved to be a rapid, a sensitive, and an excellent option for screening numerous samples obtained from DBS. Furthermore, it resulted in the consistent detection of clearly distinguishable profiles of the common p.Trp402Ter IDUA mutation with an advantageous balance of cost and technical requirements. Description: DR and AJD were FCT research grantees (PIC/IC/82822/2007)

Doenças genéticas da hipertensão

2013-04-11T00:00:00Z

Title: Doenças genéticas da hipertensão Authors: Jordan, Peter