HFE gene mutations in Brazilian thalassemic patients

Hereditary hemochromatosis is a disorder of iron metabolism characterized by increased iron intake and progressive storage and is related to mutations in the HFE gene. Interactions between thalassemia and hemochromatosis may further increase iron overload. The ethnic background of the Brazilian population is heterogeneous and studies analyzing the simultaneous presence of HFE and thalassemia-related mutations have not been carried out. The aim of this study was to evaluate the prevalence of the H63D, S65C and C282Y mutations in the HFE gene among 102 individuals with alpha-thalassemia and 168 beta-thalassemia heterozygotes and to compare them with 173 control individuals without hemoglobinopathies. The allelic frequencies found in these three groups were 0.98, 2.38, and 0.29% for the C282Y mutation, 13.72, 13.70, and 9.54% for the H63D mutation, and 0, 0.60, and 0.87% for the S65C mutation, respectively. The chi-square test for multiple independent individuals indicated a significant difference among groups for the C282Y mutation, which was shown to be significant between the beta-thalassemia heterozygote and the control group by the Fisher exact test (P value = 0.009). The higher frequency of inheritance of the C282Y mutation in the HFE gene among beta-thalassemic patients may contribute to worsen the clinical picture of these individuals. In view of the characteristics of the Brazilian population, the present results emphasize the need to screen for HFE mutations in beta-thalassemia carriers.

Advances in sickle cell disease treatment: From drug discovery until the patient monitoring

Sickle Cell Disease (SCD) is one of the most prevalent hematological diseases in the world. Despite the immense progress in molecular knowledge about SCD in last years few therapeutical sources are currently available. Nowadays the treatment is performed mainly with drugs such as hydroxyurea or other fetal hemoglobin inducers and chelating agents. This review summarizes current knowledge about the treatment and the advancements in drug design in order to discover more effective and safe drugs. Patient monitoring methods in SCD are also discussed. © 2011 Bentham Science Publishers Ltd.

Frequência dos mutantes C282Y e H63D do gene HFE e sua influência no metabolismo do ferro e na expressão da beta talassemia heterozigota

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo Doppler-ecocardiográfico de pacientes com talassemia major em regime de hipertransfusão sanguínea

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Influência dos níveis de ferritina sérica, das mutações no gene da beta globina e dos valores das enzimas de detoxificação Catalase e Glutationa S -Transferase na expressão fenotípica de Beta Talassemia Heterozigota

Pós-graduação em Genética - IBILCE

Memory-rescuing effects of cannabidiol in an animal model of cognitive impairment relevant to neurodegenerative disorders

Rationale Cannabidiol, the main nonpsychotropic constituent of Cannabis sativa, possesses a large number of pharmacological effects including anticonvulsive, sedative, hypnotic, anxiolytic, antipsychotic, anti-inflammatory, and neuroprotective, as demonstrated in clinical and preclinical studies. Many neurodegenerative disorders involve cognitive deficits, and this has led to interest in whether cannabidiol could be useful in the treatment of memory impairment associated to these diseases. Objectives We used an animal model of cognitive impairment induced by iron overload in order to test the effects of cannabidiol in memory-impaired rats. Methods Rats received vehicle or iron at postnatal days 12-14. At the age of 2 months, they received an acute intraperitoneal injection of vehicle or cannabidiol (5.0 or 10.0 mg/kg) immediately after the training session of the novel object recognition task. In order to investigate the effects of chronic cannabidiol, iron-treated rats received daily intraperitoneal injections of cannabidiol for 14 days. Twenty-four hours after the last injection, they were submitted to object recognition training. Retention tests were performed 24 h after training. Results A single acute injection of cannabidiol at the highest dose was able to recover memory in iron-treated rats. Chronic cannabidiol improved recognition memory in iron-treated rats. Acute or chronic cannabidiol does not affect memory in control rats. Conclusions The present findings provide evidence suggesting the potential use of cannabidiol for the treatment of cognitive decline associated with neurodegenerative disorders. Further studies, including clinical trials, are warranted to determine the usefulness of cannabidiol in humans suffering from neurodegenerative disorders.

Non-HFE hemochromatosis

Hereditary hemochromatosis (HH) is an autosomal recessive disorder classically related to HFE mutations. However, since 1996, it is known that HFE mutations explain about 80% of HH cases, with the remaining around 20% denominated non-HFE hemochromatosis. Nowadays, four main genes are implicated in the pathophysiology of clinical syndromes classified as non-HFE hemochromatosis: hemojuvelin (HJV, type 2Ajuvenile HH), hepcidin (HAMP, type 2B juvenile HH), transferrin receptor 2 (TFR2, type 3 HH) and ferroportin (SLC40A1, type 4 HH). The aim of this review is to explore molecular, clinical and management aspects of non-HFE hemochromatosis.

Precipitating factors of porphyria cutanea tarda in Brazil with emphasis on hemochromatosis gene (HFE) mutations. Study of 60 patients

BACKGROUND: Porphyria cutanea tarda is the most common form of porphyria, characterized by the decreased activity of the uroporphyrinogen decarboxylase enzyme. Several reports associated HFE gene mutations of hereditary hemochromatosis with porphyria cutanea tarda worldwide, although up to date only one study has been conducted in Brazil. OBJECTIVES: Investigation of porphyria cutanea tarda association with C282Y and H63D mutations in the HFE gene. Identification of precipitating factors (hepatitis C, HIV, alcoholism and estrogen) and their link with HFE mutations. METHODS: An ambispective study of 60 patients with PCT was conducted during the period from 2003 to 2012. Serological tests for hepatitis C and HIV were performed and histories of alcohol abuse and estrogen intake were investigated. HFE mutations were identified with real-time PCR. RESULTS: Porphyria cutanea tarda predominated in males and alcohol abuse was the main precipitating factor. Estrogen intake was the sole precipitating factor present in 25% of female patients. Hepatitis C was present in 41.7%. All HIV-positive patients (15.3%) had a history of alcohol abuse. Allele frequency for HFE mutations, i.e., C282Y (p = 0.0001) and H63D (p = 0.0004), were significantly higher in porphyria cutanea tarda patients, compared to control group. HFE mutations had no association with the other precipitating factors. CONCLUSIONS: Alcohol abuse, hepatitis C and estrogen intake are prevalent precipitating factors in our porphyria cutanea tarda population; however, hemochromatosis in itself can also contribute to the outbreak of porphyria cutanea tarda, which makes the research for HFE mutations necessary in these patients

Development and Validation of a Fast and Homogeneous Cell-Based Fluorescence Screening Assay for Divalent Metal Transporter 1 (DMT1/SLC11A2) Using the FLIPR Tetra.

Divalent metal ion transporter 1 (DMT1) is a proton-coupled Fe(2+) transporter that is essential for iron uptake in enterocytes and for transferrin-associated endosomal iron transport in many other cell types. DMT1 dysfunction is associated with several diseases such as iron overload disorders and neurodegenerative diseases. The main objective of the present work is to develop and validate a fluorescence-based screening assay for DMT1 modulators. We found that Fe(2+) or Cd(2+) influx could be reliably monitored in calcium 5-loaded DMT1-expressing HEK293 cells using the FLIPR Tetra fluorescence microplate reader. DMT1-mediated metal transport shows saturation kinetics depending on the extracellular substrate concentration, with a K0.5 value of 1.4 µM and 3.5 µM for Fe(2+) and Cd(2+), respectively. In addition, Cd(2+) was used as a substrate for DMT1, and we find a Ki value of 2.1 µM for a compound (2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea) belonging to the benzylisothioureas family, which has been identified as a DMT1 inhibitor. The optimized screening method using this compound as a reference demonstrated a Z' factor of 0.51. In summary, we developed and validated a sensitive and reproducible cell-based fluorescence assay suitable for the identification of compounds that specifically modulate DMT1 transport activity.

Tab. 3: Substrate pH values of crypthoendolithic microbial communities

Cryploendolithic microbial communities in the Ross Desert (McMurdo Dry Valleys) are characterized on the basis of photosymlictic microorganisms and fungi. Two eukaryotic communities (the lichen-dominated and Hemichloris communities) and three cyanobacterial communities (the red Gloeocapsa, Hormathonema-Gloeocapsa and Chroococcidiopsis communities) are described. Eleven coccoid. ohne pleurocapsoid, and five filamentous cyanobacteria occurring in these communities are characterized and illustrated. The moisture grade of the rock substrate seems to affect pH. Formation of primary iron stain, and the distribution of microbial communities.

Magnetic stratigraphy of ODP Leg 133 sites

This study is a synthesis of paleomagnetic and mineral magnetic results for Sites 819 through 823 of Ocean Drilling Program (ODP) Leg 133, which lie on a transect from the outer edge of the Great Barrier Reef (GBR) down the continental slope to the bottom of the Queensland Trough. Because of viscous remagnetization and pervasive overprinting, few reversal boundaries can be identified in these extremely high-resolution Quaternary sequences. Some of the magnetic instability, and the differences in the quality of the paleomagnetic signal among sites, can be explained in terms of the dissolution of primary iron oxides in the high near-surface geochemical gradients. Well-defined changes in magnetic properties, notably susceptibility, reflect responses to glacio-eustatic sea-level fluctuations and changes in slope sedimentation processes resulting from formation of the GBR. Susceptibility can be used to correlate between adjacent holes at a given site to an accuracy of about 20 cm. Among-site correlation of susceptibility is also possible for certain parts of the sequences and permits (tentative) extension of the reversal chronology. The reversal boundaries that can be identified are generally compatible with the calcareous nannofossil biostratigraphy and demonstrate a high level of biostratigraphic consistency among sites. A revised chronology based on an optimum match with the susceptibility stratigraphy is presented. Throughout most of the sequences there is a strong inverse correlation both between magnetic susceptibility and calcium carbonate content, and between susceptibility and d18O. In the upper, post-GBR, sections a more complicated type of magnetic response occurs during glacial maxima and subsequent transgressions, resulting in a positive correlation between susceptibility and d18O. Prior to and during formation of the outer-reef barrier, the sediments have relatively uniform magnetic properties showing multidomain behavior and displaying cyclic variations in susceptibility related to sea-level change. The susceptibility oscillations are controlled more by carbonate dilution than by variation in terrigenous influx. Establishment of the outer reef between 1.01 and 0.76 Ma restricted the supply of sediment to the slope, causing a four-fold reduction in sedimentation rates and a transition from prograding to aggrading seismic geometries (see other chapters in this volume). The Brunhes/Matuyama boundary and the end of the transition period mark a change to lower and more subdued susceptibility oscillations with higher carbonate contents. The major change in magnetic properties comes at about 0.4 Ma in the aggrading sequence, which contains prominent sharp susceptibility peaks associated with glacial cycles, with distinctive single-domain magnetite and mixed single-domain/superparamagnetic characteristics. Bacterial magnetite has been found in the sediments, particularly where there are high susceptibility peaks, but its importance has not yet been assessed. A possible explanation for the characteristic pattern of magnetic properties in the post-GBR glacial cycles can be found in terms of fluvio-deltaic processes and inter-reefal lagoonal reservoirs that develop when the shelf becomes exposed at low sea-level.

Association of the transferrin receptor in human placenta with HFE, the protein defective in hereditary hemochromatosis

Hereditary hemochromatosis (HH) is a common autosomal recessive disease associated with loss of regulation of dietary iron absorption and excessive iron deposition in major organs of the body. Recently, a candidate gene for HH (also called HFE) was identified that encodes a novel MHC class I-like protein. Most patients with HH are homozygous for the same mutation in the HFE gene, resulting in a C282Y change in the HFE protein. Studies in cultured cells show that the C282Y mutation abrogates the binding of the recombinant HFE protein to β2-microglobulin (β2M) and disrupts its transport to the cell surface. The HFE protein was shown by immunohistochemistry to be expressed in certain epithelial cells throughout the human alimentary tract and to have a unique localization in the cryptal cells of small intestine, where signals to regulate iron absorption are received from the body. In the studies presented here, we demonstrate by immunohistochemistry that the HFE protein is expressed in human placenta in the apical plasma membrane of the syncytiotrophoblasts, where the transferrin-bound iron is normally transported to the fetus via receptor-mediated endocytosis. Western blot analyses show that the HFE protein is associated with β2M in placental membranes. Unexpectedly, the transferrin receptor was also found to be associated with the HFE protein/β2M complex. These studies place the normal HFE protein at the site of contact with the maternal circulation where its association with transferrin receptor raises the possibility that the HFE protein plays some role in determining maternal/fetal iron homeostasis. These findings also raise the question of whether mutations in the HFE gene can disrupt this association and thereby contribute to some forms of neonatal iron overload.

A mouse model for beta 0-thalassemia.

We have used a "plug and socket" targeting technique to generate a mouse model of beta 0-thalassemia in which both the b1 and b2 adult globin genes have been deleted. Mice homozygous for this deletion (Hbbth-3/Hbbth-3) die perinatally, similar to the most severe form of Cooley anemia in humans. Mice heterozygous for the deletion appear normal, but their hematologic indices show characteristics typical of severe thalassemia, including dramatically decreased hematocrit, hemoglobin, red blood cell counts, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, as well as dramatically increased reticulocyte counts, serum bilirubin concentrations, and red cell distribution widths. Tissue and organ damage typical of beta-thalassemia, such as bone deformities and splenic enlargement due to increased hematopoiesis, are also seen in the heterozygous animals, as is spontaneous iron overload in the spleen, liver, and kidneys. The mice homozygous for the b1 and b2 deletions should be of great value in developing therapies for the treatment of thalassemias in utero. The heterozygous animals will be useful for studying the pathophysiology of thalassemias and have the potential of generating a model of sickle cell anemia when mated with appropriate transgenic animals.

Ferroportin Disease (Haemochromatosis-type IV): a case report

Introdution: Haemochromatosis-type IV, the ferroportin disease, is characterized by an autosomal-dominant transmission and early iron accumulation in macrophages. It is caused by mutations in the transmembrane iron exporter protein ferroportin1 (SLC40A1 gene). In form A (classic), ferroportin loss of function mutants are unable to export iron from cells leading to cellular iron accumulation with decreased availability of iron for serum transferrin (TS). We present a Portuguese rare clinical case of HH-IV. Materials and Methods: A 41-year-old woman with hyperferritinemia and normal TS. Causes of hyperferritinemia (inflammation, chronic alcohol consumption, metabolic syndrome, cell necrosis, non-alcoholic fatty liver disease and aceruloplasminemia) were assessed. Liver iron, evaluated by magnetic resonance imaging (MRI) was carried out. Screening for mutation in HFE and SCL40A1 genes were performed by Sanger sequencing. Baseline: Ferritin:708ng/ml; TS: 27%; MRI:85µmol/g; Hb:13,6g/dl. Therapy: weekly 450ml Therapeutic Phlebotomies (TP) until ferritin≤50ng/ml. Results: Hyperferritinemia comorbidities and common genetic mutations for haemochromatosis were negative. However, sequencing of the patient SLC40A1 gene has revealed the presence in heterozygosity of the variant c.238G>A; p.Gly80Ser. Due to low tolerance to TP, we adopted smaller phlebotomies every three weeks. Conclusion: This patient has a rare autosomal-dominant Ferroportin disease due to a mutated ferroportin which is predicted to be defective in iron cellular export. In agreement, she presents hyperferritinemia, with normal TS and liver iron overload. The genotype/phenotype association allowed to diagnosis this rare FD case. Although a mild form A, we decided to start TP. Her father also has been treated for iron overload.

Hemochromatosis and alcoholic liver disease

The close association of excessive alcohol consumption and clinical expression of hemochromatosis has been of widespread interest for many years. In most populations of northern European extraction, more than 90% of patients with overt hemochromatosis are homozygous for the C282Y mutation in the HFE gene. Nevertheless, the strong association of heavy alcohol intake with the clinical expression of hemochromatosis remains. We (individually or in association with colleagues from our laboratories) have performed three relevant studies in which this association was explored. In the first, performed in 1975 before the cloning of the HFE gene, the frequency of clinical symptoms and signs was compared in patients with classical hemochromatosis who consumed 100 g or more of alcohol per day versus in nondrinkers or moderate drinkers who consumed less than 100 g of alcohol per day. The results showed no difference between the two groups except for features of complications of alcoholism in the first group, especially jaundice, peripheral neuritis, and hepatic failure. Twenty-five percent of those with heavy alcohol consumption showed histologic features of alcoholic liver disease (including cirrhosis) together with heavy iron overload. It was concluded that these patients had the genetic disease complicated by alcoholic liver disease. In the second study (2002), 206 subjects with classical HFE-associated hemochromatosis in whom liver biopsy had been performed were evaluated to quantify the contribution of excess alcohol consumption to the development of cirrhosis in hemochromatosis. Cirrhosis was approximately nine times more likely to develop in subjects with hemochromatosis who consumed more than 60 g of alcohol per day than in those who drank less than this amount. In the third study (2002), 371 C282Y-homozygous relatives of patients with HFE-associated hemochromatosis were assessed. Eleven subjects had cirrhosis on liver biopsy and four of these drank 60 g or more of alcohol per day. The reason why heavy alcohol consumption accentuates the clinical expression of hemochromatosis is unclear. Increased dietary iron or increased iron absorption is unlikely. The most likely explanation would seem to be the added co-factor effect of iron and alcohol, both of which cause oxidative stress, hepatic stellate cell activation, and hepatic fibrogenesis. In addition, the cumulative effects of other forms of liver injury may result when iron and alcohol are present concurrently. Clearly, the addition of dietary iron in subjects homozygous for hemochromatosis would be unwise. (C) 2003 Elsevier Inc. All rights reserved.