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The neurodegeneration of Alzheimer's disease (AD) affects not only brain structures associate with cognition early in the progression of the disease, but other areas such as the hypothalamus, a region involved in the control of metabolism and appetite. In this context, we evaluated the effects of benfotiamine (BFT), a vitamin B1 analog that is being proposed as a therapeutical approach for AD-related cognitive alterations, which were induced by intracerebroventricular injection of streptozotocin (STZ). In addition to the already described effect of STZ on cognition, we show that this drug also causes metabolic changes which are linked to changes in hypothalamic insulin signaling and orexigenic and anorexigenic circuitries, as well as a decreased cellular integrated stress response. As expected, the supplementation with 150 mg/kg of BFT for 30 days increased blood concentrations of thiamine and its phosphate esters. This led to the prevention of body weight and fat loss in STZ-ICV-treated animals. In addition, we also found an improvement in food consumption, despite hypothalamic gene expression linked to anorexia after STZ exposure. Additionally, decreased apoptosis signaling was observed in the hypothalamus. In in vitro experiments, we noticed a high ability of BFT to increase insulin sensitivity in hypothalamic neurons. Furthermore, we also observed that BFT decreases the mitochondrial unfolded stress response damage by preventing the loss of HSP60 and reversed the mitochondria dysfunction caused by STZ. Taken together, these results suggest that benfotiamine treatment is a potential therapeutic approach in the treatment of hypothalamic dysfunction and metabolic disturbances associated with sporadic AD.

Identification of biomarkers associated with severity in sickle cell anemia is desirable. Circulating serum microRNAs (miRNA) are targets studied as diagnostic or prognostic markers, but few studies have been conducted in sickle cell anemia. The purpose of this study is to identify specific signatures of miRNAs in plasma samples from sickle cell anemia patients according to severity indexes. Screening of the miRNAs expression was performed in 8 patients, classified by tricuspid regurgitation velocity (TRV) measure: 4 with TRV ≥ 2.5 m/s and 4 with TRV

Patients with hereditary spherocytosis (HS) have increased rates of erythropoiesis and higher folate requirements. In a case-control study of patients with HS, we evaluated the associations between the use of 5 mg folic acid (FA) daily and serum concentrations of folate, unmetabolized folic acid (UMFA), interleukin (IL)-6, IL-8, IL-10, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α); and mRNA expression of dihydrofolate reductase ( DHFR), methylene tetrahydrofolate reductase ( MTHFR), IL8, IFNG and TNFA genes. Total serum folate and folate forms were measured in 27 patients with HS (21 users [HS-U] and 6 non-users [HS-NU] of supplemental FA) and 54 healthy controls not consuming 5 mg/day supplemental FA. Each patient was matched to two controls based on age, sex and body mass index. The mononuclear leucocyte mRNA expression of relevant genes and their products were determined. Serum folate, UMFA, 5-methyl-tetrahydrofolate (5-methyl-THF) and tetrahydrofolate (THF) concentrations were significantly higher in HS-U compared with matched healthy controls (p

Embora alguns estudos já tenham demonstrado a relação entre a deficiência de vitamina B12 e muitas síndromes geriátricas, a associação entre a deficiência dessa vitamina e a fragilidade biológica ainda, não foi claramente estabelecida. Nesse contexto, o presente estudo, teve como objetivo investigar a frequência da deficiência de vitamina B12 em idosos e sua associação com a fragilidade biológica. Trata-se de um estudo observacional, transversal, realizado com 64 idosos, de ambos os sexos, atendidos no ambulatório de Geriatria de um Hospital Universitário. Para classificar a fragilidade foram utilizados os critérios perda de peso, fraqueza, exaustão, lentidão e baixo nível de atividade. A análise da vitamina B12 foi realizada pela técnica quantitativa de eletroquimioluminescência através do Kit Elecsys® B12 Imunoensaio (Roche, Mannheim, Germany). Os resultados apontaram que o fenótipo de fragilidade na maioria dos idosos caracterizou-se como frágil ou risco de fragilidade. As concentrações séricas de vitamina B12 na amostra estudada apresentou-se adequada e não mostraram associação significativa com os parâmetros e fenótipos da fragilidade. Assim, novos estudos são necessários, a fim de explorar o impacto da deficiência de vitamina B12 nas condições de saúde da pessoa idosa com fragilidade. Although some studies have already shown the relationship between vitamin B12 deficiency and many geriatric syndromes, the association between vitamin B12 deficiency and biological fragility has not been clearly established. In this context, the present study aimed to investigate the frequency of vitamin B12 deficiency in the elderly and its association with biological fragility. This is an observational, cross-sectional study of 64 elderly men and women attending the Geriatrics outpatient clinic of a University Hospital. To classify the fragility, we used the criteria weight loss, weakness, exhaustion, slowness, and low level of activity. Vitamin B12 analysis was performed by quantitative electrochemiluminescence using the Elecsys® B12 Immunoassay Kit (Roche, Mannheim, Germany). The results showed that the fragility phenotype in most of the elderly was characterized as fragile or frailty risk. The serum concentrations of vitamin B12 in the sample studied were adequate and showed no significant association with the parameters and fragility phenotypes. Thus, further studies are needed to explore the impact of vitamin B12 deficiency on the health conditions of the elderly person with fragility. Aunque algunos estudios ya han demostrado la relación entre la deficiencia de vitamina B12 y muchos síndromes geriátricos, aún no se ha establecido claramente la asociación entre la deficiencia de vitamina B12 y la fragilidad biológica. En ese contexto, el presente estudio tuvo como objetivo investigar la frecuencia de la deficiencia de vitamina B12 en ancianos y su asociación con la fragilidad biológica. Se trata de un estudio observacional, transversal, realizado con 64 ancianos, de ambos sexos, atendidos en la consulta externa de Geriatría de un Hospital Universitario. Para clasificar la fragilidad se utilizaron los criterios de pérdida de peso, debilidad, agotamiento, lentitud y bajo nivel de actividad. El análisis de vitamina B12 se realizó mediante la técnica de electroquimioluminiscencia cuantitativa utilizando el kit de inmunoensayo Elecsys® B12 (Roche, Mannheim, Alemania). Los resultados mostraron que el fenotipo de fragilidad en la mayoría de los ancianos se caracterizó como frágil o en riesgo de fragilidad. Las concentraciones séricas de vitamina B12 en la muestra estudiada fueron adecuadas y no mostraron una asociación significativa con los parámetros y fenotipos de fragilidad. Por lo tanto, se necesitan más estudios para explorar el impacto de la deficiencia de vitamina B12 en las condiciones de salud de las personas mayores con fragilidad. Embora alguns estudos já tenham demonstrado a relação entre a deficiência de vitamina B12 e muitas síndromes geriátricas, a associação entre a deficiência dessa vitamina e a fragilidade biológica ainda, não foi claramente estabelecida. Nesse contexto, o presente estudo, teve como objetivo investigar a frequência da deficiência de vitamina B12 em idosos e sua associação com a fragilidade biológica. Trata-se de um estudo observacional, transversal, realizado com 64 idosos, de ambos os sexos, atendidos no ambulatório de Geriatria de um Hospital Universitário. Para classificar a fragilidade foram utilizados os critérios perda de peso, fraqueza, exaustão, lentidão e baixo nível de atividade. A análise da vitamina B12 foi realizada pela técnica quantitativa de eletroquimioluminescência através do Kit Elecsys® B12 Imunoensaio (Roche, Mannheim, Germany). Os resultados apontaram que o fenótipo de fragilidade na maioria dos idosos caracterizou-se como frágil ou risco de fragilidade. As concentrações séricas de vitamina B12 na amostra estudada apresentou-se adequada e não mostraram associação significativa com os parâmetros e fenótipos da fragilidade. Assim, novos estudos são necessários, a fim de explorar o impacto da deficiência de vitamina B12 nas condições de saúde da pessoa idosa com fragilidade. Sebbene alcuni studi abbiano già dimostrato la relazione tra carenza di vitamina B12 e molte sindromi geriatriche, l'associazione tra carenza di vitamina B12 e fragilità biologica non è stata ancora chiaramente stabilita. In questo contesto, il presente studio mirava a indagare la frequenza della carenza di vitamina B12 negli anziani e la sua associazione con la fragilità biologica. Si tratta di uno studio osservazionale trasversale condotto su 64 anziani, di ambo i sessi, trattati presso l'ambulatorio di Geriatria di un Policlinico Universitario. Per classificare la fragilità sono stati utilizzati i criteri di perdita di peso, debolezza, esaurimento, lentezza e basso livello di attività. L'analisi della vitamina B12 è stata eseguita mediante tecnica quantitativa di elettrochemiluminescenza utilizzando il kit di immunodosaggio Elecsys® B12 (Roche, Mannheim, Germania). I risultati hanno mostrato che il fenotipo della fragilità nella maggior parte degli anziani era caratterizzato come fragile oa rischio di fragilità. Le concentrazioni sieriche di vitamina B12 nel campione studiato erano adeguate e non hanno mostrato alcuna associazione significativa con parametri di fragilità e fenotipi. Pertanto, sono necessari ulteriori studi per esplorare l'impatto della carenza di vitamina B12 sulle condizioni di salute degli anziani con fragilità.

BackgroundThe 2019 coronavirus disease pandemic (COVID-19) spread rapidly across Brazil. The country has 27 federative units, with wide regional differences related to climate, lifestyle habits, socioeconomic characteristics and population density. Therefore, we aimed to document and monitor the increase in COVID-19 cases across each federative unit in Brazil, by tracking its progression from inception to 15 May 2020.MethodsObservational study.ResultsThe first confirmed COVID-19 case in the country was notified in São Paulo on 26 February, while the first death occurred on 17 March, in Rio de Janeiro. Since then, there has been a dramatic increase in both confirmed cases and deaths from the disease. São Paulo, in the Southeast region, was initially considered the COVID-19 epidemic epicentre in Brazil. However, 10 states in the North and Northeast regions were ranked among the 14 highest incidences (over 100 cases per 100,000 people) observed on 15 May. Higher incidence rates (>100 cases per 100,000) were associated to higher rates of inadequate water supply and sewerage (OR, 5.83 (95% CI, 1.08–29.37, P=0.041)). North and Northeast states with the highest social vulnerability index scores had higher increases in the incidence rate between 14 April and 15 May. States with medium human development index (HDI) showed higher incidence increases from 14 April to 15 May, being seven of them with ratios in the range from 27.49 to 63.73 times.ConclusionSpreading of COVID-19 in Brazil differs across both regions and federative units, being influenced by different socioeconomic contexts.

To investigate the association between serum unmetabolized folic acid (UMFA) concentrations and folic acid from fortified foods and nutrients known as dietary methyl-group donors (folate, methionine, choline, betaine and vitamins B2, B6 and B12) in participants exposed to mandatory fortification of wheat and maize flours with folic acid.Cross-sectional study carried out with 144 healthy Brazilian participants, both sexes, supplement nonusers. Serum folate, UMFA, vitamin B12 and total plasma homocysteine (tHcy) were biochemically measured. Dietary intake was assessed by 2 non-consecutive 24-hour dietary recalls (24-HRs) and deattenuated energy-adjusted nutrient data were used for statistical analysis.Ninety eight (68.1%) participants were women. Median (interquartile range) age was 35.5 (28.0-52.0) years. Elevated serum folate concentrations (45 nmol/L) were found in 17 (11.8%), while folate deficiency (7 nmol/L) in 10 (6.9%) participants. No one had vitamin B12 deficiency (148 pmol/L). An elevated serum UMFA concentration was defined as1 nmol/L (90th percentile). UMFA concentrations were positively correlated with folic acid intake and negatively correlated to choline, methionine and vitamin B6 intakes. Participants in the lowest quartile of UMFA concentrations had lower dietary intake of total folate (DFEs) and folic acid, and higher dietary intake of methionine, choline and vitamin B6 than participants in the highest quartile of UMFA. Folic acid intake (OR [95% CI] = 1.02 [1.01-1.04)] and being a male (OR [95% CI] = 0.40 [0.19-0.87) were associated with increased and reduced odds for UMFA concentrations0.55 nmol/L (median values), respectively.UMFA concentrations were directly influenced by folic acid intake from fortified foods in a healthy convenience sample of adult Brazilians exposed to mandatory flour fortification with folic acid.

International audience; PURPOSE:The consumption of Brazil nuts has been associated with benefits to lipid metabolism and reductions in total cholesterol and LDL concentrations. They are the richest natural source of selenium which has essential functions in human physiology. Genetic polymorphisms in Selenoprotein P could impair lipid and glucose metabolisms. The aim of this work was to verify the influence of polymorphisms in genes for selenoproteins on blood lipid levels after dietary supplementation with Brazil nuts in healthy adults.METHODS:The study included 130 healthy volunteers selected at the University of São Paulo, Brazil. They were supplemented with one nut a day for 8 weeks, followed by 8 weeks without intervention. The following analyses were performed: anthropometric measurements, serum fasting glucose, lipid profile, C-reactive protein and plasma MDA levels. The volunteers were genotyped for SNPs rs1050450, rs3811699, rs1800699, rs713041, rs3877899, rs7579, rs34713741, and rs5845 in genes for selenoproteins.RESULTS:The concentrations of total cholesterol and fasting glucose levels decreased after 8 weeks of supplementation (p < 0.05). Glucose levels were modulated by rs3877899 in SEPP1, with significantly lower levels observed for individuals with the GA + AA genotype (p = 0.025). In addition, rs7579 was associated with cholesterol concentrations, which were significantly lower for individuals with the GG genotype (p = 0.053).CONCLUSIONS:Supplementation with one Brazil nut a day for 8 weeks reduced total cholesterol and glucose levels. Furthermore, our results suggest that rs3877899 might be associated with glucose concentrations and rs7579 with cholesterol concentrations. Therefore, the effect of genetic variations should be considered in future nutritional interventions evaluating the response to Brazil nut supplementation.

INTRODUCTION Folate deficiency is commonly reported in β-thalassemia. Individuals heterozygous for β-thalassemia may have higher folate requirements than normal individuals. OBJECTIVES To document the concentration of serum total folate and its forms in β-thalassemia heterozygote users (β-TmU) and nonusers (β-TmN) of 5 mg folic acid/d; to determine whether folic acid (FA) consumption from fortified foods allows beta-Tm patients, who do not take FA supplements, to meet their dietary folate requirements; and to investigate the association between higher serum unmetabolized folic acid (UMFA) and inflammatory cytokine concentrations. METHODS Serum total folate and forms were measured in 42 β-Tm (13 β-TmU and 29 β-TmN) and 84 healthy controls. The mononuclear leucocyte mRNA expression of relevant genes and their products and hematological profiles were determined. RESULTS β-TmU had higher serum total folate, 5-methyltetrahydrofolate, UMFA, and tetrahydrofolate (THF) compared with β-TmN. The β-TmN had lower serum total folate and THF than controls. Plasma total homocysteine (tHcy) was lower in β-TmU compared with both β-TmN and controls, while β-TmN had higher tHcy than controls. β-TmU had higher IL-8 than their controls while β-TmN had higher IL-6 and IL-8 than their controls. β-TmU have higher levels of serum total folate, 5- methyltetrahydrofolate, UMFA, and THF than controls. There was no association between UMFA concentrations and cytokine levels. CONCLUSIONS Mandatory flour fortification with FA in Brazil may be insufficient for β-TmN, since they have higher tHcy and lower serum total folate than controls. Furthermore, β-TmN have higher IL-6 levels than β-TmU. UMFA was not associated with inflammatory cytokine levels.

Summary Background The beneficial effects of selenium (Se) to human health are exerted by selenoproteins, which can be quantified in blood and used as biomarkers of Se status. Different responses of Se biomarkers after supplementation with selenomethionine and sodium selenite have been observed and some of them could be due to genetic polymorphisms, mainly single nucleotide polymorphisms (SNPs). Brazil nuts are known to be the richest natural source of Se. Objective Investigate how genetic variations in selenoprotein genes modulate biomarkers of Se status in response to Brazil nut supplementation. Methods The SU.BRA.NUT study was a four month interventional trial which involved healthy volunteers of both genders, selected in University of Sao Paulo. The supplementation was done with one Brazil nut a day for 8 weeks, followed by 8 weeks of washout. Blood samples were collected at 5 time points: baseline, 4 and 8 weeks of supplementation and 4 and 8 weeks of washout for analysis of five biomarkers of Se status – erythrocyte GPx1 (Glutathione Peroxidase 1) activity, plasma GPx3 activity, plasma Se, erythrocyte Se, and plasma selenoprotein P. The gene expression of GPX1, SELENOP, SELENOF and SELENOS was done before and after 8 weeks of supplementation. The volunteers were genotyped for SNPs in GPX1 (rs1050450, rs3811699 and rs1800699), GPX4 (rs713041), SELENOP (rs3877899 and rs7579), SELENOF (rs5845) and SELENOS (rs34713741). Results A total of 130 volunteers finished the protocol. The concentrations of four biomarkers of Se status increased significantly after 4 and 8 weeks of supplementation, being modulated by gender. In addition, erythrocyte GPx1 activity was associated with rs1050450, rs713041 and rs5845. Plasma Se was associated with rs7579 and selenoprotein P with plasma Se at baseline. Nut supplementation significantly increased GPX1 mRNA expression only in subjects with CC genotype at rs1050450. SELENOP mRNA expression was significantly lower in subjects with GG genotype at rs7579 before and after supplementation. Conclusion Genetic variations in GPX1 and SELENOP genes are associated with different responses of molecular and biochemical biomarkers of Se status after Brazil nut supplementation in healthy Brazilians. The SU.BRA.NUT study was registred at www.clinicaltrials.gov as NCT 03111355.

Background: The effects of high-dose folic acid (FA) supplementation in healthy individuals on blood folate concentrations and immune response are unknown.Objective: The aim of the study was to evaluate the effects of daily consumption of a tablet containing 5 mg FA on serum folate; number and cytotoxicity of natural killer (NK) cells; mRNA expression of dihydrofolate reductase (DHFR), methylenetetrahydrofolate reductase (MTHFR), interferon γ (IFNG), tumor necrosis factor α (TNFA), and interleukin 8 (IL8) genes; and concentrations of serum inflammatory markers.Methods: This prospective clinical trial was conducted in 30 healthy Brazilian adults (15 women), aged 27.7 y (95% CI: 26.4, 29.1 y), with a body mass index (in kg/m2) of 23.1 (95% CI: 22.0, 24.3). Blood was collected at baseline and after 45 and 90 d of the intervention. Serum folate concentrations were measured by microbiological assay and HPLC-tandem mass spectrometry [folate forms, including unmetabolized folic acid (UMFA)]. We used real-time polymerase chain reaction to assess mononuclear leukocyte mRNA expression and flow cytometry to measure the number and cytotoxicity of NK cells.Results: Serum folate concentrations increased by ∼5-fold after the intervention (P 1.12 nmol/L) in 29 (96.6%) participants at day 45 and in 26 (86.7%) participants at day 90. We observed significant reductions in the number (P < 0.001) and cytotoxicity (P = 0.003) of NK cells after 45 and 90 d. Compared with baseline, DHFR mRNA expression was higher at 90 d (P = 0.006) and IL8 and TNFA mRNA expressions were higher at 45 and 90 d (P = 0.001 for both).Conclusion: This noncontrolled intervention showed that healthy adults responded to a high-dose FA supplement with increased UMFA concentrations, changes in cytokine mRNA expression, and reduced number and cytotoxicity of NK cells. This trial was registered at www.ensaiosclinicos.gov.br as RBR-2pr7zp.

Genetic variations in membrane transporters may contribute to imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML). Objective To investigate the relationship between SLCO1B3, SLCO1A2, and ABCA3 polymorphisms and IM response in CML patients.Patients in chronic phase CML (N = 118) were studied. All patients were treated with a standard dose of IM (400 mg/day) and classified into one of the two groups according to their responses. Major molecular response (MMR) and complete molecular response (CMR) were evaluated. Criteria for response failure were established according to European LeukemiaNet (2009). Analysis of the SLCO1B3 c.334TG (rs4149117) and c.699GA (rs7311358), SLCO1A2 c.516AC (rs11568563) and c.-62-361GA (rs3764043), and ABCA3 c.1755CG (rs323043) and c.4548-191CA (rs150929) polymorphisms was carried out by real-time polymerase chain reaction.SLCO1A2 and ABCA3 polymorphisms have similar frequencies between responders and non-responders. SLCO1B3 699GG and 344TT genotypes were more frequent in the responder group (63.8%) than in the non-responder group (44.7%, P = 0.042). Furthermore, carriers of 699GA/AA and 334TG/GG genotypes presented a higher probability of not responding to the standard dose of IM (odds ratio: 2.17; 95% confidence interval: 1.02-4.64, P = 0.04). Poor CMR for ABCA3 4548-91CA was observed in patients with the CC/CA genotype when compared to AA carriers in the responder group (P = 0.014).SLCO1B3 699GG and 344TT genotypes are associated with non-response to IM, while ABCA3 4548-91 CC/CA genotypes are related to poor CMR in CML patients treated with standard-dose imatinib.

The steady increase in the incidence and mortality of hepatocellular carcinoma (HCC) signifies a crucial need to understand better its pathogenesis to improve clinical management and prevention of the disease. The aim of this study was to investigate molecular mechanisms for the chemopreventive effects of folic acid and tributyrin alone or in combination on rat hepatocarcinogenesis. Male Wistar rats were subjected to a classic “resistant hepatocyte” model of liver carcinogenesis and treated with folic acid and tributyrin alone or in combination for 5 weeks during promotion stage. Treatment with folic acid and tributyrin alone or in combination strongly inhibited the development of glutathione-S-transferase placental form (GSTP)-positive foci. Microarray analysis showed significant changes in gene expression. A total of 501, 655, and 940 of differentially expressed genes, involved in cell cycle, p53-signaling, angiogenesis, and Wnt pathways, was identified in the livers of rats treated with folic acid, tributyrin or folic acid and tributyrin. A detailed analysis of these differentially expressed genes revealed that treatments inhibited angiogenesis in the preneoplastic livers. This was evidenced by the fact that 30 out of 77 differentially expressed genes common to all three treatments are involved in the regulation of the angiogenesis pathway. The inhibition of angiogenesis was confirmed by reduced levels of CD34 protein. In conclusion, the tumor-suppressing activity of folic acid and tributyrin is associated with inhibition of angiogenesis at early stages of rat liver carcinogenesis. Importantly, the combination of folic acid and tributyrin has stronger chemopreventive effect than each of the compounds alone.

Selenium (Se) status varies worldwide as a result of natural variation of Se content in soils, dietary pattern, and the presence of SNPs. Further, Se status in Brazilians and its relationship between genetic variation and Se biomarkers is unknown. This work investigated the association between SNPs in glutathione peroxidase genes and biomarkers of Se status in healthy Brazilians. The study was conducted in 116 healthy adults in São Paulo, Brazil. Plasma and erythrocyte Se were measured by HGFAAS. Erythrocyte GPx (eGPx) activity was measured spectrometrically in a biochemical analyzer. Genotypes were determined by real-time PCR using Taqman(®) Assays. eGPx activity was higher in females compared with males. Lower erythrocyte Se concentrations were found in heterozygous GC carriers for GPX1 rs8179169. eGPx activity was higher in females with the common genotypes, except for rs8179169. GC carriers for rs8179169 had lower erythrocyte Se in both genders, and only male carriers of the variant alleles of both rs1050450 and rs1800668 had higher eGPx activity. In conclusion, the genotype for SNPs in GPX1 and gender affected biomarkers of Se status in this pilot study with healthy Brazilians.

Background p.C282Y mutation and rare variants in the HFE gene have been associated with hereditary hemochromatosis (HH). HH is also caused by mutations in other genes, such as the hemojuvelin ( HJV ), hepcidin ( HAMP ), transferrin receptor 2 ( TFR2 ) and ferroportin ( SLC40A1 ). The low rate homozygous p.C282Y mutation in Brazil is suggestive that mutations in non- HFE genes may be linked to HH phenotype. Aim To screen exon-by-exon DNA sequences of HFE , HJV , HAMP , TFR2 and SLC40A1 genes to characterize the molecular basis of HH in a sample of the Brazilian population. Materials and methods Fifty-one patients with primary iron overload (transferrin saturation ≥ 50% in females and ≥ 60% in males) were selected. Subsequent bidirectional DNA sequencing of HFE , HJV , HAMP , TFR2 and SLC40A1 exons was performed. Results Thirty-seven (72.5%) out of the 51 patients presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation ( n = 11, 21.6%). In addition, heterozygous HFE p.S65C mutation was found in combination with p.H63D in two patients and homozygous HFE p.H63D was found in two patients as well. Sequencing in the HJV and HAMP genes revealed HJV p.E302K, HJV p.A310G, HJV p.G320V and HAMP p.R59G alterations. Molecular and clinical diagnosis of juvenile hemochromatosis (homozygous form for the HJV p.G320V) was described for the first time in Brazil. Three TFR2 polymorphisms (p.A75V, p.A617A and p.R752H) and six SLC40A1 polymorphisms (rs13008848, rs11568351, rs11568345, rs11568344, rs2304704, rs11568346) and the novel mutation SLC40A1 p.G204S were also found. Conclusions The HFE p.C282Y in homozygosity or in heterozygosity with p.H63D was the most frequent mutation associated with HH in this sample. The HJV p.E302K and HAMP p.R59G variants, and the novel SLC40A1 p.G204S mutation may also be linked to primary iron overload but their role in the pathophysiology of HH remain to be elucidated.

Background: Sickle cell anemia (SCA) is a complex disease, associated with hemolysis, vaso-occlusion, vascular inflammation and endothelial activation. Significant morbidity and premature mortality are hallmarks of the disease and elevation of tricuspide regurgitant velocity (TRV), determined by doppler-echocardiography, has been associated with higher risk. The identification of biomarkers associated with severity in SCA is desirable. Circulating serum microRNAs (miRNA) are molecular targets studied in different diseases as diagnostic or prognostic markers, however there are few studies in SCA. Purpose: to identify specific signatures of miRNAs in plasma samples of SCA patients according to severity indexes. Methods: Screening of the miRNAs expression was performed in 8 patients. These patients were classified by TRV measure (referred as TRV Score): 4 samples with TVR ≥ 2.5 m/s and 4 with TRV < 2.5 m/s. After extraction of total RNA, the samples were analyzed by real-time PCR using Megaplex RT Human Pool A and Megaplex RTHuman Pool Blife (Thermofisher) comprising 667 distinct miRNAs. Expression Suite Software (Thermofisher) and SPSS were used for analysis. Seventeen miRNAs were differentially expressed between the two groups (p < 0.05) and miR16 was considered as the endogenous candidate. Five differentially expressed miRNAs (miR15b, miR502, miR510, miR544, miR629) were selected for validation in 56 patient samples using TRV Score. Another two severity scores were also used: (a) Organ Injury Score (SLO) - based on the presence or absence of 5 lesions: stroke, TRV ≥ 2.5 m/s, leg ulcers, osteonecrosis, and microalbuminuria (adapted from Afenyi-Annan et al. Transfusion 2008; 48:197) and (b) NIH Bayesian score - available online (http://bios.ugr.es/dss-calculator/). The ROC curve was used to analyze the data of relative expression (2-ΔCT) of each miRNA. Results: Two out of five miRNA, miR510 and miR629, were significantly decreased in more severe patients. The miR510 expression allowed the discrimination of the patients according to TRV Score at the cutoff 0.000331043, sensitivity 71.4%, specificity 73.3% and AUC of 0.825 (95% CI: 0.689-0.962, p = 0.001). The same miRNA was also a good discriminant in the SLO at a cutoff of 0.000331043, sensitivity 77.8%, specificity 72.2% and area under the curve (AUC) of 0.769 (95% CI: 0.666-0.931), p = 0.008. The miR629 was related to severity according to the Bayesian Score at the cutoff of 0.0009854449, sensitivity 66.7%, specificity 75% and AUC of 0.729 (95% CI: 0.552-0.907, p = 0.027). The other miRNAs, miR15b, miR502 and miR544, showed no significant results. Discussion and Conclusions: This is the first study which looks into plasma miRNA as a biomarker of SCA severity. The miR510 regulate Peroxirredoxin-1 (PRDX1), a protein involved in the stress-oxidation. Increased oxidative stress presented in SCA might imply that miR510 has a role in this mechanism. The miR629 appears to be involved in the AKT1 signaling pathway related to Endothelin-1. As it is known, endothelin-1 is increased in SCA and is important in pulmonary hypertension. The miR510 and miR629 appear to be hypoexpressed in patients with more severe SCA, probably with greater importance in the regulation of clinical manifestations associated with vascular disease, although more studies seem to be necessary. Disclosures No relevant conflicts of interest to declare.

Background The pathophysiology of spontaneous abortion is complex and may involve the interaction of genetic and environmental factors. We evaluated the predictors of spontaneous abortion in Brazilian pregnant women. The effects of age, gestational age, body mass index (BMI), cigarette smoking, alcohol ingestion, use of multivitamins and concentrations of vitamins (folate, cobalamin and vitamin B6) and vitamin-dependent metabolites were analyzed. Methods Study population included 100 healthy women that attended pre-natal care in 2 health centers of Sao Paulo, Brazil, and in whom pregnancy outcome was known. Folate and cobalamin status was measured in blood specimens collected between 4 and 16 weeks. The genotypes for 8 gene polymorphisms were evaluated by PCR-RFLP. Results Eighty-eight women had normal pregnancy outcome (Group 1), while 12 experienced a miscarriage after blood collection (Group 2). Increased methylmalonic acid (MMA) concentrations were found in Group 2 (median [25th–75th percentile] = 274 [149–425] nmol/l) relative to Group 1 (138 [98–185]) ( P Conclusions Increased serum MMA and BMI concentrations are associated with spontaneous abortion in Brazilian women.

Background Cobalamin (Cbl) and folate deficiencies and gene polymorphism of key enzymes or carriers can impair homocysteine metabolism and may change the serum values of S -adenosylmethionine (SAM) and S -adenosylhomocysteine (SAH). We investigated the nutritional and genetic determinants for total homocysteine (tHcy), methylmalonic acid (MMA) and SAM/SAH in healthy Brazilian childbearing-age women. Methods Serum concentrations of Cbl, folate, red blood cell folate, ferritin, tHcy, MMA, SAM, SAH and other metabolites were measured in 102 healthy unrelated women. The genotypes for MTHFR C677T, MTHFR A1298C, MTR A2756G, MTRR A66G, TC2 C776G, TC2 A67G and RFC1 A80G gene polymorphisms were identified by PCR-RFLP. Results Serum folate and Cbl were inversely correlated with tHcy and serum MMA, respectively. Cbl deficiency was associated with increased MMA and reduced alpha-aminobutyrate, serine and N -methylglycine concentrations. No variable was associated with SAM/SAH ratio. In addition, gene polymorphisms were not selected as determinants for tHcy, MMA and SAM/SAH ratio. Iron, Cbl and folate deficiencies were found respectively in 30.4%, 22.5% and 2.0% of individuals studied. Conclusions There was a high frequency of Cbl and iron deficiency in this group of childbearing-age women. Serum folate and Cbl were the determinants of serum tHcy and MMA concentration, respectively.

O termo trombofilia foi usado pela primeira vez em 1965 por Egeberg para designar uma enfermidade associada à trombose venosa por deficiência de antitrombina III. Desde então este termo tem sido amplamente utilizado para descrever os estados pré-trombóticos ou de hipercoagulação que predispõem aa ocorrência e recorrência da trombose arterial ou venosa.

Impaired methylation due to accumulation of S-adenosylhomocysteine (SAH) may contribute to the pathophysiology of cobalamin-deficient anemia. We assayed serum S-adenosylmethionine (SAM), SAH, total homocysteine (tHcy), and methylmalonic acid (MMA) in 15 subjects with cobalamin-deficient megaloblastic anemia and compared results with those of 19 subjects with anemia/pancytopenia due to other causes. Cobalamin-deficient subjects had a median hematocrit level of 20% and mean cell volume of 111.7 fL. The median serum cobalamin level was 37 pg/mL, MMA 3030 nmol/L, and tHcy 62.0 micromol/L. SAH was elevated in 13 of 15 subjects (median, 42 nmol/L) and the median SAM value was normal (103 nmol/L), but SAM/SAH ratio was low (2.5). The SAH was higher and SAM/SAH ratio was lower in cobalamin-deficient subjects compared with those with other anemias after excluding 4 patients with renal insufficiency. SAM concentrations were not low in cobalamin deficiency. Cobalamin injections corrected anemia, MMA, tHcy, SAM/SAH ratio, and SAH. Some hematologic variables were inversely correlated with SAH and cobalamin but not tHcy or MMA. In conclusion, serum SAH is elevated in cobalamin-deficient subjects with megaloblastic anemia and corrects with parenteral cobalamin therapy.

Background: Soluble transferrin receptor (sTfR) concentration is high in iron deficiency and in conditions of increased erythropoiesis. In developing countries like Brazil, pregnant women usually have concurrent iron, vitamin B12, and folate deficiencies. This study investigated the relationship between serum sTfR concentration and iron, vitamin B12, and folate status in pregnant women. Methods: The concentration of the sTfR, hematocrit (Hct), hemoglobin (Hb), red blood cell (RBC) and white blood cell (WBC) counts, serum iron (SI), total iron-binding capacity (TIBC), transferrin saturation, serum ferritin, zinc protoporphyrin (ZPP), vitamin B12, and serum and RBC folate were determined in 40 healthy pregnant women who delivered term babies. Results: sTfR concentration was significantly higher when the women had iron deficiency (serum ferritin

Human accidental envenomation caused by skin contact with the bristles of Lonomia obliqua caterpillar causes coagulation and fibrinolysis disorders. Alterations of hematologic parameters are observed only in severe cases of envenomation, but with no clinical evidence of intravascular hemolysis. However, since we have observed intravascular hemolysis in preliminary studies using Wistar rats as an experimental model for investigating L. obliqua envenomation, the objective of the present study was to investigate the in vitro hemolytic activity of the bristle extract of L. obliqua caterpillars on human and rat erythrocytes. Our results showed that the bristle extract has indirect and direct hemolytic activity on human and rat erythrocytes, although direct hemolytic activity was only observed at higher bristle extract concentrations. We also observed that the bristle extract has a proteolytic activity on band 3 of human and rat erythrocyte membranes. Thus, crude L. obliqua bristle extract was found to contain at least two components with hemolytic activity on erythrocytes, a phospholipase enzyme and another protein with a direct activity on the erythrocyte membrane.

Some myeloproliferative neoplasm (MPN) patients harbor JAK2(V617F) mutation, and CALR mutations were recently discovered in wild type (WT) JAK2(V617F). We evaluated the frequency and type of CALR mutations, and clinical and hematological characteristics in WT JAK2(V617F) and MPL(W515K/L) MPN patients. Sixty-five patients were included: 21 with primary myelofibrosis (PMF), 21 with myelofibrosis post-essential thrombocythemia (MPET) and 23 with essential thrombocythemia (ET). Screening for JAK2(V617F) and MPL(W515K/L) were performed using real-time PCR, while CALR mutations were analyzed by fragment analysis and Sanger sequencing. JAK2(V617F) was the most frequent mutation (54.5%) and one patient (1.5%) harbored MPL(W515L). CALR mutations were present in 38.1% of PMF, 12.5% of ET and 33.3% of MPET patients. Five types of CALR mutations were detected, among which type 1 (32.1%) and type 2 (21.4%) were found to be the most common. A novel CALR mutation in a PMF patient was found. Patients carrying CALR mutations had higher platelet count and less presence of splenomegaly than JAK2(V617F), while triple negatives had higher C-reactive protein levels than CALR mutant carriers. Screening for CALR mutations and its correlation with clinical features could be useful for the characterization of MPN patients and result in its incorporation into a new prognostic score.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) AFIP Epidemiological and experimental studies indicate that the altered fetal and neonatal environment influences physiological functions and may increase the risk of developing chronic diseases in adulthood. Because homocysteine (Hcy) metabolic imbalance is considered a risk factor for neurodegenerative diseases, we investigated whether maternal Vitamin B deficiency during early development alters the offspring's methionine-homocysteine metabolism in their brain. To this end, the dams were submitted to experimental diet one month before and during pregnancy or pregnancy/lactation. After birth, the offspring were organized into the following groups: control (CT), deficient diet during pregnancy and lactation (DPL) and deficient diet during pregnancy (DP). the mice were euthanized at various stages of development. Hcy, cysteine, glutathione (GSH), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), folate and cobalamin concentrations were measured in the plasma and/or brain. At postnatal day (PND) 0, total brain of female and male offspring exhibited decreased SAM/SAH ratios. Moreover, at PND 28, we observed decreased GSH/GSSG ratios in both females and males in the DPL group. Exposure to a Vitamin B-deficient diet during the ontogenic plasticity period had a negative impact on plasma folate and brain cortex SAM concentrations in aged DPL males. We also observed decreased plasma GSH concentrations in both DP and DPL males (PND 210). Additionally, this manipulation seemed to affect the female and male offspring differently. the decreased plasma GSH concentration may reflect redox changes in tissues and the decreased brain cortex SAM may be involved in changes of gene expression, which could contribute to neurodegenerative diseases over the long term. Universidade Federal de São Paulo, Dept Pediat, São Paulo, Brazil Universidade Federal de São Paulo, Dept Psychobiol, São Paulo, Brazil Univ São Paulo, Sch Pharmaceut Sci, São Paulo, Brazil Univ Pernambuco, Pediat Hematol & Oncol Ctr, Inst Biol Sci, Recife, PE, Brazil Universidade Federal de São Paulo, Dept Pediat, São Paulo, Brazil Universidade Federal de São Paulo, Dept Psychobiol, São Paulo, Brazil FAPESP: 2010/00075-2 Web of Science

A fim de estabelecer valores de referência de certos parâmetros hematológicos e a curva de fragilidade osmótica do sangue de Colossoma macropomum, as amostras de sangue foram coletadas de 38 peixes, provenientes de fazendas do Estado do Tocantins, Brasil. Foram determinados o número de eritrócitos e o conteúdo de hemoglobina e hematócrito. A curva de fragilidade osmótica foi determinada com a incubação dos tubos por 30 minutos a 30ºC, incubadas a 37, 30 e 27ºC por 24 horas e incubadas por 12 horas a 27ºC. Os valores hematimétricos obtidos foram: número de eritrócitos= 1,58 ± 0,08 x1012/L; hemoglobina= 7,2 ± 0,32g/dL; hematócrito= 34,0 ± 1,6%; VCM= 217,6 ± 13,2fL; HCM= 46,3 ± 2,6pg e CHCM= 21,5 ± 1,4%. A fragilidade osmótica dos eritrócitos de peixes foi conduzida de acordo com os padrões de hemólise. A hemólise iniciou-se a 85,5 mM de NaCl e a hemólise total ocorreu a 17 mM. As amostras da curva incubada por 24 horas demonstraram hemólise total para todas as concentrações testadas, independentemente da temperatura. Nas amostras da curva incubada a 27ºC por 12 horas a hemólise inicial foi observada a 154 mM de NaCl e a hemólise total em 17 mMIn order to establish reference values for certain haematological parameters and osmotic fragility curve for the blood of Colossoma macropomum, blood samples were collected from 38 fish bred on farms in the state of Tocantins, Brazil. Red blood cell count, haemoglobin content and haematocrit were determined. Osmotic fragility curves were determined for 30 minutes under resting conditions at 30ºC, incubated curves at 37, 30 and 27ºC for 24 hours and incubated curves for 12 hours at 27ºC. The haematimetric values obtained were: red blood cell count= 1.58 ± 0.08 x1012/L; haemoglobin= 7.2 ± 0.32 g/dL; haematocrit= 34.0 ± 1.6%; MCV= 217.6 ± 13.2fL; MCH= 46.3 ± 2.6 pg and MCHC= 21.5 ± 1.4%. Osmotic fragility of fish erythrocytes was assessed according to haemolysis rate. Haemolysis began at 85.5 mM NaCl and total haemolysis occurred at 17 mM NaCl. Samples from incubated curve for 24 hours showed total haemolysis in all concentrations, regardless of temperature. In the samples from incubated curve at 27ºC for 12 hours initial haemolysis was observed at 154 mM and total haemolysis at 17 mM NaCl

El estado nutricional pregestacional y durante el embarazo fue evaluado en 372 gestantes, a través del indice de masa corporal y la Gráfica de Rosso, prospectivamente. Mitad de las mujeres presentó peso pregestacional normal, aún así 17,7% tenía bajo peso y 31,3% sobrepeso. En el tercer trimestre, 18,8% tenía bajo peso y 28,2% sobrepeso. El peso al nacer aumentó conforme la gestante fue nutricionalmente mejor natada. Estos resultados señalan que un control antropométrico adecuado posibilita monitorear nutricionalmente las embarazadas, preveniendo y/o controlando la ocurrencia de condiciones materno-fetales indeseables, hecho que reafirma la importancia de la ejecución de esta actividad como rutina en todo el control prenatal. Nutritional conditions in the pre-gestacional period and during pregnancy were evaluated in 372 pregnant women based on the body mass index and Rosso's Graph. Approximately half (51%) the pregnant women presented normal pre-gestacional weight; however, 17.7% were underweight and 31.3% overweight. In the third trimester, 18.8% were underweight and 28.2% overweight. The weight at birth increased as nutritional conditions during pregnancy improved. These results show the importance of appropriate anthropometric control in order to prevent a bad neonatal prognosis and reaffirms the importance of such activity during the whole routine of pre-natal control. O estado nutricional antes e durante a gestação foi avaliado prospectivamente em 372 gestantes através do índice de massa corporal e ou gráfico de Rosso. Metade das mulheres apresentou peso pré-gestacional normal, porém 17,7% estava abaixo e 31,3% acima do peso normal. No terceiro trimestre, 18,8% tinha peso abaixo e 28,2% acima do normal. O peso ao nascer aumentou de acordo com o melhor provimento nutricional da gestante. Estes resultados indicam que um controle antropométrico adequado permite monitorar nutricialmente as gestantes, prevenindo e/ou controlando a ocorrência de condições materno-fetais indesejáveis, fato que reafirma a importância da execução desta atividade como rotina no controle pré-natal.

A avaliação do estado nutricional de ferro em indivíduos e populações tem sido alvo de muitas pesquisas científicas, uma vez que existem algumas questões ainda indefinidas. Através de um levantamento bibliográfico, mediante consulta às bases de dados Medline, Lilacs e Dedalus, foram selecionadas publicações científicas em português e inglês, no período de 1972 a 1998, que se referiam aos parâmetros hematológicos e bioquímicos utilizados na avaliação do estado nutricional de ferro. Os parâmetros disponíveis refletem os três diferentes estágios da carência de ferro, a qual se manifesta de forma gradual e progressiva no organismo, até o desenvolvimento da anemia ferropriva. De forma geral, não possuem boa sensibilidade e especificidade quando considerados isoladamente, apresentando vantagens e limitações que devem ser examinadas no momento da escolha. No sentido de se aumentar a sensibilidade e a especificidade do diagnóstico do estado nutricional de ferro, têm-se utilizado combinações dos diferentes parâmetros disponíveis, considerando-se a contribuição de cada um, de acordo com as características dos indivíduos estudados, as facilidades metodológicas e o custo do processo.

Erythrocyte membrane proteins from 44 representative mammals were studied. Protein 4.2 was not detected in guinea pigs (Cavia porcellus) (N = 14), Southern Brazilian swamp large rats (Myocastor coypus) (N = 2), cutias (Dasyprocta sp) (N = 4), and horses (Equus caballus) (N = 13). These animals also presented high ankyrin concentrations except for the horse which did not exhibit a sharp band, although minor components located between proteins 2 and 3 could account for the ankyrin family. The rodents studied did present band 6, which was not detectable in other common rodents such as white rats (Rattus norvegicus) (N = 9) and mice (Mus musculus) (N = 12). Since the absence of protein 4.2 does not disrupt the cytoskeleton membrane, we suggest that it is not an essential protein. Its absence may be compensated physiologically by the higher ankyrin concentration observed.

Despite the high efficacy of imatinib mesylate (IM) treatment for chronic myeloid leukemia (CML) patients, some individuals develop resistance due to impaired bioavailability. It has been previously demonstrated that the haplotypes for ATP‑binding cassette subfamily B member 1 (ABCB1)with c.1236C>T, c.3435C>T and c.2677G>T/A polymorphisms markedly affect the secondary structure of ABCB1 mRNA and its activity. These modifications may affect efflux transporter activity and response to treatment with IM. The aim of the present study was to investigate the influence of ABCB1 haplotypes on P‑glycoprotein (P-gp) activity, IM plasma levels and IM response. In total, 28 chronic‑phase CML patients treated with a standard dose of IM (400 mg/day) were studied. The patients were selected according to the haplotypes of ABCB1, with c.1236C>T, c.3435C>T and c.2677G>T polymorphisms, and were clas- sified into two groups based on the presence of the mutated allele in each genotype for the three ABCB1 polymorphisms. In addition, expression of P‑gp and breakpoint cluster region‑abelson 1 (BCR‑ABL1), ABCB1 and solute carrier family 22 member 1 (SLC22A1) mRNA were evaluated. The P‑gp activity in the wild‑type group was found to be higher than that in the mutated group (59.1 vs. 38.3%; P=0.001). Furthermore, the patients who did not achieve major molecular response (MMR) showed a higher rate of efflux mediated by P‑gp when compared with individuals who achieved MMR (64.7 vs. 45.7%; P=0.001). All patients without MMR demon- strated effluxes of >60%. In addition, patients without MMR exhibited lower plasma concentrations of IM compared with those with MMR (0.51 vs. 1.42 µg/ml; P=0.001). Higher levels of SLC22A1 mRNA were observed in patients who achieved MMR and complete molecular response (P

SummaryRecurrent pregnancy loss (RPL) is a multifactorial condition. The effect of antithrombin (SERPINC1), protein C (PROC), thrombomodulin (THBD) and tissue factor pathway inhibitor (TFPI) single nucleotide polymorphisms (SNPs) on the risk of RPL is thus far unknown. Our objective was to determine the association of SNPs in the above mentioned genes with RPL. We included 117 non-pregnant women with three or more consecutive losses prior to 20 weeks of pregnancy without a previous history of carrying a fetus to viability, and 264 healthy fertile non-pregnant women who had at least two term deliveries and no known pregnancy losses. The PROC (rs1799809 and rs1799808), SERPINC1 (rs2227589), THBD (rs1042579) and TFPI (rs10931292, rs8176592 and rs10153820) SNPs were analysed by Real Time PCR. Genotype frequencies for PROC 2418A>G, PROC 2405C>T, THBD 1418C>T, TFPI (T-33C and TFPI C-399T) SNPs were similar in cases and controls. The carriers of SERPINC1 786A allele (GA + AA genotypes) had an increased risk for RPL (odds ratio [OR]: 1.77, 95% confidence interval [CI]: 1.05 – 3.00, p= 0.034) while women carrying the TFPI –287C allele (TC + CC genotypes) had a protection effect on having RPL (OR: 0.46, 95% CI: 0.26 – 0.83, p= 0.009). The TCC haplotype for TFPI T-33C/ TFPI T-287C/ TFPI C-399T SNPs was less frequent in cases (5.7%) than in controls (11.6%) (OR: 0.45, 95% CI: 0.23 – 0.90, p= 0.025). In conclusion, our data indicate that SERPINC1 786G>A variant increases the risk for RPL, while TFPI T-287C variant is protective; however, further studies are required to confirm our findings.

Imatinib mesylate (IM) is a selective tyrosine kinase inhibitor used for treating chronic myeloid leukemia (CML). IM has high efficacy, however some individuals develop a resistance due to impaired bioavailability. Polymorphisms in genes encoding membrane transporters such as ABCB1 have been associated with differences in protein expression and function that influence the response to several drugs.To investigate the relationship of ABCB1 polymorphisms with markers of response to IM in patients with CML.One hundred eighteen CML patients initially treated with a standard dose of IM (400mg/day) for 18months were selected at two health centers in Sao Paulo City, Brazil. The response criteria were based on the European LeukemiaNet recommendations. ABCB1 polymorphisms c.1236CT (rs1128503), c.3435CT (rs1045642) and c.2677GT/A (rs2032582) were evaluated by PCR-RFLP.ABCB1 polymorphisms were not related with a risk for CML in this sample population (p0.05). In the CML group, frequencies of ABCB1 SNPs were similar between responder and non-responder patients (p0.05). In the responder group, the frequency of ABCB11236CT/2677GT/3435CT haplotype was higher in patients with major molecular response (MMR) (51.7%) than in patients without MMR (8.3%, p=0.010). Furthermore, carriers of this haplotype had increased the probability of reaching the MMR compared with the non-carriers (OR: 11.8; 95% CI: 1.43-97.3, p=0.022).The ABCB1 1236CT/2677GT/3435CT haplotype is positively associated with the major molecular response to IM in CML patients.

BACKGROUND The genotyping of HFE p.C282Y and p.H63D mutations is one of the most requested molecular analyses in the laboratorial routine. In this scenario, the main aim was to develop a genotyping assay that has advantages compared to other methods. METHODS Genotypes for the HFE p.C282Y (c.G845A; rs1800562) and p.H63D (c.C187G, rs1799945) mutations were assessed by polymerase chain reaction (PCR) followed by high resolution melting (HRM) analysis with the Rotor-Gene 6000(®) instrument. Validation studies were conducted in samples bi-directionally sequenced. RESULTS The melting assay was developed in a unique procedure and to ensure the result in approximately 112 min (31 min for sample preparation and 81 min for the PCR-HRM step). Genotypes for the HFE p.C282Y mutation were easily distinguished in the region of 80-86°C. For the HFE p.H63D, genotypes were also easily distinguished in the region of 76-82°C, but using the addition of known wild-type genotype DNA in all unknown samples plus a reaction without addition. In validation, genotypes were 100% concordant between methods. CONCLUSIONS Our genotyping assay with the Rotor-Gene 6000(®) instrument applies to the laboratorial routine with several advantages, especially in large-scale demand. The main advantages were the non-dependence on gel electrophoresis and on mutagenic reagents for visualization of fragments, reduction of the chances for contamination due to sample preparation, the lack of use of probe-based methods and cost-effectiveness.