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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

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.

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.

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.

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

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

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.

summary. Limited data are available about iron deficiency (ID) in Brazilian blood donors. This study evaluated the frequencies of ID and iron-deficiency anaemia (IDA) separately and according to frequency of blood donations. The protective effect of the heterozygous genotype for HFE C282Y mutation against ID and IDA in female blood donors was also determined. Five hundred and eight blood donors were recruited at the Blood Bank of Santa Casa in Sao Paulo, Brazil. Haemoglobin and serum ferritin concentrations were measured. The genotype for HFE C282Y mutation was determined by polymerase chain reaction followed by restriction fragment length polymorphism analysis. The ID was found in 21·1% of the women and 2·6% of the men whereas the IDA was found in 6·8 and 0·3%, respectively. The ID was found in 11·9% of the women in group 1 (first-time blood donors) and the frequency increased to 38·9% in women of the group 3 (blood donors donating once or more times in the last 12 months). No ID was found in men from group 1; however the ID frequency increased to 0·9% in group 2 (who had donated blood before but not in the last 12 months) and 5·0% in group 3. In summary, the heterozygous genotype was not associated with reduction of ID or IDA frequencies in both genders, but in male blood donors it was associated with a trend to elevated ferritin levels (P = 0·060). ID is most frequent in Brazilian women but was also found in men of group 3.

Mutations of the HFE and TFR2 genes have been associated with iron overload. HFE and TFR2 mutations were assessed in blood donors, and the relationship with iron status was evaluated. Subjects (N = 542) were recruited at the Hemocentro da Santa Casa de Sao Paulo, Sao Paulo, Brazil. Iron status was not influenced by HFE mutations in women and was independent of blood donation frequency. In contrast, men carrying the HFE 282CY genotype had lower total iron-binding capacity (TIBC) than HFE 282CC genotype carriers. Men who donated blood for the first time and were carriers of the HFE 282CY genotype had higher transferrin saturation values and lower TIBC concentrations than those with the homozygous wild genotype for the HFE C282Y mutation. Moreover, in this group of blood donors, carriers of HFE 63DD plus 63HD genotypes had higher serum ferritin values than those with the homozygous wild genotype for HFE H63D mutation. Multiple linear regression analysis showed that HFE 282CY leads to a 17.21% increase (P = 0.018) and a 83.65% decrease (P = 0.007) in transferrin saturation and TIBC, respectively. In addition, serum ferritin is influenced by age (3.91%, P = 0.001) and the HFE 63HD plus DD genotype (55.84%, P = 0.021). In conclusion, the HFE 282Y and 65C alleles were rare, while the HFE 63D allele was frequent in Brazilian blood donors. The HFE C282Y and H63D mutations were associated with alterations in iron status in blood donors in a gender-dependent manner.

Background The methylenetetrahydrofolate reductase (MTHFR), glutamate carboxypeptidase II (GCPII) and reduced folate carrier (RFC1) gene polymorphisms were associated with folate status. We investigated the effects of these polymorphisms on serum folate (SF) and folate-related metabolites in mothers and their neonates. Methods Cobalamin (Cbl), SF, total homocysteine (tHcy), methylmalonic acid (MMA), S -adenosylmethionine (SAM), and S -adenosylhomocysteine (SAH) were measured in 275 healthy women and their neonates. MTHFR C677T, GCPII C1561T and RFC1 A80G polymorphisms were determined by PCR-RFLP. Results Maternal tHcy was affected individually by MTHFR C677T and GCPII C1561T polymorphisms and by combined genotypes MTHFR 677TT/ GCPII 1561CC and MTHFR 677TT/ RFC1 80AG. The MTHFR and RFC1 polymorphisms were not associated with variations in vitamins or SAM, SAH and MMA in neonates. Neonatal tHcy was predicted directly by maternal tHcy and inversely by maternal SF, neonatal Cbl and neonatal RFC1 80G allele (AG+GG genotypes). Maternal MMA and SAM/SAH were predicted by creatinine and Cbl, respectively. Neonatal MMA was predicted by maternal MMA and GCPII 1561T allele (CT+TT genotypes) and by neonatal Cbl. Conclusions Maternal tHcy was affected by MTHFR C677T, RFC1 A80G and GCPII C1561T polymorphisms. Maternal GCPII C1561T variant was associated with neonatal MMA. Neonatal RFC1 A80G polymorphism influenced tHcy in neonates.

To examine the association between methylenetetrahydrofolate reductase (MTHFR) (C677T and A1298C), methionine synthase (MTR) A2756G and methionine synthase reductase (MTRR) A66G gene polymorphisms and total homocysteine (tHcy), methylmalonic acid (MMA) and S-adenosylmethionine/S-adenosylhomocysteine (SAM/SAH) levels; and to evaluate the potential interactions with folate or cobalamin (Cbl) status.Two hundred seventy-five healthy women at labor who delivered full-term normal babies. Cbl, folate, tHcy, MMA, SAM and SAH were measured in serum specimens. The genotypes for polymorphisms were determined by PCR-restriction fragment length polymorphism (RFLP).Serum folate, MTHFR 677T allele and MTR 2756AA genotypes were the predictors of tHcy levels in pregnant women. Serum Cbl and creatinine were the predictors of SAM/SAH ratio and MMA levels, respectively. The gene polymorphisms were not determinants for MMA levels and SAM/SAH ratios. Low levels of serum folate were associated with elevated tHcy in pregnant women, independently of the gene polymorphisms. In pregnant women carrying MTHFR 677T allele, or MTHFR 1298AA or MTRR 66AA genotypes, lower Cbl levels were associated with higher levels of tHcy. Lower SAM/SAH ratio was found in MTHFR 677CC or MTRR A2756AA genotypes carriers when Cbl levels were lower than 142 pmol/l.Serum folate and MTHFR C677T and MTR A2576G gene polymorphisms were the determinants for tHcy levels. The interaction between low levels of serum Cbl and MTHFR (C677T or A1298C) or MTRR A66G gene polymorphisms was associated with increased tHcy.

Background In Brazil, wheat and corn flour is fortified with 150 µg of folic acid (FA), the synthetic form of folate. Individuals with increased cell duplication, including pregnant women and patients with hemolytic anemia need increased amounts of folate. The effects of amounts of FA higher than the defined tolerable upper intake of 1 mg/day are poorly understood. Some Brazilian patients with hemolytic anemia, such as hereditary spherocytosis (HS), have been receiving 5mg/day supplemental FA, in addition to being exposed to mandated food fortification with FA. Our previous data has shown that patients with HS have higher serum folate levels than healthy controls, as well as higher mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes (1). However, it was not clear whether the increased mRNA expression resulted from folic acid use or underlying disease. Objective The aim of this study was to verify the effects of an intervention with 5mg/day FA on folate levels (serum and whole blood), serum inflammatory markers levels, mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes and cytotoxicity of NK cells in healthy Brazilian volunteers. Material and methods Fifteen male and fifteen female healthy subjects were given 5mg/day FA for 90 days. Blood was collected at baseline, day 45 and day 90 for blood count, including reticulocytes, C-reactive protein and lactate dehydrogenase (LDH). Folate (serum and whole blood) and vitamin B12 were determined by a microbiological method. Serum cytokines levels were measured using a Milliplex Map kit. The mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes in mononuclear cells were performed using Real Time PCR. Cytotoxicity of lymphocytes and NK cell number were measured by flow cytometry. Results All blood count parameters were unaffected by FA intervention, whereas there was a slight increase in concentrations of LDH (P = 0.001) after 90 days compared with baseline and 45 day measurements. The folate levels (serum and whole blood) were higher at 45 and 90 days of intervention with 5mg/day of FA (P0.05). The mRNA expression of IL8 was higher at 45 days of intervention (Fig 1), while mRNA expressions of TNF-α were elevated at 45 and 90 days compared with baseline (Fig 1). No difference was found in mRNA of DHFR, MTHFR and IFNγ in this study. The data are median and interquartile intervals. The groups were compared using the Friedman test, when significant was performed for multiple comparisons Dunn`s test. Different letters show significant differences between groups After 5 mg FA daily there was a reduction in the number and cytotoxic capacity of NK cells (Table 1). The data are median and interquartile intervals. The groups were compared using the Friedman test, when significant was performed for multiple comparisons Dunn`s test. Different letters show significant differences between groups. Conclusions Intervention with 5 mg/day of FA in healthy people was associated with around 4-fold increase in serum and whole blood folate, accompanied by increased mRNA expression of proinflammatory cytokines IL8 and TNF-α and a reduction in NK cell number and cytotoxicity. High dose FA fortification may result in changes in innate immune parameters that could perturb immune surveillance pathways. Financing: FAPESP 2012/12912-1, CNPq 4826412012-6 and CNPq 401586/2014-6 References 1. Paniz, C et al. Blood 2014;124:4005. Presented at the ASH 2014. Figure 1. mRNA expression of IL8 (A) and TNFα (B) genes in healthy subjects before and 45 and 90 days after 5 mg FA daily Figure 1. mRNA expression of IL8 (A) and TNFα (B) genes in healthy subjects before and 45 and 90 days after 5 mg FA daily Tabel 1 Number, lytic activity and cytotoxic capacity of NK cells after intervention with 5 mg/day of folic acid Tabel 1. Number, lytic activity and cytotoxic capacity of NK cells after intervention with 5 mg/day of folic acid Disclosures No relevant conflicts of interest to declare.

Background: Pregnant women with low cobalamin concentrations are unable to provide the necessary amount of cobalamin to their fetuses. The effect of low maternal cobalamin concentrations on transmethylation metabolism in pregnant women and their newborns is unknown. Objective: We investigated the relation between maternal and neonatal cobalamin concentrations and changes in total homocysteine (tHcy), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH). Design: Hematologic data and concentrations of cobalamin, red blood cell folate, serum folate, tHcy, methylmalonic acid, SAM, SAH, and other metabolites were measured in 119 serum specimens from pregnant Brazilian women (gestational age: 37-42 wk) and their newborns' placental veins at the time of delivery. Results: The tHcy concentrations were higher in placental vein serum from newborns whose mothers had low cobalamin. Serum SAH concentrations were elevated and serum SAM and methionine concentrations were decreased in pregnant women with lower cobalamin concentrations. SAM:SAH was significantly decreased in both cobalamin-deficient pregnant women and their newborns. Conclusions: Lower maternal cobalamin concentrations are associated with higher tHcy and lower SAM:SAH in newborns. Because SAM:SAH is closely linked with the activity of numerous enzymatic methylation reactions, these results suggest that methylation could be impaired in cobalamin-deficient pregnant women and their newborns.

Background: A large body of evidence links plasma concentrations of homocysteine and cardiovascular disease. Several genetic and environmental variables may modulate such relationship. We investigated the influence of methylenetetrahydrofolate reductase (MTHFR) gene variants C677T, A1298C, and T1317C on homocysteine, folate, and cobalamin concentrations in a sample of individuals from a mild folate deficiency population to better clarify the complex interactions existing among these variables. Methods: In the present study, 209 individuals belonging to an admixed urban population characterized by mild folate deficiency were investigated. MTHFR gene variants C677T, A1298C, and T1317C were genotyped and homocysteine-, folate-, and cobalamin-determined for each individual. Results: Univariate analyses showed a significant association between the C677T variant with homocysteine (P

Objective To determine total homocysteine and folate levels in pregnant women according to vitamin B12 (B12) levels, and to analyse the impact of maternal B12 levels on the nutritional status of their newborn babies. Design Cross sectional observational study. Setting Two public hospitals in Jundiai City, Sao Paulo, Brazil. Sample Sixty-nine pregnant women and their respective newborn babies at the time of delivery. Methods Maternal blood was collected up to 8 hours before delivery. Umbilical cord blood was collected after the expulsion of the placenta. Total homocysteine was measured by high perfomance liquid chromatography, folate by ion capture methodology and B12 by enzyme immunoassay. Main outcome measures Relationship between low maternal vitamin B12 levels and total homocysteine and folate levels in pregnant women and new born babies. Results There was a significant correlation between maternal and neonatal B12 levels, but not between maternal B12 and neonatal red blood cell (RBC) folate. There was a weak correlation between maternal B12 levels and neonatal serum folate. However, there were significant correlations between maternal and neonatal total homocysteine levels and between neonatal B12 and neonatal total homocysteine levels. Mean (±SD) umbilical cord blood B12, RBC folate, serum folate and total homocysteine levels were 1.7 ± 0.8, 1.8 ± 0.8, 2.2 ± 0.8 and 0.9 ± 0.3 times higher than maternal B12, RBC folate, serum folate and total homocysteine values, respectively. Conclusions These data suggest that pregnant women with low B12 levels are unable to provide the necessary amount of B12 to their fetuses. The clinical consequences could be the presence of neurological abnormalities as well as the lack of utilisation of homocysteine for methionine transformation.

Background: Methylenetetrahydrofolate reductase (MTHFR) deficiency leads to impairment in folate metabolism and is implicated as a risk factor for neural tube defects (NTDs). Both C677T and A1298C MTHFR mutations are associated with NTDs, in some populations. Methods: The frequencies of the C677T and A1298C MTHFR mutations were determined in 25 children with NTDs, case mothers and 75 healthy individuals from Sao Paulo City. Both C677T and A1298C mutations were analyzed by PCR-FLRP. The effects of MTHFR mutations on folate, vitamin B12 and homocysteine concentrations were also evaluated. Results: C677T and A1298C allele frequencies in NTDs children and mothers were similar to that found in controls. Eleven in 23 NTDs patients and 10 in 21 NTDs mothers had folate or vitamin B12 concentrations in the lower end of the normal range. In NTDs children, C677T MTHFR genotypes did not affect vitamins and homocysteine concentrations, but plasma homocysteine was higher (p=0.028) in patients with 1298AA MTHFR genotype. Moreover, 677CT/1298AA haplotype was associated with lower vitamin B12 concentrations (p

Background Folic acid (FA) is the synthetic form of folate, a B complex vitamin which plays an important role in several reactions in the body. Folate deficiency produces several clinical complications including anemia and is associated with an increased risk of cancer, cardiovascular disease and neural tube defects. With the advent of FA and iron fortification of wheat and corn flour (150 µg of folic acid and 4.2 mg of iron/100 grams) in Brazil since 2004, the population was exposed to amounts of folate beyond that normally present in foods. The form present in fortified food is taken up by cells and reduced by the enzyme dihydrofolate reductase (DHFR) first to dihydrofolate (DHF) and then tetrahydrofolate (THF). DHF is the preferred natural substrate. Amounts of FA higher than the defined tolerable upper intake of 1 mg/day could impair the ability of DHFR to convert DHF to THF. Some patients with hemolytic anemia, such as hereditary spherocytosis (HS), need larger amounts of FA to compensate for their increase in erythropoiesis and have been receiving 5mg/day of supplemental FA, the only formulation available in Brazil, in addition to being exposed to compulsory food fortification with FA. Objective The aim of this study was to compare the effects of 5mg/day FA on serum folate levels, mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes; and cytotoxicity of lymphocytes and NK cells in patients with HS and healthy individuals not receiving supplemental FA. Material and Methods Twenty-five patients with HS exposed to mandatory fortification and in use or not of 5 mg/day of FA were included in this study. Forty-five healthy people were recruited as a control group, and matched with HS patients according to age, gender, body mass index and self-reported skin color. Blood count, including reticulocytes, C-reative protein and lactic dehydrogenase (LDH) were performed. Serum folate (SF) and vitamin B12 were determined by a microbiological method. The mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes in mononuclear cells were performed in duplicate, using Real Time PCR. Cytotoxicity of lymphocytes and NK cells were also carried out using a flow cytometric assay. Results Eight HS patients did not use FA 5 mg everyday (4 declared no use anytime and 4 reported intermittent use) and none of control group used FA supplementation. Reticulocytes and LDH were higher in HS group (P Conclusions Elevated SF concentrations were associated with higher mRNA expression of DHFR gene in HS patients, suggesting that the use of higher amounts of FA might influence the expression and activity of DHFR and thus affect folate metabolism in these patients. It is not known whether normal subjects receiving similar high doses of FA show the same effects. Financing: FAPESP 2012/12912-1 and CNPq 4826412012-6 Figure 1 – Serum folate levels (A) and DHFR mRNA (B) in hereditary spherocytosis and control group. The DHFR mRNA expressions in HS patients according to serum folate levels were shown (C). Figure 1 – Serum folate levels (A) and DHFR mRNA (B) in hereditary spherocytosis and control group. The DHFR mRNA expressions in HS patients according to serum folate levels were shown (C). The line in each graphic is the median. The median and percentiles 25 and 75 (P25 – P75) for control and hereditary spherocytosis groups were, respectively: A- serum folate: 22.1 (14.5 – 36.6 nmolL) and 51.5 (19.5 – 95.2 nmolL), B- DHFR mRNA expression: 1.59 (1.00 – 2.48) and 35.8 (19.0 – 52.7). In Figure C, the median and percentiles 25 and 75 (P25 – P75) for DHFR mRNA expression, according to serum folate levels (SF Disclosures No relevant conflicts of interest to declare.

Background: Myelofibrosis (MF) and essential thrombocythemia (ET) are clonal hematopoietic stem cell malignancies classified as myeloproliferative neoplasms (MPNs). FGF2 and TGF-β were associated with pathophysiology and progression of these diseases. Although FGF2 and TGF-β roles in angiogenesis and fibrosis are known, their mRNA expression in leukocytes of MPNs patients was not yet evaluated. Furthermore, the detailed mechanism of TGF-β signaling in the pathophysiology of these diseases remains unclear. In regular signaling process, Smads are a group of proteins critical for transmitting signals from TGF-β superfamily of the cell surface to the nucleus. Thus, Smads signaling plays an important role in the regulation of hematopoiesis and it could modulate TGF-β action in MPNs. Aims: The aim of this study was to investigate if there are differences in mRNA expression of FGF2, TGFB1 and SMADS 1 to 7 in MPNs patients and healthy controls and if it is related to JAK2 mutation status and allele burden. Methods: Fifteen primary myelofibrosis (PMF), 15 myelofibrosis post essential thrombocytemia (MPET), and 22 essential trombocythemia (ET) patients diagnosed according to the World Health Organization criteria (2008) were selected from the Hematology Department of the Federal University of de Sao Paulo and from the Hematology Division of the Catholic University of Sao Paulo. Other group with healthy subjects was also included. The control group was matched by age and gender with MPNs patients: 30 for PMF, 17 for MPET, and 34 for ET. Expressions of FGF2, TGFB1 and SMADs 1 to 7 mRNA were evaluated in total leukocytes by Real Time PCR, using Taqman assays. JAK2V617F mutation status and allele burden and MPL W515K/L analysis were performed in total leukocytes DNA by Real Time PCR, using Taqman MGB probes. Results: No difference was found between frequencies of JAK2V617F mutated subjects (P=0.520) and in allele burden percentage (P=0.415) in MPNs groups. Allele burden was also not associated with mRNA levels of studied genes in each group of MPNs, and none of the patients presented MPL W515K/L mutations. MF and ET patients had higher TGFB1 and FGF2 mRNA levels than its controls (P Conclusions: Our findings suggest that FGF mRNA is overexpressed in leukocytes of MF, MPET and ET patients. It seems that higher TGFB1 mRNA levels do not affect the molecular signaling by SMADs in MF patients, although SMAD6 and SMAD1 mRNA levels in ET and MPET patients might be upregulated. Financing: FAPESP 2012/ 12957-5 Disclosures No relevant conflicts of interest to declare.

Background The methylenetetrahydrofolate reductase (MTHFR) and methylenetetrahydrofolate dehydrogenase (MTHFD1) enzymes play important roles in the metabolism of folate. MTHFR catalyses the reduction of 5,10-methylene-tetrahydrofolate (5,10-methylene-THF) to 5-methyl-THF and present a crucial role in the regulation of available folate to homocysteine remethylation, in a cobalamin (Cbl) dependent reaction. MTHFD1 has three distinct enzymatic activities in folate metabolism: the conversion of THF to 10-formyl-THF; the reversible conversion of 10-formyl-THF to 5,10-methenyl-THF; and the conversion of 5,10-methenyl-THF to 5,10-methylene-THF. The two enzymes were related with DNA synthesis and single nucleotides polymorphisms (SNPs) in their genes have been associated with lower folate levels. People who have increased cellular duplication could be affected by low folate levels. Hereditary spherocytosis (HS) patients had an increased erythropoiesis and need more amounts of acid folic. In Brazil, HS patients are usually treated with 5mg/day of folic acid, the only formulation available. The use of folic acid by β-thalassemia heterozygotes (β-TH) subjects is not common, and depends of the individual clinical feature. Furthermore, in Brazil, HS patients and β-TH people have been exposed to food fortification (wheat and corn flours) with 150 µg of FA since 2004. The influences of variants of MTHFR and MTHFD1 SNPs in people with increased erythropoiesis, especially in HS patients, are not known. Objective The aim of the study was to evaluate the interaction between MTHFR c. 677C>T and c. 1298A>C and MTHFD1 c. 1958G>A SNPs and the use or not of FA supplementation in HS patients. It was also our objective to compare the folate and Cbl levels according to genotypes for MTHFR and MTHFD1 SNPs in β-TH subjects and their controls. Methods Twenty-five patients with HS, 49 β-TH subjects and 98 healthy people exposed to mandatory fortification of wheat and corn flours with FA were included in this study. Serum folate and Cbl were determined by microbiologic assays. Genomic DNA was extracted from whole blood using commercial kit. The genotypes for MTHFR c. 1298A>C and MTHFD1 c.1958G>A SNPs were determined by Real Time PCR using TaqMan assays. PCR-RFLP was used for genotyping MTHFR c. 677C>T SNP. Haplotype frequencies (MTHFR c. 677C>T and c. 1298 A>G) and the standardized disequilibrium coefficient (D’) for pair-wise linkage disequilibrium were assessed by the Expectation-Maximization (EM) algorism using the Haploview Software. We considered linkage disequilibrium when D’ ≥0.50. The study of linkage disequilibrium was performed by calculating the coefficient D' of Lewontin. Results The frequencies of genotypes for MTHFR (c. 677C>T and c. 1298A>C) and MTHFD1 c. 1958G>A SNPs were similar in HS, β-TH and control groups (p>0.05). When genotypes were grouped according to the presence of the mutated allele (CC vs. CT+TT for MTHFR c. 677C>T, AA vs. AC+CC for MTHFR c. 1298A>C and GG vs. GA+AA for MTHFD1 c. 1958G>A), no differences were also found (p>0.05). The MTHFR c.677C>T and c. 1298A>C SNPs are in complete linkage disequilibrium (D'=1.0). Regarding the correlation coefficient between the two loci (r2), the value found in this analysis was equal to 0.169 in HS versus control group and 0.205 in β-TH versus control group. The haplotype CA, which is formed by the ancestral allele of MTHFR c. 677C>T and c. 1298A>C SNPs respectively, was more frequent in controls (41.9%) than in the β-TH group (28.1%). However, this finding was not observed in HS group comared with control group. No association was found between genotypes for MTHFR c. 677C>T and c. 1298A>C and MTHFD1 c. 1958G>A and alterations in folate and Cbl levels in three groups. ANCOVA was used for evaluating the interaction between the use of FA supplementation and MTHFR c.677C>T SNP on folate levels in HS patients. Only FA supplementation was associated with increased serum folate, and no associations were observed with MTHFR SNP or interaction between both. Conclusion The SNPs were not associated with reduced serum folate levels in the three groups studied. This finding suggests that FA flour fortification in β-TH and controls, as well as, the supplementation plus fortification in HS group, could offset the effect of variants of SNPs, especially for MTHFR c.677C>T. Disclosures No relevant conflicts of interest to declare.

Introduction Recurrent pregnancy loss (RPL) is defined as three or more consecutive spontaneous abortions prior to the 20th week. RPL is a multifactorial condition that could be influenced by several factors, including haemostasis disorders. Indeed, pregnancy is characterized by a hypercoagulable state in which levels of some coagulation factors and fibrinolytic inhibitors are increased, whereas levels of the natural anticoagulant free protein S are decreased. Therefore, an unbalance in the fibrinolytic system might be involved in the pathophysiology of RPL. Plasminogen activator inhibitor type 1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI) are two fundamental inhibitors of fibrinolysis, whose levels could be influenced by genetic polymorphisms. Aims: To investigate the frequencies of polymorphisms in PAI-1 (SERPINE1 4G/5G) and TAFI (CPB2 c.505G>A) in women with primary RPL and in healthy women. Materials and Methods We included 116 unrelated 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 (primary RPL) who were evaluated in the Prenatal Care Unit at the Hospital of the University of Sao Paulo. The control group was comprised of 263 unrelated healthy fertile women who were users of Public Health Service or employees of the University of Sao Paulo, who had at least two term deliveries and no known pregnancy losses as well as known medical history of thrombosis, diabetes, hypertension, cancer and autoimmune disorders. Both cases and controls were from the general urban population of Sao Paulo City. The genotypes for CPB2 c.505G>A polymorphism were performed by Real Time PCR, and genotypes for SERPINE1 4G/5G polymorphism were performed by RFLP-PCR. The effect of these polymorphisms on RPL risk was assessed by calculating Odds ratios (OR) and their 95% confidence intervals (CI) by logistic regression. Results and Conclusions The genotype distribution of both polymorphisms was in Hardy-Weinberg equilibrium in cases and in controls (p>0.05). The genotype and allele frequencies for SERPINE1 4G/5G and CPB2 c.505G>A polymorphisms were similar in both groups (p>0.05). For SERPINE1 4G/5G polymorphism, 70 cases (60.4%) and 152 controls (57.8%) were carriers of the 4G allele, yielding an OR of RPL of 1.11 (95% CI: 0.71 - 1.74, p= 0.642) (genotype 5G5G vs. 4G5G + 4G4G). For CPB2 c.505G>A polymorphism, 64 cases (55.2%) and 153 controls (58.2%) were carriers of the A allele, yielding an OR of RPL of 0.88 (95% CI: 0.57 - 1.37, p= 0.586) (genotype GG vs. GA + AA). Furthermore, similar frequencies of minor allele were found in groups when the number of losses was considered in RPL group (women with four or more RPL vs. women with three, p >0.05) and when the period of pregnancy loss was considered (women with early losses vs. women with late losses, p>0.05). Our data suggest that polymorphisms in SERPINE1 and CPB2 are not associated with a risk of having RPL. Financing: FAPESP (Proc 2007/57605-0), CAPES. Disclosures: No relevant conflicts of interest to declare.

Abstract 1422 Background: Imatinib Mesylate (IM) used in the treatment of CML, interacts with membrane efflux transporters such as ABCB1 and ABCG2, whereas the active uptake of IM into the cells is mediated by SLC22A1. The predictive value of these markers is still controversial. The altered expression of these genes could impact on intracellular concentration of IM and contribute to resistance. Aims: The aim of this study was to investigate ABCB1, ABCG2 and SLC22A1 gene expression as potential sources of resistance to imatinib in patients with CML Methods: One hundred and eighteen patients in chronic phase of CML, both genders with age range 18 to 80 were studied. All patients were initially treated with a standard dose of IM (400 mg/day) and divided in two groups according to response. The responder group comprised 70 patients who had a complete cytogenetic response within 18 months of treatment. The non-responder group comprised 48 patients who did not have a complete cytogenetic response with the initial dose (400 mg/day) of IM or who relapsed during treatment and were submitted to higher doses of 600 or 800 mg/day. Criteria of failed response to treatment were established by European LeukemiaNet. Patients with cytogenetic patterns other than the Philadelphia chromosome and patients with mutations in the BCR-ABL1 gene were excluded from this study. Major molecular response (MMR) was defined as a reduction of BCR-ABL1 transcripts levels to ≤ 0.1% in the peripheral blood standardized on the International scale. Complete molecular response (CMR) was defined as a reduction ≤ 0.032% BCR-ABL1 transcripts levels. Primary resistance and secondary resistance also were evaluated. Real-Time PCR was performed to evaluate the ABCB1, ABCG2 and SLC22A1 mRNA relative expression to control gene GAPDH. Results: Expression of ABCG2 in the non-responder group was higher than in the responder group (P=0.028). This result was influenced by patients with primary resistance (n= 34 p=0.029) but not secondary resistance (n=14 p=0.249) when compared with responders (n=70). ABCB1 and SLC22A1 expression were similar between responder and non-responder groups. Higher levels of SLC22A1 mRNA were found in patients who achieved MMR in the responder group (p=0.009). The elevated ABCG2 expression was also found in those who did not achieve MMR (p=0.027) when all patients were analyzed. None of studied genes was associated with CMR. Conclusions: The high expression of ABCG2 is related to primary resistance and SLC22A1 is positively associated with major molecular response to treatment with IM. Our data suggests that ABCG2 may be a mediator of IM resistance, whereas SLC22A1 could be a good predictor of response to IM therapy. Financing: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2009/54184-0). Disclosures: No relevant conflicts of interest to declare.

Abstract 5136 Introduction: Folic acid deficiency has been associated with obstetric complications such as preeclampsia, placental abruption and recurrent miscarriages (RM). Folic acid is a methyl group donating in various reactions. Low concentrations of 5,10-methylenetetrahydrofolate, the cofactor of enzyme thymidylate synthase, decrease the synthesis of thymidylate, which results in increased ratio deoxyuridylate monophosphate/deoxytimidylate monophosphate (dUMP/dTMP) and increased incorporation of deoxyuridylate triphosphate (dUTP) to DNA. The removal of dUTP by DNA-glycosylase can cause permanent damage to DNA, which may lead to apoptosis or increase the risk of developing cancer. Polymorphisms in genes of enzymes (MTHFR - methylene tetrahydrofolate reductase, MTR - methionine synthase and MTRR - methionine synthase reductase) and also in the gene of the reduced folate carrier (RFC1) were related to reduced folate and increased total homocysteine concentrations and have been associated as risk factors for RM. Material and Methods: 171 women with a history of three or more recurrent miscarriages and 95 healthy women with no history of abortion and having two or more normal babies were included. Weight and height of women were obtained and the body mass index (BMI) was calculated. The presence of antibodies (ANA and anti-DNA) was evaluated using immunofluorescence kits. The genotypes of the polymorphisms MTHFR c. 677C>T, MTR c. 2756A>G and RFC1 c.80G>A were obtained by PCR-RFLP, while genotyping for polymorphisms MTRR c. 66A>G and MTHFR c. 1298A>C was made by real time PCR. Multivariate logistic regression model (forward conditional) was used to obtain the odds ratio and its 95% confidence intervals of having MR (dependent variable). The independent variables were: quartiles of BMI, age range, positive ANA (titer of 1/40), positive anti-DNA (titre 1/10), genotypes for the MTHFR c. 677C>T, MTHFR c.1298A>C, MTR c. 2756A>G, MTRR c. 66A>G and RFC1 c. 80G>A. Results: No differences between the groups were observed for serum total homocysteine or allele frequencies for MTHFR c. 677C>T, MTHFR c. 1298A>C, MTR c. 2756A>G and RFC1 c.80G>A polymorphisms. In a conditional logistic regression analysis the risk of RM was significantly associated with BMI OR [95% CI] = 1.40 [1.02, 1.93] per quartile increase in BMI), positive anti-DNA OR [95% CI] = 7.24 [0.92, 57.25], positive ANA OR [95% CI] = 2.48 [1.21, 5.08], and AA genotype for MTRR c. 66A>G polymorphism (OR [95% CI] = 2.19 [1.16, 4.12]. Conclusion: The etiology of RM is multifactorial and it is associated with increasing of BMI, presence of autoantibodies and AA genotype for MTRR c. 66A>G polymorphism. Disclosures: No relevant conflicts of interest to declare.

Hereditary hemochromatosis (HH) is a disorder characterized by increased intestinal iron absorption, which leads to a progressive accumulation of iron in the body. This iron overload has been associated with mutations in HFE gene (C282Y, H63D and S65C) and other genes. The objectives of this study were to assess the frequencies of functional mutations in HFE and TFR2 genes and to investigate their relationship with the iron status in a sample of blood donors. Blood donors (n=542) were recruited at the Hemocenter of the Santa Casa Hospital, Sao Paulo, Brazil. The genotypes for HFE (C282Y, H63D and S65C) TFR2 (Y250X and Q690P) gene mutations were evaluated by PCR-RFLP. The concentrations of serum iron and total iron-binding capacity (TIBC) were measured by automation system Advia®(Bayer Diagnostics) and serum ferritin by Axsym System®(Abbott Laboratories). The frequencies of HFE 282Y, HFE 63D and HFE 65C alleles were 2.1, 13.6 and 0.6%, respectively. The frequency C282Y allele (2.1%) in Brazilian blood donors is lower than that observed in blood donors from Northern Europe (5.1 to 8.2%, P

Methionine synthase (MS) is a cobalamin dependent enzyme that catalyses the remethylation of homocysteine to methionine. The methionine synthase reductase (MSR) maintains adequate levels of methylcob(III)alamin, the activated cofactor for MS. The aim of this study was to investigate the effect of MS A2756G and MSR A66G polymorphisms on total homocysteine (tHcy), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) levels in 390 pregnant women and their 292 newborns, from Sorocaba city, Brazil. Genotypes of two polymorphisms were determined by PCR-RFLP. Pregnant women with MS 2756AA genotype have higher tHcy and lower Cbl levels than those with 2756G alleles. The MMA values were increased in neonates with MS 2756AA genotypes (Table 1). There are no difference between the maternal values of Cbl, serum folate, tHcy, MMA and SAM according to MSR A66G genotypes.The values of SAM were lower in neonates with MSR 66G alleles than those with AA genotypes (Table 2). We conclude that MS 2756AA genotypes are associated with higher tHcy levels in pregnant women and higher MMA levels in neonates. The MSR 66GG genotypes is associated with lower SAM levels in neonates. Table 1- Distribution of geometric means and confidence intervals 95% (CI 95%) and numbers of subjects for maternal and neonatal values of cobalamin (Cbl), serum folate, total homocysteine (tHcy), methymalonic acid (MMA) and S- adenosylmethionine (SAM) according to genotypes for the polymorphism MS A2756G. | Variables | Genotypes for MS A2756G | Student t Test | |:-------------- | ----------------------- | ---------------------- | -------- | | | AA | AG + GG | | | Pregnant Women | | | | | Cbl (pmol/L) | 139 (133 – 144) 235 | 156 (146 – 166) 129 | P= 0.001 | | SF (nmol/L) | 14.3 (13.6 – 15.0) 234 | 14.5 (13.6 – 15.5) 129 | P= 0.667 | | tHcy( μmol/L) | 6.8 (6.5 – 7.1) 235 | 6.2 (5.9 – 6.6) 128 | P= 0.036 | | MMA(nmol/L) | 234 (219 – 245) 194 | 241 (219 – 265) 106 | P= 0.610 | | SAM(nmol/L) | 81.8 (77.9 – 86,0) 229 | 83.1 (79.1 – 87.4) 124 | P= 0.663 | | Neonates | | | | | Cbl (pmol/L) | 227 (212 – 244) 188 | 234 (213 – 257) 101 | P= 0.646 | | SF (nmol/L) | 32.0 (31.0 – 33.0) 186 | 32.0 (30.8 – 33.2) 99 | P= 0.967 | | tHcy μmol/L) | 5.8 (5.5 – 6.1) 185 | 5.5 (5.1 – 5.9) 100 | P= 0.229 | | MMA(nmol/L) | 383 (364 – 402) 183 | 342 (317 – 369) 100 | P= 0.011 | | SAM(nmol/L) | 188 (181 – 196) 178 | 182 (168 – 197) 98 | P= 0.491 | Table 2 - Distribution of geometric means and confidence intervals 95% (CI (%%) and number of subjects for neonatal values of cobalamin (Cbl), serum folate, total homocysteine (tHcy), methylmalonic acid (MMA) and S- adenosylmethionine (SAM) according to genotypes for the polymorphism MSR A66G. | Variables | Genotypes MSR A66G | Student t Test | |:---------------------------------------------------------------------------------------------------------- | --------------------- | ---------------------- | --------------------- | | | AA | AG | GG | | | Groups not sharing a common superscript letter are significantly different at P

In the homocysteine metabolic pathway, several key enzymes, including methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR), have been implicated in abnormal homocysteine accumulation in the presence of rare alleles. In previous study, we showed that lower maternal Cbl levels were associated with higher tHcy and lower S-adenosylmethionine/S-adenosylhomocysteine ratio in pregnant women and their neonates.The aim of this study is to investigate whether MTHFR and MTRR polymorphisms are involved in the risk for elevated total homocysteine (tHcy) and its interaction with low cobalamin (Cbl) or serum folate (SF) levels. Genotypes for polymorphisms MTHFR C677T and MTRR A66G were determined by PCR-FLRP. The serum levels of Cbl, SF and tHcy were determined in 377 pregnant women (37–42 weeks of gestational age), and cutoff values for Cbl and SF were considered the first quartile (low values). Four models of univariate logistic regression analyses were used (Table 1). Pregnant women with MTHFR 677T allele have high risk for elevated tHcy that is increased when 677T allele is associated with low Cbl. Increased risk for elevated tHcy is also met when MTRR 66G allele and low Cbl levels were associated. Women with low SF and common MTHFR and MTRR alleles have high risk for elevated tHcy, that is increased when in association with 677T allele or with 66G allele. Interaction between MTHFR C677T and MTRR A66G polymorphisms and vitamins levels in pregnant women Dependent variables Comparation levels (N) P value Odd Ratios 95% CI P for trend: (a) P8.3μmol/L MTHFR 677CC genotype and Cbl> 115.8 pmol/L (ref) (136) a 1.00 MTHFR 677CC genotype and≤Cbl 115.8 pmol/L (45) 0.298 1.57 0.67 – 3.63 MTHFR 677CT and 677TT genotypes and Cbl>115.8 pmol/L (145) 0.015 2.09 1.16 – 3.77 MTHFR 677CT and 677TT genotypes and≤Cbl 115.8 pmol/L (48) 0.001 4.63 2.22 – 9.65 tHcy>8.3μmol/L MTHFR 677CC genotype and SF > 10.9 nmol/L (ref) (148) b 1.00 MTHFR 677CC genotype and≤SF 10.9 nmol/L (33) 0.008 3.20 1.35 – 7.59 MTHFR 677CT and 677TT genotypes and SF > 10.9 nmol/L (133) 0.035 1.95 1.05 – 3.61 MTHFR 677CT and 677TT genotypes and≤SF 10.9 nmol/L (59) 0.001 6.62 3.31 – 13.26 tHcy>8.3μmol/L MTRR 66AA genotype and Cbl> 115.8 pmol/L (ref) (96) c 1.00 MTRR 66AA genotype and ≤Cbl 115.8 pmol/L (23) 0.222 1.90 0.68 – 5.29 MTRR 66AG and 66GG genotypes and Cbl>115.8 pmol/L (183) 0.418 1.29 0.70 – 2.39 MTRR 66AG and 66GG genotypes and ≤Cbl 115.8 pmol/L (69) 0.013 2.46 1.21 – 5.01 tHcy>8.3μmol/L MTRR 66AA genotype and SF > 10.9 nmol/L (ref) (92) d 1.00 MTRR 66AA genotype and ≤SF 10.9 nmol/L (27) 0.006 3.83 1.47 – 9.96 MTRR 66AG and 66GG genotypes and SF > 10.9 nmol/L (186) 0.399 1.34 0.68 – 2.63 MTRR 66AG and 66GG genotypes and≤SF 10.9 nmol/L (65) 0.001 4.78 2.26 – 10.10 In conclusion, the interaction between MTHFR and MTRR polymorphisms and low folate and cobalamin serum levels may explain the increased risk for elevated tHcy found in pregnant women.

Background An increased concentration of plasma homocysteine (Hcy) has toxic effects on vascular endothelium. This seems to be a risk factor of cardiovascular disease, premature stroke and venous thrombosis. The risk is higher in coincidence with other factors like chronic diseases and familiar hypercholesterolemia. The aim of our study was to evaluate plasma Hcy concentration in patients with juvenile idiopathic arthritis (JIA) and its correlation with methotrexate (MTX) therapy, serum folate and B12 vitamin, and hyperlipidemia. Methods Fifty-one patients (37 females; mean age 11.3 years, range 2.3–17 years) with JIA and 52 healthy controls (42 females; mean age 12.5 years; range 3–18 years) were included in the study. Thirty-two patients were using weekly MTX (mean doses: 0.1–1 mg/kg). For statistical analysis both JIA and control groups were distributed in three subgroups according to age (3 – 7, 8 – 12 and 13 – 18 years). The laboratory investigation included measurement of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), plasma Hcy, serum folate, vitamin B12, triglycerides, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL). For data analysis, we considered raised Hcy values ≥ 12.56 μmol/L, which corresponds to the 90th percentile observed in the control group. Results The mean plasma Hcy concentration was 9.3 ± 3.16 μmol/L in JIA patients and 8.9 ± 2.42 μmol/L in healthy controls (p = 0.615). Higher concentration of Hcy was observed in the subgroup of 13 – 18 years (patients and controls, p < 0.001). We did not find correlation between MTX use and plasma Hcy concentration. With regard to vitamin B12 concentration, we detected normal values in both patients and controls while serum folate concentration was higher in patients (p < 0.001). With regard to the lipidogram, lower concentration of HDL was found in patients (p = 0.007) and higher levels of VLDL (p = 0.014) and triglycerides (p = 0.001) were observed in controls. We did not observe correlation among plasma Hcy concentration, clinical findings, ESR and CRP. Conclusion JIA patients do not present significant increased concentration of Hcy despite the use of MTX, probably due to the folate supplementation. The mild abnormalities in the lipidogram may reflect a current concern with diet and health.

O presente estudo avaliou o perfil farmacogenômico de 338 pacientes, sob terapia antiagregante. Os pacientes foram submetidos a tratamento prévio com AAS (100mg/dia) e clopidogrel (75mg/dia) por no mínimo cinco dias antes da angioplastia coronária. Os indivíduos com resposta considerada indesejada 550 ARU (do inglês, Aspirin Reaction Unit), foram considerados como não respondedores. As concentrações plasmáticas dos antiagregantes foram determinadas por cromatografia líquida acoplada à espectrometria de massa do tipo triploquadrupolo (LC-MS/MS). A taxa da inibição da agregação plaquetária foi medida utilizando-se o sistema VerifyNow®. A expressão gênica global das células totais do sangue periférico foi avaliada pela tecnologia de microarranjos de DNA Human Exon ST 1.0 Array. Características genotípicas dos pacientes também foram avaliadas pelo sistema Sequenom®. Assim, foi possível obter como resultados a identificação de 64% e 10% para pacientes não respondedores ao clopidogrel e AAS respectivamente, sendo que para o primeiro foi possível identificar a associação desta não resposta a variáveis clínicas como diabetes (p = 0,003), hipertensão (p = 0,011) e hábito de fumar (p = 0,041) e sexo (p = 0,022) e idade dos pacientes (p = 0,004) em relação à resposta ao AAS. O método de quantificação simultânea do clopidogrel, seu metabólito majoritário e do AS (metabólito do AAS), apresentou limites de quantificação entre de 2 a 500 ng/mL, 2 a 2000 ng/mL e de 20 a 2000 ng/mL, respectivamente. O estudo de associação encontrou uma relação significante da presença dos SNPs presentes nos genes CYP5A1 (rs2299890) e CYP2C19 (rs4244285 e rs3758580), com a variação na resposta ao clopidogrel, obtendo um valor de p corrigido pelo teste de permutação inferior a 0,001. Como também, uma fraca associação da variação na resposta do AAS com o SNP rs9605030 do gene COMT (p = 0,009). Os resultados do microarranjos relacionaram a resposta terapêutica ao clopidogrel com os genes CA2, MKRN1, ABCC3 e MBP seguido dos genes NFIA e IGF1R para a resposta ao AAS. Concluindo que o estudo farmacogenômico apresentou todo o seu potencial para relacionar variáveis como resposta, concentração farmacológica plasmática, SNPs e expressão global de RNAm, possibilitando assim compreender melhor a variação no tratamento antiagregante. This study investigated the pharmacogenomics profile of 338 patients under antiplatelet therapy. Patients undergoing pretreatment with ASA (100 mg/day) and clopidogrel (75mg/day) for at least five days prior to coronary angioplasty. Individuals with response 550 of ARU (aspirin reaction unit), were considered as non responders for ASA. Plasma concentrations of the antiagregation drugs were determined by liquid chromatography followed mass spectrometry of triple quadrupole detection (LC-MS/MS). The rate of inhibition of platelet aggregation was measured using the VerifyNow® system. The global gene expression of total cells in blood was assessed by DNA microarray technology Human Exon 1.0 ST Array. Genotypic characteristics of the patients were also evaluated by the Sequenom® system. Thus it was possible to obtain results such as identification of 64% and 10% for patients non responders to clopidogrel and aspirin respectively, and for the first could identify the association of this response to variables such as diabetes (p = 0.003), hypertension (p = 0.011) and smoking (p = 0.041) for clopidogrel and sex and age in relation to response to ASA (p = 0.022 and p = 0.004, respectively). The method of simultaneous quantification of clopidogrel and its major metabolite of AS (metabolite of ASA), had quantification limits between 200 to 500 ng/mL 2000-2000 ng/mL and 20 to 2000 ng/mL, respectively. The association study found a significant grating presence of SNPs present in genes CYP5A1 (rs2299890) and CYP2C19 (rs4244285 and rs3758580), with the variation in the response to clopidogrel, obtaining a corrected p value by permutation test below 0.001. As well, a weak association of variation in the response of ASA with the SNP rs9605030 of the gene COMT (p = 0.009). The results of microarray related therapeutic response to clopidogrel with genes CA2, MKRN1, ABCC3 and MBP followed by NFIA and IGF1R genes for response to ASA. Concluding that the pharmacogenomics study showed its potential to relate variables such as response, plasma drug concentration, SNPs and global expression of mRNA, thus enabling better understand the variation in antiplatelet treatment.