Elvira Maria Guerra-Shinohara

Relationship between total homocysteine and folate levels in pregnant women and their newborn babies according to maternal serum levels of vitamin B12

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.

Parameters for the assessment of iron status.

The assessment of iron status at individual and populational levels has been the target of many studies because some issues still need to be defined. A Medline, Lilacs and Dedalus literature review was carried out for the period of 1972 to 1998 and scientific publications in both English and Portuguese relating hematological and biochemical parameters were used in the assessment of iron status. The parameters reflect the three different stages of iron storage deficiency, which occur in a gradual and progressive way leading to the development of anaemia. In general, when used alone, these parameters are neither sensitive nor specific for detecting iron deficiency anaemia. Moreover, some advantages and limitations should be taken into consideration for the choice of the appropriate parameter. To improve both sensitivity and specificity, different parameters have been used in association and their specific contribution is determined according to the characteristics of the study population, methodological issues and the costs involved in the process.

Association between decreased vitamin levels and MTHFR, MTR and MTRR gene polymorphisms as determinants for elevated total homocysteine concentrations in pregnant women.

Objectives: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.Subjects/Methods: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).Results: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.Conclusions: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.

Metabolic effects of C677T and A1298C mutations at the MTHFR gene in Brazilian children with neural tube defects

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<0.05) in NTDs children. CONCLUSIONS MTHFR gene mutations may affect vitamin B12 and homocysteine metabolism in Brazilian children with NTDs.

ABCB1 haplotype is associated with major molecular response in chronic myeloid leukemia patients treated with standard-dose of imatinib

BACKGROUND 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. AIM To investigate the relationship of ABCB1 polymorphisms with markers of response to IM in patients with CML. METHODS 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.1236C>T (rs1128503), c.3435C>T (rs1045642) and c.2677G>T/A (rs2032582) were evaluated by PCR-RFLP. RESULTS ABCB1 polymorphisms were not related with a risk for CML in this sample population (p<0.05). In the CML group, frequencies of ABCB1 SNPs were similar between responder and non-responder patients (p>0.05). 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). CONCLUSIONS The ABCB1 1236CT/2677GT/3435CT haplotype is positively associated with the major molecular response to IM in CML patients.

Hereditary hemochromatosis: mutations in genes involved in iron homeostasis in Brazilian patients.

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.

Homocysteine and lipid profile in children with Juvenile Idiopathic Arthritis

BackgroundAn 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.MethodsFifty-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.ResultsThe 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.ConclusionJIA 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.

ABCB1 haplotypes are associated with P-gp activity and affect a major molecular response in chronic myeloid leukemia patients treated with a standard dose of imatinib

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 classified 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 demonstrated 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<0.05). In conclusion, the ABCB1 1236CT/3435CT/2677GT and 1236TT/3435TT/2677TT haplotypes are associated with reduced P-gp activity and MMR in chronic-phase CML patients treated with a standard dose of IM.

Genotyping of the hemochromatosis HFE p.H63D and p.C282Y mutations by high-resolution melting with the Rotor-Gene 6000® instrument

Abstract 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.

HFE gene mutations and iron status of Brazilian blood donors

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 São Paulo, São 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.