Genetic Variants Involved in One-Carbon Metabolism: Polymorphism Frequencies and Differences in Homocysteine Concentrations in the Folic Acid Fortification Era.

Folate and other B vitamins are essential co-factors of one-carbon metabolism, and genetic variants, such as polymorphisms, can alter the metabolism. Furthermore, the adoption of food fortification with folic acid showed a decrease of homocysteine concentration. The aim of this study was to investigate the frequencies of the polymorphisms of enzymes and carrier proteins involved in one-carbon metabolism, and to evaluate homocysteine concentrations in the presence of these genetic variants in a population exposed to mandatory food fortification with folic acid. Using data from a population-based cross-sectional study in São Paulo, Brazil, the study population comprised 750 participants above 12 years of age of both genders. A linear regression model was used to evaluate the homocysteine concentrations according to genetic variants and folate level. The results showed that the minor allelic frequencies were 0.33 for MTHFR (rs1801133), 0.24 for MTHFR (rs1801131), 0.19 for MTR (rs1805087), 0.42 for MTRR (rs1801394), 0.46 for RFC1 (rs1051266), and 0.47 for DHFR (19-bp deletion). The genetic variants of MTHFR 677C>T, MTRR 66A>G and RFC-1 80G>A were different according to race. The homocysteine concentrations increased in the CT and TT compared to CC genotypes of polymorphism MTHFR 677C>T in all populations, and differences between the homocysteine concentrations according to the genotypes of MTHFR 677C>T were observed regardless of folate level.

Impact of genetic variants of RFC1, DHFR and MTHFR in osteosarcoma patients treated with high-dose methotrexate.

Osteosarcoma patients are commonly treated with high doses of methotrexate (MTX). MTX is an analog of folate, which is essential for DNA synthesis. Genetic polymorphism at single nucleotide can be indicative to the prognostic outcome in patients. Germ-line variants in candidate genes, coding for enzymes active in the metabolism of MTX, were studied in 62 osteosarcoma patients. Patients harboring the GG genotype in reduced folate carrier 1 (RFC1) rs1051266 had significantly better survival in comparison with patients having the AA genotype (P=0.046). These patients also had a lower frequency of metastasis (15%, P=0.029). Also patients homozygous for the G allele of rs1053129 in the dihydrofolate reductase (DHFR) gene were more likely to have a metastasis (45%, P= 0.005), and the methylenetetetrahydrofolate reductase (MTHFR) 677C allele was associated with higher degree of liver toxicity (88%, P=0.007). The study suggests that germ-line variants in the MTX metabolic pathway are associated with survival and side effects in patients treated with MTX.

Plasma micronutrient levels and telomere length in children.

OBJECTIVE: Telomeres are long hexamer (TTAGGG) repeats at the ends of chromosomes, and contribute to maintenance of chromosomal stability. Telomere shortening has been linked to cancers and other chronic diseases in adults, although evidence for causal associations is limited. The aim of this study was to determine whether nutritional factors are associated with telomere length (TL) in children. METHODS: We conducted a cross-sectional study of nutritional factors and TL in 437 children between 2009 and 2011. Healthy children ages 3, 6, and 9 y provided blood samples, and their parents completed a food frequency questionnaire and a telephone interview about relevant environmental exposures. TL and blood micronutrient levels were measured, and genotyping at 10 loci was undertaken. Associations between the micronutrients and other variables were assessed using linear regression. RESULTS: No significant main or interactive effects of age or sex were seen. After adjustment for age, sex, parental education, and month of blood collection, TL was inversely associated with plasma zinc, and shorter in children with the homozygous mutant genotype of the RFC G80A (rs1051266) polymorphism. CONCLUSIONS: To the best of our knowledge, this is the first investigation of the association between telomere length and micronutrients in healthy children. The reason for the inverse relationship of TL with zinc is unknown but could be the result of an increase in telomere sequence deletions caused by labile zinc induction of oxidative stress. These findings should be corroborated in other studies before nutritional recommendations might be considered.

RFC-1 80G>A polymorphism in case-mother/control-mother dyads is associated with risk of nephroblastoma and neuroblastoma.

AIM: Embryonic tumors are associated with an interruption during normal organ development; they may be related to disturbances in the folate pathway involved in DNA synthesis, methylation, and repair. Prenatal supplementation with folic acid is associated with a decreased risk of neuroblastoma, brain tumors, retinoblastoma, and nephroblastoma. The aim of this study was to investigate the association between MTHFR rs1801133 (C677T) and RFC-1 rs1051266 (G80A) genotypes with the risk of developing nephroblastoma and neuroblastoma. MATERIALS AND METHODS: Case-mother/control-mother dyad study. Samples from Brazilian children with nephroblastoma (n=80), neuroblastoma (n=66), healthy controls (n=453), and their mothers (case n=93; control n=75) were analyzed. Genomic DNA was isolated from peripheral blood cells and/or buccal cells and genotyped to identify MTHFR C677T and RFC-1 G80A polymorphisms. Differences in genotype distribution between patients and controls were tested by multiple logistic regression analysis. RESULTS: Risk for nephroblastoma and neuroblastoma was two- to fourfold increased among children with RFC-1 polymorphisms. An increased four- to eightfold risk for neuroblastoma and nephroblastoma was seen when the child and maternal genotypes were combined. CONCLUSION: Our results suggest that mother and child RFC-1 G80A genotypes play a role on the risk of neuroblastoma and nephroblastoma since this polymorphism may impair the intracellular levels of folate, through carrying fewer folate molecules to the cell interior, and thus, the intracellular concentration is not enough to maintain regular DNA synthesis and methylation pathways.

Association of MTHFR and RFC1 gene polymorphism with hyperhomocysteinemia and its modulation by vitamin B12 and folic acid in an Indian population.

BACKGROUND/OBJECTIVES: Unlike most Western populations, MTHFR 677T is a rare allele and a risk factor for a variety of disorders in India. What kind of nutritional (environmental) and/or genetic factors could contribute to the genetic risk is not known. To assess the incidence of hyperhomocysteinemia and its correlation with the polymorphism in homocysteine (Hcy)-pathway genes and associated cofactors in the native populations of eastern India. SUBJECTS/METHODS: Healthy population from four eastern states of India. Genotyping of SNPs, HPLC and chemiluminescence-based assay for homocysteine, vitamin B12 and folic acid. RESULTS: Approximately 30% of the population has hyperhomocysteinemia (>15 µmol/lit; hypHcy) with varying frequencies in the four states from where samples were collected (n=1426). Polymorphisms of MTR and CBS do not affect hypHcy. 677T and 1298C alleles of MTHFR and G80 RFC-1 show association with hypHcy. In contrast, RFC-1 80AA is protective even in presence of 677T MTHFR. Addition of each mutant allele has an additive effect on Hcy level. Vitamin B12 (cofactor in methionine synthesis) clearly modulates the genotypic effect on Hcy level. Although frequency of individuals with low folic acid is ≈11, 49% of the population is vitamin B12 deficient (<220 pg/lit) and has a significant negative correlation with Hcy. Individuals with optimum vitamin B12 and folic acid generally have low Hcy, even in risk genotypes. CONCLUSIONS: One of the plausible reasons for susceptibility of individuals with MTHFR C677T in the studied population to various disorders is the high frequency of hyperhomocysteinemia and vitamin B12 deficiency in the 'healthy population'. Apparently, supplementation of vitamin B(12) to this health-impoverished community may help lessen the risk of several multifactorial disorders.

Effect of pistachio oil on gene expression of IFN-induced protein with tetratricopeptide repeats 2: a biomarker of inflammatory response.

When incorporated into the diet, pistachios have a beneficial effect on lipid and lipoprotein profiles. However, little is known about potential anti-inflammatory properties. This study was conducted to determine whether pistachio oil and an organic extract from pistachio oil extract (PE) regulated expression of inflammation-related genes. A mouse macrophage cell line (RAW 264.7 cells) was treated with pistachio oil and gene expression microarray analyses were performed. Pistachio oil significantly affected genes involved in immune response, defense response to bacteria, and gene silencing, of which INF-induced protein with tetratricopeptide repeats 2 (Ifit-2) was the most dramatically reduced. PE reduced the LPS-induced Ifit-2 by 78% and the bioactive molecules contained in PE, linoleic acid, and beta-sitosterol recapitulated this inhibition. Promoter analysis identified two adjacent IFN-stimulated response elements, which lie between -110 and -85bp of the 5'-flanking region of the Ifit-2 promoter, as being responsive to LPS activation and inhibition by PE. Our results indicate that pistachio oil and bioactive molecules present therein decrease Ifit-2 expressions, and due to the sensitivity of this effect, this gene is a potential biomarker for monitoring diet-induced changes in inflammation.

Microarray analysis of E9.5 reduced folate carrier (RFC1; Slc19a1) knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex.

BACKGROUND: The reduced folate carrier (RFC1) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Targeted inactivation of the murine RFC1 gene results in post-implantation embryolethality, but daily folic acid supplementation of pregnant dams prolongs survival of homozygous embryos until mid-gestation. At E10.5 RFC1-/- embryos are developmentally delayed relative to wildtype littermates, have multiple malformations, including neural tube defects, and die due to failure of chorioallantoic fusion. The mesoderm is sparse and disorganized, and there is a marked absence of erythrocytes in yolk sac blood islands. The identification of alterations in gene expression and signaling pathways involved in the observed dysmorphology following inactivation of RFC1-mediated folate transport are the focus of this investigation. RESULTS: Affymetrix microarray analysis of the relative gene expression profiles in whole E9.5 RFC1-/- vs. RFC1+/+ embryos identified 200 known genes that were differentially expressed. Major ontology groups included transcription factors (13.04%), and genes involved in transport functions (ion, lipid, carbohydrate) (11.37%). Genes that code for receptors, ligands and interacting proteins in the cubilin-megalin multiligand endocytic receptor complex accounted for 9.36% of the total, followed closely by several genes involved in hematopoiesis (8.03%). The most highly significant gene network identified by Ingenuitytrade mark Pathway analysis included 12 genes in the cubilin-megalin multiligand endocytic receptor complex. Altered expression of these genes was validated by quantitative RT-PCR, and immunohistochemical analysis demonstrated that megalin protein expression disappeared from the visceral yolk sac of RFC1-/- embryos, while cubilin protein was widely misexpressed. CONCLUSION: Inactivation of RFC1 impacts the expression of several ligands and interacting proteins in the cubilin-amnionless-megalin complex that are involved in the maternal-fetal transport of folate and other nutrients, lipids and morphogens such as sonic hedgehog (Shh) and retinoids that play critical roles in normal embryogenesis.