Reduced folate carrier gene is a risk factor for neural tube defects in a Chinese population

BACKGROUND: There is a considerable body of data demonstrating that periconceptional supplementation of folic acid can prevent a significant proportion of neural tube defects (NTDs). At present, the mechanism by which folic acid exerts its beneficial effect remains unknown. Folate transporter genes, including the reduced folate carrier gene (RFC1), have been proposed as NTD risk factors. METHODS: The study population included 104 nuclear families with NTDs and 100 nonmalformed control families. We investigated the possible association between a common RFC1 polymorphism (A80G) and NTD risk among offspring, as well as potential gene-environment interactions between the infant RFC1 genotype and maternal periconceptional use of folic acid through a population-based case-control study. RESULTS: We observed that the infants of the GG genotype were associated with a 2.56-fold increased risk of NTDs when compared to the AA genotype (odds ratio [OR], 2.56; 95% confidence interval [CI], 1.04-6.36) in our study population. Among mothers who did not utilize folic acid supplements, the risk for having a child with an NTD was 3.30 (95% CI, 1.15-9.65) for offspring with the GG genotype, compared to the reference (AA) genotype. Children who had the GG genotype and whose mothers did not take folic acid had an elevated risk for NTDs (OR, 8.80; 95% CI, 2.83-28.69), compared to offspring with the AA and GA genotypes whose mothers utilized folic acid supplements. CONCLUSIONS: Our findings suggest that the RFC1 G allele is likely to be an important genetic factor in determining folate transport and subsequently may be a risk factor for NTDs in this Chinese population.     

G80A reduced folate carrier SNP modulates cellular uptake of folate and affords protection against thrombosis via a non homocysteine related mechanism

Dietary folate is absorbed in the jejunum by the 'Reduced Folate Carrier' binding protein. This protein also sequesters extracellular folate for use by many cells in the body. As several biosynthetic pathways require folate for critical life processes, any change in the properties of this protein could lower folate bioavailability, cellular levels of the vitamin, and thus influence health. Since folate lowers thrombogenic homocysteine, we examined the prevalence of a common genetic polymorphism encoding the Reduced Folate Carrier (G80A RFC) to see if it acts as a risk factor for thrombotic vascular disease via an effect on homocysteine disposition in a cohort of 156 patients. The odds ratio indicates a significant protective effect of the mutant A allele against thrombosis: OR = 0.56(95% CI; 0.34-0.92). chi2; p = 0.022 (Yates corrected chi2; p = 0.031). The polymorphism had no impact on homocysteine, but did increase the level of extracellular to intracellular folate as might be predicted by the biological role of the expressed protein. This, and not homocysteine level, may be what affords protection against thrombosis.     

Effect of genetic polymorphisms involved in folate metabolism on the concentration of serum folate and plasma total homocysteine (p-tHcy) in healthy subjects after short-term folic acid supplementation: a randomized,double blind,crossover study

Data on the effect of combined genetic polymorphisms, involved in folate metabolism, on the concentration of serum folate after folic acid supplementation are scarce. Therefore, we investigated the impact of seven gene polymorphisms on the concentration of serum folate and p-tHcy in healthy subjects after short-term folic acid supplementation. In a randomized, double blind, crossover study, apparently healthy subjects were given either 0.8 mg folic acid per day (n = 46) or placebo (n = 45) for 14 days. The washout period was 14 days. Fasting blood samples were collected on day 1, 15, 30 and 45. Data on subjects on folic acid supplementation (n = 91) and on placebo (n = 45) were used for the statistical analysis. The concentration of serum folate increased higher in subjects with higher age (53.5 ± 7.0 years) than in subjects with lower age (24.3 ± 3.2 years) after folic acid supplementation (p = 0.006). The baseline concentration of serum folate in subjects with polymorphism combination, reduced folate carrier protein, RFC1-80 GA and methylenetetrahydrofolate reductase, MTHFR677 CT+TT, was lower than RFC1-80 AA and MTHFR677 CT+TT (p = 0.002). After folic acid supplementation, a higher increase in the concentration of serum folate was detected in subjects with polymorphism combination RFC1-80 GA and MTHFR677 CC than RFC1-80 GG and MTHFR CT+TT combination (p < 0.0001). The baseline concentration of plasma total homocysteine (p-tHcy) was altered by combined polymorphisms in genes associated with folate metabolism. After folic acid supplementation, in subjects with combined polymorphisms in methylenetetrahydrofolate dehydrogenase, MTHFD1-1958 and MTHFR-677 genes, the concentration of p-tHcy was changed (p = 0.002). The combination of RFC1-80 and MTHFR-677 polymorphisms had a profound affect on the concentration of serum folate in healthy subjects before and after folic acid supplementation.     

Additional food folate derived exclusively from natural sources improves folate status in young women with the MTHFR 677 CC or TT genotype

The effectiveness of additional food folate in improving folate status in humans is uncertain particularly in people with the common genetic variant (677 C-->T) in the methylenetetrahydrofolate reductase (MTHFR) gene. To examine the effect of a doubling of food folate consumption on folate status response variables, women (n=32; 18-46 years) with the MTHFR 677 CC or TT genotype consumed either 400 (n=15; 7 CC and 8 TT) or 800 (n=17; 8 CC and 9 TT) microg/day of dietary folate equivalents (DFE) derived exclusively from naturally occurring food folate for 12 weeks. A repeated measures two-factor ANOVA was used to examine the effect of the dietary treatment, the MTHFR C677T genotype and their interactions on serum folate, RBC folate and plasma total homocysteine (tHcy) during the last 3 weeks of the study. Consumption of 800 microg DFE/day resulted in serum folate concentrations that were 67% (P=.005) higher than consumption of 400 microg DFE/day (18.6+/-2.9 vs. 31.0+/-2.7 nmol/L, respectively) and RBC folate concentrations that were 33% (P=.001) higher (1172+/-75 vs. 1559+/-70 nmol/L, respectively). Serum folate (P=.065) and RBC folate (P=.022) concentrations were lower and plasma tHcy was higher (P=.039) in women with the MTHFR 677 TT genotype relative to the CC genotype. However, no genotype by dietary treatment interaction was detected. These data suggest that a doubling of food folate intake will lead to marked improvements in folate status in women with the MTHFR 677 CC or TT genotype.     

A80G polymorphism of reduced folate carrier 1 (<Emphasis Type="Italic">RFC1</Emphasis>) gene and head and neck squamous cell carcinoma etiology in Brazilian population

Reduced folate carrier is an essential folate transporter and the A80G polymorphism in reduced folate carrier 1 gene (rs1051266) has been shown to be associated with alterations in folate metabolism and consequently cancer development. We evaluated the association of this polymorphism with head and neck squamous cell carcinoma risk in a case–control study of 322 head and neck carcinoma patients and 531 individuals without cancer in a Brazilian population and association of this polymorphism with clinical histopathological parameters, and gender and lifestyle factors. The PCR-RFLP technique was used to genotype the polymorphism and multiple logistic regression was used for comparation between the groups and for interaction between the polymorphism and risk factors and clinical histopathological parameters. We observed association between the RFC1 A80G polymorphism and the risk of this disease. Male gender, tobacco habit and RFC1 AG or GG genotypes may be predictors of this disease (P < 0.05). The genotype, 80AG or GG was associated with for >50 years, male gender and non alcohol consumption (P ≤ 0.05). The polymorphism did not show any association with the primary site, aggressiveness, lymph node involvement or extension of the tumor. In conclusion tobacco and male gender are associated with etiology of this disease and our data provide evidence that supports an association between the RFC1 A80G polymorphism and head and neck squamous cell carcinoma risk, male gender, alcohol non consumption and age over 50 years. However, further studies of folate and plasma concentrations may contribute to the better understanding of the factors involved in the head and neck squamous cell carcinoma etiology.     

The maternal folate hydrolase gene polymorphism is associated with neural tube defects in a high-risk Chinese population

Folate hydrolase 1 (FOLH1) gene encodes intestinal folate hydrolase, which regulates intestinal absorption of dietary folate. Previous studies on the association between polymorphisms rs202676 and rs61886492 and the risk of neural tube defects (NTDs) were inconclusive. A case–control study of women with NTD-affected pregnancies (n = 160) and controls (n = 320) was conducted in the Chinese population of Lvliang, a high-risk area for NTDs. We genotyped the polymorphic sites rs202676 and rs61886492 and assessed maternal plasma folate and total homocysteine (tHcy). Our results showed that in case group, plasma folate concentrations were 18 % lower compared with those of control group (8.32 vs. 6.79 nmol/L, p = 0.033) and tHcy concentrations were 17 % higher (10.47 vs. 12.65 μmol/L, p = 0.047). Almost all samples had the rs61886492 GG genotype (99.78 %). The result showed that the frequency of GG genotype in rs202676 was significantly higher in group with multiple NTDs than in controls (p = 0.030, OR = 2.157, 95 % CI, 1.06–4.38). The multiple-NTD group showed higher maternal plasma concentrations of tHcy (10.47 vs. 13.96 μmol/L, p = 0.024). The GG genotype of rs202676 had a lower maternal folate and higher tHcy concentrations than other genotypes with no significant differences. The result of structural prediction indicated that this variation might change the spatial structure of the protein. These results suggested that the maternal polymorphism rs202676 was a potential risk factor for multiple NTDs in this Chinese population. The allele G might affect maternal plasma folate and tHcy concentration.     

Thymidylate synthase: a novel genetic determinant of plasma homocysteine and folate levels

The thymidylate synthase gene ( TYMS or TS) encodes a tightly regulated enzyme that catalyzes the conversion of deoxyuridylate to thymidylate, and contains a tandem repeat polymorphism that affects expression of the enzyme. We have investigated the relationship between TYMS genotype and plasma concentrations of homocysteine and folate in a cohort of 505 Chinese from Singapore. TYMS 3/3 genotype was associated with reduced plasma folate and, among individuals with low dietary folate intake, with elevated plasma homocysteine levels. These associations were independent of the well-established methylenetetrahydrofolate reductase ( MTHFR) C677T genotype effects on plasma folate and homocysteine levels. Our results suggest that TYMS and MTHFR compete for limiting supplies of folate required for the remethylation of homocysteine. These genetic determinants of plasma folate and homocysteine levels may be useful in identifying individuals at increased risk for cardiovascular disease.     

Red blood cell folate vitamer distribution in healthy subjects is determined by the methylenetetrahydrofolate reductase C677T polymorphism and by the total folate status

BACKGROUND: Red blood cells (RBCs) represent a storage pool for folate. In contrast to plasma, RBC folate can appear in different biochemical isoforms. So far, only the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype has been identified as a determinant of RBC folate vitamer distribution. OBJECTIVE: The purpose of this study is to identify clinical and biochemical determinants of RBC folate vitamer distribution in healthy subjects. DESIGN: In an observational study, 109 subjects, aged 18 to 65 years, were studied. Red blood cell folate vitamers were analyzed using a liquid chromatography-tandem mass spectrometry method. Other variables recorded included vitamin B(2), B(6) and B(12) status, homocysteine, plasma and RBC S-adenosylhomocysteine and S-adenosylmethionine, renal function and the MTHFR C677T polymorphism. RESULTS: The MTHFR C677T genotype was the dominant determinant of nonmethylfolate accumulation. The median (range) nonmethylfolate/total folate ratio was 0.58% (0-12.2%) in the MTHFR CC group (n=55), 0.99% (0-14.3%) in the CT group (n=39) and 30.3% (5.7-73.3%) in the TT genotype group (n=15), P<.001. The 95th percentile for the nonmethylfolate/total folate ratio was 2.8% for the CC group, 9.1% for the CT group and 73.3% for the TT group. In the CC and CT genotype subjects, the T-allele and total folate status were positively and independently correlated with nonmethylfolate accumulation, but the degree of nonmethylfolate accumulation in these subjects was usually minor compared with those with the TT genotype. None of the other studied variables was associated with nonmethylfolate accumulation. CONCLUSIONS: The MTHFR C677T genotype is the dominant determinant of nonmethylfolate accumulation in RBCs. In addition, high total folate status may contribute to minor to moderate nonmethylfolate accumulation in MTHFR CC and CT subjects.     

The reduced folate carrier-1 (RFC1 696T>C) polymorphism is associated with spontaneously aborted embryos in Koreans

The major cause of early spontaneous abortion is believed to be chromosomal abnormality. However, the genetic etiologies of spontaneous abortion are still unknown. A central feature of fetal development is widespread rapid cell division. Due to its role in DNA synthesis, the need for folate increases during periods of rapid fetal growth. Folate transport across cell membranes is mediated by reduced folate carrier-1 (RFC1). Variants within SLC19A1 may influence folate and homocysteine concentrations. The aim of this study was to investigate the association of RFC1 mutations with spontaneous abortion in aborted embryos. We studied 115 spontaneously aborted embryos at <20 weeks of gestational age, 102 child controls, and 353 adult controls. The genotype frequencies of RFC1 polymorphisms, 80A>G and 696T>C, in spontaneously aborted embryos were measured. The RFC1 696T>C mutation was significantly increased in spontaneously aborted embryos compared to child controls. Further studies will be required to examine the functional significance of the RFC1 696T>C polymorphism.     

Tetra primer ARMS-PCR relates folate/homocysteine pathway genes and ACE gene polymorphism with coronary artery disease

Cardiovascular disorders and coronary artery disease (CAD) are significant contributors to morbidity and mortality in heart patients. As genes of the folate/homocysteine pathway have been linked with the vascular disease, we investigated association of these gene polymorphisms with CAD/myocardial infarction (MI) using the novel approach of tetraprimer ARMS-PCR. A total of 230 participants (129 MI cases, 101 normal subjects) were recruited. We genotyped rs1801133 and rs1801131 SNPs in 5'10' methylenetetrahydrofolate reductase (MTHFR), rs1805087 SNP in 5' methyltetrahydrofolate homocysteine methyltransferase (MTR), rs662 SNP in paroxanse1 (PON1), and rs5742905 polymorphism in cystathionine beta synthase (CBS). Angiotensin converting enzyme (ACE) insertion/deletion polymorphism was detected through conventional PCR. Covariates included blood pressure, fasting blood sugar, serum cholesterol, and creatinine concentrations. Our results showed allele frequencies at rs1801133, rs1801131, rs1805087 and the ACE insertion/deletion (I/D) polymorphism varied between cases and controls. Logistic regression, after adjusting for covariates, demonstrated significant associations of rs1801133 and rs1805087 with CAD in the additive, dominant, and genotype model. In contrast, ACE I/D polymorphism was significantly related with CAD where recessive model was applied. Gene-gene interaction against the disease status revealed two polymorphism groups: rs1801133, rs662, and rs1805087; and rs1801131, rs662, and ACE I/D. Only the latter interaction maintained significance after adjusted for covariates. Our study concludes that folate pathway variants exert contributory influence on susceptibility to CAD. We further suggest that tetraprimer ARMS-PCR successfully resolves the genotypes in selected samples and might prove to be a superior technique compared to the conventional approach.     

The promoter region of the adiponectin gene is a determinant in modulating insulin sensitivity in childhood obesity

We investigated the association of the -11,391G>A, -11,377G>C, +45T>G, and +276G>T adiponectin single-nucleotide polymorphisms (SNPs) and expected haplotypes with the insulin resistance (IR) state in overweight/obese children; by using the haplotype background analysis, we also assessed the effect of each SNP independently. GG genotype at the -11,391 locus was associated with higher fasting insulin levels and homeostasis model assessment-IR index and lower adiponectin levels compared with GA + AA genotypes (p = 0.01, 0.002, and 0.03, respectively). Those heterozygous and homozygous for G allele at the -11,377 locus showed higher fasting glucose (p = 0.001 for both), fasting insulin (p = 0.001 for both), homeostasis model assessment-IR index (p < 0.001 for both), and triglyceride levels (p = 0.02 and 0.03, respectively) and lower adiponectin levels (p = 0.002 and 0.02, respectively) compared with C homozygotes. The +45G carriers showed higher fasting and 2-hour glucose levels (p = 0.01 for both) and lower adiponectin levels (p = 0.02) compared with non-carriers. Haplotype analysis suggested that, considering the same haplotypic background, each of the three polymorphisms exerted an independent effect on investigated parameters. The -11,391G>A, -11,377C>G, and +45T>G SNPs are associated with IR syndrome in overweight/obese children; they independently influence the investigated variables. The effect of +45T>G SNP seems to be marginal compared with the promoter SNPs. The GGT haplotype is associated with the highest degree of IR.     

Modulatory effect of plasma folate and polymorphisms in one-carbon metabolism on catecholamine methyltransferase (COMT) H108L associated oxidative DNA damage and breast cancer risk

The present study was aimed to investigate the modulatory role of plasma folate and eight putatively functional polymorphisms of one-carbon metabolism on catecholamine methyltransferase (COMT)-mediated oxidative DNA damage and breast cancer risk. Plasma folate and 8-oxo-2'-deoxyguanosine (8-oxodG) were estimated by commercially available kits, while polymorphisms were screened by PCR-RFLP and PCR-AFLP methods. COMT H108L polymorphism showed independent association with breast cancer (OR: 1.73, 95% CI: 1.31-2.30). No significant interaction was observed between folate status and COMT genotype. Multifactor dimensionality reduction (MDR) analysis gave evidence for the significant epistatic (gene-gene) interactions (p<0.0001) of COMT H108L with reduced folate carrier 1 (RFC1) G80A, thymidylate synthase (TYMS) 5'-UTR 3R2R, TYMS 3'-UTR ins6/de16. Increased plasma 8-oxodG were observed in cases compared to controls (mean +/- SE: 5.59 +/- 0.60 vs. 3.50 +/- 0.40 ng/ml, p<0.004). Plasma folate deficiency alone was not a significant predictor of 8-oxodG elevation. The genotype combinations namely, RFC1 G80A/methionine synthase reductase (MTRR) A66G, RFC1 G80A/SHMT C1420T/TYMS 3R2R and serine hydroxymethyltransferase (SHMT) C1420T/TYMS 3R2R/methionine synthase (MTR) A2756G/COMT H108L were strong predictors of 8-oxodG elevation in the order of risk. To conclude, the current study provides substantial evidence for a cross talk between one-carbon metabolism and COMT catalysis that might influence oxidative DNA damage and breast cancer risk.     

Association of IL8 -105G/A with Mastitis Somatic Cell Score in Chinese Holstein Dairy Cows

The single nucleotide polymorphisms (SNPs) in the 5′ upstream of bovine IL8 gene were investigated in 810 Chinese Holstein cows from 35 bull families in a dairy farm in Shanghai using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique. The Real-time PCR and Western blot were used to detect the mRNA and protein levels of genotype Chinese Holstein dairy cows. The results showed that one SNP -105G>A was detected, designating three genotypes (GG, GA and AA) with respective frequencies of 0.38, 0.46, and 0.16. The significant association of the SNP -105G>A with somatic cell score (SCS) was identified. Genotype GG had a significantly lower SCS than genotype GA or AA (P < 0.01), and the relative mRNA expression and protein level of GG was found to be the highest. These results suggest that the genotype GG may be a useful genetic marker for mastitis resistance selection and breeding in Chinese Holstein dairy cows.     

Epigenetic alterations in folate transport genes in placental tissue from fetuses with neural tube defects and in leukocytes from subjects with hyperhomocysteinemia

The objectives of this study were to identify tissue-specific differentially methylated regions (T-DMR’s) in the folate transport genes in placental tissue compared with leukocytes, and from placental tissues obtained from normal infants or with neural tube defects (NTDs). Using pyrosequencing, we developed methylation assays for the CpG islands (CGIs) and the CGI shore regions of the folate receptor α (FOLR1), proton-coupled folate transporter (PCFT) and reduced folate carrier 1 (RFC1) genes. The T-DMRs differed in location for each gene and the difference in methylation ranged between 2 and 54%. A higher T-DMR methylated fraction was associated with a lower mRNA level of the FOLR1 and RFC1 genes. Methylation fractions differed according to RFC1 80G > A genotype in the NTD cases and in leukocytes from subjects with high total plasma homocysteine (tHcy). There were no differences in methylated fraction of folate transporter genes between NTD cases and controls. We suggest that T-DMRs participate in the regulation of expression of the FOLR1 and RFC1 genes, that the RFC1 80G > A polymorphism exerts a gene-nutrition interaction on DNA methylation in the RFC1 gene, and that this interaction appears to be most prominent in NTD-affected births and in subjects with high tHcy concentrations.     

Reduced levels of folate transporters (PCFT and RFC) in membrane lipid rafts result in colonic folate malabsorption in chronic alcoholism

We studied the effect of chronic ethanol ingestion on folate transport across the colonic apical membranes (CAM) in rats. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20%) solution orally for 3 months and folate transport was studied in the isolated colon apical membrane vesicles. The folate transport was found to be carrier mediated, saturable, with pH optima at 5.0. Chronic ethanol ingestion reduced the folate transport across the CAM by decreasing the affinity of transporters (high K_m) for the substrate and by decreasing the number of transporter molecules (low V_max) on the colon luminal surface. The decreased transport activity at the CAM was associated with down‐regulation of the proton‐coupled folate transporter (PCFT) and the reduced folate carrier (RFC) which resulted in decreased PCFT and RFC protein levels in the colon of rats fed alcohol chronically. Moreover, the PCFT and the RFC were found to be distributed in detergent insoluble fraction of the CAM in rats. Floatation experiments on Optiprep density gradients demonstrated the association of the PCFT and the RFC protein with lipid rafts (LR). Chronic alcoholism decreased the PCFT and the RFC protein levels in the CAM LR in accordance with the decreased synthesis. Hence, we propose that downregulation in the expression of the PCFT and the RFC in colon results in reduced levels of these transporters in colon apical membrane LR as a mechanism of folate malabsorption during chronic alcoholism. J. Cell. Physiol. 226: 579–587, 2011. © 2010 Wiley‐Liss, Inc.     

脂联素基因单核苷酸多态性T45G与湖北汉族人2型糖尿病的遗传关联研究

采用病例.家系对照和随机病例.对照两种设计,分析了603例样本脂联素基因(Adiponectin,APMl)单核苷酸多态性(SNP)rs13061862(T45G)与湖北汉族人群2型糖尿病的相关性.在所有样本中,2型糖尿病病人的G等位基因及GG基因型频率显著高于正常人(G:42.0%比21.7%,P<0.001;GG:13.6%比4.5%,P=0.032);在180个病例.家系对照中,2型糖尿病患者的GG基因型频率显著高于对照组(GG:17.8%比5.6%,P=0.011);在423个随机病例.对照中,2型糖尿病患者GG基因型频率也显著高于对照组(GG:12.2%比3.9%,P=0.025);单因素Logistic回归分析显示,GG基因型是2型糖尿病的危险因子(OR=3.58,95%C/=1.70-7.54).这些结果表明,脂联素基因SNPT45G多态性与湖北汉族人群2型糖尿病的发生发展相关,GG基因型是中国湖北汉族人2型糖尿病的遗传危险因素.     

Effect of folate deficiency on placental DNA methylation in hyperhomocysteinemic rats

We report that the maternal folate status can influence folate-mediated one-carbon metabolism and DNA methylation in the placenta. Thirty-six female Sprague-Dawley rats were divided into the following three dietary groups: folate-supplemented (FS; 8 mg/kg folic acid, n=12), homocystine- and folate-supplemented (HFS; 0.3% homocystine and 8 mg/kg folic acid, n=12) and homocystine-supplemented and folate-deficient (HFD; 0.3% homocystine and no folic acid, n=12). The animals were fed their experimental diets from 4 weeks prior to mating until Day 20 of pregnancy (n=7-9 per group). The HFS diet increased the plasma homocysteine and placental DNA methylation but did not affect plasma folate, vitamin B-12, S-adenosyl methionine (SAM) or S-adenosyl homocysteine (SAH) levels, or the SAM/SAH ratio in the liver and placenta compared with the FS diet. The HFD diet induced severely low plasma folate concentrations, with plasma homocysteine levels increasing up to 100 micromol/L, and increased hepatic SAH and decreased placental SAM levels and SAM/SAH ratio in both tissues, with a concomitant decrease in placental DNA methylation. Placental DNA methylation was significantly correlated with placental (gamma=0.819), hepatic (gamma=0.7) and plasma (gamma=0.752) folate levels; plasma homocysteine level (gamma=-0.688); hepatic SAH level (gamma=-0.662) and hepatic SAM/SAH ratio (gamma=0.494). These results suggest that the maternal folate status in hyperhomocysteinemic rats influences the homeostasis of folate-mediated one-carbon metabolism and the methyl pool, which would, in turn, affect placental DNA methylation by altering the methylation potential of the liver.     

Use of a novel genetic mouse model to investigate the role of folate in colitis-associated colon cancer

Inflammatory bowel disease (IBD) patients are at high risk for developing folate deficiency and colon cancer. Since it is difficult to study the subtle global and gene-specific epigenetic mechanisms involved in folate-mediated tumor initiation and promotion, we have generated genetically modified mouse models by targeting the reduced folate carrier (RFC1) and folate-binding protein (Folbp1) genes. The transgenic mice were fed semi-purified diets for 8 weeks containing either normal (2 mg) or deficient (0.1 mg folate/kg diet) levels of folate. Compound heterozygous mice (Folbp1(+/-); RFC1(+/-)) fed an adequate folate diet exhibited a reduction in plasma folate concentrations compared to heterozygous (Folbp1(+/-)) and littermate wild-type mice (P<.05). In contrast, no differences were observed in colonic mucosa. Consumption of a low folate diet significantly reduced (three- to fourfold) plasma and tissue folate levels in all animal models, although plasma homocysteine levels were not altered. In order to elucidate the relationship between folate status and inflammation-associated colon cancer, animals were injected with azoxymethane followed by dextran sodium sulphate treatment in the drinking water. Mice were fed a normal folate diet and were terminated 5 weeks after carcinogen injection. The number of high multiplicity aberrant crypt foci per centimeter of colon was significantly elevated (P<.05) in compound Folbp1(+/-); RFC1(+/-) (3.5+/-0.4) mice as compared to Folbp1(+/-) (1.9+/-0.3) and wild-type control mice (1.1+/-0.1). These data demonstrate that the ablation of two receptor/carrier-mediated pathways for folate transport increases the risk for developing inflammation-associated colon cancer.     

GNMT expression increases hepatic folate contents and folate-dependent methionine synthase-mediated homocysteine remethylation

Glycine N-methyltransferase (GNMT) is a major hepatic enzyme that converts S-adenosylmethionine to S-adenosylhomocysteine while generating sarcosine from glycine, hence it can regulate mediating methyl group availability in mammalian cells. GNMT is also a major hepatic folate binding protein that binds to, and, subsequently, may be inhibited by 5-methyltetrafolate. GNMT is commonly diminished in human hepatoma; yet its role in cellular folate metabolism, in tumorigenesis and antifolate therapies, is not understood completely. In the present study, we investigated the impacts of GNMT expression on cell growth, folate status, methylfolate-dependent reactions and antifolate cytotoxicity. GNMT-diminished hepatoma cell lines transfected with GNMT were cultured under folate abundance or restriction. Folate-dependent homocysteine remethylation fluxes were investigated using stable isotopic tracers and gas chromatography/mass spectrometry. Folate status was compared between wild-type (WT), GNMT transgenic (GNMT(tg)) and GNMT knockout (GNMT(ko)) mice. In the cell model, GNMT expression increased folate concentration, induced folate-dependent homocysteine remethylation, and reduced antifolate methotrexate cytotoxicity. In the mouse models, GNMT(tg) had increased hepatic folate significantly, whereas GNMT(ko) had reduced folate. Liver folate levels correlated well with GNMT expressions (r = 0.53, P = 0.002); and methionine synthase expression was reduced significantly in GNMT(ko), demonstrating impaired methylfolate-dependent metabolism by GNMT deletion. In conclusion, we demonstrated novel findings that restoring GNMT assists methylfolate-dependent reactions and ameliorates the consequences of folate depletion. GNMT expression in vivo improves folate retention and bioavailability in the liver. Studies on how GNMT expression impacts the distribution of different folate cofactors and the regulation of specific folate dependent reactions are underway.