Improving maternal folate status to prevent infant neural tube defects: working group conclusions and a framework for action.

As infectious disease control programs achieve increasing success, further reductions in child mortality in low- and middle-income countries (LMICs) will require focused prevention strategies for birth defects and other noninfectious diseases. Neural tube defects (NTDs) can cause early death or lifelong disability. Preventing NTDs provides a feasible, significant opportunity to decrease the toll of birth defects and contribute to further reducing child mortality globally. The Micronutrient Forum convened a technical consultation on Folate Status in Women and Neural Tube Defects Prevention to develop a roadmap to inform and prioritize investments in NTD prevention in LMICs; help guide implementation efforts in terms of the feasibility of interventions and the potential for acceleration; and identify research and knowledge gaps. Here, we describe the impetus for and approach to the consultation and present the conclusions and a framework for developing a roadmap for action to accelerate NTD prevention in LMICs. The framework (1) provides options for action on folate status assessment; (2) outlines a way forward to develop and implement a time-bound global action plan for NTD prevention; and (3) identifies common impediments to NTD prevention, broad strategies to overcome or minimize these impediments, and basic building blocks necessary to accelerate action.

A public health approach for preventing neural tube defects: folic acid fortification and beyond.

In this paper we review the evidence basis for prevention of folic acid-sensitive neural tube defects (NTDs) through public health interventions in women of reproductive age (WRA), the proven vehicles for delivery of folic acid, and what is needed to effectively scale these, and provide a snapshot of potential innovations that require future research. Our primary focus is on the global situation affecting large-scale food fortification (LSFF) with folic acid, in particular the fortification of wheat flour and maize meal. Our overarching conclusion is that folic acid fortification is an evidence-based intervention that reduces the prevalence of NTDs, and that LSFF with folic acid is underutilized. Thus, food fortification with folic acid should be a component of most national public health strategies, in particular where folate status is insufficient and a fortifiable food vehicle, processed by a centralized industry, is consumed regularly by WRA. The evidence shows that there is still much work needed (1) to build the enabling environment and expand programs where there is currently no legislation, (2) to improve the low quality of delivery of existing programs, and (3) to measure and sustain programs by generating new coverage data and demonstrating evidence of impact in low- and middle-income countries.

Distinctions in gene-specific changes in DNA methylation in response to folic acid supplementation between women with normal weight and obesity.

BACKGROUND/OBJECTIVES: Obesity and maternal folate deficiency are associated with increased risk for neural tube defects (NTDs). Limited knowledge exists on the impact of folate status or obesity on DNA methylation of genes related to NTD risk and folate metabolism. SUBJECTS/METHODS: Women (18-35y) with normal weight (NW; BMI 18.5-24.9kg/m2; n=12) and obesity (OB; BMI >30kg/m2; n=6) were provided FA (800µg/d) for 8-weeks. Serum folate concentration and changes in DNA methylation across 2098 CpG sites in 91 genes related to NTD risk and folate metabolism were examined. RESULTS: Serum folate concentration increased in both groups following FA supplementation, but OB maintained a relative lower concentration (NW; 38.36±2.50-71.41±3.02nmol/L and OB; 27.12±3.09-56.85±3.90nmol/L). Methylation of 56 and 99 CpG sites changed in response to supplementation in NW and OB, respectively, and majority of these sites decreased in methylation in both groups. Only 4 CpG sites responded to supplementation in both groups. Gene ontology analysis revealed a response to supplementation in 61 biological processes (BPs) from the selected genes. Five of the 61 BPs were identified only in NW, including neural tube closure, while 13 of the 61 BPs were enriched only in OB, including folate metabolism, vitamin B12 metabolism and methylation related processes. CONCLUSIONS: Changes in DNA methylation in genes related to NTD risk and folate metabolism in response to FA supplementation were different in NW and OB. Increased NTD risk and abnormal folate metabolism in obesity may be due to a distinctive epigenetic response to folate status in these genes.

DNA Methylation Changes in Whole Blood and CD16+ Neutrophils in Response to Chronic Folic Acid Supplementation in Women of Childbearing Age.

Folate, a water-soluble vitamin, is a key source of one-carbon groups for DNA methylation, but studies of the DNA methylation response to supplemental folic acid yield inconsistent results. These studies are commonly conducted using whole blood, which contains a mixed population of white blood cells that have been shown to confound results. The objective of this study was to determine if CD16+ neutrophils may provide more specific data than whole blood for identifying DNA methylation response to chronic folic acid supplementation. The study was performed in normal weight (BMI 18.5 - 24.9 kg/m2) women (18 - 35 y; n = 12), with blood samples taken before and after 8 weeks of folic acid supplementation at 800 µg/day. DNA methylation patterns from whole blood and isolated CD16+ neutrophils were measured across >485,000 CpG sites throughout the genome using the Infinium HumanMethylation450 BeadChip. Over the course of the 8-week supplementation, 6746 and 7513 CpG sites changed (p < 0.05) in whole blood and CD16+ neutrophils, respectively. DNA methylation decreased in 68.4% (whole blood) and 71.8% (CD16+ neutrophils) of these sites. There were only 182 CpG sites that changed in both the whole blood and CD16+ neutrophils, 139 of which changed in the same direction. These results suggest that the genome-wide DNA methylation response to chronic folic acid supplementation is different between whole blood and CD16+ neutrophils and that a single white blood cell type may function as a more specific epigenetic reporter of folate status than whole blood.

Biomarkers of Nutrition for Development-Folate Review.

The Biomarkers of Nutrition for Development (BOND) project is designed to provide evidence-based advice to anyone with an interest in the role of nutrition in health. Specifically, the BOND program provides state-of-the-art information and service with regard to selection, use, and interpretation of biomarkers of nutrient exposure, status, function, and effect. To accomplish this objective, expert panels are recruited to evaluate the literature and to draft comprehensive reports on the current state of the art with regard to specific nutrient biology and available biomarkers for assessing nutrients in body tissues at the individual and population level. Phase I of the BOND project includes the evaluation of biomarkers for 6 nutrients: iodine, iron, zinc, folate, vitamin A, and vitamin B-12. This review represents the second in the series of reviews and covers all relevant aspects of folate biology and biomarkers. The article is organized to provide the reader with a full appreciation of folate's history as a public health issue, its biology, and an overview of available biomarkers (serum folate, RBC folate, and plasma homocysteine concentrations) and their interpretation across a range of clinical and population-based uses. The article also includes a list of priority research needs for advancing the area of folate biomarkers related to nutritional health status and development.

Impact of folic acid fortification on global DNA methylation and one-carbon biomarkers in the Women's Health Initiative Observational Study cohort.

DNA methylation is an epigenetic mechanism that regulates gene expression and can be modified by one-carbon nutrients. The objective of this study was to investigate the impact of folic acid (FA) fortification of the US food supply on leukocyte global DNA methylation and the relationship between DNA methylation, red blood cell (RBC) folate, and other one-carbon biomarkers among postmenopausal women enrolled in the Women's Health Initiative Observational Study. We selected 408 women from the highest and lowest tertiles of RBC folate distribution matching on age and timing of the baseline blood draw, which spanned the pre- (1994-1995), peri- (1996-1997), or post-fortification (1998) periods. Global DNA methylation was assessed by liquid chromatography-tandem mass spectrometry and expressed as a percentage of total cytosine. We observed an interaction (P = 0.02) between fortification period and RBC folate in relation to DNA methylation. Women with higher (vs. lower) RBC folate had higher mean DNA methylation (5.12 vs. 4.99%; P = 0.05) in the pre-fortification period, but lower (4.95 vs. 5.16%; P = 0.03) DNA methylation in the post-fortification period. We also observed significant correlations between one-carbon biomarkers and DNA methylation in the pre-fortification period, but not in the peri- or post-fortification period. The correlation between plasma homocysteine and DNA methylation was reversed from an inverse relationship during the pre-fortification period to a positive relationship during the post-fortification period. Our data suggest that (1) during FA fortification, higher RBC folate status is associated with a reduction in leukocyte global DNA methylation among postmenopausal women and; (2) the relationship between one-carbon biomarkers and global DNA methylation is dependent on folate availability.

Does obesity modify the association of supplemental folic acid with folate status among nonpregnant women of childbearing age in the United States?

BACKGROUND: Obesity is associated with an increased risk of having a pregnancy affected by a neural tube defect (NTD). It is not clear whether the amount of folic acid required by obese women to protect against NTDs is the same as that for nonobese women. METHODS: We analyzed data from the National Health and Nutrition Examination Survey, representative of the noninstitutionalized civilian U.S. population, to assess whether body mass index (BMI; normal weight, overweight, and obese categories) modified the association between supplemental folic acid intake and folate status. We estimated the geometric mean concentration among nonpregnant women of childbearing age (15-44 years) during the postfortification period of: serum folate (2003-2008); red blood cell (RBC) folate (2007-2008); and plasma total homocysteine (tHcy; 2003-2006), adjusted for age, race and ethnicity, and total dietary folate expressed as dietary folate equivalents for strata of supplement use and BMI. RESULTS: BMI was inversely associated with serum folate among women who did not use supplements containing folic acid; no differences between women in different BMI categories were observed among supplement users. Regardless of supplement use, obese women had the highest RBC folate concentrations. There were no differences in tHcy by BMI, regardless of supplement use. CONCLUSIONS: These results do not support a straightforward modification of the relationship between supplemental folic acid intake and folate status by BMI. In this population, BMI may affect the body distribution of folate, as reflected by lower serum and higher RBC folate levels in obese women who do not use supplements.

Genomic DNA methylation changes in response to folic acid supplementation in a population-based intervention study among women of reproductive age.

Folate is a source of one-carbons necessary for DNA methylation, a critical epigenetic modification necessary for genomic structure and function. The use of supplemental folic acid is widespread however; the potential influence on DNA methylation is unclear. We measured global DNA methylation using DNA extracted from samples from a population-based, double-blind randomized trial of folic acid supplementation (100, 400, 4000 µg per day) taken for 6 months; including a 3 month post-supplementation sample. We observed no changes in global DNA methylation in response to up to 4,000 µg/day for 6 months supplementation in DNA extracted from uncoagulated blood (approximates circulating blood). However, when DNA methylation was determined in coagulated samples from the same individuals at the same time, significant time, dose, and MTHFR genotype-dependent changes were observed. The baseline level of DNA methylation was the same for uncoagulated and coagulated samples; marked differences between sample types were observed only after intervention. In DNA from coagulated blood, DNA methylation decreased (-14%; P<0.001) after 1 month of supplementation and 3 months after supplement withdrawal, methylation decreased an additional 23% (P<0.001) with significant variation among individuals (max+17%; min-94%). Decreases in methylation of ≥25% (vs. <25%) after discontinuation of supplementation were strongly associated with genotype: MTHFR CC vs. TT (adjusted odds ratio [aOR] 12.9, 95%CI 6.4, 26.0). The unexpected difference in DNA methylation between DNA extracted from coagulated and uncoagulated samples in response to folic acid supplementation is an important finding for evaluating use of folic acid and investigating the potential effects of folic acid supplementation on coagulation.

MTHFR 677C->T genotype is associated with folate and homocysteine concentrations in a large, population-based, double-blind trial of folic acid supplementation.

BACKGROUND: The methylenetetrahydrofolate reductase (MTHFR) genotype is associated with modification of disease and risk of neural tube defects. Plasma and red blood cell (RBC) folate and plasma homocysteine concentrations change in response to daily intakes of folic acid supplements, but no large-scale or population-based randomized trials have examined whether the MTHFR genotype modifies the observed response. OBJECTIVE: We sought to determine whether the MTHFR 677C→T genotype modifies the response to folic acid supplementation during and 3 mo after discontinuation of supplementation. DESIGN: Northern Chinese women of childbearing age were enrolled in a 6-mo supplementation trial of different folic acid doses: 100, 400, and 4000 µg/d and 4000 µg/wk. Plasma and RBC folate and plasma homocysteine concentrations were measured at baseline; after 1, 3, and 6 mo of supplementation; and 3 mo after discontinuation of supplementation. MTHFR genotyping was performed to identify a C→T mutation at position 677 (n = 932). RESULTS: Plasma and RBC folate and homocysteine concentrations were associated with MTHFR genotype throughout the supplementation trial, regardless of folic acid dose. MTHFR TT was associated with lower folate concentrations, and the trend of TT < CC was maintained at even the highest doses. Folic acid doses of 100 µg/d or 4000 µg/wk did not reduce high homocysteine concentrations in those with the MTHFR TT genotype. CONCLUSION: MTHFR genotype was an independent predictor of plasma and RBC folate and plasma homocysteine concentrations and did not have a significant interaction with folic acid dose during supplementation. This trial was registered at clinicaltrials.gov as NCT00207558.

Micronutrients and women of reproductive potential: required dietary intake and consequences of dietary deficiency or excess. Part II--vitamin D, vitamin A, iron, zinc, iodine, essential fatty acids.

Part II of this review considers additional micronutrients. Vitamin D is a fat soluble vitamin found in foods of animal origins (fatty fish, liver oil) or fortified products (milk, cheese). Vitamin D deficiency is common in African-American women living in northern latitudes. Vitamin D supplementation may be needed to reach desired 25-(OH)D3 concentrations of >50 nmol/L. In foods of animal origin, preformed Vitamin A is present; in plants (fruits and vegetables) vitamin A precursors (ß-carotenoids) are present. Vitamin A supplementation is usually not warranted, and in developing countries should not exceed 3000 µg (10,000 IU)/day. Iron in the form of haem-iron is found in meat, fish and poultry; non-haem (inorganic) iron is found in vegetables, fruits and grains. Iron supplementation may be necessary in the third trimester, earlier in pregnancy or in non-pregnant states if serum ferritin is <20 µg/L or haemoglobin <10.9 g/dL. Zinc is available in red meat, seafood including oysters and unpolished grains; supplementation is not necessary. To assure adequate iodine, food is fortified worldwide with iodated salt. If urinary iodine levels are low, supplementation is needed. Essential fatty acids requirements can be met by one to two portions of fish per week.

Contributions of enriched cereal-grain products, ready-to-eat cereals, and supplements to folic acid and vitamin B-12 usual intake and folate and vitamin B-12 status in US children: National Health and Nutrition Examination Survey (NHANES), 2003-2006.

BACKGROUND: US children consume folic acid from multiple sources. These sources may contribute differently to usual intakes above the age-specific tolerable upper intake level (UL) for folic acid and to folate and vitamin B-12 status. OBJECTIVE: We estimated usual daily folic acid intakes above the UL and adjusted serum and red blood cell folate, serum vitamin B-12, homocysteine, and methylmalonic acid (MMA) concentrations in US children by age group and by the following 3 major folic acid intake sources: enriched cereal-grain products (ECGP), ready-to-eat cereals (RTE), and supplements containing folic acid (SUP). DESIGN: We analyzed data in 4 groups of children aged 1-3, 4-8, 9-13, and 14-18 y from the National Health and Nutrition Examination Survey (NHANES), 2003-2006 (n = 7161). RESULTS: A total of 19-48% of children consumed folic acid from ECGP only. Intakes above the UL varied from 0-0.1% of children who consumed ECGP only to 15-78% of children who consumed ECGP+RTE+SUP. In children aged 1-8 y, 99-100% of those who consumed ≥ 200 µg folic acid/d from supplements exceeded their UL. Although < 0.5% of children had folate deficiency or low vitamin B-12 status, the consumption of RTE or SUP with folic acid was associated with higher mean folate and vitamin B-12 concentrations and, in some older children, with lower homocysteine and MMA concentrations. CONCLUSIONS: Our data suggest that the majority of US children consume more than one source of folic acid. Postfortification, the consumption of RTE or SUP increases usual daily intakes and blood concentrations of folate and vitamin B-12.

Folic acid food fortification-its history, effect, concerns, and future directions.

Periconceptional intake of folic acid is known to reduce a woman's risk of having an infant affected by a neural tube birth defect (NTD). National programs to mandate fortification of food with folic acid have reduced the prevalence of NTDs worldwide. Uncertainty surrounding possible unintended consequences has led to concerns about higher folic acid intake and food fortification programs. This uncertainty emphasizes the need to continually monitor fortification programs for accurate measures of their effect and the ability to address concerns as they arise. This review highlights the history, effect, concerns, and future directions of folic acid food fortification programs.

Micronutrients and women of reproductive potential: required dietary intake and consequences of dietary deficiency or excess. Part I--Folate, Vitamin B12, Vitamin B6.

This two-part review highlights micronutrients for which either public health policy has been established or for which new evidence provides guidance as to recommended intakes during pregnancy. One pivotal micronutrient is folate, the generic name for different forms of a water-soluble vitamin essential for the synthesis of thymidylate and purines and, hence, DNA. For non-pregnant adult women the recommended intake is 400 µg/day dietary folate equivalent. For women capable of becoming pregnant an additional 400 µg/day of synthetic folic acid from supplements or fortified foods is recommended to reduce the risk of neural tube defects (NTD). The average amount of folic acid received through food fortification (grains) in the US is only 128 µg/day, emphasising the need for the supplemental vitamin for women of reproductive age. Vitamin B12 (cobalamin) is a cofactor required for enzyme reactions, including generation of methionine and tetrahydrofolate. B12 is found almost exclusively in foods of animal origin (meats, dairy products); therefore, vegetarians are at greatest risk for dietary vitamin B12 deficiency and should be supplemented. Vitamin B6 is required for many reactions, primarily in amino acid metabolism. Meat, fish and poultry are good dietary sources. Supplementation beyond routine prenatal vitamins is not recommended.

Folic acid source, usual intake, and folate and vitamin B-12 status in US adults: National Health and Nutrition Examination Survey (NHANES) 2003-2006.

BACKGROUND: US adults have access to multiple sources of folic acid. The contribution of these sources to usual intakes above the tolerable upper intake level (UL) (1000 microg/d) and to folate and vitamin B-12 status is unknown. OBJECTIVE: The objective was to estimate usual folic acid intake above the UL and adjusted serum and red blood cell folate, vitamin B-12, methylmalonic acid, and homocysteine concentrations among US adults by 3 major folic acid intake sources-enriched cereal-grain products (ECGP), ready-to-eat cereals (RTE), and supplements (SUP)-categorized into 4 mutually exclusive consumption groups. DESIGN: We used data from the National Health and Nutrition Examination Survey (NHANES) 2003-2006 (n = 8258). RESULTS: Overall, 2.7% (95% CI: 1.9%, 3.5%) of adults consumed more than the UL of folic acid. The proportions of those who consumed folic acid from ECGP only, ECGP+RTE, ECGP+SUP, and ECGP+RTE+SUP were 42%, 18%, 25%, and 15%, respectively. Of 60% of adults who did not consume supplements containing folic acid (ECGP only and ECGP+RTE), 0% had intakes that exceeded the UL. Of 34% and 6% of adults who consumed supplements with an average of < or = 400 and >400 microg folic acid/d, <1% and 47.8% (95% CI: 39.6%, 56.0%), respectively, had intakes that exceeded the UL. Consumption of RTE and/or supplements with folic acid was associated with higher folate and vitamin B-12 and lower homocysteine concentrations, and consumption of supplements with vitamin B-12 was associated with lower methylmalonic acid concentrations (P < 0.001). CONCLUSION: At current fortification levels, US adults who do not consume supplements or who consume an average of < or =400 microg folic acid/d from supplements are unlikely to exceed the UL in intake for folic acid.

Folate status and homocysteine response to folic acid doses and withdrawal among young Chinese women in a large-scale randomized double-blind trial.

BACKGROUND: There are no large randomized trials of the effect of folic acid dosing regimens on blood folate and homocysteine concentrations. OBJECTIVE: We aimed to evaluate the changes in folate and homocysteine concentrations in response to different folic acid doses and to withdrawal in young women not exposed to other sources of folic acid. DESIGN: Women (n = 1108) were randomly assigned to 1 of 6 intervention groups for which daily intakes of folic acid for 6 mo were 100 microg 1 time/d, 25 microg 4 times/d, 400 microg 1 time/d, 100 microg 4 times/d, 4000 microg 1 time/d, or 4000 microg 1 time/wk. Plasma and red blood cell folate and homocysteine concentrations were measured at baseline; at 1, 3, and 6 mo; and 3 mo after the discontinuation of folic acid. RESULTS: Folate and homocysteine concentrations were not different at baseline between the groups who had the same daily intake of folic acid as a single dose or multiple doses (P = 0.058). Plasma folate concentrations plateaued at 3 mo with 108% (95% CI: 97.7%, 120%), 259% (95% CI: 240%, 279%), 460% (95% CI: 417%, 503%), and 142% (95% CI: 123%, 162%) observed increases for the folic acid groups receiving 100, 400, and 4000 microg/d and 4000 microg/wk, respectively. The rate of reduction in folate concentrations during the 3 mo after cessation of folic acid was dose-dependent-higher intakes were associated with faster reductions. CONCLUSIONS: Changes in folate and homocysteine concentrations were unaffected by different dosing schedules. After folic acid cessation, blood folate declined rapidly, which indicated that the intervention-enhanced folate status was rapidly diminished.

Transcobalamin 776C->G polymorphism negatively affects vitamin B-12 metabolism.

BACKGROUND: A common genetic polymorphism [transcobalamin (TC) 776C-->G] may affect the function of transcobalamin, the protein required for vitamin B-12 cellular uptake and metabolism. Remethylation of homocysteine is dependent on the production of 5-methyltetrahydrofolate and adequate vitamin B-12 for the methionine synthase reaction. OBJECTIVES: The objectives were to assess the influence of the TC 776C--> G polymorphism on concentrations of the transcobalamin-vitamin B-12 complex (holo-TC) and to determine the combined effects of the TC 776C-->G and methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphisms and vitamin B-12 status on homocysteine concentrations. DESIGN: Healthy, nonpregnant women (n = 359; aged 20-30 y) were screened to determine plasma vitamin B-12, serum holo-TC, and plasma homocysteine concentrations and TC 776C-->G and MTHFR 677C-->T genotypes. RESULTS: The serum holo-TC concentration for women with the variant TC 776 GG genotype was significantly different (P = 0.0213) from that for subjects with the CC genotype (74 +/- 37 and 87 +/- 33 pmol/L, respectively). An inverse relation was observed between plasma homocysteine concentrations and both serum holo-TC (P G polymorphism negatively affects the serum holo-TC concentration and provide additional evidence that vitamin B-12 status modulates the homocysteine concentration in this population.

Folic acid supplementation and the occurrence of congenital heart defects, orofacial clefts, multiple births, and miscarriage.

Key research findings relative to the question of whether maternal use of folic acid before and during pregnancy reduces the chance that offspring will be born with a congenital heart defect or an orofacial cleft are reviewed in this paper. Observational studies in general support an association between maternal use of multivitamins containing folic acid and a reduction in the occurrence of congenital heart defects and orofacial clefts. Results from one randomized controlled trial (RCT) provide the strongest evidence that multivitamins prevent congenital heart defects, but this RCT did not provide evidence that multivitamins prevent orofacial clefts. In addition, most observational and interventional studies are not designed to detect an independent effect from folic acid. Early studies suggested that periconceptional multivitamin use was associated with an increased occurrence of both miscarriages and multiple births, which has resulted in a great deal of controversy about the safety of folic acid use during pregnancy. We also review reports that were designed to answer these questions with more definitive data. When more substantial evidence about the effect of periconceptional folic acid on the occurrence of congenital heart defects and orofacial clefts is reported, we will have additional support for promoting folic acid intervention programs. All women capable of becoming pregnant should continue to consume 400 mug/d of folic acid in addition to a healthy diet as advised.

Methionine synthase reductase 66A->G polymorphism is associated with increased plasma homocysteine concentration when combined with the homozygous methylenetetrahydrofolate reductase 677C->T variant.

Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) are important for homocysteine remethylation. This study was designed to determine the influence of genetic variants (MTHFR 677C-->T, MTHFR 1298A-->C, and MTRR 66A-->G), folate, and vitamin B-12 status on plasma homocysteine in women (20-30 y; n = 362). Plasma homocysteine was inversely (P < 0.0001) associated with serum folate and plasma vitamin B-12 regardless of genotype. Plasma homocysteine was higher (P < 0.05) for women with the MTHFR 677 TT/1298 AA genotype combination compared with the CC/AA, CC/AC, and CT/AA genotypes. Women with the MTHFR 677 TT/MTRR 66 AG genotype had higher (P < 0.05) plasma homocysteine than all other genotype combinations except the TT/AA and TT/GG genotypes. There were 5.4-, 4.3-, and 3.8-fold increases (P < 0.001) in risk for plasma homocysteine in the top 5, 10, and 20%, respectively, of the homocysteine distribution for subjects with the MTHFR 677 TT compared with the CC and CT genotypes. Predicted plasma homocysteine was inversely associated with serum folate (P = 0.003) and plasma vitamin B-12 (P = 0.002), with the degree of correlation dependent on MTHFR 677C-->T genotype. These data suggest that coexistence of the MTHFR 677 TT genotype with the MTRR 66A-->G polymorphism may exacerbate the effect of the MTHFR variant alone. The potential negative effect of combined polymorphisms of the MTHFR and MTRR genes on plasma homocysteine in at-risk population groups with low folate and/or vitamin B-12 status, such as women of reproductive potential, deserves further investigation.