Influence of maternal folate depletion on Art3 DNA methylation in the murine adult brain; potential consequences for brain and neurocognitive health.

The developmental origins of health and disease hypothesis suggest early-life environment impacts health outcomes throughout the life course. In particular, epigenetic marks, including DNA methylation, are thought to be key mechanisms through which environmental exposures programme later-life health. Adequate maternal folate status before and during pregnancy is essential in the protection against neural tube defects, but data are emerging that suggest early-life folate exposures may also influence neurocognitive outcomes in childhood and, potentially, thereafter. Since folate is key to the supply of methyl donors for DNA methylation, we hypothesize that DNA methylation may be a mediating mechanism through which maternal folate influences neurocognitive outcomes. Using bisulphite sequencing, we measured DNA methylation of five genes (Art3, Rsp16, Tspo, Wnt16, and Pcdhb6) in the brain tissue of adult offspring of dams who were depleted of folate (n = 5, 0.4 mg folic acid/kg diet) during pregnancy (~19-21 days) and lactation (mean 22 days) compared with controls (n = 6, 2 mg folic acid/kg diet). Genes were selected as methylation of their promoters had previously been found to be altered by maternal folate intake in mice and humans across the life course, and because they have potential associations with neurocognitive outcomes. Maternal folate depletion was significantly associated with Art3 gene hypomethylation in subcortical brain tissue of adult mice at 28 weeks of age (mean decrease 6.2%, P = .03). For the other genes, no statistically significant differences were found between folate depleted and control groups. Given its association with neurocognitive outcomes, we suggest Art3 warrants further study in the context of lifecourse brain health. We have uncovered a potential biomarker that, once validated in accessible biospecimens and human context, may be useful to track the impact of early-life folate exposure on later-life neurocognitive health, and potentially be used to develop and monitor the effects of interventions.

Environmental microbiome in the home and daycare settings during the COVID-19 pandemic, and potential risk of non-communicable disease in children.

An exposure to diverse microbial population early in life is important for the development of immunity against various non-communicable diseases including asthma, childhood leukaemia and other cancers. Social mixing in daycare settings helps with exposure to a variety of microbes. However, social isolation and a high emphasis on workplace hygiene during the COVID pandemic may have affected children's exposure to diverse microbiota. The structure of microbial communities and their role in developing immunity to various diseases are not well understood. In this study, we investigated the structure of microbial communities in daycare and home settings during the pandemic. Interestingly, microbial diversity was relatively higher in dust samples collected from homes, with human-associated taxa being more prevalent compared to those from daycare settings. Environmental microbes were more abundant in dust samples from daycare providers. These results potentially suggest that cleaning practices during the pandemic may have influenced the diversity and microbial abundance of the daycare samples. Several bacterial taxa detected in both the environments are known to induce anti-inflammatory and immunomodulatory responses, conferring protection from various diseases. Therefore, exposure to diverse microbial population in early childhood may play an important role in developing immunity against various non-communicable and infectious diseases.

Intake and biomarkers of folate and folic acid as determinants of chemotherapy-induced toxicities in patients with colorectal cancer: a cohort study.

BACKGROUND: Capecitabine is an oral chemotherapeutic drug showing antitumor activity through inhibition of thymidylate synthase, an enzyme involved in folate metabolism. There are concerns about the high intake of certain vitamins, and specifically folate, during chemotherapy with capecitabine. Whether folate or folic acid, the synthetic variant of the vitamin, impact treatment toxicity remains unclear. OBJECTIVE: We studied associations between intake and biomarkers of folate as well as folic acid and toxicities in patients with colorectal cancer (CRC) receiving capecitabine. METHODS: Within the prospective COLON (Colorectal cancer: Longitudinal, Observational study on Nutritional and lifestyle factors that influence recurrence, survival, and quality of life) cohort, 290 patients with stage II to III CRC receiving capecitabine were identified. Dietary and supplemental intake of folate and folic acid were assessed at diagnosis and during chemotherapy using questionnaires (available for 280 patients). Plasma folate and folic acid levels were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) and were available for 212 patients. Toxicities were defined as toxicity-related modifications of treatment, including dose reductions, regimen switches, and early discontinuation. Associations of intake and biomarkers of folate and folic acid with toxicities were determined using Cox proportional hazards regression adjusted for age and sex. RESULTS: In total, 153 (53%) patients experienced toxicities leading to modification of capecitabine treatment. Folate intake and plasma folate levels were not associated with risk of toxicities. However, use of folic acid-containing supplements during treatment (hazard ratio (HR) 1.81 and 95% confidence interval (CI) 1.15-2.85) and presence of folic acid in plasma at diagnosis (HR 2.09, 95% CI: 1.24, 3.52) and during treatment (HR 2.31, 95% CI: 1.29, 4.13) were associated with an increased risk of toxicities. CONCLUSIONS: This study suggests a potential association between folic acid and capecitabine-induced toxicities, providing a rationale to study diet-drug interactions and raise further awareness of the use of dietary supplements during oncological treatment. CLINICAL TRIAL DETAILS: This trial was registered at clinicaltrials.gov as NCT03191110.

Folate, folic acid, and chemotherapy-induced toxicities: A systematic literature review.

Folate metabolism is a target for various chemotherapeutic drugs. Folate and its synthetic variant folic acid are B-vitamins. To what extent these vitamins impact treatment tolerance in patients with cancer remains unclear. A systematic literature review was conducted on intake and status of folate and folic acid in relation to chemotherapy-induced toxicities in children and adults with cancer. A total of 6231 publications were identified, of which 40 publications met the inclusion criteria. In 12 out of 22 studies focusing on antifolates, a deficient folate status and lower folate and folic acid intake were associated with a higher risk of toxicities. In 8 out of 14 studies focusing on fluoropyrimidine treatments, a higher folate status and intake were associated with a higher risk of toxicities. These findings might explain interindividual differences in treatment tolerance and highlight the importance of evaluating nutritional status in oncology care.

Anti-cancer therapy is associated with long-term epigenomic changes in childhood cancer survivors.

BACKGROUND: Childhood cancer survivors (CCS) exhibit significantly increased chronic diseases and premature death. Abnormalities in DNA methylation are associated with development of chronic diseases and reduced life expectancy. We investigated the hypothesis that anti-cancer treatments are associated with long-term DNA methylation changes that could be key drivers of adverse late health effects. METHODS: Genome-wide DNA methylation was assessed using MethylationEPIC arrays in paired samples (before/after therapy) from 32 childhood cancer patients. Separately, methylation was determined in 32 samples from different adult CCS (mean 22-years post-diagnosis) and compared with cancer-free controls (n = 284). RESULTS: Widespread DNA methylation changes were identified post-treatment in childhood cancer patients, including 146 differentially methylated regions (DMRs), which were consistently altered in the 32 post-treatment samples. Analysis of adult CCS identified matching methylation changes at 107/146 of the DMRs, suggesting potential long-term retention of post-therapy changes. Adult survivors also exhibited epigenetic age acceleration, independent of DMR methylation. Furthermore, altered methylation at the DUSP6 DMR was significantly associated with early mortality, suggesting altered methylation may be prognostic for some late adverse health effects in CCS. CONCLUSIONS: These novel methylation changes could serve as biomarkers for assessing normal cell toxicity in ongoing treatments and predicting long-term health outcomes in CCS.

Impact of In Utero Folate Exposure on DNA Methylation and Its Potential Relevance for Later-Life Health-Evidence from Mouse Models Translated to Human Cohorts.

SCOPE: Persistent DNA methylation changes may mediate effects of early-life exposures on later-life health. Human lifespan is challenging for prospective studies, therefore data from longitudinal studies are limited. Projecting data from mouse models of early-life exposure to human studies offers a tool to address this challenge. METHODS AND RESULTS: C57BL/6J mice were fed low/normal folate diets before and during pregnancy and lactation. Genome-wide promoter methylation was measured in male offspring livers at 17.5 days gestation and 28 weeks. Eight promoters were concurrently hypermethylated by folate depletion in fetuses and adults (>1.10 fold-change; p < 0.05). Processes/pathways potentially influenced by global changes, and function of these eight genes, suggest neurocognitive effects. Human observational and randomized controlled trial data were interrogated for translation. Methylation at birth was inversely associated with maternal plasma folate in six genes (-1.15% to -0.16% per nmol L-1 ; p < 0.05), while maternal folic acid supplementation was associated with differential methylation of four genes in adulthood. Three CpGs were persistently hypermethylated with lower maternal folate (p = 0.04). CONCLUSION: Some persistent folate-induced methylation changes in mice are mirrored in humans. This demonstrates utility of mouse data in identifying human loci for interrogation as biomarkers of later-life health.

The Biological and Social Determinants of Childhood Obesity: Comparison of 2 Cohorts 50 Years Apart.

OBJECTIVE: To determine whether the same relationships between early-life risk factors and socioeconomic status (SES) with childhood body mass index (BMI) are observed in a modern cohort (2000) compared with a historic cohort (1947). STUDY DESIGN: The relationships between early-life factors and SES with childhood BMI were examined in 2 prospective birth cohorts from the same region, born 50 years apart: 711 children in the 1947 Newcastle Thousand Families Study (NTFS) and 475 from the 2000 Gateshead Millennium Study (GMS). The associations between birth weight, breastfeeding, rapid infancy growth (0-12 months), early-life adversity (0-12 months), and parental SES (birth and childhood) with childhood BMI z-scores and whether overweight/obese (BMI >91st percentile using UK 1990 reference) aged 9 years were examined using linear regression, path analyses, and logistic regression. RESULTS: In the NTFS, the most advantaged children were taller than the least (+0.91 height z-score, P = .001), whereas in GMS they had lower odds of overweight/obese than the least (0.35 [95% CI 0.14-0.86]). Rapid infancy growth was associated with increased BMI z-scores in both cohorts, and with increased likelihood of overweight/obese in GMS. CONCLUSIONS: This study suggests that children exposed to socioeconomic disadvantage or who have rapid infancy growth in modern environments are now at lower risk of growth restriction but greater risk of overweight.

DNA methylation and the hygiene hypothesis: connecting respiratory allergy and childhood acute lymphoblastic leukemia.

Aim: The hygiene hypothesis states that a lack of infection in early-life suppresses immune system development, and is linked to respiratory allergy (RA) and childhood acute lymphoblastic leukemia (ALL) risk. Little is known about underlying mechanisms, but DNA methylation is altered in RA and ALL, and in response to infection. We investigated if aberrant methylation may be in common between these diseases and associated with infection. Materials & methods: RA and ALL disease-associated methylation signatures were compared and related to exposure-to-infection signatures. Results: A significant number of genes overlapped between RA and ALL signatures (p = 0.0019). Significant overlaps were observed between exposure-to-infection signatures and disease-associated signatures. Conclusion: DNA methylation may be a mediating mechanism through which the hygiene hypothesis is associated with RA and ALL risk.

Exploring a potential mechanistic role of DNA methylation in the relationship between in utero and post-natal environmental exposures and risk of childhood acute lymphoblastic leukaemia.

The aetiology of childhood acute lymphoblastic leukaemia (ALL) is unclear. Genetic abnormalities have been identified in a number of ALL cases, although these alone are not sufficient for leukaemic transformation. Various in utero and post-natal environmental exposures have been suggested to alter risk of childhood ALL. DNA methylation patterns can be influenced by environmental exposures, and are reported to be altered in ALL, suggesting a potential mediating mechanism between environment and ALL disease risk. To investigate this, we used a 'meet in the middle' approach, investigating the overlap between exposure-associated and disease-associated methylation change. Genome-wide DNA methylation changes in response to possible ALL-risk exposures (i.e. breast feeding, infection history, day care attendance, maternal smoking, alcohol, caffeine, folic acid, iron and radiation exposure) were investigated in a sub-population of the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort using an epigenome-wide association study (EWAS) approach (n = 861-927), and compared to a list of ALL disease-associated methylation changes compiled from published data. Hypergeometric probability tests suggested that the number of directionally concordant gene methylation changes observed in ALL disease and in response to the following exposures; maternal radiation exposure (p = 0.001), alcohol intake (p = 0.006); sugary caffeinated drink intake during pregnancy (p = 0.045); and infant day care attendance (p = 0.003), were not due to chance. Data presented suggests that DNA methylation may be one mediating mechanism in the multiple hit pathway needed for ALL disease manifestation.

Effect of methotrexate/vitamin B12 on DNA methylation as a potential factor in leukemia treatment-related neurotoxicity.

Methotrexate (MTX) is administered to treat childhood acute lymphoblastic leukemia (ALL). It acts by inhibiting dihydrofolate reductase which reduces methyltetrahydrofolate, a key component in one carbon metabolism, thus reducing cell proliferation. Further perturbations to one carbon metabolism, such as reduced vitamin B12 levels via the use of nitrous oxide for sedation during childhood ALL treatment, may increase neurotoxicity risk. With B12 as an enzymatic cofactor, methyltetrahydrofolate is essential to produce methionine, which is critical for DNA methylation. We investigated global and gene specific DNA methylation in neuronal cell lines in response to MTX treatment and vitamin B12 concentration individually, and in combination. RESULTS: MTX treatment alone significantly increased LINE-1 methylation in SH-SY5Y (p = 0.040) and DAOY (p < 0.001), and increased FKBP5 methylation in MO3.13 cells (p = 0.009). CONCLUSION: We conclude that altered DNA methylation of brain/central nervous system cells could be one mechanism involved in MTX treatment-related neurotoxicities and neurocognitive late effects in ALL survivors.

Maternal folate depletion during early development and high fat feeding from weaning elicit similar changes in gene expression, but not in DNA methylation, in adult offspring.

SCOPE: The 'Predictive Adaptive Response' hypothesis suggests that the in utero environment when mismatched with the post-natal environment can influence later life health. Underlying mechanisms are poorly understood, but may involve gene transcription changes regulated via epigenetic mechanisms. METHODS AND RESULTS: In a 2 × 2 factorial design, female C57Bl/6 mice were randomised to low or normal folate diets (0.4 mg/2 mg folic acid/kg diet) prior to and during pregnancy and lactation with offspring randomised to high- or low-fat diets at weaning. Genome-wide gene expression and promoter DNA methylation were measured using microarrays in adult male livers. Maternal folate depletion and high fat intake post-weaning influenced gene expression (1859 and 1532 genes, respectively) and promoter DNA methylation (201 and 324 loci, respectively) but changes in expression and methylation were poorly matched for both dietary interventions. Expression of 642 genes was altered in response to both maternal folate depletion and post-weaning high fat feeding, treatments imposed separately. In addition, there was evidence that the combined dietary insult (i.e. maternal folate depletion followed by high fat post-weaning) caused the largest expression change for most genes. CONCLUSION: Our observations align with, and provide evidence in support of, a potential underlying mechanism for the 'Predictive Adaptive Response' hypothesis.

Organ-Specific Gene Expression Changes in the Fetal Liver and Placenta in Response to Maternal Folate Depletion.

Growing evidence supports the hypothesis that the in utero environment can have profound implications for fetal development and later life offspring health. Current theory suggests conditions experienced in utero prepare, or "programme", the fetus for its anticipated post-natal environment. The mechanisms responsible for these programming events are poorly understood but are likely to involve gene expression changes. Folate is essential for normal fetal development and inadequate maternal folate supply during pregnancy has long term adverse effects for offspring. We tested the hypothesis that folate depletion during pregnancy alters offspring programming through altered gene expression. Female C57BL/6J mice were fed diets containing 2 mg or 0.4 mg folic acid/kg for 4 weeks before mating and throughout pregnancy. At 17.5 day gestation, genome-wide gene expression was measured in male fetal livers and placentas. In the fetal liver, 989 genes were expressed differentially (555 up-regulated, 434 down-regulated) in response to maternal folate depletion, with 460 genes expressed differentially (250 up-regulated, 255 down-regulated) in the placenta. Only 25 differentially expressed genes were common between organs. Maternal folate intake during pregnancy influences fetal gene expression in a highly organ specific manner which may reflect organ-specific functions.

Gene promoter DNA methylation patterns have a limited role in orchestrating transcriptional changes in the fetal liver in response to maternal folate depletion during pregnancy.

SCOPE: Early-life exposures are critical in fetal programming and may influence function and health in later life. Adequate maternal folate consumption during pregnancy is essential for healthy fetal development and long-term offspring health. The mechanisms underlying fetal programming are poorly understood, but are likely to involve gene regulation. Epigenetic marks, including DNA methylation, regulate gene expression and are modifiable by folate supply. We observed transcriptional changes in fetal liver in response to maternal folate depletion and hypothesized that these changes are concomitant with altered gene promoter methylation. METHODS AND RESULTS: Female C57BL/6J mice were fed diets containing 2 or 0.4 mg folic acid/kg for 4 wk before mating and throughout pregnancy. At 17.5-day gestation, genome-wide gene expression and promoter methylation were measured by microarray analysis in male fetal livers. While 989 genes were differentially expressed, 333 promoters had altered methylation (247 hypermethylated, 86 hypomethylated) in response to maternal folate depletion. Only 16 genes had both expression and methylation changes. However, most methylation changes occurred in genomic regions neighboring expression changes. CONCLUSION: In response to maternal folate depletion, altered expression at the mRNA level was not associated with altered promoter methylation of the same gene in fetal liver.

DNA methylation as a potential mediator of environmental risks in the development of childhood acute lymphoblastic leukemia.

5-year survival rate for childhood acute lymphoblastic leukemia (ALL) has risen to approximately 90%, yet the causal disease pathway is still poorly understood. Evidence suggests multiple 'hits' are required for disease progression; an initial genetic abnormality followed by additional secondary 'hits'. It is plausible that environmental influences may trigger these secondary hits, and with the peak incidence of diagnosis between 2 and 5 years of age, early life exposures are likely to be key. DNA methylation can be modified by many environmental exposures and is dramatically altered in cancers, including childhood ALL. Here we explore the potential that DNA methylation may be involved in the causal pathway toward disease by acting as a mediator between established environmental factors and childhood ALL development.

Maternal folate deficiency and metabolic dysfunction in offspring.

The importance of folate during pregnancy was established more than 80 years ago by Lucy Wills' ground-breaking studies of tropical macrocytic anaemia. More recently, it has become apparent that the adverse consequences of inadequate nutrient supply during early developmental may be exacerbated by over-nutrition postnatally. The present paper aims to review recent evidence that maternal methyl donor (notably folate) supply peri-conceptually and during pregnancy has long-term effects on offspring (metabolic) health. In addition, we propose the hypothesis that epigenetic mechanisms, especially DNA methylation, may mediate the effects of these early life nutritional insults. We discuss evidence from a natural experiment in human subjects which provides proof of principle for the hypothesis. We describe an attempt to test this hypothesis using a mouse model in which female C57Bl/6 mice were randomised to low or normal folate diets prior to, and during, pregnancy and lactation. Low maternal folate supply resulted in offspring that were more susceptible to detrimental metabolic effects of a high-fat diet fed from weaning, manifested as increased circulating TAG concentration. Interestingly, this metabolic phenotype in adult offspring occurred without any detectable change in adiposity, suggesting a different aetiological origin from the more commonly reported observation that maternal undernutrition leads to increased offspring adiposity and to symptoms of the Metabolic Syndrome. The widespread prevalence of overweight and obesity and of folate deficiency among women of child-bearing age highlights the possibility that this double nutritional insult may exacerbate the risk of metabolic disease in their offspring.

SIRT1 affects DNA methylation of polycomb group protein target genes, a hotspot of the epigenetic shift observed in ageing.

BACKGROUND: SIRT1 is likely to play a role in the extension in healthspan induced by dietary restriction. Actions of SIRT1 are pleiotropic, and effects on healthspan may include effects on DNA methylation. Polycomb group protein target genes (PCGTs) are suppressed by epigenetic mechanisms in stem cells, partly through the actions of the polycomb repressive complexes (PRCs), and have been shown previously to correspond with loci particularly susceptible to age-related changes in DNA methylation. We hypothesised that SIRT1 would affect DNA methylation particularly at PCGTs. To map the sites in the genome where SIRT1 affects DNA methylation, we altered SIRT1 expression in human intestinal (Caco-2) and vascular endothelial (HuVEC) cells by transient transfection with an expression construct or with siRNA. DNA was enriched for the methylated fraction then sequenced (HuVEC) or hybridised to a human promoter microarray (Caco-2). RESULTS: The profile of genes where SIRT1 manipulation affected DNA methylation was enriched for PCGTs in both cell lines, thus supporting our hypothesis. SIRT1 knockdown affected the mRNA for none of seven PRC components nor for DNMT1 or DNMT3b. We thus find no evidence that SIRT1 affects DNA methylation at PCGTs by affecting the expression of these gene transcripts. EZH2, a component of PRC2 that can affect DNA methylation through association with DNA methyltransferases (DNMTs), did not co-immunoprecipitate with SIRT1, and SIRT1 knockdown did not affect the expression of EZH2 protein. Thus, it is unlikely that the effects of SIRT1 on DNA methylation at PCGTs are mediated through direct intermolecular association with EZH2 or through effects in its expression. CONCLUSIONS: SIRT1 affects DNA methylation across the genome, but particularly at PCGTs. Although the mechanism through which SIRT1 has these effects is yet to be uncovered, this action is likely to contribute to extended healthspan, for example under conditions of dietary restriction.

Metabolic effects of a high-fat diet post-weaning after low maternal dietary folate during pregnancy and lactation.

SCOPE: Investigate the influence of low-folate supply during pregnancy and lactation on obesity and markers of the metabolic syndrome in offspring, and how provision of a high-fat diet post weaning may exacerbate the resultant phenotype. METHODS AND RESULTS: Female C57Bl/6 mice were randomized to low or normal folate diets (0.4 or 2 mg folic acid/kg diet) prior to and during pregnancy and lactation. At 4 wk of age, offspring were randomized to high- or low-fat diets, weighed weekly and food intake assessed at 9 and 18 wk old. Adiposity was measured at 3 and 6 months. Plasma glucose and triacylglycerol (TAG) concentrations were measured at 6 months. Maternal folate supply did not influence adult offspring body weight or adiposity. High-fat feeding post weaning increased body weight and adiposity at 3 and 6 months (p > 0.001). Maternal low folate lowered plasma glucose (p = 0.010) but increased plasma TAG (p = 0.048). High-fat feeding post weaning increased plasma glucose and TAG (p = 0.023, p = 0.049 respectively). Offspring from folate-depleted (but not folate-adequate) dams had 30% higher TAG concentration when fed the high-fat diet from weaning (p = 0.005 for interaction). CONCLUSION: Inadequate maternal folate intake has long-term effects on offspring metabolism, manifested as increased circulating TAG, particularly in offspring with high-fat intake post weaning.

Maternal folate depletion and high-fat feeding from weaning affects DNA methylation and DNA repair in brain of adult offspring.

The mechanisms through which environmental and dietary factors modulate DNA repair are still unclear but may include dysregulation of gene expression due to altered epigenetic markings. In a mouse model, we investigated the effect of maternal folate depletion during pregnancy and lactation, and high-fat feeding from weaning, on base excision repair (BER) and DNA methylation and expression of selected BER-related genes in the brain of adult offspring. While folate depletion did not affect BER activity of the mothers, BER increased in the offspring at weaning (P=0.052). In the long term, as observed in 6-mo-old offspring, the double insult, i.e., maternal low-folate supply and high-fat feeding from weaning, decreased BER activity significantly in the cortex, cerebellum, hippocampus, and subcortical regions (P≤0.017). This fall in BER activity was associated with small changes in methylation or expression of BER-related genes. Maternal folate depletion led to slightly increased oxidative DNA damage levels in subcortical regions of adult offspring, which may increase sensitivity to oxidative stress and predispose to neurological disorders. In summary, our data suggest that low-folate supply during early life may leave an epigenetic mark that can predispose the offspring to further dietary insults, causing adverse effects during adult life.

Genetic and non-genetic influences during pregnancy on infant global and site specific DNA methylation: role for folate gene variants and vitamin B12.

Inter-individual variation in patterns of DNA methylation at birth can be explained by the influence of environmental, genetic and stochastic factors. This study investigates the genetic and non-genetic determinants of variation in DNA methylation in human infants. Given its central role in provision of methyl groups for DNA methylation, this study focuses on aspects of folate metabolism. Global (LUMA) and gene specific (IGF2, ZNT5, IGFBP3) DNA methylation were quantified in 430 infants by Pyrosequencing®. Seven polymorphisms in 6 genes (MTHFR, MTRR, FOLH1, CßS, RFC1, SHMT) involved in folate absorption and metabolism were analysed in DNA from both infants and mothers. Red blood cell folate and serum vitamin B(12) concentrations were measured as indices of vitamin status. Relationships between DNA methylation patterns and several covariates viz. sex, gestation length, maternal and infant red cell folate, maternal and infant serum vitamin B(12), maternal age, smoking and genotype were tested. Length of gestation correlated positively with IGF2 methylation (rho = 0.11, p = 0.032) and inversely with ZNT5 methylation (rho = -0.13, p = 0.017). Methylation of the IGFBP3 locus correlated inversely with infant vitamin B(12) concentration (rho = -0.16, p = 0.007), whilst global DNA methylation correlated inversely with maternal vitamin B(12) concentrations (rho = 0.18, p = 0.044). Analysis of common genetic variants in folate pathway genes highlighted several associations including infant MTRR 66G>A genotype with DNA methylation (χ(2) = 8.82, p = 0.003) and maternal MTHFR 677C>T genotype with IGF2 methylation (χ(2) = 2.77, p = 0.006). These data support the hypothesis that both environmental and genetic factors involved in one-carbon metabolism influence DNA methylation in infants. Specifically, the findings highlight the importance of vitamin B(12) status, infant MTRR genotype and maternal MTHFR genotype, all of which may influence the supply of methyl groups for DNA methylation. In addition, gestational length appears to be an important determinant of infant DNA methylation patterns.