White blood cell global methylation and IL-6 promoter methylation in association with diet and lifestyle risk factors in a cancer-free population

Altered levels of global DNA methylation and gene silencing through methylation of promoter regions can impact cancer risk, but little is known about their environmental determinants. We examined the association between lifestyle factors and levels of global genomic methylation and IL-6 promoter methylation in white blood cell DNA of 165 cancer-free subjects, 18–78 years old, enrolled in the COMIR (Commuting Mode and Inflammatory Response) study, New York, 2009–2010. Besides self-administrated questionnaires on diet and physical activity, we measured weight and height, white blood cell (WBC) counts, plasma levels of high sensitivity C-reactive protein (hs-CRP), and genomic (LINE-1) and gene-specific methylation (IL-6) by pyrosequencing in peripheral blood WBC. Mean levels of LINE-1 and IL-6 promoter methylation were 78.2% and 57.1%, respectively. In multivariate linear regression models adjusting for age, gender, race/ethnicity, body mass index, diet, physical activity, WBC counts and CRP, only dietary folate intake from fortified foods was positively associated with LINE-1 methylation. Levels of IL-6 promoter methylation were not significantly correlated with age, gender, race/ethnicity, body mass index, physical activity or diet, including overall dietary patterns and individual food groups and nutrients. There were no apparent associations between levels of methylation and inflammation markers such as WBC counts and hs-CRP. Overall, among several lifestyle factors examined in association with DNA methylation, only dietary folate intake from fortification was associated with LINE-1 methylation. The long-term consequence of folate fortification on DNA methylation needs to be further evaluated in longitudinal settings.     

Significant differences in global genomic DNA methylation by gender and race/ethnicity in peripheral blood

Reduced levels of global DNA methylation are associated with genomic instability and are independent predictors of cancer risk. Little is known about the environmental determinants of global DNA methylation in peripheral blood. We examined the association between demographic and lifestyle factors and levels of global leukocyte DNA methylation in 161 cancer-free subjects enrolled in the North Texas Healthy Heart Study aged 45–75 years in 2008. We used in-person interviews for demographics and lifestyle factors, a self-administrated Block food frequency questionnaire for diet, and bioelectrical impedance analysis and CT-scan for body composition. We measured genomic DNA methylation using bisulfite conversion of DNA and pyrosequencing for LINE-1. Body composition measures including body mass index, waist circumference, areas of subcutaneous fat and visceral fat, percent of fat mass and fat-free mass were not associated with global genomic DNA methylation after controlling the effect of age, gender and race/ethnicity. Instead, female gender was significantly associated with a reduced level of global methylation (β = −2.77, 95% CI: −4.33, −1.22). Compared to non-Hispanic whites, non-Hispanic blacks (β = −2.02, 95% CI: −3.55, −0.50) had significantly lower levels of global methylation. No association was found with age, cigarette smoking, alcohol drinking and dietary intake of nutrients in one-carbon metabolism. Global leukocyte DNA methylation differs by gender and race/ethnicity, suggesting these variables need to be taken into consideration in studies of global DNA methylation as an epigenetic marker for cancer.Key words: gender, race/ethnicity, DNA methylation     

LINE-1 methylation is positively associated with healthier lifestyle but inversely related to body fat mass in healthy young individuals

With the goal of investigating if epigenetic biomarkers from white blood cells (WBC) are associated with dietary, anthropometric, metabolic, inflammatory and oxidative stress parameters in young and apparently healthy individuals. We evaluated 156 individuals (91 women, 65 men; age: 23.1±3.5 years; body mass index: 22.0±2.9 kg/m²) for anthropometric, biochemical and clinical markers, including some components of the antioxidant defense system and inflammatory response. DNA methylation of LINE-1, TNF-α and IL-6 and the expression of some genes related to the inflammatory process were analyzed in WBC. Adiposity was lower among individuals with higher LINE-1 methylation. On the contrary, body fat-free mass was higher among those with higher LINE-1 methylation. Individuals with higher LINE-1 methylation had higher daily intakes of calories, iron and riboflavin. However, those individuals who presented lower percentages of LINE-1 methylation reported higher intakes of copper, niacin and thiamin. Interestingly, the group with higher LINE-1 methylation had a lower percentage of current smokers and more individuals practicing sports. On the other hand, TNF-α methylation percentage was negatively associated with waist girth, waist-to-hip ratio and waist-to-stature ratio. Plasma TNF-α levels were lower in those individuals with higher TNF-α methylation. This study suggests that higher levels of LINE-1 and TNF-α methylation are associated with better indicators of adiposity status in healthy young individuals. In addition, energy and micronutrient intake, as well as a healthy lifestyle, may have a role in the regulation of DNA methylation in WBC and the subsequent metabolic changes may affect epigenetic biomarkers.     

Significant differences in global genomic DNA methylation by gender and race/ethnicity in peripheral blood

Reduced levels of global DNA methylation are associated with genomic instability and are independent predictors of cancer risk. Little is known about the environmental determinants of global DNA methylation in peripheral blood. We examined the association between demographic and lifestyle factors and levels of global leukocyte DNA methylation in 161 cancer-free subjects enrolled in the North Texas Healthy Heart Study aged 45–75 years in 2008. We used in-person interviews for demographics and lifestyle factors, a self-administrated Block food frequency questionnaire for diet, and bioelectrical impedance analysis and CT-scan for body composition. We measured genomic DNA methylation using bisulfite conversion of DNA and pyrosequencing for LINE-1. Body composition measures including body mass index, waist circumference, areas of subcutaneous fat and visceral fat, percent of fat mass and fat-free mass were not associated with global genomic DNA methylation after controlling the effect of age, gender and race/ethnicity. Instead, female gender was significantly associated with a reduced level of global methylation (β = -2.77, 95% CI: -4.33, -1.22). Compared to non-Hispanic whites, non-Hispanic blacks (β = -2.02, 95% CI: -3.55, -0.50) had significantly lower levels of global methylation. No association was found with age, cigarette smoking, alcohol drinking and dietary intake of nutrients in one-carbon metabolism. Global leukocyte DNA methylation differs by gender and race/ethnicity, suggesting these variables need to be taken into consideration in studies of global DNA methylation as an epigenetic marker for cancer.     

Correlations in global DNA methylation measures in peripheral blood mononuclear cells and granulocytes

Alterations in global DNA methylation levels have been associated with chronic diseases. Despite the increase in the number of studies measuring markers of global methylation, few have adequately examined within-individual differences by source of DNA and whether within-individual differences by source of DNA differ by age, race and other lifestyle factors. We examined correlations between peripheral mononuclear cell (PBMC) and granulocyte DNA methylation levels measured by the luminometric methylation assay (LUMA), and in LINE-1, Sat2, and Alu by MethyLight and pyrosequencing, in the same individual in 112 women participating in The New York City Multiethnic Breast Cancer Project. Levels of DNA methylation of Sat2 by MethyLight (r = 0.57; P < 0.01) and LINE-1 by pyrosequencing (r = 0.30; P < 0.01) were correlated between PBMC and granulocyte DNA of the same individuals, but LUMA and Alu levels were not. The magnitude of the correlations for Sat2 and LINE-1 varied when stratified by selected demographic and lifestyle factors, although the study sample size limited our comparisons across subgroups. These results lend further support to the importance of considering the source of DNA in epidemiologic studies of white blood cell DNA methylation. Results from studies that combine individuals with different available DNA sources need to be interpreted with caution.     

Metabolic,hormonal and immunological associations with global DNA methylation among postmenopausal women

DNA methylation is an epigenetic modification essential for the regulation of gene expression that has been implicated in many diseases, including cancer. Few studies have investigated the wide range of potential predictors of global DNA methylation, including biomarkers. Here, we investigated associations between DNA methylation and dietary factors, sex-steroid hormones, metabolic, lipid, inflammation, immune and one-carbon biomarkers. Data and baseline biomarker measurements were obtained from 173 overweight/obese postmenopausal women. Global DNA methylation in lymphocyte DNA was measured using the pyrosequencing assay for LINE-1 repeats. We used correlations and linear regression analyses to investigate associations between continuous data and DNA methylation, while t-tests were used for categorical data. Secondary analyses stratified by serum folate levels and multivitamin use were also conducted. There was little variability in LINE-1 methylation (66.3–79.5%). Mean LINE-1 methylation was significantly higher among women with elevated glucose levels. Mean LINE-1 methylation was also higher among women with high CD4+/CD8+ ratio, and lower among women with elevated vitamin B6, but neither reached statistical significance. In analyses stratified by folate status, DNA methylation was negatively associated with sex hormone concentrations (estrone, estradiol, testosterone and sex hormone binding globulin) among women with low serum folate levels (n = 53). Conversely, among women with high serum folate levels (n = 53), DNA methylation was positively associated with several immune markers (CD4/CD8 ratio, NK1656/lymphocytes and IgA). Results from this screening suggest that global DNA methylation is generally stable, with differential associations for sex hormones and immune markers depending on one-carbon status.     

Metabolic,hormonal and immunological associations with global DNA methylation among postmenopausal women

DNA methylation is an epigenetic modification essential for the regulation of gene expression that has been implicated in many diseases, including cancer. Few studies have investigated the wide range of potential predictors of global DNA methylation, including biomarkers. Here, we investigated associations between DNA methylation and dietary factors, sex-steroid hormones, metabolic, lipid, inflammation, immune and one-carbon biomarkers. Data and baseline biomarker measurements were obtained from 173 overweight/obese postmenopausal women. Global DNA methylation in lymphocyte DNA was measured using the pyrosequencing assay for LINE-1 repeats. We used correlations and linear regression analyses to investigate associations between continuous data and DNA methylation, while t-tests were used for categorical data. Secondary analyses stratified by serum folate levels and multivitamin use were also conducted. There was little variability in LINE-1 methylation (66.3–79.5%). Mean LINE-1 methylation was significantly higher among women with elevated glucose levels. Mean LINE-1 methylation was also higher among women with high CD4+/CD8+ ratio, and lower among women with elevated vitamin B6, but neither reached statistical significance. In analyses stratified by folate status, DNA methylation was negatively associated with sex hormone concentrations (estrone, estradiol, testosterone and sex hormone binding globulin) among women with low serum folate levels (n = 53). Conversely, among women with high serum folate levels (n = 53), DNA methylation was positively associated with several immune markers (CD4/CD8 ratio, NK1656/lymphocytes and IgA). Results from this screening suggest that global DNA methylation is generally stable, with differential associations for sex hormones and immune markers depending on one-carbon status.     

Correlation of DNA methylation levels in blood and saliva DNA in young girls of the LEGACY Girls study

Many epidemiologic studies of environmental exposures and disease susceptibility measure DNA methylation in white blood cells (WBC). Some studies are also starting to use saliva DNA as it is usually more readily available in large epidemiologic studies. However, little is known about the correlation of methylation between WBC and saliva DNA. We examined DNA methylation in three repetitive elements, Sat2, Alu, and LINE-1, and in four CpG sites, including AHRR (cg23576855, cg05575921), cg05951221 at 2q37.1, and cg11924019 at CYP1A1, in 57 girls aged 6–15 years with blood and saliva collected on the same day. We measured all DNA methylation markers by bisulfite-pyrosequencing, except for Sat2 and Alu, which were measured by the MethyLight assay. Methylation levels measured in saliva DNA were lower than those in WBC DNA, with differences ranging from 2.8% for Alu to 14.1% for cg05575921. Methylation levels for the three repetitive elements measured in saliva DNA were all positively correlated with those in WBC DNA. However, there was a wide range in the Spearman correlations, with the smallest correlation found for Alu (0.24) and the strongest correlation found for LINE-1 (0.73). Spearman correlations for cg05575921, cg05951221, and cg11924019 were 0.33, 0.42, and 0.79, respectively. If these findings are replicated in larger studies, they suggest that, for selected methylation markers (e.g., LINE-1), methylation levels may be highly correlated between blood and saliva, while for others methylation markers, the levels may be more tissue specific. Thus, in studies that differ by DNA source, each interrogated site should be separately examined in order to evaluate the correlation in DNA methylation levels across DNA sources.     

Correlations in global DNA methylation measures in peripheral blood mononuclear cells and granulocytes

Alterations in global DNA methylation levels have been associated with chronic diseases. Despite the increase in the number of studies measuring markers of global methylation, few have adequately examined within-individual differences by source of DNA and whether within-individual differences by source of DNA differ by age, race and other lifestyle factors. We examined correlations between peripheral mononuclear cell (PBMC) and granulocyte DNA methylation levels measured by the luminometric methylation assay (LUMA), and in LINE-1, Sat2, and Alu by MethyLight and pyrosequencing, in the same individual in 112 women participating in The New York City Multiethnic Breast Cancer Project. Levels of DNA methylation of Sat2 by MethyLight (r = 0.57; P < 0.01) and LINE-1 by pyrosequencing (r = 0.30; P < 0.01) were correlated between PBMC and granulocyte DNA of the same individuals, but LUMA and Alu levels were not. The magnitude of the correlations for Sat2 and LINE-1 varied when stratified by selected demographic and lifestyle factors, although the study sample size limited our comparisons across subgroups. These results lend further support to the importance of considering the source of DNA in epidemiologic studies of white blood cell DNA methylation. Results from studies that combine individuals with different available DNA sources need to be interpreted with caution.     

Associations between genetic variation in one-carbon metabolism and LINE-1 DNA methylation in histologically normal breast tissues

Genome-wide DNA hypomethylation is an early event in the carcinogenic process. Percent methylation of long interspersed nucleotide element-1 (LINE-1) is a biomarker of genome-wide methylation and is a potential biomarker for breast cancer. Understanding factors associated with percent LINE-1 DNA methylation in histologically normal tissues could provide insight into early stages of carcinogenesis. In a cross-sectional study of 121 healthy women with no prior history of cancer who underwent reduction mammoplasty, we examined associations between plasma and breast folate, genetic variation in one-carbon metabolism, and percent LINE-1 methylation using multivariable regression models (adjusting for race, oral contraceptive use, and alcohol use). Results are expressed as the ratio of LINE-1 methylation relative to that of the referent group, with the corresponding 95% confidence intervals (CI). We found no significant associations between plasma or breast folate and percent LINE-1 methylation. Variation in MTHFR, MTR, and MTRR were significantly associated with percent LINE-1 methylation. Variant allele carriers of MTHFR A1289C had 4% lower LINE-1 methylation (Ratio 0.96, 95% CI 0.93–0.98), while variant allele carriers of MTR A2756G (Ratio 1.03, 95% CI 1.01–1.06) and MTRR A66G (Ratio 1.03, 95% CI 1.01–1.06) had 3% higher LINE-1 methylation, compared to those carrying the more common genotypes of these SNPs. DNA methylation of LINE-1 elements in histologically normal breast tissues is influenced by polymorphisms in genes in the one-carbon metabolism pathway. Future studies are needed to investigate the sociodemographic, environmental and additional genetic determinants of DNA methylation in breast tissues and the impact on breast cancer susceptibility.     

Differences in DNA methylation by extent of breast cancer family history in unaffected women

Breast cancer clusters within families but genetic factors identified to date explain only a portion of this clustering. Lower global DNA methylation in white blood cells (WBC) has been associated with increased breast cancer risk. We examined whether WBC DNA methylation varies by extent of breast cancer family history in unaffected women from high-risk breast cancer families. We evaluated DNA methylation levels in LINE-1, Alu and Sat2 in 333 cancer-free female family members of the New York site of the Breast Cancer Family Registry, the minority of which were known BRCA1 or BRCA2 mutation carriers. We used generalized estimated equation models to test for differences in DNA methylation levels by extent of their breast cancer family history after adjusting for age. All unaffected women had at least one sister affected with breast cancer. LINE-1 and Sat2 DNA methylation levels were lower in individuals with 3 or more (3+) first-degree relatives with breast cancer relative to women with only one first-degree relative. For LINE-1, Alu, and Sat2, having 3+ affected first-degree relatives was associated with a decrease of 23.4% (95%CI = ?46.8%, 0.1%), 17.9% (95%CI = ?39.5%, 3.7%) and 11.4% (95% CI = ?20.3%, ?2.5%), respectively, relative to individuals with only one affected first-degree relative, but the results were only statistically significant for Sat2. Individuals having an affected mother had 17.9% lower LINE-1 DNA methylation levels (95% CI = ?28.8%, ?7.1%) when compared with those not having an affected mother. No associations were observed for Alu or Sat2 by maternal breast cancer status. If replicated, these results indicate that lower global WBC DNA methylation levels in families with extensive cancer histories may be one explanation for the clustering of cancers in these families. Family clustering of disease may reflect epigenetic as well as genetic and shared environmental factors.     

Correlation of DNA methylation levels in blood and saliva DNA in young girls of the LEGACY Girls study

Many epidemiologic studies of environmental exposures and disease susceptibility measure DNA methylation in white blood cells (WBC). Some studies are also starting to use saliva DNA as it is usually more readily available in large epidemiologic studies. However, little is known about the correlation of methylation between WBC and saliva DNA. We examined DNA methylation in three repetitive elements, Sat2, Alu, and LINE-1, and in four CpG sites, including AHRR (cg23576855, cg05575921), cg05951221 at 2q37.1, and cg11924019 at CYP1A1, in 57 girls aged 6–15 years with blood and saliva collected on the same day. We measured all DNA methylation markers by bisulfite-pyrosequencing, except for Sat2 and Alu, which were measured by the MethyLight assay. Methylation levels measured in saliva DNA were lower than those in WBC DNA, with differences ranging from 2.8% for Alu to 14.1% for cg05575921. Methylation levels for the three repetitive elements measured in saliva DNA were all positively correlated with those in WBC DNA. However, there was a wide range in the Spearman correlations, with the smallest correlation found for Alu (0.24) and the strongest correlation found for LINE-1 (0.73). Spearman correlations for cg05575921, cg05951221, and cg11924019 were 0.33, 0.42, and 0.79, respectively. If these findings are replicated in larger studies, they suggest that, for selected methylation markers (e.g., LINE-1), methylation levels may be highly correlated between blood and saliva, while for others methylation markers, the levels may be more tissue specific. Thus, in studies that differ by DNA source, each interrogated site should be separately examined in order to evaluate the correlation in DNA methylation levels across DNA sources.     

Low fruit consumption and folate deficiency are associated with LINE-1 hypomethylation in women of a cancer-free population

Several dietary agents, such as micronutrient and non-nutrient components, the so-called bioactive food components, have been shown to display anticancer properties and influence genetic processes. The most common epigenetic change is DNA methylation. Hypomethylation of long interspersed elements (LINE-1) has been associated with an increased risk of several cancers, although conflicting findings have also been observed. The aim of the present study was to test the hypothesis that a low adherence to the Mediterranean diet (MD) and folate deficiency may cause LINE-1 hypomethylation in blood leukocytes of healthy women, and thus genomic instability. One hundred and seventy-seven non-pregnant women were enrolled. Mediterranean diet score (MDS) and folate intake were calculated using a food frequency questionnaire. LINE-1 methylation level was measured by pyrosequencing analysis in three CpG sites of LINE-1 promoter. According to MDS, only 9.6 % of subjects achieved a high adherence to MD. Taking into account the use of supplements, there was a high prevalence of folate deficiency (73.4 %). Women whose consumption of fruit was below the median value (i.e., <201 gr/day) were 3.7 times more likely to display LINE-1 hypomethylation than women whose consumption was above the median value (OR 3.7; 95 % CI 1.4–9.5). Similarly, women with folate deficiency were 3.6 times more likely to display LINE-1 hypomethylation than women with no folate deficiency (OR 3.6; 95 % CI 1.1–12.1). A dietary pattern characterized by low fruit consumption and folate deficiency is associated with LINE-1 hypomethylation and with cancer risk.     

LINE-1 DNA methylation is inversely correlated with cord plasma homocysteine in man: A preliminary study

Folic acid supplementation during pregnancy has known beneficial effects. It reduces risk of neural tube defects and low birth weight. Folate and other one-carbon intermediates might secure these clinical effects via DNA methylation. However, most data on the effects of folate on the epigenome is derived from animal or in vitro models. We examined the relationship between cord blood methylation and maternal folic acid intake, cord blood folate and homocysteine using data from 24 pregnant women. Genome-wide methylation was determined by the level of methylation of LINE-1 repeats using Pyrosequencing. We show that cord plasma homocysteine (p = 0.001, r = -0.688), but not serum folate or maternal folic acid intake, is inverse correlated with LINE-1 methylation. This remained significant after correction for potential confounders (p = 0.004). These data indicate that levels of folate-associated intermediates in cord blood during late pregnancy have significant consequences for the fetal epigenome.     

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.     

Micronutrient status and global DNA methylation in school-age children

Aberrations in global LINE-1 DNA methylation have been related to risk of cancer and cardiovascular disease. Micronutrients including methyl-donors and retinoids are involved in DNA methylation pathways. We investigated associations of micronutrient status and LINE-1 methylation in a cross-sectional study of school-age children from Bogotá, Colombia. Methylation of LINE-1 repetitive elements was quantified in 568 children 5–12 years of age using pyrosequencing technology. We examined the association of LINE-1 methylation with erythrocyte folate, plasma vitamin B12, vitamin A ferritin (an indicator of iron status) and serum zinc concentrations using multivariable linear regression. We also considered associations of LINE-1 methylation with socio-demographic and anthropometric characteristics. Mean (± SD) LINE-1 methylation was 80.25 (± 0.65) percentage of 5-mC (%5-mC). LINE-1 methylation was inversely related to plasma vitamin A. After adjustment for potential confounders, children with retinol levels higher than or equal to 1.05 µmol/L showed 0.19% 5-mC lower LINE-1 methylation than children with retinol levels lower than 0.70 µmol/L. LINE-1 methylation was also inversely associated with C-reactive protein, a marker of chronic inflammation, and female sex. We identified positive associations of maternal body mass index and socioeconomic status with LINE-1 methylation. These associations were not significantly different by sex. Whether modification of these exposures during school-age years leads to changes in global DNA methylation warrants further investigation.     

Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens

We evaluated the promoter methylation levels of the APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in 107 colorectal cancer (CRC) samples and 80 healthy adjacent tissues. We searched for correlation with both physical and pathological features, polymorphisms of folate metabolism pathway genes (MTHFR, MTRR, MTR, RFC1, TYMS, and DNMT3B), and data on circulating folate, vitamin B12 and homocysteine, which were available in a subgroup of the CRC patients. An increased number of methylated samples were found in CRC respect to adjacent healthy tissues, with the exception of APC, which was also frequently methylated in healthy colonic mucosa. Statistically significant associations were found between RASSF1A promoter methylation and tumor stage, and between hMLH1 promoter methylation and tumor location. Increasing age positively correlated with both hMLH1 and MGMT methylation levels in CRC tissues, and with APC methylation levels in the adjacent healthy mucosa. Concerning gender, females showed higher hMLH1 promoter methylation levels with respect to males. In CRC samples, the MTR 2756AG genotype correlated with higher methylation levels of RASSF1A, and the TYMS 1494 6bp ins/del polymorphism correlated with the methylation levels of both APC and hMLH1. In adjacent healthy tissues, MTR 2756AG and TYMS 1494 6bp del/del genotypes correlated with APC and MGMT promoter methylation, respectively. Low folate levels were associated with hMLH1 hypermethylation. Present results support the hypothesis that DNA methylation in CRC depends from both physiological and environmental factors, with one-carbon metabolism largely involved in this process.     

Micronutrient status and global DNA methylation in school-age children

Aberrations in global LINE-1 DNA methylation have been related to risk of cancer and cardiovascular disease. Micronutrients including methyl-donors and retinoids are involved in DNA methylation pathways. We investigated associations of micronutrient status and LINE-1 methylation in a cross-sectional study of school-age children from Bogotá, Colombia. Methylation of LINE-1 repetitive elements was quantified in 568 children 5–12 years of age using pyrosequencing technology. We examined the association of LINE-1 methylation with erythrocyte folate, plasma vitamin B12, vitamin A ferritin (an indicator of iron status) and serum zinc concentrations using multivariable linear regression. We also considered associations of LINE-1 methylation with socio-demographic and anthropometric characteristics. Mean (± SD) LINE-1 methylation was 80.25 (± 0.65) percentage of 5-mC (%5-mC). LINE-1 methylation was inversely related to plasma vitamin A. After adjustment for potential confounders, children with retinol levels higher than or equal to 1.05 µmol/L showed 0.19% 5-mC lower LINE-1 methylation than children with retinol levels lower than 0.70 µmol/L. LINE-1 methylation was also inversely associated with C-reactive protein, a marker of chronic inflammation, and female sex. We identified positive associations of maternal body mass index and socioeconomic status with LINE-1 methylation. These associations were not significantly different by sex. Whether modification of these exposures during school-age years leads to changes in global DNA methylation warrants further investigation.     

Global Leukocyte DNA Methylation is Similar in African American and Caucasian Women Under Conditions of Controlled Folate Intake

DNA methylation is an epigenetic feature that may modify disease risk, and can be influenced by folate status as well as by methylenetetrahydrofolate reductase (MTHFR) C677T genotype. The aim of this study was to investigate the influence of ethnicity/race on global leukocyte DNA methylation under conditions of controlled folate intake. Caucasian (n=14) and African American (n-14) women (18-45y) possessing the MTHFR 677CC genotype consumed a folate restricted diet (135 μg/d as dietary folate equivalents, DFE) for 7 week followed by folate treatment with 400 or 800 μg DFE/d for 7 week. Global leukocyte DNA methylation was assessed via the cytosine extension assay at baseline (wk 0), after folate restriction (wk 7) and after folate treatment (wk 14). Ethnicity/race was not a determinant of global leukocyte DNA methylation. No differences (P>0.05) were detected in DNA methylation between African American and Caucasian women at baseline or any other study time point. In addition, folate intake did not modify global leukocyte DNA methylation. These data suggest that global leukocyte DNA methylation does not differ between Caucasian and African American women and that short-term folate restriction is not sufficient to modify methylation content in young women with the MTHFR 677CC genotype.     

Erythrocyte folate concentrations,CpG methylation at genomically imprinted domains,and birth weight in a multiethnic newborn cohort

Epigenetic mechanisms are proposed to link maternal concentrations of methyl group donor nutrients with the risk of low birth weight. However, empirical data are lacking. We have examined the association between maternal folate and birth weight and assessed the mediating role of DNA methylation at nine differentially methylated regions (DMRs) of genomically imprinted genes in these associations. Compared with newborns of women with folate levels in the lowest quartile, birth weight was higher in newborns of mothers in the second (β = 143.2, se = 63.2, P = 0.02), third (β = 117.3, se = 64.0, P = 0.07), and fourth (β = 133.9, se = 65.2, P = 0.04) quartiles, consistent with a threshold effect. This pattern of association did not vary by race/ethnicity but was more apparent in newborns of non-obese women. DNA methylation at the PLAGL1, SGCE, DLK1/MEG3 and IGF2/H19 DMRs was associated with maternal folate levels and also birth weight, suggestive of threshold effects. MEG3 DMR methylation mediated the association between maternal folate levels and birth weight (P =0.06). While the small sample size and partial scope of examined DMRs limit our conclusions, our data suggest that, with respect to birth weight, no additional benefits may be derived from increased maternal folate concentrations, especially in non-obese women. These data also support epigenetic plasticity as a key mechanistic response to folate availability during early fetal development.