Gestational intake of methyl donors and global LINE-1 DNA methylation in maternal and cord blood: Prospective results from a folate-replete population

Maternal diet affects offspring DNA methylation in animal models, but evidence from humans is limited. We investigated the extent to which gestational intake of methyl donor nutrients affects global DNA methylation in maternal and umbilical cord blood. Among mother-infant pairs in Project Viva, a folate-replete US population, we estimated maternal intakes of vitamin B12, betaine, choline, folate, cadmium, zinc and iron periconceptionally and during the second trimester. We examined associations of these nutrients with DNA methylation, measured as %5-methyl cytosines (%5mC) in Long Interspersed Nuclear Element-1 (LINE-1), in first trimester (n = 830) and second trimester (n = 671) maternal blood and in cord blood at delivery (n = 516). Cord blood methylation was higher for male than female infants {mean [standard deviation (SD)] 84.8 [0.6] vs. 84.4 [0.7]%}. In the multivariable-adjusted model, maternal intake of methyl donor nutrients periconceptionally and during the second trimester of pregnancy was not positively associated with first trimester, second trimester or cord blood LINE-1 methylation. Periconceptional betaine intake was inversely associated with cord blood methylation [regression coefficient = -0.08% (95% confidence interval (CI): -0.14,-0.01)] but this association was attenuated after adjustment for dietary cadmium, which itself was directly associated with first trimester methylation and inversely associated with cord blood methylation. We also found an inverse association between periconceptional choline [-0.10%, 95% CI: -0.17,-0.03 for each SD (~63 mg/d)] and cord blood methylation in males only. In this folate-replete population, we did not find positive associations between intake of methyl donor nutrients during pregnancy and DNA methylation overall, but among males, higher early pregnancy intakes of choline were associated with lower cord blood methylation.     

Birthweight, maternal weight trajectories and global DNA methylation of LINE-1 repetitive elements

Low birthweight, premature birth, intrauterine growth retardation, and maternal malnutrition have been related to an increased risk of cardiovascular disease, type 2 diabetes mellitus, obesity, and neuropsychiatric disorders later in life. Conversely, high birthweight has been linked to future risk of cancer. Global DNA methylation estimated by the methylation of repetitive sequences in the genome is an indicator of susceptibility to chronic diseases. We used data and biospecimens from an epigenetic birth cohort to explore the association between trajectories of fetal and maternal weight and LINE-1 methylation in 319 mother-child dyads. Newborns with low or high birthweight had significantly lower LINE-1 methylation levels in their cord blood compared to normal weight infants after adjusting for gestational age, sex of the child, maternal age at delivery, and maternal smoking during pregnancy (p = 0.007 and p = 0.036, respectively), but the magnitude of the difference was small. Infants born prematurely also had lower LINE-1 methylation levels in cord blood compared to term infants, and this difference, though small, was statistically significant (p = 0.004). We did not find important associations between maternal prepregnancy BMI or gestational weight gain and global methylation of the cord blood or fetal placental tissue. In conclusion, we found significant differences in cord blood LINE-1 methylation among newborns with low and high birthweight as well as among prematurely born infants. Future studies may elucidate whether chromosomal instabilities or other functional consequences of these changes contribute to the increased risk of chronic diseases among individuals with these characteristics.     

Dietary and supplemental maternal methyl-group donor intake and cord blood DNA methylation

Maternal nutrition is critically involved in the development and health of the fetus. We evaluated maternal methyl-group donor intake through diet (methionine, betaine, choline, folate) and supplementation (folic acid) before and during pregnancy in relation to global DNA methylation and hydroxymethylation and gene specific (IGF2 DMR, DNMT1, LEP, RXRA) cord blood methylation. A total of 115 mother-infant pairs were enrolled in the MAternal Nutrition and Offspring's Epigenome (MANOE) study. The intake of methyl-group donors was assessed using a food-frequency questionnaire. LC-MS/MS and pyrosequencing were used to measure global and gene specific methylation, respectively. Dietary intake of methyl-groups before and during pregnancy was associated with changes in LEP, DNMT1, and RXRA cord blood methylation. Statistically significant higher cord blood LEP methylation was observed when mothers started folic acid supplementation more than 6 months before conception compared with 3–6 months before conception (34.6 ± 6.3% vs. 30.1 ± 3.6%, P = 0.011, LEP CpG1) or no folic acid used before conception (16.2 ± 4.4% vs. 13.9 ± 3%, P = 0.036 for LEP CpG3 and 24.5 ± 3.5% vs. 22.2 ± 3.5%, P = 0.045 for LEP mean CpG). Taking folic acid supplements during the entire pregnancy resulted in statistically significantly higher cord blood RXRA methylation as compared with stopping supplementation in the second trimester (12.3 ± 1.9% vs. 11.1 ± 2%, P = 0.008 for RXRA mean CpG). To conclude, long-term folic acid use before and during pregnancy was associated with higher LEP and RXRA cord blood methylation, respectively. To date, pregnant women are advised to take a folic acid supplement of 400 µg/day from 4 weeks before until 12 weeks of pregnancy. Our results suggest significant epigenetic modifications when taking a folic acid supplement beyond the current advice.     

Type 2 diabetes mellitus in relation to global LINE-1 DNA methylation in peripheral blood: A cohort study

In the last years, epigenetic processes have emerged as a promising area of complex diseases research. DNA methylation measured in Long Interspersed Nucleotide Element 1 (LINE-1) sequences has been considered a surrogate marker for global genome methylation. New findings have suggested the potential involvement of epigenetic mechanisms in Type 2 diabetes (T2DM) as a crucial interface between the effects of genetic predisposition and environmental influences. Our study evaluated whether global DNA methylation predicted increased risk from T2DM or other carbohydrate metabolism disorders in a cohort study. We used a prospective cohort intervention study and a control group. We collected phenotypic, anthropometric, biochemical, and nutritional information from all subjects. Global LINE-1 DNA methylation was quantified by pyrosequencing technology. Subjects that did not improve their carbohydrate metabolism status showed lower levels of global LINE-1 DNA methylation (63.9 ± 1.7 vs. 64.7 ± 2.4) and they practiced less intense physical activity (5.8% vs. 21.5%). Logistic regression analyses showed a significant association between LINE-1 DNA methylation and metabolic status after adjustment for sex, age, BMI, and physical activity. Our study showed that lower LINE-1 DNA methylation levels were associated with a higher risk metabolic status worsening, independent of other classic risk factors. This finding highlights the potential role for epigenetic biomarkers as predictors of T2DM risk or other related metabolic disorders.     

Type 2 diabetes mellitus in relation to global LINE-1 DNA methylation in peripheral blood: A cohort study

In the last years, epigenetic processes have emerged as a promising area of complex diseases research. DNA methylation measured in Long Interspersed Nucleotide Element 1 (LINE-1) sequences has been considered a surrogate marker for global genome methylation. New findings have suggested the potential involvement of epigenetic mechanisms in Type 2 diabetes (T2DM) as a crucial interface between the effects of genetic predisposition and environmental influences. Our study evaluated whether global DNA methylation predicted increased risk from T2DM or other carbohydrate metabolism disorders in a cohort study. We used a prospective cohort intervention study and a control group. We collected phenotypic, anthropometric, biochemical, and nutritional information from all subjects. Global LINE-1 DNA methylation was quantified by pyrosequencing technology. Subjects that did not improve their carbohydrate metabolism status showed lower levels of global LINE-1 DNA methylation (63.9 ± 1.7 vs. 64.7 ± 2.4) and they practiced less intense physical activity (5.8% vs. 21.5%). Logistic regression analyses showed a significant association between LINE-1 DNA methylation and metabolic status after adjustment for sex, age, BMI, and physical activity. Our study showed that lower LINE-1 DNA methylation levels were associated with a higher risk metabolic status worsening, independent of other classic risk factors. This finding highlights the potential role for epigenetic biomarkers as predictors of T2DM risk or other related metabolic disorders.     

The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma

Gliomas are the most frequently occurring primary brain tumor in the central nervous system of adults. Glioblastoma multiformes (GBMs, WHO grade 4) have a dismal prognosis despite the use of the alkylating agent, temozolomide (TMZ), and even low grade gliomas (LGGs, WHO grade 2) eventually transform to malignant secondary GBMs. Although GBM patients benefit from promoter hypermethylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) that is the main determinant of resistance to TMZ, recent studies suggested that MGMT promoter methylation is of prognostic as well as predictive significance for the efficacy of TMZ. Glioma-CpG island methylator phenotype (G-CIMP) in the global genome was shown to be a significant predictor of improved survival in patients with GBM. Collectively, we hypothesized that MGMT promoter methylation might reflect global DNA methylation. Additionally in LGGs, the significance of MGMT promoter methylation is still undetermined. In the current study, we aimed to determine the correlation between clinical, genetic, and epigenetic profiles including LINE-1 and different cancer-related genes and the clinical outcome in newly diagnosed 57 LGG and 54 GBM patients. Here, we demonstrated that (1) IDH1/2 mutation is closely correlated with MGMT promoter methylation and 1p/19q codeletion in LGGs, (2) LINE-1 methylation levels in primary and secondary GBMs are lower than those in LGGs and normal brain tissues, (3) LINE-1 methylation is proportional to MGMT promoter methylation in gliomas, and (4) higher LINE-1 methylation is a favorable prognostic factor in primary GBMs, even compared to MGMT promoter methylation. As a global DNA methylation marker, LINE-1 may be a promising marker in gliomas.     

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.     

Cardiovascular disease risk factors and DNA methylation at the LINE-1 repeat region in peripheral blood from Samoan Islanders

Lower levels of LINE-1 methylation in peripheral blood have been previously associated with risk of developing non-communicable conditions, the most well-explored of these being cancer, although recent research has begun to link altered LINE-1 methylation and cardiovascular disease. We examined the relationship between LINE-1 methylation and factors associated with metabolic and cardiovascular diseases through quantitative bisulfite pyrosequencing in DNA from peripheral blood samples from participants of the Samoan Family Study of Overweight and Diabetes (2002–03). The sample included 355 adult Samoans (88 men and 267 women) from both American Samoa and Samoa. In a model including all sample participants, men had significantly higher LINE-1 methylation levels than women (p = 0.04) and lower levels of LINE-1 methylation were associated with higher levels of fasting LDL (p = 0.02) and lower levels of fasting HDL (p = 0.009). The findings from this study confirm that DNA “global” hypomethylation (as measured by methylation at LINE-1 repeats) observed previously in cardiovascular disease is associated with altered levels of LDL and HDL in peripheral blood. Additionally, these findings strongly argue the need for further research, particularly including prospective studies, in order to understand the relationship between LINE-1 DNA methylation measured in blood and risk factors for cardiovascular disease.Key words: cardiovascular disease, HDL, LDL, LINE-1, DNA methylation, Samoa     

Physical activity and global genomic DNA methylation in a cancer-free population

Changes in DNA methylation may represent an intermediate step between the environment and human diseases. Little is known on whether behavioral risk factors may modify gene expression through DNA methylation. To assess whether DNA methylation is associated with different levels of physical activity, we measured global genomic DNA methylation using bisulfite-converted DNA and real-time PCR (MethyLight) for LINE-1 in peripheral blood of 161 participants aged 45–75 years enrolled in the North Texas Healthy Heart Study and levels of physical activity using an accelerometer (Actigraph GT1M Monitor). We found that individuals with physical activity 26–30 min/day had a significantly higher level of global genomic DNA methylation compared to those with physical activity ≤10 min/day (β = 2.52, 95% CI: 0.70, 4.35). However, the association was attenuated and became statistically insignificant after multivariate adjustment (β = 1.24, 95% CI: −0.93, 3.40). There were some suggestions of a positive association between physical activity and global genomic DNA methylation in non-Hispanics (β = 1.50, 95% CI: −0.08, 3.08) that warrants further investigation.Key words: DNA methylation, physical activity, peripheral blood     

Physical activity and global genomic DNA methylation in a cancer-free population

Changes in DNA methylation may represent an intermediate step between the environment and human diseases. Little is known on whether behavioral risk factors may modify gene expression through DNA methylation. To assess whether DNA methylation is associated with different levels of physical activity, we measured global genomic DNA methylation using bisulfite converted DNA and real time PCR (MethyLight) for LINE-1 in peripheral blood of 161 participants aged 45-75 years enrolled in the North Texas Healthy Heart Study and levels of physical activity using an accelerometer (Actigraph GT1M Monitor). We found that individuals with physical activity 26-30 min/day had a significantly higher level of global genomic DNA methylation compared to those with physical activity ≤ 10 min/day (β=2.52, 95%CI: 0.70, 4.35) However, the association was attenuated and became statistically insignificant after multivariate adjustment (β=1.24, 95%CI:-0.93, 3.40). There were some suggestions of a positive association between physical activity and global genomic DNA methylation in non-Hispanics (β=1.50, 95%CI: -0.08, 3.08) that warrants further investigation.     

Cardiovascular disease risk factors and DNA methylation at the LINE-1 repeat region in peripheral blood from Samoan Islanders

Lower levels of LINE-1 methylation in peripheral blood have been previously associated with risk of developing non-communicable conditions, the most well-explored of these being cancer, although recent research has begun to link altered LINE-1 methylation and cardiovascular disease. We examined the relationship between LINE-1 methylation and factors associated with metabolic and cardiovascular diseases through quantitative bisulfite pyrosequencing in DNA from peripheral blood samples from participants of the Samoan Family Study of Overweight and Diabetes (2002-03). The sample included 355 adult Samoans (88 men and 267 women) from both American Samoa and Samoa. In a model including all sample participants, men had significantly higher LINE-1 methylation levels than women (p=0.04), and lower levels of LINE-1 methylation were associated with higher levels of fasting LDL (p=0.02) and lower levels of fasting HDL (p=0.009). The findings from this study confirm that DNA "global" hypomethylation, (as measured by methylation at LINE-1 repeats) observed previously in cardiovascular disease is associated with altered levels of LDL and HDL in peripheral blood. Additionally, these findings strongly argue the need for further research, particularly including prospective studies, in order to understand the relationship between LINE-1 DNA methylation measured in blood and risk factors for cardiovascular disease.     

LINE-1 methylation in plasma DNA as a biomarker of activity of DNA methylation inhibitors in patients with solid tumors

Multiple clinical trials are investigating the use of the DNA methylation inhibitors azacitidine and decitabine for the treatment of solid tumors. Clinical trials in hematological malignancies have shown that optimal activity does not occur at their maximum tolerated doses but selection of an optimal biological dose and schedule for use in solid tumor patients is hampered by the difficulty of obtaining tumor tissue to measure their activity. Here we investigate the feasibility of using plasma DNA to measure the demethylating activity of the DNA methylation inhibitors in patients with solid tumors. We compared four methods to measure LINE-1 and MAGE-A1 promoter methylation in T24 and HCT116 cancer cells treated with decitabine treatment and selected Pyrosequencing for its greater reproducibility and higher signal to noise ratio. We then obtained DNA from plasma, peripheral blood mononuclear cells, buccal mucosa cells and saliva from ten patients with metastatic solid tumors at two different time points, without any intervening treatment. DNA methylation measurements were not significantly different between time point 1 and time point 2 in patient samples. We conclude that measurement of LINE-1 methylation in DNA extracted from the plasma of patients with advanced solid tumors, using Pyrosequencing, is feasible and has low within patient variability. Ongoing studies will determine whether changes in LINE-1 methylation in plasma DNA occur as a result of treatment with DNA methylation inhibitors and parallel changes in tumor tissue DNA.     

LINE-1 methylation levels in leukocyte DNA and risk of renal cell cancer

Purpose

Leukocyte global DNA methylation levels are currently being considered as biomarkers of cancer susceptibility and have been associated with risk of several cancers. In this study, we aimed to examine the association between long interspersed nuclear elements (LINE-1) methylation levels, as a biomarker of global DNA methylation in blood cell DNA, and renal cell cancer risk.

Experimental Design

LINE-1 methylation of bisulfite-converted genomic DNA isolated from leukocytes was quantified by pyrosequencing measured in triplicate, and averaged across 4 CpG sites. A total of 328 RCC cases and 654 controls frequency-matched(2∶1) on age(±5years), sex and study center, from a large case-control study conducted in Central and Eastern Europe were evaluated.

Results

LINE-1 methylation levels were significantly higher in RCC cases with a median of 81.97% (interquartile range[IQR]: 80.84–83.47) compared to 81.67% (IQR: 80.35–83.03) among controls (p = 0.003, Wilcoxon). Compared to the lowest LINE-1 methylation quartile(Q1), the adjusted ORs for increasing methylation quartiles were as follows: OR(Q2) = 1.84(1.20−2.81), OR(Q3) = 1.72(1.11−2.65) and OR(Q4) = 2.06(1.34−3.17), with a p-trend = 0.004. The association was stronger among current smokers (p-trend<0.001) than former or never smokers (p-interaction = 0.03). To eliminate the possibility of selection bias among controls, the relationship between LINE-1 methylation and smoking was evaluated and confirmed in a case-only analysis, as well.

Conclusions

Higher levels of LINE-1 methylation appear to be positively associated with RCC risk, particularly among current smokers. Further investigations using both post- and pre-diagnostic genomic DNA is warranted to confirm findings and will be necessary to determine whether the observed differences occur prior to, or as a result of carcinogenesis.     

Genome-wide DNA methylation associations with spontaneous preterm birth in US blacks: findings in maternal and cord blood samples

Preterm birth (PTB) affects one in six Black babies in the United States. Epigenetics is believed to play a role in PTB; however, only a limited number of epigenetic studies of PTB have been reported, most of which have focused on cord blood DNA methylation (DNAm) and/or were conducted in white populations. Here we conducted, by far, the largest epigenome-wide DNAm analysis in 300 Black women who delivered early spontaneous preterm (sPTB, n = 150) or full-term babies (n = 150) and replicated the findings in an independent set of Black mother-newborn pairs from the Boston Birth Cohort. DNAm in maternal blood and/or cord blood was measured using the Illumina HumanMethylation450 BeadChip. We identified 45 DNAm loci in maternal blood associated with early sPTB, with a false discovery rate (FDR) <5%. Replication analyses confirmed sPTB associations for cg03915055 and cg06804705, located in the promoter regions of the CYTIP and LINC00114 genes, respectively. Both loci had comparable associations with early sPTB and early medically-indicated PTB, but attenuated associations with late sPTB. These associations could not be explained by cell composition, gestational complications, and/or nearby maternal genetic variants. Analyses in the newborns of the 110 Black women showed that cord blood methylation levels at both loci had no associations with PTB. The findings from this study underscore the role of maternal DNAm in PTB risk, and provide a set of maternal loci that may serve as biomarkers for PTB. Longitudinal studies are needed to clarify temporal relationships between maternal DNAm and PTB risk.     

Nutrition and epigenetics: an interplay of dietary methyl donors, one-carbon metabolism and DNA methylation

DNA methylation is the most extensively studied mechanism of epigenetic gene regulation. Increasing evidence indicates that DNA methylation is labile in response to nutritional and environmental influences. Alterations in DNA methylation profiles can lead to changes in gene expression, resulting in diverse phenotypes with the potential for increased disease risk. The primary methyl donor for DNA methylation is S-adenosylmethionine (SAM), a species generated in the cyclical cellular process called one-carbon metabolism. One-carbon metabolism is catalyzed by several enzymes in the presence of dietary micronutrients, including folate, choline, betaine and other B vitamins. For this reason, nutrition status, particularly micronutrient intake, has been a focal point when investigating epigenetic mechanisms. Although animal evidence linking nutrition and DNA methylation is fairly extensive, epidemiological evidence is less comprehensive. This review serves to integrate studies of the animal in vivo with human epidemiological data pertaining to nutritional regulation of DNA methylation and to further identify areas in which current knowledge is limited.