Effect of folic acid and vitamin B12 supplementation on DNA methylation

Ellen Kampman's project showed that long-term supplementation with folic acid and vitamin B12 did not have pronounced effects on global or genome-wide DNA methylation in elderly subjects with mildly elevated homocysteine levels

  • Topic: Combination of cancers
  • Institution: Wageningen University and Research Centre
  • Country: Netherlands
  • Status: Completed

Scientific abstract

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Background

It has been estimated that approximately 30% of all cancer cases are attributable to diet and lifestyle. One of the mechanisms by which diet may affect cancer risk is through epigenetic processes such as DNA methylation. Folate, and its synthetic form folic acid, function as donor of one-carbon units and have been implicated in regulation of DNA methylation as well as DNA synthesis. Epidemiological studies suggest that suboptimal levels of folate and other B-vitamins may affect DNA methylation and thereby influence genomic stability and cancer risk, although the exact underlying mechanisms have not been clarified.

Aim

The aim of this project was to identify the effects of long-term supplementation with physiological doses of folic acid and vitamin B12 on global and genome-wide DNA methylation in elderly subjects.

Methods

The current project was implemented as part of a randomised and placebo-controlled trial on the effects of supplemental intake of folic acid and vitamin B12 on fracture incidence. A total of 2919 subjects with mildly elevated homocysteine concentrations (≥12 μM), aged 65 years and older, participated in this study. Participants were randomly assigned to take 400 μg folic acid and 500 μg vitamin B12 per day or a placebo during an intervention period of two years. Blood was collected at baseline and after two years of intervention. DNA was isolated from buffy coats and bisulfite converted. For the current project, global and genome-wide DNA methylation were determined for 74 and 89 participants, respectively. The LINE-1 assay was used to assess global DNA methylation, whereas DNA methylation arrays were used to study genome-wide DNA methylation. These arrays allow determination of the DNA methylation status of more than 450,000 sites, also called CpG sites, simultaneously.

Results

The findings of our study show that long-term supplementation with folic acid and vitamin B12 did not result in pronounced changes in global or genome-wide DNA methylation as compared to the placebo group. Changes in global DNA methylation in participants receiving folic acid and vitamin B12 did not statistically significantly differ from DNA methylation changes in participants receiving a placebo. Genome-wide analysis of DNA methylation revealed that after the intervention with folic acid and vitamin B12, 33 of the 451,047 CpG sites were significantly differentially methylated as compared to baseline (p<1E-05). Likewise, in the placebo group, 13 CpG sites were significantly differentially methylated as compared to baseline.

Conclusions

In conclusion, this project showed that long-term supplementation with folic acid and vitamin B12 did not have pronounced effects on global or genome-wide DNA methylation in elderly subjects with mildly elevated homocysteine levels.

Plain language abstract

Background

It has been estimated that approximately 30% of all cancer cases are attributable to diet and lifestyle. Previous studies suggest that specific B-vitamins, such as folate, might prevent the development of several forms of cancer, among which colorectal cancer. Folate is mainly present in green leafy vegetables, while its synthetic form folic acid is used for the fortification of foods and as a constituent in dietary supplements. The exact mechanisms underlying the suggested effects of folate on cancer risk have not been clarified. One of the possible mechanisms refers to a biological process which is called DNA methylation. During DNA methylation a methyl group is attached to the DNA, a process which controls the stability of the DNA as well as the expression of genes. DNA methylation plays an essential role during normal development, however, it is also considered to be a critical process in the development of several forms of cancer.

Hypothesis

It is hypothesised that specific B-vitamins such as folate or folic acid may affect DNA methylation and thereby influence cancer risk. The aim of the current project was to identify the effects of long-term supplementation with folic acid and vitamin B12 (another B-vitamin) on DNA methylation in elderly subjects.

Methods

For the current project, blood was collected from 89 elderly (65-75 years of age) men and women. These participants received a daily dietary supplement containing folic acid and vitamin B12 or a placebo during a period of two years. DNA was isolated from the blood, collected at the beginning and the end of the intervention period, and analysed using DNA methylation arrays. These arrays allow the assessment of the DNA methylation status of more than 450,000 sites among the entire genome, simultaneously. Comparisons between the groups of participants who did or did not receive folic acid and vitamin B12 were performed to reveal specific DNA sites or genes which respond to these B-vitamins.

Key findings

This study shows that long-term supplementation with folic acid and vitamin B12 does not result in pronounced effects on DNA methylation. In comparison to the placebo group, only few sites (n=33) were more extensively methylated as a consequence of supplementation with folic acid and vitamin B12. Future studies should elucidate whether B-vitamins such as folic acid and vitamin B12 may have an effect on other biological processes that could be involved in the development or progression of cancer.

Grant publications