The effects of long-term daily folic acid and vitamin B12 supplementation on genome-wide DNA methylation in elderly subjects

Abstract

Background: Folate and its synthetic form folic acid function as donor of one-carbon units and have been, together with other B-vitamins, implicated in programming of epigenetic processes such as DNA methylation during early development. To what extent regulation of DNA methylation can be altered via B-vitamins later in life, and how this relates to health and disease, is not exactly known. The aim of this study was to identify effects of long-term supplementation with folic acid and vitamin B12 on genome-wide DNA methylation in elderly subjects. This project was part of a randomized, placebo-controlled trial on effects of supplemental intake of folic acid and vitamin B12 on bone fracture incidence (B-vitamins for the PRevention Of Osteoporotic Fractures (B-PROOF) study). Participants with mildly elevated homocysteine levels, aged 65-75 years, were randomly assigned to take 400 μg folic acid and 500 μg vitamin B12 per day or a placebo during an intervention period of 2 years. DNA was isolated from buffy coats, collected before and after intervention, and genome-wide DNA methylation was determined in 87 participants (n = 44 folic acid/vitamin B12, n = 43 placebo) using the Infinium HumanMethylation450 BeadChip.

Results: After intervention with folic acid and vitamin B12, 162 (versus 14 in the placebo group) of the 431,312 positions were differentially methylated as compared to baseline. Comparisons of the DNA methylation changes in the participants receiving folic acid and vitamin B12 versus placebo revealed one single differentially methylated position (cg19380919) with a borderline statistical significance. However, based on the analyses of differentially methylated regions (DMRs) consisting of multiple positions, we identified 6 regions that differed statistically significantly between the intervention and placebo group. Pronounced changes were found for regions in the DIRAS3, ARMC8, and NODAL genes, implicated in carcinogenesis and early embryonic development. Furthermore, serum levels of folate and vitamin B12 or plasma homocysteine were related to DNA methylation of 173, 425, and 11 regions, respectively. Interestingly, for several members of the developmental HOX genes, DNA methylation was related to serum levels of folate.

Conclusions: Long-term supplementation with folic acid and vitamin B12 in elderly subjects resulted in effects on DNA methylation of several genes, among which genes implicated in developmental processes.

Keywords: B-vitamins; Cancer; DNA methylation; Development; Elderly; Epigenetics; Folic acid; Infinium 450k BeadChip; Intervention trial; One-carbon metabolism; Vitamin B12.

Fig. 1

Flow diagram for the selection of participants and the analysis of samples. ^aSelf-reported change in use of dietary supplements containing folic acid or vitamin B₁₂. ^bSerum levels of folate or vitamin B₁₂ and plasma levels of homocysteine. Abbreviations: CRP C-reactive protein, MTHFR methylenetetrahydrofolate reductase, SNP single nucleotide polymorphism, SWAN subset-quantile within array normalization

Fig. 2

Changes in serum levels of folate, vitamin B₁₂, and plasma levels of homocysteine. Individual changes in serum levels of folate and vitamin B₁₂ and plasma levels of homocysteine after the 2-year intervention with folic acid and vitamin B₁₂ or placebo. Horizontal lines represent median ± interquartile ranges

Fig. 3

Differentially methylated positions after the intervention with folic acid and vitamin B₁₂. Volcano plots show the statistical significance versus the changes in DNA methylation after the intervention with (a) the placebo or (b) folic acid and vitamin B₁₂. Dashed lines represent 2 % methylation changes and a p value of 1.0E-05. Features of the positions (n = 162) that were differentially methylated after the intervention with folic acid and vitamin B₁₂ are presented in (c) percentages of positions expressed per relationship to CpG islands for the differentially methylated positions (n = 162) as well as for all considered positions on the Infinium HumanMethylation450 Beadchip (n = 431,312), and (d) percentage of positions expressed per relationship to the nearest gene(s). e Number of positions according to the presented categories for absolute changes in DNA methylation for the 162 positions that were differentially methylated after the intervention with folic acid and vitamin B₁₂. f Individual changes in DNA methylation for probe cg06191076 located within DIRAS3. Horizontal lines represent median ± interquartile ranges of DNA methylation, which is expressed as a beta value (0–100 %). Abbreviations: TSS200 200 base pairs around the transcription start site, TSS1500 1500 base pairs around the transcription start site, 3′UTR 3′ untranslated region, 5′UTR 5′ untranslated region, IGR intergenic region

Fig. 4

The differentially methylated region within DIRAS3. Individual (dots) and median (lines) DNA methylation values for the positions within the identified differentially methylated regions (DMRs) for DIRAS3 (chromosome 1) before (black) and after (red) the intervention with folic acid and vitamin B₁₂ (n = 44) or placebo (n = 43). The DMRs were identified using the DMRcate package [81]. Coordinates for the human genome are based on h19/GRCh37

Fig. 5

The effect of folic acid and vitamin B₁₂ on DNA methylation of HOX genes. Mean DNA methylation changes after a 2-year intervention with folic acid and vitamin B₁₂ (red, n = 44) or placebo (black, n = 43) for the 1052 positions located within one of the 39 HOX (homeobox) genes located on cluster A (chromosome 7), cluster B (chromosome 17), cluster C (chromosome 12), or cluster D (chromosome 2). Only positions annotated to one of these HOX genes were depicted; intergenic regions were not included in this figure. Bars are superimposed, meaning that red (folic acid and vitamin B₁₂) and black (placebo) bars are presented together, whenever applicable behind each other, and both reflect the actual values on the y-axis

Fig. 6

The relation between serum levels of folate, vitamin B₁₂ and plasma homocysteine and DNA methylation. The top-3 of the positions (based on lowest p values derived from the limma regression analyses) related to a serum folate levels, b serum vitamin B₁₂ levels, and c plasma homocysteine levels. Linear regression lines are presented in blue and the Pearson correlation coefficient (r) is presented. P values refer to non-adjusted p values from the limma regression analyses. All samples (n = 174) were included in the analyses, except for plasma homocysteine for which two samples were excluded because of missing values