and studies over the past two decades13,15,241,242. Still, the biological basis through which folic
acid exerts its beneficial effects is still relatively unknown, and concern is growing over studies that have linked elevated folate levels with tumourigenesis21,24. Although a recent
meta-analysis has found no significant link between folic acid supplementation and cancer25,
it is still of paramount importance that these biological mechanisms are elucidated.
S-adenosylmethionine is a methyl donor for many methylation reactions throughout the cell, including DNA methylation, produced from folate-mediated one carbon metabolism6. A
number of studies have shown that changes in one-carbon metabolism, such as increased levels of folate, can have a direct effect on the methylation of different genes throughout the genome109,111. DNA methylation is an epigenetic modification that is intricately associated
with gene expression. As a result, it is plausible that increased folate levels may cause changes in DNA methylation and gene expression, thus establishing a biological mechanism by which folic acid supplementation can impact on the health status of an individual. This was the basis of this study.
Recently, an intervention study was carried out by the University of Ulster to investigate the effect of folic acid supplementation during the second and third trimester of pregnancy on folate status and homocysteine levels4. Blood samples were taken from 119 individuals before
and after intervention, where one group received a daily supplement of folic acid, while participants from the other group received a placebo. A post-intervention sample was taken after 36 weeks of pregnancy, along with another blood sample from the umbilical cord upon delivery. Biochemical data was recorded from these blood samples, and DNA was extracted. These DNA samples made up the foundation of this study.
DNA methylation alone is a difficult modification to measure, so various strategies have been employed to measure and quantify it on both a genome-wide and gene-specific basis5. An in-
depth review was carried out on these methods, and one that suited the aims, objectives, and resources of this study was selected: Modified Methylation Specific Digital Karyotyping (MMSDK)133.
In a series of pilot studies using DNA from the bacteriophage lambda, MMSDK was found to work effectively on a small genome. However, scaling this up to human genomic DNA proved to be technically challenging, so an alternative genome-wide method was considered.
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Methylated DNA immunoprecipitation (MeDIP) was found to efficiently enrich for methylated DNA though QC tests using qPCR, and the DNA microarray approach was selected as a route of analysis instead of MMSDK.
The DNA microarray analysis produced twelve large datasets, each of which was analysed in excel using macros scripted in Visual Basic 6. After this analysis, a preliminary list of 19 novel FS-DMRs was produced, along with 11 regions observed to change their methylation status in response to pregnancy, independent of folate status. Through examination of the microarray’s raw data – the specific probe enrichment score that make up each analysed region – the preliminary list of FS-DMRs was narrowed down to five, three of which were selected for further analysis with a gene-specific method of analysis – Sensitive Melting after Real Time Methylation-Specific PCR (SMART-MSP)146 – over the rest of the FASSTT cohort.
One of the regions analysed was adjacent to the coding region of IP6K1, a gene encoding a kinase responsible for synthesis of inositol pyrophosphates involved in multiple cellular processes such as chromatin remodelling and cell aging176,177. The second was adjacent to the
coding region of RASA4, a small GTP binding signalling molecule involved in gene expression and cell cycle control183, hypomethylation of which has been correlated with poor prognosis
in juvenile myelomonocytic leukemia184. The third region, annotated as Chromosome 9, Open-
Reading Frame 44, does not lie adjacent to a known protein coding region.
When analysed across the rest of the FASSTT cohort using SMART-MSP, changes in DNA methylation did not reach statistical significance. In a cell culture model treated with demethylating agent 5’azacytidine, an increase in gene expression for IP6K1 and RASA4 were observed in response to demethylation of their associated promoter region. GPS2, a gene with an FS-DMR increasing in methylation from the preliminary list of 19 regions found from the MeDIP analysis, also exhibited increased expression in response to demethylation at the region examined by the microarray.
In this investigation into the effects of folic acid supplementation on DNA methylation, no significant impact was found. Most studies from the literature using MeDIP and promoter microarray hybridisation have investigated the effect of conditions more dramatic than folic acid supplementation – healthy cells versus tumour cells, differences across species, etc.169– 173,175. Regarding the FASSTT study, all women recruited were healthy, with folic acid
supplementation being a relatively minor experimental condition imposed upon the participants. If we had the resources available to use a more sensitive method initially, i.e., next generation bisulfite sequencing producing single base-pair resolution data, robust FS-
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DMR may have been found. However, using the MeDIP microarray approach did not yield consistent FS-DMRs in the samples studied.
Various studies have examined the effect of dramatic folate deficiency on mice. When examining changes in global DNA methylation – total DNA methylation across the genome without regional context – Caudill et al., (2001) found no significant changes in liver hypomethylation in response to folate deficiency243. Using a more sensitive technique, Mejos
et al., (2013) found a significant correlation between hepatic folate and global DNA methylation levels in rats244. Of course, these methods of global analysis tell us very little
about DNA methylation on a practical scale. Using gene-specific bisulfite sequencing, Geo et al., (2012) found that folate deficiency in mice caused a decrease in DNA methylation of Esr1, the gene encoding estrogen receptor 1245.
In human studies, examining folate deficiency is more difficult due to ethical concerns. In some instances, researchers have examined the effect of the polymorphism MTHFR 677 C>T on DNA methylation; a variation known to inhibit folate metabolism246. Across a numerous
candidate genes, including ESR1, Hanks et al., (2013) found no methylation changes attributed to the MTHFR 677 C>T genotype204, mirroring results previously found by the same research
group247,248. A recent study by Bollati et al., (2014) examined the impact of nutrient intake on
DNA methylation in obese subjects, finding a negative correlation between TNFα methylation and folic acid intake determined from a standardised food frequency questionnaire. DNA methylation levels were determined using Pyrosequencing technology249.
With respect to folic acid supplementation during pregnancy, Fryer et al., (2009) found no correlation between folic acid intake and methylation of LINE-1 repetitive elements (representative of global methylation), but saw an inverse correlation between plasma homocysteine levels and LINE-1 methylation250. The same group later examined the
relationship between homocysteine levels and genome-wide methylation using the Illumina 27k methylation bead array111, and found 5 CpG sites directly correlated with homocysteine
levels, and 12 CpG with inverse correlation. No confirmation analysis was carried out on these regions obtained from a sample size of n=12.
More recently, Amarasekera et al., (2014) found 7 FS-DMRs in neonatal cord blood correlated with foetal folate status. Of the regions found, the promoter upstream of the gene ZFP57, its product a regulator of DNA methylation during development251. In both the high folate and
low folate groups, the study found a significant correlation between maternal serum folate levels measured at the third trimester of pregnancy, and those of the umbilical cord measured
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upon delivery. This study suggests that folate levels during pregnancy – even as late as the third trimester – can have a significant impact on DNA methylation. These results mirror those of previous studies, suggesting that periconceptional folic acid supplementation – as reported by the mother after birth – is correlated with IGF2 methylation in children108, and inversely
correlated with HI9 methylation under similar conditions252.
Although our MeDIP and SMART-MSP analysis found no correlation between folic acid supplementation and DNA methylation, it is likely that the changes in methylation levels were too subtle to analyse using the methods described here. Sensitive methods examining DNA methylation at a single-base pair resolution – like next generation sequencing and Pyrosequencing following sodium bisulfite treatment – may overcome this challenge.