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The influence of folate supplementation on global gene expression in normal colonic mucosa of subjects with colorectal adenoma

ABSTRACT: Background: We tested the hypothesis that short-term supplementation with a physiological dose of folate can alter global gene expression in the colon of subjects with colorectal adenoma using a randomised double blind placebo controlled trial design. Materials and Methods: Fourteen subjects with histologically confirmed colorectal adenoma, randomised to receive folic acid (400µg/d, n=6) or placebo (n=8) for 10 weeks, had blood samples and colonic tissue biopsies collected before and after the intervention. Blood samples were used to determine serum and red cell folate, plasma homocysteine, and genetic polymorphisms in methylenetetrahydrofolate reductase (MTHFR C677T) and methionine synthase (MS A2756G). RNA extracted from normal-appearing colonic tissue samples was used to determine global gene expression in the colon using Affymetrix Microarray GeneChips. Microarray results were confirmed by quantitative real time RT-PCR. Two methods were used to analyse the microarray data: (1) differences between pre- and post-intervention gene expression values in the folic acid and placebo groups separately using paired-sample t tests; (2) differences between the folic acid and placebo groups in the ratio of post-intervention to pre-intervention gene expression values using independent sample t-tests. Results: (1) Following intervention, sixty seven genes were up-regulated and 13 genes were down-regulated in the folic acid group, while 21 genes were up-regulated and none were down-regulated in the placebo group (P<0.05, adjusted for multiple testing). (2) Thirty six genes were up-regulated and 18 genes were down-regulated in the folic acid group when compared with placebo, but none of these were statistically significant after adjustment for multiple testing. These genes are involved in multiple pathways, including cell cycle, signal transduction, cell differentiation, transport, cell division, cell motility, protein transport and immune response. There were no genes involved in 1-carbon metabolism that were altered in expression, although several genes involved in neoplasia were up-regulated. Conclusions: These results indicate that while folic acid can modify gene expression, it is difficult to separate its effects from the natural variability in gene expression in the colon.

ORGANISM(S): Homo sapiens

PROVIDER: GSE40928 | GEO | 2013/10/01

SECONDARY ACCESSION(S): PRJNA175379

REPOSITORIES: GEO

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Project description:Background: We tested the hypothesis that short-term supplementation with a physiological dose of folate can alter global gene expression in the colon of subjects with colorectal adenoma using a randomised double blind placebo controlled trial design. Materials and Methods: Fourteen subjects with histologically confirmed colorectal adenoma, randomised to receive folic acid (400M-BM-5g/d, n=6) or placebo (n=8) for 10 weeks, had blood samples and colonic tissue biopsies collected before and after the intervention. Blood samples were used to determine serum and red cell folate, plasma homocysteine, and genetic polymorphisms in methylenetetrahydrofolate reductase (MTHFR C677T) and methionine synthase (MS A2756G). RNA extracted from normal-appearing colonic tissue samples was used to determine global gene expression in the colon using AffymetrixM-oM-^CM-^R Microarray GeneChips. Microarray results were confirmed by quantitative real time RT-PCR. Two methods were used to analyse the microarray data: (1) differences between pre- and post-intervention gene expression values in the folic acid and placebo groups separately using paired-sample t tests; (2) differences between the folic acid and placebo groups in the ratio of post-intervention to pre-intervention gene expression values using independent sample t-tests. Results: (1) Following intervention, sixty seven genes were up-regulated and 13 genes were down-regulated in the folic acid group, while 21 genes were up-regulated and none were down-regulated in the placebo group (P<0.05, adjusted for multiple testing). (2) Thirty six genes were up-regulated and 18 genes were down-regulated in the folic acid group when compared with placebo, but none of these were statistically significant after adjustment for multiple testing. These genes are involved in multiple pathways, including cell cycle, signal transduction, cell differentiation, transport, cell division, cell motility, protein transport and immune response. There were no genes involved in 1-carbon metabolism that were altered in expression, although several genes involved in neoplasia were up-regulated. Conclusions: These results indicate that while folic acid can modify gene expression, it is difficult to separate its effects from the natural variability in gene expression in the colon. Fourteen patients with colorectal adenoma were treated with either folate supplementation (6 subjects) or a placebo (8 subjects) for 10 weeks in a randomised double-blind trial. Colonic biopsies of normal tissue were taken before and after the intervention, and analysed for gene expression.

Project description: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-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 two 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. 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. 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.

Project description:Defects in homocysteine and folate metabolism are associated with increased risks for neural tube and congenital heart defects, cardiovascular disease and stroke, cancers, and neurodegeneration. In many but not all cases, dietary supplementation with folate significantly reduces the severity and incidence of these conditions. Common polymorphisms modulate these metabolic pathways and disease risks, but do not fully account for the particular birth defects and adult diseases that occur in at-risk individuals. To test whether other pathways contribute to disease pathogenesis, we analyzed global and pathway-specific changes in gene expression and levels of selected metabolites after depletion and repletion of dietary folate in two genetically distinct inbred strains of mice. Compared to the C57BL/6J strain, A/J showed greater homeostatic response to folate perturbation by retaining a higher serum folate level and minimizing global gene expression changes. Remarkably, folate perturbation led to systematic strain-specific differences only in the expression profile of the cholesterol biosynthesis pathway and translated to changes in levels of serum and liver total cholesterol. By genetically increasing serum and liver total cholesterol levels in APOE deficient mice, we modestly but significantly improved folate retention during folate depletion, suggesting an interplay between homocysteine and folate metabolism and cholesterol metabolism. Absence of measurable changes in global methylation patterns or amelioration of effects with supplementation with an alternative methyl donor suggest that dietary folate perturbations do not act through large-scale or general changes in methylation. These results suggest that homeostatic responses in cholesterol metabolism contribute to the beneficial effects of dietary folate supplementation. Keywords: time course, stress response, diet, genetic, homeostasis Six-week old female A/J and C57BL/6J mice were purchased from the Jackson Laboratory. All mice were raised on a control diet containing four ppm folic acid (Basal Diet 5755, TestDiet) for one week before the start of studies. Selected mice were then placed on folic acid deficient diet (58C3, TestDiet) containing 1% succinylsulfathiazole, a non-absorbable antibiotic commonly used to suppress folate production by bacteria in the intestine. We had nine different treatment plans per strain with eight replicate mice per treatment. There were four folic acid depletion treatment in which mice were placed on folic acid deficient diet for 1, 2, 7, or 14 days. There were two folic acid repletion treatment in which mice were placed on folic acid deficient diet for 14 days followed by 1 day on control diet and another set of mice on 14 days of folic acid deficient diet followed by 7 days of control diet. There were three control time points in which mice were placed on the control diet for 0, 9, or 22 days. Eight biological replicate liver tissue from each treatment was pooled and total RNA from each pool and total RNA from Universal Mouse Reference RNA (Stratagene) were aminoallyl labeled with Cy3 and Cy5 in duplicate, with reversing of dyes.

Project description:Study question: Do short-term and long-term exposures to low dose folic acid supplementation alter DNA methylation in sperm? Summary answer: No alterations in sperm DNA methylation patterns were found following the administration of low dose folic acid supplements of 400 μg/day for 90 days (short-term exposure) or when pre-fortification of food with folic acid and post-fortification sperm samples (long-term exposure) were compared. What is known already: Excess dietary folate may be detrimental to health and DNA methylation profiles due to folate’s role in one carbon metabolism and the formation of S-adenosyl methionine, the universal methyl donor. DNA methylation patterns are established in developing male germ cells and have been suggested to be affected by high dose (5 mg/day) folic acid supplementation. Participants/materials, setting, methods: Blood and seminal plasma folate levels were measured in participants before and following the 90-day treatment with placebo or supplement. Sperm DNA methylation was assessed using the whole genome and genome-wide techniques, MassArray epityper, restriction landmark genomic scanning, methyl-CpG immunoprecipitation and Illumina HumanMethylation450 Bead Array. Main results and the role of chance: Following treatment, supplemented individuals had significantly higher levels of blood and seminal plasma folates compared to placebo. Initial first generation genome-wide analyses of sperm DNA methylation showed little evidence of changes when comparing pre- and post-treatment samples. With Illumina HumanMethylation450 BeadChip arrays, no significant changes were observed in individual probes following low-level supplementation; when compared with those of the post-fortification cohort, there were also few differences in methylation despite exposure to years of fortified foods. Limitations, reasons for caution: This study was limited to the number of participants available in each cohort, in particular those who were not exposed to early (pre-1998) fortification of food with folic acid. While genome-wide DNA methylation was assessed with several techniques that targeted genic and CpG rich regions, intergenic regions were less well interrogated. Wider implications of the findings: Overall, our findings provide evidence that short term exposure to low dose folic acid supplements of 400 μg/day, over a period of 3 months, a duration of time that might occur during infertility treatments, has no major impact on the sperm DNA methylome.

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The influence of folate supplementation on global gene expression in normal colonic mucosa of subjects with colorectal adenoma

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