Folate is a source of one-carbons necessary for DNA methylation, a critical epigenetic modification necessary for genomic structure and function. The use of supplemental folic acid is widespread however; the potential influence on DNA methylation is unclear. We measured global DNA methylation using DNA extracted from samples from a population-based, double-blind randomized trial of folic acid supplementation (100, 400, 4000 μg per day) taken for 6 months; including a 3 month post-supplementation sample. We observed no changes in global DNA methylation in response to up to 4,000 μg/day for 6 months supplementation in DNA extracted from uncoagulated blood (approximates circulating blood). However, when DNA methylation was determined in coagulated samples from the same individuals at the same time, significant time, dose, and MTHFR genotype-dependent changes were observed. The baseline level of DNA methylation was the same for uncoagulated and coagulated samples; marked differences between sample types were observed only after intervention. In DNA from coagulated blood, DNA methylation decreased (？14%; P<0.001) after 1 month of supplementation and 3 months after supplement withdrawal, methylation decreased an additional 23% (P<0.001) with significant variation among individuals (max+17%; min-94%). Decreases in methylation of ≥25% (vs. <25%) after discontinuation of supplementation were strongly associated with genotype: MTHFR CC vs. TT (adjusted odds ratio [aOR] 12.9, 95%CI 6.4, 26.0). The unexpected difference in DNA methylation between DNA extracted from coagulated and uncoagulated samples in response to folic acid supplementation is an important finding for evaluating use of folic acid and investigating the potential effects of folic acid supplementation on coagulation.
References
[1]
Dolinoy DC, Jirtle RL (2008) Environmental epigenomics in human health and disease. Environmental and Molecular Mutagenesis 49: 4–8.
[2]
Robertson KD (2005) DNA methylation and human disease. Nat Rev Genet 6: 597–610.
[3]
Okano M, Bell DW, Haber DA, Li E (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99: 247–257.
[4]
Botto L, Yang Q (2000) 5,10-Methylenetetrahydrofolate reductase gene variants and congenital anomalies: a HuGE review. Am J Epidemiol 151: 862–877.
[5]
Christensen B, Arbour L, Tran P, Leclerc D, Sabbaghian N, et al. (1999) Genetic polymorphisms in methylenetetrahydrofolate reductase and methionine synthase, folate levels in red blood cells, and risk of neural tube defects. Am J Med Genet 84: 151–157.
[6]
Den Heijer M, Lewington S, Clarke R (2005) Homocysteine, MTHFR and risk of venous thrombosis: a meta-analysis of published epidemiological studies. J Thromb Haemost 3: 292–299.
[7]
Hao L, Yang Q-H, Li Z, Bailey LB, Zhu J-H, et al. (2008) Folate status and homocysteine response to folic acid doses and withdrawal among young Chinese women in a large-scale randomized double-blind trial. Am J Clin Nutr 88: 448–457.
[8]
Crider KS, Zhu JH, Hao L, Yang QH, Yang TP, et al. (2011) MTHFR 677C→T genotype is associated with folate and homocysteine concentrations in a large, population-based, double-blind trial of folic acid supplementation. Am J Clin Nutr 93: 1365–1372.
[9]
Green R, Miller JW (1999) Folate deficiency beyond megaloblastic anemia: hyperhomocysteinemia and other manifestations of dysfunctional folate status. Semin Hematol 36: 47–64.
[10]
Hoffbrand AV, Jackson BF (1993) Correction of the DNA synthesis defect in vitamin B12 deficiency by tetrahydrofolate: evidence in favour of the methyl-folate trap hypothesis as the cause of megaloblastic anaemia in vitamin B12 deficiency. Br J Haematol 83: 643–647.
[11]
Quinlivan EP, Davis SR, Shelnutt KP, Henderson GN, Ghandour H, et al. (2005) Methylenetetrahydrofolate Reductase 677C→T Polymorphism and Folate Status Affect One-Carbon Incorporation into Human DNA Deoxynucleosides. J Nutr 135: 389–396.
[12]
Shelnutt KP, Kauwell GP, Chapman CM, Gregory JF, Maneval DR, et al. (2003) Folate status response to controlled folate intake is affected by the methylenetetrahydrofolate reductase 677C→T polymorphism in young women. J Nutr 133: 4107–4111.
[13]
Rampersaud GC, Kauwell GP, Hutson AD, Cerda JJ, Bailey LB (2000) Genomic DNA methylation decreases in response to moderate folate depletion in elderly women. Am J Clin Nutr 72: 998–1003.
[14]
Jacob RA, Gretz DM, Taylor PC, James SJ, Pogribny IP, et al. (1998) Moderate Folate Depletion Increases Plasma Homocysteine and Decreases Lymphocyte DNA Methylation in Postmenopausal Women. J Nutr 128: 1204–1212.
[15]
Berry RJ, Li Z, Erickson JD, Li S, Moore CA, et al. (1999) Prevention of neural-tube defects with folic acid in China. China-U.S. Collaborative Project for Neural Tube Defect Prevention. N Engl J Med 341: 1485–1490.
[16]
Centers for Disease Control and Prevention (1992) Recommendations for the use of folic acid to reduce the number of cases of spina bifida and other neural tube defects. MMWR Recomm Rep 41: 1–7.
[17]
Food and Drug Administration (1996) Food standards: amendment of standards of identity for enriched grain products to require addition of folic acid, , Final Rule. 21 CFR Parts 136, 137, and 139. Federal Register 64: 8781–8797.
[18]
Smith AD, Kim YI, Refsum H (2008) Is folic acid good for everyone? Am J Clin Nutr 87: 517–533.
[19]
von Castel-Dunwoody KM, Kauwell GP, Shelnutt KP, Vaughn JD, Griffin ER, et al. (2005) Transcobalamin 776C→G polymorphism negatively affects vitamin B-12 metabolism. Am J Clin Nutr 81: 1436–1441.
[20]
Prince JA, Feuk L, Howell WM, Jobs M, Emahazion T, et al. (2001) Robust and Accurate Single Nucleotide Polymorphism Genotyping by Dynamic Allele-Specific Hybridization (DASH): Design Criteria and Assay Validation. Genome Res 11: 152–162.
[21]
Quinlivan EP, Gregory JF III (2008) DNA methylation determination by liquid chromatography-tandem mass spectrometry using novel biosynthetic [U-15N]deoxycytidine and [U-15N]methyldeoxycytidine internal standards. Nucl Acids Res gkn534.
[22]
Quinlivan EP, Gregory JF 3rd (2008) DNA digestion to deoxyribonucleoside: a simplified one-step procedure. Anal Biochem 373: 383–385.
[23]
Balaghi M, Wagner C (1993) DNA methylation in folate deficiency: use of CpG methylase. Biochem Biophys Res Commun 193: 1184–1190.
[24]
Gonzalgo ML, Jones PA (1997) Mutagenic and epigenetic effects of DNA methylation. Mutat Res 386: 107–118.
Weber M, Schubeler D (2007) Genomic patterns of DNA methylation: targets and function of an epigenetic mark. Curr Opin Cell Biol 19: 273–280.
[27]
Adkins KK, Strom DA, Jacobson TE, Seemann CR, O'Brien DP, et al. (2002) Utilizing genomic DNA purified from clotted blood samples for single nucleotide polymorphism genotyping. Arch Pathol Lab Med 126: 266–270.
[28]
Gama-Sosa MA, Midgett RM, Slagel VA, Githens S, Kuo KC, et al. (1983) Tissue-specific differences in DNA methylation in various mammals. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression 740: 212–219.
[29]
Pai AA, Bell JT, Marioni JC, Pritchard JK, Gilad Y (2011) A genome-wide study of DNA methylation patterns and gene expression levels in multiple human and chimpanzee tissues. PLoS Genet 7: e1001316.
[30]
Jackson-Grusby L, Beard C, Possemato R, Tudor M, Fambrough D, et al. (2001) Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation. Nat Genet 27: 31–39.
[31]
Khan R, Schmidt-Mende J, Karimi M, Gogvadze V, Hassan M, et al. (2008) Hypomethylation and apoptosis in 5-azacytidine-treated myeloid cells. Exp Hematol 36: 149–157.
[32]
Feng S, Jacobsen SE, Reik W (2010) Epigenetic reprogramming in plant and animal development. Science 330: 622–627.
[33]
Yamagata K, Okada Y (2011) Understanding paternal genome demethylation through live-cell imaging and siRNA. Cell Mol Life Sci 2011: 15.
[34]
Bezemer ID, Doggen CJ, Vos HL, Rosendaal FR (2007) No association between the common MTHFR 677C→T polymorphism and venous thrombosis: results from the MEGA study. Arch Intern Med 167: 497–501.
[35]
Guzman N, Salazar LA (2010) Frequency of Prothrombotic Risk Factors in Patients with Deep Venous Thrombosis and Controls: Their Implications for Thrombophilia Screening in Chilean Subjects. Genet Test Mol Biomarkers 2010: 14.
[36]
Hultberg B, Andersson A, Isaksson A (2000) Hypomethylation as a cause of homocysteine-induced cell damage in human cell lines. Toxicology 147: 69–75.
[37]
Duthie SJ, Horgan G, de Roos B, Rucklidge G, Reid M, et al. (2010) Blood folate status and expression of proteins involved in immune function, inflammation, and coagulation: biochemical and proteomic changes in the plasma of humans in response to long-term synthetic folic acid supplementation. J Proteome Res 9: 1941–1950.
[38]
Deol PS, Barnes TA, Dampier K, John Pasi K, Oppenheimer C, et al. (2004) The effects of folic acid supplements on coagulation status in pregnancy. Br J Haematol 127: 204–208.
