Schematic of the One-Carbon cycle. Vitamin B12 is a co- factor in the transfer of methyl-groups (CH 3 2 ) from folate to methionine for use in situ methylation of deoxycytidine (dC) to in 5- 

Contexts in source publication

Context 1

... methylation (dC 5 mdC) patterns are transmitted through the germ line [1] and occur primarily at the 5 9 -dC of the 5 9 -deoxycytidine-deoxyguanosine-3 9 (CpG) motif in somatic cells [2,3,4]. Methylation of transcriptional start sites can regulate gene expression [5] by directly inhibiting transcription factor binding [6] and by initiating chromatin recruitment [7]. This suppression may play a critical role in regulating autosomal gene inheritance [8], X-chromosome inactivation [9,10], and inactivation of repetitive elements [10,11,12] such as long interspersed nuclear elements (LINEs), endogenous retroviruses, and satellite sequences which comprise , 35% of the genome [13]. Conversely, methylation of the gene body [14,15,16] and subsequent chromatin recruitment may promote transcription [17]. Centromere methylation may increase chromosomal stability [18,19], while the greater methyl-density [3,20] and nucleosome- occupancy [21] found in exons, and the sharp transition in methyl- density at the intron-exon boundary [3,20], may act as a marker for splicing [22]. Differences in epigenetic patterns between individuals with similar genetic backgrounds may account for differences in health outcomes [23,24,25] and, as a consequence, can contribute to the etiology of numerous health-related conditions including infertility, stroke, atherosclerosis, obesity, insulin resistance, kidney disease, cancer, neural tube defects and autoimmunity [26,27,28,29,30,31]. Meanwhile, nutritional alteration of the epigenome in utero may be persistent [32,33,34] and may contribute to the health [34,35,36,37] of the offspring in later life. The methyl-groups used for DNA methylation [38,39] are derived from preformed dietary sources (choline, betaine, methionine) or are generated de novo via the folate cycle (Fig. 1) – with vitamin B12 (cobalamin) acting as co-factor in the transfer of methyl-groups from the folate cycle to the methylation cycle. Data from a number of small-scale human studies [40,41,42] suggest that folate depletion and/or repletion can affect DNA methylation in peripheral blood cells. Likewise, Friso et al. [43] reported that methylenetetrahydrofolate reductase (MTHFR) 677 C T homozygous subjects with a sub-optimum folate status had significantly less 5-methyldeoxycytidine (5 mdC) than wild-genotype subjects (regardless of folate status) or folate-replete homozygous subjects. Our research group previously reported differences in DNA methylation level between DNA isolated from coagulated blood clots (which exhibited decreased 5 mdC) vs. uncoagulated EDTA- blood cell pellets (which exhibited no change in 5 mdC) that were dependent on folic acid supplementation and the discontinuation of supplementation in a population-based trial of folic acid supplementation [44]. In humans, prolonged vitamin B12-deficiency can result in numerous clinical ramifications including nerve damage, anemia, digestive and cognitive problems [45]. Additionally, vitamin B12- deficiency might be a risk factor for neural tube defects [46,47]. Despite the important role vitamin B12 plays in one-carbon metabolism (Fig. 1) and DNA methylation, relatively little has been reported on the effect of vitamin B12-deficiency on DNA methylation. Rodents fed vitamin B12-deficient diets [48,49,50] or a diet deficient in several B-vitamins [51] exhibited decreased DNA methylation. In a single case study, Smulders et al. [52] reported that DNA methylation increased 22% in a vitamin B12- deficient human after vitamin B12 intervention. The primary objective of the present study was to describe the effect of vitamin B12-deficiency on global DNA methylation in a female Northern Chinese population of reproductive age. This population has a high ( , 21%) prevalence of vitamin B12- deficiency ( , 148 pmol/L) [53,54]. Participants provided oral informed consent. This was permis- sible as the study posed no more than minimal risk of harm to the participant and was documented with a signature of the consenting investigator as was culturally appropriate for the research setting and involved no procedure for which written consent was normally required outside of a research setting. The study and consent procedures including a waiver for the documentation of informed consent as set forth in 45CFR46.117(c) were approved by the Centers for Disease Control and Prevention Institutional Review Board and the Ethical Committee on Biomedical Research Involving Human Subjects of the Health Science Center, Peking University. The samples used in this study were screening samples from a folic acid intervention study [44,55,56] - the screening/baseline samples were used to identify suitable subjects who met the inclusion criteria to participate in the intervention study (the intervention trial is registered at clinicaltrials.gov: NCT00207558). No intervention was performed on the subjects prior to collection of the samples used for this manuscript. All of the vitamin B12-deficient (plasma vitamin B12 , 148 pmol/L) subjects, identified from the screening samples, were excluded from participation in the subsequent intervention trial and were referred for treatment. Women were recruited from six townships of Xianghe County, Hebei Province, in Northern China, for a population-based folic acid intervention study [44,55,56]. Participants were not exposed to dietary sources of folic acid since folic acid-fortified foods were not available in China. Eligibility requirements included: residence in the townships; not pregnant or breastfeeding, and not plan to become pregnant in the next 9 months; use an IUD for contraception; have a child 2–4 y of age; have no chronic diseases; no supplement use within the last 3 months, and no current prescription medication use [55]. Fasting blood samples from each participant were collected into 7 ml tubes with EDTA, and 3 ml tubes containing no anticoagulants (Vacutainer; Becton Dickinson). Coagulated blood clots (blood clots) were prepared by allowing the blood tubes, containing no anticoagulant, to stand at room temperature for 1–2 hours, as previously described [44]. Sera were separated by centrifugation at 2000 6 g for 15 minutes at 4 u C. After the sera were removed the blood clots were frozen and stored at 2 20 u C. Uncoagulated blood cell pellets (EDTA-blood cell pellets) were prepared within 1 hour of collection by centrifuging the EDTA blood tubes at 2,000 6 g for 15 minutes at 4 u C. After the plasma was removed the uncoagulated EDTA-blood cell pellet was frozen at 2 20 u C. Blood clots and EDTA-blood cell pellets were stored at 2 20 u C before being transported on dry ice to the central laboratory of Peking University Health Science Center where they were stored at 2 70 C. Plasma vitamin B12 was measured in duplicate ...

Context 2

... methylation (dC 5 mdC) patterns are transmitted through the germ line [1] and occur primarily at the 5 9 -dC of the 5 9 -deoxycytidine-deoxyguanosine-3 9 (CpG) motif in somatic cells [2,3,4]. Methylation of transcriptional start sites can regulate gene expression [5] by directly inhibiting transcription factor binding [6] and by initiating chromatin recruitment [7]. This suppression may play a critical role in regulating autosomal gene inheritance [8], X-chromosome inactivation [9,10], and inactivation of repetitive elements [10,11,12] such as long interspersed nuclear elements (LINEs), endogenous retroviruses, and satellite sequences which comprise , 35% of the genome [13]. Conversely, methylation of the gene body [14,15,16] and subsequent chromatin recruitment may promote transcription [17]. Centromere methylation may increase chromosomal stability [18,19], while the greater methyl-density [3,20] and nucleosome- occupancy [21] found in exons, and the sharp transition in methyl- density at the intron-exon boundary [3,20], may act as a marker for splicing [22]. Differences in epigenetic patterns between individuals with similar genetic backgrounds may account for differences in health outcomes [23,24,25] and, as a consequence, can contribute to the etiology of numerous health-related conditions including infertility, stroke, atherosclerosis, obesity, insulin resistance, kidney disease, cancer, neural tube defects and autoimmunity [26,27,28,29,30,31]. Meanwhile, nutritional alteration of the epigenome in utero may be persistent [32,33,34] and may contribute to the health [34,35,36,37] of the offspring in later life. The methyl-groups used for DNA methylation [38,39] are derived from preformed dietary sources (choline, betaine, methionine) or are generated de novo via the folate cycle (Fig. 1) – with vitamin B12 (cobalamin) acting as co-factor in the transfer of methyl-groups from the folate cycle to the methylation cycle. Data from a number of small-scale human studies [40,41,42] suggest that folate depletion and/or repletion can affect DNA methylation in peripheral blood cells. Likewise, Friso et al. [43] reported that methylenetetrahydrofolate reductase (MTHFR) 677 C T homozygous subjects with a sub-optimum folate status had significantly less 5-methyldeoxycytidine (5 mdC) than wild-genotype subjects (regardless of folate status) or folate-replete homozygous subjects. Our research group previously reported differences in DNA methylation level between DNA isolated from coagulated blood clots (which exhibited decreased 5 mdC) vs. uncoagulated EDTA- blood cell pellets (which exhibited no change in 5 mdC) that were dependent on folic acid supplementation and the discontinuation of supplementation in a population-based trial of folic acid supplementation [44]. In humans, prolonged vitamin B12-deficiency can result in numerous clinical ramifications including nerve damage, anemia, digestive and cognitive problems [45]. Additionally, vitamin B12- deficiency might be a risk factor for neural tube defects [46,47]. Despite the important role vitamin B12 plays in one-carbon metabolism (Fig. 1) and DNA methylation, relatively little has been reported on the effect of vitamin B12-deficiency on DNA methylation. Rodents fed vitamin B12-deficient diets [48,49,50] or a diet deficient in several B-vitamins [51] exhibited decreased DNA methylation. In a single case study, Smulders et al. [52] reported that DNA methylation increased 22% in a vitamin B12- deficient human after vitamin B12 intervention. The primary objective of the present study was to describe the effect of vitamin B12-deficiency on global DNA methylation in a female Northern Chinese population of reproductive age. This population has a high ( , 21%) prevalence of vitamin B12- deficiency ( , 148 pmol/L) [53,54]. Participants provided oral informed consent. This was permis- sible as the study posed no more than minimal risk of harm to the participant and was documented with a signature of the consenting investigator as was culturally appropriate for the research setting and involved no procedure for which written consent was normally required outside of a research setting. The study and consent procedures including a waiver for the documentation of informed consent as set forth in 45CFR46.117(c) were approved by the Centers for Disease Control and Prevention Institutional Review Board and the Ethical Committee on Biomedical Research Involving Human Subjects of the Health Science Center, Peking University. The samples used in this study were screening samples from a folic acid intervention study [44,55,56] - the screening/baseline samples were used to identify suitable subjects who met the inclusion criteria to participate in the intervention study (the intervention trial is registered at clinicaltrials.gov: NCT00207558). No intervention was performed on the subjects prior to collection of the samples used for this manuscript. All of the vitamin B12-deficient (plasma vitamin B12 , 148 pmol/L) subjects, identified from the screening samples, were excluded from participation in the subsequent intervention trial and were referred for treatment. Women were recruited from six townships of Xianghe County, Hebei Province, in Northern China, for a population-based folic acid intervention study [44,55,56]. Participants were not exposed to dietary sources of folic acid since folic acid-fortified foods were not available in China. Eligibility requirements included: residence in the townships; not pregnant or breastfeeding, and not plan to become pregnant in the next 9 months; use an IUD for contraception; have a child 2–4 y of age; have no chronic diseases; no supplement use within the last 3 months, and no current prescription medication use [55]. Fasting blood samples from each participant were collected into 7 ml tubes with EDTA, and 3 ml tubes containing no anticoagulants (Vacutainer; Becton Dickinson). Coagulated blood clots (blood clots) were prepared by allowing the blood tubes, containing no anticoagulant, to stand at room temperature for 1–2 hours, as previously described [44]. Sera were separated by centrifugation at 2000 6 g for 15 minutes at 4 u C. After the sera were removed the blood clots were frozen and stored at 2 20 u C. Uncoagulated blood cell pellets (EDTA-blood cell pellets) were prepared within 1 hour of collection by centrifuging the EDTA blood tubes at 2,000 6 g for 15 minutes at 4 u C. After the plasma was removed the uncoagulated EDTA-blood cell pellet was frozen at 2 20 u C. Blood clots and EDTA-blood cell pellets were stored at 2 20 u C before being transported on dry ice to the central laboratory of Peking University Health Science Center where they were stored at 2 70 C. Plasma vitamin B12 was measured in duplicate samples by using the Quantaphase II radioassay (Bio-Rad Laboratories; Product # 191–1044). Subjects were characterized as vitamin B12-deficient (plasma vitamin B12 , 148 pmol/L; n = 305) or vitamin B12-replete (plasma vitamin B12 . 148 pmol/L; n = 1139). Plasma and RBC folate concentrations were measured by microbiological assay [57] while plasma homocysteine was assayed by HPLC with fluorometric detection [58]. Eligible subjects (n = 1,702) were enrolled in the study and provided blood samples. Subjects with B12-deficiency ( , 148 pmol/L) and clot DNA available were included as B12- deficient (n = 248). Subjects with normal B12 status (plasma B12 . 148 pmol/L) and normal hemoglobin concentration ( . 120 g/L) were included as non-deficient controls (n = 128) and were selected to have an even distribution of MTHFR genotypes as previously reported [44]. MTHFR TT genotype was not associated with global DNA methylation at enrollment in either the control [44] or B12-deficient participants (not shown) in clots and therefore was not controlled for statistically in these analyses. Blood clots were shipped to the University of Florida on dry ice for genotyping and for percentage deoxycytidine methylation determination. After the initial blood clot % 5 mdC analysis was completed, a subset of the EDTA-blood ...