Hypomethylation of Serum Blood Clot DNA, but Not Plasma EDTA-Blood Cell Pellet DNA, from Vitamin B12-Deficient Subjects

<div><p>Vitamin B12, a co-factor in methyl-group transfer, is important in maintaining DNA (deoxycytidine) methylation. Using two independent assays we examined the effect of vitamin B12-deficiency (plasma vitamin B12<148 pmol/L) on DNA methylation in women of childbearing age. Coagulated blood clot DNA from vitamin B12-deficient women had significantly (p<0.001) lower percentage deoxycytidine methylation (3.23±0.66%; n = 248) and greater [3 H]methyl-acceptance (42,859±9,699 cpm; n = 17) than DNA from B12-replete women (4.44±0.18%; n = 128 and 26,049±2,814 cpm; n = 11) [correlation between assays: r = –0.8538; p<0.001; n = 28]. In contrast, uncoagulated EDTA-blood cell pellet DNA from vitamin B12-deficient and B12-replete women exhibited similar percentage methylation (4.45±0.15%; n = 77 vs. 4.47±0.15%; n = 47) and [3 H]methyl-acceptance (27,378±4,094 cpm; n = 17 vs. 26,610±2,292 cpm; n = 11). Therefore, in simultaneously collected paired blood samples, vitamin B12-deficiency was associated with decreased DNA methylation only in coagulated samples. These findings highlight the importance of sample collection methods in epigenetic studies, and the potential impact biological processes can have on DNA methylation during collection.</p></div

Putative mechanisms for the conversion of 5 mdC to dC in DNA.

<p>Reactions: 1) 5 mdC nucleases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Steinberg1" target="_blank">[91]</a>; 2) 5 mdC glycosylase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Vairapandi1" target="_blank">[92]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Vairapandi2" target="_blank">[93]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Kress1" target="_blank">[94]</a>; 3) 5 mdC deaminase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Metivier1" target="_blank">[84]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Kangaspeska1" target="_blank">[87]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Rai1" target="_blank">[95]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Wijesinghe1" target="_blank">[96]</a>; 4) thymine-DNA glycosylases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Neddermann1" target="_blank">[97]</a>; 5) 5 mdC hydroxylase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Ito1" target="_blank">[63]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Tahiliani1" target="_blank">[64]</a>; 6) 5 hmC glycosylase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Cannon1" target="_blank">[98]</a>; 7) AP endonuclease/phosphodiesterase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Demple1" target="_blank">[99]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Lindahl1" target="_blank">[100]</a>; 8) DNA polymerase/DNA ligase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Lindahl1" target="_blank">[100]</a>; 9) 5 hmdC dehydroxymethylase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Liutkeviciute1" target="_blank">[88]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Chen2" target="_blank">[89]</a>; 10) DNA demethylase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Bhattacharya1" target="_blank">[101]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Cervoni1" target="_blank">[102]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Ramchandani1" target="_blank">[103]</a>.</p

Schematic of the One-Carbon cycle.

<p>Vitamin B12 is a co-factor in the transfer of methyl-groups (CH₃−) from folate to methionine for use <i>in situ</i> methylation of deoxycytidine (dC) to in 5-methyldeoxycytidine (5 mdC) in DNA. Choline, betaine and methionine can be derived from the diet or synthesized <i>in vivo</i>. SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine.</p

Effect of vitamin B12-deficiency on % 5 mdC¹ and [3 H]methyl-acceptance capacity² for DNA extracted from coagulated blood clots³ and uncoagulated EDTA-blood cell pellets⁴.

1<p>Methyldeoxycytidine as a percentage of total deoxycytidine in DNA digests, measured by LC-MS/MS (see Methods).</p>2<p>Capacity of DNA to incorporate [3 H] from [3 H-methyl]SAM (cpm incorporated per 250 ng DNA) in the presence of Sssi DNA methyltransferase (see Methods).</p>3<p>Post-centrifugation, blood clots were retained after serum was removed from plain blood tubes.</p>4<p>Post-centrifugation, blood pellets were retained after plasma was removed from EDTA blood tubes.</p>5<p>Subjects with plasma vitamin B12>148 pmol/L.</p>6<p>Subjects with plasma vitamin B12<148 pmol/L.</p><p>Power analysis: >80% power to detect a *4%, **10%, or ***15% difference in means <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065241#pone.0065241-Lenth1" target="_blank">[62]</a>.</p

Plot of % 5-methyldeoxycytidine versus [³H]methyl-acceptance capacity of DNA extracted from coagulated blood clots.

<p>DNA samples were from (•) vitamin B12-deficient (plasma vitamin B12<148 pmol/L) or (□) vitamin B12-replete subjects (plasma vitamin B12>148 pmol/L). % 5-methyldeoxycytidine: 5-methyldeoxycytidine as a percentage of total deoxycytidine in DNA digests as measured by LC-MS/MS (see Methods). [3 H]Methyl-acceptance: [3 H] (cpm) from [3 H-methyl]SAM incorporated into 250 ng of DNA in the presence of Sssi DNA methyltransferase.</p

Basic Biochemical Characteristics of Subjects.

1<p>Subjects with plasma vitamin B12 more than 148 pmol/L.</p>2<p>Subjects with plasma vitamin B12 less than 148 pmol/L.</p