Supplementary Material for: Impact of methyl-donor micronutrient supplementation on DNA methylation patterns: A systematic review and meta-analysis of in vitro, animal and human studies

Background: DNA methylation patterns are directly associated with diverse metabolic disorders. The status of methyl-donor micronutrients has been associated with DNA methylation levels, and altered ingestion of folate, choline, betaine, B vitamins and methionine may impact genes both globally and at the level of promoter regions. Despite this, the role of methyl-donor micronutrient supplementation on DNA methylation profiles is currently unclear. Objectives: The aims of this systematic review and meta-analysis were to identify and synthesize the evidence about methyl-donor nutrients supplementation on DNA methylation profile. Methods: A systematic literature search was performed in MEDLINE, EMBASE, SCOPUS and Web of Sciences databases with a combination of terms related to DNA methylation assessment, supplementation and methyl-donor nutrients. Studies (in vitro, animal models or human clinical trials) were included if DNA methylation levels after any kind of methyl-donor micronutrient supplementation or treatment was investigated. Studies were assessed for bias using Revised Cochrane risk-of-bias tool for randomized trials, Risk Of Bias in Non-randomized Studies of Interventions or Systematic Review Centre for Laboratory Animal Experimentation tools. Data was extracted from studies measuring DNA methylation level in any sample or tissue, following any kind of methyl-donor micronutrient supplementation or treatment. Separate random-effects meta-analyses were performed for animal model studies and human clinical trials, which examined the effects of folic acid supplementation on DNA methylation. Results: Fifty-seven studies were included in the systematic review: 18 human clinical trials, 35 in animal model and 4 in vitro studies. Concerning overall risk of bias, most of the studies were classified as “high risk” or “some concerns”. Meta-analysis with meta-regression from studies in animal models showed that folic acid dose significantly affected DNA methylation and that high and very high dose showed increases in DNA methylation when compared to low doses. However, meta-analysis from human clinical trials showed that folic acid supplementation did not promote significant changes in DNA methylation when compared to placebo. Conclusion: Folic acid supplementation may change global DNA methylation levels in animals supplemented with high, as compared to low, doses. Heterogeneity in studies and supplementation protocols make it difficult to establish clinical recommendations. However, these effects, even if small, might be of clinical importance in the management of patients with diseases related to DNA hypomethylation.

背景：DNA甲基化（DNA methylation）谱与多种代谢紊乱直接相关。甲基供体微量营养素（methyl-donor micronutrients）的状态与DNA甲基化水平密切相关，而叶酸（folate）、胆碱（choline）、甜菜碱（betaine）、B族维生素（B vitamins）及甲硫氨酸（methionine）的摄入变化，可能在全局层面及启动子区域（promoter regions）水平影响基因表达。尽管如此，目前甲基供体微量营养素补充对DNA甲基化谱的作用仍不明确。
研究目的：本系统评价与荟萃分析旨在识别并整合有关甲基供体营养素补充对DNA甲基化谱影响的相关证据。
研究方法：本研究在MEDLINE、EMBASE、SCOPUS及Web of Sciences数据库中开展系统文献检索，检索词组合涵盖DNA甲基化评估、补充干预及甲基供体营养素相关术语。纳入所有涉及任意形式甲基供体微量营养素补充或干预后DNA甲基化水平检测的研究，包括体外实验、动物模型研究及人体临床试验。研究偏倚风险采用修订版Cochrane随机试验偏倚风险工具、非随机干预研究偏倚风险工具，以及实验动物系统评价中心工具进行评估。从所有涉及任意形式甲基供体微量营养素补充或干预后，任意样本或组织中DNA甲基化水平检测的研究中提取数据。针对评估叶酸补充对DNA甲基化影响的动物模型研究及人体临床试验，分别开展随机效应荟萃分析。
研究结果：本系统评价共纳入57项研究：18项人体临床试验、35项动物模型研究及4项体外实验研究。就整体偏倚风险而言，大多数研究被归类为“高偏倚风险”或“存在一定偏倚风险担忧”。针对动物模型研究的荟萃分析及元回归结果显示，叶酸剂量对DNA甲基化具有显著影响；相较于低剂量组，高剂量及极高剂量组的DNA甲基化水平显著升高。然而，针对人体临床试验的荟萃分析结果显示，与安慰剂组相比，叶酸补充并未使DNA甲基化水平发生显著变化。
研究结论：与低剂量补充相比，高剂量叶酸补充可改变动物的全局DNA甲基化水平。由于研究及补充方案存在异质性，难以制定明确的临床推荐意见。但即便此类影响较为微弱，在DNA低甲基化相关疾病患者的临床管理中仍可能具有重要临床价值。