Shiwa Yuh, Hachiya Tsuyoshi, Furukawa Ryohei, Ohmomo Hideki, Ono Kanako, Kudo Hisaaki, Hata Jun, Hozawa Atsushi, Iwasaki Motoki, Matsuda Koichi, Minegishi Naoko, Satoh Mamoru, Tanno Kozo, Yamaji Taiki, Wakai Kenji, Hitomi Jiro, Kiyohara Yutaka, Kubo Michiaki, Tanaka Hideo, Tsugane Shoichiro, Yamamoto Masayuki, Sobue Kenji, Shimizu Atsushi
Division of Biobank and Data Management, Iwate Tohoku Medical Megabank Organization, Iwate Medical University Disaster Reconstruction Center, 2-1-1 Nishitokuda, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan.
Division of Biomedical Information Analysis, Iwate Tohoku Medical Megabank Organization, Iwate Medical University Disaster Reconstruction Center, 2-1-1 Nishitokuda, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan.
PLoS One. 2016 Jan 22;11(1):e0147519. doi: 10.1371/journal.pone.0147519. eCollection 2016.
Differences in DNA collection protocols may be a potential confounder in epigenome-wide association studies (EWAS) using a large number of blood specimens from multiple biobanks and/or cohorts. Here we show that pre-analytical procedures involved in DNA collection can induce systematic bias in the DNA methylation profiles of blood cells that can be adjusted by cell-type composition variables. In Experiment 1, whole blood from 16 volunteers was collected to examine the effect of a 24 h storage period at 4°C on DNA methylation profiles as measured using the Infinium HumanMethylation450 BeadChip array. Our statistical analysis showed that the P-value distribution of more than 450,000 CpG sites was similar to the theoretical distribution (in quantile-quantile plot, λ = 1.03) when comparing two control replicates, which was remarkably deviated from the theoretical distribution (λ = 1.50) when comparing control and storage conditions. We then considered cell-type composition as a possible cause of the observed bias in DNA methylation profiles and found that the bias associated with the cold storage condition was largely decreased (λ adjusted = 1.14) by taking into account a cell-type composition variable. As such, we compared four respective sample collection protocols used in large-scale Japanese biobanks or cohorts as well as two control replicates. Systematic biases in DNA methylation profiles were observed between control and three of four protocols without adjustment of cell-type composition (λ = 1.12-1.45) and no remarkable biases were seen after adjusting for cell-type composition in all four protocols (λ adjusted = 1.00-1.17). These results revealed important implications for comparing DNA methylation profiles between blood specimens from different sources and may lead to discovery of disease-associated DNA methylation markers and the development of DNA methylation profile-based predictive risk models.
在全表观基因组关联研究（EWAS）中，使用来自多个生物样本库和/或队列的大量血液样本时，DNA采集方案的差异可能是一个潜在的混杂因素。我们在此表明，DNA采集过程中涉及的分析前程序可在血细胞的DNA甲基化谱中诱导系统性偏差，而这种偏差可通过细胞类型组成变量进行调整。在实验1中，采集了16名志愿者的全血，以研究4℃下24小时储存期对使用Infinium HumanMethylation450 BeadChip阵列测量的DNA甲基化谱的影响。我们的统计分析表明，在比较两个对照重复样本时，超过45万个CpG位点的P值分布与理论分布相似（在分位数-分位数图中，λ = 1.03），而在比较对照和储存条件时，该分布与理论分布有显著偏差（λ = 1.50）。然后，我们将细胞类型组成视为观察到的DNA甲基化谱偏差的一个可能原因，并发现通过考虑细胞类型组成变量，与冷藏条件相关的偏差大幅降低（调整后的λ = 1.14）。因此，我们比较了日本大规模生物样本库或队列中使用的四种样本采集方案以及两个对照重复样本。在未调整细胞类型组成的情况下，对照与四种方案中的三种之间观察到了DNA甲基化谱的系统性偏差（λ = 1.12 - 1.45），而在对所有四种方案都调整细胞类型组成后，未观察到明显偏差（调整后的λ = 1.00 - 1.17）。这些结果揭示了在比较来自不同来源的血液样本之间的DNA甲基化谱时的重要意义，并可能有助于发现与疾病相关的DNA甲基化标记以及基于DNA甲基化谱的预测风险模型的开发。
