PLoS ONE (Jan 2016)

Adjustment of Cell-Type Composition Minimizes Systematic Bias in Blood DNA Methylation Profiles Derived by DNA Collection Protocols.

  • Yuh Shiwa,
  • Tsuyoshi Hachiya,
  • Ryohei Furukawa,
  • Hideki Ohmomo,
  • Kanako Ono,
  • Hisaaki Kudo,
  • Jun Hata,
  • Atsushi Hozawa,
  • Motoki Iwasaki,
  • Koichi Matsuda,
  • Naoko Minegishi,
  • Mamoru Satoh,
  • Kozo Tanno,
  • Taiki Yamaji,
  • Kenji Wakai,
  • Jiro Hitomi,
  • Yutaka Kiyohara,
  • Michiaki Kubo,
  • Hideo Tanaka,
  • Shoichiro Tsugane,
  • Masayuki Yamamoto,
  • Kenji Sobue,
  • Atsushi Shimizu

DOI
https://doi.org/10.1371/journal.pone.0147519
Journal volume & issue
Vol. 11, no. 1
p. e0147519

Abstract

Read online

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.