PLoS One. 2016 Jan 22;11(1):e0147519. doi: 10.1371/journal.pone.0147519. eCollection 2016.

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

Shiwa Y^1,², Hachiya T^1,², Furukawa R², Ohmomo H², Ono K², Kudo H³, Hata J^4,⁵, Hozawa A⁶, Iwasaki M⁷, Matsuda K⁸, Minegishi N³, Satoh M^1,^2,^9,¹⁰, Tanno K¹¹, Yamaji T⁷, Wakai K¹², Hitomi J^13,¹⁴, Kiyohara Y¹⁵, Kubo M¹⁶, Tanaka H¹⁷, Tsugane S⁷, Yamamoto M^18,¹⁹, Sobue K^20,²¹, Shimizu A².

Author information

1: 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.
2: 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.
3: Department of Biobank, Tohoku Medical Megabank Organization, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8573, Japan.
4: Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
5: Center for Cohort Studies, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
6: Preventive Medicine and Epidemiology, Tohoku Medical Megabank Organization, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8573, Japan.
7: Epidemiology and Prevention Group, Research Center for Cancer Prevention and Screening, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
8: Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
9: Community Medical Supports and Health Record Informatics, Iwate Tohoku Medical Megabank Organization, Iwate Medical University Disaster Reconstruction Center, 2-1-1 Nishitokuda, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan.
10: Division of Biomedical Information Analysis, Institute for Biomedical Science, Iwate Medical University, 2-1-1 Nishitokuda, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan.
11: Department of Hygiene and Preventive Medicine, Iwate Medical University, 2-1-1 Nishitokuda, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan.
12: Department of Preventive Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
13: Deputy Executive Director, Iwate Tohoku Medical Megabank Organization, Disaster Reconstruction Center, Iwate Medical University, 2-1-1 Nishitokuda, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan.
14: Department of Anatomy, School of Medicine, Iwate Medical University, 2-1-1 Nishitokuda, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan.
15: Department of Environmental Medicine, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
16: Laboratory for Genotyping Development, Center for Genomic Medicine, RIKEN, Yokohama, Japan.
17: Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, Nagoya, Japan.
18: Department of Integrative Genomics, Tohoku Medical Megabank Organization, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8573, Japan.
19: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Seiryo-machi 2-1, Aoba-ku, Sendai 980-8575, Japan.
20: Executive Director, Iwate Tohoku Medical Megabank Organization, Disaster Reconstruction Center, Iwate Medical University, 2-1-1 Nishitokuda, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan.
21: Department of Neuroscience, Institute for Biomedical Science, Iwate Medical University, 2-1-1 Nishitokuda, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan.

Abstract

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.