title: Adjustment of Cell-Type Composition Minimizes Systematic Bias in Blood DNA Methylation Profiles Derived by DNA Collection Protocols.
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.
authors.affiliation: 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.
authors.forename: Yuh
authors.initials: Y
authors.lastname: Shiwa
authors.affiliation: 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.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Hachiya
authors.affiliation: 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.
authors.forename: Ryohei
authors.initials: R
authors.lastname: Furukawa
authors.affiliation: 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.
authors.forename: Hideki
authors.initials: H
authors.lastname: Ohmomo
authors.affiliation: 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.
authors.forename: Kanako
authors.initials: K
authors.lastname: Ono
authors.affiliation: Department of Biobank, Tohoku Medical Megabank Organization, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8573, Japan.
authors.forename: Hisaaki
authors.initials: H
authors.lastname: Kudo
authors.affiliation: Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.|Center for Cohort Studies, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Hata
authors.affiliation: Preventive Medicine and Epidemiology, Tohoku Medical Megabank Organization, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8573, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Hozawa
authors.affiliation: Epidemiology and Prevention Group, Research Center for Cancer Prevention and Screening, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
authors.forename: Motoki
authors.initials: M
authors.lastname: Iwasaki
authors.affiliation: Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Matsuda
authors.affiliation: Department of Biobank, Tohoku Medical Megabank Organization, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8573, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Minegishi
authors.affiliation: 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.|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.|Division of Biomedical Information Analysis, Institute for Biomedical Science, Iwate Medical University, 2-1-1 Nishitokuda, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan.
authors.forename: Mamoru
authors.initials: M
authors.lastname: Satoh
authors.affiliation: Department of Hygiene and Preventive Medicine, Iwate Medical University, 2-1-1 Nishitokuda, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan.
authors.forename: Kozo
authors.initials: K
authors.lastname: Tanno
authors.affiliation: Epidemiology and Prevention Group, Research Center for Cancer Prevention and Screening, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
authors.forename: Taiki
authors.initials: T
authors.lastname: Yamaji
authors.affiliation: Department of Preventive Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Wakai
authors.affiliation: 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.|Department of Anatomy, School of Medicine, Iwate Medical University, 2-1-1 Nishitokuda, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan.
authors.forename: Jiro
authors.initials: J
authors.lastname: Hitomi
authors.affiliation: Department of Environmental Medicine, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kiyohara
authors.affiliation: Laboratory for Genotyping Development, Center for Genomic Medicine, RIKEN, Yokohama, Japan.
authors.forename: Michiaki
authors.initials: M
authors.lastname: Kubo
authors.affiliation: Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, Nagoya, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Tanaka
authors.affiliation: Epidemiology and Prevention Group, Research Center for Cancer Prevention and Screening, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
authors.forename: Shoichiro
authors.initials: S
authors.lastname: Tsugane
authors.affiliation: Department of Integrative Genomics, Tohoku Medical Megabank Organization, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8573, Japan.|Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Seiryo-machi 2-1, Aoba-ku, Sendai 980-8575, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamamoto
authors.affiliation: 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.|Department of Neuroscience, Institute for Biomedical Science, Iwate Medical University, 2-1-1 Nishitokuda, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Sobue
authors.affiliation: 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.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Shimizu
mesh_terms: D015982:Bias; D001773:Blood Cells / Q000378:metabolism*; D001800:Blood Specimen Collection / Q000379:methods* / Q000592:standards; D018899:CpG Islands / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D019175:DNA Methylation; D005260:Female; D005434:Flow Cytometry; D055106:Genome-Wide Association Study / Q000379:methods / Q000592:standards; D006801:Humans; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods / Q000592:standards
chemical_list: D004247:DNA