title: Self-reported smoking, serum cotinine, and blood DNA methylation.
abstract: BACKGROUND
  Epigenome-wide profiling of DNA methylation pattern with respect to tobacco smoking has given rise to a new measure of smoking exposure. We investigated the relationships of methylation markers with both cotinine, an established marker of internal smoking exposure, and self-reported smoking.
  
  
  METHODS
  Blood DNA methylation levels across the genome and serum cotinine were measured in 1000 older adults aged 50-75 years. Epigenome-wide scans were performed to identify methylation markers associated with cotinine. The inter-dose-response relationships between the number of cigarettes smoked per day, cotinine concentration, and DNA methylation were modeled by restricted cubic spline regression.
  
  
  RESULTS
  Of 61 CpGs that passed the genome-wide significance threshold (p<1.13×10(-7)), 40 CpGs in 25 chromosomal regions were successfully replicated, showing 0.2-3% demethylation per 10ng/ml increases in cotinine. The strongest associations were observed for several loci at AHRR, F2RL3, 2q37.1, 6p21.33, and GFI1 that were previously identified to be related to self-reported smoking. One locus at RAB34 was newly discovered. Both cotinine and methylation markers exhibited non-linear relationships with the number of cigarettes smoked per day, where the highest rates of increase in cotinine and decreases in methylation were observed at low smoking intensity (1-15 cigarettes/day) and plateaued at high smoking intensity (>15-20 cigarettes/day). A clear linear relationship was observed between cotinine concentration and methylation level. Both cotinine and methylation markers showed similar accuracy in distinguishing current from never smoker, but only methylation markers distinguished former from never smoker with high accuracy.
  
  
  CONCLUSIONS
  Our study corroborates and expands the list of smoking-associated DNA methylation markers. Methylation levels were linearly related to cotinine concentration and provided accurate measures for both current and past smoking exposure.
authors.affiliation: Division of Clinical Epidemiology and Aging Research, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Electronic address: y.zhang@dkfz-heidelberg.de.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Division of Clinical Epidemiology and Aging Research, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Electronic address: ines.florath@curtin.edu.au.
authors.forename: Ines
authors.initials: I
authors.lastname: Florath
authors.affiliation: Division of Clinical Epidemiology and Aging Research, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Electronic address: k.saum@dkfz-heidelberg.de.
authors.forename: Kai-Uwe
authors.initials: KU
authors.lastname: Saum
authors.affiliation: Division of Clinical Epidemiology and Aging Research, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany; Division of Preventive Oncology, German Cancer Research Center (DKFZ) and National Center for Tumor Diseases (NCT), Im Neuenheimer Feld 460, D-69120 Heidelberg, Germany; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Electronic address: h.brenner@dkfz-Heidelberg.de.
authors.forename: Hermann
authors.initials: H
authors.lastname: Brenner
mesh_terms: D000368:Aged; D015415:Biomarkers / Q000097:blood; D015331:Cohort Studies; D003367:Cotinine / Q000097:blood*; D004247:DNA / Q000097:blood*; D019175:DNA Methylation; D005260:Female; D005858:Germany / Q000453:epidemiology; D006801:Humans; D008297:Male; D008875:Middle Aged; D057566:Self Report; D012907:Smoking / Q000453:epidemiology*
chemical_list: D015415:Biomarkers; D004247:DNA; D003367:Cotinine
keywords: Cotinine; DNA methylation; Dose-response relationship; Epigenome-wide association study; Smoking