Tobacco-Smoking-Related Differential DNA Methylation: 27K Discovery and Replication Lutz P. Breitling, Rongxi Yang, Bernhard Korn, Barbara Burwinkel,

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Tobacco-Smoking-Related Differential DNA Methylation: 27K Discovery and Replication Lutz P. Breitling, Rongxi Yang, Bernhard Korn, Barbara Burwinkel, Hermann Brenner The American Journal of Human Genetics Volume 88, Issue 4, Pages 450-457 (April 2011) DOI: 10.1016/j.ajhg.2011.03.003 Copyright © 2011 The American Society of Human Genetics Terms and Conditions

Figure 1 Mixed Linear Regression p Values of the Sex-Adjusted Association of Smoking Status with Methylation Intensities at 27,578 CpG Sites –log10(P) versus –log10(rank) plot. The American Journal of Human Genetics 2011 88, 450-457DOI: (10.1016/j.ajhg.2011.03.003) Copyright © 2011 The American Society of Human Genetics Terms and Conditions

Figure 2 Distribution of Methylation Intensities by Smoking Status at the CpG Site cg03636183 Located in F2RL3 (A) Histograms of “beta values” obtained in the 27K genome-wide screening (discovery measurements). (B) cg03636183 methylation extent as determined by the Sequenom MassArray, validating the discovery measurements in the same samples. (C) cg03636183 methylation measured by the Sequenom MassArray in a nonoverlapping replication sample set. The American Journal of Human Genetics 2011 88, 450-457DOI: (10.1016/j.ajhg.2011.03.003) Copyright © 2011 The American Society of Human Genetics Terms and Conditions

Figure 3 Example Results of the Sequenom MassArray Analysis of cg03636183 Methylation Extents The graphs show the detection intensity (y axis) of amplicon fragments after base-specific cleavage. The latter results in fragments of different masses (x axis) for originally methylated and nonmethylated CpG sites. The proportion of methylated CpG at this locus can thus be determined by the ratio of detection intensities at masses corresponding to fragments originating from methylated and nonmethylated CpG sites (blue and red vertical lines, respectively). The graphs originating from samples with 17%, 59%, and 98% methylation at cg03636183 (from left to right) show clearly distinguishable peaks. The American Journal of Human Genetics 2011 88, 450-457DOI: (10.1016/j.ajhg.2011.03.003) Copyright © 2011 The American Society of Human Genetics Terms and Conditions