For annotation of the plausible role of the highlighted SNPs, we considered three alternative hypotheses: (A) SNPs directly affect the function of CYP2A6 enzyme, or are in LD with functional variants, resulting in changes in NMR, (B) DNA methylation mediates the effect of SNPs on NMR, and (C) NMR mediates the effect of SNPs on methylation. To test hypothesis A, the potential functional consequences of the associating SNPs were predicted using the Ensembl Variant Effect Predictor database (http://www.ensembl.org/index.html) (includes predictions PolyPhen and SIFT). LD patterns were estimated by using the ‘solid spine of LD’ method of the program Haploview [45] from GWAS data generated in the FINRISK 1992, 1997, 2002, and 2007 cohorts (N = 19857). Similarly, minor allele frequencies representative of the Finnish population were obtained from the FINRISK sample (N = 19857). To test hypotheses B and C we proceeded with methylation quantitative loci analyses and Causal Inference Test (CIT).
