We proceeded with a CIT with the 16 CpG sites highlighted in the meQTL analyses to establish the direction of relationship between a causal factor (G, genotype), a potential mediator (M, methylation), and an outcome (Y, NMR). Briefly, the conditions for CIT are (1) G and Y are associated, (2) G is associated with M after adjusting for Y, (3) M is associated with Y after adjusting for G, and (4) G is independent of Y after adjusting for M [51]. We performed CIT using the R script provided by Millstein and colleagues [51]. This CIT R script gives a formal p-value for causal model (‘DNA methylation mediates the effect of SNPs on NMR’), reactive model (‘NMR mediates the effect of SNPs on methylation’), and a causal call based on the two p-values obtained. To account for multiple testing we considered FDR corrected p-values below 0.05 as statistically significant.
