Normalized ratios of gene expression levels from the single OPRM1 probe available in BrainCloud (Figure S1) were log-transformed and tested for association with additive three-level SNP genotypes using the BrainCloud software (32). Genotyped SNP association results were generated from the best fit general linear model with race, sex, age, life stage (infant, child, teen, and adult), and an age-by-life stage interaction included as the range of covariates. Inclusion of age, life stage, and the corresponding interaction was used to account for nonlinear trajectories of age-dependent gene expression over the lifespan (33). We evaluated associations overall and stratified by ancestry for the 103 SNPs genotyped across OPRM1 and its 100kb flanking regions. A P value threshold of 0.00125, which takes into account the correlations among the 103 OPRM1 SNPs (α=0.05/40 independent tests) (35, 36), was used to declare statistical significance. Given the hypothesis-generating nature of our cis-eQTL mapping, SNPs associated with OPRM1 expression at the nominal significance threshold of P<0.05 were carried forward for association testing with heroin addiction.
