To understand a potential mechanism of action responsible for the ASE, we tested 3′UTR SNPs of the genes with ASE to determine whether they affected gene regulation. Because both heterozygous alleles exist in the same cell and are exposed to the same environment, ASE differences arise due to regulation in cis. Among the genes with AUD-associated differences in ASE in at least one of the four brain regions, we identified 565 SNPs in the 3′UTR regions; 437 SNPs in 61 genes had at least one heterozygote in either the AUD or control group (Supplementary Table 3). We adapted a high-throughput reporter assay, PASSPORT-seq [13], to identify 3′UTR variants that affected gene expression (RNA levels) in two human neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2) (Supplementary Table 4). We detected expression of the reference and alternative alleles in both SH-SY5Y and SK-N-BE(2) cells in 362 (82.8%) of the 437 SNPs screened. UMIs were counted to quantify the expression of the reference and alternative alleles for each SNP, and a generalized linear model was applied to identify the variants that showed ASE.
