To identify molecular biological mechanisms whereby associated SNPs may affect OPRM1 function, we first examined its position relative to newly identified OPRM1 structural elements. SNP rs563649, the strongest contributor to the pain-sensitivity phenotype, was located in the evolutionarily conserved footprint of the OPRM1 gene downstream from the predicted human exon 13, which is not represented in known human and mouse transcripts. Comparative sequence analysis showed the absence of conserved 3′-splicing sites and intronic/exonic enhancers in the vicinity of predicted human exon 13 region, suggesting that rs563649 may be situated within a novel human exon that extends downstream in primate lineages relative to rodents (Fig. 1B, Table 1). To test this hypothesis, we performed RT–PCR using RNA isolated from the human brain tissues (Supplementary Material, Table S3), which express high levels of OPRM1 and are known to respond to opioid treatment. The forward primers were designed to predicted exon 13 and reverse primers to exon 2 (Fig. 2A–C; Supplementary Material, Table S4). RT–PCR results showed that the human exon 13 is ∼0.8 kb longer than the mouse exon 13 and carries
