To gain insight into the potential molecular mechanisms linking PCDH-α, -β and -γ gene cluster SNPs to NicW risk, we surveyed the three NicW-associated SNPs and their flanking sequences for evidence of potential regulatory effects. As shown in Figure 4A, rs31746 mapped to a region with marked DNAse1 hypersensitivity (HS), while rs31743 and rs246592 did not. The DNAse1 HS site presented in Figure 4A contains HS5-1a and HS5-1b, components of a previously characterized neuron-specific enhancer element (HS5-1) that regulates the expression of PCDH-α, -β and -γ genes.4856 Rs31746 mapped to within a 113 base-pair element that corresponds to HS5-1a.47 CCCTC-binding factor (CTCF), which is required for the regulatory function of HS5-1, binds to HS5-1a (Figure 4A). Based on the position of rs31746 within this regulatory element, we hypothesized that rs31746 could affect PCDH-α, -β and -γ gene expression. To test this, we used BrainCloud human PFC gene expression data to query the association of rs31746 with the expression of 55 different PCDH-α, -β and -γ gene cluster mRNAs that were targeted by 63 probes. We observed a robust association between rs31746 and PCDHβ8 mRNA expression (Figure 4B). The withdrawal risk allele, rs31746*A, was associated with lower PCDHβ8 mRNA expression (p=5.32×10−6).
