Genetic and Epigenetic Analysis Revealing Variants in the NCAM1-TTC12-ANKK1-DRD2 Cluster Associated Significantly With Nicotine Dependence in Chinese Han Smokers.

Backgrounds: Although studies have demonstrated that the NCAM1-TTC12-ANKK1-DRD2 gene cluster plays essential roles in addictions in subjects of European and African origin, study of Chinese Han subjects is limited. Further, the underlying biological mechanisms of detected associations are largely unknown.

Methods: Sixty-four single-nucleotide polymorphisms (SNPs) in this cluster were analyzed for association with Fagerstrőm Test for Nicotine Dependence score (FTND) and cigarettes per day (CPD) in male Chinese Han smokers (N = 2616). Next-generation bisulfite sequencing was used to discover smoking-associated differentially methylated regions (DMRs). Both cis-eQTL and cis-mQTL analyses were applied to assess the cis-regulatory effects of these risk SNPs.

Results: Association analysis revealed that rs4648317 was significantly associated with FTND and CPD (p = .00018; p = .00072). Moreover, 14 additional SNPs were marginally significantly associated with FTND or CPD (p = .05-.01). Haplotype-based association analysis showed that one haplotype in DRD2, C-T-A-G, formed by rs4245148, rs4581480, rs4648317, and rs11214613, was significantly associated with CPD (p = .0005) and marginally associated with FTND (p = .003). Further, we identified four significant smoking-associated DMRs, three of which are located in the DRD2/ANKK1 region (p = .0012-.00005). Finally, we found five significant CpG-SNP pairs (p = 7.9 × 10-9-6.6 × 10-6) formed by risk SNPs rs4648317, rs11604671, and rs2734849 and three methylation loci.

Conclusions: We found two missense variants (rs11604671; rs2734849) and an intronic variant (rs4648317) with significant effects on ND and further explored their mechanisms of action through expression and methylation analysis. We found the majority of smoking-related DMRs are located in the ANKK1/DRD2 region, indicating a likely causative relation between non-synonymous SNPs and DMRs.

Implications: This study shows that there exist significant association of variants and haplotypes in ANKK1/DRD2 region with ND in Chinese male smokers. Further, this study also shows that DNA methylation plays an important role in mediating such associations.