Abstract
Variants in serotonergic genes are implicated in nicotine dependence (ND) in subjects of European and African origin, but their involvement with smoking in Asians is largely unknown. Moreover, mechanisms underlying the ND risk-associated single-nucleotide polymorphisms (SNPs) in these genes are rarely investigated. The Fagerström Test for Nicotine Dependence (FTND) score was used to assess ND in 2616 male Chinese Han smokers. Both association and interaction analysis were used to examine the association of variants in the serotonergic genes with FTND. Further, expression and methylation quantitative trait loci (cis-mQTL) analysis was employed to determine the association of individual SNPs with the extent of methylation of each CpG locus. Individual SNP-based association analysis revealed that rs1176744 in HTR3B was marginally associated with FTND (p = 0.042). Haplotype-based association analysis found that one major haplotype, T-T-A-G, formed by SNPs rs3758987-rs4938056-rs1176744-rs2276305, located in the 5′ region of HTR3B, showed a significant association with FTND (p = 0.00025). Further, a significant genetic interactive effect affecting ND was detected among SNPs rs10160548 in HTR3A, and rs3758987, rs2276305, and rs1672717 in HTR3B (p = 0.0074). Finally, we found four CpG sites (CpG_4543549, CpG_4543464, CpG_4543682, and CpG_4546888) to be significantly associated with three cis-mQTL SNPs (i.e., rs3758987, rs4938056, and rs1176744) located in our detected haplotype within HTR3B. In sum, we showed SNP rs1176744 (Tyr129Ser) to be associated with ND. Together with the SNPs rs3758987 and rs4938056 in HTR3B, they formed a major haplotype, which had significant association with ND. We further showed these SNPs contribute to ND through four methylated sites in HTR3B. All these findings suggest that variants in the serotonergic system play an important role in ND in the Chinese Han population. More importantly, these findings demonstrated that the involvement of this system in ND is through gene-by-gene interaction and methylation.
Highlights
Cigarette smoking is still a serious issue all over the world
We identified four methylation sites (i.e., CpG_4543549, CpG_4543464, CpG_4543682, and CpG_4546888) associated significantly with three expression and methylation quantitative trait loci single-nucleotide polymorphisms (SNPs), which are located within the major haplotype detected in HTR3B
Together with rs3758987, rs4938056, and rs2276305, they consist of a major haplotype, T-T-A-G, which is associated significantly with ND. We found that this SNP acts as a cis-mQTL to alter the extent of methylation of CpG_4546888 in blood and cis-eQTL to modulate the mRNA expression of HTR3B in the substantia nigra
Summary
Cigarette smoking is still a serious issue all over the world. Each year, smoking causes about six million deaths worldwide, of which more than five million result directly from cigarette smoking[1]. As a result of 5-HT3 receptor activation, the serotonin-gated ion channel undergoes rapid depolarization and desensitization, releases stored neurotransmitter, suggesting an important role of the serotonergic system in neuronal circuitry involved in drug abuse[30]. There were some genetic studies on serotonergic genes in drug abuse, for instance, smoking[32], alcohol dependence[33,34,35], and other drugs dependencies[33,36]. There have been limited genetic studies on the relation of serotonergic genes to smoking in Chinese populations. In this study, we applied various genetic analyses to investigate the role of serotonergic genes in ND in a less commonly investigated population, Chinese Han smokers. To explore the underlying mechanisms of ND, we established a link for those risk variants for ND with differential DNA methylation loci by performing methylation quantitative trait locus (cis-mQTL) analysis
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