Our previous genetic studies demonstrated that variants of the g-Aminobutyric acid B receptor subunit 2 (GPR51) and neurotrophic tyrosine kinase receptor type 2 (NTRK2) genes are significantly associated with nicotine dependence (ND) in smokers. However, whether such genetic associations lead to changes in the expression of the two genes in response to nicotine remains undetermined. In this study, we investigated the regulatory effect of nicotine on the expression of Gpr51 and Ntrk2 in seven rat brain regions during the administration of nicotine in a daily dose of 3.15 mg/kg for 7 days. With quantitative real-time RT-PCR, we found that nicotine increased the mRNA of Gpr51 by 70, 78, and 32% in the amygdala, striatum, and prefrontal cortex (PFC), respectively, but decreased by 54% in the nucleus accumbens (NA). The Gpr51 protein was upregulated by nicotine in the amygdala (26%), striatum (73%), PFC (28%), and medial basal hypothalamus (MBH; 19%) but downregulated in the NA (72%). Similarly, the mRNA level of Ntrk2 was enhanced by nicotine in the striatum (86%) and PFC (38%), but decreased in the NA (46%) and ventral tegmental area (VTA; 49%). A significant change in protein expression was also obtained for Ntrk2 in the PFC (24%), MBH (33%), NA (33%), and VTA (70%). Interestingly, these two genes showed a closely coordinated expression pattern in response to nicotine in most of the brain regions examined. In summary, our results demonstrate that the expression of Gpr51 and Ntrk2 is significantly regulated by nicotine at both the mRNA and protein levels in various brain regions, which provides further evidence that these two genes are involved in the etiology of ND, as reported in our previous genetic association studies in humans.

Our previous linkage study demonstrated that the 9q22-q23 chromosome region showed a 'suggestive' linkage to nicotine dependence (ND) in the Framingham Heart Study population. In this study, we provide further evidence for the linkage of this region to ND in an independent sample. Within this region, the gene encoding Src homology 2 domain-containing transforming protein C3 (SHC3) represents a plausible candidate for association with ND, assessed by smoking quantity (SQ), the Heaviness of Smoking Index (HSI) and the Fagerstrom Test for ND (FTND). We utilized 11 single-nucleotide polymorphisms within SHC3 to examine the association with ND in 602 nuclear families of either African-American (AA) or European-American (EA) origin. Individual SNP-based analysis indicated three SNPs for AAs and one for EAs were significantly associated with at least one ND measure. Haplotype analysis revealed that the haplotypes A-C-T-A-T-A of rs12519-rs3750399-rs4877042–rs2297313-rs1547696-rs1331188, with a frequency of 27.8 and 17.6%, and C-T-A-G-T of rs3750399-rs4877042- rs2297313-rs3818668-rs1547696, at a frequency of 44.7 and 30.6% in the AA and Combined samples, respectively, were significantly inversely associated with the ND measures. In the EA sample, another haplotype with a frequency of 10.6%, A-G-T-G of rs1331188–rs1556384-rs4534195–rs1411836, showed a significant inverse association with ND measures. These associations remained significant after Bonferroni correction. We further demonstrated the SHC3 contributed 40.1-59.2% (depending on the ND measures) of the linkage signals detected on chromosome 9. As further support, we found that nicotine administered through infusion increased the Shc3 mRNA level by 60% in the rat striatum, and decreased it by 22% in the nucleus accumbens (NA). At the protein level, Shc3 was decreased by 38.0% in the NA and showed no change in the striatum. Together, these findings strongly implicate SHC3 in the etiology of ND, which represents an important biological candidate for further investigation. Molecular Psychiatry advance online publication, 19 December 2006; doi:10.1038/sj.mp.4001933

以2个品种鸡亲本及其F1代基因组为实验材料，使用荧光标记替代甲基敏感扩增片段多态性（methylation sensitive amplified polymorphism, MSAP）方法中的同位素杨，优化了实验条件，建立了荧光标记的甲基敏感扩增片段多态性方法（fluorescent labeled methylation sensitive amplified polymorphism, F-MASP）。结果显示，鸡个体基因组的甲基化模式分为3种，甲基化片段约占40%；F1代与亲本比较，F1代甲基化多态模式约有95%来自亲本，变异的甲基化位点约5%，虽然变异的甲基化点数量少但种类多，共发现14种甲基化程度减弱型，12种甲基化程度增强型，结果表明，F-MSAP是一种有效地检测鸡基因组甲基化的方法，可以用于其他真核生物尤其是基因组复杂、甲基化多态性丰富的高等动物植物基因组甲基化研究。

采用PCR-RFLP方法对蒙古绵羊和哈萨克绵羊MHC-DRB3基因第2外显子285bp的扩增产物进行多态性分析，共检测到,17种基因型，由A、B、C、D、E、F和H共7个复等位基因控制。通过酶切图谱分析表明，蒙古绵羊和哈萨克绵羊的MHC-DRB3基因第2外显子的第154、168和220位的碱基表现出多态性。统计分析表明，MHC-DRB3基因的部分基因型频率和等位基因频率在两个群体之间差异显著或极显著（P<0.10、P<0.05或P<0.01）。X2适合性检验结果表明，蒙古绵羊和哈萨克绵羊的MHC-DRB3基因第2外显子的HaeIII酶切位点均未达到Hardy-Weinberg平衡状态（P<0.01）。

采用限制性内切核酸酶HaeIII对蒙古山羊和哈萨克山羊GOLA-DRB3基因外显子2的285bp扩增产物进行了PCR-RFLP多态性分析，共检测到17种基因型，由A、B、C、D、E、F和H等7个复等位基因控制；通过酶切图谱分析发现蒙古山羊和哈萨克山羊的GOLA-DRB3基因外显子2的154、168和220位碱基表现出多态性。并对基因型频率和等位基因频率进行了统计分析，结果表明，GOLA-DRB3基因的部分基因型频率和等位基因频率在两个群体之间差异显著(P<0.10或P<0.05)或极显著(P<0.01)；X2适合性检验结果表明，蒙古山羊和哈萨克山羊的GOLA-DRB3基因外显子2的HaeIII酶切位点均未达到Hardy-Weiberg平衡状态(P<0.01)。