Association and Function of Opioid Receptor Gene Variants to Substance Dependence

描述(由申请人提供)：这份K99/Roo申请寻求支持额外的研究培训，使申请者能够成为神经精神病学遗传学的独立研究员。将通过一个项目实现培训目标，该项目旨在查明三个阿片受体基因(OPRM1、OPRD1和OPRK1)中与物质(药物和/或酒精)依赖(SD)相关的变异，并解释这种联系的机制。在指导阶段(2007-2009年)，我将使用基于人群和家庭的方法来研究在欧洲裔美国人(EAs)和非裔美国人(AAs)中阿片受体基因(OPR)变异与SD之间的可能联系。将对跨越OPR的紧密间隔单核苷酸多态(SNPs)进行基因分型，并将通过包括结构化关联分析在内的适当统计程序来分析OPR变异与SD之间的关联。此外，还将筛选感兴趣的基因区域以寻找新的变异，并将进一步分析它们与SD的关联。在独立阶段(2009-2012年)，我将重点研究那些被发现与SD相关的OPR变体的功能。对于编码区变体，将通过受体结合分析(看看OPR变体是否改变受体亲和力)和Western blotting(看看OPR变体是否调节受体蛋白水平)进行功能研究。对于非编码区变体，将通过四种不同的方法进行功能研究：(1)实时定量PCR，检测OPR变体是否导致基因表达(或mRNA)水平的增加或降低；(2)等位基因表达失衡(AEI)分析，这是检测一个变体的两个等位基因是否导致不同表达(或mRNA)水平的替代方法；(3)电迁移率改变分析(EMSA)，检测OPR变体是否位于转录因子(TF)结合部位，以及它们是否改变了TF与其DNA序列的结合；(4)荧光素酶报告基因检测，检测OPR变异体是否调节基因表达。这四种方法是相辅相成的。这项拟议的研究将加深我们对EAS和AAS中OPR变体对SD的影响的理解。这项工作将为未来的R01项目奠定基础，该项目旨在利用一组OPR标记建立预测SD的遗传模型，并调查OPR变异对药物或酒精依赖治疗结果的贡献或对治疗结果的种族差异的影响。申请者由Joel Gelernter博士(初级导师)和Jeffrey Gruen博士(共同导师)指导，他们都是优秀的导师，在复杂疾病的遗传学研究方面拥有丰富的经验，两人都曾指导过许多以前的实习生。

DESCRIPTION (provided by applicant): This K99/ROO application seeks support for additional research training which will enable the applicant to become an independent investigator in neuropsychiatric genetics. The training goal will be achieved through a project aimed to identify substance (drug and/or alcohol) dependence (SD)-associated variants in three opioid receptor genes (OPRM1, OPRD1 and OPRK1) and interpret the mechanism of the association. In the mentored phase (year 2007-2009), I will use population- and family-based approaches to study the possible association between opioid receptor gene (OPR) variants and SD in both European Americans (EAs) and African Americans (AAs). Tightly-spaced single nucleotide polymorphisms (SNPs) spanning OPRs will be genotyped and the association between OPR variants and SD will be analyzed by appropriate statistical programs including structured association analysis. Moreover, gene regions of interest will be screened for new variants, which will be further analyzed for their association with SD. In the independent phase (year 2009-2012), I will focus on functional study of those OPR variants which are found to be associated with SD. For coding region variants, functional study will be conducted by receptor binding assay (to see if OPR variants alter receptor affinity) and Western Blotting (to see if OPR variants modulate receptor protein levels). For non-coding region variants, functional study will be performed by four different approaches: (1) real-time quantitative PCR, which examines whether OPR variants result in increased or decreased gene expression (or mRNA) levels; (2) allelic expression imbalance (AEI) assay, which is an alternative approach to examine whether the two alleles of a variant lead to different expression (or mRNA) levels; (3) Electrophoretic mobility shift assay (EMSA), which examines whether OPR variants are located in transcription factor (TF) binding sites and if they change the binding of a TF to its DNA sequence; (4) luciferase reporter gene assay, which examines whether OPR variants regulate gene expression. These four approaches are complementary. The proposed study will improve our understanding about the influence of OPR variants on SD in EAs and AAs. This work will lay the groundwork for a future R01 project aimed to establish a genetic model for prediction of SD with a set of OPR markers, and to investigate the contribution of OPR variants to the outcome of drug or alcohol dependence treatment or to the racial difference in outcomes of treatment. The applicant is mentored by Dr. Joel Gelernter (primary) and Dr. Jeffrey Gruen (co-mentor), who are both excellent supervisors with substantial experience in genetic studies of complex disorders and who have both mentored numerous previous trainees.