Association and Function of Opioid Receptor Gene Variants to Substance Dependence

描述（由申请人提供）：此K99/ROO申请寻求额外研究培训的支持，这将使申请人成为神经精神遗传学的独立研究者。培训目标将通过一个项目来实现，该项目旨在确定三种阿片受体基因（OPRM1、OPRD1和OPRK1）中与物质（药物和/或酒精）依赖（SD）相关的变异，并解释这种关联的机制。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欧裔美国人（EAs）和非洲裔美国人（AAs）中阿片受体基因（OPR）变异与SD之间可能的关联。跨OPR的紧密间隔单核苷酸多态性（snp）将进行基因分型，OPR变异与SD之间的关联将通过适当的统计程序进行分析，包括结构化关联分析。此外，将筛选感兴趣的基因区域，以寻找新的变异，并进一步分析其与SD的关联。在独立阶段（2009-2012年），我将重点研究与SD相关的OPR变异的功能。对于编码区变异，将通过受体结合试验（观察OPR变异是否改变受体亲和力）和Western Blotting（观察OPR变异是否调节受体蛋白水平）进行功能研究。对于非编码区变异，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变异是否导致基因表达（或mRNA）水平升高或降低；(2)等位基因表达不平衡（AEI）测定，这是一种检测变异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)电泳迁移位移测定（EMSA），检测OPR变异是否位于转录因子（TF）结合位点，以及它们是否改变TF与其DNA序列的结合；(4)荧光素酶报告基因检测，检测OPR变异是否调控基因表达。这四种方法是互补的。本研究将提高我们对ea和aa中OPR变异对SD的影响的理解。这项工作将为未来的R01项目奠定基础，该项目旨在建立一套OPR标记物预测SD的遗传模型，并研究OPR变异对药物或酒精依赖治疗结果的影响，以及对治疗结果的种族差异的影响。申请人由Joel Gelernter博士（主要导师）和Jeffrey Gruen博士（联合导师）指导，他们都是优秀的导师，在复杂疾病的遗传研究方面有丰富的经验，并且都指导过许多以前的学员。

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.