Systems genetics of premorbid and cocaine use traits in a rat reduced complexity cross

项目摘要 兴奋剂使用障碍（例如，可卡因、甲基安非他明）是一个主要的公共卫生问题。尽管 遗传率约为40- 50%，全基因组关联研究（GWAS）只确定了很少的位点，包括一个命中位点 可卡因（COC）依赖，映射到FAM 53 B，一个基因也通过表达数量性状鉴定 基因座（QTL）分析与小鼠中COC自我给药相关。主要目标是迅速 在大鼠中确定有助于发病前风险（强迫性，冲动性）的新遗传因素， 自发性高血压大鼠（SHR）可卡因使用性状与复杂性降低交叉（RCC）。啮齿动物 系统遗传学方法对基于发现的遗传学和多水平功能基因组分析进行三角测量 并且可以为药物作用和神经可塑性提供更快速的遗传和神经生物学见解， 成瘾几年来，接触PI一直采用小鼠降低复杂性杂交（RCC） 近等基因近交亚系之间的基因定位，以促进基因定位，验证和机制。因为 啮齿类动物亚系的遗传一致性> 99%，并且包含的变异少了几个数量级 与经典的自交系相比，定位RCC中的数量性状位点（QTL）产生了数量级 更少的因果候选基因要考虑。当与功能基因组学相结合时，RCCs可以迅速导致 致病基因和变体鉴定。我们的初步研究建立了发病前的强大的，可遗传的差异， SHR/NCrl和NCrl之间冲动性和强迫性、蔗糖奖赏敏感性和多种COC使用特征 SHR/NHsd亚株，包括COC诱导的自发活动，COC IVSA服用，寻找和摄入周期， 证明了RCC中基因定位的可行性。在目标1和2中，我们将率先使用大鼠RCC 在那里我们将进行全基因组测序（WGS）和定位行为QTL和表达QTL 在转录本和外显子水平上对来自额叶皮层（PFC）和前额叶核（NAC）的eQTL进行了分析。 F2杂交，包括COC训练的大鼠与轭化盐水（SAL）训练的大鼠。在目标3中，我们将进行蛋白质组学研究， 分析COC与轭合SAL训练大鼠的PFC和NAC，以在高置信度候选者上进行三角测量 数量性状基因（QTG）和变异体（QTVs），因为我们建立了DNA变异体之间的功能连接， 转录调控，蛋白质翻译和细胞信号适应潜在的发病前和可卡因 利用特质这些研究开创了使用大鼠RCC与深层行为表型相结合， 鉴定影响发病前风险高可信度候选新遗传因子和分子机制 因素和可卡因使用特征。未来对候选致病基因变体的基因编辑将以这两个为模型 近等基因SHR背景，以证明必要性（突变校正;“拯救”）和充分性 （突变诱导）。样本包括SHR亚株的WGS，用于将来RCC的复杂性状分析 以及中皮质边缘关键脑区的自适应大鼠转录组和蛋白质组数据集， 研究人员可以进一步挖掘这些回路，并有望为治疗提供信息。

PROJECT SUMMARY Stimulant use disorders (e.g., cocaine, methamphetamine) are a major public health concern. Despite a heritability of ~40-50%, genome-wide association studies (GWAS) have identified very few loci, including one hit for cocaine (COC) dependence that maps to FAM53B, a gene also identified via expression quantitative trait locus (QTL) analysis to be associated with COC self-administration in mice. The primary objective is to rapidly identify novel genetic factors in rats that contribute to premorbid risk (compulsivity, impulsivity) and cocaine use traits in a spontaneously hypertensive rat (SHR) reduced complexity cross (RCC). A rodent systems genetics approach triangulates on discovery-based genetic and multi-level functional genomic analysis and can provide a more rapid genetic and neurobiological insight into drug action and neuroplasticity underlying addiction. For several years, the contact PI has been employing mouse reduced complexity crosses (RCCs) between near-isogenic inbred substrains to facilitate gene mapping, validation, and mechanisms. Because rodent substrains are > 99% genetically identical and contain several orders of magnitude fewer variants compared to classical inbred strains, mapping quantitative trait loci (QTLs) in RCCs yields orders of magnitude fewer causal candidate genes to consider. When combined with functional genomics, RCCs can rapidly lead to causal gene and variant identification. Our preliminary studies establish robust, heritable differences in premorbid impulsivity and compulsivity, sucrose reward sensitivity, and multiple COC use traits between SHR/NCrl and SHR/NHsd substrains, including COC-induced locomotor activity, COC IVSA taking, seeking, and intake cycles, demonstrating feasibility for gene mapping in an RCC. In Aims 1 and 2, we will pioneer the use of a rat RCC where we will conduct whole genome sequencing (WGS) and map behavioral QTLs and expression QTLs (eQTLs) from nucleus accumbens (NAC) and prefrontal cortex (PFC) at the whole transcript and exon levels in an F2 cross comprising COC-trained versus yoked saline (SAL)-trained rats. In Aim 3, we will conduct proteomic analysis of PFC and NAC from COC vs. yoked SAL-trained rats to triangulate on high confidence candidate quantitative trait genes (QTGs) and variants (QTVs) as we build functional connections between DNA variants, transcriptional regulation, protein translation, and cell signaling adaptations underlying premorbid and cocaine use traits. These studies pioneer the use of a rat RCC combined with deep behavioral phenotyping to rapidly identify high-confidence candidate novel genetic factors and molecular mechanisms influencing premorbid risk factors and cocaine use traits. Future gene editing of candidate causal gene variants will be modeled on the two near-isogenic SHR backgrounds to demonstrate necessity (mutation correction; “rescue”) and sufficiency (mutation induction). Deliverables include WGS’s of SHR substrains for future RCCs for complex trait analysis as well as adaptive rat transcriptomic and proteomic datasets in key brain regions of the mesocorticolimbic circuitry that can be further mined by investigators and hopefully inform therapeutics.