Identification of natural variants that influence responses to ethanol in C. elegans

项目摘要 人类的遗传变异有助于个体发展酒精使用障碍的可能性。的 因此，影响酒精滥用倾向的遗传变异是一个重要的研究目标，但它一直是 难以识别特定的易感基因。动物模型的实验室研究在以下方面非常有用： 阐明酒精（乙醇）的分子药理学，但实验室衍生的遗传操作 很少模拟在野生种群中观察到的自然发生的遗传变异。因此，预测 相关的人类等位基因变异是困难的。在这里，我们研究了野生型中发现的自然等位基因变异， 线虫秀丽隐杆线虫的菌株，以鉴定在野外耐受的等位基因， 可以调节影响乙醇生理反应的途径的功能。C. elegans是一个 与人类相关的重要模式物种; 蠕虫和人类神经系统功能的机制，以及影响乙醇反应的基因 蠕虫的行为也会影响人类酒精使用障碍的可能性。 这一合作提案汇集了两个实验室的不同专业知识。我们利用一个 独特的资源，重组近交系（RILs）来自四个遗传多样性的野生菌株，以确定 可以调节乙醇作用的天然等位基因变异。我们已经证明，亲本野生型 菌株和衍生的RIL在不同的乙醇行为反应中显示出一系列表型。我们将 利用C. elegans模型进行高通量分析， 将识别改变对乙醇的行为和/或转录反应的遗传变异。我们会直接 测试通过数量性状基因座鉴定的候选乙醇反应等位基因变体的因果性质 通过使用基因编辑技术（CRISPR-Cas9）将等位基因导入菌株， 携带不同的等位基因比较和对比遗传变异对不同个体的影响的能力 对乙醇的反应为我们的分析提供了进一步的动力。我们将识别出影响 每种行为的生理反应都是独特的，其次，我们将确定影响这种反应的基因。 乙醇反应表型的行为反应。我们还将评估等位基因的影响， 调节乙醇对行为反应的转录。这些不同的分析可以告诉我们 这些对乙醇不同反应的分子机制。 总之，这些研究将提供具体的和更普遍的新见解的神经遗传学， 乙醇我们将确定已知或新的生物学途径中的遗传变异的程度， 介导或调节乙醇的作用可以改变对乙醇的反应， 在野生环境中是可以容忍的。这些数据将使我们了解人类对虐待的责任 酒精

PROJECT SUMMARY Genetic variation in humans contributes to an individual's likelihood to develop an alcohol use disorder. The genetic variation that influences alcohol abuse liability is therefore an important target for study, but it has been difficult to identify specific liability genes. Laboratory studies in animal models have been extremely useful in elucidating the molecular pharmacology of alcohol (ethanol), but laboratory derived genetic manipulations rarely model the naturally occurring genetic variation that is observed in wild populations. As such, predicting relevant human allelic variation has been difficult. Here, we study the natural allelic variation found in wild strains of the the nematode worm Caenorhabditis elegans to identify alleles that are tolerated in the wild and can modulate the function of pathways that impact physiological responses to ethanol. C. elegans is an important model species with demonstrated relevance to humans; there is striking conservation between the machinery of nervous system function in worms and humans, and genes that influence ethanol response behaviors in worms also influence the likelihood to develop alcohol use disorders in humans. This collaborative proposal brings together the diverse expertise of two laboratories. We take advantage of a unique resource, recombinant inbred lines (RILs) derived from four genetically diverse wild strains to identify natural allelic variation that can modulate the effects of ethanol. We have shown that the parent wild-type strains and the derived RILs display a range of phenotypes in different behavioral responses ethanol. We will exploit the efficiency and ease of manipulating the C. elegans model to carry out high throughput analyses that will identify genetic variation that alters behavioral and/or transcriptional responses to ethanol. We will directly test the causal nature of candidate ethanol response allelic variants, identified by quantitative trait locus mapping, through the use of gene editing techniques (CRISPR-Cas9) to introduce the allele into strains that carry different alleles. The ability to compare and contrast the influence of genetic variants on different responses to ethanol brings further power to our analyses. We will identify the genes that impact the physiological responses in each behavior uniquely, and second, we will identify genes that affect the behavioral responses across ethanol response phenotypes. We will also assess the impact of alleles that modulate transcription in response to ethanol on behavioral responses. These different analyses can inform us of the molecular mechanisms underlying these different responses to ethanol. Together, these studies will provide both specific and more general novel insights into the neurogenetics of ethanol. We will establish the degree to which genetic variation in known or novel biological pathways that mediate or modulate the effect of ethanol can change responses to ethanol and the degree to which variation in those genes is tolerated in the wild. These data will inform our understanding of human liability to abuse alcohol.