Tabakoff Boris, Hoffman Paula L, Saba Laura M
Lohocla Research Corporation, Aurora, CO, USA.
Institute for Behavioral Genetics, University of Colorado Boulder, CO, USA.
J Neurobiol Physiol. 2024;5(1):15-22. doi: 10.46439/neurobiology.5.026.
At the invitation of the Journal, we are providing a summary of our published work that has followed the publication in 2009 of our manuscript entitled "Genetical Genomic Determinants of Alcohol Consumption in Rats and Humans". Our initial premise, which has been maintained throughout, is that knowledge regarding gene transcription would greatly enhance GWAS of alcohol-related phenotypes. We chose to concentrate our studies on the quantitative phenotype of alcohol consumption since high levels of alcohol consumption are a prerequisite for the development of alcohol use disorder (AUD). We also structured our studies to focus on "predisposition" to higher levels of alcohol consumption. We defined predisposition as a genetic structure and transcriptional pattern that is inherent in an organism and present prior to exposure to an environmental stimulus that engenders a physiological/behavioral response. In studies using humans, this interest in predisposition usually requires prolonged periods of cohort follow-up. On the other hand, studies with animals can use resources such as panels of recombinant inbred (RI) animals (in our case, the HXB/BXH rat panel) to capture the transcriptional landscape of animals not exposed to alcohol and compare this transcriptional landscape to levels of alcohol consumption collected from a different cohort of animals that are the same age, have an identical genetic composition, and are raised in an identical environment. The other benefit is that the stable genetic structure of inbred strains allows for a chronological expansion of information on these animals. This characteristic of the HXB/BXH RI rats allowed us to add important information as technology and analytical methods developed over time.
Our initial studies relied on hybridization arrays for RNA quantification in brain, an initial set of polymorphic markers for the rat genome, and a standard behavioral (b)QTL analysis for alcohol consumption. What we added to the conceptual basis for analysis and interpretation was the calculation of transcript expression (e)QTLs and the requirements that: 1. the eQTL overlapped the location of the bQTL; and 2. the transcript levels were significantly correlated with the quantitative levels of alcohol consumption across rat strains. These criteria were used to identify genes (transcripts) as "candidate" contributors to the alcohol consumption phenotype. We soon realized that the search for candidate genes as unique determinants of a complex trait is irrational, since these phenotypes are best characterized by differences in genetic networks. Therefore, we incorporated Weighted Gene Coexpression Network Analysis (WGCNA) in our further work. We also realized the limitations of hybridization arrays for breadth of transcriptome coverage and quantification, and in the more current work used total RNA-Seq-derived data for characterizing nearly all of the brain transcriptome. Finally, we participated in the efforts for whole genome sequencing of the strains of the HXB/BXH panel, generating an extensive new panel of markers for remapping of the QTLs. We also realized that the biological determinants of a behavioral phenotype do not have to reside in brain and, by examining the liver transcriptome, we found that the gut-liver-brain axis was, in part, involved in predisposition to higher levels of free-choice alcohol consumption. In all, from the first exploration of the genetical genomics of the alcohol consumption phenotype, to the current status of our work, the function of the brain immune system, with emphasis on microglia and astrocytes, even prior to the animal being offered alcohol, has emerged as a most significant genetic contributor to the amount of alcohol an animal will consume on a daily basis. Particularly prominent was a cluster of inflammasome (NLRP3)-modulating transcripts () and a long noncoding transcript that repeatedly appeared within a gene coexpression module associated with alcohol consumption levels. Interestingly, data from post-mortem tissue from brain of humans suffering from AUD also indicates a hyperactive neuroimmune function. The data from studies with animals may indicate that neuroimmune hyperactivity may be a trait rather than a state marker for AUD.
应《期刊》邀请，我们提供已发表工作的总结，此项工作是在我们2009年发表题为《大鼠和人类酒精消费的遗传基因组决定因素》的手稿之后开展的。我们始终秉持的最初前提是，有关基因转录的知识将极大地提升与酒精相关表型的全基因组关联研究（GWAS）。我们选择将研究集中在酒精消费的定量表型上，因为高酒精消费量是酒精使用障碍（AUD）发展的先决条件。我们还将研究结构设定为专注于较高水平酒精消费的“易感性”。我们将易感性定义为一种遗传结构和转录模式，它是生物体固有的，在暴露于引发生理/行为反应的环境刺激之前就已存在。在使用人类的研究中，对易感性的这种关注通常需要长时间的队列随访。另一方面，动物研究可以利用诸如重组近交（RI）动物品系（在我们的研究中是HXB/BXH大鼠品系）等资源，来捕捉未接触酒精的动物的转录图谱，并将此转录图谱与从另一组年龄相同、遗传组成相同且在相同环境中饲养的动物收集的酒精消费量水平进行比较。另一个好处是近交系的稳定遗传结构允许按时间顺序扩展有关这些动物的信息。HXB/BXH RI大鼠的这一特性使我们能够随着技术和分析方法的发展不断添加重要信息。
方法、研究结果与结论：我们最初的研究依赖于用于脑内RNA定量的杂交阵列、大鼠基因组的一组初始多态性标记，以及用于酒精消费的标准行为（b）数量性状基因座（QTL）分析。我们在分析和解释的概念基础上增加的内容是转录本表达（e）QTL的计算以及以下要求：1. eQTL与bQTL的位置重叠；2. 转录本水平与大鼠品系间酒精消费的定量水平显著相关。这些标准用于将基因（转录本）鉴定为酒精消费表型的“候选”贡献因素。我们很快意识到，将候选基因作为复杂性状的唯一决定因素进行搜索是不合理的，因为这些表型最好通过遗传网络的差异来表征。因此，我们在后续工作中纳入了加权基因共表达网络分析（WGCNA）。我们还意识到杂交阵列在转录组覆盖广度和定量方面的局限性，在当前的工作中使用了源自全RNA测序的数据来表征几乎整个脑转录组。最后，我们参与了HXB/BXH品系全基因组测序的工作，生成了用于QTL重新定位的大量新标记。我们还意识到行为表型的生物学决定因素不一定存在于脑中，通过检查肝脏转录组发现，肠 - 肝 - 脑轴在一定程度上参与了较高水平自由选择酒精消费的易感性。总之，从对酒精消费表型的遗传基因组学的首次探索到我们当前的工作状态，即使在动物未接触酒精之前，以小胶质细胞和星形胶质细胞为重点的脑免疫系统功能已成为动物每日酒精消费量的最重要遗传贡献因素。特别突出的是一组调节炎性小体（NLRP3）的转录本（）以及一个在与酒精消费水平相关的基因共表达模块中反复出现的长链非编码转录本。有趣的是，来自AUD患者脑的尸检组织数据也表明神经免疫功能亢进。动物研究数据可能表明神经免疫功能亢进可能是AUD的一种特质而非状态标记。
