Single cell transcriptomic study of alcohol use

/项目摘要\ 酒精使用障碍（AUD）是一种以停止或控制酒精的能力受损为特征的医学疾病 尽管有不利的社会、职业或健康后果，仍使用。澳元通常是12个月的， 美国的终生患病率分别为13.9%和29.1%。基因表达研究提供了有希望的 有机会更好地了解AUD病因。由于细胞功能不同，基因表达通常 在不同的细胞类型中也是不同的。当研究大块组织时，不能考虑细胞多样性会对细胞的生长产生不利影响。 对检测疾病关联的能力的影响。例如，病例对照差异将被“稀释”， 它们只影响一种细胞类型，如果细胞类型之间的差异具有相反的符号，则可以抵消， 如果差异涉及低丰度细胞，则可能检测不到。此外，识别特定的细胞- 关联信号来源的类型是制定AUD病因学的精确假设的关键， 设计适当的后续实验，并最终开发新的临床干预措施。 我们的总体目标是在死后的大脑样本中进行严格的研究， 在细颗粒细胞水平上表达AUD中的基因，并确定调节这些基因的遗传元件。 差异为了实现这一目标，我们提出了一个战略，已成为可用的最新发展， 单核RNA测序（snRNA-seq）技术，可以表征表达水平， 成千上万的原子核产生一次反应我们的试验数据显示，我们的实验室能够生产 最高质量的snRNA-seq数据，并说明了snRNA-seq检测生物相关性的承诺 调查结果。通过仔细的选择程序，我们获得了一组独特的相对严重的病例， 在诊断上是同质的，同时具有最小的合并症，并且与对照组匹配， 关键变量我们将重点关注与AUD密切相关的三个大脑区域，并捕捉不同的 疾病的各个方面。研究设计特征由一系列功效分析指导， 从我们的试点研究的经验观察。为了确保稳健的结果，将使用snRNA复制发现- seq数据来自独立个人，并使用不同的技术进行验证。最后，我们提出了一系列 后续分析，旨在确定复制结果的潜在调节剂，（功能）表征 存在稳健的AUD GWAS关联，并检测脑中AUD疾病过程的血液生物标志物。 该项目的成功完成将产生（i）对AUD疾病机制的前所未有的见解， (ii)可能的血液生物标志物的基因差异表达的大脑，和（iii）奠定了基础， 功能性随访研究，最终，用于改善AUD的新型高度特异性临床干预措施 治疗

/// PROJECT SUMMARY \\\ Alcohol use disorder (AUD) is a medical condition characterized by an impaired ability to stop or control alcohol use despite adverse social, occupational, or health consequences. AUD is common with a 12-month and lifetime prevalence in the US of 13.9% and 29.1%, respectively. Gene expression studies provide promising opportunities to better understand AUD etiology. As cells differ in their function, gene expression will typically also differ across cell-types. When studying bulk tissue, failure to account for cell diversity has a detrimental impact on the ability to detect disease associations. For example, case-control differences will be “diluted” if they affect only one cell-type, may cancel out if the differences are of opposite signs across cell-types, and may be undetectable if the differences involve low abundance cells. Furthermore, identifying the specific cell- types from which the association signals originate is key to formulating refined hypotheses of AUD etiology, designing proper follow-up experiments and, eventually, developing novel clinical interventions. Our overall goal is to perform a rigorous study in post-mortem brain samples to detect differentially expressed genes in AUD at a fine-grained cellular level, and identify the genetic elements that regulate these differences. To achieve this goal, we propose a strategy that has become available with the recent evolution of single nucleus RNA sequencing (snRNA-seq) technology that can characterize the expression levels of thousands of individual nuclei with a single reaction. Our pilot data shows that our lab is able to produce snRNA-seq data of the highest quality and illustrates the promise of snRNA-seq to detect biologically relevant findings. Using careful selection procedures, we obtained a unique collection of relatively severe cases that were diagnostically homogenous while having minimal comorbidities, and that were matched to the controls on key variables. We will focus on three brain regions that are heavily implicated in AUD and capture different aspects of the disease. Study design features were guided by a series of power analyses that are grounded in empirical observations from our pilot study. To ensure robust results, findings will be replicated using snRNA- seq data from independent individuals and validated with a different technology. Finally, we propose a series of follow-up analyses aimed at identifying potential regulators of replicating findings, (functionally) characterizing existing robust AUD GWAS associations, and detecting blood biomarkers of AUD disease processes in brain. Successful completion of this project will yield (i) unprecedented insights into AUD disease mechanisms, (ii) possible blood biomarkers of genes differentially expressed in brain, and (iii) lay the foundation for functional follow-up studies and, eventually, novel highly specific clinical interventions for improving AUD treatment.