Genomic Analyses for Elucidating Novel Targets for Symptoms Management

大多数症状是多因素的起源与遗传和环境因素有助于个体差异。基于生物学假设的候选基因研究已经被用来鉴定复杂性状（如疼痛）中的相关遗传变异。然而，复杂的影响因素和参与不同症状的数千种分子使得很难检测出个体对疾病独特易感性的遗传变异。单个基因的共同变异不太可能对复杂性状起主导作用;相反，每个基因的作用似乎很微妙，作用于多个生物途径中的一个，使其信号难以检测。 疾病知识的迅速增长和应用强大和高容量技术的能力的综合影响，提高了人们对用于各种症状管理的更有效和更安全的药物的期望。开发用于症状管理的新治疗策略也关键地依赖于识别新的靶分子和定义特定类型的病症的表型。因此，该项目的第一步是确定实验和临床表型的特征。 最近，我们启动了多个采用下一代测序（NGS）技术的项目，该技术使我们能够进行整个和/或靶向基因组测序。将继续研究遗传和表观遗传因素对各种疾病和/或病症症状的作用。例如，在神经系统疾病，如急性和慢性疼痛，轻度创伤性脑损伤和创伤后应激障碍，我们正在使用NGS基因分型，基因和蛋白质表达，以及患者报告的结果，以更好地了解这些因素之间的相互作用和众多的生物/机械途径，包括炎症级联反应。 在多次会议上介绍了使用无偏见的RNA-seq技术从遭受爆炸伤的士兵中获得的基因表达谱。我们还展示了从战区回来的士兵的微RNA分析。这些基于NGS数据的基因表达谱结果也已发表。我们最近启动了一个针对多种慢性疼痛疾病的RNA-seq项目，以破译这些症状中的遗传作用，目前正在测序中。基于全基因组方法产生的数据，我们还缩小并使用了靶向分析，如NanoString和验证的冲击伤患者的NGS结果。我们开始有针对性地分析移植患者脂肪组织中的转录本，以调查体重和感觉之间的关联。两个表观遗传学研究使用DNA甲基化染色质免疫沉淀，然后全基因组规模测序进行了从平民和军事群体。研究结果还在多次会议上介绍并发表。 发表了军事创伤后应激障碍患者的表观遗传变化，而运动相关脑震荡患者的表观遗传研究已提交出版，目前正在审查中。 特殊感觉障碍患者和对照同胞的全基因组测序被测序并在ASHG会议上发表。利用靶向下一代测序技术，我们还完成了一个微生物组项目，以表征不同疾病状态下人体内的微生物种群。使用下一代半导体测序仪，以2种不同的方式对再生障碍性贫血患者的口腔微生物组的16 s rRNA区域进行广泛测序，对16 s rRNA区域中的短但多个片段进行测序，并对16 s rRNA全长测序。这些数据在IADR会议上提交并发表。分析了患者组的以下微生物组数据，目前手稿正在准备发表。该项目主要发现微生物组组成在特定疾病条件下发生变化。 考虑到蛋白质是DNA和RNA的最终产物，最近启动了对这些多个项目的蛋白质分析。我们特别关注仅显示痕量的蛋白质，使得难以用常规蛋白质测定法如ELISA检测。到目前为止，我们已经成功地检测到血浆中的tau和p-tau蛋白，这还没有用ELISA测量。并发表了其在军队TBI患者中tau蛋白的研究结果。多重分析现在用于更有效和快速的数据收集。 这些项目的另一个目标是为下一代测序仪产生的高通量大数据开发新的分析方法，包括全基因组测序，基因表达特征，表观遗传学及其与蛋白质和环境因素等其他因素的相互作用。此外，利用基因编辑技术，我们将尝试开发基于基因组学的新治疗策略。近年来的研究表明，中枢神经系统和外周神经系统之间通过exosomes进行信息交流。因此，我们使用Nanosight 300开始外泌体分析，然后进行微RNA分析或蛋白质分析。此外，最近获得了使用2种不同平台（例如Fluidigm的C1和BioRad的ddSeq）的单细胞测定，并正在优化过程中以备将来使用。 从这些结果，沿着生物学知识的多个途径的神经系统疾病，我们将能够建议这些疾病的分子遗传机制在个人的水平。最后，我们可以建议综合基因组分析来开发新药，并根据个体遗传信息进行测试。

Most symptoms are multifactorial in origin with both genetic and environmental factors contributing to individual variations. Candidate gene studies on the basis of biological hypotheses have been performed to identify relevant genetic variation in complex traits such as pain. However, the complicated mesh of contributing factors and the thousands of molecules involved in different symptoms makes it difficult to detect responsible genetic variations for an individuals unique susceptibility to disorders. It is unlikely that common variations in a single gene act dominantly on complex traits; rather, the contribution of each gene seems to be subtle, acting on one of multiple biological pathways, making its signal difficult to detect. The combined impact of the rapid increase in knowledge of diseases and the ability to apply powerful and high capacity technology has raised expectations for more effective and safer medicines for various types of symptoms management. Developing new treatment strategies for symptoms management is also critically dependent on identifying new target molecules and defining phenotypes for specific types of disorders. Therefore, the first step of this project has been to define the characteristics of experimental and clinical phenotypes. Recently, we launched multiple projects with next generation sequencing (NGS) technology, which allows us to perform entire and/or targeted genome sequencing. The role of genetic and epigenetic factors on symptoms in various disorders and/or conditions will continue to be studied. For example, in neurological disorders such as acute and chronic pain, mild traumatic brain injury and PTSD, we are using NGS for genotyping, gene and protein expression, and patient reported outcomes to better understand the reciprocal interplay between these factors and the numerous biologic/mechanistic pathways including the inflammatory cascade. Gene expression profiles using unbiased RNA-seq technology from soldiers who suffered from blast injury were presented at multiple meetings. We also presented micro RNA analysis from soldiers back from war zone. These results of gene expression profiles based on NGS data were also published. We recently launched a project of RNA-seq with multiple chronic pain conditions to decipher genetic roles in those symptoms and currently under sequencing. Based on the data generated from whole genome approaches, we also narrowed and used targeted analysis such as NanoString and validated NGS results of blast injury patients. We started targeted analysis of transcripts in adipose tissues in transplant patients to investigate the association between weight and sensory. Two epigenetic studies using DNA methylation chromatin immunoprecipitation followed by whole genome scale sequencing were conducted from civilian and military groups. The results were also presented at multiple meetings and published. Epigenetic changes in military PTSD patients was published while the epigenetic study in sports related concussion patients was submitted for publication and is currently under review. Whole genome sequencing of special sensory disorder patient and control sibling were sequenced and presented at the ASHG meeting. Using the targeted next generation sequencing technology, we also have finished a microbiome project to characterize population of microorganisms in human from different disease status. Using a next generation semiconductor sequencer, microbiome was analyzed with extensive sequencing of 16s rRNA region for oral microbiome from the aplastic anemia patients in 2 different ways, sequencing short but multiple sections in 16s rRNA region and full length of 16s rRNA sequencing. The data was presented at the IADR meeting and published. Following microbiome data of patient group was analyzed and currently the manuscript is in preparation for publication. Mainly the project found that the microbiome composition is altered in specific disease conditions. Considering that the protein is the final product from DNA and RNA, protein analysis from those multiple projects has been recently launched. We especially focus on proteins showing trace amount only so that it makes difficult to detect with conventional protein assays such as ELISA. So far, we have successfully detected tau and p-tau protein in plasma, which has not been measured with ELISA. And its result of tau protein in military TBI patients was published. Multiplexing assays are now used for more effective and fast data collection. Developing new analyzing methods for the high throughput big data generated by the next generation sequencers including whole genome sequencing, gene expression signatures, epigenetics and their interactions with other factors such as proteins and environment factors is another goal of these projects. Also, using gene editing technologies, we will try to develop a new treatment strategies based on genomics. Recent studies suggest central nervous system communicates with peripheral nervous system via exosomes. Therefore, we started exosome analysis using Nanosight 300 followed by either micro RNA analysis or protein analysis. Also, single cell assays using 2 different platforms, such as C1 from Fluidigm and ddSeq from BioRad were recently acquired and under the optimization process for future use. From these results, along with biological knowledge of multiple pathways in neurological disorders, we will be able to suggest molecular-genetic mechanisms of those diseases at the level of the individual. Finally, we can suggest integrative genomic analysis to develop new drugs and test them based on individual genetic information.