Genomic Analyses for Elucidating Novel Targets for Symptoms Management

大多数症状是由多因素引起的，遗传和环境因素都会导致个体差异。在生物学假说的基础上进行了候选基因研究，以确定疼痛等复杂特征的相关遗传变异。然而，复杂的致病因素和涉及不同症状的数千个分子，使得很难检测到一个独特的疾病易感性个体的负责任的基因变异。单个基因中常见的变异不太可能主要作用于复杂的特征；相反，每个基因的贡献似乎很微妙，作用于多个生物途径中的一个，使其信号很难被检测到。 疾病知识的迅速增加以及应用强大和高容量技术的能力的共同影响，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。为症状管理开发新的治疗策略也严重依赖于识别新的靶分子和确定特定类型疾病的表型。因此，这个项目的第一步是确定实验和临床表型的特征。 最近，我们启动了多个使用下一代测序(NGS)技术的项目，该技术允许我们执行完整和/或有针对性的基因组测序。将继续研究遗传和表观遗传因素在各种疾病和/或条件下的症状中的作用。例如，在急慢性疼痛、轻度创伤性脑损伤和创伤后应激障碍等神经疾病中，我们正在使用NGS进行基因分型、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与包括炎症级联在内的众多生物/机制途径之间的相互作用。 在多个会议上介绍了使用无偏RNA-SEQ技术从遭受冲击伤的士兵那里获得的基因表达谱。我们还对从战区归来的士兵进行了MicroRNA分析。这些基于NGS数据的基因表达谱结果也发表了。我们最近启动了一个具有多种慢性疼痛症状的rna-seq项目，以破译在这些症状中的基因作用，目前正在测序中。基于全基因组方法产生的数据，我们还缩小了范围，并使用了纳米串和验证冲击伤患者的NGS结果等靶向分析。我们开始对移植患者脂肪组织中的转录本进行有针对性的分析，以调查体重和感觉之间的关系。两项表观遗传学研究使用DNA甲基化、染色质免疫沉淀和全基因组规模测序，分别来自平民和军事群体。研究结果还在多个会议上公布并发表。 军队创伤后应激障碍患者的表观遗传学变化已发表，而运动相关脑震荡患者的表观遗传学研究已提交发表，目前正在审查中。 对特殊感觉障碍患者和对照同胞的全基因组测序进行了测序，并在ASHG会议上公布。利用定向下一代测序技术，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类的微生物种群。利用新一代半导体测序仪，对再生障碍性贫血患者口腔微生物组的16S rRNA区进行了广泛的测序，分别进行了短而多段的16S rRNA测序和全长16S rRNA测序。这些数据已在机构间反兴奋剂机构会议上提出并公布。随后对患者组的微生物组数据进行了分析，目前手稿正在准备发表。该项目主要发现，在特定的疾病条件下，微生物组的组成会发生变化。 考虑到蛋白质是DNA和RNA的最终产物，这些多个项目的蛋白质分析最近已经启动。我们特别关注的是只显示微量的蛋白质，这使得传统的蛋白质检测方法如ELISA法很难检测到。到目前为止，我们已经成功地检测到了血浆中的tau和p-tau蛋白，而这还没有用ELISA法进行检测。并发表了军用颅脑损伤患者中tau蛋白的研究结果。多路分析现在被用于更有效和更快的数据收集。 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、基因表达签名、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的治疗策略。最近的研究表明，中枢神经系统与周围神经系统之间通过外周小体进行联系。因此，我们开始使用Nanosight 300进行外显子分析，然后是Micro RNA分析或蛋白质分析。此外，最近还获得了使用两种不同平台的单细胞分析，例如来自Fluidigm的C1和来自BioRad的ddSeq，并处于优化过程中，以供未来使用。 根据这些结果，结合神经系统疾病多途径的生物学知识，我们将能够在个体水平上提出这些疾病的分子遗传机制。最后，我们可以建议进行综合基因组分析来开发新药，并基于个人遗传信息对其进行测试。

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.