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，在急性疼痛、轻度创伤性脑损伤和创伤后应激障碍等神经系统疾病中，我们正在使用基因分型、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。 从战区返回的士兵的基因表达谱在多次会议(CNRM和ASN会议)上公布，并已发表。我们还从战区士兵的类似样本中开始了SNP分析和Micro RNA分析。研究结果还在多个会议上公布并发表。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。 在ASHG会议上介绍了运动相关脑震荡患者的表观遗传学变化。这一结果已提交出版，目前正在审查中。军队创伤后应激障碍患者的第二项表观遗传学研究完成了第一组样本的测序。在初步数据分析之后，我们增加了样本量，并将进行更多的排序。 对特殊感觉障碍患者和对照同胞的全基因组测序并进行分析。利用定向下一代测序技术，我们还有两个正在进行的微生物组项目，以表征来自不同疾病状态的人体和/或动物体内的微生物种群。使用新一代半导体测序仪，对肠易激综合征动物模型和人类患者的微生物组进行了广泛的16S rRNA区域测序。研究结果在科学会议上发表，并提交发表。另一个针对再生障碍性贫血患者口腔微生物组的微生物组项目也在进行中，并完成了样本采集。到目前为止，初步数据分析已经完成，其结果已在机构间非正式报告会议上提交并提交出版。主要是这两个项目都发现，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蛋白，而这还没有用ELISA法进行检测。 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。 根据这些结果以及神经系统疾病中多个途径的生物学知识，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 pain, mild traumatic brain injury and PTSD, we are using 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 from the soldiers back from war zone were presented at the multiple meetings (CNRM and ASN meeting) and have been published. We also started SNP assays and micro RNA analysis from similar samples from war zone soldiers. The results were also presented at multiple meetings and published. Two epigenetic studies using DNA methylation chromatin immunoprecipitation followed by whole genome scale sequencing were conducted from civilian and military groups. Epigenetic changes in the sports related concussion patients was presented at the ASHG meeting. This result was submitted for publication and currently under review. The second epigenetic study in military PTSD patients finished sequencing of first set of samples. After the primary data analysis, we increased sample size and will sequence more. Whole genome sequencing of special sensory disorder patient and control sibling were sequenced and will be analyzed. Using the targeted next generation sequencing technology, we also have 2 on-going microbiome projects to characterize population of microorganisms in human and/or animal body from different disease status. Using a next generation semiconductor sequencer, microbiome was analyzed with extensive sequencing of 16s rRNA region in an animal model of irritable bowel syndrome and human patients. The results were presented at the scientific meeting and submitted for publication. Another microbiome project for oral microbiome from the aplastic anemia patients was also on-going and finished the sample collection. So far, preliminary data analysis was finished and its result was presented at the IADR meeting and submitted for publication. Mainly both projects 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 detect tau protein in plasma, which has not been measured with ELISA. 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. 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.