Genomic Approaches for Elucidating Novel Targets for Pain and Symptom Management

神经性疾病是由多因素引起的，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疼痛表型的数千个分子使得很难检测到一个人对疼痛和其他神经疾病的独特易感性的负责任的基因变异。单个基因的常见变异不太可能主要作用于疼痛；相反，每个基因的贡献似乎都很微妙，作用于多条疼痛途径中的一条，使其信号很难被检测到。 尽管几千年来，疼痛一直是影响生活质量的最重要和最常见的问题之一，但止痛治疗在很大程度上仍然局限于阿片类药物和阿司匹林类药物，这些药物类别的局限性。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综合影响，提高了人们对更有效和更安全的止痛药物的期望。为疼痛和其他神经疾病的管理开发新的治疗策略也至关重要地依赖于确定新的靶分子和确定特定类型神经疾病的表型。因此，这个项目的第一步是确定实验和临床表型的特征。在评估患者组时，性别、种族和心理因素等促成因素在疼痛和止痛反应中的作用超过遗传因素(Kim等人。2004a；Kim等人。2004b)。但遗传变异在个体水平上可能是重要的(Dionne等人。2005)。 在这个项目的基础上，我们从每个主要种族人群的候选疼痛基因中找到了单倍体。人类疼痛相关基因的单倍型数据为疼痛敏感性和止痛药反应的遗传关联研究提供了基本信息。我们还应用这些单倍型数据来寻找与实验性疼痛敏感性的关联，并验证了这种方法在研究遗传在疼痛敏感性中的作用是有用的(Kim等人。2006年，JMG)。我们还对候选基因进行了单独的SNP关联研究，并报告了与之前发表的研究相反的结果，这些研究可能与种群分层和小样本大小存在偏见(Kim等人。2006年，分子疼痛)。同时，我们在口腔外科模型中研究了前列腺素合成的遗传变异对临床诱导的疼痛和止痛反应的影响。由此，我们发现COX-2基因启动子区域(-765)SNP纯合子G/G患者与G/C杂合子患者和C/C纯合子患者对常用止痛药的反应显著不同(Lee等。2006年，CPT)。我们最近发表了一项与临床疼痛敏感性相关的全基因组扫描研究，在口腔外科模型中使用了500,000个SNPs分析。基于全基因组规模的研究，我们进一步确定了具有密集基因分型的候选区域，并在锌指蛋白中确定了导致小手术后止痛药物反应的个体间差异的遗传位点(Kim等人)。2009年，药物基因组学)。我们已经将检测人类基因组的SNPs的数量扩大到100万个，并正在对数百名患有纤维肌痛、急性冠脉综合征和蛛网膜下腔出血的患者进行基因分型。这些研究的结果发表在美国疼痛学会、美国人类遗传学学会和国际中风会议的年度会议上。来自这些项目的候选区域的详细信息和功能基因组研究可能提供关于个体患者在对组织损伤、疼痛和止痛以及其他神经症状的反应中的遗传作用的知识。此外，基于蛛网膜下腔出血患者经颅多普勒信号的脑血管痉挛证据的遗传关联结果也于2012年发表(Kim等人。2012年，Int J of Stroke)。 最近，我们启动了使用下一代测序技术的项目，该技术允许我们对具有独特表型的个人进行全基因组测序，例如辣椒素不敏感的患者，以及长期环境变化后的表观遗传变化，例如士兵暴露在战斗中，以及创伤后应激障碍导致的创伤性脑损伤。遗传和表观遗传因素在临床疼痛中的作用将继续在神经疾病中进行研究，如急性疼痛和创伤后应激障碍，使用基因分型、基因和蛋白表达以及患者报告的结果，以更好地了解这些因素和炎症级联反应之间的相互作用。这些数据将通过全基因组测序、微阵列、酶联免疫吸附试验、SNP基因分型和实时聚合酶链式反应进行分析。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。癌症疲劳患者的警示变化也将在今年的ASHG会议上公布。 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。

Neurological disorders 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 pain phenotypes makes it difficult to detect responsible genetic variations for an individuals unique susceptibility to pain and other neurological disorders. It is unlikely that common variations in a single gene act dominantly on pain; rather, the contribution of each gene seems to be subtle, acting on one of multiple pain pathways, making its signal difficult to detect. Even though pain has been one of the most significant and frequent problems affecting quality of life for thousands of years, analgesic therapy is still largely limited to opioids and aspirin-like drugs, with the limitations of these drug classes. 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 pain management. Developing new treatment strategies for the pain and other neurological disorders management is also critically dependent on identifying new target molecules and defining phenotypes for specific types of neurological disorders. Therefore the first step of this project has been to define the characteristics of experimental and clinical phenotypes. Contributing factors such as gender, ethnicity and psychological factors predominate over the role of genetic factors in pain and analgesic responses when evaluating groups of patients (Kim et al. 2004a; Kim et al. 2004b). But genetic variability may be important at the level of the individual (Dionne et al. 2005). Based on this project, we found haploblocks from candidate pain genes for each major ethnic population. Human haplotype data of pain related genes provide basic information for the genetic association studies of pain sensitivity and responses to analgesics. We also applied this haplotype data to find the association with experimental pain sensitivity and verified that this method is useful in investigating the role of genetics in pain sensitivity (Kim et al. 2006, JMG). We also have performed individual SNP association studies of candidate genes and reported results contrary to previous published studies, which may have been biased with population stratification and small sample size (Kim et al. 2006, Molecular Pain). Meanwhile we investigated the influence of the genetic variations in prostaglandin synthesis on the clinically induced pain and analgesic responses in the oral surgery model. From this, we found that homozygous G/G patients of SNP in the promoter region (-765) of COX-2 gene showed significantly different responses to common analgesic drugs compared to G/C heterozygous and C/C homozygous patients (Lee et al. 2006, CPT). We recently published a whole genome scan study related to clinical pain sensitivity using 500,000 SNPs assay in the oral surgery model. Based on whole genome scale investigation, we have further characterized a candidate region with dense genotyping and identified a genetic locus in a zinc finger protein that contributes to interindividual variability in analgesic drug responses following minor surgery (Kim et al. 2009, Pharmacogenomics). We have expanded the number of testing SNPs up to 1 million from human genome and are genotyping hundreds of patients with fibromyalgia, acute coronary syndrome and subarrachnoid hemorrhage. Results from those studies were reported at the annual meetings of American Pain Society, American Society of Human Genetics and International Stroke Conference. Detailed information and functional genomic studies of the candidate regions from those projects may provide knowledge for the genetic role in responses to tissue injury, pain and analgesia and other neurological symptoms on an individual patient basis. Also the result of genetic association in the evidence of cerebral vasospasm based on transcranial doppler signals in subarrachnoid hemorrhage patients was publishced in 2012 (Kim et al. 2012, Int J of Stroke). Recently, we launched projects with next generation sequencing technology, which allows us perform entire genome sequencing for individuals with unique phenotypes such as capsaicin non-sensitive patients, and epigenetic changes following long term environmental changes such as soldiers exposed to combat, and traumatic brain injury resulted to the post traumatic stress disorder. The role of genetic and epigenetic factors on clinical pain will continue to be studied in neurological disorders such as acute pain and PTSD using genotyping, gene and protein expression, and patient reported outcomes to better understand the reciprocal interplay between these factors and the inflammatory cascade. These data will be analyzed with whole genome sequencing, microarray, ELISA, SNP genotyping, and real time PCR. Gene expression profiles from the soldiers back from war zone were presented at the multiple meetings (CNRM and ASN meeting). Epigeetic changes in the cancer fatigue patients will also be presented at the ASHG meeting this year. 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.