Genomic Approaches for Elucidating Novel Targets for Pain and Symptom Management

神经系统疾病的起源是多因素的，遗传和环境因素都有助于个体差异。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，参与不同疼痛表型的因素和数千种分子的复杂网络使得难以检测个体对疼痛和其他神经系统疾病的独特易感性的负责任的遗传变异。单个基因的共同变异不太可能对疼痛起主导作用;相反，每个基因的作用似乎很微妙，作用于多个疼痛通路中的一个，使其信号难以检测。 尽管疼痛是数千年来影响生活质量的最重要和最常见的问题之一，但镇痛治疗仍然主要限于阿片类药物和阿司匹林类药物，这些药物类别具有局限性。疾病知识的快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对更有效和更安全的疼痛管理药物的期望。为疼痛和其他神经系统疾病管理开发新的治疗策略也关键取决于识别新的靶分子和定义特定类型神经系统疾病的表型。因此，该项目的第一步是确定实验和临床表型的特征。在评价患者组时，性别、种族和心理因素等促成因素在疼痛和镇痛反应中的作用优于遗传因素（Kim et al. 2004 a; Kim et al. 2004 b）。但是遗传变异在个体水平上可能很重要（Dionne et al. 2005）。 基于这个项目，我们发现了来自每个主要种族人群的候选疼痛基因的单倍体。人类疼痛相关基因的单倍型数据为疼痛敏感性与镇痛药反应的遗传关联研究提供了基础信息。我们还应用该单倍型数据来发现与实验性疼痛敏感性的关联，并证实该方法可用于研究遗传学在疼痛敏感性中的作用（Kim等人，2006，JMG）。我们还进行了候选基因的个体SNP关联研究，并报告了与先前发表的研究相反的结果，这些研究可能因群体分层和小样本量而存在偏倚（Kim et al. 2006，Molecular Pain）。同时，我们研究了前列腺素合成的遗传变异对口腔手术模型中临床诱导的疼痛和镇痛反应的影响。由此，我们发现，与G/C杂合子和C/C纯合子患者相比，考克斯-2基因的启动子区域（-765）中SNP的纯合子G/G患者显示出对常见镇痛药物的显著不同的反应（Lee et al. 2006，CPT）。我们最近发表了一项在口腔手术模型中使用500，000个SNP检测的与临床疼痛敏感性相关的全基因组扫描研究。基于全基因组规模研究，我们进一步表征了具有密集基因分型的候选区域，并鉴定了锌指蛋白中的遗传基因座，该基因座有助于小手术后镇痛药物反应的个体间变异性（Kim et al. 2009，Pharmacogenomics）。我们已经将人类基因组中检测SNP的数量扩大到100万个，并对数百名纤维肌痛、急性冠状动脉综合征和蛛网膜下腔出血患者进行基因分型。这些研究的结果在美国疼痛学会、美国人类遗传学学会和国际中风会议的年会上报告。这些项目提供的候选区域的详细信息和功能基因组研究可提供关于个体患者在组织损伤、疼痛和止痛以及其他神经症状反应中的遗传作用的知识。此外，2012年发表了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（Kim等人，2012，Int J of Stroke）。 最近，我们启动了下一代测序技术的项目，该技术使我们能够对具有独特表型的个体进行全基因组测序，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，例如士兵暴露于战斗中，以及创伤性脑损伤导致的创伤后应激障碍。遗传和表观遗传因素对临床疼痛的作用将继续在神经系统疾病中进行研究，如急性疼痛和PTSD，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、ELISA、SNP基因分型和真实的时间PCR进行分析。从战区回来的士兵的基因表达谱在多个会议上（CNRM和ESTA会议）。癌症疲劳患者的发病变化也将在今年的ASHG会议上提出。 从这些结果沿着的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。

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.