Quantitative Genetic Study of Seizures

描述(由申请人提供)：本申请中提出的研究代表了继续表征自然发生的基因变异的请求，该变异决定了小鼠的癫痫易感性。研究中的主要模型涉及两种常见的近交系小鼠，它们对实验诱导的癫痫的敏感性存在很大差异，C57BL/6J(B6)相对抗癫痫，DBA/2J(D2)相对容易癫痫发作。虽然该项目研究的短期目标与小鼠癫痫易感基因的鉴定有关，但长期目标包括将这些研究转化为有重点的假设和涉及人类癫痫患者的研究。因此，在过去的资助期间，我们使用B6-D2小鼠模型来确定Kcnj10是潜在的癫痫易感基因，该基因位于Chr1远端的数量性状位点(QTL)。在翻译到临床研究后，我们发现KCNJ10在人类中的基因变异与常见形式癫痫的风险有关。重要的是，后一项发现得到了独立实验室的证实，是文献中为数不多的重复癫痫遗传关联之一。因此，过去的资助期使我们能够证明“原则证明”和我们的翻译研究战略的整体效用。由于在B6-D2模型中成功地分离和翻译了远端Chr1 QTL(Szs1)，我们最近(2008年2月)提交了一项新的拨款申请，以研究Kcnj10基因变异在癫痫易感性中的机制，因此我们不建议在这个项目中进一步研究该基因。相反，我们将集中分析B6-D2模型中下一个最优先的癫痫易感性QTL，这些QTL也在上一个资助期进行了研究。因此，在目标1和目标2中，我们建议分别在Chr 5和Chr 15的关键QTL区间对候选基因进行精细定位和分析。这项工作将包括在B6和D2小鼠之间创建重组同源菌株，使用包括最大电休克发作阈值(MEST)和化学惊厥的成熟范例来确定癫痫敏感性，生物信息学以及候选基因的mRNA和蛋白质分析。目的3将重点介绍在上一个资助期建立的一个新的遗传模型，该模型涉及C57BL/KSJ(Ks)和C57BL/10SnJ(B10S)近交系小鼠。在这个模型中，我们建议通过在快速且相互平行的同源菌株中捕获MEST QTL来确认在CHRAS 3、4、6和8上发现的MEST QTL。在MEST QTL被捕获的同源菌株中，将通过用化学惊厥剂戊四唑(PTZ)、海人酸(KA)和匹罗卡品(PC)进行测试，来评估MEST QTL对癫痫敏感性的影响程度。QTL将根据所有范例的结果进行优先顺序的进一步剖析。本项目提出的研究将对癫痫遗传学研究领域产生直接影响。由于我们的实验室致力于翻译研究策略，结果将直接用于为癫痫患者的临床研究提供信息。我们保持着1000多个癫痫患者DNA样本的不断增长的存储库，并使用它们来研究小鼠研究提出的候选基因。在过去的资金支持期间，该项目完成的工作为这一策略的使用提供了“原则证明”，因此，我们建议将其作为一个原型，以进一步阐明定义人类癫痫的复杂遗传结构。最终，更好地了解癫痫易感性和癫痫遗传易感性的生物学基础将有助于为患者开发新的和更有效的治疗方案，并有一天可能导致治愈这一毁灭性的疾病组。与公共卫生相关：该项目涉及识别小鼠的基因，这些基因决定了它们对实验诱导的癫痫发作的易感性。虽然该项目的短期目标包括识别癫痫易感性基因，但长期目标是试图了解它们如何影响癫痫易感性。最终，从小鼠身上获得的知识将在我们的实验室应用于人类癫痫患者，从而对癫痫遗传学研究领域产生立竿见影的影响。由于我们的实验室致力于转化性研究策略，因此在小鼠身上的研究结果将直接用于临床研究。我们维护着一个超过1000个来自常见形式癫痫患者的DNA样本的储存库，我们使用它们来研究我们的小鼠研究提出的基因，比如这个项目中提出的那些。最终，更好地了解小鼠癫痫易感性的生物学基础和人类对癫痫的遗传易感性将有助于为癫痫患者开发新的、更有效的治疗方案，并有一天可能导致治愈这种毁灭性的脑部疾病。

DESCRIPTION (provided by applicant): The studies proposed in this application represent a request to continue characterizing naturally-occurring gene variation that determines seizure susceptibility in mice. The primary model under study involves two common strains of inbred mice that differ robustly with respect to their susceptibility to experimentally-induced seizures, C57BL/6J (B6), which is relatively seizure resistant, and DBA/2J (D2), which is relatively seizure susceptible. Whereas the short term goal of studies in this project is related to the identification of seizure susceptibility genes in mice, the long term goal involves translation of these studies into focused hypotheses and studies involving human patients with epilepsy. Thus, in the past funding period, we used the B6-D2 mouse model to identify Kcnj10 as a putative seizure susceptibility gene underlying the quantitative trait locus (QTL) on distal Chr 1. Upon translation to a clinical study, we showed that KCNJ10 genetic variation in humans is associated with risk for common forms of epilepsy. Importantly, this latter finding was confirmed by an independent laboratory and exists as one of the few replicated epilepsy genetic associations in the literature. Thus the past funding period enabled us to demonstrate "proof of principle" and the overall utility of our translational research strategy. As a result of the success achieved in the dissection and translation of the distal Chr 1 QTL (Szs1) in the B6- D2 model, we have recently (February 2008) submitted a new grant application to study the mechanism of Kcnj10 genetic variation in seizure susceptibility and thus do not propose further study of that gene in this project. Rather, we will focus on analysis of seizure susceptibility QTLs of next highest priority in the B6-D2 model which were also studied in the previous funding period. Thus in Aims 1 and 2 we propose to fine-map and analyze candidate genes in critical QTL intervals on Chr 5 and Chr 15, respectively. This work will include creation of recombinant congenic strains between B6 and D2 mice, determination of seizure susceptibility using well-established paradigms involving maximal electroshock seizure threshold (MEST) and chemical convulsants, bioinformatics and mRNA and protein analysis of candidate genes. Aim 3 will focus on a new genetic model developed in the last funding period involving C57BL/KsJ (Ks) and C57BL/10SnJ (B10S) inbred strains of mice. In this model, we propose to confirm MEST QTLs identified on Chrs 3, 4, 6 and 8 by capturing them in congenic strains which will be created rapidly and in parallel with each other. The extent of the influence of MEST QTLs on seizure susceptibility will be evaluated in those congenic strains in which MEST QTLs are captured by testing them with chemoconvulsants pentylenetetrazol (PTZ), kainic acid (KA) and pilocarpine (PC). QTLs will be prioritized for further dissection based upon the results from all paradigms. The studies proposed in this project will have immediate impact on the field of epilepsy genetics research. Since our laboratory is dedicated to a translational research strategy, results will be used directly to inform clinical studies of patients with epilepsy. We maintain a growing repository of over 1000 DNA samples from patients with epilepsy and use them to investigate candidate genes that are suggested by the mouse studies. Work completed in this project during the past funding period provides "proof of principle" for the utility of this strategy and thus serves as a prototype that we propose to follow for further elucidation of the complex genetic architecture that defines human epilepsy. Ultimately, a better understanding of the biological basis of seizure susceptibility and genetic predisposition to epilepsy will facilitate the development of novel and more effective therapeutic options for patients and may one day lead to a cure for this devastating group of diseases. PUBLIC HEALTH RELEVANCE: This project involves the identification of genes in mice that determine their susceptibility to experimentally- induced seizures. Whereas the short term goal of the project involves the identification of seizure susceptibility genes, the longer term goal is to try to understand how they influence seizure susceptibility. Ultimately, the knowledge gained from these studies on mice will be applied in our laboratory to human patients with epilepsy and thus will have immediate impact on the field of epilepsy genetics research. Since our laboratory is dedicated to a translational research strategy, results from work on mice will be used directly to inform clinical studies. We maintain a repository of over 1000 DNA samples from patients with common forms of epilepsy and we use them to investigate genes that are suggested by our mouse research such as those proposed in this project. Ultimately, a better understanding of the biological basis of seizure susceptibility in mice and genetic predisposition to epilepsy in humans will facilitate the development of novel and more effective therapeutic options for patients with epilepsy and may one day lead to a cure for this devastating group of brain diseases.