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决定小鼠癫痫易感性的自然发生的基因变异的要求。本研究的主要模型涉及两种常见的近交系小鼠，C57BL/6J （B6）相对耐癫痫发作，DBA/2J （D2）相对易癫痫发作。这两种小鼠对实验诱导的癫痫发作的易感性存在显著差异。鉴于本项目研究的短期目标与小鼠癫痫易感基因的鉴定有关，长期目标涉及将这些研究转化为重点假设和涉及人类癫痫患者的研究。因此，在过去的资助期内，我们使用B6-D2小鼠模型确定Kcnj10是位于远端Chr 1上的数量性状位点（QTL）的推定癫痫易感基因。在转化为临床研究后，我们发现人类KCNJ10遗传变异与常见癫痫形式的风险相关。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的癫痫遗传关联之一。因此，过去的资助期使我们能够展示“原理证明”和我们的转化研究策略的整体效用。由于B6- D2模型中远端Chr 1 QTL （Szs1）的解剖和翻译取得成功，我们最近（2008年2月）提交了一份新的资助申请，用于研究Kcnj10基因变异在癫痫易感性中的机制，因此本项目不建议进一步研究该基因。相反，我们将重点分析B6-D2模型中次优先级的癫痫易感性qtl，这些qtl也在之前的资助期内进行了研究。因此，在目标1和目标2中，我们建议在Chr 5和Chr 15的关键QTL区间分别精细定位和分析候选基因。这项工作将包括在B6和D2小鼠之间建立重组同源菌株，使用包括最大电休克发作阈值（MEST）和化学惊厥药在内的成熟范例确定癫痫易感性，生物信息学以及候选基因的mRNA和蛋白质分析。Aim 3将重点研究在上一个资助期内开发的涉及小鼠近交系C57BL/KsJ （Ks）和C57BL/10SnJ （B10S）的新遗传模型。在该模型中，我们建议在同源菌株中捕获Chrs 3,4,6和8上鉴定的MEST qtl，这些qtl将快速且彼此平行地创建。用化学惊厥药戊四唑（PTZ）、kainic酸（KA）和匹罗卡平（PC）检测捕获到MEST qtl的同源菌株，评估MEST qtl对癫痫易感性的影响程度。将根据所有范例的结果对qtl进行优先级排序，以便进一步剖析。本项目提出的研究成果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转化研究策略，结果将直接用于癫痫患者的临床研究。我们维护着一个不断增长的存储库，其中有来自癫痫患者的1000多个DNA样本，并利用它们来研究小鼠研究中提出的候选基因。在过去的资助期内，该项目完成的工作为该策略的实用性提供了“原理证明”，从而作为我们建议遵循的原型，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的生物学基础和癫痫的遗传易感性将有助于为患者开发新的和更有效的治疗方案，并可能有一天导致治愈这一毁灭性疾病组。公共卫生相关性：该项目涉及确定小鼠对实验性诱发癫痫易感性的基因。该项目的短期目标是确定癫痫易感性基因，而长期目标是试图了解它们如何影响癫痫易感性。最终，从这些小鼠研究中获得的知识将在我们的实验室应用于人类癫痫患者，从而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转化研究策略，因此小鼠研究的结果将直接用于临床研究。我们保存了来自常见癫痫患者的1000多个DNA样本，我们用它们来研究我们的小鼠研究中提出的基因，例如本项目中提出的基因。最终，更好地了解小鼠癫痫易感性的生物学基础和人类癫痫的遗传易感性，将有助于开发新的和更有效的癫痫患者治疗方案，并可能有一天导致治愈这一毁灭性的脑部疾病。

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.