Mechanisms of Genetic Seizure Susceptibility in Juvenile Myoclonic Epilepsy

描述（由申请人提供）：青少年肌阵挛性癫痫（JME）是一种常见的癫痫综合征，始于青春期或青年期，需要终身治疗。BRD 2是一种基因，在某些人群中，是JME和相关脑电图现象的致病因素，这是通过重复的连锁和关联研究证明的。基于家系和群体遗传学分析，它是目前常见癫痫的最佳候选基因。我们对Brd 2基因敲除小鼠的研究进一步加强了这一证据。敲除数据表明，BRD 2的缺失与生命不相容。在杂合子基因敲除小鼠（Brd 2），神经基板被改变：有一个缺陷的抑制性神经元在两个大脑区域，我们已经研究。最令人信服的是，杂合子显示出增加的癫痫发作易感性，具有差异的男性/女性模式，让人想起人类JME的模式。我们假设，在人类中，错误表达导致BRD 2蛋白的轻微不足，导致大脑组织的微妙变化。几行人类成像数据报告观察到JME患者神经基质的破坏，支持群体遗传学和小鼠研究。我们提出了合作，多学科的研究，以确定改变BRD 2表达对大脑基质和功能的影响，并确定这种干扰导致大脑过度兴奋和癫痫发作的机制。我们的目标是：1）充分表征Brd 2小鼠中由鼠Brd 2蛋白不足引起的神经底物、行为、癫痫发作易感性和电生理学的变化。这包括使用脑切片实验来研究Brd 2小鼠与野生型相比的神经回路行为，以及调查单个神经元的反应。2)鉴定导致脑过度兴奋的人BRD 2基因表达的特异性变化。我们将在组织培养中表达与JME相关的人类BRD 2等位基因，研究其转录、剪接和翻译的变化，并将这些变化与特定的多态性相关联。我们将研究BRD 2启动子、3 'UTR和5' UTR以及其他基因区域的多态性。特别是，我们确定了一个高度保守的，交替香料外显子位于内含子2。我们假设CA重复多态性，发现在内含子2和相关的JME，影响可变剪接形式的平衡。新的初步实验表明，外显子间DNA的组成改变了交替外显子的剪接特征。我们将在该位点插入不同大小的CA重复多态性，以观察对选择性剪接的BRD 2转录物水平的影响，最终研究正常和选择性剪接的RNA的命运。公共卫生相关性：项目叙述我们建议定义基因BRD 2导致青少年肌阵挛性癫痫易感性的遗传、神经解剖学和电路机制，青少年肌阵挛性癫痫是一种常见的青少年发作的癫痫，需要终身药物治疗来抑制癫痫发作。我们通过对人类癫痫的研究发现的BRD 2基因对神经结构和电路有着深远的影响。BRD 2是少数几个被证实的常见癫痫基因之一。这项工作最终可能导致理解影响数百万美国人的癫痫的基本原因和机制，并给患者，他们的家庭和医疗保健系统带来沉重的负担。

DESCRIPTION (provided by applicant): Juvenile Myoclonic Epilepsy (JME) is a common epilepsy syndrome that starts in adolescence or young adulthood and requires lifelong treatment. BRD2 is a gene that, in some populations, is a causal factor in JME and in related electroencephalographic phenomena, which is demonstrated by replicated linkage and association studies. Based on family and population genetic analysis, it is currently the best candidate gene for a common epilepsy. This evidence is further strengthened by our studies of a Brd2 knockout mouse. The knockout data show that absence of BRD2 is incompatible with life. In heterozygote knockout mice (Brd2), the neural substrate is altered: there is a deficit of inhibitory neurons in two brain regions we have studied. Most compellingly, the heterozygotes show increased seizure susceptibility with a differential male/female pattern reminiscent of the pattern for JME in humans. We hypothesize that, in humans, mis- expression results in a slight insufficiency of BRD2 protein, leading to subtle changes in brain organization. Several lines of human imaging data report observing disruptions of neural substrate in JME patients, supporting the population genetic and mouse studies. We propose collaborative, multidisciplinary studies to determine the effect of altered BRD2 expression on brain substrate and function, and to identify the mechanisms by which such disturbances lead to brain hyperexcitability and seizures. Our aims are: 1) To fully characterize changes to the neural substrate, behavior, seizure susceptibility, and electrophysiology caused by insufficiency of murine Brd2 protein in Brd2 mice. This includes using brain- slice experiments to study the behavior of neural circuits in Brd2 mice compared to wild type, as well as investigating the responses of individual neurons. 2) To identify the specific change(s) in the expression of the human BRD2 gene that lead to brain hyperexcitability. We will express human BRD2 alleles associated with JME in tissue culture, study changes in their transcription, splicing, and translation, and correlate those changes with specific polymorphisms. We will examine polymorphisms in the BRD2 promoters, 3'UTR and 5'UTR, and other gene regions. In particular, we identified a highly conserved, alternately-spiced exon located in intron 2. We hypothesize that CA-repeat polymorphisms, found in intron 2 and associated with JME, affect the balance of alternatively spliced forms. New preliminary experiments show that the composition of the inter-exon DNA alters the splicing characteristics of the alternate exon. We will insert varying sizes of CA-repeat polymorphisms at that locus to observe the effect on levels of alternatively spliced BRD2 transcripts, eventually studying the fate of both the normal and alternately spliced RNA. PUBLIC HEALTH RELEVANCE: Project Narrative We propose to define the genetic, neuroanatomical, and circuitry mechanisms by which the gene BRD2 leads to susceptibility to Juvenile Myoclonic Epilepsy, a common epilepsy of adolescent onset and one that requires life-long medication to suppress seizures. The BRD2 gene, which we identified through studies of human epilepsy, has a profound effect on neural structure and circuitry. BRD2 is one of the few proven genes for a common epilepsy. This work could, at last, lead to understanding the basic cause and mechanism of an epilepsy that affects millions of Americans and places a difficult burden on the patient, their families, and the health care system.