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。BRD2是一个基因，在一些人群中，是JME和相关脑电图现象的一个因果因素，这已被重复的连锁和关联研究证明。基于家族和群体遗传分析，它是目前常见癫痫的最佳候选基因。我们对Brd2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BRD2的缺失与生命是不相容的。在杂合子基因敲除小鼠（Brd2）中，神经基质发生了改变：我们研究的两个大脑区域中存在抑制性神经元的缺陷。最令人信服的是，杂合子表现出增加的癫痫易感性，其不同的男性/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BRD2蛋白的轻微不足，导致大脑组织的微妙变化。几组人类成像数据报告在JME患者中观察到神经基质的破坏，支持群体遗传和小鼠研究。我们建议开展多学科合作研究，以确定BRD2表达改变对脑底物和功能的影响，并确定这种紊乱导致大脑高兴奋性和癫痫发作的机制。我们的目标是：1)充分表征Brd2小鼠Brd2蛋白不足引起的神经基质、行为、癫痫易感性和电生理的变化。这包括使用脑切片实验来研究Brd2小鼠与野生型小鼠的神经回路行为，以及调查单个神经元的反应。2)确定导致大脑高兴奋性的人类BRD2基因表达的具体变化。我们将在组织培养中表达与JME相关的人类BRD2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多态性联系起来。我们将研究BRD2启动子、3'UTR和5'UTR以及其他基因区域的多态性。特别是，我们发现了一个高度保守的，位于内含子2的交替香料外显子。我们假设在内含子2中发现的ca重复多态性与JME相关，影响了可选剪接形式的平衡。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性。我们将在该位点插入不同大小的ca重复多态性，以观察对BRD2交替剪接转录本水平的影响，最终研究正常和交替剪接RNA的命运。我们建议定义BRD2基因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，这是一种青少年发作的常见癫痫，需要终身药物来抑制癫痫发作。我们通过对人类癫痫的研究发现了BRD2基因，它对神经结构和神经回路有着深远的影响。BRD2是少数被证实与常见癫痫有关的基因之一。这项工作最终可能导致了解癫痫的基本原因和机制，这种疾病影响着数百万美国人，给患者、他们的家庭和卫生保健系统带来了沉重的负担。

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.