Role of Intronic Variants Affecting Splicing in Juvenile Myoclonic Epilepsy

描述(申请人提供)：青少年肌阵挛癫痫(JME)是特发性全身性癫痫(IGE)最常见的综合征类型之一，具有复杂的遗传。虽然已经鉴定了几个可能的JME易感基因，但这些基因在大脑中的功能及其在癫痫发病机制中的作用尚不清楚。连锁和关联分析使我们找到了含溴结构域的基因BRD2。Brd2是双溴结构域基因的BET亚家族成员，参与基因表达调控。我们已经证明，BRD2在发育中的大脑和成年大脑中都有表达，最近，BrD2缺失的胚胎在妊娠中期死亡，并伴有中枢神经系统畸形。此外，我们的初步数据显示，杂合的Brd2小鼠显示出额叶皮质中GABA能中间神经元的数量减少，令人惊讶的是，杂合的Brd2小鼠对化学诱导的癫痫发作表现出更高的敏感性。我们的工作假设是BRD2的错误表达与癫痫易感性有关。我们还发现BRD2的内含子2含有一个高度保守的、选择性剪接的外显子，它引入了一个提前终止密码子，我们还发现了与JME相关的内含子2中CA重复序列长度的多态。在其他实验模型中，CA-重复序列长度的变化已被证明影响选择性剪接事件。我们现在希望检验这样的假设，即内含子2的多态，特别是我们在患者DNA中发现的与JME相关的CA重复长度变异，影响替代外显子的剪接和相应转录本的水平，从而影响BRD2产物的功能。在目标1中，我们将通过瞬时表达含有不同CA-重复长度的人BRD2的[外显子2]-[内含子2]-[外显子3]的‘mini基因’结构，并测量产生的两个转录本的比例，初步检测可变的CA-重复长度对两个选择性剪接的BRD2转录本的相对水平的影响。在目标2中，我们将通过确定选择性转录本是否被翻译以及如果是的话，所产生的蛋白质是否影响同源二聚化或亚细胞定位来阐明选择性剪接转录本的功能意义。 公共卫生相关性：青少年肌阵挛癫痫(JME)是特发性全身性癫痫(IGE)最常见的综合征类型之一，其特点是青春期发作和终生痛苦，需要持续用药来抑制癫痫发作。拟议的实验将证明BRD2内含子2中的JME相关变体是否在替代剪接的频率中发挥直接作用，并进一步提供对替代转录本可能的生理意义的洞察，这些结果与RFA和R03资助机制的声明目标高度相关。

DESCRIPTION (provided by applicant): Juvenile Myoclonic Epilepsy (JME) is among the most common types of syndromes of Idiopathic Generalized Epilepsy (IGE) and has a complex genetic inheritance. Although several putative JME susceptibility genes have been identified, the functions of these genes in the brain and their roles in the pathogenesis of seizures are unknown. Linkage and association analysis led us to the bromodomain-containing gene BRD2. BRD2 is a member of BET subfamily of double bromodomain-containing genes implicated in regulating gene expression. We have shown that BRD2 is expressed in developing and adult brain, and recently, that Brd2-null embryos die at mid-gestation with central nervous system malformations. Further, our preliminary data show that heterozygous Brd2 mice exhibit a reduction in the number of GABAergic interneurons in the frontal cortex and, strikingly, heterozygous Brd2 mice exhibit an enhanced sensitivity to chemically-induced seizures. Our working hypothesis is that mis-expression of BRD2 contributes to seizure susceptibility. We have also discovered that intron 2 of BRD2 contains a highly conserved, alternatively spliced exon, which introduces a premature termination codon and we have identified polymorphisms in the lengths of CA-repeats in intron 2 that are associated with JME. Variation in the length of CA-repeats has been shown to affect alternative splicing events in other experimental models. We now wish to test the hypothesis that the polymorphisms in intron 2, in particular variants in CA repeat-length that we have identified in patient DNA that associate with JME, affect splicing of the alternative exon and the levels of the corresponding transcripts, and hence, function of BRD2 products. In Aim 1, we will initially examine the effect of variable CA-repeat lengths on the relative levels of the two alternatively spliced BRD2 transcripts by transient expression of 'mini-gene' constructs containing [exon 2]-[intron 2]-[exon3] of human BRD2 with varying CA-repeat lengths and measuring the ratio of the resulting two transcripts. In Aim 2, we will begin to elucidate the functional significance of the alternatively spliced transcripts by determining whether the alternative transcripts are translated and if so, whether the proteins made affect homodimerization or sub-cellular localization. PUBLIC HEALTH RELEVANCE: Juvenile Myoclonic Epilepsy (JME) is among the most common types of syndromes of Idiopathic Generalized Epilepsy (IGE) and is characterized by adolescent onset and life-long affliction, requiring continual medication to suppress seizures. The proposed experiments will demonstrate whether the JME- associated variants in intron 2 of BRD2 play a direct role in the frequency of alternative splicing, and further, will provide insight into the possible physiological significance of the alternative transcripts, outcomes that are highly relevant to the stated goals of the RFA and the R03 funding mechanism.