The Role of Genome-Coded ME2 in Epilepsy

描述(由申请人提供)：特发性全身性癫痫(IGE)是最常见的癫痫形式之一，约占所有癫痫的30%；其病因被认为主要是遗传的。连锁和关联研究已经确定了几个免疫球蛋白E的候选基因，即：苹果酸酶2(ME2)和含溴结构域2(BRD2)。此外，以往的研究也表明，IgE的易感性是基因-基因相互作用的结果。我们将专注于在ME2和IgE之间建立明确的生物学联系，我们还将测试ME2和BRD2之间的相互作用。18号染色体上的连锁和关联结果表明，ME2是一个通用的免疫球蛋白E易感基因座，遵循隐性遗传方式。对20例IgE病例和18例对照进行序列分析，发现在ME2内含子1有一个区域，病例中的多态位点在对照中大多是单态的。此外，ME2是免疫球蛋白E易感性的极佳生物候选者，因为：1)它在脑神经元中含量丰富；2)它定位于线粒体；最引人注目的是，3)它催化苹果酸脱羧基，为谷氨酸和GABA的合成产生关键的丙酮酸供应。GABA是中枢神经系统中的主要抑制性神经递质，参与癫痫样棘波活动的调节。抗癫痫药物机制也调节GABA神经递质系统，GABA受体的突变已被证明在某些形式的癫痫的易感性中发挥作用。在这项资助申请中，我们将根据我们选择的可获得序列数据的病例样本来确定ME2内含子1的不同单倍型。然后，我们将使用报告构建法来检测不同内含子1单倍型对剪接和转录水平的单独影响。注意，我们的报告构建体将只包含近端的启动子元件和内含子1。接下来，我们将检查内含子1变体在神经细胞和/或神经胶质细胞中的影响；与我们的报告构建体不同，这些实验将涉及整个ME2基因。最后，使用标准的重采样方法和一种新的统计方法，我们将测试ME2和BRD2之间的相互作用与青少年肌阵挛发作--一种IGE亚型--的相互作用。总体而言，这项拟议的研究应该有助于阐明ME2作为免疫球蛋白E易感基因的一般作用，以及它在其他促成遗传因素网络中的特殊作用。与公共卫生相关：仅在美国就有270万人患有癫痫。特发性全身性癫痫(IGE)占所有癫痫的30%，始于儿童或青春期，被认为有遗传病因。我们的研究发现一个基因(ME2)与几种不同的、常见的IGE综合征密切相关，包括青少年肌阵挛癫痫(JME)、青少年失神癫痫(JAE)和癫痫伴广泛性强直-阵挛发作(EGTCS)。我们建议确定在ME2基因中发现的多态如何增加对一大类相关癫痫综合征的易感性。

DESCRIPTION (provided by applicant): Idiopathic Generalized Epilepsy (IGE) is one of the most common forms of epilepsy, representing about 30% of all epilepsies; and, its etiology is considered to be mostly genetic. Both linkage and association studies have identified several candidate genes for IGE, namely: malic enzyme 2 (ME2) and bromodomain containing 2 (BRD2). In addition, previous studies have also suggested that IGE susceptibility results from gene-gene interaction. We will focus on establishing a clear biological link between ME2 and IGE, and we will also test for an interaction between ME2 and BRD2. The strong linkage and association results on chromosome 18 suggest that ME2 is a general IGE susceptibility locus, and that it follows a recessive mode of inheritance. Sequence analysis in 20 IGE cases and 18 controls, revealed a region in intron 1 of ME2 where polymorphic sites in cases are mostly monomorphic in controls. Moreover, ME2 is an excellent biological candidate for susceptibility to IGE due to: 1) its abundance in brain neurons; 2) its localization to the mitochondria; and most compelling, 3) its catalytic function of malate decarboxylation, producing a critical supply of pyruvate for glutamate and GABA synthesis. GABA is the main inhibitory neurotransmitter in the central nervous system, and is involved in the modulation of epileptiform spike-wave activity. Anti-epilepsy drug mechanisms also modulate the GABA neurotransmitter system, and mutations in GABA receptors have been shown to play a role in susceptibility to some forms of epilepsy. In this grant application, we will determine the different haplotypes of ME2 intron 1 based on our selected sample of cases for whom sequence data is available. Then, we will use reporter constructs to examine the isolated effects of different intron 1 haplotypes on splicing and levels of transcription. Note that our reporter constructs will only contain proximal promoter elements and intron 1. Next, we will examine the effects of intron 1 variants in neuronal and/or glial cells; and unlike our reporter constructs, these experiments will involve the entire ME2 gene. Finally, using standard resampling methods and a novel statistical approach, we will test for an interaction between ME2 and BRD2 with respect to juvenile myclonic epilepsy-an IGE subtype. Overall, this propose research should help to elucidate the general role of ME2 as a susceptibility gene for IGE, as well as its particular role in a network of other contributing genetic factors. PUBLIC HEALTH RELEVANCE: Epilepsy affects 2.7 million people in the US alone. Idiopathic Generalized Epilepsy (IGE) accounts for 30% of all epilepsy, starts in childhood or adolescence, and, is thought to have a genetic etiology. Our studies have identified a gene (ME2) as both linked and strongly associated with several different, common IGE syndromes, including Juvenile Myoclonic Epilepsy (JME), Juvenile Absence Epilepsy (JAE), and Epilepsy with Generalized Tonic-Clonic Seizures (EGTCS). We propose to determine how polymorphisms found in the ME2 gene increase susceptibility to a broad class of related epilepsy syndromes.