Role of Intronic Variants Affecting Splicing in Juvenile Myoclonic Epilepsy

影响剪接的内含子变异在青少年肌阵挛性癫痫中的作用

• 批准号: 7941948
• 负责人: DEBRA J. WOLGEMUTH
• 金额: $ 15.94万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7941948
• 关键词: 6p21; Adolescent; Adult; Affect; Alternative Splicing; BRD2 gene; Brain; Bromodomain; Complex; Congenital neurologic anomalies; DEFB1 gene; DNA; Data; Development; Dimerization; Embryo; European; Event; Exhibits; Exons; Experimental Models; Family; Frequencies; Funding Mechanisms; Gene Expression; Generalized Epilepsy; Genes; Genetic; Genetic Polymorphism; Goals; Homo; Homodimerization; Human; Interneurons; Introns; Juvenile Myoclonic Epilepsy; Length; Life; Measures; Mediating; Molecular; Mus; Nature; Neuraxis; Nonsense Codon; Outcome; Pathogenesis; Patients; Pharmaceutical Preparations; Phenotype; Physiological; Play; Population; Predisposition; Pregnancy; Production; Proteins; RNA Splicing; Relative (related person); Role; Seizures; Site; Structure; Susceptibility Gene; Syndrome; Testing; Transcript; Translating; Translation Initiation; Variant; Work; frontal lobe; gene function; genetic linkage analysis; insight; mRNA Decay; member; novel; public health relevance; research study

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.

描述（由申请人提供）：少年肌阵挛性癫痫（JME）是特发性概括性癫痫（IGE）最常见的综合症类型之一，并且具有复杂的遗传遗传。尽管已经鉴定出了几种推定的JME敏感性基因，但这些基因在大脑中的功能及其在癫痫发病机理中的作用尚不清楚。连锁和关联分析使我们进入了含溴a的基因BRD2。 BRD2是与调节基因表达有关的含双溴脱域基因的BET亚科的成员。我们已经表明，BRD2在发育和成人大脑中表达，最近，BRD2-NULL胚胎在中枢神经系统畸形中死亡。此外，我们的初步数据表明，杂合BRD2小鼠表现出额叶皮质中GABA能中间神经元数量的减少，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增强的敏感性。我们的工作假设是，BRD2的错误表达有助于癫痫发作。我们还发现，BRD2的内含子2包含高度保守的剪接外显子，该外显子引入了过早的终止密码子，并且我们在内含子2中与JME相关的CA-重复的长度鉴定了多态性。 CA-重复长度的变化已显示出在其他实验模型中影响替代剪接事件的变化。现在，我们希望检验以下假设：内含子2中的多态性，特别是我们在患者DNA中鉴定出的与JME相关的患者DNA中鉴定出的变体，会影响替代外显子的剪接以及相应的转录本的水平，以及BRD2产物的功能。在AIM 1中，我们最初将检查可变长度长度对两个剪接的BRD2转录本的相对水平的影响，这是通过含有[EXON 2]的“迷你基因”构建体的短暂表达，[exon 2] - [interon 2] - [interon 2] - [exon3] - [外exon3]，人类BRD2，人类BRD2具有不同的ca-repeat ca-repeat长度和测量的两次转录的影响。在AIM 2中，我们将开始通过确定是否翻译替代转录物来阐明剪接的转录本的功能意义，如果是，是否会影响蛋白质会影响同构化或亚细胞定位。 公共卫生相关性：少年肌阵挛性癫痫（JME）是特发性概括性癫痫（IGE）最常见的综合症类型之一，其特征是青少年发作和终身苦难，需要连续的药物来抑制癫痫发作。提出的实验将证明BRD2内含子2中的JME相关变体在替代剪接的频率中是否起着直接的作用，此外，还将洞悉替代转录本的可能生理意义，与RFA和RFA和R03资助机制高度相关的结果。