Molecular Regulation of an Epilepsy Modifier Gene

癫痫修饰基因的分子调控

• 批准号: 8691435
• 负责人: BENJAMIN JORGE
• 金额: $ 1.24万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2014-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8691435
• 关键词: 3' Untranslated Regions; Address; Benign; Biological Assay; Brain; Clinical; Code; Complementary DNA; Data; Diagnostic; Dose; Elements; Epilepsy; Equilibrium; Family member; Febrile Convulsions; Future; Gated Ion Channel; Gene Transfer Techniques; Generalized Epilepsy; Genes; Genetic; Genetic Polymorphism; Genetic Transcription; Genomics; Goals; Hippocampus (Brain); Human; Initiator Codon; Knowledge; Length; Luciferases; Maps; Messenger RNA; Molecular; Mouse Strains; Mus; Mutation; Neonatal; Patients; Phenotype; Predisposition; Promoter Regions; RNase protection assay; Regulation; Regulatory Element; Reporter; Resistance; SJL Mouse; Seizures; Severities; Sodium Channel; Syndrome; System; Testing; Tetanus Helper Peptide; Transcript; Transcription Initiation Site; Transgenes; Transgenic Mice; Transgenic Organisms; Untranslated Regions; Variant; Voltage-Gated Potassium Channel; base; improved; in vivo; infancy; mRNA Stability; mRNA Transcript Degradation; mouse model; new therapeutic target; novel therapeutics; overexpression; promoter; public health relevance; screening; stability testing; voltage

DESCRIPTION (provided by applicant): Mutations in voltage-gated ion channels are one of the leading causes of monogenic epilepsies. A common feature of monogenic epilepsies is variable expressivity among family members with the same mutation, suggesting that genetic modifiers may influence susceptibility. The Scn2aQ54 transgenic mouse model of epilepsy has an epilepsy phenotype with strain-dependent expressivity. Kcnv2, which encodes the voltage- gated potassium channel subunit Kv8.2, was identified as a genetic modifier of the Scn2aQ54 phenotype. Screening of epilepsy patients identified two non-synonymous coding sequence variants that altered channel function in a heterologous expression system, suggesting that KCNV2 also contributes to human epilepsy susceptibility. Mouse data indicates that Kcnv2 is a quantitative modifier, with increased steady-state levels of Kcnv2 expression associated with an increase in Scn2aQ54 phenotype severity. These data suggest that regulatory sequence variation in the promoter and 3'UTR of KCNV2 may influence seizure susceptibility by altering steady-state expression levels. The proposed studies will characterize the Kcnv2 promoter and 3' UTR in mouse and human and determine the effects of mouse and human regulatory variants on Kcnv2 transcription rates and mRNA stability. In Specific Aim 1, rapid amplification of cDNA ends (RACE) analysis and RNase protection assays will be used to determine the transcription start site (TSS) region and 3'UTR organization of mouse and human KCNV2. Luciferase promoter assays will then be used to identify the minimal promoter regions. In Specific Aim 2, luciferase reporter constructs will be used to determine the effects of promoter sequence variation on mouse and human KCNV2 promoter activity. Tet-off reporter constructs will be used to test the influence of mouse and human 3' UTR sequence variation on mRNA stability. As a complementary approach, primary hippocampal cultures will be used to test the stability of mouse Kcnv2 mRNA following transcriptional inhibition. Next, the in vivo effects of selected Kcnv2 regulatory element(s) will be evaluated by BAC transgenesis. Elucidating the effects of regulatory sequence variants on Kcnv2 expression will help us to determine the molecular basis of the Kcnv2 modifier effect and advance our knowledge of the mechanisms by which genetic modifiers influence epilepsy susceptibility.

描述（由申请人提供）：电压门控离子通道突变是单基因癫痫的主要原因之一。单基因癫痫的一个共同特征是具有相同突变的家庭成员之间的不同表达，这表明基因修饰可能影响易感性。Scn2aQ54转基因癫痫小鼠模型具有具有株系依赖性表达的癫痫表型。Kcnv2编码电压门控钾通道亚基Kv8.2，被鉴定为Scn2aQ54表型的遗传修饰子。对癫痫患者的筛查发现了两个非同义编码序列变体，它们改变了异源表达系统中的通道功能，这表明KCNV2也有助于人类癫痫易感性。小鼠数据表明，Kcnv2是一种定量修饰因子，Kcnv2稳态表达水平的增加与Scn2aQ54表型严重程度的增加相关。这些数据表明，KCNV2启动子和3'UTR的调控序列变化可能通过改变稳态表达水平来影响癫痫易感性。拟建的研究将表征小鼠和人类的Kcnv2启动子和3' UTR，并确定小鼠和人类调控变异体对Kcnv2转录率和mRNA稳定性的影响。在Specific Aim 1中，将使用cDNA末端快速扩增（RACE）分析和RNase保护实验来确定小鼠和人KCNV2的转录起始位点（TSS）区域和3'UTR组织。荧光素酶启动子试验将用于鉴定最小启动子区域。在Specific Aim 2中，荧光素酶报告基因构建将用于确定启动子序列变异对小鼠和人类KCNV2启动子活性的影响。测试报告基因构建将用于测试小鼠和人类3' UTR序列变异对mRNA稳定性的影响。作为一种补充方法，原代海马培养将用于测试小鼠Kcnv2 mRNA转录抑制后的稳定性。接下来，将通过BAC转基因来评估选定的Kcnv2调控元件的体内效应。阐明调控序列变异对Kcnv2表达的影响将有助于我们确定Kcnv2修饰因子作用的分子基础，并进一步了解遗传修饰因子影响癫痫易感性的机制。