Molecular Regulation of an Epilepsy Modifier Gene

癫痫修饰基因的分子调控

• 批准号: 8521688
• 负责人: BENJAMIN JORGE
• 金额: $ 2.69万
• 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 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8521688
• 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可变表达性，这表明遗传修饰剂可能影响易感性。Scn 2aQ 54转基因癫痫小鼠模型具有应变依赖性表达的癫痫表型。编码电压门控钾通道亚基Kv8.2的Kcnv 2被鉴定为Scn 2aQ 54表型的遗传修饰剂。癫痫患者的筛选确定了两个非同义的编码序列变体，它们改变了异源表达系统中的通道功能，这表明KCNV 2也有助于人类癫痫易感性。小鼠数据表明，Kcnv 2是一种定量修饰剂，Kcnv 2表达的稳态水平增加与Scn 2aQ 54表型严重程度增加相关。这些数据表明，在KCNV 2的启动子和3 'UTR的调控序列的变化可能会影响癫痫发作的易感性，通过改变稳态表达水平。拟议的研究将表征小鼠和人中的Kcnv 2启动子和3' UTR，并确定小鼠和人调节变体对Kcnv 2转录速率和mRNA稳定性的影响。在特定目标1中，将使用cDNA末端快速扩增（RACE）分析和RNA酶保护试验来确定小鼠和人KCNV 2的转录起始位点（TSS）区域和3 'UTR组织。然后将使用荧光素酶启动子测定来鉴定最小启动子区域。在特定目标2中，将使用荧光素酶报告基因构建体来确定启动子序列变异对小鼠和人KCNV 2启动子活性的影响。Tet-off报告基因构建体将用于测试小鼠和人3' UTR序列变异对mRNA稳定性的影响。作为补充方法，原代海马培养物将用于测试小鼠Kcnv 2 mRNA在转录抑制后的稳定性。接下来，将通过BAC转基因来评价所选Kcnv 2调节元件的体内作用。阐明调控序列变体对Kcnv 2表达的影响将有助于我们确定Kcnv 2修饰剂效应的分子基础，并提高我们对遗传修饰剂影响癫痫易感性的机制的认识。