Project 1 - High-throughput functional evaluation of ion channel variants in epilepsy

项目 1 - 癫痫离子通道变异的高通量功能评估

• 批准号: 10247556
• 负责人: Alfred L. George
• 金额: $ 75.63万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10247556
• 关键词: Alleles; Amino Acid Substitution; Animal Model; Anticonvulsants; Benign; Biological Assay; Categories; Cell model; Cells; Classification; Clinical Medicine; Data; Data Analyses; Databases; Diagnostic; Drug Compounding; Drug Targeting; Electrophysiology (science); Epilepsy; Etiology; Evaluation; Evaluation Studies; Exposure to; Future; Genes; Genetic; Genome; Genotype; Goals; Growth; Hot Spot; Human; Institutes; Investigation; Ion Channel; Laboratories; Lead; Libraries; Manuals; Mus; Mutagenesis; Mutation; Ontology; Pathogenicity; Pharmaceutical Preparations; Pharmacology; Phenotype; Pilot Projects; Potassium; Precision therapeutics; Property; Protocols documentation; Reporting; Reproducibility; Research; SCN8A gene; Sodium; Standardization; Syndrome; Techniques; Technology; Testing; Time; Universities; Variant; cohort; experimental study; feasibility testing; flexibility; genetic variant; loss of function; mouse model; nervous system disorder; next generation sequencing; patch clamp; response; screening; voltage

PROJECT SUMMARY – PROJECT 1 Channelopathies, particularly those involving voltage-gated sodium (NaV) and potassium (KV) channels, are responsible for a diverse group of epilepsy syndromes. Collectively, genes encoding NaV and KV channels have the greatest cumulative variant burden among all epilepsy-associated genes, representing >30% of all reported variants in genetic epilepsies, and variants in just four genes (SCN1A, KCNQ2, SCN2A, SCN8A) account for 40% of variant-positive cases tested for epilepsy by one large commercial laboratory (GeneDx). Functional assessments of ion channel function using patch clamp electrophysiological recording are the cornerstone of research determining the pathogenicity of channel variants and establishing genotype- phenotype relationships. However, the technique in its typical embodiment has limited throughput, is extremely time- and labor-intensive, and suffers from a lack of standardization that hampers reproducibility. In Project 1, we will exploit high throughput automated patch clamp recording platforms available at two academic centers (Northwestern University, Broad Institute) to determine the functional consequences of ion channel variants associated with epilepsy at an unprecedented scale. For Aim 1, specific human NaV and KV channel variants will be prioritized for functional analyses by the Variant Prioritization and Curation Core (Core A), and cells expressing the variants will be provided by the Mutagenesis and Cell Expression Core (Core B). The enormous capacity of the combined automated patch clamp facilities at Northwestern and Broad Institute will enable functional evaluation of up to 1,000 epilepsy-associated variants over 5 years. Additional functional studies of prototypical variants will be performed with orthologous murine channels to validate and prioritize variants for generating mouse models of channelopathy-associated epilepsy (Project 3). In Aim 2, prototypical variants (e.g., those with functional properties representative of a larger cohort of variants) will be tested against panels of approved and investigational anticonvulsant drugs/compounds to determine which agents are best able to correct the observed functional abnormality. In Aim 3, we will develop and test an orthogonal strategy to predetermine which amino acid substitutions in SCN1A lead to loss of function (LOF) in a high- throughput pooled screen. This strategy will couple saturation mutagenesis of a genetic ‘hot spot’ and next- generation sequencing with an assay to determine the ability of cells transfected with a missense variant in SCN1A to survive when exposed to a potent NaV channel activator. The goal of this pilot study is to demonstrate proof-of-principle that we can create a comprehensive database for all possible LOF substitutions in SCN1A. If successful, this approach will generate an allele characterization framework that can be scaled to accomplish genome-guided genotype-phenotype mapping.

项目摘要-项目1 糖尿病，特别是涉及电压门控钠（NaV）和钾（KV）通道的糖尿病， 导致多种癫痫综合征总的来说，编码NaV和KV通道的基因 在所有癫痫相关基因中具有最大的累积变异负担，占所有癫痫相关基因的30%以上。 报告了遗传性癫痫的变异，以及仅四种基因（SCN 1A，KCNQ 2，SCN 2A，SCN 8A）的变异 占一家大型商业实验室（GeneDx）检测癫痫变异阳性病例的40%。 使用膜片钳电生理记录的离子通道功能的功能评估是 确定通道变体致病性和建立基因型的研究基石- 表型关系然而，该技术在其典型的实施例中具有有限的吞吐量， 时间和劳动密集型，并且缺乏标准化，这妨碍了可重复性。 在项目1中，我们将利用高通量自动膜片钳记录平台， 学术中心（西北大学，布罗德研究所），以确定离子的功能后果 与癫痫相关的通道变异在前所未有的规模。对于目标1，特异性人NaV和KV 通道变体将由变体优先级和固化核心（核心）优先进行功能分析 突变和细胞表达核心（Core B）提供表达变体的细胞。 西北大学和布罗德研究所联合自动膜片钳设备的巨大容量 将能够在5年内对多达1,000种癫痫相关变异进行功能评价。附加功能 原型变异体的研究将使用正向小鼠通道进行，以验证和确定优先级 用于产生通道病相关癫痫的小鼠模型的变体（项目3）。在目标2中，原型 变体（例如，具有代表较大变体群的功能特性的那些）将被测试 与已批准的和研究性抗惊厥药物/化合物组进行比较，以确定哪些药物 能够最好地纠正观察到的功能异常。在目标3中，我们将开发和测试一个正交 预先确定SCN 1A中哪些氨基酸取代导致高表达蛋白中的功能丧失（LOF）的策略 通量合并筛选。这种策略将结合遗传“热点”的饱和诱变，然后- 用测定法进行世代测序以确定用错义变体转染的细胞在 SCN 1A在暴露于有效的NaV通道激活剂时存活。这项试点研究的目标是 证明了我们可以为所有可能的LOF替换创建一个全面的数据库 在SCN 1A中。如果成功，这种方法将产生一个等位基因表征框架，可以扩展到 完成基因组引导的基因型-表型作图。