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

项目摘要--项目1 通道病，特别是涉及电压门控钠(NAV)和钾(KV)通道的那些，是 对不同类型的癫痫综合征负责。总的来说，编码NAV和KV通道的基因 在所有癫痫相关基因中，累积变异负担最大，占所有基因的30% 报道了遗传性癫痫的变异，以及仅有四个基因(SCN1A、KCNQ2、SCN2A、SCN8A)的变异 占一家大型商业实验室(GeneDx)检测的癫痫变异阳性病例的40%。 使用膜片钳电生理记录对离子通道功能进行的功能评估包括 确定通道变异体的致病性和建立基因分型的研究的基石- 表型关系。然而，该技术在其典型实施例中具有有限的吞吐量，非常 时间和劳动力密集，并且缺乏标准化，阻碍了可再生性。 在项目1中，我们将开发两个可用的高通量自动膜片钳记录平台 学术中心(西北大学、博德研究所)确定离子的功能后果 与癫痫相关的渠道变异达到了前所未有的规模。对于目标1，特定的人NAV和KV 渠道变体将根据变体优先级和配置核心(核心)确定功能分析的优先顺序 A)，表达变异体的细胞将由突变和细胞表达核心(核心B)提供。 西北大学和布罗德研究所联合自动化膜片钳设备的巨大容量 将在5年内对多达1,000个癫痫相关变种进行功能评估。附加功能 对原型变异的研究将与同源小鼠通道一起进行，以验证和区分优先顺序 用于产生经络病相关癫痫小鼠模型的变体(项目3)。在目标2中，原型 将测试变种(例如，具有代表更大变种队列的功能特性的变种) 对照已批准的和正在研究的抗惊厥药物/化合物小组，以确定哪些药物 最能纠正观察到的功能异常。在目标3中，我们将开发并测试一个正交表 预测SCN1a上哪些氨基酸替换导致高血压病患者功能丧失的策略 吞吐量池屏幕。这一策略将结合一个基因热点的饱和突变和下一个- 用一种检测方法来确定转导错义突变体的细胞的能力 当暴露在有效的NAV通道激活剂下时，SCN1A能够存活。这项初步研究的目标是 演示原则证明，我们可以为所有可能的LOF替换创建一个全面的数据库 在SCN1a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-表型图谱。

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.