High-throughput discovery of disease-associated ion channel variants

项目总结 离子通道以一种受调节的方式使带电离子通过类脂膜。离子通道 在调节电兴奋组织、感知和响应 环境，以及维持细胞的动态平衡。已有70多个离子通道与孟德尔有关 “经络”紊乱，影响一系列不同的器官系统。作为基因检测和基因组学 医学变得突出，一个重要的挑战是了解其中的光谱 离子通道基因的突变会导致疾病。不幸的是，很大一部分变种目前 被注释为“不确定意义的变种”，这限制了 基因组医学。 这项提议试图破译通道病基因中的哪些变异会导致疾病。我们会 首先使用与基因组测序和表型数据相关联的大型生物库数据集。我们将研究 76个经络病基因的遗传变异与相关疾病表型的相关性。 使用对照致病变异体，我们将首先确定哪些基因-表型对是相关的 在生物库数据集中，然后发现存在于 有相关疾病表型的携带者。接下来，我们将使用高通量的自动补丁 钳制以研究离子通道基因的数百个变异。我们最初的关注点将是5键离子 跨越一系列离子类型和器官系统的通道基因，以及从 生物库遗传分析。接下来，我们将进行深度突变扫描(全面的 突变研究)所选离子通道基因的每一种突变，从GABRA1开始，一种配体- 门控离子通道基因(受体)参与GABA感觉，并与癫痫发作障碍有关。我们会 利用简并突变反应生成所有可能的突变，整合突变文库 然后测量每个突变对细胞表面运输和通道功能的影响 高通量测序。最后，我们将把这些患者和体外功能数据集整合到 学习离子通道生物学和疾病的基本特征。通过对2D和3D的分析 蛋白质结构，我们将破译蛋白质突变热点。从对突变基因的分析 同源基因的影响我将决定突变信息是否可以移植到 同源基因。最后，我们将把不同的数据整合到美国医学院 遗传学分类框架，用于临床对变异进行重新分类。总体而言，这些实验 帮助解决离子通道的VUS问题和破译新的离子通道的巨大潜力 生物学。

PROJECT SUMMARY Ion channels pass charged ions through lipid membranes in a regulated manner. Ion channels play major roles in regulating electrically excitable tissues, sensing and responding to the environment, and maintaining cell homeostasis. Over 70 ion channels have been linked to Mendelian “channelopathy” disorders, affecting a diverse set of organ systems. As genetic testing and genomic medicine become prominent, an important challenge is to understand the spectrum of which mutations in ion channel genes cause disease. Unfortunately, a large fraction of variants are currently annotated as “Variants of Uncertain Significance,” which limits the effectiveness and potential of genomic medicine. This proposal seeks to decipher which variants in channelopathy genes cause disease. We will first use large biobank datasets with linked genome sequencing and phenotype data. We will examine associations between genetic variants in 76 channelopathy genes and relevant disease phenotypes. Using control pathogenic variants, we will first determine which gene-phenotype pairs are associated in biobank datasets, then discover novel candidate disease-associated variants that are present in carriers with relevant disease phenotypes. Next, we will use high-throughput automated patch clamping to study hundreds of variants in ion channel genes. Our initial focus will be 5 key ion channel genes that span a range of ion types and organ systems, as well as selected variants from the biobank genetic analyses. Next, we will perform deep mutational scans (a comprehensive mutational study) of every mutation in selected ion channel genes, starting with GABRA1, a ligand- gated ion channel gene (receptor) involved in GABA sensing and linked to seizure disorders. We will generate all possible mutations with degenerate mutagenesis reactions, integrate the mutation library into cells, then measure each mutation's impact on cell surface trafficking and channel function using high-throughput sequencing. Finally, we will integrate these patient and in vitro functional datasets to learn fundamental features of ion channel biology and disease. Through an analysis of the 2D and 3D protein structures, we will decipher protein mutational hotspots. From an analysis of mutational impacts from homologous genes I will determine whether mutation information can be ported to homologous genes. Finally, we will integrate variant data into the American College of Medical Genetics classification framework to clinically reclassify variants. Overall, these experiments have great potential to help resolve the VUS problem for ion channels and decipher novel ion channel biology.