Department of Medicine, Division of Clinical Pharmacology, Vanderbilt Center for Arrhythmia Research and Therapeutics (A.M.G., B.M.K., T.Y., Y.W., T.S., J.-E.S., D.M.R.), Vanderbilt University Medical Center, Nashville, TN.
Department of Genome Sciences, University of Washington, Seattle (K.A.M., D.M.F.).
Circ Genom Precis Med. 2020 Feb;13(1):e002786. doi: 10.1161/CIRCGEN.119.002786. Epub 2020 Jan 12.
Variants in ion channel genes have classically been studied in low throughput by patch clamping. Deep mutational scanning is a complementary approach that can simultaneously assess function of thousands of variants.
We have developed and validated a method to perform a deep mutational scan of variants in , which encodes the major voltage-gated sodium channel in the heart. We created a library of nearly all possible variants in a 36 base region of in the S4 voltage sensor of domain IV and stably integrated the library into HEK293T cells.
In preliminary experiments, challenge with 3 drugs (veratridine, brevetoxin, and ouabain) could discriminate wild-type channels from gain- and loss-of-function pathogenic variants. High-throughput sequencing of the pre- and postdrug challenge pools was used to count the prevalence of each variant and identify variants with abnormal function. The deep mutational scan scores identified 40 putative gain-of-function and 33 putative loss-of-function variants. For 8 of 9 variants, patch clamping data were consistent with the scores.
These experiments demonstrate the accuracy of a high-throughput in vitro scan of variant function, which can be used to identify deleterious variants in and other ion channel genes.
离子通道基因突变经典的研究方法是通过膜片钳技术进行低通量筛选。深度突变扫描是一种互补的方法，可以同时评估数千种变体的功能。
我们开发并验证了一种方法，可以对编码心脏主要电压门控钠离子通道的 基因中的变体进行深度突变扫描。我们在 基因的 IV 域 S4 电压传感器的 36 个碱基区域创建了一个几乎所有可能变体的文库，并将文库稳定整合到 HEK293T 细胞中。
在初步实验中，用 3 种药物（藜芦碱、布雷菲德菌素和哇巴因）挑战，可以区分野生型通道和功能获得性及功能丧失性致病性变体。在药物挑战前后的池进行高通量测序，以计算每种变体的流行率，并鉴定具有异常功能的变体。深度突变扫描评分确定了 40 个可能的功能获得性变体和 33 个可能的功能丧失性变体。在 9 个变体中的 8 个中，膜片钳数据与评分一致。
这些实验证明了高通量体外 变体功能扫描的准确性，可用于识别 和其他离子通道基因中的有害变体。
