Mark Cowley Lidwill Research Program in Cardiac Electrophysiology, Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia; Faculty of Medicine and Health, UNSW Sydney, Kensington, NSW, Australia.
Centre for Population Genomics, Garvan Institute of Medical Research and UNSW Sydney, Sydney, Australia; Centre for Population Genomics, Murdoch Children's Research Institute, Melbourne, Australia.
Am J Hum Genet. 2022 Jul 7;109(7):1199-1207. doi: 10.1016/j.ajhg.2022.05.002. Epub 2022 Jun 9.
Modern sequencing technologies have revolutionized our detection of gene variants. However, in most genes, including KCNH2, the majority of missense variants are currently classified as variants of uncertain significance (VUSs). The aim of this study was to investigate the utility of an automated patch-clamp assay for aiding clinical variant classification in KCNH2. The assay was designed according to recommendations proposed by the Clinical Genome Sequence Variant Interpretation Working Group. Thirty-one variants (17 pathogenic/likely pathogenic, 14 benign/likely benign) were classified internally as variant controls. They were heterozygously expressed in Flp-In HEK293 cells for assessing the effects of variants on current density and channel gating in order to determine the sensitivity and specificity of the assay. All 17 pathogenic variant controls had reduced current density, and 13 of 14 benign variant controls had normal current density, which enabled determination of normal and abnormal ranges for applying evidence of moderate or supporting strength for VUS reclassification. Inclusion of functional assay evidence enabled us to reclassify 6 out of 44 KCNH2 VUSs as likely pathogenic. The high-throughput patch-clamp assay can provide moderate-strength evidence for clinical interpretation of clinical KCNH2 variants and demonstrates the value of developing automated patch-clamp assays for functional characterization of ion channel gene variants.
现代测序技术彻底改变了我们对基因突变的检测。然而，在大多数基因中，包括 KCNH2，大多数错义变异目前被归类为意义不明的变异（VUS）。本研究旨在探讨自动化膜片钳检测在 KCNH2 临床变异分类中的应用。该检测根据临床基因组序列变异解读工作组提出的建议设计。31 个变异（17 个致病性/可能致病性，14 个良性/可能良性）被内部归类为变异对照。它们在 Flp-In HEK293 细胞中异质表达，以评估变异对电流密度和通道门控的影响，从而确定该检测的敏感性和特异性。17 个致病性变异对照的电流密度均降低，14 个良性变异对照中有 13 个的电流密度正常，这为 VUS 重新分类提供了中等或支持强度的证据确定了正常和异常范围。纳入功能检测证据使我们能够将 44 个 KCNH2 VUS 中的 6 个重新归类为可能致病性。高通量膜片钳检测可为临床 KCNH2 变异的临床解读提供中等强度的证据，并证明开发自动化膜片钳检测用于离子通道基因突变的功能特征分析的价值。
