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变异的检测。然而，在包括KCNH2在内的大多数基因中，大多数错义变异目前被归类为意义不确定的变异 (VUS)。本研究的目的是研究自动膜片钳测定在辅助KCNH2临床变异分类中的效用。该检测是根据临床基因组序列变异解释工作组提出的建议设计的。 31 个变异（17 个致病/可能致病，14 个良性/可能良性）在内部被分类为变异对照。它们在 Flp-In HEK293 细胞中杂合表达，用于评估变体对电流密度和通道门控的影响，从而确定测定的灵敏度和特异性。所有 17 个致病性变异对照均具有降低的电流密度，14 个良性变异对照中的 13 个具有正常电流密度，这使得能够确定正常和异常范围，以便为 VUS 重新分类应用中等或支持强度的证据。纳入功能分析证据使我们能够将 44 个KCNH2 VUS 中的 6 个重新分类为可能致病。高通量膜片钳测定可为临床KCNH2变异的临床解释提供中等强度的证据，并证明开发自动化膜片钳测定用于离子通道基因变异功能表征的价值。