Many genes, including KCNH2, contain “hotspot” domains associated with a high density of variants associated with disease. This has led to the suggestion that variant location can be used as evidence supporting classification of clinical variants. However, it is not known what proportion of all potential variants in hotspot domains cause loss of function. Here, we have used a massively parallel trafficking assay to characterize all single-nucleotide variants in exon 2 of KCNH2, a known hotspot for variants that cause long QT syndrome type 2 and an increased risk of sudden cardiac death. Forty-two percent of KCNH2 exon 2 variants caused at least 50% reduction in protein trafficking, and 65% of these trafficking-defective variants exerted a dominant-negative effect when co-expressed with a WT KCNH2 allele as assessed using a calibrated patch-clamp electrophysiology assay. The massively parallel trafficking assay was more accurate (AUC of 0.94) than bioinformatic prediction tools (REVEL and CardioBoost, AUC of 0.81) in discriminating between functionally normal and abnormal variants. Interestingly, over half of variants in exon 2 were found to be functionally normal, suggesting a nuanced interpretation of variants in this “hotspot” domain is necessary. Our massively parallel trafficking assay can provide this information prospectively.

许多基因，包括KCNH2 ，都包含与疾病相关的高密度变异相关的“热点”结构域。这导致建议变异位置可以用作支持临床变异分类的证据。然而，尚不清楚热点域中所有潜在变异中有多少比例导致功能丧失。在这里，我们使用大规模并行运输分析来表征KCNH2外显子 2 中的所有单核苷酸变异，这是导致 2 型长 QT 综合征和心源性猝死风险增加的已知变异热点KCNH2外显子的 42% 2 个变体导致蛋白质运输至少减少 50%，并且当使用校准的膜片钳电生理学测定评估时，这些运输缺陷变体中的 65% 在与 WT KCNH2等位基因共表达时产生显性负效应。在区分功能正常和异常变异方面，大规模并行运输测定（AUC 为 0.94）比生物信息学预测工具（REVEL 和 CardioBoost，AUC 为 0.81）更准确。有趣的是，发现外显子 2 中超过一半的变异在功能上是正常的，这表明有必要对这个“热点”域中的变异进行细致入微的解释。我们的大规模并行贩运分析可以前瞻性地提供这些信息。