Mark Cowley Lidwill Research Program in Cardiac Electrophysiology, Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia; School of Clinical Medicine, UNSW Sydney, Darlinghurst, NSW, Australia.
Vanderbilt Center for Arrhythmia Research and Therapeutics, Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
Am J Hum Genet. 2022 Jul 7;109(7):1208-1216. doi: 10.1016/j.ajhg.2022.05.003. Epub 2022 Jun 9.
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，都含有与疾病相关的高变异密度的“热点”结构域。这导致了这样一种观点，即变异位置可用作支持临床变异分类的证据。然而，目前尚不清楚热点结构域中所有潜在变异中有多少会导致功能丧失。在这里，我们使用大规模平行运输测定法来表征 KCNH2 外显子 2 中的所有单核苷酸变异，KCNH2 是导致 2 型长 QT 综合征和心脏性猝死风险增加的变异热点。KCNH2 外显子 2 变异的 42%导致蛋白转运至少减少 50%，并且当使用校准的膜片钳电生理学测定法评估时，这些转运缺陷变异中的 65%与 WT KCNH2 等位基因共表达时表现出显性负效应。大规模平行运输测定法比生物信息学预测工具（REVEL 和 CardioBoost，AUC 为 0.81）更准确（AUC 为 0.94），可区分功能正常和异常变异。有趣的是，外显子 2 中的一半以上变异被发现是功能正常的，这表明需要对该“热点”结构域中的变异进行细致的解释。我们的大规模平行运输测定法可以前瞻性地提供这些信息。
