Trafficking dataset for: A massively parallel assay accurately discriminates between functionally normal and abnormal variants in a hotspot domain of KCNH2

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.

包括KCNH2在内的诸多基因，均携带有与疾病相关变异高密度聚集的「热点结构域（hotspot domain）」。这一现象提示，变异位点可作为支持临床变异分类的依据。然而目前尚不明确，热点结构域内所有潜在变异中，有多大比例会导致蛋白质功能丧失。本研究采用大规模并行转运检测实验，对KCNH2基因外显子2的所有单核苷酸变异进行了功能表征——该外显子是已知的致2型长QT综合征及心源性猝死风险升高变异的热点区域。本研究中，42%的KCNH2外显子2变异可导致蛋白质转运效率至少降低50%；其中65%的转运缺陷变异，在与野生型（Wild Type, WT）KCNH2等位基因共表达时，经校准膜片钳电生理实验检测后显示出显性负效应。在区分功能正常与异常变异方面，大规模并行转运检测实验的准确性（曲线下面积AUC为0.94）优于生物信息学预测工具REVEL与CardioBoost（二者AUC均为0.81）。有趣的是，本研究发现外显子2中超过半数的变异功能正常，这提示针对该「热点结构域」内的变异需进行精细化解读。本研究采用的大规模并行转运检测实验，可前瞻性地提供此类功能表征信息。