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.

Methods
The parallel trafficking dataset was collected by sequencing flow-sorted cells that express a pool of different KCNH2 homozygous variants. The dataset contains the sequencing files and its associated barcoded key for identifying different KCNH2 variants.

包括KCNH2在内的诸多基因，均携带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，变异的所在位置可作为支持临床变异分类的证据。然而，目前尚不清楚热点结构域内所有潜在变异中，有多大比例会导致功能丧失（loss of function）。

本研究采用大规模并行转运实验（massively parallel trafficking assay），对KCNH2基因外显子2的所有单核苷酸变异（single-nucleotide variants）进行了表征；该外显子是引发2型长QT综合征（long QT syndrome type 2）并增加心源性猝死（sudden cardiac death）风险的变异的已知热点区域。KCNH2外显子2的变异中，有42%可使蛋白质转运效率降低至少50%；经校准后的膜片钳电生理实验（patch clamp electrophysiology assay）评估显示，这类转运缺陷变异中，有65%在与野生型（WT）KCNH2等位基因共表达时会产生显性负效应（dominant-negative effect）。在区分功能正常与功能异常变异方面，该大规模并行转运实验的准确性（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）。值得注意的是，外显子2中超过半数的变异被证实功能正常，这提示对该热点结构域内的变异需进行精细化解读。本研究所用的大规模并行转运实验可前瞻性地提供此类信息。

方法
本平行转运数据集通过对表达一组不同KCNH2纯合变异的流式分选细胞进行测序获得。该数据集包含测序文件及其配套的带条形码识别密钥，用于区分不同的KCNH2变异。