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.

The patch-clamp dataset was collected using the Nanion Syncropatch 384PE automated patch clamp system by assessing the electrophysiology parameters of 458 KCNH2 SNVs expressed in HEK293 as heterozygote. Three voltage protocols were used to interrogate steady-state activation, steady-state deactivation (and recovery from inactivation) as well as rates of onset of inactivation. The raw data sets obtained from the PatchControl software (Nanion) are included as 'Patch_Clamp_Dataset_1.zip, Patch_Clamp_Dataset_2.zip, Patch_Clamp_Dataset_3.zip and Patch_Clamp_Dataset_4.zip’.

The raw SyncroPatch datafiles (obtained after unzipping the Patch_Clamp_Dataset_1.zip, Patch_Clamp_Dataset_2.zip, Patch_Clamp_Dataset_3.zip and Patch_Clamp_Dataset_4.zip file) can be opened using DataControl software (Nanion). Data can be reanalysed directly in DataControl and a copy of these data for each protocol have been exported as csv files and can be analysed using Python scripts available at https://git.victorchang.edu.au/projects/SADA/repos/syncropatch_automated_analysis/browse. The list of variants assayed on each plate are available in the “compound editor” tab of each dataset. This information is also provided in the excel spreadsheet: 'Patch-clamp_variants_location.xlsx‘’ so that users can identify which plate dataset they need to open to look at specific variants.