Modern sequencing technologies have revolutionised our detection of gene variants. However, in most genes, including KCNH2, the majority of missense variants are currently classified as variants of uncertain significance (VUS). The aim of this study was to investigate the utility of an automated patch-clamp assay for aiding clinical variant classification in the KCNH2 gene. The assay was designed according to recommendations proposed by the ClinGen Sequence Variant Interpretation Working Group. Thirty-one variants (17 pathogenic/likely pathogenic, 14 benign/likely benign) were classified internally as variant controls. They were heterozygously expressed in Flp-In HEK293 cells for assessing the effects of variant on current density and channel gating in order to determine the sensitive and specificity of the assay. All 17 pathogenic variant controls had reduced current density and 13/14 benign variant controls had normal current density, which enabled determination of normal and abnormal ranges for applying moderate or supporting evidence strength for VUS reclassification. Inclusion of KCNH2 functional assay evidence enabled us to reclassify 6 out of 44 VUS as likely pathogenic. The high-throughput patch clamp assay can provide moderate strength evidence for clinical interpretation of clinical KCNH2 variants and demonstrates the value of developing automated patch clamp assays for functional characterisation of ion channel gene variants.

The dataset was collected using the Nanion Syncropatch 384PE automated patch clamp system. 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 in the ‘SyncroPatch_data.zip’ file, which contains additional variants that were not part of this study. The datasets obtained for variants that were investigated in this study after assessment for QC (seal resistances > 300 MΩ, capacitance: 5-50 pF) are included as separate csv files ‘CSV_files.zip’.

The raw SyncroPatch datafiles (obtained after unzipping the SyncroPatch_data.zip file) can be opened using DataControl software (Nanion). Data can be reanalysed directly in DataControl or have been exported as csv files, which 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: ‘variant locations.xlsx’ so that users can identify which plate dataset they need to open to look at specific variants.