Heterozygous variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology.

is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any missense variant. The key components of the assay include quick and cost-effective generation of a bi-cistronic vector to co-express Wild-type (WT) and any variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch clamp electrophysiology analysis of channel function. Stable cell lines take 3-4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 h of channel expression. This high-throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any variant.