# Large-scale functional assessment of variants of the potassium channel Kir2.1: Clinical and comparative insights

## TL;DR

This study evaluates the function of many Kir2.1 gene variants to better understand their role in Andersen-Tawil Syndrome and improve clinical classifications.

## Contribution

The study introduces a yeast-based functional assay to validate and complement deep mutational scan data for Kir2.1 variants.

## Key findings

- About 30% of Kir2.1 variants showed reduced surface expression when co-expressed with wild-type.
- Approximately 70% of variants with reduced surface expression also had reduced thermal stability.
- The yeast assay identified 7 out of 10 VUS as loss-of-function, contradicting some DMS results.

## Abstract

Hundreds of KCNJ2 (Kir2.1) variants of uncertain significance (VUS) have been associated with Andersen-Tawil Syndrome (ATS). Remarkably, most Kir2.1 variants' surface expression and function have been described via deep mutational scans (DMS). These results have provided an unprecedented picture of Kir2.1 structure-function relationships and insights into VUS. However, these studies are limited by the lack of robust validation. We performed a flow cytometry-based Kir2.1 surface expression assay for 70 variants (61 ATS-linked) distributed across the potassium inward rectifier channel, a thermal stability assay of 20 variants with reduced surface expression, a yeast-based functional assay for 20 variants (10 pathogenic or likely pathogenic (P/LP) and 10 VUS), as well as whole-cell patch clamp for 13 variants (4 P/LP and 9 VUS). Kir2.1 cell surface expression results showed that ∼30% of variants have reduced surface expression when co-expressed with WT, and that ∼25% disagreed with the DMS datasets. ∼70% of variants with reduced surface tested had reduced thermal stability. Our yeast assay showed all 10 P/LP variants exhibiting LOF, and 7 out of 10 VUS were LOF, in contrast to the DMS method. Patch clamp data further validated the yeast assay. Our data underscores the limitations to interpreting the Kir2.1 DMS datasets, demonstrates a proof-of-principle yeast assay as a complementary method to better inform ClinVar classifications, and provides several lines of evidence for LOF of 9 Kir2.1 VUS in the process.