Title : Functional characterization of KCNH2 genetic variants, encoding HERG potassium channel, as a clinically-relevant information for type 2 LQTS syndrome
Abstract:
Long QT syndrome type 2 (LQTS2) is a potentially fatal cardiac arrhythmia disorder, often resulting from rare loss-of-function variations in the KCNH2 gene, encoding the hERG cardiac channel. The identification of hERG variants is essential for diagnosing LQTS2 and providing appropriate management strategies. To date, more than 3000 variants of the KCNH2 gene have been identified in the ClinVar database. However, the potential pathogenicity of most of them remains unknown, they are classified as variants of uncertain significance or VUS. There remains an urgent clinical need for high-throughput functional characterization of VUS variants to allow optimal stratification of the rhythmic risk for each patient.
To meet this clinical need, in this work we have implemented a large-scale multiparametric evaluation of the 303 KCNH2 variants identified within the French CARDIOGEN network. We have optimized and accelerated the entire process of functional characterization of hERG from low throughput to high throughput. (i) We increased the success rate of mutagenesis to 99% by implementing the Gibson assembly strategy, (ii) by using an electroporation system, we increased the efficiency of transient expression to 80%, (iii) we optimized a protocol for acquiring all the biophysical properties of hERG in less than 5 minutes, (iv) we switched to an automatic patch-clamp system that performs simultaneous recording of 384 cells, which allowed us to increase by 30 times the number of variants analyzed per month and improve the statistical value of the results obtained, (v) we also implemented automated data analysis in the R language. For the membrane trafficking studies, we used the pHluorin tag and confocal microscopy. To assess the structural impact of hERG variants, we used the structure resolved by Cryo-EM and UCSF Chimera software to perform a structural characterisation of the variants in their near-regional environment.
We have characterized 303 hERG variants, including 201 VUS. Our strategy confirmed the pathogenicity or benignity of the previously classified 102 variants. We observed that variants causing a significant decrease in current or presenting major defects in membrane localization also exhibited substantial structural perturbations. Combined, the trafficking approach and exhaustive biophysical protocols allow us to understand the mechanisms related to hERG loss or gain of function. Finally, we validated our approach for direct clinical application by reclassifying the 201 VUS variants.
In conclusion, functional studies, including electrophysiological assays, play a crucial role in assessing the pathogenicity of hERG variants. Our data will be used to develop a new database integrating functional, clinical, and genetic information, aimed at improving the pathogenic classification of each KCNH2 variant. This database will serve as a diagnostic and prevention tool for clinicians ensuring appropriate management of affected individuals and their families.
