Jairam, N. | bioRxiv Science

bioRxiv2025

Functional profiling of KCNE1 variants informs population carrier frequency of Jervell and Lange-Nielsen syndrome type 2

Congenital long-QT syndrome (LQTS) is most often associated with pathogenic variants in KCNQ1 encoding the pore-forming voltage-gated potassium channel subunit of the slow delayed rectifier current (IKs). Generation of IKs requires assembly of KCNQ1 with an auxiliary subunit encoded by KCNE1, which is also associated with LQTS but causality of autosomal dominant disease is disputed. By contrast, KCNE1 is an accepted cause of recessive type 2 Jervell and Lange-Nielson syndrome (JLN2). The functional consequences of most KCNE1 variants have not been determined and the population prevalence of JLN2 is unknown. Methods: We determined the functional properties of 95 KCNE1 variants co-expressed with KCNQ1 in heterologous cells using high-throughput voltage-clamp recording. Experiments were conducted with each KCNE1 variant expressed in the homozygous state and then a subset was studied in the heterozygous state. The carrier frequency of JLN2 was estimated by considering the population prevalence of dysfunctional variants. Results: There is substantial overlap between disease-associated and population KCNE1 variants. When examined in the homozygous state, 68 KCNE1 variants exhibited significant differences in at least one functional property compared to WT KCNE1, whereas 27 variants did not significantly affect function. Most dysfunctional variants exhibited loss-of-function properties. We observed no evidence of dominant-negative effects. Most variants were scored as variants of uncertain significance (VUS) and inclusion of functional data resulted in revised classifications for only 14 variants. The population carrier frequency of JLN2 was calculated as 1 in 1034. Peak current density and activation voltage-dependence but no other biophysical properties were correlated with findings from a mutational scan of KCNE1. Conclusions: Among 95 disease-associated or population KCNE1 variants, many exhibit abnormal functional properties but there was no evidence of dominant-negative behaviors. Using functional data, we inferred a population carrier frequency for recessive JLN2. This work helps clarify the pathogenicity of KCNE1 variants.

genetics↗

bioRxiv2021

High-throughput Evaluation of Epilepsy-associated KCNQ2 Variants Reveals Functional and Pharmacological Heterogeneity

Hundreds of KCNQ2 variants have been identified by genetic testing of children with early onset epilepsy and/or developmental disability. Voltage-clamp recording from heterologous cells has proved useful for establishing deleterious functional effects of KCNQ2 variants, but procedures adapting these assays for standardized, higher throughput data collection and reporting are lacking. In this study, we employed automated patch clamp recording to assess in parallel the functional and pharmacological properties of 79 missense and 2 in-frame deletion variants of KCNQ2. Among the variants we studied were a training set of 18 pathogenic variants previously studied by voltage-clamp recording, 24 mostly rare population variants, and 39 disease-associated variants with unclear functional effects. Variant KCNQ2 subunits were transiently expressed in a cell line stably expressing KCNQ3 to reconstitute the physiologically relevant channel complex. Variants with severe loss-of-function were also co-expressed 1:1 with WT KCNQ2 in the KCNQ3 cell line to mimic the heterozygous genotype and assess dominant-negative behavior. In total, we analyzed electrophysiological data recorded from 9,480 cells. The functional properties of WT KCNQ2/KCNQ3 channels and pharmacological responses to known blockers and activators determined by automated patch clamp recording were highly concordant with previous findings. Similarly, functional properties of 18 known pathogenic variants largely matched previously published results and the validated automated patch clamp assay. Many of the 39 previously unstudied disease-associated KCNQ2 variants exhibited prominent loss-of-function and dominant-negative effects, providing strong evidence in support of pathogenicity. All variants, exhibit response to retigabine (10 {micro}M), although there were differences in maximal responses. Variants within the ion selectivity filter exhibited the weakest responses whereas retigabine had the strongest effect on gain-of-function variants in the voltage-sensor domain. Our study established a high throughput method to detect deleterious functional consequences of KCNQ2 variants. We demonstrated that dominant-negative loss-of-function is a common mechanism associated with missense KCNQ2 variants but this does not occur with rare population variation in this gene. Importantly, we observed genotype-dependent differences in the response of KCNQ2 variants to retigabine.

neuroscience↗