KCNQ2 R144 variants cause neurodevelopmental disability with language impairment and autistic features without neonatal seizures through a gain-of-function mechanism

Abstract

Background: Prior studies have revealed remarkable phenotypic heterogeneity in KCNQ2-related disorders, correlated with effects on biophysical features of heterologously expressed channels. Here, we assessed phenotypes and functional properties associated with KCNQ2 missense variants R144W, R144Q, and R144G. We also explored in vitro blockade of channels carrying R144Q mutant subunits by amitriptyline.

Methods: Patients were identified using the RIKEE database and through clinical collaborators. Phenotypes were collected by a standardized questionnaire. Functional and pharmacological properties of variant subunits were analyzed by whole-cell patch-clamp recordings.

Findings: Detailed clinical information on fifteen patients (14 novel and 1 previously published) was analyzed. All patients had developmental delay with prominent language impairment. R144Q patients were more severely affected than R144W patients. Infantile to childhood onset epilepsy occurred in 40%, while 67% of sleep-EEGs showed sleep-activated epileptiform activity. Ten patients (67%) showed autistic features. Activation gating of homomeric Kv7.2 R144W/Q/G channels was left-shifted, suggesting gain-of-function effects. Amitriptyline blocked channels containing Kv7.2 and Kv7.2 R144Q subunits.

Interpretation: Patients carrying KCNQ2 R144 gain-of-function variants have developmental delay with prominent language impairment, autistic features, often accompanied by infantile- to childhood-onset epilepsy and EEG sleep-activated epileptiform activity. The absence of neonatal seizures is a robust and important clinical differentiator between KCNQ2 gain-of-function and loss-of-function variants. The Kv7.2/7.3 channel blocker amitriptyline might represent a targeted treatment.

Funding: Supported by FWO, GSKE, KCNQ2-Cure, Jack Pribaz Foundation, European Joint Programme on Rare Disease 2020, the Italian Ministry for University and Research, the Italian Ministry of Health, the European Commission, the University of Antwerp, NINDS, and Chalk Family Foundation.

Keywords: Amitriptyline; Autism; Developmental and epileptic encephalopathy; Gain-of-function; KCNQ2.

Figure 1

Bar graphs showing the main clinical features shared by 9 patients with Kv7.2 R144W variants (age at last follow up between 3.5 and 27 years) and 6 patients with R144Q variants (age at last follow up between 3 years 9 months and 26 years). Distribution of (a) grade of intellectual disability, (b) language outcome, (c) highest level of motor functioning reached at 3 years of age, (d) behavioral phenotype, and (e) seizure phenotype. Persons presenting only paroxysmal episodes (such as staring) of unclear origin are not included in the epilepsy group.

Figure 2

Topological location of the R144 residue and functional characterization of the Kv7.2 R144 W/Q/G variants.a. Homology model of a wtKv7.2 channel, built upon the Kv1.2/2.1 chimera crystal structure (PDB code: 2R9R), showing the localization of the R144 position (side chain colored in red using a space-filling model). For clarity only two of the four subunits are shown. b. Macroscopic currents from wtKv7.2 (Kv7.2), Kv7.2 R144Q, Kv7.2 R144W, and Kv7.2 R144G homomeric channels, in response to the indicated voltage protocol. Current scale, 200 pA; time scale, 0.2 s. c. Conductance/voltage curves for the indicated channels. Continuous lines are Boltzmann fits to the experimental data. Each data point is the Mean±S.E.M. of 13–21 cells recorded in at least 3 separate experimental sessions. d. Relative amplitudes of the fast and slow current activation components (expressed as A_f/A_f+_As), for wtKv7.2, and Kv7.2 R144Q/W/G homomeric channels as a function of membrane voltage.

Figure 3

An enlarged view of the resting state configuration of the region where R144 is located in wtKv7.2 (a), Kv7.2 R144W (b), Kv7.2 R144Q (c) and Kv7.2 R144G (d) channel.

Figure 4

Effect of changes in PIP₂ availability on channels incorporating Kv7.2 R144Q mutant subunits. a. Representative current traces from cells expressing wtKv7.2 and Kv7.2 R144Q homomeric channels in the absence or in the presence of PIP5K in response to the voltage protocols shown in each respective panel. Current scale, 100 pA; time scale, 0.2 s. b. Conductance/voltage curves for the indicated channels with or without PIP5K co-expression. Continuous lines are Boltzmann fits to the experimental data. c. Current inhibition upon time-dependent activation of Dr-VSP. Asterisks (*) indicate values significantly different from each respective control (p<0.05).

Figure 5

Effect of amitriptyline on heteromeric channels incorporating Kv7.2 R144Q mutant subunits. a. Representative current traces recorded from cells transfected with the indicated cDNA combinations in response to the voltage ramp protocol reported, both before and after amitriptyline (AMI) exposure (1 and 10 µM). Current density scale, 20 pA/pF; time scale 0.2 s. b. Percent of current inhibition by amitriptyline. Asterisks (*) indicate values significantly different from each respective control recorded before drug exposure (p<0.05).

Figure 6

Correlation between the shift in voltage-dependent activation midpoint (V1/2) and phenotypes of pathogenic KCNQ2 variants in the VSD. Listed are 6 variants (at 5 codons) found recurrently, the shift observed under three informative and physiologically relevant subunit expression ratios (indicated schematically at top), and the associated phenotypes. The voltage shifts are taken from the publications listed. Remarkably, phenotypic severity appears correlated with the magnitude of the V1/2 shift, but even small shifts (R144W, R214W) can be pathogenic. These correlations are not interpreted as a necessarily complete pathogenic mechanism; and for LoF variants, additional disease pathomechanisms have been described (reviewed in 40).