High-throughput evaluation of epilepsy-associated KCNQ2 variants reveals functional and pharmacological heterogeneity

Hundreds of genetic variants in KCNQ2 encoding the voltage-gated potassium channel KV7.2 are associated with early onset epilepsy and/or developmental disability, but the functional consequences of most variants are unknown. Absent functional annotation for KCNQ2 variants hinders identification of individuals who may benefit from emerging precision therapies. We employed automated patch clamp recordings to assess at, to our knowledge, an unprecedented scale the functional and pharmacological properties of 79 missense and 2 inframe deletion KCNQ2 variants. Among the variants we studied were 18 known pathogenic variants, 24 mostly rare population variants, and 39 disease-associated variants with unclear functional effects. We analyzed electrophysiological data recorded from 9,480 cells. The functional properties of 18 known pathogenic variants largely matched previously published results and validated automated patch clamp for this purpose. Unlike rare population variants, most disease-associated KCNQ2 variants exhibited prominent loss-of-function with dominant-negative effects, providing strong evidence in support of pathogenicity. All variants responded to retigabine, although there were substantial differences in maximal responses. Our study demonstrated that dominant-negative loss-of-function is a common mechanism associated with missense KCNQ2 variants. Importantly, we observed genotype-dependent differences in the response of KCNQ2 variants to retigabine, a proposed precision therapy for KCNQ2 developmental and epileptic encephalopathy.


Supplemental Figures
Sequence of mutagenic primers used to generate KCNQ2 variants   Average XE-991-sensitive whole-cell currents recorded by automated patch clamp from CHO-Q3 cells electroporated with KCNQ2 variants from the literature set and normalized to wild type channel peak current recorded in parallel. For variant R201C, whole-cell currents were recorded from CHO-K1 cells co-electroporated with KCNQ3-WT plus KCNQ2-variant. Scale bars are 200 ms (horizontal) and 25% of WT channel current density (vertical).

Figure S4. Whole-cell currents from literature KCNQ2 variants expressed as heterozygous channels.
Average XE-991-sensitive whole-cell currents recorded by automated patch clamp from CHO-Q3 cells co-electroporated with wild type plus variant KCNQ2 cDNA from the literature set and normalized to wild type channel peak current recorded in parallel. Scale bars are 200 ms (horizontal) and 25% of WT channel current density (vertical).

Figure S5. Manual and automated voltage clamp analyses of KCNQ2 variants expressed in the heterozygous state yield similar biophysical properties. A.
Average whole-cell currents recorded at +40 mV from CHO-Q3 cells co-expressing variant + wild type KCNQ2 and normalized to WT channel peak current that was measured in parallel. B. Change () in voltage-dependence of activation V½ determined for heterozygous KCNQ2 variants relative to the WT channel V½ measured in parallel. Black symbols represent mean ± SEM voltageclamp data from literature reported variants (error bars are smaller than data symbol in some cases), while automated patch clamp results are shown as blue for BFNE, red for DEE, or purple symbols for BFNE/DEE pathogenic variants. All experimental data are presented as open circles with filled circles representing mean values. na = not available in the literature. Figure S6. Average whole-cell currents recorded from CHO-Q3 cells electroporated with population KCNQ2 variants. Average XE-991-sensitive whole-cell currents recorded by automated patch clamp from CHO-Q3 cells electroporated with rare population KCNQ2 variants and normalized to wild type channel peak current measured in parallel. Scale bars are 200 ms (horizontal) and 25% of WT channel current density (vertical).

Figure S7. Average whole-cell currents recorded from CHO-Q3 cells co-electroporated with selected population variants plus wild type KCNQ2.
Average XE-991-sensitive wholecell currents recorded by automated patch clamp from CHO-Q3 cells co-electroporated with rare population variants plus wild type KCNQ2 and normalized to wild type channel peak current recorded in parallel. Scale bars are 200 ms (horizontal) and 25% of WT channel current density (vertical).

Figure S8. Whole-cell currents from epilepsy-associated KCNQ2 variants expressed as homozygous channels.
Average XE-991-sensitive whole-cell currents recorded by automated patch clamp from CHO-Q3 cells electroporated with epilepsy-associated KCNQ2 variants and normalized to wild type channel peak current recorded in parallel. Variant labels: Blue = BFNEassociated; Red = DEE-associated; Black = unknown phenotype category (Q586P). For A193D and P335L, whole-cell currents were recorded from CHO-K1 cells co-electroporated with KCNQ3-WT plus KCNQ2-variant. Scale bars are 200 ms (horizontal) and 25% of WT channel current density (vertical). Figure S9. Average whole-cell currents recorded from CHO-Q3 cells co-electroporated with epilepsy-associated variants plus wild type KCNQ2. Average XE-991-sensitive wholecell currents recorded by automated patch clamp from CHO-Q3 cells co-electroporated with epilepsy-associated KCNQ2 variants plus WT KCNQ2 and normalized to wild type channel peak current recorded in parallel. Variant labels: Blue = BFNE-associated; Red = DEEassociated; Black = unknown phenotype category (Q586P). Scale bars are 200 ms (horizontal) and 25% of WT channel current density (vertical).   Table S7. Figure S11 -continued. Retigabine effects on whole-cell currents recorded from epilepsyassociated KCNQ2 variants expressed in the heterozygous state. Normalized current-voltage relationships for each variant expressed in the heterozygous state recorded in the absence of retigabine (WT|variant, open squares) compared with heterozygous variants recorded in the absence of retigabine (WT|variant +retigabine, orange filled diamonds). Currents were first normalized to cell capacitance, then re-normalized to the peak current for WT channels recorded in parallel (WT|WT, filled circles). Variant labels: Blue = BFNE-associated; Red = DEE-associated; Purple = BFNE/DEE; Black = unknown phenotype category. Complete data sets are presented in Table S7. Figure S11 -continued. Retigabine effects on whole-cell currents recorded from epilepsyassociated KCNQ2 variants expressed in the heterozygous state. Normalized current-voltage relationships for each variant expressed in the heterozygous state recorded in the absence of retigabine (WT|variant, open squares) compared with heterozygous variants recorded in the absence of retigabine (WT|variant +retigabine, orange filled diamonds). Currents were first normalized to cell capacitance, then re-normalized to the peak current for WT channels recorded in parallel (WT|WT, filled circles). Variant labels: Blue = BFNE-associated; Red = DEE-associated; Purple = BFNE/DEE; Black = unknown phenotype category. Complete data sets are presented in Table S7.