Abstracts

Rationale: KCNQ2 variants found in benign familial neonatal epilepsy are structurally diverse, including complete gene deletions, frame shifting insertions and deletions, and single nucleotide substitutions causing loss of the start codon, single amino acid changes (missense) and termination (nonsense). By contrast, the large majority of variants reported in KCNQ2 encephalopathy have been single nucleotide substitutions causing missense. We encountered several early infantile epileptic encephalopathy patients where gene sequencing revealed a KCNQ2 variant of a type not previously reported in this disorder: trinucleotide deletions leading to loss of a single amino acid. Functional characterization of the novel variants could assist in assessing whether they were pathogenic.Methods: We used site-directed mutagenesis to introduce Val136del or Phe305del into KCNQ2 cDNA. We transfected cultured Chinese hamster ovary cells with wild-type (WT) and mutant KCNQ2 cDNA, and, in some experiments, WT KCNQ3. We used whole cell voltage-clamp recording to compare the functional properties of the expressed channels and their responsiveness to the novel KCNQ2/3 opener, SF0034 (Kalappa, JNeurosci 2015;35:8829).Results: Co-expression of WT and V136del KCNQ2 in a 1:1 ratio reduced peak current density to 57.7±3.82% (n =15) of WT control levels. Though significant, this reduction was much smaller than that produced by several other KCNQ2 encephalopathy mutations (Thr274Met, Ala294Val) studied previously (Li et al., AES abstract 3.022; 2013). Co-expression of KCNQ3, KCNQ2, and V136del resulted both in loss of current density (to 45.02±5.98% of WT controls, n =10) and a strong (26± 6.2 mV, n=6) depolarizing shift in activation voltage-dependence. Co-expression of F305del along with WT subunits also reduced currents strongly through combination of reduced peak density and depolarized voltage-dependence. In cells expressing V136del or F305del in combination with WT subunits to mimic the heterozygosity seen in affected patients, SF0034 (1 uM) increased currents significantly.Conclusions: The two KCNQ2 deletion variants studied both strongly suppressed channel currents to a greater degree than usually seen in studies of KCNQ2 variants causing benign familial neonatal epilepsy. These finding provide evidence that both Val136del and Phe305del are pathogenic in cases of KCNQ2 encephalopathy. Although the extent of subthreshold current suppression produced was similar to that observed in previous studies of KCNQ2 encephalopathy variants, the electrophysiological basis was novel. Whereas previously studied KCNQ2 variants suppressed currents by either shifting voltage-dependence or reducing maximal current, Val136del and Phe305del affected both parameters. In combination, this produced strong, dominant-negative effects. The selective KCNQ2/3 opener, SF0034, shifted voltage-sensing and increased current density, thus tending to reverse the effects of the pathogenic deletion variants. Support: NINDS R01 NS49119, CURE Pediatric Epilepsy Award.