Authors :
Presenting Author: Nicole Hawkins, PhD – Northwestern University Feinberg School of Medicine
Erin Corbett, BS – Feinberg School of Medicine, Northwestern University
Jennifer Kearney, PhD – Northwestern University Feinberg School of Medicine
Rationale:
Pathogenic variants in KCNH1 are responsible for a range of neurodevelopmental disorders, including DEEs. The recurrent KCNH1-p.R357Q variant is associated non-syndromic and syndromic DEEs, including Zimmermann-Laband and Temple-Baraitser. In addition to refractory epilepsy, individuals with KCNH1-related disorders have elevated risk for status epilepticus (SE) and SE-related mortality. To investigate, we generated a novel Kcnh1R357Q mouse model. While transmission of the R357Q (Q/+) variant was successful on the C57BL/6J (B6J) strain, juvenile Q/+ mice exhibited continuous, prolonged seizure activity and 100% mortality. To rescue the severe mortality phenotype and enable propagation of the Kcnh1R357Q mouse line, we trialed crossing to multiple inbred strains and treating with the antiseizure compound GS967. Methods:
Kcnh1R357Q mosaic founders on the B6J strain (Jackson Lab, #000064) were generated using CRISPR/Cas9. For the genetic approach, mosaic B6J founders were bred to C3HeB/FeJ (C3H; Jackson, #000658 or 129S6/SvEvTac (129; Taconic, 129SVE) to generate F1.C3H or F1.129, respectively. F1.C3H mice were intercrossed to generate F2.[B6JxC3H] mice. F1.129 mice were backcrossed to 129 (n2.F1) and then intercrossed to generate F2.[B6Jx129] mice. To re-establish Q/+ on B6J, F1.129 mice were backcrossed to B6J. Concurrent with strain modification, the antiseizure compound GS967 (CAS # 126261-39-2, Cayman Chemical) was introduced. Q/+ offspring and breeding pairs were maintained on rodent chow Open Diet (D11112201, Research Diets) formulated with GS967 at 8 mg per kg chow, resulting in an estimated daily dose of ~1.5 mg/kg. Offspring were either continued on GS967 chow or weaned onto standard diet. Survival was monitored and compared by Kaplan Meier analysis with p-values determined by Logrank.
Results:
Two founder females (17% and 45%) were crossed to B6J resulting in 3 B6J.Q/+ that exhibited continuous seizure activity (SE) and 100% mortality between postnatal days 19-29 (P19-29). Crosses of mosaic females to C3H and 129 strains did not improve survival with 100% mortality observed by P26 and P31, respectively. Treatment with GS967 from birth resulted in significant improvement in survival independent of strain background. F1.C3H Q/+ (n=2) and F1.129.Q/+ (n=1) mice continuously maintained on GS967 chow survived more than 6 months; however, homozygous F2.Q/Q mice did not survive past weaning despite GS967 treatment. Subsequent backcrosses to B6J (n1.F1-n3.F1) generated n=44 Q/+ mice for survival analysis. Heterozygous Q/+ mice maintained on GS967 chow (n=19) exhibited 100% survival to >7 weeks of age, while untreated Q/+ mice (n=25) exhibited 100% mortality between P24-31 (p< 0.0001).