Hlf is a genetic modifier of epilepsy caused by voltage-gated sodium channel mutations

Abstract

Mutations in voltage-gated sodium channel genes cause several types of human epilepsies. Often, individuals with the same sodium channel mutation exhibit diverse phenotypes. This suggests that factors beyond the primary mutation influence disease severity, including genetic modifiers. Mouse epilepsy models with voltage-gated sodium channel mutations exhibit strain-dependent phenotype variability, supporting a contribution of genetic modifiers in epilepsy. The Scn2a(Q54) (Q54) mouse model has a strain-dependent epilepsy phenotype. Q54 mice on the C57BL/6J (B6) strain exhibit delayed seizure onset and improved survival compared to [B6xSJL/J]F1.Q54 mice. We previously mapped two dominant modifier loci that influence Q54 seizure susceptibility and identified Hlf (hepatic leukemia factor) as a candidate modifier gene at one locus. Hlf and other PAR bZIP transcription factors had previously been associated with spontaneous seizures in mice thought to be caused by down-regulation of the pyridoxine pathway. An Hlf targeted knockout mouse model was used to evaluate the effect of Hlf deletion on Q54 phenotype severity. Hlf(KO/KO);Q54 double mutant mice exhibited elevated frequency and reduced survival compared to Q54 controls. To determine if direct modulation of the pyridoxine pathway could alter the Q54 phenotype, mice were maintained on a pyridoxine-deficient diet for 6 weeks. Dietary pyridoxine deficiency resulted in elevated seizure frequency and decreased survival in Q54 mice compared to control diet. To determine if Hlf could modify other epilepsies, Hlf(KO/+) mice were crossed with the Scn1a(KO/+) Dravet syndrome mouse model to examine the effect on premature lethality. Hlf(KO/+);Scn1a(KO/+) offspring exhibited decreased survival compared to Scn1a(KO/+) controls. Together these results demonstrate that Hlf is a genetic modifier of epilepsy caused by voltage-gated sodium channel mutations and that modulation of the pyridoxine pathway can also influence phenotype severity.

Keywords: Epilepsy; Genetics; Mouse model; Seizure; Voltage-gated sodium channel.

Figure 1. Loss of Hlf exacerbates the Scn2a^Q54 phenotype

A) Mice were observed for focal motor seizures (FMS) and generalized tonic clonic seizures (GTCS) in 30 minute sessions at 3 and 6 weeks of age. The percentages of mice exhibiting FMS or GTCS during the observation period are shown (n=30–32 per group). Significant differences between groups were determined by Fisher’s exact test. *p < 0.0134 compared to Q54 littermate controls. B) The average number of FMS (±S.E.M) and total number of GTCS observed is shown (n=30–32 per group). C) Kaplan-Meier plot of survival to 6 weeks of age. Hlf ^KO/KO;Q54 had a significant decrease in survival compared to Q54 mice (p=0.0215, LogRank Mantel-Cox). D) Representative EEG recording from a Hlf ^KO/KO;Q54 during a seizure. Time span of plot is 3 minutes. Top trace is right posterior to left posterior; bottom trace is right anterior to left posterior.

Figure 2. Pyridoxine deficiency exacerbates the Scn2a^Q54 phenotype

A) Average FMS frequency of Q54 mice maintained on pyridoxine deficient or control diets from 3 to 9 weeks of age. The number of FMS was counted in a 30 minute session at 9 weeks of age. Significant differences were determined by Student’s T-test (*p < 0.03). Data are represented as mean ± SEM. B) Survival of Q54 mice maintained on pyridoxine deficient or control diets was monitored until nine weeks of age. Kaplan Meier survival plot is shown. Q54 mice on the deficient diet had a significant decrease in survival compared to control diet (p<0.002, LogRank Mantel-Cox).

Figure 3. Loss of Hlf exacerbates premature lethality phenotype of Scn1a^KO/− Dravet mice

Survival of Hlf ^KO/+, Scn1a^KO/+ and Hlf ^KO/+;Scn1a^KO/+ mice was monitored until nine weeks of age. Kaplan Meier survival plot is shown. Hlf ^KO/+;Scn1a^KO/+ mice had reduced survival compared to Scn1a^KO/+ mice (p<0.006, LogRank Mantel-Cox).