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

Highlights

• Genetic deletion of Hepatic leukemia factor (Hlf) exacerbated epilepsy in Scn2a^Q54 mice.

• Pyridoxine deficiency exacerbated the epilepsy phenotype of Scn2a^Q54 mice.

• Genetic deletion of Hlf worsened survival in the Scn1a^+/− model of Dravet syndrome.

• Hlf acts as a genetic modifier of epilepsy, possibly by pyridoxine pathway modulation.

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.

Introduction

Epilepsy is a common neurological disorder affecting approximately 50 million people worldwide (World Health Organization, 2015). Over 1000 mutations identified in voltage-gated sodium channel genes result in several human epilepsy syndromes (Meisler et al., 2010). Often, individuals with the same mutation can exhibit strikingly different clinical severity. This suggests that the effect of the primary mutation is influenced by other factors, which may include genetic modifiers.

Several mouse models have been generated in order to study genetic epilepsies. Frequently, strain background alters the disease phenotype, supporting a contribution of genetic modifiers in epilepsy. The Scn2a^Q54 (Q54) mouse model has a gain-of-function mutation that results in persistent sodium current and epilepsy (Kearney et al., 2001). On the C57BL/6J (B6) strain, Q54 mice have infrequent, adult-onset focal motor seizures and a 75% survival rate beyond six months of age (Bergren et al., 2005). When crossed with the SJL/J (SJL) strain, resulting F1.Q54 mice experience high seizure frequency with juvenile onset and less than 25% survival at six months of age (Bergren et al., 2005). We mapped two dominant loci that modify seizure susceptibility of Q54, designated Moe1 (Modifier of Epilepsy) on chromosome 11 and Moe2 on chromosome 19 (Bergren et al., 2005). In contrast to the overall effect, at the Moe1 locus B6 alleles confer increased seizure risk (Hawkins and Kearney, 2012). Fine-mapping and candidate gene analysis by RNA-seq suggested hepatic leukemia factor (Hlf) as a candidate modifier at the Moe1. B6 mice express lower levels of Hlf transcript compared to SJL (Hawkins and Kearney, 2012), leading us to hypothesize that deletion of Hlf would increase seizure susceptibility.

Hlf is a member of the PAR bZIP transcription factor family, which includes Hlf, Dbp and Tef. Deletion of this family in a knock-out mouse model resulted in spontaneous generalized tonic-clonic and absence seizures, and premature lethality (Gachon et al., 2004). Two-fold reduction in expression of pyridoxal kinase (PDXK) was implicated as a likely contributor to the epilepsy phenotype. PDXK is essential for conversion of pyridoxine to pyridoxal 5′ phosphate (PLP), a key coenzyme involved in amino acid and neurotransmitter metabolism, including GABA and glutamate (John, 1995). Pyridoxine deficiency in humans results in epilepsy that is successfully treated by PLP administration (Plecko and Stockler, 2009). Based on its prior association with seizures and the pyridoxine pathway, we hypothesized that genetic variation in Hlf could modify the Q54 epilepsy phenotype (Hawkins and Kearney, 2012).

To determine if Hlf could modify the Q54 phenotype, we evaluated the effect of Hlf deletion on seizures and survival in Q54 mice. Additionally, we tested whether manipulation of the pyridoxine pathway could modify the Q54 phenotype. Finally, to determine whether Hlf would modify epilepsy in another model, we evaluated the effect of Hlf deletion on phenotype of the Scn1a^+/− Dravet syndrome model.