Abstracts

Rationale: Mutations in voltage-gated ion channels are responsible for several types of inherited epilepsies. A common feature of genetic epilepsies is variable penetrance and expressivity among family members that carry the same epilepsy mutation. Genetic modifiers may influence the epilepsy phenotype and complicate molecular diagnosis and treatment. Using mouse models of epilepsy, these genetic modifiers can be identified and characterized in order to improve treatment options for patients. The Scn2a^Q54 transgenic mouse model has a progressive epilepsy phenotype due to a gain-of-function sodium channel mutation and exhibits strain-dependent variability in phenotype severity. We previously mapped modifier loci that influence Scn2a^Q54 phenotype severity and identified Kcnv2, encoding the voltage-gated potassium channel subunit Kv8.2, as a modifier gene (Bergren et al., Mamm Genome 2009; 20:359-366). The seizure susceptible SJL/J (SJL) strain has a 3-fold increase in hippocampal Kcnv2 expression compared to the resistant C57BL/6J (B6) strain, and double transgenic Kcnv2;Q54 mouse lines expressing higher levels of Kcnv2 have an increased incidence of seizures and accelerated mortality compared to lines expressing less Kcnv2 (Jorge et al., PNAS; 20(13):5443-5448). These data suggest that differences in Kcnv2 expression may underlie the observed strain dependent phenotype variability. The purpose of this study is to identify cis-regulatory elements that influence steady-state Kcnv2 expression. Methods: To determine the degree of sequence variation in potential cis-regulatory regions between strains, SJL genomic DNA was sequenced approximately 3kb upstream and 3kb downstream of the putative Kcnv2 coding region and compared to the B6 reference sequence. Rapid amplification of cDNA ends (RACE) analysis was performed on whole brain B6 and SJL mRNA to determine the transcription start site(s) (TSS) and organization of the 3 UTR of the Kcnv2 brain transcript(s). Results: Sequencing revealed a high degree of polymorphism between SJL and B6 strains, with 12 upstream and 13 downstream SNPs that differed between the strains. RACE analysis suggests multiple Kcnv2 transcription start sites and alternative splicing with strain-dependent variation in usage. We are currently performing RNase protection assays in order to confirm the RACE results. Conclusions: Based on our present data, it appears likely that strain variation in cis-regulatory elements alters Kcnv2 expression. Future studies of promoter activity and mRNA stability assays will determine how these cis-regulatory elements affect steady-state Kcnv2 expression. These studies will contribute to a better understanding of the molecular etiology of epilepsy.