Mutations in voltage-gated sodium channels have been implicated in several types of human epilepsy with varying degrees of clinical severity. Mutations in SCN1A were first identified in Generalized Epilepsy with Febrile Seizures Plus (GEFS+), a benign, childhood-onset syndrome in which family members have febrile seizures in childhood and may go on to develop other seizure types as adults. SCN1A mutations have also been identified in Severe Myoclonic Epilepsy of Infancy (SMEI), an infant-onset syndrome characterized by generalized tonic-clonic or hemiclonic seizures. As the syndrome progresses, SMEI patients develop other seizure types including myoclonic, absence and partial seizures, and a decline of psychomotor and mental development. Overall more than 300 mutations of SCN1A have been reported in patients with epilepsy, making it the most common genetic cause of epilepsy. In vitro expression studies of SCN1A mutations have revealed a variety of functional defects. However, there is not an obvious correlation between Nav1.1 dysfunction in heterologous expression systems and severity of the clinical phenotype. The lack of a clear genotype-phenotype correlation may reflect a limitation of in vitro expression systems to evaluate neuronal sodium channel mutations. The most reliable data on functional consequences of mutations can be obtained from mice engineered to carry the mutations. However, the resources and time required for generating knock-in mice by homologous recombination is prohibitive. Recombination-mediated cassette exchange (RMCE) allows for rapid and efficient production of an allele series of mice carrying mutant DNAs at the target locus. In this method, a cassette acceptor containing a selectable marker flanked by lox sites is targeted to the endogenous mouse locus by homologous recombination. Subsequent exchange of the cassette acceptor for the sequence of interest occurs by cre-mediated recombination in the ES cells, which is much more efficient than homologous recombination. The gain in efficiency decreases the time and resources required to generate multiple variants, allowing for parallel generation of an allelic series of mice.
Specific aim 1 will generate a mouse ES cell line in which Scn1a exon 1 containing the translation start site is replaced by a loxed cassette acceptor via homologous recombination. Subsequent exchange with SCN1A cDNAs will allow expression of the cDNA under the endogenous regulatory control while ablating expression of the mouse gene.
Specific aim 2 will generate mice by cre-mediated cassette exchange in which mouse Scn1a is replaced with the human SCN1A cDNA as a critical proof-of-principle experiment for this approach. Development and validation of this approach will enable in vivo characterization of human epilepsy mutations and provides a valuable resource for understanding the mechanisms underlying epilepsy.

Public Health Relevance

The goal of this proposal is to develop a system for rapid and efficient production of mouse models carrying human epilepsy mutations. Mouse models will provide insight into the molecular and genetic events that underlie epilepsy and will be valuable tools for developing novel therapeutic strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21NS063097-01A1
Application #
7660178
Study Section
Genetics of Health and Disease Study Section (GHD)
Program Officer
Silberberg, Shai D
Project Start
2009-03-01
Project End
2011-02-28
Budget Start
2009-03-01
Budget End
2010-02-28
Support Year
1
Fiscal Year
2009
Total Cost
$201,469
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Miller, A R; Hawkins, N A; McCollom, C E et al. (2014) Mapping genetic modifiers of survival in a mouse model of Dravet syndrome. Genes Brain Behav 13:163-72
Mistry, Akshitkumar M; Thompson, Christopher H; Miller, Alison R et al. (2014) Strain- and age-dependent hippocampal neuron sodium currents correlate with epilepsy severity in Dravet syndrome mice. Neurobiol Dis 65:1-11
Kearney, Jennifer A (2011) Genetic modifiers of neurological disease. Curr Opin Genet Dev 21:349-53