Abstracts

RATIONALE: Teetering [italic](tn)[/italic] is an autosomal recessive mouse mutation first identified in the 1960s. The homozygous mutant mice are recognisable by their ataxic gait from around two weeks of age. In addition, pathological examination shows that they display selective dysgenesis of the hindbrain. More recently, teetering mice have also been shown to exhibit bilaterally symmetrical, synchronous spike-wave discharges on cortical EEG (J. Noebels, pers. comm.) - a pattern characteristic of mouse models of absence epilepsy. We set out to identify the gene underlying the teetering phenotype by a positional cloning strategy. The gene had previously been mapped to the distal end of mouse chromosome 11 in a region displaying conserved synteny with human chromosome 17q25.3. We have used an interspecific intercross to refine the location of the [italic]tn[/italic] gene and are currently investigating candidate genes within this defined region. The eventual identification of this gene will shed further light on the molecular basis of seizure generation.
METHODS: An interspecific intercross has been established (B6C3Fe-[italic]a/a/tn[/italic] x CAST/Ei) to produce recombinant meioses allowing the area containing the [italic]tn[/italic] gene to be precisely delineated. Affected F2 progeny from this cross will each contain two recombinant chromosomes 11and these are then typed for a number of markers which display polymorphisms between the two parental strains. Genomic DNA has been extracted from 250 offspring (i.e. 500 meioses) and genotyped using the polymerase chain reaction for a panel of seven simple sequence length polymorphic markers (SSLPs) spanning the most distal 10cM of chromosome 11.
RESULTS: We have narrowed the [italic]tn[/italic] region to a 1cM interval on distal mouse chromosome 11 between the markers[italic] D11Mit104[/italic] and [italic]D11Mit69[/italic] - representing a physical distance of approximately 2Mb of DNA. To refine the gene location further, novel polymorphic markers (including microsatellites and single-nucleotide polymorphisms) are being developed within the intronic sequences and 3[ssquote] untranslated regions of the many genes in this genomic region. Calcium channel subunit genes are obvious candidates for teetering as four other mouse models with a similar EEG phenotype (tottering, lethargic, stargazer and ducky) have been found to possess mutations in such genes. Several calcium channel subunits map to mouse chromosome 11 including the brain expressed [italic]Cacng4[/italic] and [italic]Cacna1g[/italic]. However, analysis of mouse draft genome assembly resources (http://genome.cse.ucsc.edu and http//www.ensembl.org/Mus_musculus) and comparison with the corresponding human genomic region suggest that both of these genes lie outside the current region of interest.
CONCLUSIONS: Further analysis of this region is now being undertaken to identify candidate genes which will be evaluated using RT-PCR, DNA sequencing and northern analysis. It is anticipated that the identification of the teetering gene and knowledge of the role of the gene product will further inform the process of seizure generation in the mammalian brain and may lead to the development of novel AEDs.
[Supported by: The Medical Research Council of the United Kingdom]