DBA/2J (D2) and C57BL/6j (B6) mice differ significantly in susceptibility to seizures induced by various physical and chemical stimuli. In general, D2 mice are seizure sensitive whereas B6 mice are seizure resistant and these traits are controlled by multiple genes interacting with the environment. This is a renewal application for a project aimed at characterizing gene sequence variation which underlines this strain difference. The objectives of this proposal are 1) To confirm loci of major and minor effect using new seizure screening paradigms; 2)to develop congenic strains for confirmed QTLs of significant influence; and 3) to study candidate genes for major QTL on chromosome 1 (Kaszl). In two parallel QTL studies, populations of F2 intercross mice (n=500) derived from D2 and B6 parental strain will be screened for seizure sensitivity with a benzodiazepine inverse agonist (Beta-CCM) or an opiod antagonist (naloxone). Both of these drugs distinguish responses between D2 and B6 mice. Quantitative phenotypes based on seizure latencies will be established in all F2 mice along with genotypes at microsatellite loci spaced evenly at 15-20 cM intervals. Mapmaker/QTL and multivariate regression analysis will be used as complementary statistical mapping tools to confirm QTLs at loci detected in previous screening models and to evaluate new QTLs. Fine mapping of QTLs of significant effect confirmed in at least two different seizure paradigms will proceed by developing congenic strains to study the influence of each QTL separately . Reciprocal congenic strains will be generated by the method of introgression and marker selection across a region that includes the seizure QTLs and will be tested for seizure susceptibility in comparison to each other and parental strains. QTLs that interact through epistasis (determined by multilocus mapping procedures) will be placed in congenic strains together and compound congenic strains will be used to evaluate combined QTL effects. Finally, a systematic screening strategy for evaluating candidate genes for Kaszl, a major seizure QTL in this model, will include cDNA, mRNA and protein studies. Candidate genes mapped to the approximate location of Kaszl include genes for a serotonin receptor (Htr5b), a neurotransmitter synaptic vesicle protein (Syt2) and ATPase subunits (Arpla2, Atplb1, Atp2b4). Identification and characterization of seizure QTLs in this model will provide new insight into the relationship between gene and seizure susceptibility. Progress: This is an application for continuation of funding for a 3 year project. In the original proposal, the PI proposed to use QTL mapping methods to identify loci involved in chemical-induced seizures. During the initial funding period, the KA response was explored using a seizure score which is based on the following 3 measures: latencies to partial clonus, generalized clonus and status epilepticus. Analysis of these 4 measures in the F1 and F2 generations demonstrated that each had a significant inheritable component (0.65-0.46). Subsequent genome-scanning work identified significant evidence for a seizure-susceptibility locus on chromosome 1 (Kasz1) near markers D1Mit30 and D1Mit16. Also detected with varying degrees of certainty were 7 other QTL loci. In addition, interactions were detected between Kasz1 and Kasz4 and between Kasz2 and Kasz3. This work is significant evidence of completion of the initial goals of this proposal. This work is in press in Mammalian Genome. Work has begun on the second seizure paradigm, PTZ-induced seizures. Seizure trait data includes latency to general clonus, to partial clonus, to maximal seizure and a combined seizure score. Data from F2 experiments indicate heritability estimates of 0.3 to 0.84. A genome-scan has been partially completed with testing of the loci detected with the KA paradigm completed. The PTZ mapping efforts with D1Mit30 and close markers indicate that the Kasz1 locus detected by KA experiments is also involved in the PTZ response. This confirms the KA localization using a different chemical method of inducing seizures. Also, loci on chromosomes 4 and 5 first detected with KA (Kasz4 and Kasz8) were also detected in the PTZ mapping confirming the original KA results. Work is underway to complete the genomic scan for additional markers.
