QUANTITATIVE GENETIC STUDY OF SEIZURE SUSCEPTIBILITY

描述：DBA/2 J（D2）和C57 BL/6 j（B6）小鼠在以下方面存在显著差异： 对由各种物理和化学刺激诱发的癫痫发作的敏感性。 一般来说，D2小鼠是癫痫敏感的，而B6小鼠是癫痫敏感的 这些性状是由多个基因相互作用控制的， 环境保护 这是一个项目的更新申请， 基因序列变异的特征 差 本提案的目标是：1）确定主要的 使用新的癫痫发作筛查模式的影响较小; 2）开发 确定的具有显著影响的QTL的同源株系;和3） 研究1号染色体上主效QTL的候选基因（Kaszl）。 在两 平行QTL研究中，F2杂交小鼠群体（n=500）来自 D2和B6亲本菌株将用以下方法筛选癫痫发作敏感性： 苯二氮卓类反向激动剂（β-CCM）或阿片类拮抗剂（纳洛酮）。 这两种药物区分D2和B6小鼠之间的反应。 基于癫痫发作的定量表型将在 所有F2小鼠沿着与基因型在微卫星基因座均匀间隔， 15-20 cM间隔。 Mapmaker/QTL和多元回归分析将 可作为辅助统计作图工具，用于确定位点上的QTL 在以前的筛选模型检测和评估新的QTL。 罚款 在至少两个不同的QTL中确定了显著效应的QTL定位， 癫痫发作的范例将通过开发同类菌株来研究 每个QTL的影响。 相互同源的菌株将是 通过跨物种基因渗入和标记选择的方法产生 包含癫痫发作QTL并将进行癫痫发作检测的区域 与彼此和亲本菌株相比的易感性。 QTL， 通过上位性相互作用（通过多位点作图程序确定） 将被放置在同类菌株中，并复合同类菌株 将用于评估组合的QTL效应。 最后，系统 评估Kaszl（一种主要癫痫发作）候选基因的筛选策略 QTL在这个模型中，将包括cDNA，mRNA和蛋白质的研究。 候选 定位到Kaszl的近似位置的基因包括以下基因： 5-羟色胺受体（Htr 5 b），一种神经递质突触囊泡蛋白 （Syt 2）和ATP酶亚基（Arpla 2、Atplb 1、Atp 2b 4）。 识别和 在该模型中，癫痫发作QTL的表征将提供新的见解， 基因与癫痫易感性的关系。 进展：这是一个持续资助3年的申请 项目 在最初的提案中，PI建议使用QTL作图 方法来确定参与化学诱导癫痫发作的位点。 期间 在最初的资助期间，使用癫痫发作评分探索KA反应 其基于以下3种测量：轻瘫至部分阵挛， 全身性阵挛和癫痫持续状态。 对这4项措施的分析 F_1和F_2代都表现出显著的 遗传成分（0.65-0.46）。 随后的基因组扫描工作 发现了一个重要的证据， 1号染色体（Kasz 1）靠近标记D1 Mit 30和D1 Mit 16。 还检测到 其它7个QTL位点的确定性不同。 此外，本发明还提供了一种方法， Kasz 1和Kasz 4之间以及Kasz 2和Kasz 4之间检测到相互作用。 Kasz 3. 这项工作是初步目标完成的重要证据 这一提议。 这项工作正在出版的哺乳动物基因组。 第二种癫痫发作模式，戊四氮诱发癫痫发作的工作已经开始。 癫痫发作特征数据包括全身阵挛、部分阵挛、 最大癫痫发作和综合癫痫发作评分。 F2实验数据 表明遗传力估计值为0.3至0.84。 基因组扫描已经 部分完成了对KA范式检测到的基因座的测试 完成 使用D1 Mit 30和接近标记物的PTZ标测工作表明， KA实验检测到的Kasz 1位点也参与PTZ反应。 这证实了KA定位使用不同的化学方法， 诱发癫痫 此外，第4和第5号染色体上的位点首先检测到KA （Kasz 4和Kasz 8）也在PTZ映射中检测到，证实了 原始KA结果。 目前正在进行的工作是完成基因组扫描， 额外的标记。

DESCRIPTION: 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.