QUANTITATIVE GENETIC STUDY OF SEIZURE SUSCEPTIBILITY

描述：DBA/2J(D2)和C57BL/6J(B6)小鼠在 对各种物理和化学刺激引起的癫痫的敏感性。 一般来说，D2小鼠是癫痫敏感的，而B6小鼠是癫痫敏感的 抗性，这些性状是由多个基因相互作用控制的 环境。这是一个项目的续签申请，目标是 表征该菌株的基因序列变异 不同。这项建议的目标是：1)确定主要工作地点 使用新的癫痫筛查范例和轻微效果；2)开发 确认有显著影响的QTL的同源菌株；以及3)对 研究1号染色体上主要QTL(Kaszl)的候选基因。分成两份 平行QTL研究，F2杂交小鼠群体(n=500)来自 将对D2和B6亲本菌株进行癫痫敏感性筛选 苯二氮卓类反向激动剂(Beta-CCM)或阿片拮抗剂(纳洛酮)。 这两种药物都区分了D2和B6小鼠的反应。 基于发作潜伏期的定量表型将在#年确定 所有F2小鼠以及微卫星座位上的基因型均分布在 间隔15-20厘米。MapMaker/QTL和多元回归分析将 作为互补的统计作图工具来确定座位上的QTL 在以前的筛选模型中检测到，并评估新的QTL。很好 在至少两个不同的QTL中确认显著效应的QTL的定位 癫痫的范例将通过开发同源菌株来研究 各QTL的影响分别存在。相互同源的菌株将是 通过导入和标记选择的方法生成 包括癫痫QTL的区域，并将接受癫痫检测 对彼此和亲本菌株的敏感性进行比较。QTL 通过上位性相互作用(由多位点作图程序确定) 将放在一起的同源菌株和复合同源菌株 将用于评估组合QTL效应。最后，一个系统的 癫痫大发作Kaszl候选基因的筛选策略 在这个模型中，QTL将包括cDNA、mRNA和蛋白质的研究。侯选人 被定位到Kaszl大致位置的基因包括 5-羟色胺受体(Htr5b)，一种神经递质突触泡蛋白 (Syt2)和ATPase亚基(Arpla2、Atplb1、Atp2b4)。身份识别和 这一模型中癫痫发作QTL的特征将提供新的见解 基因与癫痫易感性的关系 进展：这是一份申请继续资助3年的申请书 项目。在最初的建议中，PI建议使用QTL定位 方法寻找与化学致痫相关的基因座。在.期间 在最初的筹资期间，使用癫痫评分来探索KA的反应 其基于以下3个测量：部分阵挛的潜伏期， 全身性阵挛和癫痫持续状态。对这四项措施的分析 F1和F2世代均表现出显著的 可继承分量(0.65-0.46)。后续的基因组扫描工作 发现了癫痫易感性基因座的重要证据 1号染色体(Kasz1)位于标记D1Mit30和D1Mit16附近。还检测到 另外7个QTL座位也有不同程度的确定度。此外, 检测到Kasz1和Kasz4之间以及Kasz2和Kasz4之间的相互作用 Kasz3.这项工作是初步目标完成的重要证据 这项提议。这项研究正在《哺乳动物基因组》杂志上发表。 关于第二种癫痫发作范式的工作已经开始，即PTZ诱导的癫痫发作。 发作特征数据包括全身性阵挛、部分性阵挛、 最大癫痫发作和综合癫痫评分。来自F2实验的数据 表示遗传力估计在0.3到0.84之间。一项基因组扫描已经完成 部分完成了对KA范例检测到的基因座的测试 完成。 用D1Mit30和CLOSE标记进行的PTZ作图工作表明 KA实验检测到的Kasz1基因座也参与了PTZ反应。 这证实了KA的定位使用了不同的化学方法 导致癫痫发作。此外，4号和5号染色体上的基因座首次用KA检测到 (Kasz4和Kasz8)也在PTZ图谱中检测到，证实了 原创KA结果。完成基因组扫描的工作正在进行中 其他标记。

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.