Mapping and Characterization of Epilespy Genes

描述(由申请人提供)：这项研究提案的主要目标是绘制和表征导致癫痫的基因。这是通过研究具有平衡的染色体重排的癫痫患者实现的，这些患者的染色体重排在与癫痫有关的基因座上有一个断裂点。本提案正在调查三起此类案件，即DGAP095、097和131(dgap.atherard.edu)。对DGAP095基因断裂点的分析表明，DGKD基因在2q37基因座的重排中被破坏，该基因位于人类癫痫易感基因座附近。我们认为DGKD的中断是患者表现出的癫痫表型的致病因素，因为(1)二酰甘油激酶在神经信号转导中的意义，(2)小鼠DGKD在癫痫易感位点附近的定位，以及(3)DGKD在模式生物发育中的中枢神经系统中的表达。DGKD同源基因的表达将在小鼠大脑的不同部分进行评估，确认DGKD病因学作用的研究将包括构建小鼠模型。在DGAP097和DGAP131中，Xp22.1和5q13断裂点位于癫痫发病部位附近。它们将通过荧光原位杂交定位在人类基因组图谱上，并将分析断裂点上的序列以寻找潜在的候选基因，如果断裂点落在基因编码区内，将使用Northern印迹分析来评估由于重排而产生的新转录物(S)的存在或不存在。在癫痫家系和常染色体显性遗传的癫痫患者中进行前瞻性突变扫描是这项研究的未来方向，可以使用单链构象多态或变性梯度凝胶电泳法，随后对异常的PCR片段进行测序。

DESCRIPTION (provided by applicant): The primary objective of this research proposal is mapping and characterization of genes that contribute to epilepsy. This is achieved by studying epileptic individuals who have balanced chromosomal rearrangement with a breakpoint in a locus implicated in epilepsy. Three such cases, DGAP095, 097 and 131 (dgap.harvard.edu), are being investigated in this proposal. Analysis of breakpoints in DGAP095 showed that the DGKD gene is disrupted by rearrangement at the 2q37 locus, which maps near one of the seizure susceptibility loci in humans. We propose that DGKD disruption is pathogenetic to the seizure phenotype exhibited by the patient in light of the (1) implication of diacylglycerol kinases in neural signaling, (2) murine DGKD localization near a seizure susceptibility locus, and (3) DGKD expression in developing CNS in model organisms. DGKD ortholog expression will be assessed in various sections of the mouse brain, and studies confirming the etiologic role of DGKD will include construction of a mouse model. In DGAP097 and 131, breakpoints Xp22.1 and 5q13 lie near epilepsy implicated loci. They will be positioned on the human genome map using fluorescence in situ hybridization and the sequence at the breakpoints will be analyzed in a search for potential candidate genes, if the breakpoint falls within a gene-coding region, Northern blot analyses will be used to assess the presence or absence of novel transcript(s) due to the rearrangement. A prospective mutation scan in families with epilepsy /inked to the locus of interest as well as in patients with epilepsy inherited in an autosomal dominant fashion is a future direction of this research, and could be done using single-stranded conformation polymorphism or denaturing gradient gel electrophoresis with subsequent sequencing of aberrant PCR fragments.