Mapping and Characterization of Epilespy Genes

描述（由申请人提供）：本研究计划的主要目标是绘制和表征导致癫痫的基因。这是通过研究癫痫患者谁有平衡的染色体重排与一个断点涉及癫痫位点。三个这样的案例，DGAP095, 097和131 (dgap.harvard.edu)，正在调查中。DGAP095的断点分析表明，DGKD基因在2q37位点重排而中断，该位点位于人类癫痫易感性位点之一附近。我们认为DGKD的破坏对患者的癫痫表型具有致病作用，原因如下：(1)二酰基甘油激酶在神经信号传导中的作用；(2)小鼠DGKD定位在癫痫易感性位点附近；(3)DGKD在模型生物发育中的中枢神经系统中的表达。DGKD的同源表达将在小鼠大脑的各个部分进行评估，确认DGKD的病因学作用的研究将包括构建小鼠模型。在DGAP097和131中，断点Xp22.1和5q13位于癫痫相关位点附近。使用荧光原位杂交技术将它们定位在人类基因组图谱上，并分析断点处的序列以寻找潜在的候选基因，如果断点位于基因编码区域内，则使用Northern blot分析来评估由于重排而导致的新转录物的存在或缺失。对癫痫家族/感兴趣位点相关以及常染色体显性遗传的癫痫患者进行前瞻性突变扫描是本研究的未来方向，可以使用单链构象多态性或变性梯度凝胶电泳，随后对异常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.