Mapping and Characterization of Epilespy Genes

描述（由申请人提供）：本研究提案的主要目标是绘制和表征导致癫痫的基因。这是通过研究癫痫个体实现的，这些癫痫个体具有平衡的染色体重排，其中在涉及癫痫的基因座中具有断点。本提案正在调查三个此类案件，DGAP 095、097和131（dgap.harvard.edu）。DGAP 095中断裂点的分析表明，DGKD基因通过2 q37位点的重排而被破坏，该位点位于人类癫痫发作易感性位点之一附近。我们认为，DGKD破坏是患者表现出的癫痫发作表型的致病因素，这是根据（1）二酰基甘油激酶在神经信号传导中的作用，（2）小鼠DGKD定位在癫痫发作易感基因座附近，以及（3）模型生物体中发育中CNS中的DGKD表达。将在小鼠脑的各个部分中评估DGKD直系同源物表达，并且确认DGKD的病因学作用的研究将包括小鼠模型的构建。在DGAP 097和131中，断点Xp22.1和5 q13位于癫痫相关位点附近。将使用荧光原位杂交将其定位在人类基因组图谱上，并分析断裂点处的序列以寻找潜在的候选基因，如果断裂点福尔斯落在基因编码区内，则将使用北方印迹分析来评估是否存在重排导致的新转录本。一个前瞻性的突变扫描，在家庭与癫痫/墨到感兴趣的基因座，以及在癫痫患者的常染色体显性遗传的方式是未来的研究方向，可以使用单链构象多态性或变性梯度凝胶电泳与随后的测序异常PCR片段。

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.