ObjectiveRecent studies have identified brain somatic variants as a cause of focal epilepsy. These studies relied on resected tissue from epilepsy surgery, which is not available in most patients. The use of trace tissue adherent to depth electrodes used for stereo electroencephalography (EEG) has been proposed as an alternative but is hampered by the low cell quality and contamination by nonbrain cells. Here, we use our improved depth electrode harvesting technique that purifies neuronal nuclei to achieve molecular diagnosis in a patient with focal cortical dysplasia (FCD).MethodsDepth electrode tips were collected, pooled by brain region and seizure onset zone, and nuclei were isolated and sorted using fluorescence‐activated nuclei sorting (FANS). Somatic DNA was amplified from neuronal and astrocyte nuclei using primary template amplification followed by exome sequencing of neuronal DNA from the affected pool, unaffected pool, and saliva. The identified variant was validated using droplet digital polymerase chain reaction (PCR).ResultsAn 11‐year‐old male with drug‐resistant genetic–structural epilepsy due to left anterior insula FCD had seizures from age 3 years. Stereo EEG confirmed seizure onset in the left anterior insula. The two anterior insula electrodes were combined as the affected pool and three frontal electrodes as the unaffected pool. FANS isolated 140 neuronal nuclei from the affected and 245 neuronal nuclei from the unaffected pool. A novel somatic missense MTOR variant (p.Leu489Met, CADD score 23.7) was identified in the affected neuronal sample. Droplet digital PCR confirmed a mosaic gradient (variant allele frequency = .78% in affected neuronal sample; variant was absent in all other samples).SignificanceOur findings confirm that harvesting neuronal DNA from depth electrodes followed by molecular analysis to identify brain somatic variants is feasible. Our novel method represents a significant improvement compared to the previous method by focusing the analysis on high‐quality cells of the cell type of interest.

中文翻译：

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使用新型深度电极采集技术鉴定局灶性皮质发育不良患者纯化神经元 DNA 中的马赛克 MTOR 变异

目的最近的研究已确定脑体细胞变异是局灶性癫痫的一个原因。这些研究依赖于癫痫手术切除的组织，而大多数患者无法获得这些组织。已提出使用粘附在深度电极上的微量组织用于立体脑电图（EEG）作为替代方案，但由于细胞质量低和非脑细胞污染而受到阻碍。在这里，我们使用改进的深度电极采集技术来纯化神经元细胞核，以实现局灶性皮质发育不良 (FCD) 患者的分子诊断。方法收集深度电极尖端，按大脑区域和癫痫发作区域汇集，并对细胞核进行分离和分类使用荧光激活核分选（FANS）。使用初级模板扩增从神经元和星形胶质细胞核中扩增体细胞 DNA，然后对受影响池、未受影响池和唾液中的神经元 DNA 进行外显子组测序。使用液滴数字聚合酶链式反应 (PCR) 验证了所识别的变异。结果 一名 11 岁男性因左前岛叶 FCD 而患有耐药性遗传性结构性癫痫，从 3 岁时开始癫痫发作。立体脑电图证实左前岛叶癫痫发作。两个前岛叶电极组合为受影响池，三个额电极作为未受影响池。 FANS 从受影响的池中分离出 140 个神经元核，从未受影响的池中分离出 245 个神经元核。一种新颖的体细胞错义磁导率在受影响的神经元样本中鉴定出变异（p.Leu489Met，CADD 评分 23.7）。液滴数字 PCR 证实了镶嵌梯度（受影响的神经元样本中变异等位基因频率 = 0.78%；所有其他样本中不存在变异）。意义我们的研究结果证实，从深度电极收获神经元 DNA，然后进行分子分析来识别脑体细胞变异是可行的。通过将分析重点放在感兴趣的细胞类型的高质量细胞上，我们的新方法比以前的方法有了显着的改进。