Somatic Mutation in Intractable Focal Epilepsy

项目摘要 患有难治性局灶性癫痫和正常结构成像（非病灶性局灶性癫痫，NLFE）的个体 治疗挑战。当没有发现病灶时，癫痫病灶的定位是困难的， 成功难以捉摸。目前尚不清楚放射学正常结构的皮质如何出现局灶性癫痫发作， 低于标准3 T MRI分辨率的病理学是否是常见的罪魁祸首。我们最近证明， 编码UDP半乳糖转运蛋白的SLC 35 A2中的体细胞突变导致了一些NLFE。一些 放射学正常的病例显示FCD 1a，表明体细胞SLC 35 A2突变可以解释FCD 1a的表达。 在结构正常和异常皮质中癫痫发作，并将SLC 35 A2鉴定为第一个相关基因。 FCD 1a和NLFE。我们最近在正常成像的病例中发现了PLXNB 1和BRAF的体细胞脑变体 但FCD 2a对病理学的影响PLXNB 1可能代表FCD 2中的第一个非mTOR基因。我们由此证明 脑体细胞变异可导致放射学NLFE，有些伴有异常病理。作为基因鉴定 阐明了局灶性癫痫的生物学并为治疗提供信息，这些发现可能具有重大的临床意义。 我们将进一步探讨放射学NLFE的体细胞遗传学，包括1）基因发现的复制 在新病例中，对较低频率的致病等位基因进行额外的基因鉴定和更深入的测序; 2） 阐明与特定体细胞突变相关的表型，3）开发成像技术， 变异等位基因频率（VAF）与病理和特定活检部位EEG的相关性 在切除的脑内和5）当突变不存在时，区域病理学与EEG的关联。 在目标1中，我们将定义NLFE的体细胞遗传景观。在外科癫痫样本中，我们将进行 从NLFE患者癫痫病灶切除的脑组织的超高深度全外显子组测序 以鉴定SLC 35 A2、PLXNB 1、BRAF和其他基因中的其他体细胞突变。在目标2中，我们将定义 NLFE中的基因型-表型相关性。我们将确定与体细胞突变相关的表型 在一组NLFE患者的特定基因中，关注癫痫发作时的年龄， 术前先进的3 T和7 T MRI与计算后处理，和组织病理学分析， 切除的组织在目标3中，我们将定义区域EEG，病理和致病性等位基因负担， NLFE内的体细胞变异。在癫痫手术期间，我们将从组织中进行4-6次活检， 使用MRI定位、电生理引导的手术进行切除。我们会用病理学和脑电图 每次活检中的放电模式，区分EEG指定的癫痫发作病灶（核心）与周围（半影）。 我们将对每个活检组织进行定量基因分型，以确定被认为是致病性的变异， 当突变不存在时，将EEG模式与组织病理学异常相关联。这将定义 VAF、EEG放电模式和病理之间的关联。

PROJECT SUMMARY Individuals with intractable focal epilepsy and normal structural imaging (non-lesional focal epilepsy, NLFE) pose treatment challenges. When no lesion is detected, localization of the seizure focus is difficult and surgical success elusive. It is unknown how focal seizures arise in cortex with radiographically normal structure, and whether pathology below the resolution of standard 3T MRI is a common culprit. We recently demonstrated that somatic mutations in SLC35A2, which encodes a UDP galactose transporter, account for some NLFE. Some radiographically normal cases exhibited FCD1a, demonstrating that somatic SLC35A2 mutations can account for seizure onset in structurally normal and abnormal cortex, and identifying SLC35A2 as the first gene underlying FCD1a and NLFE. We recently found somatic brain variants in PLXNB1 and BRAF in cases with normal imaging but FCD2a on pathology. PLXNB1 may represent the first non-mTOR gene in FCD2. We thereby demonstrated brain somatic variants can result in radiographic NLFE, some with abnormal pathology. As gene identification illuminates the biology of focal epilepsy and informs therapy, discoveries can have major clinical implications. We will further explore the somatic genetics of radiographic NLFE, including 1) replication of gene discoveries in new cases, additional gene identification, and deeper sequencing for lower-frequency pathogenic alleles; 2) clarifying phenotypes associated with specific somatic mutations, 3) development of imaging to detect subtle abnormalities; 4) correlation of variant allele frequency (VAF) with pathology and EEG from specific biopsy sites within resected brain and 5) association of regional pathology with EEG when mutations are absent. In Aim 1 We will define the somatic genetic landscape of NLFE. In surgical epilepsy samples, we will conduct ultra-high-depth whole exome sequencing of brain resected from the seizure focus from individuals with NLFE to identify additional somatic mutations in SLC35A2, PLXNB1, BRAF, and other genes. In Aim 2 we will define genotype-phenotype correlations in NLFE. We will determine phenotypes associated with somatic mutations in specific genes in a cohort of individuals with NLFE, focusing on age at onset of epilepsy, abnormalities on presurgical advanced 3T and 7T MRI with computational post-processing, and histopathologic analysis of resected tissue. In Aim 3 we will define the regional EEG, pathological, and allelic burden of pathogenic somatic variants within NLFE. During epilepsy surgery, we will perform 4-6 biopsies from tissue destined for resection using an MRI-localized, electrophysiology-guided procedure. We will characterize pathology and EEG firing pattern in each biopsy, distinguishing the EEG-designated seizure focus (core) vs. surround (penumbra). We will quantitatively genotype each biopsy for variants deemed pathogenic to establish per-biopsy VAF and when mutations are absent, correlate EEG pattern with histopathologic abnormalities. This will define associations among VAF, EEG firing pattern, and pathology.