Genes and pathways underlying brain overgrowth and focal cortical malformations

 描述(申请人提供)：巨头症(或MEG)是一种以广泛性脑过度生长为特征的发育性大脑疾病。它发生在越来越多的发育和代谢障碍中，并与严重的儿童神经并发症有关，包括癫痫、智力残疾、自闭症、脑积水和Chiari畸形。脑磁图显示与更严重的节段性脑疾病有显着重叠，包括半巨脑(HMEG)和局灶性皮质发育不良(FCD)，这些共同构成了儿童难治性癫痫的最常见原因。最近在MEG、HMEG和FCD中发现了关键的PI3K-AKT-mTOR信号网络(包括PIK3CA、PIK3R2、AKT3和CCND2)中关键基因的激活突变，表明这些表型构成了具有共同的分子病因和神经病理特征的单一广泛的发育性脑疾病。这项建议的目的是在受影响的人类来源的细胞和组织中询问这些疾病的分子基础，以确定这些疾病的突变谱、嵌合体水平、组织分布和途径失调。首先，我建议对从癫痫手术中获得的HMEG和FCD人脑样本中受影响的神经元进行单细胞测序，以测试已知和候选基因。其次，我将使用高通量蛋白质组学方法，在受影响的人脑组织中全面分析关键的PI3K-AKT途径基因突变的PI3K-AKT途径蛋白，以识别途径失调，并定位特定的亚途径和下游靶点。第三，到目前为止发现的PI3K AKT基因可以解释两种最常见的MEG综合征中约75%的儿童，约25%的HMEG-FCD儿童，以及没有罕见的MEG综合征。我建议进行研究，以确定更多的MEG-HMEG-FCD致病基因，使用全外显子组和全基因组测序从受影响的个人的多个组织。我的职业目标是成为一名内科科学家，致力于将人类脑组织的尖端基因组和蛋白质组技术，包括单细胞测序，与准确和定量的表型鉴定相结合，以进一步了解人类发育性大脑疾病的分子和生物学基础。我提出了一个为期五年的研究计划，在整合脑研究中心(Cibr)的威廉·B·多比恩斯博士的指导下，将教学和研究培训结合起来。我的顾问委员会和合作者网络，加上西雅图儿童研究所和华盛顿大学的资源，将为我成功完成这项提案并过渡到一名独立资助的内科科学家提供必要的环境。

 DESCRIPTION (provided by applicant): Megalocephaly (or MEG) is a developmental brain disorder characterized by generalized brain overgrowth. It occurs in a growing number of developmental and metabolic disorders and is associated with severe childhood neurological complications including epilepsy, intellectual disability, autism, and hydrocephalus and Chiari malformations. MEG shows significant overlap with more severe but segmental brain disorders including hemimegalencephaly (HMEG) and focal cortical dysplasia (FCD) that collectively constitute the most common cause of intractable epilepsy in children. Activating mutations in key genes within the critical PI3K- AKT-mTOR signaling network (including PIK3CA, PIK3R2, AKT3 and CCND2) have been recently identified in MEG, HMEG and FCD, suggesting that these phenotypes constitute a single broad spectrum of developmental brain disorders with shared molecular etiologies and neuropathological features. The goal of this proposal is to interrogate the molecular basis of these disorders in affected human-derived cells and tissues to define the mutational spectrum, levels of mosaicism, tissue distribution and pathway dysregulation in these disorders. First, I propose to perform single cell sequencing of affected neurons from HMEG and FCD human brain samples obtained from epilepsy surgery to test for known and candidate genes. Second, I will globally assay PI3K-AKT pathway proteins in affected human brain tissues with mutations in key PI3K-AKT pathway genes using high throughput proteomics to identify pathway dysregulation and localize specific sub-pathways and downstream targets. Third, the PI3K AKT genes discovered so far explain ~75% of children with the two most common MEG syndromes, ~25% with HMEG-FCD and none with rare MEG syndromes. I propose studies to identify additional MEG-HMEG-FCD causative genes using whole exome and whole genome sequencing on multiple tissues from affected individuals. My career goal is to become a physician-scientist devoted to integrating cutting-edge genomic and proteomic techniques on human-derived brain tissues, including single cell sequencing, with accurate and quantitative phenotyping to further our understanding of the molecular and biologic basis of human developmental brain disorders. I propose a five-year research program that will incorporate didactic and research training under the mentorship of Dr. William B. Dobyns at the Center for Integrative Brain Research (CIBR). My advisory committee and network of collaborators, combined with the resources at the Seattle Children's Research Institute and University of Washington, will provide the environment necessary for my successful completion of this proposal and transition to an independently funded physician-scientist.