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Defining disease mechanisms in SLC35A2 epilepsy

定义 SLC35A2 癫痫的疾病机制

基本信息

批准号：
10609219
负责人：
Peter B Crino
金额：
$ 6.8万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10609219
关键词：
3-Dimensional Adult Age of Onset Alleles Astrocytes Biological Assay Brain CRISPR/Cas technology Cell Fraction Cell Line Cell Nucleus Cells Clustered Regularly Interspaced Short Palindromic Repeats Complement Complementary DNA Conceptions Congenital disorders of glycosylation Cortical Dysplasia Cortical Malformation Coupled Data Defect Dendritic Spines Development Disease Electroencephalogram Electrophysiology (science)Electroporation Embryonic Development Epilepsy Event FDA approved Freezing Functional disorder Galactose Galactosyltransferases Genes Genotype Glutamates Glycoconjugates Glycosphingolipids Golgi Apparatus Guide RNA Human Individual Induced pluripotent stem cell derived neurons Intellectual impairment Intractable Epilepsy Kinetics Knock-out Lead Length Link Lipids Liquid Chromatography Mass Spectrum Analysis Metabolic Diseases Microscopy Modeling Molecular Monitor Morphology Mosaicism Mus Mutation Neurites Neurons Oligodendroglia Organoids Parents Partial Epilepsies Pathogenicity Pathologic Pathology Pathway interactions Patients Phenotype Polysaccharides Population Process Production Protein Glycosylation Proteoglycan Refractory Resected Role Sampling Scanning Seizures Series Severities Severity of illness Somatic Mutation Specimen Supplementation Syndrome Therapeutic Time Translating Uridine Diphosphate Galactose Variant Western Blotting Work X Chromosome brain cell brain dysfunction brain tissue cell type clinical phenotype de novo mutation druggable target early onset effectiveness evaluation epileptic encephalopathies fetal genetic variant girls glycosylation hemimegalencephaly in utero induced pluripotent stem cell insertion/deletion mutation loss of function mRNA Expression migration mouse model multi-electrode arrays mutant neocortical nerve stem cell neural network neurodevelopment neurogenesis neuron development neuronal cell body novel offspring patch clamp precision medicine prevent protein transport radiological imaging relating to nervous system time use transcriptome sequencing

项目摘要

ABSTRACT Many epilepsy syndromes associated with severe, early-onset seizures result from de novo variants in genes involved in early brain development. Recent studies have also identified somatic variants in focal epilepsy associated with cortical malformations, including hemimegalencephaly and the more common focal cortical dysplasia (FCD) type 2. These post-zygotically acquired variants arise during neurogenesis and are therefore present in only a fraction of cells. Expanding on these early discoveries implicating somatic variants in epilepsy, we recently identified brain-specific somatic mutations in SLC35A2 in individuals with refractory neocortical epilepsy. Germline variants in SLC35A2 were previously implicated in a rare X-linked developmental and epileptic encephalopathy. Our data suggest that somatic variants in SLC35A2 may also be responsible for approximately 17% of intractable non-lesional focal epilepsy cases. The number of cells harboring a pathogenic SLC35A2 variant allele appears to correlate with disease severity, and several of the cases have FCD type 1a (FCD1a) pathology. Pathogenic variants in SLC35A2, both somatic and germline, prevent the Golgi-localized transporter from moving UDP-Galactose (UDP-Gal) into the Golgi apparatus for use in the formation of essential galactosylated glycans. There is theoretical, experimental, and observational data suggesting that Gal supplementation may be able to restore glycosylation to the cell to provide therapeutic benefit. In this study, we will define the functional consequences of SLC35A2 variants in epilepsy. Given that not all cells carry the variant allele in the individuals with a somatic SLC35A2 variant, in Aim 1 we seek to use resected human brain tissue specimens to identify the specific cell types in the brain harboring the variant alleles. This will allow us to determine which cell types contribute to SLC35A2 epilepsy and whether cell-type-specific burden dictates the pathological observations. In Aim 2 we will evaluate the effects of the variants on cell-type-specific glycosylation in human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells and mature glutamatergic neurons, a cell type that we have preliminary data to support involvement in the epileptogenic processes. Since nearly all patients with an SLC35A2 variant have seizures, and a significant fraction has FCD1a, in Aim 3 we will also characterize the effects of the variants on individual and neural network activity, neural migration, and neurodevelopment in both human (e.g., hiPSC-derived neurons, 3-D organoids) and mouse models (e.g., mouse neural progenitors and in utero electroporation). In Aims 2 and 3, we will assess the effectiveness of Gal to restore glycosylation and reverse effects on neuronal development or activity. Collectively, these studies will translate our exciting initial discovery implicating a novel pathway underlying a significant fraction of individuals suffering from intractable seizures into studies of the role of glycosylation defects underlying localized brain dysfunction in focal epilepsy. Given that the glycosylation pathway represents a potentially druggable target, this work may inform precision medicine approaches to the treatment of refractory epilepsy.

摘要许多与严重的早发性癫痫发作相关的癫痫综合征是由基因的新生变异引起的参与大脑的早期发育。最近的研究还确定了局灶性癫痫的体细胞变异与皮质畸形相关，包括半侧巨脑畸形和更常见的局灶性皮质畸形异型增生（FCD）2型。这些合子后获得的变异出现在神经发生过程中，只存在于一小部分细胞中。在这些早期发现的基础上，进一步探讨癫痫的体细胞变异，我们最近在难治性新皮层神经病患者中发现了SLC 35 A2的脑特异性体细胞突变，癫痫SLC 35 A2中的种系变异先前与罕见的X连锁发育和癫痫性脑病我们的数据表明，SLC 35 A2的体细胞变异也可能是导致约17%的难治性非病灶性局灶性癫痫病例。携带病原体的细胞数量 SLC 35 A2变异等位基因似乎与疾病严重程度相关，其中几例患者为FCD 1a型（FCD 1a）病理学。SLC 35 A2中的致病性变体，体细胞和生殖细胞，阻止高尔基体定位的将UDP-半乳糖（UDP-Gal）移动到高尔基体中用于形成必需的半乳糖基化聚糖。有理论、实验和观测数据表明，补充可能够恢复细胞的糖基化以提供治疗益处。本研究将定义SLC 35 A2变体在癫痫中的功能后果。鉴于并非所有细胞都携带这种变异在具有体细胞SLC 35 A2变体的个体中，在目的1中，我们寻求使用切除的人脑组织，标本，以确定特定的细胞类型，在大脑窝藏变异等位基因。这将使我们能够确定哪些细胞类型有助于SLC 35 A2癫痫，以及细胞类型特异性负荷是否决定了病理观察。在目标2中，我们将评估变体对细胞类型特异性糖基化的影响。在人诱导多能干细胞（hiPSC）衍生的神经祖细胞和成熟的神经胶质细胞中，神经元，我们有初步数据支持参与癫痫过程的细胞类型。以来几乎所有携带SLC 35 A2变异体的患者都有癫痫发作，并且很大一部分患有FCD 1a，在目标3中，我们将还描述了变异对个体和神经网络活动、神经迁移的影响，在两个人中的神经发育（例如，hiPSC衍生的神经元、3D类器官）和小鼠模型（例如，鼠标神经祖细胞和子宫内电穿孔）。在目标2和3中，我们将评估Gal的有效性，恢复糖基化并逆转对神经元发育或活性的影响。这些研究将将我们激动人心的初步发现转化为一种新的途径，患有顽固性癫痫发作的研究中的糖基化缺陷的作用，局灶性癫痫的功能障碍。考虑到糖基化途径代表了潜在的药物靶点，这项工作可能为治疗难治性癫痫的精确医学方法提供信息。