Defining disease mechanisms in SLC35A2 epilepsy

定义 SLC35A2 癫痫的疾病机制

基本信息

批准号：
10191063
负责人：
Peter B Crino
金额：
$ 68.96万
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/10191063
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 Diagnostic radiologic examination 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 Immunoblotting Impairment Individual Induced pluripotent stem cell derived neurons 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 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 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.

抽象的许多与重度早发作相关的癫痫综合征是由基因的从头变异引起的参与早期大脑发育。最近的研究还确定了局灶性癫痫中的体细胞变异与皮质畸形有关，包括半脑畸形和更常见的局灶性皮质异常增生（FCD）2型。这些后杂合的变体在神经发生过程中出现，因此是仅存在于一小部分细胞中。扩展这些早期发现，暗示癫痫中的躯体变体，我们最近在患有新皮质的个体中发现了SLC35A2中脑特异性的体细胞突变癫痫。 SLC35A2中的种系变体以前与罕见的X连锁发育和癫痫性脑病。我们的数据表明，SLC35A2中的体细胞变体也可能负责约有17％的顽固性非局灶性癫痫病例。具有致病性的细胞数量 SLC35A2变体等位基因似乎与疾病的严重程度相关，其中一些病例具有FCD 1A型（FCD1A）病理学。 SLC35A2中的致病变异，包括体细胞和种系，防止高尔基体定位从移动的UDP-半乳糖（UDP-GAL）到高尔基体的转运蛋白，用于形成必需品半乳糖基化的糖。有理论，实验和观察数据表明GAL 补充可能能够将糖基化恢复到细胞上以提供治疗益处。在这项研究中，我们将定义癫痫中SLC35A2变体的功能后果。鉴于并非所有细胞都带有变体具有体细胞SLC35A2变体的个体的等位基因，在AIM 1中，我们寻求使用切除的人脑组织标本以识别具有变体等位基因的大脑中的特定细胞类型。这将使我们能够确定哪种细胞类型有助于SLC35A2癫痫以及细胞型特异性负担是否决定病理观察。在AIM 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类诱导的多能干细胞（HIPSC）衍生的神经祖细胞和成熟的谷氨酸能神经元，我们具有初步数据的细胞类型，以支持参与癫痫发作。自从几乎所有患有SLC35A2变体的患者都有癫痫发作，而很大的部分具有FCD1A，在AIM 3中，我们将还表征了变体对个体和神经网络活动，神经迁移以及人（例如，hipsc衍生的神经元，3-D器官）和小鼠模型（例如，小鼠）的神经发育神经祖细胞和子宫电穿孔）。在目标2和3中，我们将评估GAL的有效性恢复对神经元发育或活性的糖基化和反向影响。这些研究总的来说翻译我们令人兴奋的最初发现，暗示了大量个人的新途径在局部大脑基础的糖基化缺陷的作用研究中遭受了棘手的癫痫发作局灶性癫痫功能障碍。鉴于糖基化途径代表了一个潜在的可药物靶标，这工作可以为治疗难治性癫痫的治疗方法提供精确的医学方法。