Defining disease mechanisms in SLC35A2 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从头变异引起的 参与早期的大脑发育。最近的研究也发现了局灶性癫痫的躯体变异。 与皮质畸形相关，包括半巨脑和更常见的局灶性皮质 发育不良(FCD)2型。这些合子后获得性变异发生在神经发生过程中，因此 只存在于一小部分细胞中。对这些早期发现涉及癫痫的体细胞变异进行了扩展， 我们最近在难治性新皮质患者中发现了SLC35A2的脑特异性体细胞突变 癫痫。SLC35A2的生殖系变异先前被认为与一种罕见的X连锁发育和 癫痫性脑病。我们的数据表明SLC35A2的体细胞变异也可能是 约17%的难治性非局灶性癫痫病例。携带致病病毒的细胞的数量 SLC35A2变异等位基因似乎与疾病严重程度相关，其中几个病例患有FCD1a型 (FCD1a)病理学。SLC35A2的致病变异，无论是体细胞还是生殖系，都阻止了高尔基体定位 从移动的UDP-半乳糖(UDP-Gal)到高尔基体的转运体用于形成必需的 半乳糖化的多聚糖。有理论、实验和观察数据表明，Gal 补充剂可能能够恢复细胞的糖基化，从而提供治疗益处。在这项研究中，我们 将定义SLC35A2变体在癫痫中的功能后果。鉴于并不是所有的细胞都携带该变体 SLC35A2体细胞变异个体的等位基因，在目标1中，我们试图使用切除的人脑组织 以确定大脑中含有变异等位基因的特定细胞类型。这将使我们能够 确定哪些细胞类型导致SLC35A2癫痫，以及特定细胞类型的负担是否决定了SLC35A2癫痫 病理观察。在目标2中，我们将评估这些变体对细胞类型特异性糖基化的影响。 人诱导多能干细胞来源的神经前体细胞和成熟谷氨酸能 神经元，一种我们有初步数据支持参与癫痫形成过程的细胞类型。自.以来 几乎所有SLC35A2变异患者都有癫痫发作，其中相当一部分患者患有FCD1a，在目标3中，我们将 还描述了变异对个体和神经网络活动、神经迁移和 人类(例如，HiPSC衍生的神经元、3-D有机体)和小鼠模型(例如，小鼠)的神经发育 神经前体细胞和宫内电穿孔)。在目标2和目标3中，我们将评估Gal to的有效性 恢复糖基化，逆转对神经元发育或活动的影响。总的来说，这些研究将 我们令人兴奋的初步发现暗示了一种新的途径，在相当一部分人的基础上 难治性癫痫发作对局部脑糖基化缺陷作用的研究 局灶性癫痫的功能障碍。鉴于糖基化途径代表了一个潜在的可用药靶点，这 这项工作可能会为治疗难治性癫痫的精确医学方法提供参考。

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.