Exploring the range of seizure and behavioral phenotypes due to SCN8A mutations

探索 SCN8A 突变引起的癫痫发作和行为表型的范围

• 批准号: 9978424
• 负责人: Andrew P Escayg
• 金额: $ 15.07万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978424
• 关键词: Affect; Agonist; Amino Acids; Ataxia; Ataxic Gait; Behavior; Behavioral Assay; CRISPR/Cas technology; Clinical; Data; Development; Developmental Delay Disorders; Disease; Dyskinetic syndrome; Electroencephalography; Epilepsy; Exhibits; Exposure to; Flurothyl; Frequencies; Functional disorder; Gene Family; Generations; Genes; Genetic Variation; Glutamate Receptor; Goals; Heterozygote; Human; Individual; Intellectual functioning disability; Intractable Epilepsy; Kainic Acid; Knock-in; Knockout Mice; Knowledge; Lead; Longevity; Missense Mutation; Motor; Mus; Muscle hypotonia; Mutate; Mutation; Neural Conduction; Neuraxis; Neurons; Pathogenicity; Patients; Phenotype; Play; Point Mutation; Positioning Attribute; Predisposition; Proteins; Publishing; Recurrence; Reporting; Research Project Grants; Resistance; Role; SCN8A gene; Seizures; Series; Severities; Sodium Channel; Tremor; United States National Institutes of Health; autism spectrum disorder; autistic behaviour; behavioral phenotyping; childhood epilepsy; clinical phenotype; clinically relevant; comorbidity; experimental study; improved; insertion/deletion mutation; motor disorder; motor neuron function; mouse model; mutant; nervous system disorder; neuronal excitability; next generation sequencing; programs; receptor; research study; sensor; voltage

Epilepsy, characterized by recurrent spontaneous seizures, affects over 50 million people worldwide and is one of the most common neurological disorders. Mutations in the voltage-gated sodium channel gene, SCN8A, have recently been identified as an important cause of severe pediatric epilepsy. With the advent of next-generation sequencing, over 150 pathogenic SCN8A mutations have been identified in patients. At least twenty of these mutations are located in the S4 voltage-sensor domains (VSDs) that are critical for voltage-dependent gating of the SCN8A channel protein, Nav1.6. Patients with VSD mutations exhibit a broad spectrum of clinical phenotypes, including intellectual disability, ataxic gait, hypotonia, and treatment-resistant epilepsy. Some patients also present with mild or no seizures but with neurodevelopmental abnormalities such as autism, intellectual disability and developmental delay. To better understand how mutations in the Scn8a VSDs can cause this broad range of clinical phenotypes, we utilized CRISPR/Cas9 to knock-in human SCN8A epilepsy mutations into the mouse Scn8a DIIS4 and DIVS4 VSDs. One unplanned but exciting consequence of this strategy was the generation of four mouse lines with small in-frame insertions/deletions in Scn8a: [ΔRVF] a 9bp deletion in DIIS4 that removes three amino acid residues (R848_F850del), [849D] a 3bp insertion in DIIS4 that introduces one amino acid residue (R848_V849_InsD), [ΔVIR] a 9bp deletion in DIVS4 that removes three amino acid residues (V1616_R1618del), and [ΔIRL] a 9bp deletion in DIVS4 that removes three amino acid residues (I1617_L1619del). Since most published SCN8A mutations are missense mutations, these mouse lines will provide a unique opportunity to 1) contribute to our understanding of the range of clinically-relevant phenotypes associated with SCN8A dysfunction, 2) further our understanding of the impact of mutations in the VSD on SCN8A function, and 3) identify phenotypes associated with small in-frame SCN8A insertions/deletions. We hypothesize that this class of SCN8A mutations (small insertions and deletions) will lead to a range of seizure and behavioral phenotypes. It is also possible that distinct phenotypes might be associated with these types of mutations when compared to point mutations. Consistent with this, our preliminary data demonstrate that heterozygous ΔRVF mutants exhibit increased resistance to induced seizures, whereas heterozygous ΔVIR mutants exhibit increased seizure susceptibility and spontaneous seizures. In addition, homozygous ΔRVF mutants exhibit reduced nerve conduction velocity, while nerve conduction velocity was unaltered in homozygous Scn8a null mice. Thus, the goal of this R03 proposal is to characterize and compare the seizure and behavioral phenotypes of these four Scn8a mouse lines. In Aim 1, we will establish the effect of each mutation on seizure susceptibility. In Aim 2, we will determine the effect of each mutation on mouse behavior and motor coordination. The proposed experiments align with the scope of the NIH Small Research Grant Program in that they are 1) feasible, and 2) small self-contained, research studies.

癫痫以反复发作的自发性癫痫发作为特征，影响着全世界超过 5000 万人，是一种 电压门控钠通道基因 SCN8A 发生突变。 随着下一代的出现，最近被确定为严重小儿癫痫的重要原因。 通过测序，已在患者中发现了超过 150 种致病性 SCN8A 突变，其中至少有 20 种。 突变位于 S4 电压传感器域 (VSD)，这对于电压依赖性门控至关重要 SCN8A 通道蛋白 Nav1.6 突变的患者表现出广泛的临床症状。 表型，包括智力障碍、共济失调步态、肌张力低下和难治性癫痫。 患者也有轻度或无癫痫发作，但有神经发育异常，如自闭症、 更好地了解 Scn8a VSD 突变如何影响智力障碍和发育迟缓。 导致如此广泛的临床表型，我们利用 CRISPR/Cas9 敲入人类 SCN8A 癫痫 小鼠 Scn8a DIIS4 和 DIVS4 VSD 发生突变，这是一个意想不到但令人兴奋的结果。 策略是在 Scn8a 中生成四个具有小框内插入/删除的小鼠品系：[ΔRVF] a DIIS4 中的 9bp 删除去除了三个氨基酸残基 (R848_F850del)，[849D] DIIS4 中的 3bp 插入 引入一个氨基酸残基 (R848_V849_InsD)，[ΔVIR] DIVS4 中的 9bp 删除，删除了三个 氨基酸残基 (V1616_R1618del)，以及 [ΔIRL] DIVS4 中的 9bp 删除，删除了三个氨基酸 由于大多数已发表的 SCN8A 突变都是错义突变，因此这些小鼠品系。 将提供一个独特的机会：1）有助于我们了解临床相关的范围 与 SCN8A 功能障碍相关的表型，2）进一步加深了我们对突变影响的理解 SCN8A 功能上的 VSD，以及 3) 识别与小型框内 SCN8A 插入/删除相关的表型。 我们发现此类 SCN8A 突变（小插入和缺失）将导致一系列癫痫发作 和行为表型也可能与这些类型相关。 与点突变相比，我们的初步数据表明。 杂合子 ΔRVF 突变体表现出对诱导癫痫发作的抵抗力增强，而杂合子 ΔVIR 此外，纯合子 ΔRVF 突变体表现出癫痫易感性和自发性癫痫发作增加。 突变体表现出神经传导速度降低，而纯合子中神经传导速度没有改变 因此，R03 提案的目标是表征和比较癫痫发作和行为。 这四种 Scn8a 小鼠品系的表型 在目标 1 中，我们将确定每种突变对癫痫发作的影响。 在目标 2 中，我们将确定每种突变对小鼠行为和运动协调的影响。 拟议的实验符合 NIH 小型研究资助计划的范围，因为它们是 1) 可行，以及 2) 小型独立研究。