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.

癫痫病以复发赞助商为特征，在全球范围内影响超过5000万人 最常见的神经系统疾病。电压门控钠通道基因SCN8A中的突变具有 最近被确定为严重小儿癫痫的重要原因。随着下一代冒险 在患者中已经确定了超过150种致病性SCN8A突变的测序。其中至少二十个 突变位于S4电压传感器结构域（VSD）中，这对于电压依赖性门控至关重要 SCN8A通道蛋白，NAV1.6。 VSD突变的患者暴露了一系列临床 表型，包括智力障碍，共同步态，低骨和耐药性癫痫。一些 患者还出现轻度或没有癫痫发作，但具有神经发育异常，例如自闭症， 智力残疾和发育延迟。更好地了解SCN8A VSD中的突变如何可以 由于这种广泛的临床表型，我们利用CRISPR/CAS9来敲击人类SCN8A癫痫 突变到小鼠SCN8A DIIS4和DIVS4 VSD中。一个计划外的但令人兴奋的结果 策略是在SCN8A中产生的四个小鼠线插入/缺失：[ΔRVF] a DIIS4中的9BP删除删除了三个氨基酸残基（R848_F850DEL），[849d] DIIS4中的3BP插入 这引入了一个氨基酸住宅（R848_V849_INSD），[ΔVIR] DIVS4中的9bp删除，可去除三个 氨基酸残基（V1616_R1618DEL）和[ΔIrl] DIVS4中的9bp缺失，该删除去除三个氨基酸 残留物（I1617_L1619DEL）。由于大多数已发表的SCN8A突变是错义突变，因此这些小鼠线 将为我们提供独特的机会1）为我们理解与临床相关的范围做出贡献 与SCN8A功能障碍相关的表型，2）进一步的理解 SCN8A函数上的VSD和3）识别与小框架内SCN8A插入/缺失相关的表型。 我们假设这类SCN8A突变（小插入和缺失）将导致一系列癫痫发作 和行为表型。不同的表型也可能与这些类型的 与点突变相比，突变。与此一致，我们的初步数据表明 杂合ΔRVF突变体暴露了对诱导性癫痫发作的抗性增加，而杂合ΔVir 突变体暴露于癫痫发作的敏感性增加并赞助癫痫发作。另外，纯合ΔRVF 突变体暴露了降低神经传导速度，而神经传导速度在纯合中未改变 SCN8A无效老鼠。这是该R03提案的目标是表征和比较癫痫发作和行为 这四种SCN8A小鼠系的表型。在AIM 1中，我们将建立每个突变对癫痫发作的影响 敏感性。在AIM 2中，我们将确定每个突变对小鼠行为和运动配位的影响。 提出的实验与NIH小型研究赠款计划的范围保持一致，因为它们是1） 可行的，以及2）小型独立研究研究。