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，以反复的自发性发作为特征，影响着全世界超过5000万人，是 最常见的神经系统疾病。电压门控钠通道基因SCN8A的突变 最近被确定为儿童严重癫痫的重要原因。随着下一代的到来 通过测序，已经在患者中发现了150多个致病性SCN8A突变。至少有20个这样的 突变位于S4电压传感结构域(VSD)中，该结构域对电压依赖门控至关重要 SCN8A通道蛋白，Nav1.6。室间隔缺损突变患者表现出广泛的临床症状 表型，包括智力残疾、共济失调步态、低眼压和难治性癫痫。一些人 患者也有轻微或没有癫痫发作，但有神经发育异常，如自闭症， 智力残疾和发育迟缓。为了更好地了解Scn8a VSD中的突变如何 引起如此广泛的临床表型，我们利用CRISPR/Cas9敲入人类SCN8A癫痫 突变为小鼠Scn8a DIIS4和DIVS4 VSD。这件事的一个意外但令人兴奋的后果 策略是在Scn8a：[ΔRVF]a中产生四个带有少量框内插入/缺失的小鼠品系 DIIS4缺失9bp，去除3个氨基酸残基(R848_F850del)，[849D]DIIS4插入3bp 这在DIVS4中引入了一个氨基酸残基(R848_V849_INSD)，[ΔVIR]一个9bp的缺失，删除了三个 氨基酸残基(V1616_R1618del)和[ΔIRL]在DIVS4中缺失9bp，去掉了三个氨基酸 残留物(I1617_L1619del)。由于大多数已发表的SCN8A突变都是错义突变，这些小鼠品系 将提供一个独特的机会来1)促进我们对临床相关疾病范围的理解 与SCN8A功能障碍相关的表型，2)进一步了解突变对 SCN8A功能上的VSD，以及3)识别与框内小的SCN8A插入/缺失相关的表型。 我们假设这种类型的SCN8A突变(微小的插入和缺失)将导致一系列癫痫发作 和行为表型。也有可能不同的表型可能与这些类型的 突变与点突变相比。与此一致，我们的初步数据表明 杂合子Δrvf突变体对诱发癫痫的抵抗力增强，而杂合子Δvir 突变体表现出更高的癫痫敏感性和自发性癫痫发作。此外，纯合子Δrvf 突变体表现出神经传导速度降低，而纯合子患者的神经传导速度没有改变 Scn8a基因缺失小鼠。因此，本R03提案的目标是描述和比较癫痫发作和行为 这四个Scn8a小鼠品系的表型。在目标1中，我们将确定每个突变对癫痫发作的影响。 敏感度。在目标2中，我们将确定每个突变对小鼠行为和运动协调的影响。 建议的实验与NIH小型研究资助计划的范围一致，因为它们是1) 可行的；2)自给自足的小型研究性研究。