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

癫痫，其特征在于反复自发性癫痫发作，影响全世界超过5000万人，并且是一种 最常见的神经系统疾病电压门控钠通道基因SCN 8A的突变， 最近被确定为严重小儿癫痫的重要原因。随着新一代 通过测序，已经在患者中鉴定出超过150种致病性SCN 8A突变。至少有二十个这样的 突变位于S4电压传感器结构域（VSD），这对电压依赖性门控是至关重要的。 SCN 8A通道蛋白，Nav1.6。VSD突变患者表现出广泛的临床表现， 表型，包括智力残疾、共济失调步态、张力减退和难治性癫痫。一些 患者还表现为轻度或无癫痫发作但具有神经发育异常如自闭症， 智力残疾和发育迟缓。为了更好地理解Scn 8a VSD中的突变如何 由于这种广泛的临床表型，我们利用CRISPR/Cas9敲入人SCN 8A癫痫 突变为小鼠Scn 8a DIIS 4和DIVS 4 VSD。一个意外但令人兴奋的结果是， 策略是产生在Scn 8a中具有小的框内插入/缺失的四个小鼠品系：[ΔRVF] a DIIS 4中删除3个氨基酸残基的9 bp缺失（R848_F850del），[849 D] DIIS 4中3bp插入 引入一个氨基酸残基（R848_V849_InsD），[ΔVIR]在DIVS 4中缺失9 bp， 氨基酸残基（V1616_R1618del），和[ΔIRL]在DIVS 4中去除三个氨基酸残基的9 bp缺失， 残基（I1617_L1619del）。由于大多数已发表的SCN 8A突变是错义突变，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SCN 8A功能障碍相关的表型，2）进一步了解突变对SCN 8A功能障碍的影响。 VSD对SCN 8A功能的影响，以及3）鉴定与小的框内SCN 8A插入/缺失相关的表型。 我们假设这类SCN 8A突变（小的插入和缺失）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。与此一致，我们的初步数据表明， 杂合ΔRVF突变体表现出对诱导癫痫发作的抵抗力增加，而杂合ΔVIR突变体表现出对诱导癫痫发作的抵抗力增加， 突变体表现出增加的癫痫发作易感性和自发性癫痫发作。此外，纯合子ΔRVF 突变体表现出降低的神经传导速度，而纯合子的神经传导速度没有改变， Scn 8a无效小鼠。因此，本R 03提案的目标是描述和比较癫痫发作和行为 这四种Scn 8a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。 易感性在目标2中，我们将确定每个突变对小鼠行为和运动协调的影响。 拟议的实验与NIH小型研究资助计划的范围一致，因为它们是1） 可行的，和2）小型独立的研究。

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.