Genetic Modifiers of Childhood Epilepsy

癫痫是一种常见的神经系统疾病，每26个美国人中就有1个会在一生中受到影响。SCN 1A突变， 编码神经元电压门控钠通道Nav1.1，是癫痫最常见的遗传原因。 在不同严重程度的癫痫患者中报告了超过1600种SCN 1A突变，范围从 Dravet综合征是一种严重的婴儿发作性癫痫性脑病， 杂合性功能丧失突变。Dravet综合征的特征是多种癫痫发作类型， 发育迟缓和死亡风险升高。单基因癫痫的一个共同特点是可变的 在携带相同突变的个体中的表达率，表明临床严重程度受遗传因素的影响。 修饰语Scn 1a杂合缺失（Scn 1a +/-）小鼠重现Dravet综合征的核心特征 表型，包括自发性癫痫发作和死亡风险增加。Scn 1a的缺失导致 海马GABA能中间神经元中的钠电流，导致抑制和兴奋/抑制失败 大脑的不平衡。Scn 1a +/-小鼠的表型严重程度强烈依赖于品系背景。Scn1a+/- 耐药129品系（129.Scn1a+/-）的小鼠没有明显的表型，并且寿命正常。与此相反， [129 xB 6]F1品系（F1.Scn1a+/-）的Scn 1a +/-小鼠表现出自发性癫痫发作和过早致死， 50%在1个月大时死亡。应变依赖性差异在神经元亚型水平上也很明显。 从易感的F1.Scn1a+/-小鼠分离的GABA能中间神经元表现出降低的钠电流密度 与野生型同窝仔相比，虽然钠电流密度在分离自 129.Scn1a+/-相对于野生型同窝仔。这表明，来自菌株129的中间神经元补偿了 Nav1.1缺失，而F1中间神经元没有。基于表型的菌株依赖性差异， 在整个动物和细胞水平上，我们假设遗传修饰剂影响Scn 1a +/-表型的严重程度， 由于在Scn 1a杂合子的情况下神经元亚型之间的代偿能力的差异， 删除。我们以前绘制了几个Dravet生存修饰因子（Dsm）基因座，这些基因座影响了早产儿的致死率。 Scn 1a +/-小鼠。在当前的建议中，我们将在三个目标中解决我们的假设。第一，我们会好好表现 在小鼠7号和8号染色体上的两个Dsm基因座的定位和候选基因分析。第二，我们将执行 单细胞RNA-seq分析以表征特定细胞中细胞组成和基因表达差异 癫痫易感F1.Scn1a+/-和耐药F1.Scn1a+/-的表型发作关键期亚群 129.Scn1a+/-小鼠。第三，我们将评估候选基因在体内的修饰潜力，使用转录本。 调节以上调和下调候选基因表达。这些研究的结果将确定改性剂 影响Scn 1a +/-小鼠表型严重程度的基因和途径。鉴定修饰基因， 影响Dravet综合征的严重程度将提供对癫痫病理生理学的深入了解，并将建议 用于改善人类患者治疗的新治疗方法。

Epilepsy is a common neurological that will affect 1 in 26 Americans during their lifetime. Mutations in SCN1A, encoding the neuronal voltage-gated sodium channel Nav1.1, are the most common genetic cause of epilepsy. Over 1600 SCN1A mutations have been reported in individuals with epilepsy of varying severity, ranging from mild febrile seizures to Dravet syndrome, a severe infant-onset epileptic encephalopathy caused by heterozygous loss-of-function mutations. Dravet syndrome is characterized by a variety of seizure types, developmental delay and elevated mortality risk. A common feature of monogenic epilepsies is variable expressivity in individuals carrying the same mutation, suggesting that clinical severity is influenced by genetic modifiers. Mice with heterozygous deletion of Scn1a (Scn1a+/-) recapitulate core features of Dravet syndrome phenotypes, including spontaneous seizures and increased mortality risk. Loss of Scn1a results in reduced sodium current in hippocampal GABAergic interneurons, resulting in failure of inhibition and excitatory/inhibitory imbalance in the brain. Phenotype severity in Scn1a+/- mice is strongly dependent on strain background. Scn1a+/- mice on the resistant 129 strain (129.Scn1a+/-) have no overt phenotype and live a normal lifespan. In contrast, Scn1a+/- mice on a [129xB6]F1 strain (F1.Scn1a+/-) exhibit spontaneous seizures and premature lethality, with 50% dying by 1 month of age. Strain-dependent differences are also evident at the level of neuron subtypes. GABAergic interneurons isolated from the susceptible F1.Scn1a+/- mice exhibit decreased sodium current density compared to wildtype littermates, while sodium current density is preserved in interneurons isolated from 129.Scn1a+/- relative to wildtype littermates. This suggests that interneurons from strain 129 compensate for the loss of Nav1.1, while F1 interneurons do not. Based on the strain-dependent difference in phenotypes at the whole animal and cellular levels, we hypothesize that genetic modifiers influence Scn1a+/- phenotype severity due to differences in compensatory capacity among neuronal subtypes in the context of Scn1a heterozygous deletion. We previously mapped several Dravet survival modifier (Dsm) loci that influence premature lethality of Scn1a+/- mice. In the current proposal, we will address our hypothesis in three aims. First, we will perform fine mapping and candidate gene analysis at two Dsm loci on mouse chromosomes 7 and 8. Second, we will perform single cell RNA-seq analysis to characterize differences in cell composition and gene expression in specific cell subpopulations during the critical phase of phenotype onset in epilepsy susceptible F1.Scn1a+/- and resistant 129.Scn1a+/- mice. Third, we will evaluate the modifier potential of candidate genes in vivo using transcriptional modulation to up- and down-regulate candidate gene expression. Results from these studies will identify modifier genes and pathways that influence phenotype severity in Scn1a+/- mice. Identification of modifier genes that influence severity of Dravet syndrome will provide insight into the pathophysiology of epilepsy and will suggest novel therapeutic approaches for improved treatment of human patients.