Rare genetic factors in epileptogenesis

导致遗传性全身性癫痫（GGE）的因素在很大程度上是未知的，已知的因素只能解释其高遗传性的一小部分。可能的解释是大量尚未确定的贡献罕见变异和更常见的风险赋予变异。项目P3中讨论了常见变量。在这里，我们将运行的基因发现的罕见变异的完整范围，从分析最大的组装收集的外显子组序列- 6400 GGE和3200癫痫性脑病（EE）的情况下-功能解释癫痫的背景下。为此，我们将使用广泛的方法，从组学数据建模到细胞和动物模型中基因和变体的验证。首先，来自全球合作的全外显子组序列的样本量大幅增加将使我们能够从无假设分析中识别新的变体。罕见变异的识别将通过最先进的统计模型作为负荷或崩溃检验进行。癫痫的异质性无疑是导致基因发现失败的原因，负荷试验不能利用表型严重程度的信息。因此，我们将探索多效性模型，用于分析GGE解决表型细节，但也包括EE以及与GGE相比的局灶性癫痫（FE）。其次，我们将通过整合蛋白质-蛋白质相互作用、基因表达和遗传变异的富集来寻找GGE中介导癫痫发生的基因网络。来自单细胞RNA-seq实验的新数据将使我们能够对单个细胞类型进行变异解释。在系统生物学方法中，将与其他项目合作验证此类网络的预测关键基因，例如斑马鱼。第三，将在细胞和小鼠模型中进行遗传变异的生理测试，以完成我们对其影响的理解。非洲爪蟾卵母细胞的自动筛选和哺乳动物细胞系的膜片钳研究将确定离子通道和受体变异的生物物理后果。将研究尚未参与GGE的基因中的因果变异，如KCNA 2，KCNQ 5和KCND 2。我们产生的在GABRA 5中携带人GGE相关突变的敲入（KI）小鼠将允许通过与其他癫痫小鼠模型杂交来评估修饰效应。通过这种多方面的协调一致的方式，该项目将导致识别新的和经验证的易感性变体，并对GGE癫痫发生进行深入的定性了解。

The factors contributing to genetic generalized epilepsies (GGE) are largely uncharted and known factors explain only a minor component of its high heritability. Possible explanations are large numbers of not yet identified contributing rare variants and also more common risk-conferring variants. Common variants are addressed in project P3. Here, we will run the complete gamut of gene discovery for rare variants, from analysing the largest assembled collection of exome sequences – 6400 GGE and 3200 epileptic encephalopathies (EE) cases – to functional interpretation in the context of epileptogenesis. To this end, we will use a broad spectrum of methods, from omics data modeling to validation of genes and variants in cellular and animal models. Firstly, substantially increased sample sizes of whole-exome sequences from global collaborations will put us in a position to identify new variants from hypothesis-free analyses. The identification of rare variants will be run by state-of-the-art statistical models as burden or collapsing tests. The heterogeneity of epilepsies is undoubtedly contributing to prior failures in gene discovery and burden tests are not able to utilize information on the severity of the phenotype. We will therefore explore pleiotropic models for analysing GGE addressing phenotype details but also including EE as well as focal epilepsies (FE) in comparison to GGE. Secondly, we will search for gene networks mediating epileptogenesis in GGE by integration of protein-protein interactions, gene expression and enrichment of genetic variants. New data from single-cell RNA-seq experiments will allow us to take variant interpretation to individual cell types. In a systems biology approach, predicted key genes of such networks will be validated in cooperation with other projects, e.g. in zebrafish. Thirdly, physiological testing of genetic variants in cellular and mouse models will be performed to complete our understanding of their impact. Automated screening in Xenopus oocytes and patch clamp studies in mammalian cell lines will identify biophysical consequences of variants in ion channels and receptors. Causal variants in genes not yet involved in GGE, such as KCNA2, KCNQ5, and KCND2, will be investigated. A knock-in (KI) mouse which we generated carrying a human GGE-associated mutation in GABRA5 will allow assessing modifying effects by cross-breeding with other epilepsy mouse models. In this multifaceted, concerted way, the project will lead to the identification of new and validated susceptibility variants and an in-depth qualitative understanding of GGE epileptogenesis.