PCDH19-related Epilepsy-Human Genotype-Phenotype Studies Lead to Zebrafish Studies

癫痫是一种常见的疾病，每26个人中就有一个受到影响，对于这一点，遗传学的作用是众所周知的。 自2008年发现以来，PCDH19一直是导致癫痫的最突出的单基因原因之一。 PCDH19的突变导致X连锁的“女性局限性癫痫”，伴随着难治性的儿童期癫痫发作， 智力残疾和自闭症。PCDH19编码一种中枢神经系统原钙粘附素，预测 介导细胞黏附，但其在神经发育中的作用尚未确定。PCDH19突变可以从头开始 或者遗传自受轻微影响/未受影响的母亲，或者奇怪的是，来自未受影响的父亲。女性 这种优势被认为是“专制的马赛克主义”造成的。罕见的有症状的马赛克雄性病例报告 与这一假设是一致的。然而，最近注意到“携带者”父亲的微妙行为特征 提示并非所有的表型表现都需要马赛克状态。我们将使用这些解决方案 以异常遗传学为线索，PCDH19的功能作用及其在癫痫中的作用。我们将进行基因分型- 表型研究以了解PCDH19-突变位置、类型和遗传的重要性 相关功能障碍。然后我们将利用一组精心挑选的人类PCDH19突变，来自 我们建立的PCDH19注册表，变成了斑马鱼机械模型。 目前还没有建立PCDH19诱发癫痫的动物模型。斑马鱼代表着一种 建立了具有遗传可控性和可识别癫痫发作的脊椎动物模型系统。我们的初步数据 来自CRISPR/Cas9基因组编辑的斑马鱼表明，pcdh19功能的丧失会导致癫痫发作。使用 这些模型，我们将研究所涉及的细胞类型和丢失或改变的具体机制 PCDH19会导致疾病。我们推测PCDH19突变的位置和类型与 人类的表型严重性以及基于人类突变的斑马鱼模型将显示 癫痫发作。因此，我们寻求一种新的方法来研究PCDH19功能障碍的机制。 基于我们通过我们的登记处积累的突变的模型。我们将致力于将 PCDH19相关表型的严重性与患者突变的位置和类型；建立PCDH19斑马鱼 基于人类突变的模型，并表征其癫痫表型；并确定 突变pcdh19斑马鱼对细胞迁移、黏附和兴奋性的影响。 我们的研究将建立首个PCDH19相关癫痫的活体动物模型。通过我们的斑马鱼 模型，我们将研究PCDH19的神经发育作用，并开发PCDH19的药物筛选- 与癫痫相关的精准医学时代正在兴起。这项研究的更广泛的影响是，它将(1) 提供对原钙粘附素相关癫痫的洞察，(2)建立人到斑马鱼的范例 遗传性癫痫的翻译研究，以及(3)为其他马赛克神经疾病的建模提供信息。

Epilepsy is a common condition, affecting 1 in 26 individuals, for which the role of genetics is well recognized. Since its discovery in 2008, PCDH19 has been amongst the most prominent single gene causes of epilepsy. Mutations in PCDH19 cause X-linked “female limited epilepsy,” with refractory childhood-onset seizures, intellectual disability, and autism. PCDH19 encodes a central nervous system protocadherin predicted to mediate cell adhesion, but its role in neurodevelopment is not established. PCDH19 mutations can be de novo or inherited from mildly affected/unaffected mothers or, curiously, unaffected fathers. The female predominance is hypothesized to be due to ‘obligate mosaicism.’ Reports of rare symptomatic mosaic males are consistent with this hypothesis. However, recently noted subtle behavioral features in ‘carrier’ fathers suggest that a mosaic state is not required for all phenotypic manifestations. We will address, using these unusual genetics as a clue, the functional role of PCDH19 and its role in epilepsy. We will conduct genotype- phenotype studies to understand the importance of mutation location, type, and inheritance on PCDH19- related dysfunction. We will then harness a carefully curated set of human PCDH19 mutations, from the PCDH19 Registry that we founded, into zebrafish mechanistic models. There are currently no established animal models of PCDH19-induced epilepsy. Zebrafish represent an established vertebrate model system with genetic tractability and identifiable seizures. Our preliminary data from CRISPR/Cas9 genome-edited zebrafish suggest that loss of function of pcdh19 results in seizures. With these models, we will study the cell types involved and the specific mechanisms by which loss or alteration of pcdh19 results in disease. We hypothesize that the location and type of PCDH19 mutation correlates with phenotypic severity in humans and that zebrafish models based on human mutations will display seizures. We thus pursue a novel approach to study the mechanisms involved in PCDH19 dysfunction, with models based on mutations that we have accrued through our Registry. We will aim to correlate the severity of PCDH19-related phenotypes with location and type of patient mutations; establish pcdh19 zebrafish models based on human mutations and characterize their epilepsy phenotypes; and identify the effects of mutations on cell migration, adhesion, and excitability in mutant pcdh19 zebrafish. Our research will develop the first in vivo animal models for PCDH19-related epilepsy. Through our zebrafish models, we will investigate the neurodevelopmental role of PCDH19 and develop drug screens for PCDH19- related epilepsy in the emerging era of precision medicine. The broader impacts of this study are that it will (1) provide insight into protocadherin-related epilepsies, (2) establish a human-to-zebrafish paradigm for translational research in genetic epilepsy, and (3) inform the modeling of other mosaic neurological disorders.