Identification and molecular characterization of somatic mutations in MCD

项目摘要 引起疾病的基因突变可能是遗传的，在父母配子中新获得的，并存在于 受精卵，或在受精后发育的某个时刻作为体细胞事件获得（受精后）。的 任何获得性突变的负担和定位取决于突变何时出现。的情况一样 遗传的遗传变异，有越来越多的证据表明，体细胞突变可导致严重的组织- 具体疾病。皮质发育畸形（MCD）是一组特征为 大脑皮质的一系列形态和结构异常反映了胚胎发育的错误 皮质发育MCD与智力残疾以及难治性癫痫有关， 需要手术切除受影响的组织遗传性基因突变参与神经元发育 只能解释一小部分MCD病例。这些观察结果导致了一种假设，即某些MCD结果 来自神经胶质祖细胞中发生的体细胞突变引起异常皮质发育。在 事实上，我们和其他人已经表明，在半侧巨脑畸形中，一种严重的半球MCD亚型， 患者在PI 3 K-AKT-mTOR信号传导通路中具有体细胞突变，仅通过直接 研究异常皮层组织因此，在这个项目中，我们将评估体细胞突变的假设， 破坏胚胎皮质发育，并导致MCD的大部分。我们， 合作者网络，获得了大量切除的癫痫手术组织标本。使用 从异常脑中提取的DNA中的蛋白质编码区的高覆盖度下一代测序 组织和未受影响的组织（白细胞）从患有三种形式的MCD的患者，我们将确定体细胞 每个MCD患者体内的突变（Aim 1）。体细胞突变被证实选择性地存在于 将使用以下方法评估受影响组织导致皮质异常的可能性： 比较病例和对照之间的体细胞突变模式， 在同一基因中具有体细胞候选者的个体，病理异常的突变模式， 正常细胞群，并评估候选突变对大脑皮层的影响。 体内发育（目标2）。最后，初步描述疾病的影响和表现- 将使用体外测定来研究引起皮质突变的基因，以评估突变对 关键的皮质发育过程和突变谱系追踪，以确定携带 突变（目的3）。我们的项目汇集了临床，遗传学和神经生物学专业知识，并建立在 癫痫表型组/基因组计划（EPGP），Epi 4K和多个NINDS资助的计划在 体细胞遗传学这些研究将：（i）提供对体细胞突变作用的首次详细评估 在MCD亚型中，（ii）鉴定皮层发育的新基因/途径，和（iii）建立 并测试体外和体内模型，以了解基因在皮质发育中的作用。

PROJECT SUMMARY Genetic mutations causing disease may be inherited, newly acquired in parental gametes and present in the zygote, or acquired as somatic events at some point in development after fertilization (post-zygotic). The burden and localization of any acquired mutation depends on when the mutation arises. As is the case for inherited genetic variation, there is accumulating evidence that somatic mutations can lead to severe tissue- specific disease. Malformations of cortical development (MCD) represent a group of disorders characterized by a range of morphological and structural abnormalities of the cerebral cortex reflecting errors in embryonic cortical development. MCD are associated with intellectual disability, as well as refractory epilepsy, and may require the surgical removal of the affected tissue. Inherited gene mutations involved in neuronal development explain only a fraction of MCD cases. These observations have led to the hypothesis that some MCD result from somatic mutations occurring in neuroglial progenitor cells give rise to abnormal cortical development. In fact, we and others have shown that in hemimegalencephaly, a severe hemispheric MCD subtype, ~30% of patients have somatic mutations in the PI3K-AKT-mTOR signaling pathway, detectable only by directly studying abnormal cortical tissue. Thus, in this project we will evaluate the hypothesis that somatic mutations disrupt embryonic cortical development and are responsible for a substantial fraction of MCD. We, with a network of collaborators, have access to a large number of resected epilepsy surgical tissue specimens. Using high-coverage next-generation sequencing of protein-coding regions in DNA extracted from abnormal brain tissue and unaffected tissue (leukocytes) from patients with three forms of MCD, we will identify somatic mutations within each patient with MCD (Aim 1). Somatic mutations confirmed to be selectively present in affected tissue will be assessed for their likelihood of being responsible for the cortical abnormality using comparisons of patterns of somatic mutations between cases and controls, phenotypic comparisons between individuals with somatic candidates in the same gene, patterns of mutations in pathologically abnormal and normal cell populations, and evaluations of the effects of the candidate mutation on cerebral cortical development in vivo (Aim 2). Finally, initial characterization of the effects and presentation of the disease- causing mutation in the cortex will be studied using in vitro assays to evaluate the effects of the mutation on key cortical developmental processes, and mutation lineage tracing to identify cell populations carrying the mutation (Aim 3). Our project brings together clinical, genetic, and neurobiological expertise and builds on the activities of the Epilepsy Phenome/Genome Project (EPGP), Epi4K, and multiple NINDS-funded initiatives in somatic genetics. These studies will: (i) provide the first detailed assessment of the role of somatic mutations across MCD subtypes, (ii) identify novel genes/pathways underlying cortical development, and (iii) establish and test in vitro and in vivo models to understand the role of genes in cortical development.