Identification and molecular characterization of somatic mutations in MCD

项目总结 致病的基因突变可能是遗传的，新获得的亲本配子，并存在于 受精卵，或在受精后的某个发育阶段获得的。职位的负担和本地化- 合子获得性突变取决于突变发生的时间。皮质发育畸形 (MCD)是一组以一系列的形态和结构异常为特征的疾病。 大脑皮层反映胚胎皮质发育的错误。MCD与耐火材料有关 癫痫以及智力残疾，可能需要手术切除受影响的组织以进行癫痫发作 控制力。越来越多的人认识到，受精卵后获得的体细胞突变发生在神经胶质细胞中 祖细胞可能会导致大脑皮质畸形。在这笔赠款的上一个资助期，我们 在鉴定和分子表征一系列合子后致病病毒方面取得了重大进展 癫痫相关皮质脑患者切除的脑组织中的获得性体细胞变异 畸形。最值得注意的是，我们鉴定了第一个与焦点相关的基因并对其进行了功能鉴定 皮质发育不良I型，SLC35A2，并在体细胞的理解方面取得了重大进展 横跨MCD的遗传景观。下一个供资周期的总体目标是继续确定 MCD上的体细胞变异并在功能上表征与以下相关的新变异的影响 不同类型的MCD对皮质发育的影响。在目标1中，我们将继续收集切除的脑组织 来自MCD患者的样本，用于高深度外显子组或靶向基因测序。目标1的目标是 识别与MCD相关的新基因，并确定外显子组阴性病例的子集，以用于AIM 2。 在目标2中，我们将使用高灵敏度的双重测序来检测极低水平的体细胞变异和不含聚合酶链式反应的 全基因组测序检测体细胞短串联重复序列变异体和中等大小的体细胞 外显子阴性的MCD病例中的拷贝数变异。目标2的目标是确定 由于标准短读外显子组的限制而经常遗漏的这些体细胞变异体 MCD总体躯体遗传风险的测序。最后，目标3将评估功能后果 敲除神经元上新发现的含有躯体功能丧失变体的MCD基因 发育中小鼠大脑的形态、神经元迁移以及最终的大脑皮层发育。 这些研究将：(I)继续深入评估体细胞突变在MCD中的作用 亚型，(Ii)识别涉及皮质发育的新基因/途径，(Iii)使用体外互补， 体外和体内模型，以了解涉及皮质发育的新基因的作用，以及(Iv) 建立未来可用于化合物筛选的生物标志物和平台，以可能 预防或改善MCD的预后。

PROJECT SUMMARY Genetic mutations causing disease may be inherited, newly acquired in parental gametes and present in the zygote, or acquired at some point in development after fertilization. The burden and localization of a post- zygotically acquired mutation depends on when the mutation arises. Malformations of cortical development (MCD) are 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 refractory epilepsy as well as intellectual disability and may require the surgical removal of the affected tissue for seizure control. There is increasing recognition that post-zygotically acquired somatic mutations occurring in neuroglial progenitor cells can result in a cortical brain malformation. In the previous funding period of this grant, we made significant progress identifying and molecularly characterizing a series of pathogenic post-zygotically acquired somatic variants in the resected brain tissue of individuals with an epilepsy-associated cortical brain malformation. Most notably we identified and functionally characterized the first gene associated with focal cortical dysplasia type I, SLC35A2, and made significant advancements in the understanding of the somatic genetic landscape across MCD. The overarching objective for the next funding cycle is to continue to identify somatic variants across MCD and to functionally characterize the effects of novel variants associated with different types of MCD on cortical development. In Aim 1, we will continue to collect resected brain tissue specimens from individuals with MCD for high-depth exome or targeted gene sequencing. The goal of Aim 1 is to identify novel genes involved in MCD and to ascertain the subset of exome-negative cases for use in Aim 2. In Aim 2, we will use highly sensitive duplex sequencing to detect very low-level somatic variants and PCR-free whole-genome sequencing to detect somatic short tandem repeat variants and intermediately sized somatic copy number variants in exome-negative MCD cases. The goal of Aim 2 is to determine the contribution of these classes of somatic variants that are routinely missed due to the limitations of standard short-read exome sequencing in the overall somatic genetic risk of MCD. Finally, Aim 3 will evaluate the functional consequences of knocking out newly identified MCD genes harboring somatic loss-of-function variants on neuronal morphology, neuronal migration, and ultimately cerebral cortical development in the developing mouse brain. These studies will: (i) continue our in-depth assessment of the role of somatic mutations across MCD subtypes, (ii) identify novel genes/pathways involved in cortical development, (iii) use complementary in vitro, ex vivo, and in vivo models to understand the role of novel genes implicated cortical development, and (iv) establish biomarkers and platforms that can be used in the future for screening of compounds to possibly prevent or improve the prognosis of MCD.