Deciphering brain mosaicism in drug-resistant epilepsy at cellular resolution

项目概要项目概要 虽然体细胞嵌合现象是一个已知的原因畸形的皮层发育（MCD），它仍然是困难的 检测患者组织中的稀疏体细胞变异并研究其分子和功能效应。很长的- 本项目的长期目标是揭示MCD的细胞和分子复杂性，并将这些知识转化为 为患者提供更好的诊断和治疗机会。本申请的总体目标是 (i)改进体细胞变体检测，（ii）鉴定体细胞变体的转录相关物，和（iii）测试 体细胞变体在相关细胞类型中的功能效应。核心假设是体细胞变异是 在MCD中富集特定的细胞谱系，并且细胞身份是变异的重要决定因素。 分子和功能效应。这个项目的基本原理是，了解体细胞变异及其 细胞类型分辨率的结果将最终改善MCD的基因发现，并有助于推动该领域的发展 与MCD相关的耐药性癫痫的靶向治疗。中心假设将被检验 通过追求三个具体目标：（1）改善局灶性皮质骨疾病患者的体细胞变异检测 畸形，（2）评估体细胞突变的细胞类型特异性转录效应，以及（3）功能性 验证体细胞突变对疾病特征的细胞类型特异性贡献。在第一个目标下， 细胞类型将从受影响的患者组织中富集并测序，作为改善体细胞变异的策略 侦测对于第二个目标，整合患者组织的单核基因分型和转录组学分析 将用于评估体细胞嵌合现象的基因表达特征。第三个目标将利用一部小说 MCD转基因小鼠模型，以驱动特定细胞类型的体细胞变异，并确定细胞特性 导致疾病的特定成分。本申请中提出的研究具有创新性，因为 它采用了一种新的方法来发现基因的MCD，利用领先的技术，如集成的单， 核基因分型/转录组学，并利用由申请人的 实验室拟议的研究是重要的，因为它有望产生新的因果体细胞变异的MCD 以及它们在适当的细胞环境中的分子和功能效应的新知识。最终，这样的 知识有可能为开发或应用靶向治疗提供新的机会。

Project Summary PROJECT SUMMARY Although somatic mosaicism is a known cause of malformations of cortical development (MCD), it is still difficult to detect sparse somatic variants in patient tissue and study their molecular and functional effects. The long- term goal of this project is to unravel the cellular and molecular complexity of MCD and translate this knowledge into improved diagnostic and treatment opportunities for patients. The overall objectives in this application are to (i) improve somatic variant detection, (ii) identify transcriptional correlates of somatic variants, and (iii) test functional effects of somatic variants in relevant cell types. The central hypothesis is that somatic variants are enriched in particular cell lineages in MCD and that cell identity is an important determinant of a variant’s molecular and functional effects. The rationale for this project is that understanding somatic variation and its consequences at cell-type resolution will ultimately improve gene discovery for MCD and help advance the field toward targeted therapies for drug-resistant epilepsy associated with MCD. The central hypothesis will be tested by pursuing three specific aims: (1) Improve somatic variant detection in patients with focal cortical malformations, (2) Evaluate cell-type-specific transcriptional effects of somatic mutations, and (3) Functionally validate the cell-type-specific contribution of somatic mutations to disease features. Under the first aim, relevant cell types will be enriched from affected patient tissue and sequenced as a strategy to improve somatic variant detection. For the second aim, integrated single-nuclei genotyping and transcriptomic analyses of patient tissue will be used to evaluate gene expression signatures of somatic mosaicism. The third aim will leverage a novel transgenic mouse model of MCD to drive somatic variation in particular cell types and determine how cell identity contributes to specific components of disease. The research proposed in this application is innovative because it takes a new approach to gene discovery in MCD, leverages leading-edge techniques such as integrated single- nuclei genotyping/transcriptomics, and takes advantage of a unique mouse model generated by the applicant’s lab. The proposed research is significant because it is expected to yield new causal somatic variants for MCD and new knowledge of their molecular and functional effects in appropriate cellular contexts. Ultimately, such knowledge has the potential to provide new opportunities to develop or apply targeted treatments.