Origins of Brain Somatic Mosaicism in Developmental Brain Disease

摘要 脑体细胞嵌合体(BSM)指的是在大脑中数十亿个细胞中的任何一个细胞内积累突变 人类的大脑，从胚胎发育到成年都可能发生。其程度、影响和 BSM治疗脑部疾病的机制尚不清楚。来自大脑体细胞的先前工作 PI所服务的镶嵌网络(BSMN)在镶嵌的可靠性方面取得了关键突破 检测，但也提出了新的问题，包括BSM在健康大脑中的存在程度，以及 BSM突变解释疾病的机制。 局灶性皮质发育不良(FCD)与实质性的神经精神障碍有关，是 儿童时期顽固性癫痫最常见的原因。15%-59%的人有神经精神病学特征 患者5-7例，神经病理显示神经发生、迁移、分化和改变 神经兴奋性。我们和其他人之前在少数人中发现了mTOR途径的镶嵌突变 大多数病例仍未解决，缺乏基本的机制。 我们假设：1]FCD突变在模式上类似于中性体细胞突变，并且 分布由发育过程决定，但其功能效应不同。2]BSM模式，等位基因 细胞间的片段(AFs)和等位基因共享可以重建细胞谱系和迁徙历史。3] 对FCD切除组织的研究可以发现新的疾病原因，如果存在于 每一个细胞。4]在小鼠体内建立BSM模型可以解开复杂的嵌合突变的信号网络。 我们的初步数据显示：1]从一具死后对照身体上，我们验证了259具体细胞 变异体使用300X基因组测序，并开始使用这些变异体作为条形码重建 血统历史。2]对314例FCD患者的脑切除进行了深度测序，确定了12个新的候选 基因，突出信号和突触功能障碍，以及一种新的“两次打击”的疾病机制。3]我们 在宫内建立小鼠电穿孔模型，以评估可能的FCD变体功能的获得或丧失， 并评估“一击”和“两击”突变的影响。 我们提出了三个目标：1]从对照身体出发，我们将跨解剖重建细胞谱系 使用BSM作为条形码的域。2]有了这些谱系信息，我们将研究BSM的起源 FCD的突变，通过招募新患者，进行有针对性和无偏倚的测序，以及 找出新的原因。3]我们将在动物模型中对推测的有害等位基因进行功能性验证 “一击”和“两击”都是原因。我们的目标是从机械上理解 BSM在对照个体中的程度，以重建神经谱系并识别新的机制 发育性脑部疾病。

Abstract Brain somatic mosaicism (BSM) refers to the accumulation of mutations within any of the billions of cells in the human brain, which can occur from embryogenesis through adulthood. The extent, impact and mechanisms of BSM on brain disease remain poorly understood. Prior work from the Brain Somatic Mosaicism Network (BSMN), on which the PI served, made critical breakthroughs in reliability of mosaicism detection, but also raised new questions, including the degree to which BSM exists in the healthy brain, and the mechanisms by which BSM mutations explain disease. Focal cortical dysplasia (FCD) is associated with substantial neuropsychiatric disability, and is the most common cause of intractable epilepsy in childhood. Neuropsychiatric features are seen in 15-59% of patients 5-7, and neuropathologically shows disrupted neurogenesis, migration, differentiation, and altered neural excitability. We and others previously identified mosaic mutations in the mTOR pathway in a minority of FCD cases, but most cases remain unsolved, and fundamental mechanisms are lacking. We hypothesize that: 1] FCD mutations are similar to neutral somatic mutations in their patterns and distributions, dictated by developmental processes, but differ in their functional effect. 2] BSM patterns, allelic fractions (AFs) and allele sharing between cells can reconstruct cellular lineages and migratory histories. 3] Study of FCD resected tissue can uncover novel causes of disease that would not be tolerated if present in every cell. 4] BSM modeling in mouse can unravel disrupted signaling networks of complex mosaic mutations. Our preliminary data shows: 1] From a post-mortem control cadaver, we validated 259 somatic variants using 300X genome sequencing, and started to use these variants as ‘barcodes’ to reconstruct lineage histories. 2] Deep sequencing from 314 FCD patient brain resections identified 12 new candidate genes, highlighting signaling and synaptic dysfunction, and a novel ‘two-hit’ disease mechanisms. 3] We established in utero mouse electroporation models to assess putative FCD variants as gain or loss of function, and to assess effects of ‘single-hit’ and ‘two-hit’ mutations. We propose three aims: 1] From control cadavers, we will reconstruct cell lineage across anatomical domains using BSM as barcodes. 2] With this lineage information, we will study the origins of BSM mutations in FCD, by recruiting new patients, performing both targeted and unbiased sequencing, and identifying novel causes. 3] We will functionally validate putative deleterious alleles in animal models for both ‘single-hit’ and ‘two-hit’ causes. The goal is to achieve a mechanistic understanding of the extent of BSM in control individuals, to reconstruct neural lineages and to identify novel mechanisms in developmental brain disease.