Somatic Mosaicism in the Brain of Tourette Syndrome

 描述(申请人提供)：多发性抽动症(TS)是一种发育中的端脑疾病，通过对血液样本的检查，尚未出现明显的致病基因变异。在这项建议中，我们调查体细胞突变是否可能是TS发病的部分基础。现有证据表明，细胞在受精卵形成后积累体细胞突变，这意味着人体细胞没有相同的DNA序列。除了单核苷酸变异(SNV)和小插入/缺失(INDel)外，细胞还可以积累拷贝数变异(CNV，即复制和缺失)、可转座元件的插入、倒置和易位，所有这些都涉及数百到数百万个核苷酸。脑细胞中出现的体细胞嵌合体可以解释为什么未能在TS等神经精神疾病中发现一致的、可复制的遗传危险因素，并至少部分地解释了经常观察到的血型和总表型之间的差异。无论是在正常发育中还是在疾病中，都没有体细胞嵌合体的估计。为了验证体细胞突变可能是TS出现的基础的假设，在这个建议中，我们将发现并量化TS和正常对照组大脑中的体细胞基因组变异，然后探索这种变异的潜在功能后果。在目标1中，我们将使用先进的测序技术，全面发现谱系特定和区域特定的体细胞基因组变异：SNV、INDELS、CNV、反转录转座子插入、倒位和易位。这项分析将涉及20个TS大脑和20个匹配的正常对照组大脑。在前额叶皮质(PFC)、运动前皮质(PMC)和纹状体(STR)这三个与TS密切相关的区域以及从这些区域分离出的特定细胞系中，包括锥体神经元、中棘神经元、中间神经元和小胶质细胞，将发现并验证镶嵌变体。在目标2中，我们将选择10个基因组变异体，利用CRISPR技术将它们改造成ipscs和转基因小鼠，并表征它们对分子、组织和行为水平的影响。总而言之，这些特定的目标将提供对TS大脑中体细胞基因组变异(数量、类型、频率)的第一次估计，并将产生关于它们对大脑发育意义的假设。

 DESCRIPTION (provided by applicant): Tourette Syndrome (TS) is a disorder of the developing telencephalon for which no significant causative genetic variant has yet emerged through the examination of blood samples. In this proposal we investigate whether somatic mutations might underlie in part the pathogenesis of TS. Existing evidence suggests that cells accumulate somatic mutations after the formation of the zygote, implying that cells of the human body do not have identical DNA sequence. Besides single nucleotide variation (SNV) and small insertion/deletions (InDels), cells can accumulate copy number variations (CNVs, i.e., duplications and deletions), insertions of transposable elements, inversions and translocations, all involving from few hundred to several millions of nucleotides. Somatic mosaicism arising in brain cells could explain the failure to discover consistent, replicable genetic risk factors in neuropsychiatric disorders like TS, and underlie at least in part the frequently observed variability between blood genotype and overall phenotype. There is no estimate of somatic mosaicism in either normal development or in disease. To test the hypothesis that somatic mutations might underlie the emergence of TS, in this proposal we will discover and quantify somatic genome variation in TS and normal control brains, followed by exploration of potential functional consequences of this variation. In Aim 1, we will perform using advanced sequencing techniques comprehensive discovery of lineage-specific and region-specific somatic genomic variations: SNVs, InDels, CNVs, retrotransposon insertions, inversion and translocations. The analysis will involve 20 TS brains and matched 20 normal control brains. Mosaic variants will be discovered and validated in prefrontal cortex (PFC), premotor cortex (PMC) and striatum (STR), three regions strongly implicated in TS, as well as in specific cell lineages isolated from these regions, including pyramidal neurons, medium spiny neurons, interneurons and microglial cells. In Aim 2, we will select 10 genomic variants, engineer them into iPSCs and in transgenic mice using CRISPR technologies, and characterize their impact on the molecular, tissue and behavior level. Together, these specific aims will provide the first estimate of somatic genomic variation (number, type, frequency) in the brain of TS and will yield hypotheses about their significance for brain development.