Genome-wide investigation of somatic mutation in the developing and aging brain

描述（由申请人提供）： 体细胞谱系中受精后出现的突变与癌症和衰老有关，并已被证明有助于越来越多的人类疾病。类似地，神经元基因中的从头生殖系突变是自闭症、精神分裂症和智力残疾病例的原因，这表明类似类型的体细胞突变可能通过在特定细胞类型中提供大效应突变而导致这些和其他神经系统疾病，这些突变可能单独或与遗传变异一起起作用。尽管越来越多的人意识到基因组嵌合体对人类健康的重要性，但我们目前对身体和大脑不同细胞谱系中的体细胞突变的了解很少。这个问题一直难以解决，因为传统的全基因组方法无法检测细胞群体中罕见的变异，并且大多数组织由不同的细胞类型和混合的谱系组成。单细胞测序提供了一种解决方案，然而，目前的方法存在高错误率和低分辨率的问题，并且不允许仅在一个细胞中检测到的突变的独立验证。从单细胞扩增基因组的第二种方法是通过克隆扩增。这对于癌症和一些自我更新的细胞类型是可行的，但对于许多有趣的或老化的细胞类型，如有丝分裂后的神经元，则不可行。在这里，我们建议使用两种创新的策略来分析来自单个神经元的基因组，并控制来自年轻和老年小鼠的成纤维细胞。首先，我们利用唯一已知的方法来扩增神经元细胞，而不使用致癌因子：体细胞核移植克隆。这将使神经元衍生细胞系的深度全基因组测序和全面的突变分析成为可能。其次，我们将使用核移植来产生来自单个神经元的成对姐妹细胞。复制的姐妹细胞的单细胞测序将使得能够在真实突变可以与DNA扩增伪影清楚区分的情况下灵敏且准确地检测从头拷贝数变异。这些互补技术的应用将揭示发育和衰老过程中不同细胞谱系中出现的基因组变异的全谱，并将有助于解决长期存在的关于神经元基因组多样性的程度，影响和起源的假设。

DESCRIPTION (provided by applicant): Mutations that arise after fertilization in somatic cell lineages are linked to cancer and aging an have been shown to contribute to an increasing number of human disorders. Similarly, de novo germline mutations in neuronal genes are responsible for cases of autism, schizophrenia and intellectual disability, suggesting that similar types of somatic mutations could contribute to these and other neurological disorders by providing large-effect mutations in specific cell types, that may act alone or in concert with inherited variants. Despite the growing awareness of the importance of genomic mosaicism for human health, our present understanding of somatic mutation in different cellular lineages of the body and brain is minimal. This question has been difficult to address because conventional genome-wide methods cannot detect variants that are rare within a cell population, and most tissues are composed of diverse cell types and intermixed lineages. Single cell sequencing offers one solution, however, current methods suffer from high error-rates and low resolution, and do not allow for independent validation of mutations detected in merely one cell. A second means to amplify genomes from single cells is through clonal expansion. This is feasible for cancer and some self-renewing cell types, but not for many interesting or aged cell types such as post-mitotic neurons. Here, we propose to use two innovative strategies to profile genomes from individual neurons and control fibroblasts from young and aged mice. First, we take advantage of the only known method to amplify neuronal cells without use of oncogenic factors: cloning by somatic cell nuclear transfer. This will enable deep whole genome sequencing and comprehensive mutational profiling of neuron-derived cell lines. Second, we will use nuclear transfer to produce pairs of sister cells derived from single neurons after one division. Single cell sequencing of replicate sister cells will enable sensitive and accurate detection of de novo copy number variation in a context where bona-fide mutations can be clearly distinguished from DNA amplification artifacts. Our application of these complementary technologies will reveal the full spectrum of genome variation that arises in different cell lineages during development and aging, and will help resolve longstanding hypotheses regarding the extent, impact and origins of neuronal genome diversity.