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

描述(由申请人提供)： 体细胞受精后出现的突变与癌症和衰老有关，已被证明与越来越多的人类疾病有关。同样，神经元基因的新生胚系突变是导致自闭症、精神分裂症和智力残疾的原因，这表明类似类型的体细胞突变可能会导致这些和其他神经系统疾病，因为它们提供了特定细胞类型的大效应突变，这些突变可能是单独作用的，也可能是与遗传变异一起起作用的。尽管人们越来越意识到基因组嵌合体对人类健康的重要性，但目前我们对身体和大脑不同细胞系中的体细胞突变的了解还很少。这个问题一直很难解决，因为传统的全基因组方法无法检测到细胞群体中罕见的变异，而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，目前的方法存在错误率高和分辨率低的问题，并且不允许对仅在一个细胞中检测到的突变进行独立验证。从单细胞中扩增基因组的第二种方法是克隆扩增。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，如有丝分裂后神经元，这是可行的。在这里，我们建议使用两种创新的策略来分析单个神经元的基因组和控制年轻和老年小鼠的成纤维细胞。首先，我们利用了唯一已知的不使用致癌因子扩增神经细胞的方法：体细胞核移植克隆。这将使神经来源细胞系的深度全基因组测序和全面的突变图谱成为可能。其次，我们将使用核移植，在一个分裂后产生来自单个神经元的姐妹细胞对。复制姐妹细胞的单细胞测序将能够在真正的突变与DNA扩增伪像明显区分的情况下，灵敏而准确地检测从头开始的拷贝数变化。我们应用这些互补技术将揭示不同细胞系在发育和衰老过程中出现的基因组变异的全谱，并将有助于解决有关神经元基因组多样性的程度、影响和起源的长期假说。

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.