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.
The purpose of this project is to gain a thorough understanding of the prevalence and origins of genome variation among the trillions of single somatic cells that comprise each individual, and to assess the role of age and cell type on the process of somatic genome mutation. This work will inform models of cancer and aging, and will enable future investigations of the role of somatic variation in human neurological disease.