Understanding genomic stability betweengenerations by assessing mutational burdens in single sperms

项目摘要/摘要 胚胎发育、衰老、细胞新陈代谢和环境暴露期间的突变 永久记录在每个细胞及其子体的基因组中。这取决于突变是否 可以在常规的下一代测序中检测到，它们在性质上被识别为克隆或非克隆 并呈现出不同的特点。阐明这些突变的模式及其传播的可能性 后代是了解先天性新生突变(DNM)疾病和 人类世世代代。随着父母年龄的增长，他们生殖细胞中的DNM数量增加，随之而来的是 增加了DNMS及其在后代中引起的疾病的风险。尽管与年龄相关的DNM风险一直是 在大量人口中报道，我们对父系特有的克隆和非克隆嵌合体的理解 对后代的贡献以及自然选择如何塑造突变模式仍然有限。在我的前一次 在研究生和博士后的研究中，我确立了这样一个概念，即相当一部分儿童中的DNM 由精子中的克隆性马赛克突变引起的神经和精神障碍(Yang，et al.细胞 2021年)。我开发了实验和计算管道来准确检测克隆马赛克突变 大量样本进行深度全基因组测序(Yang，et al.自然生物技术出版社；布鲁斯，杨， 共同第一，等人。自然2022)。在这个K99/R00应用程序中，我的目标是解开非克隆性腺的特征 单细胞水平的突变负担，采用跨越被指导对象的多学科方法[K99] 和独立的[R00]奖励阶段。我会比较700个人类精子的基因组序列 从35名健康年轻男性的大量精子序列中，分析非克隆的基因组位置 与克隆突变相比，突变倾向于驻留，并研究这些突变的影响(目标1)。这就做 开发新的计算软件，准确检测单个细胞的马赛克突变，而不仅仅是 单倍体和二倍体基因组，并开发实验方法来准确验证体细胞突变 来自单细胞扩增的DNA(目标2)。最后，我将测量单细胞DNA突变率和突变率 使用另外45名年轻捐赠者和75名老年捐赠者的2300个单一精子的模式来寻找与年龄相关的线索 突变机制及其对下一代基因组稳定性的影响 自然选择(目标3)。总体而言，这一提议的结果将有助于我们理解非克隆镶嵌 人类精子的突变负荷、突变分布和突变功能，以及与年龄相关的 遗传对下一代基因组稳定性的影响。我的职业目标是领导一项独立的研究 专注于人类基因组中的体细胞突变及其原因，并预测其后果的小组 儿童健康。在K99阶段，我将继续接受学科招募，生殖科学， 我的博士后导师格里森博士提供的实验、计算和职业发展培训， 威尔金森博士和塞巴特博士的导师，以及加州大学圣地亚哥分校和其他研究所的外部导师。严谨的 导师的支持将大大提高我在人类学科处理和生殖科学方面的知识，以及 让我为工作申请做好准备。在K99阶段取得的成果将有助于我过渡到 R00阶段的独立调查员，为我未来的职业生涯奠定了基础。

PROJECT SUMMARY/ABSTRACT Mutations during embryonic development, aging, cellular metabolism, and environmental exposure are permanently recorded in the genomes of each cell and its daughters. Depending upon whether the mutations can be detected in regular next-generation sequencing, they are recognized as clonal or non-clonal in nature and present different features. Elucidating the patterns of these mutations and their potential to transmit to offspring is key to understanding congenital de novo mutation (DNM) disorders and genetic variability across human generations. As parents age, the number of DNMs in their germ cells increases, and with this, an increased risk of DNMs and the disease they cause in offspring. Although age-related DNM risks have been reported in large populations, our understanding of how paternal-specific clonal and non-clonal mosaicism contribute to offspring and how natural selection shapes the mutation pattern is still limited. During my previous graduate and postdoctoral research, I established the concept that a considerable portion of DNMs in children with neurological and psychiatric disorders arise from clonal mosaic mutations in the sperm (Yang, et al. Cell 2021). I developed experimental and computational pipelines to accurately detect clonal mosaic mutations in bulk samples with deep whole-genome sequencing (Yang, et al. Nature Biotechnology, in press; Breuss, Yang, co-firsts, et al. Nature 2022). In this K99/R00 application, I aim to unravel the feature of the non-clonal gonadal mutation burden at the single-cell level, employing multidisciplinary approaches spanning the mentored [K99] and independent [R00] award phases. I will compare the genomic sequences from 700 single human sperm from bulk sperm sequences in 35 healthy young men, analyze the genomic positions where the non-clonal mutations tend to reside compared to the clonal ones, and study the impact of those mutations (Aim 1). I will develop new computational software to accurately detect mosaic mutations from single cells not only from haploid and diploid genomes, and develop experimental approaches to accurately validate the somatic mutations from single-cell amplified DNA (Aim 2). Finally, I will measure the single-cell DNA mutation rate and mutation patterns using 2300 single sperm from an additional 45 young versus 75 aged donors for clues on age-related mutational mechanisms and how they will impact the genome stability in the next generation before and after natural selection (Aim 3). Overall, the results from this proposal will help us to understand the non-clonal mosaic mutational burden, mutation distributions, as well as mutational functions in human sperm, and the age-related genetic impacts on the genome stability of the next generation. My career goal is to lead an independent research group focusing on somatic mutations in the human genome, their causes, and predicting their consequences on child health. During the K99 phase, I will continue to receive subject recruitment, reproductive science, experimental, computational, and career development training from my postdoctoral advisor Dr. Gleeson, co- mentors Dr. Wilkinson and Sebat, as well as external mentors at UC San Diego and other institutes. The rigorous mentored support will greatly my knowledge in human subject handling and reproductive science, as well as getting me prepared for job applications. The results obtained in the K99 phase will facilitate my transition to an independent investigator in the R00 phase and lay the foundation for my future career.