Understanding genomic stability betweengenerations by assessing mutational burdens in single sperms

通过评估单个精子的突变负担来了解代际基因组稳定性

• 批准号: 10740598
• 负责人: Xiaoxu Yang
• 金额: $ 11.38万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10740598
• 关键词: Address; Age; Aging; Algorithms; Award; Benchmarking; Biological; Biotechnology; Brain; Categories; Cells; Chemical Exposure; Child; Child Health; Computer software; Congenital Disorders; DNA Sequence Alteration; DNA amplification; DNA sequencing; Data; Data Set; Daughter; Detection; Diploidy; Disease; Embryonic Development; Environmental Exposure; Fathers; Focus Groups; Foundations; Future; Generations; Genetic; Genetic Risk; Genome; Genome Stability; Genomics; Germ Cells; Goals; Haploidy; Haplotypes; Human; Human Biology; Human Genome; Human Subject Research; Individual; Job Application; Knowledge; Lead; Measures; Mental disorders; Mentors; Metabolism; Methods; Modeling; Mosaicism; Mutation; Mutation Detection; Natural Selections; Nature; Outcome; Parents; Paternal Age; Pattern; Performance; Phase; Population; Positioning Attribute; Postdoctoral Fellow; Publishing; Recording of previous events; Reporting; Reproductive Medicine; Reproductive Sciences; Research; Research Personnel; Risk; Sampling; Sensitivity and Specificity; Shapes; Somatic Mutation; Source; Technology; Training; Variant; age effect; age related; aged; base; burden of illness; career; career development; computational pipelines; de novo mutation; deep learning; density; experimental study; genome sequencing; human subject; improved; interdisciplinary approach; learning strategy; male; men; mosaic; mosaic variant; nervous system disorder; new technology; next generation; next generation sequencing; novel; offspring; pressure; recruit; recurrent neural network; sperm cell; transmission process; whole genome; young man

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.

项目总结/摘要 在胚胎发育、衰老、细胞代谢和环境暴露过程中的突变， 永久记录在每个细胞及其子细胞的基因组中。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 并呈现不同的特征。阐明这些突变的模式及其传播到 后代是理解先天性从头突变（DNM）疾病和遗传变异的关键。 人类世代。随着父母年龄的增长，生殖细胞中DNM的数量增加， 增加了DNM及其在后代中引起的疾病的风险。尽管与年龄相关的DNM风险已经被 在大群体中的报道，我们对父系特异性克隆和非克隆嵌合现象如何 自然选择如何塑造突变模式仍然有限。在我以前的 通过研究生和博士后的研究，我建立了一个概念，即相当一部分的DNMs在儿童 神经和精神障碍由精子中的克隆镶嵌突变引起（Yang等人，Cell 2021年）。我开发了实验和计算管道，以准确检测克隆镶嵌突变， 用深度全基因组测序的大量样品（Yang，等人，Nature Biotechnology，in press; Breuss，Yang， co-firsts等人，Nature 2022）。在这个K99/R 00应用程序中，我的目标是揭示非克隆性腺的特征， 突变负担在单细胞水平，采用跨指导的多学科方法[K99] 和独立的[R 00]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，分析非克隆精子的基因组位置， 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）。我会 开发新的计算软件，以准确地检测单细胞的嵌合突变，不仅从 单倍体和二倍体基因组，并开发实验方法，以准确验证体细胞突变 从单细胞扩增的DNA（Aim 2）。最后，我将测量单细胞DNA突变率和突变 使用来自另外45名年轻和75名老年捐赠者的2300个单精子， 突变机制以及它们将如何影响下一代基因组的稳定性， 自然选择（目标3）。总的来说，这一建议的结果将有助于我们了解非克隆镶嵌 突变负担，突变分布，以及人类精子中的突变功能，以及与年龄相关的 对下一代基因组稳定性的遗传影响。我的职业目标是领导一个独立的研究 该小组专注于人类基因组中的体细胞突变，其原因，并预测其对人类的影响。 儿童健康。在K99阶段，我将继续接受学科招募，生殖科学， 实验，计算和职业发展培训，从我的博士后导师格里森博士，合作， 导师威尔金森博士和Sebat，以及在加州大学圣地亚哥分校和其他机构的外部导师。严谨的 指导支持将大大提高我在人类主题处理和生殖科学方面的知识，以及 帮我准备工作申请在K99阶段取得的成果将有助于我过渡到一个 在R 00阶段成为独立调查员，并为我未来的职业生涯奠定基础。