Understanding genomic stability between generations by assessing mutational burdens in single sperms - 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.
