AbstractPostzygotic mutations are acquired in all of the normal tissues throughout an individual’s lifetime and hold clues for identifying mutagenesis causing factors. The process and underlying mechanism of postzygotic mutations in normal tissues is still poorly understood. In this study, we investigated postzygotic mutation spectra in healthy individuals by optimized ultra-deep exome sequencing of time series samples from the same volunteer and samples from different individuals. In cells of blood, sperm, and muscle, we resolved three common types of mutational signature. Two of them are known to represent clock-like mutational processes, and their proportions in mutation profiles associated with polymorphisms of epigenetic regulation genes, suggesting the contribution of personal genetic backgrounds to underlying biological process. Notably, the third signature, characterized by C>T transitions at GpCpN sites, tends to be a feature of diverse normal tissues. Mutations of this type were likely to occur early in embryo development even before the tissue differentiation, as indicated by their relatively high allele frequencies, sharing variants between multiple tissues, and lacking of age-related accumulation. Almost all tumors shown in public datasets did not have this signature detected except for 19.6% of clear cell renal cell carcinoma samples, which featured by activation of the hypoxia-induced signaling pathway. Moreover, in vitro activation of HIF signaling pathway successfully introduced the corresponding mutation profile of this signature in a culture-expanded human embryonic stem cell line. Therefore, embryonic hypoxia may explain this novel signature across multiple normal tissues. Our study suggest hypoxic conditions in the early stage of embryo development may be a crucial factor for the C>T transitions at GpCpN sites and individual genetic background also related to shaping human postzygotic mutation profiles.Author SummaryThe process and related mechanism of post-zygotic mutations in normal tissues is still poorly understood. By analyzing post-zygotic mutations in blood, sperm and muscle from healthy individuals, we found a normal tissues specific mutation type characterized by C>T transitions at GpCpN sites. Almost none of tumors in The Cancer Genome Atlas project harbors this type of mutations, except for a subset of clear cell renal cell carcinoma samples with higher activity of hypoxia inducible signaling pathways. We further reproduce the enrichment of this type of mutations in human embryonic stem cells by specific activating hypoxia inducible factor 1α. Taken together, we propose that hypoxic conditions are one crucial factor responsible for the occurrence of post-zygotic mutations, especially the C>T transition in GpCpN sites, in the early stage of embryo development in healthy individuals.