Transcription start sites experience a high influx of heritable variants fuelled by early development

Mutations drive evolution and genetic diversity, but the impact of transcription on germline mutagenesis remains poorly understood. Here, we identify a hypermutation phenomenon at transcription start sites in the human germline, spanning several hundred base pairs in both directions. We link this TSS mutational hotspot to divergent transcription, RNA polymerase II stalling, R-loops, and mitotic—but not meiotic—double-strand breaks, revealing a recombination-independent mechanism distinct from known processes. Notably, the hotspot is absent in de novo mutation data. We reconcile this by showing that TSS mutations are significantly enriched with early mosaic variants often filtered out in de novo mutation calls, indicating that the hotspot arises during early embryogenesis. Mutational signature analysis reinforces these findings and implicates alternative non-homologous end joining and maternal mutation clusters. Our study provides the first detailed description of a germline TSS mutation hotspot, with broad evolutionary and biomedical implications.