Age-related mutagenesis of driver genes in the male germline

New germline mutations are directly transmitted to our children, and therefore have profound consequences for future generations, but we still know surprisingly little about germline mutation processes. Recent advances in individual genome sequencing showed that the majority of de novo mutations (DNMs) originate in the male germline, and firmly established that the rate of DNMs increases with paternal age. To date, however, we lack information on individual mutagenic events necessary to study a unique type of mutagenesis the selfish expansion of driver mutations in the male germline due to the limitations of current technologies in studying DNMs. As a result, only a handful of driver mutations are known and characterized so far. This is unfortunate, as these might represent the tip of an iceberg given that in cancer hundreds of driver mutations are found within single genes. Moreover, they cause congenital disorders, self-propagate, are highly recurrent, and drastically increase with paternal age. We have adapted a new ultra-sensitive sequencing technology with extremely low error-rates to identify accurately DNMs in selected candidate driver genes in sperm that can then be analyzed by our high-throughput digital PCR method. By combining these technologies, we have, for the first time, the capability to study DNMs in candidate driver genes, and investigate their expansion and increase with paternal age in the male germ line. Using our unique expertise in ultrasensitive methodologies to detect rare mutations, in this project we propose to 1) sequence exons of driver genes to discover high frequency DNMs; 2) test selection by examining the spatial expansion of the discovered DNMs in dissected testes, and 3) analyze their increase and transmission patterns in sperm of differently aged men. We envision that this project will generate a comprehensive and powerful analysis of driver mutations in the male germline, and establish a basis for understanding this type of mutagenesis and the associated risks of delayed parenthood in our society.

Investigating mutations in the male germline can yield insights into human diseases, their impact on future generations, and evolutionary processes. In particular, driver mutations increase in frequency in the male germline and often result in congenital disorders in the offspring. To date, however, the knowledge on this unique type of mutagenesis has been limited to a handful of driver mutations. Identifying these mutations is challenging due to their low frequencies. This study developed an ultra-sensitive sequencing technology with low error rates to identify candidate driver mutations and combined this technology with high-throughput digital PCR to explore the spatial mutation expansion in testes and sperm from donors of different ages. The study resulted in an extensive dataset of mutations enriched in sperm DNA from donors of varying ages, facilitating characterization of mutational distribution, spectra, and specific signatures related to this mutagenesis. Further, this study identified novel substitutions in the FGFR3, Erbb2, and p53 gene, all well-known oncogenes, revealing distinct accumulation patterns with age. Some mutations increased with age, while others emerged before sexual maturity and remained constant, potentially impacting offspring regardless of the age of conception. Another notable outcome was the observed discordance between mutation frequency in the testis and sperm for two mutations with different severity outcomes, emphasizing the intricate interplay between activation levels, aging, and meiotic differentiation. Furthermore, the impact of mutations on protein function was analyzed using biophysical methods, providing insights into variations in responsiveness to ligand binding among mutations. This lays the foundation for a deeper understanding of the molecular mechanisms behind the expansion and transmission of pathogenic driver mutations in the male germline and the risks for the transmission and recurrence of these type of mutagenesis and their associated disorders.