Discovery of an unusual high number of de novo mutations in sperm of older men using duplex sequencing

De novo mutations (DNMs) are an important player in heritable diseases and evolution, yet little is known about the different mutagenic processes in our germline given the difficulty to reliably identify ultra-low frequency variants. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultra-deep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined ~4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified highly mutable sites with mutation frequencies 4-5 orders of magnitude higher than the genome average. Multiple mutations were found at a higher frequency, or exclusively, in older donors, suggesting that these mutations are testis exclusive mosaics expanding in the male germline with age. Also, older donors harbored more mutations associated with congenital disorders. Some mutations were found in both age groups with no significant difference, suggesting that these might result from a different mechanism (e.g., post-zygotic mosaicism). We also observed that independently of age, the frequency and deleteriousness of the mutations in sperm were elevated compared to reports in the population. Our approach is an important strategy to identify mutations that could be associated with aberrant receptor tyrosine kinase activity, with unexplored consequences in a society with delayed fatherhood.