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. 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 sites with variant frequencies of 10-4 to 10-5, with an overall mutation frequency of the region of [~]6x10-7. Some of the substitutions were re-current and were found at a higher variant frequency in older donors than in younger ones, or exclusively, in older donors. Also, older donors harbored more mutations associated with congenital disorders. Other mutations were present in both age groups suggesting that these might result from a different mechanism (e.g., post-zygotic mosaicism). We also observed that independent of age, the frequency and deleteriousness of the mutational spectra was more similar to COSMIC than to gnomAD variants. Our approach is an important strategy to identify mutations that could be associated with a gain-of-function of the receptor tyrosine kinase activity, with unexplored consequences in a society with delayed fatherhood.