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

Abstract

De novo mutations (DNMs) are important players 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 ultradeep 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 allele frequencies (VAFs) of 10−4 to 10−5, with an overall mutation frequency of the region of ∼6 × 10−7. Some of the substitutions are recurrent and are found at a higher VAF in older donors than in younger ones or are found exclusively in older donors. Also, older donors harbor more mutations associated with congenital disorders. Other mutations are present in both age groups, suggesting that these might result from a different mechanism (e.g., postzygotic mosaicism). We also observe that independent of age, the frequency and deleteriousness of the mutational spectra are 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.

Activating mutations in FGFR3 are associated with clonal expansion events and high de novo rates in the male germline

“…For the majority of the donors (63%) we counted mutations for this variant, but almost one-third of the donors had a VAF of 0 (Figure 2C). Overall, the measured frequencies in sperm DNA for the c.1620C>A variant were in accordance with the VAF recently published (Salazar et al, 2022) and reported in Table 2.…”

“…Five variants were reported in the literature to be associated with a congenital disorder and categorized to be of clinical significance by ClinVar (pathogenic or benign/uncertain). The other three have not been reported so far, yet were found at a higher frequency in sperm DNA (Salazar et al, 2022) and were predicted in silico to have a strong effect on modifying the protein structure (CADD score). In order to assess if the variant resulted in sub-clonal expansions in the male germline, we screened the VAF in two different cell types of the male germline: the spermatogonial stem cells and the differentiated sperm.…”

High prevalence of somatic PIK3CA and TP53 pathogenic variants in the normal mammary gland tissue of sporadic breast cancer patients revealed by duplex sequencing

“…UM, PT, BL, and SK samples of four individuals (P10, P28, P51, and P52) were selected for detection of variants by duplex sequencing based on the presence of PIK3CA or TP53 hotspot variants in PT, but not UM tissue, according to standard NGS. The protocols used here are based on the ones described in more detail in Salazar et al 53 .…”

“…Size selected genomic DNA was end-repaired and A-tailed using the NEBNext® Ultra™ II End Repair/dA-Tailing Module (New England Biolabs) according to the manufacturer’s instructions followed by adapter ligation with the NEBNext® Ultra™ II Ligation Module (New England Biolabs) following the manufacturer’s instructions. The adapters ligated to the A-tailed DNA were synthesized as previously described (Adapter 2) 53 . The ligation reaction was then purified using 1.2 volumes of Sera-Mag Select beads (Cytiva).…”

“…Targeted captures and post-capture amplification. Two rounds of targeted captures followed by PCR amplification were performed as described in Salazar et al, with minor modifications on the post-capture amplification (Supplementary Table 10) 53 . The biotinylated probes used to target exonic regions of TP53, and PIK3CA are detailed on Supplementary Table 10.…”