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

Delayed fatherhood results in a higher risk to inherit a new germline mutation that might result in a congenital disorder in the offspring. In particular, some FGFR3 mutations increase in frequency with age, but there are still a large number of uncharacterized FGFR3 mutations that could be expanding in the male germline with potentially early or late-onset effects in the offspring. Here, we investigated the mutation frequency in the DNA of human testis and sperm and the activation state of the expressed mutant protein of eight different FGFR3 variants categorized by ClinVar as deleterious, benign, or not reported. Overall, the ligand-independent activation of the mutant protein resulted in a increased number of mutant sperm; although, strong activating mutations did not necessarily result in the highest frequencies. Moreover, only two mutants c.952G>A and c.1620C>A showed an increase with the donor’s age; the latter also forming larger clonal expansions in the testis. We also showed that the prediction of deleteriousness of a mutation is not always accurate, and similar in silico scores can reflect either a gain-of-function or loss-of-function. Our approach led to the discovery of two novel variants c.1261G>A and c.952G>A to have promiscuous FGFR3 activation and increased mutation frequencies in the male germline. The large fraction of donors with mutations suggests a high de novo rate potentially explained by a selective advantage before the maturation of the male germline. This sequence-function study provides important data for the evaluation and interpretation of variants with relevant clinical implications. ### Competing Interest Statement The authors have declared no competing interest. * ACH : Achondroplasia ddPCR : droplet digital Polymerase Chain Reaction DNMs : De novo mutations FGFR3 : Fibroblast growth factor 3 HCH : Hypochondroplasia ORF : Open Reading Frame PAE : Paternal Age Effect PZM : Post-zygotic Mosaic SSCs : Spermatogonial stem cells TDI : Thanatophoric Dysplasia Type I TIRF : Total internal reflection fluorescence