High-throughput analysis of driver mutations in FGFR3 by digital PCR
Sprache des Vortragstitels:
European Human Genetics Conference
Sprache des Tagungstitel:
Introduction: Mutations occurring at increased frequencies in the germline of older men in the FGFR3 gene were suggested to confer a selective growth advantage to spermatogonial stem cells. These mutations have been associated with several congenital disorders. Many more mutations have been reported in FGFR3 for cancer, and it is possible that these expand in the male germline with age.
Materials and Methods: To test an age-related expansion of driver mutations, we are screening sperm and testes of different aged donors with two high-throughput techniques: bead emulsion amplification (BEA) and allele-specific PCR. We are focusing on 13 de novo mutations occurring within a ~3kb region of the FGFR3, identified in sperm DNA by duplex sequencing (DS) in our laboratory.
Results: With our approach, we verified with BEA similar levels of the c.1118A>G mutation in sperm (~1.65x10-5) as measured with DS. We observed an increased mutation frequency with donor?s age in 118 sperm donors of different ages. Moreover, there is a ~5-fold difference between young (?30 years) and middle-aged donors (>45 years). Interestingly, analysis of ~30% of one testis from an 80-year-old man did not show any high-frequency clusters previously observed for other mutations in the FGFR3 (TDII and ACH).
Conclusions: BEA can validate mutations discovered by DS, and more importantly be used for a more detailed and high-throughput screening of age-related expansions of these in the male germline (sperm and testes) to gain further knowledge about the mechanisms in germline mutagenesis and their potential consequences.
Project funded by FWF (P30867-B26).