Content:
De novo mutations (DNMs) in germline cells are an important source of genetic variation. However, several DNMs are known players in congenital growth disorders associated with a paternal age effect. Activating DNMs that accumulate in spermatogonia of ageing men have been reported in FGFR3 and other genes that take part in the receptor tyrosine kinase pathway. Even though these mutations can have a major impact in the offspring, the available information on the mutational landscape of the human male germline and its underlying mechanisms is very limited. The direct detection of DNMs expanding in the male germline is extremely challenging due to their ultra-low frequencies, estimated to be as low as 10-6 – 10-4. To overcome that, we used duplex sequencing (DS), a massive parallel sequencing-based technique that has the power to reliably call one mutant in millions of sequenced reads. Due to the stringent nature of this method, reads with potentially valuable information are discarded at different stages of the bioinformatics pipeline. To improve this method, we developed an error correction approach to mitigate the effects of PCR and sequencing errors within the duplex tags. We have also developed a bioinformatic toolset that monitors data loss and maximizes DS data output by analyzing the tag and family composition. Making use of these novel tools, and adapting DS, we targeted ~4.5 kb of the FGFR3 coding region in sperm DNA from younger and older donors to detect prospective gain-of-function variants expanding in the male germline. The overall mutation frequency of the analyzed region was calculated to be ~6 × 10-7 and we successfully identified variants with frequencies of 10-5 – 10-4. Some of these were identified in different donor pools and were detected at higher frequencies or exclusively in older donors. Additionally, more variants associated with congenital disorders were found in this group. We also identified several variants in both age groups that could be associated with a distinct mutational mechanism (e.g., post-zygotic mosaicism). Finally, our variant allele frequency and deleteriousness data suggest the germline variants identified here are comparable to the driver variants reported in tumors for FGFR3. Our work has contributed to further develop the power of DS and has proven to be a relevant strategy for the direct detection of variants potentially associated with the dysregulation of the receptor tyrosine kinase activity. In a society with delayed fatherhood, our approach is particularly important to shed light into the different mutational mechanisms acting in the male germline. ; Author Renato Salazar, MSc ; Dissertation Universität Linz 2022
Note:
Dissertation 2022
Language:
English
URN:
urn:nbn:at:at-ubl:1-48400
