Functional analysis of driver mutations in the FGFR3 expanding with paternal age
Sprache des Vortragstitels:
European Society of Human Genetics Conference 2019
Sprache des Tagungstitel:
Certain point mutations in the FGFR3 gene have a very high mutation frequency in sperm compared to the genome average that increases with age. Studies proposed this effect to be linked with single nucleotide substitutions causing the expansion of spermatogonial stem cells following a germline selection model. As a tyrosine receptor kinase (RTK) family member, FGFR3 is involved in the regulation of cell proliferation, differentiation and apoptosis. Therefore, severe phenotypes are associated with aberrant receptor signaling caused by these mutations, which have also been detected in numerous human cancers.
Our lab is currently screening for yet undescribed de novo mutations in the FGFR3 occurring at increased levels in human sperm with duplex sequencing. These newly discovered mutations are tested for their functional changes in a structure-function-analysis. Specifically, we are determining potentially altered signaling of different mutated FGFR3 in comparison to the wild-type receptor at three different levels: 1) Analysis of tyrosine phosphorylation in the kinase region of the receptor via phospho-specific Western blotting; 2) Measuring the dynamics of receptor stoichiometry on the cell surface using single molecule microscopy (TOCCSL); 3) Evaluation of intracellular calcium kinetics by ratiometric analysis of Fura-2 loaded cells.
In this study we are functionally characterizing newly described rare mutations arising in the male germline using highly quantitative biophysical methods. Therefore, this study will provide new insights in the processes underlying increased signaling of mutated RTKs coupled with aberrant signaling in mutant cells.