The fibroblast growth factor receptor 3 (FGFR3) activation leads to dwarfism with a spectrum of severity, hypochondroplasia (HCH), severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN), and thanatophoric dysplasia (TD). Interestingly, FGFR3 mutations localized at the same position in the tyrosine kinase domain are responsible for HCH (p. Lys650Asn), SADDAN (p. Lys650Met) and TD (p. Lys650Glu).
The mechanisms of FGFR3 activation for these three mutants are unknown. To decipher these mechanisms, we developed in silico, in vitro and in vivo studies.
Computational studies were conducted to get an atomic description of the p. Lys650Met, p. Lys650Glu and p. Lys650Asn. FGFR3 activation loop mutation-related changes have been quantified by measuring the distance between the activation loop and the C-terminal domain. We demonstrated a correlation within the major modifications and the severity of the dwarfism (Lys650Asn 12.2 Æ; Lys650Glu 16.4 Æ; Lys650Met 16.8 Æ).
To evaluate these structural changes, we transfected three DNA mutants in chondrocyte, we observed a gradient of phosphorylation levels correlated with the severity of the disease. A higher activation of the ERK1/2, AKT, ADAMTS5 and β-catenin signalling pathways was observed in the more severe dwarfism.
We complete these analyses with in vivo transient overexpression of fgfr3 wild type and mutant in zebrafish. At 96hpf injected zebrafish (1 cell stage) present a gradient of skeletal development anomalies correlated with the severity of the dwarfism. 45% of larvae injected with TD mRNA fgfr3Lys650Glu (TD) are highly malformed with axial and craniofacial anomalies whereas mRNA fgfr3Lys650Asn (HCH) injections induce mild skeletal anomalies.
Altogether, the data confirm that the mutation of lysine 650 alters differently the conformation of the kinase domain thus leading to activate unusual signalling pathways and to modify the ossification process. Various biological mechanisms seem to be responsible for mild and lethal dwarfism.
14 - 17 May 2016
European Calcified Tissue Society