Best understanding of structural and functional impact of FGFR3 mutations at the same position (K650N, K650M, K650E) leading to both mild and lethal dwarfism. D. Komla Ebri1, C. Benoist-Lasselin1, N. Kaci1, P. Busca2, G. Prestat2, A. Munnich1, F. Barbault3, L. Legeai-Mallet1 1) INSERM U781, UniversitÚ Paris Descartes - Sorbonne Paris CitÚ, Institut Imagine, Paris, France; 2) UMR CNRS 8601, UniversitÚ Paris Descartes, France; 3) 3ITODYS - UniversitÚ Paris Diderot, France.

   Three different missense mutations in the tyrosine kinase domain of fibroblast growth factor receptor 3 (FGFR3) affecting a lysine residue at position 650 lead to dwarfism with a spectrum of severity, hypochondroplasia (p. Lys650Asn), severe achondroplasia with developmental delay and acanthosis nigricans (p. Lys650Met), and thanatophoric dysplasia (p. Lys650Glu). Fgfr3 mutations induce a constitutive activation of the receptor characterized by a sustained phosphorylation responsible of growth plate defects. To understand the link between the severity of the clinical phenotype and a single amino acid change localized in the tyrosine kinase domain (TKD), we developed in silico and in vitro studies. Computational studies were conducted to get an atomic description of the p. Lys650Met, p. Lys650Glu and p. Lys650Asn built using a validated structural model of the FGFR3 kinase domain. Structural analyses indicate that a salt bridge between R655 and E686 is the cornerstone of the Tyr647 solvent exposition. This salt bridge is, significantly, disturbed with Lys650Glu mutation and is destroyed with Lys650Met and Lys650Asn mutants (p< 0.01). The activation of FGFR3 is initiated with the transphosphorylation of specific tyrosines in TKD, and induces conformation changes in FGFR3 activation loop. These FGFR3 activation loop mutation-related changes have been quantified by measuring the distance between the backbone gravity center of 650-656 aa (activation loop) and the backbone gravity center of 562-565 aa and 620-623 aa. We demonstrated a correlation within the major modifications and the severity of the chondrodysplasias (Lys650Asn 12.2 ┼; Lys650Glu 16.4 ┼; Lys650Met 16.8 ┼). To confirm these structural changes, we studied in vitro the impact of the FGFR3 mutants on signaling pathways. Transient transfections of three DNA mutants at position 650 in chondrocyte show a gradient of phosphorylation levels correlated with the severity of the disease. Using HEK cells transfections, we noted an increased proliferation associated with the severity of the phenotype. Interestingly, we observed a higher activation of the Mapk, STAT and -catenin signalling pathways related to the disease severity. In conclusion, the mutation of lysine 650 alters differently the conformation of the kinase domain thus leading to activate unusual signalling pathways. Various biological mechanisms seem to be responsible for mild and lethal dwarfism.

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