Structural and Biological Characterization of FGFR2(C342Y) Protein Associated with Crouzon Craniosynostosis Syndrome

Abstract

Craniofacial anomalies are among the most common of all birth defects. Craniofacial anomalies can occur in isolation (with no other defects and no established genetic basis), as part of an established syndrome with a known constellation of defects due to single gene mutation or chromosomal abnormality, or in association with additional development defects, but without a known genetic basis. One such craniofacial anomaly that occurs sporadically or with an identified syndrome is craniosynostosis, the premature loss of cranial suture tissue leading to cranial bone fusion and altered craniofacial growth. Craniosynostosis occurs in approximately one in 2000 live births. Decades of research have identified various genetic mutations and cell populations involved in the mechanism of cranial suture fusion. However, mechanisms of craniosynostosis and corresponding craniofacial skeletal malformations, particularly in craniosynostosis syndromes, remain incompletely understood. Crouzon syndrome is the one of the most common craniosynostosis syndromes, with an estimated incidence of 1 to 9 affected infants out of 100,000 live births. In the 1990s, Crouzon syndrome was mapped to an autosomal dominant mutation in the fibroblast growth factor receptor 2 (FGFR2) gene on chromosome 10q25-26. Point mutations in the third immunoglobulin-like domain involving the loss of a cysteine residue are most common, including C278F, C342Y, and C342R. Individuals with Crouzon syndrome have distinct craniofacial features, including bicoronal suture fusion with occasional pansynostosis, severe midface hypoplasia, hypertelorism, and severe ocular proptosis. Unlike other craniosynostosis syndromes linked to FGFR2 mutations, individuals with Crouzon syndrome have no limb or digit abnormalities and generally have no intellectual deficits. FGFR2C342Y is traditionally described as an activating/gain-of-function mutation in the literature based on in vitro studies showing the mutant receptor exhibits ligand independent dimerization and autophosphorylation. However, results from in vivo studies suggest the opposite. Fgfr2C342Y/+ mice exhibit phenotypic similarities to that of Fgfr2IIIc-/- mice, including coronal suture fusion and midface hypoplasia. Fgfr2C342Y/– mice have a more severe craniofacial phenotype than Fgfr2C342Y/+ mice. Overexpression of Fgfr2IIIc in Fgfr2C342Y/+ mice prevents coronal suture fusion. While not explicitly stated in these studies, this in vivo evidence suggests a mechanism other than classic gain-of-function. These discrepancies will be discussed in detail throughout this dissertation. The global purpose of this dissertation is to identify mechanisms underlying the Crouzon craniosynostosis syndrome phenotype. In Chapter 2, I review neural crest cells in craniofacial development, craniofacial anomalies, and the pathogenesis of coronal craniosynostosis. I discuss the various possible mechanisms of craniosynostosis and offer perspectives of how craniosynostosis-linked mutations can differentially affect cells from different embryological lineages. In Chapter 3, I specifically focus on the FGFR2C342Y mutation associated with Crouzon syndrome. I use computational modeling to predict wildtype–mutant heterodimer protein structures and verify this interaction experimentally in Chapter 4. To my knowledge, this is the first time an interaction between wildtype FGFR2 and FGFR2C342Y has been shown. Chapter 4 also includes discussion of biological consequences due to the FGFR2C342Y mutation. Chapter 5 offers an expanded discussion on the implications of my findings on the mechanisms underlying Crouzon syndrome craniofacial development and proposes additional studies for continued investigation. Overall, this dissertation contributes a unique perspective on how we characterize the FGFR2 mutations associated with Crouzon and other craniosynostosis syndromes. Considering the substantial inconsistencies of experimental data published on Crouzon syndrome FGFR2 mutations, this work aims to provide some clarity in our understanding of this human condition.