FLNA Variants Associated with Single Suture Craniosynostosis

Abstract

Craniosynostosis (CS) is a common birth defect affecting the skull vault. It is characterized by premature ossification of one or more of the cranial sutures. Single suture craniosynostosis (SSC) accounts for approximately 85% of all CS cases and affects approximately 1 in 2000- 2500 live births worldwide, with a higher rate in males than females. It is a significant craniofacial disorder associated with increased intracranial pressure, distortion of the skull shape, facial deformities, misalignment of the teeth, and mortality. Patients with CS may undergo invasive surgical procedures and experience neuropsychological trauma associated with self-image. Although several genetic mutations have been attributed to the pathogenesis of syndromic craniosynostosis, the etiology and molecular mechanisms of SSC remain largely unknown. Thus, identification of candidate genes or signaling pathways involved in intramembranous ossification and suture development is critical in understanding the underlying biology of SSC. Through generation of primary cell lines from individuals with SSC and the use of Next Generation RNA sequencing (RNA-Seq), our lab has identified a set of rare genetic variants we propose are associated with craniosynostosis. Among these are nine variants in Filamin A (FLNA), a gene which codes for a mechanosensitive, actin-binding extracellular matrix protein. FLNA is one of the most abundant proteins in the cytoskeleton and regulates cell polarization, cell shape, cell adhesion, cell contraction and cell migration and protects the cell from mechanical shear stress. FLNA also modulates the functional activities of several other matrix proteins. It binds and crosslinks actin filaments and remodels the cytoskeleton by interacting with the adhesive transmembrane receptor Integrin b1, vimentin, and the Rho family GTPases (Rac1, Cdc42, RaIA and RhoA). In doing so, FLNA coordinates a variety of cellular processes and cell signaling. In the context of osteogenesis, FLNA plays an important role in the regulation of calvarial mesenchymal stem cells and osteoblasts as it acts on pivotal signaling pathways. FLNA influences osteogenic gene expression by binding transcription factor FOXC1 and mediating BMP signaling. It also binds SMAD2 and SMAD5 regulating both TGFb and BMP signaling, respectively. Missense mutations in FLNA have been described in otopalatodigital spectrum disorders which are associated with bone dysplasia and craniosynostosis. In this dissertation, I isolated and characterized primary human calvarial MSCs (cMSCs) from primary calvarial osteoblasts (cOBs). Then, I tested the hypothesis that the FLNA variants we identified in patients with SSC contribute to craniosynostosis through dysregulating cellular proliferation and differentiation, and attenuating cell mechanics. I assessed the contribution of FLNA mutations in the pathogenesis of craniosynostosis through characterizing the impact of nine rare variants on the development and cellular mechanics of cMSCs and cOBs. This work has revealed that cMSCs have a distinct gene expression profile from bone morrow mesenchymal stem cells (BMMSCs). We have also found that the identified rare FLNA variants affected biological properties, attenuated cellular mechanics, and altered osteogenic gene .expression pattern in SSC patients