Apert p.Ser252Trp mutation in FGFR2 alters osteogenic potential and gene expression of cranial periosteal cells

Abstract

Apert syndrome (AS), a severe form of craniosynostosis, is caused by dominant gain-of-function mutations in FGFR2. Because the periosteum contribution to AS cranial pathophysiology is unknown, we tested the osteogenic potential of AS periosteal cells (p.Ser252Trp mutation) and observed that these cells are more committed toward the osteoblast lineage. To delineate the gene expression profile involved in this abnormal behavior, we performed a global gene expression analysis of coronal suture periosteal cells from seven AS patients (p.Ser252Trp), and matched controls. We identified 263 genes with significantly altered expression in AS samples (118 upregulated, 145 downregulated; SNR >or= |0.4|, P <or= 0.05). Several upregulated genes are involved in positive regulation of cell proliferation and nucleotide metabolism, whereas several downregulated genes are involved in inhibition of cell proliferation, gene expression regulation, cell adhesion, and extracellular matrix organization, and in PIK3-MAPK cascades. AS expression profile was confirmed through real-time PCR of a selected set of genes using RNAs from AS and control cells as well as from control cells treated with high FGF2 concentration, and through the analysis of genes involved in FGF-FGFR signaling. Our results allowed us to: (a) suggest that AS periosteal cells present enhanced osteogenic potential, (b) unravel a specific gene expression signature characteristic of AS periosteal cells which may be associated with their osteogenic commitment, (c) identify a set of novel genes involved in the pathophysiology of AS or other craniosynostotic conditions, and (d) suggest for the first time that the periosteum might be involved in the pathophysiology of AS.

Figure 1

(A) Morphology of undifferentiated AS periosteal cells. (B and C) Osteogenic differentiation of periosteal cells after 21 days of culture in osteogenic medium. Secretion of a calcified extracellular matrix was clearly observed. (D) Control cells after 21 days of osteogenic induction without signals of osteogenic differentiation (mineralized matrix are absent). Differentiation was accessed by von Kossa staining.

Figure 2

Hierarchical clustering of a 263 gene expression signature of AS samples relative to normal control samples. Individual genes are represented in lines and different samples are represented in rows. Expression level of each gene is represented by the number of standard deviations above (red) or below (blue) the average value for that gene across all samples. Color intensity is proportional to the number of standard deviations in the range −1.5 to 1.5, as indicated by the color-coded bar at the bottom of the figure.

Figure 3

Real-time PCR showing expression levels for six genes identified as upregulated in AS cells by microarray analysis: STMN1, SPAG5, RRM2, HIP2, CENPN, and EEF1B2. The expression levels for these genes are enhanced in AS samples when compared with controls.

Figure 4

Real-time PCR showing expression levels for six genes (STMN1, SPAG5, RRM2, HIP2, CENPN, and EEF1B2) in response to FGF2 treatment. The bars are representing the expression levels of each gene before administration of 0.5 percent FBS and FGF2 (0 h), after 24 h of administration of 0.5 percent FBS (0.5 percent FBS) and after addition of 0.5 percent FBS and FGF2 (0.5 percent FBS + FGF2).