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.
Highlights
Craniosynostosis, the premature fusion of one or more cranial sutures, is a relatively common malformation with an incidence of 1:2.500 births
The present study aimed to evaluate if p.Ser252Trp fibroblast growth factor 2 receptor (FGFR2) mutant periosteal cells present a greater commitment toward osteogenic differentiation, which could contribute to the pathophysiology of Apert syndrome (AS), and to address if these cells present a transcriptional signature that would be involved in the molecular mechanisms of this syndrome
Cultured coronal suture periosteal cells from wild type controls and AS patients appeared microscopically to be a homogeneous population of adherent fibroblast-like cells, which exhibited neither morphology alteration nor significant cell death after several passages
Summary
Craniosynostosis, the premature fusion of one or more cranial sutures, is a relatively common malformation with an incidence of 1:2.500 births. A transcriptional gene expression signature in periosteal AS cells has been previously reported based on the analysis of only one patient harboring the p.Pro253Arg mutation and two controls [20]. Expression Analysis of FGFR2 by RT-PCR One step RT-PCR (Invitrogen Carlsbad, CA, USA) was performed with 2 μg of total RNA samples from five AS patients and four control subjects and specific primer pairs for each of the two major isoforms of FGFR2 (FGFR2b and FGFR2c). Intensity values from the probes common to both microarray platforms were highly correlated between sample replicates (average Pearson correlation = 0.95 ± 0.01) among the three sets of hybridizations This result confirms that intensity measurements obtained in the different platforms can be compared without any significant loss in accuracy. Transcription of two μg of total RNA using Superscript II (Invitrogen). qRTPCR was used to measure expression levels of STMN1, SPAG5, RRM2, HIP2, CENPN, EEF1B2 after FGF2 stimulation
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