Integrated Strategy for Fast and Automated Molecular Characterization of Genes Involved in Craniosynostosis

Background: Craniosynostosis, the premature fusion of 1 or more sutures of the skull, is a common congenital defect, with a prevalence of 1 in 2500 live births. Untreated progressive craniosynostosis leads to inhibition of brain growth and increased intracranial and intraorbital pressure. The heterogeneity of clinical phenotypes and the overlap of the various associated syndromes render the correct diagnosis of the different craniosynostoses particularly difficult. Methods: To identify 10 common mutations in the genes for fibroblast growth factor receptors 2 and 3 (FGFR2 and FGFR3), we developed a microelectronic microchip assay that exploited the PCR multiplexing format and coupled it with serial addressing and probe hybridization on the same pad. For the molecular characterization of patients who tested negative in the microchip screening, we also developed conditions for denaturing HPLC (DHPLC) analysis of the most mutated regions of FGFR2 and FGFR3 and the entire coding region of the TWIST1 gene. Results: In our cohort of 159 patients with various craniosynostosis syndromes, mutations were found in 100% of patients with Apert syndrome, 83.3% with Pfeiffer syndrome, 72.7% with Crouzon syndrome, 50.0% with Saethre-Chotzen syndrome, 27.7% with plagiocephaly, 31.8% with brachicephaly, 20% of complex cases, and 6.9% of mixed cases. No mutations were found in syndromic cases. Conclusions: The combined microchip-DHPLC strategy allows rapid and specific molecular diagnosis of craniosynostosis and is an effective tool for the medical and surgical management of these common congenital anomalies in a newborn or an infant with a developmental defect of the cranial vault.