Rapid Detection of FGFR Mutations in Syndromic Craniosynostosis by Temporal Temperature Gradient Gel Electrophoresis

Several autosomal dominant craniosynostosis syndromes, including Crouzon, Pfeiffer, Apert, and Jackson-Weiss syndromes, are caused by mutations in the fibroblast growth factor receptor (FGFR) gene family (1). Mutations are not uniformly dispersed among these genes, and most mutations described to date are in exons IIIa and IIIc of the FGFR2 gene (2)(3). The advent of molecular analysis of these genes has allowed confirmatory testing in ambiguous cases and for those families desiring prenatal diagnosis. However, molecular analysis is difficult at present, especially for Crouzon syndrome, where current methods rely on the sequencing of the aforementioned exons. We report the rapid and inexpensive detection of FGFR mutations by a relatively novel screening assay, temporal temperature gradient gel electrophoresis (TTGE). TTGE is a heteroduplex detection method similar to denaturing gradient gel electrophoresis (DGGE). Like DGGE, but often unlike single-strand conformation polymorphism and heteroduplex analysis using mutation detection enhancement gels, TTGE is highly sensitive (4)(5)(6). However, TTGE is simpler than DGGE in avoiding the use of a chemical denaturing gradient gel and GC clamps (4). In TTGE, a homogeneous gel is bathed in a tank of buffer in which the temperature of the entire unit increases linearly throughout the electrophoretic run (5). After PCR, the products are heated and allowed to cool gradually, which leads to the formation of heteroduplex DNA if sequence heterogeneity is present. In dominant diseases, such as FGFR mutations that cause craniosynostosis syndromes, the sequence difference between the two alleles in the heteroduplex causes a physical bulge at the site of the sequence mismatch, …