High-Resolution Melting Analysis for Rapid Detection of Mutations in Patients with FGFR3 -Related Skeletal Dysplasias

Mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are related to skeletal dysplasias (SDs): acondroplasia (ACH), hypochodroplasia (HCH) and type I (TDI) and II (TDII) tanatophoric dysplasias. This study was designed to standardize and implement a high-resolution melting (HRM) technique to identify mutations in patients with these phenotypes. Initially, gene segments from 84 patients were PCR amplified and subjected to Sanger sequencing. Samples from 29 patients positive for mutations were analyzed by HRM. Twelve of the patients mutations had ACH (six g.16081 G > A, three g.16081 G > C and three g.16081 G > A + g.16002 C > T); thirteen of patients with HCH had mutations (eight g.17333 C > A, five g.17333 C > G and five were negative); and four patients with DTI had mutations (three g.13526 C > T and one g.16051G > T and two patients with DTII (presented mutation g.17852 A > G). When analyzing the four SDs altogether, an overlap of the dissociation curves was observed, making genotyping difficult. When analyzed separately, however, the HRM analysis method proved to be efficient for discriminating among the mutations for each SD type, except for those patients carrying additional polymorphism concomitant to the recurrent mutation. We conclude that for recurrent mutations in the gene, that the HRM technique can be used as a faster, reliable and less expensive genotyping routine for the diagnosis of these pathologies than Sanger sequencing.