Simple and efficient screening of patients with Fabry disease with high resolution melting

Fabry disease (FD [MIM: 301500]) is a disorder caused by mutations in the alpha-galactosidase gene (GLA), which presents great allelic heterogeneity. The development of fast screening methods may reduce costs and length of diagnosis, being particularly important for screening programs of high-risk female patients. Therefore, the purpose of this study was to develop a pre-sequencing genetic screening method based on high resolution melting (HRM) analysis. We performed HRM analysis in one hundred and three individuals, 79 females and 24 males, with a total of 27 different variants in 30 different genotypes. We standardized a protocol using EvaGreen, a release-on-demand dye specific for HRM, added to the PCR reaction. Amplification was performed in a conventional real-time system with HRM capability. All genotypes in all amplicons were distinguishable from wild type. In most amplicons it was even possible to differentiate each genotype from the others. We developed a simple, fast and highly sensitive HRM based protocol that may facilitate genetic screening of FD. •The described HRM protocol is a simple screening method suitable for FD patients.•All genotypes in all amplicons were distinguishable from wild type.•This is the largest cohort of FD patients analyzed by HRM, covering the whole gene.