Gabriela Pasqualim1, Bruna Almeida Dos Santos2, Roberto Giugliani3, Ursula Matte4. 1. Post-Graduation Program on Genetics and Molecular Biology, UFRGS, Porto Alegre, RS 91501-970, Brazil; Gene Therapy Center, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS 90035-903, Brazil. 2. Gene Therapy Center, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS 90035-903, Brazil. 3. Post-Graduation Program on Genetics and Molecular Biology, UFRGS, Porto Alegre, RS 91501-970, Brazil; Gene Therapy Center, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS 90035-903, Brazil; Medical Genetics Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS 90035-903, Brazil; Department of Genetics, UFRGS, Porto Alegre, RS 91501-970, Brazil; INAGEMP, Porto Alegre, RS 90035-903, Brazil. 4. Post-Graduation Program on Genetics and Molecular Biology, UFRGS, Porto Alegre, RS 91501-970, Brazil; Gene Therapy Center, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS 90035-903, Brazil; Department of Genetics, UFRGS, Porto Alegre, RS 91501-970, Brazil. Electronic address: umatte@hcpa.edu.br.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: All genotypes in all amplicons were distinguishable from wild type. In most amplicons it was even possible to differentiate each genotype from the others. CONCLUSION: We developed a simple, fast and highly sensitive HRM based protocol that may facilitate genetic screening of FD.
BACKGROUND: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. METHODS: 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. RESULTS: All genotypes in all amplicons were distinguishable from wild type. In most amplicons it was even possible to differentiate each genotype from the others. CONCLUSION: We developed a simple, fast and highly sensitive HRM based protocol that may facilitate genetic screening of FD.