BACKGROUND: Age-related macular degeneration (AMD) is a complex disorder causing irreversible central vision loss. Complement Factor H (CFH) and age-related maculopathy susceptibility 2 (ARMS2) are now widely accepted as important AMD susceptibility genes. In particular, two specific variants, CFH p.Y402H and ARMS2 p.A69S, have been reported as strongly AMD associated. In order to perform the genetic screening of these single nucleotide polymorphisms (SNPs), we describe a high resolution melting analysis (HRM) as a rapid closed tube mutation scanning assay. METHODS: To validate HRM genotyping, 94 DNA samples from AMD patients (previously genotyped by sequence analysis) were analyzed. PCR amplification and melting curve analysis were performed in the LightCycler 480 Real-Time PCR System. In order to evaluate the accuracy of the HRM assay, we performed a blinded study of 20 unknown independent samples. RESULTS: We correctly genotyped all samples. In fact, all samples corresponded to the previous genotype assignments. CONCLUSIONS: Early identification of individuals with genetic risk variants CFH p.Y402H and ARMS2 p.A69S is clinically important for the definition of AMD status. High-resolution DNA melting is homogenous, accurate and rapid method for CFH and ARMS2 genotyping.
BACKGROUND: Age-related macular degeneration (AMD) is a complex disorder causing irreversible central vision loss. Complement Factor H (CFH) and age-related maculopathy susceptibility 2 (ARMS2) are now widely accepted as important AMD susceptibility genes. In particular, two specific variants, CFHp.Y402H and ARMS2p.A69S, have been reported as strongly AMD associated. In order to perform the genetic screening of these single nucleotide polymorphisms (SNPs), we describe a high resolution melting analysis (HRM) as a rapid closed tube mutation scanning assay. METHODS: To validate HRM genotyping, 94 DNA samples from AMDpatients (previously genotyped by sequence analysis) were analyzed. PCR amplification and melting curve analysis were performed in the LightCycler 480 Real-Time PCR System. In order to evaluate the accuracy of the HRM assay, we performed a blinded study of 20 unknown independent samples. RESULTS: We correctly genotyped all samples. In fact, all samples corresponded to the previous genotype assignments. CONCLUSIONS: Early identification of individuals with genetic risk variants CFHp.Y402H and ARMS2p.A69S is clinically important for the definition of AMD status. High-resolution DNA melting is homogenous, accurate and rapid method for CFH and ARMS2 genotyping.