A real-time ARMS PCR/high-resolution melt curve assay for the detection of the three primary mitochondrial mutations in Leber's hereditary optic neuropathy.

PURPOSE: Approximately 95% of patients who are diagnosed with Leber's hereditary optic neuropathy (LHON) have one of three mitochondrial point mutations responsible for the disease, G3460A, G11778A, and T14484C. The purpose of this study was to develop a novel multiplex real-time amplification-refractory mutation system (ARMS) PCR combined with high-resolution melt curves to identify the individual mutations involved. The study aimed to provide a more robust, cost- and time-effective mutation detection strategy than that offered with currently available methods. The assay reported in this study will allow diagnostic laboratories to avoid costly next-generation sequencing (NGS) assays for most patients with LHON and to focus resources on patients with unknown mutations that require further analysis. METHODS: The test uses a combination of multiplex allele-specific PCR (ARMS PCR) in combination with a high-resolution melt curve analysis to detect the presence of the mutations in G3460A, G11778A, and T14484C. PCR primer sets were designed to produce a control PCR product and PCR products only in the presence of the mutations in 3460A, 11778A, and 14484C in a multiplex single tube format. Products produce discrete well-separated melt curves to clearly detect the mutations. RESULTS: This novel real-time ARMS PCR/high-resolution melt curve assay accurately detected 95% of the mutations that cause LHON. The test has proved to be robust, cost- and time-effective with the real-time closed tube system taking approximately 1 h to complete. CONCLUSIONS: A novel real-time ARMS PCR/high-resolution melt curve assay is described for the detection of the three primary mitochondrial mutations in LHON. This test provides a simple, robust, easy-to-read output that is cost- and time-effective, thus providing an alternative method to individual endpoint PCR-restriction fragment length polymorphism (RFLP), PCR followed by Sanger sequencing or pyrosequencing, and next-generation sequencing.

Development and validation of a novel PCR-RFLP based method for the detection of 3 primary mitochondrial mutations in Leber's hereditary optic neuropathy patients.

BACKGROUND: Leber's Hereditary Optic Neuropathy (LHON; MIM 535000) is one of the most commonly inherited optic neuropathies and it results in significant visual morbidity among young adults with a peak age of onset between the ages of 15-30. The worldwide incidence of LHON is approximately 1 in 31,000. 95 % of LHON patients will have one of 3 primary mitochondrial mutations, G3460A (A52T of ND1), G11778A (R340H of ND4) and T14484C (M64V of ND6). There is incomplete penetrance and a marked gender bias in the development of visual morbidity with approximately 50 % of male carriers and 10 % of female carriers developing optic neuropathy. Visual recovery can occur but is dependent on the mutation present with the highest level of visual recovery seen in patients who have the T14484C mutation. The 3 primary mutations are typically identified by individual end-point PCR-restriction fragment length polymorphism (RFLP) or individual targeted bi-directional Sanger sequencing reactions. The purpose of this study was to design a simple multiplex PCR-RFLP that could detect these 3 primary LHON mutations in one assay. METHODS: PCR primers were designed to incorporate a MaeIII restriction site in the presence of 3460A and 14484C mutations with the 11778A mutation naturally incorporating a MaeIII site. A multiplex PCR-RFLP assay was developed to detect the 3 common mutations in a single assay. Synthetic LHON controls based on the mitochondrial genome harbouring the 3 common mutations were synthesized and cloned into plasmids to act as reliable assay controls. DNA from previously tested patients and the synthetic LHON controls were subjected to the multiplex PCR-RFLP assay. The RFLP products were detected by agarose gel electrophoresis. RESULTS: The novel PCR-RFLP assay accurately detects the 3 primary mutations both in patient DNA and in synthesized DNA control samples with a simple visual mutation detection procedure. The synthesized DNA was demonstrated to be a robust control for the detection of LHON Mutations. CONCLUSION: In this paper, we describe a novel, robust and simple PCR-RFLP based method for the detection of mutations causing LHON, and report the generation of a series of LHON DNA controls suitable for all currently published assays.