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.