Development Of Oligonucleotide Biochips For Detection Of Mitochondrial DNA Mutations In Chinese Pedigree

OBJECTIVES Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes(MELAS), myoclonic epilepsy with ragged red fibers(MERRF), and Leber's hereditary optic neuropathy(LHON) are among the most common human diseases primarily caused by mitochondrial DNA point mutations. So far, more than 60 point mutations have been identified to be associated with MELAS, MERRF or LHON. The heterogeneous clinical manifestations often make the clinical diagnosis of these entities difficult, confirming a specific diagnosis remains a great challenge. Molecular genetic studies of mtDNA are helpful in the understanding and classification of mitochondrial diseases. Therefore, detection of disease-causing mutations using modern techniques is no doubt important. This study aimed to develop novel oligonucleotide biochip methods enabling rapid detection of pathogenic point mutations in MELAS, MERRF and LHON.METHODS A set of probes sharing a given allele-specific sequence with a single base substitution near the middle of the sequence was covalently immobilized on aldehyde modified glass slides, and then hybridized with Cy5-labled targets amplified from sample DNAs by a multiplex asymmetric PCR (MAP) method(MITO-MAP-Chip). To discriminate genotype at a given site and to minimize the bench affect between experiments, a diagnostic factor was used based on the fluorescence signal intensity ratio between each wild type and mutant probe after hybridizing with Cy5-labled targets. Sample with the factor over 3 was referred to as a wild-type and the factor below 0.3 was interpreted as a homoplasmic mutant, and the mutation indicated a heteroplasmy if the factor varied between 0.3 and 3. Primers for PCR and ASO probes for genotyping mtDNA point muations were strictly designed, and detection parameters for the oligonucleotide chip-based assay were accordingly optimized before sample screening.RESULTS Two novel oligonucleotide biochips were successfully developed . The first biochip format was used for synchronous detection of 31 known base substitutions in mitochondrial DNA causing MELAS and/or MERRF, and was briefly called MELAS-MERRF Biochip. The second format was specialized in screening of additional 32 known base substitutions in mitochondrial DNA associated with LHON pathogenesis, and was referred to as LHON Biochip. Seven clinically confirmed patients with MELAS, 5 patients with MERRF, 1 suspected MERRF case and 25 healthy controls were validated by MELAS-MERRF Biochip. Nine clinically confirmed patients with LHON, 8 suspected LHON cases and 25 healthy controls were tested by LHON Biochip. The results showed that all the cases with MELAS had a causal heteroplasmic A3243G tRNALeu(UUR) mutation. In the MERRF patients, four cases were found to be a homoplasmic A8344G tRNALys mutation and one case was a heteroplasmic T8356C tRNALys mutation. The suspected MERRF case and the control subjects do carried none of the 31 potential point mutations. On the other hand, in the LHON patients, six cases carried a homoplasmic G11778A MTND4 mutation, one case carried a heteroplasmic G11778A MTND4 mutation, and one case carried a homoplasmic T14484C MTND6 mutation. A bi-base mutation in MTND1 gene was luckily detected in one LHON case: a homoplasmic T3394C mutation and a homoplasmic G3460A mutation. Six cases suspected with LHON were found to have a homoplasmic G11778A MTND1 mutation. Two suspected LHON cases and the control subjects exhibited wild type mtDNA. The results of the MELAS-MERRF Biochip and the LHON Biochip were evaluated, and were completely consistent with those by DNA sequencing.CONCLUSION We developed two biochip- based genotyping methods for screening and discriminating of single base substitution in a panel of known mtDNA mutations of MELAS, MERRF and LHON and evaluated the feasibility of the biochip format in diagnostics of the entities. The oligonucleotide biochips combined with MAP method (Mito-MAP-Chip) were robust and reproducible and would become an available tool in clinical multiplex detecting of point mutations in mtDNA leading to MELAS, MERRF or LHON. The results also showed that the biochips were simple, fast, low-cost and could be easily implemented in standard clinical laboratories. This biochip format, once modified, would potentially extend the screening spectrum of SNPs for any other mitochondrial diseases, improving quality of life and prognosis of those still often neglected or overlooked entities.