Time-resolved fluorometry in the diagnosis of Leber hereditary optic neuroretinopathy

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Abstract
We have applied time‐resolved fluorometry (TRF) to construct a DNA hybridization assay for the diagnosis of Leber hereditary optic neuroretinopathy (LHON). A rapid and reliable detection of the most prevalent mitochondrial DNA (mtDNA) point mutation associated with LHON is demonstrated. In addition, the TRF‐method can be used in the quantification of heteroplasmy, a phenomenon commonly present in mtDNA mutations. The assay includes PCR amplification of a fragment encompassing the mutation site followed by hybridization reactions with allele‐specific europium (Eu)‐labelled oligonucleotide probes. A time‐resolved fluorometer is used to measure the bound label. The TRF assay was succesfully used to demonstrate the ND4/11778 mutation in patient samples. For quantification of heteroplasmy, synthetic target oligonucleotide mixtures with known ratios of wild‐type and mutated sequences were used as standards to control the hybridization step. The assay allowed the detection of heteroplasmy ranging from 5 to 95%. This was also shown in a family with several heteroplasmic members.