TIME-RESOLVED FLUOROMETRY IN THE DIAGNOSIS OF LEBER HEREDITARY OPTIC NEURORETINOPATHY

We have applied time-reserved fluorometry (TRF) to construct a DNA hyb ridization assay for the diagnosis of Leber hereditary optic neuroreti nopathy (LHON). A rapid and reliable detection of the most prevalent m itochondrial DNA (mtDNA) point mutation associated with LHON is demons trated. 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 mutat ion site followed by hybridization reactions with allele-specific euro pium (Eu)-labelled oligonucleotide probes, A time-resolved fluorometer is used to measure the bound label. The TRF assay was successfully us ed to demonstrate the ND4/11778 mutation in patient samples, For quant ification of heteroplasmy, synthetic target oligonucleotide mixtures w ith known ratios of wild-type and mutated sequences were used as stand ards to control the hybridization step. The assay allowed the detectio n of heteroplasmy ranging from 5 to 95%. This was also shown in a fami ly with several heteroplasmic members. (C) 1994 Wiley-Liss, Inc.