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

Abstract

Communicated by Lena PeltonenWe 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 mitochondria1 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 The recent identification of the two most prevalent mutations associated with the disease has opened possibilities for specific laboratory diagnosis. These mutations are located in two mitochondrial genes encoding subunits of NADH:ubiquinone oxidoreductase, or complex I. A G-to-A transition at nt 11778 was first found by Wallace et al. (1988), and subsequently in about half of all LHON families (Holt et al., 1989;Vilkki et al., 1989;Bolhuis et al., 1990;Poulton et al., 1991).The second mutation, a G-to-A transition at nt 3460 in the ND1 gene, was first detected by Huoponen et al. (1991) and it accounts for about 13% of LHON cases (Howell et al., 1991;Johns, 1992;Norby et al., 1992 by Tanno et al. (1991), there is n o Southern hybridization and problems caused by heteroduplexes, since they have used a radioactively labelled PCR primer in the last cycle of PCR. By using PCR with allele-specific priming, the restriction enzyme treatment can be omitted (Norby et al., 1991). Furthermore, a technique called solidphase minisequencing (Syvanen et al., 1990) has turned out to be a promising tool for diagnostic purposes and for the detection of heteroplasmy in mitochondrial diseases (Suomalainen et al. , 1993;Juvonen et al., 1993).In order to introduce a specific and simple diagnostic test for LHON, we have applied a timeresolved fluorometry (TRF) method (Soini and Lovgren, 1987) for the identification of the ND4/ 11778 mutation and for the quantification of heteroplasmy. TRF has earlier been used for the detection of viral DNA in clinical specimes (Dahlen et al., 1988(Dahlen et al., , 1991 and of the mutations in nuclear genes (Iitia et al., 1992a, b). Here we demonstrate that the TRF method can also be used in the detection of mitochondrial point mutations and quantification of heteroplasmy. MATERIALS AND METHODS The Principle of the TRF AssaySchematic presentation of the TRF assay is shown in Figure 1. First, the DNA fragment encomp...