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

✍ Scribed by Kirsi Huoponen; Vesa Juvonen; Antti Iitiä; Patrik Dahlen; Harri Siitari; Pertti Aula; Eeva Nikoskelainen; Marja-Liisa Savontaus

• Publisher: John Wiley and Sons
• Year: 1994
• Tongue: English
• Weight: 633 KB
• Volume: 3
• Category: Article
• ISSN: 1059-7794
• DOI: 10.1002/humu.1380030106

✦ Synopsis

Communicated by Lena Peltonen

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 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 members.