QUANTIFICATION OF POINT MUTATIONS ASSOCIATED WITH LEBER HEREDITARY OPTIC NEURORETINOPATHY BY SOLID-PHASE MINISEQUENCING

About two-thirds of patients with Leber hereditary optic neuroretinopathy (LHON) harbor mutations in mitochondrial DNA at positions 11778 (ND4) or 3460 (ND1). Thus, the clinical diagnosis of LHON can often be confirmed with mutation analysis. Detection of pathogenic mutations and quantification of heteroplasmy has mainly relied on PCR and restriction site analysis and densitometric scanning. We applied the recently developed solid-phase minisequencing method, based on primer-guided nucleotide incorporation, to the simultaneous detection and quantitation of the ND4/11778 and ND1/3460 mutations. The method was highly sensitive, heteroplasmy as low as 1.5% being easily detected. Rapid, reproducible, and accurate results prove solid-phase minisequencing to be the method of choice for quantitative analysis of LHON mutations.