Quantification of point mutations associated with Leber hereditary optic neuroretinopathy by solid-phase minisequencing

Abstract

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.

Accurate Detection and Quantitation of Heteroplasmic Mitochondrial Point Mutations by Pyrosequencing

“…Therefore, diagnostic techniques must be capable of reliably detecting low levels of heteroplasmy in such samples. A number of established techniques have been used to genotype and quantitate the level of heteroplasmy for a variety of mitochondrial mutations; denaturing gradient gel electrophoresis (Tully et al, 2000), single-stranded conformational polymorphism (Mashima et al, 1995;Tanno et al, 1995), real-time fluorescent polymerase chain reaction (PCR; Szuhai et al, 2001;Bai and Wong, 2004;He et al, 2002), temporal temperature gradient gel electrophoresis (Boles et al, 2001), Invader technology (Mashima et al, 2004), denaturing high-performance liquid chromatography (DHPLC) (Conley et al, 2003), solid-phase minisequencing (Juvonen et al, 1994;Suomalainen and Syvanen, 2000), and PCR-restriction fragment length polymorphism (RFLP); (e.g. Holt et al, 1990).…”

mtDNA Haplotype Analysis in Finnish Families with Leber Hereditary Optic Neuroretinopathy

“…Family I belonged to the cluster of families which share 5 mutations (nts 4216,7028,10398, 13708 and 16069), but unlike other families in this cluster, family I does not har bor the ND4/11778 mutation. This was intriguing and therefore we studied the proband of family I for possible low level heteroplasmy of the ND4/11778 mutation in blood, hair, urinary epithelium and buccal mucosa sam ples by restriction site analysis and solid-phase minise quencing [39]. No sign of heteroplasmy for the ND4/ 11778 mutation was observed in any of the tissues studied…”

“…Thirdly, among 11 ND4/11778-positive families there are heteroplasmic families, indicating recent occurrences of the mutation. In families G and P the primary mutation can be traced back to the maternal relative in whom the mutation has arisen [39].…”

Human Mitochondrial Genetics

“…For example, a DGGE assay designed to examine heteroplasmy in hypervariable region I (HV 1) demonstrated sufficient sensitivity to detect a heteroplasmic species present at concentrations as low as I% (Tully, 1998;Steighner et al, 1999). Detection and quantification of the 11,778 Inutation (LHON) at levels as low as 1.50/0 has been achieved using DNA minisequencing (Juvonen et al, 1994). Constant denaturant capillary electrophoresis (CDCE)) a capillary-based modification of DGGE, has been shown to detect heteroplaslnic sequences present at less than 0.03% (Khrapko et al, 1994).…”