Rapid identification of mitochondrial DNA (mtDNA) mutations in neuromuscular disorders by using surveyor strategy.
ABSTRACT Mutations of mitochondrial genome are responsible for respiratory chain defects in numerous patients. We have used a strategy, based on the use of a mismatch-specific DNA endonuclease named " Surveyor Nuclease", for screening the entire mtDNA in a group of 50 patients with neuromuscular features, suggesting a respiratory chain dysfunction. We identified mtDNA mutations in 20% of patients (10/50). Among the identified mutations, four are not found in any mitochondrial database and have not been reported previously. We also confirm that mtDNA polymorphisms are frequently found in a heteroplasmic state (15 different polymorphisms were identified among which five were novel).
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ABSTRACT: High-sensitivity and high-throughput mutation detection techniques are useful for screening the homoplasmy or heteroplasmy status of mitochondrial DNA (mtDNA), but might be susceptible to interference from nuclear mitochondrial DNA sequences (NUMTs) co-amplified during polymerase chain reaction (PCR). In this study, we first evaluated the platform of SURVEYOR Nuclease digestion of heteroduplexed DNA followed by the detection of cleaved DNA by using the WAVE HS System (SN/WAVE-HS) for detecting human mtDNA variants and found that its performance was slightly better than that of denaturing high-performance liquid chromatography (DHPLC). The potential interference from co-amplified NUMTs on screening mtDNA heteroplasmy when using these 2 highly sensitive techniques was further examined by using 2 published primer sets containing a total of 65 primer pairs, which were originally designed to be used with one of the 2 techniques. We confirmed that 24 primer pairs could amplify NUMTs by conducting bioinformatic analysis and PCR with the DNA from 143B-ρ0 cells. Using mtDNA extracted from the mitochondria of human 143B cells and a cybrid line with the nuclear background of 143B-ρ0 cells, we demonstrated that NUMTs could affect the patterns of chromatograms for cell DNA during SN-WAVE/HS analysis of mtDNA, leading to incorrect judgment of mtDNA homoplasmy or heteroplasmy status. However, we observed such interference only in 2 of 24 primer pairs selected, and did not observe such effects during DHPLC analysis. These results indicate that NUMTs can affect the screening of low-level mtDNA variants, but it might not be predicted by bioinformatic analysis or the amplification of DNA from 143B-ρ0 cells. Therefore, using purified mtDNA from cultured cells with proven purity to evaluate the effects of NUMTs from a primer pair on mtDNA detection by using PCR-based high-sensitivity methods prior to the use of a primer pair in real studies would be a more practical strategy.PLoS ONE 03/2014; 9(3):e92817. · 3.53 Impact Factor
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ABSTRACT: We report an unusual case of Leigh syndrome due to the m.10191T>C mutation in the complex I gene MT-ND3. This mutation has been associated with a spectrum of clinical phenotypes ranging from infant lethality to adult onset. Despite infantile onset and severe symptoms, our patient has survived to early adulthood because of a strict dietary regimen and parental care. This patient is an extreme example of the frequently prolonged course of Leigh syndrome due to this particular mutation.Journal of child neurology 11/2013; · 1.59 Impact Factor
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ABSTRACT: Sticky rice–lime mortar is one of the most important technological inventions in the Chinese architectural history. In this work, an experimental study on the application of sticky rice–lime mortar in conservation of the stone tower in the Xiangji Temple was performed by means of the property characterization, SEM, XRD and DSC-TG analysis. Our Results indicate that paper fiber is the most effective admixture to endure freezing–thawing cycles and increase the compressive strength of sticky rice–lime mortar, and the use of aluminum sulfate reduces the drying shrinkage values and increases the compressive strength because of the formation of ettringite in sticky rice–lime mortar. It is suggested that 6% aluminum sulfate or 3% paper fiber may be the appropriate admixture of sticky rice–lime mortar in conservation of cultural relics. Based on the results of laboratory experiment, the sticky rice–lime mortar with 3% paper fiber is selected as the grouting material for the conservation of the Xiangji Temple tower.Construction and Building Materials 03/2012; · 2.27 Impact Factor