Quantitative Mitochondrial DNA Mutation Analysis by Denaturing HPLC

Abstract Background: In recent years, denaturing HPLC (DHPLC) has been widely used to screen the whole mitochondrial genome or specific regions of the genome for DNA mutations. The quantification and mathematical modeling of DHPLC results is, however, underexplored. Methods: We generated site-directed mutants containing some common mutations in the mitochondrial DNA (mtDNA) tRNA(leu) region with different mutation loads and used PCR to amplify the gene segment of interest in these mutants. We then performed restriction digestion followed by slow reannealing to induce heteroduplex formation and analyzed the samples by use of DHPLC. Results: We observed a quadratic relationship between the heteroduplex peak areas and mutant loads, consistent with the kinetics of heteroduplex formation reported by others. This was modeled mathematically and used to quantify mtDNA mutation load. The method was able to detect a mutation present in a concentration as low as 1% and gave reproducible measurements of the mutations in the range of 2.5%–97.5%. Conclusion: The quantitative DHPLC assay is well suited for simultaneous detection and quantification of DNA mutations.

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Oxidative Insults and Mitochondrial DNA Mutation Promote Enhanced Autophagy and Mitophagy Compromising Cell Viability in Pluripotent Cell Model of Mitochondrial Disease

Cells ◽

10.3390/cells8010065 ◽

2019 ◽

Vol 8 (1) ◽

pp. 65 ◽

Cited By ~ 9

Author(s):

Dar-Shong Lin ◽

Yu-Wen Huang ◽

Che-Sheng Ho ◽

Pi-Lien Hung ◽

Mei-Hsin Hsu ◽

...

Keyword(s):

Mitochondrial Dna ◽

Cell Viability ◽

Mitochondrial Disease ◽

Cell Model ◽

Ips Cells ◽

Mitochondrial Dna Mutation ◽

Dna Mutation ◽

Dna Mutations ◽

Cell Death Pathway ◽

Induced Pluripotent

Dysfunction of mitochondria causes defects in oxidative phosphorylation system (OXPHOS) and increased production of reactive oxygen species (ROS) triggering the activation of the cell death pathway that underlies the pathogenesis of aging and various diseases. The process of autophagy to degrade damaged cytoplasmic components as well as dysfunctional mitochondria is essential for ensuring cell survival. We analyzed the role of autophagy inpatient-specific induced pluripotent stem (iPS) cells generated from fibroblasts of patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) with well-characterized mitochondrial DNA mutations and distinct OXPHOS defects. MELAS iPS cells recapitulated the pathogenesis of MELAS syndrome, and showed an increase of autophagy in comparison with its isogenic normal counterpart, whereas mitophagy is very scarce at the basal condition. Our results indicated that the existence of pathogenic mtDNA alone in mitochondrial disease was not sufficient to elicit the degradation of dysfunctional mitochondria. Nonetheless, oxidative insults induced bulk macroautophagy with the accumulation of autophagosomes and autolysosomes upon marked elevation of ROS, overload of intracellular calcium, and robust depolarization of mitochondrial membrane potential, while mitochondria respiratory function was impaired and widespread mitophagy compromised cell viability. Collectively, our studies provide insights into the dysfunction of autophagy and activation of mitophagy contributing to the pathological mechanism of mitochondrial disease.

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`Secondary' 4216/ND1 and 13708/ND5 Leber's hereditary optic neuropathy mitochondrial DNA mutations do not further impair in vivo mitochondrial oxidative metabolism when associated with the 11778/ND4 mitochondrial DNA mutation

Brain ◽

10.1093/brain/123.9.1896 ◽

2000 ◽

Vol 123 (9) ◽

pp. 1896-1902 ◽

Cited By ~ 39

Author(s):

R. Lodi

Keyword(s):

Mitochondrial Dna ◽

Optic Neuropathy ◽

Oxidative Metabolism ◽

Mitochondrial Dna Mutation ◽

Dna Mutation ◽

Mitochondrial Dna Mutations ◽

Dna Mutations ◽

Mitochondrial Oxidative Metabolism ◽

Hereditary Optic Neuropathy

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Genetic mosaic analysis of a deleterious mitochondrial DNA mutation in Drosophila reveals novel aspects of mitochondrial regulation and function

Molecular Biology of the Cell ◽

10.1091/mbc.e14-11-1513 ◽

2015 ◽

Vol 26 (4) ◽

pp. 674-684 ◽

Cited By ~ 31

Author(s):

Zhe Chen ◽

Yun Qi ◽

Stephanie French ◽

Guofeng Zhang ◽

Raúl Covian Garcia ◽

...

Keyword(s):

Mitochondrial Dna ◽

Cytochrome C ◽

Cytochrome C Oxidase ◽

Mtdna Mutation ◽

Mitochondrial Dna Mutation ◽

Tissue Specific ◽

Dna Mutation ◽

Mtdna Mutations ◽

Genetic Mosaic ◽

Age Related

Various human diseases are associated with mitochondrial DNA (mtDNA) mutations, but heteroplasmy—the coexistence of mutant and wild-type mtDNA—complicates their study. We previously isolated a temperature-lethal mtDNA mutation in Drosophila, mt:CoIT300I, which affects the cytochrome c oxidase subunit I (CoI) locus. In the present study, we found that the decrease in cytochrome c oxidase (COX) activity was ascribable to a temperature-dependent destabilization of cytochrome a heme. Consistently, the viability of homoplasmic flies at 29°C was fully restored by expressing an alternative oxidase, which specifically bypasses the cytochrome chains. Heteroplasmic flies are fully viable and were used to explore the age-related and tissue-specific phenotypes of mt:CoIT300I. The proportion of mt:CoIT300I genome remained constant in somatic tissues along the aging process, suggesting a lack of quality control mechanism to remove defective mitochondria containing a deleterious mtDNA mutation. Using a genetic scheme that expresses a mitochondrially targeted restriction enzyme to induce tissue-specific homoplasmy in heteroplasmic flies, we found that mt:CoIT300I homoplasmy in the eye caused severe neurodegeneration at 29°C. Degeneration was suppressed by improving mitochondrial Ca2+ uptake, suggesting that Ca2+ mishandling contributed to mt:CoIT300I pathogenesis. Our results demonstrate a novel approach for Drosophila mtDNA genetics and its application in modeling mtDNA diseases.

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Arsenic Trioxide Induces Mitochondrial DNA Mutation on D-Loop Region in Patients with Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v108.11.4356.4356 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4356-4356

Author(s):

Jin Zhou ◽

Ran Meng

Keyword(s):

Acute Myeloid Leukemia ◽

Mitochondrial Dna ◽

Myeloid Leukemia ◽

Mtdna Mutation ◽

Mitochondrial Dna Mutation ◽

Dna Mutation ◽

D Loop ◽

Acute Myeloid

Abstract Objective:To investigate the effect of arsenic trioxide (As2O3) on mitochondrial DNA (mtDNA) in acute myeloid leukemia cells. Methods: NB4 cell line and primary leucocytes, isolated from peripheral blood of eight cases with newly diagnosed acute myeloid leukemia (the ratio of the blast cell was more than 80%), were incubated in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, and treated with 0.1, 1.0, 2.0 micromol/L of As2O3 for 48hrs respectively in vitro. Blood samples in eight cases above were collected before and 14 days after As2O3 treatment with the dosage of 0.16mg/kg.d in vivo. Genome DNA was isolated from the cells and blood samples by salt fractionation, and the D-loop fragment of mtDNA was isolated from the genome DNA and amplified by polymerase chain reaction, and the sequencing techniques were applied to identify positive clones. MTT assay, electrophoresis of genomic DNA, and protein/DNA dual parameter flow cytometry were used to examine the effect of As2O3 on cell proliferation and apoptosis. Results: mtDNA mutations were found on D-loop fragment of mtDNA in both NB4 cells and primary leucocytes, the number of single nucleotide polymorphism (SNP) or mutation were increased remarkably after 1.0, 2.0 micromol/L of As2O3 treatment both in vitro and in vivo than non-As2O3 treated baseline. The types of mutation were included the base transversion, transition, depletion and insertion. The 1.0 and 2.0 micromol/L of As2O3-treated samples revealed apoptosis, reduced proliferation and mtDNA mutation, while in the 0.1 micromol/L of As2O3-treated samples, showed reduced proliferation, differentiation and mtDNA mutation, but no apoptosis was appeared. Conclusions: As2O3 promoted leucocytes’ mtDNA mutation on D-loop fragment both in NB4 cell line and in primary leucocytes isolated from patients with acute myeloid leukemia. Mitochondrial DNA might be one of the targets of As2O3 act on leukemia cells. Mitochondrial DNA mutation as well as nuclear DNA might participate the process of As2O3 inducing differentiation and reducing proliferation.

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The mitochondrial DNA mutation T12297C affects a highly conserved nucleotide of tRNALeu(CUN) and is associated with dilated cardiomyopathy

European Journal of Human Genetics ◽

10.1038/sj.ejhg.5200622 ◽

2001 ◽

Vol 9 (4) ◽

pp. 311-315 ◽

Cited By ~ 32

Author(s):

Maurizia Grasso ◽

Marta Diegoli ◽

Agnese Brega ◽

Carlo Campana ◽

Luigi Tavazzi ◽

...

Keyword(s):

Mitochondrial Dna ◽

Dilated Cardiomyopathy ◽

Mitochondrial Dna Mutation ◽

Dna Mutation

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Intramyocellular lipid excess in the mitochondrial disorder MELAS

Neurology Genetics ◽

10.1212/nxg.0000000000000160 ◽

2017 ◽

Vol 3 (3) ◽

pp. e160 ◽

Cited By ~ 5

Author(s):

Sailaja Golla ◽

Jimin Ren ◽

Craig R. Malloy ◽

Juan M. Pascual

Keyword(s):

Mitochondrial Dna ◽

Fat Metabolism ◽

Mitochondrial Disorder ◽

Scientific Investigation ◽

Resonance Spectroscopy ◽

Lipid Deposition ◽

Metabolic Dysfunction ◽

Intramyocellular Lipid ◽

Mitochondrial Dna Mutation ◽

Dna Mutation

Objective:There is a paucity of objective, quantifiable indicators of mitochondrial disease available for clinical and scientific investigation.Methods:To this end, we explore intramyocellular lipid (IMCL) accumulation noninvasively by 7T magnetic resonance spectroscopy (MRS) as a reporter of metabolic dysfunction in MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). We reasoned that mitochondrial dysfunction may impair muscle fat metabolism, resulting in lipid deposition (as is sometimes observed in biopsies), and that MRS is well suited to quantify these lipids.Results:In 10 MELAS participants and relatives, IMCL abundance correlates with percent mitochondrial DNA mutation abundance and with disease severity.Conclusions:These results indicate that IMCL accumulation is a novel potential disease hallmark in MELAS.

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