11 research outputs found
Accurate Measurement of Mitochondrial DNA Deletion Level and Copy Number Differences in Human Skeletal Muscle
<div><p>Accurate and reliable quantification of the abundance of mitochondrial DNA (mtDNA) molecules, both wild-type and those harbouring pathogenic mutations, is important not only for understanding the progression of mtDNA disease but also for evaluating novel therapeutic approaches. A clear understanding of the sensitivity of mtDNA measurement assays under different experimental conditions is therefore critical, however it is routinely lacking for most published mtDNA quantification assays. Here, we comprehensively assess the variability of two quantitative Taqman real-time PCR assays, a widely-applied <i>MT-ND1</i>/<i>MT-ND4</i> multiplex mtDNA deletion assay and a recently developed <i>MT-ND1</i>/<i>B2M</i> singleplex mtDNA copy number assay, across a range of DNA concentrations and mtDNA deletion/copy number levels. Uniquely, we provide a specific guide detailing necessary numbers of sample and real-time PCR plate replicates for accurately and consistently determining a given difference in mtDNA deletion levels and copy number in homogenate skeletal muscle DNA.</p></div
Clinical details of patients with single, large-scale mtDNA deletions and control individuals.
<p>CPEO, chronic progressive external ophthalmoplegia.</p>1<p>Age at time of biopsy.</p>2<p>GenBank accession number NC_012920.1.</p>3<p>Patient 1 published in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114462#pone.0114462-Murphy1" target="_blank">[7]</a>.</p>4<p>Patient 3 published in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114462#pone.0114462-Taivassalo1" target="_blank">[8]</a>.</p><p>Clinical details of patients with single, large-scale mtDNA deletions and control individuals.</p
Inter- and intra-plate variability in the measurement of mtDNA copy number differences between DNA samples.
<p>Replicate (<i>n</i>β=β12) <i>MT</i>-<i>ND1</i>/<i>B2M</i> copy number ratios were obtained for five homogenate skeletal muscle DNA samples of varying mtDNA copy number (samples S1 to S5) on each of four replicate <i>MT</i>-<i>ND1</i>/<i>B2M</i> real time PCR plate pairs at different DNA concentrations (low β corresponding to an average C<sub>q<i>MT-ND1</i></sub> of 19 and an average C<sub>q<i>B2M</i></sub> of 27, moderate - C<sub>q<i>MT-ND1</i></sub>β=β24 and C<sub>q<i>B2M</i></sub>β=β30, high - C<sub>q<i>MT-ND1</i></sub>β=β25.5 and C<sub>q<i>B2M</i></sub>β=β33). Independent relative copy number values were then calculated for each replicate of S2, S3, S4 and S5 compared to S1 for each <i>MT</i>-<i>ND1</i>/<i>B2M</i> plate. Intra-plate standard deviations are shown as circles (relative copy number values: S2:S1β=β1.10 (green circles), S3:S1β=β1.25 (blue circles), S4:S1β=β1.50 (orange circles), S5:S1β=β1.9 (red circles)). Inter-plate standard deviations are shown as grey crosses. As relative copy number is a multiplicative relationship, geometric standard deviations are shown.</p
Number of plates required to identify a given heteroplasmy difference between two homogenate skeletal muscle DNA samples at various <i>MT</i>-<i>ND4</i> deletion levels with the <i>MT</i>-<i>ND1</i>/<i>MT</i>-<i>ND4</i> Taqman deletion assay.
<p>Standard deviation (SD) is expressed in percentage points.</p><p>Actual power (P) and number of plates (<i>n</i>) are shown. C<sub>q</sub><23 ([DNA]<sub>PCR</sub>>1 pg/Β΅l).</p><p>Number of plates required to identify a given heteroplasmy difference between two homogenate skeletal muscle DNA samples at various <i>MT</i>-<i>ND4</i> deletion levels with the <i>MT</i>-<i>ND1</i>/<i>MT</i>-<i>ND4</i> Taqman deletion assay.</p
Effects of DNA concentration and heteroplasmy level on the accuracy of the <i>MT</i>-<i>ND1</i>/<i>MT</i>-<i>ND4</i> deletion assay.
<p>Percentage standard deviations of DNA sample replicates (<i>n</i>β=β84) are represented relative to average C<sub>q</sub> for homogenate skeletal muscle DNA samples (<i>n</i>β=β6) varying in <i>MT</i>-<i>ND4</i> deletion levels from 10β80%. Confidence intervals at 95% are shown. The y axis is log<sub>10</sub> scaled.</p
Number of <i>MT</i>-<i>ND1</i> and <i>B2M</i> real time PCR plates required to detect a specific relative difference in mtDNA copy number between two homogenate skeletal muscle DNA samples for a given number of replicate sample wells.
<p>Each cell defines the number of plates (<i>n</i>) and actual power (P; minimum power 0.8 at Ξ±β=β0.05) for a two sample <i>t</i>-test to detect, with the specified number of sample replicates per plate, a given percentage difference in mtDNA copy number. Estimates are calculated at high (approximate C<sub>q<i>MT-ND1</i></sub> of 19 ([DNA]<sub>PCR</sub>β=β20 pg/Β΅l) and C<sub>q<i>B2M</i></sub> of 27 ([DNA]<sub>PCR</sub>β=β0.6 ng/Β΅l)), moderate (approximate C<sub>q<i>MT-ND1</i></sub> of 24 ([DNA]<sub>PCR</sub>β=β0.6 pg/Β΅l) and C<sub>q<i>B2M</i></sub> of 30 ([DNA]<sub>PCR</sub>β=β0.1 ng/Β΅l)) and low (approximate C<sub>q<i>MT-ND1</i></sub> of 25 ([DNA]<sub>PCR</sub>β=β0.3 pg/Β΅l) and C<sub>q<i>B2M</i></sub> of 33 ([DNA]<sub>PCR</sub>β=β10 pg/Β΅l)) PCR DNA concentration levels.</p><p>Number of <i>MT</i>-<i>ND1</i> and <i>B2M</i> real time PCR plates required to detect a specific relative difference in mtDNA copy number between two homogenate skeletal muscle DNA samples for a given number of replicate sample wells.</p
Inter- and intra-plate variability in the measurement of <i>MT</i>-<i>ND4</i> deletion level differences between DNA samples.
<p>Independent values for the difference in deletion level (in percentage points) between two homogenate skeletal muscle DNA samples harbouring either low (20β25%; samples AβC) or high (70β80%; samples DβF) <i>MT</i>-<i>ND4</i> deletion levels are shown for each sample replicate (<i>n</i>β=β24; grey dots) per replicate real time PCR plate (<i>n</i>β=β3). Data are normalised by subtracting the best estimate of the true deletion level difference, determined as the mean of all replicates on all plates. Mean values for each dataset are displayed (horizontal bars). The mean values from individual plates are within 1% of the actual deletion level, though individual replicates vary by up to Β±9% at low deletion level and Β±6% at high deletion level.</p
Comparison of Mitochondrial Mutation Spectra in Ageing Human Colonic Epithelium and Disease: Absence of Evidence for Purifying Selection in Somatic Mitochondrial DNA Point Mutations
<div><p>Human ageing has been predicted to be caused by the accumulation of molecular damage in cells and tissues. Somatic mitochondrial DNA (mtDNA) mutations have been documented in a number of ageing tissues and have been shown to be associated with cellular mitochondrial dysfunction. It is unknown whether there are selective constraints, which have been shown to occur in the germline, on the occurrence and expansion of these mtDNA mutations within individual somatic cells. Here we compared the pattern and spectrum of mutations observed in ageing human colon to those observed in the general population (germline variants) and those associated with primary mtDNA disease. The pathogenicity of the protein encoding mutations was predicted using a computational programme, MutPred, and the scores obtained for the three groups compared. We show that the mutations associated with ageing are randomly distributed throughout the genome, are more frequently non-synonymous or frameshift mutations than the general population, and are significantly more pathogenic than population variants. Mutations associated with primary mtDNA disease were significantly more pathogenic than ageing or population mutations. These data provide little evidence for any selective constraints on the occurrence and expansion of mtDNA mutations in somatic cells of the human colon during human ageing in contrast to germline mutations seen in the general population.</p> </div
Gene location and types of mutations observed in ageing, population, and disease.
<p>A: Gene location of mutations. Data are represented as the percentage of the total coding region mutations. Contingency analysis with Bonferroni correction for multiple testing was carried out on the frequencies of the changes in each gene type (ageing nβ=β117, population nβ=β182, disease nβ=β176). Thresholds for statistical significance are; ***<0.0003, ** 0.003, *β=β0.017. B: Types of changes observed in ageing, population and disease. Data are represented as the percentage of the total coding region mutations. Contingency analysis with Bonferroni correction for multiple testing was carried out on the frequencies of the changes in each mutational category. Thresholds for statistical significance are; ***<0.0003, ** 0.003, *β=β0.017.</p
Genetic consequences of mutations observed in ageing, population, and disease.
<p>A: The percentage of changes which predict synonymous, non-synonymous and premature termination codons or frameshifts in protein encoding genes. Contingency analysis with Bonferroni correction for multiple testing was carried out on the frequencies of the changes in each gene type (ageing nβ=β81, population nβ=β155, disease nβ=β76). Thresholds for statistical significance are; ***<0.0003, ** 0.003, *β=β0.017. B: Frequency of termination codon and frameshift mutations in inherited and sporadic disease-causing mtDNA mutations. Chi-squared analysis showed a significantly higher frequency such mutations in sporadic than inherited cases (pβ=β0.003).</p