A single-tube multiplex qPCR assay for mitochondrial DNA (mtDNA) copy number assessment

Abstract Objective Detection of mtDNA copy number is required for diagnosis of mtDNA depletion. Multiplex quantification of mtDNA in blood samples was claimed via normalizing to a nuclear single copy gene using qPCR. This is not possible in high mtDNA samples due to template abundance. Multiplex qPCR assays cannot be normalized to single copy sequences of the nuclear genome. Methods mtDNA quantification was tested normalizing to a single copy nuclear gene via singleplex and multiplex reactions. Failure in normalization directed to design and test targeting multi-copy 18S rDNA gene with success. mtDNA quantification was standardized both in separate and multiplexed single-tube reactions based on molecular beacon technology. Results mtDNA copy number assessment cannot be normalized to a single copy sequence in high-copy-number tissues. However, normalizing mtDNA to the nuclear 18S rDNA multiple copy sequence is amenable to be standardized in single tube. When compared, multiplexing exhibited higher resolution power for quantification of mtDNA in various samples from the most abundant to the scant ones. Conclusion We describe a multiplex assay that can be translated as a standard technique for single-tube quantification of mtDNA copy number. Our findings show higher accuracy and reproducibility over canonical approach, reducing cost and error rate.

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A nuclear encoded tRNA of Trypanosoma brucei is imported into mitochondria.

Molecular and Cellular Biology ◽

10.1128/mcb.14.4.2317 ◽

1994 ◽

Vol 14 (4) ◽

pp. 2317-2322 ◽

Cited By ~ 70

Author(s):

A Schneider ◽

J Martin ◽

N Agabian

Keyword(s):

Trna Gene ◽

Point Mutations ◽

Nuclear Genome ◽

Nuclear Gene ◽

Single Copy ◽

Mitochondrial Fraction ◽

Site Directed Mutagenesis ◽

Micrococcal Nuclease ◽

Cell Fractionation

The mitochondrial genome of trypanosomes, unlike that of most other eukaryotes, does not appear to encode any tRNAs. Therefore, mitochondrial tRNAs must be either imported into the organelle or created through a novel mitochondrial process, such as RNA editing. Trypanosomal tRNA(Tyr), whose gene contains an 11-nucleotide intron, is present in both the cytosol and the mitochondrion and is encoded by a single-copy nuclear gene. By site-directed mutagenesis, point mutations were introduced into this tRNA gene, and the mutated gene was reintroduced into the trypanosomal nuclear genome by DNA transfection. Expression of the mutant tRNA led to the accumulation of unspliced tRNA(Tyr) (A. Schneider, K. P. McNally, and N. Agabian, J. Biol. Chem. 268:21868-21874, 1993). Cell fractionation revealed that a significant portion of the unspliced mutant tRNA(Tyr) was recovered in the mitochondrial fraction and was resistant to micrococcal nuclease treatment in the intact organelle. Expression of the nuclear integrated, mutated tRNA gene and recovery of its gene product in the mitochondrial fraction directly demonstrated import. In vitro experiments showed that the unspliced mutant tRNA(Tyr), in contrast to the spliced wild-type form, was no longer a substrate for the cognate aminoacyl synthetase. The presence of uncharged tRNA in the mitochondria demonstrated that aminoacylation was not coupled to import.

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A nuclear encoded tRNA of Trypanosoma brucei is imported into mitochondria

Molecular and Cellular Biology ◽

10.1128/mcb.14.4.2317-2322.1994 ◽

1994 ◽

Vol 14 (4) ◽

pp. 2317-2322

Author(s):

A Schneider ◽

J Martin ◽

N Agabian

Keyword(s):

Trna Gene ◽

Point Mutations ◽

Nuclear Genome ◽

Nuclear Gene ◽

Single Copy ◽

Mitochondrial Fraction ◽

Site Directed Mutagenesis ◽

Micrococcal Nuclease ◽

Cell Fractionation

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Mitonuclear interactions affect locomotor activity and sleep in Drosophila melanogaster

10.1101/2021.10.30.464953 ◽

2021 ◽

Author(s):

Lucy Anderson ◽

M. Florencia Camus ◽

Katy M Monteith ◽

Tiina S. Salminen ◽

Pedro F Vale

Keyword(s):

Drosophila Melanogaster ◽

Copy Number ◽

Nuclear Gene ◽

Life History Trait ◽

Activity Levels ◽

Mtdna Copy Number ◽

Mtdna Variation ◽

Geographic Origins ◽

Nuclear Background ◽

Mtdna Variants

Mitochondria are organelles that produce cellular energy in the form of ATP through oxidative phosphorylation, and this primary function is conserved between many taxa. Locomotion is a trait that is highly reliant on metabolic function and expected to be greatly affected by disruptions to mitochondrial performance. To this end, we aimed to examine how activity and sleep vary between Drosophila melanogaster strains with different geographic origins, how these patterns are affected by mitochondrial DNA (mtDNA) variation, and how breaking up co-evolved mito-nuclear gene combinations affect the studied activity traits. The results demonstrate that Drosophila strains from different locations differ in sleep and activity, and the extent of variation differs between sexes, females in general being more active. By comparing activity and sleep of mtDNA variants introgressed onto a common nuclear background in cytoplasmic hybrid (cybrid) strains, we establish that mtDNA variation affects both traits, sex specifically. Furthermore, by using previously published mtDNA copy number data, we detected a positive correlation between mtDNA copy number and the activity levels of the cybrid flies. Altogether, our study shows that both mtDNA variation and mitonuclear interactions affect activity and sleep patterns, highlighting the important role that both genomes play on life-history trait evolution.

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Quasi-Mendelian Paternal Inheritance of mitochondrial DNA: A notorious artifact, or anticipated mtDNA behavior?

10.1101/660670 ◽

2019 ◽

Author(s):

Sofia Annis ◽

Zoe Fleischmann ◽

Mark Khrapko ◽

Melissa Franco ◽

Kevin Wasko ◽

...

Keyword(s):

Mitochondrial Dna ◽

Nuclear Genome ◽

Nuclear Gene ◽

Selective Advantage ◽

Somatic Tissue ◽

Biparental Inheritance ◽

Mtdna Copy Number ◽

Mendelian Segregation ◽

Paternal Haplotype ◽

Mtdna Haplotype

AbstractA recent report by Luo et al (2018) in PNAS (DOI:10.1073/pnas.1810946115) presented evidence of biparental inheritance of mitochondrial DNA. The pattern of inheritance, however, resembled that of a nuclear gene. The authors explained this peculiarity with Mendelian segregation of a faulty gatekeeper gene that permits survival of paternal mtDNA in the oocyte. Three other groups (Vissing, 2019; Lutz-Bonengel and Parson, 2019; Salas et al, 2019), however, posited the observation was an artifact of inheritance of mtDNA nuclear pseudogenes (NUMTs), present in the father’s nuclear genome. We present justification that both interpretations are incorrect, but that the original authors did, in fact, observe biparental inheritance of mtDNA. Our alternative model assumes that because of initially low paternal mtDNA copy number these copies are randomly partitioned into nascent cell lineages. The paternal mtDNA haplotype must have a selective advantage, so ‘seeded’ cells will tend to proceed to fixation of the paternal haplotype in the course of development. We use modeling to emulate the dynamics of paternal genomes and predict their mode of inheritance and distribution in somatic tissue. The resulting offspring is a mosaic of cells that are purely maternal or purely paternal – including in the germline. This mosaicism explains the quasi-Mendelian segregation of the paternal mDNA. Our model is based on known aspects of mtDNA biology and explains all of the experimental observations outlined in Luo et. al., including maternal inheritance of the grand-paternal mtDNA.

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Mitochondrial respiratory gene expression is suppressed in many cancers

eLife ◽

10.7554/elife.21592 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 48

Author(s):

Ed Reznik ◽

Qingguo Wang ◽

Konnor La ◽

Nikolaus Schultz ◽

Chris Sander

Keyword(s):

Gene Expression ◽

Copy Number ◽

Respiratory Activity ◽

Nuclear Genome ◽

Sequencing Data ◽

Mtdna Copy Number ◽

A Cell ◽

Cancer Types ◽

Respiratory Proteins ◽

Tumor Types

The fundamental metabolic decision of a cell, the balance between respiration and fermentation, rests in part on expression of the mitochondrial genome (mtDNA) and coordination with expression of the nuclear genome (nuDNA). Previously we described mtDNA copy number depletion across many solid tumor types (Reznik et al., 2016). Here, we use orthogonal RNA-sequencing data to quantify mtDNA expression (mtRNA), and report analogously lower expression of mtRNA in tumors (relative to normal tissue) across a majority of cancer types. Several cancers exhibit a trio of mutually consistent evidence suggesting a drop in respiratory activity: depletion of mtDNA copy number, decreases in mtRNA levels, and decreases in expression of nuDNA-encoded respiratory proteins. Intriguingly, a minority of cancer types exhibit a drop in mtDNA expression but an increase in nuDNA expression of respiratory proteins, with unknown implications for respiratory activity. Our results indicate suppression of respiratory gene expression across many cancer types.

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Can mtDNA copy number assessment be a valid embryo selection tool over morphology-based embryo scoring?

10.26226/morressier.5912d9ead462b802923861e1 ◽

2017 ◽

Author(s):

Meral Gultomruk

Keyword(s):

Copy Number ◽

Embryo Selection ◽

Mtdna Copy Number ◽

Selection Tool

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Risk of cardiac manifestations in adult mitochondrial disease caused by nuclear genetic defects

Open Heart ◽

10.1136/openhrt-2020-001510 ◽

2021 ◽

Vol 8 (1) ◽

pp. e001510

Author(s):

Albert Zishen Lim ◽

Daniel M Jones ◽

Matthew G D Bates ◽

Andrew M Schaefer ◽

John O'Sullivan ◽

...

Keyword(s):

Mitochondrial Disease ◽

Nuclear Dna ◽

Laboratory Data ◽

Nuclear Genome ◽

Nuclear Gene ◽

Left Ventricular ◽

Adult Patients ◽

Limited Information ◽

Cardiac Abnormalities ◽

Cardiac Manifestations

ObjectiveRegular cardiac surveillance is advocated for patients with primary mitochondrial DNA disease. However, there is limited information to guide clinical practice in mitochondrial conditions caused by nuclear DNA defects. We sought to determine the frequency and spectrum of cardiac abnormalities identified in adult mitochondrial disease originated from the nuclear genome.MethodsAdult patients with a genetically confirmed mitochondrial disease were identified and followed up at the national clinical service for mitochondrial disease in Newcastle upon Tyne, UK (January 2009 to December 2018). Case notes, molecular genetics reports, laboratory data and cardiac investigations, including serial electrocardiograms and echocardiograms, were reviewed.ResultsIn this cohort-based observational study, we included 146 adult patients (92 women) (mean age 53.6±18.7 years, 95% CI 50.6 to 56.7) with a mean follow-up duration of 7.9±5.1 years (95% CI 7.0 to 8.8). Eleven different nuclear genotypes were identified: TWNK, POLG, RRM2B, OPA1, GFER, YARS2, TYMP, ETFDH, SDHA, TRIT1 and AGK. Cardiac abnormalities were detected in 14 patients (9.6%). Seven of these patients (4.8%) had early-onset cardiac manifestations: hypertrophic cardiomyopathy required cardiac transplantation (AGK; n=2/2), left ventricular (LV) hypertrophy and bifascicular heart block (GFER; n=2/3) and mild LV dysfunction (GFER; n=1/3, YARS2; n=1/2, TWNK; n=1/41). The remaining seven patients had acquired heart disease most likely related to conventional cardiovascular risk factors and presented later in life (14.6±12.8 vs 55.1±8.9 years, p<0.0001).ConclusionsOur findings demonstrate that the risk of cardiac involvement is genotype specific, suggesting that routine cardiac screening is not indicated for most adult patients with nuclear gene-related mitochondrial disease.

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Geographic Structure of Mitochondrial and Nuclear Gene Polymorphisms in Australian Green Turtle Populations and Male-Biased Gene Flow

Genetics ◽

10.1093/genetics/147.4.1843 ◽

1997 ◽

Vol 147 (4) ◽

pp. 1843-1854 ◽

Cited By ~ 8

Author(s):

Nancy N FitzSimmons ◽

Craig Moritz ◽

Colin J Limpus ◽

Lisa Pope ◽

Robert Prince

Keyword(s):

Gene Flow ◽

Genetic Structure ◽

Green Turtle ◽

Nuclear Dna ◽

Nuclear Gene ◽

Chelonia Mydas ◽

Single Copy ◽

Microsatellite Data ◽

Ecological Data ◽

Infinite Allele Model

Abstract The genetic structure of green turtle (Chelonia mydas) rookeries located around the Australian coast was assessed by (1) comparing the structure found within and among geographic regions, (2) comparing microsatellite loci vs. restriction fragment length polymorphism analyses of anonymous single copy nuclear DNA (ascnDNA) loci, and (3) comparing the structure found at nuclear DNA markers to that of previously analyzed mitochondrial (mtDNA) control region sequences. Significant genetic structure was observed over all regions at both sets of nuclear markers, though the microsatellite data provided greater resolution in identifying significant genetic differences in pairwise tests between regions. Inferences about population structure and migration rates from the microsatellite data varied depending on whether statistics were based on the stepwise mutation or infinite allele model, with the latter being more congruent with geography. Estimated rates of gene flow were generally higher than expected for nuclear DNA (nDNA) in comparison to mtDNA, and this difference was most pronounced in comparisons between the northern and southern Great Barrier Reef (GBR). The genetic data combined with results from physical tagging studies indicate that the lack of nuclear gene divergence through the GBR is likely due to the migration of sGBR turtles through the courtship area of the nGBR population, rather than male-biased dispersal. This example highlights the value of combining comparative studies of molecular variation with ecological data to infer population processes.

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Characterization and evolution of a single-copy sequence from the human Y chromosome

Molecular and Cellular Biology ◽

10.1128/mcb.5.3.576-581.1985 ◽

1985 ◽

Vol 5 (3) ◽

pp. 576-581

Author(s):

R D Burk ◽

P Ma ◽

K D Smith

Keyword(s):

Y Chromosome ◽

Primate Evolution ◽

Single Copy ◽

Homologous Sequence ◽

Old World Monkeys ◽

Chromosomal Distribution ◽

Human Dna ◽

Single Copy Sequence ◽

Copy Sequence ◽

Human Y Chromosome

To study the evolution and organization of DNA from the human Y chromosome, we constructed a recombinant library of human Y DNA by using a somatic cell hybrid in which the only cytologically detectable human chromosome is the Y. One recombinant (4B2) contained a 3.3-kilobase EcoRI single-copy fragment which was localized to the proximal portion of the Y long arm. Sequences homologous to this human DNA are present in male gorilla, chimpanzee, and orangutan DNAs but not in female ape DNAs. Under stringent hybridization conditions, the homologous sequence is either a single-copy or a low-order repeat in humans and in the apes. With relaxed hybridization conditions, this human Y probe detected several homologous DNA fragments which are all derived from the Y in that they occur in male DNAs from humans and the apes but not in female DNAs. In contrast, this probe hybridized to highly repeated sequences in both male and female DNAs from old world monkeys. Thus, sequences homologous to this probe underwent a change in copy number and chromosomal distribution during primate evolution.

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Urea Memory: Transient Cell Exposure to Urea Causes Persistent Mitochondrial ROS Production and Endothelial Dysfunction

Toxins ◽

10.3390/toxins10100410 ◽

2018 ◽

Vol 10 (10) ◽

pp. 410 ◽

Cited By ~ 2

Author(s):

Maria d’Apolito ◽

Anna Colia ◽

Enrica Manca ◽

Massimo Pettoello-Mantovani ◽

Michele Sacco ◽

...

Keyword(s):

Fusion Proteins ◽

Copy Number ◽

Electron Transport Chain ◽

Mitochondrial Fission ◽

Functional Decline ◽

Cell Types ◽

Mtdna Copy Number ◽

Mitochondrial Ros ◽

Transport Chain ◽

Arterial Endothelial Cells

Urea at post-dialysis levels induces increased ROS in a number of cell types. The aim of this study was to determine whether urea-induced production of ROS remains elevated after urea is no longer present, and, if it does, to characterize its origin and effects. Human arterial endothelial cells were incubated with 20 mM urea for two days, and then cells were incubated for an additional two days in medium alone. Maximal ROS levels induced by initial urea continued at the same level despite urea being absent. These effects were prevented by either MnSOD expression or by Nox1/4 inhibition with GKT13781. Sustained urea-induced ROS caused a persistent reduction in mtDNA copy number and electron transport chain transcripts, a reduction in transcription of mitochondrial fusion proteins, an increase in mitochondrial fission proteins, and persistent expression of endothelial inflammatory markers. The SOD-catalase mimetic MnTBAP reversed each of these. These results suggest that persistent increases in ROS after cells are no long exposed to urea may play a major role in continued kidney damage and functional decline despite reduction of urea levels after dialysis.

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