Assessment of precision and concordance of quantitative mitochondrial DNA assays: a collaborative international quality assurance study

Hammond, E.; Sayer, D.; Nolan, David; Walker, Ulrich A.; Ronde, Anthony de; Montaner, Julio S. G.; Côté, Hélène; Gahan, Michelle E.; Cherry, Catherine L.; Wesselingh, Steven Lodewyk; Reiss, Peter; Mallal, S.

doi:10.1016/s1386-6532(02)00134-8

Cited by 45 publications

(30 citation statements)

References 7 publications

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“…The second aliquot of the liver biopsy (4 mm) was immediately frozen and stored at Ϫ70°C until shipment on dry ice for centralized and blinded mtDNA measurements by quantitative Southern blot analysis as described previously. 5,14,15 mtDNA was probed with a 12.9-kb pair, random-prime digoxigenin-labeled fragment, spanning nucleotide positions 3470 and 16379 of human mtDNA; nuclear DNA (nDNA) was simultaneously detected with a second probe, directed against the multicopy 18S ribosomal DNA gene. The intensities of the mtDNA and nDNA signals were densitometrically quantified using Scion-image (Scion Corporation, Frederick, MD), and mtDNA was normalized for nDNA-content by calculating the mtDNA/nDNA ratio.…”

Section: Methodsmentioning

confidence: 99%

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Depletion of mitochondrial DNA in liver under antiretroviral therapy with didanosine, stavudine, or zalcitabine

et al. 2004

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A ntiretroviral therapy (ART) has significantly decreased the HIV-associated morbidity and mortality in industrialized countries. 1 ART usually consists of a combination of two nucleoside analogue reverse transcriptase inhibitors (NRTIs) with either protease inhibitors (PIs) or a nonnucleoside reverse transcriptase inhibitor (NNRTI), or of three NRTIs. 2 With prolonged exposure to antiretroviral drugs, clinicians became aware of long-term side effects of individual ART components. Many adverse effects of the NRTI class of anti-HIV drugs are now related to the fact that NRTIs undergo intracellular triphosphorylation, then inhibit the replication of mitochondrial DNA (mtDNA) by interacting with gamma-polymerase. 3 In vitro studies point toward differences between the potencies of the individual NRTIs in depleting mtDNA, with the socalled "D drugs" zalcitabine (ddC), didanosine (ddI), and stavudine (d4T) being relatively strong inhibitors of polymerase-gamma compared with the other currently licensed nucleoside analogues (so-called "non-D drugs"). 4,5 Studies performed in vitro and in animals suggest that depletion of mtDNA may represent an underlying mechanism of NRTI-related hepatic side effects in HIV patients. [5][6][7] Cell models and animal data, however, have limitations in predicting clinical toxicities, partly because Abbreviations: ART, antiretroviral therapy; NRTI, nucleoside analogue reverse transcriptase inhibitor; PI, protease inhibitor; NNRTI, nonnucleoside reverse transcriptase inhibitor; mtDNA, mitochondrial DNA; ddC, zalcitabine; ddI, didanosine; d4T, stavudine; HCV, hepatitis C virus; nDNA, nuclear DNA; ULN, upper limit of normal; ALT, alanine aminotranferase. From

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Section: Methodsmentioning

confidence: 99%

“…The mtDNA/nDNA measurements were reliable with an interrun variation of 20%; large variations in the amount of DNA loaded onto the gel do not influence the result. 14,15 Southern blot analysis was also used to screen for large-scale mtDNA deletions.…”

Section: Methodsmentioning

confidence: 99%

Depletion of mitochondrial DNA in liver under antiretroviral therapy with didanosine, stavudine, or zalcitabine

et al. 2004

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“…In addition, while fresh tissue is preferable for study, this is often not an option due to geographical constraints [6]. Molecular diagnostic techniques have greater inter-laboratory reproducibility [7]. However, mtDNA analysis presents a particular diagnostic challenge in that false negative results may result from low level heteroplasmy in peripheral blood samples [8].…”

Section: Introductionmentioning

confidence: 99%

The in-depth evaluation of suspected mitochondrial disease

Haas

Parikh

Falk

et al. 2008

Molecular Genetics and Metabolism

311

300

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Mitochondrial disease confirmation and establishment of a specific molecular diagnosis requires extensive clinical and laboratory evaluation. Dual genome origins of mitochondrial disease, multiorgan system manifestations, and an ever increasing spectrum of recognized phenotypes represent the main diagnostic challenges. To overcome these obstacles, compiling information from a variety of diagnostic laboratory modalities can often provide sufficient evidence to establish an etiology. These include blood and tissue histochemical and analyte measurements, neuroimaging, provocative testing, enzymatic assays of tissue samples and cultured cells, as well as DNA analysis. As interpretation of results from these multifaceted investigations can become quite complex, the Diagnostic Committee of the Mitochondrial Medicine Society developed this review to provide an overview of currently available and emerging methodologies for the diagnosis of primary mitochondrial disease, primarily focusing on disorders characterized by impairment of oxidative phosphorylation. The aim of this work is to facilitate the diagnosis of mitochondrial disease by geneticists, neurologists, and other metabolic specialists who face the challenge of evaluating patients of all ages with suspected mitochondrial disease.

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“…2 Using a sensitive polymerase chain reaction (PCR) method specific to the JAK2V617F mutation, Hammond et al 3 reported also very low copies of this mutation in granulocyte populations of normal individuals that they assumed to represent nonspecific amplification of the wild-type JAK2 allele. It is difficult to comment accurately on this matter as no technical details are given, but their assumption is probably based on the fact that the …”

Section: E Hammond 1 K Shaw 2 R Herrmann 3mentioning

confidence: 99%

“…We also detected the presence of the mutation to less then 0.05% clinical dilution (30 copies), with a linear range extending from 10 and 300 000 copies per reaction (Figure 1). By combining this assay with another TaqMan assay for determining cell copy number, 3 we have been able to determine the average number of JAK2 V617F mutant copies per cell and per 20 ng of DNA in absolute terms from granulocyte fractions (Figure 2). Quantitative determination of JAK2 V617F by either means was extremely low among healthy controls (n ¼ 18), and we assumed this to represent non-specific amplification of the wild-type allele, as shown in Figure 1.…”

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confidence: 99%

The JAK2 V617F mutation is detectable in granulocyte populations at greater than two copies per cell among individuals with myeloproliferative disorders

Housni¹,

Sidon²,

Dessars³

et al. 2007

Leukemia

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Huang et al. used an allele-specific PCR 6-8 followed by separation and detection with capillary electrophoresis and this test has a sensitivity up to 200 pg mutated DNA; they only sequenced selected cases. The method used has the same sensitivity as our method (allele-specific pcr), but the result is displayed by capillary electrophoresis instead of agarose gel electrophoresis.This study confirms our previous report 4 bringing out the absence of a significant involvement of JAK2 V617F mutation in the pathogenesis of MM. 4

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Assessment of precision and concordance of quantitative mitochondrial DNA assays: a collaborative international quality assurance study

Cited by 45 publications

References 7 publications

Depletion of mitochondrial DNA in liver under antiretroviral therapy with didanosine, stavudine, or zalcitabine

Depletion of mitochondrial DNA in liver under antiretroviral therapy with didanosine, stavudine, or zalcitabine

The in-depth evaluation of suspected mitochondrial disease

The JAK2 V617F mutation is detectable in granulocyte populations at greater than two copies per cell among individuals with myeloproliferative disorders

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