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Quantification of mitochondrial DNA in peripheral blood mononuclear cells and subcutaneous fat using real-time polymerase chain reaction
Abstract
With decreased rates of HIV mortality and disease progression attributable to treatment with nucleoside analogue reverse transcriptase inhibitors (NRTIs), attention has now become focused on the toxicities of these forms of treatment. It is believed NRTIs cause a decrease in mitochondrial DNA (mtDNA) synthesis due to their inhibition of DNA polymerase gamma. This hypothesis is supported by in vitro data from muscle biopsies and human lymphoblastic cell lines. The resulting mitochondrial toxicity is thought to manifest itself in a variety of clinical symptoms including fatigue, fat wasting and peripheral neuropathy. A non-invasive test of mitochondrial toxicity is needed to assess toxicity and optimise HIV treatment strategies. Peripheral blood mononuclear cells (PBMC) and subcutaneous fat could be ideal and accessible sources of mtDNA for examining toxicity.
The objectives of this study were (a) to develop an assay to quantify the mtDNA copy number of PBMC and obtain reproducible results and (b) to establish the utility of subcutaneous fat as a source of mtDNA for quantification.
PBMC were isolated from blood by centrifugation over Ficoll-Paque and subcutaneous fat was obtained from two 3 mm punch skin biopsies. Following DNA extraction, the mtDNA copy number in each sample was quantified by real-time polymerase chain reaction (PCR).
The real-time PCR assay was found to generate consistent and reproducible results with replicates of samples undertaken within the same run, and in two or more different runs, having a mean coefficient of variation of 11.3 and 17.2%, respectively. PBMC and subcutaneous fat contained 409+/-148 and 2042+/-391 copies of mtDNA per cell, respectively.
From the work carried out it can be concluded that firstly, the real-time PCR assay generates consistent and reproducible results, and secondly that mtDNA can be extracted and quantified from PBMC and subcutaneous fat.
... We have developed a real-time polymerase chain reaction (PCR) assay to accurately quantify copy numbers of mtDNA per cell in human tissue. This assay has been optimized for use on PBMCs and subcutaneous fat and has been shown to give consistent and reproducible results (23). We hypothesize that quantifying mtDNA in readily accessible tissues will prove a sensitive marker of NRTI (and more particularly ddN) exposure in vivo. ...
... Chain Reaction mtDNA copy number was quantified by amplifying a 131-base pair (bp) region of the highly conserved cytochrome b gene of the human mitochondrial genome using a real-time PCR/molecular beacon assay (23). To normalize results for variation in DNA recovery, a 239-bp region of the CCR5 gene was amplified using a separate real-time PCR/molecular beacon assay (25). ...
... The primers G2F (5Ј-CCAACATCTCCGCATGATGAAAC-3Ј) and H14949 (5Ј-GTGGGCGATTGATGAAAAGG-3Ј) (Gibco BRL) were used to amply the mtDNA and a molecular beacon MIT01 (5Ј FAM-CGCGTCCCTAGCCATGCACTACTCACCAGACGCG-DABCYL 3Ј) (Midland Certified Reagent Company) was included to serve as a real-time detector for the amplified product. Reaction components, concentrations, and cycle conditions were as previously described (23). ...
Nucleoside reverse transcriptase inhibitors (NRTIs), particularly dideoxynucleoside analogs (ddNs), used in the treatment of HIV, inhibit mitochondrial DNA polymerase gamma in vitro. Mitochondrial DNA (mtDNA) depletion is proposed as the underlying mechanism of many of the in vivo side effects of these agents. A reliable and valid laboratory test to detect this is not yet available. The objective of this study was to correlate tissue mtDNA quantification in HIV-infected patients with exposure to nucleoside analogs.
60 HIV-infected adults underwent detailed clinical assessment and blood and tissue sampling. Clinical and antiretroviral treatment details were correlated with results of plasma lactate assays, and real-time polymerase chain reaction quantification of mtDNA in peripheral blood mononuclear cells (PBMCs) and subcutaneous fat from the lower limb.
Forty-nine (82%) subjects were on combination antiretroviral therapy. Of these, 33 (55%) were currently receiving one or more ddNs (stavudine, didanosine, or zalcitabine). mtDNA in subcutaneous fat was lower in subjects currently on ddNs than in those not taking ddNs (mean [log10] 2.47 vs. 2.74, p =.002). Plasma lactate was somewhat higher in subjects currently taking ddNs than those on no antiretroviral treatment (median 1.5 vs. 1.0, p =.03), but was not significantly different in either of these groups compared with subjects on other NRTIs. mtDNA in PBMCs did not vary with treatment status.
mtDNA in subcutaneous fat was significantly reduced in patients currently taking ddNs. mtDNA in PBMCs was independent of patient exposure to NRTIs.
... Thus, mtDNA quantification requires assessment of both mtDNA and genomic copy numbers. Relative quantification of mtDNA was previously investigated by others using canonical qPCR assay, targeting the amplification of mitochondrial and genomic DNA in separate tubes to provide a relative copy number [13][14][15][16][17]. Comparison of two amplicons in separate tubes is open to technical pipetting errors due to the nature of the PCR reaction or DNA quality. ...
... When skeletal muscle DNA samples from the individuals that consisted the pool of controls were individually assessed, this ~8-cycledifference was observed to be consistent throughout the samples (Figure 2A . This is much before the initial detection of any of the ALB gene amplicons (cycles [16][17]. ...
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.
... Somatic cells usually contain between 10 3 and 10 4 copies of mtDNA, with 2 to 10 copies per organelle (Satoh & Kuroiwa, 1991), correlating to each particular cell's requirement for OXPHOS. For example, it has been reported that human subcutaneous fat cells contain more mtDNA copies than peripheral blood mononuclear cells (Gahan et al., 2001) and that liver tissue contains more mtDNA than various muscle tissues (Wiesner et al., 1992). Replication of mtDNA and ETC activity can be influenced by the expression of nuclear-encoded transcription and replication factors, including TFAM, PolG and the nuclear respiratory factors (NRF). ...
... For example, there is a significant difference between skeletal and cardiac muscle, with 3,650 ± 620 and 6,790 ± 920 mtDNA copies per diploid nuclear genome, respectively (P = 0.006; Miller et al., 2003). Similar differences have been observed in, for example, peripheral blood mononuclear cells and subcutaneous fat (Gahan et al., 2001); cultured fibroblasts (Zhang et al., 1994); bovine oocytes and bovine foetal heart fibroblasts (Michaels et al., 1982). Despite this variation, the vast majority of differentiated cell types have significantly more mtDNA molecules than the ten copies reported in the relatively undifferentiated PGCs (Jansen & de Boer, 1998). ...
... Several qPCR assays for the quantification of human mtDNA have been reported (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25), and a number have been specifically developed for forensic purposes (14,20,21,(23)(24)(25). Many, although not all, of these utilize highly specific fluorogenic probes and mtDNA-specific standard curves, which enable the absolute quantification of mtDNA down to 10 copies of mtDNA or less (14,17,25). ...
... The calculated values were found to be highly consistent, exhibiting a CV of 17% (Table 3). This value was comparable to, or more favorable than, interassay CVs reported for alternative qPCR assays described elsewhere (16,18,23). One alternative assay yielded lower CV values (19), however, these were calculated among five independent assays. ...
Successful mitochondrial DNA (mtDNA) forensic analysis depends on sufficient quantity and quality of mtDNA. A real-time quantitative PCR assay was developed to assess such characteristics in a DNA sample, which utilizes a duplex, synthetic DNA to ensure optimal quality assurance and quality control. The assay's 105-base pair target sequence facilitates amplification of degraded DNA and is minimally homologous to nonhuman mtDNA. The primers and probe hybridize to a region that has relatively few sequence polymorphisms. The assay can also identify the presence of PCR inhibitors and thus indicate the need for sample repurification. The results show that the assay provides information down to 10 copies and provides a dynamic range spanning seven orders of magnitude. Additional experiments demonstrated that as few as 300 mtDNA copies resulted in successful hypervariable region amplification, information that permits sample conservation and optimized downstream PCR testing. The assay described is rapid, reliable, and robust.
... Mitochondrial DNA -CN was measured by amplifiying ND1 gene in mitochondrial DNA and nuclear gene 18s by Real Time PCR technique. 10,11 Two pairs of primers were designed and used in two steps for quantification of mitochondrial DNA. One primer pair was to amplify MT-ND1 gene in mitochondria and other primer sequence to amplify single-copy nuclear gene 18s.The ND1 to18s CN ratio was determined from standard curve for each sample. ...
Mitochondria have its own DNA called Mitochondrial DNA (mtDNA)). There are many reasons behind Mitochondrial dysfunction, may be due to point mutations, deletions, and variations in Mitochondrial DNA copy number. Pregnant women affected with GDM are associated with some alterations in mitochondria DNA copy number (mtDNA-CN). The aim of this study is to check the changes in mtDNA-CN using real-time PCR in pregnant women who are diagnosed with GDM. 25 GDM affected pregnant women and 25 healthy pregnant women were enrolled for this study. Peripheral mitochondrial copy number was checked by Real Time PCR . The result shown a mild decrease in mtDNA-CN in the pregnant women who are affected with GDM when compared to the healthy pregnant women. This reduction or variation in mitochondria DNA CN can leads to mitochondrial dysfunction.
... The qPCR technique enables analysis over an extremely wide dynamic range, from single-molecule-input copy number of target DNA up to very high concentrations of DNA [24]. However, qPCR provides only a relative analysis, since quantitation is based on interpolation of a sample signal against a standard curve [10,25,26]. Long-amplicon PCR has been used for measuring mtDNA and nDNA, both in toxicology and ecotoxicology studies [24,27]. ...
Mitochondria are vulnerable to the effects of ionizing radiation; damage to mitochondrial DNA (mtDNA) may be more extensive and persistent than damage to nuclear DNA (nDNA). Variation in mtDNA copy number has been proposed as a marker for mitochondrial dysfunction in response to ionizing radiation. We have developed a precise and sensitive duplex droplet digital PCR (ddPCR) method for quantitation of the mtDNA/nDNA ratio in the model organism Caenorhabditis elegans. The effect on this ratio was investigated over a wide range of doses (0.03 to 72 Gy) of chronic gamma irradiation. Five mitochondrial targets and two nuclear reference genes were amplified pairwise in duplex PCR format (one mitochondrial and one nuclear target per PCR) by both ddPCR and quantitative PCR (qPCR). The results showed that ddPCR but not qPCR enabled detection of a significant increase in mtDNA copy number (1.6 ± 0.1-fold) for nematodes exposed to high doses (≥24 Gy). Thus, ddPCR provided higher precision and greater sensitivity than qPCR for detection of mtDNA copy number variation. The variation followed a Hill-type dose response with threshold 10.3 ± 1 Gy. This strongly suggests that chronic genotoxic stress affects mtDNA replication. The duplex ddPCR method is a novel, high-precision, sensitive tool for determination of mitochondrial DNA copy number variation and function in C. elegans.
... The mtDNA copy number or content reflects the mitochondrial function through the mitochondrial enzyme activity and ATP production [79]. Quantification of the mtDNA copy number of PBMCs using real-time polymerase chain reaction (PCR) was found to produce consistent and reproducible results [80]. ...
Cardiovascular diseases (CVDs) are devastating disorders and the leading cause of mortality worldwide. The pathophysiology of cardiovascular diseases is complex and multifactorial and, in the past years, mitochondrial dysfunction and excessive production of reactive oxygen species (ROS) have gained growing attention. Indeed, CVDs can be considered as a systemic alteration, and understanding the eventual implication of circulating blood cells peripheral blood mononuclear cells (PBMCs) and or platelets, and particularly their mitochondrial function, ROS production, and mitochondrial DNA (mtDNA) releases in patients with cardiac impairments, appears worthwhile. Interestingly, reports consistently demonstrate a reduced mitochondrial respiratory chain oxidative capacity related to the degree of CVD severity and to an increased ROS production by PBMCs. Further, circulating mtDNA level was generally modified in such patients. These data are critical steps in term of cardiac disease comprehension and further studies are warranted to challenge the possible adjunct of PBMCs’ and platelets’ mitochondrial dysfunction, oxidative stress, and circulating mtDNA as biomarkers of CVD diagnosis and prognosis. This new approach might also allow further interesting therapeutic developments.
... Others have reported similar values [56][57][58][59][60]. In contrast, Anderson reported an average of 16,569 base pairs per copy of human mtDNA [61], and Qiu reported an average of 409 (range observed: 223-854; n = 16) copies of mitochondrial DNA per PBMC in normal healthy individuals [62,63]. The number of base pairs for mtDNA may be lower in individuals with specific diseases, such as HIV, depending on treatments [64]. ...
... Saiki et al. and Lo et al. reported conversion factor of 6.6 pg of DNA per fetal cell [115,116]. In contrast, Anderson et al. reported an average of 16,569 base pairs per copy of human mtDNA [117], and Qiu et al. reported an average of 409 (range observed: 223-854; n = 16) copies of mtDNA per PBMC in normal healthy individuals [118,119]. The numbers may be lower in individuals with specific diseases, such as HIV, depending on treatments [120]. ...
Introduction: PBMCs are a critical component of the immune system and the target cells for HIV-1 infection. Nucleoside/nucleotide analogs for the treatment of HIV infection are prodrugs that require cellular activation to triphosphate (TP) metabolites for antiviral activity. A reliable method of PBMC isolation and subsequent cell counting, as well as an accurate bioanalytical determination of the TPs in PBMCs are important for understanding the intracellular PK of the TPs and its correlation with plasma PK, the drug effect and dose determination. This review article summarizes the challenges and solutions to the PBMC sample collection, sample treatment, analyte recovery, quantitative analyte determination, and cell counting.
Areas Covered: The authors review the challenges and solutions in PBMC sample collection, sample processing, cell lysis, cell counting methods, analyte extraction, and LC-MS/MS quantitative analysis of the NRTI triphosphate metabolites (NRTI-TPs) and analogs.
Expert Opinion: Analyzing large numbers of clinical PBMC samples for determination of NRTI-TPs and analogs in PBMCs requires not only a validated LC-MS/MS bioanalytical method but also reliable methods for PBMC isolation, counting, cell lysis, and analyte recovery, as well as an approach for assessing analyte stability. Furthermore, a simple, consistent, and validated cell counting method often involves DNA quantitation of the PBMCs samples collected from clinical studies.
... Indeed, oxidative stress and mtDNA damage have been found to be associated with increased mtDNAcn in leukocytes (18,19). In various somatic cells, mitochondria possess 2-10 000 copies of their genomes (20)(21)(22)(23). Further, population-based estimates of leukocyte mtDNAcn vary from ~50 copies per cell in Danish adults (24), to about 110-120 copies per cell in Sardinian and U.S. adults (25,26). ...
Households in Xuanwei and Fuyuan, China, possess hazardous levels of fine particulate matter with an aerodynamic diameter < 2.5 microns (PM2.5) and polycyclic aromatic hydrocarbons (PAHs) from coal combustion. Previous studies found that increased exposure to PM2.5 and benzo[a]pyrene (BaP; a PAH) were associated with decreased mitochondrial DNA copy number (mtDNAcn), a marker of oxidative stress. We further evaluated these associations in a cross-sectional study of 148 healthy non-smoking women from Xuanwei and Fuyuan. Personal exposure to PM2.5 and BaP was measured using portable devices. MtDNAcn was measured using qPCR amplification of leukocyte DNA that was collected after air measurements. Linear regression models were used to estimate the associations between personal exposure to PM2.5 and BaP, and mtDNAcn adjusted for age, body mass index (BMI) and fuel type. We found inverse associations between exposure to PM2.5 and BaP, and mtDNAcn. Each incremental log-μg/m³ increase in PM2.5 was associated with a significant decrease in mtDNAcn of -10.3 copies per cell [95% confidence interval (95% CI): -18.6, -2.0; P = 0.02]. Additionally, each log-ng/m³ increase in BaP was associated with a significant decrease in mtDNAcn of -5.4 copies per cell (95% CI: -9.9, -0.8, P = 0.02). Age, BMI, fuel type and coal mine type were not significantly associated with mtDNAcn. Exposure to PM2.5 and BaP may alter mitochondrial dynamics in non-smoking Chinese women. MtDNAcn may be a potential mediator of indoor air pollution on chronic disease development.
... The amount of mtDNA per cell, or mtDNA copy number (mtDNAcn), can be expressed as a ratio of mtDNA to nDNA copies, i.e. using nDNA as reference, assuming that all quantified cells are nucleated and diploid [6]. Nowadays, quantitative real-time PCR (qPCR) is a suitable method to quantify mtDNAcn [7][8][9]. ...
Alterations of mitochondrial DNA copy number (mtDNAcn) in the blood (mitochondrial to nuclear DNA ratio) appear associated with several systemic diseases, including primary mitochondrial disorders, carcinogenesis, and hematologic diseases. Measuring mtDNAcn in DNA extracted from whole blood (WB) instead of from peripheral blood mononuclear cells or buffy coat may yield different results due to mitochondrial DNA present in platelets. The aim of this work is to quantify the contribution of platelets to mtDNAcn in whole blood [mtDNAcn(WB)] and to propose a correction formula to estimate leukocytes' mtDNAcn [mtDNAcn(L)] from mtDNAcn(WB). Blood samples from 10 healthy adults were combined with platelet-enriched plasma and saline solution to produce artificial blood preparations. Aliquots of each sample were combined with five different platelet concentrations. In 46 of these blood preparations, mtDNAcn was measured by qPCR. MtDNAcn(WB) increased 1.07 (95%CI 0.86, 1.29; p<0.001) per 1000 platelets present in the preparation. We proved that leukocyte count should also be taken into account as mtDNAcn(WB) was inversely associated with leukocyte count; it increased 1.10 (95%CI 0.95, 1.25, p<0.001) per unit increase of the ratio between platelet and leukocyte counts. If hematological measurements are available, subtracting 1.10 the platelets/leukocyte ratio from mtDNAcn(WB) may serve as an estimation for mtDNAcn(L). Both platelet and leukocyte counts in the sample are important sources of variation if comparing mtDNAcn among groups of patients when mtDNAcn is measured in DNA extracted from whole blood. Not taking the platelet/leukocyte ratio into account in whole blood measurements, may lead to overestimation and misclassification if interpreted as leukocytes' mtDNAcn.
... This number is not only dependent on the amount of mitochondria per cell but also on the number of mtDNA molecules per mitochondrion. Broad ranges in mtDNA content have been reported, from a few molecules in embryonic and pluripotent stem cells [2,3] up to several thousands in subcutaneous adipocytes [4] or cardiac myocytes [5]. The cell-specific mtDNA content is assumed to be fairly stable under physiological conditions but can be altered by stress such as exogenous toxins [6], viral infection [7] and by genetic mutations [8]. ...
Reduced mitochondrial DNA (mtDNA) content in breast cancer cell lines has been associated with transition towards a mesenchymal phenotype, but its clinical consequences concerning breast cancer dissemination remain unidentified. Here, we aimed to clarify the link between mtDNA content and a mesenchymal phenotype and its relation to prognosis of breast cancer patients. We analyzed mtDNA content in 42 breast cancer cell lines and 207 primary breast tumor specimens using a combination of quantitative PCR and array-based copy number analysis. By associating mtDNA content with expression levels of genes involved in epithelial-to-mesenchymal transition (EMT) and with the intrinsic breast cancer subtypes, we could not identify a relation between low mtDNA content and mesenchymal properties in the breast cancer cell lines or in the primary breast tumors. In addition, we explored the relation between mtDNA content and prognosis in our cohort of primary breast tumor specimens that originated from patients with lymph node-negative disease who did not receive any (neo)adjuvant systemic therapy. When patients were divided based on the tumor quartile levels of mtDNA content, those in the lowest quarter (≤ 350 mtDNA molecules per cell) showed a poorer 10-year distant metastasis-free survival than patients with > 350 mtDNA molecules per cell (HR 0.50 [95% CI 0.29-0.87], P = 0.015). The poor prognosis was independent of established clinicopathological markers (HR 0.54 [95% CI 0.30-0.97], P = 0.038). We conclude that, despite a lack of evidence between mtDNA content and EMT, low mtDNA content might provide meaningful prognostic value for distant metastasis in breast cancer.
... The DNA lysate volume was adjusted to approximately 1.5 mL using Solution A:Solution B 1:1 (vol/vol). Based on the assumption that each WBC contains about 400 copies of mtDNA, 23 the concentration of the DNA lysate stock solution used from spiking studies was approximately 4 3 10 10 mtDNA copies/mL. ...
Background:
Pathogen inactivation methods are increasingly used to reduce the risk of infections after transfusion of blood products. Photochemical treatment (PCT) of platelets (PLTs) and plasma with amotosalen and ultraviolet A (UVA) light inactivates pathogens and white blood cells through formation of adducts between amotosalen and nucleic acid that block replication, transcription, and translation. The same adducts block the amplification of nucleic acids using polymerase chain reaction (PCR) in a manner that correlates with the number of adducts formed, providing a direct quality control (QC). Current QC measures for PCT rely on indirect methods that measure the delivered UVA dose or percent residual amotosalen after illumination, rather than directly measuring nucleic acid modification.
Study design and methods:
Endogenous mitochondrial DNA (mtDNA), which is detectable in PLT and plasma units, was chosen as a target for the quantification of photochemically induced modifications. DNA was extracted from untreated or amotosalen and UVA-treated PLTs or plasma, and mtDNA fragments of variable lengths were quantified using a real-time PCR inhibition assay.
Results:
PCT induced increasing real-time PCR inhibition of mtDNA amplification for larger amplicon sizes. Amplification was unaffected by treatment with amotosalen or UVA alone, whereas up to 3 log inhibition was observed after PCT. Blinded PCR testing of a panel of 110 samples each, from PLT or plasma components prepared for routine use within a blood center, allowed 100% discrimination between untreated and treated units.
Conclusion:
Our initial findings indicate that an adequately sensitive, quantitative real-time PCR inhibition assay targeting mtDNA could provide a valuable tool to confirm and monitor PCT.
... 13 Subcutaneous fat is a rich source of mitochondria. We use the subcutaneous fat, which is usually trimmed and discarded from punch skin biopsies, to measure levels of mitochondrial DNA with real-time PCR 23 in HIV-seropositive patients receiving antiretrovirals. Specific antiretrovirals, the dideoxynucleosides, reduce mitochondrial DNA levels by inhibition of gamma DNA polymerase, 60 and thus this technique may provide a convenient measure of the severity of dideoxynucleoside toxicity and the metabolic complications common in individuals treated with these drugs longterm. ...
... When coupled to reverse transcription, qPCR is the most popular method for the quantification of gene expression because of its high sensitivity and accuracy (Bustin 2000). In recent years, this technique has also been increasingly used for quantitative analysis of mtDNA contents (Gahan, Miller et al. 2001;Cote, Brumme et al. 2002;von Wurmb-Schwark, Higuchi et al. 2002;Liu, Tsai et al. 2003). ...
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... We found that the mean copy number in CRC was significantly lower than the copy number in adjacent tissues. The copy numbers obtained from the present study were significantly lower than the copy number of myocardial and peripheral blood mononuclear cells reported from other studies[5,20]. We also found that the decrease in mtDNA copy number was significantly correlated with lymph-node metastasis but not with sex, age, pathological type, or TNM stage. ...
Background
Experimental data suggest that mitochondria is involved in tumorigenesis. However, little is known about the qualitative and quantitative changes of mtDNA in colorectal cancer tissues. We therefore conducted possible correlations of the mitochondrial DNA (mtDNA) copy number in colorectal cancer (CRC) with clinical and pathological findings and CRC prognosis.
Methods
mtDNA copy numbers in CRC cancer tissue and adjacent non-cancerous tissue samples were measured using quantitative real-time polymerase chain reaction analyses from 60 patients admitted to our hospital. We examined the correlation of mtDNA copy numbers and clinicopathologic parameters of CRC patients. The correlation between mtDNA copy number and three-year survival was analyzed.
Results
The mtDNA copy number was lower in CRC tissue compared with the corresponding non-cancerous colorectal tissue (mean: 108.60 ± 20.11 vs. 153.68 ± 25.72) and was significantly correlated with lymph-node metastasis. Patients with a lower mtDNA copy number tended to have lower 3-year survival than patients with a higher mtDNA copy number assessed by Kaplan–Meier curves, but the correlation was not significant (overall survival, 63.0 vs 83%).
Conclusions
These results suggest that a reduced copy number of mtDNA is correlated with malignant potential in CRC.
... e relatively low mtDNA copy number revealed in isolated lymphocytes is consistent with very limited data reported in the literature. For example, one study detected ∼87 to 579 copies/cell with a different real-time PCR method [36] and the other ∼70 to 320 copies/cell with competitive PCR in lymphocytes [37]. Many studies report mtDNA content on a relative scale [19,[38][39][40][41]. ...
Systemic oxidative stress is associated with a wide range of pathological conditions. Oxidative DNA damage is frequently measured in circulating lymphocytes. Mitochondrial DNA (mtDNA) is known to be more sensitive to oxidative damage than nuclear DNA but is rarely used for direct measurement of DNA damage in clinical studies. Based on the supercoiling-sensitive real-time PCR method, we propose a new approach for the noninvasive monitoring of systemic oxidative stress by quantifying the mtDNA structural damage and copy number change in isolated lymphocytes in a single test. We show that lymphocytes have significantly less mtDNA content and relatively lower baseline levels of damage than cancer cell lines. In an ex vivo challenge experiment, we demonstrate, for the first time, that exogenous H2O2 induces a significant increase in mtDNA damage in lymphocytes from healthy individuals, but no repair activity is observed after 1 h recovery. We further demonstrate that whole blood may serve as a convenient alternative to the isolated lymphocytes in mtDNA analysis. Thus, the blood analysis with the multiple mtDNA end-points proposed in the current study may provide a simple and sensitive test to interrogate the nature and extent of systemic oxidative stress for a broad spectrum of clinical investigations.
... The genetics of mitochondrial disorders is complex in that disease can be caused by mutations in either nuclear-encoded genes or maternally inherited mitochondrial DNA (mtDNA), which are present in hundreds to thousands of copies per cell. [1][2][3] Pathogenic mtDNA mutations, ranging from point mutations to large deletions, are often present in mixed proportions with wildtype mtDNA molecules within the same cell, a biological phenomenon termed heteroplasmy. The degree of mtDNA mutant heteroplasmy can vary significantly across different tissues of the same individual, and the percentage of a mutation is an important contributor to the clinical phenotype. ...
Purpose:
The application of massively parallel sequencing technology to the analysis of the mitochondrial genome has demonstrated great improvement in the molecular diagnosis of mitochondrial DNA-related disorders. The objective of this study was to investigate the performance characteristics and to gain new insights into the analysis of the mitochondrial genome.
Methods:
The entire mitochondrial genome was analyzed as a single amplicon using a long-range PCR-based enrichment approach coupled with massively parallel sequencing. The interference of the nuclear mitochondrial DNA homologs was distinguished from the actual mitochondrial DNA sequences by comparison with the results obtained from conventional PCR-based Sanger sequencing using multiple pairs of primers.
Results:
Our results demonstrated the uniform coverage of the entire mitochondrial genome. Massively parallel sequencing of the single amplicon revealed the presence of single-nucleotide polymorphisms and nuclear homologs of mtDNA sequences that cause the erroneous and inaccurate variant calls when PCR/Sanger sequencing approach was used. This single amplicon massively parallel sequencing strategy provides an accurate quantification of mutation heteroplasmy as well as the detection and mapping of mitochondrial DNA deletions.
Conclusion:
The ability to quantitatively and qualitatively evaluate every single base of the entire mitochondrial genome is indispensible to the accurate molecular diagnosis and genetic counseling of mitochondrial DNA-related disorders. This new approach may be considered as first-line testing for comprehensive analysis of the mitochondrial genome.Genet Med 2013:15(5):388-394.
... While evidence of the important role mitochondria and glycolysis play in maintaining hESC pluripotency is emerging, there is currently little known about the role mitochondria play in hESC differentiation. It is known that mitochondria levels vary in different cell types [23], [24] and similarly their role in differentiation has been implicated in multiple human lineages including mesenchymal stem cells [25], [26], cardiac mesangioblasts [27] and embryonic stem cells [20]. Based on recent evidence, which indicates that hESC pluripotency status can be influenced by shifts in oxidative phosphorylation and glycolysis, we examined the molecular changes in mitochondrially associated genes in response to mitochondrial biogenesis agents. ...
Human embryonic stem cell (hESC) derivatives show promise as viable cell therapy options for multiple disorders in different tissues. Recent advances in stem cell biology have lead to the reliable production and detailed molecular characterisation of a range of cell-types. However, the role of mitochondria during differentiation has yet to be fully elucidated. Mitochondria mediate a cells response to altered energy requirements (e.g. cardiomyocyte contraction) and, as such, the mitochondrial phenotype is likely to change during the dynamic process of hESC differentiation. We demonstrate that manipulating mitochondrial biogenesis alters mesendoderm commitment. To investigate mitochondrial localisation during early lineage specification of hESCs we developed a mitochondrial reporter line, KMEL2, in which sequences encoding the green fluorescent protein (GFP) are targeted to the mitochondria. Differentiation of KMEL2 lines into the three germ layers showed that the mitochondria in these differentiated progeny are GFP positive. Therefore, KMEL2 hESCs facilitate the study of mitochondria in a range of cell types and, importantly, permit real-time analysis of mitochondria via the GFP tag.
... It is well known that the number of mitochondria varies by a wide range in the various cell types. This is manifested as different numbers of mtDNA molecules: the variation range is 1600-2000 for fibroblasts (Legros et al., 2004), 240-420 for lymphocytes (Szuhai et al., 2001) and the number is estimated as about 400 for peripheral human blood monocytes (Gahan et al., 2001). There are no data on the number of mitochondria and mtDNA in ES cells. ...
In hybrid cells, not only are the nuclear genomes of parent cells fused, but their cytoplasm is as well. Mitochondrial DNA
(mtDNA) is a convenient marker of cytoplasm that allows us to gain insight into the organization of hybrid-cell cytoplasm.
We analyzed the parental mtDNA in hybrid cells resulting from the fusion of Mus musculus embryonic stem (ES) cells with splenocytes and fetal fibroblasts of DD/c mice or with splenocytes of M. caroli. Identification of parental mtDNA in hybrid cells was based on polymorphism among parental mtDNA for certain restriction
endonucleases. We found that intra- and interspecific ES cell-splenocyte hybrid cells either entirely or partially lost mtDNA
derived from a somatic partner, whereas ES cell-fibroblast hybrids retained mtDNA from both parents in similar ratios with
a slight bias. The loss of somatic mitochondria by ES-splenocyte hybrids implies a nonrandom segregation of parental mitochondria,
which was supported by a computer simulation of genetic drift. In contrast, ES cell-fibroblast hybrids show bilateral random
segregation of the parental mitochondria judging from the analysis of mtDNA in single cells. Preferential segregation of somatic
mitochondria does not depend on the differences in sequences of the parental mtDNA, but rather on the replicative state of
parental cells.
... Real-time PCR has been used to positively identify small quantities of mtDNA from human peripheral blood and subcutaneous fat cells [30] ...
The ivory industry is the single most serious threat to global elephant populations. A highly sensitive, species-specific real-time PCR assay has been developed to detect and quantify African elephant (Loxodonta africana), Asian elephant (Elephas maximus) and Woolly Mammoth (Mammuthus primigenius) mitochondrial DNA from highly processed samples involved in the international ivory trade. This assay is especially useful for highly processed samples where there are no distinguishing morphological features to identify the species of origin. Using species-specific Taqman(®) probes targeting a region of the mitochondrial cytochrome b gene, we developed an assay that can be used to positively identify samples containing elephant or Woolly mammoth DNA faster and more cost-effectively than traditional sequencing methods. Furthermore, this assay provides a diagnostic result based on probe hybridization that eliminates ambiguities associated with traditional DNA sequence protocols involving low template DNA. The real-time method is highly sensitive, producing accurate and reproducible results in samples with as few as 100 copies of template DNA. This protocol can be applied to the enforcement of the Convention on the International Trade of Endangered Species (CITES), when positive identification of species from illegally traded products is required by conservation officers in wildlife forensic cases.
... There is currently no method able to quantify total nonhuman mammalian mtDNA. Most methods to quantify mtDNA are limited to human samples and are generally focused on medical implications [18] such as identifying single nucleotide polymorphisms to quantify mutant versus wild type mitochon-dria [19][20][21] and the effects of HIV treatment drugs on mitochondrial synthesis [22]. There have been several recent advances in detecting copy number of human mtDNA for forensic purposes [23,24]. ...
The number of mitochondria per cell varies by cell type and the number of mitochondrial DNA (mtDNA) genomes varies per mitochondrion. Biological samples from unknown species are encountered frequently in forensic science investigations and are often contaminated with human mtDNA making analysis difficult. Currently, no techniques to quantify non-human mtDNA are available. We report on a method to accurately quantify, sensitive to 100 copies (1.7fg), mtDNA from human and non-human sources when present as a mixture. The test developed uses the cytochrome b (cytb) and the ribosomal 12S genes on the mitochondrial genome. Universal and human specific fragments of similar size are amplified and quantified using SYBR Green. We validate the test with 24 human samples and 27 non-human mammalian samples. The human fraction of a sample can then be subtracted from the universal fraction for an accurate estimation of non-human mtDNA copy number.
... Several laboratories have developed methods of directly quantifying cellular mtDNA content (Gahan et al., 2001) and are in the process of assessing the validity of these assays in the clinical context. Work undertaken at the Alfred Hospital, Melbourne, has demonstrated that mtDNA content in subcutaneous fat is significantly lower in patients currently prescribed at least one DDx compared with those who are either not currently on treatment, or those who are on treatment including only non-DDx NRTIs (Cherry et al., 2001a) (Fig. 3). ...
Sensory neuropathies occur commonly in the setting of HIV infection. Sensory neuropathy (SN) is clearly associated with HIV itself, and in this context develops in association with increased macrophage activation in the peripheral nervous system. A clinically identical SN may also occur as a consequence of exposure to some HIV treatments. In this setting, impaired mitochondrial function is thought to play a role in the development of neurological dysfunction.
This review explores the evidence for the neurotoxicity of HIV and HIV treatments, the effect of nucleoside reverse transcriptase inhibitors on mitochondria, and the likely associations between these.
Dideoxynucleotide drugs are commonly associated with SN. The nucleoside reverse transcriptase inhibitors inhibit mitochondrial DNA synthesis and may thus exacerbate existing viral-induced nerve damage.
... Because of the difficulties in accessing skeletal muscle, which is known to be the best source for evaluation of mitochondrial cytotoxicity by quantifying mtDNA, researchers are focusing on the more accessible biological samples. Therefore, it is believed that peripheral blood mononuclear cells (PBMCs) and subcutaneous adipose tissues can reflect the toxicity of antiretroviral therapy on mitochondria [11][12][13][14]. In this study, we describe a real-time polymerase-chainreaction (PCR) assay for mtDNA quantification associating DNA extraction procedures applied on PBMCs and subcutaneous adipose tissues, and study the antiretroviral effect on mitochondria. ...
Known for their ability to inhibit the human DNA polymerase-gamma, nucleoside analogues induce toxic effects on mitochondria ranging from increased serum lactate levels to fatal lactic acidosis. DNA polymerase-gamma ensures the mitochondrial DNA (mtDNA) replication and, thus, its inhibition leads to the decrease of the mtDNA. We describe a real-time PCR assay for mtDNA quantification associating DNA extraction procedures applied on peripheral blood mononuclear cells (PBMCs) and subcutaneous adipose tissues and to study the antiretroviral effect on mitochondria.
Total DNA was extracted from PBMCs and subcutaneous adipose tissues. Nuclear and mitochondrial genes were amplified to determine the number of copies of mtDNA per cell using a cyt-b recombinant plasmid as standard control. We analysed eight HIV-infected asymptomatic patients never treated, four patients who had been treated for 6 months with highly active antiretroviral therapy (HAART) and six non-infected donors.
The mtDNA quantification gave rise to reproducible results as the mean coefficients of variation were 1.09% for replicates of samples undertaken 10 times within the same run, and 5.78% and 3.7% for replicates tested in five different runs at 1:100 and 1:1000 dilutions, respectively. Median levels of mtDNA in PBMCs of healthy donors, naive and treated HIV-infected patients were 2.94, 2.78 and 1.93 log HIV-1 RNA copies/mL, respectively. Whereas DNA from PBMCs was shown to be devoid of inhibitors, subcutaneous adipose tissues needed an extra treatment as they were found to be highly inhibited.
The method generated consistent and reproducible results and was successfully applied to DNAs extracted from PBMCs and subcutaneous adipose tissues with adapted extraction. The mtDNA changes in PBMCs were found to be fast as they fall off after 6 months' therapy, decreasing from 2.78 to 1.93 log copies/mL.
... For example, in skeletal and cardiac muscle there are 3650^620 and 6790^920 mtDNA copies per diploid nuclear genome respectively, representing a significant difference of P ¼ 0.006 (Miller et al. 2003). The mtDNA copy number in peripheral blood mononuclear cells is 409^148 copies per cell and in subcutaneous fat it is 2049^391 (Gahan et al. 2001), whilst cultured fibroblasts possess 823^71 copies/cell (Zhang et al. 1994). Interestingly, there are 2.6 £ 10 5 copies per bovine oocyte whilst bovine fetal heart fibroblasts possess 2.6 £ 10 3 copies/cell (Michaels et al. 1982). ...
The introduction of nuclear transfer (NT) and other technologies that involve embryo reconstruction require us to reinvestigate patterns of mitochondrial DNA (mtDNA) transmission, transcription and replication. MtDNA is a 16.6 kb genome located within each mitochondrion. The number of mitochondria and mtDNA copies per organelle is specific to each cell type. MtDNA is normally transmitted through the oocyte to the offspring. However, reconstructed oocytes often transmit both recipient oocyte mtDNA and mtDNA associated with the donor nucleus. We argue that the transmission of two populations of mtDNA may have implications for offspring survival as only one allele might be actively transcribed. This could result in the offspring phenotypically exhibiting mtDNA depletion-type syndromes. A similar occurrence could arise when nucleo-cytoplasmic interactions fail to regulate mtDNA transcription and replication, especially as the initiation of mtDNA replication post-implantation is a key developmental event. Furthermore, failure of the donor somatic nucleus to be reprogrammed could result in the early initiation of replication and the loss of cellular mtDNA specificity. We suggest investigations should be conducted to enhance our understanding of nucleo-cytoplasmic interactions in order to improve NT efficiency.
... We developed a real-time polymerase chain reaction (PCR) assay to accurately quantify the mtDNA copy numbers per cell in peripheral blood mononuclear cells (PBMCs). This assay has been shown to give consistent and reproducible results, through internal studies conducted in our laboratory (Neurovirology Research Unit, Infectious Diseases Unit, Alfred Hospital, Melbourne, Australia) [6] and participation in an international quality-control program [7]. ...
It has been suggested that lipoatrophy associated with exposure to nucleoside analogues is caused by depletion of mitochondrial
DNA (mtDNA). The aim of the present study was to determine whether switching treatment from a thymidine analogue to abacavir
was associated with an increase in the mtDNA copy number in peripheral blood mononuclear cells (PBMCs). Of 111 patients with
lipoatrophy who were randomized to have treatment switched to abacavir or to continue treatment with thymidine analogues,
94 patients had PBMCs obtained at baseline and at weeks 4, 12, and 24, for quantification of the mtDNA copy number. During
the 24-week study, there was no significant change in mtDNA copy numbers in PBMCs in either treatment group, despite improvement
in peripheral lipoatrophy among patients whose treatment was switched to abacavir.
... Furthermore, these PCR methods use an endpoint determination that may not be truly quantitative because of plateau effects. Several methods have been developed using real-time PCR (either TaqMan or molecular beacons) to monitor either mtDNA copy number or the amount of the common deletion in human cells [Heid et al., 1996; Steuerwald et al., 2000; Gahan et al., 2001; Lim et al., 2001; Reynier et al., 2001; Rodriguez-Santiago et al., 2001; Szuhai et al., 2001; He et al., 2002; Miller et al., 2003]. Rodriguez-Santiago et al. [2001] determined mtDNA copy number in the brains of Alzheimer patients using qPCR of the mitochondrial ND2 gene vs. the nuclear 18S gene. ...
Changes in mitochondrial DNA copy number and increases in mitochondrial DNA mutations, especially deletions, have been associated with exposure to mutagens and with aging. Common deletions that are the result of recombination between direct repeats in human and rat (4,977 and 4,834, bp, respectively) are known to increase in tissues of aged individuals. Previous studies have used long-distance PCR and Southern blot or quantitative PCR to determine the frequency of deleted mitochondrial DNA. A quantitative PCR (TaqMan) assay was developed to detect both mitochondrial DNA copy number and deletion frequency in the rat. This methodology allows not only the determination of changes in the amount of mitochondrial DNA deletion relative to total mitochondrial DNA but also to determine changes in total mitochondrial DNA relative to genomic DNA. As a validation of the assay in rat liver, the frequency of the common 4,834 bp deletion is shown to increase with age, while the relative mitochondrial DNA copy number rises at a young age (3-60 days), then decreases and holds fairly steady to 2 years of age.
The past thirty years have seen a surge in interest in pathophysiological roles of mitochondrial, and the accurate quantification of mitochondrial DNA copy number (mCN) in cells and tissue samples is a fundamental aspect of assessing changes in mitochondrial health and biogenesis. Quantification of mCN between studies is surprisingly variable due to a combination of physiological variability and diverse protocols being used to measure this endpoint. The advent of novel methods to quantify nucleic acids like digital polymerase chain reaction (dPCR) and high throughput sequencing offer the ability to measure absolute values of mCN. We conducted an in-depth survey of articles published between 1969 - 2020 to create an overview of mCN values, to assess consensus values of tissue-specific mCN, and to evaluate consistency between methods of assessing mCN. We identify best practices for methods used to assess mCN, and we address the impact of using specific loci on the mitochondrial genome to determine mCN. Current data suggest that clinical measurement of mCN can provide diagnostic and prognostic value in a range of diseases and health conditions, with emphasis on cancer and cardiovascular disease, and the advent of means to measure absolute mCN should future clinical applications of mCN measurements.
Mitochondria are semiautonomous organelles that contain double-stranded (a-helix) circular DNA molecules (mtDNAs) of about 16300 to 16500 base pairs (bp). The mtDNA encodes only 13 proteins, 22 tRNAs and 2 rRNAs. Up to 95% of proteins involved in biogenesis and functions of mitochondria are encoded by the cell nucleus. The copy number of mitochondrial genome in a typical mammalian somatic cell ranges from approximately 2 x 10(3) to about 5 x 1033, whereas the number of mtDNA molecules in a single mature (Metaphase II) oocyte is about 1.6 x 10(5) in mice, 2.5 x 10(5) in cattle and 3-8 x 10(5) in humans. In the procedure of somatic or embryo cell cloning (nuclear transfer), mitochondria of nuclear donor cells, together with the nucleus, are transplanted to the enucleated recipient oocyte (ooplast). Thus, the cloned embryo should harbour the mtDNAs from both the donor cell and the recipient oocyte. In cloned animals, mitochondria are inherited primarily from recipient oocytes, and mitochondria from donor cells appear to be rapidly eliminated during the first few cleavage divisions and are almost undetectable by the blastocyst stage. This selective segregation of the donor mitochondrial genome, which takes place in the preimplantation development of nuclear transfer-derived embryos, leads gradually to cellular mtDNA homoplasmy. Only in some cases does the mitochondria originating from both donor cells and recipient oocytes coexist (the so-called rntDNA heteroplasmy). When cloning both embryos and adult individuals, what is often forgotten is the presence in the cytoplasm of donor- and recipient-cells of mtDNA. This contains small (approximately 0.01%) amounts of total cell genetic information, but it is different from information recorded in the nuclear DNA (99.99% of cellular genome). Thus, introduction of donor cell nucleus into the oocyte derived from another individual leads to generation of hybridic (in terms of genetic mitochondrial material) reconstructed oocytes, which can have a certain influence on the genotype and phenotype identity of offspring produced as a result of cloning. The "ideal" clone can be obtained only with donor cell nucleus transplanted into the oocyte originating from the same individual. Therefore "ideal mammalian clones" can only be the clones of females, whose overall mitochondrial genome has a completely homogenous pattern of regulatory and coding nucleotide sequences of all cellular mtDNA copies in all the somatic and germ cell lines.
Various side effects have been associated to HIV antiretroviral therapy (ART) containing nucleoside reverse transcriptase inhibitors (NRTIs) as a result of mitochondrial toxicity. Mitochondrial DNA (mtDNA) depletion has important clinical consequences: lipodystrophy, hepatic toxicity, lactic acidosis, myopathy and polyneuropathy. Several methods have been developed in recent years in order to prevent the clinical and biological side effects during NRTIs treatment. These methods aim to predict the metabolic risk using non invasive tools. The present study aims to evaluate the current laboratory methods for mitochondrial toxicity and to assess their utility for monitoring ART toxicity.
This chapter intends to provide an overview of sample treatment techniques, and liquid chromatography—mass spectrometry (LC-MS) instrumental setups for the bioanalyis of intracellular drugs. The sample pretreatment includes isolation, counting and lysis of the cells of interest, and cleanup of the harvested subcellular fraction of cells to remove proteins, carbohydrates, salts, lipids, and other endogenous compounds. Various interface and MS detection techniques for intracellular drug LC-MS bioanalysis are discussed along with optimization of experimental conditions and method validation. Finally, application examples were summarized according to cell types, and method information, such as LC conditions, MS conditions, validation status, limit of quantitation (LOQ), and limit of detection (LOD).
Mitochondrial DNA (mtDNA) content has been shown to be associated with cancer susceptibility. We identified 926 bladder cancer patients and compared these to 926 healthy-controls frequency matched on age, gender, and ethnicity. Patients diagnosed with bladder cancer had significantly decreased mtDNA content when compared to control subjects (median: 0.98 vs. 1.04, p<0.001). Low mtDNA content (ie, less than the median in control subjects) was associated with a statistically significant increased risk of bladder cancer, when compared with high mtDNA content (Odds Ratio (OR) = 1.37, 95% CI = 1.13 to 1.66, p<0.001). In a trend analysis, a statistically significant dose-response relationship was detected between lower mtDNA content and increasing risk of bladder cancer (P for trend <0.001). When stratified by host characteristics, advanced age (>65 years), male/female sex and positive smoking history were all significantly associated with low mtDNA content and increased risk of bladder cancer. We identified two unique mtDNA polymorphisms significantly associated with risk of bladder cancer: mitot10464c (OR=1.39 95%CI: 1.00-1.93, p=0.048) and mitoa4918g (OR=1.40, 95% CI: 1.00-1.95, p=0.049). Analysis of the joint effect of low mtDNA content and unfavorable mtDNA polymorphisms revealed a 2.5 fold increased risk of bladder cancer (OR=2.50, 95% CI: 1.60-3.94, p<0.001). Significant interaction was observed between mitoa4918g and mtDNA content (p for interaction = 0.028). Low mtDNA content was associated with increased risk of bladder cancer and we identified new susceptibility mtDNA alleles associated with increased risk that require further investigation into the biological underpinnings of bladder carcinogenesis.
Copyright © 2015, American Association for Cancer Research.
Asian box turtles (genus Cuora, family Geoemydidae) comprise a clade of 12 aquatic and semiaquatic nominate species distributed across southern China and
Southeast Asia. Over the last two decades, turtles throughout Asia have been harvested at an unsustainable rate to satisfy
demands for food, traditional Chinese medicine, and the pet trade. Consequently, all species of Cuora were recently placed on the IUCN Red List, nine are currently listed as critically endangered by the IUCN, and all species
are listed in Appendix II of CITES. We compiled a 67-specimen mitochondrial (∼1,650 base pairs (bp) from two mitochondrial
genes) and a 40-specimen nuclear-plus-mitochondrial (∼3,900bp total, three nuclear introns plus an additional ∼860bp mitochondrial
gene fragment) DNA data set to reconstruct the phylogeny of Cuora species and to assess genetic diversity and species boundaries for several of the most problematic taxa. Our sampling included
23 C. trifasciata, 17 C. zhoui and 1–4 individuals of the remaining 10 species of Cuora. Maximum likelihood, maximum parsimony and Bayesian analyses all recovered similar, well resolved trees. Within the Cuora clade, mitochondrial and nuclear sequence data indicated that both C. zhoui and C. mccordi represent old lineages with little or no history of interspecific gene flow, suggesting that they are good genealogical species.
Based on mtDNA, Cuora pani was paraphyletic and C. trifasciata was composed of two highly divergent lineages that were not each other’s closest relatives; both cases of non-monophyly were
due to a mtDNA sequence that was widespread and identical in C. aurocapitata, C. pani and C. trifasciata. However, when combined with nuclear DNA results, our data indicate that C. trifasciata is a single, monophyletic taxon, and that mitochondrial introgression and nuclear-mitochondrial pseudogenes have led to a
complex pattern of mitochondrial gene relationships that does not reflect species-level histories. Our results imply that
captive “assurance colonies” of both C. trifasciata and C. pani should be genotyped to identify pure, non-hybrid members of both taxa, and we recommend that introgressed and pure taxa be
managed as independent entities until the full evolutionary histories of these critically endangered turtles are resolved.
HIV therapy with nucleoside analogue reverse transcriptase inhibitors (NRTI) such as stavudine remains in widespread use in resource-limited nations due to potent efficacy, convenience of formulation and lack of practical alternatives. However, it remains unclear whether adverse side effects with NRTI include reduced mitochondrial respiratory function, particularly in peripheral blood lymphocytes (PBLs). The aim of this study was to determine whether stavudine-based highly active antiretroviral therapy (HAART) is associated with impaired mitochondrial respiratory transport chain function in patient derived CD4+PBLs. CD4+PBLs were isolated from asymptomatic HIV-infected patients treated with stavudine-HAART for 3 months (n=10), HIV-infected patients not on treatment (n=9) and uninfected controls (n=18). The basal mitochondrial oxygen consumption of CD4+PBLs from stavudine-treated patients was reduced relative to that of untreated HIV-infected patients and controls (stavudine treated group, 4.22 (25% 2.16, 75% 8.84); control uninfected, 11.2 (25% 3.95, 75% 16.6); untreated 18.1 (25% 11.8, 75% 37.9)ng oxygen atoms/min/ml). Maximal oxygen consumption (stimulated with the proton ionophore FCCP) in cells from stavudine treated patients was also reduced relative to that of untreated patients and controls (stavudine treated, 24.4+/-10.5; control uninfected, 50.6+/-39.5; untreated, 68.8+/-41.1 ng oxygen atoms/min/ml). Citrate synthase activities, relative mitochondrial volume (by electron microscopy) and mtDNA copy numbers per cell were not different between groups. Therapy with stavudine results in impaired mitochondrial function in CD4+PBLs that does not appear to be due to reduced mitochondrial volume or DNA content and cannot be attributed to infection with HIV.
Increasing evidence suggests that oxidative stress plays a causative role in prostate cancer initiation and progression. Furthermore, enhanced levels of systemic oxidative stress detected in lymphocytes are also associated with prostate cancer risk. Due to mtDNA's high sensitivity to oxidative stress, we hypothesize that mtDNA may serve as a surrogate to oxidative stress in cancer cells and lymphocytes. First, we improved the sensitivity of a novel method for mtDNA damage analysis and proposed a standardized protocol. Secondly, we developed an approach for quantifying systemic oxidative stress using mtDNA responses in circulating lymphocytes. Finally, we demonstrated differential mtDNA responses induced by oxidative stress in two isogenic prostate cancer cell lines with different metastatic potential. The more metastatic C4-2 was more susceptible to oxidative stress and prone to mtDNA damage and copy number change. These results offer new insights into prostate cancer progression. De plus en plus d'études suggèrent que le stress oxydatif joue un rôle déterminant dans l'initiation et la progression du cancer de la prostate. Aussi, des niveaux élevés de stress oxydatif systémique mesurés dans les lymphocytes ont été associés à un risque accru du cancer de la prostate. Puisque le génome mitochondrial (ADNmt) est très sensible au stress oxydatif, nous postulons que ce dernier peut servir de marqueur pour le stress oxydatif dans les cellules cancéreuses ainsi que dans les lymphocytes. Dans un premier temps, nous avons accru la sensibilité d'une nouvelle méthode d'analyse du dommage de l'ADNmt. Ceci nous a permis de proposer un protocole de mesure standardisé. Deuxièmement, nous avons développé une méthode afin de quantifier le stress oxydatif systémique en utilisant les réponses de l'ADNmt des lymphocytes. Finalement, nous avons démontré que deux lignées cellulaires prostatiques isogéniques, LNCaP et C4-2, ayant un potentiel métastatique distinct, répondaient différemment au stress oxydatif induit expérimentalement. La lignée C4-2, à potentiel plus métastatique, était plus sensible au stress oxydatif, se traduisant par une plus grande susceptibilité de l'ADNmt à être endommagé. Ces résultats apportent donc une nouvelle avenue dans la compréhension de la progression du cancer de la prostate.
Title proper from title frame. Thesis (Ph. D.)--University of Hong Kong, 2005. Text (Electronic book) Mode of access: World Wide Web.
Im Rahmen dieser Promotion wurden mehrere Projekte aus dem Bereich humaner autosomaler, Y-chromosomaler und mitochondrialer DNA-Marker bearbeitet. U.a. wurde eine Meiosenstudie für das bislang kaum eingesetzte autosomale short tandem repeat (STR) System D10S2325 in einer deutschen und kurdischen Population durchgeführt. Dieses Pentanukleotid-System erwies sich als nützlicher Marker für die Verwandtschaftsanalyse. Mittels der Sublocus-spezifischen Amplifikation konnte die Individualisierungseffizienz des Y-STR-Systems DYS385 gesteigert und ein Mutationsfall mit einer sehr seltenen 3-Schritt-Mutation aufgeklärt werden. Außerdem konnten zwei neuartige real-time PCR-Assays zur Quantifizierung von Zellkern und mitochondrialer DNA entwickelt werden. Diese Assays zeichnen sich durch eine duale Nutzung aus: sie erlauben neben der Quantifizierung eine direkt anschließende Genotypisierung mittels Amplikonlängenbestimmung bzw. direkter Sequenzierung.
Antiretroviral therapy (ART) has improved life expectancy with HIV infection, but long-term toxicities associated with these medications are now a major global disease burden. There is a clear need to develop useful methods for monitoring patients on antiretroviral drugs for early signs of toxicity. Assays with predictive utility -- allowing therapy to be changed before serious end organ damage occurs -- would be ideal. Attempts to develop biochemical methods of monitoring ART toxicity have concentrated on the mitochondrial toxicity of nucleoside analogue reverse transcriptase inhibitors and have not generally lead to assays with widespread clinical applications. For example, plasma lactate and peripheral blood measurements of mitochondrial DNA associate with exposure to potentially toxic nucleoside analogue reverse transcriptase inhibitors but have not reliably predicted clinical toxicity. Better assays are needed, including markers of toxicity from additional drug classes. Apoptosis may be a potential marker of ART toxicity. Increased apoptosis has been demonstrated both in vitro and in vivo in association with various antiretroviral drug classes and a range of clinical toxicities. However, quantifying apoptosis on biopsy specimens of tissue (such as adipose tissue) is impractical for patient monitoring. Novel assays have been described that can quantify apoptosis using minute tissue samples and initial results from clinical samples suggest peripheral blood may have utility in predicting ART toxicities. The limitations and potential of such techniques for monitoring patients for drug side effects will be discussed.
Nucleoside Reverse Transcriptase Inhibitors (NRTIs) used for the treatment of HIV-1 inhibit the replication of mitochondrial DNA (mtDNA), which may contribute to severe mitochondrial toxicity including lipodystrophy, through the inhibition of polymerase gamma (POLG). Polymorphisms of POLG could explain the variation in mitochondrial toxicity in HIV-1-infected patients. We explored the relationship between selected polymorphisms of POLG and lipodystrophy related to NRTIs. We studied single nucleotide polymorphisms (SNP) at three amino acid residues (R1142, E1143 and R1146) and the CAG repeats of POLG in a case-control study including HIV-1 treated patients with lipodystrophy (n=69) and 2 controls (without lipodystrophy) per case matched by age, race and sex (n=138). Compared with matched controls, the polymorphisms in E1143 were significantly more frequent in case patients with lipodystrophy (aOR=4.7; p=0.048), and this was associated with a significant decrease of mtDNA in PBMC. In addition, among the parameters tested, the conditional logistic regression showed that the lipodystrophy has a strong link with E1143 polymorphisms, associated with D4T treatment (aOR=9.29, p=0.002). In conclusion, patients harbouring the changes of E1143 in the catalytic site of POLG exhibit a 4-fold increased risk to develop lipodystrophy than HIV-1 treated patients who do not have changes in E1143 and this risk can increase if the patient presenting the SNP received D4T. These could be due to decreased content of mtDNA in PBMC in these patients. Therefore, the toxicity of NRTIs leading to lipodystrophy in some HIV-1 infected patients could be explained in part by the occurrence of POLG polymorphisms.
The extent to which mitochondrial DNA (mtDNA) content (also termed mtDNA copy number) in normal human cells is influenced by genetic factors has yet to be established. In addition, whether inherited variation of mtDNA content in normal cells contributes to cancer susceptibility remains unclear. Renal cell carcinoma accounts for 85% of all renal cancers. No studies have investigated the association between mtDNA content and the risk of renal cell carcinoma.
We first used a classic twin study design to estimate the genetic contribution to the determination of mtDNA content. mtDNA content was measured by quantitative real-time polymerase chain reaction in peripheral blood lymphocytes from 250 monozygotic twins, 92 dizygotic twins, and 33 siblings (ie, individual siblings of a pair of twins). We used biometric genetic modeling to estimate heritability of mtDNA content. We then used a case-control study with 260 case patients with renal cell carcinoma and 281 matched control subjects and multivariable logistic regression analysis to examine the association between mtDNA content in peripheral blood lymphocytes and the risk of renal cell carcinoma. All statistical tests were two-sided.
The heritability (ie, proportion of phenotypic variation in a population that is attributable to genetic variation among individuals) of mtDNA content was 65% (95% confidence interval [CI] = 50% to 72%; P < .001). Case patients with renal cell carcinoma had a statistically significantly lower mtDNA content (1.18 copies) than control subjects (1.29 copies) (difference = 0.11, 95% CI = 0.03 to 0.17; P = .006). Low mtDNA content (ie, less than the median in control subjects) was associated with a statistically significantly increased risk of renal cell carcinoma, compared with high content (odds ratio = 1.53, 95% CI = 1.07 to 2.19). In a trend analysis, a statistically significant dose-response relationship was detected between lower mtDNA content and increasing risk of renal cell carcinoma (P for trend <.001).
mtDNA content appears to have high heritability. Low mtDNA content appears to be associated with increased risk of renal cell carcinoma.
A number of international research groups have developed DNA quantitation assays in order to investigate the role of mitochondrial DNA depletion in anti-retroviral therapy-induced toxicities.
A collaborative study was undertaken to evaluate intra-assay precision and between laboratory concordance of measurements of mitochondrial DNA quantity, as a component of a comprehensive quality assurance project.
Four laboratories were asked to measure and report mitochondrial DNA and nuclear DNA genome copy number, as well as mitochondrial DNA copy number/cell, for 17 coded aliquots of DNA derived from serial dilutions of pooled DNA from a lymphoblastoid cell line. Samples included masked replicates and five standards. All samples had similar mitochondrial DNA/nuclear DNA ratios. Precision within laboratories was assessed by determining the coefficient of variation of replicates. Concordance between laboratories was assessed by determining the average coefficient of variation of the mean replicate values for each sample. The effect of standardising the assay for these three measurements was also assessed for laboratories A, B and C.
Measurements of mitochondrial DNA and nuclear DNA content for replicate samples varied by an average of less than 6% (based on log(10) values, 72% non-logged values), and measurements of mitochondrial DNA/cell for replicates varied by less than 12% (based on log(10) values, 32% non-logged values), with no improvement of precision after standardisation. Standardisation did significantly improve the concordance of results for measurements of mitochondrial DNA content and mitochondrial DNA/cell. Non-standardised measurements of mitochondrial DNA content for the same sample set varied by 19% between laboratories (based on log(10) values, 96% non-logged values), and after standardisation results varied by less than 3% (based on log(10) values, 54% non-logged values). There was no significant improvement for concordance of measures of nuclear DNA content after standardisation, with results varying by 4.56% between laboratories (based on log(10) values, 45% non-logged values) before standardisation, and by 2.49% (based on log(10) values, 50% non-logged values) after standardisation. Derived values of mitochondrial DNA/cell varied between laboratories by an average of 91% (non-logged, 56% log(10) values) before and by 56% (non-logged, 13% log(10) values) after standardisation.
All assays demonstrated good precision. The use of common standards is an important step in improving the comparability of data between laboratories.
In the past few years, mitochondria have been carefully studied to ascertain whether and how in patients affected by HIV antiretroviral therapy is able to alter their functionality and exert a toxic effect on immune cells, as well as on cells present in other districts.
A variety of in-vivo and ex-vivo models have been developed to investigate the functionality of mitochondria and DNA during a variety of physiopathological situations, including HIV infection and its treatment. Numerous technologies are available to study at the single-cell or at the single-organelle level a variety of parameters, such as membrane potential, the activity of respiratory chain enzymes, and DNA content or its sequence. As far as in-vitro studies are concerned, a substantial homogeneity of data exists, and several changes in different mitochondrial parameters have been described that depend upon the drug used, the cell model and the parameter investigated. On the other hand, different results have been reported on biological material collected from HIV-positive patients and immediately analysed. Ex-vivo studies showed that changes in mitochondrial DNA content or in the functionality of the organelle exist in some tissues or cells, but not in others.
One of the possible causes of the discrepancies is the technologies used to investigate mitochondria, and this paper summarizes some of the pros and cons of the main methods used to study mitochondrial function or DNA.
To investigate the impact of antiretroviral treatment on the mitochondrial DNA (mtDNA) content of peripheral blood mononuclear cells (PBMCs) from HIV-1-infected patients.
As absolute mtDNA copy numbers widely differ between individuals, we performed a longitudinal analysis where the patient's first historical specimen was obtained as a baseline reference for relative comparison with subsequent samples from that patient.
mtDNA and nuclear DNA quantitation per cell (beta-globin gene copies) were both measured by real-time polymerase chain reaction analysis of whole DNA extracts of 361 serial live-cryopreserved PBMCs collected in former trials and clinical follow-ups from 60 individuals with established or recently acquired HIV-1 infections before and during administration of various antiviral combination therapies.
mtDNA amounts were stable or increasing over years of natural HIV-1 infection in untreated patients (n = 7), consistent with our finding of a lack of differences in mtDNA copy numbers in patients with either a long established or recent lentivirus infection. Our quantitation system revealed significant changes in mtDNA copy number depending on the designated triple, quadruple, or quintuple anti-HIV drug combinations. Zidovudine + zalcitabine + ritonavir and zidovudine + lamivudine + didanosine regularly lead to mtDNA depletion in each of the treated patients, whereas none of 7 patients (and 35 cell specimens) receiving a stavudine + lamivudine + indinavir combination had any significant mtDNA content variations. In 7 patients, mtDNA copy numbers returned to pretreatment levels and/or higher levels without any interruption of the previously mtDNA-depleting antiretroviral drug combination.
Our assay system allowed the detection of significant changes in the mtDNA content of PBMCs from HIV-1-infected patients taking antiretroviral drugs, as has been reported in the literature with other detection systems. Yet, mtDNA copy numbers regularly diminished during administration of some but not all nucleoside analog-containing combinations. This, plus the occasional finding that depleted mtDNA contents spontaneously increased to baseline levels and/or higher levels during uninterrupted treatment, should raise a note of caution about resorting to the PBMC mtDNA marker for monitoring of antiretroviral drug-related mitochondrial toxicities.
Available evidence suggests that a number of important clinical events in individuals with HIV infection are related to mitochondrial dysfunction. Several factors may contribute to the development of these events and the tissue(s) in which the event occurs. Some individuals are likely to have important genetic predispositions for mitochondrial disease, which may be unmasked by the presence of HIV infection or the introduction of NRTI antiretroviral drugs. HIV infection per se is associated with reduction in mtDNA content and changes in mitochondrial morphology and function, which in some cases leads to clinical events such as myopathy or distal symmetrical neuropathy. NRTI antiretroviral agents may impact mtDNA content and function through a number of different mechanisms and have been demonstrated to be causative of a number of clinical toxicities. A range of other clinical events occurring in individuals with HIV infection, and particularly those on therapy, have also been suggested to be associated with mitochondrial dysfunction. Newer nucleoside and nucleotides agents such as lamivudine, abacavir, and tenofovir appear in vitro and in limited clinical data to be less likely to inhibit mitochondrial DNA polymerase-γ or other mitochondrial functions and appear to be associated with a lower risk of events thought to be related to mitochondrial toxicity. Avoidance of mitochondrial toxicity is the better option. While drug choice plays an important role in the avoidance of mitochondrial toxicity, for many individuals treatment options are limited. Simple, non-invasive tests for mitochondrial function are not routinely available at present, and assays of mtDNA content in blood cells may miss key aspects of mitochondrial function, require careful sample handling and may not reflect events occurring in other tissues. There remains a need for the development of rapid, cheap and clinically applicable assays that would enable the prediction of increased likelihood of mitochondrial events. Additionally, international collaborative studies are required to look at interventions that may be used to enable individuals to remain on specific drug therapies but with a diminished risk of mitochondrial toxicity events.
Highly active antiretroviral therapy (HAART) can cause mitochondrial toxicity. The concentration of mitochondrial DNA (mtDNA) in peripheral blood cells has been reported to be a marker of this toxicity. However, these observations are controversial and were drawn from small series. Thus, we analysed the value of blood mtDNA as a marker of mitochondrial toxicity in a large cohort of human immunodeficiency virus (HIV)-infected out-patients during routine clinical evaluations. Real-time quantitative PCR was used to determine the mtDNA to nuclear DNA (nDNA) ratio in peripheral blood mononuclear cells from 157 consecutive HIV-1-infected patients (13 naive, 144 receiving HAART) and 30 HIV-1-uninfected patients. The mtDNA to nDNA ratio was significantly lower in both groups of HIV-infected patients than in the control group. No significant difference was observed between treated and naive HIV-infected patients. Lactataemia was significantly lower in controls than in the group of HIV-treated patients. None of the treated patients had lactataemia >5 mmol/l or bicarbonates <20 mmol/l. Triglyceride levels were significantly higher in the HAART-treated patients than in the nontreated patients. Clinical symptoms of lipodystrophy were observed in 62 HAART-treated patients. These symptoms were not associated with an abnormal mtDNA to nDNA ratio or plasma triglyceride concentration. The mtDNA to nDNA ratio was lower in DDI/D4T-treated patients than in AZT/3TC-treated patients. In conclusion, there are no obvious links between the mtDNA to nDNA ratio in peripheral mononuclear cells and any clinical symptoms or lactate level. Thus, the mtDNA to nDNA ratio in leukocytes does not seem to be an accurate marker of mild and/or long-term mitochondrial toxicity.
Idiopathic Parkinson's disease (PD) involves a systemic loss of activity of complex I of the mitochondrial electron transport chain. This biochemical lesion plays a key pathogenic role. Transfer of PD mitochondrial DNA recapitulates this loss of activity and several other pathogenic features of PD suggesting that this lesion may arise, at least in part, from mitochondrial DNA. We investigated this possibility by an extensive clonal sequencing of the seven mitochondrial genes encoding complex I subunits in PD and age-matched control frontal cortex. Each gene was completely sequenced an average of 94.4 times for each subject. Aminoacid-changing mutations were found at the frequency of 59.3 per million bases in both PD and controls, corresponding to approximately 32% of the mitochondrial genomes in the average sample having at least one mutation in a complex I gene. Individual low frequency mutations had an abundance of 1-10%. Significant interindividual variation in mutation frequency was observed. Several aminoacid-changing mutations were identified and multiple PD brains but not in controls. Genetic algorithm analysis detected areas in ND genes with a higher mutation frequency in PD that allowed differentiation of PD from controls. Total mutational burden due to low-abundance heteroplasmy is high and may play a role in human disease.
The role of the thymus in HIV-1 pathogenesis remains unclear. We developed an assay to quantify the number of recent thymic
emigrants in blood based on the detection of a major excisional DNA byproduct (termed α1 circle) of T cell receptor rearrangement.
By studying 532 normal individuals, we found that α1 circle numbers in blood remain high for the first 10–15 yr of life, a
sharp drop is seen in the late teen years, and a gradual decline occurs thereafter. Compared with age-matched uninfected control
individuals, α1 circle numbers in HIV-1–infected adults were significantly reduced; however, there were many individuals with
normal α1 circle numbers. In 74 individuals receiving highly active antiretroviral therapy, we found no appreciable effect
on α1 circle numbers in those whose baseline values were already within the normal range, but significant increases were observed
in those with a preexisting impairment. The increases in α1 circle numbers were, however, numerically insufficient to account
for the rise in levels of naive T lymphocytes. Overall, it is difficult to invoke thymic regenerative failure as a generalized
mechanism for CD4 lymphocyte depletion in HIV-1 infection, as α1 circle numbers are normal in a substantial subset of HIV-1–infected
individuals.
The complete sequence of the 16,569-base pair human mitochondrial genome is presented. The genes for the 12S and 16S rRNAs, 22 tRNAs, cytochrome c oxidase subunits I, II and III, ATPase subunit 6, cytochrome b and eight other predicted protein coding genes have been located. The sequence shows extreme economy in that the genes have none or only a few noncoding bases between them, and in many cases the termination codons are not coded in the DNA but are created post-transcriptionally by polyadenylation of the mRNAs.
We have developed a novel "real time" quantitative PCR method. The method measures PCR product accumulation through a dual-labeled fluorogenic probe (i.e., TaqMan Probe). This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays. The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude). Real-time quantitative PCR is extremely accurate and less labor-intensive than current quantitative PCR methods.
To examine trends in disease progression and survival among patients enrolled in the Swiss HIV cohort study during 1988-96 and to assess the influence of new antiretroviral combination therapies.
Prospective multicentre study, with follow up visits planned at six monthly intervals.
Seven HIV units at university centres and cantonal hospitals in Switzerland.
3785 men (mean age 35.0 years) and 1391 women (30.3 years) infected with HIV. 2023 participants had a history of intravenous drug misuse; 1764 were men who had sex with men; 1261 were infected heterosexually; and 164 had other or unknown modes of transmission. 601 participants had had an AIDS defining illness.
During more than 15,000 years of follow up, there were 1456 first AIDS defining diagnoses and 1903 deaths. Compared with those enrolled during 1988-90, the risk of progression to a first AIDS diagnosis was reduced by 18% (relative risk 0.82 (95% confidence interval 0.73 to 0.93)) among participants enrolled in 1991-2, by 23% (0.77 (0.65 to 0.91)) among those enrolled in 1993-4, and by 73% (0.27 (0.18 to 0.39)) among those enrolled in 1995-6. Mortality was reduced by 19% (0.81 (0.73 to 0.90)), 26% (0.74 (0.63 to 0.87)), and 62% (0.38 (0.25 to 0.97)) respectively. Compared with no antiretroviral treatment, the risk of an initial AIDS diagnosis after CD4 lymphocyte counts fell to < 200 cells x 10(6)/1 was reduced by 16% (0.84 (0.73 to 0.97)) with monotherapy, 24% (0.76 (0.63 to 0.91)) with dual therapy, and 42% (0.58 (0.37 to 0.92)) with triple therapy. Mortality was reduced by 23% (0.77 (0.68 to 0.88)), 31% (0.69 (0.60 to 0.80)), and 65% (0.35 (0.20 to 0.60)) respectively.
The introduction of antiretroviral combination therapies outside the selected patient groups included in clinical trials has led to comparable reductions in disease progression and mortality.
We have designed a novel, precise, and sensitive assay to measure unspliced (US) human immunodeficiency virus type 1 (HIV-1) mRNA in peripheral blood mononuclear cells of HIV-1-infected individuals by using real-time PCR and molecular beacons. Individuals were classified as either well suppressed (WS) or partially suppressed, based on longitudinal measurements of plasma HIV-1 RNA. The proportion of individuals with US mRNA undetectable over time was significantly higher among WS individuals; however, 30% of WS subjects still had detectable US mRNA after 24 months of effective antiviral therapy.
The role of the thymus in HIV-1 pathogenesis remains unclear. We developed an assay to quantify the number of recent thymic emigrants in blood based on the detection of a major excisional DNA byproduct (termed alpha1 circle) of T cell receptor rearrangement. By studying 532 normal individuals, we found that alpha1 circle numbers in blood remain high for the first 10-15 yr of life, a sharp drop is seen in the late teen years, and a gradual decline occurs thereafter. Compared with age-matched uninfected control individuals, alpha1 circle numbers in HIV-1-infected adults were significantly reduced; however, there were many individuals with normal alpha1 circle numbers. In 74 individuals receiving highly active antiretroviral therapy, we found no appreciable effect on alpha1 circle numbers in those whose baseline values were already within the normal range, but significant increases were observed in those with a preexisting impairment. The increases in alpha1 circle numbers were, however, numerically insufficient to account for the rise in levels of naive T lymphocytes. Overall, it is difficult to invoke thymic regenerative failure as a generalized mechanism for CD4 lymphocyte depletion in HIV-1 infection, as alpha1 circle numbers are normal in a substantial subset of HIV-1-infected individuals.
Objective
To examine the effect of recent developments in antiretroviral therapy on HIV disease progression and survival.
Design
Retrospective cohort study.
Participants and setting
Two cohorts of people with HIV were defined retrospectively from the records of a large immunology laboratory. The first cohort were subjects whose CD4+ T cell counts had dropped to 200 · 10⁶/L during 1990, and the second were subjects whose CD4+ T cell counts had dropped to 200 · 10⁶/L in 1994.
Main outcome measures
HIV disease progression and survival was determined over a minimum three years of follow‐up for each cohort (ie, 1990–1993; 1994–1997).
Results
346 subjects were included in the analysis (193 subjects from 1990 and 153 from 1994). The relative risk of progression to AIDS in the 1994 cohort compared with the 1990 cohort was 0.57 (95% confidence interval, 0.35–0.91; P=0.018) and the relative risk of death was 0.20 (95% confidence interval, 0.08–0.49; P<0.001).
Conclusions
There were 43% fewer AIDS cases and 80% fewer deaths in the time following the increased availability of combination antiretroviral therapy in Australia.
BACKGROUND & METHODS: National surveillance data show recent, marked reductions in morbidity and mortality associated with the acquired immunodeficiency syndrome (AIDS). To evaluate these declines, we analyzed data on 1255 patients, each of whom had at least one CD4+ count below 100 cells/mm' who were seen at 9 clinics specializing in the treatment of human immunodeficiency virus (HIV) infection in 8 U.S. cities from January 1994 through June 1997. RESULTS: Mortality among the patients declined from 29.4/100 person-yrs in the first quarter of 1995 to 8.8/100 in the second quarter of 1997. There were reductions in mortality regardless of sex, race, age, and risk factors for transmission of HIV. The incidence of any of 3 major opportunistic infections (Pneumocystis curinii pneumonia, Mycohacterium avium complex disease, and cytomegalovirus retinitis) declined from 21.9 /100 person-yrs in 1994 to 3.7/100 person-yrs by mid-1997. In a failure-rate model, increases in the intensity of antiretroviral therapy (classified as none, monotherapy, combination therapy without a protease inhibitor, and combination therapy with a protease inhibitor) were associated with stepwise reductions in morbidity and mortality. Combination antiretroviral therapy was associated with the most benefit; the inclusion of protease inhibitors in such regimens conferred additional benefit. Patients with private insurance were more often prescribed protease inhibitors and had lower mortality rates than those insured by Medicare or Medicaid. CONCLUSIONS: The recent declines in morbidity and mortality due to AIDS are attributable to the use of more intensive antiretroviral therapies.
Objective: To assess the clinical and economic consequences of the use of protease inhibitors in the treatment of HIV infection.
Design: Multicentric, observational, retrospective cohort study.
Setting: Ten AIDS reference centres in France.
Patients: All patients followed in each centre from September 1995 through October 1996.
Main outcome measures: AIDS-defining events, death, health-care resources use, administration of antiretroviral therapy.
Results: Data from 7749 patients in 10 centres showed a drop in hospitalization days by 35%, new AIDS cases by 35%, and deaths by 46%. In the same period, the proportion of patients receiving antiretrovirals rose from 36 to 53% including highly active antiretroviral therapy (HAART), which rose from 0.3 to 18%. Overall cost evaluation showed a slight increase of monthly treatment cost of US$ 12 per patient. Comparison of the three centres that used HAART earliest to the three centres that used it latest showed a clear benefit to early HAART with a drop in hospitalization days by 41%, new AIDS cases by 41% and deaths by 69%. The proportion of patients with HAART rose to 27% and monthly health-care cost decreased by US$ 248 852 (i.e., by US$ 101 per patient per month). Late prescribing centres experienced a less marked effect with a drop in hospitalization days by 22%, new AIDS cases by 31%, and deaths by 32.5%. Proportion of patients with HAART rose to 12% and monthly health-care costs increased by US$ 113 578 (i.e., by US$ 38 per patient per month).
Conclusions: This study supports the extensive use of HAART in HIV-infected patients.
National surveillance data show recent, marked reductions in morbidity and mortality associated with the acquired immunodeficiency syndrome (AIDS). To evaluate these declines, we analyzed data on 1255 patients, each of whom had at least one CD4+ count below 100 cells per cubic millimeter, who were seen at nine clinics specializing in the treatment of human immunodeficiency virus (HIV) infection in eight U.S. cities from January 1994 through June 1997.
Mortality among the patients declined from 29.4 per 100 person-years in the first quarter of 1995 to 8.8 per 100 in the second quarter of 1997. There were reductions in mortality regardless of sex, race, age, and risk factors for transmission of HIV. The incidence of any of three major opportunistic infections (Pneumocystis carinii pneumonia, Mycobacterium avium complex disease, and cytomegalovirus retinitis) declined from 21.9 per 100 person-years in 1994 to 3.7 per 100 person-years by mid-1997. In a failure-rate model, increases in the intensity of antiretroviral therapy (classified as none, monotherapy, combination therapy without a protease inhibitor, and combination therapy with a protease inhibitor) were associated with stepwise reductions in morbidity and mortality. Combination antiretroviral therapy was associated with the most benefit; the inclusion of protease inhibitors in such regimens conferred additional benefit. Patients with private insurance were more often prescribed protease inhibitors and had lower mortality rates than those insured by Medicare or Medicaid.
The recent declines in morbidity and mortality due to AIDS are attributable to the use of more intensive antiretroviral therapies.
Long-term zidovudine therapy in patients with human immunodeficiency virus (HIV) infection can cause a destructive mitochondrial myopathy with histological features of ragged-red fibres (RRF) and proliferation of abnormal mitochondria. In 9 zidovudine-treated patients with this myopathy we found severely reduced amounts (up to 78% reduction vs normal adult controls) of mitochondrial DNA (mtDNA) in muscle biopsy specimens by means of Southern blotting. In 2 HIV-positive patients who had not received zidovudine, muscle mtDNA content did not differ from that in the 4 controls. Depletion of mtDNA seems to be reversible, since 1 patient showed a substantial reduction in RRF and a concomitant pronounced increase in muscle mtDNA content after zidovudine therapy was discontinued. Depletion of muscle mtDNA is probably due to zidovudine-induced inhibition of mtDNA replication by DNA polymerase gamma and is not a secondary effect of HIV infection.
The anti-human immunodeficiency virus (-HIV) nucleoside analogs azidothymidine (AZT), dideoxycytidine (ddC), dideoxyinosine (ddl), dideoxydidehydrothymidine (D4T), and dideoxydidehydrocytidine (D4C) and the anticancer drug cytosine arabinoside (AraC) were compared for their effects on the mitochondrial DNA (mtDNA) content in a human lymphoblastoid cell line, CEM. The potency of these compounds in reducing mtDNA content was in the order of ddC greater than D4C greater than D4T greater than AZT greater than ddl. AraC did not have a significant effect on mtDNA content. All of the compounds tested, except AraC, stimulated lactic acid production at concentrations that inhibited mtDNA synthesis. The action of ddC and ddl occurred at concentrations that did not affect cell growth significantly in 4 days but retarded cell growth by day 6. D4T and D4C decreased mtDNA content by 50% at doses lower than those that inhibited cell growth by 50% in 4 days (ID50). However, AZT required a dose higher than the ID50 to exert similar effects on mtDNA content. The decrease of mtDNA content caused by ddC also occurred in nerve growth factor-treated PC12 cells, which differentiate to neuron-like cells upon treatment with nerve growth factor. The preferential inhibition of mtDNA, compared with cell growth, by some of these anti-HIV nucleoside analogs correlates well with their ability to cause drug-limiting delayed toxicity, such as peripheral neuropathy, in patients. These data suggest that the selective mitochondrial toxicity could be responsible for the delayed toxicity caused by these anti-HIV analogs.
With increasing awareness of the mitochondrial toxicity associated with certain 2',3'-dideoxynucleosides used in anti-human immunodeficiency virus therapy, procedures for quantitative analyses of drug effects on mitochondrial DNA (mtDNA) have assumed enhanced importance. For this reason we have developed a method to measure the copy numbers of mtDNA in cultured MOLT-4 cells. First a hybrid competitive DNA template was synthesized by conventional polymerase chain reaction (PCR), using two custom-synthesized 40-mer composite primers incorporating mitochondrial displacement loop sequences linked by a non-mitochondrial cDNA template (a 76-base pair sequence from the tat/rev region of human immunodeficiency virus cDNA). For the competitive assay, increasing known copy numbers of the hybrid competitive template were added as an internal control to samples containing total cellular DNA. With this approach, two competitive PCR products were generated, 1) a mitochondrial displacement loop-derived fragment (182 base pairs) and 2) a competitive DNA template-derived fragment (156 base pairs). Absolute quantitation was achieved by radiometric comparison of the relative amounts of the two products. To test the versatility of this method, varying amounts of competitive template (6.6 x 10(4) to 6.6 x 10(9) copies) were used with a fixed quantity of total cellular DNA taken from cells cultured for 9 days in the presence or absence of selected pyrimidine and purine dideoxynucleosides. The results showed that the copy number of cellular mtDNA is 823 +/- 71 copies/cell in MOLT-4 cells. Little selective depletion of mtDNA, compared with total cellular DNA, was seen with the purine dideoxynucleosides examined; however, when the cells were exposed to the pyrimidine dideoxynucleoside 2',3'-dideoxycytidine (50 nM) for 9 days, mtDNA content was specifically depleted, although total cellular DNA decreased by only 10%. Thus, in addition to the presently used methods of assessing mitochondrial impairment, i.e., Southern blot analysis and electron microscopy, the competitive PCR method provides a third and convenient assay, with particular applicability to determination of mtDNA in very small numbers of cells.
To assess the clinical and economic consequences of the use of protease inhibitors in the treatment of HIV infection.
Multicentric, observational, retrospective cohort study.
Ten AIDS reference centres in France.
All patients followed in each centre from September 1995 through October 1996.
AIDS-defining events, death, health-care resources use, administration of antiretroviral therapy.
Data from 7749 patients in 10 centres showed a drop in hospitalization days by 35%, new AIDS cases by 35%, and deaths by 46%. In the same period, the proportion of patients receiving antiretrovirals rose from 36 to 53% including highly active antiretroviral therapy (HAART), which rose from 0.3 to 18%. Overall cost evaluation showed a slight increase of monthly treatment cost of US$ 12 per patient. Comparison of the three centres that used HAART earliest to the three centres that used it latest showed a clear benefit to early HAART with a drop in hospitalization days by 41%, new AIDS cases by 41% and deaths by 69%. The proportion of patients with HAART rose to 27% and monthly health-care cost decreased by US$ 248852 (i.e., by US$ 101 per patient per month). Late prescribing centres experienced a less marked effect with a drop in hospitalization days by 22%, new AIDS cases by 31%, and deaths by 32.5%. Proportion of patients with HAART rose to 12% and monthly health-care costs increased by US$ 113578 (i.e., by US$ 38 per patient per month).
This study supports the extensive use of HAART in HIV-infected patients.
After zidovudine (ZDV), a 3′-azido analogue of thymi-dine, was found to be an effective antiretroviral drugagainst HIV [1,2], other nucleoside analogues inhibit-ing reverse transcriptase (RT) soon followed: didano-sine (ddI), zalcitabine (ddC), lamivudine (3TC),stavudine (D4T), and recently abacavir (1592U89)[3–7]. These drugs have demonstrated efficacy inreduction of morbidity and mortality, especially incombination therapy [8–10]. A special feature of someof these drugs is the protection against AIDS dementiacomplex, which appears to be related to good penetra-tion of the blood–brain barrier [11–13]. Although theintroduction of protease inhibitors has changed themanagement of HIV infection drastically, this cerebro-protective property will assert the role of these nucleo-side RT inhibitors (NRTI) as a cornerstone ofantiretroviral therapy [9,10].More than 10 years of experience with NRTI therapyhas revealed important adverse effects ranging frommild (myopathy) to fatal in some cases (pancreatitis,liver failure and lactic acidosis). Behind most of theseside-effects there appears to be a common mechanism:a decreased mitochondrial energy-generating capacity.In this review we will summarize the literature inwhich this mechanism is analysed and will emphasizethe importance of acquired mitochondrial dysfunctionthat will accumulate during long-term treatment withantiretroviral nucleoside analogues.
To examine the effect of recent developments in antiretroviral therapy on HIV disease progression and survival.
Retrospective cohort study.
Two cohorts of people with HIV were defined retrospectively from the records of a large immunology laboratory. The first cohort were subjects whose CD4+ T cell counts had dropped to 200 x 10(6)/L during 1990, and the second were subjects whose CD4+ T cell counts had dropped to 200 x 10(6)/L in 1994.
HIV disease progression and survival was determined over a minimum three years of follow-up for each cohort (i.e., 1990-1993; 1994-1997).
346 subjects were included in the analysis (193 subjects from 1990 and 153 from 1994). The relative risk of progression to AIDS in the 1994 cohort compared with the 1990 cohort was 0.57 (95% confidence interval, 0.35-0.91; P = 0.018) and the relative risk of death was 0.20 (95% confidence interval, 0.08-0.49; P < 0.001).
There were 43% fewer AIDS cases and 80% fewer deaths in the time following the increased availability of combination antiretroviral therapy in Australia.
Highly active antiretroviral therapy (HAART) can induce a characteristic lipodystrophy syndrome of peripheral fat wasting and central adiposity. HIV-1 protease inhibitors are generally believed to be the causal agents, although the syndrome has also been observed with protease-inhibitor-sparing regimens. Here, we postulate that the mitochondrial toxicity of the nucleoside-analogue reverse-transcriptase inhibitors plays an essential part in the development of this lipodystrophy, similar to the role of mitochondrial defects in the development of multiple symmetrical lipomatosis.
Lipodystrophy (LD; peripheral lipoatrophy, central adiposity) hyperlipidaemia and insulin resistance often complicate protease inhibitor-containing antiretroviral therapy. Lipoatrophy and abdominal distension were observed in protease inhibitor-naive nucleoside analogue reverse transcriptase inhibitor (NRTI) recipients with lactic acidaemia and hepatic impairment, which are known NRTI-induced mitochondrial toxicities.
Case-control study in a university-based outpatient clinic.
The patients studied included 14 NRTI recipients with lipoatrophy, 32 antiretroviral-naive patients without LD, 28 NRTI recipients without LD, 44 combined NRTI-protease inhibitor recipients without LD, and 102 NRTI-protease inhibitor recipients with LD. Data was obtained on body composition (questionnaire, physical examination, dual-energy x-ray absorptiometry and abdominal computerized tomography), with biochemical, lipid and glycaemic parameters.
The NRTI-LD syndrome was characterized by recent onset fatigue and nausea, peripheral lipoatrophy (6 kg loss over 4 months), abdominal distension (ascites +/- hepatomegaly) and elevated lactate (4.6, 1.1, 1.2, 1.4 and 1.7 mmol/l, respectively; P< 0.0001) and liver enzymes. Cases without hepatic involvement also had lower body fat and greater lactate than unaffected controls. Metabolic disturbances and weight improved after cessation. The NRTI-LD syndrome differed from protease inhibitor-related LD syndrome by the presence of recent onset symptoms and weight loss, higher lactate and alanine aminotransferase, and lower albumin, cholesterol, triglycerides, glucose and insulin. In treated controls, current stavudine therapy, protease inhibitor duration, and lactic acidaemia were independently associated with both lipoatrophy and abdominal obesity; total NRTI duration was also associated with lipoatrophy, and lamivudine and protease inhibitor duration with buffalo hump.
A syndrome of lipoatrophy, constitutional illness, lactic acidaemia and hepatic dysfunction can complicate NRTI therapy. Both protease inhibitor and NRTI therapies, particularly if associated with lactic acidaemia, contribute to LD syndrome, but have some distinguishable clinical and metabolic effects.
Decrease in mitochondrial DNA content in adipose tissue of HIV-1-infected patients
- U A Walker
- M Lü
- S I Volksbeck
- H Schö
- B Setzer
Walker U.A., Bickel M., Lü tke Volksbeck S.I., Schö fer H., Setzer B., Rickerts V. et al. Decrease in mitochondrial DNA content in adipose tissue of HIV-1-infected patients
Subcutaneous adipose tissue mitochondrial DNA analy-sis from individuals with HAART-associated lipodystro-phy Impact of combination therapy for HIV infection on inpatient census
- C Shikuma
- N Hu
- C Milne
- F Yost
- S Shimizu
- Shiramizu B Torres Ra
- Barr
Shikuma C., Hu N., Milne C., Yost F., Shimizu S., Shiramizu B. Subcutaneous adipose tissue mitochondrial DNA analy-sis from individuals with HAART-associated lipodystro-phy. Abstract 07, 2nd International Workshop on Adverse Drug Interactions and Lipodystrophy, Toronto, September 2000. Torres RA, Barr M. Impact of combination therapy for HIV infection on inpatient census. New Engl J Med 1997;336:1531 – 2.
Adverse effects of reverse transcriptase inhibitors: mitochondrial toxicity as common pathway
- K Brinkman
- Hjm Ter Hofstede
- D M Burger
- Jam Smeitink
- P P Koopmans
Brinkman K, ter Hofstede HJM, Burger DM, Smeitink JAM,
Koopmans PP. Adverse effects of reverse transcriptase
inhibitors: mitochondrial toxicity as common pathway.
AIDS 1998;12:1735 -44.
Quantitation of mitochondrial DNA in human lymphoblasts by a competitive polymerase chain reaction method: application to the study of inhibitors of mitochondrial DNA content
- H Zhang
- D A Cooney
- A Sreenath
- Q Zhan
- R Agbaria
- E E Stowe
Zhang H, Cooney DA, Sreenath A, Zhan Q, Agbaria R,
Stowe EE, et al. Quantitation of mitochondrial DNA in
human lymphoblasts by a competitive polymerase chain
reaction method: application to the study of inhibitors of
mitochondrial
DNA
content.
Mol
Pharmacol
1994;46:1063 -9.
Decrease in mitochondrial DNA content in adipose tissue of HIV-1-infected patients treated with NRTIs
- U A Walker
- M Bickel
- S I Lü Tke Volksbeck
- H Schö Fer
- B Setzer
- V Rickerts
Walker U.A., Bickel M., Lü tke Volksbeck S.I., Schö fer H.,
Setzer B., Rickerts V. et al. Decrease in mitochondrial
DNA content in adipose tissue of HIV-1-infected patients
treated with NRTIs. Abstract 06, 2nd International Workshop on Adverse Drug Interactions and Lipodystrophy,
Toronto, September 2000.
Subcutaneous adipose tissue mitochondrial DNA analysis from individuals with HAART-associated lipodystrophy
- C Shikuma
- N Hu
- C Milne
- F Yost
- S Shimizu
- B Shiramizu
Shikuma C., Hu N., Milne C., Yost F., Shimizu S., Shiramizu
B. Subcutaneous adipose tissue mitochondrial DNA analysis from individuals with HAART-associated lipodystrophy. Abstract 07, 2nd International Workshop on Adverse
Drug Interactions and Lipodystrophy, Toronto, September
2000.
Decrease in mitochondrial DNA content in adipose tissue of HIV-1-infected patients treated with NRTIs. Abstract 06
- U A Walker
- M Bickel
- S I Lü Tke Volksbeck
- H Schö
- B Setzer
- V Rickerts
Walker U.A., Bickel M., Lü tke Volksbeck S.I., Schö fer H.,
Setzer B., Rickerts V. et al. Decrease in mitochondrial
DNA content in adipose tissue of HIV-1-infected patients
treated with NRTIs. Abstract 06, 2nd International Workshop on Adverse Drug Interactions and Lipodystrophy,
Toronto, September 2000.
Decrease in mitochondrial DNA content in adipose tissue of HIV-1-infected patients treated with NRTIs. Abstract 06
- U A Walker
- M Bickel
- Lütke Volksbeck
- S I Schöfer
- H Setzer
- B Rickerts
Adverse effects of reverse transcriptase inhibitors: mitochondrial toxicity as common pathway
- Brinkman
Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection
- Palella
Quantitation of mitochondrial DNA in human lymphoblasts by a competitive polymerase chain reaction method: application to the study of inhibitors of mitochondrial DNA content
- Zhang