Amount Of mtDNA Research Articles

The Role of Mitochondria in Oocyte Maturation

With the nucleus as an exception, mitochondria are the only animal cell organelles containing their own genetic information, called mitochondrial DNA (mtDNA). During oocyte maturation, the mtDNA copy number dramatically increases and the distribution of mitochondria changes significantly. As oocyte maturation requires a large amount of ATP for continuous transcription and translation, the availability of the right number of functional mitochondria is crucial. There is a correlation between the quality of oocytes and both the amount of mtDNA and the amount of ATP. Suboptimal conditions of in vitro maturation (IVM) might lead to changes in the mitochondrial morphology as well as alternations in the expression of genes encoding proteins associated with mitochondrial function. Dysfunctional mitochondria have a lower ability to counteract reactive oxygen species (ROS) production which leads to oxidative stress. The mitochondrial function might be improved with the application of antioxidants and significant expectations are laid on the development of new IVM systems supplemented with mitochondria-targeted reagents. Different types of antioxidants have been tested already on animal models and human rescue IVM oocytes, showing promising results. This review focuses on the recent observations on oocytes’ intracellular mitochondrial distribution and on mitochondrial genomes during their maturation, both in vivo and in vitro. Recent mitochondrial supplementation studies, aiming to improve oocyte developmental potential, are summarized.

Effect of the Non-Immunosuppressive MPT Pore Inhibitor Alisporivir on the Functioning of Heart Mitochondria in Dystrophin-Deficient mdx Mice.

Supporting mitochondrial function is one of the therapeutic strategies that improve the functioning of skeletal muscle in Duchenne muscular dystrophy (DMD). In this work, we studied the effect of a non-immunosuppressive inhibitor of mitochondrial permeability transition pore (MPTP) alisporivir (5 mg/kg/day), reducing the intensity of the necrotic process and inflammation in skeletal muscles on the cardiac phenotype of dystrophin-deficient mdx mice. We found that the heart mitochondria of mdx mice show an increase in the intensity of oxidative phosphorylation and an increase in the resistance of organelles to the MPT pore opening. Alisporivir had no significant effect on the hyperfunctionalization of the heart mitochondria of mdx mice, and the state of the heart mitochondria of wild-type animals did not affect the dynamics of organelles but significantly suppressed mitochondrial biogenesis and reduced the amount of mtDNA in the heart muscle. Moreover, alisporivir suppressed mitochondrial biogenesis in the heart of wild-type mice. Alisporivir treatment resulted in a decrease in heart weight in mdx mice, which was associated with a significant modification of the transmission of excitation in the heart. The latter was also noted in the case of WT mice treated with alisporivir. The paper discusses the prospects for using alisporivir to correct the function of heart mitochondria in DMD.

QUANTIFICATION OF EMBRYONIC MITOCHONDRIAL DNA CONTENT ONLY REFLECTS BLASTOCYST DEVELOPMENTAL STAGE AND NOT CLINICAL OUTCOMES

Mitochondria biogenesis is critical for appropriate energy production during embryogenesis and implantation. Investigations into the predictive value of measuring mitochondrial DNA (mtDNA) content from trophectoderm (TE) biopsies has been contentious. The aim of this study was to further investigate the potential of TE biopsy mtDNA quantification as a tool for the determination of embryo quality and subsequent embryo selection.

P–212 Mitochondrial DNA content shows a significant association with timing of human embryo development and fertility diagnosis in euploid embryos

Abstract Study question Does mitochondrial DNA content (mtDNA) correlate with clinical parameters and embryo morphokinetics using advanced time-lapse technology? Summary answer mtDNA correlated with embryo morphokinetics and the growth trajectory of euploid embryos. Maternal age, anti-mullerian hormone level and fertility diagnosis were significantly associated with mtDNA. What is known already With the push towards single embryo transfers, laboratories are working to improve embryo selection. In addition to conventional microscopy, preimplantation genetic testing and time-lapse microscopy have been utilized to aid in embryo selection. More recently, as mtDNA may represent the energy potential of an embryo, some data have supported the use of mtDNA as an additional tool. Limited studies have suggested that a lower amount of mtDNA is associated with higher rates of implantation and improved embryo quality. Study design, size, duration This is a retrospective chart review. All embryos that underwent preimplantation genetic testing for aneuploidy (PGT-A) between January to December of 2020 were studied. Participants/materials, setting, methods Women undergoing in vitro fertilization (IVF) with intracytoplasmic sperm injection undergoing PGT-A were studied. All patients were from a single academic institution. This study exclusively examined the characteristics of euploid embryos. Mitochondrial DNA content was expressed as a ratio of mtDNA:nDNA (MitoScore). Time-lapse imaging was utilized to evaluate embryo development every 15 minutes in 5–7 focal planes. Chi square test and Spearman correlation analysis were performed with a p-value of < 0.05 considered significant. Main results and the role of chance A total of 494 embryos from 52 women who underwent 58 IVF cycles were cultured to blastocyst and 331 embryos were biopsied for PGT-A evaluation. Of these, 132 embryos were diagnosed as euploid. A moderate positive correlation was found between MitoScore and time to morula, time to blast and time to expanded blast (correlation value 0.54, 0.50 and 0.54, respectively; p < 0.001). Consistent with this trend, day 5 blastocysts had a significantly lower MitoScore values than day 6 blastocysts (20.2 v. 29.2; p < 0.001). When examining all biopsied euploid embryos, no significant association was found between MitoScore, blastocyst maturity, trophectoderm or inner cell mass scores. Our data also demonstrated a positive correlation between MitoScore and maternal age (correlation factor 0.33; p < 0.001). A negative association between MitoScore and serum anti-mullerian hormone levels (correlation factor –0.20; p < 0.021) was also noted. Of particular interest was the significant association between fertility diagnosis and mitochondrial score (p < 0.001). Even amongst euploid embryos, mtDNA content varied widely, potentially reflecting differences in embryo potential and quality. Additionally, the significant difference in MitoScore between that day 5 and day 6 blastocysts may reflect a fundamental difference in cytoplasmic characteristics and requires further study. Limitations, reasons for caution Due to the study cohort of euploid embryos undergoing PGT-A, this study was biased for the selection of high grade embryos. This limited diversity in embryo quality may have masked other potential associations between mitochondrial content and blastocyst quality. Wider implications of the findings: mtDNA may be additional tool aiding in embryo selection as IVF labs work to improve pregnancy rates while minimizing the risks of transferring multiple embryos. To our knowledge, this is the largest study assessing the relationship of mtDNA content of blastocysts and the timing of embryo development using time-lapse imaging. Trial registration number None

Increased Mitochondrial DNA in an ECMO Model Is Associated with Loss of Platelet Function

BackgroundExtracorporeal Membrane Oxygenation (ECMO) represents a promising technology for resuscitating trauma patients with severe lung and/or cardiac injury. Common ECMO-related complications of thrombosis and bleeding result in significant rates of mortality and morbidity. Multiple studies with ex-vivo ECMO devices using blood demonstrate an increased proinflammatory response. Mitochondrial DNA (mtDNA) is a known damage associated molecular pattern (DAMP) that has been shown to have proinflammatory effects. Recent studies have correlated poor clinical outcomes to increased levels of plasma mtDNA. The objective of this study was to determine the levels of mtDNA in an ECMO model as a function of shear stress and duration of pump flow and evaluate the possible impact of mtDNA on platelet function.MethodsBlood was collected from healthy donors under a US Army Institute of Surgical Research approved standard operating procedure. We constructed an ex-vivo ECMO circuit that circulates heparinized human blood for six hours at differing flow rates (static control-0 L/min, 0.3, and 0.7 L/min). Quantification of mtDNA and assays of platelet function were performed on samples obtained from each group and were compared to static controls at 0, 60, 120, 240, and 360 min. mtDNA levels were assayed by measuring the copy number of the NADH dehydrogenase 1 gene using quantitative real-time PCR. Additional tests were performed on platelet count, aggregation, viability (as measured by calcein/CD41a), TEG, and Multiplate.ResultsIncreased flow rates and duration resulted in significantly higher levels of plasma mtDNA compared to static controls (static control, 0 L/min flow rate: 120 min = 3 x 105 ± 1.8 x 105, 240 min = 4 x 105 ± 2 x 105 copies/µL; 0.3 L/min flow rate: 0 min = 4 x 105 ± 0.8 x 105, 120 min = 8 x 105± 0.2 x 105, 240 min = 8 x 105 ± 1.6 x 105 copies/µL; 0.7 L/min flow rate: 0 min = 3 x 105 ± 0.9x 105, 120 min = 19 x 105± 4 x 105, 240 min = 35 x 105 ± 6 x 105 copies/µL; p < 0.001). Platelet count and viability were not significantly different. In contrast, both TEG platelet mapping and Multiplate demonstrated significant losses in ADP and collagen aggregation response (Multiplate at 120 min: static control 1564± 265, 0.3 L/min = 830 ± 474, 0.7 L/min = 1010 ± 261; 240 min: static control 874± 381, 0.3 L/min = 527 ± 384, 0.7 L/min = 405 ± 268; p < 0.01) when compared to static controls.ConclusionsIncreased mtDNA in surgical patients has been linked to poor outcomes. It has recently been shown that circulating mtDNA can trigger the innate immune system through toll-like receptor 9 (TLR9) and NF-κB activation, causing deleterious effects. In this study we found that mtDNA levels significantly increase as a function of shear stress and increased duration of pump flow. These increases were shown to be associated with loss in platelet function in both TEG and Multiplate measures of ADP and collagen agonist stimulated responses. Future ECMO studies will explore the correlation between circulating mtDNA levels and TLR9. mtDNA may be a clinically relevant biomarker and therapeutic target for efforts to mitigate life-threatening ECMO coagulation complications. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Oxygen tension modulates the mitochondrial genetic bottleneck and influences the segregation of a heteroplasmic mtDNA variant in vitro

Most humans carry a mixed population of mitochondrial DNA (mtDNA heteroplasmy) affecting ~1–2% of molecules, but rapid percentage shifts occur over one generation leading to severe mitochondrial diseases. A decrease in the amount of mtDNA within the developing female germ line appears to play a role, but other sub-cellular mechanisms have been implicated. Establishing an in vitro model of early mammalian germ cell development from embryonic stem cells, here we show that the reduction of mtDNA content is modulated by oxygen and reaches a nadir immediately before germ cell specification. The observed genetic bottleneck was accompanied by a decrease in mtDNA replicating foci and the segregation of heteroplasmy, which were both abolished at higher oxygen levels. Thus, differences in oxygen tension occurring during early development likely modulate the amount of mtDNA, facilitating mtDNA segregation and contributing to tissue-specific mutation loads.

Mitochondrial DNA Depletion in Granulosa Cell Derived Nuclear Transfer Tissues.

Somatic cell nuclear transfer (SCNT) is a key technology with broad applications that range from production of cloned farm animals to derivation of patient-matched stem cells or production of humanized animal organs for xenotransplantation. However, effects of aberrant epigenetic reprogramming on gene expression compromise cell and organ phenotype, resulting in low success rate of SCNT. Standard SCNT procedures include enucleation of recipient oocytes before the nuclear donor cell is introduced. Enucleation removes not only the spindle apparatus and chromosomes of the oocyte but also the perinuclear, mitochondria rich, ooplasm. Here, we use a Bos taurus SCNT model with in vitro fertilized (IVF) and in vivo conceived controls to demonstrate a ∼50% reduction in mitochondrial DNA (mtDNA) in the liver and skeletal muscle, but not the brain, of SCNT fetuses at day 80 of gestation. In the muscle, we also observed significantly reduced transcript abundances of mtDNA-encoded subunits of the respiratory chain. Importantly, mtDNA content and mtDNA transcript abundances correlate with hepatomegaly and muscle hypertrophy of SCNT fetuses. Expression of selected nuclear-encoded genes pivotal for mtDNA replication was similar to controls, arguing against an indirect epigenetic nuclear reprogramming effect on mtDNA amount. We conclude that mtDNA depletion is a major signature of perturbations after SCNT. We further propose that mitochondrial perturbation in interaction with incomplete nuclear reprogramming drives abnormal epigenetic features and correlated phenotypes, a concept supported by previously reported effects of mtDNA depletion on the epigenome and the pleiotropic phenotypic effects of mtDNA depletion in humans. This provides a novel perspective on the reprogramming process and opens new avenues to improve SCNT protocols for healthy embryo and tissue development.

Increased Sirtuin expression, senescence regulating miRNAs, mtDNA, and Bifidobateria correlate with wellbeing and skin appearance after Sirtuin- activating drink

Background: Sirtuins attract high attention considering their properties to reverse molecular hallmarks of aging and age-related disorders. Many secondary plant ingredients (SPI) are known for their sirtuin-activating activities as well as epigenetic regulation of telomers, autophagy, senolysis, DNA repair but also improvement of gut microbiota. Furthermore, prebiotics enhanced butyrate was shown to interact with SIRT pathways. This study investigated effects of a drink containing a mix of different SPIs in combination with galactooligosaccharides (GOS) and their effect on SIRT activation, markers of aging relevant mechanisms and gut microbiota composition in correlation with subjective wellbeing and skin structure appearance. Methods: We analyzed gene expression, mtDNA amount, and microbial composition in response to a sirtuin-activating drink in humans compared to a control group consuming a placebo. Food frequency, beauty, and general health questionnaires were asked, and a set of mRNAs and miRNAs were assessed using qPCR analysis. The gut composition was analyzed using Illumnia sequencing.Results: SPI increased SIRT1, SIRT3 and modulated cell cycle relevant miR16 and senescence regulating miR34 expression. Additionally, mtDNA amount was higher in the group consuming the active supplement indicating an improved mitochondrial activity. The combined effect of SPI and GOS lead to an increase of Actinobacteria, especially Bifidobacterium, but also Veillonellaceae which was not observed in the control group. Significant correlations between SIRT3 expression and the gut microbiota Bifidobacterium and Veillonellaceae were observed. Additionally, statistical analysis of subjects self-reporting indicated beneficial effects regarding beauty and wellbeing.Conclusion: Our results show that the combination of sirtuins inducing SPI and prebiotic GOS influences molecular pathways counteracting aging, senescence, inflammation, and enhanced groups of gut microbiota which are known to improve the innate and adaptive immune system. Keywords: secondary plant ingredients, prebiotic, Sirtuins, subjective wellbeing, Bifidobacterium

Effect of maternal aging and vitrification on mitochondrial DNA copy number in embryos and spent culture medium

Maternal aging and vitrification affect mitochondrial quality and quantity in embryos. The present study investigated the effects of maternal aging on mitochondrial DNA (mtDNA) copy number in embryos, and the amount of cell-free mtDNA (cf-mtDNA) in spent culture medium (SCM) of embryos. Moreover, we examined the effects of vitrification on mtDNA copy number in embryos of young and aged cows, and on cf-mtDNA abundance in SCM. Oocytes collected from ovaries of young (20–40 months old) and aged cows (> 140 months old) were used to produce early stage embryos (8–12 cell-stage, 48 h after insemination). These embryos were individually cultured for 5 days, and mtDNA copy number in blastocysts and cf-mtDNA content in SCM, were evaluated by real-time PCR. At 48 h post-insemination, mtDNA copy number in embryos was greater for young cows compared with that of aged cows, whereas no significant difference was observed in cf-mtDNA in the SCM. Next, we addressed whether zona pellucida (ZP) may mask the difference in cf-mtDNA content in SCM. Using ZP-free embryos, we found significantly greater cf-mtDNA content in the SCM of blastocysts derived from aged cows. Furthermore, when embryos were vitrified and warmed, mtDNA copy number in blastocysts derived from young cows was lower, whereas cf-mtDNA content in SCM was greater than in those derived from aged cows. In conclusion, maternal aging affects mitochondrial kinetics and copy number in embryos following vitrification.

Mitochondria-DNA copy-number and incident venous thromboembolism among middle-aged women: a population-based cohort study

Venous thromboembolism (VTE) is the third most common cardiovascular disease. Low amount of mitochondrial DNA copy number (mtDNA-CN) has been associated with arterial cardiovascular disease (CVD) and reflects mitochondrial dysfunctions. However, whether mtDNA-CN is associated with VTE has not been determined. To examine the association between mtDNA-CN and incident VTE among middle-aged women. 6917 women aged 50–64 years, followed for 20 years in the Women’s Health In the Lund Area (WHILA) study. DNA samples for mtDNA quantification were available from 2521 women. Quantification of mtDNA-CN was performed using a well-optimized droplet digital PCR method. After exclusions of women with anticoagulant treatment, women living in nursing homes, and women who were diagnosed with cancer, stroke, VTE, or coronary heart disease at baseline, a cohort of 2117 women remained for analysis. Cox regression was used to analyze the relationship between mtDNA-CN and time to VTE (hazard ratio = HR). In total, 87 women were diagnosed with VTE during follow-up, corresponding to an incidence rate of 2.8 per 1000 person-years. Neither crude nor adjusted HR for mtDNA-CN were significantly associated with incident VTE. A sensitivity analysis with inclusion of excluded women did not change the results. MtDNA-CN was not significantly associated with VTE. The present study suggests that mtDNA-CN, reflecting mitochondrial dysfunction, should not be considered a biomarker that plays a major role for developing VTE. However, due to limited study size we may not exclude minor associations.

Skeletal muscle mitochondrial DNA copy number and mitochondrial DNA deletion mutation frequency as predictors of physical performance in older men and women.

Mitochondrial DNA (mtDNA) quality and quantity relate to two hallmarks of aging-genomic instability and mitochondrial dysfunction. Physical performance relies on mitochondrial integrity and declines with age, yet the interactions between mtDNA quantity, quality, and physical performance are unclear. Using a validated digital PCR assay specific for mtDNA deletions, we tested the hypothesis that skeletal muscle mtDNA deletion mutation frequency (i.e., a measure of mtDNA quality) or mtDNA copy number predicts physical performance in older adults. Total DNA was isolated from vastus lateralis muscle biopsies and used to quantitate mtDNA copy number and mtDNA deletion frequency by digital PCR. The biopsies were obtained from a cross-sectional cohort of 53 adults aged 50 to 86 years. Before the biopsy procedure, physical performance measurements were collected, including VO2max, modified physical performance test score, 6-min walk distance, gait speed, grip strength, and total lean and leg mass. Linear regression models were used to evaluate the relationships between age, sex, and the outcomes. We found that mtDNA deletion mutation frequency increased exponentially with advancing age. On average from ages 50 to 86, deletion frequency increased from 0.008 to 0.15%, an 18-fold increase. Females may have lower deletion frequencies than males at older ages. We also measured declines in VO2max and mtDNA copy number with age in both sexes. The mtDNA deletion frequency measured from single skeletal muscle biopsies predicted 13.3% of the variation in VO2max. Copy number explained 22.6% of the variation in mtDNA deletion frequency and 10.4% of the lean mass variation. We found predictive relationships between age, mtDNA deletion mutation frequency, mtDNA copy number, and physical performance. These data are consistent with a role for mitochondrial function and genome integrity in maintaining physical performance with age. Analyses of mtDNA quality and quantity in larger cohorts and longitudinal studies could extend our understanding of the importance of mitochondrial DNA in human aging and longevity.

Mitochondria: their role in spermatozoa and in male infertility.

The best-known role of spermatozoa is to fertilize the oocyte and to transmit the paternal genome to offspring. These highly specialized cells have a unique structure consisting of all the elements absolutely necessary to each stage of fertilization and to embryonic development. Mature spermatozoa are made up of a head with the nucleus, a neck, and a flagellum that allows motility and that contains a midpiece with a mitochondrial helix. Mitochondria are central to cellular energy production but they also have various other functions. Although mitochondria are recognized as essential to spermatozoa, their exact pathophysiological role and their functioning are complex. Available literature relative to mitochondria in spermatozoa is dense and contradictory in some cases. Furthermore, mitochondria are only indirectly involved in cytoplasmic heredity as their DNA, the paternal mitochondrial DNA, is not transmitted to descendants. This review aims to summarize available literature on mitochondria in spermatozoa, and, in particular, that with respect to humans, with the perspective of better understanding the anomalies that could be implicated in male infertility. PubMed was used to search the MEDLINE database for peer-reviewed original articles and reviews pertaining to human spermatozoa and mitochondria. Searches were performed using keywords belonging to three groups: 'mitochondria' or 'mitochondrial DNA', 'spermatozoa' or 'sperm' and 'reactive oxygen species' or 'calcium' or 'apoptosis' or signaling pathways'. These keywords were combined with other relevant search phrases. References from these articles were used to obtain additional articles. Mitochondria are central to the metabolism of spermatozoa and they are implicated in energy production, redox equilibrium and calcium regulation, as well as apoptotic pathways, all of which are necessary for flagellar motility, capacitation, acrosome reaction and gametic fusion. In numerous cases, alterations in one of the aforementioned functions could be linked to a decline in sperm quality and/or infertility. The link between the mitochondrial genome and the quality of spermatozoa appears to be more complex. Although the quantity of mtDNA, and the existence of large-scale deletions therein, are inversely correlated to sperm quality, the effects of mutations seem to be heterogeneous and particularly related to their pathogenicity. The importance of the role of mitochondria in reproduction, and particularly in gamete quality, has recently emerged following numerous publications. Better understanding of male infertility is of great interest in the current context where a significant decline in sperm quality has been observed.

Suboptimal trophectoderm mitochondrial DNA level is associated with delayed blastocyst development.

To provide a comprehensive analysis of mtDNA quantity in D5 and D6 blastocysts, as well as a further insight to the origin of delayed blastocyst development. A retrospective cohort analysis of 829 D5 and 472 D6 blastocysts from 460 patients who underwent in vitro fertilization (IVF) with next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A). The quantity of trophectoderm mtDNA was extrapolated from the NGS data, followed by the analysis of mean mtDNA levels between D5 and D6 blastocysts of the same ploidy (aneuploid/euploid) and transfer outcomes (positive/negative clinical pregnancy). D5 blastocysts had significantly higher euploidy rate and clinical pregnancy rate when compared with D6 blastocysts. The proportion of blastocysts derived from patients ≧40years old were similar between the D5 and D6 cohorts. When blastocysts with identical ploidy were analyzed, the D5 cohorts all had significantly higher mean mtDNA levels than their D6 counterparts. Similarly, when embryo transfers with identical outcome were analyzed, the D5 cohorts also had significantly higher mean mtDNA levels than the D6 cohorts. Trophectoderm mtDNA level was independent of maternal age and blastocyst morphology grades. Our data provided further evidence D5 blastocysts contained significantly greater mtDNA quantity than D6 blastocysts, and mtDNA quantity could be a key factor that affects the development rate of blastocysts. Furthermore, one must avoid using an arbitrary threshold when incorporating mtDNA quantity into the embryo selection criteria, as the observed value may have vastly different clinical implication when blastulation rate is also considered.

Mitochondrial DNA in Fresh versus Frozen Embryo Culture Media of Polycystic Ovarian Syndrome Patients Undergoing Invitro Fertilization: A Possible Predictive Marker of a Successful Pregnancy

PurposeFrozen embryos transfer (ET) may improve the live-birth and reduce rates of ovarian hyperstimulation in polycystic ovary syndrome (PCOS) patients. Morphological criteria are the classical way for embryo selection, yet recently, many biochemical and genetic markers have been developed. This study aimed to compare fresh and frozen ET using the mtDNA/gDNA ratio of embryo secretome and the possibility of using this ratio as a predictive marker of PCOS pregnancy rate.Subjects and MethodsOne hundred PCOS patients undergoing IVF were chosen according to Rotterdam criteria and divided into two groups. Group I (50 with fresh ET), group II (50 with frozen ET), and otherwise 33 apparently healthy women as a control group with fresh ET. We then carried out absolute quantification of embryo culture media mtDNA and gDNA by real-time PCR.ResultsmtDNA/gDNA ratio was significantly low in PCOS embryo culture media in comparison with control. Additionally, while the mtDNA/gDNA ratio was significantly high in pregnant PCOS embryo culture media, it was high, though not statistically significant, in the fresh ET than frozen ET group. mtDNA/gDNA ratio sensitivity and specificity in PCOS embryo culture media as a predictive value of pregnancy rate were (86% and 96%, respectively).ConclusionmtDNA/gDNA ratio measurement in PCOS embryo culture media is a novel marker that can be clinically applied as a predictive value of the quality of the morphologically good embryo.

Generation of two human iPSC lines, FINCBi002-A and FINCBi003-A, carrying heteroplasmic macrodeletion of mitochondrial DNA causing Pearson's syndrome.

Pearson marrow pancreas syndrome (PMPS) is a sporadic mitochondrial disease, resulting from the clonal expansion of a mutated mitochondrial DNA (mtDNA) molecule bearing a macro-deletion, and therefore missing essential genetic information. PMPS is characterized by the presence of deleted (Δ) mtDNA that co-exist with the presence of a variable amount of wild-type mtDNA, a condition termed heteroplasmy. All tissues of the affected individual, including the haemopoietic system and the post-mitotic, highly specialized tissues (brain, skeletal muscle, and heart) contain the large-scale mtDNA deletion in variable amount. We generated human induced pluripotent stem cells (hiPSCs) from two PMPS patients, carrying different type of large-scale deletion.

Accurate Measurement of Cellular and Cell-Free Circulating Mitochondrial DNA Content from Human Blood Samples Using Real-Time Quantitative PCR.

Changes in circulating mitochondrial DNA (mtDNA) are widely used to indicate mitochondrial dysfunction in common non-genetic diseases where mitochondrial dysfunction may play a role. However, the methodology being used is not always specific and reproducible, and most studies use whole blood rather than evaluating cellular and cell-free mtDNA separately. Cellular mtDNA is contained within the mitochondrion and encodes vital subunits of the OXPHOS machinery. Conversely, cell-free mtDNA can have harmful effects, triggering inflammatory responses and potentially contributing to pathogenic processes. In this chapter, we describe a protocol to accurately measure the amount of cellular and cell-free human mtDNA in peripheral blood. Absolute quantification is carried out using real-time quantitative PCR (qPCR) to quantify cellular mtDNA, measured as the mitochondrial genome to nuclear genome ratio (designated the Mt/N ratio) in whole blood and peripheral blood mononuclear cells (PBMCs) and the number of mtDNA copies per μL in plasma and serum. We describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions, (2) prepare DNA from each of these fractions, (3) prepare dilution standards for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.

Mitochondrial DNA as a Sensitive Biomarker of UV-Induced Cellular Damage in Human Skin.

Mitochondrial DNA (mtDNA) has been demonstrated to be a reliable biomarker of UV-induced genetic damage in both animal and human skin. Properties of the mitochondrial genome which allow for its use as a biomarker of damage include its presence in multiple copies within a cell, its limited repair mechanisms, and its lack of protective histones. To measure UV-induced mtDNA damage (particularly in the form of strand breaks), real-time quantitative PCR (qPCR) is used, based on the observation that PCR amplification efficiency is decreased in the presence of high levels of damage. Here, we describe the measurement of UV-induced mtDNA damage which includes the extraction of cellular DNA, qPCR to determine the relative amount of mtDNA, qPCR to determine UV-induced damage within a long strand of mtDNA, and the verification of the amplification process using gel electrophoresis.

Muscle Mitochondrial DNA Copy Number, Deletion Mutation Frequency, and Physical Performance in Older Adults

Mitochondrial DNA (mtDNA) quantity and quality influence hallmarks of aging – mitochondrial dysfunction and genomic instability. The interactions between mtDNA quantity and quality and physical performance have not been extensively examined in humans. The aim of this study was to test the interactions between skeletal muscle mtDNA copy number, mtDNA deletion mutation frequency, and physical performance measures in older adults. Total DNA was isolated from muscle biopsies and used for quantitation of mtDNA copy number and mutation frequency by digital PCR. The biopsies were obtained from a cross-sectional cohort of 53 adults aged 50 to 86 years. Before the biopsy, physical performance measures were collected. MtDNA deletions increased exponentially with advancing age. On average, mtDNA deletion frequency increased 18-fold between 50 and 80, with a trend toward lower deletion frequency in females. MtDNA deletion frequency predicted declines in VO2 max, where 4.7% of the variation in VO2 max was explained by mtDNA deletion frequency. MtDNA copy number was negatively correlated with age and mtDNA deletion frequency, but positively correlated with lean mass. There was a trend to lower mtDNA deletion frequency in females, consistent with increased longevity in females. Larger studies may better delineate sex effects. These data are consistent with a role for mitochondrial function and genome integrity in the maintenance of physical performance with age. Analyses of mtDNA quality and quantity in longitudinal studies could extend our understanding of the importance of mitochondria in human aging and longevity.

Sample adequacy controls for infectious disease diagnosis by oral swabbing.

Oral swabs are emerging as a non-invasive sample type for diagnosing infectious diseases including Ebola, tuberculosis (TB), and COVID-19. To assure proper sample collection, sample adequacy controls (SACs) are needed that detect substances indicative of samples collected within the oral cavity. This study evaluated two candidate SACs for this purpose. One detected representative oral microbiota (Streptococcus species DNA) and the other, human cells (human mitochondrial DNA, mtDNA). Quantitative PCR (qPCR) assays for the two target cell types were applied to buccal swabs (representing samples collected within the oral cavity) and hand swabs (representing improperly collected samples) obtained from 51 healthy U.S. volunteers. Quantification cycle (Cq) cutoffs that maximized Youden’s index were established for each assay. The streptococcal target at a Cq cutoff of ≤34.9 had 99.0% sensitivity and specificity for oral swab samples, whereas human mtDNA perfectly distinguished between hand and mouth swabs with a Cq cutoff of 31.3. The human mtDNA test was then applied to buccal, tongue, and gum swabs that had previously been collected from TB patients and controls in South Africa, along with “air swabs” collected as negative controls (total N = 292 swabs from 71 subjects). Of these swabs, 287/292 (98%) exhibited the expected Cq values. In a paired analysis the three oral sites yielded indistinguishable amounts of human mtDNA, however PurFlockTM swabs collected slightly more human mtDNA than did OmniSwabsTM (p = 0.012). The results indicate that quantification of human mtDNA cannot distinguish swabs collected from different sites within the mouth. However, it can reliably distinguish oral swabs from swabs that were not used orally, which makes it a useful SAC for oral swab-based diagnosis.

Doxorubicin-Induced Translocation of mtDNA into the Nuclear Genome of Human Lymphocytes Detected Using a Molecular-Cytogenetic Approach.

Translocation of mtDNA in the nuclear genome is an ongoing process that contributes to the development of pathological conditions in humans. However, the causal factors of this biological phenomenon in human cells are poorly studied. Here we analyzed mtDNA insertions in the nuclear genome of human lymphocytes after in vitro treatment with doxorubicin (DOX) using a fluorescence in situ hybridization (FISH) technique. The number of mtDNA insertions positively correlated with the number of DOX-induced micronuclei, suggesting that DOX-induced chromosome breaks contribute to insertion events. Analysis of the odds ratios (OR) revealed that DOX at concentrations of 0.025 and 0.035 µg/mL significantly increases the rate of mtDNA insertions (OR: 3.53 (95% CI: 1.42–8.76, p < 0.05) and 3.02 (95% CI: 1.19–7.62, p < 0.05), respectively). Analysis of the distribution of mtDNA insertions in the genome revealed that DOX-induced mtDNA insertions are more frequent in larger chromosomes, which are more prone to the damaging action of DOX. Overall, our data suggest that DOX-induced chromosome damage can be a causal factor for insertions of mtDNA in the nuclear genome of human lymphocytes. It can be assumed that the impact of a large number of external and internal mutagenic factors contributes significantly to the origin and amount of mtDNA in nuclear genomes.