Measurement Of mtDNA Copy Number Research Articles

Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy using Digital Droplet Polymerase Chain Reaction.

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.

Measurement of mitochondrial DNA copy number in dried blood spots: A pilot study

BackgroundWe evaluated the feasibility of mitochondrial DNA (mtDNA) copy number measurement in dried blood spots (DBS), its comparability with measurement in whole blood samples, and stability of mtDNA copy number from DBS over time. MethodsWomen in this pilot study were participants in the Sister Study, a large prospective cohort. Sister Study participants provided a whole blood sample and DBS at enrollment. A second DBS sample was collected 5–10 years later from a subcohort of women with and without an incident breast cancer diagnosis between collections. Among 54 women (27 with breast cancer, 27 without) we measured mtDNA copy number from whole blood at enrollment and from DBS at both time points. ResultsThe average age at enrollment was 58.7 years (range:50–69). Values of mtDNA copy number measured in whole blood samples and DBS from enrollment were moderately correlated (Spearman R = 0.45; p = 0.005). Stability of mtDNA copy number in DBS from the two time points was moderate overall (ICC = 0.50) and similar between women with (ICC = 0.50) and without (ICC = 0.51) a breast cancer diagnosis between the two collections. ConclusionsOur results suggest that measurement of mtDNA copy number in DBS is feasible and may be a valid alternative to measurement in whole blood samples.

Blastocysts with disproportionally high mtDNA copy number can result in healthy babies

Introduction Mitochondrial DNA (mtDNA) copy number has been proposed as a biomarker of implantation in IVF embryos. Some studies suggest that blastocysts containing disproportionately elevated levels of mtDNA per cell always fail to implant, and blastocysts implanting successfully have lower mtDNA levels on average ( Fragouli et al., 2015 , Diez-Juan et al., 2015 ). Other studies do not report such a trend, including one from our group ( Treff et al., 2017 , Victor et al., 2017 ). An explanation for the conflicting reports couldbe that the method for quantitation of mtDNA copy number varies across studies. As a result, technicalguidelines have been proposed to increase uniformity of mtDNA quantitation ( Wells, 2017 ). Here, weadhere to those guidelines in analyzing mtDNA copy number in a large number of blastocysts used fortransfer, to determine whether it is a valid predictor of implantation. Materials & Methods We quantified mtDNA levels in surplus product of the PGT-A process from blastocysts used in IVF withknown outcomes. In order to satisfy the suggested guidelines for mtDNA quantitation, we used qPCRmeasuring a locus in the mtDNA sequence and a multicopy locus in the nuclear DNA sequence, andcomputed the ratio between the two values. All molecular material was fresh and devoid of degradation. Results In our hands, unlike in previous reports, blastocysts with extremely high mtDNA levels successfully implanted and led to healthy births. If using mtDNA copy number to deselect embryos, these samples would not have been chosen for transfer. In addition, analysis of >100 blastocysts showed a statistically insignificant difference between mtDNA levels in implanted versus non-implanted blastocysts. Conclusions These findings suggest that in a single-clinic setting, measurement of mtDNA copy number might not provide any advantage to embryo ranking, and might even lead to de-selection of blastocysts that result to healthy pregnancies. The validity of measurement of mtDNA copy number requires further investigation, given different findings in individual centers.

Interrelation of chemerin and TNF-\u03b1 with mtDNA copy number in adipose tissues and blood cells in obese patients with and without type 2 diabetes

BackgroundInflammatory response plays a key role in the development of insulin resistance (IR) in obesity. Oxidative stress triggers the replication of the mitochondrial genome and division of the organelle. The purpose of this study was to identify the relationship of chemerin and TNF-α with mitochondrial DNA (mtDNA) copy number in subcutaneous adipose tissue (SAT) and visceral adipose tissue (mesentery of the small intestine (Mes), greater omentum (GO) and blood mononuclear cells (MNCs)) in patients with obesity with/without type 2 diabetes mellitus (T2DM).MethodsThe study included 142 obese patients and 34 healthy donors. The samples used for the study were peripheral venous blood (MNCs) and ATs (SAT, Mes and GO). The measurement of mtDNA copy number was done by droplet digital PCR. Quantitative determination of insulin, adiponectin, TNF-α and chemerin in serum/plasma was performed by flow-through fluorometry and commercial ELISA kit. Statistical analysis and graphs were obtained in R Statistical Software (version 3.3.1).ResultsThe increase in body mass index (BMI) was accompanied by an increase in TNF-α production, an increase in mtDNA copy number in SAT and a decrease in mtDNA copy number in MNCs. The negative association between plasma chemerin and mtDNA copy number in the Mes, as well as between mtDNA copy number and chemerin expression in the Mes, in the group with BMI > 40 kg/m2 without T2DM demonstrates the protective effect of chemerin against the development of IR via the regulation of mtDNA copy number in adipose tissues.ConclusionsWe thus speculated the existence of a compensatory mechanism in which leads to the increased number of mtDNA copies under the influence of proinflammatory factors. Based on the obtained data, we propose that reducing mtDNA copy number in cases of morbid obesity without T2DM has a positive effect on carbohydrate metabolism, which may help maintain glucose levels within reference values.Obesity, type 2 diabetes, mtDNA, cytokines, TNF-a, chemerin.

Mitochondrial DNA depletion in sporadic inclusion body myositis

Sporadic inclusion body myositis (sIBM) is a late onset disorder of unkown aetiology. Mitochondrial changes such as cytochrome oxidase deficient fibres are a well recognised feature and mitochondrial DNA (mtDNA) deletions have also been reported, but not consistently. Since mtDNA deletions are not present in all cases, we investigated whether other types of mtDNA abnormality were responsible for the mitochondrial changes. We studied 9 patients with sIBM. To control for fibre loss or replacement with inflammatory cells, we compared sIBM patients with necrotising myopathy (n = 4) as well as with healthy controls. Qualitative anlysis for mtDNA deletions and quantitative measurement of mtDNA copy number showed that muscle from patients with sIBM contained on average 67% less mtDNA than healthy controls (P = 0.001). The level of mtDNA was also significantly depleted in sIBM when compared to necrotising myopathy. No significant difference in copy number was seen in patients with necrotising myopathy compared to controls. Deletions of mtDNA were present in 4 patients with sIBM, but not all. Our findings suggest that mtDNA depletion is a more consistent finding in sIBM, and one that may be implicated in the pathogenesis of the disease.

Mitochondrial DNA copy number, but not haplogroup is associated with keratoconus in Han Chinese population

Oxidative stress may play a role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is closely related to mitochondrion function, and variations may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. To test whether mtDNA background and copy number confer genetic susceptibility to KC in the Han Chinese population, we performed this association study. We analyzed mtDNA sequence variations in 210 KC patients and 309 matched individuals from China, and classified each subject by haplogroup. Mitochondrial DNA copy number was measured in a subset of these subjects (193 patients and 103 controls). Comparison of matrilineal components of the cases and control populations revealed no significant difference. However, measurement of mtDNA copy number showed that KC patients had significantly lower mtDNA copy numbers than controls (P = 0.0002), even when age, gender, and mtDNA background were considered. Our results suggest that mtDNA copy number, but not haplogroup, is associated with keratoconus, and may contribute to its pathogenesis.

ASSA13-06-1 New Findings from Myocardial Mitochondria DNA Sequencing of Patients with Left Ventricular Non-Compaction Cardiomyopathy

Background Left ventricular non-compaction cardiomyopathy (LVNC) is a rare congenital cardiomyopathy that is widely believed to be arrest of cardiac development during embryogenesis. Mitochondria, as critical regulators, play an important role in cardiac myocyte differentiation and cardiac development. MtDNA mutations or deletions have been associated with cardiomyopathies. However, no study of mtDNA from cardiac tissue of LVNC patients has been reported. Methods and Results To identify novel candidate mtDNA variants that may be responsible for the pathogenesis of LVNC, myocardial specimens were examined to research for pathogenic mtDNA variants. Samples from six patients who were diagnosed with LVNC and underwent heart transplantation were analysed. The sequence and copy number of mtDNA from these samples were determined by Sanger sequencing and fluorescence-based quantitative polymerase chain reaction, respectively. Myocardial mtDNA sequences analysis revealed 227 substitution variants, including 157 coding variants and 70 non-coding variants. An m.9856T > C (Ile217Thr) mutation in MT-CO3 from one LVNC patient was found to be a non-haplogroups associated variant, and was rare in the mtDB Human Mitochondrial Genome Database, suggesting that the variant may be pathogenic. And there was statistically significant difference in mtDNA copy number between LVNC patients and normal control subjects. Conclusions The identification of mtDNA sequence variants in myocardial specimens may be helpful for further investigation of the underlying pathogenic implications of myocardial mtDNA mutations in LVNC. However, measurement of mtDNA copy number showed that LVNC patients had a lower mtDNA content than controls (P = 0.003). Lower mtDNA copy number suggested that mitochondria dysfunction may be associated with aetiology of LVNC.

Do mitochondria contribute to left ventricular non-compaction cardiomyopathy? New findings from myocardium of patients with left ventricular non-compaction cardiomyopathy

BackgroundLeft ventricular non-compaction cardiomyopathy (LVNC) is a rare congenital cardiomyopathy that is associated with mutations in mitochondrial DNA (mtDNA), however, no study of myocardium mtDNA of LVNC patients has been reported. To identify novel candidate mtDNA variants that may be responsible for the pathogenesis of LVNC, myocardial specimens were examined to investigate pathogenic mtDNA variants. Materials and methodsSamples from six patients who were diagnosed with LVNC and underwent heart transplantation were analyzed. The sequence and copy number of mtDNA from these samples were determined by Sanger sequencing and fluorescence-based quantitative polymerase chain reaction, respectively. ResultsMyocardial mtDNA sequences analysis revealed 227 substitution variants, including 157 coding variants and 70 non-coding variants. An m.9856T>C (Ile217Thr) mutation in MT-CO3 from one LVNC patient was found to be a non-haplogroup associated variant, and was rare in the mtDB Human Mitochondrial Genome Database, suggesting that the variant may be pathogenic. And there was statistically significant difference in mtDNA copy number between LVNC patients and normal control subjects. Electron microscopy (EM) of left ventricular myocardium showed abnormality in mitochondrial morphology and disordered sarcomeric organization. ConclusionThe identification of mtDNA sequence variants in myocardial specimens may be helpful for further investigation of the underlying pathogenic implications of myocardial mtDNA mutations in LVNC. However, measurement of mtDNA copy number showed that there was lower mtDNA content in myocardium of LVNC patients than in normal controls (P<0.01). Lower mtDNA copy number and morphological abnormalities of mitochondria suggested mitochondrial dysfunction that may be associated with etiology of LVNC.

Mitochondrial DNA Copy Number, but Not Haplogroup, Confers a Genetic Susceptibility to Leprosy in Han Chinese from Southwest China

BackgroundLeprosy is a chronic infectious disease caused by Mycobacterium leprae, an unculturable pathogen with an exceptionally eroded genome. The high level of inactivation of gene function in M. leprae, including many genes in its metabolic pathways, has led to a dependence on host energy production and nutritional products. We hypothesized that host cellular powerhouse - the mitochondria - may affect host susceptibility to M. leprae and the onset of clinical leprosy, and this may be reflected by mitochondrial DNA (mtDNA) background and mtDNA copy number.MethodsWe analyzed the mtDNA sequence variation of 534 leprosy patients and 850 matched controls from Yunnan Province and classified each subject by haplogroup. mtDNA copy number, taken to be proportional to mtDNA content, was measured in a subset of these subjects (296 patients and 231 controls) and 12 leprosy patients upon diagnosis.ResultsComparison of matrilineal components of the case and control populations revealed no significant difference. However, measurement of mtDNA copy number showed that lepromatous leprosy patients had a significantly higher mtDNA content than controls (P = 0.008). Past medical treatments had no effect on the alteration of mtDNA copy number.ConclusionsOur results suggested that mtDNA content, but not haplogroup, affects leprosy and this influence is limited to the clinical subtype of lepromatous leprosy.

Obesity and Hepatosteatosis in Mice with Enhanced Oxidative DNA Damage Processing in Mitochondria

Mitochondria play critical roles in oxidative phosphorylation and energy metabolism. Increasing evidence supports that mitochondrial DNA (mtDNA) damage and dysfunction play vital roles in the development of many mitochondria-related diseases, such as obesity, diabetes mellitus, infertility, neurodegenerative disorders, and malignant tumors in humans. Human 8-oxoguanine-DNA glycosylase 1 (hOGG1) transgenic (TG) mice were produced by nuclear microinjection. Transgene integration was analyzed by PCR. Transgene expression was measured by RT-PCR and Western blot analysis. Mitochondrial DNA damage was analyzed by mutational analyses and measurement of mtDNA copy number. Total fat content was measured by a whole-body scan using dual-energy X-ray absorptiometry. The hOGG1 overexpression in mitochondria increased the abundance of intracellular free radicals and major deletions in mtDNA. Obesity in hOGG1 TG mice resulted from increased fat content in tissues, produced by hyperphagia. The molecular mechanisms of obesity involved overexpression of genes in the central orexigenic (appetite-stimulating) pathway, peripheral lipogenesis, down-regulation of genes in the central anorexigenic (appetite-suppressing) pathway, peripheral adaptive thermogenesis, and fatty acid oxidation. Diffuse hepatosteatosis, female infertility, and increased frequency of malignant lymphoma were also seen in these hOGG1 TG mice. High levels of hOGG1 expression in mitochondria, resulting in enhanced oxidative DNA damage processing, may be an important factor in human metabolic syndrome, infertility, and malignancy.

Quantification of mitochondrial DNA copy number: Pre-analytical factors

Mitochondrial DNA (mtDNA) content is important for understanding many cellular processes. Several pre-analytical factors, from sample collection to DNA extraction can affect measurement of mtDNA copy number. In the present study, whole blood samples yielded a higher mtDNA copy number than buffy coat samples. mtDNA content is affected by the cell separation method used and the time between blood withdrawal and cell separation. Thus, reference values must be established with the same type of sample. As to the DNA isolation and purification method, the manual phenol method can give randomly false high values. The QIAamp DNA Mini Kit provided the most highly reproducible mtDNA/nDNA yield.