Global Genetic Determinants of Mitochondrial DNA Copy Number

Many human diseases including development of cancer is associated with depletion of mitochondrial DNA (mtDNA) content. These diseases are collectively described as mitochondrial DNA depletion syndrome (MDS). High similarity between yeast and human mitochondria allows genomic study of the budding yeast to be used to identify human disease genes. In this study, we systematically screened the pre-existing respiratory-deficient Saccharomyces cerevisiae yeast strains using fluorescent microscopy and identified 102 nuclear genes whose deletions result in a complete mtDNA loss, of which 52 are not reported previously. Strikingly, these genes mainly encode protein products involved in mitochondrial protein biosynthesis process (54.9%). The rest of these genes either encode protein products associated with nucleic acid metabolism (14.7%), oxidative phosphorylation (3.9%), or other protein products (13.7%) responsible for bud-site selection, mitochondrial intermembrane space protein import, assembly of cytochrome-c oxidase, vacuolar protein sorting, protein-nucleus import, calcium-mediated signaling, heme biosynthesis and iron homeostasis. Thirteen (12.7%) of the genes encode proteins of unknown function. We identified human orthologs of these genes, conducted the interaction between the gene products and linked them to human mitochondrial disorders and other pathologies. In addition, we screened for genes whose defects affect the nuclear genome integrity. Our data provide a systematic view of the nuclear genes involved in maintenance of mitochondrial DNA. Together, our studies i) provide a global view of the genes regulating mtDNA content; ii) provide compelling new evidence toward understanding novel mechanism involved in mitochondrial genome maintenance and iii) provide useful clues in understanding human diseases in which mitochondrial defect and in particular depletion of mitochondrial genome plays a critical role.     

mtDNA拷贝数的生物学意义及其调控

线粒体是细胞能量和自由基代谢中心,并在细胞凋亡、钙调控、细胞周期和信号转导中发挥重要作用,维持线粒体功能正常对于细胞正常行使职能意义重大。线粒体的功能与线粒体DNA（mitochondrial DNA,mtDNA）的数量和质量紧密相关,mtDNA的数量即mtDNA拷贝数又受到mtDNA质量的影响,因此mtDNA拷贝数可作为线粒体功能的重要表征。mtDNA拷贝数变异引起线粒体功能紊乱,进而导致疾病发生。本文综述了mtDNA拷贝数变异与神经退行性疾病、心血管疾病、肿瘤等疾病的发生发展和个体衰老之间的关系,以及mtDNA复制转录相关因子、氧化应激、细胞自噬等因素介导mtDNA拷贝数变异的调控机制。以期为进一步深入探究mtDNA拷贝数调控的分子机制,以及未来治疗神经退行性疾病、肿瘤及延缓衰老等提供一定的理论基础。     

Mitochondrial DNA Copy Number in Peripheral Blood and Melanoma Risk

Mitochondrial DNA (mtDNA) copy number in peripheral blood has been suggested as risk modifier in various types of cancer. However, its influence on melanoma risk is unclear. We evaluated the association between mtDNA copy number in peripheral blood and melanoma risk in 500 melanoma cases and 500 healthy controls from an ongoing melanoma study. The mtDNA copy number was measured using real-time polymerase chain reaction. Overall, mean mtDNA copy number was significantly higher in cases than in controls (1.15 vs 0.99, P<0.001). Increased mtDNA copy number was associated with a 1.45-fold increased risk of melanoma (95% confidence interval: 1.12-1.97). Significant joint effects between mtDNA copy number and variables related to pigmentation and history of sunlight exposure were observed. This study supports an association between increased mtDNA copy number and melanoma risk that is independent on the known melanoma risk factors (pigmentation and history of sunlight exposure).     

Mitochondrial DNA Copy Number in Cleavage Stage Human Embryos—Impact on Infertility Outcome

A retrospective case control study was undertaken at the molecular biology department of a private center for reproductive medicine in order to determine whether any correlation exists between mitochondrial DNA (mtDNA) content of cleavage-stage preimplantation embryos and their developmental potential. A total of 69 couples underwent IVF treatment (averaged women age: 36.5, SD 4.9) and produced a total of 314 embryos. A single blastomere was biopsied from each embryo at the cleavage stage (day-3 post-fertilization) subjected to low-pass next generation sequencing (NGS), for the purpose of detecting aneuploidy. For each sample, the number of mtDNA reads obtained after analysis using NGS was divided by the number of reads attributable to the nuclear genome. The mtDNA copy number amount was found to be higher in aneuploid embryos than in those that were euploid (mean mtDNA ratio ± SD: 6.3 ± 7.5 versus 7.1 ± 5.8, p < 0.004; U Mann–Whitney test), whereas no statistically significant differences in mtDNA content were seen in relation to embryo morphology (6.6 ± 4.8 vs. 8.5 ± 13.6, p 0.09), sex (6.6 ± 4.1 vs. 6.2 ± 6.8, p 0.16), maternal age (6.9 ± 7.8 vs. 6.7 ± 4.5, p 0.14) or its ability to implant (7.4 ± 6.6 vs. 5.1 ± 4.6, p 0.18). The mtDNA content cannot serve as a useful biomarker at this point in development. However, further studies investigating both quantitative and qualitative aspects of mtDNA are still required to fully evaluate the relationship between mitochondrial DNA and human reproduction.     

Mitochondrial DNA Mutations Induce Mitochondrial Dysfunction,Apoptosis and Sarcopenia in Skeletal Muscle of Mitochondrial DNA Mutator Mice

Background

Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established.

Methodology/Principal Findings

We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase γ, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Δψ_m). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage.

Conclusions/Significance

These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.     

Mitochondrial DNA Copy Number Is Associated with Breast Cancer Risk

Mitochondrial DNA (mtDNA) copy number in peripheral blood is associated with increased risk of several cancers. However, data from prospective studies on mtDNA copy number and breast cancer risk are lacking. We evaluated the association between mtDNA copy number in peripheral blood and breast cancer risk in a nested case-control study of 183 breast cancer cases with pre-diagnostic blood samples and 529 individually matched controls among participants of the Singapore Chinese Health Study. The mtDNA copy number was measured using real time PCR. Conditional logistic regression analyses showed that there was an overall positive association between mtDNA copy number and breast cancer risk (P_trend = 0.01). The elevated risk for higher mtDNA copy numbers was primarily seen for women with <3 years between blood draw and cancer diagnosis; ORs (95% CIs) for 2^nd, 3^rd, 4^th, and 5^th quintile of mtDNA copy number were 1.52 (0.61, 3.82), 2.52 (1.03, 6.12), 3.12 (1.31, 7.43), and 3.06 (1.25, 7.47), respectively, compared with the 1^st quintile (P_trend = 0.004). There was no association between mtDNA copy number and breast cancer risk among women who donated a blood sample ≥3 years before breast cancer diagnosis (P_trend = 0.41). This study supports a prospective association between increased mtDNA copy number and breast cancer risk that is dependent on the time interval between blood collection and breast cancer diagnosis. Future studies are warranted to confirm these findings and to elucidate the biological role of mtDNA copy number in breast cancer risk.     

Quantitative 3D Mapping of the Human Skeletal Muscle Mitochondrial Network

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Leukocyte Mitochondrial DNA Copy Number Is Associated with Chronic Obstructive Pulmonary Disease

Background

Oxidative stress is known to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Evidence suggests that leukocytes mitochondria DNA (mtDNA) is susceptible to undergo mutations, insertions, or depletion in response to reactive oxidative stress (ROS). We hypothesize that mtDNA copy number is associated with the development of COPD.

Methodology/Principal Findings

Relative mtDNA copy number was measured by a quantitative real-time PCR assay using DNA extracted from peripheral leukocytes. MtDNA copy number of peripheral leukocytes in the COPD group (n = 86) is significantly decreased compared with non-smoker group (n = 77) (250.3± 21.5 VS. 464.2± 49.9, P<0.001). MtDNA copy number in the COPD group was less than that in the healthy smoking group, but P value nearly achieved significance (250.3± 21.5 VS. 404.0± 76.7, P = 0.08) MtDNA copy number has no significance with age, gender, body mass index, current smoking, and pack-years in COPD group, healthy smoker group and no smoker group, respectively. Serum glutathione level in the COPD group is significantly decreased compared with healthy smoker and non-smoker groups (4.5± 1.3 VS. 6.2± 1.9 and 4.5± 1.3 VS. 7.1±1.1 mU/mL; P<0.001 respectively). Pearson correlation test shows a significant liner correlation between mtDNA copy number and serum glutathione level (R = 0.2, P = 0.009).

Conclusions/Significance

COPD is associated with decreased leukocyte mtDNA copy number and serum glutathione. COPD is a regulatory disorder of leukocytes mitochondria. However, further studies are needed to determine the real mechanisms about the gene and the function of mitochondria.     

The Relationship between Leukocyte Mitochondrial DNA Copy Number and Telomere Length in Community-Dwelling Elderly Women

Purpose

Both telomere length and mitochondrial function are accepted as reflective indices of aging. Recent studies have shown that telomere dysfunction may influence impaired mitochondrial biogenesis and function. However, there has been no study regarding the possible association between telomere and mitochondrial function in humans. Therefore, the purpose of the study was to identify any relationships between mitochondrial and telomere function.

Methods

The present study included 129 community-dwelling, elderly women. The leukocyte mitochondrial DNA copy number and telomere length were measured using a quantitative real-time polymerase chain reaction method. Anthropometric measurement, biochemical blood testing, a depression screening questionnaire using a 15-question geriatric depression scale (GDS-15), and a cognitive function test using the Korean version of the mini mental state examination (K-MMSE) were performed.

Results

Leukocyte mtDNA copy number was positively associated with telomere length (r=0.39, p=<0.0001) and K-MMSE score (r=0.06, p=0.02). Additionally, leukocyte mtDNA copy number was negatively correlated with GDS-15 score (r=-0.17, p=0.04). Age (r=-0.15, p=0.09), waist circumference (r=-0.16, p=0.07), and serum ferritin level (r=-0.13, p=0.07) tended to be inversely correlated with leukocyte mtDNA copy number. With a stepwise multiple regression analysis, telomere length was found to be an independent factor associated with leukocyte mtDNA copy number after adjustment for confounding variables including age, body mass index, waist circumference, total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, hs-CRP, serum ferritin, HOMA-IR, K-MMSE, GDS-15, hypertension, diabetes, dyslipidemia, currently smoking, alcohol drinking, and regular exercise.

Conclusions

This study showed that leukocyte mtDNA copy number was positively correlated with leukocyte telomere length in community-dwelling elderly women. Our findings suggest that telomere function may influence mitochondrial function in humans.     

Mitochondrial DNA Copy Number and Risk of Oral Cancer: A Report from Northeast India

Background

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer globally. Tobacco consumption and HPV infection, both are the major risk factor for the development of oral cancer and causes mitochondrial dysfunction. Genetic polymorphisms in xenobiotic-metabolizing enzymes modify the effect of environmental exposures, thereby playing a significant role in gene–environment interactions and hence contributing to the individual susceptibility to cancer. Here, we have investigated the association of tobacco - betel quid chewing, HPV infection, GSTM1-GSTT1 null genotypes, and tumour stages with mitochondrial DNA (mtDNA) content variation in oral cancer patients.

Methodology/Principal Findings

The study comprised of 124 cases of OSCC and 140 control subjects to PCR based detection was done for high-risk HPV using a consensus primer and multiplex PCR was done for detection of GSTM1-GSTT1 polymorphism. A comparative ΔCt method was used for determination of mtDNA content. The risk of OSCC increased with the ceased mtDNA copy number (P_trend = 0.003). The association between mtDNA copy number and OSCC risk was evident among tobacco – betel quid chewers rather than tobacco – betel quid non chewers; the interaction between mtDNA copy number and tobacco – betel quid was significant (P = 0.0005). Significant difference was observed between GSTM1 - GSTT1 null genotypes (P = 0.04, P = 0.001 respectively) and HPV infection (P<0.001) with mtDNA content variation in cases and controls. Positive correlation was found with decrease in mtDNA content with the increase in tumour stages (P<0.001). We are reporting for the first time the association of HPV infection and GSTM1-GSTT1 null genotypes with mtDNA content in OSCC.

Conclusion

Our results indicate that the mtDNA content in tumour tissues changes with tumour stage and tobacco-betel quid chewing habits while low levels of mtDNA content suggests invasive thereby serving as a biomarker in detection of OSCC.     

Wild-Type Mitochondrial DNA Copy Number in Urinary Cells as a Useful Marker for Diagnosing Severity of the Mitochondrial Diseases

The genotype-phenotype relationship in diseases with mtDNA point mutations is still elusive. The maintenance of wild-type mtDNA copy number is essential to the normal mitochondrial oxidative function. This study examined the relationship between mtDNA copy number in blood and urine and disease severity of the patients harboring A3243G mutation. We recruited 115 A3243G patients, in which 28 were asymptomatic, 42 were oligo-symptomatic, and 45 were poly-symptomatic. Increase of total mtDNA copy number without correlation to the proportion of mutant mtDNA was found in the A3243G patients. Correlation analyses revealed that wild-type mtDNA copy number in urine was the most important factor correlated to disease severity, followed by proportion of mutant mtDNA in urine and proportion of mutant mtDNA in blood. Wild-type copy number in urine negatively correlated to the frequencies of several major symptoms including seizures, myopathy, learning disability, headache and stroke, but positively correlated to the frequencies of hearing loss and diabetes. Besides proportion of mutant mtDNA in urine, wild-type copy number in urine is also an important marker for disease severity of A3243G patients.     

基于数字PCR的不同品种鸭组织中线粒体与核DNA拷贝数差异研究

肉和肉制品是人类生活的重要营养来源,但近年来肉制品中发生的掺假使假事件屡见不鲜,使得肉品的质量安全问题已经成为全世界关注的热点话题。以核酸为目标的动物源鉴定是当前普遍使用的方法。在核酸检测中,常用线粒体基因或核基因作为靶标,缺乏统一标准。以绍兴鸭和北京鸭等不同品种及生鲜组织(鸭血、鸭胸肉、鸭肝、鸭皮、鸭心和鸭腿肉)为实验材料,提取DNA后利用微滴式数字PCR开展线粒体和核DNA拷贝数的比较研究,以两者拷贝数及其比值的变异系数为判定依据。结果显示,核DNA的拷贝数在不同品种鸭组织间相对稳定,且变异系数小于线粒体DNA,表明核DNA是开展鸭肉制品掺假定量检测的最适DNA来源。鸭腿肉中线粒体/核DNA拷贝数比值的变异系数最小,表明线粒体DNA作为靶基因的鸭肉掺假比例定量检测时,鸭腿肉来源的肉制品是最佳选择。     

Peripheral Blood Mitochondrial DNA Copy Number Is Associated with Prostate Cancer Risk and Tumor Burden

Alterations of mitochondrial DNA (mtDNA) have been associated with the risk of a number of human cancers; however, the relationship between mtDNA copy number in peripheral blood leukocytes (PBLs) and the risk of prostate cancer (PCa) has not been investigated. In a case-control study of 196 PCa patients and 196 age-paired healthy controls in a Chinese Han population, the association between mtDNA copy number in PBLs and PCa risk was evaluated. The relative mtDNA copy number was measured using quantitative real-time PCR; samples from three cases and two controls could not be assayed, leaving 193 cases and 194 controls for analysis. PCa patients had significantly higher mtDNA copy numbers than controls (medians 0.91 and 0.82, respectively; P<0.001). Dichotomized at the median value of mtDNA copy number in the controls, high mtDNA copy number was significantly associated with an increased risk of PCa (adjusted odds ratio  = 1.85, 95% confidence interval: 1.21–2.83). A significant dose-response relationship was observed between mtDNA copy number and risk of PCa in quartile analysis (P _trend = 0.011). Clinicopathological analysis showed that high mtDNA copy numbers in PCa patients were significantly associated with high Gleason score and advanced tumor stage, but not serum prostate-specific antigen level (P = 0.002, 0.012 and 0.544, respectively). These findings of the present study indicate that increased mtDNA copy number in PBLs is significantly associated with an increased risk of PCa and may be a reflection of tumor burden.     

Age-Associated Impairments in Mitochondrial ADP Sensitivity Contribute to Redox Stress in Senescent Human Skeletal Muscle

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首发未服药精神分裂症患者中线粒体DNA拷贝数及其与血脂指标的相关性分析

精神分裂症是一种重度的精神疾病,伴发严重的能量代谢失衡。其中,脂代谢的异常近年来受到越来越多的关注。多项研究表明,线粒体在精神分裂症患者代谢异常的过程中发挥重要的作用。线粒体DNA拷贝数是线粒体含量及完整性的重要指标,直接参与线粒体的多项重要功能。本研究旨在通过考察首发精神分裂症患者线粒体DNA拷贝数的变化特征及其与相关代谢指标之间的相关性,为阐明精神分裂症代谢异常的机制提供支持。本研究共计纳入82例首发且未服用抗精神病药物的精神分裂症患者和77例健康对照,采用荧光定量PCR技术进行线粒体DNA拷贝数的检测,并进行了临床信息和血脂指标的采集。结果表明,精神分裂症患者组的线粒体DNA拷贝数显著低于健康对照组（P=0.000572,FC=-1.22）。血脂指标中,患者组的HDL-c显著低于健康对照组（P=0.001, FC=-1.12）,LDL-c（P=0.009, FC=1.09）、CHOL/HDL-c（P=0.000019, FC=1.17）、TG/HDL-c（P=0.000656, FC=1.31）和LDL-c/HDL-c（P=0.000004, FC=1.30）均显著高于健康对照组。相关性分析表示,在健康对照组中,mtDNA拷贝数与TG显著负相关（r=-0.232, P=0.0499）,与TG/HDL-c（r=-0.235, P=0.052）呈负相关,但不显著;在精神分裂症患者组,mtDNA拷贝数与各血脂指标的关系都不显著。本研究为精神分裂症患者的线粒体功能障碍、脂代谢异常提供了证据,提示了线粒体在精神分裂症患者并发心血管代谢疾病高风险中可能的重要作用。