Real-time detection and quantification of mitochondrial mutations with oligonucleotide primers containing locked nucleic acid

BACKGROUND: The phenotypic expression of disorders caused by point mutations, deletions or depletions within the mitochondrial genome (mtDNA) is heterogeneous. This relates to the phenomena of heteroplasmy, tissue threshold as well as the distribution of mutant DNA among tissues. Hence, the diagnostics of these disorders demands highly specific, sensitive and quantitative methods. METHODS: We have developed an allele-specific quantitative real-time PCR method for the detection of two of the most prevalent disease causing mitochondrial mutations, m.3243A>G (MELAS) and m.8993T>G (NARP). Locked Nucleic Acid (LNA) modified primers were used to obtain high allele specificity. In order to monitor mtDNA depletion a real-time method for mtDNA/nuclear DNA copy number ratio determination was developed. RESULTS: Rapid and sensitive detection and quantification of MELAS and NARP mtDNA alleles were achieved. Heteroplasmy levels as low as 0.01% could be detected, and the mtDNA/nuclear DNA ratio could be determined. CONCLUSIONS: The present method that allows simultaneous determination of heteroplasmy levels and mtDNA/nuclear DNA copy number ratio, will provide a useful tool in molecular diagnostics and in future epidemiological studies of mitochondrial diseases.     

Simultaneous detection and quantification of mitochondrial DNA deletion(s), depletion, and over-replication in patients with mitochondrial disease

Heterogeneous clinical expression of mitochondrial DNA (mtDNA) disorders depends on both qualitative and quantitative changes in mtDNA. We developed a sensitive and effective method that simultaneously detects mtDNA deletion(s) and quantifies total mtDNA content. The percentage of deletions and mtDNA content of 19 patients with single or multiple deletions were analyzed by real-time quantitative polymerase chain reaction (real-time qPCR) using TaqMan probes specific for mtDNA (tRNA leu(UUR), ND4, ATPase8, and D-loop regions) and nuclear DNA (AIB1, beta-2-microglobulin, and beta-actin). The proportion of deletion mutants determined by real-time qPCR was consistent with that determined by Southern analysis. Most patients with mtDNA deletions also demonstrated compensatory mtDNA over-replication. Multiple mtDNA deletions that were not detectable by Southern analysis due to low percentage of each deletion molecule were readily detected and quantified by real-time qPCR. Furthermore, 12 patients with clinical features and abnormal biochemical/histopathological results consistent with mitochondrial respiratory chain disorders without identified mtDNA mutations had either substantially depleted or significantly over-replicated mtDNA content, supporting the diagnosis of mitochondrial disease. Our results demonstrate that both qualitative and quantitative analyses are important in molecular diagnosis of mitochondrial diseases. The presence of deletion(s) and mtDNA depletion or compensatory over-replication can be determined simultaneously by real-time qPCR.     

Acute ethanol administration oxidatively damages and depletes mitochondrial dna in mouse liver, brain, heart, and skeletal muscles: protective effects of antioxidants.

Ethanol metabolism causes oxidative stress and lipid peroxidation not only in liver but also in extra-hepatic tissues. Ethanol administration has been shown to cause oxidative degradation and depletion of hepatic mitochondrial DNA (mtDNA) in rodents, but its in vivo effects on the mtDNA of extra-hepatic tissues have not been assessed. We studied the effects of an acute intragastric ethanol administration (5 g/kg) on brain, heart, skeletal muscle, and liver mtDNA in mice. Ethanol administration caused mtDNA depletion and replacement of its supercoiled form by linearized forms in all tissues examined. Maximal mtDNA depletion was about similar (ca. 50%) in all organs studied. It occurred 2 h after ethanol administration in heart, skeletal muscle, and liver but after 10 h in brain. This mtDNA depletion was followed by increased mtDNA synthesis. A secondary, transient increase in mtDNA levels occurred 24 h after ethanol administration in all organs. In hepatic or extra-hepatic tissues, mtDNA degradation and depletion were prevented by 4-methylpyrazole, an inhibitor of ethanol metabolism, and attenuated by vitamin E, melatonin, or coenzyme Q, three antioxidants. In conclusion, our study shows for the first time that ethanol metabolism also causes oxidative degradation of the mitochondrial genome in brain, heart, and skeletal muscles. These effects could contribute to the development of (cardio)myopathy and brain injury in some alcoholic patients. Antioxidants prevent these effects in mice and could be useful in persevering drinkers.     

Mitochondrial DNA depletion in HIV-infected patients is more pronounced with chronic hepatitis C and enhanced following treatment with pegylated interferon plus ribavirin

BACKGROUND: Mitochondrial DNA (mtDNA) damage seems to be responsible for many of the toxicities associated with the long-term use of nucleoside analogues in HIV-infected patients. These adverse effects, mainly lipoatrophy, seem to be even more pronounced in subjects with hepatitis C virus (HCV) co-infection. However, there is no information about a possible additive effect of HCV on mtDNA depletion nor about the impact of ribavirin use in HIV/HCV-coinfected individuals. PATIENTS AND METHODS: mtDNA was measured in peripheral blood mononuclear cells (PBMC) collected from 192 individuals classified into 4 groups: HIV-neg/HCV-neg (control group, n = 11), HIV-pos/HCV-neg (56), HIV-neg/HCV-pos (18) and HIV-pos/HCV-pos (107). A duplex real-time NASBA assay was used to quantify mtDNA on maximal platelet-depleted specimens and all experiments were run in duplicate. The mtDNA copy number per cell was estimated taking as reference the nuclear DNA copy number. RESULTS: The mean mtDNA values in the control group was 757 copies/cell, while it was 428, 349 and 296 for HIV-pos, HCV-pos and HIV/HCV-coinfected individuals, respectively (P < 0.001 for all groups relative to the control group). No significant differences were observed when comparing patients with HIV or HCV infections alone, but coinfected individuals showed a lower mtDNA copy number than patients infected with HIV (P < 0.001) or with HCV (P = 0.089). In a subset of 18 patients with HIV/HCV-coinfection, treatment with pegylated interferon plus ribavirin produced a further reduction in mtDNA (mean value, 189 copies/cell; P = 0.009). CONCLUSIONS: HIV and HCV may independently cause mtDNA depletion in PBMC. Coinfection may result in more pronounced mtDNA depletion. The administration of interferon plus ribavirin may further enhance mtDNA depletion. These findings may explain the greater risk of lipoatrophy of antiretroviral therapy in HIV-infected patients with HCV coinfection and why anti-HCV therapy may aggravate this effect.     

Mitochondrial effects of antiretroviral therapies in asymptomatic patients

BACKGROUND: A decrease in the mitochondrial (mt) DNA to nuclear DNA ratio has gained acceptance as a marker of mitochondrial toxicity in treated HIV-infected patients, but the functional meaning of this alteration is unclear. METHODS: We assessed mtDNA content, mitochondrial content and function in peripheral blood mononuclear cells (PBMCs) of consecutive asymptomatic HIV-infected patients. Patients selected had been receiving a first-line highly active antiretroviral therapy (HAART) regimen for at least 6 months, consisting of zidovudine plus lamivudine or stavudine plus didanosine plus either nelfinavir or nevirapine, or were antiretroviral-naive. The mtDNA content was assessed by quantitative real-time PCR, mitochondrial content by citrate synthase activity, enzyme activity of complexes III and IV (both partially encoded by mtDNA) of the electron transport chain by spectrophotometry, oxygen consumption by polarography, and oxidative damage in cell membranes by monitoring cis-parinaric acid fluorescence. RESULTS: Mitochondrial content was significantly lower in all treated groups. Patients receiving stavudine plus didanosine had mtDNA depletion and a decrease in complex IV activity. However, oxygen consumption capacity and lipid peroxidation were unaffected in all groups. CONCLUSION: Long-term HAART may induce mitochondrial abnormalities in PBMC mitochondria, which do not necessarily translate into functional abnormalities, at least in asymptomatic patients.     

血细胞线粒体DNA拷贝数变化与高原红细胞增多症易感性的关联研究

目的探讨血细胞线粒体DNA(mtDNA)拷贝数的变化与高原红细胞增多症易感性的关联性。方法分别采集年龄、职业等相匹配的高原红细胞增多症患者37例及对照组42例的静脉血2 mL,提取总DNA,通过定量PCR法(染料法)检测白细胞的mtDNA相对拷贝数。结果高原红细胞增多症患者中,mtDNA拷贝数显著低于对照组(P<0.05)。通过线性回归分析显示,在对照组中,随血红蛋白浓度的增加mtDNA拷贝数增加(P<0.05);然而在高原红细胞增多症病例组中,随着血红蛋白浓度的增加,mtDNA拷贝数降低(P<0.05)。结论高原红细胞增多症患者中,mtDNA拷贝数较低,可能作为该病的分子标记。     

Decreased copy number of mitochondrial DNA in Ewing's sarcoma

BackgroundSomatic mutations and germline variations in mitochondrial DNA (mtDNA) have been widely detected in a range of human malignancies including Ewing's sarcoma (EWS). However, it still remains unknown whether quantitative alterations in mtDNA level occur during the initiation and/or progression of EWS.MethodsTo test this possibility, we determined mtDNA copy number from 17 cases of EWS tumor tissues and 5 normal bone tissue samples using a quantitative real-time PCR assay.ResultsOur results showed that the average mtDNA content was significantly reduced in cancerous specimens as compared to that in normal bone controls. mtDNA copy number was statistically associated with tumor metastasis. There was an over 2-fold decrease in tumors with metastasis than in low-grade ones without metastasis. In addition, change in mtDNA content was related to somatic mutations in the D-loop control region. Tumors harboring D-loop mutations, at the polycytidine stretch between nucleotide positions 303 and 309 or close to the replication origin sites of the heavy strand, exhibited significantly lowered mtDNA levels in comparison with those without D-loop alterations.ConclusionsThe mtDNA content reduction may be an important genetic event in the carcinogenesis of EWS. This study provides evidence that somatic D-loop mutation is likely one of the key factors contributing to quantitative changes of mtDNA in EWS.     

Mitochondrial DNA copy number in peripheral blood is associated with cognitive function in apparently healthy elderly women

BackgroundThe identification of early markers of dementia is of increasing clinical importance. Recently, impaired mitochondrial function has emerged as a potential marker for age-related diseases and the maintenance of mtDNA copy number is essential for the preservation of mitochondrial function. We investigated the association between mtDNA copy number and cognitive function in elderly women.MethodsA total of 107 apparently healthy elderly women were included. Cognitive abilities were assessed using the Mini-Mental State Examination (MMSE). We measured mtDNA copy number in peripheral leukocytes using real-time polymerase chain reaction (PCR) methods. Additionally, cardiometabolic risk factors and physical function were measured.ResultsMMSE scores were negatively correlated with the homeostasis model of insulin resistance (HOMA-IR) scores and positively correlated with gait speed as well as mtDNA copy number. After adjusting for age and level of education, the mean values of MMSE scores gradually increased with mtDNA copy number when divided into quartiles. Using step-wise multiple regression analysis, gait speed, mtDNA copy number, and age were determined to be the strongest predictors of MMSE score.ConclusionsThese data suggest that reduced mtDNA content may be a possible early marker of dementia, and this finding warrants further study in large, prospective investigations.     

建立RT-ARMS-qPCR系统定量测定含A1555G突变的线粒体DNA拷贝数

目的建立RT-ARMS-qPCR（real time-amplification refractory mutation system-quantitative PCR）系统定量测定含线粒体DNA（mitochondrial DNA,mtDNA）A1555G位点突变的片段的拷贝数,为阐明线粒体耳聋临床表型多样性的分子生物学基础奠定基础。方法根据ARMS的基本原理设计引物,在引物3’端插入错配碱基AC建立RT-ARMS-qPCR系统,优化定量PCR体系,并进行方法学评价。经PCR分别扩增相应突变型和野生型的片段,将其克隆到pGEM-T Easy载体上,构建质粒标准品;同时对含突变型和/或野生型mtDNA 1555位点的片段的拷贝数进行定量检测。结果RT-ARMS-qPCR系统用于检测含mtDNA 1555位点的片段,线性检测范围为102-108拷贝数/μl,检测灵敏度为1×102拷贝数/μl,其批内变异系数（CV）为1.34%,批间CV为1.96%,重复性好;突变型和野生型引物分别只扩增相应片段,特异性好。结论RT-ARMS-qPCR系统适合于定量检测含mtDNA A1555G点突变的线粒体DNA片段,结果稳定、准确,有助于阐明线粒体耳聋严重程度的分子基础。     

Effect of stavudine on mitochondrial genome and fatty acid oxidation in lean and obese mice

Like other antihuman immunodeficiency virus dideoxynucleosides, stavudine may occasionally induce lactic acidosis and perhaps lipodystrophy in metabolically or genetically susceptible patients. We studied the effects of stavudine on mitochondrial DNA (mtDNA), fatty acid oxidation, and blood metabolites in lean and genetically obese (ob/ob) mice. In lean mice, mtDNA was depleted in liver and skeletal muscle, but not heart and brain, after 6 weeks of stavudine treatment (500 mg/kg/day). With 100 mg/kg/day, mtDNA transiently decreased in liver, but was unchanged at 6 weeks in all organs, including white adipose tissue (WAT). Despite unchanged mtDNA levels, lack of significant oxidative mtDNA lesions (as assessed by long polymerase chain reaction experiments), and normal blood lactate/pyruvate ratios, lean mice treated with stavudine for 6 weeks had increased fasting blood ketone bodies, due to both increased hepatic fatty acid beta-oxidation and decreased peripheral ketolysis. In obese mice, basal WAT mtDNA was low and was further decreased by stavudine. In conclusion, stavudine can decrease hepatic and muscle mtDNA in lean mice and can also cause ketoacidosis during fasting without altering mtDNA. Stavudine depletes WAT mtDNA only in obese mice. Fasting and ketoacidosis could trigger decompensation in patients with incipient lactic acidosis, whereas WAT mtDNA depletion could cause lipodystrophy in genetically susceptible patients.     

Prkdc participates in mitochondrial genome maintenance and prevents Adriamycin-induced nephropathy in mice

Adriamycin (ADR) is a commonly used chemotherapeutic agent that also produces significant tissue damage. Mutations to mitochondrial DNA (mtDNA) and reductions in mtDNA copy number have been identified as contributors to ADR-induced injury. ADR nephropathy only occurs among specific mouse inbred strains, and this selective susceptibility to kidney injury maps as a recessive trait to chromosome 16A1-B1. Here, we found that sensitivity to ADR nephropathy in mice was produced by a mutation in the Prkdc gene, which encodes a critical nuclear DNA double-stranded break repair protein. This finding was confirmed in mice with independent Prkdc mutations. Overexpression of Prkdc in cultured mouse podocytes significantly improved cell survival after ADR treatment. While Prkdc protein was not detected in mitochondria, mice with Prkdc mutations showed marked mtDNA depletion in renal tissue upon ADR treatment. To determine whether Prkdc participates in mtDNA regulation, we tested its genetic interaction with Mpv17, which encodes a mitochondrial protein mutated in human mtDNA depletion syndromes (MDDSs). While single mutant mice were asymptomatic, Prkdc/Mpv17 double-mutant mice developed mtDNA depletion and recapitulated many MDDS and ADR injury phenotypes. These findings implicate mtDNA damage in the development of ADR toxicity and identify Prkdc as a MDDS modifier gene and a component of the mitochondrial genome maintenance pathway.     

AAV-mediated Liver-specific MPV17 Expression Restores mtDNA Levels and Prevents Diet-induced Liver Failure

Mutations in human MPV17 cause a hepatocerebral form of mitochondrial DNA depletion syndrome (MDS) hallmarked by early-onset liver failure, leading to premature death. Liver transplantation and frequent feeding using slow-release carbohydrates are the only available therapies, although surviving patients eventually develop slowly progressive peripheral and central neuropathy. The physiological role of Mpv17, including its functional link to mitochondrial DNA (mtDNA) maintenance, is still unclear. We show here that Mpv17 is part of a high molecular weight complex of unknown composition, which is essential for mtDNA maintenance in critical tissues, i.e. liver, of a Mpv17 knockout mouse model. On a standard diet, Mpv17^−/− mouse shows hardly any symptom of liver dysfunction, but a ketogenic diet (KD) leads these animals to liver cirrhosis and failure. However, when expression of human MPV17 is carried out by adeno-associated virus (AAV)-mediated gene replacement, the Mpv17 knockout mice are able to reconstitute the Mpv17-containing supramolecular complex, restore liver mtDNA copy number and oxidative phosphorylation (OXPHOS) proficiency, and prevent liver failure induced by the KD. These results open new therapeutic perspectives for the treatment of MPV17-related liver-specific MDS.     

Mitochondrial DNA analysis in clinical laboratory diagnostics

Mitochondrial disorders are increasingly being diagnosed, especially among patients with multiple, seemingly unrelated, neuromuscular and multi-sytem disorders. The genetics are complex, in particular as the primary mutation can be either on the nuclear or the mitochondrial DNA (mtDNA). mtDNA mutations are often maternally inherited, but can be sporadic or secondary to autosomally inherited mutations in nuclear genes that regulate mtDNA biosynthesis. mtDNA mutations demonstrate extreme variable expressivity in terms of clinical manifestations and severity, even within a family. Disease is often episodic. Several well-defined clinical syndromes associated with specific mutations are described, yet the genotype-phenotype correlation is fair at best and most patients do not fit within any defined syndrome and have rare or novel mutations. In most patients, mutant and wild-type mtDNA coexist ("heteroplasmy"), although homoplasmic mtDNA mutations also are known. "Standard" mtDNA clinical diagnostics usually consists of a PCR-based assay to detect a small number of relatively common point mutations and Southern blotting (or PCR) for large (>500 bp) rearrangements. In selected cases testing negative, additional analyses can include real-time PCR for mtDNA depletion, and full mtDNA genome screening for the detection of rare and novel point mutations by a variety of methods. Prenatal diagnosis is problematic in most cases.     

Mitochondrial DNA deletion associated with the reduction of adenine nucleotides in human atrium and atrial fibrillation

BACKGROUND: Structural changes in the number, size, and shape of mitochondria (mt) have been observed in the atrial muscles of patients with atrial fibrillation (AF) and of animals with rapid atrial pacing, however, it is not known whether the mitochondrial function is impaired in human atrium with AF. MATERIALS AND METHODS: We determined adenine nucleotides concentrations and mtDNA deletions in 26 human right atria obtained at the time of cardiac surgery, using HPLC and PCR amplification, and studied the relationship between mtDNA deletions and clinical manifestations, the haemodynamic parameters of the patients and adenine nucleotide concentrations in their atrium. RESULTS: The age and the prevalence of AF were significantly higher in the patients with a mtDNA deletion of 7.4 kb than in those without a deletion; there were no significant differences regarding haemodynamic parameters between the two groups. The concentrations of ATP, ADP, AMP and total adenine nucleotides in the right atrium were significantly lower in the patients with mtDNA deletions than the patients without a deletion. In a gender- and diseased-matched population, the mtDNA deletion was still significantly associated with age and a decreased concentration of adenine nucleotides in the atrium. Using quantitative PCR analysis, the proportion of mtDNA deletion to normal mtDNA of the atrium, was estimated to be 0.3-2% in four cases. CONCLUSION: These results suggest that the deletion of mtDNA associated with ageing or AF can lead to a bioenergetic deficiency due to an impaired ATP synthesis in the human atrium; however, no conclusion can be made whether mtDNA deletion were the result or the cause of an impaired ATP synthesis, ageing, hemodynamic deterioration, or AF.     

Fall in circulating mononuclear cell mitochondrial DNA content in human sepsis

Purpose  

Loss of mitochondrial DNA (mtDNA) has been described in whole blood samples from a small number of patients with sepsis, but the underlying mechanism and clinical implications of this observation are not clear. We have investigated the cellular basis of the mtDNA depletion in sepsis, and determined clinical correlates with mtDNA depletion.     

RT-ARMS-qPCR定量测定线粒体耳聋患者mtDNA A1555G位点突变

目的 探讨RT-ARMS-qPCR(real time amplification refractory mutation system quantitativePCR)系统定量测定线粒体DNA(mitochondrial DNA,mtDNA)A1555G位点突变在线粒体耳聋发病机制研究中的价值.方法 以PeR扩增含mtDNA 1555位点的片段,并将其克隆到pGEM-T Easy载体上,构建质粒标准品;在引物3'端插入错配碱基AC建立RT-ARMS-qPCR系统,对含突变型和野生型mtDNA 1555位点片段的12例线粒体耳聋患者进行定量检测.结果 RT-ARMS-qPCR系统在检测1个含野生型mtDNA 1555的重组质粒DNA模板时,其批内变异系数(CV)为1.34%,批间CV为1.96%,线性范围为102-108拷贝/ul;突变型或野生型引物只特异扩增相对应的序列,特异性好;突变型/野生型mtDNA拷贝数及突变型所占的百分比与耳聋的严重程度相关(r=0.771,P=0.003),突变型mtDNA所占的比例越高,耳聋程度越严重.结论 RT-ARMS-qPCR系统适合于定量检测mtDNAA1555G点突变的线粒体DNA片段,结果特异、稳定、准确.线粒体耳聋的严重程度与含突变型和野生型mtDNA 1555位点的拷贝数比例有关.