Detection of mtDNA with 4977 bp deletion in blood cells and atherosclerotic lesions of patients with coronary artery disease

Recent evidence suggests that somatic mutations in nuclear and mitochondrial DNA accumulated during aging, may significantly contribute to the pathogenesis of chronic-degenerative illness such as coronary artery disease (CAD). Mitochondrial DNA with 4977 bp deletion mutation (mtDNA4977) is a common type of mtDNA alteration in humans. However, little attempt has been made to detect the presence of mtDNA4977 deletion in cells and tissues of cardiovascular patients. This study investigated the presence of mtDNA4977 in blood samples of 65 cardiovascular patients and 23 atherosclerotic plaques of human coronaries with severe atherosclerosis. Moreover, the presence of the deletion has been investigated in blood cells from 22 healthy age-matched subjects. The detection of mtDNA4977 has been performed by using a nested polymerase chain reaction (PCR) protocol and normalized to wild-type mtDNA. A significant higher incidence of mtDNA4977 was observed in CAD patients with respect to healthy subjects (26.2% versus 4.5%; P=0.03). Furthermore, the relative amount of the deletion was significantly higher in the patients compared to the control group (P=0.02). The mtDNA4977 was detected in 17 of the 65 patients blood samples (26.2%) and deletion levels ranged from 0.18 to 0.46% of the total mtDNA (mean: 0.34+/-0.02%). For what concerns atherosclerotic lesions, 5 patients (21.7%) showed the deletion ranging from 0.13 to 0.45% of the total mtDNA (mean: 0.35+/-0.06%). In both samples from patients, the incidence and the relative amount of mtDNA4977 was not significantly influenced by atherogenic risk factors and clinical parameters. The obtained results may suggest that the increase of oxidative stress in cardiovascular disease may be responsible for the accumulation of mtDNA damage in coronary artery disease patients.     

Differential accumulations of 4,977 bp deletion in mitochondrial DNA of various tissues in human ageing

Several types of deletions in mitochondrial DNA (mtDNA) have been recetly identified in various tissues of old humans. In order to determine whether there are differences in the incidence and proportion of deleted mtDNAs in different tissues during human ageing, we examined tha 4,977 bp deletion in mtDNA of various tissues from subjects of different ages. Total DNA was extracted from each of the biopsied tissues and was serially diluted by two-fold with distilled water. A 533 bp DNA fragment was amplified by PCR from total mtDNA using a pair of primers L3304-3323 and H3817-3836, and another 524 bp PCR product was amplified from 4,977 bp deleted mtDNA by identical conditions using another pair of primers L8150-8166 and H13631-13650. The maximum dilution fold of each sample that still allowed the ethidium bromide-stained PCR product (533 bp or 524 bp) in the agarose gel to be visible under UV light illumination was taken as the relative abundance of the mtDNA (wild-type or mutant) in the original sample. By this method, we were able to determine the proportion of deleted mtDNA in human tissues. We found that the 4,977 bp deletion started to appear in the second and third decades of life in human muscle and liver tissues. But the deletion was not detectable in the testis until the age of 60 years. Moreover, the proportion of deleted mtDNA varied greatly in different tissues. Among the tissues examined, muscle was found to harbor higher proportin of deleted mtDNA than the other tissues. The average proportion of the 4,977 bp depleted mtDNA of the muscle from subjects over 70 years old was approximately 0.06%, and that of the liver and the testis was 0.0076% and 0.05%, respectively. These findings suggest that the frequency and proportion of the deleted mtDNA in human tissues increase with age and that the mtDNA deletions occur more frequently and abundantly in high energy-demanding tissues during the ageing process of the human.     

Absolute quantitation of a heteroplasmic mitochondrial DNA deletion using a multiplex three-primer real-time PCR assay

Quantitation of wild-type and deleted mitochondrial DNA (mtDNA) coexisting within the same cell (a.k.a., heteroplasmy) is important in mitochondrial disease and aging. We report the development of a multiplex three-primer PCR assay that is capable of absolute quantitation of wild-type and deleted mtDNA simultaneously. Molecular beacons were designed to hybridize with either type of mtDNA molecule, allowing real-time detection during PCR amplification. The assay is specific and can detect down to six copies of mtDNA, making it suitable for single-cell analyses. The relative standard deviation in the threshold cycle number is approximately 0.6%. Heteroplasmy was quantitated in individual cytoplasmic hybrid cells (cybrids), containing a large mtDNA deletion, and bulk cell samples. Individual cybrid cells contained 100-2600 copies of wild-type mtDNA and 950-4700 copies of deleted mtDNA, and the percentage of heteroplasmy ranged from 43+/-16 to 95+/-16%. The average amount of total mtDNA was 3800+/-1600 copies/cybrid cell, and the average percentage of heteroplasmy correlated well with the bulk cell sample. The single-cell analysis also revealed that heteroplasmy in individual cells is highly heterogeneous. This assay will be useful for monitoring clonal expansions of mtDNA deletions and investigating the role of heteroplasmy in cell-to-cell heterogeneity in cellular models of mitochondrial disease and aging.     

Maternal inheritance of deleted mitochondrial DNA in a family with mitochondrial myopathy

Skeletal muscles from a mother and her daughter both with chronic progressive ophthalmoplegia were analyzed. Histological and biochemical analyses of their muscle samples showed typical features of this type of mitochondrial myopathy. Southern blot analysis revealed that, in both patients, there were two species of mitochondrial DNA (mtDNA): normal one and partially deleted one. The sizes of the deletion were different; the mutant mtDNAs from the mother and the daughter had about 2.5- and 5-kilobase deletions, respectively. The two mutant mtDNAs shared a common deleted region of 1.2-kilobase. However, both the start and the end of deletion were different between them, implying a novel mode of inheritance. This is the first report that the mutant mtDNA is responsible for the maternal inheritance of a human disease.     

Absence of the 9-bp deletion of mitochondrial DNA in pre-Hispanic inhabitants of Argentina

We investigated the incidence of the Region V mitochondrial DNA 9-base-pair (bp) deletion from human remains recovered from several archaeological sites and contexts throughout Argentina. Of the 34 samples analyzed, 24 yielded DNA extractions that gave clear amplification results. All of the individuals carried two repeats of the 9 bp, one of which has been shown to be deleted in some individuals of Asian origin and defines mitochondrial lineage B. Although most of the modern Amerindian groups in the region exhibit the deletion in high frequencies, the absence of the 9-bp deletion among ancient populations of South America seems to be the rule rather than the exception, as was reported by several studies involving extinct populations. The evidence gathered until now suggests that the earliest settlers of this region of South America did not carry mitochondrial lineage B.     

Specific amplification of deleted mitochondrial DNA from a myopathic patient and analysis of deleted region with S1 nuclease

Heteroplasmy of the normal-sized and the deleted mitochondrial genome has been observed in mitochondrial myopathy. The deleted region of the genome in the skeletal muscle of a patient was analyzed both by the conventional Southern blot method and by the novel method of employing the combination of polymerase chain reaction and S1 nuclease digestion. The results obtained by these methods were compared. Southern hybridization using various mitochondrial DNA fragments localized the deletion from at least position 9020 to 14,955, but regions of uncertainty of 1 kb remained on both ends of the deletion. Using the polymerase chain reaction, a fragment from the deleted genome was specifically amplified by choosing a pair of primers surrounding the deletion, and two fragments adjacent to the starting and end of the deletion were amplified from the normal-sized genome. S1 nuclease analysis of the heteroduplexes formed among these fragments demonstrated that the deletion extended from positions 8650 +/- 50 to 15,660 +/- 60. This method does not require radioisotopes and, moreover, can determine the deleted region within 5 h, in contrast to the 2 days required by the conventional Southern blot analysis. These results indicate that the novel method is faster and more accurate than the conventional method for the determination of the deleted region of genome.     

Multiple mitochondrial DNA deletions in an elderly human individual.

We have used the polymerase chain reaction (PCR) to study deletions in the mitochondrial DNA (mtDNA) of an elderly human individual. An extended set of PCR primers has been utilised to identify 10 mitochondrial DNA deletions in a 69-year-old female subject with no known mitochondrial disease. The particular deletions visualised as PCR products depended on the primer pairs used, such that the more distantly separated PCR primers enabled visualisation of larger deletions. Some deletions were common to the heart, brain and skeletal muscle, whereas others were apparently specific to individual tissues. DNA sequencing analysis of PCR products showed that short direct repeat sequences (5 to 13 bp) flanked all deletion breakpoints; in most cases one copy of the repeat was deleted. It is proposed that the accumulation of such multiple deletions is a general phenomenon during the ageing process.     

The effect of chronic alcohol consumption on mitochondrial DNA mutagenesis in human blood

The 4977bp deletion of mitochondrial DNA (mtDNA) is known to accumulate with increasing age in post mitotic tissues. Recently, studies came out detecting this specific alteration also in fast replicating cells, e.g. in blood or skin tissue, often in correlation to specific diseases or -- specifically in skin -- external stressors such as UV radiation. In this study, we investigated mitochondrial mutagenesis in 69 patients with a chronic alcoholic disease and 46 age matched controls with a moderate drinking behavior. Two different fragments, specific for total and for deleted mtDNA (dmtDNA) were amplified in a duplex-PCR. A subsequent fragment analysis was performed and for relative quantification, the quotient of the peak areas of amplification products specific for deleted and total mtDNA was determined. Additionally, a real time PCR was performed to quantify mtDNA copy number. The relative amount of 4977bp deleted mtDNA in alcoholics was significantly increased compared to controls. On the other hand, no difference regarding the mtDNA/nuclear DNA ratio in both investigated groups was detected. Additionally, no age dependence could be found nor in alcoholics, neither in the control group. These findings indicate that mtDNA mutagenesis in blood can be influenced by stressors such as alcohol. Ethanol seems to be a significant factor to alter mitochondrial DNA in blood and might be an additional contributor for the cellular aging process.     

Equine connective tissue tumors contain unintegrated bovine papilloma virus DNA

Bovine papilloma virus (BPV) appears to be the etiological agent of common equine connective tissue tumors. We investigated the physical state of the viral DNA within such tumors and found no indication for integration into the host genome. The BPV genomes were present as free circular episomes. Two equine sarcoids were shown to contain multiple copies of free circular BPV type 1 (BPV-1) DNA. When the tumors were digested with several single-cut restriction enzymes, there were only form III BPV-1 DNA sequences could be revealed. One of the sarcoids contained, apart from wild-type BPV-1 DNA, a class of smaller BPV-1 circular DNA molecules bearing a deletion of approximately 9% of the BPV-1 genome. This deletion is located in the physical map between the relative units 0 and 0.32.     

Preferential amplification and molecular characterization of junction sequences of a pathogenetic deletion in human mitochondrial DNA

Deletions of mitochondrial DNA have been detected in skeletal muscle of some patients with mitochondrial encephalomyopathies, but their junctions have been defined only approximately. We developed a procedure, using widely spaced primers for the polymerase chain reaction, that amplifies preferentially the sequences bracketing the deletion. This procedure permits detection of minor proportions, not detectable by Southern analysis, of deleted mitochondrial DNA species in a heteroplasmic mixture. Different proportions of intact mitochondrial DNA and species deleted from nucleotide 8708 to 13,722 were found in skeletal muscle, blood, and urinary epithelial cells from a patient with chronic progressive external ophthalmoplegia. These data indicate that the mutation occurred at or before early embryonic development and provide the first definition at the nucleotide level of a human disease caused by a deletion of mitochondrial DNA.     

Detection of deletions flanked by short direct repeats in mitochondrial DNA of aging Drosophila

Cumulative damage due to reactive oxygen species (ROS) in mitochondria, especially in mitochondrial DNA (mtDNA), would result in a decrease in mitochondrial respiratory function and contributes to the age-related decline in the physiological functioning of organisms. Previously, we reported the tissue-specific accumulation of deleted mtDNA with age in Drosophila melanogaster. In the present study, to understand the mechanism by which mtDNA deletion is generated with age, nucleotide sequences of deleted mtDNA were determined. Consequently, 33 different sequences each containing a deletion were obtained from flies that were more than 55-day-old. Most of the deletions were found to be flanked by short direct repeats. The present results, together with those from other animals, suggest that there is a common mechanism generating mtDNA deletions through direct repeats.     

Accumulation of mitochondrial DNA deletions in human oral tissues -- effects of betel quid chewing and oral cancer

Accumulation of mitochondrial DNA (mtDNA) mutations in human tissues has been associated with intrinsic aging and environmental insult. Recently, mtDNA mutations have been detected in various tumors, including head and neck tumors. However, the factors affecting the occurrence and accumulation of mtDNA deletions in tumor tissues are poorly understood. In Taiwan, betel quid chewing is a major risk factor for oral cancer. Using polymerase chain reaction (PCR) techniques, we examined large-scale deletions of mtDNA in 53 pairs of tumor and non-tumor oral tissues from the patients with or without betel quid chewing history. The results revealed that irrespective of the history of betel quid chewing, the incidences of the 4977bp deletion and other deletions of mtDNA were lower in the tumor portion as compared with the non-tumor portion. The average proportions of the 4977bp deleted mtDNA in the tumor tissues of the betel quid chewers and non-betel quid chewers were 13- and 5-fold, respectively, lower than those in the corresponding non-tumor tissues. Moreover, the average proportion of 4977bp deleted mtDNA was significantly higher (P<0.05) in the non-tumor oral tissues of the patients with betel quid chewing history than that of the patients without the history of betel quid chewing. These results suggest that betel quid chewing may increase mtDNA mutation in human oral tissues and that accumulation of mtDNA deletions and subsequent cytoplasmic segregation of these mutations during cell division could be an important contributor to the early phase of oral carcinogenesis.     

Molecular analyses of mtDNA deletion mutations in microdissected skeletal muscle fibers from aged rhesus monkeys

Mitochondrial DNA (mtDNA) deletion mutations co-localize with electron transport system (ETS) abnormalities in rhesus monkey skeletal muscle fibers. Using laser capture microdissection in conjunction with PCR and DNA sequence analysis, mitochondrial genomes from single sections of ETS abnormal fibers were characterized. All ETS abnormal fibers contained mtDNA deletion mutations. Deletions were large, removing 20-78% of the genome, with some to nearly all of the functional genes lost. In one-third of the deleted genomes, the light strand origin was deleted, whereas the heavy strand origin of replication was conserved in all fibers. A majority (27/39) of the deletion mutations had direct repeat sequences at their breakpoints and most (36/39) had one breakpoint within or in close proximity to the cytochrome b gene. Several pieces of evidence support the clonality of the mtDNA deletion mutation within an ETS abnormal region of a fiber: (a) only single, smaller than wild-type, PCR products were obtained from each ETS abnormal region; (b) the amplification of mtDNA from two regions of the same ETS abnormal fiber identified identical deletion mutations, and (c) a polymorphism was observed at nucleotide position 16103 (A and G) in the wild-type mtDNA of one animal (sequence analysis of an ETS abnormal region revealed that mtDNA deletion mutations contained only A or G at this position). Species-specific differences in the regions of the genomes lost as well as the presence of direct repeat sequences at the breakpoints suggest mechanistic differences in deletion mutation formation between rodents and primates.     

Different copy numbers of apparently identically deleted mitochondrial DNA in tissues from a patient with Kearns-Sayre syndrome detected by PCR

An apparently identical deletion of 4.977 bp in length (position 8,483-13,459) was detectable in the mitochondrial DNA from skeletal muscle, heart muscle, kidney, and liver of a patient with Kearns-Sayre syndrome. The proportion of deleted genome varied from 60% for the skeletal muscle to 15% for heart muscle and kidney, and was below 5% in the liver. The mtDNA heteroplasmy of the liver was only detectable after amplification by PCR. In skeletal and heart muscle histochemical and immunocytochemical findings concerning cytochrome c oxidase were in good correlation with the proportion of deleted mitochondrial DNA.     

Accumulation of deletions in human mitochondrial DNA during normal aging: analysis by quantitative PCR

We have developed a quantitative PCR technique to measure the amount of a specific mitochondrial DNA deletion (ΔmtDNA), the so-called ‘common deletion’, in human tissues. Using this method, we estimate that there is a 10 000-fold increase in this ΔmtDNA species in muscle during the course of the normal human lifespan. The maximum amount of common deletion observed in aged muscle was approx 0.1%. Tissues that turn-over slowly, such as skeletal muscle and heart, contained more ΔmtDNA than more rapidly dividing tissues, such as a liver, in agreement with studies performed by others.     

Relationship of an unstable argG gene to a 5.7-kilobase amplifiable DNA sequence in Streptomyces lividans 66.

The relationship between an unstable argG gene and a 5.7-kilobase (kb) amplifiable DNA sequence in Streptomyces lividans 66 was investigated. Spontaneous, high-frequency Arg mutants deleted for this gene typically contain 200 to 300 copies of the tandemly reiterated sequence. A library of S. lividans 66 (strain 1326) wild-type genomic DNA was prepared in the vector lambda Charon 35. Chromosome walking over 44 kb established that argG is located 25 kb distant from a duplicated amplifiable DNA structure. A sequence was characterized, located farther distal from the amplifiable structure, containing strong homology with an internal sequence of the amplifiable DNA, which may have a role in the deletion of argG. Genetic mapping showed that argG and the 5.7-kb amplifiable sequence are linked to another unstable gene, determining chloramphenicol resistance (Camr) and that together these genes may be located in a silent chromosomal arc.