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1.
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.  相似文献   

2.
Defects of mitochondrial DNA (mtDNA) are an important cause of disease and play a role in the ageing process. There are multiple copies of the mitochondrial genome in a single cell. In many patients with acquired or inherited mtDNA mutations, there exists a mixture of mutated and wild type genomes (termed heteroplasmy) within individual cells. As a biochemical and clinical defect is only observed when there are high levels of mutated mtDNA, a crucial investigation is to determine the level of heteroplasmic mutations within tissues and individual cells. We have developed an assay to determine the relative amount of deleted mtDNA using real-time fluorescence PCR. This assay detects the vast majority of deleted molecules, thus eliminating the need to develop specific probes. We have demonstrated an excellent correlation with other techniques (Southern blotting and three- primer competitive PCR), and have shown this technique to be sensitive to quantify the level of deleted mtDNA molecules in individual cells. Finally, we have used this assay to investigate patients with mitochondrial disease and shown in individual skeletal muscle fibres that there exist different patterns of abnormalities between patients with single or multiple mtDNA deletions. We believe that this technique has significant advantages over other methods to quantify deleted mtDNA and, employed alongside our method to sequence the mitochondrial genome from single cells, will further our understanding of the role of mtDNA mutations in human disease and ageing.  相似文献   

3.
In this work, we present the results of the screening of human mitochondrial DNA (mtDNA) heteroplasmy in the control region of mtDNA from 210 unrelated Spanish individuals. Both hypervariable regions of mtDNA were amplified and sequenced in order to identify and quantify point and length heteroplasmy. Of the 210 individuals analyzed, 30% were fully homoplasmic and the remaining presented point and/or length heteroplasmy. The prevalent form of heteroplasmy was length heteroplasmy in the poly(C) tract of the hypervariable region II (HVRII), followed by length heteroplasmy in the poly(C) tract of hypervariable region I (HVRI) and, finally, point heteroplasmy, which was found in 3.81% of the individuals analyzed. Moreover, no significant differences were found in the proportions of the different kinds of heteroplasmy in the population when blood and buccal cell samples were compared. The pattern of heteroplasmy in HVRI and HVRII presents important differences. Moreover, the mutational profile in heteroplasmy seems to be different from the mutational pattern detected in population. The results suggest that a considerable number of mutations and, particularly, transitions that appear in heteroplasmy are probably eliminated by drift and/or by selection acting at different mtDNA levels of organization. Taking as a whole the results reported in this work, it is mandatory to perform a broad-scale screening of heteroplasmy to better establish the heteroplasmy profile which would be important for medical, evolutionary, and forensic proposes.  相似文献   

4.
The area of somatic mtDNA mutation measurement is in a crisis because the methods used to quantify mtDNA mutations produce results varying by multiple orders of magnitude. The reason for these discrepancies is not clear, but given that most methods involve PCR, the prime suspect is PCR artifacts (e.g. spontaneous errors by the DNA polymerases used). In addition to simple misincorporation, another important source of artificial mutations is the conversion of chemically modified (e.g. damaged) nucleotides into mutations when bypassed by a thermostable DNA polymerase. These latter mutations are particularly difficult to account for because appropriate controls are not available. Here, we argue that single molecule PCR (smPCR) is uniquely positioned to account for these bypass-related artificial mutations and discuss the methodology involved in employing this technique.  相似文献   

5.
Mitochondrial DNA (mtDNA) is widely used to clarify phylogenetic relationships among and within species, and to determine population structure. Due to the linked nature of mtDNA genes it is expected that different genes will show similar results. Phylogenetic incongruence using mtDNA genes may result from processes such as heteroplasmy, nuclear integration of mitochondrial genes, polymerase errors, contamination, and recombination. In this study we used sequences from two mitochondrial genes (cytochrome b and cytochrome oxidase subunit I) from the wild vectors of Chagas disease, Triatoma eratyrusiformis and Mepraia species to test for topological congruence. The results showed some cases of phylogenetic incongruence due to misplacement of four haplotypes of four individuals. We discuss the possible causes of such incongruence and suggest that the explanation is an intra-individual variation likely due to heteroplasmy. This phenomenon is an independent evidence of common ancestry between these taxa.  相似文献   

6.
刘艳  胡婧  黄原 《动物学杂志》2006,41(5):120-126
线粒体DNA的异质性在生物学、医学、法医、生物技术等领域有重要的应用。本文从自然发生(包括体细胞突变、父系渗入和杂交)和人工产生(包括转基因、细胞融合、核移植和转线粒体)两大方面系统地介绍了线粒体DNA异质性的产生机制及其遗传。并介绍了线粒体DNA异质性的检测方法,已知突变位点mtDNA异质性的检测方法有原位PCR技术、PCR-RFLP和实时荧光定量PCR技术,未知突变位点mtDNA异质性的检测方法有长PCR技术、时相温度梯度凝胶电泳(TTGE)和变性高效液相色谱(DHPLC)等。最后对克隆生物中线粒体异质性检测的应用实例作了介绍。  相似文献   

7.
In patients with mitochondrial disease a continuously increasing number of mitochondrial DNA (mtDNA) mutations and polymorphisms have been identified. Most pathogenic mtDNA mutations are heteroplasmic, resulting in heteroduplexes after PCR amplification of mtDNA. To detect these heteroduplexes, we used the technique of denaturing high performance liquid chromatography (DHPLC). The complete mitochondrial genome was amplified in 13 fragments of 1–2 kb, digested in fragments of 90–600 bp and resolved at their optimal melting temperature. The sensitivity of the DHPLC system was high with a lowest detection of 0.5% for the A8344G mutation. The muscle mtDNA from six patients with mitochondrial disease was screened and three mutations were identified. The first patient with a limb-girdle-type myopathy carried an A3302G substitution in the tRNALeu(UUR) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy carried a T3271C mutation in the tRNALeu(UUR) gene (80% heteroplasmy) and the third patient with Leigh syndrome carried a T9176C mutation in the ATPase6 gene (93% heteroplasmy). We conclude that DHPLC analysis is a sensitive and specific method to detect heteroplasmic mtDNA mutations. The entire automatic procedure can be completed within 2 days and can also be applied to exclude mtDNA involvement, providing a basis for subsequent investigation of nuclear genes.  相似文献   

8.
线粒体 DNA(mitochondrial DNA,mtDNA)是线粒体内最重要的遗传物质。mtDNA 突变普 遍存在,突变型 mtDNA 与野生型 mtDNA 共存的现象被称为 mtDNA 异质性。mtDNA 异质性与衰老和多种疾病密切相关。mtDNA异质性特性、mtDNA 异质性与衰老和疾病相关性以及线粒体疾病的治疗等都是近年来遗传学研究的热点。本文从 mtDNA 异质性的动态变化、组织特异性、mtDNA 异质性与疾病以及线粒体疾病的治疗等方面对 mtDNA 异质性进行综述。  相似文献   

9.
Mouse models are widely employed to study mitochondrial inheritance, which have implications to several human diseases caused by mutations in the mitochondrial genome (mtDNA). These mouse models take advantage of polymorphisms between the mtDNA of the NZB/BINJ and the mtDNA of common inbred laboratory (i.e., C57BL/6) strains to generate mice with two mtDNA haplotypes (heteroplasmy). Based on PCR followed by restriction fragment length polymorphism (PCR-RFLP), these studies determine the level of heteroplasmy across generations and in different cell types aiming to understand the mechanisms underlying mitochondrial inheritance. However, PCR-RFLP is a time-consuming method of low sensitivity and accuracy that dependents on the use of restriction enzyme digestions. A more robust method to measure heteroplasmy has been provided by the use of real-time quantitative PCR (qPCR) based on allelic refractory mutation detection system (ARMS-qPCR). Herein, we report an ARMS-qPCR assay for quantification of heteroplasmy using heteroplasmic mice with mtDNA of NZB/BINJ and C57BL/6 origin. Heteroplasmy and mtDNA copy number were estimated in germline and somatic tissues, providing evidence of the reliability of the approach. Furthermore, it enabled single-step quantification of heteroplasmy, with sensitivity to detect as low as 0.1% of either NZB/BINJ or C57BL/6 mtDNA. These findings are relevant as the ARMS-qPCR assay reported here is fully compatible with similar heteroplasmic mouse models used to study mitochondrial inheritance in mammals.  相似文献   

10.
The accumulation of somatic mutations in mtDNA is correlated with aging. In this work, we sought to identify somatic mutations in the HVS-1 region (D-loop) of mtDNA that might be associated with aging. For this, we compared 31 grandmothers (mean age: 63 ± 2.3 years) and their 62 grandchildren (mean age: 15 ± 4.1 years), the offspring of their daughters. Direct DNA sequencing showed that mutations absent in the grandchildren were detected in a presumably homoplasmic state in three grandmothers and in a heteroplasmic state in an additional 13 grandmothers; no mutations were detected in the remaining 15 grandmothers. However, cloning followed by DNA sequencing in 12 grandmothers confirmed homoplasia in only one of the three mutations previously considered to be homoplasmic and did not confirm heteroplasmy in three out of nine grandmothers found to be heteroplasmic by direct sequencing. Thus, of 12 grandmothers in whom mtDNA was analyzed by cloning, eight were heteroplasmic for mutations not detected in their grandchildren. In this study, the use of genetically related subjects allowed us to demonstrate the occurrence of age-related (> 60 years old) mutations (homoplasia and heteroplasmy). It is possible that both of these situations (homoplasia and heteroplasmy) were a long-term consequence of mitochondrial oxidative phosphorylation that can lead to the accumulation of mtDNA mutations throughout life.  相似文献   

11.
Mutations of the mitochondria DNA (mtDNA) displacement loop (D-loop) were investigated to clarify different changes of exogenous and endogenous liver carcinogenesis in rats. We induced hepatocellular carcinomas (HCCs) in rats with N-nitrosodiethylamine (DEN) and a choline-deficient l-amino acid-defined (CDAA) diet. DNAs were extracted from 10 HCCs induced by DEN and 10 HCCs induced by the CDAA diet. To identify mutations in mtDNA D-loop, polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing, was performed. Mutations were detected in 5 out of 10 HCCs (50%) induced by DEN. Four out of 5 mutations were G/C to A/T transitions at positions 15707, 15717, 15930, and 16087, and one T/A to C/G transition at position 15559. By contrast, no mutations were found in 10 HCCs induced by the CDAA diet. These results demonstrated that mutations in mtDNA D-loop occur in rat HCCs induced by DEN but not by the CDAA diet, suggesting that mtDNA D-loop is a target of exogenous liver carcinogenesis in rats.  相似文献   

12.
Mutations in mitochondrial DNA (mtDNA) may result in various pathological processes. Detection of mutant mtDNAs is a problem for diagnostic practice that is complicated by heteroplasmy – a phenomenon of the inferring presence of at least two allelic variants of the mitochondrial genome. Also, the level of heteroplasmy largely determines the profile and severity of clinical manifestations. Here we discuss detection of mutations in heteroplasmic mtDNA using up-todate methods that have not yet been introduced as routine clinical assays. These methods can be used for detecting mutations in mtDNA to verify diagnosis of “mitochondrial disease”, studying dynamics of mutant mtDNA in body tissues of patients, as well as investigating structural features of mtDNAs. Original data on allele-specific discrimination of m.11778G>A mutation by droplet digital PCR are presented, which demonstrate an opportunity for simultaneous detection and quantitative assessment of mutations in mtDNAs.  相似文献   

13.
Mutations of mitochondrial DNA (mtDNA) cause a wide array of multisystem disorders, particularly affecting organs with high energy demands. Typically only a proportion of the total mtDNA content is mutated (heteroplasmy), and high percentage levels of mutant mtDNA are associated with a more severe clinical phenotype. MtDNA is inherited maternally and the heteroplasmy level in each one of the offspring is often very different to that found in the mother. The mitochondrial genetic bottleneck hypothesis was first proposed as the explanation for these observations over 20 years ago. Although the precise bottleneck mechanism is still hotly debated, the regulation of cellular mtDNA content is a key issue. Here we review current understanding of the factors regulating the amount of mtDNA within cells and discuss the relevance of these findings to our understanding of the inheritance of mtDNA heteroplasmy.  相似文献   

14.
The gradual accumulation of mitochondrial DNA (mtDNA) mutations is implicated in aging and may contribute to the accelerated aging phenotype seen with tobacco smoking and HIV infection. mtDNA mutations are thought to arise from oxidative damage; however, recent reports implicate polymerase γ errors during mtDNA replication. Investigations of somatic mtDNA mutations have been hampered by technical challenges in measuring low‐frequency mutations. We use primer ID‐based next‐generation sequencing to quantify both somatic and heteroplasmic blood mtDNA point mutations within the D‐loop, in 164 women and girls aged 2–72 years, of whom 35% were smokers and 56% were HIV‐positive. Somatic mutations and the occurrence of heteroplasmic mutations increased with age. While transitions are theorized to result from polymerase γ errors, transversions are believed to arise from DNA oxidative damage. In our study, both transition and transversion mutations were associated with age. However, transition somatic mutations were more prevalent than transversions, and no heteroplasmic transversions were observed. We also measured elevated somatic mutations, but not heteroplasmy, in association with high peak HIV viremia. Conversely, heteroplasmy was higher among smokers, but somatic mutations were not, suggesting that smoking promotes the expansion of preexisting mutations rather than de novo mutations. Taken together, our results are consistent with blood mtDNA mutations increasing with age, inferring a greater contribution of polymerase γ errors in mtDNA mutagenesis. We further suggest that smoking and HIV infection both contribute to the accumulation of mtDNA mutations, though in different ways.  相似文献   

15.
The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype–phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes. As disease expression of mitochondrial tRNA mutations is mostly in postmitotic tissues, studies to elucidate disease mechanisms need to be performed on patient material. Heteroplasmy quantitation and copy number estimation using small patient biopsy samples has not been reported before, mainly due to technical restrictions. In order to resolve this problem, we have developed a robust assay that utilizes Molecular Beacons to accurately quantify heteroplasmy levels and determine mtDNA copy number in small samples carrying the A8344G tRNALys mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.  相似文献   

16.
Tissue specific somatic mutations occurring in the mtDNA control region have been proposed to provide a survival advantage. Data on twins and on relatives of long-lived subjects suggested that the occurrence/accumulation of these mutations may be genetically influenced. To further investigate control region somatic heteroplasmy in the elderly, we analyzed the segment surrounding the nt 150 position (previously reported as specific of Leukocytes) in various types of leukocytes obtained from 195 ultra-nonagenarians sib-pairs of Italian or Finnish origin collected in the frame of the GEHA Project. We found a significant correlation of the mtDNA control region heteroplasmy between sibs, confirming a genetic influence on this phenomenon. Furthermore, many subjects showed heteroplasmy due to mutations different from the C150T transition. In these cases heteroplasmy was correlated within sibpairs in Finnish and northern Italian samples, but not in southern Italians. This suggested that the genetic contribution to control region mutations may be population specific. Finally, we observed a possible correlation between heteroplasmy and Hand Grip strength, one of the best markers of physical performance and of mortality risk in the elderly. Our study provides new evidence on the relevance of mtDNA somatic mutations in aging and longevity and confirms that the occurrence of specific point mutations in the mtDNA control region may represent a strategy for the age-related remodelling of organismal functions.  相似文献   

17.
In embryos derived by nuclear transfer (NT), fusion, or injection of donor cells with recipient oocytes caused mitochondrial heteroplasmy. Previous studies have reported varying patterns of mitochondrial DNA (mtDNA) transmission in cloned calves. Here, we examined the transmission of mtDNA from NT pigs to their progeny. NT pigs were created by microinjection of Meishan pig fetal fibroblast nuclei into enucleated oocytes (maternal Landrace background). Transmission of donor cell (Meishan) mtDNA was analyzed using 4 NT pigs and 25 of their progeny by PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, PCR-RFLP, and a specific PCR to detect Meishan mtDNA single nucleotide polymorphisms (SNP-PCR). In the blood and hair root of NT pigs, donor mtDNAs were not detected by PCR-SSCP and PCR-RFLP, but detected by SNP-PCR. These results indicated that donor mtDNAs comprised between 0.1% and 1% of total mtDNA. Only one of the progeny exhibited heteroplasmy with donor cell mtDNA populations, ranging from 0% to 44% in selected tissues. Additionally, other progeny of the same heteroplasmic founder pig were analyzed, and 89% (16/18) harbored donor cell mtDNA populations. The proportion of donor mtDNA was significantly higher in liver (12.9 +/- 8.3%) than in spleen (5.0 +/- 3.9%), ear (6.7 +/- 5.3%), and blood (5.8 +/- 3.7%) (P < 0.01). These results demonstrated that donor mtDNAs in NT pigs could be transmitted to progeny. Moreover, once heteroplasmy was transmitted to progeny of NT-derived pigs, it appears that the introduced mitochondrial populations become fixed and maternally-derived heteroplasmy was more readily maintained in subsequent generations.  相似文献   

18.
哺乳动物线粒体DNA(mitochondrial DNA, mtDNA)位于线粒体.当细胞中mtDNA发生突变时,就会出现野生型与突变型mtDNA的共存.这种情况被称为mtDNA异质性.从mtDNA异质性的形成到在表型上引起相应的病变是一个复杂的过程.mtDNA异质性是如何形成和其在特异组织的增殖复制,mtDNA异质性的变化对个体的影响,如何提高mtDNA突变负荷检测的精度和灵敏度都是近些年的研究热点.本文对mtDNA异质性的检测、遗传、组织特异性以及其相关的疾病等方面进行了阐述.  相似文献   

19.
For identification of somatic mitochondrial DNA (mtDNA) mutations, the mtDNA major noncoding region (D-loop) sequence in blood samples and carotid atherosclerosis plaques from patients with atherosclerosis was analyzed. Five point heteroplasmic positions were observed in 4 of 23 individuals (17%). Only in two cases could heteroplasmy have resulted from somatic mutation, whereas three heteroplasmic positions were found in both vascular tissue and blood. In addition, length heteroplasmy in a polycytosine stretches was registered at nucleotide positions 303–315 in 16 individuals, and also in the 16184–16193 region in four patients. The results suggest that somatic mtDNA mutations can occur during atherosclerosis, but some heteroplasmic mutations may appear in all tissues, possibly being inherited.  相似文献   

20.
Temporal temperature gradient gel electrophoresis was used to screen 70% of the mtDNA, including all 22 tRNA genes, for heteroplasmy in 75 children with neuromuscular and/or multi-system dysfunction and elevated lactate levels, and in 95 controls. Standard PCR/ASO (allele specific oligonucleotide) and Southern analyses were also employed. Excluding common length variants, heteroplasmy was found in 22 patients and two controls (P < 0.001), with four patients demonstrating heteroplasmy in two locations each. Of the 23 heteroplasmic variants sequenced among the patients, 17 were novel point variants in the control region (CR) and only two involved tRNA genes. Heteroplasmy is highly associated with the disease group, and is predominately found in the CR, an area rarely studied in patient populations. These variants may be pathological mutations or disease markers.  相似文献   

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