Profound Length Heteroplasmic Alterations in Mitochondrial DNA Control Region From Primary AML Cells: Implication of Impaired Mitochondrial Replication and Demonstration of ‘vicious cycle’ Hypothesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3116-3116
Author(s):  
Myung-Geun Shin ◽  
Hye Ran Kim ◽  
Hyeoung-Joon Kim ◽  
Hoon Kook ◽  
Tai Ju Hwang ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Control Region ◽  
Copy Number ◽  
Regulatory Region ◽  
Mtdna Control Region ◽  
Vicious Cycle ◽  
Mtdna Copy Number ◽  
Length Heteroplasmy ◽  
Region Length ◽  
D Loop

Abstract Abstract 3116 Poster Board III-53 Mitochondrial DNA (mtDNA) control region (displacement (D)-loop including HV1 and HV2) is a non-coding region of 1124 bp (nucleotide positions, np 16 024–576), which acts as a promoter for both the heavy and light strands of mtDNA, and contains essential transcription and replication elements (Blood 2004;103:4466-77). Importantly, mutations in the D-loop regulatory region might change mtDNA replication rate by modifying the binding affinity of significant trans-activating factors (Eur J Cancer 2004;40:2519-24). Thus, length heteroplasmic alterations of mtDNA control region may be related with mitochondrial dysfunction resulting in ‘vicious cycle’ (Mol Med Today 2000;6:425-32). In an attempt to investigate profiling of mtDNA length heteroplasmic alterations in primary AML cells, we carried out a quantitative size-based PCR product separation by capillary electrophoresis (ABI 3130XL Genetic Analyzer and ABI Prism Genotyper version 3.1) using six targets (np 303-315 poly C, np 16184-16193 poly C, np 514-511 CA repeats, np 3566-3572 poly C, np 12385-12391 poly C and np 12418-12426 poly A). Length heteroplasmy was further confirmed by cloning and sequencing. Quantitative analysis of mtDNA molecules was performed using the QuantiTect SYBR Green PCR kit (Qiagen) and Rotor-Gene 3000 (Corbett Research). Forty-eight AML bone marrow samples were collected after receiving Institutional Review Board approval and informed consent. There were profound alterations of mtGI in 303 poly C, 16184 poly C and 514 CA repeats. The length heteroplasmy pattern of 303 poly C tract in the HV2 region disclosed mixture of 7C, 8C, 9C and 10C mtDNA types. In the HV2 region, length heteroplasmy in poly-C tract at np 303 - 309 exhibited 5 variant peak patterns: 7CT6C+8CT6C (50.0%), 8CT6C+9CT6C (14.0%), 8CT6C+ 9CT6C+ 10CT6C (10.4%), 9CT6C+10CT6C+11CT6C (8.3%) 9CT6C + 10CT6C + 11CT6C+12CT6C (2.1%). The length heteroplasmy pattern of 514-523 CA repeats in the HV2 region exhibited 2 variant peak patterns: CACACACACA (56.3%) and CACACACA (43.7%). In the HV1 region, length heteroplasmy in the poly-C tract at np 16184 - 16193 exhibited 9 variant peak patterns: 5CT4C+5CT3C (31.0%), 6CT4C+6CT3C (2.1%), 9C+10C+11C+12C (16.7%), 9C+10C+11C (2.1%), T4CT4C+5CT3C (4.2%), 9C+10C+11C+12C+13C (2.1%), 3CTC4C+5CT3C (2.1%), 10C+11C+12C+13C (4.2%), 8C+9C+10+11C (2.1%). Primary AML cells revealed decreased enzyme activity in respiratory chain complex I, II and III. AML cells had about a two-fold decrease in mtDNA copy number compared with normal blood mononuclear cells. Current study demonstrates that profound length heteroplasmic alterations in mtDNA control region of primary AML cells may lead to impairment of mitochondrial biogenesis (reduction of mtDNA copy number) and derangement of mitochondrial ATP synthesis. During this perturbation, mitochondria in primary AML cells might produce a large amount of reactive oxygen species, which causes the vicious cycle observed in chronic inflammatory diseases and cancers as well. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mitochondrial DNA structure and expression in specialized subtypes of mammalian striated muscle.

1990 ◽  
Vol 10 (11) ◽  
pp. 5671-5678 ◽  
Author(s):  
B H Annex ◽  
R S Williams
Keyword(s):  
Mitochondrial Dna ◽  
Copy Number ◽  
Striated Muscle ◽  
Dna Structure ◽  
Triplex Dna ◽  
Mtdna Copy Number ◽  
Loop Region ◽  
D Loop ◽  
Heavy Strand ◽  
Light Strand

Mitochondrial DNA (mt DNA) in cells of vertebrate organisms can assume an unusual triplex DNA structure known as the displacement loop (D loop). This triplex DNA structure forms when a partially replicated heavy strand of mtDNA (7S mtDNA) remains annealed to the light strand, displacing the native heavy strand in this region. The D-loop region contains the promoters for both heavy- and light-strand transcription as well as the origin of heavy-strand replication. However, the distribution of triplex and duplex forms of mtDNA in relation to respiratory activity of mammalian tissues has not been systematically characterized, and the functional significance of the D-loop structure is unknown. In comparisons of specialized muscle subtypes within the same species and of the same muscle subtype in different species, the relative proportion of D-loop versus duplex forms of mtDNA in striated muscle tissues of several mammalian species demonstrated marked variation, ranging from 1% in glycolytic fast skeletal fibers of the rabbit to 65% in the mouse heart. There was a consistent and direct correlation between the ratio of triplex to duplex forms of mtDNA and the capacity of these tissues for oxidative metabolism. The proportion of D-loop forms likewise correlated directly with mtDNA copy number, mtRNA abundance, and the specific activity of the mtDNA (gamma) polymerase. The D-loop form of mtDNA does not appear to be transcribed at greater efficiency than the duplex form, since the ratio of mtDNA copy number to mtRNA was unrelated to the proportion of triplex mtDNA genomes. However, tissues with a preponderance of D-loop forms tended to express greater levels of cytochrome b mRNA relative to mitochondrial rRNA transcripts, suggesting that the triplex structure may be associated with variations in partial versus full-length transcription of the heavy strand.

Mitochondrial DNA structure and expression in specialized subtypes of mammalian striated muscle

1990 ◽  
Vol 10 (11) ◽  
pp. 5671-5678
Author(s):  
B H Annex ◽  
R S Williams
Keyword(s):  
Mitochondrial Dna ◽  
Copy Number ◽  
Striated Muscle ◽  
Dna Structure ◽  
Triplex Dna ◽  
Mtdna Copy Number ◽  
Loop Region ◽  
D Loop ◽  
Heavy Strand ◽  
Light Strand

Mitochondrial DNA (mt DNA) in cells of vertebrate organisms can assume an unusual triplex DNA structure known as the displacement loop (D loop). This triplex DNA structure forms when a partially replicated heavy strand of mtDNA (7S mtDNA) remains annealed to the light strand, displacing the native heavy strand in this region. The D-loop region contains the promoters for both heavy- and light-strand transcription as well as the origin of heavy-strand replication. However, the distribution of triplex and duplex forms of mtDNA in relation to respiratory activity of mammalian tissues has not been systematically characterized, and the functional significance of the D-loop structure is unknown. In comparisons of specialized muscle subtypes within the same species and of the same muscle subtype in different species, the relative proportion of D-loop versus duplex forms of mtDNA in striated muscle tissues of several mammalian species demonstrated marked variation, ranging from 1% in glycolytic fast skeletal fibers of the rabbit to 65% in the mouse heart. There was a consistent and direct correlation between the ratio of triplex to duplex forms of mtDNA and the capacity of these tissues for oxidative metabolism. The proportion of D-loop forms likewise correlated directly with mtDNA copy number, mtRNA abundance, and the specific activity of the mtDNA (gamma) polymerase. The D-loop form of mtDNA does not appear to be transcribed at greater efficiency than the duplex form, since the ratio of mtDNA copy number to mtRNA was unrelated to the proportion of triplex mtDNA genomes. However, tissues with a preponderance of D-loop forms tended to express greater levels of cytochrome b mRNA relative to mitochondrial rRNA transcripts, suggesting that the triplex structure may be associated with variations in partial versus full-length transcription of the heavy strand.

Rare instances of non-random dropout with the monochrome multiplex qPCR assay for mitochondrial DNA copy number

2021 ◽  
Author(s):  
Stephanie Y Yang ◽  
Charles E Newcomb ◽  
Stephanie L Battle ◽  
Anthony YY Hsieh ◽  
Hailey L Chapman ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Copy Number ◽  
Whole Genome Sequencing Data ◽  
Whole Genome ◽  
Dna Copy Number ◽  
Loop Primer ◽  
Mitochondrial Dna Copy Number ◽  
D Loop

Mitochondrial DNA copy number (mtDNA-CN) is a proxy for mitochondrial function and has been of increasing interest to the mitochondrial research community. There are several ways to measure mtDNA-CN, ranging from whole genome sequencing to qPCR. A recent article from the Journal of Molecular Diagnostics described a novel method for measuring mtDNA-CN that is both inexpensive and reproducible. However, we show that certain individuals, particularly those with very low qPCR mtDNA measurements, show poor concordance between qPCR and whole genome sequencing measurements. After examining whole genome sequencing data, this seems to be due to polymorphisms within the D-loop primer region. Non-concordant mtDNA-CN was observed in all instances of polymorphisms at certain positions in the D-loop primer regions, however, not all positions are susceptible to this effect. In particular, these polymorphisms appear disproportionately in individuals with the L, T, and U mitochondrial haplogroups, indicating non-random dropout.

scholarly journals mtDNA in the Pathogenesis of Cardiovascular Diseases

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Lili Wang ◽  
Qianhui Zhang ◽  
Kexin Yuan ◽  
Jing Yuan
Keyword(s):  
Heart Failure ◽  
Cardiovascular Disease ◽  
Mitochondrial Dna ◽  
Copy Number ◽  
Heart Diseases ◽  
Mtdna Copy Number ◽  
Cardiac Inflammation ◽  
Ischemic Heart Diseases ◽  
Metabolic Functions ◽  
Underlying Mechanisms

The incidence rate of cardiovascular disease (CVD) has been increasing year by year and has become the main cause for the increase of mortality. Mitochondrial DNA (mtDNA) plays a crucial role in the pathogenesis of CVD, especially in heart failure and ischemic heart diseases. With the deepening of research, more and more evidence showed that mtDNA is related to the occurrence and development of CVD. Current studies mainly focus on how mtDNA copy number, an indirect biomarker of mitochondrial function, contributes to CVD and its underlying mechanisms including mtDNA autophagy, the effect of mtDNA on cardiac inflammation, and related metabolic functions. However, no relevant studies have been conducted yet. In this paper, we combed the current research status of the mechanism related to the influence of mtDNA on the occurrence, development, and prognosis of CVD, so as to find whether these mechanisms have something in common, or is there a correlation between each mechanism for the development of CVD?

scholarly journals Targeting reduced mitochondrial DNA quantity as a therapeutic approach in pediatric high-grade gliomas

2019 ◽  
Vol 22 (1) ◽  
pp. 139-151 ◽  
Author(s):  
Han Shen ◽  
Man Yu ◽  
Maria Tsoli ◽  
Cecilia Chang ◽  
Swapna Joshi ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Glucose Metabolism ◽  
Mitochondrial Function ◽  
Copy Number ◽  
Pyruvate Dehydrogenase Kinase ◽  
High Grade ◽  
Mtdna Copy Number ◽  
Mitochondrial Oxidation ◽  
High Grade Gliomas

Abstract Background Despite increased understanding of the genetic events underlying pediatric high-grade gliomas (pHGGs), therapeutic progress is static, with poor understanding of nongenomic drivers. We therefore investigated the role of alterations in mitochondrial function and developed an effective combination therapy against pHGGs. Methods Mitochondrial DNA (mtDNA) copy number was measured in a cohort of 60 pHGGs. The implication of mtDNA alteration in pHGG tumorigenesis was studied and followed by an efficacy investigation using patient-derived cultures and orthotopic xenografts. Results Average mtDNA content was significantly lower in tumors versus normal brains. Decreasing mtDNA copy number in normal human astrocytes led to a markedly increased tumorigenicity in vivo. Depletion of mtDNA in pHGG cells promoted cell migration and invasion and therapeutic resistance. Shifting glucose metabolism from glycolysis to mitochondrial oxidation with the adenosine monophosphate–activated protein kinase activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) or the pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA) significantly inhibited pHGG viability. Using DCA to shift glucose metabolism to mitochondrial oxidation and then metformin to simultaneously target mitochondrial function disrupted energy homeostasis of tumor cells, increasing DNA damage and apoptosis. The triple combination with radiation therapy, DCA and metformin led to a more potent therapeutic effect in vitro and in vivo. Conclusions Our results suggest metabolic alterations as an onco-requisite factor of pHGG tumorigenesis. Targeting reduced mtDNA quantity represents a promising therapeutic strategy for pHGG.

264 MITOCHONDRIAL DNA COPY NUMBER IN OOCYTES OF PREPUBERTAL AND CYCLIC GILTS

2011 ◽  
Vol 23 (1) ◽  
pp. 230
Author(s):  
P. Pawlak ◽  
E. Pers-Kamczyc ◽  
D. Lechniak-Cieslak
Keyword(s):  
Mitochondrial Dna ◽  
Copy Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Published Data ◽  
Mtdna Copy Number ◽  
Final Size ◽  
Domestic Species ◽  
Mitochondrial Dna Copy Number ◽  
Wide Range

In many domestic species (pig, cow, sheep), oocytes from prepubertal females show impaired quality when compared with those from adult animals. Incomplete cytoplasmic maturation is thought to be the main factor responsible for reduced developmental competence of embryos derived from prepubertal oocytes. The status of ooplasm maturation is also reflected by the copy number of mitochondrial DNA (mtDNA). Because replication of mtDNA ceases when oocytes reach their final size and occurs again at the blastocyst stage, the mtDNA copy number is a proved marker of oocyte quality in the pig (El Shourbagy et al. 2006 Reproduction 131, 233–245). The number of mtDNA copies in the grown oocyte is crucial to sustain the first embryonic divisions. To increase the rate of good-quality blastocysts, oocytes of domestic animals have been evaluated by the brilliant cresyl blue test (BCB). According to El Shourbagy et al. (2006), more competent BCB+ oocytes possess higher copy number of mtDNA (on average 222 446) than do their BCB– counterparts (115 352). However, there are no published data on the variation in mtDNA copy number in oocytes derived from ovaries of prepubertal (NCL) and cyclic (CL) gilts. Ovaries of NCL and CL gilts were collected in a local slaughterhouse. Cumulus–oocyte complexes (COC) were aspirated from nonatretic follicles 2 to 6 mm in diameter and evaluated morphologically. Only COC with a proper morphology were subjected to the BCB test. A group of non-BCB-treated COC served as control. Four groups of COC were collected: BCB+ (CL, NCL) and control (CL, NCL). Follicular cells attached to oocytes were removed by pipetting, and completely denuded gametes were individually frozen in liquid nitrogen. Analysis of the mtDNA copy number included isolation of the total DNA followed by amplification of the Cytochrome b (CYTB) gene by real-time PCR (one copy per one mitochondrial genome). Differences in mtDNA copy number among experimental groups were evaluated by Student’s t-test. To date, 30 BCB+ oocytes have been analysed individually (15 CL and 15 NCL). The analysed parameter varied in a wide range from 79 852 to 522 712 copies in CL oocytes and from 52 270 to 287 852 copies in NCL oocytes. Oocytes from cyclic gilts contained significantly more mtDNA copies (on average 267 524) than did gametes of prepubertal females (179 339; P < 0.05). The data on the mtDNA copy number in the control oocytes are currently under investigation. The preliminary results indicate that impaired oocytes quality of prepubertal gilts may be also attributed to the reduced copy number of mtDNA. This project was sponsored by MSHE Poland (grant no. 451/N-COST/2009/0).

scholarly journals Unusual mtDNA Control Region Length Heteroplasmy in the COS-7 Cell Line

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 607
Author(s):  
Nataliya Kozhukhar ◽  
Sunil Mitta ◽  
Mikhail F. Alexeyev
Keyword(s):  
Mitochondrial Genome ◽  
Cell Line ◽  
Control Region ◽  
Tandem Repeats ◽  
Variable Number ◽  
Length Heteroplasmy ◽  
Repeat Units ◽  
Chlorocebus Aethiops ◽  
Region Length ◽  
Transcription And Replication

The COS-7 cell line is a workhorse of virology research. To expand this cell line’s utility and to enable studies on mitochondrial DNA (mtDNA) transcription and replication, we determined the complete nucleotide sequence of its mitochondrial genome by Sanger sequencing. In contrast to other available mtDNA sequences from Chlorocebus aethiops, the mtDNA of the COS-7 cell line was found to contain a variable number of perfect copies of a 108 bp unit tandemly repeated in the control region. We established that COS-7 cells are heteroplasmic with at least two variants being present: with four and five repeat units. The analysis of the mitochondrial genome sequences from other primates revealed that tandem repeats are absent from examined mtDNA control regions of humans and great apes, but appear in lower primates, where they are present in a homoplasmic state. To our knowledge, this is the first report of mtDNA length heteroplasmy in primates.

Biological Significance and Profile of Length Heteroplasmy in the Hypervariable Segments of the Human Mitochondrial DNA Control Regions from Blood Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3815-3815
Author(s):  
Myung-Geun Shin ◽  
Hyeoung-Joon Kim ◽  
Hye-Ran Kim ◽  
Hee-Nam Kim ◽  
Il-Kwon Lee ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Control Region ◽  
Blood Cells ◽  
Biological Significance ◽  
Length Heteroplasmy ◽  
Conserved Sequence ◽  
Healthy Donors ◽  
Mtdna Sequence ◽  
Mtdna Replication ◽  
Two Peaks

Abstract A high incidence of mitochondrial DNA (mtDNA) variations was observed in both hypervariable region (HV) 1 and HV2; most mtDNA sequence variations were localized at poly C tract at nucleotides (nt) 303-315 (CCCCCCCTCCCCC, 7CT5C) in the HV2. Another poly C tract variant in HV1 at nt 16184-6193 have been suggested to be related with diabetes, dilated cardiomyopathy and some cancers. Poly C tract in HV2 is part of the conserved sequence block II located in 92-bp from the heavy strand replication origin. It is not yet clear whether poly C variants at nt 303–315 would lead to alterations in mtDNA replication. We hypothesized that some severe alterations in poly C tracts may lead to impairment of mtDNA replication. Here we present the profile of length heteroplasmy in HV from blood cells and its biological significance. A total of 57 maternally unrelated healthy donors were included and heparinized bloods were obtained from five age groups including 12 cord bloods. We amplified and sequenced the 1,121-bp control region including HV1 and HV2. In an attempt to investigate mtDNA length heteroplasmy, we carried out a qualitative and quantitative profiling length heteroplasmy using size-based PCR product separation by capillary electrophoresis (ABI 3100 Genetic Analyzer and ABI Prism Genotyper version 3.1). Length heteroplasmy was further confirmed by cloning and sequencing. Quantitative analysis of mtDNA molecules was performed using the QuantiTect SYBR Green PCR kit (Qiagen) and Rotor-Gene 3000 (Corbett Research) and standard plot was obtained from cloned cytochrome b gene. The mtDNA control region sequences showed 57 different haplotypes resulting from 77 polymorphic positions. Common polymorphisms were 73A>G (98%), 263A>G (91%), 16223C>T (47%), 16189T>C (35%), 150C>T (25%) and 152T>C (18%). The patterns of length heteroplasmy in the HV2 region were classified into 6 types. In the HV1 region, length heteroplasmy showed 8 variant peak patterns. The distribution of length heteroplasmy in poly C tracts at nt 303 – 315 was mtDNA mixture of 7CT6C+8CT6C (53%), 8CT6C+9CT6C (26%), 8CT6C+9CT6C+10CT6C (11%), 9CT6C+10CT6C +11CT6C (5%), 9CT6C+10CT6C (3%) and 7CT6C+6CT6C (2%). The distribution of length heteroplasmy pattern in poly C tract at nt 16184 – 16193 was 5CT4C+5CT3C (60%), 9C+10C+11C+12C (21%), 9C+10C+11C (5%), 3CT6C+3CT5C (3%), 9C+10C+11C+12C+13C (3%), 3CT4C+3CT3C (3%), 10C+11C+12C (2%), and 8C+9C+10C+11C+12C (2%). Interestingly, this study revealed that all healthy subjects showed length heteroplasmy in the HV1 and HV2 regions in contrast to previous studies. Length heteroplasmy in poly C 303–315 showed two groups of two peaks (n = 48) and more than three peaks (n = 9). MtDNA content from group with three peaks in poly C 303–315 (61,983,373 molecules/ul ± 33,219,871, mean±SD) was markedly lower than those with two peaks (133,777,955 molecules/ul ± 87,209,377). In conclusion, significantly higher rate of length heteroplasmy was observed in HV1 and HV2 from healthy donors and the presence of more than three mtDNA types in poly C at nt 303 – 315 might be associated with impairment of mtDNA replication.

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