Direct detection of multiple point mutations in mitochondrial DNA

Abstract Mitochondrial defects can be caused by mutations in nuclear or mitochondrial DNA. Large deletion/duplication and point mutations are the two major types of mitochondrial DNA (mtDNA) mutations. Comprehensive molecular diagnosis requires the analysis of multiple point mutations. We developed an effective multiplex PCR/allele-specific oligonucleotide (ASO) method to simultaneously screen multiple point mutations in mtDNA. The system involved three pairs of primers to amplify mutation “hot spots” at tRNAleu(UUR), tRNAlys/ATPase, and ND4 regions, followed by detection of point mutations with ASO probes. Over 2000 specimens were analyzed and the results were compared with those from previous studies with the PCR/restriction fragment length polymorphism method. Our data demonstrate that the multiplex PCR/ASO method is much more sensitive in the detection of low mutant heteroplasmy. It is simple and cost effective, especially if a large number of samples are to be screened for multiple point mutations.

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Mitochondrial DNA spectra of single human CD34+ cells, T cells, B cells, and granulocytes

Blood ◽

10.1182/blood-2005-01-0150 ◽

2005 ◽

Vol 106 (9) ◽

pp. 3271-3284 ◽

Cited By ~ 15

Author(s):

Yoji Ogasawara ◽

Kazutaka Nakayama ◽

Magdalena Tarnowka ◽

J. Philip McCoy ◽

Sachiko Kajigaya ◽

...

Keyword(s):

T Cells ◽

Mitochondrial Dna ◽

B Cells ◽

Single Cells ◽

Point Mutations ◽

Isolated Cells ◽

Mtdna Mutations ◽

Human Cd34 ◽

Cd34 Cells ◽

Mtdna Sequence

Abstract Previously, we described the age-dependent accumulation of mitochondrial DNA (mtDNA) mutations, leading to a high degree of mtDNA heterogeneity among normal marrow and blood CD34+ clones and in granulocytes. We established a method for sequence analysis of single cells. We show marked, distinct mtDNA heterogeneity from corresponding aggregate sequences in isolated cells of 5 healthy adult donors—37.9% ± 3.6% heterogeneity in circulating CD34+ cells, 36.4% ± 14.1% in T cells, 36.0% ± 10.7% in B cells, and 47.7% ± 7.4% in granulocytes. Most heterogeneity was caused by poly-C tract variability; however, base substitutions were also prevalent, as follows: 14.7% ± 5.7% in CD34+ cells, 15.2% ± 9.0% in T cells, 15.4% ± 6.7% in B cells, and 32.3% ± 2.4% in granulocytes. Many poly-C tract length differences and specific point mutations seen in these same donors but assayed 2 years earlier were still present in the new CD34+ samples. Additionally, specific poly-C tract differences and point mutations were frequently shared among cells of the lymphoid and myeloid lineages. Secular stability and lineage sharing of mtDNA sequence variability suggest that mutations arise in the lymphohematopoietic stem cell compartment and that these changes may be used as a natural genetic marker to estimate the number of active stem cells.

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Simultaneous Detection of Multiple Point Mutations Using Allele‐Specific Oligonucleotides

Current Protocols in Human Genetics ◽

10.1002/0471142905.hg0904s41 ◽

2004 ◽

Vol 41 (1) ◽

Cited By ~ 3

Author(s):

Nichole M. Napolitano ◽

Elizabeth M. Rohlfs ◽

Ruth A. Heim

Keyword(s):

Simultaneous Detection ◽

Point Mutations ◽

Multiple Point ◽

Allele Specific

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Do Somatic Mitochondrial DNA Mutations Contribute to Parkinson's Disease?

Parkinson s Disease ◽

10.4061/2011/659694 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Joanne Clark ◽

Ying Dai ◽

David K. Simon

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Mitochondrial Dna ◽

Mitochondrial Dysfunction ◽

Point Mutations ◽

Premature Aging ◽

Mtdna Mutations ◽

Dna Mutations ◽

Mitochondrial Dna Polymerase ◽

Mitochondrial Defect

A great deal of evidence supports a role for mitochondrial dysfunction in the pathogenesis of Parkinson's disease (PD), although the origin of the mitochondrial dysfunction in PD remains unclear. Expression of mitochondrial DNA (mtDNA) from PD patients in “cybrid” cell lines recapitulates the mitochondrial defect, implicating a role for mtDNA mutations, but the specific mutations responsible for the mitochondrial dysfunction in PD have been difficult to identify. Somatic mtDNA point mutations and deletions accumulate with age and reach high levels in substantia nigra (SN) neurons. Mutations in mitochondrial DNA polymeraseγ(POLG) that lead to the accumulation of mtDNA mutations are associated with a premature aging phenotype in “mutator” mice, although overt parkinsonism has not been reported in these mice, and with parkinsonism in humans. Together these data support, but do not yet prove, the hypothesis that the accumulation of somatic mtDNA mutations in SN neurons contribute to the pathogenesis of PD.

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Simultaneous Detection of Multiple Point Mutations Using Allele‐Specific Oligonucleotides

Current Protocols in Human Genetics ◽

10.1002/0471142905.hg0904s06 ◽

1995 ◽

Vol 6 (1) ◽

Author(s):

Barbara Handelin ◽

Anthony P. Shuber

Keyword(s):

Simultaneous Detection ◽

Point Mutations ◽

Multiple Point ◽

Allele Specific

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Mitochondrial DNA Mutations Induced by Carbon Ions Radiation: A Preliminary Study

Dose-Response ◽

10.1177/1559325818789842 ◽

2018 ◽

Vol 16 (3) ◽

pp. 155932581878984

Author(s):

Yong Chen ◽

Haining Gao ◽

Wenling Ye

Keyword(s):

Mitochondrial Dna ◽

Nuclear Dna ◽

Ion Irradiation ◽

Direct Sequencing ◽

Point Mutations ◽

Heavy Ion ◽

X Rays ◽

Carbon Ions ◽

Mtdna Mutations ◽

Heavy Ion Irradiation

Heavy-ion irradiation-induced nuclear DNA damage and mutations have been studied comprehensively. However, there is no information about the deleterious effect of heavy-ion irradiation on mitochondrial DNA (mtDNA). In this study, 2 typical mtDNA mutations were examined, including 4977 deletions and D310 point mutations. The 4977 deletions were quantified by real-time polymerase chain reaction, and D310 point mutations were analyzed by direct sequencing and a specific enzyme digestion genotyping method. Results showed that carbon ions radiation can induce temporal fluctuation of mtDNA 4977 deletions in 72 hours after irradiation, while survived clones were free from this deletion. Carbon ions induced more D310 mutations than X-rays, and the single-cell heteroplasmy was eliminated. This is the first study investigating mtDNA mutations induced by carbon ions irradiation in vitro. These findings would provide fundamental information for further investigation of radiation-induced mitochondrial biogenesis.

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PCR-Based Method to Create a Specific Mutation as a Reference for Oligomelting

Clinical Chemistry ◽

10.1093/clinchem/37.12.2137 ◽

1991 ◽

Vol 37 (12) ◽

pp. 2137-2138 ◽

Cited By ~ 1

Author(s):

Vilmundur Gudnason ◽

Steve Humphries

Keyword(s):

Direct Detection ◽

Low Density Lipoprotein ◽

Point Mutations ◽

Density Lipoprotein ◽

Positive Control ◽

Restriction Sites ◽

Density Lipoprotein Receptor ◽

Allele Specific ◽

Polymerase Chain ◽

Artery Disease

Abstract In screening selected populations for known mutations in genes relevant to risk for hyperlipidemia, atherosclerosis, and coronary artery disease, one gene we have examined is the low-density-lipoprotein receptor (LDLR) gene in patients with familial hypercholesterolemia (FH), which has a frequency of 1:500 in most populations(1). More than 30 mutations in this gene have been described, with gross alterations accounting for about 2-6& of defects in the populations investigated (2), leaving the majority to be point mutations. Some mutations create a change of a recognition site for a restriction enzyme, allowing direct detection; the remainder can be detectedby the oligomelting technique with allele-specific oligonucleotides(ASOs) (3). One problem in using ASOs to screen for mutations is the unavailability of a DNA samplefrom a patient with the mutation to use as a positive control in the test. To overcome this problem, we have used a polymerase chain reaction (PCR)-based method to create mutations of interest, using primers containingthe mutated DNA sequence [this method is frequently used to create new restriction sites in PCR fragments via 3' mutated primers (4)].

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Do mitochondrial DNA mutations play the key role in chronification of sterile inflammation? Special focus on atherosclerosis

Current Pharmaceutical Design ◽

10.2174/1381612826666201012164330 ◽

2020 ◽

Vol 26 ◽

Author(s):

Alexander N. Orekhov ◽

Elena V. Gerasimova ◽

Vasily N. Sukhorukov ◽

Anastasia V. Poznyak ◽

Nikita G. Nikiforov

Keyword(s):

Innate Immunity ◽

Mitochondrial Dna ◽

Low Density Lipoprotein ◽

Atherosclerotic Lesion ◽

Density Lipoprotein ◽

Sterile Inflammation ◽

Special Focus ◽

Mitochondrial Dysfunctions ◽

Mtdna Mutations ◽

Dna Mutations

Background: The elucidation of mechanisms implicated in the chronification of inflammation is able to shed the light on the pathogenesis of disorders that are responsible for the majority of the incidence of disease and deaths, and also causes of ageing. Atherosclerosis is an example of the most significant inflammatory pathology. The inflammatory response of innate immunity is implicated in the development of atherosclerosis arising locally or focally. Modified low-density lipoprotein (LDL) was regarded as the trigger for this response. No atherosclerotic changes in the arterial wall occur due to the quick decrease in inflammation rate. Nonetheless, the atherosclerotic lesion formation can be a result of the chronification of local inflammation, which, in turn, is caused by alteration of the response of innate immunity. Objective: In this review, we discussed potential mechanisms of the altered response of the immunity in atherosclerosis with a particular emphasis on mitochondrial dysfunctions. Conclusion: A few mitochondrial dysfunctions can be caused by the mitochondrial DNA (mtDNA) mutations. Moreover, mtDNA mutations were found to affect the development of defective mitophagy. Modern investigations have demonstrated the controlling mitophagy function in the response of the immune system. Therefore, we hypothesized that impaired mitophagy, as a consequence of mutations in mtDNA, can raise a disturbed innate immunity response resulting in the chronification of inflammation in atherosclerosis.

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