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Methodology for Accurate Detection of Mitochondrial DNA Methylation
Published on: May 20, 2018
Detection of the '4977 bp' mitochondrial DNA deletion in human atherosclerotic lesions
M Bogliolo1, A Izzotti, S De Flora
1Laboratory of Mutagenesis-CSTA, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy.
Abstract:
The presence of the 4977 bp deletion ('common deletion') in the mitochondrial DNA (mtDNA) is associated with defects in the metabolic machinery acquired during ageing as a hallmark of a degenerative phenotype. We analysed 27 samples (18 from surgical patients and nine from autopsy cases) of DNA extracted from smooth muscle cells of abdominal aorta fragments affected by atherosclerotic lesions. The deletion was detected by PCR amplification gel electrophoresis and characterized by sequencing of the PCR product. The mtDNA 'common deletion' was detected in all analysed samples. However, its levels were not particularly high, which may be ascribed to the fact that smooth muscle cells in atherosclerotic lesions have a lower energy requirement and an appreciable proliferation rate, as compared for instance with cardiac myocytes. When the subjects were divided into two numerically equivalent age classes (60-72 years plus a 45-year-old subject versus 73-95 years), the deletion had significantly higher levels in the older subjects. Conversely, its presence did not correlate with source (surgical or autoptic), sex, cigarettes consumption, other clinical and anamnestic parameters or with the levels of adducts and 8-hydroxy-2'-deoxyguanosine measured in the nuclear DNA of the same samples. A previously unreported deletion of 5111 bp was additionally found in the mtDNA from a 45-year-old woman. The origin of this lesion seems to be compatible with the slipped mispairing model proposed for the 'common deletion'.
Insights
The common deletion in mitochondrial DNA (mtDNA) accumulates with age in aortic smooth muscle cells. This age-related increase in mtDNA deletions may contribute to degenerative conditions like atherosclerosis.
Area of Science:
- Molecular Biology
- Genetics
- Gerontology
Background:
- The 4977 bp deletion, or 'common deletion', in mitochondrial DNA (mtDNA) is linked to aging-related metabolic defects and degenerative conditions.
- Atherosclerosis involves changes in vascular smooth muscle cells, where mtDNA integrity may play a role.
Purpose of the Study:
- To investigate the presence and levels of the mtDNA 'common deletion' in atherosclerotic lesions of the abdominal aorta.
- To determine the correlation between mtDNA deletion levels and age, as well as other clinical factors.
Main Methods:
- DNA was extracted from smooth muscle cells of abdominal aorta fragments from 27 individuals.
- The mtDNA 'common deletion' was detected using PCR amplification and gel electrophoresis, followed by sequencing.
- Levels of nuclear DNA adducts and 8-hydroxy-2'-deoxyguanosine were also measured.
Main Results:
- The mtDNA 'common deletion' was detected in all analyzed samples.
- Significantly higher levels of the deletion were found in older subjects (73-95 years) compared to younger subjects (45-72 years).
- No correlation was found between deletion presence and surgical/autoptic origin, sex, smoking, or nuclear DNA damage markers.
Conclusions:
- The mtDNA 'common deletion' accumulates with age in aortic smooth muscle cells, potentially contributing to atherosclerosis.
- The lower levels in smooth muscle cells compared to other cell types may relate to their metabolic and proliferative characteristics.
- A novel 5111 bp deletion was identified, suggesting potential shared mechanisms with the common deletion.
