Effects of chronological age on mitochondrial DNA (mtDNA) deletion in muscle and blood of older individuals : a maternal line study

Since Harman ' fîrst proposed that the mitochondria plays a role in the aging process, several lines of research have been undertaken to support his theory. According to the theory, oxygen free radicals are responsible for the age-associated decline in function at the cellular, tissue and organ levels. The mitochondria use oxygen at a high rate, but due to a dysfunction in its usage of oxygen the mitochondria releases oxygen free radicals, which exceeds the cellular antioxidant defense. The mitochondrial genome, however, is extremely susceptible to oxidative damage and DNA mutations, including deletions when compared to nuclear DNA. One of the causative agents of this deletion(s) includes oxygen free radicals, by-product of ATP (energy) production. The most frequent deletion associated with aging and diseases is known as “the common deletion” or 4977 base pair (bp) deletion. In recent years a number of studies have been able to detect mitochondrial DNA (mtDNA) deletions in various tissues from individuals over 20 years of age. However, these studies were all cross-sectional in nature and the DNA sources were all post-mitotic tissues such as muscle and brain. Mitotic tissues, such as blood were not used even though acquiring blood is usually less invasive and would be more convenient to study the aging process. Also, since aging occurs over time a longitudinal study design would be the most appropriate model for the detection and quantification of mtDNA deletion(s). It has been well documented that identical mtDNA is transmitted from mother to child at the point of conception, and therefore monitoring the incidence of mtDNA deletion(s) by employing a maternal line across several generations would mimic a longitudinal study model. Therefore, the first purpose of this study is to mimic the benefits of a longitudinal study design by employing maternal lines (3 & 4 generations) to establish mtDNA deletion(s) as a sensitive, specific and stable biomarker for studying the aging process. The second purpose of this study is to determine the differences in mtDNA deletions when comparing muscle and blood. There were 75 healthy participants with age ranging from 8 months to 99 years. The participants provided 71 blood samples and 27 muscle biopsies. --from abstract

The pseudo-mitochondrial genome influences mistakes in heteroplasmy interpretation

BACKGROUND: Nuclear mitochondrial pseudogenes (numts) are a potential source of contamination during mitochondrial DNA PCR amplification. This possibility warrants careful experimental design and cautious interpretation of heteroplasmic results. RESULTS: Here we report the cloning and sequencing of numts loci, amplified from human tissue and rho-zero (ρ(0)) cells (control) with primers known to amplify the mitochondrial genome. This paper is the first to fully sequence 46 paralogous nuclear DNA fragments that represent the entire mitochondrial genome. This is a surprisingly small number due primarily to the primer sets used in this study, because prior to this, BLAST searches have suggested that nuclear DNA harbors between 400 to 1,500 paralogous mitochondrial DNA fragments. Our results indicate that multiple numts were amplified simultaneously with the mitochondrial genome and increased the load of pseudogene signal in PCR reactions. Further, the entire mitochondrial genome was represented by multiple copies of paralogous nuclear sequences. CONCLUSION: These findings suggest that mitochondrial genome disease-associated biomarkers must be rigorously authenticated to preclude any affiliation with paralogous nuclear pseudogenes. Importantly, the common perception that mitochondrial template "swamps" numts loci precluding detectable amplification, depends on the region of the mitochondrial genome targeted by the PCR reaction and the number of pseudogene loci that may co-amplify. Cloning and relevant sequencing data will facilitate the correct interpretation. This is the first complete, wet-lab characterization of numts that represent the entire mitochondrial genome

Facile whole mitochondrial genome resequencing from nipple aspirate fluid using MitoChip v2.0

<p>Abstract</p> <p>Background</p> <p>Mutations in the mitochondrial genome (mtgenome) have been associated with many disorders, including breast cancer. Nipple aspirate fluid (NAF) from symptomatic women could potentially serve as a minimally invasive sample for breast cancer screening by detecting somatic mutations in this biofluid. This study is aimed at 1) demonstrating the feasibility of NAF recovery from symptomatic women, 2) examining the feasibility of sequencing the entire mitochondrial genome from NAF samples, 3) cross validation of the Human mitochondrial resequencing array 2.0 (MCv2), and 4) assessing the somatic mtDNA mutation rate in benign breast diseases as a potential tool for monitoring early somatic mutations associated with breast cancer.</p> <p>Methods</p> <p>NAF and blood were obtained from women with symptomatic benign breast conditions, and we successfully assessed the mutation load in the entire mitochondrial genome of 19 of these women. DNA extracts from NAF were sequenced using the mitochondrial resequencing array MCv2 and by capillary electrophoresis (CE) methods as a quality comparison. Sequencing was performed independently at two institutions and the results compared. The germline mtDNA sequence determined using DNA isolated from the patient's blood (control) was compared to the mutations present in cellular mtDNA recovered from patient's NAF.</p> <p>Results</p> <p>From the cohort of 28 women recruited for this study, NAF was successfully recovered from 23 participants (82%). Twenty two (96%) of the women produced fluids from both breasts. Twenty NAF samples and corresponding blood were chosen for this study. Except for one NAF sample, the whole mtgenome was successfully amplified using a single primer pair, or three pairs of overlapping primers. Comparison of MCv2 data from the two institutions demonstrates 99.200% concordance. Moreover, MCv2 data was 99.999% identical to CE sequencing, indicating that MCv2 is a reliable method to rapidly sequence the entire mtgenome. Four NAF samples contained somatic mutations.</p> <p>Conclusion</p> <p>We have demonstrated that NAF is a suitable material for mtDNA sequence analysis using the rapid and reliable MCv2. Somatic mtDNA mutations present in NAF of women with benign breast diseases could potentially be used as risk factors for progression to breast cancer, but this will require a much larger study with clinical follow up.</p

Somatic Mitochondrial DNA Mutations in Prostate Cancer and Normal Appearing Adjacent Glands in Comparison to Age-Matched Prostate Samples without Malignant Histology

Studies of somatic mitochondrial DNA mutations have become an important aspect of cancer research because these mutations might have functional significance and/or serve as a biosensor for tumor detection. Here we report somatic mitochondrial DNA mutations from three specific tissue types (tumor, adjacent benign, and distant benign) recovered from 24 prostatectomy samples. Needle biopsy tissue from 12 individuals referred for prostate biopsy, yet histologically benign (symptomatic benign), were used as among individual control samples. We also sampled blood (germplasm tissue) from each patient to serve as within individual controls relative to the somatic tissues sampled (malignant, adjacent, and distant benign). Complete mitochondrial genome sequencing was attempted on each sample. In contrast to both control groups [within patient (blood) and among patient (symptomatic benign)], all of the tissue types recovered from the malignant group harbored significantly different mitochondrial DNA (mtDNA) mutations. We conclude that mitochondrial genome mutations are an early indicator of malignant transformation in prostate tissue. These muta-tions occur well before changes in tissue histo-pathology, indicative of prostate cancer, are evident to the pathologist