Large-scale mtDNA Deletions Research Articles

Developmental and Pathological Changes in the Human Cardiac Muscle Mitochondrial DNA Organization, Replication and Copy Number

Adult human heart mitochondrial DNA (mtDNA) has recently been shown to have a complex organization with abundant dimeric molecules, branched structures and four-way junctions. In order to understand the physiological significance of the heart-specific mtDNA maintenance mode and to find conditions that modify human heart mtDNA structure and replication, we analyzed healthy human heart of various ages as well as several different heart diseases, including ischemic heart disease, dilated as well as hypertrophic cardiomyopathies, and several mitochondrial disorders. By using one- and two-dimensional agarose gel electrophoresis, various enzymatic treatments and quantitative PCR we found that in human newborns heart mtDNA has a simple organization, lacking junctional forms and dimers. The adult-type branched forms are acquired in the early childhood, correlating with an increase in mtDNA copy number. Mitochondrial disorders involving either mutations in the mtDNA polymerase γ (PolGα) or mtDNA helicase Twinkle, while having no obvious cardiac manifestation, show distinct mtDNA maintenance phenotypes, which are not seen in various types of diseased heart or in mitochondrial disorders caused by point mutations or large-scale deletions of mtDNA. The findings suggest a link between cardiac muscle development, mtDNA copy number, replication mode and topological organization. Additionally, we show that Twinkle might have a direct role in the maintenance of four-way junctions in human heart mtDNA.

The investigation and diagnosis of pathogenic mitochondrial DNA mutations in human urothelial cells

Patients with mitochondrial DNA disease are amongst the most challenging to diagnose and manage given the striking phenotypic and genetic heterogeneity, which characterise these conditions. Recently, we and others have demonstrated the m.3243A>G mutation, one of the most common mitochondrial DNA pathogenic mutations, is present at clinically relevant levels in urinary epithelium, thus providing a practical, non-invasive test for diagnosis and mutation screening. In this study we further evaluate the use of these cells in detecting the m.3243A>G mutation, other mtDNA tRNA gene point mutations including the m.8344A>G mutation and single large-scale mtDNA deletions. We observe a robust relationship between m.3243A>G levels in urothelial cells and clinically affected tissues that does not change with time. Conversely, single large-scale mtDNA deletions can be detected in urothelial cells, with higher levels present in younger patients with more severe disease, but generally mtDNA deletion levels are not representative of those seen in a clinically affected tissue. Our results have implications for the diagnosis, management and counselling of families with mtDNA disease.

Accurate prediction of repeat prostate biopsy outcomes by a mitochondrial DNA deletion assay

Several cancers are characterized by large-scale mtDNA deletions. We previously provided evidence that one of these deletions has potential utility in resolving false from true-negative prostate needle biopsies. This study was to assess the clinical value of this deletion in predicting re-biopsy outcomes. We used a quantitative polymerase chain reaction assay to measure the levels of the deletion in individual negative needle biopsies from 101 patients who had a repeat biopsy within a year with known outcomes. Using an empirically established cycle threshold (Ct) cutoff of 31, and the lowest Ct for each patient as diagnostic of prostate cancer, as well as the histopathologic diagnosis on second biopsy, we calculated the clinical performance of the deletion. The Ct cutoff at 31 gave a sensitivity and specificity of 84 and 54%, respectively, with the area under a receiver-operating characteristics curve of 0.749. The negative predictive value was 91%. The assay was able to predict the presence of a missed tumor in 17 out of 20 men a year before diagnosis. This ancillary test appears to identify men who do not require a repeat biopsy with a high degree of certainty. The results suggest that the majority of men with atypical small acinar proliferation have a concurrent missed tumor and therefore require close monitoring for early detection.

Limited diagnostic value of enzyme analysis in patients with mitochondrial tRNA mutations

We evaluated the diagnostic value of respiratory chain (RC) enzyme analysis of muscle in adult patients with mitochondrial myopathy (MM). RC enzyme activity was measured in muscle biopsies from 39 patients who carry either the 3243A>G mutation, other tRNA point mutations, or single, large-scale deletions of mtDNA. Findings were compared with those obtained from asymptomatic relatives with the 3243A>G mutation, myotonic dystrophy patients, and healthy subjects. Plasma lactate concentration, maximal oxygen uptake, and ragged-red fibers/cytochrome c-negative fibers in muscle were also determined. Only 10% of patients with the 3243A>G point mutation had decreased enzyme activity of one or more RC complexes, whereas this was the case for 83% of patients with other point mutations and 62% of patients with deletions. Abnormal muscle histochemistry was found in 65%, 100%, and 85% of patients, respectively, in these three groups. The results indicate that RC enzyme analysis in muscle is not a sensitive test for MM in adults. In these patients, abnormal muscle histochemistry appears to be a better predictor ofMM.

Suppression of Mitochondrial DNA Instability of Autosomal Dominant Forms of Progressive External Ophthalmoplegia-AssociatedANT1Mutations inPodospora anserina

Maintenance and expression of mitochondrial DNA (mtDNA) are essential for the cell and the organism. In humans, several mutations in the adenine nucleotide translocase gene ANT1 are associated with multiple mtDNA deletions and autosomal dominant forms of progressive external ophthalmoplegia (adPEO). The mechanisms underlying the mtDNA instability are still obscure. A current hypothesis proposes that these pathogenic mutations primarily uncouple the mitochondrial inner membrane, which secondarily causes mtDNA instability. Here we show that the three adPEO-associated mutations equivalent to A114P, L98P, and V289M introduced into the Podospora anserina ANT1 ortholog dominantly cause severe growth defects, decreased reactive oxygen species production (ROS), decreased mitochondrial inner membrane potential (Deltapsi), and accumulation of large-scale mtDNA deletions leading to premature death. Interestingly, we show that, at least for the adPEO-type M106P and A121P mutant alleles, the associated mtDNA instability cannot be attributed only to a reduced membrane potential or to an increased ROS level since it can be suppressed without restoration of the Deltapsi or modification of the ROS production. Suppression of mtDNA instability due to the M106P and A121P mutations was obtained by an allele of the rmp1 gene involved in nucleo-mitochondrial cross- talk and also by an allele of the AS1 gene encoding a cytosolic ribosomal protein. In contrast, the mtDNA instability caused by the S296M mutation was not suppressed by these alleles.

The Relationship between the Aging- and Photo-Dependent T414G Mitochondrial DNA Mutation with Cellular Senescence and Reactive Oxygen Species Production in Cultured Skin Fibroblasts

Mutations in the mitochondrial genome (mtDNA) are thought to be one of the causes of age-dependent cellular decline through their detrimental effects on respiration or reactive oxygen species (ROS) production. However, for many mutations, this link has not been clearly established. This study aimed to further investigate the phenotypic importance of a T414G mutation within the control region of mtDNA, previously shown to accumulate in both chronologically and photoaged human skin. We demonstrate that during dermal skin fibroblast replication in vitro in five separate cultures obtained from elderly individuals, the T414G mutant load can either increase or decrease during progressive cell division, implying the absence of consistent selection against the mutation in this context. In support of this, by utilizing a cell-sorting approach, we demonstrate that the level of the T414G mutation does not directly correlate with increased or decreased mtDNA copy number, or markers of cellular ageing including lipofuscin accumulation or ROS production. By consequence, the mutation can be distributed with a bias towards either the proliferating or senescent cell populations depending on the cell line. In conclusion, we propose that this particular mutation may have little effect on ROS production and the onset of cellular senescence in cultured fibroblasts.

Response To Prolonged Endurance Training and Detraining In Mitochondrial Myopathy

Patients with mitochondrial myopathies (MM) due to mtDNA mutations in skeletal muscle often suffer disabling exercise intolerance. Our previous studies suggest that short duration (14 weeks) endurance training improves exercise capacity and increases the activity of rate-limiting enzymes. The effect of a longer duration exercise training has not been studied. PURPOSE: To assess the effect of 6 months of regular endurance training on exercise capacity and to determine if training adaptations are maintained after a prolonged period of self-selected activity in a patient with severe muscle oxidative impairment due to a mitochondrial myopathy. METHODS: A 26-year male with a heteroplasmic single large-scale deletion of mtDNA underwent 26 weeks of cycle exercise training followed by a 52 week period of self-selected activity level. Peak work, oxygen uptake (VO2), and submaximal heart rate and blood lactate were determined at baseline, after training and following 52 weeks of self-determined activity. RESULTS: Compared to baseline, training increased peak work from 110 to 130 watts and VO2 from 1.527 to 1.837 L/min. During submaximal (50 watt) exercise, peak heart rate decreased from 153 to 120 bpm and venous lactate from 6.41 to 3.25 mM. After 52 weeks of self-selected activity following 6 months of endurance training, peak work and VO2 fell to 110 watts and 1.684 L/min respectively. During submaximal exercise, after the self-selected activity period compared to post training, heart rate (133 vs. 120 bpm) and lactate levels (4.36 vs. 3.25 mM) were higher. CONCLUSION: Endurance training in MM improved work and oxidative capacity supporting the efficacy of this therapy in patients with mitochondrial myopathy due to single large-scale mtDNA deletions. Despite the substantial benefits of exercise training, the patient did not choose to maintain regular exercise training and after the 52 week period of self-selected activity, exercise capacity declined to near pre-training levels indicating that deconditioning contributes to exercise limitations in these patients and that the applicability of exercise training as therapy is limited by patient motivation.

Increased levels of large scale deletions of mtDNA of skin fibroblasts result in increased collagen degradation in dermal skin equivalents

Aged tissues contain increased levels of large-scale deletions of the mitochondrial (mt) DNA. Especially extrinsically aged skin has been shown to carry a significant burden of mtDNA deletions along with several other structural and functional impairments. Until now the functional role of mtDNA mutations for functional and structural changes of aged skin is not understood. Therefore we studied how human skin cells harbouring mtDNA mutations affect their microenvironment by comparing normal human fibroblasts (NHF) to dermal fibroblasts derived from patients suffering from Kearns-Sayre syndrome (KSS) in 3D collagen gels resembling human skin (dermal equivalents, DE). KSS fibroblasts carry a 10,000 fold higher amount of mtDNA deletions exemplified by quantitative measurement of the 4977 bp Common Deletion. We here examined the degradation of components of the extracellular matrix in DE during cultivation. During six weeks we detected more fragmented collagen measured by the marker hydroxyproline (HYP) via GC-MS. Moreover we observed a less robust collagen lattice structure in dermal equivalents with KSS fibroblasts in comparison to NHF by picrosirius red staining of histological sections. In line with that, we could measure higher mRNA expression levels of the dominant collagen degrading enzyme matrixmetalloproteinase-1 (MMP-1) in KSS DE using realtime PCR and increased MMP-1 protein amounts in histological sections over the whole cultivation time. We conclude that increased levels of photo-inducible mtDNA deletions are functionally relevant for intensified matrix degradation and are therefore responsible for detrimental changes in photoaged skin.

Partial Depletion of Mitochondrial DNA from Human Skin Fibroblasts Induces a Gene Expression Profile Reminiscent of Photoaged Skin

In photoaged skin, wrinkles result from an increased degradation and a decreased de novo synthesis of collagen fibers. At the molecular level, photoaged skin is characterized by increased amounts of large-scale deletions of the mitochondrial (mt) genome such as the 4,977 bp common deletion. The common deletion can be generated in dermal fibroblasts through repetitive ultraviolet (UV) A irradiation, and this was found to be associated with an increased expression of the collagen-degrading enzyme matrix metalloproteinase-1 (MMP-1). These studies did not clarify whether increased MMP-1 expression was caused by a disturbance of mtDNA integrity or whether it occurred independently. We have therefore generated a phenocopy of cells bearing large-scale deletions of mtDNA by gradually depleting the mtDNA from unirradiated human skin fibroblasts. Gradual depletion of mtDNA caused a gene expression profile, which was reminiscent of that observed in photoaged skin. Accordingly, in these cells an increased expression of MMP-1 without a concomitant change in tissue inhibitor metalloproteinase-1 as well as a decreased expression of collagen type 1 alpha-1, that is, a gene involved in collagen de novo synthesis, was observed. This altered gene expression resulted from intracellular, mitochondria-derived oxidative stress. Our results support the concept that disruption of mt integrity, for example, by UV-induced mtDNA mutagenesis, is of pathogenetic relevance for photoaging of human skin.

Resistance training in patients with single, large-scale deletions of mitochondrial DNA

Dramatic tissue variation in mitochondrial heteroplasmy has been found to exist in patients with sporadic mitochondrial DNA (mtDNA) mutations. Despite high abundance in mature skeletal muscle, levels of the causative mutation are low or undetectable in satellite cells. The activation of these typically quiescent mitotic cells and subsequent shifting of wild-type mtDNA templates to mature muscle have been proposed as a means of restoring a more normal mitochondrial genotype and function in these patients. Because resistance exercise is known to serve as a stimulus for satellite cell induction within active skeletal muscle, this study sought to assess the therapeutic potential of resistance training in eight patients with single, large-scale mtDNA deletions by assessing: physiological determinants of peak muscle strength and oxidative capacity and muscle biopsy-derived measures of damage, mtDNA mutation load, level of oxidative impairment and satellite cell numbers. Our results show that 12 weeks of progressive overload leg resistance training led to: (i) increased muscle strength; (ii) myofibre damage and regeneration; (iii) increased proportion of neural cell adhesion molecule (NCAM)-positive satellite cells; (iv) improved muscle oxidative capacity. Taken together, we believe these findings support the hypothesis of resistance exercise-induced mitochondrial gene-shifting in muscle containing satellite cells which have low or absent levels of deleted mtDNA. Further investigation is warranted to refine parameters of the exercise training protocol in order to maximize the training effect on mitochondrial genotype and treatment potential for patients with selected, sporadic mutations of mtDNA in skeletal muscle.

A persistent mitochondrial deletion reduces fitness and sperm performance in heteroplasmic populations of C. elegans

BackgroundMitochondrial DNA (mtDNA) mutations are of increasing interest due to their involvement in aging, disease, fertility, and their role in the evolution of the mitochondrial genome. The presence of reactive oxygen species and the near lack of repair mechanisms cause mtDNA to mutate at a faster rate than nuclear DNA, and mtDNA deletions are not uncommon in the tissues of individuals, although germ-line mtDNA is largely lesion-free. Large-scale deletions in mtDNA may disrupt multiple genes, and curiously, some large-scale deletions persist over many generations in a heteroplasmic state. Here we examine the phenotypic effects of one such deletion, uaDf5, in Caenorhabditis elegans (C. elegans). Our study investigates the phenotypic effects of this 3 kbp deletion.ResultsThe proportion of uaDf5 chromosomes in worms was highly heritable, although uaDf5 content varied from worm to worm and within tissues of individual worms. We also found an impact of the uaDf5 deletion on metabolism. The deletion significantly reduced egg laying rate, defecation rate, and lifespan. Examination of sperm bearing the uaDf5 deletion revealed that sperm crawled more slowly, both in vitro and in vivo.ConclusionWorms harboring uaDf5 are at a selective disadvantage compared to worms with wild-type mtDNA. These effects should lead to the rapid extinction of the deleted chromosome, but it persists indefinitely. We discuss both the implications of this phenomenon and the possible causes of a shortened lifespan for uaDf5 mutant worms.

Endurance training and detraining in mitochondrial myopathies due to single large-scale mtDNA deletions

At present there are limited therapeutic interventions for patients with mitochondrial myopathies. Exercise training has been suggested as an approach to improve physical capacity and quality of life but it is uncertain whether it offers a safe and effective treatment for patients with heteroplasmic mitochondrial DNA (mtDNA) mutations. The objectives of this study were to assess the effects of exercise training and detraining in eight patients with single, large-scale mtDNA deletions to determine: (i) the efficacy and safety of endurance training (14 weeks) in this patient population; (ii) to determine the effect of more prolonged (total of 28 weeks) exercise training upon muscle and cardiovascular function and (iii) to evaluate the effect of discontinued training (14 weeks) upon muscle and cardiovascular function. Our results show that: (i) 14 weeks of exercise training significantly improved tolerance of submaximal exercise and peak capacity for work, oxygen utilization and skeletal muscle oxygen extraction with no change in the level of deleted mtDNA; (ii) continued training for an additional 14 weeks maintained these beneficial adaptations; (iii) the cessation of training (detraining) resulted in loss of physiological adaptation to baseline capacity with no overall change in mutation load. Patients' self assessment of quality of life as measured by the SF-36 questionnaire improved with training and declined with detraining. Whilst our findings of beneficial effects of training on physiological outcome and quality of life without increases in the percentage of deleted mtDNA are encouraging, we did not observe changes in mtDNA copy number. Therefore there remains a need for longer term studies to confirm that endurance exercise is a safe and effective treatment for patients with mitochondrial myopathies. The effects of detraining clearly implicate physical inactivity as an important mechanism in reducing exercise capacity and quality of life in patients with mitochondrial myopathy.

Stability and Association with the Cytomatrix of Mitochondrial DNA in Spontaneously Immortalized Mouse Embryo Fibroblasts Containing or Lacking the Intermediate Filament Protein Vimentin

To extend previous observations demonstrating differences in number, morphology, and activity of mitochondria in spontaneously immortalized vim(+) and vim(-) fibroblasts derived from wild-type and vimentin knockout mice, some structural and functional aspects of mitochondrial genome performance and integrity in both types of cells were investigated. Primary Vim(+/+) and Vim(-/-) fibroblasts, which escaped terminal differentiation by immortalization were characterized by an almost twofold lower mtDNA content in comparison to that of their primary precursor cells, whereby the average mtDNA copy number in two clones of vim(+) cells was lower by a factor of 0.6 than that in four clones of vim(-) cells. However, during serial subcultivation up to high passage numbers, the vim(+) and vim() fibroblasts increased their mtDNA copy number 1.5- and 2.5-fold, respectively. While early-passage cells of the vim(+) and vim(-) fibroblast clones differed only slightly in the ratio between mtDNA content and mitochondrial mass represented by mtHSP70 protein, after ca. 300 population doublings the average mtDNA/mtmass ratio in the vim(+) and vim() cells was increased by a factor of 2 and 4.5, respectively. During subcultivation, both types of cells acquired the fully transformed phenotype. These findings suggest that cytoskeletal vimentin filaments exert a strong influence on the mechanisms controlling mtDNA copy number during serial subcultivation of immortalized mouse embryo fibroblasts, and that vimentin deficiency causes a disproportionately enhanced mtDNA content in high-passage vim(-) fibroblasts. Such a role of vimentin filaments was supported by the stronger retention potential for mtDNA and mtDNA polymerase (gamma) detected in vim(+) fibroblasts by Triton X-100 extraction of mitochondria and agaroseembedded cells. Moreover, although the vim(+) and vim(-) fibroblasts were equally active in generating free radicals, the vim(-) cells exhibited higher levels of immunologically detectable 8-oxoG and mismatch repair proteins MSH2 and MLH1 in their mitochondria. Because in vim(-) fibroblasts only one point mutation was detected in the mtDNA D-loop control region, these cells are apparently able to efficiently remove oxidatively damaged nucleobases. On the other hand, a number of large-scale mtDNA deletions were found in high-passage vim(-) fibroblasts, but not in low-passage vim(-) cells and vim(+) cells of both low and high passage. Large mtDNA deletions were also induced in young vim(-) fibroblasts by treatment with the DNA intercalator ethidium bromide, whereas no such deletions were found after treatment of vim(+) cells. These results indicate that in immortalized vim(-) fibroblasts the mitochondrial genome is prone to large-scale rearrangements, probably due to insufficient control of mtDNA repair and recombination processes in the absence of vimentin.

Attenuation of UV‐Induced Apoptosis by Coenzyme Q10 in Human Cells Harboring Large‐Scale Deletion of Mitochondrial DNA

Chronic progressive external ophthalmoplegia (CPEO) syndrome is one of the mitochondrial diseases caused by large-scale deletions in mitochondrial DNA (mtDNA) that impair the respiratory function of mitochondria and result in decreased production of ATP in affected tissues. In order to investigate whether CPEO-associated mtDNA mutations (i.e., 4,366-bp and 4,977-bp large-scale deletions) render human cells more vulnerable to apoptosis, we constructed cybrids carrying the deleted mtDNA. Assays for cell viability, DNA fragmentation, cytochrome c release, and caspase 3 activation revealed that UV irradiation at 20 J/m2 triggered apoptosis in all the cybrids. This treatment also produced elevated intracellular levels of reactive oxygen species (ROS). The rate of UV-induced cell death was more pronounced in the cybrids harboring mtDNA deletions than in the control cybrid with wild-type mtDNA. Subsequently, we evaluated the effect of coenzyme Q10 on the UV-triggered apoptosis. The results showed that after pretreatment of the cybrids with 100 microM coenzyme Q10 the UV-induced cell damage (i.e., ROS production and activation of caspase 3) was significantly reduced. Taken together, these findings suggest that large-scale deletions of mtDNA increased the susceptibility of human cells to the UV-triggered apoptosis and that coenzyme Q10 mitigated the damage; hence, it might potentially serve as a therapeutic agent to treat mitochondrial diseases resulting from mtDNA deletions.

Prevalence of large-scale mitochondrial DNA deletions in an adult Finnish population

Large-scale mitochondrial DNA (mtDNA) deletions are associated with clinical conditions such as Kearns-Sayre syndrome and chronic progressive external ophthalmoplegia in adults and Pearson syndrome in children. Reported case series have suggested that deletions are not uncommon in the population, but their prevalence has not been documented. The authors ascertained patients with clinical features associated with mtDNA deletions in a defined adult population in northern Finland. Buccal epithelial samples were requested from each patient fulfilling the selection criteria, and full-length mtDNA was amplified using the long PCR method. Deletion breakpoints were identified using sequencing. Patients with deletions were examined clinically. The authors identified four patients with single large-scale mtDNA deletions. The prevalence of deletions was calculated to be 1.6/100,000 in the adult population in the province of Northern Ostrobothnia (0.0 to 3.2; 95% CI). Analysis of incident cases from a neighboring province revealed two patients with deletions and yielded a similar population frequency. The frequency of large-scale mitochondrial DNA deletions is similar among populations, suggesting that there is a constant rate of new deletions.

How are Mitochondrial Disorders Inherited?

Mitochondrial disorders are complex diseases that are due to defects in genes encoding for the respiratory chain. These genes are located not only in the maternally-inherited mitochondrial genome (mtDNA), but also in the nuclear genome as the majority of proteins that are important for respiratory chain function are encoded by nuclear DNA and subsequently imported into mitochondria. Thus, in addition to a maternal mode of inheritance, autosomal recessive and dominant traits are apparent while some cases are sporadic. Mitochondrial disorders encompass a wide spectrum of clinical presentations commonly affecting the muscle and nervous systems. One syndrome can be due to different gene defects. For example, a common clinical presentation is progressive external ophthalmoplegia (PEO) that is frequently associated with single, large-scale deletions of mtDNA that are generally sporadic. However, PEO can also be caused by defects in nuclear genes (POLG, C10Orf2, ANT1) that are important in mtDNA maintenance resulting in autosomal inheritance, or by maternally-inherited point mutations of mtDNA such as the 3243A>G mutation that was originally identified in patients with MELAS (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). MERRF syndrome (myoclonic epilepsy with ragged red fibers) is commonly due to the maternally-inherited 8344A>G mutation of mtDNA. The 3243A>G and 8344A>G mutations belong to the group of tRNA mutations that show remarkable phenotypical variability with occurrence of asymptomatic or oligosymptomatic maternal relatives with low levels of mutant DNA (heteroplasmy). Another common mitochondrial disorder is Leber's hereditary optic neuropathy (LHON) that is also due to point mutations of mtDNA. There are three common mutations (11778G>A, 14484T>C, 3460A>G) that are found in 95% of the LHON patients. Families with LHON also show a maternal mode of inheritance but women are rarely affected, thus an X-chromosomal modifier gene has been postulated. Finally, Leigh syndrome (subacute necrotizing encephalopathy) is a frequent presentation of mitochondrial disease in children that is genotypically heterogeneous. Mutations of mtDNA (e.g., 8993G>T/C, 9176T>C, 13513G>A) result in maternally-inherited Leigh syndrome, while many cases harbor mutations in nuclear genes that are inherited recessively, notably in structural subunits of respiratory chain complex I and assembly proteins required for the biosynthesis of complex IV.

Mitochondrial DNA and human thyroid diseases.

Cells of the thyroid tissue, either diseased or normal, can accumulate altered mitochondrial genomes in primary lesions and in surrounding parenchyma. Depending on the experimental approaches and the extent of the mutational process, it has been possible to demonstrate the occurrence of homoplasmic or heteroplasmic point mutations, presence of a common deletion and random large-scale mtDNA aberrations in various pathological states. Point somatic mutations documented in 5-60% of thyroid tumors do not concentrate in obvious hotspots but tend to cluster in certain regions of the mitochondrial genome and their distribution may differ between carcinomas and controls. Large-scale deletions in mtDNA are quite prevalent in healthy and diseased thyroid; however, the proportion of aberrant mtDNA molecules accounts for a very small part of total mtDNA and does not seem to correlate with pathological characteristics of thyroid tumors. Common deletion is most abundant in Hurthle cell tumors, yet it also occurs in other thyroid diseases as well as in normal tissue. The principal difference between the common deletion and other deletion-type mtDNA molecules is that the former does not depend on the relative mtDNA content in the tissue whereas in a subset of thyroid tumors, such as radiation-associated papillary carcinomas and follicular adenomas, there is a strong correlation between mtDNA levels and prevalence of large-scale deletions. Relative mtDNA levels by themselves are elevated in most thyroid tumors compared to normal tissue. Distinct differential distribution and prevalence of mutational mtDNA burden in normal tissue and thyroid lesions are suggestive of the implication of altered mtDNA in thyroid diseases, especially in cancer.

Large-scale mitochondrial DNA deletion mutations and nuclear genome instability in human breast cancer

Deletion mutations in mitochondrial (mt) DNA (mtDNA) as well as microsatellite instability (MSI) and loss of heterozygosity (LOH) in nuclear DNA (nDNA) exist in human cancer. We determined if: (1) large-scale mtDNA deletion mutations were present in cancerous and not in normal breast tissue, and (2) combining mt- and nDNA findings would provide complementary information to identify breast cancer. Thirty-nine matched breast cancer/histologically normal and 23 “true” normal tissue samples from women without breast cancer were microdissected and DNA extracted. 4977, ∼3938, ∼4388 and ∼4576 bp deletions were observed, with the 4576 bp deletion being present in 0% of true normal, 13% of histologically normal specimens from a cancerous breast and 77% of breast cancers. The other three deletions were not specific to a breast containing cancer. LOH was found in 66.7% and MSI in 38.5% of samples. 38/39 (97.4%) tumors had at least one nDNA or 4576 bp mtDNA alteration, suggesting that mt- and nDNA analysis provides complementary information in breast cancer detection. The 4576 bp deletion appears to indicate cancer in the breast. The higher mtDNA copy number in cancer coupled with a mtDNA deletion mutation which appears specific for breasts which contain cancer may prove to be a good target to screen for cancer in the breast, including specimens of low and/or mixed cellularity.

Characteristics of mitochondrial DNA in the peripheral blood cells of residents of Kazakhstan around Semipalatinsk Nuclear Test Site

The study was undertaken to address the question whether there are differences in the molecular features of mitochondrial DNA (mtDNA) extracted from peripheral blood lymphocytes between the individuals possibly exposed to radiation around Semipalatinsk Nuclear Testing Site and those who lived in noncontaminated areas in Kazakhstan. Subjects were residents from three villages considered to be highly contaminated with radioactive fallout. Control individuals were from one settlement in Kazakhstan and a Japanese noncontaminated area. Two parameters, the relative mtDNA content and abundance of large-scale deletions in mtDNA were estimated by real-time PCR and fluorescence-mediated PCR, respectively. The number of large-scale deletions generally decreased with the increase of mtDNA content both in Kazakhstan and in the control Kazakhstan and Japanese individuals. The character of relationship between the number of large-scale deletions and the mtDNA content was similar in any group tested irrespective of the radiation history of a blood donor, and this tendency was preserved between different age groups. We conclude that examination of large-scale mtDNA deletions in nonstimulated peripheral blood lymphocytes is unlikely to be informative as a bioindicator of radiation exposure.

Molecular features of mitochondrial DNA in radiation-associated thyroid tumors

DNA samples from tumor and normal thyroid tissues of adult patients with papillary thyroid carcinoma (PTC) and follicular adenoma (FA), who lived in the Chernobyl radioactive zones of Russia and control groups of Japanese patients with PTC, were quantitatively analyzed for the mitochondrial DNA (mtDNA) content and prevalence of the common deletion (CD) and large-scale deletions (LSD). In most tumor tissues, level of mtDNA was elevated. CD and LSD were detected in every sample examined. The quantity of LSD in mtDNA was elevated in most tumor tissues, especially in the radiation-associated group of samples. Also in this group, in the tumor tissue of both PTC and FA, there was a highly significant positive correlation between the number of LSD and the level of relative mtDNA content. In contrast, in sporadic PTCs, this correlation did not occur. Normal tissue samples displayed a statistically insignificant tendency to negative association with relative mtDNA content. Thus, determination of the LSD levels and mtDNA content may be useful for the molecular distinction of radiation-associated and spontaneous thyroid tumors.