Mitochondrial DNA Deletions Research Articles

Prevalence of the 4977-bp and 4408-bp mitochondrial DNA deletions in mesenteric arteries from patients with colorectal cancer

Mitochondrial DNA (mtDNA) deletions are found in many diseased tissues and lead to impairment of mitochondrial functions. In this study, we found wide presence of the common 4977-bp and a novel 4408-bp deletion in the mtDNA of mesenteric arteries from patients with colorectal cancer. These two deletions were also detected in samples from healthy individuals. The content of mtDNA with the 4977-bp deletion was significantly lower in healthy controls than cancer-associated samples, and there was no significant difference for the 4408-bp deletion between the two groups. These results suggest that mtDNA in blood vessels around cancer cells may be strongly affected by oxidative stress and tend to accumulate more large-scale variations.

Undergraduate research describes the effect of large mitochondrial DNA deletions on Caenorhabditis elegans nematode health and fitness

Undergraduate research describes the effect of large mitochondrial DNA deletions on Caenorhabditis elegans nematode health and fitness

Mitochondrial DNA deletions detected by Multiplex Ligation-dependent Probe Amplification

The genetic diagnosis algorithm for mitochondrial (mt) diseases starts looking for deletions and common mutations in mtDNA. MtDNA’s special features, such as large and variable genome copies, heteroplasmy, polymorphisms, and its duplication in the nuclear genome as pseudogenes (NUMTs), make it vulnerable to diagnostic misleading interpretations. Multiplex Ligation-dependent Probe Amplification (MLPA) is used to detect copy number variations in nuclear genes and its application on mtDNA has not been widely spread. We report three Kearns Sayre Syndrome patients and one Chronic Progressive External Ophthalmoplegia adult, whose diagnostic mtDNA deletions were detected by MLPA using a very low amount of DNA. This managed to “dilute” the NUMT interference as well as enhance MLPA’s efficiency. By this report, we conclude that when MLPA is performed upon a reduced amount of DNA, it can detect effectively mtDNA deletions. We propose MLPA as a possible first step method in the diagnosis of mt diseases.

A novel mitochondrial DNA deletion in a patient with Pearson syndrome and neonatal diabetes mellitus provides insight into disease etiology, severity and progression

Pearson syndrome (PS) is a rare, mitochondrial DNA (mtDNA) deletion disorder mainly affecting hematopoietic system and exocrine pancreas in early infancy, which is characterized by multi-organ involvement, variable manifestations and poor prognosis. Since the clinical complexity and uncertain outcome of PS, the ability to early diagnose and anticipate disease progression is of great clinical importance. We described a patient with severe anemia and hyperglycinemia at birth was diagnosed with neonatal diabetes mellitus, and later with PS. Genetic testing revealed that a novel mtDNA deletion existed in various non-invasive tissues from the patient. The disease course was monitored by mtDNA deletion heteroplasmy and mtDNA/nucleus DNA genome ratio in different tissues and at different time points, showing a potential genotype–phenotype correlation. Our findings suggest that for patient suspected for PS, it may be therapeutically important to first perform detailed mtDNA analysis on non-invasive tissues at the initial diagnosis and during disease progression.

Extra-ocular muscle MRI in genetically-defined mitochondrial disease.

ObjectivesConventional and quantitative MRI was performed in patients with chronic progressive external ophthalmoplegia (CPEO), a common manifestation of mitochondrial disease, to characterise MRI findings in the extra-ocular muscles (EOMs) and investigate whether quantitative MRI provides clinically relevant measures of disease.MethodsPatients with CPEO due to single mitochondrial DNA deletions were compared with controls. Range of eye movement (ROEM) measurements, peri-orbital 3 T MRI T1-weighted (T1w) and short-tau-inversion-recovery (STIR) images, and T2 relaxation time maps were obtained. Blinded observers graded muscle atrophy and T1w/STIR hyperintensity. Cross-sectional areas and EOM mean T2s were recorded and correlated with clinical parameters.ResultsNine patients and nine healthy controls were examined. Patients had reduced ROEM (patients 13.3°, controls 49.3°, p < 0.001), greater mean atrophy score and increased T1w hyperintensities. EOM mean cross-sectional area was 43 % of controls and mean T2s were prolonged (patients 75.6 ± 7.0 ms, controls 55.2 ± 4.1 ms, p < 0.001). ROEM correlated negatively with EOM T2 (rho = −0.89, p < 0.01), whilst cross-sectional area failed to correlate with any clinical measures.ConclusionsMRI demonstrates EOM atrophy, characteristic signal changes and prolonged T2 in CPEO. Correlation between elevated EOM T2 and ROEM impairment represents a potential measure of disease severity that warrants further evaluation.Key Points• Chronic progressive external ophthalmoplegia is a common clinical manifestation of mitochondrial disease.• Existing extra-ocular muscle MRI data in CPEO reports variable radiological findings.• MRI confirmed EOM atrophy and characteristic signal changes in CPEO.• EOM T2 was significantly elevated in CPEO and correlated negatively with ocular movements.• EOM T2 represents a potential quantitative measure of disease severity in CPEO.

Mutation analysis of Chinese sporadic congenital sideroblastic anemia by targeted capture sequencing.

BackgroundCongenital sideroblastic anemias (CSAs) comprise a group of heterogenous genetic diseases that are caused by the mutation of various genes involved in heme biosynthesis, iron-sulfur cluster biogenesis, or mitochondrial solute transport or metabolism. However, approximately 40 % of patients with CSA have not been found to have pathogenic gene mutations. In this study, we systematically analyzed the mutation profile in 10 Chinese patients with sporadic CSA.FindingsWe performed targeted deep sequencing analysis in ten patients with CSA using a panel of 417 genes that included known CSA-related genes. Mitochondrial genomes were analyzed using next-generation sequencing with a mitochondria enrichment kit and the HiSeq2000 sequencing platform. The results were confirmed by Sanger sequencing. The ALAS2 mutation was detected in one patient. SLC25A38 mutations were detected in three patients, including three novel mutations. Mitochondrial DNA deletions were detected in two patients. No disease-causing mutations were detected in four patients.ConclusionTo our knowledge, the pyridoxine-effective mutation C471Y of ALAS2, the compound heterozygous mutation W87X, I143Pfs146X, and the homozygous mutation R134C of SLC25A38 were found for the first time. Our findings add to the number of reported cases of this rare disease and to the CSA pathogenic mutation database. Our findings expand the phenotypic profile of mitochondrial DNA deletion mutations. This work also demonstrates the application of a congenital blood disease assay and targeted capture sequencing for the genetic screening analysis and diagnosis of heterogenous genetic CSA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0154-0) contains supplementary material, which is available to authorized users.

Triplex real-time PCR--an improved method to detect a wide spectrum of mitochondrial DNA deletions in single cells.

Mitochondrial DNA (mtDNA) mutations are commonly found in the skeletal muscle of patients with mitochondrial disease, inflammatory myopathies and sarcopenia. The majority of these mutations are mtDNA deletions, which accumulate to high levels in individual muscle fibres causing a respiratory defect. Most mtDNA deletions are major arc deletions with breakpoints located between the origin of light strand (OL) and heavy strand (OH) replication within the major arc. However, under certain disease conditions, rarer, minor arc deletions are detected. Currently, there are few techniques which would allow the detection and quantification of both types of mtDNA deletions in single muscle fibres. We have designed a novel triplex real-time PCR assay which simultaneously amplifies the MT-ND4 gene in the major arc, the MT-ND1 gene in the minor arc, and the non-coding D-Loop region. We demonstrate that this assay is a highly sensitive and reliable tool for the detection and quantification of a broad range of major and minor arc mtDNA deletions with the potential to investigate the molecular pathogenesis in both research and diagnostic settings.

Influence of Antioxidant SkQ1 on Accumulation of Mitochondrial DNA Deletions in the Hippocampus of Senescence-Accelerated OXYS Rats.

Reduction of efficiency of oxidative phosphorylation associated with aging and the development of neurodegenerative diseases including Alzheimer's disease is thought to be linked to the accumulation of deletions in mitochondrial DNA (ΔmtDNA), which are seen as a marker of oxidative damage. Recently, we have shown that mitochondria-targeted antioxidant SkQ1 (10-(6'-plastoquinonyl)decyltriphenylphosphonium) can slow the development of signs of Alzheimer's disease in senescence-accelerated OXYS rats. The purpose of this study was to explore the relationship between the development of neurodegenerative changes in the brain of OXYS rats and changes in the amount of mtDNA and the 4834-bp mitochondrial DNA deletion (ΔmtDNA4834) as well as the effect of SkQ1. We studied the relative amount of mtDNA and ΔmtDNA4834 in the hippocampus of OXYS and Wistar (control) rats at ages of 1, 2, 6, 10, and 20 days and 3, 6, and 24 months. During the period crucial for manifestation of the signs of accelerated aging of OXYS rats (from 1.5 to 3 months of age), we evaluated the effects of administration of SkQ1 (250nmol/kg) and vitamin E (670mmol/kg, reference treatment) on the amount of mtDNA and ΔmtDNA4834 and on the formation of the behavioral feature of accelerated senescence in OXYS rats - passive type of behavior in the open field test. In OXYS rats, the level of ΔmtDNA4834 in the hippocampus is increased compared to the Wistar rats, especially at the stage of completion of brain development in the postnatal period. This level remains elevated not only at the stages preceding the manifestation of the signs of accelerated brain aging and the development of pathological changes linked to Alzheimer's disease, but also during their progression. However, at age of 24 months, there were no detectable differences between the two strains. SkQ1 treatment reduced the level of ΔmtDNA4834 in the hippocampus of Wistar and OXYS rats and slowed the formation of passive behavior in OXYS rats. These results support the possible use of SkQ1 for prophylaxis of brain aging.

OP46 – 2969: Novel phenotypes of childhood encephalomyopathies with mitochondrial DNA depletion or deletions

Objective To study the clinical manifestations and occurrence of mtDNA depletion and deletions in paediatric patients with neuromuscular diseases, in order to estimate the role of mtDNA rearrangements in pathogenesis of these diseases and to identify novel clinical phenotypes associated with mtDNA depletion or deletions. Methods Muscle DNA samples from patients presenting with undefined encephalomyopathies or myopathies were analysed for mtDNA content by quantitative real-time PCR and for deletions by long-range PCR. Direct sequencing of mtDNA maintenance genes and whole-exome sequencing were used to study the genetic aetiologies of diseases. Clinical and laboratory findings were collected. Results Muscle samples were obtained from 104 paediatric patients with neuromuscular diseases. MtDNA depletion was found in three patients with severe early-onset encephalomyopathy or myopathy Two of these patients presented with novel types of mitochondrial DNA depletion syndromes associated with increased serum creatine kinase and multiorgan disease without mutations in any of the known mtDNA maintenance genes; muscle electron micrograph of one patient revealed disordered myofibrillar structure, pathological endoplasmic reticulum membranes and accumulation of glycogen and extracellular collagen fibres. The third patient with mtDNA depletion and severe myopathy was diagnosed with merosine-deficient muscular dystrophy caused by a homozygous mutation in the LAMA2 gene. Two patients with an early-onset Kearns-Sayre/Pearson-like phenotype harboured a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content (4.9 and 6.9-fold increase relative to the median of age-matched controls). Conclusion We describe two novel early-onset phenotypes associated with mtDNA depletion, one of them presenting with pathologic endoplasmic reticulum membranes. Furthermore, mtDNA depletion can be a secondary finding in hereditary muscular dystrophy. We suggest that a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content associated with Kearns-Sayre/Pearson syndromes may be secondary changes caused by mutations in an unknown nuclear gene.

Leydig cell steroidogenesis unexpectedly escapes mitochondrial dysfunction in prematurely aging mice.

Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in tissues during aging in animals and humans and are the basis for mitochondrial diseases. Testosterone synthesis occurs in the mitochondria of Leydig cells. Mitochondrial dysfunction (as induced here experimentally in mtDNA mutator mice that carry a proofreading-deficient form of mtDNA polymerase γ, leading to mitochondrial dysfunction in all cells types so far studied) would therefore be expected to lead to low testosterone levels. Although mtDNA mutator mice showed a dramatic reduction in testicle weight (only 15% remaining) and similar decreases in number of spermatozoa, testosterone levels in mtDNA mutator mice were unexpectedly fully unchanged. Leydig cell did not escape mitochondrial damage (only 20% of complex I and complex IV remaining) and did show high levels of reactive oxygen species (ROS) production (>5-fold increased), and permeabilized cells demonstrated absence of normal mitochondrial function. Nevertheless, within intact cells, mitochondrial membrane potential remained high, and testosterone production was maintained. This implies development of a compensatory mechanism. A rescuing mechanism involving electrons from the pentose phosphate pathway transferred via a 3-fold up-regulated cytochrome b5 to cytochrome c, allowing for mitochondrial energization, is suggested. Thus, the Leydig cells escape mitochondrial dysfunction via a unique rescue pathway. Such a pathway, bypassing respiratory chain dysfunction, may be of relevance with regard to mitochondrial disease therapy and to managing ageing in general.

Effect of propofol on mitochondrial DNA deletion in neurons during brain ischemia-reperfusion in rats

Objective To evaluate the effect of propofol on mitochondrial DNA (mtDNA) deletion in neurons during brain ischemia-reperfusion (I/R) in rats. Methods Fifty-four male Sprague-Dawley rats, aged 3 months, weighing 250-300 g, were randomly divided into 3 groups (n=18 each) using a random number table: sham operation group (S group), I/R group and propofol group (P group). Brain I/R was produced by occlusion of bilateral common carotid arteries combined with hypotension.In group P, propofol was infused for 1 h at 1.0 mg·kg-1·min-1 via the femoral vein before I/R.At 6, 24 and 48 h of reperfusion, 6 rats were sacrificed, and brains were removed for examination of pathologic changes with light microscope.Hippocampi were isolated, and mtDNA was extracted to detect mtDNA deletion using real-time PCR. Results Compared with group S, mtDNA deletion at 6, 24 and 48 h of reperfusion in group I/R, and at 48 h of reperfusion in group P were significantly increased.Compared with group I/R, mtDNA deletion was significantly decreased at 6, 24 and 48 h of reperfusion, and the pathologic changes of brains were mitigated in group P. Conclusion The mechanism by which propofol reduces global brain I/R injury is related to reduction of mtDNA deletion in neurons of rats. Key words: Propofol; Reperfusion injury; Brain; Neurons; DNA, mitochondrial

Reduced adolescent-age spatial learning ability associated with elevated juvenile-age superoxide levels in complex I mouse mutants.

Large-scale, heteroplasmic and generally pathogenic mtDNA defects (as induced by defective mitochondrial DNA polymerase, clonal mutations or DNA deletions) are known to negatively impact on life span and can result in apoptosis and tissue loss in, e.g., skeletal muscle or reduce learning abilities. The functional impact of homoplasmic specific mtDNA point mutations, e.g., in genes coding for the electron transport chain, however, remains a matter of debate. The present study contributes to this discussion and provides evidence that a single point mutation in complex I of the respiratory chain is associated with impairment of spatial navigation in adolescent (6-month-old) mice, i.e., reduced performance in the Morris Water Maze, which goes along with increased production of reactive oxygen species (ROS) in juvenile mice (3 months) but not at the age of phenotype expression. A point mutation in complex III goes along with only a mild and non-significant negative effect on cognitive performance and no significant changes in ROS production. These findings suggest to also consider the ontogenetic development of phenotypes when studying mtDNA mutations and highlights a possible impact of complex I dysfunction on the emergence of neurological deficits.

Measurement and Detection of Mitochondrial DNA Deletions in Irradiated Human Cell Lines

The effect of ionizing radiation such as that of X‐rays on mitochondrial DNA is not well‐understood. We have been using PCR‐based methods to quantify and detect changes in human mtDNA in cell culture. We previously developed a real‐time PCR assay for measuring numbers of mitochondrial DNA copies using either plasmid standards into which mitochondrial DNA had been cloned or isolated human mitochondrial DNA as quantitative standards for mitochondrial DNA genomes. Now we investigate a different real‐time PCR quantification method using single‐copy nuclear genes as quantitative standards for cell number. We also show how x rays affect the amount of total mitochondrial DNA and mitochondrial DNA bearing the 4977‐bp common deletion. In addition, we show how long‐extension PCR (LX‐PCR) can reveal the presence of new deletions in human lymphoblasts treated with X‐ray doses up to 5 Gy.

BMPR2 Preserves Mitochondrial Function and DNA during Reoxygenation to Promote Endothelial Cell Survival and Reverse Pulmonary Hypertension

Mitochondrial dysfunction, inflammation, and mutant bone morphogenetic protein receptor 2 (BMPR2) are associated with pulmonary arterial hypertension (PAH), an incurable disease characterized by pulmonary arterial (PA) endothelial cell (EC) apoptosis, decreased microvessels, and occlusive vascular remodeling. We hypothesized that reduced BMPR2 induces PAEC mitochondrial dysfunction, promoting a pro-inflammatory or pro-apoptotic state. Mice with EC deletion of BMPR2 develop hypoxia-induced pulmonary hypertension that, in contrast to non-transgenic littermates, does not reverse upon reoxygenation and is associated with reduced PA microvessels and lung EC p53, PGC1α and TFAM, regulators of mitochondrial biogenesis, and mitochondrial DNA. Decreasing PAEC BMPR2 by siRNA during reoxygenation represses p53, PGC1α, NRF2, TFAM, mitochondrial membrane potential, and ATP and induces mitochondrial DNA deletion and apoptosis. Reducing PAEC BMPR2 in normoxia increases p53, PGC1α, TFAM, mitochondrial membrane potential, ATP production, and glycolysis, and induces mitochondrial fission and a pro-inflammatory state. These features are recapitulated in PAECs from PAH patients with mutant BMPR2.

Redefining phenotypes associated with mitochondrial DNA single deletion.

Progressive external ophthalmoplegia (PEO), Kearns-Sayre syndrome (KSS) and Pearson syndrome are the three sporadic clinical syndromes classically associated with single large-scale deletions of mitochondrial DNA (mtDNA). PEO plus is a term frequently utilized in the clinical setting to identify patients with PEO and some degree of multisystem involvement, but a precise definition is not available. The purpose of the present study is to better define the clinical phenotypes associated with a single mtDNA deletion, by a retrospective study on a large cohort of 228 patients from the database of the "Nation-wide Italian Collaborative Network of Mitochondrial Diseases". In our database, single deletions account for about a third of all patients with mtDNA-related disease, more than previously recognized. We elaborated new criteria for the definition of PEO and "KSS spectrum" (a category of which classic KSS represents the most severe extreme). The criteria for "KSS spectrum" include the resulting multisystem clinical features associated with the KSS features, and which therefore can predict their presence or subsequent development. With the new criteria, we were able to classify nearly all our single-deletion patients: 64.5% PEO, 31.6% KSS spectrum (including classic KSS 6.6%) and 2.6% Pearson syndrome. The deletion length was greater in KSS spectrum than in PEO, whereas heteroplasmy was inversely related with age at onset. We believe that the new phenotype definitions implemented here may contribute to a more homogeneous patient categorization, which will be useful in future cohort studies of natural history and clinical trials.

Chemerin-induced mitochondrial dysfunction in skeletal muscle.

Chemerin is a novel adipocyte-derived factor that induces insulin resistance in skeletal muscle. However, the effect of chemerin on skeletal muscle mitochondrial function has received little attention. In the present study, we investigated whether mitochondrial dysfunction is involved in the pathogenesis of chemerin-mediated insulin resistance. In this study, we used recombinant adenovirus to express murine chemerin in C57BL/6 mice. The mitochondrial function and structure were evaluated in isolated soleus muscles from mice. The oxidative mechanism of mitochondrial dysfunction in cultured C2C12 myotubes exposed to recombinant chemerin was analysed by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction. The overexpression of chemerin in mice reduced the muscle mitochondrial content and increased mitochondrial autophagy, as determined by the increased conversion of LC3-I to LC3-II and higher expression levels of Beclin1 and autophagy-related protein-5 and 7. The chemerin treatment of C2C12 myotubes increased the generation of mitochondrial reactive oxygen species, concomitant with a reduced mitochondrial membrane potential and increased the occurrence of mitochondrial protein carbonyls and mitochondrial DNA deletions. Knockdown of the expression of chemokine-like receptor 1 or the use of mitochondria-targeting antioxidant Mito-TEMPO restored the mitochondrial dysfunction induced by chemerin. Furthermore, chemerin exposure in C2C12 myotubes not only reduced the insulin-stimulated phosphorylation of protein kinase B (AKT) but also dephosphorylated forkhead box O3α (FoxO3α). Chemerin-induced mitochondrial autophagy likely through an AKT-FoxO3α-dependent signalling pathway. These findings provide direct evidence that chemerin may play an important role in regulating mitochondrial remodelling and function in skeletal muscle.

Postconditioning attenuates renal ischemia-reperfusion injury by mobilization of stem cells.

We recently showed that reactive oxygen species (ROS) and mitochondrial DNA damage and deletions were attenuated by postconditioning (POC). It is not known, however, whether a population of progenitor cells is recruited by POC and is responsible for repair of renal tubular epithelial cells after ischemic injury. The model of renal POC was induced by 45 min clamping of the left renal artery and right nephrectomy followed by 7 min of short-time full reperfusion and 3 cycles of 30 s ischemia and 30 s reperfusion. The lymphocyte compartment of peripheral blood was evaluated by fluorescence-activated cell sorting (FACS) to determine expression of the bone marrow-derived progenitor cell markers CXC-chemokine receptor 4 (CXCR4), c-Kit, and CD34, at 12 h, 1 day and 3 days post-ischemia. Serum and kidney tissue were collected for analysis at 3 and 7 days post-ischemia. Renal functional and structural recovery was markedly improved by POC, which increased the number of CXCR4(+) and CD34(+) stem cells in peripheral blood and kidney tissue. Inhibition of ROS burst by POC was likely associated with increased hypoxia-inducible factor-1 expression, which may further promote stromal cell-derived factor 1 (SDF-1) expression. The mechanisms of stem cell recruitment to the injured foci mobilized by POC appear to be mediated by moderate oxidative stress, which may lead to increased SDF-1 expression.

Common 4977 bp deletion and novel alterations in mitochondrial DNA in Vietnamese patients with breast cancer

Mitochondrial DNA (mtDNA) has been proposed to be involved in carcinogenesis and ageing. The mtDNA 4977 bp deletion is one of the most frequently observed mtDNA mutations in human tissues and may play a role in breast cancer (BC). The aim of this study was to investigate the frequency of mtDNA 4977 bp deletion in BC tissue and its association with clinical factors.We determined the presence of the 4977 bp common deletion in cancer and normal paired tissue samples from 106 Vietnamese patients with BC by sequencing PCR products.The mtDNA 4977 bp deletion was significantly more frequent in normal tissue in comparison with paired cancer tissue. Moreover, the incidence of the 4977 bp deletion in BC tissue was significantly higher in patients with estrogen receptor (ER) positive as compared with ER negative BC tissue. Preliminary results showed, in cancerous tissue, a significantly higher incidence of novel deletions in the group of patients with lymph node metastasis in comparison with the patients with no lymph node metastasis.We have found 4977 bp deletion in mtDNA to be a common event in BC and with special reference to ER positive BC. In addition, the novel deletions were shown to be related to lymph node metastasis. Our finding may provide complementary information in prediction of clinical outcome including metastasis, recurrence and survival of patients with BC.

Mitochondrial DNA Depletion and Deletions in Paediatric Patients with Neuromuscular Diseases: Novel Phenotypes.

To study the clinical manifestations and occurrence of mtDNA depletion and deletions in paediatric patients with neuromuscular diseases and to identify novel clinical phenotypes associated with mtDNA depletion or deletions. Muscle DNA samples from patients presenting with undefined encephalomyopathies or myopathies were analysed for mtDNA content by quantitative real-time PCR and for deletions by long-range PCR. Direct sequencing of mtDNA maintenance genes and whole-exome sequencing were used to study the genetic aetiologies of the diseases. Clinical and laboratory findings were collected. Muscle samples were obtained from 104 paediatric patients with neuromuscular diseases. mtDNA depletion was found in three patients with severe early-onset encephalomyopathy or myopathy. Two of these patients presented with novel types of mitochondrial DNA depletion syndromes associated with increased serum creatine kinase (CK) and multiorgan disease without mutations in any of the known mtDNA maintenance genes; one patient had pathologic endoplasmic reticulum (ER) membranes in muscle. The third patient with mtDNA depletion was diagnosed with merosine-deficient muscular dystrophy caused by a homozygous mutation in the LAMA2 gene. Two patients with an early-onset Kearns-Sayre/Pearson-like phenotype harboured a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content. Novel encephalomyopathic mtDNA depletion syndrome with structural alterations in muscle ER was identified. mtDNA depletion may also refer to secondary mitochondrial changes related to muscular dystrophy. We suggest that a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content associated with Kearns-Sayre/Pearson syndromes may be secondary changes caused by mutations in an unknown nuclear gene.

Effect of Lewy Bodies on Mitochondrial DNA Copy Numbers and Deletion Burden in ParkinsonÂ’s Disease Substantia nigra Neurons

Objective: Our objective was to evaluate mitochondrial DNA (mtDNA) gene copy numbers in substantia nigra neurons from post-mortem Parkinson’s Disease cases and determine if the presence of Lewy bodies (LBs) altered mtDNA copy numbers or changed apparent deletion of mtDNAs. Methods: We used laser capture micro dissection to isolate neuromelanin-containing cells with or without Lewy bodies from 6 Parkinson’s Disease cases. Anti-alpha-synuclein immunohistochemistry was used to identify Lewy bodies in flash frozen tissue. Cells were collected in triplicate groups of 15 cells (LB (+) or LB (-)) from each case, and DNA was extracted with proteinase K-containing buffer. mtDNA copy number was determined by quantitative PCR for 4 genes distributed around the mitochondrial genome (12S rRNA; ND2; CO3; ND4) and normalized to a copy number standard curve of human mtDNA run on the same plate. Results: We found an average ~4-fold higher mtDNA copy numbers in LB (+) neurons compared to LB (-) neurons from the same cases, but we observed substantial heterogeneity. ND4 appeared to be deleted in all cases. ND2 appeared to be deleted in one case as was CO3 in two cases. Conclusion: Our studies indicate that in most cases of synucleinopathy, LB presence does not reduce, and appears to increase, mtDNA copy numbers in PD nigral neuromelanin-containing neurons.