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Accumulation of Somatic Nucleotide Substitutions in Mitochondrial DNA Associated with the 3243 A-to-G tRNALeu(UUR)Mutation in Encephalomyopathy and Cardiomyopathy
Abstract
To understand the pathogenesis of mitochondrial encephalomyopathy and cardiomyopathy, we analyzed the sequence heterogeneity of the skeletal muscle mitochondrial DNA from a patient with Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (MELAS). A mtDNA segment of 347 bp amplified from the total DNA was cloned into a vector. Analysis of 60 independent clones (20,800 bp in total) revealed the 3243 A-->G transition in all the sequenced clones and additional nucleotide substitutions at 5 sites in 10 clones. The frequency of mutant clones (10/60) in the MELAS patient was significantly higher [chi2 = 10.909, P < 0.05] than that in an age-matched skeletal muscle control (0/60) as well as in a normal placenta (2/60). These results support our hypothesis that secondary somatic mtDNA mutations can be initiated by the 3243 A-->G mutation and that the accumulation of somatic mutation in individuals with deleterious inherited mitochondrial genotype can contribute to the progressive mitochondrial dysfunction in MELAS.
... Archetypal clinical symptoms in MD also comprise fatigue, muscle and brain symptoms "Encephalomyopathy" [14][15][16][17][18][19][20] is the classic manifestation of mitochondrial respiratory chain dysfunction. Over 100 distinct mtDNA mutations have been linked to brain and muscle symptoms 21 . ...
... Symptom heterogeity in MD rests on a) random somatic segregation of mitochondrial (mt) DNA mutations 47, 48 and mitochondrial heteroplasmy 49-51 : different organs bear different prior mtDNA mutation loads leading to different vulnerability; b) variable progression of mitochondrial defects: different mutations may arise in different tissues, with some more severe and/or conducive to OS that fosters more mtDNA mutations 19,52 ; and c) clinical threshold effects 21, 53 : symptoms arise when mtDNA mutations or resulting cell death surpass a threshold. The fraction of mutated mtDNA in an organ, the mutation severity, the energetic demands of the organ and the cell loss determine clinical consequences 21, 54 . ...
... Even in a kindred with heritable mtDNA defects that involved neurological problems, "the age of onset of major neurological disturbance varied from 3-70 years" 55 . The principles are similar whether initial mtDNA damage is heritable or acquired; however acquired mtDNA mutations may be typified by multiple different mutations each in a low fraction 19,49 , which may particularly dispose to classical or "nonspecific" symptoms of MD, compatible with CMI. OS may predispose to autoantibodies and vulnerability to infection (below). ...
Background
Overlapping chronic multisymptom illnesses (CMI) include Chronic Fatigue Syndrome (CFS), fibromyalgia, irritable bowel syndrome, multiple chemical sensitivity, and Gulf War illness (GWI), and subsets of autism spectrum disorder (ASD). GWI entails a more circumscribed set of experiences that may provide insights of relevance to overlapping conditions.
Objectives
To consolidate evidence regarding a role for oxidative stress and mitochondrial dysfunction (OSMD), as primary mediators in CMI, using GWI as a departure point.
Methods
Exposure relations, character, timecourse and multiplicity of symptoms, and objective correlates of GWI are compared to expectation for OSMD. Objective correlates of OSMD in GWI and overlapping conditions are examined.
Discussion
OSMD is an expected consequence of known GWI exposures; is compatible with symptom characteristics observed; and accords with objective markers and health conditions linked to GWI, extending to autoimmune disease and infection. Emergent triangulating evidence directly supports OSMD in multisymptom “overlap” CMI conditions, with similarities to, and diagnosed at elevated rates in, GWI, suggesting a common role in each.
Conclusions
GWI is compatible with a paradigm by which uncompensated exposure to oxidative/nitrative stressors accompanies and triggers mitochondrial dysfunction, cell energy compromise, and multiple downstream effects such as vulnerability to autoantibodies. This promotes a profile of protean symptoms with variable latency emphasizing but not confined to energy-demanding post-mitotic tissues, according with (and accounting for) known properties of multisystem overlap conditions. This advances understanding of GWI; health conditions attending GWI at elevated rates; and overlap conditions like CFS and ASD, providing prospects for vulnerability assessment, mitigation of progression, treatment, and future prevention – with implications germane to additive and excessive environmental oxidative stressor exposures in the civilian setting.
... Cardiomyopathy is known to be one of the important complications of several types of mitochondrial disease. [1][2][3][4][5][6][7][8][9][10][11] In patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), cardiomyopathy-especially hypertrophic cardiomyopathy (HCM)-is known to be common. [8][9][10][11] Some patients manifest cardiomyopathy with poor ventricular contraction, and this is thought to be one of the causes of death at the advanced stage of the disease. 1 3 However, neither the clinical condition nor its course has been fully assessed. ...
... [1][2][3][4][5][6][7][8][9][10][11] In patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), cardiomyopathy-especially hypertrophic cardiomyopathy (HCM)-is known to be common. [8][9][10][11] Some patients manifest cardiomyopathy with poor ventricular contraction, and this is thought to be one of the causes of death at the advanced stage of the disease. 1 3 However, neither the clinical condition nor its course has been fully assessed. In this study, we investigated cardiac function in patients with MELAS during subsequent follow up. ...
... Cardiomyopathy is a common cardiac manifestation in MELAS-like conduction abnormalities such as the WolV-Parkinson-White syndrome, 11 and it has been categorised as hypertrophic on the basis of morphological assessment with echocardiography and necropsy examination. [1][2][3][4][5][6][7][8][9][10][11] In idiopathic hypertrophic cardiomyopathy, left ventricular contractility usually remains within the normal range. Sudden cardiac arrhythmia is the major cause of death and only 10-15% of these patients show transition to dilated cardiomyopathy. ...
To investigate cardiac function in patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) and clarify the clinical features of cardiomyopathy in MELAS.
11 consecutive patients with MELAS (mean age at initial examination 11.3 years, range 4 to 16 years) were enrolled in the study. Six were followed for more than five years.
On echocardiographic examination, three patients showed increased left ventricular end diastolic posterior wall thickness (LVPWTd), exceeding 140% of the normal value. Four patients, including these three, had an ejection fraction of less than 50%, and two also had increased left ventricular end diastolic volume (LVEDV) exceeding 140% of the normal value (%N). The LVPWTd%N was correlated positively with the LVEDV%N (R = 0.669, p < 0.05) and negatively with the ejection fraction (R = -0.6701, p < 0.05). One patient died of heart failure aged 22 years.
The cardiomyopathy in MELAS is characterised by an abnormally thick left ventricular wall with progressive dilatation and poor left ventricular contraction developing over several years, indicating hypertrophic cardiomyopathy advancing to dilated cardiomyopathy.
... [5] In particular, the A3243G mutation in the tRNA Leu(UUR) gene causes mitochondrial encephalomyopathy. [6] In the present study, we report a boy diagnosed with mitochondrial encephalomyopathy carried the mitochondrial A3243G heterplastic mutation. We present detailed clinical data and further delineate the phenotype associated with this disease. ...
Rationale:
Mitochondrial mutations are associated with a wide spectrum of clinical abnormalities. More than half of these mutations are distributed in the 22 mitochondrial tRNA genes, including tRNA. In particular, the A3243G mutation in the tRNA gene causes mitochondrial encephalomyopathy.
Patient concerns:
A 12-year-old boy was admitted to Shaoxing People's Hospital because there is a reduction in the volume of speech, dysphonia, unable to write, recognize words, and unable to wear clothes, accompanied by unstable walking after treatment of unexplained fever and somnolence.
Diagnoses:
The proband underwent a thorough examination in our hospital and was diagnosed as mitochondrial encephalomyopathy. The proband carried the pathogenic heteroplasmic mutation A3243G mutation in mitochondrial 12S rRNA gene. Although his parents did not carry the mutation.
Interventions:
Intravenous acyclovir, ceftriaxone, and dexamethasone were used for the patient's antiviral, antimicrobial, and anti-inflammatory therapy, respectively. Intravenous mannitol was gradually tapered for reducing intracranial pressure with furosemide for inducing diuresis. Intravenous arginine could help to treat alkalosis and supple some essential amino acids. Oral oxiracetam capsules, vitamin B1, and coenzyme Q10 were used for providing nutrition and improving energy. His medications were 30 mg vitamin B1, 0.1 g vitamin C, and mecobalamin 750 μg daily after discharge from our hospital.
Outcomes:
The patient was able to walk and talk slowly with improved writing skills and no stroke-like episodes. The neurological examination was negative and muscle tension was identified as grade V.
Lessons:
Mitochondrial encephalomyopathy has different phenotypes, in addition to traditional examinations, it is important for clinicians to be familiar with genetic testing methodology as well as applications of these tests in clinic to get an accurate diagnosis.
... Most frequently reported is an A→G transition at position 3243 (Enter et al., 1991) and a T→C transition at position 3271 (Sakuta et al., 1993). It has been proposed that mutations at position 3243 may result in somatic mutations accumulating in the mitochondrial DNA, leading to progressive mitochondrial dysfunction (Kovalenko et al., 1996). ...
Ischaemic stroke can be caused by a number of monogenic disorders, and in such cases stroke is frequently part of a multisystem disorder. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL), due to mutations in the NOTCH: 3 gene, is increasingly appreciated as a cause of familial subcortical stroke. The genetics and phenotypes of monogenic stroke are covered in this review. However, the majority of cases of ischaemic stroke are multifactorial in aetiology. Strong evidence from epidemiological and animal studies has implicated genetic influences in the pathogenesis of multifactorial ischaemic stroke, but the identification of individual causative mutations remains problematic; this is in part limited by the number of approaches currently available. In addition, genetic influences are likely to be polygenic, and ischaemic stroke itself consists of a number of different phenotypes which may each have different genetic profiles. Almost all human studies to date have employed a candidate gene approach. Associations with polymorphisms in a variety of candidate genes have been investigated, including haemostatic genes, genes controlling homocysteine metabolism, the angiotensin-converting enzyme gene, and the endothelial nitric oxide synthase gene. The results of these studies, and the advantages and limitations of the candidate gene approach, are presented. The recent biological revolution, spurred by the human genome project, promises the advent of novel technologies supported by bioinformatics resources that will transform the study of polygenic disorders such as stroke. Their potential application to polygenic ischaemic stroke is discussed.
... This translates into a few mutants detected per 20 typical sequencing reactions, which is acceptable given low current sequencing costs. Thus, clone-by-clone analysis has been widely used in mitochondrial mutational analysis (Monnat and Loeb, 1985a), (Bodenteich et al., 1991), (Kovalenko et al., 1996), (Jazin et al., 1996), (Michikawa et al., 1999), (Simon et al., 2001), (Lin et al., 2002), (Khaidakov et al., 2003), (Del Bo et al., 2003), (Kamiya and Aoki, 2003). This technique should be applicable to nuclear DNA mutations in hypermutable regions or in cases where some kind of enrichment of mutants is feasible. ...
This chapter advocates the use of single molecule PCR (smPCR) as a tool in mutational analysis. smPCR is compared to the widely used vector-mediated cloning of PCR products. We argue that smPCR is capable of circumventing the problems inherent in post-PCR cloning, namely, the PCR-derived errors, allelic preference, and the template jumping artifact. These arguments are substantiated by examples of smPCR applications: an estimate of the error rate of smPCR, a study of linkage between multiple mutations within a single cell, and a measurement of frequencies of deletions in mitochondrial DNA in an aged human tissue. Background: The problems of clone-by-clone analysis of PCR products.
... The resulting synthesis of premature proteins due to this mis-translation could affect cells considerably, even if there is only a minor amount (usually, 0.02%-0.03%) of the 3243 A>G mutant mtDNA. Of interest, m.3243 A>G itself increases the intracellular production of ROS [56] , which could in turn cause secondary somatic mutations [57] . An increased frequency of somatic transversion mutations in two segments of mtDNA (control region and gene encoding tRNA Leu(UUR) ) was found in diabetic patients compared to sex-and age-matched healthy subjects, and the mutation incidence correlated with hyperglycemia, e.g., level of glycated hemoglobin HbA1c [58] . ...
Mitochondrial DNA (mtDNA) is particularly prone to oxidation due to the lack of histones and a deficient mismatch repair system. This explains an increased mutation rate of mtDNA that results in heteroplasmy, e.g., the coexistence of the mutant and wild-type mtDNA molecules within the same mitochondrion. In diabetes mellitus, glycotoxicity, advanced oxidative stress, collagen cross-linking, and accumulation of lipid peroxides in foam macrophage cells and arterial wall cells may significantly decrease the mutation threshold required for mitochondrial dysfunction, which in turn further contributes to the oxidative damage of the diabetic vascular wall, endothelial dysfunction, and atherosclerosis.
... Most frequently reported is an A→G transition at position 3243 (Enter et al., 1991) and a T→C transition at position 3271 (Sakuta et al., 1993). It has been proposed that mutations at position 3243 may result in somatic mutations accumulating in the mitochondrial DNA, leading to progressive mitochondrial dysfunction (Kovalenko et al., 1996). ...
Matarin M, Singleton A, Hardy J, Meschia J (Laboratory of Neurogenetics, Bethesda, MD, USA; UCL Institute of Neurology, London, UK; Mayo Clinic, Jacksonville, FL, USA). The genetics of ischaemic stroke (Review). J Intern Med 2010; 267: 139–155.
In this review, we discuss the genetic factors in both the aetiology and treatment of ischaemic stroke. We discuss candidate gene association studies, family linkage studies and the more recent whole genome association studies and whole genome expression studies. We also briefly discuss genetic testing for stroke risk and genetic analysis of treatment complications.
... h high lactate levels found within lesions under proton nuclear magnetic resonance (Hassan and Markus, 2000 ). Patients typically have mutations of mitochondrial DNA for the tRNA-leu gene at an A-G transition mutation at nucleotide position 3243 (Ciafoaloni et al., 1992; Macmillan et al., 1993) and at a T-C transition at 3271 (Sakuta et al., 1993). Kovalenko et al. (1996) speculated that as mutations accumulate, a gradual mitochondrial dysfunction develops. It is unclear how widespread such mutations are in the broader stroke population. Indeed, cases of MELAS have been reported without a family history, suggesting that these point mutations may be spontaneous (Rastenyte et al., 1998). Pharmacologic inte ...
Sequencing of the human genome is nearing completion and biologists, molecular biologists, and bioinformatics specialists have teamed up to develop global genomic technologies to help decipher the complex nature of pathophysiologic gene function. This review will focus on differential gene expression in ischemic stroke. It will discuss inheritance in the broader stroke population, how experimental models of spontaneous stroke might be applied to humans to identify chromosomal loci of increased risk and ischemic sensitivity, and also how the gene expression induced by stroke is related to the poststroke processes of brain injury, repair, and recovery. In addition, we discuss and summarise the literature of experimental stroke genomics and compare several approaches of differential gene expression analyzes. These include a comparison of representational difference analysis we have provided using an experimental stroke model that is representative of stroke evolution observed most often in man, and a summary of available data on stroke differential gene expression. Issues regarding validation of potential genes as stroke targets, the verification of message translation to protein products, the relevance of the expression of neuroprotective and neurodestructive genes and their specific timings, and the emerging problems of handling novel genes that may be discovered during differential gene expression analyses will also be addressed.
... Of interest, mtDNA A3243G itself increases the intra-cellular production of ROS [27], which could in turn cause secondary somatic mutations [28]. However, it is not clear if the degree of somatic A3243G seen in our study has a pathological effect or not. ...
A point mutation of mitochondrial DNA at nucleotide number 3243 A to G is responsible for both the major genetic aetiologies of the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and mitochondrial diabetes. Otherwise, this mutation is also reported to occur as an acquired somatic mutation, possibly due to oxidative stress. Since diabetes can cause severe oxidative stress, we hypothesize that the accumulation of the somatic 3243 A to G mutation in mitochondrial DNA can be accelerated by diabetes.
DNA was extracted from blood samples of 290 non-diabetic healthy subjects (age 20-60) including 98 newborn infants and from 383 patients with Type II (non-insulin-dependent) diabetes mellitus (age 18-80). The extent of somatic 3243 A to G mutation to total mitochondrial DNA was detected by real-time PCR using the TaqMan Probe.
Whereas the level of the 3243 A to G mutation was negligible in the newborn group, it was increased in healthy subjects who were 20 to 29 and 41 to 60 years of age, suggesting that this mutation was somatic. In the diabetic patients the mutation rate increased along with age and the duration of diabetes. In the middle-aged group (age 41-60), the 3243 A to G mutation accumulates fourfold higher in the diabetic patients than the healthy subjects. Moreover, multiple regression analysis showed that the most critical factor associated with this mutation in diabetic patients was the duration of diabetes.
Diabetes accelerates the accumulation of the somatic 3243 A to G mutation in mitochondrial DNA, which can accelerate the ageing process. This somatic mutation could possibly be a new marker for estimating the duration of diabetes.
... The clinical variability of many mtDNA mutations could be caused by different genetic backgrounds, mitochondrial or nuclear or both [30]. 'Environmental' factors could interact with genetic factors, amplifying their effect [30][31][32]. ...
The authors report the clinical, neuroimaging, muscle biopsy and mtDNA findings in a patient affected by bilateral hearing loss and mental retardation since infancy, presenting at age 31 years with a rapid deterioration of mental status and ataxia leading to vegetative condition and death at the age of 32 years. Clinical and genetic studies have been also performed in the mother, affected by neurosensorial hearing loss. Muscle biopsy showed severe mitochondrial alterations in the propositus and evidence of mitochondrial alterations in his mother. Direct mtDNA sequencing in all family members revealed the known 7472insC mutation and the recently described A7472C sequence variation in the tRNA(Ser(UCN))gene. RFLP-PCR confirmed the heteroplasmic nature of the two mutations and failed to find the second transversion in 200 controls. The percentage of mutant genomes harbouring 7472insC ranged from 3 to 7% in asymptomatic family members to 70% in the proband and his mother, whereas the percentage of A7472C mutant genomes was about 90% in all maternal relatives except the proband (56%) and his sister (5%). In conclusion, this is the first report of a rapidly progressive encephalopathy in association with the 7472insC mutation in mtDNA, combined with an A>C variation at the same nucleotide with a possible suppression effect on the pathogenic mutation.
... A10398G, C10400T) found in 80% of the controls and othermutations (A8860G, A8701G, A4769G, A1438G, A15326G, C7028T, C12705T) were found in nearly 50% of the control mtDNA sequences. However, a different genetic mitochondrial background can further determine the phenotype or as previously known that environmental factors could interact with genetic factors (mitochondrial or nuclear or both) amplifying their effect [33,34]. In conclusion, complete mitochondrial genome sequencing allowed us to detect both novel and already known variants in children who presented with unexplained encephalopathy and combination of neuromuscular/non-neuromuscular defects with OXPHOS defects. ...
Mitochondrial encephalomyopathies are a heterogeneous group of clinical disorders generally caused due to mutations in either mitochondrial DNA (mtDNA) or nuclear genes encoding oxidative phosphorylation (OXPHOS). We analyzed the mtDNA sequences from a group of 23 pediatric patients with clinical and morphological features of mitochondrial encephalopathies and tried to establish a relationship of identified variants with the disease.
Complete mitochondrial genomes were amplified by PCR and sequenced by automated DNA sequencing. Sequencing data was analyzed by SeqScape software and also confirmed by BLASTn program. Nucleotide sequences were compared with the revised Cambridge reference sequence (CRS) and sequences present in mitochondrial databases. The data obtained shows that a number of known and novel mtDNA variants were associated with the disease. Most of the non-synonymous variants were heteroplasmic (A4136G, A9194G and T11916A) suggesting their possibility of being pathogenic in nature. Some of the missense variants although homoplasmic were showing changes in highly conserved amino acids (T3394C, T3866C, and G9804A) and were previously identified with diseased conditions. Similarly, two other variants found in tRNA genes (G5783A and C8309T) could alter the secondary structure of Cys-tRNA and Lys-tRNA. Most of the variants occurred in single cases; however, a few occurred in more than one case (e.g. G5783A and A10149T).
The mtDNA variants identified in this study could be the possible cause of mitochondrial encephalomyopathies with childhood onset in the patient group. Our study further strengthens the pathogenic score of known variants previously reported as provisionally pathogenic in mitochondrial diseases. The novel variants found in the present study can be potential candidates for further investigations to establish the relationship between their incidence and role in expressing the disease phenotype. This study will be useful in genetic diagnosis and counseling of mitochondrial diseases in India as well as worldwide.
In this survey we present current views on the origin, evolution, maintenance and expression of the separate DNA genome inside mitochondria, that encodes a small subset of genes required for the functions of this organelle. Since the rest of this book is devoted to a description of the involvement of the mitochondrial genetic system (MGS) in human disease, we shall focus principally on human mitochondrial DNA (mtDNA). Notwithstanding, an enormous amount has been learned about mitochondrial function from studies in other organisms. Such model systems illuminate mechanisms that are very likely to operate in similar ways in humans and thereby provide a powerful paradigm to inform future research.
Mitochondrial (mt) DNA encodes important subunits of the mt oxidative phosphorylation system—the central cellular apparatus for bioenergy production. Mutations in mtDNA have been shown to be a crucial causative factor of myocardial dysfunction, hence this is called mt cardiomyopathy (CM).1 The majority of CMs present clinical manifestations of either dilated or hypertrophic CM (DCM or HCM, respectively). Kelly and Strauss2 classified CM into two general categories: (1) disorders of cardiac energy metabolism, which include defects in fatty-acid oxidation and disorders of mt oxidative phosphorylation; and (2) abnormalities of myocardial contractile and structural proteins, which include familial HCM and X-chromosome linked muscular dystrophies. However, based on the molecular genetics and pattern of inheritance, it is more logical to classify CM into the following two groups: (i) familial CM linked with nuclear (n) DNA mutations, which are inherited as autosomal dominant/recessive disorders in 50–56% of HCM,3 and in 20–25% of DCM;4 and (ii) mtCM caused by mtDNA point mutations, which are inherited from an affected mother but not from an affected father (maternal inheritance) or are of sporadic occurrence, and by somatically acquired mtDNA deletions. In a similar way, diabetes mellitus (DM) caused by mtDNA mutations is called mitochondrial DM (mtDM).5 The nucleotide position (np) 3243 A→G point mutation in the tRNALeu(UUR) gene has been reported to be associated with DM in about 1.5% of the diabetic population.5 Often, patients with mtCM harboring the np3243 mutation as well, express signs of insulin-deficient type DM (Fig.12.1, Table 12.1). Clinically, these patients have classified as “diabetic CM”.6
This chapter reviews the principles of mitochondrial genetics, and discusses the evidence for a role for mtDNA mutations in Parkinson's disease (PD). The chapter also explores the possibility that the accumulation of somatic mtDNA mutations may account for the mitochondrial dysfunction in PD. Following this, it presents the mtDNA mutations that have been linked to certain forms of dystonia, with a discussion of the possible role of mtDNA mutations in the more common late-onset forms of focal dystonia. The basal ganglia are a common site of pathology in relatively rare classic mitochondrial disorders such as Leigh syndrome. Mitochondrial dysfunction and mtDNA mutations also may play a role in more common basal ganglia disorders such as PD and dystonia. MtDNA mutations can be associated with parkinsonism in rare patients, but inherited pathogenic mtDNA mutations are absent in the majority of idiopathic PD patients. Further studies are needed to address the possibility that the accumulation of somatic mutations may play a role in PD. mtDNA mutations also can cause rare forms of dystonia, but the role of mitochondrial dysfunction and mtDNA mutations in more common forms of dystonia is uncertain.
The mitochondrial theory of aging proposes that mitochondrial DNA (mtDNA) accumulates mutations with age, and that these mutations contribute to physiological decline in aging and degenerative diseases. Although a great deal of indirect evidence supports this hypothesis, the aggregate burden of mtDNA mutations, particularly point mutations, has not been systematically quantified in aging or neurodegenerative disorders. Therefore, we directly assessed the aggregate burden of brain mtDNA point mutations in 17 subjects with Alzheimer's disease (AD), 10 elderly control subjects and 14 younger control subjects, using a PCR-cloning-sequencing strategy. We found that brain mtDNA from elderly subjects had a higher aggregate burden of mutations than brain mtDNA from younger subjects. The average aggregate mutational burden in elderly subjects was 2 x 10 -4 mutations/bp. The bulk of these mutations were individually rare point mutations, 60% of which changed an amino acid. Control experiments ensure that these results were not due to artifacts arising from PCR error, mistaken identification of nuclear pseudogenes or ex vivo oxidation. Cytochrome oxidase activity correlated negatively with increasing mutational burden. These findings significantly bolster the mitochondrial theory of aging.
Abnormalities in mitochondrial DNA (mtDNA) including specific deletions and point mutations have been found in an increasing number of cases of both dilated and hypertrophic cardiomyopathy. The role that these mutations may play in contributing to the cardiomyopathic phenotype is discussed in this survey of the recent literature.
This chapter describes the clinical features of the mitochondrial encephalomyopathies. Mitochondrial dysfunction is implicated in a rapidly increasing number and variety of disorders. These have been categorized into class I and class II oxidative phosphorylation defects. The former represents the archetypal mitochondrial encephalomyopathies on which this chapter focuses. These result from mutations of mitochondrial DNA (mtDNA) or mutations of nuclear genes that encode subunits of the respiratory chain and oxidative phosphorylation system (OXPHOS). The mitochondrial encephalomyopathies are a diverse group of disorders. Neurologic features may reflect dysfunction of any part of the neuraxis. Their clinical features encompass a broad range of common neurologic symptoms, including dementia, psychiatric disease, developmental delay, stroke, epilepsy, neuropathy, and muscle disease. They are multisystem disorders and may manifest with cardiac, endocrine, gastrointestinal, hepatic, renal, or hematologic involvement. Another key feature of the mitochondrial encephalomyopathies is their marked phenotypic and genotypic diversity. A specific mtDNA defect may cause markedly varied phenotypes in different individuals or even within a single family.
This chapter discusses the causes of the increased vulnerability of mitochondria in neurons to mitochondrial (mt)DNA mutations, the reasons neurons are at special risk from mitochondrial dysfunction, and some of mtDNA mutations in relation to neuropathology. It is suggested that because of the locally high oxidizing environment in the mitochondria, the relative exposure of mtDNA and the poor mitochondrial DNA repair mechanisms, mtDNA is much more susceptible to damage and mutation than nuclear DNA. This damage decreases the efficiency of adenosine triphosphate (ATP) synthesis and increases the production of toxic reactive waste products. Whereas all cells may suffer by these events, neurons are particularly vulnerable, and a number of neurological diseases have mtDNA mutations either as a cause or contributing factor. A number of neurological diseases associated with aging may have mitochondrial dysfunction as a contributing factor to the age of onset and progression of the disease. The most common neuropathies associated with aging are Alzheimer's and Parkinson's diseases, and the role of mitochondrial dysfunction, as well as possible contributions of and correlations with, mtDNA mutations have been studied.
Mitochondrial respiration is gradually uncoupled from oxidative phosphorylation and the activities of respiratory enzymes are decreased in an age-dependent manner. More severe defects in the respiratory chain are usually found in the target tissues of patients with mitochondrial diseases. These impairments affect the efficiency of ATP synthesis and cause enhanced production of reactive oxygen species (ROS) through increased electron leak in the respiratory chain. Mitochondrial DNA (mtDNA), which is not protected by histones or histone-like proteins but which is continually exposed to high steady-state levels of ROS and free radicals in the matrix, is susceptible to oxidative damage and mutation in tissue cells. In the past 8 years, more than 24 large-scale deletions and 2 point mutations of mtDNA have been demonstrated to occur in ageing human tissues. The frequency of occurrence, and the abundance, of mutated and oxidatively modified mtDNAs are generally increased with age. Several pathogenic point mutations and large-scale deletions of mtDNA have been detected in the target tissues of some patients with cardiomyopathies alone or in combination with other disorders. Evidence indicates that idiopathic dilated cardiomyopathy and hypertrophic cardiomyopathy can be caused by point mutation, or deletion, of mtDNA. Some patients with cardiomyopathy carry maternally inherited point mutations of mtDNA. Large-scale deletions of mtDNA are mostly sporadic and are rarely transmitted through maternal lineage. The mutant mtDNAs coexist with the wild-type mtDNA (heteroplasmy) in the cells in a random manner due to replicative segregation. The distribution pattern and degree of heteroplasmy of mutant mtDNA determine the severity and the organs or systems affected. Cardiomyopathy may be manifested as one of the multisystem disorders in some patients with mitochondrial myopathy, mitochondrial encephalomyopathy, diabetes mellitus, deafness or lactic acidosis. The ageing-associated 4,977 by or 7,436 by deletion of mtDNA occurs more frequently and abundantly in the target tissues (for example, skeletal and heart muscle) of patients with cardiomyopathy. These less abundant secondary mtDNA mutations, together with elevated oxidative stress-elicited damage to vital biomolecules, may contribute to the premature ageing and early death of patients with cardiomyopathy.
Rapid progress has been made in the identification of mitochondrial DNA mutations which are typically associated with diseases of the nervous system and muscle. The well established mitochondrial disorders are maternally inherited and males and females are equally affected. An exception is Leber's hereditary optic atrophy (LHON) which is observed much more frequently in males than in females. There are three common point mutations in LHON which can be homoplasmic or heteroplasmic. In mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) most mutations are single base changes and lie within the tRNA-Leu gene. Point mutations in myoclonic epilepsy with ragged red fibres (MERRF) usually occur within the tRNA-Lys gene but mutations of the tRNA-Leu gene are also observed. MELAS and MERRF mutations are heteroplasmic and there is considerable clinical overlap between these diseases. Point mutations within the ATPase6 gene result in either neuropathy, ataxia and retinitis pigmentosa (NARP) or in Leigh's syndrome. The latter occurs if the mutation is present in the majority of mitochondria (extreme heteroplasmy). Finally, mitochondrial DNA deletions are the cause underlying Kearns-Sayre syndrome (KSS). Apart from the well-established mitochondrial diseases, there is increasing evidence that mitochondrial mutations may also play a role in the neurodegenerative disorders Parkinson, Alzheimer and Huntington disease. The complex I defect found in Parkinson disease is especially interesting in this respect. However, no causative mitochondrial mutation has as yet been established in any of these three common disorders.
Mitochondrial genome integrity is an important issue in somatic mitochondrial genetics. Development of quantitative methods is indispensable to somatic mitochondrial genetics as quantitative studies are required to characterize heteroplasmy and mutation processes, as well as their effects on phenotypic developments. Quantitative studies include the identification and measurement of the load of pathogenic and non-pathogenic clonal mutations, screening mitochondrial genomes for mutations in order to determine the mutation spectra and characterize an ongoing mutation process. Single-molecule PCR (smPCR) has been shown to be an effective method that can be applied to all areas of quantitative studies. It has distinct advantages over conventional vector-based cloning techniques avoiding the well-known PCR-related artifacts such as the introduction of artificial mutations, preferential allelic amplifications, and "jumping" PCR. smPCR is a straightforward and robust method, which can be effectively used for molecule-by-molecule mutational analysis, even when mitochondrial whole genome (mtWG) analysis is involved. This chapter describes the key features of the smPCR method and provides three examples of its applications in single-cell analysis: di-plex smPCR for deletion quantification, smPCR cloning for clonal point mutation quantification, and smPCR cloning for whole genome sequencing (mtWGS).
Mitochondrial disorders are caused by mutations in either nuclear or mitochondrial genes involved in the synthesis of respiratory chain subunits or in their post-translational control. Molecular lesions of mitochondrial DNA are a frequent cause of defective oxidative phosphorylation. Although only one mutation of nuclear-encoded oxidative phosphorylation subunits has so far been reported in humans, numerous biochemically defined disorders are attributed to nuclear gene defects. The pathogenesis of these disorders has been investigated through a combination of different expertises, including keen clinical observation, classical biochemistry and muscle morphology, molecular and cellular biology, linkage analysis and population genetic studies.
During the past 16 years since the delineation of the human mitochondrial genome, substantial advances have been made in identifying pathogenic mutations causing mitochondrial disorders. However, just as we have come to accept the unexpected in the nontraditional aspects of Mendelian inheritance with the discovery of trinucleotide expansions, imprinting and uniparental disomy, unusual characteristics of mitochondrial inheritance also have been found that defy existing laws. For example, we now know that the nuclear genetic background of an individual might influence the expression and tissue specificity of mitochondrial mutations. Pathogenic mitochondrial DNA mutations contribute to the generation of new mutations by compromising mitochondrial function and increasing free radical production. Evidence for recombination raises new questions about repair mechanisms of mitochondrial DNA. It appears that the more we learn about the bases of mitochondrial disease, the more complex diagnosis, treatment, and genetic counseling become.
Mitochondrial respiration, the most efficient metabolic pathway devoted to energy production, is at the crosspoint of 2 quite different genetic systems, the nuclear genome and the mitochondrial genome (mitochondrial DNA, mtDNA). The latter encodes a few essential components of the mitochondrial respiratory chain and has unique molecular and genetic properties that account for some of the peculiar features of mitochondrial disorders. However, the perpetuation, propagation, and expression of mtDNA, the majority of the subunits of the respiratory complexes, as well as a number of genes involved in their assembly and turnover, are contained in the nuclear genome. Although mitochondrial disorders have been known for more than 30 years, a major breakthrough in their understanding has come much later, with the discovery of an impressive, ever-increasing number of mutations of mitochondrial DNA. Partial deletions or duplications of mtDNA, or maternally inherited point mutations, have been associated with well-defined clinical syndromes. However, phenotypes transmitted as mendelian traits have also been identified. These include clinical entities defined on the basis of specific biochemical defects, and also a few autosomal dominant or recessive syndromes associated with multiple deletions or tissue-specific depletion of mtDNA. Given the complexity of mitochondrial genetics and biochemistry, the clinical manifestations of mitochondrial disorders are extremely heterogenous. They range from lesions of single tissues or structures, such as the optic nerve in Leber hereditary optic neuropathy or the cochlea in maternally inherited nonsyndromic deafness, to more widespread lesions including myopathies, encephalomyopathies, cardiopathies, or complex multisystem syndromes. The recent advances in genetic studies provide both diagnostic tools and new pathogenetic insights in this rapidly expanding area of human pathology.
Mitochondrial DNA (mtDNA) is essential for the ability of mammalian cells to generate a functional oxidative phosphorylation system. Mutations in mtDNA occur in human disease and also during ageing. Here, we address three questions concerning the occurrence and accumulation of mtDNA mutations during the lifespan of the mammalian cell. What sort of mutations accumulate with age in humans and other mammals? How is the female germ line spared from the accumulation of such mutations as occurs in many somatic tissues, so that neonates normally start life with a 'clean sheet'? Is the occurrence of mtDNA mutations associated with the functional decline of cells and tissues during ageing? We argue that mtDNA mutations in somatic cells do not just reflect a passive imprint of ageing, but they are causally associated with the loss of bioenergetic function during the ageing process.
Apopotic cell death is reported to be prominent in the stable tissues of the failing heart, in cardiomyopathies (CM), in the sinus node of complete heart block, in B cells of diabetes mellitus, and in neurodegenerative diseases. Recently, mitochondrial (mt) control of nuclear apoptosis was demonstrated in the cell-free system. The mt bioenergetic crisis induced by exogenously added factors such as respiratory inhibitors leads to the collapse of mt transmembrane potential, to the opening of the inner membrane pore, to the release of the apoptotic protease activating factors into cytosol, and subsequently to nuclear DNA fragmentation. However, the endogenous factor for the mt bioenegertic crisis in naturally occurring cell death under the physiological conditions without vascular involvement has remained unknown. Recently devised, the total detection system for deletion demonstrates the extreme fragmentation of mtDNA in the cardiac myocytes of senescence, and mt CM harboring maternally inherited point mutations in mtDNA and on the cultured cell line with or without mtDNA disclosed that mtDNA is unexpectedly fragile to hydroxyl radial damage and hence to oxygen stress. The great majority of wild-type mtDNA fragmented into over two hundreds types of deleted mtDNA related to oxidative damage, resulting in pleioplasmic defects in the mt energy transducing system. The mtDNA fragmentation to this level is demonstrated in cardiac myocytes of normal subjects over age 80, of an mtCM patient who died at age 20 and one who died at age 19, of a recipient of heart transplantation at age 7 with severe mtCM, and in mtDNA of a cultured cell line under hyperbaric oxygen stress for two days, leading a majority of cells to apoptotic death on the third day. The extreme fragility of mtDNA could be the missing link in the apoptosis cascade that is the physiological basis of aging and geriatrics of such stable tissues as nerve and muscle.
This article reviews the concept, molecular genetics, and pathology of cell death and aging in relation to mitochondrial genome mutation. Accumulating evidence emphasizes the role of genetic factors in the development of naturally occurring cell death and aging. The ATP required for a cell's biological activity is almost exclusively produced by mitochondria. Each mitochondrion possesses its own DNA (mtDNA) that codes essential subunits of the mitochondrial energy-transducing system. Recent studies confirm that mtDNA is unexpectedly fragile to hydroxyl radical damage, hence to the oxygen stress. Cellular mtDNA easily fragments into over a hundred-types of deleted mtDNA during the life of an individual. Cumulative accumulation of these oxygen damages and deletions in mtDNA results in a defective energy transducing system and in bioenergetic crisis. The crisis leads cells to the collapse of mitochondrial trans-membrane potential, to the release of the apoptotic protease activating factors into cytosol, to uncontrolled cell death, to tissue degeneration and atrophy, and to aging. The total base sequencing of mtDNA among individuals revealed that germ-line point mutations transmitted from ancestors accelerate the somatic oxygen damages and mutations in mtDNA leading to phenotypic expression of premature aging and degenerative diseases. A practical survey of point mutations will be useful for genetic diagnosis in predicting the life-span of an individual.
An 11 year old male presented with headache, vomiting and weakness of right side of body. One day after admission he developed right focal seizures. He had 5 previous episodes of stroke, the first at 11 months age. His milestones were normal upto the first episode but subsequent mile stones were delayed. His serum and CSF lactic acids were raised. Muscle biopsy showed ragged red fibres on modified Gomori-trichrome staining. His EEG, CT scan and MRI were normal this time. The child improved spontaneously after 7 days. His recovery time progressively became shorter with each episode of stroke. Maximum time for recovery was noted during first episode and least in current episode. This is the first report of Melas syndrome in Indian literature.
Mitochondria are not only the major site of ATP production in cells but also an important source of reactive oxygen species (ROS) under certain pathological conditions. Because mitochondrial DNA (mtDNA) in the mitochondrial matrix is exposed to ROS that leak from the respiratory chain, this extranuclear genome is prone to mutations. Therefore, the mitochondrial genome is a rich source of single nucleotide polymorphisms (SNPs) and the functional significance of SNPs in the mitochondrial genome is comparable to that of SNPs in the entire nuclear genome. To demonstrate the contribution of mitochondrial SNPs to the susceptibility to adult-onset diseases, we analyzed the mtDNA from Japanese centenarians and identified a longevity-associated mitochondrial genotype, Mt5178A. Because this genotype was demonstrated to suppress the occurrence of mtDNA mutations in the oocytes, it also would seem to decelerate the accumulation of mtDNA mutations in the somatic cells with increasing age. This genotype is likely to confer resistance to adult-onset diseases by suppressing obesity and atherosclerosis.
Respiratory function of mitochondria is compromised in aging human tissues and severely impaired in the patients with mitochondrial disease. A wide spectrum of mitochondrial DNA (mtDNA) mutations has been established to associate with mitochondrial diseases. Some of these mtDNA mutations also occur in various human tissues in an age-dependent manner. These mtDNA mutations cause defects in the respiratory chain due to impairment of the gene expression and structure of respiratory chain polypeptides that are encoded by the mitochondrial genome. Since defective mitochondria generate more reactive oxygen species (ROS) such as O2- and H2O2 via electron leak, we hypothesized that oxidative stress is a contributory factor for aging and mitochondrial disease. This hypothesis has been supported by the findings that oxidative stress and oxidative damage in tissues and culture cells are increased in elderly subjects and patients with mitochondrial diseases. Another line of supporting evidence is our recent finding that the enzyme activities of Cu,Zn-SOD, catalase and glutathione peroxidase (GPx) decrease with age in skin fibroblasts. By contrast, Mn-SOD activity increases up to 65 years of age and then slightly declines thereafter. On the other hand, we observed that the RNA, protein and activity levels of Mn-SOD are increased two- to three-fold in skin fibroblasts of the patients with CPEO syndrome but are dramatically decreased in patients with MELAS or MERRF syndrome. However, the other antioxidant enzymes did not change in the same manner. The imbalance in the expression of these antioxidant enzymes indicates that the production of ROS is in excess of their removal, which in turn may elicit an elevation of oxidative stress in the fibroblasts. Indeed, it was found that intracellular levels of H2O2 and oxidative damage to DNA and lipids in skin fibroblasts from elderly subjects or patients with mitochondrial diseases are significantly increased as compared to those of age-matched controls. Furthermore, Mn-SOD or GPx-1 gene knockout mice were found to display neurological disorders and enhanced oxidative damage similar to those observed in the patients with mitochondrial disease. These observations are reviewed in this article to support that oxidative stress elicited by defective respiratory function and impaired antioxidant enzyme system plays a key role in the pathophysiology of mitochondrial disease and human aging.
The mitochondrial theory of aging proposes that mitochondrial DNA (mtDNA) accumulates mutations with age, and that these mutations contribute to physiological decline in aging and degenerative diseases. Although a great deal of indirect evidence supports this hypothesis, the aggregate burden of mtDNA mutations, particularly point mutations, has not been systematically quantified in aging or neurodegenerative disorders. Therefore, we directly assessed the aggregate burden of brain mtDNA point mutations in 17 subjects with Alzheimer's disease (AD), 10 elderly control subjects and 14 younger control subjects, using a PCR-cloning-sequencing strategy. We found that brain mtDNA from elderly subjects had a higher aggregate burden of mutations than brain mtDNA from younger subjects. The average aggregate mutational burden in elderly subjects was 2 x 10(-4) mutations/bp. The bulk of these mutations were individually rare point mutations, 60% of which changed an amino acid. Control experiments ensure that these results were not due to artifacts arising from PCR error, mistaken identification of nuclear pseudogenes or ex vivo oxidation. Cytochrome oxidase activity correlated negatively with increasing mutational burden. These findings significantly bolster the mitochondrial theory of aging.
We are currently sequencing the entire mitochondrial genome from 600 persons, including centenarians, patients with Parkinson's disease or diabetes, and young adults with or without obesity to search for single nucleotide polymorphisms (SNPs) associated with longevity or diseases. To test the hypothesis that centenarians are free from deleterious mitochondrial variations, we analyzed amino acid variations in cytochrome b of 64 Japanese centenarians. Although the frequencies of some variations, such as N260D and G251S, differed significantly between centenarians and patients with Parkinson's disease, the most striking feature of centenarian cytochrome b was the much greater scarceness of amino acid variations in contrast with the variety of amino acid replacements in patients with Parkinson's disease. Particular deviations from the standard amino acid sequence may be associated with increased production by mitochondria of reactive oxygen species. The absence of certain variations in centenarians and their presence in patients with Parkinson's disease indicate that these variations do not benefit long-term survival but do predispose to adult-onset diseases and that centenarians are genetically hitting the "golden mean."
Somatic mutations are present in various proportions in numerous developmental pathologies. Somatic activating missense mutations
of the GNAS gene encoding the Gsα protein have previously been shown to be the cause of fibrous dysplasia of bone (FD)/McCune‐Albright syndrome (MAS). Because
in MAS patients, tissues as diverse as melanocytes, gonads and bone are affected, it is generally accepted that the GNAS mutation in this disease must have occurred early in development. Interestingly, it has been shown that the development of
an active FD lesion may require both normal and mutant cells. Studies of the somatic mosaic states of FD/MAS and many other
somatic diseases need an accurate method to determine the ratio of mutant to normal cells in a given tissue. A new method
for quantification of the mutant:normal ratio of cells using a PNA hybridization probe‐based FRET technique was developed.
This novel technique, with a linear sensitivity of 2.5% mutant alleles, was used to detect the percentage mutant cells in
a number of tissue and cell culture samples derived from FD/MAS lesions and could easily be adapted for the quantification
of mutations in a large spectrum of diseases including cancer.
Because mitochondria are the major sources of reactive oxygen species (ROS) in cells, certain alterations in mitochondrial functions can lead to metabolic perturbation in vascular endothelial cells and smooth muscle cells, resulting in vascular dysfunction. We previously demonstrated that a C --> A transversion in mitochondrial DNA (mtDNA) at nucleotide 5178 of the NADH dehydrogenase subunit 2 (ND2) gene, which results in a Lue --> Met substitution at amino acid 237, was found more frequently in Japanese centenarians than in controls. We also demonstrated that this Mt5178C --> A polymorphism has anti-atherosclerotic effects in diabetic subjects. We have now examined whether the Mt5178C --> A (Leu237Met) polymorphism in the mitochondrial ND2 gene is associated with a low prevalence of myocardial infarction (MI) in a case-control study. The genotype of ND2 gene was determined either with a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or a colorimetry-based allele-specific DNA probe assay. Multivariate logistic regression analysis with adjustment for age, gender, body mass index, smoking status, hypertension, diabetes mellitus, hypercholesterolemia, and hyperuricemia revealed that the frequency of the Mt5178A genotype was significantly higher in controls than in subjects with MI. These results suggest that the 5178A genotype of mitochondrial ND2 gene polymorphism is protective against MI; and this effect would explain, at least in part, its contribution to longevity.
The purpose of this study was to identify novel mitochondrial deoxyribonucleic acid (mtDNA) mutations in a series of patients with clinical and/or morphological features of mitochondrial dysfunction, but still no genetic diagnosis. A heterogeneous group of clinical disorders is caused by mutations in mtDNA that damage respiratory chain function of cell energy production. We developed a method to systematically screen the entire mitochondrial genome. The sequence-data were obtained with a rapid automated system. In the six mitochondrial genomes analysed we found 20 variants of the revised Cambridge reference sequence [Nat. Genet. 23 (1999) 147]. In skeletal muscle nineteen novel mtDNA variants were homoplasmic, suggesting secondary pathogenicity or co-responsibility in determination of the disease. In one patient we identified a novel heteroplasmic mtDNA mutation which presumably has a pathogenic role. This screening is therefore useful to extend the mtDNA polymorphism database and should facilitate definition of disease-related mutations in human mtDNA.
A critical review of the clone-by-clone approach to the analysis of complex spectra of somatic mutations is presented. The study of a priori unknown somatic mutations requires painstaking analysis of complex mixtures of multiple mutant and non-mutant DNA molecules. If mutant fractions are sufficiently high, these mixtures can be dissected by the cloning of individual DNA molecules and scanning of the individual clones for mutations (e.g., by sequencing). Currently, the majority of such cloning is performed using PCR fragments. However, post-PCR cloning may result in various PCR artifacts - PCR errors and jumping PCR - and preferential amplification of certain mutations. This review argues that single-molecule PCR is a simple alternative that promises to evade the disadvantages inherent to post-PCR cloning and enhance mutational analysis in the future.
To describe the prevalence of somatic and psychiatric co-morbidity in children diagnosed with ADHD and other behavioural problems compared to this prevalence in children seen at the outpatient department without either of these conditions.
A retrospective controlled case study was conducted in 369 children. All children with ADHD were diagnosed by a clinical psychologist in a hospital setting according to the DSM IV classification. Co-morbidity was determined by pediatricians.
Somatic co-morbidity was seen in 94 % of the children. However, there was no significant difference in the prevalence of somatic co-morbidity in patients with ADHD nor in patients with behavioural problems other than ADHD when compared with the control group. Only two differences slight were observed. In the ADHD group and the group with behavioural problems motor impairment was seen more often and in the control group constipation was diagnosed more frequently.
Except for motor impairment, somatic co-morbidity of any kind does not seem to occur more frequently in children with ADHD.
Aim:
To find out whether simulated bladder voiding was able to induce arousals in sleeping infants.
Methods:
Polygraphic recordings were performed in 34 infants and voiding was simulated by administering water into the diaper.
Results:
Heart rate, respiratory frequency and electroencephalogram frequency did not change significantly during this procedure. Furthermore, simulated voiding was unable to cause an awakening or to induce body movements in sleeping infants.
Conclusion:
Simulated voiding was unable to induce arousals.
Unlabelled:
The phenotypic spectrum of the mitochondrial A3243G DKA mutation is highly variable, particularly when occuring in childhood. In contrast to the classical presentation in adulthood (MELAS syndrome; mitochondria! myopathy, encephalopathy, lactic acidosis and stroke-like episodes) children show a different pattern of symptoms, often without the typical encephalopathy or psychomotor regression. We present six children carrying the A3243G mtDNA mutation with a heteroplasmy above 50 % in muscle tissue. The age of diagnosis ranged from 2 weeks up to 14.5 years. The clinical presentation was rather non-specific including muscle weakness, developmental delay and epilepsy. In this small pediatric group we detected presymptomatic cardiac involvement in five out of six children already at an early stage of disease. The cardiac pathology included cardiomyopathy and biventricular hypertrophy with rhythm disturbances (for example long QT-syndrome). The observed cardiac changes do not always increase the risk of cardiac deterioration; however, two of our patients died early on.
Conclusion:
We hypothesize that the A3243G mutation might be underdiagnosed, as patients could suffer from an unexplained cardiac death before the diagnosis is made. We advise performing regular repeated ECGs and echocardiography in all children carrying a A3243G mtDNA mutation independently from the presence of cardiac symptoms.
Mutations in the control region (D-loop) of mitochondrial DNA (mtDNA) have been described in normal old individuals and it is suggested that they originated from oxidative damage. Respiratory chain defects may lead to increased free radical generation, increased susceptibility to oxidative damage and further increased accumulation of age-related mutations. The objective of this study was to verify whether patients with a mitochondrial disease are more predisposed to accumulate the A189G and T408A mutations in the D-loop and confirm their age-associated nature. We evaluated the presence and levels of heteroplasmy of these two mutations in muscle DNA of 52 individuals with different ages (21 age-matched controls and 31 patients with single or multiple mtDNA deletions). The frequency of both mutations was significantly increased with age, but no differences were observed comparing the group of patients with their age-matched controls. We could not observe correlation of levels of heteroplasmy with age. Our results confirm the age-related nature of the A189G and T408A mutations in the D-loop in controls and patients with mitochondrial disease, but do not suggest that patients are more predisposed to the development of age-related point mutations.
We studied 23 patients with clinically defined mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), 25 oligosymptomatic or asymptomatic maternal relatives, and 50 mitochondrial disease control subjects for the presence of a previously reported heteroplasmic point mutation at nt 3,243 in the transfer RNALeu(UUR) gene of mitochondrial DNA. We found a high concordance between clinical diagnosis of MELAS and transfer RNALeu(UUR) mutation, which was present in 21 of the 23 patients with MELAS, all 11 oligosymptomatic and 12 of 14 asymptomatic relatives, but in only five of 50 patients without MELAS. The proportion of mutant genomes in muscle ranged from 56 to 95% and was significantly higher in the patients with MELAS than in their oligosymptomatic or asymptomatic relatives. In subjects in whom both muscle and blood were studied, the percentage of mutations was significantly lower in blood and was not detected in three of 12 asymptomatic relatives. The activities of complexes I + III, II + III, and IV were decreased in muscle biopsies harboring the mutation, but there was no clear correlation between percentage of mutant mitochondrial DNAs and severity of the biochemical defect.
Transgenic mice with a lambda shuttle vector containing a lacI target gene were generated for use as a short-term, in vivo mutagenesis assay. The gene is recovered from the treated mice by exposing mouse genomic DNA to in vitro packaging extracts and plating the rescued phage on agar plates containing 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal). Phage with mutations in the lacI gene form blue plaques, whereas phage with a nonmutated lacI form colorless plaques. Spontaneous background mutant rates using this system range from 0.6 x 10(-5) to 1.7 x 10(-5), depending upon tissue analyzed, with germ cells exhibiting less than one-third the background rate of somatic tissue. Treatment of the mice with N-ethyl-N-nitrosourea (EtNU), benzo[a]pyrene (B[a]P), or cyclophosphamide caused an induction of mutations over background. Recovery of the lacI target for sequence analysis was performed by genetic excision of a plasmid from the phage using partial filamentous phage origins. The predominant mutations identified from untreated and treated populations were base substitutions. Although it has been shown by others that 70% of all spontaneous mutations within the lacI gene, when replicated in Escherichia coli, occur at a hot spot located at bases 620-632, only 1 of 21 spontaneous mutations has been identified in this region in the transgenic mouse system. In addition, 5 of 9 spontaneous transitions analyzed occur at CpG dinucleotides, whereas no transition mutations were identified at the prokaryotic deamination hot spots occurring at dcm sites (CCA/TGG) within the lacI gene. For EtNU, approximately equal amounts of transitions and transversions were observed, contrasting with B[a]P-induced mutations, in which only transversions were obtained. In addition, B[a]P mutagenesis showed a predominance of mutations (81%) involving cytosines and/or guanines, consistent with its known mode of action. The discovery of a spontaneous mutation spectrum different from that of bacterial assays, coupled with the concordance of EtNU and B[a]P base mutations with the known mechanisms of activity for these mutagens, suggests that this transgenic system will be useful as a short-term, in vivo system for mutagen assessment and analysis of mechanisms leading to mutations.
In a 24-year-old woman with mitochondrial encephalomyopathy presenting hypertrophic cardiomyopathy, microscopical examination of myocardial biopsy specimen disclosed severe vacuolar degeneration of myocardium and aggregates of enlarged mitochondria with proliferated cristae. Limb muscle biopsy specimen showed ''ragged-red fibers'' light microscopically and enlarged abnormal mitochondria with markedly increased cristae ultrastructurally, Mitochondrial DNA analysis by polymerase chain reaction (PCR) revealed an A-to-G transition in the mitochondrial transfer RNA(Leu(UUR)) gene at nucleotide position 3,243 which is reported to be associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), However, the clinical features of this case, presenting mainly cardiac abnormalities, were not consistent with the typical MELAS.
A method is presented for the isolation of highly purified mitochondrial (mt)DNA from a crude DNA extract, making use of the different mobilities of covalently closed circular mtDNA vs. endonuclease-digested nuclear DNA in agarose gels. The preparation is virtually free of any contaminating linear DNA, as judged from its electron microscopic appearance, and can be used for further procedures such as polymerase chain reaction (PCR). Since isolation of mitochondria is not a prerequisite for this method, it can be applied to tissue samples in the mg range. In principle, the method can be applied to every eukaryotic species, provided a molecular hybridization probe is available which permits the position of mtDNA to be located in an agarose gel. This probe can be a cDNA, a DNA fragment generated by PCR, or mtDNA itself, if only the approximate size of the genome is known.
DNA is subject to constant oxidative damage from endogenous oxidants. The oxidized DNA is continuously repaired and the oxidized bases are excreted in the urine. A simple routine analytical procedure is described for urinary 8-hydroxy-2'-deoxyguanosine, an oxidative DNA damage adduct, as an indicator of oxidative damage in humans and rodents. This adduct was purified from human urine and characterized. The described assay employs a series of solid-phase extraction steps that separate 8-hydroxy-2'-deoxyguanosine from other urinary constituents, followed by analysis by gradient reversed-phase HPLC coupled to a dual-electrode high-efficiency electrochemical detection system. Analysis of urine from three species by this method indicates that mice excrete approximately 3.3-fold more 8-hydroxy-2'-deoxyguanosine than humans (582 vs. 178 residues per cell per day), a result that supports the proposal that oxidative damage to DNA increases in proportion to species-specific basal metabolic rates.
A simple technique for rapid isolation of mitochondrial DNA (mtDNA) from animal cells is described. The method is based on the selective alkaline denaturation procedure of Birnboim and Doly [(1979) Nucleic Acids Res. 7, 1513-1523] and avoids the use of CsCl gradient centrifugation. The yield of mtDNA is comparable to that obtained by standard techniques. This DNA is sufficiently pure for restriction analysis and cloning of mtDNA fragments.
Oxidative damage to DNA can be caused by excited oxygen species, which are produced by radiation or are by-products of aerobic metabolism. The oxidized base, 8-hydroxydeoxyguanosine (oh8dG), 1 of approximately 20 known radiation damage products, has been assayed in the DNA of rat liver. oh8dG is present at a level of 1 per 130,000 bases in nuclear DNA and 1 per 8000 bases in mtDNA. Mitochondria treated with various prooxidants have an increased level of oh8dG. The high level of oh8dG in mtDNA may be caused by the immense oxygen metabolism, relatively inefficient DNA repair, and the absence of histones in mitochondria. It may be responsible for the observed high mutation rate of mtDNA.
Isolation and characterization of a novel radiation-induced product, i.e., the 8-hydroxyguanine residue, produced in deoxyribonucleic acid (DNA), 2'-deoxyguanosine, and 2'-deoxyguanosine 5'-monophosphate by gamma-irradiation in aqueous solution, are described. For this purpose, gamma-irradiated DNA was first hydrolyzed with a mixture of four enzymes, i.e., DNase I, spleen and snake venom exonucleases, and alkaline phosphatase. Analysis of the resulting mixture by capillary gas chromatography-mass spectrometry after trimethylsilylation revealed the presence of a product, which was identified as 8-hydroxy-2'-deoxyguanosine on the basis of the typical fragment ions of its trimethylsilyl (Me3Si) derivative. This product was then isolated by using reversed-phase high-performance liquid chromatography. The UV and proton nuclear magnetic resonance spectra taken from the isolated product confirmed the structure suggested by the mass spectrum of its Me3Si derivative. In addition, the accurate molecular mass of the Me3Si derivative of the isolated product was determined by MS. The obtained value agreed with the theoretical molecular mass within 1 millimass unit. The yield of 8-hydroxyguanine was also measured. Its mechanism of formation is believed to involve OH radical addition to the C-8 position of guanine followed by oxidation of the radical adduct.
In a 24-year-old woman with mitochondrial encephalomyopathy presenting hypertrophic cardiomyopathy, microscopical examination of myocardial biopsy specimen disclosed severe vacuolar degeneration of myocardium and aggregates of enlarged mitochondria with proliferated cristae. Limb muscle biopsy specimen showed "ragged-red fibers" light microscopically and enlarged abnormal mitochondria with markedly increased cristae ultrastructurally. Mitochondrial DNA analysis by polymerase chain reaction (PCR) revealed an A-to-G transition in the mitochondrial transfer RNA(Leu)(UUR) gene at nucleotide position 3,243 which is reported to be associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). However, the clinical features of this case, presenting mainly cardiac abnormalities, were not consistent with the typical MELAS.
Replication of mitochondrial DNA is highly asymmetric between the heavy (H) and the light (L) strands. The parental H strand is displaced by the daughter H strand and remains in a single-stranded state until the daughter L strand is synthesized. To examine the effect of this asymmetric replication on mutagenesis, we determined sequences of mtDNAs from 43 human individuals. Occurrence of nucleotide substitutions at 4-fold degenerate sites was distinctly asymmetric between the two strands: G-->A and T-->C transitions were 9- and 1.8-fold more frequent on the L strand than on the H strand, respectively. This nucleotide substitution bias is consistent with the T and G abundance of the H strand as well as the A and C abundance of the L strand.
Mitochondria were prepared from three lymphoblast cell lines from patients with high percentage copy numbers of the human mtDNA 8993 mutation and compared to those prepared from related and non-related control cell lines. Rates of ATP synthesis with pyruvate/malate, succinate/rotenone, ascorbate/N'N'N'N' tetramethyl phenylene diamine were reduced to 67%, 58% and 54% of the control rates, respectively. The backward reaction measured as oligomycin sensitive ATPase was reduced to an average of 42% of that in controls. This mutation which changes a conserved leucine to an arginine in the putative membrane proton channel of mitochondrial ATPase effectively reduces the overall rate of oxidative phosphorylation.