Mitochondrial DNA Abnormalities Research Articles

Associations between cigarette smoking and mitochondrial DNA abnormalities in buccal cells

DNA alterations in mitochondria are believed to play a role in carcinogenesis and are found in smoking-related cancers. We sought to replicate earlier findings for the association of smoking with increased mitochondrial DNA (mtDNA) content in buccal cells and further hypothesized that there would be an increased number of somatic mtDNA mutations in smokers. Buccal cells and blood lymphocytes were studied from 42 healthy smokers and 30 non-smokers. Temporal temperature gradient electrophoresis screening and sequencing was used to identify mtDNA mutations. The relative mtDNA content was determined by real-time polymerase chain reaction. Assuming that mtDNA in lymphocytes represents the inherited sequence, it was found that 31% of smokers harbored at least one somatic mtDNA mutation in buccal cells with a total of 39 point mutations and 8 short deletions/insertions. In contrast, only 23% of non-smokers possessed mutations with a total of 10 point mutations and no insertions/deletions detected. mtDNA somatic mutation density was higher in smokers (0.68/10 000 bp per person) than in non-smokers (0.2/10 000 bp per person). There was a statistically significant difference in the pattern of homoplasmy and heteroplasmy mutation changes between smokers and non-smokers. Whereas non-smokers had the most mutations in D-loop region (70%), smokers had mutations in both messenger RNA encoding gene (36%) and D-loop region (49%). The mean ratio of buccal cells to lymphocytes of mtDNA content in smokers was increased (2.81) when compared with non-smokers (0.46). These results indicate that cigarette smoke exposure affects mtDNA in buccal cells of smokers. Additional studies are needed to determine if mitochondrial mutation assays provide new or complementary information for estimating cigarette smoke exposure at the cellular level or as a cancer risk biomarker.

Mitochondrial DNA abnormalities in NAION

We reported that a group of 19 patients with non-arteritic ischaemic optic neuropathy (NAION) had an increased number of mitochondrial (mt) DNA nucleotide changes1 and significantly increased relative mtDNA content...

Multiple mitochondrial DNA deletions in monozygotic twins with OPMD

Background:Oculopharyngeal muscular dystrophy (OPMD) is caused by expansions of the poly (A) binding protein 2 (PABP2) gene. Previous histological analyses have revealed mitochondrial abnormalities in the muscles of OPMD patients...

Activities of mitochondrial complexes correlate with nNOS amount in muscle from ALS patients

The pathogenesis of amyotrophic lateral sclerosis (ALS) is poorly understood. Increased levels of free radicals derived from nitric oxide (NO), the product of nitric oxide synthase (NOS), may damage mitochondrial function leading to motor neurone death. Previous studies demonstrated a specific impairment of mitochondrial function in skeletal muscle of ALS patients. In order to verify a pathogenetic relationship between neuronal NOS (nNOS) and mitochondrial function, we studied nNOS expression by Western blot and mitochondrial enzyme activity by spectrophotometric assays in muscle biopsies of 16 sporadic ALS patients and 16 controls subjects. We observed a reduced activity of respiratory chain complexes with mitochondrial encoded subunits and a lower nNOS amount in ALS muscles. There was a direct correlation between levels of nNOS and values of mitochondrial enzymes function. In ALS muscles we found normal levels of manganese superoxide dismutase (SOD2) that is assumed as related to mitochondrial DNA abnormalities. Our data suggest a beneficial role for NO to mitochondrial function and lead to the hypothesis of a common oxidative damage in motor neurones and skeletal muscle in sporadic ALS patients.

Mitochondrial DNA and disease: What happens when things go wrong

Mitochondria are ubiquitous in eukaryotic cells and one of their important functions is to provide ATP via oxidative phosphorylation (OXPHOS). The mitochondria also host other biochemical pathways, including -oxidation, Krebs' citric acid cycle and parts of the urea cycle. Thus, the mitochondria play a pivotal role in cellular biochemistry. The relationship of mitochondria to human disease has been identified only recently, but has now become one of the most rapidly expanding areas of human pathology. Mitochondrial disorders may be a consequence of inherited defects of either the nuclear or mitochondrial genomes or, alternatively, may be due to endogenous or exogenous environmental toxins. This article will focus upon abnormalities of mitochondrial DNA (mtDNA) and human disease.

Mitochondrial D-loop mutations and deletion profiles of cancerous and noncancerous liver tissue in hepatitis B virus-infected liver

The largest single underlying cause of hepatocellular carcinoma (HCC) worldwide is hepatitis B virus (HBV) infection. Hepatitis B virus increases cellular oxidative stress and the development of HCC occurs after a long latency period. The study was carried out to determine whether mitochondrial DNA abnormalities were associated with HCC in individuals with HBV. The frequency of mutation and deletion of specific areas of the mitochondrial genome in tumour and matched normal tissue of patients with HBV infection was investigated in the current study. The percentage of control subjects harbouring D-loop mutations was 11%, which was significantly lower than that observed in both the noncancerous (49%, P=0.033) and tumour tissue (59%, P=0.014) of patients with HCC. In contrast, the number of cases in which the common 4977 bp deletion of the mitochondrial genome was detected was significantly greater in control liver and noncancerous liver tissue of subjects with HCC (100 and 95%, respectively) than in cancerous liver tissue (28%, P<0.001). These observations suggest that the inflammatory process contributes to the rate of mitochondrial mutations. However, the lower frequency of the large deletion in cancerous tissue suggests that there is selection against either mitochondria, which harbour large deletions, or against cells that contain these mitochondria during hepatocarcinogenesis.

Mitochondrial DNA (mtDNA) Sequence Heterogeneity among and within Single Human CD34 Cells, T Cells, B Cells and Granulocytes.

Abnormalities of mitochondrial DNA (mtDNA) are responsible for a variety of inherited syndromes and have been broadly implicated in aging, cancer, and autoimmunity diseases. Mutations in mtDNA have been reported in myelodysplasia and leukemia, although their pathogenic mechanism remains uncertain. We have described age-dependent accumulation of mtDNA mutations, leading to a high degree of mtDNA sequence heterogeneity among normal marrow and blood CD34 cells as well as in granulocytes (Shin M et al, Blood 101:3118[ 2003], 103:553 [2004], 103:4466 [2004]). In order to examine mtDNA heterogeneity in detail, we developed a method for analysis of the mtDNA control region from single cells that were sorted by flow cytometry. Highly purified populations of CD34 cells, T cells, B cells, and granulocytes were obtained from five healthy adult donors. The sequence of the individual cells' mtDNA was compared to the aggregate mtDNA for the respective cell type and differences were expressed as a measure of mtDNA heterogeneity among cells. Overall, heterogeneity was high: for circulating CD34 cells, 38±3.4%; for T cells, 37±14%; B cells, 36±10.8%; and for granulocytes, 48±7.2% (the value for granulocytes statistically differed from CD34 cells; p = 0.03). Most intercellular heterogeneity was due to polyC tract length variability; however, mtDNA base substitution mutations were also prevalent: 15±5.5% in CD34 cells; 15±9.0% in T cells, 15±6.7% in B cells; and 33±2.4% for granulocytes (granulocytes were significantly higher than other cells; p < 0.01). The higher rate of base substitution in granulocytes may reflect their greater exposure to reactive oxygen species. Surprisingly, for both polyC tract length differences and point mutations, the specific mtDNA abnormalities and the proportion of circulating cells characterized by these changes were similar among different cell lineages and relatively constant over time (~2 years) in the same donors. One inference from these results is that mtDNA heterogeneity during development is fixed in the primitive lymphohematopoietic stem cell compartment. In contrast to normal adults, circulating CD34 cells from patients obtained even years after successful allogeneic stem cell transplantation showed a remarkable level of mtDNA homogeneity, similar to the uniformity we have previously observed in cord blood CD34 cells and consistent with limited numbers of stem cells active in these individuals. Leukemic blast cells (from patients with AML-M2, AML evolving from CMML, and T-PLL) also showed a high degree of homogeneity. We propose that mtDNA sequence of single cells may be utilized as a natural genetic marker of hematopoietic progenitors and stem cells; to detect minimal residual disease in leukemia; and as a measure of the accumulation of mutagenic events in mammalian cells in vivo and in vitro.

Clinical spectrum, morbidity, and mortality in 113 pediatric patients with mitochondrial disease.

The aim of this study was to elucidate the frequency of major clinical manifestations in children with mitochondrial disease and establish their clinical course, prognosis, and rates of survival depending on their clinical features. We performed a retrospective review of the medical records of 400 patients who were referred for evaluation of mitochondrial disease. By use of the modified Walker criteria, only patients who were assigned a definite diagnosis were included in the study. A total of 113 pediatric patients with mitochondrial disease were identified. A total of 102 (90%) patients underwent a muscle biopsy as part of the diagnostic workup. A significant respiratory chain (RC) defect, according to the diagnostic criteria, was found in 71% of the patients who were evaluated. In this cohort, complex I deficiency (32%) and combined complex I, III, and IV deficiencies (26%) were the most common causes of RC defects, followed by complex IV (19%), complex III (16%), and complex II deficiencies (7%). Pathogenic mitochondrial DNA abnormalities were found in 11.5% of the patients. A substantial fraction (40%) of patients with mitochondrial disorders exhibited cardiac disease, diagnosed by Doppler echocardiography; however, the majority (60%) of patients had predominant neuromuscular manifestations. No correlation between the type of RC defect and the clinical presentation was observed. Overall, the mean age at presentation was 40 months. However, the mean age at presentation was 33 months in the cardiac group and 44 months in the noncardiac group. Twenty-six (58%) patients in the cardiac group exhibited hypertrophic cardiomyopathy, 29% had dilated cardiomyopathy, and the remainder (13%) had left ventricular noncompaction. Patients with cardiomyopathy had an 18% survival rate at 16 years of age. Patients with neuromuscular features but no cardiomyopathy had a 95% survival at the same age. This study gives strong support to the view that in patients with RC defects, cardiomyopathy is more common than previously thought and tends to follow a different and more severe clinical course. Although with a greater frequency than previously reported, mitochondrial DNA mutations were found in a minority of patients, emphasizing that most mitochondrial disorders of childhood follow a Mendelian pattern of inheritance.

Lactic Acidosis during Propofol–Remifentanil Anesthesia in an Adult

Lactic Acidosis during Propofol–Remifentanil Anesthesia in an Adult

Mitochondrial DNA turnover occurs during preimplantation development and can be modulated by environmental factors

There is increasing evidence in humans that abnormal mitochondrial DNA (mtDNA) is associated with common degenerative disorders of the twenty-first century. MtDNA is exclusively female in origin and abnormalities in mtDNA can either be inherited, or generated de novo by adverse environmental factors that disturb mitochondrial DNA synthesis or destabilize mtDNA. The preimplantation period of development in mammals was thought to be relatively immune from environmentally induced changes to mtDNA, since no replication of mtDNA was thought to occur at this stage. This study demonstrates that there is a very short period of mtDNA synthesis immediately after fertilization, which can be affected by environmental stress. Adverse culture conditions during this phase of development could therefore alter the mitochondrial genome, with possible long-term consequences for the health of the offspring. The findings have relevance for all assisted reproduction programmes and for the rapidly emerging field of stem cell technologies.

Mitochondrial DNA abnormalities and autistic spectrum disorders

ObjectivesTo further characterize mtDNA defects associated with autistic features, especially the A3243G mtDNA mutation and mtDNA depletion. Study designFive patients with autistic spectrum disorders and family histories of mitochondrial DNA diseases were studied. We performed mtDNA analysis in all patients and magnetic resonance spectroscopy in three. ResultsThree patients manifested isolated autistic spectrum features and two had additional neurologic symptoms. Two patients harbored the A3243G mutation. In two others, the A3243G mutation was not found in accessible tissues but was present in tissues from their mothers. The fifth patient had 72% mtDNA depletion in skeletal muscle. ConclusionsAutistic spectrum disorders with or without additional neurologic features can be early presentations of the A3243G mtDNA mutation and can be a prominent clinical manifestation of mtDNA depletion. Mitochondrial dysfunction should be considered in patients who have autistic features and associated neurologic findings or who have evidence of maternal inheritance.

Genetic characterization of a pentatricopeptide repeat protein gene, orf687, that restores fertility in the cytoplasmic male-sterile Kosena radish.

Cytoplasmic male sterility (CMS) in plants is a maternally inherited inability to produce functional pollen, and is often associated with mitochondrial DNA abnormalities. Specific nuclear loci that suppress CMS, termed as restorers of fertility (Rf), have been identified. Previously, we identified an Rf for the CMS Kosena radish and used genetic analysis to identify the locus and create a contig covering the critical interval. To identify the Rf gene, we introduced each of the lambda and cosmid clones into the CMS Brassica napus and scored for fertility restoration. Fertility restoration was observed when one of the lambda clones was introduced into the CMS B. napus. Furthermore, introduction of a 4.7-kb BamHI/HpaI fragment of the lambda clone is enough to restore male fertility. A cDNA strand isolated from a positive fragment contained a predicted protein (ORF687) of 687 amino acids comprising 16 repeats of the 35-amino acid pentatricopeptide repeat (PPR) motif. Kosena CMS radish plants were found to express an allele of this gene possessing four substituted amino acids in the second and third repeats of the PPR suggesting that the domains formed by these repeats in ORF687 are essential for fertility restoration. Protein levels of the Kosena CMS-associated mitochondrial protein ORF125 were considerably reduced in plants in which fertility was restored, although mRNA expression was normal. Regarding the possible role for PPR-containing proteins in the regulation of the mitochondrial gene, we propose that ORF687 functions either directly or indirectly to lower the levels of ORF125, resulting in the restoration of fertility in CMS plants.

Elevated plasma deoxyuridine in patients with thymidine phosphorylase deficiency

Mutations in the nuclear gene encoding thymidine phosphorylase (TP) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disease with mitochondrial dysfunction and mitochondrial DNA abnormalities. We have demonstrated alterations of thymidine (dThd) metabolism in MNGIE patients. Here, we report the accumulation of another substrate of TP, deoxyuridine (dUrd), whose circulating levels ranged from 5.5 to 24.4 μM (average 14.2) in MNGIE and were undetectable (<0.05 μM) in both TP mutation carriers and controls. The dramatic accumulation of dUrd may contribute to nucleotide pool imbalances and, together with the increased levels of dThd, is likely to contribute to the pathogenesis of MNGIE.

Marin-Garcia et al: Striated Muscle Mitochondrial Phenotype: Probing striated muscle mitochondrial phenotype in neuromuscular disorders

Multisystemic disorders with predominantly neurologic manifestations often present with mitochondrial abnormalities in striated muscle biopsies. Decreased respiratory complex activities and abnormalities in mitochondrial structure and DNA constitute the spectrum of mitochondrial changes used as diagnostic and prognostic indicators in patients with neuromuscular disorders. This study assessed mitochondrial defects present in a cohort of 154 young patients to determine diagnostic efficiency and probe the relationship of mitochondrial to clinical phenotype. Striated muscle biopsies were analyzed for mitochondrial structure and number, levels of enzyme activities of complex I-V and citrate synthase, mitochondrial DNA and specific mitochondrial DNA deletions, and presence of 15 pathogenic mitochondrial DNA point mutations. Reduced complex I, III, IV, and V activities were the most ubiquitous finding, with complex III most commonly affected. Mitochondrial structural defects (39%) included changes in mitochondria sizes/shapes and number and aberrant cristae formation. Mitochondrial DNA deletions were evident in 15 patients, three displayed mitochondrial DNA depletion, and only two harbored pathogenic point mutations. Reductions in specific enzyme activities may be the most sensitive diagnostic indicator, whereas defects in ultrastructure and mitochondrial DNA integrity were frequently accompanied by the full spectrum of mitochondrial abnormalities. Some phenotypes displayed specific mitochondrial abnormalities; however, most clinical phenotypes displayed little specificity with regard to mitochondrial phenotype.

Rebuttal to Jacobs: the mitochondrial theory of aging: alive and well.

Rebuttal to Jacobs: the mitochondrial theory of aging: alive and well.

Alteration of nucleotide metabolism: a new mechanism for mitochondrial disorders.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). TP deficiency alters the metabolism of the nucleosides thymidine and deoxyuridine, which, in turn, produces abnormalities of mitochondrial DNA (mtDNA) including depletion, deletions, and point mutations. MNGIE is the best characterized of the expanding number of mitochondrial disorders caused by alterations in the metabolism of nucleosides/nucleotides. Because mitochondria contain their own machinery for nucleoside and nucleotide metabolism and have physically separate nucleotide pools, it is not surprising that disorders of these pathways cause human diseases. Other diseases in this group include mtDNA depletion syndromes caused by mutations on the nuclear genes encoding the mitochondrial thymidine kinase and deoxyguanosine kinase; autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA due to mutations in the genes encoding the muscle-isoform of mitochondrial ADP/ATP translocator; and mitochondrial DNA depletion due to toxicities of nucleoside analogues. Mutations in the deoxynucleotide carrier, a transporter of deoxynucleoside diphosphates, have been identified as a cause of congenital microcephaly. However, alterations of mtDNA have not yet been established in this disorder. Future studies are likely to reveal additional diseases and provide further insight into this new subject.

Mitochondrial gene abnormalities as a cause of psychiatric diseases.

(Summary) Genetic predisposition has been implicated in the pathogenesis of mental disorders including schizophrenia. Mitochondrial dysfunction has been shown to be associated with mitochondrial myopathies, type 2 diabetes. We encountered with a diabetic woman who was associated with mitochondrail myopathy and schizophrenia. To know an involvement of mitochondrial DNA abnormalities in the pathogenesis of psychiatric diseases, we screened patients with schizophrenia for mitochondrial DNA abnormalities. By screening for an A3243G mutation of the tRNA(LEU) of the mitochondrial DNA, no one carried this mutation. Other mutations were also analysed. Although some mitochondrial DNA abnormalities may be associated with schizophrenia, the A3 2 43 G mutation is unlikely to play a major role in the pathogenesis of schizophrenia in

Multiple symmetric lipomatosis may be the consequence of defective noradrenergic modulation of proliferation and differentiation of brown fat cells.

Multiple symmetric lipomatosis (MSL) is an inherited disorder in which enlarging and unencapsulated lipomas symmetrically develop in the subcutaneous tissue of the neck, shoulders, mammary, and truncal regions. In some cases, it is associated with mitochondrial DNA abnormalities. The pathogenesis of MSL is completely unknown, although the fat deposits may be due to a neoplastic-like proliferation of functionally defective brown adipocytes. It has recently been demonstrated that the beta(3)-adrenergic receptor is the functionally relevant adrenergic receptor subtype in brown adipocytes and that its stimulation by noradrenaline (NA) modulates the expression of genes, such as uncoupling protein (UCP)-1 and inducible nitric oxide synthase (iNOS), involved in fat cell proliferation and differentiation. Furthermore, Trp64Arg mutation of the beta(3)-adrenoceptor has been implicated in lower NA activity in adipose tissues. The aim of this study was to investigate the molecular and functional characteristics of MSL adipocytes and to analyse the effects of nitric oxide (NO) on the proliferation/differentiation of MSL adipocytes in culture, and the relevance of putative noradrenergic deficit in the development of lipomas in MSL patients. Cultured MSL adipocytes were able to synthesize UCP-1 (the selective marker of brown adipocytes), but unlike that of normally functioning brown fat cells, the expression of the UCP-1 gene was not significantly induced by NA. NA is also defective in inducing iNOS gene expression, thus leading to reduced NO production and a consequent reduction in the anti-proliferative, adipogenic (mitochondrial biogenesis) effects of NA on MSL cells. Furthermore, the transcriptional peroxisome proliferator-activated receptor gamma co-activator-1 (PGC-1), which plays a key role in the sympathetic-stimulated mitochondrial biogenesis of brown adipocytes, is expressed but not induced by NA in MSL cells, as it is in brown adipocytes. The study did not find any association between beta(3)-adrenoceptor gene polymorphism and noradrenergic signalling defects in MSL subjects with or without mitochondrial DNA mutations.

A practical approach to the diagnosis and management of MELAS: case report and review.

Mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS) is a mitochondrial disorder and an important diagnostic consideration in the young patient with nonhemorrhagic stroke. Its presentation is varied and diagnosis is based on early recognition of the clinical features and correct interpretation of laboratory and radiologic studies. In this article, we report a patient with MELAS and review the clinical, laboratory, and neuroradiologic features of the condition. In the young patient with multiple stroke-like episodes in different vascular territories and neuroradiologic features of transient abnormalities in varying regions, laboratory testing for MELAS must be performed. The presence of ragged red fibers in skeletal muscle and biochemical demonstration of defects in mitochondrial respiratory enzymes strongly support the diagnosis. Molecular genetic testing for abnormalities in mitochondrial DNA will confirm the diagnosis. The importance of a thorough assessment of family history is also emphasized. The basic principles of mitochondrial genetics and the common point mutations and rearrangements of mitochondrial DNA associated with MELAS are reviewed. Although treatment options are limited, several therapeutic agents have been studied. The diagnosis of MELAS should be considered in the young patient with stroke, especially when accompanied by other clinical features such as seizures, encephalopathy, and muscle weakness. Laboratory evaluation can provide an accurate diagnosis, especially when the appropriate mitochondrial DNA studies are performed. Genetic counseling should be provided to patients with MELAS associated with mitochondrial DNA point mutations. Better understanding of the molecular basis of the condition may result in the development of effective treatment strategies.

Mitochondrial dysfunction in patients with hypotonia, epilepsy, autism, and developmental delay: HEADD syndrome.

A group of 12 children clinically presenting with hypotonia, intractable epilepsy, autism, and developmental delay, who did not fall into previously described categories of mitochondrial encephalomyopathy, were evaluated for mitochondrial respiratory enzyme activity levels, mitochondrial DNA, and mitochondrial structural abnormalities. Reduced levels in specific respiratory activities were found solely in enzymes with subunits encoded by mitochondrial DNA in seven of eight biopsied skeletal muscle specimens evaluated. Five cases exhibited increased levels of large-scale mitochondrial DNA deletions, whereas pathogenic point mutations previously described in association with mitochondrial encephalomyopathies were not found. Mitochondrial structural abnormalities were present in three of four patients examined. Our findings suggest that mitochondrial dysfunction, including extensive abnormalities in specific enzyme activities, mitochondrial structure, and mitochondrial DNA integrity, may be present in children with a clinical constellation including hypotonia, epileptic seizures, autism, and developmental delay. The acronym HEADD is presented here to facilitate pursuit of mitochondrial defects in patients with this clinical constellation after other causes have been excluded.