Mitochondrial DNA Rearrangements Research Articles

Exploiting synteny in Cucumis for mapping of Psm: a unique locus controlling paternal mitochondrial sorting

The three genomes of cucumber show different modes of transmission, nuclear DNA bi-parentally, plastid DNA maternally, and mitochondrial DNA paternally. The mosaic (MSC) phenotype of cucumber is associated with mitochondrial DNA rearrangements and is a valuable tool for studying mitochondrial transmission. A nuclear locus (Psm) has been identified in cucumber that controls sorting of paternally transmitted mitochondrial DNA. Comparative sequencing and mapping of cucumber and melon revealed extensive synteny on the recombinational and sequence levels near Psm and placed this locus on linkage group R of cucumber and G10 of melon. However, the cucumber genomic region near Psm was surprisingly monomorphic with an average of one SNP every 25 kb, requiring that a family from a more diverse cross is produced for fine mapping and eventual cloning of Psm. The cucumber ortholog of Arabidopsis mismatch repair (MSH1) was cloned and it segregated independently of Psm, revealing that this candidate gene is not Psm.

Structural implications of mitochondrial dynamics

Mitochondrial components are continuously distributed throughout the whole chondriome of a cell by fusion and fission. Thus, a single mitochondrion represents a transient fraction of the chondriome. Mitochondrial dynamics are responsible for intracellular distribution and reaction of mitochondria to functional requirements. Dynamics occur on different levels: overall morphology, inner membrane-matrix compartment, turnover and rearrangements of mitochondrial proteins and DNA. Electron micrographs of serial sections of human umbilical vein endothelial cells reveal perinuclear mitochondria of extreme length and with branches in those cells that also have short peripheral mitochondria. Interactions of mitochondria with cytoskeletal elements are revealed in cells treated with cytochalasin D to destroy actin fibrillar structures or after disassembling microtubule by nocodazole. In the latter case mitochondria not only become immobilized, they also acquire a multiple ring structure. In F-actin-disturbed cells, motility (shape changes in particular) is increased and the mitochondria become elongated. Mechanisms of how F-actin might render mitochondria immobile may involve dynamin-related protein 1 (DRP1) or interaction with anion channels. This may be responsible for the lack of mitochondrial motility in senescent cells. Fusion between mitochondria revealed local fluctuations of mitochondrial red fluorescent protein (mtRFP), indicating novel fast inner membrane reorganizations. Mitochondrial dynamics result from a complex interplay between the molecular organization of the inner membrane-matrix complex and cytoskeletal elements outside.

Variable outcome of growth hormone administration in respiratory chain deficiency

Genetic defects of oxidative phosphorylation (OXPHOS) are known to account for a variety of neuromuscular and non-neuromuscular symptoms in childhood, including growth hormone (GH) deficiency. However GH administration for GH deficiency is controversial in OXPHOS deficiencies as GH is a mitosis-stimulator which may increase energy demand for cell proliferation. Here, we report the observation of four unrelated children with OXPHOS deficiency or bearing a mitochondrial DNA rearrangement and growth retardation, who required GH therapy. The first patient had no GH deficiency while the other three had low GH response to test stimulations. The condition of the first two patients quickly deteriorated under GH administration, GH was then stopped and subsequent clinical improvement was noted. In the other two patients, no adverse event was noted but various additional organs were involved following GH administration. In all patients, no benefit was observed concerning growth response as growth speed remained unchanged. These observations question the use of GH as a treatment of growth retardation for patients with OXPHOS deficiency.

Liver Disease in Mitochondrial Disorders

Liver involvement, a common feature in childhood mitochondrial hepatopathies, particularly in the neonatal period, may manifest as neonatal acute liver failure, hepatic steatohepatitis, cholestasis, or cirrhosis with chronic liver failure of insidious onset. There are usually significant neuromuscular symptoms, multisystem involvement, and lactic acidemia. The liver disease is usually progressive and eventually fatal. Current medical therapy of mitochondrial hepatopathies is largely ineffective, and the prognosis is usually poor. The role of liver transplantation in patients with liver failure remains poorly defined because of the systemic nature of the disease that does not respond to transplantation. Several specific molecular defects (mutations in nuclear genes such as SCO1, BCS1L, POLG, DGUOK, and MPV17 and deletion or rearrangement of mitochondrial DNA) have been identified in recent years. Prospective, longitudinal multicenter studies will be needed to address the gaps in our knowledge in these rare liver diseases.

The yeast Holliday junction resolvase, CCE1, can restore wild-type mitochondrial DNA to human cells carrying rearranged mitochondrial DNA

Rearrangements of mitochondrial DNA (mtDNA) are a well-recognized cause of human disease; deletions are more frequent, but duplications are more readily transmitted to offspring. In theory, partial duplications of mtDNA can be resolved to partially deleted and wild-type (WT) molecules, via homologous recombination. Therefore, the yeast CCE1 gene, encoding a Holliday junction resolvase, was introduced into cells carrying partially duplicated or partially triplicated mtDNA. Some cell lines carrying the CCE1 gene had substantial amounts of WT mtDNA suggesting that the enzyme can mediate intramolecular recombination in human mitochondria. However, high levels of expression of CCE1 frequently led to mtDNA loss, and so it is necessary to strictly regulate the expression of CCE1 in human cells to ensure the selection and maintenance of WT mtDNA.

The selection of mosaic (MSC) phenotype after passage of cucumber (Cucumis sativus L.) through cell culture — a method to obtain plant mitochondrial mutants

Mosaic (MSC) mutants of cucumber (Cucumis sativus L.) appear after passage through cell cultures. The MSC phenotype shows paternal transmission and is associated with mitochondrial DNA rearrangements. This review describes the origins and phenotypes of independently produced MSC mutants of cucumber, including current knowledge on their mitochondrial DNA rearrangements, and similarities of MSC with other plant mitochondrial mutants. Finally we propose that passage of cucumber through cell culture can be used as a unique and efficient method to generate mitochondrial mutants of a higher plant in a highly homozygous nuclear background.

Transgenic induction of mitochondrial rearrangements for cytoplasmic male sterility in crop plants

Stability of the mitochondrial genome is controlled by nuclear loci. In plants, nuclear genes suppress mitochondrial DNA rearrangements during development. One nuclear gene involved in this process is Msh1. Msh1 appears to be involved in the suppression of illegitimate recombination in plant mitochondria. To test the hypothesis that Msh1 disruption leads to the type of mitochondrial DNA rearrangements associated with naturally occurring cytoplasmic male sterility in plants, a transgenic approach for RNAi was used to modulate expression of Msh1 in tobacco and tomato. In both species, these experiments resulted in reproducible mitochondrial DNA rearrangements and a condition of male (pollen) sterility. The male sterility was, in each case, heritable, associated with normal female fertility, and apparently maternal in its inheritance. Segregation of the transgene did not reverse the male sterile phenotype, producing stable, nontransgenic male sterility. The reproducible transgenic induction of mitochondrial rearrangements in plants is unprecedented, providing a means to develop novel cytoplasmic male sterile lines for release as non-GMO or transgenic materials.

Mitochondrial hepatopathies: Advances in genetics and pathogenesis

Hepatic involvement is a common feature in childhood mitochondrial hepatopathies, particularly in the neonatal period. Respiratory chain disorders may present as neonatal acute liver failure, hepatic steatohepatitis, cholestasis, or cirrhosis with chronic liver failure of insidious onset. In recent years, specific molecular defects (mutations in nuclear genes such as SCO1, BCS1L, POLG, DGUOK, and MPV17 and the deletion or rearrangement of mitochondrial DNA) have been identified, with the promise of genetic and prenatal diagnosis. The current treatment of mitochondrial hepatopathies is largely ineffective, and the prognosis is generally poor. The role of liver transplantation in patients with liver failure remains poorly defined because of the systemic nature of the disease, which does not respond to transplantation. Prospective, longitudinal, multicentered studies will be needed to address the gaps in our knowledge in these rare liver diseases.

Dominant inheritance of premature ovarian failure associated with mutant mitochondrial DNA polymerase gamma

Premature ovarian failure (POF) results in menopause before the age of 40. Recently, mutations in the catalytic subunit of mitochondrial DNA polymerase gamma (POLG) were shown to segregate with POF in families with progressive external ophthalmoplegia (PEO) and multiple large-scale rearrangements of mitochondrial DNA (mtDNA). A patient, mother and maternal grandmother are described, all presenting with POF and PEO. The mother developed parkinsonism in her sixth decade. Normal mtDNA sequence excluded mitochondrial inheritance. Sequence analysis of polymerase gamma revealed a dominant Y955C mutation that segregated with disease. Southern blot analysis demonstrated mtDNA depletion in fibroblasts (43% of controls). In contrast, multiple rearrangements of mtDNA were seen in skeletal muscle, consistent with the relative sparing of nuclear-encoded complex II activity compared with other respiratory chain enzymes. Immunoblotting of native gels showed that DNA polymerase gamma stability was not affected, whereas a reverse-transcriptase primer-extension assay suggested a trend towards reduced polymerase activity in fibroblasts. This study confirms that POLG mutations can segregate with POF and parkinsonism and demonstrates for the first time that the Y955C mutation can lead to mtDNA depletion. Future screening projects will determine the frequency with which POLG is involved in the aetiology of POF and its impact on reproductive counselling.

Renal pathology in children with mitochondrial diseases

We studied renal involvement in 42 children with mitochondrial diseases (MDs). The diagnosis of MD was established by morphological, biochemical, and molecular genetic criteria. Renal disease was considered when patients had renal failure, nephrotic syndrome, Fanconi's syndrome or any symptomatic renal alteration. Mild tubular disorder was established if they had abnormal laboratory findings with no apparent clinical symptom. Renal involvement was found in 21 children (50%), of whom 8 had an apparent clinical picture and 13 a mild tubular disorder. Five patients with renal disease showed Debré-Toni-Fanconi's syndrome, 2 of them with decreased glomerular filtration rate (GFR). One case had nephrotic syndrome, another one presented decreased GFR, and the last one had a neurogenic bladder and bilateral hydronephrosis. Patients with mild renal disease showed tubular dysfunction with normal GFR. Renal involvement is frequent and present in about half of the children with MD. Thus, studies for evaluating kidney function should be performed on children with MD. Conversely, patients with tubulopathy of unknown origin or progressive renal disease should be investigated for the existence of MD, especially if associated with involvement of other organs or tissues. Southern blot analysis to search for large-scale mitochondrial DNA (mtDNA) rearrangements should be performed for patients with MD and kidney involvement.

Mitochondrial DNA as a Cancer Biomarker

As part of a national effort to identify biomarkers for the early detection of cancer, we developed a rapid and high-throughput sequencing protocol for the detection of sequence variants in mitochondrial DNA. Here, we describe the development and implementation of this protocol for clinical samples. Heteroplasmic and homoplasmic sequence variants occur in the mitochondrial genome in patient tumors. We identified these changes by sequencing mitochondrial DNA obtained from tumors and blood from the same individual. We confirmed previously identified primary lung tumor changes and extended these findings in a small patient cohort. Eight sequence variants were identified in stage I to stage IV tumor samples. Two of the sequence variants identified (22%) were found in the D-loop region, which accounts for 6.8% of the mitochondrial genome. The other sequence variants were distributed throughout the coding region. In the forensic community, the sequence variations used for identification are localized to the D-loop region because this region appears to have a higher rate of mutation. However, in lung tumors the majority of sequence variation occurred in the coding region. Hence, incomplete mitochondrial genome sequencing, designed to scan discrete portions of the genome, misses potentially important sequence variants associated with cancer or other diseases.

Deleted Mitochondrial DNA in Human Luteinized Granulosa Cells

The rearrangement of mitochondrial DNA in luteinized granulosa cells was determined in order to evaluate the fertilization capacity of oocytes and the development of embryos. Multiple deletions of mtDNA were found in luteinized granulosa cells from in vitro fertilization (IVF) patients. The 4977-base pair (bp) deletion was the most frequent deletion found in human granulosa cells. No significant difference was noted between mtDNA deletions of granulosa cells based on the fertilization capacity of oocytes and the development of embryos. To determine the relationship of proportions of mtDNA rearrangements with the aging process, granulosa cells were grouped into three different cohorts according to maternal age: younger than 32 years, between 32 and 37 years, and older than 37 years. No statistical correlation was noted between patient age and the frequency of occurrence of multiple mtDNA deletions. However, an increase in granulosa cell apoptosis was associated with an increase in mtDNA deletions. Accumulation of mtDNA deletions may contribute to mitochondrial dysfunction and impaired ATP production. We concluded that the accumulation of rearranged mtDNA in granulosa cells might not interfere with fertilization of human oocytes and further embryonic development; it was, however, associated with apoptosis processes.

Oxidative Damage and Mitochondrial DNA Mutations with Endometriosis

Endometriosis, a frequently encountered disease in gynecology, is a considerable threat to the physical, psychological, and social integrity of women. Moreover, up to 50% of infertile patients have this disease. The etiology and pathogenesis of this important disease are poorly understood; it is defined as an ectopic location for endometrium-like glandular epithelium and stroma outside of the uterine cavity. It still remains an open question as to what extent the peritoneal environment influences the establishment and/or progression of endometriosis. As a result of such stress, a sterile, inflammatory reaction with the secretion of growth factors, cytokines, and chemokines is generated, which is especially deleterious to successful reproduction. Significantly higher amounts of oxidative damage were detected in endometriotic lesions than in controlled normal endometrium, including mitochondrial DNA (mtDNA) rearrangement, 8-OH-deoxyguanosine (8-OH-dG), and lipoperoxide contents. There were approximately sixfold increases in 8-OH-dG and lipoperoxides in chocolate cysts compared with normal endometrial tissues. A novel 5,335-bp deletion of mtDNA was identified in endometriotic tissue. According to these results, we propose that oxidative stress and mtDNA mutations might be anticipated in the initiation or progression of endometriosis. Only by understanding the mechanisms involved in the pathogenesis of endometriosis can we develop a basis for new diagnostic and therapeutic approaches.

Mitochondrial tuning fork in nuclear homeotic functions

Homeotic-like flower morphologies in plants with cytoplasmic male sterility (CMS) are maternally inherited and associated with rearrangements in mitochondrial DNA. Recent studies allow an interpretation of dramatic CMS morphologies in the light of the floral ABC model. They uncover new nuclear targets for interactions with mitochondrial genes. GLOBOSA-, DEFICIENS- and APETALA3-like genes were transcriptionally down-regulated in carpelloid CMS flowers of tobacco, carrot and wheat. These results allow cooperation between nuclear and cytoplasmic genetic compartments to be considered as a developmental function and an evolutionary mechanism of speciation.

Peripheral Benzodiazepine Binding Sites in Platelets of Patients Affected by Mitochondrial Diseases and Large Scale Mitochondrial DNA Rearrangements

The peripheral-type benzodiazepine receptors (PBR) are localized on the outer mitochondrial membrane, as a constituent of mitochondrial permeability transition (MPT)-pore. Among its hypothesized functions, the regulation of the mitochondrial respiratory chain and apoptosis have been suggested; in addition alterations of PBR site density have been shown in some neuropathologic conditions with putative mitochondrial involvement. The aim of this work has been to evaluate PBR kinetic binding parameters in platelets from patients affected by mitochondrial disorders (MD) with large-scale mitochondrial DNA deletions and reduced cytochrome c oxidase activity. Using the specific PBR radioligand [(3) H] PK 11195, the kinetic binding parameters of PBR sites were determined in platelet membrane of 15 healthy subjects and 11 patients affected by different form of MD. Significant changes of dissociation constant (K(d)) and maximal number of binding sites (B(max)) values were evidenced in platelets of patients versus controls. In all patients the B(max) values were decreased (2,387.0 +/- 305.6 fmol/ mg proteins versus 4889.0 +/- 357.8 fmol/mg proteins, p< 0.05), whereas the K(d) values were higher in patients than controls (13.18 +/- 2.06 nM versus 5.63 +/- 0.46 nM, p< 0.05). These data suggest that the kinetic binding parameters of PBR are altered in MD and that the observed changes might be related to the mitochondrial dysfunction associated with MD.

Differential expression of alternative oxidase genes in maize mitochondrial mutants.

We have examined the expression of three alternative oxidase (aox) genes in two types of maize mitochondrial mutants. Nonchromosomal stripe (NCS) mutants carry mitochondrial DNA deletions that affect subunits of respiratory complexes and show constitutively defective growth. Cytoplasmic male-sterile (CMS) mutants have mitochondrial DNA rearrangements, but they are impaired for mitochondrial function only during anther development. In contrast to normal plants, which have very low levels of AOX, NCS mutants exhibit high expression of aox genes in all nonphotosynthetic tissues tested. The expression pattern is specific for each type of mitochondrial lesion: the NADH dehydrogenase-defective NCS2 mutant has high expression of aox2, whereas the cytochrome oxidase-defective NCS6 mutant predominantly expresses aox3. Similarly, aox2 and aox3 can be induced differentially in normal maize seedlings by specific inhibitors of these two respiratory complexes. Translation-defective NCS4 plants show induction of both aox2 and aox3. AOX2 and AOX3 proteins differ in their ability to be regulated by reversible dimerization. CMS mutants show relatively high levels of aox2 mRNAs in young tassels but none in ear shoots. Significant expression of aox1 is detected only in NCS and CMS tassels. The induction pattern of maize aox genes could serve as a selective marker for diverse mitochondrial defects.

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.

The Mitochondrial Genome And Human Mitochondrial Diseases

To date, more than 100 point mutations and several hundreds of structural rearrangements of mitochondrial DNA (mtDNA) are known too be connected with characteristic neuromuscular and other mitochondrial syndromes varying from those causing death at the neonatal stage to diseases with late ages of onset. The immediate cause of mitochondrial disorders is a defective oxidative phosphorylation. Wide phenotypic variation and the heteroplasmy phenomenon, which some authors include in mutation load, are characteristic of human mitochondrial diseases. As the numbers of cases identified and pedigrees described increase, data on the genotype‐phenotype interaction and the structure and frequency of pathogenic and conditionally pathogenic mtDNA mutations in human populations are rapidly accumulated. The data on the genetics and epidemiology of mitochondrial diseases are not only important for differential diagnosis and genetic counseling. Since both neutral and mildly pathogenic mutations of mtDNA are progressively accumulated in maternal phyletic lines, molecular analysis of these mutations permits not only reconstruction of the genealogical tree of modern humans, but also estimation of the role that these mutations play in natural selection.

Manipulation of mitochondrial DNA gene expression in the mouse

Mitochondrial dysfunction due to impaired respiratory chain function is increasingly recognized as an important cause of human disease. Mitochondrial disorders are relatively common and have an estimated incidence of 1:10,000 live births. There are more than 100 different point mutations and numerous large rearrangements of mitochondrial DNA (mtDNA; mainly single deletions) that cause human disease. We aimed at obtaining an animal model to study physiological aspects of mtDNA mutation disorders. There are as yet unsolved technical problems associated with transfection of mammalian mitochondria. We therefore choose to manipulate mtDNA expression by targeting of the nuclear gene encoding Tfam. We utilised the cre- loxP recombination system to disrupt Tfam since this system allows manipulation of respiratory chain function in selected mouse tissues. We have found increased cell death or apoptosis induction in both germ line and tissue-specific Tfam knockouts. Our results further suggest that increased production of reactive oxygen species (ROS) is not a prominent feature in cells with impaired mtDNA expression.

Novel rearrangements of arthropod mitochondrial DNA detected with long-PCR: applications to arthropod phylogeny and evolution.

Rearrangements of mitochondrial DNA gene order have been suggested as a tool for defining the pattern of evolutionary divergence in arthropod taxa. We have employed a combination of highly conserved insect-based polymerase chain reaction (PCR) primers with long-PCR to survey 14 noninsect arthropods for mitochondrial gene rearrangements. The size of the amplified fragments was used to order the primer containing genes. Five chelicerates exhibit amplicons that are consistent with the presumptive ancestral arthropod mtDNA gene order. These five species comprise two soft ticks, two prostriate hard ticks, and an opilionid. Six other chelicerates, all metastriate hard ticks, have a different arrangement that was originally discovered by this procedure and has been previously detailed in a complete mtDNA sequence. Three new arthropod mtDNA gene arrangements are described here. They were discovered in a terrestrial crustacean (Isopoda) and two myriapods (Chilopoda, centipede; Diplopoda, millipede). These rearrangements include major realignments of some of the large coding regions and two possible new positions for the tRNA(Met) (M) gene in arthropods. The long-PCR approach affords an opportunity to quickly screen divergent taxa for major rearrangements. Taxa exhibiting rearrangements can be targeted for DNA sequencing of gene boundaries to establish the details of the mtDNA organization.