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

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

Intimate Relationship between mtDNA, Plasmids, and the Fusion of Mitochondria

Physarum polycephalum. The conformation of Physarum mtDNA is currently thought to be circular. The inheritance of its mtDNA depends on the multiallelic mating type loci, matA. In a cross with ordinary matA combinations, the strain that has the higher matA status transmits its mtDNA to the progeny (uniparental inheritance). The mF plasmid promotes the fusion of mitochondria in the zygote and during sporulation. When it exists in a strain with a lower status matA, the mF plasmid overcomes the force of uniparental inheritance and is preferentially transmitted to the progeny via mitochondrial fusion. Moreover, the conformation of mtDNA is changed from circular to linear by recombination with the mF plasmid. Since biparental inheritance usually occurs in a cross involving a combination of matA1 and matA15, two types of inheritance of Physarum mtDNA exist. Considering the existence of the mF plasmid, there are four patterns of cytoplasmic inheritance in P. polycephalum: 1) uniparental inheritance of mtDNA, 2) uniparental inheritance of mtDNA and preferential transmission of the mF plasmid, 3) biparental inheritance of mtDNA, and 4) biparental inheritance of mtDNA and the mF plasmid. This article describes the events involved in each pattern. Finally, we discuss a hypothetical mechanism for mitochondrial fusion. The essential protein may be the ORF640 protein encoded in the mF plasmid. Received 8 March 2000/ Accepted in revised form 23 March 2000     

Human aging and somatic point mutations in mtDNA: A comparative study of generational differences (grandparents and grandchildren)

The accumulation of somatic mutations in mtDNA is correlated with aging. In this work, we sought to identify somatic mutations in the HVS-1 region (D-loop) of mtDNA that might be associated with aging. For this, we compared 31 grandmothers (mean age: 63 ± 2.3 years) and their 62 grandchildren (mean age: 15 ± 4.1 years), the offspring of their daughters. Direct DNA sequencing showed that mutations absent in the grandchildren were detected in a presumably homoplasmic state in three grandmothers and in a heteroplasmic state in an additional 13 grandmothers; no mutations were detected in the remaining 15 grandmothers. However, cloning followed by DNA sequencing in 12 grandmothers confirmed homoplasia in only one of the three mutations previously considered to be homoplasmic and did not confirm heteroplasmy in three out of nine grandmothers found to be heteroplasmic by direct sequencing. Thus, of 12 grandmothers in whom mtDNA was analyzed by cloning, eight were heteroplasmic for mutations not detected in their grandchildren. In this study, the use of genetically related subjects allowed us to demonstrate the occurrence of age-related (> 60 years old) mutations (homoplasia and heteroplasmy). It is possible that both of these situations (homoplasia and heteroplasmy) were a long-term consequence of mitochondrial oxidative phosphorylation that can lead to the accumulation of mtDNA mutations throughout life.     

Polyphyly of mtDNA lineages in the Russian sturgeon, Acipenser gueldenstaedtii: forensic and evolutionary implications

Molecular species identification methods are an important component of CITES monitoring programs for trade in sturgeon and caviar. To date, obtaining molecular evidence for distinguishing caviar from four closely related Eurasian sturgeon species Acipenser baerii (Siberian sturgeon), A. gueldenstaedtii (osetra), A. persicus (Persian sturgeon), A. naccarii (Italian sturgeon) remains problematic. Using approximately 2.3 kb of mtDNA sequence data (cytochrome b, NADH5, control region), we find this to be attributable to the polyphyletic nature of these mitochondrial DNA markers in the Russian sturgeon, A. gueldenstaedtii. Two mitochondrial lineages are present within this species: one is phylogenetically affiliated with A. persicus and A. naccarii, while the other clusters with A. baerii. These findings have a direct impact on molecular testing of commercial caviar and demonstrate the necessity of using large sample sizes when constructing forensic databases. Furthermore, the results affect current taxonomic designations for these species as well as hypotheses concerning their evolutionary origins.     

Deciphering OPA1 mutations pathogenicity by combined analysis of human,mouse and yeast cell models

OPA1 is the major gene responsible for Dominant Optic Atrophy (DOA) and the syndromic form DOA “plus”. Over 370 OPA1 mutations have been identified so far, although their pathogenicity is not always clear. We have analyzed one novel and a set of known OPA1 mutations to investigate their impact on protein functions in primary skin fibroblasts and in two “ad hoc” generated cell systems: the MGM1/OPA1 chimera yeast model and the Opa1?/? MEFs model expressing the mutated human OPA1 isoform 1. The yeast model allowed us to confirm the deleterious effects of these mutations and to gain information on their dominance/recessivity. The MEFs model enhanced the phenotypic alteration caused by mutations, nicely correlating with the clinical severity observed in patients, and suggested that the DOA “plus” phenotype could be induced by the combinatorial effect of mitochondrial network fragmentation with variable degrees of mtDNA depletion. Overall, the two models proved to be valuable tools to functionally assess and define the deleterious mechanism and the pathogenicity of novel OPA1 mutations, and useful to testing new therapeutic interventions.     

Intracellular heteroplasmy for disease-associated point mutations in mtDNA: implications for disease expression and evidence for mitotic segregation of heteroplasmic units of mtDNA

Studies in vitro have shown that a respiratorydeficient phenotype is expressed by cells when the proportion of mtDNA with a disease-associated mutation exceeds a threshold level, but analysis of tissues from patients with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) have failed to show a consistent relationship between the degree of heteroplasmy and biochemical expression of the defect. One possible explanation for this phenomenon is that there is variation of heteroplasmy between individual cells that is not adequately reflected by the mean heteroplasmy for a tissue. We have confirmed this by study of fibroblast clones from subjects heteroplasmic for the MELAS 3243 (A G) mtDNA mutation. Similar observations were made with fibroblast clones derived from two subjects heteroplasmic for the 11778 (GA) mtDNA mutation of Leber's hereditary optic neuropathy. For the MELAS 3243 mutation, the distribution of mutant mtDNA between different cells was not randomly distributed about the mean, suggesting that selection against cells with high proportions of mutant mtDNA had occurred. To explore the way in which heteroplasmic mtDNA segregates in mitosis we followed the distribution of heteroplasmy between clones over approximately 15 generations. There was either no change or a decrease in the variance of intercellular heteroplasmy for the MELAS 3243 mutation, which is most consistent with segregation of heteroplasmic units of multiple mtDNA molecules in mitosis. After mitochondria from one of the MELAS 3243 fibroblast cultures were transferred to a mitochondrial DNA-free (⁰) cell line derived from osteosarcoma cells by cytoplast fusion, the mean level and intercellular distribution of heteroplasmy was unchanged. We interpret this as evidence that somatic segregation (rather than nuclear background or cell differentiation state) is the primary determinant of the level of heteroplasmy.     

植物细胞质雄性不育分子机理研究进展

本文从线粒体基因组、线粒体基因、线粒体转录RNA、线粒体蛋白、转基因植物以及花粉败育机理六个方面详细介绍了植物细胞质雄性不育分子生物学研究的技术和方法。综述了植物细胞质雄性不育分子机理研究的进展 ,并对植物细胞质雄性不育分子机理的前景作了展望。     

Human settlement history between Sunda and Sahul: a focus on East Timor (Timor-Leste) and the Pleistocenic mtDNA diversity

Background

Distinct, partly competing, “waves” have been proposed to explain human migration in(to) today’s Island Southeast Asia and Australia based on genetic (and other) evidence. The paucity of high quality and high resolution data has impeded insights so far. In this study, one of the first in a forensic environment, we used the Ion Torrent Personal Genome Machine (PGM) for generating complete mitogenome sequences via stand-alone massively parallel sequencing and describe a standard data validation practice.

Results

In this first representative investigation on the mitochondrial DNA (mtDNA) variation of East Timor (Timor-Leste) population including >300 individuals, we put special emphasis on the reconstruction of the initial settlement, in particular on the previously poorly resolved haplogroup P1, an indigenous lineage of the Southwest Pacific region. Our results suggest a colonization of southern Sahul (Australia) >37 kya, limited subsequent exchange, and a parallel incubation of initial settlers in northern Sahul (New Guinea) followed by westward migrations <28 kya.

Conclusions

The temporal proximity and possible coincidence of these latter dispersals, which encompassed autochthonous haplogroups, with the postulated “later” events of (South) East Asian origin pinpoints a highly dynamic migratory phase.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-014-1201-x) contains supplementary material, which is available to authorized users.     

The common deletion found in patient reexamined after 33 years and comparison with complete mtDNA sequences of maternal relatives

In 1966, a male (17 years old) was clinically examined at the National Institutes of Health (NIH) and diagnosed with Idiopathic Progressive External Ophthalmoplegia (IPEO). A muscle biopsy showing ragged-red fibers implicated mitochondrial involvement. Since the sequence of human mitochondrial DNA (mtDNA) was not determined until 1981, no genetic confirmation of the disease was possible at that time. In 1999, clinical reexamination and sequencing the entire mtDNA of the patient and living maternal relatives (mother and brother) indicated a progressive mitochondrial myopathy and the presence of the 4977 base pair (bp) deletion (the common deletion) in the patient.     

Voltage-dependent anion channel involved in the mitochondrial calcium cycle of cell lines carrying the mitochondrial DNA A4263G mutation

In this study, we investigated the effects of the voltage-dependent anion channel (VDAC) on the mitochondrial calcium cycle in cell lines carrying the mitochondrial DNA A4263G mutation. We established lymphoblastoid cell lines from three symptomatic individuals and one asymptomatic individual from the large Chinese Han family carrying the A4263G mutation; these were compared with three control cell lines. The mitochondrial Ca²⁺ concentration and membrane potential were detected by loading cells with Rhod-2 and JC-1, respectively. Confocal imagines showed the average Rhod-2 and JC-1 fluorescence levels of individuals carrying the tRNA^Ile A4263G mutation were lower than those of the control group (P < 0.05). The baseline Rhod-2 fluorescence in the control group increased after exposure to atractyloside (an opener of the adenine nucleotide translocator, P < 0.05), but no significant change was detected in the cell line harboring the A4263G mutation (P > 0.05). The baseline JC-1 fluorescence in both the mutated and control cell lines decreased after subsequent exposure to atractyloside (P < 0.05), whereas this effect of atractyloside was inhibited by Cyclosporin A (CsA, a VDAC blocker). We conclude that the mitochondrial VDAC is involved in both the increase of mitochondrial permeability to Ca²⁺ and the decrease of mitochondrial membrane potential in cell lines carrying the mtDNA A4263G mutation.     

In vivo conformation of mitochondrial DNA revealed by pulsed-field gel electrophoresis in the true slime mold, Physarum polycephalum.

Pulsed-field gel electrophoresis (PFGE) was used to examine the in vivo and in vitro conformations of Physarum polycephalum mitochondrial DNA (mtDNA). We used plugs containing isolated mitochondria, isolated mitochondrial nucleoids (mt-nuclei), and isolated mtDNA, in addition to whole cells. The mtDNA contained in the myxamoebae, plasmodia, isolated mitochondria, and isolated mt-nuclei was circular, but most of the isolated mtDNA had been site-specifically fragmented and linearized during DNA preparation and storage under low ionic strength conditions. Restriction mapping of Physarum mtDNA by the direct digestion of the isolated mt-nuclei from two different strains, DP89 x AI16 and KM88 x AI16, resulted in the circular form. A linear mitochondrial plasmid, mF, is known to promote mitochondrial fusion and integration of itself into the mtDNA in Physarum. Linearization of mtDNA by the integration of the mF plasmid was demonstrated when we used PFGE to analyze isolated mitochondria from the plasmodial strain DP89 x NG7 carrying the mF plasmid (mF+). The PFGE system can be used not only to determine whether the form of mtDNA is linear or circular but also to analyze the dynamic conformational changes of mtDNA.     

20 years of human mtDNA pathologic point mutations: Carefully reading the pathogenicity criteria

Despite the strong purifying selection that occurs during embryonic development, the particular location and features of mitochondrial DNA make it especially susceptible to accumulating point mutations, giving rise to a large number of mitochondrial DNA variants. Many of these will have moderate or no phenotypic effects but others will be the cause of very dramatic diseases, usually known as mitochondriopathies. Because of the abundance of different mitochondrial DNA variants, it is not easy to determine whether a new mutation is pathogenic. To facilitate this task, different criteria have been proposed, but they are often either too severely or too loosely applied. Citing examples from the literature, in this paper we discuss some critical aspects of these criteria.     

Genetics and mitochondrial abnormalities in autism spectrum disorders: a review

We review the current status of the role and function of the mitochondrial DNA (mtDNA) in the etiology of autism spectrum disorders (ASD) and the interaction of nuclear and mitochondrial genes. High lactate levels reported in about one in five children with ASD may indicate involvement of the mitochondria in energy metabolism and brain development. Mitochondrial disturbances include depletion, decreased quantity or mutations of mtDNA producing defects in biochemical reactions within the mitochondria. A subset of individuals with ASD manifests copy number variation or small DNA deletions/duplications, but fewer than 20 percent are diagnosed with a single gene condition such as fragile X syndrome. The remaining individuals with ASD have chromosomal abnormalities (e.g., 15q11-q13 duplications), other genetic or multigenic causes or epigenetic defects. Next generation DNA sequencing techniques will enable better characterization of genetic and molecular anomalies in ASD, including defects in the mitochondrial genome particularly in younger children.     

Founder effects and phenotypic variation in Adelges cooleyi, an insect pest introduced to the eastern United States

Introduced organisms experience founder effects including genetic bottlenecks that result in significant reductions in genetic variation. Genetic bottlenecks may constrain the evolution of phenotypic traits that facilitate success in novel habitats. We examined the effect of introduction into novel environments on genetic diversity of an insect pest, Adelges cooleyi, which was introduced into the eastern United States during the mid nineteenth century. We compared variation in mitochondrial and nuclear genomes in native and introduced samples to determine the effect of introduction on genetic variation experienced by this insect. We also measured an ecologically important phenotype, variation in host preference, in both native and introduced samples to compare variation in that trait with molecular genetic variation. To further investigate the relationship between genetic and phenotypic variation, we examined the degree to which mtDNA haplotypes provide information about host preference. Adelges cooleyi in eastern North America has significantly reduced genetic and phenotypic variation, but this low variation does not appear to have prevented persistence in a novel environment. Introduced insects appear to have retained host preference phenotypes similar to those of insects found where introductions likely originated.     

Migratory connectivity and temporal segregation of dunlin (Calidris alpina) in Portugal: evidence from morphology, ringing recoveries and mtDNA

Migratory connectivity plays an important role in conservation of long-distance migrant birds. Here, we study migratory links of dunlin (Calidris alpina), focusing on a stopover and wintering region (Portugal) where it is known that migration routes of dunlin from a broad geographic range (three subspecies) converge, and populations occur simultaneously or separated in time. We combine three methods (ringing recoveries, morphometrics and molecular genetics) to assess breeding origins and extent of temporal segregation of dunlin assemblages. Ringing recoveries show temporal separation of dunlin from different migration routes. Birds found in Portugal during August and September, migrating via Britain, reveal links to breeding areas in Iceland and Greenland. In October, a clear shift to more eastern migration routes occurs, with most Portuguese winter records from stopover sites along migration routes of populations from northern Scandinavia and Russia. Mitochondrial DNA (mtDNA) of Portuguese dunlin was compared with breeding populations. Spring and autumn migrants in Portugal corresponded to C. a. schinzii and C. a. arctica populations, while the Portuguese winter population clearly differs by including mtDNA haplotypes of C. a. alpina. For genetically sexed individuals, we found significant differences in morphology (bill and tarsus length) supporting the temporal separation of populations/subspecies revealed by recoveries and mtDNA. Our results give evidence for migratory connectivity of dunlin populations between geographic areas previously not considered connected. They confirm the existence of clear differences in breeding origin between birds in Portugal at different times of year. These results are important in the consideration of future long-term conservation plans.     

TFAM knockdown-triggered mtDNA-nucleoid aggregation and a decrease in mtDNA copy number induce the reorganization of nucleoid populations and mitochondria-associated ER-membrane contacts

The correct organization of mitochondrial DNA (mtDNA) in nucleoids and the contacts of mitochondria with the ER play an important role in maintaining the mitochondrial genome distribution within the cell. Mitochondria-associated ER membranes (MAMs) consist of interacting proteins and lipids located in the outer mitochondrial membrane and ER membrane, forming a platform for the mitochondrial inner membrane-associated genome replication factory as well as connecting the nucleoids with the mitochondrial division machinery. We show here that knockdown of a core component of mitochondrial nucleoids, TFAM, causes changes in the mitochondrial nucleoid populations, which subsequently impact ER-mitochondria membrane contacts. Knockdown of TFAM causes a significant decrease in the copy number of mtDNA as well as aggregation of mtDNA nucleoids. At the same time, it causes significant upregulation of the replicative TWNK helicase in the membrane-associated nucleoid fraction. This is accompanied by a transient elevation of MAM proteins, indicating a rearrangement of the linkage between ER and mitochondria triggered by changes in mitochondrial nucleoids. Reciprocal knockdown of the mitochondrial replicative helicase TWNK causes a decrease in mtDNA copy number and modifies mtDNA membrane association, however, it does not cause nucleoid aggregation and considerable alterations of MAM proteins in the membrane-associated fraction. Our explanation is that the aggregation of mitochondrial nucleoids resulting from TFAM knockdown triggers a compensatory mechanism involving the reorganization of both mitochondrial nucleoids and MAM. These results could provide an important insight into pathological conditions associated with impaired nucleoid organization or defects of mtDNA distribution.     

普通小麦三种细胞质雄性不育系线粒体DNA的比较研究

对细胞质分别来源于粘果山羊草（Ａｅ．ｋｏｔｓｃｈｙｉ）、偏凸山羊草（Ａｅ．ｖｅｎｔｒｉｃｏｓａ）、提莫菲维（Ｔ．ｔｉｍｏｐｈｅｅｖｉ）的３种普通小麦雄性不育系,其相应保持系和共有的一种恢复系的ｍｔＤＮＡ进行了ＲＦＬＰ比较分析。发现Ｋ型和Ｖ型不育系的ｍｔＤＮＡ在组织结构上不同于Ｔ型,说明Ｋ、Ｖ型不育系是有别于Ｔ型的两种新不育类型。Ｋ型、Ｖ型不育系的ｍｔＤＮＡ与保持系和恢复系显著不同,推测ｍｔＤＮＡ与小麦细胞质雄性不育性有关。实验同时发现Ｔ型不育系与其保持系的ｍｔＤＮＡ非常相似,对这种相似性的原因进行了讨论。     

小麦胞质突变型雄性不育系85EA与其保持系线粒体DNA的比较研究

８５ＥＡ是通过电子束辐照获得的胞质突变型小麦不育要用ＲＦＬＰ和ＲＡＰＤ技术对８５ＥＡ及春保持系的线粒体ＤＮＡ进行了比较研究。ＲＦＬＰ分析表明８５ＥＡ线粒体基因组中ｃｏｘⅡ基因的位置结构与保持系发生了变化；ＲＡＰＤ分析中引物ＯＰＤ－１５扩增产物在不育系和保持系间有明显差异，不育系的扩增产物比保持系多１条分子量为０．６ｋｂ的特展览 要带，用Ｔ－ｅａｓｙ ｖｅｃｔｏｒ克隆该不育系特异条带并命名为ＯＰＤ－     

Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations.     

Parasites and deleterious mutations: interactions influencing the evolutionary maintenance of sex

The restrictive assumptions associated with purely genetic and purely ecological mechanisms suggest that neither of the two forces, in isolation, can offer a general explanation for the evolutionary maintenance of sex. Consequently, attention has turned to pluralistic models (i.e. models that apply both ecological and genetic mechanisms). Existing research has shown that combining mutation accumulation and parasitism allows restrictive assumptions about genetic and parasite parameter values to be relaxed while still predicting the maintenance of sex. However, several empirical studies have shown that deleterious mutations and parasitism can reduce fitness to a greater extent than would be expected if the two acted independently. We show how interactions between these genetic and ecological forces can completely reverse predictions about the evolution of reproductive modes. Moreover, we demonstrate that synergistic interactions between infection and deleterious mutations can render sex evolutionarily stable even when there is antagonistic epistasis among deleterious mutations, thereby widening the conditions for the evolutionary maintenance of sex.