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.     

The co‐occurrence of mtDNA mutations on different oxidative phosphorylation subunits,not detected by haplogroup analysis,affects human longevity and is population specific

To re‐examine the correlation between mtDNA variability and longevity, we examined mtDNAs from samples obtained from over 2200 ultranonagenarians (and an equal number of controls) collected within the framework of the GEHA EU project. The samples were categorized by high‐resolution classification, while about 1300 mtDNA molecules (650 ultranonagenarians and an equal number of controls) were completely sequenced. Sequences, unlike standard haplogroup analysis, made possible to evaluate for the first time the cumulative effects of specific, concomitant mtDNA mutations, including those that per se have a low, or very low, impact. In particular, the analysis of the mutations occurring in different OXPHOS complex showed a complex scenario with a different mutation burden in 90+ subjects with respect to controls. These findings suggested that mutations in subunits of the OXPHOS complex I had a beneficial effect on longevity, while the simultaneous presence of mutations in complex I and III (which also occurs in J subhaplogroups involved in LHON) and in complex I and V seemed to be detrimental, likely explaining previous contradictory results. On the whole, our study, which goes beyond haplogroup analysis, suggests that mitochondrial DNA variation does affect human longevity, but its effect is heavily influenced by the interaction between mutations concomitantly occurring on different mtDNA genes.     

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.     

Mitochondria: Biogenesis and mitophagy balance in segregation and clonal expansion of mitochondrial DNA mutations

Mitochondria are cytoplasmic organelles containing their own multi-copy genome. They are organized in a highly dynamic network, resulting from balance between fission and fusion, which maintains homeostasis of mitochondrial mass through mitochondrial biogenesis and mitophagy. Mitochondrial DNA (mtDNA) mutates much faster than nuclear DNA. In particular, mtDNA point mutations and deletions may occur somatically and accumulate with aging, coexisting with the wild type, a condition known as heteroplasmy. Under specific circumstances, clonal expansion of mutant mtDNA may occur within single cells, causing a wide range of severe human diseases when mutant overcomes wild type. Furthermore, mtDNA deletions accumulate and clonally expand as a consequence of deleterious mutations in nuclear genes involved in mtDNA replication and maintenance, as well as in mitochondrial fusion genes (mitofusin-2 and OPA1), possibly implicating mtDNA nucleoids segregation. We here discuss how the intricacies of mitochondrial homeostasis impinge on the intracellular propagation of mutant mtDNA.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

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.     

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.     

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。     

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.     

宁夏固原地区农村回、汉族近亲结婚的调查

固原地区位于宁夏南部山区,共辖６县,总人口１６２．２万,其中回族占４６．２％。当地经济、文化等条件都非常落后,加之风俗习惯的影响,致使本地近亲婚配多见,尤其回族较多,出生遗传缺陷也较多。在加拿大基金会的援助下,我所于１９９２年８月对本地区农村的回、汉...     

Liver mtDNA content increases during development: a comparison of methods and the importance of age- and tissue-specific controls for the diagnosis of mtDNA depletion

BACKGROUND: The quantitative loss of mitochondrial DNA (mtDNA) known as mtDNA depletion, often gives rise to liver disease. The diagnosis of mtDNA depletion syndrome is frequently imprecise, both for technical reasons and because of the lack of established age-adjusted normal ranges. We aimed to refine quantitative methods for diagnosing the hepatic type of mtDNA depletion syndrome, firstly by establishing an age-matched reference range for mitochondrial to nuclear DNA ratio (henceforth "mtDNA content") and secondly by investigating mtDNA in fibroblasts. METHODS: By comparing realtime PCR with an established method for quantifying mtDNA content we established a reference range for young children using biopsy and post-mortem material from patients <15 years. In addition, we investigated the arrangement of mtDNA in nucleoids from fibroblasts using fluorescence microscopy. RESULTS: Both methods showed that the mtDNA content of liver increases rapidly over the perinatal period. In a patient whose liver mtDNA content fell, but remained within the reference range, early investigation and age-matched controls were essential, as we found a progressive increase in muscle mtDNA copy number, respiratory chain activity and muscle power with age. In three further patients, fluorescence microscopy of the fibroblasts proved diagnostic. In one case a movement disorder was an important pointer. CONCLUSIONS: These cases highlight the (i) need for comparing mtDNA copy number data generated from patients to DNA isolated from an age-matched normal range from the tissue of interest and (ii) the utility of mtDNA staining with PicoGreen as a method to detect aberrant nucleoid morphology in mtDNA depletion patient fibroblast lines when affected tissues are not available for measuring mtDNA copy number.     

线粒体DNA用作分子标记的可靠性和研究前景

mtDNA作为分子标记的广泛应用,使得系统进化、生物地理、群体遗传、人类学及法医鉴定等领域的研究得到前所未有的发展.但随着对线粒体基因组遗传特性的了解不断加深,以mtDNA作为分子标记的潜在问题便逐渐暴露出来.本文从7个方面对mtDNA应用的可靠性问题进行了讨论,并对今后线粒体基因的应用前景做了初步分析。 Abstract:With some special features,mtDNA is used widely as molecular marker in the fields of systematic evolution,biogeography,population genetics,anthropology,and forensic medicine.However,more and more evidences that challenge the traditional concept on mtDNA features re found increasingly.In this paper,seven facts that could affect the reliability of mtDNA used as molecular marker are reviewed,then the perspective on application of mtDNA is discussed.     

猕猴桃属植物线粒体DNA片段PCR-RFLP研究初报

猕猴桃属 ( Actinidia)植物 ,全世界有 66个种 ,约 1 1 8个种下分类单位 (变种、变型 ) [1] 。近来通过对猕猴桃属植物的细胞质 DNA研究证实 ,该属植物细胞质 DNA为单亲遗传 ,其线粒体 DNA为严格的母性遗传 [2 ] ,这一单亲遗传特性为研究猕猴桃属植物分类及系统学提供了新的途径。在高等植物中 ,由于线粒体 DNA的高度保守 ,以及重排率高而突变率低 ,加之在植物组织中的含量较低 ,限制了它在系统学研究中的应用 ,不适合属以上高阶元间的比较研究[3 ] ,但线粒体 DNA序列进化速度快 ,在近缘种内或种间显示出了更大的变异性 ,并且线粒体 …     

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.     

中国大陆若干群体的黑果蝇的线粒体DNA多态性研究

本文研究了果蝇Ｄ．ｖｉｒｉｌｉｓ种群Ｄ．ｖｉｒｉｌｉｓ线粒体ＤＮＡ（ｍｉｔｏｃｈｏｎｄｒｉａｌＤＮＡ,ｍｔＤＮＡ）的多态性。用９种限制性内切酶ＸｂａⅠ,ＥｃｏＲⅠ,ＰｓｔⅠ,ＨｉｎｄⅢ,ＢｇｌⅡ,ＳａｃⅠ,ＳｃａⅠ,ＥｃｏＲＶ和ＰｕｖⅡ,对青岛、南京、上海、宁波与泉州５个Ｄ．ｖｉｒｉｌｉｓ群体的ｍｔＤＮＡ进行了限制性片段长度多态性（ｒｅｓｔｒｉｃｔｉｏｎｆｒａｇｍｅｎｔｓｌｅｎｇｔｈｐｏｌｙｍｏｒｐｈｉｓｍ,ＲＦＬＰ）的研究。在５群体中,发现５种不同的酶切图谱,它们彼此之间的遗传差异π为０．４６％－１．７６％,群体内遗传差异πｉｊ为０．００％－０．３３％,群体间的差异ｄｘｙ,为０．００％－０．８２％。分布于中国大陆的Ｄ．ｖｉｒｉｌｉｓ的群体间遗传差异在总遗传差异中所占比例γｓｔ值为２４．６２％。我们发现,Ｄ．ｖｉｒｉｌｉｓ的栖息环境对ｍｔＤＮＡ的遗传变异有十分明显的影响,而不同地理纬度的群体之间其遗传距离并无倾群（ｃｌｉｎｅ）表现。     

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.