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The mutational spectrum of the mitochondrial DNA (mtDNA) does not resemble any of the known mutational signatures of the nuclear genome and variation in mtDNA mutational spectra between different organisms is still incomprehensible. Since mitochondria are responsible for aerobic respiration, it is expected that mtDNA mutational spectrum is affected by oxidative damage. Assuming that oxidative damage increases with age, we analyse mtDNA mutagenesis of different species in regards to their generation length. Analysing, (i) dozens of thousands of somatic mtDNA mutations in samples of different ages (ii) 70053 polymorphic synonymous mtDNA substitutions reconstructed in 424 mammalian species with different generation lengths and (iii) synonymous nucleotide content of 650 complete mitochondrial genomes of mammalian species we observed that the frequency of AH > GH substitutions (H: heavy strand notation) is twice bigger in species with high versus low generation length making their mtDNA more AH poor and GH rich. Considering that AH > GH substitutions are also sensitive to the time spent single-stranded (TSSS) during asynchronous mtDNA replication we demonstrated that AH > GH substitution rate is a function of both species-specific generation length and position-specific TSSS. We propose that AH > GH is a mitochondria-specific signature of oxidative damage associated with both aging and TSSS., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The presence of granular swollen epithelial cells (GSECs) in tubular cells was recently reported to be a specific change associated with mitochondrial cytopathy. However, at present, GSEC is not routinely evaluated. We, in this study, present a case of glomerulosclerosis, in which the presence of GSECs should provide us one clue to understand the pathogenesis of its progressive decline of renal function. A 54-year-old Japanese female, who had been diagnosed with Graves' disease, was referred for the examination and treatment of her proteinuria (5.4 g/gCre at the first visit to our hospital). A kidney biopsy showed 28.6% of the glomeruli to be globally sclerosed and 10.7% of the glomeruli to have completely collapsed. However, according to a light microscopic analysis, all other glomeruli showed an almost normal appearance, except for some slight enlargement. Almost 30% of the interstitium was damaged by fibrosis. Characteristically, GSECs were observed in the medulla collecting ducts. Although she had no symptoms of either myopathy or encephalopathy, no history of stroke-like episodes or difficulty in hearing, her serum concentrations of lactate and pyruvate were both elevated. Therefore, mitochondrial DNA sequencing was performed to assess the etiopathogenesis of her nephropathy. Consequently, a homoplasmic 7501 T > A replacement, which has not been previously reported in patients with renal diseases, was detected. This case suggests that the routine evaluation of GSECs can provide important clues to assess the etiopathogenesis of cryptogenic glomerulosclerosis.

The purpose of the present study was to identify mitochondrial DNA (mtDNA) polymorphisms and rare variants that associate with elite Japanese athletic status. Subjects comprised 185 elite Japanese athletes who had represented Japan at international competitions (that is, 100 endurance/middle-power athletes: EMA; 85 sprint/power athletes: SPA) and 672 Japanese controls (CON). The entire mtDNA sequences (16 569 bp) were analyzed by direct sequencing. Nucleotide variants were detected at 1488 sites in the 857 entire mtDNA sequences. A total of 311 variants were polymorphisms (minor allele frequency 1% in CON), and the frequencies of these polymorphisms were compared among the three groups. The EMA displayed excess of seven polymorphisms, including subhaplogroup D4e2- and D4g-specific polymorphisms, compared with CON (P<0.05), whereas SPA displayed excess of three polymorphisms and dearth of nine polymorphisms, including haplogroup G- and subhaplogroup G2a-specific polymorphisms, compared with CON (P<0.05). The frequencies of 10 polymorphisms, including haplogroup G- and subhaplogroup G2a-specific polymorphisms, were different between EMA and SPA (P<0.05): although none of these polymorphisms differed significantly between groups after correcting for multiple comparison (false discovery rate q-value 0.05). The number of rare variants in the 12S ribosomal RNA and NADH dehydrogenase subunit I genes were also higher in SPA than in CON (P<0.05). Analysis of the entire mtDNA of elite Japanese athletes revealed several haplogroup- and subhaplogroup-specific polymorphisms to be potentially associated with elite Japanese athletic status.

Hearing loss (HL) is the most common sensory disorder in humans. Many patients with mitochondrial diseases have sensorineural HL (SNHL). The HL of these patients manifests as a consequence of either syndromic or nonsyndromic mitochondrial diseases. Furthermore, the phenotypes vary among patients even if they are carrying the same mutation. Therefore, these features make it necessary to analyze every presumed mutation in patients with hereditary HL, but the extensive analysis of various mutations is laborious. We analyzed 373 patients with suspected hereditary HL by using an extended suspension-array screening system for major mitochondrial DNA (mtDNA) mutations, which can detect 32 other mtDNA mutations in addition to the previously analyzed 29 mutations. In the present study, we detected 2 different mtDNA mutations among these 373 patients; m.7444G>A in the MT-CO1 gene and m.7472insC in the MT-TS1 gene in 1 patient (0.3%) for each. As these two patients had no clinical features other than HL, they had not been suspected of having mtDNA mutations. This extended screening system together with the previous one is useful for the genetic diagnosis and epidemiological study of both syndromic and nonsyndromic HL.

Conclusion: Haplogroup D4b, especially subhaplogroup D4b2, may be one of the modifiers associated with the phenotypic expression of hereditary hearing loss (HL)., Objectives: The present study investigated the association between suspected hereditary HL and 12 major mtDNA haplogroups in a Japanese population. Besides the mutations of mitochondrial DNA, many modifiers including environmental factors and genetic polymorphisms are involved in HL., Methods: The subjects comprised 373 unrelated Japanese patients with suspected hereditary HL and 480 controls. Twenty of the 373 patients were excluded from the study because the m.1555A>G or the m.3243A>G mutation had been detected in them. The mitochondrial haplotypes were classified into 12 major Japanese haplogroups (i.e. F, B, A, N9a, N9b, M7a, M7b, G1, G2, D4a, D4b, and D5). The frequency of each haplogroup in patients with HL was compared with that of the controls using the chi-squared test., Results: The frequency of the HL patients carrying the mitochondrial haplogroup D4b was significantly higher than that of the controls (37/353 [10.5%] vs 31/480 [6.5%]; OR 1.70 [95% CI 1.03-2.79, p = 0.036]) and evidence for enhancement was found in subhaplogroup D4b2 (32/353 [9.1%] vs 24/480 [5%], OR 1.89 [95% CI 1.09-3.28, p = 0.021]).

We have previously reported a heteroplasmic mtDNA mutation (T1095C) in the 12SrRNA gene of an Italian family with features of maternally-inherited parkinsonism, antibiotic-mediated deafness and peripheral neuropathy. In the present study, we demonstrate that a transmitochondrial cybrid line derived from the proband of this family shows selective depletion of mitochondrial glutathione and decreases in the activity of complex II/III. Moreover, when exposed to an aminoglycoside antibiotic these cells responded with a ten-fold increase in the number of apoptotic cells compared to controls. These results support a pathogenic role for the T1095C mutation and indicate that the mutation increases the risk for aminoglycoside-induced toxicity., (Copyright © 2012 © Elsevier B.V. and Mitochondria Research Society. All rights reserved. All rights reserved.)

Background: Mitochondrial DNA depletion syndromes are a group of heterogeneous autosomal recessive disorders associated with a severe reduction in mitochondrial DNA in the affected tissues. Sodium pyruvate has been reported to have a therapeutic effect in mitochondrial diseases., Methods: We analyzed the effects of 0.5g/kg of sodium pyruvate administered through a nasogastric tube in a one-year-old patient with myopathic mitochondrial DNA depletion syndrome. To evaluate the improvement, we used the Newcastle Paediatric Mitochondrial Disease Scale (NPMDS) and manual muscle testing. As the improvement of motor functions in this severely disabled infant could not be comprehensively detected by NPMDS, we also observed the infant's ability to perform several tasks such as pouting, winking, and number of times she could tap a toy xylophone with a stick. Blood lactate and pyruvate levels were also monitored., Results: After one month's treatment, the NPMDS score in section IV, the domain for the quality of life, improved from 17 to13. The infant became capable of raising her forearm, lower leg and wrist against gravity. The maximum number of times she could repeat each task increased and the movements became brisker and stronger. No significant change of the blood lactate level or lactate-to-pyruvate ratio, both of which were mildly increased at the initiation of the therapy, was observed despite the clinical improvement., Conclusion: Sodium pyruvate administered at 0.5g/kg improved the muscle strength and the NPMDS score of an infant with myopathic mitochondrial DNA depletion syndrome., General Significance: Sodium pyruvate may be effective for ameliorating the clinical manifestations of mitochondrial diseases. This article is part of a Special Issue entitled: Biochemistry of Mitochondria., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Mitochondrial dynamics including morphological fission and mitochondrial movement are essential to normal mitochondrial and cellular physiology. This study investigated how mtDNA T8993G (NARP)-induced inhibition of mitochondrial complex V altered mitochondrial dynamics in association with a protective mitochondrial phospholipid, cardiolipin (CL), as a potential therapeutic target. NARP cybrids harboring 98% of mtDNA T8993G genes and its parental osteosarcoma 143B cells were studied for comparison, and protection provided by melatonin, a potent mitochondrial protector, was explored. We demonstrate for the first time that NARP mutation significantly enhances apoptotic death as a result of three distinct lethal mitochondrial apoptotic insults including oxidative, Ca(2+), and lipid stress. In addition, NARP significantly augmented pathological depletion of CL. NARP-augmented depletion of CL results in enhanced retardation of mitochondrial movement and fission and later swelling of mitochondria during all insults. These results suggest that CL is a common and crucial pathological target for mitochondrial apoptotic insults. Furthermore, CL possibly plays a central role in regulating mitochondrial dynamics that are associated with NARP-augmented mitochondrial pathologies. Intriguingly, melatonin, by differentially preserving CL during various stresses (oxidation > Ca(2+) > lipid), rescues differentially CL-altered mitochondrial dynamics and cell death (oxidation > Ca(2+) > lipid). Thus, melatonin, in addition to being a mitochondrial antioxidant to antagonize mitochondrial oxidative stress, a mitochondrial permeability transition modulator to antagonize mitochondrial Ca(2+) stress, may stabilize directly CL to prevent its oxidization and/or depletion and, therefore, exerts great potential in rescuing CL-dependent mitochondrial dynamics-associated mitochondrial pathologies for treatment of NARP-induced pathologies and diseases., (© 2011 John Wiley & Sons A/S.)

After several years of research, there is now a consensus that America was populated from Asia through Beringia, probably at the end of the Pleistocene. But many details such as the timing, route(s), and origin of the first settlers remain uncertain. In the last decade genetic evidence has taken on a major role in elucidating the peopling of the Americas. To study the early peopling of South America, we sequenced the control region of mitochondrial DNA from 300 individuals belonging to indigenous populations of Chile and Argentina, and also obtained seven complete mitochondrial DNA sequences. We identified two novel mtDNA monophyletic clades, preliminarily designated B2l and C1b13, which together with the recently described D1g sub-haplogroup have locally high frequencies and are basically restricted to populations from the extreme south of South America. The estimated ages of D1g and B2l, about ~15,000 years BP, together with their similar population dynamics and the high haplotype diversity shown by the networks, suggests that they probably appeared soon after the arrival of the first settlers and agrees with the dating of the earliest archaeological sites in South America (Monte Verde, Chile, 14,500 BP). One further sub-haplogroup, D4h3a5, appears to be restricted to Fuegian-Patagonian populations and reinforces our hypothesis of the continuity of the current Patagonian populations with the initial founders. Our results indicate that the extant native populations inhabiting South Chile and Argentina are a group which had a common origin, and suggest a population break between the extreme south of South America and the more northern part of the continent. Thus the early colonization process was not just an expansion from north to south, but also included movements across the Andes.

Purpose: It has been hypothesised that certain mitochondrial haplogroups, which are defined by the presence of a characteristic cluster of tightly linked mitochondrial DNA polymorphisms, would be associated with elite Japanese athlete status. To examine this hypothesis, the frequencies of mitochondrial haplogroups found in elite Japanese athletes were compared with those in the general Japanese population., Methods: Subjects comprised 139 Olympic athletes (79 endurance/middle-power athletes (EMA), 60 sprint/power athletes (SPA)) and 672 controls (CON). Two mitochondrial DNA fragments containing the hypervariable sequence I (m16024-m16383) of the major non-coding region and the polymorphic site at m.5178C>A within the NADH dehydrogenase subunit 2 gene were sequenced, and subjects were classified into 12 major mitochondrial haplogroups (ie, F, B, A, N9a, N9b, M7a, M7b, M*, G2, G1, D5 or D4). The mitochondrial haplogroup frequency differences among EMA, SPA and CON were then examined., Results: EMA showed an excess of haplogroup G1 (OR 2.52, 95% CI 1.05 to 6.02, p=0.032), with 8.9% compared with 3.7% in CON, whereas SPA displayed a greater proportion of haplogroup F (OR 2.79, 95% CI 1.28 to 6.07, p=0.007), with 15.0% compared with 6.0% in CON., Conclusions: The results suggest that mitochondrial haplogroups G1 and F are associated with elite EMA and SPA status in Japanese athletes, respectively.

To clarify the colonizing process of East/Northeast Asia as well as the peopling of the Americas, identifying the genetic characteristics of Paleolithic Siberians is indispensable. However, no genetic information on the Paleolithic Siberians has hitherto been reported. In the present study, we analyzed ancient DNA recovered from Jomon skeletons excavated from the northernmost island of Japan, Hokkaido, which was connected with southern Siberia in the Paleolithic period. Both the control and coding regions of their mitochondrial DNA (mtDNA) were analyzed in detail, and we confidently assigned 54 mtDNAs to relevant haplogroups. Haplogroups N9b, D4h2, G1b, and M7a were observed in these individuals, with N9b being the predominant one. The fact that all these haplogroups, except M7a, were observed with relatively high frequencies in the southeastern Siberians, but were absent in southeastern Asian populations, implies that most of the Hokkaido Jomon people were direct descendants of Paleolithic Siberians. The coalescence time of N9b (ca. 22,000 years) was before or during the last glacial maximum, implying that the initial trigger for the Jomon migration in Hokkaido was increased glaciations during this period. Interestingly, Hokkaido Jomons lack specific haplogroups that are prevailing in present-day native Siberians, implying that diffusion of these haplogroups in Siberia might have been after the beginning of the Jomon era, about 15,000 years before present., (Copyright © 2011 Wiley-Liss, Inc.)

Aim: We previously reported significant associations between mitochondrial single nucleotide polymorphisms (mtSNPs) and myocardial infarction, atherothrombotic cerebral infarction, metabolic syndrome and type 2 diabetes. Here, we assessed the hypothesis that mtSNPs may confer a risk for atherosclerosis, the most important intermediate phenotype of ischemic cardiovascular events., Methods: The subjects were 1,536 consecutive autopsy cases (827 men and 709 women). The average age at death was 80 years. The severity of coronary atherosclerosis was semi-quantitatively examined on cut sections. We examined 149 mtSNPs using the PCR-Luminex method, with a success rate of 97%. Phylogenetic tree analysis yielded 36 haplogroups. Multiple logistic regression analysis was performed after adjustments for sex, age, and conventional cardiovascular risk factors., Results: Among the 45 mtSNPs with minor genotype frequencies >0.05, 6 mtSNPs were associated with coronary atherosclerosis. Among 10 haplogroups with frequencies >0.04, haplogroups A and M7a were significantly associated with coronary atherosclerosis, with odds ratios (95% confidence intervals) of 1.80 (1.09-2.97; p=0.023) and 1.92 (1.23-3.01; p=0.004), respectively. Haplogroup D4a, which was previously reported to be associated with extreme longevity in a Japanese population, was associated with pathological myocardial infarction in men with an odds ratio of 2.05 (1.01-4.14; p=0.046)., Conclusions: The mitochondrial haplogroups A and M7a confer a significant risk for coronary atherosclerosis in the Japanese. The mitochondrial haplogroup may contribute some genetic risk for coronary heart disease.

Recently published results on the association between metabolic syndrome, type 2 diabetes, myocardial infarction or atherothrombotic cerebral infarction and Japanese major haplogroups based on the comprehensive analysis of mitochondrial genome polymorphisms (mtSNP) in the coding region of human mitochondrial DNA (mtDNA), and longevity-related haplogroups are described in the present review. Our aim was to provide information that would allow us to predict the genetic risk for lifestyle-related diseases and thereby contribute to the primary prevention of these conditions. The mitochondrial genome variation is so large that a given haplogroup might consist of various subhaplogroups carrying unique and presumably functional mtSNP. The frequency of each subhaplogroup is sometimes only a few percent. Therefore, large-scale association study is necessary for elucidating the impact of each subhaplogroup on the susceptibility to various common diseases.

We established an extensive and rapid system using suspension array to detect 61 representative mitochondrial DNA (mtDNA) heteroplasmic or homoplasmic point mutations (29 for Series A and 32 for Series B) in 22 genes: 1 each in MT-RNR1, -TV, -ND1, -TQ, -TW, -TC, and -TH genes; 2 each in MT-TN, -TG, -ND4, -TL2, -TE, and -CYB genes; 3 each in MT-ATP6, -ND3, and -ND5 genes; 4 each in MT-CO1 and -TK genes; 5 each in MT-TI, -TS1, and -ND6 genes; and 10 in the MT-TL1 gene. We carefully selected 5'-biotinylated primers and pooled primers for use in two sets of multiplex-PCR amplifications. To detect both mutant and wild-type mtDNA, even when polymorphisms were present near the target mutation sites, we designed specific oligonucleotide probes. By using the mtDNA point mutation detection system of Series A (29 mutations) and Series B (32 mutations), we screened a total of 3103 mutant sites in 107 DNA samples for Series A and 13,101 mutant sites in 397 DNA samples for Series B. We succeeded in determining 99.4% (Series A) and 99.6% (Series B) of the targeted mutant sites by use of the system. The 22 samples with the m.3243A>G heteroplasmic mutation revealed positive signals with both mutant- and wild-type-specific probes in this detection system with a detection limit of approximately 2%. This genetic screening platform is useful to reach a definitive diagnosis for mitochondrial diseases., (Copyright 2010 Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.)

Sensorineural hearing loss (HL) is one of the most frequent clinical features in patients with mitochondrial diseases caused by mitochondrial DNA (mtDNA) mutations, and hearing is impaired in over half of all cases with mitochondrial disorders. This study analyzed 373 patients with suspected hereditary HL using an extensive and rapid suspension-array screening system for 29 major mtDNA mutations, including the m.1555A>G homoplasmic mutation in the MT-RNR1 gene, which causes non-syndromic sensorineural HL and aminoglycoside-induced HL, and the m.3243A>G heteroplasmic mutation in the MT-TL1 gene. This method is rapid and suitable for large-scale screening because universal 96-well plates are available for use, and because an analysis of each plate can be completed within 1 h. This system detected five different mtDNA mutations in 24 of the 373 (6.4%) patients. The m.1555A>G and m.3243A>G mutations were detected in 11 (2.9%) and 9 (2.7%) patients, respectively. In addition, three mutations, that is, m.8348A>G in the MT-TK gene, m.11778G>A in the MT-ND4 gene and 15498G>A in the MT-CYB gene were detected in one patient for each. This screening system is useful for the genetic diagnosis and epidemiological study of both syndromic and non-syndromic HL.

Mutant mitochondrial (mt) DNA variants are related to human disease and have been investigated using cytoplasmic hybrid (cybrid) cells generated from human tumor cells in which mutant mt maintenance depends on the cell line. It is, however, unclear whether human intercellular fusion of non-tumorous cells influences the maintenance of disease-related mutant mt. A preliminary experiment of cell-cell fusion between a human skin fibroblast cell line from a Lesch-Nyhan syndrome patient and an osteosarcoma cybrid cell line harboring the mitochondrial tRNALeu(UUR)A3243G mutation showed a decrease of A3243G mutant mtDNA in fused cells during passages. In order to confirm the decrease of mutant mtDNA, we performed cell-cell fusion experiments using another human lung fibroblastic cell line. When the hygromycin-resistant osteosarcoma cybrid cell line was fused with the fibroblasts without any A3243G mtDNA mutations, the proportion of A3243G mutant mtDNA in the hybrid cells gradually decreased during cell culture and almost completely disappeared in all hybrid clones at the end of 15 passages. These results indicated that A3243G mutant specific mtDNA decreases in the hybrid background when normal fibroblast-derived cell contents, including the nucleus and mt, were introduced. Thus, we are hypothesizing that the non-tumorigenic fibroblast cellular components induce a difference in replication efficacy between the mtDNAs with and without the A3243G mutant sequence, which may be related to the decrease of disease-related mutant mtDNA in the hybrid cells.

The evidence for the role of mitochondria in Alzheimer's disease (AD) has been well investigated, based on the amyloid hypothesis and its relation to the mitochondrial dysfunction due to oxidative stress. However, contrasting reports describe an unclear picture on the relationship between AD and mitochondrial DNA (mtDNA) variations. Therefore, we analyzed complete mtDNA sequences from 153 AD patients and 129 normal control subjects to determine if inherited mtDNA polymorphisms or rare variants, or both contribute to the etiology of late-onset AD. The results reported herein indicate that inherited mtDNA common polymorphisms could not be the single major causes of AD but that some rare variants in the protein-coding-region may have protective effects for high-risk populations with the APOE e4 allele. Furthermore, our results support the idea that the np956-965 poly-c insertion and 856A>G variant might be a riskfactor for AD.

To test the hypothesis that mitochondrial DNA (mtDNA) variants contribute to the susceptibility to schizophrenia, we sequenced the entire mtDNAs from 93 Japanese schizophrenic patients. Three non-synonymous homoplasmic variants in subunit six of the ATP synthase (MT-ATP6) gene that were detected only in patients but not in controls were suggested to be slightly deleterious, because (1) their original amino acid residues (AA) were highly conserved and (2) the physicochemical differences between the original and altered AA were relatively high. In addition, we detected three novel heteroplasmic variants that were potentially pathogenic. Although functional analysis is needed, rare variants in the mtDNA may convey susceptibility to schizophrenia.

In humans, mitochondrial DNA (mtDNA) is a 16,569-bp double-stranded circular molecule, encoding 37 genes, and is exclusively transmitted from the mother. According to the recent findings from many studies of mitochondrial diseases caused by nuclear gene mutations, the accumulation of somatic mtDNA mutations in tissues has been expected to contribute toward age-associated mitochondrial dysfunction and a life span.

Unlabelled: The maternal inheritance of mitochondrial DNA (mtDNA) has enabled construction of detailed phylogenies. Analysis of key polymorphisms from these phylogenies allows mtDNA to be assigned to haplogroups, which have been associated with elite endurance performance., Purpose: To compare the frequencies of mtDNA haplogroups found in elite Kenyan athletes with those in the general Kenyan population., Methods: DNA samples were obtained from 221 national level Kenyan athletes (N), 70 international Kenyan athletes (I), and 85 members of the general Kenyan population (C). mtDNA haplogroups were classified by sequencing 340 bases of hypervariable section (HVS I) and by genotyping known restriction sites. Frequency differences between groups were assessed using exact tests of population differentiation., Results: The haplogroup distribution of national (P = 0.023) and international athletes (P < 0.001) differed significantly from controls, with international athletes showing a greater proportion of L0 haplogroups (C = 15%, N = 18%, I = 30%) and lower proportion of L3* haplogroups (C = 48%, N = 36%, I = 26%). Although a high number of international athletes originated from the Rift Valley province relative to controls (C = 20%, N = 65%, I = 81%), subjects from this province did not differ in haplogroup distribution from other regions (P = 0.23). Nor did Bantu subjects differ from Nilotic (P = 0.12) despite an overrepresentation of Nilotic languages among the athletes., Conclusions: International athletes differed in their mtDNA haplogroup distribution relative to the general Kenyan population. They displayed an excess of L0 haplogroups and a dearth of L3* haplogroups. These findings suggest that mtDNA haplogroups are influential in elite Kenyan distance running, although population stratification cannot be ruled out.

Background: Large-scale genome sequencing poses enormous problems to the logistics of laboratory work and data handling. When numerous fragments of different genomes are PCR amplified and sequenced in a laboratory, there is a high imminent risk of sample confusion. For genetic markers, such as mitochondrial DNA (mtDNA), which are free of natural recombination, single instances of sample mix-up involving different branches of the mtDNA phylogeny would give rise to reticulate patterns and should therefore be detectable., Methodology/principal Findings: We have developed a strategy for comparing new complete mtDNA genomes, one by one, to a current skeleton of the worldwide mtDNA phylogeny. The mutations distinguishing the reference sequence from a putative recombinant sequence can then be allocated to two or more different branches of this phylogenetic skeleton. Thus, one would search for two (or three) near-matches in the total mtDNA database that together best explain the variation seen in the recombinants. The evolutionary pathway from the mtDNA tree connecting this pair together with the recombinant then generate a grid-like median network, from which one can read off the exchanged segments., Conclusions: We have applied this procedure to a large collection of complete human mtDNA sequences, where several recombinants could be distilled by our method. All these recombinant sequences were subsequently corrected by de novo experiments--fully concordant with the predictions from our data-analytical approach.

We report results from the analysis of complete mitochondrial DNA (mtDNA) sequences from 112 Japanese semi-supercentenarians (aged above 105 years) combined with previously published data from 96 patients in each of three non-disease phenotypes: centenarians (99-105 years of age), healthy non-obese males, obese young males and four disease phenotypes, diabetics with and without angiopathy, and Alzheimer's and Parkinson's disease patients. We analyze the correlation between mitochondrial polymorphisms and the longevity phenotype using two different methods. We first use an exhaustive algorithm to identify all maximal patterns of polymorphisms shared by at least five individuals and define a significance score for enrichment of the patterns in each phenotype relative to healthy normals. Our study confirms the correlations observed in a previous study showing enrichment of a hierarchy of haplogroups in the D clade for longevity. For the extreme longevity phenotype we see a single statistically significant signal: a progressive enrichment of certain "beneficial" patterns in centenarians and semi-supercentenarians in the D4a haplogroup. We then use Principal Component Spectral Analysis of the SNP-SNP Covariance Matrix to compare the measured eigenvalues to a Null distribution of eigenvalues on Gaussian datasets to determine whether the correlations in the data (due to longevity) arises from some property of the mutations themselves or whether they are due to population structure. The conclusion is that the correlations are entirely due to population structure (phylogenetic tree). We find no signal for a functional mtDNA SNP correlated with longevity. The fact that the correlations are from the population structure suggests that hitch-hiking on autosomal events is a possible explanation for the observed correlations.

Although an association between mitochondrial DNA (mtDNA) subhaplogroups and complex traits has been suggested, few functional analyses have been reported. To identify the mtDNA subhaplogroups that alter intracellular calcium dynamics, we analysed data on intracellular calcium dynamics in 35 transmitochondrial hybrid cells (cybrids). One cybrid showing decreased calcium levels had mtDNA subhaplogroup G3 or G4, characterised by 1413T>C, 2109A>T, 3434A>G, 5460G>A, 7521G>A, 9011C>T, 9670A>G and 15940T>C. The cybrid having higher calcium levels was subhaplogroup D4a, characterised by a non-synonymous polymorphism, 13651A>G. These mtDNA subhaplogroups might have functional effects.

We report new results from the re-analysis of 672 complete mitochondrial (mtDNA) genomes of unrelated Japanese individuals stratified into seven equal sized groups by the phenotypes: diabetic patients, diabetic patients with severe angiopathy, healthy non-obese young males, obese young males, patients with Alzheimer's disease, patients with Parkinson's disease and centenarians. Each phenotype had 96 samples over 27 known haplogroups: A, B4a, B4b, B4c, B*, B5, D*, F1, F2, M*, M7a, M7b, M8, M9, D4a, D4b1, D4b2, D4d, D4e, D4g, D4h, D5, G, Z, M*, N9a, and N9b. A t-test comparing the fraction of samples in a haplogroup to healthy young males showed a significant enrichment of haplogroups D4a, D5, and D4b2 in centenarians. The D4b2 enrichment was limited to a subgroup of 40 of 61 samples which had the synonymous mutation 9296C > T. We identified this cluster as a distinct haplogroup and labeled it as D4b2b. Using an exhaustive procedure, we constructed the complete list of "mutation patterns" for centenarians and showed that the most significant patterns were in D4a, D5, and D4b2b. We argue that if a selection for longevity appeared only once, it was probably an autosomal event which could be dated to after the appearance of the D mega-group but before the coalescent time of D4a, D5, and D4b2b. Using a simple procedure, we estimated that this event occurred 24.4 +/- 0.9 kYBP.

Because mitochondria play pivotal roles in both insulin secretion from the pancreatic beta cells and insulin resistance of skeletal muscles, we performed a large-scale association study to identify mitochondrial haplogroups that may confer resistance against or susceptibility to type 2 diabetes mellitus (T2DM). The study population comprised 2,906 unrelated Japanese individuals, including 1,289 patients with T2DM and 1,617 controls, and 1,365 unrelated Korean individuals, including 732 patients with T2DM and 633 controls. The genotypes for 25 polymorphisms in the coding region of the mitochondrial genome were determined, and the haplotypes were classified into 10 major haplogroups (i.e., F, B, A, N9a, M7a, M7b, G, D4a, D4b, and D5). Multivariate logistic-regression analysis with adjustment for age and sex revealed that the mitochondrial haplogroup N9a was significantly associated with resistance against T2DM (P=.0002) with an odds ratio of 0.55 (95% confidence interval 0.40-0.75). Even in the modern environment, which is often characterized by satiety and physical inactivity, this haplogroup might confer resistance against T2DM.

Mitochondrion-derived reactive oxygen species possibly play an important role in the pathogenesis of atherosclerosis and atherothrombotic cerebral infarction, because mitochondria in vascular endothelial cells are the major site of superoxide production. In the present study, we surveyed mitochondrial haplogroups associated with atherothrombotic cerebral infarction in 1081 Japanese subjects. Twenty-six mitochondrial single nucleotide polymorphisms of 11 major mitochondrial haplogroups (F, B, A, N9a, M7a, M7b, M7c, G1, G2, D4, and D5) were determined by use of 28-plex PCR and fluorescent beads combined with sequence-specific oligonucleotide probes. Multivariate logistic regression analysis with adjustment for conventional risk factors revealed that mitochondrial haplogroup A was associated with atherothrombotic cerebral infarction in female subjects (P< 0.05). However, no significant association was detected for males. Our study shows that haplogroup A confers an increased risk of atherothrombotic cerebral infarction in Japanese females. Validation of our findings will require additional studies with independent subject panels.

Superoxide, which mitochondria mainly produce in vascular endothelial cells, plays an important role in the pathogenesis of atherosclerosis and coronary artery disease. Accordingly, mitochondrial functional differences are thought to be one of the most important factors for the risk of myocardial infarction among various individuals. In the present study, we surveyed mitochondrial haplogroups associated with myocardial infarction in Japanese subjects. The study population comprised 2,137 unrelated Japanese individuals, including 1,181 subjects with a first myocardial infarction (920 males, 261 females) and the control subjects (522 males, 434 females). Twenty-eight mitochondrial single nucleotide polymorphisms of 12 major mitochondrial haplogroups (A, B, D4, D5, F, G1, G2, M7a, M7b, M7c, N9a, and N9b) were determined by use of 28-plex PCR and fluorescent beads combined with sequence-specific oligonucleotide probes. After adjustment for age, sex, body mass index, and prevalence of smoking, hypertension, hypercholesterolemia, and type 2 diabetes, a significantly (P = 0.0019) lower prevalence of haplogroup N9b was detected in subjects with myocardial infarction than in the controls. Especially, the prevalence of this haplogroup was significantly lower (P = 0.0007) in the male subjects with the disease than in the male controls. In contrast, there were trends towards higher prevalence of the disease in haplogroup G1 for males (P < 0.05). No significant haplogroup-related associations were detected for females. Our data suggest that haplogroup N9b confers resistance against myocardial infarction in Japanese males.

Mitochondrial dysfunction caused by mutations in mitochondrial DNA (mtDNA) is related to a variety of diseases including MELAS and NARP syndromes. However, little is known about the intracellular responses induced by mtDNA mutations. In order to identify genes whose expression is altered as a result of the presence of mtDNA mutations, DNA microarray analysis was performed using human 143B osteosarcoma cells harboring 3243A>G [tRNA-Leu (UUR)] and 8993T>G [ATPase6 Leu156Arg] mtDNA mutations associated with MELAS and NARP syndromes (2SD and NARP3-1 cybrid cells), respectively. We found that mRNA and protein levels of ATF4, CHOP and ASNS were upregulated in 2SD and NARP3-1 cells as compared with parental cells. Reporter assays demonstrated that transcription of CHOP and ASNS genes was upregulated through the AARE (amino acid regulatory element) and NSRE-1 (nutrient-sensing response element-1) enhancer elements to which ATF4 binds, respectively. Furthermore, knockdown of ATF4 by RNA interference reduced CHOP and ASNS transcription in 2SD and NARP3-1 cells. These results suggest that the presence of mtDNA mutations elicits upregulation of CHOP and ASNS genes through the elevation of ATF4 expression and its binding to the AARE and NSRE-1, respectively.

Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function as well as susceptibility to various diseases such as Parkinson disease, Alzheimer disease, bipolar disorder, and cancer. However, it is unclear whether and how mtDNA polymorphisms affect intracellular function, such as calcium signaling or pH regulation. Here we searched for mtDNA polymorphisms that have intracellular functional significance using transmitochondrial hybrid cells (cybrids) carrying ratiometric Pericam (RP), a fluorescent calcium indicator, targeted to the mitochondria and nucleus. By analyzing the entire mtDNA sequence in 35 cybrid lines, we found that two closely linked nonsynonymous polymorphisms, 8701A and 10398A, increased the basal fluorescence ratio of mitochondria-targeted RP. Mitochondrial matrix pH was lower in the cybrids with 8701A/10398A than it was in those with 8701G/10398G, suggesting that the difference observed by RP was mainly caused by alterations in mitochondrial calcium levels. Cytosolic calcium response to histamine also tended to be higher in the cybrids with 8701A/10398A. It has previously been reported that 10398A is associated with an increased risk of Parkinson disease, Alzheimer disease, bipolar disorder, and cancer, whereas 10398G associates with longevity. Our findings suggest that these mtDNA polymorphisms may play a role in the pathophysiology of these complex diseases by affecting mitochondrial matrix pH and intracellular calcium dynamics., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Although classical psychopathological studies have shown the presence of an independent diagnostic category, 'atypical psychosis', most psychotic patients are currently classified into two major diagnostic categories, schizophrenia and bipolar disorder, by the Diagnostic and Statistical Manual of Mental Disorders (4th edn; DSM-IV) criteria. 'Atypical psychosis' is characterized by acute confusion without systematic delusion, emotional instability, and psychomotor excitement or stupor. Such clinical features resemble those seen in organic mental syndrome, and differential diagnosis is often difficult. Because patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) sometimes show organic mental disorder, 'atypical psychosis' may be caused by mutations of mitochondrial DNA (mtDNA) in some patients. In the present study whole mtDNA was sequenced for seven patients with various psychotic disorders, who could be categorized as 'atypical psychosis'. None of them had known mtDNA mutations pathogenic for mitochondrial encephalopathy. Two of seven patients belonged to a subhaplogroup F1b1a with low frequency. These results did not support the hypothesis that clinical presentation of some patients with 'atypical psychosis' is a reflection of subclinical mitochondrial encephalopathy. However, the subhaplogroup F1b1a may be a good target for association study of 'atypical psychosis'.

Although a strong correlation between type-2 diabetes and obesity has been found, no comparative analysis between diabetes and obesity has been performed with respect to mitochondrial DNA (mtDNA) polymorphisms. To test the hypothesis that certain mitochondrial single nucleotide polymorphisms (mtSNPs) might be associated with obesity or type-2 diabetes, we determined the entire sequences of the mitochondrial genomes from 96 patients with type-2 diabetes and those from 96 young obese adults by direct sequencing and compared the frequencies of mtSNPs between these two groups. A mtSNP, 8684C > T (T53I) in the mitochondrial ATP synthase subunit 6 gene (ATP6), was detected in 5 of the 96 patients with type-2 diabetes, whereas this substitution was not detected in any of the 96 young obese adults. Two mtSNPs, 3497C > T (A64V) in NADH dehydrogenase subunit 1 gene (ND1) and 1119T > C (472U > C) in the 12S rRNA gene, were detected in 5 of the 96 young obese adults, whereas these substitutions were not detected in any of the 96 diabetic patients. The 8684C > T transition associated with type-2 diabetes represents haplogroup M8a, and the 3497C > T and 1119T > C transitions predisposing to obesity represent haplogroup B4c. These results suggest that distinct mtSNPs contribute to susceptibility to type-2 diabetes or obesity, pointing out the necessity of large-scale case control studies.

Mitochondrial DNA (mtDNA) coding region polymorphisms, as well as the 150T polymorphism in the noncoding region, have been associated with longevity. We have studied here the association of 150T with longevity further and assessed differences in this association between various mtDNA haplogroups. We analysed a sample of 321 very old subjects and 489 middle-aged controls from Finland and Japan. 150T was more frequent among the very old than among the controls in both the Finnish and Japanese subjects. Interestingly, the association was not similar in all haplogroups, and a stratified analysis revealed that two additional common polymorphisms, 489C and 10398G, modified the association between 150T and longevity. These findings suggest that longevity is partly determined by epistatic interactions involving these three mtDNA loci.

We present a reliable, rapid, and economical multiplex amplified product-length polymorphism (APLP) method for analyzing the haplogroup-diagnostic mitochondrial single-nucleotide polymorphisms (mtSNPs) in East Asian populations. By examining only 36 haplogroup-specific mtSNPs in the coding region by using four 9-multiplex polymerase chain reaction (PCR) and subsequent electrophoresis, we could safely assign 1815 individuals from 8 populations of Japanese, Korean, Chinese, and Germans to 45 relevant haplogroups. This multiplex APLP analysis of coding-region mtSNPs for haplogrouping is especially useful not only for molecular phylogenetic studies but also for large-scale association studies due to its rapid and economical nature. This is the first panel of mtSNPs in the coding region to be used for haplogrouping of East Asian populations.

Mitochondrial dysfunction associated with mutant mitochondrial DNA (mtDNA) has been suggested in bipolar disorder, and comorbidity with neurodegenerative diseases was often noted. We examined the entire sequence of mtDNA in six subjects with bipolar disorder having comorbid somatic symptoms suggestive of mitochondrial disorders and found several uncharacterized homoplasmic nonsynonymous nucleotide substitutions of mtDNA. Of these, 3644C was found in 5 of 199 patients with bipolar disorder but in none of 258 controls (p = 0.015). The association was significant in the extended samples [bipolar disorder, 9/630 (1.43%); controls, 1/734 (0.14%); p = 0.007]. On the other hand, only 5 of 25 family members with this mutation developed bipolar disorder, of which 4 patients with 3644C had comorbid physical symptoms. The 3644T-->C mutation converts amino acid 113, valine, to alanine in the NADH-ubiquinone dehydrogenase subunit I, a subunit of complex I, and 113 valine is well conserved from Drosophila to 61 mammalian species. Using transmitochondrial cybrids, 3644T-->C was shown to decrease mitochondrial membrane potential and complex I activity compared with haplogroup-matched controls. According to human mitochondrial genome polymorphism databases, 3644C was not found in centenarians but was found in 3% of patients with Alzheimer disease and 2% with Parkinson disease. The result of modest functional impairment caused by 3644T-->C suggests that this mutation could increase the risk for bipolar disorder.

To construct an East Asia mitochondrial DNA (mtDNA) phylogeny, we sequenced the complete mitochondrial genomes of 672 Japanese individuals (http://www.giib.or.jp/mtsnp/index&#95;e.html). This allowed us to perform a phylogenetic analysis with a pool of 942 Asiatic sequences. New clades and subclades emerged from the Japanese data. On the basis of this unequivocal phylogeny, we classified 4713 Asian partial mitochondrial sequences, with <10% ambiguity. Applying population and phylogeographic methods, we used these sequences to shed light on the controversial issue of the peopling of Japan. Population-based comparisons confirmed that present-day Japanese have their closest genetic affinity to northern Asian populations, especially to Koreans, which finding is congruent with the proposed Continental gene flow to Japan after the Yayoi period. This phylogeographic approach unraveled a high degree of differentiation in Paleolithic Japanese. Ancient southern and northern migrations were detected based on the existence of basic M and N lineages in Ryukyuans and Ainu. Direct connections with Tibet, parallel to those found for the Y-chromosome, were also apparent. Furthermore, the highest diversity found in Japan for some derived clades suggests that Japan could be included in an area of migratory expansion to Continental Asia. All the theories that have been proposed up to now to explain the peopling of Japan seem insufficient to accommodate fully this complex picture.

Mitochondrial DNA (mtDNA) mutations observed in both non-coding control and coding regions are being used widely to characterize human evolution and for identification. Several methodological approaches have been available for detection of single nucleotide polymorphisms (SNPs). SNPs of the non-coding region were mainly analyzed by means of direct sequencing. Especially, sequence analysis of D-loop is often the final resort in forensic and ancient personal identification. However, sequencing is relatively expensive and time-consuming. Furthermore, because the mtDNA molecule is a single linked unit, the statistical significance in forensic cases requires mtDNA matching in comparison with large sequence data. In this study, we developed a procedure of technique simultaneous typing to 11 SNPs and 2 tandem repeat sites in the non-coding and coding region by use of allele-specific amplification. In an analysis to 631 unrelated Japanese individuals the 180 haplotypes were defined in this study, and the level of haplotypic diversity and random mach probability was similar to that obtained by sequencing of the human hypervariable region 1 in mtDNA. These results show the usefulness of mtDNA haplotype analysis by the presented method for personal identification.

Because mitochondria are the major sources of reactive oxygen species (ROS) in cells, certain alterations in mitochondrial functions can lead to metabolic perturbation in vascular endothelial cells and smooth muscle cells, resulting in vascular dysfunction. We previously demonstrated that a C --> A transversion in mitochondrial DNA (mtDNA) at nucleotide 5178 of the NADH dehydrogenase subunit 2 (ND2) gene, which results in a Lue --> Met substitution at amino acid 237, was found more frequently in Japanese centenarians than in controls. We also demonstrated that this Mt5178C --> A polymorphism has anti-atherosclerotic effects in diabetic subjects. We have now examined whether the Mt5178C --> A (Leu237Met) polymorphism in the mitochondrial ND2 gene is associated with a low prevalence of myocardial infarction (MI) in a case-control study. The genotype of ND2 gene was determined either with a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or a colorimetry-based allele-specific DNA probe assay. Multivariate logistic regression analysis with adjustment for age, gender, body mass index, smoking status, hypertension, diabetes mellitus, hypercholesterolemia, and hyperuricemia revealed that the frequency of the Mt5178A genotype was significantly higher in controls than in subjects with MI. These results suggest that the 5178A genotype of mitochondrial ND2 gene polymorphism is protective against MI; and this effect would explain, at least in part, its contribution to longevity., (Copyright 2004 Elsevier Ireland Ltd)

Polymorphisms in the human mitochondrial genome have been used for the elucidation of phylogenetic relationships among various ethnic groups. Because analysis by mitochondrial genetics has detected pathogenic mutations causing mitochondrial encephalomyopathy or cardiomyopathy, most of the mitochondrial single nucleotide polymorphisms (mtSNPs) found in control subjects have been regarded as merely normal variants. However, we cannot exclude the possibility that the mitochondrial functional differences among individuals are ascribable at least in part to the mtSNPs of each individual. Human lifespan in ancient history was much shorter than that at the present time. Therefore, it is reasonable to speculate that certain mtSNPs that predispose one toward susceptibility to adult- or elderly-onset diseases, such as Parkinson's disease and Alzheimer's disease, have never been a target for natural selection in the past. Similarly, thrifty mtSNPs that had been advantageous for survival under severe famine or cold climate conditions might turn out to be related to satiation-related diseases, such as diabetes mellitus and obesity. To examine these hypotheses, we have constructed a mtSNP database by sequencing the entire mitochondrial genomes of 672 subjects: 96 in each of seven groups (i.e., centenarians, young obese or non-obese subjects, diabetic patients with or without major vascular involvement, patients with Parkinson's disease, and those with Alzheimer's disease).

Although polymorphism of the mitochondrial DNA 15497guanine/adenine (Mt15497G-->A) leads to the Gly251Ser amino acid replacement on human cytochrome b, it is unknown whether functional alteration of the mitochondrion is induced by the Gly251Ser replacement. To see if an association exists between the Mt15497G-->A polymorphism and obesity, we examined differences in body size, body composition, and regional body fat distribution between the two genotypes in middle-aged and elderly Japanese individuals (825 women and 906 men). The Mt15497 genotype was determined with an automated colorimetric allele-specific DNA probe assay system using the polymerase chain reaction (PCR) method. The Mt15497G-->A polymorphism was detected in 3.5% ( n=60) of all subjects: 2.8% ( n=23) among women and 4.1% ( n=37) among men. After adjusting for age and smoking, we found that body weight, body mass index, waist and hip circumferences, fat mass, fat-free mass, intra-abdominal fat and triglycerides were significantly greater in women with the A allele compared with the G allele ( p=0.001-0.025). For men, waist to hip ratio was significantly greater ( p=0.032), and waist circumference, intra-abdominal fat and triglycerides had a trend to be significantly greater ( p=0.062-0.087) in subjects with the A allele compared with the G allele. These data suggest that the Mt15497 polymorphism may be associated with obesity-related variables and lipid metabolism.

To examine an association between the mitochondrial DNA (mt5178) genotype and various cancers, we genotyped 1120 non-cancer controls and 930 cancer cases including esophageal, stomach, colorectal, lung, breast and malignant lymphoma in a sample of Japanese patients. The mt5178A/C was genotyped by the polymerase chain reaction with confronting two-pair primers (PCR-CTPP). The frequency of mt5178A/C within the non-cancer and cancer groups, and age distribution of subjects with mt5178A and C were investigated. Odd ratios (ORs) of the mt5178A and C genotypes were also examined. The frequency of mt5178A was 39.1% in non-cancer subjects while frequencies in those having cancer included 39.0% in breast, 37.4% in colorectal, 45.1% in esophageal, 38.0% in lung, 41.5% in malignant lymphoma, and 38.8% in stomach cancer. There was no significant difference in the frequency of the mt5178 genotype among the six types of cancer studied. There was also no significant difference in the frequency of the mt5178 genotype between non-cancer and cancer subjects regardless of total age with the exception that ages 40-49 years (the frequency of the mt5178A was higher in cancer subjects). There was a significant interaction term between age and the mt5178 genotype in older (age>=60) lung cancer patients. The cumulative frequency of mt5178C increased more markedly than that of mt5178A after age 40 in non-cancer subjects, and after age 50 in cancer subjects ORs of the genotype were not significant for all cancers combined or for any individual site of cancer. In the present study, the mt5178 genotype seems to have no association with any of the cancers examined here. But an interaction term between the mt5178 genotype and aging on cancer was suggested with the Japanese population under study.

To test the hypothesis that centenarians are free from deleterious mitochondrial variations, we analyzed amino acid variations in the cytochrome b molecule of 64 Japanese centenarians. Although the frequencies of some variations, such as N260D and G251S, differed significantly between centenarians and patients with Parkinson's disease, the most striking feature of centenarian cytochrome b was the rareness of amino acid variations in contrast to the variety of amino acid replacements in patients with Parkinson's disease. These results suggest that centenarians are genetically hitting the "golden mean" (less variation from the consensus cytochrome b sequence or less mismatch with other subunits). A multiplex detection system for various deleterious variations in combination with genetic tests for longevity-associated genotypes will be necessary to predict longevity or age-related diseases., (Copyright 2002 Wiley-Liss, Inc.)

The restriction endonuclease SmaI has been used for the diagnosis of neurogenic muscle weakness, ataxia and retinitis pigmentosa disease or Leigh's disease, caused by the Mt8993T-->G mutation which results in a Leu156Arg replacement that blocks proton translocation activity of subunit a of F(0)F(1)-ATPase. Our ultimate goal is to apply SmaI to gene therapy for this disease, because the mutant mitochondrial DNA (mtDNA) coexists with the wild-type mtDNA (heteroplasmy), and because only the mutant mtDNA, but not the wild-type mtDNA, is selectively restricted by the enzyme. For this purpose, we transiently expressed the SmaI gene fused to a mitochondrial targeting sequence in cybrids carrying the mutant mtDNA. Here, we demonstrate that mitochondria targeted by the SmaI enzyme showed specific elimination of the mutant mtDNA. This elimination was followed with repopulation by the wild-type mtDNA, resulting in restoration of both the normal intracellular ATP level and normal mitochondrial membrane potential. Furthermore, in vivo electroporation of the plasmids expressing mitochondrion-targeted EcoRI induced a decrease in cytochrome c oxidase activity in hamster skeletal muscles while causing no degenerative changes in nuclei. Delivery of restriction enzymes into mitochondria is a novel strategy for gene therapy of a special form of mitochondrial diseases., (Copyright 2002 National Science Council, ROC and S. Karger AG, Basel)