Mitochondrial DNA analysis of Sporothrix schenckii]

Restriction fragment length polymorphism (RFLP) in mitochondrial DNA (mtDNA) of clinical and environmental isolates of Sporothrix schenckii was investigated. Isolates of S. schenckii were classified into 24 mtDNA types (Types 1-24) based on mtDNA RFLP patterns with HaeIII and clustered into two major groups by phylogeny, Group A and Group B. Group A isolates are predominant in South Africa, North America, Central America and South America, while Group B isolates are predominant in Australia, Japan and China. Based on the mtDNA-RFLP patterns with HaeIII, most environmental isolates morphologically identified as S. schenckii were confirmed to be species distinct from S. schenckii and S. schenckii isolates were few, while all of more than 500 clinical isolates were confirmed as S. schenckii. Therefore, RFLP analysis of mtDNA is essential for the identification of environmental, but not clinical isolates of S. schenckii.     

线粒体DNA与人精子活力间的相关性分析

目的 探讨线粒体ＤＮＡ与人精子活力间的关系。方法 用长链ＰＣＲ技术,对６０例精子活力正常和４０例精子活力异常不育患者的精子线粒体ＤＮＡ（ｍｔＤＮＡ）进行了多重缺失的分析。结果 两组不育患者中共有８例具有ｍｔＤＮＡ的多重缺失（其中精子活力正常不育患者６名,精子活力异常不育患者２名）,但缺失型ｍｔＤＮＡ（Ｓ５除外）在总ｍｔＤＮＡ中所占比例很小（０．１６％～１．８５％）,１例精子活力正常的不育患者（Ｓ５     

Molecular epidemiology of Sporothrix schenckii]

Restriction fragment length polymorphism (RFLP) in mitochondrial DNA (mtDNA) of clinical and environmental isolates of Sporothrix schenckii was investigated. Among mtDNA RFLP patterns with Hae III, of 14 environmental isolates morphologically identified as S. schenckii, only 2 isolates were confirmed as S. schenckii, while of more than 500 clinical isolates, all were confirmed to have this condition. Therefore, RFLP analysis of mtDNA is essential for the identification of environmental, but not clinical, isolates of S. schenckii. Isolates of Sporothrix schenckii were classified into 23 mtDNA types (Types l-23) based on mtDNA RFLP patterns with HaeIII and clustered into two major groups by phylogeny, Group A (Types 1-3, 11, 14-19, 22 and 23) and Group B (Types 4-10, 12, 13, 20 and 21). Group A isolates are predominant in South Africa, North America, Central America and South America, while Group B isolates are predominant in Australia and Japan. In Japan, the relative distribution of the mtDNA types varied with geographic region: Types 4, 6 are comparatively abundant in West Japan (Kansai and Kyushu districts), Type 5 is comparatively abundant in East Japan (Tokai, Kanto and Tohoku districts) and Type 2 is abundant in the Hokuriku district. Type 1 is found only in the Hokuriku district.     

Respiratory chain complex I deficiency caused by mitochondrial DNA mutations

Defects of the mitochondrial respiratory chain are associated with a diverse spectrum of clinical phenotypes, and may be caused by mutations in either the nuclear or the mitochondrial genome (mitochondrial DNA (mtDNA)). Isolated complex I deficiency is the most common enzyme defect in mitochondrial disorders, particularly in children in whom family history is often consistent with sporadic or autosomal recessive inheritance, implicating a nuclear genetic cause. In contrast, although a number of recurrent, pathogenic mtDNA mutations have been described, historically, these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency from 101 families. Pathogenic mtDNA mutations were found in 29 of 101 probands (29%), 21 in MTND subunit genes and 8 in mtDNA tRNA genes. Nuclear gene defects were inferred in 38 of 101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. The median age at onset was higher in mtDNA patients (12 months) than in patients with a nuclear gene defect (3 months). However, considerable overlap existed, with onset varying from 0 to >60 months in both groups. Our findings confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children. In the absence of parental consanguinity, we recommend whole mitochondrial genome sequencing as a key approach to elucidate the underlying molecular genetic abnormality.     

Characterization of a novel mitochondrial DNA deletion in a patient with a variant of the Pearson marrow-pancreas syndrome

We have recently diagnosed a patient with anaemia, severe tubulopathy, and diabetes mellitus. As the clinical characteristics resembled Pearson marrow-pancreas syndrome, despite the absence of malfunctioning of the exocrine pancreas in this patient, we have performed DNA analysis to seek for deletions in mtDNA. DNA analysis showed a novel heteroplasmic deletion in mtDNA of 8034bp in length, with high proportions of deleted mtDNA in leukocytes, liver, kidney, and muscle. No deletion could be detected in mtDNA of leukocytes from her mother and young brother, indicating the sporadic occurrence of this deletion. During culture, skin fibroblasts exhibited a rapid decrease of heteroplasmy indicating a selection against the deletion in proliferating cells. We estimate that per cell division heteroplasmy levels decrease by 0.8%. By techniques of fluorescent in situ hybridisation (FISH) and mitochondria-mediated transformation of rho(o) cells we could show inter- as well as intracellular variation in the distribution of deleted mtDNA in a cell population of cultured skin fibroblasts. Furthermore, we studied the mitochondrial translation capacity in cybrid cells containing various proportions of deleted mtDNA. This result revealed a sharp threshold, around 80%, in the proportion of deleted mtDNA, above which there was strong depression of overall mitochondrial translation, and below which there was complementation of the deleted mtDNA by the wild-type DNA. Moreover, catastrophic loss of mtDNA occurred in cybrid cells containing 80% deleted mtDNA.     

Molecular insight into mitochondrial DNA depletion syndrome in two patients with novel mutations in the deoxyguanosine kinase and thymidine kinase 2 genes

Thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) are the two key enzymes in mitochondrial DNA (mtDNA) precursor synthesis. Deficiencies in TK2 or dGK activity, due to genetic alteration, have been shown to cause tissue-specific depletion of mtDNA. In the case of TK2 deficiency, affected individuals suffer severe myopathy and, in the case of dGK deficiency, devastating liver or multi-systemic disease. Here, we report clinical and biochemical findings from two patients with mtDNA depletion syndrome. Patient A was a compound heterozygote carrying the previously reported T77M mutation and a novel mutation (R161K) in the TK2 gene. Patient B carried a novel mutation (L250S) in the dGK gene. The clinical symptoms of patient A included muscular weakness and exercise intolerance due to a severe mitochondrial myopathy associated with a 92% reduction in mtDNA. There was minimal involvement of other organs. Patient B suffered from rapidly progressive, early onset fatal liver failure associated with profoundly decreased mtDNA levels in liver and, to a lesser extent, in skeletal muscle. Site-directed mutagenesis was used to introduce the mutations detected in patients A and B into the TK2 and dGK cDNAs, respectively. We then characterized each of these recombinant enzymes. Catalytic activities of the three mutant enzymes were reduced to about 2-4% for TK2 and 0.5% for dGK as compared to the wild-type enzymes. Altered competition between dCyd and dThd was observed for the T77M mutant. The residual activities of the two mitochondrial enzymes correlated directly with disease development.     

Role of mitochondrial mutations in cancer

A role for mitochondria in cancer causation has been implicated through identification of mutations in the mitochondrial DNA (mtDNA) and in nuclear-encoded mitochondrial genes. Although many mtDNA mutations were detected in common tumors, an unequivocal causal link between heritable mitochondrial abnormalities and cancer is provided only by the germ line mutations in the nuclear-encoded genes for succinate dehydrogenase (mitochondrial complex II) and fumarate hydratase (fumarase). The absence of evidence for highly penetrant tumors caused by inherited mtDNA mutations contrasts with the frequent occurrence of mtDNA mutations in many different tumor types. Thus, either the majority of diverse mtDNA mutations observed in tumors are not important for the process of carcinogenesis or that they play a common oncogenic role.     

Mitochondrial DNA analysis of Sporothrix schenckii in South Africa and Australia.

Clinical isolates of Sporothrix schenckii were investigated for mitochondrial DNA (mtDNA) types using restriction fragment length polymorphism patterns with HaeIII. The 62isolates in South Africa comprised Types 3 (9.7%), 4 (1.6%), 11 (9.7%), 17 (77.4%) and 23 (1.6%) while the 23 Australian isolates comprised Types 3 (26.0%), 4 (56.5%), 7 (8.6%) and 21 (8.6%). In a phylogenetic tree based on the sequence divergence of mtDNA, the mtDNA types were clustered into two groups, A and B. The results suggested that isolates in South Africa mainly belong to Group A and isolates in Australia mainly belong to Group B.     

The Amerindian mtDNA haplogroup B2 enhances the risk of HPV for cervical cancer: de-regulation of mitochondrial genes may be involved

Although human papillomavirus (HPV) infection is the main causal factor for cervical cancer (CC), there are data suggesting that genetic factors could modulate the risk for CC. Sibling studies suggest that maternally inherited factors could be involved in CC. To assess whether mitochondrial DNA (mtDNA) polymorphisms are associated to CC, HPV infection and HPV types, a case-control study was performed in the Mexican population. Polymorphism of mtDNA D-loop was investigated in 187 CC patients and 270 healthy controls. HPV was detected and typed in cervical scrapes. The expression of 29 mitochondrial genes was analyzed in a subset of 45 tumor biopsies using the expression microarray ST1.0. The Amerindian haplogroup B2 increased the risk for CC (odds ratio (OR)=1.6; 95% confidence interval (CI): 1.05-2.58) and enhanced 36% (OR=208; 95% CI: 25.2-1735.5) the risk conferred by the HPV alone (OR=152.9; 95% CI: 65.4-357.5). In cases, the distribution of HPV types was similar in all haplogroups but one (D1), in which is remarkable the absence of HPV18, a very low frequency of HPV16 and high frequencies of HPV45, HPV31 and other HPV types. Two mtDNA genes (mitochondrial aspartic acid tRNA (MT-TD), mitochondrial lysine tRNA (MT-TK)) could be involved in the increased risk conferred by the haplogroup B2, as they were upregulated exclusively in B2 tumors (P<0.01, t-test). Although the association of mtDNA with CC and HPV infection is clear, other studies with higher sample size will be needed to elucidate the role of mtDNA in cervical carcinogenesis.     

Ancient mitochondrial DNA from Malaysian hair samples: some indications of Southeast Asian population movements.

The late Pleistocene and early Holocene population history of Southeast Asia is not well-known. Our study provides new data on mitochondrial DNA (mtDNA) lineages of the aboriginal inhabitants of the Malay Peninsula, and through an extensive comparison to the known mtDNA diversity in Southeast and East Asia, provides some new insights into the origins and historical geography of certain mtDNA lineages in the region. We extracted DNA from hair samples (dating back 100 years) preserved in the Duckworth Collection and belonging to two Peninsular Malaysian individuals identified as "Negrito." Ancient DNA was analyzed by sequencing hypervariable region I (HVS-I) of the mtDNA control region and the mtDNA region V length polymorphism. The results show that the maternal lineages of these individuals belong to a recently defined haplogroup B sub-branch called B4c2. A comparison of mitochondrial haplotypes and haplogroups with those of 10,349 East Asian individuals indicates their very restricted geographical distribution (southwestern China, Southeast Asia Peninsula, and Indonesia). Recalculation of the B4c2 age across all of East Asia ( approximately 13,000 years) and in different subregions/populations suggests its rapid diffusion in Southeast Asia between the end of the Last Glacial Maximum and the Neolithic expansion of the Holocene.     

A comparison of mitochondrial and nuclear DNA status in testicular sperm from fertile men and those with obstructive azoospermia

BACKGROUND: Mitochondria are vital to sperm as their motility powerhouses. They are also the only animal organelles with their own unique genome; encoding subunits for the complexes required for the electron transfer chain. METHODS: A modified long PCR technique was used to study mitochondrial DNA (mtDNA) in ejaculated and testicular sperm samples from fertile men undergoing vasectomy (n = 11) and testicular sperm from men with obstructive azoospermia (n = 25). Nuclear DNA (nDNA) fragmentation was measured by an alkaline gel electrophoresis (comet) assay. RESULTS: Wild-type mtDNA was detected in only 60% of fertile men's testicular sperm, 50% of their ejaculated sperm and 46% of testicular sperm from men with obstructive azoospermia. The incidence of mitochondrial deletions in testicular sperm of fertile and infertile men was not significantly different, but the mean size of the deletions was significantly less in testicular sperm from fertile men compared with men with obstructive azoospermia (P < 0.02). NDNA fragmentation in testicular sperm from fertile men and men with obstructive azoospermia was not significantly different. CONCLUSION: Multiple mtDNA deletions are common in testicular and ejaculated sperm from both fertile and infertile men. However, in males with obstructive azoospermia, the mtDNA deletions in testicular sperm are of a larger scale.     

Spontaneous and artificial changes in human ooplasmic mitochondria

Our research has focused on promoting the development of compromised embryos by transferring presumably normal ooplasm, including mitochondria, to oocytes during intracytoplasmic insemination. Because of the enigma of mitochondrial heteroplasmy, the mixing of populations of oocyte cytoplasm has provoked considerable debate. We are currently investigating oocyte mitochondrial (mt) DNA mutations and the effects of ooplasmic transplantation on mitochondrial inheritance and mitochondrial functionality. Ageing human oocytes could accumulate mtDNA deletions, which might lead to detrimental development. Elimination of abnormal, rearranged mtDNA, such that the offspring inherit only normal mitochondria, is postulated to occur by a mtDNA 'bottleneck'. Among compromised human oocytes (n = 74) and early embryos (n = 137), investigations have shown the occurrence of deltamtDNA4977, the so-called common deletion, to be 33% among oocytes and 8% among embryos. Using a nested polymerase chain reaction (PCR) strategy of long followed by short PCR, another 23 novel mtDNA rearrangements were found: various rearrangements were present in 51% of the oocytes (n = 295) and 32% of early embryos (n = 197). The difference in the percentage of mtDNA rearrangements between oocytes and embryos was significant (P < 0.0001) and implies that there could be a process of selection as fertilized oocytes become embryos. There was no significant relationship between the percentage of human oocytes or embryos that contained mtDNA rearrangements and age. The first series of ooplasmic transfers have been performed in women with repeated implantation failure associated with slow and morphologically abnormal development of their embryos. In a total of 23 attempts in 21 women, eight healthy babies have been born and other pregnancies are ongoing. By examining the donor and recipient blood samples it is possible to distinguish differences in their mtDNA fingerprint. A small proportion of donor mitochondrial DNA was detected in samples with the following frequencies: embryos (six out of 13), amniocytes (one out of four), placenta (two out of four), and fetal cord blood (two out of four). Ooplasmic transfer can thus result in sustained mtDNA heteroplasmy representing both the donor and recipient.     

Decreased mtDNA, oxidative stress, cardiomyopathy, and death from transgenic cardiac targeted human mutant polymerase gamma

POLG is the human gene that encodes the catalytic subunit of DNA polymerase gamma (Pol gamma), the replicase for human mitochondrial DNA (mtDNA). A POLG Y955C point mutation causes human chronic progressive external ophthalmoplegia (CPEO), a mitochondrial disease with eye muscle weakness and mtDNA defects. Y955C POLG was targeted transgenically (TG) to the murine heart. Survival was determined in four TG (+/-) lines and wild-type (WT) littermates (-/-). Left ventricle (LV) performance (echocardiography and MRI), heart rate (electrocardiography), mtDNA abundance (real time PCR), oxidation of mtDNA (8-OHdG), histopathology and electron microscopy defined the phenotype. Cardiac targeted Y955C POLG yielded a molecular signature of CPEO in the heart with cardiomyopathy (CM), mitochondrial oxidative stress, and premature death. Increased LV cavity size and LV mass, bradycardia, decreased mtDNA, increased 8-OHdG, and cardiac histopathological and mitochondrial EM defects supported and defined the phenotype. This study underscores the pathogenetic role of human mutant POLG and its gene product in mtDNA depletion, mitochondrial oxidative stress, and CM as it relates to the genetic defect in CPEO. The transgenic model pathophysiologically links human mutant Pol gamma, mtDNA depletion, and mitochondrial oxidative stress to the mtDNA replication apparatus and to CM.     

Mitochondrial DNA polymorphisms in the human pathogenic fungus Cryptococcus neoformans

This study examined mitochondrial DNA (mtDNA) restriction site polymorphisms among 416 strains of the human pathogenic yeast Cryptococcus neoformans from the United States and Japan. The strains included 378 serotype A, 14 serotype D, 18 serotype AD, two serotype B, and two strains whose serotype could not be determined using current commercial monoclonal antibodies. Portions of two genes were examined: (1) the mitochondrial large ribosomal RNA gene (mtLrRNA) and (2) the NADH dehydrogenase subunit 2 ( ND2). To screen for polymorphisms among the 416 strains, the endonuclease MaeIII was used to digest the PCR-amplified mtLrRNA gene fragment and three endonucleases ( BanI, AluI, MseI) were used to digest the PCR-amplified ND2 gene fragment. Four mtDNA haplotypes were identified among these strains. All strains of serotype A had mtDNA haplotype I, strains of serotype D had haplotype II, and strains of serotype B had haplotypes III and IV. Of the two non-typable strains, one was haplotype I while the other was haplotype II. Among the strains of serotype AD, 14 were haplotype I and the other four were haplotype II. These results were discussed in the context of recent findings regarding the origins of serotype AD strains and the observed uniparental mtDNA inheritance in laboratory crosses between strains of serotypes A and D.     

Histological heterogeneity and somatic mtDNA mutations in gastric intraepithelial neoplasia.

Somatic mutations of mitochondrial DNA (mtDNA) are associated with various types of human cancer. To elucidate their role in gastric carcinogenesis, we analyzed mutations in the displacement loop region of mtDNA in 24 paraffin-embedded gastric intraepithelial neoplasias (formerly dysplasia) from a high gastric cancer risk area in northern Italy. Helicobacter pylori infection was assessed by histological examination (Giemsa staining). Gastritis was classified according to the guidelines of the Updated Sydney System. The mtDNA displacement loop region was amplified and sequenced from gastric intraepithelial neoplasia samples and adjacent non-neoplastic gastric mucosa. The gastric intraepithelial neoplasias were divided into two groups by their association with H. pylori gastritis. Group A with lesions arising on a background of H. pylori-positive gastritis contained 7 patients, and group B with lesions associated with H. pylori-negative gastritis contained 17 patients. Group A had a larger proportion of high-grade lesions than group B and showed a foveolar phenotype (type II dysplasia). Group B had a larger proportion of cases with mtDNA displacement loop region mutations than group A (P=0.004, Fisher's exact test) and exhibited an intestinal phenotype. No evidence of heteroplasmic variants in the mtDNA displacement loop, suggestive of mutations, was detected in gastric biopsies from 25 H. pylori-negative subjects and 60 cancer-unaffected H. pylori-positive patients. These results provide further evidence for the morphologic and mtDNA biomolecular differences of gastric intraepithelial neoplasias, and suggest the existence of two distinct pathways to gastric cancer--corpus-dominant H. pylori gastritis and the atrophy-metaplasia pathway.     

Tigecycline-induced inhibition of mitochondrial DNA translation may cause lethal mitochondrial dysfunction in humans

BackgroundA 65-year-old patient developed an unexplained and ultimately lethal metabolic acidosis under prolonged treatment with tigecycline. Tigecycline is known to have a selective inhibitory effect on eukaryotic mitochondrial translation. The underlying molecular mechanisms of the metabolic acidosis in this patient were explored.MethodsOxidative phosphorylation system (OXPHOS) analysis, blue native polyacrylamide gel electrophoresis followed by in-gel activity staining in mitochondria, molecular analysis of mitochondrial DNA (mtDNA) for genomic rearrangements and sequencing of the rRNA genes was performed on the subject's skeletal muscle.ResultsOXPHOS analysis revealed a combined deficiency of the complexes I, III, IV and V, with a preserved function of complex II (encoded by nuclear DNA), thus demonstrating a defective mtDNA translation. There were no known underlying mitochondrial genetic defects. The patient had a (m.1391T>A) variant within the 12SrRNA gene in heteroplasmy (50–60%).ConclusionsThis patient developed an ultimately lethal mitochondrial toxicity while receiving prolonged treatment with tigecycline, which was caused by a defective translation of the mtDNA. Tigecycline is known to suppress eukaryotic mitochondrial DNA translation, but until now this effect has been considered to be clinically insignificant. The observations in this patient suggest a clinically significant mitochondrial toxicity of tigecycline in this patient, and warrant further investigation.     

Exercise intolerance due to mutations in the cytochrome b gene of mitochondrial DNA.

BACKGROUND: The mitochondrial myopathies typically affect many organ systems and are associated with mutations in mitochondrial DNA (mtDNA) that are maternally inherited. However, there is also a sporadic form of mitochondrial myopathy in which exercise intolerance is the predominant symptom. We studied the biochemical and molecular characteristics of this sporadic myopathy. METHODS: We sequenced the mtDNA cytochrome b gene in blood and muscle specimens from five patients with severe exercise intolerance, lactic acidosis in the resting state (in four patients), and biochemical evidence of complex III deficiency. We compared the clinical and molecular features of these patients with those previously described in four other patients with mutations in the cytochrome b gene. RESULTS: We found a total of three different nonsense mutations (G15084A, G15168A, and G15723A), one missense mutation (G14846A), and a 24-bp deletion (from nucleotide 15498 to 15521) in the cytochrome b gene in the five patients. Each of these mutations impairs the enzymatic function of the cytochrome b protein. In these patients and those previously described, the clinical manifestations included progressive exercise intolerance, proximal limb weakness, and in some cases, attacks of myoglobinuria. There was no maternal inheritance and there were no mutations in tissues other than muscle. The absence of these findings suggests that the disorder is due to somatic mutations in myogenic stem cells after germ-layer differentiation. All the point mutations involved the substitution of adenine for guanine, but all were in different locations. CONCLUSIONS: The sporadic form of mitochondrial myopathy is associated with somatic mutations in the cytochrome b gene of mtDNA. This myopathy is one cause of the common and often elusive syndrome of exercise intolerance.     

Placing mitochondrial DNA mutations within the progression model of type I endometrial carcinoma

Mitochondrial DNA (mtDNA) mutations have been described in almost all types of cancer. However, their exact role and timing of occurrence during tumor development and progression are still a matter of debate. A Vogelstein-like model of progression is well established for endometrial carcinoma (EC), however, mtDNA has been scarcely investigated in these tumors despite the fact that mitochondrial biogenesis increase has been shown to be a hallmark of type I EC. Here, we screened a panel of 23 type I EC tissues and matched typical hyperplasia for mutations in mtDNA and in four oncosupressors/oncogenes, namely PTEN, KRAS, CTNNB1 and TP53. Overall, mtDNA mutations were identified in 69% of cases, while mutational events in nuclear genes occurred in 56% of the cases, indicating that mtDNA mutations may precede the genetic instability of these genes canonically involved in progression from hyperplasia to tumor. Protein expression analysis revealed an increase in mitochondrial biogenesis and activation of oxidative stress response mechanisms in tumor tissues, but not in hyperplasia, in correlation with the occurrence of pathogenic mtDNA mutations. Our results point out an involvement of mtDNA mutations in EC progression and explain the increase in mitochondrial biogenesis of type I EC. Last, since mtDNA mutations occur after hyperplasia, their potential role in contributing to genetic instability may be envisioned.     

Analysis of the trinucleotide CAG repeat from the human mitochondrial DNA polymerase gene in healthy and diseased individuals

The human nuclear gene (POLG) for the catalytic subunit of mitochondrial DNA polymerase (DNA polymerase gamma) contains a trinucleotide CAG microsatellite repeat within the coding sequence. We have investigated the frequency of different repeat-length alleles in populations of diseased and healthy individuals. The predominant allele of 10 CAG repeats was found at a very similar frequency (approximately 88%) in both Finnish and ethnically mixed population samples, with homozygosity close to the equilibrium prediction. Other alleles of between 5 and 13 repeat units were detected, but no larger, expanded alleles were found. A series of 51 British myotonic dystrophy patients showed no significant variation from controls, indicating an absence of generalised CAG repeat instability. Patients with a variety of molecular lesions in mtDNA, including sporadic, clonal deletions, maternally inherited point mutations, autosomally transmitted mtDNA depletion and autosomal dominant multiple deletions showed no differences in POLG trinucleotide repeat-length distribution from controls. These findings rule out POLG repeat expansion as a common pathogenic mechanism in disorders characterised by mitochondrial genome instability.