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.     

Contrasting phenotypes in three patients with novel mutations in mitochondrial tRNA genes

We studied three patients, each harboring a novel mutation at a highly conserved position in a different mitochondrial tRNA gene. The mutation in patient 1 (T5543C) was associated with isolated mitochondrial myopathy, and occurred in the anticodon loop of tRNA(Trp). In patient 2, with mitochondrial myopathy and marked retinopathy, the mutation (G14710A) resulted in an anticodon swap (Glu to Lys) in tRNA(Glu). Patient 3, who manifested mitochondrial encephalomyopathy and moderate retinal dysfunction, harbored a mutation (C3287A) in the TpsiC loop of tRNA(Leu(UUR)). The mutations were heteroplasmic in muscle in all cases, and sporadic in two cases. PCR-RFLP analysis in all patients showed much higher amounts of mutated mtDNA in affected tissue (muscle) than unaffected tissue (blood), and significantly higher levels of mutated mtDNA in cytochrome c oxidase (COX)-negative muscle fibers than in COX-positive fibers, confirming the pathogenicity of these mutations. The mutation was also detected in single hair roots from all three patients, indicating that each mutation must have arisen early in embryonic development or in maternal germ cells. This suggests that individual hair root analyses may reflect a wider tissue distribution of mutated mtDNA than is clinically apparent, and might be useful in predicting prognosis and, perhaps, the risk of transmitting the mutation to offspring. Our data suggest a correlation between clinical phenotype and distribution of mutated mtDNA in muscle versus hair roots. Furthermore, the high threshold for phenotypic expression in single muscle fibers (92-96%) suggests that therapies may only need to increase the percentage of wild-type mtDNA by a small amount to be beneficial.     

Depletion of the other genome-mitochondrial DNA depletion syndromes in humans

We present the current knowledge on the genetic and phenotypic aspects of mitochondrial DNA depletion syndromes. The human mitochondrial DNA encodes 13 of the 82 structural proteins of the mitochondrial electron transport chain. The replication and maintenance of the mtDNA require a large number of nuclear encoded enzymes and balanced nucleotide pools. Mitochondrial nucleotide synthesis is of major importance because of the constant need for nucleotides for mtDNA maintenance even in quiescent cells. As de novo enzymes are not present in the mitochondria, synthesis is accomplished via the salvage pathway. Defective mtDNA synthesis and maintenance manifest by multiple deletions or by depletion of the mitochondrial genome. Patients with multiple deletions typically present with progressive external ophthalmoplegia, ptosis and, exercise intolerance after the first decade of life. mtDNA depletion is usually an infantile disease characterized by severe muscle weakness, hepatic failure, or renal tubulopathy with fatal outcome. Linkage analysis in families with multiple mtDNA deletions reveal mutations in proteins that participate in mtDNA replication, the mitochondrial DNA polymerase gene, and the Twinkle gene, a putative mitochondrial helicase and in factors which play a role in mitochondrial nucleotide metabolism, the adenine nucleotide translocator, and the thymidine phosphorylase gene. We have recently identified mutations in an additional two essential proteins in the nucleotide salvage pathway, the mitochondrial deoxyribonucleoside kinases. The phenotype was distinctive for each gene, with hepatic failure and encephalopathy associated with mutations in the deoxyguanosine kinase gene and isolated devastating myopathy as the sole manifestation of thymidine kinase 2 deficiency. The tissue selectivity of these disorders and especially the exclusive muscle involvement in thymidine kinase 2 mutations is puzzling. The normal sequence of the remaining mtDNA copies in spite of a serious mitochondrial nucleotide imbalance is also unexpected. We propose several tissue-specific protective mechanisms and a time window, likely encompassing fetal life and even early infancy, during which nuclear nucleotide synthesis provides mitochondrial needs in all organs. We also speculate on future genes to be discovered in other phenotypes of mtDNA depletion.     

Adult onset limb-girdle type mitochondrial myopathy with a mitochondrial DNA np8291 A-to-G substitution

We analyzed mitochondrial DNA (mtDNA) from 7 patients in four families with adult onset limb-girdle type mitochondrial myopathy to clarify their genetic background. The patients, 2 men and 5 women, showed common clinical features, characterized by isolated skeletal myopathy, high serum creatine kinase level, ragged-red fibers and cytochrome c oxidase-defective fibers. Analysis of muscle biopsy specimens indicated that cytochrome c oxidase activity was decreased relative to that of citrate synthase in 5 of the 7 patients. Southern blotting and direct sequence analyses showed an A-to-G homoplasmic transition at np8291 and intergenic COII/tRNA(Lys) 9bp deletion in all patients. This substitution was detected in only 2 of 600 control individuals including healthy subjects and patients with other neuromuscular disorders; these 2 individuals had diabetes mellitus and myotonic dystrophy, respectively. Consequently, the mtDNA transition at np8291 was a rare polymorphism. However, the 7 patients we studied had identical clinical, pathological, biochemical, and genetic features. Therefore, limb-girdle type mitochondrial myopathy with this rare polymorphism may form a subgroup of adult onset mitochondrial myopathy. Received: December 3, 1998 / Accepted: February 18, 1999     

Functional characterization of novel mutations in the human cytochrome b gene.

The great variability of the human mitochondrial DNA (mtDNA) sequence induces many difficulties in the search for its deleterious mutations. We illustrate these pitfalls by the analysis of the cytochrome b gene of 21 patients affected with a mitochondrial disease. Eighteen different sequence variations were found, five of which were new mutations. Extensive analysis of the cytochrome b gene of 146 controls found 20 supplementary mutations, thus further demonstrating the high variability of the cytochrome b sequence. We fully evaluated the functional relevance of 36 of these 38 mutations using indirect criteria such as the nature of the mutation, its frequency in controls, or the phylogenetic conservation of the mutated amino acid. When appropriate, the mtDNA haplotype, the heteroplasmic state of the mutation, its tissue distribution or its familial transmission were also assessed. The molecular consequences of the mutations, which appeared possibly deleterious in that first step of evaluation, were evaluated on the complex III enzymological properties and protein composition using specific antibodies that we have generated against four of its subunits. Two original deleterious mutations were found in the group of seven patients with overt complex III defect. Both mutations (G15150A (W135X) and T15197C (S151P)) were heteroplasmic and restricted to muscle. They had significant consequences on the complex III structure. In contrast, only two homoplasmic missense mutations with dubious clinical relevance were found in the patients without overt complex III defect.     

POLG mutations in sporadic mitochondrial disorders with multiple mtDNA deletions

The accumulation of multiple mitochondrial DNA (mtDNA) deletions in stable tissues is a distinctive feature of several autosomal disorders, characterized by Progressive External Ophthalmoplegia (PEO), ptosis, and proximal myopathy. At least three nuclear genes are responsible for these disorders: ANT1 and C10orf2 cause autosomal dominant PEO, while mutations of DNA polymerase gammaA (POLG1 or POLG) gene on chromosome 15q25 causes both autosomal dominant and recessive forms of PEO. To investigate the contribution of these genes to the sporadic cases of PEO with multiple mtDNA deletions, we studied 31 mitochondrial myopathy patients without any family history for the disorder: 23 had PEO with myopathy, with or without the additional features of pigmentary retinopathy, ataxia, neurosensorial hypoacusia and diabetes mellitus, 7 presented isolated myopathy and one a peripheral neuropathy with ptosis. In all patients Southern blot of muscle DNA showed multiple mtDNA deletions; screening for ANT1 and C10ORF2 genes was negative. POLG analysis revealed mutations in eight patients; in six of them the mutations were allelic, while two patients were heterozygous. Five mutations were new, namely one stop codon (c.2407C>T/p.R709X) and four missense mutations (c.1085G>C/p.G268A; c.1967G>A/p.R562Q; c.2702G>C/p.R807P; c.3076C>T/p.H932W). A high degree of conservation was observed for all the new missense mutations. Only patients presenting PEO as part of their clinical phenotype had POLG mutations, in seven of them together with myopathic signs and in one with a sensori-motor peripheral neuropathy.     

线粒体肌病患者线粒体DNA的突变分析

目的分析线粒体肌病患者线粒体DNA的突变情况，为疾病诊断提供依据。方法用常规HE、酶组化染色和电镜检查等病理形态学方法对3例线粒体肌病疑似患者进行诊断，并用聚合酶链反应-单链构象多态和DNA测序等方法对患者线粒体DNA中全部22个tRNA基因进行突变筛查。结果3例患者均被确诊为线粒体肌病，其中例1tRNA—VaI基因发生A1627G纯合突变，例2tRNA—Val基因发生A1627G／A杂合突变，例3tRNA—Trp基因发生T5554C突变、tRNA—Arg基因发生A10412C／A杂合突变。结论线粒体DNA中的tRNA基因突变是线粒体肌病的重要病因之一。     

Novel mitochondrial DNA mutations associated with myopathy, cardiomyopathy, renal failure, and deafness

Patients with mitochondrial disease usually manifest multisystemic dysfunction with a broad clinical spectrum. When the tests for common mitochondrial DNA (mtDNA) point mutations are negative and the mtDNA defects are still hypothesized, it is necessary to screen the entire mitochondrial genome for unknown mutations in order to confirm the diagnosis. We report an 8-year-old girl who had a long history of ragged-red fiber myopathy, short stature, and deafness, who ultimately developed renal failure and fatal cardiac dysfunction. Respiratory chain enzyme analysis on muscle biopsy revealed deficiency in complexes I, II/III, and IV. Whole mitochondrial genome sequencing analysis was performed. Three novel changes: homoplasmic 15458T > C and 15519T > C in cytochrome b, and a near homoplasmic 5783G > A in tRNA(cys), were found in the proband in various tissues. Her mother and asymptomatic sibling also carry the two homoplasmic mutations and the heteroplasmic 5783G > A mutation in blood, hair follicles, and buccal cells, at lower percentage. The 5783G > A mutation occurs at the T arm of tRNA(cys), resulting in the disruption of the stem structure, which may reduce the stability of the tRNA. 15458T > C changes an amino acid serine to proline at a conserved alpha-helix, which may force the helix to bend. These two mutations may have pathogenic significance. This case emphasizes the importance of pursuing more extensive mutational analysis of mtDNA in the absence of common mtDNA point mutations or large deletions, when there is a high suspicion of a mitochondrial disorder.     

A new pathologic mitochondrial DNA mutation in the cytochrome oxidase subunit I (MT-CO1)

A disorder of mitochondrial energy metabolism may be missed in children with a very mild phenotype. Here, we described a patient with a moderate mental retardation and a mild exercise intolerance. This child harboured a mtDNA transition (m.6955G>A) in the subunit I of the cytochrome oxidase (MT-CO1) that fulfils most of the requirements to be pathologic. Despite this subunit is the second longest polypeptide encoded in the mtDNA, only one other missense mutation associated with a myopathy has been described. This suggests that we are missing other phenotypes and that the mitochondrial pathology field is broader that previously thought.     

A patient with two mitochondrial DNA mutations causing PEO and LHON

We report a 22-year-old man with PEO and optic atrophy. PEO developed before the onset of optic atrophy. The patient showed mitochondrial myopathy with cytochrome c oxidase deficient fibers.In skeletal muscle the patient was homoplasmic for the mtDNA G11778A Leber hereditary optic neuropathy (LHON) mutation and heteroplasmic for the mtDNA 5 kb “common” deletion mutation. In blood only the homoplasmic LHON mutation was identified.The occurrence of two pathogenic mtDNA mutations is exceedingly rare. The clinical findings in this patient indicate that the combination of the two mtDNA mutations resulted in the expected combined phenotype since the mtDNA deletion mutation accounted for the PEO and the mtDNA G11778A point mutation for the optic atrophy.     

Gastrointestinal dysmotility in mitochondrial neurogastrointestinal encephalomyopathy is caused by mitochondrial DNA depletion

Chronic intestinal pseudo-obstruction is a life-threatening condition of unknown pathogenic mechanisms. Chronic intestinal pseudo-obstruction can be a feature of mitochondrial disorders, such as mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a rare autosomal-recessive syndrome, resulting from mutations in the thymidine phosphorylase gene. MNGIE patients show elevated circulating levels of thymidine and deoxyuridine, and accumulate somatic mitochondrial DNA (mtDNA) defects. The present study aimed to clarify the molecular basis of chronic intestinal pseudo-obstruction in MNGIE. Using laser capture microdissection, we correlated the histopathological features with mtDNA defects in different tissues from the gastrointestinal wall of five MNGIE and ten control patients. We found mtDNA depletion, mitochondrial proliferation, and smooth cell atrophy in the external layer of the muscularis propria, in the stomach and in the small intestine of MNGIE patients. In controls, the lowest amounts of mtDNA were present at the same sites, as compared with other layers of the gastrointestinal wall. We also observed mitochondrial proliferation and mtDNA depletion in small vessel endothelial and smooth muscle cells. Thus, visceral mitochondrial myopathy likely causes gastrointestinal dysmotility in MNGIE patients. The low baseline abundance of mtDNA molecules may predispose smooth muscle cells of the muscularis propria external layer to the toxic effects of thymidine and deoxyuridine, and exposure to high circulating levels of nucleosides may account for the mtDNA depletion observed in the small vessel wall.     

High mutational burden in the mtDNA control region from aged muscles: a single-fiber study

The ageing process is associated with the accumulation of somatic mutations of mitochondrial DNA (mtDNA). The aged human skeletal muscle tissue presents a mosaic of fibers when stained histochemically for cytochrome c oxidase (COX) activity with a proportion of COX negative fibers. Given the potential relevance of any alteration in the mtDNA control region for replication, we analysed the correlation between the presence of mutations and their degree of heteroplasmy and the COX phenotype in individual muscle fibers of aged healthy donors.A region of the mtDNA D-loop was cloned from single fiber-derived DNA and multiple clones were analysed. This strategy showed that a high level of mutational burden is present in all fibers and that several types of mtDNA rearrangements are detectable: recurrent (A189G, T408A and T414G) and rare point mutations, length variations affecting the homopolymeric tract and the (CA)(n) repeat and macrodeletions. The aggregate mutational load in the D-loop region correlated with the single fiber COX phenotype, suggesting that the cumulative burden of multiple, individually rare, mtDNA alterations might functionally impair the mitochondrial genetic machinery.     

A novel heteroplasmic tRNAleu(CUN) mtDNA point mutation in a sporadic patient with mitochondrial encephalomyopathy segregates rapidly in skeletal muscle and suggests an approach to therapy

A novel mtDNA point mutation was detected in the tRNAleu(CUN) gene (G to A at position 12315) in a sporadic patient with chronic progressive external ophthalmoplegia, ptosis, limb weakness, sensorineural hearing loss and a pigmentary retinopathy. The mutation disrupts base pairing in the T psi C stem at a site which has been conserved throughout evolution. Although the other mtDNA tRNAleu gene (UUR) is a hotspot for mutation, this is the first pathogenic mutation to be reported in the gene coding for tRNAleu(CUN). MtDNAs carrying the mutation constituted 94% of total mtDNAs in two separate muscle biopsies. Single fibre analysis showed that skeletal muscle fibres without detectable cytochrome c oxidase activity (COX-ve fibres) contained predominantly mutant mtDNAs (93-98%) while fibres with apparently normal COX activity had up to 90% mutant mtDNAs, demonstrating that the G12315A mutation is functionally recessive. Immunofluorescence studies with specific antibodies to mtDNA- or nuclear-encoded subunits of COX were consistent with a defect in mitochondrial protein translation. The mutation was not present in blood cells or cultured fibroblasts and surprisingly, it could not be detected in satellite cells cultured from the patient's muscle. This pattern, which may by typical of patients who have inherited new germline pathogenic mtDNA mutations, possibly reflects loss of the mutation by random genetic drift in mitotic tissues and proliferation of mitochondria containing the mutant mtDNA in post- mitotic cells. The absence of mtDNA carrying the mutation in satellite cells suggests that regeneration of skeletal muscle fibres from satellite cells could restore a wild-type mtDNA genotype and normal muscle function.      

Complete loss-of-function of the heart/muscle-specific adenine nucleotide translocator is associated with mitochondrial myopathy and cardiomyopathy

Multiple mitochondrial DNA deletions are associated with clinically heterogeneous disorders transmitted as mendelian traits. Dominant missense mutations were found in the gene encoding the heart and skeletal muscle-specific isoform of the adenine nucleotide translocator (ANT1) in families with autosomal dominant progressive external opthalmoplegia and in a sporadic patient. We herein report on a sporadic patient who presented with hypertrophic cardiomyopathy, mild myopathy with exercise intolerance and lactic acidosis but no ophthalmoplegia. A muscle biopsy showed the presence of numerous ragged-red fibers, and Southern blot analysis disclosed multiple deletions of muscle mitochondrial DNA. Molecular analysis revealed a C to A homozygous mutation at nucleotide 368 of the ANT1 gene. The mutation converted a highly conserved alanine into an aspartic acid at codon 123 and was absent in 500 control individuals. This is the first report of a recessive mutation in the ANT1 gene. The clinical and biochemical features are different from those found in dominant ANT1 mutations, resembling those described in ANT1 knockout mice. No ATP uptake was measured in proteoliposomes reconstituted with protein extracts from the patient's muscle. The equivalent mutation in AAC2, the yeast ortholog of human ANT1, resulted in a complete loss of transport activity and in the inability to rescue the severe Oxidative Phosphorylation phenotype displayed by WB-12, an AAC1/AAC2 defective strain. Interestingly, exposure to reactive oxygen species (ROS) scavengers dramatically increased the viability of the WB-12 transformant, suggesting that increased redox stress is involved in the pathogenesis of the disease and that anti-ROS therapy may be beneficial to patients.     

Clinical heterogeneity in mitochondrial DNA deletion disorders: a diagnostic challenge of Pearson syndrome

The clinical presentation of mitochondrial DNA (mtDNA) disorders is quite diverse. Very often, the initial symptoms do not fit a specific disease, and diagnosis is difficult to make. We describe a patient who presented with macrocytic anemia. Extensive biochemical and clinical work-up failed to provide an etiology for the macrocytic anemia. The patient over the course of 6 years developed gait problems, exercise intolerance, episodic vomiting, short stature, dermatological problems, and recurrent infection. At age 8 years she had encephalopathy with ataxia and dysphagia. The presence of elevated lactate, bilateral basal ganglia calcification, and ragged red fibers led to mtDNA mutational analysis. A novel 4.4-kb deletion from nucleotide position 10,560 to nucleotide position 14, 980 was identified in muscle biopsy. The same heteroplasmic mtDNA deletion was present in blood, buccal cells, and hair follicles, but not in mother's blood, consistent with sporadic mutation in the patient. This case emphasizes the importance of considering mtDNA disorder in patients with multisystemic symptoms that cannot be explained by a specific diagnosis.     

Complete restoration of a wild-type mtDNA genotype in regenerating muscle fibres in a patient with a tRNA point mutation and mitochondrial encephalomyopathy

Replicative segregation of mitochondrial DNA (mtDNA) can produce large differences in the proportions of wild-type and mutant mtDNAs in different cell types of patients with mitochondrial encephalomyopathy. This is particularly striking in the skeletal muscle of patients with Kearns-Sayre syndrome (KSS), a sporadic disease associated with large- scale mtDNA deletions, and in sporadic patients with tRNA point mutations. Although the skeletal muscle fibres of these patients invariably contain a large proportion of mutant mtDNAs, mutant mtDNAs are rare or undetectable in satellite cells cultured from the same muscle biopsy specimens. Since satellite cells are responsible for muscle fibre regeneration, restoration of the wild-type mtDNA genotype might be achieved in these patients by encouraging muscle regeneration. To test this concept, we re-biopsied a patient with a KSS phenotype and a mtDNA point mutation in the tRNAleu(CUN)gene and analysed muscle fibres regenerating at the site of the original muscle biopsy. Regenerating fibres were identified by morphological criteria and by expression of neural cell adhesion molecule (NCAM). All such fibers were positive for cytochrome c oxidase (COX) activity by cytochemistry and essentially homoplasmic for wild-type mtDNA, while the majority of non-regenerating fibres were COX-negative and contained predominantly mutant mtDNAs. These results demonstrate that it may be possible to improve muscle function in similar patients by methods that promote satellite cell incorporation into existing myofibres.      

Noninvasive diagnosis of the 3243A > G mitochondrial DNA mutation using urinary epithelial cells

The 3243A > G mutation is one of the most frequently observed mutations of mitochondrial DNA (mtDNA), and is associated with numerous clinical presentations including mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), progressive external ophthalmoplegia (PEO) and diabetes and deafness. The routine diagnosis of the 3243A > G mutation in blood is difficult as mutation levels are known to decrease in this tissue over time, while in some patients it may be absent. We have directly compared the levels of the 3243A > G mutation in skeletal muscle, blood and urinary epithelial cells in 18 patients and observed a striking correlation between the mutation load in postmitotic muscle and urinary epithelium, a mitotic tissue. These data strongly support the use of urinary epithelial cells as the tissue of choice in the noninvasive diagnosis of the 3243A > G mutation.     

Somatic mitochondrial DNA mutations in cortex and substantia nigra in aging and Parkinson's disease

Oxidative damage to mitochondrial DNA (mtDNA) increases with age in the brain and can induce G:C to T:A and T:A to G:C point mutations. Though rare at any particular site, multiple somatic mtDNA mutations induced by oxidative damage or by other mechanisms may accumulate with age in the brain and thus could play a role in aging and neurodegenerative diseases. However, no prior study has quantified the total burden of mtDNA point mutation subtypes in the brain. Using a highly sensitive cloning and sequencing strategy, we find that the aggregate levels of G:C to T:A and T:A to G:C transversions and of all point mutations increase with age in the frontal cortex (FCtx). In the substantia nigra (SN), the aggregate levels of point mutations in young controls are similar to the levels in the SN or FCtx of elderly subjects. Extrapolation from our data suggests an average of 2.7 (FCtx) to 3.2 (SN) somatic point mutations per mitochondrial genome in elderly subjects. There were no significant differences between Parkinson's disease (PD) patients and age-matched controls in somatic mutation levels. These results indicate that individually rare mtDNA point mutations reach a high aggregate burden in FCtx and SN of elderly subjects.     

ND5 is a hot-spot for multiple atypical mitochondrial DNA deletions in mitochondrial neurogastrointestinal encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive multisystem disorder associated with depletion, multiple deletions and site-specific point mutations of mitochondrial DNA (mtDNA). MNGIE is caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP; endothelial cell growth factor 1). Deficiency of TP leads to dramatically elevated levels of circulating thymidine and deoxyuridine. The alterations of pyrimidine nucleoside metabolism are hypothesized to cause imbalances of mitochondrial nucleotide pools that, in turn, may cause somatic alterations of mtDNA. We have now identified five major forms of mtDNA deletions in the skeletal muscle of MNGIE patients. While direct repeats and imperfectly homologous sequences appear to mediate the formation of mtDNA deletions, the nicotinamide adenine dinucleotide dehydrogenase 5 gene is a hot-spot for these rearrangements. A novel aspect of the mtDNA deletions in MNGIE is the presence of microdeletions at the imperfectly homologous breakpoints.     

Dominant membrane uncoupling by mutant adenine nucleotide translocase in mitochondrial diseases

Adenine nucleotide translocase (Ant) is the most abundant protein on the mitochondrial inner membrane (MIM) primarily involved in ADP/ATP exchange. Ant also possesses a discrete membrane uncoupling activity. Specific mis-sense mutations in the human Ant1 cause autosomal dominant Progressive External Ophthalmoplegia (adPEO), mitochondrial myopathy and cardiomyopathy, which are commonly manifested by fractional mitochondrial DNA (mtDNA) deletions. It is currently thought that the pathogenic mutations alter substrate preference (e.g. ATP versus ADP) thereby dominantly disturbing adenine nucleotide homeostasis in mitochondria. This may interfere with mtDNA replication, consequently affecting mtDNA stability and oxidative phosphorylation. Here, we showed that the adPEO-type A128P, A106D and M114P mutations in the yeast Aac2p share the following common dominant phenotypes: electron transport chain damage, intolerance to moderate over-expression, synthetic lethality with low Deltapsi(m) conditions, hypersensitivity to the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and mtDNA instability. More interestingly, the aac2(A137D) allele mimicking ant1(A123D) in mitochondrial myopathy and cardiomyopathy exhibits similar dominant phenotypes. Because Aac2(A137D) is known to completely lack transport activity, it is strongly argued that the dominant mitochondrial damages are not caused by aberrant nucleotide transport. The four pathogenic mutations occur in a structurally dynamic gating region on the cytosolic side. We provided direct evidence that the mutant alleles uncouple mitochondrial respiration. The pathogenic mutations likely enhance the intrinsic proton-conducting activity of Ant, which excessively uncouples the MIM thereby affecting energy transduction and mitochondrial biogenesis. mtDNA disintegration is a phenotype co-lateral to mitochondrial damages. These findings provide mechanistic insights into the pathogenesis of the Ant1-induced diseases.