Lessons from mitochondrial DNA mutations

seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 9, 2001: pp. 397–405 doi:10.1006/scdb.2001.0277, available online at http://www.idealibrary.com on

Lessons from mitochondrial DNA mutations Salvatore DiMauro

(cytochrome c oxidase), and two subunits of complex V (ATP synthetase). The respiratory complexes also contain nuclear-DNA- (nDNA) encoded subunits, which are imported into the mitochondria from the cytosol and assembled, together with their mtDNA-encoded counterparts, into the respective holoenzymes in the mitochondrial inner membrane. Complex II (succinate dehydrogenase–ubiquinone oxidoreductase) is entirely encoded by nDNA. Mitochondrial genetics differs from Mendelian genetics in three major aspects. 1. Maternal inheritance. At fertilization, all mitochondria (and all mtDNAs) in the zygote derive from the oöcyte. Therefore, a mother carrying an mtDNA mutation is expected to pass it on to all her children, but only her daughters will transmit it to their progeny. 2. Heteroplasmy/threshold effect. In contrast to nuclear genes, each consisting of one maternal and one paternal allele, there are hundreds or thousands of mtDNA molecules in each cell. Deleterious mutations of mtDNA usually affect some but not all genomes, such that cells, tissues, in fact whole individuals, will harbor two populations of mtDNA: normal (wild type) and mutant, a situation known as heteroplasmy. The situation of normal subjects, in whom all mtDNAs are identical, is called homoplasmy. Non-deleterious mutations of mtDNA (neutral polymorphisms) are homoplasmic, whereas pathogenic mutations are usually, but not invariably, heteroplasmic. Not surprisingly, a minimum critical number of mutant mtDNAs must be present before oxidative dysfunction and clinical signs become apparent (threshold effect). Also not surprisingly, the pathogenic threshold will be lower in tissues that are highly dependent on oxidative metabolism than in tissues with higher capacity for anaerobic metabolism. 3. Mitotic segregation. At cell division, the proportion of mutant mtDNAs in daughter cells can shift: if and when the pathogenic threshold for a given tissue is surpassed, the phenotype can also change. This explains the time-related variability of clinical

The small, maternally inherited mitochondrial DNA (mtDNA) has turned out to be a hotbed of pathogenic mutations: 13 years into the era of ‘mitochondrial medicine’, over 100 pathogenic point mutations and countless rearrangements have been associated with a variety of multisystemic or tissue-specific human diseases. MtDNArelated disorders can be divided into two major groups: those due to mutations in genes affecting mitochondrial protein synthesis in toto and those due to mutations in specific protein-coding genes. Pathogenesis is only partially explained by the rules of mitochondrial genetics and remains largely uncharted territory. Therapy is still woefully inadequate, but a number of promising approaches are being developed. Key words: mitochondrial DNA / mitochondrial genetics / mitochondrial encephalomyopathies / mitochondrial respiratory chain c 2001 Academic Press

Mitochondrial genetics Human mtDNA is a 16 569 bp circle of doublestranded DNA. 1 It is highly compact, and contains only 37 genes (Figure 1): two genes encode ribosomal RNAs (rRNAs), 22 encode transfer RNAs (tRNAs), and 13 encode polypeptides. All 13 polypeptides are components of the respiratory chain, including seven subunits of complex I (NADH dehydrogenase–ubiquinone oxidoreductase), one subunit of complex III (ubiquinone–cytochrome c oxidoreductase), three subunits of complex IV

From the Department of Neurology, Columbia University College of Physicians and Surgeons, New York, NY, USA. Corresponding address. 4-420 College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA. E-mail: . c

2001 Academic Press 1084–9521 / 01 / 060397+ 09 / $35.00 / 0

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Figure 1. Morbidity map of the human mitochondrial genome as of January 1, 2000. The map of the 16.5 kb mtDNA shows differently shaded areas representing the protein coding genes for seven subunits of complex I (ND), the three subunits of cytochrome oxidase (COX), cytochrome b (Cyt b), and the two subunits of ATP synthetase (ATPase 6 and 8), the 12S and 16S ribosomal RNA (rRNA), and the 22 transfer RNAs (tRNA) identified by one-letter codes for the corresponding amino acids.

features frequently observed in mtDNA-related disorders. Mitochondria and mtDNA are ubiquitous, which explains why every tissue in the body can be affected by mtDNA mutations. This is illustrated by Table 1, a compilation of all symptoms and signs reported in patients with three different types of mtDNA mutation, including single deletions, point mutations in two distinct tRNA genes, and point mutations in a protein-coding gene. These mutations are associated with the most common mtDNA-related clinical syndromes, known by the acronyms KSS (KearnsSayre syndrome), MERRF (myoclonus epilepsy and ragged-red fibers), MELAS (mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes), and NARP/MILS (neuropathy, ataxia,

retinitis pigmentosa/maternally inherited Leigh syndrome). Certain constellations of symptoms and signs are characteristic of these syndromes and the diagnosis in typical patients is relatively easy. On the other hand, depending on heteroplasmy and on the threshold effect, different tissues harboring the same mtDNA mutation may be affected to different degrees or not at all, which explains the frequent occurrence of oligosymptomatic or asymptomatic individuals within a single family. It is often stated that any patient having multiple organ involvement and evidence of maternal inheritance should be suspected of harboring a pathogenic mtDNA mutation until proven otherwise. While this generalization has some practical value, there are exceptions, some of which are discussed below. 398

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Table 1. Clinical features of mitochondrial diseases associated with mtDNA mutations. Boxes highlight typical features of different syndromes, except for maternally inherited Leigh syndrome, which is defined on the basis of neuroradiologic or neuropathologic criteria. 1-mtDNA, deleted mtDNA; RNA, ribonucleic acid; KSS, Kearns-Sayre syndrome; MERRF, myoclonic epilepsy and ragged-red fibers; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes; NARP, neuropathy, ataxia, retinitis pigmentosa; MILS, maternally inherited Leigh syndrome. +, present; −, absent. Modified from DiMauro and Bonilla 66 1-mtDNA KSS Pearson

Tissue

Symptom/Sign

tRNA MERRF MELAS

Central Nervous System

Seizures Ataxia Myoclonus Psychomotor retardation Psychomotor regression Hemiparesis/hemianopia Cortical blindness Migraine-like headaches

− + − − + − − −

− − − − − − − −

+ + + − ± − − −

Dystonia

−

−

Peripheral Nervous System

Peripheral neuropathy

±

Muscle

Weakness Ophthalmoplegia Ptosis

Eye

Pigmentary retinopathy Optic atrophy Cataracts

ATPase6 NARP MILS − + − − − − − −

+ ± − + − − − −

−

+ + ± − + + + + +

−

+

−

±

±

+

−

+ + +

− ± −

+ − −

+ − −

+ − −

+ − −

+ − −

− − −

− − −

− − −

+ ± −

± ± −

Blood

Sideroblastic anemia

±

+

−

−

−

−

Endocrine

Diabetes mellitus Short stature Hypoparathyroidism

± ± ±

− − −

− + −

± ± −

− − −

− − −

Heart

Conduction block Cardiomyopathy

+ ±

− −

− −

± ±

− −

− ±

Gastrointestinal

Exocrine pancreatic dysfunction Intestinal pseudo-obstruction

± −

+ −

− −

− −

− −

− −

Ear, Nose, and Throat

Sensorineurol hearing loss

−

−

+

+

±

−

Kidney

Fanconi’s syndrome

±

±

−

±

−

−

Laboratory

Lactic acidosis Muscle biopsy: ragged-red fibers

+ +

+ ±

+ +

+ +

− −

± −

Inheritance

Maternal Sporadic

− +

− +

+ −

+ −

+ −

+ −

Mutations affecting mitochondrial protein synthesis

disorders, lactic acidosis, and massive mitochondrial proliferation in muscle, resulting in the ‘raggedred’ appearance of fibers in the muscle biopsy. 2 Histochemical studies have shown that the raggedred fibers (RRF) in these disorders react intensely with the succinate dehydrogenase (SDH) stain but weakly or not at all with the cytochrome c oxidase (COX) stain (see Bonilla). This staining pattern is

Mutations affecting protein synthesis include single deletions (which always encompass one or more tRNA genes), and point mutations in rRNA or in tRNA genes. Clinical experience suggests that these mutations are usually associated with multisystem 399

S. DiMauro

explained by the fact that SDH is entirely encoded by the nuclear genome (and is therefore unaffected by mtDNA mutations) whereas the three catalytic subunits of COX are encoded by mtDNA. The main exception to this ‘rule’ is seen in biopsies from typical MELAS patients, in which RRF are usually COX positive. The reason for this exception may be that the mutational load in muscle of patients with typical MELAS does not surpass the extremely high threshold needed to impair COX activity. 3 As far as inheritance is concerned, conditions associated with point mutations, such as MELAS, MERRF, and NARP/MILS (Table 1), are transmitted maternally, whereas single mtDNA deletions are sporadic events in almost all cases. The sporadic nature of all three disorders associated with single mtDNA deletions, that is, Pearson syndrome, KSS, and progressive external ophthalmoplegia (PEO), may be due to the ‘bottleneck’ that exists between oöcyte and embryo. Of the approximately 100 000 mtDNAs present in a fertilized oöcyte, only about 1000 will eventually repopulate the fetus. As a few rearranged mtDNAs are present in oöcytes from normal women, 4 it is conceivable, though extremely rare, that one such rearranged mtDNA molecule may slip through the bottleneck and appear in the child. This scenario would explain not only the lack of maternal transmission but also the clonal nature of each patient’s deletion, and the generalized or tissue-specific clinical involvent. The few rearranged mtDNAs present at the blastocyst stage could enter all three germ layers and result in KSS, segregate to the hematopoetic lineage and cause Pearson syndrome, or segregate to muscle and cause PEO. 5 While mutations in tRNA genes are usually associated with multisystem disorders, in some cases there is involvement of a single tissue, most commonly skeletal muscle. Family history was negative in some of the patients with pure myopathy, suggesting that the mutations occurred ex novo. However, in most patients the mutation was also present in blood or cultured skin fibroblasts, implying ‘skewed heteroplasmy’, with preferential accumulation of the pathogenic mutation in skeletal muscle. 6 Pathogenic mutations in rRNA genes are rare. A homoplasmic mutation in the 12S rRNA gene (A1555G) has been described in patients with deafness 7 or cardiomyopathy, 8 and a heteroplasmic mutation in the same gene has been identified in a family with maternally inherited Parkinsonism, deafness, and peripheral neuropathy. 9

Table 2. Myopathy associated with mutations in mtDNA protein-coding genes Nucleotide/Mutation

Gene

Family HX

Reference

7 nt inversion G11832A

ND1 ND4

Negative Negative

17 16

G15615G G15242A G15762A G15059A 24 bp deletion G14846A G15168A G15084A G15723A C15800T

Cyt b Cyt b Cyt b Cyt b Cyt b Cyt b Cyt b Cyt b Cyt b Cyt b

Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

52 20 53 54 19 19 19 19 19 55

G5920A T7671A 15 bp deletion

COX I COX II COX III

Negative Negative Negative

22 56 21

Mutations in protein-coding genes Generalizations regarding mutations in mtDNA protein-coding genes have been based on the two better known disorders of this type, Leber hereditary optic neuropathy (LHON) and NARP/MILS. LHON is usually associated with mutations in complex I (NADH dehydrogenase, or ND) genes and manifests as a painless optic neuropathy, often associated with Wolff–Parkinson-White syndrome. 10–12 The NARP/MILS syndrome is associated with mutations in the ATPase 6 gene. 13–15 Because both conditions are multisystemic, maternally inherited, inconsistently accompanied by lactic acidosis, and never associated with RRF in muscle, the syllogistic conclusion was drawn that all mtDNA mutations in protein-coding genes would have the same characteristics. However, recent experience from patients with exercise intolerance has taught us that all three generalizations were incorrect. Although exercise intolerance is a common complaint in mitochondrial encephalomyopathies, it is often overshadowed by other symptoms and signs. Only recently have we come to appreciate that exercise intolerance, myalgia, and myoglobinuria can be the sole presentation of respiratory chain defects, affecting complex I, complex III, or complex IV (Table 3). Exercise intolerance (without myoglobinuria) was the predominant clinical feature in two sporadic patients with complex I deficiency and COX-positive 400

Lessons from mitochondrial DNA mutations

Table 3.

Encephalomyopathies associated with pathogenic mutations in mtDNA protein-coding genes

Nucleotide/Mutation

Gene

Phenotype

Family Hx

G3460A T10191C G11778A G13513A T3308C G13513A T14484C G14459A

ND1 ND3 ND4 ND5 ND5 ND5 ND6 ND6

LHON Encephalopathy LHON MELAS MELAS/BSN LHON/MELAS LHON LHON/Dystonia

Positive Negative Positive Positive Positive Negative Positive Positive

11 58 10 23 59 24 12 60

4 bp deletion

Cyt b

MELAS/Parkinsonism

Negative

61

G6930A 5 bp deletion T7587C T9957C G9952A 9537Cins

COX I COX I COX II COX III COX III COX III

Deafness/ataxia/blindness Motor neuron disease Myopathy/ataxia/dementia/O.A. MELAS Exercise intol./E.M. LS

Negative Negative Positive Negative Negative Negative

62 27 63 25 64 26

T8851C T8993G T8993C T9176C

ATPase 6 ATPase 6 ATPase 6 ATPase 6

BSN NARP/MILS NARP/MILS BSN

Positive Positive Positive Positive

15 13 14 65

RRF in their muscle biopsies. One had a nonsense mutation (G11832A) in the ND4 gene; 16 the other had an intragenic inversion of seven nucleotides within the ND1 gene, resulting in the alteration of three amino acids. 17 Nine patients with isolated complex III deficiency in muscle complained of exercise intolerance, but only two had myoglobinuria. 18,19 All patients in whom muscle histochemistry was performed showed COXpositive RRF. The nine mutations in the cytochrome b gene were different from one another but, except for a single deletion, they were all G-to-A transitions. Most patients had no detectable mutant mtDNA in blood or fibroblasts, but one patient had low levels (0.7%) of the mutation in non-muscle tissues, suggesting skewed heteroplasmy. 20 The first mtDNA molecular defect identified in a patient with complex IV (COX) deficiency was a 15 bp microdeletion in the gene-encoding subunit III of COX (COX III). The patient was a 16-year-old woman with recurrent myoglobinuria triggered by prolonged exercise or viral illness. 21 Between attacks, both physical and neurological exams were normal, as were routine laboratory tests, including serum creatine kinase (CK) and lactate. No tissue other than muscle was affected, and family history was entirely negative. Muscle biopsy showed many SDHpositive, COX-negative RRF and marked isolated

Reference

COX deficiency. We have identified a nonsense mutation (G5920A) in the COX I gene of muscle mtDNA in a 34-year-old man with life-long exercise intolerance and recurrent myoglobinuria induced by intense or repetitive exercise. 22 His muscle biopsy showed scattered COX-negative RRF and numerous COX-negative non-RRF, and isolated COX deficiency. The mutation was not present in blood or fibroblasts from the patient nor in blood from his asymptomatic mother and sister. However, numerous mutations in protein-coding genes have been associated with encephalomyopathies other than LHON and NARP/MILS (Table 3). It is especially noteworthy that two distinct mutations in ND5 and one in COX III were associated with typical MELAS, 23–25 and still another mutation in COX III was identified in a child with sporadic Leigh syndrome. 26 It is also interesting that one patient with a mutation in COX I had the typical features of sporadic amyotrophic lateral sclerosis (ALS): 27 though this is probably a rare association, it reinforces the prevailing view that mitochondrial dysfunction may well be involved in the pathogenesis of ALS. Other phenotypes varied greatly, from predominantly myopathic syndromes to severe encephalopathies (Table 3). Finally, and not surprisingly, mutations in protein-coding genes have also been associated with selective involvement 401

S. DiMauro

of other tissues, resulting in cardiomyopathy 28,29 or acquired sideroblastic anemia. 30

A slightly more complicated strategy was employed by Sligh et al. 37 to generate mice harboring a point mutation for chloramphenicol resistance (CAPR ). The mutation in homoplasmic or heteroplasmic CAPR mutants was severe enough to cause death in utero or within 11 days of birth, and affected animals showed dilated cardiomyopathy and abnormal mitochondria in both cardiac and skeletal muscle, features often observed in human mtDNA diseases. As pointed out by Hirano in a thoughtful editorial to Sligh’s article, 38 the papers by Inoue et al. and Sligh et al. prove the principle that heteroplasmic mtDNA mutations can be transmitted through germlines and will produce phenotypic abnormalities. These pioneering studies are starting a new wave of research into the pathogenesis and therapy of human mitochondrial diseases.

Pathogenesis Until recently, the lack of animal models for mtDNA mutations had prevented detailed studies of pathogenesis. As an alternative, the biochemical and functional consequences of mtDNA point mutations and deletions have been studied in cybrid cell cultures, that is, in established human cell lines that are first depleted of their mtDNA, then repopulated with various percentages of mutated or deleted genomes. 31 This ingenious system has been of great value, but caution has to be used in extrapolating data to patients. For example, the high threshold shown by cybrid cells harboring the A3243G MELAS mutation appears to be much lower in vivo. In one study, maximal ATP production measured by magnetic resonance spectroscopy in the calf muscle from an oligosymptomatic patient was markedly decreased despite the fact that the mutational load was very low in muscle. 32 In another study, proton magnetic resonance spectroscopy showed that brain lactate in carriers of the A3243G mutation was increased and linearly related to the proportion of mutant mtDNAs. 33 These data agree with our own observation that lactate levels were increased in the CSF of the lateral ventricles in oligosymptomatic or asymptomatic relatives of MELAS patients. 34 Since the first description of pathogenic mutations in human mtDNA, one of the goals of clinical scientists was the generation of animal models. A formidable obstacle to the generation of the so called ‘mito-mice’ was our inability to introduce mutant mtDNA into mitochondria of a mammalian cell (see Nakada). To solve this problem, Inoue et al. 35 first prepared synaptosomes from the brain of aging mice, which contained a certain percentage of deleted mtDNAs. They then fused the synaptosomes with mtDNA-less rho0 cells, thus obtaining cybrid cell lines. One such cell line harboring mtDNA deletions was enucleated, fused with donor embryos, and implanted in pseudopregnant females. Heteroplasmic founder females were bred and mtDNA deletions were transmitted through three generations. Although there are significant differences between these mitomice and human patients with mtDNA deletions, the fact remains that the animals show mitochondrial dysfunction in various organs. 36

Therapy Therapy of mtDNA-related diseases is still woefully inadequate. Besides palliative pharmacological and surgical interventions directed at alleviating symptoms, approaches to therapy include (i) removing toxic products, especially lactic acid; (ii) administration of artificial electron acceptors, such as vitamin K3 and vitamin C; (iii) administration of metabolites and cofactors, such as L-carnitine and coenzyme Q10 (CoQ10); (iv) administration of oxygen radical scavengers, such as CoQ10. 39 Gene therapy is daunting in these conditions for much the same reasons that made creation of animal models such a challenge. However, if we could cause even a small shift in the relative proportion of mutant and normal mtDNAs, thus lowering the mutant load below the pathogenic theshold, we probably would improve the clinical expression dramatically. Various strategies are being considered, including the use of peptide nucleic acids (PNAs) to inhibit the replication of complementary mutant mtDNAs, 40 or pharmacologic approaches directed to eliminate mitochondria with high proportions of mutations. 41 The observation that myoblasts often contain lesser amounts of pathogenic mtDNA mutations than mature muscle fibers 42–44 has led to the use of myotoxic agents 45 or isometric exercise 46 to induce limited muscle damage, which would be followed by regeneration of muscle fibers harboring lower mutational loads. While these therapies have met with mixed results, they show our ability to affect mutational loads, at least in skeletal muscle. 402

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Acknowledgements

A recent paper has reported ‘rejuvenation’ of old oöcytes by replacing aliquots (5% to 10%) of their cytoplasm with that of young oöcytes. 47 Although this technique was used to improve the success rate of in vitro fertilization, it could be applied to the oöcytes of oligosymptomatic or asymptomatic carriers of tRNA mutations to reduce their chances of having affected children. This approach, which had already been discussed in the past, 48 raises delicate ethical questions regarding germline genetic manipulation. 49

Part of the work presented here was supported by NIH grants NS11766 and PO1HD32062, and by a grant from the Muscular Dystrophy Association.

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Prenatal diagnosis This poses special problems in mtDNA-related disorders because of two main concerns: (1) that the mutant load in amniocytes or chorionic villi will not correspond to that of other fetal tissues; (2) that the mutant load in prenatal samples may shift in utero or after birth due to mitotic segregation. These concerns still impede prenatal diagnosis for tRNA mutations, including the common causes of MELAS and MERRF. However, there is good evidence that mutations in the ATPase 6 gene (T8993G and T8993C), commonly associated with MILS, do not show tissue- or age-related variations, 50 thus making prenatal diagnosis an option for families with this devastating condition. 51

Conclusions Are we scraping the bottom of the mtDNA barrel? 6 Although the mtDNA circle is certainly crowded with pathogenic mutations (Figure 1), there is room for more, and new mutations are still being described, especially in protein-coding genes. In addition, as mentioned above, we still do not fully understand the pathophysiology of mtDNA-related diseases, although the advent of ‘mito-mice’ will undoubtedly help us in this endeavor. Finally, our therapeutic armamentarium is still totally inadequate, although some interesting novel approaches are being considered. Here, again, the availability of animal models will be of great help. So, the ‘mtDNA barrel’ is far from empty. In fact, we have probably just skimmed the top by answering the easier questions. The more difficult, and juicier, questions still challenge us. 403

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31. King MP, Attardi G (1989) Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science 246:500–503 32. Chinnery PF, Taylor DJ, Brown DT, Manners D, Styles P, Lodi R (2000) Very low levels of the mtDNA A3243G mutation associated with mitochondrial dysfunction in vivo. Ann Neurol 47:381–384 33. Dubeau F, De Stefano N, Zifkin BG, Arnold DL, Shoubridge EA (2000) Oxidative phosphorylation defect in the brains of carriers of the tRNAleu (UUR) A3243G mutation in a MELAS pedigree. Ann Neurol 47:179–185 34. Shungu DC, Sano M, Millar WS, Polanco Y, Kaufmann P, DeLaPaz RL, DiMauro S et al. (1999) Metabolic, structural and neuropsychological deficits in mitochondrial encephalomyopathies assessed by 1H MRSI, MRI and neuropsychological testing. Proc Intl Soc Mag Reson Med 7:49 35. Inoue K, Nakada K, Ogura A, Isobe K, Goto Y-i, Nonaka I, Hayashi J-I (2000) Generation of mice with mitochondrial dysfunction by introducing mouse mtDNA carrying a deletion into zygotes. Nat Genet 26:176–181 36. Shoubridge EA (2000) A debut for mito-mouse. Nat Genet 26:132–134 37. Sligh JE, Levy SE, Waymire KG, Allard P, Dillehay DL, Nusinowitz S, Heckenlively JR et al. (2000) Maternal germline transmission of mutant mtDNAs from embryonic stem cell-derived chimeric mice. Proc Natl Acad Sci USA 97:14461–14466 38. Hirano M (2001) Transmitochondrial mice: proof of principle and promises. Proc Nat Acad Sci USA 98:401–403 39. DiMauro S, Hirano M, Schon EA (2000) Mitochondrial encephalomyopathies: therapeutic approaches. Neurol Sci 21:S901–S908 40. Taylor RW, Chinnery PF, Turnbull DM, Lightowlers RN (1997) Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids. Nat Genet 15:212–215 41. Manfredi G, Gupta N, Vazquez-Memije ME, Sadlock JE, Spinazzola A, De Vivo DC, Schon EA (1999) Oligomycin induces a decrease in the cellular content of a pathogenic mutation in the human mitochondrial ATPase 6 gene. J Biol Chem 274:9386–9391 42. Clark KM, Bindoff LA, Lightowlers RN, Andrews RM, Griffiths PG, Johnson MA, Brierly EJ et al. (1997) Reversal of a mitochondrial DNA defect in human skeletal muscle. Nat Genet 16:222–224 43. Fu K, Hartlen R, Johns T, Genge A, Karpati G, Shoubridge EA (1996) 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. Hum Mol Genet 5:1835–1840 44. Shoubridge EA, Johns T, Karpati G (1997) Complete restoration of a wild-type mtDNA genotype in regenerating muscle fibers in a patient with a tRNA point mutation and mitochondrial encephalomyopathy. Hum Mol Genet 6:2239–2242 45. Andrews RM, Griffiths PG, Chinnery PF, Turnbull DM (1999) Evaluation of bupivacaine-induced muscle regeneration in the treatment of ptosis in patients with chronic progressive external ophthalmoplegia and Kearns-Sayre syndrome. Eye 13:769–772 46. Taivassalo T, Fu K, Johns T, Arnold D, Karpati G, Shoubridge EA (1999) Gene shifting: a novel therapy for mitochondrial myopathy. Hum Mol Genet 8:1047–1052 47. Barritt JA, Brenner CA, Malter HE, Cohen J (2001)

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