Mitochondrial encephalomyopathies

Mitochondrial encephalomyopathies are diseases caused by defective oxidative phosphorylation (OXPHOS), and affect the nervous system and/or skeletal muscle. They have emerged as a major entity among the neurometabolic diseases of childhood with an incidence of 1 in 11,000 children, and also have a high prevalence in adults. The first pathogenic mutation of human mitochondrial DNA (mtDNA) was discovered in 1988. Since then more than 100 mutations of mtDNA have been reported, including point mutations of genes encoding transfer RNA, ribosomal RNA, and proteins, as well as large-scale deletions. The first nuclear-DNA gene mutation causing OXPHOS disease was described in 1995. Mutations in nuclear genes may affect the respiratory chain by various mechanisms. Pathogenic mutations of nuclear-DNA-encoded subunits of complex I and II have been demonstrated as have mutations of respiratory chain assembly proteins. Several nuclear genes associated with mtDNA maintenance have been found to be associated with mitochondrial disorders since mutations in these genes predispose to multiple mtDNA deletions and/or reduced copy number of mtDNA. The genotype-phenotype correlation is not yet entirely clear, but new animal models will enhance our ability to study the pathophysiology of OXPHOS disorders.     

Amyotrophic lateral sclerosis with ragged-red fibers

BACKGROUND: Motor neuron diseases (amyotrophic lateral sclerosis [ALS] and spinal muscular atrophy [SMA]) have been rarely associated with mitochondrial respiratory chain defects. OBJECTIVES: To describe a patient with typical ALS and the finding of ragged-red fibers in muscle biopsy specimens and to review the literature on respiratory chain defects in ALS and SMA. DESIGN: Case report and review of the literature. SETTING: Collaboration between tertiary care academic hospitals. PATIENT: A 65-year-old man with typical ALS. MAIN OUTCOME MEASURES: The patient had 10% ragged-red fibers and 3% cytochrome-c oxidase-negative fibers in muscle biopsy specimens but no biochemical defects of respiratory chain enzymes or alterations of mitochondrial DNA (mtDNA). RESULTS: Amyotrophic lateral sclerosis with ragged-red fibers has been reported in 5 families and is associated with mtDNA mutations in some subjects. Spinal muscular atrophy without mutations in the survival motor neuron gene (SMN; OMIM 600354) has been associated with mtDNA depletion or with mutations in the cytochrome-c oxidase assembly gene (SCO2; OMIM 604377). CONCLUSION: Respiratory chain defects can mimic ALS or SMA and should be considered in the differential diagnosis.     

Mitochondrial diseases

Mitochondrial diseases, and particularly mitochondrial myopathies or encephalomyopathies, have drawn increasing attention in the past decade. Initially defined by morphologic changes in muscle ("ragged red fibers" and ultrastructural abnormalities of mitochondria), mitochondrial encephalomyopathies can now be classified according to biochemical defects involving: (1) mitochondrial transport; (2) substrate oxidation; (3) Krebs cycle; (4) respiratory chain; and (5) oxidation-phosphorylation coupling. For each biochemical group of disorders, the authors describe clinical presentations and biochemical findings. These disorders are especially interesting from the genetic point of view because mitochondria have their own DNA (mtDNA), which encodes 13 polypeptides, all of them subunits of respiratory chain complexes. Other mitochondrial proteins are encoded by nuclear DNA, synthesized in the cytoplasm, and imported into the mitochondria by a complex mechanism. Because mtDNA is inherited strictly by maternal, cytoplasmic inheritance, mitochondrial diseases can be transmitted by Mendelian or by non-Mendelian, maternal inheritance, as illustrated by human pathology.     

Lack of paternal inheritance of muscle mitochondrial DNA in sporadic mitochondrial myopathies

In 2002, paternal inheritance of muscle mitochondrial DNA (mtDNA) was reported in a patient with exercise intolerance and a mitochondrial DNA (mtDNA) mutation restricted to skeletal muscle. To evaluate whether paternal inheritance is a common phenomenon, we studied 10 sporadic patients with skeletal muscle-restricted mtDNA mutations: five harbored mtDNA point mutations in protein-coding genes and five had single mtDNA deletions. We performed haplotype analysis and direct sequencing of the hypervariable regions 1 and 2 of the D-loop in muscle and blood from the patients and, when available, in blood from their parents. We did not observe paternal inheritance in any of our patients.     

Neuromuscular implications in CADASIL

OBJECTIVES: Recent studies indicate that Notch3 gene mutations not only manifest as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) but also in the peripheral nerves and skeletal muscles. METHODS: A MEDLINE search with appropriate terms was carried out. Six articles, dealing with neuromuscular involvement in CADASIL, were selected and reviewed. RESULTS: Several case studies presented CADASIL patients with clinical features of myopathy. Neurological diagnostic workup in these patients revealed weakness, wasting, reduced/exaggerated tendon reflexes, abnormal nerve conduction and electromyography, muscle biopsy with ragged red muscle fibers, reduced COX staining, decreased complex I respiratory chain activity, abnormally structured mitochondria, or mitochondrial DNA (mtDNA) mutations, such as G5650A in the tRNAAla gene, or various other mtDNA substitutions. Additionally, fibroblasts in skin biopsy may show reduced complex V respiratory chain activity. CONCLUSIONS: These findings suggest Notch3 mutations to be associated with mitochondrial disease, particularly affecting the skeletal muscle. Whether mtDNA mutations were induced by Notch3 mutations, by oxidative stress due to chronic hypoxia, resulting from arteriopathy, or occurred spontaneously remains elusive. Patients carrying Notch3 mutations should be systematically investigated for neuromuscular involvement, which may have therapeutic and prognostic implications for these patients.     

Exercise intolerance and the mitochondrial respiratory chain

The syndrome of exercise intolerance, cramps, and myoglobinuria is a common presentation of metabolic myopathies and has been associated with several specific inborn errors of glycogen or lipid metabolism. As disorders in fuel utilization presumably impair muscle energy production, it was more than a little surprising that exercise intolerance and myoglobinuria had not been associated with defects in the mitochondrial respiratory chain, the terminal energy-yielding pathway. Recently, however, specific defects in complex I, complex III, and complex IV have been identified in patients with severe exercise intolerance with or without myoglobinuria. All patients were sporadic cases and all harbored mutations in protein-coding genes of muscle mtDNA, suggesting that these were somatic mutations not affecting the germ-line. Another respiratory chain defect, primary coenzyme Q10 (CoQ10) deficiency, also causes exercise intolerance and recurrent myoglobinuria, usually in conjunction with brain symptoms, such as seizures or cerebellar ataxia. Primary CoQ10 deficiency is probably due to mutations in nuclear gene(s) encoding enzymes involved in CoQ10 biosynthesis.     

Mitochondrial DNA depletion in Leigh syndrome

Leigh syndrome is a heterogenous neurologic disease characterized by seizures, developmental delay, muscle weakness, respiratory abnormalities, optic abnormalities, including atrophy and ophthalmoplegia, and progressive cranial nerve degeneration with early onset in infants and children. Diagnosis can be confirmed by characteristic pathologic findings of necrosis in the basal ganglia, thalamus, and brainstem. Severe dysfunction of mitochondrial energy metabolism is generally present and involved in the etiology of this degenerative central nervous system disease. At the molecular level, a number of point mutations have been located in mitochondrial DNA genes, including ATPase6 and tRNA(Lys) genes, and in nuclear genes encoding subunits of oxidative enzymes, such as pyruvate dehydrogenase. Biochemically these mutations are responsible for enzymatic defects in either respiratory complexes (I, IV, or V) or pyruvate dehydrogenase. We describe here the first case of Leigh syndrome with marked depletion of mitochondrial DNA levels in skeletal muscle and abnormal activities in skeletal muscle of mitochondrial respiratory complexes I, III, IV, and V.     

Mitofusin 2 mutations affect mitochondrial function by mitochondrial DNA depletion

Charcot–Marie–Tooth neuropathy type 2A (CMT2A) is associated with heterozygous mutations in the mitochondrial protein mitofusin 2 (Mfn2) that is intimately involved with the outer mitochondrial membrane fusion machinery. The precise consequences of these mutations on oxidative phosphorylation are still a matter of dispute. Here, we investigate the functional effects of MFN2 mutations in skeletal muscle and cultured fibroblasts of four CMT2A patients applying high-resolution respirometry. While maximal activities of respiration of saponin-permeabilized muscle fibers and digitonin-permeabilized fibroblasts were only slightly affected by the MFN2 mutations, the sensitivity of active state oxygen consumption to azide, a cytochrome c oxidase (COX) inhibitor, was increased. The observed dysfunction of the mitochondrial respiratory chain can be explained by a twofold decrease in mitochondrial DNA (mtDNA) copy numbers. The only patient without detectable alterations of respiratory chain in skeletal muscle also had a normal mtDNA copy number. We detected higher levels of mtDNA deletions in CMT2A patients, which were more pronounced in the patient without mtDNA depletion. Detailed analysis of mtDNA deletion breakpoints showed that many deleted molecules were lacking essential parts of mtDNA required for replication. This is in line with the lack of clonal expansion for the majority of observed mtDNA deletions. In contrast to the copy number reduction, deletions are unlikely to contribute to the detected respiratory impairment because of their minor overall amounts in the patients. Taken together, our findings corroborate the hypothesis that MFN2 mutations alter mitochondrial oxidative phosphorylation by affecting mtDNA replication.     

Infantile-onset disorders of mitochondrial replication and protein synthesis

Most inherited mitochondrial diseases in infants result from mutations in nuclear genes encoding proteins with specific functions targeted to the mitochondria rather than primary mutations in the mitochondrial DNA (mtDNA) itself. In the past decade, a growing number of syndromes associated with dysfunction resulting from tissue-specific depletion of mtDNA have been reported in infants. MtDNA depletion syndrome is transmitted as an autosomal recessive trait and causes respiratory chain dysfunction with prominent neurological, muscular, and hepatic involvement. Mendelian diseases characterized by defective mitochondrial protein synthesis and combined respiratory chain defects have also been described in infants and are associated with mutations in nuclear genes that encode components of the translational machinery. In the present work, we reviewed current knowledge of clinical phenotypes, their relative frequency, spectrum of mutations, and possible pathogenic mechanisms responsible for infantile disorders of oxidative metabolism involved in correct mtDNA maintenance and protein production.     

Early-onset encephalomyopathy associated with tissue-specific mitochondrial DNA depletion: A morphological,biochemical and molecular-genetic study

A male infant, born from consanguineous parents, suffered from birth with a progressive neuromuscular disorder characterized by psychomotor delay, hypotonia, muscle weakness and wasting, deep-tendon areflexia and spastic posture. High levels of lactic acid in blood and cerebrospinal fluid suggested a mitochondrial respiratory chain defect. Muscle biopsy revealed raggedred and cytochromec oxidase-negative fibres, lipid accumulation and dystrophic changes. Multiple defects of respiratory complexes were detected in muscle homogenate, but cultured fibroblasts, myoblasts and myotubes were normal. Southern blot analysis showed markedly reduced levels of mitochondrial DNA (mtDNA) in muscle, while lymphocytes, fibroblasts and muscle precursor cells were normal. Neither depletion of mtDNA nor abnormalities of the respiratory complexes were observed in innervated muscle fibres cultured for as long as 4 months. No mutations were observed in two candidate nuclear genes,mtTFA andmtSSB, retro-transcribed, amplified and sequenced from the proband's mRNA. Sequence analysis of the mtDNA D-loop and of the origin of replication of the mtDNA light strand failed to identify potentially pathogenic mutations of these replicative elements in the proband's muscle mtDNA. Our findings indicate that mtDNA depletion is due to a nuclear encoded gene and suggest that the abnormality underlying defective mtDNA propagation must occur after muscle differentiation in vivo.     

Complex neurologic syndrome associated with the G1606A mutation of mitochondrial DNA

OBJECTIVES: To confirm the pathogenicity of the G-to-A substitution at nucleotide 1606 (G1606A) mutation in the mitochondrial DNA (mtDNA) tRNA(Val) gene, and to characterize genotype-phenotype correlation. PATIENT AND METHODS: A 37-year-old man since childhood developed a complex clinical picture characterized by hearing loss, migraine, ataxia, seizures, cataracts, retinitis pigmentosa, mental deterioration, and hypothyroidism. Magnetic resonance imaging revealed diffuse calcification of the basal ganglia and cerebral cortical atrophy. Morphologic and biochemical studies of respiratory chain complexes were performed in skeletal muscle. All 22 mitochondrial tRNA genes were screened for mutations by direct sequencing. RESULTS: Biochemical analysis showed normal activities of respiratory chain enzymes and citrate synthase; morphologic examination showed scattered ragged-red fibers and poor or absent cytochrome c oxidase staining in 10% of the fibers. A heteroplasmic G1606A transition in the mtDNA tRNA(Val) gene was found. Mutant DNA was 70% of the total in the proband's muscle. The mutation was absent in blood samples and urinary sediment from his healthy brother and mother. CONCLUSION: This second patient with the G1606A mutation confirms both the pathogenicity of the mutation and its association with a characteristic complex neurologic phenotype.     

Aerobic conditioning in patients with mitochondrial myopathies: physiological, biochemical, and genetic effects

Aerobic training has been shown to increase work and oxidative capacity in patients with mitochondrial myopathies, but the mechanisms underlying improvement are not known. We evaluated physiological (cycle exercise, 31P-MRS), biochemical (enzyme levels), and genetic (proportion of mutant/wild-type genomes) responses to 14 weeks of bicycle exercise training in 10 patients with heteroplasmic mitochondrial DNA (mtDNA) mutations. Training increased peak work and oxidative capacities (20-30%), systemic arteriovenous O2 difference (20%), and 31P-MRS indices of metabolic recovery (35%), consistent with enhanced muscle oxidative phosphorylation. Mitochondrial volume in vastus lateralis biopsies increased significantly (50%) and increases in deficient respiratory chain enzymes were found in patients with Complex I (36%) and Complex IV (25%) defects, whereas decreases occurred in 2 patients with Complex III defects (approximately 20%). These results suggest that the cellular basis of improved oxygen utilization is related to training-induced mitochondrial proliferation likely resulting in increased levels of functional, wild-type mtDNA. However, genetic analysis indicated the proportion of wild-type mtDNA was unchanged (3/9) or fell (6/9), suggesting a trend toward preferential proliferation of mutant genomes. The long-term implications of training-induced increases in mutant relative to wild-type mtDNA, despite positive physiological and biochemical findings, need to be assessed before aerobic training can be proposed as a general treatment option.     

Diseases of oxidative phosphorylation due to mtDNA mutations

Mitochondrial encephalomyopathies are disorders due to biochemical defects in the respiratory chain, which is under dual genetic control: 13 proteins are encoded by mitochondrial DNA (mtDNA), while all others are encoded by nuclear DNA. In the past 12 years, the small circle of mtDNA has proven to be a Pandora's box of pathogenic mutations, associated with a bewildering variety of multisystemic or tissue-specific disorders. After summarizing the principles of mitochondrial genetics, we attempt to provide general principles and practical clues to the diagnosis of mtDNA-related disorders by reviewing sequentially clinical presentation, family history, laboratory data, neuroradiology, exercise physiology, muscle morphology, muscle biochemistry, and molecular genetics.     

A novel mitochondrial tRNA(Leu(UUR)) mutation in a patient with features of MERRF and Kearns-Sayre syndrome

In a patient with clinical features of both myoclonus epilepsy ragged-red fibers (MERRF) and Kearns-Sayre syndrome (KSS), we identified a novel guanine-to-adenine mitochondrial DNA (mtDNA) mutation at nucleotide 3255 (G3255A) of the tRNA(Leu(UUR)) gene. Approximately 5% of the skeletal muscle fibers had excessive mitochondria by succinate dehydrogenase histochemistry while a smaller proportion showed cytochrome c oxidase (COX) deficiency. In skeletal muscle, activities of mitochondrial respiratory chain complexes I, I + III, II + III, and IV were reduced. The G3255A transition was heteroplasmic in all tissues tested: muscle (53%), urine sediment (67%), peripheral leukocytes (22%), and cultured skin fibroblasts (< 2%). The mutation was absent in 50 control DNA samples. Single-fiber analysis revealed a higher proportion of mutation in COX-deficient RRF (94% +/- 5, n = 25) compared to COX-positive non-RRF (18% +/- 9, n = 21). The identification of yet another tRNA(Leu(UUR)) mutation reinforces the concept that this gene is a hot-spot for pathogenic mtDNA mutations.     

Mitochondrial syndromes with leukoencephalopathies

White matter involvement has recently been recognized as a common feature in patients with multisystem mitochondrial disorders that may be caused by molecular defects in either the mitochondrial genome or the nuclear genes. It was first realized in classical mitochondrial syndromes associated with mitochondrial DNA (mtDNA) mutations, such as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), Leigh's disease, and Kearns-Sayre's syndrome. Deficiencies in respiratory chain complexes I, II, IV, and V often cause Leigh's disease; most of them are due to nuclear defects that may lead to severe early-onset leukoencephalopathies. Defects in a group of nuclear genes involved in the maintenance of mtDNA integrity may also affect the white matter; for example, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) caused by thymidine phosphorylase deficiency, Navajo neurohepatopathy (NNH) due to MPV17 mutations, and Alpers syndrome due to defects in DNA polymerase gamma (POLG). More recently, leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) has been reported to be caused by autosomal recessive mutations in a mitochondrial aspartyl-tRNA synthetase, DARS2 gene. A patient with leukoencephalopathy and neurologic complications in addition to a multisystem involvement warrants a complete evaluation for mitochondrial disorders. A definite diagnosis may be achieved by molecular analysis of candidate genes based on the biochemical, clinical, and imaging results.     

A novel mitochondrial DNA deletion producing progressive external ophthalmoplegia associated with multiple sclerosis

We report a previously undescribed 7676 base pair mitochondrial (mt)DNA deletion involving genes of complex I, complex IV subunits 2 and 3 (cytochrome oxidase [Cox] II, III), adenosine triphosphatase 8 and 6, cytochrome b and 8 transfer (t)RNA genes producing myopathy and progressive external ophthalmoplegia (PEO) in a 44-year-old right-handed Caucasian man with features of multiple sclerosis (MS). We performed complete mtDNA sequencing and deletion analysis, spectrophotometric analysis of muscle and platelet respiratory chain activity, measurement of platelet mitochondrial membrane potential with the potentiometric dye JC-1 and magnetic resonance spectroscopy (MRS) and MRI studies of normal-appearing and lesional cerebral tissue. The deletion resulted in significant respiratory chain deficiency in muscle and blood and abnormalities of the platelet mitochondrial membrane potential. However, cerebrospinal fluid analysis, magnetic resonance spectroscopy and MRI features suggested inflammatory central nervous system demyelination rather than a primary respiratory chain disorder. We conclude that this novel mtDNA deletion causing myopathy and PEO is associated with severe muscle and platelet cellular energetic abnormalities. Furthermore, clinical and paraclinical features of multiple sclerosis were found. The potential pathomechanistic interaction between mtDNA variation and multiple sclerosis is reviewed.     

Deletions of mitochondrial DNA in Kearns-Sayre syndrome and ocular myopathies: genetic, biochemical and morphological studies.

Genetic, biochemical and morphological investigations were conducted on skeletal muscle mitochondria from 6 cases of ocular myopathy: 4 cases with Kearns-Sayre syndrome (KSS) and 2 with chronic progressive external ophthalmoplegia. All of these 6 cases showed mitochondrial DNA (mtDNA) deletions in addition to normal sized DNA in the quadriceps muscle. The deletions ranging from 3 to 8 kbp were also mapped between nucleotides 5500 and 16000 by Southern blot. The deleted genes encoded for some subunits of complexes I, IV, V and 5-10 tRNAS. The boundaries of the deletions have been sequenced in three patients. Five patients had mitochondrial respiratory chain deficiency in complex I as shown by the low oxygen consumption in isolated mitochondria using three NAD(+)-linked substrates. Mitochondria with an abnormal ultrastructure were also observed in 2 cases. A good relationship between the cytochrome c oxidase deficiency and the amount of deleted mtDNA was shown in our present investigations.     

The molecular pathology of human respiratory chain defects

Deletions of the mitochondrial genome were identified in 21 out of 58 patients (36 percent) with mitochondrial myopathies, 47 of whom had defects in the mitochondrial respiratory chain. In cases with Complex I defects, the deleted regions of mtDNA, were confined to structural genes encoding Complex I subunits but additionally involved the intervening tRNA genes and in one case included the large and small rRNA genes. In cases with more extensive loss of respiratory chain function, the deletions eliminated genes encoding subunits of Complexes I, IV and V, as well as several tRNAs. Complex I and Complex IV polypeptides were usually normal in deleted cases. This was in contrast to 7 out of 22 patients without detectable mtDNA deletions, who showed specific deficiencies of subunits encoded by nuclear genes. Further studies in one of these cases pointed to defective translocation of the Rieske precursor from the cytosol into the mitochondria. The genetic basis of the disease in 15 cases without detectable deletions or specific subunit deficiencies, remains unknown. The multiple biochemical abnormalities encountered in these cases would be consistent with more subtle alterations of the mitochondrial genome.