In 1988, a major breakthrough in our understanding of mitochondrial disorders occurred with the report of the association of sporadic human encephalomyopathies with large deletions of the mtDNA (Holt et al., 1988) and of Leber hereditary optic neuropathy (LHON) with a point mutation at nucleotide pair (np) 11778 in the mtDNA. More than 170 pathogenic point mutations of the mtDNA, large-scale mtDNA deletions, and rearrangements have been subsequently reported (Schon and DiMauro, 2007). More than 85% of mitochondrial proteins are encoded by the nuclear DNA (nDNA), and therefore many unknown mitochondrial disorders due to nDNA defects may exist. The list of nDNA defects affecting mitochondrial function, including mtDNA replication and integrity, has been growing in recent years (DiMauro and Hirano, 2005). The delineation of human mtDNA variation and genetics has also provided startling new insights into the evolution and migration of human populations (Wallace, 1995). Mitochondria may also play an important role in neurodegenerative diseases and the aging process (Schapira, 2006, 2008; Turner and Schapira, 2010).

The diseases included under the term mitochondrial disorders are so diverse and involve so many parts of the nervous system and other organs that the whole spectrum cannot be easily addressed in any one chapter. Therefore the disorders related to intermediate metabolism and mitochondrial Krebs cycle are discussed with inborn errors of metabolism in Chapter 62. The syndrome that combines epilepsy and ragged-red muscle fibers (myoclonic epilepsy and ragged-red fibers, MERRF) is discussed in Chapters 40 and 67. The syndrome of progressive external ophthalmoplegia (PEO) is discussed with other abnormalities of eye movement (Chapter 16), and LHON with other causes of vision loss (Chapter 14). This chapter overviews the principles of mitochondrial genetics and mitochondrial pathophysiology and provides a summary of the clinical features and management of patients with mitochondrial disorders.

Genetics of Mitochondrial Disorders

MtDNA is a 16,569-np double-stranded, closed, circular molecule located within the matrix of the double-membrane mitochondrion. Each human cell contains a dynamic network of mitochondria and hundreds of mtDNA molecules. Human mtDNA encodes 13 of the 89 subunits of the mitochondrial respiratory chain, as well as the small (12S) and large (16S) ribosomal RNAs (rRNAs) and 22 tRNAs necessary for intramitochondrial protein synthesis (Fig. 63.1, A).

Fig. 63.1 A, Human mtDNA map. The mtDNA encompasses 16,569 np, with numbering starting at OH and proceeding counterclockwise around the circular map. Each gene is identified by shading, and the tRNA gene is identified by the letter of its cognate amino acid. Mutation sites of common mitochondrial diseases are indicated. B, Schematic representation of the mitochondrial respiratory chain located in the inner mitochondrial membrane. Electrons are transferred from complexes I and II to complex III via the mobile lipid carrier, coenzyme Q10 (CoQ10 [ubiquinone]). Electrons are then transferred to complex IV through the protein mobile carrier, cytochrome c (CYT c). Electrons are finally transferred to molecular oxygen by complex IV. Three of the respiratory complexes (I, III, and IV) have a proton pumping mechanism coupled to the electron transfer. Pumping of protons to the intermembrane space generates a membrane potential and a proton gradient that flows back through complex V. The flow of protons through complex V enables ATP synthesis. LHON, Leber hereditary optic neuropathy; MELAS, mitochondrial encephalomyopathy with lactic acidosis and strokelike episodes; MERRF, myoclonic epilepsy with ragged-red fibers; MILS, maternally inherited Leigh syndrome; NARP, neuropathy, ataxia, and retinitis pigmentosa; PEO, progressive external ophthalmoplegia.

The mtDNA is replicated and transcribed by using an origin and promoter for each of the two DNA strands, the G-rich heavy (H) strand and the C-rich light (L) strand. The H- and L-strand replication origins (OH and OL; see Fig. 63.1, A) are relatively distant within the molecule, but the H- and L-strand promoters (PH and PL) are closely spaced and located adjacent to OH in the approximately 1000-np noncoding control region. This region also encompasses the D-loop, which is formed by replication initiation events from the OH. Research involving evolution of mammalian species and origin and migration of humans on earth has focused on the variation in a small hypervariable noncoding region within the D-loop. The replication mechanism of mtDNA may be more complex than originally suggested, in that the mtDNA can also replicate by the extension of both leading and lagging strands, a process resembling nDNA replication (Holt et al., 2000).

The respiratory chain (see Fig. 63.1, B) is located within the mitochondrial inner membrane and is composed of five multimeric enzyme complexes whose genes are dispersed between the mtDNA and nDNA. Complex I (NADH–ubiquinone oxidoreductase) accepts electrons from the reduced form of nicotinamide adenine dinucleotide (NADH), whereas complex II (succinate–ubiquinone oxidoreductase) collects electrons from succinate. Both NADH and succinate are the products of the Krebs cycle. Enzyme complexes I and II transfer electrons to coenzyme Q10 (CoQ10). From CoQ10, the electrons flow through complex III (ubiquinone–cytochrome c oxidoreductase) to cytochrome c, then to complex IV (cytochrome c oxidase), and finally to oxygen to yield water. The electron transfer is coupled to proton (H⁺) pumping by complexes I, III, and IV from the matrix to the intermembrane space, creating an electrochemical gradient across the inner membrane. This electrochemical gradient is utilized by complex V (adenosine triphosphate [ATP] synthase) as a source of energy to condense adenosine diphosphate (ADP) and inorganic phosphate (Pi) to synthesize ATP. ATP and ADP are then exchanged across the mitochondrial membrane by the adenine nucleotide translocator (ANT).

Complex I comprises approximately 46 subunits, 7 of which (ND-1, -2, -3, -4, -4L, -5, and -6) are encoded by the mtDNA; complex II has 4 subunits, none from the mtDNA; complex III includes 11 subunits, 1 (cytochrome b) from the mtDNA; complex IV contains 13 subunits, 3 (COX-1, -2, and -3) from the mtDNA; and complex V contains 13 subunits, 2 (ATPase-6 and -8) from the mtDNA. The remaining subunits of complexes I, III, IV, and V; the entire complex II; the 2 small electron carriers, CoQ10 and cytochrome c, and ANT are encoded by the nDNA. The mitochondrial rRNA and tRNA genes provide the structural RNAs for mitochondrial protein synthesis (i.e., for the expression of 13 mtDNA-encoded polypeptides). The majority of mitochondrial respiratory chain proteins are encoded by the nDNA in the cytosol. A complex mitochondrial importation process therefore enables the cytosolically synthesized nuclear-encoded mitochondrial respiratory chain subunits to be co-assembled with mtDNA encoded counterparts in the inner mitochondrial membrane. There are over 1000 other nuclear genes which express mitochondrial proteins that are important for mitochondrial function.

Mitochondrial diseases therefore can arise from defects in the mtDNA (sporadic or maternal inheritance, see following section) or nDNA (sporadic or Mendelian inheritance). Nuclear DNA-related mitochondrial disorders result from defects involving nDNA-encoded mitochondrial polypeptides including respiratory chain complexes, respiratory chain assembly, mtDNA maintenance, protein import, lipid dynamics, and biosynthesis of CoQ10. Table 63.1 summarizes a simplified clinical and genetic classification of mitochondrial diseases.

Table 63.1 Major Gene Mutations in Mitochondrial Disorders

Maternal Inheritance of Mitochondrial DNA

Patients inherit mtDNA from their mothers, and therefore the mode of transmission of mtDNA, including pathogenic mtDNA mutations, follows maternal line inheritance. A single case report has demonstrated paternal transmission of a mtDNA mutation (Schwartz and Vissing, 2002). Maternal inheritance implies maternal transmission of mtDNA to all offspring but subsequent transmission only by females. Thus a disease expressed in all children of both sexes of an affected individual, without evidence of paternal inheritance, strongly suggests an mtDNA point mutation. However, exceptions to this general rule are encountered in clinical practice. First, a de novo point mutation in the mtDNA of the maternal germ cell line will not necessarily be transmitted to all children. Second, for unknown reasons, mtDNA mutations involving large-scale deletions, rearrangements, and point mutations in some protein-coding genes may occur sporadically and be due to mutations arising in the oocyte. Bottleneck expression of an mtDNA mutation may be enhanced in the fetus. Multiple mtDNA deletions and depletions are autosomally transmitted, as they are the consequence of mutations in nuclear-encoded factors involved in mtDNA metabolism or replication. Third, maternal inheritance may not always be clinically evident because of extreme variability of clinical expression among family members due to heteroplasmy and the threshold effect (described in the following section).

Heteroplasmy and Mitotic Segregation of Mitochondrial DNA

Each cell contains thousands of mtDNA copies in a network of cytoplasmic mitochondria. MtDNA is constantly undergoing replication, fission, and fusion even in terminally differentiated cells. When all mtDNA molecules are identical, the mtDNA is homoplasmic. If pathogenic mtDNA mutations exist with normal (wild-type) mtDNA, then there are two populations of mtDNA in the system, and this is heteroplasmy. The severity of the phenotypic effects of the mtDNA mutations in heteroplasmic cells is determined by the proportion of the wild-type and mutant mtDNA. Neutral polymorphic sites (nonpathogenic mutations) of mtDNA are generally homoplasmic, whereas pathogenic mutations are mostly, but not invariably, heteroplasmic.

Heteroplasmy may occur at the level of the cell or at the level of the individual mitochondrion (intramitochondrial heteroplasmy). At cell division, mitochondria and mtDNA are randomly partitioned to be carried into the two daughter cells (mitotic segregation). Therefore, mtDNA mutation is typically represented in a variable proportion of mitochondrial genomes, with the consequence that cells, tissues, and the whole individual would harbor variable proportions of mutant and wild-type mtDNA. Mitotic segregation explains how certain patients with mtDNA-related disorders may present with one manifestation at an early age and shift to another as they grow older as the proportions of heteroplasmy change with time in different tissues. For a disease to manifest in a tissue, the proportion of mutant mtDNA must be greater than the threshold level.

Threshold Effect of Mitochondrial DNA Mutations

Threshold effect denotes the minimal critical number of mutated mtDNA molecules that would cause mitochondrial dysfunction in one or more tissues or organ systems. Variable somatic load of mtDNA mutation in different tissues and organ systems, or skewed heteroplasmy is a universal finding in heteroplasmic states. It often changes over time, particularly in postmitotic cells such as neurons, and it increases with age. This in part explains the age-dependent penetration of many mitochondrial clinical phenotypes and age-related variability in their clinical features. The mtDNA mutation load needed for clinical expression is typically high and in the range of 70% to 90% for both point mutations and deletions. The mutation threshold effect is believed to be affected by the oxidative metabolic requirement of a particular tissue. For example, the threshold effect may manifest itself at lower concentrations of mutated mtDNA in tissues that are inherently dependent on high oxidative metabolism, such as brain, eye, myocardium, and skeletal muscle.

Examples of threshold effect and mitotic segregation include a patient with MELAS (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) who may present with only episodic headache in childhood but with stroke-like episodes and neurological deficits as they age and the pathogenic mutations accumulate in brain and cerebral vasculature. Infants with Pearson syndrome (a hematopoietic disease) may survive their disease because the proportion of mtDNA with deletions may decrease as bone marrow cells with heavy mutation loads are selected against. Following this process of outgrowing the hematopoietic disease in childhood, individuals with Pearson syndrome may later develop a different clinical phenotype such as Kearns-Sayre syndrome (KSS) as the mutation accumulates in their postmitotic neurons and muscle fibers.

Pathophysiology of Mitochondrial Disorders

Mitochondria have several functions in maintaining cellular homeostasis, such as transient storage of intracellular calcium, fatty acid oxidation, the Krebs cycle, and iron metabolism (McBride et al., 2006; Schapira, 2006). Mitochondria also have a key role in the regulation of apoptosis (Wang, 2001). One of the most important roles of mitochondria is to catalyze the phosphorylation of the majority of cellular ADP to ATP. ATP is generated by oxidation of intermediates such as NADH and FADH2 via the process of oxidative phosphorylation within mitochondria by the respiratory chain enzymes. Several intermediary metabolic pathways from carbohydrates, fatty acids, and amino acids converge in mitochondria at the level of acetyl-CoA for the final conversion of the fuel into ATP. Pyruvate is the terminal product of anaerobic glycolysis and is transported across the inner mitochondrial membrane to the mitochondrial matrix where it is converted to acetyl CoA. The transportation of pyruvate is coupled with the influx of hydrogen ions down their electrochemical gradient across the inner mitochondrial membrane. Pyruvate can also be generated during the catabolism of the amino acids, alanine, serine, glycine, and cysteine. Transport of free fatty acids across the mitochondrial membrane requires two enzymes (carnitine palmitoyltransferases [CPT] I and II), a carrier molecule (l-carnitine), and a translocase (carnitine-acylcarnitine translocase). Fatty acids are also metabolized into acetyl-CoA. Acetyl-CoA enters the Krebs cycle, where three molecules of NADH and one molecule of the reduced form of flavin adenine dinucleotide (FADH₂) are produced from each acetyl-CoA. Molecules of NADH and FADH₂ donate electrons to the electron transport chain (NADH to complex I and succinate to FAD in complex II). The functional unit comprising the electron transport chain (complex I to IV) and complex V (ATP synthase) constitutes the oxidative phosphorylation (OXPHOS) system (see Fig. 63.1, B).

Lactic acid is the end product of glycolytic anaerobic metabolism and acts as a reservoir for excess pyruvate. Physiological states such as exercise may cause transient lactic acidosis. Pathological lactic acidosis occurs during anoxia/ischemia, in metabolic failure from liver disease and diabetes mellitus (secondary lactic acidosis), and in defects of OXPHOS (primary lactic acidosis) such as mitochondrial disorders. The blood lactate-to-pyruvate ratio reflects the NADH to NAD⁺ ratio, or redox state, and is useful in the diagnosis of primary lactic acidosis. Defects in pyruvate dehydrogenase, which catalyses the conversion of pyruvate to acetyl CoA with the conversion of NAD⁺ to NADH, are associated with a low redox state (due to increased NAD⁺) and usually produce elevated levels of lactate and pyruvate with normal or slightly low lactate/pyruvate ratios (<20). OXPHOS defects often cause a high redox state (due to increased NADH accumulation) and generally produce a high lactate/pyruvate ratio (>20) as NADH and pyruvate are converted to lactate by lactate dehydrogenase.

Neurons, myocardial and skeletal muscle, liver, and renal tissues are highly dependent on oxidative metabolism and are most commonly involved in mitochondrial diseases. Neurons especially require high levels of ATP production to maintain ion homeostasis following controlled flux of ions across the cell membrane during electrical signaling. Within the brain, the high metabolic activity of the basal ganglia makes them vulnerable to oxidative metabolic defects. Necrosis of the basal ganglia and brainstem is an early feature of Leigh syndrome and is common in other mitochondrial cytopathies.

In skeletal muscle, some fibers are severely involved and others may appear normal on histological analysis. With more severe involvement, the combination of patchy myofibrillar degeneration along with mitochondrial proliferation gives rise to the so-called ragged-red appearance of fibers on modified Gomori trichrome staining (Fig. 63.2, A). Defective OXPHOS may result in compensatory mitochondrial proliferation, particularly in type I and IIA muscle fibers. Ultrastructural analysis can demonstrate abnormal mitochondrial morphology such as intramitochondrial paracrystalline inclusions (see Fig. 63.2, B). Patients with mitochondrial disorders can show a wide range of symptoms, including any combination of developmental delay, short stature, small muscle bulk, seizures, vision loss, hearing impairment, peripheral neuropathy, autonomic nervous system difficulties, gastrointestinal dysfunction, endocrine problems, hematopoietic disease, and failure to thrive (Box 63.1). The presentation of a particular mitochondrial disorder can be variable, even among affected individuals in the same family. Conversely, more than one pathogenic mutation can give rise to similar clinical phenotypes.

Fig. 63.2 A, Modified Gomori trichrome section of a biopsy showing ragged-red muscle fibers. Increased mitochondrial staining in the subsarcolemmal and intermyofibrillar regions of the muscle fibers imparts the ragged-red appearance. B, Mitochondrial myopathy. Electron micrograph of a part of the muscle fiber showing mitochondrial proliferation; many mitochondria contain typical “parking lot” paracrystalline inclusions.

Box 63.1 Clinical Features in Mitochondrial Diseases

Peripheral Nervous System Abnormalities

Sensorimotor polyneuropathy

Autonomic polyneuropathy

Myopathy

Progressive external ophthalmoplegia

Proximal limb myopathy

Exercise intolerance, myalgia, and fatigue

Rhabdomyolysis, myoglobinuria

Cardiomyopathy

Hypertrophic cardiomyopathy

Dilated cardiomyopathy

Cardiac conduction defects

Liver Dysfunction

Hepatic steatosis

Liver failure

Renal Dysfunction

Renal tubular acidosis

Fanconi syndrome

Myoglobinuric renal failure

Gastrointestinal Disturbance

Gastroparesis

Intestinal pseudo-obstruction

Constipation, diarrhea

Pancreatitis

Endocrine Dysfunction

Diabetes mellitus

Hypothalamic hypogonadism

Hypothyroidism

Hypoparathyroidism

Hypoadrenalism

Miscellaneous

Failure to thrive

Developmental delay or regression

Approach to the Diagnosis of Mitochondrial Disorders

The complex inheritance patterns and clinical heterogeneity of mitochondrial diseases often result in incorrect or delayed diagnosis of the affected individuals. This delay can result in increased morbidity or mortality. For example, patients with specific metabolic defects such as β-oxidation of fatty acids can benefit from appropriate manipulations of diet and physical activity. CoQ10 (ubiquinone) and l-carnitine replacements are often effective in rare metabolic disorders of CoQ10 and primary systemic carnitine deficiencies. Failure to make a precise genetic diagnosis and, in turn, the lack of appropriate genetic counseling, can lead to the subsequent birth of affected children in unsuspecting families. Leigh syndrome is a good example in that the phenotype can vary among members of the same family and can be inherited in the maternal line (mtDNA-related mutations) or in Mendelian (autosomal or sex-linked) inheritance patterns.

A detailed clinical history and examination in conjunction with experienced interpretation of a battery of complex laboratory results is often required to make an accurate diagnosis. This process is often best carried out in a specialist mitochondrial clinic and subsequently discussed at a multidisciplinary meeting involving neurologists, pediatricians, geneticists, pathologists, and biochemists. Consensus diagnostic criteria for mitochondrial disorders in infants and children (Morava et al., 2006; Wolf and Smeitink, 2002) and adults (Bernier et al., 2002) have been proposed.

Although the overall clinical spectrum of mitochondrial disorders is broad, recognized patterns of clinical presentations, clinical signs, and investigations have emerged. A detailed extended family history is essential in deciphering subtle clues suggesting a maternal line of inheritance. Any patient with unexplained multisystem problems, particularly affecting the nervous system, skeletal muscle, liver, kidney, and heart may have mitochondrial disease. Rare presentations can be of mitochondrial origin, such as stroke-like episodes with MELAS, chronic ophthalmoplegia in KSS, and a movement disorder in children or young adults with Leigh syndrome. Patients and families often report a history of periods of severe fatigue with intercurrent illnesses, trauma, or surgery. Affected individuals may develop exacerbations, such as an increase in seizures, or new symptoms, such as an episode of lactic acidosis, during a seemingly minor illness. The patient may develop a permanent neurological deficit following these physiological stressors.

Laboratory Findings

The mitochondrial metabolic test battery includes serum creatine kinase (CK), lactate and pyruvate, plasma and urine acylcarnitines, blood and urine amino acids, urine organic acids, and cerebrospinal fluid (CSF) lactate and pyruvate (if the CNS is involved). The lactate/pyruvate ratio may differentiate disorders of the OXPHOS system in comparison to more proximal metabolic defects such as pyruvate dehydrogenase deficiency. However, normal values for the lactate and lactate/pyruvate ratio in a patient do not exclude mitochondrial disease; mtDNA-related diseases are generally associated with normal or only mildly elevated resting blood lactate levels but often with a significant rise with exercise. Serum CK is usually normal or mildly elevated in patients with mitochondrial myopathies. Blood levels of free carnitine are often decreased in mtDNA-related disorders, with a relative increase in acylcarnitine levels. The interpretation of test results of free and total carnitine, blood and urine amino acids, and urine organic acids is discussed in Chapter 62.

A controlled muscle exercise test may offer a useful noninvasive tool to investigate muscle oxidative metabolism. Lactate level, oxygen extraction from hemoglobin (near-infrared spectroscopy), and the ratio of phosphocreatine (PCr) to inorganic phosphate (Pi) (31P magnetic resonance spectroscopy [MRS]) have been measured in muscle at rest and during exercise and recovery. In patients with mitochondrial dysfunction, PCr : Pi ratios are lower than normal at rest, decreased excessively during exercise, and return to baseline values more slowly than normal controls. However, normative PCr : Pi values overlap with those in patients with mitochondrial disorders, and this test is not suitable for infants and young children because it requires a high degree of patient cooperation.

Neuro-ophthalmology

The four most common neuro-ophthalmological mitochondrial disorders are LHON, progressive external ophthalmoplegia (PEO), pigmentary retinopathy, and retrochiasmal visual loss. Standard flash electroretinograms are typically normal in LHON. Visual evoked responses are predictably abnormal in patients with visual loss. KSS is a subset of PEO associated with ataxia, cognitive dysfunction, cardiac conduction defects, and elevated protein in CSF. The most common pigmentary retinopathy appearance in mtDNA-related diseases is that of “salt-and-pepper” retinopathy that typically becomes more prominent with advancing age. Macular involvement and vascular attenuation are common in pigmentary retinopathy. Fluorescein angiography in LHON reveals vasculopathy without leakage, whereas rod-and-cone–specific electroretinography may help confirm subtle changes of pigmentary retinopathy. Disruption of retrochiasmal visual pathways, generally due to stroke-like episodes, causes homonymous hemianopic defects or cortical blindness in patients with MELAS.

Neuroradiology

The use of neuroimaging, especially brain magnetic resonance imaging (MRI), has greatly facilitated the detection of CNS involvement in mitochondrial disorders. Brain atrophy is common in children with mitochondrial disease. Developmental delay and basal ganglia calcification are common in KSS and MELAS, and diffuse signal abnormalities of the white matter are characteristic of KSS and myoneurogastrointestinal encephalopathy (MNGIE) (Fig. 63.3). The diagnosis of MELAS can be aided by the clinical association of stroke-like episodes with radiological lesions that do not conform to the anatomical territories of blood vessels and predominantly involve cortical gray matter (Fig. 63.4). The initial or predominant lesions in MELAS are characteristically in the parietal-occipital region, and new lesions generally appear with acute illness and elevated CSF lactate levels. Leigh syndrome characteristically shows bilateral hyperintense signals on T2-weighted and fluid-attenuated inversion recovery (FLAIR) MRIs in the putamen, globus pallidus (Fig. 63.5), and thalamus. 1H-MRS often detects lactate accumulation in the CSF and in specific areas of the brain.

Fig. 63.3 Mitochondrial neurogastrointestinal syndrome (MNGIE). T2-weighted axial magnetic resonance image showing diffuse white-matter signal changes in cerebral hemispheres.

Fig. 63.4 Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. Proton density axial magnetic resonance image showing ischemic lesions in the left occipital and posterior parietal regions (note predominantly cerebral cortical lesions).

Fig. 63.5 Leigh syndrome. T2-weighted magnetic resonance image showing symmetrical signal changes in the globus pallidus and putamen.

Muscle Biopsy

An open muscle biopsy under local anesthesia provides material for histochemistry, ultrastructural studies, biochemistry, myoblast culture, and molecular genetic studies.

Histochemistry

Many of the histopathological abnormalities found in muscle biopsies from patients with mitochondrial diseases are nonspecific and include excessive variability of muscle fiber size, fiber type-specific atrophy, scattered myofibrillar necrosis and regeneration, and intermyofibrillar lipid or glycogen accumulation. Mild peripheral nerve involvement is common in mitochondrial diseases, and the muscle biopsy may reveal evidence of partial denervation. Oxidative enzyme staining may show core targetoid fibers and fiber type grouping.

The hallmark feature in mitochondrial diseases is the ragged-red fiber (RRF) (see Fig. 63.2, A). In frozen sections stained with modified Gomori trichrome, subsarcolemmal and intermyofibrillar accumulation of mitochondria appear as bright red masses on at least three sides of the fiber, against the background of the blue myofibrils. These abnormal accumulations represent a compensatory proliferation of mitochondria, some of which are ultrastructurally normal and others dystrophic (see Fig. 63.2, B). The same fibers stain intensely blue with the histochemical reaction for succinate dehydrogenase (SDH), an OXPHOS enzyme encoded entirely by the nDNA. SDH staining is more sensitive than the modified Gomori trichrome in detecting mitochondrial proliferation. NADH-tetrazolium reductase (NADH-TR) stains mitochondria-rich fibers even more intensely, but the enzyme reaction is less specific than SDH for mitochondria. RRFs are seen in most patients with mtDNA defects and in some patients with nDNA mutations, but RRFs are neither a universal feature in mitochondrial disorders, nor are they specific for primary mitochondrial diseases. RRFs can occur in other neuromuscular diseases such as inflammatory myopathies or inclusion body myositis, as well as in normal aging. They also occur in the toxic myopathy caused by the drug, zidovudine, where the underlying pathogenesis is drug-induced damage of the mtDNA. Mitochondrial proliferation in the smooth muscle of intramuscular vessels results in strongly SDH-reactive vessels in MELAS. The histochemical stain for cytochrome oxidase (COX) activity may be helpful. COX or complex IV of the mitochondrial respiratory chain has 13 subunits; three—COX I, II, and III—are encoded in mtDNA and the others are encoded in the nuclear genome. COX may be absent in myofibers from patients with defects of mtDNA, mitochondrial transcription and translation, or assembly of complex IV. RRFs which are COX-negative suggest impaired mitochondrial protein synthesis in the face of mitochondrial proliferation and are typically seen in mtDNA deletions. In the combined COX-SDH histochemical stain, COX-negative fibers with normal or high concentrations of SDH stain blue against a background of normal brown fibers that have both COX and SDH. Single-fiber polymerase chain reaction (PCR) from these fibers (RRF/SDH-rich and COX-negative) shows higher levels of mutated mtDNA molecules, suggesting that these mutations are deleterious. Discordance between RRF status and COX activity is not uncommon in muscle fibers in mitochondrial diseases. In patients with mutations in mtDNA protein-coding genes and defects in complex IV, RRF and many non-RRFs are COX negative or deficient, whereas in patients with defects of complex I or III and tRNA mtDNA point mutations, many RRFs with normal COX activity may be seen. SCO2 and SURF1 nDNA mutations, which cause complex IV deficiency, are generally associated with diffuse COX deficiency but without RRFs. The absence of either RRFs or COX-negative fibers does not rule out mitochondrial disease. For instance, patients with NARP-MILS syndrome (neuropathy, ataxia, retinitis pigmentosa and maternally inherited Leigh syndrome) or LHON do not have RRF or COX-negative fibers.

Electron Microscopy

Electron microscopy of muscle biopsy specimens from patients with mitochondrial diseases may reveal subsarcolemmal and intermyofibrillar proliferation of mitochondria and the presence of abnormal mitochondria in muscle fibers. Enlarged, elongated, irregular, and dumbbell-shaped mitochondria with hypoplastic and dystrophic cristae and paracrystalline inclusions (see Fig. 63.2, B) may be seen in a patient’s muscle biopsy with a diagnosis of mitochondrial disease. However, they are nonspecific and can be present in other neuromuscular disorders. Significant intermyofibrillar mitochondrial proliferation should be detectable by light microscopy in the modified Gomori trichrome, NADH-TR, and SDH stains. Isolated focal collections of mitochondria in the subsarcolemmal space or near the A-I junction in muscle fiber can be normal and should not be mistaken for pathological accumulation.

Biochemistry

In specialized mitochondrial laboratories, expertise for biochemical analysis of OXPHOS enzymes in muscle, cultured skin fibroblasts, and peripheral blood lymphocytes can be performed. Muscle tissue is generally preferred for biochemical analysis because it has high oxidative metabolism, and it is more often affected in mitochondrial diseases. Biochemical analysis can be performed in fresh or frozen muscle tissue. The advantage of fresh muscle is that functionally intact mitochondria can be isolated for polarographic analysis, but the disadvantage is that the patient has to travel to the site of the study laboratory. Frozen muscle sample can be stored and shipped to specialized laboratories for biochemical analysis of the OXPHOS enzymes in the muscle homogenate. The activities of specific respiratory complexes should be compared with the activity of an unrelated nuclear encoded mitochondrial enzyme such as citrate synthase in order to compensate for mitochondrial mass in the sample.

Isolated defects of complex I, III, or IV, each of which incorporate subunits encoded by mtDNA and nDNA, can occur in sporadic, Mendelian, or maternal line mutations. Combined defects of complexes I, III, and IV suggest either a single large deletion or tRNA mutations in mtDNA, or an nDNA defect that secondarily alters mtDNA (multiple mtDNA deletions, mtDNA depletion). The activities of specific respiratory complexes may be abnormal even when the muscle histochemistry is normal, especially in children.

The combined defect of complexes II/III when assayed together as succinate cytochrome c reductase with normal activities of the individual complexes suggests a possible diagnosis of CoQ10 deficiency. The combined complex II+III assay requires endogenous CoQ10, whereas the individual complex II and complex III assays are independent of CoQ10. CoQ10 levels can be determined by high-performance liquid chromatography (HPLC) in muscle and peripheral blood mononuclear cells.

Blue Native Polyacrylamide Gel Electrophoresis

One of the most powerful tools to appear in the last few years for the analysis of the OXPHOS complexes is the resolution of the individual fully assembled complexes in blue native polyacrylamide gel electrophoresis (BN-PAGE). By using mild detergents, the OXPHOS complexes normally remain intact in the electrophoretic field, but they begin separating from each other if components of the OXPHOS are abnormal (Ugalde et al., 2004). The complexes and their components can be detected by direct staining of proteins, enzyme activity, or antibodies.

DNA-Based Diagnosis

A large number of mtDNA and nDNA mutations are now known to cause mitochondrial disorders, and their number (especially nDNA gene mutations) has continued to rise in recent years (DiMauro and Hirano, 2005) Further details about mtDNA mutations can be found in the MitoMap database, http://www.mitomap.org. If a patient has a clearly defined clinical syndrome such as MELAS, MERRF, LHON, or NARP, the most common mutations associated with the syndrome can be initially screened in blood (see Table 63.1). Genetic tests for mutations at positions 3243, 8344, and 8993 are now widely available in diagnostic laboratories. The majority of LHON cases will have one of the three common point mutations at positions 11778, 14484, or 3460. The level of np-3243A>G in blood declines with age and may become undetectable over the age of 30.

However, especially in adult mitochondrial disease, lymphocyte DNA may be normal, and skeletal muscle will have to be analysed, such as in sporadic patients with isolated PEO. This is particularly true for mtDNA deletions and mtDNA depletion syndromes. The entire mitochondrial genome can be sequenced for a pathogenic mutation if there is a strong suspicion of a mitochondrial disease.

There are only a few reported pathogenic mutations in the nuclear-encoded protein subunits of the mitochondrial respiratory chain enzymes. Most of the nDNA mutations reported occur in gene products that regulate the assembly of OXPHOS complexes or control of mtDNA replication. The majority of nDNA mitochondrial disorders present with neonatal or early-childhood onset. Mutations in the nuclear gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma (POLG) have been described as presenting with a variety of phenotypes in childhood and adulthood (see later discussion). Nuclear gene tests can be performed from the DNA sample isolated from the peripheral lymphocytes.

Fig. 63.6 demonstrates an algorithm for the DNA diagnosis of a patient with suspected mitochondrial disease.

Fig. 63.6 Queen Square mitochondrial disease investigation pathway. 1. Mitochondrial DNA (mtDNA) deletion screen can be performed on blood from patients younger than 20 years of age. 2. Perform respiratory chain enzyme assays even if histochemistry normal if strong clinical suspicion. 3. Sequence mtDNA even if respiratory chain enzyme assays normal if strong clinical suspicion. CI, Complex I; CII, complex II; CIII, complex III; HCM, hypertrophic cardiomyopathy; LS, Leigh syndrome; MNGIE, mitochondrial neurogastrointestinal encephalomyopathy; PEO, progressive external ophthalmoplegia; POLG, polymerase gamma; RRF, ragged-red fiber; SDHA, gene encoding succinate dehydrogenase subunit A.

(From Rahman, S., Hanna, M.G., 2009. Diagnosis and therapy in neuromuscular disorders: diagnosis and new treatments in mitochondrial diseases. J Neurol Neurosurg Psychiatry 80, 943-953.)

Mitochondrial Clinical Syndromes

Progressive External Ophthalmoplegia and Kearns-Sayre Syndrome

Clinically isolated PEO with progressive ptosis is a common manifestation of mitochondrial disease. Onset is often before 20 years of age or after 50. There is a slow evolution of symmetrical extraocular muscle weakness, and diplopia is uncommon. Ptosis progresses over time and often needs treatment such as eyelid props or eyelid surgery. A sporadic single clonal deletion of mtDNA is the most common genetic defect in patients with PEO, although point mutations in tRNA genes (e.g., A3243G mutation) and a duplication of mtDNA have also been reported.

Autosomal dominant or recessive PEO due to defects in nuclear genes involved in mtDNA maintenance results in multiple mtDNA deletions. It tends to present in adulthood and may be associated with multisystem involvement such as neuropathy, ataxia, tremor, parkinsonism, depression, cataracts, pigmentary retinopathy, deafness, rhabdomyolysis, and hypogonadism. Mutations in POLG1 is one of the more common nuclear genes to cause this syndrome.

KSS is defined by the triad of PEO and onset before age 20, with at least one of the following: pigmentary retinopathy, cerebellar ataxia, heart block, and/or elevated CSF protein (>100 mg/dL). Patients often have a progressive limb myopathy and frequently require a pacemaker for AV block. Many patients with KSS have delayed motor milestones, are small of stature, and have cognitive impairment. Some clinical features of MELAS and MERRF may overlap with KSS. The clinical course in KSS is progressive, and many patients with CNS or cardiac complications die in the third or fourth decade. Nearly all cases of KSS are sporadic and usually caused by a single large clonal mtDNA deletion that arises in the mother’s oocyte.

Mitochondrial Myopathies without Progressive External Ophthalmoplegia

The clinical spectrum of isolated mitochondrial myopathy varies from mild nondisabling proximal limb weakness to severe infantile myopathy with lactic acidosis and death by 1 year. Exercise intolerance is common. Some of these cases present in adult life, but careful questioning usually elicits a history of lifelong exercise intolerance (weakness, fatigue, shortness of breath, and tachycardia). A sporadic form of myopathy related to somatic mutations in the cytochrome b gene of mtDNA is associated with progressive exercise intolerance and weakness and, in some cases, attacks of rhabdomyolysis (Andreu et al., 1999). Rare patterns associated with a cytochrome b mutation include weakness and exertional myoglobinuria. Less frequently, sporadic patients with exercise intolerance have been found to have mtDNA mutations in genes encoding subunits of complexes I or IV (DiMauro and Hirano, 2005). Some patients with mitochondrial myopathy without PEO will develop progressive PEO in later life, and others may have overlapping deficits with MERRF and MELAS.

Mitochondrial Peripheral Neuropathy

Patients with complex mtDNA-associated mitochondrial phenotypes involving the CNS (e.g., MERRF, MELAS) often have a mild axonal sensorimotor neuropathy that may be subclinical. A peripheral neuropathy can be the dominant clinical feature in some patients. Mutations in the mitofusin 2 (MFN2) gene, which encodes a protein that influences mitochondrial dynamics, is a cause of Charcot-Marie-Tooth disease (CMT). Some families with MFN2 mutations have additional clinical features including optic atrophy (Züchner et al., 2006). Mutations in POLG can cause a prominent large-fiber sensory neuropathy with significant proprioceptive loss in the SANDO (sensory ataxic neuropathy dysarthria and ophthalmoplegia) syndrome. Axonal sensorimotor polyneuropathy may also be a feature of dominant optic atrophy caused by mutations in the OPA1 gene encoding a mitochondrial dynamin-like protein.

Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes

MELAS is a maternally inherited encephalomyopathy clinically characterized by short stature, stroke-like episodes, migrainous headaches, vomiting, seizures, and lactic acidosis. The stroke-like deficits are sometimes transient but can be permanent and cause progressive encephalopathy with dementia. Ataxia, deafness, muscle weakness, cardiomyopathy, and diabetes are common as the disease progresses.

A typical radiological feature is that the stroke involves the cerebral cortex, spares the white matter (see Fig. 63.4), mostly affects the parietal and occipital cortices, and does not conform to vascular territories. Neuroimaging may show additional lesions that have no clinical correlates. The onset is generally in childhood or early adult life. Most patients have RRF on muscle biopsy. Roughly 80% of patients with MELAS have an A-to-G point mutation at np-3243 (tRNA^Leu[UUR] gene, MT-TL1). Another point mutation at np-3271 of the tRNA^Leu(UUR) gene accounts for 10% of cases of MELAS.

Myoclonic Epilepsy with Ragged-Red Fiber Myopathy

MERRF is a maternally inherited encephalomyopathy characterized by myoclonus, epilepsy, cerebellar ataxia, and myopathy with RRF. Onset is usually in childhood or early adulthood. The syndrome begins with stimulus-sensitive myoclonic epilepsy in childhood, which may be photosensitive. Worsening ataxia and mental retardation is seen in later childhood. Patients may also develop cardiomyopathy, short stature, deafness, optic atrophy, PEO, cutaneous lipomas, and neuropathy. Overlapping clinical features of MERRF and MELAS can occur in the same patient or among different members of the same family (Verma et al., 1996). The clinical course in MERRF is variable, but it is typically progressive.

Approximately 80% of MERRF cases have a point mutation at np-8344 of the tRNA^Lys gene (MT-TK2). As with other mtDNA mutations, the time of onset and severity of the disease has been related to the quantitative burden of mutant mtDNA.

Mitochondrial Neurogastrointestinal Encephalomyopathy

MNGIE is an autosomal recessive disease with secondary alterations of mtDNA. There is typically a combination of ptosis, PEO, severe gastrointestinal dysmotility leading to episodes of pseudoobstruction and cachexia, peripheral neuropathy, leukoencephalopathy on brain MRI (see Fig. 63.3), and evidence of mitochondrial dysfunction (e.g., lactic acidosis or RRF in muscle biopsy) (Hirano et al., 2004). Onset is usually in the late teens, and most patients die before age 40. MNGIE is caused by mutations in the gene encoding thymidine phosphorylase. The disease can be diagnosed by blood tests demonstrating loss of thymidine phosphorylase activity or elevation of plasma thymidine and deoxyuridine.

Neuropathy, Ataxia, Retinitis Pigmentosa Syndrome

NARP syndrome is a relatively rare disorder due to a point mutation at np-8993 of the mitochondrial ATPase-6 gene encoding subunit 6 of complex V, giving rise to two maternally inherited and clinically related phenotypes even within the same family: NARP and maternally inherited Leigh syndrome (MILS). Other point mutations in the same gene have been reported with the NARP and MILS. The severity of the syndrome corresponds to the mutant mtDNA load in the tissues. A mutation load over 90% of mtDNA tends to cause the more severe phenotype of MILS.

Subacute Necrotizing Encephalomyelopathy (Leigh Syndrome)

Leigh syndrome is a familial or sporadic mitochondrial disorder characterized by psychomotor regression and lesions in the basal ganglia and brainstem (see Fig. 63.5). Some cases display a maternal inheritance, such as mtDNA np-8993 and np-8344 mutations (MILS). Others follow an autosomal (pyruvate carboxylase, SURF1 gene mutations with COX deficiency, complex I deficiencies) or sex-linked (pyruvate dehydrogenase E1 gene mutations) pattern of inheritance. Over 50% of cases present in the first year of life, usually before 6 months of age. Late-onset varieties with a greater degree of clinical heterogeneity are also reported. The precise clinical boundaries of Leigh syndrome have not been defined; there is clinical heterogeneity even among members of the same family. Leigh syndrome and congenital lactic acidosis are described further in Chapter 62.

Leber Hereditary Optic Neuropathy

Patients with LHON usually present with a subacute bilaterally sequential and isolated optic neuropathy. LHON is expressed predominantly in males of the maternal lineage, and the greater susceptibility of males to vision loss in LHON remains unexplained. The age of onset is typically between 15 and 35 years, and the vision loss is painless, central, and usually occurs in one eye weeks or months before involvement of the other eye. Funduscopic abnormalities may be seen in patients with LHON and in their asymptomatic relatives. During the acute phase of vision loss, there may be hyperemia of the optic nerve head, dilatation and tortuosity of peripapillary vessels, circumpapillary telangiectasia, nerve-fiber edema, and focal hemorrhage. Vision loss in LHON affects central or centrocecal fields and is usually permanent. A minority of patients show objective improvement, sometimes to a dramatic degree. Three primary point mutations at mtDNA np-11778 (69%), np-14484 (14%), and np-3460, all within coding regions for complex I subunits, account for 80% to 95% of cases of LHON worldwide. These mutations are found in blood and are often homoplasmic. Patients with np-14484T>C have a better chance of some visual recovery. Some families have additional members with associated cardiac conduction abnormalities, especially preexcitation syndromes. There may also be a movement disorder such as dystonia or other mild neurological or skeletal abnormalities. Occasionally LHON is associated with an MS-like illness.

Sensorineural Deafness

Sensorineural hearing loss (SNHL) is a feature of many mitochondrial diseases and commonly occurs in MELAS, maternally inherited diabetes and deafness (MIDD), MERRF, or KSS. The presence of SNHL in a patient with a complex multisystem phenotype suggests a possible mitochondrial disease. SNHL may also occur in isolation. The np-1555A>G mitochondrial mutation (which confers sensitivity to aminoglycoside-induced deafness and may cause nonsyndromic deafness) is present in 1 in 500 of the general population. Mutations in the tRNA gene for serine (UCN) may also cause isolated deafness. SNHL is also a feature of some nuclear-encoded mitochondrial disorders such as dominant optic atrophy associated with OPA1 mutations.

Mitochondrial DNA Depletion Syndrome

Mitochondrial DNA depletion syndrome (MDDS) is an autosomal recessive disorder caused by a quantitative reduction in the amount of mtDNA. A myopathic and a hepatocerebral form have been described. Both forms are usually fatal in childhood, although patients with Navajo neurohepatopathy may survive into their late teens. MDDS may be caused by recessive defects of the mtDNA replication machinery (POLG or PEO1) or from defects of maintaining the deoxyribonucleoside triphosphate pool necessary for mtDNA replication.

Management of Mitochondrial Diseases

Treatment of Associated Complications

Treatment of mitochondrial disease is mainly symptomatic, empirical, and often palliative (DiMauro and Mancuso, 2007). Patients and families with confirmed mitochondrial disease require management and support in a multidisciplinary clinical team setting. This is often coordinated by a neurologist with close links to a range of different disciplines such as rehabilitation medicine, physiotherapy, occupational therapy, cardiology, endocrinology, ophthalmology, audiology, and speech therapy. There is usually no specific treatment for most mitochondrial disorders, and therefore monitoring and treatment of complications arising from the disease is vital for improving quality of life and reducing morbidity.

Genetic Counseling, Prenatal Diagnosis, and Reproductive Options

If a nuclear gene mutation is identified, genetic counseling and prenatal diagnosis can be offered to the patient. Primary mtDNA mutations present in the male will not be transmitted. If an mtDNA mutation is identified in a woman with mitochondrial disease, it is more difficult to provide accurate genetic counseling advice. Most large-scale deletions of mtDNA are sporadic, and the risk of transmission is relatively low. Some mtDNA point mutations are also sporadic. For heteroplasmic mtDNA point mutations, the factors that determine the amount of a particular point mutation that will be transmitted to offspring are poorly understood. Although a heteroplasmic point mutation will be transmitted in the maternal line, because of the genetic bottleneck for mtDNA (where only a small number of mtDNA molecules in the mother are passed on to the next generation), large shifts in the proportion of mutant from mother to offspring may occur. It is therefore not possible to offer women who harbor heteroplasmic disease-causing point mutations accurate advice regarding the risk of transmission.

The offspring of a patient with a homoplasmic point mutation such as in LHON will be homoplasmic for the mutation, but they may not all develop the disease. Unknown non-mtDNA factors may be important in determining disease expression. At present, the only method of preventing disease transmission is ovum donation. Some preliminary studies of preimplantation genetic diagnosis for the NARP mutation have shown encouraging results. Oocyte manipulation techniques to replace maternal mutant mtDNA with donor mtDNA—for example, by transferring the pronuclei from an oocyte carrying an mtDNA mutation into an enucleated donor egg—may provide a more satisfactory method, although these studies are still at a preliminary stage. Individuals at risk of inheriting an mtDNA mutation may request predictive genetic testing. If they have any symptoms suggestive of mitochondrial phenotype, diagnostic genetic testing is appropriate.

Pharmacological Approaches

Coenzyme Q10 Deficiency

CoQ10 is a lipophilic mobile electron carrier and antioxidant located in the inner mitochondrial membrane. Disorders of CoQ10 biosynthesis are clinically heterogeneous. Presentations include recurrent rhabdomyolysis with seizures, multisystem disorder of infancy with prominent nephropathy, ataxia with or without seizures, Leigh syndrome, and pure myopathy. These disorders respond well to CoQ10 supplementation if treatment is started early, but very large doses may be necessary because of poor uptake into the mitochondrion. The results of randomized controlled trials using CoQ10 in other mitochondrial disorders have yielded conflicting results. Many specialists recommend CoQ10 to all patients with a proven diagnosis of mitochondrial disease, initially at a dose of 100 mg 3 times daily, increasing to 400 mg 3 times daily in adults.

Other Pharmacological Approaches

A number of other pharmacological agents have been tried in mitochondrial disease but with limited benefit. A recent Cochrane systematic review concluded that there was insufficient evidence to recommend any standard treatment. There are anecdotal reports of benefit of various agents (e.g., riboflavin, succinate, l-carnitine, α-lipoic acid creatine (Tarnopolsky et al., 1997), and vitamins C, E, and K), but the clinical heterogeneity and unpredictable natural history of mitochondrial disease, with a frequently relapsing-remitting course, makes interpretation of the effectiveness of any given agent in a single individual difficult. The few randomized double-blind clinical trials that have been performed yielded inconclusive or conflicting results. Recently some novel pharmacological approaches have emerged, aimed at stimulating mitochondrial biogenesis via the transcriptional coactivator, PGC-1. Drugs that may stimulate this pathway include bezafibrate and resveratrol.