Chapter 28 Mitochondrial diseases

Clinical Neurophysiology of Disorders of Muscle and Neuromuscular Junction, Including Fatigue Handbook of Clinical Neurophysiology, Vol. 2 Erik Stalberg (Ed.) © 2003 Elsevier B. V. All rights reserved

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CHAPTER 28

Mitochondrial diseases Emma Ciafaloni" and Enrica Arnaudo'':" h

a University of Rochester, Rochester, NY, USA Thomas Jefferson University, Medical College Building, 1025 Walnut Street, Suite 310. Philadelphia. PA 19107. USA

28.1. Introduction The mitochondrial diseases are a heterogeneous group of disorders with widely diverse clinical features, defined by deficits of the mitochondrial respiratory chain. The main function of the respiratory chain is to generate adenosine triphosphate (ATP), essential to all nucleated cells for survival, by oxidative phosphorylation. The respiratory chain is composed of five enzyme complexes, each containing multiple polypeptide subunits, mostly encoded by the nuclear DNA (nDNA). However, the mitochondrial genome encodes a few essential subunits of the respiratory chain, as well as RNAs needed for their translation. Biosynthesis of a functional mitochondrion requires the coordinate expression of genes in both mitochondrial DNA (mtDNA) and nONA. Mitochondrial diseases can thus be caused by either a nuclear or a mitochondrial gene defect, and occasionally by nuclear-mitochondrial signaling defects. Primary disease of the mitochondrial respiratory chain is estimated to occur with an incidence between 6 and 16 per 100,000 individuals (Chinnery and Turnbull, 200 I). Populations with limited genetic diversity are thought to be at higher risk for rare mitochondrial diseases (Fosslien, 2001). This is possibly related to the higher mutation rate of the mtDNA compared to the nONA. Differences in the genetic background and population structure may influence the prevalence of specific mutations. For example, the A3243G mutation appears to be very common in Finland, but rare in the African American population.

* Correspondence to: Dr. Enrica Amaudo, MD, Thomas Jefferson University, Medical College Building, 1025 Walnut Street, Suite 310, Philadelphia, PA 19107, USA. E-mail address: [email protected] Tel.: 215-955-7952; fax: 215-503-4307.

The mitochondrial disorders display a great variety of clinical symptoms, age of onset, severity of progression, and mode of inheritance. Patients may display a heterogeneous spectrum of chronic symptoms resulting from a failure of cellular oxidative metabolism. They usually present with neurological symptoms and signs but any body tissue or organ can be affected, resulting in diffuse multisystemic involvement. Tissues that rely on oxidative phosphorylation for production of ATP, such as muscle and nervous system, are usually affected more severely. Neuropathy and myopathy are common and often presenting features in these disorders. In some cases, however, the muscle and nerve involvement is subclinical, or a minor part of a diffuse, non-specific clinical presentation (such as isolated deafness, cardiomyopathy, or diabetes mellitus), posing a greater diagnostic challenge for the clinician.

28.2. Mitochondrial biogenesis Mitochondria are cytoplasmic organelles within eukaryotic cells that supply energy for most cellular functions in the form of ATP, generated by oxidative phosphorylation. They are unusual among the cellular organelles in that they contain DNA. However, the mtDNA contributes only to the synthesis of a portion of the mitochondrial proteins needed for oxidative phosphorylation. The synthesis of the majority of the respiratory chain subunits, as well as the biogenesis and other functions of the mitochondrion are directed by the nucleus. Most mitochondrial proteins are encoded by nuclear genes, synthesized in the cytoplasm and then imported into the organelle. The mitochondrial genome is a double-stranded DNA circle of 16,569 base pairs with only few noncoding nucleotides. The mtDNA encodes for 13 polypeptide subunits of the respiratory chain and a

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small portion of the factors necessary to express them, including 2 ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs). A mitochondria-specific DNA polymerase and associated nuclear replication factors are responsible for mtDNA replication.

28.3. Mitochondrial genetics There are several underlying principles of mitochondrial genetics, some of which influence the phenotypic expression of any mtDNA defect. They include maternal inheritance, sequence heterogeneity, mtDNA heteroplasmy, mitotic segregation, and the "threshold effect". 28.3.1. Maternal inheritance

Because of its extranuclear location, mitochondrial genetics differs in significant respects from nuclear, Mendelian genetics. The oocyte and spermatocyte contribute the same amount of nDNA information to the human zygote. However, mammalian mtDNA is inherited via the oocyte cytoplasm; the sperm does not contribute to the zygote's complement of mitochondria. Therefore, mtDNA exhibits a maternal pattern of inheritance. A mother carrying a mtDNA mutation will transmit it to all her children, males and females, but only her daughters will pass it on to their progeny. 28.3.2. Sequence heterogeneity

In contrast with nuclear genes, which are represented by two alleles, most cells contain several thousand mtDNA molecules within several hundred mitochondria. There is extensive sequence heterogeneity of mtDNA among humans. The mtDNAs of randomly selected individuals differ by an estimated 0.32%. This corresponds to approximately 50 base changes, approximately 10 of which result in replacement changes in protein coding genes. Thus, functional differences are expected among mtDNAencoded proteins in humans. 28.3.3. mtDNA heteroplasmy If there is a polymorphism or a mutation in mtDNA, it may result in the coexistence of more than one population of mtDNA in the same cell. The presence of more than one type of mtDNA in a cell is termed heteroplasmy. The presence of only one

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genotype of mtDNA in a cell or individual is termed homoplasmy. Since each mitochondrion contains between 2 and 10 mtDNAs, heteroplasmy can exist even within an individual mitochondrion. 28.3.4. Mitotic segregation

At cell division, mitochondria and therefore mtDNAs are distributed to the daughter cells. This can result in the daughter cells receiving differing amounts of wild-type and mutated mtDNA. This partitioning of mtDNA to daughter cells, termed mitotic segregation, can result in the relative proportion of wild-type and mutated mtDNAs varying from one cell to another in the same tissue and between different tissues in the body. 28.3.5. Threshold effect

Because there can be an infinite number of different proportions of mutated and wild-type mtDNA within a cell or tissue, the terms dominant or recessive that are frequently used with nuclear genes usually do not apply to mitochondrial genetics. The phenotypic expression of a pathogenic mutation of mtDNA is largely determined by the "threshold effect" instead. This is the minimum critical relative proportion of mutated vs. wild-type mtDNAs that is required to express a respiratory chain defect. The threshold required to express a defect and cause mitochondrial dysfunction is dependent upon the type of mtDNA mutation. Also, the proportion of mutated mtDNAs resulting in cell dysfunction will vary from tissue to tissue depending upon the requirements of a given tissue on oxidative metabolism. In addition, this metabolic vulnerability may vary within the same tissue with respect to time or functional demands. As a result of heteroplasmy and threshold effect, the phenotypic expression of a pathogenic mutation depends on the proportion of mutated to wild-type mtDNAs, the metabolic vulnerability of the tissue, and the functional demands of the tissue. Further, these factors can change during the course of time (cell growth and division, development, and aging), and therefore the phenotype may change over time.

28.4. Clinical manifestations The classification of mitochondrial diseases is complicated by the fact that many phenotypes have

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Table I Systems and symptoms frequently involved in mitochondrial diseases. Centra/nervous system

Ataxia, myoclonus, seizures, episodic coma, migraines, stroke, cortical blindness, psychomotor retardation or regression, dystonia, dementia, sensorineural hearing loss

Peripheral nervous system

Peripheral neuropathy, proximal myopathy, fatigue, recurrent myoglobinuria

Eye

Retinitis pigmentosa (RP), ptosis, ophtalmoparesis, cataracts, optic atrophy

Heart

Cardiomyopathy, conduction defects: AVB, RBBB, sudden cardiac death, LAHB

Endocrine system

Diabetes mellitus, short stature, hypoparathyroidism

G/

Cyclic nausea and vomiting, pseudo-obstruction, exocrine pancreatic dysfunction, weight loss

Skin

Multiple lipomas

B/ood

Sideroblastic anemia, pancytopenia

Kidney

Fanconi's syndrome, glomerulopathy

been associated with more than one genetic defect and mtDNA defects, including point mutations and single or multiple deletions, can cause different clinical manifestations. The clinician should therefore use a combination of clinical, biochemical and genetic classification when working toward the diagnosis of a mitochondrial disorder. Because all cells depend on the mitochondria for energy production, it is not surprising that defects affecting mitochondrial function manifest with a multitude of symptoms and signs in many different tissues and organs. Clinical manifestations in a given system are determined by the ratio of wild and mutant mtDNA and by the vulnerability to impaired oxidative metabolism of each specific tissue. Muscle, heart and brain are frequently affected because of their high dependence on oxidative metabolism. A mitochondrial disorder should be suspected whenever a combination of symptoms and signs listed in Table I are found in an individual patient or in members of a pedigree after other most common etiologies have been ruled out. Certain combinations of symptoms and signs are very characteristic and define specific mitochondrial syndromes, such as myoclonic epilepsy with ragged red fibers (MERRF) or mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS). 28.4.1. Kearn-Sayre syndrome (KSS)

KSS is characterized by the triad of progressive external ophthalmoplegia (PED), onset before age

20 and retinitis pigmentosa (RP), plus one of the following: ataxia, cerebrospinal fluid (CSF) proteins > 100 mg/dl, heart conduction abnormalities (Keams and Sayre, 1958). Frequently associated features also include muscle weakness, short stature, peripheral neuropathy (PN), lactic acidosis, ragged red fibers (RRF) on muscle biopsy, and low serum and CSF folic acid levels. It is a sporadic disorder caused by a single mtDNA deletion, which in the vast majority of cases is 4,977 base pair long ("common deletion"). Tandem duplications and a point mutation in the ATPase 6 gene have also been reported in rare cases (Holt et aI., 1989). Deletions and duplications may coexist in a subgroup of KSS patients (Poulton et al., 1994; Brockington et al., 1995) Pearson's syndrome is a usually fatal combination of sideroblasic anemia and exocrine pancreas dysfunction in children and, like KSS, is linked to a large-scale mtDNA deletion. The rare patients with Pearson's syndrome who survive, eventually develop KSS. This shift from one phenotype to another is an example of the mitotic segregation phenomenon, typical of mitochondrial disorders. 28.4.2. MELAS: Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes

MELAS is the most common of the maternally inherited mitochondrial disorders. Typical cases manifest with intermittent lactic acidosis and encephalopathy and acute onset of hemiparesis and hemianopsia or cortical blindness. The stroke-like

552 episodes occur before age 40 in most cases. The underlying pathophysiological mechanism seems to be metabolic rather than ischemic. This hypothesis is supported by the observation that the stroke-like lesions are not usually in the distribution of major cerebral blood vessels. The proposed underlying mechanism for these lesions is impaired metabolic activity of mitochondria affecting the endothelium and smooth muscle cells of blood vessels, termed mitochondrial angiopathy (5.2). Patients frequently manifest recurrent nausea, vomiting and vascular headaches in conjunction with muscle weakness, seizures and ataxia. The full syndrome may be found only in one member of a family where maternal relatives carry a low percentage of mutant mtDNA and display fewer or no symptoms. This is frequently the case, due to heteroplasmy and the threshold effect. The MELAS syndrome is commonly associated with a A3243G missense mutation in the tRNALeu (UUR) gene of the mtDNA, which is found in about 80% of cases (Ciafaloni et aI., 1992; Goto et aI., 1992). Nine other point mutations in the tRNALeu have been reported, and rarely other mtDNA gene mutations including a 4bp deletion in the mitochondrial cytochrome b gene have been associated to MELAS. 28.4.3. MERRF: Myoclonic epilepsy with ragged red fibers

Typically, patients with MERRF present with a clinical combination of myoclonus, seizures, ataxia and myopathy. Their maternal relatives may have a partial syndrome or isolated sensorineural hearing loss, short stature, peripheral neuropathy or multiple lipomas. Most patients with MERRF harbor the A8344G mutation in the tRNALys gene (Shoffner et aI., 1990; Silvestri et al., 1993). Two other mutations in the tRNALys gene, T8356C and G8363A, have also been linked with this syndrome. Overlap MELASIMERRF phenotypes are not uncommon and have been associated with mutations T8356C, T75l2C andA3243G. 28.4.4. NARP: Neuropathy, ataxia and retinitis pigmentosa

In this maternally inherited disease, ataxia, RP and PN are frequently associated with muscle weakness, sensorineural hearing loss and optic atrophy.

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This syndrome is associated with a missense, heteroplasmic mutation at base pair 8993 in the ATPase 6 gene. Interestingly, when about 70% of mtDNA is mutated, the patients display the NARP phenotype. If the mtDNA mutant percentage is greater than 90%, patients present with a more severe, infantile disease characterized by hypotonia, psychomotor regression, seizures, dystonia, ataxia, brainstem dysfunction, optic atrophy and PN, called Leigh's syndrome or maternally inherited Leigh's syndrome (MILS). MILS is characterized radiologically by bilateral signal hyperintensities in the globus pallidus, putamen, and caudate nucleus (Holt et al., 1990; Tatuch et aI., 1992). 28.4.5. Chronic progressive external ophtalmoplegia: CPEO

CPEO is probably the most common symptom in mitochondrial disorders and is characterized by slowly progressive bilateral ocular dysmotility and ptosis. The ophthalmoparesis affects the eyes symmetrically and relentlessly leading to complete ophthalmoplegia, and patients only rarely complain of double vision. The onset is insidious ranging from infancy to late adulthood. CPEO may occur as an isolated manifestation of mitochondrial dysfunction or be associated with other neurologic or systemic symptoms, such as proximal muscle weakness, ataxia, cardiac conduction defects, hearing loss, diabetes mellitus and short stature ("CPEO plus"). CPEO represents a good example of the genetic heterogeneity frequently seen in mitochondrial disorders. Several genetic defects of the mtDNA have been associated with CPEO: sporadic, single, large deletions; partial duplications; maternally inherited tRNAs point mutations and multiple deletions with either autosomal recessive or dominant inheritance. Autosomal dominant CPEO (AD-PEO) has been linked to at least two different loci on chromosome lOq24 and 3p (Suomalainen et aI., 1995; Kaukonen et al 1996; Kaukonen et al., 2000). AD-PEO, like MNGIE, may be due to a defect of intergenomic communication, where nuclear mutations cause replication errors in the mtDNA. 28.4.6. Leber's hereditary optic neuropathy: LHON

LHON is the first disease to be linked to specific mtDNA defects (Wallace et aI., 1988; Singh et aI., 1989). Bilateral optic neuropathy with subacute

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onset of painless, central visual loss occurring between 15 and 35 years of age is usually the only clinical manifestation. In some families, associated cardiac conduction abnormalities, "multiple sclerosis"-like illness, dystonia and encephalopathy have been described. Inheritance is maternal but with marked male predominance in most pedigrees, possibly due to nuclear genetic factors (Newman, 1993; Kerrison and Newman, 1997). The primary pathogenetic mutations accounting for the majority of LHON cases are mtDNA mutations at G11778A, G3460A and Tl4484C in the ND4, NOI and N06 genes respectively. Depending on the mtDNA point mutation, some degree of vision improvement can occur: the 14484 mutation is associated with visual improvement in up to 60% while the 11778 only in 5% of cases. 28.4.7. Mitochondrial neurogastrointestinal encephalomyopathy: MNGIE MNGIE is characterized by PEO, gastrointestinal dysmotility, PN and leukoencephalomyopathy. Cachexia and severe weight loss are frequently seen (Hirano et al., 1994). The disorder is inherited in an autosomal recessive manner and frequently associated with multiple deletions of the mtDNA. The disease has been mapped to chromosome 22q13.32 and mutations causing loss of function have been documented in the gene coding for thymidine phosphorilase (Hirano et al., 1998; Nishino et al., 1999). It is hypothesized that altered plasma thyamine levels might affect mitochondrial deoxynucleotide pools resulting in mtDNA replication abnormalities and multiple deletions. MNGIE, like AD-PEO, represents an example of nuclear-mitochondrial inter-genomic communication defect, one of the possible mechanisms underlying some of the mitochondrial disorders which do not yet have a molecular diagnosis.

28.5. Diagnostic evaluation and investigations The investigation of a mitochondrial disease can be challenging and requires an integrated approach including clinical, biochemical and genetic tests looking for evidence of mitochondrial dysfunction at multiple levels, as no single finding is pathognomonic. The clinical evaluation should always include a detailed review of systems to uncover symptoms and

involvement of other organs beside CNS and PNS. A family history of maternal inheritance is suggestive of mitochondrial disorder but it's important to remember that mendelian inheritance and sporadic occurrence are also common. The physical examination should always include funduscopy, hearing test. and cardiac auscultation. 28.5.1. Laboratory and biochemistry Elevated serum lactate and pyruvate at rest or after aerobic exercise are important clues in the diagnosis of a mitochondrial disorder, although normal levels do not rule out such diagnosis. Elevated lactate and pyruvate levels are a consequence of defective carbohydrate aerobic oxidation, and may also be found in CSF. One must be cautious in interpreting elevated serum lactic acid levels in the setting of intercurrent sepsis, dehydration, congestive heart failure and valproic acid use. CSF lactate may be elevated for days after seizures or acute brain ischemia. The lactate: pyruvate ratio is also a useful diagnostic tool and indicates a defect of respiratory chain function when greater than 25: 1. Serum creatine kinase (CK) is usually normal or only mildly elevated in most mitochondrial myopathies. A normal CK in the presence of muscle weakness and myopathic EMG findings should therefore raise concern for a mitochondrial disorder. Decreased folic acid in serum and CSF has been reported in patients with KSS, and supplementation should be considered in these patients (Allen et al., 1983). Other metabolic abnormalities described in mitochondrial disorders include increased blood concentration of alanine, generalized aminoaciduria, organic aciduria and decreased blood levels of free carnitine due to partial impairment of l3-oxidation (Jackson et al., 1995; Zeviani et al., 1996). Biochemical analysis of respiratory chain enzymes is used to characterize the underlying molecular defect and to help directing molecular genetic analysis. It can be performed in lymphocytes, cultured skin fibroblasts and fresh or frozen muscle. Functionally intact mitochondria can been extracted from fresh muscle and used for polarographic analysis in specialized laboratories. Frozen muscle specimens are adequate to measure the individual activities of complexes I to IV, and the

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combined activities of I + III and II + III and are used most frequently for diagnostic purpose in specialized laboratories. Muscle biochemistry is almost invariably affected in multisystem mitochondrial disorders due to the high oxidative metabolism in this tissue. Because mitochondrial proliferation is a frequent feature of mitochondrial disorders, enzyme activities should be corrected based on the citrate synthase activity, which is a good marker of mitochondrial mass. Complex II is entirely encoded by nDNA and therefore patients with a complex II defect must have a nuclear gene defect. Patients who have an isolated complex I, III or IV deficiency may have a gene defect in specific encoding regions of either nONA or mtDNA, because 13 of the 78 subunits that make up these complexes are encoded in the mtDNA. Adult patients with mitochondrial disease often have a partial biochemical defect of multiple respiratory chain complexes, reflecting a more global disorder of mitochondrial protein synthesis usually associated with mutations in the mtDNA tRNA genes, mtDNA single or multiple deletions and mtDNA duplications.

28.5.2. Radiology testing

Central nervous system (CNS) involvement is common in mitochondrial diseases, and brain MRI or CT abnormalities are useful tools for diagnosis. Brain MRI is particularly useful in Leigh syndrome, typically showing bilateral signal hyperintensities in the caudate nucleus, putamen and globus pallidus. Prior to the MRI, this disease needed confirmation by postmortem studies. Diffuse central white matter signal abnormalities and basal ganglia calcifications are frequently seen in KSS patients. In MELAS the brain MRI usually shows signal changes involving both gray and white matter, predominantly in the occipital and parietal lobes. These infarct-like lesions typically do not follow the distribution of a vascular territory and no pathologic lesions are found in the main vessels. T2 and diffusion weighted images may show hyperintense lesions indistinguishable from those seen in acute ischemic strokes, however, the apparent diffusion coefficient (ADC) values are normal or increased, indicating a different mechanism under-

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lying the stroke-like episodes in MELAS. ADC is typically decreased for several days after an acute ischemic stroke, and it may help differentiating ischemic strokes from stroke-like episodes early after the onset of neurological symptoms (Clark et aI., 1996; Ohshita et aI., 2000; Oppenheim et al., 2000; Yonemura et aI., 2001). Neuroradiological disappearance of the stroke-like lesions and their reappearance in a different region is also typical of MELAS (Abe et aI., 1990; Pavlakis et al., 1998). Phosphorus magnetic resonance spectroscopy (31P-MRS) can measure the ratio of phosphocreatine (PCr) to inorganic phosphate (Pi) in muscle at rest, during exercise and during recovery. In patients with mitochondrial myopathy this ratio is lower than normal at rest, decreases excessively during exercise and returns to baseline more slowly than normal (Argov and Bank, 1991). 28.5.3. Electrophysiological testing

Nerve conduction studies (NCS) and electromyography are an important part of the evaluation of mitochondrial patients, who often present with myopathy and/or neuropathy. Typically, patients with mitochondrial myopathy and weakness show mild myopathic abnormalities (i.e. short duration polyphasic motor units with reduced amplitude) in their proximal muscles (DiMauro et aI., 1985). However, mitochondrial muscle disease may be clinically evident and yet give rise to only few or no EMG abnormalities. Even patients with proximal muscle weakness may show no EMG abnormalities of muscle dysfunction. Mitochondrial myopathies only rarely show fibrillations and positive sharp waves (Petty et aI., 1986). In the literature one finds reports of individual cases or family but no large series of electrophysiological findings in patients with each type of mitochondrial myopathy, but rather reports of individual cases or families. A retrospective study of a large series of Italian patients with various mitochondrial disorders (Sciacco et aI., 200 I) showed that nerve conduction and EMG studies were abnormal in 86% of cases. Peripheral neuropathy, when present, was always axonal. Axonal neuropathy has been associated with various mitochondrial disorders, including NARP, MELAS. MERRF, MNGIE, KSS and CPEO. Reduced sensory

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amplitudes are the most common finding (Melberg et al., 1996). The neuropathy may be clinically asymptomatic, and frequently undiagnosed. Attempts to characterize the neuropathy and establish a genotype-phenotype correlation in patients with various mitochondrial diseases have not been successful (Colomer et aI., 2000; Bouillot et al., 2002). Mixed myopathic and neurogenic findings have been described in patients with various mitochondrial diseases (Melberg et aI., 1996; Girlanda et aI., 1998; Sciacco, 2001; Sladky, 2001; Torbergsen et aI., 1991). The electrophysiological findings usually correlate with the clinical diagnosis of myopathy and/or neuropathy. Therefore, patients who display concomitant electrophysiological evidence of neuropathy by NCS (i.e. low sural amplitude) and myopathy by EMG should be evaluated for possible mitochondrial disease. In 199I, Torbergsen and colleagues reported a multisystem mitochondrial disorder due to a metabolic defect of the respiratory complex! In a large family including 13 patients with muscle fatigability and neuropathy spanning two generations (Torbergsen et aI., 1991). Using NCS, concentric needle EMG, single-fiber EMG (SFEMG), and macroEMG, they found electrical evidence of axonal neuropathy combined with myopathy in most patients. The EMG showed myopathic changes in the proximal muscles and neuropathic changes in the distal leg muscles, especially when the disease was clinically evident and advanced. Patients with clinical symptoms of neuropathy showed reduced motor and sensory amplitudes by NCS, compatible with an axonal neuropathy. The SFEMG showed increased fiber density and the macro-EMG showed increased motor unit potential (MUP) amplitude in the anterior tibial muscle of most patients. The authors postulated that younger and/or mildly affected individuals begin displaying muscle membrane abnormalities, leading eventually to a myopathy. As the disease progresses, in addition to myopathy, peripheral axonal nerve dysfunction ensues, affecting mainly distal muscles. A rapidly progressive and fatal mitochondrial neuro-myopathy has been reported (Farrell et aI., 1992; King et al., 1996), and characterized by elevated serum CK and CSF protein, onion bulbs and axonal loss in the sural nerve and subsarcolemmal accumulation of mitochondria in the quadriceps muscle.

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Neuro-myopathy by NCSIEMG may rarely be found in other conditions, including inclusion body myopathy (IBM). Of note, some of the IBM patients demonstrate respiratory chain dysfunction, and multiple deletions of muscle mtDNA have been reported in these patients (Oldfors et aI., 1993). Electrophysiological evidence of central nervous system (CNS) involvement is reported in a high percentage of patients with mitochondrial disorders, and the threshold for electrophysiological CNS abnormalities is well below that for clinical and/or radiological manifestations. In a series of 39 mitochondrial diseases studied with somatosensory evoked potentials (Di Lazzaro et al., 1997) the overall incidence of electrophysiological abnormalities was 64%. Abnormal evoked potentials were found in 33% of patients with pure myopathic forms of mitochondrial diseases and one asymptomatic carrier of MERRF mutation. Of the individual tests, somatosensory evoked potentials were abnormal in 49% of the patients and motor evoked potentials were abnormal in 46% of the patients. 28.5.3.1. Kearn-Sayre syndrome (KSS) Nerve conduction studies. Not specifically reported. However, axonal sensory neuropathy has been clinically reported in KSS. Electromyography. Not specifically reported. Single-fiber EMG. Not reported. Motor unit estimates. Not reported. Evoked potentials. Not reported. Electroencephalography. Not reported. Autonomic studies. Not reported. 28.5.3.2. MELAS: Mitochondrial encephalomyopathy. lactic acidosis and stroke-like episodes Nerve conduction studies. Peripheral neuropathy may be part of this syndrome and is usually axonal (Sciacco et aI., 2001). Demyelinating polyneuropathy has also been reported in MELAS patients with the A3243G mutation (Rusanen, 1995; Fang, 1996). In 1995, Rusanen and colleagues described a patient with the A3243G mutation and a uniform, demyelinating, mixed (motor greater than sensory) polyneuropathy with prolonged F-waves and no partial motor conduction block or temporal disper-

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sion. The electrical features resembled the hereditary more than the acquired demyelinating polyneuropathies. The authors postulated a defect in the mitochondrial function of the Schwarm cells. In other cases, the motor nerve conduction studies showed CMAP temporal dispersion, and teased-fiber nerve preparations showed segmental demyelination and remyelination (Pezeshkpour et al., 1987; Mizusawa, 1991). Mixed axon loss and demyelinating sensorimotor neuropathy was the most common type of polyneuropathy in a recent report of 32 patients with MELAS 3243 from Finland (Karppa et al., 2003). One patient presented with uniform demyelinating sensorimotor polyneuropathy. Four patients had sensory greater than motor neuropathy. Three patients (9.4%) had clinical and electrophysiological evidence of carpal tunnel syndrome (CTS), suggesting a higher prevalence of CTS than in the general population. Electromyography. EMG usually shows "myopathic" changes (Arpa et al., 1994). In the large Italian series of mitochondrial patients (Sciacco et al., 2001) all patients with the A3243G point mutation had an abnormal EMG and pure myopathic changes were the most frequent findings (59%). Single-fiber EMG. Not reported. Motor unit estimates. Not reported. Evoked potentials. Visual evoked potentials (VEP) may show sporadic disturbances, even changing dramatically within a matter of weeks (Pachalska, 2002). YEP showed delayed PlOD responses in four of eight MELAS A3243G patients with retinal pigmentary abnormalities, detected by retinal photography (Sue et al., 1997). Somatosensory-evoked potentials (SEPs) and averaged EMG for long loop reflexes, revealed socalled "giant SEP" and enhanced long loop reflexes reflecting cortical hyperexicitability in a 9-year-old MELAS patient with myoclonus (Saitoh et al., 1992). Jerk-locked averaging yielded no myoclonus related spikes, but myoclonus-contingent 4-5 Hz theta bursts appeared. These findings suggest that some types of MELAS may be associated with cortical types of myoclonus. Electroencephalography. EEG may show posteriorly predominant abnormalities in patients with MELAS and epileptic seizure (Hori et al., 1989). Autonomic studies. Not reported.

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28.5.3.3. MERRF: Myoclonic epilepsy with ragged red fibers Nerve conduction studies. Peripheral neuropathy may be part of this syndrome and is usually axonal. Chu and colleagues from Taiwan reported 3 MERRF patients with clinical symptoms and signs of polyneuropathy where NCS showed decreased amplitudes of the compound muscle or nerve action potentials (Chu et al., 1997). Sural nerve biopsy from one of these patients showed axonal degeneration with reduction of large myelinated fibers, and mtDNA analysis of the sural nerve showed 80% Ato-G 8344 mtDNA mutation. Electromyography. Myopathic EMG abnormalities are typically never seen in MERRF patients with the A8344G mutation (Sciacco, 2001). Single-fiber EMG. Not reported. Motor unit estimates. Not reported. Evoked potentials. Somatosensory evoked potentials may be abnormally enlarged (giant SSEP) in patients with myoclonus and MERRF-8344 mtDNA mutation (Thompson et al., 1994). The findings are those of cortical reflex myoclonus, with enlarged cortical SSEP and late reflex responses to peripheral nerve stimulation. This pattern of electrophysiological abnormalities was uniform in patients with various degree of myoclonus (Thompson et al., 1994). Abnormal giant SSEP have been reported also in asymptomatic carriers of MERRF mutation (Di Lazzaro et al., 1997), and in the asymptomatic sister of a MERRF patient (Acharya et al., 1995). Electroencephalography. Electroencephalography showed paroxysmal spike and polyspike and wave discharges, with photic sensitivity (Thompson et al., 1994). In a 5-year clinical and electrophysiologic follow-up study of two siblings with MERRF-8344 (Ohtsuka et al., 1993) the EEGs showed slowing of basic patterns, diffuse spike-and-wave complexes, occipital dominant wave-and-spike phantoms, 6- and 14-Hz positive spikes, and photosensitivity. No definite deterioration of basic patterns was seen, and diffuse spike-and-wave complexes and photosensitivity gradually disappeared during the slowly progressive clinical course. P2 latencies of patternreversal visual evoked potentials throughout the clinical course and III through V interpeak latencies of auditory brainstem responses were prolonged at

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follow-up without giant sensory evoked potentials in both cases. Autonomic studies. Not reported. 28.5.3.4. NARP: Neuropathy, ataxia and retinitis pigmentosa Nerve conduction studies. Neuropathy is a cardinal clinical feature of this specific genotype, as sensory polyneuropathy is required for diagnosis (Holt et al., 1990). The neuropathy is usually axonal and reduced sensory amplitudes are the most common finding. Electromyography. Not specifically reported. Single-fiber EMG. Not reported. Motor unit estimates. Not reported. Evoked potentials. Not reported. Electroencephalography. Not reported. Autonomic studies. Not reported. 28.5.3.5. Chronic progressive external ophthalmoplegia: CPEO Nerve conduction studies. Generally, electrophysiological tests are a valuable supplement to the clinical examination in patients with mitochondrial CPEO. Schubert and colleagues studied 28 patients with histologically and biochemically proven mitochondrial CPEO using motor and sensory nerve conduction tests, somatosensory, auditory and visual evoked potentials and transcranial magnetic stimulation. Nervous system involvement was demonstrated in 24 patients, affecting the peripheral and central nervous systems in 18 and 10 patients, respectively. Electromyography. In CPEO patients, myopathic EMG changes may be most evident in the frontalis and orbicularis oculi muscles (Melberg et al., 1996). Older CPEO patients with proximal limb weakness and fatigue of long duration frequently show myopathic EMG changes in their proximal muscles (Melberg et aI., 1996). In 1987, Krendel and Sanders reviewed the EMG studies of 17 patients with CPEO. In 13 of 17 patients, conventional concentric needle EMG demonstrated a "myopathic" pattern, usually predominating in the shoulder muscles. Single-fiber EMG. Single-fiber EMG may show increased fiber density or increased jitter in CPEO patients (Fawcett et aI., 1982; Krendel et al., 1987; Torbergsen et aI., 1991; Bertorini et aI., 1994). SFEMG may not distinguish ocular myasthenia from mitochondrial myopathy, since the jitter is increased

557 in both conditions (Krendel et aI., 1987; Ukachoke et aI., 1994). Single-fiber EMG showed increased jitter and/or blocking in at least one muscle in 13 of 16 CPEO patients (Krendel et aI., 1987). Jitter was increased in the frontalis muscle in 10 of 13 patients and in an arm muscle in 5 of 12. When both muscles were tested, jitter was greater in the frontalis muscle in 5 patients and in the arm muscle in 2. These observations demonstrate that it may be difficult to distinguish myasthenia gravis from CPEO by EMG. For these reasons Sanders and colleagues postulated that in addition to a mild generalized myopathy. a primary defect in neuromuscular transmission may be present in CPEO patients (Krendel et al., 1987). Other authors showed that the increased jitter seen in some mitochondrial patients correlates with the neurogenic changes in these patients (Torbergsen et aI., 1991; Girlanda et aI., 1999). Bertorini et al. (1994) reported slightly increased fiber density by SFEMG in 50% of patients with mitochondrial myopathy, possibly due to myogenic denervation followed by reinnervation. Motor unit estimates. Not reported. Evoked potentials. In the study by Schubert and colleagues, evidence of cortico-spinal tract involvement was found in 4 CPEO patients using somatosensory, auditory and visual evoked potentials. This was clinically evident in only 2 patients, raising suspicion that dysfunction of the corticospinal tract in CPEO may occur more frequently than clinically assumed (Schubert et al., 1994). Electroencephalography. Not reported Autonomic studies. Not reported. 28.5.3.6. Mitochondrial neurogastrointestinal encephalomyopathy: MNGIE Nerve conduction studies. MNGIE by definition includes a sensorimotor polyneuropathy. The polyneuropathy of MNGIE is mainly characterized by areflexia, distal greater than proximal limb weakness and sensory loss (Hirano et aI., 1994), and it is typically axonal, although some cases showed clear demyelinating features (Uncini et aI., 1994). Electromyography. Not specifically reported. Single-fiber EMG. Not reported. Motor unit estimates. Not reported. Evoked potentials. Not reported Electroencephalography. Not reported. Autonomic studies. Not reported.

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28.5.4. Muscle biopsy

Because muscle is so often clinically or subclinically affected in mitochondrial disorders, muscle biopsy is frequently used for diagnosis and provides useful morphological, biochemical and genetic information. Abnormal proliferation of mitochondria is a hallmark of mitochondrial dysfunction and is identified as irregular subsarcolemmal patchy aggregates staining purple with the modified Gomori trichrome and intensely dark with the succinate dehydrogenase (SDH). These stainings give rise to the so-called "ragged red fibers" (RRF). Although RRF are commonly considered a hallmark for abnormal oxidative phosphorylation, they are not specific and not even required for diagnosis of a mitochondrial disorder. They are more commonly seen in diseases due to mtDNA defects, but may be found also in mitochondrial diseases of Mendelian inheritance. RRF are frequently associated with mtDNA deletions, depletion and tRNA mutations (MELAS, MERRF) affecting mitochondrial protein synthesis. RRF are almost never seen in association with mutations of structural genes like ND and ATPase genes, causing NARP, Leigh syndrome and LHON. RRF have occasionally been described in other neuromuscular diseases, including inclusion body myositis, polymyositis and zidovudine (AZT)induced myopathy, and in endurance-trained athletes. With aging, the mtDNA accumulates increasing number of point mutations and deletions and therefore, the presence of a few RRF in the muscles of older people may be a normal occurrence. In AZT-induced myopathy, the mitochondrial dysfunction is due to depletion of mtDNA as a result of AZT-induced inhibition of mtDNA replication (Arnaudo et al., 1991). Cytochrome C oxidase (COX) staining is a useful histochemical staining for the identification of complex IV, which is partly encoded by the mtDNA. The presence of COX-negative fibers usually indicates impaired mitochondrial protein synthesis, and may be the only detectable abnormality. COX negative fibers are found predominantly in primary COX deficiencies, but scattered COX-negative fibers are often seen in disorders associated with mtDNA deletions and tRNA mutations. RRF are usually but not always COX-negative. The absence of RRF or

E. CIAFALONI AND E. ARNAUDO

COX-negative fibers does not rule out a mtDNArelated disease. In specialized laboratories, Immunohistochemistry using antibodies against mtDNA- and nDNAencoded proteins is utilized to characterize the underlying mitochondrial defect. In general, mutations of mtDNA protein-coding genes show abnormal stain limited to the specific complex affected, where mutations of tRNAs and large mtDNA deletions typically cause a more diffuse impairment of mitochondrial protein synthesis. Electron microscopy (EM) may show enlarged, bizarre mitochondria with abundant and distorted cristae, containing paracrystalline inclusions. These findings are considered the EM hallmark of mitochondrial myopathies but do not have a specific correlation with the clinical phenotype or the biochemical and genetic defect. Single-fiber PCR has been used in specialized laboratories to document the pathogenicity of novel mtDNA mutations (Moraes et al., 1993). A high level of mutant mtDNA in dissected RRF or COXnegative fibers as compared to normal fibers is considered strong evidence for a mutation to be pathogenic.

28.5.5. Genetic testing and counseling

Southern blotting and polymerase chain reaction (PCR) are commercially available techniques to detect mtDNA deletions and duplications, and the most common mitochondrial point mutations. They can be performed on DNA extracted from any tissue. Research laboratories employ restriction fragment length polymorphism (RFLP) screening of PCR products and direct sequencing of the entire mitochondrial genome to search for rare or new mutations. Genetic screening for the mutations most commonly associated with each particular syndrome can be the first step toward a definite diagnosis in those patients with a typical clinical presentation. Genetic screening on blood DNA is not always adequate to detect mutations and caution in interpreting the results is therefore needed. A muscle biopsy is frequently necessary for definite diagnosis and genetic test should be pursued in muscle DNA if the clinical suspicion is strong and blood test negative.

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In most LHON patients it's usually adequate to send blood for gene testing because these patients have a very high level of mutated mtDNA in blood and one of the three point mutations is found in 95% of cases. The tRNALys 8344 mutation associated with MERRF is also highly detectable in blood, while the tRNA Leu 3243 can be missed in some MELAS patients. Muscle DNA testing is also more sensitive for detecting point mutations in olygosymptomatic and asymptomatic maternal relatives of MELAS patients and single deletions in sporadic CPEO and KSS patients. Males with mtDNA mutations will not transmit the disease. Females with single mtDNA deletions as found in CPEO and KSS have a less than 5% chance of transmission. Recurrence risks in LHON's families with a pathogenic mtDNA mutation are 30% for brothers, 8% for sisters, 46% for nephews, 10% for nieces and 31% and 6% for male and female cousins respectively (Harding et al., 1995). Females carrying the MERRF G8344A mutation have a less than 5% risk of transmission if their mutant load in blood is less than 35%. The percentage of affected offspring of females carrying the A3423G MELAS mutation increases with the mutant load in maternal blood with 50% being affected if the blood maternal mutant load is 20% or greater (Chinnery et al., 1998). Identification of a genetic mutation is helpful in guiding genetic counseling in family members but precise risk assessment in these disorders remains challenging due to the frequently poor correlation between phenotypic severity and level of mutant genome. Genetic counseling is further complicated by the difficulty in predicting mutant levels and expression in different tissues and the change of mutant mtDNA load over time due to the mitotic segregation and heteroplasmy phenomena (Chinnery et aI., 1999). Prenatal diagnosis and counseling of mtDNA diseases is difficult because little is known regarding the tissue distribution of mtDNA mutants in the developing fetus and the mutation load in amnyocytes and chorionic villi may not correspond to that of other fetal tissues and may shift in utero or after birth due to random mitotic segregation. Recommendations regarding prenatal diagnosis for maternally inherited mtDNA diseases have been outlined and depend on the particular mutation (Poulton and Marchington, 2000).

28.6. Treatment There are currently no curative treatments for mitochondrial disorders, and the management of these patients is largely supportive. However, a definitive diagnosis is important for prognostic and genetic counseling, as well as watchful monitoring of likely complications. For example, the use of cardiac pacing may prevent sudden death in the patients with Kearns-Sayre syndrome, and the early diagnosis and treatment of mitochondrial diabetes may reduce its complications. Aminoglycoside antibiotics must be avoided in all patients with mtDNA mutations, because of higher risk of ototoxicity. Treatments for patients with mtDNA mutations have focused primarily on metabolic therapies designed to increase ATP production in affected tissues (Shoffner and Wallace et al., 1994; Walker and Byrne, 1995). Examples of metabolic supplements that have been used include coenzyme QIIJ' menadione, succinate, ascorbate, carnitine, thiamine, and riboflavin. The small number of patients subjected to therapies and the highly variable symptomatogy of individual patients during disease progression make it difficult to assess the efficacy of these treatments. Clinical improvement has been reported in isolated cases, but also a lack of response has been reported for each agent. In some cases there was laboratory evidence for improvement (i.e. reduction in lactate levels, improvement of 31P MRS) without any change in the clinical status. In other cases clinical improvement was not quantified by metabolic studies. The largest and the only double-blind trial using coenzyme Coenzyme Q/o, a lipid soluble quinone that may act by improving electron transport through defective respiratory complexes, showed only a modest clinical improvement in some patients (Bresolin et al., 1990). A dose of 4.3 mg/kg is recommended in mitochondrial diseases (Shoffner and Wallace, 1994). Riboflavin (vitamin B2) acts as precursor for flavin co-factors for complexes I and II, and it was successfully used in patients with complex I deficiency, and other mitochondrial disorders affecting lipid metabolism, at the dose of 100 mg/day. Although there is no hard evidence of clinical improvement, there are good theoretical reasons for using these agents, and they have no major side effects (Chinnery and Turnbull, 2001).

560

Some metabolic therapies are treatments devised to bypass a specific deficiency. For example, a patient with an isolated complex III deficiency was successfully treated with menadione and ascorbate, a therapy designed to bypass the blockade at complex III and to donate electrons directly to cytochrome c (Eleff et al., 1984; Argov et al., 1986). However, mutations in tRNA genes and large-scale deletions of mtDNA affect the biosynthesis of all the respiratory chain complexes, therefore it is not possible to bypass the defect. Thus, metabolic therapies are expected to have limited success for patients harboring these mutations. Dichloroacetate (DCA) indirectly stimulates the pyruvate dehydrogenase complex, and has been shown to lower lactic acid concentrations in various conditions of congenital and acquired lactic acidemia (Stacpoole, 1989). Short-term improvement in muscle and brain oxidative metabolism has been reported (DeStefano et aI., 1995), but potential complications include a painful peripheral neuropathy (Kurlemann et al., 1995). In a series of 27 patients treated with DCA for over one year, more than 50% developed electrical signs of peripheral neuropathy (Spruijt et al., 2001). The neuropathy affected motor more than sensory fibers and the legs more than the arms, and it was not prevented by coadministration of daily thiamine. Another approach to therapy is myoblast transfer. Skeletal muscle is composed of multi-nucleated fibers formed from the fusion of multiple myoblasts. Wild-type mitochondrial genomes could be transferred to affected muscle via myoblasts. However, myoblast transfer would be limited to skeletal muscle and not to other affected tissues. A further limitation includes the limited success of myoblast transfer in humans (Coovert and Burghes, 1994; Pagel and Morgan, 1995; Smythe et aI., 2000). In addition, since most mtDNA mutations are heteroplasmic, wild-type mtDNA is already present in affected tissues. Therefore, myoblast transfer would need to transfer sufficient quantities of wild-type mtDNA to increase the ratio of wild-type to mutated mtDNA to enable normal mitochondrial function. It is not known if sufficient numbers of myoblasts could be transferred to alter these ratios significantly. Increasing the amount of wild-type mtDNA in muscle by 'gene shifting' has shown promise as a therapy. In several studies where high levels of a

E. CIAFALONI AND E. ARNAUDO

mtDNA mutation were found in skeletal muscle, the mutant mtDNA could not be detected in skeletal muscle satellite cells. Satellite cells are dormant muscle progenitor cells that are activated during muscle growth or repair. A stimulation of satellite cell incorporation into muscle fibers following a biopsy (Shoubridge et al., 1997) or chemically induced necrosis (Clark et aI., 1997) showed that the regenerated muscle fibers contained predominantly wild-type mtDNA. These studies led to a protocol to increase the ratio of wild-type to mutated mtDNA by resistance exercise training (Taivassalo et al., 1999). However, this method has limitations since it is restricted to skeletal muscle. Alternatively it may be possible to directly alter the ratios of wild-type to mutated mtDNA in cells. Since mtDNA is continually replicating and turning over, it has been proposed to treat patients by selectively inhibiting the replication of the mutated mtDNA, thereby allowing propagation of only the wild-type molecule. Over time, therefore, the biochemical and, potentially, the clinical deficiency could be reversed. Taylor and colleagues (Taylor et al., 2000) have proposed the use of sequence-specific antigenomic peptide nucleic acids (PNAs) to hybridize and specifically inhibit the replication of mutated mtDNA to achieve these goals. Unfortunately, the feasibility of this approach has not been demonstrated. Gene therapy would provide another approach to treatment. Direct manipulation of the mtDNA is presently not possible and would present many of the same difficulties as other therapies because of the issue of heteroplasmy. However, rapid advances are being made in gene therapy in a great number of tissues based upon manipulation of the nuclear genome and its expression. This approach may offer the best prospects for the eventual treatment of patients suffering from mitochondrial diseases.

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