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KEY POINT

Mitochondria are present in every cell in the human body, except red blood cells. In addition to their main role providing aerobically derived ATP, they are also involved in reactive oxygen species generation, apoptosis, and calciumbuffering.

MITOCHONDRIAL CYTOPATHIES IN CHILDREN AND ADULTS


Mark A. Tarnopolsky ABSTRACT
Mitochondrial cytopathies represent a heterogenous group of disorders with abnormal mitochondrial structure or function as the defining entity and with clinical onset across all age groups. Given that the mitochondria provide the majority of cellular adenosine 50 -triphosphate (ATP), the clinical symptoms can manifest with a myriad of presentations. Cellular pathology includes a reduction in aerobic energy transduction, an increase in anaerobic energy utilization (lactate production), and, in some cases, higher oxidative stress. Diagnostic criteria include history (classic multisystem involvement), blood test abnormalities (elevated lactate, alanine, and creatine kinase activity), urine organic acids (3-methylglutaconic acid, ethylmalonic acid, fumarate), muscle enzymology (reduction in activity), exercise testing (low maximum oxygen consumption [VO2max], early lactate production) and other ancillary tests, including MRI, MR spectroscopy, and near-infrared spectroscopy. Treatment is multifactorial and supportive, including optimization of nutrition and deficiency (folate and vitamin B12) replacement, gastrostomy tube if necessary (failure to thrive), treatment of secondary neurologic issues, vitamin and cofactor therapy (creatine monohydrate, coenzyme Q10, -lipoic acid, etc), and exercise.
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INTRODUCTION Mitochondria are essential to the functional integrity and energy metabolism of all cells (except red blood cells) and can be directly implicated in human disease (mitochondrial cytopathies) or as contributors to health (mitochondrial proliferation in muscle cells of elite athletes) and disease (mitochondrial dysfunction in ischemic tissue, neurodegenerative diseases, and critical illness). Mitochondrial diseases manifest across the full spectrum from infancy to old age. Children with severe early-

onset mitochondrial cytopathies are surviving into adulthood, and intrafamilial variability in age at onset and clinical manifestations of inherited mitochondrial conditions have implications for the care of all family members. This chapter will provide an overview of mitochondrial biology and will discuss the most common forms of mitochondrial cytopathies in children and adults. Discussion of the contributions of secondary mitochondrial dysfunction in other neurologic or systemic diseases is beyond the scope of this chapter.

Relationship Disclosure: Dr Tarnopolsky has received personal compensation for speaking engagements with Transgenomic, Inc., has received personal compensation in an editorial capacity from Medicine & Science in Sports & Exercise, and has received research support from ApoPharma, Inc., and Gilead. Unlabeled Use of Products/Investigational Use Disclosure: Dr Tarnopolsky has nothing to disclose.

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MITOCHONDRIAL BIOLOGY PRIMER Mitochondria are the main powerhouse for all cells in the body except red blood cells. The mitochondria are considered an evolutionary parasite evolved from purple photosynthetic bacteria that invaded proto-eukaryotic cells approximately 11/2 billion years ago. This symbiotic relationship is felt to confer selective advantage through enhanced aerobic adenosine 50 -triphosphate (ATP) delivery and oxygen detoxification given the steep oxygen gradient evolving in the earths atmosphere. Over 11/2 billion years of evolution, all of the proteins involved in mitochondrial DNA (mtDNA) replication and maintenance were transferred to the nuclear genome, and the bacterialike circular mitochondrial genome retains only 16,569 base pairs encoding for 13 polypeptide subunits, 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and a small noncoding region. The circular genome is composed of an outer heavy strand (predominately purines [adenine and guanine]) and an inner light strand (predominantly pyrimidines [cytosine and thymine]). Bacterialike polycistronic replication of mitochondria is triggered by cellular signals sensed through a variety of mechanisms (reactive oxygen species, protein kinase, energy charge) that increase gene expression of a variety of nuclear-encoded mitochondrial proteins and mitochondrial transcription factor A (mtTFA) under coregulation with peroxisome proliferator-activated receptor -coactivator-1 ( Wu et al, 1999). mtTFA binds to regulatory elements in the mitochondrial genome, initiating replication of mtDNA. mtDNA encodes for seven subunits of complex I, one subunit of complex III, three subunits of complex IV, and two subunits of complex V. Consequently, mtDNA encodes for a component of each of the major enzyme complexes involved

in the core series circuit of the electron transport chain (I, III, IV) and the adenosine triphosphatase (ATPase) function ( V ). There can be two to several dozens of mtDNA copies per mitochondria, and in certain cells, such as a multinucleate muscle cell, there may be many thousands of mitochondria. The purpose of the mitochondria is to provide aerobically derived ATP for a variety of cellular energy processes. Fat, protein, and carbohydrate are all oxidized in the mitochondria through a variety of different pathways that ultimately converge on the electron transport chain. Nicotinamide adenine dinucleotide (reduced form) (NADH) + H+ ( proton) derived from -oxidation or the tricarboxylic acid (TCA) cycle provides reducing equivalents to complex I, where a proton is pumped from the mitochondrial matrix to the intermembrane of space, and electrons are passed onto coenzyme Q10. Flavin adenine dinucleotide (reduced)2 provides reducing equivalents to complex II from -oxidation or the TCA cycle, and electrons are also passed to coenzyme Q10. Electrons then flow to complex III, where proton pumping occurs, and onto complex IV, which is the terminal oxygen acceptor, and a final proton is pumped. The potential energy from the reducing equivalents is created by the relatively impermeable inner mitochondrial membrane leading to a buildup of a proton motive force and the development of a membrane potential ($ M). The accumulated protons in the intermembrane space flow through a dynamic turbinelike system at complex V (ATPase synthase) toward the matrix and generate ATP, analogous to the potential energy from a waterfall captured by a turbine. mtDNA is maternally inherited, and both male and female offspring will inherit mtDNA from their mother. A unique aspect of mtDNA is a concept termed heteroplasmy, referring to the relative proportion of mutant to wild
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" MITOCHONDRIAL CYTOPATHIES

KEY POINT

It is estimated that the minimal prevalence of mitochondrial cytopathies is 1:6000 individuals and this is likely to increase as new genes are being discovered for phenotypes not previously considered to be mitochondrial in origin.

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type mitochondrial genomes within a cell. For example, a mother with a specific transition mutation (A>G) at position 3243 in the mtDNA that was 30% mutant and 70% wild type would pass on the 3243 mutation in varying ratios to her offspring and each child would have different ratios in each tissue. The latter process is called replicative segregation, in which the heteroplasmy in the oocyte undergoes expansion, contraction, and random expansion in different tissues within the developing embryo and fetus after going through an embryologic bottleneck. Consequently, the proportion of mutant:wild type heteroplasmy in different tissues can vary from 99:1 to 1:99. Higher levels of mutant heteroplasmy are generally associated with more severe mitochondrial dysfunction; however, the percent of heteroplasmy that results in mitochondrial dysfunction is highly variable and mutation specific, and consequently a set level of mutant heteroplasmy cannot be uniformly ascribed as the threshold. Any somatic or mtDNA gene mutation that leads to a breakdown in the complex and carefully orchestrated electron transport chain energy circuit can lead to a reduction in energy transduction and have tissue-specific deleterious consequences. To some extent, the clinical symptoms of mitochondrial dysfunction can be predicted from the relative reliance of different tissues on aerobic energy transduction. The latter fact provides some rationale for the frequent involvement of the CNS (strokes, seizures, developmental delay, blindness, hypoacusis) and muscle (weakness, fatigue) as common symptoms of mitochondrial cytopathies. MITOCHONDRIAL DNA MUTATIONS AND DISEASE The explosion of knowledge in the area of mitochondrial cytopathies and the emergence of mitochondrial medicine began in 1988 to 1989 with a discovery
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of large-scale mtDNA deletions as the cause of Kearns-Sayre syndrome, a transition mutation (3243 A>G) as a cause for mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS), and a transition mutation (11778 G>A) as a cause of Leber hereditary optic neuropathy (Holt et al, 1988; Singh et al, 1989). Since these original descriptions, and with the ability to rapidly sequence mtDNA, the identification of pathogenic point mutations in mtDNA has exploded in recent years, with 165 rRNA/tRNA mutations reported on Mitomap (www.mitomap. org/cgi-bin/tbl9gen.pl) and 194 reported mutations in coding and control regions (www.mitomap.org/cgi-bin/tbl8gen.pl). Many of the mitochondrial disorder names have followed in the tradition of an acronym describing the clinical phenotype (eg, IOSCA = infantile onset spino cerebellar ataxia, MERRF = myoclonus epilepsy and ragged red fibers; MILS = maternally inherited Leigh disease). An increasing number of nuclearencoded mitochondrial function and structural genes are clearly associated with mitochondrial cytopathies (www. mitomap.org/rimtab5.html). Although once considered rare, the incidence and prevalence of mitochondrial cytopathies is increasing dramatically because of increased recognition of these conditions and the discovery of genes responsible for the disorders. It is estimated that the minimal prevalence of mitochondrial cytopathies is 1:6000 individuals (Chinnery et al, 2000; Schaefer et al, 2008), and again this is likely to increase as new genes are being discovered for phenotypes not previously considered to be mitochondrial in origin (eg, mfn2 resulting in Charcot-MarieTooth disease type 2) (Zuchner et al, 2004). The largest representation of nuclear mutations directly affecting the electron transport chain subunits are

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the complex I defects resulting in the clinical picture of Leigh disease (Hoefs et al, 2008; Lebon et al, 2007). Other autosomal recessively inherited mutations have been described in association with complex II subunit (SDHA) and complex III subunit (UQCRB)associated Leigh disease. Autosomal dominant mutations have been described in the succinate dehydrogenase (SDHB, C, D) gene, usually resulting in paraganglionoma and pheochromocytoma (Astuti et al, 2001). A large number of mutations characterized to date affecting nonstructural nuclear genomes implicated mitochondrial disease. For example, a number of genes have been implicated in Leigh disease (SURF-1, LRPPRC, COX15, and BCS1L) or cardioencephalomyopathy and hypertrophic cardiomyopathy (SCO2) and hypertrophic cardiomyopathy (COX10, COX15) (Antonicka et al, 2003a; Antonicka et al, 2003b; Horvath et al, 2006; Jaksch et al, 2001a). Mitochondrial genes implicated in mtDNA stability represent the largest group of nuclear mutations involved in mitochondrial cytopathy. The largest subgroup is the polymerase gamma (POLG) mutations responsible for a variety of syndromes including Alpers syndrome (hepatic encephalomyopathy), chronic progressive external ophthalmoplegia (CPEO), spinocerebellar ataxia, and sensory ataxic neuropathy with dysarthria and ophthalmoplegia (SANDO). Other stability factors including ANT1 and C10ORF2 (Twinkle) are associated with CPEO, and mutations in the ECGF1 (thymidine phosphorylase) gene result in a severe disorder called myoneurogastrointestinal encephalomyopathy (MNGIE). Two genes implicated in maintenance of the deoxynucleotide triphosphate pool include DGUOK and TK2, which result in hepatocerebral and myopathic mtDNA depletion syndrome, respectively. Genes affecting mitochondrial integrity include OPA1, which is a dynamin-related protein resulting in autosomal dominant

optic atrophy and associated with encephalopathy (Amati-Bonneau et al, 2008). Tafazzin gene mutations result in an X-linked condition associated with cardiolipin defects causing Barth syndrome (noncompaction cardiomyopathy, 3-methylglutatonic aciduria, failure to thrive), and mitofusin 2 (mfn2) gene mutations result in an autosomal dominant predominately axonal myopathy called Charcot-Marie-Tooth disease type 2 ( Verhoeven et al, 2006). It is likely that the number of nuclear genes responsible for mitochondrial disorders will increase dramatically in the years to come. A significant advantage derived from the discovery of nuclear gene mutations is the ability to provide accurate prenatal diagnosis and genetic counseling, as the transmission follows mendelian genetics. Although prenatal counseling can be given for some of the mtDNA mutations, such as maternally inherited Leigh disease (MILS, 8993 T>C/G) (Dahl et al, 2000; White et al, 1999), heteroplasmic transmission from mother to child always leads to uncertainty regarding the relative risk to the fetus of inheriting a high proportion of mutated to wild type mitochondria. Some of the more common mitochondrial disorders are summarized in Table 6-1. MITOCHONDRIAL MYOPATHY DIAGNOSIS History Given that the mitochondria are very important in terminally differentiated tissues, including brain, heart, and muscle, mitochondrial cytopathies commonly involve these tissues. Mitochondrial dysfunction in brain can result in developmental delay, hypotonia, seizures, strokelike episodes, visual loss, and hypoacusis. Involvement in skeletal muscle can lead to hypotonia in younger children with gross motor delay, while manifestations in older children, adolescents, and adults tend to include muscle weakness and
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KEY POINT

A history of fluctuating symptoms affecting multiple tissues or organs should prompt consideration of a mitochondrial cytopathy in any age group.

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" MITOCHONDRIAL CYTOPATHIES

TABLE 6-1 Disease


MELAS

Examples of Common Types of Mitochondrial Disorders in Children and Adults Genetics


3423 A>G 3271 T>C 3260 A>G (Maternal)

Clinical Presentation/Features
Infancy: rare (less than 1% of cases) Childhood and adult: (variable pattern and age of onset) encephalopathy, strokelike episodes, lactic acidosis, myopathy, cognitive decline, hypoacusis, type 2 diabetes, ataxia, optic atrophy Childhood and adult: (variable pattern and age of onset) generalized epilepsy (myoclonic and tonic-clonic), myoclonus, cognitive decline, hypoacusis, ataxia, optic atrophy, head and neck lipomas, muscle weakness/atrophy, neuropathy, psychiatric disorders Childhood and adult: progressive external ophthalmoplegia, pigmentary retinopathy, and cardiac conduction block; can also have early-onset cognitive decline, hypoacusis, ataxia, proximal muscle weakness, and multiple endocrinopathies Adult: slowly progressive weakness of muscles involved in horizontal eye movements and eyelid opening, resulting in a progressive and symmetric external ophthalmoplegia with bilateral ptosis Infancy and childhood: (often fatal at less than 5 years of age) hypotonia failure to thrive due to poor feeding and suck reflex, and recurrent vomiting; also noted are psychomotor retardation, ophthalmoplegia, optic atrophy, muscle weakness, spasticity, abnormal movements (dystonia, chorea), and ataxia

MERRF

8344 A>G 8356 T>C 8363 G>A (Maternal)

Kearns-Sayre syndrome

Deletion (1.3 kb8.8 kb) of mtDNA (Often sporadic)

CPEO

Deletions of mtDNA (AD and AR; polymerase gamma, Twinkle and ANT mutations; sporadic)

Leigh disease

Complex I, II, IV, pyruvate dehydrogenase deficiency, 8993 mutations

MELAS = mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes; MERRF = myoclonic epilepsy and ragged red fibers; CPEO = chronic progressive external ophthalmoplegia; mtDNA = mitochondrial DNA; AR = autosomal recessive; AD = autosomal dominant.

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exercise intolerance. Cardiac involvement can manifest in infancy with cyanosis and cardiac failure (SCO2 gene mutations), contribute to exercise intolerance at any age, and manifest as cardiomyopathy later in life. Cardiomyopathy is usually hypertrophic, but dilated forms or left ventricular noncompaction can also occur (Scaglia et al, 2004). Liver involvement is most commonly seen with Alpers syndrome (POLG mutations) and mtDNA depletion due to dGUOK gene mutations (Mandel et al, 2001; Naviaux et al, 1999; Naviaux and Nguyen, 2004). GastroinContinuum Lifelong Learning Neurol 2009;15(6)

testinal symptoms can be mild and nonspecific, including irritable bowellike symptoms and constipation in MELAS syndrome and other mitochondrial cytopathies, or can be more severe, including intestinal pseudoobstruction (MELAS syndrome) or severe gastrointestinal dysfunction (MNGIE syndrome). Symptoms of peripheral neuropathy (numbness, tingling, distal weakness) are seen frequently in MNGIE syndrome and may be misdiagnosed as Charcot-Marie-Tooth disease. Numbness, tingling, and sensory ataxia are common in SANDO syndrome seen

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with POLG and Twinkle gene mutations (Gago et al, 2006; Hudson et al, 2005). Psychiatric symptoms of dementia (MELAS), depression (Twinkle), and pervasive developmental symptoms, including autism, have been described. We have seen a number of patients with adolescent-onset or later-onset psychosis as part of the clinical manifestations of mitochondrial disease (MELAS 3271, MERRF 8363, Twinkle). Ataxia is a common symptom in children with Leigh disease and is particularly prominent in a syndrome termed infantile-onset spinocerebellar ataxia (IOSCA), and in mitochondrial recessive ataxia syndrome (MIRAS). IOSCA has been associated with mutations in Twinkle (Hakonen et al, 2007; Nikali et al, 2005), and MIRAS is usually associated with mutations in the POLG gene (Hakonen et al, 2008). Both of these conditions lead to mtDNA depletion in the brain and liver with complex I deficiency seen only in large neurons (Hakonen et al, 2008). The family history is also helpful in diagnosing mitochondrial myopathy. A strong maternal inheritance pattern is suggestive of mtDNA mutation. Absence of maternal inheritance does not rule out mitochondrial cytopathy. Nuclear inheritance patterns are represented in all types of mitochondrial cytopathy, including autosomal recessive (NDUF-and SURF-1-associated Leigh disease, SCO2 mutations, mtDNA depletion, MNGIE syndrome), autosomal dominant (OPA-1, CMT2A2 [mfn2 gene], POLG- and ANT1associated CPEO, Twinkle-associated SANDO and CPEO), and X-linked (PDHA1associated Leigh disease, Barth syndrome, and DDP gene mutations causing deafness and dystonia). It is extremely important to specifically ask for a history of deafness, short stature, early cardiac death, muscle discomfort or exercise intolerance, or early-onset diabetes. Many families do not appreciate these conditions as manifestations of inherited mitochondrial

disease and do not mention them in the context of an ill child. Neurologic examination of both parents and all siblings is always an important aspect of the family investigation. Blood and Urine Testing An elevated serum lactate level raises the suspicion of mitochondrial cytopathy; however, the sensitivity is only approximately 60% (Tarnopolsky et al, 2003). Thus, a normal lactate level cannot be used to exclude mitochondrial cytopathy. An elevated lactate level is also not specific for mitochondrial disease and can be seen in adults with type 2 diabetes or if the individual has not been fasting. Lactate elevation in blood is also very common in upset or struggling children who not only may have their arm held firmly (tourniquet effect), but who use excessive isometric muscle contraction, leading to an increase in the generation of lactate through anaerobic pathways. Finally, accurate determination of lactate requires proper sample handling: samples must be collected and placed immediately on ice (as red blood cells are obligate anaerobes and thus will generate lactate unless their metabolism is inhibited by cooling) followed by prompt centrifugation. The lactate/ pyruvate ratio can also be obtained in CSF or in culture medium from tissue culture where the absolute lactate level and the lactate/pyruvate ratio may be helpful in differentiating pyruvate dehydrogenase deficiency from primary mitochondrial cytopathies. Plasma amino acid profiles are helpful in ruling out primary aminoacidopathies and may show elevated alanine in mitochondrial cytopathies. Serum creatine kinase activity may be elevated in some mitochondrial cytopathies, especially those with a myopathic presentation; however, they cannot be used for ruling in or ruling out mitochondrial cytopathies. A very high level of creatine
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KEY POINT

Mitochondrial dysfunction in the brain can result in developmental delay, hypotonia, seizures, strokelike episodes, visual loss, and hypoacusis. Involvement of skeletal muscle can lead to hypotonia/ motor delay in children and weakness/ exercise intolerance in adolescents and adults. Cardiac involvement can manifest in infancy with cyanosis and cardiac failure (SCO2 mutations) and as cardiomyopathy anytime later in life.

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" MITOCHONDRIAL CYTOPATHIES

KEY POINT

A muscle biopsy for suspected mitochondrial disease must include microscopic analysis (light and electron) with two other pieces collected into liquid nitrogen or dry ice and stored at 808C for enzyme and molecular or protein analysis.

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kinase in the absence of rhabdomyolysis would, however, point one more toward primary dystrophy evaluation. Elevation of liver aminotransferase (alanine aminotransferase and aspartate aminotransferase) bilirubin, and glutamyl transferase can be seen in Alpers syndrome and hepatic mtDNA depletion (dGUOK mutations). An early sign of Alpers syndrome may be the elevation of -fetoprotein (Robert Naviaux, MD, PhD; oral communication, 2008), but high levels should also prompt consideration of ataxia telangiectasia in children and/or an underlying tumor in children or adults. Specific testing for thymidine levels showing massive elevations is characteristic of MNGIE syndrome. Urine analysis for organic acids can also be quite helpful with a pattern showing 3-methylglutaconic acid and various TCA cycle intermediates (fumarate, malate) (Barshop, 2004). High levels of isolated ethylmalonic acid should prompt investigation into the ETHE1 gene responsible for encephalopathy. High 3-methylglutaconic acid levels should prompt investigation into tafazzin mutations responsible for Barth syndrome. Cerebral folate deficiency has also been seen as a nonspecific consequence of mitochondrial cytopathy (Garcia-Cazorla et al, 2008; Pineda et al, 2006). CSF studies are also helpful in ruling out mitochondrial mimics such as CSF neurotransmitter defects or disorders of CSF folate metabolism. In my experience, approximately 10% to 13% of the patients with mitochondrial cytopathy have a coincidental deficiency of vitamin B12, red blood cell folate, or vitamin E in serum. Given that deficiencies in each of these vitamins can cause neuropathy and ataxia, it is prudent to check levels in patients, particularly in teenagers in whom diet may not be optimal. Muscle Biopsy A muscle biopsy is important in the investigation of suspected mitochonContinuum Lifelong Learning Neurol 2009;15(6)

drial cytopathy. A muscle biopsy can be obtained with a modified needle technique or an open technique (Bourgeois and Tarnopolsky, 2004). Modifications to needle biopsy tools have aided in the ability to obtain adequate sample sizes for light and electron microscopic evaluation as well as DNA and enzyme studies. A needle biopsy can be obtained in less than 20 minutes in the clinic from most people aged 12 or older; in younger children or young adults who are very anxious, a short propofol infusion (children) or lorazepam (young adults) is often required. Irrespective of whether an open or needle biopsy technique is used, it is imperative that the sample be handled appropriately. Muscle biopsies for suspected mitochondrial disease should be performed in a facility with a metabolic laboratory, or at least with well-trained staff familiar with proper technique. In addition to preparing a frozen section with an optimal cutting temperature embedding medium and placing a piece of tissue into chilled glutaraldehyde, another piece must be placed in liquid nitrogen or immediately on dry ice and transported to the laboratory for enzyme assessment. A detailed description of all of the pathologic consequences of mitochondrial cytopathies is beyond the scope of this chapter, and readers may refer to specific reviews (Bourgeois and Tarnopolsky, 2004; DiMauro, 2006; Moraes et al, 1992). In general, alterations apparent on light microscopy are less prevalent in children than in adults. Specifically, ragged red fibers (RRFs) tend to accumulate with age. While the presence of RRFs is one of the classic patterns of mitochondrial disease (visualized on routine muscle histochemistry by the modified Gomori trichrome stain), RRFs can be present in otherwise healthy older adults. RRFs often indicate a tRNA defect in mtDNA. A uniform reduction in cytochrome c oxidase (COX) staining

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can be seen in SCO2 deficiency (cardiomyopathy, spinal muscular atrophylike syndrome) or SURF1 mutations (Leigh disease). COX-negative fibers are seen in aging (older than 65 years), CPEO, MELAS, and MERFF syndrome and highlight as ragged blue fibers when combined with succinate dehydrogenase (SDH) staining (see later discussion). COX-negative RRFs are characteristic of MERFF, Kearns-Sayre syndrome, and CPEO, while in MELAS the RRFs are COX positive. Other stains, including SDH and NADH, usually parallel the changes with Gomori trichrome, although the SDH stain is useful in highlighting mitochondrial proliferation in blood vessels, and strongly SDH-positive blood vessels are seen predominately in MELAS syndrome. Electron microscopy is imperative in the evaluation of pediatric mitochondrial cytopathies. Significant abnormalities may not be apparent with any histologic stains, and changes such as paracrystalline inclusions, pleomorphism, and alterations of mitochondrial cristae structure may be very evident on electron microscopy without any histopathologic correlate. Although paracrystalline inclusion is one of the hallmarks of mitochondrial dysfunction, it is more likely in older patients and may be absent in the pediatric age group. Paracrystalline inclusions represent upregulation of mitochondrial creatine kinase for dimeric crystalline structures in the presence of peroxynitrite (oxidative stress). These crystalline structures accumulate in the intermembrane space and disrupt the three-dimensional relationship between the inner and outer mitochondrial membrane and alter contact sites important for energy transduction. Paracrystalline inclusions can also be seen in nonmitochondrial cytopathies due to secondary mitochondrial dysfunction (HIV medications, type 2 diabetes). Markedly pleomorphic mitochondria are seen in POLG muta-

tions, and large mitochondria with homogenous matrix staining with paucity of cristae can be seen in the mtDNA depletion syndrome (Bourgeois and Tarnopolsky, 2004). The electron transport chain enzyme analysis is an essential component of the workup for suspected mitochondrial disease. Skeletal muscle is an ideal tissue for sampling, given that histologic analysis, heteroplasmy determinations for specific mutations and deletion/ depletion analysis can also be done on the sample. Some laboratories have found that fibroblast analysis is helpful; however, the diagnostic yield in skeletal muscle is usually higher. In spite of the ongoing debate about fresh isolated mitochondrial preparations and frozen muscle, the most important issue is that the enzyme analysis is done in an experienced laboratory and that normal reference ranges are available for that specific laboratory. For frozen sample analysis, it is important that the tissue is frozen rapidly on dry ice or liquid nitrogen and stored in either liquid nitrogen or at less than 808C before analysis. Dr David Thorburn has extensive experience with frozen tissue analysis and has presented data showing longterm (greater than 5 years) stability of electron transport chain enzyme activity in a single large muscle sample kept at less than 808C (David Thorburn, PhD; oral communication, 2006). The complex V assay is the only one that must be done on fresh mitochondria; however, this aspect of the analysis can be performed from fibroblasts (it is suggested that one obtain a small skin biopsy from the muscle biopsy site, place the skin in sterile fibroblast culture medium, and then grow the fibroblasts for analysis). Furthermore, many of the conditions affecting complex V have characteristic features, and clinical suspicion would lead to mutation analysis of ATPase 6 and 8 genes in such cases (eg, maternally inherited Leigh disease,
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KEY POINT

A skin biopsy taken at the time of the muscle biopsy can be used for fibroblast analysis if muscle samples are limited.

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" MITOCHONDRIAL CYTOPATHIES

KEY POINT

Exercise testing may reveal a low maximal oxygen consumption (V O2max) and/or a high respiratory exchange ratio. Laboratories must establish their own normative data.

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neurogenic muscle weakness, ataxia, and retinitis pigmentosa). An isolated complex I deficiency is the most common abnormality in pediatric enzyme assessment. In most cases of isolated complex I deficiency, a definitive pathogenic mutation is often not found. The exception is in a Leighlike presentation in which enzymatic evidence of complex I deficiency is associated with an increasing number of nuclear mutations (NDUF nuclearencoded complex I subunits). Muscle biopsy results often guide genetic investigations. A severe COX deficiency in a child with cardiomyopathy or spinal muscular atrophy pattern would be consistent with a SCO-2, SCO-1, or COX-15 mutation; and an isolated COX deficiency in a child with a Leighlike presentation would be most consistent with a SURF-1 mutation. Global reductions in many enzymes can be seen in the mtDNA depletion syndromes, while complex I and IV deficiencies are seen in the mtDNA-encoded tRNA mutations. Deletions in mtDNA can be seen in a variety of conditions and be detected using long-range PCR or Southern blotting. Large single deletions can be seen in Kearns-Sayre syndrome or cases of later-onset CPEO with and without other neurologic features (CPEO plus). Multiple deletions are often seen in POLG, Twinkle, ANT1, thymidine phosphorylase (ECGF1), or OPA1 mutations. For most neurologists, the key to diagnosis of a patient with suspected mitochondrial disease rests on direct communication with the pathology department staff member obtaining the specimen, with the medical director of the mitochondrial laboratory, and with a metabolic genetics team. Miscellaneous Tests A variety of other ancillary tests, including exercise testing, near-infrared spectroscopy, MRI, and MR spectroscopy
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(MRS) may be useful in the diagnosis of mitochondrial cytopathies. The principle of exercise testing is that a primary mitochondrial disorder will generally reduce the maximal oxygen consump tion per unit time ( VO2) of a person and lower the VO2max during exercise and/or increase the rate of lactate/CO2 production ( high peak respiratory ratio during exercise). A variety of testing protocols available have been developed and validated in adults, and extensive details regarding principles and practice of exercise testing have been described elsewhere (Taivassalo and Haller, 2005; Tarnopolsky, 2004; Tarnopolsky and Raha, 2005). Near-infrared spectroscopy evaluates the oxygenation/deoxygenation status of hemoglobin and myoglobin. Consequently, the peripheral extraction of oxygen by the mitochondria during exercise normally leads to deoxygenation but fails to do so in patients with mitochondrial disease (Taivassalo and Haller, 2005). A downside to the use of exercise testing in children with mitochondrial disease is that most children under 6 years of age cannot comply with testing or the equipment is not suitable for them. Furthermore, children with cognitive issues and major neurologic impairments, such as ataxia or spasticity, often cannot complete exercise protocols. The MRI evaluation of the brain is extremely helpful in the diagnostic evaluation of a child with suspected mitochondrial disease. The patterns of Leigh disease on MRI are fairly characteristic with high T2 signal in the basal ganglia ( primarily the putamen) and the brainstem as the more common findings (Saneto et al, 2008; Savoiardo et al, 2002), with progression to bilateral striatal necrosis sometimes seen. A pattern of strokelike lesions crossing vascular territories with a propensity for the occipital cortex should prompt investigations for MELAS syndromes. Most patients with mitochondrial encephalopathy, however, have nonspecific periventricular white

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matter high signal on T2 and fluidattenuated inversion recovery imaging. MRS using proton (1H) spectroscopy can show elevated lactate in brain parenchyma, particularly if the MRS voxel is placed in an area of involvement (basal ganglia or abnormal white matter) (Sijens et al, 2008). Importantly, a normal scan does not rule out mitochondrial disease, and children with a previously normal scan may demonstrate abnormalities only during acute decompensation. Phosphorus31 MRS ( 31P-MRS) can show a reduced brain phosphocreatine and/or a low phosphocreatine/inorganic phosphate ratio. Comprehensive analyses of the MRI and MRS findings associated with mitochondrial cytopathies have been published (Farina et al, 2002; Munoz et al, 1999; Saneto et al, 2008; Sijens et al, 2008; Valanne et al, 1998). The diagnosis of mitochondrial disorders is ultimately based on a composite of several tests, and sole reliance on a single test, such as electron transport chain enzyme activities, can be misleading. Consequently, individuals have proposed several diagnostic criteria for mitochondrial disease (Bernier et al, 2002; Thorburn et al, 2004). It is clear that the diagnostic criteria for each of the aforementioned templates is influenced by the strengths of the clinic/ laboratory making the proposal; consequently, it is important for a given center to adopt the criteria that best fit with the available testing methods. For example, if the local laboratory uses frozen muscle for electron transport chain assessment, it would not be useful to use criteria based on isolated mitochondria from a fresh biopsy. Given the treatment and counseling implications, we have found it useful to use the labels possible, probable, and definite mitochondrial disease for these patients. It is very important to entertain the possibility of alternative diagnoses until arriving at a definitive diagnosis. For example, many pediatric neurologic disorders can

have clinical features that mimic mitochondrial disease (Rett syndrome, leukodystrophies [eg, metachromatic, Krabbe], chromosomal anomalies, etc), and we have seen many children who have had abnormal mitochondrial electron transport chain enzyme tests (especially complex I deficiency) in whom subsequent testing confirmed an alternative diagnosis (eg, Rett syndrome, Prader-Willi syndrome, chromosomal deletion, congenital muscular dystrophy). SPECIFIC PEDIATRIC MITOCHONDRIAL DISORDERS Leigh Disease Leigh disease was first described by Dennis Leigh in 1951 based on neuropathologic findings of cystic cavitation, demyelination, vascular proliferation, and neuronal loss predominately in the thalamus, brainstem, and posterior columns of the spinal cord in a child with a neurodegenerative disease (Morava et al, 2006; Vilarinho et al, 1997; Zhang et al, 2007). Leigh disease is a rapidly progressive encephalopathy occurring in infancy or childhood with a mean age of onset of approximately 2 years. Manifestations include developmental delay or psychomotor regression, optic atrophy, ophthalmoplegia, dystonia, tremor, spasticity, ataxia, and/or respiratory abnormalities (apnea/hypopnea). The neurologic deterioration is often more apparent during periods of concurrent illness and other metabolic stressors. The progression of neurologic dysfunction can be rapid, with compromised respiratory status being the main indication for intensive care admission. Leigh disease is associated with complex I (approximately 35%), complex II (approximately 5%), complex III (approximately 5%), complex IV (approximately 20%), or pyruvate dehydrogenase (approximately 15%) deficiencies, and mtDNA mutations (approximately 20%) (Berger et al, 2008; Kirby et al, 2003;
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KEY POINT

Leigh disease often presents before age 2 with psychomotor regression, optic atrophy, ophthalmoplegia, dystonia, spasticity, ataxia, and respiratory changes. Most children with Leigh disease do not survive beyond age 5.

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" MITOCHONDRIAL CYTOPATHIES

Schiff et al, 2006; Zhang et al, 2007; Zhu et al, 1998). A muscle biopsy with electron transport chain enzyme activity analysis is helpful to target definitive mutational analysis (complex I = NDUF mutations, mtDNA (10158 T>C, 10191 T>C, 11777 C>A, 14459 G>A); complex IV = SURF-1, LRPPRC). If the electron transport chain enzyme activity for complexes I through IV are normal, pyruvate dehydrogenase analysis should be considered, as should point mutation analysis for ATPase mutations associated with Leigh disease (9176 T>G/C, 9185 T>C, 8993 T>C/G). Specific mutation analysis is also very helpful from a genetic counseling and prenatal diagnostic perspective (Case 6-1).

Cardiomyopathies Cardiomyopathy is a common finding in pediatric and adult mitochondrial cytopathy, with a recent study showing that 40% of children with mitochondrial cytopathies had cardiomyopathy (Scaglia et al, 2004). Most patients had hypertrophic cardiomyopathy (58%), 29% had dilated cardiomyopathy, and 13% showed echocardiographic evidence of left ventricular noncompaction. The diagnosis of cardiomyopathy in a child with mitochondrial cytopathy implies a poor prognosis, with a survival rate of only 18% by age 16. In contrast, children with mitochondrial cytopathy manifesting solely with skeletal

Case 6-1
A 13-month-old boy presented with a 2-month history of decreased eating and falling off the growth curve for weight, height, and head circumference. Developmental history indicated that he started to cruise on furniture at 12 months but appeared weak, never attained the ability to ambulate, and subsequently lost his ability to stand. Impaired swallowing necessitated a percutaneous gastrostomy tube, which was associated with rapid weight gain and improved neurologic function and alertness. On neurologic examination, he was alert and demonstrated age-appropriate stranger anxiety. He would look at his mother and father to command but did so by moving his head to gain visual fixation. He was unable to initiate voluntary eye movements to an object of interest, consistent with ocular motor apraxia. Motor examination demonstrated mild hypotonia and thin muscle bulk. Muscle stretch reflexes were normal with no clonus, and the plantar response was flexor. Serum lactate levels were mildly elevated on two of three blood draws (3.2 mmol/L and 2.8 mmol/L, normal is less than 2.2 mmol/L). Cranial MRI demonstrated symmetric hyperintensity and increased fluid-attenuated inversion recovery signal bilaterally in the brainstem, particularly in the ventral aspect of the medulla. MRS demonstrated a lactate peak in the basal ganglia. Muscle biopsy demonstrated COX enzyme activity less than 5% of normal. A diagnosis of complex IV deficiency Leigh disease was confirmed by the finding of a homozygous SURF1 mutation 845-846 delCT (Ser272cys). Parents were devastated by the diagnosis and then revealed that the mother was 10 weeks pregnant with their second child. Comment. This case demonstrates the often ominous outcome in children presenting with developmental regression. The nonspecificity of the weight loss and the absence of neurologic symptoms until several months into the disease course diverted diagnostic attention initially toward more of a gastrointestinal-focused investigation. The marked changes on MRI showing much more extensive changes than are revealed by the clinical examination are a common feature in patients with Leigh disease. Prenatal counseling for the family was offered. Optimism was very high for prenatal diagnosis given that the SURF1 mutation is nuclear. At our institution we have provided prenatal testing for previously documented Leigh diseaseassociated mutations in three separate families and have had successful outcomes (not affected) in all three cases.

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myopathy experienced a survival rate by age 16 of 95% (Scaglia et al, 2004). Hypertrophic cardiomyopathy may be the predominant and/or exclusive feature in children with defects in COX assembly, including SCO2, COX10, and COX15 mutations (Antonicka et al, 2003a; Jaksch et al, 2001b). Most children with SCO2 deficiency also show significant hypotonia owing to alpha motor neuron involvement. These children can be indistinguishable at onset from those with Werdnig-Hoffman disease (spinal muscular atrophy type I). A major distinguishing feature between spinal muscular atrophy type 1 and SCO2 mutations is presence of high lactate concentration in serum/plasma seen in SCO2. Most children with SCO2 deficiency die before 2 years of age, and many succumb in the first few months of life ( Jaksch et al, 2001b). Cardiomyopathy can be a part of the phenotype in MELAS 3243 A>G or the predominant symptom in other mtDNA mutations (1555 A>G, 3260 A>G, 4295 and 4269 A>G, 4320 C>T, 4330 A>G, 8296 A>G, 8363 G>A, 9997 T>C, and 15243 G>A) (Case 6-2). Although conduction disturbances can be seen in the cardiomyopathies, patients rarely require pacemakers, although those with Kearns-Sayre syndrome often show progressive conduction block requiring pacing in the absence of cardiomyopathy. A cardiomyopathy is often asymptomatic and usually picked up as part of the workup in a child with suspected mitochondrial disease. Every child with mitochondrial cytopathy should be screened for cardiomyopathy using echocardiography given the opportunity for therapeutic intervention (ie, afterload reduction) and prognostication. Mitochondrial DNA Depletion mtDNA depletion syndromes are categorized into two groups: an enceph-

alomyopathic form and a hepatoencephalopathic form. mtDNA depletion is characterized by a reduction in mtDNA copy number, and mutations have been demonstrated in the deoxyguanosine kinase (dGUOK) gene in patients with the hepato-encephalopathic form (Mandel et al, 2001), and thymidine kinase (TK2) in children with the encephalomyopathic form of mitochondrial depletion syndrome (Saada et al, 2001). These mutations interrupt the deoxyribose nucleotide pathway and inhibit incorporation of the deoxynucleotide triphosphates into the mtDNA, resulting in mtDNA depletion. Mutations in dGUOK are found in approximately 10% to 15% of all patients with the hepato-encephalopathic form of mtDNA depletion, and approximately 20% of patients with the encephalomyopathic form have TK2 mutations (Carrozzo et al, 2003; Mandel et al, 2001; Saada et al, 2001). More recently, mutations in POLG, Twinkle, and OPA1 (Amati-Bonneau et al, 2008; Hakonen et al, 2007; Hudson et al, 2005; Hudson et al, 2008) have also been associated with mtDNA depletion, and the clinical features can overlap with TK2-associated mtDNA depletion. The discovery of these specific mutations is helpful in understanding pathogenesis of the syndrome and for accurate genetic and prenatal counseling. The mtDNA depletion syndromes often present in the first few months of life and frequently have a rapidly progressive and fatal outcome. Significant clinical heterogeneity occurs for any given mutation. For example, mutations in TK2 can present with encephalopathy and hypotonia, and some can present with a spinal muscular atrophy (lower motor neuron disease) picture (Mancuso et al, 2002; Saada et al, 2001). The dGUOK mutations usually present with progressive hepatic failure, and POLG mutations manifest with Alpers syndrome (Mandel et al, 2001). In addition,
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KEY POINT

Cardiomyopathy may be a subclinical comorbidity in some mitochondrial disorders (ie, MELAS) or can be the defining feature (ie, 1555 A>G, 3260 A>G, SCO2). Most cases of cardiomyopathy in mitochondrial diseases are hypertrophic, but dilated and noncompacted forms of cardiomyopathy may also occur.

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" MITOCHONDRIAL CYTOPATHIES

Case 6-2
An 8-year-old girl presented with shortness of breath on exertion and cyanosis. Cardiovascular workup revealed a hypertrophic cardiomyopathy with very low ejection fraction. In spite of afterload reduction, heart failure worsened rapidly and she received a cardiac transplant. In the postoperative period she was encephalopathic. Serum lactate levels rose to over 20 mmol/L (normal is less than 2 mmol/L). Pathologic examination of the native heart demonstrated mitochondrial proliferation of pleomorphic mitochondria with paracrystalline inclusions. Genetic analysis revealed a 3260 A>G transition mutation with high heteroplasmy in heart tissue and skeletal muscle (mutant 90%). Evaluation of family members revealed two siblings with lactic acidemia (6 mmol/L to 8 mmol/L) and moderate to severe exercise intolerance. The mother experienced exercise-induced deafness, had resting lactic acidemia, and had suffered a severe strokelike episode at age 42. The maternal grandmother died at age 42 with encephalitis. One maternal uncle had severe migraines and exercise-induced rhabdomyolysis, a maternal aunt also had migraines and had experienced a single strokelike episode at age 38, and another aunt also had migraines. All children born to the maternal aunts had lactic acidemia and exercise intolerance, while the two children of the maternal uncle were asymptomatic and had normal resting lactate levels. The proband went on to develop multiple strokelike episodes with incomplete neurologic recovery, seizures, and progressive emaciation. She had a percutaneous gastrostomy tube placed and over the ensuing year had marked improvement in neurologic function, gained 1.8 kg of weight, had less abdominal pain, and increased exercise capacity with activities of daily living. Comment. The comprehensive family pedigree highlights the extreme clinical heterogeneity that can arise from a single point mutation (cardiomyopathy, strokelike episodes, seizures, hypoacusis, migraines, exercise-induced rhabdomyolysis, and intestinal pseudo-obstruction). Of particular relevance is the fact that it was not until the proband presented with cardiac failure that the true nature of the familial disorder was revealed. Migraines are a common neurologic disorder, and the early onset of strokes was thought to be related to cholesterol or blood pressure, although no family members were hypertensive or hypercholesterolemic. Exercise-induced deafness was reported by the mother to her family physician, who found her hearing to be normal at rest and dismissed the symptom as odd, without further investigation. This case also illustrates the clinical improvement that is often seen in patients with mitochondrial cytopathy when adequate nutrition and hydration are provided. In addition, such treatment ensures timely and complete delivery of prescribed medications. Seizures were managed with carbamazepine, as valproic acid is contraindicated in patients with mitochondrial disease (valproic acid impairs mitochondrial carnitine transport).

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children can present with a renal Fanconi syndrome, seizures, congestive heart failure, and progressive external ophthalmoContinuum Lifelong Learning Neurol 2009;15(6)

plegia. Occasionally, the disease can present with psychomotor regression and/or progressive weakness in childhood.

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mtDNA depletion is measured using real time PCR for the copy number of mtDNA relative to a nuclear DNA housekeeping gene (eg, 18S). mtDNA depletion is very often tissue specific with liver being the criterion standard for the hepato-encephalopathic form, and skeletal muscle showing depletion in the encephalomyopathic form. Mitochondrial DNA Disorders mtDNA disorders are maternally inherited and arise from specific point mutations in either tRNA or specific subunit regions of the mtDNA. Mutations exist in essentially every region of the mtDNA with over 100 specific point mutations now considered pathogenic and over 200 more reported and awaiting confirmation. An updated list of all reported pathogenic and polymorphic mtDNA sequence variants can be found on Mitomap (www.mitomap.org/). In general, more adults than children are identified with mtDNA point mutations. MELAS syndrome is the most common of the mtDNA point mutations and can show an extremely wide range of phenotypic manifestations, including hypoacusis, ataxia, dementia, ophthalmoplegia, encephalopathy, strokelike episodes, exercise intolerance, proximal myopathy, and type 2 diabetes. The progression of MELAS can resemble multiple sclerosis and be chronically progressive or relapsing and remitting. The age at onset of symptoms is as variable as the clinical phenotypes with presentations starting in infancy and up to adult age groups (Case 6-2). MERRF can present as isolated myoclonic epilepsy with and without ataxia, myopathy, peripheral neuropathy, and multiple lipomas ( particularly in the head and neck). In our experience, neuropsychiatric manifestations, including severe obsessive-compulsive disorder, depression, and psychotic features, as well as type 2 diabetes, are common. The age of

onset can range from childhood to middle age (forties) and usually a progressive decline in function occurs. Leber hereditary optic neuropathy (LHON) usually results in painless, rapidly progressive, visual loss with centrocecal scotoma in teenagers or young adults. A marked sex difference is found, with more men carrying the mutation developing symptoms (40% to 70%) as compared with women (20% to 30%). The three common mutations (11778 G>A = 69%; 3460 G>A = 13%; and 14484 T>C = 13%) account for the majority of cases. Some of the LHON mutations can present with a multiple sclerosislike syndrome, including the most common 11778 G>A mutation (Vanopdenbosch et al, 2000). Other LHON mutations have been associated with dystonia, especially the 14459 G>A mutation (Tarnopolsky et al, 2004). Several other mtDNA mutations that can present in the pediatric age group include lethal infantile mitochondrial disorder (15923 A>G, 15924 A>G), complex I deficiency Leigh disease (10191 T>C, 10158 T>C, 12706 T>C, 13513 G>A, 14459 G>A), isolated myopathy (618 T>CC, 3250 T>C, 3302 A>G, 5521 G>A, 12320 A>G, 15990 C>T), and exercise intolerance (predominately cytochrome b mutations such as 14846 G>A, 15059 G>A, 15084 G>A, and 15615 G>A) (www. mitomap.org/cgi-bin/tbl8gen.pl). Point mutations in the tRNA regions of mtDNA often result in variable combinations of electron transport chain enzyme activity reduction, whereas isolated enzyme activity reductions can point to specific protein subunit DNA regions (eg, complex I = ND subunits, complex III = cytochrome b). Targeted PCR-restriction fragment length polymorphism is the most common method used to screen for the common mutations, whereas exploratory analysis for new mutations can be done with DNA sequencing, denaturing high-performance
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KEY POINT

mtDNA disorders are maternally inherited and arise from specific point mutations in either tRNA or specific subunit regions of the mtDNA. Mutations exist in essentially every region of the mtDNA with over 100 specific point mutations now considered pathogenic and over 200 more reported and awaiting confirmation.

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" MITOCHONDRIAL CYTOPATHIES

KEY POINT

Most POLG mutations are associated with progressive external ophthalmoplegia in adults, Alpers syndrome, myocerebrohepatopathy, and other infantile hepatocerebral syndromes associated with mtDNA depletion in children.

liquid chromatography, and sequencing microarrays. Polymerase Gamma Mutations and Alpers Syndrome The explosion of information regarding polymerase gamma mutations began when Naviaux discovered low POLG activity in a child with fatal seizures, hepatopathy, coma, and lactic acidosis (Alpers syndrome) (Naviaux et al, 1999). POLG is the sole polymerase for mtDNA replication. The POLG protein has a polymerase domain, a linker region, and an exonuclease domain. Close to 100 mutations have been reported in each of the three main regions and have been listed in the Human DNA Polymerase Gamma Database (http://tools.niehs. nih.gov/polg/). Most of the reported mutations have been associated with progressive external ophthalmoplegia in adults, Alpers syndrome, myocerebrohepatopathy, and other infantile hepatocerebral syndromes associated with mtDNA depletion in children. Mutations in the POLG gene have also been seen in children with Leigh disease, Charcot-Marie-Tooth neuropathy, MIRAS, SANDO, and other ataxia-neuropathy syndromes resembling Friedreich ataxia. A teenage-onset disorder with seizures, ataxia, headache, neuropathy, myoclonus, and ophthalmoplegia with the A467T and W748S POLG mutations has also been described (Tzoulis et al, 2006) (Case 6-3). Mutations in POLG result in mtDNA depletion and sometimes deletions (especially in adult-onset cases), but the ETC enzyme activity in skeletal muscle may be normal. Myoneurogastrointestinal Encephalomyopathy Syndrome MNGIE is an autosomal recessive condition associated with severe abdominal pain, malabsorption, and weight loss. The associated peripheral neuropathy may be the presenting feature, but the associated leukoencephalopathy may
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be variably symptomatic or even asymptomatic (Hirano et al, 1994). The age of onset is usually in the early to late teenage years with progression of the gastrointestinal problems leading to a feeding tube and eventually total parental nutritional requirement. The leukodystrophy may become apparent during periods of concurrent illness in which a delirium/encephalopathic picture can appear. The evaluation of these patients will show variably elevated serum lactate levels and a demyelinating peripheral neuropathy, leukodystrophic changes on MRI, and often multiple mtDNA deletions or depletion in skeletal muscle. The gene responsible for this disorder is ECGF1, which encodes thymidine phosphorylase involved in the degradation pathway of thymidine (Nishino et al, 1999). Patients will show very elevated levels of plasma thymidine, and white blood cell preparations can be used to demonstrate the reduced thymidine phosphorylase activity (Marti et al, 2004) (Case 6-4). Miscellaneous Conditions Kearns-Sayre syndrome is a multisystem mitochondrial disorder characterized by progressive external ophthalmoplegia, retinitis pigmentosa, hypoacusis, ataxia, myopathy, heart block, elevated CSF protein, and onset before the age of 25. These patients usually have high lactate levels in plasma, and muscle biopsy shows RRFs, a variable reduction in electron transport chain enzyme activity, and a single large-scale deletion (4,977 bp) in muscle (not white blood cells) detected by long-range PCR or Southern blot. The specific nuclear gene responsible for this condition has not yet been identified, and the diagnosis is based on the clinical and muscle biopsy features. Coenzyme Q10 (CoQ10) deficiency has been reported in a variety of clinical syndromes, including childhood-onset ataxia with cerebellar atrophy, encephalopathy and seizures, encephalomyopathy,

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Case 6-3
A 56-year-old man presented with a several-year history of feeling a bit off balance. He first noticed this while playing soccer when he had difficulty turning suddenly and would bump into people. He also had a difficult time suddenly trying to get out of the way of the ball when it was coming at him. In the preceding 2 months he had noted some slurring of his words. He was fine otherwise with a negative functional inquiry; his past history was positive for a three-vessel bypass in 1995 for atherosclerotic heart disease secondary to hypercholesterolemia. He had been on statin medications for 14 years prior to presentation. He had no allergies, did not smoke, rarely consumed ethanol, and worked in a university employee relations office. Family history revealed that his mother had balance problems for as long as he could remember. One brother died from cancer at age 65 and had no reported problems. Two other brothers were reportedly without symptoms. His 29-year-old son and 31-year-old daughter had no neurologic symptoms. Examination showed normal mental status, horizontal nystagmus, and scanning speech; cranial nerves were otherwise normal. Motor examination showed normal bulk, power, and tone proximally and distally in the upper and lower extremities. Muscle stretch reflexes were normal, and pathologic reflexes were absent. Sensory examination was normal. Dysmetria was noted in both arms and legs. He could not perform tandem gait. EMG and nerve conduction studies were normal. Amino acids, C-reactive protein, autoimmune workup, antigliadin antibodies, aprataxin and SCA 1, 2, 3, 6, 7, 8, 17 gene mutations, vitamin B12, red blood cell, folate, vitamin E, thyrotropin, and complete blood count values were normal. Serum lactate was elevated at 3.0 mmol/L (normal is less than 2.2 mmol/L). Urine organic acid showed a large elevation of 3-methylglutaconic acid and the moderate elevation of ethylmalonic acid. Cranial MRI showed nonspecific high signal and periventricular white matter with no cerebellar atrophy. Vastus lateralis muscle biopsy showed borderline type II fiber atrophy and prominent lipid droplets on electron microscopy. Electron transport chain enzyme activities showed a slight reduction in complex I + III relative to citrate synthase (8% ratio) with complex II + III, IV normal and citrate synthase slightly high, indicative of mitochondrial proliferation. Point mutation analysis from muscle-derived DNA was normal for MELAS 3243, 3271, NARPP 8993 and LHON 11778. During the course of the workup, his brother reported similar symptoms when asked specifically and was evaluated by a neurologist at another center who found similar clinical findings. Mutation analysis revealed a 2243 G>C heterozygous mutation in POLG1 gene causing a Trp748Ser mutation (W748S +/). The sequence variant was not found in either of the two asymptomatic children, and his symptomatic brother was being screened for the mutation. Comment. This case describes the value of muscle biopsy with enzyme workup as well as full mitochondrial screening, including lactate and urine organic acids in atypical cases of ataxia. Diagnosis can be elusive as highlighted by the relatively normal MRI and the absence of any classic mitochondrial changes on muscle biopsy with histology and electron microscopy. Of interest also is the fact that subsequent lactates have been normal (hence the value of three lactates taken at different times in suspected cases of mitochondrial cytopathy). This also shows that having a single allele mutation for the W748S mutation can lead to later-onset neurologic symptoms, and previous reports have demonstrated ataxia and epilepsy in an adult (age 55) positive for the W748S +/ mutation (Tzoulis et al, 2006). Several reports have documented pediatric-onset seizures in ataxia with W748S homozygous state (Engelsen et al, 2008). This also shows the value of ongoing follow-up in patients, for initially his brother was reported to have no symptoms but after 1 year he clearly had identical symptoms.

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cardiomyopathy and renal failure, and isolated myopathy (Horvath et al, 2006; Lamperti et al, 2003) CoQ10 is the electron acceptor for complexes I and II and transfers electrons to complex III of the electron transport chain. Several genes

have been found in association with CoQ10 deficiency, including mitochondrial parahydroxybenzoid-polyprenyltransferase (CoQ2), APTX, CABC1, and prenyl (decaprenyl) diphosphate synthase subunit 2 gene (PDSS2) or PDSS1 (DiMauro et al,
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" MITOCHONDRIAL CYTOPATHIES

Case 6-4
A 12-year-old boy presented with a several-month history of weakness and clumsiness. In the preceding few months he had increasing problems tripping, stubbing his toe, and feeling off balance. He also described some nausea and had lost approximately 0.91 kg over the same time period. Examination revealed a thin boy with normal mental status and cranial nerve examination. His muscles were generally thin with mild distal atrophy in the intrinsic foot muscles and 4/5 weakness of dorsiflexion and eversion. He was areflexic in the upper and lower extremities. Vibration sensation was reduced in the lower extremities, and pain and temperature sensation were reduced in a stocking distribution. Nerve conduction study confirmed a motor and sensory axonal neuropathy. Family history was negative for confirmed mitochondrial disease, although his mother had absent ankle reflexes, mild pes cavus, and borderline slowing of nerve conduction studies in the peroneal nerve. Genetic testing for CMT1A and CMTX was negative. Over the following 6 weeks, the patient continued to lose weight and developed persistent abdominal pain, and his teachers noted that he had difficulty in maintaining concentration. An MRI revealed confluent white matter high signal on T2-weighted images. Investigations for common forms of leukodystrophy were negative, as were lactate, vitamin B12, and very long-chain free fatty acids. A muscle biopsy of the vastus lateralis showed normal histology and ultrastructure and no mtDNA deletions. Plasma thymidine levels were markedly elevated (5.7, normal range less than 0.05 mol/L), and thymidine phosphorylase activity in white blood cells was very low (50, normal range = 634 +/ 237 ng/h/mg protein). A diagnosis of MNGIE was confirmed with mutation analysis showing homozygosity for the c.770 A>C, p.254 T>P mutation in the ECGF1 gene. Over the ensuing 3 years he developed severe abdominal pain and gastroparesis requiring total parenteral nutrition. His parents requested transfer of care to the pediatric facility, where he received all care until he reached the age of 18. At age 18, he weighed 34 kg, was profoundly weak in all four limbs, and was dependent for all aspects of care. He developed increasing respiratory distress, confirmed to be pneumonia at his local emergency department. The pediatric facility on-call physician, who was not familiar with the family, stated that the patient should be admitted to the adult care hospital. The attending adult care physician told the family that he had never even heard of MNGIE. The parents and the patient were frantic and called their neurologist at home. He arranged compassionate admission to the pediatric care facility, where the patient died from sepsis following a prolonged stay in the intensive care unit. Comment. This case discussion highlights the characteristic picture of MNGIE syndrome, as well as the diagnostic challenges. Of particular relevance are the issues faced in caring for a young terminally ill adult with a rare pediatric-onset disease. Many pediatric facilities will extend their age of care for patients with such disorders, but this care plan must be discussed prior to age 18 years, and letters documenting approval for admission as an adult must be in the medical record as well as in the familys possession. If admission for young adults is not permissible at the pediatric facility, then transition of care should occur well before the 18th birthday to ensure that the adult facility will have a comprehensive medical record that includes detailed plans of care. The case also highlights the challenges in making a diagnosis of mitochondrial disorder, because common tests such as lactate and muscle structure were normal.

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2007; Lopez-Martin et al, 2007). This condition is suspected when linked muscle enzyme assays (I and III, and II and III) show low activity, yet the individual components do not show reductions and are confirmed by showing reduced CoQ10 concenContinuum Lifelong Learning Neurol 2009;15(6)

tration in skeletal muscle to below 50% of healthy controls. The importance of making the diagnosis comes from the fact that some of the children have shown a clinical response to very high doses of CoQ10 (Lamperti et al, 2003).

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Recently CPEO plus ataxia phenotypes and multiple sclerosislike phenotypes have been described in adults with mutations in OPA1 (Hudson et al, 2008; Verny et al, 2008). OPA1 is a dynamin-related GTPase that is involved in mitochondrial maintenance (fission, cristae organization). Although traditionally associated with isolated optic atrophy (Alavi et al, 2007), several groups have described hypoacusis, axonal motor-sensory neuropathy, optic atrophy, progressive external ophthalmoplegia, ataxia, and myopathy starting in childhood or young adulthood (Amati-Bonneau et al, 2008). The myopathy is characterized by RRFs and COXve fibers and multiple mtDNA deletions (Amati-Bonneau et al, 2008). Several disorders have been associated with mutations in the BCS1L gene involved in complex III assembly (Blazquez et al, 2009; Fellman et al, 2008; Hinson et al, 2007). In addition to Leigh disease described earlier in this chapter, cases of growth retardation, amino aciduria, cholestasis, iron overload, lactic acidosis, early death (GRACILE syndrome), and Bjornstad syndrome (sensorineural hearing loss and pili torti) have been reported (Hinson et al, 2007). These conditions are all associated with a reduced complex III activity and mutations in the BCS1L gene.

TREATMENT OF MITOCHONDRIAL DISORDERS General Care Issues From a nutritional perspective, most children do not tolerate prolonged periods of fasting, and, in general, more frequent meals throughout the day are preferable. In some cases, the addition of complex carbohydrates such as Polycose or cornstarch may be required. Polycose is generally much easier to blend into food but does not have as long a duration of action as cornstarch.

Cornstarch, however, is difficult to add to formula but can be mixed into baby foods. Care must be taken not to clog percutaneous endoscopic and gastrostomy tubes when using cornstarch, Polycose, or creatine monohydrate. A high-fat diet may be of benefit in complex I deficiency, especially when severe seizures are a feature, given the potential benefit from the ketosis. Since many infants and children have limited exercise capacity, energy expenditure can be quite low and care must be taken to not overfeed children and induce obesity, which can further impair mobility and lead to other chronic health care issues. From an anesthetic perspective, one of the most important issues is to avoid prolonged periods of fasting. The provision from 10% aqueous dextrose solution (D10W) and/or total parenteral nutrition is important to prevent a fasting-induced metabolic stress. It is also important to postpone any surgical procedure during a period of even minor concurrent illness. With respect to specific anesthetic agents, there does not appear to be a particular predisposition toward true malignant hyperthermia in patients with mitochondrial cytopathy; however, malignant hyperthermia-like situations can arise with a metabolic crisis induced by additional metabolic stress that then can mimic some aspects of malignant hyperthermia. It is important to maintain normothermia throughout the anesthetic period to avoid an increase in shiveringinduced thermogenesis. For brief procedures such as a needle muscle biopsy, EMG, or lumbar puncture we have found that propofol and local anesthesia have been well tolerated without a single adverse reaction in several hundred procedures. Given that propofol only induces conscious sedation, we always use local anesthesia, often with preapplication of a topical anaesthetic such as a eutectic mixture of local anesthetics (EMLA) cream 60 minutes before the procedure.
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KEY POINTS

Nutritional support for all patients with mitochondrial disease is critical. Gastrostomy tube placement should be considered early in any child or adult with weight loss, as persistently inadequate caloric intake directly compromises residual mitochondrial function and exacerbates neurologic and multiorgan failure. In patients with mitochondrial disease, it is important to postpone any surgical procedure during a period of even minor concurrent illness.

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Peripheral neuropathy and other types of neurogenic pain can be a feature of mitochondrial cytopathy, especially in MNGIE syndrome and SANDO. Gabapentin is often well tolerated and one of the first-line therapies for neuropathic pain; however, it is best to start gabapentin in the evening and gradually titrate the dose to avoid somnolence. More recently, we have had success with pregabalin in patients who cannot tolerate gabapentin. We have also found that a combination of capsaicin cream and EMLA (50/50 mix) is often effective for mild to moderate dysesthesias from peripheral neuropathy and has the advantage of being nonsystemic. For severe spasticity, occupational therapy and physiotherapy may help to prevent and treat contractures. Baclofen is a good first-line medication for spasticity, and in children with a seizure disorder, benzodiazepines are quite helpful. Some patients may benefit from tizanidine with varying levels of success. The use of dantrolene should be avoided in patients with mitochondrial disorders because it can result in muscle weakness and fatigue. In children with severe spasticity limiting hygiene or activities of daily living, botulinum toxin injections can be of benefit. Seizures are common in children with mitochondrial cytopathy. In general, standard anticonvulsants for the given seizure types should be employed albeit with a few caveats. Valproic acid should not be used except as a last resort because it may trigger Reye syndrome like hepatic dysfunction in children with mitochondrial cytopathy, most notably in children with POLG mutations (McFarland et al, 2008; Uusimaa et al, 2008). Topiramate is particularly sedating in children with mitochondrial cytopathy but still can be used in selective cases. A number of patients experience gastrointestinal problems particularly, with MNGIE syndrome in which abdominal
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pain, cramps, and diarrhea predominate. These symptoms may be improved with elemental diets or total parenteral nutrition if necessary. Buscopcan may be helpful in alleviating abdominal cramps and pain, and if excess gas production is present, simethicone can be introduced. In a variety of other conditions ( particularly MELAS), severe constipation, in some cases leading to intestinal pseudo-obstruction, can be seen. High-fluid and high-fiber intake is the first-line therapy, followed by stool softeners, lactulose, glycerin suppositories if no bowel movement occurs in 3 days. Mineral oilbased laxatives should not be used because reflux, with the potential for aspiration, is quite common and can lead to serious chemical pneumonitis. Psychological support is important for patients with mitochondrial cytopathy and their families. A high degree of anxiety and marital strife may occur in families affected by maternally inherited disorders, with individuals blaming themselves or their spouse for the condition. A high degree of stress in the families of children with severe mitochondrial cytopathy is also posed by the need for medical appointments, multiple hospitalizations, and the complexities and intensity of care. Psychological and emotional support for the caregivers is very important, and often people find comfort and support through a group such as the United Mitochondrial Disease Foundation, which has a great deal of educational information (eg, MITO 101: A Primer for Physicians and Patients) and conducts a yearly conference for scientists, patients, and their families. As children get older and gain insight into their disorder, depression and despair may develop, particularly in those who experienced the death from mitochondrial cytopathy of an older sibling or other family members. Early intervention with counseling and possibly antidepressant medications may

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be of benefit. Some general suggestions for care are found in Table 6-2. Cofactors, Antioxidants, and Drugs The rationale for treatment of mitochondrial disorders is derived from the pathologic consequences of the disease as described earlier in this chapter: (1) Increase antioxidant capacity (vitamin E, vitamin C, CoQ10, -lipoic acid). (2) Enhance electron transport chain flux and/or bypass a specific defect (succinate, CoQ10, riboflavin, and thiamine). (3) Alternative energy sources such as creatine monohydrate increase intracellular phosphocreatine stores with some evidence of success in vivo and in vitro (Hultman et al, 1996; Tarnopolsky et al, 1997). (4) A high-fat diet may increase the flux through complex II in patients with a complex I defect (Roef et al, 2002), and a high-fat diet may also be of some benefit in pyruvate dehydrogenase mutations, particularly with difficult-to-control seizures, in which ketogenesis may be secondarily useful as an antiseizure intervention. (5) Levocarnitine is sometimes used because a secondary carnitine deficiency can occur in the absence of primary carnitine deficiency. In general, most clinicians recommend a combination of several of the above-mentioned compounds as a mitochondrial cocktail, with the rationale of providing compounds that target the final common pathways of cellular dysfunction in mitochondrial cytopathies (Tarnopolsky and Beal, 2001). A list of some of the treatments and their doses is presented in Table 6-3. A more complete discussion of each of these compounds can be found in review papers (Murphy et al, 2008; Tarnopolsky, 2008; Tarnopolsky and Raha, 2005) and in MITO-101: A Primer for Physicians and Patients links from the United Mitochondrial Disease Foundation (www.umdf.org/site/

TABLE 6-2

General Principles of Treatment

"

Treat Underlying Neurologic Issues Seizures (antiepileptic drugs, avoid valproic acid). Spasticity (baclofen, botulinum toxin [focal]; avoid dantrolene if liver is involved). Dystonia (diazepam, botulinum toxin [focal], trihexyphenidyl). Headache (acute: nonsteroidal anti-inflammatory drugs and acetaminophen; avoid aspirin and triptans in mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes [MELAS]; chronic: amitriptyline, calcium blockers, riboflavin, coenzyme Q10, -lipoic acid).

"

Identify and Treat Nutritional Deficiencies Growth curves are imperative to identify suboptimal nutrition. Identify and treat deficiencies in vitamins (vitamins A, B12, E, D, folate for red blood cells), minerals (iron, zinc, selenium, calcium, magnesium), and protein calorie (albumin, prealbumin). If children fall off growth curves and show signs or symptoms of undernutrition and do not respond to a nutritionists suggestions, consider a feeding tube (also helpful for medications and mitochondrial cocktail).

"

Avoid Metabolic Stressors Extremes of heat and cold are not well tolerated. Fever should be treated with acetaminophen (10 mg/kg every 4 hours to 15 mg/kg every 4 hours). Shivering is metabolically expensive and should be avoided. Patients should avoid unaccustomed strenuous exercise. They should not exercise in the fasted state or with a concomitant illness. Avoid prolonged (greater than 12 hours) fasting.

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c.dnJEKLNqFoG/b.4585961/k.C6AC/MITO_ 101.htm). Dichloroacetate has been proposed as a treatment for mitochondrial cytopathies since it reduces serum lactate concentrations by activating the pyruvate dehydrogenase complex. Several trials have been completed; however, the overall efficacy has not been dramatic,
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" MITOCHONDRIAL CYTOPATHIES

TABLE 6-3

Examples of Commonly Used Vitamins, Cofactors, and Other Nutraceuticals Mechanism of Action
Bypass complexes I and II (Coenzyme Q10 deficiency) Energy source, neuroprotection Pyruvate dehydrogenase cofactor Bypass complex I defects Antioxidant Antioxidant Treating secondary deficiencies Bypass complex I defects Antioxidant

Medication
Coenzyme Q10 Coenzyme Q10 Creatine monohydrate Thiamine Riboflavin Vitamin E Vitamin C Levocarnitine Succinate -Lipoic acid

Dose Range
5 mg/kg/d to 10 mg/kg/d 75 mg/kg/d to 150 mg/kg/d 100 mg/kg/d 3 mg/kg/d to 9 mg/kg/d 3 mg/kg/d to 5 mg/kg/d 5 mg/kg/d to 10 mg/kg/d 7 mg/kg/d to 15 mg/kg/d 30 mg/kg/d to 50 mg/kg/d 30 mg/kg/d to 70 mg/kg/d 5 mg/kg/d to 7 mg/kg/d

It is important to start with a single compound at the lower end of the dosing range for a few days and to add in a new compound one at a time, titrating the dose to the desired effect as tolerated. The main side effect with all of the above medications is gastrointestinal upset, which can often be treated by dividing the dose into a 2-times-perday or 3-times-per-day dosing regimen and taking the medication with meals. Liquid or hydrosoluble forms of coenzyme Q10 appear to be better absorbed than powdered capsule forms.

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and one of the side effects is the development of peripheral neuropathy (Kaufmann et al, 2006). Our approach has been to use dichloroacetate in cases of acute metabolic crisis for short periods of time, only when serum lactate concentrations are elevated above 10 mmol/L. In summary, most of the constituents of the mitochondrial cocktail are based on biological rationale (Tarnopolsky and Beal, 2001), and many of the components have been well tolerated (Haas, 2007; Mahoney et al, 2002; Matthews et al, 1993; Rodriguez et al, 2007). As single agents, CoQ10 and creatine monohydrate probably have been most extensively investigated (Haas, 2007; Klopstock et al, 2000; Tarnopolsky et al, 1997), and, on balance, there does appear to be some degree of efficacy when the two are combined with -lipoic acid (Rodriguez et al, 2007). Further studies are required to develop
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a complete mitochondrial cocktail strategy capable of targeting multiple final common pathways of cellular dysfunction induced by mitochondrial disorders. The difficulty, however, will be to evaluate the infinite possible combinations in a heterogeneous group of individuals, using a prospective randomized double-blind methodology. The development of animal models of mitochondrial disease will certainly be useful in identifying combination therapies for mitochondrial cytopathies to advance the development of clinical studies (Torraco et al, 2009; Wenz et al, 2009). Other Therapies No genetic therapies for mitochondrial cytopathies are currently available, although in vitro approaches using mutation-specific peptide nucleic acids for mtDNA mutations and deletions have

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shown some promise (Taylor et al, 1997). With an increasing recognition of nuclear DNA mutations, the potential for viral vector and other DNA-based strategies as well as possible short interfering RNA strategies may be greater in the future. Both endurance and resistance exercise has also been demonstrated to be of benefit in mitochondrial cytopathies in adults (Taivassalo et al, 2006; Taivassalo and Haller, 2005). At the genetic level, the sporadic mitochondrial mutation may be more amenable to therapy for exercise-induced muscle damage and can activate satellite cells that often show no or very low levels of mutational burden. The activation of the satellite cells results in mtDNA shifting, which can reduce the percentage of mutant heteroplasmy in the skeletal muscle (Murphy et al, 2008; Taivassalo et al, 1999). The difficulty with exercise in the pediatric population is that compliance and accessibility to appropriate exercise methods are challenging. In general, we recommend that children be as active as possible in school-based and play-based activities, provided that they work within their limit and not push to the point where they get nausea, vomiting, or encephalopathic features. We have had several patients who have experienced vertigo, nausea, vomiting, and even exerciseinduced deafness with overexertion. For these reasons, we advise people to listen to their body and progress slowly and cautiously. With careful and

progressive increases in activity, the threshold for the onset of these symptoms is increased, and most people are better able to tolerate activities of daily living without adverse affects. We usually advise that they do not perform any vigorous physical activity if they are experiencing a migraine headache or have a concurrent illness such as an upper respiratory tract infection. Activities such as tai chi, karate, and ballet are particularly helpful in children with dyscoordination and/or ataxia. In summary, the diagnosis, treatment, and long-term care of patients with mitochondrial cytopathies is extremely complicated and requires a dedicated team of specialists, including physicians (geneticists, neurologists, pulmonologists, cardiologists, physiatrists, gastroenterologists, psychiatrists), nurses, social workers, occupational and physical therapists, genetic counselors, and specifically trained nutritionists, in order to deal with all of the complexities of mitochondrial cytopathies. The age of onset of the mitochondrial cytopathies can range from infancy to adults older than 65 years, with considerable clinical and genetic heterogeneity. A patient who presents with neurologic symptoms commonly seen in mitochondrial cytopathies (eg, ptosis, ophthalmoplegia, hypoacusis, optic atrophy, muscle fatigue) or shows evidence of both neurologic and other systemic involvement should be investigated for the possibility of a mitochondrial cytopathy.

KEY POINT

Exercise has been demonstrated to be of benefit in mitochondrial cytopathies, with endurance and resistance types of exercise showing evidence of efficacy.

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