Gastrointestinal manifestations of mitochondrial disease

Gastroenterol Clin N Am 32 (2003) 789–817

Gastrointestinal manifestations of mitochondrial disease Lynette A. Gillis, MDa,b, Ronald J. Sokol, MDc,d,* a

Division of Gastroenterology and Nutrition, Department of Pediatrics; University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, 34th St. and Civic Center Blvd., Philadelphia, PA, 19104, USA b Section of Biochemical Genetics, Department of Human Genetics and Molecular Biology, University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, 34th St. and Civic Center Blvd, Philadelphia, PA, 19104, USA c Pediatric Liver Center and Liver Transplantation Program, Department of Pediatrics University of Colorado School of Medicine, The Children’s Hospital, 1056 E. 19th Ave. Denver, CO 80218, USA d Pediatric General Clinical Research Center, University of Colorado School of Medicine, The Children’s Hospital, 1056 E. 19th Ave. Denver, CO 80218-1007, USA

Over recent years it has become apparent that the hepatocyte mitochondrion functions as a cause and as a target of liver injury. Resultant dysfunction of mitochondria yields deficient oxidative phosphorylation (OXPHOS), increased generation of reactive oxygen species (ROS), accumulation of hepatocyte lipid, impairment of other metabolic pathways and activation of both apoptotic and necrotic pathways of cellular death. Thus, mitochondria are important ‘‘control switches’’ that are involved in determining the ultimate fate of many cell types. Since the mitochondria are under dual control of nuclear DNA and mitochondrial DNA (mtDNA), mutations in genes of both classes have been associated with inherited mitochondrial myopathies, encephalopathies, and hepatopathies. The autosomal nuclear gene defects affect a variety of mitochondrial processes such as protein assembly, mtDNA synthesis and replication, and transport of nucleotides or metals. Drug interference with mtDNA replication is now recognized as a cause of acquired mtDNA depletion resulting in liver failure, lactic acidosis, and myopathy in HIV and HBV patients treated with

This work was supported in part by grants from the National Institutes of Health (RO1 DK38446 and 5 MO1 RR00069) and the Abbey Bennet Liver Research Fund. * Corresponding author. Department of Pediatrics, The Children’s Hospital, 1056 E. 19th Ave., Denver, CO 80218. E-mail address: [email protected] (R.J. Sokol). 0889-8553/03/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0889-8553(03)00052-9

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nucleoside analogs. Finally, mitochondrial dysfunction has been implicated in alcoholic and non-alcoholic fatty liver diseases, drug and toxin-induced liver injury, and chronic cholestasis, and is an important target for future therapeutic intervention. In this review, emphasis is placed on describing newly discovered genes that control mitochondrial DNA synthesis, clinical and pathophysiologic descriptions of inherited mitochondrial gastrointestinal disorders and hepatopathies, and important clinical aspects related to nucleoside analogs and acquired mtDNA depletion in HIV and HBV patients. Lessons learned from infants with these ‘‘experiments of nature’’ have major bearing on understanding the more common acquired mtDNA diseases now being identified in older children and adults.

Mitochondrial genetics Mitochondrial DNA was identified by Nass and Nass in 1963 [1], and 2 decades later, Anderson [2] reported the complete sequence of the human mitochondrial genome. Mitochondria are able to replicate, transcribe, and translate their DNA independently. The mtDNA genome is a circular, double-stranded molecule that encodes for 2 ribosomal RNAs; 22 transfer RNAs; and 13 polypeptides of complexes I, III, IV, and V of the respiratory chain. Oxidative phosphorylation, the process of energy production in the form of adenosine triphosphate (ATP), occurs in the respiratory chain of the mitochondria in every cell. The respiratory chain (Fig. 1) is divided into five multi-enzymatic complexes embedded in the inner mitochondrial membrane, the first four of which are (1) reduced nicotinamide adenine dinucleotide (NADH)-coenzyme Q (CoQ) reductase (complex I), (2) succinate-CoQ reductase (complex II), (3) reduced CoQ-cytochrome c reductase (complex III), and (4) cytochrome c oxidase (complex IV). Electrons are transferred to oxygen from substrates of the tricarboxylic acid cycle (through NADH) and of the fatty acid oxidation cycle (through NADH and FADH2). CoQ and cytochrome c act as ‘‘shuttles’’ between the complexes. Complex V (ATP synthase) allows protons to flow back into the mitochondrial matrix and uses the released energy to synthesize ATP [3]. The respiratory chain is under dual genetic control of both the mitochondrial and nuclear genomes; mutations in either genome can result in abnormalities of OXPHOS. The majority of the estimated 1000 different mitochondrial proteins are encoded by nuclear DNA, translated in the cytoplasm and transported into the mitochondria. Key concepts of mitochondrial genetics and mtDNA include:  Maternal inheritance: Mitochondrial DNA does not follow the rules of Mendelian inheritance. At fertilization, all mtDNA derives from the oocyte, and is not capable of recombination. Thus, a point mutation of the mtDNA is passed from a mother to all of her children, but only the female children will pass the mutation on to their children. Maternal

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Fig. 1. The mitochondrial respiratory electron transport chain consists of 5 multi-enzymatic complexes that transfer electrons to molecular oxygen. The distribution of redox centers and proton translocating activities of the complexes effect a charge separation such that the potential energy derived from oxidations drives ATP synthesis and other energy-requiring processes. The principal reductants of the respiratory chain are NADH and succinate, but electrons from acyl CoA thioesters and several other low-molecular weight substrates enter through a number of ‘‘side-chains.’’ NADH acts as a carrier for reducing equivalents from glycolysis into the mitochondrial matrix, whereas NADH and FADH2 shuttle reducing equivalents produced by fatty acid oxidation and the tricarboxylic acid (TCA) cycle. Coenzyme Q and cytochrome c act as ‘‘shuttles’’ between the complexes. cyt, cytochrome; Fe-S, iron sulfur cluster; FP, flavoprotein; Q, coenzyme Q.

inheritance is not always evident at a clinical level because of variable expressivity of the phenotype.  Heteroplasmy and threshold effect: Each cell has hundreds to thousands of mtDNA copies. In normal tissues, all mtDNA molecules are identical (homoplasmy). A mutation of mtDNA segregates randomly into daughter cells leading to a mixture of normal (wild-type) and mutant mtDNA coexisting within the same cell or tissue (heteroplasmy). The threshold effect, or minimum critical number of mutated mtDNAs necessary to impair cellular function, depends on the balance between oxidative supply and demand for a particular tissue. The threshold of mutant genomes needed for clinical expression (phenotype) of a pathogenic mtDNA mutation varies among persons, among organ systems, and within a given tissue.  Random segregation: During cell division, mitochondria are segregated randomly between daughter cells, shifting the proportion of mutant and wild-type mtDNA. This explains how some patients with mitochondrial disorders may actually shift from one clinical phenotype to another as they age.  Mutability: MtDNA, with its high information density and lack of redundancy, mutates more than 10 times as frequently as nuclear DNA. The lack of introns renders the coding region of mtDNA sequence

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vulnerable to random mutations [4]. The mtDNA genome lacks protective histones and an effective repair system, rendering it more prone to oxidative injury. It is continuously exposed to oxygen free radicals generated by OXPHOS. The gradual accumulation of mtDNA mutations over time may contribute to the process of aging and a number of neurodegenerative diseases [5].

Epidemiology of mitochondrial disorders Pathogenic mitochondrial DNA (mtDNA) mutations have been associated with an expansive catalog of pediatric and adult mitochondrial diseases, and more than 200 pathogenic point mutations, deletions, insertions, and rearrangements have been identified since the first mitochondrial mutations were reported in 1988 [6,7]. The past decade has brought forth the dawn of ‘‘molecular mitochondrial medicine,’’ with discoveries of numerous genetic defects associated with mitochondrial diseases. Disease expression is complex and depends on interactions between nuclear genes and the mtDNA; an estimated 90% of mitochondrial diseases are caused by mutations in nuclear genes, with only a small fraction of these nuclear mutations having been identified at this time. As a general rule, point mutations of mtDNA genes are usually maternally inherited (mtDNA genes) whereas deletions or rearrangements of mtDNA are either sporadic or inherited in an autosomal recessive manner (caused by mutations in nuclear genes). Mitochondrial disorders pose particular problems for the genetic epidemiologist. Many factors influence the prevalence of mitochondrial disorders, including the mutation rate, inheritance pattern, population structure, and the genetic background. Accurate diagnosis can be difficult, requiring the proper collection and handling of tissue specimens; it is not always possible to identify the underlying mitochondrial molecular genetic defect in the blood [8]. The clinical presentations vary considerably and many patients present with non-specific features, leading to significant delays in diagnosis; moreover, transmission of a pathogenic mutation does not always produce a clinical phenotype. Most epidemiologic studies were designed to investigate the frequency of a particular mtDNA mutation in patients with a specific disease presentation, failing to account for phenotypic variability. Recently, there have been a number of studies designed to determine the true prevalence of mitochondrial disorders at a population level [9–12]. The results of these studies are summarized in Table 1. Based on the available studies, Chinnery [13] has calculated and estimated a minimum prevalence of mitochondrial disease of 11.5 cases per 100,000 individuals, or 1 in 8500 of the general population. This conservative estimate corresponds to the point prevalence in adults at any one time and to the birth incidence of individuals who will go on to develop mtDNA disease later in life. As the spectrum of mtDNA disease continues to rapidly

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Table 1 Population-based studies of mitochondrial disease

Study population

Mutations or disease

Majamaa et al (1998) [10] Adult point prevalence Northern Finland of A3243G mutation N = 245,201 adults Identified 615 patients Chinnery et al (2000) [9] Adult point prevalence of Northern England all mtDNA mutations Identified 104 patients N = 1,582,584 and 161 maternal adults relatives Uusimaa et al (2000) [11] All mtDNA mutations in Finland children with respiratory N = 146,482 chain disorder children Identified 26 children Darin et al (2001) [12] Pediatric point prevalence Western Sweden of pediatric mitochondrial N = 358,616 encephalomyopathies children Identified 32 children

Disease prevalence/ 100,000 (95% CI)

Mutation prevalence/ 100,000 (95% CI)

5.71 (4.53–6.89) 16.3 (11.3–21.4)

6.57 (5.30–7.83) 12.48 (10.75–14.23)

Not able to be calculated from data

Not able to be calculated from data

4.76 (2.80–7.60)

Not able to be calculated from data

expand, with novel genotypes and phenotypes, epidemiologic data will accordingly require future revisions.

Clinical presentations of mitochondrial disorders Mitochondrial disorders were once regarded as neuromuscular diseases, with most mutations found in mtDNA. Defects in OXPHOS clearly can affect any tissue to a variable degree, with the most energy-dependent organs seeming most vulnerable, resulting in heterogeneous clinical presentations. Symptoms involving every organ system have been reported, and a frequent feature is an increasing number of organs involved as the disease progresses over time. Initial symptoms usually persist and gradually worsen, but sometimes they improve or even disappear as other organs become involved. It is important to consider the diagnosis of a mitochondrial disorder in patients with progressive, multi-system symptoms that cannot be explained by a specific diagnosis. Neuromuscular manifestations are common, but are not a requirement for diagnosis or an invariable outcome of mitochondrial disorders. Thirty-three to 56 percent of patients with mitochondrial disorders have non-neuromuscular symptoms at presentation [14]. Nonspecific gastrointestinal symptoms, such as vomiting, diarrhea, constipation, failure to thrive, and abdominal pain are commonly found in many mitochondrial disorders. Other gastrointestinal manifestations, such as chronic intestinal pseudo-obstruction and pancreatic exocrine insufficiency are among the cardinal manifestations of mitochondrial neurogastrointestinal

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encephalomyopathy (MNGIE) and Pearson syndrome, respectively. Other inherited or acquired mitochondrial diseases, such as mtDNA depletion syndrome, are notable for their hepatic presentations, with or without neuromuscular involvement, and typically result in death from liver failure. Chronic hepatic involvement has also been reported, and includes cholestasis, steatosis, and hepatomegaly. Classification of mitochondrial hepatopathies and gastrointestinal disorders In a variety of gastrointestinal and liver disorders, defects in specific biochemical pathways or more general dysfunction of mitochondria have been described. Sokol and Treem [15] have proposed a classification scheme for mitochondrial hepatopathies (Box 1). The disorders are divided into primary, in which the mitochondrial defect is the primary cause of the liver disorder, and secondary, in which a secondary insult to mitochondria is caused by either a gene defect that affects non-mitochondrial proteins (eg, Wilson’s disease) or by an acquired (exogenous) injury to mitochondria. Leonard and Schapira [16] have divided primary mitochondrial diseases into those caused by mutations affecting mtDNA genes (class 1a) and those caused by mutations in nuclear genes that encode mitochondrial respiratory chain proteins or cofactors (class 1b). Mutations in nuclear genes coding for non-respiratory chain mitochondrial proteins also cause mitochondrial disorders. Two patients have been described with multiple deficiencies of heat-shock protein 60, a chaperone protein [17]. Truncation of a mitochondrial protein homologous to a yeast mitochondrial intermembranous space protein (Tim8) has been associated with the X-linked deafness–dystonia syndrome (MohrTranebjaerg syndrome) [18]. Many patients with Leigh’s syndrome have now been shown to have mutations in the nuclear gene encoding Surf1, a mitochondrial protein involved in cytochrome C oxidase assembly [19]. An interesting group of mitochondrial diseases caused by nuclear genes that affect mtDNA stability has been described: mitochondrial neurogastrointestinal encephalomyopathy (TP: thymidine phosphorylase gene); mtDNA depletion syndrome (dGK: deoxyguanosine kinase; and TK2: thymidine kinase-2 genes); and autosomal dominant progressive external ophthalmoplegia (ANT1: adenine nucleotide translocator 1). These phenotypically and genotypically heterogeneous disorders seem to share a common mechanism of disturbed mitochondrial nucleoside pools. The identification and pathogenesis of many additional nuclear genes regulating mitochondrial function are the focus of exhaustive research efforts and are anticipated discoveries in mitochondrial medicine. The secondary disorders, in which hepatic mitochondria undergo injury or dysfunction caused by another pathologic process, include diseases of uncertain etiology but clearly involving hepatic mitochondria (eg, Reye’s syndrome), conditions caused by endogenous and exogenous mitochondrial

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Box 1. Classification of mitochondrial hepatopathies A. Primary disorders 1. Electron transport (respiratory chain) defects  Neonatal liver failure Complex I deficiency (NADH: ubiquinone oxidoreductase) Complex IV deficiency (cytochrome c oxidase) Complex III deficiency (ubiquinol: cytochrome c oxidoreductase) Multiple Complex deficiencies  Mitochondrial DNA depletion syndrome  Delayed-onset liver failure: Alpers disease (complex I deficiency)  Pearson’s marrow–pancreas syndrome (mtDNA deletion)  Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)  Chronic diarrhea (villous atrophy) with hepatic involvement (complex III deficiency)  Navajo neurohepatopathy  Long-chain hydroxyacyl CoA dehydrogenase deficiency  Acute fatty liver of pregnancy (AFLP) 2. Fatty acid oxidation and transport defects 3. Carnitine palmitoyltransferase I and II deficiencies 4. Carnitine–acylcarnitine translocase deficiency 5. Urea cycle enzyme deficiencies 6. Electron-transfer flavoprotein (ETF) and ETF-dehydrogenase deficiencies 7. Phosphoenolpyruvate carboxykinase (PEPCK) deficiency (mitochondrial) 8. Nonketotic hyperglycinemia (glycine cleavage enzyme deficiency) B. Secondary disorders 1. Reye’s syndrome 2. Hepatic copper overload  Wilson’s disease  Indian Childhood cirrhosis  Idiopathic infantile copper toxicosis  ? cholestasis 3. Hepatic iron overload  Hereditary hemochromatosis  Neonatal iron storage disease (continued on next page)

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Box 1. (continued)  Tyrosinemia, type I  Zellweger syndrome 4. Drugs and toxins  Drugs: Valproic acid, salicylic acid, nucleoside analogs (FIAU, DDI, AZT,d4T), amiodarone, tetracycline, chloramphenicol, barbiturates.  Chemical toxins: Iron, ethanol, cyanide, antimycin A, rotenone, others.  Bacterial toxins: Cereulide (B cereus emetic toxin), Ekiri 5. Conditions causing mitochondrial oxidative stress:  Cholestasis and bile acid synthesis/transport defects  Hydrophobic bile acid toxicity (cholestasis, bile acid synthesis defects)  Non-alcoholic steatohepatitis – associated with obesity, diabetes mellitus, drugs, parenteral nutrition, bacterial contamination of small bowel, J-I bypass, or idiopathic  Alpha-1 antitrypsin deficiency (PiZZ) 6. Cirrhosis (Adapted from Sokol RJ, Treem WR. Mitochondrial hepatopathies. In: Suchy FJ, Sokol RJ, Balistreri WF, editors. Liver Disease in Children, 2nd edition Philadelphia: Lippincott, Williams & Wilkins; 2001. p. 787–810; with permission.)

toxins, drugs (such as nucleoside analogs) or metals, and other conditions in which mitochondrial oxidative injury or abnormal electron transport may be involved in the pathogenesis of liver injury (eg, cholestasis, NASH). Primary mitochondrial hepatopathies and gastrointestinal disorders The liver and the gastrointestinal tract are major target organs in inherited defects of mitochondrial function (Box 1). Neonatal and early childhood presentations predominate, however, it is postulated that milder abnormalities or polymorphisms may predispose to later presentation during adulthood or serve as disease modifying genes. Neonatal liver failure One of the more common presentations of respiratory chain defects in childhood is severe liver failure in the first months of life, characterized by lactic acidosis, jaundice, conjugated hyperbilirubinemia, serum ALT values of 2 to 12 times normal, coagulopathy, ketotic hypoglycemia, renal tubulopathy, and hyperammonemia [14,20–22]. Symptoms include lethargy,

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hypotonia, and vomiting. The key biochemical features are the markedly elevated plasma lactate concentration, an elevated molar ratio of plasma lactate to pyruvate (>20 mol/mol), and elevation of beta-hydroxybutyrate and the arterial ketone body ratio (beta-hydroxybutyrate: acetoacetate >2.0 mol/mol) and elevated cerebrospinal fluid lactate concentration. Plasma lactate may increase during the provision of intravenous glucose; an unusual finding that should raise suspicion of a respiratory chain defect. Liver biopsy shows microvesicular with or without macrovesicular steatosis, canalicular and hepatocellular cholestasis and bile ductular proliferation. Periportal and centrilobular fibrosis can develop into micronodular cirrhosis. Glycogen depletion and iron overload are additional features [22]. Ultrastructural evidence of mitochondrial injury may be observed as swollen mitochondria, abnormal cristae, paracrystalline arrays, and a fluffy matrix, although normal mitochondrial morphology does not exclude these disorders. Increased numbers of mitochondria may be seen in hepatocytes. The clinical course in most patients is rapid progression to death from liver failure or sepsis in the first months of life despite standard treatment. Importantly, most patients have severe neurologic involvement in infancy with a weak cry, poor suck, hypotonia, recurrent apnea, or myoclonic epilepsy, which precludes consideration for liver transplantation [23,24]. However, because the heteroplasmy for mtDNA mutations is not uniform in all tissues, and other modifying genes may affect the degree of expression of the underlying defect in different tissues, in the absence of detectable extrahepatic manifestations several affected infants have undergone successful liver transplantation [25]. Other patients have developed neuromuscular symptoms following liver transplantation (see later). The hepatic activity of respiratory chain complex IV, complex I, complex III and occasionally of complex II, is very low in these infants. Among these, cytochrome C oxidase (Complex IV) deficiency is the most common. In some cases, the use of valproic acid to treat myoclonic seizures has seemingly precipitated hepatic failure, even if no prior liver involvement was evident. MtDNA mutations are not present. De Lonlay et al [26] reported mutations in the nuclear gene BCS1L in infants presenting with hepatic failure, lactic acidosis, renal tubulopathy and variable degrees of encephalopathy, who were found to have deficient activity of complex III of the respiratory chain in liver, fibroblasts or muscle. This nuclear gene codes for proteins involved in the assembly of respiratory complex III, and may be responsible for a substantial portion of infants who present with neonatal liver failure and lactic acidosis. Later onset progressive liver failure in early childhood Deficiencies of respiratory chain complex I, complex IV, or combinations of respiratory chain enzymes have been associated with a later onset of

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recognizable liver disease in infancy and early childhood. The onset of symptoms occurs between 2 months and 8 years of life and is characterized by hepatomegaly and jaundice with hepatic failure evolving over time [21]. In most cases liver failure is preceded by hypotonia, feeding difficulties, symptoms of gastroesophageal reflux or intractable vomiting, failure to thrive, and ataxia followed by the onset of refractory partial motor epilepsy or multifocal myoclonus. The seizure disorder may necessitate the use of multiple anticonvulsants, including valproic acid, which may further impair respiratory chain activity. In addition to mild to moderate elevation of aminotransferases, evidence of hepatic synthetic failure is present (low serum albumin, prolonged prothrombin time, depressed clotting factor 5 or 7 levels). Progressive neurologic deterioration may ensue rapidly, however, in other children, the neurologic features are less severe or delayed in onset. Children with this presentation are said to have Alpers–Huttenlocher syndrome (Alpers progressive infantile poliodystrophy) [24,27–29]. Neurologic evaluation may reveal elevated blood or CSF lactate and pyruvate levels, characteristic electroencephalogram, asymmetric abnormal visual evoked responses, and low density areas or atrophy in the occipital or temporal lobes on CT scanning of the brain [29]. A family history of an affected sibling has been reported in up to 50% of cases. In some patients, NADH oxidoreductase (complex I) deficiency has been found in liver or muscle mitochondria [30]. Early in the course of the liver disease, liver pathology may only be notable for microvesicular steatosis, focal hepatocyte degeneration, and portal fibrosis; later massive hepatocyte dropout (probably caused by apoptosis), parenchymal collapse, and bile ductular proliferation within broad bands of fibrous tissue are present. Mitochondrial morphology varies from only showing an increased number in each hepatocyte to swollen, pleomorphic mitochondria with less dense matrix and few cristae. Occasionally, Alpers disease is not recognized before liver transplantation and neurologic deterioration progresses following transplantation [23,24]. Mitochondrial DNA depletion syndrome Several reports [22,31–36] have described infants with severe or fatal hepatic disease of early onset caused by the inherited mtDNA depletion syndrome (MDS), which is characterized by tissue-specific reduction in mtDNA copy number. Early reports of this condition focused on the myopathic presentation in infancy or later in childhood. Phenotypic heterogeneity has been reported, with both myopathic and hepatocerebral presentations of MDS occurring within the same family [37]. Infants with the hepatocerebral form of MDS present within the first few weeks or months of life and display progressive liver failure, neurologic abnormalities, hypoglycemia, and lactic acidosis. Severe mtDNA depletion may also present with nonspecific symptoms such as vomiting, failure to thrive, and

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developmental delay [38]. Lactic acidemia and hypoglycemia predominate, yet serum AST and ALT may be only modestly elevated. In all reported patients neurologic abnormalities developed before death, although the initial hypotonia may have been attributed to the lactic acidosis. Death usually occurs from liver failure by one year of age [39]. Histologic findings in MDS liver biopsies include microvesicular steatosis, cholestasis and focal cytoplasmic biliary necrosis, and cytosiderosis in hepatocytes and sinusoidal cells. Ultrastructural findings [22,40] include mitochondria having aspects of ‘‘oncocytic transformation,’’ associated with numerous changes in shape, size, cristae, and matrix. Although the individual histologic and ultrastructural findings are non-specific, when taken together in the appropriate clinical context, are suggestive of a respiratory chain disorder [41]. Diagnosis is established by showing a low ratio of mtDNA to nuclear DNA in affected tissues. MDS is characterized by increased numbers of mitochondria in hepatocytes, decreased activities of the mtDNA-encoded electron transport chain complexes (I, III, IV, V), and a depletion of mtDNA to less than 10% of normal in the affected tissues or organs [31,36]. The sequence of the mtDNA genome is normal. The severity of mtDNA depletion correlates with the severity of tissue involvement and biochemical defects. Heteroplasmy of mtDNA with differential tissue involvement diseases suggested that MDS is a mitochondrial disease [37], but no cases of mtDNA mutations or maternal transmission have been reported. The consanguineous origin of several of these children further suggested an autosomal recessive form of inheritance, indicating that a primary nuclear gene defect is involved that secondarily causes the mtDNA depletion. The factors known to be responsible for mtDNA maintenance are all encoded by nuclear genes, and transported into the mitochondria. The mtDNA processing enzyme activities are dependent on several factors, including deoxyribonucleotide (dNTP) concentrations within the mitochondria, availability of ATP, and several metal cofactors. Imbalance of any of these factors or enzymes could affect mtDNA stability. The mitochondrial pool is maintained by either import of cytosolic dNTPs through dedicated transporters or by salvaging deoxynucleosides within the mitochondria. The mitochondrial deoxynucleoside salvage pathway is regulated by nuclear-encoded enzymes, including deoxyguanosine kinase (dGK) and thymidine kinase-2 (TK2) [42,43]. Human dGK phosphorylates deoxyguanosine and deoxyadenosine, whereas TK2 phosphorylates deoxythymidine, deoxycytidine, and deoxyuridine. Mitochondrial dNTP pool imbalance has been proposed as the underlying pathogenesis of the hepatocerebral and myopathic forms of MDS [44]. In 2001, mutations in these two genes were identified in patients with MDS: dGK in the hepatocerebral form [44] and TK2 in the myopathic form [45]. The frequency of dGK mutations in 21 patients with hepatocerebral MDS was only 14% in a recent study, suggesting that dGK is not the only gene responsible for mitochondrial depletion in the liver [46]. No genotype-phenotype correlation was demonstrated.

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Mitochondrial neurogastrointestinal encephalomyopathy A multi-system syndrome affecting muscle, peripheral and central nervous systems, and the GI tract was first described in 1983 [47]. In 1994, Hirano et al [48] called the syndrome mitochondrial neurogastrointestinal encephalomyopathy, thus preserving the acronym MNGIE originally coined by Bardosi et al [49] as myoneurogastrointestinal encephalopathy. MNGIE is characterized by myopathy with ragged-red fibers, peripheral sensorimotor neuropathy, progressive external ophthalmoplegia, ptosis, leukoencephalopathy, and chronic intestinal pseudo-obstruction [48]. The disease onset ranges from five months to 43 years of age [50]. Non-specific gastrointestinal signs and symptoms of nausea, vomiting, abdominal pain, borborygmi, diarrhea, constipation and abdominal distention often lead to a diagnosis of intestinal dysmotility or ‘‘pseudo-obstruction’’ [47,51,52]. GI symptoms typically have onset in childhood, and were the presenting complaint in 45% to 67% of patients [53]. Neurologic manifestations, hearing loss, or ocular symptoms were the initial manifestations in 42% to 49% of patients. Thin body habitus and short stature are constant findings, presumably secondary to chronic malnutrition and malabsorption. Small bowel diverticulosis, presumably secondary to markedly delayed intestinal motility, appears in early adult years in 30% to 67% of patients. The chronic intestinal pseudoobstruction has been attributed to a visceral myopathy with atrophic fibrotic longitudinal smooth muscle in the intestinal wall but normal ganglion cells in some reported patients. In other cases, autopsy findings have suggested an autonomic neuropathy in other patients with fibrosis and vacuolization of autonomic ganglia in the myenteric plexus and decreased nerve fibers innervating intestinal smooth muscle. MNGIE is an autosomal recessive disease associated with multiple mtDNA deletions or depletion in skeletal muscle. In the limited number of patients studied, skeletal muscle respiratory-chain defects consisting of complex IV, complex I, or combination defects have been identified. MNGIE was mapped to chromosome 22q13.32-qter region in 4 kindreds [54], and Nishino et al [55] showed loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). Sixteen different mutations in ethnically diverse MNGIE pedigrees have been found [56]. TP is a multifunctional enzyme that has an important role in the nucleoside salvage pathway, catalyzing the breakdown of thymidine to be reused for mtDNA synthesis as dexoxythymidine triphosphate (dTTP). TP also produces 2deoxyribose, which is an endothelial cell chemoattractant in angiogenesis induction [57]. None of the MNGIE patients have had vascular abnormalities, suggesting that the absence of TP activity does not interfere with normal angiogenesis. Impaired thymidine metabolism has been demonstrated by biochemical analysis in 27 MNGIE patients [58], with elevated plasma levels of thymidine and depletion of the mitochondrial dTTP pool used for DNA synthesis. The pathogenic mechanism of MNGIE, like mitochondrial

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depletion syndrome, appears to be related to an imbalance of the mitochondrial nucleoside pool. Pearson marrow–pancreas syndrome Pearson marrow–pancreas syndrome was described in 1979 in four unrelated children with neonatal-onset severe refractory macrocytic anemia with variable neutropenia and thrombocytopenia, and a bone marrow characterized by the presence of ringed-sideroblasts and marked vacuolization of erythroid and myeloid precursors [59]. Later in infancy or early childhood, chronic diarrhea and fat malabsorption developed and the patients were found to have exocrine pancreatic insufficiency. Some patients also have endocrine involvement with insulin-dependent diabetes mellitus in the neonatal period. Extensive pancreatic fibrosis and acinar atrophy, a reduction in the size and number of islets, and splenic atrophy have been found at autopsy [60]. Marked hepatomegaly, steatosis, hemosiderosis, and cirrhosis have been associated with liver failure and death in some cases by 3 months of age [61]. Other GI manifestations occur in early childhood, including dysmotility, vomiting, gastroparesis, and pseudo-obstruction. Renal Fanconi syndrome is variably present, with pathologic glomerulosclerosis and vacuolization of the renal tubules. Neurologic involvement includes variable degrees of hypotonia and developmental delays, and skeletal muscle biopsy may demonstrate the characteristic ragged red fibers seen in many mitochondrial disorders. All patients have an impaired redox status in the plasma (see section on Diagnosis), and 3-methylglutaconic aciduria [62] may be an additional organic acid marker. Pearson syndrome is fatal before 3 years of age in 62% of cases [63]. Many patients who survive spontaneously recover from their myelodysplasias but subsequently develop Kearns–Sayre syndrome [64,65], a severe encephalomyopathy characterized by visual impairment, tremor, ataxia, proximal muscle weakness, external ophthalmoplegia, and a pigmentary retinopathy. Large-scale heteroplasmic mtDNA rearrangements are constantly observed in affected and non-affected organs. In a series of 21 patients, Rotig et al [66] demonstrated that all patients had large deletions of mtDNA, with direct repeat sequences consistently found at their boundaries, suggesting that these repeats might have triggered molecular mitochondrial recombinations. Nine patients shared the common 4977 bp deletion, and associated duplications were identified both in lethal cases and in patients who survived and developed Kearns–Sayre syndrome. Complex I is the most severely affected by the 4977 bp deletion, however it also encompasses genes that encode two subunits of complex V, one subunit of complex IV, and five transfer RNA genes. The clinical severity is not correlated with the size, type, or location of the mitochondrial rearrangements; however, the tissue distribution and relative proportion of abnormal mtDNA molecules (heteroplasmy and threshold effect) appear to contribute to the phenotype.

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In the clinically more severely affected tissues such as bone marrow, PMNs, lymphocytes, pancreas, and gut, mtDNA deletions are found in 80% to 90% of cells, but several unaffected organs have been found to contain large amounts of deleted mtDNA. The lack of maternal inheritance or positive family histories, and the absence of mtDNA rearrangements in the lymphocytes of parents or siblings of cases, suggests that many cases are caused by de novo mutations occurring during oogenesis or the early development of fertilized eggs. Poulton et al [67] and Bernes et al [68] reported transmission of the common 4977 bp deletion from a mother with chronic progressive external ophthalmoplegia (CPEO) to a child with Pearson syndrome, although in both cases the authors did not exclude the possibility of an additional mtDNA duplication being present. The risk for transmission of mtDNA deletions seems to be low, but in the absence of substantive data, Warner et al [69] have estimated a 5 percent recurrence risk for genetic counseling. Chronic diarrhea and intestinal pseudo-obstruction Severe anorexia, vomiting, chronic diarrhea, and villous atrophy have recently been described as the initial manifestations of another mtDNA rearrangement syndrome appearing late in the first year or during the second year of life [70]. Hepatic involvement includes mild elevations of liver enzymes, hepatomegaly, and steatosis. Diarrhea, vomiting, and lactic acidosis worsened in these patients with high dextrose intravenous infusions or enteral nutrition. Diarrhea improved or resolved completely by 5 years of age with normalization of intestinal biopsies. However, retinitis pigmentosa, cerebellar ataxia, sensorineural deafness, and proximal muscle weakness became evident late in the first decade of life and patients died soon thereafter. Respiratory-chain enzyme assays were normal in circulating lymphocytes but were abnormal in skeletal muscle tissue, revealing a complex III deficiency. Like Pearson syndrome, large-scale heteroplasmic mtDNA rearrangements are found in all reported patients, with direct repeat sequences consistently found at boundaries of the mtDNA deletions. Chronic intestinal pseudo-obstruction, even in the absence of neurologic changes, may reflect an underlying mitochondrial disorder. A novel mtDNA G8313A point mutation has been described in a child with prominent GI symptoms that preceded a progressive encephalopathy [71]. Santorelli et al [72] and Muehlenberg et al [73] reported the mitochondrial A3242G point mutation in the tRNALeu gene in 2 adult women with chronic pseudoobstruction. Both patients had lactic acidosis, and their gastrointestinal symptoms preceded the neurologic manifestations by several years. Ragged red fibers were identified in the muscle biopsy of only one of the 2 patients, while the rectal biopsy from the other patient showed abnormal mitochondria on ultrastructural examination. Heteroplasmy in both of these patients favored a higher percentage of mutant mtDNA in the gastrointestinal tract,

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consistent with the phenotype of pseudo-obstruction. However, Chinnery et al [74] found no morphologic or molecular genetic abnormalities in either the intestinal mucosal or muscle layers of the resected bowel from a 20-yearold woman with the A3243G mutation and pseudo-obstruction, and suggested that the gastrointestinal symptoms were because of autonomic nervous system involvement. Ischemic colitis [75] and chronic diarrhea [76] have also been described in association with the A3243G mutation. It has also been associated with intestinal pseudo-obstruction in diabetic patients [76,77]. The A3243G mutation is associated with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), which usually presents in childhood with headaches, limb weakness, lactic acidosis, and recurrent neurologic symptoms resembling strokes. Buzzi et al [78] found no A3243G mutations in peripheral blood leukocytes from 21 consecutively enrolled female migraine patients with a matri-linear, multigenerational family history of migraines with or without auras. This does not exclude that mitochondrial energy impairment might still have a role in the pathogenesis of migraines, and various mitochondrial drugs (see Treatment section) are being assessed in clinical trials for migraine prophylaxis. No studies have evaluated the prevalence of the A3243G mutation in children with idiopathic, chronic pseudo-obstruction. Navajo neurohepatopathy Navajo neurohepatopathy (NNH) is a sensorimotor neuropathy with progressive liver disease confined to Navajo children and is manifested by the development of weakness, hypotonia, areflexia, loss of sensation in the extremities, acral mutilation, corneal ulceration, poor growth, short stature, and serious systemic infections [79,80]. Cerebral magnetic resonance scanning further demonstrated the presence of progressive white matter lesions, and peripheral nerve biopsies showed severe loss of myelinated fibers [81]. The inheritance is autosomal recessive. Holve et al [80] demonstrated three clinical presentations of NNH, including an infantile presentation, in which failure to thrive and jaundice progress to hepatic failure and death within the first 2 years of life, with or without neurologic findings; a childhood form presenting between 1 and 5 years of age with rapid development of liver failure; and the classical form in which progressive neurologic findings dominate although liver dysfunction (and even cirrhosis) was present in all patients. Elevation of AST, ALT, alkaline phosphatase, and gamma glutamyl transpeptidase were present in all cases. Liver histology demonstrated portal fibrosis or micronodular cirrhosis, macrovesicular and microvesicular steatosis, pseudacinar formation, multinucleated giant cells, cholestasis, and periportal inflammation. Nonspecific mitochondrial changes, such as swollen mitochondria and ringed cristae, were seen in several patients. Blood lactate and pyruvate levels were normal in patients tested, and skin fibroblasts had normal respiration from one

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patient. Neurologic symptoms progressed after liver transplant in one patient. There has been no effective treatment to date for affected children. The etiology of Navajo neuropathy remains unknown, however, Vu et al [82] recently reported findings consistent with mtDNA depletion in two patients with NNH. MtDNA levels compared with nuclear DNA levels were markedly depressed in liver tissue. Further genetic and metabolic investigations in this disorder will be needed to clearly define its etiology.

Secondary mitochondrial hepatopathies and gastrointestinal disorders Secondary mitochondrial hepatopathies are caused by an injurious metal, drug, toxin, xenobiotic or endogenous metabolite (see Table 1). Acquired abnormalities of mitochondrial respiration caused by these factors may be involved in the pathogenesis of these disorders. Selected diseases will be reviewed. Reye’s syndrome Reye’s syndrome is the best known secondary mitochondrial hepatopathy, and is caused by the interaction of a viral infection (influenza, varicella, enteroviruses, other viruses) and salicylate use or some underlying undefined metabolic or genetic predisposition. Liver and brain electron microscopy in Reye’s syndrome patients reveals striking abnormalities in mitochondrial structure and their function is perturbed, resulting in defective ureagenesis and ketogenesis, hyperammonemia, hypoglycemia, elevated serum free fatty acids, and lactate and dicarboxylic acids. Salicylates have been shown to impair mitochondrial fatty acid oxidation in vitro and recent studies indicate that this may be by reversible inhibition of LCHAD activity. Cells from Reye’s syndrome patients were found to be more susceptible to inhibition by low concentrations of salicylates than controls. Glasgow et al [83] proposed that this increased sensitivity could potentially be caused by a lack of or reduced activity of an uncoupling protein in mitochondria. This study was the first biochemical evidence that might help to explain why only certain individuals react to aspirin in a manner that precipitates Reye’s syndrome. Most cases of Reye’s syndrome had traditionally occurred in the autumn and winter (influenza season) with the peak age between 5 and 15 years. Symptoms developed several days following onset of influenza A or B infections, or varicella. There was a strong association of aspirin use during these illnesses and the development of Reye’s syndrome. Frequently, the child appeared to be recovering from a viral illness after 3 to 5 days when sudden, unremitting vomiting developed. After several hours of vomiting, and not uncommonly dehydration, variable degrees of encephalopathy developed. Liver dysfunction was always present when vomiting developed and was characterized by elevated AST, ALT and blood ammonia with mild

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to moderate prolongation of prothrombin time, variable hypoglycemia but normal serum bilirubin values [84]. Metabolic support with hypertonic dextrose infusions and control of cerebral edema and intracranial pressure became the most important facets of clinical treatment, until spontaneous recovery occurred or irreversible brain injury developed. Mortality was high when patients presented in deeper stages of coma and correlated with levels of blood ammonia at presentation. Abnormal mitochondrial morphology is present in liver, brain, muscle, and kidney. Although patients present with hepatic dysfunction, hyperammonemia, and coma, the encephalopathy may be caused by direct involvement of CNS mitochondria and the accumulating metabolic toxins. Liver biopsies show microvesicular steatosis in the absence of hepatic inflammation or necrosis and characteristic swelling and pleomorphism of mitochondria under electron microscopy. The liver makes a full recovery in this disease, despite progressive and sometimes fatal cerebral edema. Many young patients once thought to have Reye’s syndrome have been found subsequently to have defects in fatty acid oxidation, a form of primary mitochondrial hepatopathy [85]. Since the incidence of Reye’s syndrome has decreased dramatically in western countries following the public warnings of salicylate use in children with viral infections, it is imperative to thoroughly evaluate all children who are diagnosed with Reye’s syndrome for FAO and fatty acid transport defects, particularly those children under 5 years of age. Wilson’s disease Mitochondrial involvement in Wilson’s disease has been implicated since the early observation by Sternlieb [86] of abnormal mitochondrial morphology so characteristic of this disorder of copper metabolism. Changes include decreased matriceal density, enlarged intermembranous spaces, dilatation and vacuolization of cristae, crystalline inclusions, and vacuoles in the matrix. Recent studies in experimental animals, naturally occurring copper toxicity in dogs and in humans have shown that the mitochondrion is a major intracellular target for copper toxicity. The accumulation of copper in the hepatic mitochondria leads to oxidant stress (increased free radical generation) with subsequent lipid peroxidation and oxidative alterations of thiol-containing proteins. In a rat model of copper overload, a 60% decrease of cytochrome c oxidase activity was demonstrated in hepatic mitochondria in conjunction with significant lipid peroxidation. Similar increased lipid peroxidation has been demonstrated in hepatic mitochondria isolated from copper-overloaded dogs and from patients with Wilson’s disease undergoing liver transplantation [87]. Recently, Mansouri et al [88] have shown that this oxidant damage in hepatic mitochondria also leads to deletions in mtDNA in young adults with Wilson’s disease. The underlying defect in Wilson’s disease is caused by

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mutations in the P type ATPase, ATP7B, which is present in the trans-Golgi and seems to be transported to hepatic mitochondria. The function of ATP7B in mitochondria and how this may relate to the pathogenesis of Wilson’s disease has not been determined. In addition to copper chelation therapy, attempts to reduce the oxidative stress (eg, with antioxidants) in the liver of patients with Wilson’s disease could potentially help protect the mitochondria from injury. Drugs and toxins Acquired abnormalities of mitochondrial respiration may be caused by several drugs and toxins (see Table 1). Valproic acid is an 8-carbon branched fatty acid that can be metabolized into a possible mitochondrial toxin, 4envalproic acid. Children with underlying respiratory chain defects (Complex I deficiency) seem to be more sensitive to valproic acid; its use has been associated with precipitating liver failure in patients with Alpers syndrome [24] and cytochrome c oxidase deficiency [89]. For other drugs, the mechanism causing inhibition of mitochondrial respiration is still unclear. Several toxins inhibit specific protein complexes of the respiratory chain (eg, cyanide, antimycin A, and rotenone) and lead to reduced ATP production and increased oxidative stress. Recently, the emetic toxin of Bacillus cereus, cereulide, has been demonstrated to cause inhibition of respiratory chain activity, and as a causative agent for fulminant liver failure. Nucleoside analogs (reverse transcriptase inhibitors) Several hepatotoxic drugs directly inhibit protein complexes of the intramitochondrial respiratory chain or b-oxidation enzymes. A recent example of drug-induced mitochondrial toxicity was the unexpected fatal lactic acidosis and liver failure that developed in seven of the adults with chronic hepatitis B who were treated with the experimental antiviral nucleoside analog, fialuridine (FIAU) [90]. Most of these patients presented with fatigue, nausea, constipation and abdominal pain, coagulopathy, hyperammonemia, and profound lactic acidosis with only mild jaundice and minimal increases in serum aminotransferase values. Pancreatitis, peripheral neuropathy, and myopathy, reminiscent of inherited mitochondrial disorders of the respiratory chain, also developed. Liver tissue from the five patients who underwent liver transplantation showed marked microvesicular and macrovesicular steatosis, cholestasis and swollen dysmorphic mitochondria. FIAU toxicity is caused by its incorporation directly into mtDNA replacing thymidine and directly inhibiting DNA at the transcriptional level leading to acquired mtDNA depletion. This has a profound effect on mtDNA-encoded proteins, with impaired mitochondrial respiration and fatty acid oxidation causing microvesicular steatosis, morphologic changes in mitochondria, and severe lactic acidosis.

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Other nucleoside analogs (reverse transcriptase inhibitors), zidovudine, didanosine (DDI), stavudine (d4T). and zalcitabine, have been shown to inhibit the DNA polymerase-c of mitochondria and may block the elongation of mtDNA, potentially resulting in mtDNA depletion over time [91]. Zidovudine has been occasionally associated with a toxic myopathy characterized by the depletion of mtDNA in myocytes, and has occasionally caused lactic acidosis, hepatic steatosis, and hepatic failure in HIV-infected patients treated with the drug. Peripheral neuropathy has been reported with the use of zalcitabine, and pancreatitis occurs as a toxic consequence of didanosine (DDI) in up to 50% of HIV-infected patients treated for prolonged periods. Lamivudine, another nucleoside analog used to treat chronic HBV infection, is not incorporated into mtDNA (as is FIAU) and shows little inhibition of mtDNA synthesis at concentrations that block the synthesis of HBV DNA. Consequently, lamivudine has not been associated with myopathy or significant hepatic toxicity. Recent studies have focused on characterizing mtDNA levels, mitochondrial morphology, and lactic acidemia in HIV patients on various nucleoside analogs. There is evidence for decreased mtDNA content in adipose tissue, nerve, muscle, and liver of treated HIV patients. Cote et al [92] showed that venous whole blood mtDNA levels were low in untreated HIV patients and fall further before onset of hyperlactatemia, increasing back to baseline after the nucleoside analog was discontinued. Falco et al [93] showed that the mortality rate is 33% to 57% for patients who develop lactic acidosis, a serum lactate of >10 mM was associated with higher mortality (Odds Ratio, 13.23) and that treatment with cofactors was associated with lower mortality (OR 0.17). Treatment frequently used includes discontinuing the presumed offending medication and administering L-carnitine, coenzyme Q, thiamine, vitamin E and riboflavin, and intravenous dextrose for hypoglycemia. Improved means of detecting early significant lactic acidemia and liver dysfunction may lead to timely discontinuation of the offending agent and prevention of more sever lactic acidosis, pancreatitis, and liver failure. Hydrophobic bile acid toxicity Hydrophobic bile acids [94] and metals that accumulate in the liver during cholestasis may lead to perturbations of mitochondrial membrane function and cellular apoptosis or necrosis. During experimental cholestasis induced by bile duct ligation in the rat, Krahenbuhl et al [95] have demonstrated reduced activity of the electron transport chain in hepatic mitochondria with an increased density of mitochondria per hepatocyte. This group further showed that hydrophobic bile acids led to a reduction of Complexes I and III activities in isolated hepatic mitochondria. Physiologic concentrations of hydrophobic bile acids induce the mitochondrial membrane permeability transition and hydroperoxide generation [96]. Thus, it has been proposed that, during cholestasis and in patients with bile acid

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synthesis and transport defects, increased concentrations of hydrophobic bile acids within the hepatocyte induce generation of reactive oxygen species from the altered mitochondrial electron transport chain, with the resultant opening of the permeability pore, reduced OXPHOS acitivity, release of cytochrome c, and the onset of cellular necrosis or apoptosis. Indeed, the antioxidant, vitamin E, has provided significant protection against bile acid toxicity in an in vivo rat model [97]. Possible interventions in humans with cholestasis to improve mitochondrial function and prevent triggering of these pathways are under investigation.

Diagnosis of respiratory-chain disorders Diagnosing a mitochondrial respiratory chain defect in patients with liver disease requires a high index of suspicion. Clinical scenarios that should suggest these disorders include (a) association of neuromuscular symptoms with liver dysfunction, (b) multi-system involvement in a patient with acute or chronic liver disease, and (c) a rapidly progressive course of liver disease, particularly in the presence of lactic acidosis or ketonemia. Although CNS and neuromuscular syndromes have been the predominant findings in many cases, a recent report of 100 patients with respiratory chain deficiencies at one European center showed that 56% of patients presented with an extraneuromuscular problem with only 44% being referred for a neuromuscular problem [14]. Laboratory findings in the blood and urine of an altered redox status, suggestive of a respiratory-chain defect [98], are listed in Box 2. Persistent elevation of plasma lactate (>2.5 mM) with an elevated molar ratio of plasma lactate to pyruvate (L/P > 20) and elevated ketone body ratio of b-hydroxybutyrate to acetoacetate (>2) is suggestive of a respiratory chain disorders [98]. It should be stressed, however, that lactic acid and these ratios are not elevated in all patients with respiratory chain defects; for example, proximal tubule disorders can result in a falsely normal plasma lactate because the increased loss of lactate in the urine. A struggling child who actively resists phlebotomy can reproduce the ‘‘ischemic forearm test’’ resulting in a falsely elevated lactate; instead, blood samples should be taken with the patient at rest through a heparinized venous catheter and immediately deprotonated with perchloric acid [99]. Elevated ratios are indicative of an increase in reducing equivalents (excess of NADH and lack of NAD) caused by impaired transfer of electrons from NADH to oxygen as a result of disrupted OXHPOS. The L/P ratio is a reflection of the NADH to NAD balance in the cytosol, and the b-OHB/AA ratio is a reflection of the NADH to NAD ratio within the mitochondrion. The elevated ketone body ratio is a consequence of functional impairment of the citric acid cycle, with ketone body synthesis increasing (particularly after meals) because of the channeling of acetyl CoA away from coalescence with oxalate to form citrate

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Box 2. Screening tests for mitochondrial disorders Plasma lactate/pyruvate ratio (L/P) molar ratio Often > 20 in respiratory chain disorders Often \ 10 in defects of pyruvate dehydrogenase Plasma lactate typically > 2.5 mM (repeatedly) Oral glucose load (2 g/kg): repeat plasma L/P testing each 15 minutes for 90 minutes Plasma ketone body (3-OH butyrate/acetoacetate) molar ratio Often > 2 in OXPHOS defects Often \ 1 in disorders of pyruvate and Krebs cycle metabolism Plasma amino acid quantitation Can have elevations of alanine and proline in situations of lactic acidosis Plasma acylcarnitine profile To evaluate for fatty acid oxidation disorders Can also measure total and free carnitine Urine organic acids To evaluate for urine lactate, succinate, fumarate, malate, 3-methylglutaconate, and 3-methylglutarate by gas chromatography-mass spectroscopy Cerebrospinal fluid (CSF) studies L/P molar ratio Ketone body molar ratio Amino acid quantitation Skeletal muscle biopsy Light microscopy for presence of ragged red fibers Electron microscopy for mitochondria ultrastructure Respiratory chain analysis (polarographic with or without spectroscopy)

and into the ketogenic pathway. After feeding, the exaggerated paradoxical production of ketones is even more evident, as ketone production should normally decrease after meals because the suppressive effect of insulin on ketogenesis. Similarly, the abnormal L/P ratio is particularly apparent in the postprandial period when more NAD is required for adequate oxidation of glycolytic substrates. In the presence of decreased NAD, pyruvate will be diverted by anaerobic metabolism to lactate. Thus, to fully evaluate the patient, some have recommended that the concentration of these substrates and their molar ratios, and blood glucose and free fatty acids, should be

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determined before and 1 hour after meals. Occasionally, it is necessary to load a fasted patient with oral glucose (2 g/kg) to provoke lactic acidemia and abnormal ratios, if the values are normal under baseline conditions [100]. Substrates and ratios should be measured every 15 minutes for 90 minutes after the load. Lactate/pyruvate molar ratios in the CSF may be helpful when no elevation in plasma lactate is observed, particularly in the patient with CNS involvement. Urine gas chromatography-mass spectrometry (GC-MS) can detect elevated urinary lactate, Krebs cycle intermediates (succinate, fumarate, and malate), and at times, 3-methyl-glutaconic acid in patients with mitochondrial disorders. These urine organic acids may confirm diagnostic suspicion, but are non-specific in respiratory chain disorders. Urine amino acids are used to assess for the presence of a proximal tubulopathy. Plasma amino acid chromatography frequently shows indirect evidence of lactic acidosis, with an elevated alanine, proline, and occasionally, methionine. Hypocitrullinemia is a non-specific finding of impaired OXPHOS; the synthesis of citrulline by enterocytes might be limited by the reduced availability of mitochondrial ATP. A plasma acylcarnitine profile is the best screening test for a mitochondrial fatty acid oxidation disorder, along with characteristic urinary dicarboxylic acid excretion detected by GC-MS of urine. More recent non-invasive techniques that are under investigation include nuclear magnetic resonance, the 2-keto [1-(13)C] isocaproic acid breath test, and the 13C-methionine breath test [101]. As research tools, both the ketoisocaproic acid and 13C-methionine breath tests have been shown to provide an estimate of hepatic mitochondrial function in vivo [102,103]. It should be emphasized that these are screening tests and may not be abnormal if the respiratory chain defect is confined to one or two organs. Therefore, searching for dysfunction or abnormal histology/biochemistry of the target organs is also important. Definitive diagnostic tests for mitochondrial disorders include direct measurement of mitochondrial respiration; quantitation of enzymatic activity of respiratory chain complexes in affected tissues; histopathologic investigations of muscle or liver, including histochemical stains for cytochrome c oxidase activity; genotyping for mtDNA and nuclear DNA mutations or deletions; and testing for mtDNA depletion. Investigations should be performed on the liver and more standard tissues, such as muscle, since defects can be tissuespecific. Further information about these tests can be found in referenced texts [15]. Treatment of respiratory chain disorders Unfortunately, there is no ideal effective therapy for most patients with respiratory chain disorders, including those with gastrointestinal dysmotility, liver failure, and more slowly progressive liver disease. It is not clear

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that any currently available medical therapy significantly alters the course of severe disease; however, there are anecdotal reports suggesting an improvement of neuromuscular symptoms in some patients. Based on an understanding of the enzymatic and biochemical derangements, several treatment strategies have been proposed (Table 2). ‘‘Mitochondrial cocktails,’’ alleged to promote mitochondrial health, are empiric combinations of various antioxidants, vitamins, cofactors, and electron acceptors. To date, no controlled trials have been performed on any of these ‘‘mitochondrial cocktails,’’ and the few controlled clinical trials of individual pharmacologic agents have not been encouraging. Isolated reports of ubiquinone (coenzyme Q10) supplementation have shown clinical improvements in mitochondrial cardiomyopathies and neuromuscular symptoms. However, a 6-month, double-blind trial with coenzyme Q10 showed no objective improvement in 44 patients with mitochondrial myopathies [104]. Significant problems encountered in clinical trials of mitochondrial disorders include: genotypic and phenotypic heterogeneity; variable natural history within an individual and among family members; uncertainty of best measurements of outcome and therapeutic response. There is a need for well-performed double-blind placebo-controlled randomized clinical trialswith comparable groups of patients and with sufficient follow-up periods.

Table 2 Proposed pharmacologic treatments for mitochondrial disorders Electron acceptors and cofactors Coenzyme Q10 Redox bypass of complex I; Free radical scavenger (antioxidant) Idebenone Redox bypass of complex I; Free radical scavenger (antioxidant) Thiamine (vitamin B1) Cofactor of pyruvate dehydrogenase Riboflavin (vitamin B2) Acts as flavin precursor for complexes I and II Menadione (vitamin K3) Bypass complex III (with vitamin C) Antioxidants Vitamin E (TPGS) Antioxidant Ascorbic acid (vitamin C) Other Mechanisms Succinate Carnitine Creatine monohydrate Dichloroacetate

Antioxidant

Adult: 60–300 mg/day a Ped: 3–5 mg/kg/day Adult: 90–270 mg/day a Ped: 5 mg/kg/day Adult: 150–300 mg/day Adult: 50–200 mg/day Adult: 40–160 mg/day Adult: 400–800 IU/day a Ped: 25 IU/kg/day Adult: 2–4 g/day

Donates electrons directly to complex II Adult: 6–16 g/day Replace secondary carnitine deficiency Adult: up to 3 g/day a Ped: 50–100 mg/kg/day Enhances muscle phosphocreatine Adult: Up to 10 g/day a Ped: 0.1–0.2 g/kg/day Reduces lactic acidosis by enhancing Adult: 25 mg/kg/day a pyruvate dehydrogenase activity Ped: 25–50 mg/kg/day

Abbreviations: TPGS: D-alpha tocopheryl polyethylene glycol-1000 succinate. a Pediatric (Ped) doses are estimates and have not been subjected to clinical dosage trials.

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Despite significant advances in our understanding of mitochondrial biochemistry and genetics, current treatment strategies for mitochondrial disorders are generally supportive. Management of these heterogeneous disorders includes the empiric supplementation with various ‘‘mitochondrial cocktails,’’ supportive therapies, and avoidance of drugs and conditions known to have a detrimental effect on the respiratory chain. Examples of symptomatic therapies include: sodium bicarbonate for acute and chronic metabolic acidosis; transfusions for anemia and thrombocytopenia; exogenous pancreatic enzyme replacement for chronic pancreatic insufficiency; and electrolyte replacement to compensate for renal losses. Dietary measures, such as avoidance of fasting, have also been advocated, but have not been subjected to clinical trials. Hypermetabolic states, such as exhaustive exercise or fever, should also be avoided. Infections can precipitate rapid metabolic deterioration, and require prompt attention and treatment. Interestingly, sustained aerobic exercise may ameliorate symptoms of exercise intolerance caused by mitochondrial dysfunction. Many drugs (see earlier) interfere with mitochondrial metabolism, and should be avoided in these patients. Seizure control should not include phenobarbital because it can inhibit OXPHOS. Valproic acid should likewise be used with caution because of its effects on respiration and fatty acid metabolism. Several nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit or uncouple OXPHOS, and may result in clinical deterioration. Aminoglycoside antibiotics must be avoided in patients with mitochondrial mutations, particularly the A1555G rRNA mutation, because of the significant risk for aminoglycoside-induced ototoxicity [105]. Liver transplantation Although the presence of neuromuscular or extra-hepatic involvement in respiratory chain disorders should preclude the use of liver transplantation, several patients with defects isolated to the liver have now successfully undergone liver transplantation with excellent long-term outcomes and no extra-hepatic disease expression. Extra-hepatic disease, especially neurologic disease, should be ruled out before liver transplantation, but it may be difficult to differentiate mitochondrial neurologic manifestations from signs accompanying liver failure. In a series of 11 patients who underwent liver transplantation before 7 months of age for primary OXPHOS deficiencies, only 5 were alive and well at follow up between 5 months and 8 years posttransplantation [25]. Three of the 6 patients who died developed neurologic features after liver transplantation. All three patients with initial liver failure and associated gastrointestinal disease died shortly after liver transplantation. For patients with acquired mtDNA depletion caused by nucleoside analogs, successful liver transplantation has been performed without recurrence of disease, as long as the offending agent has been discontinued [90].

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