Defects of Substrate Transport

Several genetically determined defects of substrate transport have been described. Perhaps the best examples are the defects involving the carnitine cycle. There are four such defects, involving:

The carnitine cycle is illustrated in Figure 37-4. Carnitine is actively transported from the blood across the plasma membrane. Approximately 90 percent of carnitine body stores reside in skeletal muscle, with skeletal muscle concentration of carnitine being about 60-fold higher than the plasma concentration. Long-chain fatty acids are translocated across the inner mitochondrial membrane as the carnitine ester. Medium-chain fatty acids enter the mitochondrial matrix directly before being activated to the thioester (see Figure 37-4).

A common defect in this cycle involves the carnitine transporter system [Tein, 2003]. This condition is inherited as an autosomal-recessive trait, and is clinically manifested in infancy or early childhood as a carnitine-responsive cardiomyopathy, associated with hypotonia, weakness, failure to thrive, hypoketotic hypoglycemia, and altered consciousness or coma. The defect is generalized, with involvement of skeletal muscle, heart, and kidney. Studies of cultured skin fibroblasts define the condition and identify the heterozygote asymptomatic carriers. Molecular genetic studies have identified pathogenic mutations in the gene (OCTN2) encoding the sodium-dependent, high-affinity carnitine transporter, OCN2 [Lamhonwah et al., 2009]. Oral carnitine supplementation is life-saving, with resolution of clinical symptoms and restoration of normal cardiac function.

Carnitine palmitoyltransferase I deficiency is a clinical condition of infancy manifested by nonketotic hypoglycemic coma [DiDonato and Taroni, 2003; Tein, 2003]. The patients have marked hepatomegaly and hypertriglyceridemia, even when they are clinically asymptomatic. The clinical syndrome mimics Reye’s syndrome, but dicarboxylicaciduria is absent and the serum carnitine values are normal or high. Hyperammonemia, disturbed liver function test results, and marked hepatomegaly are distinctive; persistent renal tubular acidosis may be present. Medium-chain triglycerides may be used to document a ketotic response and as treatment for this autosomal-recessive condition. Muscle from these patients has normal activities for both carnitine palmitoyltransferase I and carnitine palmitoyltransferase II, suggesting the existence of tissue-specific isoenzymes for carnitine palmitoyltransferase I. This finding has been confirmed by molecular studies, demonstrating that there are two distinct genes for carnitine palmitoyltransferase I. One encodes the muscle–heart isoform; the other encodes the liver–fibroblast isoform.

Several infants with carnitine–acylcarnitine translocase deficiency have been described, combining hypoketotic hypoglycemia, hepatomegaly, cardiomyopathy, seizures, lethargy, and coma [DiDonato and Taroni, 2003; Tein, 2003]. The gene for carnitine–acylcarnitine translocase is located on chromosome 3p21.31, and several mutations have been identified in patients.

Carnitine palmitoyltransferase II was the first primary biochemical defect of fatty acid oxidation to be described [DiMauro and DiMauro, 1973]. The adult form is relatively benign and consistent with a normal life expectancy. A disproportionate number of affected males have been reported, despite the fact that it is an autosomal-recessive trait. The clinical picture is that of a metabolic myopathy, with recurrent muscle pain and myoglobinuria. Symptoms are provoked by fasting, prolonged exercise, cold exposure, infection, or emotional stress. Fixed limb weakness develops in about 10 percent of patients at an older age, and a similar percentage have evidence of a lipid storage myopathy [DiDonato and Taroni, 2003; Tein, 2003]. Carnitine concentrations are normal in this clinical syndrome. Several mutations compatible with some residual activity (“leaky mutations”) in the CPT2 gene have been associated with the myopathic form. A common mutation (Ser113Leu) has been identified in at least 50 percent of patients and may be used for diagnostic screening in genomic DNA isolated from blood.

Carnitine palmitoyltransferase II deficiency in newborns is a generalized lethal disease, with reduced enzyme activity in multiple organs and corresponding signs of liver failure, cardiomyopathy, hypoketotic hypoglycemia, and mild myopathy. There are reduced concentrations of free and total carnitine and increased concentrations of lipids and long-chain acylcarnitines. It remains unclear what distinguishes the relatively benign adult, myopathic form of carnitine palmitoyltransferase II deficiency from the lethal infantile form. The residual enzyme activity is similar in these two phenotypes, but oxidation of long-chain fatty acids may be more severely impaired in the infantile form [DiDonato and Taroni, 2003].

Some patients with the mild form of multiple acyl coenzyme A dehydrogenase deficiency have a riboflavin-responsive syndrome, suggesting a genetic defect in flavin adenine nucleotide biosynthesis or transport [DiDonato and Taroni, 2003, 2004]. Riboflavin supplementation, together with l-carnitine and a low-protein, low-fat diet, represents effective treatment in this subgroup of patients with the glutaricaciduria type II phenotype. A mostly myopathic presentation of glutaricaciduria type II, due to mutations in the gene (ETFDH) encoding the electron-transferring flavoprotein dehydrogenase, is associated with coenzyme Q₁₀ (CoQ₁₀) deficiency in muscle, and responds to CoQ₁₀ and flavoprotein administration [Gempel et al., 2007].

Defects of Substrate Utilization

Pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, and defects of beta oxidation are examples of substrate utilization defects. The sites of these biochemical defects are illustrated in Figure 37-4 (beta-oxidation defects) and Figure 37-5.

Classic pyruvate carboxylase deficiency, an autosomal-recessive condition, is life-threatening in infancy and is associated with hypotonia, psychomotor retardation, failure to thrive, and metabolic acidosis [De Vivo et al., 1977; Wang and De Vivo, 2009; Wang et al., 2008]. Approximately 50 patients with pyruvate carboxylase deficiency have been described. The severe French phenotype is associated with absence of the enzyme protein. These patients die in early infancy and have lactic acidosis, ketoacidosis, citrullinemia, hyperlysinemia, hyperammonemia, and aspartate depletion. The less severe, although equally fatal, North American phenotype is associated with severe mental and motor developmental delay, and death in late infancy or early childhood. A mutated protein is present in tissue with some residual enzyme activity. Three patients have had the laboratory features of the North American phenotype but were clinically spared, with normal mental and motor development between recurrent attacks of metabolic crisis. Consanguinity and mosaicism are quite evident in this group of patients, and mosaicism may mute the severity of the clinical phenotype [Wang et al., 2008].

Biotin deficiency affects pyruvate carboxylase, together with the other three biotin-dependent carboxylase reactions: propionyl-coenzyme A carboxylase, β-methylcrotonyl-coenzyme A carboxylase, and acetyl coenzyme A carboxylase. All carboxylation reactions occur in the mitochondria, except for the carboxylation of acetyl coenzyme A. Multiple carboxylase deficiency, an autosomal-recessive trait in all of its clinical presentations, is seen in early infancy as a result of holocarboxylase synthetase deficiency, or later in infancy as the result of biotinidase deficiency. These patients may have irregular breathing patterns, failure to thrive, alopecia, rash, immunologic disturbances, optic atrophy, hearing loss, seizures, and metabolic acidosis; they respond to high-dose biotin supplementation, particularly if they are treated early in the disease course.

Several hundred patients with defects involving the pyruvate dehydrogenase complex have been described [De Meirleir, 2002]. Most patients have a defect involving the E1 alpha subunit, and a male predominance is expected because the gene is located on the X chromosome. Two patients had mutations in the E1 beta subunit, inherited as an autosomal-recessive trait [Brown et al., 2004]. The neonatal presentation includes hypotonia, episodic apnea, convulsions, weak suck, dysmorphic features, lethargy, low birth weight, failure to thrive, and coma. Dysmorphic features include broad nasal bridge, upturned nose, micrognathia, low-set and posteriorly rotated ears, short fingers, short arms, simian creases, hypospadias, and anteriorly placed anus. The infantile phenotype has been described in about 100 patients, and usually is seen between 3 and 6 months of age, with psychomotor retardation, hypotonia, convulsions, episodic apnea, ataxia, pyramidal tract signs, lethargy, dysmorphic features, deceleration of head growth, ophthalmoplegia, optic atrophy, peripheral neuropathy, ptosis, dysphagia, deceleration of somatic growth, extrapyramidal signs, and cranial nerve palsies. Postmortem examinations were performed in 21 cases of the infantile form; of these, 81 percent were consistent with Leigh’s syndrome [DeVivo and Van Coster, 1991]. Seven male children had a benign phenotype, with fluctuating ataxia, post-exercise fatigue, transient paraparesis, and thiamine responsiveness. These children had normal mental and motor development between episodes. A high-fat diet has been recommended as an alternative source of acetyl coenzyme A in patients with pyruvate dehydrogenase deficiency [Wexler et al., 1997]. Thiamine, lipoic acid, and l-carnitine supplementation may be helpful in selected cases.

Seven genetically determined conditions have been described involving enzyme deficiencies in the beta-oxidation pathway (see Figure 37-4) [DiDonato and Taroni, 2004; Tein, 2003]. All are transmitted as autosomal-recessive traits. Five of the genetic defects (deficiencies of medium-chain acyl coenzyme A, short-chain acyl coenzyme A, long-chain acyl coenzyme A, electron transfer flavoprotein, and electron transfer flavoprotein-coenzyme Q oxidoreductase) involve the first step in beta oxidation. The other two conditions (long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency and short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency) involve the third step in beta oxidation. Two more enzyme defects involve the two membrane-bound enzymes of beta oxidation, very-long-chain acyl-CoA dehydrogenase (VLCAD), and the trifunctional enzyme protein. VLCAD deficiency, as carnitine palmitoyltransferase II deficiency, can present in infancy with hypoketotic hypoglycemia, hepatopathy, and cardiomyopathy, or in adult life as recurrent myoglobinuria [DiDonato and Taroni, 2003]. The gene (ACADVL) for VLCAD has been mapped to chromosome 17p12, and a relatively good genotype–phenotype relationship is emerging. The severe infantile form is associated with nonsense mutations, whereas the benign adult form is due to missense mutations compatible with some residual VLCAD activity.

Trifunctional enzyme protein is a hetero-octamer composed of four alpha subunits catalyzing the long-chain L-3-hydroxyacyl-CoA dehydrogenase (LCHAD) and the long-chain 2-enoyl-CoA hydratase (LCEH) activities, and four beta subunits catalyzing the long-chain 3-ketoacyl-CoA thiolase (LCKT) activity. Trifunctional enzyme protein deficiency had been diagnosed as LCHAD deficiency until more careful biochemical analysis revealed combined defects of LCHAD, LCEH, and LCKT activities. In fact, most patients with mitochondrial trifunctional protein deficiency have isolated LCHAD deficiency, with relative sparing of LCEH and LCKT activities. In infancy and early childhood, clinical features include recurrent attacks of hypoketotic hypoglycemia, cardiomyopathy, limb weakness, sensory neuropathy, pigmentary retinopathy, and hepatic dysfunction. Later in childhood, recurrent attacks of myoglobinuria are common. Two syndromes associated with LCHAD deficiency have been labeled HELLP (hemolysis, elevated liver enzymes, low platelets) and AFLP (acute fatty liver of pregnancy). The latter condition occurs in pregnant women carrying an affected fetus. A common mutation (G1528C) has been identified in the LCHAD domain of the alpha subunit. Full mitochondrial trifunctional protein deficiency, that is, combined severe deficiency of LCHAD, LCEH, and LCKT, is much rarer, and symptoms are similar but more severe than in isolated LCHAD deficiency. The molecular basis of full mitochondrial trifunctional protein deficiency is heterogeneous, with mutations in both alpha and beta subunits. Mutations have been identified in the HADHA and the HADHB genes, both located on chromosome 2p23 [DiDonato and Taroni, 2003].

Medium-chain acyl coenzyme A dehydrogenase deficiency commonly develops before 2 years of age and represents about 50 percent of the described cases associated with beta oxidation defects [Roe and Coates, 1989]. The metabolic crisis in medium-chain acyl coenzyme A dehydrogenase deficiency is provoked by fasting or intercurrent infection. The classic picture is that of episodic vomiting, hypotonia, obtundation, or coma. The patient is healthy between attacks. This condition may masquerade as systemic carnitine deficiency, recurrent Reye’s syndrome, or sudden infant death syndrome. Developmental disabilities, chronic seizure disorder, and proximal limb weakness represent long-term consequences of this condition. The molecular defect involves a gene on chromosome 1p31. In multiple studies of white patients, one mutation, A985G, was found in 90 percent of mutant medium-chain acyl coenzyme A dehydrogenase alleles. This mutation alters protein stability and assembly of the subunits into the holoenzyme tetramer.

The phenotypes of the other six defects resemble medium-chain acyl coenzyme A dehydrogenase deficiency, but there are some clinical caveats. Long-chain acyl coenzyme A dehydrogenase deficiency tends to be more severe in infancy, with a worse prognosis. Cardiac involvement is prominent, and patients may develop microcephaly, hypotonia, and pigmentary retinopathy. Limb weakness may be seen with all beta-oxidation defects except medium-chain acyl coenzyme A dehydrogenase deficiency. Short-chain acyl coenzyme A dehydrogenase deficiency may present in infancy with vomiting, hypoglycemia, developmental delay, limb weakness, muscle carnitine deficiency, and a lipid storage myopathy, or it may present in adulthood with progressive limb weakness and lipid storage myopathy [Turnbull et al., 1984]. Electron transfer flavoprotein or electron transfer flavoprotein-coenzyme Q oxidoreductase deficiency states may present the clinical picture of multiple acyl coenzyme A dehydrogenase deficiency (glutaricaciduria type II). The neonatal presentation may be associated with congenital anomalies, including facial dysmorphism with low-set ears, hypertelorism, high forehead, hypoplastic midface, rocker-bottom feet, muscle defects of the anterior abdominal wall, and anomalies of the external genitalia. These patients usually die during the first week of life. The neonatal presentation may occur without congenital anomalies, and these patients have hypotonia, tachypnea, metabolic acidosis, hepatomegaly, hypoglycemia, and a peculiar odor reminiscent of isovaleric acidemia. An adult phenotype may exhibit episodic vomiting, hypoglycemia, hepatomegaly, and limb weakness. Some patients with the milder form of multiple acyl coenzyme A dehydrogenase deficiency respond to riboflavin. Patients with LCHAD deficiency have persistent hepatic dysfunction, low serum carnitine concentrations, and a neuropathy or myopathy with recurrent myoglobinuria [Dionisi-Vici et al., 1991]. The short-chain 3-hydroxyacyl-CoA dehydrogenase phenotype appears to be similar to the LCHAD phenotype, but only three cases have been described [Tein et al., 1991].

Defects of the Krebs Cycle

Several partial defects of this cycle have been described. The first patient, described with dihydrolipoyl dehydrogenase deficiency, died at the age of 6 months with persistent metabolic acidosis and progressive neurologic symptoms, including respiratory difficulties, lethargy, hypotonia alternating with irritability, optic atrophy, and hyperreflexia [Robinson et al., 1977]. The organic acid profile includes elevations of α-ketoglutarate, branched-chain α-keto acids, pyruvate, and lactate. A few cases of α-ketoglutarate dehydrogenase deficiency have been described [Bonnefont et al., 1992]. This condition is devastating in infancy.

Fumarase deficiency is a progressive infantile encephalopathy with failure to thrive, hypotonia, microcephaly, and fumaric aciduria. This condition may be associated with enlarged cerebral ventricles and polyhydramnios in utero [De Meirleir, 2002; Zinn et al., 1986].

Succinate dehydrogenase is a critical step in the Krebs cycle and the first component of complex II in the respiratory chain (see Figure 37-5). Before the molecular era, several patients were described with defects of complex II and progressive encephalomyopathy. A mutation in the flavoprotein subunit of succinate dehydrogenase was identified in two sisters with Leigh’s syndrome [Bourgeron et al., 1995], the first example of a nuclear DNA mutation resulting in a respiratory chain disorder. A few more cases of Leigh’s syndrome were later associated with mutations in the same gene [DiMauro and Bonilla, 2004].

Defects of Oxidation–Phosphorylation Coupling

Luft’s disease (nonthyroidal hypermetabolism) is the singular example of a defect in this pathway. Only two cases have been reported, both sporadic and with negative family histories [Luft et al., 1962; DiMauro et al., 1976]. Onset is in adolescence, with fever, heat intolerance, profuse perspiration, polyphagia, polydipsia, resting tachycardia, and exercise intolerance. Both women died in middle age. Numerous ragged red fibers were present in skeletal muscle, but the underlying biochemical defect has not yet been elucidated.

Defects of the Respiratory Chain

The respiratory chain contains five functional units or complexes that are embedded in the inner mitochondrial membrane (see Figure 37-5). The respiratory chain is unique in that it is controlled by the nuclear and mitochondrial genomes. Complex II, unlike the other four complexes, is under the control of the nuclear genome only. As a result, any defects involving this complex are inherited as mendelian traits. Box 37-1 represents a molecular classification of defects of the respiratory chain. However, patients studied before the “molecular era” (1988) cannot be classified by these criteria. Classification is also complicated by the diversity of clinical presentations. Some patients with neurologic or neuromuscular symptoms are grouped together in the neurologic literature as examples of mitochondrial encephalomyopathies. Other patients have non-neurologic symptoms resulting from primary involvement of other organs, such as the liver, heart, kidney, bone marrow, pancreas, or gastrointestinal tract [DiMauro and Schon, 2003].

Complex I (reduced nicotinamide adenine dinucleotide phosphate-coenzyme Q reductase) is the largest complex of the respiratory chain, containing about 46 polypeptides and several nonprotein components. Seven of the polypeptides are encoded by mtDNA. Patients with complex I deficiency can be subclassified as examples of a myopathy or a multisystem disorder. The myopathy may develop in childhood or early adult life, and is manifested by exercise intolerance and limb weakness. The weakness may become prevalent as the disease progresses. These patients are often sporadic and harbor “somatic mutations” in mtDNA-encoded genes of complex I – that is, spontaneous de novo mutations in the mitochondrial genome occurring in the oocyte or in the embryo, but affecting myoblasts after germ-layer differentiation [DiMauro et al., 2003].

Patients with multisystem involvement of complex I deficiency can be subdivided into three clinical conditions. The first is manifested by a fatal infantile disorder with congenital lactic acidosis, weakness and hypotonia, developmental delay, and death resulting from cardiopulmonary failure [Hoppel et al., 1987; Moreadith et al., 1984; Robinson et al., 1986]. The molecular basis of this rapidly fatal infantile presentation is not known but may relate to mutations in nuclear genes (see later). The second presentation is associated with the clinical and neuroradiologic features of Leigh’s syndrome. In fact, complex I deficiency is one of the major causes of Leigh’s syndrome, although it may be overlooked because of the difficulty of documenting the biochemical defect in frozen tissues. This concept seems confirmed by studies of nuclear genes, revealing a number of mutations in children with Leigh’s syndrome or leukodystrophy [Smeitink and van den Heuvel, 1999; Triepels et al., 2001; Distelmaier et al., 2009]. Attention has recently been drawn to mutations in mtDNA-encoded complex I (ND) subunits, especially mutations in the ND5 gene, which appears to be a hotspot. These mutations have been associated with maternally inherited MELAS, MERRF, and Leigh’s syndrome phenotypes, or various overlap syndromes of these phenotypes [Liolitsa et al., 2003; Naini et al., 2005; Shanske et al., 2008].

Complex II (succinate-coenzyme Q reductase) contains only polypeptides encoded by the nuclear genome. There are few examples of complex II deficiency, and again, the clinical picture is that of an encephalomyopathy. Haller and colleagues [1991] and Schapira and associates [1990] described two patients with lack of succinate dehydrogenase stain in muscle biopsy specimens. One was a 22-year-old man with exercise intolerance and recurrent myoglobinuria; the other was a 14-year-old girl with exercise intolerance but no myoglobinuria. The complex II deficiency in these patients was accompanied by partial defects of complex I, complex III, and aconitase, and was attributed to defective importation of nonheme iron–sulfur clusters into mitochondria. As mentioned before, a few patients with Leigh’s syndrome and complex II deficiency had mutations in the gene encoding the flavoprotein subunit of the complex [Bourgeron et al., 1995; Parfait et al., 2000; Taylor et al., 1996]. The first defect in an assembly factor of complex II, a LYR-motif protein, has been associated with infantile leukoencephalopathy characterized by progressive psychomotor delay starting at 6–12 months of age, spastic quadriparesis, and dystonia. Proton nuclear MR spectroscopy reveals lactate peaks in the white matter [Ghezzi et al., 2009].

Coenzyme Q₁₀ serves as an electron shuttle between complexes I and II and complex III. There are three clinical presentations of what appears to be primary, autosomal-recessive coenzyme Q₁₀ deficiency. The myopathic form, first described in 1989 in two sisters [Ogasahara et al., 1989], has been reported in three more patients [DiGiovanni et al., 2001; Sobreira et al., 1997]. It is characterized by the triad:

The ataxic form is dominated by cerebellar ataxia and cerebellar atrophy, inconsistently accompanied by seizures, pyramidal signs, and mental retardation [Gironi et al., 2004; Lamperti et al., 2003; Musumeci et al., 2001]. Some of these patients have mutations in a gene involved in CoQ₁₀ biosynthesis and should be considered primary CoQ₁₀ defects [Lagier-Tourenne et al., 2008; Mollet et al., 2008]. Several primary infantile encephalomyopathic forms have been attributed to mutations in biosynthetic genes (COQ1, COQ2, and COQ9) and are often accompanied by nephrosis [Quinzii et al., 2006; López et al., 2006; Mollet et al., 2007; Duncan et al., 2009]. It is important to consider these syndromes because all patients, but especially those with the myopathic and the infantile forms, often respond dramatically to oral supplementation with coenzyme Q₁₀.

Complex III (coenzyme Q-cytochrome-c oxidoreductase) contains 11 subunits, one of which is encoded by mtDNA. Defects involving complex III are subdivided into generalized multisystem disorders manifested by limb weakness, exercise intolerance, and various neurologic signs, or a tissue-specific syndrome manifested either as a pure myopathy with onset in childhood or adolescence or as a pure cardiopathy (“histiocytoid cardiomyopathy”) manifested in early infancy. Some patients with the myopathic phenotype and without apparent maternal inheritance have mutations (“somatic” mutations; see earlier) in the cytochrome-b gene of mtDNA [Andreu et al., 1999; DiMauro and Bonilla, 2004]. A rapidly fatal infantile disorder, observed in Finland and dubbed GRACILE (growth retardation, aminoaciduria, cholestasis, iron overload, lactacidosis, and early death), was associated with mutations in an assembly protein (BCS1L) needed for the correct synthesis and function of complex III [Visapaa et al., 2002]. Mutations in the same nuclear gene (BCS1L) have also been found in children with Leigh’s syndrome [de Lonlay et al., 2001].

Complex IV (cytochrome-c oxidase, COX) contains 13 subunits, three of which are encoded by mtDNA. In keeping with the clinical presentations of respiratory chain defects, complex IV deficiency can be divided into a myopathic form and a multisystemic form [DiMauro and Bonilla, 2004].

There are two infantile myopathic presentations, a fatal infantile syndrome and a “benign” (reversible) infantile syndrome. The fatal infantile syndrome may be restricted to skeletal muscle and manifested by congenital lactic acidosis and progressive respiratory insufficiency [Bresolin et al., 1985]. These patients die before 1 year of age. Some of these patients may have kidney dysfunction with the de Toni–Fanconi syndrome, whereas others may have abnormalities of cardiac function. The cause of this condition is still unclear but some cases may be due to unrecognized mtDNA depletion. The reversible infantile myopathy is virtually indistinguishable from the fatal infantile form in the newborn period [DiMauro et al., 1983]. These patients also have respiratory insufficiency and severe lactic acidosis. Patients with the benign syndrome, however, spontaneously improve during subsequent months and are essentially normal by 1–3 years of age. The reversible COX-deficient myopathy has been clearly associated with a homoplasmic mtDNA mutation (m.14674T>C in tRNA^Glu), but it is clear that the phenotype is conditioned by a modifier nuclear gene [Horvath et al., 2009]. On the other hand, somatic mutations in mtDNA genes encoding COX I or COX III can cause exercise intolerance and recurrent myoglobinuria [Karadimas et al., 2000; Keightley et al., 1996].

The most common clinical presentation for the partial generalized defect of complex IV is Leigh’s syndrome [DiMauro and De Vivo, 1996; Van Coster et al., 1991]. These patients are clinically asymptomatic during early infancy. Somatic and neurologic symptoms subsequently develop after 6 months of age, and patients die around 4–5 years of age. This syndrome is inherited as an autosomal-recessive trait. Cytochrome-c oxidase-associated Leigh’s syndrome can be distinguished clinically from pyruvate dehydrogenase complex-associated Leigh’s syndrome and from maternally inherited Leigh’s syndrome due to mutations in the ATPase 6 gene of mtDNA [Santorelli et al., 1993]. Patients with cytochrome-c oxidase deficiency are clinically symptomatic in the neonatal or early infantile period, and die within 6–12 months of life. Patients with pyruvate dehydrogenase complex-associated Leigh’s syndrome or maternally inherited Leigh’s syndrome commonly have seizures and recurrent apnea; in addition, patients with maternally inherited Leigh’s syndrome may have retinitis pigmentosa, which – when present – is pathognomonic. Patients with cytochrome-c oxidase-associated Leigh’s syndrome have a slower clinical course, deceleration of head growth, and peripheral neuropathy. Seizures are uncommon in patients with cytochrome-c oxidase-associated Leigh’s syndrome. In 1998, two groups discovered that most cases of cytochrome-c oxidase-associated Leigh’s syndrome were due to mutations in a cytochrome-c oxidase assembly gene, SURF1 [Tiranti et al., 1998; Zhu et al., 1998]. This finding has greatly improved genetic counseling and has made prenatal diagnosis possible. Mutations in several other ancillary proteins needed for cytochrome-c oxidase assembly have been found in children with Leigh-like encephalopathy and involvement of one other tissue, liver, heart, or kidney. Thus, mutations in the SCO2 gene cause cardioencephalopathy [Papadopoulou et al., 1999]; in general, the cardiopathy predominates and causes early death, but a few cases presented as spinal muscular atrophy with cardiopathy [Salviati et al., 2001]. Mutations in COX15 also cause cardioencephalopathy [Antonicka et al., 2003], whereas mutations in SCO1 cause hepatoencephalopathy [Valnot et al., 1999], and mutations in COX10 cause nephroencephalopathy [Valnot et al., 2000]. The reasons for the selective involvement of specific tissues besides brain remain unclear.

Alpers’ disease [Alpers, 1931] has been associated with complex IV deficiency, but it is now clear that typical cases of Alpers–Huttenlocher syndrome with hepatocerebral presentation are due to mtDNA depletion due to mutations in the POLG gene, which encodes the mitochondrial polymerase-γ [Naviaux et al., 1999; Kurt et al., 2010].

Complex V (mitochondrial ATP synthase) is composed of 12–14 subunits, two of which are encoded by mtDNA. The most important causes of complex V deficiency seem to be mutations affecting the ATPase 6 gene of mtDNA. Two mutations at the same nucleotide, G8993T and G8993C, cause syndromes of different severity. Depending on the abundance of mutant mtDNAs (mutation load), the T8993G mutation can cause maternally inherited Leigh’s syndrome in infants (mutation load around 90 percent), or a milder encephalomyopathy called neuropathy, ataxia, and retinitis pigmentosa in young adults. (The two conditions can coexist in the same family.) The T8993C mutation causes milder symptoms in both children and adults, often associated with familial bilateral striatal necrosis. In accordance with the more severe presentation, there is biochemical evidence of severely impaired ATP production in cells harboring the T8993G mutation, whereas ATP production is essentially normal in cells harboring the T8993C mutation (whose pathogenic mechanism remains unclear) [Schon et al., 2002]. A few mutations elsewhere in the ATPase 6 gene have been associated with encephalomyopathy and familial bilateral striatal necrosis [DiMauro and Schon, 2003].

The first nuclear defect impairing complex V function has been described. It involves a gene (ATP12) encoding an assembly protein, and it is accompanied by congenital lactic acidosis and a fatal infantile multisystemic disease [De Meirleir et al., 2004]. Mutations in another nuclear gene (TMEM70) have been associated with complex V deficiency in children of Roma ethnic origin, with multisystem involvement, lactic acidosis, and 3-methylglutaconic aciduria [Cizkova et al., 2008].

Defects of Protein Importation

The protein importation process is energy-dependent and needs the proteins to be unfolded before traversing the mitochondrial membranes, then refolded before reaching their final destination. This process requires a complex machinery involving leader peptides, chaperonins, translocases in the outer and inner membranes, and proteases [Bolender et al., 2008]. Relatively few human diseases have been attributed to dysfunction of mitochondrial protein import, possibly because of the devastating consequences that disruption of the general import machinery would have. Most of these mutations involve leader peptides. One affected the enzyme methylmalonyl-CoA mutase and accounted for one form of methylmalonicaciduria [Ledley et al., 1990]. Another affected the leader sequence of the E1 alpha subunit of the pyruvate dehydrogenase complex in a child with pyruvate dehydrogenase deficiency and Leigh’s syndrome [Takakubo et al., 1995]. One cause of primary hyperoxaluria type I was associated with the misdirecting of a mutated alanine glyoxylate aminotransferase enzyme protein to the mitochondrial matrix rather than to the peroxisome [Purdue et al., 1990]. The enzyme, although catalytically intact, was unable to perform its biologic function because it had been misdirected to the wrong intracellular compartment. Some defects of ornithine transcarbamoylase and carbamylphosphate synthetase I, the two urea cycle enzymes located in the mitochondrion, may be additional examples of mitochondrial protein importation defects. Two children have been described with a defect of heat-shock protein 60, a chaperonin needed for the correct folding and assembly of imported proteins. One infant female, the product of a consanguineous marriage, presented at birth with dysmorphic features, hypotonia, respiratory insufficiency, and severe lactic acidosis, and died 2 days later [Agsteribbe et al., 1993]. The second child also had dysmorphic facial features, congenital hypotonia, and failure to thrive, and she died at 4.5 years of age [Briones et al., 1997]. The molecular bases of these biochemical defects were not established. However, two disorders have been associated with mutations in components of the transport machinery. The first is an X-linked disease called deafness-dystonia syndrome (Mohr–Tranebjaerg syndrome), characterized by neurosensory hearing loss, dystonia, cortical blindness, and psychiatric symptoms; it is due to mutations in the TIMM8A gene, which encodes the deafness-dystonia protein (DDP1), a component of the transport machinery [Roesch et al., 2002]. The second is an autosomal-dominant form of hereditary spastic paraplegia due to mutations in the chaperonin heat-shock protein 60 [Hansen et al., 2002].

Defects of Intergenomic Signaling

Two clinical syndromes result from primary molecular defects involving the nuclear genome but affecting either the quality or the quantity of mtDNA. The first condition is associated with multiple deletions of mtDNA and was first described by Zeviani and colleagues [1989]. Patients with multiple mtDNA deletions have progressive external ophthalmoplegia, which can be the dominating feature or can be associated with multisystemic involvement. Patients with autosomal-dominant progressive external ophthalmoplegia usually become symptomatic in the third decade of life, with ophthalmoplegia, progressive limb weakness, bilateral cataracts, depression, parkinsonism, and precocious death. At least four genes affect mtDNA maintenance and have been associated with multiple mtDNA deletions:

ANT1, encoding the adenine nucleotide translocator

PEO1 (Twinkle), encoding an mtDNA helicase

POLG, encoding the mtDNA polymerase γ

OPA1, encoding a protein involved in mitochondrial fusion [Kaukonen et al., 2000; Spelbrink et al., 2001; Van Goethem et al., 2001; Hudson et al., 2008].

A special form of autosomal-recessive progressive external ophthalmoplegia is the mitochondrial neurogastrointestinal encephalomyopathy syndrome, in which gastrointestinal dysfunction (chronic diarrhea, intestinal pseudo-obstruction), peripheral neuropathy, and leukodystrophy are prominent [Nishino et al., 2000]. In both conditions, there are ragged red fibers in the muscle biopsy specimen. The molecular bases of these conditions are being understood at a rapid pace. Mutations in the gene for thymidine phosphorylase (TP) are responsible for mitochondrial neurogastrointestinal encephalomyopathy [Nishino et al., 1999].

The second example of an intergenomic signaling defect is the mtDNA depletion syndrome with variable tissue expression. This condition was first described by Moraes and colleagues [1991] as an autosomal-recessive trait associated with a quantitative reduction of mtDNA copy number. Two major phenotypes have emerged, one dominated by myopathy, the other by liver and brain involvement. The myopathic form of mtDNA depletion syndrome may present as a congenital and rapidly fatal myopathy (generally associated with very low residual mtDNA in muscle) or as a childhood progressive myopathy (associated with higher residual amounts of mtDNA). The “hepatocerebral” form can present as fulminant hepatopathy of infancy or as a multisystem disorder affecting predominantly brain and liver (not unlike Alpers’ syndrome). However, symptoms overlap; for example, infants with the “myopathic” phenotype sometimes mimic spinal muscular atrophy, and mtDNA depletion should be considered in children with spinal muscular atrophy but without mutations in the SMN gene. Mutations in nine nuclear genes, all but one involved in mitochondrial nucleotide homeostasis, have been associated with mtDNA depletion syndrome, although they do not explain all cases [Poulton et al., 2009]. Mutations in the gene encoding thymidine kinase (TK2) are more common in patients with the myopathic presentation [Saada et al., 2001], whereas mutations in the gene encoding deoxyguanosine kinase (dGK) predominate in patients with the hepatocerebral presentation [Mandel et al., 2001].

Defects of the Lipid Milieu of the Inner Mitochondrial Membrane

This pathogenetic mechanism is illustrated by Barth’s syndrome, an X-linked recessive disorder characterized by mitochondrial myopathy, cardiopathy, and leukopenia [Barth et al., 1999]. Mutations in the tafazzin (TAZ) gene are responsible for Barth’s syndrome, and TAZ encodes a family of proteins, named tafazzins, that are homologous to phospholipid acyltransferases [Bione et al., 1996]. Analysis of phospholipids in target tissues from patients with genetically proven Barth’s syndrome found markedly decreased levels of cardiolipin, as well as altered cardiolipin composition [Schlame et al., 2002, 2003]. Cardiolipin is the main phospholipid component of the inner mitochondrial membrane, where it not only functions as a “scaffold” but also interacts with respiratory chain complexes. Thus, alterations of cardiolipin amount and composition can affect respiratory function.

Defects of Mitochondrial Motility, Fusion, and Fission

Mitochondria are dynamic organelles forming complex tubular structures in many cells, which requires constant fusion of the mitochondrial tubules, balanced by fission. Also, individual mitochondria move in the cell – sometimes covering considerable distances, as in axons – propelled by energy-requiring dynamins along cytoskeletal microtubular rails [Chan, 2006].

Mutations in a gene (OPA1) encoding a dynamin-related guanosine triphosphatase have been associated with an autosomal-dominant form of optic atrophy (DOA) resulting in young-onset blindness, similar to Leber’s hereditary optic neuropathy [Alexander et al., 2000; Delettre et al., 2002], as well as with progressive external ophthalmoplegia and multiple mtDNA deletions (see above).

Mutations in the gene (MFN2) encoding mitofusin 2, an outer membrane fusion protein, cause Charcot–Marie–Tooth 2A, a severe and early-onset form of axonal neuropathy [Zuchner et al., 2004; DiMauro and Schon, 2008].

Sporadic Large-Scale Rearrangements

Kearns–Sayre syndrome is the prototype of this group of diseases [Moraes et al., 1989; Zeviani et al., 1988]. Males and females are affected in roughly equal numbers, and the risk of maternal transmission is exceedingly small [Chinnery et al., 2004]. The dominant clinical features include progressive eye signs, such as ptosis, restricted eye movements, and pigmentary retinopathy. Neurologic symptoms include cerebellar ataxia, mental retardation, and episodic coma. Seizures are infrequent and are usually associated with hypoparathyroidism. Complete heart block may lead to sudden death. Insertion of a pacemaker is life-saving. Short stature and hearing loss are common. Endocrine disturbances include diabetes mellitus, hypoparathyroidism, and isolated growth hormone deficiency. Ragged red fibers are found in almost all affected patients. Calcification of the basal ganglia usually occurs in the setting of hypoparathyroidism. The surprisingly rare incomplete forms of Kearns–Sayre syndrome demonstrate progressive external ophthalmoparesis as an invariant clinical sign. Some cases have unusual clinical presentations, such as renal tubular acidosis, whereas others may overlap with clinically distinct syndromes, such as MELAS [Eviatar et al., 1990; Zupanc et al., 1991].

Pearson’s syndrome is a non-neurologic disorder of infancy characterized by pancytopenia, disturbed pancreatic exocrine function, and liver abnormalities [Pearson et al., 1979; Rötig et al., 1990]. The few survivors of this infantile condition may later develop clinical features of Kearns–Sayre syndrome [Blaw and Mize, 1990; Larsson et al., 1990; McShane et al., 1991]. The phenotypic transformation from Pearson’s syndrome to Kearns–Sayre syndrome is an example of tissue-specific modifications of mitochondrial heteroplasmy (mitotic segregation).

Point Mutations Affecting Protein-Coding Genes

There are six clinical conditions that are maternally inherited and associated with point mutations in mtDNA protein-coding genes. Two of these, Leber’s hereditary optic neuropathy (LHON) and the syndrome of neuropathy, ataxia, and retinitis pigmentosa (NARP), affect structural genes in the mitochondrial genome. Lactic acidosis is usually absent in LHON and may be absent in NARP; ragged red fibers are conspicuously absent in the skeletal muscle biopsy specimens from patients with both conditions.

LHON is dominated clinically by the sudden onset of visual loss in a young adult and is more common in men [Carelli et al., 2006]. The peak age at onset is between 20 and 24 years, although children as young as 5 years have been described. Visual loss is bilateral in the majority of cases, although the condition may first develop in one eye, followed by involvement of the second eye in weeks or months. The presentation is that of a retrobulbar neuropathy, often associated with disc edema and subtle alterations of retinal vessels. The male predominance suggests an X-linked factor that modulates the expression of the mtDNA point mutation [Hudson et al., 2005; Shankar et al., 2008]. The condition is relatively static after the subacute visual loss. Other neurologic and psychiatric symptoms have been described in patients and their family members, signifying the global nature of the molecular defect. Variable findings have included hyperreflexia, Babinski signs, incoordination, peripheral neuropathy, and cardiac conduction abnormalities. Three mutations, all of them in genes encoding subunits of complex I (G3460A in ND1, G1178A in ND4, and T14484C in ND6) are considered “primary”: that is, capable of causing LHON in and by themselves.

The second point mutation affecting an mtDNA structural gene results in NARP (see earlier). Holt and associates [1990] described four family members of three successive generations with a variable combination of retinitis pigmentosa, ataxia, developmental delay, dementia, seizures, proximal limb weakness, and sensory neuropathy. This maternally transmitted condition was associated with an mtDNA point mutation involving the gene for subunit 6 of mitochondrial H⁺-ATPase. The most severely affected member was 3 years old. She had exhibited reduced fetal movements. Development was delayed, and a pigmentary retinopathy was noted. She had increased limb tone, hyperreflexia, bilateral Babinski signs, and generalized ataxia. As already mentioned, the T8993G mutation in the ATPase 6 gene of mtDNA has been recognized as one important cause of Leigh’s syndrome [DiMauro and Schon, 2003]. Curiously, a different mutation at the very same nucleotide (T8993C) causes a milder clinical phenotype and a less severe impairment of ATP synthesis in mitochondria isolated from cultured fibroblasts [Santorelli et al., 1996]. Mutations in the ATPase 6 gene of mtDNA appear to be associated with various syndromes characterized by the neuroradiologic features of Leigh’s syndrome or bilateral striatal necrosis [DiMauro and Schon, 2003].

Point Mutations Affecting Synthetic Genes

There are more than 200 pathogenic point mutations but the two most common clinical conditions that are maternally inherited and associated with mtDNA point mutations affecting transfer RNA genes are mitochondrial encephalopathy with lactic acidosis and strokelike episodes (MELAS), and myoclonus epilepsy and ragged red fibers (MERRF). MELAS was first described in 1984 [Pavlakis et al., 1984] and appears to be the most common mtDNA-related disease. Most patients become symptomatic before the age of 40 years, and 90–100 percent of patients have normal early development, followed by the onset of exercise intolerance, strokelike episodes, seizures, and dementia. Almost all patients have lactic acidosis and had ragged red fibers in skeletal muscle biopsy specimens [Kaufmann et al., 2009]. Recurrent migraine-like headaches preceded by nausea and vomiting are common, as are hearing loss, short stature, learning difficulties, hemiparesis and hemianopia, and limb weakness. The cerebrospinal fluid protein concentration is normal in half of the patients and only mildly elevated in the other half. One-third of patients have basal ganglia calcifications. Seizures are common and often precede the strokelike events. Progressive external ophthalmoparesis was noted in approximately 10 percent of cases. The A3243G mutation in the tRNA^leu(uur) gene of mtDNA is responsible for about 80 percent of MELAS cases worldwide [Goto et al., 1990]. Several other mutations in the same gene have been associated with MELAS [Kaufmann et al., 2004], as well as increasing numbers of mutations in structural genes of mtDNA [Liolitsa et al., 2003; Manfredi et al., 1995; Naini et al., 2005; Shanske et al., 2008].

MERRF was first described in 1980 [Fukuhara et al., 1980]. The major clinical features include cerebellar syndrome, generalized convulsions, myoclonus, dementia, hearing loss, impaired deep sensation, and a positive family history consistent with maternal inheritance. Other findings may include optic atrophy and short stature [DiMauro et al., 2002]. In 1990, Shoffner and colleagues [1990] described a point mutation (A8344G) involving the mtDNA gene for tRNA^lys. Two more mutations in the same gene (T8356C and G8363A) are less frequent causes of typical MERRF. Only one mutation in a different gene (G611A in the tRNAPhe gene) has been reported to cause typical MERRF [Mancuso et al., 2004].