Clinical features

Acid maltase deficiency (AMD, glycogenosis type II, GSD type II) results in three very different clinical presentations [DiMauro and Lamperti 2001]:

Slonim et al. [2000] have described a subgroup of patients who share muscle morphological features with the neonatal-onset infantile form patients, but differ from typical Pompe’s disease patients in that the heart is not involved and survival is longer.

Laboratory data

All forms of AMD demonstrate an increase in serum CK. Electromyography (EMG) reveals myopathic abnormalities and may also show fibrillation potentials, positive waves, complex repetitive discharges, and myotonic discharges [DiMauro and Lamperti, 2001]. These are more frequently demonstrated in the paraspinal muscles. The electrocardiogram (EKG) demonstrates characteristic, although not specific, changes, including a short PR interval, giant QRS complexes, and signs of biventricular hypertrophy in infantile AMD. In adults, there is markedly reduced vital capacity.

Pathology

In infantile AMD, glycogen accumulation is evident in all tissues, most notably the heart. There is significant involvement of the anterior horn cells of the spinal cord and of brainstem nuclei, leading to severe weakness and fasciculations. Glycogen accumulation in Schwann cells is seen in peripheral nerve biopsies. There is a vacuolar myopathy on muscle biopsy in all three forms of AMD [DiMauro et al., 1992]. In the infantile form, all muscle fibers contain many vacuoles, which often coalesce into a lacework pattern and contain periodic acid–Schiff (PAS)-positive, diastase-digestible material and stain for acid phosphatase. Electron microscopy reveals excess glycogen either within lysosomal vacuoles or free in the cytoplasm.

Inheritance

The three forms of AMD are allelic disorders that are transmitted as autosomal-recessive traits. The gene encoding acid maltase is assigned to chromosome 17. Acid maltase activity can be measured in cultured amniocytes for the purposes of prenatal diagnosis.

Biochemistry and molecular genetics

Although the enzyme defect is generalized and can be documented in lymphocytes [Shanske and DiMauro, 1981] or in cultured skin fibroblasts, biochemical measurement of acid maltase in muscle establishes the diagnosis. It is not entirely clear why symptoms are mainly confined to muscle in the childhood and adult forms. However, differences in clinical expression may relate, in part, to the amounts of residual activity [Mehler and DiMauro, 1977; Reuser et al., 1987; Van Der Ploeg et al., 1988]. Second, genetic heterogeneity has also been demonstrated. In a study of 14 patients, Martiniuk et al. [1990a, 1990b] found that messenger RNA (mRNA) was lacking in 5 of 10 infantile AMD cases and was present in all 4 adult cases, although it was shorter in 2 cases. The defective lysosomal enzyme, α-glucosidase, is encoded by a gene on chromosome 17 [D’Ancona et al. 1979; Weil et al., 1979; Solomon et al., 1979], and over 55 mutations have been identified in patients with the three variants [DiMauro and Lamperti, 2001]. The genotype–phenotype correlation is hard to establish due to the frequent compound heterozygosity. However, there is good correlation between the severity of the mutation and the severity of the clinical phenotype. Therefore, deletions and nonsense mutations are usually associated with the infantile variant, whereas “leaky” mutations, such as the IVS1(-13T>G) splice-site mutations, are associated with the adult-onset variant [Hirschhorn, 1995]. Childhood AMD is often the result of compound heterozygosity [Huie et al., 1998], but has also been associated with at least one homozygous, apparently specific mutation [Adams et al., 1997]. A reliable and rapid first-tier test for screening patients suspected of having Pompe’s disease has been provided, using a fluorometric dried blood spot-based α-glucosidase activity assay [Goldstein et al., 2009]. The measurement of acid α-glucosidase activities in dried blood spots using tandem mass spectrometry has been found suitable for high-throughput analysis and newborn screening for Pompe’s disease [Dajnoki et al., 2008].

Treatment

Clinical trials with lysosome-stabilizing agents and activators of glycogenolysis have been unsuccessful in Pompe’s disease [DiMauro et al., 1992]. Recent experimental work with small molecule pharmacological chaperones may hold promise for future clinical trials [Flanagan, 2009]. Enzyme replacement looks promising, particularly for patients with childhood and adult AMD. Recombinant human α-glucosidase, obtained from milk of transgenic rabbits and injected into knockout mice lacking acid maltase, corrected the enzyme defect fully in all tissues but brain [Bijvoet et al., 1999]. Amalfitano et al. [2001] reported the results of a phase I/II open-label, single-dose study of recombinant human α-glucosidase infused intravenously twice weekly in three infants with infantile GSD type II. The results of more than 250 infusions showed that recombinant human α-glucosidase was generally well tolerated. There were steady decreases in heart size and maintenance of normal cardiac function for more than 1 year observed in all three infants. These infants lived past the critical age of 1 year and also show improvements in skeletal muscle function. In a 52-week trial with acid glucosidase alpha, there was marked improvement in the cardiomyopathy, ventilatory function, and overall survival among 18 children under 7 months of age with infantile Pompe’s disease [Kishnani et al., 2009]. Sixteen of the 18 patients enrolled in an extension study continued to receive alglucosidase alpha for up to a total of 3 years and showed reduction in the risk of death by 95 percent, and reduced risk of invasive ventilation or death by 91 percent, compared to an untreated historical control group, over the entire study period. The cardiomyopathy continued to improve and 11 patients learned and sustained substantial motor skills. In another study of late-onset AMD, three patients (aged 11, 16, and 32 years), all wheelchair-dependent and two ventilator-dependent with a history of deteriorating pulmonary function, received 3 years of treatment with weekly infusions of recombinant α-glucosidase from rabbit milk, and had stabilized pulmonary function and reported less fatigue [Winkel et al., 2004]. The youngest and least affected patient showed significant improvement of skeletal muscle strength and function, and, after 72 weeks of treatment, could walk without support. There have also been promising gene therapy results, both in vitro and in vivo, using E1-deleted recombinant adenovirus encoding human α-glucosidase. Transduction of the viral construct into enzyme-deficient human fibroblasts in culture resulted in a dose-dependent return of acid maltase activity, which was localized within lysosomes [Pauly et al., 1998]. Equally encouraging results were obtained by injecting a similar viral construct into the pectoral muscle of acid maltase-deficient Japanese quails that are an avian model of AMD [Tsujino et al., 1998]. Individuals with childhood or adult AMD may require ventilatory assistance. A high-protein diet has improved strength and respiratory function in some but not all patients [DiMauro et al., 1992; Slonim et al., 1983].

Clinical features

Phosphorylase b kinase (PBK) deficiency (glycogenosis type VIII) may be divided into four clinical syndromes [DiMauro and Tsujino, 1994]. These can be differentiated by inheritance and tissue distribution as follows:

Laboratory data

The resting serum CK is variably increased in most patients. The EMG may be normal or may show nonspecific myopathic changes. Muscle biopsy may be normal or show subsarcolemmal accumulations of glycogen, primarily located in type 2B fibers. The PPL stain is normal. There are pools of free, normal-looking glycogen particles on electron microscopy [DiMauro and Tsujino, 1994].

Biochemistry and molecular genetics

PBK participates in glycogen metabolism regulation by acting on the two main enzymes involved in glycogen synthesis and degradation: namely, glycogen synthetase and PPL [Heilmeyer, 1991]. PBK converts PPL from the less active b form to the more active a form, and simultaneously converts glycogen synthetase from a more active, dephosphorylated form to a less active, phosphorylated form. Thus, when glycogen synthesis is turned on, glycogen degradation is turned off, and vice versa. PBK is a multimeric enzyme composed of four different subunits (alpha, beta, gamma, delta) [DiMauro and Tsujino, 1994]. The alpha and beta subunits are regulatory, the gamma subunit is catalytic, and the delta subunit is identical to calmodulin and confers calcium sensitivity to the enzyme [DiMauro and Lamperti, 2001]. In addition, there are two isoforms for the alpha subunit (muscle and liver), which are encoded by genes on the X chromosome. The two isoforms for the gamma subunit (muscle and testis) and the beta subunit are encoded by autosomal genes. Glycogen is normal or moderately increased in patients with myopathy, and the PBK activity is absent or markedly decreased [DiMauro and Tsujino, 1994].

It is not easy to account for the tissue specificity in the different clinical variants. Two distinct genes have been identified. One gene (PHKA1) is on the proximal long arm at Xq13, which encodes a muscle isozyme; the other gene (PHKA2) is on the distal short arm of the X chromosome at Xp22.2–p22.1, which encodes a nonmuscle isozyme, and is in the same region to which the gene for X-linked liver glycogenosis resulting from PBK deficiency has been mapped [Willems et al., 1991]. Tissue-specific isozymes may be the result of alternative RNA splicing. DiMauro and Tsujino [1994] suggest that mutations in tissue-specifically expressed exons of the beta subunit [Harmann et al., 1991] could explain the apparently autosomal-recessive forms of myopathy and fatal infantile cardiomyopathy.

Treatment

Although there is no specific therapy for PBK deficiency, a high-protein diet may be helpful.

Clinical features

Myophosphorylase deficiency (type V glycogenosis, or McArdle’s disease) is a rare disease but is an important cause of recurrent myoglobinuria. Exercise intolerance generally starts in childhood, but overt episodes of muscle cramping and myoglobinuria develop later in adolescence. Brief isometric contraction (e.g., lifting heavy objects) and less intense but sustained dynamic exercise (e.g., climbing stairs) are the two primary precipitants. Approximately 50 percent of these patients experience episodes of muscle necrosis and myoglobinuria after exercise, while 27 percent develop acute renal failure. Although there is usually complete functional recovery after the episode of myoglobinuria, about one-third of patients have fixed mild proximal greater than distal weakness, which is seen more commonly in older patients [DiMauro and Bresolin, 1986]. The diagnosis is usually made in the second or third decade of life. There may be a marked variation in the severity of symptoms. In some, progressive weakness may begin late in life (sixth decade), with no history of cramps or pigmenturia [DiMauro and Bresolin, 1986; Pourmand et al., 1983]. The cumulative effect of recurrent muscle damage over the years may explain the appearance of fixed weakness in older individuals. In contrast, there have been four unique childhood cases with severe generalized muscle and respiratory weakness noted at or soon after birth, with death in infancy [DiMauro and Hartlage, 1978; DiMauro and Tsujino, 1994; Milstein et al., 1989]. A hypotonic 1-year-old infant has been described with repeated episodes of hyper-creatine kinase-emia during febrile episodes [Ito et al., 2003].

Laboratory data

Between episodes of myoglobinuria, the serum CK is variably increased in 93 percent of cases. This serves as a differentiating feature from CPT deficiency, in which the resting CK generally is normal. EMG between episodes of myoglobinuria demonstrates fibrillations, myotonic discharges, and positive waves in up to 50 percent of patients, suggesting a mild myopathy [DiMauro and Tsujino, 1994]. There is a lack of cytoplasmic acidification on phosphorus-31 nuclear magnetic resonance (³¹P-NMR) spectroscopy during aerobic or ischemic exercise, as well as a greater than normal drop of the phosphocreatine/inorganic phosphate ratio [Argov and Bank, 1991; Ross et al., 1981].

Inheritance

There is an unexplained marked male predominance, despite the autosomal-recessive pattern of inheritance. The muscle isoform gene has been localized to chromosome 11 [Lebo et al., 1984]. Two families with apparent autosomal-dominant transmission [Chui and Munsat, 1976; Schimrigk et al., 1967] may be explained on the basis of subsequent generations of homozygotes and manifesting heterozygotes, in whom the residual activity was below a critical threshold [Papadimitriou et al., 1990; Schmidt et al., 1987]. In a third family, Tsujino et al. [1993a] demonstrated that the affected mother was a compound heterozygote carrying two different point mutations in the PPL gene, the unaffected father was heterozygous for a third point mutation, and the three affected children were compound heterozygotes.

Muscle biopsy

There may or may not be focal accumulations of glycogen between myofibrils and in the subsarcolemmal regions on PAS stain. The PPL stain [Takeuchi and Kuriaki, 1955] shows no staining in muscle fibers, in contrast to normal staining of the smooth muscle in the walls of intramuscular vessels. DiMauro and Tsujino [1994] pointed out that a positive histochemical reaction may be seen in McArdle’s disease under the following two conditions:

A false-positive reaction in regenerating fibers is due to expression of a different isozyme in immature muscle cells [DiMauro et al., 1978; Sato et al., 1977]. Accumulation of normal-looking glycogen beta particles under the sarcolemma and between myofibrils and myofilaments may be seen on electron microscopy [DiMauro and Tsujino, 1994].

Biochemical considerations

PPL (alpha-1,4-glucan orthophosphate glycosyl transferase) initiates glycogen breakdown by removing 1,4-glucosyl residues phosphorolytically from the outer branches of the glycogen molecule, with liberation of glucose-1-phosphate until the peripheral chains have been shortened to approximately four glucosyl units. The debranching enzyme catalyzes further breakdown. The combined action of these two enzymes degrades glycogen to glucose-1-phosphate (approximately 93 percent) and glucose (approximately 7 percent). Muscle PPL exists as an active phosphorylated alpha form and a less active dephosphorylated beta form. The muscle PPL activity is undetectable in most or up to 10 percent of normal residual activity in affected patients [DiMauro and Tsujino, 1994]. Glycogen accumulation is moderate (about 2-fold) or normal. The glycogen structure is normal. There is no enzyme protein in muscle immunologically detectable by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblot, and enzyme-linked immunosorbent assay (ELISA) in the majority of patients [McConchie et al., 1991; Servidei et al., 1988b]. Normal mature human muscle has a single PPL isozyme, whereas cardiac muscle and brain have three different isozymes on PAGE, including:

Lack of the muscle isozyme therefore should cause partial defects in the heart and brain. A defect of the brain isozyme would be expected to cause heart and brain involvement [DiMauro and Tsujino, 1994].

Pathophysiology

The pathophysiology of exercise intolerance in McArdle’s disease involves a combination of interconnected factors related to impaired ATP production from both aerobic and anaerobic glycolysis. The decrease of ATP generated during anaerobic glycolysis may affect muscle ATPases selectively [James et al., 1996], including the Na⁺K⁺-ATPase. This inhibition, together with a decrease in Na⁺K⁺ pump numbers, may lead to excessive increases in blood and extracellular potassium, which typically are seen during exercise in McArdle’s disease [Haller et al., 1998a]. These changes can explain, in turn, the sarcolemmal inexcitability during repetitive neural stimulation [Dyken et al., 1967]. Furthermore, the lack of lactic acid production and intracellular acidification during exercise alters the equilibrium of the CK reaction, resulting in an exaggerated rise of ADP during exercise. The excessive intracellular ADP is converted to AMP, inosine monophosphate, ammonia, and adenine nucleotide degradation products, including inosine, hypoxanthine, and uric acid [Mineo et al., 1987]. Premature fatigue may be due to impaired excitation–contraction coupling, mediated by excessive accumulation of ADP [Lewis and Haller, 1991a]. Oxidative metabolism is also affected in McArdle’s disease because the block in glycogenolysis impairs production of pyruvate, which is a major anaplerotic substrate for the Krebs cycle [Sahlin et al., 1995]. In patients with McArdle’s disease, oxygen extraction and maximal oxygen uptake are decreased; these can be restored partially by administration of intravenous glucose [Haller et al., 1985].

Molecular genetics

The three PPL isozyme genes have been cloned, sequenced, and localized to different chromosomes [Billingsley et al., 1994; Lebo et al., 1984; Newgard et al., 1986, 1988; Rao et al., 1992] (see Table 94-1). Tsujino et al. [1993a] identified three different point mutations in the muscle isozyme gene: a C to G mutation in codon 49 of exon 1 (converting an arginine to a stop codon), a G to A mutation in codon 204 of exon 5 (converting a glycine to a serine), and an A to C mutation in codon 542 of exon 14 (converting a lysine to a threonine). In an analysis of 32 patients, 15 were homozygous for the first mutation and 12 were compound heterozygotes [Tsujino et al., 1993a]. More than 100 mutations have been identified in the muscle isozyme gene, which will facilitate carrier identification [DiMauro and Tsujino, 1994; Tsujino et al., 1993a, 1994a, 1994b, 1994c; DiMauro and Lamperti, 2001; Vissing et al., 2009]. These include missense, nonsense, and splice-junction mutations. The most common mutation in Europe and North America is the Arg49Stop, which accounts for 81 percent of the alleles in British patients [Bartram et al., 1993] and 63 percent of alleles in a survey of U.S. patients [El-Schahawi et al., 1996]. The high frequency of this mutation, on at least one allele, has facilitated molecular diagnosis of blood samples from patients with suspected McArdle’s disease. However, it is important to realize that the frequency of different mutations varies in different ethnic groups. The frequency of the Arg49Stop mutation appears to decrease from north to south in Europe, from 81 percent of alleles in the United Kingdom [Bartram et al., 1993] to 56 percent in Germany [Vorgerd et al., 1998] and 32 percent in Italy [Martinuzzi et al., 1996].

To date, there has been no clear genotype–phenotype correlation in McArdle’s disease. For example, the common homozygous Arg49Stop mutation was also present in an infant with the fatal myopathic variant [Tsujino et al., 1993a], and in a child who died of sudden infant death syndrome [El-Schahawi et al., 1997]. This lack of clear genotype–phenotype correlation was also documented in a study of 54 Spanish patients with McArdle’s disease from 40 unrelated families [Martin et al., 2001]. One possible explanation for increased severity of clinical presentation would be the presence of additional gene defects, such as myoadenylate deaminase deficiency [Rubio et al., 1997a; Tsujino et al., 1995a]. Recently, two patients with atypical McArdle’s disease, who carried common mutations on one allele and novel splice mutations in introns 3 and 5 on the other allele, demonstrated minimal phosphorylase activity that ameliorated the typical phenotype by augmenting muscle oxidative capacity [Vissing et al., 2009].

Treatment

A key therapeutic strategy has been the attempt to bypass the metabolic block by providing the muscle with glycolytic substrates [DiMauro and Tsujino, 1994]. The oral administration of glucose or fructose to raise the blood glucose level has been helpful but has resulted in weight gain. Glucagon injections have been impractical, with inconsistent results. Although exercise tolerance was increased by raising the serum FFA concentration through the use of fat emulsions, fasting, and the administration of norepinephrine or heparin, more practical regimens, such as a high-fat, low-carbohydrate diet, have not been effective [DiMauro and Bresolin, 1986]. In a study of intralipid infusion to increase FFA availability during 70 percent maximal oxygen consumption cycle ergometry, FFA levels more than tripled, but heart rate was significantly higher during exercise compared with placebo and glucose infusions trials, suggesting that, although lipids are an important source of fuel for exercising muscle in McArdle’s disease, the maximal rates of fat oxidation appeared limited and could not be increased above physiological normal rates during exercise [Andersen et al., 2009]. It was suggested that the limitation was probably caused by a metabolic bottleneck in the tricarboxylic acid cycle due to impaired glycolytic flux, and that therapies aimed at enhancing fat use in McArdle’s disease should be combined with interventions targeting expansion of the tricarboxylic acid cycle. An alternative strategy was to supply branched-chain amino acids, which are taken up, rather than released, by tissue of patients with McArdle’s disease during exercise [Wahren et al., 1973]. Slonim and Goans [1985] demonstrated an improvement of muscle endurance and strength in a patient with weakness after institution of a high-protein diet. Direct administration of branched-chain amino acids to six patients, however, resulted in impairment rather than improvement of bicycle exercise capacity in 5 of 6 patients, possibly due to a lowering of FFA by the amino acids [MacLean et al., 1998]. In a more recent study, patients with McArdle’s disease were randomized to follow either a carbohydrate-rich or protein-rich diet for 3 days before testing; patients on the carbohydrate-rich diet improved their maximal work capacity and exercise tolerance to submaximal workloads [Andersen and Vissing, 2008]. Aerobic training of four McArdle’s patients improved peak cycle exercise capacity, circulatory capacity, and oxygen uptake [Haller et al., 1998b].

Vitamin B₆ is another potential therapeutic aid, as overall body stores of pyridoxal phosphate are depleted in McArdle’s disease due to the lack of enzyme protein to which pyridoxal phosphate is bound [Haller et al., 1983]. One patient has been shown to have a beneficial effect with vitamin B₆ supplementation [Phoenix et al., 1998], but further studies need to be done. In addition, oral creatine monohydrate supplementation in a placebo-controlled crossover trial involving nine patients has been shown to alleviate symptoms and increase their capacity for ischemic, isometric forearm exercise [Vorgerd et al., 2000]. Another strategy has been the ingestion of sucrose prior to exercise to increase the availability of glucose [Vissing and Haller, 2003]. In a single-blind, randomized, placebo-controlled crossover study, 12 patients were studied. Ingestion of sucrose prior to exercise improved exercise tolerance and sense of well-being. In another comparative study, ingestion of 37 g of sucrose 5 minutes before exercise had a marked and prolonged effect on exercise tolerance in patients with McArdle’s disease. This was a more sustained effect than that seen with 75 g of sucrose 40 minutes before exercise, probably related to more continuous glucose uptake from the intestine and correspondingly higher circulating glucose levels later during exercise [Andersen et al., 2008].

Finally, a first-generation adenoviral recombinant containing the full-length human myophosphorylase complementary DNA (cDNA) has been efficiently transduced into phosphorylase-deficient sheep and human myoblasts, resulting in restoration of phosphorylase activity [Pari et al., 1999].

Clinical features

Debrancher deficiency (glycogenosis type III, GSD type III) is characterized by childhood-onset liver dysfunction with hepatomegaly, growth failure, fasting hypoglycemia, and, infrequently, hypoglycemic seizures. Spontaneous resolution may occur around puberty with normal adult liver function [Smit et al., 1990], although cirrhosis and liver failure may develop later [Fellows et al., 1983]. The myopathy tends to present late in the third or fourth decade in 70 percent of cases and involves primarily distal leg and intrinsic hand muscles. It is slowly progressive and rarely incapacitating. Childhood onset has been documented in 7 of 22 patients. Two patients had exercise intolerance, cramps, and premature fatigue [Murase et al., 1973; Ozand et al., 1967], and 5 others had diffuse weakness and wasting, growth failure, and gross motor delay [Badurska et al., 1970; Slonim et al., 1982]. Myoglobinuria has been reported in one case [Brown, 1986]. Enzymatic deficiency was documented in the muscle of 16 patients, 11 of whom had no weakness [Moses et al., 1986]. However, given the young age of these patients (2–27 years), clinical myopathy may have developed later. Peripheral neuropathy has also been documented [Moses et al., 1986]. Although clinical cardiomyopathy is infrequent, cardiac involvement has been demonstrated in most patients with myopathy [Brunberg et al., 1971; Miller et al., 1972; Moses et al., 1989]. Of interest, Cleary et al. [2002] reported consistent facial features in seven patients with GSD type III, which included midfacial hypoplasia with a depressed nasal bridge and a broad upturned nasal tip, indistinct philtral pillars, and bow-shaped lips with a thin vermillion border. In addition, younger patients had deep-set eyes. Several children had clinical problems, such as persistent otitis media or recurrent sinusitis.

Laboratory data

Glucagon or epinephrine in the fasting state does not increase blood glucose, which indicates liver involvement [DiMauro and Tsujino, 1994]. All patients with myopathy have an increase in serum CK. The EMG is myopathic but also may demonstrate fibrillation activity, and the nerve conduction velocities frequently are decreased [Brunberg et al., 1971; Moses et al., 1986]. Evidence of left ventricular or biventricular hypertrophy may be seen on EKG and echocardiography [Brunberg et al., 1971; Moses et al., 1989; Smit et al., 1990].

Inheritance

There is a male predominance. The gene has been assigned to chromosome 1p21 [Yang-Feng et al., 1992]. Prenatal diagnosis can be made by a qualitative assay based on the persistence of the abnormal polysaccharide in cultured amniocytes exposed to a glucose-free medium [Yang et al., 1990].

Pathology

A severe vacuolar myopathy, which contains PAS-positive material that is digested by diastase, is seen on muscle biopsy. This corresponds to large pools of free and apparently normal glycogen particles by electron microscopy [DiMauro and Tsujino, 1994]. Excessive glycogen is also seen in skin biopsies [Sancho et al., 1990], cultured muscle [Miranda et al., 1981], endomyocardial biopsies [Olson et al., 1984], intramuscular nerves [Powell et al., 1985], Schwann cells and axons of sural nerve [Ugawa et al., 1986], and brain [Hug and Schubert, 1966].

Biochemistry

The debrancher enzyme is a single 160-kDa polypeptide with two distinct catalytic functions. After digestion of glycogen to four glucosyl units by PPL, the residual stubs are removed by the debrancher enzyme in two steps – a transferase and a glucosidase – which are located in different domains of the polypeptide [Bates et al., 1975]. Based on enzymatic and immunologic assays, debrancher deficiency has been classified into three groups [Chen et al., 1987; Ding et al., 1990]:

Molecular genetics

Human debrancher cDNA has been isolated and sequenced [Yang et al., 1992]. A number of mutations have been described in GSD type IIIa [Okubo et al., 1996, 1999, 2000; Parvari et al., 1998; Shen et al., 1996, 1997; Shaiu et al., 2000], as well as in GSD type IIIb [Shen et al., 1996]. It is interesting to note that most patients with the IIIb variant have mutations in exon 3 of the debrancher gene, which are expected to result in truncated proteins [Shen et al., 1996]. Shaiu et al. [2000] reported two frequent mutations, each of which was found in homozygous state in multiple patients, and each of which was associated with a subset of clinical phenotype in those patients with that mutation. The first mutation was a novel point mutation of a single T deletion at cDNA position 3964 (3964 delT), which was first detected in an African American patient who had a severe phenotype and early onset of clinical symptoms. The second mutation was an A to G transition at position −12 upstream of the 3′ splice site of intron 32 (IVS32-12A>G) which was confirmed in a Caucasian GSD IIIa patient presenting with mild clinical symptoms. These two mutations together accounted for more than 12 percent of the molecular defects in the GSD type III patients tested at that time.

Whereas the overall incidence of GSD type III in the United States is about 1 in 100,000 live births, it is unusually frequent among North African Jews in Israel (prevalence of 1 in 5400, carrier prevalence 1 in 35) [Parvari et al., 1997]. Another ethnic group with a high prevalence of GSD type III (1 in 3600 with a carrier frequency of 1 in 30) are individuals from the Faroe Islands [Santer et al., 2001]. The population of 45,000 of this small archipelago in the North Atlantic has its roots in colonization by the Norwegians in the 8th century and throughout the Viking age. It was concluded that, due to a founder effect, the Faroe Islands have the highest prevalence of GSD type IIIa worldwide.

Treatment

Fasting hypoglycemia should be avoided in infants and young children with debrancher deficiency through the implementation of frequent feedings and nocturnal gastric glucose infusions and uncooked cornstarch [Fernandes, 1990]. A marked improvement was noted in a 7-year-old child with severe weakness and wasting, following institution of a high-protein diet overnight for 6 months [Slonim et al., 1982]. However, a 6-month high-protein diet had no significant effect in an adult with myopathy and distal wasting [DiMauro and Tsujino, 1994].

Clinical features

Muscle PFK deficiency (Tarui’s disease) (glycogenosis type VII) has been reported in more than 100 patients [Toscano and Musumeci, 2007]. Exercise intolerance with cramps and compensated hemolysis is the main clinical feature, often associated with hyperuricemia. Fixed weakness of late adult onset was prominent in three patients. Myoglobinuria has been documented in 10 of 25 patients. Six children have been reported with early severe myopathy, with or without contractures [Amit et al., 1992; DiMauro and Tsujino, 1994]. One boy also had a cardiomyopathy and died at 21 months of age [Amit et al., 1992]. None of the infantile cases had hemolysis, suggesting different molecular etiologies. Other associated multisystem signs in infants or very young children have included seizures, cortical blindness, corneal opacifications, and cardiomyopathy [DiMauro and Lamperti, 2001; Al-Hassnan et al., 2007]. Four patients had hemolysis without myopathy, and two had normal or only mildly decreased muscle activity and about half-normal erythrocyte PFK activity. This was attributed to instability of the muscle-specific subunit of PFK [Etiemble and Kahn, 1976; Kahn et al., 1975]. Minor clinical differences from McArdle’s disease include more common reports of nausea and vomiting during exercise-induced crises of myalgia, cramps, weakness, and lower frequency of attacks of myoglobinuria [DiMauro and Lamperti, 2001]. As PFK deficiency blocks the metabolism of both muscle glycogen and blood glucose, Haller and Vissing [2004] studied 5 individuals with muscle PFK deficiency and 29 patients with McArdle’s disease during continuous cycle exercise; they found that no PFK-deficient patient developed a spontaneous second-wind phenomenon under conditions that consistently produced one in patients with McArdle’s disease, and concluded that the ability to metabolize blood glucose was critical to the development of a typical spontaneous second wind.

Laboratory data

The serum CK level is usually increased in patients with muscle disease. Hemolysis is indicated by an increased serum bilirubin and moderate reticulocytosis [DiMauro and Tsujino, 1994]. Most patients have an increase in uric acid. EMG may be normal or myopathic. ³¹P-NMR spectroscopy shows a different pattern from that obtained in McArdle’s disease because, in PFK deficiency, as well as in other defects of distal glycolysis, glycolytic intermediates accumulate in muscle as phosphorylated monoesters and can be detected as a distinct spectral peak [Argov and Bank, 1991].

Inheritance

The gene encoding the muscle (M) subunit is located on chromosome 12 [Howard et al., 1996].

Muscle biopsy

Diagnosis can be made by a negative histochemical PFK stain [Bonilla and Schotland, 1970]. The definitive diagnosis comes from the biochemical analysis, but only if the muscle specimen is snap-frozen at the time of biopsy, as PFK is highly labile and its activity declines rapidly in specimens kept at room temperature or on wet ice; this is why partial PFK deficiencies with residual activities above 10 or 20 percent of normal should be considered skeptically [DiMauro and Lamperti, 2001]. An important differentiating feature from McArdle’s disease is the presence of an abnormal polysaccharide in some cases, particularly older patients [Hays et al., 1981]. This abnormal polysaccharide has the characteristics of polyglucosan and stains intensely with the PAS reaction, but is resistant to diastase digestion; ultrastructurally, it is composed of finely granular and filamentous material, similar to the storage material seen in branching enzyme deficiency and in Lafora’s disease [DiMauro and Lamperti, 2001].

Biochemistry

PFK (ATP:d-fructose 1-phosphotransferase) is a tetrameric enzyme under the control of three structural loci that encode three distinct subunits: namely, M (muscle), L (liver), and P (platelet) [Vora, 1983; Vora et al., 1983a]. The three subunits show variable expression in different tissues. Mature human muscle expresses only the M subunit, whereas erythrocytes express both the M and L subunits, thereby containing five isozymes. In the typical form of PFK deficiency, genetic defects of the M subunit cause total lack of activity in muscle and partial enzyme deficiency in erythrocytes, where the homotetramer L4 is responsible for the residual activity. Other tissues, such as liver, that express predominantly the L subunit are not affected.

Pathophysiology

The functional consequences of PFK deficiency are similar to those observed in McArdle’s disease and are related to the inability of muscle to generate pyruvate [DiMauro and Tsujino, 1994]. With the ischemic exercise test, venous lactate fails to rise [Tarui et al., 1965]. However, PFK deficiency differs significantly from PPL deficiency because of the inability of PFK-deficient muscle to use glucose. Due to this inability, maximal oxygen uptake has been obtained by increasing FFA availability [Haller and Lewis, 1991]. Exercise intolerance is not decreased by glucose administration, which is potentially harmful because it reduces the concentration of FFAs and ketones. This explains why high-carbohydrate meals exacerbate exercise intolerance in patients with PFK deficiency, a situation referred to as the “out-of-wind” phenomenon [Haller and Lewis, 1991]. The excessive exercise-induced degradation of muscle purine nucleotides likely accounts for the hyperuricemia seen in PFK, PPL, and debrancher-deficient patients [Mineo et al., 1987]. Finally, in PFK deficiency, as in McArdle’s disease, exercise triggers increases in heart rate, cardiac output, and blood flow, which are exaggerated relative to the muscle capacity to use oxygen [Lewis et al., 1991].

Molecular genetics

The genes encoding the subunits M, P, and L have been assigned to chromosomes 12, 10, and 21 (see Table 94-1) [Van Keuren et al., 1986; Vora et al., 1982, 1983b]. The M subunit cDNA has been isolated and sequenced [Nakajima et al., 1987], and evidence for tissue-specific alternative mRNA splicing has been documented [Nakajima et al., 1990]. At least 20 mutations responsible for muscle PFK deficiency have now been identified [DiMauro et al., 1995; Sherman et al., 1994; Tsujino et al., 1994d; DiMauro and Lamperti, 2001]. Raben and Sherman [1995] tabulated 15 disease-inducing mutations of the muscle PFK gene and several polymorphisms. These included splicing defects, frameshifts, and missense mutations in patients from six different ethnic backgrounds, supporting genetic heterogeneity of the disease. This disorder appeared to be particularly prevalent among individuals of Ashkenazi Jewish descent. The most frequent mutation in this group was an exon 5 splicing defect that accounted for approximately 68 percent of mutant alleles in the Ashkenazim. Patients with muscle PFK deficiency diagnosed in the United States have been of Ashkenazi Jewish origin, and the several mutations identified in them differ from those described in the non-Ashkenazi Italian or Japanese patients [DiMauro et al., 1995].

Treatment

Glucose is not an alternative substrate in PFK deficiency. A high-protein diet may be beneficial, although this has not been tried [DiMauro and Tsujino, 1994]. A 2-year old boy with the severe infantile form of PFK deficiency, including arthrogryposis multiplex congenita, respiratory insufficiency, slowed motor nerve conduction velocities, and abnormal electroencephalogram (EEG), had a significant improvement in strength, EMG features, and EEG pattern on the ketogenic diet [Swoboda et al., 1997]. Unfortunately, the child died of complications of pneumonia at 35 months.

Clinical features

PGK deficiency (glycogenosis type IX) presents with nonspherocytic hemolytic anemia and dysfunction of the central nervous system [Boivin et al., 1974; Valentine et al., 1960]. Neurologic problems have included behavioral abnormalities, mental retardation, seizures, and strokes. In hemizygous males, the severe hemolytic anemia manifests soon after birth with jaundice, splenomegaly, and hemoglobinuria. Childhood mortality has been reported in four members of one family [Valentine et al., 1960]. Heterozygous females may be normal or may have mild chronic hemolytic anemia. Myopathic features have included myopathy in males [DiMauro et al., 1981a, 1983a; Rosa et al., 1982; Tonin et al., 1993; Morimoto et al., 2003], with exercise intolerance, cramps, and myoglobinuria. Twenty PGK variants with reduced PGK activity have been identified; myopathy was present in eight of these variants [Tsujino et al., 1995b]. Spiegel et al. [2009] reported an 18-year-old man of Arab Bedouin descent with onset of muscle cramps and recurrent myoglobinuria at age 7 years, having elevated CK, without hemolytic anemia or brain dysfunction, in whom PGK deficiency was documented in muscle and erythrocytes due to a novel mutation, T378P. This was the ninth case presenting with isolated myopathy, whereas most other patients show nonspherocytic hemolytic anemia alone or associated with brain dysfunction and a few patients have myopathy and brain involvement.

Laboratory data

Myopathic patients demonstrate an inconsistent increase in resting serum CK.

Inheritance

PGK is located on the long arm of the X chromosome (see Table 94-1). The defect is expressed in fibroblasts, and prenatal diagnosis is possible [DiMauro and Tsujino, 1994].

Muscle biopsy

PAS stain for glycogen was mildly increased in one patient. Electron microscopy demonstrated glycogen accumulation in all patients [DiMauro and Tsujino, 1994].

Biochemistry

Human PGK is a single polypeptide encoded on the X chromosome for all tissues except spermatogenic cells [DiMauro and Tsujino, 1994]. The complete amino-acid sequence of normal human PGK has been determined [Huang et al., 1980a, 1980b], and mutations have been identified. Patients with hemoglobinuria and those with myopathy demonstrate genetic heterogeneity in enzyme activities. PGK mutants associated with myopathy have been named Creteil [Rosa et al., 1982], New Jersey [DiMauro et al., 1983a], Alberta [Tonin et al., 1993], Hamamatsu [Sugie et al., 1989, 1998], Shizuoka [Fujii et al., 1992], and North Carolina [Tsujino et al., 1994e]. As PGK is a monomer, there are no tissue-specific isoforms, with the exception of PGK2, which is confined to spermatogenic cells; therefore, the defect should be expressed in all tissues. In three patients with myopathy, PGK was significantly decreased in erythrocytes, leukocytes, platelets, fibroblasts, and muscle culture [DiMauro et al., 1983a; Rosa et al., 1982; Tonin et al., 1993].

Pathophysiology

It is difficult to explain the lack of clinical myopathy in many patients with hemolytic anemia, and, conversely, the selective involvement of muscle in other patients [DiMauro and Tsujino, 1994]. However, one patient with PGK Shizuoka has been described with chronic hemolysis and myoglobinuria [Fujii et al., 1992], and a second patient has been described with hemolytic anemia, central nervous system dysfunction, and myopathy [Sugie et al., 1994]. Systematic correlation of molecular genetic, biochemical, and clinical data in future patients may provide insight into the explanation for the striking clinical heterogeneity of PGK deficiency [Tsujino et al., 1995b].

Molecular genetics

A full-length cDNA for the normal PGK gene on the X chromosome has been isolated [Michelson et al., 1983], and genetic heterogeneity has been documented [Tonin et al., 1993]. Seven missense mutations and one splice-junction mutation have been identified in eight patients, three who had myopathy [Fujii et al., 1992; Tsujino et al., 1994e, 1995b; Morimoto et al., 2003]. The testicular isozyme, PGK2, is encoded by a gene on chromosome 19 [DiMauro and Lamperti, 2001].

Clinical features

PGAM deficiency (glycogenosis type X) has been reported in four men and two women [DiMauro et al., 1981b; DiMauro and Tsujino, 1994]. Exercise intolerance, cramps, and myoglobinuria are the main clinical features.

Laboratory data

Four patients demonstrated an increase in serum CK between attacks. EMG and nerve-conduction studies were normal.

Muscle biopsy

A variable increase in the PAS reaction has been demonstrated on histochemical examination, and electron microscopy has confirmed mild glycogen accumulation in most cases [DiMauro and Tsujino, 1994].

Biochemistry

PGAM catalyzes the conversion of 3-phosphoglycerate to 2-phosphoglycerate, and is very active in normal human muscle [DiMauro et al., 1981b, 1982]. The glycogen concentration of muscle was normal in four patients and moderately increased in two [DiMauro and Tsujino, 1994]. Insufficient in vitro lactate production has been demonstrated in three patients during anaerobic glycolysis [DiMauro et al., 1981b; Servidei and DiMauro, 1989]. The residual PGAM activity varied from 2.1 to 6 percent of normal. Human PGAM is a dimeric enzyme that consists of different proportions of a slow-migrating muscle (MM) isozyme, a fast-migrating brain (BB) isozyme, and an intermediate hybrid (MB) form in different tissues [Omenn and Cheung, 1974; Omenn and Hermodson, 1975]. The electrophoretic pattern of normal adult human muscle PGAM reveals a marked predominance of the MM band, with only faint BB and MB bands [DiMauro et al., 1981b; DiMauro et al., 1982; Omenn and Cheung, 1974; Omenn and Hermodson, 1975]. The only other tissues containing substantial amounts of the M subunit are heart and sperm, although there is no evidence of cardiomyopathy or male infertility in PGAM deficiency [DiMauro and Lamperti, 2001]. In cardiac muscle, the MM isozyme predominates, although all three bands are seen [DiMauro et al., 1981b; Omenn and Hermodson, 1975]. In most other tissues, such as brain, liver, leukocytes, and erythrocytes, PGAM-BB is the only isozyme on electrophoresis [Omenn and Cheung, 1974; Omenn and Hermodson, 1975].

Pathophysiology

Normal lactate production was demonstrated in one patient during maximal aerobic exercise [Kissel et al., 1985]. ³¹P-NMR studies revealed exercise-induced accumulation of PME and mild intracellular acidosis [Argov et al., 1987].

Molecular genetics

A full-length cDNA [Shanske et al., 1987] and the genomic clone for PGAM-M [Tsujino et al., 1989] have been isolated and sequenced, and the gene has been localized to chromosome 7 [Edwards et al., 1989]. Tsujino et al. [1993b] identified three distinct point mutations in five patients with PGAM-M deficiency. A full-length cDNA has been identified for PGAM-B [Sakoda et al., 1988]. The gene has been localized to chromosome 10 [Junien et al., 1982].

Clinical features

LDH deficiency (glycogenosis type XI) has been reported in two young men with exercise intolerance and recurrent myoglobinuria [Bryan et al., 1990; Kanno et al., 1980]. It has also been reported in an asymptomatic woman through blood screening [Maekawa et al., 1984]. In addition to muscle symptoms, three affected Japanese women suffered from dystocia, necessitating cesarian sections, and a few patients had dermatologic problems [Kanno and Maekawa, 1995]. Takayasu et al. [1991] reported a 16-year-old Japanese girl who had desquamating erythematosquamous lesions, primarily on the extensor surface of the extremities. The epidermis of the diseased skin and scalp hair follicles were virtually devoid of LDH activity.

Laboratory data

The forearm ischemic exercise test demonstrated an abnormally low increase in venous lactate, contrasting with an excessive increase in pyruvate, in all three patients studied. The muscle biopsy was normal in one patient [Bryan et al., 1990].

Biochemistry

LDH is a tetrameric enzyme composed of two subunits, M (or A) and H (or B), which result in five isozymes: the two homotetramers, M4 and H4, and the three hybrid forms. The M subunit-containing isozymes predominate in skeletal muscle. The H subunit-containing isozymes predominate in heart and other tissues [DiMauro and Tsujino, 1994].

Molecular genetics

The gene encoding LDH-M has been assigned to chromosome 11 [Boone et al., 1972]. Scrable et al. [1990] demonstrated that LDH-M is located in band 11p15.4. The gene encoding LDH-H has been assigned to chromosome 12 [Chen et al., 1973]. Five mutations have been described that lead to muscle LDH deficiency [DiMauro et al., 1995; Maekawa et al., 1990, 1991, 1994].

Clinical features

Branching enzyme (brancher) deficiency (glycogenosis type IV) presents in infancy as a rapidly progressive disease dominated by liver dysfunction with hepatosplenomegaly and cirrhosis, death from hepatic failure or gastrointestinal bleeding supervening by 4 years of age [DiMauro and Tsujino, 1994]. Other clinical features include hypotonia, muscle wasting, and contractures [Fernandes and Huijing, 1968; Zellweger et al., 1972]. Cardiomyopathy has also been documented in several children [Farrans et al., 1966; Servidei et al., 1987]. Isolated myopathy has been reported in six patients: three siblings with delayed motor development and chronic proximal weakness [Reusche et al., 1992], a child with myopathy and hepatic involvement [Bruno et al. 1999], and two adults in whom proximal weakness had started at 26 and 49 years of age [Bornemann et al., 1996; Ferguson et al., 1983]. Schroder et al. [1993] reported a case of juvenile type IV glycogenosis, with total branching enzyme deficiency in skeletal muscle and liver, in a male who presented with severe myopathy, dilated cardiomyopathy, heart failure, dysmorphic features, and subclinical neuropathy; the patient died at age 19 of a sudden cardiac death. Alegria et al. [1999] reported hydrops fetalis as a presenting manifestation of GSD type IV. The infant was delivered by cesarean section at 34 weeks and had generalized edema, severe hypotonia, and arthrogryposis of the lower limbs at birth, and died at 4 days of age. Cox et al. [1999] reported three sibling fetuses who were shown to have type IV GSD by pathologic and biochemical studies; they had onset of hydrops, limb contractures, and akinesia in the early second trimester.

This disease shows considerable clinical heterogeneity. The neuromuscular presentation of GSD type IV can be distinguished by age at onset into four groups: perinatal, presenting as fetal akinesia deformation sequence and perinatal death; congenital, with hypotonia, neuronal involvement, and death in early infancy; childhood, with myopathy or cardiomyopathy; and adult, with isolated myopathy or adult polyglucosan body diseases [Bruno et al., 2004; 2007]. Brancher deficiency has been identified in the leukocytes of two Israeli patients with adult polyglucosan body disease [Lossos et al., 1991]. Adult polyglucosan body disease has been described in approximately 15 patients and is characterized by progressive upper and lower motor neuron involvement, sensory loss, sphincter problems, neurogenic bladder, and in some cases, dementia [Bruno et al., 1993; Cafferty et al., 1991]. Burrow et al. [2006] reported a 30-month-old girl with GSD type IV, who had stable congenital hypotonia, gross motor delay, and severe fibro-fatty replacement of the muscles, but no hepatic or cardiac involvement, secondary to compound heterozygosity for two missense mutations in the glycogen branching enzyme gene.

Laboratory data

Carbohydrate tolerance tests and the response of blood glucose to glucagon or epinephrine are normal in patients with liver disease [DiMauro and Tsujino, 1994]. The serum CK is inconsistently increased. Echocardiography showed dilatation and impaired shortening of the left ventricular ejection fraction in one patient [Servidei et al., 1987]. Sensorimotor axonopathy has been demonstrated in patients with adult polyglucosan body disease [Bruno et al., 1993; Cafferty et al., 1991].

Inheritance

Prenatal diagnosis is possible in fibroblasts and amniocytes [Brown and Brown, 1989].

Muscle biopsy

Muscle biopsy demonstrates deposits of basophilic, intensely PAS-positive material, which is only partially sensitive to diastase digestion. Electron microscopy reveals storage material consisting of filamentous and finely granular material, often associated with normal-looking glycogen beta particles [DiMauro and Tsujino, 1994]. The abnormal polysaccharide is found in skin, liver, muscle, heart, and the central nervous system. In adult polyglucosan body disease, polyglucosan bodies have been found in both gray and white matter in the processes of neurons and astrocytes. In sural nerve biopsies, the bodies are in the axoplasm, primarily of myelinated fibers and in Schwann cells [Cafferty et al., 1991].

Biochemistry

Branching enzyme catalyzes the last step in glycogen biosynthesis by attaching a short glucosyl chain in an alpha-1,6-glucosidic link to a naked peripheral chain of nascent glycogen [DiMauro and Tsujino, 1994]. The polysaccharide that accumulates has longer than normal peripheral chains and fewer branching points than normal glycogen.

Molecular genetics

Several mutations have been identified in patients with different clinical presentations [Bao et al., 1996; Bruno et al., 1999]. One of these – namely, Tyr329Ser – was present in seven patients with adult polyglucosan body disease [Lossos et al., 1998]. It was concluded that adult polyglucosan body disease represents an allelic variant of GSD type IV. In a non-Ashkenazi patient with adult polyglucosan body disease, Ziemssen et al. [2000] identified compound heterozygosity for the R515H mutation and the R524Q mutation in the glycogen branching enzyme gene, both of which had been found in patients with GSD type IV. The findings confirmed that adult polyglucosan body disease and GSD IV are allelic disorders. In a series of 8 patients with GSD type IV with four different neuromuscular presentations – namely, 3 with fetal akinesia deformation sequence, 3 with congenital myopathy, 1 with juvenile myopathy, and 1 with combined myopathic and hepatic features – nine novel mutations were identified, including nonsense, missense, deletion, insertion, and splice-junction mutations [Bruno et al., 2004]. The three cases with fetal akinesia deformation sequence were homozygous, whereas all the other cases were compound heterozygotes.

Treatment

A number of strategies have been used, including administration of glucagon; long-term use of steroids; a high-protein, low-carbohydrate diet; and administration of fungal alpha-1,4-glucosidase and alpha-1,6-glucosidase. These strategies have been ineffective [DiMauro and Tsujino, 1994]. Liver transplantation was thought to be beneficial in ten children [Selby et al., 1991], although one child developed intractable cardiomyopathy 2 years after transplantation [Sokal et al., 1992].

Clinical features

This disorder was discovered in 1996 in a 4½-year-old boy who presented with episodic exercise intolerance and weakness following febrile illnesses [Kreuder et al., 1996]. This boy was considered to have myoglobinuria, even though his highest serum CK was 6480 U/L (normal <60 U/L). This boy also had proximal muscle wasting with weakness and episodes of nonspherocytic hemolytic anemia and jaundice.

Biochemistry

Biochemical assays revealed a profound reduction in muscle and red cell aldolase levels and a decrease in the thermostability of the residual enzyme. The enzyme is a tetramer of identical 40,000-dalton subunits. Vertebrates have three aldolase isozymes that are distinguished by their electrophoretic and catalytic properties [Charlesworth, 1972].

Molecular genetics

The aldolase A gene has been mapped to chromosome 16q22–q24 and two pseudogenes were found on chromosomes 3 and 10 [Kukita et al., 1987; Serero et al., 1988]. The patient described by Kreuder et al. [1996] was found to be homozygous for germline mutation in which a negatively charged glutamic acid is changed to a positively charged lysine at residue 206, a residue that is highly conserved within the subunit interface region. Subsequently, Amberger [2008] mapped the ALDOA gene to chromosome 16p11.2, based on an alignment of the ALDOA sequence with the genomic sequence.

Metabolic decompensation in association with fasting

Children with FAO defects are most prone to decompensation, within the context of depleted glycogen and glucose reserves, during conditions that place stress on the FAO pathway for fuel generation. These conditions include prolonged exercise (particularly after 1 hour of mild to moderate aerobic exercise), fasting, infection with vomiting, and cold-induced shivering thermogenesis. In cold exposure, ketogenesis is stimulated in normal individuals [Johnson et al., 1961], and shivering, which is an involuntary form of muscle activity, is also dependent on long-chain FAO [Bell and Thompson, 1979]. After an overnight fast, children are most likely to be found comatose in the early morning hours. During infection, there may be an added problem with vomiting and decreased oral intake. Children may also present with a Reye-like syndrome. Infants and younger children are at greater risk during fasting because of their decreased abilities for fasting adaptation. Prolonged fasting for an infant of less than 1 year of age would be 6–10 hours, compared with 12 hours for a child between 1 and 4 years of age [Hale and Bennett, 1992].

Involvement of fatty acid oxidation-dependent tissues

Tissues such as skeletal muscle, heart, and liver have high energy demands and are therefore dependent on efficient FAO. When there is a defect in hepatic ketogenesis, glucose becomes the only available fuel and thus becomes rate-limiting under conditions of FAO stress when glycogen and glucose stores have been depleted. As a result, FFAs, which are liberated during fasting and cannot be metabolized because of the block, may be stored in the cytosol as triglycerides. This produces a progressive lipid storage myopathy with weakness, a hypertrophic and/or dilatative cardiomyopathy, and a fatty liver. Increased content of short- or medium-chain fatty acids and, in particular, their dicarboxylic metabolites, from compensatory ω-oxidation, may cause a variety of secondary biochemical abnormalities, including an impairment of gluconeogenesis, β-oxidation, and the citric acid cycle [Corkey et al., 1988; Tonsgard and Getz, 1985; Tonsgard, 1986], leading to a further decrease in cellular ATP production. In addition, in the long-chain FAO disorders, which may have recurrent episodes of acute muscle breakdown or myoglobinuria (e.g., CPT II, VLCAD, trifunctional enzyme deficiencies), the accumulation of long-chain fatty acids and long-chain acylcarnitines may have detergent-like actions on muscle membranes. Excessive amounts of palmitoyl-CoA and palmitoylcarnitine have detergent properties on isolated canine myocytic sarcolemmal membranes and potentiate free radical-induced lipid membrane peroxidative injury in ischemia [Mak et al., 1986]. Long-chain acylcarnitines also activate calcium channels in cardiac [Inoue and Pappano, 1983; Spedding, 1985] and smooth muscle myocytes [Spedding, 1985; Spedding and Mir, 1987]. They may thus potentiate the increase in cytosolic calcium associated with arrhythmogenesis, as seen in ischemic myocardium [Lee et al., 1987].

Hypoketotic hypoglycemia

The pattern of hypoketotic hypoglycemia reflects the accelerated rate of glucose utilization that occurs when fatty acids cannot be used as fuels, and ketone bodies are not generated to spare glucose/glycogen stores. An increase in the ratio of serum FFAs to ketones, from the normal ratio of 1:1 to greater than 2:1, therefore, would suggest a block in β-oxidation.

Alterations in plasma and tissue concentrations of carnitine

In most cases of intramitochondrial β-oxidation defects, the total carnitine concentration is decreased (<50 percent of normal), and the acylcarnitine fraction is increased (>50 percent esterified; normal is 10–25 percent in the fed state and 30–50 percent in the fasted state) [Hale and Bennett, 1992]. In the intramitochondrial β-oxidation disorders, the excessive acyl-CoAs that accumulate proximal to the metabolic block may be converted into acylcarnitines by chain length-specific carnitine acyltransferases [Bremer, 1983]. These acylcarnitines, when filtered through the kidneys, compete with free carnitine at the renal tubular reabsorptive site. As the longer chain-length acylcarnitines have an increasingly higher affinity for the carnitine transporter than free carnitine [Stanley et al., 1991], the free carnitine will be excreted, leading to a decrease in free carnitine in the serum. In the case of the plasmalemmal carnitine transporter defect, the total carnitine is markedly reduced (e.g., <5 percent of normal) and the esterified fraction is normal [Tein et al., 1990a] because the transport defect, which is also expressed in kidney, leads to a decreased renal threshold for carnitine reabsorption.

Fatty acid oxidation studies

Provided the defect is expressed in cultured skin fibroblasts, a useful screening tool is the measurement of the oxidation rates of [1-¹⁴C]-labeled palmitate (C16), octanoate (C8), and butyrate (C4) and deuterium-labeled oleic acid in the fibroblasts to establish the chain-length specificity of the defect [Rhead, 1990; He et al., 2007]. Another useful screening test to suggest an underlying defect in FAO is to culture skin fibroblasts in a glucose-free medium, which will simulate fasting and exacerbate microvesicular steatosis in the fibroblasts expressing a genetic defect in FAO [Renaud et al., 2002]. Another method has been developed for quantitative acylcarnitine profiling by ESI-MS/MS in human skin fibroblasts, using unlabeled palmitic acid as substrate; this has revealed pathognomonic acylcarnitine profiles in a variety of short-, medium-, and long-chain FAO defects [Okun et al., 2002]. In addition, this method delineates different variants of MCAD deficiency, e.g., mild and classical. A rapid method for acylcarnitine profiling has also been described [Tyni et al., 2002], using stable isotopically labeled palmitate and intact mitochondria prepared from fresh-biopsied muscle homogenates, which reduces the delay in diagnosis related to tissue culture.

Enzymatic assays

Depending on the suspected site of defect, a direct enzymatic assay may then be performed for the specific enzyme (Table 94-5) [Tein, 1995]. These assays can be performed in cultured skin fibroblasts or in biopsied muscle specimens for CPT I, CPT II, SCAD, LCAD, SCHAD, and trifunctional enzyme deficiencies, among others. However, certain of these defects may be tissue-specific, such as muscle SCHAD deficiency [Tein et al., 1991]; in this case, the defect is expressed in biopsied muscle and not in fibroblasts, and therefore may be missed unless the affected tissue is examined. Carnitine acylcarnitine translocase deficiency [Pande et al., 1993] and MCAD deficiency can be measured in cultured skin fibroblasts. Evidence for a defect in ETF or ETF-CoQ relies on demonstration of a combined deficiency in the activities of the SCAD, MCAD, and LCAD enzymes. CPT II deficiency has been rapidly diagnosed prenatally by enzyme assay in 10 mg of chorionic villus sampling at the 11th week of gestation, in combination with haplotyping using polymorphic markers linked to the CPT II gene [Vekemans et al., 2003].

Carnitine uptake studies

For the carnitine uptake defect or OCTN2 deficiency, diagnosis is confirmed by in vitro studies of carnitine uptake in cultured skin fibroblasts. This study demonstrates negligible or minimal uptake of carnitine in the affected homozygote or compound heterozygote patients, thereby precluding the calculation of K_m and V_max values [Tein et al., 1990a; Treem et al., 1988; Lamhonwah et al., 2002]. This supports the concept that primary carnitine deficiency is due to a defect in the specific high-affinity, plasmalemmal carrier-mediated carnitine transporter (OCTN2). Heterozygotes demonstrated normal K_m values but reduced V_max values ranging from 13 to 44 percent of control values [Stanley et al., 1991; Tein et al., 1990a], suggesting that the heterozygotes in this series had a reduced number of normally functioning transporters. This is the most sensitive study for detection of the carrier state, as serum carnitine concentrations in the heterozygotes may be normal. Negligible carnitine uptake has also been demonstrated in the cultured lymphoblasts [Tein and Xie, 1996] and myoblasts [Pons et al., 1997] of affected children.

Molecular studies

Molecular characterization of the specific defects includes Western blotting to determine whether the defects are crossreacting material-positive, suggesting a kinetic deficiency, or whether they are crossreacting material-negative, suggesting a decrease in the production of the affected protein. Western blotting has also been used in the determination of the amounts of the alpha and beta subunits of ETF [Finocchiaro et al., 1990]. A number of the enzymes (e.g., CPT I, CPT II, VLCAD, LCAD, SCAD, MCAD, trifunctional protein) and transporters (e.g., high-affinity plasmalemmal carnitine OCTN2 transporter) have now been cloned (see Table 94-4). This has led to the discovery of specific mutations resulting in the defective activity, which has given rise to the development of specific molecular probes. These probes can now be used for the precise and rapid detection of certain specific defects. Specific mutations have been documented for CPT II [Taroni et al., 1993; Verderio et al., 1995], VLCAD [Andresen et al., 1996a, 1996b; Souri et al., 1996; Strauss et al., 1995], MCAD [Blakemore et al., 1991; Gregersen et al., 1991], SCAD [Corydon et al., 1996; Gregersen et al., 1996; Naito et al., 1990], trifunctional protein [Sims et al., 1995; Strauss, 1997], and carnitine OCTN2 transporter defects [Lamhonwah et al., 2002], among others. The types of mutations may include missense mutations, small amino acid deletions and insertions, truncating mutations, nonsense mutations, out-of-frame deletions and insertions, and splice-site mutations [Gregersen et al., 2001]. The most straightforward application of mutation analysis is to confirm the diagnosis in suspected patients, particularly in the context of family studies and for prenatal/preimplantation analysis.

Medium-chain triglyceride oil

Medium-chain triglyceride oil as a nutritional source could be useful in long-chain FAO disorders because the medium-chain fatty acids would circumvent the block in long-chain FAO and thereby facilitate ATP production from the remainder of the patent FAO pathway. The medium-chain triglyceride oil could be started at a dose of 0.5 g/kg/day, divided in three daily doses, and could be increased up to 1 or 1.5 g/kg/day as tolerated. The major side effect is diarrhea. The usefulness of this approach, however, may be limited because excess medium-chain triglyceride would ultimately be stored as long-chain fats in adipocytes. The success of medium-chain triglyceride oil supplementation in LCHAD deficiency has been variable [Tein et al., 1995]. When a high percentage of energy from fat is provided by medium-chain triglyceride oil, patients are at risk for essential fatty acid deficiency and their diet should therefore be supplemented with essential fatty acid (1–2 percent of total energy intake) [Solis and Singh, 2002].

Riboflavin

Certain cases of the multiple acyl-CoA dehydrogenase deficiencies (e.g., ETF or ETF-CoQ-linked deficiencies) are responsive to riboflavin supplementation [Gregersen et al., 1982b]. The dosage is approximately 50 mg 3 times a day for infants and young children, and 100 mg 3 times a day for older children.

Carnitine

The essential indication for carnitine therapy is the carnitine transporter (OCTN2) defect, which is characterized by carnitine-responsive cardiomyopathy and very low plasma and tissue concentrations of carnitine (generally below 5 percent of normal) [Stanley et al., 1991; Tein et al., 1990a]. There was a dramatic improvement in the cardiomyopathy and myopathy in all 22 patients treated with high-dose oral carnitine supplementation within the first few weeks, as well as a reduction of heart size toward normal within a few months of therapy. In addition, three children with significant failure to thrive before therapy demonstrated a marked improvement in growth after therapy [Tein et al., 1990a]. Of 19 patients treated with carnitine therapy for 5–20 years, 18 continued to be healthy [Stanley et al., 1991; Cederbaum et al., 2002]. Thus, in the carnitine transporter defect, high-dose oral carnitine supplementation at 100 mg/kg/day in four divided daily doses is critical and life-saving, significantly reversing the pathology in this otherwise progressive and lethal disease. Furthermore, if a child is diagnosed prospectively from birth, early carnitine therapy from birth has been shown to prevent the development of the clinical phenotype [Lamhonwah et al., 2002].

In the intramitochondrial β-oxidation defects with secondary carnitine deficiency, the results of carnitine therapy have been highly variable and insufficiently evaluated. Theoretically, carnitine has been given to limit the intracellular concentrations of potentially toxic acyl-CoA intermediates within the cell through transesterification and thereby to liberate CoA, which is a critical intracellular co-factor [Hale and Bennett, 1992]. However, there has been no objective prospective study to prove that carnitine administration has had a beneficial effect. In one study of a patient with MCAD deficiency, Stanley et al. [1990] demonstrated that the associated carnitine deficiency was not the cause of the defect in FAO, as shown by the lack of effect of carnitine replacement on fasting ketogenesis. After 3 months of oral carnitine therapy, this patient had no increase in plasma ketones and still became ill and hypoglycemic after 14 hours of fasting. Furthermore, there is increasing evidence to suggest that carnitine administration may have deleterious effects in the long-chain FAO disorders. In these disorders, there is an accumulation of long-chain acyl-CoAs proximal to the metabolic block, which, upon esterification, become long-chain acylcarnitines. Excessive palmitoylcarnitine may have detergent effects on membranes and arrhythmogenic effects, as previously discussed [Inoue and Pappano, 1983; Lee et al., 1987; Mak et al., 1986; Spedding, 1985; Spedding and Mir, 1987]. This field warrants further investigation. In view of potential adverse effects, supplementation at times of severe metabolic derangement should be avoided.

Specific therapies for lchad/trifunctional protein deficiency

Oral prednisone has been shown to lead to a dramatic reversal of the limb-girdle myopathy and marked reduction in the episodic myoglobinuria in one boy with the myoneuropathic form of LCHAD deficiency [Tein et al., 1995]. Several children with LCHAD deficiency who had associated pigmentary retinopathy were shown to have a deficiency of the n-3-polyunsaturated fatty acid, docosahexaenoic acid (DHA), and subsequent supplementation with DHA led to some improvement in visual function [Gillingham et al., 1997; Harding et al., 1999]. Furthermore, the daily oral administration of a cod liver oil extract containing high amounts of DHA led to a marked clinical and electrophysiological recovery of the progressive peripheral sensorimotor axonopathy in one boy with the myoneuropathic form of LCHAD deficiency [Tein et al., 1999b]. The dose of DHA supplementation recommended by a European consensus panel for trifunctional protein complex, including LCHAD, deficiency is 60 mg/day in children weighing less than 20 kg, and 120 mg/per day in children of over 20 kg body weight [Spiekerkoetter et al., 2009a, b]. Higher amounts of DHA were used to reverse the neuropathy [Tein et al., 1999b].

Triheptanoin

Use of the anaplerotic odd-chain triglyceride, triheptanoic acid, has been reported to be of value in the therapy of long-chain FAO defects [Roe et al., 2002]. In three patients with very long-chain acyl-CoA dehydrogenase deficiency, who were fed controlled diets in which the fat component was switched from medium-even-chain triglycerides to triheptanoin, this treatment led rapidly to clinical improvement that included the resolution of chronic cardiomyopathy, myoglobinuria, and muscle weakness for more than 2 years in one child, and resolution of myoglobinuria and weakness in the others.

Peroxisome proliferator-activated receptor agonists

The peroxisome proliferator-activated receptor agonists (PPARs) have been shown to upregulate mitochondrial FAO [Lee et al., 2003; Feige et al., 2006]. Three different isoforms of these nuclear receptors have been described in mammalian cells – namely, PPARα, PPARδ and PPARγ – which exhibit distinct tissue distributions, ligand specificities, and physiological functions [Lee et al., 2003]. Bezafibrate, a hypolipidemic agent, is the only fibrate capable of activating the PPARδ isoform together with PPARα [Peters et al., 2003; Tenenbaum et al., 2005]. It has been found to increase palmitate oxidation in fibroblasts from patients with a typical mild myopathic phenotype of CPT II or VLCAD deficiency, but had no effect in cells from severely affected patients in whom the mutations were severe and likely involved residues that were essential for the architecture of the active site [Djouadi and Bastin, 2008]. One of the reported adverse effects that should be monitored is possible drug-induced increase in serum CK; however, reports of bezafibrate-induced myoglobinuria are rare, and it has only been described in patients with renal insufficiency who tend to accumulate the drug [Monk and Todd, 1987]. Djouadi and Bastin [2008] recommend that FAO tests in patient fibroblasts may offer a reliable way to assess responsiveness to the drug and, with the combination of clinical, biochemical, and molecular analysis, should provide a rational framework for patient stratification in any future clinical bezafibrate trials in CPT II- or VLCAD-deficient patients. The practical applications of bezafibrate require future studies.

mtDNA rearrangements: single deletions and duplications

Single deletions of mtDNA have been associated with three conditions that are usually sporadic [DiMauro and Hirano, 2003]:

Duplications of mtDNA can occur in isolation or together with single deletions, and have been documented in patients with Kearns–Sayre syndrome or with diabetes mellitus and deafness [DiMauro, 2004]. Duplications and duplications/deletions are rare and are usually transmitted by maternal inheritance.

mtDNA point mutations

Point mutations have been identified in mtDNA from a variety of disorders, most of which are maternally inherited and multisystemic, though some are sporadic and tissue-specific. Two of the more common syndromes among the maternally inherited encephalomyopathies are the MERRF and MELAS syndromes. Syndromes associated with tRNA mutations may be multisystemic, affecting the eye (optic atrophy, retinitis pigmentosa, cataracts), hearing (sensorineural hearing loss), endocrine system (short stature, diabetes mellitus, hypoparathyroidism), heart (hypertrophic cardiomyopathies, cardiac conduction blocks), gastrointestinal tract (exocrine pancreatic dysfunction, intestinal pseudo-obstruction, gastroesophageal reflux), and kidney (renal tubular acidosis).

ATPase 6 mutation

These maternally inherited disorders present with either Leigh’s syndrome or developmental delay, retinitis pigmentosa, dementia, seizures, ataxia, proximal weakness, and sensory neuropathy, and are caused by a point mutation at nt 8993 within the ATPase 6 gene [Holt et al., 1990]. The severity of disease presentation appears to correlate with the percentage of mutant mtDNA. When the degree of heteroplasmy is moderate (around 70 percent), the clinical expression is NARP, a subacute or chronic disease of young adults; but when the degree of heteroplasmy is very high (about 90 percent), the clinical expression is a rapidly progressive encephalopathy of infancy or childhood – Leigh’s syndrome [Tatuch et al., 1992]. MILS is a more severe infantile encephalopathy with characteristic symmetric lesions in the basal ganglia and the brainstem. MILS patients present with dysmorphism, developmental delay, epilepsy, strokelike episodes, dystonia, ophthalmoparesis, myopathy, or polyneuropathy [Finsterer et al., 2009]. NARP and MILS may coexist in the same family. There are two recognized mutations: namely, T8993G and T8993C, with the T to G transversion being both clinically and biochemically more deleterious than the T to C change [Vázquez-Memije et al., 1998].

Leber’s hereditary optic neuroretinopathy

LHON is characterized by the onset, usually between 18 and 30 years of age, of acute or subacute loss of vision resulting from severe bilateral optic atrophy. There is a marked male predominance [Newman and Wallace, 1990]. The classic ophthalmologic features include circumpapillary telangiectatic microangiopathy and pseudoedema of the optic disc. Associated features may include cardiac conduction abnormalities (pre-excitation syndrome), cerebellar ataxia, hyperreflexia, and peripheral neuropathy. About a dozen different mtDNA point mutations in structural genes have been associated with LHON, but only three appear to be pathogenic, even when present in isolation. A homoplasmic G to A transition at nt 11778, which causes the replacement of a highly conserved arginine residue by histidine at position 340 of the ND4 protein of complex 1, was first described in 11 American pedigrees [Wallace et al., 1988a]. Another point mutation was found, in which a G to A transition at nt 3460 in the ND1 gene was identified in three Finnish families [Huoponen et al., 1991]. A third pathogenic mutation – namely, T14484C in ND6 – has been identified [Carelli, 2002]. All three mutations affect genes of complex I. Predominance of affected men (approximately 50 percent of males but only approximately 20 percent of females have optic atrophy) has been attributed to the influence of the nuclear gene on the X chromosome [DiMauro, 1993]. Complex I deficiency was demonstrated in platelets from four affected members of an Australian family in which the molecular genetic defect was not documented [Parker et al., 1989].

Interestingly, point mutations in mtDNA protein-coding genes often escape the rules of mitochondrial genetics, in that they affect single individuals and single tissues, and most commonly involve skeletal muscle [DiMauro et al., 2003]. For example, patients with exercise intolerance, myalgia, and, occasionally, recurrent myoglobinuria, may have isolated defects of complex I, complex III, or complex IV, due to pathogenic mutations in genes encoding ND subunits, COX subunits, and, in particular, cytochrome b [Andreu et al., 1999]. The lack of maternal inheritance and the involvement of muscle alone would suggest that the mutations arose de novo in myogenic stem cells after germ-layer differentiation (“somatic mutations”) [DiMauro, 2004]. mtDNA COX gene mutations are important causes of exercise intolerance and myoglobinuria. Three young adults have been reported with life-long exercise intolerance, exertional myalgia, and at least one episode of myoglobinuria. All three cases were sporadic, harboring presumably somatic, heteroplasmic mutations confined to skeletal muscle and affecting COX 1 (G6708A) [Kollberg et al., 2005], COX II (T7989C) [McFarland et al., 2004], and COX III (T9789C) [Horvath et al., 2005].

In complex I deficiency, mutations in ND1 and ND4 have been associated with pure myopathy [Hays et al., 2006]. Mutation in ND2 (G4810A) may cause severe exercise intolerance, followed by mild ptosis and progressive external ophthalmoplegia [Pulkes et al., 2005].