Glycogen Storage Diseases of Muscle

Phosphorylase Kinase Deficiency

Phosphorylase b kinase (PhK) is a regulatory enzyme in the activation cascade of glycogenolysis. By phosphorylating and thus activating glycogen phosphorylase, PhK stimulates glycogen degradation in response to various neural and hormonal signals.

The enzyme is a decahexameric protein composed of four subunits (α,β,γ,δ). Subunits α and β are regulatory, γ is catalytic, and δ is identical to calmodulin. Two isoforms are known for subunit α (muscle and liver, α _M and α _L ) and for subunit γ (muscle and testis, γ _M and γ _T ). The genes encoding α _Μ ( PHKA1 ) and α _L ( PHKA2 ) are both on the X chromosome, whereas the β ( PHKB ), γ _M ( PHKG1 ), and γ _T ( PHKG2 ) genes are autosomal.

PhK deficiency is associated with four different phenotypes, distinguished by tissue involvement (liver, muscle, liver and muscle, heart), and mode of inheritance (X-linked or autosomal).

• 1.

  Liver disease, the most frequent type, is usually a benign condition of infancy or childhood, characterized by hepatomegaly, growth retardation, delayed motor development, and fasting hypoglycemia. Inheritance is X-linked recessive, due to mutations in PHKA2 . The enzyme defect is also expressed in erythrocytes and leukocytes, but not in muscle.

• 2.

  Liver and muscle disease is characterized by hepatomegaly, usually resolving with age, and nonprogressive myopathy. Transmission appears to be autosomal recessive in most cases and due to mutations in PHKB . The enzyme defect is expressed in muscle and liver.

• 3.

  Myopathy, inherited as an autosomal recessive or as an X-linked recessive trait, has been reported in about 20 cases. The clinical picture consists of exercise intolerance, with cramps, myalgia, and weakness of exercising muscle. Myoglobinuria after strenuous exercise has been described in less than 50% of the cases. Onset is usually in childhood or adolescence. Although fixed weakness was only present in a few cases, one of the patients reported by Clemens and associates, a 58-year-old man, had progressive weakness starting at age 46 and affecting distal more than proximal muscles. In most patients, serum CK level is variably increased.

  The FIET is often normal or only mildly altered, and electromyography (EMG) can be normal or show nonspecific myopathic changes. Muscle biopsies may be normal or show subsarcolemmal accumulation of glycogen. Although PhK activity in muscle homogenate is absent or markedly decreased, the generally mild presentation of this myopathy has raised the question of whether this is indeed a disease or merely a metabolic variant.

• 4.

  Cardiomyopathy has been described in a few infants and is well defined in terms of clinical pathology and biochemistry. The clinical phenotype is uniformly severe and survival has not exceeded months. The isolated involvement of the heart was a riddle because there is no cardiac isoform of PhK. Now we know that the PhK deficiency in these cases is secondary to de novo mutations in the gene ( PRKAG2 ) encoding the γ2 subunit of the AMP-activated protein kinase (AMPK).

Myophosphorylase Deficiency

Muscle phosphorylase (PYGM) initiates glycogen breakdown by removing 1,4-glucosyl residues phosphorolytically from the outer branches of the glycogen molecule, with liberation of glucose-1-phosphate: this goes on until the peripheral chains are shortened to about four glucosyl units. The degraded polysaccharide is known as phosphorylase-limit dextrin (PLD), and its short peripheral chains are removed by the debranching enzyme. In muscle, phosphorylase exists in two forms: a more active, phosphorylated a form, and a less active, dephosphorylated b form. The phosphorylation of myophosphorylase is catalyzed by PhK.

Human phosphorylase has three isozymes, muscle (PYGM), liver (PYGL), and brain/heart (PYGB), encoded by different genes that have been mapped on different chromosomes. Normal adult human muscle expresses only the muscle isozyme, whereas heart and brain express, in different amounts, both the brain and the muscle isoforms. In particular, heart contains the muscle isozyme (13%), the brain isozyme (58%), and a hybrid of the two isozymes (29%). Brain contains the muscle isozyme (8%), the brain isozyme (64%), and a hybrid of the two (28%), whereas liver contains exclusively the liver isozyme. Therefore, patients with the muscle isozyme deficiency must also have partial defects in heart and brain; however, symptoms of heart or brain involvement have not been reported.

A defect of the muscle isoform underlies glycogenosis type V (McArdle disease), whereas phosphorylase deficiency in liver is referred to as glycogenosis type VI (Hers’ disease).

McArdle disease is characterized by exercise intolerance with premature fatigue, myalgia, and cramps (see Case Example 39.1 ). Symptoms usually occur during brief and intense exercise (pushing or lifting heavy objects) or during sustained, but less intense exercise (climbing stairs). Most patients experience the so-called second-wind phenomenon: if they rest briefly at the onset of symptoms, they can resume exercising without discomfort. Myoglobinuria occurs in about 50% of patients and half of these develop renal failure. Although exercise intolerance is present from childhood, cramps and myoglobinuria develop later in life, and the diagnosis is usually established in young adult life.

Although the clinical phenotype is rather uniform, a few variants have been reported, including a mild form with excessive tiredness and poor stamina, a late-onset form with fixed weakness in the fifth to sixth decade without cramps or myoglobinuria, and a rare fatal infantile form characterized by weakness, severe respiratory insufficiency, and early death. Congenital myopathy and mental retardation were reported in a 4-year-old boy.

Laboratory tests show increased resting serum CK, flat venous lactate response to ischemic exercise, and myopathic EMG. The diagnosis is confirmed by the histochemical or biochemical documentation of phosphorylase deficiency in muscle and by molecular analysis of DNA. The presence of subsarcolemmal or intermyofibrillar deposits of glycogen gives muscle a vacuolar appearance ( Figure 39.3A,B ).

In patients with myophosphorylase deficiency, phosphorylase activity is normal in erythrocytes, leukocytes, platelets, and fibroblasts. Therefore, the diagnosis relies on measurement of phosphorylase activity in muscle or on genetic testing.

The histochemical reaction for phosphorylase is a valuable diagnostic tool because in most patients muscle fibers have no enzyme activity ( Figure 39.3D,E ). However, the phosphorylase reaction can be positive in rare cases with some residual enzyme activity and in biopsy specimens with abundant regenerating fibers. This false-positive reaction, which is due to the expression of PYGMB, may be misleading in muscle biopsy specimens taken too soon after an episode of myoglobinuria, when regeneration is at its most active.

The myophosphorylase gene has been cloned, assigned to chromosome 11q1, and its genomic structure has been described. The disease is transmitted as an autosomal recessive trait, but pseudo-dominant transmission has been documented in a few families. The molecular heterogeneity of myophosphorylase deficiency is striking: in the 20 years from the description of the first molecular defects, more than 100 different mutations have been reported. The most common mutation in North America and Northern Europe is the R50X. This has allowed the use of molecular genetic analysis in leukocytes for diagnostic purposes, thus circumventing the need for a muscle biopsy. However, as leukocytes are increasingly used for diagnosis, it becomes important to keep in mind the relative frequency of distinct mutations in different ethnic groups. For example, the R50X mutation has never been observed in Japan, where the most common mutation appears to be a 3-bp deletion, TTC at codon 708/709. The genotype/phenotype relationship in McArdle disease remains unclear. For example, the common R49X mutation was also found in an infant with the fatal variant, in a child who died of sudden infant death syndrome, and in two virtually asymptomatic children with persistent and unusually high levels of serum CK.

The exercise intolerance in McArdle disease is due to two main mechanisms. First, the block of anaerobic glycolysis deprives muscle of the energy required for isometric exercise. Second, the block of aerobic glycogen utilization and the consequent shortage of pyruvate and acetyl-CoA impairs dynamic exercise above a certain intensity (about 50% VO _2max ). The impairment of oxidative phosphorylation is evidenced by the decrease in oxygen extraction and maximum oxygen uptake documented in patients with myophosphorylase deficiency, which can at least partially be restored by intravenous glucose infusion.

There is no specific therapy for McArdle disease, but regular moderate aerobic training is effective because it favors alternate fuel delivery and utilization. In cases with residual phosphorylase activity, vitamin B6 should be tried because the overall body stores of pyridoxal phosphate (PLP) are depleted in McArdle disease due to the frequent lack of enzyme protein (to which PLP is bound ). Patients should be warned about the risks of strenuous exercise and advised to seek medical attention at the first appearance of pigmenturia, especially if accompanied by oliguria. Sucrose ingestion before exercise is beneficial but may lead to weight gain.

The two spontaneous animal models of McArdle disease—Charolais cattle and Merino sheep —are not practical for experimentation. However, a knock-in mouse model of the R50X mutation recapitulates faithfully the human disease and will provide valuable information on pathophysiology and experimental therapy.

Debranching Deficiency

Debranching enzyme is a monomeric protein encoded by a single gene and expressed ubiquitously in all tissues. It has two independent catalytic activities: transferase and α-glucosidase. After phosphorylase has shortened the peripheral chains of glycogen to about four glucosyl units, these residual stubs are removed by the debranching enzyme in two steps, catalyzed by an oligo-1,4-1,4-glucantransferase and by an amylo-1,6-glucosidase. The transferase and glucosidase activities can be assessed separately or together by using appropriate substrates.

Debrancher deficiency (glycogenosis type III) typically presents as a childhood-onset liver disease with hepatomegaly, growth failure, fasting hypoglycemia, and, less frequently, hypoglycemic seizures. Liver symptoms improve with age and often resolve completely around puberty.

Although clinical cardiopathy is infrequent, cardiac involvement is demonstrable by laboratory tests in most patients with myopathy and a child was reported to have died of cardiac failure at 4 years of age.

Myopathy is not always manifest in infants or children, who may show hypotonia, delayed motor milestones, or mild weakness. Myopathy often appears in adult life, long after liver symptoms have remitted or in patients without any history of hepatopathy. However, in 7 of 22 patients reviewed by Cornelio and colleagues onset was in childhood and the diagnosis was facilitated by the coexistence of liver disease. Two of these children had exercise intolerance, cramps, and premature fatigue, and five had diffuse weakness and wasting, growth retardation, and delayed motor development.

Adult-onset myopathies can be predominantly distal or generalized. Patients with distal myopathy develop wasting of distal leg and intrinsic hand muscles, often leading to the diagnosis of motor neuron disease or peripheral neuropathy: the “mixed” myopathic and neurogenic EMG pattern and the often slowed nerve conduction velocity suggests that weakness in these patients may have a neurogenic component. Patients with generalized myopathy tend to have more severe weakness, often affecting respiratory muscles.

It may seem paradoxical that the typical presentation of debrancher deficiency is so different from that of McArdle disease, although the two defective enzymes act sequentially in the first step of glycogenolysis. However, cycle ergometry with or without glucose infusion in six young patients with debrancher deficiency and no or only mild proximal weakness has documented that peak oxygen consumption was below normal and glucose improved work capacity by lowering heart rate and increasing peak work rate. Thus, even before developing overt weakness, patients with debrancher deficiency have exercise intolerance, not unlike patients with McArdle disease, albeit to a much lower extent.

Muscle biopsy specimens typically show large PAS-positive vacuoles containing diastase-digestible glycogen under the sarcolemma and between myofibrils ( Figure 39.4A,B ). Ultrastructurally, the vacuoles are collections of free and apparently normal glycogen β-particles ( Figure 39.4C ).

The gene encoding human debrancher ( AGL ) has been cloned, sequenced, and assigned to chromosome 1p2, and several different mutations have been identified.

Although there is no specific therapy for debrancher deficiency, various interventions, including dietary regimens, liver transplantation, physical therapy, and precautions with anesthesia and pregnancy, are discussed in a review article.

A Pediatrician’s Perspective

The disorders of glycogenolysis described earlier, and especially those affecting muscle, tend to affect adults more than children. However, the following exceptions are worthy of note.

The “liver and muscle” variant of PhK deficiency is typically seen in childhood. An interesting phenomenon is illustrated by a large family that we reported in 1985. Seven male children had an X-linked disorder characterized by hepatomegaly, hypotonia, and weakness in childhood (but resolving with age), delay in growth and sexual maturation, and gouty arthritis. Glycogen storage was severe in liver but also present in muscle, both histochemically and biochemically. We considered PhK deficiency a likely diagnosis, but found normal enzyme activities in both liver and muscle. In 1997, molecular analysis in the propositus by Drs. Jan Hendrickx and Patrick Willems (University of Groningen, the Netherlands) revealed a pathogenic mutation in the gene encoding the liver α-subunit of PhK ( PHKA2 ). As these authors have documented, there are two subgroups of X-linked glycogenosis, XLGI and XLGII, distinguished by the fact that PhK deficiency is demonstrable in liver from patients with XLGI but not in liver from patients with XLGII. Mutational analysis has shown that molecular defects in XLGI affect the stability of the protein whereas those in XLGII affect the regulation of enzyme activity, which probably explains the normal PhK activity that we had observed in two tissues. Although hypotonia in these children can be attributed to liver dysfunction, weakness and glycogen storage in muscle are more difficult to explain. Irrespective of the exact pathogenesis, mutations in PHKA2 should be considered in the differential diagnosis of children with liver and muscle glycogenosis, even in the absence of enzyme deficiency.

Myophosphorylase deficiency (McArdle disease) is typically a disease of young adults and is usually diagnosed in children only when older siblings are affected. In addition to the rare fatal infantile variant, pediatricians should also remember that early onset of symptoms, including myoglobinuria, can be due to genetic “double-trouble”—that is, the coexistence of homozygous mutations in the myophosphorylase gene and in the gene encoding myoadenylate deaminase.