Galactose-1-Phosphate Uridyltransferase Deficiency

Galactose-1-phosphate uridyltransferase (GALT) deficiency is by far the most common cause of galactosemia. The incidence of galactosemia in Western Europe varies between 1:23,000 and 1:44,000 [Bosch, 2006]. Neonatal screening programs have found population incidence rates as high as 1 in 19,700 in Estonia [Ounap et al., 2010].

Biochemistry

The primary abnormality in galactosemia is the deficiency of activity of galactose-1-phosphate uridyltransferase (Figure 34-1) that leads to accumulation of galactose-1-phosphate in red blood cells, liver, and brain [Lai et al., 2009].

Fig. 34-1 Pathway of galactose metabolism depicting sites of metabolic block that lead to galactosemia.

ATP, adenosine triphosphate; NAD, nicotinamide adenine dinucleotide; UDP, uridine diphosphate.

Galactose is metabolized through the following four possible pathways [Fridovich-Keil, 2006]:

1. the reduction of galactose to galactitol

2. oxidation of galactose to galactono-γ-lactone

3. the reaction of galactose-1-phosphate with uridine triphosphate to form uridine diphosphate galactose and eventually glucose-1-phosphate

4. the reaction of galactose-1-phosphate with uridine diphosphate-glucose to form uridine diphosphate-galactose and glucose-1-phosphate.

In classic galactosemia, the fourth pathway is obstructed; the other pathways function normally.

Shih and colleagues assigned the GALT locus to 9p13 by gene dosage [Shih et al., 1984]. A number of mutations produce abnormal enzymes with little or no galactose-1-phosphate uridyltransferase activity [Reichardt, 1991; Reichardt et al., 1992]. It is therefore not surprising that various genetic forms of galactosemia result from the presence of inefficient isoenzymes of galactose-1-phosphate uridyltransferase (Table 34-1). Most patients are compound heterozygotes, not true molecular homozygotes [Elsas et al., 1995].

Table 34-1 Biochemical Criteria for Assigning Phenotypes of Galactosemia*

The isoenzymes are separated and identified by electrophoresis. Cross-reactivity patterns for the enzyme variants have been studied using rabbit antibodies to purify human placental galactose-1-phosphate uridyltransferase [Andersen et al., 1984]. Transferase activity is absent in homozygous classic galactosemia (Q188R – the most common mutation in the United States, arising in Western Europe about 20,000 years ago [Flanagan et al., 2010]) and the African American variant (S135L – accounting for 8.4 percent of U.S. cases [Lai and Elsas, 2001]), virtually absent in the Rennes variant [Schapira and Kaplan, 1969], and abnormally low in the Chicago, Indiana, and Duarte (N314D) variants [Beutler et al., 1965; Lai et al., 1998; Levy et al., 1978]. Activity is normal in the Los Angeles variant (L218L) [Applegarth et al., 1976; Ng et al., 1973]. A screening method utilizing a standard newborn screening blood spot has been described that detects the four most common GALT alleles (Q188R, S135L, K285N, and L195P) and the N314D Duarte variant with a turnaround time of less than 2 hours [Dobrowolski et al., 2003]. By January 2010, 239 sequence variants had been described in the GALT mutation database [Calderon et al., 2007], most of which were missense mutations. The precise molecular mechanisms of galactosemia are not yet known, but a computational biology approach has yielded insights into the structure–function relationships of many mutations [Facchiano and Marabotti, 2010].

The precise mechanisms of central nervous system dysfunction and developmental aberrations remain unexplained. Hypoglycemia associated with galactosemia may be responsible for some of the neurologic (including higher cortical function) abnormalities observed in these patients, but the accumulation of unusual metabolites also may contribute to the pathogenesis of the disorder. Hypoglycemia may be the result of inhibition of phosphoglucomutase and accompanying failure of hepatic glycogenesis. The high concentration of galactose may increase both insulin production and release, and the increased galactose also may compete with glucose for transport sites and carrier mechanisms across the blood–brain barrier.

Galactose-1-phosphate accumulates in erythrocytes, liver, lenses, and kidneys, and may be influential in disruption of both tissue structure and function, but galactitol accumulation likely is a more important factor in cataract development and perhaps other tissue disruptions and cerebral dysfunction.

Galactokinase or transferase activity appears to be lower than expected in many women who give birth to nongalactosemic children with cataracts [Harley et al., 1974].

Uridine Diphosphogalactose Epimerase Deficiency

Uridine diphosphogalactose epimerase (GALE) deficiency has conventionally been separated into peripheral and generalized forms, implying a dichotomy between levels of enzyme activity in blood and other tissues. Ten children in a study were diagnosed with peripheral GALE deficiency as neonates were found to have a range of GALE activity in lymphoblasts (i.e., nonperipheral tissue), that was correlated with metabolic abnormalities in some patients, implying that this is a spectrum disorder, and not a binary condition as suggested in the older literature [Openo et al., 2006]. Children previously recognized with generalized (severe) deficiency of GALE (see Figure 34-1) [Bowling et al., 1986; Garibaldi et al., 1986; Sardharwalla et al., 1988; Walter et al., 1999] had manifestations resembling those in classic galactosemia. Most survivors were dysmorphic and deaf. The GALE locus is at 1p36–p35; the human GALE gene is about 4 kilobases (kb) in length and contains 11 exons [Maceratesi et al., 1998]. The coding sequence of the GALE gene and screening for mutations in epimerase-deficient persons have been reported by the same investigators. The patients are either homozygotes or compound heterozygotes for mutations. Two forms of enzyme deficiency were originally described, one type benign (with expression restricted to the lens) and the other severe [Quimby et al., 1997], but an intermediate form is now recognized [Openo et al., 2006]. Three mutations (S81R, T150M, and P293L) have been reported in children with this intermediate form of GALE deficiency [Chhay et al., 2008]. Patients with GALE deficiency require exogenous galactose for the synthesis of glycolipids and glycoproteins.

Galactokinase Deficiency

Galactokinase deficiency was first detected in the Bulgarian gypsy (Romany) population. Its birth incidence varies, ranging from a high of 1 in 52,000 in Bulgaria to 1 in 2,200,000 in Switzerland [Kalaydjieva et al., 1999]. Deficiency of galactokinase activity causes a clinical condition similar to that in classic galactosemia. Patients have cataracts and accumulation of galactose. As in galactosemia, galactitol, a reduction metabolite of galactose, is found in the urine and tissues [Egan and Wells, 1966]. The existence of two GALK genes is likely. The GALK1 gene is located at 17q24 [Bergsma et al., 1996]. GALK2 may reside on chromosome 15 [Lee et al., 1992]; its metabolic role is unknown, but it does not appear to be necessary for galactose metabolism. No mutations in GALK2 have been described. The structure of GALK1 has been solved and its relationship to other members of the GHMP kinase superfamily defined, as well as its role as an essential componenet of the ‘switch’ permitting expression of the Leloir pathway genes in the presence of galactose [Holden et al., 2004]. More than 20 mutations in GALK1 have been described, most in compound heterozygotes for private mutations [Sangiuolo et al., 2004]. Only one common mutation, P28T, has been recognized in the Romany population [Kalaydjieva et al., 1999].

Biochemistry

Fructose is rapidly absorbed from the gut, facilitated by the glucose transporters GLUT 2 and GLUT5, and is metabolized in the liver by the fructokinase pathway, through which it is linked to glycolysis, gluconeogenesis, glycogenolysis, and lipid metabolism. Fructose can also be synthesized endogenously from sorbitol, an important point in management [Bouteldja and Timson, 2010]. Hereditary fructose intolerance was first described by Chambers and Pratt [1956]. This condition results from a deficiency of hepatic fructose-1-phosphate aldolase B [Hers and Joassin, 1961]. (Isoenzyme A is found in most vertebrate tissues and isoenzyme C in brain.) The gene encoding this enzyme maps to 9q22 [Henry et al., 1985]. Normal fructokinase activity results in the accumulation of large amounts of fructose-1-phosphate in the liver and kidneys. Fibroblasts from patients with hereditary fructose intolerance consume less glucose, produce less lactate, and contain less glycogen compared with control cells [Lemonnier et al., 1987]. A radioisotopic method for fructose-1-phosphate assay is available [Shin et al., 1983]. The enzyme deficiency is inherited as an autosomal-recessive trait and has an estimated prevalence in central Europe of 1:26,100 (95 percent confidence interval 1: 12,600–79,000) [Santer et al., 2005].

Urinary fructose excretion also is present in a harmless metabolic variant resulting from fructokinase deficiency that should not be confused with hereditary fructose intolerance.

Clinical Characteristics and Differential Diagnosis

Patients with hereditary fructose intolerance who ingest fructose experience nausea and vomiting; with continued exposure, weight gain is poor. Hypoglycemia begins immediately and reaches its low point 30–90 minutes after ingestion [Froesch et al., 1963]. Subsequent clinical and neuropathologic alterations result primarily from the hypoglycemia. Immoderate fructose feedings lead to albuminuria, jaundice, and a generalized aminoaciduria. Examination reveals jaundice and hepatomegaly.

Neurologic impairment is relatively uncommon but may result from hypoglycemia, cardiovascular collapse, or liver failure. Central nervous system complications include seizures with subsequent epilepsy, increased intracranial pressure, mental retardation, quadriplegia, and deafness [Labrune et al., 1990; Rennert and Greer, 1970]. Imaging findings include cortical atrophy, hydrocephalus, and parenchymal hemorrhage [Labrune et al., 1990].

Microscopic examination of the brain demonstrates neuronal loss and decreased myelination, attributed to hypoglycemia. Cirrhosis of the liver usually is marked.

Clinical Laboratory Tests and Diagnosis

The diagnosis may be corroborated by intravenous fructose challenge (0.1–0.2 g/kg, with adequate precautions to manage hypoglycemia). A positive response includes hypoglycemia, hypomagnesemia, hypouricemia, and hyperphosphatemia. Assay of fructose-1-phosphate aldolase B in liver tissue permits definitive diagnosis, but liver biopsy may be avoided by direct genotyping in most patients. Although more than 20 mutations of the aldolase B gene have been described, two alleles (A149P and A174D) account for 70 percent or more of cases in Western Europe and North America, and may be readily detected in leukocytes by restriction fragment length polymorphism (RFLP) analysis [Kullberg-Lindh et al., 2002]. Fructosemia causes abnormal glycosylation of transferrin, leading to misdiagnosis of CDG1x on occasion [Quintana et al., 2009].

Management

Fructose elimination from the diet is accomplished by limited selection of vegetables and cereal products. Sorbitol should also be avoided, as it is an endogenous source of fructose. Dietary counseling is required for proper therapy [Bell and Sherwood, 1987].

Pathology

In 1929, Von Gierke described the pathology of “hepatonephromegalia glycogenica,” and in 1952 the Coris described glucose-6-phosphatase deficiency as its cause (and established an enzyme deficiency as the cause of an inborn error of metabolism for the first time) [Moses 2002]. Patients with von Gierke’s disease, now known as glycogen storage disease type I, have hepatomegaly and renomegaly. Light microscopy reveals enormous amounts of glycogen in liver cells and in the cells of the renal convoluted tubules. No increase in the concentration of glycogen is found in skeletal muscle, tongue, or heart. Best’s stain marks the presence of glycogen. Tissue must be fixed in a nonaqueous medium such as absolute alcohol; otherwise, the glycogen dissolves, and only vacuolar spaces will remain to mark the areas of deposit.

Electron microscopy of glycogen storage disease type I liver reveals loss of microvilli of the membranes lining Disse’s spaces and bile canaliculi. Changes in endoplasmic reticulum include the development of doubly contoured vesicles that appear to help ribosomes only on the innermost surface of the inner membrane. Glucose-6-phosphatase is located in or on the membranes of endoplasmic reticulum, and these findings parallel a disturbance of the phospholipid environment of the enzyme as a consequence of the mutation that affects enzyme activity [Spycher and Gitzelmann, 1971].

Biochemistry

Two distinct subgroups of glycogen storage disease type I have been identified: those with primary glucose-6-phosphatase deficiency (type Ia) and those phenocopies with additional features of immune impairment (neutropenia and neutrophil adherence defects), now designated as glycogen storage disease type I non-a [Moses, 2002]. Glycogen storage disease type I non-a disorders originally were thought to result from defects in a multicomponent translocase system responsible for transporting glucose-6-phosphatase into microsomes [Annabi et al., 1998]. This model postulated three transport proteins, T1, T2, and T3, to chaperone glucose, glucose-6-phosphatase, phosphate, and pyrophosphate across the endoplasmic reticulum membrane. Cloning of the glucose-6-phosphatase translocase gene (G6PT) demonstrated that the previously proposed subtypes b, c, and d all were associated with mutations in G6PT, producing different kinetic variants [Matern et al., 2002; Moses and Parvari, 2002]. The G6PC gene that codes for glucose-6-phosphatase is located at 17q21 [Brody et al., 1995]. A number of allelic variants have been described. Glucose-6-phosphatase comprises at least five different polypeptides. The G6P locus is at 11q23 [Annabi et al., 1998]; several mutations have been characterized [Matern et al., 2002].

All forms share common clinical manifestations that are attributable to abnormal metabolism of glucose-6-phosphate. In type Ia, glucose-6-phosphatase deficiency results in storage of glycogen of normal configuration in the liver and kidneys. The glycogen concentration usually exceeds 4 percent by weight. The enzyme activity frequently is absent or extremely low [Cori and Cori, 1952]. Glucose-6-phosphatase is important in regulating the entry of free glucose into the circulation from the liver. Because of this pivotal role, deficiency of the enzyme produces hypoglycemia.

Ethanol causes decreased blood lactate and pyruvate content, presumably by diverting carbon to triglyceride formation [Sadeghi-Nejad et al., 1975]. A study of insulin secretion in five adult patients with glucose-6-phosphatase deficiency found that their capacity to increase blood insulin was significantly less than normal. As patients with this condition mature, they become normoglycemic and characteristically have abnormal glucose tolerance curves. These studies suggest that the increasing clinical stability noted with age and the associated tendency toward normoglycemia reflect a decrease in insulin responsiveness that may develop as an adaptive process [Lockwood et al., 1969]. Diabetes mellitus has been reported in GSD type 1 [Spiegel et al., 2005]. The intricacies of the glucose-6-phosphatase system and its role in glucose metabolism have been reviewed [Foster and Nordlie, 2002].

Clinical Characteristics

Hypoglycemia causes much of the morbidity during the first year of life. Seizures are frequent and almost invariably are the presenting complaint of affected children. Hypoglycemia may result in severe, chronic neurologic impairment, including hemiplegia [Fine et al., 1969]. Hepatomegaly and the failure to thrive syndrome are commonly present. An association with moyamoya disease has been described [Goutières et al., 1997]. A study of 19 patients with glycogen storage disease type I (median age 11 years) in one center found prevalence rates for epilepsy, deafness, and neuroradiologic abnormalities of 10.5 percent, 15 percent, and 57 percent, respectively, far in excess of the rates in the general population, or in children with other causes of neonatal hypoglycemia. MRI abnormalities included dilatation of occipital horns and/or hyperintensity of subcortical white matter in the occipital lobes in all patients [Melis et al., 2004]. Subcutaneous fat often is increased, especially over the buttocks, breasts, and cheeks. Xanthomas of the skin occur over the extensor surfaces of the limbs and at times over the buttocks [Hou et al., 1996; Hou and Wang, 2003]. Affected children frequently have a protuberant abdomen because of massive enlargement of the liver. Hepatomegaly may be present at birth. The liver edge is hard and not tender. Careful palpation may reveal enlarged kidneys. Hepatic adenomas develop in between one-half and three-quarters of adults with glycogen storage disease I; about 10 percent undergo malignant transformation. Some data suggest that lower frequencies are associated with better dietary control [Lee, 2002]. Hepatocellular carcinoma has been reported as complicating hepatic adenomas, and may reflect poor metabolic control [Franco et al., 2005]. Patients carrying mutations that cause relatively mild expression of the disease in childhood, often without hypoglycemia (such as 727 G>T), are associated with adult presentation of hepatocellular carcinoma [Matern et al., 2002].

Type I non-a patients typically have recurrent stomatitis, frequent infections, and chronic inflammatory bowel disease secondary to neutropenia and neutrophil dysfunction [de Parscau et al., 1988]. The neutropenia seen in GSD1b has been attributed to endoplasmic reticulum and oxidative stress secondary to the G6PT deficiency [Chou et al., 2010]. Seventy-five percent of 36 GSD type I non-a patients had chronic gastrointestinal complaints, and 28 percent had proven inflammatory bowel disease. A further 22 percent had a highly suggestive history [Dieckgraefe et al., 2002].

Clinical Laboratory Tests

The diagnosis can be made by assaying the enzyme activity in liver and peripheral white blood cells [Maire et al., 1991]. Direct assay of hepatic glucose-6-phosphatase activity in liver remains the definitive diagnostic procedure but can be replaced by mutational analysis in many patients. Just five mutant alleles account for almost 70 percent of cases of glucose-6-phosphatase deficiency, so that mutation screening is a reasonable initial diagnostic approach, avoiding the risks and discomfort of liver biopsy [Matern et al., 2002]. Molecular analysis of the G6P and G6PT genes permits rapid confirmation of the diagnosis in most cases [Janecke et al., 2001].

Severe hypoglycemia frequently occurs because of the failure of glucose formation from glucose-6-phosphate. Postprandial blood glucose concentration may be exceedingly high. Severe acidosis, which may vary in degree but usually is associated with lacticacidemia and pyruvicacidemia, and hyperuricemia are frequent. The presence of ketoacidosis has been unduly stressed.

Hyperuricemia has often been documented, but is poorly understood. Adenosine triphosphate depletion has been postulated as a causative factor [Greene et al., 1978]. Ketoacidosis and hyperthermia during anesthesia in a child with GSD type I has been described [Edelstein and Hirshman, 1980]. Blood cholesterol, fatty acids, and triglycerides are elevated; overt lipemia may be present.

Decreased bone density documented by radiography may be related to chronic metabolic acidosis. Serum inorganic phosphate may be decreased in the presence of normal alkaline phosphatase activity.

Provocation with epinephrine or glucagon demonstrates failure of the patient’s blood glucose concentration to increase within 10–20 minutes, although intracellular glucose-6-phosphate is formed quickly after the induced activation of liver phosphorylase.

Management

The goal of therapy is to provide sufficient free glucose to maintain a normal blood glucose concentration. Continuous nocturnal intragastric infusion of glucose has been relatively successful [Greene et al., 1976]. Subsequently, the use of cornstarch suspensions given during the day obviated the need for nocturnal infusion in some children [Chen et al., 1984; Wolfsdorf et al., 1990]. The dietary carbohydrate must be monitored because excess glucose leads to glycogen storage in the liver and kidneys. Frequent small feedings of carbohydrates are provided. Severity of the disease reaches a plateau after the fourth or fifth year of life. Vigorous treatment is, therefore, worthwhile until the plateau is reached. A long-term study of 15 children with GSD type I, beginning in infancy, found that careful metabolic control, aiming for high to normal plasma glucose levels and normal urine lactate, was associated with normal growth and lowering, but not normalization, of plasma lipids. Hepatic adenomas or renal impairment developed in none of the patients who reached adolescence [Daublin et al., 2002; Weinstein et al., 2002].

Dietary substitution of medium-chain triglycerides for long-chain triglycerides was attempted in glucose-6-phosphatase deficiency, to alter the hyperlipemic state by means of the unique absorptive and metabolic properties of medium-chain triglycerides. The results suggested that substitution of medium-chain triglycerides for long-chain triglycerides in the diet, along with normal carbohydrate consumption, leads to significant decrease in serum lipid levels, disappearance of eruptive xanthomas, and decrease in liver mass [Cuttino et al., 1970].

The hyperglycemic agent diazoxide has been beneficial [Rennert and Mukhopadhyay, 1968]. The drug’s action is not well understood, but normal blood glucose concentration has been maintained with this drug. Phenytoin also has been used [Jubiz and Rallison, 1974].

Surgical treatment for glucose-6-phosphatase deficiency involves creation of a portacaval shunt, which increases the peripheral blood glucose by allowing portal blood to bypass the liver after absorption of glucose from the gut; excellent metabolic control can be achieved over the long term, and the operation does not preclude subsequent liver transplantation [Corbeel et al., 2000]. The postoperative course has been complicated by severe hypoglycemia, hypocalcemia, acidosis, and respiratory impairment, the last primarily because of hepatomegaly. Preoperative intravenous hyperalimentation appears to eliminate these metabolic problems, reduce the size of the liver, and provide a smoother and shorter postoperative course [Folkman et al., 1972]. Liver transplantation was reported to produce beneficial results [Malatack et al., 1983]. One report suggested that this procedure, usually indicated for management of multiple hepatic adenomas, does not itself benefit metabolic control, and indeed, careful systemic management is essential to prevent graft complications [Labrune, 2002]. A more recent report of living donor liver transplantation in four children with GSD1b described markedly improved quality of life [Kasahara et al., 2009]. Another case report described a 47-year-old woman with GSD1a, whose fasting tolerance was significantly improved after infusion of hepatocytes [Muraca et al., 2002].

Chronic inflammatory bowel disease similar to Crohn’s disease has been associated with GSD type I non-a [Dieckgraefe et al., 2002]. Initial studies suggested benefit from therapy with colony-stimulating factors [Roe et al., 1992]. A retrospective study of 57 patients with GSD type I non-a found evidence of less frequent infections and diminished severity of inflammatory bowel disease in those who received granulocyte colony-stimulating factor [Visser et al., 2002]. Splenomegaly was associated with granulocyte colony-stimulating factor therapy in this group. Renal disease also may ensue in older patients [Chen, 1991].

Brain abscess has been reported in a patient with type I non-a disease [Park et al., 1991]. Renal transplantation has been used for terminal renal failure, and occasionally, combined hepatic and renal grafting has been used. In both circumstances, meticulous systemic metabolic management is essential to successful outcome [Labrune, 2002].

Pathology

Infants with GSD type II have a severe vacuolar myopathy, with accumulation of large amounts of periodic acid–Schiff-positive material within cardiac, skeletal, and smooth muscle fibers and in liver, renal tubules, lymphocytes, glial cells, anterior horn cells, and brainstem nuclei, in infantile cases. Storage in later-onset cases is largely restricted to skeletal muscle. Large amounts of metachromatic material are found within the muscle fibers in the infantile cases. Metachromasia is not seen as often in the adult cases. The metachromasia reflects glycolipid or glycoprotein accumulation. Glycogen also accumulates in anterior horn cells. Scattered, sparse, perivascular lymphocytic infiltrates are seen in the interstitial tissue [Hudgson and Fulthorpe, 1975].

Biochemistry

Hers [1963] first reported the deficiency of activity of the lysosomal enzyme acid maltase (α-1,4-glucosidase), located at 17q25.2–q25.3 [Kuo et al., 1996; Martiniuk et al., 1985]. Glycogen structure has consistently been normal, and its accumulation is restricted primarily to lysosomes, although lysosomal breakdown and cytoplasmic accumulation with disruption of muscle fibers occur in severe cases. Although direct injury of muscle fibers by glycogen leaking from lysosomes was thought to be the major cause of contractile dysfunction, experimental and pathological evidence suggests that the accumulation of autophagosomes is the major culprit, and that these pathologic orgenelles impair the effectiveness of enzyme replacement therapy by acting as a sink for infused enzyme [Shea and Raben, 2009; Raben et al., 2009]. Hypoglycemia is not a feature of this condition, but increased protein turnover with increased leucine flux and oxidation and increased resting energy expenditure has been found in late-onset cases [Bodamer et al., 2000].

Attempts have been made to differentiate biochemically among the infantile, late infantile, and adult-onset forms of acid maltase deficiency. Activity of α-1,4-glucosidase (acid maltase) at various pH values in infants, children, and adults with acid maltase deficiency has been studied. Only traces of neutral maltase are found in the heart, and significantly decreased neutral maltase activity was measured in the skeletal muscle and liver of an affected infant. In the late infantile form, neutral acid maltase activity is decreased only in the liver; in the adult form, neutral maltase is not deficient in any tissue. An absolute decrease of leukocyte acid maltase was found in four adults and a relative decrease in 1 of 5 adults with acid maltase deficiency. Decrease in the pH ratio of acid to neural maltase activity in leukocytes may be of diagnostic importance in adult acid maltase deficiency [Angelini and Engel, 1972].

A number of allelic variations have been described and may explain the differences in age at onset. The theoretical abnormalities that could result in a deficiency of α-glucosidase include synthesis of catalytically inactive protein, absence of messenger RNA (mRNA) for the enzyme, decreased synthesis of the precursor, lack of phosphorylation of the precursor, impaired conversion of the precursor to the mature enzyme, and synthesis of unstable precursor [Tager et al., 1987; Zhong et al., 1991]. In general, the location and nature of mutations predict the phenotype, but exceptional cases are described in which relatively mild phenotypes occur despite low levels of α-glucosidase expression in cultured cells and in the patient’s tissues [Hermans et al., 2004; Kroos et al., 2004]. Thus far, unidentified genetic modifiers and environmental factors are presumed to account for such variability.

Unexplained storage of increased neutral lipid is coupled with low carnitine concentration and reduced β-hydroxyacyl-CoA dehydrogenase in muscle [Verity, 1991].

Clinical Characteristics

Development usually is normal for several weeks to several months; then the affected infant presents with feeding difficulties, weakness, or respiratory impairment. The median age at presentation was 1.6 and 1.9 months, respectively, in 20 Dutch patients and 133 patients described in the literature [Van den Hout et al., 2003] (Figure 34-4). Little spontaneous movement occurs, and the cry is short-lived and weak. Swallowing is grossly limited, and secretions pool in the posterior oropharynx. Respiratory difficulty reflects weakness of the accessory muscles of respiration [Tanaka et al., 1979]. Massive cardiomegaly develops, and a soft systolic murmur is often heard along the left sternal border [Pompe, 1932]. Obstruction to ventricular outflow and impairment of inflow may develop [Seifert et al., 1992], and serial echocardiography reveals progressive left ventricular posterior wall diastolic thickening [Van den Hout et al., 2003]. Hepatomegaly is almost universally present. The liver has a sharp edge and a firm consistency on palpation. Subcutaneous fat over all areas of the body is sparse, and the muscles are small and firm. The tongue often is enlarged and may protrude. Intermittent cyanosis reflects respiratory and cardiac embarrassment. Deep tendon reflexes are lost by the age of 6 months. Affected infants undergo progressive debilitation, and most die, at median ages of 6 and 7.7 months in the literature cases and Dutch series, respectively. Fewer than 10 percent survive beyond 1 year; only two patients have been described who survived 18 months [Van den Hout et al., 2003], and almost all die by 2 years. A subgroup of children has been described who present later in infancy, with lesser degrees of weakness and cardiac impairment, who have survived for periods as long as 13 years with ventilatory and nutritional support [Slonim et al., 2000].

Fig. 34-4 This 8-month-old infant with infantile acid maltase deficiency (Pompe’s disease) is profoundly hypotonic and weak.

The child is draped over the examiner’s hand while the infant’s arms and legs are immobile against gravity.

(From Swaiman KF, Wright FS. Pediatric neuromuscular diseases. St. Louis: Mosby, 1979.)

Clinical Laboratory Tests

The quantity of glycogen storage and activity of the enzyme involved in glycogen metabolism can be monitored by using skin removed with a vacuum skin-blistering technique or fibroblasts grown from skin biopsy material. Among the enzymes in skin are phosphorylase, acid maltase, and the debranching enzyme amylo-1,6-glucosidase. No glucose-6-phosphatase activity is present in skin.

Both skin fibroblasts and amniotic fluid cells can be used for assay of acid maltase (α-1,4-glucosidase) activity [Butterworth and Broadhead, 1977; Leathwood and Ryman, 1971]. Prenatal diagnosis by biochemical study of uncultured amniotic fluid cells and chorionic villus biopsy material using maltose as a substrate have been reported [Hug et al., 1984; Park et al., 1992]. Mutational analysis has superseded these techniques in some cases, and complemented enzyme analysis and ultrasound examinations in others, as in a report of a fetus with this deficiency diagnosed in the second trimester, in which glycogen accumulation was detectable in muscle, as well as a visibly enlarged tongue on prenatal ultrasound examination [Chen et al., 2004].

A simple differential immunoprecipitation assay of urinary acid and neutral α-glucosidases has been developed [Tsuji et al., 1987].

The chest x-ray reveals massive cardiomegaly [Ruttenberg et al., 1964]. The electrocardiogram contains depressed ST segments, inverted T waves, and a shortened P-R interval. These changes may be confused with those of myocarditis. Electromyography (EMG) shows myopathic changes; polyphasic potentials and a reduced interference pattern with low voltage are the usual findings. Unusual high-frequency discharges, best described as myotonic-like, are very common [Gutman et al., 1967; Hogan et al., 1969]. Muscle and liver biopsy specimens contain large amounts of structurally normal glycogen when studied by both light and electron microscopy. Changes in peripheral nerve also have been reported [Araoz et al., 1974].

Genetics

GAA deficiency is inherited as an autosomal-recessive trait. The gene for human acid α-glucosidase is contained on chromosome 17 (segment q21–q23) [Martiniuk et al., 1986]. The structural gene for human acid α-glucosidase is undergoing intensive study; it is approximately 28 kb in length and contains 20 exons [Martiniuk et al., 1991]. Various mutations may result in the phenotype; missense mutations and failure of an allele to manifest mRNA expression have been reported [Zhong et al., 1991]. Prenatal diagnosis has been available since the 1970s [Hug et al., 1974]. The use of chorionic villus assay allows first-trimester diagnosis [Chowers et al., 1986]. Both adult-onset and infantile glycogenosis type II have been detected in one family. Two types of mutant alleles were identified; one leads to complete deficiency of the enzyme, and the other results in reduced net production of active α-glucosidase, resulting in partial enzyme deficiency [Hoefsloot et al., 1990].

Management

Before the advent of enzyme replacement therapy (ERT), no practical treatment was available. Therapies previously studied included epinephrine administration, which reduced liver, but not muscle, glycogen content to normal [Hug, 1974]. Dietary supplementation with l-alanine, designed to reduce the elevated protein turnover characteristic of acid maltase deficiency, has apparently slowed progression of weakness and even reversed cardiomyopathy in some patients with late infantile and juvenile forms [Bodamer et al., 1997, 2000, 2002].

Attempts to replace the deficient enzyme date back to 1964, but effective ERT was not possible until suitable sources of receptor-targeted human recombinant acid glucosidase became available in the late 1990s. This substance was derived from both rabbit milk and Chinese hamster ovary (CHO) cells. The first clinical trial began in 1998 [Reuser et al., 2002; Winkel et al., 2004], and in 2006, ERT received Food and Drug Administration (FDA) approval for treatment of acid maltase deficiency [Koeberl et al., 2007]. Infants who received treatment early in the course of their illness demonstrated improved strength and cardiac function, with survival now extending over several years. It has become apparent that ERT is most effective at reversing cardiomyopathy and extending the life span of infants, but that skeletal muscle disease is relatively resistant to this modality [Schoser et al., 2008]. The follow-up interval has been too short to determine if anterior horn cell and glial storage of glycogen will lead to chronic weakness and impairment of cerebral function in long-term survivors.

Clinical Laboratory Tests

Light and electron microscopy of muscle biopsy material displays moderate glycogen storage (Figure 34-6). In muscles stained with hematoxylin and eosin, the glycogen-containing areas appear vacuolated. In one report, only type I fibers were involved [Papapetropoulos et al., 1984]. A few patients with glycogen storage in lysosomes have been described who appear to have normal acid maltase enzyme activity [Tachi et al., 1989].

Fig. 34-6 Infantile acid maltase deficiency.

A, Ballooned muscle cells with dispersed myofibrils (arrow) resulting from glycogen appearing as clear areas. (Verhoeff–van Gieson stain; × 170.) B, Ultrastructure showing excessive glycogen beneath the sarcolemma and between myofibrillar bundles. (× 10,050.)

(A and B, Courtesy of Dr. Stephen A Smith.)

Electron microscopy depicts monogranular and multigranular deposits of glycogen free in the sarcoplasm, as well as membrane bound in lysosomes. EMG documents polyphasic potentials and a reduced, low-voltage interference pattern. The bizarre, myotonic-type potentials described in early infantile acid maltase deficiency also occur in the late infantile form.

Biochemistry

Aside from the accumulation of glycogen and its possible abnormal architecture, the most prominent abnormality described is a deficiency of acid maltase activity. Quantitation of glycogen reveals increased content. The liver may or may not contain increased glycogen stores [Smith et al., 1966, 1967]. Two patients have been described who had glycogen of abnormal configuration because of shortened outer chains.

Management

Attempts to manage patients by dietary means enjoyed modest success after initially disappointing results (see earlier) [Bodamer et al., 1997, 2000, 2002].

At present, ERT appears to offer the best hope for definitive treatment in this group of patients [Reuser et al., 2002]. Investigational studies are on-going.

Pathology

Electron microscopy of skeletal muscle of persons with glycogenoses has demonstrated glycogen deposits just inside the sarcolemmal membrane and between the filaments of the I and A bands, as well as between the myofibrils. These abnormalities are not pathognomonic for this glycogenosis [Neustein, 1969], now classified as glycogen storage disease type III. Glycogen storage in liver is indistinguishable from glycogen storage in other glycogenoses that involve the liver.

Biochemistry

A complementary DNA encoding the human muscle glycogen debranching enzyme (AGL) was used to localize the gene to 1p21 by somatic cell hybrid analysis and in situ hybridization [Yang-Feng et al., 1992]. The AGL gene was cloned and found to encode six isoforms that manifested tissue-specific distribution and two distinct functions, both as a debranching enzyme and as a transferase [Bao et al., 1996]. Polymorphic markers within the gene can be used for linkage analysis for prenatal diagnosis and carrier detection [Shen et al., 1997], although direct mutational analysis is frequently used. GSD type III has marked genetic heterogeneity, with almost 70 mutations described by 2004 [Lam et al., 2004; HGMD, 2010]. Although genotype–phenotype correlations are difficult in rare recessive phenotypes such as GSD type III, in which most patients are compound heterozygotes for private mutations, it appears that GSD type IIIa is associated with mutations downstream to exon 3, whereas GSD type IIIb is associated with mutations in exon 3 [Lucchiari et al., 2002, 2003]. There is considerable allelic heterogeneity in different ethnic groups harboring mutations in this gene [Endo et al., 2006; Aoyama et al., 2009].

Several designated biochemical categories of type III glycogenosis have been identified. In type IIIa deficiency (both transferase and glucosidase deficiency), debranching enzyme activity is either absent or greatly reduced in liver and muscle. When the enzyme activity is deficient in liver alone, the condition is designated type IIIb. Type IIIc patients have deficient glucosidase but not transferase activity. A 12-year-old girl homozygous for p.R1147G has been diagnosed with isolated glucosidase deficiency [Aoyama et al., 2009]. Some patients have the reverse: that is, isolated transferase deficiency with retention of glucosidase activity (type IIId disease) [Ding et al., 1990]. The likelihood of myopathy and cardiomyopathy can be determined from assay of debranching enzyme and debranching enzyme transferase activity [Coleman et al., 1992]. Approximately 70 percent of the patients have no activity in all tissues studied. In another 10 percent of patients, enzyme activity is absent in liver but present to a small degree in muscle tissue. In yet another group, some activity of the debranching enzyme is present in either or both liver and skeletal muscle. The use of skin fibroblasts for study of debrancher enzyme activity is the usual initial approach to enzyme studies [Brown et al., 1978]. Oligo-1,4 α1,4-transglucosylase (transferase) activity may be present in muscle and liver of patients with type III glycogenosis but absent in their leukocytes. Electron microscopy of skin indicates glycogen storage in eccrine sweat glands [Sancho et al., 1990].

Characterization of the enzyme indicates immunochemical similarity of debranching enzyme in liver and in muscle. The evidence also suggests that deficiency of debranching enzyme activity in GSD type III is the result of the absence of debrancher protein [Chen et al., 1987].

Assay of liver tissue of a patient with debrancher enzyme deficiency revealed increased activity of fructose-1,6-diphosphatase. Lack of enzymatic activity limits the breakdown of glycogen, and during fasting, the release of glucose from the liver stems from gluconeogenesis. The increase in fructose-1,6-diphosphatase activity likely reflects increased gluconeogenesis. Administration of galactose [Hers, 1959], dihydroxyacetone [Brombacher et al., 1964], fructose [Hers, 1959], casein [Fernandes and van de Kamer, 1968], and glycerol [Senior and Loridan, 1968] has resulted in increased blood glucose concentrations; these findings support the critical compensatory role of gluconeogenesis in this condition [Sadeghi-Nejad et al., 1970]. In patients with deficient muscle enzyme activity, incorporation of uridine-¹⁴C-glucose into red cell glycogen is either very low or absent.

Myogenic hyperuricemia is common in this condition but is not unique; hyperuricemia also accompanies glycogenosis type V and type VII [Mineo et al., 1987].

Clinical Characteristics

Clinical Laboratory Tests

Pseudomyotonic discharges are present on EMG. Serum creatine kinase activity may increase before and after exercise. Blood studies demonstrate mild fasting hypoglycemia, hyperlipidemia, fasting ketonuria, and diabetic glucose tolerance curves. Blood glucose concentration usually is not responsive to epinephrine or glucagon, but at times a mild response may occur. Results on galactose, fructose, and glycerol tolerance testing are normal. Blood lactic acid does not increase on ischemic exercise. Abnormally structured glycogen containing short outer chains has been demonstrated in liver, skeletal muscle, and red and white cells [Brandt and DeLuca, 1966; Van Hoof, 1967; Van Hoof and Hers, 1967].

Genetics

GSD type III is inherited as an autosomal-recessive trait. The use of cultured amniotic fluid cells and chorionic villus assay allows first-trimester diagnosis [Chowers et al., 1986; Yang et al., 1990]. Heterozygotes cannot be diagnosed with certainty using enzyme analysis [Cohn et al., 1975], but mutational analysis can accurately identify both affected persons and carriers when two mutant alleles have been detected in a proband.

Management

Patients with growth failure and hepatic dysfunction, including hypoglycemia, appear to benefit from the administration of oral cornstarch [Borowitz and Greene, 1987; Gremse et al., 1990]. It may be important to avoid overtreatment with carbohydrate; cardiomyopathy was reversed in a 16-year-old patient by increasing the protein content of the diet from 20 to 30 percent of caloric intake, with corresponding reduction of cornstarch to the minimum level required to avoid hypoglycemia [Dagli et al., 2009].

Pathology

Glycogen may accumulate disproportionately in the tongue and diaphragm in comparison with other striated muscle groups. The characteristic lesion is the polyglucosan body, a periodic acid–Schiff-positive inclusion that also is seen in phosphofructokinase deficiency, Lafora body disease, double athetosis (Bielschowsky bodies), and aging (corpora amylacea) [Cavanagh, 1999]. Electron microscopy of the deposits reveals branched filaments, osmiophilic granules, and electron-dense amorphous material. Autopsy of a neonate who died at 1 month of life of cardiorespiratory failure showed vacuoles filled with periodic acid–Schiff-positive diastase-resistant materials in cells including neurons. Electron microscopy demonstrated polyglucosan bodies in all tissues examined. GBE1 activity was markedly reduced in muscle and fibroblasts, and absent in liver and heart, as well as glycogen synthase activity. The patient was homozygous for p.E152X in GBE1 [Lamperti et al., 2009].

Biochemistry

The first patient described with deficiency of brancher enzyme activity manifested cirrhosis of the liver and glycogen accumulation [Anderson, 1956], but patients with normal [Holleman et al., 1966] and decreased muscle glycogen concentrations [Sidbury et al., 1962] also have been described. Brancher enzyme deficiency results in the synthesis of unbranched glycogen composed of elongated chains of glucose molecules joined together in 1,4 linkages. As a result, the glycogen is composed of long outer chains, has few branch points, and resembles the pattern of starch also known as amylopectin.

The glycogen brancher enzyme has been purified beyond 3000-fold from rabbit skeletal muscle. The enzyme appears to have a molecular weight of 92–103 kilodaltons (kDa), depending on the choice of reference protein. Amylopectin polysaccharide isolated from the liver of a patient with branching deficiency is branched in the presence of the purified enzyme and α-d-glucose-1-phosphate at pH 7 [Gibson et al., 1971].

Study of the fine structure of glycogen from a patient with brancher enzyme deficiency found that the similarity of abnormal glycogen to amylopectin is in some ways superficial. The abnormal glycogen contains a significant number of short branches. This finding is consistent with the hypothesis that a normal debranching enzyme system in these patients can participate in a reverse reaction, with a resultant small degree of branching activity. The short chains are explained further by the supposition that the glycogen debranching enzyme system would form branch points by the apposition of 1→6 bonded α-glucose units by amylo-1,6-glucosidase. Further elongation of this chain would occur by transfer of oligosaccharide by the oligo-1,4→1,4-transferase component of the debranching system. The transferase favors transfer of maltotriosyl residue, which creates a four-unit branch. Brancher enzyme from muscle or liver ordinarily transfers glucose units containing seven glucose molecules. The shorter branches formed by a reversal of the debranching enzyme system are not as readily extended by glycogen synthetase. If the units are shorter than four glucose units, it may be impossible for them to be extended by synthetase [Mercier and Whelan, 1970]. The presence of short branches suggests that reversal of the debranching mechanism is operative.

Clinical Characteristics

Manifestations of the disease – failure to thrive, hepatosplenomegaly, and liver failure with cirrhosis – usually appear in the first 6 months of life. Affected infants exhibit delayed motor and social development, hypotonia, weakness, and muscle atrophy, accompanied by absent or decreased deep tendon reflexes [McMaster et al., 1979; Zellweger et al., 1972]. The most severe phenotype presents in the fetus. Manifestations of this lethal disorder include cervical cystic hygroma, fetal hydrops, and fetal akinesia in differing combinations [L’Hermine-Coulomb et al., 2005]. A more benign form with clinical onset at the age of 2 years manifested as hepatomegaly and elevated liver enzyme activity. The patient had no neurologic abnormalities, and the liver disease was not progressive [Greene et al., 1988]. Another patient, a 3-year-old male, had mild glycogen storage, as well as dicarboxylicaciduria and secondary carnitine deficiency. Notable clinical improvement occurred with administration of oral l-carnitine [Maaswinkel-Moody et al., 1987]. Yet another patient had mild clinical symptoms at 8 years of age despite profound deficiency of glycogen branching enzyme [Guerra et al., 1986]. Adult myopathic variants have been described [Bornemann et al., 1996].

Clinical Laboratory Tests

Diagnosis of brancher deficiency by assay of peripheral white blood cells, skin fibroblasts, and amniotic cell activity is feasible [Howell et al., 1971]. Confirmation of the diagnosis by mutational analysis is now possible, and is often preferable, given that enzyme analysis may sometimes be difficult to interpret [Li et al., 2010].

Genetics

Early studies confirmed an autosomal-recessive mode of inheritance [Legum and Nitowsky, 1969]. Enzyme activity in cultured fibroblasts is less than control levels in patients and both parents, corroborating the presence of an autosomal-recessive mode of inheritance. Prenatal testing using cultured amniocytes and chorionic villi is feasible [Brown and Brown, 1989] but has been superseded by molecular analysis when available. The gene encoding brancher enzyme, GBE1, was identified in 1993 [Thon et al., 1993]. By 2010, 34 mutations had been described [Li et al., 2010]. Most are missense, but nonsense, intronic donor and acceptor splice-site mutations, small deletion frameshift mutations, small insertion frameshift mutations, and large deletions have all been reported. Although genotype–phenotype correlations are imperfect, missense mutations are more likely to be associated with milder phenotypes, and truncating mutations or large deletions with severe forms of the disease.

Management

Treatment with a combination of zinc-glucagon and α-glucosidase decreased liver glycogen concentration, but the infant died at 11 months of age from an infection [Fernandes and Huijing, 1968]. Liver transplantation has been successful in a number of patients [Selby et al., 1991]. Orthotopic liver transplantation has been attempted with varied success [Selby et al., 1991]. In one report, cardiac amylopectinosis occurred 9 months after successful transplantation [Sokal et al., 1992].

Pathology

Light microscopic studies of muscle reveal moderately increased stores of glycogen beneath the sarcolemmal membrane. Electron microscopy demonstrates disorganization of the I band region and distortion of the myofibrils secondary to glycogen deposition [Rowland et al., 1963]. Histochemical study of muscle suggests the absence of myophosphorylase activity, but only quantitative biochemical studies are reliable to confirm the diagnosis. Critical and definitive diagnosis depends on assay for the enzymatic deficiency in the affected muscle tissue.

Biochemistry

Glycogen breakdown to lactate begins with the initial disruption of the 1,4 linkage between glucosyl units. The enzyme myophosphorylase facilitates this reaction in skeletal muscle. After this linkage is cleaved, glucose-1-phosphate is freed and metabolized to lactate through the Embden–Meyerhof pathway. The myophosphorylase enzyme is regenerated in a complex reaction involving a number of other enzymes, including phosphorylase kinase (see Figure 34-3).

Absence of myophosphorylase activity results in decreased glucose-1-phosphate production; as a result, lactic acid is not formed in exercised muscle, and serum lactic acid concentration is not appropriately elevated (Figure 34-7). Structure of the excess glycogen stored is normal. Mitochondrial metabolism is normal [Argov et al., 1987].

Fig. 34-7 This line graph reflects the failure of increase in blood lactic acid concentration during ischemic exercise of the arm in a patient with McArdle’s disease.

Histochemical stains of fresh frozen sections of skeletal fibers demonstrate absence of phosphorylase. Studies of early multinucleated fibers and striated myofibers grown in vitro from these tissues reveal definite evidence of phosphorylase activity. Genetic coding for developing a form of myophosphorylase activity must be present in the precursor cells of regenerating skeletal muscle. The observation suggests the presence of a mechanism for loss of activity during maturation of tissues. Feasible explanations include the following possibilities: muscle maturation may result in loss of an enzyme that maintains phosphorylase production, survival, or activity; an abnormal specific protease may develop with maturity and inactivate myophosphorylase; a normally repressed myophosphorylase repressor gene may be “de-repressed”; and a normally present but inactive myophosphorylase-inhibiting or destroying enzyme may be activated and inhibit myophosphorylase enzyme activity or survival [DiMauro et al., 1978; Roelofs et al., 1972]. Occasionally, patients are found to have no immunologic cross-reactive material to normal myophosphorylase [Koster et al., 1979].

Clinical Characteristics

Affected children have decreased stamina and tire easily. Fatigue may be mediated in part by ammonia accumulation [Coakley et al., 1992]. Severe cramping pain after minimal exercise is noted in the involved skeletal muscles and primarily affects distal muscles. Cardiac symptoms have not been reported, but cardiac muscle is involved [Ratinov et al., 1965]. Myoglobinuria occurs with moderate or strenuous exercise. In adolescence and adulthood, persistent weakness may develop, with moderate loss of muscle bulk [Schmid and Hammaker, 1961]. A “second wind” phenomenon has been described in which the patient appears to recover after a 15-minute period of weakness and fatigue [Braakhekke et al., 1986]. This phenomenon has been attributed to improved energy production when metabolic dependence switches from glycogen stores to blood-borne fuels, including glucose and fatty acids, and is consistently seen in GSD type V but not in GSD type VII, whose phenotype is otherwise indistinguishable [Haller and Vissing, 2004]. Prolonged or frequent repetitive episodes of myoglobinuria should be avoided because they may result in both acute and chronic renal failure.

Renal failure occurs primarily in men, especially those who perform unusually vigorous exercise, thereby inducing myoglobinuria. These men generally are aware that they are exceeding their usual exercise tolerance. Acute renal failure may not be reversible in these patients [Bank et al., 1972]. One patient with recurrent myoglobinuria and renal failure was found to have had previously unrecognized convulsive seizures as the precipitating events for his episodes [Walker et al., 2003]. In another case, the presence of sickle cell trait and bulimia were likely significant stressors [Pillarisetti and Ahmed, 2007].

Onset usually is in childhood; neonatal onset has been reported [Milstein et al., 1989]. Adult-onset McArdle’s disease also has been reported. One patient had onset at 60 years of age [Felice et al., 1992]. Frequent ingestion of glucose or fructose has had little therapeutic effect. Muscle damage after prolonged exercise may be demonstrated by means of radionuclide scanning techniques [Swift and Brown, 1978].

A 4-week-old female manifested diffuse, progressive muscle weakness and died at 13 weeks of age. She subsequently was demonstrated to have myophosphorylase deficiency and glycogen storage in the muscles. A female sibling died at 4 months of age from the same condition [Miranda et al., 1979]. A very severe phenotype, lethal in infancy, was reported in a child born to consanguineous parents with mutations in both PYGM and dGK, the gene encoding deoxyguanosine kinase, whose deficiency causes the hepatic form of mitochondrial depletion syndrome [Mancuso et al., 2003].

Clinical Laboratory Tests

Exercise results in elevated serum creatine kinase activity and increase in activity of other serum enzymes released from muscle, ostensibly a result of loss of sarcolemmal membrane integrity. The EKG may demonstrate an increased QRS amplitude, a prolonged R-S interval, T wave inversion, and bradycardia [Ratinov et al., 1965].

Electromyographic study of contracted muscles after exercise reveals a decreased interference pattern; after ischemic exercise, the contracted muscles may demonstrate no electrical activity.

Ischemic exercise effects may be studied using two blood pressure cuffs, one at the wrist and one just above the elbow. The cuffs are inflated to above systolic pressure, and the pressure is maintained for 3 minutes. Blood is removed from the antecubital vein at 0, 3, 5, 10, 15, and 20 minutes. After the initial blood specimen is drawn (at time 0), the patient is asked to contract and extend the fingers over a rubber ball or rod while the cuff pressure is maintained. A patient with McArdle’s disease, or with any of the glycolytic abnormalities that interfere with lactate production, will experience severe contractures and complain bitterly of pain within 30 seconds after the initiation of ischemic exercise. The contracture phenomenon may be related to delayed reaccumulation of calcium ions in the sarcoplasmic reticulum [Gruener et al., 1968]. The exercise test may not identify patients with low levels of myophosphorylase, in contradistinction to those patients with absence of the enzyme [Taylor et al., 1987].

A test in which subjects cycle with moderate intensity for 15 minutes revealed a consistent decrease in heart rate from 7 to 15 minutes in GSD type V patients, in contrast with an elevation in heart rate in control subjects and patients with other glycogen storage diseases. The test appears specific and sensitive in this population [Vissing and Haller, 2003a]. Near-infrared spectroscopy (NIRS) is another noninvasive approach to screening for GSD V. This technique involves measurement of deoxyhemoglobin and deoxymyoglobin levels in the vastus lateralis muscle as surrogates for oxygen extraction. Patients with GSD V show reduced oxygen extraction compared to controls [Grassi et al., 2007]. Pulmonary oxygen uptake kinetics are negatively correlated with NIRS findings in these patients [Grassi et al., 2009].

Relaxing factor (which controls accumulation of calcium ions by the sarcoplasmic reticulum) appears to be normal in patients with McArdle’s disease [Brody et al., 1970]. Spectroscopy studies are useful in the detection of excessive muscle glycogen [deKerviler et al., 1991; Jehenson et al., 1991].

Genetics

The gene encoding synthesizing myophosphorylase, PYGM, is located at 11q13 [Lebo et al., 1990]. A number of mutations have been described [Vorgerd et al., 1998] but do not appear to explain the clinical heterogeneity of GSD type V. A study of potential genetic modifiers found a strong association between angiotensin-converting enzyme genotype and clinical phenotype, suggesting that angiotensin-converting enzyme is a modifier of PYGM [Martinuzzi et al., 2003]. A further study of 99 Spanish patients confirmed this relationship, and also found that female gender conferred a more severe phenotype [Rubio et al., 2007]. GSD V is transmitted as an autosomal-recessive trait and may manifest in a heterozygote [Schmidt et al., 1987]. Study of muscle biopsy material demonstrates molecular heterogeneity, including near-normal expression of phosphorylase mRNA concentration and size in some patients [McConchie et al., 1990].

Management

A fat-rich diet was administered to a 21-year-old male patient; he subsequently had a shortened recovery period from the acute physical load and suffered no induration of the deltoid muscle after sustained abduction to 90°. Maximal strength did not appear to be improved by the fat-rich diet, but tolerance of submaximal loads appeared to be increased, and recovery from muscle discomfort was accelerated [Viskoper et al., 1975]. The interrelationship of pyridoxal phosphate and glycogen phosphorylase offered some hope for nutritional therapy [Beynon et al., 1996], but this has not been supported by more recent studies. A controlled trial of oral sucrose loading showed improved exercise tolerance and stable glucose levels in 12 adults with GSD type V [Vissing and Haller, 2003b]. Although not suitable for continuous use owing to its tendency to induce weight gain, this regimen, combined with aerobic conditioning, appears likely to be useful in improving performance under stressful conditions and may protect against acute rhabdomyolysis [Amato, 2003]. A review of published trials cited this study and one other in which oral creatine improved ischemic performance, suggesting these as the only therapies with evidence of benefit [Quinlivan and Beynon, 2004]. A more recent review noted that, while low doses of creatine monohydrate improved performance, higher doses could produce worsening of function [Tarnopolsky, 2007]. A trial of aerobic conditioning in eight adults led to increased exercise capacity without adverse effects [Haller et al., 2006]. A short-term trial of gentamicin as “read through” therapy in four patients with stop mutations showed no evidence of benefit [Schroers et al., 2006].

Biochemistry

GSD type VI was described by Hers in 1959, and is characterized by increased glycogen stores of normal configuration in the liver. Hepatic phosphorylase (hepatophosphorylase) activity is diminished or absent.

Because of the possibility of abnormalities in the complex activating mechanism of hepatophosphorylase, systematic study of enzyme activity in suspected hepatophosphorylase deficiency is necessary to exclude phosphorylase kinase deficiency and other metabolic errors in the activating sequence. One patient had hepatomegaly with increased glycogen content and low activity of hepatophosphorylase. No abnormalities of muscle tissue enzymes or glycogen configuration were noted. A liver homogenate prepared from the patient’s biopsy specimen converted rabbit muscle phosphorylase b to phosphorylase a. Hepatic homogenate from the patient manifested no phosphorylase activity under the same conditions in which control human liver homogenate demonstrated phosphorylase activity through activation procedures. No activity was observed in the patient’s hepatic homogenate after the addition of phosphorylase kinase [Hug and Schubert, 1970].

GSD VI could only be diagnosed by enzymology of liver tissue [Hug et al., 1974] until the gene was identified. This identification was accomplished in a Mennonite kindred, whose affected members harbored a single base pair change in a splice donor site of intron 13 in the PYGL gene [Chang et al., 1998]. The technique is particularly helpful in distinguishing patients with relatively high residual enzyme activity from heterozygotes [Tang et al., 2003]. A series of eight patients with GSD VI from seven families were studied, and found to harbor 11 novel mutations, most of which were missense [Beauchamp et al., 2007b]. The patients’ symptoms ranged from hepatomegaly and subclinical hypoglycemia, to severe hepatomegaly with recurrent severe hypoglycemia and postprandial lactic acidosis.

Clinical Characteristics

The patients display various degrees of growth retardation, hypoglycemia, ketosis, and hepatomegaly. Specific neurologic findings are absent. Muscle and cardiovascular tissues are not primarily involved.

Genetics

The gene coding for the enzyme liver glycogen phosphorylase is located on chromosome 14 [Newgard et al., 1987] at 14q21–22. The condition is transmitted as an autosomal-recessive trait [Wallis et al., 1966]. Most mutations are missense; there are no common mutations [Beauchamp et al., 2007b].

Management

Symptoms can be controlled with frequent small carbohydrate meals. No other forms of therapy have been necessary or recommended.

Biochemistry

The enzyme phosphofructokinase transforms fructose-6-phosphate to fructose-1,6-diphosphate. Decreased activity of this enzyme results in increased muscle glycogen stores of normal structure and increased concentration of glucose-6-phosphate and fructose-6-phosphate [Tarui et al., 1965; Layzer et al., 1967; Thomson et al., 1963; Vora et al., 1987]. The history of GSD type VII, since its discovery as the first enzymatic disorder of glycolysis in 1965, has been reviewed [Nakajima et al., 2002]. Phosphofructokinase exists in five different isoforms with tissue-specific distribution. The gene consists of varying combinations of liver (L), muscle (M), and platelet (P) subunits. Muscle phosphofructokinase is a homotetramer of M subunits [Vora et al., 1980].

Clinical Characteristics

Motor development is normal during the first decade. Nevertheless, patients experience decreased exercise tolerance and easy fatigability during childhood. They perform poorly in games requiring physical stamina and complain of muscle stiffness and weakness, and occasionally of muscle cramps. Myoglobinuria may follow moderate to strenuous exercise and has precipitated acute renal failure [Exantus et al., 2004]. The clinical pattern is reminiscent of McArdle’s disease, except for the absence of a “second wind” phenomenon in GSD type VII [Haller and Vissing, 2004]. One patient with hyperuricemia and gout has been described [Agamanolis et al., 1980].

An unusual infantile syndrome characterized by limb weakness, seizures, cortical blindness, and corneal opacifications has been reported. The infant died at 7 months of age. Microscopic studies of the brain revealed typical features of neuroaxonal dystrophy [Servidei et al., 1986]. Another infant has been reported with infantile seizures and a relatively mild course; two of his sisters died in infancy with hypotonia, delayed milestones, and epilepsy [Al-Hassnan et al., 2007]. A 70-year-old male with progressive weakness of the legs has also been described [Vora et al., 1987]. An Ashkenazi kindred with GSD type VII has been described whose members had clinical manifestations of diabetes in addition to abnormal results on glucose tolerance testing, confirming that PFKM mutations can cause impaired glucose responses to insulin [Ristow et al., 1997]. Affected persons in this family had first become symptomatic in childhood, with easy fatigability after exercise.

A 66-year-old female with epilepsy and cardiac disease attributed to GSD type VII manifested slow progression of symptoms over an 8-year period, but showed marked improvement after effective treatment of her seizures and cardiac disease [Finsterer et al., 2002]. This report emphasizes the importance of meticulously treating (or preferably preventing) complications, rather than adopting a nihilistic approach.

Physical examination in patients with GSD type VII is unremarkable, except for variable weakness and loss of skeletal muscle bulk.

Clinical Laboratory Tests

After exercise, serum creatine kinase and other serum enzymes released from muscle may be elevated. EMG findings may be normal. Ischemic exercise testing, as described earlier for McArdle’s disease, results in muscle contracture and decreased lactic acid production. The definitive diagnosis is made by enzymatic assay of muscle tissue.

Phosphofructokinase activity is about 50 percent of the expected value in the erythrocytes of these patients. A decrease from normal levels of activity also is noted in other patients [Sivakumar et al., 1996]. Immunologic study suggests that half of the total activity of erythrocyte phosphofructokinase is derived from an enzyme form identical to phosphofructokinase. One patient with phosphofructokinase deficiency was found to have (incidental) Gilbert’s syndrome (hepatic glucuronyltransferase deficiency) [Fogelfeld et al., 1990].

Genetics

The phosphofructokinase gene (PFKM) is encoded at 12q13.3 [Howard et al., 1996]. GSD type VII is prevalent in Ashkenazim; 68 percent of mutant alleles in this population are accounted for by a splicing mutation in exon 5 [Raben and Sherman, 1995]. About 20 disease-causing mutations in PFKM have been described [Toscano et al., 2007].