BB i~ll!t'~l.' II Biochi~ic~a et Biophysica A~ta ELSEVIER Biochimica et Biophysica Acta 1272 (1995) 1-13

Review McArdle's disease-muscle glycogen phosphorylase deficiency

Clare Bartram a,*, Richard H.T. Edwards b, Robert J. Beynon a

a Department of Biochemistry and Applied Molecular Biology, UMIST, PO Box 88, Manchester M60 IQD, UK b . . Department of Medwme, Universi~ of Liverpool, PO Box 147, Liverpool 1.,69 3BX, UK

Received 13 February 1995; accepted 23 February 1995

Keywords: Glycogen phosphorylase; McArdle's disease; Muscle glycolysis

Contents

I. Introduction ...... 2 1.1. Discovery and history of the first defect in muscle glycolysis ...... 2 1.2. Identification of the enzyme defect ...... 2 1.3. Glycogen phosphorylase ...... 2 Isozymic forms of glycogen phosphorylase ...... 2 Activation of muscle glycogen phosphorylase ...... 2 Pyridoxal 5' phosphate ...... 3

2. Clinical presentation in McArdle's disease ...... 3 2.1. Symptomatology ...... 3 2.2. The 'second wind' phenomenon ...... 3 2.3. Clinical heterogeneity ...... 3 2.4. Histology and the diagnosis of McArdle's disease ...... 3 2.5. Exercise analysis ...... 4

3. Glycogen utilisation in skeletal muscle ...... 4 3.1. Induction of glycogen breakdown during exercise ...... 4 3.2. Correlation of symptomatology with an absence of muscle glycogen phosphorylase ...... 4 3.3. Other biochemical effects of the glycogen block ...... 4

4. Molecular pathology of McArdle's disease ...... 5 4.1. Molecular phenotypes ...... 5 4.2. Muscle glycogen phosphorylase gene ...... 5 4.3. Mutational analysis of McArdle's disease ...... 7 4.4. Effect of mutations on molecular phenotypes ...... 7

5. Clinical management and therapy ...... 9 5.1. Exercise management ...... 9 5.2. Dietary management ...... 9 Carbohydrate supplementation ...... 9 Fatty acid supplementation ...... 9 Amino acid/protein supplementation ...... 9 Vitamin B-6 supplementation ...... 10 5.3. Gene therapy ...... 11

* Corresponding author. Fax: +44 161 2360409.

0925-4439/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0925-4439(95)00060-7 C. Bartram et al. / Biochimica et Biophysica Acta 1272 (1995) 1-13

Acknowledgements...... 11

References ...... l I

1. Introduction in the glycogen molecule. The combined action of these enzymes results in approx. 93% glucose-l-phosphate and 1.1. Discovery and history of the first defect in muscle 7% glucose. glycolysis Isozymic forms of glycogen phosphorylase In 1951, Dr. Brian McArdle reported physiological Glycogen phosphorylase has three isozymic forms: these studies of a patient who presented with an 'exercise intol- are known as the muscle, liver and brain isozymes. The erance' [1]: "For as long as the patient could remember, muscle form has been assigned to the long arm of chromo- light exercise of any muscle had always led to pain in the some 11 by high resolution chromosome sorting and DNA muscle and, if the exercise were continued, to weakness spot-blot analysis [4]. The liver form is encoded on chro- and stiffness." [1] mosome 14 [5] and the brain form maps to chromosome 20 In contrast to a normal individual, this patient failed to and to an homologous sequence on chromosome 10 al- produce a rise in blood lactate in response to ischaemic though it is not known whether the latter is an expressed forearm exercise. This suggested that there was a block in gene or a pseudogene [6]. There is a high degree of the glycolytic pathway preventing the breakdown of glyco- homology between the amino acid sequences of the three gen to lactate. It was inferred that skeletal muscle alone isozymes of glycogen phosphorylase [6] and in particular, was affected because the normal hyperglycaemic response residues involved in the activity of these enzymes are to adrenaline indicated that liver glycogen was broken highly conserved [7]. This homology at the amino acid down to glucose. Additionally, normal glycolysis was seen level is reflected in the nucleotide sequences. However, in blood cells [1]. both the amino acid and nucleotide sequences of the muscle and brain forms show greater similarity to each 1.2. Identification of the enzyme defect other than to the liver sequences [6]. Although the three isozymes of glycogen phosphorylase It was not until 8 years later that the defect in glycolysis are highly conserved, each is generally more similar to its was identified [2,3]. The alleviation of the symptoms of corresponding isozyme in another species than to the other this disorder by glucose suggested that the metabolic block isozymes in the same species [8] and each has a distinct occurred early in the glycolytic pathway. This was subse- physiological role. The muscle isozyme provides ATP for quently confirmed by the absence of glycogen phospho- muscle contraction whereas the liver form has a homeo- rylase activity in powdered muscle from a patient and static function in the regulation of glucose release from similarly, by a lack of histochemical staining for activity in hepatic glycogen stores. The brain isozyme is thought to a muscle biopsy [2]. In another study, the addition of provide an emergency supply of glucose during periods of crystalline phosphorylase to a muscle homogenate from an anoxia or hypoglycaemia [8]. As their different functions affected individual resulted in a 3-fold increase in lactate would imply, isozymic expression of phosphorylase is production, whereas no lactate was generated in the ab- tissue-specific and the brain, liver and muscle isozymes are sence of exogenous phosphorylase [3]. McArdle's disease the major forms expressed in the brain, liver and skeletal was thus identified as a lack of muscle glycogen phospho- muscle, respectively. Expression in other tissues varies rylase. It has subsequently been classified as Glycogen between species but in man, the liver form predominates; Storage Disease Type V (GSD V). the muscle isozyme is found only in skeletal muscle, heart and possibly as a hybrid in brain [9]. It is thought that the 1.3. Glycogen phosphorylase brain isozyme is the foetal form of phosphorylase and that this is completely or partially replaced during development Glycogen phosphorylase (1, 4-a-D-glucan: orthophos- by the liver and muscle isozymes. This isozyme-switching phate a-D-glucosyltransferase, E.C. 2.4.1.1) catalyses the during development is seen in rats [10] and in human phosphorolytic cleavage of glycogen to glucose-l-phos- skeletal muscle [11]. However, northern blot analysis of phate at a-l, 4-glycosidic linkages. This glucose-l-phos- phosphorylase mRNA from a 24 week old human foetal phate enters the glycolytic pathway to generate ATP with liver indicated that the liver transcript predominated in this the concomitant formation of pyruvate or lactate. How- tissue and only low levels of brain mRNA were seen [6]. ever, glycogen phosphorylase cannot completely degrade glycogen to its constituent glucose units and a second Activation of muscle glycogen phosphorylase enzyme, debrancher enzyme, is required for the cleavage Glycogen phosphorylase exists as a dimer of identical of a-1, 6-glycosidic linkages which form the branch points 97 kDa subunits. There are two forms of glycogen phos- C. Bartram et al. / Biochimica et Biophysica Acta 1272 (1995) 1-13 3 phorylase: phosphorylase a (higher activity) and phospho- myoglobinuria may result and this can cause renal failure rylase b (lower activity) and conversion from b to a can [17,18]. Symptoms are typically less severe during child- be achieved by phosphorylation of Ser-14 [12,13]. hood and as a consequence, the disease is often not Phosphorylation of the muscle isozyme is under neural and recognised until individuals reach their second or third hormonal control. Neural control is mediated by calcium decade of life (Section 5.1). However, patients frequently flux via calmodulin. Calcium ions released from the sarco- recall incidences during childhood when they had prob- plasmic reticulum, upon initiation of muscle contraction, lems with exercise although, at the time, a clinical disorder bind to the calmodulin y-subunit of phosphorylase kinase was not suspected. thus activating it. This enzyme phosphorylates glycogen phosphorylase resulting in more of the high-activity phos- 2.2. The 'second wind' phenomenon phorylase a and hence, glycogen breakdown is induced. Hormonal control of glycogen phosphorylase activity is Patients frequently find that they can continue to exer- mediated via the glycogenolytic cascade in response to cise with increased endurance if they rest briefly at the adrenaline which activates the cAMP-dependent protein first signs of muscle pain. This is termed the 'second kinase. This enzyme activates phosphorylase kinase by wind' phenomenon [19] and it is attributed to either a shift phosphorylation and this in turn results in an increase in in a metabolic pathway or to a circulatory adjustment phosphorylase a. which results in enhanced peffusion of intramuscular capil- Glycogen phosphorylase is also a complex allosteric laries [19-21]. The metabolic shift is facilitated by mobili- enzyme with two conformers: an active 'R' state and an sation of reserves, such as fatty acids [20,21]. This phe- inactive 'T' state and interconversion between these two nomenon has latterly been studied by 31P-NMR techniques states is via phosphorylation or binding of allosteric lig- [22,23] and it is clear, although not always feasible, that ands at distinct sites on the enzyme. The muscle isozyme increasing the availability of substrate can improve exer- can be activated to 80% of its full activation capacity by cise tolerance (Section 5.2). AMP [8]. The phosphorylation site and the AMP binding site are in close proximity in the three-dimensional struc- 2.3. Clinical heterogeneity ture of the rabbit muscle isozyme and may activate the enzyme in the same way. Thus, the muscle isozyme is In addition to the normal clinical picture of McArdle's sensitive to both external signals and to changes in the disease, a number of atypical presentations have been concentrations of intracellular ligands which indicate the described. These include: a very mild form of the disease energy balance within the cell. with symptoms such as excessive tiredness and poor stamina [16]; a late-onset form of the disease (> 40 years Pyridoxal 5' phosphate old) in which no exercise-induced symptoms were noted Each subunit of the phosphorylase dimer binds, in a previously [24-27]; a fatal infantile form which is charac- tight complex, one molecule of the cofactor pyridoxal-5'- terised by progressive weakness soon after birth and severe phosphate [14] to an active site lysine residue (Lys-680 in breathing difficulties followed by death before 4 months, rabbit muscle phosphorylase) via a Schiff base. The cofac- as seen in two sisters [28,29]. An unrelated infant also tor is known to play a role in catalysis and although the exhibited this form of the disease but died at 16 days [30]; proposed mechanism is not conclusive, it is thought that it mild congenital weakness seen in a 4 year old boy [31]; serves as a proton donor and also has a role in maintaining exercise-induced myoglobinuria in an 8 year old boy with the correct electrostatic environment of the catalytic site no previous history of exercise intolerance [32]; an attack [15]. of myoglobinuria in a 42 year old man which was appar- ently unrelated to a triggering effect. The second episode of myoglobinuria resulted in severe renal failure [33]. 2. Clinical presentation in McArdle's disease 2.4. Histology and the diagnosis of McArdle's disease 2.1. Symptomatology McArdle's disease is typically diagnosed by negative McArdle's disease is characterised by myalgia, severe histochemical staining for phosphorylase activity in a mus- cramps, early fatigue and muscle weakness brought on by cle biopsy [34]. This stain relies on the ability of phospho- exercise, although the severity of the symptoms does vary rylase to work in the reverse direction and synthesise between individuals. Progressive weakness of the muscle glycogen from glucose-l-phosphate -the newly formed may occur later in life. Patients are generally most affected glycogen is detected by an iodine stain. Added insulin, by either high intensity exercise of short duration, such as AMP (activators) and glycogen (primer) enhance the reac- sprinting or carrying heavy loads, or by less intense but tion to the extent that even low levels of phosphorylase sustained activity, such as walking uphill or riding a activity result in a deep blue stain. In the absence of bicycle [16]. If exercise is continued once pain occurs, phosphorylase activity, the section remains brown. In a 4 C. Bartram et al./Biochimica et Biophysica Acta 1272 (1995) 1-13 biopsy from McArdle's patients, the muscle fibres do not tion is of limited use during intense exercise because the usually take up stain but the smooth muscle of the intra- circulation cannot provide substrates (fatty acids and muscular blood vessels does stain. In addition, McArdle's carbohydrates) and oxygen quickly enough to meet the patients often show subsarcolemmal deposits of glycogen energy demands [19]. Muscle glycogen phosphorylase which can be detected with periodic acid-Schiff (PAS) breaks down glycogen to glucose-l-phosphate which en- stain on a portion of a muscle biopsy. These increases are ters the glycolytic pathway to generate ATP with the typically 3-5-fold compared to the glycogen content of formation of pyruvate and, under anaerobic conditions, normal individuals. lactate. Use of glycogen for glycolysis instead of intra- cellular glucose results in greater net ATP generation and 2.5. Exercise analysis delays muscle fatigue [44].

Although histochemical staining of a muscle biopsy 3.2. Correlation of symptomatology with an absence of from patients is the most reliable method for the diagnosis muscle glycogen phosphorylase of McArdle's disease (Section 2.4), the ischaemic forearm test may also be performed on patients. This test is based Due to a lack of muscle phosphorylase activity, McAr- on the failure of blood lactate levels to rise in McArdle's dle's patients cannot mobilise their muscle glycogen stores. disease in response to exercise (Section 1.1). However, this Therefore, depletion of intracellular glucose and ATP, test is not specific for glycogen phosphorylase and may during the first few minutes of exercise, results in onset of indicate a block in another enzyme of the glycolytic symptoms. To exacerbate the situation, oxidative phospho- pathway. rylation is impaired in patients due to an abnormally low Muscle cell membrane damage frequently occurs in substrate flux through the TCA cycle. This is probably McArdle's patients due to over-exertion of the muscles because the virtual absence of pyruvate from glycolysis is and this releases intracellular components into the circula- likely to reduce the rate of acetyl-CoA formation and this tion. The most significant of these is myoglobin (Section affects the TCA cycle [23,45]. Acetyl-CoA can be gener- 2.1) but creatine kinase activity is almost always raised in ated from the breakdown of fatty acids but unless trained, the plasma of McArdle's patients. In addition to the release most individuals have a limited capacity for fatty acid of these intracellular contents, acute local muscle damage oxidation during exercise. This decline in oxidative phos- appears as a painful hardening and shortening of the phorylation results in a decreased oxygen consumption in muscle. This contracture is electrically silent indicating patients [23] and in fact, maximal oxygen uptake into that it is not caused by active contraction of the muscle McArdle's muscle is 35-50% of normal values [46]. In fibres. It could be due to the same kind of failure of addition, McArdle's patients exhibit a disproportionate actin-myosin crossbridge relaxation as is known to occur increase in heart rate and ventilation rate compared to in 'rigor mortis' when intramuscular ATP is depleted [35]. normal individuals [21,23]. However, needle biopsy studies have shown that the mus- cle content of phosphocreatine is not depleted when the 3.3. Other biochemical effects of the glycogen block muscle is fatigued [36] and this has been confirmed by 31P-NMR [37]. The absence of glycolysis and reduction in oxidative Leakage of potassium is also consequential to muscle phosphorylation in McArdle's patients means that in- damage. During whole body exercise, the large increases creased reliance is placed on the creatine kinase and in the plasma potassium concentration offers a possible adenylate kinase reactions. Both reactions regenerate ATP explanation of the cause of fatigue [38]. Induction of very rapidly to provide an immediate short-term source of fatigue by electrical stimulation showed that muscle of a energy [47]. The action of these enzymes in combination McArdle's patient was more fatiguable than that of a with decreased ATP generation from carbohydrate and normal individual and this was attributed in part to 'fad- fatty acids results in increased levels of ADP and AMP ing' of the muscle action potential [39-41] and partly to within the muscle. This affects normal metabolism of myofibrillar activation failure [42]. adenine nucleotides resulting in excessive purine degrada- tion [48,49]: 3. Glycogen utilisation in skeletal muscle ATP ~ ADP ",~ "" AMP --~ ~ ~ Inosine ~ Hypoxanthine

3.1. Induction of glycogen breakdown during exercise NH3

In the resting state, fatty acid oxidation predominates in the skeletal muscle of normal individuals but during exer- Inosine, hypoxanthine and ammonia enter the blood- cise, the energy requirements are largely met by glycogen stream and elevated plasma levels of all three have been breakdown and anaerobic glycolysis [43]. Aerobic oxida- detected in some McArdle's patients following exercise C. Bartram et al. / Biochimica et Biophysica Acta 1272 (1995) 1-13 5

[48-50]. However, excessive release of hypoxanthine and cle phosphorylase (molecular mass 97 kDa) was not seen inosine has not been observed in all McArdle's patients but two spots with molecular masses 60 kDa and 70 kDa [51,52]. Elevated ammonia output is seen consistently but were detected. However, these two spots were not recog- cannot be solely attributed to purine nucleotide degrada- nised by antibodies to phosphorylase [62]. These spots may tion use of amino acids as an alternative energy source in be completely unrelated to phosphorylase or alternately, McArdle's muscle also releases ammonia into the blood- the antibodies may have been sensitive to a conformational stream (Section 5.2.). epitope that was altered by misfolding or by sample prepa- McArdle's patients also show an enhanced response to ration. various hormones compared to normal individuals which helps to minimise the effect of the glycolytic block. Neural 4.2. Muscle glycogen phosphorylase gene feedback from working muscle causes an increase in the concentrations of growth hormone, cortisol, noradrenaline, Although there have been a number of studies on the adrenaline and adrenocorticotrophic hormone and a de- molecular phenotypes of McArdle's patients, until re- crease in insulin concentration which results in mobilisa- cently, nothing was known of the mutations causing the tion of extramuscular fuel, such as glucose and free fatty disease. The human muscle glycogen phosphorylase gene acids, to meet the energy requirements of the muscle [53]. is approx. 14 kb [63]. Sequencing of the 5' region identi- fied several promoter regions [11] including a TATA-Iike homology, TTTAAA, located at position -29 (based on 4. Molecular pathology of McArdle's disease the designation of the transcription start site as + 1) and a potential CAAT box, CCAAGAC, at -80. There is a 4.1. Molecular phenotypes region at -592 with the sequence CTCCAAAAGG which is necessary for efficient transcription of the phosphorylase McArdle's disease is characterised by an absence of gene. This sequence superficially resembles the CArG functional muscle glycogen phosphorylase but patients motif CC(A/T)rGG present in the promoter regions of show heterogeneity in their protein and mRNA expression skeletal and cardiac muscle a-actin genes and has been (Table 1). The majority of McArdle's patients do not have implicated in their tissue-specific expression [64,65]. It is detectable muscle glycogen phosphorylase [54-61]; of 93 repeated at -252 in the 5' region of the phosphorylase patients, only 13 expressed low levels of protein [54,57-61] gene, but expression studies indicate that this later se- and a further two expressed inactive protein [55,61]. Of 22 quence is not necessary for transcription so sequence patients whose mRNA has been determined, twelve lacked context may be critical [11]. detectable mRNA [54,57,59] whereas nine expressed the Even before the elucidation of the mutations causing 3.4 kb phosphorylase mRNA at normal [59,61] or reduced McArdle's disease, it was generally held to be an autoso- levels [56]. Another patient expressed a truncated form mal recessive disease [66,16]. Autosomal dominant inheri- (1.2 kb) of the transcript [61]. Bidimensional protein maps tance was suggested in a family in which four generations of muscle extract from three McArdle's patients identified were reportedly affected with McArdle's disease [67]. potential degradative intermediates of phosphorylase. Mus- However, the disease was only confirmed in two individu-

Table 1 Molecular phenotypes in McArdle's patients Molecular phenotypes [54] [55] [56] [57] [58] [59] [60] [61 ] No protein, no mRNA 2 5 5 No protein, normal mRNA 3 1 No protein, decreased mRNA 3 No protein, truncated mRNA 1 No protein, mRNA n.d. 5 2 2 2 4 2 2 39 Decreased protein, normal mRNA 1 1 Decreased protein, mRNA n.d. 2 I 2 1 5 Normal levels of inactive protein 1 1

Total number of patients 9 3 10 3 6 11 3 48

The column headings indicate the citations from which the information was obtained. n.d. = not determined. A number of studies have focused on expression of muscle phosphorylase protein and mRNA in McArdle's patients [54-61]. This has demonstrated the heterogeneity in the molecular phenotypes of patients with the same clinical condition. There is much more information available on the expression of phosphorylase protein than on mRNA expression. The majority of patients do not express protein at all but of those that do, the protein is generally present at very low levels. Normal levels of phosphorylase protein are seen in two patients but in both cases, the protein is inactive or virtually inactive [55-61]. 6 C. Bartram et al./Biochimica et Biophysica Acta 1272 (1995) 1-13

1844+ 1G->A NcoI

L291P 122G-> "IT SacI 1606delG AF708/709

t'N\\\lll II • Ill nl Im nm nnn 1/ nit niL.~ llHH

R49X G204S K542T Nla 111 HaeBI MaeII

77A->C MallI

Key: Untraml~ted regions Exons Introns

Mutation Base change Exon Effect of mutation

R49X CGA->TGA 1 Nonsense

77A->C ATG->CTG 1 Missense

122G->TT G->TT 1 Frameshift

G204S GGC->AGC 5 Missense

L291P CTG->CCG 8 Missense

1606delG GAG->GA 13 Frameshift

K542T AAG->ACG 14 Missense

1844+ 1G->A G->A '14' Abnormal splicing

AF708/709 ATTC 17 Amino acid deletion

Fig. 1. Sites of mutation and their detection in the muscle phosphorylase gene. At present, there are nine known mutations across the muscle phosphorylase gene which cause McArdle's disease [71,72,74-77]. The rapid diagnosis of some of these mutations is facilitated by digestion of a PCR product with the restriction enzyme indicated. The G204S mutation disrupts a recognition site for HaelII but all the other mutations create an extra restriction site and so an additional band results from digestion with the enzyme. Rapid detection of 1844 + 1G --* A is dependent upon the introduction of a base change into the PCR product by a primer which binds adjacent to the possible site of mutation; in the presence of the mutation, the mismatch primer creates a recognition site for NcoI. The effects of each mutation are shown in the table. Note: Mutation 1844 + 1G ~ T has previously been defined as 1778de167 [109] but whilst a deletion of 67 bp in the transcript does result from this mutation, the primary defect is in fact alteration of the consensus splice site sequence and so the nomenclature has been reviewed. C. Bartram et aL / Biochimica et Biophysica Acta 1272 (1995) 1-13 7 als from two generations and a dominant mechanism of [74]. Again the truncated peptide would probably be unsta- inheritance in this family is questionable. In most cases, ble and therefore, rapidly degraded. heterozygous carriers for McArdle's disease are unaffected There are four missense mutations and a single amino because it seems that one normal allele of the muscle acid deletion which may have functional or structural phosphorylase gene is adequate for normal levels of the consequences on muscle glycogen phosphorylase (Section protein. However, there are a number of reports of mani- 4.4). The G204S mutation occurs in a domain involved in festing heterozygotes who do have phosphorylase activity glycogen binding whereas K542T affects the glucose-bind- upon histochemical staining but who show clinical symp- ing domain [72]. Both L291P and AF708/709 may alter toms of McArdle's disease [68,69]. A threshold value of normal protein folding and possibly, intracellular stability 20-45% of normal has been suggested to be the minimum [75]. The initial methionine codon is altered by the 77A --* activity below which symptoms occur [69] although as- C mutation thus abolishing translation initiation [76]. An- signment is tentative. other mutation (1844+ IG~A) alters the consensus splice site sequence at the 5' end of intron 14 and as a 4.3. Mutational analysis of McArdle's disease consequence, an alternative sequence in exon 14 is used. This results in abnormal splicing which produces a 67 bp Initial work focused on restriction fragment length poly- deletion in the transcript. The resulting shift in the reading morphism of the muscle phosphorylase gene of McArdle's frame generates a short sequence of missense polypeptide patients but met with little success [56,61]. The muscle containing 14 amino acids before translation is prema- phosphorylase gene is notable for its lack of polymor- turely terminated after 580 amino acids [75]. Another phisms and restriction enzyme analysis with 94 enzyme- frameshift is caused by the deletion of a single base in probe combinations in 20 unrelated patients revealed just a codon 509 (1606delG) and this again results in premature single polymorphism at a CpG site associated with McAr- termination of translation, this time after 536 amino acids dle's disease [70]. This analysis did, however, preclude the [77]. The allele frequencies of the above mutations are possibility of gene deletions greater than 100 bp. shown in Table 2. Since these initial studies, sequencing has revealed a number of mutations in the muscle phosphorylase gene 4.4. Effect of mutations on molecular phenotypes which cause McArdle's disease (Fig. 1). The most com- mon base change is a nonsense mutation [71,72], desig- The R49X mutation occurs in the coding region of the nated R49X (nomenclature proposed in Ref. [73]). This muscle phosphorylase gene and causes premature termina- results in premature termination of translation; the result- tion of protein translation. However, of the patients whose ing truncated peptide is so short that it is non-functional biochemical phenotype is known, all those homozygous and probably rapidly degraded. A second mutation in exon for the R49X mutation have no detectable mRNA on 1, 122G ~ TT, shifts the reading frame such that a short northern blots (Fig. 2). Therefore, premature termination of sequence of missense protein containing 11 amino acids is protein translation affects the stability of the mRNA [71,72] synthesised before translation is prematurely terminated and there are a number of precedents for this in other

N M5 M6 M2 N M7 N M8 M1 M3 M4 M9 N

3.4kb phosphorylase mRNA

Mutation __+ R49X R49X R49X +_ R49X + R49X R49X n.d. Rg9X R49X + analysis + ? R49X R49X + R49X + ? 122G->TT ? R49X +

Key: N = normal individual M1-9 = McArdle's patients [numbering as in ref. 71] +/+ = normal sequence i.e. no mutations n.d. = not determined Fig. 2. Comparison of northern blot analysis and mutational analysis. Northern blot analysis has shown heterogeneity in the expression of phosphorylase mRNA in McArdle's patients [59]. Correlation of this information with the mutation analysis for these patients [71,74] shows that those individuals who are homozygous for the R49 × mutation lack detectable mRNA. Therefore, a premature termination codon in the muscle phosphorylase gene not only affects protein synthesis but decreases transcript stability. However, detectable mRNA is seen with individual M1 although premature termination of translation also occurs from both alleles in this individual. (Reprinted from 'McArdle's disease: molecular genetics and metabolic consequences of the phenotype' by R.J. Beynon et al. in Muscle and Nerve. Copyright: 1995, John Wiley and Sons, Inc.) 8 C. Bartram et al./Biochimica et Biophysica Acta 1272 (1995) 1-13

Table 2 saemias [78-81], mRNA of cystic fibrosis transmembrane Allele frequency of the mutations causing McArdle's disease conductance regulator which is implicated in cystic fibro- Mutation Allele frequency in Allele frequency in USA sis [82] and mRNA of human /3-hexosaminidase A which European population and Japanese population is defective in Sandhoff disease [83]. [71,74,109] [72,75-77] Patient M 1 carries the R49X mutation on one allele of R49X 46 5 l the muscle phosphorylase gene and the 122G ~ TT on the 77A ~ C 0 1 122G ~ TT 1 n.d. other, both of which cause premature termination of trans- G204S 2 8 lation. However, this individual does seem to express low L291P 0 1 levels of mRNA as seen by northern blot analysis (Fig. 2) 1606delG n.d. 2 which is unexpected given that termination of translation K542T 0 3 in exon 1 occurs from both alleles. The mechanism by 1844+1G ~ A 0 1 AF708/709 n.d. 7 which mRNA stability could be differentially affected by these two different mutations is not clear but it is possible n.d. = not determined. that the mRNA carrying the frameshift mutation is not The most common mutation is R49X which is present in 46 out of a total translatable. Degradation of aberrant transcripts is thought of 76 available alleles from European McArdle's patients, The majority to be co-translational [84], such that a complete inability to of these patients are Caucasians although there are a number of patients whose ethnic origins are not known. The USA patients are Caucasian and translate the mRNA would cause it to have increased R49X is the predominant mutation in these patients. However, the stability. AF708/709 mutation has only been found in Japanese patients (7 out of Analysis of molecular phenotypes of patients has sug- 14 alleles) suggesting that this might be a common mutation in this gested that the missense mutations destabilise the protein population. None of the Japanese patients carry R49X and so, it would [75]. Rabbit muscle glycogen phosphorylase remains the appear that McArdle's disease has occurred independently in these two populations. The other mutations are much less frequently observed. only isozyme for which the three-dimensional structure has been determined and thus the structural interpretation of amino acid changes must be based upon this protein. The diseases. For example, decreased stability of the transcript overall amino acid sequence identity for rabbit and human in response to premature termination of translation occurs muscle phosphorylase is 97% and there is no variation in in human /3-globin mRNA as illustrated by the /30 thalas- any catalytic or allosteric sites [63,85]. All three of the

G204S L29 IP I I Human muscle VHFYGHVEH Human muscle EGKELRLKQ Rabbit muscle VHFYGRVEH Rabbit muscle EGKELRLKQ Rat muscle VHFYORVEH Rat muscle EGKELRLKQ Human liver VHFYGKVEH Human liver EGKELRLKQ Rat liver VHFYGRVEH Rat liver EGKELRLKQ Human brain VHFYGRVEH Human brain EGKELRLKQ Rat brain VHFYORVEH Rat brain EGKELRLKQ Yeast VTFYGYVDR Yeast QGKELRLKQ Potato type H IRFFGHVEV Potato type H NGKLLRLKQ

K542T AF'/08/709 I I Human muscle KQENKLKFA Human muscle GEENFFIFG Rabbit muscle KQEN]KLKFA Rabbit muscle GEENFFIFG Rat muscle KQENKLKFS Rat muscle GEDNFFIFG Human liver KQENKLKFS Human liver GEENLFIFG Rat liver KQENKLKFS Rat liver GEENLFIFG Human brain KQENIKLKFS Human brain GAENLFIFG Rat brain KQENKLKFS Rat brain GEENLFIFG Yeast KLNNI~IRLV Yeast GEDNVFLFG Potato type H KMANKQRLA Potato type H GEDNFFLFG

Fig. 3. Sequence conservation at the sites of mutation in McArdle's disease. The above figure shows short segments of the amino acid sequence alignments for nine glycogen pbosphorylases [7] in the vicinity of the G204S, L291P, K542T and AF708/709 mutations. The mammalian sequences include liver, brain and muscle isozymes from human and rat plus the muscle isozyme from rabbit. Yeast phosphorylase and potato phosphorylase type H are shown to indicate the high degree of sequence conservation even in non-mammalian phosphorylases. All three missense mutations occur at a position at which there is absolute conservation of the amino acid between the nine sequences. Both pbenylalanine residues in the region 708/709 are highlighted, because the codons are the same and it is not possible to be more specific about the deletion. c. Bartram et al. / Biochimica et Biophysica Acta 1272 (1995) 1-13 9 missense mutations (G204S, L291P and K542T) change a creased by heparin [21], noradrenaline [20,21] or by infu- highly conserved amino acid (Fig. 3). The sequence con- sion of emulsified fat, particularly in combination with servation is such that the amino acid at these positions glucose [19]. The same effect was produced by fasting remains unchanged across the species [7] including two which increases plasma concentrations of free fatty acid non-mammalian phosphorylases (yeast and potato type H). [91]. However, a high fat diet gave more variable results This suggests that each of these amino acids is critical for because one patient reported no beneficial effect [89] the normal function of glycogen phosphorylase; their alter- whereas another noted subjective improvement after 3 ation could decrease the activity or the stability of the days although there was no increase in measured muscle mutant protein. There is also strong conservation in the strength [92]. The benefits of a high fat intake on muscle region of the AF708/709 mutation (Fig. 3). performance must be weighed up against the potentially detrimental effect on an individual's long-term health.

5. Clinical management and therapy Amino acid/protein supplementation There is evidence for increased utilisation of amino 5.1. Exercise management acids in skeletal muscle of McArdle's patients [52,93]. Therefore, provision of adequate amino acids by direct Exercise management and weight control have an im- supplementation, or via a high protein diet might improve portant role to play in the management of McArdle's muscle function. This was confirmed in one patient whose disease. Weight determines oxygen consumption during protein intake was increased to 25-30% protein from 14% walking, especially when walking uphill. It is therefore [94]. In a subsequent study, three patients were provided advantageous for a patient with McArdle's disease to with branched chain amino acids in amounts calculated to avoid excess weight. Patients should also try to keep active exceed the levels expected from a high protein diet but within the capability of their muscles as reduced activity in none experienced either an immediate or long-term (1-2 patients with muscle disorders can diminish the mitochon- months) subjective improvement or an objective benefit as drial function in muscle [86]. The greater the mitochondrial assessed by their maximal strength and endurance [95]. oxidative capacity, the less dependent an individual is on The first step in amino acid catabolism is donation of glycogenolysis. Children are more active than adults with a the a-amino group to 2-oxoglutarate (a TCA cycle inter- higher aerobic capacity per kilogram body weight [87] and mediate) in a transamination reaction which generates the this may explain why McArdle's disease is usually not cognate 2-oxo acid and glutamate. The 2-oxoglutarate may diagnosed in young children. be regenerated by deamination with the release of ammo- nia. Alternately, a second transamination reaction with 5.2. Dietary management pyruvate regenerates 2-oxoglutarate and results in the cor- The aim of any current therapy for McArdle's disease is responding formation of alanine. In the normal individual, to enable patients to exercise with increased tolerance and alanine and glutamine (formed by the reaction of gluta- to prevent the occurrence of muscle damage or myoglobin- mate with ammonia) provide a mechanism for the elimina- uria which can lead to renal failure. The provision of tion of nitrogen from the muscle and its transport to the alternative or additional energy fuels to the skeletal muscle liver for detoxification. However, in McArdle's disease, can accelerate the onset of the 'second wind' phenomenon the supply of precursors for alanine and glutamine is and thus alleviate glycolytic deprivation. compromised. In exercising McArdle's muscle, glycolytic flux is suppressed and it is unlikely that adequate pyruvate Carbohydrate supplementation is synthesised to sustain the levels of alanine required for Glucose infusion enables patients to exercise with in- ammonia removal. Likewise, any reaction that drains TCA creased endurance [19,21,22,50] and prevents exercise-in- cycle intermediates, such as 2-oxoglutarate, would ulti- duced ATP degradation as measured by a decrease in the mately impair energy metabolism. Thus, a decreased abil- degradative metabolites [50] and by a decline in plasma ity to remove ammonia, in the form of alanine and glu- ammonia [23]. Similarly, fructose infusion improves exer- tamine might explain the elevated plasma ammonia levels cise endurance [ 19,21 ]. However, long-term oral adminis- during exercise in McArdle's disease [38]. These increased tration of glucose or fructose has been largely unsuccessful ammonia levels may be responsible in part for the muscle in improving the exercise capabilities of patients [16,89]. pain and fatigue [96]. The ability of glucose to diminish Benefits are only seen when carbohydrate is given immedi- ammonia output by McArdle's muscle [23] is probably due ately before exercise and in practice, it is not always to an increased supply of pyruvate which can be transami- possible to predict a bout of exertion [90]. nated to alanine. Uptake and oxidation of the branched chain amino acids Fatty acid supplementation occurs in both normal individuals and at a higher rate in McArdle's patients could exercise with increased en- McArdle's patients during exercise [52]. Consistent with durance when plasma levels of free fatty acid were in- this suggestion is the observation of exercise-induced acti- 10 c. Bartram et al. / Biochimica et Biophysica Acta 1272 (1995) 1-13 vation of the branched chain 2-oxo acid dehydrogenase, a) the key enzyme in the oxidation of branched chain amino 8O acids [97]. The complex was activated to a greater extent, and at a lower work load in McArdle's patients compared to normal individuals. Amino acid supplements (10 g each 6O of leucine, isoleucine and valine) were initially given to patients prior to exercise in an effort to improve their performance. However, supplementation did not have the ~" 40 expected beneficial effect and in fact, exercise perfor- mance actually deteriorated [96]. This may be due to the increased ammonia resulting from transamination of these g' 20 amino acids. Any benefits gained from branched chain amino acid oxidation must be offset against the additional nitrogen burden. 0 In contrast, supplements of 10 g of the ornithine salts of 0 20 40 60 80 the 2-oxo acids of leucine and valine (4-methyl-2-oxopen- Exercise duration (rain) tanoate and 3-methyl-2-oxobutanoate, respectively) and 5 g of the sodium salt of the 2-oxo acid of isoleucine b) (3-methyl-2-oxopentanoate), if given 20 min prior to exer- 160 cise, improved exercise performance, resulted in smaller 120 ~ Release increases in heart rate and suppressed plasma ammonia output [38,96]. The 2-oxo acids provide an alternative energy source but they do not contribute to the nitrogen 80 load of the muscle cells, sparing pyruvate and 2-oxogluta- rate. If the supplements of 2-oxo acids were given 90 min ~ 40 II prior to exercise, this beneficial effect was no longer seen [52] which may have been due to re-amination. The most consistent increase in endurance was achieved when the 0 2 ~40 60- 80 -40 - Time (rain) branched chain oxo acids were combined with 75 g glu- = cose given orally 30 rain before exercise [38,96]. ~ -80 - Uptake of alanine by exercising muscle was described in one McArdle's patient [93] although it is usually re- -120 + leased from normal muscle. A later study with a number of Uptake McArdle's patients initially appeared to be at variance - 160 l with this finding as alanine was released by muscle [52]. Fig. 4. Alanine fluxes in McArdle's disease. This figure collates the data from two studies on alanine flux in McArdle's disease. They used very However, when the exercise protocols were examined in different exercise protocols, which are illustrated in panel a. The study by more detail, the two opposing results can be related to the Wagenmakers and colleagues (study I) [52] used short periods of higher work pattern (Fig. 4a). In both studies, similar total work intensity exercise, whereas that of Wahren and colleagues (study II) [93] was performed but in the earlier study, the work was was a longer duration, low intensity programme. The comparisons of the gradually increased and so, it is likely that the patient two protocols are approximate, and are based on our interpretation of the experimental details given. The data for alanine flux, obtained from both actually reached the 'second wind'. It is possible to gain studies (including those from normal individuals), are placed on the same some insight into alanine metabolism by combining the time scale, but must be interpreted in the light of the different exercise information from these two studies although the conclu- protocols. McArdle's patients, []; Normal individuals, I. sions should be treated with caution (Fig. 4b). Release of alanine from exercising McArdle's muscle peaks earlier, and total alanine output is lower than in normal individu- might reduce their availability for the synthesis of new als. This is consistent with an initially increased demand muscle protein [98] or elicit enhanced protein degradation. on pyruvate for transamination and a subsequent decrease If this imbalance in protein turnover is prolonged it could in the pyruvate pool which cannot be replenished. It then ultimately lead to long term muscle wasting and weakness. appears that McArdle's muscle actually takes up alanine which may be driven by gluconeogenesis. However, the Vitamin B-6 supplementation magnitude of alanine uptake into muscle is substantially Glycogen phosphorylase requires PLP as a cofactor and lower than initial release. apo-phosphorylase cannot be detected in skeletal muscle Amino acid supplementation may have a second effect [99]. It is not clear whether this is due to a marked on muscle protein metabolism. Increased oxidation of instability of apo-enzyme or whether the apo-enzyme has amino acids by skeletal muscle in McArdle's disease such a high affinity for its cofactor that it 'scavenges' PLP C. Bartram et al. / Biochimica et Biophysica Acta 1272 (1995) 1-13 11 from other body pools. However, because phosphorylase is the cofactor saturation of the aminotransferases in the such an abundant protein (2-5% of the total soluble muscles is improved. Supplementation of two patients with protein in skeletal muscle), and because of the large mass vitamin B-6 in earlier studies did not prove effective of the musculature, 80% of the body pool of vitamin B-6 is [ 18,11 1], but both patients had some phosphorylase protein attached to this enzyme [100]. We have used [G-3H] on an SDS polyacrylamide gel. The presence of protein, pyridoxine as a specific label for phosphorylase in vivo albeit inactive, means that it could still function as a and have observed that the rate of loss of labelled PLP 'buffer' for PLP and vitamin B-6 supplementation may not from the enzyme parallels the rate of phosphorylase degra- be beneficial in these patients. Mild sensory neuropathy dation [101-103]. Therefore, release of PLP from associated with vitamin B-6 overdosage is only seen at phosphorylase is dependent on degradation of the protein levels of 2-4 g/d over several months [112]. Therefore, [104]. Vitamin B-6 metabolism studies have consistently vitamin B-6 at 50 mg/d may potentially improve the identified a large, slowly accessible vitamin B-6 pool in exercise capacity of the majority of McArdle's patients the body [105,106] which is undoubtedly muscle glycogen without having a detrimental effect on their health. In phosphorylase. The other body pools of vitamin B-6 ap- addition, as aminotransferases require PLP as a cofactor, pear to have a much higher turnover rate and are probably we suggest that any increased protein or amino acid regi- more readily depleted when vitamin B-6 intake is inade- men must ensure that vitamin B-6 intake is adequate. If the quate. In contrast, even extended periods of vitamin B-6 activity of the aminotransferases is limited by inadequate deprivation do not significantly diminish the adult muscle PLP then the increased protein may have little benefit. phosphorylase pool [107,108]. This has led to the sugges- tion that the daily vitamin B-6 requirements are different 5.3. Gene therapy in growth and maintenance; during growth, vitamin B-6 is incorporated into the slow muscle phosphorylase pool, Interest is now being focused on gene therapy in the whereas in the adult, the daily requirement is that which is treatment of genetically inherited diseases. A recombinant needed to sustain the fast pools [106]. We therefore pro- adenovims has been used to drive expression of rabbit pose that muscle phosphorylase could function as a muscle phosphorylase cDNA in a C2C12 myoblast cell 'buffer', compensating for day-to-day variation in dietary line [1 13]. Adenoviral delivery into myoblasts, which were intake of vitamin B-6 in normal individuals. subsequently induced to differentiate, resulted in a moder- The majority of McArdle's patients lack muscle glyco- ate and transient increase in phosphorylase activity. How- gen phosphorylase, which means that this slow pool of ever, introduction into non-proliferating myotubes resulted vitamin B-6 is absent and consequently, its 'buffering' in higher phosphorylase activity which was sustained over capacity is lost. McArdle's patients may therefore be more the period of the experiment (20 days). The heterologously reliant on a regular intake of vitamin B-6 and vitamin B-6 expressed phosphorylase was subject to both hormonal and status, as assessed by an erthyrocyte transaminase assay, allosteric control which suggests that glycogen breakdown does seem to be compromised in some of our patients will occur in a regulated manner in the treated muscle of [109]. Furthermore, McArdle's patients may be more re- patients. It might be anticipated that when suitable delivery liant on adequate vitamin B-6 compared to normal individ- vehicles are developed for muscle, probably driven by uals because they are dependent on alternative energy other severe myopathies, such therapy in McArdle's dis- pathways which require PLP. Aminotransferases play a ease will also be successful. key role in amino acid catabolism (Section 5.2.) and these enzymes are PLP-dependent. In addition, there is evidence that carnitine biosynthesis requires PLP. Carnitine is re- Acknowledgements quired to facilitate the transport of fatty acyI-CoA across We would like to acknowledge The Muscular Dystro- the inner mitochondrial membrane prior to oxidation. Inad- phy Group of Great Britain and Northern Ireland for their equate vitamin B-6 status results in reduced carnitine financial support. We would also like to thank the McAr- biosynthesis and as a consequence, reduced fatty acid dle's patients, their families and our colleagues for their oxidation [ 110]. continuing contribution to this work. We have postulated that the exercise performance of those McArdle's patients who lack glycogen phospho- rylase protein might be improved by supplementation with References relatively high daily doses (50 mg) of vitamin B-6 (the recommended daily dose for normal individuals is 2 mg). [1] McArdle, B. (1951) Clin. Sci. 10, 13-33. Vitamin B-6 supplementation did improve the muscle fa- [2] Mommaerts, W.F.H.M., Illingworth, B., Pearson, C.M., Guillory, tiguability of three McArdle's patients by increasing the R.J. and Serayolarian, K. (1959) Proc. Natl. Acad. Sci. USA 45, 791-797. force generation of the muscle [109]. This implies that [3] Schmid, R. and Mahler, R. (1959) J. Clin. Invest. 38, 2044-2058. improvement is due to an increase in fuel [4] Lebo, R.V., Gorin, F., Fletterick, R.J., Kao, F.-T., Cheung, M.-C., mobilisation/utilisation and the inference from this is that Bruce, B.D. and Kan, Y.W. (1984) Science 225, 57-59. 12 C. Bartram et al. / Biochimica et Biophysica Acta 1272 (1995) 1-13

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