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BiBBochi~ic~a
i~ll!t'~l.' II
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; M c A r d l e ' s disease; Muscle glycolysis
Contents
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. Discovery and history of the first defect in muscle glycolysis . . . . . . . . . . . . . . . . . . . . . . .
1.2. Identification of the enzyme defect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3. Glycogen phosphorylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isozymic forms of glycogen phosphorylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Activation of muscle glycogen phosphorylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pyridoxal 5' phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
2
2
2
2
2
3
2. Clinical presentation in M c A r d l e ' s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Symptomatology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. The 'second wind' phenomenon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Clinical heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Histology and the diagnosis of M c A r d l e ' s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5. Exercise analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
3
3
3
3
4
3. Glycogen utilisation in skeletal muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Induction of glycogen breakdown during exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Correlation of symptomatology with an absence of muscle glycogen phosphorylase . . . . . . . . . . .
3.3. Other biochemical effects of the glycogen block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
4
4
4
4. Molecular pathology of M c A r d l e ' s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Molecular phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Muscle glycogen phosphorylase gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Mutational analysis of M c A r d l e ' s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Effect of mutations on molecular phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
5
5
7
7
5. Clinical management and therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Exercise management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Dietary management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Carbohydrate supplementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fatty acid supplementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A m i n o a c i d / p r o t e i n supplementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Vitamin B-6 supplementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
* Corresponding author. Fax: + 4 4 161 2360409.
0 9 2 5 - 4 4 3 9 / 9 5 / $ 0 9 . 5 0 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0 9 2 5 - 4 4 3 9 ( 9 5 ) 0 0 0 6 0 - 7
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C. Bartramet al. / Biochimicaet BiophysicaActa 1272 (1995) 1-13
Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
lI
1. I n t r o d u c t i o n
1.1. Discovery and history of the first defect in muscle
glycolysis
In 1951, Dr. Brian McArdle reported physiological
studies of a patient who presented with an 'exercise intolerance' [1]: " F o r as long as the patient could remember,
light exercise of any muscle had always led to pain in the
muscle and, if the exercise were continued, to weakness
and stiffness." [1]
In contrast to a normal individual, this patient failed to
produce a rise in blood lactate in response to ischaemic
forearm exercise. This suggested that there was a block in
the glycolytic pathway preventing the breakdown of glycogen to lactate. It was inferred that skeletal muscle alone
was affected because the normal hyperglycaemic response
to adrenaline indicated that liver glycogen was broken
down to glucose. Additionally, normal glycolysis was seen
in blood cells [1].
1.2. Identification of the enzyme defect
It was not until 8 years later that the defect in glycolysis
was identified [2,3]. The alleviation of the symptoms of
this disorder by glucose suggested that the metabolic block
occurred early in the glycolytic pathway. This was subsequently confirmed by the absence of glycogen phosphorylase activity in powdered muscle from a patient and
similarly, by a lack of histochemical staining for activity in
a muscle biopsy [2]. In another study, the addition of
crystalline phosphorylase to a muscle homogenate from an
affected individual resulted in a 3-fold increase in lactate
production, whereas no lactate was generated in the absence of exogenous phosphorylase [3]. McArdle's disease
was thus identified as a lack of muscle glycogen phosphorylase. It has subsequently been classified as Glycogen
Storage Disease Type V (GSD V).
1.3. Glycogen phosphorylase
Glycogen phosphorylase (1, 4-a-D-glucan: orthophosphate a-D-glucosyltransferase, E.C. 2.4.1.1) catalyses the
phosphorolytic cleavage of glycogen to glucose-l-phosphate at a-l, 4-glycosidic linkages. This glucose-l-phosphate enters the glycolytic pathway to generate ATP with
the concomitant formation of pyruvate or lactate. However, glycogen phosphorylase cannot completely degrade
glycogen to its constituent glucose units and a second
enzyme, debrancher enzyme, is required for the cleavage
of a-1, 6-glycosidic linkages which form the branch points
in the glycogen molecule. The combined action of these
enzymes results in approx. 93% glucose-l-phosphate and
7% glucose.
Isozymic forms of glycogen phosphorylase
Glycogen phosphorylase has three isozymic forms: these
are known as the muscle, liver and brain isozymes. The
muscle form has been assigned to the long arm of chromosome 11 by high resolution chromosome sorting and DNA
spot-blot analysis [4]. The liver form is encoded on chromosome 14 [5] and the brain form maps to chromosome 20
and to an homologous sequence on chromosome 10 although it is not known whether the latter is an expressed
gene or a pseudogene [6]. There is a high degree of
homology between the amino acid sequences of the three
isozymes of glycogen phosphorylase [6] and in particular,
residues involved in the activity of these enzymes are
highly conserved [7]. This homology at the amino acid
level is reflected in the nucleotide sequences. However,
both the amino acid and nucleotide sequences of the
muscle and brain forms show greater similarity to each
other than to the liver sequences [6].
Although the three isozymes of glycogen phosphorylase
are highly conserved, each is generally more similar to its
corresponding isozyme in another species than to the other
isozymes in the same species [8] and each has a distinct
physiological role. The muscle isozyme provides ATP for
muscle contraction whereas the liver form has a homeostatic function in the regulation of glucose release from
hepatic glycogen stores. The brain isozyme is thought to
provide an emergency supply of glucose during periods of
anoxia or hypoglycaemia [8]. As their different functions
would imply, isozymic expression of phosphorylase is
tissue-specific and the brain, liver and muscle isozymes are
the major forms expressed in the brain, liver and skeletal
muscle, respectively. Expression in other tissues varies
between species but in man, the liver form predominates;
the muscle isozyme is found only in skeletal muscle, heart
and possibly as a hybrid in brain [9]. It is thought that the
brain isozyme is the foetal form of phosphorylase and that
this is completely or partially replaced during development
by the liver and muscle isozymes. This isozyme-switching
during development is seen in rats [10] and in human
skeletal muscle [11]. However, northern blot analysis of
phosphorylase mRNA from a 24 week old human foetal
liver indicated that the liver transcript predominated in this
tissue and only low levels of brain mRNA were seen [6].
Activation of muscle glycogen phosphorylase
Glycogen phosphorylase exists as a dimer of identical
97 kDa subunits. There are two forms of glycogen phos-
C. Bartramet al. / Biochimica et BiophysicaActa 1272 (1995) 1-13
phorylase: phosphorylase a (higher activity) and phosphorylase b (lower activity) and conversion from b to a can
be achieved by phosphorylation of Ser-14 [12,13].
Phosphorylation of the muscle isozyme is under neural and
hormonal control. Neural control is mediated by calcium
flux via calmodulin. Calcium ions released from the sarcoplasmic reticulum, upon initiation of muscle contraction,
bind to the calmodulin y-subunit of phosphorylase kinase
thus activating it. This enzyme phosphorylates glycogen
phosphorylase resulting in more of the high-activity phosphorylase a and hence, glycogen breakdown is induced.
Hormonal control of glycogen phosphorylase activity is
mediated via the glycogenolytic cascade in response to
adrenaline which activates the cAMP-dependent protein
kinase. This enzyme activates phosphorylase kinase by
phosphorylation and this in turn results in an increase in
phosphorylase a.
Glycogen phosphorylase is also a complex allosteric
enzyme with two conformers: an active 'R' state and an
inactive 'T' state and interconversion between these two
states is via phosphorylation or binding of allosteric ligands at distinct sites on the enzyme. The muscle isozyme
can be activated to 80% of its full activation capacity by
AMP [8]. The phosphorylation site and the AMP binding
site are in close proximity in the three-dimensional structure of the rabbit muscle isozyme and may activate the
enzyme in the same way. Thus, the muscle isozyme is
sensitive to both external signals and to changes in the
concentrations of intracellular ligands which indicate the
energy balance within the cell.
Pyridoxal 5' phosphate
Each subunit of the phosphorylase dimer binds, in a
tight complex, one molecule of the cofactor pyridoxal-5'phosphate [14] to an active site lysine residue (Lys-680 in
rabbit muscle phosphorylase) via a Schiff base. The cofactor is known to play a role in catalysis and although the
proposed mechanism is not conclusive, it is thought that it
serves as a proton donor and also has a role in maintaining
the correct electrostatic environment of the catalytic site
[15].
3
myoglobinuria may result and this can cause renal failure
[17,18]. Symptoms are typically less severe during childhood and as a consequence, the disease is often not
recognised until individuals reach their second or third
decade of life (Section 5.1). However, patients frequently
recall incidences during childhood when they had problems with exercise although, at the time, a clinical disorder
was not suspected.
2.2. The 'second wind' phenomenon
Patients frequently find that they can continue to exercise with increased endurance if they rest briefly at the
first signs of muscle pain. This is termed the 'second
wind' phenomenon [19] and it is attributed to either a shift
in a metabolic pathway or to a circulatory adjustment
which results in enhanced peffusion of intramuscular capillaries [19-21]. The metabolic shift is facilitated by mobilisation of reserves, such as fatty acids [20,21]. This phenomenon has latterly been studied by 31P-NMR techniques
[22,23] and it is clear, although not always feasible, that
increasing the availability of substrate can improve exercise tolerance (Section 5.2).
2.3. Clinical heterogeneity
In addition to the normal clinical picture of McArdle's
disease, a number of atypical presentations have been
described. These include: a very mild form of the disease
with symptoms such as excessive tiredness and poor
stamina [16]; a late-onset form of the disease ( > 40 years
old) in which no exercise-induced symptoms were noted
previously [24-27]; a fatal infantile form which is characterised by progressive weakness soon after birth and severe
breathing difficulties followed by death before 4 months,
as seen in two sisters [28,29]. An unrelated infant also
exhibited this form of the disease but died at 16 days [30];
mild congenital weakness seen in a 4 year old boy [31];
exercise-induced myoglobinuria in an 8 year old boy with
no previous history of exercise intolerance [32]; an attack
of myoglobinuria in a 42 year old man which was apparently 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 characterised by myalgia, severe
cramps, early fatigue and muscle weakness brought on by
exercise, although the severity of the symptoms does vary
between individuals. Progressive weakness of the muscle
may occur later in life. Patients are generally most affected
by either high intensity exercise of short duration, such as
sprinting or carrying heavy loads, or by less intense but
sustained activity, such as walking uphill or riding a
bicycle [16]. If exercise is continued once pain occurs,
McArdle's disease is typically diagnosed by negative
histochemical staining for phosphorylase activity in a muscle biopsy [34]. This stain relies on the ability of phosphorylase to work in the reverse direction and synthesise
glycogen from glucose-l-phosphate -the newly formed
glycogen is detected by an iodine stain. Added insulin,
AMP (activators) and glycogen (primer) enhance the reaction to the extent that even low levels of phosphorylase
activity result in a deep blue stain. In the absence of
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
usually take up stain but the smooth muscle of the intramuscular blood vessels does stain. In addition, McArdle's
patients often show subsarcolemmal deposits of glycogen
which can be detected with periodic acid-Schiff (PAS)
stain on a portion of a muscle biopsy. These increases are
typically 3-5-fold compared to the glycogen content of
normal individuals.
2.5. Exercise analysis
Although histochemical staining of a muscle biopsy
from patients is the most reliable method for the diagnosis
of McArdle's disease (Section 2.4), the ischaemic forearm
test may also be performed on patients. This test is based
on the failure of blood lactate levels to rise in McArdle's
disease in response to exercise (Section 1.1). However, this
test is not specific for glycogen phosphorylase and may
indicate a block in another enzyme of the glycolytic
pathway.
Muscle cell membrane damage frequently occurs in
McArdle's patients due to over-exertion of the muscles
and this releases intracellular components into the circulation. The most significant of these is myoglobin (Section
2.1) but creatine kinase activity is almost always raised in
the plasma of McArdle's patients. In addition to the release
of these intracellular contents, acute local muscle damage
appears as a painful hardening and shortening of the
muscle. This contracture is electrically silent indicating
that it is not caused by active contraction of the muscle
fibres. It could be due to the same kind of failure of
actin-myosin crossbridge relaxation as is known to occur
in 'rigor mortis' when intramuscular ATP is depleted [35].
However, needle biopsy studies have shown that the muscle content of phosphocreatine is not depleted when the
muscle is fatigued [36] and this has been confirmed by
31P-NMR [37].
Leakage of potassium is also consequential to muscle
damage. During whole body exercise, the large increases
in the plasma potassium concentration offers a possible
explanation of the cause of fatigue [38]. Induction of
fatigue by electrical stimulation showed that muscle of a
McArdle's patient was more fatiguable than that of a
normal individual and this was attributed in part to 'fading' of the muscle action potential [39-41] and partly to
myofibrillar activation failure [42].
tion is of limited use during intense exercise because the
circulation cannot provide substrates (fatty acids and
carbohydrates) and oxygen quickly enough to meet the
energy demands [19]. Muscle glycogen phosphorylase
breaks down glycogen to glucose-l-phosphate which enters the glycolytic pathway to generate ATP with the
formation of pyruvate and, under anaerobic conditions,
lactate. Use of glycogen for glycolysis instead of intracellular glucose results in greater net ATP generation and
delays muscle fatigue [44].
3.2. Correlation of symptomatology with an absence of
muscle glycogen phosphorylase
Due to a lack of muscle phosphorylase activity, McArdle's patients cannot mobilise their muscle glycogen stores.
Therefore, depletion of intracellular glucose and ATP,
during the first few minutes of exercise, results in onset of
symptoms. To exacerbate the situation, oxidative phosphorylation is impaired in patients due to an abnormally low
substrate flux through the TCA cycle. This is probably
because the virtual absence of pyruvate from glycolysis is
likely to reduce the rate of acetyl-CoA formation and this
affects the TCA cycle [23,45]. Acetyl-CoA can be generated from the breakdown of fatty acids but unless trained,
most individuals have a limited capacity for fatty acid
oxidation during exercise. This decline in oxidative phosphorylation results in a decreased oxygen consumption in
patients [23] and in fact, maximal oxygen uptake into
McArdle's muscle is 35-50% of normal values [46]. In
addition, McArdle's patients exhibit a disproportionate
increase in heart rate and ventilation rate compared to
normal individuals [21,23].
3.3. Other biochemical effects of the glycogen block
The absence of glycolysis and reduction in oxidative
phosphorylation in McArdle's patients means that increased reliance is placed on the creatine kinase and
adenylate kinase reactions. Both reactions regenerate ATP
very rapidly to provide an immediate short-term source of
energy [47]. The action of these enzymes in combination
with decreased ATP generation from carbohydrate and
fatty acids results in increased levels of ADP and AMP
within the muscle. This affects normal metabolism of
adenine nucleotides resulting in excessive purine degradation [48,49]:
3. Glycogen utilisation in skeletal muscle
ATP ~
3.1. Induction of glycogen breakdown during exercise
In the resting state, fatty acid oxidation predominates in
the skeletal muscle of normal individuals but during exercise, the energy requirements are largely met by glycogen
breakdown and anaerobic glycolysis [43]. Aerobic oxida-
ADP ",~ "" AMP - - ~
~
~
Inosine ~
Hypoxanthine
NH3
Inosine, hypoxanthine and ammonia enter the bloodstream and elevated plasma levels of all three have been
detected in some McArdle's patients following exercise
C. Bartram et al. / Biochimica et Biophysica Acta 1272 (1995) 1-13
[48-50]. However, excessive release of hypoxanthine and
inosine has not been observed in all McArdle's patients
[51,52]. Elevated ammonia output is seen consistently but
cannot be solely attributed to purine nucleotide degradation use of amino acids as an alternative energy source in
McArdle's muscle also releases ammonia into the bloodstream (Section 5.2.).
McArdle's patients also show an enhanced response to
various hormones compared to normal individuals which
helps to minimise the effect of the glycolytic block. Neural
feedback from working muscle causes an increase in the
concentrations of growth hormone, cortisol, noradrenaline,
adrenaline and adrenocorticotrophic hormone and a decrease in insulin concentration which results in mobilisation of extramuscular fuel, such as glucose and free fatty
acids, to meet the energy requirements of the muscle [53].
4. Molecular pathology of McArdle's disease
4.1. Molecular phenotypes
McArdle's disease is characterised by an absence of
functional muscle glycogen phosphorylase but patients
show heterogeneity in their protein and mRNA expression
(Table 1). The majority of McArdle's patients do not have
detectable muscle glycogen phosphorylase [54-61]; of 93
patients, only 13 expressed low levels of protein [54,57-61]
and a further two expressed inactive protein [55,61]. Of 22
patients whose mRNA has been determined, twelve lacked
detectable mRNA [54,57,59] whereas nine expressed the
3.4 kb phosphorylase mRNA at normal [59,61] or reduced
levels [56]. Another patient expressed a truncated form
(1.2 kb) of the transcript [61]. Bidimensional protein maps
of muscle extract from three McArdle's patients identified
potential degradative intermediates of phosphorylase. Mus-
5
cle phosphorylase (molecular mass 97 kDa) was not seen
but two spots with molecular masses 60 kDa and 70 kDa
were detected. However, these two spots were not recognised by antibodies to phosphorylase [62]. These spots may
be completely unrelated to phosphorylase or alternately,
the antibodies may have been sensitive to a conformational
epitope that was altered by misfolding or by sample preparation.
4.2. Muscle glycogen phosphorylase gene
Although there have been a number of studies on the
molecular phenotypes of McArdle's patients, until recently, nothing was known of the mutations causing the
disease. The human muscle glycogen phosphorylase gene
is approx. 14 kb [63]. Sequencing of the 5' region identified several promoter regions [11] including a TATA-Iike
homology, TTTAAA, located at position - 2 9 (based on
the designation of the transcription start site as + 1) and a
potential CAAT box, CCAAGAC, at - 8 0 . There is a
region at - 5 9 2 with the sequence CTCCAAAAGG which
is necessary for efficient transcription of the phosphorylase
gene. This sequence superficially resembles the CArG
motif CC(A/T)rGG present in the promoter regions of
skeletal and cardiac muscle a-actin genes and has been
implicated in their tissue-specific expression [64,65]. It is
repeated at - 2 5 2 in the 5' region of the phosphorylase
gene, but expression studies indicate that this later sequence is not necessary for transcription so sequence
context may be critical [11].
Even before the elucidation of the mutations causing
McArdle's disease, it was generally held to be an autosomal recessive disease [66,16]. Autosomal dominant inheritance was suggested in a family in which four generations
were reportedly affected with McArdle's disease [67].
However, the disease was only confirmed in two individu-
Table 1
Molecular phenotypes in McArdle's patients
Molecular phenotypes
[54]
No protein, no mRNA
No protein, normal mRNA
No protein, decreased mRNA
No protein, truncated mRNA
No protein, mRNA n.d.
Decreased protein, normal mRNA
Decreased protein, mRNA n.d.
Normal levels of inactive protein
2
Total number of patients
[55]
[56]
[57]
[58]
5
[59]
[60]
5
3
[61 ]
1
3
5
2
2
2
2
4
I
2
3
6
2
1
2
1
1
9
3
10
11
3
1
39
1
5
1
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
SacI
122G-> "IT
t'N\\\lll
llHH
R49X
N l a 111
II
•
Ill
1606delG
AF708/709
I 1
nl m nm nnn
/
nit niL.~
K542T
MaeII
G204S
HaeBI
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'
AF708/709
ATTC
17
Abnormal splicing
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
als from two generations and a dominant mechanism of
inheritance in this family is questionable. In most cases,
heterozygous carriers for McArdle's disease are unaffected
because it seems that one normal allele of the muscle
phosphorylase gene is adequate for normal levels of the
protein. However, there are a number of reports of manifesting heterozygotes who do have phosphorylase activity
upon histochemical staining but who show clinical symptoms of McArdle's disease [68,69]. A threshold value of
2 0 - 4 5 % of normal has been suggested to be the minimum
activity below which symptoms occur [69] although assignment is tentative.
[74]. Again the truncated peptide would probably be unstable and therefore, rapidly degraded.
There are four missense mutations and a single amino
acid deletion which may have functional or structural
consequences on muscle glycogen phosphorylase (Section
4.4). The G204S mutation occurs in a domain involved in
glycogen binding whereas K542T affects the glucose-binding domain [72]. Both L291P and A F 7 0 8 / 7 0 9 may alter
normal protein folding and possibly, intracellular stability
[75]. The initial methionine codon is altered by the 77A --*
C mutation thus abolishing translation initiation [76]. Another mutation ( 1 8 4 4 + I G ~ A )
alters the consensus
splice site sequence at the 5' end of intron 14 and as a
consequence, an alternative sequence in exon 14 is used.
This results in abnormal splicing which produces a 67 bp
deletion in the transcript. The resulting shift in the reading
frame generates a short sequence of missense polypeptide
containing 14 amino acids before translation is prematurely terminated after 580 amino acids [75]. Another
frameshift is caused by the deletion of a single base in
codon 509 (1606delG) and this again results in premature
termination of translation, this time after 536 amino acids
[77]. The allele frequencies of the above mutations are
shown in Table 2.
4.3. Mutational analysis o f McArdle's disease
Initial work focused on restriction fragment length polymorphism of the muscle phosphorylase gene of McArdle's
patients but met with little success [56,61]. The muscle
phosphorylase gene is notable for its lack of polymorphisms and restriction enzyme analysis with 94 enzymeprobe combinations in 20 unrelated patients revealed just a
single polymorphism at a CpG site associated with McArdle's disease [70]. This analysis did, however, preclude the
possibility of gene deletions greater than 100 bp.
Since these initial studies, sequencing has revealed a
number of mutations in the muscle phosphorylase gene
which cause McArdle's disease (Fig. 1). The most common base change is a nonsense mutation [71,72], designated R49X (nomenclature proposed in Ref. [73]). This
results in premature termination of translation; the resulting truncated peptide is so short that it is non-functional
and probably rapidly degraded. A second mutation in exon
1, 122G ~ TT, shifts the reading frame such that a short
sequence of missense protein containing 11 amino acids is
synthesised before translation is prematurely terminated
N
M5
M6
M2
N
M7
N
7
4.4. Effect o f mutations on molecular phenotypes
The R49X mutation occurs in the coding region of the
muscle phosphorylase gene and causes premature termination of protein translation. However, of the patients whose
biochemical phenotype is known, all those homozygous
for the R49X mutation have no detectable m R N A on
northern blots (Fig. 2). Therefore, premature termination of
protein translation affects the stability of the m R N A [71,72]
and there are a number of precedents for this in other
M8
M1
M3
M4
M9
N
3.4kb phosphorylase
mRNA
Mutation
analysis
__+ R49X R49X R49X +_ R49X
+
?
R49X R49X + R49X
+ R49X R49X n.d. Rg9X R49X +
+
? 122G->TT
? R49X +
K e y : 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
Allele frequency of the mutations causing McArdle's disease
Mutation
Allele frequency in
European population
[71,74,109]
Allele frequency in USA
and Japanese population
[72,75-77]
R49X
77A ~ C
122G ~ TT
G204S
L291P
1606delG
K542T
1844+1G ~ A
AF708/709
46
0
1
2
0
n.d.
0
0
n.d.
5l
1
n.d.
8
1
2
3
1
7
n.d. = not determined.
The most common mutation is R49X which is present in 46 out of a total
of 76 available alleles from European McArdle's patients, The majority
of these patients are Caucasians although there are a number of patients
whose ethnic origins are not known. The USA patients are Caucasian and
R49X is the predominant mutation in these patients. However, the
AF708/709 mutation has only been found in Japanese patients (7 out of
14 alleles) suggesting that this might be a common mutation in this
population. None of the Japanese patients carry R49X and so, it would
appear that McArdle's disease has occurred independently in these two
populations. The other mutations are much less frequently observed.
diseases. For example, decreased stability of the transcript
in response to premature termination of translation occurs
in human /3-globin mRNA as illustrated by the /30 thalas-
saemias [78-81], mRNA of cystic fibrosis transmembrane
conductance regulator which is implicated in cystic fibrosis [82] and mRNA of human /3-hexosaminidase A which
is defective in Sandhoff disease [83].
Patient M 1 carries the R49X mutation on one allele of
the muscle phosphorylase gene and the 122G ~ TT on the
other, both of which cause premature termination of translation. However, this individual does seem to express low
levels of mRNA as seen by northern blot analysis (Fig. 2)
which is unexpected given that termination of translation
in exon 1 occurs from both alleles. The mechanism by
which mRNA stability could be differentially affected by
these two different mutations is not clear but it is possible
that the mRNA carrying the frameshift mutation is not
translatable. Degradation of aberrant transcripts is thought
to be co-translational [84], such that a complete inability to
translate the mRNA would cause it to have increased
stability.
Analysis of molecular phenotypes of patients has suggested that the missense mutations destabilise the protein
[75]. Rabbit muscle glycogen phosphorylase remains the
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
overall amino acid sequence identity for rabbit and human
muscle phosphorylase is 97% and there is no variation in
any catalytic or allosteric sites [63,85]. All three of the
G204S
L29 IP
I
Human muscle
Rabbit muscle
Rat muscle
Human liver
Rat liver
Human brain
Rat brain
Yeast
Potato type H
VHFYGHVEH
VHFYGRVEH
VHFYORVEH
VHFYGKVEH
VHFYGRVEH
VHFYGRVEH
VHFYORVEH
VTFYGYVDR
IRFFGHVEV
I
Human muscle
Rabbit muscle
Rat muscle
Human liver
Rat liver
Human brain
Rat brain
Yeast
Potato type H
K542T
KQENKLKFA
KQEN]KLKFA
KQENKLKFS
KQENKLKFS
KQENKLKFS
KQENIKLKFS
KQENKLKFS
KLNNI~IRLV
KMANKQRLA
EGKELRLKQ
EGKELRLKQ
EGKELRLKQ
EGKELRLKQ
EGKELRLKQ
EGKELRLKQ
QGKELRLKQ
NGKLLRLKQ
AF'/08/709
I
Human muscle
Rabbit muscle
Rat muscle
Human liver
Rat liver
Human brain
Rat brain
Yeast
Potato type H
EGKELRLKQ
I
Human muscle
Rabbit muscle
Rat muscle
Human liver
Rat liver
Human brain
Rat brain
Yeast
Potato type H
GEENFFIFG
GEENFFIFG
GEDNFFIFG
GEENLFIFG
GEENLFIFG
GAENLFIFG
GEENLFIFG
GEDNVFLFG
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
highly conserved amino acid (Fig. 3). The sequence conservation is such that the amino acid at these positions
remains unchanged across the species [7] including two
non-mammalian phosphorylases (yeast and potato type H).
This suggests that each of these amino acids is critical for
the normal function of glycogen phosphorylase; their alteration could decrease the activity or the stability of the
mutant protein. There is also strong conservation in the
region of the AF708/709 mutation (Fig. 3).
creased by heparin [21], noradrenaline [20,21] or by infusion of emulsified fat, particularly in combination with
glucose [19]. The same effect was produced by fasting
which increases plasma concentrations of free fatty acid
[91]. However, a high fat diet gave more variable results
because one patient reported no beneficial effect [89]
whereas another noted subjective improvement after 3
days although there was no increase in measured muscle
strength [92]. The benefits of a high fat intake on muscle
performance must be weighed up against the potentially
detrimental effect on an individual's long-term health.
5. Clinical management and therapy
Amino a c i d / p r o t e i n supplementation
5.1. Exercise management
Exercise management and weight control have an important role to play in the management of McArdle's
disease. Weight determines oxygen consumption during
walking, especially when walking uphill. It is therefore
advantageous for a patient with McArdle's disease to
avoid excess weight. Patients should also try to keep active
within the capability of their muscles as reduced activity in
patients with muscle disorders can diminish the mitochondrial function in muscle [86]. The greater the mitochondrial
oxidative capacity, the less dependent an individual is on
glycogenolysis. Children are more active than adults with a
higher aerobic capacity per kilogram body weight [87] and
this may explain why McArdle's disease is usually not
diagnosed in young children.
5.2. Dietary management
The aim of any current therapy for McArdle's disease is
to enable patients to exercise with increased tolerance and
to prevent the occurrence of muscle damage or myoglobinuria which can lead to renal failure. The provision of
alternative or additional energy fuels to the skeletal muscle
can accelerate the onset of the 'second wind' phenomenon
and thus alleviate glycolytic deprivation.
Carbohydrate supplementation
Glucose infusion enables patients to exercise with increased endurance [19,21,22,50] and prevents exercise-induced ATP degradation as measured by a decrease in the
degradative metabolites [50] and by a decline in plasma
ammonia [23]. Similarly, fructose infusion improves exercise endurance [ 19,21 ]. However, long-term oral administration of glucose or fructose has been largely unsuccessful
in improving the exercise capabilities of patients [16,89].
Benefits are only seen when carbohydrate is given immediately before exercise and in practice, it is not always
possible to predict a bout of exertion [90].
Fatty acid supplementation
McArdle's patients could exercise with increased endurance when plasma levels of free fatty acid were in-
There is evidence for increased utilisation of amino
acids in skeletal muscle of McArdle's patients [52,93].
Therefore, provision of adequate amino acids by direct
supplementation, or via a high protein diet might improve
muscle function. This was confirmed in one patient whose
protein intake was increased to 25-30% protein from 14%
[94]. In a subsequent study, three patients were provided
with branched chain amino acids in amounts calculated to
exceed the levels expected from a high protein diet but
none experienced either an immediate or long-term (1-2
months) subjective improvement or an objective benefit as
assessed by their maximal strength and endurance [95].
The first step in amino acid catabolism is donation of
the a-amino group to 2-oxoglutarate (a TCA cycle intermediate) in a transamination reaction which generates the
cognate 2-oxo acid and glutamate. The 2-oxoglutarate may
be regenerated by deamination with the release of ammonia. Alternately, a second transamination reaction with
pyruvate regenerates 2-oxoglutarate and results in the corresponding formation of alanine. In the normal individual,
alanine and glutamine (formed by the reaction of glutamate with ammonia) provide a mechanism for the elimination of nitrogen from the muscle and its transport to the
liver for detoxification. However, in McArdle's disease,
the supply of precursors for alanine and glutamine is
compromised. In exercising McArdle's muscle, glycolytic
flux is suppressed and it is unlikely that adequate pyruvate
is synthesised to sustain the levels of alanine required for
ammonia removal. Likewise, any reaction that drains TCA
cycle intermediates, such as 2-oxoglutarate, would ultimately impair energy metabolism. Thus, a decreased ability to remove ammonia, in the form of alanine and glutamine might explain the elevated plasma ammonia levels
during exercise in McArdle's disease [38]. These increased
ammonia levels may be responsible in part for the muscle
pain and fatigue [96]. The ability of glucose to diminish
ammonia output by McArdle's muscle [23] is probably due
to an increased supply of pyruvate which can be transaminated to alanine.
Uptake and oxidation of the branched chain amino acids
occurs in both normal individuals and at a higher rate in
McArdle's patients during exercise [52]. Consistent with
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,
the key enzyme in the oxidation of branched chain amino
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
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
expected beneficial effect and in fact, exercise performance actually deteriorated [96]. This may be due to the
increased ammonia resulting from transamination of these
amino acids. Any benefits gained from branched chain
amino acid oxidation must be offset against the additional
nitrogen burden.
In contrast, supplements of 10 g of the ornithine salts of
the 2-oxo acids of leucine and valine (4-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate, respectively) and 5
g of the sodium salt of the 2-oxo acid of isoleucine
(3-methyl-2-oxopentanoate), if given 20 min prior to exercise, improved exercise performance, resulted in smaller
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
load of the muscle cells, sparing pyruvate and 2-oxoglutarate. If the supplements of 2-oxo acids were given 90 min
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
branched chain oxo acids were combined with 75 g glucose given orally 30 rain before exercise [38,96].
Uptake of alanine by exercising muscle was described
in one McArdle's patient [93] although it is usually released from normal muscle. A later study with a number of
McArdle's patients initially appeared to be at variance
with this finding as alanine was released by muscle [52].
However, when the exercise protocols were examined in
more detail, the two opposing results can be related to the
work pattern (Fig. 4a). In both studies, similar total work
was performed but in the earlier study, the work was
gradually increased and so, it is likely that the patient
actually reached the 'second wind'. It is possible to gain
some insight into alanine metabolism by combining the
information from these two studies although the conclusions 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 individuals. This is consistent with an initially increased demand
on pyruvate for transamination and a subsequent decrease
in the pyruvate pool which cannot be replenished. It then
appears that McArdle's muscle actually takes up alanine
which may be driven by gluconeogenesis. However, the
magnitude of alanine uptake into muscle is substantially
lower than initial release.
Amino acid supplementation may have a second effect
on muscle protein metabolism. Increased oxidation of
amino acids by skeletal muscle in McArdle's disease
a)
8O
6O
~" 40
g' 20
0
0
20
40
60
80
Exercise duration (rain)
b)
160
120 ~
Release
80
~
40
II
0
= -40 ~
-80
2
~40
Time (rain)
60-
80
-
-120 +
Uptake
- 160
l
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
different exercise protocols, which are illustrated in panel a. The study by
Wagenmakers and colleagues (study I) [52] used short periods of higher
intensity exercise, whereas that of Wahren and colleagues (study II) [93]
was a longer duration, low intensity programme. The comparisons of the
two protocols are approximate, and are based on our interpretation of the
experimental details given. The data for alanine flux, obtained from both
studies (including those from normal individuals), are placed on the same
time scale, but must be interpreted in the light of the different exercise
protocols. McArdle's patients, []; Normal individuals, I .
might reduce their availability for the synthesis of new
muscle protein [98] or elicit enhanced protein degradation.
If this imbalance in protein turnover is prolonged it could
ultimately lead to long term muscle wasting and weakness.
Vitamin B-6 supplementation
Glycogen phosphorylase requires PLP as a cofactor and
apo-phosphorylase cannot be detected in skeletal muscle
[99]. It is not clear whether this is due to a marked
instability of apo-enzyme or whether the apo-enzyme has
such a high affinity for its cofactor that it 'scavenges' PLP
C. Bartram et al. / Biochimica et Biophysica Acta 1272 (1995) 1-13
from other body pools. However, because phosphorylase is
such an abundant protein (2-5% of the total soluble
protein in skeletal muscle), and because of the large mass
of the musculature, 80% of the body pool of vitamin B-6 is
attached to this enzyme [100]. We have used [G-3H]
pyridoxine as a specific label for phosphorylase in vivo
and have observed that the rate of loss of labelled PLP
from the enzyme parallels the rate of phosphorylase degradation [101-103]. Therefore, release of PLP from
phosphorylase is dependent on degradation of the protein
[104]. Vitamin B-6 metabolism studies have consistently
identified a large, slowly accessible vitamin B-6 pool in
the body [105,106] which is undoubtedly muscle glycogen
phosphorylase. The other body pools of vitamin B-6 appear to have a much higher turnover rate and are probably
more readily depleted when vitamin B-6 intake is inadequate. In contrast, even extended periods of vitamin B-6
deprivation do not significantly diminish the adult muscle
phosphorylase pool [107,108]. This has led to the suggestion that the daily vitamin B-6 requirements are different
in growth and maintenance; during growth, vitamin B-6 is
incorporated into the slow muscle phosphorylase pool,
whereas in the adult, the daily requirement is that which is
needed to sustain the fast pools [106]. We therefore propose that muscle phosphorylase could function as a
'buffer', compensating for day-to-day variation in dietary
intake of vitamin B-6 in normal individuals.
The majority of McArdle's patients lack muscle glycogen phosphorylase, which means that this slow pool of
vitamin B-6 is absent and consequently, its 'buffering'
capacity is lost. McArdle's patients may therefore be more
reliant on a regular intake of vitamin B-6 and vitamin B-6
status, as assessed by an erthyrocyte transaminase assay,
does seem to be compromised in some of our patients
[109]. Furthermore, McArdle's patients may be more reliant on adequate vitamin B-6 compared to normal individuals because they are dependent on alternative energy
pathways which require PLP. Aminotransferases play a
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 required to facilitate the transport of fatty acyI-CoA across
the inner mitochondrial membrane prior to oxidation. Inadequate vitamin B-6 status results in reduced carnitine
biosynthesis and as a consequence, reduced fatty acid
oxidation [ 110].
We have postulated that the exercise performance of
those McArdle's patients who lack glycogen phosphorylase protein might be improved by supplementation with
relatively high daily doses (50 mg) of vitamin B-6 (the
recommended daily dose for normal individuals is 2 mg).
Vitamin B-6 supplementation did improve the muscle fatiguability of three McArdle's patients by increasing the
force generation of the muscle [109]. This implies that
i m p r o v e m e n t is due to an increase in fuel
mobilisation/utilisation and the inference from this is that
11
the cofactor saturation of the aminotransferases in the
muscles is improved. Supplementation of two patients with
vitamin B-6 in earlier studies did not prove effective
[ 18,11 1], but both patients had some phosphorylase protein
on an SDS polyacrylamide gel. The presence of protein,
albeit inactive, means that it could still function as a
'buffer' for PLP and vitamin B-6 supplementation may not
be beneficial in these patients. Mild sensory neuropathy
associated with vitamin B-6 overdosage is only seen at
levels of 2 - 4 g / d over several months [112]. Therefore,
vitamin B-6 at 50 m g / d may potentially improve the
exercise capacity of the majority of McArdle's patients
without having a detrimental effect on their health. In
addition, as aminotransferases require PLP as a cofactor,
we suggest that any increased protein or amino acid regimen must ensure that vitamin B-6 intake is adequate. If the
activity of the aminotransferases is limited by inadequate
PLP then the increased protein may have little benefit.
5.3. Gene therapy
Interest is now being focused on gene therapy in the
treatment of genetically inherited diseases. A recombinant
adenovims has been used to drive expression of rabbit
muscle phosphorylase cDNA in a C2C12 myoblast cell
line [1 13]. Adenoviral delivery into myoblasts, which were
subsequently induced to differentiate, resulted in a moderate and transient increase in phosphorylase activity. However, introduction into non-proliferating myotubes resulted
in higher phosphorylase activity which was sustained over
the period of the experiment (20 days). The heterologously
expressed phosphorylase was subject to both hormonal and
allosteric control which suggests that glycogen breakdown
will occur in a regulated manner in the treated muscle of
patients. It might be anticipated that when suitable delivery
vehicles are developed for muscle, probably driven by
other severe myopathies, such therapy in McArdle's disease will also be successful.
Acknowledgements
We would like to acknowledge The Muscular Dystrophy Group of Great Britain and Northern Ireland for their
financial support. We would also like to thank the McArdle's patients, their families and our colleagues for their
continuing contribution to this work.
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