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Figure 1.
Sequence analysis of the missense mutation in the myophosphorylase gene. Antisense sequence electropherogram of the myophosphorylase gene shows the A→G change at codon 797 in 1 homozygous patient. The mutated nucleotide is indicated by an arrow.

Sequence analysis of the missense mutation in the myophosphorylase gene. Antisense sequence electropherogram of the myophosphorylase gene shows the A→G change at codon 797 in 1 homozygous patient. The mutated nucleotide is indicated by an arrow.

Figure 2.
Restriction analysis of mutation W797R, showing digestion with Bsr BI in a normal subject (lane 6), in 2 homozygous patients (lanes 3 and 5), and in 1 heterozygous patient (lane 4). Lane 2 corresponds to a nondigested fragment, and lane 1 shows DNA molecular-weight markers. In the presence of the mutation, the 903–base pair (bp) fragment is cleaved into 2 fragments of 664 and 239 bp.

Restriction analysis of mutation W797R, showing digestion with Bsr BI in a normal subject (lane 6), in 2 homozygous patients (lanes 3 and 5), and in 1 heterozygous patient (lane 4). Lane 2 corresponds to a nondigested fragment, and lane 1 shows DNA molecular-weight markers. In the presence of the mutation, the 903–base pair (bp) fragment is cleaved into 2 fragments of 664 and 239 bp.

1.
DiMauro  SServidei  STsujino  S Disorders of carbohydrate metabolism: glycogen storage diseases. Rosenberg  RNPrusiner  SBDiMauro  SBarchi  RLeds.The Molecular and Genetic Basis of Neurological Disease. 2nd ed. Boston, Mass Butterworth-Heinemann1997;1067- 1097
2.
DiMauro  SHartlage  PL Fatal infantile form of muscle phosphorylase deficiency. Neurology. 1978;281124- 1129Article
3.
El-Schahawi  MBruno  CTsujino  S  et al.  Sudden infant death syndrome (SIDS) in a family with myophosphorylase deficiency. Neuromuscul Disord. 1997;781- 83Article
4.
Lebo  RVGorin  FFletterick  RJ  et al.  High-resolution chromosome sorting and DNA spot-blot analysis assign McArdle's syndrome to chromosome 11. Science. 1984;22557- 59Article
5.
Burke  JHwang  PAnderson  LLebo  RGorin  FFletterick  R Intron/exon structure of the human gene for the muscle isozyme of glycogen phosphorylase. Proteins. 1987;2177- 187Article
6.
Kubisch  CWicklein  EMJentsch  TJ Molecular diagnosis of McArdle's disease: revised genomic structure of the myophosphorylase gene and identification of a novel mutation. Hum Mutat. 1998;1227- 32Article
7.
Vorgerd  MKubisch  CBurwinkel  B  et al.  Mutation analysis in myophosphorylase deficiency (McArdle's disease). Ann Neurol. 1998;43326- 331Article
8.
Bruno  CTamburino  LKawashima  N  et al.  A nonsense mutation in the myophosphorylase gene in a Japanese family with McArdle's disease. Neuromuscul Disord. 1999;934- 37Article
9.
Tsujino  SShanske  SDiMauro  S Molecular heterogeneity of myophosphorylase deficiency (McArdle's disease). N Engl J Med. 1993;329241- 245Article
10.
El-Schahawi  MTsujino  SShanske  SDiMauro  S Diagnosis of McArdle's disease by molecular genetic analysis of blood. Neurology. 1996;47579- 580Article
11.
Bartram  CEdwards  RHTClague  JBeynon  RJ McArdle's disease: a nonsense mutation in exon 1 of the muscle glycogen phosphorylase gene explains some but not all cases. Hum Mol Genet. 1993;21291- 1293Article
12.
Martinuzzi  ATsujino  SVergani  L  et al.  Molecular characterization of myophosphorylase deficiency in a group of patients from northern Italy. J Neurol Sci. 1996;13714- 19Article
13.
Andreu  ALBruno  CGamez  J  et al.  Molecular genetic analysis of McArdle's disease in Spanish patients. Neurology. 1998;51260- 262Article
14.
Shanske  SSakoda  SHermodson  MADiMauro  SSchon  EA Isolation of a cDNA encoding the muscle-specific subunit of human phosphoglycerate mutase. J Biol Chem. 1987;26214612- 14617
15.
Hudson  JWGolding  GBCrerar  MM Evolution of allosteric control in glycogen phosphorylase. J Mol Biol. 1993;234700- 721Article
16.
Sugie  HSugie  YIto  MFukuda  TNonaka  YIgarashi  Y Genetic analysis of Japanese patients with myophosphorylase deficiency (McArdle's disease): single-codon deletion in exon 17 is the predominant mutation. Clin Chim Acta. 1995;23681- 86Article
17.
Tsujino  SShanske  SGoto  YNonaka  IDiMauro  S Two mutations, one novel and one frequently observed, in Japanese patients with McArdle's disease. Hum Mol Genet. 1994;31005- 1006Article
18.
Tsujino  SRubin  LAShanske  SDiMauro  S An A to C substitution involving the translation initiation codon in a patient with myophosphorylase deficiency (McArdle's disease). Hum Mutat. 1994;473- 75Article
19.
Tsujino  SShanske  SNonaka  I  et al.  Three new mutations in patients with myophosphorylase deficiency (McArdle's disease). Am J Hum Genet. 1994;5444- 52Article
20.
Tsujino  SShanske  SMartinuzzi  AHeiman-Patterson  TDiMauro  S Two novel missense mutations (E654K, L396P) in Caucasian patients with myophosphorylase deficiency (McArdle's disease). Hum Mutat. 1995;6276- 277Article
21.
Bartram  CEdwards  RHTClague  JBeynon  RJ McArdle's disease: a rare frameshift mutation in exon 1 of the muscle glycogen phosphorylase gene. Biochim Biophys Acta. 1994;1226341- 343Article
22.
Olmos  JMZarrabeitia  MTValero  MCFigols  JMatorras  PRiancho  JA McArdle's disease in adults: clinical and genetic study [in Spanish]. Med Clin (Barc). 1997;109753- 755
23.
Andreu  ALBruno  CTamburino  L  et al.  A new mutation in the myophosphorylase gene (Asn684Tyr) in a Spanish patient with McArdle's disease. Neuromuscul Disord. 1999;9171- 173Article
24.
Gamez  JFernandez  RBruno  C  et al.  A new mutation in the regulatory domain of the myophosphorylase gene affecting protein dimer contact. Muscle Nerve. 1999;221136- 1138Article
25.
Martiniuk  FMehler  MTzall  SMeredith  GHirschhorn  R Extensive genetic heterogeneity in patients with acid alpha glucosidase deficiency as detected by abnormalities of DNA and mRNA. Am J Hum Genet. 1990;4773- 78
26.
Raben  NSherman  JB Mutations in muscle phosphofructokinase gene. Hum Mutat. 1995;61- 6Article
Original Contribution
February 2000

A Novel Missense Mutation (W797R) in the Myophosphorylase Gene in Spanish Patients With McArdle Disease

Author Affiliations

From the H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases, Department of Neurology, Columbia University College of Physicians and Surgeons, New York, NY (Mr Fernández and Drs Andreu, Bruno, Shanske, Musumeci, and DiMauro); Departments of Pathology and Neuropathology (Messrs Fernández and Teijeiro, Dr Navarro, and Ms Teijeira) and Rheumatology (Dr Hernández), Hospital do Meixoeiro, Vigo, Spain; Centre d'Investigacions en Bioquimica i Biología Molecular (Dr Andreu) and Department of Neurology (Dr Gámez), Hospitals Vall d' Hebron, Barcelona, Spain; Department of Pediatrics, University of Genova, Istituto G. Gaslini, Genova, Italy (Dr Bruno); and Department of Clinical Neurophysiology, Hospital Xeral-Cíes, Vigo, Spain (Dr Fernández).

Arch Neurol. 2000;57(2):217-219. doi:10.1001/archneur.57.2.217
Abstract

Objective  To investigate the degree of genetic heterogeneity of myophosphorylase deficiency (McArdle disease) in Spain through molecular studies of 10 new patients.

Design  The coding sequence of the entire myophosphorylase gene was sequenced in DNA extracted from muscle and blood. Restriction fragment length polymorphism analysis of polymerase chain reaction fragments was used to confirm and simplify detection of a novel mutation.

Setting  A collaborative study between 2 university laboratories in Spain and the United States.

Results  Five of the 10 patients harbored a novel missense mutation in exon 20, converting a tryptophan to an arginine (W797R). Three patients were homozygous for the "common" R49X mutation, and the remaining 2 patients were compound heterozygotes for R49X and a previously described missense mutation, G204S.

Conclusions  The W797R missense mutation is the third novel mutation to be identified among Spanish patients. Its relative frequency suggests that it should be added to the R49X mutation in the molecular screening of McArdle disease in Spain.

MCARDLE disease (glycogenosis type V), one of the most common metabolic myopathies, is caused by the deficiency of myophosphorylase (α-1,4-glucan orthophosphate glycosyltransferase), a specific skeletal muscle enzyme that initiates glycogen breakdown.1 The disease is characterized clinically by exercise intolerance, myalgia, muscle cramps, and, in some patients, recurrent myoglobinuria. These symptoms typically appear in adolescence or early adulthood, although a rare fatal infantile form of the disease has also been described.13

The myophosphorylase gene, located on chromosome 11q13,4,5 has been cloned and sequenced, and a recent revision of its structure6 has made it possible to identify several new mutations,7,8 for a total of 16 different mutations in patients from the United States, Japan, and several European countries, including Spain. The most frequent mutation, at least among patients of Anglo-Saxon origin, is a nonsense mutation in exon 1 (R49X), which causes the substitution of an arginine (CGA) with a stop codon (TGA).911 However, this mutation appears to be less frequent in southern European countries (32% frequency in Italian and Spanish patients), suggesting a decreasing north-south gradient.12,13 Also, increasing numbers of private mutations have been reported in specific ethnic groups, which underscores the genetic heterogeneity of this disease.

We now describe molecular studies in a series of Spanish patients with McArdle disease that revealed a new and relatively frequent missense mutation (W797R) in exon 20.

PATIENTS AND METHODS

We studied 10 patients from 9 families, who had typical symptoms, including exercise intolerance with cramps and myalgia but no overt pigmenturia. Only 1 patient had a positive family history. Laboratory tests showed increased serum creatine kinase levels (range, 138-20 000 U/L; reference range, <80 U/L). Electromyography showed myopathic features, and forearm ischemic exercise caused no rise in venous lactate levels. Open muscle biopsy was performed in all cases.

Genomic DNA was extracted by conventional methods from peripheral-blood leukocytes and muscle tissue of patients and 40 healthy controls.14 The coding sequence of the entire myophosphorylase gene was amplified by polymerase chain reaction (PCR) from genomic DNA as described by Kubisch et al.6 The PCR products were purified by electrophoresis in 2% agarose gel and sequenced directly, with the same set of primers as for amplification, by means of a terminator cycle sequencing kit and a genetic analyzer (ABI Prism Big-Dye Terminator Cycle Sequencing Kit and ABI Prism 310 Genetic Analyzer, respectively; Perkin Elmer, Foster City, Calif).

To simplify the detection method of mutation T→C in exon 20, a 903–base pair (bp) genomic DNA fragment was amplified by PCR with primers 18F and 20B.6 The PCR conditions were 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute. In the presence of the mutation, the PCR product was cleaved by Bsr BI restriction endonuclease (New England Biolabs, Beverly, Mass) into 2 fragments of 664 and 239 bp, after incubation at 37°C overnight. Digestion products were analyzed in 2% agarose gel.

RESULTS

Muscle biopsies showed subsarcolemmal vacuoles containing periodic acid–Schiff–positive, amylase-sensitive material in the majority of fibers. The histochemical reaction for myophosphorylase was absent.

Direct sequencing of the entire coding region of the myophosphorylase gene showed a new missense mutation (T→C) at codon 797, exon 20 (W797R), which resulted in the change of tryptophan (TGG) to arginine (CGG) (Figure 1). To confirm the mutation and develop a rapid detection method, a 903-bp DNA fragment spanning exons 18, 19, and 20 was amplified and its product digested with Bsr BI restriction endonuclease. In the presence of the mutation, the 903-bp fragment was cleaved into 2 segments of 664 and 239 bp.

Restriction length fragment polymorphism analysis with the enzyme Bsr BI (Figure 2) showed that 2 patients were homozygous for the W797R mutation, while 1 patient was a compound heterozygote for this mutation and R49X. In 2 other patients, the new mutation was identified only in 1 allele. Three patients were homozygous for the common R49X mutation, and 2 siblings were compound heterozygotes for R49X and mutation G204S. The W797R mutation was not detected in a series of 40 normal individuals of the same ethnic origin.

COMMENT

We have identified a novel mutation in exon 20 (W797R) of the myophosphorylase gene in 5 of 10 Spanish patients with McArdle disease. We believe this mutation to be pathogenic for the following reasons: (1) the T→C at codon 797 was the only nucleotide change found in the entire coding region and exon-intron boundaries of the myophosphorylase gene; (2) the tryptophan in amino acid position 797 is highly conserved, not only in human liver and brain isozymes, but also in glycogen phosphorylases of species phylogenetically far removed from humans, such as yeast, potato, and Escherichia coli 15; (3) substitution of an aromatic amino acid (Trp) with a basic amino acid (Arg) in codon 797 may affect the C-terminal catalytic domain of the enzyme, changing its secondary structure and possibly inducing rapid breakdown of the protein15; and (4) this mutation was not found in 40 normal Spanish controls.

McArdle disease is genetically highly heterogeneous, with 16 specific mutations described to date in patients from different countries. The most frequent among these is a nonsense mutation at codon 49 (R49X), observed in a high percentage of patients from the United States (64%)9,10 and Great Britain (81%)11; however, the frequency of this mutation declines to 56% in Germany7 and to 32% in Italy and Spain,12,13 suggesting a north-south gradient through the European continent.

The missense mutation G204S located in exon 5 of the gene, which changes a glycine to a serine, is thought to affect the N-terminal regulatory domain, which contains the majority of ligand binding residues.15 This mutation was found in several American families (12%) and in 2 different Spanish families (1 of which is described here), but always in heterozygous form. A frequent mutation confined to the Japanese population is a deletion of codon 708/709, found in 72% of cases.16 A few other mutations have been described in Japanese patients,8,17 but the R49X, which is so common in western countries, has never been found. The remaining mutations are authentic rarities,1821 described only in isolated cases.

Previous genetic studies in Spanish patients had established the R49X mutation as the most frequent in this population,13,22 although private mutations are also being described.23,24 In our series, the frequency of the R49X mutation was significantly higher than in earlier series, whereas we found none of the other mutations described in the United States, United Kingdom, or Japan. On the other hand, in 5 patients we identified a new pathogenic missense mutation (W797R); considering that 2 of these patients were homozygous, W797R appears to be the second most frequent mutation in Spanish patients with McArdle disease. The fact that 2 patients who were heterozygous for this mutation did not harbor any known mutation in the second allele suggests the existence of more undefined mutations.

Our findings confirm the allelic heterogeneity of myophosphorylase deficiency, a characteristic shared with other glycogenoses, such as acid maltase and phosphofructokinase deficiencies.25,26

McArdle disease can be easily diagnosed in blood cells, especially in ethnic groups with specific mutations. However, since there are other metabolic myopathies with similar clinical picture and the number of known mutations is relatively large, it seems advisable to perform a muscle biopsy in at least 1 member of the family before embarking on molecular analysis. Finally, the fact that many patients are compound heterozygotes may indicate a high frequency of asymptomatic carriers in the general population.

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Article Information

Accepted for publication July 21, 1999.

This study was supported by grant FIS 96/0229 and fellowship BAE 98/5144 (Dr Andreu) from the Fondo de Investigación Sanitaria, Madrid, Spain, and by a grant from the Muscular Dystrophy Association, Tucson, Ariz. Dr Bruno was supported by a fellowship from Telethon-Italy, Rome.

We are grateful to Christine O'Hara for her help with the English version of this article.

Reprints: Salvatore DiMauro, MD, 4-420 College of Physicians and Surgeons, 630 W 168th St, New York, NY 10032 (e-mail: sd12@columbia.edu).

References
1.
DiMauro  SServidei  STsujino  S Disorders of carbohydrate metabolism: glycogen storage diseases. Rosenberg  RNPrusiner  SBDiMauro  SBarchi  RLeds.The Molecular and Genetic Basis of Neurological Disease. 2nd ed. Boston, Mass Butterworth-Heinemann1997;1067- 1097
2.
DiMauro  SHartlage  PL Fatal infantile form of muscle phosphorylase deficiency. Neurology. 1978;281124- 1129Article
3.
El-Schahawi  MBruno  CTsujino  S  et al.  Sudden infant death syndrome (SIDS) in a family with myophosphorylase deficiency. Neuromuscul Disord. 1997;781- 83Article
4.
Lebo  RVGorin  FFletterick  RJ  et al.  High-resolution chromosome sorting and DNA spot-blot analysis assign McArdle's syndrome to chromosome 11. Science. 1984;22557- 59Article
5.
Burke  JHwang  PAnderson  LLebo  RGorin  FFletterick  R Intron/exon structure of the human gene for the muscle isozyme of glycogen phosphorylase. Proteins. 1987;2177- 187Article
6.
Kubisch  CWicklein  EMJentsch  TJ Molecular diagnosis of McArdle's disease: revised genomic structure of the myophosphorylase gene and identification of a novel mutation. Hum Mutat. 1998;1227- 32Article
7.
Vorgerd  MKubisch  CBurwinkel  B  et al.  Mutation analysis in myophosphorylase deficiency (McArdle's disease). Ann Neurol. 1998;43326- 331Article
8.
Bruno  CTamburino  LKawashima  N  et al.  A nonsense mutation in the myophosphorylase gene in a Japanese family with McArdle's disease. Neuromuscul Disord. 1999;934- 37Article
9.
Tsujino  SShanske  SDiMauro  S Molecular heterogeneity of myophosphorylase deficiency (McArdle's disease). N Engl J Med. 1993;329241- 245Article
10.
El-Schahawi  MTsujino  SShanske  SDiMauro  S Diagnosis of McArdle's disease by molecular genetic analysis of blood. Neurology. 1996;47579- 580Article
11.
Bartram  CEdwards  RHTClague  JBeynon  RJ McArdle's disease: a nonsense mutation in exon 1 of the muscle glycogen phosphorylase gene explains some but not all cases. Hum Mol Genet. 1993;21291- 1293Article
12.
Martinuzzi  ATsujino  SVergani  L  et al.  Molecular characterization of myophosphorylase deficiency in a group of patients from northern Italy. J Neurol Sci. 1996;13714- 19Article
13.
Andreu  ALBruno  CGamez  J  et al.  Molecular genetic analysis of McArdle's disease in Spanish patients. Neurology. 1998;51260- 262Article
14.
Shanske  SSakoda  SHermodson  MADiMauro  SSchon  EA Isolation of a cDNA encoding the muscle-specific subunit of human phosphoglycerate mutase. J Biol Chem. 1987;26214612- 14617
15.
Hudson  JWGolding  GBCrerar  MM Evolution of allosteric control in glycogen phosphorylase. J Mol Biol. 1993;234700- 721Article
16.
Sugie  HSugie  YIto  MFukuda  TNonaka  YIgarashi  Y Genetic analysis of Japanese patients with myophosphorylase deficiency (McArdle's disease): single-codon deletion in exon 17 is the predominant mutation. Clin Chim Acta. 1995;23681- 86Article
17.
Tsujino  SShanske  SGoto  YNonaka  IDiMauro  S Two mutations, one novel and one frequently observed, in Japanese patients with McArdle's disease. Hum Mol Genet. 1994;31005- 1006Article
18.
Tsujino  SRubin  LAShanske  SDiMauro  S An A to C substitution involving the translation initiation codon in a patient with myophosphorylase deficiency (McArdle's disease). Hum Mutat. 1994;473- 75Article
19.
Tsujino  SShanske  SNonaka  I  et al.  Three new mutations in patients with myophosphorylase deficiency (McArdle's disease). Am J Hum Genet. 1994;5444- 52Article
20.
Tsujino  SShanske  SMartinuzzi  AHeiman-Patterson  TDiMauro  S Two novel missense mutations (E654K, L396P) in Caucasian patients with myophosphorylase deficiency (McArdle's disease). Hum Mutat. 1995;6276- 277Article
21.
Bartram  CEdwards  RHTClague  JBeynon  RJ McArdle's disease: a rare frameshift mutation in exon 1 of the muscle glycogen phosphorylase gene. Biochim Biophys Acta. 1994;1226341- 343Article
22.
Olmos  JMZarrabeitia  MTValero  MCFigols  JMatorras  PRiancho  JA McArdle's disease in adults: clinical and genetic study [in Spanish]. Med Clin (Barc). 1997;109753- 755
23.
Andreu  ALBruno  CTamburino  L  et al.  A new mutation in the myophosphorylase gene (Asn684Tyr) in a Spanish patient with McArdle's disease. Neuromuscul Disord. 1999;9171- 173Article
24.
Gamez  JFernandez  RBruno  C  et al.  A new mutation in the regulatory domain of the myophosphorylase gene affecting protein dimer contact. Muscle Nerve. 1999;221136- 1138Article
25.
Martiniuk  FMehler  MTzall  SMeredith  GHirschhorn  R Extensive genetic heterogeneity in patients with acid alpha glucosidase deficiency as detected by abnormalities of DNA and mRNA. Am J Hum Genet. 1990;4773- 78
26.
Raben  NSherman  JB Mutations in muscle phosphofructokinase gene. Hum Mutat. 1995;61- 6Article
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