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Background and Methods Myophosphorylase deficiency (McArdle's disease) is one of the most common causes of exercise intolerance, muscle cramps, and recurrent myoglobinuria. The myophosphorylase gene has been sequenced and assigned to chromosome 11, but the molecular basis of McArdle's disease is not known. We sequenced complementary DNA in 4 patients and studied genomic DNA by restriction-endonuclease analysis in 40 patients with McArdle's disease.
Results Sequence analysis revealed three distinct point mutations: the substitution of thymine for cytosine at codon 49 in exon 1, changing an encoded arginine to a stop codon; the substitution of adenine for guanine at codon 204 in exon 5, changing glycine to serine; and the substitution of cytosine for adenine at codon 542 in exon 14, changing lysine to threonine. Analysis of restriction-fragment-length polymorphisms of appropriate fragments of genomic DNA after amplification with the polymerase chain reaction showed that 18 patients were homozygous for the stop-codon mutation, 6 had different mutations in the two alleles (compound heterozygotes), and 11 were presumed to be compound heterozygotes for a known mutation and an unknown one; only 5 patients had none of the three mutations. All three mutations were present in various combinations in five members of a family in which transmission appeared to be autosomal dominant.
Conclusions McArdle's disease is genetically heterogeneous, but the most common mutation is the substitution of thymine for cytosine at codon 49. These results suggest that in about 90 percent of patients the diagnosis of McArdle's disease can be made from a patient's leukocytes, thus avoiding the need for muscle biopsy.
Most patients with myophosphorylase deficiency have exercise intolerance, with premature fatigue, myalgia, and cramps in exercising muscles. About half the patients have acute muscle necrosis and myoglobinuria after exercise, and about half these patients have acute renal failure4. About 90 percent of patients lack immunologically reactive myophosphorylase protein in muscle6,7. This biochemical homogeneity contrasts with an apparent genetic heterogeneity: in Northern blot analysis of muscle RNA isolated from 21 patients, myophosphorylase messenger RNA (mRNA) was absent in 12 muscle-biopsy specimens, decreased in 4, and truncated in 16,7,8.
Approximately half the patients have a family history of McArdle's disease, and transmission is autosomal recessive4. Although the gene for human myophosphorylase, which is on chromosome 11,9 has been isolated and sequenced,10 the molecular genetic basis of the disease is not known. We have identified three point mutations in the myophosphorylase gene in 35 of 40 patients with McArdle's disease, confirming the genetic heterogeneity of the disorder.
Methods
Study Population
We studied 40 patients with McArdle's disease (22 male and 18 female), ranging in age from 13 weeks to 62 years, from 36 families. In 38 patients, myophosphorylase deficiency was documented biochemically by a muscle biopsy. The other two patients were symptomatic relatives of patients with confirmed disease.
Thirty-three patients were adults with the typical clinical manifestations of the disease -- exercise intolerance, myalgia, and muscle cramps. One infant had diffuse limb weakness and respiratory insufficiency; she died at the age of 13 weeks11. Six patients were children: an 8-year-old boy, a 9-year-old boy, a 14-year-old girl, and three siblings -- two boys, 12 and 10 years of age, and a 9-year-old girl -- in a family in which McArdle's disease was apparently transmitted in an autosomal dominant fashion. The 30-year-old mother of the three siblings had had exercise intolerance and cramps since childhood and had had at least five episodes of myoglobinuria after exercise; one episode resulted in acute renal failure. The 31-year-old father was asymptomatic. All three children had exercise intolerance and cramps, but no myoglobinuria. A muscle biopsy in the older boy showed glycogen storage and a lack of phosphorylase activity both histochemically and biochemically. The other two children did not have biopsies.
Control muscle specimens were obtained from biopsies performed for diagnostic purposes in three patients ultimately deemed to have no neuromuscular diseases. Blood was obtained from 5 patients with McArdle's disease, 12 normal subjects, and 8 patients with various metabolic myopathies: 2 with myoclonic epilepsy and "ragged-red" muscle fibers; 2 with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; 1 with phosphofructokinase deficiency; 1 with phosphoglycerate kinase deficiency; 1 with phosphoglycerate mutase deficiency; and 1 with phosphorylase b kinase deficiency.
Extraction and Amplification of RNA
Total RNA was extracted from 30 to 70 mg of muscle from four patients with McArdle's disease with a modified cesium chloride centrifugation method12. Six DNA fragments encompassing the entire coding region of myophosphorylase complementary DNA (cDNA) were directly amplified from each RNA sample (about 500 ng) with primers 1 and 3, 2 and 5, 4 and 7, 6 and 9, 8 and 11, and 10 and 12 (Figure 1), with the Gene Amp Thermostable rTth Reverse Transcriptase RNA polymerase-chain-reaction (PCR) kit (Perkin-Elmer Cetus, Norwalk, Conn.), according to the manufacturer's specifications.
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Genomic DNA was extracted from muscle or white cells as described previously13. Appropriate fragments for restriction-enzyme analysis were amplified with Taq polymerase and reagents obtained from Boehringer-Mannheim (Indianapolis), according to the manufacturer's instructions, and the primers shown in Figure 1. For primers 1 through 13 and 16, 17, 18, and 19, PCR was performed for 30 cycles consisting of denaturation at 94 °C for one minute, annealing at 60 °C for one minute, and extension at 72 °C for two minutes. For the short fragment between primers 14 and 15, the PCR was performed for 2 cycles consisting of denaturation at 94 °C for one minute, annealing at 60 °C for one minute, and extension at 72 °C for one minute, followed by 38 cycles consisting of denaturation at 91 °C for one minute, annealing at 65 °C for one minute, and extension at 72 °C for one minute.
Sequencing
Amplified DNA fragments were sequenced directly with the same primers used for amplification and the BRL Sequencing kit (GIBCO BRL Life Technologies, Gaithersburg, Md.), according to the manufacturer's specifications. DNA fragments were subjected to a cycle-sequencing PCR program in the presence of 2 pmol of a primer whose 5' end was labeled with [32P]ATP. The PCR products were subjected to electrophoresis on a gel consisting of 6 percent polyacrylamide and 7 M urea. The gel was vacuum-dried for 1 hour and exposed to Kodak XAR film (Eastman Kodak, Rochester, N.Y.) for 12 hours. The sequence of the entire coding region in the four patients was compared with the previously reported sequence of the myophosphorylase gene9. Differences were confirmed by comparison with control DNA sequenced at the same time.
Restriction-Enzyme Assays
Restriction endonucleases NlaIII (New England Biolabs, Beverly, Mass.), HaeIII (Boehringer-Mannheim), and MaeIII (Boehringer-Mannheim) were used with the buffers provided by the manufacturers and according to their specifications. Restriction digests were analyzed by electrophoresis on either 2 percent NuSieve agarose gels (FMC Bioproducts, Rockland, Me.) or 12 percent polyacrylamide gels.
Results
We extracted RNA from muscle-biopsy specimens from four patients with McArdle's disease and amplified the entire cDNA of the myophosphorylase gene. The six partially overlapping DNA fragments so obtained were sequenced directly (Figure 1). Comparison with the published sequence of the human myophosphorylase gene10 and with the sequences of DNA fragments from control muscle obtained at the same time revealed three point mutations. To exclude artifacts, we sequenced both DNA strands in the mutated regions. Two patients were homozygous for a single-base mutation, the substitution of thymine for cytosine (C to T) at codon 49 within the first exon of the myophosphorylase gene, changing an encoded arginine (CGA) to a stop codon (TGA) (mutation 1 in Table 1). Another patient was heterozygous for two distinct mutations (compound heterozygote), the substitution of adenine for guanine (G to A) at codon 204 in exon 5, changing an encoded glycine (GGC) to serine (AGC) (mutation 2 in Table 1), and the substitution of cytosine for adenine (A to C) at codon 542 in exon 14, changing an encoded lysine (AAG) to threonine (ACG) (mutation 3 in Table 1). The fourth patient, the mother in a family in which the disease was apparently transmitted in autosomal dominant fashion (Figure 2), had mutation 2 and was also found to be a compound heterozygote (see below).
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Discussion
The genes for the three human phosphorylase isoenzymes, characteristic of mature skeletal muscle, liver, and brain, have been cloned, sequenced, and localized to chromosomes 11, 14, and 10 or 209,14,15. In contrast to the gene for the liver isoenzyme, which is highly polymorphic,14 the muscle-specific gene contains no known neutral restriction-fragment-length polymorphisms (RFLPs), and only by extending the analysis to flanking regions was it possible to detect two RFLPs, sufficient to identify 75 percent of the subjects at risk; in that analysis, Lebo et al. found no deletions in patients with McArdle's disease16. In this study, we found no deletions in the four patients in whom the entire myophosphorylase cDNA was sequenced. Instead, there were three distinct point mutations in exons 1, 5, and 14.
The most common mutation was a C-to-T mutation at codon 49 in exon 1, changing an encoded arginine to a stop codon. This mutation was present in both alleles in 18 patients and in one allele in 12, thus accounting for 75 percent of all patients. The other two mutations were less frequent and were encountered only in the heterozygous state; the G-to-A mutation at codon 204 in exon 5 was present in eight patients, and the A-to-C mutation at codon 542 in exon 14 was found in three patients. In an intact protein, each of these missense mutations would be predicted to have deleterious functional consequences. The mutation at codon 204 sits in a domain involved in glycogen binding and tetramerization, and the mutation at codon 542 affects a glucose-binding domain9,17.
The occurrence of multiple mutations explains the relatively large number of patients who had different mutations in the two alleles: 6 patients were proved and 11 were presumed to be compound heterozygotes. Altogether, 60 percent of our patients had two mutant alleles, and only 12 percent did not have any of the three mutations. Therefore, nearly 90 percent of the patients could have been identified by analysis of genomic DNA from leukocytes, which could be a useful alternative to muscle biopsy as a first diagnostic approach in patients suspected of having McArdle's disease.
Although clinical heterogeneity is uncommon, four children with myophosphorylase deficiency have had severe generalized weakness at or soon after birth and died in infancy of respiratory failure11,18,19,20. In one of these children, neither morphologic nor biochemical studies explained the unusual early onset and clinical severity11. Genetic analysis also failed to provide an immediate explanation; this infant was homozygous for the most common mutation and was no different, in this respect, from 17 adults with typical McArdle's disease. Knowledge of the genetic error, however, offers the possibility of prenatal diagnosis.
The finding of a high frequency of the mutation that results in a stop codon in the first exon of the myophosphorylase gene agrees with the observation that most patients with McArdle's disease lack immunologically detectable enzyme protein in muscle6,7. However, the finding that almost all patients lacked enzyme protein, whereas only 45 percent were homozygous for the stop codon mutation, implies that missense mutations must also contribute to this phenomenon. This has been verified by the lack of detectable myophosphorylase protein on electrophoresis or immunoblotting of muscle extracts from seven of our patients who were heterozygous or compound heterozygous for the two missense mutations (data not shown). The association of a lack of enzyme protein with missense mutations has been documented in other metabolic diseases, such as pyruvate dehydrogenase deficiency21 and carnitine palmitoyltransferase deficiency,22 and has been attributed to the rapid degradation of unstable mutant proteins.
The lack of mRNA in about 50 percent of patients in previous series6,7,8 is more difficult to explain but may be related to the nonsense mutation that we found in a similar proportion of patients, because markedly decreased levels of mRNA have been associated with nonsense mutations in several diseases of humans23,24.
McArdle's disease is inherited as an autosomal recessive trait, but there have been a few reports of autosomal dominant transmission25,26. Some of these have been attributed to the presence in subsequent generations of homozygotes and symptomatic heterozygotes, in whom phosphorylase activity in muscle fell below a critical threshold needed for normal muscle function27,28. We describe a different genetic mechanism underlying what appeared to be autosomal dominant inheritance in a family with McArdle's disease. All three mutations were present in this family -- a dramatic illustration of genetic heterogeneity; the affected mother was a compound heterozygote for mutations 1 and 2, and the asymptomatic father was heterozygous for mutation 3. The three affected children were compound heterozygotes for mutations 1 and 3 or 2 and 3.
This study documents the genetic heterogeneity of McArdle's disease and demonstrates that in the majority of patients, the disease can be diagnosed from a patient's white cells, a procedure that eliminates the need for a muscle biopsy.
Supported by a center grant (NS-11766) from the National Institute of Neurological and Communicative Disorders and Stroke and by a grant from the Muscular Dystrophy Association. Dr. Tsujino was supported by a postdoctoral fellowship from the Muscular Dystrophy Association.
We are indebted to the many colleagues who, through the years, have sent us muscle-biopsy specimens from patients with suspected McArdle's disease and to Drs. Eric A. Schon and Lewis P. Rowland for their support and advice.
Source Information
From the H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases, Department of Neurology, Columbia-Presbyterian Medical Center, New York.
Address reprint requests to Dr. DiMauro at 4-420 College of Physicians and Surgeons, Columbia-Presbyterian Medical Center, 630 W. 168th St., New York, NY 10032.
References
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