In Exon 17 of the PYGM Gene Causing Mcardle Disease

Home , Missense mutation, Myophosphorylase

OBSERVATION A New Rare Mutation (691delCC/insAAA) in Exon 17 of the PYGM Gene Causing McArdle Disease

Beatriz Quintans, MS; Amalia Sanchez-Andrade, MD; Susana Teijeira, MS; Roberto Fernandez-Hojas, PhD; Eloy Rivas, MD; Marı´a Jose´Lo´pez, MD; Carmen Navarro, MD, PhD

Objective: To investigate the genetic effect of a new mu- of exon 17. This is an insertion/deletion (indel) and con- tation found in exon 17 of the myophosphorylase (PYGM) sists simultaneously of a deletion of 2 bases and an in- gene as a cause of McArdle disease (also known as type sertion of 3 bases (691delCC/insAAA). A restriction analy- 5 glycogenosis). sis was designed to simplify the detection method.

Patients: A Spanish patient with McArdle disease was Conclusions: The 691delCC/insAAA is the third indel screened for 3 common mutations in the PYGM gene (R49X, described in the PYGM gene. Indels represent 0.95% of W797R, and G204S), as previously described. The patient the total reported mutations in the Human Gene Muta- was heterozygous for R49X. To find other mutations, the tion Database. The molecular origin of this mutation is coding sequence of the entire PYGM gene was sequenced. not fully understood. These findings point again to the The carrier status of his relatives was also studied. allelic heterogeneity of McArdle disease.

Results: A novel rare mutation was found in codon 691 Arch Neurol. 2004;61:1108-1110

YPE 5 GLYCOGENOSIS, OR mutation has only been reported in Span- McArdle disease, is one of ish patients and seems to be the second the most common meta- most frequent mutation in this popu- bolic myopathies. It is an lation.18 autosomal recessive disor- Single base pair substitutions are the derT that affects the glycolytic phase of most frequent type of mutations in the muscle glycogen metabolism. The dis- PYGM gene, but 5 small deletions and 2 ease is clinically characterized by exer- insertions/deletions (indels) have been re- cise intolerance, premature fatigue, my- ported as well. This last mutation is an un- algia, muscle cramps, and recurrent usual complex lesion that seems to com- myoglobinuria. Symptoms typically ap- bine microdeletion and microinsertion. pear in adolescence or early adulthood. Herein, we report a new indel in the The genetic cause is a deficiency of PYGM gene at codon 691 in exon 17 myophosphorylase due to inherited mu- (691delCC/insAAA) that causes the pre- tations in the myophosphorylase (PYGM) mature termination of translation 29 amino gene. Myophosphorylase (␣-1,4-glucan or- acids downstream of the mutation site. thophosphate glycosyltransferase [En- From the Department of zyme Commission 2.4.1.1]) is a specific METHODS Pathology and Neuropathology, skeletal muscle enzyme that initiates gly- Hospital Meixoeiro, Vigo cogen breakdown. The PYGM gene has PATIENTS (Mss Quintans and Teijeira, been cloned, sequenced, and assigned to Drs Fernandez-Hojas, Rivas, chromosome 11q131,2 (GenBank U94774). A 34-year-old man of nonconsanguineous par- and Navarro); the Molecular Allelic heterogeneity has been dem- ents had increased creatine kinase levels in a Medicine Unit (SERGAS), onstrated by the identification of 41 dif- routine laboratory analysis. University of Santiago de ferent mutations in the coding region and The patient had exercise-related myalgia Compostela, Santiago de splice sites of the gene, most of which are and early fatigue since infancy. He described Compostela (Ms Quintans); and 2-19 a characteristic “second-wind phenomenon,” the Department of quoted by other researchers. The most being able to continue to exercise if he rests Rheumatology, Hospital common mutation in white persons is briefly after the first signs of muscle pain. He 5 Xeral-Calde, Lugo R49X, and nearly 70% of Spanish pa- did not have pigmenturia and has never con- (Drs Sanchez-Andrade and tients harbor the R49X, G204S, or W797R sulted for these symptoms. His family history Lo´pez), Spain. mutation.8 To our knowledge, the W797R was negative for neuromuscular diseases.

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Overlapped Sequence

Figure 1. Forward (A) and reverse (B) sequences of the insertion/deletion in exon 17 of the myophosphorylase (PYGM) gene in a heterozygous patient. Overlapping sequences are originated by a mixture of wild-type and mutant alleles. This pattern was also found in the proband’s mother and sister. The varying colors represent a different nucleotide: green, adenine; blue, cytosine; red, thymine; and black, guanine.

The results of his physical examination were normal, and he had normal muscular balance. The results of bio- A chemistry showed elevated aspartate aminotransferase (189 IU/L; normal, 0-35 IU/L), alanine aminotransferase (97 IU/L; normal, 0.35 IU/L), creatine kinase (6826 IU/L; normal, 150 IU/L), and uric acid (9.73 mg/dL [0.58 mmol/L]; normal, 2.5-8.0 mg/dL [0.15-0.48 mmol/L]) levels. His electrocardio- gram showed no abnormalities. Electromyography showed a reduced amplitude and duration of motor units. Study of a muscle biopsy revealed typical periodic acid– B 123456789 10 11 12 13 Schiff–positive amylase-sensitive subsarcolemmal vacuoles and absent myophosphorylase. The diagnosis of type 5 gly- 400 bp cogenosis, or McArdle disease, was established. 200 bp MUTATION SCREENING

After informed consent was obtained, genomic DNA was extracted from peripheral blood leukocytes by the conven- Figure 2. A, Family pedigree. Squares indicate males; circles, females; the arrow, the proband; black symbols, the presence of an insertion/deletion; gray tional method of phenol-chloroform. Screening for the R49X, symbol, the presence of the R49X mutation; and white symbol, a healthy G204S, and W797R mutations was performed by polymerase subject. B, Restriction analysis of mutation 691delCC/insAAA, showing chain reaction–restriction fragment length polymorphism.5,18 digestion with NcoI. The wild-type allele (392 base pairs [bp]) is cleaved by The patient was heterozygous for the R49X mutation. To NcoI into 2 fragments (198 and 184 bp) that are visualized as a unique band. search for mutations in the other allele, the coding sequence Lane 1 indicates the ladder; lanes 2 through 7, uncut samples; lanes 8 through of the entire PYGM gene was amplified by polymerase chain 13, digested samples; lanes 2 and 8, the proband; lanes 3 and 9, the proband’s 2 mother; lanes 4 and 10, the proband’s sister; lanes 5 and 11, the proband’s reaction, and products were checked by electrophoresis in brother; and lanes 6 and 7 and 12 and 13, healthy control subjects. 3% agarose gel. After purification with a polymerase chain reaction kit (QIAquick PCR Purification kit; QIAGEN GmbH, Hilden, Germany), a sequence reaction was performed using RESULTS another kit (ABI Prism BigDye Terminator kit; Applied Bio- systems, Foster City, Calif) and running the sequences in an The proband was heterozygous for the R49X mutation, the analyzer (ABI Prism 310 Genetic Analyzer; Applied most common defect in the PYGM gene in the white popu- Biosystems). lation. By sequencing the entire coding region of the PYGM To simplify the detection of the 691delCC/insAAA gene, we identified an overlap sequence downstream at mutation, a restriction fragment length polymorphism codon 691 in exon 17 in the forward and reverse direc- method was optimized because indel destroys a restriction tions (Figure 1). This suggests the presence of 2 alleles: site for the NcoI endonuclease (New England Biolabs, Bev- one is a wild type, and the other presents a deletion of CC erly, Mass). Enzyme restriction was performed with 1-hour incubation at 37°C, and products were visualized in a 2.5% and an insertion of AAA (691delCC/insAAA). The pro- agarose gel. band’s mother and sister carry this mutation (Figure 2A). We also analyzed the proband’s family members and 40 The mutation was confirmed by restriction fragment healthy control subjects, who all provided informed consent, length polymorphism analysis after digestion with NcoI to demonstrate the pathogenic role of the mutation. (Figure 2B).

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 COMMENT tent (Drs Sanchez-Andrade, Fernandez-Hojas, Rivas, Lo´pez, and Navarro, and Ms Teijeira); obtained funding (Dr We have identified a novel rare mutation in exon 17 Navarro); administrative, technical, and material support (Mss (691delCC/insAAA) in a Spanish patient with McArdle dis- Quintans and Teijeira and Dr Fernandez-Hojas); study su- ease. The pathogenic effect of this mutation is supported pervision (Ms Quintans); patient evaluation (Dr Lo´pez). by the following: (1) It was the only nucleotide change This study was supported by grant 01/1049 from Fondo detected, apart from the R49X mutation, in the entire cod- de Investigacio´n Sanitaria, Madrid; and by Convenio Univer- ing region and splice junctions of the PYGM gene. (2) It sidad Santiago de Compostela–Hospital Meixoeiro, Vigo, Spain. predicts a frameshift and premature termination of the myo- We thank Nadia Chuzhanova, PhD, for her help with phosphorylase protein 29 amino acids downstream of the the interpretation of possible mutagenesis mechanisms; and mutation, resulting in a 720–amino acid peptide instead James Stilwell-Stronge, BA, for his help with the English ver- of the normal 841-residue protein. (3) This truncated pro- sion of the manuscript. tein has possibly affected the C-terminal catalytic do- Correspondence: Carmen Navarro, MD, PhD, Depart- main that extends from amino acid 482 to amino acid 841.20 ment of Pathology and Neuropathology, Hospital Meix- This peptide may be unstable and enzymatically inactive, oeiro, 36215 Vigo, Spain ([email protected]). inducing rapid breakdown of the protein. (4) This mutation was not found in the 40 healthy Spanish controls. 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