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Manifesting heterozygotes in a Japanese family with a novel mutation in the muscle-specific phosphoglycerate mutase (PGAM-M) gene
Neuromuscular Disorders 9 (1999) 399±402 www.elsevier.com/locate/nmd
Manifesting heterozygotes in a Japanese family with a novel mutation in the muscle-speci®c phosphoglycerate mutase (PGAM-M) gene Georgios M. Hadjigeorgiou a, Noriko Kawashima b, Claudio Bruno a, Antonio L. Andreu a, c, Carolyn M. Sue a, Daniel J. Rigden d, Atsushi Kawashima b, Sara Shanske a, Salvatore DiMauro a,* a
Department of Neurology, H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases, Columbia University College of Physicians and Surgeons, New York, NY, USA b Department of Neurology, General Hospital, Sapporo, Japan c Centre d'Investigacions en Bioquimica i Biologia Molecular, Hospitals Vall d'Hebron, Barcelona, Spain d GENARGEN/EMBRAPA, Brazilia, Brazil Received 7 December 1998; received in revised form 8 February 1999; accepted 4 March 1999
Abstract Muscle-speci®c phosphoglycerate mutase (PGAM-M) de®ciency results in a metabolic myopathy (glycogenosis type X). Three mutations in the PGAM-M gene have been described thus far, two in African-American families and one in a Caucasian family. In two of them, manifesting heterozygotes were documented. We found a new PGAM-M mutation in a Japanese family with partial PGAM de®ciency: a Gto-A transition at nucleotide position 209, resulting in the substitution of a highly conserved glycine at codon 97 with aspartic acid (G97D). Two heterozygous family members for the G97D mutation presented with exercise intolerance and muscle cramps. We describe the ®rst PGAM-M mutation in the Japanese population and con®rm that heterozygous individuals can be symptomatic. q 1999 Elsevier Science B.V. All rights reserved. Keywords: heterozygotes; phosphoglycerate mutase; Japanese
1. Introduction Phosphoglycerate mutase (PGAM) (EC 2.7.5.3) is a dimeric glycolytic enzyme that catalyzes the interconversion of 2-phosphoglycerate and 3-phosphoglycerate using 2,3-bisphosphoglycerate as a cofactor [1,2]. There are two subunits of mammalian PGAM, a muscle-speci®c subunit (PGAM-M) and a non-muscle speci®c, or brain, subunit (PGAM-B) [3], with mature human skeletal muscle containing almost exclusively the MM homodimer. PGAM-M de®ciency results in an autosomal recessive metabolic myopathy (glycogenosis type X) characterized by exercise intolerance and cramps [4]. The tissue-speci®c expression of PGAM-M has been attributed to a single myocyte-speci®c enhancer-binding factor (MEF-2) in the 5 0 -untranslated region of the PGAM-M gene [5]. The cDNA encoding PGAM-M has been cloned [6], and the gene has been isolated [7,8], and assigned to chromosome 7p12-7p13 [9,10]. Three point mutations have been reported in families with PGAM-M * Corresponding author. 4-420 College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA.
de®ciency [11±13] (Table 1). A nonsense mutation (W78X) was predominant among African-Americans; a missense mutation (Q89A) was also found in one AfricanAmerican family; and a missense mutation (R90W) was reported in a Caucasian family. Although all reported patients were homozygotes or compound heterozygotes for mutations in PGAM-M, in at least two families manifesting heterozygotes have been described [11,14]. We report a new missense mutation, Gly97Asp (G97D), in the PGAM-M gene in a Japanese family with two heterozygous patients presenting muscle symptoms.
2. Patients and methods 2.1. Patients The proband (II-2) has been described in detail elsewhere [15]. Brie¯y, this 55-year-old obese Japanese man, born of nonconsanguineous parents, was diagnosed with diabetes mellitus at the age of 49 years. At 51 years, he developed painful muscle cramps after physical work and was admitted to the hospital. On one occasion, after a motorcycle ride, he
0960-8966/99/$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0960-896 6(99)00039-5
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G.M. Hadjigeorgiou et al. / Neuromuscular Disorders 9 (1999) 399±402
Table 1 Pathogenic mutations in the PGAM-M gene Codon
Nucleotide change
Exon
Amino acid change
Ethnic group
Reference
78 89 90 97
G-to-A A-to-C C-to-T G-to-A
1 1 1 1
Trp . stop Glu . Ala Arg . Trp Gly . Asp
African-American African-American Italian Japanese
11 11 11
a
a
Present report.
had painful cramps in the calves, thighs, and arms, which persisted for half a day and did not disappear with passive stretching. There was no pigmenturia. Clinical neurophysiologic examination was consistent with diabetic polyneuropathy. Serum CK was 433 U/l (normal: 35±185). A muscle biopsy of the gastrocnemius muscle was performed about 2 months after a bout of severe muscle cramps. Histological examination showed regenerating ®bers and increased PASpositive material in the subsarcolemmal area. Ultrastructural studies con®rmed the presence of excessive glycogen particles in the subsarcolemmal area and between myo®brils. Biochemical studies showed slightly increased glycogen content (1.9 g/100g; normal: 1.03 ^ 0.18) and partial decrease of PGAM activity (170.3 mmol/min per g; normal: 363.5 ^ 92.2). Myophosphorylase and other glycolytic enzymes were normal. Carnitine palmitoyltransferase activity was also normal. The proband has three children, ages
22, 19, and 18 years (Fig. 1A). All but one (patient III-1) are asymptomatic at this time. Patient III-1, a 22-year-old man, complained of exercise intolerance and had muscle cramps after intense exercise. Serum CK was normal. Neurophysiological examination was normal. Muscle biopsy was not performed. 2.2. PCR ampli®cation and sequencing Total genomic DNA was extracted from blood of all available family members (II 1-3, III 1-3) (Fig. 1) and from 20 normal Japanese individuals. All three exons, including exon-intron boundaries and MEF-2 in the 5 0 untranslated region, were ampli®ed by polymerase chain reaction (PCR) as previously reported [11]. In brief, 35 PCR cycles were performed with denaturation at 958C for 15 s, annealing at 608C for 15 s, and extension at 728C for 30 s. Three sets of primers were used: for exon 1 and MEF-2 site, primer 713±734 forward (F) and 1418±1398 reverse (R); for exon 1 and 2, primer 930±958F and 1719±1692R; and for exon 3, primer 3413±3435F and 3758±3737R. The nucleotide (nt) positions correspond to the published sequence [7]. All PCR-ampli®ed fragments were sequenced, using the primers described above, in an ABI Prism 310 Genetic Analyser using Big Dye Terminator Cycle Sequencing Reaction Kits (Perkin-Elmer Applied Biosystems, Foster City, CA). 2.3. Mutation screening
Fig. 1. (A) Pedigree of the family with the G97D PGAM-M mutation. Solid symbols, manifesting heterozygotes; stippled symbols, asymptomatic heterozygotes; open symbols, clinically and genetically unaffected individuals. (B) Restriction enzyme analysis of the G-to-A transition at nt position 209 using the restriction endonuclease StuI. The 467-bp PCR-ampli®ed fragment containing the mutation is not digested by Stu.I. M, Molecular weight markers (Gibco BRL products, Life technologies, Gaithersburg, MD). (C) Illustration of the 467-bp PCR-ampli®ed fragment, showing the restriction site for StuI in normal DNA.
To facilitate screening for the new mutation (G-to-A at nt position 209) in family members and controls individuals, a 467 bp DNA fragment was ampli®ed using primers 930± 958F and 1396±1376R and PCR conditions as described above. In the presence of the normal sequence, the PCR product is cleaved by the restriction endonuclease StuI (New England Biolabs, Beverly, MA) into two fragments, 320 and 147 bp (Fig. 1C). In the presence of the mutation, the 467 bp fragment is not cut. To exclude partial digestion, the uncut bands (Fig. 1B) were removed, re-ampli®ed, and re-digested with EcoNI (New England Biolabs Inc., Beverly, MA), which cleaves the DNA fragment at nt position 209 in the presence of the new G-to-A mutation. Complete digestion with EcoNI would rule out partial digestion with StuI.
G.M. Hadjigeorgiou et al. / Neuromuscular Disorders 9 (1999) 399±402
401
Fig. 2. (A) Direct sequencing of PCR-ampli®ed genomic DNA from patient II-2 showing the G-to-A transition at nt position 209. (B) Aligment of the amino acid sequences (one letter code) around the mutated residue of human PGAM-M (HM), human PGAM-B (HB), rat PGAM-M (RM), yeast PGAM (Y), human diphosphoglycerate mutase (DPGAM) (HD), and rabbit DPGAM (RD). Arrow indicates the G97D mutation.
3. Results Using direct sequence analysis of PCR-ampli®ed DNA fragments, we found a novel G-to-A transition at nt position 209, converting an encoded Gly (GGC) to an Asp (GAC) at codon 97 (G97D) (Fig. 2A). This mutation abolishes a StuI site and provides a simple screening test using this restriction enzyme (Fig. 1C). Analysis with StuI con®rmed the presence of this mutation and showed that the proband (II2) and all three siblings (III-1-3) were heterozygous for the G209A transition (Fig. 1B). An artifact due to partial digestion was excluded as described in Section 2 (data not shown). We did not ®nd the G209A transition in 20 ethnically matched controls.
4. Discussion PGAM-M de®ciency, ®rst reported in 1981 [4], results in a partial block of terminal glycolysis and nine patients have been reported so far [12]. All patients had exercise intolerance, with myalgia and cramps, and six had pigmenturia. Age at onset ranged from 8 to 20 years. The proband in the family we report had no pigmenturia and the only notable difference from previously reported cases was the later onset (51 years) and the presence of diabetic polyneuropathy with obesity
[15]. In six of the nine previously reported patients, the diagnosis was established both by biochemical and by molecular genetic analyses, in one only by PGAM determination in muscle, and in two only by molecular genetic analysis [12]. All patients in whom muscle biochemistry was studied showed little residual PGAM activity (,10%), which was due to the normal expression of the BB dimer. The PGAM activity in our proband's muscle was 46.9% of the normal mean [15], far too high to be explained by the expression of the BB dimer, and rather suggesting a heterozygous condition in which one allele of the PGAM-M gene was unaffected. This is precisely what we found. PGAM-M is encoded by a single gene encompassing three exons and two introns [7], and the genetic basis of PGAM-M de®ciency has been established with the description of three mutations [11] (Table 1). All three mutations are close together in exon 1 and they affect well conserved amino acids [12]. Two of them, including the most common W78X, were reported in African-American families [11,12] while the third was found in an Italian family [12,13]. We now report a novel PGAM-M mutation in a Japanese family with partial de®ciency of PGAM-M. The mutation is a G-to-A transition at nt position 209 affecting a wellconserved amino acid (Fig. 2B) and resulting in the substitution of Gly by Asp at codon 97 (G97D) in exon 1, the same exon in which all three previous mutations were found [12]. The proband was heterozygous for the G97D mutation, in
402
G.M. Hadjigeorgiou et al. / Neuromuscular Disorders 9 (1999) 399±402
agreement with the degree of PGAM-M de®ciency (about 50% of normal) found in muscle [15]. All three children of the proband are heterozygous for the mutation but only the oldest one has muscle symptoms. Since the proband did not become symptomatic until he was in his ®fties, we can not exclude that the younger siblings may become symptomatic later in life. Although PGAM-M de®ciency is a relatively benign autosomal recessive disorder, manifesting heterozygotes were noted in at least two families [11,14]. In the ®rst family, the proband was a 17-year-old African-American girl with exercise intolerance, myalgia and cramps: she was homozygous for the W78X mutation. Her full brother was heterozygous but he complained of exercise intolerance and cramps, and had persistently elevated serum CK. A half brother on the maternal side was also heterozygous for the W78X mutation but had no symptoms [11]. In the second family, muscle biopsies from the parents of an AfricanAmerican patient homozygous for the W78X mutation showed not only the expected half-normal activity of PGAM but also rare necrotic ®bers and decreased number of type I ®bers in the mother, and occasional atrophic and degenerating type II ®bers in the father [14]. These observations indicate that clinical or morphological manifestations do occur in individuals who are heterozygous for PGAM-M mutations. Manifesting heterozygosity has been reported in several families with another glycogenosis, myophosphorylase de®ciency (McArdle's disease) [16±19]. Ê X-ray crystal structure of PGAM in Recently, the 2.3 A Saccharomyces cerevisiae (S.c.) was published [20]. The Gly97 residue of human PGAM-M that is mutated in our family corresponds to Gly94 in the S.c. PGAM crystal structure. Although the Gly94 residue is not close to the active site of the enzyme, its replacement with Asp would introduce suf®cient strain into the S.c. crystal structure model to affect the active site. This strain could also lead to partial unfolding of the protein, which might then become susceptible to aggregation. We believe that the G97D mutation is pathogenic in this family for the following reasons: (1) the residue that is mutated is conserved as Gly in all of the PGAM sequences that are currently available (Genbank database-National Center for Biotechnology); (2) no other nucleotide alteration was found in the entire coding region, in all splice junctions, or in the regulatory region including the MEF2; (3) computer modeling of the crystal structure derived from S.c. PGAM showed that the G97D might cause structural disruption; (4) no other biochemical defect was detected in the glycolytic pathway in muscle tissue; and (5) we did not ®nd this nucleotide alteration in 20 ethnically matched controls. Our report describes the ®rst mutation in the PGAM-M gene in the Japanese population, expands the genetic heterogeneity of PGAM-M de®ciency, and con®rms that exon 1 is a `hot spot' for mutations in the PGAM-M gene.
References [1] Grisolia S, Joyce BK. Distribution of two types of phosphoglyceric acid mutase, diphosphoglycerate mutase, and D-2,3-diphosphoglyceric acid. J Biol Chem 1959;234:1335±1340. [2] Grisolia S, Carreras J. Diphosphoglycerate mutase from yeast, chicken breast muscle, and kidney (2,3-PGA-dependent). Meth Enzymol 1975;42:435±450. [3] Omenn GS, Cheung SCY. Phosphoglycerate mutase: isoenzyme marker for tissue differentiation in man. Am J Hum Genet 1974;26:393±399. [4] DiMauro S, Miranda AF, Khan S, Gitlin K, Friedman R. Human muscle phosphoglycerate mutase de®ciency: a newly discovered metabolic myopathy. Science 1981;212:1277±1279. [5] Nakatsuji Y, Hidaka K, Tsujino S, et al. A single MEF-2 site is a major positive regulatory element required for transcription of the muscle-speci®c subunit of the human phosphoglycerate mutase gene in skeletal and cardiac muscle cells. Mol Cell Biol 1992;12:4384±4390. [6] Shanske S, Sakoda S, Hermodson MA, DiMauro S, Schon EA. Isolation of a cDNA encoding the muscle-speci®c subunit of human phosphoglycerate mutase. J Biol Chem 1987;262:14612±14617. [7] Tsujino S, Sakoda S, Mizuno R, et al. Structure of the gene encoding the muscle-speci®c subunit of human phosphoglycerate mutase. J Biol Chem 1989;264:15334±15337. [8] Castella-Escola J, Ojcius DM, Le Boulch P, et al. Isolation and characterization of the gene coding for the muscle-speci®c isozyme of human phosphoglycerate mutase. Gene 1990;91:225±232. [9] Edwards YH, Sakoda S, Schon EA, Povey S. The gene for human muscle-speci®c phosphoglycerate mutase. PGAM2, mapped to chromosome 7 by polymerase chain reaction. Genomics 1989;5:948±951. [10] Castella-Escola J, Mattei MG, Ojcius DM, Passage E, Valentin C, Cohen-Solal M. In situ mapping of the muscle-speci®c form of phosphoglycerate mutase gene to human chromosome 7p12-7p13. Hum Genet 1990;84:210±212. [11] Tsujino S, Shanske S, Sakoda S, Fenichel G, DiMauro S. The molecular genetic basis of muscle phosphoglycerate mutase (PGAM) de®ciency. Am J Hum Genet 1993;52:472±477. [12] Tsujino S, Shanske S, Sakoda S, Toscano A, DiMauro S. Molecular genetic studies in muscle phosphoglycerate mutase (PGAM-M) de®ciency. Muscle Nerve 1995;3:S50±S53. [13] Toscano A, Tsujino S, Vita G, Shanske S, Messina C, DiMauro S. Molecular basis of muscle phosphoglycerate mutase (PGAM-M) de®ciency in the Italian kindred. Muscle Nerve 1996;19:1134±1137. [14] Bresolin N, Ro JI, Reyes M, Miranda AF, DiMauro S. Muscle phosphoglycerate mutase (PGAM) de®ciency: a second case. Neurology 1983;33:1049±1053. [15] Kawashima N, Mishima M, Shindo R, et al. Partial de®ciency of phosphoglycerate mutase with diabetic polyneuropathy: the ®rst Japanese patient. Intern Med 1996;35:799±802. [16] Schmidt B, Servidei S, Gabbai AA, Silva AC, Sousa Bulle de Oliveira A, DiMauro S. McArdle's disease in two generations: Autosomal recessive transmission with manifesting heterozygote. Neurology 1987;37:1558±1561. [17] Papadimitriou A, Manta P, Divari R, Karabetsos A, Papadimitriou E, Bresolin N. McArdle's disease: two clinical expression in the same pedigree. J Neurol 1990;237:267±270. [18] Tsujino S, Shanske S, DiMauro S. Molecular genetic heterogeneity of myophosphorylase de®ciency (McArdle's disease). N Engl J Med 1993;329:241±245. [19] DiMauro S, Tsujino S, Shanske S, Rowland LP. Biochemistry and molecular genetics of human glycogenoses: an overview. Muscle Nerve 1995;3:S10±S17. [20] Rigden DJ, Alexeev D, Phillips SEV, Fothergill-Gilmore LA. The 2.3 Ê X-ray crystal structure of S. cerevisiae phosphoglycerate mutase. J A Mol Biol 1998;276:449±459.
Manifesting heterozygotes in a Japanese family with a novel mutation in the muscle-speci®c phosphoglycerate mutase (PGAM-M) gene Georgios M. Hadjigeorgiou a, Noriko Kawashima b, Claudio Bruno a, Antonio L. Andreu a, c, Carolyn M. Sue a, Daniel J. Rigden d, Atsushi Kawashima b, Sara Shanske a, Salvatore DiMauro a,* a
Department of Neurology, H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases, Columbia University College of Physicians and Surgeons, New York, NY, USA b Department of Neurology, General Hospital, Sapporo, Japan c Centre d'Investigacions en Bioquimica i Biologia Molecular, Hospitals Vall d'Hebron, Barcelona, Spain d GENARGEN/EMBRAPA, Brazilia, Brazil Received 7 December 1998; received in revised form 8 February 1999; accepted 4 March 1999
Abstract Muscle-speci®c phosphoglycerate mutase (PGAM-M) de®ciency results in a metabolic myopathy (glycogenosis type X). Three mutations in the PGAM-M gene have been described thus far, two in African-American families and one in a Caucasian family. In two of them, manifesting heterozygotes were documented. We found a new PGAM-M mutation in a Japanese family with partial PGAM de®ciency: a Gto-A transition at nucleotide position 209, resulting in the substitution of a highly conserved glycine at codon 97 with aspartic acid (G97D). Two heterozygous family members for the G97D mutation presented with exercise intolerance and muscle cramps. We describe the ®rst PGAM-M mutation in the Japanese population and con®rm that heterozygous individuals can be symptomatic. q 1999 Elsevier Science B.V. All rights reserved. Keywords: heterozygotes; phosphoglycerate mutase; Japanese
1. Introduction Phosphoglycerate mutase (PGAM) (EC 2.7.5.3) is a dimeric glycolytic enzyme that catalyzes the interconversion of 2-phosphoglycerate and 3-phosphoglycerate using 2,3-bisphosphoglycerate as a cofactor [1,2]. There are two subunits of mammalian PGAM, a muscle-speci®c subunit (PGAM-M) and a non-muscle speci®c, or brain, subunit (PGAM-B) [3], with mature human skeletal muscle containing almost exclusively the MM homodimer. PGAM-M de®ciency results in an autosomal recessive metabolic myopathy (glycogenosis type X) characterized by exercise intolerance and cramps [4]. The tissue-speci®c expression of PGAM-M has been attributed to a single myocyte-speci®c enhancer-binding factor (MEF-2) in the 5 0 -untranslated region of the PGAM-M gene [5]. The cDNA encoding PGAM-M has been cloned [6], and the gene has been isolated [7,8], and assigned to chromosome 7p12-7p13 [9,10]. Three point mutations have been reported in families with PGAM-M * Corresponding author. 4-420 College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA.
de®ciency [11±13] (Table 1). A nonsense mutation (W78X) was predominant among African-Americans; a missense mutation (Q89A) was also found in one AfricanAmerican family; and a missense mutation (R90W) was reported in a Caucasian family. Although all reported patients were homozygotes or compound heterozygotes for mutations in PGAM-M, in at least two families manifesting heterozygotes have been described [11,14]. We report a new missense mutation, Gly97Asp (G97D), in the PGAM-M gene in a Japanese family with two heterozygous patients presenting muscle symptoms.
2. Patients and methods 2.1. Patients The proband (II-2) has been described in detail elsewhere [15]. Brie¯y, this 55-year-old obese Japanese man, born of nonconsanguineous parents, was diagnosed with diabetes mellitus at the age of 49 years. At 51 years, he developed painful muscle cramps after physical work and was admitted to the hospital. On one occasion, after a motorcycle ride, he
0960-8966/99/$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0960-896 6(99)00039-5
400
G.M. Hadjigeorgiou et al. / Neuromuscular Disorders 9 (1999) 399±402
Table 1 Pathogenic mutations in the PGAM-M gene Codon
Nucleotide change
Exon
Amino acid change
Ethnic group
Reference
78 89 90 97
G-to-A A-to-C C-to-T G-to-A
1 1 1 1
Trp . stop Glu . Ala Arg . Trp Gly . Asp
African-American African-American Italian Japanese
11 11 11
a
a
Present report.
had painful cramps in the calves, thighs, and arms, which persisted for half a day and did not disappear with passive stretching. There was no pigmenturia. Clinical neurophysiologic examination was consistent with diabetic polyneuropathy. Serum CK was 433 U/l (normal: 35±185). A muscle biopsy of the gastrocnemius muscle was performed about 2 months after a bout of severe muscle cramps. Histological examination showed regenerating ®bers and increased PASpositive material in the subsarcolemmal area. Ultrastructural studies con®rmed the presence of excessive glycogen particles in the subsarcolemmal area and between myo®brils. Biochemical studies showed slightly increased glycogen content (1.9 g/100g; normal: 1.03 ^ 0.18) and partial decrease of PGAM activity (170.3 mmol/min per g; normal: 363.5 ^ 92.2). Myophosphorylase and other glycolytic enzymes were normal. Carnitine palmitoyltransferase activity was also normal. The proband has three children, ages
22, 19, and 18 years (Fig. 1A). All but one (patient III-1) are asymptomatic at this time. Patient III-1, a 22-year-old man, complained of exercise intolerance and had muscle cramps after intense exercise. Serum CK was normal. Neurophysiological examination was normal. Muscle biopsy was not performed. 2.2. PCR ampli®cation and sequencing Total genomic DNA was extracted from blood of all available family members (II 1-3, III 1-3) (Fig. 1) and from 20 normal Japanese individuals. All three exons, including exon-intron boundaries and MEF-2 in the 5 0 untranslated region, were ampli®ed by polymerase chain reaction (PCR) as previously reported [11]. In brief, 35 PCR cycles were performed with denaturation at 958C for 15 s, annealing at 608C for 15 s, and extension at 728C for 30 s. Three sets of primers were used: for exon 1 and MEF-2 site, primer 713±734 forward (F) and 1418±1398 reverse (R); for exon 1 and 2, primer 930±958F and 1719±1692R; and for exon 3, primer 3413±3435F and 3758±3737R. The nucleotide (nt) positions correspond to the published sequence [7]. All PCR-ampli®ed fragments were sequenced, using the primers described above, in an ABI Prism 310 Genetic Analyser using Big Dye Terminator Cycle Sequencing Reaction Kits (Perkin-Elmer Applied Biosystems, Foster City, CA). 2.3. Mutation screening
Fig. 1. (A) Pedigree of the family with the G97D PGAM-M mutation. Solid symbols, manifesting heterozygotes; stippled symbols, asymptomatic heterozygotes; open symbols, clinically and genetically unaffected individuals. (B) Restriction enzyme analysis of the G-to-A transition at nt position 209 using the restriction endonuclease StuI. The 467-bp PCR-ampli®ed fragment containing the mutation is not digested by Stu.I. M, Molecular weight markers (Gibco BRL products, Life technologies, Gaithersburg, MD). (C) Illustration of the 467-bp PCR-ampli®ed fragment, showing the restriction site for StuI in normal DNA.
To facilitate screening for the new mutation (G-to-A at nt position 209) in family members and controls individuals, a 467 bp DNA fragment was ampli®ed using primers 930± 958F and 1396±1376R and PCR conditions as described above. In the presence of the normal sequence, the PCR product is cleaved by the restriction endonuclease StuI (New England Biolabs, Beverly, MA) into two fragments, 320 and 147 bp (Fig. 1C). In the presence of the mutation, the 467 bp fragment is not cut. To exclude partial digestion, the uncut bands (Fig. 1B) were removed, re-ampli®ed, and re-digested with EcoNI (New England Biolabs Inc., Beverly, MA), which cleaves the DNA fragment at nt position 209 in the presence of the new G-to-A mutation. Complete digestion with EcoNI would rule out partial digestion with StuI.
G.M. Hadjigeorgiou et al. / Neuromuscular Disorders 9 (1999) 399±402
401
Fig. 2. (A) Direct sequencing of PCR-ampli®ed genomic DNA from patient II-2 showing the G-to-A transition at nt position 209. (B) Aligment of the amino acid sequences (one letter code) around the mutated residue of human PGAM-M (HM), human PGAM-B (HB), rat PGAM-M (RM), yeast PGAM (Y), human diphosphoglycerate mutase (DPGAM) (HD), and rabbit DPGAM (RD). Arrow indicates the G97D mutation.
3. Results Using direct sequence analysis of PCR-ampli®ed DNA fragments, we found a novel G-to-A transition at nt position 209, converting an encoded Gly (GGC) to an Asp (GAC) at codon 97 (G97D) (Fig. 2A). This mutation abolishes a StuI site and provides a simple screening test using this restriction enzyme (Fig. 1C). Analysis with StuI con®rmed the presence of this mutation and showed that the proband (II2) and all three siblings (III-1-3) were heterozygous for the G209A transition (Fig. 1B). An artifact due to partial digestion was excluded as described in Section 2 (data not shown). We did not ®nd the G209A transition in 20 ethnically matched controls.
4. Discussion PGAM-M de®ciency, ®rst reported in 1981 [4], results in a partial block of terminal glycolysis and nine patients have been reported so far [12]. All patients had exercise intolerance, with myalgia and cramps, and six had pigmenturia. Age at onset ranged from 8 to 20 years. The proband in the family we report had no pigmenturia and the only notable difference from previously reported cases was the later onset (51 years) and the presence of diabetic polyneuropathy with obesity
[15]. In six of the nine previously reported patients, the diagnosis was established both by biochemical and by molecular genetic analyses, in one only by PGAM determination in muscle, and in two only by molecular genetic analysis [12]. All patients in whom muscle biochemistry was studied showed little residual PGAM activity (,10%), which was due to the normal expression of the BB dimer. The PGAM activity in our proband's muscle was 46.9% of the normal mean [15], far too high to be explained by the expression of the BB dimer, and rather suggesting a heterozygous condition in which one allele of the PGAM-M gene was unaffected. This is precisely what we found. PGAM-M is encoded by a single gene encompassing three exons and two introns [7], and the genetic basis of PGAM-M de®ciency has been established with the description of three mutations [11] (Table 1). All three mutations are close together in exon 1 and they affect well conserved amino acids [12]. Two of them, including the most common W78X, were reported in African-American families [11,12] while the third was found in an Italian family [12,13]. We now report a novel PGAM-M mutation in a Japanese family with partial de®ciency of PGAM-M. The mutation is a G-to-A transition at nt position 209 affecting a wellconserved amino acid (Fig. 2B) and resulting in the substitution of Gly by Asp at codon 97 (G97D) in exon 1, the same exon in which all three previous mutations were found [12]. The proband was heterozygous for the G97D mutation, in
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G.M. Hadjigeorgiou et al. / Neuromuscular Disorders 9 (1999) 399±402
agreement with the degree of PGAM-M de®ciency (about 50% of normal) found in muscle [15]. All three children of the proband are heterozygous for the mutation but only the oldest one has muscle symptoms. Since the proband did not become symptomatic until he was in his ®fties, we can not exclude that the younger siblings may become symptomatic later in life. Although PGAM-M de®ciency is a relatively benign autosomal recessive disorder, manifesting heterozygotes were noted in at least two families [11,14]. In the ®rst family, the proband was a 17-year-old African-American girl with exercise intolerance, myalgia and cramps: she was homozygous for the W78X mutation. Her full brother was heterozygous but he complained of exercise intolerance and cramps, and had persistently elevated serum CK. A half brother on the maternal side was also heterozygous for the W78X mutation but had no symptoms [11]. In the second family, muscle biopsies from the parents of an AfricanAmerican patient homozygous for the W78X mutation showed not only the expected half-normal activity of PGAM but also rare necrotic ®bers and decreased number of type I ®bers in the mother, and occasional atrophic and degenerating type II ®bers in the father [14]. These observations indicate that clinical or morphological manifestations do occur in individuals who are heterozygous for PGAM-M mutations. Manifesting heterozygosity has been reported in several families with another glycogenosis, myophosphorylase de®ciency (McArdle's disease) [16±19]. Ê X-ray crystal structure of PGAM in Recently, the 2.3 A Saccharomyces cerevisiae (S.c.) was published [20]. The Gly97 residue of human PGAM-M that is mutated in our family corresponds to Gly94 in the S.c. PGAM crystal structure. Although the Gly94 residue is not close to the active site of the enzyme, its replacement with Asp would introduce suf®cient strain into the S.c. crystal structure model to affect the active site. This strain could also lead to partial unfolding of the protein, which might then become susceptible to aggregation. We believe that the G97D mutation is pathogenic in this family for the following reasons: (1) the residue that is mutated is conserved as Gly in all of the PGAM sequences that are currently available (Genbank database-National Center for Biotechnology); (2) no other nucleotide alteration was found in the entire coding region, in all splice junctions, or in the regulatory region including the MEF2; (3) computer modeling of the crystal structure derived from S.c. PGAM showed that the G97D might cause structural disruption; (4) no other biochemical defect was detected in the glycolytic pathway in muscle tissue; and (5) we did not ®nd this nucleotide alteration in 20 ethnically matched controls. Our report describes the ®rst mutation in the PGAM-M gene in the Japanese population, expands the genetic heterogeneity of PGAM-M de®ciency, and con®rms that exon 1 is a `hot spot' for mutations in the PGAM-M gene.
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