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A new mutation in PRKAG2 gene causing hypertrophic cardiomyopathy with conduction system disease and muscular glycogenosis
Neuromuscular Disorders 16 (2006) 178–182 www.elsevier.com/locate/nmd
Case report
A new mutation in PRKAG2 gene causing hypertrophic cardiomyopathy with conduction system disease and muscular glycogenosis Pascal Laforeˆt a,*, Pascale Richard b, Mina Ait Said c, Norma Beatriz Romero a, Emmanuelle Lacene a, Jean-Paul Leroy d, Christiane Baussan e, Jean-Yves Hogrel a, Thomas Lavergne c, Karim Wahbi f, Bernard Hainque b, Denis Duboc f b
a Institut de Myologie, Baˆtiment Babinski, Groupe Hospitalier Pitie´-Salpeˆtrie`re, Paris, France UF Cardioge´ne´tique et Myoge´ne´tique, Service de Biochimie B, Groupe Hospitalier Pitie´-Salpeˆtrie`re, 47-83 boulevard de l’Hoˆpital, 75651 Paris cedex 13, France c Service de Cardiologie 2, Hoˆpital Europe´en Georges Pompidou, Paris, France d Laboratoire de Neuropathologie, CHU de Brest, Brest, France e Service de Biochimie, Hoˆpital de Biceˆtre, Kremlin-Biceˆtre, France f Service de Cardiologie, Hoˆpital Cochin, Paris, France
Received 1 August 2005; received in revised form 2 December 2005; accepted 13 December 2005
Abstract Mutations in the gene encoding the g2 subunit of AMP-activated protein kinase (PRKAG2) cause familial cardiac hypertrophy and electrophysiological abnormalities, with glycogen accumulation in the heart of affected patients. The authors describe a 38-year-old man with a new heterozygous PRKAG2 mutation (Ser548Pro) manifesting by hypertrophic cardiomyopathy, severe conduction system abnormalities, and skeletal muscle glycogenosis. Considering those results, PRKAG2 gene could be a potential candidate for unexplained muscle glycogenosis associated with cardiac abnormalities. q 2006 Elsevier B.V. All rights reserved. Keywords: PRKAG2; Glycogenosis; AMPK
1. Introduction Glycogenosis or glycogen storage diseases (GSD) are rare inherited disorders caused by genetic deficiencies of various enzymes involved in the synthesis or breakdown of glycogen. Twelve specific enzyme defects affect skeletal muscle alone or in combination with other tissues, and cause two main clinical syndromes: short exercise intolerance with recurrent rhabdomyolysis; or fixed, progressive muscle weakness [1]. Cardiac muscle may also be involved, essentially in glycogenosis with a permanent muscle weakness as acid maltase, brancher and debrancher deficiencies. In particular, a life-threatening hypertrophic cardiomyopathy may be the main clinical manifestation of * Corresponding author. Tel.: C33 1 42 16 37 91; fax: C33 1 42 16 37 93. E-mail address: [email protected] (P. Laforeˆt).
0960-8966/$ - see front matter q 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2005.12.004
infantile-onset acid maltase deficiency (Pompe’s disease) and brancher deficiency. Recently, an autosomal dominant cardiac syndrome combining cardiac hypertrophy, ventricular pre-excitation (Wolff–Parkinson–White syndrome, WPW) and progressive atrioventricular conduction block, has been related to consequences of mutations in the g2 subunit of AMPactivated protein kinase (AMPK) [2]. Skeletal muscle involvement with myalgias or muscle weakness may also occur in a minority of patients [3]. AMPK is a cellular energy sensor, that is activated by exercise in muscle and increase in AMP: ATP ratio, stimulating fatty acid oxidation, glycolysis and glucose oxidation. This enzyme form a heterotrimeric complex comprising a catalytic subunit (a) and two regulatory subunits (b and g). Three isoforms of the gamma subunits are known (g1, g2 and g3) with different tissue expression [4], and each contain four repeats of a structural module known as a cystathionine b-synthase (CBS) domains. The function of g subunits is
P. Laforeˆt et al. / Neuromuscular Disorders 16 (2006) 178–182
uncertain although they could play an important role in the binding of adenosine moieties of AMP and ATP. Pathological examinations of hearts revealed vacuoles containing polysaccharide in patients with PRKAG2 mutations [5], and an abundant glycogen accumulation is also observed in the myocardium of transgenic mice overexpressing mutant PRKAG2, with a disruption of the atrioventricular connections providing a possible explanation for the electrophysiological abnormalities. Unexpectedly, histopathological aspect of skeletal muscle recently reported in two patients who complained of myalgia exhibited mitochondrial proliferation with only minimal glycogen excess [3]. We report here the observation of a patient with a new mutation in PRKAG2 gene presenting a skeletal muscle involvement leading to the demonstration of a glycogen accumulation in muscle.
179
cardiomyopathy with an important basal diastolic septal thickness of 18 mm (N!13 mm), a left ventricular diastolic diameter of 42 mm (N!55 mm), and a normal left ventricular ejection fraction (60%). At clinical examination, the limbs muscle bulk and strength were normal. Upper and lower limbs muscle CT scan was normal. A forearm exercise test was performed twice with an isometric exercise at 70% of the maximal voluntary contraction during 30 s in non-ischemic conditions [6]. The grip strength was within the normal ranges considering the patient age, gender, weight and forearm circumference (43.3 and 42.0 daN for the first and second test, respectively). Despite a sufficient amount of physical work (80% of predicted mechanical values), a moderate increase of lactate levels was observed after exercise (1.5 and 1.6 mmol/L at rest, and maxima of 2.8 and 2.6 mmol/L after exercise for the first and second test, respectively) with a relative steadiness of ammonia concentration (Fig. 1).
2. Case report 2.1. Muscle morphology and biochemical analysis A 38-year-old-man was admitted in cardiological intensive care unit after the occurrence of four episodes of faintness after swimming. The patient had a first episode of unexplained faintness 10 years before, and complained since 15 years of lasting muscle stiffness in arms and legs the days after prolonged exercises, without important muscle pain at the beginning of physical efforts. He never experienced episodes of myoglobinuria. His 41-year-old brother and 45-year-old sister were asymptomatic, but his paternal aunt died suddenly at age 65 years. Electrocardiogram at admission showed sinusal bradycardia (35/mn), high degree ventricular block, and left bundle branch block. Coronarography was normal and a pacemaker was implanted the next day. Serum creatine kinase levels were elevated to three times the upper normal value. Echocardiography revealed a hypertrophic 7.0
120.0
Grip Test 1 Grip Test 2
6.0
100.0 Ammonia (µmol/l)
Lactate (mmol/l)
Deltoid muscle biopsy showed subsarcolemmal vacuoles in 10% of muscle fibers with H–E preparations with intense staining in periodic acid-Schiff (Fig. 2A and B), and a normal myophosphorylase histochemical reaction. Muscle sections stained with PAS after diastase digestion were negative (Fig. 2C). Ultrastructural analysis confirmed the presence of a non-lysosomal glycogen accumulation, with abundant granular non-membrane bound electron-dense material within vacuoles (Fig. 2D). Gomori trichrome and succinic dehydrogenase reactions did not show mitochondrial proliferation. Histochemical reaction for adenylate deaminase, and immunohistochemical analysis performed with an anti-LAMP-2 antibody were normal. All values of enzymatic activities measured in muscle of the patient were in the normal ranges, acid maltase: 0.27 U/g (NO0.2);
5.0 4.0 3.0 2.0
80.0 60.0 40.0 20.0
1.0 0.0
0.0 –5
0
5 t (min)
exercise
10
–5
0
5
10
t (min) exercise
Fig. 1. Results of the forearm exercise test. Grip test was performed twice (Grit Test 1 and Grip Test 2). Lactate values were within the normal range but increase of lactate after exercise was very moderate. The ammonia concentration did not change after exercise. The shaded areas represent the normal ranges computed from 55 healthy subjects.
180
P. Laforeˆt et al. / Neuromuscular Disorders 16 (2006) 178–182
Fig. 2. (A) Histo-enzymological and ultrastructural analysis of deltoid biopsy. Subsarcolemmal vacuoles are visualized with Hematoxylin and eosin staining in some fibers (arrow). (B) Periodic acid-Schiff (PAS) shows excessive staining in vacuoles (arrows), and (C) negativity after diastase digestion. (D) Electron microscopy shows presence of a non-lysosomal glycogen accumulation, with abundant granular non-membrane bound electron-dense material (arrows).
debranching enzyme: 1.07 (NO0.2); branching enzyme: 69 (N 50/150); phosphorylase: 132 (100/150); phosphorylase kinase: 44 (50/100). 2.2. Molecular analysis Genetic analysis was performed on genomic DNA after informed written consent. According to the notion of cardiac conduction defect, the LMNA gene encoding lamin A/C was first tested but no mutation was identified. Then, each exon and flanking intronic regions of PRKAG2 (Ref. Seq: ENSG1066178) was then sequenced on both strands
with the BigDye terminator sequencing kit, run on a ABI 3100 capillary electrophoresis and analysed with the SeqScape software (Applied Biosystems). This allowed to reveal a heterozygous T–C transition at nucleotide 1732 of the cDNA sequence (NM 016203) leading to the new Serine 548 to Proline missense mutation (Fig. 3). This S548P mutation is located in the fourth CBS domain, the serine at position 548 is a highly conserved residue among species and isoforms. Analysis of controls revealed that this variant was not detected on 200 normal chromosomes. The mutation was not found in his brother’s blood sample.
Fig. 3. (A) Pedigree of the patient showing the dominant mode of inheritance of the disease. (B) Mutation analysis. Sequencing analysis allowed detection of a heterozygous T to C transition at nucleotide 1732, leading to a S356P mutation in the fourth CBS domain. Arrowheads mark the site of the base alteration.
P. Laforeˆt et al. / Neuromuscular Disorders 16 (2006) 178–182
3. Discussion We identified a new mutation in PRKAG2 gene in a patient exhibiting a hypertrophic cardiomyopathy revealed by severe atrioventricular conduction defect, and associated with a skeletal muscle glycogenosis. Muscle symptoms were limited to muscle pain after exercise, without muscle weakness, and forearm exercise test was suggestive of a possible partial blockage of glycogenolysis due to the very mild increase of lactate after exercise. The initial diagnosis was a hypertrophic cardiomyopathy, most often related to sarcomere-protein gene mutations, but high CK levels and symptoms of exercise intolerance prompted the muscle biopsy. Searching for mutations in PRKAG2 gene was subsequently carried out because of the presence of histological features suggestive of a muscle glycogenosis, concurrently with normal glycogenosis enzymatic activities. Eight mutations have been so far reported in PRKAG2 gene, but mainly in patients with apparently isolated cardiac phenotype [3,7]. Interestingly, although not yet reported in humans, mutations in the CBS domains of the AMPK g3 subunit which has a skeletal musclespecific expression [4], also cause an abnormally high glycogen content in skeletal muscle of pigs [8]. Deleterious mutations were not found in the six others AMPK subunit isoforms in a large panel of patients with hypertrophic cardiomyopathies in whom contractile protein mutations had not been found [9]. In the present case, there is a striking discrepancy between the severity of cardiac complications and the muscle symptoms which were limited to occasional pain after exercise and high CK levels. This could be explained by the variability in tissue distribution of AMPK isoforms, g2 isoform being more abundant in heart than in skeletal muscle [4]. Moreover we could speculate that the presence of g3 isoform in skeletal muscle, but not in the heart could compensate the effects of the mutation in PRKAG2. Initial reports of functional effect of PRKAG2 mutations concluded to a constitutive activation of AMPK increasing AMP kinase activity and stimulating carbohydrate accumulation [5]. However, subsequent studies describing the effects of different mutations, demonstrated that the major effect of the mutations is to reduce activation of AMPK in response to stress [10]. The most clearly identified physiological role of AMPK on carbohydrate metabolism is the stimulation of glucose transporter GLUT4 induced by muscle contraction or exercise, which increases glucose intake and hexokinase activity, thus accelerating glycolytic flux and lactate formation [11]. AMPK probably also phosphorylates glycogen synthase, inducing an inhibition of this enzyme. The normal glycogen appearance at the optical and ultrastructural levels in the skeletal muscle of our patient, contrasting with PAS diastase-resistant amylopectin-like deposits which have been showed in hearts of patients with PRKAG2 mutations, could be the result of
181
a different effect in heart and muscle tissue of AMPK deficiency. The clinical triad including either alone or in combination, cardiac hypertrophy, progressive conduction system disease and ventricular pre-excitation is also encountered in two muscle disorders in which heart involvement is predominant, infantile-onset acid maltase deficiency (Pompe’s disease) and Danon’s disease. Cardiomyopathy is always associated with a severe hypotonia in Pompe’s disease, in contrast with Danon’s disease in which skeletal muscle weakness may be absent [12]. Thus far, the glycogen storage cardiomyopathy due to PRKAG2 gene was not considered as a muscle disorder, but this observation advocate that mutations in PRKAG2 gene mutations should be searched in unexplained muscle glycogenosis associated with hypertrophic cardiomyopathy or conduction effects. The subsarcolemmal localization of the vacuoles that we observed in the muscle biopsy of our patient could help to differentiate this disorder from Pompe and Danon’s diseases, which are generally associated with a diffuse intracytoplasmic vacuolation of muscle fibres. However, a recent study showing mitochondrial proliferation on muscle biopsy of patients with a different PRKAG2 mutation, suggest that the consequences of PRKAG2 mutations on skeletal muscle metabolism may differ according to the type of mutation. Further clinical, morphological and biochemical studies remains necessary for a better knowledge of muscle symptoms related to this disease and a precise analysis of the consequences of PRKAG2 mutations on muscle glycogen metabolism.
References [1] DiMauro S, Hays AP, Tsujino S. Nonlysosomal glycogenoses. In: Engel AG, Franzini-Armstrong C, editors. Myology, 3rd ed., vol. 2. New York, NY: McGraw-Hill; 2004. p. 1535–57. [2] Gollob MH, Green MS, Tang AS, et al. Identification of a gene responsible for familial Wolff–Parkinson–White syndrome. N Engl J Med 2001;344:1823–31. [3] Murphy RT, Mogensen J, McGarry K, et al. Adenosine monophosphate-activated protein kinase disease mimicks hypertrophic cardiomyopathy and Wolff–Parkinson–White syndrome: natural history. J Am Coll Cardiol 2005;45:922–30. [4] Cheung PC, Salt IP, Davies SP, et al. Characterization of AMPactivated protein kinase gamma-subunit isoforms and their role in AMP binding. Biochem J 2000;346(Pt 3):659–69. [5] Arad M, Benson DW, Perez-Atayde AR, et al. Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy. J Clin Invest 2002;109:357–62. [6] Hogrel JY, Laforet P, Ben Yaou R, et al. A non-ischemic forearm exercise test for the screening of patients with exercise intolerance. Neurology 2001;56:1733–8. [7] Burwinkel B, Scott JW, Bu¨hrer C, et al. Fatal congenital heart glycogenosis caused by a recurrent activating R531Q mutation in the g2-subunit of AMP-activated protein kinase (PRKAG2), not by phosphorylase kinase deficiency. Am J Hum Genet 2005;76:1034–49.
182
P. Laforeˆt et al. / Neuromuscular Disorders 16 (2006) 178–182
[8] Milan D, Jeon JT, Looft C, et al. A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle. Science 2000; 288:1248–51. [9] Oliveira SM, Ehtisham J, Redwood CS, et al. Mutation analysis of AMP-activated protein kinase subunits in inherited cardiomyopathies: implications for kinase function and disease pathogenesis. J Mol Cell Cardiol 2003;35:1251–5. [10] Daniel T, Carling D. Functional analysis of mutations in the gamma 2 subunit of AMP-activated protein kinase associated with cardiac
hypertrophy and Wolff–Parkinson–White syndrome. J Biol Chem 2002;277:51017–24. [11] Holmes BF, Kurth-Kraczek EJ, Winder WW. Chronic activation of 5 0 -AMP-activated protein kinase increases GLUT-4, hexokinase, and glycogen in muscle. J Appl Physiol 1999;87:1990–5. [12] Arad M, Maron BJ, Gorham JM, et al. Glycogen storage diseases presenting as hypertrophic cardiomyopathy. N Engl J Med 2005;352: 362–72.
Correction In the article “The early history of myasthenia gravis” by Dr. Trevor Hughes and published in Neuromuscular Disorders 15 (2005) 878-886, the following footnote was inadvertently omitted: “This article is based on the Meryon Society Lecture at Worcester College, Oxford, given by the author on July 22nd, 2005.”
Case report
A new mutation in PRKAG2 gene causing hypertrophic cardiomyopathy with conduction system disease and muscular glycogenosis Pascal Laforeˆt a,*, Pascale Richard b, Mina Ait Said c, Norma Beatriz Romero a, Emmanuelle Lacene a, Jean-Paul Leroy d, Christiane Baussan e, Jean-Yves Hogrel a, Thomas Lavergne c, Karim Wahbi f, Bernard Hainque b, Denis Duboc f b
a Institut de Myologie, Baˆtiment Babinski, Groupe Hospitalier Pitie´-Salpeˆtrie`re, Paris, France UF Cardioge´ne´tique et Myoge´ne´tique, Service de Biochimie B, Groupe Hospitalier Pitie´-Salpeˆtrie`re, 47-83 boulevard de l’Hoˆpital, 75651 Paris cedex 13, France c Service de Cardiologie 2, Hoˆpital Europe´en Georges Pompidou, Paris, France d Laboratoire de Neuropathologie, CHU de Brest, Brest, France e Service de Biochimie, Hoˆpital de Biceˆtre, Kremlin-Biceˆtre, France f Service de Cardiologie, Hoˆpital Cochin, Paris, France
Received 1 August 2005; received in revised form 2 December 2005; accepted 13 December 2005
Abstract Mutations in the gene encoding the g2 subunit of AMP-activated protein kinase (PRKAG2) cause familial cardiac hypertrophy and electrophysiological abnormalities, with glycogen accumulation in the heart of affected patients. The authors describe a 38-year-old man with a new heterozygous PRKAG2 mutation (Ser548Pro) manifesting by hypertrophic cardiomyopathy, severe conduction system abnormalities, and skeletal muscle glycogenosis. Considering those results, PRKAG2 gene could be a potential candidate for unexplained muscle glycogenosis associated with cardiac abnormalities. q 2006 Elsevier B.V. All rights reserved. Keywords: PRKAG2; Glycogenosis; AMPK
1. Introduction Glycogenosis or glycogen storage diseases (GSD) are rare inherited disorders caused by genetic deficiencies of various enzymes involved in the synthesis or breakdown of glycogen. Twelve specific enzyme defects affect skeletal muscle alone or in combination with other tissues, and cause two main clinical syndromes: short exercise intolerance with recurrent rhabdomyolysis; or fixed, progressive muscle weakness [1]. Cardiac muscle may also be involved, essentially in glycogenosis with a permanent muscle weakness as acid maltase, brancher and debrancher deficiencies. In particular, a life-threatening hypertrophic cardiomyopathy may be the main clinical manifestation of * Corresponding author. Tel.: C33 1 42 16 37 91; fax: C33 1 42 16 37 93. E-mail address: [email protected] (P. Laforeˆt).
0960-8966/$ - see front matter q 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2005.12.004
infantile-onset acid maltase deficiency (Pompe’s disease) and brancher deficiency. Recently, an autosomal dominant cardiac syndrome combining cardiac hypertrophy, ventricular pre-excitation (Wolff–Parkinson–White syndrome, WPW) and progressive atrioventricular conduction block, has been related to consequences of mutations in the g2 subunit of AMPactivated protein kinase (AMPK) [2]. Skeletal muscle involvement with myalgias or muscle weakness may also occur in a minority of patients [3]. AMPK is a cellular energy sensor, that is activated by exercise in muscle and increase in AMP: ATP ratio, stimulating fatty acid oxidation, glycolysis and glucose oxidation. This enzyme form a heterotrimeric complex comprising a catalytic subunit (a) and two regulatory subunits (b and g). Three isoforms of the gamma subunits are known (g1, g2 and g3) with different tissue expression [4], and each contain four repeats of a structural module known as a cystathionine b-synthase (CBS) domains. The function of g subunits is
P. Laforeˆt et al. / Neuromuscular Disorders 16 (2006) 178–182
uncertain although they could play an important role in the binding of adenosine moieties of AMP and ATP. Pathological examinations of hearts revealed vacuoles containing polysaccharide in patients with PRKAG2 mutations [5], and an abundant glycogen accumulation is also observed in the myocardium of transgenic mice overexpressing mutant PRKAG2, with a disruption of the atrioventricular connections providing a possible explanation for the electrophysiological abnormalities. Unexpectedly, histopathological aspect of skeletal muscle recently reported in two patients who complained of myalgia exhibited mitochondrial proliferation with only minimal glycogen excess [3]. We report here the observation of a patient with a new mutation in PRKAG2 gene presenting a skeletal muscle involvement leading to the demonstration of a glycogen accumulation in muscle.
179
cardiomyopathy with an important basal diastolic septal thickness of 18 mm (N!13 mm), a left ventricular diastolic diameter of 42 mm (N!55 mm), and a normal left ventricular ejection fraction (60%). At clinical examination, the limbs muscle bulk and strength were normal. Upper and lower limbs muscle CT scan was normal. A forearm exercise test was performed twice with an isometric exercise at 70% of the maximal voluntary contraction during 30 s in non-ischemic conditions [6]. The grip strength was within the normal ranges considering the patient age, gender, weight and forearm circumference (43.3 and 42.0 daN for the first and second test, respectively). Despite a sufficient amount of physical work (80% of predicted mechanical values), a moderate increase of lactate levels was observed after exercise (1.5 and 1.6 mmol/L at rest, and maxima of 2.8 and 2.6 mmol/L after exercise for the first and second test, respectively) with a relative steadiness of ammonia concentration (Fig. 1).
2. Case report 2.1. Muscle morphology and biochemical analysis A 38-year-old-man was admitted in cardiological intensive care unit after the occurrence of four episodes of faintness after swimming. The patient had a first episode of unexplained faintness 10 years before, and complained since 15 years of lasting muscle stiffness in arms and legs the days after prolonged exercises, without important muscle pain at the beginning of physical efforts. He never experienced episodes of myoglobinuria. His 41-year-old brother and 45-year-old sister were asymptomatic, but his paternal aunt died suddenly at age 65 years. Electrocardiogram at admission showed sinusal bradycardia (35/mn), high degree ventricular block, and left bundle branch block. Coronarography was normal and a pacemaker was implanted the next day. Serum creatine kinase levels were elevated to three times the upper normal value. Echocardiography revealed a hypertrophic 7.0
120.0
Grip Test 1 Grip Test 2
6.0
100.0 Ammonia (µmol/l)
Lactate (mmol/l)
Deltoid muscle biopsy showed subsarcolemmal vacuoles in 10% of muscle fibers with H–E preparations with intense staining in periodic acid-Schiff (Fig. 2A and B), and a normal myophosphorylase histochemical reaction. Muscle sections stained with PAS after diastase digestion were negative (Fig. 2C). Ultrastructural analysis confirmed the presence of a non-lysosomal glycogen accumulation, with abundant granular non-membrane bound electron-dense material within vacuoles (Fig. 2D). Gomori trichrome and succinic dehydrogenase reactions did not show mitochondrial proliferation. Histochemical reaction for adenylate deaminase, and immunohistochemical analysis performed with an anti-LAMP-2 antibody were normal. All values of enzymatic activities measured in muscle of the patient were in the normal ranges, acid maltase: 0.27 U/g (NO0.2);
5.0 4.0 3.0 2.0
80.0 60.0 40.0 20.0
1.0 0.0
0.0 –5
0
5 t (min)
exercise
10
–5
0
5
10
t (min) exercise
Fig. 1. Results of the forearm exercise test. Grip test was performed twice (Grit Test 1 and Grip Test 2). Lactate values were within the normal range but increase of lactate after exercise was very moderate. The ammonia concentration did not change after exercise. The shaded areas represent the normal ranges computed from 55 healthy subjects.
180
P. Laforeˆt et al. / Neuromuscular Disorders 16 (2006) 178–182
Fig. 2. (A) Histo-enzymological and ultrastructural analysis of deltoid biopsy. Subsarcolemmal vacuoles are visualized with Hematoxylin and eosin staining in some fibers (arrow). (B) Periodic acid-Schiff (PAS) shows excessive staining in vacuoles (arrows), and (C) negativity after diastase digestion. (D) Electron microscopy shows presence of a non-lysosomal glycogen accumulation, with abundant granular non-membrane bound electron-dense material (arrows).
debranching enzyme: 1.07 (NO0.2); branching enzyme: 69 (N 50/150); phosphorylase: 132 (100/150); phosphorylase kinase: 44 (50/100). 2.2. Molecular analysis Genetic analysis was performed on genomic DNA after informed written consent. According to the notion of cardiac conduction defect, the LMNA gene encoding lamin A/C was first tested but no mutation was identified. Then, each exon and flanking intronic regions of PRKAG2 (Ref. Seq: ENSG1066178) was then sequenced on both strands
with the BigDye terminator sequencing kit, run on a ABI 3100 capillary electrophoresis and analysed with the SeqScape software (Applied Biosystems). This allowed to reveal a heterozygous T–C transition at nucleotide 1732 of the cDNA sequence (NM 016203) leading to the new Serine 548 to Proline missense mutation (Fig. 3). This S548P mutation is located in the fourth CBS domain, the serine at position 548 is a highly conserved residue among species and isoforms. Analysis of controls revealed that this variant was not detected on 200 normal chromosomes. The mutation was not found in his brother’s blood sample.
Fig. 3. (A) Pedigree of the patient showing the dominant mode of inheritance of the disease. (B) Mutation analysis. Sequencing analysis allowed detection of a heterozygous T to C transition at nucleotide 1732, leading to a S356P mutation in the fourth CBS domain. Arrowheads mark the site of the base alteration.
P. Laforeˆt et al. / Neuromuscular Disorders 16 (2006) 178–182
3. Discussion We identified a new mutation in PRKAG2 gene in a patient exhibiting a hypertrophic cardiomyopathy revealed by severe atrioventricular conduction defect, and associated with a skeletal muscle glycogenosis. Muscle symptoms were limited to muscle pain after exercise, without muscle weakness, and forearm exercise test was suggestive of a possible partial blockage of glycogenolysis due to the very mild increase of lactate after exercise. The initial diagnosis was a hypertrophic cardiomyopathy, most often related to sarcomere-protein gene mutations, but high CK levels and symptoms of exercise intolerance prompted the muscle biopsy. Searching for mutations in PRKAG2 gene was subsequently carried out because of the presence of histological features suggestive of a muscle glycogenosis, concurrently with normal glycogenosis enzymatic activities. Eight mutations have been so far reported in PRKAG2 gene, but mainly in patients with apparently isolated cardiac phenotype [3,7]. Interestingly, although not yet reported in humans, mutations in the CBS domains of the AMPK g3 subunit which has a skeletal musclespecific expression [4], also cause an abnormally high glycogen content in skeletal muscle of pigs [8]. Deleterious mutations were not found in the six others AMPK subunit isoforms in a large panel of patients with hypertrophic cardiomyopathies in whom contractile protein mutations had not been found [9]. In the present case, there is a striking discrepancy between the severity of cardiac complications and the muscle symptoms which were limited to occasional pain after exercise and high CK levels. This could be explained by the variability in tissue distribution of AMPK isoforms, g2 isoform being more abundant in heart than in skeletal muscle [4]. Moreover we could speculate that the presence of g3 isoform in skeletal muscle, but not in the heart could compensate the effects of the mutation in PRKAG2. Initial reports of functional effect of PRKAG2 mutations concluded to a constitutive activation of AMPK increasing AMP kinase activity and stimulating carbohydrate accumulation [5]. However, subsequent studies describing the effects of different mutations, demonstrated that the major effect of the mutations is to reduce activation of AMPK in response to stress [10]. The most clearly identified physiological role of AMPK on carbohydrate metabolism is the stimulation of glucose transporter GLUT4 induced by muscle contraction or exercise, which increases glucose intake and hexokinase activity, thus accelerating glycolytic flux and lactate formation [11]. AMPK probably also phosphorylates glycogen synthase, inducing an inhibition of this enzyme. The normal glycogen appearance at the optical and ultrastructural levels in the skeletal muscle of our patient, contrasting with PAS diastase-resistant amylopectin-like deposits which have been showed in hearts of patients with PRKAG2 mutations, could be the result of
181
a different effect in heart and muscle tissue of AMPK deficiency. The clinical triad including either alone or in combination, cardiac hypertrophy, progressive conduction system disease and ventricular pre-excitation is also encountered in two muscle disorders in which heart involvement is predominant, infantile-onset acid maltase deficiency (Pompe’s disease) and Danon’s disease. Cardiomyopathy is always associated with a severe hypotonia in Pompe’s disease, in contrast with Danon’s disease in which skeletal muscle weakness may be absent [12]. Thus far, the glycogen storage cardiomyopathy due to PRKAG2 gene was not considered as a muscle disorder, but this observation advocate that mutations in PRKAG2 gene mutations should be searched in unexplained muscle glycogenosis associated with hypertrophic cardiomyopathy or conduction effects. The subsarcolemmal localization of the vacuoles that we observed in the muscle biopsy of our patient could help to differentiate this disorder from Pompe and Danon’s diseases, which are generally associated with a diffuse intracytoplasmic vacuolation of muscle fibres. However, a recent study showing mitochondrial proliferation on muscle biopsy of patients with a different PRKAG2 mutation, suggest that the consequences of PRKAG2 mutations on skeletal muscle metabolism may differ according to the type of mutation. Further clinical, morphological and biochemical studies remains necessary for a better knowledge of muscle symptoms related to this disease and a precise analysis of the consequences of PRKAG2 mutations on muscle glycogen metabolism.
References [1] DiMauro S, Hays AP, Tsujino S. Nonlysosomal glycogenoses. In: Engel AG, Franzini-Armstrong C, editors. Myology, 3rd ed., vol. 2. New York, NY: McGraw-Hill; 2004. p. 1535–57. [2] Gollob MH, Green MS, Tang AS, et al. Identification of a gene responsible for familial Wolff–Parkinson–White syndrome. N Engl J Med 2001;344:1823–31. [3] Murphy RT, Mogensen J, McGarry K, et al. Adenosine monophosphate-activated protein kinase disease mimicks hypertrophic cardiomyopathy and Wolff–Parkinson–White syndrome: natural history. J Am Coll Cardiol 2005;45:922–30. [4] Cheung PC, Salt IP, Davies SP, et al. Characterization of AMPactivated protein kinase gamma-subunit isoforms and their role in AMP binding. Biochem J 2000;346(Pt 3):659–69. [5] Arad M, Benson DW, Perez-Atayde AR, et al. Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy. J Clin Invest 2002;109:357–62. [6] Hogrel JY, Laforet P, Ben Yaou R, et al. A non-ischemic forearm exercise test for the screening of patients with exercise intolerance. Neurology 2001;56:1733–8. [7] Burwinkel B, Scott JW, Bu¨hrer C, et al. Fatal congenital heart glycogenosis caused by a recurrent activating R531Q mutation in the g2-subunit of AMP-activated protein kinase (PRKAG2), not by phosphorylase kinase deficiency. Am J Hum Genet 2005;76:1034–49.
182
P. Laforeˆt et al. / Neuromuscular Disorders 16 (2006) 178–182
[8] Milan D, Jeon JT, Looft C, et al. A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle. Science 2000; 288:1248–51. [9] Oliveira SM, Ehtisham J, Redwood CS, et al. Mutation analysis of AMP-activated protein kinase subunits in inherited cardiomyopathies: implications for kinase function and disease pathogenesis. J Mol Cell Cardiol 2003;35:1251–5. [10] Daniel T, Carling D. Functional analysis of mutations in the gamma 2 subunit of AMP-activated protein kinase associated with cardiac
hypertrophy and Wolff–Parkinson–White syndrome. J Biol Chem 2002;277:51017–24. [11] Holmes BF, Kurth-Kraczek EJ, Winder WW. Chronic activation of 5 0 -AMP-activated protein kinase increases GLUT-4, hexokinase, and glycogen in muscle. J Appl Physiol 1999;87:1990–5. [12] Arad M, Maron BJ, Gorham JM, et al. Glycogen storage diseases presenting as hypertrophic cardiomyopathy. N Engl J Med 2005;352: 362–72.
Correction In the article “The early history of myasthenia gravis” by Dr. Trevor Hughes and published in Neuromuscular Disorders 15 (2005) 878-886, the following footnote was inadvertently omitted: “This article is based on the Meryon Society Lecture at Worcester College, Oxford, given by the author on July 22nd, 2005.”