J. Inher. Metab. Dis. 20 (1997) 186 –192 © SSIEM and Kluwer Academic Publishers. Printed in the Netherlands
Clinical heterogeneity and molecular mechanisms in inborn muscle AMP deaminase deficiency M. GROSS Medizinische Poliklinik, Klinikum Innenstadt, Ludwig-Maximilians-Universität München, Pettenkoferstraße 8a, D-80336 München, Germany
Summary: Lack of the muscle-specific isoform of AMP deaminase (myoadenylate deaminase deficiency) can cause a metabolic myopathy, with exercise-induced muscle symptoms such as early fatigue, cramps and/or myalgia. It is the most common muscle enzyme defect in man, found in about 2 – 3% of all muscle biopsies. The genetic basis of the inherited defect is the nonsense mutation C34-T in the AMPD1 gene encoding myoadenylate deaminase. The mutation results in a premature stop of the enzyme synthesis. In a healthy German population, the frequency of the mutant allele was 0.1, and 1% of this population is expected to be homozygous for the mutation. In people with muscle symptoms, the allele frequency was significantly higher (0.145). The correlation between allele frequency and muscle symptoms underscores the clinical significance of this defect. However, the vast majority of homozygous subjects do not develop a metabolic myopathy. This clinical heterogeneity may be due to molecular genetic factors such as alternative splicing of the exon harbouring the mutation, or due to metabolic conditions such as pathways compensating for the defect. The real basis for the high percentage of asymptomatic homozygous subjects remains to be revealed. Myoadenylate deaminase (MAD) is the muscle-specific isoform of AMP deaminase. Lack of activity of this enzyme causes a metabolic myopathy. It was first described in 1978 as a new enzyme defect in patients with exercise-induced muscle symptoms (Fishbein et al 1978). Within the following few years, this turned out to be the most common enzyme defect in skeletal muscle. It is found in about 2 – 3% of the population in large series of muscle biopsies (Goebel and Bardosi 1987). AMP deaminase (EC 3.5.4.6) is part of the purine nucleotide cycle (Figure 1). It catalyses the deamination of AMP to IMP with liberation of ammonia. This reaction is the source of ammonia production in exercising skeletal muscle. IMP can be converted into adenylosuccinate by adenylosuccinate synthetase (EC 6.3.4.4). The third enzyme in this cycle is adenylosuccinate lyase (EC 4.3.2.2), which converts adenylosuccinate into AMP with liberation of fumarate. The net effect of this cycle is the conversion of aspartate into fumarate and ammonia (Van den Berghe et al 1992). 186 J. Inher. Metab. Dis. 20 (1997)
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this confusion may be due to the high prevalence of MAD deficiency. Since this enzyme defect is very common, there will be patients with symptoms due to this defect as well as patients with a coincidence of (asymptomatic) MAD deficiency and other causes of muscular symptoms. METABOLIC BASIS OF SYMPTOMS IN PATIENTS The metabolic basis of the symptoms is not yet completely understood. Based on preliminary studies (Sabina et al 1980), it was initially thought that the enzyme defect causes an accumulation of AMP in myocytes during exercise, with subsequent dephosphorylation to adenosine. Since adenosine can easily diffuse across cell membranes, the muscle cells in patients with MAD deficiency were expected to be depleted of adenine nucleotides. Subsequent studies, however, came to contradictory results. During exercise, in patients with MAD deficiency, the muscle cells not not lose more adenine nucleotides than muscle cells of healthy controls (Sabina et al 1982, 1984). Because of symptoms, patients had to stop exercising earlier than the control subjects. Although the patients exercised less, more creatine phosphate and more ATP was degraded. Therefore, an impaired energy production in these patients seems to be the most likely explanation for the symptoms. This impairment in energy production may at least partially be due to a lack of fumarate production during exercise. THERAPY On the basis of the initial hypothesis of an increased loss of adenine nucleotides from exercising muscle cells in these patients, ribose was given to increase de novo synthesis of adenine nucleotides. Some but not all patients reported an improvement of muscular performance during ribose intake (Patten 1982; Lecky 1983). However, only 2g of ribose was given daily in these first studies. Since this is a relatively small amount of ribose, subsequent studies with up to 100g of ribose daily were performed (Zöllner et al 1986). Results showed most successful improvement with 10 – 20g of ribose per hour given at the time of heavy exercise. About half of the patients reported an improvement under such a treatment. For reasons that are not yet understood, about half of the patients did not experience any improvement even with high doses of ribose (Reimers et al 1988). Ribose may be used in myocytes as an additional source of energy. It can be degraded into lactate with the beneficial effect of ATP production. Furthermore, after synthesis into phosphoribosyl pyrophosphate, it may stimulate both the salvage pathway (from hypoxanthine to ATP) and the de novo synthesis of adenine nucleotides (Zimmer et al 1984; Gross et al 1989, 1991). GENETIC BASIS All patients reported so far are homozygous for the same nonsense mutation C34-T in the last position of exon 2 of the AMPD1 gene encoding myoadenylate deaminase (Morisaki et al 1992). This mutation changes the codon CAA into the stop-codon TAA, resulting in
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Muscle AMP deaminase deficiency Table 1
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Frequency of the C34-T mutation in the AMPD1 gene in various populations
Expected Sequence at position 34 of prevalence of the AMPD1 cDNA homozygous Total Wild-type Heterozygous Homozygous Allele subject Population number C34 C34-T T34 frequency (%) Reference Randomly selected subjects Caucasian US 59 47 citizens Black US 13 9 citizens German 106 83
10
2
0.119
1.4
3
12
0.192
3.7
20
3
0.123
1.5
0
0
0
0
Subjects without muscle symptoms German 290 236 50
4
0.100
1.0
Gross et al (1995b)
Subjects with muscle symptoms German 320 233
6
0.145
2.1
Gross et al (1995b)
Japanese
106
106
81
Morisaki et al (1992) Morisaki et al (1992) Gross et al (1992) Morisaki et al (1992)
premature stop of protein synthesis. The patients are also homozygous for another mutation C143-T. This mutation would cause an amino acid substitution of Pro-48 by Leu. However, because of the location of this mutation downstream of the nonsense mutation, it does not have any effect. Since the genetic basis of myoadenylate deaminase deficiency is so homogeneous, it is easy to screen for this defect (Gross 1994). To estimate the prevalence of patients with MAD deficiency in the population, the allele frequency of the C34-T mutant allele was determined in various groups of subjects. Both randomly selected Germans, US citizens and Japanese were studied (Gross et al 1992; Morisaki et al 1992). In addition, German patients with muscular complaints with or without obvious explanation as well as subjects free of any kind of muscular symptoms were studied (Gross et al 1995b). The results are shown in Table 1. There are obvious differences in the allele frequency in various populations. Among the Japanese patients, not a single mutant allele was found. In contrast, a high frequency was observed among black US citizens. More importantly, there is a correlation between the allele frequency and the prevalence of muscular symptoms. The lowest allele frequency was found in Germans without muscle symptoms. Among patients with muscle symptoms, the allele frequency was significantly higher (χ 2 test, df = 1, p <0.025). The frequency of the mutant allele among randomly selected subjects (some of them may have muscle symptoms) was intermediate. These data support the clinical significance of myoadenylate deaminase deficiency. However, even among those without muscle symptoms, 1% were homozygous for the mutation. Therefore, there must be mechanisms to compensate for the effect of the nonsense C34-T mutation in the AMPD1 gene. J. Inher. Metab. Dis. 20 (1997)
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POSSIBLE MOLECULAR EXPLANATIONS FOR THE CLINICAL HETEROGENEITY About 1% of the total population is expected to be homozygous for the nonsense C34-T mutation and therefore expected to be MAD deficient. This frequency is clearly higher than the prevalence of a known metabolic myopathy. Several hypotheses may explain this discrepancy: 1. MAD may not be critical for energy metabolism. However, considering the central position of this enzyme in energy metabolism and the fact that its expression is highly regulated, this does not seem very likely. In addition, the enzyme was highly conserved in evolution, which underscores its importance. 2. The expression of one of the two other genes encoding other isoforms of AMP deaminase may compensate for MAD deficiency. The AMPD2 gene encodes the liver isoform, the AMPD3 gene encodes the erythrocyte isoform. The expression of these genes in muscle cells of patients with MAD deficiency has been studied, but no compensatory increase in gene expression was found in these patients (Mahnke-Zizelman and Sabina 1992). 3. There may be mechanisms to overcome the deleterious effects of this mutation. It is possible that other metabolic pathways can compensate for MAD deficiency. However, there is no evidence to support this speculation. A more likely explanation may be alternative splicing of exon 2 harbouring the mutation. Since exon 2 is only 12 nucleotides in length (4 times 3), skipping of exon 2 would not affect the reading frame. Furthermore, only four amino acids close to the amino terminus would be missing. These amino acids are not necessary for the enzymatic activity of this protein. On in vitro protein expression of normal and exon 2-deficient cDNA, no difference in enzyme activity could be found (Gross et al 1995a). Therefore, alternative splicing of exon 2 could explain the high percentage of homozygous subjects not suffering from a metabolic myopathy. The third hypothesis seems to be the most likely explanation for the high prevalence of homozygous asymptomatic subjects. Therefore, studies are ongoing to investigate the effect of the C34-T mutation of the splicing pattern of exon 2. In rat, alternative splicing of this exon is highly controlled in a tissue- and stage-specific pattern (Mineo et al 1990; Sabina et al 1990). For example, in embryonic skeletal muscle, the majority of the AMPD1 mRNA lacks the exon 2 sequence. In adult skeletal muscle, the vast majority of the mRNA contains exon 2 (Mineo and Holmes 1991). In human skeletal muscle, alternative splicing of exon 2 occurs in 0.6– 2% of the message (Morisaki et al 1993). Since the C34-T mutation affects the last nucleotide of exon 2, thus altering the 5′ splicing donor site, it may cause an even higher percentage of alternatively spliced message. To test this hypothesis, the AMPD1 mRNA was extracted from skeletal muscle of healthy subjects and patients with MAD deficiency. The message was analysed for the C34-T mutation as well as for alternative splicing of exon 2 (Rötzer and Gross, unpublished results). Preliminary results indicate that the C34-T muta-tion does not seem to significantly alter the percentage of alternatively spliced message. Homozygous patients with a myopathy due to MAD deficiency did not show a difference
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in the percentage of alternatively spliced AMPD1 message in comparison to subjects with wild-type sequence. However, it still cannot be excluded that in myocytes of homozygous asymptomatic subjects more AMPD1 message may be alternatively spliced in comparison to patients with symptomatic MAD deficiency. This study remains to be performed. Since alternative splicing may not be the main explanation for the prevalence of homozygous asymptomatic subjects, the molecular basis for this phenomenon remains to be revealed. The clinical heterogeneity found in symptomatic MAD deficiency also cannot yet be fully explained by molecular mechanisms. The genotype – phenotype correlation seems to be low, as it is in most other enzyme defects studied. The vast majority of homozygous subjects do not develop a metabolic myopathy. However, it may be possible that the homozygous asymptomatic subjects are not completely free of symptoms. Maybe these subjects are simply not very successful in sports. It would be worth undertaking a study to investigate the prevalence of homozygous subjects among professional athletes in comparison to those not participating in any sporting activity. CONCLUSION In conclusion, MAD deficiency is the most common muscle enzyme defect in man. About 2– 3% of all muscle biopsies show this defect. In patients with exercise-induced muscular symptoms, MAD deficiency has to be considered as a possible explanation. The genetic basis of this enzyme defect is homogeneous: all patients reported so far are homozygous for a single nonsense-mutation, C34-T, in the AMPD1 gene. The presence of the C34-T mutation can easily be analysed in genomic DNA as a screening test for MAD deficiency in patients with evidence of a muscular disorder. However, the vast majority of C34-T homozygous subjects do not suffer from a metabolic myopathy. The metabolic or genetic basis for this phenomenon remains to be revealed. The finding of homozygosity for this mutation therefore does not establish the diagnosis of myoadenylate deaminase deficiency. In patients with atypical symptoms, it is difficult or sometimes impossible to determine whether the patient is indeed suffering from MAD deficiency or whether there is a coincidence with another (not yet diagnosed) disorder. REFERENCES Atkinson DE (1968) The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7: 4030 – 4034. Fishbein WN, Armbrustmacher VW, Griffin JL (1978) Myoadenylate deaminase deficiency: a new disease of muscle. Science 200: 545 – 548. Goebel HH, Bardosi A (1987) Myoadenylate deaminase deficiency. Klin Wochenschr 65: 1023 – 1033. Gross M (1994) New method for detection of C34-T mutation in the AMPD1 gene causing myoadenylate deaminase deficiency. Ann Rheum Dis 53: 353 – 354. Gross M, Kormann B, Kamilli I, Gresser U, Reiter S (1989) Influence of D-ribose administration on the exercise-induced increase in hypoxanthine excretion. Ann Nutr Metab 33: 215 – 216. Gross M, Reiter S, Zöllner N (1989) Metabolism of D-ribose administered continuously to healthy persons and to patients with myoadenylate deaminase deficiency. Klin Wochenschr 67: 1205 – 1213. Gross M, Kormann B, Zöllner N (1991) Ribose administration during exercise: effects on substrates and products of energy metabolism in healthy subjects and a patient with myoadenylate deaminase deficiency. Klin Wochenschr 69: 151 – 155.
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