Myoadenylate Deaminase Deficiency Research Articles

Adenosine inhibits depolarization-induced Ca(2+) release in mammalian skeletal muscle.

In normal skeletal muscle, prolonged stimulation results in some cellular adenosine triphosphate (ATP) being converted to adenosine monophosphate (AMP) and then deaminated to inosine monophosphate (IMP). Here, we investigate whether the build-up of IMP contributes to muscle fatigue and also determine what happens if AMP is instead hydrolyzed to adenosine. Rat skeletal muscle fibers were mechanically skinned, allowing rapid manipulation of the cytoplasmic conditions, while still retaining the normal excitation-contraction coupling mechanism. Inosine monophosphate (3 mM) had no noticeable effect on either depolarization-induced or caffeine-induced Ca(2+) release from the sarcoplasmic reticulum. In contrast, 3 mM adenosine substantially inhibited depolarization-induced force responses and completely abolished caffeine activation of Ca(2+) release in a reversible fashion, with noticeable inhibition occurring even at 0.4 mM adenosine. These results indicate that IMP does not appreciably inhibit excitation-contraction coupling in normal muscle, and further suggest that the build up of adenosine may be at least partly responsible for the early onset of fatigue occurring in subjects with myoadenylate deaminase deficiency.

Exertional Myalgia Syndrome Associated with Diminished Serum Ammonia Elevation in Ischemic Exercise Testing

A 36-year-old man with chronic severe exertional myalgias had a normal serum lactate elevation and diminished serum ammonia elevation on an ischemic forearm exercise test (IFET). The IFET is commonly performed in the evaluation of patients with complaints of exertional myalgias, cramps, and rhabdomyolysis. The finding of a normal serum lactate elevation and a diminished serum ammonia elevation after ischemic exercise is usually considered indicative of myoadenylate deaminase deficiency. However, myoadenylate deaminase activity was normal in this man's muscle biopsy specimen. This case suggests that a diminished serum ammonia elevation in the IFET is not always indicative of myoadenylate deaminase deficiency, a disorder of ammonia production. A diminished serum ammonia elevation in the IFET could also reflect an impairment of net ammonia efflux from muscle into blood.

Gene mutations responsible for human erythrocyte AMP deaminase deficiency in Poles.

In human, there are three fundamental tissue-specific isozymes of AMP deaminase (EC 3.5.4.6),1,2 and three different genes for the isozymes have been identified:3–6 AMPD1 encoding isozyme M found in skeletal muscle, AMPD2 encoding L in liver, and AMPD3 encoding erythrocyte AMP deaminase (El). The erythrocyte AMP deaminase deficiency that we initially identified,7,8 is clinically asymptomatic. Erythrocyte ATP contents in the complete deficiency are about 150% of normal,8 which can be explained by impairment of the degradation of AMP by way of AMP deaminase. The inheritance of the deficiency was autosomal recessive and the heterozygote frequency was estimated at about 1/30 in Japan, Korea and Taiwan.7,8 The deficiency was also reported in Europe,9 and the frequency in northern Poland was almost the same as that in east Asia. The nonsense mutation on AMPD1 responsible for human myoadenylate deaminase (muscle type AMP deaminase) deficiency was found in 12% of Caucasians and 19% of African-Americans, whereas none of the 106 Japanese subjects surveyed has the mutant allele.10 Human erythrocyte AMP deaminase deficiency in Japanese is associated with 75% of the same mutation (R573C) and 25% of other heterogeneous mutations on AMPD3.11,12 It is of interest to know whether the same mutation occurs in Poles, since their frequency of the enzyme deficiency was not significantly different from Japanese.KeywordsPolish SampleDirect Sequencing AnalysisComplete DeficiencyHeterogeneous MutationHeterozygote FrequencyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The genetic basis of myoadenylate deaminase deficiency is heterogeneous.

Myoadenylate deaminase (MAD) is the muscle specific isoform of adenosine monophosphate deaminase (AMPD). AMPD (EC 3.5.4.6.) is an aminohydrolase that catalyzes the deamination of AMP to inosine monophosphate (IMP) with liberation of ammonia from position 6 of the adenine ring. As part of the purine nucleotide cycle,1,2 AMPD is involved in the regulation of the relative concentrations of intracellular purine nucleotides, the stabilization of the adenylate energy charge, the replenishment of citric acid cycle intermediates by the formation of fumarate, the deamination of amino acids (aspartate) and the regulation of the activity of the glycolytic enzymes phosphofructokinase and phosphorylase b.3–5 The fact that AMPD is an ubiquitous enzyme found in eukaryotes and in all tissues of vertebrates6–8 underlines the important role of this enzyme in energy metabolism. In man there are at least three isozymes. The isozyme with the highest activity is myoadenylate deaminase found in skeletal muscle.6,9

Myoadenylate deaminase deficiency, hypertrophic cardiomyopathy and gigantism syndrome

We report a 20-year-old man with gigantism syndrome, hypertrophic cardiomyopathy, muscle weakness, exercise intolerance, and severe psychomotor retardation since childhood. Histochemical and biochemical analysis of skeletal muscle biopsy revealed myoadenylate deaminase deficiency; molecular genetic analysis confirmed the diagnosis of primary (inherited) myoadenylate deaminase deficiency. Plasma, urine, and muscle carnitine concentrations were reduced.l-Carnitine treatment led to gradual improvement in exercise tolerance and cognitive performance; plasma and tissue carnitine levels returned to normal, and echocardiographic evidence of left ventricular hypertrophy disappeared. The combination of inherited myoadenylate deaminase deficiency, gigantism syndrome and carnitine deficiency has not previously been described.

Clinical heterogeneity and molecular mechanisms in inborn muscle AMP deaminase deficiency.

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.

A competitive allele-specific oligomers polymerase chain reaction assay for the cis double mutation in AMPD1 that is the major cause of myo-adenylate deaminase deficiency

A competitive allele-specific oligomers polymerase chain reaction assay for the cis double mutation in AMPD1 that is the major cause of myo-adenylate deaminase deficiency

Myoadenylate deaminase deficiency myopathy in pregnancy

Myoadenylate deaminase deficiency myopathy in pregnancy

Maximizing residual AMP deaminase expression in myoadenylate deaminase deficiency through the formation of heterotetramers of isoforms M and E

The activity of dihydroorotate dehydrogenase (DHO-dehase) has been reported to decrease both in vitro and in vivo in hepatocellular carcinomas. DHO-dehase, the fourth enzyme of the de novo pyrimidine biosynthetic pathway, is a mitochondrial enzyme which is both a potential rate-limiting reaction in the de novo pyrimidine biosynthetic pathway and a potential therapeutic target for tumor inhibitors. This paper reports results on a series of pyrimidine analogs of dihydroorotate (DHO) and orotic acid (OA) as inhibitors of DHO-dehase. The enzyme test results established that the intact amide and imide groups of the pyrimidine ring and the 6-carboxylic acid are required for significant enzyme inhibition. The testing of several functional groups similar in characteristics to that of the carboxylic acid, such as sulfonamide, tetrazole and phosphate, indicated that the carboxylic acid group is preferred by the enzyme. Using various 5-substituted OA and DHO derivatives, it was shown that there is a steric limitation of a methyl group at this position. The compound d,l-5-trans-methyl DHO (7) (Ki of 45 μM) was both an inhibitor and a weak substrate for the enzyme, demonstrating that mechanism-based enzyme inhibitors should be effective. The testing results further suggest that a negatively charged enzyme substituent may be present near the 5-position of the pyrimidine ring and that there may be an enzymesubstrate metal coordination site near the N-1 and carboxylic acid positions of the pyrimidine ring. The combined testing results were then used to define both conformational and steric substrate enzyme binding requirements from which a model was proposed for the binding of DHO and OA to the DHO-dehase active site.

Human adenosine receptors

Myo-adenylate deaminase deficiency (mADD) is the most common enzyme deficiency restricted to skeletal muscle, with a frequency of 1–2% in frozen muscle biopsies and complaints of easy fatigue and muscle cramping on exertion. A double C > T transition at coding bases 34 in exon 2 and 143 in exon 3 is the main cause of mADD. A 1-day assay using allele-specific oligomers and no isotope would be valuable for single cases.Downstream primers with penultimate mismatch and 3′ terminus matching the mutant or the normal base in exons 2 and 3 are used with a common upstream primer for each exon, to give amplimers of 150 bp for exon 2 and 200 bp for exon 3. A short common primer further downstream in exon 3 provides a competing 300-bp amplimer whose product contribution is readily controlled by adjusting the annealing temperature. The entire procedure could be done in 1 day: DNA isolation, polymerase chain reaction (PCR), electrophoresis in agarose gel with ethidium bromide, and visualization by ultraviolet light. Deficient individuals have bands only with the mutant primers, normal persons have bands only with the normal primers, and heterozygotes (carriers) show bands with both primer sets. The empty slots show the 300-bp competing band, proving the PCR amplified the correct template. Allele-specific oligomers PCR results were verified by dot blots and by restriction endonuclease analysis of exon 2.A simple and reliable allele-specific PCR assay using DNA from blood (or muscle) is now available for the diagnosis of individual cases of mADD caused by the common double-mutant AMPD1 gene, including the rare instances arising from homologous recombination between the two mutations.

The genetic basis of myoadenylate deaminase deficiency is heterogeneous

Inosine 5′-monophosphate dehydrogenase (IMPDH) is a target of the immunosuppressant mycophenolic acid (MPA) which is used for the prevention of acute rejection. We evaluated the concentration-dependent effects of MPA and its phenolic (MPAG) and acyl (AcMPAG) glucuronides on IMPDH activity in erythrocytes in an initial and a stable phase.Eight kidney transplant recipients treated with mycophenolate mofetil (MMF) in an initial phase and 104 recipients [56 received MMF (MMF+) and 48 did not (MMF−)] in a stable phase were enrolled.IMPDH activity in erythrocytes was inhibited at the peak plasma concentration of MPA in initial kidney transplant recipients. However, median IMPDH activity in erythrocytes was 1.73 times higher in MMF+ than in MMF− kidney transplant recipients (38.7 to 22.4 nmol/g hemoglobin/h, P < 0.01). The median values of trough plasma concentrations (C12) of MPA, MPAG, and AcMPAG in stable kidney transplant recipients were 2.86, 49.9, and 0.256 μg/ml, respectively. In a multivariate analysis, AcMPAG C12, MPAG C12, MPA C12, serum creatinine, age, lactate dehydrogenase, and serum albumin were significant predictors, accounting for interindividual variability of IMPDH (R2 = 0.537, P = 0.02).IMPDH activity in erythrocytes may be useful indicators of short-term immunosuppression and long-term exposure of MPA.

Malignant hyperthermia (MH) susceptibility and myoadenylate deaminase (MAD) deficiency

To date a clear association between MH susceptibility and underlying muscle disease has been shown for three clinical myopathies: King Denborough syndrome, central core disease and Evans myopathy. However, other more common myopathies such as Myoadenylate-deaminase (MAD) deficiency have also been thought to be associated with an increased risk of MH [1]. Thus we investigated in this study the occurrence of MAD deficiency in individuals who proved MH susceptible in the in vitro contracture test (IVCT). Muscle specimens from 50 individuals clinically suspicious for MH were used for diagnostic IVCTs using halothane and caffeine. Specimens of all biopsies were referred to detailed light and electron microscopic examination, additionally molecular biological techniques were used. These examinations were under-taken by a blinded investigator, who did not know the result of the IVCT. According to the protocol of the European MH Study Group 18 out of 50 individuals were diagnosed MH susceptible (MHS) and 17 out of 50 halothane equivocal (MHEh). Fourteen out of these MHS/MHEh individuals showed morphological signs of metabolic disorders. Two MHS and 3 MHEh individuals revealed a MAD deficiency that was proved by negative enzymatic biochemical and histochemical reaction. Two of the MHEh individuals were siblings. None of these individuals was aware of his enzyme deficiency, but all were suffering from subtile clinical symptoms (i.e. recurrent episodes of muscle pain and muscle weakness). None of the MHN individuals was MAD deficient. Our data show that patients with MAD deficiency have an increased risk of MH as revealed by IVCT. Because MAD deficiency is one of the most frequent muscle diseases [2] it has to be taken into serious consideration in MH suspicious individuals with symptoms of muscle weakness and/or pain. In conclusion, our results strongly suggest that additionally to IVCT the muscle biopsies obtained from such patients should be examined by histological and biochemical methods.

Tension myalgia versus myoadenylate deaminase deficiency: A case report

Myalgia is a complaint associated with numerous medical conditions such as metabolic or hormonal abnormalities, toxic myopathies, tetanus, electrolyte disturbances, inflammatory diseases, and exertion-related pain. A diagnosis of tension myalgia or myofascial-type pain is often considered when no objective findings are seen in the evaluation. This is a report of a patient who was treated unsuccessfully for fibromyalgia for many years and who ultimately was diagnosed with a rare benign skeletal muscle metabolic disorder caused by myoadenylate deaminase deficiency. We discuss this enzyme deficiency and its importance for the physiatric community.

Metabolic myopathies

Disorders of glycogen, lipid or mitochondrial metabolism may cause two main clinical syndromes, namely (1) progressive weakness (eg, acid maltase, debrancher enzyme, and brancher enzyme deficiencies among the glycogenoses; long- and very-long-chain acyl-CoA dehydrogenase (LCAD, VLCAD), and trifunctional enzyme deficiencies among the fatty acid oxidation (FAO) defects; and mitochondrial enzyme deficiencies) or (2) acute, recurrent, reversible muscle dysfunction with exercise intolerance and acute muscle breakdown or myoglobinuria (with or without cramps) (eg, phosphorylase (PPL), phosphorylase b kinase (PBK), phosphofructokinase (PFK), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGAM), and lactate dehydrogenase (LDH) among the glycogenoses and carnitine palmitoyltransferase II (CPT II) deficiency among the disorders of FAO or (3) both (eg, PPL, PBK, PFK among the glycogenoses; LCAD, VLCAD, short-chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD), and trifunctional enzyme deficiencies among the FAO defects; and multiple mitochondrial DNA (mtDNA) deletions). Myoadenylate deaminase deficiency, a purine nucleotide cycle defect, is somewhat controversial and is characterized by exercise-related cramps leading rarely to myoglobinuria.

Immunolocalization of AMP-deaminase isozymes in human skeletal muscle and cultured muscle cells: concentration of isoform M at the neuromuscular junction.

The three major isoforms of AMP-deaminase (AMPda) were localized in human skeletal muscle and cultured muscle cells by immunocytochemistry. The M isoform was mainly located in Type II muscle fibers and showed a clear cross-striation. Particularly strong staining was present at the neuromuscular junction. Capillaries were also immunoreactive. The L isoform was predominantly observed in nerve bundles and to a minor extent in smooth muscle cells and endothelial cells. The E isoform was predominantly present in smooth muscle cells, and to a lesser extent in Type I muscle fibers and nerve bundles. In quadriceps muscle of patients with myoadenylate deaminase deficiency, no immunostaining for the M isozyme was observed, whereas reactivity for the L and E isoforms was unaltered. In human muscle cell cultures, mononuclear cells, including myoblasts, were immunoreactive for the L isoform and to a lesser extent the E isoform, whereas the M isoform was absent. In myotubes, diffuse or fibrillar staining was present for all three isoforms, but only the M isoform showed a clear cross-striation pattern in highly differentiated myotubes.

Molecular biology of AMP deaminase deficiency.

In man, there are at least four isoforms of adenosine monophosphate deaminase (AMPD): myoadenylate deaminase in skeletal muscle, the L isoform in liver, and the E1 and E2 isoforms in erythrocytes. Myoadenylate deaminase is encoded by the AMPD1 gene located on chromosome 1 p13-p21, the L isoform by the AMPD2 gene, and both isoforms in erythrocytes by the AMPD3 gene. Myoadenylate deaminase deficiency is found in 2-3% of all muscle biopsies. The inborn type of myoadenylate deaminase deficiency is caused by a single mutant allele harbouring two mutations: C34-->T (Gln-->Stop) and C143-->T (Pro-48-->Leu). Population studies revealed a frequency of the mutant allele of 0.12 in Caucasian Americans and Germans. The C34-->T mutation is located in exon 2, which is alternatively spliced in part of the AMPD1 transcript in human muscle. Since the second mutation does not affect enzyme function, alternatively spliced mRNA encodes a catalytically active enzyme. Only one patient with a disorder linked to liver AMPD has been described so far. In this patient the decreased inhibition of this enzyme by GTP resulted in uric acid overproduction and gout. A complete lack of erythroyte AMPD activity is found in asymptomatic subjects. The molecular basis of both disorders is not yet known.

Ergometer exercise in myoadenylate deaminase deficient patients.

Three patients with primary myoadenylate deaminase deficiency were subjected to exercise on a bicycle ergometer at 125 W for 30 minutes. Blood samples prior to, during, and at the end of exercise were analyzed for lactate, ammonia, and hypoxanthine. In addition, urinary hypoxanthine excretion was measured. In these patients the serum lactate level increased to concentrations between 7.9 and 9.0 mmol/l at the end of exercise whereas the mean lactate level in nine control subjects at the end of exercise was 3.3 mmol/l (range 1.1-8.1 mmol/l). There was no difference to control subjects in the exercise-induced increase in plasma levels of ammonia and hypoxanthine or in the increase in urinary hypoxanthine excretion. The findings support the hypothesis of a reduced substrate supply to the citric acid cycle in myoadenylate deaminase deficiency. The normal formation of ammonia and hypoxanthine excludes a marked loss of adenine nucleotides in working muscles in these patients.

Influence on myoadenylate deaminase function in rat skeletal muscle after homologous and heterologous immunization with the purified enzyme.

The aim of the study was to shed some light on the possibility of anti-MAD autoantibodies in cases of rhabdomyolysis or microtraumatic muscular damage causing the symptoms of the acquired myoadenylate deaminase deficiency. Therefore a homologous and a heterologous immunization of rats was carried out with myoadenylate deaminase (MAD; EC 3.5.4.6.) isolated from rat white gastrocnemius muscle (type IIb) and rabbit skeletal muscle. Antibody response to homologous and heterologous antigens was quantified by ELISA and by the inhibition of MAD activity in vitro. Anti-MAD IgG antibodies obtained by homologous immunization caused an inhibition rate of 80% when using MAD from rat white gastrocnemius muscle. Furthermore, the function of MAD in homologously and heterologously immunized rats was investigated after maximal swimming exercise in comparison to non-immunized animals by determination of muscle ammonia and by HPLC detection of inosine 5'-monophosphate (IMP) in skeletal muscle tissues. Both IMP and ammonia accumulation were significantly reduced in type IIb muscle fibers after homologous immunization of rats compared to non-immunized controls (n = 7), whereas the concentration of muscle lactate showed no differences. According to these results we propose that by homologous immunization with purified MAD, anti-MAD IgG impairs the function of cytoplasmic MAD during vigorous exercise in vivo.

Expression of different isoenzymes of adenylate deaminase in cultured human muscle cells. Relation to myoadenylate deaminase deficiency

Adenylate deaminase activity was determined in cultured muscle cells of different maturation grades and muscle biopsies from normal subjects and four patients with a primary myoadenylate deaminase (MAD) deficiency. Adenylate deaminase activity was much lower in cultured human muscle cells than in normal muscle. .The activity increased with maturation. The ratio of activities measured at 5 and 2 mM AMP decreased in the order: immature muscle cells > more mature muscle cells > muscel. Adenylate deaminase activity was detectable in muscle cell cultures of MAD-deficient patients. However, both at 2 and 5 mM AMP this activity was significantly lower than in cultured cells with the same high maturation grade obtained from control sujects, whereas the ratio between the activities at 5 and 2 mM AMP was higher. The observations indicate that transition from a fetal to an adult muscle isoenzyme deaminase takes place in human cultured muscle cells during maturation. In cultures obtained from MAD-deficient patients this transition does not occur and only the fetal isoenzyme is present.

Molecular analysis of the myoadenylate deaminase deficiencies.

Myoadenylate deaminase (mAMPD) deficiency in a clinically heterogeous metabolic myopathy consisting of primary (inherited) and secondary (acquired) forms based on a variety of clinical and laboratory findings. To provide a basis for delineating the underlying molecular defects in mAMPD deficiency, and as a means to test the proposal for multiple forms of the resulting disease, Northern blot analyses were performed with RNA isolated from individual patients with classified primary and secondary deficiency utilizing human mAMPD cDNA probes isolated from adult skeletal muscle libraries. Analysis of nine patients with primary mAMPD deficiency indicates normal abundance of mAMPD transcript. No immunoreactive mAMPD polypeptide is detected in Western blot analyses of skeletal muscle extracts prepared from these patients. Specificity to mAMPD is demonstrated by normal creatine kinase (CK) activities and M-creatine kinase (M-CK) transcript abundance. Similar analyses of four individuals with secondary mAMPD deficiency reveal heterogeneity in this subgroup of patients. Whereas two of these patients exhibit normal mAMPD transcript abundance, two others associated with inflammatory myopathy display reductions in mAMPD and M-CK transcript abundance. Examination of tissue sections derived from the same biopsies utilized in the isolation of RNA demonstrates the integrity of the skeletal muscle in those patients with associated inflammatory myopathy. Combined, these data support the proposal for multiple forms of mAMPD deficiency, and indicate that the primary condition is most commonly characterized by specific point mutations or small deletions/rearrangements in the ampd1 gene, whereas some patients with secondary mAMPD deficiency display more generalized aberrations in gene expression.