Guanidinoacetate Methyltransferase Deficiency Research Articles

Diagnóstico y tratamiento de los síndromes de deficiencia de creatina cerebral

To review the clinical, biochemical and genetic aspects of brain creatine deficiency syndromes, as well as the therapeutic options available. Brain creatine deficiency syndrome has recently been described as a series of inborn errors of metabolism that affect the synthesis and transport of creatine. Three metabolic defects are known: two affect synthesis -guanidinoacetate methyltransferase (GAMT) and arginine:glycine amidinotransferase (AGAT)- and one affects the transport of creatine. Clinically, these patients can display mental retardation, language disorders, epilepsy, autistic behaviour, neurological impairment and movement disorders. After the clinical selection, the different defects can be identified by a biochemical study involving the analysis of metabolites in biological fluids (guanidinoacetate and creatine/ creatinine ratio). Before continuing with the molecular studies, it is important to confirm the deficiency of brain creatine by means of magnetic resonance imaging with spectroscopy. Diagnostic confirmation of AGAT and GAMT deficits is carried out by determining the enzymatic activity in fibroblasts or lymphoblasts, or the incorporation of creatine in the case of studies of transport defects. The study of mutations in AGAT, GAMT (autosomal recessive inheritance) and SLC6A8 (X-linked) genes completes the diagnosis. Brain creatine deficiency syndromes are mainly associated to mental retardation and autism. GAMT and AGAT deficiencies respond to treatment with creatine, whereas patients with transport defects do not respond to this therapy; new therapeutic approaches are therefore being evaluated for this disease.

A mutation on exon 6 of guanidinoacetate methyltransferase (GAMT) gene supports a different function for isoform a and b of GAMT enzyme

A new patient affected by Guanidinoacetate methyltransferase (GAMT) deficiency was reported. This 13-year-old girl presented with mental retardation, as main symptom, associated with a typical pattern of biochemical and neurochemical (brain magnetic resonance spectroscopy) alterations. Molecular study detected a L197P transition on exon 6 of the GAMT gene. Since this mutation leaves the isoform B of the GAMT enzyme unaffected, the occurrence of biochemical alterations and disease in this subject testifies against the possibility that isoform b had GAMT activity.

Quantitative determination of guanidinoacetate and creatine in dried blood spot by flow injection analysis-electrospray tandem mass spectrometry

Background Guanidinoacetate (GAA) and creatine (Cr) are reliable biochemical markers of primary creatine disorders. The aim of this study was to develop and validate a method for the determination of GAA and Cr in dried blood spot through the use of stable isotope dilution and flow injection analysis (FIA)-ESI-MS/MS. Methods Dried blood spots were extracted using methanol–water solution containing D3-Cr. After evaporation and formation of butyl esters, samples were analyzed using multiple reaction monitoring mode ( m/ z 174.2→101.1 for GAA, 188.3→90.1 for Cr and 191.3→93.1 for D3-Cr). Results The analysis was very fast (1 min). The detection limits were 0.34 μmol/l of blood and 0.30 μmol/l of blood for Cr and GAA, respectively, and the response was linear over the range 0.25–12.5 μmol/l of blood for GAA and 3.57–624.7 μmol/l of blood for Cr. Recovery range was 93–101% for Cr and 94–105% for GAA and between-run CVs were 5.3% for GAA and 4.5% for Cr. Ion suppression effect was also studied. The method was applied to spots obtained from two patients affected by GAMT deficiency, four patients affected by AGAT deficiency (including a newborn) as well as 282 healthy subjects. Conclusions The detection of GAA in dried blood spot by FIA-ESI-MS/MS is a highly reliable and high throughput method for the diagnosis of GAMT and AGAT deficiencies and a possible tool for newborn screening of both these tractable disorders.

Use of denaturing HPLC to provide efficient detection of mutations causing guanidinoacetate methyltransferase deficiency

Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive error of creatine synthesis characterized by cerebral creatine deficiency, accumulation of guanidinoacetate, mental retardation, epilepsy, and extrapyramidal symptoms. To date, 14 mutations of the GAMT gene in 27 patients have been reported. Mutation analysis was done using direct sequencing of PCR products and denaturing gradient gel electrophoresis in combination with direct sequencing. In contrast, we evaluated the efficiency of a newly developed DHPLC method to detect mutations in the GAMT gene by analysing DNA from 14 GAMT patients with known mutations. PCR amplification of both patient and control DNA was followed by formation of homoduplices and heteroduplices, and their detection by DHPLC. DHPLC identified all mutations tested and is the preferred choice of analytical method.

1H chemical shift imaging of the brain in guanidino methyltransferase deficiency, a creatine deficiency syndrome; guanidinoacetate accumulation in the gray matter

MR spectroscopy results in a mild case of guanidinoacetate methyltransferase (GAMT) deficiency are presented. The approach differs from previous MRS studies in the acquisition of a chemical shift imaging spectral map showing gray and white matter with the corresponding spectra in one overview. MR spectroscopy revealed guanidinoacetate (GAA) in the absence of creatine. New is that GAA signals are more prominent in gray matter than in white. In the prevailing view, that enzyme deficiency is localized in liver and pancreas and that all GAA is transported into the brain from the blood and the cerebrospinal fluid, this would be compatible with a more limited uptake and/or better clearance of GAA from the white matter compared to the grey matter.

Les syndromes de déficit en créatine

Creatine deficiency syndromes are a newly described group of inborn errors of metabolism affecting creatine metabolism. Three diseases have been described: deficiency of arginine: glycine amidinotransferase (AGAT), deficiency of guanidinoacetate methyltransferase (GAMT) and creatine transporter defect (CRTR). These syndromes are characterized by a depletion of creatine/phosphocreatine in the brain. Clinically, most of the patients develop a variable mental retardation and a severe speech delay associated with epilepsy, extra-pyramidal syndrome and behavior disturbances. These diseases are often diagnosed during infancy but a few adult cases have been reported recently. Diagnosis is established by measurement of guanidinoacetate and creatine in biologic fluids and in vivo proton magnetic resonance spectroscopy by the total lack of intra-cerebral creatine/phosphocreatine demonstrating. GAMT and AGAT deficiencies are treatable by oral creatine supplementation whereas patients with CRTR do not respond to the treatment. Better knowledge of these syndromes is necessary to optimize diagnosis and patient management of these rare but potentially treatable disorders.

Biochemical and behavioural phenotyping of a mouse model for GAMT deficiency

Biochemical and behavioural phenotyping of a mouse model for GAMT deficiency

Guanidinoacetate methyltransferase deficiency identified in adults and a child with mental retardation

Our study describes the adult clinical and biochemical spectrum of guanidinoacetate methyltransferase (GAMT) deficiency, a recently discovered inborn error of metabolism. The majority of the previous reports dealt with pediatric patients, in contrast to the present study. A total of 180 institutionalized patients with a severe mental handicap were investigated for urine and plasma uric acid and creatinine. Patients with an increased urinary uric acid/creatinine ratio and/or decreased creatinine were subjected to the analysis of guanidinoacetate (GAA). Four patients (three related and one from an unrelated family) were identified with GAMT-deficiency. A fifth patient had died before a biochemical diagnosis could be made. They all had shown a normal psychomotor development for the first year of life, after which they developed a profound mental retardation. Three out of four had convulsions and all four totally lacked the development of speech. Their GAMT activity in lymphoblasts was impaired and two novel mutations were identified: the 59 G > C and 506 G > A missense mutations. Urinary GAA was increased, but highly variable 347-1,624 mmol/mol creat (Controls <150 mmol/mol creat). In plasma and CSF the GAA levels were fairly constant at 17.3-27.0 mumol/L (Controls 1.33-3.33) and 11.0-12.4 mumol/L, respectively (Controls 0.068-0.114). GAMT deficiency in adults is associated with severe mental retardation and absence or limited speech development. Convulsions may be prominent. The nonspecific nature of the clinical findings as well as the limited availability of GAA assays and/or in vivo magnetic resonance spectroscopy of the brain may mean that many more patients remain undiagnosed in institutions for persons with mental handicaps.

Evaluation of the mechanism underlying the inhibitory effect of guanidinoacetate on brain Na+, K+-ATPase activity

Guanidinoacetate methyltransferase deficiency (GAMT-deficiency) is an inherited neurometabolic disorder clinically characterized by epilepsy and mental retardation and biochemically by accumulation of guanidinoacetate (GAA) and depletion of creatine. Although the neurological symptoms are predominant, the pathogenesis of the brain dysfunction in this disorder is not yet established. In the present study we investigated the in vitro effect of GAA on Na+, K+-ATPase and Mg2+-ATPase activities in synaptic plasma membrane from hippocampus of young rats. Results showed that GAA significantly inhibited Na+, K+-ATPase activity without affecting Mg2+-ATPase activity. We also evaluated the effect of glutathione (GSH), trolox, Nϖ-nitro-l-arginine methyl ester (L-NAME) and taurine (Tau) on the inhibition elicited by GAA on Na+, K+-ATPase activity. GSH, trolox, L-NAME and Tau per se did not alter Na+, K+-ATPase activity. However, L-NAME and taurine prevented the inhibitory effect of GAA on this enzyme activity. Our findings suggest that the inhibition of Na+, K+-ATPase activity caused by GAA is possibly mediated by nitric oxide (NO) formation and/or synaptic membrane alteration. The present data may contribute to the understanding of the neurological dysfunction characteristic of GAMT-deficient patients.

Guanidinoacetate methyltransferase deficiency: differences of creatine uptake in human brain and muscle

Deficiency of guanidinoacetate methyltransferase (GAMT), the first described creatine biosynthesis defect, leads to depletion of creatine and phosphocreatine, and accumulation of guanidinoacetate in brain. This results in epilepsy, mental retardation, and extrapyramidal movement disorders. Investigation of skeletal muscle by proton and phosphorus magnetic resonance spectroscopy before therapy demonstrated the presence of considerable amounts of creatine and phosphocreatine, and accumulation of phosphorylated guanidinoacetate in a 7-year-old boy diagnosed with GAMT deficiency, suggesting separate mechanisms for creatine uptake and synthesis in brain and skeletal muscle. The combination of creatine supplementation and a guanidinoacetate-lowering therapeutic approach resulted in improvement of clinical symptoms and metabolite concentrations in brain, muscle, and body fluids.

Guanidinoacetate methyltransferase deficiency: differences of creatine uptake in human brain and muscle

Deficiency of guanidinoacetate methyltransferase (GAMT), the first described creatine biosynthesis defect, leads to depletion of creatine and phosphocreatine, and accumulation of guanidinoacetate in brain. This results in epilepsy, mental retardation, and extrapyramidal movement disorders. Investigation of skeletal muscle by proton and phosphorus magnetic resonance spectroscopy before therapy demonstrated the presence of considerable amounts of creatine and phosphocreatine, and accumulation of phosphorylated guanidinoacetate in a 7-year-old boy diagnosed with GAMT deficiency, suggesting separate mechanisms for creatine uptake and synthesis in brain and skeletal muscle. The combination of creatine supplementation and a guanidinoacetate-lowering therapeutic approach resulted in improvement of clinical symptoms and metabolite concentrations in brain, muscle, and body fluids.

Clinical, biochemical and molecular findings in 16 patients with GAMT deficiency

Objective: Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive disorder of creatine synthesis, resulting in creatine deficiency and in accumulation of guanidinoacetate (GAA) in brain and body fluids. The symptoms and the severity of this disease are variable. In this study we analyzed clinical, biochemical and molecular findings in 16 patients with GAMT deficiency.

Enzyme assay for diagnosis of guanidinoacetate methyltransferase deficiency.

Creatine in the human body is derived from two sources: the diet and endogenous synthesis. Biosynthesis involves two enzymes: l-arginine:glycine amidinotransferase (EC 2.1.4.1) and guanidinoacetate N -methyltransferase (GAMT; EC 2.1.1.2). Defects in both of these enzymes lead to inborn errors of metabolism (1)(2). Deficiency of l-arginine:glycine amidinotransferase has been reported very recently in two patients by Item et al. (3) and Bianchi et al. (4). GAMT deficiency was first described in 1994 (1)(5). The clinical presentation of the disease varies widely (2)(6). Some patients present with intractable epilepsy, extrapyramidal movement disorder, and severe retardation, whereas other patients have much milder symptoms and show only mild retardation and autistic behavior. The aspecific clinical picture of GAMT deficiency and the relatively good treatment options makes a good diagnostic strategy important. Magnetic resonance spectroscopy of the brains of affected patients shows absence of the creatine/creatine phosphate peak. However, magnetic resonance spectroscopy may not be performed on all patients with mild symptoms. Screening for metabolic disorders may also aid in the diagnosis because generalized increases in amino acids, organic acids, and other metabolites can be found when their concentrations are calculated relative to the creatinine concentration (2). The relatively low creatinine excretion in GAMT patients, which is attributable to the creatine deficiency, gives falsely increased values for a range of metabolites when they are calculated relative to creatinine. When GAMT deficiency is suspected, analysis of guanidinoacetate in plasma should be performed (7)(8). Confirmation of the defect can be obtained by enzymatic and DNA analysis (9)(10). Ilas et al. (11) described an assay for the determination of GAMT in fibroblasts and lymphoblasts. This method uses 14C-labeled guanidinoacetate as substrate. Product formation ([14C]creatine) is determined by HPLC with detection …

Biochemical and clinical characteristics of creatine deficiency syndromes.

Creatine deficiency syndromes are a newly described group of inborn errors of creatine synthesis (arginine:glycine amidinotransferase (AGAT) deficiency and guanidinoacetate methyltransferase (GAMT) deficiency) and of creatine transport (creatine transporter (CRTR) deficiency). The common clinical feature of creatine deficiency syndromes is mental retardation and epilepsy suggesting main involvement of cerebral gray matter. The typical biochemical abnormality of creatine deficiency syndromes is cerebral creatine deficiency, which is demonstrated by in vivo proton magnetic resonance spectroscopy. Measurement of guanidinoacetate in body fluids may discriminate between the GAMT (high concentration), AGAT (low concentration) and CRTR (normal concentration) deficiencies. Further biochemical characteristics include changes in creatine and creatinine concentrations in body fluids. GAMT and AGAT deficiency are treatable by oral creatine supplementation, while patients with CRTR deficiency do not respond to this type of treatment. The creatine deficiency syndromes are underdiagnosed, so their possibility should be considered in all children affected by unexplained mental retardation, seizures and speech delay.

MR spectroscopy of muscle and brain in guanidinoacetate methyltransferase (GAMT)-deficient mice: validation of an animal model to study creatine deficiency.

As a model for guanidinoacetate methyltransferase (GAMT) deficiency in humans, a gene knockout mouse model was generated. Here we report on several metabolic abnormalities in these mice, observed by in vivo and in vitro MR spectroscopy. In (1)H MR spectra of brain and hindleg muscle a clearly reduced signal of creatine (Cr) was observed in GAMT-deficient (GAMT-/-) animals. Analysis of the (1)H MR spectra of GAMT-/- brain indicated little or no increase of a signal for guanidinoacetate (Gua). In proton MR spectra of muscle, a broad signal of low intensity was observed for Gua. However, substantial Gua accumulation in intact muscle tissue was unequivocally confirmed in high-resolution magic angle spinning spectra, in which the Gua signal was resolved as one clear sharp singlet. In (31)P MR analysis of brain and hindleg muscle a strongly reduced phosphocreatine (PCr) content was shown. In addition, a signal of phosphorylated Gua at 0.5 ppm upfield of PCr was observed, with much higher intensity in muscle than in brain. This signal decreased when ischemia was applied to the muscle and recovered after ischemia was released. Overall, the in vivo (31)P and (1)H MR spectroscopy of GAMT-/- mice is similar to that of human GAMT deficiency. This opens up new avenues for the fundamental study of tissue-type dependence of creatine synthesis and transport and for diagnostic and therapeutic aspects of creatine deficiencies in humans.

Activation of GABA A Receptors by Guanidinoacetate: A Novel Pathophysiological Mechanism

Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessively inherited disorder of creatine biosynthesis. The disease occurs in early life with developmental delay or arrest and several neurological symptoms, e.g., seizures and dyskinesia. Both the deficiency of high-energy phosphates in neurons and the neurotoxic action of the accumulated metabolite guanidinoacetate (GAA) are candidate mechanisms for the pathophysiology of this disease. To examine a potential role of GAA accumulation, we analyzed the electrophysiological responses of neurons induced by GAA application in primary culture and acute murine brain slices. GAA evoked picrotoxin- and bicuculline-sensitive GABA A receptor-mediated chloride currents with an EC 50 of 167 μM in cortical neurons. Pathophysiologically relevant GAA concentrations hyperpolarized globus pallidus neurons and reduced their spontaneous spike frequency with an EC 50 of 15.1 μM. Furthermore, GAA acted as a partial agonist at heterologously expressed GABA A but not GABA B receptors. The interaction of GAA with neuronal GABA A receptors represents a candidate mechanism explaining neurological dysfunction in GAMT deficiency.

Guanidinoacetate and Creatine plus Creatinine Assessment in Physiologic Fluids: An Effective Diagnostic Tool for the Biochemical Diagnosis of Arginine:Glycine Amidinotransferase and Guanidinoacetate Methyltransferase Deficiencies

Disorders of creatine metabolism arise from genetic alterations of arginine:glycine amidinotransferase (AGAT), guanidinoacetate methyltransferase (GAMT), and the creatine transporter. We developed a strategy for the detection of AGAT and GAMT defects by measurement of guanidinoacetate (GAA) and creatine plus creatinine (Cr+Crn) in biological fluids. Three patients with AGAT deficiency from the same pedigree and their eight relatives, as well as a patient affected by a GAMT defect and his parents were analyzed by a new HPLC procedure in comparison with 90 controls. The method, which uses precolumn derivatization with benzoin, separation with a reversed-phase column, and fluorescence detection, has shown good precision and sensitivity and requires minimal sample handling. In the three AGAT patients, plasma GAA was 0.01-0.04 micro mol/L [mean (SD) for neurologically normal controls was 1.16 (0.59) micromol/L], Cr+Crn was 15-29 micro mol/L [reference limit in our laboratory, 79 (38) micromol/L]. Urinary GAA was 2.4-5.8 micro mol/L [reference, 311 (191) micromol/L], and Cr+Crn was 2.1-3.3 mmol/L [reference, 9.9 (4.1) mmol/L]. We found a smaller decrease in GAA and Cr+Crn in some carriers of an AGAT defect. In the patient with GAMT deficiency, plasma and urine GAA was increased (18.6 and 1783 micromol/L, respectively), and Cr+Crn was decreased in plasma (10.7 micromol/L) and urine (2.1 mmol/L). GAA was increased in the parents' plasmas and in the mother's urine. The assessment of GAA is a new tool for the detection of both GAMT and AGAT deficiencies.

Denaturing Gradient Gel Electrophoresis for the Molecular Characterization of Six Patients with Guanidinoacetate Methyltransferase Deficiency

Guanidinoacetate methyltransferase [(GAMT); EC 2.1.1.2] deficiency is the first recognized inborn error of creatine biosynthesis, manifesting in infancy with severe neurologic symptoms such as epilepsy, mental retardation, muscular hypotonia, and progressive extrapyramidal movement disorder (1). Patients with GAMT deficiency have shown favorable responses to oral supplementation of creatine-monohydrate, but complete reversal of symptoms has not been observed (2). Biochemical findings include high urinary excretion of guanidinoacetate (the immediate precursor of creatine and substrate of GAMT), low urinary excretion of creatinine [conversion product of intracellular creatine; see Ref. (3)], and depletion of creatine in brain and muscle (4). After assessing two index patients (5)(6), we aimed to establish methods for the noninvasive molecular diagnosis of GAMT deficiency. We recently developed a radiochemical method for the determination of GAMT activity in cultured skin fibroblasts and in virus-transformed lymphoblasts (7). Here we report a denaturing gradient gel electrophoresis (DGGE) technique for the screening of mutations in the GAMT gene in DNA extracted from dried-blood spot filter-paper samples and from fibroblasts and virus-transformed lymphoblasts. Three index patients with mutations confirmed by techniques other than DGGE [P1, P2, and P6; see Refs. (5), (8)] and three new patients (P3, P4, and P5) with undetectable GAMT activity but unknown GAMT deficiency mutations (9)(10) were investigated. Details of the patient studies are summarized in Table 1⇓ , and the locations and frequencies of the mutations identified in these patients are shown in Fig. 1⇓ . We extracted DNA from dried-blood spot, filter-paper samples and from fibroblasts and lymphoblasts using …

Improving Treatment of Guanidinoacetate Methyltransferase Deficiency: Reduction of Guanidinoacetic Acid in Body Fluids by Arginine Restriction and Ornithine Supplementation

Guanidinoacetate methyltransferase (GAMT) deficiency (McKusick 601240), an inborn error of creatine biosynthesis, is characterized by creatine depletion and accumulation of guanidinoacetate (GAA) in the brain. Treatment by oral creatine supplementation had no effect on the intractable seizures. Based on the possible role of GAA as an epileptogenic agent, we evaluated a dietary treatment with arginine restriction and ornithine supplementation in order to achieve reduction of GAA. In an 8-year-old Kurdish girl with GAMT deficiency arginine intake was restricted to 15 mg/kg/day (0.4 g natural protein/kg/day) and ornithine was supplemented with 100 mg/kg/day over a period of 14 months. The diet was enriched with 0.4 g/kg/day of arginine-free essential amino acid mixture and creatine treatment remained unchanged (1.1 g/kg/day). Guanidino compounds in blood, urine, and CSF were measured by means of cation-exchange chromatography. The combination of arginine restriction and ornithine supplementation led to a substantial and permanent decrease of arginine without disturbance of nitrogen detoxification. Formation of GAA was effectively reduced after 4 weeks of treatment and sustained thereafter. Biochemical effects were accompanied by a marked clinical improvement. Distinctly reduced epileptogenic activities in electroencephalography accompanied by almost complete disappearance of seizures demonstrates the positive effect of GAA reduction. This indicates for the first time that GAA may exert an important epileptogenic potential in man. Arginine restriction in combination with ornithine supplementation represents a new and rationale therapeutic approach in GAMT deficiency.

Analysis of guanidinoacetate and creatine by isotope dilution electrospray tandem mass spectrometry

Guanidinoacetate methyltransferase (GAMT) deficiency is a disorder of creatine metabolism characterized by low plasma creatine concentrations in combination with elevated guanidinoacetate (GAA) concentrations. Although rare, GAMT deficiency has been identified in children with seizures, extrapyramidal movements, developmental delay, myopathies and behavioral abnormalities. Treatment with creatine monohydrate has been proven to be effective. We describe an isotope dilution electrospray tandem mass spectrometry (ES-MS/MS) assay for the simultaneous determination of plasma GAA and creatine using multiple reaction monitoring (MRM), d 3-creatine as the internal standard and derivatization of GAA and creatine as butyl-esters. We analysed plasma of 16 healthy adults and 20 healthy children as well as three affected children. Plasma GAA concentrations were 5.02±1.84 μmol/l (mean±S.D.) in adults, 3.91±0.76 μmol/l in children age 5–10 years and 11.57, 15.16, 14.36 μmol/l in children with GAMT deficiency. Plasma creatine concentrations were 34.7±15.25 μmol/l in adults, 58.96±22.30 μmol/l in children and 5.37, 8.15, 403.5 μmol/l in two untreated children and one treated child with GAMT deficiency, respectively. GAA can also be reliably measured from filter cards, which is sufficient to make the correct diagnosis while creatine is consistently falsely elevated probably secondary to liberation of red cell creatine. In nine healthy newborn infants, GAA concentrations from filter cards were 4.83±1.43 and 5.04±1.84 μmol/l in 16 healthy adults. We conclude that isotope dilution ES-MS/MS is ideal for rapid high-throughput diagnosis of GAMT deficiency both from plasma and filter paper cards. Using this technique neonatal screening is feasible for this treatable inborn error of creatine metabolism.