ClinGen variant curation expert panel recommendations for classification of variants in GAMT, GATM and SLC6A8 for cerebral creatine deficiency syndromes.

Cerebral creatine deficiency syndromes (CCDS) are inherited metabolic phenotypes of creatine synthesis and transport. There are two enzyme deficiencies, guanidinoacetate methyltransferase (GAMT), encoded by GAMT and arginine-glycine amidinotransferase (AGAT), encoded by GATM, which are involved in the synthesis of creatine. After synthesis, creatine is taken up by a sodium-dependent membrane bound creatine transporter (CRTR), encoded by SLC6A8, into all organs. Creatine uptake is very important especially in high energy demanding organs such as the brain, and muscle. To classify the pathogenicity of variants in GAMT, GATM, and SLC6A8, we developed the CCDS Variant Curation Expert Panel (VCEP) in 2018, supported by The Clinical Genome Resource (ClinGen), a National Institutes of Health (NIH)-funded resource. We developed disease-specific variant classification guidelines for GAMT-, GATM-, and SLC6A8-related CCDS, adapted from the American College of Medical Genetics/Association of Molecular Pathology (ACMG/AMP) variant interpretation guidelines. We applied specific variant classification guidelines to 30 pilot variants in each of the three genes that have variants associated with CCDS. Our CCDS VCEP was approved by the ClinGen Sequence Variant Interpretation Working Group (SVI WG) and Clinical Domain Oversight Committee in July 2022. We curated 181 variants including 72 variants in GAMT, 45 variants in GATM, and 64 variants in SLC6A8 and submitted these classifications to ClinVar, a public variant database supported by the National Center for Biotechnology Information. Missense variants were the most common variant type in all three genes. We submitted 32 new variants and reclassified 34 variants with conflicting interpretations. We report specific phenotype (PP4) using a points system based on the urine and plasma guanidinoacetate and creatine levels, brain magnetic resonance spectroscopy (MRS) creatine level, and enzyme activity or creatine uptake in fibroblasts ranging from PP4, PP4_Moderate and PP4_Strong. Our CCDS VCEP is one of the first panels applying disease specific variant classification algorithms for an X-linked disease. The availability of these guidelines and classifications can guide molecular genetics and genomic laboratories and health care providers to assess the molecular diagnosis of individuals with a CCDS phenotype.

Creatine-mediated crosstalk between adipocytes and cancer cells regulates obesity-driven breast cancer.

Obesity is a major risk factor for adverse outcomes in breast cancer; however, the underlying molecular mechanisms have not been elucidated. To investigate the role of crosstalk between mammary adipocytes and neoplastic cells in the tumor microenvironment (TME), we performed transcriptomic analysis of cancer cells and adjacent adipose tissue in a murine model of obesity-accelerated breast cancer and identified glycine amidinotransferase (Gatm) in adipocytes and Acsbg1 in cancer cells as required for obesity-driven tumor progression. Gatm is the rate-limiting enzyme in creatine biosynthesis, and deletion in adipocytes attenuated obesity-driven tumor growth. Similarly, genetic inhibition of creatine import into cancer cells reduced tumor growth in obesity. In parallel, breast cancer cells in obese animals upregulated the fatty acyl-CoA synthetase Acsbg1 to promote creatine-dependent tumor progression. These findings reveal key nodes in the crosstalk between adipocytes and cancer cells in the TME necessary for obesity-driven breast cancer progression.

Cardiac expression and location of hexokinase changes in a mouse model of pure creatine deficiency.

Creatine kinase (CK) is considered the main phosphotransfer system in the heart, important for overcoming diffusion restrictions and regulating mitochondrial respiration. It is substrate limited in creatine-deficient mice lacking l-arginine:glycine amidinotransferase (AGAT) or guanidinoacetate N-methyltranferase (GAMT). Our aim was to determine the expression, activity, and mitochondrial coupling of hexokinase (HK) and adenylate kinase (AK), as these represent alternative energy transfer systems. In permeabilized cardiomyocytes, we assessed how much endogenous ADP generated by HK, AK, or CK stimulated mitochondrial respiration and how much was channeled to mitochondria. In whole heart homogenates, and cytosolic and mitochondrial fractions, we measured the activities of AK, CK, and HK. Lastly, we assessed the expression of the major HK, AK, and CK isoforms. Overall, respiration stimulated by HK, AK, and CK was ∼25, 90, and 80%, respectively, of the maximal respiration rate, and ∼20, 0, and 25%, respectively, was channeled to the mitochondria. The activity, distribution, and expression of HK, AK, and CK did not change in GAMT knockout (KO) mice. In AGAT KO mice, we found no changes in AK, but we found a higher HK activity in the mitochondrial fraction, greater expression of HK I, but a lower stimulation of respiration by HK. Our findings suggest that mouse hearts depend less on phosphotransfer systems to facilitate ADP flux across the mitochondrial membrane. In AGAT KO mice, which are a model of pure creatine deficiency, the changes in HK may reflect changes in metabolism as well as influence mitochondrial regulation and reactive oxygen species production.NEW & NOTEWORTHY In creatine-deficient AGAT-/- and GAMT-/- mice, the myocardial creatine kinase system is substrate limited. It is unknown whether subcellular localization and mitochondrial ADP channeling by hexokinase and adenylate kinase may compensate as alternative phosphotransfer systems. Our results show no changes in adenylate kinase, which is the main alternative to creatine kinase in heart. However, we found increased expression and activity of hexokinase I in AGAT-/- cardiomyocytes. This could affect mitochondrial regulation and reactive oxygen species production.

Altered calcium handling in cardiomyocytes from arginine-glycine amidinotransferase-knockout mice is rescued by creatine.

The creatine kinase system facilitates energy transfer between mitochondria and the major ATPases in the heart. Creatine-deficient mice, which lack arginine-glycine amidinotransferase (AGAT) to synthesize creatine and homoarginine, exhibit reduced cardiac contractility. We studied how the absence of a functional CK system influences calcium handling in isolated cardiomyocytes from AGAT-knockouts and wild-type littermates as well as in AGAT-knockout mice receiving lifelong creatine supplementation via the food. Using a combination of whole cell patch clamp and fluorescence microscopy, we demonstrate that the L-type calcium channel (LTCC) current amplitude and voltage range of activation were significantly lower in AGAT-knockout compared with wild-type littermates. Additionally, the inactivation of LTCC and the calcium transient decay were significantly slower. According to our modeling results, these changes can be reproduced by reducing three parameters in knockout mice when compared with wild-type: LTCC conductance, the exchange constant of Ca2+ transfer between subspace and cytosol, and SERCA activity. Because tissue expression of LTCC and SERCA protein were not significantly different between genotypes, this suggests the involvement of posttranslational regulatory mechanisms or structural reorganization. The AGAT-knockout phenotype of calcium handling was fully reversed by dietary creatine supplementation throughout life. Our results indicate reduced calcium cycling in cardiomyocytes from AGAT-knockouts and suggest that the creatine kinase system is important for the development of calcium handling in the heart.NEW & NOTEWORTHY Creatine-deficient mice lacking arginine-glycine amidinotransferase exhibit compromised cardiac function. Here, we show that this is at least partially due to an overall slowing of calcium dynamics. Calcium influx into the cytosol via the L-type calcium current (LTCC) is diminished, and the rate of the sarcoendoplasmic reticulum calcium ATPase (SERCA) pumping calcium back into the sarcoplasmic reticulum is slower. The expression of LTCC and SERCA did not change, suggesting that the changes are regulatory.

Increased creatine demand during pregnancy in Arginine: Glycine Amidino-Transferase deficiency: a case report.

BACKGROUND: Creatine (Cr), an amino acid derivative, is one of the most important sources of energy acting as both a spatial and temporal energy buffer through its phosphorylated analogue phosphocreatine (PCr) and creatine kinase (CK). Maternal Cr biosynthesis and metabolism seem to play an important role in pregnancy, as shown in preclinical and in healthy human pregnancy studies. Patients with Arginine:Glycine Amidino-Transferase deficiency (AGAT-d), due to the deficit of the first enzyme involved in Cr synthesis, are at a disadvantage due to their failure to synthesize Cr and their dependence on external intake, in contrast to normal subjects, where changes in Cr biosynthesis supply their needs. We report the outcomes of a pregnancy in an AGAT-d woman, and the challenge we faced in managing her treatment with oral Cr to ensure optimal conditions for her fetus. CASE PRESENTATION: A 22-year-old AGAT-d woman referred to our Institute for the management of her first conception at 11 weeks of fetal gestational age. Sonographic monitoring at 20 w GA indicated a reduction of fetal growth, in particular of the head circumference that was below the 3rd centile. Biochemical monitoring of Cr in biological fluids of the mother revealed a decline of the Cr concentrations, in particular in the urine sample, requiring prompt correction of the Cr dose. At 35 weeks of gestation the patient delivered a male infant, heterozygous for GATM mutation, with normal brain Cr levels; at one year the baby achieved typical developmental milestones. CONCLUSIONS: This rare pregnancy demonstrates that Cr levels in the blood and urine of the mother with AGAT-d decreased since the first months of gestation. The increase of the Cr daily dose administered to the mother seems to have produced beneficial effects also on the fetus.

Creatine, guanidinoacetate and homoarginine in statin-induced myopathy.

Our study evaluated the effect of creatine and homoarginine in AGAT- and GAMT-deficient mice after simvastatin exposure. Balestrino and Adriano suggest that guanidinoacetate might explain the difference between AGAT- and GAMT-deficient mice in simvastatin-induced myopathy. We agree with Balestrino and Adriano that our data shows that (1) creatine possesses a protective potential to ameliorate statin-induced myopathy in humans and mice and (2) homoarginine did not reveal a beneficial effect in statin-induced myopathy. Third, we agree that guanidinoacetate can be phosphorylated and partially compensate for phosphocreatine. In our study, simvastatin-induced damage showed a trend to be less pronounced in GAMT-deficient mice compared with wildtype mice. Therefore, (phospo) guanidinoacetate cannot completely explain the milder phenotype of GAMT-deficient mice, but we agree that it might contribute to ameliorate statin-induced myopathy in GAMT-deficient mice compared with AGAT-deficient mice. Finally, we agree with Balestino and Adriano that AGAT metabolites should further be evaluated as potential treatments in statin-induced myopathy.

Marker enzyme activities in hindleg from creatine-deficient AGAT and GAMT KO mice - differences between models, muscles, and sexes.

Creatine kinase (CK) functions as an energy buffer in muscles. Its substrate, creatine, is generated by L-arginine:glycine amidinotransferase (AGAT) and guanidinoacetate N-methyltransferase (GAMT). Creatine deficiency has more severe consequences for AGAT than GAMT KO mice. In the present study, to characterize their muscle phenotype further, we recorded the weight of tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius (GAS), plantaris (PLA) and soleus (SOL) from creatine-deficient AGAT and GAMT, KO and WT mice. In GAS, PLA and SOL representing glycolytic, intermediate and oxidative muscle, respectively, we recorded the activities of pyruvate kinase (PK), lactate dehydrogenase (LDH), citrate synthase (CS) and cytochrome oxidase (CO). In AGAT KO compared to WT mice, muscle atrophy and differences in marker enzyme activities were more pronounced in glycolytic than oxidative muscle. In GAMT KO compared to WT, the atrophy was modest, differences in PK and LDH activities were minor, and CS and CO activities were slightly higher in all muscles. SOL from males had higher CS and CO activities compared to females. Our results add detail to the characterization of AGAT and GAMT KO skeletal muscle phenotypes and illustrate the importance of taking into account differences between muscles, and differences between sexes.

Homoarginine- and Creatine-Dependent Gene Regulation in Murine Brains with l-Arginine:Glycine Amidinotransferase Deficiency.

l-arginine:glycine amidinotransferase (AGAT) and its metabolites homoarginine (hArg) and creatine have been linked to stroke pathology in both human and mouse studies. However, a comprehensive understanding of the underlying molecular mechanism is lacking. To investigate transcriptional changes in cerebral AGAT metabolism, we applied a transcriptome analysis in brains of wild-type (WT) mice compared to untreated AGAT-deficient (AGAT-/-) mice and AGAT-/- mice with creatine or hArg supplementation. We identified significantly regulated genes between AGAT-/- and WT mice in two independent cohorts of mice which can be linked to amino acid metabolism (Ivd, Lcmt2), creatine metabolism (Slc6a8), cerebral myelination (Bcas1) and neuronal excitability (Kcnip3). While Ivd and Kcnip3 showed regulation by hArg supplementation, Bcas1 and Slc6a8 were creatine dependent. Additional regulated genes such as Pla2g4e and Exd1 need further evaluation of their influence on cerebral function. Experimental stroke models showed a significant regulation of Bcas1 and Slc6a8. Together, these results reveal that AGAT deficiency, hArg and creatine regulate gene expression in the brain, which may be critical in stroke pathology.

Benefits and drawbacks of guanidinoacetic acid as a possible treatment to replenish cerebral creatine in AGAT deficiency.

Arginine-glycine amidinotransferase (AGAT) deficiency is a rare inherited metabolic disorder that severely affects brain bioenergetics. Characterized by mental retardation, language impairment, and behavioral disorders, AGAT deficiency is a treatable condition, where long-term creatine supplementation usually restores brain creatine levels and improves its clinical features. In some cases of AGAT deficiency, creatine treatment might be somewhat limited due to possible shortcomings in performance and transport of creatine to the brain. Guanidinoacetic acid (GAA), a direct metabolic precursor of creatine, has recently been suggested as a possible alternative to creatine to tackle brain creatine levels in experimental medicine. AGAT patients might benefit from oral GAA due to upgraded bioavailability and convenient utilization of the compound, while possible drawbacks (e.g. brain methylation issues, neurotoxicity, and hyperhomocysteinemia) should be accounted as well.

Impaired cardiac contractile function in arginine:glycine amidinotransferase knockout mice devoid of creatine is rescued by homoarginine but not creatine.

AIMS: Creatine buffers cellular adenosine triphosphate (ATP) via the creatine kinase reaction. Creatine levels are reduced in heart failure, but their contribution to pathophysiology is unclear. Arginine:glycine amidinotransferase (AGAT) in the kidney catalyses both the first step in creatine biosynthesis as well as homoarginine (HA) synthesis. AGAT-/- mice fed a creatine-free diet have a whole body creatine-deficiency. We hypothesized that AGAT-/- mice would develop cardiac dysfunction and rescue by dietary creatine would imply causality. METHODS AND RESULTS: Withdrawal of dietary creatine in AGAT-/- mice provided an estimate of myocardial creatine efflux of ∼2.7%/day; however, in vivo cardiac function was maintained despite low levels of myocardial creatine. Using AGAT-/- mice naïve to dietary creatine we confirmed absence of phosphocreatine in the heart, but crucially, ATP levels were unchanged. Potential compensatory adaptations were absent, AMPK was not activated and respiration in isolated mitochondria was normal. AGAT-/- mice had rescuable changes in body water and organ weights suggesting a role for creatine as a compatible osmolyte. Creatine-naïve AGAT-/- mice had haemodynamic impairment with low LV systolic pressure and reduced inotropy, lusitropy, and contractile reserve. Creatine supplementation only corrected systolic pressure despite normalization of myocardial creatine. AGAT-/- mice had low plasma HA and supplementation completely rescued all other haemodynamic parameters. Contractile dysfunction in AGAT-/- was confirmed in Langendorff perfused hearts and in creatine-replete isolated cardiomyocytes, indicating that HA is necessary for normal cardiac function. CONCLUSIONS: Our findings argue against low myocardial creatine per se as a major contributor to cardiac dysfunction. Conversely, we show that HA deficiency can impair cardiac function, which may explain why low HA is an independent risk factor for multiple cardiovascular diseases.

Fifteen-year follow-up of Italian families affected by arginine glycine amidinotransferase deficiency.

BACKGROUND: Arginine:glycine amidinotransferase deficiency (AGAT-d) is a very rare inborn error of creatine synthesis mainly characterized by absence of brain Creatine (Cr) peak, intellectual disability, severe language impairment and behavioural disorder and susceptible to supplementary Cr treatment per os. Serial examinations by magnetic resonance spectroscopy are required to evaluate Cr recovery in brain during treatment of high doses of Cr per os, which have been proved beneficial and effective in treating main clinical symptoms. A long term study with detailed reports on clinical, neurochemical and neuropsychological outcomes of the first Italian patients affected by AGAT-d here reported can represent a landmark in management of this disorder thus enhancing medical knowledge and clinical practice. RESULTS: We have evaluated the long term effects of Cr supplementation management in four Italian patients affected by AGAT-d, correlating specific treatments with serial clinical, biochemical and magnetic resonance spectroscopy examinations as well as the neuropsychological outcome by standardized developmental scales. Consecutive MRS examinations have confirmed that Cr depletion in AGAT-d patients is reversible under Cr supplementation. Cr treatment is considered safe and well tolerated but side effects, including weight gain and kidney stones, have been reported. CONCLUSIONS: Early treatment prevents adverse developmental outcome, while patients diagnosed and treated at an older age showed partial but significant cognitive recovery with clear improvements in adaptive functioning.

Laboratory diagnosis of creatine deficiency syndromes: a technical standard and guideline of the American College of Medical Genetics and Genomics.

Disclaimer: These ACMG Standards and Guidelines are intended as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these standards and guidelines is voluntary and does not necessarily assure a successful medical outcome. These Standards and Guidelines should not be considered inclusive of all proper procedures and tests or exclusive of others that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, clinical laboratory geneticists should apply their professional judgment to the specific circumstances presented by the patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether or not it is in conformance with these Standards and Guidelines. They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Cerebral creatine deficiency syndromes are neurometabolic conditions characterized by intellectual disability, seizures, speech delay, and behavioral abnormalities. Several laboratory methods are available for preliminary and confirmatory diagnosis of these conditions, including measurement of creatine and related metabolites in biofluids using liquid chromatography-tandem mass spectrometry or gas chromatography-mass spectrometry, enzyme activity assays in cultured cells, and DNA sequence analysis. These guidelines are intended to standardize these procedures to help optimize the diagnosis of creatine deficiency syndromes. While biochemical methods are emphasized, considerations for confirmatory molecular testing are also discussed, along with variables that influence test results and interpretation.Genet Med 19 2, 256-263.

Arginine-Glycine Amidinotransferase Deficiency and Functional Characterization of Missense Variants in GATM.

Arginine-glycine amidinotransferase (GATM) deficiency is an autosomal-recessive disorder caused by pathogenic variants in GATM. Clinical features include intellectual disability, hypotonia, and myopathy. Due to normal neurodevelopment in asymptomatic individuals on creatine monotherapy, GATM deficiency is a good candidate for newborn screening. To determine the carrier frequency of GATM deficiency, we performed functional characterization of rare missense variants in GATM reported as heterozygous in the Exome Variant Server database. To assess phenotype and genotype correlation, we developed a clinical severity scoring system. Two patients with mild phenotype had a nonsense missense variant. Severe phenotype was present in patients with missense as well as truncating variants. There seems to be no phenotype and genotype correlation. We cloned a novel GATM transcript. We found seven missense variants retaining 0% of wild-type GATM activity indicating putative pathogenicity. Based on our study results, high Genomic Evolutionary Rate Profiling conservation score, conserved amino acid substitution in species, and low allele frequency in exome databases would be the most sensitive in silico analysis tools to predict pathogenicity of missense variants. We present first study of the functional characterization of missense variants in GATM as well as clinical severity score of patients with GATM deficiency.

Transcriptomic and metabolic analyses reveal salvage pathways in creatine-deficient AGAT(-/-) mice.

Skeletal muscles require energy either at constant low (e.g., standing and posture) or immediate high rates (e.g., exercise). To fulfill these requirements, myocytes utilize the phosphocreatine (PCr)/creatine (Cr) system as a fast energy buffer and shuttle. We have generated mice lacking L-arginine:glycine amidino transferase (AGAT), the first enzyme of creatine biosynthesis. These AGAT(-/-) (d/d) mice are devoid of the PCr/Cr system and reveal severely altered oxidative phosphorylation. In addition, they exhibit complete resistance to diet-induced obesity, which is associated with a chronic activation of AMP-activated protein kinase in muscle and white adipose tissue. The underlying metabolic rearrangements have not yet been further analyzed. Here, we performed gene expression analysis in skeletal muscle and a serum amino acid profile of d/d mice revealing transcriptomic and metabolic alterations in pyruvate and glucose pathways. Differential pyruvate tolerance tests demonstrated preferential conversion of pyruvate to alanine, which was supported by increased protein levels of enzymes involved in pyruvate and alanine metabolism. Pyruvate tolerance tests suggested severely impaired hepatic gluconeogenesis despite increased availability of pyruvate and alanine. Furthermore, enzymes of serine production and one-carbon metabolism were significantly up-regulated in d/d mice, indicating increased de novo formation of one-carbon units from carbohydrate metabolism linked to NAD(P)H production. Besides the well-established function of the PCr/Cr system in energy metabolism, our transcriptomic and metabolic analyses suggest that it plays a pivotal role in systemic one-carbon metabolism, oxidation/reduction, and biosynthetic processes. Therefore, the PCr/Cr system is not only an energy buffer and shuttle, but also a crucial component involved in numerous systemic metabolic processes.

Creatine synthesis and exchanges between brain cells: What can be learned from human creatine deficiencies and various experimental models?

While it has long been thought that most of cerebral creatine is of peripheral origin, the last 20 years has provided evidence that the creatine synthetic pathway (AGAT and GAMT enzymes) is expressed in the brain together with the creatine transporter (SLC6A8). It has also been shown that SLC6A8 is expressed by microcapillary endothelial cells at the blood-brain barrier, but is absent from surrounding astrocytes, raising the concept that the blood-brain barrier has a limited permeability for peripheral creatine. The first creatine deficiency syndrome in humans was also discovered 20 years ago (GAMT deficiency), followed later by AGAT and SLC6A8 deficiencies, all three diseases being characterized by creatine deficiency in the CNS and essentially affecting the brain. By reviewing the numerous and latest experimental studies addressing creatine transport and synthesis in the CNS, as well as the clinical and biochemical characteristics of creatine-deficient patients, our aim was to delineate a clearer view of the roles of the blood-brain and blood-cerebrospinal fluid barriers in the transport of creatine and guanidinoacetate between periphery and CNS, and on the intracerebral synthesis and transport of creatine. This review also addresses the question of guanidinoacetate toxicity for brain cells, as probably found under GAMT deficiency.

Creatine biosynthesis and transport in health and disease.

Creatine is physiologically provided equally by diet and by endogenous synthesis from arginine and glycine with successive involvements of arginine glycine amidinotransferase [AGAT] and guanidinoacetate methyl transferase [GAMT]. A specific plasma membrane transporter, creatine transporter [CRTR] (SLC6A8), further enables cells to incorporate creatine and through uptake of its precursor, guanidinoacetate, also directly contributes to creatine biosynthesis. Breakthrough in the role of creatine has arisen from studies on creatine deficiency disorders. Primary creatine disorders are inherited as autosomal recessive (mutations affecting GATM [for glycine-amidinotransferase, mitochondrial]) and GAMT genes) or X-linked (SLC6A8 gene) traits. They have highlighted the role of creatine in brain functions altered in patients (global developmental delay, intellectual disability, behavioral disorders). Creatine modulates GABAergic and glutamatergic cerebral pathways, presynaptic CRTR (SLC6A8) ensuring re-uptake of synaptic creatine. Secondary creatine disorders, addressing other genes, have stressed the extraordinary imbrication of creatine metabolism with many other cellular pathways. This high dependence on multiple pathways supports creatine as a cellular sensor, to cell methylation and energy status. Creatine biosynthesis consumes 40% of methyl groups produced as S-adenosylmethionine, and creatine uptake is controlled by AMP activated protein kinase, a ubiquitous sensor of energy depletion. Today, creatine is considered as a potential sensor of cell methylation and energy status, a neurotransmitter influencing key (GABAergic and glutamatergic) CNS neurotransmission, therapeutic agent with anaplerotic properties (towards creatine kinases [creatine-creatine phosphate cycle] and creatine neurotransmission), energetic and antioxidant compound (benefits in degenerative diseases through protection against energy depletion and oxidant species) with osmolyte behavior (retention of water by muscle). This review encompasses all these aspects by providing an illustrated metabolic account for brain and body creatine in health and disease, an algorithm to diagnose metabolic and gene bases of primary and secondary creatine deficiencies, and a metabolic exploration by (1)H-MRS assessment of cerebral creatine levels and response to therapeutic measures.

Arginine:glycine amidinotransferase (AGAT) deficiency: Clinical features and long term outcomes in 16 patients diagnosed worldwide.

BACKGROUND: Arginine:glycine aminotransferase (AGAT) (GATM) deficiency is an autosomal recessive inborn error of creative synthesis. OBJECTIVE: We performed an international survey among physicians known to treat patients with AGAT deficiency, to assess clinical characteristics and long-term outcomes of this ultra-rare condition. RESULTS: 16 patients from 8 families of 8 different ethnic backgrounds were included. 1 patient was asymptomatic when diagnosed at age 3 weeks. 15 patients diagnosed between 16 months and 25 years of life had intellectual disability/developmental delay (IDD). 8 patients also had myopathy/proximal muscle weakness. Common biochemical denominators were low/undetectable guanidinoacetate (GAA) concentrations in urine and plasma, and low/undetectable cerebral creatine levels. 3 families had protein truncation/null mutations. The rest had missense and splice mutations. Treatment with creatine monohydrate (100-800 mg/kg/day) resulted in almost complete restoration of brain creatine levels and significant improvement of myopathy. The 2 patients treated since age 4 and 16 months had normal cognitive and behavioral development at age 10 and 11 years. Late treated patients had limited improvement of cognitive functions. CONCLUSION: AGAT deficiency is a treatable intellectual disability. Early diagnosis may prevent IDD and myopathy. Patients with unexplained IDD with and without myopathy should be assessed for AGAT deficiency by determination of urine/plasma GAA and cerebral creatine levels (via brain MRS), and by GATM gene sequencing.

Biosynthesis of homoarginine (hArg) and asymmetric dimethylarginine (ADMA) from acutely and chronically administered free L-arginine in humans.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis, whereas L-arginine (Arg) and L-homoarginine (hArg) serve as substrates for NO synthesis. ADMA and other methylated arginines are generally believed to exclusively derive from guanidine (N (G))-methylated arginine residues in proteins by protein arginine methyltransferases (PRMTs) that use S-adenosylmethionine (SAM) as the methyl donor. L-Lysine is known for decades as a precursor for hArg, but only recent studies indicate that arginine:glycine amidinotransferase (AGAT) is responsible for the synthesis of hArg. AGAT catalyzes the formation of guanidinoacetate (GAA) that is methylated to creatine by guanidinoacetate methyltransferase (GAMT) which also uses SAM. The aim of the present study was to learn more about the mechanisms of ADMA and hArg formation in humans. Especially, we hypothesized that ADMA is produced by N (G)-methylation of free Arg in addition to the known PRMTs-involving mechanism. In knockout mouse models of AGAT- and GAMT-deficiency, we investigated the contribution of these enzymes to hArg synthesis. Arg infusion (0.5 g/kg, 30 min) in children (n = 11) and ingestion of high-fat protein meals by overweight men (n = 10) were used to study acute effects on ADMA and hArg synthesis. Daily Arg ingestion (10 g) or placebo for 3 or 6 months by patients suffering from peripheral arterial occlusive disease (PAOD, n = 20) or coronary artery disease (CAD, n = 30) was used to study chronic effects of Arg on ADMA synthesis. Mass spectrometric methods were used to measure all biochemical parameters in plasma and urine samples. In mice, AGAT but not GAMT was found to contribute to plasma hArg, while ADMA synthesis was independent of AGAT and GAMT. Arg infusion acutely increased plasma Arg, hArg and ADMA concentrations, but decreased the plasma hArg/ADMA ratio. High-fat protein meals acutely increased plasma Arg, hArg, ADMA concentrations, as well as the plasma hArg/ADMA ratio. In the PAOD and CAD studies, plasma Arg concentration increased in the verum compared to the placebo groups. Plasma ADMA concentration increased only in the PAOD patients who received Arg. Our study suggests that in humans a minor fraction of free Arg is rapidly metabolized to ADMA and hArg. In mice, GAMT and N (G)-methyltransferases contribute to ADMA and hArg synthesis from Arg, whereas AGAT is involved in the synthesis of hArg but not of ADMA. The underlying biochemical mechanisms remain still elusive.