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.

Evidence of an intracellular creatine-sensing mechanism that modulates creatine biosynthesis via AGAT expression in human HAP1 cells.

Cellular homeostasis of creatine (CT), integral part of the energy buffering and transducing system connecting intracellular sites of ATP production and utilization, comprises of mechanisms that increase CT, i.e., biosynthesis and cellular uptake, and CT-lowering processes, such as export and non-enzymatic conversion to creatinine. The biosynthesis of CT is controlled by negative feedback loop via suppression of the rate-limiting enzyme arginine:glycine amidinotransferase (AGAT). Although the regulatory mechanism involved is not well understood, AGAT suppression is successfully used in patients with guanidinoacetate methyltransferase (GAMT) deficiency to reduce the neurotoxic accumulation of the AGAT-mediated guanidinoacetate production by supplementing patients with CT. Utilizing the CT-dependent feedback loop for the upregulation of AGAT expression may well represent a therapeutic target for an additional CT deficiency syndrome, the CT transporter (CrT) defect, for which no effective treatment option is available so far. We have used CRISPR to tag the C-terminus of AGAT with a nanoluc luciferase (NLuc) reporter in HAP1 cells. A biphasic decay of AGAT-NLuc in response to increasing extracellular CT was observed, whereas the decrease in AGAT-NLuc expression was directly proportional to the rise in intracellular CT levels with an approximate IC50 of 1-2 mM. CRISPR generated HAP1 CrT null cells and HAP1 CrT null cells stably expressing a CrT-GFP fusion protein further demonstrated that the biphasic response to extracellular CT is mediated by a high-affinity (Km 9-10 µM) CrT dependent, saturable mechanism and a CrT independent, unsaturable uptake process. The direct response to intracellular CT suggests the existence of an intracellular CT sensing system enabling a dynamic cell response to changing CT concentration that is relevant for cellular CT homeostasis.

Evidence and Recommendation for Guanidinoacetate Methyltransferase Deficiency Newborn Screening.

Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive disorder of creatine biosynthesis due to pathogenic variants in the GAMT gene that lead to cerebral creatine deficiency and neurotoxic levels of guanidinoacetate. Untreated, GAMT deficiency is associated with hypotonia, significant intellectual disability, limited speech development, recurrent seizures, behavior problems, and involuntary movements. The birth prevalence of GAMT deficiency is likely between 0.5 and 2 per million live births. On the basis of small case series and sibling data, presymptomatic treatment with oral supplements of creatine, ornithine, and sodium benzoate, and a protein-restricted diet to reduce arginine intake, appear to substantially improve health and developmental outcomes. Without newborn screening, diagnosis typically happens after the development of significant impairment, when treatment has limited utility. GAMT deficiency newborn screening can be incorporated into the tandem-mass spectrometry screening that is already routinely used for newborn screening, with about 1 per 100 000 newborns screening positive. After a positive screen, diagnosis is established by finding an elevated guanidinoacetate concentration and low creatine concentration in the blood. Although GAMT deficiency is significantly more rare than other conditions included in newborn screening, the feasibility of screening, the low number of positive results, the relative ease of diagnosis, and the expected benefit of presymptomatic dietary therapy led to a recommendation from the Advisory Committee on Heritable Disorders in Newborns and Children to the Secretary of Health and Human Services that GAMT deficiency be added to the Recommended Uniform Screening Panel. This recommendation was accepted in January 2023.

Identification of novel variations in SLC6A8 and GAMT genes causing cerebral creatine deficiency syndrome.

Cerebral creatine deficiency syndromes (CCDSs) are a group of rare mendelian disorders mainly characterized by intellectual disability, movement anomaly, behavior disorder and seizures. SLC6A8, GAMT, and GATM are known genes responsible for CCDS. In this study, seven pediatric patients with developmental delay were recruited and submitted to a series of clinical evaluation, laboratory testing, and genetic analysis. The clinical manifestations and core biochemical indications of each child were basically consistent with the diagnosis of CCDS. Genetic diagnosis determined that all patients were positive for SLC6A8 or GAMT variation. A total of 12 variants were identified in this cohort, including six novel ones. The frequency of these variants, the Revel scores and the conservatism of the affected amino acids support their pathogenicity. Our findings expanded the mutation spectrum of CCDS disorders, and provided solid evidence for the counseling to affected families.

Method modification to reduce false positives for newborn screening of guanidinoacetate methyltransferase deficiency.

Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive disorder that results in reduced activity of guanidinoacetate methyltransferase, an accumulation of guanidinoacetate (GUAC), and a lack of cerebral creatine (CRE). Lack of CRE in the brain can cause intellectual disability, autistic-like behavior, seizures, and movement disorders. Identification at birth and immediate therapy can prevent intellectual disability and seizures. If started early in life, treatment with creatine supplements is highly effective. Because there are reliable biomarkers for GAMT deficiency, GUAC and CRE, and because the disorder is readily treatable with a significant improvement in outcomes, GAMT deficiency is an excellent candidate for newborn screening. Several programs have conducted pilot programs or started screening. An isobaric interferant of the GUAC marker has been reported which may cause false positive results. To reduce the number of false positives, a second-tier HPLC test to separate GUAC from unknown, isobaric interferants may be incorporated into the screening algorithm. New York State began screening for GAMT deficiency in October 2018 using a three-tiered screening approach. Quantification of GUAC and CRE were incorporated into routine screening for amino acids and acylcarnitines. In the first year of screening a total of 263,739 samples were tested for GAMT deficiency. Of these, 3382 required second tier testing. After second tier testing, 210 repeat specimens were requested for borderline results and 10 referrals were made to specialty care centers for confirmatory testing. In the first year of screening there were no confirmed cases of GAMT deficiency detected. To reduce the number of samples needing second tier testing and the number false positives we explored the use of a second MS transition to confirm the identity of the GUAC marker. GUAC and its internal standard are detected as butylated esters after sample preparation and derivatization. The original method used transition of the GUAC molecular ion of m/z 174.1 to a reactant ion of m/z 101.1. To confirm the identity of the GUAC marker we selected a qualifier ion of 174.1 > 73. The alternative product ion results were found to agree more closely with the second tier HPLC-MS/MS results for GUAC. It was found that the alternative transition may be used for quantification of the GUAC marker with acceptable analytical performance (linearity, accuracy, and precision). On March 5, 2020, the method of analysis for GUAC was modified to use the alternative product ion. For a comparable 6-month period, the modified method reduced the number of samples requiring second tier testing by 98%, reduced the number of borderline results requiring a repeat sample by 87.5%, and reduced the number of referrals to specialty care centers by 85%. Using the modified method, the correlation (r-squared) of the first and second tier screening results for GUAC is greater than 0.95. Since the first-tier results correlate well with the second-tier results, the second-tier screening is no longer necessary with the modified method.

Cerebral creatine deficiency disorders - A clinical, genetic and follow up study from India.

INTRODUCTION: Cerebral creatine deficiency syndromes (CCDS) are a group of potentially treatable neurometabolic disorders. The clinical, genetic profile and follow up outcome of Indian CCDS patients is presented. MATERIALS AND METHODS: This was a retrospective cohort of CCDS patients seen over six-years. Diagnosis was based either on low creatine peak on proton magnetic resonance spectroscopy (MRS) and/or genetic evaluation. RESULTS: Thirteen patients were eligible [8 creatine transporter deficiency (CTD), 4 guanidinoacetate methyltransferase (GAMT) deficiency and 1 could not be classified]. The mean (±SD) age at diagnosis was 7.2(±5.0) years. Clinical manifestations included intellectual disability (ID) with significant expressive speech delay in all. Most had significant behavior issues (8/13) and/or autism (8/13). All had history of convulsive seizures (11/13 had epilepsy; 2 patients only had febrile seizures) and 2/13 had movement disorder. Constipation was the commonest non-neurological manifestation (5/13 patients). Cranial MRI was normal in all CTD patients but showed globus pallidus hyperintensity in all four with GAMT deficiency. MRS performed in 11/13 patients, revealed abnormally low creatine peak. A causative genetic variant (novel mutation in nine) was identified in 12 patients. Three GAMT deficiency and one CTD patient reported neurodevelopmental improvement and good seizure control after creatine supplementation. CONCLUSION: Intellectual disability, disproportionate speech delay, autism, and epilepsy, were common in our CCDS patients. A normal structural neuroimaging with easily controlled febrile and/or afebrile seizures differentiated CTD from GAMT deficiency patients who had abnormal neuroimaging and often difficult to control epilepsy and movement disorder.

Creatine transporter-deficient rat model shows motor dysfunction, cerebellar alterations, and muscle creatine deficiency without muscle atrophy.

Creatine (Cr) is a nitrogenous organic acid and plays roles such as fast phosphate energy buffer to replenish ATP, osmolyte, antioxidant, neuromodulator, and as a compound with anabolic and ergogenic properties in muscle. Cr is taken from the diet or endogenously synthetized by the enzymes arginine:glycine amidinotransferase and guanidinoacetate methyltransferase, and specifically taken up by the transporter SLC6A8. Loss-of-function mutations in the genes encoding for the enzymes or the transporter cause creatine deficiency syndromes (CDS). CDS are characterized by brain Cr deficiency, intellectual disability with severe speech delay, behavioral troubles, epilepsy, and motor dysfunction. Among CDS, the X-linked Cr transporter deficiency (CTD) is the most prevalent with no efficient treatment so far. Different animal models of CTD show reduced brain Cr levels, cognitive deficiencies, and together they cover other traits similar to those of patients. However, motor function was poorly explored in CTD models, and some controversies in the phenotype exist in comparison with CTD patients. Our recently described Slc6a8Y389C knock-in rat model of CTD showed mild impaired motor function, morphological alterations in cerebellum, reduced muscular mass, Cr deficiency, and increased guanidinoacetate content in muscle, although no consistent signs of muscle atrophy. Our results indicate that such motor dysfunction co-occurred with both nervous and muscle dysfunctions, suggesting that muscle strength and performance as well as neuronal connectivity might be affected by this Cr deficiency in muscle and brain.

Intellectual Disability and Brain Creatine Deficit: Phenotyping of the Genetic Mouse Model for GAMT Deficiency.

Guanidinoacetate methyltransferase deficiency (GAMT-D) is one of three cerebral creatine (Cr) deficiency syndromes due to pathogenic variants in the GAMT gene (19p13.3). GAMT-D is characterized by the accumulation of guanidinoacetic acid (GAA) and the depletion of Cr, which result in severe global developmental delay (and intellectual disability), movement disorder, and epilepsy. The GAMT knockout (KO) mouse model presents biochemical alterations in bodily fluids, the brain, and muscles, including increased GAA and decreased Cr and creatinine (Crn) levels, which are similar to those observed in humans. At the behavioral level, only limited and mild alterations have been reported, with a large part of analyzed behaviors being unaffected in GAMT KO as compared with wild-type mice. At the cerebral level, decreased Cr and Crn and increased GAA and other guanidine compound levels have been observed. Nevertheless, the effects of Cr deficiency and GAA accumulation on many neurochemical, morphological, and molecular processes have not yet been explored. In this review, we summarize data regarding behavioral and cerebral GAMT KO phenotypes, and focus on uncharted behavioral alterations that are comparable with the clinical symptoms reported in GAMT-D patients, including intellectual disability, poor speech, and autistic-like behaviors, as well as unexplored Cr-induced cerebral alterations.

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.

[Genetic analysis and treatment for an infant with cerebral creatine deficiency syndrome type 2].

OBJECTIVE: To carry out genetic and metabolite analysis for an infant with cerebral creatine deficiency syndrome type 2 (CCDS2). METHODS: Clinical data of the child was collected. Whole-exome sequencing was carried out to identify potential variants by next generation sequencing. Candidate variants were confirmed by Sanger sequencing. Metabolites were determined by tandem mass spectrometry and magnetic resonance spectroscopy. Treatment was carried out following the diagnosis and genetic counseling for the affected family. RESULTS: Two novel heterozygous variants (c.289delC and c.392-1G>C) of the GAMT gene were identified in the proband, which were respectively inherited from her father and mother. In silico analysis suggested both variants to be pathogenic. Creatine (Cr) level of the child was very low, and cerebral guanidinoacetate (GAA) level was slightly increased. But both had recovered to normal in two weeks, and cerebral Cr level was significantly improved after two months. Intellectual and motor development of the child were significantly improved. CONCLUSION: The child was diagnosed with CCDS type 2, for which pathogenic variants of the GAMT gene may be accountable. Treatment has attained a satisfactory effect for the patient.

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.

Inhibition of myostatin prevents microgravity-induced loss of skeletal muscle mass and strength.

The microgravity conditions of prolonged spaceflight are known to result in skeletal muscle atrophy that leads to diminished functional performance. To assess if inhibition of the growth factor myostatin has potential to reverse these effects, mice were treated with a myostatin antibody while housed on the International Space Station. Grip strength of ground control mice increased 3.1% compared to baseline values over the 6 weeks of the study, whereas grip strength measured for the first time in space showed flight animals to be -7.8% decreased in strength compared to baseline values. Control mice in space exhibited, compared to ground-based controls, a smaller increase in DEXA-measured muscle mass (+3.9% vs +5.6% respectively) although the difference was not significant. All individual flight limb muscles analyzed (except for the EDL) weighed significantly less than their ground counterparts at the study end (range -4.4% to -28.4%). Treatment with myostatin antibody YN41 was able to prevent many of these space-induced muscle changes. YN41 was able to block the reduction in muscle grip strength caused by spaceflight and was able to significantly increase the weight of all muscles of flight mice (apart from the EDL). Muscles of YN41-treated flight mice weighed as much as muscles from Ground IgG mice, with the exception of the soleus, demonstrating the ability to prevent spaceflight-induced atrophy. Muscle gene expression analysis demonstrated significant effects of microgravity and myostatin inhibition on many genes. Gamt and Actc1 gene expression was modulated by microgravity and YN41 in opposing directions. Myostatin inhibition did not overcome the significant reduction of microgravity on femoral BMD nor did it increase femoral or vertebral BMD in ground control mice. In summary, myostatin inhibition may be an effective countermeasure to detrimental consequences of skeletal muscle under microgravity conditions.

Magnetic resonance imaging reveals specific anatomical changes in the brain of Agat- and Gamt-mice attributed to creatine depletion and guanidinoacetate alteration.

Arginine:glycine amidinotransferase- and guanidinoacetate methyltransferase deficiency are severe neurodevelopmental disorders. It is not known whether mouse models of disease express a neuroanatomical phenotype. High-resolution magnetic resonance imaging (MRI) with advanced image analysis was performed in perfused, fixed mouse brains encapsulated with the skull from male, 10-12 week old Agat -exc and B6J.Cg-Gamt tm1Isb mice (n = 48; n = 8 per genotype, strain). T2-weighted MRI scans were nonlinearly aligned to a 3D atlas of the mouse brain with 62 structures identified. Local differences in brain shape related to genotype were assessed by analysis of deformation fields. Creatine (Cr) and guanidinoacetate (GAA) were measured with high-performance liquid chromatography (HPLC) in brain homogenates (n = 24; n = 4 per genotype, strain) after whole-body perfusion. Cr was decreased in the brain of Agat- and Gamt mutant mice. GAA was decreased in Agat-/- and increased in Gamt-/- . Body weight and brain volume were lower in Agat-/- than in Gamt-/- . The analysis of entire brain structures revealed corpus callosum, internal capsule, fimbria and hypothalamus being different between the genotypes in both strains. Eighteen and fourteen significant peaks (local areas of difference in relative size) were found in Agat- and Gamt mutants, respectively. Comparing Agat-/- with Gamt-/- , we found changes in three brain regions, lateral septum, amygdala, and medulla. Intra-strain differences in four brain structures can be associated with Cr deficiency, while the inter-strain differences in three brain structures of the mutant mice may relate to GAA. Correlating these neuroanatomical findings with gene expression data implies the role of Cr metabolism in the developing brain and the importance of early intervention in patients with Cr deficiency syndromes.

Primary creatine deficiency syndrome as a potential missed diagnosis in children with psychomotor delay and seizure: case presentation with two novel variants and literature review.

Creatine is the main source of energy for the brain. Primary creatine deficiency syndromes (PCDSs) are inborn error of metabolism of creatine synthesis. Symptoms of central nervous system involvement are the most common clinical manifestations in these disorders. We reviewed medical records of all genetically confirmed patients diagnosed by whole exome sequencing who were referred to Myelin and Neurodegenerative Disorders Clinic, Children's Medical Center, Tehran, Iran, from May 2016 to Dec 2018. A literature review was conducted on clinical and genomic variability of PCDS to compare our patients with previously reported cases. We report two patients with creatine deficiency among a cohort of 550 registered cases out of which 200 patients had a genetically confirmed neurodegenerative disorder diagnosis. The main complain in the first patient with creatine transporter (CRTR) deficiency was seizure and genetic study in this patient identified a novel hemizygote variant of "c.92 > T; p.Pro31Leu" in the first exon of SLC6A8 gene. The second patient with guanidinoacetate methyltransferase (GAMT) deficiency had an unknown motor and speech delay as the striking manifestation and molecular assay revealed a novel homozygote variant of "c.134G > A; p.Trp45*" in the first exon of GAMT gene. PCDSs usually are associated with nonspecific neurologic symptoms. The first presented case had a mean delayed diagnosis of 5 years. Therefore, in children with unexplained neurologic features including developmental delay and/or regression, mental disability and repeated seizures without any significant findings in metabolic studies, PCDSs can be considered as a differential diagnosis and molecular analysis can be helpful for the precise diagnosis and treatment.

Muscle phenotype of AGAT- and GAMT-deficient mice after simvastatin exposure.

Statin-induced myopathy affects more than 10 million people worldwide. But discontinuation of statin treatment increases mortality and cardiovascular events. Recently, L-arginine:glycine amidinotransferase (AGAT) gene was associated with statin-induced myopathy in two populations, but the causal link is still unclear. AGAT is responsible for the synthesis of L-homoarginine (hArg) and guanidinoacetate (GAA). GAA is further methylated to creatine (Cr) by guanidinoacetate methyltransferase (GAMT). In cerebrovascular patients treated with statin, lower hArg and GAA plasma concentrations were found than in non-statin patients, indicating suppressed AGAT expression and/or activity (n = 272, P = 0.033 and P = 0.039, respectively). This observation suggests that statin-induced myopathy may be associated with AGAT expression and/or activity in muscle cells. To address this, we studied simvastatin-induced myopathy in AGAT- and GAMT-deficient mice. We found that simvastatin induced muscle damage and reduced AGAT expression in wildtype mice (myocyte diameter: 34.1 ± 1.3 µm vs 21.5 ± 1.3 µm, P = 0.026; AGAT expression: 1.0 ± 0.3 vs 0.48 ± 0.05, P = 0.017). Increasing AGAT expression levels of transgenic mouse models resulted in rising plasma levels of hArg and GAA (P < 0.01 and P < 0.001, respectively). Simvastatin-induced motor impairment was exacerbated in AGAT-deficient mice compared with AGAT-overexpressing GAMT-/- mice and therefore revealed an effect independent of Cr. But Cr supplementation itself improved muscle strength independent of AGAT expression (normalized grip strength: 55.8 ± 2.9% vs 72.5% ± 3.0%, P < 0.01). Homoarginine supplementation did not affect statin-induced myopathy in AGAT-deficient mice. Our results from clinical and animal studies suggest that AGAT expression/activity and its product Cr influence statin-induced myopathy independent of each other. The interplay between simvastatin treatment, AGAT expression and activity, and Cr seems to be complex. Further clinical pharmacological studies are needed to elucidate the underlying mechanism(s) and to evaluate whether supplementation with Cr, or possibly GAA, in patients under statin medication may reduce the risk of muscular side effects.

A novel mutation in two cousins with guanidinoacetate methyltransferase (GAMT) deficiency presented with autism.

Aydin HI, Sönmez FM. A novel mutation in two cousins with guanidinoacetate methyltransferase (GAMT) deficiency presented with autism. Turk J Pediatr 2019; 61: 92-96. Guanidinoacetate methyltransferase (GAMT) deficiency is a rare autosomal recessive disorder of creatine biosynthesis. Here, we report 9 and 10-year-old cousins with GAMT deficiency caused by a novel mutation who both exhibited neurodevelopmental retardation, seizures, behavioral problems, and autism that began during early infancy. The patients were diagnosed as having only autism and followed for years without a specific diagnosis although they had very low levels of serum creatinine for several times. A novel nonsense mutation in the GAMT gene that caused cessation of synthesis of the protein encoded by this gene was identified in these patients. GAMT deficiency is a treatable inborn error of metabolism and should be considered for all patients with hypotonia, developmental delay, seizures and autism, particularly if low serum creatinine levels are observed.

Placental creatine metabolism in cases of placental insufficiency and reduced fetal growth.

Creatine is a metabolite involved in cellular energy homeostasis. In this study, we examined placental creatine content, and expression of the enzymes required for creatine synthesis, transport and the creatine kinase reaction, in pregnancies complicated by low birthweight. We studied first trimester chorionic villus biopsies (CVBs) of small for gestational age (SGA) and appropriately grown infants (AGA), along with third trimester placental samples from fetal growth restricted (FGR) and healthy gestation-matched controls. Placental creatine and creatine precursor (guanidinoacetate-GAA) levels were measured. Maternal and cord serum from control and FGR pregnancies were also analyzed for creatine concentration. mRNA expression of the creatine transporter (SLC6A8); synthesizing enzymes arginine:glycine aminotransferase (GATM) and guanidinoacetate methyltransferase (GAMT); mitochondrial (mtCK) and cytosolic (BBCK) creatine kinases; and amino acid transporters (SLC7A1 & SLC7A2) was assessed in both CVBs and placental samples. Protein levels of AGAT (arginine:glycine aminotransferase), GAMT, mtCK and BBCK were also measured in placental samples. Key findings; total creatine content of the third trimester FGR placentae was 43% higher than controls. The increased creatine content of placental tissue was not reflected in maternal or fetal serum from FGR pregnancies. Tissue concentrations of GAA were lower in the third trimester FGR placentae compared to controls, with lower GATM and GAMT mRNA expression also observed. No differences in the mRNA expression of GATM, GAMT or SLC6A8 were observed between CVBs from SGA and AGA pregnancies. These results suggest placental creatine metabolism in FGR pregnancies is altered in late gestation. The relevance of these changes on placental bioenergetics should be the focus of future investigations.

Creatine and phosphocreatine mapping of mouse skeletal muscle by a polynomial and Lorentzian line-shape fitting CEST method.

PURPOSE: To obtain high-resolution Cr and PCr maps of mouse skeletal muscle using a polynomial and Lorentzian line-shape fitting (PLOF) CEST method. METHODS: Wild-type mice and guanidinoacetate N-methyltransferase-deficient (GAMT-/-) mice that have low Cr and PCr concentrations in muscle were used to assign the Cr and PCr peaks in the Z-spectrum at 11.7 T. A PLOF method was proposed to simultaneously extract and quantify the Cr and PCr by assuming a polynomial function for the background and 2 Lorentzian functions for the CEST peaks at 1.95 ppm and 2.5 ppm. RESULTS: The Z-spectra of phantoms revealed that PCr has 2 CEST peaks (2 ppm and 2.5 ppm), whereas Cr only showed 1 peak at 2 ppm. Comparison of the Z-spectra of wild-type and GAMT-/- mice indicated that, contrary to brain, there was no visible protein guanidinium peak in the skeletal-muscle Z-spectrum, which allowed us to extract clean PCr and Cr CEST signals. High-resolution PCr and Cr concentration maps of mouse skeletal muscle were obtained by the PLOF CEST method after calibration with in vivo MRS. CONCLUSIONS: The PLOF method provides an efficient way to map Cr and PCr concentrations simultaneously in the skeletal muscle at high MRI field.

PFN2 and GAMT as common molecular determinants of axonal Charcot-Marie-Tooth disease.

BACKGROUND: Charcot-Marie-Tooth type 2 (CMT2) neuropathy is characterised by a vast clinical and genetic heterogeneity complicating its diagnosis and therapeutic intervention. Identification of molecular signatures that are common to multiple CMT2 subtypes can aid in developing therapeutic strategies and measuring disease outcomes. METHODS: A proteomics-based approach was performed on lymphoblasts from CMT2 patients genetically diagnosed with different gene mutations to identify differentially regulated proteins. The candidate proteins were validated through real-time quantitative PCR and western blotting on lymphoblast samples of patients and controls, motor neurons differentiated from patient-derived induced pluripotent stem cells (iPSCs) and sciatic nerves of CMT2 mouse models. RESULTS: Proteomic profiling of patient lymphoblasts resulted in the identification of profilin 2 (PFN2) and guanidinoacetate methyltransferase (GAMT) as commonly downregulated proteins in different genotypes compared with healthy controls. This decrease was also observed at the transcriptional level on screening 43 CMT2 patients and 22 controls, respectively. A progressive decrease in PFN2 expression with age was observed in patients, while in healthy controls its expression increased with age. Reduced PFN2 expression was also observed in motor neurons differentiated from CMT2 patient-derived iPSCs and sciatic nerves of CMT2 mice when compared with controls. However, no change in GAMT levels was observed in motor neurons and CMT2 mouse-derived sciatic nerves. CONCLUSIONS: We unveil PFN2 and GAMT as molecular determinants of CMT2 with possible indications of the role of PFN2 in the pathogenesis and disease progression. This is the first study describing biomarkers that can boost the development of therapeutic strategies targeting a wider spectrum of CMT2 patients.

Variability of Creatine Metabolism Genes in Children with Autism Spectrum Disorder.

Creatine deficiency syndrome (CDS) comprises three separate enzyme deficiencies with overlapping clinical presentations: arginine:glycine amidinotransferase (GATM gene, glycine amidinotransferase), guanidinoacetate methyltransferase (GAMT gene), and creatine transporter deficiency (SLC6A8 gene, solute carrier family 6 member 8). CDS presents with developmental delays/regression, intellectual disability, speech and language impairment, autistic behaviour, epileptic seizures, treatment-refractory epilepsy, and extrapyramidal movement disorders; symptoms that are also evident in children with autism. The objective of the study was to test the hypothesis that genetic variability in creatine metabolism genes is associated with autism. We sequenced GATM, GAMT and SLC6A8 genes in 166 patients with autism (coding sequence, introns and adjacent untranslated regions). A total of 29, 16 and 25 variants were identified in each gene, respectively. Four variants were novel in GATM, and 5 in SLC6A8 (not present in the 1000 Genomes, Exome Sequencing Project (ESP) or Exome Aggregation Consortium (ExAC) databases). A single variant in each gene was identified as non-synonymous, and computationally predicted to be potentially damaging. Nine variants in GATM were shown to have a lower minor allele frequency (MAF) in the autism population than in the 1000 Genomes database, specifically in the East Asian population (Fisher's exact test). Two variants also had lower MAFs in the European population. In summary, there were no apparent associations of variants in GAMT and SLC6A8 genes with autism. The data implying there could be a lower association of some specific GATM gene variants with autism is an observation that would need to be corroborated in a larger group of autism patients, and with sub-populations of Asian ethnicities. Overall, our findings suggest that the genetic variability of creatine synthesis/transport is unlikely to play a part in the pathogenesis of autism spectrum disorder (ASD) in children.