Creatine Transporter Deficiency Research Articles

Cell-specific vulnerability to metabolic failure: the crucial role of parvalbumin expressing neurons in creatine transporter deficiency

Mutations in the solute carrier family 6-member 8 (Slc6a8) gene, encoding the protein responsible for cellular creatine (Cr) uptake, cause Creatine Transporter Deficiency (CTD), an X-linked neurometabolic disorder presenting with intellectual disability, autistic-like features, and epilepsy. The pathological determinants of CTD are still poorly understood, hindering the development of therapies. In this study, we generated an extensive transcriptomic profile of CTD showing that Cr deficiency causes perturbations of gene expression in excitatory neurons, inhibitory cells, and oligodendrocytes which result in remodeling of circuit excitability and synaptic wiring. We also identified specific alterations of parvalbumin-expressing (PV+) interneurons, exhibiting a reduction in cellular and synaptic density, and a hypofunctional electrophysiological phenotype. Mice lacking Slc6a8 only in PV+ interneurons recapitulated numerous CTD features, including cognitive deterioration, impaired cortical processing and hyperexcitability of brain circuits, demonstrating that Cr deficit in PV+ interneurons is sufficient to determine the neurological phenotype of CTD. Moreover, a pharmacological treatment targeted to restore the efficiency of PV+ synapses significantly improved cortical activity in Slc6a8 knock-out animals. Altogether, these data demonstrate that Slc6a8 is critical for the normal function of PV+ interneurons and that impairment of these cells is central in the disease pathogenesis, suggesting a novel therapeutic venue for CTD.

Creatine Deficiency Disorders: Phenotypes, Genotypes, Diagnosis, and Treatment Outcomes

Creatine is synthetized from arginine and glycine. There are two enzymes in the synthesis: l-arginine:glycine amidinotransferase and guanidinoacetate methyltransferase. After the synthesis, it is taken up by high-energy-requiring organs using creatine transporter. Biallelic pathogenic variants in GAMT result in guanidinoacetate methyltransferase deficiency and biallelic pathogenic variants in GATM result in l-arginine:glycine amidinotransferase deficiency. Hemizygous pathogenic variant in males and heterozygous pathogenic variant in females in SLC6A8 result in creatine transporter deficiency. Patients with these disorders present with a wide range of symptoms, including developmental delay, seizures, movement disorder, behavioral problems, and hypotonia. The diagnosis can be suspected by elevated guanidinoacetate and low creatine levels in body fluids in guanidinoacetate methyltransferase deficiency, low guanidinoacetate and low creatine levels in body fluids in l-arginine:glycine amidinotransferase deficiency, and elevated creatine-to-creatinine ratio in urine in creatine transporter deficiency in males as well as absent or significantly decreased creatine level in brain proton magnetic resonance spectroscopy. Genetic investigations such as targeted next-generation sequencing panel or exome sequencing can also identify these disorders; however, metabolite measurements and creatine in proton magnetic resonance spectroscopy are crucial to confirm the diagnosis. While all 3 disorders are currently treated with creatine supplementation, guanidinoacetate methyltransferase deficiency is also treated with ornithine supplementation and a protein- or arginine-restricted diet, and creatine transporter deficiency is treated with arginine and glycine supplementation (with no proven improvements).

Guanidinoacetate (GAA) is a potent GABAA receptor GABA mimetic: Implications for neurological disease pathology.

Impairment of excretion and enzymatic processing of nitrogen, for example, because of liver or kidney failure, or with urea cycle and creatine synthesis enzyme defects, surprisingly leads to primarily neurologic symptoms, yet the exact mechanisms remain largely mysterious. In guanidinoacetate N-methyltransferase (GAMT) deficiency, the guanidino compound guanidinoacetate (GAA) increases dramatically, including in the cerebrospinal fluid (CSF), and has been implicated in mediating the neurological symptoms in GAMT-deficient patients. GAA is synthesized by arginine-glycine amidinotransferase (AGAT), a promiscuous enzyme that not only transfers the amidino group from arginine to glycine, but also to primary amines in, for example, GABA and taurine to generate γ-guanidinobutyric acid (γ-GBA) and guanidinoethanesulfonic acid (GES), respectively. We show that GAA, γ-GBA, and GES share structural similarities with GABA, evoke GABAA receptor (GABAA R) mediated currents (whereas creatine [methylated GAA] and arginine failed to evoke discernible currents) in cerebellar granule cells in mouse brain slices and displace the high-affinity GABA-site radioligand [3 H]muscimol in total brain homogenate GABAA Rs. While γ-GBA and GES are GABA agonists and displace [3 H]muscimol (EC50 /IC50 between 10 and 40 μM), GAA stands out as particularly potent in both activating GABAA Rs (EC50 ~6μM) and also displacing the GABAA R ligand [3 H]muscimol (IC50 ~3μM) at pathophysiologically relevant concentrations. These findings stress the role of substantially elevated GAA as a primary neurotoxic agent in GAMT deficiency and we discuss the potential role of GAA in arginase (and creatine transporter) deficiency which show a much more modest increase in GAA concentrations yet share the unique hyperexcitability neuropathology with GAMT deficiency. We conclude that orthosteric activation of GABAA Rs by GAA, and potentially other GABAA R mimetic guanidino compounds (GCs) like γ-GBA and GES, interferes with normal inhibitory GABAergic neurotransmission which could mediate, and contribute to, neurotoxicity.

Cardiac involvement in creatine deficiency syndrome

Abstract Background Previous reports have demonstrated electrocardiographic and echocardiographic abnormalities consistent with early-stage cardiomyopathy in patients with creatine transporter (CrT) deficiency, but cardiac involvement has never been accurately characterized. Purpose To investigate the cardiac phenotype associated with creatine deficiency in a murine transgenic model and in patients with creatine deficiency syndrome (CDS). Methods Wild type and transgenic CrT−/y mice (n=20) were serially evaluated in vivo by electrocardiogram (EKG) and echocardiography for six months, and ex vivo by histological and biochemical analyses. Nine patients with CDS (n=4 with L-arginine:glycine amidinotransferase - AGAT- deficiency, n=5 with CrT deficiency) underwent blood samples, including cardiac biomarkers, EKG, Holter monitoring, echocardiography and cardiac magnetic resonance (CMR). Results Compared to wild type, CRT−/y mice showed a prolongation of QT interval (p=0.008) (Fig. 1), as well as significant reduction in cardiac ATP production from mitochondria (p<0.001) (Fig. 2). No differences were detected in the left ventricular systolic function, in terms of ejection fraction and global longitudinal strain, along the whole follow-up. Similarly, 3 patients with CrT deficiency also showed a prolonged QTc interval (median QTc 515 ms), while other 2 had a borderline QTc at Holter monitoring, while no tachyarrhythmias could be observed. No subject had abnormal systolic and diastolic function. At CMR, an increase of native T1 value was reported in the 4 subjects (median 1076 ms) with AGAT deficiency, 1 with an increase of extracellular volume matching with the detection of late gadolinium enhancement. Finally, subjects with AGAT deficiency showed an increase level of plasma norepinephrine. Familiar carriers (3 for AGAT and 1 for CrT) showed no notable cardiac functional alterations, except for QTc prolongation in 2 subjects. Conclusion Prolonged QT interval is observed in the murine model and in subjects with CrT deficiency. Biohumoral signs of neurohormonal activation and an initial replacement fibrosis – the latter suggested by the increase of native T1 – are identified in subjects with AGAT deficiency, but their clinical significance remains to be determined. Funding Acknowledgement Type of funding sources: None.

Abstract P217: Cardiac Involvement In Creatine Deficiency Syndrome: From Transgenic Model To Humans

Background: Electrocardiographic/echocardiographic abnormalities consistent with early-stage cardiomyopathy were previously reported in creatine transporter (CrT) deficiency. Nevertheless, cardiac involvement has never been accurately characterized. Aim: widen the current knowledge of the cardiac phenotype associated with creatine deficiency in a murine transgenic model and in patients with creatine deficiency syndrome (CDS). Methods: Wild type and transgenic CrT -/y mice (n=20) were serially evaluated in vivo by electrocardiogram (EKG) and echocardiography for 6 months, and ex vivo by histological/biochemical analyses. Nine patients with CDS (n=4 with L-arginine:glycine amidinotransferase - AGAT- deficiency, n=5 with CrT deficiency) underwent blood samples, including cardiac biomarkers, EKG, Holter monitoring, echocardiography and cardiac magnetic resonance (CMR). Results: Compared to wild type, CrT -/y mice showed a prolongation of QT interval (p=0.008) associated with a lower expression of ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 2, involved in turn in intracellular Ca 2+ homeostasis. The significant reduction in mitochondrial ATP rate production (p<0.001) was attributed to a limited availability of ketone bodies, whereas ATP synthase F1 subunit alpha was not affected. No differences were detected in the left ventricular systolic function, neither by 2D-echo nor by speckle tracking. A prolonged QTc interval, detected in 7 patients at Holter monitoring, was mainly detected in case of CrT deficiency, without evidence of tachyarrhythmias. Although no impairment of cardiac function was detected at CMR, only subjects with AGAT deficiency showed an increase level of plasmatic norepinephrine and of native T1 value - 1 with an increase of extracellular volume, matching with the detection of late gadolinium enhancement. Conclusion: Prolonged QT interval is observed in murine model - even with a slightly impaired metabolism - and in subjects with CrT deficiency. Biohumoral signs of neurohormonal activation and an initial replacement fibrosis - the latter suggested by the increase of native T1 - are identified in subjects with AGAT deficiency, but their clinical significance remains to be determined.

An International Cross-Sectional Survey to Characterize the Humanistic Burden of Creatine Transporter Deficiency

Aim: The purpose of this research was to collect caregiver-reported data on the burden of creatine transporter deficiency (CTD). Materials & methods: Following institutional review board approval, caregivers of individuals with CTD were recruited through advocacy networks and clinical sites. Interested caregivers were consented, screened and sent a survey link. The survey included questions about CTD diagnosis, management and characteristics. Results: Thirty-seven caregivers provided data for 40 patients with CTD. Results demonstrated delays in developmental milestones and deficits in speech, communication, and fine and gross motor skills. Patients saw on average six specialists before receiving a CTD diagnosis. Conclusion: The burden of CTD is substantial and there is a need for effective management and treatment.

P241 CARDIAC INVOLVEMENT IN CREATINE DEFICIENCY SYNDROME

Abstract Background Previous reports have demonstrated electrocardiographic and echocardiographic abnormalities consistent with early–stage cardiomyopathy, in patients with creatine transporter deficiency (CRT–D), but cardiac involvement has never been accurately characterized. Our aim was to widen the current knowledge of the cardiac phenotype associated with creatine deficiency in a murine transgenic model and in patients with creatine deficiency syndrome (CDS). Methods Wild type (n = 6) and transgenic CRT–/y mice (n = 6) were evaluated in vivo by electrocardiogram (EKG) and echocardiography for six months, and ex vivo by histological and biochemical analyses. Nine patients with CDS (4 with L–arginine:glycine amidinotransferase deficiency – AGAT–D, 5 with CRT–D) underwent blood samples, including cardiac biomarkers, EKG, Holter monitoring, echocardiography and cardiac magnetic resonance (CMR). Results Compared to wild–type, CRT–/y mice showed a prolongation of QT interval (p = 0.008) (Fig. 1), as well as a trend of reduction of mitochondrial function (Fig. 2). No differences were dected in the left ventricular systolic function, in terms of ejection fraction and global longitudinal strain, at different times. Aldosterone levels were also not different between the two groups. Three patients with CDS also showed a prolonged QTc interval (median QTc 515 ms), while other 3 had a borderline QTc at Holter monitoring, without tachyarrhythmias. No subject had abnormal systolic and diastolic function. At CMR, an increase of native T1 value was reported in 4 subjects (median 1076 ms), 1 with an increase of extracellular volume matching with the detection of late gadolinium enhancement. Finally, 7 subjects with CDS presented with biohumoral signs of neurohormonal activation (2 with elevated renin, 1 with increased aldosterone and 4 with increased norepinephrine levels). Familiar carriers (3 for AGAT–D and 1 for CRT–D) showed no notable cardiac functional alterations, except for QTc prolongation in 2 AGAT–D carriers. Conclusion Prolonged QT interval is observed in a murine model of CRT–D deficiency and in patients with CDS, in absence of an arrhythmic burden. No alterations in cardiac function were detected in the two setting, except for an increase of native T1 in patients with CDS suggesting an interstitial and replacement fibrosis.

EP194: Three generations of females with a heterozygous likely pathogenic variant in SLC6A8 causative of X-linked creatine transporter defect

SLC6A8-Related Disorders falls under the umbrella of cerebral creatine deficiency syndromes (CCDS). CCDS are inborn errors of creatine metabolism. SLC6A8-Related Disorders is specifically a creatine transporter deficiency. The SLC6A8 is located at Xq28 and encodes for the sodium- and chloride-dependent creatine transporter 1 that assists in transporting creatine into cells. Pathogenic variants in SLC6A8 impair the ability of the transporter protein to bring creatine into cells, resulting in a creatine shortage.

EP235: Interim results of the Vigilan observational study: clinical characteristics of creatine transporter deficiency

eP235: Interim results of the Vigilan observational study: clinical characteristics of creatine transporter deficiency

Effects of Delivering Guanidinoacetic Acid or Its Prodrug to the Neural Tissue: Possible Relevance for Creatine Transporter Deficiency.

The creatine precursor guanidinoacetate (GAA) was used as a dietary supplement in humans with no adverse events. Nevertheless, it has been suggested that GAA is epileptogenic or toxic to the nervous system. However, increased GAA content in rodents affected by guanidinoacetate methyltransferase (GAMT) deficiency might be responsible for their spared muscle function. Given these conflicting data, and lacking experimental evidence, we investigated whether GAA affected synaptic transmission in brain hippocampal slices. Incubation with 11.5 μM GAA (the highest concentration in the cerebrospinal fluid of GAMT-deficient patients) did not change the postsynaptic compound action potential. Even 1 or 2 mM had no effect, while 4 mM caused a reversible decrease in the potential. Guanidinoacetate increased creatine and phosphocreatine, but not after blocking the creatine transporter (also used by GAA). In an attempt to allow the brain delivery of GAA when there was a creatine transporter deficiency, we synthesized diacetyl guanidinoacetic acid ethyl ester (diacetyl-GAAE), a lipophilic derivative. In brain slices, 0.1 mM did not cause electrophysiological changes and improved tissue viability after blockage of the creatine transporter. However, diacetyl-GAAE did not increase creatine nor phosphocreatine in brain slices after blockage of the creatine transporter. We conclude that: (1) upon acute administration, GAA is neither epileptogenic nor neurotoxic; (2) Diacetyl-GAAE improves tissue viability after blockage of the creatine transporter but not through an increase in creatine or phosphocreatine. Diacetyl-GAAE might give rise to a GAA–phosphoGAA system that vicariates the missing creatine–phosphocreatine system. Our in vitro data show that GAA supplementation may be safe in the short term, and that a lipophilic GAA prodrug may be useful in creatine transporter deficiency.

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

IntroductionCerebral 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 methodsThis 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. ResultsThirteen 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. ConclusionIntellectual 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.

Impedimetric Sensors for Cyclocreatine Phosphate Determination in Plasma Based on Electropolymerized Poly(o-phenylenediamine) Molecularly Imprinted Polymers.

Cyclocreatine and its water-soluble derivative, cyclocreatine phosphate (CCrP), are potent cardioprotective drugs. Based on recent animal studies, CCrP, FDA-awarded Orphan Drug Designation, has a promising role in increasing the success rate of patients undergoing heart transplantation surgery by preserving donor hearts during transportation and improving the recovery of transplanted hearts in recipient patients. In addition, CCrP is under investigation as a promising treatment for creatine transporter deficiency, an X-linked inborn error resulting in a poor quality of life for both the patients and the caregiver. A newly designed molecularly imprinted polymer (MIP) material was fabricated by the anodic electropolymerization of o-phenylenediamine on screen-printed carbon electrodes and was successfully applied as an impedimetric sensor for CCrP determination to dramatically reduce the analysis time during both the clinical trial phases and drug development process. To enhance the overall performance of the proposed sensor, studies were performed to optimize the electropolymerization conditions, incubation time, and pH of the background electrolyte. Scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry were used to characterize the behavior of the developed ultrathin MIP membrane. The CCrP-imprinted polymer has a high recognition affinity for the template molecule because of the formation of 3D complementary cavities within the polymer. The developed MIP impedimetric sensor had good linearity, repeatability, reproducibility, and stability within the linear concentration range of 1 × 10–9 to 1 × 10–7 mol/L, with a low limit of detection down to 2.47 × 10–10 mol/L. To verify the applicability of the proposed sensor, it was used to quantify CCrP in spiked plasma samples.

X-linked creatine transporter deficiency results in prolonged QTc and increased sudden death risk in humans and disease model

PurposeCreatine transporter deficiency (CTD) is a rare X-linked disorder of creatine transport caused by pathogenic variants in SLC6A8 (Xq28). CTD features include developmental delay, seizures, and autism spectrum disorder. This study was designed to investigate CTD cardiac phenotype and sudden death risk. MethodsWe performed a cross-sectional analysis of CTD males between 2017 and 2020. Subjects underwent evaluation with electrocardiogram (ECG), echocardiography, and ambulatory ECG with comparable analysis in creatine transporter deficient mice (Slc6a8−/y) using ECG, echocardiography, exercise testing, and indirect calorimetry. ResultsEighteen subjects with CTD (18 males, age 7.4 [3.8] years) were evaluated: seven subjects (39%) had QTc ≥ 470 milliseconds: 510.3±29.0 vs. 448.3 ± 15.9, P < 0.0001. The QTc ≥ 470 milliseconds cohort had increased left ventricular internal dimension (diastole) ([LVIDd] Z-score: 0.22 ± 0.74, n = 7 vs. −0.93 ± 1.0, n = 11, P = 0.0059), and diminished left ventricular posterior wall dimension (diastole) ([LVPWDd, in mm]: 5.0 ± 0.6, n = 7 vs. 5.7 ± 0.8, n = 11, P = 0.0183), when compared to subjects with normal or borderline QTc prolongation. Similar ECG and echocardiographic abnormalities were seen in Slc6a8−/y mice. Additionally, Slc6a8−/y mice had diminished survival (65%). ConclusionProlonged QTc and abnormal echocardiographic parameters consistent with developing cardiomyopathy are seen in some male subjects with CTD. Slc6a8−/y mice recapitulated these cardiac abnormalities. Male CTD subjects may be at increased risk for cardiac dysfunction and sudden death.

Creatine transporter deficiency impairs stress adaptation and brain energetics homeostasis.

The creatine transporter (CrT) maintains brain creatine (Cr) levels, but the effects of its deficiency on energetics adaptation under stress remain unclear. There are also no effective treatments for CrT deficiency, the second most common cause of X-linked intellectual disabilities. Herein, we examined the consequences of CrT deficiency in brain energetics and stress-adaptation responses plus the effects of intranasal Cr supplementation. We found that CrT-deficient (CrT–/y) mice harbored dendritic spine and synaptic dysgenesis. Nurtured newborn CrT–/y mice maintained baseline brain ATP levels, with a trend toward signaling imbalance between the p-AMPK/autophagy and mTOR pathways. Starvation elevated the signaling imbalance and reduced brain ATP levels in P3 CrT–/y mice. Similarly, CrT–/y neurons and P10 CrT–/y mice showed an imbalance between autophagy and mTOR signaling pathways and greater susceptibility to cerebral hypoxia-ischemia and ischemic insults. Notably, intranasal administration of Cr after cerebral ischemia increased the brain Cr/N-acetylaspartate ratio, partially averted the signaling imbalance, and reduced infarct size more potently than intraperitoneal Cr injection. These findings suggest important functions for CrT and Cr in preserving the homeostasis of brain energetics in stress conditions. Moreover, intranasal Cr supplementation may be an effective treatment for congenital CrT deficiency and acute brain injury.

Evaluation of chronic toxicity of cyclocreatine in beagle dogs after oral gavage administration for up to 23 weeks

Cyclocreatine (LUM-001) was evaluated for chronic toxicity (23 weeks) in beagle dogs to support clinical development in patients with creatine transporter deficiency (CTD) disorder. Deionized water (vehicle control) or cyclocreatine was administered by oral gavage twice daily (12 ± 1 h apart) at 20, 40 and 75 mg/kg/dose followed by a recovery period. Due to severe toxicity, the study was terminated earlier than the planned 39 weeks of dosing. Animals in the 20, 40 and 75 mg/kg/dose groups completed 160, 106, and 55 days of dosing, respectively, followed by 30, 55 and 106 days of a recovery period, respectively. Three (25%), 7 (58%), and 7 (58%) animals were euthanized and/or found dead in the 40, 80, and 150 mg/kg/day dose groups, respectively. Clinical signs observed were inappetence, frequent emesis, stool abnormalities, weight loss, lethargy and respiratory distress. Histopathological evaluation revealed congestion, edema, cellular infiltration, fibrin, and/or hemorrhage in the lungs of all dose groups. Additionally, animals in all cyclocreatine treatment groups had perinuclear cytoplasmic vacuoles in the heart, kidneys, skeletal and smooth muscles. After the recovery period, the vacuoles were still observed in the cardiac and renal tissues. Cyclocreatine was absorbed rapidly with mean Tmax within 1 to 2 h and half-life ranged between 2.17 and 2.79 h on Day 1, however, on the final day of dosing, it ranged between 5.80 and 8.77 h (males) and 10.3 to 13.1 h (females). To conclude, in this study the lungs, kidneys, heart, skeletal and smooth muscles were identified as the target organs of cyclocreatine toxicity in beagle dogs.

The Role of Preclinical Models in Creatine Transporter Deficiency: Neurobiological Mechanisms, Biomarkers and Therapeutic Development.

Creatine (Cr) Transporter Deficiency (CTD) is an X-linked metabolic disorder, mostly caused by missense mutations in the SLC6A8 gene and presenting with intellectual disability, autistic behavior, and epilepsy. There is no effective treatment for CTD and patients need lifelong assistance. Thus, the research of novel intervention strategies is a major scientific challenge. Animal models are an excellent tool to dissect the disease pathogenetic mechanisms and drive the preclinical development of therapeutics. This review illustrates the current knowledge about Cr metabolism and CTD clinical aspects, with a focus on mainstay diagnostic and therapeutic options. Then, we discuss the rodent models of CTD characterized in the last decade, comparing the phenotypes expressed within clinically relevant domains and the timeline of symptom development. This analysis highlights that animals with the ubiquitous deletion/mutation of SLC6A8 genes well recapitulate the early onset and the complex pathological phenotype of the human condition. Thus, they should represent the preferred model for preclinical efficacy studies. On the other hand, brain- and cell-specific conditional mutants are ideal for understanding the basis of CTD at a cellular and molecular level. Finally, we explain how CTD models might provide novel insight about the pathogenesis of other disorders, including cancer.

PRO39 Caregiver Perspectives on the Humanistic Burden of Creatine Transporter Deficiency

Creatine Transporter Deficiency (CTD) is a rare, X-linked, recessive disorder resulting in creatine deficiency, primarily in the brain. Patients with CTD can experience significant developmental delays in motor skills, speech, and cognition, as well as seizures and behavioral issues. The objective of this research was to gather data on the burden of CTD from the caregiver perspective.

Targeted urine metabolomics with a graphical reporting tool for rapid diagnosis of inborn errors of metabolism.

The current diagnostic work‐up of inborn errors of metabolism (IEM) is rapidly moving toward integrative analytical approaches. We aimed to develop an innovative, targeted urine metabolomics (TUM) screening procedure to accelerate the diagnosis of patients with IEM. Urinary samples, spiked with three stable isotope‐labeled internal standards, were analyzed for 258 diagnostic metabolites with an ultra‐high performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (UHPLC‐QTOF‐MS) configuration run in positive and negative ESI modes. The software automatically annotated peaks, corrected for peak overloading, and reported peak quality and shifting. Robustness and reproducibility were satisfactory for most metabolites. Z‐scores were calculated against four age‐group‐matched control cohorts. Disease phenotypes were scored based on database metabolite matching. Graphical reports comprised a needle plot, annotating abnormal metabolites, and a heatmap showing the prioritized disease phenotypes. In the clinical validation, we analyzed samples of 289 patients covering 78 OMIM phenotypes from 12 of the 15 society for the study of inborn errors of metabolism (SSIEM) disease groups. The disease groups include disorders in the metabolism of amino acids, fatty acids, ketones, purines and pyrimidines, carbohydrates, porphyrias, neurotransmitters, vitamins, cofactors, and creatine. The reporting tool easily and correctly diagnosed most samples. Even subtle aberrant metabolite patterns as seen in mild multiple acyl‐CoA dehydrogenase deficiency (GAII) and maple syrup urine disease (MSUD) were correctly called without difficulty. Others, like creatine transporter deficiency, are illustrative of IEM that remain difficult to diagnose. We present TUM as a powerful diagnostic screening tool that merges most urinary diagnostic assays expediting the diagnostics for patients suspected of an IEM.

Use of an animal model of disease for toxicology enables identification of a juvenile no observed adverse effect level for cyclocreatine in creatine transporter deficiency

In standard general toxicology studies in two species to support clinical development, cyclocreatine, a creatine analog for the treatment of creatine transporter deficiency, caused deaths, convulsions, and/or multi-organ pathology. The potential translatability of these findings to patients was evaluated by comparing toxicity of cyclocreatine in wild-type mice to creatine transporter-deficient mice, a model of the human disease. A biodistribution study indicated greater accumulation of cyclocreatine in the brains of wild-type mice, consistent with its ability to be transported by the creatine transporter. Subsequent toxicology studies confirmed greater sensitivity of wild-type mice to cyclocreatine-induced toxicity. Exposure at the no observed adverse effect level in creatine transporter-deficient (554 μg*hr/ml) mice exceeded exposure at the maximum tolerated dose in wild-type (248 μg*hr/ml) mice. When dosed at 300 mg/kg/day for 3 months, cyclocreatine-related mortality, convulsions, and multi-organ pathology were observed in wild-type mice whereas there were no adverse findings in creatine transporter-deficient mice. Brain vacuolation was common to both strains. Although transporter-deficient mice appeared to be more sensitive, the finding had no functional correlates in this strain. The results highlight the importance of considering models of disease for toxicology in cases where they may be relevant to assessing safety in the intended patient population.

EP118 - Path to diagnosis and the caregiver’s perspective on the impact of creatine transporter deficiency

eP118 - Path to diagnosis and the caregiver’s perspective on the impact of creatine transporter deficiency