Slc6a8-Mediated Creatine Uptake and Accumulation Reprogram Macrophage Polarization via Regulating Cytokine Responses.

Macrophage polarization is accompanied by drastic changes in L-arginine metabolism. Two L-arginine catalytic enzymes, iNOS and arginase 1, are well-characterized hallmark molecules of classically and alternatively activated macrophages, respectively. The third metabolic fate of L-arginine is the generation of creatine that acts as a key source of cellular energy reserve, yet little is known about the role of creatine in the immune system. Here, genetic, genomic, metabolic, and immunological analyses revealed that creatine reprogrammed macrophage polarization by suppressing M(interferon-γ [IFN-γ]) yet promoting M(interleukin-4 [IL-4]) effector functions. Mechanistically, creatine inhibited the induction of immune effector molecules, including iNOS, by suppressing IFN-γ-JAK-STAT1 transcription-factor signaling while supporting IL-4-STAT6-activated arginase 1 expression by promoting chromatin remodeling. Depletion of intracellular creatine by ablation of the creatine transporter Slc6a8 altered macrophage-mediated immune responses in vivo. These results uncover a previously uncharacterized role for creatine in macrophage polarization by modulating cellular responses to cytokines such as IFN-γ and IL-4.

Cardiac function and energetics in mice with combined genetic augmentation of creatine and creatine kinase activity.

Improving energy provision in the failing heart by augmenting the creatine kinase (CK) system is a desirable therapeutic target. However, over-expression of the creatine transporter (CrT-OE) has shown that very high creatine levels result in cardiac hypertrophy and dysfunction. We hypothesise this is due to insufficient endogenous CK activity to maintain thermodynamically favourable metabolite ratios. If correct, then double transgenic mice (dTg) overexpressing both CrT and the muscle isoform of CK (CKM-OE) would rescue the adverse phenotype. In Study 1, overexpressing lines were crossed and cardiac function assessed by invasive haemodynamics and echocardiography. This demonstrated that CKM-OE was safe, but too few hearts had creatine in the toxic range. In Study 2, a novel CrT-OE line was generated with higher, homogeneous, creatine levels and phenotyped as before. Myocardial creatine was 4-fold higher in CrT-OE and dTg hearts compared to wildtype and was associated with hypertrophy and contractile dysfunction. The inability of dTg hearts to rescue this phenotype was attributed to downregulation of CK activity, as occurs in the failing heart. Nevertheless, combining both studies in a linear regression analysis suggests a modest positive effect of CKM over a range of creatine concentrations. In conclusion, we confirm that moderate elevation of creatine is well tolerated, but very high levels are detrimental. Correlation analysis lends support to the theory that this may be a consequence of limited CK activity. Future studies should focus on preventing CKM downregulation to unlock the potential synergy of augmenting both creatine and CK in the heart.

SLC6A8 creatine transporter deficiency can be detected by plasma creatine and creatinine concentrations.

Creatine transporter deficiency has been described with normal or uninformative levels of creatine and creatinine in plasma, while urine has been the preferred specimen type for biochemical diagnosis. We report a cohort of untreated patients with creatine transporter deficiency and abnormal plasma creatine panel results, characterized mainly by markedly decreased plasma creatinine. We conclude that plasma should be considered a viable specimen type for the biochemical diagnosis of this disorder, and abnormal results should be followed up with further confirmatory testing.

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.

Characterization of seizures and EEG findings in creatine transporter deficiency due to SLC6A8 mutation.

Seizures occur in up to 59% of boys with creatine transporter deficiency (CTD). While seizure phenotypes have been previously described, electroencephalogram (EEG) findings have only been reported in several case reports. In this prospective observational study, we report seizure characteristics and EEG findings in combination with neurobehavioral and SLC6A8 pathogenic variants in twenty males with CTD. Eighteen study participants (SP) underwent video-EEG, and seven had follow-up EEG recordings. Seizures typically occurred by age of 2 years. Thirteen (65%) had non-febrile seizures, requiring anti-seizure medications in nine. Four had febrile seizures. Seizures were bilateral tonic-clonic in 7 SP and focal impaired awareness in 5 SP; often responding to 1 to 2 antiseizure medications. EEG showed slowing in 5 SP, beta activity in 6 SP, and focal/multifocal, and/or generalized epileptiform activity in 9 SP. Follow-up EEGs in 7 SP showed emergence of epileptiform activity in 1 SP, and increased activity in 2 SP. In conclusion, seizures were frequent in our cohort but tended to respond to antiseizure medications. Longitudinal follow up provided further insight into emergence of seizures and EEG abnormalities soliciting future studies with long term follow up. Biomarkers of epileptogenicity in CTD are needed to predict seizures in this population.

Molecular Mechanism of SLC6A8 Dysfunction with c.1699T > C (p.S567P) Mutation in Cerebral Creatine Deficiency Syndromes.

Cerebral creatine deficiency syndromes (CCDS) are neurodevelopmental disorders caused by a decrease in creatine levels in the central nervous system (CNS) due to functional mutations in creatine synthetic enzymes or creatine transporter (CRT/SLC6A8). Although SLC6A8 mutations have been reported to be the most frequent cause of CCDS, sufficient treatment for patients with CCDS harboring SLC6A8 mutations has not yet been achieved. This study aimed to elucidate the molecular mechanism of SLC6A8 dysfunction caused by the c. 1699T > C missense mutation, which is thought to induce dysfunction through an unidentified mechanism. A study on SLC6A8-expressing oocytes showed that the c.1699T > C mutation decreased creatine uptake compared to that in wild-type (WT) oocytes. In addition, a kinetics study of creatine uptake revealed that the c.1699T > C mutation reduced the maximum uptake rate but not Michaelis-Menten constant. In contrast, the c.1699T > C mutation did not attenuate SLC6A8 protein levels or alter its cellular localization. Based on the SLC6A8 structure in the AlphaFold protein structure database, it is possible that the c.1699T > C mutation alters the interaction between the S567 and Y143 residues of SLC6A8, leading to decreased creatine transport function. These findings contribute to the understanding of the pathology of CCDS and to the development of strategies for CCDS treatment.

Effect of creatine administration on locomotor activity and stress response in olive flounder (Paralichthys olivaceus).

The creatine kinase system is crucial for maintaining cellular energy homeostasis and plays a role in regulating locomotor behavior in organisms, but its significance in the regulating the motionless behavior in olive flounder is limited. In the first experiment of this study, elevated levels of creatine kinase (CK) activity in the spinal cord were detected in the juvenile group (JG) flounder compared to the adult group (AG) flounder. In the second experiment, to further confirm the involvement of CK in the locomotor behavior, the adult flounder was given an intraperitoneal injection of creatine (150 mg/kg), while the flounder in the control group received a saline solution. After one week post-injection, the behavioral analysis revealed that the flounder in the creatine-treated group displayed higher levels of locomotor activity and a greater number of escape attempts in response to external stimuli when compared to the control group. However, the acute stress response, induced by intraperitoneal injection and characterized by tail beating, was significantly alleviated in the flounder in the creatine-treated group. Additionally, there was an upregulation of the UII and AchR genes in the spinal cord, as well as increased levels of UII and AchR in the muscle tissues of the creatine-treated flounder. However, a reduction in UI mRNA levels was observed in the brain of the flounder. Collectively, our data provide the evidence that the elevated enzyme activity and gene expression of creatine kinase play important roles in off-bottom swimming behavior in the JG flounder. Furthermore, administration of creatine improved the locomotor activity and alleviated the stress response in flounder, which is associated with regulation of the locomotor- and stress-related gene in the brain, spinal cord, and muscle.

Rare disease variant curation from literature: assessing gaps with creatine transport deficiency in focus.

BACKGROUND: Approximately 4-8% of the world suffers from a rare disease. Rare diseases are often difficult to diagnose, and many do not have approved therapies. Genetic sequencing has the potential to shorten the current diagnostic process, increase mechanistic understanding, and facilitate research on therapeutic approaches but is limited by the difficulty of novel variant pathogenicity interpretation and the communication of known causative variants. It is unknown how many published rare disease variants are currently accessible in the public domain. RESULTS: This study investigated the translation of knowledge of variants reported in published manuscripts to publicly accessible variant databases. Variants, symptoms, biochemical assay results, and protein function from literature on the SLC6A8 gene associated with X-linked Creatine Transporter Deficiency (CTD) were curated and reported as a highly annotated dataset of variants with clinical context and functional details. Variants were harmonized, their availability in existing variant databases was analyzed and pathogenicity assignments were compared with impact algorithm predictions. 24% of the pathogenic variants found in PubMed articles were not captured in any database used in this analysis while only 65% of the published variants received an accurate pathogenicity prediction from at least one impact prediction algorithm. CONCLUSIONS: Despite being published in the literature, pathogenicity data on patient variants may remain inaccessible for genetic diagnosis, therapeutic target identification, mechanistic understanding, or hypothesis generation. Clinical and functional details presented in the literature are important to make pathogenicity assessments. Impact predictions remain imperfect but are improving, especially for single nucleotide exonic variants, however such predictions are less accurate or unavailable for intronic and multi-nucleotide variants. Developing text mining workflows that use natural language processing for identifying diseases, genes and variants, along with impact prediction algorithms and integrating with details on clinical phenotypes and functional assessments might be a promising approach to scale literature mining of variants and assigning correct pathogenicity. The curated variants list created by this effort includes context details to improve any such efforts on variant curation for rare diseases.

X-linked creatine transporter (SLC6A8) deficiency in females: Difficult to recognize, but a potentially treatable disease.

Creatine transporter deficiency (CTD), caused by pathogenic variants in SLC6A8, is the second most common cause of X-linked intellectual disability. Symptoms include intellectual disability, epilepsy, and behavioral disorders and are caused by reduced cerebral creatine levels. Targeted treatment with oral supplementation is available, however the treatment efficacy is still being investigated. There are clinical and theoretical indications that heterozygous females with CTD respond better to supplementation treatment than hemizygous males. Unfortunately, heterozygous females with CTD often have more subtle and uncharacteristic clinical and biochemical phenotypes, rendering diagnosis more difficult. We report a new female case who presented with learning disabilities and seizures. After determining the diagnosis with molecular genetic testing confirmed by proton magnetic resonance spectroscopy (1H-MRS), the patient was treated with supplementation treatment including creatine, arginine, and glycine. After 28 months of treatment, the patient showed prominent clinical improvement and increased creatine levels in the brain. Furthermore, we provide a review of the 32 female cases reported in the current literature including a description of phenotypes, genotypes, diagnostic approaches, and effects of supplementation treatment. Based on this, we find that supplementation treatment should be tested in heterozygous female patients with CTD, and a prospective treatment underlines the importance of diagnosing these patients. The diagnosis should be suspected in a broad clinical spectrum of female patients and can only be made by molecular genetic testing. 1H-MRS of cerebral creatine levels is essential for establishing the diagnosis in females, and especially valuable when assessing variants of unknown significance.

Current and potential new treatment strategies for creatine deficiency syndromes.

Creatine deficiency syndromes (CDS) are inherited metabolic disorders caused by mutations in GATM, GAMT and SLC6A8 and mainly affect central nervous system (CNS). AGAT- and GAMT-deficient patients lack the functional brain endogenous creatine (Cr) synthesis pathway but express the Cr transporter SLC6A8 at blood-brain barrier (BBB), and can thus be treated by oral supplementation of high doses of Cr. For Cr transporter deficiency (SLC6A8 deficiency or CTD), current treatment strategies benefit one-third of patients. However, as their phenotype is not completely reversed, and for the other two-thirds of CTD patients, the development of novel more effective therapies is needed. This article aims to review the current knowledge on Cr metabolism and CDS clinical aspects, highlighting their current treatment possibilities and the most recent research perspectives on CDS potential therapeutics designed, in particular, to bring new options for the treatment of CTD.

Creatine transport and pathological changes in creatine transporter deficient mice.

The severe impact on brain function and lack of effective therapy for patients with creatine (Cr) transporter deficiency motivated the generation of three ubiquitous Slc6a8 deficient mice (-/y). While each mouse knock-out line has similar behavioral effects at 2 to 3 months of age, other features critical to the efficient use of these mice in drug discovery are unclear or lacking: the concentration of Cr in brain and heart differ widely between mouse lines, there are limited data on histopathologic changes, and no data on Cr uptake. Here, we determined survival, measured endogenous Cr and uptake of its deuterium-labeled analogue Cr-d3 using a liquid chromatography coupled with tandem mass spectrometry assay, and performed comprehensive histopathologic examination on the Slc6a8-/y mouse developed by Skelton et al. Our results show that Slc6a8-/y mice have widely varying organ-specific uptake of Cr-d3, significantly diminished growth with the exception of brain, progressive vacuolar myopathy, and markedly shortened lifespan.

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.

Functional assessment of creatine transporter in control and X-linked SLC6A8-deficient fibroblasts.

Creatine transporter is currently the focus of renewed interest with emerging roles in brain neurotransmission and physiology, and the bioenergetics of cancer metastases. We here report on amendments of a standard creatine uptake assay which might help clinical chemistry laboratories to extend their current range of measurements of creatine and metabolites in body fluids to functional enzyme explorations. In this respect, short incubation times and the use of a stable-isotope-labeled substrate (D3-creatine) preceded by a creatine wash-out step from cultured fibroblast cells by removal of fetal bovine serum (rich in creatine) from the incubation medium are recommended. Together, these measures decreased, by a first order of magnitude, creatine concentrations in the incubation medium at the start of creatine-uptake studies and allowed to functionally discriminate between 4 hemizygous male and 4 heterozygous female patients with X-linked SLC6A8 deficiency, and between this cohort of eight patients and controls. The functional assay corroborated genetic diagnosis of SLC6A8 deficiency. Gene anomalies in our small cohort included splicing site (c.912G > A [p.Ile260_Gln304del], c.778-2A > G and c.1495 + 2 T > G), substitution (c.407C > T) [p.Ala136Val] and deletion (c.635_636delAG [p.Glu212Valfs*84] and c.1324delC [p.Gln442Lysfs*21]) variants with reduced creatine transporter function validating their pathogenicity, including that of a previously unreported c.1324delC variant. The present assay adaptations provide an easy, reliable and discriminative manner for exploring creatine transporter activity and disease variations. It might apply to drug testing or other evaluations in the genetic and metabolic horizons covered by the emerging functions of creatine and its transporter, in a way, however, requiring and completed by additional studies on female patients and blood-brain barrier permeability properties of selected compounds. As a whole, the proposed assay of creatine transporter positively adds to currently existing measurements of this transporter activity, and determining on a large scale the extent of its exact suitability to detect female patients should condition in the future its transfer in clinical practice.

A novel SLC6A8 mutation associated with intellectual disabilities in a Chinese family exhibiting creatine transporter deficiency: case report.

BACKGROUND: X-linked creatine transporter deficiency (OMIM#300036,CRTR-D) is characterized by cerebral creatine deficiency, intellectual disabilities, severe speech impairment, seizures and behavioral problems. Mutations in the creatine transporter gene SLC6A8, a member of the solute-carrier family 6 mapped to Xq28, have been reported to cause the creatine transporter deficiency. CASE PRESENTATION: The proband presented at 5 yrs. 1 month of age with delays in intellectual and development, seizures and behavioral problems. A novel missense mutation, c.1181C > A (p.Thr394Lys), in the SLC6A8 gene (NM_005629.3) was detected via targeted exome sequencing, and then validated by Sanger sequencing. Multiple in silico variant effect analysis methods, including SIFT, PolyPhen2, PROVEAN, and Mutation Taster predicted that this variant was likely damaging or diseasing-causing. This hemizygous variation was also identified in the affected brother with the same clinical condition and inherited from the heterozygous carrier mother. The diagnosis was suggested by increased urinary creatine/creatinine (Cr:Crn) ratio and markedly reduced creatine content peak by brain proton magnetic resonance spectroscopy (MRS). The proband's mother became pregnant with a 3rd sibling, in whom the Sanger sequencing result of c.1181C > A was negative. CONCLUSION: The novel mutation c.1181C > A in the SLC6A8 gene reported in a Chinese family has expanded the mutation spectrum of CRTR-D. The combination of powerful new technologies such as targeted exome sequencing with thorough systematic clinical evaluation of patients will improve the diagnostic yield, and assist in genetic counselling and prenatal diagnosis for suspected genetic disorders.

Treatment outcome of creatine transporter deficiency: international retrospective cohort study.

To evaluate the outcome of current treatment for creatine transporter (CRTR) deficiency, we developed a clinical severity score and initiated an international treatment registry. An online questionnaire was completed by physicians following patients with CRTR deficiency on a treatment, including creatine and/or arginine, and/or glycine. Clinical severity score included 1) global developmental delay/intellectual disability; 2) seizures; 3) behavioural disorder. Phenotype scored 1-3 = mild; 4-6 = moderate; and 7-9 = severe. We applied the clinical severity score pre- and on-treatment. Seventeen patients, 14 males and 3 females, from 16 families were included. Four patients had severe, 6 patients had moderate, and 7 patients had a mild phenotype. The phenotype ranged from mild to severe in patients diagnosed at or before 2 years of age or older than 6 years of age. The phenotype ranged from mild to severe in patients with mildly elevated urine creatine to creatinine ratio. Fourteen patients were on the combined creatine, arginine and glycine therapy. On the combined treatment with creatine, arginine and glycine, none of the males showed either deterioration or improvements in their clinical severity score, whereas two females showed improvements in the clinical severity score. Creatine monotherapy resulted in deterioration of the clinical severity score in one male. There seems to be no correlation between phenotype and degree of elevation in urine creatine to creatinine ratio, genotype, or age at diagnosis. Combined creatine, arginine and glycine therapy might have stopped disease progression in males and improved phenotype in females.

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.

RNA sequencing of creatine transporter (SLC6A8) deficient fibroblasts reveals impairment of the extracellular matrix.

Creatine transporter (SLC6A8) deficiency is the most common cause of cerebral creatine syndromes, and is characterized by depletion of creatine in the brain. Manifestations of this X-linked disorder include intellectual disability, speech/language impairment, behavior abnormalities, and seizures. At the moment, no effective treatment is available. In order to investigate the molecular pathophysiology of this disorder, we performed RNA sequencing on fibroblasts derived from patients. The transcriptomes of fibroblast cells from eight unrelated individuals with SLC6A8 deficiency and three wild-type controls were sequenced. SLC6A8 mutations with different effects on the protein product resulted in different gene expression profiles. Differential gene expression analysis followed by gene ontology term enrichment analysis revealed that especially the expression of genes encoding components of the extracellular matrix and cytoskeleton are altered in SLC6A8 deficiency, such as collagens, keratins, integrins, and cadherins. This suggests an important novel role for creatine in the structural development and maintenance of cells. It is likely that the (extracellular) structure of brain cells is also impaired in SLC6A8-deficient patients, and future studies are necessary to confirm this and to reveal the true functions of creatine in the brain.

Distal Xq28 microdeletions: clarification of the spectrum of contiguous gene deletions involving ABCD1, BCAP31, and SLC6A8 with a new case and review of the literature.

The contiguous ABCD1/DXS1375E (BCAP31) deletion syndrome (CADDS) is a rare X-linked contiguous gene deletion syndrome with a severe clinical phenotype that includes marked delays, significant growth failure, liver dysfunction, and early death. The X-linked creatine transporter deficiency is a considerably more common and a cause of X-linked intellectual disability; however, multi-exon deletions of the creatine transporter are rare. We report the fifth case of CADDS, who also has a deletion of the X-linked creatine transporter. We also review reported cases of deletions in this region in order to clarify the clinical spectrum of contiguous microdeletions in this region.

Phenotype and genotype in 101 males with X-linked creatine transporter deficiency.

BACKGROUND: Creatine transporter deficiency is a monogenic cause of X-linked intellectual disability. Since its first description in 2001 several case reports have been published but an overview of phenotype, genotype and phenotype--genotype correlation has been lacking. METHODS: We performed a retrospective study of clinical, biochemical and molecular genetic data of 101 males with X-linked creatine transporter deficiency from 85 families with a pathogenic mutation in the creatine transporter gene (SLC6A8). RESULTS AND CONCLUSIONS: Most patients developed moderate to severe intellectual disability; mild intellectual disability was rare in adult patients. Speech language development was especially delayed but almost a third of the patients were able to speak in sentences. Besides behavioural problems and seizures, mild to moderate motor dysfunction, including extrapyramidal movement abnormalities, and gastrointestinal problems were frequent clinical features. Urinary creatine to creatinine ratio proved to be a reliable screening method besides MR spectroscopy, molecular genetic testing and creatine uptake studies, allowing definition of diagnostic guidelines. A third of patients had a de novo mutation in the SLC6A8 gene. Mothers with an affected son with a de novo mutation should be counselled about a recurrence risk in further pregnancies due to the possibility of low level somatic or germline mosaicism. Missense mutations with residual activity might be associated with a milder phenotype and large deletions extending beyond the 3' end of the SLC6A8 gene with a more severe phenotype. Evaluation of the biochemical phenotype revealed unexpected high creatine levels in cerebrospinal fluid suggesting that the brain is able to synthesise creatine and that the cerebral creatine deficiency is caused by a defect in the reuptake of creatine within the neurones.

Rescue of myocytes and locomotion through AAV2/9-2YF intracisternal gene therapy in a rat model of creatine transporter deficiency.

Creatine deficiency syndromes (CDS), caused by mutations in GATM (AGAT), GAMT, and SLC6A8, mainly affect the central nervous system (CNS). CDS show brain creatine (Cr) deficiency, intellectual disability with severe speech delay, behavioral troubles, epilepsy, and motor dysfunction. AGAT/GAMT-deficient patients lack brain Cr synthesis but express the Cr transporter SLC6A8 at the blood-brain barrier and are thus treatable by oral supplementation of Cr. In contrast, no satisfactory treatment has been identified for Cr transporter deficiency (CTD), the most frequent of CDS. We used our Slc6a8Y389C CTD rat model to develop a new AAV2/9-2YF-driven gene therapy re-establishing the functional Slc6a8 transporter in rat CNS. We show, after intra-cisterna magna AAV2/9-2YF-Slc6a8-FLAG vector injection of postnatal day 11 pups, the transduction of Slc6a8-FLAG in cerebellum, medulla oblongata, and spinal cord as well as a partial recovery of Cr in these brain regions, together with full prevention of locomotion defaults and impairment of myocyte development observed in Slc6a8Y389 C/y male rats. While more work is needed to correct those CTD phenotypes more associated with forebrain structures, this study is the first demonstrating positive effects of an AAV-driven gene therapy on CTD and thus represents a very encouraging approach to treat the so-far untreatable CTD.