ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Amidinotransferases , Amidinotransferases/deficiency , Amino Acid Metabolism, Inborn Errors , Creatine , Creatine/deficiency , Guanidinoacetate N-Methyltransferase , Intellectual Disability , Language Development Disorders , Movement Disorders/congenital , Nerve Tissue Proteins , Plasma Membrane Neurotransmitter Transport Proteins , Plasma Membrane Neurotransmitter Transport Proteins/deficiency , Speech Disorders , Humans , Guanidinoacetate N-Methyltransferase/deficiency , Guanidinoacetate N-Methyltransferase/genetics , Creatine/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Amidinotransferases/genetics , Amidinotransferases/metabolism , Mental Retardation, X-Linked/genetics , Mental Retardation, X-Linked/diagnosis , Mutation , Brain Diseases, Metabolic, Inborn/genetics , Brain Diseases, Metabolic, Inborn/diagnosis , Phenotype , Data Curation , Developmental DisabilitiesABSTRACT
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.
Subject(s)
Brain Diseases, Metabolic, Inborn , Creatine , Creatine/deficiency , Creatinine , Mental Retardation, X-Linked , Plasma Membrane Neurotransmitter Transport Proteins , Plasma Membrane Neurotransmitter Transport Proteins/deficiency , Humans , Creatine/blood , Creatine/urine , Creatinine/blood , Creatinine/urine , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Plasma Membrane Neurotransmitter Transport Proteins/blood , Male , Female , Mental Retardation, X-Linked/genetics , Mental Retardation, X-Linked/blood , Mental Retardation, X-Linked/diagnosis , Child , Child, Preschool , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/blood , Nerve Tissue Proteins/deficiency , Infant , Adolescent , Membrane Transport Proteins/genetics , Membrane Transport Proteins/deficiency , Membrane Transport Proteins/blood , AdultABSTRACT
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.
Subject(s)
Creatine , Mutation, Missense , Humans , Creatine/metabolism , Mutation , Biological Transport , Nerve Tissue Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Plasma Membrane Neurotransmitter Transport Proteins/metabolismABSTRACT
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.
Subject(s)
Brain Diseases, Metabolic, Inborn , Creatine/deficiency , Electroencephalography , Mental Retardation, X-Linked , Male , Humans , Child, Preschool , Mutation , Seizures/diagnosis , Seizures/drug therapy , Seizures/genetics , Nerve Tissue Proteins , Plasma Membrane Neurotransmitter Transport Proteins/geneticsABSTRACT
(1) Background: X-linked creatine transporter deficiency (CTD) (OMIM 300036) is a rare group of inherited metabolic disorders characterized by global developmental delay/intellectual disability (GDD/ID), seizures, autistic behavior, and movement disorders. Pathogenic variants in the SLC6A8 gene, located at Xq28, are causative of the disease, leading to impaired creatine transport into the brain. Supplementation with creatine and its precursors, glycine and arginine, has been attempted, yet the treatment efficacy remains controversial. (2) Methods: Here we report a de novo SLC6A8 variant in a boy aged 3 years 9 months presenting with GDD, autistic behavior, and epilepsy. Elevated urinary creatine/creatinine ratio and diminished creatine peak on brain MR spectroscopy suggested the diagnosis of CTD. Genetic sequencing revealed a de novo hemizygous frameshift variant (NM_005629: c.1136_1137del, p. Glu379ValfsTer85). Creatine supplementation therapy was initiated after definitive diagnosis. Electroencephalography and MR spectroscopy were monitored during follow-up in concurrence with neuropsychological evaluations. The clinical phenotype and treatment response of CTD were summarized by systematic view of the literature. (3) Results: In silico analysis showed this variant to be deleterious, probably interfering with substrate binding and conformational changes during creatine transport. Creatine supplementation therapy led to seizure cessation and modest cognitive improvement after half-year's treatment. (4) Conclusions: This case highlights the importance of MR spectroscopy and metabolic screening in males with GDD/ID, allowing for early diagnosis and therapeutic intervention. Mechanistic understanding and case-per-se analysis are required to enable precision treatment for the patients.
ABSTRACT
A range of experiments suggests that creatine, a molecule known for recycling ATP in muscle and brain tissue, may also function as a neurotransmitter in the central nervous system.
Subject(s)
Central Nervous System , Creatine , Brain , MusclesABSTRACT
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.
Subject(s)
Brain Diseases, Metabolic, Inborn , Intellectual Disability , Mental Retardation, X-Linked , Male , Humans , Female , Intellectual Disability/genetics , Creatine , Brain Diseases, Metabolic, Inborn/diagnosis , Brain Diseases, Metabolic, Inborn/genetics , Brain Diseases, Metabolic, Inborn/drug therapy , Mental Retardation, X-Linked/diagnosis , Mental Retardation, X-Linked/genetics , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Nerve Tissue ProteinsABSTRACT
Background: Creatine transporter deficiency (CTD) is a rare X-linked disorder of creatine transport caused by pathogenic variants in SLC6A8 (Xq28). The disorder is marked by developmental delay, especially speech delay. The biomarkers Aß40, Aß42 and total tau are abnormal in Alzheimer disease (AD), a common neurodegenerative disorder pathologically characterized by Aß peptide containing amyloid plaques and tau neurofibrillary tangles. Although CTD results in neuronal energy deficiency, the pathological processes underlying the CTD phenotype are not fully characterized. Methods: Cerebral spinal fluid (CSF) was collected as an optional part of a natural history study of CTD. Aß40, Aß42 and total tau levels were quantified in CSF from individuals with CTD and from age-appropriate comparison samples. Neuro3-Plex enzyme-linked immunoassay was performed on a Quanterix SR-X instrument. The Vineland Adaptive Behavior Scale, 3rd Edition was used to determine an overall Adaptive Behavior Composite (ABC) standard score. Results: CSF from 12 individuals with CTD and 23 age appropriate non-CTD comparison samples were analyzed. We found that levels of total tau [t(32) = 4.05, p = 0.0003], Aß40 [t(31) = 6.11, p < 0.0001], and Aß42 [t(32) = 3.20, p = 0.003] were elevated in the participants with CTD relative to the comparison group. Additionally, except for one individual that we considered an outlier, all three biomarkers correlated inversely with the adaptive behavior score (total tau: ρ = -0.60 [-0.88, 0.005]; Aß40: ρ = -0.67 [-0.91, -0.12]; Aß42: ρ = -0.62 [-0.89, -0.02]). Conclusion: We describe here the novel finding of elevated protein biomarkers in the CSF of individuals with CTD. Aß40, Aß42 and total tau are markedly elevated in individuals with CTD compared to comparison samples, and increased levels of these biomarkers inversely correlated with ABC scores. We hypothesize that elevated CSF levels of Aß40 and Aß42 are due to cellular energy deficiency. Elevated CSF total tau levels may indicate ongoing neuronal damage. The observed inverse correlation of Vineland ABC scores with increased biomarker levels needs to be confirmed in a larger CTD cohort; however, our observation of increased Aß40, Aß42 and total tau levels in CSF from individuals with CTD may provide insight into pathological mechanisms contributing to the CTD phenotype and may prove useful as supportive data in future therapeutic trials.
ABSTRACT
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.
Subject(s)
Creatine , Rare Diseases , Humans , Rare Diseases/genetics , Introns , Algorithms , NucleotidesABSTRACT
Pancreatic cancer (PC) is featured with low survival rate and poor outcomes. Herein, we found that the expression of caspase-recruitment domain-containing protein 9 (CARD9), predominantly expressed in innate immune cells, was positively related to the prognosis of PC patients. CARD9-deficient PC mice exhibited rapider cancer progression and poorer survival rate. CARD9 knockout decreased dendritic cell (DC) maturation and impaired DC ability to activate T cells in vivo and in vitro. Adoptive DC transfer confirmed that the role of CARD9 deficiency in PC relied on DCs. Creatine was identified as the most significant differential metabolite between WT DCs and CARD9-/- DCs wherein it played an essential role in maintaining DC maturation and function. CARD9 deficiency led to decreased creatine levels in DCs by inhibiting the transcription of the creatine-specific transporter, solute carrier family 6 member 8 (SLC6A8). Furtherly, CARD9 deletion blocked p65 activation by abolishing the formation of CARD9-BCL10-MALT1 complex, which prevented the binding between p65 and SLC6A8 promoter. These events decreased the creatine transport into DCs, and led to DC immaturity and impairment in antitumor immunity, consequently promoting PC progression.
Subject(s)
Pancreatic Neoplasms , Signal Transduction , Animals , Mice , Creatine/metabolism , Pancreatic Neoplasms/genetics , Dendritic Cells , CARD Signaling Adaptor Proteins/metabolismABSTRACT
There is mounting evidence on the implication of SLC6A8 in the initiation and progression of human cancers. However, a comprehensive understanding of the role of SLC6A8 in pan-cancer remains elusive yet. Bioinformatics analysis was performed to investigate the expression and mutation profiles of SLC6A8 in cancers, and the association of SLC6A8 expression with cancer patients' survival and immune cell infiltration. In general, SLC6A8 is significantly upregulated across multiple cancers. SLC6A8 expression was inconsistently prognostic in different types of cancer, albeit associated with favorable survival in the vast majority of cancers. The receiver operating characteristic curves showed that SLC6A8 was relatively accurate in identifying possible cancer patients. The genetic alteration of SLC6A8, including mutation, amplification and deletion, was frequently present across various types of cancer. Mechanistically, SLC6A8 might be involved in tumorigenesis through "carbon metabolism" and "HIF-1 signaling pathway." Besides, SLC6A8 expression had significant correlation with immune checkpoints genes and tumor-infiltrating immune cell abundances. The present study offers a novel insight into the roles of SLC6A8 in the oncogenesis and development of multiple common cancers.
ABSTRACT
Background: Recent studies have demonstrated that creatine can promote tumor metastasis and has implications for immune cell function. SLC6A8 encodes a membrane protein that can transport creatine inside and outside the cell. However, there are currently no studies of SLC6A8 in lung adenocarcinoma (LUAD). Methods: In this study, the expression of SLC6A8 in LUAD was analyzed using the Oncomine database, the Cancer Genome Atlas (TCGA) database, and immunohistochemical staining analysis. Survival analysis of patients with LUAD was performed using the cBioPortal and the Kaplan-Meier Plotter websites and clinical follow-up data. An analysis of the association between SLC6A8 and the tumor immune microenvironment (TIME) of LUAD was performed through the TISIDB database and estimation of stromal and immune cells in malignant tumor tissues using expression data (ESTIMATE) algorithm. Then, based on the curated list of SLC6A8-related immunomodulators, three genes (NT5E, CD40LG, CD80) were selected to construct SLC6A8-related immune signatures to further evaluate the immune aspect of LUAD prognosis. Results: Our studies indicated that SLC6A8 was overexpressed in LUAD, and the high expression of SLC6A8 was associated with poor survival. Genetic alteration of SLC6A8 was also associated with a poorer prognosis. Furthermore, multivariate Cox analysis indicated that SLC6A8 could be used as an independent risk prognostic factor. Then, immune infiltration analysis indicated that SLC6A8 was also strongly associated with poor prognosis in the TIME of LUAD. A multivariate Cox proportional hazard model was then constructed, and was shown effective at identifying high-risk patients. Univariate and multivariate Cox analysis showed that the risk scoring of the model was an independent prognostic risk factor in LUAD. Conclusion: SLC6A8 may serve as a biomarker for poor prognosis in LUAD.
ABSTRACT
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.
Subject(s)
Brain Diseases, Metabolic, Inborn , Guanidinoacetate N-Methyltransferase , Intellectual Disability , Nerve Tissue Proteins , Plasma Membrane Neurotransmitter Transport Proteins , Brain Diseases, Metabolic, Inborn/diagnosis , Brain Diseases, Metabolic, Inborn/genetics , Child , Creatine/deficiency , Guanidinoacetate N-Methyltransferase/genetics , Humans , Intellectual Disability/genetics , Nerve Tissue Proteins/genetics , Plasma Membrane Neurotransmitter Transport Proteins/genetics , SyndromeABSTRACT
[This corrects the article DOI: 10.3389/fonc.2022.781903.].
ABSTRACT
BACKGROUND: Studies have demonstrated that the regulatory role of competitive endogenous RNA (ceRNA) networks is closely related to tumorigenesis, which provides new targets for tumor therapy. In this study, the focus was to explore the ceRNA networks that regulate SLC6A8 expression and their prognosis in non-small cell lung cancer (NSCLC). METHODS: Firstly, the Cancer Genome Atlas (TCGA) data combined with immunohistochemical staining was used to compare SLC6A8 expression in NSCLC tissues and normal tissues. Thereafter, samples from the immunohistochemical staining of NSCLC were integrated with clinical follow-up data for prognostic analysis. The Starbase database was employed to search for SLC6A8-targeted miRNAs and lncRNAs, and survival analysis was performed using clinical data from TCGA to obtain SLC6A8 expression and prognosis-related ceRNA networks. Finally, the prognostic and therapeutic prospects of SLC6A8 in NSCLC were further analyzed from methylation sites and the immune microenvironment. RESULTS: The study results revealed that SLC6A8 was significantly overexpressed in NSCLC tissues compared to normal tissues, and clinical follow-up data showed that the overexpression group was associated with poor prognosis. In addition, the Starbase data combined with TCGA clinical data analysis demonstrated that the AL513318.2/hsa-miR-26a-5p/SLC6A8 network regulates SLC6A8 overexpression in NSCLC and is associated with poor prognosis. Methylation analysis revealed that 11 methylation sites were closely associated with the prognosis of NSCLC. In addition, the immune prognostic risk model showed that the high-risk group was associated with a poorer prognosis than the low-risk group, despite showing a better immunotherapy outcome. CONCLUSION: In summary, the AL513318.2/hsa-miR-26a-5p/SLC6A8 network upregulates SLC6A8 expression in NSCLC and is associated with poor prognosis. Therefore it may be a prognostic biomarker of NSCLC and a potential therapeutic target.
ABSTRACT
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.
Subject(s)
Brain Diseases, Metabolic, Inborn , Mental Retardation, X-Linked , Brain/metabolism , Brain Diseases, Metabolic, Inborn/drug therapy , Brain Diseases, Metabolic, Inborn/genetics , Creatine , Guanidinoacetate N-Methyltransferase , Humans , Mental Retardation, X-Linked/drug therapy , Mental Retardation, X-Linked/genetics , SyndromeABSTRACT
The urea cycle generates arginine that is one of the major precursors for creatine biosynthesis. Here we evaluate levels of creatine and guanidinoacetate (the precursor in the synthesis of creatine) in plasma samples (ns = 207) of patients (np = 73) with different types of urea cycle disorders (ornithine transcarbamylase deficiency (ns = 22; np = 7), citrullinemia type 1 (ns = 60; np = 22), argininosuccinic aciduria (ns = 81; np = 31), arginase deficiency (ns = 44; np = 13)). The concentration of plasma guanidinoacetate positively correlated (p < 0.001, R2 = 0.64) with levels of arginine, but not with glycine in all patients with urea cycle defects, rising to levels above normal in most samples (34 out of 44) of patients with arginase deficiency. In contrast to patients with guanidinoacetate methyltransferase deficiency (a disorder of creatine synthesis characterized by elevated guanidinoacetate concentrations), creatine levels were normal (32 out of 44) or above normal (12 out of 44) in samples from patients with arginase deficiency. Creatine levels correlated significantly, but poorly (p < 0.01, R2 = 0.1) with guanidinoacetate levels and, despite being overall in the normal range in patients with all other urea cycle disorders, were occasionally below normal in some patients with argininosuccinic acid synthase and lyase deficiency. Creatine levels positively correlated with levels of methionine (p < 0.001, R2 = 0.16), the donor of the methyl group for creatine synthesis. The direct correlation of arginine levels with guanidinoacetate in patients with urea cycle disorders explains the increased concentration of guanidino compounds in arginase deficiency. Low creatine levels in some patients with other urea cycle defects might be explained by low protein intake (creatine is naturally present in meat) and relative or absolute intracellular arginine deficiency.
ABSTRACT
BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with poor prognosis and limited treatment options. Hypoxia is a key hallmark of TNBC. Metabolic adaptation promotes progression of TNBC cells that are located within the hypoxic tumor regions. However, it is not well understood regarding the precise molecular mechanisms underlying the regulation of metabolic adaptions by hypoxia. METHODS: RNA sequencing was performed to analyze the gene expression profiles in MDA-MB-231 cell line (20% O2 and 1% O2). Expressions of Slc6a8, which encodes the creatine transporter protein, were detected in breast cancer cells and tissues by quantitative real-time PCR. Immunohistochemistry was performed to detect SLC6A8 protein abundances in tumor tissues. Clinicopathologic correlation and overall survival were evaluated by chi-square test and Kaplan-Meier analysis, respectively. Cell viability assay and flow cytometry analysis with Annexin V/PI double staining were performed to investigate the impact of SLC6A8-mediated uptake of creatine on viability of hypoxic TNBC cells. TNBC orthotopic mouse model was used to evaluate the effects of creatine in vivo. RESULTS: SLC6A8 was aberrantly upregulated in TNBC cells in hypoxia. SLC6A8 was drastically overexpressed in TNBC tissues and its level was tightly associated with advanced TNM stage, higher histological grade and worse overall survival of TNBC patients. We found that SLC6A8 was transcriptionally upregulated by p65/NF-κB and mediated accumulation of intracellular creatine in hypoxia. SLC6A8-mediated accumulation of creatine promoted survival and suppressed apoptosis via maintaining redox homeostasis in hypoxic TNBC cells. Furthermore, creatine was required to facilitate tumor growth in xenograft mouse models. Mechanistically, intracellular creatine bolstered cell antioxidant defense by reducing mitochondrial activity and oxygen consumption rates to reduce accumulation of intracellular reactive oxygen species, ultimately activating AKT-ERK signaling, the activation of which protected the viability of hypoxic TNBC cells via mediating the upregulation of Ki-67 and Bcl-2, and the downregulation of Bax and cleaved Caspase-3. CONCLUSIONS: Our study indicates that SLC6A8-mediated creatine accumulation plays an important role in promoting TNBC progression, and may provide a potential therapeutic strategy option for treatment of SLC6A8 high expressed TNBC.
Subject(s)
Creatine/metabolism , Nerve Tissue Proteins/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Triple Negative Breast Neoplasms/metabolism , Animals , Cell Hypoxia/physiology , Cell Line, Tumor , Cell Proliferation/physiology , Female , Heterografts , Humans , MCF-7 Cells , Mice , Mice, Nude , Middle Aged , Oxidative Stress , Triple Negative Breast Neoplasms/pathologyABSTRACT
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.