1. Functional assessment of creatine transporter in control and X-linked SLC6A8-deficient fibroblasts
- Author
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Jean-Marie Cuisset, Isabelle Kim, Marie-Adélaïde Bout, Stéphanie Moortgat, David Cheillan, Marie Joncquel-Chevalier Curt, Monique Fontaine, Soumeya Bekri, Alexandre Moerman, Guillemette Huet, Joseph Vamecq, Gilles Morin, Centre de Génétique Humaine (Institut de Pathologie et de Génétique, Charleroi), Institut de Pathologie et de Génétique, Charleroi, Cardiovasculaire, métabolisme, diabétologie et nutrition (CarMeN), Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hospices Civils de Lyon (HCL), Service Maladies Héréditaires du Métabolisme, Centre de Biologie Est, Hospices Civils de Lyon (HCL), Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National de la Recherche Agronomique (INRA)
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0301 basic medicine ,Male ,Bioenergetics ,diagnosis ,guanidinoacetate ,UPLC/tandem MS ,Endocrinology, Diabetes and Metabolism ,[SDV]Life Sciences [q-bio] ,Biochemistry ,Plasma Membrane Neurotransmitter Transport Proteins ,Cohort Studies ,chemistry.chemical_compound ,0302 clinical medicine ,Endocrinology ,tandem mass-spectrometry ,Female carriers ,Child ,Incubation ,metabolites ,chemistry.chemical_classification ,Genetics & Heredity ,slc6a8 deficiency ,D-3-creatine ,SLC6A8 gene ,Research & Experimental ,Prognosis ,urine ,3. Good health ,Child, Preschool ,RNA splicing ,Medicine ,Female ,inborn-errors ,medicine.medical_specialty ,Adolescent ,Nerve Tissue Proteins ,Creatine ,03 medical and health sciences ,Endocrinology & Metabolism ,Guanidino ,Internal medicine ,Genetics ,medicine ,Humans ,Molecular Biology ,Gene ,plasma ,Brain Diseases, Metabolic, Inborn ,Infant ,Transporter ,Fibroblasts ,X-linked disorder ,030104 developmental biology ,Enzyme ,chemistry ,Case-Control Studies ,methyltransferase gamt deficiency ,Mutation ,mental-retardation ,Mental Retardation, X-Linked ,body-fluids ,Creatine transporter ,Male hemizygotes ,030217 neurology & neurosurgery ,Fetal bovine serum ,Follow-Up Studies - Abstract
International audience; 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 (D-3-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 \textgreater A [p.Ile260_Gln304del], c.778-2A \textgreater G and c.1495 + 2 T \textgreater G), substitution (c.407C \textgreater 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.
- Published
- 2018
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