title: Functional characterization of missense variants in the creatine transporter gene (SLC6A8): improved diagnostic application.
abstract: Creatine transporter deficiency is an X-linked mental retardation disorder caused by mutations in the creatine transporter gene (SLC6A8). So far, 20 mutations in the SLC6A8 gene have been described. We have developed a diagnostic assay to test creatine uptake in fibroblasts. Additionally, we expanded the assay to characterize novel SLC6A8 missense variants. A total of 13 variants were introduced in the SLC6A8 cDNA by site-directed mutagenesis. All variants were transiently transfected in SLC6A8-deficient fibroblasts and tested for restoration of creatine uptake in deficient primary fibroblasts. Thus, we proved that nine variants (p.Gly87Arg, p.Phe107del, p.Tyr317X, p.Asn336del, p.Cys337Trp, p.Ile347del, p.Pro390Leu, p.Arg391Trp, and p.Pro554Leu) are pathogenic mutations and four variants (p.Lys4Arg, p.Gly26Arg, p.Met560Val, and p.Val629Ile) are nonpathogenic. The present study provides an improved diagnostic tool to classify sequence variants of unknown significance.
authors.affiliation: Department of Clinical Chemistry, Metabolic Unit, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Efraim H
authors.initials: EH
authors.lastname: Rosenberg
authors.forename: Cristina
authors.initials: C
authors.lastname: Martínez Muñoz
authors.forename: Ofir T
authors.initials: OT
authors.lastname: Betsalel
authors.forename: Silvy J M
authors.initials: SJ
authors.lastname: van Dooren
authors.forename: Matilde
authors.initials: M
authors.lastname: Fernandez
authors.forename: Cornelis
authors.initials: C
authors.lastname: Jakobs
authors.forename: Ton J
authors.initials: TJ
authors.lastname: deGrauw
authors.forename: Tjitske
authors.initials: T
authors.lastname: Kleefstra
authors.forename: Charles E
authors.initials: CE
authors.lastname: Schwartz
authors.forename: Gajja S
authors.initials: GS
authors.lastname: Salomons
mesh_terms: D000465:Algorithms; D002478:Cells, Cultured; D003401:Creatine / Q000493:pharmacokinetics; D003947:Diagnostic Techniques, Radioisotope; D008401:Gas Chromatography-Mass Spectrometry; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D038901:X-Linked Intellectual Disability / Q000175:diagnosis* / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000172:deficiency / Q000235:genetics*; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000172:deficiency / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012016:Reference Values; D014162:Transfection
chemical_list: D009419:Nerve Tissue Proteins; D050480:Plasma Membrane Neurotransmitter Transport Proteins; D011993:Recombinant Fusion Proteins; C094770:SLC6A8 protein, human; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003401:Creatine