In order toprovide final proof that SLC6A8 deficiency is caused by a de-fect of the creatine transporter, we transfected the wild-typeSLC6A8 open reading frame (ORF) into primary fibroblastsof a SLC6A8-deficient patient, and tested the creatine uptakecapacity
RescuePatientCell: Primary fibroblasts from a SLC6A8-deficient patient.
GeneAlteration: c.1540C>T (p.Arg514X) (previously described by Salomons et al, 2001, Am J Hum Genet, 68: 1497-1500)
RescuePhenotype: Ability to transport creatine into the cell.
RescueMethod: The wild type SLC6A8 open reading frame was cloned into an expression vector with EGFP. This construct was stably transfected into primary fibroblasts from patient with creatine transporter deficiency due to a nonsense variant, c.1540C>T (p.Arg514X) in SLC6A8 (previously described in PMD 11326334). Expression of the SLC6A8 fusion protein or EGFP protein (empty vector) was confirmed by the presence of green fluorescence. Expression was also confirmed by RT-PCR. Transfected cells were incubated for 24 hours with 25 umol/L creatine, 500 umol/L creatine, or 500 umol/L creatine plus 500 umol/L guanidinoproprionate (creatine transporter inhibitor). Intracellular creatine level was measured by GC-MS.
RescueEvidence: When the wild type SLC6A8 open reading frame was expressed in primary fibroblasts from patient with creatine tarnsporter deficiency, due to a nonsense variant, c.1540C>T (p.Arg514X) in SLC6A8 (previously described in PMID 11326334), creatine uptake was restored to a level comparable with controls.
RescueVariants: ClinVarID: 11696