X-linked creatine-transporter gene (SLC6A8) defect: a new creatine-deficiency syndrome

Abstract

We report the first X-linked creatine-deficiency syndrome caused by a defective creatine transporter. The male index patient presented with developmental delay and hypotonia. Proton magnetic-resonance spectroscopy of his brain revealed absence of the creatine signal. However, creatine in urine and plasma was increased, and guanidinoacetate levels were normal. In three female relatives of the index patient, mild biochemical abnormalities and learning disabilities were present, to various extents. Fibroblasts from the index patient contained a hemizygous nonsense mutation in the gene SLC6A8 and were defective in creatine uptake. The three female relatives were heterozygous for this mutation in SLC6A8, which has been mapped to Xq28.

Figure 1

Identification of SLC6A8 nonsense mutations in a white family with creatine-transporter deficiency. a, Pedigree. Affected individuals are indicated by completely blackened symbols, symptomatic carriers by half-blackened symbols, and the asymptomatic carrier by the dot within a circle. b, Sequence analysis of DNA of the normal control, the mother of the index patient (individual “2-1” in panel a), and the index patient (individual “3-1” in panel a). DNA was isolated from fibroblasts by DNAzol (GibcoBRL). PCR reactions were performed with HotstarTaq (Qiagen), in a PE Applied Biosystems model 9700. PCR products were purified with PCR cleanup columns (Millipore). Subsequently, these purified PCR products were sequenced by capillary electrophoresis, on an ABI PRISM 310 with the Big Dye terminator-cycle sequencing kit (Perkin Elmer). Sequence analysis of part of exon 11—codons 512–516 (GenBank accession number NM_005629)—shows that the C→T transition, 15239C→T, results in a 514R→X replacement. The index patient (individual “3-1” in panel a) is hemizygous for this nonsense mutation, and the three females (individuals “1-1,” “2-1,” and “2-3” in panel a) are heterozygous for this nonsense mutation.

Figure 2

Creatine uptake in fibroblasts of the index patient, in fibroblasts of two carriers (expressing only the wild-type SLC6A8) and in cells of controls. a, Fibroblasts from the index patient (black), two carriers (gray), and two controls (white), cultured for 24 h in medium (HAM/F10 supplemented with 10% fetal bovine serum, penicillin, and streptavidin) that contains 25 μM creatine. Creatine was added to obtain final concentrations of 25, 125, and 500 μM, as indicated. b, Cells from the index patient, as well as cells from two carriers and from two controls, were incubated with 500 μM creatine for 24 h, in either the absence (white) or the presence (gray) of 500 μM 3-guanidinopropionate, an inhibitor of the creatine transporter. Prior to creatine measurement, the cells were washed twice with Hanks' balanced salt solution. Cell pellets were frozen until used. Protein concentrations were measured in 10% of the lysates, by the method used by Lowry. The lysates were used to measure creatine concentrations after the addition of stable isotope–labeled creatine as the internal standard. Each value represents the mean ± SD of duplicate incubations from the index patient, from two carriers, or from two controls.