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SLC6A8 deficiency is caused by mutations in the X-linked creatine transporter gene (SLC6A8), which leads to cerebral creatine deficiency, mental retardation, speech and language delay, autistic-like behaviour and epilepsy. Insight in the mechanism of how the transporter is regulated is largely unknown and it is of importance for the development of successful treatment strategies of cerebral creatine deficient syndromes. Our goal was to characterize CRT2 (SLC6A8B), a published splice variant of the creatine transporter. Surprisingly, using RT-PCR we found a novel splice variant, SLC6A8C, which is predominantly found in human tissues with a high energy requirement such as brain, kidney, heart, small intestines and skeletal muscle, where SLC6A8 transporter is most required. The 5' untranslated region (UTR) of the SLC6A8C mRNA was identified using the Smart Race cDNA amplification kit. The SLC6A8C mRNA contains intron 4 and exons 5 through 13 of SLC6A8, including part of the 3' UTR. An open reading frame was found, which predicts a truncated protein identical to the SLC6A8 transporter, comprising the five last C-terminal transmembrane domains of the SLC6A8 transporter. SLC6A8C open reading frame was cloned as a fusion protein with EGFP and the SLC6A8C protein expression was detected by Western Blot. RT-PCR and sequence analysis showed that this splice variant is conserved in evolution, since we also detected it in mouse. This study reveals the presence of a novel SLC6A8 splice variant, SLC6A8C in human and mouse.

A novel X-linked mental retardation (XLMR) syndrome was recently identified, resulting from creatine deficiency in the brain caused by mutations in the creatine transporter gene, SLC6A8. We have studied the prevalence of SLC6A8 mutations in a panel of 290 patients with nonsyndromic XLMR archived by the European XLMR Consortium. The full-length open reading frame and splice sites of the SLC6A8 gene were investigated by DNA sequence analysis. Six pathogenic mutations, of which five were novel, were identified in a total of 288 patients with XLMR, showing a prevalence of at least 2.1% (6/288). The novel pathogenic mutations are a nonsense mutation (p.Y317X) and four missense mutations. Three missense mutations (p.G87R, p.P390L, and p.P554L) were concluded to be pathogenic on the basis of conservation, segregation, chemical properties of the residues involved, as well as the absence of these and any other missense mutation in 276 controls. For the p.C337W mutation, additional material was available to biochemically prove (i.e., by increased urinary creatine : creatinine ratio) pathogenicity. In addition, we found nine novel polymorphisms (IVS1+26G-->A, IVS7+37G-->A, IVS7+87A-->G, IVS7-35G-->A, IVS12-3C-->T, IVS2+88G-->C, IVS9-36G-->A, IVS12-82G-->C, and p.Y498) that were present in the XLMR panel and/or in the control panel. Two missense variants (p.V629I and p.M560V) that were not highly conserved and were not associated with increased creatine : creatinine ratio, one translational silent variant (p.L472), and 10 intervening sequence variants or untranslated region variants (IVS6+9C-->T, IVS7-151_152delGA, IVS7-99C-->A, IVS8-35G-->A, IVS8+28C-->T, IVS10-18C-->T, IVS11+21G-->A, IVS12+15C-->T, *207G-->C, IVS12+32C-->A) were found only in the XLMR panel but should be considered as unclassified variants or as a polymorphism (p.M560V). Our data indicate that the frequency of SLC6A8 mutations in the XLMR population is close to that of CGG expansions in FMR1, the gene responsible for fragile-X syndrome.