Creatine Transporter (CrT; Slc6a8) Knockout Mice as a Model of Human CrT Deficiency

Mutations in the creatine (Cr) transporter (CrT; Slc6a8) gene lead to absence of brain Cr and intellectual disabilities, loss of speech, and behavioral abnormalities. To date, no mouse model of CrT deficiency exists in which to understand and develop treatments for this condition. The purpose of this study was to generate a mouse model of human CrT deficiency. We created mice with exons 2–4 of Slc6a8 flanked by loxP sites and crossed these to Cre:CMV mice to create a line of ubiquitous CrT knockout expressing mice. Mice were tested for learning and memory deficits and assayed for Cr and neurotransmitter levels. Male CrT−/y (affected) mice lack Cr in the brain and muscle with significant reductions of Cr in other tissues including heart and testes. CrT−/y mice showed increased path length during acquisition and reversal learning in the Morris water maze. During probe trials, CrT−/y mice showed increased average distance from the platform site. CrT−/y mice showed reduced novel object recognition and conditioned fear memory compared to CrT+/y. CrT−/y mice had increased serotonin and 5-hydroxyindole acetic acid in the hippocampus and prefrontal cortex. Ubiquitous CrT knockout mice have learning and memory deficits resembling human CrT deficiency and this model should be useful in understanding this disorder

X-Linked Creatine-Transporter Gene (SLC6A8) Defect: A New Creatine-Deficiency Syndrome

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

High Prevalence of SLC6A8 Deficiency in X-Linked Mental Retardation

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

High Prevalence of SLC6A8 Deficiency in X-Linked Mental Retardation

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

Acoustic Startle with prepulse inhibition.

<p>Startle response was measured following exposure to a 120 dB tone. For prepulse (PP) inhibition, tone was preceded by a softer tone of either 70 or 76 dB. As the size of the animal affects the ability to move the chamber, weight was used a covariate in the analysis of startle response. No differences were noted in startle response with weight as a covariate. Data are Mean ± SEM; n = 16/genotype.</p

Generation of the CrT^flox/flox mice.

<p>(A) Schematic of mouse Slc6a8 locus (top), targeting vector (middle), and targeted locus (bottom). Exons 2-4 were flanked by LoxP sites and the neomyocin cassette was flanked by FRT sites. Neo cassette was excised prior to breeding to any Cre recombinase expressing mice. (B) Southern blot showing successful recombination in ES cells used for generation of the CrT^flox mice. (C) PCR products are shown for the respective genotypes. Due to the CrT being located on the X-chromosome, males have one copy of the CrT gene and are designated as −/y (left), +/y (center-left) or flox/y (center) while females (center right and right) have two copies of the CrT. Female −/− mice are not generated as there are no CrT deficient females.</p

CrT^−/y mice have spatial learning and memory deficits.

<p>Path length for the (A) Acquisition, (C) Reversal, and (E) Shift phases of the MWM; each phase consisted of 4 (90 s) trials/day for 6 consecutive days. Probe trials of 30 s with the platform removed were conducted on the 7^th day following the (B) Acquisition, (D) Reversal, and (F) Shift phases. Data are presented as LSMeans ± SEM. ***P<0.001, **P<0.01; n = 16/genotype.</p

Body weight is reduced in CrT^−/y mice while brain weights were unchanged.

<p>(A) Body weights were collected prior to behavioral testing and (B) brain weights were collected from animals following the end of behavioral testing. Data are presented as Mean ± SEM. *** P<0.001, n = 16/genotype.</p