Estimated carrier frequency of creatine transporter deficiency in females in the general population using functional characterization of novel missense variants in the SLC6A8 gene

Gene. 2015 Jul 10;565(2):187-91. doi: 10.1016/j.gene.2015.04.011. Epub 2015 Apr 8.


Creatine transporter deficiency (CRTR-D) is an X-linked inherited disorder of creatine transport. All males and about 50% of females have intellectual disability or cognitive dysfunction. Creatine deficiency on brain proton magnetic resonance spectroscopy and elevated urinary creatine to creatinine ratio are important biomarkers. Mutations in the SLC6A8 gene occur de novo in 30% of males. Despite reports of high prevalence of CRTR-D in males with intellectual disability, there are no true prevalence studies in the general population. To determine carrier frequency of CRTR-D in the general population we studied the variants in the SLC6A8 gene reported in the Exome Variant Server database and performed functional characterization of missense variants. We also analyzed synonymous and intronic variants for their predicted pathogenicity using in silico analysis tools. Nine missense variants were functionally analyzed using transient transfection by site-directed mutagenesis with In-Fusion HD Cloning in HeLa cells. Creatine uptake was measured by liquid chromatography tandem mass spectrometry for creatine measurement. The c.1654G>T (p.Val552Leu) variant showed low residual creatine uptake activity of 35% of wild type transfected HeLa cells and was classified as pathogenic. Three variants (c.808G>A; p.Val270Met, c.942C>G; p.Phe314Leu and c.952G>A; p.Ala318Thr) were predicted to be pathogenic based on in silico analysis, but proved to be non-pathogenic by our functional analysis. The estimated carrier frequency of CRTR-D was 0.024% in females in the general population. We recommend functional studies for all novel missense variants by transient transfection followed by creatine uptake measurement by liquid chromatography tandem mass spectrometry as fast and cost effective method for the functional analysis of missense variants in the SLC6A8 gene.

Keywords: Carrier frequency; Creatine transporter deficiency; Exome Variant Server; Liquid chromatography tandem mass spectrometry; Site-directed mutagenesis with In-Fusion HD Cloning.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Brain Diseases, Metabolic, Inborn / genetics*
  • Cell Line, Tumor
  • Creatine / deficiency*
  • Creatine / genetics
  • Creatinine / metabolism
  • Female
  • Gene Frequency / genetics*
  • Genotype
  • HeLa Cells
  • Heterozygote
  • Humans
  • Male
  • Mental Retardation, X-Linked / genetics*
  • Mutagenesis, Site-Directed / methods
  • Mutation, Missense / genetics*
  • Nerve Tissue Proteins / genetics*
  • Plasma Membrane Neurotransmitter Transport Proteins / deficiency*
  • Plasma Membrane Neurotransmitter Transport Proteins / genetics


  • Nerve Tissue Proteins
  • Plasma Membrane Neurotransmitter Transport Proteins
  • SLC6A8 protein, human
  • Creatinine
  • Creatine

Supplementary concepts

  • Creatine deficiency, X-linked