title: Detection of a novel intragenic rearrangement in the creatine transporter gene by next generation sequencing.
abstract: Deficiency caused by mutations in the creatine transporter gene (SLC6A8/CT1) is an X-linked form of intellectual disability. The presence of highly homologous pseudogenes and high GC content of SLC6A8 genomic sequence complicates the molecular diagnosis of this disorder. To minimize the pseudogene interference, exons 2 to 13 of SLC6A8 were amplified as a single PCR product using gene-specific long-range PCR (LR-PCR) primers. The GC-rich exon 1 and its flanking intronic sequences were amplified separately in a short fragment under GC-rich conditions and a touchdown PCR program. Traditional Sanger sequence analysis of all coding exons of SLC6A8 from a 3-year-old boy with creatine transporter deficiency did not detect deleterious mutations. The long-range PCR product was used as template followed by massively parallel sequencing (MPS) on HiSeq2000. We were able to detect a tandem duplication involving part of exons 11 and 12 in the SLC6A8 gene. The deduced c.1592_1639dup133 mutation was confirmed to be a hemizygous insertion by targeted genomic DNA and cDNA Sanger sequencing. Combination of deep sequencing technology with long-range PCR revealed a novel intragenic duplication in the SLC6A8 gene, providing a definitive molecular diagnosis of creatine transporter deficiency in a male patient.
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Yu
authors.forename: Clara
authors.initials: C
authors.lastname: van Karnebeek
authors.forename: Graham
authors.initials: G
authors.lastname: Sinclair
authors.forename: Alan
authors.initials: A
authors.lastname: Hill
authors.forename: Hong
authors.initials: H
authors.lastname: Cui
authors.forename: Victor Wei
authors.initials: VW
authors.lastname: Zhang
authors.forename: Lee-Jun
authors.initials: LJ
authors.lastname: Wong
mesh_terms: D020739:Brain Diseases, Metabolic, Inborn / Q000175:diagnosis / Q000235:genetics*; D002675:Child, Preschool; D003401:Creatine / Q000172:deficiency* / Q000235:genetics; D005091:Exons; D020440:Gene Duplication; D040181:Genetic Diseases, X-Linked / Q000175:diagnosis / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008607:Intellectual Disability / Q000235:genetics*; D008297:Male; D038901:X-Linked Intellectual Disability / Q000175:diagnosis / Q000235:genetics*; D008961:Models, Structural; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics*; D057089:Pathology, Molecular / Q000379:methods*; D010375:Pedigree; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000172:deficiency* / Q000235:genetics; D011544:Pseudogenes / Q000235:genetics
chemical_list: D009419:Nerve Tissue Proteins; D050480:Plasma Membrane Neurotransmitter Transport Proteins; C094770:SLC6A8 protein, human; D003401:Creatine
keywords: Creatine transporter deficiency; Duplication; Gene rearrangement; Massively parallel sequencing; Molecular diagnostics; Pseudogenes