title: Twenty-one-base-pair insertion polymorphism creates an enhancer element and potentiates SLC6A1 GABA transporter promoter activity.
abstract: OBJECTIVE
  Sodium-dependent and chloride-dependent gamma-aminobutyric acid (GABA) transporter 1 (SLC6A1) is the target of a number of drugs of clinical importance and is a major determinant of synaptic GABA concentrations. We resequenced the human SLC6A1 gene previously and discovered a novel 21 bp insertion in the predicted promoter region that creates a second tandem copy of the sequence. Here we sought to determine the functional relevance of this variation.
  
  
  METHODS
  We used reporter assays, mobility shift assays, quantitative PCR, and proteomics methods as well as postmortem expression analysis for this work.
  
  
  RESULTS
  Reporter assays showed that the insertion allele significantly increases promoter activity in multiple cell lines. The zinc finger transcription factor ZNF148 was found to significantly transactivate the promoter and increase expression when overexpressed but could not account for the differences in activity between the two alleles of the promoter. Copy number of the insertion sequence was associated with exponentially increasing activity of a downstream promoter, suggesting that the insertion sequence has enhancer activity when present in multiple copies. SLC6A1 promoter genotype was found to predict SLC6A1 RNA expression in human postmortem hippocampal samples. These results suggest that the insertion polymorphism leads to increased SLC6A1 promoter activity because, in part, of creation of an enhancer element when present as multiple copies. Genotyping individuals from Tanzania in this study suggested that the insertion allele has its origin in Africa.
  
  
  CONCLUSIONS
  On account of the effect of the insertion on promoter activity, this relatively common polymorphism may prove useful in predicting clinical response to pharmacological modulators of SLC6A1 as well as GABAergic function in individuals of African descent.
authors.affiliation: Department of Psychiatry and the Yale Keck Center, Yale University School of Medicine, New Haven, CT 06511-6624, USA.
authors.forename: Rungnapa
authors.initials: R
authors.lastname: Hirunsatit
authors.forename: Elizabeth D
authors.initials: ED
authors.lastname: George
authors.forename: Barbara K
authors.initials: BK
authors.lastname: Lipska
authors.forename: Hani M
authors.initials: HM
authors.lastname: Elwafi
authors.forename: Lisa
authors.initials: L
authors.lastname: Sander
authors.forename: Carolyn M
authors.initials: CM
authors.lastname: Yrigollen
authors.forename: Joel
authors.initials: J
authors.lastname: Gelernter
authors.forename: Elena L
authors.initials: EL
authors.lastname: Grigorenko
authors.forename: Jaakko
authors.initials: J
authors.lastname: Lappalainen
authors.forename: Shrikant
authors.initials: S
authors.lastname: Mane
authors.forename: Angus C
authors.initials: AC
authors.lastname: Nairn
authors.forename: Joel E
authors.initials: JE
authors.lastname: Kleinman
authors.forename: Arthur A
authors.initials: AA
authors.lastname: Simen
mesh_terms: D000293:Adolescent; D000328:Adult; D001741:Black or African American / Q000235:genetics; D000368:Aged; D000818:Animals; D001483:Base Sequence; D016022:Case-Control Studies; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005260:Female; D050485:GABA Plasma Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D008875:Middle Aged; D018598:Minisatellite Repeats; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010597:Pharmacogenetics; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012559:Schizophrenia / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D015533:Transcriptional Activation; D055815:Young Adult
chemical_list: D017931:DNA Primers; D050485:GABA Plasma Membrane Transport Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C493413:SLC6A1 protein, human