title: Functional analysis of the role of the TPMT gene promoter VNTR polymorphism in TPMT gene transcription.
abstract: OBJECTIVE
  Thiopurine S-methyltransferase (TPMT) activity is polymorphic, and a trimodal distribution has been demonstrated in Caucasians (low, intermediate and high methylator groups). The TPMT gene promoter contains a variable number of three GC-rich tandem repeats, namely A, B and C, ranging from three to nine in length in a A(n)B(m)C architecture.
  
  
  METHODS
  Here, we investigated the influence of number and type of TPMT gene promoter tandem repeats on human TPMT gene transcription in K562 cells transiently transfected with reporter constructs bearing various variable number of tandem repeats (VNTR) and addressed the interaction of transcription factor binding to the VNTRs by electrophoretic mobility shift assays.
  
  
  RESULTS
  We found that the distribution patterns of VNTR alleles do not significantly differ among acute lymphoblastic leukemia patients, acute myeloid leukemia patients and normal individuals. We also demonstrated that the A repeat has a negative effect in TPMT gene transcription and that a positive regulatory element, identified immediately upstream to the VNTR region of the TPMT gene promoter, is indispensable for TPMT gene transcription. Our electrophoretic mobility shift assay analysis indicated that the Sp1 and Sp3 transcription factors bind to the VNTR repeats.
  
  
  CONCLUSIONS
  Overall, our data underline that both the number and type of VNTRs, as well as the upstream regulatory region of the TPMT gene promoter, determine the overall level of TPMT gene transcription. It remains to be seen whether these VNTRs can be employed as pharmacogenetic markers to individualize thiopurine therapy.
authors.affiliation: Institute for Molecular Genetics & Genetic Engineering, Laboratory of Molecular Hematology, Vojvode Stepe 444a, 11010 Belgrade, Serbia.
authors.forename: Branka
authors.initials: B
authors.lastname: Zukic
authors.forename: Milena
authors.initials: M
authors.lastname: Radmilovic
authors.forename: Maja
authors.initials: M
authors.lastname: Stojiljkovic
authors.forename: Natasa
authors.initials: N
authors.lastname: Tosic
authors.forename: Farzin
authors.initials: F
authors.lastname: Pourfarzad
authors.forename: Lidija
authors.initials: L
authors.lastname: Dokmanovic
authors.forename: Dragana
authors.initials: D
authors.lastname: Janic
authors.forename: Natasa
authors.initials: N
authors.lastname: Colovic
authors.forename: Sjaak
authors.initials: S
authors.lastname: Philipsen
authors.forename: George P
authors.initials: GP
authors.lastname: Patrinos
authors.forename: Sonja
authors.initials: S
authors.lastname: Pavlovic
mesh_terms: D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D020014:K562 Cells; D008780:Methyltransferases / Q000235:genetics*; D018598:Minisatellite Repeats; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000502:physiology*; D014162:Transfection; D044465:White People / Q000235:genetics
chemical_list: D008780:Methyltransferases; C022745:thiopurine methyltransferase