title: Molecular pathogenesis of Gilbert's syndrome: decreased TATA-binding protein binding affinity of UGT1A1 gene promoter.
abstract: OBJECTIVE
  Gilbert's syndrome is a congenital, nonhemolytic, unconjugated hyperbilirubinemia. The most common genotype of Gilbert's syndrome is the homozygous polymorphism, A(TA)7TAA, in the promoter of the gene for UDP-glucuronosyltransferase 1A1 (UGT1A1), with a thymine adenine insertion in the TATA-box-like sequence, which results in a decrease in UGT1A1 activity. The mechanism responsible for this decrease in UGT1A1 activity, however, has not been elucidated. To clarify the mechanism underlying this deficiency in UGT1A1 activity in patients with Gilbert's syndrome.
  
  
  METHODS
  The promoter activity assay using the wild-type A(TA)6TAA or the mutant A(TA)7TAA promoter and a luciferase reporter was performed in two different hepatoma cell lines. The binding affinity for a nuclear protein complex or for TATA-binding protein was evaluated by a competitive electophoretic mobility shift assay using wild-type or mutant TATA-box-like oligonucleotide probes and nuclear extract or TATA-binding protein. The formation of complexes between TATA-binding protein and wild-type or mutant oligonucleotide probes was also studied by a quantitive electophoretic mobility shift assay.
  
  
  RESULTS
  A TA insertion in the TATA-box-like sequence of the promoter activity of UGT1A1 gene. A competitive electrophoretic mobility shift assay showed a decrease in nuclear protein complex binding affinity and TATA-binding protein binding affinity of the mutant TATA-box-like sequence A(TA)7TAA. When the mutants A(TA)5TAA and A(TA)8TAA were also compared, quantitative electrophoretic mobility shift assay demonstrated that the TATA-binding protein binding affinity progressively decreased as the number of TA repeats in the TATA-box-like sequence increased.
  
  
  CONCLUSIONS
  TA insertion in the TATA-box-like sequence of the UGT1A1 promoter affected its binding affinity for TATA-binding protein, causing a decrease in its activity. This explains the pathogenesis of Gilbert's syndrome.
authors.affiliation: Department of Internal Medicine, Tri-Service General Hospital, Taipei, Taiwan, ROC.
authors.forename: Tsai-Yuan
authors.initials: TY
authors.lastname: Hsieh
authors.forename: Tzu-Yue
authors.initials: TY
authors.lastname: Shiu
authors.forename: Shih-Ming
authors.initials: SM
authors.lastname: Huang
authors.forename: Hsuan-Hwai
authors.initials: HH
authors.lastname: Lin
authors.forename: Tai-Chi
authors.initials: TC
authors.lastname: Lee
authors.forename: Peng-Jen
authors.initials: PJ
authors.lastname: Chen
authors.forename: Heng-Cheng
authors.initials: HC
authors.lastname: Chu
authors.forename: Wei-Kuo
authors.initials: WK
authors.lastname: Chang
authors.forename: King-Song
authors.initials: KS
authors.lastname: Jeng
authors.forename: Michael M C
authors.initials: MM
authors.lastname: Lai
authors.forename: You-Chen
authors.initials: YC
authors.lastname: Chao
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D005878:Gilbert Disease / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D016254:Mutagenesis, Insertional; D010597:Pharmacogenetics; D011401:Promoter Regions, Genetic; D035181:TATA-Box Binding Protein / Q000378:metabolism*; D000099148:UGT1A1 Enzyme
chemical_list: D017931:DNA Primers; D014453:Glucuronosyltransferase; D035181:TATA-Box Binding Protein; D000099148:UGT1A1 Enzyme