Influence of mutations associated with Gilbert and Crigler-Najjar type II syndromes on the glucuronidation kinetics of bilirubin and other UDP-glucuronosyltransferase 1A substrates

Pharmacogenet Genomics. 2007 Dec;17(12):1017-29. doi: 10.1097/FPC.0b013e328256b1b6.

Abstract

Objectives: UGT1A1 coding region mutations, including UGT1A1*6 (G71R), UGT1A1*7 (Y486D), UGT1A1*27 (P229Q) and UGT1A1*62 (F83L), have been linked to Gilbert syndrome in Asian populations, whereas homozygosity for UGT1A1*7 is associated with the Crigler-Najjar syndrome type II. This work compared the effects of (a) the individual UGT1A1 mutations on the glucuronidation kinetics bilirubin, beta-estradiol, 4-methylumbelliferone (4MU) and 1-naphthol (1NP), and (b) the Y486 mutation, which occurs in the conserved carboxyl terminal domain of UGT1A enzymes, on 4MU, 1NP and naproxen glucuronidation by UGT1A3, UGT1A6 and UGT1A10.

Methods: Mutant UGT1A cDNAs were generated by site-directed mutagenesis and the encoded proteins were expressed in HEK293 cells. The glucuronidation kinetics of each substrate with each enzyme were characterized using specific high-performance liquid chromatography (HPLC) methods.

Results: Compared with wild-type UGT1A1, in-vitro clearances for bilirubin, beta-estradiol, 4MU and 1NP glucuronidation by UGT1A1*6 and UGT1A1*27 were reduced by 34-74%, most commonly as a result of a reduction in Vmax. However, the magnitude of the decrease in the in-vitro clearances varied from substrate to substrate with each mutant. The glucuronidation activities of UGT1A1*7 and UGT1A1*62 were reduced by >95%. Introduction of the Y486D mutation essentially abolished UGT1A6 and UGT1A10 activities, and resulted in 60-90% reductions in UGT1A3 in-vitro clearances.

Conclusions: The glucuronidation of all UGT1A1 substrates is likely to be impaired in subjects carrying the UGT1A1*6 and UGT1A1*62 alleles, although the reduction in metabolic clearance might vary with the substrate. The Y486D mutation appears to greatly reduce most, but not all, UGT1A activities.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Bilirubin / metabolism*
  • Cell Line
  • Crigler-Najjar Syndrome / enzymology*
  • Crigler-Najjar Syndrome / genetics*
  • Estradiol / metabolism
  • Gilbert Disease / enzymology*
  • Gilbert Disease / genetics*
  • Glucuronides / metabolism
  • Glucuronosyltransferase / genetics*
  • Glucuronosyltransferase / metabolism*
  • Humans
  • Hymecromone / analogs & derivatives
  • Hymecromone / metabolism
  • Kinetics
  • Mutagenesis, Site-Directed
  • Naphthols / metabolism
  • Pharmacogenetics
  • Point Mutation
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity

Substances

  • Glucuronides
  • Naphthols
  • Recombinant Proteins
  • 1-naphthol
  • Hymecromone
  • Estradiol
  • UGT1A1 enzyme
  • Glucuronosyltransferase
  • Bilirubin