The UDP-glucuronosyltransferase (UGT) 1A polymorphism c.2042C>G (rs8330) is associated with increased human liver acetaminophen glucuronidation, increased UGT1A exon 5a/5b splice variant mRNA ratio, and decreased risk of unintentional acetaminophen-induced acute liver failure

Abstract

Acetaminophen is cleared primarily by hepatic glucuronidation. Polymorphisms in genes encoding the acetaminophen UDP-glucuronosyltransferase (UGT) enzymes could explain interindividual variability in acetaminophen glucuronidation and variable risk for liver injury after acetaminophen overdose. In this study, human liver bank samples were phenotyped for acetaminophen glucuronidation activity and genotyped for the major acetaminophen-glucuronidating enzymes (UGTs 1A1, 1A6, 1A9, and 2B15). Of these, only three linked single nucleotide polymorphisms (SNPs) located in the shared UGT1A-3'UTR region (rs10929303, rs1042640, rs8330) were associated with acetaminophen glucuronidation activity, with rs8330 consistently showing higher acetaminophen glucuronidation at all the tested concentrations of acetaminophen. Mechanistic studies using luciferase-UGT1A-3'UTR reporters indicated that these SNPs do not alter mRNA stability or translation efficiency. However, there was evidence for allelic imbalance and a gene-dose proportional increase in the amount of exon 5a versus exon 5b containing UGT1A mRNA spliced transcripts in livers with the rs8330 variant allele. Cotransfection studies demonstrated an inhibitory effect of exon 5b containing cDNAs on acetaminophen glucuronidation by UGT1A1 and UGT1A6 cDNAs containing exon 5a. In silico analysis predicted that rs8330 creates an exon splice enhancer site that could favor exon 5a (over exon 5b) utilization during splicing. Finally, the prevalence of rs8330 was significantly lower (P = 0.027, χ(2) test) in patients who had acute liver failure from unintentional acetaminophen overdose compared with patients with acute liver failure from other causes or a race- or ethnicity-matched population. Together, these findings suggest that rs8330 is an important determinant of acetaminophen glucuronidation and could affect an individual's risk for acetaminophen-induced liver injury.

Figures

Fig. 1.

Association of UGT1A-3′UTR rs8330 SNP genotype (CC, CG, and GG) with microsomal acetaminophen glucuronidation activities (40 mM acetaminophen concentration) measured in a bank of human livers obtained from 48 white donors. Shown are activities for each liver (filled circle), geometric mean values for each genotype group (horizontal dashed line), and also P values for post-hoc pairwise testing on log transformed data by the Student-Newman-Keuls multiple comparisons test (P = 0.002, ANOVA). *P < 0.05; **P < 0.01.

Fig. 2.

Results of pairwise linkage disequilibrium and haplotype block analysis of 9 UGT1A gene SNPs and one UGT2B15 gene SNP genotyped using DNA extracted from human liver bank samples obtained from 48 white donors. Shown above are the approximate locations of each of the nine SNPs (identified by their dbSNP rs number) relative to the unique exons 1 and the shared exons 2 to 5 in the UGT1A gene. Shown below is a matrix of linkage disequilibrium r-squared values (as percent) for each pairwise comparison. Two distinct haplotype blocks were identified, including one block incorporating the UGT1A6 and UGT1A1 SNPs (block 1) and another block incorporating the UGT1A-3′UTR SNPs (block 2).

Fig. 3.

Effect of the UGT1A-3′UTR reference (CCC) and variant (TGG) haplotype sequences on luciferase-UGT1A-3′UTR reporter activities when transfected into human kidney (A), liver (B), intestinal (C) cell lines, and primary human hepatocytes (D). Bars indicate the mean and S.D. of results of either four independent assays (cell lines) or three different donors (hepatocytes). Luciferase data were normalized to a renilla transfection control and expressed as a ratio relative to wells transfected with a plasmid construct lacking the UGT1A-3′UTR (reference or variant). Also shown for each set of data are the results of ANOVA with post-hoc pairwise testing by the Student-Newman-Keuls multiple comparisons test. n.s., not significant.

Fig. 4.

Repressive effect of UGT1A_i2 variant on UGT1A_i1-mediated acetaminophen glucuronidation activity. Top panel (A) shows acetaminophen glucuronidation activities measured in HEK293 cells transiently transfected with plasmids encoding UGT1A6_i1 (2 µg) and increasing amounts of UGT1A6_i2 (0 to 2 µg). Data were expressed as a percentage of activities in control wells that lacked UGT1A6_i2. Bars represent the mean and S.D. of three independent experiments conducted in duplicate. Also shown are the P values for significant (P < 0.05) pairwise multiple comparisons to the control group using the Holm-Sidak test (P = 0.02, ANOVA). Bottom panel (B) shows acetaminophen glucuronidation activities measured in HEK293 cell lines stably expressing either UGT1A1_i1 alone or both UGT1A1_i1 and UGT1A1_i2 (ratio of 1:0.33 by immunoblot). Data were expressed as a percentage of activities in cell lines expressing only UGT1A1_i1 and were adjusted for the relative UGT1A1_i1 content of each cell line. Bars represent the mean and SD of four independent experiments. Also shown is the P value for the comparison in activities between cell lines by t test.

Fig. 5.

SNPs identified by resequencing DNA from human livers with high (n = 8) and low (n = 8) exon 5a to exon 5b variant mRNA ratios. Shown below are a graphic depiction of the UGT1A shared exons 2 to 5 region (5′ to 3′ with introns), the locations of primer pairs (given in Table 1) used for resequencing, and the locations of SNPs (and their dbSNP accession numbers) that were identified. Shown above are the P values for a Fisher's exact test comparing SNP genotype frequencies in the low mRNA ratio versus the high mRNA ratio livers. Only the UGT1A-3′UTR SNPs (rs10929303, rs1042640, and rs8330) were significantly associated with mRNA ratios (P < 0.05).

Fig. 6.

Putative effect of the UGT1A-3′UTR SNPs on gene splicing. The top panel (A) shows the results of binding site consensus sequence analysis for the Srp family of splicing regulatory proteins using the ESEfinder program (release 3.0). Shown are the sequences surrounding the reference (above) and variant (below) sequences surrounding each SNP and the predicted consensus binding sites and scores in parentheses. No ESE elements were predicted for the rs8330 reference sequence. The bottom panel (B) is a proposed model explaining the effect of the UGT1A3′-UTR SNPs on glucuronidation by UGT1A isoforms through effects on gene splicing. The presence of the UGT1A-3′UTR SNPs would favor utilization of exon 5a over the exon 5b splicing donor site, resulting in more UGT1A_i1 relative to UGT1A_i2 protein variants and less post-translation repression of UGT1A_i1 enzyme activities.