Identification of polymorphisms in the 3'-untranslated region of the human pregnane X receptor (PXR) gene associated with variability in cytochrome P450 3A (CYP3A) metabolism

Abstract

Single nucleotide polymorphisms in the 3'-untranslated region (3'UTR) of the human pregnane X receptor (PXR) gene might contribute to interindividual variability in cytochrome P450 3A (CYP3A) activity. Genotype-phenotype associations involving PXR-3'UTR single nucleotide polymorphisms were investigated through in vitro (53 human livers from primarily White donors) and in vivo (26 mainly White or African-American volunteers) studies using midazolam 1'-hydroxylation and midazolam apparent oral clearance (CL/F), respectively, as CYP3A-specific probes. PXR-3'UTR resequencing identified twelve single nucleotide polymorphisms, including two that were novel. Although none of the single nucleotide polymorphisms evaluated were associated with altered midazolam 1'-hydroxylation in the liver bank, both rs3732359 homozygotes and rs3732360 carriers showed 80% higher (p < 0.05) CL/F compared with homozygous reference individuals. These differences in CL/F were even larger (100% and 120% higher, respectively; p < 0.01) when only African-American subjects (n = 14) were considered. Five major haplotypes were identified containing the PXR-3'UTR single nucleotide polymorphisms and previously identified intron single nucleotide polymorphisms. Although CL/F differences were not statistically significant within the entire study cohort, African-American carriers of Haplotype-1 (which includes both rs3732359 and rs3732360 variants) exhibited 70% higher median CL/F compared with African-American non-carriers (p = 0.036). The results identify rs3732359 and rs3732360 as PXR-3'UTR single nucleotide polymorphisms associated with higher CYP3A activity in vivo in African-Americans.

Figure 1

Graphical representation of the structure of human PXR gene including locations of all SNPs evaluated in this study. Also shown are the PXR DNA binding domain (DBD) and ligand binding domain (LBD), as well as an expanded 3′-UTR region.

Figure 2

MDZ CL/F (mL/min/kg) in 26 healthy pharmacokinetic study subjects (panels A and B) and 1′OH MDZ formation rate (nmole/min/mg protein) in 53 human livers (panels C and D) grouped by PXR-3′UTR genotype for rs3732359g>a and rs3732360c>t. Bars indicate the median value for each group. Also shown on each plot are the results of statistical analyses using ANOVA followed by Student Newman Keuls test (A), Kruskal-Wallis ANOVA on ranks test followed by Dunn’s multiple comparisons test (B), or Kruskal-Wallis ANOVA on ranks (C and D).

Figure 3

MDZ CL/F (mL/min/kg) in 26 pharmacokinetic study subjects grouped by self-identified race (African-American or white) and PXR genotype for variants rs3732359g>a and rs3732360c>t. Bars indicate the median value for each group. Also shown on each plot are the results of statistical analyses using Mann Whitney Rank Sum test (A, B), Kruskal-Wallis ANOVA on ranks test followed by Dunn’s multiple comparisons test (C), or Kruskal-Wallis ANOVA on ranks (D).

Figure 4

Linkage disequilibrium matrix plot showing pair-wise relationships between SNPs located in the PXR gene intron 2 (rs1464603 and rs1464602), intron 4 (rs3732357) and 3′UTR regions identified in African-American (n = 18) and white (n= 56) individuals. Genotype data from human liver bank samples and pharmacokinetic study subjects were combined for this analysis. The plot was created using the LDPlotter software available at https://www.pharmgat.org/Tools/pbtoldplotform. Colored boxes represent the approximate regression value (r²) as shown in the legend for the pair of SNPs being compared.

Figure 5

Association of PXR haplotype carrier status with MDZ CL/F in pharmacokinetic study subjects. Shown are results for all 26 subjects (A), the 14 African-Americans (B) and the 9 whites (C). Haplotypes (shown in Table 3) were inferred using PHASE 2.0 software (Stephens et al. 2001) with genotype data from eight PXR-3′UTR SNPs and three upstream intron 2 and 4 SNPs may predict MDZ CL/F in pharmacokinetic study subjects. Bars indicate the median values for each group. Also shown are the P values of statistical comparisons between carrier and non-carrier groups using Mann Whitney Rank Sum test. N/A denotes that the statistical test was not performed since there were no carrier individuals. None of the pharmacokinetic study subjects carried Haplotype-4.

Figure 6

Relationship between total 3′UTR PXR mRNA levels, MDZ-1′OH rate, and PXR genotype. (A). Lack of correlation between total 3′UTR mRNA PXR levels and 1-OH formation MDZ rate (Spearman rank correlation, R_s=0.184, P>0.05). (B). Liver samples carrying the rs3814057a>c and rs3810458t>c genotype have 140% greater median total 3′UTR mRNA levels than those homozygous reference (Mann Whitney Rank Sum test, P=0.048). Bar indicates the median values for each group. qPCR results are presented as relative to the lowest liver expression, which was assigned a value of 1.

Figure 7

Predicted PXR-3′UTR mRNA secondary structures and calculated free energies for the Ancestral haplotype (A), Haplotype-1 (B), and Haplotype-2 (C). Images were generated using the RNA GeneBee secondary structure predictor software (Brodsky 1995). The circled areas indicate secondary structure differences in Haplotype-1 and Haplotype-2 compared with the Ancestral haplotype.

Figure 8

HEK293T (A), COS-7 (B), and LS180 (C) cell transfection experiments comparing the effect of the PXR-3′UTR of the Ancestral haplotype, Haplotype-1, and Haplotype-2 on normalized luciferase activity (Luciferase activity/ β-Galactosidase activity) relative to the pMIR-DEST activity. Columns represent the mean of 4 experiments performed in triplicate and error bars denote standard error of the mean.