Intronic polymorphism in CYP3A4 affects hepatic expression and response to statin drugs

Abstract

Cytochrome P450 3A4 (CYP3A4) metabolizes ∼50% of all clinically used drugs. Although CYP3A4 expression varies widely between individuals, the contribution of genetic factors remains uncertain. In this study, we measured allelic CYP3A4 heteronuclear RNA (hnRNA) and mRNA expression in 76 human liver samples heterozygous for at least one of eight marker SNPs and found marked allelic expression imbalance (1.6-6.3-fold) in 10/76 liver samples (13%). This was fully accounted for by an intron 6 SNP (rs35599367, C>T), which also affected mRNA expression in cell culture on minigene transfections. CYP3A4 mRNA level and enzyme activity in livers with CC genotype were 1.7- and 2.5-fold, respectively, greater than in CT and TT carriers. In 235 patients taking stable doses of atorvastatin, simvastatin, or lovastatin for lipid control, carriers of the T allele required significantly lower statin doses (0.2-0.6-fold, P=0.019) than non-T carriers for optimal lipid control. These results indicate that intron 6 SNP rs35599367 markedly affects expression of CYP3A4 and could serve as a biomarker for predicting response to CYP3A4-metabolized drugs.

Figure 1

Allelic mRNA/hnRNA expression ratios of CYP3A4 in human livers measured with a primer extension assay (SNaPshot) using seven marker SNPs (SNP 5, 7, 9, 10, 11, 12, and 13 in Table 1). Allelic RNA ratios were normalized to genomic DNA ratios set at 1. Data represent the average of three measurements per marker using single or multiple marker SNPs (mean±s.d.). Arrow indicates samples with AEI ratios significant different from 1 (analysis of variance with Dunnett post-test, P<0.05).

Figure 2

Association between genotypes and allelic RNA expression imbalance (AEI) status (AEI positive or AEI negative). Only intron 6 SNP rs35599367 scored with high significance, whereas SNP 7 (rs2246709 in intron 7) was marginally significant. The solid line indicates P=0.05 level for the association, without adjusting for multiple comparisons. 1. rs34401238 (TGT insertion); 2. rs2737418; 3. rs2740574(*1B); 4. rs2687105; 5. rs28988579; 6. rs35599367 (intron 6); 7. rs2246709; 8. rs4646437; 9. rs2242480; 10. rs3735451; 11. rs28988604; 12. rs28969391; 13. rs28371763.

Figure 3

Allelic hnRNA expression ratios of CYP3A4 in human livers (a) and intestines (b) measured with a primer extension assay (SNaPshot) using intron 6 SNP as marker. Allelic RNA ratios were normalized to genomic DNA ratios set at 1. Data represent the average of three independent measurements for each sample (mean±s.d.). All allelic RNA ratios in panel (a) were significantly different from 1 (analysis of variance with Dunnett post-test, P<0.05), whereas allelic RNA ratios in panel (b) were all close to 1.

Figure 4

Correlation between mRNA expression of four transcription factors PXR (a), RXRα (b), CAR (c), and HNF4α (d) and CYP3A4 in 93 human livers (cohort 2). CYP3A4 mRNA levels were positively correlated with mRNA levels of each of the four transcription factors, with correlation coefficients R ranging from 0.37 to 0.53 (P≤0.001). Data are in Log₁₀ scale.

Figure 5

Box plots of CYP3A4 mRNA levels (a) (cohort 2) and enzyme activity (b) (cohort 1) in human liver samples, grouped by intron 6 SNP genotype. The y axis shows adjusted CYP3A4 mRNA levels or enzyme activities. mRNA levels were adjusted for sex and mRNA levels of PXR and RXR transcription factors, whereas enzyme activities were adjusted for age, sex, and inducer exposure.

Figure 6

Allelic RNA ratios in liver samples heterozygous for SNPs rs2740574 (CYP3A4*1B) (a), rs34401238 (TGT insertion) (b), rs2737418 (c), and rs4646437 (d). None of the samples showed allelic RNA ratios deviating from 1, except A30 in panel (d), which was also heterozygous for intron 6 SNP.

Figure 7

In vitro cell transfection assays. (a) Effect of intron 6 region on CYP3A4-promoter activity tested in a luciferase reporter gene assay. The intron 6 region was inserted upstream of the proximal CYP3A4-promoter region, and both C and T alleles were tested using bioluminescence output. (b) Effects of intron 6 SNP on CYP3A4 minigene RNA expression. Minigene constructs, consisting of exon 6, intron 6, and exon 7, harboring the C or T allele of intron 6 SNP, were co-transfected into HepG2 or HEK293 cells, and allelic DNA and RNA ratios measured at 24, 48, and 96 h post-transfection, using intron 6 SNP as the marker. The intron 6 SNP plasmid DNA ratio was normalized to 1 for each experiment. Compared with plasmid DNA ratio, **P<0.01, ***P<0.001 analysis of variance with Bonferroni post-test.