Relative to other large population groups, the contribution of genetic polymorphisms to interindividual differences in CYP2C9 expression in Hispanics is poorly understood. In the current study, five of the nine novel SNPs identified in the 24PDR (g.-8422A>G, g.-5146G>C, g.-5143A>C, g.-5140A>T, and g.-4302C>T) were present in the Hispanics but were absent in the non-Latino Whites. A total of 22 SNPs were present in both populations, five of which (g.-620G>T, g.-1096A>G, g.-1188T>C, g.-2663delTG and g.-3089G>A) occurred at significantly different frequencies (). Thus, similar to what has been observed for CYP2C9 structural variants, ethnic differences exist in the presence and frequency of CYP2C9 regulatory polymorphisms, which may contribute to interpopulation differences in CYP2C9-dependent metabolism.
The observed frequency of two previously reported CYP2C9
structural variants, g.3608C>T (CYP2C9*2
) and g.42614A>C (CYP2C9*3
) were not significantly different between the Hispanics and non-Latino Whites, nor were the determined frequencies different than those reported by Llerena et al. (2004)
for Hispanic Americans of Mexican descent. In contrast, there was a significant difference when compared to the frequencies reported by Xie et al. (2002)
. However, the ancestral background of the Hispanic population was not defined in the latter study. Thus, the discrepancy in these data is likely explained by a population of mixed ancestry.
Based upon the observed minor allele frequencies of 32 upstream and five structural variants, 10 common (>1%) CYP2C9 haplotypes were inferred. The observed haplotype 1 (reference) frequency in Hispanics was 60.53%, compared to 63.64% in non-Latino Whites, consistent with a relatively simple haplotype structure. Four CYP2C9 haplotypes (1A, 1B, 1E, and 2A) were inferred in Hispanics, but not in non-Latino Whites ().
The possible contribution of upstream CYP2C9
genetic variability to interindividual pharmacokinetic differences is more controversial. Variant effects on promoter activity in vitro
have been observed, but failed to correlate with differences in in vivo
activity (Shintani et al., 2001
). Other studies have failed to show an independent effect of CYP2C9
upstream haplotype variants on mean warfarin clearance or dose (King et al., 2004
; Takahashi et al., 2004
; Veenstra et al., 2005
). However, the design of these studies precluded the ability to test the impact of the CYP2C9
upstream polymorphisms independently of the variants impacting CYP2C9 catalytic activity. In the current report, haplotype 1B, consisting of only the g.-4302C>T variant, exhibited a significant decrease in promoter activity in vitro
. Combined with its relatively high frequency (i.e.,
10.0%), these data would be consistent with reduced constitutive CYP2C9
expression in this population. It is interesting that the frequency of the g.-4302C>T SNP was not in Hardy-Weinberg equilibrium, suggesting a possible selective pressure for the presence of the variant allele, although this deviation may also be due to recent population admixture. Significant decreases in in vitro
constitutive promoter activity also were observed with haplotypes 3A and 3B. Thus, the results of our study suggest that, in addition to the defective enzymatic function of CYP2C9.3, a decrease in basal CYP2C9
transcription also may contribute to the overall observed CYP2C9*3
studies using primary hepatocytes have shown that CYP2C9 mRNA, protein, and catalytic activity are all increased by drugs such as rifampicin, hyperforin, and phenobarbital through a PXR-dependent mechanism (Chen et al., 2004
). Rifampicin treatment also has been reported to enhance the clearance of CYP2C9 substrates, indicative of CYP2C9 induction in vivo
(Williamson et al., 1998
; Niemi et al., 2001
). Co-transfection studies in HepG2 cells with a human PXR expression plasmid and CYP2C
9 reporter constructs were used to investigate the possible functional effects of CYP2C9
haplotype variants on rifampicin-dependent enhancement of CYP2C9
promoter activity. With haplotype 1 (reference construct), addition of human PXR alone had no effect on basal CYP2C9
promoter activity, consistent with results reported by Ferguson et al. (2002)
. These results, however, conflict with those of Chen et al. (2004)
, who reported a 3.8-fold increase in CYP2C9
promoter activity upon addition of human PXR alone. When the co-transfected cells were treated with 10 μM rifampicin, a 3.2-fold increase in promoter activity was observed, similar to the approximate 3-fold increase reported by Chen et al. (2004)
. Yet Ferguson et al. (2002)
failed to see an effect with rifampicin. The discrepancies in these data may result from differences in the amount of CYP2C9
upstream sequences present in the reporter construct or differences in transfection conditions. When co-transfection studies were performed with the CYP2C9
promoter variants, two of the inferred CYP2C9
haplotypes exhibited an altered induction profile; haplotype 1D resulted in a 1.6-fold reduction in inducibility, whereas haplotype 3B resulted in a 1.4-fold increase in inducibility. Given the frequency of these two haplotypes (6.6% and 2.4%, respectively) and the magnitude of this observed effect, it is possible these variants contribute to observed interindividual differences in CYP2C9 phenotype.
Previous studies have identified two constitutive androstane receptor (CAR)-responsive elements within the CYP2C9
promoter at positions -2,898 and -1,839 that bind human CAR in vitro
and transactivate reporter constructs (Ferguson et al., 2002
). Further, Gerbal-Chaloin et al. (2001)
described a 4-fold increase in CYP2C9
mRNA following treatment with phenobarbital, a known human CAR ligand. CAR-mediated phenobarbital induction of CYP2C9
promoter activity was not observed in the current study, although a two-fold, PXR-mediated phenobarbital enhancement of CYP2C9
promoter activity was observed. This latter observation supports the recent findings of Chen et al. (2004)
who determined that human PXR is responsible for the induction of human CYP2C9
by both rifampicin and phenobarbital.
To gain some insight into which SNP, or combination of SNPs, might be responsible for the observed altered activities, a careful comparison between all observed haplotypes was made. Reduced constitutive activity was observed with haplotypes 1B, 3A and 3B. As indicated earlier, the g.-4302C>T SNP is unique to haplotype 1B and as such, is assumed to be causative, yet does not alter any known or putative transcription factor binding sites. Several SNPs are found in common between haplotypes 3A and 3B, but not in other haplotypes (g.-8897C>A, g.-7419A>G, g.-5813A>G, g.-5661C>A, g.-4877G>A, -1911T>C, -1885C>G, g.-1537G>A, and g.-981G>A). However, similar to the haplotype 1B g.-4302C>T SNP, none of these variants alter known or putative regulatory elements. Altered PXR-mediated inducibility was observed with both haplotypes 1D and 3B. In the case of haplotype 1D (g.-3089G>A, g.-2663delTG, g.-1188T>C), two other inferred haplotypes (1C and 1E) also contain g.-3089G>A and/or g.-1188T>C but exhibit promoter activities no different from the reference construct. The remaining SNP in haplotype 1D, g.-2663delTG, is not present in any other inferred haplotype and as such, may be causative for the reduced inducibility of this promoter variant. Haplotype 3B resulted in an increase in rifampicin-induced CYP2C9
promoter activity, whereas haplotype 3A exhibited no difference compared to the reference control. Yet, the only difference between these two haplotypes is the presence of the g.-1188T>C SNP in haplotype 3B. This observation suggests that the g.-1188T>C transition might be responsible for the observed difference in induction profiles. However, two other haplotypes (1C and 1E) also contain g.-1188T>C, yet exhibit no difference relative to the control construct. Further, a recent study by Sandberg et al. (2004)
found that the g.-1188T>C variant did not affect gene expression in vitro
. Finally, haplotype 1D also contains the g.-1188T>C SNP, and, in contrast to haplotype 3B, was associated with decreased induction of CYP2C9
promoter activity. These findings suggest that g.-1188T>C cannot solely be responsible for the observed increase in activity, but rather contributes to a combinatorial effect. The simplest explanation as to how individual or clusters of SNPs might function to alter gene regulation is by modifying or eliminating transcription factor binding. However, this does not appear to be the case for the variants identified herein and is clearly not the case for many regulatory polymorphisms. A survey of 247 known promoters and 647 haplotype variants by Buckland et al. (2005)
revealed that only 35% of the functional regulatory variants identified were localized within predicted transcription factor binding sites. Thus, many regulatory polymorphisms may impact gene expression through other yet to be identified sequence-specific mechanisms.
In summary, this is the first extensive study of CYP2C9 haplotype and in vitro functional analysis in a Hispanic population of Mexican descent. Interindividual and interethnic differences were observed in the incidence and frequency of CYP2C9 regulatory polymorphisms. Further, based on in vitro assays, several of the inferred haplotypes are predicted to significantly decrease and/or alter PXR-mediated rifampicin-dependent CYP2C9 induction. These observations suggest that genetic variation within CYP2C9 regulatory sequences likely contributes to differences in CYP2C9 phenotype both within and among different populations. However, in vivo studies will be required to determine the relevance of these haplotype variants, if any, to clinical outcomes.