Genetic polymorphisms of PXR in Brazilians Moreira RPP et al. obtained from all subjects. Healthy volunteers consisted of 76 females and 41 males with a mean age 25.8 ¡ 11.8 years. The self-reported ethnicity of this cohort was 87.2% Caucasian and 12.8% African. In addition, 88% were from the southeast, 8.5% from the northeast, and 3.5% from the southern region of Brazil. METHODS DNA samples from all subjects were obtained from peripheral blood leukocytes using a salting out procedure. All coding regions of the PXR gene were analyzed in addition to three variants in non-coding regions that had a minor allele frequency of at least $ 0.10 in other ethnic groups. We also looked for three variants, which modified the PXR and/or CYP3A4 expression, previously described in non-coding regions with a minor allele frequency $ 0.10 in other ethnic groups.4,17 The PXR gene was amplified as previously described.4,18 The amplified products were sequenced using the Big Dye Terminator Sequencing KitTM (Applied Biosystem, Inc., Foster City, CA, USA) and capillary electro- phoresis was conducted on an ABI PRISM 3100 sequencer (Applied Biosystem, Inc.). Sequence traces were analyzed using Sequencher (version 4.5 build 1416) and assembled to the reference (NCBI accession number AF364606). Statistical Analysis When appropriate, identified allele frequencies were compared to the expected normal distribution in a popula- tion (Hardy-Weinberg equilibrium) by using a goodness-of- fit x2 test. Differences in allele frequencies between ethnic groups were analyzed using the x2 test. Values of p , 0.05 were considered to be significant. RESULTS The allelic frequencies of PXR polymorphisms that we identified in this study are shown in Table 1 along with previously published data from Asian, Caucasian, and African populations. The genotypic frequencies that we found are listed in Table 2. All polymorphisms were in Hardy-Weinberg equilibrium. We identified 11 polymorphisms (Tables 1 and 2), one of which is a new synonymous variant located in exon 4 CLINICS 2011;66(6):1041-1044 (4306 C.G) that was present in 1.3 % of Brazilian alleles. Eight polymorphisms were identified in the coding regions of the PXR gene and 3 polymorphisms in non-coding regions. Of the variants identified, 8055 C.T, -25913 C.T, and -6994 C.T are known to be associated with functionality. The 8055 C.T (rs2276707) variant was found in 12.5% of alleles, which is similar to its frequencies in Caucasian and African alleles of 15% and 18% (p . 0.05),12 respectively, but lower than its frequency in Chinese alleles of 45% (p , 0.05).19 The -25913 C.T (rs1523130) variant occurred in 50% of the Brazilian alleles, which is less frequent than in Caucasians (70%, p = 0.003, 95% CI: 0.66-0.76) but more frequent than in Africans (28%, p , 0.001, 95% CI: 0.49-0.61). The -6994 C.T (rs2472677) polymorphism was found in 46% of the Brazilian alleles, which is a higher frequency than in Africans (38.1%, p = 0.03, 95% CI: 0.80-0.85) but a lower frequency than in Caucasians (62%, p = 0.01, 95% CI: 0.70-0.79). The 79 C.T (rs12727613) polymorphism was found in 2.1% of Brazilian alleles. This variant is significantly more frequent in African alleles (20%, p , 0.001) and not present in Caucasians or Asians. The 106 G.A (rs12721607) polymorphism, which is absent in Asians, was found in 1.3% of alleles in this population, similar to its frequencies in Caucasians and Africans of 5% and 4%, respectively (p , 0.05). The 4321 G.A (rs12721608) polymorphism is absent in Africans but present in Brazilians at a similarly low frequency as that found in Caucasians (p , 0.05). The other five polymorphisms were detected at a frequency lower than 1% [4447 T.C (rs12721611), 4499 C.T (rs12721600), 4773 G.A (rs12721612), and 8528 G.A (rs59152710)]. DISCUSSION PXR polymorphisms have been implicated in modulating CYP3A4 and PXR expression, potentially accounting for interindividual differences in drug metabolism.20 These variants impact drug response and some types of drug interactions by affecting serum levels of the medicines and endogenous compounds, which can include PXR ligands and substrates of enzymes and drug transporters.13 The distribution of allelic variants in the PXR gene varies widely among different ethnic groups,12,14 which helps to explain variations in the pharmacokinetics and pharmacodynamics of drugs that are PXR ligands. Table 1 - Allelic frequency of polymorphisms in the PXR gene in Brazilians compared to Asians, Caucasians, and Africans. Location dbSNP *Position in AF364606 Amino Acid Change 59UTR Intron 1 Exon 2 Exon 2 Exon 4 Exon 4 Exon 4 Exon 5 Exon 6 Intron 6 Exon 8 rs1523130 rs2472677 rs12727613 rs12721607 - rs12721608 rs12721611 rs12721600 rs12721612 rs2276707 rs59152710 -25913 C.T -6994 C.T 79 C.T 106 G.A 4306 C.G 4321 G.A 4447 T.C 4499 C.T 4773 G.A 8055 C.T 8528 G.A - - Pro27Ser Gly36Arg Arg156Arg Gln122Arg Thr164Thr Gly220Gly Gly317Gly - Ala370Thr n˚ 801 801 801 801 801 Frequency 0.67 0 0 0 0.51 1042 aAfrican-American population. n˚: number of alleles. Data from Asians, Caucasians, and Africans was obtained from 1King et al. 2007, 2Hustert et al. 2001, 3Wang et al. 2008, 4Lamba et al. 2008, and 5Zhang et al. 2008. #Data from sub-Saharan Africans (Svard et al. 2010). Italics: new variant. *Position in relation to translation start site of PXR (NR1I2): GenBank Accession AF364606. Asians Caucasians Africansa This study Frequency Frequency Frequency n˚ 801 924 3002 801 1443 801 1505 3122 0.70 0.62 0 0.05 0.01 0 0.15 0 n˚ 801 714# 663 801 714# 801 225 642 0.28 0.381 0.20 0.04 0 0.03 0.18 0.016 n˚ 234 234 234 234 234 234 234 234 234 234 234 0.50 0.457 0.021 0.013 0.013 0.012 0.008 0.008 0.004 0.125 0.004