Three novel single nucleotide polymorphisms (SNPs) of the CYP2B6 gene in Japanese individuals.
ABSTRACT We sequenced all exons and exon-intron junctions of the CYP2B6 gene from 200 Japanese individuals. We found three novel single nucleotide polymorphisms (SNPs) (1375A>G, 1427G>A and 1454A>T) causing amino acid substitutions (Met(459)Val, Gly(476)Asp and Gln(485)Leu in exon 9), respectively. The detected SNP was as follows: 1) SNP, 031226Hiratsuka01; GENE NAME, CYP2B6; ACCESSION NUMBER, AC023172; LENGTH, 25 base; 5'-CAGAACTTCTCCA/GTGGCCAGCCCCG-3'. 2) SNP, 031226Hiratsuka02; GENE NAME, CYP2B6; ACCESSION NUMBER, AC023172; LENGTH, 25 base; 5'-CCCAGGAGTGTGG/ATGTGGGCAAAAT-3'. 3) SNP, 031226Hiratsuka03; GENE NAME, CYP2B6; ACCESSION NUMBER, AC023172; LENGTH, 25 base; 5'-CCCCAACATACCA/TGATCCGCTTCCT-3'.
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ABSTRACT: Cytochrome P450 (CYP) is a supergene family of metabolizing enzymes involved in the phase I metabolism of drugs and endogenous compounds. CYP oxidation often leads to inactive drug metabolites or to highly toxic or carcinogenic metabolites involved in adverse drug reactions (ADR). During the last decade, the impact of CYP polymorphism in various drug responses and ADR has been demonstrated. Fifty six percent of drugs involved in ADR are metabolized by polymorphic phase I metabolizing enzymes, 86% among them being CYP. Here, we review the major CYP polymorphic forms, their impact for drug response and current advances in molecular modeling of CYP polymorphism. We focus on recent studies exploring CYP polymorphism performed by the use of sequence- and/or protein structure- based computational approaches. The importance of understanding the molecular mechanisms related to CYP polymorphism and drug response at the atomic level is outlined.Journal of Molecular Biology 07/2013; · 3.91 Impact Factor
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ABSTRACT: Using a multiple alignment of 175 cytochrome P450 (CYP) family 2 sequences, 20 conserved sequence motifs (CSMs) were identified with the program PCPMer. Functional importance of the CSM in CYP2B enzymes was assessed from available data on site-directed mutants and genetic variants. These analyses suggested an important role of the CSM 8, which corresponds to(187)RFDYKD(192) in CYP2B4. Further analysis showed that residues 187, 188, 190, and 192 have a very high rank order of conservation compared with 189 and 191. Therefore, eight mutants (R187A, R187K, F188A, D189A, Y190A, K191A, D192A, and a negative control K186A) were made in an N-terminal truncated and modified form of CYP2B4 with an internal mutation, which is termed 2B4dH/H226Y. Function was examined with the substrates 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC), 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and testosterone and with the inhibitors 4-(4-chlorophenyl)imidazole (4-CPI) and bifonazole (BIF). Compared with the template and K186A, the mutants R187A, R187K, F188A, Y190A, and D192A showed > or =2-fold altered substrate specificity, k(cat), K(m), and/or k(cat)/K(m) for 7-MFC and 7-EFC and 3- to 6-fold decreases in differential inhibition (IC(50,BIF)/IC(50,4-CPI)). Subsequently, these mutants displayed 5-12 degrees C decreases in thermal stability (T(m)) and 2-8 degrees C decreases in catalytic tolerance to temperature (T(50)) compared with the template and K186A. Furthermore, when R187A and D192A were introduced in CYP2B1dH, the P450 expression and thermal stability were decreased. In addition, R187A showed increased activity with 7-EFC and decreased IC(50,BIF)/IC(50,4-CPI) compared with 2B1dH. Analysis of long range residue-residue interactions in the CYP2B4 crystal structures indicated strong hydrogen bonds involving Glu(149)-Asn(177)-Arg(187)-Tyr(190) and Asp(192)-Val(194), which were significantly-reduced/abolished by the Arg(187)-->Ala and Asp(192)-->Alasubstitutions, respectively.Journal of Biological Chemistry 06/2008; 283(31):21808-16. · 4.65 Impact Factor
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ABSTRACT: Identification of poor and rapid metabolizers for the category of drugs metabolized by cytochrome P450 2B6 (CYP2B6) is important for understanding the differences in clinical responses of drugs metabolized by this enzyme. This study reports the prevalence of poor and rapid metabolizers in North Indian population residing in the National Capital Territory. The prevalence of poor and rapid metabolizers was determined in the target population for the category of drugs metabolized by CYP2B6 by measuring plasma bupropion, a drug metabolized by CYP2B6, and its metabolite. Bupropion (75 mg) was administered to 107 volunteers, and the drug (bupropion) and its metabolite (hydroxybupropion) were determined simultaneously by LCMS/MS in the plasma. CYP2B6 activity was measured as hydroxybupropion/bupropion ratio, and volunteers were categorized as rapid or poor metabolizers on the basis of cutoff value of log (hydroxybupropion/bupropion). Significant differences were observed between the mean metabolite/drug ratio of rapid metabolizers (Mean = 0.59) and poor metabolizers (Mean = 0.26) with p<0.0001. Results indicate that 20.56% individuals in the target population were poor metabolizers for the category of drugs metabolized by CYP2B6. Cutoff value defined in this study can be used as a tool for evaluating the status of CYP2B6 using bupropion as a probe drug. The baseline information would be clinically useful before administering the drugs metabolized by this isoform.SpringerPlus 01/2012; 1:34.
Received; December 20, 2003, Accepted; January 17, 2004
*To whom correspondence should be addressed: Michinao MIZUGAKI, Ph.D., Department of Clinical Pharmaceutics, Tohoku Pharmaceutical
University, 4-4-1, Komatsushima, Aoba-ku, Sendai 981-8558, Japan. Tel. ＋81-22-234-4181, Fax. ＋81-22-275-2013, E-mail: mizugaki＠tohoku-
On December 26, 2003, the SNP did not appear either in the ``Human
Cytochrome P450 (CYP) Allele Nomenclature Committee database
(http:W W www.imm.ki.seW CYPallelesW )'' or the ``JSNPs database
(http:W W snp.ims.u-tokyo.ac.jpW )''.
Drug Metab. Pharmacokin. 19 (2): SNP1 (155)–SNP4 (158) (2004).
Three Novel Single Nucleotide Polymorphisms (SNPs) of the CYP2B6
Gene in Japanese Individuals
Masahiro HIRATSUKA1, Yudai HINAI1, Yumiko KONNO1, Hisayoshi NOZAWA1,2,
Shoetsu KONNO3and Michinao MIZUGAKI1,*
1Department of Clinical Pharmaceutics, Tohoku Pharmaceutical University, Sendai, Japan
2Department of Pharmacy, NTT East Tohoku Hospital, Sendai, Japan
3Department of Pharmacy, Hoshi General Hospital, Koriyama, Japan
Full text of this paper is available at http:/ /www.jssx.org
Summary: We sequenced all exons and exon-intron junctions of the CYP2B6 gene from 200 Japanese
individuals. We found three novel single nucleotide polymorphisms (SNPs) (1375AÀG, 1427GÀA and
1454AÀT) causing amino acid substitutions (Met459Val, Gly476Asp and Gln485Leu in exon 9), respec-
The detected SNP was as follows:
1)SNP, 031226Hiratsuka01; GENE NAME, CYP2B6; ACCESSION NUMBER, AC023172;
LENGTH, 25 base; 5?-CAGAACTTCTCCAW GTGGCCAGCCCCG-3?.
2)SNP, 031226Hiratsuka02; GENE NAME, CYP2B6; ACCESSION NUMBER, AC023172;
LENGTH, 25 base; 5?-CCCAGGAGTGTGGW ATGTGGGCAAAAT-3?.
3)SNP, 031226Hiratsuka03; GENE NAME, CYP2B6; ACCESSION NUMBER, AC023172;
LENGTH, 25 base; 5?-CCCCAACATACCAW TGATCCGCTTCCT-3?.
Key words: CYP2B6; genetic polymorphism; Japanese
CYP2B6 is involved in the metabolism of clinically
important drugs; including cyclophosphamide,1)ifos-
The capacity of the activation has been reported to vary
among individuals from four to nine times. Recent
publications indicate that the extensive interindividual
variability of CYP2B6 expression and function is
due not only to regulatory phenomena, but also to a
common genetic polymorphism.7)In addition to the
wild-type allele CYP2B6*1, to date there are eight
variants, designated CYP2B6*2 (64CÀT), CYP2B6*3
(1459CÀT), CYP2B6*6 (516GÀT and 785AÀG),
CYP2B6*8 (415AÀG), and CYP2B6*9 (516GÀT) (see
http:W W www.imm.ki.seW CYPallelesW ).7,8)Recently, we
have reported allele frequencies for CYP2B6*2–7 in 256
Japanese subjects using genotyping methods.9)In this
study, we sequenced all exons and exon-intron junctions
of the CYP2B6 gene from 200 Japanese individuals. We
identiˆed three novel SNPs of the CYP2B6 gene.
Materials and Methods
Venous blood was obtained from 200 unrelated
Japanese volunteers of Tohoku University Hospital.
The local Ethics Committee of Tohoku University
Hospital and TohokuPharmaceutical
approved the study and written, informed consent was
obtained from all blood donors. DNA was isolated
from anticoagulated (with K2EDTA) peripheral blood
using a DNA Extractor WB-Rapid Kit (Wako Pure
Chemical Industries, Osaka, Japan) or a QIAamp DNA
Mini Kit (QIAGEN, Hilden, Germany) according to the
Table 1 lists the primer pairs (PCR) used to amplify
Table 1. Primers used for ampliˆcation and sequencing of the CYP2B6 gene
(5? to 3?)
(5? to 3?)
Temp. (9 C)
Exon 1 ccacacacccacacattcacttgctcacct
tgtttggtgaagcttccccaagtaccaagg60.0PCR and sequence
65.0 PCR and sequence
Exon 4 gcctctcggtctgcccatctataaac tgttctgggtgttcacctcacccccacacc60.0 PCR and sequence
64.3PCR and sequence
tctcgttgtttttctcaagttggggatagt60.0 PCR and sequence
Exon 9 cttatgcaaatctgttgcagtggacatttg
60.0PCR and sequence
Masahiro HIRATSUKA, et al.SNP2 (156)
CYP2B6 exons. These primers were designed based on
the genomic sequence reported in the Genbank with
accession number: AC023172. Amplicons for exons 1,
4, 7–8, and 9 were generated with the AmpliTaq Gold
PCR Master Mix by Applied Biosystems (Foster City,
CA, USA). The PCR reactions proceeded using a
BIO-RAD iCycler (Hercules, CA, USA). The thermal
proˆle consisted of denaturation at 959 C for 10
minutes, followed by 30 cycles of denaturation at 959 C
for 30 seconds, annealing for 30 seconds, extension at
729 C for 30 seconds, and a ˆnal extension at 729 C for 7
minutes. The annealing temperatures are indicated in
Table 1. Amplicons for exons 2–3 were generated with
the LA-Taq DNA polymerase by TaKaRa Co. (Kyoto,
Japan). The thermal proˆle consisted of denaturation at
949 C for 5 minutes, followed by 30 cycles of denatura-
tion at 949 C for 30 seconds, annealing at 659 C for 60
seconds, extension at 729 C for 90 seconds, and a ˆnal
extension at 729 C for 7 minutes. Amplicons for exons
5–6 were generated with the Ex-Taq DNA polymerase
by TaKaRa Co. (Kyoto, Japan). The thermal proˆle
consisted of denaturation at 949 C for 5 minutes, fol-
lowed by 30 cycles of denaturation at 949 C for 30
seconds, annealing at 64.39 C for 60 seconds, extension
at 729 C for 90 seconds, and a ˆnal extension at 729 C for
Sequences were determined using the CEQ8000}
automated DNA sequencer (Beckman-Coulter Inc.,
Fullerton, CA, USA). The PCR products were se-
quenced by the ‰uorescent dideoxy termination reaction
method using the DTCS DNA Sequencing Kit (Beck-
man-Coulter Inc.) according to manufacturer's instruc-
tions with the primers (sequence) listed in Table 1.
Results and Discussion
We found three novel SNPs as follows:
LENGTH, 25 base; 5?-CAGAACTTCTCCAW GTGGC-
LENGTH, 25 base; 5?-CCCAGGAGTGTGGW ATGT-
3) SNP, 031226Hiratsuka03;
LENGTH, 25 base; 5?-CCCCAACATACCAW TGAT-
The electropherograms of the novel SNPs are shown
in Fig. 1. The positions of these SNPs were 459
(031226Hiratsuka01), 476 (031226Hiratsuka02), and
485 (031226Hiratsuka03) in exon 9. All of the SNPs
were heterozygous, and their frequencies were 0.0075
for 031226Hiratsuka01, 0.005 for 031226Hiratsuka02,
and 0.0025 for 031226Hiratsuka03 in the Japanese
population. Sequences from each sample were obtained
from at least three diŠerent PCR ampliˆcations.
Ariyoshi et al. have reported that the 516GÀT
O-deethylase activity of the CYP2B6 enzyme in an in
vitro assay.10)Further, Jinno et al. have also reported
that compared with CYP2B6*1, the alleles CYP2B6*4
(Gln172His; Lys262Arg), and CYP2B6*7 (Gln172His;
Lys262Arg; Arg487Cys) are associated with a higher in-
trinsic clearance of 7-ethoxy-4-tri‰uoromethylcouma-
rin.11)Xie et al. have reported that the CYP2B6*6 carri-
ers has signiˆcantly higher cyclophosphamide 4-hydrox-
ylation.12)In contrast Lang et al. have reported that
The nucleotide sequences of the CYP2B6 gene at exon 9. The sequences are shown for sense strands. Arrows indicate the variant nucleo-
Novel SNPs of CYP2B6 in Japanese
signiˆcantly reduced CYP2B6 protein expression and
S-mephenytoin N-demethylase activity were found in
carriers of the 1459CÀT (Arg487Cys) mutation (alleles
CYP2B6*5 and CYP2B6*7).7)Furthermore, it has been
reported that the 1459CÀT (Arg487Cys) are associated
with the lowest level of CYP2B6 activity in livers of
females.8)The 1459CÀT SNP in exon 9 are closely
located in the substrate recognition site.13)These three
SNPs in this study are also located in the site. In particu-
lar, the Gly476 is conserved in the CYP2 family.13)
Thus, these amino acid substitutions are expected to
alter the catalytic properties of the CYP2B6.
Acknowledgements: This work was supported in part by
a Grant-in-Aid for Research on Advanced Medical
Technology from the Ministry of Health, Labor and
Welfare of Japan.
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