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Pharmacogenetics of CYP1A2, Novel Polymorphisms and Haplotypes in Three Distinct Asian Populations
Drug Metab. Pharmacokinet. 25 (6): 616–623 (2010).
SNP Communication Pharmacogenetics of CYP1A2, Novel Polymorphisms and Haplotypes in Three Distinct Asian Populations Joanne Siok Liu LIM1,2, Onkar SINGH1, Rathi Devi RAMASAMY1, Saminathan RAMASAMY1, Koilan SUBRAMANIAN1, Edmund JD LEE2 and Balram CHOWBAY1,* 1Laboratory of Clinical Pharmacology, Division of Medical Sciences, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore 2Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: CYP1A2 play an important role in the metabolism of many carcinogens and clinically important drugs. CYP1A2 activity has been found to be influenced by the presence of polymorphic variants which were reported to display wide interethnic variation. This study investigates the frequency distribution and linkage disequilibrium patterns of CYP1A2 genetic polymorphisms, and characterize their haplotype structures in three healthy Asian populations in Singapore (Chinese, Malay, and Indian). The entire CYP1A2 gene was screened in 126 healthy subjects from all three ethnic groups (N＝42 each). A total of 25 polymorphisms was identified, of which nine were novel. The polymorphisms, －2467delT and －163CÀA were detected at high frequencies in all Asian ethnic groups. Significant interethnic differences were observed in the genotypic frequency distribution of IVS2－99GÀA (Pº0.01) and 1548CÀT (P＝ 0.05) across the three ethnic groups while －163CÀA (P＝0.02) was found to differ between Chinese and Malays. Haplotype analyses revealed four to six major haplotypes in each ethnic population which accounted for more than 60z of the cumulative haplotype frequencies. Future studies should be done to investigate the functional roles of these haplotypes. Keywords: CYP1A2; pharmacogenetics; polymorphism; linkage disequilibrium; haplotypes; Asians
ticularly those with narrow therapeutic indices. It has been shown that approximately 28% of the variation in CYP1A2 activity is due to environmental factors such as induction of CYP1A2 by cigarette smoking and inhibition by grapefruit juice1,4) while an estimated 39% to 72% of the variation in CYP1A2 activity are due to genetic factors.5) The CYP1A2 gene is mapped onto chromosome 15, spans 7.8 kb and consists of 7 exons including a non-coding exon 1. To date, more than 150 variant alleles have been reported,6,7) of which approximately 26 are nonsynonymous polymorphisms (SNPs)1) present at frequencies of less than 1%. The polymorphic variants present in the 5? transcriptional regulatory region [－3860GÀA (*1C; rs2069514), －3113AÀG (rs2069521), －
Introduction CYP1A2 belongs to the cytochrome P450 (CYP450) superfamily of phase I metabolic enzymes and represents approximately 15% of the total P450 protein content in the liver. It is constitutively expressed in the liver and is involved in the metabolism of 8–10% of clinically important drugs such as caffeine, olanzapine, and theophylline.1,2) In addition, this enzyme is also involved in the catalytic activation of several carcinogenic heterocyclic amines to reactive metabolites that are implicated in the development of various cancers.3) A wide degree of interindividual as well as interethnic variability in CYP1A2 activity exists, which may affect the efficacy and toxicity profiles of putative drugs metabolized by CYP1A2, par-
Received; May 31, 2010, Accepted; August 10, 2010, J-STAGE Advance Published Date; October 1, 2010 *To whom correspondence should be addressed: Balram CHOWBAY, PhD, Principal Pharmacologist, Clinical Pharmacology Laboratory, Division of Medical Sciences, Humphrey Oei Institute of Cancer Research, National Cancer Centre, 11 Hospital Drive, Singapore 169610. Tel. (65) 64368321, Fax. (65) 63720161, E-mail: ctebal＠nccs.com.sg This study was supported by National Medical Research Council (NMRCB1011 and NMRC/1159/2008) and Singapore Cancer Syndicate (PS00123R) grants.
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2467delT (*1D; rs35694136)] and intron 1 [－739TÀG (*1E; rs2069526), －729CÀT (rs12720461) and － 163CÀA (*1F; rs762551)] have been found to be present at higher frequencies and associated with functional changes in enzyme activity.8–12) As the N3-demethylation of caffeine to 1,7-dimethylxanthine is predominantly mediated by CYP1A2, the metabolic ratio of 1,7-dimethylxanthine to caffeine has been widely used as an indicator of CYP1A2 catalytic activity.1) Sachse et al.10) first associated the －163CÀA (*1F; rs762551) polymorphism with higher caffeine metabolic ratio in smokers of Caucasian ethnic origin. A subsequent study by Aklillu et al.8) identified 4 haplotypes (CYP1A2*1A, *1F, *1J and *1K) based on the linkage disequilibrium (LD) between －163CÀA (*1F; rs762551), －739TÀG (*1E; rs2069526) and －729CÀT (rs12720461) in the Ethiopian population. CYP1A2*1K, which comprised － 163CÀA (*1F; rs762551), －739TÀG (*1E; rs2069526) and －729CÀT (rs12720461) was associated with 40% lower enzymatic activity compared to the reference *1A haplotype. There were no significant differences in the activity of CYP1A2 among individuals harboring the haplotypes *1A, *1F (－163CÀA; rs762551) and *1J [*1F (－163CÀA; rs762551) and *1E (－739TÀG; rs2069526)].13) It has been suggested that the differences in results reported by Sachse et al.10) and Aklillu et al.8) may be due to the incomplete screening of SNPs in the CYP1A2 gene.14) The studies carried out thus far have focused on selected CYP1A2 SNPs and comprehensive screening for polymorphisms across the whole gene is lacking. As the pattern of LD has been shown to differ between ethnic populations14) and a comprehensive screening of the CYP1A2 gene in a diverse group of Asian populations has not been reported previously, this study was conducted to investigate the frequencies of CYP1A2 polymorphisms and the patterns of LD, followed by characterization of haplotype structures in three distinct healthy Asian populations in Singapore, namely, the Chinese, Malay, and Indian populations.
Materials and Methods Healthy subjects: A total of 126 healthy Asian subjects [Chinese (N＝42), Malay (N＝42), Indians (N＝42)] were recruited for the study. The ethnicities of all subjects were traced back two generations and confirmed by verbal questioning and verified against their National Registry Identification Cards. The study was approved by the Institutional Review Board of the National Cancer Centre, Singapore. Informed consent was obtained from all subjects for participation in the study. Pharmacogenetic analysis of CYP1A2 polymorphisms: The phenol-chloroform extraction method was employed to extract genomic DNA from the peripheral blood of healthy subjects (N＝126). Poly-
merase chain reactions (PCRs) were carried out to amplify the entire CYP1A2 gene (UCSC Reference Sequence: NM_000761) spanning approximately 11.8 kb. The amplified PCR products were purified with Exonuclease 1 and Shrimp Alkaline Phosphatase followed by direct sequencing using Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, CA). Statistical, linkage disequilibrium (LD) and haplotype analysis: Hardy-Weinberg equilibrium between the genotype frequencies was confirmed using Chi-square test. Pairwise LD between the polymorphisms was analyzed using HelixTree software (HelixTree} Genetics Analysis Software, Golden Helix Inc.) and quantified by `D?` and rho square (R2) values. The haplotypes for each ethnic group were inferred by application of the expectation-maximization algorithm (Haploview 4.0).15)
Results and Discussion Pharmacogenetics of CYP1A2 in healthy Asian populations: In the present study, 126 healthy individuals from the 3 Asian ethnic groups (Chinese, Malay and Indians) were screened for the presence of polymorphic variants in the CYP1A2 gene and 25 polymorphisms were detected. There were 11 polymorphisms detected in the 5? transcriptional regulatory region, four in the exonic regions, nine in the intronic regions and one in the 3? untranslated region (Table 1). Nine polymorphisms (－2602insA, －202CÀA, －114GÀA, IVS4＋ 92GÀA, 1049TÀG, IVS5＋199CÀT, IVS6＋418CÀ T, 1459GÀA and 1503CÀT) were novel SNPs (Table 1) with minor allele frequencies of 5% or less in each of the three ethnic groups (Table 2). Among the 25 SNPs, 8 variants were ethnic-specific and present at low allelic frequencies ranging between 0.01 and 0.05: －2602insA, －202CÀA, －114GÀA and 1503CÀT were present in the Chinese population; 1049TÀG and IVS5 ＋199CÀT in the Malay population; IVS2－99GÀA (rs34264399) and IVS4＋92GÀA in the Indian population. The －2808AÀC (rs12592480) and －733GÀC (rs28399417) SNPs were present in both the Chinese and Malay populations but monomorphic in the Indian population. Genotypic distributions of IVS2－99GÀA (rs34264399) and 1548CÀT (*1B; rs2470890) were observed to exhibit significant interethnic differences across all three Asian populations (Pº0.01 and P＝0.05, respectively). The －2467delT (*1D; rs35694136) and －163CÀA (*1F; rs762551) SNPs were detected at high frequencies in the Asian ethnic groups. The allelic frequency of －163CÀA (*1F; rs762551) was significantly higher in the Malay population (0.78) compared to the Indian population (0.58; P＝0.02). This highly polymorphic variant has previously been shown to be associated with increased inducibility of CYP1A2.10,16) Subsequently, the allele was correlated to higher caffeine metabolic ratio10)
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Table 1. Polymorphisms present in human CYP1A2 gene
SNPs
Location
Position from the translational initiation site or from the end of the nearest exonb
－2847TÀC －2808AÀC －2602insA# －2467delT (*1D) －1804AÀC/G －1708TÀC －739TÀG (*1E) －733GÀC －202CÀA# －163CÀA (*1F) －114GÀA# IVS2－249CÀT IVS2－99GÀA IVS4＋43GÀA IVS4＋92GÀA# IVS4－65GÀC 1049TÀG# IVS5＋199CÀT# IVS5－318GÀC IVS6＋81TÀC IVS6＋418CÀT# 1459GÀA# 1503CÀT# 1548CÀT (*1B) *1324CÀG
5? upstream 5? upstream 5? upstream 5? upstream 5? upstream 5? upstream intron 1 intron 1 intron 1 intron 1 intron 1 intron 2 intron 2 intron 4 intron 4 intron 4 exon 4 intron 5 intron 5 intron 6 intron 6 exon 7 exon 7 exon 7 3? untranslated region
－2847 －2808 －2602 －2467 －1804 －1708 －739 －733 －202 －163 －114 IVS2－249 IVS2－99 IVS4＋43 IVS4＋92 IVS4－65 1049 IVS5＋199 IVS5－318 IVS6＋81 IVS6＋418 1459 1503 1548 2875
SNP ID This study LCP_P1CP12_001 LCP_P1CP12_002 LCP_P1CP12_003 LCP_P1CP12_004 LCP_P1CP12_005 LCP_P1CP12_006 LCP_P1CP12_007 LCP_P1CP12_008 LCP_P1CP12_009 LCP_P1CP12_010 LCP_P1CP12_011 LCP_P1CP12_012 LCP_P1CP12_013 LCP_P1CP12_014 LCP_P1CP12_015 LCP_P1CP12_016 LCP_P1CP12_017 LCP_P1CP12_018 LCP_P1CP12_019 LCP_P1CP12_020 LCP_P1CP12_021 LCP_P1CP12_022 LCP_P1CP12_023 LCP_P1CP12_024 LCP_P1CP12_025
dbSNP (NCBI) rs2069522 rs12592480 rs35694136 rs2069524 rs2069525 rs2069526 rs28399417 rs762551 rs4646425 rs34264399 rs2472304 rs3743484
rs55711332 rs4646427
rs2470890 rs17861162
Nucleotide change
ACATGCTCTGTTT/CTCTATTGGATTC CTTGGCATCTTCA/CCAGGTTGATCCC CTCTACAAAAAA-/AATGTTTAAAAAT GATTGTGGCACAT/-GAACCCCAACCTG GGTGATCCGCCCA/C/GTCTCGGCCTCTC AAAAGGAGGTTGT/CGGGGATCATGAC TAGGTGTAGGGGT/GCCTGAGTTCCGG TAGGGGTCCTGAG/CTTCCGGGCTTTG TCCTTTCCAGCTC/ATCAGATTCTGTG AGCTCTGTGGGCC/ACAGGACGCATGG ATCCAGCTGGGAG/ACCAAGCACAGAA TGTTCACACTAAC/TCTTTTCCTTCTT TGGGAGGATAGGG/AGGGTACCCAGCC GAGAAGCCTTGAG/AACCCAGGTTGTT TGTGCCTGCTGTG/ATGCAAGCCCTGG CATGGGGTATAAG/CAGGGGATAATTC CTACAGACACTGT/GGATTGGCAGGGA TTGTGATCTCAGC/TGCTCATTCCCCT CCCAGGCTGCCTG/CCTGCTTTTTTTT AGGAACTGTTTAT/CATAATGAAAGGA GTGAGCCGAGATC/TGCTCCACTGCAC GTGCCGCCGGGCG/ATGAAAGTCGACC GACCATGAAGCAC/TGCCCGCTGTGAA CTTCTCCATCAAC/TTGAAGAAGACAC TGAGCCACGGTGC/GCCGGCCCACAAT
a. #denotes novel polymorphism not reported in the National Center for Biotechnology Information dbSNP (http://www.ncbi.nlm.nih.gov/SNP/). Information correct as of May 10, 2010. b. NM_000761 was used as the reference sequence
Table 2. Genotypic and allelic frequencies of polymorphisms in CYP1A2 Genotype frequency, n (%) SNPs
Allele Chinese
－2847TÀC
－2808AÀC
－2602insA#
－2467delT (*1D) －1804AÀC/G
－1708TÀC
Allele Frequency
Genotype TT TC TT AA AC CC wt/wt wt/insA insA/insA wt/wt wt/delT delT/delT AA AC AG CC CG GG TT TC CC
38 4 0 41 1 0 40 2 0 4 18 16 33 0 3 0 0 0 33 5 1
(90.5) (9.5) (0) (97.6) (2.4) (0) (95.2) (4.8) (0) (10.5) (47.4) (42.1) (91.7) (0) (8.3) (0) (0) (0) (84.6) (12.8) (2.6)
Malays 33 7 0 36 3 1 42 0 0 7 19 16 31 0 3 0 0 0 30 4 1
(82.5) (17.5) (0) (90) (7.5) (2.5) (100) (0) (0) (16.7) (45.2) (38.1) (91.2) (0) (8.8) (0) (0) (0) (85.7) (11.4) (2.9)
Indians 32 7 0 40 0 0 41 0 0 12 20 9 31 1 5 0 1 0 33 8 0
(82.1) (17.9) (0) (100) (0) (0) (100) (0) (0) (29.3) (48.8) (22) (81.6) (2.6) (13.2) (0) (2.6) (0) (80.5) (19.5) (0)
Chinese
Malays
Indians
T C
0.95 0.05
0.91 0.09
0.91 0.09
A C
0.99 0.01
0.94 0.06
1.00 0.00
wt insA
0.98 0.02
1.00 0.00
1.00 0.00
wt delT
0.34 0.66
0.39 0.61
0.54 0.46
A C G
0.96 0.00 0.04
0.96 0.00 0.04
0.90 0.01 0.09
T C
0.91 0.09
0.91 0.09
0.90 0.10
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Table 2. Continuation Genotype frequency, n (%) SNPs
Allele Chinese
－739TÀG (*1E) －733GÀC
－202CÀA#
－163CÀA (*1F) －114GÀA#
IVS2－249CÀT
IVS2－99GÀA
IVS4＋43GÀA
IVS4＋92GÀA
#
IVS4－65GÀC
1049TÀG
#
IVS5＋199CÀT
#
IVS5－318GÀC
IVS6＋81TÀC
IVS6＋418CÀT
1459GÀA
1503CÀT
#
#
1548CÀT (*1B) *1324CÀG
a.
#
Allele Frequency
Genotype
#
TT TG GG GG GC CC CC CA AA CC CA AA GG GA AA CC CT TT GG GA AA GG GA AA GG GA AA GG GC CC TT TG GG CC CT TT GG GC CC TT TC CC CC CT TT GG GA AA CC CT TT CC CT TT CC CG GG
denotes novel polymorphism
39 3 0 38 3 1 37 0 2 4 14 20 40 0 1 38 4 0 41 0 0 33 2 1 41 0 0 23 9 1 38 0 0 42 0 0 24 15 1 31 4 0 36 1 0 39 1 0 39 1 0 30 10 0 20 16 2
(92.9) (7.1) (0) (90.5) (7.1) (2.4) (94.9) (0) (5.1) (10.5) (36.8) (52.6) (97.6) (0) (2.4) (90.5) (9.5) (0) (100) (0) (0) (91.7) (5.6) (2.8) (100) (0) (0) (69.7) (27.3) (3) (100) (0) (0) (100) (0) (0) (60) (37.5) (2.5) (88.6) (11.4) (0) (97.3) (2.7) (0) (97.5) (2.5) (0) (97.5) (2.5) (0) (75) (25) (0) (52.6) (42.1) (5.3)
Malays 36 6 0 37 4 1 41 0 0 1 15 23 41 0 0 35 6 0 40 0 0 28 2 6 40 0 0 22 13 2 37 0 2 41 1 0 26 12 4 31 4 0 38 2 0 37 1 0 38 0 0 28 6 4 19 12 6
(85.7) (14.3) (0) (88.1) (9.5) (2.4) (100) (0) (0) (2.6) (38.5) (59) (100) (0) (0) (85.4) (14.6) (0) (100) (0) (0) (77.8) (5.6) (16.7) (100) (0) (0) (59.5) (35.1) (5.4) (94.9) (0) (5.1) (97.6) (2.4) (0) (61.9) (28.6) (9.5) (88.6) (11.4) (0) (95) (5) (0) (97.4) (2.6) (0) (100) (0) (0) (73.7) (15.8) (10.5) (51.4) (32.4) (16.2)
Indians 33 8 0 42 0 0 42 0 0 6 23 13 42 0 0 34 8 0 35 6 0 34 3 5 39 1 0 22 13 5 40 0 0 41 0 0 21 13 8 33 8 0 38 4 0 39 3 0 42 0 0 32 10 0 17 17 7
(80.5) (19.5) (0) (100) (0) (0) (100) (0) (0) (14.3) (54.8) (31) (100) (0) (0) (81) (19) (0) (85.4) (14.6) (0) (81) (7.1) (11.9) (97.5) (2.5) (0) (55) (32.5) (12.5) (100) (0) (0) (100) (0) (0) (50) (31) (19) (80.5) (19.5) (0) (90.5) (9.5) (0) (92.9) (7.1) (0) (100) (0) (0) (76.2) (23.8) (0) (41.5) (41.5) (17.1)
Chinese
Malays
Indians
T G
0.96 0.04
0.93 0.07
0.90 0.10
G C
0.94 0.06
0.93 0.07
1.00 0.00
C A
0.95 0.05
1.00 0.00
1.00 0.00
C A
0.29 0.71
0.22 0.78
0.42 0.58
G A
0.98 0.02
1.00 0.00
1.00 0.00
C T
0.95 0.05
0.93 0.07
0.90 0.10
G A
1.00 0.00
1.00 0.00
0.93 0.07
G A
0.94 0.06
0.81 0.19
0.85 0.15
G A
1.00 0.00
1.00 0.00
0.99 0.01
G C
0.83 0.17
0.77 0.23
0.71 0.29
T G
1.00 0.00
0.95 0.05
1.00 0.00
C T
1.00 0.00
0.99 0.01
1.00 0.00
G C
0.79 0.21
0.76 0.24
0.65 0.35
T C
0.94 0.06
0.94 0.06
0.90 0.10
C T
0.99 0.01
0.98 0.02
0.95 0.05
G A
0.99 0.01
0.99 0.01
0.96 0.04
C T
0.99 0.01
1.00 0.00
1.00 0.00
C T
0.88 0.12
0.82 0.18
0.88 0.12
C G
0.74 0.26
0.68 0.32
0.62 0.38
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and increased levels of carcinogen formation leading to greater susceptibility to several cancers.17–19) Recent studies have, however reported conflicting findings with regard to the －163CÀA (*1F; rs762551) allele being a cancer susceptibility allele.20–22) This could probably be attributed to wide interethnic differences in allelic frequency of the －163CÀA (*1F; rs762551) polymorphism and the varying linkage and haplotype patterns of －163CÀA (*1F; rs762551) with other CYP1A2 polymorphic variants.8,23) Similarly, the －2467delT (*1D; rs35694136) allele was noted to occur at a higher frequency in Chinese and Malay populations (0.66 and 0.61, respectively) compared to the Indian population (0.46) (Table 2). In a recent study, Pavanello et al.9) reported a significant association of this polymorphism with an increase in the metabolic ratio of caffeine as well as urinary mutagenicity (indicator of genotoxic effect), suggesting that －2467delT (*1D; rs35694136) may be associated with greater CYP1A2 enzymatic activity. With regards to the four exonic SNPs, two were synonymous polymorphisms [1503CÀT and 1548CÀT (*1B; rs2470890)] while the other two polymorphisms, 1049TÀG and 1459GÀA, were non-synonymous variants resulting in V350G and V487M amino acid changes, respectively. The residue V350 is located two amino acids from D348 in the N-terminal of helix J which is positioned on the surface of the CYP1A2 protein (3,24). The polymorphism 1042GÀA (*3; rs56276455) leads to the amino acid substitution D348N and has been previously reported to be associated with decreased expression of CYP1A2 in E. coli.25) In addition, helix J has been found to be conserved within the cytochrome P450 proteins.24,26) Hence, it is postulated that V350G may possibly exert an influence on the expression of CYP1A2. On the other hand, V487 is located between 2 beta sheets near the N-terminal of the CYP1A2 polypeptide and is 4 amino acids upstream of substrate binding site 6.27) Future studies should be done to investigate the phenotypic effect of these polymorphisms though the impact may be limited by the low variant allelic frequencies (0.05). Linkage disequilibrium (LD) & haplotype analyses: Pairwise LD analysis for the identified CYP1A2 polymorphisms suggested strong LD between the following polymorphisms across all three ethnic groups (Fig. －2847TÀC －1804AÀC/G 1): (rs2069522), (rs2069524), －1708TÀC (rs2069525), －739TÀG (*1E; rs2069526), IVS2－249CÀT (rs4646425) and IVS6＋81TÀC (rs4646427) with each other (`D?`＝ 1.00; R2À0.60) except IVS6＋81TÀC (rs4646427) with －1804AÀC/G (rs2069524) and －739TÀG (*1E; rs2069526) in Malays. The polymorphism IVS6＋ 81TÀC (rs4646427) was found to have moderate linkage with －1804AÀC/G (rs2069524) (`D?`＝0.63; R2 ＝0.54) and －739TÀG (*1E; rs2069526) (`D?`＝
Fig. 1. Pairwise LD of CYP1A2 polymorphisms in Chinese (A), Malay (B) and Indian (C) populations
0.77; R2＝0.75) in the Malay population. Close associations were observed among these polymorphisms in two ethnic groups: －2467delT (*1D; rs35694136) with －163CÀA (*1F; rs762551) and IVS4－65GÀC (rs2472304) (`D?`Æ0.86; R2Æ0.28) among Malays and Indians; *1324CÀG (rs17861162) with IVS4－65GÀC (rs2472304) and IVS5－318GÀC (rs55711332) (`D?`Æ0.89; R2Æ0.67) in Chinese and Malays. The polymorphisms found in strong linkage in one ethnic group were: *1324CÀG (rs17861162), －1708TÀC (rs2069525) and －2467delT (`D?`＝1; R2Æ0.35) among Indian population and *1324CÀG (rs17861162) with －733GÀC (rs28399417) (`D?`＝1; R2＝0.43) in the Chinese population. A total of 16 haplotypes was identified in the Chinese
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a. b. c. d. e.
－739TÀG
－733GÀC
－202CÀA
－163CÀA
－114GÀA
IVS2－249CÀT
IVS2－99GÀA
IVS4＋43GÀA
IVS4＋92GÀA
IVS4－65GÀC
1049TÀG
IVS5＋199CÀT
IVS5－318GÀC
W W W W W W W W W
W W D D D W W W D
A A A A G A A A A
T T T T C T T T T
T T T T G T T T T
G G G G G G G G C
C C C C C C C C C
C C A A A C A A A
G G G G G G G G G
C C C C T C C C C
G G G G G G G G G
G G G G G A G A G
G G G G G G G G G
G G G C G G G G C
T T T T T T T T T
C C C C C C C C C
G G G C G G G G C
*1324CÀG
－1708TÀC
A A A A A A A A A
1548CÀT
－1804AÀC/G
0.21 — 0.21 0.07 0.07 0.06 — —
1503CÀT
－2467delT
Indians
0.11 0.26 0.13 — — 0.06 0.05 0.05
1459GÀA
－2602InsA
Malays
Chinese
Average Frequency
Common Haplotypes
Reference Allelesc H1a 0.18 0.20 H2 0.28 0.31 H3 0.15 0.11 H4 0.07 — H5 0.07 — H6 0.06 — H7 0.05 — H8 0.05 0.05
IVS6＋81TÀC
－2808AÀC
T T T T C T T T T
Frequency
IVS6＋418CÀT
－2847TÀC
Table 3. CYP1A2 haplotypes and their frequencies among Asian ethnic groups
T T T T C T T T T
C C C C C C C C C
G G G G G G G G G
C C C C C C C C C
C C C C C C T T C
C C C G G C C C G
H1 is the reference haplotype. Polymorphisms selected as htTAG SNPs are bolded. Abbreviations: W-wild type, D-deletion and I-insertion. Average Frequency refers to average of the observed frequencies in each ethnic group. `—' refers to haplotype frequency of less than 0.05 in the ethnic group.
Fig. 2. Network diagram illustrating the relationship between CYP1A2 common haplotypes in healthy Asian populations Size of nodes indicates average frequencies of haplotypes; H, haplotype; mv, median vector.
population and four high frequency haplotypes (Æ5.0%) accounted for a cumulative frequency of 66.7%. There were 22 haplotypes identified in the Malay population with six high frequency haplotypes occurring with a cumulative frequency of 66.9%. Haplotype structure diversity was greatest in the Indian population, with five of 24 high frequency haplotypes that constituted 61.6% of all haplotypes. The cumulative frequencies of minor haplotypes were observed to be 9.4%, 4.0% and 1.5% in Chinese, Malay and Indian populations, respectively. The high frequency haplotypes in each ethnic population were further aligned to identify eight common haplotypes (H1-H8; Table 3). Haplotype H1, the reference haplotype, and H3 were common in all ethnic groups. Other haplotypes were ethnic-specific: H2 and H8 in Malays and Chinese; H6 in Malays and Indians; H4 and H5 in Indians and H7 in Malay. The haplotype H2,
tagged by the polymorphisms －2467delT (*1D; rs35694136) and －163CÀA (*1F; rs762551), displayed the highest average frequency at 28.4% while the reference haplotype H1 was observed in 17.5% of the populations. The haplotype network between the common haplotypes was calculated using the median joining algorithm (Fig. 2).28) Six polymorphisms were identified as htTAG SNPs: －739TÀG (*1E; rs2069526), *1324CÀ G (rs17861162), －2467delT (*1D; rs35694136), －163CÀA (*1F; rs762551), IVS4＋43GÀA (rs2472304) and －733GÀC (rs28399417). The haplotypes H6 and H7 as well as H1 and H5 were closely related and both pairs of haplotypes could be distinguished by IVS4＋43GÀA (rs2472304), while haplotypes H3 and H8 could be differentiated by －733GÀC (rs28399417). Haplotype H4 was observed to display the greatest diversity from other common haplotypes. H4
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was connected to other haplotypes by a median vector (mv2) that was tagged by 6 polymorphic variants (Fig. 2) and －739TÀG (*IE; rs2069526) was chosen as htTAG SNP. The －2467delT (*1D; rs35694136) and －163CÀA (*1F; rs762551) alleles were found to be in strong LD in the present study and was represented by H2. This haplotype was present in the Chinese and Malay populations at a frequency consistent with the Korean population (Chinese: 0.31; Malay: 0.26; Korean:0.26) but absent in the Indian population. The frequency of H2 is much lower in the Swedish population at 0.12. This haplotype was previously reported as CYP1A2*1V29) and has been found to be associated with higher plasma caffeine metabolic ratio in Turkish subjects.13) However, this conclusion differs from an earlier study by Ghobti et al.23) This discrepancy may be attributed to the different polymorphic variants included in the two studies. In the study by Ghobti et al.,23) the 1548CÀT (*1B; rs2470890) polymorphism was not included in the construction of the haplotypes while Gunes et al.13) investigated the genotypic distribution of this polymorphism and included it in the derivation of haplotypes in their population. This suggest that the 1548CÀT (*1B; rs2470890) allele may alter the phenotypic effect of the H2 haplotype. In view of the high frequency of H2 haplotype in Asian populations and that both alleles are associated with increased enzymatic activity, further studies evaluating the effects of the haplotype on the in vitro activity of CYP1A2 as well as the pharmacokinetics of substrates metabolized by CYP1A2 are warranted. In summary, this study is the first comprehensive report on the pharmacogenetics of CYP1A2 in three healthy Asian populations in Singapore. The pharmacogenetic profiles of our study populations were found to be similar to other Asian populations (Koreans and Japanese).14,29) The phenotypic effects of the nine novel polymorphisms remain to be investigated in future studies. Haplotype analyses revealed great inter-ethnic diversity in haplotype structures which may explain the differences in the distribution of CYP1A2 metabolic status between different populations. The impact of these haplotypes on cancer susceptibility and pharmacology of CYP1A2 substrates should be further evaluated in the future.
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SNP Communication Pharmacogenetics of CYP1A2, Novel Polymorphisms and Haplotypes in Three Distinct Asian Populations Joanne Siok Liu LIM1,2, Onkar SINGH1, Rathi Devi RAMASAMY1, Saminathan RAMASAMY1, Koilan SUBRAMANIAN1, Edmund JD LEE2 and Balram CHOWBAY1,* 1Laboratory of Clinical Pharmacology, Division of Medical Sciences, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore 2Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: CYP1A2 play an important role in the metabolism of many carcinogens and clinically important drugs. CYP1A2 activity has been found to be influenced by the presence of polymorphic variants which were reported to display wide interethnic variation. This study investigates the frequency distribution and linkage disequilibrium patterns of CYP1A2 genetic polymorphisms, and characterize their haplotype structures in three healthy Asian populations in Singapore (Chinese, Malay, and Indian). The entire CYP1A2 gene was screened in 126 healthy subjects from all three ethnic groups (N＝42 each). A total of 25 polymorphisms was identified, of which nine were novel. The polymorphisms, －2467delT and －163CÀA were detected at high frequencies in all Asian ethnic groups. Significant interethnic differences were observed in the genotypic frequency distribution of IVS2－99GÀA (Pº0.01) and 1548CÀT (P＝ 0.05) across the three ethnic groups while －163CÀA (P＝0.02) was found to differ between Chinese and Malays. Haplotype analyses revealed four to six major haplotypes in each ethnic population which accounted for more than 60z of the cumulative haplotype frequencies. Future studies should be done to investigate the functional roles of these haplotypes. Keywords: CYP1A2; pharmacogenetics; polymorphism; linkage disequilibrium; haplotypes; Asians
ticularly those with narrow therapeutic indices. It has been shown that approximately 28% of the variation in CYP1A2 activity is due to environmental factors such as induction of CYP1A2 by cigarette smoking and inhibition by grapefruit juice1,4) while an estimated 39% to 72% of the variation in CYP1A2 activity are due to genetic factors.5) The CYP1A2 gene is mapped onto chromosome 15, spans 7.8 kb and consists of 7 exons including a non-coding exon 1. To date, more than 150 variant alleles have been reported,6,7) of which approximately 26 are nonsynonymous polymorphisms (SNPs)1) present at frequencies of less than 1%. The polymorphic variants present in the 5? transcriptional regulatory region [－3860GÀA (*1C; rs2069514), －3113AÀG (rs2069521), －
Introduction CYP1A2 belongs to the cytochrome P450 (CYP450) superfamily of phase I metabolic enzymes and represents approximately 15% of the total P450 protein content in the liver. It is constitutively expressed in the liver and is involved in the metabolism of 8–10% of clinically important drugs such as caffeine, olanzapine, and theophylline.1,2) In addition, this enzyme is also involved in the catalytic activation of several carcinogenic heterocyclic amines to reactive metabolites that are implicated in the development of various cancers.3) A wide degree of interindividual as well as interethnic variability in CYP1A2 activity exists, which may affect the efficacy and toxicity profiles of putative drugs metabolized by CYP1A2, par-
Received; May 31, 2010, Accepted; August 10, 2010, J-STAGE Advance Published Date; October 1, 2010 *To whom correspondence should be addressed: Balram CHOWBAY, PhD, Principal Pharmacologist, Clinical Pharmacology Laboratory, Division of Medical Sciences, Humphrey Oei Institute of Cancer Research, National Cancer Centre, 11 Hospital Drive, Singapore 169610. Tel. (65) 64368321, Fax. (65) 63720161, E-mail: ctebal＠nccs.com.sg This study was supported by National Medical Research Council (NMRCB1011 and NMRC/1159/2008) and Singapore Cancer Syndicate (PS00123R) grants.
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2467delT (*1D; rs35694136)] and intron 1 [－739TÀG (*1E; rs2069526), －729CÀT (rs12720461) and － 163CÀA (*1F; rs762551)] have been found to be present at higher frequencies and associated with functional changes in enzyme activity.8–12) As the N3-demethylation of caffeine to 1,7-dimethylxanthine is predominantly mediated by CYP1A2, the metabolic ratio of 1,7-dimethylxanthine to caffeine has been widely used as an indicator of CYP1A2 catalytic activity.1) Sachse et al.10) first associated the －163CÀA (*1F; rs762551) polymorphism with higher caffeine metabolic ratio in smokers of Caucasian ethnic origin. A subsequent study by Aklillu et al.8) identified 4 haplotypes (CYP1A2*1A, *1F, *1J and *1K) based on the linkage disequilibrium (LD) between －163CÀA (*1F; rs762551), －739TÀG (*1E; rs2069526) and －729CÀT (rs12720461) in the Ethiopian population. CYP1A2*1K, which comprised － 163CÀA (*1F; rs762551), －739TÀG (*1E; rs2069526) and －729CÀT (rs12720461) was associated with 40% lower enzymatic activity compared to the reference *1A haplotype. There were no significant differences in the activity of CYP1A2 among individuals harboring the haplotypes *1A, *1F (－163CÀA; rs762551) and *1J [*1F (－163CÀA; rs762551) and *1E (－739TÀG; rs2069526)].13) It has been suggested that the differences in results reported by Sachse et al.10) and Aklillu et al.8) may be due to the incomplete screening of SNPs in the CYP1A2 gene.14) The studies carried out thus far have focused on selected CYP1A2 SNPs and comprehensive screening for polymorphisms across the whole gene is lacking. As the pattern of LD has been shown to differ between ethnic populations14) and a comprehensive screening of the CYP1A2 gene in a diverse group of Asian populations has not been reported previously, this study was conducted to investigate the frequencies of CYP1A2 polymorphisms and the patterns of LD, followed by characterization of haplotype structures in three distinct healthy Asian populations in Singapore, namely, the Chinese, Malay, and Indian populations.
Materials and Methods Healthy subjects: A total of 126 healthy Asian subjects [Chinese (N＝42), Malay (N＝42), Indians (N＝42)] were recruited for the study. The ethnicities of all subjects were traced back two generations and confirmed by verbal questioning and verified against their National Registry Identification Cards. The study was approved by the Institutional Review Board of the National Cancer Centre, Singapore. Informed consent was obtained from all subjects for participation in the study. Pharmacogenetic analysis of CYP1A2 polymorphisms: The phenol-chloroform extraction method was employed to extract genomic DNA from the peripheral blood of healthy subjects (N＝126). Poly-
merase chain reactions (PCRs) were carried out to amplify the entire CYP1A2 gene (UCSC Reference Sequence: NM_000761) spanning approximately 11.8 kb. The amplified PCR products were purified with Exonuclease 1 and Shrimp Alkaline Phosphatase followed by direct sequencing using Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, CA). Statistical, linkage disequilibrium (LD) and haplotype analysis: Hardy-Weinberg equilibrium between the genotype frequencies was confirmed using Chi-square test. Pairwise LD between the polymorphisms was analyzed using HelixTree software (HelixTree} Genetics Analysis Software, Golden Helix Inc.) and quantified by `D?` and rho square (R2) values. The haplotypes for each ethnic group were inferred by application of the expectation-maximization algorithm (Haploview 4.0).15)
Results and Discussion Pharmacogenetics of CYP1A2 in healthy Asian populations: In the present study, 126 healthy individuals from the 3 Asian ethnic groups (Chinese, Malay and Indians) were screened for the presence of polymorphic variants in the CYP1A2 gene and 25 polymorphisms were detected. There were 11 polymorphisms detected in the 5? transcriptional regulatory region, four in the exonic regions, nine in the intronic regions and one in the 3? untranslated region (Table 1). Nine polymorphisms (－2602insA, －202CÀA, －114GÀA, IVS4＋ 92GÀA, 1049TÀG, IVS5＋199CÀT, IVS6＋418CÀ T, 1459GÀA and 1503CÀT) were novel SNPs (Table 1) with minor allele frequencies of 5% or less in each of the three ethnic groups (Table 2). Among the 25 SNPs, 8 variants were ethnic-specific and present at low allelic frequencies ranging between 0.01 and 0.05: －2602insA, －202CÀA, －114GÀA and 1503CÀT were present in the Chinese population; 1049TÀG and IVS5 ＋199CÀT in the Malay population; IVS2－99GÀA (rs34264399) and IVS4＋92GÀA in the Indian population. The －2808AÀC (rs12592480) and －733GÀC (rs28399417) SNPs were present in both the Chinese and Malay populations but monomorphic in the Indian population. Genotypic distributions of IVS2－99GÀA (rs34264399) and 1548CÀT (*1B; rs2470890) were observed to exhibit significant interethnic differences across all three Asian populations (Pº0.01 and P＝0.05, respectively). The －2467delT (*1D; rs35694136) and －163CÀA (*1F; rs762551) SNPs were detected at high frequencies in the Asian ethnic groups. The allelic frequency of －163CÀA (*1F; rs762551) was significantly higher in the Malay population (0.78) compared to the Indian population (0.58; P＝0.02). This highly polymorphic variant has previously been shown to be associated with increased inducibility of CYP1A2.10,16) Subsequently, the allele was correlated to higher caffeine metabolic ratio10)
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Table 1. Polymorphisms present in human CYP1A2 gene
SNPs
Location
Position from the translational initiation site or from the end of the nearest exonb
－2847TÀC －2808AÀC －2602insA# －2467delT (*1D) －1804AÀC/G －1708TÀC －739TÀG (*1E) －733GÀC －202CÀA# －163CÀA (*1F) －114GÀA# IVS2－249CÀT IVS2－99GÀA IVS4＋43GÀA IVS4＋92GÀA# IVS4－65GÀC 1049TÀG# IVS5＋199CÀT# IVS5－318GÀC IVS6＋81TÀC IVS6＋418CÀT# 1459GÀA# 1503CÀT# 1548CÀT (*1B) *1324CÀG
5? upstream 5? upstream 5? upstream 5? upstream 5? upstream 5? upstream intron 1 intron 1 intron 1 intron 1 intron 1 intron 2 intron 2 intron 4 intron 4 intron 4 exon 4 intron 5 intron 5 intron 6 intron 6 exon 7 exon 7 exon 7 3? untranslated region
－2847 －2808 －2602 －2467 －1804 －1708 －739 －733 －202 －163 －114 IVS2－249 IVS2－99 IVS4＋43 IVS4＋92 IVS4－65 1049 IVS5＋199 IVS5－318 IVS6＋81 IVS6＋418 1459 1503 1548 2875
SNP ID This study LCP_P1CP12_001 LCP_P1CP12_002 LCP_P1CP12_003 LCP_P1CP12_004 LCP_P1CP12_005 LCP_P1CP12_006 LCP_P1CP12_007 LCP_P1CP12_008 LCP_P1CP12_009 LCP_P1CP12_010 LCP_P1CP12_011 LCP_P1CP12_012 LCP_P1CP12_013 LCP_P1CP12_014 LCP_P1CP12_015 LCP_P1CP12_016 LCP_P1CP12_017 LCP_P1CP12_018 LCP_P1CP12_019 LCP_P1CP12_020 LCP_P1CP12_021 LCP_P1CP12_022 LCP_P1CP12_023 LCP_P1CP12_024 LCP_P1CP12_025
dbSNP (NCBI) rs2069522 rs12592480 rs35694136 rs2069524 rs2069525 rs2069526 rs28399417 rs762551 rs4646425 rs34264399 rs2472304 rs3743484
rs55711332 rs4646427
rs2470890 rs17861162
Nucleotide change
ACATGCTCTGTTT/CTCTATTGGATTC CTTGGCATCTTCA/CCAGGTTGATCCC CTCTACAAAAAA-/AATGTTTAAAAAT GATTGTGGCACAT/-GAACCCCAACCTG GGTGATCCGCCCA/C/GTCTCGGCCTCTC AAAAGGAGGTTGT/CGGGGATCATGAC TAGGTGTAGGGGT/GCCTGAGTTCCGG TAGGGGTCCTGAG/CTTCCGGGCTTTG TCCTTTCCAGCTC/ATCAGATTCTGTG AGCTCTGTGGGCC/ACAGGACGCATGG ATCCAGCTGGGAG/ACCAAGCACAGAA TGTTCACACTAAC/TCTTTTCCTTCTT TGGGAGGATAGGG/AGGGTACCCAGCC GAGAAGCCTTGAG/AACCCAGGTTGTT TGTGCCTGCTGTG/ATGCAAGCCCTGG CATGGGGTATAAG/CAGGGGATAATTC CTACAGACACTGT/GGATTGGCAGGGA TTGTGATCTCAGC/TGCTCATTCCCCT CCCAGGCTGCCTG/CCTGCTTTTTTTT AGGAACTGTTTAT/CATAATGAAAGGA GTGAGCCGAGATC/TGCTCCACTGCAC GTGCCGCCGGGCG/ATGAAAGTCGACC GACCATGAAGCAC/TGCCCGCTGTGAA CTTCTCCATCAAC/TTGAAGAAGACAC TGAGCCACGGTGC/GCCGGCCCACAAT
a. #denotes novel polymorphism not reported in the National Center for Biotechnology Information dbSNP (http://www.ncbi.nlm.nih.gov/SNP/). Information correct as of May 10, 2010. b. NM_000761 was used as the reference sequence
Table 2. Genotypic and allelic frequencies of polymorphisms in CYP1A2 Genotype frequency, n (%) SNPs
Allele Chinese
－2847TÀC
－2808AÀC
－2602insA#
－2467delT (*1D) －1804AÀC/G
－1708TÀC
Allele Frequency
Genotype TT TC TT AA AC CC wt/wt wt/insA insA/insA wt/wt wt/delT delT/delT AA AC AG CC CG GG TT TC CC
38 4 0 41 1 0 40 2 0 4 18 16 33 0 3 0 0 0 33 5 1
(90.5) (9.5) (0) (97.6) (2.4) (0) (95.2) (4.8) (0) (10.5) (47.4) (42.1) (91.7) (0) (8.3) (0) (0) (0) (84.6) (12.8) (2.6)
Malays 33 7 0 36 3 1 42 0 0 7 19 16 31 0 3 0 0 0 30 4 1
(82.5) (17.5) (0) (90) (7.5) (2.5) (100) (0) (0) (16.7) (45.2) (38.1) (91.2) (0) (8.8) (0) (0) (0) (85.7) (11.4) (2.9)
Indians 32 7 0 40 0 0 41 0 0 12 20 9 31 1 5 0 1 0 33 8 0
(82.1) (17.9) (0) (100) (0) (0) (100) (0) (0) (29.3) (48.8) (22) (81.6) (2.6) (13.2) (0) (2.6) (0) (80.5) (19.5) (0)
Chinese
Malays
Indians
T C
0.95 0.05
0.91 0.09
0.91 0.09
A C
0.99 0.01
0.94 0.06
1.00 0.00
wt insA
0.98 0.02
1.00 0.00
1.00 0.00
wt delT
0.34 0.66
0.39 0.61
0.54 0.46
A C G
0.96 0.00 0.04
0.96 0.00 0.04
0.90 0.01 0.09
T C
0.91 0.09
0.91 0.09
0.90 0.10
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Table 2. Continuation Genotype frequency, n (%) SNPs
Allele Chinese
－739TÀG (*1E) －733GÀC
－202CÀA#
－163CÀA (*1F) －114GÀA#
IVS2－249CÀT
IVS2－99GÀA
IVS4＋43GÀA
IVS4＋92GÀA
#
IVS4－65GÀC
1049TÀG
#
IVS5＋199CÀT
#
IVS5－318GÀC
IVS6＋81TÀC
IVS6＋418CÀT
1459GÀA
1503CÀT
#
#
1548CÀT (*1B) *1324CÀG
a.
#
Allele Frequency
Genotype
#
TT TG GG GG GC CC CC CA AA CC CA AA GG GA AA CC CT TT GG GA AA GG GA AA GG GA AA GG GC CC TT TG GG CC CT TT GG GC CC TT TC CC CC CT TT GG GA AA CC CT TT CC CT TT CC CG GG
denotes novel polymorphism
39 3 0 38 3 1 37 0 2 4 14 20 40 0 1 38 4 0 41 0 0 33 2 1 41 0 0 23 9 1 38 0 0 42 0 0 24 15 1 31 4 0 36 1 0 39 1 0 39 1 0 30 10 0 20 16 2
(92.9) (7.1) (0) (90.5) (7.1) (2.4) (94.9) (0) (5.1) (10.5) (36.8) (52.6) (97.6) (0) (2.4) (90.5) (9.5) (0) (100) (0) (0) (91.7) (5.6) (2.8) (100) (0) (0) (69.7) (27.3) (3) (100) (0) (0) (100) (0) (0) (60) (37.5) (2.5) (88.6) (11.4) (0) (97.3) (2.7) (0) (97.5) (2.5) (0) (97.5) (2.5) (0) (75) (25) (0) (52.6) (42.1) (5.3)
Malays 36 6 0 37 4 1 41 0 0 1 15 23 41 0 0 35 6 0 40 0 0 28 2 6 40 0 0 22 13 2 37 0 2 41 1 0 26 12 4 31 4 0 38 2 0 37 1 0 38 0 0 28 6 4 19 12 6
(85.7) (14.3) (0) (88.1) (9.5) (2.4) (100) (0) (0) (2.6) (38.5) (59) (100) (0) (0) (85.4) (14.6) (0) (100) (0) (0) (77.8) (5.6) (16.7) (100) (0) (0) (59.5) (35.1) (5.4) (94.9) (0) (5.1) (97.6) (2.4) (0) (61.9) (28.6) (9.5) (88.6) (11.4) (0) (95) (5) (0) (97.4) (2.6) (0) (100) (0) (0) (73.7) (15.8) (10.5) (51.4) (32.4) (16.2)
Indians 33 8 0 42 0 0 42 0 0 6 23 13 42 0 0 34 8 0 35 6 0 34 3 5 39 1 0 22 13 5 40 0 0 41 0 0 21 13 8 33 8 0 38 4 0 39 3 0 42 0 0 32 10 0 17 17 7
(80.5) (19.5) (0) (100) (0) (0) (100) (0) (0) (14.3) (54.8) (31) (100) (0) (0) (81) (19) (0) (85.4) (14.6) (0) (81) (7.1) (11.9) (97.5) (2.5) (0) (55) (32.5) (12.5) (100) (0) (0) (100) (0) (0) (50) (31) (19) (80.5) (19.5) (0) (90.5) (9.5) (0) (92.9) (7.1) (0) (100) (0) (0) (76.2) (23.8) (0) (41.5) (41.5) (17.1)
Chinese
Malays
Indians
T G
0.96 0.04
0.93 0.07
0.90 0.10
G C
0.94 0.06
0.93 0.07
1.00 0.00
C A
0.95 0.05
1.00 0.00
1.00 0.00
C A
0.29 0.71
0.22 0.78
0.42 0.58
G A
0.98 0.02
1.00 0.00
1.00 0.00
C T
0.95 0.05
0.93 0.07
0.90 0.10
G A
1.00 0.00
1.00 0.00
0.93 0.07
G A
0.94 0.06
0.81 0.19
0.85 0.15
G A
1.00 0.00
1.00 0.00
0.99 0.01
G C
0.83 0.17
0.77 0.23
0.71 0.29
T G
1.00 0.00
0.95 0.05
1.00 0.00
C T
1.00 0.00
0.99 0.01
1.00 0.00
G C
0.79 0.21
0.76 0.24
0.65 0.35
T C
0.94 0.06
0.94 0.06
0.90 0.10
C T
0.99 0.01
0.98 0.02
0.95 0.05
G A
0.99 0.01
0.99 0.01
0.96 0.04
C T
0.99 0.01
1.00 0.00
1.00 0.00
C T
0.88 0.12
0.82 0.18
0.88 0.12
C G
0.74 0.26
0.68 0.32
0.62 0.38
620
Joanne Siok Liu LIM, et al.
and increased levels of carcinogen formation leading to greater susceptibility to several cancers.17–19) Recent studies have, however reported conflicting findings with regard to the －163CÀA (*1F; rs762551) allele being a cancer susceptibility allele.20–22) This could probably be attributed to wide interethnic differences in allelic frequency of the －163CÀA (*1F; rs762551) polymorphism and the varying linkage and haplotype patterns of －163CÀA (*1F; rs762551) with other CYP1A2 polymorphic variants.8,23) Similarly, the －2467delT (*1D; rs35694136) allele was noted to occur at a higher frequency in Chinese and Malay populations (0.66 and 0.61, respectively) compared to the Indian population (0.46) (Table 2). In a recent study, Pavanello et al.9) reported a significant association of this polymorphism with an increase in the metabolic ratio of caffeine as well as urinary mutagenicity (indicator of genotoxic effect), suggesting that －2467delT (*1D; rs35694136) may be associated with greater CYP1A2 enzymatic activity. With regards to the four exonic SNPs, two were synonymous polymorphisms [1503CÀT and 1548CÀT (*1B; rs2470890)] while the other two polymorphisms, 1049TÀG and 1459GÀA, were non-synonymous variants resulting in V350G and V487M amino acid changes, respectively. The residue V350 is located two amino acids from D348 in the N-terminal of helix J which is positioned on the surface of the CYP1A2 protein (3,24). The polymorphism 1042GÀA (*3; rs56276455) leads to the amino acid substitution D348N and has been previously reported to be associated with decreased expression of CYP1A2 in E. coli.25) In addition, helix J has been found to be conserved within the cytochrome P450 proteins.24,26) Hence, it is postulated that V350G may possibly exert an influence on the expression of CYP1A2. On the other hand, V487 is located between 2 beta sheets near the N-terminal of the CYP1A2 polypeptide and is 4 amino acids upstream of substrate binding site 6.27) Future studies should be done to investigate the phenotypic effect of these polymorphisms though the impact may be limited by the low variant allelic frequencies (0.05). Linkage disequilibrium (LD) & haplotype analyses: Pairwise LD analysis for the identified CYP1A2 polymorphisms suggested strong LD between the following polymorphisms across all three ethnic groups (Fig. －2847TÀC －1804AÀC/G 1): (rs2069522), (rs2069524), －1708TÀC (rs2069525), －739TÀG (*1E; rs2069526), IVS2－249CÀT (rs4646425) and IVS6＋81TÀC (rs4646427) with each other (`D?`＝ 1.00; R2À0.60) except IVS6＋81TÀC (rs4646427) with －1804AÀC/G (rs2069524) and －739TÀG (*1E; rs2069526) in Malays. The polymorphism IVS6＋ 81TÀC (rs4646427) was found to have moderate linkage with －1804AÀC/G (rs2069524) (`D?`＝0.63; R2 ＝0.54) and －739TÀG (*1E; rs2069526) (`D?`＝
Fig. 1. Pairwise LD of CYP1A2 polymorphisms in Chinese (A), Malay (B) and Indian (C) populations
0.77; R2＝0.75) in the Malay population. Close associations were observed among these polymorphisms in two ethnic groups: －2467delT (*1D; rs35694136) with －163CÀA (*1F; rs762551) and IVS4－65GÀC (rs2472304) (`D?`Æ0.86; R2Æ0.28) among Malays and Indians; *1324CÀG (rs17861162) with IVS4－65GÀC (rs2472304) and IVS5－318GÀC (rs55711332) (`D?`Æ0.89; R2Æ0.67) in Chinese and Malays. The polymorphisms found in strong linkage in one ethnic group were: *1324CÀG (rs17861162), －1708TÀC (rs2069525) and －2467delT (`D?`＝1; R2Æ0.35) among Indian population and *1324CÀG (rs17861162) with －733GÀC (rs28399417) (`D?`＝1; R2＝0.43) in the Chinese population. A total of 16 haplotypes was identified in the Chinese
621
CYP1A2 Pharmacogenetics in Asians
a. b. c. d. e.
－739TÀG
－733GÀC
－202CÀA
－163CÀA
－114GÀA
IVS2－249CÀT
IVS2－99GÀA
IVS4＋43GÀA
IVS4＋92GÀA
IVS4－65GÀC
1049TÀG
IVS5＋199CÀT
IVS5－318GÀC
W W W W W W W W W
W W D D D W W W D
A A A A G A A A A
T T T T C T T T T
T T T T G T T T T
G G G G G G G G C
C C C C C C C C C
C C A A A C A A A
G G G G G G G G G
C C C C T C C C C
G G G G G G G G G
G G G G G A G A G
G G G G G G G G G
G G G C G G G G C
T T T T T T T T T
C C C C C C C C C
G G G C G G G G C
*1324CÀG
－1708TÀC
A A A A A A A A A
1548CÀT
－1804AÀC/G
0.21 — 0.21 0.07 0.07 0.06 — —
1503CÀT
－2467delT
Indians
0.11 0.26 0.13 — — 0.06 0.05 0.05
1459GÀA
－2602InsA
Malays
Chinese
Average Frequency
Common Haplotypes
Reference Allelesc H1a 0.18 0.20 H2 0.28 0.31 H3 0.15 0.11 H4 0.07 — H5 0.07 — H6 0.06 — H7 0.05 — H8 0.05 0.05
IVS6＋81TÀC
－2808AÀC
T T T T C T T T T
Frequency
IVS6＋418CÀT
－2847TÀC
Table 3. CYP1A2 haplotypes and their frequencies among Asian ethnic groups
T T T T C T T T T
C C C C C C C C C
G G G G G G G G G
C C C C C C C C C
C C C C C C T T C
C C C G G C C C G
H1 is the reference haplotype. Polymorphisms selected as htTAG SNPs are bolded. Abbreviations: W-wild type, D-deletion and I-insertion. Average Frequency refers to average of the observed frequencies in each ethnic group. `—' refers to haplotype frequency of less than 0.05 in the ethnic group.
Fig. 2. Network diagram illustrating the relationship between CYP1A2 common haplotypes in healthy Asian populations Size of nodes indicates average frequencies of haplotypes; H, haplotype; mv, median vector.
population and four high frequency haplotypes (Æ5.0%) accounted for a cumulative frequency of 66.7%. There were 22 haplotypes identified in the Malay population with six high frequency haplotypes occurring with a cumulative frequency of 66.9%. Haplotype structure diversity was greatest in the Indian population, with five of 24 high frequency haplotypes that constituted 61.6% of all haplotypes. The cumulative frequencies of minor haplotypes were observed to be 9.4%, 4.0% and 1.5% in Chinese, Malay and Indian populations, respectively. The high frequency haplotypes in each ethnic population were further aligned to identify eight common haplotypes (H1-H8; Table 3). Haplotype H1, the reference haplotype, and H3 were common in all ethnic groups. Other haplotypes were ethnic-specific: H2 and H8 in Malays and Chinese; H6 in Malays and Indians; H4 and H5 in Indians and H7 in Malay. The haplotype H2,
tagged by the polymorphisms －2467delT (*1D; rs35694136) and －163CÀA (*1F; rs762551), displayed the highest average frequency at 28.4% while the reference haplotype H1 was observed in 17.5% of the populations. The haplotype network between the common haplotypes was calculated using the median joining algorithm (Fig. 2).28) Six polymorphisms were identified as htTAG SNPs: －739TÀG (*1E; rs2069526), *1324CÀ G (rs17861162), －2467delT (*1D; rs35694136), －163CÀA (*1F; rs762551), IVS4＋43GÀA (rs2472304) and －733GÀC (rs28399417). The haplotypes H6 and H7 as well as H1 and H5 were closely related and both pairs of haplotypes could be distinguished by IVS4＋43GÀA (rs2472304), while haplotypes H3 and H8 could be differentiated by －733GÀC (rs28399417). Haplotype H4 was observed to display the greatest diversity from other common haplotypes. H4
622
Joanne Siok Liu LIM, et al.
was connected to other haplotypes by a median vector (mv2) that was tagged by 6 polymorphic variants (Fig. 2) and －739TÀG (*IE; rs2069526) was chosen as htTAG SNP. The －2467delT (*1D; rs35694136) and －163CÀA (*1F; rs762551) alleles were found to be in strong LD in the present study and was represented by H2. This haplotype was present in the Chinese and Malay populations at a frequency consistent with the Korean population (Chinese: 0.31; Malay: 0.26; Korean:0.26) but absent in the Indian population. The frequency of H2 is much lower in the Swedish population at 0.12. This haplotype was previously reported as CYP1A2*1V29) and has been found to be associated with higher plasma caffeine metabolic ratio in Turkish subjects.13) However, this conclusion differs from an earlier study by Ghobti et al.23) This discrepancy may be attributed to the different polymorphic variants included in the two studies. In the study by Ghobti et al.,23) the 1548CÀT (*1B; rs2470890) polymorphism was not included in the construction of the haplotypes while Gunes et al.13) investigated the genotypic distribution of this polymorphism and included it in the derivation of haplotypes in their population. This suggest that the 1548CÀT (*1B; rs2470890) allele may alter the phenotypic effect of the H2 haplotype. In view of the high frequency of H2 haplotype in Asian populations and that both alleles are associated with increased enzymatic activity, further studies evaluating the effects of the haplotype on the in vitro activity of CYP1A2 as well as the pharmacokinetics of substrates metabolized by CYP1A2 are warranted. In summary, this study is the first comprehensive report on the pharmacogenetics of CYP1A2 in three healthy Asian populations in Singapore. The pharmacogenetic profiles of our study populations were found to be similar to other Asian populations (Koreans and Japanese).14,29) The phenotypic effects of the nine novel polymorphisms remain to be investigated in future studies. Haplotype analyses revealed great inter-ethnic diversity in haplotype structures which may explain the differences in the distribution of CYP1A2 metabolic status between different populations. The impact of these haplotypes on cancer susceptibility and pharmacology of CYP1A2 substrates should be further evaluated in the future.
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