Investigation of the major cytochrome P450 1A2 genetic variant in a healthy Tibetan population in China
- Authors:
- Published online on: May 29, 2017 https://doi.org/10.3892/mmr.2017.6645
- Pages: 573-580
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Copyright: © Ren et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Interethnic differences in drug-metabolizing enzyme activity have been associated with inter-individual differences in the efficacy and toxicity of many medications (1). Among drug-metabolizing enzymes, the cytochrome P450 (CYP), a supergene family involved in the phase I reactions of the metabolism of several drugs and endogenous compounds, has increasingly been recognized to have clinically significant consequences (2). Cytochrome P450 1A2 (CYP1A2), one of the CYP enzyme isoforms, is of particular interest because it exhibits a genetic polymorphism.
CYP1A2, mapped to the positive strand of the long arm of chromosome 15 at 15q24.1, is predominantly expressed in the human liver and at lower levels in intestine, pancreas, lung and brain (3). The human CYP1A2 enzyme has been demonstrated to be responsible for many commonly used drugs, including caffeine, imipramine, paracetamol, clozapine, theophylline, tacrine, phenacetin and some neurotoxins (4). In addition, CYP1A2 is known to gain further importance in the metabolic activation of numerous carcinogens (5). Therefore, any alteration to CYP1A2 activity has been suggested to be a susceptibility factor for drug metabolism and the etiology of developing cancers and other diseases (6).
Like other drug metabolizing enzymes, numerous factors have been presented to elucidate the mechanisms underlying the inter-individual differences in CYP1A2 activity, such as race, gender, environmental exposure to inducers or inhibitors and genetic factors (7). With respect to genetic factors, several alleles and additional haplotype variants have been identified in coding and non-coding regions of the CYP1A2 gene, in particular in the CYP1A2 upstream sequence and the intron 1 region (CYP allele nomenclature website at http://www.cypalleles.ki.se/). The frequencies of these polymorphisms display interethnic variability particularly between those of European and East Asian ancestry (8).
Tibet, as a part of China, contains a large number of high altitude populations that have a distinctive suite of physiological traits that enable them to tolerate environmental hypoxia. Because few data are available on the investigation of the CYP1A2 genotype in the Tibetan population, the aim of the present study was to determine the CYP1A2 genotype profile of a random Tibet population by screening for the main allelic variants and compare to the allelic frequencies of those previously reported from other ethnic groups. It is hoped that the results will prospectively offer a preliminary basis for more rational usage of drugs that are substrates for CYP1A2.
Materials and methods
Subjects and DNA extraction
A total of 96 unrelated Chinese healthy volunteers (48 males and 48 females) of Tibetan origin, mostly students or employees at Xizang Minzu University (Xi'an, China), were enrolled in the study. All of the individuals lived in the same region at the time of the study and were of Tibetan ancestry without any known ancestry from other ethnicities. The study protocol was approved by The Human Research Committee of Xizang Minzu University (Xi'an, China), and each volunteer gave written informed consent to participate in the study. Peripheral blood samples were collected and stored after centrifugation at −70°C until analysis, and genomic DNA was isolated and purified using a commercial blood Genomic DNA extraction kit (Xi'an GoldMag Nanobiotech Co., Ltd., Xi'an, China) according to the manufacturer's recommendations.
Polymerase chain reaction (PCR) and DNA sequencing
The primer pairs designed to amplify the 5′ flanking regions, all exons and all introns of the CYP1A2 gene are listed in Table I. The PCR was conducted in a total volume of 10 µl consisting of 1 µl genomic DNA (20 ng/µl), 0.5 µl each primer pair (5 µM), 5 µl HotStart TaqMasterMix (Qiagen China Co., Ltd., Shanghai, China), and 3 µl deionized water. PCR amplification consisted of an initial denaturation step at 95°C for 15 min followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 55–64°C for 30 sec, extension at 72°C for 1 min. The final extension step was performed at 72°C for 3 min. The PCR products were purified and sequenced on an ABI Prism 3100 sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using a BigDye Terminator Cycle Sequencing kit (version, 3.1; Applied Biosystems; Thermo Fisher Scientific, Inc.).
Statistical analysis
The sequences were edited and assembled using Sequencher software (version, 4.10.1; Gene Codes Corporation, Ann Arbor, MI, USA). Allele nomenclature was assigned according to the Human Cytochrome P450 (CYP) Allele Nomenclature Committee (http://www.cypalleles.ki.se/). Differences in allele frequencies between Tibet and other ethnic populations were measured by Fisher exact test. P<0.05 was considered to indicate a statistically significant difference. The observed genotype frequencies of CYP1A2 were also estimated by the Hardy Weinberg law for the predicted frequencies. The linkage equilibrium (LD) coefficient (D') between each genetic variant was analyzed by Haploview software (version, 4.1; Daley Lab at the Broad Institute, Cambridge, MA, USA).
Protein prediction of novel mutations
PolyPhen-2 (http://genetics.bwh.harvard.edu/pph/) and SIFT (http://blocks.fhcrc.org/sift/SIFT.html) software were performed to predict the effect of missense variants on the protein function. Based on the SIFT score, SIFT scores ≤0.05 were predicted by the algorithm to be evolutionary conservation and intolerance to substitution, whereas scores >0.05 were considered tolerant (not likely to affect protein function) (9). The PolyPhen-2 score ranges from 0 to 1, and PolyPhen-2 scores >0.85, between 0.85 and 0.15, and <0.15 were coded as ‘probably damaging’, ‘possibly damaging’ and ‘benign’, respectively (10).
Results
Single nucleotide polymorphism (SNP) discovery
In the current study, the authors used direct sequencing to analyze sequence variation within the CYP1A2 gene among 96 healthy Tibetans. The analyses covered the proximal promoter region, all exons as well as surrounding intronic regions and variable lengths of the flanking regions. Table II presented all the CYP1A2 mutation variations in this population. The most frequent polymorphism was the C-163A change in intron 1 which had 88.54% frequency, followed by G2321C change in intron 4 which had 37.5% frequency and T-739G change in intron 1 which had 20.83% frequency in the healthy group. Both 2159G>A and 5347C>T had similar results (13.5%), correspondingly. Additionally, among a total of 14 nucleotide variants detected, the authors detected three novel CYP1A2 variants (795G>C, 1690G>A and 2896C>T) in exon 2 and intron 5 region with minor allele frequency of 1.04%, of which one variant (795G>C) resulted in an amino acid change from glutamine to histidine at position 265.
Allele & genotype frequencies
A total of eight different CYP1A2 alleles and genotypes were determined based on the polymorphisms identified in the current study (Table III). Hardy-Weinberg equilibriums were assessed and all CYP1A2 allele and genotype frequencies were in accordance with the Hardy-Weinberg equilibrium. The wild-type allele, CYP1A2* 1A, with a frequency of 6.77%, was classified as normal enzyme activity. Besides the wild-type allele, CYP1A2* 1B (58.33%) and CYP1A2*1F (14.58%) were the best-characterized defect alleles in the Chinese Tibetan population, of which CYP1A2*1F alleles were putatively linked to higher inducibility of the enzyme. CYP1A2*1G, CYP1A2*1J, CYP1A2*1M, CYP1A2*13 and CYP1A2*14 alleles have been included in the table, as these were the most scarce alleles in the study population. They occurred at a frequency of 1.56–5.21% in the current study population.
In relation to genotypes, the most frequent genotypes were * 1A/* 1B (13.54%), * 1B/* 1B (16.67%) and * 1B/* 1F (29.17%) (Table III). All five other genotypes presented frequencies of <10.5% in the study. In addition, individuals with the * 1B/* 1B genotype have been associated with a higher activity of the enzyme.
Interethnic variability
In order to better understand the occurrence and distributional patterns of the common mutation allele amongst different ethnic groups, the data were compared with those from previous investigations in different countries and ethnic groups in Caucasians, Africans, Arabs and Asians (Table IV). C-163A (88.54%) was most frequent among the Tibetan population, when compared with T-739 G (20.83%) and C5347T (13.54%). The allele frequency of C-163A and T-739G was significantly higher than that in Caucasians, Africans, Arabs and Asians, but allelic distributions of C-163A were relatively equal to that in Malays (78%), and T-739G was relatively similar to Tunisia (13.5%), Southern Chinese (9.3%) and Indians (10%). For C5347T, Tibetans demonstrated a relatively lower frequency of mutation compared with Caucasians (48–64.4%), but was similar to that in Africans (20.9%) and Asians (12.0–20.4%) with the only exception of South Asians (35%), which was significantly higher than Tibetans.
 | Table IV.Distribution of mutant allele frequencies of CYP1A2 −739T>G, −163C>A and 5347C>T in different ethnicities. |
LD analysis
To identify relationships between the SNPs identified in the polymorphism screening, linkage disequilibrium (LD) analysis was evaluated in Haploview (http://www.broad.mit.edu/mpg/haploview/) using coefficient of linkage disequilibrium D' values (Fig. 1). Even though no distinct LD blocks or extended haplotypes could be detected in the sequenced data, some SNPs were identified (−739T>G and 1202C>T, −163C>A and 2321G>C, 1202C>T and 3613T>C, −739T>G and 3613T>C, −739T>G and 5112C>T) seemed to be linked with high D'.
Protein function prediction of non-synonymous mutation
The SIFT scores for the amino acid substitutions Val75Ile (223G>A), Gln265His (novel variant 795G>C), Gly299Ser (1514G>A) and Arg356Gln (2410G>A), ranged between 0.07 and 0.72 and were predicted as being tolerated. In contrast, the Thr438Ile (5112C>T) mutations gave SIFT scores of 0.00, predicting they were highly likely to affect protein function. To validate the prediction of SIFT scores, the PolyPhen-2 algorithm was used to predict variations Val75Ile, Gln265His, Gly299Ser and Arg356Gln as benign with scores of 0.415, 0.039, 0.045 and 0.002, respectively, and Thr438Ile as possibly damaging, with a score of 0.281. Four substitutions (Gln265His, Gly299Ser, Arg356Gln and Thr438Ile) were consistently computationally predicted using both PolyPhen-2 and SIFT, while Val75Ile was not consistent. The protein function prediction of variants 5112C>T and 795G>C (novel variant) is presented Fig. 2 (PolyPhen-2).
Discussion
CYP1A2, one of the major P450 isoforms, accounts for ~5–20% of the total hepatic CYP content and contributes to the metabolism of 10% of clinically relevant drugs, including clozapine and caffeine (3). It has been demonstrated that CYP1A2 activity has been influenced by the presence of polymorphic variants, which displays wide interindividual and interethnic variability. In the present study, the CYP1A2 gene polymorphisms were systematically screened in 96 healthy Chinese Tibetan subjects. To the best of the authors' knowledge, these efforts are the first to investigate allelic variants of CYP1A2 among the Tibetan population to date.
A total of 14 SNPs were detected in the current study. There were eight SNPs detected in the intron region. The −163 C>A (* 1F/* 1J/* 1K/* 1M allele) in intron 1 is the most common CYP1A2 polymorphism in various population studies (Table IV). In Tibetans, −163C>A is the most frequently observed SNP, with an overall frequency of 88.54%, which is significantly higher than that in Caucasians, Africans, Arabs and Asians (except Malays). Possible explanations for these differences include: Genetic background, cultural variants and other factors, such as living environment, medication use, body composition and dietary habits (11,12). In addition, much confusion and controversy still arises as to the available data in literature about the functional consequences and allele frequencies of CYP1A2 variants, mainly because of limitation of sample size and the differing designations of the CYP1A2* 1F allele (defined as having-163A by The HumanCytochrome P450 Allele Nomenclature Committee). Sachse et al (4) first reported that smokers homozygous for the C-allele had, on average, 40% lower CYP1A2 activity in comparison with those with the A/A genotype. In contrast, some inconsistent studies have reported that CYP1A2 * 1F mutation was associated with a high inducibility of CYP1A2 in smokers as well as in nonsmokers (13). It is tempting to speculate the divergence may be the possibility of the −163C>A occurring in linkage disequilibrium with another mutation that is responsible for the increased CYP1A2 inducibility (14). The present study identified a strong linkage disequilibrium between −163C>A and 2321G>C polymorphisms (Fig. 1), providing researchers in the field with abundant clues, however, more studies are required to shed more light on this idea. Another most prevalent polymorphism in intron 1 region, −739T>G, was first reported in in a Japanese population (5). −739T>G is located on the CYP1A2* 1E, * 1G, * 1J or * 1K allele, and previous research demonstrated that this polymorphism has no effect on the enzyme activity (6). −739T>G is the most common variant among Asians and the frequency of 20.83% found in the present study is significantly higher than other Asians (Table IV), Caucasians (0.77–5%) (6,8), Africans (6.6–13.5%) (15,16) and Arabs studied elsewhere (3–10%) (17,18). Interethnic differences in the prevalence of −739T>G may be one of the major factors to consider in large pharmacogenetic studies and clinical applications in populations of Asian ancestry, such as Chinese Tibetans, since the proportion of high expressers due to the presence of −739T>G varies depending on the ethnic background. Among the six SNPs identified in the exons, the synonymous 5347T>G (* 1B/* 1G/* 1H), was the most common variant among Caucasians and the frequency of 13.54% identified in the present study presented a frequency significantly lower than Caucasians, but it was quite similar to Asians (except South Asians) (Table IV). This may be because these populations are distributed in different geographical regions, which may result in the formation of numerous, small, genetically isolated groups.
In the tested Chinese Tibetan population, CYP1A2* 1A is referred to as the wild-type allele with a frequency of 6.77%, which is significantly less when compared with Swedes (24.4%), Koreans (21.7%), Japanese (34.8%), Caucasians (33.4%) and Serbs (33.4%) (19–21). The occurrence of the most prevalent defective alleles, CYP1A2* 1B (5347T>G), evaluated in Chinese Tibetan subjects (58.3%) in the present study is slightly lower compared to the occurrence reported in Caucasians (61.8%), but is higher than other Chinese population (20.4%) (22). However, the genotype frequencies observed for * 1B, * 1B in Tibetans (16.67%) was slightly higher than that in Caucasian (6.19%), Japanese (7.5%), Korean (10.75%) and other Chinese population (9%). Currently, only Chen et al (22) reported that CYP1A2*1B homozygotes demonstrated marginally higher CYP1A2 activity, when compared with CYP1A2* 1A/* 1A homozygotes (22). Because the * 1B, * 1B genotype is relatively common in Chinese Tibetan subjects, this genotype may have a major influence in altered CYP1A2 activity, of course, this requires further investigation. CYP1A2* 1F resulted from a C>A substitution at −163 in intron 1 of the promoter region. The haplotype *1F allele is common with high and comparable frequencies in various studies. However, the frequencies of CYP1A2* 1F (−163A allele) in Tibetans is 14.58%, which was far less frequent compared with Caucasians (73.7%) (23), Africans (61%) (24), Arabs (68%) (17) and Asians (69.3%) (23). Since CYP1A2* 1F is reported to be associated with an effect on enzyme inducibility, the estimates of their frequencies in the Tibetan population may be of extreme importance. Compared with the alleles CYP1A2* 1B and CYP1A2* 1F, * 1G, * 1J, * 1M, * 13 and * 14 are relatively rare in Tibetans, thus the clinical applicability of this pharmacogenetic testing seems to be limited to a small number of individuals. In addition, the-163C>A variant is present in the CYP1A2*1F allele, but it is also presented in several other CYP1A2 haplotypes, two of which (CYP1A2* 1J and * 1M) were identified in the sample population. Therefore, it is informative to take the complete haplotypes into consideration when investigating associations of phenotype rather than focusing on single SNPs.
After systematically screening the polymorphisms of the CYP1A2 gene in the healthy population of Chinese Tibetan subjects, three novel variants were detected that included one nonsynonymous change at position G795C in exon 2. These variants are rare but not absent, occurring in <1.04% of the population, but the current study is the first to report these variants in Chinese Tibetan subjects. Although the c.795 G>C variation is predicted to not have an affect on protein function by the SIFT or PolyPhen algorithms, further functional studies are still necessary to clarify the role of their clinical significance.
It should be acknowledged that the current research was designed to investigate the unique distribution of the CYP1A2 alleles in the Tibetan population. The characterization of CYP1A2 genetic polymorphisms among different races may contribute to the outcome and risks to certain drug therapies.
Acknowledgements
This work was supported by the Key Program of Natural Science Foundation of Xizang (Tibet) Autonomous Region (grant no. 20152R-13-11), Major Training Program of Tibet University for Nationalities (grant no. 13myZP06), Natural Science Foundation of Xizang (Tibet) Autonomous Region (grant no. 20152R-13-11), and Major Science and Technology Research Projects of Chinese Ministry of Education (grant no. 211176).
References
|
Evans WE and Relling MV: Pharmacogenomics: Translating functional genomics into rational therapeutics. Science. 286:487–491. 1999. View Article : Google Scholar : PubMed/NCBI | |
|
Alexov E and Sternberg M: Understanding molecular effects of naturally occurring genetic differences. J Mol Biol. 425:3911–3913. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Klein K, Winter S, Turpeinen M, Schwab M and Zanger UM: Pathway-Targeted Pharmacogenomics of CYP1A2 in Human Liver. Front Pharmacol. 1:1292010. View Article : Google Scholar : PubMed/NCBI | |
|
Sachse C, Brockmöller J, Bauer S and Roots I: Functional significance of a C->A polymorphism in intron 1 of the cytochrome P450 CYP1A2 gene tested with caffeine. Br J Clin Pharmacol. 47:445–449. 1999. View Article : Google Scholar : PubMed/NCBI | |
|
Chida M, Yokoi T, Fukui T, Kinoshita M, Yokota J and Kamataki T: Detection of three genetic polymorphisms in the 5′-flanking region and intron 1 of human CYP1A2 in the Japanese population. Jpn J Cancer Res. 90:899–902. 1999. View Article : Google Scholar : PubMed/NCBI | |
|
Sachse C, Bhambra U, Smith G, Lightfoot TJ, Barrett JH, Scollay J, Garner RC, Boobis AR, Wolf CR and Gooderham NJ: Colorectal Cancer Study Group: Polymorphisms in the cytochrome P450 CYP1A2 gene (CYP1A2) in colorectal cancer patients and controls: Allele frequencies, linkage disequilibrium and influence on caffeine metabolism. Br J Clin Pharmacol. 55:68–76. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Dobrinas M, Cornuz J, Pedrido L and Eap CB: Influence of cytochrome P450 oxidoreductase genetic polymorphisms on CYP1A2 activity and inducibility by smoking. Pharmacogenet Genomics. 22:143–151. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Perera V, Gross AS and McLachlan AJ: Influence of environmental and genetic factors on CYP1A2 activity in individuals of South Asian and European ancestry. Clin Pharmacol Ther. 92:511–519. 2012.PubMed/NCBI | |
|
Ng PC and Henikoff S: SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res. 31:3812–3814. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Liu X, Jian X and Boerwinkle E: dbNSFP: A lightweight database of human nonsynonymous SNPs and their functional predictions. Hum Mutat. 32:894–899. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Pavanello S, Fedeli U, Mastrangelo G, Rota F, Overvad K, Raaschou-Nielsen O, Tjønneland A and Vogel U: Role of CYP1A2 polymorphisms on lung cancer risk in a prospective study. Cancer Genet. 205:278–284. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Chida M, Yokoi T, Fukui T, Kinoshita M, Yokota J and Kamataki T: Detection of three genetic polymorphisms in the 5′-flanking region and intron 1 of human CYP1A2 in the Japanese population. Jpn J Cancer Res. 90:899–902. 1999. View Article : Google Scholar : PubMed/NCBI | |
|
Han XM, Ou-Yang DS, Lu PX, Jiang CH, Shu Y, Chen XP, Tan ZR and Zhou HH: Plasma caffeine metabolite ratio (17X/137X) in vivo associated with G-2964A and C734A polymorphisms of human CYP1A2. Pharmacogenetics. 11:429–435. 2001. View Article : Google Scholar : PubMed/NCBI | |
|
Gunes A and Dahl ML: Variation in CYP1A2 activity and its clinical implications: Influence of environmental factors and genetic polymorphisms. Pharmacogenomics. 9:625–637. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Browning SL, Tarekegn A, Bekele E, Bradman N and Thomas MG: CYP1A2 is more variable than previously thought: A genomic biography of the gene behind the human drug-metabolizing enzyme. Pharmacogenet Genomics. 20:647–664. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
B'Chir F, Pavanello S, Knani J, Boughattas S, Arnaud MJ and Saguem S: CYP1A2 genetic polymorphisms and adenocarcinoma lung cancer risk in the Tunisian population. Life Sci. 84:779–784. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Hamdy SI, Hiratsuka M, Narahara K, Endo N, El-Enany M, Moursi N, Ahmed MS and Mizugaki M: Genotyping of four genetic polymorphisms in the CYP1A2 gene in the Egyptian population. Br J Clin Pharmacol. 55:321–324. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Aklillu E, Carrillo JA, Makonnen E, Hellman K, Pitarque M, Bertilsson L and Ingelman-Sundberg M: Genetic polymorphism of CYP1A2 in Ethiopians affecting induction and expression: Characterization of novel haplotypes with single-nucleotide polymorphisms in intron 1. Mol Pharmacol. 64:659–669. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Djordjevic N, Ghotbi R, Jankovic S and Aklillu E: Induction of CYP1A2 by heavy coffee consumption is associated with the CYP1A2 −163C>A polymorphism. Eur J Clin Pharmacol. 66:697–703. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Ghotbi R, Christensen M, Roh HK, Ingelman-Sundberg M, Aklillu E and Bertilsson L: Comparisons of CYP1A2 genetic polymorphisms, enzyme activity and the genotype-phenotype relationship in Swedes and Koreans. Eur J Clin Pharmacol. 63:537–546. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Soyama A, Saito Y, Hanioka N, Maekawa K, Komamura K, Kamakura S, Kitakaze M, Tomoike H, Ueno K, Goto Y, et al: Single nucleotide polymorphisms and haplotypes of CYP1A2 in a Japanese population. Drug Metab Pharmacokinet. 20:24–33. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Chen X, Wang L, Zhi L, Zhou G, Wang H, Zhang X, Hao B, Zhu Y, Cheng Z and He F: The G-113A polymorphism in CYP1A2 affects the caffeine metabolic ratio in a Chinese population. Clin Pharmacol Ther. 78:249–259. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Myrand SP, Sekiguchi K, Man MZ, Lin X, Tzeng RY, Teng CH, Hee B, Garrett M, Kikkawa H, Lin CY, et al: Pharmacokinetics/genotype associations for major cytochrome P450 enzymes in native and first- and third-generation Japanese populations: Comparison with Korean, Chinese, and Caucasian populations. Clin Pharmacol Ther. 84:347–361. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Dandara C, Lombard Z, Du Plooy I, McLellan T, Norris SA and Ramsay M: Genetic variants in CYP (−1A2, −2C9, −2C19, −3A4 and −3A5), VKORC1 and ABCB1 genes in a black South African population: A window into diversity. Pharmacogenomics. 12:1663–1670. 2011. View Article : Google Scholar : PubMed/NCBI |
