Identification of a Single Nucleotide Polymorphism in the TATA Box of the CYP2A6 Gene: Impairment of Its Promoter Activity

doi:10.1006/bbrc.2001.4990

Biochemical and Biophysical Research Communications

Volume 284, Issue 2, 8 June 2001, Pages 455-460

https://doi.org/10.1006/bbrc.2001.4990 Get rights and content

Abstract

Human cytochrome P450 2A6 (CYP2A6) constitutes the major nicotine oxidase, and large interindividual differences are seen in the levels of this enzyme, to a great extent caused by the distribution of several different polymorphic gene variants mainly located in the open reading frame (ORF). In the present study, we report a common polymorphism located in the 5′ flanking region of CYP2A6 affecting its expression. DHPLC analysis and complete sequence of the open reading frame of the gene from a Turkish individual revealed a −48T > G substitution disrupting the TATA box. Using dynamic allele-specific hybridization (DASH), genotyping of this novel variant (named CYP2A6*9) was carried out in 116 Swedish, 132 Turkish, and 102 Chinese subjects, and the allele frequencies were found to be 5.2, 7.2, and 15.7%, respectively. The significance of the polymorphism was investigated by the construction of luciferase reporter plasmids containing 135 or 500 bp of the 5′-upstream region of the gene transfected into human hepatoma B16A2 cells. The constructs carrying the −48T > G mutation were only expressed at about 50% of the wild-type alleles. It is concluded that the CYP2A6*9 allele might be one of the most common CYP2A6 variants in Caucasians that alters the levels of enzyme expression.

References (36)

M. Oscarson et al.
FEBS Lett.
(1998)
O. Pelkonen et al.
Toxicology
(2000)
M. Miyamoto et al.
Biochem. Biophys. Res. Commun.
(1999)
N. Ariyoshi et al.
Biochem. Biophys. Res. Commun.
(2001)
A. Westlind et al.
Biochem. Biophys. Res. Commun.
(1999)
M. Oscarson et al.
FEBS Lett.
(1999)
B.F. Pugh
Gene
(2000)
H. Peltoketo et al.
Genomics
(1994)
R. Jover et al.
Hepatology
(2001)
J.S. Miles et al.
Biochem. J.
(1990)

S. Yamano et al.

Biochemistry

(1990)

C.H. Yun et al.

Mol. Pharmacol.

(1991)

M. Nakajima et al.

Drug Metab. Dispos.

(1996)

E.S. Messina et al.

J. Pharmacol. Exp. Ther.

(1997)

M. Nakajima et al.

J. Pharmacol. Exp. Ther.

(1996)

S.E. Murphy et al.

Chem. Res. Toxicol.

(1999)

M. Oscarson

Drug Metab. Dispos.

(2001)

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With the absence of effective reinforcement of tobacco control laws in the country, the Lebanese population has been vulnerable to the health burden of smoking. Smoking and quitting behaviors are influenced by genetic factors, mainly CYP2A6 polymorphisms. To date, the assessment of the genetic profile and its effect on the behavior of Lebanese smokers has not yet been studied. The aim of this study was to determine the CYP2A6 polymorphisms and its relation to smoking and quitting behavior in a cohort of Lebanese smokers.
Healthy adult Lebanese smokers were recruited from the American University of Beirut and its Medical Center. Their nicotine metabolite (NM) levels and CYP2A6 alleles (2, 4, 9 and 12) were determined. The smoking behavior, Nicotine Dependence Score (NDS) and quitting behavior of the participants were assessed after filling a “smoking assessment questionnaire”.
53 healthy adult smokers participated in our study (M: F = 4:1, median age = 38.47 +/− 14.15 years). Mutated CYP* 2 was present in 33.9% (18/53 cases), CYP* 4 in 11.3% (6/53 cases), CYP* 9 in 100% (53/53 cases) and CYP* 12 in 86.7% (46/53 cases). Slow metabolizers constituted 90.6% of our group while 9.4% were intermediate metabolizers. Slow metabolizers smoked a smaller number of cigarettes per day (CPD) (median = 15 CPD vs 25 CPD) and had higher NM levels (median = 23 ng/mL vs 10 ng/mL or undetectable) than intermediate metabolizers. A statistically significant correlation with NDS and quitting behavior was not found in either group.
In this first study from Lebanon, a unique distribution of CYP2A6 alleles (high prevalence of CYP2A6*9 and CYP2A6*12) was detected. In accordance with previously published data, slow metabolizers smoked a lower number of CPD and had higher NM levels in their serum. Larger studies are required to fully elucidate the CYP2A6 polymorphisms of Lebanese smokers and include them in personalized smoking cessation programs.
Genetic polymorphisms in CYP2A6 are associated with a risk of cigarette smoking and predispose to smoking at younger ages
2017, Gene
Nicotine is the main component of cigarettes that causes addiction, which is considered a complex disease, and genetic factors have been proposed to be involved in the development of addiction. The CYP2A6 gene encodes the main enzyme responsible for nicotine metabolism. Depending on the study population, different genetic variants of CYP2A6 associated with cigarette smoking have been described. Therefore, we evaluated the possible association between SNPs in CYP2A6 with cigarette smoking and nicotine addiction-related variables in Mexican mestizo smokers. We performed a genetic association study comparing light smokers (LS, n = 349), heavy smokers (HS, n = 351) and never-smokers (NS, n = 394). SNPs rs1137115, rs4105144, rs1801272 and rs28399433 were genotyped in the CYP2A6 gene. We found that the A allele of rs1137115 (OR = 1.41) in exon 1 of CYP2A6 and the T allele of rs4105144 (OR = 1.32) in the 5′ UTR of the gene are associated with the risk of cigarette smoking (p < 0.05); rs1137115 affects the level of alternative splicing, resulting in a CYP2A6 isoform with low enzymatic activity, whereas rs4105144 is likely to be in a binding site for the transcription factor for glucocorticoids receptor (GR) and regulates the expression of CYP2A6. In addition, having a greater number of risk alleles (rs1137115 (A), rs4105144 (T) and rs28399433 (G)) is associated with a younger age at onset. The present study shows that in Mexican mestizos, the analyzed SNPs confer greater risk in terms of consumption and age of onset.
Genetic Aspects of Smoking Behavior in the Japanese Population
2016, Neuropathology of Drug Addictions and Substance Misuse
Smoking behavior is a multifactorial trait influenced by genetic and environmental components. Several genome-wide association studies (GWASs) have identified candidate genes or loci for smoking behavior. However, most of these results were from European studies. Genetic differences are known to exist between ethnic groups or samples from different geographic locations. This chapter focuses on the genetic aspects of Japanese smoking behavior. Focusing on the CYP2A6 polymorphism, many studies have reported that a mutant allele with an impaired or null enzyme activity was a negative risk factor for nicotine addiction; however, some studies have indicated negative associations. Regarding neuronal nicotinic acetylcholine receptors, we showed that the CHRNA5 rs16969968 polymorphism was associated with smoking cessation and a high Tobacco Dependence Screener score. We have also confirmed that the TaqIA polymorphism near the DRD2 gene was related to current smoking, which was consistent with the results of previous studies examining Japanese populations. The further accumulation of investigations, including GWASs, and functional studies is warranted to establish a wider view of the genetic factors affecting smoking behavior.
Genetic Aspects of Smoking Behavior in the Japanese Population
2016, Neuropathology of Drug Addictions and Substance Misuse Volume 2: Stimulants, Club and Dissociative Drugs, Hallucinogens, Steroids, Inhalants and International Aspects
Smoking behavior is a multifactorial trait influenced by genetic and environmental components. Several genome-wide association studies (GWASs) have identified candidate genes or loci for smoking behavior. However, most of these results were from European studies. Genetic differences are known to exist between ethnic groups or samples from different geographic locations. This chapter focuses on the genetic aspects of Japanese smoking behavior. Focusing on the CYP2A6 polymorphism, many studies have reported that a mutant allele with an impaired or null enzyme activity was a negative risk factor for nicotine addiction; however, some studies have indicated negative associations. Regarding neuronal nicotinic acetylcholine receptors, we showed that the CHRNA5 rs16969968 polymorphism was associated with smoking cessation and a high Tobacco Dependence Screener score. We have also confirmed that the TaqIA polymorphism near the DRD2 gene was related to current smoking, which was consistent with the results of previous studies examining Japanese populations. The further accumulation of investigations, including GWASs, and functional studies is warranted to establish a wider view of the genetic factors affecting smoking behavior.
Single tube genotyping of CYP2A6 gene deletion based on copy number determination by quantitative real-time PCR
2014, Experimental and Molecular Pathology
Citation Excerpt :
The enzymatic activity of CYP2A6 varies considerably between individuals and is mainly caused by the extensive genetic polymorphism of the CYP2A6 gene (Di et al., 2009; Satarug et al., 2006). So far, over 35 different alleles and additional detailed subgroups of the CYP2A6 gene were reported, including alleles with single nucleotide polymorphisms (SNPs), whole gene deletions and gene conversions that are distributed at variable frequencies across different ancestral groups (Hoffman et al., 2001; Nakajima et al., 2006; Nurfadhlina et al., 2006; Pitarque et al., 2001). The CYP2A6*4 allele, which is characterized as the whole or partial deletion of the gene, was found at a high frequency of approximately 15% to 24% in Asian subjects but at much lower frequencies in White or Black individuals (Huang et al., 2005; Nakajima et al., 2006; Oscarson et al., 1999; Schoedel et al., 2004).
The CYP2A6*4 allele, characterized as the whole deletion of this gene, is closely associated with nicotine dependence, cancer susceptibility, and drug responsiveness. It has long been a significant challenge for pharmacogenetics scientists to develop a reliable method to detect this molecular variant due to its high homology with its homologous genes CYP2A6 and CYP2A3 in the clinical setting. Here, we introduce a quantitative real-time PCR assay that specifically amplifies CYP2A6 by designing a specific set of primers and the probe, which effectively prevent the amplification of the CYP2A7 and CYP2A13 alleles. CYP2A6 gene copy numbers were normalized to albumin (ALB) which was co-amplified simultaneously in a single-tube duplex reaction and at a setting as the internal reference gene. The established assay was validated with a selection of previously genotyped DNA samples, which harbored none, one or two CYP2A6 gene copies. The results were in complete concordance with previously published data and no overlap between the three groups was observed. Further analysis of a cohort of 120 samples revealed high specificity and sensitivity of this assay as demonstrated by the agreement of determined gene copy numbers in all of the cases. In conclusion, this novel assay allows reliable and sensitive detection of the CYP2A6 gene deletion, which will be useful for pharmacogenetics studies and routine clinical settings.
Impact of Genetic Variants in the Nicotine Metabolism Pathway on Nicotine Metabolite Levels in Smokers
2023, Cancer Epidemiology Biomarkers and Prevention

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^☆: Abbreviations used: DASH, dynamic allele specific hybridisation; SNP, single nucleotide polymorphism; ORF, open reading frame; dHPLC, denaturing high-performance liquid chromatography.

¹: To whom correspondence should be addressed at Division of Molecular Toxicology, IMM, Karolinska Institutet, Box 210, 171 77 Stockholm, Sweden. Fax: +46-8-33 73 27. E-mail: [email protected].

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Regular ArticleIdentification of a Single Nucleotide Polymorphism in the TATA Box of the CYP2A6 Gene: Impairment of Its Promoter Activity☆

Abstract

FEBS Lett.

Toxicology

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

FEBS Lett.

Gene

Genomics

Hepatology

Biochem. J.

Biochemistry

Mol. Pharmacol.

Drug Metab. Dispos.

J. Pharmacol. Exp. Ther.

J. Pharmacol. Exp. Ther.

Chem. Res. Toxicol.

Drug Metab. Dispos.

Regular Article
Identification of a Single Nucleotide Polymorphism in the TATA Box of the CYP2A6 Gene: Impairment of Its Promoter Activity☆