Comparisons of CYP2A6 genotype and enzyme activity between Swedes and Koreans

1. Title page

Title:

Comparisons of CYP2A6 genotype and enzyme activity

between Swedes and Koreans

Authors:

Natasa Djordjevic a, b, Juan Antonio Carrillo c, Marieke PJ van den Broek a, Junko Kishikawa a ,

Hyung-Keun Roh d, Leif Bertilsson a, Eleni Aklillu a

Primary laboratory of origin:

Division of Clinical Pharmacology, Department of Laboratory Medicine, Karolinska University

Hospital, Huddinge, Karolinska Institutet, SE-14186 Stockholm, Sweden

The study was financially supported by the Swedish Research Council, Medicine, 3902,

the Swedish Institute, Plan Nacional de Investigación Científica, Desarrollo e Innovación

Tecnológica (I+D+I), Instituto de Salud Carlos III, Subdirección General de Evaluación y

Fomento de la Investigación, PI071152; and Ayudas para la consolidación y apoyo a

grupos de investigación de Extremadura, GRU09015 (Orden de 17 de diciembre de

2008, DOE 5 de enero de 2009).

1

Copyright 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)C

Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGEReceived; March 15, 2012Accepted; July 10, 2012

Published online; July 24, 2012doi; 10.2133/dmpk.DMPK-12-RG-029

2. Running title page

Running title: CYP2A6 genotype and activity in Swedes and Koreans

Corresponding author:

Eleni Aklillu, B.Pharm, MSc., Ph.D., Associate professor,

Division of Clinical Pharmacology, Department of Laboratory Medicine,

Karolinska Institutet, Karolinska University Hospital, Huddinge, C1: 68, SE-141 86

Stockholm, Sweden

Tel: + 46 8 58 58 78 82

Fax: + 46 8 58 58 10 70

E-mail: [email protected]

Number of text pages: 17

Number of tables: 1

Number of figures: 2

2

Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

mailto:[email protected]

3. Summary

The aim was to compare cytochrome P450 2A6 (CYP2A6) genotype and enzyme activity

between Swedes and Koreans, and to investigate the influence of genotype, sex, age, cigarette

smoking and oral contraceptive (OC) use on enzyme activity. The study involved 190 Swedes

and 144 Koreans. Genotyping for CYP2A6*1B, *1x2, *4, *5, *7, *8, *9, *10, *18 and *19 alleles

were done. Using caffeine as a probe, in vivo CYP2A6 activity was estimated by the 17U/17X

urinary ratio. Multiple regression analysis indicated ethnicity (p=0.0001) and CYP2A6 genotype

(p=0.006), but not sex, age, cigarette smoking or OC use as predictors of CYP2A6 activity.

There were significant differences in CYP2A6 genotype distribution and enzyme activity between

Swedes and Koreans. Functional CYP2A6 alleles and rapid genotypes were more frequent in

Swedes, whereas the defective alleles and slow genotypes were more frequent in Koreans

(p≤0.0001). Distribution of log 17U/17X was bimodal in Koreans but unimodal in Swedes with a

common antimode at 0.01, classifying 3.16% of Swedes and 18.75% of Koreans as slow

metabolizers. CYP2A6 activity was higher in Swedes compared to Koreans (p<0.0001), even

among carriers of rapid genotypes. We report major differences in CYP2A6 enzyme activity

between Swedes and Koreans mainly due to CYP2A6 genetic variation but not exclusively.

Key words: CYP2A6 · Swedes · Koreans · phenotype · genotype

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5. Introduction

Cytochrome P450 2A6 (CYP2A6) is involved in the metabolism of carcinogens such

nitrosamines and aflatoxin B1, as well as in biotransformation of some drugs, including nicotine,

disulfiram, halothane and valproic acid 1,2). As observed in previous studies, enzyme activity of

CYP2A6 significantly depends on CYP2A6 genetic variability 3,4). Namely, carriers of CYP2A6*4

(gene deletion) and CYP2A6*5 (Gly479Val) completely lack CYP2A6 3,5-8). Similarly, CYP2A6*7

(Ile471Thr), CYP2A6*8 (Arg485Leu), CYP2A6*9 (single nucleotide polymorphism in the TATA

box), CYP2A6*10 (combination of *7 and *8 allele), CYP2A6*18 (Tyr392Phe) and CYP2A6*19

(combination of *7 and *18 allele) all lead to decreased enzymatic activity 3,6,8-11). On the other

hand, CYP2A6*1B1 (gene conversion in 3′-UTR) is mostly associated with increased enzyme

activity 2,12-15), while CYP2A6 duplication corresponds to the presence of three copies of the gene

and thus provides about 1.4-fold higher enzyme activity 16,17).

In addition to genetic variations, activity of CYP2A6 enzyme seems to be affected by a

variety of non-genetics factors, including several antiepileptic agents as inducers, and

pilocarpine and tranylcypromine as inhibitors 2,7). Therefore, it is not surprising that liver content

and activity of CYP2A6 vary considerably among individuals 1,7). Due to potential clinical

importance, this pronounced variation and its possible causes have been widely investigated.

Numerous factors affecting CYP2A6 enzyme activity have been described, including ethnicity 18),

genotype 9,19), sex 18,20), age 12,21), cigarette smoking 20,22) and oral contraceptive (OC) use 21,23).

However, the findings were not always consistent 22,24-26), warranting further investigations.

Following our previous reports on inter-ethnic differences in activities of drug

metabolizing enzymes CYP1A2 and NAT2 between Swedes and Koreans 27,28), in the present

study we aimed to explore the possible effect of ethnicity, as well as other potential influencing

factors, on CYP2A6 activity in the same populations. Our results indicate higher CYP2A6

enzyme activity in Swedes compared to Koreans, regardless of genotype.

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6. Methods

Subjects

Three hundred thirty four unrelated healthy volunteers (190 Swedes and 144 Koreans)

participated in the study. Swedes were 18 to 60 years old (median age: 29 years), and in the

group there were 79 men, 39 smokers and 44 oral contraceptive (OC) users. Korean participants

were 20 to 46 years old (median age: 25 years), and of those 74 were men, 28 were smokers

and only 1 was OC user. The study protocol was approved by the ethics committees at the

Karolinska Institutet, Stockholm, Sweden, and at the Inha University Hospital, Korea. The study

was conducted in accordance with the Declaration of Helsinki and its subsequent revisions.

Caffeine phenotyping

The caffeine urinary test was performed according to Carrillo et al. 29), with modifications

as previously described 22). High-performance liquid chromatography was used to determine

molar concentrations of caffeine metabolites 17X (1,7-dimethylxanthine or paraxanthine) and

17U (1,7-dimethyluric acid) in the urine samples, and the CYP2A6 enzyme activity was

assessed by the ratio of 17U/17X 22,30).

CYP2A6 genotyping

Genomic DNA was isolated from whole blood samples using QIAamp DNA Mini Kit

(QIAGEN GmbH, Hilden, Germany). PCR reactions were performed on GeneAmp PCR System

2700 (Applied Biosystems, Foster City, CA), using the primers purchased from Invitrogen,

Carlsbad, CA. All other reagents used for PCR amplification were from Roche Applied Science,

Mannheim, Germany. Genotyping for gene deletion (CYP2A6*4), gene conversion in the 3′

region (CYP2A6*1B1), -48T>G (rs28399433, CYP2A6*9) and c.1436G>T (rs5031017,

CYP2A6*5) were performed as previously described 5,16,22). In addition, only Swedes were

genotyped for gene duplication (CYP2A6*1x2), as this polymorphism is known to be extremely

rare in Asians 31,32). For the similar reasons 32,33), the detection of c.1412T>C (rs5031016,

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CYP2A6*7), c.1454G>T (rs28399468, CYP2A6*8) and c.1175A>T (rs1809810, CYP2A6*18)

was performed only in Koreans as described previously 11,34).

Statistical analysis

Chi-square test was used to compare observed and expected allele frequencies

according to Hardy-Weinberg equilibrium. Haplotype analysis was done using the population

genetic software program Arlequin, version 3.11 (http://cmpg.unibe.ch/software/arlequin3). The

17U/17X ratio was log-transformed before statistical analyses. Consistency of the data with the

normal distribution was assessed by Shapiro-Wilk test. In all study participants, effects of

ethnicity, genotype, sex, age, cigarette smoking and OC use on enzyme activity were evaluated

using multiple regression analysis. Chi-square test or Fisher exact test were used to compare

frequencies of polymorphisms, haplotypes, genotypes and genotype groups between Swedes

and Koreans. Statistical analyses were performed using Statistica, version 10 (StatSoft Inc,

Tulsa, OK, USA). P<0.05 was considered as significant.

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7. Results

There were significant differences in the distribution of most of the CYP2A6

polymorphisms, haplotypes and genotypes between Swedes and Koreans (Table 1, χ2≥10.3,

df=1, p≤0.001). Based on the expected effects of specific CYP2A6 genotypes on enzyme

activity, i.e. on a presence of functional (*1A, *1x2, or *1B1) or less functional/non-functional

CYP2A6 alleles (*4, *7, *8, *9, *10, *18 or *19), study participants were assigned to either rapid,

intermediate or slow genotype group. The distribution of these genotype groups was significantly

different between the two populations (Table 1, χ2≥33.2, df=1, p<0.0001). The rapid genotype

group was the most prevalent among Swedes (95% CI of the difference in proportions 0.40,

0.70), whereas the slow metabolizer genotypes were more frequent in Koreans (95% CI of the

difference in proportions -0.46, -0.06). None of the study participants were homozygous for

CYP2A6 gene deletion (CYP2A6*4/*4).

The frequency distributions and the probit plots of the log-transformed 17U/17X ratio in

Swedes and Koreans are presented in Figure 1. The distribution of 17U/17X ratio was distinctly

bimodal in Koreans whereas in Swedes it was unimodal but skewed. The metabolic ratios

ranged from 0.00 to 2.81 in Swedes and from 0.00 to 1.96 in Koreans, with the median values of

0.50 and 0.22, respectively. According to the Shapiro-Wilk test (W≥0.75, p<0.0001), the data

were not normally distributed. The probit transformation indicated a clear cut off point, with the

common antimode at 0.01 describing 3.16% of Swedes (95% CI: 0.01, 0.07) and 18.75% of

Koreans (95% CI: 0.13, 0.26) as slow metabolizers (p<0.0001, 95% CI of the difference in

proportions -0.22, -0.09).

Overall multiple linear regression analysis revealed ethnicity (p=0.0001, R2=0.36) and

CYP2A6 genotype (p=0.006, R2=0.26), but not sex (p=0.14), age (p=0.32), cigarette smoking

(p=0.39) or OC use (p=0.58), as predictors of CYP2A6 activity. Overall, Swedes displayed

higher CYP2A6 enzyme activity than Koreans (P=0.0001, 95% CI of the median difference -0.46,

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-0.23) which remained significant when considering rapid (p=0.0013, 95% CI of the median

difference -0.09, -0.37) and intermediate genotype groups separately (p=0.029, 95% CI of the

median difference -0.01, 0.48) (Fig. 2).

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8. Discussion

The present study compared CYP2A6 genotype distribution and enzyme activity between

Swedes and Koreans. We also examined possible effect of sex, age, cigarette smoking and OC

use on CYP2A6 enzyme activity. We found major significant differences in the distribution of

CYP2A6 genotype and enzyme activity between the two populations. Swedes displayed higher

CYP2A6 enzyme activity than Koreans, mainly due to lower frequencies of CYP2A6 defective

variant alleles in Swedes. Nevertheless, having the same CYP2A6 genotype, the enzyme

activity remained significantly higher in Swedes compared to Koreans, indicating contribution of

ethnicity or other non-genetic factors on CYP2A6 enzyme activity.

CYP2A6 gene is highly polymorphic with more than 80 different alleles described so far

(http://www.cypalleles.ki.se). In the present study, subjects were genotyped for functional variant

alleles known to be existing in Caucasians and/or Orientals, namely 2A6*4, *5, *7, *8, *9, *18,

*1x2, and *1B. Selection of variant alleles for genotyping was based on functional importance

and reported frequency in whites and Asians. Our results corresponded well with the earlier

reports of approximately 1% Caucasians 5,8,9,16,32,35) and 11% Koreans 13,31,36) having CYP2A6

gene deleted. Similarly, the frequencies we obtained for 2A6*9 allele were in accordance with

previous observations of up to 8% in Caucasians 9,10,22,31,32,35,36) and 22% in Koreans 31,37). In

regard to 2A6*5 and CYP2A6 duplication, our observations in Swedes and Koreans were as

expected, as both variants were already proved to be either extremely rare or completely absent

in most populations 4,9,13,16,22,31,32,35,36). Based on the previous studies, 2A6*7, *8 and *10 alleles

appears to be completely absent in Caucasians 8,31-33,35,36), whereas in Koreans they are found at

the frequencies of up to 10%, 1.2% and 1%, respectively 31,37). Frequencies of 2A6*7, *8 and *10

alleles in our study were also in agreement with the earlier findings, as well as the frequencies

obtained for 2A6*18 and 2A6*19 alleles among Koreans 11). The most frequently observed

variant allele in our study in both Swedes and Koreans was CYP2A6*1B, which corresponded

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http://www.cypalleles.ki.se/

well with the previous reports of up to 34% frequency distribution in Caucasians 14,22,32) and up to

43% in Koreans 13). We grouped subjects into rapid, intermediate or slow genotype group based

on presence of two, one or no functional CYP2A6 alleles (CYP2A6*1A or *1B). Carriers of

CYP2A6*1x2 allele were assigned to rapid genotype group. There were significant differences in

the observed frequencies of different genotype groups between Swedes and Koreans (Table 1).

The rapid genotypes were more prevalent in Swedes while the slow genotype group was more

prevalent in Koreans.

CYP2A6 enzyme activity can be determined by several phenotyping strategies, including

coumarin 6,25), nicotine 13,31), cotinine 38), and caffeine 21,22) methods. In the present study, we

used caffeine as a probe drug, and estimated enzyme activity using urine 17X/17U ratio as an

index. In both Swedes and Koreans, the data implied the lack of population homogeneity and the

presence of factors influencing enzyme activity. Since CYP2A6 genetic polymorphism is usually

considered to be largely responsible for variation in enzyme activity 3,4), we investigated its

potential influence. As expected, the effect of genotype on CYP2A6 activity was confirmed.

It is widely accepted that ethnicity represents an important component of inter-individual

variability in response to drugs 39). Due to its crucial role in metabolism of many drugs, toxins and

endogenous compounds 1,3,40), potential inter-ethnic differences in CYP2A6 levels and activity

have been studied in numerous populations. Significantly lower CYP2A6 activity was observed

in African Americans and Asians compared to Caucasians 18) as well as in Japanese compared

to black, white or Korean population 13,31). Among carriers of CYP2A6*1/*1 genotype, Koreans

were reported to display significantly higher CYP2A6 activity compared to black, White American

or Japanese using nicotine as a probe13,31). In contrast, using the same methodology and

caffeine as probe drug, we observed generally higher enzyme activity in Swedes compared to

Koreans regardless of CYP2A6 genotype group. Our finding is line with Kandel et.al, 18,21), where

a higher nicotine metabolite ratio among Whites than Asians is reported. Supporting our

observation, rapid CYP2A6 genotypes were more frequent in Swedes and slow genotypes more

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frequent in Koreans, which corresponds well with the observed inter-ethnic variation in enzyme

activity. Consequently, the observed higher CYP2A6 activity in Swedes than in Koreans could

mainly be due to genotype effect. However, when we stratified study subjects according to the

genotype, the observed difference in enzyme activity between the two populations remained

significant. In both rapid and intermediate genotype groups, Swedes still displayed higher

enzyme activity. Based on these results, CYP2A6 genetic polymorphisms significantly contribute

to, but cannot fully explain, the observed inter-ethnic variability in CYP2A6 enzyme activity. As

suggested previously 13,31,41), apart from investigated genetic variations, other factors, such as

diet, environmental factors, uncharacterized alleles, or epigenetics may possibly affect CYP2A6

enzyme activity too.

In addition to genotype and ethnicity, sex has been frequently associated with CYP2A6

activity. So far, higher enzyme activity in women compare to men was observed in European–

Americans 9,18,23), Africans 20,31), Spanish 18,21), British 12), and Thais 6). Some of the previous

studies also reported increased enzyme activity in OC users 21,23), suggesting estrogen as the

mechanism of CYP2A6 induction in women 23). Yet, the findings on association between

CYP2A6 and sex were not always consistent, as no significant sex differences in enzyme activity

was discovered in Chinese 42), Greeks 26), Serbs 22), or Caucasian in general 24,31). In the present

study, we did not observe any effect of either sex or OC use on CYP2A6 activity in Swedes or in

Koreans.

Unlike other potential determinants of CYP2A6 activity, age was not widely investigated,

but some of the studies described significantly higher enzyme activity in older individuals 12,21,24).

However, the results of the present study support opposite reports 20), as we did not observe any

association of CYP2A6 activity with age neither in Swedes nor in Koreans. On the other hand,

several studies investigated the effect of cigarette smoking on CYP2A6 activity, assuming

competitive enzyme inhibition by nicotine 1,22). Decreased enzyme activity in smokers was

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observed in some 9,20,22,35), but not in all studies 21,24,26), and the results of the present study

comply with the latter.

In conclusion, our study reports major ethnic differences in CYP2A6 genotype distribution

and enzyme activity between Swedes and Koreans. The observed higher CYP2A6 activity in

Swedes compared to Koreans is mainly, but not entirely due to lower frequency of defective

variants alleles in former compared to latter. The influence of sex, age, cigarette smoking and

OC use on CYP2A6 activity is not significant.

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9. Acknowledgments

We thank all volunteers who participated in the study.

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of novel polymorphisms in the CYP2A locus: implications for nicotine metabolism. FEBS

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19


11. Footnotes

Authors:

Natasa Djordjevic a, b, Juan Antonio Carrillo c, Marieke PJ van den Broek a, Junko Kishikawa a,

Hyung-Keun Roh d, Leif Bertilsson a, Eleni Aklillu a

Affiliations:

a Division of Clinical Pharmacology, Department of Laboratory Medicine, Karolinska

University Hospital, Huddinge, Karolinska Institutet, SE-14186 Stockholm, Sweden

b Department of Pharmacology and Toxicology, Medical Faculty, University of Kragujevac,

Svetozara Markovica 69, 34 000 Kragujevac, Serbia

c Division of Clinical Pharmacology, Department of Medical and Surgical Therapeutics,

Medical School, University of Extremadura, E-06071-Badajoz, Spain

d Department of Internal Medicine, Division of Clinical Pharmacology, Inha University

Hospital, 7-206, 3-Ga, Shinheung-Dong, Jung-Gu, Incheon 400–711, South Korea

20


12. Tables

Table 1. Frequency comparison of CYP2A6 polymorphisms, haplotypes, genotypes and

genotype groups between Swedes and Koreans

Swedes Koreans

Polymorphisms

-48T>G ** 0.079 (30/380) 0.219 (63/288)

Gene deletion ** 0.011 (4/380) 0.094 (27/288)

Gene conversion in 3' region * 0.326 (124/380) 0.441 (127/288)

c.1175A>T 0.017 (5/288)

c.1412T>C 0.167 (48/288)

c.1436G>T 0.000 (0/380) 0.000 (0/288)

c.1454G>T 0.049 (14/288)

Gene duplication 0.008 (3/380)

Haplotypes

CYP2A6*1A ** 0.576 (219/380) 0.243 (70/288)

CYP2A6*1B 0.326 (124/380) 0.267 (77/288)

CYP2A6*4 ** 0.011 (4/380) 0.094 (27/288)

CYP2A6*5 0.000 (0/380) 0.000 (0/288)

CYP2A6*7 0.111 (32/288)

CYP2A6*8 0.007 (2/288)

CYP2A6*9 ** 0.079 (30/380) 0.219 (63/288)

CYP2A6*10 0.042 (12/288)

CYP2A6*18 0.003 (1/288)

CYP2A6*19 0.014 (4/288)

CYP2A6*1x2 0.008 (3/380)

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Genotypes

CYP2A6*1A/*1A ** 0.332 (63/190) 0.056 (8/144)

CYP2A6*1A/*1B ** 0.374 (71/190) 0.174 (25/144)

CYP2A6*1A/*4 0.021 (4/190) 0.049 (7/144)

CYP2A6*1A/*7 0.035 (5/144)

CYP2A6*1A/*8 0.007 (1/144)

CYP2A6*1A/*9 0.095 (18/190) 0.076 (11/144)

CYP2A6*1A/*10 0.028 (4/144)

CYP2A6*1A/*18 0.007 (1/144)

CYP2A6*1B/*1B 0.111 (21/190) 0.056 (8/144)

CYP2A6*1B/*4 0.049 (7/144)

CYP2A6*1B/*7 0.076 (11/144)

CYP2A6*1B/*8 0.007 (1/144)

CYP2A6*1B/*9 0.047 (9/190) 0.083 (12/144)

CYP2A6*1B/*10 0.007 (1/144)

CYP2A6*1B/*19 0.028 (4/144)

CYP2A6*4/*7 0.035 (5/144)

CYP2A6*4/*9 0.028 (4/144)

CYP2A6*4/*10 0.028 (4/144)

CYP2A6*7/*9 0.069 (10/144)

CYP2A6*7/*10 0.007 (1/144)

CYP2A6*9/*9 ** 0.005 (1/190) 0.083 (12/144)

CYP2A6*9/*10 0.014 (2/144)

CYP2A6*1x2/*1B 0.011 (2/190)

CYP2A6*1x2/*9 0.005 (1/190)

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Genotype groups

Rapid metabolizer a ** 0.832 (158/190) 0.285 (41/144)

Intermediate metabolizer b ** 0.163 (31/190) 0.451 (65/144)

Slow metabolizer c ** 0.005 (1/190) 0.264 (38/144)

*p=0.002, **p<0.0001

a - CYP2A6*1A/*1A, CYP2A6*1A/*1B, CYP2A6*1B/*1B, CYP2A6*1x2/*1B, or CYP2A6*1x2/*9

b - CYP2A6*1A/*4, CYP2A6*1A/*7, CYP2A6*1A/*8, CYP2A6*1A/*9, CYP2A6*1A/*10,

CYP2A6*1A/*18, CYP2A6*1B/*4, CYP2A6*1B/*7, CYP2A6*1B/*8, CYP2A6*1B/*9,

CYP2A6*1B/*10, or CYP2A6*1B/*19

c - CYP2A6*4/*7, CYP2A6*4/*9, CYP2A6*4/*10, CYP2A6*7/*9, CYP2A6*7/*10, CYP2A6*9/*9, or

CYP2A6*9/*10

23


13. Legends for figures

Figure 1:

The frequency distributions and probit plots of the log transformed 17U/17X ratio in Swedes and

Koreans.

The broken line indicates the common antimode at 0.01.

Figure 2:

Comparison of mean 17U/17X ratio between Swedes (n = 190) and Koreans (n = 144) in

different CYP2A6 genotype groups.

Rapid, intermediate or slow acetylator genotype group was assigned based on the presence of

functional or less functional/non-functional CYP2A6 alleles.

24


14. Figures

0.00 0.02 1.00

17U/17X

0

5

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45

Num

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f sub

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s

Swedesn=190

-3

-2

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0.00 0.02 1.00

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No

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Figure 1

17U/17X17U/17X

Num

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-3

-2

-1

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25


CYP2A6 genotype group

17U

/17X

Rapid Intermediate Slow

0.00

0.02

1.00

Swedes Koreans

Figure 2

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Documents

Comparisons of CYP2A6 genotype and enzyme activity between Swedes and Koreans