canadian native indians exhibit unique cyp2a6 and cyp2c19 mutant allele frequencies*

Canadian Native Indians exhibit unique CTP2A6 and CTP2C19 mutant allele frequencies

Many human cytochromes P450 (CYI’) enzymes are polymorphically expressed, resulting in interindividual and interethnic differences in the metabolism of substrate drugs. Little is known about the genetic variation of CYP enzymes in Canadian Native Indians. We therefore determined the CYP2Cl9 and CTP2A6 mutant allele frequencies in 159 Canadian Native Indians and compared them with white and Asian subjects. Canadian Native Indians differed significantly from both white and Asian populations in allelic patterns of both CYP2C19 (19.1% CTP2C19*2 and 0% CYP2C19*3) and CYP2A6 (0.9% CYP2A6”2 and 13.9% CYP2A6*3). In addition, analysis of the Canadian Native Indian data suggested that there may be an association between the presence of the CYP2CI9 and CYP2A6 mutant alleles such that the co-occur- rence of these 2 alleles is higher than would be predicted on the basis of their individual frequencies in this population. (Clin Pharmacol Ther 1998;64:378-83.)

Maciej I’. Nowak, MSc, Edward M. Sellers, MD, PhD, and Rachel F. Tpdale, PhD Toronto, Ontario, Canada

Cytochromes P450 (CYP) metabolize a wide variety of endogenous and exogenous compounds such as drugs, steroids, prostaglandins, and pheromones.l,* A number of CYPs are genetically polymorphic, exhibit- ing both interindividual and interethnic differences in the metabolism of substrates.

Little is known about CYP allelic variation among Canadian Native Indians. Mitochondrial DNA, anthro- pology, archaeology, linguistics, taxonomy, and genetic studies suggest that Canadian Native Indians are descendants of Asians who crossed the Bering Strait 13,000 to 30,000 years ago.s-5 However, Canadian Native Indians differ significantly from both Asians and white subjects with respect to allelic frequencies for CYP2D6.6 For example, in contrast to Chinese subjects,

From the Departments of Pharmacology, Medicine, and Psychiatry, Centre for Research in Women’s Health, University of Toronto, the Women’s College Hospital, and the Addiction Research Foun- dation.

Supported in part by grant DA06889 from the National Institute on Drug Abuse (Washington, DC) and by the Addiction Research Foundation of Ontario (Toronto, Ontario).

Received for publication March 19, 1998; accepted June 24, 1998. Reprint requests: Rachel F. Tyndale, PhD, Department of Pharma-

cology, Room 4336 Medical Sciences Bldg. University of Toronto, King’s College Circle, Toronto, Ontario, M5S lA8, Canada. E- mail: [email protected]

Copyright 0 1998 by Mosby, Inc. 0009-9236/98/$5.00 + 0 13/l/92751

378

Canadian Native Indians have low CYP2D6*10 allele frequencies (3% versus 51%; P < .OOl) resulting in higher (P < .OOl) CYP2D6 activity.6 In addition, Cana- dian Native Indians have a CYP2D6 poor metabolizer (PM) frequency of 1 .l%, which is lower (P = .03) than that in white subjects (6.2%), principally because of the much lower CYP2D6*4 allele frequencies (3% versus 23%; P < .OOl).

CYP2C19 and CYP2A6 also exhibit interethnic differences in poor metabolizer frequencies (2% to 5% of white subjects and 18% to 23% of Asians for CYP%C19,7-9 and 2% of white subjects, 2.6% of Chi- nese subjects, and 23% of Japanese subjects for CYP2A6).1@12 Because both CYP2C19 and CYP2A6 are involved in the metabolism of many clinically used drugs and toxins, interethnic differences may have important pharmacotherapeutic and toxicologic implications.‘*-‘* To determine whether the differences observed in CYP2D6 allele frequencies among the Canadian Native Indians, Asians, and white subjects extend to other polymorphic CYPs, we characterized CYP2C19 and CYP2A6 mutant allele frequencies in 159 Canadian Native Indians.

SUBJECTS AND METHODS Ethics approval. All study protocols were approved

by the Addiction Research Foundation Ethics Review Committee (Toronto, Ontario).

Now&, Sellers, and Tjmdab 379

Table I. CYP2C19 mutant allele frequencies in Canadian Native Indians

Blood quantum factor Subjects analyzed CYP2C19*2

(male/female) .frequency CYP2Cl9”S frequency

CYP2CIY poor metabolizer,y

100 7s 50 25

TOTAL

Chinese subjects Chinese subjects* Japanese subjects5 Inuit subjectsI/ White subjects?

85/30 19.1% IO/8 8.3% 1202 18.8% 2/o 0% 159 17.6% 69 297ot

202 26%‘ci 217 27%i

90 12% 173 13%:

0% 7.0% (n = 8) 0% 0% (n = 0) 0% 4.2% (n = I) 0% 0% (n = 0) 0% 5.7% (n = 9) 7Q.F 17.8% (n = 12)1 5.5%1 13.4% (n = 27)t

107c’c: 17.3%, (n = 59)t 0% 3.3% (n = 3) 0.3% 3.3% (n = 16)t

Data from $Xiao et al,** $Takakubo et al ,23 /~Jurima-Remet et al,‘x and ‘#Ferguaon et al.“’ tSigniticantly (P < 45) different from rerpective Canadian Native Indian (BQF 100) wlue.

Canadian Native Indian subjects. One hundred fifty-nine Canadian Native Indian volunteers partici- pated in the study. Subjects were recruited from Native Indian community centers in the Toronto area through the Mental Health Unit of the Addiction Research Foundation of Ontario.

Chinese subjects. Unrelated Chinese subjects with 4 Chinese grandparents were genotyped for both CYP2C19 (n = 70) and CYP2A6 (n = 67) mutant alleles as allelic comparisons.

Inclusion and exclusion criteria. All subjects were required to meet the following criteria before accep- tance into the study: (1) at least 1 grandparent of Cana- dian Native Indian descent, (2) healthy male or female, (3) from 16 to 70 years old, and (4) willingness to sign the consent form and abide by the rules of the study. To ensure that study subjects were Indian with respect to race, their Indian ancestry was determined as fol- lows: (1) subjects’ own declaration of whether or not he or she was a Canadian Indian, (2) a family history provided by the subjects, which included the ethnic background and names of parents, grandparents, and great-grandparents, and (3) calculation of the Blood Quantum Factor (BQF) as determined by the subjects’ family history (the BQF is a factor used for racial quan- tification by the United States Government to settle land claims and distinguish Indians from non-Indians’“). In this study, the BQF was used to determine the propor- tion of Canadian Native Indian ancestry for each subject. The BQF ranges from 0 to 100 depending on the individual’s ancestry. For example, an individual who had 3 grandparents of full Canadian Native Indian ancestry and 1 white grandparent would be assigned a BQF of 75.

Blood sample collection. Venous blood samples (12 mL) were collected from all study subjects in Vacu-

tainer tubes (Becton Dickinson, Rutherford, NJ) that contained ACD anticoagulant and stored at -20°C. DNA extraction was carried out as described previously.6

Primer preparation. Primers for all polymerase chain reaction (PCR) assays were synthesized by the Hospital for Sick Children Biotech Service in Toronto, Ontario. Primer sequences were identical to those described previously.l0.2().?’

CYP2C19 genotyping. The CYP2ClY*2 and CYP2C19*3 mutant alleles were identified by use of the method described by de Morais et al.*O.*’ In addition to the SmaI digestion described by de Morais et al,*().21 exon 4 PCR product was also digested with BstNI. This provided a mutant-positive digestion in addition to the wild-type-positive %a1 digestion. Sam- ples, positive (previously characterized controls), and negative (water blanks) were digested with BstNI in a 25 FL mixture that contained NEBuffer 2 (50 mmol/L sodium chloride, 10 mmol/L Tris-hydrochloric acid, 10 mmol/L magnesium chloride, and 1 mmol/L dithio- threitol, pH 7.9 at 25”(Z), 3 units BstNI, and 20 FL PCR product at 60°C for 5 hours.

CYP2A6genotyping. The CYP2A6*2 and CYP2A6*3 mutant alleles were identified with use of nested PCR and restriction fragment length polymorphism.l() PCR products were digested with XcmI at 37°C for 2 hours in a 30 yL reaction mixture containing 1X NEBuffer 3 (10 mmol/L sodium chloride, 50 mmol/L Tri-hydrochloric acid, 10 mmol/L magnesium chloride, and 1 mmol/L DTT, pH 7.9 at 25”(Z), dH,O, and 2 units XcmI. L)deI digestions were carried out at 37°C in a 30 yL reaction mixture that contained One-Phor-All buffer (Pharmacia Biotech) and 2 units DdeI. Positive mutant allele control samples were provided by Drs P. Fernando-Salguero, F.J. Gonzalez, A. Rautio, and H. Raunio. Negative control samples were water blanks.

380 Nowak, Sellers, and Tyndab CLINICAL I’ HARMACOLOGY & THERAPEUTICS

OCTOBER 1998

Table II. CYP2A6 mutant allele frequencies in Canadian Native Indians

Blood quantum factor Subjects analyzed

(male/female) CYP2A6*2

frequency (%) CYP2A6 *3

frequency (70) CYP2A6 poor metabolizers

100 75 50 25

Total Taiwanese$ Japanese+ Caucasian§

80/28 0.9% 7/8 0%

1102 6% 2/o 0%

148 1.3% 89 ll%t 20 20%t

237 4%$

13.9% 10.4% 17.6%

0% 13.6%

6%t 28%t

9%

0% (n = 0) 0% (n = 0) 4% (n = 1) 0% (n = 0) 0.7% (n = 1) 2.9% (NA)

23% (NA)‘F 2.5% (n = 6)

NA, Not available. Poor metabolizer frequencies are derived from allele frequencies. Data from $Femandez-Salguero et al,“’ and &Mlst&n et al.” tSigniticantly (P < .05) different from respective Canadian Native Indian (BQF 100) value

Statistical analysis. Allele frequencies were statisti- n = 140; P < .OOl), Chinese subjects studied by Xiao cally analyzed and compared with use of the x2 test et a122 (5.5%; n = 404; P < .OOl), and Japanese subjects (SAS version 6.12, SAS Institute, Cary, NC). In cases described by Takakubo et a123 (10%; n = 434; P < .OOl) in which the ~2 test could not be used because of a low In contrast, this frequency was not significantly differ- number of observations (n < 5) in any given cell, the ent from the frequency observed in white subjects Fisher exact test was used. (0.3%; n = 173; P = .9) by Ferguson et a1.z4

RESULTS Of the 159 unrelated Canadian Native Indian volun-

teers, 115 had BQF values of 100, indicating that they had 4 Canadian Native Indian grandparents. The remaining 44 volunteers had BQF values that ranged from 25 to 87.5, indicating that at least 1 great-grandparent was not of Canadian Native Indian ancestry (Table I). Thirty-eight of the mixed-ancestry subjects reported partial white ancestry, 4 did not know the ethnic background of their non-Native ancestors, and 2 reported mixed Canadian Native Indian, white, and unknown ancestry. The study volunteers consisted of 109 males and 50 females whose ages ranged from 16 to 62 years (mean f SD age, 33.8 + 9.9 years).

Genotype results indicated that 8 of the Canadian Native Indians (7%; n = 115) were CYP2C19 poor metabolizers, which was in agreement with the Hardy- Weinberg equation (P = .264). A poor metabolizer frequency of 7% is significantly lower (P < .03) than the CYP2C19 poor metabolizer genotype frequency of 17.8% observed in Chinese subjects in our laboratory (Table I) and in Japanese populations23 (17.3%; n = 434; P < .OOl). In contrast, the CYP2C19 poor metabolizer frequency of 7% among Canadian Native Indi- ans is not significantly different from the frequency observed previously in white subjects (3.3%; n = 173; P = .247; Ferguson et a124; Table I).

CYP2C19 genotype. The CYP2C19*2 mutant allele frequency in full Canadian Native Indians (n = 230 alleles) was 19.1% (95% confidence intervals [CI], 16.3% to 21.9%). This frequency was lower than that observed in unrelated Chinese subjects in our laboratory (29%; n = 138; P < .03), Chinese subjects described by Xiao et al22 (26%; n = 404; P < .05), and Japanese subjects described by Takakubo et al*3 (27%; n = 434; P < .03). However, it was significantly higher than the frequency reported by Ferguson et a124 in white subjects (13%; n = 346; P < .05).

CYP2A6 genotype. The CYP2A6*2 mutant allele frequency in full Canadian Native Indians (n = 216 alleles) was 0.9% (95% CI, 0% to 2.2%; Table II), which was significantly lower than in Taiwanese subjects (11%; n = 178; P < .OOl), Japanese subjects (20%; n = 40; P < .OOl),ru and white subjects (4%; n = 237; P < .03).”

No CYP2C19*3 mutant alleles were found in the full Canadian Native Indian subjects (0%; n = 230 alleles). This was significantly lower than the frequencies observed in Chinese subjects in our laboratory (7%;

A frequency of 13.9% (95% CI, 9.3% to 18.5%) for the CYP2A6*3 mutant allele in the Canadian Native Indian subjects (n = 216 alleles) was significantly higher than observed by Femandez-Salguero et alto in Taiwanese populations (6%; n = 178; P < .02) but was lower than the frequency observed among Japanese subjects (28%; n = 40; P c .04). This frequency (13.9%) was not significantly different (9%; n = 474; P = .056) from the frequencies observed in white subjects by Gullsten et al.1

Genotype results indicated that none of the Cana- dian Native Indians (0%; n = 108) were CYP2A6 poor

Nowab, Sellen, and Tjndale 381

metabolizers, which is in agreement with the frequency derived from the Hardy-Weinberg equation (P = .119). A poor metabolizer frequency of 0% is lower than the CYP2A6 poor metabolizer genotype frequency observed in Japanese (23%; n = 20; P < .OO1)lo but not significantly different from the frequency observed previously in white subjects (2.5%; n = 237; P = .182)tt or Taiwanese subjects (2.9%; n = 89; P = .09; Table I).]”

Effects of white admixture on mutant allele frequencies. We have previously reported significant differences in CYP2D6 mutant allele frequencies between full and mixed-ancestry Canadian Native Indian subjects.6 Statistical analysis of the CYP2Cl9 and CYP2A6 data revealed that there were no significant differences in either CYP2C19*2 (P = .2) or CYP2A6 (P = .3 for 2A6*2 and P = .5 for 2A6*3) mutant allele frequencies between the full and mixed-ancestry Canadian Native Indians. This lack of difference may be attributable to the small sample size of the mixed ethnicity study group or to the similarity of allele frequencies found in Canadian Native Indians and in white subjects (the main contributing ethnic group in the mixed ethnicity Canadian Native Indian subjects).

Because CYP2A6 metabolizes nicotine,t2$‘* CYP2A6*1 homozygous (n = 74) and heterozygous (n = 25) smokers were compared with respect to the number of cigarettes smoked per week. However, no statistically significant differences were observed, which is most likely the result of the small numbers of subjects and the phenotypic overlap between homozygous and heterozygous subjects.

Association between CYP2C19 and CYP2A6 genotype. The allele frequency results were analyzed in 108 Canadian Native Indian subjects to determine whether there was an association between the presence of CYP2C19 and CYP2A6 mutant alleles. These genes reside on different chromosomes and are therefore expected to be inherited independently. Because the frequencies of CYP2C19 and CYP2A6 mutant allele frequencies in full Canadian Native subjects were 19.1% (19.1 + 0) and 14.8% (13.9 + 0.9), respectively, one would expect that 2.8% (19.1% x 14.8%) of subjects would carry mutant alleles at both the CYP2A6 and CYP2C19 genes. Our results show that 13 of 108 (12.0%) full Canadian Native Indian subjects exhibit this combination of genotypes, significantly higher (P < .Ol) than that predicted from the allele frequencies. This suggests that there may be an association between the presence of CYP2A6 and CYP2C19 mutant alleles in the Canadian Native Indians and possibly in other populations.

DISCUSSION Because polymorphically expressed CYP enzymes

are involved in the metabolism of many commonly used drugs and toxins, understanding the genotype distribu- tion in different populations may have important clinical implications. Moreover, understanding the patterns of CYPs in Canadian Native Indians may also provide insight into the evolutionary histories of these peoples.

Canadian Native Indians are thought to be Asian descendants who migrated to North America 13,000 to 30,000 years ago.3-5 They have been genetically isolated for thousands of years in an environment that differs from Asia in climate, flora, and fauna. Environmental differences, including diet,* may have produced unique selective pressures on Canadian Native Indians, resulting in altered CYP allelic frequencies. According to the Hardy-Weinberg principle, “both the allele and genotype frequencies in a large, random-mating population will remain constant from generation to generation if there is no mutation, no migration, and no selection.“”

The factors that might best explain the unique allelic frequencies in the Canadian Native Indians include mutation, genetic drift, and selection. Mutation is unlikely to play an important role because mutation rates occur so slowly that they have little impact on the frequencies of common alleles.25 For example, the mutation rate of mammalian P450 genes is approxi- mately 1% in amino acid sequence every 4 million years.26 Genetic drift is “a random change in the frequency of alleles at a locus “25; in small populations it may lead to the loss of specific alleles. One form of genetic drift, the founder principle, results in the frequencies of certain alleles becoming enhanced or decreased when small populations migrate and become founders of a new population. Another form of genetic drift is the bottleneck effect, which describes populations that experience drastic reductions in size as a result of events such as floods, fires, earthquakes, dis- ease, or other progressive changes in environment. These events can result in a small surviving population that has been selected by chance from the original pop- ulation25; certain alleles present in the surviving population may be overpropagated or underpropagated relative to the original population. Finally, differences in CYP mutant allele frequencies may involve natural selection. It is possible that environmental factor(s) in North American (absent in Asia) conferred a genetic advantage to individuals with certain phenotypes resulting in decreased or increased frequencies of certain alleles. In contrast to mutation and genetic drift, natural selection acts to increase fitness and survival poten- tial of the affected populations.

382 Nowak, Sellers, and Tyndale

In a previous study, we examined CYP2D6 in Cana- dian Native Indians and found a low CYP2D6*10 mutant allele frequency relative to Asians, suggesting that a divergence in allele frequency from Asian populations had occurred.6 In addition, beyond that self-reported, there was no indication of white admixture in either our Canadian Native Indian or the Inuit data from Jurima- Romet et a1.27,2* For example, the frequency of CYP2D6*4 in both the Canadian Native Indian and the Inuit populations was not significantly different from the frequency observed in Chinese populations and was significantly lower than that in white subjects. Our data indicate that Canadian Native Indians resemble white subjects with respect to the CYP2C19*3 mutant allele frequency and are intermediate between white and Asian populations with respect to CYP2C19*2 and CYP2C19 poor metabolizer frequencies. A recent study of CYP2C19 in Canadian Inuit subjects (n = 90) revealed CYP2C19*2 and CYP2C19*3 mutant allele frequencies of 12% and O%, respectively,27 which are not significantly different from those observed among Canadian Native Indian subjects. The fact that these 2 populations (1) are both descendants of North Asian populations, (2) arrived in North America independently during 2 sep- arate migratory waves, and (3) both exhibit CYP2C19 mutant allele frequencies that are significantly different from those observed in other Asian populations, all suggest that the differences in CYP2C19 gene frequencies between Asians and Native North Americans might be attributed to natural selection. It is possible that individuals with a higher CYP2C19 activity have an evolutionary advantage in the North American environment compared with individuals with decreased or deficient CYP2C19 activity. Unlike Asians subjects, who may require lower doses of some CYP2C19-substrate drugs, Canadian Native Indians could take CYP2C19-substrate drugs at doses comparable to those administered to white patients as the frequency of the CYP2C19 mutant alleles more closely resembles white subjects than Asians.

CYP2A6 has been implicated in the bioactivation of a number of carcinogens such as nitrosamines, suggesting that interindividual differences in CYP2A6 activity may be associated with altered cancer risk.16,17,29 Given that nitrosamines are present in tobacco smoke, it is possible that smokers with high CYP2A6 activity may be at significantly increased risk of developing tobacco- related cancers compared with smokers with decreased or normal CYP2A6 activity. This suggests that Cana- dian Native Indians may be at higher risk than Asian populations because they carry fewer mutant alleles.

Our study revealed an unexpectedly high co-occur- rence of mutant alleles for the CYP2C19 and CYP2A6

CLINICAL PHARMA COLOGY &THERAPEUTICS OCTOBER 1998

loci in Canadian Native Indians (12.0% versus the predicted 2.8%; P < .Ol), suggesting an advantage may be conferred on individuals who carry mutant alleles at both loci or wild-type alleles at both loci. One expla- nation could be that CYP2A6 and CYP2C19 activate a benign xenobiotic through different pathways to a toxic intermediate, followed by inactivation by the alterna- tive CYP pathway; it would be advantageous to have either wild-type or mutant alleles at both loci rather than having an accumulation of a toxic intermediate.

In summary, our study shows that Canadian Native Indian subjects exhibit CYP2C19 and CYP2A6 genotype patterns that are significantly different from those of both white and Asians subjects. The fact that Cana- dian Native Indians do not resemble Asians with respect to the mutant allele frequencies of CYP2C19 and CYP2A6 is interesting, given that both populations share common genetic ancestral histories. These differences in CYP2C19 and CYP2A6 mutant allele frequencies might be explained by distinct selective pressures unique to North America. These findings may have significant consequences with respect to clinical drug use among Canadian Native Indians.

We thank Linda Gorthy for her help in subject recruitment and Ewa Hoffman, Yushu Rao, and Michael Pianezza for their technical assistance with PCR.

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canadian native indians exhibit unique cyp2a6 and cyp2c19 mutant allele frequencies*

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