Rasmus S Pedersen
email: rpedersen@health.sdu.dk.

Charlotte Brasch-Andersen

Sarah C Sim

Troels K Bergmann

Jónrit Halling

Maria S Petersen

Pál Weihe

Hege Edvardsen

Vessela N Kristensen

Kim Brøsen

Magnus Ingelman-Sundberg

Steen Rasmus

Pedersen

Linkage disequilibrium between the allele and wildtype and alleles: identification of haplotypes in healthy Nordic populations

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Human cytochrome P450 2C (CYP2C) enzymes are responsible for the metabolism of approximately 25% of clinically used drugs [1] and some endogenous substances such as arachidonic acid [2]. The human CYP2C subfamily includes CYP2C8, CYP2C9, CYP2C18 and CYP2C19 that are homologous and share more than 80% amino acid sequence identity [3]. However, only CYP2C8, CYP2C9 and CYP2C19 are considered of clinical importance [4], since CYP2C18 mRNA is detected at relatively low amounts [5]. The entire CYP2C gene locus spans approximately 500 kb on chromosome 10 with the gene order Cen-2C18-2C19-2C9-2C8-Tel (Figure 1) and each of the genes consist of nine exons with conserved exon/intron boundaries [6].

CYP2C8, CYP2C9 and CYP2C19 exhibit clinically important genetic polymorphism as a result of single nucleotide polymorphisms (SNPs) (www.cypalleles.ki.se).

CYP2C8 catalyzes the metabolism of several clinically used drugs such as the anti-cancer drug paclitaxel, the anti-arrhythmic drug amiodarone, the antidiabetic drug repaglinide, and the thiazolidinediones rosiglitazone and troglitazone [7].

Fourteen allelic variants of CYP2C8 have been described of which some cause poor metabolism [8]. CYP2C8*3 is a quantitatively and functionally relevant variant allele in the Caucasian population with a reported frequency of 10-23% but rare or absent in Asian and African populations [5]. CYP2C8*3 is defined by two amino acid substitutions in exon 3 and exon 8 (Table 1), respectively, causing decreased paclitaxel turnover [9] and decreased paclitaxel clearance in vivo [10].

CYP2C9 is among the most important drug metabolizing enzymes in humans.

It is involved in the metabolism of several important drugs including phenytoin, losartan, (S)-warfarin, tolbutamide and numerous NSAIDs [11]. Of the 34 allelic variants that have been described [12], the two clinically most important variant alleles are CYP2C9*2 and CYP2C9*3, which both cause a defective CYP2C9 enzyme (Table 1) [START_REF] King | Upstream and coding region CYP2C9 polymorphisms: correlation with warfarin dose and metabolism[END_REF]. The allele frequencies of CYP2C9*2 and CYP2C9*3 are approximately 10-15% and 5-10%, respectively, in European populations [START_REF] Pedersen | Cytochrome P4502C9 (CYP2C9) genotypes in a Nordic population in Denmark[END_REF].

CYP2C19 is another important drug metabolizing enzyme encoded by a highly polymorphic gene [15]. The racemic anticonvulsant drug mephenytoin is the classic probe drug where the 4-hydroxylation of the (S)-enantiomer is catalysed by CYP2C19. Furthermore, a range of clinically important drugs including diazepam, omeprazole and other proton pump inhibitors, the antidepressants citalopram, escitalopram and imipramine, the antimalarial drugs proguanil and chlorproguanil, the β-adrenoceptor blocker propranolol, and the antiplatelet drug clopidogrel are metabolized or bioactivated by CYP2C19. Population studies have shown that individuals that are homozygous for the inactivating allele CYP2C19*2 that leads to a splicing defect (Table 1), are phenotypic poor metabolizers of CYP2C19 substrates [5,[START_REF] Wilkinson | Genetic polymorphism of Smephenytoin hydroxylation[END_REF][START_REF] De Morais | The major genetic defect responsible for the polymorphism of Smephenytoin metabolism in humans[END_REF]. The CYP2C19 poor metabolizers only represent 3-5% of Caucasian populations, but is found in 12-23% of Asian populations [3]. In recent years, much attention has focused on the allele CYP2C19*17 that correlates with high in vivo CYP2C19 activity and is present at an allele frequency of 18% in a Swedish healthy population [START_REF] Sim | A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants[END_REF]. The initial finding of a rapid drug metabolism phenotype was later supported by a clear association of lower plasma concentration of omeprazole and escitalopram in subjects homozygous for CYP2C19*17 [START_REF] Baldwin | Increased omeprazole metabolism in carriers of the CYP2C19*17 allele; a pharmacokinetic study in healthy volunteers[END_REF][START_REF] Rosenborg | Kinetics of omeprazole and escitalopram in relation to the CYP2C19*17 allele in healthy subjects[END_REF]. Interestingly, CYP2C19*17 also leads to increased bioactivation of the antiplatelet drug clopidogrel that causes an almost 4-fold higher risk of bleeding complications in CYP2C19*17

homozygotes, but no beneficial antithrombotic effect for patients undergoing percutaneous coronary intervention [START_REF] Sibbing | Cytochrome 2C19*17 allelic variant, platelet aggregation, bleeding events, and stent thrombosis in clopidogreltreated patients with coronary stent placement[END_REF]. As opposed to subjects homozygous for the CYP2C19*1 allele, subjects homozygous for CYP2C19*17 display a very low interindividual variation in CYP2C19 metabolism [START_REF] Baldwin | Increased omeprazole metabolism in carriers of the CYP2C19*17 allele; a pharmacokinetic study in healthy volunteers[END_REF][START_REF] Rudberg | Impact of the ultrarapid CYP2C19*17 allele on serum concentration of escitalopram in psychiatric patients[END_REF]. The rapid phenotype caused by CYP2C19*17 has been explained by a recruitment of transcription factor(s) by the -806C>T conversion (Table 1) in the gene promotor region that causes an increased transcriptional rate [START_REF] Sim | A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants[END_REF]. A hypothesis is that CYP2C19*17 is also a haplotype marker within the CYP2C locus [START_REF] Baldwin | Increased omeprazole metabolism in carriers of the CYP2C19*17 allele; a pharmacokinetic study in healthy volunteers[END_REF]. In fact, haplotype block structures at the CYP2C locus have been described [START_REF] Ahmadi | A single-nucleotide polymorphism tagging set for human drug metabolism and transport[END_REF][START_REF] Walton | Haplotype block structure of the cytochrome P450 CYP2C gene cluster on chromosome 10[END_REF]. Almost all subjects carrying CYP2C8*3 also carry CYP2C9*2 and three quarters of all subjects carrying CYP2C9*2 also carry CYP2C8*3 [START_REF] Yasar | Linkage between the CYP2C8 and CYP2C9 genetic polymorphisms[END_REF]. CYP2C19*2 and CYP2C19*3 has also been shown to be linked with CYP2C18 mutations in Japanese subjects [START_REF] Mamiya | Association of polymorphisms in the cytochrome P450 (CYP) 2C19 and 2C18 genes in Japanese epileptic patients[END_REF].

The aim of this study was to determine important CYP2C genotypes with special focus on CYP2C19*17 in healthy Nordic populations, and to investigate the linkage disequilibrium between them. [START_REF] Halling | Polymorphsims of CYP2D6, CYP2C19, CYP2C9, CYP2C8 in the Faroese population[END_REF] were included in this study. Furthermore, DNA samples from 328 Norwegian Caucasian blood donors that had not been genotyped for CYP2C variants were included [START_REF] Heimdal | Association studies of estrogen receptor polymorphisms in a Norwegian testicular cancer population[END_REF][START_REF] Tefre | Genotyping of the CYP2D6 gene in Norwegian lung cancer patients and controls[END_REF].

Methods

Comment [RP2]: Detailed info about the allellic variant has been moved to

Genotyping

A total of 915 DNA samples from three different populations were available for genotyping and the aim was to cover the five clinically important CYP2C allelic variants; CYP2C8*, CYP2C9*2, CYP2C9*3, CYP2C19*2 and CYP2C19*17. All 276 DNA samples from Denmark [START_REF] Pedersen | Cytochrome P4502C9 (CYP2C9) genotypes in a Nordic population in Denmark[END_REF] were successfully genotyped for CYP2C8*3, CYP2C19*2 and CYP2C19*17 and all the 311 Faroese samples were successfully genotyped for CYP2C19*17. Of the 328 Norwegian DNA samples, 309 were successfully genotyped for all five CYP2C variants. The analyses were performed using Applied Biosystems TaqMan SNP genotyping assays (Table 1) according to the guidelines and conducted on an Applied Biosystems StepOne Plus apparatus or an Applied Biosystems 7500 Real Time PCR apparatus.

Statistics

Possible deviation from Hardy Weinberg equilibrium was tested for each SNP with Pearsons Chi-Square test with a level of significance of 5%. Overall differences of allele frequencies between the populations were also tested by Chi-Squared test with a level of significance of 5%. "Head to head" comparison between the populations was tested with Fisher´s exact test with a level of significance of 5%.

Linkage disequilibrium and haplotype inference

The software Haploview was used to visualize the structure of pair-wise linkage disequilibrium (LD) between the SNPs (in terms of Lewontin's D') [START_REF] Barrett | Haploview: analysis and visualization of LD and haplotype maps[END_REF]. Individual haplotypes were inferred by use of the five CYP2C SNPs and software package PHASE version 2.1 [START_REF] Stephens | A new statistical method for haplotype reconstruction from population data[END_REF][START_REF] Stephens | Accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation[END_REF]. This software infers haplotypes from population genotype data using a Bayesian statistical method. We performed PHASE analyses Ten CYP2C haplotypes were inferred (Table 3). The 2). The previously reported linkage disequilibrium between CYP2C9*2 and CYP2C8*3 presented by Yasar et al. [START_REF] Yasar | Linkage between the CYP2C8 and CYP2C9 genetic polymorphisms[END_REF] was also supported by our data. In detail, 94% (144 out of 154) of the haplotypes with CYP2C8*3 were also predicted to carry CYP2C9*2, and 79% (144 out of 183) of the haplotypes with CYP2C9*2 were also predicted to carry CYP2C8*3.

Discussion

This study provides support to the hypothesis of a linkage disequilibrium between CYP2C19*17 and functional CYP2C alleles. This hypothesis was first expressed by Baldwin et al. [START_REF] Baldwin | Increased omeprazole metabolism in carriers of the CYP2C19*17 allele; a pharmacokinetic study in healthy volunteers[END_REF] but was based on relatively limited data. Further indications can be found by reviewing the genotype information presented by Rudberg et al. [START_REF] Rudberg | Impact of the ultrarapid CYP2C19*17 allele on serum concentration of escitalopram in psychiatric patients[END_REF]. In the present study, we found that CYP2C19*17 is predicted to be almost exclusively present together with wild type alleles of CYP2C8 and CYP2C9, thus mediating rapid metabolism of CYP2C19 substrates and normal (mostly referred to as extensive) metabolism of CYP2C8 and CYP2C9 substrates.

The prediction of one haplotype out of the 343 CYP2C19*17-containing haplotypes that exists in combination with CYP2C8*3 must be recognized. However the existence of these two variants on separate haplotypes is still a theoretic possibility, although predicted to be rare. Nevertheless, the clinical picture is clear showing that subjects with CYP2C19*17 that confers a rapid metabolism of [START_REF] Yasar | Linkage between the CYP2C8 and CYP2C9 genetic polymorphisms[END_REF]. Thus, poor metabolisers of CYP2C9 substrate drugs are also likely to poorly metabolise CYP2C8 substrates. It is interesting to note that across the three genes CYP2C19, CYP2C9 and CYP2C8, the five SNPs examined occur in six distinct haplotypes 99% of the time and further that these haplotypes exclusively carry none or just a single SNP except for haplotype No.9 characterized by two SNPs. This is in accordance with the notion that SNPs are introduced to the genome as a single mutation occurring at a single point in time in a single individual and vertically propagated to the population. In future pharmacogenetic studies it might be worthwhile to consider these haplotypes as an alternative to the individual SNPs as this approach might offer a more powerful design that can possibly reveal effects of interplay between cis variants, which is impossible in simple analysis of individual SNPs.

The current study supports the general assumption that the Nordic populations have a relatively similar distribution of cytochrome P450 genotypes. The CYP2C19*17 allele was first reported in Swedes with a frequency of 18% [START_REF] Sim | A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants[END_REF], but a recent study report a Swedish allele frequency of 20% [START_REF] Ramsjö | CYP2C19 activity comparison between Swedes and Koreans: effect of genotype, sex, oral contraceptive use, and smoking[END_REF]. The Norwegian CYP2C19*17 allele frequency of 22% in the current study of healthy volunteers is identical with the reported frequency in 166 Norwegian psychiatric patients [START_REF] Rudberg | Impact of the ultrarapid CYP2C19*17 allele on serum concentration of escitalopram in psychiatric patients[END_REF], and similar to that observed in the Danish subjects of the present study (20%). The Faroese population had a statistically significant lower allele frequency of 15%, but no correction for multiple testing was done. A similar allele frequency of 20% was found in Greek population [START_REF] Ragia | Need for reassessment of reported CYP2C19 allele frequencies in various populations in view of CYP2C19*17 discovery: the case of Greece[END_REF] and in African populations: 18% in Ethiopians [START_REF] Sim | A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants[END_REF] and 17% in Ugandans [START_REF] Miura | Cytochrome P450 2C19 genetic polymorphisms in Ugandans[END_REF]. In Asian populations very low frequencies of CYP2C19*17 has been reported: 4% in Chinese [START_REF] Sim | A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants[END_REF], 1% in Japanese [START_REF] Sugimoto | Limited frequency of the CYP2C19*17 allele and its minor role in a Japanese population[END_REF] and 0.3% in Koreans [START_REF] Ramsjö | CYP2C19 activity comparison between Swedes and Koreans: effect of genotype, sex, oral contraceptive use, and smoking[END_REF]. On the other hand, the frequency is apparently higher in Central Europe; 25% in a German breast cancer population [START_REF] Justenhoven | CYP2C19*17 is associated with decreased breast cancer risk[END_REF] and 28% in a population of Polish peptic ulcer patients [START_REF] Gawrońska-Szklarz | Effects of CYP2C19, MDR1, and interleukin 1-B gene variants on the eradication rate of Helicobacter pylori infection by triple therapy with pantoprazole, amoxicillin, and metronidazole[END_REF]. Overall, CYP2C19*17 must be considered prevalent in all the Nordic populations studied here, and the frequency of subjects homozygous for CYP2C19*17 only ranged from 3.5 to 5.1 % in the studied material.

In conclusion, we have in the current study identified a common haplotype in the Nordic population carrying the CYP2C19*17, CYP2C8*1 and CYP2C9*1 alleles, thus representing a haplotype encoding efficient metabolism of all drugs that are

Introduction

Human cytochrome P450 2C (CYP2C) enzymes are responsible for the metabolism of approximately 25% of clinically used drugs [1] and some endogenous substances such as arachidonic acid [2]. The human CYP2C subfamily includes CYP2C8, CYP2C9, CYP2C18 and CYP2C19 that are homologous and share more than 80% amino acid sequence identity [3]. However, only CYP2C8, CYP2C9 and CYP2C19 are considered of clinical importance [4], since CYP2C18 mRNA is detected at relatively low amounts [5]. The entire CYP2C gene locus spans approximately 500 kb on chromosome 10 with the gene order Cen-2C18-2C19-2C9-2C8-Tel (Figure 1) and each of the genes consist of nine exons with conserved exon/intron boundaries [6].

CYP2C8, CYP2C9 and CYP2C19 exhibit clinically important genetic polymorphism as a result of single nucleotide polymorphisms (SNPs) (www.cypalleles.ki.se).

CYP2C8 catalyzes the metabolism of several clinically used drugs such as the anti-cancer drug paclitaxel, the anti-arrhythmic drug amiodarone, the antidiabetic drug repaglinide, and the thiazolidinediones rosiglitazone and troglitazone [7].

Fourteen allelic variants of CYP2C8 have been described of which some cause poor metabolism [8]. CYP2C8*3 is a quantitatively and functionally relevant variant allele in the Caucasian population with a reported frequency of 10-23% but rare or absent in Asian and African populations [5]. CYP2C8*3 is defined by two amino acid substitutions in exon 3 and exon 8 (Table 1), respectively, causing decreased paclitaxel turnover [9] and decreased paclitaxel clearance in vivo [10].

CYP2C9 is among the most important drug metabolizing enzymes in humans.

It is involved in the metabolism of several important drugs including phenytoin, losartan, (S)-warfarin, tolbutamide and numerous NSAIDs [11]. Of the 34 allelic variants that have been described [12], the two clinically most important variant alleles are CYP2C9*2 and CYP2C9*3, which both cause a defective CYP2C9 enzyme (Table 1) [START_REF] King | Upstream and coding region CYP2C9 polymorphisms: correlation with warfarin dose and metabolism[END_REF]. The allele frequencies of CYP2C9*2 and CYP2C9*3 are approximately 10-15% and 5-10%, respectively, in European populations [START_REF] Pedersen | Cytochrome P4502C9 (CYP2C9) genotypes in a Nordic population in Denmark[END_REF]. 1), are phenotypic poor metabolizers of CYP2C19 substrates [5,[START_REF] Wilkinson | Genetic polymorphism of Smephenytoin hydroxylation[END_REF][START_REF] De Morais | The major genetic defect responsible for the polymorphism of Smephenytoin metabolism in humans[END_REF]. The CYP2C19 poor metabolizers only represent 3-5% of Caucasian populations, but is found in 12-23% of Asian populations [3]. In recent years, much attention has focused on the allele CYP2C19*17 that correlates with high in vivo CYP2C19 activity and is present at an allele frequency of 18% in a Swedish healthy population [START_REF] Sim | A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants[END_REF]. The initial finding of a rapid drug metabolism phenotype was later supported by a clear association of lower plasma concentration of omeprazole and escitalopram in subjects homozygous for CYP2C19*17 [START_REF] Baldwin | Increased omeprazole metabolism in carriers of the CYP2C19*17 allele; a pharmacokinetic study in healthy volunteers[END_REF][START_REF] Rosenborg | Kinetics of omeprazole and escitalopram in relation to the CYP2C19*17 allele in healthy subjects[END_REF]. Interestingly, CYP2C19*17 also leads to increased bioactivation of the antiplatelet drug clopidogrel that causes an almost 4-fold higher risk of bleeding complications in CYP2C19* 17homozygotes, but no beneficial antithrombotic effect for patients undergoing percutaneous coronary intervention [START_REF] Sibbing | Cytochrome 2C19*17 allelic variant, platelet aggregation, bleeding events, and stent thrombosis in clopidogreltreated patients with coronary stent placement[END_REF]. As opposed to subjects homozygous for the CYP2C19*1 allele, subjects homozygous for CYP2C19*17 display a very low interindividual variation in CYP2C19 metabolism [START_REF] Baldwin | Increased omeprazole metabolism in carriers of the CYP2C19*17 allele; a pharmacokinetic study in healthy volunteers[END_REF][START_REF] Rudberg | Impact of the ultrarapid CYP2C19*17 allele on serum concentration of escitalopram in psychiatric patients[END_REF]. The rapid phenotype caused by CYP2C19*17 has been explained by a recruitment of transcription factor(s) by the -806C>T conversion (Table 1) in the gene promotor region that causes an increased transcriptional rate [START_REF] Sim | A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants[END_REF]. A hypothesis is that CYP2C19*17 is also a haplotype marker within the CYP2C locus [START_REF] Baldwin | Increased omeprazole metabolism in carriers of the CYP2C19*17 allele; a pharmacokinetic study in healthy volunteers[END_REF]. In fact, haplotype block structures at the CYP2C locus have been described [START_REF] Ahmadi | A single-nucleotide polymorphism tagging set for human drug metabolism and transport[END_REF][START_REF] Walton | Haplotype block structure of the cytochrome P450 CYP2C gene cluster on chromosome 10[END_REF]. Almost all subjects carrying CYP2C8*3 also carry CYP2C9*2 and three quarters of all subjects carrying CYP2C9*2 also carry CYP2C8*3 [START_REF] Yasar | Linkage between the CYP2C8 and CYP2C9 genetic polymorphisms[END_REF]. CYP2C19*2 and CYP2C19*3 has also been shown to be linked with CYP2C18 mutations in Japanese subjects [START_REF] Mamiya | Association of polymorphisms in the cytochrome P450 (CYP) 2C19 and 2C18 genes in Japanese epileptic patients[END_REF].

The aim of this study was to determine important CYP2C genotypes with special focus on CYP2C19*17 in healthy Nordic populations, and to investigate the linkage disequilibrium between them. previously genotyped for CYP2C9*2 and CYP2C9*3 [START_REF] Pedersen | Cytochrome P4502C9 (CYP2C9) genotypes in a Nordic population in Denmark[END_REF], and 311 Faroese samples from healthy volunteers previously genotyped for CYP2C8*3, CYP2C9*2, CYP2C9*3 and CYP2C19*2 [START_REF] Halling | Polymorphsims of CYP2D6, CYP2C19, CYP2C9, CYP2C8 in the Faroese population[END_REF] were included in this study. Furthermore, DNA samples from 328 Norwegian Caucasian blood donors that had not been genotyped for CYP2C variants were included [START_REF] Heimdal | Association studies of estrogen receptor polymorphisms in a Norwegian testicular cancer population[END_REF][START_REF] Tefre | Genotyping of the CYP2D6 gene in Norwegian lung cancer patients and controls[END_REF].

Methods

Genotyping

A total of 915 DNA samples from three different populations were available for genotyping and the aim was to cover the five clinically important CYP2C allelic variants; CYP2C8*, CYP2C9*2, CYP2C9*3, CYP2C19*2 and CYP2C19*17. All 276 DNA samples from Denmark [START_REF] Pedersen | Cytochrome P4502C9 (CYP2C9) genotypes in a Nordic population in Denmark[END_REF] were successfully genotyped for CYP2C8*3, CYP2C19*2 and CYP2C19*17 and all the 311 Faroese samples were successfully genotyped for CYP2C19*17. Of the 328 Norwegian DNA samples, 309 were successfully genotyped for all five CYP2C variants. The analyses were performed using Applied Biosystems TaqMan SNP genotyping assays (Table 1) according to the guidelines and conducted on an Applied Biosystems StepOne Plus apparatus or an Applied Biosystems 7500 Real Time PCR apparatus.

Statistics

Possible deviation from Hardy Weinberg equilibrium was tested for each SNP with Pearsons Chi-Square test with a level of significance of 5%. Overall differences of allele frequencies between the populations were also tested by Chi-Squared test with a level of significance of 5%. "Head to head" comparison between the populations was tested with Fisher´s exact test with a level of significance of 5%.

Linkage disequilibrium and haplotype inference

The software Haploview was used to visualize the structure of pair-wise linkage disequilibrium (LD) between the SNPs (in terms of Lewontin's D') [START_REF] Barrett | Haploview: analysis and visualization of LD and haplotype maps[END_REF]. Individual haplotypes were inferred by use of the five CYP2C SNPs and software package PHASE version 2.1 [START_REF] Stephens | A new statistical method for haplotype reconstruction from population data[END_REF][START_REF] Stephens | Accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation[END_REF]. This software infers haplotypes from population genotype data using a Bayesian statistical method. We performed PHASE analyses Ten CYP2C haplotypes were inferred (Table 3). The CYP2C haplotype frequencies predicted by PHASE and STATA were almost identical. Six of the 2). The previously reported linkage disequilibrium between CYP2C9*2 and CYP2C8*3 presented by Yasar et al. [START_REF] Yasar | Linkage between the CYP2C8 and CYP2C9 genetic polymorphisms[END_REF] was also supported by our data. In detail, 94% (144 out of 154) of the haplotypes with CYP2C8*3 were also predicted to carry CYP2C9*2, and 79% (144 out of 183) of the haplotypes with CYP2C9*2 were also predicted to carry CYP2C8*3.

Discussion

This study provides support to the hypothesis of a linkage disequilibrium between CYP2C19*17 and functional CYP2C alleles. This hypothesis was first expressed by Baldwin et al. [START_REF] Baldwin | Increased omeprazole metabolism in carriers of the CYP2C19*17 allele; a pharmacokinetic study in healthy volunteers[END_REF] but was based on relatively limited data. Further indications can be found by reviewing the genotype information presented by Rudberg et al. [START_REF] Rudberg | Impact of the ultrarapid CYP2C19*17 allele on serum concentration of escitalopram in psychiatric patients[END_REF]. In the present study, we found that CYP2C19*17 is predicted to be almost exclusively present together with wild type alleles of CYP2C8 and CYP2C9, thus mediating rapid metabolism of CYP2C19 substrates and normal (mostly referred to as extensive) metabolism of CYP2C8 and CYP2C9 substrates.

The prediction of one haplotype out of the 343 CYP2C19*17-containing haplotypes that exists in combination with CYP2C8*3 must be recognized. However the existence of these two variants on separate haplotypes is still a theoretic possibility, although predicted to be rare. Nevertheless, the clinical picture is clear showing that subjects with CYP2C19*17 that confers a rapid metabolism of CYP2C19 substrates, are likely to display extensive metabolic capacity toward other CYP2C substrates.

This study also confirms the linkage between CYP2C9*2 and CYP2C8*3 with similar [START_REF] Yasar | Linkage between the CYP2C8 and CYP2C9 genetic polymorphisms[END_REF]. Thus, poor metabolisers of CYP2C9 substrate drugs are also likely to poorly metabolise CYP2C8 substrates. It is interesting to note that across the three genes CYP2C19, CYP2C9 and CYP2C8, the five SNPs examined occur in six distinct haplotypes 99% of the time and further that these haplotypes exclusively carry none or just a single SNP except for haplotype No.9 characterized by two SNPs. This is in accordance with the notion that SNPs are introduced to the genome as a single mutation occurring at a single point in time in a single individual and vertically propagated to the population. In future pharmacogenetic studies it might be worthwhile to consider these haplotypes as an alternative to the individual SNPs as this approach might offer a more powerful design that can possibly reveal effects of interplay between cis variants, which is impossible in simple analysis of individual SNPs.

The current study supports the general assumption that the Nordic populations have a relatively similar distribution of cytochrome P450 genotypes. The CYP2C19*17 allele was first reported in Swedes with a frequency of 18% [START_REF] Sim | A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants[END_REF], but a recent study report a Swedish allele frequency of 20% [START_REF] Ramsjö | CYP2C19 activity comparison between Swedes and Koreans: effect of genotype, sex, oral contraceptive use, and smoking[END_REF]. The Norwegian CYP2C19*17 allele frequency of 22% in the current study of healthy volunteers is identical with the reported frequency in 166 Norwegian psychiatric patients [START_REF] Rudberg | Impact of the ultrarapid CYP2C19*17 allele on serum concentration of escitalopram in psychiatric patients[END_REF], and similar to that observed in the Danish subjects of the present study (20%). The Faroese population had a statistically significant lower allele frequency of 15%, but no correction for multiple testing was done. A similar allele frequency of 20% was found in Greek population [START_REF] Ragia | Need for reassessment of reported CYP2C19 allele frequencies in various populations in view of CYP2C19*17 discovery: the case of Greece[END_REF] and in African populations: 18% in Ethiopians [START_REF] Sim | A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants[END_REF] and 17% in Ugandans [START_REF] Miura | Cytochrome P450 2C19 genetic polymorphisms in Ugandans[END_REF]. In Asian populations very low frequencies of CYP2C19*17 has been reported: 4% in Chinese [START_REF] Sim | A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants[END_REF], 1% in Japanese [START_REF] Sugimoto | Limited frequency of the CYP2C19*17 allele and its minor role in a Japanese population[END_REF] and 0.3% in Koreans [START_REF] Ramsjö | CYP2C19 activity comparison between Swedes and Koreans: effect of genotype, sex, oral contraceptive use, and smoking[END_REF]. On the other hand, the frequency is apparently higher in Central Europe; 25% in a German breast cancer population [START_REF] Justenhoven | CYP2C19*17 is associated with decreased breast cancer risk[END_REF] and 28% in a population of Polish peptic ulcer patients [START_REF] Gawrońska-Szklarz | Effects of CYP2C19, MDR1, and interleukin 1-B gene variants on the eradication rate of Helicobacter pylori infection by triple therapy with pantoprazole, amoxicillin, and metronidazole[END_REF]. Overall, CYP2C19*17 must be considered prevalent in all the Nordic populations studied here, and the frequency of subjects homozygous for CYP2C19*17 only ranged from 3.5 to 5.1 % in the studied material.

In conclusion, we have in the current study identified a common haplotype in the Nordic population carrying the CYP2C19*17, CYP2C8*1 and CYP2C9*1 alleles, thus representing a haplotype encoding efficient metabolism of all drugs that are The linkage disequilibrium (LD) map of the 5 SNPs analysed in the current study was created using Haploview. The dark squares without numbers show complete LD (D'=1, LOD >3.6).

F o r P e e r R e v i e w

1

  ten times with different random seed. Additionally, haplotype frequencies were estimated using the expectation maximation algorithm (EM) in STATA10 (StataCorp, Texas, USA).ResultsWeperformed genotyping of CYP2C8*3, CYP2C9*2, CYP2C9*3, CYP2C19*2 and CYP2C19*17 in three different Nordic populations. The genotype and allele frequencies in the Danish, Faroese and Norwegian samples are presented in Table 2. All SNPs in all three populations were in Hardy-Weinberg equilibrium displaying χ 2values less than 3.84. An overall significant difference in allele frequency of CYP2C19*17 was found between the populations (P=0.010). The allele frequency of 15 % in the Faroese population (n=311) was lower than the 20% in the Danish population (n=276) (P=0.038) and the 22% in the Norwegian population (n=309, P=0.003). No statistically significant difference between the Danish and the Norwegian population was found. None of the four other SNPs displayed significantly different allele frequencies between the populations. When analyzing all three populations, none of the 40 subjects homozygous for the rapid CYP2C19*17 allele carried the defective CYP2C8*3, CYP2C9*2 or CYP2C9*3 alleles. In addition, none of the 9 subjects homozygous for CYP2C8*3 carried CYP2C19*17 and none of the 20 subjects homozygous for CYP2C9*2 and/or CYP2C9*3 carried CYP2C19*17. The linkage disequilibrium between the five analysed SNPs as based on Haploview calculations is visualized in Figure 2. The calculations show complete LD (D'=1) between CYP2C19*17 and the four other allelic variants. Complete linkage disequilibrium was also observed between CYP2C19*2 and the two CYP2C9 alleles CYP2C9*2 and CYP2C9*3. The analysis also displays strong linkage disequilibrium between CYP2C9*2 and CYP2C8*3 (D´=0.92).

  CYP2C haplotype frequencies predicted by PHASE and STATA were almost identical. Six of the .1-5 and 9) account for 99% of the haplotypes. The most frequent haplotype (No. 1) with an inferred frequency of 49% carries the CYP2C8*1, CYP2C9*1 and CYP2C19*1 alleles. The second most frequent haplotype (No. 2) with an inferred frequency of 19% is composed by CYP2C19*17, CYP2C8*1 and CYP2C9*1 wildtype alleles, and is representing 99.7% (342 out of 343) of all predicted CYP2C19*17-containing haplotypes. In one Norwegian subject, a single variant haplotype with CYP2C19*17 in combination with CYP2C8*3 was inferred, thus leading to an estimated haplotype frequency of <0.001 in the total material of 896 subjects (Table

  CYP2C19 substrates, are likely to display extensive metabolic capacity toward other CYP2C substrates.This study also confirms the linkage between CYP2C9*2 and CYP2C8*3 with similar previously shown byYasar et al.

  Comment [RP4]: More info of CYP2C19*17 allele freq in other populations enzymes. The CYP2C19*17 genotype could thus be used as a determinant for extensive metabolism of CYP2C substrates in Nordic subjects. BP, Khan TI, Aithal GP, Kamali F, Daly AK (2004) Upstream and coding region CYP2C9 polymorphisms: correlation with warfarin dose and metabolism. Pharmacogenetics 14:813-22. 14. Pedersen RS, Verstuyft C, Becquemont L, Jaillon P, Brøsen K (2004) Cytochrome P4502C9 (CYP2C9) genotypes in a Nordic population in Denmark. Basic Clin Pharmacol Toxicol 94:151-152 15. http://www.cypalleles.ki.se/cyp2c19.htm assessed in June 2010 16. Wilkinson GR, Guengerich FP, Branch RA (1989) Genetic polymorphism of Smephenytoin hydroxylation. Pharmacol Ther 43:53-76 17. de Morais SM, Wilkinson GR, Blaisdell J, Nakamura K, Meyer UA, Goldstein JA (1994) The major genetic defect responsible for the polymorphism of Smephenytoin metabolism in humans. J Biol Chem 269:15419-22 18. Sim SC, Risinger C, Dahl ML, Aklillu E, Christensen M, Bertilsson L, Ingelman-Sundberg M (2006) A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants. Clin Pharmacol Ther 79:103-13 19. Baldwin RM, Ohlsson S, Pedersen RS, Mwinyi J, Ingelman-Sundberg M, Eliasson E, Bertilsson L (2008) Increased omeprazole metabolism in carriers of the CYP2C19*17 allele; a pharmacokinetic study in healthy volunteers. Br J Clin Pharmacol 65:767-74 20. Ohlsson Rosenborg S, Mwinyi J, Andersson M, Baldwin RM, Pedersen RS, Sim SC, Bertilsson L, Ingelman-Sundberg M, Eliasson (2008) Kinetics of omeprazole and escitalopram in relation to the CYP2C19*17 allele in healthy subjects. Eur J Clin Pharmacol 64:1175-9 21. Sibbing D, Koch W, Gebhard D, Schuster T, Braun S, Stegherr J, Morath T, Schömig A, von Beckerath N, Kastrati A. (2010) Cytochrome 2C19*17 allelic variant, platelet aggregation, bleeding events, and stent thrombosis in clopidogreltreated patients with coronary stent placement. Circulation.;121:512-8. 22. Rudberg I, Mohebi B, Hermann M, Refsum H, Molden E (2008) Impact of the ultrarapid CYP2C19*17 allele on serum concentration of escitalopram in psychiatric patients. Clin Pharmacol Ther 83:322-7 23. Ahmadi KR, Weale ME, Xue ZY, Soranzo N, Yarnall DP, Briley JD, Maruyama Y, Kobayashi M, Wood NW, Spurr NK, Burns DK, Roses AD, Saunders AM, 2005) A single-nucleotide polymorphism tagging set for human drug metabolism and transport. Nat Genet 37:84-9 24. Walton R, Kimber M, Rockett K, Trafford C, Kwiatkowski D, Sirugo G (2005) Haplotype block structure of the cytochrome P450 CYP2C gene cluster on chromosome 10. Nat Genet 37:915-6 25. Yasar U, Lundgren S, Eliasson E, Bennet A, Wiman B, de Faire U, Rane A (2002) Linkage between the CYP2C8 and CYP2C9 genetic polymorphisms. Biochem Biophys Res Commun 299:25-8 26. Mamiya K, Ieiri I, Miyahara S, Imai J, Furuumi H, Fukumaki Y, Ninomiya H, Tashiro N, Yamada H, Higuchi S (1998) Association of polymorphisms in the cytochrome P450 (CYP) 2C19 and 2C18 genes in Japanese epileptic patients. Pharmacogenetics 8:87-90 27. Halling J, Petersen MS, Damkier P, Nielsen F, Grandjean P, Weihe Pál, Lundgren S, Lundblad MS, Brøsen K (2005) Polymorphsims of CYP2D6, CYP2C19, CYP2C9, CYP2C8 in the Faroese population. Eur J Clin Pharmacol 61:491-497 28. Heimdal K, Andersen TI, Skrede M, Fosså SD, Berg K, Børresen AL (1995) Association studies of estrogen receptor polymorphisms in a Norwegian testicular cancer population. Cancer Epidemiol Biomarkers Prev 4(2):123-6 29. Tefre T, Daly AK, Armstrong M, Leathart JB, Idle JR, Brøgger A, Børresen AL (1994) Genotyping of the CYP2D6 gene in Norwegian lung cancer patients and controls. Pharmacogenetics 4(2):47-57 30. Barrett JC, Fry B, Maller J, Daly MJ (2005) Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21:263-265 31. Stephens M, Smith NJ, Donnelly P (2001) A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 68: 978-989 32. Stephens M, Scheet P (2005) Accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation. Am J Hum Genet 76:449-462 33. Ramsjö M, Aklillu E, Bohman L, Ingelman-Sundberg M, Roh HK, Bertilsson L (2010) CYP2C19 activity comparison between Swedes and Koreans: effect of genotype, sex, oral contraceptive use, and smoking. Eur J Clin Pharmacol (Epub ahead of print) G, Arvanitidis KI, Tavridou A, Manolopoulos VG (2009) Need for reassessment of reported CYP2C19 allele frequencies in various populations in view of CYP2C19*17 discovery: the case of Greece. Pharmacogenomics 10:43-9. 35. Miura J, Obua C, Abbo C, Kaneko S, Tateishi T (2009) Cytochrome P450 2C19 genetic polymorphisms in Ugandans. Eur J Clin Pharmacol 65: 319-20 36. Sugimoto K, Uno T, Yamazaki H, Tateishi T (2008) Limited frequency of the CYP2C19*17 allele and its minor role in a Japanese population. Br J Clin Pharmacol 65:437-439. 37. Justenhoven C, Hamann U, Pierl CB, Baisch C, Harth V, Rabstein S, Spickenheuer A, Pesch B, Brüning T, Winter S, Ko YD, Brauch H (2009) CYP2C19*17 is associated with decreased breast cancer risk. Breast Cancer Res Treat 115:391-6 38. Gawrońska-Szklarz B, Siuda A, Kurzawski M, Bielicki D, Marlicz W, Droździk M (2010) Effects of CYP2C19, MDR1, and interleukin 1-B gene variants on the eradication rate of Helicobacter pylori infection by triple therapy with pantoprazole, amoxicillin, and metronidazole. Eur J Clin Pharmacol 66:681-7 Comment [RP5]: Six more ref.

  To determine the distribution of clinically important CYP2C genotypes and allele frequencies in healthy Nordic populations with special focus on linkage disequilibrium. Methods. 896 healthy subjects from three Nordic populations (Faroese, Danish and Norwegian) were genotyped for five frequent and clinically important CYP2C allelic variants: the defective CYP2C8*3, CYP2C9*2, CYP2C9*3 and CYP2C19*2 alleles, and the CYP2C19*17 allele that causes rapid drug metabolism. Linkage disequilibrium was evaluated and CYP2C haplotypes were inferred in the entire population. Results. Ten CYP2C haplotypes were inferred of which the most frequent (49%) was the CYP2C wildtype haplotype carrying CYP2C8*1, CYP2C9*1 and CYP2C19*1. The second most frequent haplotype (19%) is composed by CYP2C19*17, CYP2C8*1 and CYP2C9*1. This predicted haplotype accounts for 99.7% of the CYP2C19*17 alleles found in the 896 subjects. Conclusion. CYP2C19*17 is a frequent genetic variant in Nordic populations that exists in strong linkage disequilibrium with wild type CYP2C8*1 and CYP2C9*1 alleles, which effectively makes it a determinant for a haplotype exhibiting efficient CYP2C substrate metabolism.

  CYP2C19 is another important drug metabolizing enzyme encoded by a highly polymorphic gene[15]. The racemic anticonvulsant drug mephenytoin is the classic probe drug where the 4-hydroxylation of the (S)-enantiomer is catalysed by CYP2C19. Furthermore, a range of clinically important drugs including diazepam, proton pump inhibitors, the antidepressants citalopram, escitalopram and imipramine, the antimalarial drugs proguanil and chlorproguanil, the β-adrenoceptor blocker propranolol, and the antiplatelet drug clopidogrel are metabolized or bioactivated by CYP2C19. Population studies have shown that individuals that are homozygous for the inactivating allele CYP2C19*2 that leads to a splicing defect (Table

5 Samples

 5 DNA samples from 276 healthy volunteers living around Odense in Denmark and

  ten times with different random seed. Additionally, haplotype frequencies were estimated using the expectation maximation algorithm (EM) in STATA10 (StataCorp, Texas, USA).ResultsWeperformed genotyping of CYP2C8*3, CYP2C9*2, CYP2C9*3, CYP2C19*2 and CYP2C19*17 in three different Nordic populations. The genotype and allele frequencies in the Danish, Faroese and Norwegian samples are presented in Table 2. All SNPs in all three populations were in Hardy-Weinberg equilibrium displaying χ 2values less than 3.84. An overall significant difference in allele frequency of CYP2C19*17 was found between the populations (P=0.010). The allele frequency of 15 % in the Faroese population (n=311) was lower than the 20% in the Danish population (n=276) (P=0.038) and the 22% in the Norwegian population (n=309, P=0.003). No statistically significant difference between the Danish and the Norwegian population was found. None of the four other SNPs displayed significantly different allele frequencies between the populations. When analyzing all three populations, none of the 40 subjects homozygous for the rapid CYP2C19*17 allele carried the defective CYP2C8*3, CYP2C9*2 or CYP2C9*3 alleles. In addition, none of the 9 subjects homozygous for CYP2C8*3 carried CYP2C19*17 and none of the 20 subjects homozygous for CYP2C9*2 and/or CYP2C9*3 carried CYP2C19*17. The linkage disequilibrium between the five analysed SNPs as based on Haploview calculations is visualized in Figure 2. The calculations show complete LD (D'=1) between CYP2C19*17 and the four other allelic variants. Complete linkage disequilibrium was also observed between CYP2C19*2 and the two CYP2C9 alleles CYP2C9*2 and CYP2C9*3. The analysis also displays strong linkage disequilibrium between CYP2C9*2 and CYP2C8*3 (D´=0.92).

  .1-5 and 9) account for 99% of the haplotypes. The most frequent haplotype (No. 1) with an inferred frequency of 49% carries the CYP2C8*1, CYP2C9*1 and CYP2C19*1 alleles. The second most frequent haplotype (No. 2) with an inferred frequency of 19% is composed by CYP2C19*17, CYP2C8*1 and CYP2C9*1 wildtype alleles, and is representing 99.7% (342 out of 343) of all predicted CYP2C19*17-containing haplotypes. In one Norwegian subject, a single variant haplotype with CYP2C19*17 in combination with CYP2C8*3 was inferred, thus leading to an estimated haplotype frequency of <0.001 in the total material of 896 subjects (Table

  previously shown byYasar et al.

Figure 1

 1 Figure 1

  Figure 2

Table

Table 1

 1 The studied CYP2C allelic variants

			Gene	Protein	TaqMan   
	CYP allele	rs number	nucleotide change	Effect	Assay Number
	CYP2C19*17	rs12248560	-806C>T	Ile 331 →Val	C__469857_10
	CYP2C19*2	rs4244285	19154G>A	Splicing defect	C__25986767_70
	CYP2C9*2	rs1799853	3608C>T	Arg 144 →Lys	C__25986767_10
	CYP2C9*3	rs1057910	42614A>C	Ile 359 →Leu	C__27104892_10
	CYP2C8*3	rs10509681	40411A>G	Lys 399 →Arg	C__25625782_20
	CYP2C8*3	rs11572080	2130G>A	Arg 139 →Lys	Not tested

Table 2

 2 Genotype and allele frequencies of CYP2C8, CYP2C9 and CYP2C19 allelic SNPs in Danish, Faroese and Norwegian subjects.

		Danish	Faroese	Norwegian
		Number of	Frequency Number of	Frequency Number of	Frequency
	CYP2C8	subjects		subjects		subjects
	CYP2C8*1/*1	226	0.819	268	0.862	257	0.832
	CYP2C8*1/*3	45	0.163	43	0.138	48	0.155
	CYP2C8*3/*3	5	0.018	0	0.000	4	0.013
		Number of	Frequency Number of	Frequency Number of	Frequency
	Allele	alleles		alleles		alleles
	CYP2C8*1	497	0.900	579	0.931	562	0.909
	CYP2C8*3	55	0.100	43	0.069	56	0.091
		Number of	Frequency Number of	Frequency Number of	Frequency
	CYP2C9	subjects		subjects		subjects
	CYP2C9*1/*1	190	0.688	228	0.733	213	0.689
	CYP2C9*1/*2	53	0.192	50	0.161	55	0.178
	CYP2C9*1/*3	23	0.083	28	0.090	36	0.117
	CYP2C9*2/*2	4	0.014	0	0.000	2	0.007
	CYP2C9*3/*3	0	0.000	0	0.000	1	0.003
	CYP2C9*2/*3	6	0.022	5	0.016	2	0.007
		Number of	Frequency Number of	Frequency Number of	Frequency
	Allele	alleles		alleles		alleles
	CYP2C9*1	456	0.826	534	0.859	517	0.837
	CYP2C9*2	67	0.121	55	0.088	61	0.099
	CYP2C9*3	29	0.053	33	0.053	40	0.065

CYP2C19 Number of subjects Frequency Number of subjects Frequency Number of subjects

							Frequency
	CYP2C19*1/*1	122	0.442	143	0.460	122	0.395
	CYP2C19*1/*2	51	0.185	73	0.235	62	0.201
	CYP2C19*1/*17	63	0.228	51	0.164	82	0.265
	CYP2C19*2/*2	6	0.022	10	0.032	4	0.013
	CYP2C19*17/*17	14	0.051	11	0.035	15	0.049
	CYP2C19*2/*17	20	0.073	23	0.074	24	0.078
		Number of	Frequency Number of	Frequency Number of	Frequency
	Allele	alleles		alleles		alleles
	CYP2C19*1	358	0.649	410	0.659	388	0.628
	CYP2C19*2	83	0.150	116	0.187	94	0.152
	CYP2C19*17	111	0.201	96	0.154	136	0.220

European Journal of Clinical Pharmacology

Acknowledgements

This research was supported in part by grants from The Swedish Research Council, Torsten and Ragnar Söderbergs Stiftelser, Karolinska Institutet, The Danish Research Council for Health and Disease, and The Lundbeck Foundation. We thank Pernille Jordan for analytical expertise.

Acknowledgements

This research was supported in part by grants from The Swedish Research Council, Torsten and Ragnar Söderbergs Stiftelser, Karolinska Institutet, The Danish Research Council for Health and Disease, and The Lundbeck Foundation. We thank Pernille Jordan for analytical expertise.