Phenotype-genotype analysis of CYP2C19 in Colombian mestizo individuals
© Isaza et al; licensee BioMed Central Ltd. 2007
Received: 23 February 2007
Accepted: 11 July 2007
Published: 11 July 2007
Omeprazole is metabolized by the hepatic cytochrome P450 (CYP) 2C19 enzyme to 5-hydroxyomeprazole. CYP2C19 exhibits genetic polymorphisms responsible for the presence of poor metabolizers (PMs), intermediate metabolizers (IMs) and extensive metabolizers (EMs). The defective mutations of the enzyme and their frequencies change between different ethnic groups; however, the polymorphism of the CYP2C19 gene has not been studied in Colombian mestizos. The aim of this study was to evaluate the genotype and phenotype status of CYP2C19 in Colombian mestizos, in order to contribute to the use of appropriate strategies of drug therapy for this population.
189 subjects were genotyped using the multiplex SNaPshot technique and a subgroup of 44 individuals received 20 mg of omeprazole followed by blood collection at 3 hours to determine the omeprazole hydroxylation index by HPLC.
83.6%, 15.3% and 1.1% of the subjects were genotyped as EMs, IMs and PMs, respectively. The frequencies of the CYP2C29*1 and CYP2C19*2 alleles were 91.3% and 8.7% respectively whereas the *3, *4, *5, *6 and *8 alleles were not found. No discrepancies were found between the genotype and phenotype of CYP2C19.
The frequency of poor metabolizers (1.1%) in the Colombian mestizos included in this study is similar to that in Bolivian mestizos (1%) but lower than in Mexican-Americans (3.2%), West Mexicans (6%), Caucasians (5%) and African Americans (5.4%). The results of this study will be useful for drug dosage recommendations in Colombian mestizos.
The CYP2C19 isoenzyme, a member of the superfamily of xenobiotic enzymes of cytochrome P-450, is responsible for the metabolism of several therapeutically important drugs, such as proton-pump inhibitors (omeprazole, lansoprazole, pantoprazole), antidepressants (citalopram, imipramine), benzodiazepines (diazepam, flunitrazepam), propranolol and proguanil [1–3].
The CYP2C19 gene polymorphism divides populations in three phenotypic subgroups: extensive metabolizers (EMs), intermediate metabolizers (IMs), and poor metabolizers (PMs). The enzymatic deficiency is inherited as an autosomal recessive trait .
The frequencies and types of alleles vary between ethnic groups. Thus, 13 to 23% of Orientals are PMs and the CYP2C19*2 and *3 alleles account for 99% of them in this ethnic group , whereas in Caucasians, with a percentage of PMs near to 5% of the population, only the *2 allele is common although other variants have been described [6–9]. In Black population some new mutations (*9, *10, *12) have been reported and the frequency of PMs individuals is 5.4% [10, 11]. The clinical consequences of the CYP2C19 gene polymorphisms have not been fully understand but can be illustrated with the case of proton-pump inhibitors. The EMs metabolizes these drugs at a speed that requires doses up to four times greater than PMs to reach similar serum concentrations and effects [12, 13].
Several studies carried out in different ethnic groups have shown that the efficacy of proton-pump inhibitors is related to the CYP2C19 genotype, explaining the differences in terms of therapeutic efficacy observed between EMs and PMs individuals [14–19]. Nevertheless, the resistance of H. pylori to antimicrobial and others non-genetic factors such as age, liver disease and enzymatic inhibition or induction by drugs are also important causes of therapeutic failure [3, 20, 21].
Although the genotype-phenotype correlation for CYP2C19 using omeprazole as "probe drug" has been widely studied, there are no data regarding South American Mestizos [22, 23]. Therefore, the aim of this study was to evaluate the genotype and phenotype status of CYP2C19 in Colombian mestizos, in order to contribute to the use of appropriate strategies of drug therapy for this population. Colombian population is divided ethnically into four main groups: the Mestizo (56%), White (30.1%), Black (10.5%) and Amerindian (3.4%) people. The Colombian mestizo population is an admixture between Amerindian, Hispanic and African descent .
Healthy unrelated Colombian mestizos (n = 189) participated in the study. None of the volunteers included in the phenotyping test (n = 44) had a history of allergy to omeprazole or other drugs, alcohol abuse, drug addiction or a smoking habit of more than 15 cigarettes/day. The participants were not allowed to take any medication during the previous week before and during the study . The experimental protocol was approved by the Ethics Committee of Universidad Tecnológica de Pereira, Colombia. Written informed consent was obtained from all participating subjects.
A multiplex primer-extension assay that simultaneously detects the six CYP2C19 alleles using the ABI Prism® SNaPshot™ ddNTP Primer-Extension Assay was used . Buccal swabs were obtained from experimental subjects and the material was deposited onto FTA blood cards, dried at room temperature and stored for DNA extraction as reported . The DNA was used to amplify five fragments of the CYP2C19 gene corresponding to exons 1 (410 bp), 2–3 (719 bp), 4 (310 bp), 5 (410 bp) and 9 (529 bp); which include the six Single Nucleotide Polymorphisms (SNPs) studied. Amplification was carried out on a DNA thermal cycler PBX2 (Thermo Electron Corporation), according to previously described techniques . PCR products were electrophoresed in 2% agarose gels and the bands purified using the GFX purification kit (Amersham Pharmacia Biotech). The amplification products were used as templates for the multiplex reactions in order to detect the wild-type allele CYP2C19*1 and the *2, *3, *4, *5, *6 and *8 mutations.
The minisequencing was based on the method proposed by Bender et al , with detection primers present in the reaction at a concentration of 1.5 pmoles. The amplification products were injected onto the DNA ABI prism 3100 Avant Genetic Analyzer ant the results were analyzed using the GeneMapper Analysis Software (PE Applied Biosystems).
The omeprazole hydroxylation index (HI) was determined in 44 subjects previously genotyped. After an overnight fast, each subject took 20 mg omeprazole (Tecnoquímicas, Cali, Colombia) orally. Then, 3 h after ingestion of the drug, 5 ml of venous blood was collected from an antecubital vein into tubes with anticoagulant. Plasma was separated after centrifugation and stored frozen at -20°C until analysis. The concentration of omeprazole and 5-hydroxy omeprazole in plasma was measured by HPLC as described by Gonzalez et al , with some modifications. Omeprazole and 5-hydroxy omeprazole were purchased from Sigma-Aldrich Co (St. Louis, USA) and SynFine Research Inc. (Ontario, Canada), respectively. To avoid recovery problems, standards were prepared by spiking blank plasma with omeprazole and 5-hydroxy omeprazole to get concentrations of 0.1, 0.25, 1.0, 3.0 and 5.0 μg/mL and processed by solid phase extraction (SPE) in the same way as samples. Standards and samples (2 mL) were submitted to SPE using 500 mg RP-18 cartridges (Merck) previously activated with AcCN (3 mL), followed by three portions of 3 mL of phosphate buffer (0.05 M, pH 7.2). After sample application the cartridge was washed sequentially by three 0.5 mL portions of phosphate buffer and a 0.5 mL portion of phosphate buffer-AcCN (80:20), drying after each washing. Analytes were eluted with phosphate buffer-AcCN (10:90). HPLC analysis was carried out in a Jasco HPLC 2000 plus Series System equipped with a PU-2089 Plus Quaternary Gradient Pump, an AS-2059 Plus Intelligent Autosampler, a CO-2065 Plus Column Oven, a MD-2015 Plus Intelligent Diode Array Detector, and a LC Net II/ADC, controlled by EZChrom Elite Software version 3.16. Each standard and sample were injected (25 μL) into an Ultra Aqueous RP-18 Restek analytical column (3 μm particle size, 100 × 3.2 mm I.D.), eluting in isocratic mode with phosphate buffer-AcCN (70:30) as mobile phase at 0.5 mL/min flow rate. Calibration curve and quantification were performed at 298 nm by external standard, with linear fit showing a coefficient of determination (r 2) of 0.9993 for omeprazole and 0.9992 for 5-hydroxyomeprazole in the range from 0.1 to 5 μg/mL. Omeprazole and its metabolite 5-hydroxyomeprazole eluted at 9.80 min and 3.67 min, respectively. Detection limits were 0.026 μg/mL for omeprazole and 0.01 μg/mL for 5-hydroxyomeprazole. Intraday coefficients of variation for omeprazole and 5-hydroxyomeprazole were 5% and 4%, respectively. Individuals with a metabolic ratio (MR) ≥ 0.63 were phenotyped as PMs.
Allelic frequencies were calculated according to the number of alleles observed and the number of chromosomes examined. The Hardy-Weinberg equilibrium was established using the Chi-square test. The MR was calculated as log10 (omeprazole/hydroxyomeprazole) [21, 29, 30]. The normality of the MR distribution was evaluated by the D'Agostino test. A probit plot was used to confirm the bimodal distribution of the MR. 95% confidence intervals were used and the level of statistical significance was set at p < 0.05. Statistical analysis was conducted using SPSS 10.0 for Windows (SPSS, USA) and the GraphPad Prism 5.0 software (GraphPad Software Inc. San Diego, Ca, USA).
Allelic and genotypic frequencies of CYP2C19 in Colombian Mestizo individuals (n = 189). CI: confidence interval.
Number of alleles
Frequency (%) (95% CI)
Number of subjects
Frequency (%) (95% CI)
Correlation between CYP2C19 genotype and MR in Colombian Mestizo individuals (n = 44). MR: Metabolic ratio, n: number of subjects, SD standard deviation.
MR (mean ± SD)
-0.01 ± 0.15
0.23 ± 0.15
0.99 ± 0.35
Discussion and conclusion
Genetic polymorphisms are the more influential factors for individual and interethnic variations in drug responses . The metabolic activity of CYP2C19 is genetically controlled and individuals can be characterized as EMs, IMs or PMs.
This is the first study evaluating the genotype and phenotype status of CYP2C19 in Colombian mestizos. The CYP2C19 genetic profile found in this population, with the *2 allele responsible for most of the deficient metabolizers, and the absence of *3, *4, *5, *6 and *8 variants, is similar to Mexican-Americans profile , however, the PMs frequency is lower (1,1% vs. 3,2%). Also, the frequency of PMs found in our study is lower that the ones reported in Caucasians (5%) , Blacks (5.4%)  and West Mexicans , which are genetically related to American mestizos [5, 6]. Since Amerindians are also part of the American mestizo ancestor, it is interesting to mention that PMs were not found in Cuna Amerindians of Panama , whereas in Bolivian mestizos (an admixture between white and Amerindian populations), the prevalence of PMs is 1% .
The pharmacogenetic characterization becomes one of the therapeutic options more cost-effective , because it is helpful for both to prevent adverse drug reactions as well as to enhance therapeutic efficiency in the case of drugs with narrow therapeutic index. The genotype-phenotype correlation found in this study, allows us to state that genotyping only the *2 allele would have a high predictive value of the CYP2C19 phenotype between Colombian mestizos. Since pharmacogenetic allows the introduction of personalized pharmacotherapy, according to individual genetic data, the results of this study will be useful for drug dosage recommendations in Colombian mestizos.
For CYP2C19 genotyping we used the minisequencing Multiplex SnaPshot technique , which could be a safer and more cost-effective strategy than the conventional genotyping techniques based on allele-specific PCR or RFLP (restriction fragment length polymorphism).
This research was supported by COLCIENCIAS and Universidad Tecnológica de Pereira, Colombia.
- Meyer UA, Zanger UM: Molecular mechanisms of genetic polymorphisms of drug metabolism. Annu Rev Pharmacol Toxicol. 1997, 37: 269-296. 10.1146/annurev.pharmtox.37.1.269.View ArticlePubMedGoogle Scholar
- Desta Z, Zhao X, Shin JG, Flockhart DA: Clinical significance of the cytochrome P450 2C19 genetic polymorphism. Clin Pharmacokinet. 2002, 41 (12): 913-958. 10.2165/00003088-200241120-00002.View ArticlePubMedGoogle Scholar
- Yamazaki H, Inoue K, Shaw PM, Checovich WJ, Guengerich FP, Shimada T: Different contributions of cytochrome P450 2C19 and 3A4 in the oxidation of omeprazole by human liver microsomes: effects of contents of these two forms in individual human samples. J Pharmacol Exp Ther. 1997, 283 (2): 434-442.PubMedGoogle Scholar
- Brosen K, de Morais SM, Meyer UA, Goldstein JA: A multifamily study on the relationship between CYP2C19 genotype and s-mephenytoin oxidation phenotype. Pharmacogenetics. 1995, 5 (5): 312-317. 10.1097/00008571-199510000-00007.View ArticlePubMedGoogle Scholar
- Garcia-Barcelo M, Chow LY, Kum Chiu HF, Wing YK, Shing Lee DT, Lam KL, Waye MM: Frequencies of defective CYP2C19 alleles in a Hong Kong Chinese population: detection of the rare allele CYP2C19*4. Clin Chem. 1999, 45 (12): 2273-2274.PubMedGoogle Scholar
- Ibeanu GC, Blaisdell J, Ferguson RJ, Ghanayem BI, Brosen K, Benhamou S, Bouchardy C, Wilkinson GR, Dayer P, Goldstein JA: A novel transversion in the intron 5 donor splice junction of CYP2C19 and a sequence polymorphism in exon 3 contribute to the poor metabolizer phenotype for the anticonvulsant drug S-mephenytoin. J Pharmacol Exp Ther. 1999, 290 (2): 635-640.PubMedGoogle Scholar
- Ferguson RJ, De Morais SM, Benhamou S, Bouchardy C, Blaisdell J, Ibeanu G, Wilkinson GR, Sarich TC, Wright JM, Dayer P: A new genetic defect in human CYP2C19: mutation of the initiation codon is responsible for poor metabolism of S-mephenytoin. J Pharmacol Exp Ther. 1998, 284 (1): 356-361.PubMedGoogle Scholar
- Ibeanu GC, Goldstein JA, Meyer U, Benhamou S, Bouchardy C, Dayer P, Ghanayem BI, Blaisdell J: Identification of new human CYP2C19 alleles (CYP2C19*6 and CYP2C19*2B) in a Caucasian poor metabolizer of mephenytoin. J Pharmacol Exp Ther. 1998, 286 (3): 1490-1495.PubMedGoogle Scholar
- Xiao ZS, Goldstein JA, Xie HG, Blaisdell J, Wang W, Jiang CH, Yan FX, He N, Huang SL, Xu ZH: Differences in the incidence of the CYP2C19 polymorphism affecting the S-mephenytoin phenotype in Chinese Han and Bai populations and identification of a new rare CYP2C19 mutant allele. J Pharmacol Exp Ther. 1997, 281 (1): 604-609.PubMedGoogle Scholar
- Xie HG, Kim RB, Stein CM, Wilkinson GR, Wood AJ: Genetic polymorphism of (S)-mephenytoin 4'-hydroxylation in populations of African descent. Br J Clin Pharmacol. 1999, 48 (3): 402-408. 10.1046/j.1365-2125.1999.00009.x.View ArticlePubMedPubMed CentralGoogle Scholar
- Blaisdell J, Mohrenweiser H, Jackson J, Ferguson S, Coulter S, Chanas B, Xi T, Ghanayem B, Goldstein JA: Identification and functional characterization of new potentially defective alleles of human CYP2C19. Pharmacogenetics. 2002, 12 (9): 703-711. 10.1097/00008571-200212000-00004.View ArticlePubMedGoogle Scholar
- Furuta T, Shirai N, Xiao F, Ohashi K, Ishizaki T: Effect of high-dose lansoprazole on intragastic pH in subjects who are homozygous extensive metabolizers of cytochrome P4502C19. Clin Pharmacol Ther. 2001, 70 (5): 484-492.View ArticlePubMedGoogle Scholar
- Kita T, Sakaeda T, Aoyama N, Sakai T, Kawahara Y, Kasuga M, Okumura K: Optimal dose of omeprazole for CYP2C19 extensive metabolizers in anti-Helicobacter pylori therapy: pharmacokinetic considerations. Biol Pharm Bull. 2002, 25 (7): 923-927. 10.1248/bpb.25.923.View ArticlePubMedGoogle Scholar
- Sapone A, Vaira D, Trespidi S, Perna F, Gatta L, Tampieri A, Ricci C, Cantelli-Forti G, Miglioli M, Biagi GL: The clinical role of cytochrome p450 genotypes in Helicobacter pylori management. Am J Gastroenterol. 2003, 98 (5): 1010-1015. 10.1111/j.1572-0241.2003.07427.x.View ArticlePubMedGoogle Scholar
- Kawamura M, Ohara S, Koike T, Iijima K, Suzuki J, Kayaba S, Noguchi K, Hamada S, Noguchi M, Shimosegawa T: The effects of lansoprazole on erosive reflux oesophagitis are influenced by CYP2C19 polymorphism. Aliment Pharmacol Ther. 2003, 17 (7): 965-973. 10.1046/j.1365-2036.2003.01539.x.View ArticlePubMedGoogle Scholar
- Kawabata H, Habu Y, Tomioka H, Kutsumi H, Kobayashi M, Oyasu K, Hayakumo T, Mizuno S, Kiyota K, Nakajima M: Effect of different proton pump inhibitors, differences in CYP2C19 genotype and antibiotic resistance on the eradication rate of Helicobacter pylori infection by a 1-week regimen of proton pump inhibitor, amoxicillin and clarithromycin. Aliment Pharmacol Ther. 2003, 17 (2): 259-264. 10.1046/j.1365-2036.2003.01406.x.View ArticlePubMedGoogle Scholar
- Klotz U: Clinical impact of CYP2C19 polymorphism on the action of proton pump inhibitors: a review of a special problem. Int J Clin Pharmacol Ther. 2006, 44 (7): 297-302.View ArticlePubMedGoogle Scholar
- Furuta T, Shirai N, Sugimoto M, Ohashi K, Ishizaki T: Pharmacogenomics of proton pump inhibitors. Pharmacogenomics. 2004, 5 (2): 181-202. 10.1517/phgs.188.8.131.52483.View ArticlePubMedGoogle Scholar
- Take S, Mizuno M, Ishiki K, Nagahara Y, Yoshida T, Inaba T, Yamamoto K, Okada H, Yokota K, Oguma K: Interleukin-1beta genetic polymorphism influences the effect of cytochrome P 2C19 genotype on the cure rate of 1-week triple therapy for Helicobacter pylori infection. Am J Gastroenterol. 2003, 98 (11): 2403-2408.PubMedGoogle Scholar
- Miki I, Aoyama N, Sakai T, Shirasaka D, Wambura CM, Maekawa S, Kuroda K, Tamura T, Kita T, Sakaeda T: Impact of clarithromycin resistance and CYP2C19 genetic polymorphism on treatment efficacy of Helicobacter pylori infection with lansoprazole- or rabeprazole-based triple therapy in Japan. Eur J Gastroenterol Hepatol. 2003, 15 (1): 27-33. 10.1097/00042737-200301000-00006.View ArticlePubMedGoogle Scholar
- Kimura M, Ieiri I, Wada Y, Mamiya K, Urae A, Iimori E, Sakai T, Otsubo K, Higuchi S: Reliability of the omeprazole hydroxylation index for CYP2C19 phenotyping: possible effect of age, liver disease and length of therapy. Br J Clin Pharmacol. 1999, 47 (1): 115-119. 10.1046/j.1365-2125.1999.00858.x.View ArticlePubMedPubMed CentralGoogle Scholar
- Gonzalez HM, Romero EM, Peregrina AA, de JCT, Escobar-Islas E, Lozano F, Hoyo-Vadillo C: CYP2C19- and CYP3A4-dependent omeprazole metabolism in West Mexicans. J Clin Pharmacol. 2003, 43 (11): 1211-1215. 10.1177/0091270003258170.View ArticlePubMedGoogle Scholar
- Chong E, Ensom MH: Pharmacogenetics of the proton pump inhibitors: a systematic review. Pharmacotherapy. 2003, 23 (4): 460-471. 10.1592/phco.23.4.460.32128.View ArticlePubMedGoogle Scholar
- Arango R, Sanchez E: Los pueblos indígenas de Colombia (Guía etnográfica de Colombia para actividades de planeación). 1988, DNP P: Departamento Nacional de PlaneaciónGoogle Scholar
- Tamminga WJ, Wemer J, Oosterhuis B, Brakenhoff JP, Gerrits MG, de Zeeuw RA, de Leij LF, Jonkman JH: An optimized methodology for combined phenotyping and genotyping on CYP2D6 and CYP2C19. Eur J Clin Pharmacol. 2001, 57 (2): 143-146. 10.1007/s002280100273.View ArticlePubMedGoogle Scholar
- Bender K: SNaPshot for pharmacogenetics by minisequencing. Methods Mol Biol. 2005, 297: 243-252.PubMedGoogle Scholar
- Tanigawara Y, Kita T, Hirono M, Sakaeda T, Komada F, Okumura K: Identification of N-acetyltransferase 2 and CYP2C19 genotypes for hair, buccal cell swabs, or fingernails compared with blood. Ther Drug Monit. 2001, 23 (4): 341-346. 10.1097/00007691-200108000-00004.View ArticlePubMedGoogle Scholar
- Morita J, Kobayashi K, Wanibuchi A, Kimura M, Irie S, Ishizaki T, Chiba K: A novel single nucleotide polymorphism (SNP) of the CYP2C19 gene in a Japanese subject with lowered capacity of mephobarbital 4'-hydroxylation. Drug Metab Pharmacokinet. 2004, 19 (3): 236-238. 10.2133/dmpk.19.236.View ArticlePubMedGoogle Scholar
- Gonzalez HM, Romero EM, Chavez Tde J, Peregrina AA, Quezada V, Hoyo-Vadillo C: Phenotype of CYP2C19 and CYP3A4 by determination of omeprazole and its two main metabolites in plasma using liquid chromatography with liquid-liquid extraction. J Chromatogr B Analyt Technol Biomed Life Sci. 2002, 780 (2): 459-465. 10.1016/S1570-0232(02)00573-1.View ArticlePubMedGoogle Scholar
- Kortunay S, Basci NE, Bozkurt A, Isimer A, Sayal A, Kayaalp SO: The hydroxylation of omeprazole correlates with S-mephenytoin and proguanil metabolism. Eur J Clin Pharmacol. 1997, 53 (3–4): 261-264. 10.1007/s002280050373.View ArticlePubMedGoogle Scholar
- Relling MV, Giacomini KM: Pharmacogenetics. Goodman and Gilman's, The Pharmacological Basis of Therapeutics. Edited by: Brunton LL, Lazo JS, Parker KL. 2006, McGraw Hill, 93-117. 11Google Scholar
- Luo HR, Poland RE, Lin KM, Wan YJ: Genetic polymorphism of cytochrome P450 2C19 in Mexican Americans: a cross-ethnic comparative study. Clin Pharmacol Ther. 2006, 80 (1): 33-40. 10.1016/j.clpt.2006.03.003.View ArticlePubMedGoogle Scholar
- Xie HG, Stein CM, Kim RB, Wilkinson GR, Flockhart DA, Wood AJ: Allelic, genotypic and phenotypic distributions of S-mephenytoin 4'-hydroxylase (CYP2C19) in healthy Caucasian populations of European descent throughout the world. Pharmacogenetics. 1999, 9 (5): 539-549.PubMedGoogle Scholar
- Inaba T, Jorge LF, Arias TD: Mephenytoin hydroxylation in the Cuna Amerindians of Panama. Br J Clin Pharmacol. 1988, 25 (1): 75-79.View ArticlePubMedPubMed CentralGoogle Scholar
- Bravo-Villalta HV, Yamamoto K, Nakamura K, Baya A, Okada Y, Horiuchi R: Genetic polymorphism of CYP2C9 and CYP2C19 in a Bolivian population: an investigative and comparative study. Eur J Clin Pharmacol. 2005, 61 (3): 179-184. 10.1007/s00228-004-0890-5.View ArticlePubMedGoogle Scholar
- Lehmann DF, Medicis JJ, Franklin PD: Polymorphisms and the pocketbook: the cost-effectiveness of cytochrome P450 2C19 genotyping in the eradication of Helicobacter pylori infection associated with duodenal ulcer. J Clin Pharmacol. 2003, 43 (12): 1316-1323. 10.1177/0091270003259389.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6904/7/6/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.