Prevalence of CYP2C19 polymorphisms in the Lebanese population
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- Djaffar Jureidini, I., Chamseddine, N., Keleshian, S. et al. Mol Biol Rep (2011) 38: 5449. doi:10.1007/s11033-011-0700-y
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Clopidogrel is one of the most commonly prescribed drugs, as its combination with low-dose aspirin is the recommended oral anti-platelet therapy, to prevent ischaemic events following coronary syndromes or stent placement. Numerous recent studies have shown that polymorphisms in the gene encoding the cytochrome P450 (CYP450) 2C19 enzyme (CYP2C19) contribute to variability in response to clopidogrel; patients with certain common genetic variants of CYP2C19 (*2, *3) have a reduced metabolism of clopidogrel and have a higher rate of cardiovascular events or stent thrombosis compared to patients with the CYP2C19 (*1) allele. CYP2C19*2 is most common in Caucasians, Africans and Asians while CYP2C19*3 has been found mostly in Asians. Since the prevalence of these variants in the Lebanese population has not yet been reported, our aim was to determine the genotypes of CYP2C19 in our population. CYP2C19 (*1/*2/*3) variants were assessed by Polymerase Chain Reaction-Restriction Length Polymorphism (PCR–RFLP) assays in a representative sample of 161 unrelated healthy Lebanese volunteers. The allele frequencies of CYP2C19 *2 and *3 were 0.13 and 0.03. Carriers of the CYP2C19 *2 or *3 represented 24.2% of the subjects. Our data show no significant difference in the frequency of CYP2C19 allelic variants when compared to Caucasian populations and demonstrate that the application of the recent FDA recommendations would also be beneficial in Lebanon, allowing physicians to identify patients at high risk for atherothrombotic events, and eventually advising them to consider other antiplatelet medications or alternative dosing strategies in poor metabolizers.
Interpatient variability in drug response is multifactorial and depends on clinical as well as genetic factors. Clopidogrel is one of the most commonly prescribed drugs, as its combination with low-dose aspirin is the recommended oral anti-platelet therapy, to prevent ischaemic events following coronary syndromes or stent placement [1–8]. Clinical factors affecting the variability in response to Clopidogrel include obesity, insulin resistance, drug interaction and the nature of the coronary event [9–11]. Genetic factors, on the other hand, influence the pharmacokinetics and pharmacodynamics of the drug, such as intestinal absorption and metabolic activation in the liver [12–19]. Indeed, Clopidogrel, like a variety of other drugs, is metabolized by CYP2C19, a member of the cytochrome P450 enzyme superfamily . The CYP2C19 gene is polymorphic, the two main variant forms of CYP2C19 being CYP2C19*2 (a G > A splice site mutation at position 681 in exon 5) and CYP2C19*3 (a G > A mutation at position 636 in exon 4). CYP2C19*2 is the most common in Caucasians, Africans and Asians while CYP2C19*3 has been found mostly in Asians. Fully functional metabolism corresponds to the CYP2C19*1 allele, while reduced metabolism corresponds to the CYP2C19*2 and CYP2C19*3 alleles. The CYP2C19*2 and CYP2C19*3 alleles account for 85% of reduced-function alleles in whites and 99% in Asians. Other alleles associated with reduced metabolism include CYP2C19*4, *5, *6, *7, and *8, but these are less frequent in the general population [Reviewed in 21]. A number of studies have shown that those variants are also associated with adverse clinical outcomes in patients with acute coronary syndromes treated with Clopidogrel, including higher rates of stent thrombosis and death from cardiovascular causes, myocardial infarction or stroke [14, 15, 22–25].
The high frequency of those alleles and their strong association with prognosis in treated patients means that a number of patients are at risk of coronary events despite treatment with Clopidogrel. The frequencies of these two alleles have been established in various populations but such a study has not yet been performed in Lebanon, a country at the crossroads between Europe, Asia and Africa. Among neighboring Middle Eastern countries, studies have been performed in Egypt, Saudi Arabia and the Gaza strip and showed that the frequency of CYP2C19*2 allele varied between 0.11 and 0.15 . Therefore the aim of this study was to assess the prevalence of the *2 and *3 alleles and their associated genotypes in the Lebanese population and to draw comparisons with studies from other countries.
Materials and methods
Knowing that the prevalence for the less frequent allele (*3) ranges from 0 to 7% in distinctive Caucasians populations (21) and is around 1% in other Middle East populations (26), we calculated the theoretical Sample size needed for a prevalence of 1% (95% Confidence) and found it to be 150. The study therefore included 161 young healthy unrelated subjects (81 females and 80 males, 18–40 years old) selected among a representative population (blood bank donors, students, volunteers), and geographically originating from the five major areas of Lebanon (Beirut, Mount Lebanon, North, Bekaa, South). Numbers in each region were calculated in order to cover proportionally the whole geographic distribution of the population :(Beirut : N = 48, Mount Lebanon : N = 35, North : N = 25, Bekaa : N = 23, South : N = 20).
Informed consent was obtained from all subjects. The study was approved by the Research and Ethical Committees of the Faculty of Medicine and Medical Sciences at Saint George Hospital University Medical Center.
For all subjects, 2 ml of whole blood was collected on EDTA.
CYP2C19 *1, *2 and *3 genotyping
Genomic DNA was extracted from peripheral blood leukocytes using the High Pure PCR Template Kit (ROCHE, Mannheim, Germany) according to the standard protocol, and stored at −80°C.
DNA was amplified by Polymerase Chain Reaction using the primers described by Zand et coll. . Two PCR reactions specific for *2 and *3 were conducted in parallel for each specimen in a final volume of 20 μl. Allele 2 PCR product (10 μl) was digested with SmaI1 and Allele 3 PCR product with BamH1 for 2 h at 37°C. The digested products were visualized on a 3% agarose gel stained with ethidium bromide. The CYP2C19*2 abolishes a restriction site for the SmaI enzyme and therefore *2 was defined by a non-cut 169 bp band, whereas the *1 will produce 2 bands of 120 and 49 bps. The CYP2C19*3 abolishes a restriction site for the BamH1 Enzyme and therefore *3 was defined by a non-cut 329 bp band, whereas the *1 produced 2 bands of 233 and 96 bps.
Genotype frequencies were tested for deviations from the Hardy–Weinberg disequilibrium (HWE) through Chi-square analysis.
Genotype and allele frequencies of CYP2C19
N = 161
The importance of genotyping CYP2C19 *2 and *3 has been highlighted by the United States Food and Drug Administration (FDA) which recommends that Clopidogrel therapy in populations with a high prevalence of those “risk” alleles be carefully administered . Indeed, in May 2009, the US labeling for Clopidogrel was updated  to mention that patients with genetically reduced CYP2C19 function have diminished antiplatelet responses, and generally exhibit higher cardiovascular event rates following myocardial infarction than do patients with normal CYP2C19 function. Reduced CYP2C19 metabolism in intermediate and poor metabolizers decreased the maximum concentration of the active metabolite by 30 to 50% resulting in lower platelet inhibition. More recently (March 2010), the FDA required a boxed warning for Clopidogrel to (i) caution that poor metabolizers of the drug may not receive full protection from heart attacks, stroke, and cardiovascular death, (ii) draw attention about poor metabolism of Clopidogrel when the drug is taken together with the heartburn medicine omeprazole (Prilosec, Proctor & Gamble), (iii) state that tests are available to determine the genetic profile of the CYP2C19 and predict whether a patient will ineffectively convert Clopidogrel to its active form .
In order to provide physicians in Lebanon with an estimate of the potential number of poor metabolizers among their patients, we set out to study the prevalence of the CYP2C19 *2 and *3 risk alleles in the Lebanese population. To our knowledge, this is the first study on the prevalence of those CYP2C19 genotypes in our country. Our results show that the prevalence of CYP2C19 *2 (0.13) and *3 (0.03) is similar to the one reported for the Caucasian population (range 0.11– 0.16 and 0.0–0.7 respectively)  or other Middle Eastern populations (Egyptian : 0.11 and 0.002; Israeli Jewish : 0.15 and 0.01 respectively) . Five individuals (3.1%) were found to be homozygous for CYP2C19*2 alleles, and could thus be classified as poor metabolizers, while 33 subjects (20.5%) carried one mutant *2 allele (CYP2C19*1/*2) and one subject (0.6%) the mutant *3 allele (CYP2C19*1/*3). The very low prevalence of CYP2C19*3 in our study further illustrates the ethnic difference between Caucasian and Oriental populations, by confirming the Asian specificity of this allelic variant . Nevertheless, studies on a larger sample size would be required to confirm those frequencies.
Our results demonstrate that the application of FDA recommendations would also be beneficial in Lebanon, allowing physicians to identify patients at high risk for atherothrombotic events when undergoing Clopidogrel therapy, and eventually advising them to consider other antiplatelet medications or alternative dosing strategies in poor metabolizers. In addition, our data could be extrapolated to other drugs metabolized by the CYP2C19 .
Conflict of interest