anna tavridou and vangelis g. manolopoulos* frequency...

Drug Metab Drug Interact 2014; 29(4): 235–248

Review

Georgia Ragia, Efstathia Giannakopoulou, Makrina Karaglani, Ioanna-Maria Karantza, Anna Tavridou and Vangelis G. Manolopoulos*

Frequency of CYP450 enzyme gene polymorphisms in the Greek population: review of the literature, original findings and clinical significance

Abstract: The cytochrome P450 (CYP450) enzyme family is involved in the oxidative metabolism of many therapeutic drugs and various endogenous substrates. These enzymes are highly polymorphic. Prevalence of CYP450 enzyme gene polymorphisms vary among different populations and substantial inter- and intra-ethnic variability in fre-quency of CYP450 enzyme gene polymorphisms has been reported. This paper provides an overview and investiga-tion of CYP450 genotypic and phenotypic reports pub-lished in the Greek population.

Keywords: CYP450; CYP2D6; CYP2C19; CYP2C9; CYP3A5; CYP3A4; CYP1A2; Greece; polymorphisms.

DOI 10.1515/dmdi-2014-0006Received January 24, 2014; accepted March 26, 2014; previously published online April 21, 2014

IntroductionThe cytochrome P450 (CYP450) enzyme family is involved in the oxidative metabolism of many therapeutic drugs, carcinogens and various endogenous substrates. Among

all CYP450 isoenzymes in humans, CYP2D6, CYP2C19, CYP2C9, CYP3A4, CYP3A5 and CYP1A2 enzymes are the major CYP450 isoenzymes mediating the metabolism of the majority of clinically prescribed drugs.

Activity of the above-mentioned isoenzymes is geneti-cally determined. Polymorphisms in CYP450 genes that influence enzyme activity lead to the appearance of dis-tinct phenotypic responses to the drug, ranging from: a) loss of catalytic enzyme activity in individuals possessing two defective alleles (termed poor metabolizers, PMs); b) decreased enzyme activity in individuals possessing one defective and one functional allele (termed intermedi-ate metabolizers, IMs); c) normal function of the enzyme in individuals possessing two functional alleles (termed extensive metabolizers, EMs); and d) increased enzyme activity in individuals either expressing multiple copies of the functional allele or possessing increased activity alleles (termed ultrarapid metabolizers, UMs) [1].

All identified polymorphisms in CYP450 enzyme encoding genes are listed in The Human Cytochrome P450 (CYP) Allele Nomenclature Database [http://www.cypalleles.ki.se/]. It should be noted, however, that not all CYP450 gene polymorphisms have a proven significant effect on enzyme activity and, as a consequence, a clinical significance in drug response.

Prevalence of CYP450 enzyme gene polymorphisms vary among different populations and substantial inter- and intra-ethnic variability in frequency of CYP450 enzyme gene polymorphisms has been reported [2]. In general, genomic structure differs among European popu-lations, including within the European continent, where genetic variation has been shown to be distributed across two major axes, namely from north to south and from east to west [3]. Greece is located in Southern Europe sur-rounded to the south by the Mediterranean Sea approxi-mately 3450 km from Equator. The latitude and longitude of Greece is 39° 00′ N and 22° 00′ E [4]. Geographic loca-tion of a country is often associated with the prevalence

*Corresponding author: Dr. Vangelis G. Manolopoulos, Laboratory of Pharmacology, Medical School, Democritus University of Thrace, Dragana Campus, 68100 Alexandroupolis, Greece, Phone/Fax: +30-2551-030523, E-mail: [email protected]; Clinical Pharmacology Unit, Academic General Hospital of Alexandroupolis, Alexandroupolis, GreeceGeorgia Ragia, Efstathia Giannakopoulou, Makrina Karaglani and Ioanna-Maria Karantza: Laboratory of Pharmacology, Medical School, Democritus University of Thrace, Alexandroupolis, GreeceAnna Tavridou and Vangelis G. Manolopoulos: Laboratory of Pharmacology, Medical School, Democritus University of Thrace, Alexandroupolis, Greece; Clinical Pharmacology Unit, Academic General Hospital of Alexandroupolis, Alexandroupolis, Greece

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236 Ragia et al.: CYP450 enzyme gene polymorphisms in the Greek population

of variant alleles that follow an evolutionary gradient in their distribution in cases of thrifty genes [5]. For CYP450 isoenzymes, it has also been postulated that specific alleles such as CYP2D6*2xN and CYP3A5*1 alleles could confer a selective advantage in equatorial populations experiencing either food or water shortage [6]. Knowledge of the ethnic distribution of genetically controlled altered metabolism of several important drugs has the potential to optimize pharmacotherapy and help in the selection of the panel of clinically significant gene polymorphisms based on the ethnic background.

In the present study, we review all available informa-tion on the frequency of major CYP2D6, CYP2C19, CYP2C9 and CYP3A5 gene polymorphisms in the Greek population and we also report novel results from our group on the fre-quency of CYP3A4*22 and CYP1A2*1F gene polymorphisms in a sample of Greek population. Published papers were retrieved from PubMed and Scopus by use of the keywords CYP450, gene polymorphism, Greece, Greek, Hellenic, CYP2D6, CYP2C19, CYP2C9, CYP3A4, CYP3A5, CYP1A2. The potential for CYP450 pharmacogenomics application in routine clinical practice in Greece and current approaches are also discussed.

CYP2D6CYP2D6 (debrisoquine 4-hydroxylase) comprises approx-imately 1.5% of the total hepatic CYP450 content and is involved in the elimination of approximately 25% of all prescribed drugs including tricyclic antidepressants, selective serotonin re-uptake inhibitors, neuroleptic agents, β-blockers, antiarrythmics, tamoxifen and opiates [7, 8]. The CYP2D6 gene is highly polymorphic with more than 80 different allelic variants reported to date on the webpage of CYP450 Allele Nomenclature Committee [http://www.cypalleles.ki.se/index.htm]. The clinical effect of most annotated genetic variants still needs to be specified; however, carriage of the most common defec-tive variants CYP2D6*3/*4/*5/*6 predicts the majority of PMs and IMs in European populations, whereas *2 allele is often met in multi duplicated CYP2D6 genes and pre-dicts the UM phenotype. Using the standard prescribed dose of a drug primarily metabolized by CYP2D6, the UMs will have low plasma concentrations of parent drug and thus inadequate response to therapy, while the PMs could develop harmful side-effects [1]. In the case of prescribed prodrugs, PMs will have low potency of activating the parent drug to its active metabolite, while the UMs could develop harmful side-effects [1]. In European populations, analysis of the most common CYP2D6 defective alleles,

i.e., CYP2D6*3, CYP2D6*4, allows for the prediction of more than 90% of the PMs and IMs [9].

Most of the data on the frequency of CYP450 isoen-zyme gene polymorphisms in the Greek population have been generated by our research group in the Laboratory of Pharmacology of Democritus University of Thrace in Alexandroupolis. Specifically, the frequency of the most common polymorphisms in CYP2D6, CYP2C19, CYP2C9 and CYP3A5 genes was determined in a sample of 283 individu-als residing in Alexandroupolis but originating from all parts of Greece, and results were published on 2007 and 2009 [10, 11]. In the case of CYP2D6, we have estimated the frequency of CYP2D6*3 and *4 alleles at 2.3 and 17.8%, respectively, while gene duplications were detected in 21 subjects (incidence 7.4%) (Table 1). The genotype-derived PM phenotype (CYP2D6*4/*4 and *3/*4 genotypes) was estimated at 3.9%. To date, there is no other published study on the frequency of CYP2D6 gene polymorphisms. Neverthe-less, CYP2D6 phenotype was determined in 102 unrelated Greek volunteers by means of dextromethorphan metabolic ratio concentration in urine. CYP2D6 PM phenotype fre-quency was estimated at 6.9% [12]. It should be noted that the CYP2D6 PM phenotype may often occur in greater fre-quency than the genotype-determined PM phenotype, since CYP2D6 enzyme is subjected to inhibition by several drugs and this inhibition leads to the acquired PM phenotype [13].

The frequencies reported for CYP2D6 polymorphic alleles vary among different populations. For Europeans, the frequency of CYP2D6*4 and *3 allele ranges from 15 to 21% and from 0.9 to 2.8%, respectively. The frequency of these alleles described in the Greek population is in accordance with relative results in Europeans (Table 2). Interestingly, the frequency of reported CYP2D6 gene duplications presents a gradient of increasing percent-age from north to south: 1% in Swedes, 2% in Germans, 6% in Spaniards, 8% in Turks, 20% in Arabs and 29% in Ethiopians [10]. The high frequency of functional gene duplications in Black Ethiopians is possibly due to a selec-tive pressure associated with diet: subjects with a higher content of CYP2D6 could have a higher capacity for detox-ification of plant toxins (alkaloids), known to have an inhibitory effect on CYP2D6, thereby able to digest more vegetation as food, offering a higher probability for sur-vival in a region with severe food shortage [56].

CYP2C19CYP2C19 metabolizes several important drugs such as clopidogrel, S-mephenytoin, diazepam, omeprazole,



Ragia et al.: CYP450 enzyme gene polymorphisms in the Greek population 237

Table 1 Frequency of CYP450 enzyme gene polymorphisms genotypes and alleles in the Greek population.

Gene Population (n), study design Genotype and allele frequency, %

Phenotype frequency, % References

CYP2D6 283 Individuals, prevalence of gene polymorphisms Genotypes *1/*1 57.6 EM 57.6 [10] *1/*4 27.6 IM 31.1 *1/*3 3.5 *4/*4 3.2 PM 3.9 *3/*4 0.7 *3/*3 0 *2xN 6.0 UM 6.0 *2xN/*4 1.1 Undetermined 1.4 *2xN/*3 0.3 Alleles *4 17.8 *3 2.3 *2 7.4

CYP2C19 283 Individuals, prevalence of gene polymorphisms Genotypes *1/*1 44.2 EM 44.2 [10, 11] *1/*2 17.7 IM 17.7 *1/*3 0 *2/*2 2.1 PM 2.1 *2/*17 4.2 Undetermined 4.2 *1/*17 28.6 hetUM 28.6 *17/*17 3.2 UM 3.2 Alleles *2 13.1 *3 0 *17 19.6

CYP2C9 283 Individuals, prevalence of gene polymorphisms Genotypes *1/*1 62.2 EM 62.2 [10] *1/*2 20.1 IM 33.6 *1/*3 13.4 *2/*2 1.4 PM 4.2 *2/*3 2.8 *3/*3 0 Alleles *2 12.9 *3 8.1

CYP3A5 283 Individuals, prevalence of gene polymorphisms Genotypes *3/*3 88.7 Non- expressors 88.7 [10] *1/*3 11.3 Expressors 11.3 *1/*1 0 Alleles *3 94.4 *1 5.6

CYP3A4 521 Individuals, prevalence of gene polymorphisms Genotypes *1/*1 89.6 EM 89.6 Present study *1/*22 10.2 IM 10.2 *22/*22 0.2 PM 0.2 Alleles *1 94.7 *22 5.3

CYP1A2 247 Individuals, prevalence of gene polymorphisms Genotypes *1A/*1A 10.1 Normal metabolizers

55.5 Present study

*1A/*1F 45.4 *1F/*1F 44.5 Fast

metabolizers 44.5

Alleles *1A 32.8 *1F 67.2

proguanil, R-warfarin and many antidepressants. Until 2006, studies on CYP2C19 gene polymorphisms included the defective CYP2C19*2 and *3 alleles, that predict the PM and IM phenotype. The discovery of a novel CYP2C19 allele, namely CYP2C19*17, also led to the introduction of UM phenotype for CYP2C19 enzyme.

In the Greek population, CYP2C19*2 allele frequency was estimated in a sample of 283 individuals at 13.1%, while CYP2C19*3 allele was absent from the studied popu-lation [10]. In this initial study, we estimated that the pro-portion of genotype-derived phenotypes was 2.1% for PMs, 21.9% for IMs and 75.9% for EMs. Upon the identification




of CYP2C19*17 allele, we re-genotyped the same popula-tion and re-assessed the frequency of EMs. Specifically, the novel CYP2C19*17 allele was found at a frequency as high as 19.6%, whereas 31.8% of the population carried at least one CYP2C19*17 allele (*1/*17 and *17/*17 geno-types) [11] (Table 1). As a consequence, the frequency of CYP2C19*1/*1 EMs was re-calculated to a lower figure of 44.2% from the previously described 75.9%. It should be recalled that, as it was proven for CYP2C19, all CYP450 ‘wild type’ subjects designated as *1 allele carriers, are potentially a mixture of subjects carrying *1 alleles and all other non-genotyped and presently undiscovered alleles. The frequency of CYP2C19*2, *3 and *17 alleles as described in the Greek population is in accordance with relative results in Europeans (Table 2).

Genotyping of CYP2C19 gene polymorphisms has received much attention due to its involvement in the metabolism of the widely-used antiplatelet agent, clopi-dogrel. Clopidogrel is administered as an inactive prodrug that is mainly metabolized by CYP2C19 to its active metab-olite. Intense study on clopidogrel pharmacogenomics has shown that carriers of CYP2C19*2 defective allele are at an increased risk of experiencing cardiovascular events due to ameliorated activation of clopidogrel. The growing body of literature implicating CYP2C19*2 (and prob-ably other loss-of-function alleles) in adverse clopidogrel responses prompted the US Food and Drug Administra-tion (FDA) to implement a boxed warning on the clopi-dogrel label describing the relationship between CYP2C19 pharmacogenetics and drug response among acute coro-nary syndrome (ACS)/percutaneous intervention (PCI) patients, particularly noting the diminished effectiveness in PMs [57]. More recently, the Clinical Pharmacogenetics Implementation Consortium updated the guidelines for CYP2C19 and clopidogrel therapy and proposed an algo-rithm for suggested clinical actions based on CYP2C19 genotype when considering clopidogrel therapy in ACS/PCI patients. Based on this algorithm, an alternative anti-platelet therapy should be considered in CYP2C19 IMs and PMs [58].

In the Greek population, several studies have assessed the association of CYP2C19*2 allele with clopidogrel response. In 2012, Kassimis and colleagues estimated the effect of CYP2C19*2 and *17 genetic variants on-clopidog-rel platelet reactivity (HTRP) in 146 patients undergoing PCI who were on a standard clopidogrel maintenance dose (75 mg/day) for at least 7 days before PCI, or received a 600 mg loading dose before PCI in the case of 0 or < 7 days pre-treatment [59]. The authors have found that carriers of at least one CYP2C19*2 allelic variant had higher on-clopidogrel platelet reactivity compared to non-carriers. Ta

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In multivariate logistic regression analysis for HTRP in the entire study population adjusted for demographic characteristics, the presence of at least one CYP2C19*2 allelic variant was independently associated with HTPR. However, no association of CYP2C19*17 allele with HTRP was found. In the studied population, the frequencies of alleles were 13.4% and 24.7%, respectively, while 26% of patients carried at least one CYP2C19*2 allele (25.3 IMs and 0.7% PMs) and 44.5% of patients carried at least one CYP2C19*17 allele, and were presumed to be UMs [59].

In a more recent study, the effect of both CYP2C19 inhibition and CYP2C19*2 allele on platelet response to clopidogrel, as assessed by platelet function assays, was estimated in 95 patients with documented coronary artery disease (CAD) treated with clopidogrel 75 mg/day in combination with daily aspirin therapy (100 mg) [60]. Thirty-seven patients (39% of the study cohort) were also receiving omeprazole, a strong CYP2C19 inhibitor. The authors have shown that, when platelet reactivity was assessed by measuring platelet phosphorylated-VASP, car-riers of CYP2C19*2 allele had a significant higher platelet reactivity index compared to non-carriers. In this study, concomitant treatment with omeprazole was not associ-ated with platelet reactivity. The frequency of CYP2C19*2 allele was 27.3% and the prevalence of CYP2C19 geno-type-derived PM phenotype was 2% [60]. The impact of CYP2C19 genotype on cardiovascular events and platelet reactivity was also assessed in a study consisted of 353 CAD patients receiving clopidogrel 75 mg/day [61]. In 126 subjects, platelet function was evaluated. All subjects were evaluated prospectively up to 24 months, or until they discontinued clopidogrel treatment according to their physician’s suggestion, to record any primary end points (composite of death from cardiovascular causes, nonfatal myocardial infarction, nonfatal stroke or hospitalization for cardiovascular causes including stroke, severe recur-rent cardiac ischemia, recurrent cardiac ischemia, tran-sient ischemic attack, or other arterial thrombotic event). The authors found that the rate of occurrence of primary end point differs marginally between CYP2C19*2 carriers and non carriers (for carriers hazard ratio = 1.91, p = 0.05). In the total study population, 131 patients (37.1%) were carriers of at least one CYP2C19*2 reduced-function allele and 222 patients (62.9%) were non carriers [61].

CYP2C9CYP2C9 represents the most abundant among the human CYP2C isoform and comprises approximately 20% of total

hepatic CYP450 content [62]. It participates in the metabo-lism of several drugs, including nonsteroid anti-inflamma-tory agents, S-warfarin, antidiabetics, phenytoin, losartan and fluoxetine. CYP2C9*2 and *3 polymorphisms occur in approximately 85% of PMs and the frequency reported for white populations varies from 2 to 6%. CYP2C9*2 effect on enzyme activity appears less severe than the effect of CYP2C9*3 allele [63, 64], however, both alleles cause a reduction in CYP2C9 metabolized drug clear-ance with a 10-fold variation observed from the genotype with the highest (CYP2C9*1/*1) to the one with the lowest (CYP2C9*3/*3) activity (*1/*1 > *1/*2 > *1/*3 > *2/*2 > *2/*3 > *3/*3).

In the Greek population, CYP2C9*2 and *3 allele fre-quency was estimated in a sample of 283 individuals at 12.9% and 8.1%, respectively, while the proportion of gen-otype-derived phenotypes was 4.2% for PMs and 33.6% for IMs [10] (Table 1). The frequency of these alleles described in the Greek population is in agreement with relative results in Europeans (Table 2).

The clinical significance of CYP2C9 gene polymor-phisms has been assessed in the Greek population in two different drug classes; the coumarinic anticoagu-lant (COAs) acenocoumarol and the antidiabetic agents sulfonylureas.

In the case of COAs, evidence accumulated during the last decade suggests that interindividual COAs dose variability is influenced significantly by variations in the CYP2C9 gene that metabolizes COAs, and the vitamin K epoxide reductase (VKORC1) gene, the pharmacologic target enzyme of these drugs. It has been estimated that polymorphisms in CYP2C9 and VKORC1 genes together account for 35–50% of the variability in COAs initiation and maintenance dose requirements [65]. Efforts are focusing on incorporating this knowledge to currently used dosing regimens by use of pharmacogenetic-based dosing algorithms that include CYP2C9 and VKORC1 geno-type information. Results of two randomised clinical trials conducted in European populations that prospectively compared the effect of a genotype-guided dosing algo-rithm, have been recently released [29, 66]. Both studies showed that patients who received genotype-guided dosing of COAs had increased percentage of time in the therapeutic range compared to controls monitored during the first 4 weeks after treatment initiation for acenocou-marol and phenprocoumon and during the 12 weeks after treatment initiation for warfarin.

In the Greek population, Markatos and colleagues have investigated in a retrospective study the effect of CYP2C9*2, CYP2C9*3 and VKORC1 –1639G > A gene polymorphisms on the interindividual variability of acenocoumarol dose




requirements [67]. A total of 98 patients of Greek origin receiving chronic oral anticoagulant treatment with acen-ocoumarol who were stable for at least 4 weeks within a therapeutic international normalized ratio (INR) were included in the study. Patients were classified as low dosers if receiving < 1 mg/day, moderate dosers if receiving > 1 mg/day and < 4 mg/day, and high dosers if receiving > 4 mg/day. The authors have shown that the mean maintenance dose of acenocoumarol was related to CYP2C9 genotype. Specifically, CYP2C9*1/*1 patients required a mean dose of 2.91 mg/day, while CYP2C9*1/*3 and *2/*3 patients required lower doses, 1.73 mg/day and 1.28 mg/day, respec-tively. Even though carriers of both CYP2C9*2 and *3 alleles required a lower acenocoumarol dose, the difference was statistically significant only for CYP2C9*3 allele [67]. The authors have also constructed a dosing algorithm for acenocoumarol that included both demographic (age) and genetic information (CYP2C9 and VKORC1 genotypes). The frequency of CYP2C9*2 and *3 alleles was 15.5% and 7.5%, respectively, while the prevalence of CYP2C9 genotype-derived IM and PM phenotype was 37.7% and 4.1%, respec-tively [67]. VKORC1 –1639G > A polymorphism frequency has also been adequately described in the Greek popula-tion [68, 69], but presentation of these results is beyond the scope of the present article.

Sulfonylurea oral hypoglycemic agents both of the first (tolbutamide, tolazamide, chloropropamide and acetohexamine) and of the second generation (glibencla-mide or glyburide, glipizide, gliclazide and glimepiride) are extensively metabolized by CYP2C9 enzyme. CYP2C9 *2 and *3 alleles are associated with impaired metabolism and reduced oral clearance of sulfonylureas that leads to increased sulfonylurea plasma levels and may lead to improved therapeutic effect of these drugs [68]. Further-more, in carriers of CYP2C9 defective alleles, increased plasma concentrations of sulfonylureas decreased clear-ance and prolonged action due to diminished drug metabolism might also be associated with incidence of hypoglycemia, sulfonylurea major and most common adverse event. In a previous study in the Greek popula-tion, we have investigated the association of CYP2C9*2 and *3 alleles with sulfonylurea induced mild hypoglyce-mia [70]. The study group consisted of 176 type 2 diabetes mellitus (T2DM) patients treated with the sulfonylureas glimepiride or gliclazide. Ninety two sulfonylurea-treated patients reported drug-associated hypoglycemia, while 84 sulfonylurea-treated patients had never experienced a hypoglycemic event. The frequency of CYP2C9 genotypes that lead to impaired CYP2C9 function (*1/*2, *1/*3 and *2/*3) was higher in hypoglycemic patients than in non-hypoglycemic controls.

Logistic regression analysis, with hypoglycemia status as the dependent variable and CYP2C9 genotype as contributing variable, estimated that CYP2C9*1/*3 geno-type increases significantly the risk of hypoglycemia in a model adjusted for age, sex, body mass index, T2DM duration and renal impairment (OR 1.687, p = 0.011) [70]. More recently, following the hypothesis that P450 oxi-doreductase (POR) *28 allele is associated with increased CYP2C9 activity, we have also shown that, additionally to CYP2C9*3 allele, CYP2C9*2 allele is a strong predictor of sulfonylurea induced hypoglycemia in patients having the wild type genotype (*1/*1) in POR enzyme [71]. These results suggest that CYP2C9 genotyping might be a useful tool for predicting adverse effects caused by sulfonylureas and it might help clinicians in the safer prescribing of oral hypoglycemic agents. The association of other gene poly-morphisms (Inward Rectifier Potassium Channel Kir6.2, KCNJ11) with sulfonylurea hypoglycemia incidence has also been studied in the Greek population [72], but the presentation of these results is beyond the scope of the present article.

CYP3A5CYP3A5 is the major extrahepatic isoform of CYP3A gene family and in association with CYP3A4 are responsible for the metabolism of over 50% of all clinically used drugs, including steroids, antidepressants, immunosuppressive agents and antibiotics. CYP3A4 and CYP3A5 isoenzymes show overlapping substrate specificity. In many cases, CYP3A5 has an increased affinity compared with CYP3A4, for CYP3A metabolized drugs such as midazolam and tacrolimus. The primary causal mutation for its polymor-phic expression (CYP3A5*3) confers low CYP3A5 protein expression as a result of improper mRNA splicing and reduced translation of a functional protein. The CYP3A5*3 polymorphism is widely detectable in European popula-tions and homozygosity for the allelic variant is strongly correlated with decreased enzymatic activity. In contrast, CYP3A5*1 allele is associated with high CYP3A5 expres-sion. Carriers of CYP3A5*1 allele are designated as CYP3A5 expressors and have increased metabolism of CYP3A4/5 substrates.

In the Greek population, CYP3A5*3 allele is abun-dantly present at an allelic frequency of 94.4% [10] (Table 1). This frequency derives from the seminal study on the frequency of major CYP450 gene polymorphisms in a sample of 283 non-related Greek volunteers. In this study, 32 individuals (11.3%) were carriers of CYP3A5*1




allele (CYP3A5 expressors). The frequency of CYP3A5*3 allele as described in the Greek population is in accord-ance with relative results in Europeans (Table 2).

Interestingly, similarly to CYP2D6 gene duplication, the hypothesis that CYP3A5*1 allele ethnic distribution is governed by a selective pressure has been made. The frequency of the CYP3A5*3 allele varies broadly across populations, and this variation is positively correlated with geographical distance from the equator [73–75], with the equatorial populations presenting with the highest frequency of CYP3A5*1 allele [76]. One potential expla-nation is that CYP3A5 isoenzyme is also expressed in the kidney. It has been postulated that the CYP3A5*1 allele could confer a selective advantage in equatorial popula-tions experiencing water shortage by increasing sodium retention [77].

CYP3A5*3 allele has also been assessed in association with pharmacokinetics and response to the immunosup-pressant tacrolimus. In 2010, Katsakiori and colleagues investigated the impact of CYP3A5*1 and CYP3A5*3 alleles on the kinetics of tacrolimus in 40 renal transplant recipi-ents [78]. Tacrolimus was given twice a day in individu-ally adjusted doses and its trough levels were measured 12 h post dose. The authors have found that CYP3A5*1 variant was associated with significant lower tacrolimus dose adjusted concentration at 3, 6, 12 and 36 months after transplantation, and that carriers of CYP3A5*1 allele had lower predicted measures for tacrolimus dose adjusted concentration and higher predicted measures for volume of distribution [78]. The frequency of CYP3A5*1 allele was 6.3% and the prevalence of CYP3A5 expressors was 12.5%. One year later, the same research group investi-gated the possible pharmacological interaction between tacrolimus and statins in CYP3A5 non-expressors, renal transplant recipients [79]. A total of 24 tacrolimus treated patients who, after an observation time of approximately 4 months, were co-administered a statin were included in the study. The authors did not observe any statistically sig-nificant difference in tacrolimus pharmacokinetic param-eters after the initiation of statin treatment [79].

CYP3A5*3 allele has also been studied in association with lipid-lowering response to statins. Results of previous studies have showed that CYP3A5 expression in CYP3A5*1 allele carriers, is associated with lower decreases in low density lipoprotein cholesterol (LDL-C) and total choles-terol (TChol), or need higher dosages for optimal lipid lowering response. In the seminal study, Kivisto et al. showed that lovastatin, simvastatin and atorvastatin were significantly less effective in CYP3A5 expressors than in non-expressors [80]. In this study, CYP3A5 expressors had higher concentration at 1 year of both TChol (23% higher)

and LDL-C (24% higher), while the mean percentage reduction in serum TChol from baseline was significantly smaller in CYP3A5 expressors than in non-expressors (17 vs. 31%, p = 0.026). In the case of simvastatin, CYP3A5*1 carriers had significantly lower mean simvastatin AUC and higher oral clearance than CYP3A5*3/*3 individuals [81]. The results of a study of CYP3AP1 pseudogene –44G > A polymorphism with statin response in a patient cohort consisted of 202 simvastatin-treated and 177 atorvastatin treated Chinese hyperlipidemic patients also showed that in the simvastatin treatment group, the percentage reduc-tion of LDL-C was greater in female CYP3AP1*3/*3 carriers than CYP3AP1*1 carriers [82]. In the Greek population, in a more recent study in 191 simvastatin treated patients, Kolovou et al. have shown that CYP3A5 non-expressors display a trend towards higher LDL-C reductions com-pared with CYP3A5 expressors [83]. In this study, the fre-quency of CYP3A5 expressors was estimated at 15.6%, in accordance with the prevalence already reported for the Greek population.

CYP3A4CYP3A4 is the most abundant hepatic and intestinal CYP450 isoenzyme that metabolizes approximately 50% of all marketed drugs, such as erythromycin, midazolam, cyclosporine, tacrolimus, sirolimus, everolimus, statins, several anticancer agents, antibiotics, and anti-HIV drugs [30]. Interindividual variability in CYP3A expres-sion in liver is very high (20–40 fold). This variability has been suggested to be associated with variants in CYP3A4 and CYP3A5 encoding genes that may contribute to the differences found in response to several drugs. It has been suggested that approximately 90% of interin-dividual differences in hepatic CYP3A4 activity is due to genetic variation. To date, more than 39 single nucleotide polymorphisms (SNPs) of the CYP3A4 gene have been published on the Human Cytochrome P450 Allele Nomen-clature Committee homepage, however, the majority of CYP3A4 functional SNPs is infrequent and accounts only for a small portion of the observed CYP3A4 interindividual variability. Furthermore, the most common CYP3A4 SNP reported to be associated with drug response, CYP3A4*1B, appears in strong linkage disequlibrium with CYP3A5*1 allele that leads to expression of CYP3A5 and increased total CYP3A content.

Recently, a novel functional SNP located in intron 6 of CYP3A4 (CYP3A4*22, rs35599367) was identified and fully correlated with the observed allelic mRNA pattern,




CYP3A4 total mRNA level and activity in human livers [84, 85]. CYP3A4*22 allele has emerged as a novel impor-tant biomarker for identifying reduced metabolism of CYP3A4 drugs and has been associated with reduced dose requirements for optimal pharmacotherapy with the drugs simvastatin, tacrolimus and cyclosporine [30].

For the purposes of the present paper, we have ana-lyzed the frequency of CYP3A4*22 allele in a sample of 521 unrelated volunteers of Greek origin. The mean age of the subjects was 46 ( ± 22) years. All participants were of Greek ethnic origin residing in the Alexandroupolis urban area but originating from all parts of Greece and gave their informed consent. The study protocol was approved by the Ethics Committee of the Democritus University of Thrace Medical School.

Three millilitres of venous blood were obtained from each participant and genomic DNA was extracted from peripheral blood leucocytes by Puregene DNA Purifica-tion System (Gentra Systems Inc, Minneapolis, MN, USA), according to the instructions of the manufacturer. Deter-mination of CYP3A4*22 genotype was performed by use of TaqMan (Applied Biosystems, Carlsbad, CA, USA) geno-typing assays (C_59013445_10) on the ABI PRISM 7500 Fast real-time PCR Systems (Applied Biosystems, Carls-bad, CA, USA) according to manufacturer instructions, using 50 ng of genomic DNA.

In the total population, 53 individuals (10.2%) were *1/*22 heterozygous, whereas only one individual (0.2%) carried the homozygous *22/*22 genotype (Table 1). The frequency of CYP3A4*22 allele was estimated at 5.3% and is in line with the frequency described in other European populations (Table 2).

CYP1A2Human CYP1A2 is one of the major CYPs in human liver and metabolizes a variety of clinically important drugs such as clozapine, tacrine, tizanidine, and theophyl-line, a number of procarcinogens, and several important endogenous compounds (e.g., steroids and arachidonic acids) [86]. CYP1A2 primarily metabolizes caffeine that is used as a probe model substrate for CYP1A2 activity evaluation in vivo. There are wide interindividual differ-ences (10- to 200-fold) in CYP1A2 (also called phenacetin O-deethylase) expression and activity. Approximately 15- and 40-fold interindividual variations in CYP1A2 mRNA and protein expression levels have been observed in human livers. It has been suggested that approximately 35 to 75% of the interindividual variability in CYP1A2 activity is due to genetic factors. To date, more than 15

variant alleles (*1B to *16) and a series of subvariants of the CYP1A2 gene have been identified [86]. The most extensively studied polymorphism is –163C > A located in intron 1 (*1F allele). Homozygous carriers of *1F allele (*1F/*1F genotype) are termed as fast metabolizers and have increased CYP1A2 activity. Interestingly, CYP1A2 is the most inducible enzyme among CYP450 isoenzymes and CYP1A2*1F encoded isoform is highly inducible [87]. Among other factors, smoking is the strongest inducer of CYP1A2 enzyme [88].

For the purposes of the present paper, we have ana-lyzed the frequency of CYP1A2*1F allele in a sample of 247 unrelated volunteers individuals of Greek origin. The mean (SD) age of the subjects was 52 ( ± 22.3) years. All par-ticipants were of Greek ethnic origin residing in the Alex-androupolis urban area but originating from all parts of Greece and they gave their informed consent. The study protocol was approved by the Ethics Committee of the Democritus University of Thrace Medical School.

Three mililiters of venous blood were obtained from each participant and genomic DNA was extracted from peripheral blood leucocytes by Puregene DNA Purifi-cation System (Gentra Systems Inc, Minneapolis, MN, USA), according to the instructions of the manufacturer. CYP1A2*1F allele was analyzed by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) method, as described elsewhere [89, 90]. In the total population, 25 individuals (10.1%) were *1A/*1A, 112 (45.4%) were *1A/*1F and 110 (44.5%) were *1F/*1F (Table 1). The frequency of CYP1A2*1F allele was estimated at 67.2%. Homozygous *1F/*1F individu-als are expected to be fast metabolizers of CYP1A2. The frequency of CYP1A2*1F allele as described in the Greek population is in agreement with relative results in Euro-peans (Table 2).

In the Greek population, the activity of CYP1A2 enzyme was first measured in 2004 in terms of molar ratio (MR) of caffeine metabolites in urine and saliva in a sample of 40 patients with schizophrenia. In this study, the authors found that smokers (30 patients) had significant higher MR in urine (p < 0.001) as well as in saliva (p = 0.001) than nonsmokers (10 patients), suggesting a higher activity of CYP1A2 dependent on smoking [91]. CYP1A2 activity has been also evaluated in the Greek population by an RP-HPLC-based method for quantification of caffeine and its metabolites in urine samples [92]. In a sample of 44 vol-unteers, Begas and colleagues have shown that smoking significantly increases CYP1A2 index. The median values of CYP1A2 activities were significantly higher in smokers (metabolic ratio 5.83) than non-smokers (metabolic ratio 3.57) (p < 0.001) [92].




Other CYP450 enzymes associated with potential pharmacogenomic interestCYP2D6, CYP2C19, CYP2C9, CYP3A5, CYP3A4 and CYP1A2 gene polymorphisms gather the greatest pharmacog-enomic interest for implementation in routine clinical practice. However, other CYP450 enzymes associated with potential pharmacogenomic interest or with disease risk prognostic value, such as CYP2B6, CYP1A1, CYP11A1, CYP11B2 and CYP17, have also been studied in the Greek population.

CYP2B6 participates in the oxidative metabolism of endogenous chemicals, pesticides and environmen-tal chemicals, and several clinically important drugs, including the anesthetic propofol, the antiretroviral agent efavirenz, the antidepressant bupropion, and the antie-pileptic agent mephobarbital. The association of CYP2B6 516G > T polymorphism with the distribution of propofol concentrations, has been studied in 44 Greek women undergoing oocyte retrieval [93]. CYP2B6 516 T allele was strongly associated with high propofol concentrations determined in whole blood, shortly after a single bolus dose. The frequency of CYP2B6 516 T allele was estimated at 29.5%. Another CYP2B6 polymorphism, 1459C > T (*5 allele), was associated, additionally to CYP2C19*2 allele, with clopidogrel metabolism and action [59]. Specifically, among CYP2C19*2 non-carriers, when adjusting for demo-graphic characteristics, carriers of at least one CYP2B6*5 allelic variant had higher on-clopidogrel platelet reactivity compared to non-carriers of this particular allele. 71.9% of the studied population carried at least one CYP2B6*5 allele.

CYP1A1 is involved in detoxification of pesticide chemicals and carcinogens such as polycyclic aromatic hydrocarbons and aromatic amines. CYP1A1 gene poly-morphisms have been studied in association with the occurrence of several cancers in different populations. In the Greek population, CYP1A1 T3801C (*2A allele or MspI) polymorphism was not associated with smoking-related lung cancer risk in a study that included 100 patients with histologically or cytologically documented advanced lung cancer [94] or with the development of cervical intraepithelial neoplasia, in a study in 166 women with history of cervical intraepithelial neoplasia [95]. Similarly, CYP1A1*2A allele was not associated with breast cancer risk in a study in 207 female breast cancer patients [96]. No association of CYP1A1*2A allele with pathological diseases was the main finding of a study conducted in 220 partici-pants of an agricultural cohort exposed to pesticides in

the south of Greece [97]. In a different study conducted in 492 individuals exposed to pesticides, CYP1A1*2A allele was found to have significant association with chronic obstructive pneumonopathy, peripheral circulatory prob-lems, arteritis, allergies, hemorrhoids, allergic dermatitis and miscarriages [98]. Finally, in a study including 275 sporadic endometriosis patients, CYP1A1*2A allele was associated with endometriosis risk in patients also car-rying glutathione S-transferase μ1 (GSTM1) null deletion [99]. The frequency of CYP1A1*2A allele has been esti-mated at 10.7%.

CYP11A1, CYP11B2 and CYP17 enzymes mediate steroid hormone synthesis. CYP11A microsatellite polymorphism (tttta)n was associated with both polycystic ovary syn-drome (PCOS) and total testosterone levels in 170 women with polycystic ovary syndrome [100], whereas CYP11B2 C-344T polymorphism was associated with higher values of intima-media thickness in 318 untreated, newly diag-nosed essential hypertensive patients [101]. CYP17 –34C/T promoter polymorphism (MspI or A1/A2 polymorphism) was not associated with the risk of recurrent spontane-ous abortions in 148 Greek women with a relative medical history [102]. However, in a study that included 50 Greek polycystic ovary syndrome patients, CYP17 A2 homozygo-sity was associated with PCOS [103].

Technical advances in CYP450 gene polymorphism identification reported by Greek research groupsVarious assays, such as allele-specific PCR, real time PCR, multiplex PCR followed by ARMS, RFLP, single-strand conformation polymorphism (SSCP), denaturing HPLC, oligonucleotide ligation assay, pyrosequencing, invader assay, mass spectrometry and DNA arrays, have been developed for the genotyping of the CYP450 gene poly-morphisms. There is, however, a constant need for devel-opment of new, simple and rapid genotyping assays of CYP450 gene polymorphisms to facilitate the introduction of pharmacogenomics in routine clinical practice.

Several Greek research teams have developed dif-ferent genotyping techniques for the identification of CYP2D6 and CYP2C19 gene polymorphisms. More specifi-cally, in 2007, a rapid genotyping approach was described for CYP2D6*3 and *4 and CYP2C19*2 and *3 alleles by primer extension reaction in a dipstick format [104]. In this approach, genomic DNA is isolated from whole blood and the segments that span the SNP of interest are




amplified by PCR. The products are subjected directly to two primer extension reactions (PEXT), each consisting from three cycles, using normal and mutant primers in the presence of biotin-dUTP. The PEXT products are visu-alized through a dry-reagent dipstick-type assay. In this assay, the biotinylated extension products are captured from immobilized streptavidin on the test zone of the strip and detected by hybridization with oligo(dT)-functional-ized gold nanoparticles [104].

One year later, same alleles were genotyped with a modified oligonucleotide ligation reaction (OLR) approach [105]. In this approach, the authors describe a simple and low-cost disposable biosensor in dry-reagent format, which allows visual genotyping with no need for instru-mentation [105]. Specifically, after OLR product denatura-tion, products are applied to the biosensor next to gold nanoparticles that are decorated with oligo(dT) strands. The OLR product is captured by immobilized streptavidin at the sensor and hybridizes with the oligo(dT) strands of the nanoparticles. A characteristic red line is generated due to the accumulation of nanoparticles, while the excess nanoparticles are captured by immobilized oligo(dA) at the control zone of the strip, giving a second red line [105].

The same research group, has also developed a dip-stick test that enables visual detection of tetra-primer PCR products within minutes without electrophoresis or instruments for the detection of CYP2D6*4 and CYP2C19*2 alleles [106]. In this approach, a pair of external primers amplifies the segment of interest that encompasses the SNPs, while biotinylated inner primers have a 3-mis-match and pair off with the external primers to guide a bidirectional amplification that generates allele-specific fragments. The products are hybridized briefly with poly(dA)-tailed probes and applied to the DNA biosensor, in which, when immersed in the appropriate buffer, the hybrids are captured from streptavidin and interact with oligo(dT)-functionalized gold nanoparticles leading to the formation of a red line. Proper function of the sensor is indicated by another formed red line [106].

Pharmacogenomics implementation in routine clinical practice in GreeceImplementation of pharmacogenomics in routine clini-cal practice remains a world wide matter of concern, with only a few applications applied so far prior to therapy initiation. CYP450 isoenzyme pharmacogenomics still lacks behind in clinical implementation even though the

benefit of genotyping in fields such as psychiatry, oncol-ogy and cardiology has been proven in several studies. In Greece, to the best of our knowledge, our research group in Alexandroupolis (Laboratory of Pharmacology, Medical School, Democritus University of Thrace) is the only group applying pharmacogenetic analysis of CYP450 isoenzymes as a clinical service for physicians.

Several steps are required for clinical implementation of pharmacogenomics in routine clinical practice. A main step is the education of clinicians and all other parties involved in the use and benefits of pharmacogenomics. To achieve this goal, a course of Pharmacogenomics directed to undergraduate Medical School and Molecular Biology students has been taught since 2000, extensively cover-ing the range of applied pharmacogenomics and specifi-cally focusing on CYP450 gene polymorphisms and their effect on drug response. Furthermore, several seminars addressed to physicians take place into the clinics, spe-cifically in Psychiatric and Cardiology Department.

Apart from education, during the last 6 years, our laboratory collaborates with the Psychiatry Unit of Aca-demic General Hospital of Alexandroupolis in an effort to prospectively guide the pharmacotherapy of psychiatric patients. More than 300 patients who have been admit-ted to the Psychiatric Unit or treated at its Outpatient Clinic have been genotyped for the most common CYP450 genetic variants and an extensive report describing the genotype-derived predicted patient response is incorpo-rated in each patient medical record. Efforts are focused on recording both genotype-based changes in pharma-cotherapy in association with both clinical response and rates of re-admission or hospitalization length after midterm and long-term patient follow-up.

ConclusionsFrequencies of all major CYP450 isoenzyme gene poly-morphisms have been reported in the Greek population. Frequencies of CYP2D6*3,*4 and *2xN, CYP2C19*2,*3 and *17, CYP3A4*22, CYP3A5*3, CYP2C9*2 and *3 and CYP1A2*1F polymorphic alleles are in accordance with the ones reported in other European populations. In the case of CYP2D6*2xN and CYP3A5*1 the frequencies in the Greek population follow the reported north to south gra-dient. Research results on the clinical impact of CYP450 pharmacogenomics have been extensively reported for CYP2C19 and clopidogrel, CYP2C9/VKORC1 and acenocou-marol and CYP3A5 and immunosuppressants, whereas no reports are yet available in the field of psychiatry.




CYP450 superfamily is responsible for the oxidation of almost 90% currently used drugs. CYP450 gene poly-morphisms can be responsible for various drug effects and even adverse effects and therefore constitute the core of pharmacogenomics in an effort to personalize pharma-cotherapy. Indeed, genotyping of CYP450 gene polymor-phisms can be widely applied in a range of fields to guide the optimal drug dosages so that physicians both achieve the desired response and ameliorate the incidence of serious adverse events. Currently, detailed guidelines for the use of genetic testing with antidepressants and antip-sychotics are available to introduce psychiatrists to the clinical application of genetic information. Additionally, both FDA and European Medicines Agency (EMA) are con-stantly updating the drug package inserts and propose dose genotype-based recommendations. Such recommen-dations already exist for aripiprazole, atomoxetine and clobazam. Furthermore, pharmacogenomics research in cardiology has urged the integration of pharmacogenom-ics information on inserts of two commonly prescribed drugs, namely clopidogrel and warfarin. Additionally, strong evidence exists for the implementation of CYP450 gene polymorphisms in the therapy with the immuno-suppressant tacrolimus and the adjuvant breast cancer therapy with tamoxifen. Research on the association of CYP450 gene polymorphisms with drug pharmacokinet-ics and clinical response continues in almost every field of medicine. Results of such pharmacogenomics research are expected to provide relevant information for genotype-guided dosage administration for all drugs, prescribed in different fields of medicine, that are extensively metabo-lized by CYP450 enzymes.

The future of CYP450 gene polymorphism implemen-tation in routine clinical practice is highly depended on the education of clinicians and all other parties involved in the use and benefits of pharmacogenomics. It is expected that, in the next 5 years, results from prospective studies on the application of pharmacogenomics in psy-chiatry and from original research in several other fields will boost CYP450 pharmacogenomic analysis and con-tribute to improved clinical decisions in pharmacotherapy success with CYP450 metabolized drugs.

Conflict of interest statement

Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article.Research funding: None declared.Employment or leadership: None declared.Honorarium: None declared.

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