DISTRIBUTION OF THE MOST COMMON GENETIC VARIANTS ASSOCIATED WITH A VARIABLE DRUG RESPONSE IN THE POPULATION OF THE REPUBLIC OF MACEDONIA
Kapedanovska Nestorovska A1, Jakovski K2, Naumovska Z1, Hiljadnikova Bajro M1, Sterjev Z1, Eftimov A1, Matevska Geskovska N1, Suturkova L1, Dimitrovski K3, Labacevski N3, Dimovski AJ
*Corresponding Author: Aleksandar J. Dimovski, MD., Ph.D., Center for Biomolecular and Pharmaceutical Analysis, Faculty of Pharmacy, University Ss Cyril and Methodius, Mother Theresa 47, Skopje 1000, Republic of Macedonia. Tel: +389-2-3217-580; +389-2-3119-694. Fax: +389-2-3290-830; +389-2-3123 054. E-mail: adimovski@ff.ukim.edu.mk
page: 5

GENETIC VARIANTS ASSOCIATED WITH A VARIABLE DRUG RESPONSE

Phase I: Oxidation, CYP450 Family (CYP2D6, CYP2C19, CYP2C9, CYP3A5). The CYP450 isoform activity has emerged as a major determinant of the variability in drug metabolism and response as these enzymes are responsible for about 80.0% of all Phase I DMEs. Despite the large number of CYP genes and enzymes, 57 individual CYP genes organized in 18 gene families comprising 44 subfamilies, only the members of the CYP2 and CYP3 gene families have a significant importance since they contribute to the metabolism of the majority of clinically important drugs [5,6]. The CYP2D6 enzyme, al-though expressed at low levels, constituting only 4.0-6.0% of the CYP content in the liver, metabolizes 25.0 to 30.0% of all clinically used drugs including anti-arithmics, antidepressants, anti-psychotics, beta blockers and codeine. The enzyme is the only one among the CYPs DMEs that is not inducible, and therefore, the genetic variation contributes largely to the inter-individual variation in the enzyme activity [7]. The CYP2C19 enzyme plays a critical role in the oxidative biotransformation of approximately 10.0% of the commonly used drugs belonging to drug classes such as proton pump inhibitors, tricyclic anti-depressants, selective serotonin reuptake inhibitors, benzodiazepines, barbiturates, phenytoin, voriconazole, nelfinavir and proguanil [7,8], whereas, the CYP2C9 enzyme contributes to the metabolism of 15.0% of clinically important drug classes, such as nonsteroidal anti-inflamatory drugs, angiotensin II receptor antagonist, anti-diabetic drugs, diuretic torsemide as well as the narrow therapeutic index drugs such as phenytoin and warfarin [7,9]. The CYP3A enzymes are responsible for the metabolism of approximately 50.0% of the currently marketed drugs, including steroids, anti-depressants, immunosuppressive agents and anti-biotics. The CYP3A5 is the primary extrahepatic CYP3A isoform, and typically, displays decreased catalytic activity compared to CYP3A4 [5,9]. The CYP450 allelic variant and numerous subvariants reported to date are summarized at the Human CYP Allele Nomenclature Committee website (http:// www. cypalleles.ki.se/). Currently, over 100 allelic variants and numerous subvariants have been identified within the CYP2D6 gene. The most important non functional alleles that are predominantly responsible for poor metabolizers (PMs) and account for 90.0-95.0% of PMs in indigenous Europeans are CYP2D6*3, CYP2D6*4 and CYP2D6*6 [10]. The CYP2D6*4 with an allele frequency of 12.0-21.0%, is the most common allele associated with PMs. Other detrimental alleles, CYP2D6*3 and CYP2D6*6, are frequently found in indigenous Europeans (1.0-2.0%), but are very rare or absent in other populations. The most common alleles with severely reduced activity are represented by CYP2D6*9, CYP2D6*10, CYP2D6*17 and CYP2D6*41. The frequency of CYP2D6 deletions ranges from 2.0-7.0% in indigenous Europeans, 6.0% in Blacks and less than 1.0% in Asians and 2.0-10.0% of the populations carry multiple copies of the functional CYP2D6 allele [7,11]. The majority of PMs of CYP2C19 are due to the CYP2C19*2 (681G>A) and CYP2C19*3 (636G>A) allelic variants that are non functional alleles, resulting in a complete loss of enzyme activity. They account for 95.0% of PMs. The frequency of the CYP2C19*2 allele ranges between 15.0-17.0% in indigenous Europeans and Blacks and up to 30.0% in the Asian population. The most common allele associated with ultrarapid enzyme activity, CYP2C19*17 (806C>T), has a similar allelic frequency of 15.0-25.0% in both the indigenous European and Black populations, and much reduced frequency (4.0%) in Asians [7,12]. The most prevalent CYP2C9 alleles in the European population are CYP2C9*2 (430C>T) and CYP2C9*3 (1075A>T). Both are decreased-function alleles, lead to significant reduction in the enzymatic activity of the CYP2C9 enzyme and account for 85.0% of PM phenotypes. The reported frequencies of CYP2C9* 2 and CYP2C9*3, ranges between 13.0-22.0% and 4.0-6.0%, respectively, in indigenous Europeans, but are much lower in Blacks and Asians [7,9,12]. CYP3A5 is a highly polymorphic enzyme as a result of mutations that severely diminish the synthesis of the functional CYP3A5 protein. The CYP3A5*3C (6986A>G) mutant is the major defective allele, leading to alternative splicing and protein truncation, which in turn results in the absence of the enzyme activity. It is widely detectable in the indigenous European population (85.0-98.0%) with a lower frequency in the Black (27.0-48.0%) and Asian (60.0-75.0%) populations [7,12,13]. Distribution of the CYP450 (CYP2D6, CYP2C19, CYP2C9, CYP3A5) Genetic Variants in the Population of the Republic of Macedonia. The allelic and genotype frequency distributions in our population and the correlated CYP450 phenotype status for the investigated DMEs are presented in Table 1. The CYP2D6 gene is considered a challenge for genotyping because its genetic variants include not only single nucleotide polymorphisms (SNPs), but copy number variations (CNVs) as well. The optimum amount of genotyping required to accurately predict the CYP2D6 phenotype remains debatable. A total of 11 different CYP2D6 variant alleles have been identified in our population. The prevalence of both inactive (*3,*4 and *6) and decreased activity )*9, *10 and *41) alleles was estimated to be 0.324. The most frequently observed variant allele was *4, accounting for 0.575 of all variant alleles. A total of 33 different genotypes were identified. The geno-typephenotype association was made according to the CYP2D6 DPWG Genotype Phenotype Classification System (http://www.pharmgkb.org). Table 2 summarizes the resulting CYP2D6 genotypes and corresponding phenotypes recognized in the population of the Republic of Macedonia. Our results regarding the CYP2D6 profile were generally in accordance with the frequencies reported for other European populations (Supplementary Table 1A; references 5-18). Statistically significant differences were found in comparison to the prevalence of the CYP2D6 gene duplication and deletion. We report a higher frequency of CYP2D6 duplications/deletions in the Republic of Macedonia compared to that of Germany, France, Russia and Croatia [5,14,17]. These findings regarding the distribution of the CYP2D6 CNV status corresponds to the north-south gradient theory, suggesting a decreasing prevalence of CYP2D6 CNV from the Mediterranean countries towards Northern Europe. The frequency distribution of CYP2D6 PMs is similar to that of the other European populations [10,11,15-23]. All six possible CYP2C19*2 and *17 genotypes with the corresponding phenotypes [*1/*1 and *1/*2, both specifying extensive metabolizers (EMs); *1/*17 and *17/*17 indicative for ultrarapid metabolizers (UMs); *2/*2 predicting PM and the *2/*17 combined heterozygous genotype with a difficult to predict phenotype] have been identified [23] (Table 1). The detected frequency of the most common CYP2C19 variant alleles in the population of the Republic of Macedonia is within the range comparable with other European populations (Supplementary Table 1A; references 7,11-14,18-36). According to the previously reported data [24], the CYP2C19*3 allele has a very rare occurrence in the population of the Republic of Macedonia and therefore, it was not a subject of comparison in this study. The allelic frequencies for CYP2C9*2 and CYP2C9* 3 found in our population correspond to the general frequencies reported for the various ethnic groups throughout Europe (Supplementary Table 1B; references 11-14,20,23, 34-47). The frequencies of the CYP2C9 genotypes associated with PM phenotypes (0.297), *2/*2 and *3/*3 were found to be 0.026 and 0.010 [23], respectively (Table 1). The CYP3A5*3 allele was abundantly present in the specimens of our study with a frequency of 0.908. We found no statistically significant difference compared to the frequencies reported for other Europeans (Supplementary Table 1B; references 13,36,48-52). Since heterozygotes and wild types for CYP3A5*3 may have a CYP3A5 activity, we estimate that 0.82 of the population of the Republic of Macedonia do not have a CYP3A5 enzymatic activity [25]. This is in agreement with the finding that 10.0% of the indigenous European population are high expressers of CYP3A [13]. Phase II: Conjugation (GSTT1, SULT1A1; UGT1A1). Extensive polymorphism also occurs in the majority of Phase II drug metabolizing enzymes. Sulfo-transferases and UDP-glucuronosyltransferases are two important phase II enzyme families that catalyze the sulfate and glucuronide conjugation of many endogenous and exogenous substances. The most frequent polymorphism in the SULT1A1 gene is the SULT1A1*2 (638G>A) that is correlated with a diminished capacity to sulphate SULT1A1 substrates. The greatest clinical significance of SULT1A1 is its role in the metabolism of estrogens and tamoxifen [26]. UGT1A1 pharmacogenetics has been studied more extensively than that of other uridine 5-diphospho- glucuronosyltransferases (UGTs). The greatest clinical significance has the UGT1A1*28 variant allele correlated with an altered level of the enzyme expression and therefore a low enzymatic activity. It has been associated with adverse drug reactions (ADRs) due to reduced clearance, most notably lifethreatening toxicity of chemotherapy with a high-dose irinotecan [27]. Ethnic variations in allele frequencies observed for SULT1A1*2 ranges from 0.332 in indigenous Europeans, 0.080 in Asians and 0.294 in African- Americans. The frequency of the UGT1A1*28 allele varies among ethnicities, being the highest in those of African (43.0%) or European (39.0%) descent and the lowest in those of Asian (16.0%) descent [26,27]. Glutathione S-transferases (GSTs), are active in the detoxification of a wide variety of potentially toxic and carcinogenic electrophiles in conjugation with glutathione (GSH). The most common polymorphism is caused by a gene deletion that results in the absence of GSTT1 enzyme activity and has been associated with a reduction of the relapse risk in several tumor types (acute lymphoblastic leukemia, acute myeloblastic leukemia, breast cancer, ovarian cancer and lung cancer) treated with chemotherapy. Reported frequencies for the GSTT1 null genotype from different ethnic groups show a range of 16.0-60.0% for Asians, 15.0-26.0% for Africans and 10.0-20.0% for the European population [26,28]. Distribution of the Phase II Conjugation (GSTT1, SULT1A1; UGT1A1) Genetic Variants in the Population of the Republic of Macedonia. For the SULT1A1 (638G>A) polymorphism, approximately one-third of the population from the Republic of Macedonia carry the low-activity SULT1A1*2 allele and 17.0% are predicted to be homozygous for the SULT1A1*2 genetic variant [29] (Table 3). The distributions of the UDP-glucuronosyltransferase (UGT)1A1*28 allele and genotype frequencies in our population are summarized in Table 3 [30]. Regarding the GSTT1 genotype distribution, 16 (0.13) out of 128 study subjects were predicted to carry the null genotype. We found no significant difference in SULT1A1*2 allelic and genotype distribution in our population compared to the one reported for other Europeans (Supplementary Table 2; references 53-57). Our findings regarding allelic and genotypic frequencies for UGT1A1*28 genetic variant are comparable to those observed in the Framingham Heart Study that comprises subjects of almost entirely European descent from England, Ireland, France and Italy, and to the variant allele frequency reported for the Turkish population (Supplementary Table 2; references 58-63). The results concerning the population frequency of GSTT1 null genotypes corroborate the regional frequency gradients of the GSTT1 null allele that exist within the European populations (Supplementary Table 2; references 18, 64-70). Drug Target Genetic Variants (MTHFR, TYMS-TSER, VKORC1). Polymorphisms in drug targets are also an important area for pharmacogenetic studies, since an over-expression or under-expression of drug targets could also lead to resistance or toxicity in standard chemotherapy regimens. Two polymorphisms of folate cycle enzymes, methylenetetrahydrofolate reductase (MTHFR) C677T and thymidylate synthase (TYMS) enhancer region (TSER) 28 bp tandem repeat, are the two most important intracellular targets for several chemotherapeutic drugs, including antifolates (metotrexate) and fluoropirimidines (5FU, capecitabine), respectively [31]. The most common MTHFR 677C>T variant is associated with a decreased enzyme activity and increased metotrexate intolerance. The polymorphism of a 28 bp sequence in the 5 promoter enhancer region of the TS gene (TSER) varies from two (TSER*2) to nine (TSER*9) copies of the tandem repeat, with TSER*2 and TSER*3 being the most common alleles. The second SNP within the second repeat of TSER*3 (TSER*3RG>C), upregulates the TS expression and is associated with resistance to TS-targeted chemotherapy. The MTHFR C677T polymorphism shows wide regional and ethnic variations among the European population (23.0- 41.0%), Asian countries (40.0-44.0%) and Africans (12.0, 6.0%) [31,32]. Inter-population differences have also been documented for TYMS-TSER genetic variants; the frequencies reported for TSER*3 allele within the Asian, indigenous European and African populations were 0.81, 0.60 and 0.52, respectively [32,33]. The VKORC1 gene encodes for the enzyme vitamin K epoxide reductase that is the pharmacodynamic target of coumarinic oral anticoagulants. The VKORC1 (1639 G>A) variant, the one SNP included in the International Warfarin Pharmacogenetics Consortium (IWPC) dosing algorithm, has been shown to confer a high risk of bleeding, predisposing the individuals carrying these polymorphisms to hemorrhagic incidents. In general, the A allele has an increasing frequency from Western Europe (40.0%) towards Eastern Asia (90.0%) [34]. Distribution of the Drug Target Genetic Variants in the Population of the Republic of Macedonia. The allelic frequencies and the genotype distributions of the MTHFR 677C>T [35], TYMS-TSER *2/*3 (2R/3R; 3C/3G) [36] and VKORC1 1639G>A genetic variants in our population are presented in Table 4. Both MTHFR C677T and TYMS-TSER genetic variants show wide regional and ethnic variations among the European population (Supplementary Table 2; references 71-90). Significantly higher frequencies of the MTHFR 677T allele was observed when compared to the frequencies reported in the populations of the Nordic countries, Turkey and Croatia. There are few population studies of the TYMS-TSER locus in the literature and the frequencies reported for the TSER*3 allele correspond to that found in our population (Supplementary Table 2; references 80-90). The prevalence of VKORC1 1639G>A in the population of the Republic of Macedonia is in accordance with the values reported for other European populations (Supplementary Table 2; references 42,91-94).



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