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Aims The goal of this study was to determine the frequencies of important allelic variants of CYP2C9, CYP2C19, CYP2E1 and DPYD in the Egyptian population and compare them with the frequencies in other ethnic populations.
Methods Genotyping of CYP2C9 (*2 and *3), CYP2C19 (*2 and *3), c2 variant of CYP2E1 and DPYD alleles (*2 A-*6 ) was carried out in a total of 247 unrelated Egyptian subjects. An allele-specific fluorogenic 5′ nuclease chain reaction assay was applied for detection of CYP2C9 and CYP2C19 variants. Other variants of the CYP2E1 and DPYD genes were determined using polymerase chain reaction (PCR)-restriction fragment length polymorphism and allele-specific PCR based assays.
Results CYP2C9 allele frequencies in 247 Egyptian subjects were 0.820 for CYP2C9*1, 0.120 for CYP2C9*2 and 0.060 for CYP2C9*3. For CYP2C19, the frequencies of the wild type (CYP2C19*1) and the nonfunctional (*2 and *3) alleles were 0.888, 0.110 and 0.002, respectively. CYP2C19*3, which is considered an Asian mutation, was detected in one subject (0.40%) who was heterozygous (*1/*3). Two subjects (0.80%) were homozygous for *2/*2, while no compound heterozygotes (*2/*3) or homozygotes for *3 were detected. For CYP2E1, only four subjects (1.70%) had the rare c2 variant, expressed heterozygously, giving an allele frequency of 0.009. Five variants of DPYD were analysed, with no splice sites (*2 A) or ΔC1897 (*3) found in this population. The frequencies of other variants were 0.028, 0.115 and 0.090 for *4, *5 and *6, respectively.
Conclusions Comparing our data with that obtained in several Caucasian, African-American and Asian populations, we found that Egyptians resemble Caucasians with regard to allelic frequencies of the tested variants of CYP2C9, CYP2C19, CYP2E1 and DPYD. Our results may help in better understanding the molecular basis underlying ethnic differences in drug response, and contribute to improved individualization of drug therapy in the Egyptian population.
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The cytochrome P450 enzymes (CYP) play a central role in the metabolism of many therapeutic agents. Differences in the activities of these enzymes are thought to be responsible for individual variability in drug response and/or toxicity. Among the CYP enzymes many isoforms exhibit genetic polymorphism, examples include 2C9, 2C19 and 2E1.
CYP2C9 catalyses the oxidation of clinically important drugs including phenytoin, tolbutamide, warfarin and a large number of nonsteroidal anti-inflammatory drugs . Previous studies have shown that changes in amino acid composition of this enzyme can affect both its activity and substrate specificity, and thereby produce individual variability in the elimination and/or dosage requirements of prototypic CYP2C9 substrates [2, 3]. Eleven CYP2C9 variant alleles have been reported to date ; however, only two amino acids substitutions, CYP2C9*2 (Arg144 to Cys) and CYP2C9*3 (Ile359 to Leu), are recognized in humans as main CYP2C9 variants [5, 6]. CYP2C9*2 and CYP2C9*3 have reduced catalytic activity compared with wild type (CYP2C9*1) [7, 8].
CYP2C19 metabolizes several therapeutically important drugs, namely omeprazole, lansoprozole, propranolol, imipramine, mephenytoin, chloroguanide, hexabarbitone and diazepam . Carriers of two CYP2C19 non-functional alleles have a severely impaired capacity to metabolize drugs that are substrates for this enzyme and are hence designated poor metabolizers (PMs). Nine CYP2C19 variant alleles have been reported to date . Among these variants, two alleles with separate mutations have been associated with the defective enzyme in Caucasian and Asian populations. The first is CYP2C19*2, which has a mutation in exon 5 causing an aberrant splice site. The other variant allele is CYP2C19*3 with a point mutation in exon 4 producing a premature stop codon. The most commonly mutated allele is CYP2C19*2 in both Asians and Caucasian PMs . The second discovered mutation, CYP2C19*3, is rare among Caucasian subjects but accounts for the remaining defective alleles in Asian subjects .
Human CYP2E1 is involved in the oxidation of drugs such as ethanol, chlorzoxazone, paracetamol and fluorinated anaesthetics . It can be induced by ethanol, and studies have shown an approximately 50-fold individual variability in its expression [14, 15]. In addition, the c2 mutant allele in the regulatory 5′ flanking region has been associated with higher transcriptional activity .
For CYP2C9, CYP2C19 and CYP2E1 the prevalence of non-functional alleles varies substantially in populations of different racial origin. While the frequencies of mutant alleles of these clinically important enzymes have been studied extensively in all major human races, only limited information has been available for the Egyptian population .
Aside from the importance of the CYP system in the biotransformation of drugs, the dihydropyrimidine dehydrogenase (DPD) enzyme is of equal importance, especially in cancer chemotherapy. DPD is the initial and rate-limiting enzyme in the metabolic pathway for the catabolism of the pyrimidine bases uracil and thymine . It is also the key enzyme that degrades the structurally related pyrimidine antimetabolite 5-FU. Patients with low DPD activity are characterized by a decreased elimination of 5-FU from plasma associated with severe haematological, gastrointestinal and neurological toxicity, and often death . Among Caucasian populations DPD activity is highly variable and subject to polymorphic regulation of the DPYD gene . Few studies on the frequency of DPD deficiency in general populations have been performed, so the population distribution of the variant alleles and their contribution to reduced DPD activity in vivo is not well characterized . Moreover, no data has been reported regarding the frequency of the DPYD alleles in the Egyptian population.
We report here the allele frequencies of CYP2C9 (*2 and *3), CYP2C19 (*2 and *3), the c2 variant of CYP2E1, and DPYD allelic variants (*2 A, *3-*6), in a sufficiently large sample of the Egyptian population (247 subjects). To our knowledge it is the largest genotyping study of the Egyptian population reported to date.
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Egypt is unique geographically, as it is located centrally to the three continents of Africa, Europe and Asia. Throughout history, the Greeks, Romans, Arabs, Turks, French and British have all ruled Egypt and mixed with its people, such that modern Egypt now is an amalgam of all these legacies. Because of the clear heterogeneity and genetic admixture in the Egyptian population, we considered it especially important to investigate the allele frequencies and genotype distributions of some variants of a pharmacogenetic interest in this population. This study was carried out in 247 unrelated Egyptian subjects, and is among the first to describe many allelic variants in this population.
The frequency of the CYP2C9*2 allele in the Egyptian population was 0.120, which is in a range comparable with other Caucasian populations; 0.080 in American , 0.125 in British , 0.107 in Swedish  and 0.106 in Turkish . In the study of Sullivan-Klose et al., it was reported that the CYP2C9*2 allele occurs at a significantly lower frequency in the African-American population (0.010). On the other hand, the CYP2C9*2 allele was reported to be absent or at least very rare in the East Asian populations [6, 26, 27] (P values are listed in Table 2).
The CYP2C9*3 allele occurred with a frequency of 0.060 in the Egyptian subjects. This finding is similar to that of other Caucasian populations in which the frequency of the CYP2C9*3 allele was reported to be 0.060 in American , 0.085 in British , 0.074 in Swedish  and 0.100 in Turkish . Lower frequencies were reported for the CYP2C9*3 variant in East Asian populations (0.011–0.021) in Japanese , Korean  and Chinese-Taiwanese . The lowest frequency of CYP2C9*3 was reported in African-Americans (0.005)  (P values are listed in Table 2).
The catalytic activity of the CYP2C9*3 encoded enzyme is much lower than those of CYP2C9*1 and *2. Moreover, the clearance of orally ingested S-warfarin in vivo was reduced by 66% and 90% among subjects heterozygous and homozygous for the CYP2C9*3 allele, respectively, compared with homozygous CYP2C9*1 subjects . In our study only one subject was homozygous for *3/*3 (0.40%), which is in a range comparable with British (0.10%) , Swedish (0.70%)  and Turkish (0.80%) . On the other hand, no subjects homozygous for CYP2C9*3 were identified in Eastern populations [6, 26, 27].
The genetic polymorphism of CYP2C19 has been shown to have the most striking interethnic variation of a CYP so far. The PM frequency ranges from 2 to 7% in Caucasians, 14–25% in Asians, and 60% in the Vanuatu [30, 31]. It has been shown that a higher concentration of omeprazole in PMs results in great gastric acid suppression as compared with extensive metabolizers (EMs) . Cure rates for Helicobacter pylori in patients receiving omeprazole and amoxicillin were found to be 28% in homozygous EMs (CYP2C19*1/*1), 60% in heterozygous EMs (CYP2C19*1/*2 and *1/*3), and 100% in PMs (CYP2C19*2/*2 and *2/*3), indicating the importance of dose adjustment in the case of EMs .
CYP2C19 genotyping was conducted in 1356 Caucasian individuals throughout the world . In the present study, we found that the incidence of CYP2C19*2 in Egyptians (0.110) was similar to that found in other Caucasian populations (0.110–0.161) residing in America , Canada , Europe [37–40], Australia  and Saudi Arabia . Higher frequencies of CYP2C19*2 were reported for Asian populations (0.209–0.390) [35, 42], North Indians (0.300) , Africans (0.131–0.217) [42, 44, 45] and African Americans (0.250)  (P values are listed in Table 3).
On the other hand, the discovered presence of CYP2C19*3 in one Egyptian subject (0.002) was an interesting finding because limited numbers of studies have reported CYP2C19*3 in populations other than the Asians. CYP2C19*3 was reported in the Swedish population (0.003) , (0.007) , the Bantu-Tanzanian (0.006)  and the Ethiopian (0.018)  (P values are listed in Table 3). CYP2C19*3 has been regarded as an Asian mutation and accounted for the remaining alleles in Asian PMs after genotyping for CYP2C19*2. An explanation for our finding of CYP2C19*3 in the Egyptian subjects may be the emigration and possible genetic admixture between the Egyptians and Asian populations.
Significant interethnic difference exists in the CYP2E1 polymorphism among different populations [23, 46]. The frequency of the mutant c2 allele is about 20% in Japanese, while it is present only in 2–4% of Caucasians and 1–2% of African-Americans . The frequency of the c2 allele was previously detected in 42 Egyptian subjects (0.010) by Anwar et al.. In the present study we obseved a frequency of 0.009 of the c2 allele by analysing a larger number of Egyptian subjects confirming the rare occurrence of this mutant allele in the Egyptian population. This rare occurrence may prove that any influence of the CYP2E1 gene on diseases such as cancer or alcoholic liver disease, or on hepatotoxicity following the use of halogenated anaesthetics is likely to be minimal in this population.
Polymorphic drug metabolizing enzymes often have a limited number of variant genotypes that account for most individuals with reduced or deficient phenotypes. In the case of DPD, although 13 variant DPYD alleles have been identified to date in DPD-deficient subjects, the majority of these alleles are not associated with the DPD deficiency . However, because of the severity of 5-FU induced toxicity, it would be advantageous to prescreen cancer patients for levels of DPD activity and/or DPYD genotype before 5-FU therapy and modify the dose appropriately. The present study was the first aimed to detect the frequencies of five DPYD variants in the Egyptian population and compare them with those of other ethnic populations.
No exon 14 splice site mutations (DPYD*2 A) were found among the 239 subjects studied. This allele was not found among 60 British , 50 Japanese, 131 Taiwanese, or 105 African-Americans , but had a 0.011 frequency in the Finnish population . Previous studies [47–49] demonstrated that intron 14 G1A (DPYD*2 A) results in the production of a truncated mRNA, and has been consistently associated with low DPD activity and 5-FU toxicity. However, one patient with a heterozygous intron 14 G1A mutation had normal DPD activity . This fact was explained by the possible allelic regulation of the DPYD gene that may occur to allow a normal DPD phenotype through increased expression of the wild-type allele .
No subject with ΔC1897 (DPYD*3) was detected. This allele results in premature termination of translation prior to the uracil binding site, and no DPD activity was detectable in a homozygous mutant individual . However, other studies suggested that ΔC1897 is a relatively rare allele in Caucasian populations [21, 24].
The presence of a G to A transition at codon 534 (DPYD*4) was detected with a frequency of 0.028. This is similar to that found in Finnish (0.033) , British (0.008) , Japanese (0.011) and African-Americans (0.005) . In the study of Ridge et al., one individual with a codon 534 mutation had the lowest DPD activity, whereas two previously described colorectal cancer patients with codon 534 mutations had DPD activities greater than the average value , making the impact of this mutation unclear.
Mutations at codon 543 (DPYD*5) were found with a frequency of 0.115, also similar to that found in the Finnish subjects (0.072) . However, this allele was more common in British (0.280) , Japanese (0.350), Taiwanese (0.210) and African-Americans (0.227)  (P values are listed in Table 4). This mutation was associated with a wide range of mononuclear cell DPD activity [22, 51], suggesting that this mutation is a common polymorphism, and alone is not associated with impaired DPD activity.
Mutations at codon 732 (DPYD*6) were also a relatively common finding (0.090). This is similar to that found in Finnish (0.067) , British (0.058)  and Japanese (0.014)  but is more frequent than that found in Taiwanese (0.014) and African-Americans (0.019)  (P values are listed in Table 4). This mutation was also found in individuals with a wide range of DPD activity [21, 24], confirming that it is not a functional mutation.
In the present study, 15 subjects (6.7%) had more than one mutation in the DPYD gene. In the study of Ridge et al., the three individuals having more than one mutation in the DPYD gene had DPD activity values lower than 10% of the population, suggesting that accumulation of DPYD mutations may contribute to low activity phenotype of the enzyme.
In conclusion, the tested allelic variants of CYP2C9, CYP2C19, CYP2E1 and DPYD existed in Egyptian people with frequencies comparable to other Caucasian populations with some differences among Asians and African Americans. It is hoped that our results will aid in understanding the ethnic diversity of the Egyptian population, and offer a preliminary basis for more rational use of drugs that are substrates for CYP2C9, CYP2C19, CYP2E1 and DPD in this population.