Genetic Polymorphism of Cytochrome p450 (2C9) Enzyme in Iranian Baluch Ethnic Group

Genetic Polymorphism of Cytochrome p450 (2C9) Enzyme in Iranian Baluch Ethnic Group

The Open Biochemistry Journal 09 Jun 2015 RESEARCH ARTICLE DOI: 10.2174/1874091X20150609E001


The aim of the present study is to assess and compare the frequencies of the cytochrome P450 CYP2C9 variations in the Baluch ethnic group (n=110) with other ethnic groups. The allele frequencies of CYP2C9*1, CYP2C9*2 and CYP2C9*3 were 80.90%, 11.82% and 7.27%, respectively. 70.90%, 11.82%, 8.18%, 4.55%, 2.73% and 1.82% of subjects were with CYP2C9*1/*1, CYP2C9*1/*2, CYP2C9*1/*3, CYP2C9*2/*2, CYP2C9*2/*3 and CYP2C9*3/*3 genotypes, respectively. Different mutants may effect on prediction of drug dose requirements in different ethnic groups. Thus, CYP2C9 variants to be determined for findings high risk groups use optimal dosage of drugs metabolized by this polymorphic enzyme.

Keywords: Baluch ethnic group, CYP2C9 genetic polymorphism, polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP).


The cytochrome P-450 (CYP) is responsible for the metabolism of many drugs, such as warfarin, phenytoin, losartan, irbesartan, tolbutamide, glipizide, torsemide, and different nonsteroidal anti-inflammatory drugs [1]. Various single nucleotide genetic polymorphisms for CYP2C9 have been recognized [2, 3]. There are 3 alleles, (CYP2C9*1 (wild-type allele), *2 and *3) in most different ethnic groups. People with the *2 and *3 alleles show decreased enzymatic activity, and these subjects have been indicated as “poor metabolizers” [4, 5] that they may tolerate drug toxicity. Therefore, it is necessary to determine CYP2C9 genotype in different ethnic groups to prevent drug toxicity. Study on the genotype distribution among Caucasian populations varies for the *1/*1, *1/*2 *1/*3*, 2/*2, *2/*3 and*3/*3 genotypes [2]. Caucasians populations have shown more allele frequencies of CYP2C9*2 and CYP2C9*3 when compared to African-American and Asian populations while Chinese and Japanese have not indicated the CYP2C9*2 allele [6]. There has not been any investigation on the genotype of CYP2C9 allelic variants in Baluch ethnic group in Iran. Different studies have shown that there are variation in the distribution of polymorphic alleles of CYP2C9 between Iranian ethnic groups in North and South of Iran. These studies have indicated that the frequency of CYP2C9*3 among southern Iranians (9.8%) and Caucasians (9.7%) were similar. The frequency of CYP2C9*3 among Africans (1%), Japanese (2.3%), and northern Iranians (0%) were lower than southern Iranians [7]. Study of Azarpira et al. also showed that the frequency of CYP2C9*2 (25.3%) was higher than Caucasian (10%–13%), African (2%), and Asian (0%) populations [7]. The aim of the study was to assess and compare the frequencies of the cytochrome P450 CYP2C9 alleles and genotypes in the Baluch ethnic group with the frequencies in other different ethnic groups.


The present study contained 110 unrelated healthy Baluch people (who speak Baluch as a native language and population inbreeding people) referred to Health Center in Chabahar, in Sistan and Baluchestan province (located in South East of Iran). The age ranges of Baluch ethnic group was 14-53 years old. _The mean ages of subjects were 30/76 ±11/63 years old. A five milliliters venous blood was collected into EDTA tubes. DNA extraction from peripheral white blood cells was carried out by the method of salting out [8]. Sterilized distilled water was used to dissolve DNA extract and samples were stored in -20 oC until analyzed by polymerase chain reaction (PCR). Genotyping of CYP2C9 alleles was done by Polymerase Chain Reaction (PCR)-Restriction Fragment Length Polymorphism (RFLP) technique [9]. The PCR was performed in a 25 microliter reaction mixture containing PCR buffer (2.5 μl (10 mM Tris–HCl, pH 9), 2 μl (40 Mm) MgCl2 (Fermentas), 50 mM KCl (Fermentas), 12.5 mM deoxyribonucleotide triphosphate (dNTP) mix, 0.2 μl (5 U/μl) Taq polymerase (Fermentas), 1 μl (0.4 μM) of each primer (Bioneer), 2 μl (100 ng/μl) DNA (Genomic) and sterile distillated water). Genetix CG palm-thermocycler (India) was used to perform PCR. Restriction enzymes (Fermentas) were utilized to digest PCR products (10μl). Restriction enzymes, Ava II and Kpn I were used for CYP2C9*2 and CYP2C9*3 at 37C for 16 hrs for complete digestion. Amplification of primers was done by De Morais et al. method [10]. Electrophorese is of the DNA fragments (Apelex, France) was performed on a 3% agarose gel and Ethidium bromide was utilized to stain the gel. Detected Bands were photographed using a Polaroid Gel Camera with black and white film (not shown). For detection of the CYP2C9*2 mutation, sense primer (5'-CACTGGCTGAAAGAGCTAACAGAG-3' and antisense primer (5'-GTGATATGGAGTAGGGTCACCCAC-3') were used. The CYP2C9*3 mutation detection was done using sense primer 5'-TGCACGAGGTCCAGAGGTAC-3'and antisense primer 5'-ACAAACTTACCTTGGGAATGAGA-3'. The conditions of PCR amplification were as follows: For CYP2C9*2: Initial denaturation, Number of cycle(s), Denaturation, annealing, Extention and final extention step were 95oC, 10 min.; 40; 95oC, 5 sec.; 65 oC, 30 sec.; 72oC, 30 sec. and 72oC, 5 min. and for CYP2C9*3: 94C, 5 min.; 30; 94 oC, 45 sec.; 63 oC, 45 sec.; 72 oC, 30 sec. and 72oC, 5 min., respectively. 95% confidence intervals (95% CI) were performed to determine the frequency of the variant alleles of each gene. Fisher exact test was used to evaluate variations in allele and genotype frequencies between Baluch ethnic groups and different other population.


The distribution of genotype and allelic frequencies of CYP2C9 among Baluch ethnic group are shown in Table 1. The allele frequency of CYP2C9*1 (Wild type), CYP2C9*2 and CYP2C9*3 were 80.90% (95% CI: 73.55-88.24), 11.82% (95% CI: 5.78-17.82) and 7.27% (95% CI: 2.44-12.16), respectively (Table 1). The frequencies of CYP2C9 genotypes in Baluch ethnic group were found to be 70.90% of subjects with CYP2C9*1/*1 genotype (95% CI: 62.41-79.38). 11.82%, 8.18%, 4.55%, 2.73 and 1.82 subjects with CYP2C9*1/*2 (95% CI: 5.78-17.84), CYP2C9*1/*3(95% CI: 3.06-13.30), CYP2C9*2/*2(95% CI: 0.65-8.43), CYP2C9*2/*3 (95% CI: 0-5.77) and CYP2C9*3/*3(95% CI: 0-4.30) genotypes, respectively (Table 1). Tables 2 and 3 show the distribution of CYP2C9 genotype and allele frequency in Baluch ethnic group and in different populations.

Table 1.

Genotype and Allelic frequencies of CYP2C9 in the Baluch ethnic group.

CYP2C9 Genotypes Number (%) 95% CI CYP2C9 Alleles Number (%) 95% CI
*1/*1 78(70.90) 62.41-79.38 *1 89(80.90) 73.55-88.24
*1/*2 13(11.82) 5.78-17.84 *2 13(11.82) 5.78-17.82
*1/*3 9(8.18) 3.06-13.30 *3 8(7.27) 2.44-12.16
*2/*2 5(4.55) 0.65-8.43
*2/*3 3(2.73) 0-5.77
*3/*3 2(1.82) 0-4.30
Total 110 (100)

*Means separation of effective alleles.

Table 2.

Comparison of allele frequencies of CYP2C9 in Baluch ethnic group with different populations.

Population Study Sample Size (n) Allele Frequency of *1(%) Allele Frequency of *2(%) Allele Frequency of *3(%) References
Iranian Baluch 110 80.90 11.82 7.27 Present study
Iranian Fars 140 83 11(NS) 6(NS) [16]
Iranian Turkmen 140 88 8(NS) 4(NS) [16]
Italian 360 77.7 12.5(NS) 9.7(NS) [17]
Greek 283 79 12.8(NS) 8.1(NS) [18]
Russians 290 82.7 10.5(NS) 6.7 (NS) [19]
Swedish 430 81.9 10.6(NS) 7.4(NS) [20]
Slovenia 129 81.7 12(NS) 6.2(NS) [21]
UK 561 84.1 10.6(NS) 5.2 (NS) [22]
Egyptians 247 81.7 11.8(NS) 6.2(NS) [23]
Japan 828 97.6 0(0.001) 2.3(0.004) [24]
African 47 9 2(0.004) 1 (<0.0001) [25]
Pakistan 120 91.6 0.8(0.001) 7.5(NS) [26]
Tamilians 135 90.7 2.6(0.0045) 6.7(NS) [27]
Kerala 120 90 2.0(0.0011) 8.0 (NS) [28]
Chinese 115 98.3 0(0.001) 1.7(<0.0001) [29]
Korean 574 98.9 0 (0.001) 1.1(<0.0001) [30]

Differences in the allele frequencies were determined by Fisher exact test. NS: No significant differences.

P-value versus Baluch ethnic group.

*Means separation of effective alleles.

Table 3.

Comparison of CYP2C9 genotype frequency in Baluch ethnic group with different populations.

Population Study Sample Size
Frequency of *1/*1(%)
Frequency of *1/*2(%)
Frequency of *1/*3(%)
Frequency of *2/*2(%)
Genotype Frequency of *2/*3(%) Genotype Frequency of *3/*3(%) References
Iranian Baluch 110 70.90 11.82 8.18 4.55 2.73 1.82 Present study
Iranian Fars 140 70(NS) 14.55(NS) 10.91(NS) 2.73(0.011) 1.82(NS) 0(0.001) [16]
Iranian Turkmen 140 37.9(<0.0001) 42.1(<0.0001) 9.3(NS) 9.3(<0.0001) 0 1.4(NS) [16]
Italian 360 62(NS) 17.2(NS) 14.5(0.014) 2.7(0.011) 2.2(NS) 1.3(NS) [17]
Greek 283 62(NS) 20(NS) 13.5(0.014) 1.5(0.001) 2.8(NS) 0(0.001) [18]
Russians 290 68(NS) 18.2(NS) 10.3(NS) 0.6(<0.0001) 1.2(0.011) 0.3(0.0085) [19]
Swedish 430 66.7(NS) 18.6(NS) 11.6(NS) 0.4(<0.0001) 1.6(0.011) 0.6(0.0095) [20]
Slovenia 129 86.6(<0.0016) 19.3(NS) 10.8(NS) 1.5(0.001) 1.5(0.011) 0(0.001) [21]
UK 561 69.9(NS) 19(NS) 0.06(<0.0001) 0.003(<0.0001) 0(<0.0001) 0(0.001) [22]
Egyptians 247 66.3(NS) 19(NS) 12(NS) 2.4(0.011) 0(<0.0001) 0.4(0.0088) [23]
Japan 828 95(<0.0001) 0(<0.0001) 4(<0.001) 0(<0.0001) 0(<0.0001) 1(NS) [24]
African 47 93.6(<0.0001) 4.2(0.014) 2.1(<0.001) 0(<0.0001) 0(<0.0001) 0(0.001) [25]
Pakistan 120 85.8(<0.0011) 0(<0.0001) 11.7(NS) 0.8(<0.0001) 0(<0.0001) 1.7(NS) [26]
Tamilians 135 82.3(NS) 4.4(0.017) 12.7(NS) 0(<0.0001) 0.7(<0.0001) 0(0.001) [27]
Kerala 120 81.0(NS) 4.0(0.011) 14.0(0.014) 0(<0.0001) 0(<0.0001) 1.0(NS) [28]
Chinese 115 97.0(<0.0001) 0(<0.0001) 3.0(<0.001) 0(<0.0001) 0(<0.0001) 0(0.001) [29]
Korean 574 97.7(<0.0001) 0(<0.0001) 2.3(<0.001) 0(<0.0001) 0(<0.0001) 0(0.001) [30]

Differences in the genotype frequencies were determined by Fisher exact test. NS: No significant differences. P-value versus Baluch ethnic group.

*Means separation of effective alleles.


Variations in CYP expression among different ethnic groups make it important in drug response, activity and detoxification. It is been shown that there are three major variants of the CYP2C9 gene in different ethnic groups. It is reported that there are three major variants of the CYP2C9 gene in Caucasian populations [11]. CYP2C9*2 and CYP2C9*3 allelic variants show almost 10–40% and 5–15% of the activity of CYP2C9*1 for various drug, respectively [12-15]. The allelic frequency of CYP2C9*2 found in the present study was 11.82% (almost similar to the Iranian Fars) [16] which was higher than Japanese [24], African [25], Pakistanis [26], Tamilians [27], Keralans [28], Chinese [29] and Korean [30] populations (Table 2). Our results show that the CYP2C9*2 variant was more frequent than above mentioned populations. In our study group, the allelic frequency of CYP2C9*3 was 7.27%. The frequency of CYP2C9*3 was almost similar to the frequencies found in Italians [17], Greeks [18], Swedish [20], Pakistanis [26] and Keralans [28] (Table 2). Genetic differences in the CYP2C9 make it possible that some drugs such as warfarin metabolize differently. Single-nucleotide polymorphisms in CYP2C9 influence metabolism of this drug. Clinicians can appraise optimal warfarin dose by genotyping subjects for single-nucleotide polymorphisms. Subjects who have the CYP2C9*2 and/or CYP2C9*3 variants metabolize warfarin more in a different manner than subjects without these variants. It has been shown that CYP2C9 genotype estimates warfarin optimal dosage in subjects on warfarin treatment [30]. Distribution of CYP2C9 genotypes in Baluch and different ethnic groups has been shown in Table 3. Our genotype results have shown that frequency of *1/*2 and *1/*3 was 11.82% and 8.18% which was higher [24-30] and lower [16-21, 23 and 26-28] than other populations, respectively. The prevalence of CYP2C9*2/*2 (except Iranian Turkmen [16]), *2/*3 and *3/*3 genotypes in Iranian Baluch ethnic group were higher when compared with different other ethnic groups [16-30]. Many studies have indicated that*2/*2 [24-29, 6], *2/*3 [16, 22-29 and 6] and *3/*3 [16, 18, 21, 22, 25, 27 and 29, 6] genotype frequency was not detectable in some populations. CYP2C9*3/*3 genotype is contributed with some important clinical variations in the pharmacokinetics of CYP2C9 substrates.


In conclusion, our results show that there are ethnic variations in the CYP2C9 allele and genotype frequencies. Genetic differences in the CYP2C9 were seen in Iranian Baluch. The presence of different mutant genotypes and alleles may effect on prediction of drug dose requirements in different ethnic groups. Thus, it requires that CYP2C9 variants determination in different populations may help high risk groups to use optimal dosage of drugs metabolized by this polymorphic enzyme.


The authors confirm that this article content has no conflict of interest.


Declared none.


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