1. Introduction
Personalized medicine addresses the terms covered by targeted therapies, related diagnostics, and the medical intervention’s safety [Reference De Andrés, Teran, Hernandez, Teran and Llerena1]. Following the evolution of the Human Genome Project, the association between personalized medicine and genes encoding drug-metabolizing proteins became more outstanding [Reference Chen and Wei2]. Amongst all those proteins, Cytochrome P450, known as CYP, is responsible for the metabolization of roughly 90% of currently prescribed medications, apart from its intrinsic activities [Reference Chen, Zhang, Wang and Wei3].
Cytochrome P450 enzymes play a crucial role in the metabolism of different agents, including food and drugs, with various enzymatic activities leading to various drug responses among individuals [Reference Hamdy, Hiratsuka, Narahara, El-Enany, Moursi, Ahmed and Mizugaki4]. Many highly polymorphic genes encode cytochrome P450, and several Single Nucleotide Polymorphisms (SNPs) have been identified in each gene [Reference Zhou5]. SNPs may result in various enzymatic activities in people, eventually leading to insufficient efficacy of medical regimens or their adverse effects in a constant amount of drugs [Reference Lauschke and Ingelman-Sundberg6].
Ethnicity is among those factors influencing the SNP frequencies and various drug responses [Reference Helgadottir, Manolescu and Helgason7]. Although there are worldwide databases providing the frequency of multiple SNPs, they still need some data on specific ethnicities. Based on the latest studies, Iran’s population was calculated at 83.9 million in 2020 and accounted for the 18th most populated country in the world [8]. Based on available data, 65% of the Iranian population are Fars, 16% Turk, 7% Kurd, 6% Lor, and 6% other ethnicities. In other words, Fars, Turk, and Kurd together account for 88% of the Iranian population [Reference Amar9].
CYP2B6 belongs to the Cytochromes P450 (CYPs) superfamily of enzymes that are essential for the clearance of various compounds, as well as for hormone synthesis [Reference Rotondo, Giari and Guerranti10, Reference McDonnell and Dang11]. CYP2B6 participates in the metabolism of drugs, including many antibiotics, antiretroviral drugs, antimalarial, and first-lineanti-Tuberculosis drugs [Reference Simonsson, Jansson and Hai12]. CYP2B6 is polymorphic and contains significant interindividual variability in the human population [Reference Ward, Gorski, Jones, Hall, Flockhart and Desta13]. Hence, the analysis of CYP2B6 SNPs may improve drug efficacy and also decrease medical regimens’ adverse effects.
Based on Lang et. al, CYP2B6 primarily consisted of 9 SNPs in 6 allelic variations; CYP2B6 c.64C > T as ∗2, CYP2B6 c.777C > A as ∗3, CYP2B6 c.785A > G as ∗4, CYP2B6 c.1459C > T as ∗5, CYP2B6 c.516G > T and c.785A > G as ∗6, and CYP2B6 c.516G > T and c.785A > G and c.1459C > T as ∗7 [Reference Lang, Klein and Fischer14]. To date, the last CYP2B6 constellation is named ∗38 [Reference Anagnostopoulos, Rotger and Aouri15].
Studies on the CYP2B6 isozyme have expanded significantly since 2003, at the same time as the discovery of this isozyme effect in the clearance of the antiviral drug, especially efavirenz [Reference Ward, Gorski, Jones, Hall, Flockhart and Desta13]. Besides, in another study conducted in 2003, the clearance of bupropion drug in people with the CYP2B6∗4 variant was 1.6 times more than in patients of other variants [Reference Kirchheiner, Klein and Meineke16]. A higher concentration of anti-HIV drugs, including efavirenz, and its toxicity in CYP2B6∗2 [Reference Usami, Ashino, Komaki and Hattori17] have been reported.
Various drugs are metabolized by CYP2B6 [Reference Yamada, Ishii, Yamamoto and Oguri18, Reference Hodgson and Rose19]; among them, two drugs that are more specifically metabolized by a CYP2B6 isoenzyme are methadone and bupropion, and these two drugs are even utilized as probes to detect the function and investigate the behavior of the CYP2B6 enzyme [Reference Kharasch, Mitchell and Coles20, Reference Eap, Crettol and Rougier21]. Reports show that Iran has a considerable rate of opium use, especially methadone [Reference Rostam-Abadi, Gholami and Noroozi22]. To the extent of our knowledge, CYP2B6∗2 and ∗3 have not been evaluated in Iranian populations, except for Southern Iran [Reference Zakeri, Amiri, Pirahmadi and Dinparast Djadid23], which is previously reported to have a distinguished genetic pool from other Iranian populations [Reference Mehrjoo, Fattahi and Beheshtian24].
In this study, due to the scarcity of data on the distribution of CYP2B6 c.64C > T (rs8192709) and CYP2B6 c.777C > A (rs45482602) in the Iranian population, we aimed to evaluate the mentioned polymorphism in three dominant Iranian ethnicities, Fars, Turk, and Kurd.
2. Methods and Materials
2.1. Samples Collection and Ethical Consideration
One hundred and seventy-four samples from unrelated healthy donors, aged 18–60, of three ethnicities of the Iranian population from various provinces, were achieved. Patients with a history of cancer, metabolic disorders, and any disease affecting DNA were excluded from the study. Of this number, 65 (37.4%) cases were males and 109 (62.6%) were females. The average age of cases was 40.69 ± 11.87 (mean ± SD) and 39.06 ± 11.63 (mean ± SD) for males and females, respectively. Two milliliters of blood samples encompassing 80 Fars, 69 Turk, and 25 Kurd were collected in ethylenediaminetetraacetic acid (EDTA) enriched tubes. An informed written consent form was obtained from each person to permit genetic analysis and publication. Besides, the Medical ethics committee has approved the study.
2.2. DNA Extraction and Primer Sequencing
DNA extraction was performed using an MBST kit (salting-out method) in two milliliters of blood samples. For DNA isolation, lysis buffer for the digestion of non-nucleic acid components of the cell, precipitation buffer for protein isolation, washing buffer, and eventually elusion buffer for DNA resuspension were utilized. Therefore, we utilized Oligonucleotide F (forward) and R (reverse) primers for CYP2B6 ∗2 and ∗3 amplification, as shown in Table 1.
TABLE 1: Oligonucleotide sequences of primers in real-time, rapid-cycle PCR.
2.3. ARMS and Tetra ARMS-PCR
Genotyping analysis was conducted utilizing an amplification refractory mutation system polymerase chain reaction (ARMS PCR) for CYP2B6∗3 and tetra ARMS PCR for CYP2B6∗2. The total volume of each PCR reaction was 25 microliters consisting of 0.6 μl of each F and R primers, two μl DNA template, and 11 μl EmeraldAmp PCR master mix. The PCR cycling was set at 96°C for 4 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing for 45 seconds, extension at 72°C for 45 seconds, and final extension at 72°C for 8 minutes. The 3% agarose gel was used to rub the PCR products. Eventually, visualization was performed by staining with ethidium bromide and analyzed by the gel documentation system (Figure 1).
Figure 1 (a) A heterozygous, wild type, and homozygous mutant, left to right, in rs8192709 in three cases after tetra ARMS-PCR. (b) A heterozygous, wild type, and homozygous mutant, left to right, in rs45482602 in three cases after ARMS-PCR.
2.4. Sequencing Analysis
Similar to our previous studies [Reference Dehbozorgi, Kamalidehghan and Hosseini25], forwards and reverse primers sequenced PCR products on an automated ABI 3100 sequencing machine (Applied Biosystems, Kavosh Fanavaran Kawsar Company, Iran). Then, we used the Finch TV program for sequencing and analyzing to confirm the results of nucleotide variations.
2.5. Statistical Analysis
The statistical analyses were performed using SPSS (version 24; IBM) software. A confidence interval test (95%) was considered for the frequency of alleles and genotypes. A P value of less than 0.05 was considered statistically significant [Reference Dehbozorgi, Kamalidehghan and Hosseini25].
3. Results
3.1. Demographic Distribution
In this study, 174 samples of whole blood cells were collected from three dominant Iranian ethnicities consisting of 80 (46%) Fars, 69 (39.7%) Turk, and 25 (14.4%) Kurd to investigate the allelic and phenotypic distribution of CYP2B6∗2 and ∗3. There was no significant correlation between the samples’ gender and allelic genotypes.
3.2. CYP2B6 (c.64 > T) Allelic and Genotype Frequency
CYP2B6 wild-type homozygote (C/C) was 87% (95% CI (6.7–10.7)), while mutated homozygote (T/T) and heterozygote (C/T) genotypes were 5.2% (95% CI (3.9–6.5)) and 86% (95% CI (83.2–88.8)), respectively. C/C genotype highest and lowest frequencies were observed in Kurd (12% (95% CI (10.3–13.7))) and Fars (6.3% (95% CI (5.7–6.9))), respectively. T/T genotype was highest in Turk (10.3% (95% CI (5.2–15.4))) and lowest in Kurd (0%). Furthermore, C/T genotype ranged from 91.1% (95% CI (87.5–94.7)) in Fars to 79.4% (95% CI (73.5–85.5)) in Turk. The CYP2B6∗2 (c.64 C > T) allele was 48.2% (95% CI (37.8–58.6)), ranging from 44% (95% CI (36.8–51.2)) in Kurd to 50% (95% CI (41.6–58.4)) in Turk. Tables 2 and 3 summarize the genotype and allelic frequency, respectively.
TABLE 2: Analysis of the CYP2B6∗2 allelic frequencies (%) among three dominant ethnicities in Iran; the correlation between ethnicity genotype frequency ethnicity and the each ethnicity is shown.
TABLE 3: Analysis of the CYP2B6∗2 genotypic frequencies (%) among three dominant ethnicities in Iran; the correlation between ethnicity genotype frequency ethnicity and the each ethnicity is shown.
* value of less than 0.05 is statistically significant. ** value of less than 0.01 is statistically highly significant.
3.3. CYP2B6∗3 (c.777C > A) Allelic and Genotype Frequency
CYP2B6 wild-type homozygote (C/C) was 75.3% (95% CI (70.1–80.5)), while mutated homozygote (A/A) and heterozygote (C/A) genotypes were 13.6% (95% CI (12.5–14.7)) and 11% (95% CI (8.3–13.7)), respectively. C/C genotype highest and lowest frequencies were observed in Turk (77.8% (95% CI (74.6–81))) and Kurd (72% (95% CI (65.3–78.7))), respectively. A/A genotype was highest in Fars (16% (95% CI (13.7–18.3))) and lowest in Turk (11.1% (955 CI (7.4–14.8))). Furthermore, C/A genotype ranged from 9.3% (95% CI (7.2–11.4)) in Fars to 16% (95% CI (11.8–20.2)) in Kurd. The CYP2B6∗3 (c.777C > A) allele was 19.1% (95% CI (17.5–20.7)), ranging from 16.6% (95% CI(12.8–20.4)) in Turk to 20.6 (95% CI (16.2–25)) in Fars. Tables 2 and 3 summarize the genotype and allelic frequency, respectively.
4. Discussion
CYP2B6 plays a crucial role in the metabolism of antiretroviral drugs, including efavirenz, immunosuppressant drugs, including cyclophosphamide, antidepressants, e.g., bupropion, antiepileptics, e.g., valproic acid, and antiarrhythmic, e.g., mexiletine, among other drugs, by changing to their metabolic forms [Reference Ward, Gorski, Jones, Hall, Flockhart and Desta13, Reference Chang, Weber, Crespi and Waxman26–Reference Labbe, Abolfathi, Lessard, Pakdel, Beaune and Turgeon29]. Also, it is believed that CYP2B6 expression is higher in smokers and alcoholics compared to the normal population [Reference Miksys, Lerman, Shields and Mash30].
To the extent of our knowledge, this is the first study investigating the frequency of CYP2B6∗2 and CYP2B6∗3 in the three main Iranian populations: Fars, Turk, and Kurd. In this study, CYP2B6∗2 frequency was 48.2%, ranging from 44% in Kurd to 50% in Turk ethnicities; besides, CYP2B6∗3 frequency was 19.1% on average, ranging from 16.6% in Turk to 20% in Kurd. In a survey in Japan by Hiratsuka et al., CYP2B6∗2 and CYP2B6∗3 frequencies were 4.7% and 0%, respectively; besides, the same alleles frequencies in the Caucasian population were reported at 5.3% and 0.5%, respectively [Reference Hiratsuka, Takekuma and Endo31].
In another study evaluating the CYP2B6 allelic distribution in 631 West African populations, the frequency of CYP2B6∗3 was 0%, while the frequency of CYP2B6∗2 was 2.82% [Reference Mehlotra, Ziats, Bockarie and Zimmerman32]. In a study by Musa et al., CYP2B6∗2 allelic distribution was 0.8% in Malaysian and 4.1% in Indians [Reference Musa, Musa, Talib, Mohamad and Zulkafli33]. Another study investigating the allelic frequency of CYP2B6 in ethnicity in Southern Iran, known as the Baluch population, reported a 3.9% allelic distribution of CYP2B6∗2 [Reference Zakeri, Amiri, Pirahmadi and Dinparast Djadid23]. In a study in 2021, CYP2B6∗3 allelic distribution was reported at 6.5% of the Pakistan population [Reference Ahmed, Khan, Janjua, Imran and Khan34].
As demonstrated in Tables 4 and 5, the CYP2B6∗2 and ∗3 allelic frequency is remarkably higher in the three main Iranian populations than in other ethnicities, even those in Southern Iran. It is believed that due to a considerable number of consanguineous marriages in Iran, autosomal recessive diseases highly manifest in this country; furthermore, it is said that Iranian allele frequency is noticeably different from those in Europeans, while Iranian Baluch seemed to have a tendency towards south Asians in allele frequency [Reference Zakeri, Amiri, Pirahmadi and Dinparast Djadid23, Reference Mehrjoo, Fattahi and Beheshtian24]. Besides, CYP2B is believed to have the most frequent interpersonal variety, which accounted for 20–250 times more than other Cytochrome P450 isoenzymes [Reference Wang and Tompkins40].
TABLE 4: Comparison of allele frequency of CYP2B6∗2 reported in various populations.
TABLE 5: Comparison of allele frequency of CYP2B6∗3 reported in various populations.
Our results, alongside those from other studies, provide further evidence for ethnic heterogeneity in drug metabolism. We hope our findings contribute to a better understanding of various drug responses in different populations.
5. Study Limitations
This study was conducted on 173 cases from three various major Iranian ethnicities. Further studies are needed to evaluate the allelic polymorphism in larger populations and other ethnicities.
6. Conclusion
Allelic distribution in three main Iranian ethnicities, i.e., Turk, Kurd, and Fars, is remarkably different from other populations, even those in Southern Iran. High frequencies of CYP2B6∗2 and ∗3 in the Iranian population remarkably affect drug responsiveness, including higher efavirenz toxicity. Understanding such variability could help to increase drug efficacy and reduce its toxicity.
Data Availability
We have presented all the necessary data in this manuscript. The data would be available from the corresponding author on reasonable request.
Ethical Approval
This study is approved by the Ethics Board Committee of Alborz University of Medical Sciences with the approval ID: IR.ABZUMS.REC.1397.169.
Consent
Informed consent forms were obtained from all participants for participation in the study.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Authors’ Contributions
A.Kh contributed to the study design and methodology. R.J. and M.H. supervised the whole process and modified it. B.T. contributed to manuscript provision, data analysis, and submission process. S.A. and P.V. contributed to writing the original draft. F.Gh and S.Sh helped in study coordination. All authors have read and approved the manuscript.
Acknowledgments
The authors are grateful to all residents who participated in this study.
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