Allelic and Genotype Frequencies of CYP2B6∗2 (64C > T) and CYP2B6∗3 (777C > A) in Three Dominant Ethnicities of the Iranian Population

Background Cytochrome P450 complex plays a key role in drug metabolism. CYP2B6 has an essential part in Cytochrome P450 complex metabolism. This study aims to determine the allelic distribution of CYP2B6∗2 and CYP2B6∗3 in three main Iranian ethnicities: Fars, Turk, and Kurd. Methods The study was conducted on 174 unrelated healthy volunteers from three main Iranian ethnicities. After DNA extraction from peripheral blood samples, genotyping of CYP2B6∗2 and ∗3 was performed using tetra ARMS and ARMS PCR, respectively. Results The average age of 174 cases was 40.69 ± 11.87 (mean ± SD) and 39.06 ± 11.63 (mean ± SD) for males and females. In the CYP2B6∗2 variant, the genotyping frequency of wild type (C/C), heterozygous (C/T), and homozygous mutant (T/T) was 8.7%, 86%, and 5.2%, respectively. The CYP2B6∗2 (c.64C > T) allele frequency was 48.2% (95% CI: (37.8–58.6)). In the CYP2B6∗3 variant, the frequency of wild type (C/C), heterozygous (C/T), and homozygous mutant (T/T) was 75.3%, 11%, and 13.6%, respectively. The CYP2B6∗3 (c.777C > A) allelic frequency was 19.1% (95% CI: (17.5–20.7)). Conclusion Allelic distribution in three main Iranian ethnicities, i.e., Turk, Kurd, and Fars, is remarkably higher than that in other populations, even that in Southern Iran. High frequencies of CYP2B6∗2 and ∗3 in the Iranian population highly affect drug responsiveness. Understanding such variability could help to increase drug efficacy and reduce its toxicity.


Introduction
Personalized medicine addresses the terms covered by targeted therapies, related diagnostics, and the medical intervention's safety [1]. Following the evolution of the Human Genome Project, the association between personalized medicine and genes encoding drug-metabolizing proteins became more outstanding [2]. 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 [3].
Cytochrome P450 enzymes play a crucial role in the metabolism of diferent agents, including food and drugs, with various enzymatic activities leading to various drug responses among individuals [4]. Many highly polymorphic genes encode cytochrome P450, and several Single Nucleotide Polymorphisms (SNPs) have been identifed in each gene [5]. SNPs may result in various enzymatic activities in people, eventually leading to insufcient efcacy of medical regimens or their adverse efects in a constant amount of drugs [6].
Ethnicity is among those factors infuencing the SNP frequencies and various drug responses [7]. Although there are worldwide databases providing the frequency of multiple SNPs, they still need some data on specifc ethnicities. Based on the latest studies, Iran's population was calculated at 83.9 million in 2020 and accounted for the 18 th 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 [9].
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 [10,11]. CYP2B6 participates in the metabolism of drugs, including many antibiotics, antiretroviral drugs, antimalarial, and frst-lineanti-Tuberculosis drugs [12]. CYP2B6 is polymorphic and contains signifcant interindividual variability in the human population [13]. Hence, the analysis of CYP2B6 SNPs may improve drug efcacy and also decrease medical regimens' adverse efects.
Studies on the CYP2B6 isozyme have expanded significantly since 2003, at the same time as the discovery of this isozyme efect in the clearance of the antiviral drug, especially efavirenz [13]. 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 [16]. A higher concentration of anti-HIV drugs, including efavirenz, and its toxicity in CYP2B6 * 2 [17] have been reported.
Various drugs are metabolized by CYP2B6 [18,19]; among them, two drugs that are more specifcally 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 [20,21]. Reports show that Iran has a considerable rate of opium use, especially methadone [22]. To the extent of our knowledge, CYP2B6 * 2 and * 3 have not been evaluated in Iranian populations, except for Southern Iran [23], which is previously reported to have a distinguished genetic pool from other Iranian populations [24].
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.

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 afecting DNA were excluded from the study. Of this number, 65 (37.4%) cases were males and 109 (62.6%) were females. Te 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.

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 bufer for the digestion of non-nucleic acid components of the cell, precipitation bufer for protein isolation, washing bufer, and eventually elusion bufer for DNA resuspension were utilized. Terefore, we utilized Oligonucleotide F (forward) and R (reverse) primers for CYP2B6 * 2 and * 3 amplifcation, as shown in Table 1.

ARMS and Tetra ARMS-PCR.
Genotyping analysis was conducted utilizing an amplifcation refractory mutation system polymerase chain reaction (ARMS PCR) for CYP2B6 * 3 and tetra ARMS PCR for CYP2B6 * 2. Te 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. Te 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 fnal extension at 72°C for 8 minutes. Te 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).

Sequencing Analysis.
Similar to our previous studies [25], forwards and reverse primers sequenced PCR products on an automated ABI 3100 sequencing machine (Applied Biosystems, Kavosh Fanavaran Kawsar Company, Iran). Ten, we used the Finch TV program for sequencing and analyzing to confrm the results of nucleotide variations.

Statistical Analysis.
Te statistical analyses were performed using SPSS (version 24; IBM) software. A confdence interval test (95%) was considered for the frequency of alleles and genotypes. A P value of less than 0.05 was considered statistically signifcant [25].
To the extent of our knowledge, this is the frst 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 [31].
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% [32]. In a study by Musa et al., CYP2B6 * 2 allelic distribution was 0.8% in Malaysian and 4.1% in Indians [33]. 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 [23]. In a study in 2021, CYP2B6 * 3 allelic distribution was reported at 6.5% of the Pakistan population [34].
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 diferent from those in Europeans, while Iranian Baluch seemed to have a tendency towards south Asians in allele frequency [23,24]. Besides, CYP2B is believed to have the most frequent interpersonal variety, which accounted for 20-250 times more than other Cytochrome P450 isoenzymes [40].
Our results, alongside those from other studies, provide further evidence for ethnic heterogeneity in drug metabolism. We hope our fndings contribute to a better understanding of various drug responses in diferent populations.

Study Limitations
Tis 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.

Conclusion
Allelic distribution in three main Iranian ethnicities, i.e., Turk, Kurd, and Fars, is remarkably diferent from other populations, even those in Southern Iran. High frequencies of CYP2B6 * 2 and * 3 in the Iranian population remarkably afect drug responsiveness, including higher efavirenz toxicity. Understanding such variability could help to increase drug efcacy and reduce its toxicity.

Data Availability
We have presented all the necessary data in this manuscript. Te data would be available from the corresponding author on reasonable request.

Ethical Approval
Tis study is approved by the Ethics Board Committee of Alborz University of Medical Sciences with the approval ID: IR.ABZUMS.REC.1397.169. Genetics Research