1. Introduction
Penalized medicine is an approach that evaluates and manages patients based on their predicted response to treatment. This approach is also beneficial to predict and prevent therapeutic resistance, which can enhance patients’ outcomes. One of the main components of this approach is to predict the drugs’ metabolism pace and consequently drug response and toxicity [Reference Goetz and Schork1, Reference Sabnis and Bivona2]. Different pathways have been identified for the metabolism of drugs. One of the crucial pathways is cytochrome P450, which comprises different enzymes [Reference Micaglio, Locati, Monasky, Romani, Heilbron and Pappone3, Reference Rotondo, Giari and Guerranti4]. One of the major components of cytochrome P450 is CYP2B6, which is responsible for the metabolism of drugs such as antiretrovirals such as efavirenz [Reference Langmia, Just, Yamoune, Brockmöller, Masimirembwa and Stingl5, Reference Hashemi-Soteh, Hosseini, Fazelnia, Ghasemian-Sorbeni, Madahian and Shiran6].
Efavirenz ((S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one) is one of the nonnucleoside reverse transcriptase inhibitors, which is prescribed as first-line therapy in patients diagnosed with human immunodeficiency virus (HIV) infection. Antiretroviral medications have transformed HIV infection into a chronic disease, which can be managed effectively [Reference Benzaken, Pereira, Costa, Tanuri, Santos and Soares7]. Efavirenz, which is a noncompetitive inhibitor of HIV-1 reverse transcriptase, is one of the most effective drugs prescribed for HIV infection [Reference Vrouenraets, Wit, Tongeren and Lange8].
Efavirenz is highly potent; nevertheless, it is attributed to several adverse effects including, QT interval (the time from the start of the Q wave to the end of the T wave in electrocardiogram) prolongation, dyslipidemia, hepatotoxicity, and neuropsychiatric side effects [Reference Vrouenraets, Wit, Tongeren and Lange8]. Although the drug is no longer recommended as the preferred initial antiretroviral therapy (ART) regimen for nonpregnant adults due to its neuropsychiatric adverse effects, it is still recommended in selected patients based on the New York State Department of Health (NYSDOH) acquired immunodeficiency syndrome (AIDS) institute guideline (AI) [Reference Merrick9]. Furthermore, unlike in the United States, efavirenz is a vital part of the first-line therapeutic guideline in middle and low-income countries such as Iran and it is prescribed in almost all patients diagnosed with HIV infection [10]. Side effects of efavirenz are one of the biggest challenges for health services in managing HIV-positive patients [Reference Abutika11]. Evaluation of the pharmacokinetics of efavirenz is the key in managing its adverse drug reactions (ADRs) [Reference Apostolova, Blas-Garcia, Galindo and Esplugues12].
This drug initially transforms to its primary metabolite, 8-hydroxyefavirenz, in the liver mainly by cytochrome P450 2B6 (CYP2B6) enzyme, followed by the formation of 8,14-dihydroxyefavirenz chiefly by the same drug-metabolizing P450 2B6 [Reference McDonagh, Lau, Alvarellos, Altman and Klein13]. Figure 1 concisely shows the metabolic pathway of efavirenz pharmacokinetics [Reference McDonagh, Lau, Alvarellos, Altman and Klein13]. CYP2B6 is encoded by the CYP2B6 gene, and its various polymorphisms, including CYP2B6*6 (516G > T), CYP2B6*4 (785A > G), and CYP2B6*5 (1459C > T), are clinically relevant for HIV-infected patients treated with efavirenz. For instance, the TT genotype for CYP2B6*6 (516G > T) is associated with increased EFV plasma concentrations, reduced clearance, and consequently increased efavirnez exposure compared to the GG or GT genotype [Reference Wyen, Hendra and Vogel14, Reference Leger, Dillingham and Beauharnais15]. In other words, patients’ genotype for some of the CYP2B6 polymorphisms predicts the pace of drug metabolism in their bodies.
FIGURE 1: Schematic representation of efavirenz metabolism in the liver. It should be noted that the main route of efavirenz metabolism is 8-hydroxyefavirenz, which is predominately formed by CYP2B6. UGT, uridine 5′-diphospho-glucuronosyltransferase.
A recent study indicated that CYP2B6 polymorphisms are ethnically and geographically diverse among different populations, which results in differences in drugs, especially antiretrovirals, pharmacokinetics, and therapy outcomes [Reference Li, Menard and Benish16]. Iran is a geographically diverse, and multiethnic country; Fars, Turk (Azerbaijanis), and Kurds comprise about 80 percent of the country’s population [17]. The genotype and allele frequency of CYP2B6 polymorphisms have not been studied in any of its major ethnicities. This study aims to evaluate the frequency of three clinically important CYP2B6 polymorphisms (CYP2B6*6 (516G > T), CYP2B6*4 (785A > G), and CYP2B6*5 (1459C > T)) in three major Iranian ethnicities.
2. Methods
2.1. Ethical Compliance
All patients consented to participate in genetic and molecular analyses and consented to publish the results. This study was verified by the Alborz University of Medical Sciences Ethical Committee (IR.ABZUMS.REC.1398.121).
2.2. Sample Collection and DNA Extraction
One hundred and forty seven participants were included in this study, comprising 26 Kurd, 52 Turk, and 69 Fars participants. Included participants aged between 18 and 77 years (median = 43, interquartile range (IQR) = 30 to 55). It should be noted that the number of participants from each ethnic group was calculated based on the ethnic composition of Iran [18]. Two milliliters of venous blood were gathered from each participant and drawn into a tube that contains ethylenediaminetetraacetic acid as an anticoagulant followed by DNA extraction using a molecular biological system transfer (MBST) salting-out kit (CinnaGen, Tehran, Iran) from the blood. Extracted DNA was stored at −20°C before genotyping.
2.3. Genotyping
CYP2B6*6 (516G > T), CYP2B6*4 (785A > G), and CYP2B6*5 (1459C > T) were genotyped using tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS–PCR). The amplification was conducted using 60 ng of extracted genomic DNA, 0.4 U Taq DNA polymerase (CinnaGen), six pmol of each primer, 10X PCR buffer, 0.5 mM dNTP, and 1.5 mM MgCl2. The mixture was initially denatured at 95°C for 3 minutes, followed by 32 cycles of 95°C for 1 minute, 56°C for 1 min, and 72°C for 2 min for CYP2B6 516G > T; 32 cycles of 95°C for 1 minute, 64°C for 50 seconds, and 72°C for 1 minute for CYP2B6 785A > G; 35 cycles of 95°C for 1 minute, 63°C for 1 min, and 72°C for 1 min for CYP2B6 1459C > T; followed by a final extension at 72°C for 10 min. Afterward, amplified fragments were run on a 1.5% agarose gel electrophoresis for 1 hour at 80 volt, ahead of staining using silver nitrate (CinnaGen) [Reference Dehbozorgi, Kamalidehghan and Hosseini19, Reference Haghshenas, Kamalidehghan and Bagheri20]. Table 1 provides information on the primer sequences [Reference Hashemi-Soteh, Hosseini, Fazelnia, Ghasemian-Sorbeni, Madahian and Shiran6]. The DNA bands in the agarose gel were visualized under ultraviolet (UV) rays, and images were captured [Reference Dehbozorgi, Kamalidehghan and Hosseini19, Reference Haghshenas, Kamalidehghan and Bagheri20]. Figure 2 schematically summarizes the genotyping process using Tetra-ARMS-PCR for CYP2B6*6 (516G > T).
TABLE 1: Polymorphism details and primers.
FIGURE 2: Process of SNP genotyping using Tetra-ARMS-PCR for CYP2B6*6 (516G > T) in not-mutated (a) and mutated (b) DNAs. PCR product lengths of CYP2B6*6 (516G > T) were FU + RU = 600, FU + RN = 353, and FU + RM = 353.
2.4. Statistical Analysis
Genotype and allele frequencies were carried out according to tetra-primer ARMS–PCR findings. The frequency of alleles and genotypes was accompanied by confidence intervals (CI) proportions, calculated based on the formula
. Chi-square analysis was used for comparing allele frequencies in different ethnicities [Reference Ott and Rao21, Reference Balram, Sharma, Sivathasan and Lee22]. A p value less than 0.05 was considered statistically significant.
3. Results
3.1. Allelic and Genotype Frequency of CYP2B6*6 (516G > T)
Table 2 demonstrates the frequencies of CYP2B6*6 (516G > T) genotypes in the Iranian population. The frequency of wild-type homozygotes is estimated to be 35.37% (95% CI: 27.67, 43.68), while the frequency of heterozygotes and mutated homozygotes was 46.26% (95% CI: 38.01, 54.66) and 18.37% (95% CI: 12.47, 25.59), respectively. The highest frequency of TT genotype was measured in the Kurd population (34.62%), whereas the lowest frequency was calculated in Turk participants (5.38%) (p value <0.05). It should be emphasized that Kurds’ mutated allele frequency was 59.62% (95% CI: 45.10, 72.9), which was significantly higher than whole Iranian participants (p value <0.05). Allele frequencies of CYP2B6*6 are available in Table 3. Also, an electrophoresis gel demonstrating different polymorphisms of CYP2B6*6 is illustrated in Figure 3.
TABLE 2: Genotype frequencies for CYP2B6 polymorphism in the Kurd, Turk, and Fars population.
TABLE 3: Allele frequencies in Iranian populations.
* p value for χ2 test is statistically significant (p value <0.05).
FIGURE 3: CYP2B6*6 gene electrophoresis findings for three samples with genotypes of GG, TT, and GT from left to right. FU, forward outer primer; RU, reverse outer primer; RN, reverse nonmutant primer, RM, reverse mutant primer; the leftmost well is filled with a 100 bp DNA ladder.
3.2. Allelic and Genotype Frequency of CYP2B6*4 (785A > G)
The allelic and genotype frequency of CYP2B6*4 in three major Iranian ethnicities is illustrated in Table 2. A great proportion of Iranians had wild-type homozygote genotypes for CYP2B6*4 (43.54%, 95% CI: 35.39, 51.95). Furthermore, as shown in Table 3, mutated allele frequency was found to be 37.41% (95% CI: 31.87, 43.22) in the Iranian population. The highest and lowest G allele frequency was measured in Kurd (46.15, 95% CI: 32.23, 60.53) and Fars (28.98, 95% CI: 21.58, 37.31) participants (p value <0.05).
3.3. Allelic and Genotype Frequency of CYP2B6*5 (1459C > T)
As Table 2 depicts data regarding CYP2B6*5 (1459C > T) polymorphism, CC, CT, and TT frequency were 62.59%, 25.85%, and 11.56%, respectively. Among ethnicities, Turks showed the highest frequency for CC (76.92%, 95% CI: 63.16, 87.47), followed by 63.77% (95% CI: 51.31, 75.01) for Fars participants. On the other hand, Kurd participants showed a significantly higher frequency for the T allele (46.15%, 95% CI: 32.23, 60.53) compared to Iranians (62.59%, 95% CI: 54.23, 70.42) (p value <0.05). Frequencies of alleles in each ethnicity are summarized in Table 2.
4. Discussion
This study demonstrates the allele frequency of CYP2B6 mutations, which are clinically significant in the metabolism of efavirenz in three major Iranian ethnicities. The conclusion derived from the data obtained in the present study showed that Kurds, which comprise about 10 percent of the country’s population, are found to be attributed at higher risk of both decreased and increased efavirenz metabolism compared to other Iranian ethnicities due to their higher frequency of CYP2B6*6 and CYP2B6*5, respectively.
As demonstrated in Figure 1, efavirenz pharmacokinetic is closely linked to CYP2B6 and its polymorphisms play a crucial role in the metabolization pace of the drug [Reference McDonagh, Lau, Alvarellos, Altman and Klein13]. That is to say, the large intersubject variability of efavirenz exposure could be explained by the CYP2B6 genetic variations [Reference Burger, van Der Heiden and La Porte23]. Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline suggests CYP2B6 genotyping prior to prescription of efavirenz-containing antiretroviral therapy, as CYP2B6 is highly polymorphic and some of its variants lead to substantial differences in plasma efavirenz exposure [Reference Desta, Gammal and Gong24]. For instance, CYP2B6*6 (516G > T) results in aberrant splicing, which in turn leads to reduced CYP2B6 expression. Table 4 summarizes the effects of CYP2B6*6 (516G > T), CYP2B6*4 (785A > G), and CYP2B6*5 (1459C > T) polymorphisms in efavirenz metabolization and provides the frequencies of CYP2B6 polymorphisms.
TABLE 4: The effects of CYP2B6*6 (516G > T), CYP2B6*4 (785A > G), and CYP2B6*5 (1459C > T) polymorphisms in efavirenz metabolization and mutated allele frequency of CYP2B6 polymorphisms worldwide, especially in West Asia.
COS, CV-1 (simian) in origin and carrying the SV40 genetic material; NR, not reported.
Not only in-vitro studies demonstrated that CYP2B6*6 (516G > T) is associated with the decreased catalytic activity of CYP2B6 and decreased efavirenz metabolism [Reference Zhang, Sridar, Kenaan, Amunugama, Ballou and Hollenberg35–Reference Desta, Saussele and Ward37] but also clinical studies proved that CYP2B6*6 (516G > T) is associated with increased efavirenz level in plasma [Reference Sukasem, Chamnanphon and Koomdee32, Reference Abdelhady, Shugg and Thong38, Reference Swart, Skelton, Ren, Smith, Takuva and Dandara39]. In other words, previous studies revealed that populations with a higher frequency of CYP2B6*6 are at higher risk of efavirenz toxicity. In this study, Kurds were identified as a population with CYP2B6*6 mutated homozygote genotype (TT) frequencies as high as 34 percent.
The greatness of this percentage becomes more tangible when compared to previous studies. For instance, Haas et al.’s study reported a TT frequency of 20 percent in its African-Americans participants. They concluded that African-Americans have greater efavirenz plasma exposure during HIV therapy and should be considered a high-risk group in terms of efavirenz ADR [Reference Haas, Ribaudo and Kim40]. The findings of the Gounden et al. study in South African HIV-infected patients were also similar to Haas et al.’s study [Reference Gounden, van Niekerk, Snyman and George41]. On the other hand, studies conducted in Mozambican, Zimbabwean, and Senegalese populations reported a CYP2B6*6 allele frequency higher than Kurd participants in this study [Reference Nyakutira, Röshammar and Chigutsa42–Reference Mehlotra, Ziats, Bockarie and Zimmerman44]. To consider the study's small sample, we reported all frequencies with their 95% confidence intervals. It is noteworthy that even the lowest 95% confidence interval of TT frequency in the Kurd population is higher than the TT frequency reported in some previous studies [Reference Haas, Ribaudo and Kim40, Reference Hiratsuka, Takekuma and Endo45].
Zakeri et al.’s study, which is the single study conducted in Iran to evaluate the frequency of CYP2B6 variants, showed that CYP2B6*6 allele frequency is 10.2% in the Baloch population in southeast Iran [Reference Mehlotra, Ziats, Bockarie and Zimmerman44]. Their findings were consistent with our results in Turk and Fars populations. Conversely, the T allele was more frequent in the Kurd population than in the Baloch population. We should keep in mind that the Baloch ethnicity, which comprises only about 2 percent of the country’s population [18], is not a representative sample of whole Iranians. Moreover, there is a significant geographical distance between Balochs predominantly living in the southeast and Kurds residence in the country’s northwest.
Moreover, Zakeri et al. showed that the prevalence of mutated homozygote of CYP2B6*4 (785A > G) (GG), which is attributed to decreased CYP2B6 activity, in Baloch ethnicity is 10.4 (7.8–13.8) percent [Reference Zakeri, Amiri, Pirahmadi and Dinparast Djadid25], which is relatively lower than the results of this study. These findings can also be justified by the small share of Balochs in Iran’s population and geographic considerations. On the other hand, the frequency of the G allele reported in this study is similar to other ethnic populations such as Timorians (29.2 percent) [Reference Hananta, Astuti, Sadewa, Alice, Hutagalung and Mustofa46], Malays (37.2 percent) [Reference Ismail, Fauzi, Musa, Talib, Mohamad and Zulkafli47], and Indians (36.3 percent) [Reference Varshney, Saha, Tandon, Shrivastava and Ali48].
Similar to CYP2B6*6, the mutated allele frequency of CYP2B6*5 (1459C > T) is higher in Kurds than in other Iranian ethnicities. TT phenotype leads to the increased catalytic activity of CYP2B6 and increased efavirenz metabolism [Reference Zhang, Sridar, Kenaan, Amunugama, Ballou and Hollenberg35]. Having a high frequency of both CYP2B6*6 and *5 puts Kurds at higher risk of inadequate drug exposure and ADRs, prioritizing this ethnicity for testing CYP2B6 variants over other Iranian ethnicities. Arenaz et al. investigated the potential differences in allele frequencies of the CYP2B6 gene between Spaniards and Central Americans. They showed that the frequency of the T allele ranges from 1.0 percent in Japanese ethnicity to 14.0 in Caucasian (German) ethnicity. Although the frequency of CYP2B6*5 in their study is not as high as ours, their findings verified that CYP2B6*5 frequency is vastly different among ethnicities [Reference Arenaz, Vicente and Fanlo49].
There are several limitations to our study. First, the sample size of the current study was small, and other cross-sectional studies with larger sample sizes should be conducted to evaluate the allele frequency in Iranian ethnicities and identify high-risk ethnicity groups. Moreover, in this study, three major mutations of CYP2B6 were evaluated (CYP2B6*6 (516G > T), CYP2B6*4 (785A > G), and CYP2B6*5 (1459C > T)), while other minor mutations such as CYP2B6*15 (1172T > A), CYP2B6*11 (136A > G), CYP2B6*2 (64C > T), and CYP2B6*3 (777C > A) in CYP2B6 may also play a role in the drug metabolism. More importantly, sequence analysis was not conducted in this study. The lack of sequence analysis warns us to interpret the data of the investigated CYP2B6 polymorphisms more carefully.
In conclusion, this study revealed that a high frequency of CYP2B6*6 and *5 leads to an increase in the risk of ADRs and insufficient anti-HIV response in Kurds, respectively. This study proposes genotyping for clinically significant mutations, especially in Kurds, before anti-HIV therapy with efavirenz.
Data Availability
The raw data supporting the conclusions of this article will be made available by the corresponding authors without undue reservation.
Disclosure
The funder had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Authors’ Contributions
PM drafted the manuscript; PM and SSN conducted the experiments; BTF participated in sample collection and revised the manuscript critically. RJ and MH supervised the experiments and revised the manuscript critically. All authors read and approved the final version of the manuscript.
Acknowledgments
This study was funded by Alborz University of Medical Sciences. The authors wish to thank all staff members of the National Institute of Genetic Engineering and Biotechnology (NIGEB), especially Parisa Veiskrami and Fardis Markazi Laboratory. The authors are also grateful to Dr. Mostafa Qorbani for his scientific support.
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