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Haplotypes frequencies of CYP2B6 in Malaysia N Musa1, MI Zulkafli1, N Talib1, N Mohamad1, H Fauzi2, R Ismail11 Institute for Research in Molecular Medicine (INFORMM), School of Medicine, University Sains Malaysia, Malaysia 2 Department of Pharmacy, University Teknologi MARA, Malaysia
Correspondence Address: Source of Support: USM grant under the “Research University Program” (Grant Number: 1001/CIPPM/8130133), Conflict of Interest: None DOI: 10.4103/0022-3859.105439
Background: Drugs with complex pharmacology are used in the management of drug use disorder (DUD) and HIV/AIDS in Malaysia and in parts of South-East Asia. Their multiethnic populations suggest complexity due to the genetic polymorphism, such as CYP2B6 that metabolizes methadone and anti-retroviral. Aims: Our aim was to explore the genetic polymorphism of CYP2B6 among Malays, Chinese, Indians, and opiate-dependent individuals in Malaysia. Settings and Design: The study utilized DNA from our previous studies on CYPs and new recruitments from opiate-dependent individuals. Materials and Methods: For the new recruitment, after obtaining consent and baseline demography, 5 ml blood was obtained from patients attending methadone maintenance therapy (MMT) Clinics. Genomic DNA was extracted using standard methods. 10 nucleotide changes associated with CYP2B6*10, CYP2B6*2, CYP2B6*17, CYP2B6*11, CYP2B6*8, CYP2B6*14, CYP2B6*9, CYP2B6*4, CYP2B6*6, CYP2B6*27, and CYP2B6*20 were determined using multiplex nested allele-specific PCR. Statistical Analysis: Descriptive statistics were used to summarize demographic data. Differences in allele frequencies between populations were tested using Chi-squared test and were corrected using the Bonferroni test. Results: CYP2B6 polymorphism in Malaysia is variable with trends that suggest an ethnic difference. Reduced activity CYP2B6*6 occurred in 13% to 26% among Malays, Chinese, Indians and opiate-dependent individuals. Another 'reduced activity', CYP2B6*2 allele, was found at much lower percentages in the groups. Conclusions: The relative commonness of reduced-activity CYP2B6 alleles in our study called for attention in terms of dosage requirements for MMT and ARV in Malaysia. It also implored follow-up association studies to determine its relevance and consequences in personalized medicine for drug use disorder and HIV/AIDS. Keywords: CYP2B6 Polymorphism, HIV/AIDS, Malaysia, methadone
As HIV entered Malaysia in 1986, quickly spreading like wild fire, peaking in 2003 with about 23 new cases per 100,000 population, and representing Malaysia's only failed millennium goal. Thanks to its rapid spread fuelled by direct blood contact among opioid-dependent individuals, Malaysia now has the questionable distinction of having the fastest growing HIV epidemic in the Asia Pacific Region. [1] Opioid dependence thus continues to haunt Malaysia and almost all the nations in the world, but now with the added dimension of HIV epidemic, giving rise to the dual epidemic of drug use disorder and HIV/AIDS. Of the 81,000 cumulative HIV infections in Malaysia at the end of 2007, 58,135 were among injecting drug users. [2] This dual epidemic feature in Malaysia is shared by parts of South-East Asia and several other countries in West Asia and lately in Africa as well. Thus, challenged by this dual epidemic, our task is to develop a treatment system that can meet the needs of entire populations and regions. The cost of failure is unacceptable. No single treatment is effective in the management of an opioid dependence. With its added role in driving the HIV pandemic, an objective now is also to prevent an HIV spread rather than just solely to eliminate opioid dependence. Evidence, however, abounds that several modalities can successfully impact on outcomes, both in terms of reducing the many harms of illicit drug use as well as to stem the spread of an HIV. Millions are now on MMT world-wide and its effectiveness are legion. Another important approach in HIV prevention is the use of anti-retroviral (ARV). This reduces viral loads, simultaneously making the patients better and reducing HIV transmissions. With ARV and especially with MMT, effective dose remains contentious. What is common with both drugs is their complex metabolism, mediated by several polymorphic drug metabolizing enzymes (DMEs). [3],[4],[5] The influence on pharmacology of geography, race, and ethnicity has long been studied and recognized. The influence of ethnicity, for instance, has extensively been described. [6],[7],[8] SNPs in CYP3A4, CYP2C9, and CYP2B6 contribute significantly to inter-ethnic variations in drug response. [6],[7],[8] CYP2B6, on the other hand, has not been thought to be important in human drug metabolism [9],[10] although it is highly polymorphic. Recent data, however, shows that it is important. Its substrates include cyclophosphamide, [11] bupropion, [12] and efavirenz. [13] Studies revealed an altered efavirenz metabolism due to CYP2B6 polymorphism. [14],[15] CYP2B6 has 9 single base mutations with 5 giving non-synonymous amino acid changes and 4 silent. [16] Their alleles include: CYP2B6*2 (64C>T), CYP2B6*3 (777C>A), CYP2B6*4 (785A>G), and CYP2B6*5 (1459C>T). Other alleles include CYP2B6*16 (785A>G and 983T>C), CYP2B6*18 (983T>C), CYP2B6*27 (593T>C), and CYP2B6*28 (1132C>T) and 4 phenotypic null alleles CYP2B6*8 (415A>G), CYP2B6*11 (136A>G), CYP2B6*12 (296G>A), and CYP2B6*15 (1172T>A). [17] At least 28 alleles, over 50 haplotypes and >100 SNPs have already been documented. [17] Most are unimportant. Others however occur at high frequencies in certain populations and they may exert significant clinical impact. [16],[18],[19],[20],[21] That CYP2B6 polymorphism exhibits ethnic variations has amply been described. Thus, in some Asian populations, the frequency of CYP2B6*6 (516G>T and 785A>G) is 15%-40% as contrasted to over 50% among African-Americans. [16],[22],[23] The 516 G>T SNP, found in both CYP2B6*6 and CYP2B6*9 allele, significantly reduces CYP2B6 protein expression and enzyme activity. [24] This has important repercussions on the pharmacokinetics and therapeutic outcomes with some substrate drugs, including methadone, [25] bupropion, [26] and efavirenz. [27] The increase in CYP2B6 activity by the additional 785 A>G mutation in CYP2B6*6 haplotype appears not to overcome the decrease in CYP2B6 expression or activity produced by the 516 G>T SNP. [20] In Malaysia and elsewhere, methadone is used widely in methadone maintenance therapy (MMT). Racemic methadone, as usually prescribed, is associated with some undesired consequences. While the (R)-enantiomer mediates the narcotic effect by activating the μ-opioid receptor, (S)-methadone inhibits cardiac potassium channel, contributing to the cardiac side effects of methadone treatment. [28] Wang et al., [29] showed that CYP2B6 polymorphism was strongly associated with clearance and plasma S-methadone. Our previous data indicate heterogeneity in many of the CYP gene we studied, and our population appeared to be quite distinct compared to studies in other geographic locations. [30],[31],[32],[33],[34],[35],[36],[37] The objective of this study was, therefore, to determine the types and frequencies of CYP2B6 alleles among Malays, Chinese, Indians, and among opiate-dependent individuals in Malaysia. This, we hope, can contribute to the literature on the genetic variability of CYP2B6 across populations as well as to help guide the use of drugs like methadone and anti-retroviral used in AIDS in Malaysia.
The part of the study that explored CYP2B6 polymorphism in the major ethnic groups is Malaysia was part of our larger study on the genetic polymorphism of drug metabolizing enzymes that began in 2003 and continuing. It was approved by our local institutional ethical committee. Its protocols were in accordance with the Helsinki Declaration 1975 and 1983 involving healthy unrelated individuals enrolled from January to December 2003 from among blood donors at our local blood bank. They gave written informed consent. Also, included were local university students and residents of a nearby Indian community. All the subjects were screened against the following inclusion and exclusion criteria: Inclusion criteria
Exclusion criteria
Genomic DNA was extracted from peripheral leucocytes using methods we previously described. [30] The quantity and quality of the extracted DNA was determined on the spectrophotometer with measurements done at 260 and 280 nm. 10 nucleotide changes at positions A62T (CYP2B6*10), C64T (CYP2B6*2), A76T (CYP2B6*17), A136G (CYP2B6*11), A13072G (CYP2B6*8), G13076A (CYP2B6*14), G15631T (CYP2B6*9), A18053G (CYP2B6*4), T15708C (CYP2B6*27), C15818T (CYP2B6*20), and CYP2B6*6 were determined by multiplex nested allele-specific PCR, we previously developed to simultaneously detect CYP2B6 and mu-opiate receptor polymorphisms. This method is the subject of a separate publication. In summary, it involved the use of primers [Table 1] in the first PCR to isolate regions of interest in the CYP2B6 and M opioid receptor gene. This was followed by parallel second PCR using primers [Table 2] that were designed to have specific 3'-ends, which were manipulated to differentiate single nucleotide changes at the specific loci during PCR amplification. PCR was performed on an Eppendorf instrument.
The first PCR comprised 4 sets that yielded 8 products of sizes ranging from 222 base pair to 1020 base pair. The reactions were carried out in 25 μl-volume reaction mixtures each containing 2 μl of genomic DNA, 1X PCR buffer, 2.0 mM MgCl 2 , 1.0 U of Biotools DNA Taq Polymerase, 0.2 mM dNTPs and primers at appropriate concentrations. The cycling conditions were optimized for the different sets, and the products were analyzed on 2.5% agarose gel (LE, analytical grade; Promega) in 1X TBE (Tris, Borate, EDTA) buffer. In the second PCR, 12 sets of parallel allele-specific PCR were performed: Set 1 for A454G, C534T, G779A, and CYP2B6*14; set 2 for G24A, G820A, G942A, and G31A; set 3 for G438A, T106C, and C440G; set 4 for CYP2B6*27, T480C, and T397A; set 5 for G794A, C17T, and CYP2B6*8; set 6 for CYP2B6*17 and CYP2B6*10; set 7 for CYP2B6*2 and CYP2B6*11; set 8 for CYP2B6*20; set 9 for A118G; set 10 for T802C and CYP2B6*9; set 11 for C691G and set 12 for CYP2B6*4. CYP2B6*6 allele is made up of 2 SNPs; A18053G and G15631T as per published nomenclature [17] (see also Nomenclature [17] for CYP2B6 Alleles). For all the sets, 2 μl of the first PCR product (diluted 25×) were used as a template for the second PCR in a 25 μl reaction mixture containing 1× PCR buffer, 2.0 mM MgCl 2 , 0.2 mM dNTPs, 1.0 U DNA Taq Polymerase and appropriate amounts of primers. The cycling conditions were again optimized for the different sets, and the products were again analyzed on 3% agarose gel and 1X TBE buffer. Statistical analysis CYP2B6 allele data from this study was analyzed using the population genetic data analytical program SVS 7.3.1 (GOLDEN HELIX, SNP & VARIATION SUITE 7) based on an expectation-maximization (EM) algorithm for the following procedures:
This was an exploratory study to investigate the genetic polymorphism of CY2B6 in Malaysia. Study population consists of 424 healthy unrelated and 110 unrelated Malay opiate-dependent individuals. Details of demographic data were described in [Table 3].
Although not sufficiently powered to determine ethnic differences, there was a trend to suggest that the 3 ethnic groups in Malaysia shared some similarity and some differences in term of CYP2B6 polymorphism. Of the 10 SNPs investigated, 2 at position 15631 (G>T) and 18053 (T>C) were found common in all the 3 ethnic groups at frequencies ranging from 24% to 38% [Table 4]. On the other hand, at the population level, C64T polymorphism was less important, occurring at frequencies that ranged from less than 1% to 2.5% among Chinese Malays, and Indians. A slightly higher percentage of 3.2% was seen in opiate-dependent individuals. Population-wide, this was not expected to confer phenotypic differences among the ethnic groups, whatever expression is associated with the allele. Heterozygous A136G was rare, at 0.3% but in only with Chinese. This again was not expected to confer phenotypic differences among the ethnic groups. The genotype frequencies observed in this study were similar to those predicted by the Hardy-Weinberg equilibrium as shown in [Table 4].
Using an expectation-maximization (EM) algorithm, 9 "maximum-likelihood" haplotypes were defined in the 4 populations of Malays, Chinese, Indians and opiate-dependent individuals. They were CYP2B6*1, CYP2B6*2, CYP2B6*4, CYP2B6*6, CYP2B6*9, CYP2B6*27. 3 others were novel combinations of SNPs. The haplotype frequencies in percentages are shown in [Table 5]. If at all, there were small differences observed, but at population levels, these were not expected to be of significance.
This is the first paper that reports CYP2B6 genotype and allele frequencies for the 3 major ethnic groups in Malaysia and among its heroin-dependent individuals. Our interest in CYP2B6 stemmed from its purported importance in the metabolism of drugs like methadone and efavirenz, roles that have previously been underestimated. Coupled with its highly polymorphic nature, its importance cannot be over-estimated. Malaysia is a country in transition, both physically and economically. The country lies in the trades routes between the East and the West. Malaysia also has a potpourri of ethnicities with Malays, Chinese, and Indians making up the majority. Over the years, the transitional position of the country also subjected Malaysia to ethnic admixtures consequent to intermarriages. Furthermore, its proximity to the "Golden Triangle" avails Malaysia to illicit drug trafficking and use and all of its associated harms, including an HIV transmission. There are thus many dimensions to drug use in Malaysia. It, therefore, makes Malaysia a population that is interesting to study including in terms of the many ramification of drug use, licit or otherwise. In terms of the types and frequencies of SNP's, we documented 9 previously described haplotypes and 3 hithereto novel combinations of SNP's. The novel combination may require naming. Additionally, in parallel with our previous studies, [30],[31],[32],[33],[34],[35],[36],[37] our results suggested that the genetic polymorphism of CYP2B6 in Malaysia features marked interethnic differences. This poses a challenge in the development of personalized medicine. Of further interest, another feature observed in this study was the heterogeneity of the polymorphism. Such heterogeneity has previously also been described for the several other genes we studied. Again, this requires an attention in the development of personalized medicine using pharmacogenetics. As in our earlier studies of CYP polymorphisms, Malaysian Indians appeared to occupy an intermediate position between the East and the West. Malaysian Chinese on the other hand, seem to have maintained similar polymorphic characteristics as do most East Asian populations. [30],[31],[32],[33],[34],[35],[36],[37] Malaysian Malays appeared to occupy a position intermediate between Malaysian Chinese and Malaysian Indians. We postulated that this was probably owed to the origins of the ethnic groups and their migration to Malaysia and subsequent genetic admixing through intermarriages. [30],[31],[32] As a whole, Malaysian Indians and Malays appear to be closer to each other than they are to Malaysian Chinese. In terms of CYP2B6, it was interesting to note though that our Indians and Malays appeared similar to Japanese (Hiratsuka 2002) and other East Asian [38],[39],[40] for CYP2B6*6 but lower than among Caucasian [41] and African Americans. [21],[40] Malaysian Chinese also appeared to have lower CYP2B6*6 frequency than other East Asian ethnic populations. The discrepancy could not be explained, but it could just be a chance finding due to our relatively small sample sizes. It was also noteworthy that CYP2B6*6 tended to be more prevalent among Malays than they were among Indians and Chinese. The difference was, however, very small for us to expect an influence at the population level although it would be interesting to see if these minor differences would translate to differences in dose requirements for drugs like methadone and efavirenz among the ethnic groups in Malaysia as previously reported to occur in African Americans and Hispanics. [42] At the individual level, however, this difference may be clinically relevant. The higher prevalence of CYP2B6*6 among Malays and the similarity in the frequency when healthy Malays were compared to heroin-dependent Malays could just be a chance finding, but it may also suggest a role for this allele in heroin dependence as heroin dependence occurs most commonly among Malays in Malaysia compared to other ethnic groups. For the association to be established, however, we need to investigate the physiologic/pathophysiologic role of CYP2B6*6.
Another null allele, CYP2B6*2 was also found at a frequency similar to some [38] and lower in others. [23],[43] In several Asian populations, the frequencies were higher and ranged from 2.4% to 12.2%, [40] frequencies that were also observed among Malaysian Indians and among Malay opiate-dependent individuals as well as among other populations of Asians, African Americans and Caucasians. [40] Higher frequencies have been seen in Hispanics at 14% [42] and also in Taiwanese at 13.2%. [40] Why this was so was again a matter of conjecture. Nevertheless, it would appear that slow CYP2B6 metabolism occurring among Malaysians would be attributable to CYP2B6*6 rather than CYP2B6*2 in other populations. Other alleles like CYP2B6*27 was rarely found at percentages that were similar to Rotger's [33] who also showed that the allele exhibited an 85% decrease in enzyme activity when efavirenz was used as a substrate. Ours is the first study that investigated CYP2B6 polymorphism in Malaysia that incorporated healthy volunteers from the major ethnic groups and opiate-dependent individuals. The knowledge gained from the study would be good in planning drug therapies for the different groups, especially as it pertains to CYP2B6-substrate drugs. It should, however, be noted that we employed a convenience sampling approach rather that randomized sampling to enroll our subjects, and this may have relevance in terms of generalizing the findings. A random approach involving multiple centers in Malaysia would probably be warranted, and such studies should perhaps be done. Nevertheless, we feel that our study did reveal some important information about CYP2B6 polymorphisms. We conclude that CYP2B6 polymorphism as it occurs in Malaysia is of importance as it is associated with a relatively frequent occurrence of null alleles associated with poor enzyme activity. There was also a trend for an ethnic difference, but our small sample size was not adequately powered to unequivocally say so. A difference is indeed expected arising from the diverse routes of evolution of the ethnic groups. The finding provides a rationale for follow-up research to better understand its phenotypic association and its effect on the pharmacokinetic of substrate drugs among the different ethnic groups. Such research is important in places like Malaysia where there is a dual epidemic of HIV and opiate-dependence that use several CYP2B6-substrate drugs in their management. The widespread use of MMT in the prevention of HIV spread together with ARV in the treatment of AIDS may create unique patterns of drug interactions that are associated with ethnic variation in CYP2B6 polymorphism.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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