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Cytochrome P450 2C8*2 allele in Botswana: Human genetic diversity and public health implications
Acta Tropica 157 (2016) 54–58
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Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Cytochrome P450 2C8*2 allele in Botswana: Human genetic diversity and public health implications Thato Motshoge a,b , Leabaneng Tawe b,c , Charles Waithaka Muthoga b,c , Joel Allotey d , Rita Romano e , Isaac Quaye f , Giacomo Maria Paganotti b,c,g,∗ a
Botswana Ministry of Health, Plot 54609, 24 Amos Street, Gaborone, Botswana Medical Education Partnership Initiative (MEPI) Laboratory, Private Bag UB 00712, Gaborone, Botswana c Botswana-University of Pennsylvania Partnership, P.O. Box AC 157 ACH, Gaborone, Botswana d Department of Oncology, University of Sheffield, Beech Hill Road, S10 2RX Sheffield, UK e Department of Public Health and Infectious Diseases, University of Rome “La Sapienza”, P.le Aldo Moro 5, 00185 Rome, Italy f Department of Biochemistry, University of Namibia School of Medicine, Private Bag 13301, Windhoek, Namibia g Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6073, USA b
a r t i c l e
i n f o
Article history: Received 24 July 2015 Received in revised form 18 January 2016 Accepted 27 January 2016 Available online 1 February 2016 Keywords: Botswana Bantu ethnic group CYP2C8 Pharmacogenetics San ethnic group
a b s t r a c t Human cytochrome P450 2C8 is a highly polymorphic gene and shows variation according to ethnicity. The CYP2C8*2 is a slow drug metabolism allele and shows 10–24% frequency in Black populations. The objective of this study was to assess the prevalence of CYP2C8*2 allele in Botswana among the San (or Bushmen) and the Bantu ethnic groups. For that purpose we recruited 544 children of the two ethnicities in three districts of Botswana from primary schools, collected blood samples, extracted DNA and genotyped them through PCR-based restriction fragment length polymorphism analysis. The results demonstrated that in the San the prevalence of the CYP2C8*2 allele is significantly higher than among the Bantu-related ethnic groups (17.5% and 8.5% for San and Bantu, respectively; P = 0.00002). These findings support the evidence of a different genetic background of the San with respect to Bantu-related populations, and highlight a possible higher risk of longer drug clearance or poor level of activation of pro-drugs among the San group. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Exploring pharmacogenetics in different African ethnic groups can lead to a better understanding of the evolutionary history and the adaptation of humans to their environment. In all life forms (from bacteria to multicellular organisms) several detoxifying enzyme systems act to reduce the harmful effects of molecules that organisms introduce into the cells/body through nutrition or that are present in the environment (Gonzalez and Nebert, 1990). The detoxifying enzymes belong to several super-families of molecules and are actually of relevant importance in modern drug’s metabolism, inactivation and clearance and they are often responsible of the side effects and adverse reactions, and ultimately of the therapeutic response (Cascorbi, 2006; Bains, 2013). During the evolution of modern humans, farming, nomadic pastoralism, hunter-gathering imposed different chemical microenvironments to the populations often allowing several toxic
∗ Corresponding author at: P.O. BOX AC 157 ACH, Gaborone, Botswana. E-mail address: [email protected] (G.M. Paganotti). http://dx.doi.org/10.1016/j.actatropica.2016.01.028 0001-706X/© 2016 Elsevier B.V. All rights reserved.
molecules to be present in the diet, and this was more true in the adaptive challenge of the hunter-gatherers groups as the San or Bushmen in Southern Africa (Wyndham and Morrison, 1958; Bronte-Stewart et al., 1960; Barnard, 1983; Garruto et al., 1999; Higman, 2011). In literature, there are very few studies on San pharmacogenetics. One of such studies relates to the metabolic ratio of debrisoquine, the probe substrate for cytochrome P450 2D6 (CYP2D6) activity. It has been reported that the San population accounts for a double of the rate of debrisoquine poor metabolisers compared to Caucasians (19% vs 8–10%) (Sommers et al., 1988) and also showed different metabolic activity for metoprolol, another substrate for testing CYP2D6 activity (Sommers et al., 1989). Furthermore, a study on acetylator status among the San of North-Western Kalahari revealed that up to 20% of San may be slow inactivators of isoniazid (Jenkins et al., 1974), a drug still widely used for tuberculosis therapy and prophylaxis. Comparative studies on the pharmacogenetics of this population could help to shed light on its genetic structure and could also inform future personalised therapies, since most modern drugs, developed on European populations, could have little and/or toxic
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effect among this and other related Southern African ethnic groups, raising potential population-specific pharmacogenetic incompatibilities of certain drugs that are globally prescribed (Daar and Singer, 2005). Cytochrome P450 enzymes are a family of enzymes ubiquitously expressed and involved in the metabolism of eicosanoids, sterols, drugs and xenobiotics. Among them, cytochrome P450 2C8 enzyme (CYP2C8) is a member of the human CYP2C enzyme family, which also includes CYP2C9, CYP2C18 and CYP2C19, whose genes are located on chromosome 10q24 (Daily and Aquilante, 2009). CYP2C8 gene is known to be polymorphic, and the distribution of allelic variants differs among populations (Totah and Rettie, 2005), thus could serve as a useful marker to distinguish and discriminate among ethnic groups, ultimately providing recommendations in the perspective of personalised medicine. This is very important in Africa where human genetic variability is high (Campbell and Tishkoff, 2008; Alessandrini et al., 2013). CYP2C8*2 (Ile268Phe; rs11572103), the allelic variant most common in Africans, is related to a lower drug metabolism rate in subjects carrying at least one copy of the defective allele (Cavaco et al., 2005; Totah and Rettie, 2005). Subjects who are intermediate or poor metabolisers experience higher drug concentration in plasma, lower clearance, longer drug half-life and increased adverse side effects that potentially affect compliance and therapeutic outcomes (Wijnen et al., 2007). Several studies have reported the CYP2C8*2 allele frequency in Africa where it is known to have a distinctive inter-ethnic difference (Alessandrini et al., 2013). In the published reports on African populations the frequency varies in the range of 9.9–24.2% (Cavaco et al., 2005; Röwer et al., 2005; Parikh et al., 2007; Adjei et al., 2008; Kudzi et al., 2009; Paganotti et al., 2011, 2012; Alessandrini et al., 2013). Interestingly, CYP2C8*2 is virtually absent in non-African populations (Dai et al., 2001). Despite its effects on a range of different treatments, CYP2C8 genetic variation has been studied in African populations mainly for its impact on the antimalarial therapies based on the 4aminoquinolines, particularly amodiaquine (AQ) and chloroquine (CQ). Moreover, it was recently reported (Paganotti et al., 2011) and confirmed by an independent group (Cavaco et al., 2013), that
decreased CYP2C8 activity due to poor metaboliser alleles may expose Plasmodium falciparum parasites to a sub-therapeutic level of drug for longer time driving the selection of drug resistance. In recent years, a number of Southern African countries including Botswana have targeted malaria elimination attributed largely to P. falciparum. The National Malaria Control Program has approached malaria elimination through intensive strategies (Simon et al., 2013) including drug treatment for uncomplicated cases with artemether-lumefantrine (AL) as first line artemisinin combination therapy (ACT). Moreover, a combined regime of CQ and proguanil continues to be recommended for pregnant women in risk areas (Botswana Ministry of Health, 2012). Currently, a switch from quinine (QN) to artesunate/amodiaquine (AL/AQ) as second line ACT is under evaluation. These considerations highlight the need to examine the CYP2C8 genetic polymorphism in the population vis-à-vis the role of the enzyme in the metabolism of the 4-aminoquinolines. Human CYP2C8 enzyme is also involved in the metabolism of a variety of clinically important drugs such as trans-retinoic acid (leukemia treatment), paclitaxel (cancer), diclofenac and ibuprofen (pain and inflammation), loperamide (antidiarrheal), morphine (pain), verapamil (hypertension), carbamazepine (epilepsy) (Lai et al., 2009). These drugs are used extensively in the hospitals and clinics in Botswana. For all these reasons, we screened three different districts of Botswana for the CYP2C8*2 allele to inform decision on the use of the 4-aminoquinolines not only towards malaria elimination but also on populations at risk of toxicity during the deployment of these drugs. In the selected areas the populations are largely of Bantu (Serowe/Palapye and Chobe districts) and San (also called Basarwa or Bushmen, in Ghanzi district, Central Kalahari) ethnicity. Importantly, this report is the first CYP enzyme investigation that also includes reference to ethnicity in Botswana.
2. Material and methods The survey was performed in March 2012 in Botswana in the broader context of a Malaria Indicator Survey (Botswana Ministry of Health, 2012). A total of 544 children asymptomatic for malaria were enrolled in several primary schools: 160 from Ghanzi dis-
Fig. 1. Map of Botswana with the three districts indicated. Source: Google Earth, accessed on 11th December 2015.
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T. Motshoge et al. / Acta Tropica 157 (2016) 54–58
Table 1 CYP2C8 genotypes and T allele frequency in subjects from three districts of Botswana and two different ethnic groups. Districts (ethnicity)
CYP2C8 (rs11572103, A > T) frequencies N
Serowe/Palapye(Bantu ethnic group) (Bantu ethnic group) Chobe (Bantu ethnic group) S/P + C Ghanzi (San ethnic group)
227 157 384 160
Allele frequency ± SE
Relative and (absolute) genotype frequencies AA
AT
TT
0.855 (194) 0.802 (126) 0.833 (320) 0.675 (108)
0.145 (33) 0.192 (30) 0.164 (63) 0.300 (48)
0.000 (0) 0.006 (1) 0.003 (1) 0.025 (4)
T 0.073 ± 0.017 0.102 ± 0.024 0.085 ± 0.014 0.175 ± 0.030
S/P: Serowe/Palapye district; C: Chobe district; N: number of individual samples.
trict, 227 from Serowe/Palapye district and 157 from Chobe district (Fig. 1). The ethnicity was assessed from family names in each location according to Friscella and Fremont (2006) and Mateos (2007) and village of birth (excluding all the foreigners or born in other districts). The same protocol for enrolment was followed in all sites. Signed informed consent for multiple genetic and epidemiological surveys was obtained from all the subjects’ parents/caregivers included in the study. This study was conducted in accordance with the guidelines of the Helsinki Declaration of 2000, with the approval of the Human Research and Development Division of the Botswana Ministry of Health [PPME-13/18 V (380)] and the Institutional Review Board of the University of Pennsylvania [protocol number 820378]. Three ml of whole blood was collected into EDTA-containing tubes by physicians and trained phlebotomists. DNA was extracted with Qiagen kits according to the manufacturer’s procedure. CYP2C8*2 (rs11572103, A > T) detection was carried out using the PCR-RFLP technique (Paganotti et al., 2011). Briefly, we started from 2 l of human DNA to amplify a 107 bp fragment of the CYP2C8 gene containing the polymorphism of interest using the following primers: forward 5 -GAACACCAAGCATCACTGGA-3 and reverse 5 -GAAATCAAAATACTGATCTGTTGC-3. The PCR product was subsequently digested with BclI enzyme that cuts the wild-type allele only (A); undigested products represent the variant allele (T). To detect the size polymorphisms, the samples were ran on a 3% agarose gel (Fig. 2). Controls for human genotyping were utilized after sequencing three PCR products relative to each different genotype. Moreover, to avoid the genotyping error, the heterozygous samples were repeated. Inter-populations comparisons were obtained by Yates corrected 2 test. Odds ratios (ORs) were calculated with 95% confidence intervals (CIs). The analyses were performed with Epi info 6 statistical package (freely available at https://wwwn.cdc.gov/ epiinfo/html/ei6downloads.htm) Evaluation of Hardy-Weinberg equilibrium was performed using the HWSIM software (freely available athttp://krunch.med.yale.edu/hwsim/) and Monte-Carlo permutation test performed when genotypic classes had an expected cell size of less than five.
3. Results The frequencies of the CYP2C8*2 allele for each district included in the present study are presented in Table 1. Genotype frequencies were in Hardy-Weinberg equilibrium after a Monte Carlo permutation test (10,000 runs) in all the three districts (P = 0.158, P = 0.213 and P = 0.135 respectively). No statistically significant difference was found comparing the allele frequency between Serowe/Palapye and Chobe (Yates corrected 2 = 1.686, P = 0.1526). Considering that these districts are largely of Bantu ethnicity, we pooled together the results and evaluated Hardy-Weinberg equilibrium after a Monte Carlo permutation test (10,000 runs) (P = 0.09). A striking difference was revealed when the pooled genotypic classes of the two Bantu districts were compared with the Ghanzi district
Fig. 2. Agarose gel (3%) showing BclI digestion of the PCR products and restriction pattern. Line 1: 25 bp marker; Line 2: undigested PCR product (107 bp); Line 3: AA genotype positive control [wild-type homozygous (56 bp + 51 bp)]; Line 4: AT genotype positive control [heterozygous (107 bp + 56 bp + 51 bp)]; Line 5: TT genotype positive control [mutant homozygous (107 bp)]; Line 6: 25 bp marker; Line 7: sample 7201195 (AA genotype); Line 8: sample 50282 (AT genotype); Line 9: sample 8001881 (AA genotype); Line 10: sample 80908 (TT genotype); Line 11: sample 7201189 (AA genotype); Line 12: sample 5001165 (AT genotype); Line 13: 25 bp marker; Line 14: negative control.
(Yates corrected 2 = 18.64, P = 0.0000251; OR = 0.44[0.29–0.65]). In addition, the percentage of subjects carrying at least one copy of the CYP2C8*2 allele (T) was 16.67% and 32.50% for Banturelated subjects and San, respectively (Yates corrected 2 = 15.95, P = 0.0000652; OR = 0.42[0.27–0.65]). 4. Discussion So far our study is the first report of CYP2C8*2 allele distribution comparing two different ethnic groups, San and Bantu, of Botswana. Overall, the allele frequency of CYP2C8*2 was 17.5% in the San and 8.5% in the Bantu. In general, the allele frequency from the two Bantu-inhabited sites (Serowe/Palapye and Chobe combined) are slightly below the lower value in the documented range for Africa (9.9–24.2% frequency, according to Alessandrini et al., 2013), whereas the data from the San ethnic group falls within the reported range for the allele (Alessandrini et al., 2013). Several investigations in Africa have shown similar allele frequency as the San, e.g. in Northern Ghana (16.8%) (Röwer et al., 2005), in Madagascar (15%) (Paganotti et al., 2012), in Mozambique (15.6%) (Suarez-Kurtz, 2011) and in African-Americans (18%) (Dai et al., 2001). Instead, the Bantu of Botswana show comparable allele frequency as the Fulani of Burkina Faso (9.9%) (Paganotti et al., 2011) and two other populations of Uganda (10.5%) (Paganotti et al., 2012). Notably, even though similar allele frequencies exist among
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different populations all over the continent, it does not necessarily indicate a similar genetic background or a common ancestor, rather it is more important to evaluate the possible interethnic differences within the same country or territory as we have done for the San and Bantu in Botswana. Interestingly, when combining the genotype results from Serowe/Palapye and Chobe, the probability associated to the Monte Carlo permutation test for the Hardy-Weinberg equilibrium dropped to P = 0.09. This result could suggest a marginal tendency for deviation from equilibrium, perhaps due to another prominent allele, the known linkage between CYP2C8 and CYP2C9 genes (Speed et al., 2009) or the presence of several subgroups into the Bantu ethnicity (e.g., Tswana, Bangwato, Mbukushu, Bayei). Importantly, we observed that the percentage of the San population carrying at least one copy of this allele accounts for almost one third of the subjects (AT = 30%, TT = 2.5%; T carriers = 32.5%). In this study very few individuals were homozygous null (0–2.5%), which has already been documented by other studies in African populations (Alessandrini et al., 2013). The pharmacological implications of being heterozygous or homozygous for the defective CYP2C8*2 allele are not well studied, and few reports show that there is also substrate variability with regard to drug disposition and response. A study on AQ in Burkina Faso showed a significant increased rate of adverse events in both heterozygotes and homozygotes for the variant CYP2C8*2 genotypes compared with the wild-type genotype (52% vs 30%, P < 0.01) (Parikh et al., 2007). Moreover, the recombinant CYP2C8*2 enzyme showed a defective metabolism of AQ (3-fold higher Km and 6-fold lower intrinsic clearance than the functional enzyme) in vitro (Parikh et al., 2007). The paper from Paganotti et al. (2011) reported a statistically significant trend among the three different genotypes for carrying CQ-resistant P. falciparum parasites in Burkina Faso (38.1%, 50.0%, 54.2% for AA, AT, TT genotypes, respectively, P = 0.02), an observation that might be explained by the pharmacokinetic of CQ. In another report, the recombinant CYP2C8*2 was associated with the defective metabolism (2-fold higher Km and 2-fold lower intrinsic clearance than the wild-type) of the anticancer drug paclitaxel in vitro (Dai et al., 2001). Despite the fact that this argument needs further studies and in vivo data, it seems that the heterozygous genotype has an intermediate metaboliser phenotype for AQ, CQ and paclitaxel (Bains, 2013). Since in our study the majority of the carriers of the defective allele are the heterozygous subjects, we are aware that the metabolic status, although dependent on the drug used, will be affected and decreased to a certain level. Therefore, the relatively high prevalence of intermediate and poor metaboliser subjects for CYP2C8-metabolised drugs in the San population, predisposes them to a higher risk of side effects for 4aminoquinolines than the Bantu ethnic group in Botswana. This could impact negatively on the drug compliance and results in a lower efficacy of 4-aminoquinolines. This observation is of significant interest as the San are spread throughout the Southern African region. We suggest that strategies for malaria elimination will have to take this into consideration when dealing with this ethnic group. The same will apply for the other drugs that are substrates for the CYP2C8 enzyme. Our study has some limitations. The sample size for San ethnicity is relatively small. Moreover, pharmacokinetic and pharmacodynamic data on the participants were not available, since they were healthy subject and not under treatment.
5. Conclusions We conclude that CYP2C8*2 polymorphism in the San population of Botswana is present at higher frequency than among Bantu-related communities. In addition, we believe that CYP2C8*2,
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beside being an important marker for personalised medicine, is also a useful indicator of inter-ethnic differences among human populations. Finally, further studies are needed to assess the therapeutic impact of pharmacogenetic variants among the San in Southern Africa. Acknowledgements We thank all the children in this study, and their parents and teachers, for their understanding and assistance. Furthermore, we thank all the staff for technical assistance in the blood collection and DNA extraction. This work was based at the University of Botswana under the supervision and financial support from the Ministry of Health and in collaboration with the University of Pennsylvania. This publication was made possible through core services and support from the Penn Center for AIDS Research (CFAR), an NIH-funded program (P30 AI 045008). Finally, the authors would like to thank the four anonymous reviewers for their valuable comments and suggestions to improve the quality of the paper. They are also grateful to Dr. Federica Verra for a critical revision ofthe manuscript. References Adjei, G.O., Kristensen, K., Goka, B.Q., Hoegberg, L.C., Alifrangis, M., Rodrigues, O.P., Kurtzhals, J.A., 2008. Effect of concomitant artesunate administration and cytochrome P4502C8 polymorphisms on the pharmacokinetics of amodiaquine in Ghanaian children with uncomplicated malaria. Antimicrob. Agents Chemother. 52, 4400–4406, http://dx.doi.org/10.1128/AAC.00673-07. Alessandrini, M., Asfaha, S., Dodgen, T.M., Warnich, L., Pepper, M.S., 2013. Cytochrome P450 pharmacogenetics in african populations. Drug. Metab. Rev. 45, 253–275, http://dx.doi.org/10.3109/03602532.2013.783062. Bains, R.K., 2013. African variation at Cytochrome P450 genes: evolutionary aspects and the implications for the treatment of infectious diseases. Evol. Med. Public Health 2013, 118–134, http://dx.doi.org/10.1093/emph/eot010. Barnard, A., 1983. Contemporary hunter-gatherers: current theoretical issues in ecology and social organization. Annu. Rev. Anthropol. 12, 193–214. Botswana Ministry of Health Botswana malaria indicator survey 2012 report. Gaborone, Botswana, National Malaria Program, Ministry of Health, (2012). Bronte-Stewart, B., Budtz-Olsen, O.E., Hickley, J.M., Brock, J.F., 1960. The health and nutritional status of the Kung Bushmen of South West Africa. S. Afr. J. Lab. Clin. Med. 6, 187–216. Campbell, M.C., Tishkoff, S.A., 2008. African genetic diversity: implications for human demographic history, modern human origins, and complex disease mapping. Annu. Rev. Genomics Hum. Genet. 9, 403–433, http://dx.doi.org/10. 1146/annurev.genom.9.081307.164258. Cascorbi, I., 2006. Genetic basis of toxic reactions to drugs and chemicals. Toxicol. Lett. 162, 16–28. Cavaco, I., Strömberg-Nörklit, J., Kaneko, A., Msellem, M.I., Dahoma, M., Ribeiro, V.L., Bjorkman, A., Gil, J.P., 2005. CYP2C8 polymorphism frequencies among malaria patients in Zanzibar. Eur. J. Clin. Pharmacol. 61, 15–18. Cavaco, I., Mårtensson, A., Fröberg, G., Msellem, M., Björkman, A., Gil, J.P., 2013. CYP2C8 status of patients with malaria influences selection of Plasmodium falciparum pfmdr1 alleles after amodiaquine-artesunate treatment. J. Infect. Dis. 207, 687–688, http://dx.doi.org/10.1093/infdis/jis736. Daar, A.S., Singer, P.A., 2005. Pharmacogenetics and geographical ancestry: implications for drug development and global health. Nat. Rev. Genet. 6, 241–246. Dai, D., Zeldin, D.C., Blaisdell, J.A., Chanas, B., Coulter, S.J., Ghanayem, B.I., Goldstein, J.A., 2001. Polymorphisms in human CYP2C8 decrease metabolism of the anticancer drug paclitaxel and arachidonic acid. Pharmacogenetics 11, 597–607. Daily, E.B., Aquilante, C.L., 2009. Cytochrome P450 2C8 pharmacogenetics: a review of clinical studies. Pharmacogenomics 10, 1489–1510, http://dx.doi. org/10.2217/pgs.09.82. Friscella, K., Fremont, A.M., 2006. Use of geocoding and surname analysis to estimate race and ethnicity. Health Serv. Res. 41, 1482–1500. Garruto, R.M., Little, M.A., James, G.D., Brown, D.E., 1999. Natural experimental models: the global search for biomedical paradigms among traditional, modernizing, and modern populations. Proc. Natl. Acad. Sci. U. S. A. 96, 10536–10543. Gonzalez, F.J., Nebert, D.W., 1990. Evolution of the P450 gene superfamily: animal-plant ‘warfare’, molecular drive and human genetic differences in drug oxidation. Trends Genet. 6, 182–186. Higman, B.W., 2011. How Food Made History, first ed. Wiley-Blackwell, Chichester, UK. Jenkins, T., Lehmann, H., Nurse, G.T., 1974. Public health and genetic constitution of the San (Bushmen): carbohydrate metabolism and acetylator status of the Kung of Tsumkwe in the North-western Kalahari. Br. Med. J. 2, 23–26.
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Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Cytochrome P450 2C8*2 allele in Botswana: Human genetic diversity and public health implications Thato Motshoge a,b , Leabaneng Tawe b,c , Charles Waithaka Muthoga b,c , Joel Allotey d , Rita Romano e , Isaac Quaye f , Giacomo Maria Paganotti b,c,g,∗ a
Botswana Ministry of Health, Plot 54609, 24 Amos Street, Gaborone, Botswana Medical Education Partnership Initiative (MEPI) Laboratory, Private Bag UB 00712, Gaborone, Botswana c Botswana-University of Pennsylvania Partnership, P.O. Box AC 157 ACH, Gaborone, Botswana d Department of Oncology, University of Sheffield, Beech Hill Road, S10 2RX Sheffield, UK e Department of Public Health and Infectious Diseases, University of Rome “La Sapienza”, P.le Aldo Moro 5, 00185 Rome, Italy f Department of Biochemistry, University of Namibia School of Medicine, Private Bag 13301, Windhoek, Namibia g Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6073, USA b
a r t i c l e
i n f o
Article history: Received 24 July 2015 Received in revised form 18 January 2016 Accepted 27 January 2016 Available online 1 February 2016 Keywords: Botswana Bantu ethnic group CYP2C8 Pharmacogenetics San ethnic group
a b s t r a c t Human cytochrome P450 2C8 is a highly polymorphic gene and shows variation according to ethnicity. The CYP2C8*2 is a slow drug metabolism allele and shows 10–24% frequency in Black populations. The objective of this study was to assess the prevalence of CYP2C8*2 allele in Botswana among the San (or Bushmen) and the Bantu ethnic groups. For that purpose we recruited 544 children of the two ethnicities in three districts of Botswana from primary schools, collected blood samples, extracted DNA and genotyped them through PCR-based restriction fragment length polymorphism analysis. The results demonstrated that in the San the prevalence of the CYP2C8*2 allele is significantly higher than among the Bantu-related ethnic groups (17.5% and 8.5% for San and Bantu, respectively; P = 0.00002). These findings support the evidence of a different genetic background of the San with respect to Bantu-related populations, and highlight a possible higher risk of longer drug clearance or poor level of activation of pro-drugs among the San group. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Exploring pharmacogenetics in different African ethnic groups can lead to a better understanding of the evolutionary history and the adaptation of humans to their environment. In all life forms (from bacteria to multicellular organisms) several detoxifying enzyme systems act to reduce the harmful effects of molecules that organisms introduce into the cells/body through nutrition or that are present in the environment (Gonzalez and Nebert, 1990). The detoxifying enzymes belong to several super-families of molecules and are actually of relevant importance in modern drug’s metabolism, inactivation and clearance and they are often responsible of the side effects and adverse reactions, and ultimately of the therapeutic response (Cascorbi, 2006; Bains, 2013). During the evolution of modern humans, farming, nomadic pastoralism, hunter-gathering imposed different chemical microenvironments to the populations often allowing several toxic
∗ Corresponding author at: P.O. BOX AC 157 ACH, Gaborone, Botswana. E-mail address: [email protected] (G.M. Paganotti). http://dx.doi.org/10.1016/j.actatropica.2016.01.028 0001-706X/© 2016 Elsevier B.V. All rights reserved.
molecules to be present in the diet, and this was more true in the adaptive challenge of the hunter-gatherers groups as the San or Bushmen in Southern Africa (Wyndham and Morrison, 1958; Bronte-Stewart et al., 1960; Barnard, 1983; Garruto et al., 1999; Higman, 2011). In literature, there are very few studies on San pharmacogenetics. One of such studies relates to the metabolic ratio of debrisoquine, the probe substrate for cytochrome P450 2D6 (CYP2D6) activity. It has been reported that the San population accounts for a double of the rate of debrisoquine poor metabolisers compared to Caucasians (19% vs 8–10%) (Sommers et al., 1988) and also showed different metabolic activity for metoprolol, another substrate for testing CYP2D6 activity (Sommers et al., 1989). Furthermore, a study on acetylator status among the San of North-Western Kalahari revealed that up to 20% of San may be slow inactivators of isoniazid (Jenkins et al., 1974), a drug still widely used for tuberculosis therapy and prophylaxis. Comparative studies on the pharmacogenetics of this population could help to shed light on its genetic structure and could also inform future personalised therapies, since most modern drugs, developed on European populations, could have little and/or toxic
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effect among this and other related Southern African ethnic groups, raising potential population-specific pharmacogenetic incompatibilities of certain drugs that are globally prescribed (Daar and Singer, 2005). Cytochrome P450 enzymes are a family of enzymes ubiquitously expressed and involved in the metabolism of eicosanoids, sterols, drugs and xenobiotics. Among them, cytochrome P450 2C8 enzyme (CYP2C8) is a member of the human CYP2C enzyme family, which also includes CYP2C9, CYP2C18 and CYP2C19, whose genes are located on chromosome 10q24 (Daily and Aquilante, 2009). CYP2C8 gene is known to be polymorphic, and the distribution of allelic variants differs among populations (Totah and Rettie, 2005), thus could serve as a useful marker to distinguish and discriminate among ethnic groups, ultimately providing recommendations in the perspective of personalised medicine. This is very important in Africa where human genetic variability is high (Campbell and Tishkoff, 2008; Alessandrini et al., 2013). CYP2C8*2 (Ile268Phe; rs11572103), the allelic variant most common in Africans, is related to a lower drug metabolism rate in subjects carrying at least one copy of the defective allele (Cavaco et al., 2005; Totah and Rettie, 2005). Subjects who are intermediate or poor metabolisers experience higher drug concentration in plasma, lower clearance, longer drug half-life and increased adverse side effects that potentially affect compliance and therapeutic outcomes (Wijnen et al., 2007). Several studies have reported the CYP2C8*2 allele frequency in Africa where it is known to have a distinctive inter-ethnic difference (Alessandrini et al., 2013). In the published reports on African populations the frequency varies in the range of 9.9–24.2% (Cavaco et al., 2005; Röwer et al., 2005; Parikh et al., 2007; Adjei et al., 2008; Kudzi et al., 2009; Paganotti et al., 2011, 2012; Alessandrini et al., 2013). Interestingly, CYP2C8*2 is virtually absent in non-African populations (Dai et al., 2001). Despite its effects on a range of different treatments, CYP2C8 genetic variation has been studied in African populations mainly for its impact on the antimalarial therapies based on the 4aminoquinolines, particularly amodiaquine (AQ) and chloroquine (CQ). Moreover, it was recently reported (Paganotti et al., 2011) and confirmed by an independent group (Cavaco et al., 2013), that
decreased CYP2C8 activity due to poor metaboliser alleles may expose Plasmodium falciparum parasites to a sub-therapeutic level of drug for longer time driving the selection of drug resistance. In recent years, a number of Southern African countries including Botswana have targeted malaria elimination attributed largely to P. falciparum. The National Malaria Control Program has approached malaria elimination through intensive strategies (Simon et al., 2013) including drug treatment for uncomplicated cases with artemether-lumefantrine (AL) as first line artemisinin combination therapy (ACT). Moreover, a combined regime of CQ and proguanil continues to be recommended for pregnant women in risk areas (Botswana Ministry of Health, 2012). Currently, a switch from quinine (QN) to artesunate/amodiaquine (AL/AQ) as second line ACT is under evaluation. These considerations highlight the need to examine the CYP2C8 genetic polymorphism in the population vis-à-vis the role of the enzyme in the metabolism of the 4-aminoquinolines. Human CYP2C8 enzyme is also involved in the metabolism of a variety of clinically important drugs such as trans-retinoic acid (leukemia treatment), paclitaxel (cancer), diclofenac and ibuprofen (pain and inflammation), loperamide (antidiarrheal), morphine (pain), verapamil (hypertension), carbamazepine (epilepsy) (Lai et al., 2009). These drugs are used extensively in the hospitals and clinics in Botswana. For all these reasons, we screened three different districts of Botswana for the CYP2C8*2 allele to inform decision on the use of the 4-aminoquinolines not only towards malaria elimination but also on populations at risk of toxicity during the deployment of these drugs. In the selected areas the populations are largely of Bantu (Serowe/Palapye and Chobe districts) and San (also called Basarwa or Bushmen, in Ghanzi district, Central Kalahari) ethnicity. Importantly, this report is the first CYP enzyme investigation that also includes reference to ethnicity in Botswana.
2. Material and methods The survey was performed in March 2012 in Botswana in the broader context of a Malaria Indicator Survey (Botswana Ministry of Health, 2012). A total of 544 children asymptomatic for malaria were enrolled in several primary schools: 160 from Ghanzi dis-
Fig. 1. Map of Botswana with the three districts indicated. Source: Google Earth, accessed on 11th December 2015.
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Table 1 CYP2C8 genotypes and T allele frequency in subjects from three districts of Botswana and two different ethnic groups. Districts (ethnicity)
CYP2C8 (rs11572103, A > T) frequencies N
Serowe/Palapye(Bantu ethnic group) (Bantu ethnic group) Chobe (Bantu ethnic group) S/P + C Ghanzi (San ethnic group)
227 157 384 160
Allele frequency ± SE
Relative and (absolute) genotype frequencies AA
AT
TT
0.855 (194) 0.802 (126) 0.833 (320) 0.675 (108)
0.145 (33) 0.192 (30) 0.164 (63) 0.300 (48)
0.000 (0) 0.006 (1) 0.003 (1) 0.025 (4)
T 0.073 ± 0.017 0.102 ± 0.024 0.085 ± 0.014 0.175 ± 0.030
S/P: Serowe/Palapye district; C: Chobe district; N: number of individual samples.
trict, 227 from Serowe/Palapye district and 157 from Chobe district (Fig. 1). The ethnicity was assessed from family names in each location according to Friscella and Fremont (2006) and Mateos (2007) and village of birth (excluding all the foreigners or born in other districts). The same protocol for enrolment was followed in all sites. Signed informed consent for multiple genetic and epidemiological surveys was obtained from all the subjects’ parents/caregivers included in the study. This study was conducted in accordance with the guidelines of the Helsinki Declaration of 2000, with the approval of the Human Research and Development Division of the Botswana Ministry of Health [PPME-13/18 V (380)] and the Institutional Review Board of the University of Pennsylvania [protocol number 820378]. Three ml of whole blood was collected into EDTA-containing tubes by physicians and trained phlebotomists. DNA was extracted with Qiagen kits according to the manufacturer’s procedure. CYP2C8*2 (rs11572103, A > T) detection was carried out using the PCR-RFLP technique (Paganotti et al., 2011). Briefly, we started from 2 l of human DNA to amplify a 107 bp fragment of the CYP2C8 gene containing the polymorphism of interest using the following primers: forward 5 -GAACACCAAGCATCACTGGA-3 and reverse 5 -GAAATCAAAATACTGATCTGTTGC-3. The PCR product was subsequently digested with BclI enzyme that cuts the wild-type allele only (A); undigested products represent the variant allele (T). To detect the size polymorphisms, the samples were ran on a 3% agarose gel (Fig. 2). Controls for human genotyping were utilized after sequencing three PCR products relative to each different genotype. Moreover, to avoid the genotyping error, the heterozygous samples were repeated. Inter-populations comparisons were obtained by Yates corrected 2 test. Odds ratios (ORs) were calculated with 95% confidence intervals (CIs). The analyses were performed with Epi info 6 statistical package (freely available at https://wwwn.cdc.gov/ epiinfo/html/ei6downloads.htm) Evaluation of Hardy-Weinberg equilibrium was performed using the HWSIM software (freely available athttp://krunch.med.yale.edu/hwsim/) and Monte-Carlo permutation test performed when genotypic classes had an expected cell size of less than five.
3. Results The frequencies of the CYP2C8*2 allele for each district included in the present study are presented in Table 1. Genotype frequencies were in Hardy-Weinberg equilibrium after a Monte Carlo permutation test (10,000 runs) in all the three districts (P = 0.158, P = 0.213 and P = 0.135 respectively). No statistically significant difference was found comparing the allele frequency between Serowe/Palapye and Chobe (Yates corrected 2 = 1.686, P = 0.1526). Considering that these districts are largely of Bantu ethnicity, we pooled together the results and evaluated Hardy-Weinberg equilibrium after a Monte Carlo permutation test (10,000 runs) (P = 0.09). A striking difference was revealed when the pooled genotypic classes of the two Bantu districts were compared with the Ghanzi district
Fig. 2. Agarose gel (3%) showing BclI digestion of the PCR products and restriction pattern. Line 1: 25 bp marker; Line 2: undigested PCR product (107 bp); Line 3: AA genotype positive control [wild-type homozygous (56 bp + 51 bp)]; Line 4: AT genotype positive control [heterozygous (107 bp + 56 bp + 51 bp)]; Line 5: TT genotype positive control [mutant homozygous (107 bp)]; Line 6: 25 bp marker; Line 7: sample 7201195 (AA genotype); Line 8: sample 50282 (AT genotype); Line 9: sample 8001881 (AA genotype); Line 10: sample 80908 (TT genotype); Line 11: sample 7201189 (AA genotype); Line 12: sample 5001165 (AT genotype); Line 13: 25 bp marker; Line 14: negative control.
(Yates corrected 2 = 18.64, P = 0.0000251; OR = 0.44[0.29–0.65]). In addition, the percentage of subjects carrying at least one copy of the CYP2C8*2 allele (T) was 16.67% and 32.50% for Banturelated subjects and San, respectively (Yates corrected 2 = 15.95, P = 0.0000652; OR = 0.42[0.27–0.65]). 4. Discussion So far our study is the first report of CYP2C8*2 allele distribution comparing two different ethnic groups, San and Bantu, of Botswana. Overall, the allele frequency of CYP2C8*2 was 17.5% in the San and 8.5% in the Bantu. In general, the allele frequency from the two Bantu-inhabited sites (Serowe/Palapye and Chobe combined) are slightly below the lower value in the documented range for Africa (9.9–24.2% frequency, according to Alessandrini et al., 2013), whereas the data from the San ethnic group falls within the reported range for the allele (Alessandrini et al., 2013). Several investigations in Africa have shown similar allele frequency as the San, e.g. in Northern Ghana (16.8%) (Röwer et al., 2005), in Madagascar (15%) (Paganotti et al., 2012), in Mozambique (15.6%) (Suarez-Kurtz, 2011) and in African-Americans (18%) (Dai et al., 2001). Instead, the Bantu of Botswana show comparable allele frequency as the Fulani of Burkina Faso (9.9%) (Paganotti et al., 2011) and two other populations of Uganda (10.5%) (Paganotti et al., 2012). Notably, even though similar allele frequencies exist among
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different populations all over the continent, it does not necessarily indicate a similar genetic background or a common ancestor, rather it is more important to evaluate the possible interethnic differences within the same country or territory as we have done for the San and Bantu in Botswana. Interestingly, when combining the genotype results from Serowe/Palapye and Chobe, the probability associated to the Monte Carlo permutation test for the Hardy-Weinberg equilibrium dropped to P = 0.09. This result could suggest a marginal tendency for deviation from equilibrium, perhaps due to another prominent allele, the known linkage between CYP2C8 and CYP2C9 genes (Speed et al., 2009) or the presence of several subgroups into the Bantu ethnicity (e.g., Tswana, Bangwato, Mbukushu, Bayei). Importantly, we observed that the percentage of the San population carrying at least one copy of this allele accounts for almost one third of the subjects (AT = 30%, TT = 2.5%; T carriers = 32.5%). In this study very few individuals were homozygous null (0–2.5%), which has already been documented by other studies in African populations (Alessandrini et al., 2013). The pharmacological implications of being heterozygous or homozygous for the defective CYP2C8*2 allele are not well studied, and few reports show that there is also substrate variability with regard to drug disposition and response. A study on AQ in Burkina Faso showed a significant increased rate of adverse events in both heterozygotes and homozygotes for the variant CYP2C8*2 genotypes compared with the wild-type genotype (52% vs 30%, P < 0.01) (Parikh et al., 2007). Moreover, the recombinant CYP2C8*2 enzyme showed a defective metabolism of AQ (3-fold higher Km and 6-fold lower intrinsic clearance than the functional enzyme) in vitro (Parikh et al., 2007). The paper from Paganotti et al. (2011) reported a statistically significant trend among the three different genotypes for carrying CQ-resistant P. falciparum parasites in Burkina Faso (38.1%, 50.0%, 54.2% for AA, AT, TT genotypes, respectively, P = 0.02), an observation that might be explained by the pharmacokinetic of CQ. In another report, the recombinant CYP2C8*2 was associated with the defective metabolism (2-fold higher Km and 2-fold lower intrinsic clearance than the wild-type) of the anticancer drug paclitaxel in vitro (Dai et al., 2001). Despite the fact that this argument needs further studies and in vivo data, it seems that the heterozygous genotype has an intermediate metaboliser phenotype for AQ, CQ and paclitaxel (Bains, 2013). Since in our study the majority of the carriers of the defective allele are the heterozygous subjects, we are aware that the metabolic status, although dependent on the drug used, will be affected and decreased to a certain level. Therefore, the relatively high prevalence of intermediate and poor metaboliser subjects for CYP2C8-metabolised drugs in the San population, predisposes them to a higher risk of side effects for 4aminoquinolines than the Bantu ethnic group in Botswana. This could impact negatively on the drug compliance and results in a lower efficacy of 4-aminoquinolines. This observation is of significant interest as the San are spread throughout the Southern African region. We suggest that strategies for malaria elimination will have to take this into consideration when dealing with this ethnic group. The same will apply for the other drugs that are substrates for the CYP2C8 enzyme. Our study has some limitations. The sample size for San ethnicity is relatively small. Moreover, pharmacokinetic and pharmacodynamic data on the participants were not available, since they were healthy subject and not under treatment.
5. Conclusions We conclude that CYP2C8*2 polymorphism in the San population of Botswana is present at higher frequency than among Bantu-related communities. In addition, we believe that CYP2C8*2,
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beside being an important marker for personalised medicine, is also a useful indicator of inter-ethnic differences among human populations. Finally, further studies are needed to assess the therapeutic impact of pharmacogenetic variants among the San in Southern Africa. Acknowledgements We thank all the children in this study, and their parents and teachers, for their understanding and assistance. Furthermore, we thank all the staff for technical assistance in the blood collection and DNA extraction. This work was based at the University of Botswana under the supervision and financial support from the Ministry of Health and in collaboration with the University of Pennsylvania. This publication was made possible through core services and support from the Penn Center for AIDS Research (CFAR), an NIH-funded program (P30 AI 045008). Finally, the authors would like to thank the four anonymous reviewers for their valuable comments and suggestions to improve the quality of the paper. They are also grateful to Dr. Federica Verra for a critical revision ofthe manuscript. References Adjei, G.O., Kristensen, K., Goka, B.Q., Hoegberg, L.C., Alifrangis, M., Rodrigues, O.P., Kurtzhals, J.A., 2008. Effect of concomitant artesunate administration and cytochrome P4502C8 polymorphisms on the pharmacokinetics of amodiaquine in Ghanaian children with uncomplicated malaria. Antimicrob. Agents Chemother. 52, 4400–4406, http://dx.doi.org/10.1128/AAC.00673-07. Alessandrini, M., Asfaha, S., Dodgen, T.M., Warnich, L., Pepper, M.S., 2013. Cytochrome P450 pharmacogenetics in african populations. Drug. Metab. Rev. 45, 253–275, http://dx.doi.org/10.3109/03602532.2013.783062. Bains, R.K., 2013. African variation at Cytochrome P450 genes: evolutionary aspects and the implications for the treatment of infectious diseases. Evol. Med. Public Health 2013, 118–134, http://dx.doi.org/10.1093/emph/eot010. Barnard, A., 1983. Contemporary hunter-gatherers: current theoretical issues in ecology and social organization. Annu. Rev. Anthropol. 12, 193–214. Botswana Ministry of Health Botswana malaria indicator survey 2012 report. Gaborone, Botswana, National Malaria Program, Ministry of Health, (2012). Bronte-Stewart, B., Budtz-Olsen, O.E., Hickley, J.M., Brock, J.F., 1960. The health and nutritional status of the Kung Bushmen of South West Africa. S. Afr. J. Lab. Clin. Med. 6, 187–216. Campbell, M.C., Tishkoff, S.A., 2008. African genetic diversity: implications for human demographic history, modern human origins, and complex disease mapping. Annu. Rev. Genomics Hum. Genet. 9, 403–433, http://dx.doi.org/10. 1146/annurev.genom.9.081307.164258. Cascorbi, I., 2006. Genetic basis of toxic reactions to drugs and chemicals. Toxicol. Lett. 162, 16–28. Cavaco, I., Strömberg-Nörklit, J., Kaneko, A., Msellem, M.I., Dahoma, M., Ribeiro, V.L., Bjorkman, A., Gil, J.P., 2005. CYP2C8 polymorphism frequencies among malaria patients in Zanzibar. Eur. J. Clin. Pharmacol. 61, 15–18. Cavaco, I., Mårtensson, A., Fröberg, G., Msellem, M., Björkman, A., Gil, J.P., 2013. CYP2C8 status of patients with malaria influences selection of Plasmodium falciparum pfmdr1 alleles after amodiaquine-artesunate treatment. J. Infect. Dis. 207, 687–688, http://dx.doi.org/10.1093/infdis/jis736. Daar, A.S., Singer, P.A., 2005. Pharmacogenetics and geographical ancestry: implications for drug development and global health. Nat. Rev. Genet. 6, 241–246. Dai, D., Zeldin, D.C., Blaisdell, J.A., Chanas, B., Coulter, S.J., Ghanayem, B.I., Goldstein, J.A., 2001. Polymorphisms in human CYP2C8 decrease metabolism of the anticancer drug paclitaxel and arachidonic acid. Pharmacogenetics 11, 597–607. Daily, E.B., Aquilante, C.L., 2009. Cytochrome P450 2C8 pharmacogenetics: a review of clinical studies. Pharmacogenomics 10, 1489–1510, http://dx.doi. org/10.2217/pgs.09.82. Friscella, K., Fremont, A.M., 2006. Use of geocoding and surname analysis to estimate race and ethnicity. Health Serv. Res. 41, 1482–1500. Garruto, R.M., Little, M.A., James, G.D., Brown, D.E., 1999. Natural experimental models: the global search for biomedical paradigms among traditional, modernizing, and modern populations. Proc. Natl. Acad. Sci. U. S. A. 96, 10536–10543. Gonzalez, F.J., Nebert, D.W., 1990. Evolution of the P450 gene superfamily: animal-plant ‘warfare’, molecular drive and human genetic differences in drug oxidation. Trends Genet. 6, 182–186. Higman, B.W., 2011. How Food Made History, first ed. Wiley-Blackwell, Chichester, UK. Jenkins, T., Lehmann, H., Nurse, G.T., 1974. Public health and genetic constitution of the San (Bushmen): carbohydrate metabolism and acetylator status of the Kung of Tsumkwe in the North-western Kalahari. Br. Med. J. 2, 23–26.
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