http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, 2014; 44(6): 579–582 ! 2014 Informa UK Ltd. DOI: 10.3109/00498254.2013.866300
RESEARCH ARTICLE
Frequency and genotype distribution of ABCB1 gene polymorphisms among Maharashtrian population of Central India Sreemanta Pramanik1,2, Subin T. Surendran2, Saravana Devi2, Kannan Krishnamurthi2, and Tapan Chakrabarti2 Kolkata Zonal Laboratory, CSIR-National Environmental Engineering Research Institute, Kolkata, West Bengal, India and 2Environmental Health Division, National Environmental Engineering Research Institute, Nagpur, Maharashtra, India
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1
Abstract
Keywords
1. Apart from conferring multidrug resistance to cancer cells, P-glycoprotein (P-gp) encoded by the gene ABCB1 (also, known as Multidrug resistance gene, MDR1), plays a major role in drug disposition. Single nucleotide polymorphisms (SNPs) in the ABCB1 gene might contribute to inter-individual and ethnic differences in drug disposition and thereby, could influence the outcome and prognosis of certain diseases. 2. India is one of the most ethnically and genetically diverse regions of the world. This study was undertaken with a view to determine the allele and genotype frequencies of C3435T and C1236T polymorphisms in the ABCB1 gene among the Maharshtrian population, residing in the Vidarbha region of central India and compare them with HapMap and other Indian populations. The common synonymous C3435T polymorphism has been found to be associated with lower P-gp functional expression and drug uptake, alone or in conjunction with a few other linked SNPs like C1236T. 3. The genotypes of C3435T and C1236T SNPs were determined by PCR-RFLP in 222 healthy and unrelated Maharashtrian individuals. 4. According to the findings of this study, the Maharashtrians were found to be not significantly different from the Gujarati Indians in Houston, Texas in the HapMap database.
Drug disposition, HapMap, P-glycoprotein, SNP
Introduction One of the greatest challenges of the post-genomic sequencing era is to develop a detailed understanding of the heritable variation present in the human genome. By characterizing genetic variations among individuals and populations, we stand a better chance of understanding the differential susceptibility to diseases, differential response to pharmacological agents and complex interaction between genetic and environmental components in producing phenotypes. Single nucleotide polymorphisms (SNPs), the most common form of sequence variation, are highly abundant and account for more than 90% of all genetic variations in the human genome. More than 10 million SNPs are estimated to be present in the human genome and about half of these SNPs are expected to be common with minor allele frequency of greater than 10%. SNPs could serve as useful markers to identify genes that predispose individuals to common, multifactorial disorders by
Address for correspondence: Dr. Sreemanta Pramanik, Principal Scientist & Head, Kolkata Zonal Laboratory, CSIR-NEERI, Kolkata – 700107, West Bengal, India. Tel: 91-033-24421988. Fax: 91-033-24417608. E-mail: [email protected]/sreemanta@ gmail.com
History Received 9 October 2013 Revised 12 November 2013 Accepted 12 November 2013 Published online 6 December 2013
using linkage disequilibrium mapping (Risch & Merikangas, 1996). The human ABCB1 gene (also, known as Multidrug resistance gene, MDR1) is located on the long arm of chromosome 7 at 7q21 and has 28 exons. The ABCB1 gene encodes a 170-kDa plasma membrane glycoprotein [P-glycoprotein (P-gp)], a member of the ATP-binding cassette (ABC) super family of transporters. Acting as an energy-dependent efflux pump, P-glycoprotein plays a major role in drug disposition and protects cells against toxic xenobiotics, in particular due to its co-localization with the major drug metabolizing enzyme, CYP3A4, in the small intestine and liver (Ayrton & Morgan, 2001). A wide variety of drugs including anti-cancer drugs, cardiovascular drugs, HIV protease inhibitors, immunosuppressants and b-adrenoreceptor antagonists are transported via P-gp. Moreover, P-gp confers additional protection to sensitive organs like brain, testis or fetus from exposure to toxic compounds due to its expression in the respective blood-tissue barriers. Presently, more than 100 SNPs have been identified in the ABCB1 gene (Kerb, 2006). These genetic polymorphisms might contribute to inter-individual and inter-ethnic variability in disposition of various drugs that are P-gp substrates and consequently, could influence the outcome and prognosis of certain diseases. Widespread variation in allele frequencies has been observed for the most common polymorphisms,
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C3435T (rs1045642), C1236T (rs1128503) and G2677T/A (rs2032582) of the ABCB1 gene among different ethnic groups (Kim et al., 2001). The synonymous C3435T polymorphism in the exon 26 of ABCB1, which gives rise to a silent mutation (Ile > Ile), was found to have a statistically significant correlation with P-gp expression and activity and oral bioavailability of digoxin in Caucasians (Hoffmeyer et al., 2000). Individuals with the CC genotype had about two fold higher P-gp expression in the small intestine in comparison to the individuals with the TT genotype. Another study by Schwab et al. (2003) found significantly increased frequencies of the 3435T allele and 3435TT genotype in patients with ulcerative colitis as compared to the controls. A few studies also reported an association between the C3435T polymorphism and psychiatric and mood disorders (Turgut et al., 2009; Yasui-Furukori et al., 2006). C3435T polymorphism was found to be linked to another synonymous C1236T polymorphism in the exon 12, giving rise to a silent mutation (Gly > Gly) and a nonsynonymous G2677T/A polymorphism in the exon 21, causing amino acid substitution (Ala > Ser/Thr; Tang et al., 2002) and also, seemed to influence P-gp function in a haplotype-dependent manner (Salama et al., 2006). The silent C1236T and the non-synonymous G2677T/A polymorphisms have also been linked to several diseases like pharmacoresistant epilepsy and Parkinsons disease (KimchiSarfaty et al., 2007). India represents a vast collection of peoples with different morphological, cultural, linguistic and genetic diversity. Studies on the ABCB1 gene polymorphisms in Indian populations are scanty (Grover et al., 2010; Ramasamy et al., 2006; Vahab et al., 2009). This study was aimed at determining the allele and genotype frequencies of the C3435T and C1236T polymorphisms in the ABCB1 gene among the Maharshtrian population, residing in the Vidarbha region of central India and comparing them with HapMap and other Indian populations. To the best of our knowledge, this study is the first of its kind on the ABCB1 gene polymorphisms in a central Indian population.
Materials and methods Study population The study was approved by the Institutional Ethical Committee. The study population consisted of 222 healthy and unrelated Maharashtrian individuals in the age group of 21–50, residing in the Vidarbha region of central India. After obtaining prior informed consents, 5 ml of blood sample was collected from each subject. DNA extraction and genotyping of samples Genomic DNA samples were extracted from whole blood of subjects using a standard lysis procedure and amplified by PCR using respective primers for the ABCB1 C3435T (rs1045642; Calado et al., 2002) and C1236T polymorphisms (rs1128503; Hoffmeyer et al., 2000). The genotypes of the SNPs were determined by digesting the amplified products with appropriate restriction enzymes (DpnII for C3435T and EcoO109I for C1236T, respectively) and running the digested products in 2.5% agarose gel.
Xenobiotica, 2014; 44(6): 579–582
Statistical analysis The observed genotype distributions of the ABCB1 C3435T and C1236T polymorphisms were compared to their expected values by performing Chi square (2) test. The allele and genotype frequencies of polymorphisms in the Maharashtrian population were compared to some of the populations in the HapMap database (www.hapmap.org) e.g. Utah residents with Northern and Western European ancestry from the CEPH collection (CEU), Yoruban in Ibadan, Nigeria (YRI), Han Chinese in Beijing, China (CHB), Gujarati Indians in Houston, Texas (GIH), Japanese in Tokyo, Japan (JPT) and some other Indian populations selected from literature e.g. South Indian population from Tamilnadu State (SInd; Ramasamy et al., 2006), North Indian population from Delhi State (NInd; Grover et al., 2010), Western Indian population from Pune, Maharashtra State (WInd; Ghodke et al., 2011). Pair-wise Chi square (2) tests were performed between Maharashtrians and these populations using the allele frequencies in a 2X2 contingency table to find whether the Maharashtrian population was significantly different from others.
Results The genotypes of the C3435T and C1236T SNPs were determined in 222 healthy and unrelated Maharashtrian individuals. Allele and genotype frequencies of ABCB1 C3435T (Ile > Ile) polymorphism The observed C/C, C/T and T/T genotype frequencies of the SNP in the Maharashtrian population were 0.18, 0.473, and 0.347, respectively (Table 1). The C (wild-type) and T (variant) allele frequencies were 0.42 and 0.58, respectively. The variant allele frequency was lowest (0.107) among the Yorubans and highest (0.64) among the North Indians. Significant differences were found between the Maharashtrians and YRI, CHB and JPT populations (Table 1). Allele and genotype frequencies of ABCB1 C1236T (Gly > Gly) polymorphism The observed C/C, C/T and T/T genotype frequencies of the SNP in the Maharashtrian population were 0.135, 0.541 and 0.347, respectively (Table 2). The C (wild-type) and T (variant) allele frequencies were found to be 0.41 and 0.59, respectively. The variant allele frequency was lowest (0.122) among the Yorubans and highest (0.695) among the Han Chinese. Pair-wise Chi-square (2) test revealed a significant difference between the Maharashtrians and CEU, YRI and CHB populations (Table 2). The allele and genotype frequency data was not available for any South Indian populations.
Discussion Using mitochondrial, Y-chromosomal and limited autosomal markers, several studies have demonstrated extensive genetic diversity in India pertinent to the issues of origin and migrations of populations (Bamshad et al., 2001; Basu et al., 2003; Sengupta et al., 2006). A recent study carried out by the
ABCB1 gene polymorphisms among Maharashtrians
DOI: 10.3109/00498254.2013.866300
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Table 1. Allele and genotype frequencies of ABCB1 C3435T in HapMap and Indian populations including Maharashtrians. Genotype
Allele frequency
Population
Geno
Freq (No)
Geno
Freq (No)
Geno
Freq (No)
Allele
Freq
Allele
Freq
Pairwise 2 test value between MAH and other populations
CEU (n ¼ 113)a YRI (n ¼ 145)a CHB (n ¼ 135)a GIH (n ¼ 101)a JPT (n ¼ 113)a SInd (n ¼ 185) NInd (n ¼ 93) WInd (n ¼ 144) MAH (n ¼ 222)
C/C C/C C/C C/C C/C C/C C/C C/C C/C
0.15 (17) 0.8 (116) 0.4 (54) 0.178 (18) 0.283 (32) 0.18 (33) 0.13 (12) 0.146 (21) 0.18 (40)
C/T C/T C/T C/T C/T C/T C/T C/T C/T
0.558 (63) 0.186 (27) 0.452 (61) 0.475 (48) 0.531 (60) 0.56 (104) 0.462 (43) 0.472 (68) 0.473 (105)
T/T T/T T/T T/T T/T T/T T/T T/T T/T
0.292 (33) 0.014 (2) 0.148 (20) 0.347 (35) 0.186 (21) 0.26 (48) 0.408 (38) 0.382 (55) 0.347 (77)
C C C C C C C C C
0.429 0.893 0.626 0.416 0.549 0.46 0.36 0.38 0.42
T T T T T T T T T
0.571 0.107 0.374 0.584 0.451 0.54 0.64 0.62 0.58
0.025b 82.252 14.25 0.004b 5.011 0.656b 0.981b 0.58b –
Wild-type allele
Variant allele
a
Population data source: HapMap (www.hapmap.org). Chi-square test statistic value less than 3.841 at 5% significance level, so populations are not significantly different from MAH. Population descriptions: CEU, Utah residents with Northern and Western European ancestry from the CEPH collection; YRI, Yoruban in Ibadan, Nigeria; CHB, Han Chinese in Beijing, China; GIH, Gujarati Indians in Houston, Texas; JPT, Japanese in Tokyo, Japan; Sind, South Indian population from Tamilnadu, India (Ramasamy et al., 2006); NInd, North Indian population from Delhi, India (Grover et al., 2010); Wind, Western Indian population from Pune, India (Ghodke et al., 2011); MAH, Maharashtrian population residing in Vidarbha region of central India (this study).
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b
Table 2. Allele and genotype frequencies of ABCB1 C1236T in HapMap and Indian populations including Maharashtrians. Genotype
Allele frequency
Geno
Freq (No)
Geno
Freq (No)
Geno
Freq (No)
Allele
Freq
Allele
Freq
Pairwise 2 test value between MAH and other populations
C/C C/C C/C C/C C/C C/C C/C C/C
0.265 (30) 0.762 (112) 0.118 (16) 0.139 (14) 0.177 (20) 0.12 (12) 0.146 (21) 0.135 (30)
C/T C/T C/T C/T C/T C/T C/T C/T
0.566 (64) 0.231 (34) 0.375 (51) 0.545 (55) 0.478 (54) 0.45 (45) 0.472 (68) 0.541 (120)
T/T T/T T/T T/T T/T T/T T/T T/T
0.168 (19) 0.007 (1) 0.507 (69) 0.317 (32) 0.345 (39) 0.43 (43) 0.382 (55) 0.347 (72)
C C C C C C C C
0.549 0.878 0.305 0.411 0.416 0.345 0.38 0.41
T T T T T T T T
0.451 0.122 0.695 0.589 0.584 0.655 0.62 0.59
5.83 80.475 3.988 0.000b 0.011b 1.225b 0.328b –
Wild-type allele Population a
CEU (n ¼ 113) YRI (n ¼ 147)a CHB (n ¼ 136)a GIH (n ¼ 101)a JPT (n ¼ 113)a NInd (n ¼ 100) WInd (n ¼ 144) MAH (n ¼ 222)
Variant allele
a
Population data source: HapMap (www.hapmap.org). Chi-square test statistic value less than 3.841 at 5% significance level, so populations are not significantly different from MAH. Population descriptions: CEU, Utah residents with Northern and Western European ancestry from the CEPH collection; YRI, Yoruban in Ibadan, Nigeria; CHB, Han Chinese in Beijing, China; GIH, Gujarati Indians in Houston, Texas; JPT, Japanese in Tokyo, Japan; NInd, North Indian population from Delhi, India (Grover et al., 2010); Wind, Western Indian population from Pune, India (Ghodke et al., 2011); MAH, Maharashtrian population residing in Vidarbha region of central India (this study).
b
Indian Genome Variation Consortium, on 405 SNPs in 75 disease or drug response related genes and a 5.2 Mb genomic region on chromosome 22 spanning 49 genes, has revealed a high degree of genetic differentiation among different ethnic groups of India and found that Indian populations formed a continuing genetic spectrum, bridging two distinct HapMap populations, CEU and CHB/JPT (Consortium IGV, 2008). Vidarbha region, lying on the eastern side of the Maharashtra state, is broadly referred to as central India because of its central location. Linguistically, Marathi, spoken by the Maharashtrians, belongs to the Indo-Aryan language group of the Indo-European linguistic family. In this study, the observed genotype distributions followed Hardy–Weinberg expectations for the C3435T (p Value ¼ 0.68) and C1236T (p Value ¼ 0.07) SNPs in the Maharashtrian population. A wide variation in allele frequency and genotype distribution among different populations was observed for the ABCB1 C3435T and C1236T SNPs. For the C3435T polymorphism, the variant allele frequency in the Maharashtrians (0.58) was similar to the Utah residents with Northern and Western
European ancestry (CEU) and Gujarati Indians in Texas (GIH), but was higher than the Yorubans in Nigeria (YRI), Han Chinese in Beijing (CHB) and Japanese in Tokyo (JPT). The Maharashtrians (MAH) were found to be significantly different from the YRI, CHB and JPT, but not significantly different from the CEU and GIH in the HapMap database. For the C1236T polymorphism, the variant allele frequency in the Maharashtrians (0.59) was similar to the JPT and GIH, but lower than the CHB and higher than the YRI and CEU. The Maharashtrians were found to be significantly different from the CEU, YRI and CHB, but not significantly different from the JPT and GIH. The findings of our earlier study on DNA repair gene polymorphisms at XRCC1, XRCC3, XPD and OGG1 loci, also pointed out that the Maharashtrian population did not show significant difference to the CEU and GIH populations from the HapMap database (Pramanik et al., 2011). The Maharashtrians were found to be not significantly different from other Indian populations selected for this study (SInd, NInd, WInd for the C3435T and NInd, Wind for the C1236T, respectively). The fact that several different ethnic
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groups inhabit a vast region like North, South, East or West of India and subjects drawn from the study populations (namely, SInd, NInd and WInd) were selected randomly from the places of study rather than from a particular ethnic group could be the reason behind this finding.
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Conclusions Analyses of SNPs are becoming increasingly important in identifying genetic variants underlying susceptibility to human disease or other phenotypes. Knowledge of the basic prevalence of some of the ABCB1 gene polymorphisms in the general population, which have been directly correlated with P-gp expression and function, would be essential to estimate the efficacy of the drugs that are transported through P-gp. This study found a wide variation in allele frequency and genotype distribution of the ABCB1 C3435T and C1236T polymorphisms between the Maharashtrians and some HapMap populations. That the Maharashtrian population was not significantly different from the Gujarati Indians in Houston, Texas in the HapMap database was another major finding of the study. This study could form the basis of future investigations to explore the relationship between ABCB1 genotypes and drug efficacy, drug toxicity, disease susceptibility or other phenotypes in Indian populations.
Acknowledgements The authors wish to express their sincere thanks to the participants of the study.
Declaration of interest The study was financially supported by Council of Scientific and Industrial Research (CSIR). The authors declare that that there are no conflicts of interest in relation to this work.
References Ayrton A, Morgan P. (2001). Role of transport proteins in drug absorption, distribution and excretion. Xenobiotica 31:469–97. Bamshad M, Kivisild T, Watkins WS, et al. (2001). Genetic evidence on the origins of Indian caste populations. Genome Res 11:994–1004. Basu A, Mukherjee N, Roy S, et al. (2003). Ethnic India: a genomic view, with special reference to peopling and structure. Genome Res 13:2277–90. Calado RT, Franco RF, Zago MA, Falca˜o RP. (2002). MDR1 gene C3435T polymorphism and the risk of acquired aplastic anaemia. Br J Haematol 117:769.
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Consortium IGV. (2008). Genetic landscape of the people of India: a canvas for disease gene exploration. J Genet 87:3–20. Ghodke Y, Chopra A, Shintre P, et al. (2011). Profiling single nucleotide polymorphisms (SNPs) across intracellular folate metabolic pathway in healthy Indians. Indian J Med Res 133:274–9. Grover S, Bala K, Sharma S, et al. (2010). Absence of a general association between ABCB1 genetic variants and response to antiepileptic drugs in epilepsy patients. Biochimie 92:1207–12. Hoffmeyer S, Burk O, von Richter O, et al. (2000). Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc Natl Acad Sci USA 97:3473–8. Kerb R. (2006). Implications of genetic polymorphisms in drug transporters for pharmacotherapy. Cancer Lett 234:4–33. Kim RB, Leake BF, Choo EF, et al. (2001). Identification of functionally variant MDR1 alleles among European Americans and African Americans. Clin Pharmacol Ther 70:189–99. Kimchi-Sarfaty C, Marple AH, Shinar S, et al. (2007). Ethnicity-related polymorphisms and haplotypes in the human ABCB1 gene. Pharmacogenomics 8:29–39. Pramanik S, Devi S, Chowdhary S, et al. (2011). DNA repair gene polymorphisms at XRCC1, XRCC3, XPD, and OGG1 loci in Maharashtrian population of central India. Chemosphere 82:941–6. Ramasamy K, Sisy Sam S, Chandrasekaran A. (2006). Allele and genotype frequency of MDR1 C3435T in Tamilian population. Drug Metab Pharmacokinet 21:506–8. Risch N, Merikangas K. (1996). The future of genetic studies of complex human diseases. Science 273:1516–17. Salama NN, Yang Z, Bui T, Ho RJ. (2006). MDR1 haplotypes significantly minimize intracellular uptake and transcellular P-gp substrate transport in recombinant LLC-PK1 cells. J Pharm Sci 95: 2293–308. Schwab M, Schaeffeler E, Marx C, et al. (2003). Association between the C3435T MDR1 gene polymorphism and susceptibility for ulcerative colitis. Gastroenterology 124:26–33. Sengupta S, Zhivotovsky LA, King R, et al. (2006). Polarity and temporality of high-resolution y-chromosome distributions in India identify both indigenous and exogenous expansions and reveal minor genetic influence of Central Asian pastoralists. Am J Hum Genet 78: 202–21. Tang K, Ngoi SM, Gwee PC, et al. (2002). Distinct haplotype profiles and strong linkage disequilibrium at the MDR1 multidrug transporter gene locus in three ethnic Asian populations. Pharmacogenetics 12: 437–50. Turgut G, Kurt E, Sengul C, et al. (2009). Association of MDR1 C3435T polymorphism with bipolar disorder in patients treated with valproic acid. Mol Biol Rep 36:495–9. Vahab SA, Sen S, Ravindran N, et al. (2009). Analysis of genotype and haplotype effects of ABCB1 (MDR1) polymorphisms in the risk of medically refractory epilepsy in an Indian population. Drug Metab Pharmacokinet 24:255–60. Yasui-Furukori N, Saito M, Nakagami T, et al. (2006). Association between multidrug resistance 1 (MDR1) gene polymorphisms and therapeutic response to bromperidol in schizophrenic patients: a preliminary study. Prog Neuropsychopharmacol Biol Psychiatry 30: 286–91.
RESEARCH ARTICLE
Frequency and genotype distribution of ABCB1 gene polymorphisms among Maharashtrian population of Central India Sreemanta Pramanik1,2, Subin T. Surendran2, Saravana Devi2, Kannan Krishnamurthi2, and Tapan Chakrabarti2 Kolkata Zonal Laboratory, CSIR-National Environmental Engineering Research Institute, Kolkata, West Bengal, India and 2Environmental Health Division, National Environmental Engineering Research Institute, Nagpur, Maharashtra, India
Xenobiotica Downloaded from informahealthcare.com by University of Laval on 03/04/15 For personal use only.
1
Abstract
Keywords
1. Apart from conferring multidrug resistance to cancer cells, P-glycoprotein (P-gp) encoded by the gene ABCB1 (also, known as Multidrug resistance gene, MDR1), plays a major role in drug disposition. Single nucleotide polymorphisms (SNPs) in the ABCB1 gene might contribute to inter-individual and ethnic differences in drug disposition and thereby, could influence the outcome and prognosis of certain diseases. 2. India is one of the most ethnically and genetically diverse regions of the world. This study was undertaken with a view to determine the allele and genotype frequencies of C3435T and C1236T polymorphisms in the ABCB1 gene among the Maharshtrian population, residing in the Vidarbha region of central India and compare them with HapMap and other Indian populations. The common synonymous C3435T polymorphism has been found to be associated with lower P-gp functional expression and drug uptake, alone or in conjunction with a few other linked SNPs like C1236T. 3. The genotypes of C3435T and C1236T SNPs were determined by PCR-RFLP in 222 healthy and unrelated Maharashtrian individuals. 4. According to the findings of this study, the Maharashtrians were found to be not significantly different from the Gujarati Indians in Houston, Texas in the HapMap database.
Drug disposition, HapMap, P-glycoprotein, SNP
Introduction One of the greatest challenges of the post-genomic sequencing era is to develop a detailed understanding of the heritable variation present in the human genome. By characterizing genetic variations among individuals and populations, we stand a better chance of understanding the differential susceptibility to diseases, differential response to pharmacological agents and complex interaction between genetic and environmental components in producing phenotypes. Single nucleotide polymorphisms (SNPs), the most common form of sequence variation, are highly abundant and account for more than 90% of all genetic variations in the human genome. More than 10 million SNPs are estimated to be present in the human genome and about half of these SNPs are expected to be common with minor allele frequency of greater than 10%. SNPs could serve as useful markers to identify genes that predispose individuals to common, multifactorial disorders by
Address for correspondence: Dr. Sreemanta Pramanik, Principal Scientist & Head, Kolkata Zonal Laboratory, CSIR-NEERI, Kolkata – 700107, West Bengal, India. Tel: 91-033-24421988. Fax: 91-033-24417608. E-mail: [email protected]/sreemanta@ gmail.com
History Received 9 October 2013 Revised 12 November 2013 Accepted 12 November 2013 Published online 6 December 2013
using linkage disequilibrium mapping (Risch & Merikangas, 1996). The human ABCB1 gene (also, known as Multidrug resistance gene, MDR1) is located on the long arm of chromosome 7 at 7q21 and has 28 exons. The ABCB1 gene encodes a 170-kDa plasma membrane glycoprotein [P-glycoprotein (P-gp)], a member of the ATP-binding cassette (ABC) super family of transporters. Acting as an energy-dependent efflux pump, P-glycoprotein plays a major role in drug disposition and protects cells against toxic xenobiotics, in particular due to its co-localization with the major drug metabolizing enzyme, CYP3A4, in the small intestine and liver (Ayrton & Morgan, 2001). A wide variety of drugs including anti-cancer drugs, cardiovascular drugs, HIV protease inhibitors, immunosuppressants and b-adrenoreceptor antagonists are transported via P-gp. Moreover, P-gp confers additional protection to sensitive organs like brain, testis or fetus from exposure to toxic compounds due to its expression in the respective blood-tissue barriers. Presently, more than 100 SNPs have been identified in the ABCB1 gene (Kerb, 2006). These genetic polymorphisms might contribute to inter-individual and inter-ethnic variability in disposition of various drugs that are P-gp substrates and consequently, could influence the outcome and prognosis of certain diseases. Widespread variation in allele frequencies has been observed for the most common polymorphisms,
Xenobiotica Downloaded from informahealthcare.com by University of Laval on 03/04/15 For personal use only.
580
S. Pramanik et al.
C3435T (rs1045642), C1236T (rs1128503) and G2677T/A (rs2032582) of the ABCB1 gene among different ethnic groups (Kim et al., 2001). The synonymous C3435T polymorphism in the exon 26 of ABCB1, which gives rise to a silent mutation (Ile > Ile), was found to have a statistically significant correlation with P-gp expression and activity and oral bioavailability of digoxin in Caucasians (Hoffmeyer et al., 2000). Individuals with the CC genotype had about two fold higher P-gp expression in the small intestine in comparison to the individuals with the TT genotype. Another study by Schwab et al. (2003) found significantly increased frequencies of the 3435T allele and 3435TT genotype in patients with ulcerative colitis as compared to the controls. A few studies also reported an association between the C3435T polymorphism and psychiatric and mood disorders (Turgut et al., 2009; Yasui-Furukori et al., 2006). C3435T polymorphism was found to be linked to another synonymous C1236T polymorphism in the exon 12, giving rise to a silent mutation (Gly > Gly) and a nonsynonymous G2677T/A polymorphism in the exon 21, causing amino acid substitution (Ala > Ser/Thr; Tang et al., 2002) and also, seemed to influence P-gp function in a haplotype-dependent manner (Salama et al., 2006). The silent C1236T and the non-synonymous G2677T/A polymorphisms have also been linked to several diseases like pharmacoresistant epilepsy and Parkinsons disease (KimchiSarfaty et al., 2007). India represents a vast collection of peoples with different morphological, cultural, linguistic and genetic diversity. Studies on the ABCB1 gene polymorphisms in Indian populations are scanty (Grover et al., 2010; Ramasamy et al., 2006; Vahab et al., 2009). This study was aimed at determining the allele and genotype frequencies of the C3435T and C1236T polymorphisms in the ABCB1 gene among the Maharshtrian population, residing in the Vidarbha region of central India and comparing them with HapMap and other Indian populations. To the best of our knowledge, this study is the first of its kind on the ABCB1 gene polymorphisms in a central Indian population.
Materials and methods Study population The study was approved by the Institutional Ethical Committee. The study population consisted of 222 healthy and unrelated Maharashtrian individuals in the age group of 21–50, residing in the Vidarbha region of central India. After obtaining prior informed consents, 5 ml of blood sample was collected from each subject. DNA extraction and genotyping of samples Genomic DNA samples were extracted from whole blood of subjects using a standard lysis procedure and amplified by PCR using respective primers for the ABCB1 C3435T (rs1045642; Calado et al., 2002) and C1236T polymorphisms (rs1128503; Hoffmeyer et al., 2000). The genotypes of the SNPs were determined by digesting the amplified products with appropriate restriction enzymes (DpnII for C3435T and EcoO109I for C1236T, respectively) and running the digested products in 2.5% agarose gel.
Xenobiotica, 2014; 44(6): 579–582
Statistical analysis The observed genotype distributions of the ABCB1 C3435T and C1236T polymorphisms were compared to their expected values by performing Chi square (2) test. The allele and genotype frequencies of polymorphisms in the Maharashtrian population were compared to some of the populations in the HapMap database (www.hapmap.org) e.g. Utah residents with Northern and Western European ancestry from the CEPH collection (CEU), Yoruban in Ibadan, Nigeria (YRI), Han Chinese in Beijing, China (CHB), Gujarati Indians in Houston, Texas (GIH), Japanese in Tokyo, Japan (JPT) and some other Indian populations selected from literature e.g. South Indian population from Tamilnadu State (SInd; Ramasamy et al., 2006), North Indian population from Delhi State (NInd; Grover et al., 2010), Western Indian population from Pune, Maharashtra State (WInd; Ghodke et al., 2011). Pair-wise Chi square (2) tests were performed between Maharashtrians and these populations using the allele frequencies in a 2X2 contingency table to find whether the Maharashtrian population was significantly different from others.
Results The genotypes of the C3435T and C1236T SNPs were determined in 222 healthy and unrelated Maharashtrian individuals. Allele and genotype frequencies of ABCB1 C3435T (Ile > Ile) polymorphism The observed C/C, C/T and T/T genotype frequencies of the SNP in the Maharashtrian population were 0.18, 0.473, and 0.347, respectively (Table 1). The C (wild-type) and T (variant) allele frequencies were 0.42 and 0.58, respectively. The variant allele frequency was lowest (0.107) among the Yorubans and highest (0.64) among the North Indians. Significant differences were found between the Maharashtrians and YRI, CHB and JPT populations (Table 1). Allele and genotype frequencies of ABCB1 C1236T (Gly > Gly) polymorphism The observed C/C, C/T and T/T genotype frequencies of the SNP in the Maharashtrian population were 0.135, 0.541 and 0.347, respectively (Table 2). The C (wild-type) and T (variant) allele frequencies were found to be 0.41 and 0.59, respectively. The variant allele frequency was lowest (0.122) among the Yorubans and highest (0.695) among the Han Chinese. Pair-wise Chi-square (2) test revealed a significant difference between the Maharashtrians and CEU, YRI and CHB populations (Table 2). The allele and genotype frequency data was not available for any South Indian populations.
Discussion Using mitochondrial, Y-chromosomal and limited autosomal markers, several studies have demonstrated extensive genetic diversity in India pertinent to the issues of origin and migrations of populations (Bamshad et al., 2001; Basu et al., 2003; Sengupta et al., 2006). A recent study carried out by the
ABCB1 gene polymorphisms among Maharashtrians
DOI: 10.3109/00498254.2013.866300
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Table 1. Allele and genotype frequencies of ABCB1 C3435T in HapMap and Indian populations including Maharashtrians. Genotype
Allele frequency
Population
Geno
Freq (No)
Geno
Freq (No)
Geno
Freq (No)
Allele
Freq
Allele
Freq
Pairwise 2 test value between MAH and other populations
CEU (n ¼ 113)a YRI (n ¼ 145)a CHB (n ¼ 135)a GIH (n ¼ 101)a JPT (n ¼ 113)a SInd (n ¼ 185) NInd (n ¼ 93) WInd (n ¼ 144) MAH (n ¼ 222)
C/C C/C C/C C/C C/C C/C C/C C/C C/C
0.15 (17) 0.8 (116) 0.4 (54) 0.178 (18) 0.283 (32) 0.18 (33) 0.13 (12) 0.146 (21) 0.18 (40)
C/T C/T C/T C/T C/T C/T C/T C/T C/T
0.558 (63) 0.186 (27) 0.452 (61) 0.475 (48) 0.531 (60) 0.56 (104) 0.462 (43) 0.472 (68) 0.473 (105)
T/T T/T T/T T/T T/T T/T T/T T/T T/T
0.292 (33) 0.014 (2) 0.148 (20) 0.347 (35) 0.186 (21) 0.26 (48) 0.408 (38) 0.382 (55) 0.347 (77)
C C C C C C C C C
0.429 0.893 0.626 0.416 0.549 0.46 0.36 0.38 0.42
T T T T T T T T T
0.571 0.107 0.374 0.584 0.451 0.54 0.64 0.62 0.58
0.025b 82.252 14.25 0.004b 5.011 0.656b 0.981b 0.58b –
Wild-type allele
Variant allele
a
Population data source: HapMap (www.hapmap.org). Chi-square test statistic value less than 3.841 at 5% significance level, so populations are not significantly different from MAH. Population descriptions: CEU, Utah residents with Northern and Western European ancestry from the CEPH collection; YRI, Yoruban in Ibadan, Nigeria; CHB, Han Chinese in Beijing, China; GIH, Gujarati Indians in Houston, Texas; JPT, Japanese in Tokyo, Japan; Sind, South Indian population from Tamilnadu, India (Ramasamy et al., 2006); NInd, North Indian population from Delhi, India (Grover et al., 2010); Wind, Western Indian population from Pune, India (Ghodke et al., 2011); MAH, Maharashtrian population residing in Vidarbha region of central India (this study).
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b
Table 2. Allele and genotype frequencies of ABCB1 C1236T in HapMap and Indian populations including Maharashtrians. Genotype
Allele frequency
Geno
Freq (No)
Geno
Freq (No)
Geno
Freq (No)
Allele
Freq
Allele
Freq
Pairwise 2 test value between MAH and other populations
C/C C/C C/C C/C C/C C/C C/C C/C
0.265 (30) 0.762 (112) 0.118 (16) 0.139 (14) 0.177 (20) 0.12 (12) 0.146 (21) 0.135 (30)
C/T C/T C/T C/T C/T C/T C/T C/T
0.566 (64) 0.231 (34) 0.375 (51) 0.545 (55) 0.478 (54) 0.45 (45) 0.472 (68) 0.541 (120)
T/T T/T T/T T/T T/T T/T T/T T/T
0.168 (19) 0.007 (1) 0.507 (69) 0.317 (32) 0.345 (39) 0.43 (43) 0.382 (55) 0.347 (72)
C C C C C C C C
0.549 0.878 0.305 0.411 0.416 0.345 0.38 0.41
T T T T T T T T
0.451 0.122 0.695 0.589 0.584 0.655 0.62 0.59
5.83 80.475 3.988 0.000b 0.011b 1.225b 0.328b –
Wild-type allele Population a
CEU (n ¼ 113) YRI (n ¼ 147)a CHB (n ¼ 136)a GIH (n ¼ 101)a JPT (n ¼ 113)a NInd (n ¼ 100) WInd (n ¼ 144) MAH (n ¼ 222)
Variant allele
a
Population data source: HapMap (www.hapmap.org). Chi-square test statistic value less than 3.841 at 5% significance level, so populations are not significantly different from MAH. Population descriptions: CEU, Utah residents with Northern and Western European ancestry from the CEPH collection; YRI, Yoruban in Ibadan, Nigeria; CHB, Han Chinese in Beijing, China; GIH, Gujarati Indians in Houston, Texas; JPT, Japanese in Tokyo, Japan; NInd, North Indian population from Delhi, India (Grover et al., 2010); Wind, Western Indian population from Pune, India (Ghodke et al., 2011); MAH, Maharashtrian population residing in Vidarbha region of central India (this study).
b
Indian Genome Variation Consortium, on 405 SNPs in 75 disease or drug response related genes and a 5.2 Mb genomic region on chromosome 22 spanning 49 genes, has revealed a high degree of genetic differentiation among different ethnic groups of India and found that Indian populations formed a continuing genetic spectrum, bridging two distinct HapMap populations, CEU and CHB/JPT (Consortium IGV, 2008). Vidarbha region, lying on the eastern side of the Maharashtra state, is broadly referred to as central India because of its central location. Linguistically, Marathi, spoken by the Maharashtrians, belongs to the Indo-Aryan language group of the Indo-European linguistic family. In this study, the observed genotype distributions followed Hardy–Weinberg expectations for the C3435T (p Value ¼ 0.68) and C1236T (p Value ¼ 0.07) SNPs in the Maharashtrian population. A wide variation in allele frequency and genotype distribution among different populations was observed for the ABCB1 C3435T and C1236T SNPs. For the C3435T polymorphism, the variant allele frequency in the Maharashtrians (0.58) was similar to the Utah residents with Northern and Western
European ancestry (CEU) and Gujarati Indians in Texas (GIH), but was higher than the Yorubans in Nigeria (YRI), Han Chinese in Beijing (CHB) and Japanese in Tokyo (JPT). The Maharashtrians (MAH) were found to be significantly different from the YRI, CHB and JPT, but not significantly different from the CEU and GIH in the HapMap database. For the C1236T polymorphism, the variant allele frequency in the Maharashtrians (0.59) was similar to the JPT and GIH, but lower than the CHB and higher than the YRI and CEU. The Maharashtrians were found to be significantly different from the CEU, YRI and CHB, but not significantly different from the JPT and GIH. The findings of our earlier study on DNA repair gene polymorphisms at XRCC1, XRCC3, XPD and OGG1 loci, also pointed out that the Maharashtrian population did not show significant difference to the CEU and GIH populations from the HapMap database (Pramanik et al., 2011). The Maharashtrians were found to be not significantly different from other Indian populations selected for this study (SInd, NInd, WInd for the C3435T and NInd, Wind for the C1236T, respectively). The fact that several different ethnic
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groups inhabit a vast region like North, South, East or West of India and subjects drawn from the study populations (namely, SInd, NInd and WInd) were selected randomly from the places of study rather than from a particular ethnic group could be the reason behind this finding.
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Conclusions Analyses of SNPs are becoming increasingly important in identifying genetic variants underlying susceptibility to human disease or other phenotypes. Knowledge of the basic prevalence of some of the ABCB1 gene polymorphisms in the general population, which have been directly correlated with P-gp expression and function, would be essential to estimate the efficacy of the drugs that are transported through P-gp. This study found a wide variation in allele frequency and genotype distribution of the ABCB1 C3435T and C1236T polymorphisms between the Maharashtrians and some HapMap populations. That the Maharashtrian population was not significantly different from the Gujarati Indians in Houston, Texas in the HapMap database was another major finding of the study. This study could form the basis of future investigations to explore the relationship between ABCB1 genotypes and drug efficacy, drug toxicity, disease susceptibility or other phenotypes in Indian populations.
Acknowledgements The authors wish to express their sincere thanks to the participants of the study.
Declaration of interest The study was financially supported by Council of Scientific and Industrial Research (CSIR). The authors declare that that there are no conflicts of interest in relation to this work.
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