Clinical Pharmacogenetics and Potential Application in Personalized Medicine
Shu-Feng Zhou1,*, Yuan Ming Di1, Eli Chan2, Yao-Min Du3, Vivian Deh-Wei Chow1, Charlie Changli Xue1,Xinsheng Lai4, Jian-Cheng Wang5, Chun Guang Li1, Min Tian5 and Wei Duan6,*
School of Health Sciences, RMIT University, Victoria, Australia; 2Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore; 3Department of Internal Medicine, Guangdong Provincial People’s Hospital, Guangzhou, China; 4College of Acupuncture and Massage, Guangzhou University of Traditional Chinese Medicine, Guangzhou 510407, China; 5Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing, 100083, China; and 6School of Medicine, Deakin University, Waurn Ponds, Victoria 3216, Australia
Abstract: The current ‘fixed-dosage strategy’ approach to medicine, means there is much inter-individual variation in drug response. Pharmacogenetics is the study of how inter-individual variations in the DNA sequence of specific genes affect drug responses. This arti-cle will highlight current pharmacogenetic knowledge on important drug metabolizing enzymes, drug transporters and drug targets to un-derstand interindividual variability in drug clearance and responses in clinical practice and potential use in personalized medicine. Poly-morphisms in the cytochrome P450 (CYP) family may have had the most impact on the fate of pharmaceutical drugs. CYP2D6,CYP2C19 and CYP2C9 gene polymorphisms and gene duplications account for the most frequent variations in phase I metabolism of drugs since nearly 80% of drugs in use today are metabolised by these enzymes. Approximately 5% of Europeans and 1% of Asians lack CYP2D6 activity, and these individuals are known as poor metabolizers. CYP2C9 is another clinically significant drug metabolising en-zyme that demonstrates genetic variants. Studies into CYP2C9 polymorphism have highlighted the importance of the CYP2C9*2 and CYP2C9*3 alleles. Extensive polymorphism also occurs in a majority of Phase II drug metabolizing enzymes. One of the most important polymorphisms is thiopurine S-methyl transferases (TPMT) that catalyzesthe S-methylation of thiopurine drugs. With respect to drug transport polymorphism, the most extensively studied drug transporter is P-glycoprotein (P-gp/MDR1), but the current data on the clini-cal impact is limited. Polymorphisms in drug transporters may change drug’s distribution, excretion and response. Recent advances in molecular research have revealed many of the genes that encode drug targets demonstrate genetic polymorphism. These variations, in many cases, have altered the targets sensitivity to the specific drug molecule and thus have a profound effect on drug efficacy and toxic-ity. For example, the 2-adrenoreceptor, which is encoded by the ADRB2 gene, illustrates a clinically significant genetic variation in drug targets. The variable number tandem repeat polymorphisms in serotonin transporter (SERT/SLC6A4) gene are associated with response to antidepressants. The distribution of the common variant alleles of genes that encode drug metabolizing enzymes, drug transporters and drug targets has been found to vary among different populations. The promise of pharmacogenetics lies in its potentialto identify the right drug at the right dose for the right individual. Drugs with a narrow therapeutic index are thought to benefit more from pharmacoge-netic studies. For example, warfarin serves as a good practical example of how pharmacogenetics can be utilized prior to commencement of therapy in order to achieve maximum efficacy and minimum toxicity. As such, pharmacogenetics has the potential to achieve optimal quality use of medicines, and to improve the efficacy and safety of both prospective and licensed drugs.
1
Keywords: Pharmacogenetics, drug metabolizing enzymes, cytochrome P450, CYP3C9, CYP2D6, P-glycoprotein, single nucleotide poly-morphism, thiopurine S-methyltransferase, -adrenergic receptor. INTRODUCTION
It is a well recognised fact that individuals respond differently to drug therapy and that no single drug is 100% efficacious in all patients. While some individuals obtain the desired effects, others can have little or no therapeutic response. Additionally, certain patients might experience adverse effects that vary from mild and tolerable to bothersome and life-threatening [1]. This inter-individual variability in drug response that is so often seen is thought to be a consequence of multiple factors such as disease determinants, genetic and environmental factors, variability in drug target response or idiosyncratic response and other factors including patient age, concomitant therapies and lifestyle factors such as smoking and alcohol consumption [1]. Most drugs undergo Phase I and/or Phase II biotransformation (metabolism) in the body and their disposition may involve active transport mediated by multiple drug transporters. In addition, drugs interact with a diverse range of protein targets including enzymes and receptors to elicit a therapeu-tic response [2]. This concerted action demonstrates the multigenic nature of a majority of drug responses.
*Address correspondence to these authors at the Division of Chinese Medi-cine, School of Health Sciences, RMIT University, Bundoora, Victoria, Austrália; Tel: +61 3 9925 7794; Fax: +61 3 9925 7178; E-mail: shufeng.zhou@rmit.edu.au; and School of Medicine, Deakin University, Waurn Ponds, Victoria, 3216, Australia; Tel: 61 3 5227 1149; Fax: 61 3 5227 2945; E-mail: wduan@deakin.edu.au
One of the major reasons for inter-individual variations in drug response is genetic variations that result in proteins with variable activities. Polymorphism is the occurrence in the same population of two or more alleles at one locus, each with a frequency of >1%. Polymorphisms in genes have been found in most enzymes in-volved in drug metabolism, most drug transporters and many drug targets with substantial ethnic differences in their frequencies, and they can be important in determining clinical response [3]. Genetic variations can be a result of nucleotide repeats, insertions, deletions, and single nucleotide polymorphisms (SNPs), which can alter the amino acid sequence of the encoded proteins, RNA splicing, and gene transcription. The major types of genetic variation seen are single base changes, i.e. SNPs. SNPs are present throughout the human genome, with the frequency of approximately 1 per 1000 base pairs. The vast majority of SNPs are biologically silent (i.e. there is no effect on gene function or inherited traits). If the SNP occurs in the gene itself, it may impact protein function.
Vogel in 1959 was the first to use the term pharmacogenetics but it was not until 1962 when pharmacogenetics was defined as the study of heredity and the response to drugs in a book by Kalow [4], which covered the whole field for the first time. After this start there slowly accumulated several discoveries during this early pe-riod that led to the merger of the fields of pharmacology and genet-ics. These included the recognition that haemolytic anaemia from primaquine (an antimalarial drug) resulted from a variant form of the enzyme glucose-6-phosphate dehydrogenase. The discovery that prolonged paralysis following the administration of succinylcholine
© 2008 Bentham Science Publishers Ltd.
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Pharmacogenetics and Clinical Practicewas the result of a variant of the butyryl-cholinesterase enzyme [5], and that peripheral neuropathy resulting from isoniazid was a con-sequence of genetic diversity in the enzyme N-acetyltransferase (NAT) [6]. For these early examples, genetic components were identified through studies of families or ethnic populations in com-bination with careful testing of the phenotype. As with many fields in the biomedical sciences, advances in molecular biology have transformed pharmacogenetics such that the focus is now on the study of inter-individual variations in DNA sequences and specific genes in order to predict differential drug responses and drug-drug interactions, differences in drug efficacy and the relative risk of adverse effects occurring [7]. Functional variants caused by SNPs in genes encoding drug-metabolizing enzymes, transporters and drug receptors have been known to be associated with inter-individual and interethnic variation in drug clearance and drug re-sponse [8]. It is well known that patients can be poor metabolizers (PMs) or extensive metabolizers (EMs) when given a drug. Genetic variations in these genes are thought to play an important role in influencing the efficacy and toxicity of medications. Presently, pharmacogenetics is a well established discipline that combines important components of both pharmacology and genetics.
This article will highlight current pharmacogenetic knowledge on important drug metabolizing enzymes, drug transporters and drug targets to understand remarkable interindividual variability in drug clearance and response in clinical practice. We will address how genetic variations in the genes encoding drug metabolizing enzymes, drug transporters and drug targets affect drug response and the potential use of pharmacogenetics in personalized medicine. CLINICAL PHARMACOGENETICS OF PHASE I DRUG METABOLIZING ENZYMES
The activity of many drugs depends on their interaction with enzymes localised predominately in the liver and to a smaller extent the lungs and small intestine. Among these enzymes, the most im-portant enzymes belong to the cytochrome P450 (YP, EC1.14.14.1) superfamily. CYP represents the most important Phase I drug metabolizing enzymes that oxidize a number of en-dogenous substances and xenobiotics, including more than 60% of all medications into more hydrophilic compounds. There are as many as 57 CYPgenes in humans (http://drnelson.utmem.edu/ Cy-tochromeP450.html). Three families of genes, CYP1,CYP2 and CYP3, are the main ones contributing to the oxidative metabolism of most drugs. The liver and intestinal epithelium contains the most abundant member of the CYP subfamily – CYP3A, and these en-zymes are responsible for processing more than half of the thera-peutic drugs [9]. Activity varies among members of a given popula-tion. Genetic polymorphisms within CYPs mainly affect the me-tabolism of drugs that are substrates for those particular enzymes, probably leading to differences in drug response in addition to an altered risk for adverse drug effects [3, 10]. Most members of the CYP subfamilies are polymorphic (see http://www.imm.ki.se/ CY-Palleles) and allelic variants resulting in altered protein expression or activity have significant effects on the disposition of drugs. CYP2B6 The CYP2B6 gene has been mapped to chromosome 19between 19q12 and 19q13.2 which is located together with the pseudogene CYP2B7P and consists of nine exons [11]. CYP2B6 is mainly ex-pressed in the liver accounting for 3-5% of total microsomal CYPs and many extrahepatic tissues including the kidney, skin, brain, intestine, and lung [12]. CYP2B6 can metabolise a number of therapeutic drugs, including cyclophosphamide [13], ifosfamide [13], tamoxifen [14], ketamine [15], artemisinin [16], nevirapine andefavirenz (both HIV-1 reverse transcriptase inhibitors) [17, 18], bupropion (an antidepressant and antismoking agent) [19], propofol [20], S-mephenytoin [21] and diazepam [22]. In addition, CYP2B6 can metabolize the procarcinogens such asaflatoxin B1, 6-
Current Drug Metabolism, 2008, Vol. 9, No. 8 739
aminochrysene, and 7,12-dimethylbenz[a]anthracene [12, 23, 24]. Additional substrates known to bein part metabolized by CYP2B6 include stimulants like ecstasy(methylenedioxymethamphetamine [25] and nicotine [26]. A substantial interindividual difference has been found in thecontribution of CYP2B6 to cyclophosphamide 4-hydroxylation ina panel of human liver microsomes, and this is probably due tothe large interindividual variability in hepatic ex-pression level of CYP2B6 [13]. Large interpatient differencein the clinical pharmacokinetics and biotransformation of cyclophos-phamide has also been observed [27-30].
To date, at least 28 allelic variants of CYP2B6 (*1B through *29) have been described (Table 1) (http://www.imm.ki.se/ CYPal-leles, access date: 2 August 2008). Lang et al. [31] first identified nine SNPs of the CYP2B6 gene in a Caucasian population, with five ofthese causing amino acid substitutions in exons 1, 4, 5, and 9. In Caucasians, these SNPswere found at variant frequencies of up to 30%. They found six different alleles, termed CYP2B6*2,*3,*4,*5,*6, and *7 (wild-type, *1). Hiratsuka et al. [32] havefound a lower frequency of allelic variants of CYP2B6 in the Japanese com-pared with Caucasians. The 516G>T SNPwas reported to be the only one of the five non-synonymous SNPs of CYP2B6seen exclu-sively in combination with other amino acid mutationsin both populations. SNP 13072A>G in exon 3 was reported to occur assingle amino acid change K139E in allele CYP2B6*8 [31]. Lang etal. [33] identified five novel missense mutations in Caucasians, including 62A>T (Q21L in exon 1), 136A>G(M46V in exon 1), 12820G>A (G99E in exon 2), 13076G>A(R140Q in exon 3), and 21388T>A (I391N in exon 8). Haplotype analysis indicated thepresence of at least six novel alleles, CYP2B6*10 (Q21L, and R22C), *11 (M46V), *12(G99E), *13 (K139E, Q172H, and K262R), *14 (R140Q),and *15 (I391N). The CYP2B6 variants encoded by these alleles all showed decreased enzyme activity ex-cept for CYP2B6*10. Recently, a number of additional SNPs of CYP2B6 have been identified and its haplotype variants up to *29have been described (Table 2) [34].
Phenotype-genotype correlation studies in human liver samplesshowed that the 1459C>T SNP (R487C) in exon 9 was associatedwith an 8-fold lower enzyme expression level in Cys487homozy-gotes compared with the wild-type, whereas the other variantswere associated with moderate changes in enzyme expression andactiv-ity [31]. In contrast, a Q172H variant,more frequently found in Japanese compared with Caucasians,had increased enzyme activity [35]. Jinno et al. [36] compared the enzyme activity of the variants of CYP2B6 and its wild-type protein with 7-ethoxy-4-trifluoromethylcoumarin as the probe substrate. The CYP2B6 vari-antshaving a Lys262Arg substitution (CYP2B6*4, *6, and *7)showed increased values for Vmax and Vmax/Km, whereas the kineticparameters of CYP2B6*2 and *3 were not altered by the corre-spondingamino acid substitution [36]. The Km value of CYP2B6*6 containing both Gln172His and Lys262Arg polymorphisms wassignificantly higher than that of wild-type enzyme. Xie et al. [37] reported that CYP2B6*6 carriers had significantly higher cyclo-phosphamide 4-hydroxylation activity than wild-type. The mecha-nism of the CYP2B6*6 allele involves aberrant splicing, leading to reduced functional mRNA, protein, and activity [38]. M46V, G99E, and I391N in CYP2B6 resulted in almost unmeasurable (M46V) or undetectable (G99Eand I391N) enzyme activity, whereas K139E completely abolished proteinexpression and function [33].
Smokers with the 1459C>T (R487C) variant may be morevul-nerable to abstinence symptoms and relapse following treatmentwith bupropion as a smoking cessation agent [39]. CYP2B6*4(K262R) carriers had increased total clearance of bupropion, whereasother alleles were not different from wild-type [40]. There is a clinical report where CYP2B6 mutations are associated with altered response or survival in patients with proliferative lupus ne-phritis when treated with pulsed cyclophosphamide [41]. The pa-
740 Current Drug Metabolism, 2008, Vol. 9, No. 8Table 1
Reported Variants of Human CYP2B6 Gene
Nucleotide change
Amino acid change
Zhou et al.
CYP2B6 Reference
cDNA Gene *1A *1B *1C *1D *1E *1F *1G *1H *1J *1K *1L *1M *1N
-2320T>C
-2320T>C; 14593C>G; 15582C>T -1778A>G; -1186C>G; 12917A>T -1578C>T; -757C>T -1224A>G -750T>C
-2320T>C; -750T>C
-2320T>C; -1778A>G; -1186C>G; -750T>C -1972C>T; -1578C>T; -750T>C -1578C>T; -750T>C -1578C>T
-1456T>C; -750T>C
CR22 R22C
S259R K262R K262R
K262R K262R
CR487 R487C
[11] [353] [353] [353] [353] [353] [353] [354] [354] [354] [354] [354] [86]
C*2A 64>T 64C>T
*2B
64C>T; 216G>C
64C>T; 12740G>C
[31] [353]
C*3777>A 18045C>A *4A *4B *4C *4D
785A>G 18053A>G 785A>G
-2320T>C; -1778A>G; -1186C>G; -750T>C; 18053A>G
[31] [40] [354]
785A>G 18053A>G; 12917A>T 785A>G
-1456T>C; -750T>C; 18053A>G
[355] [356]
C*5A 1459>T 25505C>T *5B
1459C>T
-2320T>C; -750T>C; 25505C>T
[31] [31]
C*5C 1459>T -591A>G; C25505>T CR487 *6A *6B *6C *7A *7B *8*9*10*11A *11B
516G>T; 785A>G 516G>T; 785A>G 516G>T; 785A>G
516G>T; 785A>G; 1459C>T 516G>T; 785A>G; 1459C>T 415A>Ga516G>T
62A>T; 64C>T; 216G>C 136A>G136A>G
15631G>T; 18053A>G
-1456T>C; -750T>C; 15631G>T; 18053A>G -750T>C; 15631G>T; 18053A>G 15631G>T; 18053A>G; 25505C>T
-1456 T>C; -750T>C; 15631G>T; 18053A>G; 25505C>T 13072A>G
-1456T>C; 15631G>T; 21563C>T 62A>T; 64C>T; 12740G>C 136A>G
136A>G; 18273G>A
Q172H; K262R Q172H; K262R Q172H; K262R Q172H; K262R; R487C Q172H; K262R; R487C K139EQ172H Q21L; R22C
[356] [42]
[38] [354] [356] [31] [354] [353] [31] [353] [33]
M46V [33] M46V [33] Pharmacogenetics and Clinical Practice
(Table 1) contd…...
CYP2B6
Nucleotide change
Current Drug Metabolism, 2008, Vol. 9, No. 8 741
Amino acid change Reference
cDNA Gene *12 Tentative allele *13A *13B *14 Tentative allele *15A *15B *16*17A *17B *18*19*20*21*22*23*24*25*26*27*28
296G>A
415A>G; 516G>T; 785A>G 415A>G; 516G>T; 785A>G
12820G>A; 18273G>A
13072A>G; 15631G>T; 18053A>G; 18273G>A; 21563C>T
G99E [33] K139E; Q172H; K262R
[33] [33]
13072A>G; 15582C>T; 15631G>T; 18053A>G; 18273G>A;
K139E; Q172H; K262R
21563C>T
13076G>A; 18273G>A
419G>AR140Q [33] 1172T>A 15582C>T; 21388T>A
1172T>A 15582C>T; 18273G>A; 21388T>A
I391N [33] I391N [33] 785A>G; 983T>C 18053A>G, 21011T>C K262R; I328T [357] 76A>T; 83A>G; 85C>A; 86G>C;
76A>T; 83A>G; 85C>A; 86G>C; 18273G>A; 18799C>T
933C>T
76A>T; 83A>G; 85C>A; 86G>C 983T>C
516G>T; 785A>G; 1006C>T 503C>T; 516G>T; 785A>G;
76A>T; 83A>G; 85C>A; 86G>C; 18273G>A 17897C>T; 18273G>A; 18627G>A; 21011T>C
15631G>T; 18053A>G; 18273G>A; 21034C>T; 21563C>T 15618C>T; 15631G>T; 18053A>G; 18273G>A
T26S; D28G; R29T T26S; D28G; R29T
[358] [358]
I328T [358] Q172H; K262R; R336C [358] T168I; Q172H; K262R
[358]
1282C>A 18273G>A; 21498C>A
1375A>G 1427G>A 1454A>T
499C>G; 516G>T; 785A>G 593T>C
917C>G; 1132C>T
CYP2B7/CYP2B6 hybrid (cross-over in intron 4)
6A>G; 42A>C; 66C>T; 76A>T; 83A>G; 85C>A; 86G>C; 123A>G; 141T>C; 264T>G; 273G>C; 305C>T; 309G>C; 310G>A; 312C>G; 319T>G; 323T>A; 326G>A; 336T>C; 337G>A; 340A>C; 393T>C; 444G>C; 468G>A; 486G>A; 493A>G; 516G>T; 547G>A; 561A>C; 593T>C; 608A>G; 614C>G; 618T>C; 620C>T; 634G>A; 638T>C; 640G>A
CYP2B7/CYP2B6 hybrid (crossover in intron 4)
P428T [358] [356] [32] [32] [32] [45]
-1848C>A; -801G>T; -750T>C, -82T>C Incr. transcr. 25421A>G 25473G>A 25500A>T
15614C>G; 15631G>T; 18053A>G 15708T>C; 18273G>A
-1456T>C; -750T>C; 15837C>T; 18273G>A; 18627G>A; 18783C>G; 18912G>C; 21160C>T
M459V G476D Q485L
P167A; Q172H; K262R
M198T [359] T306S; R378X [359] *29
T26S; D28G; R29T; K91N; A102V; M103I; V104M;
6A>G; 42A>C; 66C>T; 76A>T; 83A>G; 85C>A; 86G>C;
F107V; F108Y; R109Q;
123A>G; 141T>C; 12788T>G; 12797G>C; 12829C>T;
V113M; I114L; E148D;
12833G>C; 12834G>A; 12836C>G; 12843T>G; 12847T>A;
M165V; Q172H; V183I;
12850G>A; 12993T>C; 12994G>A; 12997A>C; 13050T>C;
M198T; Y203C; T205S;
13101G>C; 13125G>A; 15601G>A; 15608A>G; 15631G>T;
S207L; V212I; F213S;
15662G>A; 15676A>C; 15708T>C; 15723A>G; 15729C>G;
G214S
15733T>C; 15735C>T; 15749G>A; 15753T>C; 15755G>A
[360]
Data are extracted from http://www.imm.ki.se/CYPalleles (access date: 2 August 2008). a
Nucleotide variations in bold are the major SNPs responsible for the phenotype of the corresponding allele.
742 Current Drug Metabolism, 2008, Vol. 9, No. 8Table 2.
CYP2C9
Zhou et al.
Reported Variants of the Human CYP2C9 Gene
Amino acid change
cDNA Gene Nucleotide change
-2665_-2664delTG; -1188T>C -1188T>C -2665_-2664delTG
-1188T>C, -1096A>G; -620G>T; -485T>A; -484C>A; 3608C>T
-2665_-2664delTG, -1188T>C; -1096A>G; -620G>T; -485T>A; -484C>A; 3608C>T-1096A>G; -620G>T; -485T>A; -484C>A; 3608C>T-1911T>C; -1885C>G; -1537G>A; -981G>A; 42614A>C
-1911T>C; -1885C>G; -1537G>A; -1188T>C; -981G>A; 42614A>C42615T>C 42619C>G10601delAC55>A
Reference
*1A Wild-type *1Ba
[48] [361] [361, 362] [361]
*1Ca *1Da *2Aa*2Ba*2Ca*3Aa*3Ba
430C>Tb430C>T 430C>T 1075A>C 1075A>C
R144C [51] R144C [361] R144C [361] I359L [363] I359L
[361, 362]
*4 1076T>C *5*6
1080C>G818delA
I359T [53] D360E [54] 273Frame shift [57] L19I
[364]
*7 55C>A *8
449G>A 3627G>A10535A>G 10598A>G 42542C>T
R150H [364] H251R [364] E272G [364] R335W [365] R335W [361] P489S [364] *9 752A>G *10 815A>G *11Aa*11Ba*12
1003C>T
1003C>T -2665_-2664delTG; C-1188T>; 42542C>T1465C>T
50338C>T3276T>C 3552G>A
9100C>A (linkage with -1188T>C can not be excluded)
*13 269T>C *14*15 *16
374G>A485C>A
L90P [366] R125H [367] S162X
[367]
895A>G -1188T>C; 33497A>G
C42683>T
-1911T>C; -1885C>G; -1537G>A; -1188T>C; -981G>A; 42614A>C; 47391A>C; 50298A>T -1188T>C; 50235G>C -1188T>C; 3215G>C C89>T
121A>G 3233G>A
T299A [367] P382S [367] I359L; D397A Q454H G70R
[367] [367] [367]
*17 1144C>T *18
1075A>C;
1190A>C; 1425A>T
*19 1362G>C *20
208G>C
*21 89C>T *22 121A>G *23 226G>A P30L [368] N41D [368] V76M [368] E354K [369] 118Frameshift
[370]
*24 1060G>Ac 42599G>A *25 353_362deld *26a 389C>G *27a
449G>T
3531_3540del (AGAAATGGAA)
1565C>T; -1188T>C; 3567C>G; 3856G>A; 8763C>T; 9032G>C; 10311A>G; 33349A>G;
50056A>T
-3089G>A; -2665_-2664delTG; -1188T>C; 3627G>T; 3898C>T; 47639C>T;50056A>T
T130R [370] R150L
[370]
Pharmacogenetics and Clinical Practice
(Table 2) contd…. CYP2C9
Current Drug Metabolism, 2008, Vol. 9, No. 8 743
Amino acid change
cDNA Gene Nucleotide change
9256A>T
251T>C; 3411T>C; 33437C>A; 33658A>G; 50056A>T 50302G>A
Reference
*28 641A>T *29a
835C>A
Q214L [370] P279T
[370]
*30 1429G>A ab
A477T [370] Predicted.
Nucleotide variations in bold are the major SNPs responsible for the phenotype of the corresponding allele. c
Existence of the CYP2C9*2 polymorphism 430C>T on the same allele can not be excluded. d
AGAAATGGAA (deleted).
tients received 1.0 g/m2 body surface area of cyclophosphamide as a monthly intravenous bolus injection for at least 1 year, with or without combined intravenous methylprednisolone at 1.0 g/m2 body surface area. Patients homozygous for CYP2B6*5(C1459T giving an Arg487Cys substitution) or CYP2C19*2 had a higher probability of reaching end-stage renal disease with a doubled serum creatinine level and a lower probability of achieving complete renal response [41]. Patients who were either heterozygous or homozygous for CYP2C19*2 (C430T resulting in an Arg144Cys substitution) had a significantly lower risk of developing premature ovarian failure, compared to the wild-type CYP2C19*1 [41]. However, there was no association between clinical renal outcome and allelic polymor-phisms of CYP2C9 and CYP3A4. These results suggest that both CYP2B6 and CYP2C19 play an important role in the in vivo activa-tion of cyclophosphamide in humans, while CYP2C9 and CYP3A4 appear less important. Both CYP2B6*5 and CYP2C19*2 produced proteins with decreased enzyme activity, leading to a reduced metabolic activation of cyclophosphamide and consequently a pro-tective effect on development of premature ovarian failure but a worsening renal response.
Clinical studies demonstrate that CYP2B6 polymorphisms can affect the pharmacokinetics and therapeutic outcome of anti-HIV agents that are substrates of CYP2B6. The mean plasma efavirenz concentration of patients with CYP2B6*6/*6 (Q172H and K262R) was significantly higher than that of patients with genotypes *6heterozygote or without alleles *6 [42]. Elevated plasma efavirenz concentrations were found in 40% of subjects with the homozygous genotype 516G>T and 19% of subjects with the heterozygous geno-type [43]. Homozygous 516T genotype was associated with greater plasma and intracellular exposure to efavirenz and increased neuro-psychological toxicity [44]. CYP2B6 *6/*6 and *6/*26 carriers had extremely high plasma efavirenz concentrations while receiving the standard dosage and these patients needed reduced dosage of efavirenz [45]. The 983T>C genotype (part of the CYP2B6*18haplotype) was associated with nevirapine plasma concentration [46]. In addition, the oral clearance of nevirapine in children with the 516T/T genotype (homozygous variant) was significantly de-creased compared to those with the 516G/G (wild-type) and those with the G/T genotype (heterozygous variants) [47]. Furthermore, children with the T/T genotype had a significant increase in CD4+T-cell percentage compared with those with the G/G and G/T geno-type from baseline to week 12.
CYP2C9 The CYP2C9 is mapped to the long arm of chromosome 10, located in a densely packed region also containing genes encoding CYP2C8, 2C18 and 2C19 [48]. CYP2C9 encodes a protein of 490 amino acids, with a molecular weight of 55.6 kDa. CYP2C9 is one of the most abundant CYP enzymes in the human liver (20% of hepatic CYP content), where it metabolizes approximately 15% clinical drugs, including a number of drugs with narrow therapeutic ranges [49]. Its substrates include oral hypoglycemics (e.g. tolbu-
tamide, glyburide, glimepiride and glipizide), non-steroidal anti-inflammatory drugs (e.g. diclofenac, ibuprofen, suprofen, naproxen, flurbiprofen, piroxicam, and celecoxib), diuretics (e.g. torsemide and sulfinpyrazone), antiepileptics (e.g. phenytoin and phenobarbi-tal), angiotensin II inhibitors (e.g. losartan, irbesartan, and cande-sartan), anticancer drugs (e.g. cyclophosphamide and tamoxifen), and anticoagulants (e.g. S-acenocumarol and S-warfarin) [49, 50]. To date, more than 29 variants of CYP2C9 (*1B to *30) have been identified (Table 2) (http://www.imm.ki.se/CYPalleles, access date: 2 August 2008). One of the first identified and most common allelic variants is CYP2C9*2, a missense mutation of 430T>C caus-ing the substitution of Arg144Cys [51]. Typically, this mutation causes a decrease in enzyme activity. CYP2C9*3 is a missense mutation of 1075A>C on exon 7 that leads to an Ile359Leu substi-tution [52]. CYP2C9*2 causes ~20-30% loss of enzyme activity, whereas the *3 mutation may reduce Vmax activity by as much as 70%. It is possible this loss is due to enzyme conformational changes that reduce the enzyme’s ability to bind to substrates. CYP2C9*4 is an extremely rare missense mutation of 1076T>C originally identified in a Japanese epilepsy patient with an adverse reaction to phenytoin [53]. Little is known about this variant due to its rarity; it is believed the lack of activity is due to an Ile359Thr change. CYP2C9*5 with the 1080C>G SNP on exon 7 causing Asp360Glu change has been found almost exclusively in African-Americans [54-56]. Unlike CYP2C9*2 and *3,*5 appears to affect the Michaelis-Menten constant of various drugs, substantially re-ducing the efficiency of the enzyme [55]. CYP2C9*6 is a null allele because of deletion of A at 818 nucleotide on exon 5 originally identified in an African American patient with a high sensitivity to phenytoin, which results in a shortened protein [56, 57]. Other vari-ants of CYP2C9, such as *7 (55C>A), *8 (449G>A), *9 (752A>G), *10 (815A>G), *12 (1465C>T), *22 (121A>G), *24 (226G>A), *27 (449G>T), *28 (641A>T), *29 (835>A), and *30(1429G>A), have been recently described.
There is a significant ethnic difference in the frequency of CYP2C9 variants. CYP2C9*2 is reasonably frequent among Cauca-sians with 1% of the population being homozygous carriers and a significant 22% are heterozygous [52]. The corresponding figures for the CYP2C9*3 allele are 0.4% and 15%; with another 1.4% being compound heterozygotes – CYP2C9*2*3 [58]. CYP2C9*5 is estimated to be inherited in ~3% of the African-American popula-tion as a single allele mutation of 1080C>G [55, 56]. In addition, African-Americans have a significantly lower rate of CYP2C9*2and *3 inheritance than Caucasians, with 2.5% and 1.25% fre-quency, respectively.
Mutant alleles of the CYP2C9genehave been associated with slow hydroxylation of S-warfarin [59]. There are two common alle-lic polymorphisms in the CYP2C9 gene, including CYP2C9*2 and *3 that encode enzymes that are approximately 12% and 5% as efficient as the wild-type, respectively, and both have a substantial effect on the intrinsic clearance of warfarin [60]. Subjects who were homozygous for the CYP2C9*3 allele showed a 90% reduction in
744 Current Drug Metabolism, 2008, Vol. 9, No. 8the elimination of S-warfarin in comparison to subjects who were homozygous for the wild-type allele [61]. Impaired metabolism of a low therapeutic index drug such as warfarin has important clinical implications. Carriers of such polymorphisms require both smaller loading and maintenance doses and have a 4-fold increase in risk of bleeding complications, particularly at the beginning of therapy [60]. An individual that requires a low dose of warfarin is 6-fold more likely to be positive for one or more of the variant alleles compared with the general population. Patients who are CYP2C9*3homozygous require the lowest doses [58]. Pharmacogenetic testing of CYP2C9 would be useful to identify this subgroup of patients who have difficulty at the initiation of warfarin therapy, and are potentially at a higher risk of haemorrhage. These findings clearly demonstrate the need for clinical assessment of CYP2C9 genotype when establishing optimal warfarin therapy [62].
There exists variation between ethnic groups in warfarin ther-apy with Hong Kong Chinese patients and resident Asian patients in southern California requiring only half the warfarin maintenance dose and nearly 40% decreased average dose of that of Caucasians [59]. Additionally, the adjusted mean weekly warfarin dosage needed to maintain an internationally normalized ratio between 2.0 and 3.0 was lowest in Asian-Americans and highest in African-Americans [63]. Genetic differences can account for some of the interethnic differences in warfarin dosing. The CYP2C9*2 allele appears to be absent in Chinese, but present in native Canadian Indians and Caucasian at frequencies of 0.03, 0.08 and 0.15, respec-tively [64]. The CYP2C9*3 variant occurs in native Canadian Indi-ans at a frequency similar to that in other ethnic groups. The CYP2C9*2 variant is absent and the CYP2C9*3 allele occurs in low frequency in the Japanese compared to Jewish Israelis. In Indian patients, CYP2C9*2 and *3 allelic frequencies are similar to that of Caucasians, however, the *2 allele is very rare and usually absent in East Asian populations including Malays, Koreans, Chinese and Japanese [58]. Although the CYP2C9*3 variant has been associated with re-duced warfarin dosage requirement in Chinese and Indians, this variant is uncommon in Chinese and Malays and therefore cannot account for their lower requirements of warfarin [59]. Chinese CYP2C9*3 carriers require a reduced maintenance dose of warfarin compared to Indian CYP2C9*3 carriers, while in Malays, the occur-rence of the CYP2C9*3 allele is not linked with decreased dosage requirements [59]. As such, the reduced warfarin dosage require-ments displayed in Chinese and Malay populations, strongly sug-gests that other factors are involved besides CYP2C9 polymor-phism.
Variation in warfarin dosing could be due partially to polymor-phisms in the gene encoding vitamin K epoxide reductase complex 1 (VKORC1), a target enzyme for warfarin. Recently, the gene that encodes it has been cloned and non-synonymous SNPs have been found in warfarin-resistant patients [58]. As a result of further stud-ies, a number of common polymorphisms in non-coding sequences have been identified. Several studies have shown that together with environmental factors such as age and body size, CYP2C9 and VKORC1 genotypes explain almost two-thirds of the interindividual variability in warfarin dosage requirement [65]; however, more than one-third of the variability is yet to be determined.
CYP2C19
The CYP2C19 enzyme is a protein of 490 amino acids. It is encoded by the CYP2C19 gene consisting of 9 exons which is mapped on chromosome 10 (10q24.1-q24.3) [48]. CYP2C19 is primarily located in hepatic tissue, but a significant amount is also found in the gut wall, particularly the duodenum. CYP2C19 is re-sponsible for the metabolism of approximately 10% of commonly used drugs, including proton pump inhibitors (e.g. omeprazole, lansoprazole and pantoprazole), antidepressants (e.g. imipramine, amitriptyline and nortriptyline), benzodiazepines (e.g. diazepam
Zhou et al.
and flunitrazepam), phenytoin, mephenytoin, phenobarbital and proguanil [66].
The majority of enzyme deficiency associated with PMs of S-mephenytoin has been found to be the responsibility of various variant alleles of CYP2C19. To date, at least 21 variants of CYP2C19 (*1B to *20) have been identified (Table 3)(http://www.imm.ki.se/CYPalleles, access date: 2 August 2008). CYP2C19*1 represents the wild-type. The first CYP2C19 variant allele to be discovered was CYP2C19*2containing 681G>A on exon 5 that causes splicing defect[67]. CYP2C19*3 carries the 636G>A SNP resulting in a premature stop codon in exon 4. Both CYP2C19*2 and *3 are null alleles, resulting in no enzyme activity [68]. The majority of PMs of CYP2C19 are due to these two variant alleles [66]. CYP2C19*4is an initiation codon variant of 1A>G, resulting in GTG initiation codon [69]. CYP2C19*5,*6, and *8harbour 1297C>T, 395G>A, and 358T>C, respectively, that cause single amino acid changes and affect the structure and stability of the protein [70, 71]. CYP2C19*7 has a splicing defect at intron. The CYP2C19*4,*6 and *7 result in no enzyme activity, and the *5allele causes greatly reduced enzyme activity. CYP2C19*8 encodes a protein with decreased enzyme activity. Because these variant alleles (CYP2C19*4 to *8) only account for a minor percentage of CYP2C19 defective alleles, it is unlikely that these alleles will re-sult in clinically significant consequences. More recently, new po-tentially defective variant alleles of CYP2C19 have been discovered and these alleles are CYP2C19*9 through *19. Three types of CYP2C19 genotypes of the PM phenotype exist, including two homozygous genotypes, *2/*2 and *3/*3, and one heterozygous genotype, *2/*3. The homozygous CYP2C19*2/*2genotype is by far the most frequent of the three defective/PM genotypes [66]. There are also three types of EM genotypes. For EMs, there are two genotypes that are heterozygous for the CYP2C19 wild type, *1/*2 and *1/*3, and one genotype that is homozygous for the wild-type allele, *1/*1.
The distribution of the common variant alleles of CYP2C19 has been found to vary among different populations. The allelic fre-quency of CYP2C19*2 has been shown to be 17% in African-Americans, 30% in Chinese and 15% in Caucasians [66]. CYP2C19*3 has also been shown to be more frequent in Chinese (5%) and less frequent in African-Americans (0.4%) and Cauca-sians (0.04%). CYP2C19*2 is the dominant defective allele and accounts for around 75-85% of PM phenotype in Chinese and Cau-casian populations [66]. Almost all PMs in the Asian and Black African populations can be attributed to CYP2C19*2 and CYP2C19*3. The minor CYP2C19 variant alleles also show variations in ethnic distribution. CYP2C19*4 and *5 were found at very low frequencies (<0.5%) in Chinese [69]. CYP2C19*9,*10,*12,*13,*14 and *15 have been detected at low frequencies in African-Americans and individuals with African descent [72]. CYP2C19*16is found at a frequency of 0.6% in Japanese [73]. CYP2C19*17 has been reported in the Chinese, Swedish and Ethiopian populations at frequencies of 4-19% [66]. CYP2C19*6 to *15 have not yet been detected in any Asian populations, but *18 and *19 have been found in the Japanese population at low frequencies of 0.2-0.3% [74].
The PM phenotype of CYP2C19 is inherited as an autosomal recessive trait and the distribution of PM trait varies greatly with ethnic background (Table 4). The Japanese population has an 18 to 23% incidence of PMs of S-mephenytoin [66]. 15 to 17% of the Chinese population are PMs and 13% of Koreans are PMs [75, 76]. The Asian population has a much greater frequency of PM (12-23%), when compared to Caucasians (1-6%), and Black Africans (1-7.5%) [77, 78]. The frequency of PMs in African, African-American and Middle Eastern populations is very similar to the Caucasian population. Indigenous populations, such as the Cana-
Pharmacogenetics and Clinical PracticeTable 3.
CYP2C19
Current Drug Metabolism, 2008, Vol. 9, No. 8 745
Reported Variants of Human CYP2C19
Nucleotide changes
Amino acid change
Reference
cDNA Gene *1A None *1B *1C
99C>T; 991A>G 991A>G
None 99C>T; 80161A>G 80161A>G
99C>T; 19154G>A; 80160C>T; 80161A>G
99C>T; 12460G>C; 19154G>A; 80160C>T; 80161A>G -98T>C; 99C>T; 12122G>A; 12662A>G; 12834G>C; 19154G>A; 19520A>G; 57740C>G; 79936T>A; 80160C>T; 80161A>G
17948G>A; 80161A>G; 87313A>C
None I331V I331V
Splicing defect; I331V E92D; splicing defect;I331V
A161P, splicing defect;I331V W212X; I331V
[48] [371] [72] [68] [372]
*2A 99C>T; 681G>Aa; 990C>T; 991A>G *2B
99C>T; 276G>C; 681G>A; 990C>T;
991A>G
99C>T; 481G>C; 681G>A; 990C>T; 991A>G
636G>A; 991A>G; 1251A>C 636G>A; 991A>G; 1078G>A; 1251A>C
*2C (=*21)*3A*3B (=*20)
[373]
[67]
-889T>G; 12013T>G; 12122G>A; 12306G>A; 13166T>C;
W212X; D360N; I331V 17948G>A;18911A>G; 80161A>G; 80248G>A;
87313A>C
1A>G; 99C>T; 80161A>G 90033C>T
GTG initiation codon;I331V
[373]
*4*5A
1A>G; 99C>T; 991A>G 1297C>T
[69]
R433W [70, 374] 5B 99C>T; 991A>G; 1297C>T 99C>T; 80161A>G; 90033C>T I331V; R433W [70] *6 99C>T; 395G>A; 991A>G *7 *8*9 *10 *11 *12
358T>C
99C>T; 431G>A; 991A>G 99C>T; 680C>T; 991A>G 99C>T; 449G>A; 991A>G 99C>T; 991A>G; 1473A>C
99C>T; 12748G>A; 80161A>G 19294T>A12711T>C
99C>T; 12784G>A; 80161A>G 99C>T; 19153C>T; 80161A>G 99C>T; 12802G>A; 80161A>G 99C>T; 80161A>G; 90209A>C
R132Q; I331V
[372]
Splicing defect [372] W120R [372] R144H; I331V P227L; I331V R150H; I331V I331V; X491C; 26 extra amino acid I331V; R410C L17P; I331V I19L; I331V
[72] [72] [72] [72]
*13 *14 *15 *16 *17 *18 *19
991A>G; 1228C>T 50T>C; 99C>T; 991A>G 55A>C; 991A>G
80161A>G; 87290C>T 50T>C; 99C>T; 80161A>G 55A>C; 80161A>G
[72] [72] [72]
1324C>Tb 90060C>T CR442 [73] 99C>T; 991A>G
99C>T; 986G>A; 991A>G 99C>T; 151A>G; 991A>G
-3402C>T; -1041A>G; -806C>T; 99C>T; 80161A>G 99C>T; 80156G>A; 80161A>G; 87106T>C 99C>T; 151A>G; 80161A>G; 87106T>C
I331V R329H; I331V S51G; I331V
[375,
376] [373] [373]
*20 See *3B *21 See *2C
ab
Nucleotide variations in bold are the major SNPs responsible for the phenotype of the corresponding allele. Existence of the CYP2C9*2 polymorphism 681G>A on the same allele can not be excluded.
746 Current Drug Metabolism, 2008, Vol. 9, No. 8Table 4.
Interethnic Distribution of Poor Metaboliser Phenotype of CYP2C19
Ethnic group
PM (%)
Zhou et al.
Total PM (%)
Japanese 15.1-22.5 (18.75) Korean 12.6 Philipino 23.1 Chinese 13.4-19.8 (16.6) Sri Lankan
14
Indian 11-20.8 (15.9) Asian
Indonesian 15.4 21.51
Vietnamese 21.6 South Pacific Polynesian
13.6
Greenland 2.9-9.3 (6.1) Vanuatu 79 Turkish 0.94 Jordanian 4.6 Israeli 2.9 Tanzanian 2.8-4.6 (3.7) African
Zimbabwean 4 4.3
Ethiopian 5.2 African-American 2-7 (4.5) Swedish 1.2-4.6 (2.9) French 6 Danish 2.5-3.8 (3.15) Portuguese 1.3 Spanish 1.3 Caucasian
3.03 Russian 2.3 Estonian 0.95 Australian 7 Canadian 2.4 American 2-2.7 (2.35) Causasian-American Canadian Indian
Indigenous
Canadian Inuit Panama Cuna Indian Australian Aboriginals
Data are extracted from references [66, 69-71, 74, 373].
19.1 2 0 25.6
11.65
dian Indians and the Australian Aboriginals, have a high frequency
of PMs, which is similar to that of the Asian population [66]. The Cuna Indians of Panama do not appear to have the PM trait. In con-trast, 79% of the population of Vanuatu are PMs, which is most likely to be due to a genetic founder effect [79].
The ethnic differences in the metabolism of CYP2C19 sub-strates are primarily due to the interethnic variation in distribution of the PM trait. The Asian population has approximately 65 to 70% PMs and heterozygous EMs, whereas Caucasians have only 20 to 25% [66]. The Chinese population has a two-fold greater number of heterozygous EMs than the Caucasian population. The Chinese population has been shown to have slower elimination of diazepam when compared to Caucasians, which suggests that CYP2C19 PMs are more common among the Chinese. Furthermore, Chinese and Koreans were also found to have slower clearance of omeprazole than the Caucasian population, which further suggests that Asians more frequently have the PM CYP2C19 phenotype.
CYP2D6
One of the most studied CYPs is the polymorphic CYP2D6 that accounts for only a small percentage of all hepatic P450 enzymes, however, it metabolises 25% of all medications in the human liver, with many of them having a narrow therapeutic index [80]. The CYP2D6 gene is mapped to chromosome 22q13.1 and encom-passes nine exons with an open reading frame of 1383 base pairs coding for 461 amino acids [81]. CYP2D6 belongs to a gene cluster of highly homologous inactive pseudogenes CYP2D7 and CYP2D8[82, 83]. CYP2D6 can metabolize a number of drugs, including antidepressants (e.g. clozapine, imipramine and nortriptyline), neu-roleptics (e.g. haloperidol), -blockers (e.g. atenolol and alprenol) and antiarrhythmics (e.g. debrisoquine) [83]. There is a large inter-individual variation in the enzyme activity of CYP2D6 mainly brought about by genetic polymorphisms which often presents as a barrier to reaching optimal therapeutic concentrations.
Pharmacogenetics and Clinical Practice To date, more than 63 different CYP2D6 variants are identified by the human cytochrome P450 allele nomenclature committee (Table 5) (http://www.imm.ki.se/CYPalleles, access date: 2 August 2008). Relative to the wild-type CYP2D6 allele, variant versions of the CYP2D6 gene may result in a complete absence of enzyme activity, reduced activity, normal activity or even increased activity. Null alleles of CYP2D6 do not encode a functional protein and there is no detectable residual enzymatic activity. They are respon-sible for the PM phenotype when present in homozygous or com-pound heterozygous constellations. The mechanism by which lead-ing to a total loss of function includes: a) single base mutations or small insertions/deletions that interrupt the reading frame or inter-fere with correct splicing leading to prematurely terminated pro-tein/stop codon (e.g. CYP2D6*3, *4, *6, *8, *11, *15, *19, *20, *38, *40, *42, and *44) [84]; b) non functional full length coded alleles (e.g. CYP2D6*5, *12, *14 and *18) [85]; and c) deletion of entire CYP2D6 gene as a result of large sequence deletions (e.g. CYP2D6*5. *13, and *16) [86]. On the other hand, extremely high CYP2D6 activity results from gene duplication of functional allele *1 and *2 fused in a head to tail orientation, as a result of unequal crossover events and other mechanisms. This was noted by a mo-lecular characterisation of the CYP2D6 locus in patients with ex-tremely rapid metabolisms [87]. The CYP2D6*17 allele has four mutations (1023>T, 1661G>C, 2850C>T, and 4180G>C) that result in decreased activ-ity [88]. Two of which are non-synonymous mutations (1661G>C and 2850C>T) in common with the *2 allele. This creates an altera-tion in the active site and hence an alteration in substrate specific-ity. The corresponding enzyme exhibits a five-fold higher apparent Km for codeine and the -adrenoceptor antagonist, bufuralol com-pared with the wild-type enzyme [89], and members carrying this allele exhibit a diminished capacity for drug metabolism [88].
•Between people with different ethnic backgrounds, the pattern
of CYP2D6 polymorphisms differs dramatically:
•Extremely high CYP2D6 activity occurs with allele frequencies
of 1-2% in Caucasians [90]. However, much higher frequencies have been observed in Saudi Arabians [91] and Ethiopians (up to 29%) [92].
•The sians. It occurs with a frequency of 20% to 25% and is respon-CYP2D6*4 is by far the most frequent null allele in Cauca-sible for 70% to 90% of all PMs [93]. No functional alleles are present in about 6% of Caucasians [93].
•The Asian population [94], and 6-7% in Africans and African-CYP2D6*4 allele occurs with a frequency of 1% in the
Americans [95].
•CYP2D6*5 seems to be present at similar frequencies in all
these population groups.
•The most common CYP2D6 allele in the Asian population is
CYP2D6*10occurring with a frequency exceeding 50% [94], which has a Pro34Ser substitution in the proline-rich region near the NH2-terminal. In Caucasians, it occurs at a low fre-quency of 2%, however, it accounts for 10-20% of individuals with the intermediate metabolizer phenotype [90].
•CYP2D6*17it occurs with a frequency of is virtually absent from European Caucasians but
30% in the African-American/ black population. It is the major variant in the black population and appears to explain why black Africans have a higher me-dian metabolic ratio [95].
Above data would therefore explain the apparent low frequency of the PM phenotype amongst the Asian and African populations [94]. These are demonstrated in studies highlighting the need for lower dose neuroleptics in Asian patients when compared to Cauca-sian subjects [96]. Also, studies have shown higher concentrations of S-mianserin found in Japanese patients with depression [97].
Current Drug Metabolism, 2008, Vol. 9, No. 8 747
Drugs that are most affected by CYP2D6 polymorphisms are commonly those in which CYP2D6 represents a substantial meta-bolic pathway either in the activation or clearance of the agent. For instance, CYP2D6 oxidation is a fundamental metabolic process in the activation of the anticancer agent, tamoxifen, to endoxifen, its most therapeutically active metabolite [98]. Therefore, individuals who have poor CYP2D6 enzymatic activity are likely to exhibit therapeutic failure, and those who have a high enzyme activity are likely to experience adverse effects and toxicities. PMs may also experience greater exposure to tamoxifen’s adverse effect profile which includes venous thrombosis and endometrial cancer [98]. Thus, proper dose adjustment may be needed when CYP2D6 geno-type is determined in a patient. The CYP2D6 genotype can be used to predict possible side effects associated with certain antidepressant and narcoleptic treat-ments and the likelihood of their occurrence, especially in geneti-cally PMs [99]. In a clinical study involving opiates (tramadol and codeine), the analgesic effects were seen to be substantially less in subjects found to be PMs for both medications [100]. Codeine is a prodrug which is metabolised into its active form morphine by CYP2D6-mediated O-demethylation reaction. In the case of co-deine, no analgesic effect was recorded in PMs [101, 102]. Clearly a lack of CYP2D6 enzyme would be expected to result in reduced drug plasma morphine concentrations and thus, reduce the effec-tiveness of the drug. Ironically, subjects deficient in CYP2D6 me-tabolism are protected against possible opioid dependence, as none of the poor metabolising subjects in the study populations showed symptoms or signs of dependence [103-105]. However, further research is required to establish the validity of such findings. CYP3A4
CYP3A4 has the highest abundance in the human liver (40%) and metabolizes more than 50% of clinical drugs [106]. The CYP3A4 gene is located on chromosome 7q22.1 and is about 27 kb long consisting of 13 exons and 12 introns [107]. The substrate specificity of the CYP3A4 enzymes is very broad, with an ex-tremely large number of structurally divergent chemicals being metabolized often in a regio- and stereo-selective fashion. CYP3A4 is known to metabolize a large variety of compounds varying in molecular weight from metyrapone (Mr = 226 Dal) to cyclosporine (Mr = 1203 Da) [106]. CYP3A4 exhibits a relatively large sub-strate-binding cavity that is consistent with its capacity to oxidize bulky substrates such as cyclosporine, statins, taxanes, and macrol-ide antibiotics. Although the active site volume is similar to that of CYP2C8, the shape of the active site cavity differs considerably due to differences in the folding and packing of portions of the protein that form the cavity [108]. Compared with CYP2C8, the active site cavity of CYP3A4 is much larger near the heme iron [108].
More than 19 CYP3A4 variants (*1B to *20) have been identi-fied to date (Table 6) (http://www.imm.ki.se/CYPalleles, access date: 2 August 2008). The CYP3A4*1B (i.e. CYP3A4-V) contains a -392A>G mutation in the nifedipine-specific response element of the 5’ regulatory region of the gene [109]. CYP3A4*2 containing a 664T>C SNP (leading to Ser222Pro change) was found at a fre-quency of 2.7% in Caucasians and was absent in the blacks and Chinese [110]. The CYP3A4*2 allele was found to encode a CYP with substrate-dependent altered kinetics compared with the wild-type enzyme. The allelic frequencyofCYP3A4*3 with a 1334T>C mutation and subsequently a Met445Thr change in the Caucasian was 1.1% [111]. The CYP3A4*4 and *5 alleles contain 352A>G (Ile118Val) and 653C>G (Pro218Arg) in exons 4 and 5, respec-tively [112]. CYP3A4*6 represents a frameshift mutation (831 insA) arising from an A17776 insertion in exon 9, resulting in an early stop codon. Functionally, the *4, *5 and *6 alleles are associ-ated with decreased CYP3A4 activity in Chinese [113] and Cauca-sian [114]. In addition, the alleles of CYP3A4*7 with the 167G>A SNP [115] through to *20 with an insertion of A between 1461 and
748 Current Drug Metabolism, 2008, Vol. 9, No. 8Table 5.
Reported Variants of Human CYP2D6
Zhou et al.
CYP2D6
*1A None Nucleotide change
N active genes R296C; S486T
Amino acid change Reference
[82] [377] [377] [377] [93] [93, 378] [379-381]
*1B 3828G>A C*1 1978C>T
*1D 2575C>A *1E 1869T>C *1XN *2A
-1584C>G; -1235A>G; -740C>T; -678G>A; CYP2D7 gene conversion in
intron 1; 1661G>C; 2850C>T; 4180G>C 1039C>T; 1661G>C; 2850C>T; 4180G>C 1661G>C; 2470T>C; 2850C>T; 4180G>C 2850C>T; 4180G>C
997C>G; 1661G>C; 2850C>T; 4180GC 1661G>C; 1724C>T; 2850C>T; 4180G>C
1661G>C; 2470T>C; 2575C>A; 2850C>T; 4180G>C 1661G>C; 2480C>T; 2850C>T; 4180G>C
*2B *2C *2D *2E *2F *2G *2H
R296C; S486T R296C; S486T R296C; S486T R296C; S486T R296C; S486T R296C; S486T R296C; S486T
R296C; S486T
[377] [93, 377] [377] [377] [377] [377] [377] [377] [382]
*2J See CYP2D6*59 *2K
1661G>C; 2850C>T; 4115C>T; 4180G>C
*2L (formerly *41B) -1584C; -1298G>A; -1235A>G; -740C>T; 310G>T; 746C>G; 843T>G; R296C; S486T
1513C>T; 1661G>C; 1757C>T; 2850C>T; 3384A>C; 3584G>A; 3790C>T; 4180G>C *2M
-1584C; -1237_-1236insAA; -1235A>G; -750_-749delGA; -740C>T; -678G>A; CYP2D7 gene conversion in intron 1; 310G>T; 746C>G;
843T>G; 1661G>C; 2850C>T; 2988G; 3384A>C; 3584G>A; 3790C>T; 4180G>C; 4481G>A
R296C; S486T
[383]
*2XN 1661G>C; 2850C>T; 4180G>C (N=2, 3, 4, 5 or 13) *3A *3B *4A *4B
R296C; S486T; N active genes
[92, 378, 379]
2549delAa 259Frameshift [84] 1749A>G; 2549delA
100C>T; 974C>A; 984A>G; 997C>G; 1661G>C; 1846G>A; 4180G>C 100C>T; 974C>A; 984A>G; 997C>G; 1846G>A; 4180G>C
N166D; 259frameshift
P34S; L91M; H94R; splicing defect; S486T P34S; L91M; H94R; splicing defect; S486T P34S; splicing defect; L421P; S486T P34S; splicing defect; S486T P34S; splicing defect; S486T
P34S; L91M; H94R; splicing defect; R173C; S486T
[377] [84, 384, 385]
[84] [386] [377] [377] [377]
C*4 100CC>T; 1661G>; 1846G>A; 3887T>C; 4180G>C
*4D
100C>T; 1039C>T; 1661G>C; 1846G>A; 4180G>C
*4E 100CC>T; 1661G>; 1846G>A; 4180G>C *4F
100C>T; 974C>A; 984A>G; 997C>G; 1661G>C; 1846G>A; 1858C>T; 4180G>C
100C>T; 974C>A; 984A>G; 997C>G; 1661G>C; 1846G>A; 2938C>T; 4180G>C
100C>T; 974C>A; 984A>G; 997C>G; 1661G>C; 1846G>A; 3877G>C; 4180G>C
100C>T; 974C>A; 984A>G; 997C>G; 1661G>C; 1846G>A
*4G P34S; L91M; H94R; splicing defect; P325L; S486T [377]
*4H P34S; L91M; H94R; splicing defect; E418Q; S486T [377]
*4J P34S; L91M; H94R; splicing defect [377] Pharmacogenetics and Clinical Practice
(Table 5) contd….
CYP2D6
Nucleotide change
Current Drug Metabolism, 2008, Vol. 9, No. 8 749
Amino acid change
P34S; splicing defect; R296C; S486T P34S; splicing defect; S486T
Reference [93] [387]
*4K 100CC>T; 1661G>; 1846G>A; 2850C>T; 4180G>C *4L *4M
100C>T; 997C>G; 1661G>C; 1846G>A; 4180G>C
-1235A>G; 746C>G; 843T>G 974C>A; 984A>G; 997C>G; 1661G>C;
1846G>A; 2097A>G; 3384A>C; 3582A>G; 4401C>T
-1426C>T; -1235A>G; -1000G>A; 100C>T; 310G>T; 746C>G; 843T>G; 974C>A; 984A>G; 997C>G; 1661G>C; 1846G>A; 2097A>G; 3384A>C; 3582A>G; gene conversion to CYP2D7 in exon 9; 4180G>C; 4401C>T
L91M; H94R; splicing defect [388-390] *4N (Found in a gene duplication) P34S; L91M; H94R; splicing defect; P469A; T470A; H478S; G479A; F481V; A482S; S486T
[390]
*4X2 *5*6A *6B *6C *6D *7*8 *9*10A *10B
CYP2D6 deleted
CYP2D6 deleted
[391, 392] [86, 393]
1707delT 118Frameshift [394] 1707delT; 1976G>A
1707delT; 1976G>A; 4180G>C 1707delT; 3288G>A
118Frameshift 118Frameshift 118Frameshift
[395, 396] [377] [377]
2935A>C H324P [397] 1661G>C; 1758G>T; 2850C>T; 4180G>C
G169X
[398]
2615_2617delAAG K281del [399, 400] 100C>T; 1661G>C; 4180G>C
-1426C>T; -1237_-1236insAA; -1235A>G; -1000G>A; 100C>T;
1039C>T; 1661G>C; 4180G>C
P34S; S486T P34S; S486T
[386] [401]
*10C *10D *10X2 *11*12*13
100C>T; 1039C>T; 1661G>C; 4180G>C, CYP2D7-like 3'-flanking region P34S; S486T
883G>C; 1661G>C; 2850C>T; 4180G>C 124G>A; 1661G>C; 2850C>T; 4180G>C CYP2D7P/CYP2D6 hybrid:
Exon 1 CYP2D7, exons 2-9 CYP2D6
Splicing defect; R296C; S486T G42R; R296C; S486T
[402] [403-406] [407] [408]
Frameshift [409] *14A 100C>T; 1758G>A; 2850C>T; 4180G>C *14B *15*16
intron 1 conversion with CYP2D7 (214-245); 1661G>C; 1758G>A;
2850C>T; 4180G>C
P34S; G169R; R296C; S486T G169R; R296C; S486T
[410] [404]
137_138insT 46Frameshift [411] CYP2D7P/CYP2D6 hybrid:
Exons 1-7 CYP2D7P-related, exons 8-9 CYP2D6 1023C>T; 1661G>C; 2850C>T; 4180G>C
Frameshift [412] *17*17XN *18
T107I;R296C; S486T
[88, 89] [413] [414] [377] [415]
4125_4133dupGTGCCCACT 468_470dupVPT 255Frameshift 211Frameshift
*19 1661G>C; 2539_2542delAACT; 2850C>T; 4180G>C
*20 1661G>C; 1973_1974insG; 1978C>T; 1979T>C; 2850C>T; 4180G>C *21A
-1584C>G; -1426C>T; -1258_-1257insAAAAA; -1235A>G; -740C>T; -678G>A; -629A>G; 214G>C; 221C>A; 223C>G; 227T>C; 310G>T;
601delC; 1661G>C; 2573_2574insC; 2850C>T; 3584G>A; 4180G>C
267Frameshift [416] 750 Current Drug Metabolism, 2008, Vol. 9, No. 8Zhou et al.
(Table 5) contd….
CYP2D6 Nucleotide change
-1584C>G; -1235A>G; -740C>T; -678G>A; intron 1 conversion with CYP2D7 (214-245); 1661G>C; 2573_2574insC; 2850C>T; 4180G>C
Amino acid change Reference
*21B 267Frameshift [417]
[377] [377] [377] [377] [377] [377] [377] [377, 418, 419]
[377] [377] [377]
[377] [377] [377, 420] [90] [95, 401]
*22 82C>T *23 957C>T *24 2853A>C *25 3198C>G *26 3277T>C *27 3853G>A *28 *29 *30 *31 *32
19G>A; 1661G>C; 1704C>G; 2850C>T; 4180G>C 1659G>A; 1661G>C; 2850C>T; 3183G>A; 4180G>C 1661G>C; 1863_1864insTTTCGCCCC; 2850C>T; 4180G>C 1661G>C; 2850C>T; 4042G>A; 4180G>C 1661G>C; 2850C>T; 3853G>A; 4180G>C
CR28
A85V I297L R343G I369T E410K V7M; Q151E; R296C; S486T V136M; R296C; V338M; S486T 174_175insFRP; R296C; S486T R296C; R440H; S486T R296C; E410K; S486T A237S
CR296
V11M; R296C; S486T V11M; R296C; S486T
P34S; P469A; T470A; H478S; G479A; F481V; A482S;
S486T
*33 2483G>T *34 2850C>T *35 *35X2
*36 (Duplication or tandem) *36 (Single)
-1584C>G; 31G>A; 1661G>C; 2850C>T; 4180G>C 31G>A; 1661G>C; 2850C>T; 4180G>C
-1426C>T; -1237_-1236insA; -1235A>G; -1000G>A; 100C>T; 1039C>T; 1661G>C; gene conversion to CYP2D7 in exon 9; 4180G>C
-1426C>T; -1235A>G; -1000G>A; 100C>T; 310G>T; 843T>G; 1039C>T; P34S; P469A; T470A; H478S; G479A; F481V; A482S; 1661G>C; 2097A>G; 3384A>C; 3582A>G; gene conversion to CYP2D7 in S486T exon 9
100C>T; 1039C>T; 1661G>C; 1943G>A; 4180G>C;
P34S; R201H; S486T
[390]
*37 *38
[377]
2587_2590delGACT 271Frameshift [95]
S486T
[387] [421] [381, 422-424]
*39 1661G>CC; 4180G>
*40 1023CC>T; 1661G>; 1863_1864ins(TTT CGC CCC)2; 2850C>T; 4180G>CT107I; 174_175ins(FRP)2; R296C; S486T *41*42
-1584C; -1235A>G; -740C>T; -678G>A; CYP2D7 gene conversion in intron 1; 1661G>C; 2850C>T; 2988G>A; 4180G>C -1584C; 1661G>C; 2850C>T; 3259_3260insGT; 4180G>C
R296C; splicing defect; S486T
R296C; 365Frameshift [425] R26H Splicing defect
[377] [417]
*43 77G>A *44 82C>T; 2950G>C
-1601_-1600GA>TT; -1584C; -1238_-1237delAA; -1094_-1093insA; -1011T>C; 310G>T; 746C>G; 843T>G; 1661G>C; 1716G>A; 2129A>C;
2575C>A; 2661G>A; 2850C>T; 3254T>C; 3384A>C; 3584G>A; 3790C>T; 4180G>C
-1584C; -1543G>A; -1298G>A; -1235A>G; -1094_-1093insA; -740C>T; -695_-692delTGTG; 310G>T; 746C>G; 843T>G; 1661G>C; 1716G>A; 2575C>A; 2661G>A; 2850C>T; 3254T>C; 3384A>C; 3584G>A; 3790C>T; 4180G>C
-1584C; -1543G>A; -1298G>A; -1235A>G; -740C>T; 77G>A; 310G>T; 746C>G; 843T>G; 1661G>C; 1716G>A; 2575C>A; 2661G>A; 2850C>T; 3030G>G/A*; 3254T>C; 3384A>C; 3491G>A; 3584G>A; 3790C>T; 4180G>C
*45A E155K; R296C; S486T [382]
*45B E155K; R296C; S486T [382]
*46R26H; E155K; R296C; S486T [382]
Pharmacogenetics and Clinical Practice
(Table 5) contd….
CYP2D6
Nucleotide change
Current Drug Metabolism, 2008, Vol. 9, No. 8 751
Amino acid change Reference
*47
-1426C>T; -1235A>G; -1000G>A; 73C R25W; P34S; S486T 4180G>C A90V P34S; F120I; S486T E156A R296C; E334A; S486T [426] [426] [426] [426] [426] *48 972C>T *49 -1426C>T; -1235A>G; -1000G>A; 100C>T; 1039C>T; 1611T>A; 1661G>C; 4180G>C *50 1720A>C *51 -1584C>G; -1235A>G; -740C>T; -678G>A; CYP2D7 gene conversion in intron 1; 1661G>C; 2850C>T; 3172A>C; 4180G>C -1426C>T; -1245_-1244insGA; -1235A>G; -1028T>C; -1000G>A; -377A>G; 100C>T; 1039C>T; 1661G>C; 3877G>A; 4180G>C; 4388C>T; 4401C>T 1598A>G; 1611T>A; 1617G>T 100C>T; 1039C>T; 1661G>C; 2556C>T; 4180G>C 1661G>C; 2850C>T; 3790C>T; 3835A>C; 4180G>C *52P34S; E418K http://www.imm.ki.se/CYPal-leles[427] [427] [427] *53 *54 *55 F120I; A122S P34S; T261I; S486T R296C; K404Q; S486T *56A -1584C>G; -1235A>G; -740C>T; -678G>A; CYP2D7 gene conversion in intron 1; 1661G>C; 2850C>T; 3201C>T; 3384A>C; 3584G>A; 3790C>T;R296C; R344X [428] 4180G>C -1426C>T; -1235A>G; -1000G>A; 100C>; 310G>T; 843T>G; 1039C>T; 1661G>C; 2097A>G; 3201C>T; 3384A>C; 3582A>G, 4180G>C *56B P34S; R344X [429] P34S; R62W; P469A; T470A; H478S; G479A; F481V; A482S; S486T *57 (In tandem with 100C>T; 310G>T; 843T>G; 887C>T; 1039C>T; 1661G>C; 3384A>C; *10) 3582A>G; gene conversion to CYP2D7 in exon 9; 4180G>C -1426C>T; -1235A>G; -740C>T; CYP2D7 gene conversion in intron 1; 310G>T; 843T>G; 1023C>T; 1661G>T; 1863_1864insTTTCGCCCC; 2850C>T; 3384A>C; 3584G>A; 3790C>T; 4180G>C [430] *58T107I; 174_175insFRP; R296C; S486T http://www.imm.ki.se/CYPal-leles[377, 431] *59 1661G>C; 2291G>A; 2850C>T; 2939G>A; 4180G>C *60 R296C; S486T *61 gene conversion to CYP2D7 in exon 9 P469A; T470A; H478S; G479A; F481V; A482S; S486T http://www.imm.ki.se/CYPal-leles *624044C>T R441C [432] R296C; P469A; T470A; H478S; G479A; F481V; A482S; S486T *63 2850C>T; gene conversion to CYP2D7 in exon 9 http://www.imm.ki.se/CYPal-leles *64 -1426C>T; -1235A>G; -1000G>A; 100C>T; 310G>T; 843T>G; 1023C>T; P34S; T107I; S486T 1661G>C; 2097A>G; 3384A>C; 3582A>G; 4180G>C; 4401C>T; 4722T>G 100C>T; 310G>T; 843T>G; 1661G>C; 2850C>T; 3384A>C; 3584G>A; 3790C>T; 4180G>C; 4481G>A CYP2D7P/CYP2D6 hybrid: Exons 1-6 CYP2D7, exons 7-9 CYP2D6 CYP2D7P/CYP2D6 hybrid: Exons 1-5 CYP2D7, exons 6-9 CYP2D6 P34S; R296C; S486T [433] *65[433] *66Frameshift [433] *67Frameshift http://www.imm.ki.se/CYPal-lelesData are extracted from http://www.imm.ki.se/CYPalleles (access date: 2 August 2008). a Nucleotide variations in bold are the major SNPs responsible for the phenotype of the corresponding allele. 752 Current Drug Metabolism, 2008, Vol. 9, No. 8Table 6. Reported Variants of Human CYP3A4 Gene Nucleotide change Amino acid change Zhou et al. CYP3A4 Reference cDNA Gene *1A *1B *1C *1D *1E *1F *1G *1H *1J *1K *1L *1M *1N *1P *1Q *1R *1S *1T None None -392A>G -444T>G -62C>A -369T>A -747C>G 20230G>A 20230G>A; 26206C>A 6077A>G -655A>G -630A>G -156C>A 14200T>G 15727G>A 15809T>C 16775A>G 17815_17816delAT 26013T>C C15713T> C23171T> 13871A>G C15702>G 17661_176622insA 6004G>A 13908G>A 14292G>A C14304G> C21867>T C21896>T C22026>T C44 T> 14269G>A -845_-844insATGGAGTGA; -392A>G; 14269G>A S222P M445T I118V P218R [434] [129, 435] [436] [436] [437] [437] [438] [438] [438] [438] [438] [438] [438] [438] [438] [438] [438] [438] [110] [110] [113] [113] *2 664T>C *3 1334T>C *4 352A>G *5 653C>G *6 830_831insA *7 167G>A *8 389G>A *9 508G>A *10 520G>C *11 1088C>T *12 1117C>T *13 1247C>T *14 44T>C *15A 485G>A *15B 485G>A *16A 277Frameshift [113] G56D R130Q V170I D174H T363M L373F P416L L15P R162Q [115] [115] [115] [115] [115] [115] [115] [112] [112] R162Q [112, 437] T185S [439] 554C>Ga 15603C>G Pharmacogenetics and Clinical Practice (Table 6) contd…. CYP3A4 Nucleotide change Current Drug Metabolism, 2008, Vol. 9, No. 8 753 Amino acid change Reference cDNA Gene *16B*17*18A*18B 554C>G 15603C>G; 20230G>A 566T>C 15615T>C878T>C878T>C 20070T>C 20070T>C; 20230G>A C23237>T; 20230G>A 25889_25890insA T185S [438, 439] F189S [440] L293P [440] L293P [438] P467S [440] *19 1399C>T *20 1461_1462insA 488Frameshift [116] Data are extracted from http://www.imm.ki.se/CYPalleles (access date: 2 August 2008). a Nucleotide variations in bold are the major SNPs responsible for the phenotype of the corresponding allele. 1462 nucleotides resulting in completely defective enzyme [116] have been identified and a number of additional SNPs detected in CYP3A4. Genetic polymorphisms in CYP3A4 seem to be more prevalent in Caucasian population than in Asians. In genotyping studies com-paring the frequencies of the four CYP3A4allelic variants (CYP3A4*1B, *4, *5 and *6) in healthy subjects, CYP3A4*4was detected in 2 of 110 Chinese subjects and CYP3A4*6was found in 1 of 104 Malays and 1 of 101 Indians, whereas the *1Band *5 al-leles were absent in all three ethnic groups [117]. The frequency of CYP3A4*1B carrying -392A>G was higher in white and Hispanic subjects (3.6-11.0%) and much higher in blacks (53.0-69.0%). Like other Asian ethnic groups, CYP3A4*1B is absent in Japanese [118]. There is no consensus on a direct functional correlation of CYP3A4 polymorphism, but CYP3A4 alleles may have minor to moderate clinical importance based on currently available data. These coding variants may contribute to but are not likely to be the major cause of interindividual differences in the clearance of CYP3A4 substrates, because of the low allele frequencies and lim-ited alterations in enzyme expression or catalytic function [112]. ThoughCYP3A4*1B allele was shown to be associated with lower hepatic CYP3A activity in most ethnic groups including Chinese, African and Caucasian, the differences were only modest and did not remarkably impact on the elimination of CYP3A4 substrates [119]. For example, there was no difference in the rate of CYP3A4-mediated demethylation of erythromycin or the pharmacokinetics of nifedipine between the two genotype groups (AA or GG) in black Americans [119]. CYP3A4*1B did not correlate with low enzyme activity, either in vivo or in vitro, in either homozygotes or het-erozygotes [114]. Hepatic CYP3A activity and the systemic clear-ance of midazolam were 30% lower in G/G homozygotes than in A/A homozygotes in African Americans and European descent [120]. Additionally, CYP3A4*1B did not significantly alter the pharmacokinetics of cyclosporine in humans compared to the wild-type [121, 122]. It seems that ethnic differences in CYP3A4 polymorphisms, particularly in Asians are very rare and do not appear to fully ex-plain the large interindividual variation in CYP3A4 activity. There is a significant variation in the hepatic expression of CYP3A4 based on in vitro and in vivo [106], which is considered to be the combined effects of a number of environmental, physiological and genetic factors. CYP3A4 variability might be partially explained by allelic variations in one of its master transcriptional regulators, the nuclear pregnane X receptors (PXR, also called steroid and xenobi-otic receptor or pregnane-activatedreceptor [123]. Model inducers of CYP3A4 include rifampicin, phenobarbital and dexamethasone. PXR is a major activator of CYP3A transcription and the resulting transcriptional activation of CYP3Ainvolves the formation of a heterodimer with the retinoid X receptor which binds to several PXR response elements in the CYP3A4 5’ flanking regions [123]. A number of SNPs have been identified in the PXR gene that might affect an individual’s ability to induce CYP3A4 [124]. CYP3A4*1B is associated with early puberty [125]. High activ-ity of CYP3A4, but not CYP3A5, which primarily metabolizes testosterone, showed a striking association with the onset of puberty [125]. However, the polymorphic variants of the CYP3A4 gene did not influence age of menarche [126] and a recent study did not identify an association between CYP3A4*1B and risk of breast or ovarian cancer [127]. This may reflect the complex interplay be-tween genetic and environmental factors in carcinogenesis. CYP3A4*1B has been associated with high grade and advanced stage of prostate cancers in Caucasians, Hispanic, African Ameri-cans [128]. The associations were most pronounced among men aged >65 years of age with no family history [129, 130] or in men with benign prostate hyperplasia [131]. CYP3A4 is involved in the oxidative deactivation of testosterone and thus may modify the risks of sex hormone-related tumours. There is a strong link between high levels of testosterone and prostate cancer development [132, 133]. Africans have the highest frequency of CYP3A4*1B allele that has been associated with increased prostate cancer risk. Link-age disequilibrium between CYP3A4*1B and another CYP3A4al-lele (CYP3A5*1) may be the underlying cause of the clinical pheno-type. In addition, there are several reports on the association of CYP3A4*1B with therapy-related leukemia [134, 135]. Individuals with CYP3A4*1B genotype may be at increased risk for epipodo-phyllotoxin-related leukemia and that epipodophyllotoxin metabo-lism by CYP3A4 may contribute to the secondary cancer risk [135]. The CYP3A4*1B genotype may increase production of potentially DNA-damaging reactive intermediates. The variant may decrease production of the epipodophyllotoxin catechol metabolite, which is the precursor of the potentially DNA-damaging quinone. However, CYP3A4*1B genotypes had a decreased risk of peripheral neuropa-thy in leukaemia children receiving chemotherapy [136]. This vari-ant was not associated with an increased risk of relapse. Despite large variability in the expression of CYP3A4, its contribution to the effect of anticancer drugs has not yet been elucidated. Since almost half of all anticancer drugs are CYP3A4 substrates, poly-morphisms in CYP3A4 are likely to alter the pharmacokinetics and pharmacodynamics of anticancer drugs. CLINICAL PHARMACOGENETICS OF PHASE II DRUG METABOLIZING ENZYMES Phase II metabolism, usually known as conjugation reaction, includes glucuronidation, sulfation, methylation, acetylation, glutathione conjugation and amino acid conjugation. These 754 Current Drug Metabolism, 2008, Vol. 9, No. 8reactions are usually detoxication in nature, and involve the interactions of the polar functional groups of phase I metabolites. Many Phase II conjugating enzymes exhibit polymorphic variations and some of them can result in functional changes. N-Acetyltransferase (NAT2) In humans, NAT1 and NAT2 are located on human chromosome 8p22 [137, 138]. A third pseudogene, NAT3, has been identified but it does not encode any functional protein [139]. Both NAT1 and NAT2 genes are intronless and encode a 290 amino acid protein that share 87% nucleotide homology in the coding region. NAT2 (EC 2.3.1.87) is primarily present in the liver, and in the intestine, while NAT1 is expressed in almost all tissues. NATs transfer an acetyl group from acetyl coenzyme A to the terminal nitrogen or oxygen atom of primary arylamines, hydrazines, and N-hydroxylated me-tabolites [140, 141]. NATs, however, are also involved in bioactiva-tion reactions such as O-acetylation of N-hydroxyarylamines and intramolecular N,O-acetyltranfer of arylhydroxamic acids [140]. Human NAT2 provides a major route to detoxification of other drugs including the anti-hypertensive drug hydralazine and anti-bacterial sulphonamides. Individuals can be divided into rapid, intermediate and slow acetylator phenotypes for NAT2. In humans, the genetic individual variation of NAT2 protein content is based of the well-known isoniazid acetylation polymorphism, which has significant toxicological implications. To date, at least 15 different NAT2 alleles (*5A through to *19)have been identified in human populations (Table 7) (http:// www. louisville. edu/ medschool/ pharmacology/ NAT.html,access date: 2 August 2008). Although NAT2*4 is designated as the “wild-type” for NAT2, owing to its very frequent occurrence in some eth-nic groups, this designation is somewhat arbitrary. This allele is present in ~20% Caucasians, ~30% African-Americans, and 42% Hispanics, and with Asian frequencies varying between ~66-70% [142]. The NAT2 alleles containing the 191G>A, 341T>C, 434A>C, 590G>A and/or 857G>A SNPs are known to be associ-ated with the slow acetylator phenotypes [143]. The NAT2*5A(341T>C), *6A (590G>A)and *7A (857G>A)alleles accounts for more than 95% of slow acetylators in Caucasians [144]. There are remarkable ethnic differences in the frequencies of genetic polymorphisms of NAT2 associated with the slow acetylator phenotype [481C>T (NAT2*5A), 590G>A (NAT2*6A) and 857G>A (NAT2*7A)]. The allelic frequencies for wild-type, NAT2*5A,*6Aand *7A alleles in the Chinese are 0.51, 0.075, 0.32 and 0.10, re-spectively (Table 8) [145]. These results were similar to those re-ported amongst Hong Kong Chinese [144]. The observed allelic frequencies for wild-type, NAT2*5A,*6A and *7A alleles in the Malay subjects were 0.41, 0.12, 0.38 and 0.09, respectively, and in the Indian subjects are 0.44, 0.20, 0.32 and 0.04, respectively [146]. The frequencies of rapid and slow acetylators are 72% and 23% in Chinese subjects, 56% and 43% in Malays and 62% and 38% in Indians [145, 146]. When compared to other ethnic populations, slow acetylator allele frequencies in Indians and Malays are differ-ent from those in Caucasians (58-65% slow acetylators), Koreans (11% slow acetylators), Japanese (9-20% slow acetylators) and Filipino subjects (22.7% slow acetylators). Interestingly, slow ace-tylators are found to be predominant in Indian populations, with a frequency of 74% [147]. The NAT2 acetylation polymorphism plays an important role in the metabolism and disposition of a large number of aromatic amines and clinically used hydrazine drugs [140, 141]. Genetic variation in acetylation has been shown to be responsible for drug-induced toxicity [142]. NAT2 has long been known to be polymor-phic following reports of differences observed in tuberculosis pa-tients to isoniazid toxicity, although the association of polymorphic NAT2 acetylator status and isoniazid-induced hepatitis is debatable. Early study in 1970s indicated that there was an increased incidence of isoniazid-induced hepatitis in rapid acetylators [148]. However, a Zhou et al. recent study with relatively large sample size (n = 224) has demon-strated that slow-acetylator status of NAT2 is a significant suscepti-bility risk factor for isoniazid-induced hepatitis, and slow acetyla-tors are prone to develop more severe hepatotoxicity than rapid acetylators [149]. Understanding the link between isoniazid-induced hepatitis and acetylator phenotype may help alleviate the risk of fatal hepatotoxicity. NAT2 polymorphisms are associated with disease and cancer susceptibility. Certain NAT2 genotypes, particularly when in com-bination with exposure to carcinogenic arylamine or hydrazines, increased the risk of lung, bladder, gastric, prostate, breast and co-lon cancers [150, 151]. The underlying mechanism may involve metabolic activation (usually O-acetylation) and deactivation (usu-ally N-acetylation) of aromatic and heterocyclic amines present in cigarette smoke and diet. Slow acetylators may be at higher risk for aromatic amine and smoking-induced DNA damage. Thiopurine S-Methyltransferase (TPMT) TPMT (EC 2.1.1.67) is a cytosolic enzyme that S-methylates thiopurine drugs such as 6-mercaptopurine, azathioprine and thioguanine [152]. Thiopurine drugs have been commonly used for the treatment of acute lymphoblastic and myeloid leukemia, auto-immune diseases, inflammatory bowel disease and organ transplant recipients. Thiopurine drugs as prodrugs are converted into cyto-toxic 6-thioguanine nucleotides through multi-step metabolic acti-vation initiated by hypoxanthine guanine phosphoribosyl trans-ferase [152]. The anticancer and immunosuppressive activities of thiopurine drugs rely on the incorporation of cytotoxic 6-thioguanine nucleotides into DNA and RNA to trigger cancer cellu-lar apoptosis, while S-methylation of these drugs by TPMT diverts these drugs from their route of metabolic activation by formation of inactive metabolites. There is a large inter-individual variability in the activity of TPMT [152]. Caucasians show a trimodal distribution, with 89-94% possessing high enzyme activity, 6-11% intermediate activity due to heterozygosity at the TPMT locus and 0.33% low activity [152]. However, an unimodal distribution of TPMT activity was found in the mainland Chinese population [153], which was some-what different from the findings in a population of Chinese adults from Singapore, where a bimodal distribution in TPMT activity with a clear antimode was observed [154]. TPMT gene is localized to chromosome 6p22.3 and approxi-mately 34 kb in length and possesses 10 exons and 9 introns with a cDNA of 3,000 bp. Its open readingframe is 735 bp long and encodes a 245-amino acid peptide witha molecular mass of ap-proximately 35 kDa [155]. TPMT is known to display co-dominant genetic polymorphism in Africans, African-Americans, Caucasians, Latin-Americans, Arabs and Asians. To date, at least 23 variant alleles of TPMT gene have been reported in humans (Table 9)(http://www.pharmgkb.org/; access date 2 August 2008). The common mutant alleles in Caucasian include TPMT*2 [156], TPMT*3A [157], TPMT*3B and TPMT*3C [158]. These four mu-tant alleles are detected in over 80-95% of Caucasians with low or intermediate TPMT activity [159]. The mutant allele TPMT*2 is defined by a single nucleotide transversion (238G>C) in the open reading frame, leading to an amino acid substitution of Ala80Pro and a more than 100-fold reduction in the TPMT activity relative to wild type cDNA, despite a comparable level of mRNA [160]. The second mutant allele, TPMT*3A which is and more prevalent in Caucasian,contains two nucleotide transition mutations (460G>A and 719A>G) in the open reading frame, leading to amino acid substitutions of Ala154Thr and Tyr240Cys, respectively [157]. A number of rare mutant TPMT alleles (e.g. TPMT*3D,*4,*5,*6, *7,*8,*10, *11, *12, *13, *14, *15,*16, *17, *18, *19, 20*,21*,and 22*) have been identified [152]. TPMT*4 has a GAtransition at the intron 9–exon 10 junction, which disrupts the final Pharmacogenetics and Clinical PracticeTable 7. NAT2 *4 *5A *5B *5C *5D *5E *5F *5G *5H *5I *5J d *5K *5L *5M *6A *6B *6C *6D *6E *6F *6G *6H *6I *6J *6K *6L *7A *7B *10 *11A *11B *12A *12B *12C *12D *12E *12F *12G *12H *13A *13B *14A *14B *14C *14D *14E Wild-type 341T>C; 481C>T 341T>C; 481C>T; 803A>G 341T>C; 803A>G 341T>C 341T>C; 590G>A 341T>C; 481C>T; 759C>T; 803A>G 282C>T; 341T>C; 481C>T; 803A>G 341T>C; 481C>T; 803A>G; 859Del 341T>C; 411A>T; 481C>T; 803A>G 282C>T; 341T>C; 590G>A 282C>T; 341T>C 70T>A; 341T>C; 481C>T; 803A>G 341T>C; 481C>T; 803A>G; 838G>A 282C>T; 590G>A 590G>A (rs1799930) 282C>T; 590G>A; 803A>G 111T>C; 282C>T; 590G>A 481C>T; 590G>A 590G>A; 803A>G 282C>T; 518A>G; 590G>A 282C>T; 590G>A; 766A>G 282C>T; 590G>A; 838G>A; 857G>A 282C>T; 590G>A; 857G>A 282C>T; 590G>A; 638C>T 282C>T; 345C>T; 590G>A 857G>A 282C>T; 857G>A 499G>A 481C>T 481C>T; 859Del 803A>G 282C>T; 803A>G 481C>T; 803A>G 364G>A; 803A>G 282C>T; 578C>T; 803A>G 622T>C; 803A>G 609G>T; 803A>G 403C>G; 803A>G 282C>T 282C>T; 578C>T 191G>A 191G>A; 282C>T 191G>A; 341T>C; 481C>T; 803A>G 191G>A; 282C>T; 590G>A 191G>A; 803A>G Current Drug Metabolism, 2008, Vol. 9, No. 8 755 Reported Variants of Human NAT2 Gene Nucleotide change I114T ; L161L (synonymous) I114T; L161L (synonymous); K268R I114T; K268R I114T I114T; R197Q I114T; L161L (synonymous); V253V (synonymous); K268R Y94Y (synonymous); I114T; L161L (synonymous); K268R I114T; L161L (synonymous); K268R; S287 Frameshift I114T; L137F; L161L (synonymous); K268R Y94Y (synonymous); I114T R197Q Y94Y (synonymous); I114T L24I; I114T; L161L (synonymous); K268R I114T; L161L (synonymous); K268R; V289M Y94Y (synonymous); R197Q R197Q Y94Y (synonymous); R197Q K268R F37F (synonymous); Y94Y (synonymous); R197Q L161L (synonymous); R197Q R197Q; K268R Y94Y (synonymous); K173R; R197Q Y94Y (synonymous); R197Q; K256E Y94Y (synonymous); R197Q; V280M; G286E Y94Y (synonymous); R197Q; G286E Y94Y (synonymous); R197Q; P213L Y94Y (synonymous); D115D(synonymous); R197Q G286E Y94Y (synonymous); G286E E167K L161L (synonymous) L161L (synonymous); S287 Frameshift K268R Y94Y (synonymous); K268R L161L (synonymous); K268R D122N; K268R Y94Y (synonymous); T193M; K268R Y208H; K268R E203D; K268R L135V; K268R Y94Y (synonymous) Y94Y (synonymous); T193M R64Q R64Q; Y94Y (synonymous) R64Q; I114T; L161L (synonymous); K268R R64Q; Y94Y (synonymous); R197Q R64Q; K268R Amino acid change Rapid Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow Slow (Substrate dependent?) Slow (Substrate dependent?) Slow (Substrate dependent?) Rapid Unknown Rapid Rapid Rapid Slow Rapid Slow Slow Slow Slow Slow Phenotype Reference [441] [137] [442] [443] [444] [444] [445] [147] [147] [446] [447] [448] [448, 449] [450] [137] [144] [448, 451, 452] [453] [454] [449] [449] [449] [449] [450] [450] [450] [137] [455] [456] [457] [147] [144] [144] [458] [446] [448, 449] [448] [449] [450] [144] [449] [459] [459] [444, 451, 452] [444, 452] [444, 449] 756 Current Drug Metabolism, 2008, Vol. 9, No. 8 (Table 7) contd…. NAT2 *14F *14G *14H *14I *17 *18 *19 Nucleotide change 191G>A; 341T>C; 803A>G 191G>A; 282C>T; 803A>G 191G>A; 282C>T; 683C>T 191G>A; 481C>T; 803A>G 434A>C 845A>C 190C>T R64Q; I114T; K268R R64Q; Y94Y (synonymous); K268R R64Q; Y94Y (synonymous); P228L R64Q; L161L (synonymous); K268R Q145P K282T R64W Amino acid change Slow Slow Slow Rapid Slow Phenotype Zhou et al. Reference [452] [453] [449] [460] [460] [461-463] Data are also extracted from http://www.louisville.edu/medschool/pharmacology/NAT.html (access date: 2 August 2008). Table 8. Frequencies of Common NAT2 Variants in Various Ethnic Groups Ethnicity No. of alleles *4 (WT) Asian Chinese Malay Indian Japanese Korean Polynesian Taiwanese Philipino Egyptian 374 292 278 158 170 50 200 0.51 0.41 0.44 0.64 0.69 0.60 0.51 0.07 0.12 0.20 0.02 0.02 0.04 0.03 0.32 0.38 0.32 0.23 0.18 0.34 0.31 0.10 0.09 0.04 0.11 0.11 0.02 0.15 [145] [146] [146] [464] [464] [464] [464] Allele frequency *5 *6 *7 Reference 200 0.39 0.07 0.36 0.18 [464] 398 0.21 African 0.50 0.26 0.03 [465] Gabonese Dogons African-American Tanzanian Venda Zimbabwean 104 130 428 234 192 326 0.36 0.30 0.43 0.31 0.23 0.29 Caucasian 0.40 0.30 0.30 0.34 0.39 0.31 0.22 0.37 0.23 0.20 0.22 0.21 0.02 0.03 0.04 0.03 0.05 0.06 [466] [466] [464] [454] [454] [454] Spanish Caucasian-American German Scottish Swedish Danish Portuguese 516 532 1688 0.26 0.22 0.24 0.47 0.47 0.47 0.18 0.28 0.28 0.01 0.00 0.01 [467] [464] [468] 192 0.20 0.49 0.27 0.04 [469] 140 484 256 0.19 0.25 0.21 0.51 0.47 0.43 0.28 0.25 0.33 0.02 0.02 0.03 [469] [470] [471] Table 9. TPMT Reported Alleles of Human TPMT Gene Nucleotide change Position Amino acid substitution Reference *1 Wild-type *2 238G>C *3A 460G>A 719A>G *3B 460G>A C*3 719A>G Exon 5 Ala80Pro [156] Exon 7 Exon 10 Ala154Thr Tyr240Cys [157] Exon 7 Ala154Thr [158] Exon 10 Tyr240Cys [158] Pharmacogenetics and Clinical Practice (Table 9) contd…. TPMT Nucleotide change Position Exon 7 Exon 10 Exon 5 Intron 9 and exon 10 junction Current Drug Metabolism, 2008, Vol. 9, No. 8 757 Amino acid substitution Ala154Thr Tyr240Cys Glu98Stop Reference [161] *3D 460G>A 719A>G 292G>T *4 GA transition [161] *5 146T>C *6 539A>T transversion *7 681T>G transversion *8 644G>A *10 430G>C *11 395G>A *12 374C>T *13 83A>T *14 AG transition in the start codon of exon 3 *15 GA transition Exon 4 Leu49Ser [161] Exon 8 Exon 10 Tyr180Phe His227Glu [161] [162] Exon 10 Arg215His [163] Exon 7 Gly144Arg [472] Exon 6 Cys132Tyr [473] Exon 6 Ser125Leu [472] Exon 3 Glu28Val [472] Exon 3 MetVal [474] Acceptor splice site in intron 7/exon 8 (IVS7 -1GA) [474] *16 488G>A *17 124C>G *18 211G>A *19 365A>C *20 106G>A 712A>G *21 205C>G *22 488G>C Allele nomenclature of TPMT is according to [476, 478]. Exon 7 Arg163His [475] Exon 3 Gln42Glu [476] Exon 4 Gly71Arg [476] Exon 5 Lys122Thr [475] Exon 3 Exon 10 Gly36Ser Lys238Glu [477] [478] Exon 4 Leu69Val [478] Exon 7 Arg163Pro [478] nucleotide of the intron at the 3 acceptor splice site sequence [161]. TPMT*5 was identified as a 146T>C transition in a heterozygous individual and has intermediate TPMT activity [161]. This mutation results in a Leu49Ser change. TPMT*6 results in intermediate activ-ity [161]. This 539A>T transversion in exon 8 results in a Tyr180Phe substitution. TPMT*7 results in intermediate activity [162].This allele contains a 681T>G transversion in exon 10, which results in a His227Glu substitution. TPMT*8 contains a sin-gle nucleotide transition (644G>A), leading to Arg215His [163]. The pattern and frequency of mutant TPMT alleles is different among various ethnic populations. The most prevalent TPMT mu-tant allele in the Caucasian and Latin-American population is TPMT*3A [159, 164], while TPMT*3C is predominant in Chinese, Taiwanese, Japanese, Egyptian and African-American [152]. For the Caucasian, Black and Latin American populations, trimodal or bimodal distributions have been largely observed [165]. For the East Asian and Israel populations, most studies showed that a uni-modal distribution was commonly observed. TPMT polymorphisms have been associated with the therapeu-tic efficacy and toxicity of thiopurine drugs [152]. The impact of 6-mercaptopurine dose intensity is also being clarified as an important determining factor of event-free survival in childhood leukemia. Acute lymphoblastic leukemia patients with at least one mutant TPMT allele tend to have an improved response to 6-mercaptopurine therapy and better chances of being cured, com-pared with patients who have two wild-type TPMT alleles [152]. Patients with inherited low levels of TPMT activity are at greatly increased risk for thiopurine-induced toxicity such as myelosup-pression when treated with standard doses of these drugs, and re-quire doses to be reduced to as little as a tenth of the normal dose in order to tolerate therapy [152]. The mutation, however, might be a double-edged sword, as a reduced TPMT expression will increase the risk of developing a thiopurine-related second tumor, including brain tumors and acute myelogenous leukemia. In this regard, a number of studies have indicated that there is a significant negative correlation between the intracellular concentration of 6-thioguanine nucleotides and TPMT activity in erythrocytes and 6-thioguanine nucleotide concentrations are associated with the efficacy and tox-icity of thiopurines in various diseases including leukemia and in-flammatory bowel disease [152]. It is found that only higher dose intensity of 6-mercaptopurine for acute lymphocytic leukemia is a significant predictor of event-free survivor [152]. Lower TPMT activity is associated with better outcome. Increasing the dose intensity of 6-mercaptopurine in chil-dren homozygous for the TPMT wild-type allele has been shown to increase the event-free survival rate, but caution is needed to avoid drastic increases in dose which may increase the risk of severe tox-icity and worsen the clinical outcome in an otherwise genotypically tolerable group [166]. Generally, TPMT-deficient patients or low methylators (homozygous mutant or compound heterozygote) can be treated with 6–10% of the standard dose (i.e. 10- to 15-fold de-crease of standard dose), while patients with heterozygous pheno-types/genotypes can be treated with 65% of the standard dose (i.e. 2-fold decrease of standard dose) [152]. If patients are able to toler-ate the adjusted dosage regimens without toxicity, it may be desir-able to carefully increase the dose to avoid sub-therapeutic drug exposure. Nevertheless, prospective validation for each disease indication is required before this approach can be recommended for broad application to therapy with thiopurines. The number of clinically important applications of TPMT mo-lecular genetics has increased considerably in recent years. From the initial application of TPMT polymorphism screening in acute lymphoblastic leukemia patients to prevent toxicity, current inter-ests in application of TPMT pharmacogenetics include TPMT phe- 758 Current Drug Metabolism, 2008, Vol. 9, No. 8notyping/genotyping studies in transplant patients, patients with inflammatory bowel disease, Crohn’s disease, systemic lupus erythematosus, or other autoimmune diseases receiving thiopurine-based immunosuppressive therapy. Of note, there is no known probe drug to identify TPMT deficient patients unless they are treated with thiopurine medications and TPMT genotyping may be useful to avert serious adverse events in such patients. Uridine Diphosphate Glucuronosyltransferase (UGT1A1) The human UGT (EC 2.4.1.17) family is divided into two major classes, UGT1 and UGT2, based on amino acid sequence compari-son [167]. To date, at least 15 human UGT cDNAs have been iden-tified: eight UGT1A proteins are encoded by the UGT1A locus (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9 and 1A10), located on chromosome 2q37, and seven proteins are encoded by the UGT2genes (UGT2A1, 2B4, 2B7, 2B10, 2B11, 2B15 and 2B17), located on chromosome 4q13 and 4q28 [167]. The UGT1A gene contains exon 1 complex and common exons 2 to 5. The exon 1 complex comprises at least 10 unique exons (1A1 to 1A10), each preceded by its own promoter region and encoding a unique UGT enzyme. The mRNA encoding each UGT enzyme is formed by the fusion of one type of exon 1 to the common exons 2 to 5 [167]. Conse-quently, gene mutations in the common exons 2 to 5 region can lead to changes in activity and/or expression of additional enzymes, while gene mutations in the exon 1 complex or promoter region may only affect the unique enzyme involved. UGT1 enzymes are expressed predominantly in the liver, although some enzymes have been reported to be expressed at extra-hepatic sites such as the in-testine and oesophagus [168, 169]. UGT1A1 catalyzes the glucurondiation of a number of impor-tant clinical drugs and/or their hydroxylated metabolites [170]. Drugs glcuuronidated by UGT1A1 include acetaminophen [171], carvedilol [172, 173], tranilast [174], fulvestrant [175], retigabine [176], gemfibrozil [177], frusemide [178], ezetimibe [179], mura-glitazar [180], flavopiridol [181], etoposide [182, 183], HMR1098 [184], and mycophenolic acid [185, 186]. Fulvestrant is glucuroni-dated at position 3 by UGT1A1, 1A3, 1A4, and 1A8 [175]. Gemfi-brozil, a fibrate hypolipidemic agent, is conjugated by UGT1A1, 1A3, 1A9, 2B4,2B7, and 2B17, with 2B7 showing the highest ac-tivity toward gemfibrozil [187]. The main circulating metabolite of ezetimibe, a potent cholesterol absorption inhibitor, in human plasma is SCH60663, the phenolic glucuronide of ezetimibe [188]. This drug is converted by UGT1A1, 1A3, and 2B15 to SCH60663 and by UGT2B7 to its benzylic glucuronide, SCH488128 [179]. Muraglitazar (Pargluva) is a dual / peroxisome proliferator-activatedreceptor activator, has both glucose- and lipid-lowering effectsin animal models and in patients with type II diabetes [189]. It is glucuronidated by UGT1A1, 1A3, and 1A9 [180]. Flavopiridol, a novel cyclin-dependent kinase inhibitor used for cancer treatment, is glucuronidated by UGT1A1 and 1A9 to 5- and 7-O--glucuronides [181]. FYX-051 (4-(5-pyridin-4-yl-1H-[1,2,4]triazol-3-yl) pyridine-2-carbonitrile), a novel xanthine oxidoreductase in-hibitor, is N-glucurondiated by UGT1A1, 1A7 and 1A9 [190]. 4-Methylumbelliferone is glucuronidated by UGT1A1, 1A8 and 1A9 [191]. In addition, both UGT1A1 and 1A3 can catalyse the forma-tion of the glucuronide conjugates and the corresponding lactones for simvastatin, atorvastatin, and cerivastatin [192]. There are at least 112mutant UGT1A1 alleles identified to date (http://www.pharmacogenomics.pha.ulaval.ca/sgc/ugt_alleles/; access date: 2 August 2008). There have been 195 SNPs found in human UGT1A1 gene in NCBI dbSNP (http://www.ncbi.nlm.nih. gov/, access date: 2 August 2008). Among these SNPs, there are 11 non-synonymous SNPs in exons 1-5: 211G>A (G71R); 247T>C (F83L); 674T>G (V225G); 686C>A (P229Q); 748T>C (S250P); 965T>C (I322T); 1091C>T (P364L); 1099C>G (R367G); 1231G>T (V411L); 1456T>G (Y486D); 1531G>C (A511P). Syn- Zhou et al. onymous SNPs of UGT1A1 include 141G>A (I47I); 1279C>T (L427L); 1428C>T). The 211G>A (G71R, UGT1A1*6) mutation in exon 1 is the most common nonsynonymous SNPs found in theEast Asian popu-lation. 211G>A was frequently found in the Japanese population (15.7%),but not in the Caucasians population (0.7%), and none were detected in African-Americans [193]. The allele frequency in Koreanand Chinese populations has been reported to be 23% [194]. It has been suggested thatthe 211G>A variant contributes to the high incidence of neonatalhyperbilirubinemia in Asian children [195]. A comparable allele frequency(11%) has been reported for 211G>A mutation in Taiwanese population,where 686C>A (P229Q, UGT1A1*27) in exon 1 is found at a frequency of 2.8% [196]. The 1456T>G (Y486D, UGT1A1*7) mutation in exon 1, as double homozygous with 211G>A (UGT1A1*6), isthe most abun-dant mutation in Japanese patients with Crigler-Najjarsyndrome type II [197]. UGT1A1*6 and 2B7*3 have been shown to affect the disposition of carvedilol in Japanese [198]. The variant686C>A (P229Q) (UGT1A1*27) is very rare in all the ethnicgroups examined, and only one heterozygous carrier was found in the Japanesepopulation [193]. A variation in the same position as the variant686C>A, but with a 686C>T,was found in an African-American. This SNP led toan amino acid change of P229L [193]. Exon1 is unique for each member of the UGT1A subfamily, whereasexons 2 to 5 are common to all members of the subfamily. Thevariants in the 3'-UTR of UGT1A1 in exon 5,therefore, could have distinct effects on all members of theUGT1A subfamily. Kaniwa et al. [193] compared allele frequencies of three SNPs in the 3'-UTR of exon 5, namely 1813C>T, 1941C>G and 2042C>G in Japanese, African-Americansand Caucasians. 1813C>T and 2042C>G have been reported to be in complete association with 1941C>Gin a Japanese population without exception [199], but not often in inAfrican-Americans and Caucasians [193]. The allele frequency of 1813C>Tand 2042C>G in the Japanese is expected to be equal to thatof 1941C>G (9.7%). The allelefrequencies of the three SNPs are highest in African-Americans (18.3%), followed by Caucasians (16.3%)and lowest in the Japanese (9.7%) [193]. TheSNP 1941C>G is a marker for haplotype UGT1A1*IB; Sai et al.[199] have reported the UGT1A1*IB-dependent decreasingtrend of the AUC ratio (SN-38 glucuronide/SN-38) and an increase in serumtotal bilirubin levels. Notably, the 715C>T mutation in exon 1 re-sults in a truncated protein. Genetic polymorphism in the promoter region of the UGT1A1 gene is due to variability in the number of TA repeats in the TATA-box upstream of the UGT1A1. The presence of seven TA repeats, (UGT1A1*28) is associated with reduced UGT1A1 expression compared to the wild-type (UGT1A1*1), which has six TA repeats. Homozygous individuals carrying the A(TA)7TAA allele showed significantly higher plasma levels of unconjugated bilirubin caused by a 30% reduction in UGT1A1 gene transcription [200]. There are interethnic differences in the frequency of UGT1A1*28 allele, with an incidence of about 35-40% in Caucasians and Canadian Inuit population compared with 2% in Asians [201]. The frequency of the UGT1A1*28 allele was highest in the Indian population (35%) compared to similar frequencies that were found in the Chinese (16%) and Malay (19%) populations (Table 10) [202]. The results of this study which included a total of 266 Asian healthy subjects (Chinese, N = 89; Malays, N = 93 and Indi-ans, N = 84) contrasted with previous results whereby the allelic frequency of UGT1A1*28 allele were found to be very low or ab-sent in Asians [201]. The UGT1A1*28 allelic frequency in Indians were very similar to those reported in Caucasians [200], whereas those in Chinese are comparable to the frequencies of 10-25% in the blacks [203]. Furthermore, a strong correlation was found be- Pharmacogenetics and Clinical PracticeTable 10. Current Drug Metabolism, 2008, Vol. 9, No. 8 759 Distribution of UGT1A1 Promoter Polymorphism Genotype in Different Ethnic Groups Ethnicity No. of subjects No. of genotype 5/6* 5/7* 6/6* 6/7* 6/8* 7/7* 7/8* Asian Reference C hinese Malay Indian Japanese Korean Egyptian 89 93 84 58 20 50 0 0 71 27 0 2 0 [202] 0 0 67 29 0 4 0 [202] 0 0 43 44 0 13 0 [202] 0 0 44 12 0 2 0 [479] 0 0 17 2 0 1 0 [480] 0 0 56 36 0 8 0 [481] African African Gambian African Americans Ewondo 82 36 40 10 0 0 26 37 0 19 0 14 8 19 28 3 19 8 3 10 8 10 23 30 Caucasian 43 50 10 0 13 0 3 0 [482] [483] [483] [483] Scottish C aucasian German 77 202 0 0 31 37 0 9 0 [201] 2 0 102 75 1 22 0 [482] 1000 0 0 50 42 0 8 0 [481] tween promoter length polymorphism and serum total bilirubin levels in all three Asian ethnic groups [202]. No mutations were found in all Asian subjects in the coding region of UGT1A1 [204]. This is in contrast to other studies which postulated that variations in the coding region may be responsible for the higher prevalence of hyperbilirubinaemia in Asians. Most Japanese patients with Gilbert’s syndrome had variations within the coding region rather than the promoter region [205, 206]. In another study the prevalence of Gly71Arg (due to 211G>A change) varia-tion within the coding region was higher among Chinese, Japanese, Korean and Taiwanese populations than in Caucasians [207]. Het-erozygous variation in the UGT1A1 gene were prevalent in more than 50% of healthy Taiwanese adults [208]. Of the four variant sites within the coding region, the 211G>A mutation was the most common, with an allelic frequency of 10.9% and similar to the Japanese. These findings demonstrate that both the promoter and coding regions of the UGT1A1 gene may differ greatly among eth-nic groups. Defective UGT1 gene results in unconjugated hyperbili-rubinaemia in patients. Three inherited forms of unconjugated hy-perbilirubinaemia are known to occur in humans: Crigler-Najjar syndromes type I and II and Gilbert’s syndrome [167]. All these inheritable hyperbilirubinaemias result from either mutant UGT1A1alleles [200] or UGT1A1 promoter polymorphisms [200, 201]. Clinically they can be distinguished from each other by the degree of UGT1A1 activity deficiency and the serum bilirubin level. In Crigler-Najjar syndrome Type I, which has an autosomal recessive pattern of inheritance, bilirubin glucuronidation is completely lack-ing and serum unconjugated bilirubin levels may be as high as 20 to 50 mg/dl [167]. Bilirubin may accumulate in nerve terminals and glial cells; a condition termed kernicterus and that can result in early childhood death. Patients with Crigler-Najjar syndrome Type II also display an autosomal recessive pattern of inheritance and bilirubin UGT activity is markedly reduced to 10% to 30% of nor-mal activity. Bilirubin levels are usually below 20 mg/dl. Gilbert’s syndrome manifest itself as mild chronic hyperbilirubinaemia and affects about 5-7% of population. Hepatic UGT activity is about 60% to 70% of normal activity and serum bilirubin levels are less than 3 mg/dl. The 7/7 UGT promoter polymorphism was associated with increased hemolysis as well as diminished serum total conju- gated bilirubin ratios [209], probably contributing to the pathogene-sis of increased serum total bilirubin values seen in Gilbert's syn-drome The UGT1A1 polymorphism has been shown to be responsible for modulating the disposition and toxicity of the anticancer drug irinotecan (CPT-11). Irinotecan is a potent DNA topoisomerase I inhibitor used in the treatment of advanced colorectal and lung cancer. CPT-11 is mainly converted to its active metabolite SN-38 by liver carboxylestesterases [210]. UGT1A1 inactivates SN-38 into the more polar SN-38 glucuronide which is then eliminated in bile and urine [211]. The dose-limiting toxicities of irinotecan con-sist of diarrhoea and leukopenia [211, 212], and these toxic effects are associated with excessive formation of SN-38 [211]. The pres-ence of seven TA repeats, rather than the wild-type number of six, in the UGT1A1 promoter reduces enzyme expression and conse-quently SN-38 glucuronidation. This leads to accumulation of ac-tive SN-38, and a higher chance of developing diarrhoea and/or leukopenia during irinotecan therapy than patients with a wild-type genotype [212]. Gilbert’s syndrome is also associated with the TA7/TA7 genotype [200, 213], and these patients might be at in-creased risk of irinotecan-induced toxicity [214]. CLINICAL PHARMACOGENETICS OF DRUG TRANS-PORTERS Most of drug transporters belong to one of two superfamilies: the ATP-binding cassette (ABC) and solute-linked carrier superfa-milies. The ABC superfamily of transporters consist of a large number of functionally diverse transmembrane proteins which have been subdivided into seven families designated A through G [215-218]. The human genome contains 49 ABC transporter genes [219]; 16 of these have a known function and 14 are associated with a defined human disease while two of the members lack transmem-brane domains and donot qualify as transporters. In addition to their drug efflux function, many of these transporters are now also well characterized as mediators of the elimination of a broad range of endogenous substances and xenobiotics including therapeutic drugs, metals and chemical toxicants [215-218]. The transport of endogenous compounds is particularly important for maintaining organ and cellular functions. Accumulation of these endogenous compounds in organ and cells will result in pathological conditions. Members of both superfamilies facilitate the uptake or efflux of 760 Current Drug Metabolism, 2008, Vol. 9, No. 8drugs in and out of various human tissues. Polymorphisms of drug transporters are common, but current data on their clinical impact are limited. P-glycoprotein (P-gp/MDR1/ABCB1) P-gp (MDR1/ABCB1) is a 170 kDa glycoprotein encoded by thehuman MDR1 and MDR3 genes and the murine mdr1a,mdr1b,and mdr2genes. The human MDR1 gene extends over more than 100 kb and is located on the long arm of chromosome 7 at q21.1 and consists of a core promoter region and 28 exons [220]. Regula-tion of the transcriptional activity of the human MDR1 gene de-pends on several trans-acting proteins that bind consensus cis-elements of MDR1 promoter. P-gp is constitutively expressed in several organs involved in drug absorption and elimination such as the intestine, liver, kidney and brain [221]. It is found at high levels on the apical surfaces of epithelial cells in the liver (bile canaliculi), kidney (proximal tubule), pancreas (pancreatic ductal cell), small intestine and colon (columnar mucosal cell) and adrenal gland. It has been shown that the oral absorption and brain penetration of P-gp substrates are significantly lower in normal mice compared with mdr1a-/- mice, and oral bioavailability and brain penetration of P-gp substrates can be significantly enhanced by coadministration of P-gp modulators [222]. Due to the nature of P-gp/MDR1 as an efflux pump for cell protection against a variety of substances, P-gp/MDR1 substrates vary greatly in size, structure and function, ranging from small molecules such as organic cations, carbohy-drates, amino acids and some antibiotics to macromolecules such as polysaccharides and proteins. The MDR1 gene is highly polymorphic with different frequen-cies of allelic variants amongst various ethnic groups. Hoffmeyer etal. [223] conducted the first systematic screening of SNPs in the MDR1 gene and reported the occurrence of 15 SNPs in healthy Caucasian subjects. Those silent mutations were located in introns close to exon boundaries and wobble positions that do not change amino acids. Three polymorphisms resulted in protein alterations in exon 2, 5 and 11. Exon 2 contains a polymorphism that changes Asn21 to Asp, and the mutation at exon 5 changes Phe103 to Leu. Up to date, very few studies have been conducted to illustrate the importance of these two amino acid changes and the occurrence of these mutations is very low in general population. The 1199G>A polymorphism is located at exon 11, which changes Ser400 to Asn. In an RNase protection analysis, two additional sites of genetic polymorphism were identified at residues 2677 and 2995 of the gene [224]. At 2677 residue of the MDR1 gene, a G to T change resulted in an amino acid change from Ala to Ser. Similarly, a sin-gle base pair mismatch (G to A) at 2995 switches the amino acid from Ala to Thr. The 3320A>C SNP was revealed in a screen of 461 German Caucasians for allele and genotype distribution [225]. To date, at least 2062 SNPs of MDR1 have been described in NCBI SNP database (http://www.ncbi.nlm.nih.gov/, access date: 2 August 2008). Many of these SNPs are non-synonymous located in exons and can change the amino acid sequence (Table 11). These naturally occurring genetic variants of MDR1 may affect interindi-vidual variability in the pharmacokinetics and pharmacodynamics of many drugs and account for differences in the bioavailability of various P-gp substrates. SNPs at 2677 and 3435 residues of the MDR1 gene are far more frequent than other SNPs. Africans and African-Americans have relatively low 2677G>T allelic frequency than other populations, while Caucasian, Mexican-American and Asian-American are more likely to carry this mutant allele. Overall, the presence of the corre-sponding wild-type alleles is more frequent than the one of mutant alleles. In contrast, people across the world are much more likely to be 3435C>T heterozygous or homozygous than wild-type. Black Africans and African-Americans are few populations with the dominance of 3435 wild-type while 17-27% of them have 3435C>T. In European Caucasians, 3435T frequencies of 52-57% Zhou et al. were reported. In oriental populations, this mutation accounts for 41- 47%. In general, MDR1 SNPs are found in all studied popula-tions. The chance of carrying a particular mutant allele is higher in some populations. Three high-frequency SNPs occurring at exons 12, 21 and 26 in the MDR1 gene have been genotyped in the Asian populations [117]. There were no difference in genotype and allelic variant frequencies in exon 12 between the Chinese, Malay and Indian populations. When compared with other ethnic groups, the distribu-tion of wild-type C allele in exon 12 in the Malays (34.2%) and Indians (32.8%) were relatively high and similar to the Japanese (38.5%) [226] and Caucasians (41%) [227] but different from Afri-can-Americans (15%) [228]. The frequency of wild-type TT geno-type in the Asians (43.5% to 52.1%) and Japanese (61.5%) were much higher than those found in the Caucasians (13.3%) [223]. For 2677G>A/T in exon 21, the genotype frequencies differed significantly between ethnic groups [117]. The GG variant was similar in the Chinese and Indian population (17% and 14%, re-spectively) compared with 28% in the Malays. The frequency of the TT variant was much lower in the Malays (19%) compared with the Indians (41%) and the Chinese (26%). The AA variant was found to be rare in all 3 populations, being detected in 1% of the Chinese population and missing in both the Malay and Indian populations. The frequency of the T allele was lower in Malays (44%) than in Chinese (50%) and Indians (60%). The distribution of the A and G alleles in the Malays were different from those in the Indians and Chinese. The frequencies of the A and G alleles were lowest and highest, respectively, in the Malay population compared with Chi-nese and Indian populations. Furthermore, although the A allele was a low frequency allele in the Asians, it seemed to be more common in the Asian population compared with the Caucasian (0%) and African (0%) populations [228]. The frequency of the G and T alleles in Asians was similar to that in the Caucasians (46%) but different from the African-Americans [228]. In the latter, the G allele frequency was 93.5% compared with 6.5% for the T allele. For 3435C>T in exon 26, the frequency of the homozygous TT variant was 45% in the Indian subjects compared to 18% of them carrying the homozygous CC variant. There were no statistically significant difference observed in the distribution of CC and TT genotypes among the Chinese, Malays and Indians [117]. The fre-quencies of C and T alleles were similar in the Chinese and Malay groups, while the Indians had a slightly higher frequency of T al-lele. The frequency of the C allele in the Asian population was lower (range: 37-49%) compared with the African population (range: 73-84%) [229]. Other studies also revealed that the allelic frequency of the wobble SNP in exon 26 varied among various ethnic groups, with Caucasians and Japanese having similar fre-quencies and different from the Africans [225, 230]. Several studies investigating the functional significance of the 3435C>T SNP on the disposition of digoxin and other P-gp sub-strates have revealed conflicting results. Sakaeda et al. [231] showed that the serum concentrations of digoxin after a single ad-ministration was lower in healthy Japanese subjects harbouring the variant T allele compared with wild-type subjects. However, an-other studies reported subjects with the TT genotype had higher steady-state plasma concentration of digoxin after oral administra-tion than wild-type subjects [232], demonstrating that the 3435C>T correlated with decreased enterocyte P-gp expression, higher in-vivo activity of P-gp and increased exposure to digoxin. Fellay etal. [233] reported lower plasma concentrations of antiretroviral drugs, efavirenz and nelfinavir in subjects with variant TT geno-type. Kim et al. [228] showed the plasma concentration time profile of single-dose fexofenadine was lower in subjects with TT geno-type compared with CC subjects while no genotype-phenotype relationships were seen with fexofenadine [234]. On the other hand, no associations were found between the 3435C>T SNP and cy-closporine efficacy in renal transplant patients [122]. Pharmacogenetics and Clinical PracticeTable 11. Reported Non-Synonymous SNPs of the MDR1/ABCB1 Gene. Nucleotide change rs number Current Drug Metabolism, 2008, Vol. 9, No. 8 761 Amino acid change C49T> rs28381804 F17L 61A>G rs61615398; rs9282564 N21D 131A>G rs1202183 N44S C178A> rs41315618 I60L C239>A rs9282565 A80E C266T> Rs35810889 M89T C431T> rs61607171 I144T 502G>A rs61122623 V168I 548A>G rs60419673 N183S 554G>T rs1128501 G185V 781A>G rs36008564 I261V 1199G>A rs2229109 S400N 1696G>A rs28381902 E566K C1777>T rs28381914 CR593 1778G>A rs56107566 R593H 1795G>A rs2235036 A599T 1837G>T rs57001392 D613Y 1985T>G rs61762047 L662R C2005>T rs35023033 CR669 2207A>T rs41316450 I736K 2398G>A rs41305517 D800N 2401G>A rs2235039 V801M 2485A>G rs2032581 I829V 2506A>G rs28381967 I836V 2547A>G rs36105130 I849M 2677T>A/G rs2032582 S893A/T 2975G>A rs56849127 S992N C3151>G rs28401798 P1051A C3188G> rs2707944 G1063A 3262G>A rs57521326 D1088N 3295A>G rs41309225 K1099E C3320A> rs55852620 Q1107P C3322T> rs35730308 W1108R 3410G>T rs41309228 S1137I 3421T>A rs2229107 S1141T 3502A>G rs59241388 K1168E 3669A>T rs41309231 E1223D 3751G>A rs28364274 V1251I 3767C>A Data are from NCBI dbSNP (access date: 2 August 2008). r35721439 T1256K Part of the reason for the discrepancies in the results could be due to the fact that the 3435C>T SNP is not the only polymorphism influencing P-gp expression levels and hence, the pharmacokinetics and pharmacodynamics of P-gp substrates. Polygenic rather than monogenic traits might be responsible for influencing P-gp expres-sion levels. Kim [228] indicated that cells transduced with a variant MDR1 containing Ser893 showed reduced intracellular accumula-tion of [3H]-digoxin compared with cells with the wild-type Ala893, suggesting enhanced efflux by the polymorphism of Ala893Ser (2677G>T). They also showed in vivo the concentration of fexofenadine to be lower in subjects homozygous for the T vari-ant allele at the 2677 and 3435 loci. In Asian heart transplant pa-tients haplotypic association of SNPs at exons 12, 21 and 26 were found to influence cyclosporine exposure levels [117]. This pheno-typic outcome was most significant in the Indian population. How-ever, MDR1 3435T allele carriers have enhanced oral clearance of cyclosporine compared to individuals with the CC genotype in Caucasians [235]. 762 Current Drug Metabolism, 2008, Vol. 9, No. 8 The inconsistent findings involving 3435C>T polymorphism are also reported from studies on tacrolimus. Among Turkish renal transplant recipients, tacrolimus daily doses were significantly lower among patients with the 3435 TT genotype at months 1 and 6. Interestingly, at 6 and 12 months post-transplant patients with wild type showed significantly lower dose-adjusted trough tac-rolimus concentrations compared with TT and CT genotypes [236]. However, in a study relating to tissue expression of P-gp, the 3435C>T SNP in exon 26 had no significant influence on the ex-pression level of P-gp in intestinal enterocytes and it also did not correlate with the tacrolimus concentration:dose ratio in liver trans-plant patients [237]. They, however, noted that carriers of the 3435C>T allele had reduced expression of intestinal CYP3A4 mRNA. These results indicates that unique interactions of SNPs within a haplotype or polygenic traits involving more than one gene may be responsible for influencing P-gp expression and the resul-tant phenotypic trait and that individual SNPs may have poor pre-dictive power as pharmacogenetic loci. The results from a controlled trial agreed with this concept. The study compared the allelic frequency of MDR1 polymorphism and clinical response to olanzapine in 117 schizophrenic patients [238]. Significant associations between the 2677G>T genotypes and better treatment response were found. In contrast, no significant associa-tions were found for the 3435C>T polymorphism. Additionally, saturation kinetic parameters of 2677G>T and 2677G>A were found to be considerably different from wild-type, despite similar protein expression levels [239]. In comparison with wild-type, maximal transport rates for vincristine of 2677G>T and 2677G>A increased 50% and three-fold, respectively. This further reinforces the idea that the pharmacokinetic impact of MDR 2677G>T SNP generated some false positive results for 3435C>T SNP studies. In another study, verapamil, digoxin, vinblastine and cyclosporine were examined for transcellular transport activities and intracellular accumulation in vitro [240]. No significant differences were ob-served between cells expressing five polymorphic types of the MDR1 (2677G/3435T, 2677A/3435C, 2677A/3435T, 2677T/3435C, and 2677T/3435T) and cells expressing the corre-sponding wild-type. As a result, the mechanism of MDR1 SNP-related drug resistance remains unclear due to conflicting evidence. In a study involving 746 Han Chinese epileptic patients and 179 controls, patients with drug resistance epilepsy were more likely found to be 3435C>T homozygous compared with those with drug responsive epilepsy [241]. Similar results were observed in another study in Turkish epilepsy patients [242]. However, the association was not found in several other studies [243-245]. These studies highlight the complexity of the possible role of this polymorphism in antiepileptic drug response in different ethnic populations. An-other study in British epilepsy patients challenged these findings and suggested patients with drug-resistant epilepsy were more likely to be the wild-type at MDR1 3435 than the 3435C>T homo-zygote [246]. The inconsistent and contradicting evidence further complicates the role of 3435C>T SNP in epilepsy control. Never-theless, it is important to note that epilepsy is classified clinically into several major types and that the corresponding pharmacothera-pies are often contraindicated in other types of seizures. Addition-ally, not all chemically and structurally diverse antiepileptic drugs are P-gp substrates. Unfortunately, none of those studies docu-mented the clinical nature of subjects’ epilepsy. Therefore, the as-sociation between 3435C>T SNP genotype and increased drug resistance in epileptic patients remains inconclusive at the moment. Because the changes in P-gp functions increase the exposure of various tissues and organs to potentially toxic xenobiotics, it has been suspected that P-gp activities impaired patients may have in-creased disease susceptibility. Ulcerative colitis is an inflammatory disease mainly affecting lower gastrointestinal tract. Genotyping among 249 ulcerative colitis and 179 Crohn’s disease patients and 260 healthy controls was conducted and the results were analysed Zhou et al. [247]. A highly significant association between the common haplo-types and ulcerative colitis but not Crohn’s disease was observed. Additionally, human patients with the 3435C>T polymorphism, which is associated with lower intestinal expression of P-gp, are over-represented among patients with ulcerative colitis [248, 249]. However, a lack of such association was also observed in several studies [250, 251]. Thus, more studies are needed to provide compelling evidence to support the contribution of the MDR1 gene in determining risk to ulcerative colitis. Interestingly, Mdr1a-/-knockout mice can develop an inflammatory bowel condition similar to human ulcerative colitis [252]. Those Mdr1a-/- mice tends to respond to antimicrobial therapy. Based on these observations, it is likely that toxic P-gp-binding chemicals from intestinal bacteria contribute to the disease process. Since P-gp in the blood-brain barrier protects the brain by eliminating toxins, the mutation-induced P-gp malfunction could contribute to the development of some neurological diseases. In-deed, the 3435 TT genotype was highest in the early-onset Parkin-son’s disease group, second highest in the late-onset Parkinson’s disease group and lowest in controls [253]. Another report has also found that 3435C>T allele occurred with higher frequency com-pared with the wild type patients with early-onset Parkinson’s dis-ease [254]. As the 3435C>T allele is associated with lower P-gp expression and Parkinson’s disease is thought to result from the gradual accumulation of environmental toxins in the brain, it is very possible that the MDR1 polymorphism genotype exposes the neu-rones to higher concentration of toxic P-gp substrates due to re-duced efflux capability [255]. In the early stage of Parkinson’s dis-ease, the P-gp function in the blood-brain barrier is normal [256]. P-gp is expressed in CD56+, CD8+, CD4+, CD19+ and other subpopulations in peripheral blood monocytes [257]. Because CD4+cells are a major target for the HIV virus, their P-gp activities can affect intracellular concentration of many HIV protease inhibitors [258]. In a Swiss study, patients with the 3435TT genotype experi-enced a significantly higher increase in CD4+ cell counts and marked improvement in viral infection than those patients with the CT or CC genotype [233]. Although no other long term studies have been conducted, the evidence highlights the role of MDR1 polymorphisms in retarding the spread of HIV infection and im-proving therapy outcome. Furthermore, MDR1 SNPs may decrease the susceptibility of HIV patients to HIV infection. At the moment, more evidence is needed to support the importance of MDR1 SNPs in HIV patient management. MDR1 polymorphisms have been found to influence drug expo-sure and target cell concentration. It is expected that MDR1 poly-morphisms can potentiate the drug concentration alteration found in normal P-gp-related drug-drug interaction. In one study involving 20 healthy volunteers, subjects received 24 mg loperamide suspen-sion orally and, in a double-blind randomized two-way crossover fashion, 800 mg quinidine or placebo orally 1 h before loperamide [259]. Co-administration with quinidine and the expression of 2677G>T/3435C>T genotype were two major factors contributing to higher loperamide plasma concentrations and more profound miosis. As a result, MDR1 polymorphisms indeed enhance the pharmacokinetic effects of P-gp-related drug-drug interaction. Nev-ertheless, MDR1 genetic variants could sometimes cancel the con-sequences of drug-drug interactions that would otherwise be sig-nificant in wild-type [260]. Digoxin was co-administered with clarithromycin, a P-gp inhibitor. The increase in digoxin bioavail-ability was observed in wild-type but not in mutant allele carriers. As the efflux of substrates in the intestinal wall decreases oral ab-sorption, it is reasonable to think that the P-gp inhibitor inhibited intestinal P-gp activities, leading to increased absorption of the substrate from the gut. However, this does not explain unaltered bioavailability of digoxin observed in subjects carrying mutant alleles. Perhaps, because the mutation changed the shapes of the Pharmacogenetics and Clinical Practicedrug-binding sites of P-gp, clarithromycin could no longer act as a competitive inhibitor. Breast Cancer Resistance Protein (BCRP/ABCG2) BCRP recognized as the ABCG2 is the second member of the subfamily G of the human ABC transporter superfamily. Unlike P-gp which is arranged in two repeated halves, BCRP is a half-transporter only consisting of one nucleotide-binding domain fol-lowed by one membrane-spanning domain [261]. BCRP is physio-logically present in many human tissues, with the highest level found in human placenta followed by prostate, intestine, brain, colon, liver and ovary [262]. However, BCRP mRNA is not de-tected by Northern analysis in tissues such as heart, skeletal muscle, lung, kidney, spleen and pancreas thymus, and peripheral blood leukocytes [262]. Substantial localization of BCRP in the placenta, small intestine and in the apical membrane (e.g. in the bile canalicu-lar membrane of hepatocytes) suggests that its main physiological role may be as a protective efflux pump, regulating intestinal ab-sorption and biliary secretion of drugs, their metabolites and poten-tially toxic xenobiotics [263]. Studies have shown that treatment with the BCRP inhibitor GF 120918 (also a P-gp inhibitor) de-creases plasma clearance and hepatobiliary excretion of topotecan and increases the absorption of anticancer drug from the small in-testine in P-gp knock out mice [263]. BCRP is also found in various stem cells, where this protein acts as a marker of the so-called side population cells, where it represents pluripotent stem cells [264]. Furthermore, BCRP may play an important role in basic heme me-tabolism, altering the hypoxic response in a number of cell types. BCRP was first cloned by Doyle et al. [265] from a doxorubi-cin-resistant MCF7 breast cancer cell line. Expression of BCRP is detected in a large number of hemtalogical malignancies and solid tumors, suggesting that this transporter may relate to clinical drug resistance of cancer [266]. The overexpression of BCRP confers resistance to a wide range of anticancer agents, including camp-tothecins, anthracylines, and mitoxantrone by enhancing drug efflux [266, 267]. Besides its role to confer resistance against chemothera-peutic agents, BCRP also actively transports conjugated or uncon-jugated organic molecules such as estrone-3-sulfate, and 17-estradiol 17-(-D-glucuronide). BCRP also transports a range of chemical toxicants, including pheophorbide a and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [268]. This transporter is known as a potent efflux transporter with extensive substrate speci-ficity molecule recognition of either negative or positive charge, organic anions and sulfate conjugates [268]. The human BCRP gene contains 16 exons, ranging from 60 to 532 bp, 15 introns and spanning over 66 kb located on chromosome 4q22 [269]. BCRP is a 655 amino acid ABC protein with a molecu-lar weight of 72 kDa, that contains a single NH2-terminal cytosolic nucleotide-binding domain, an ATP-binding cassette involved in ATP hydrolysis. It was discovered that the first cloned BCRPcDNA [265] encoded a mutant BCRP that differs from the wild-type BCRP at Arg482 (R482), which was substituted with either threonine (R482T) or glycine (R482G) [269]. Substrate specificity is altered for doxorubicin, methotrexate, rhodamine 123, and topo-tecan due to mutations in BCRP [270]. On the other hand, sub-stances such as mitoxantrone and Hoechst 33342 are still substrates of both wild-type BCRP and the mutant [270]. To date, at least 546 SNPs of MDR1 have been described in NCBI SNP database (http://www.ncbi.nlm.nih.gov/, access date: 2 August 2008). Only a small portion of them are non-synonymous (V12M, Q141K, Q166E, I206L, F208S, S248P, D296H, L525R, A528T, F571I, and Y590N) and there is one frameshift (1515delC) mutation observed in the coding region of ABCG2. Among the above variations, 34G>A (V12M) AND 421C>A (Q141K) have been testified to be polymorphic in numerous populations [267, 271]. Current Drug Metabolism, 2008, Vol. 9, No. 8 763 The V12M polymorphism located in exon 2 influenced the N-terminal intracellular region of the protein. Both the wild-type (Val) and the variant (Met) amino acids have uncharged, hydrophobic side chains. The V12M polymorphism was discovered in all ethnic groups tested, found with the highest allele frequency in Mexican-Indians (90%, but only 10 individuals were tested), while only 2% in a Swedish population [272]. Upon the combination of several population studies, a consistent and significant difference can be seen between the overall allele frequencies of V12M in Caucasian, African American and Japanese populations [271]. The Q141K polymorphism located in exon 5 leads to the re-placement of the negatively charged glutamic acid residue with a positively charged lysine residue. This polymorphism affects the ATP-binding domain, between the Walker A motif (located at resi-dues 83–89) and the signature region (residues 186–189). The Q141K variant was detected in all ethnic groups tested; it was found that Q141K occurs frequently in Asian populations ranging from (~30-60%) and relatively low allele frequency in Caucasians and African American subjects (~5-10%) [273]. For example, 60% are heterozygous for Q141 K in a Chinese population; 39-50% heterozygous for a Japanese population and 7% homozygous for the variant Q141K. Several other variants such as I206L, N520Y and D620N are much less frequent with allele frequencies of ~1%. In addition, a polymorphism in exon 4 that had a substitution of a stop codon occurring for Gln at position 126 has also been identified [274]. Studies have discovered that the expression levels of Q141K ABCG2 protein is lower than the wild-type, or the V12M variant when expressed in PA317 or HEK-293 cells [273]. It was also found that a portion of Q141K remained intracellular despite having a low level of expression, as both V12M and Q141K BCRP could reach the plasma membrane in the HEK-293 cells [273]. Other reports quoted that there is a 30–40% reduction in expression of cell surface Q141K variant, despite having a similar mRNA level com-pared to the wild-type. Individuals homozygous for the Q141K variant had signifi-cantly lower expression levels of BCRP in the placenta while the heterozygous samples displayed an intermediate expression level [274]. On the other hand, another investigation regarding the ex-pression of naturally allelic variants of ABCG2 in human intestine, displays no significant differences in mRNA and protein levels among subjects expressing the Q141 allele in heterozygous form, as compared to the wild-type [273]. ABCG2 is expressed in polarized LLC-PKI cells, and a study has demonstrated that the V12M variant has an intracellular local-ization whereas the wild-type ABCG2 and Q141K show mainly apical staining [275]. The localization of other variants including V12M, A149P, R163K, Q166E, P269S and S441N was also exam-ined. All polymorphisms, including V12M and Q141K, had an apical localization, and only the S441N variant displayed an intra-cellular staining [275]. The opposing expression and localization data for the ABCG2 variants implies that dissimilarities in culture conditions or other cellular determinants could result in variation influencing the cellular trafficking of these proteins. Further studies are required to clarify the mechanism of a reduced protein expres-sion for Q141K, and the change of cellular localization for the V12M and Q141K variants found under specific conditions. To be able to elucidate the possible physiological or pathologi-cal relevance of the ABCG2 polymorphism, several studies were performed to explore the functional consequence of the variants. There was a 10-fold decrease in drug resistance compared with the wild-type ABCG2 when the V12M or Q141K-transfected LLC-PKI cells were challenged by mitoxantrone or topotecan [275]. In con-trast, when compared to wild-type ABCG2-transfected cells, Q141K variant alone had a fairly lower level resistance against mitoxantrone, topotecan or SN-38. In addition, when transport ac- 764 Current Drug Metabolism, 2008, Vol. 9, No. 8tivities of wild-type are compared against variants for estrone 3-sulfate, dehydroepiandrosterone sulfate, methotrexate or p-aminohippurate, no major dissimilarities were seen, when the trans-port activities were normalized for the expression levels of ABCG2 protein [275]. In the ATP-binding cassette region of ABCG2, Q141K is be-tween the Walker A and the signature motifs; hence variant ATPase activity alteration is possible. Experiments were undertaken to ob-serve between the vanadate-sensitive ATPase activity of ABCG2 V12M and Q141K variants, using Sf9 (Spodoptera frugiperda) cell membranes [276]. There was a 1.3 and 1.8-fold lower basal ATPase activity, respectively, for V12M and Q141K compared to wild-type [276]. On the other hand, the V12M (and D620N) ABCG2 dis-played a comparable ATPase activity as the wild-type protein. No stimulation of the ATPase activity was seen by transported sub-strates of ABCG2 and ABCG2 variants [276]. These data suggest that the investigated SNPs do not alter the specificity of ABCG2 substrates. If a reduction in the expression level has occurred or their membrane localization is altered, this may modify ABCG2-dependent drug transport (rate and substrate specificity). Multidrug Resistance Associated Protein 1 (MRP1/ABCC1) MRP1 (ABCC1) is expressed in most tissues throughout the human body and is located on the basolateral membrane of epithe-lial cells. It is also located on the basolateral membrane of proximal tubular cells in the kidney thus; it acts as a pump for substrates into the interstitial space instead of secretion into the bile, urine or gut [277, 278]. MRP1 also plays a protective role in several tissues including the lungs, testes, kidneys and in several protected sites such as the blood-organ barrier. The location of MRP1 at the blood-brain barrier supports findings that MRPs act as efflux pumps from the cerebrospinal fluid into the blood and therefore acts as a protec-tive mechanism [277]. The MRP1 mRNA is highly expressed in the choroid plexus, at much higher levels than in the kidneys of rats [279]. MRP1 has a wide range of substrates; it is a significant transporter for a diverse range of organic anion conjugates such as glutathione conjugates, glucuronides, glutathione disulfide, uncon-jugated anionic drugs and dyes. Ampipathic neutral/basic drugs and oxyanions are also transported via MRP1. The presence of glu-tathione can enhance the transportation of many MRP1 substrates that can be transported without glutathione, for example, etoposide and glucuronides [280]. MRP1 is a causative factor for the resis-tance of several groups of drugs. These MRP1 mediated resistant drugs include anthracyclines (for example, doxorubicin), epipodo-phyllotoxins (for example, etoposide), vinca alkaloids (for example, vincristine) and camptothecins (for example, topotecan) [281]. MRP1 is also usually involved with inflammatory processes involv-ing leukotriene C4. This is due to studies that have shown that MRP1 is the efflux pump that is responsible for the transport of leukotriene C4 out of mast cells. Glutathione homeostasis is also mediated by MRP1. The MRP1 gene seems to be a conserved gene as many of the mutations are reasonably rare [282]. The MRP1 gene maps to chromosome 16p13.1 and MRP1 is expressed in many drug resis-tant cancer cells. The MRP1 gene was first identified in the small cell lung carcinoma cell line NCI-H69, this cell was doxorubicin resistant however, and it did not over express P-gp [283]. Distur-bance of the Mrp1 gene established that the gene is not necessary for fertility or viability [284]. Mrp1 knockout mice have an im-paired inflammatory response and display hypersensitivity to etoposide. This may be caused as MRP1 can transport leukotrienes for example, leukotriene C4, which is a signaling compound for the migration of dendritic cells. In Mrp1 knockout mice, the migration of dendritic cells to the lymphatic vessel is impaired, which impli-cates leukotriene C4 in the response of dendritic cells to chemokines [284]. It is for this reason that MRP1 is believed to have a role in the mediation of inflammatory responses involving cysteinyl leu- Zhou et al. kotrienes as well as being involved in the protection of cells from chemical toxicity and oxidative stress [285]. To date, 3981 SNPs have been identified for the MRP1 gene and about 13 of them are non-synonymous (Table 12)(http://www.ncbi.nlm.nih.gov/SNP, access date: 2 August 2008). A number of SNPs have been identified and ten non-synonymous SNPs leading to amino acid changes have been investigated in transfected HEK293T cells [286]. None of the SNPs significantly changed the expression level of MRP1, thus it can be deduced that amino acid changes do not considerably affect the production or stability of MRP1. In contrast, when the influence of these SNPs on transport capacities of 3 different substrates including leukotriene C4 was investigated, they were found to have a moderate affect. Polymorphism 989A>T which is caused by a 2965G>A variant was found to have the lowest transport capacity [286]. A haplotype con-taining a -260G>C SNP in the 5’ flanking region was found and was linked with decreased activity in a reporter gene assay. This haplotype was then studied in different ethnicities. This particular SNP was found to have frequencies of 23% in European Ameri-cans, 55% in African Americans but only up to 5% in Asians. It has been suggested that this SNP may therefore play a role in inter-individual differences and variances between ethnic groups in re-sponse to drugs transported by MRP1 [287]. A mutation in exon 16 (2012G>T) which causes the substitu-tion of a conserved Gly residue with a Val residue at position 671, did not show any functional differences when compared to the wild type gene [288]. This is in contrast to a naturally occurring, low frequency (less than 1% of general population) mutation in exon 10 (1299G>T) which substitutes a highly conserved Arg residue for a Ser at position 433. This mutation resulted in decreased transporta-tion of leukotriene C4 and increased resistance to doxorubicin [289]. A mutation, 128G>C in exon 2 results in a Cys-Ser substitu-tion at position 43 which impairs the plasma membrane location of MRP1 and leads to reduced resistance to vincristine and arsenite when compared to MRP1 wild type [290]. An in-vitro study has found that anthracycline resistance medi-ated by MRP1 can be decreased or eliminated via the substitution of Glu1089 with a neutral or positive charged amino acid without affecting leukotriene C4 transport. Thereby reducing drug resis-tance without interfering with the normal inflammatory response associated with MRP1 [291]. Multidrug Resistance Associated Protein 2 (MRP2/ABCC2) MRP2 (ABCC2) is encoded by the Mrp2 gene and is located in the apical luminal membrane of epithelial cells of most excretory organs such as the kidney, liver and intestine. It is also localized in the blood-brain barrier and placenta [292]. This particular MRP was originally known as the canalicular multispecific organic anion transporter. For the reason that before MRP1 was first discovered, it was known that there was an organic anion transporter in the ca-nalicular membrane of hepatocytes [293]. MRP2 was originally discovered in a cisplatin resistant cancer cell line. It plays an impor-tant role in the body as it secretes metabolites into the bile [294]. Substrate specificity of this protein was demonstrated by MRP2 lacking rats, it was found that these rats were deficient in bilirubin-glucuronide secretion and were known to have inactivating muta-tions in their MRP2 gene [295]. This is similar to humans with the same mutations, as these results in patients having Dubin-Johnson syndrome [296]. Like MRP1, MRP2 substrates for the transporter include alkylating agents such as chlorambucil [297]. The MRP2 gene has been mapped to chromosome 10q24 [298]. Its primary function is to export organic anions into the bile from the liver. This role of MRP2 was first discovered when the encod-ing gene was mutated in the TR- rat, which is a rat strain that pre-sents with jaundice and a deficiency in organic ion transport [296]. The mutations of the MRP2 gene include, point mutations, deletions that can cause rapid degradation of mRNA, impaired protein matu- Pharmacogenetics and Clinical PracticeTable 12. Reported Non-Synonymous SNPs of Human MRP1/ABCC1 and MRP2/ABCC2 Genes Nucleotide change rs number MRP1 Region Current Drug Metabolism, 2008, Vol. 9, No. 8 765 Amino acid change C128G> rs41395947 Exon 2 C43S C218>T rs41494447 Exon 2 C275>T rs45611740 Exon 3 689G>A rs45551631 Exon 7 1057G>A rs45585132 Exon 9 T73I S92F R230N V353M 1299G>T rs60782127 Exon10 2012G>T rs45511401 Exon 16 2168G>A rs4148356 Exon 17 2581G>A rs45517537 Exon 19 2965G>A rs45620940 Exon 22 R433S G671V R723Q A861T A989T C3140G> rs13337489 Exon 23 C1047S 3173G>A rs41410450 Exon 23 3436G>A rs28706727 Exon 24 4009A>G rs45544333 Exon 28 R1058Q V1146I T1337A C4202>T rs8057331 Exon 29 T1401M MRP2 116A>T rs927344 Exon 2 C736A> rs45462493 Exon 7 842G>A rs56131651 Exon7 998A>G rs17222674 Exon 8 1058G>A rs7080681 Exon 9 Y39F M246L S281N D333G R353H 1249G>A rs2273697 Exon 10 C1457>T rs45518933 Exon 10 1483A>G rs45592535 Exon 11 1686T>G rs45477091 Exon 13 2153A>G rs3740072 Exon 17 C2302>T rs56199535 Exon 18 C2366>T rs56220350 Exon18 2546T>G rs45494393 Exon 19 C2677G> rs3740071 Exon 20 2761G>A rs41318029 Exon 21 C2901>A rs17222547 Exon 22 2944A>G rs45483699 Exon 22 C3026T> rs57351269 Exon 22 C3107T> rs45441199 Exon 23 3188A>G rs45568535 Exon 23 V417I T486I K495E F562L N718S R768W S789F L849R E893Q G921S Y967* I982V I1009T I1036T N1063S 3542G>T rs8187692 Exon 25 R1181L 3563T>A rs17222723 Exon 25 V1188E 3817A>G rs8187699 Exon 27 T1273A C3872>T rs17216317 Exon 28 P1291L C3895A> rs4148400 Exon 28 K1299Q 4348G>A rs56296335 Exon 31 A1450T 4544G>A rs8187710 Exon 32 C1515Y Data are from NCBI dbSNP (access date: 2 August 2008). ration or inappropriate MRP2 trafficking [299]. To date, 2983 SNPs have been identified for the MRP2 gene and many of them are non-synonymous (Table 12) (http://www.ncbi.nlm.nih.gov/SNP, access date: 2 August 2008). 766 Current Drug Metabolism, 2008, Vol. 9, No. 8 It was later found that the gene is also mutated in individuals with DJS, an autosomal recessive disorder. Dubin-Johnson syn-drome is a disorder of organic ion transport that results in hyperbili-rubinemia, characterized by jaundice, liver pigmentation and ab-normal gallbladder function [300]. A very uncommon missense mutation, 2302C>T, found in the C motif within the fist nucleotide binding domain, is the main causative gene for Dubin-Johnson syndrome. The homozygous mutations normally cause a complete lack of detectable MRP2 in Dubin-Johnson syndrome patients. Heterozygous mutations occur in between 0.5 and 1 percent of the population, with differences across ethnic groups [301]. Several naturally occurring polymorphisms and mutations of MRP2 have been identified in individuals not resulting in DJS. One of these mutations is 1249G>A which involves a substitution from the Val residue to an Ile residue at position 417 in exon 10. A polymorphism which is frequently observed is 3972C>T which is a silent SNP at position 1324 in exon 28. Both of these polymor-phisms are frequently observed in cancer cell lines [302]. Mrp2 double knockout mice exhibit biochemical abnormalities that are comparable to those of Dubin-Johnson syndrome patients [303]. They also have increased levels of serum and urine bilirubin glucuronide, increased hepatocyte glutathione concentrations, de-creased bile flow rates and increased hepatocyte glutathione con-centrations. In these Mrp2 knockout mice there is a relationship that has been established that as a compensatory mechanism, MRP3 and MRP4 expression are both up regulated. This is believed to occur so that these transporters can facilitate the secretion of some MRP2 substrates into the blood and urine [303]. It has been reported that a patient receiving a high dose methotrexate infusion for B-cell lymphoma had a three fold de-crease in methotrexate elimination rate and exhibited nephrotoxicity [304]. The patient was heterozygous for a 1271A>G mutation in the Mrp2 gene and did not efflux methotrexate or other MRP2 sub-strates [304]. The SNP 3972C>T could affect MRP2 activity on irinotecan clearance, where patients carrying homozygous 3972C alleles could have an increased AUC for irinotecan and its metabo-lites [305]. However, when a study on 9-nitrocamptothecin and its metabolite was undertaken with the 3972C>T SNP there was no significant link [306]. Forty one SNPs have been found in Japanese individuals including variations that cause amino acid substitutions. It has been established that the highest frequency mutation (24C>T) has no effect on the expression of MRP2 mRNA in human duodenal enterocytes [307]. CLINICAL PHARMACOGENETICS OF DRUG TARGETS Most drugs illicit their pharmacological action at a specific target site such as a receptor or an enzyme. Recent advances in molecular research have revealed many of the genes that encode these drug receptor sites demonstrate genetic polymorphism. These variations, in many cases, have altered the targets sensitivity to the specific drug molecule and thus have a profound effect on drug efficacy. 1-Adrenergic Receptor (ADRB1) It is well known that human myocardial cells and cells from many other tissues possess three subtypes of -adrenergic receptors encoded by ADRB1, ADRB2, and ADRB3 genes, and a putative fourth subtype is yet to be confirmed [308-311]. These receptors belong to the superfamily of adrenergic receptors with seven trans-membrane domains. ADRBs are involved in the epinephrine and norepinephrine-induced activation of adenylate cyclase through the action of G proteins. Following the binding of ADRB to G-protein and adenylate cyclase, a functional complex is generated, which initiates one of the most powerful cellular signal transductions to regulate the physiological and pathological responses in the heart [312, 313]. Zhou et al. To date, 76 SNPs of ADRB1 have been reported in NCBI dbSNP and 13 of them are non-synonymous SNPs resulting in changes in the amino acid of the ADRB1 protein (Table 13)(http://www.ncbi.nlm.nih.gov/, access date: 2 August 2008). Many polymorphisms of ADRB1 are quite rare and as a result, associated studies on these SNPs have been very limited [314, 315]. Of all the SNPs described to date, there are two common SNPs that stand out and form the basis for majority of the polymorphism studies associ-ated with the cardiovascular system. The first reported ADRB1missense polymorphism is 1165G>C (resulting in Gly389Arg), with the polymorphism being localized to the intracellular cyto-plasmic tail of ADRB1 [316]. The second is 145A>G, resulting in a Ser to Gly substitution at the 49 position of the amino acid, which is localized on the extracellular N-terminal domain of the ADRB1 protein [317]. Mason et al. [316] revealed that the Arg389 variant manifested slightly higher basal levels of adenylyl cyclase activity. This variant had an enhanced sensitivity to stimulation by the -agonist, isopro-terenol. These data suggest that the Arg389 ADRB1 protein may have stronger signal transduction and coupling ability with Gs compared with the Gly389 allele. The genetic variation at this locus may be the primary reason behind differences among individuals with respect to pathophysiologic characteristics and responses to therapeutics agents such as -agonist and -blockers. In one study, differential responses of -blockade were compared in mice with Arg389 and Gly389 polymorphisms [318]. After being adminis-tered propranolol (a -blocker), hearts containing Gly389 (wild-type) showed no marked decrease in contractility except for at maximal doses [318]. Conversely, Arg389 hearts were highly sensi-tive to -blockade, resulting in a greater decrease in contractility. This allele-specific response suggests that the protective antagonist effects of -blockers against prolonged stimulation due to catecho-lamines would be more likely to occur in patients possessing the Arg389 variant. Clinically, this would translate to a greater thera-peutic response at lower doses of -blockers for those expressing the Arg389 variant, in terms of an improvement in left ventricular function. As expected, Arg389 homozygous patients treated with carvedilol showed a significantly greater improvement in heart function compared with Gly389 homozygous patients, which con-sequently supported the above studies [318]. On the contrary to these results, the Arg389 allele has been found to have negative effects associated with heart failure. One such example can be demonstrated by a study conducted, whose results indicated that the Arg389 allele predisposed its carriers to heart failure [318]. In the presence of this allele, Mialet-Perez et alshowed that the heart of a mouse began to lack contractility in re-sponse to 1 stimulation, which is a pronounced feature of human heart failure. In later months of this study, Arg389 mice began to develop reduced ventricular function and pathologic fibrosis, which were not seen to be present in Gly389 mice [318]. As a result, this is a clear indication that the Arg389 polymorphism may predispose individuals to heart failure. In support of these findings, numerous additional studies have shown analogous associations between Arg389 and predisposition to heart failure, however, this time in-volving a synergistic interaction with the 2c-adrenergic receptor polymorphism [314]. In a case-control study of heart failure, homo-zygotes for this polymorphism, which tonically releases more noradrenaline, had an increased chance of heart failure [319]. When the homozygotes for the Arg389 polymorphism were added to this model, the chances of heart failure were dramatically increased for carriers of both alleles [319]. Thus, the Arg389 polymorphism ap-pears a risk-factor allele for heart failure but seems to be associated with a favourable clinical response to cardiovascular drugs. In vitro studies have been conducted in order to investigate how molecular signaling is affected by the second of the two common ADRB1 SNPs. In these studies, it was shown that the Gly49 allele, as compared to the wild type Ser49 allele, was associated with Pharmacogenetics and Clinical PracticeTable 13. Reported Non-Synonymous SNPs of the ADRB1 and ADRB2 Genes Nucleotide change rs number ADRB1 Current Drug Metabolism, 2008, Vol. 9, No. 8 767 Amino acid change C77>T rs34844626 A26V 85G>A rs35720093 A29T 92G>A rs35230616 R31Q 145A>G rs1801252 S49G C952>A rs238741 R318S 971A>G rs622397 Lys324Arg 1027G>A rs180897 A343T 1056G>T rs189429 E352D C1165G> rs59130083 G389R 1166G>T rs17875445 G389V 1196G>A rs36052953 R399H 1199G>T rs171170 R400L C1213>T rs35705839 H405Y C1380>A rs238740 D460E ADRB2 44A>G rs33973603 N15S 46G>A rs56964295 G16R C79G> rs60374884 E27Q C171ins rs35336948 57frameshift 311insA rs35680672 104frameshift C491>T rs1800888 T164I C659>G rs3729943 CS220 C718T> rs41320345 F240L 741G>T rs41358746 Q247H 769G>A rs56100672; rs62627384 G257R Data are from NCBI dbSNP (access date: 2 August 2008). higher basal and agonist-stimulated adenylyl cyclase activity [314]. Additionally, results also showed that the Gly49 variant showed a more pronounced agonist-induced receptor desensitization and down-regulation when it was chronically stimulated with isoproter-enol for twenty minutes [320]. It has been hypothesized that a higher regulating capacity of the adrenergic system could be advan-tageous and protective for patients suffering from heart failure [321]. As a result, this down-regulation of receptors reinforces the speculation that this can be a protective mechanism for patients with heart failure [321]. Clinical studies have been conducted in humans to see the ef-fect of these polymorphisms and their positive or negative effect on the cardiovascular system. In a preliminary study, 18 single nucleo-tide polymorphisms were observed and investigated with respect to the occurrence of idiopathic dilated cardiomyopathy, a condition which can lead to congestive heart failure [322]. Observations from this study highlighted that the genotypes carrying the Gly49 variant located in the N-terminus of the molecule in a homozygous or het-erozygous form were highly prevalent in the group of patients suf-fering from idiopathic dilated cardiomyopathy. On the contrary to these findings, another study showed that patients with congestive heart failure expressing the Gly49 allele experienced less mortality, morbidity and hospitalization compared to those patients expressing the Ser49 gene [317]. This conclusion, which was contradictory to the former study, suggests that the Gly49 allele might be associated with myocardial protection, therefore supporting in vivo studies. However, the results from another study supported the fact that there was no correlation between the Gly49 allele and risk of car-diovascular failure [323]. From the above three trials involving the Ser49Gly polymorphism, it can be seen that the association of this to cardiovascular risks remains uncertain at this point in time. 2-Adrenergic Receptor (ADRB2) ADRB2 is abundantly expressed in bronchial smooth muscles, and activation of these subtype of receptors results in dilation of the bronchi, known as bronchodilation [308-311]. As a result, these receptors are commonly targeted by -agonists in the treatment of bronchospasms, and in particular, in the treatment of asthma. Addi-tionally, ADRB2 is located in cardiac myocytes and vascular smooth muscle, and upon stimulation, it mediates an increase in inotropic effects and vasodilation respectively [308-311]. Polymor-phisms present in ADRB2 have been investigated more comprehen-sively since the potential phenotypes of interest look beyond the cardiovascular traits, all the way to asthma, obesity and diabetes [314, 315]. To date, at least 20 SNPs have been described in the coding region of ADRB2; however, only seven of these SNPs cause changes in the sequence of amino acids (http://www.ncbi.nlm. nih.gov/, access date: 2 August 2008). These include Asn15Ser, Gly16Arg, Glu27Gln, Ser220Cys, Phe240Leu, Gln247His, and Arg257Gln (Table 13). The most common polymorphisms are those present in the amino terminus of the receptor at amino acid position 16, where either Arg or Gly is found, and amino acid position 27, where Gln or Glu are commonly found. Extensive research with 768 Current Drug Metabolism, 2008, Vol. 9, No. 8respect to link these polymorphisms to asthmatic phenotypes and the response to -agonist therapy has been conducted. In a clinical study by Turki et al. [324], the association of the Gly16Arg polymorphism with nocturnal asthma was found. Noc-turnal asthmatics experienced a significant down-regulation in ADRB2. On the contrary, non-nocturnal asthmatics and non-asthmatics did not experience this. This concept of receptor down-regulation was also noticed in functional studies, which were con-ducted by site-directed mutagenesis. Results showed that substitu-tion of Arg by Gly at amino acid position 16, regardless of whether a Glu or Gln is present at amino acid position 27, produced a more profound degree of agonist-induced receptor down-regulation as oppose to the wild type Arg16 [324]. Given these observations, Turki et al. examined the genotype in two groups of patients, who did and did not suffer from asthma. It was found that nocturnal asthma sufferers consisted of a significant overrepresentation of the Gly16 polymorphism, while the frequency of the polymorphism at position 27 was not dissimilar between the two groups [324]. These results which indicate the relationship of the Gly16 polymorphism to nocturnal asthma have been clarified further by a meta-analysis study in which it was concluded that the Gly16 allele predisposes patients to nocturnal asthma and severity [325]. The Gly16Arg polymorphism has undergone further extensive studies relating to asthma pharmacotherapies [314, 315]. In one such trial, albuterol was administered to child patients with or with-out a history of wheezing. This study by Martinez et al. [326] re-ported that the amino acid 16 position was associated with marked differences in the prevalence of positive responses to this bron-chodilator. Observations demonstrated that asthmatic subjects who were homozygous for the Gly16 variant were significantly more likely to show less improvement when treated with ADRB2 ago-nists [326]. As a result, these patients would require more anti-inflammatory therapy by inhaled corticosteroids as oppose to those expressing the Arg16 allele. In clinical practice, this would translate to a higher dose of albuterol for patients expressing the Gly16 vari-ant in order to achieve a therapeutically effective drug concentra-tion, as opposed to Arg16 patients who would not require this. However, the reverse was noticed in adult patients with mild asthma [327]. It was found that patients expressing the Arg/Arg genotype significantly improved once the 2-agonist was ceased and subsequently replaced by another drug [327]. On the contrary, for patients expressing the Gly/Gly genotype, therapy was consid-erably better whilst on 2-agonist therapy than when it was with-drawn [327-329]. Consequently, from the above findings, it is strongly evident that the effect of Gly16Arg polymorphism on the response to al-buterol therapy is not consistent. One reason for this may be due to the difference in the patient age groups in both studies, in that one involved children and the other involved adults. None the less, it has shown that polymorphisms in ADRB2 are associated with var-ied and differential responses to drug therapy. SerotoninTransporter (SERT) The main role of SERT in the brain is the principle site of ac-tion for many antidepressants and mediates behavioural and toxic effects of cocaine and amphetamines. SERT uptakes 5-hydrosytryptamine (5-HT) into the presynaptic neuron, which ter-minates the synaptic action and recycles it into the neurotransmitter pool [330]. SERT is encoded by the gene SLC6A4, which is local-ised to chromosome 17q11.1-q12 and spans 31kb consisting of 14 exons [331]. To date, 424 SNPs of the SERT gene have been reported in humans (http://www.ncbi.nlm.nih.gov/, access date: 2 August 2008). Ten of them are non-synonymous SNPs, resulting in amino acid changes. These include Gly41Ala, Gly56Ala, Ile108Val, Asp193Asn, Lys201Asn, Ile425Leu, Phe465Leu, Val488Met, Leu550Val, and Lys605Asn. Several mutations at nucleotide posi- Zhou et al. tions 565, 1180, and 1388 also cause frameshift changes. Little is know about the functional impact of these polymorphisms. SERT contains variable number tandem repeat polymorphisms and the long and short variants of this SERT gene-linked polymor-phic region have different transcriptional efficiencies [332]. The polymorphism that was located 1kb upstream of the transcription initiation site showed it had 16 repeat elements consisting of 44-bp insertion or deletion involving repeat elements six to eight. Lesch etal. [333] found that the basal activity of the SERT long variant (L) was more then twice that of the short form (S) of the SERT gene promoter. The L*allele has been shown to have higher levels of SERT transcription than the S*allele in vitro, resulting in reduced 5-HT reuptake in S/S homozygotes [334]. One study by Perils et al. [335] suggests that S/S homozygotes may have an increased propensity to develop agitation or insomnia from fluoxetine. Serretti et al. [336] showed that allelic variation of the SERT promoter could be related to the antidepressant response to fluvoxamine and/or augmentation with pindolol. Serretti et al.[336]did the study on 102 in-patients with a major depression with psychotic features and randomly assigned treatment with fluvoxam-ine and either placebo or pindolol for a 6 week period. Homozy-gotes for the long variant (L/L) and heterozygous (L/S) showed a better response to fluvoxamine then homozygotes for the short vari-ant (S/S). Further studies [337-342] have also shown that the S*allele was associated with a poor response to fluvoxamine treat-ment. However, a study done on an Asian population by Kim et al.[343] showed contrasting results as homozygotes subjects for the S*allele showed a better response both to fluvoxamine and paroxetine. Two other Asian studies confirm this finding [344]. Two studies in Japanese did not find the association between SERT promoter polymorphism and response to fluvoxamine [345, 346]. This shows the need to conduct pharmacogenetic studies in subjects of different racial groups as the effect of SLC6A4 promoter polymorphism on the efficacy of antidepressants seems to be dependant on ethnic background. POTENTIAL APPLICATION OF CLINICAL PHARMACO-GENETICS IN PERSONALIZED MEDICINE As already mentioned above, allelic variations in the genes encoding drug metabolizing enzymes, drug transporters and drug targets as a result of polymorphism has the potential to have a sub-stantial effect on drug clearance and response. It is expected that personalised treatments will be offered in the near future based on the genotypes of individuals therefore optimize the dosage and decreasing the frequency of adverse drug reactions. Personalized medicine is the use of detailed information about a patient's geno-type or level of gene expression and a patient's clinical data in order to select a medication, therapy or preventative measure that is par-ticularly suited to that patient at the time of administration. The benefits of this approach include accuracy, efficacy, safety and speed. The term emerged in the late 1990s with progress in the Human Genome Project. Unlike other genetic testing, pharmacogenetics does not aim to specifically determine or predict the risk of disease, but rather char-acterises an individual based on disease susceptibility, risk of se-vere adverse effects, or even efficacy of certain drugs [347]. Phar-macogenetic testing has the ability to give an estimate of the likely effectiveness, thereby removing much of the uncertainty surround-ing current pharmacotherapy [348]. The ultimate goal of pharmaco-genetic testing is to aid physicians in the prescription of the appro-priate medication at the correct dose prior to the initiation of the therapy in an attempt to minimize adverse events and toxicity and maximize efficacy by excluding those who are unlikely to benefit (non-responders) or who may be harmed (adverse responders) [347]. The promise of pharmacogenetics lies in its potentialto iden-tify the right drug at the right dose for the right individual. The application of pharmacogenetics also aims to discover better drugs Pharmacogenetics and Clinical Practiceand improve the efficacy and safety of both prospective as well as licensed drugs. Minimal pharmacogenetic testing is required for all new drug applications to the Food and Drug Administration, includ-ing a requirement for germline DNA to be prospectively collected from all subjects participating in pre-approval clinical trials and genotyping studies for drugs that are metabolized by enzymes whose genes have clearly inactivating polymorphisms [349]. There is though, increasing evidence that applied pharmacogenetics is beginning to take on a role in health care with the emergence of commercially provided services, such as AmpliChip (http:// www.amplichip.us), which claims to provide personalized, fact-based pharmacogenetic information to assist physicians in optimiz-ing individual patient’s drug therapy. AmpliChip is the first FDA-cleared test for genotype analysis of CYP2D6 and CYP2C19 using a microarray hybridization method [350]. The AmpliChip tests the DNA from patients white blood cells collected in a standard antico-agulated blood sample for 29 polymorphisms and mutations from the CYP2D6gene and two polymorphisms from the CYP2C19gene. Therefore, the paradigm of medicine is progressively chang-ing from merely treating individuals based on their symptoms, to being able to predict disease-susceptibility and patient tolerability to pharmacotherapy in order to customize diagnosis and pharma-cotherapy. In the case of TPMT, there is a Clinical Laboratory Improve-ment Act-certified test available and clinical use of this test is in-creasing as physicians become more aware of the benefits of geno-typing before treating patients with thiopurines. Additionally, the FDA-approved product labelling for azathioprine indicates that prospective TPMT genotyping might help identify those patients at risk of hematological toxicities. It is interesting to contrast the test used to determine theTPMT phenotype with that used originally to classify subjectsas having either poor or extensive metabolism of CYP2D6. Inthe case of TPMT, a blood sample is obtained and the enzymaticactivity measured directly, whereas for CYP2D6 a probe drug is administered and a urine sample collected.The fact that TPMT is expressed in an easily accessiblecell (red blood cell) fa-cilitated the introductionof this pharmacogenetic test into clinical use. The availabilityof DNA-based tests means that the clinical application of pharmacogeneticscould be greatly accelerated for a large number of genes thatencode proteins important in drug re-sponse. The pharmacogenetic approach to clinical medicine is currently minimal despite its discovery dating back to the early 1960’s. At present, prescription genetic screening is largely confined to teach-ing hospitals and specialized laboratories and is not yet a part of routine practice [351]. The strategy of prescription genotyping is seldom practiced in the clinic, even for substrates of extensively characterized SNPs such as CYP2D6 and codeine; CYP2C19 and phenytoin; and CYP2C9 and warfarin. Its limited use may be due to the fact that the CYP2C9 genotype contributes to less than 10% to the total variability in an individuals dose requirements [60]. Shah et al. [352] related the infrequent clinical use of CYP2D6 testing to the complexity of interpreting results, the availability of alternative therapies, low clinical utility and tolerable adverse events. Although there are well established techniques for molecular genotyping and phenotyping in major research institutes, the facili-ties for genetic testing and measurement of parent and metabolite concentrations are not always accessible in the diagnostic labora-tory. What’s more, mutational screening using the current state-of-the-art technology is still laborious and time-consuming. Further-more, the lack of adequate education provided to physicians in clinical practice as well as medical undergraduates and trainee phy-sicians and it is yet to be incorporated into the curriculum of medi-cal courses worldwide. Thus, the bridging between medicine and basic science requires the collaborative efforts of both clinicians and researchers. Current Drug Metabolism, 2008, Vol. 9, No. 8 769 Another obstacle impeding the advancement of pharmacoge-netic approaches to therapeutics is the precision of the genotyping results and the confidence in associating SNPs with altered drug response. The ambiguity that can potentially arise in classifying an individual’s genotype based on the laboratory results is an contrib-uting factor. It is still unclear what the exact association between SNPs of the drug target genes with therapeutic outcome is; very few mutations have been characterised to establish their potential func-tional impact. As such, the functionality of these SNPs and their causative role remain largely speculative. Although through pharmacogenetics it is currently possible to reduce pharmacokinetic-related toxicities in some situations, de-creasing the occurrence of toxicities that are not predictably associ-ated to drug concentration may be more difficult. These so-called idiosyncratic toxicities are rare but often serious. Examples include drug-induced agranulocytosis, hepatotoxicity, Torsades de Pointesand rhabdomyolysis. Efforts are presently in progress to identify the genetic basis of numerous types of these toxicities, yet this may eventually prove to be the most challenging field in which to apply pharmacogenetic information [351]. This is due to the fact that the drugs causing these types of toxicities with any frequency are not approved for use (or are withdrawn from the market). Additionally, as these toxicities are very uncommon, it is a daunting task to ac-crue adequate numbers of patients who have experienced the toxic-ity. Currently one of two general treatment approaches is typically employed in the pharmacological management of disease. The first is a trial and error approach, employed for drug treatment of dis-eases such as hypertension, diabetes, depression, schizophrenia, arrhythmias, oesophageal reflux and others. For these diseases, there are several drugs that are reasonable first line therapy. Finding the most effective drugs for a given patient is often done through trial and error, and can often take months to accomplish. The other approach to drug management of disease is a per protocol approach, where the treatment for a given disease is essentially the same for everyone with that diagnosis. Examples of diseases treated in this way include most cancers, heart failure, myocardial infarction and organ transplantation. In both scenarios, a certain percentage of patients will obtain no benefit from a given drug, or will experience serious adverse effects. Thus, there are two general goals for the clinical application of pharmacogenetics; the ability to predict those patients at high risk of toxicity (and in whom a lower dose or a different drug would be administered), and the ability to predict those patients who are most likely to obtain the desired therapeutic effect from the drug [347]. By incorporating information from the above mentioned tech-nology, health professionals can identify the patient’s polymor-phisms and disease subtypes to determine most advantageous man-agement. This can include the immediate administration of the most efficacious and least toxic drugs at the correct doses. Apart from the disease states already mentioned, the treatment of hypertension is another example of the ability to individualise drug therapy as a result understanding a person’s genetic make-up. Currently, patients are required to have regular clinical visits to adjust dosing and po-tentially add another class of antihypertensive, as many patients, especially those from specific ethnic backgrounds, respond variably to different therapies. Understanding and identifying an individual’s genetic variations has the potential to decrease both the time ex-pended in achieving effective therapy and the number of visits re-quired for proper dose adjustment [347]. In order for a pharmacogenetic test to be useful clinically, there must be enough evidence that the genetic information has sufficient predictive value to provide meaningful information to clinicians. In most cases, this is not yet the case. As research moves forward from the field of pharmacogenetics and into the realm of pharmacoge-nomics advances in clinical applications should be seen. 770 Current Drug Metabolism, 2008, Vol. 9, No. 8Zhou et al. example, instead of hypertension being a coexisting risk factor that Ethnicity must be taken into account when pharmacogenetic should be considered before starting a new therapy, the risk factor information is used to make a clinical decision. The frequency of will be patients with CYP2D6/2C9 allelic variants. variant alleles of CYP families varies among populations according to the race and ethnic background. For instance, where the fre-ABBREVIATIONS quency of the CYP2D6 PM phenotype is 7-10% among Caucasians, it is only about 1-2% in north Asians [80]. The most frequent (20%) CAB = ATP-binding cassette mutant allele of CYP2D6 in Caucasians is the variation CYP2D6*4ADRB = -Adrenergic receptor which due to a splicing defect produces an inactive enzyme. How-AhR = Aryl hydrocarbon receptor ever, in Asians its frequency is only 1-2%. Conversely, the fre-BCRP = Breast cancer resistance protein quency of the PM phenotype of CYP2C19 is only 3-5% in Cauca-sians and almost 20% in north Asian populations. CYP = Cytochrome P450 CONCLUSIONS AND FUTURE PERSPECTIVES What have we learned from pharmacogenetics? The most im-portant lesson is the fact that all drug effects vary between indi-viduals, and all drug effects are influenced by genes. The majority of drug effects are multifactorial, i.e. they are affected by numerous genes, typically with some influence by environmental factors. However, some single mutations can alter a drug response consid-erably. From the pharmacogenetic studies of single genes we learn: •Polymorphisms of the same enzyme may display several dif-ferent functional alterations, which can vary from absence to multiplication of enzyme activity. Particular attention should be paid to the fact that not all substrates are affected by a given mutation alike. •A given drug-metabolising enzyme tends to metabolise numer-ous drugs. •We can no longer consider pharmacogenetics solely as struc-tural alterations of a gene, but also have to consider differences in gene expression, i.e. gene function. However, a number of challenges remain for clinical based pharmacogenetics to become a reality. Overtime, improvements in multi-genic testing promise to increase the role of personalized medicine. However, the pharmacogenomic complexities and par-ticularly time-dependent changes of gene expression, will never allow personalized medicine to become an error-free entity [347]. One challenge for the future lies in documenting enough of the drug response variability to make the genetic information clinically pre-dictive. In some cases this might only require information on a few polymorphisms of genes; in others it might require very complex studies that involve relatively large numbers of genes or a genomic-based approach. In the future, genetic testing to identify slow and fast metabo-lisers of a wide range of drugs may be conducted early in life, on a one-time basis, with the information placed on file in an individ-ual’s medical record. Such testing could have many benefits, both for individuals and drug companies. It could identify people who are susceptible to adverse drug reactions, and could also identify those who are unlikely to benefit from a particular dug. It would also make possible the resurrection of some older drugs that are safe and effective for most people but have been taken off the mar-ket because a few people had serous reactions. Companies would be able to reduce the size of clinical trials, creating greater efficacy. Therefore, pharmacogenetics has the potential of changing the pipe-line model of drug discovery, clinical development, and mass cus-tomization marketing. Pharmacogenetic profiling may consequently affect drug label-ling to limit prescriptions only to those individuals with the appro-priate genetic profile. This means for drugs already approved by the regulatory agencies, succeeding discoveries that individuals with certain genetic profiles might experience adverse drug effects would require addition of this information to the label and as well as a warning that genetic screening is necessary [349]. One could then take the view that pharmacogenetics is not about individual-ized drug therapy but rather about re-classifying risk factors. 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