Cytochrome P450 Polymorphism Ñ Molecular, Metabolic, and Pharmacogenetic Aspects

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 65 No. 3 pp. 319ñ329, 2008 ISSN 0001-6837 Polish Pharmaceutical Society

CYTOCHROME P450 POLYMORPHISM ñ MOLECULAR, METABOLIC, AND PHARMACOGENETIC ASPECTS. III. INFLUENCE OF CYP GENETIC POLYMORPHISM ON POPULATION DIFFERENTIATION OF DRUG METABOLISM PHENOTYPE

PIOTR TOMASZEWSKI*, GRAØYNA KUBIAK-TOMASZEWSKA, JACEK £UKASZKIEWICZ and JAN PACHECKA

Department of Biochemistry and Clinical Chemistry Pharmaceutical Faculty, Medical University of Warsaw 1 Banacha Str., 02-097 Warsaw, Poland

Abstract: In the human genome 684 alleles of CYP genes, and additionally 30 complete CYP pseudogenes, have been identified. So far 388 isoforms of 58 human CYP isoenzymes have been described at the phenotypic level. The molecular forms of many CYP isoenzymes responsible for drug biotransformation show a differentiated degree of specific catalytic activity ñ from increased, through normal and decreased to various extent, to trace or even absent. Depending on the homo- or heterozygous genotype, a broad palette of phenotypic forms may be present, differentiated in respect to biotransformation dynamics of specific drugs. The progress of molecular biology with particular consideration of genotyping and DNA microarray technologies has created a basis for the dynamic progress of pharmacogenetics, allowing fast and sensitive determination of the individual pharmacogenetic profile, encompassing a large set of CYP alleles extended by allelic variants of genes encoding other enzymes participating in drug metabolism. The possibility to evaluate the pharmacogenetic profile of patients together with the increasing knowledge about the mechanisms of inhibition, repression and also induction of enzymes participating in biotransformation of xenobiotics and endogenous compounds create increasing possibilities of elaborating optimal individualized pharmacotherapeutic strategies.

Keywords: cytochrome P450, genetic polymorphism of CYP, CYP alleles, drug biotransformation, poor metabolizers, ultra-rapid metabolizers, pharmacogenetics

Abbreviations: Ad ñ adrenodoxin; ADH ñ alcohol dehydrogenases: NAD-dependent (EC 1.1.1.1) and NADP- dependent (EC 1.1.1.2); ALDH ñ aldehyde dehydrogenases: NAD-dependent (EC 1.2.1.3), NADP-dependent (EC 1.2.1.5); AR ñ adrenodoxin reductase (EC 1.18.1.2); CBR ñ cytochrome b5 reductase (EC 1.6.2.2); CPR ñ cytochrome P450 reductase (EC 1.6.2.4); CYP ñ cytochrome P450; cyt ñ cytochrome; EM ñ extensive metabolizer; FMO ñ flavin monooxygenases (EC 1.14.13.-); GST ñ glutathione S-transferaze (EC 2.5.1.18); IM ñ intermediate metabolizer; kbp ñ kilo base pairs; MDR ñ multidrug resistance protein; MRP ñ multidrug resistance-associated protein; NADH+H+ ñ nicotinamide adenine dinucleotide, reduced form; NADPH+H+ ñ nicotinamide-adenine dinucleotide phosphate, reduced form; NAT ñ N-acetyltransferase (EC 2.3.1.-); OATP ñ organic anion-transporting polypeptide; PASA ñ PCR allele-specific amplification; PCR ñ polymerase chain reaction; PM ñ poor metabolizer; RFLP ñ restriction fragment length polymorphism; RM ñ rapid metabolizer; ST ñ sulfotransferase (EC 2.8.2.-); UGT ñ UDP-glucuronosyltransferase (EC 2.4.1.17); URM ñ ultra-rapid metabolizer.

Genetic polymorphism of CYP tion into the genome of the product of reverse tran- The CYP isoenzymes present in the human scription of mRNA obtained by transcription of the organism show considerable genetic polymorphism. proper gene. Such pseudogenes do not contain So far 388 isoforms of 58 human CYP isoenzymes introns (so-called intronless pseudogenes) because have been described. In the human genome 684 alle- of their origin from the mRNA molecules formed by les of CYP genes, and additionally 30 complete CYP splicing. They often have sequences corresponding pseudogenes, have been identified (1). Pseudogenes to Ñpoly-A tailsî (2). Sporadically the transcription in general are non-expressed coding sequences of some pseudogenes is observed, however, the formed by the duplication of the proper genes, and formed mRNA in general cannot be used in transla- then mutations of the copies which in general make tion, or the product of expression is not a functional their transcription impossible. Pseudogenes may protein. Recently it was shown that one of the CYP also be formed during retroposition, i.e. incorpora- pseudogenes, designated by the CYP2D7P1 symbol

* Correspondence: e-mail: [email protected]

319 320 PIOTR TOMASZEWSKI et al.

(locus 22q13, 9 exons) is expressed. The protein are elements of a large CYP gene cluster on chro- designated by the symbol CYP2D7P1, with a mosome 7 (7q.21.1 CYP-cluster). Some authors con- molecular weight of 57.7 kDa and 516 aa. residues sider that isoenzyme CYP3A3 is formed as the result is formed, and shows a low catalytic activity, lead- of an alternatively initiated transcription of gene ing to codeine monooxygenation (1, 3-5). common for CYP3A3 and CYP3A4 (3, 5, 9-11). Besides complete CYP pseudogenes in the human genome have been identified: 24 single Influence of CYP polymorphism on drug metab- exons of CYP genes which occur individually far olism phenotype from the proper genes, 9 detritus exons in the vicin- The occurrence in the genotype of pair of alle- ity of the proper CYP genes and 1 duplicated exon les encoding the molecular forms of some important occurring within the proper CYP gene (1, 6). for drug biotransformation CYP isoenzymes with a The significantly higher number of CYP gene decreased activity leads to a considerable decrease alleles in comparison to the number of identified in the biotransformation effectiveness of drugs spe- CYP isoenzyme isoforms is due to several factors. cific for a given CYP isoenzyme. As among the Many CYP gene alleles are the result of silent point molecular forms of the same isoenzyme often sever- mutations which do not lead to a change in the al variants occur with different degrees of decrease amino acid sequence of the protein encoded by the of catalytic activity, slowing and sometimes even gene, as the codons resulting from the mutation preventing drug biotransformation by a defined encode the same amino acids as the precursor CYP isoenzyme is found not only, as was believed codons. The products of the expression of such alle- previously, in homozygotes having pairs of identical les are thus identical to those encoded by the basic alleles encoding isoforms with decreased or abol- allele. Some alleles are not expressed due to a point ished activity. A decreased effectiveness of drug mutation changing the sequence of the initiation biotransformation can also be found in heterozy- codon or a large deletion of most of the gene includ- gotes with two different alleles encoding differenti- ing the initiation codon. In the case of some of the ated isoforms with decreased or abolished catalytic alleles the product of expression is extremely differ- activity, e.g. decreased activity of isoenzyme ent from that encoded by the basic gene due to a CYP2D6 is found both in the cases of homozygous point mutation causing a frameshift, significant per- genotypes CYP2D6*9 / CYP2D6*9 and CYP2D6*17 / turbations in splicing or premature termination. The CYP2D6*17, and in the heterozygous variant products of expression of such alleles, which of CYP2D6*9 / CYP2D6*17 (7). course have no catalytic activity, are not classified Traditional classification performed in human as CYP isoforms. The highest polymorphism among populations because of the differentiated biotrans- human monooxygenases from the CYP superfamily formation of specific drugs due to genetic poly- is demonstrated by isoenzymes CYP21A2 with 85 morphisms of CYP isoenzymes, which distinguish- isoforms and CYP2D6 with 44 isoforms (1, 7). es only two phenotypic variants: poor metabolizers The genetic polymorphism of CYP isoenzymes (PM) and extensive metabolizers (EM), is greatly participating in drug biotransformation is presented simplified. In the later papers four phenotypic vari- in Table 1. In the case of some CYP isoenzymes ants are distinguished: ultra-rapid metabolizers involved in drug biotransformation, e.g. CYP3A3, (URM), rapid metabolizers (RM), intermediate CYP2C18, no cases of genetic polymorphism have metabolizers (IM) and poor metabolizers (PM) (7, been observed so far (1, 3, 4, 8). 12). In fact, the molecular forms of many CYP Genes for CYP1A1 and CYP1A2 isoenzymes isoenzymes responsible for drug biotransformation are located on chromosome 15 at a distance of about show a differentiated degree of catalytic activity ñ 25 kbp from each other. Isoenzymes CYP2A6, from normal, through decreased to various extent, CYP2B6 with all other isoenzymes from the to trace or even absent. Depending on the homo- or CYP2A, CYP2B and CYP2F subfamilies are encod- heterozygous genotype, a broad palette of pheno- ed within a large CYP gene cluster on chromosome typic forms may be present, differentiated in 19 (19q CYP-cluster). Isoenzymes CYP2C8, respect of biotransformation dynamics of specific CYP2C9, CYP2C18, CYP2C19, CYP2E1 are drugs (7, 12, 13). encoded within a large CYP gene cluster on chro- In the human population a differentiated fre- mosome 10 (10q.24 CYP-cluster). The CYP2D6 quency of particular molecular forms of CYP isoen- isoenzyme gene is located on chromosome 22 near zymes responsible for drug metabolism has been two CYP pseudogenes. Genes of isoenzymes found in different ethnic and racial groups. Among CYP3A3, CYP3A4, CYP3A5, CYP3A7, CYP3A43 genotypes showing a decreased effectiveness of Cytochrome P450 polymorphism ñ molecular, metabolic, and pharmacogenetic aspects. III... 321

Table 1a. Genetic polymorphism of CYP isoenzymes participating in drug biotransformation (1, 3-5, 9-11).

Isoenzyme Isoform Specific Alleles Locus Number activity of exons CYP1A1.1 N CYP1A1*1A, CYP1A1*1B, CYP1A1*1C, CYP1A1*2A, CYP1A1*3 CYP1A1.2 N CYP1A1*2B, CYP1A1*2C CYP1A1.4 ? CYP1A1*4 CYP1A1.5 ? CYP1A1*5 CYP1A1 CYP1A1.6 ? CYP1A1*6 15q22-q24 7 fsp 0 CYP1A1*7 CYP1A1.8 ? CYP1A1*8 CYP1A1.9 ? CYP1A1*9 CYP1A1.10 ? CYP1A1*10 CYP1A1.11 ? CYP1A1*11 CYP1A2.1 N CYP1A2*1A, CYP1A2*1B, CYP1A2*1C (decreased exp.), CYP1A2*1D, CYP1A2*1E, CYP1A2*1F (increased ind.), CYP1A2*1G, CYP1A2*1H, CYP1A2*1J, CYP1A2*1K (decreased exp.), CYP1A2*1L, CYP1A2*1M, CYP1A2*1N, CYP1A2*1P, CYP1A2*1Q, CYP1A2*1R, CYP1A2*1S, CYP1A2*1T, CYP1A2*1U CYP1A2.2 ? CYP1A2*2 CYP1A2.3 N CYP1A2*3 (decreased exp.) CYP1A2.4 N CYP1A2*4 (decreased exp.) CYP1A2.5 ? CYP1A2*5 CYP1A2 CYP1A2.6 N CYP1A2*6 (decreased exp.) 15q22-q24 7 CYP1A2.7 decreased CYP1A2*7 CYP1A2.8 decreased CYP1A2*8 CYP1A2.9 ? CYP1A2*9 CYP1A2.10 ? CYP1A2*10 CYP1A2.11 decreased CYP1A2*11 CYP1A2.12 ? CYP1A2*12 CYP1A2.13 ? CYP1A2*13 CYP1A2.14 ? CYP1A2*14 CYP1A2.15 decreased CYP1A2*15 CYP1A2.16 decreased CYP1A2*16 CYP2A6.1 N CYP2A6*1A, CYP2A6*1B1, CYP2A6*1B2, CYP2A6*1B3, CYP2A6*1B4, CYP2A6*1B5, CYP2A6*1B6, CYP2A6*1B7, CYP2A6*1B8, CYP2A6*1B9, CYP2A6*1B10, CYP2A6*1B11, CYP2A6*1B12, CYP2A6*1B13, CYP2A6*1D, CYP2A6*1F, CYP2A6*1G, CYP2A6*1H, CYP2A6*1J, CYP2A6*1X2 (duplicated), CYP2A6*9A (decreased exp.), CYP2A6*9B (decreased exp.) CYP2A6.2 0 CYP2A6*2 CYP2A6.3 0 CYP2A6*3 ldp 0 CYP2A6*4A, CYP2A6*4B, CYP2A6*4D CYP2A6.5 0 CYP2A6*5 CYP2A6.6 decreased CYP2A6*6 CYP2A6.7 decreased CYP2A6*7 CYP2A6.8 0 CYP2A6*8 CYP2A6 CYP2A6.10 decreased CYP2A6*10 19q13.2 9 CYP2A6.11 decreased CYP2A6*11 CYP2A6.12 decreased CYP2A6*12A, CYP2A6*12B, CYP2A6*12C CYP2A6.13 N CYP2A6*13 CYP2A6.14 N CYP2A6*14 CYP2A6.15 N CYP2A6*15 CYP2A6.16 N CYP2A6*16 CYP2A6.17 decreased CYP2A6*17 CYP2A6.18 decreased CYP2A6*18A, CYP2A6*18B, CYP2A6*18C CYP2A6.19 decreased CYP2A6*19 CYP2A6.20 0 CYP2A6*20 CYP2A6.21 ? CYP2A6*21 CYP2A6.22 ? CYP2A6*22 322 PIOTR TOMASZEWSKI et al.

Table 1b. Genetic polymorphism of CYP isoenzymes participating in drug biotransformation (1, 3-5, 9-11).

Isoenzyme Isoform Specific Alleles Locus Number activity of exons CYP2B6.1 N CYP2B6*1A, CYP2B6*1B, CYP2B6*1C, CYP2B6*1D, CYP2B6*1E CYP2B6*1F, CYP2B6*1G, CYP2B6*1H, CYP2B6*1J, CYP2B6*1K CYP2B6*1L, CYP2B6*1M, CYP2B6*1N CYP2B6.2 N CYP2B6*2A, CYP2B6*2B CYP2B6.3 N CYP2B6*3 CYP2B6.4 N CYP2B6*4A, CYP2B6*4B, CYP2B6*4C, CYP2B6*4D CYP2B6.5 decreased CYP2B6*5A, CYP2B6*5B, CYP2B6*5C CYP2B6.6 N CYP2B6*6A, CYP2B6*6B, CYP2B6*6C CYP2B6.7 N CYP2B6*7A, CYP2B6*7B CYP2B6.8 decreased CYP2B6*8 (decreased exp.) CYP2B6.9 N CYP2B6*9 CYP2B6.10 N CYP2B6*10 CYP2B6.11 decreased CYP2B6*11A (decreased exp.), CYP2B6*11B (decreased exp.) CYP2B6.12 decreased CYP2B6*12 (decreased exp.) CYP2B6.13 decreased CYP2B6*13A, CYP2B6*13B CYP2B6 CYP2B6.14 decreased CYP2B6*14 19q13.2 9 CYP2B6.15 N CYP2B6*15A (decreased exp.), CYP2B6*15B CYP2B6.16 decreased CYP2B6*16 (decreased exp.) CYP2B6.17 N CYP2B6*17A, CYP2B6*17B CYP2B6.18 decreased CYP2B6*18 (decreased exp.) CYP2B6.19 decreased CYP2B6*19(decreased exp.) CYP2B6.20 N CYP2B6*20 (decreased exp.) CYP2B6.21 N CYP2B6*21(decreased exp.) CYP2B6.22 N CYP2B6*22 (increased exp.) CYP2B6.23 N CYP2B6*23 CYP2B6.24 ? CYP2B6*24 CYP2B6.25 ? CYP2B6*25 CYP2B6.26 ? CYP2B6*26 CYP2B6.27 decreased CYP2B6*27 CYP2B6.28 0 CYP2B6*28 CYP2C8.1 N CYP2C8*1A, CYP2C8*1B, CYP2C8*1C CYP2C8.2 decreased CYP2C8*2 CYP2C8.3 decreased CYP2C8*3 CYP2C8.4 N CYP2C8*4 CYP2C8 fsp 0 CYP2C8*5 10q23.33 10 CYP2C8.6 N CYP2C8*6 CYP2C8.7 0 CYP2C8*7 CYP2C8.8 decreased CYP2C8*8 CYP2C8.9 N CYP2C8*9 CYP2C8.10 N CYP2C8*10 CYP2C9.1 N CYP2C9*1A, CYP2C9*1B, CYP2C9*1C, CYP2C9*1D CYP2C9.2 decreased CYP2C9*2A, CYP2C9*2B , CYP2C9*2C CYP2C9.3 decreased CYP2C9*3A, CYP2C9*3B CYP2C9.4 N CYP2C9*4 CYP2C9.5 decreased CYP2C9*5 fsp 0 CYP2C9*6 CYP2C9.7 N CYP2C9*7 CYP2C9.8 decreased ? CYP2C9*8 CYP2C9 CYP2C9.9 N CYP2C9*9 10q24.1 9 CYP2C9.10 N CYP2C9*10 CYP2C9.11 decreased CYP2C9*11A, CYP2C9*11B CYP2C9.12 decreased CYP2C9*12 CYP2C9.13 decreased CYP2C9*13 CYP2C9.14 decreased CYP2C9*14 CYP2C9.15 0 CYP2C9*15 CYP2C9.16 decreased CYP2C9*16 CYP2C9.17 N CYP2C9*17 CYP2C9.18 decreased CYP2C9*18 CYP2C9.19 ? CYP2C9*19 Cytochrome P450 polymorphism ñ molecular, metabolic, and pharmacogenetic aspects. III... 323

Table 1b. continued.

CYP2C9.20 N CYP2C9*20 CYP2C9.21 N CYP2C9*21 CYP2C9.22 N CYP2C9*22 CYP2C9.23 N CYP2C9*23 CYP2C9.24 N CYP2C9*24 CYP2C9.25 0 CYP2C9*25 CYP2C9.26 decreased CYP2C9*26 CYP2C9.27 N CYP2C9*27 CYP2C9.28 decreased CYP2C9*28 CYP2C9.29 N CYP2C9*29 CYP2C9.30 decreased CYP2C9*30 CYP2C18 CYP2C18 N CYP2C18 10q24 9 CYP2C19.1 N CYP2C19*1A, CYP2C19*1B, CYP2C19*1C, CYP2C19*17 (increased exp.) lsee 0 CYP2C19*2A, CYP2C19*2B, CYP2C19*2C syn.CYP2C19*21 CYP2C19.3 0 CYP2C19*3A, CYP2C19*3B syn.CYP2C19*20 nonexp. (icm) - CYP2C19*4 (nonexp.) CYP2C19.5 0 CYP2C19*5A, CYP2C19*5B CYP2C19.6 0 CYP2C19*6 lsee 0 CYP2C19*7 CYP2C19.8 0 CYP2C19*8 CYP2C19 CYP2C19.9 decreased CYP2C19*9 10q24 9 CYP2C19.10 decreased CYP2C19*10 CYP2C19.11 N CYP2C19*11 CYP2C19.12 decreased CYP2C19*12 CYP2C19.13 N CYP2C19*13 CYP2C19.14 N CYP2C19*14 CYP2C19.15 N CYP2C19*15 CYP2C19.16 ? CYP2C19*16 CYP2C19.18 N CYP2C19*18 CYP2C19.19 N CYP2C19*19

drug metabolism (PM) the following are predomi- alleles CYP2D6*4A, CYP2D6*4B, CYP2D6*4C, nant among Caucasians (7, 13, 14): CYP2D6*4D, CYP2D6*4E, CYP2D6*4F, CYP2D6*4G, ñ for the CYP2A6 isoenzyme: homozygous geno- CYP2D6*4H, CYP2D6*4J, CYP2D6*4K, CYP2D6*4L, type CYP2A6*4 causing the formation of an inactive CYP2D6*4M, CYP2D6*4N, CYP2D6*4X2; causing the protein with a considerably truncated polypeptide formation of an inactive protein with a considerably chain sequence because of an extensive deletion in changed polypeptide chain sequence in comparison the gene sequence; to the active isoenzyme because of splicing errors. ñ for the CYP2B6 isoenzyme: homozygous geno- The frequency of occurrence of differentiated type CYP2B6*5 causing the formation of the phenotypic and genotypic variants of poor metabo- CYP2B6.5 isoform with a considerably decreased lizers (PM) in Caucasian populations is shown catalytic activity; below for the following isoenzymes (1, 7, 15): ñ for the CYP2C9 isoenzyme: homozygous geno- CYP2B6 3-4%; types CYP2C9*2 and CYP2C9*3 or heterozygous CYP2C9 1-3%; genotypes with both mentioned alleles causing the CYP2C19 2-6%; formation of isoforms CYP2C9.2 and CYP2C9.3 CYP2D6 5-10%. with a considerably decreased catalytic activity; The most frequent (7%) genotypic variant of ñ for the CYP2C19 isoenzyme: homozygous geno- ultra-rapid metabolizer (URM) phenotype in type CYP2C19*2A causing the formation of an inac- Caucasian populations is CYP2D6*2XN (N=2,3,4,5 tive protein with a considerably changed polypep- or 13) (16). In the case of populations of other tide chain sequence in comparison with the active human races these indices may differ considerably, isoenzyme because of splicing error; e.g. the frequency of phenotypic and genotypic vari- ñ for the CYP2D6 isoenzyme: homozygous and het- ants of poor metabolizers (PM) for CYP2C19 is as erozygous genotypes encompassing the following high as 12-23% in the Oriental populations (17, 18). 324 PIOTR TOMASZEWSKI et al.

Table 1c. Genetic polymorphism of CYP isoenzymes participating in drug biotransformation (1, 3-5, 9-11).

Isoenzyme Isoform Specific Alleles Locus Number activity of exons CYP2D6.1 N CYP2D6*1A, CYP2D6*1B, CYP2D6*1C, CYP2D6*1D, CYP2D6*1E, CYP2D6*1XN (increased exp.) CYP2D6.2 N CYP2D6*2A, CYP2D6*2B, CYP2D6*2C, CYP2D6*2D, CYP2D6*2E, CYP2D6*2F, CYP2D6*2G, CYP2D6*2H, CYP2D6*2J, CYP2D6*2K, CYP2D6*2L, CYP2D6*2M, CYP2D6*2XN (increased exp.), CYP2D6*41A fsp 0 CYP2D6*3A, CYP2D6*3B lsee 0 CYP2D6*4A, CYP2D6*4B, CYP2D6*4C, CYP2D6*4D, CYP2D6*4E, CYP2D6*4F, CYP2D6*4G, CYP2D6*4H, CYP2D6*4J, CYP2D6*4K, CYP2D6*4L, CYP2D6*4M, CYP2D6*4N, CYP2D6*4X2 nonexp. (ld) 0 CYP2D6*5 (nonexp.) fsp 0 CYP2D6*6A, CYP2D6*6B, CYP2D6*6C, CYP2D6*6D CYP2D6.7 0 CYP2D6*7 pt 0 CYP2D6*8 CYP2D6.9 decreased CYP2D6*9 CYP2D6.10 decreased CYP2D6*10A, CYP2D6*10B, CYP2D6*10D, CYP2D6*10X2 lsee 0 CYP2D6*11 CYP2D6.12 0 CYP2D6*12 fsp 0 CYP2D6*13 CYP2D6.14 0 CYP2D6*14A, CYP2D6*14B fsp 0 CYP2D6*15 fsp 0 CYP2D6*16 CYP2D6.17 decreased CYP2D6*17 CYP2D6.18 0 CYP2D6*18 fsp 0 CYP2D6*19 fsp 0 CYP2D6*20 fsp 0 CYP2D6*21A, CYP2D6*21B CYP2D6.22 N CYP2D6*22 CYP2D6.23 N CYP2D6*23 CYP2D6 CYP2D6.24 N CYP2D6*24 22q13.1 9 CYP2D6.25 N CYP2D6*25 CYP2D6.26 N CYP2D6*26 CYP2D6.27 N CYP2D6*27 CYP2D6.28 N CYP2D6*28 CYP2D6.29 N CYP2D6*29 CYP2D6.30 N CYP2D6*30 CYP2D6.31 N CYP2D6*31 CYP2D6.32 N CYP2D6*32 CYP2D6.33 N CYP2D6*33 CYP2D6.34 ? CYP2D6*34 CYP2D6.35 N CYP2D6*35, CYP2D6*35X2 (increased exp.) CYP2D6.36 trace CYP2D6*36 CYP2D6.37 N CYP2D6*37 fsp 0 CYP2D6*38 CYP2D6.39 N CYP2D6*39 CYP2D6.40 0 CYP2D6*40 fsp 0 CYP2D6*42 CYP2D6.43 N CYP2D6*43 lsee 0 CYP2D6*44 CYP2D6.45 N CYP2D6*45A, CYP2D6*45B CYP2D6.46 N CYP2D6*46 CYP2D6.47 N CYP2D6*47 CYP2D6.48 N CYP2D6*48 CYP2D6.49 N CYP2D6*49 CYP2D6.50 N CYP2D6*50 CYP2D6.51 N CYP2D6*51 CYP2D6.52 N CYP2D6*52 CYP2D6.53 N CYP2D6*53 CYP2D6.54 N CYP2D6*54 Cytochrome P450 polymorphism ñ molecular, metabolic, and pharmacogenetic aspects. III... 325

Table 1c. continued.

CYP2D6.55 N CYP2D6*55 pt 0 CYP2D6*56 CYP2D6.57 N CYP2D6*57 CYP2D6.58 N? CYP2D6*58 CYP2D6.59 decreased CYP2D6*59 CYP2D6.60 ? CYP2D6*60 CYP2D6.61 ? CYP2D6*61 CYP2E1.1 N CYP2E1*1A, CYP2E1*1B, CYP2E1*1C, CYP2E1*1D, CYP2E1*5A, CYP2E1*5B, CYP2E1*6, CYP2E1*7A, CYP2E1*7B, CYP2E1*7C CYP2E1 CYP2E1.2 decreased CYP2E1*2 10q24.3-qter 9 CYP2E1.3 N CYP2E1*3 CYP2E1.4 N CYP2E1*4

Table 1d. Genetic polymorphism of CYP isoenzymes participating in drug biotransformation (1, 3-5, 9-11).

Isoenzyme Isoform Specific Alleles Locus Number activity of exons CYP3A3 CYP3A3 N CYP3A3X 7q22.1-q31.33 13 CYP3A4.1 N CYP3A4*1A, CYP3A4*1B, CYP3A4*1C, CYP3A4*1D, CYP3A4*1E, CYP3A4*1F, CYP3A4*1G, CYP3A4*1H, CYP3A4*1J, CYP3A4*1K, CYP3A4*1L, CYP3A4*1M, CYP3A4*1N, CYP3A4*1P, CYP3A4*1Q, CYP3A4*1R, CYP3A4*1S, CYP3A4*1T CYP3A4.2 N? CYP3A4*2 CYP3A4.3 N? CYP3A4*3 CYP3A4.4 N? CYP3A4*4 CYP3A4.5 N? CYP3A4*5 fsp 0 CYP3A4*6 CYP3A4.7 N? CYP3A4*7 CYP3A4.8 decreased CYP3A4*8 CYP3A4.9 N? CYP3A4*9 CYP3A4 CYP3A4.10 N? CYP3A4*10 7q21.3-q22.1 13 CYP3A4.11 decreased CYP3A4*11 CYP3A4.12 decreased CYP3A4*12 CYP3A4.13 decreased CYP3A4*13 CYP3A4.14 N? CYP3A4*14 CYP3A4.15 N CYP3A4*15A, CYP3A4*15B CYP3A4.16 decreased CYP3A4*16A, CYP3A4*16B CYP3A4.17 decreased CYP3A4*17 CYP3A4.18 increased CYP3A4*18A, CYP3A4*18B CYP3A4.19 N? CYP3A4*19 fsp 0 CYP3A4*20 CYP3A5.1 N CYP3A5*1A, CYP3A5*1B, CYP3A5*1C, CYP3A5*1D, CYP3A5*1E CYP3A5.2 N CYP3A5*2 lsee 0 CYP3A5*3A, CYP3A5*3B, CYP3A5*3C, CYP3A5*3D, CYP3A5*3E, CYP3A5*3F, CYP3A5*3G, CYP3A5*3H, CYP3A5*3I, CYP3A5*3J CYP3A5.4 N CYP3A5*4 lsee 0 CYP3A5*5 CYP3A5 lsee 0 CYP3A5*6 7q21.3-q22.1 13 fsp 0 CYP3A5*7 CYP3A5.8 decreased CYP3A5*8 CYP3A5.9 decreased CYP3A5*9 CYP3A5.10 decreased CYP3A5*10 CYP3A5.11 decreased CYP3A5*11 CYP3A7.1 N CYP3A7*1A, CYP3A7*1B, CYP3A7*1C, CYP3A7*1D, CYP3A7*1E CYP3A7 CYP3A7.2 increased ? CYP3A7*2 7q21.3-q22.1 13 CYP3A7.3 decreased ? CYP3A7*3 CYP3A43.1 N CYP3A43*1A, CYP3A43*1B CYP3A43 CYP3A43.2 trace CYP3A43*2A, CYP3A43*2B 7q21.1 13 CYP3A43.3 N CYP3A43*3 Abbreviations in Tables 1a-1d: exp. ñ expressibility; fspñ frameshift product; icm ñ initiation codon mutation; ind. ñ inductibility; ld ñ large deletion; ldp ñ large deletion product; lsee ñ large splicing error effect; N ñ normal activity; nonexp. ñ nonexpressing allel; pt ñ premature termination; 0 ñ lack of activity; ? ñ verification required. 326 PIOTR TOMASZEWSKI et al.

Table 2. The differentiation of catalytic activity of molecular forms of CYP21A2 isoenzyme (1).

Isoform Specific activity Alleles CYP21A2 CYP21A2.1-6 N CYP21A2*1A, CYP21A2*1B, CYP21A2*2, CYP21A2*3, CYP21A2*4, CYP21A2*5, CYP21A2*6 nonexp. (ld) 0 CYP21A2*7 (nonexp.) CYP21A2.8 decreased (≤50%) CYP21A2*8 CYP21A2.9-12 trace (<3%) CYP21A2*9, CYP21A2*10, CYP21A2*11, CYP21A2*12 CYP21A2.13 trace (≤0,1%) CYP21A2*13 CYP21A2.14 decreased (≤98%) CYP21A2*14 CYP21A2.15 decreased (≤50%) CYP21A2*15 fsp 0 CYP21A2*16 pt 0 CYP21A2*17 CYP21A2.18 0 CYP21A2*18 CYP21A2.19 decreased (≤68%) CYP21A2*19 CYP21A2.20 0 CYP21A2*20A, CYP21A2*20B, CYP21A2*20C, CYP21A2*20D (decreased exp.), CYP21A2*20E, CYP21A2*20F, CYP21A2*20G, CYP21A2*20H, CYP21A2*20J, CYP21A2*20K, CYP21A2*20L, CYP21A2*20M, CYP21A2*20N, CYP21A2*20P, CYP21A2*20Q, CYP21A2*20R, CYP21A2*20S, CYP21A2*20T, CYP21A2*20U, CYP21A2*20V CYP21A2.21 N ? CYP21A2*21 CYP21A2.22 decreased (≤10%) CYP21A2*22 CYP21A2.23 trace (≤0,8%) CYP21A2*23 CYP21A2.24 ? CYP21A2*24 fsp 0 CYP21A2*25 lsee 0 CYP21A2*26 pt 0 CYP21A2*27 CYP21A2.28 trace (<2,5%) CYP21A2*28 CYP21A2.29 N ? CYP21A2*29 pt 0 CYP21A2*30 lsee 0 CYP21A2*31 pt 0 CYP21A2*32 CYP21A2.33 trace (<0,2%) CYP21A2*33 CYP21A2.34 trace (<1,2%) CYP21A2*34 CYP21A2.35 decreased (≤30%) CYP21A2*35 pt 0 CYP21A2*36 fsp 0 CYP21A2*37 CYP21A2.38 decreased (≤23%) CYP21A2*38 lsee 0 CYP21A2*39 pt 0 CYP21A2*40 fsp 0 CYP21A2*4, CYP21A2*42 lsee 0 CYP21A2*43 fsp 0 CYP21A2*44 CYP21A2.45 trace (≤0,2%) CYP21A2*45 fsp 0 CYP21A2*46 CYP21A2.47 0 CYP21A2*47 pt 0 CYP21A2*48 CYP21A2.49 0 CYP21A2*49 pt 0 CYP21A2*50 CYP21A2.51 0 CYP21A2*51 CYP21A2.52 decreased (≤19%) CYP21A2*52 CYP21A2.53-54 0 CYP21A2*53, CYP21A2*54 CYP21A2.55 ? CYP21A2*55 CYP21A2.56 trace (≤1%) CYP21A2*56 CYP21A2.57 trace (<4%) CYP21A2*57 CYP21A2.58 decreased (<10%) CYP21A2*58 CYP21A2.59 decreased ? CYP21A2*59 CYP21A2.60 trace (≤0,7%) CYP21A2*60 CYP21A2.61 decreased (≤72%) CYP21A2*61 fsp 0 CYP21A2*62 CYP21A2.63 ? CYP21A2*63 CYP21A2.64 N ? CYP21A2*64 CYP21A2.65 ? CYP21A2*65 Cytochrome P450 polymorphism ñ molecular, metabolic, and pharmacogenetic aspects. III... 327

Table 2. continued.

CYP21A2.66-67 N ? CYP21A2*66, CYP21A2*67 lsee 0 CYP21A2*68 fsp, fsp 0 CYP21A2*69, CYP21A2*70 CYP21A2.71 decreased (≤46%) CYP21A2*71 CYP21A2.72 trace (≤1%) CYP21A2*72 CYP21A2.73-75 N ? CYP21A2*73, CYP21A2*74, CYP21A2*75 fsp 0 CYP21A2*76 CYP21A2.77-78 N ? CYP21A2*77, CYP21A2*78 CYP21A2.79 trace (≤1%) CYP21A2*79 CYP21A2.80 N ? CYP21A2*80 CYP21A2.81 trace (≤4%) CYP21A2*81 CYP21A2.82 trace (≤0,5%) CYP21A2*82 CYP21A2.83-85 N ? CYP21A2*83, CYP21A2*84, CYP21A2*85 fsp 0 CYP21A2*86 CYP21A2.87 N ? CYP21A2*87 fsp 0 CYP21A2*88, CYP21A2*89 pt 0 CYP21A2*90 CYP21A2.91-93 N ? CYP21A2*91, CYP21A2*92, CYP21A2*93 CYP21A2.94 trace (≤5%) CYP21A2*94 CYP21A2.95 decreased (≤14%) CYP21A2*95 CYP21A2.96 trace (≤1,2%) CYP21A2*96 fsp 0 CYP21A2*97, CYP21A2*98 CYP21A2.99 N ? CYP21A2*99 CYP21A2.100 decreased (≤80%) CYP21A2*100 fsp 0 CYP21A2*101, CYP21A2*102 CYP21A2.103 decreased ? CYP21A2*103 pt 0 CYP21A2*104 CYP21A2.105 decreased ? CYP21A2*105 CYP21A2.106 trace (≤0,5%) CYP21A2*106 CYP21A2.107 trace (≤0,1%) CYP21A2*107 CYP21A2.108 trace (≤0,4%) CYP21A2*108 CYP21A2.109 trace (≤0,1%) CYP21A2*109 CYP21A2.110 0 CYP21A2*110 CYP21A2.111-117 decreased ? CYP21A2*111, CYP21A2*112, CYP21A2*113, CYP21A2*114, CYP21A2*115, CYP21A2*116, CYP21A2*117 CYP21A2.118 trace (≤0,4%) CYP21A2*118 CYP21A2.119 decreased (≤38%) CYP21A2*119

Abbreviations in Table 2: exp. ñ expressibility; fspñ frameshift product; ld ñ large deletion; lsee ñ large splicing error effect; N ñ normal activity; nonexp. ñ nonexpressing allel; pt ñ premature termination; 0 ñ lack of activity; ? ñ verification required.

The phenotypic level of activity of molecular allele also causes the formation of an inactive CYP forms depend not only on genotypic basis. The product of expression, which is not an isoform complexity of these relations may be indicated by of a defined CYP isoenzyme because of the fact that the occasionally observed lack of activ- extreme sequence differences in respect to the ity of a given CYP isoenzyme may be due to: proper CYP, due to a considerable frameshift, 1. the presence of an isoform of a defined CYP splicing errors, premature termination of tran- isoenzyme not showing catalytic activity as the only scription or translation; molecular form of this isoenzyme, which occurs in: 2. the presence of an unexpressed allele of a defined a. homozygotes with an allele encoding an CYP gene in the homozygous state; inactive CYP isoform; 3. the presence of two different unexpressed alleles b. heterozygotes, in which besides the allele of a defined CYP gene in the heterozygous state; encoding an inactive CYP isoform the second 4. the presence in the homozygous state of an allele allele of the given CYP isoenzyme is not causing the formation of a catalytically inactive expressed; expression product, which is not an isoform of a c. heterozygotes, in which besides the allele defined CYP isomer because of extreme sequence encoding the inactive CYP isoform the second differences in respect to the proper CYP, formed as 328 PIOTR TOMASZEWSKI et al. the result of a frameshift, splicing errors, or prema- CYP polymorphism analysis as an element of the ture termination of transcription or translation; individual pharmacogenetic profile 5. the presence in the heterozygous state of two dif- Work on defining relations between scale of ferent alleles causing the formation of catalytically catalytic activity and molecular structure of CYP inactive expression products, which are not isoforms isoforms is performed in many research centers in of a defined CYP isomer because of extreme the world. At present, the best known is CYP21A2, sequence differences in respect to the proper CYP, and even though it does not participate in the bio- for reasons given above; transformation of most drugs, with the exception of 6. inhibition of the expression of alleles encoding some hormonal steroids (e.g. progesterone), the data active isoforms of a defined CYP isoenzyme by presented in Table 2 give a picture of the scale of the transcriptional repressors; possible percentwise differentiation of catalytic 7. considerable intensification of the degradation activity of molecular forms of CYP isoenzymes (1). and elimination of potentially active molecular The ongoing investigations on analogous descrip- forms of a defined CYP isoenzyme; tion of activity differentiation of molecular forms of 8. lack of the activity of existing, potentially active CYP isoenzymes participating in drug biotransfor- molecular forms of a defined CYP isoenzyme mation will allow a considerable progress in evalua- because of: tion of drug biotransformation effectiveness, a. inhibitor action; depending on the individual pharmacogenetic pro- b. lack of access of the proper substrate/sub- file of CYP alleles. strates; The progress of molecular biology with partic- c. lack of activity of enzymatic electron trans- ular consideration of genotyping techniques based

port systems (CPR; CBR, cyt b5; AR, Ad) on the polymerase chain reaction (PCR) using meth- which form complexes with CYP; ods of allele specific amplification (PASA) and d. lack of access of coenzymatic donors of reduc- restriction fragment length polymorphisms analysis tive equivalents ñ NADPH+H+ and/or NADH+H+. (RFLP) make it possible to define the profile of In cases 1, 2, 3, 4 and sometimes also 8c, the polymorphic genes encoding CYP isoenzymes par- lack of activity of a given CYP isoenzyme has a per- ticipating in drug metabolism. The development of manent character and in the remaining cases it is, in DNA microarray technology (also known as gene general, a periodic state and thus reversible. In case chips) has created a basis for the dynamic progress 8 in the analyzed cells it is possible to detect a pro- of pharmacogenetics, allowing fast and very sensi- tein corresponding to the active molecular form of a tive determination of the individual pharmacogenet- defined CYP isoenzyme. In case 1 an isoform of a ic profile, encompassing a large set of CYP alleles defined CYP isoenzyme which has no catalytic extended by allelic variants of genes encoding other activity can be detected. In cases 4, 5, and also in enzymes participating in phase I of drug metabolism case 1c an expression product or products extreme- (including FMO, ADH, ALDH, some hydrolases ly different in their amino acid sequence from the and reductases), also in phase II (including GST, proper CYP, formed as a result of a frameshift, UGT, ST, NAT) and in the so-called phase III, or splicing errors or premature protein biosynthesis ter- drug transport through biological barriers (among mination are present. In cases 2, 3, 6, 7 at the phe- others MDR, MRP, OATP proteins). notypic level no protein product of the expression of The possibility to evaluate the pharmacogenet- the proper CYP gene can be detected. ic profile of patients together with the increasing The permanent increase of total activity of a knowledge about the mechanisms of inhibition, given CYP isoenzyme is usually the result of: repression and also induction of enzymes participat- ñ amplification of a defined CYP gene (e.g. ing in biotransformation of xenobiotics and endoge- CYP2D6*1XN (N=2,3,4,5), CYP2D6*2XN (N=2,3,4,5 nous compounds create increasing possibilities of or 13), CYP2D6*35X2) causing increased expression elaborating optimal individualized pharmacothera- of a defined CYP isoenzyme showing normative spe- peutic strategies using the knowledge about individ- cific catalytic activity (1, 7, 17); ual specificities in the scope of reaction to the drug ñ the presence of an isoform of a defined CYP isoen- and about interactions occurring between the admin- zyme showing higher than normative specific cat- istered drugs, their metabolites, food components alytic activity (e.g. isoform CYP3A4.18 encoded by and environmental toxins, under conditions of natu- alleles CYP3A4*18A, CYP3A4*18B and probably ral variation of the physiological and pathological isoform CYP3A7.2 encoded by CYP3A7*2) (1, 16). state of the organism. Cytochrome P450 polymorphism ñ molecular, metabolic, and pharmacogenetic aspects. III... 329

REFERENCES 10. Human Gene Lynx: www.genelynx.org. 11. Source. Stanford University, Genetics Depart- 1. Ingelman-Sundberg M., Daly A.K., Nebert ment: www.stnford.edu/cgi-bin/source/surcese- D.W. (eds.): Human CYP Allele Nomenclature arch. Committe: Allele nomenclature for cytochrome 12. Clarke B.: Biomed. Sci. 1, 38 (2006). P450 enzymes. www.cypalleles.ki.se. 13. McKinnon R., Evans A.M.: Aust. J. Hosp. 2. Gerstein M., Zheng D.: Sci. Amer. 295, 48 (2006). Pharm. 30, 102 (2000). 3. Ensembl Human GeneViev: www.ensembl.org. 14. Coleman M.: Human Drug Metabolism: An 4. UniProt ñ The Universal Protein Knowledgeba- Introduction., pp.-286, Wiley, Hoboken 2005. se: www.expasy.org/uniprot. 15. Raimundo S., Fischer J., Eichelbaum M., Griese 5. Weizmann Institute of Science: GeneCards: E.U., Schwab M., Zanger U.M.: Pharmacoge- www.geneards.org. netics 10, 577 (2000). 6. Nelson D.: Cytochrome P450 Homepage: 16. Hasler J.A., Estabrook R., Murray M., et al.: drnelson.utmem.edu/CytochromeP450.html Mol. Aspects Med. 20, 1 (1999). 7. Ortiz de Montellano P.R. (ed.): Cytochrome 17. Meyer U.A., Zanger U.M.: Annu. Rev. Phar- P450. Structure, Mechanism, and Biochemistry. macol. Toxicol. 37, 269 (1997). 3rd ed. pp. 1-658, Kluwer Academic/ Plenum 18. Wen X: In vitro approaches in evaluation and Publishers, New York 2005. predication of drug-drug interactions involving 8. Entrez Protein. NCBI Sequence Viever v. 2.0. the inhibition of cytochrome P450 enzymes. National Center for Biotechnology Information: Dissertation,University of Helsinki, 2002, www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmddb. http://ethesis.helsinki.fi. 9. Genatlas Gene Database: www.genatlas.org. Received: 24.06.2007