Abstract
Polymorphisms and other genetic factors related to enzymes metabolizing drugs and xenobiotic chemicals are well known. This article focuses on polymorphism in the xenobiotic drug metabolizing enzymes particularly cytochromes P-450 (CYP) as example of phase I enzymes and N-acetyl transferases as example of phase II enzymes and distribution of variant forms of these enzymes in different ethnic groups.The cytochrome P450 (CYP) enzyme system is involved in the metabolism and elimination of number of widely used drugs. The capacity of this system varies from one person to another, leading to variable drug excretion rates and intersubject differences in the final serum drug concentrations. For this reason, therapeutic response and side-effects vary widely between patients treated with the same dose of drug. The intersubject variability in metabolic rate is largely determined by genetic factors. Some CYP enzymes including CYP2D6, CYP2C9 and CYP2C19 are genetically polymorphic. Similarly N-acetyltransferase enzyme is genetically polymorphic. Several mutant alleles have been described. All the major human drug-metabolizing P450 enzymes have been identified, and the major gene variants that cause inter-individual variability in drug response and are related to adverse drug reactions have been identified. This information now provides the basis for the use of predictive pharmacogenetics to yield drug therapies that are more efficient and safer.
Keywords: Genetic polymorphism, Cytochrome P450, N-acetyltransferase.
Introduction:
Inter-individual variation in drug response among patients is well known and poses a serious problem in medicine. There are no biomarkers at present that can predict which group of patients responds positively, which patients are nonresponders and who experiences adverse reactions for the same medication and dose. Many different enzymes are involved in the metabolism of chemical substances, but the focus of this overview will be on members of the cytochrome P450 family of enzymes and N-acetyltransferases (NAT). Many of the enzymes involved in the metabolism show a polymorphic distribution, and the genetic basis for some of these polymorphisms has been identified ( Herman A, 2000). Among drug metabolizing enzymes, cytochrome P450 (CYP) proteins are heme-containing enzymes. They are well known for their oxidative degradation of endogenous chemicals present in the diet, environment and medications. There are as many as 57 CYP genes and among them three families of genes - CYP1, CYP2 and CYP3-are the major genes (Wilkinson GR 2005). contributing to the oxidative metabolism of various compounds. NATs catalyze the acetylation of carcinogens and other xenobiotics including Arylamines, hydrazines and hydrazides are metabolized by NAT-mediated N-acetylation. The NAT locus on human chromosome 8p (21.3-23.1) ( Hickman D et. al.,1994) encodes three distinct NAT genes, the two active NAT genes, monomorphic NAT1(Blum M. et al., 1990) and polymorphic NAT2 (Grant DM 1989), separated by an inactive pseudogene. Large interindividual variation in these two enzymes has been observed in human which is described in this review.
Genetic polymorphism in CYP P- 450 system:
The most important enzymes accounting for variation in phase I metabolism of drugs is the cytochrome P-450 enzyme group,(Ingelman-Sundberg M. 2004; Weinshilboum R, 2003) which exists in many forms among individuals because of genetic differences. The cytochrome P450 enzymes in families 1-3 mediate 70-80% of all phase I-dependent metabolism of clinically used drugs ( Bertz R.J. and Granneman G.R,1997; Evans W.E. and Relling M.V, 1999) and participate in the metabolism of a huge number of xenobiotic chemicals. The polymorphic forms of P450s are responsible for the development of a significant number of adverse drug reactions (ADRs). According to Phillips et al. (2001) 56% of drugs that are cited in ADR studies are metabolized by polymorphic phase I enzymes, of which 86% are P450s. The major P450 forms that are important in human drug metabolism are shown, together with their properties and polymorphisms in table 1.
Table 1. Relative importance of polymorphisms in human cytochrome P450 enzymes involved in drug metabolism.
Enzymes
Major allelic variants.a
Clinical effect of the polymorphism.
Significans of the polymorphism.b
CYP1A2
CYP1A2*1K
Less enzyme expression and induciblity
+
CYP2A6
CYP2A6*4, CYP2A6*9
Altered nicotine metabolism of cancer drug.
+
CYP2B6
-
Significant for metabolism of cancer drugs.
+
CYP2C8
CYP2C8*3
Altered taxol metabolism
+
CYP2C9
CYP2C9*2, CYP2C9*3
Drug dosage
+++
CYP2C19
CYP2C19*2, CYP2C19*3
Drug dosage, drug efficacy
+++
CYP2D6
CYP2D6*2xn
No response
+++
CYP2D6*4
Drug dosage
+++
CYP2D6*10
Drug dosage
+
CYP2D6*17
Drug dosage
+
CYP2D6*41
Drug dosage
+
CYP2E1
-
No conclusive study
-
CYP3A4
No conclusive study
-
CYP3A5
CYP3A5*3
No conclusive study
-
a A description of the alleles can be found on the human cytochrome P450 allele nomenclature committee home page (http://www.imm.ki.se/CYPalleles/). b The signiï¬cance of the polymorphism is based on the number of reports showing impact of the P450 polymorphism on the pharmacokinetics of drugs that are substrates for the enzyme in question. Increasing numbers of ' + illustrate the increasing importance of the polymorphism relative to the other forms of P450.
Among CYP 450 enzyme system CYP2C9, CYP2C19 and CYP2D6 are highly polymorphic and accounts together for about 40% of hepatic human phase I metabolism. The functional importance of the variant alleles, however, differs and the frequencies of their distribution in different ethnic groups also differ. As a result, the metabolic conversion and excretion rate of drugs vary between individuals, from extremely slow to ultrafast. For many drugs, four major phenotypes can be distinguished: poor metabolizers (PMs), intermediate metabolizers (IMs), extensive metabolizers (EMs) and ultrarapid metabolizers (UMs). The intersubject variability in metabolic rate is largely determined by genetic factors.( May DG, 1994; Meyer UA et al. 1996) Genetic variation may produce normal enzyme in diminished amounts, poorly active or inactive enzyme, or massive amounts of enzyme. A number of CYP enzymes are known to be genetically polymorphic. Mutant alleles carrying certain nucleotide substitutions, deletions, insertions or gene conversions are known, which may result in CYP enzymes with abnormal activity.( Weide J and Steijns SW, 1999) Some mutants, the so-called null alleles, lead to enzyme deficiency or total absence of enzyme activity. This genetically determined variance in enzyme activity results in the different drug metabolism phenotypes.
CYP2D6 Genetic Polymorphism:
Among all CYP enzymes, the highly genetically polymorphic enzyme debrisoquine-4-hydroxylase, or CYP2D6, has been the most extensively studied. CYP2D6 is involved in the oxidative metabolism of more than 40 widely prescribed drugs. The CYP2D6 gene is localized on chromosome 22q13.1( Sachse C et al., 1997). The CYP2D6 gene contains nine exons within a total of 4378 base pairs. The locus contains two neighbouring pseudogenes, CYP2D7 and CYP2D8 ( Eim MH, Meyer UA, 1992). The evolution of the human CYP2D locus has involved elimination of three genes and inactivation of two (CYP2D7P and CYP2D8P) and partial inactivation of one (CYP2D6). At present, more than 50 known different major polymorphic CYP2D6 alleles are known ( Marez D,1997; Sachse C, 1997). The presence of the highly similar closely located pseudogenes carrying detrimental mutations have through, for example, unequal crossover reactions led to the formation of many of the variant CYP2D6 alleles, which most commonly encode defective gene products. The 'activity' in the CYP2D locus is high as compared to, for example, the CYP2C locus and as a result many variant alleles have been formed in a relatively short period of time. The variant CYP2D6 alleles can be classified into categories, which cause abolished, decreased, normal, increased or qualitatively altered cataxlytic activity. Among the most important variant ones are CYP2D6*2, CYP2D6*4, CYP2D6*5, CYP2D6*10, CYP2D6*17. For further details see table 2.
Table 2 Major human polymorphic variant CYP2D6 alleles and their global distribution.
Major variant
Alleles
Mutation
Consequence
Allele frequencies (%)
Caucasians
Asians
Black Africans
CYP2D6*2
Gene duplicatio/ multiduplication
Increased enzyme activity
1-5
0-2
2
CYP2D6*4
Defective splicing
Inactive enzyme
12-21
1
2
CYP2D6*5
Gene deletion
No enzyme
2-7
6
4
CYP2D6*10
P34S, S486T
Unstable enzyme
1-5
51
6
CYP2D6*17
T107I, R296C, S486T
Altered affinity for substrates
0
0
20-35Data is summarised from http://www.imm.ki.se/cypalleles/cyp2d6.htm and Bradford 2002 For further allele frequencies in different populations, see Bradford 2002.
The most common allele in Asians (allele frequency of 450%) and thus perhaps the most common CYP2D6 allele in the world is CYP2D6*10.( Johansson I, et al., 1994) Five to 10% of Caucasians lack CYP2D6 activity completely because of the inheritance of two mutant CYP2D6 null alleles. These subjects are classifieded as PMs, with an impaired metabolism of CYP2D6 substrates ( Alvan G, et al 1990). The majority of defective allelic variants of the CYP2D6 gene that give rise to the PM phenotype have now been identified. Up to 7% of Caucasians are UMs of CYP2D6 substrates, owing to the inheritance of alleles with duplication or amplification of functional CYP2D6 genes, causing an excessive amount of enzyme to be expressed ( Johansson I, et al., 1993 ; Agundez JAG et al ,1995). The metabolic capacity of the rest of the population lies somewhere between the extremes. IMs have mutations on the CYP2D6 gene, which cause only a partial decrease in enzyme activity. Subjects who are either homozygous for the normal-functioning alleles, or heterozygous with one active and one defect allele, are classified as EMs. Among EMs, metabolic rate can range considerably. In subjects homozygous for the active alleles, CYP2D6 drugs are metabolized more efficiently than the heterozygous genotypes. The latter are more at risk for drug - drug interaction if two or more CYP2D6 drugs have to be administered concomitantly. In general, the drug metabolism phenotype is determined by the number of functional CYP2D6 genes present( Johansson I, et al., 1993 ; Sachse C et al.,1997). In non-Caucasian populations, PM and UM phenotypes of CYP2D6 substrates may occur with different prevalences. In Orientals, for example, there are hardly any PMs and UMs, but the frequency of the IM phenotype is very high (Bertilsson L et al., 1995). In some African populations, prevalences of up to 29% for the UM phenotype have been reported( Alklillu E et al., 1996). Evaluation of the number of subjects carrying CYP2D6 gene duplications in Western Europe reveals that 5.5% of the Europeans carry more than two active CYP2D6 gene copies and are UMs (Table 3).
Table 3. An estimation of the number of ultrarapid CYP2D6 metabolisers in Western Europe carrying two or more active CYP2D6 genes on one allele. The overall percentage in the population is 5.45%
Ethnic Group
Million inhabitants
Frequency UMs
Austria
08
0.04
Belgium
10
0.03
Denmark
05
0.01
England
60
0.03
Finland
05
0.01
France
60
0.04
Germany
82
0.04
Greece
10
0.1
Holland
15
0.03
Italy
57
0.1
Norway
05
01
Portugal
10
0.1
Spain
40
0.1
Sweden
09
0.01
Total
376
CYP2C9 Genetic Polymorphism:
CYP2C9 is the major human enzyme of the cytochrome P450 2C subfamily and metabolizes approximately 10% of all therapeutically relevant drugs. It constitutes about 20% of the hepatic cytochrome P450 enzyme expressed in humans and thus is responsible for the metabolism of a wide spectrum of clinically important drug(Takahashi H and Echizen H, 2001). The human CYP2C9 gene is located on chromosome 10 between q23 and q24 with a length of approximately 55 kb ( Lee CR et al., 2002; Finta C and Zaphiropoulos PG., 2000). It has been shown to be polymorphic. CYP2C9 polymorphism has contributed to the wide inter-individual pharmacokinetic variability in terms of drug metabolism, for instance, S-warfarin ( Takahashi H et al., 2003), diclofenac ( Dorado P et al., 2003), losartan (Odani A et al., 1997), phenytoin ( Odani A et al., 1997) and tolbutamine (Shon JH et al 2002). Thus far, 12 different variants have been identified. The wild type of allele is identified as CYP2C9*1 and the mutant variants are known according to an ascending numerical order of CYP2C9*2 to CYP2C9*12 (Table 4 ). Although several allelic variants of CYP2C9 have been reported and identified, the most common variants are CYP2C9*2 and CYP2C9*3, apart from the wild type allele. CYP2C9*2 and CYP2C9*3 allelic variants differ from the wild type allele by a single nucleotide substitution. Two inhereted amino acid substitutions, Arg144Cys (2*) and Ile359Leu (3*), are know to affect catalytic functions of CYP2C9 ( Ieiri I et al., 2000; Sullivan-Klose TH et al., 1996; Tarbit MH and Wolf CR 1996). The effect of the polymorphism on warfarin metabolism has been studied extensively in the Caucasian population. CYP2C9*3 allele has much lower catalytic activity as compared to the CYP2C9*2 allele ( Dorado P et al., 2003; Aithal GP et al., 1999 ). For example, a heterozygous variant of CYP2C9*3 has caused a reduction in the clearance of oral S-warfarin in vivo by 66%, whereas the homozygote CYP2C9*3 showed a 90% reduction in warfarin clearance relative to the wild type of the genotype (CYP2C9 *1/*1 genotype)( Takahashi H et al., 2003). CYP2C9 polymorphism has been extensively studied in many major populations including the Caucasian, Russian, African, Turkish, Japanese, Korean, and Tamilian Indian. The mutants CYP2C9*2 and CYP2C9*3 are relatively more frequently found in Caucasians, whereas they are rare in the Chinese and Japanese (García-Martín E et al., 2001), in table IV allele frequencies of the most prevalent alleles are given for the major ethnic groups. Variant CYP2C9*2 is almost absent in African and Asian populations. Besides CYP2C9*2 and *3, many other genetic variants have been described within CYP2C (Lee CR et al., 2002; Dickmann LJ et al., 2003; Kidd RS et al., 2001; Zhao F et al.,2004; Sandberg M, 2004 ) but the frequency of most of these alleles is low or the polymorphisms do not lead to an amino acid exchange (www.imm.ki.se/CYPalleles/CYP2C9.htm).
Table 4. Polymorphic variant of CYP2C9 alleles and their global distribution.
Allele
Nucleotide
changes
Protein
variation
Activity
compared with
CYP2C9*1/*1
Allele frequency
Africans
Asians
Caucasian
CYP2C9*2
430C→T
Arg144Cys
Decrease
4%
0%
11%
CYP2C9*3
1075A→C
Ile359Leu
Decrease
2%
3%
7%
CYP2C9*4
1076T→C
Ile359Thr
-
0%
-
0%
CYP2C9*5
1080C→G
Asp360Glu
Decrease
1.8%
-
0%
CYP2C9*6
818delA
Null allele
No activity
0.6%
-
0%
CYP2C9*7
55C→A
Leu19Ile
-
-
-
-
CYP2C9*8
449G→A
Arg150His
Increase
6.7%
-
-
CYP2C9*9
752A→G
His251Arg
Decrease
-
-
-
CYP2C9*10
815A→G
Glu272Gly
-
-
-
-
CYP2C9*11
1003C→D
Arg335Trp
Decrease
2.7%
-
0.4%
CYP2C9*12
1465C→D
Pro489Ser
Decrease
-
2%
-
Summarized frequency data from (Lee CR et al., 2002; Blaisdell J et al., 2004; Takanashi K et al.,
2000; Haining RL et al., 2004; Xie HG et al., 2002; Bahadur N et al., 2002; Yasar U et al., 2002; Allabi AC et al.,
2003; Scordo MG et al., 2001; Allabi AC et al.,2004.) A hyphen indicates lack of sufficient data. Note that such allele frequencies may even differ within the major ethnic groups.CYP: Cytochrome P450.
CYP2C19 Genetic Polymorphism:
Cytochrome P450 2C19 (CYP2C19) plays an important role in the metabolism and elimination of a wide range of medications such as S-mephenytoin, diazepam, omeprazole, proguanil, citalopram, R-warfarin and many antidepressants ( Bertilsson L et al., 1995). The human CYP2C19 gene is located on chromosome 10. CYP2C19 is a polymorphically expressed enzyme (Goldstein JA, 2001). Thus far, 6 different variants have been identified (Table 5). The most frequent of these genetic polymorphisms are CYP2C19*2, containing a G681A point mutation in exon 5 resulting in a splicing defect, and CYP2C19*3, containing a G636A transition in exon 4, which produces a premature stop codon. The wild-type allele is referred to as *1. CYP2C19*17 is a newly identified allele carrying -806C>T and -3402C>T.
Table 5. Major human polymorphic variant CYP2C19 alleles.
Allele
Variation
Activity
CYP2C19*1A
Reference
Active
CYP2C19*1B
C99T, A991G, Ile331Val
Active
CYP2C19*2A
C99T;G681A;C990T;A991G,Ile331Val.).
Inactive
CYP2C19*2B
C99T;G276C, Glu92Asp;G681A; C990T; A991G; Ile331Val
Inactive
CYP2C19*3
G636A
Inactive
CYP2C19*4
A1G
Inactive
CYP2C19*5A
C99; A991,Ile 331; C1297T,Arg433Trp
Inactive
CYP2C19*5B
C99T; A991G, Ile331Val; C1297 T, Arg433Trp).
Inactive
CYP2C19*6
C99T;G395A, Arg132Gln;A991G, Ile331Val).
Inactive
Summarized data from (Romkes M et al.,1991; Richardson TH et al., 1995; De Morais SMF et a.,1994).
The polymorphisms of this enzyme give rise to substantial inter-individual and inter-ethnic variability (table 6). The frequency of poor metabolizers of CYP2C19 varies between 19-23% among the Japanese population, 15% among the Chinese, and 13% among Koreans, 2-5% in Caucasians and 4% in a Shona population of Zimbabwe. CYP2C19*2 accounts for 75 % of CYP2C19 defective alleles in Orientals, and 93 % in Caucasians. The other well characterized detrimental allele (CYP2C19*3) discovered in Japanese PMs , accounts for approximately 25% of all inactive forms in Orientals, being by converse extremely rare in non-Oriental populations.
Table 6. Frequency of the CYP2C19 alleles in different populations.
Ethnicity
CYP2C19*1
CYP2C19*2
CYP2C19*3
Iranian
86
14
0
Japanese
67
23
10
Filipinos
54
39
7
Chinese-Taiwanese
63
32
5
Asians
62
32
6
Saudi Arabian
85
15
0
African American
75
25
0
European American
87
13
0
Summarized frequency data from (Ozawa S et al., 2004; Goldstein JA et al., 1997)
N-acetyl -transferase (NAT):
NATs are cytosolic enzymes and are found in a large number of tissues. There exist two functional human arylamine N-acetyltransferases, the polymorphic NAT2 (Grant DM et al., 1989) and the formerly named monomorphic NAT1 (Blum M et al., 1990). Both genes have been assigned to chromosome 8p2l.3-23.1(Hickman D et al., 1994) they contain 870-bp intronless protein-coding regions and are separated by 25 ≥ kb. In addition there exists a pseudogene, NATP, which does not encode a functional protein. Both NAT1 and NAT2 genes are known to be polymorphic in humans, corresponding to slow and rapid acetylator phenotypes. Both catalyze the N-acetylation of aromatic amine and hydrazine drugs. While substrates of NAT2 enzyme are isoniazid, sulfamethazine, 2-aminofluorene and 4-aminobiphenyl, NAT1 enzyme has para-aminosalicylic acid (PAS), para-aminobenzoic acid (PABA) and sulfanilamide as substrates. Considerable ethnic variation in the NAT activity has been observed, the slow phenotype ranging from 90% in North Africans to 10%-30% in Japanese and Korean orientals.
NAT1 Genetic Polymorphism:
The polymorphism of NAT1 gene was first described about two decade ago and 26 alleles have been identified in human populations (Hein DW et al., 2000). NAT1*3, NAT1*4, NAT1*5, NAT1*10 and NAT1*11 being the most common alleles reported (table 7). NAT1*4 allele denoted as the wild-type (Vatsis KP and Weber W, 1993). The NAT1*3 allele is probably functionally comparable with the NAT1*4 allele, as the mutation does not cause an amino acid change (Hirvonen A 1999). The recently described NAT1*14 and *17 alleles encode for proteins with reduced acetylation capacity, whereas the NAT1*11 allele is now agreed to be associated with higher NAT1 activity. A single mutation or a combination of multiple nucleotide substitutions and insertions/deletions are responsible for the allelic variants of NAT1.
Table 7. Major human polymorphic variant NAT1 alleles.
NAT1 Allele
Nucleotide Change(s)
Phenotype
NAT1*4
Reference
Reference
NAT1*3
1095C>A
Equivalent to NAT1*4
NAT1*5
350,351G>C 497-499G>C 884A>G.
Unknown
NAT1*10
1088T>A, 1095C>A.
Greater than NAT1*4 "Rapid"
NAT1*11A
344C>T, 40A>T, 445G>A, 459G>A 640T>G, 1095C>A
Greater than NAT1*4 "Rapid"?
NAT1*11B
344C>T, 40A>T, 445G>A, 459G>A 640T>G
Greater than NAT1*4 "Rapid"?
NAT1*11C
-344C>T -40A>T 459G>A 640T>G
1095C>A
Unknownhttp://louisville.edu/medschool/pharmacology/consensus-human-arylamine-n-acetyltransferase-gene-nomenclature/nat_pdf_files/Human_NAT1_alleles.pdf
NAT2 Genetic Polymorphism:
Arylamine N-acetyltransferase 2 (NAT2) is a cytosolic phase II conjugation enzyme which catalyzes the transfer of an acetyl group from acetyl-CoA to the nitrogen or oxygen atom and is responsible for the acetylation of numerous xenobiotics and arylamine or hydrazine containing drugs, including tuberculosis (TB) and acquired immune deficiency syndrome (AIDS)-related therapeutics, such as isoniazid (INH) and sulphonamides, and is expressed predominantly in the liver. The human NAT2 gene is highly polymorphic and represents one of the best studied examples of the large interindividual variability of genetic control of drug or xenobiotic metabolism. Most SNPs (single nucleotide polymorphisms) reported to date are found within the 873 bp intronless coding region of NAT2 gene, which is located in chromosome 8p22. Analysis of polymorphisms at the NAT2 gene locus can identify individuals with rapid, intermediate or slow acetylator phenotypes (table 8). Among seven major different NAT2 mutations, five led to amino acid changes (Deguchi T et al., 1990). Each slow allele contains a combination of one or two nucleotide substitutions that occur at position 481 (C to T; M1 allele), 590 (G to A; M2), 857 (G to A; M3) and191 (G to A; M4) of the gene 8 which has increasingly been recognized as imposing a higher risk of clinically significant health problems. According to the consensus gene nomenclature of human NAT2 (www.louisville.edu/medschool/pharmacology/NAT.html), the NAT2 alleles or haplotypes are characterized by the combination of up to four SNPs present in the coding region. Currently, there is a total of 32 SNPs identified in the NAT2 coding region (25 in the nomenclature official site and the other seven in the Entrez SNP database) with 53 described combinations (alleles). The presence of the NAT2*4 (wild-type) allele defines the NAT2 genotype as rapid and combinations of the frequently occurring mutant alleles NAT2*5B, *5C, *6A, *7B, and *14 cluster as slow.
Table 8. Major human polymorphic variant NAT2 alleles.
NAT2 Allele
Nucleotide Change(s)
Phenotype
NAT2*4
