LEADING ARTICLE

Clin. Pharmacokinet. 23 (5): 321-327, 1992 0312-5963/92/00 I0-0321 /$03.50/0 © Adis International Limited. All rights reserved. CPK1231

Polymerase Chain Reaction and Its Potential as a Pharmacokinetic Tool Markus H. Heim Department of Pharmacology, Biocenter, University of Basel, Basel, Switzerland

1. Pharmacokinetics and Pharmacogenetics Interindividual variability of pharmacokinetic processes such as drug distribution, rates of drug metabolism and elimination results from genetic differences and nongenetic variables including age, sex, liver size, liver function, circadian rhythms, body temperature, as well as nutritional and environmental factors, such as exposure to inducers or inhibitors of drug metabolism. Genetic influence on pharmacokinetics can be either polygenic or of the single-gene type. The latter category can be subdivided into common polymorphisms such as the debrisoquine/sparteine polymorphism or the N-acetyltransferase polymorphism (Meyer et al. 1990) and rare pharmacogenetic traits such as impaired phenytoin metabolism (Inaba 1990) or serum cholinesterase defects (La Du 1989). In the last few years a number of single-gene type pharmacogenetic defects have been investigated on the molecular level and, for some of them, genotyping is already possible (table I). Identification of the 2 alleles of an individual at a specific gene locus is achieved by analysis of restriction fragment length polymorphisms (RFLPs) and with the help of the polymerase chain reaction. Restriction endonucleases recognise specific nucleotide sequences of variable length, known as restriction sites. An average of I in 500 nucleotides differs between 2 randomly selected alleles and, if restriction sites are changed, this variability can be

recognised by one of the known restriction endonucleases (Antonarakis 1989). Alleles may then be identified by characteristic fragments after digestion of genomic DNA with a specific endonuclease. These RFLPs may be generated by a primary mutation causing the altered phenotype or by allelic restriction sites in the flanking DNA that are linked to the primary mutation. RFLPs and their application in DNA diagnostics have been reviewed by Gusella (1986). In the past few years a new method has revolutionised DNA analysis: the polymerase chain reaction (PeR).

2. Polymerase Chain Reaction The polymerase chain reaction is the amplification of selected DNA sequences via repeated cycles of DNA denaturation, the annealing of oligonucleotide primers and their extension by a thermostable DNA polymerase (Erlich et al. 1991 ; Mullis & Faloona 1987; Saiki et al. 1985, 1988). Under appropriate conditions it results in the exponential accumulation of discrete DNA fragments whose termini are defined by the 5' ends of the primers to an amplification level of up to 106_fold the initial target sequence concentration. Selection of the sequence of interest is based on the specific base pairing between the oligonucleotide primers and the target sequence. The amplified DNA fragments may then be analysed by gel electrophoresis, various blotting methods or sequencing.

322

Clin. Pharmacokinet. 23 (5) 1992

Table I. Genotyping of single-gene type pharmacogenetic defects with pharmacokinetic consequences Condition Acetylation polymorphism

Drugs involved

Gene/enzyme

Isoniazid,

NAT2

sulphadiazine Debrisoquine/sparteine polymorphism

Debrisoquine,

CYP2D6

sparteine, bufuralol Mephenytoin polymorphism

Mephenytoin

Unknown

Captopril, 6-

Unknown

Genotyping

Reference

PCR and RFLP

Deguchi et al. (1990)

PCR

Blum et al. (1991)

PCR

Heim & Meyer (1990)

PCR and RFLP

Gough et al. (1990)

Methyltransferase polymorphisms S-methylation

mercaptopurine O-methylation

Norepinephrine

S-catechol-O-

Lundstrom et al.

(noradrenaline),

methyl

(1991)

levodopa, methyldopa

transferase (COMna

Bertocci et al. (1991)

Suxamethonium

S-Cholinesterase b

hCOMTB Suxamethonium sensitivity

PCRc, Dot-blot, RFLP

McGuire et al. (1989) Bartels et al. (1989) Nogueira et al. (1990) La Du (1989)d

a

Mutations not yet identified.

b

Many different mutations.

c

Combined with sequencing of amplified fragments.

d Review article. Abbreviations: PCR

= polymerase chain reaction; RFLP = restriction fragment length polymorphisms.

Since its introduction in 1985, PCR has revolutionised the analysis of genetic diseases and polymorphisms. The genes and their mutated alleles can be amplified from small amounts of genomic DNA and rapidly analysed. Nonselective amplification from both alleles of diploid genomic DNA can be circumvented by an application of a PCR technique called allele-specific amplification. The DNA to be genotyped is amplified once in a reaction containing a primer specific for the normal allele and also amplified in parallel in a reaction containing the mutation-specific primer. The conditions of the reactions are chosen to allow amplification only in the case of a perfect match between template and primer. If only the first reaction results in a amplification product, the sample DNA is concluded to be homozygous for the normal allele, i.e. both alleles are 'normal' or 'wildtype'. In the case of amplification in both reactions, the subject is heterozygous. Homozygotes for the mutated allele show amplification in the second reaction only.

Two important genetic polymorphisms of drug metabolism, the debrisoquine/sparteine oxidation and the N-acetyltransferase polymorphism, have been characterised on the molecular level and allele-specific amplification tests have been recently developed that allow genotyping of patients and study subjects (Blum et al. 1991; Heim & Meyer 1990).

3. Debrisoquine/Sparteine Polymorphism Soon after the development ofa PCR-based genotyping method it became clear that contrary to other single-gene type diseases such as cystic fibrosis or the thalassaemias, where a large number (over 100 in cystic fibrosis) of mutant alleles cause the same disease, only 4 alleles of CYP2D6, the gene coding for the hepatic cytochrome P450IlD6, are responsible for over 95% of the cases of poor metabolisers (PM) of debrisoquine (Heim & Meyer 1990). This unexpectedly small number of mutant alleles was complicated by the existence of a num-

323

PCR and Pharmacokinetics

8P

8P

8P

7(Al9)P

7(AIB)P

7(Al9)P

6WT

?

7(Al9)P

WT

44

WT

29

A

29

9

29

C

44

9

11 .5

0

6WT

6A

-D---D-----D8P

29

69

-D----D-------8P

7(Al9)P

6C

-D------D--D8P

8P

7AP

79P

69

7(AlB)P

-D-----D--

Fig. 1. Summary of known CYP2D gene clusters. The 4 alleles of CYP2D6 designated CYP2D6WT (6WT, wildtype), CYP2D6A (6A), CYP2D6B (6B) and CYP2D6C (6C) exist in various combinations with the pseudogenes CYP2D8P (8P), CYP2D7P (7P), CYP2D7 AP (7 AP) and CYP2D7BP (7BP). The left column shows the 7 combinations characterised to date, which make up more than 95% of the existing gene cluster variants of a European population. 7(A/BjP designates the occurrence of either CYP2D7P, CYP2D7 AP or CYP2D7BP. The middle column shows the genotype of these allelic gene clusters obtained by restriction fragment length polymorphism analysis after enzymatic digestion with the endonuclease Xbal. Designations are derived from the length of characteristic fragments of Il.5kb (II, 5), 29kb (29) and 44kb (44) [Skoda et a!. 1988). The right column indicates the results obtained by allele specific amplification by the polymerase chain reaction (Heim & Meyer 1990). The abbreviations are: WT '" wildtype; A '" mutation A; B = mutation B; C = mutation C. The gene cluster at the bottom lacking the entire CYP2D6 gene results in no amplification in the polymerase chain reaction test (result D).

ber of highly homologous pseudogenes located immediately adjacent to the CYP2D6 gene in the so-called CYP2D gene cluster. The pseudogenes CYP2D7P, CYP2D7 AP, CYP2D7BP and CYP2D8P are found in various combinations with the 4 major alleles of CYP2D6, namely CYP2D6wt (wildtype, functional), CYP2D6A, CYP2D6B and CYP2D6C (Heim & Meyer 1992). These different combinations of genes at the same locus represent allelic gene clusters (fig. I). The widely-used genotyping method by restriction fragment length polymorphism (RFLP) of these gene clusters has designated these alleles according to characteristic fragments after enzymatic digestion with the endonuclease XbaI as l1.5kb, 29kb and 44kb 'alleles' of the CYP2D6 gene, at that time called P450db I (Skoda et al. 1988). This terminology is now confronted with the new designations of the PCR genotyping method based on well characterised mutations in the CYP2D6 gene (Kagimoto et al. 1990; Tyndale et al. 1991). The replacement of the 'RFLP' terminology by the 'PCR' terminology is hampered by the existence of the allelic cluster designated' 11.5kb allele' in the former and CYP2D6D (D for deletion) in the latter nomenclature. This allelic cluster lacks the entire CYP2D6 gene (Gaedigk et al. 1991), and therefore yields no amplification product in the PCR method. It has been claimed that lack of amplification in a PCR-based assay can identify the 11.5kb allele (Gough et al. 1990; Nigel et al. 1991; Wolf et al. 1990). We hesitate to consider lack of amplification as an unequivocal proof for a deletion, and certainly in heterozygous situations the CYP2D6D or l1.5kb allele cannot be identified by the published PCR methods. The 'gold standard' for its identification remains the RFLP method published in 1988 (Skoda et al. 1988). Users ofPCR methods for genotyping poor metabolisers of debrisoquine must also be aware of the over 90% identity in nucleotide sequences between CYP2D6 and its pseudogenes and the presence of several of the mutations of the CYP2D6alleles CYP2D6A and CYP2D6B in the pseudogenes CYP2D7 AP and CYPD27BP (Heim & Meyer 1992). The tests therefore should be well controlled

Clin. Pharmacokinet. 23 (5) 1992

324

for false positive results derived from the pseudogenes. One possible strategy, generally known as the nested primer strategy, is a 2-step procedure with, in the case of the debrisoquine/sparteine polymorphism genotyping, the use of primers complementary to specific intron sequences of CYP2D6 in a first amplification and the use of mutationspecific primers in a second reaction (Heim & Meyer 1991). Using this method it has been shown that the phenotype of 100% of the extensive metabolisers (EM) and of 86% of the PMs was correctly predicted in a study group of 396 healthy European subjects (Broly et al. 1991). Combined with RFLP analysis for the l1.5kb allele, over 90% of individuals with the PM phenotype could be identified (Broly et al. 1991). In a recently published study on 199 White Swedish subjects using this allele-specific amplification assay a prediction rate of 100% for EMs and 96.9% for PMs was reported (Dahl et al. 1992).

1991). So far 3 mutated alleles have been identified by 3 independent groups (fig. 2) [Blum et al. 1991; Deguchi et al. 1990; Kostas et al. 1991]. An allelespecific amplification method allows the identification of these alleles in a small sample of genomic DNA and predicted over 93% ofthe phenotypes in an initial study group of 46 subjects (Blum et al. 1991). In a recently published study, the same method predicted all of 48 rapid acetylators and 33 of 35 slow acetylators (Graf et al. 1992). A slightly different genotyping method based on PCR and restriction endonuclease digests with KpnI, TaqI and BamHI was reported to have predicted correctly 10 of 11 slow acetylators and all of II fast acetylators (Hickman & Sim 1991). The existence of the closely related NAT! with 87% nucleotide identity requires PCR amplification of NAT2 to be controlled for incidental amplification from this gene with a single coding exon of 870 nucleotides, exactly the same length as NAT2 (Blum et al. 1990). HindIII is a restriction endonuclease that cuts exactly at the palindromic hexanucleotide sequence AAGCTT. A HindIII restriction site in NATI that has not been found in any of the known

4. Acetylation Polymorphism The acetylation polymorphism is caused by mutations in the single coding exon of the gene for Nacetyltransferase designated NA T2 (Blum et al. T

C

I 282

341

C

G

G

I

I

I

481

690

957

C

T

I

I

Ml

Fig. 2. Summary of the 4 N-acetyltransferase NAT2 alleles. The wildtype allele of NAT2 is shown on top. Numbers indicate the nucleotide positions (position I = A in the initiator ATG triplet) of the bases partially mutated in the mutant alleles MI, M2 and M3.

peR and Pharmacokinetics

alleles of NA T2 allows digestion of NA Tl amplification products. NAT2-specific PCR amplification should not result in any DNA fragments that can be cleaved by the endonuclease HindIII. As in the case of the debrisoquine/sparteine polymorphism it appears that a small number of mutated alleles accounts for 95% of all mutated alleles. PCR-based genotyping methods therefore can be used for phenotype prediction in population studies and clinical practice. No larger studies have been published so far, however, and conclusions about the sensitivity and specificity of this method cannot yet be made.

5. Other Pharmacogenetic Defects of Pharmacokinetic Interest Several variants of human serum cholinesterase have been analysed and for some ofthem the mutations causing impaired metabolism of succinylcholine have been identified (La Du 1989). Genotyping methods are not evaluated so far in a general population. The genes of the enzyme catechol-O-methyltransferase have been cloned and sequenced recently (Bertocci et al. 1991; Lundstrom et al. 1991). The mutations causing impaired O-methylation of catecholamines and catechol drugs such as levodopa and methyldopa (Weinshilboum 1989) are not yet characterised. No genotyping method has been described. The molecular mechanism of deficient mephenytoin hydroxylation (Kupfer et al. 1984) is not known.

6. Conclusions Two of the clinically important genetic polymorphism in drug metabolism, the debrisoquine/ sparteine oxidation polymorphism and the acetylation polymorphism, have been elucidated on the molecular level and simple and rapid genotyping assays based on the PCR/allele-specific amplification method are now available. Genotyping needs a small amount of genomic DNA and circumvents the need for administration of test drugs, collection

325

of urine and determination of the ratio between parent drug and its metabolite, and the pitfalls of phenotyping, i.e. adverse drug reactions, drug interaction and confounding effects of diseases. In a study of the National Cancer Institute designed to investigate the link between lung cancer and the debrisoquine/sparteine polymorphism, over 40% of eligible subjects had to be excluded because of limitations of the phenotyping procedure (Caporaso et al. 1990). Genotyping can predict the likelihood of normal or impaired metabolism in a given individual, but not the actual metabolic capacity, as usually determined by the urinary metabolic ratio. Thus, genotyping makes no distinction between a 'rapid' EM of debrisoquine with a urinary metabolic ratio of debrisoquine/4-hydroxydebrisoquine of 0.1 and a 'slow' EM with a ratio of 10 who is close to a 'rapid' PM with a ratio of 15. Depending on the questions asked in a study, the appropriate method can be genotyping, phenotyping or both. In clinical situations where determination of the acetylator or the debrisoquine/sparteine oxidation phenotype is recommended for individualisation of dosage (Meyer 1991) the urinary metabolic ratio is more informative for the design of a drug regimen of a substance with underlying metabolic elimination by one of the polymorphic enzymes described above. Phenotyping, however, is not possible in a patient who is already taking a drug metabolised by the same enzyme as the test drug or that inhibits the enzyme. In the case of a psychiatric patient treated with a tricyclic antidepressant (i.e. amitriptyline) or a neuroleptic (i.e. thioridazine), phenotyping by debrisoquine or sparteine will underestimate the metabolic capacity because of competitive interaction of the 2 drugs (Brosen & Gram 1989; Spina et al. 1991). Phenotyping and genotyping of slow and fast acetylators and of poor and extensive metabolisers of debrisoquine are complementary methods that allow study ofthe influence of these important genetic polymorphisms in drug metabolism in such diverse fields as the development of new drugs, the epidemiology of cancer and chemically-induced diseases, monitoring of adverse drug reactions, the

326

interethnic differences of drug effects and last but not least, clinical practice.

Acknowledgement This work was supported by Grant 32-31266.91 from the Swiss National Science Foundation. I thank Dr U.A. Meyer for reviewing the manuscript.

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Gusella JF. DNA polymorphism and human disease. Annual Review of Biochemistry 55: 831-854, 1986 Heim M, Meyer VA. Genotyping of poor metabolisers of debrisoquine by allele-specific PCR amplification. Lancet 336: 529532, 1990 Heim MH, Meyer VA. Genetic polymorphism of debrisoquine oxidation: analysis of mutant alleles ofCYP2D6 by restriction fragment analysis and by allele specific amplification. Methods in Enzymology 206: 173-183, 1991 Heim MH, Meyer VA. Evolution of a highly polymorphic gene locus for a drug metabolizing enzyme. Genomics, in press, 1992 Hickman 0, Sim E. N-acetyltransferase polymorphism: comparison of phenotype and genotype in humans. Biochemical Pharmacology 42: 1007-1014, 1991 Inaba T. Phenytoin: pharmacogenetic polymorphism of 4'-hydroxylation. Pharmacology and Therapeutics 46: 341-347, 1990 Kagimoto M, Heim M, Kagimoto K, Zeugin T, Meyer VA. Multiple mutations of the human cytochrome P450IID6 gene (CYP2D6) in poor metabolizers of debrisoquine. Journal of Biological Chemistry 265: 17209-17214, 1990 Kostas PV, Martell KJ, Weber WW. Diverse point mutations in the human gene for polymorphic N-acetyltransferase. Proceedings of the National Academy of Science of the Vnited States of America 88: 6333-6337, 1991 Kiipfer A, Desmond P, Patwardhan R, Schenker S, Branch RA. Mephenytoin hydroxylation deficiency: kinetics after repeated doses. Oinical Pharmacology and Therapeutics 35: 33-39, 1984 La Du BN. Identification of human serum cholinesterase variants using the polymerase chain reaction amplification technique. Trends in Pharmacological Sciences 10: 309-313, 1989 Lundstrom K, Salminen M, Jalanko A, Savolainen R, Vlmanen I. Cloning and characterization of human placental catecholO-methyltransferase cDNA. DNA and Cell Biology 10: 181189,1991 McGuire MC, Nogueira CP, Bartels CF, Lightstone H, Hajra A, et al. Identification of the structural mutation responsible for the dibucaine-resistant (atypical) variant form of human serum cholinesterase. Proceedings of the National Academy of Science of the Vnited States of America 86: 953-957, 1989 Meyer VA. Clinical importance of genetics in drug effects. In Melmon & Morelli (Eds) Clinical pharmacology: basic principles of therapeutics, 3rd ed., Pergamon Press, Oxford, 1991 Meyer VA, Zanger VM, Grant 0, Blum M. Genetic polymorphisms of drug metabolism. In Testa (Ed.) Advances in drug research, Vol. 19, pp. 197-241, Academic Press Limited, london, 1990 Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction. Methods in Enzymology 155: 335-350, 1987 Nigel KS, Gough AC, Smith CAD, Wolf CR. Genetic analysis of cytochrome P450 system. Methods in Enzymology 206: 149166,1991 Nogueira CP, McGuire MC, Graeser C, Bartels CF, Arpagaus M, et al. Identification of a frameshift mutation responsible for the silent phenotype of human serum cholinesterase, Gly 117 (GGT-GGAG). American Journal of Human Genetics 46: 934-942, 1990 Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, et al. Enzymatic amplification of {J globin genomic sequences and restriction site analysis for the diagnosis of sickle cell anemia. Science 230: 1350-1354, 1985 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491, 1988 Skoda RC, Gonzalez FJ, Demierre A, Meyer VA. Two mutant alleles of the human cytochrome P45Odbl-gene (P45OC2D1) associated with genetically deficient metabolism of debrisoquine and other drugs. Proceedings of the National Academy of Science of the Vnited States of America 85: 5240-5243, 1988

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Wolf CR, Moss JE, Miles JS, Gough AC, Spurr NK. Detection of debrisoquine hydroxylation phenotypes. Lancet 336: 14521453, 1990

Correspondence and reprints: Dr Markus H. Heim. Departement [nnere Medizin, Kantonsspital Basel, Petersgraben 2, 4051 Basel, Switzerland.

XIIth International Congress of Pharmacology

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