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Dassel, Germany) followed by elution with 3 M NaC1 or treatment of the PCR product with exonuclease I and shrimp alkaline phosphatase (PCR sequencing kit, Amersham, Bucks, UK) results in a template that gives good quality sequencing data with Sequenase II and 35S-dATP.

[23] G e n e t i c T e s t s W h i c h I d e n t i f y t h e P r i n c i p a l D e f e c t s i n CYP2C19 Responsible for the Polymorphism in Mephenytoin Metabolism By JOYCE A. GOLDSTEINand JOYCE BLAISDELL Introduction A genetic polymorphism in the metabolism of the anticonvulsant drug S-mephenytoin has been studied extensively in humans, m In population studies, individuals can be divided into two phenotypes, extensive metabolizers (EMs) and poor metabolizers (PMs) of mephenytoin. There are large interracial differences in the frequency of the PM phenotype, with Oriental populations having a five-fold greater frequency (13-23%) compared to Caucasians (3-5%). PMs are deficient in the ability to 4'-hydroxylate the S-enantiomer of mephenytoin, and this results in a slower rate of metabolism of the racemic mixture. This polymorphism also affects the metabolism of a number of other clinically used drugs, including omeprazole, proguanil, citalopram, barbiturates, and, to a somewhat smaller extent, that of propranolol, certain tricyclic antidepressants, and diazepam. The enzyme responsible for the metabolism of mephenytoin has been identified as CYP2C19. 3"4 Recently, we have identified the two principal genetic defects in CYP2C19 that are responsible for the PM phenotype in humansfi '6 The primary defect producing the PM phenotype is a single
1 G. R. Wilkinson, F. P. Guengerich, and R. A. Branch, Pharmacol. Ther. 43, 53 2 j. A. Goldstein and S. M. F. de Morais, Pharmacogenetics 4, 285 (1994). 3 j. A. Goldstein, M. B. Faletto, M. Romkes-Sparks, T. Sullivan, S. Kitareewan, J. J. M. Lasker, and B. I. G h a n a y e m , Biochemistry 33, 1743 (1994). 4 S. A. Wrighton, J. C. Stevens, G. W. Becker, and M. V a n d e n B r a n d e n , Arch. Biophys. 306, 240 (1993). 5 S. M. F. de Morais, G. R. Wilkinson, J. Blaisdell, K. N a k a m u r a , U. A. Meyer, Goldstein, J. Biol. Chem. 269, 15419 (1994). 6 S. M. F. de Morais, G. R. Wilkinson, J. Blaisdell, U. A. Meyer, K. N a k a m u r a , Goldstein, Mol. Pharmacol. 46, 595 (1994). (1989). L. Raucy,

Biochem.
and J. A. and J. A.

METHODS IN ENZYMOLOGY,VOL. 272

Copyright 1996by AcademicPress, Inc. All rights of reproduction in any form reserved.

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G ~ A base pair mutation in exon 5 of CYP2C19 (CYP2C19mD which produces an aberrant splice site. This splice site appears to be used exclusively in PMs metabolizers carrying this mutation. The aberrantly spliced m R N A lacks the first 40 bp of exon 5, producing a premature stop codon and a truncated 234 amino acid protein lacking the heme-binding region. This mutation also destroys a SmaI site. Polymerase chain reaction ( P C R ) restriction-based genotyping tests have shown that CYP2C19ml accounts for - 7 5 - 8 3 % of the defective alleles in both Oriental and Caucasian PMs. A second major mutation (CYP2C19m2) was subsequently identified in a Japanese PM consisting of a G ~ A mutation at position 636 of exon 4 of CYP2C19 which creates a premature stop codon. This mutation also destroys a BamHI site. This defect appears to account for the remaining defective alleles in Oriental poor metabolizers but is rare in Caucasians. This chapter describes the PCR-restriction tests for the detection of CYP2C19ml and CYP2C19m2.

General Method We recently described PCR-based restriction enzyme tests for the analysis of both CYP2C19ml and CYP2C19m2 defects. 5-7 In this chapter, we have modified the original procedures slightly by selecting more specific CYP2C19 primers (given below) for the amplification of both exon 5 (CYP2C19ml) and exon 4 (CYP2C19m2). The present primers decrease the possibility of amplifying closely related CYP2C sequences which complicate the interpretation of results. The method consists of two steps (Fig. 1). In the first step, two CYP2C19-specific pairs of primers are used to amplify exons 4 and 5 of CYP2C19, respectively. In the second step, the PCR fragments from the amplification of exon 5 are digested with Sinai which digests the normal or wild-type (CYP2C19,,) sequence but does not digest the CYP2C19mlallele. Similarly, the PCR fragments from the amplification of exon 4 are digested with BamHI which digests the normal sequence, but does not digest the CYP2C19m2 allele. It should be noted that the digestion of the PCR products of exon 5 by Sinai does not distinguish between the normal and the mutant CYP2C19me alleles since this portion of the sequence is normal in CYP2C19m2. Similarly, the digestion of exon 4 PCR products by BamHI does not distinguish between the wild-type and the CYP2CI9ml alleles. The final assignment of genotype requires the comparison of digestion patterns for both sets of PCR products.

7j. A. Goldstein and S. M. F. de Morais, U.S. Patent Appl. USSN08/238,821 (1994).

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1231

Nomenclature
We have referred to the normal CYP2C19 allele as CYP2C19,~t, the allele containing a base pair mutation that creates an aberrant splice site in exon 5 as CYP2C19ml, and the allele containing a G---~ A base mutation at position 636 producing a stop codon in exon 4 as CYP2C19m2. If additional defective alleles are discovered, these can be numbered consecutively beginning with CYP2C19,n3.

Oligonucleotide Primers
Primers were synthesized on an Applied Biosystems (Foster City, CA) synthesizer. Primer positions in introns are stated relative to the exons, and positions in CYP2C19 exons are stated relative to the translational start site of the cDNA.

Primers for ml Defect (Exon 5) Reaction A

Primer i (forward) 5 ' - C A G A G C T T G G C A T A T T G T A T C - 3 ' from 71 to 51 bases upstream of exon 5 in intron 4 Primer 2 (reverse) 5 ' - G T A A A C A C A C A A C T A G T C A A T G - 3 ' from 52 to 73 bases downstream of exon 5 in intron 5

Primers for m2 Defect (Exon 4) Reaction B

Primer 3 (forward) 5 ' - A A A T T G T T T C C A A T C A T T T A G C T - 3 ' from 21 bases upstream of exon 4 in intron 3 to base 2 in exon 4 (position 483 in the c D N A ) Primer 4 (reverse) 5 ' - A C T F C A G G G C T F G G T C A A T A - 3 ' from 70 to 89 bases downstream of exon 4 in intron 4

Stock Solutions 10 PCR Buffer. 0.67 M Tris-HC1, p H 8.8, 0.166 M ammonium sulfate, 0.05 M 2-mercaptoethanol, 67 /zM E D T A , and 0.8 mg/ml bovine serum albumin. This is aliquoted in sterile microfuge tubes and stored at - 2 0 . dNTP Mix. 2.5 m M dATP, 2.5 m M dCTP, 2.5 m M dGTP, and 2.5 m M d T T P at p H 7.0 (prepared from equal volumes of 10 m M of each dNTP from USB, Cleveland, OH, and stored frozen at -20). Enzymes. The following enzymes may be purchased from several sources. We have used Taq polymerase from USB (Cat. No. 71085) and SmaI(lO units//xl) and BamHI(20 units//zl) from New England Biolabs (Beverly, MA).

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TABLE I
FINAL CONCENTRATION OF REAGENTS IN PCR REACTIONS

Reagent PCR buffer dNTP mix MgCI2 Forward primer Reverse primer Sterile water to volume Taq polymerase

Final concentration or amount 1 100/xM each 2 mM 0.25/xM 0.25/xM 0.25 units//,1

6 Stop Buffer. 30% glycerol, 0.75% sodium dodecyl sulfate, 0.75% bromophenol blue. DNA. Genomic D N A can be prepared by different methods. We have used D N A isolated from peripheral leukocytes by standard methods. 8 However, for simplicity, we routinely purify genomic D N A for PCR from 200/xl of whole blood using the Qiamp blood kits (Cat. No. 29104, 29106) (Qiagen Inc., Chatsworth, CA) according to the manufacturer's recommendations. This yields D N A at a predicted concentration of - 3 0 ng//xl, and we use 2 - 5 / x l directly in a 20-/,1 PCR reaction. Procedure
Two sets of sterile microfuge tubes are set up for each sample of D N A s to be tested: one for reaction A (CYP2C19ml or ml) and one for reaction B (CYP2C19m2 or m2). To each tube the following is added: 2/,1 10 PCR buffer 0.8/xl dNTP mix (2.5 mM each) 1.6/xl 25 mM MgC12 0.5/xl forward primer (10 pmol//xl) 0.5/xl reverse primer (10 pmol//,l) 40 ng (20-150 ng) genomic D N A Sterile water to a final volume of 19.9/xl 0.1 ixl Taq polymerase (5 U//xl) Final volume of 20/xl The appropriate concentration of reagents is also indicated in Table I for ease in use with PCR machines requiring larger volumes. 8j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd Ed., p. 9.16. Cold Spring Harbor Lab., Cold Spring Harbor, NY, 1989.

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Tubes are then placed in the thermocycler. All conditions given are for 20/zl volumes using a GenAmp PCR System 9600 (Perkin Elmer Corp., Norwalk, CT) and it may be necessary to modify the times for thermocyclers using larger volumes. For both the m l and m2 PCR reactions, the initial denaturation is allowed to proceed at 94 for 5 min, then 37 cycles are performed with denaturation at 94 for 20 sec, annealing at 53 for 10 sec, and extension at 72 for 10 sec. The products are then extended for an additional 5 min at 72. Digestion Conditions. To test for the m l defect, add 1/xl SmaI (10 units/ /xl) to the entire 20-/zl PCR reaction A and incubate at room temperature for 3 hr (or overnight). To test for the m2 defect, add 1 /xl BamHI (20 units//zl) directly to the 20-/xl PCR reaction B and incubate at 37 for 3 hr (or overnight). Add stop buffer (4.2/xl) to both reactions, and analyze by gel electrophoresis on a 4% agarose gel containing ethidium bromide.

Interpretation of Results
The products of the PCR reaction A for exon 5 (ml defect) should yield a single band of 321 bp which will be digested completely to two smaller bands at 109 and 212 bp by SmaI in individuals who are homozygous for the normal (wild-type)(wt) sequence as shown in Fig. 1. The 321-bp fragment will not be digested in individuals homozygous for the m l defect. In contrast, DNA from individuals heterozygous for this allele will be partially digested to produce three bands of 321,212, and 109 bp. The products of the PCR reaction B for exon 4 (m2) will yield a single 271-bp fragment. This product will be completely digested to two bands of 175 and 96 bp in individuals homozygous for the wt sequence. The 271-bp fragment will not be digested in individuals homozygous for the m2 defect, but will be partially digested to produce three fragments of 96, 175, and 271 bp in individuals who are heterozygous for the m2 defect. The results of both the A and B amplification/digestions must be taken into account to determine the identity of the two alleles. Figure 2 shows an example of the restriction patterns expected for the six known genotypes. Individuals homozygous for m l / m l should show only the undigested 321bp fragment in reaction A. The presence of three bands of 321,212, and 109 bp indicates that an individual carries one m l allele, but does not indicate whether the remaining allele is normal or m2. Similarly, the complete digestion of the 321-bp fragment to 212 and 109 bp means that the individual does not carry the m l mutation, but the results of the second reaction are required to determine whether the individual is wt/wt, wt/m2, or m2/m2. In reaction B, individuals who are homozygous for m2 will show only one 271-bp band after digestion with BamHI. However, PCR products

[231

GENOTYPING MEPHENYTOIN-POORMETABOLIZERS OF
Sma[ ~

215

A
2C19wt Intron4

~n5 3'

Sma I Digestion
Wt/wt wt/ml ml/rnl ~1
~

1109bpI

212bp I

*- 321 bp
,.- 212 bp *- 109 bp

2C19ml

Intron4 5' I

~ t

Intron5
3' ~BI

321 bp

B
2C19wt 5" - - t
I

In.n3

EXON 4 I

BamH I I
I

Intron4

BamH I Digestion
3'

,,I,.
175 bp

4.
96 bp

Wt/wt

Wt/m2 m2/m2
_

I ~ 175 bp 96 bp

2C19m2

~;"

Intron3

I EXON 4 J

Intron4

3'

,,I,,
271 bp

4,

FIG. 1. Strategy used to genotype human genomic DNA utilizing PCR amplification of exon 5 followed by SmaI digestion for CYP2C19ml (A) and PCR amplification of exon 4 followed by BamHI digestion for CYP2C19m2 (B). The predicted sizes of the digested DNA fragments for the various genotypes are shown. CYP2C19wtindicates the PCR fragment from the normal (wild-type) gene.

f r o m individuals w h o have one copy of this defective allele will show three bands of 271, 175, and 96 bp. P C R p r o d u c t s f r o m individuals w h o are wt/wt will yield completely digested products in b o t h reactions, but those f r o m m l / m l or m l / w t individuals will also yield completely digested products in this reaction due to the fact that the B a m H I site is present in the m l allele.

Troubleshooting Suggestions
A blank containing no D N A should always be run with each P C R reaction. T h e a p p e a r a n c e of P C R products in the blank indicates p r o b a b l e c o n t a m i n a t i o n of one of the reagents with D N A . S t a n d a r d reactions con-

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DETERMINATION OF GENOTYPE
wt wt

[231

ml ml

wt

~_

ml Exon 5 Sma I

400k_ 300~ 200r 100

50~

<-- 321 bp <-- 212 I~)

~-- 109 bp

m2 Exon 4 BamH I

3oo~..
200~

100/,50

<'- 271 bp 1751~

,,E- 96 lop

FI6.2. Ethidium bromide-stained agarose gel showing PCR-restriction enzyme fragmentation patterns for CYP2C19,nl (top) and CYP2C19m2 (bottom) from individuals representing the six possible genotypes (wt/wt, wt/ml, ml/ml, ml/m2, m2/m2). The size of the molecular weight markers (MW) is shown on the left, and the sizes of the PCR-restriction fragments on the right.

taining five samples of known genotype, wt/wt, wt/ml, m l / m l , m2/wt, and m2/m2, are recommended with each PCR reaction. The wild-type genotypes should be digested completely. Incomplete digestion of the wildtype allele may result from excess DNA, insufficient enzyme/digestion time, or inappropriate salt concentrations in the restriction reaction. In heterozygotes, the intensity of the upper band should be brighter than that of the lower two bands. A higher intensity of the lower two bands in certain samples may suggest incomplete digestion and can occur if the concentration of DNA in the sample was too high or insufficient time was given for complete digestion. If incomplete digestion is suspected, the analysis should be repeated with a longer digestion time. Inappropriate annealing temperatures may lead to contamination of PCR products with other CYP2C fragments which could lead to the apparent partial digestion of m l / m l or m2/m2 genotypes. BamHI can give star activity (cleavage of sequences similar but not identical to its recognition sequence) at high concentrations of enzyme. This generally results in digestion of the 175-bp fragment to two pieces of 32 and 143 bp. Therefore, we recommend using low amounts of this enzyme and a longer 3-hr or overnight digestion time to avoid the appearance of spurious PCR products. The absence of PCR products in a sample may suggest inappropriate amounts of DNA or impurity of the sample. In this case, it is suggested

[23]

GENOTYPING MEPHENYTOIN-POORMETABOLIZERS OF

217

that the D N A concentration in the P C R reaction can be increased or the cycle n u m b e r can be increased to 40. Finally, we have found that treatment of the D N A with G e n e R e l e a s e r (BioVentures, Inc.) enhances the amplification of impure D N A samples. The P C R reaction can be p e r f o r m e d on whole blood treated with GeneReleaser. We have treated 1 /A blood or impure D N A with 10/~1 of G e n e R e l e a s e r according to the manufacturer's instructions and scaled up the P C R reactions to 50 t~l. When the P C R reactions are finished, we spin down the pellet and remove 20/~1 before digestion. However, we generally use D N A purified using a Q i a m p blood kit or other standard purification methods.

Sensitivity and Specificity of the Tests

In our experience to date, P C R tests for CYP2C19mI and CYP2C19m2 correctly predict 100% of the defective alleles in 65 Oriental P M s . 5'6'9'10 In Caucasians, CYP2C19ml accounts for - 8 7 % of defective alleles in 30 unrelated PMs while the CYP2C19m2 has been found in a single Caucasian PM (1/60 alleles.) 5,6,1'll All 128 Caucasian and 158 Oriental EMs contain at least one normal copy of the gene. Moreover, all individuals identified as PMs genotypically were also phenotypic PMs, indicating that the specificity of the tests for identification of PMs is 100%. However, the sensitivity of the tests in identifying PMs is - 1 0 0 % in Orientals, but somewhat lower ( - 8 0 % ) in Caucasians. This suggests that other as yet unidentified rare mutations may contribute to the PM phenotype in Caucasian individuals. It should be noted that the interpretation of phenotyping tests is not always unequivocal. PMs have been characterized phenotypically by a high urinary S/R ratio of >1.0 or by the excretion of low amounts of 4'-hydroxymephenytoin in the urine after a 100-mg dose (e.g., hydroxylation indices). 1,12 However, the prediction of phenotype is complicated by the presence of an acid-labile metabolite of S-mephenytoin in the urine of some EMs, which can hydrolyze on storage to produce a high S/R ratio and the improper designation of phenotype in some EMs. Therefore, the urinary S/R ratios of any outliers from genotype/phenotype comparisons should be repeated before and after acidification of the urine if possible. Lack of compliance with some individuals failing to take the dose of mephenytoin or incomplete urine collection can also lead to problems of interpretation. 9 S. M. F. de Morais, J. A. Goldstein, H.-G. Xie, S.-L. Huang, Y. P Lu, H. Xia, Z.-S. Xiao, N. Ile, and H. H. Zhou, Clin. Pharmacol. Ther. 58, 404 (1995). ~0S. M. F. de Morais, D. A. Price Evans, P. Krahn, K. Chiba, T. Ishizaki, and J. A. Goldstein, unpublished data. J1 K. BrCsen, S. M. F. de Morais, U. A. Meyer, and J. A. Goldstein, Pharmacogenetics 5, 312 (1995). 12p. j. Wedlund and G. R. Wilkinson, Methods EnzymoL 272, Chap. 11, 1996 (this volume).

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Acknowledgments
The authors acknowledge the work of Dr. Sonia M. F. de Morais in this laboratory which led to the identification of the defects responsible for this polymorphism. The authors also thank Dr. Douglas A. Bell and Dr. Masahiko Negishi, NIEHS, for reviewing this manuscript and for helpful suggestions.

[24] G e n e t i c P o l y m o r p h i s m o f H u m a n P450 2El

Cytochrome and

By V E S S E L A

N E D E L C H E V A , IRENE PERSSON,

MAGNUS INGELMAN-SUNDBERG

Introduction Ethanol-inducible cytochrome P450 (CYP2E1) is constitutively expressed in liver and in many other tissues. The substrate specificity is broad and includes at present at least 80 different characterized substrates, mainly small and hydrophobic substances. Among them are ethanol and other aliphatic alcohols, aldehydes like acetaldehyde, alkanes, most organic solvents, putative carcinogenic N-rlitrosoamines, aromatic heterocyclic compounds, halogenated anesthetics, ethers, aromatic hydrocarbons, fatty acids, acetone, and caffeine. 1 The enzyme is induced by a variety of different chemicals, mainly substrates, and the induction in these cases is to a major extent mediated at the posttranslational level, a It is evident that CYP2E1 plays an important toxicological role. It activates precarcinogens, organic solvents, and drugs to cytotoxic or carcinogenic products that might be harmful and of importance for the synergistic effect of ethanol on many types of human cancer as well as the potentiation of solvent and drug toxicity. 1'2 Furthermore, it has the ability to effectively reduce dioxygen with the subsequent formation of reactive oxy radicals that can initiate lipid peroxidation processes. 3 This reaction is implicated as being of importance in the etiology of alcohol liver disease. 4 In addition,
1 M. J. J. Ronis, K. O. Lindros, and M. Ingelman-Sundberg, in "Cytochromes P450: Pharmacological and Toxicological Aspects" (I. Ioannides, ed.). CRC Press, Boca Raton, PL, 1996 (in press). 2 y . Terelius, K. O. Lindros, K. O. E. Albano, and M. Ingelman-Sundberg, in "Frontiers of Biotransformation" (H. Rein and K. Ruckpaul, eds.), Vol. 8, p. 187. Akademie-Verlag, Berlin, 1992. 3 G. Ekstr6m and M. Ingelman-Sundberg, Biochem. Pharrnacol. 38, 1313 (1989). 4 M. Ingelman-Sundberg, I. Johansson, H. Yin, Y. Terelius, E. Eliasson, P. Clot, and E. Albano, Alcohol 10, 447 (1993). Copyright 1996by AcademicPress, Inc. All rights of reproductionin any form reserved.

METHODS IN ENZYMOLOGY,VOL. 272