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A novel genotyping algorithm for the CYP2D6*10 allele in Asians using real-time, rapid-cycle PCR and… Kwong, Evan Holden 2004

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A NOVEL GENOTYPING ALGORITHM FOR THE CYP2D6*10 ALLELE IN ASIANS USING REAL-TIME, RAPID-CYCLE PCR AND MULTIPLEX PCR by Evan Holden Kwong B.Sc. (Pharm.), University of British Columbia, 2002 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES 'Pharmaceutical Sciences We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 2004 © Evan Holden Kwong, 2004 Library Authorization In presenting this thesis in partial fulfil lment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his o r h e r representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Evan Holden Kwong 28/07/2004 Name of Author (please print) Date (dd/mm/yyyy) Title of Thesis: A novel genotyping algorithm for the CYP2D6*10 allele in Asians using real-time, rapid-cycle PCR and multiplex PCR Degree: Master of Science Year: 2004 Department of Faculty of Pharmaceutical Sciences The University of British Columbia Vancouver, BC Canada A B S T R A C T Purpose: The CYP2D6*10 allele (C188T) is common among Asians and is associated with decreased metabolism of various CYP2D6 substrates. Based on reported allele frequencies among Asians, to accurately genotype patients for CYP2D6*10, it is necessary to rule out CYP2D6H (C188T, G1934A) and CYP2D6*5 (gene deletion) before inferring the presence of CYP2D6*wt (CYP2D6*1 or CYP2D6*2; C at position 188). The objectives of this project were to develop and validate genotyping methods for detecting the C188T and G1934A single nucleotide polymorphisms (SNPs) and CYP2D6*5, to use these methods to genotype Asian subjects from a pilot clinical study, and to devise a genotyping algorithm for CYP2D6*10 in Asians. Methods: Long PCR was used to amplify the CYP2D6 gene. Nested real-time, rapid-cycle polymerase chain reaction (PCR) methods for detecting the C188T and G1934A SNPs were developed and validated by restriction fragment length polymorphism (RFLP) and sequence analyses of reference samples with genotypes CYP2D6*1/*1, CYP2D6*l/*4, and CYP2D6*4/*4. A multiplex PCR method for detecting CYP2D6*5 using published primer sequences was optimized and validated by analyzing reference samples with genotypes CYP2D6*1/*1, CYP2D6*l/*5, CYP2D6*5/*5. These methods were used to genotype Asian subjects from a pilot clinical study. Results: C188T and G1934A genotyping results using nested real-time, rapid-cycle PCR were consistent with results from RFLP and sequence analyses, as well as the genotypes of ii the reference samples. CYP2D6*5 genotyping results were also in agreement with the genotypes of the reference samples. CYP2D6 genotype frequencies were determined in 36 Asian subjects: CYP2D6*10/*10 (50.0%; n=18), CYP2D6*wt/*10 (33.3%; n=12), CYP2D6*wt/*wt (11.1%; n=4), and CYP2D6*wt/*5 (5.6%; n=2). Conclusions: The novel genotyping algorithm for identifying Asian patients with the CYP2D6*wt/*wt, CYP2D6*wt/*10, and CYP2D6*10/*10 genotypes involves the use of: 1) long PCR to amplify the CYP2D6 gene; 2) nested real-time, rapid-cycle PCR to detect the C188T SNP (CYP2D6*10 or CYP2D6*4); 3) nested real-time, rapid-cycle PCR to detect the G1934A SNP (CYP2D6*4) in those carrying a C188T SNP; and 4) multiplex PCR to detect CYP2D6*5 in those who appear homozygous for C at 188 (CYP2D6*wt/wt or CYP2D6*wt/*5) or T at 188 (CYP2D6*10/*10 or CYP2D6*10/*5). This algorithm can be used in future clinical studies that require genotyping Asians with respect to CYP2D6*10. iii T A B L E O F C O N T E N T S ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES viii LIST OF FIGURES x LIST OF ABBREVIATIONS xii ACKNOWLEDGEMENTS xiv DEDICATION xv CHAPTER 1 INTRODUCTION 1 1.1 Pharmacogenetics 2 1.2 Cytochrome P450 3 1.3 CYP2D6 enzyme 4 1.4 CYP2D6 phenotypes 6 1.5 CYP2D locus 9 1.6 CYP2D6 alleles 12 1.7 CYP2D6 alleles in Asians 15 1.7.1 CYP2D6*10 15 1.7.1.1 In vivo studies of CYP2D6*10 17 1.7.1.2 In vitro studies of CYP2D6*10 - human liver microsomes 21 1.7.1.3 In vitro studies ofCYP2D6*10 - recombinant CYP2D6.10 22 1.7.2 CYP2D6*1 27 1.7.3 CYP2D6*2 27 1.7.4 CYP2D6*4 28 1.7.5 CYP2D6*5.... 29 1.8 CYP2D6 genotypes and metabolizer status 29 1.9 Real-time, rapid-cycle PCR 32 1.9.1 Fluorescence resonance energy transfer 33 1.9.2 Fluorescent hybridization probes 35 1.9.3 Principle of SNP detection 37 1.10 Objectives 41 1.11 Specific aims and rationale 42 iv CHAPTER 2 MATERIALS AND METHODS 47 2.1 Equipment 48 2.2 Materials 48 2.2.1 PCR reagents 48 2.2.2 PCR primers 49 2.2.3 Fluorescent hybridization probes 50 2.2.4 DNA isolation and purification 50 2.2.5 Molecular biology reagents 51 2.2.6 Reference samples 52 2.2.7 Patient samples 53 2.3 Specific methods 54 2.3.1 Long PCR 54 2.3.2 Nested real-time, rapid-cycle PCR method for detecting the Cl88T SNP 55 2.3.3 Restriction fragment length polymorphism analysis for detecting the Cl88T SNP 57 2.3.4 Nested real-time, rapid-cycle PCR method for detecting the G1934A SNP.... 60 2.3.5 Restriction fragment length polymorphism analysis for detecting the G1934A SNP 61 2.3.6 Multiplex PCR method for detecting CYP2D6*5 62 2.3.7 Validation of the genotyping methods 63 2.3.8 Genotyping for CYP2D6*10 in subject samples 65 2.4 Statistical methods 65 2.5 General methods 66 2.5.1 Estimation of DNA concentration 66 2.5.2 Preparation of agarose gels 66 2.5.3 Gel electrophoresis 67 2.5.4 Sequence analysis 67 2.5.5 Isolation and purification of genomic DNA from subject samples 69 CHAPTER 3 RESULTS 71 3.1 CYP2D6 genotyping methods 72 3.1.1 Amplification of the CYP2D6 gene by Long PCR 72 3.1.2 Nested real-time, rapid-cycle PCR method for detecting the CYP2D6 C188T SNP 74 3.1.3 Nested real-time, rapid-cycle PCR method for detecting the CYP2D6 G1934A SNP 80 3.1.4 Multiplex PCR method for detecting CYP2D6 *5 84 3.2 CYP2D6 genotype results for subject samples 84 3.3 Genotyping algorithm for the CYP2D6*J0 allele in Asians 93 CHAPTER 4 DISCUSSION 95 4.1 Genotyping methods 96 4.1.1 Long PCR : 96 4.1.2 Development and validation of the nested real-time, rapid-cycle PCR method for detecting the C188T SNP 98 4.1.3 Development and validation of the nested real-time, rapid-cycle PCR method for detecting the G1934A SNP 101 4.1.4 Optimization and validation of the multiplex PCR method for detecting the CYP2D6 gene deletion (CYP2D6*5) 103 4.2 CYP2D6 genotype frequencies in Asian study subjects 105 4.3 CYP2D6 allele frequencies in Asian study subjects 107 4.4 Genotyping algorithm for CYP2D6*10 in Asians 108 4.5 Problems encountered and limitations 109 4.5.1 Selection of primers for the real-time, rapid-cycle PCR method for detecting theC188TSNP 109 4.5.2 DNA polymerase for multiplex PCR 110 4.5.3 Duplication of CYP2D6*10 I l l 4.5.4 Accuracy of the assignment of CYP2D6*wt 111 4.5.5 Other CYP2D6 alleles containing the C188T SNP 112 4.5.6 Linkage of C188T and G1934A SNPs 115 4.6 Future studies 116 4.6.1 Pilot clinical study of codeine metabolism 116 4.6.2 Analysis for other CYP2D6 alleles 117 4.6.3 Analysis for CYP2D6 alleles in a wider range of Asians 117 4.6.4 Further improvements to CYP2D6 genotyping in Asians 118 4.7 Conclusion 119 4.8 Significance 120 BIBLIOGRAPHY 121 APPENDIX A - CYP2D6 Numbering Systems 131 APPENDIX B - CYP2D6 Alleles 132 APPENDIX C - Detecting the CYP2D6 C188T SNP 133 APPENDIX D - Detecting the CYP2D6 G1934A SNP 134 APPENDIX E - CYP2D6 Allele Frequencies in Asians 135 APPENDIX F - English Consent Form for Pilot Clinical Study 136 APPENDIX G - Chinese Consent Form for Pilot Clinical Study 138 APPENDIX H - Certificate of Approval for Pilot Clinical Study 140 vi APPENDIX I - CYP2D6 Genotypes in Pilot Clinical Study 141 APPENDIX J - CYP2D6 Allele Frequencies in Asians (updated) 142 APPENDIX K - Accuracy of the Genotyping Algorithm 143 vii LIST OF TABLES Table 1. A selection of medications that are substrates of C YP2D6 5 Table 2. A selection of CYP2D6 alleles, some of their associated base changes, and expected CYP2D6 activities 14 Table 3. CYP2D6 allele frequencies in Asian study samples 16 Table 4. CYP2D6 genotype and expected metabolizer status 30 Table 5. Oligonucleotide sequences of primers 49 Table 6. Oligonucleotide sequences of fluorescent hybridization probes used in real-time, rapid-cycle PCR 50 Table 7. Details of Hphl and BstNl restriction enzymes 51 Table 8. Expected base changes at positions 188 and 1934 of the CYP2D6 gene for the CYP2D6*1/*J, CYP2D6*l/*5, CYP2D6*l/*4, CYP2D6*4/*4, and CYP2D*4/*5 reference samples 52 Table 9. Demographics of the 36 Asian study subjects involved in the pilot clinical study of codeine metabolism 53 Table 10. Reaction mix for amplification of the CYP2D6 gene by long PCR 54 Table 11. Thermocycling conditions for amplification of the CYP2D6 gene by long PCR with P100/P200 primers 55 Table 12. Pre-PCR mix for detecting the CYP2D6 C188T SNP by nested real-time, rapid-cycle PCR 56 Table 13. Reaction mix for detecting the CYP2D6 C188T SNP by nested real-time, rapid-cycle PCR 56 Table 14. Thermocycling and melting curve conditions for detecting the CYP2D6 C188T SNP by nested real-time, rapid-cycle PCR 56 Table 15. Reaction mix for detecting the CYP2D6 C188T SNP by restriction fragment length polymorphism analysis of PI 1/P12 amplicons 58 Table 16. Pre-PCR mix for detecting the CYP2D6 G1934A SNP by nested real-time, rapid-cycle PCR 60 Table 17. Reaction mix for detecting the CYP2D6 G1934A SNP by nested real-time, rapid-cycle PCR 60 Table 18. Thermocycling and melting curve conditions for detecting the CYP2D6 G1934A SNP by nested real-time, rapid-cycle PCR 61 Table 19. Reaction mix for detecting the CYP2D6 G1934A SNP by restriction fragment length polymorphism analysis of X3F3/X3R1 amplicons 61 Table 20. Reaction mix for detecting the CYP2D6 gene deletion by multiplex PCR 62 viii Table 21. Thermocycling conditions for detecting the CYP2D6 gene deletion by multiplex PCR 63 Table 22. Amount of agarose used in the preparation of agarose gels 66 Table 23. PCR reaction mix for sequence analysis 68 Table 24. Thermocycling conditions for sequence analysis 68 Table 25. Reagent volumes for ethanol precipitation of extension products 68 Table 26. Summary of CYP2D6 genotype assignments for subjects 07, 14, 18, and 19 ....92 Table 27. Summary of CYP2D6 genotype frequencies in 36 Asian study subjects 92 Table 28. Summary of CYP2D6 allele frequencies in 36 Asian study subjects 92 Table 29. Comparison of three different numbering systems for CYP2D6 single nucleotide polymorphisms 131 Table 30. An assortment of CYP2D6 alleles, their associated base changes, and amino acid substitutions 132 Table 31. CYP2D6 allele frequencies in study samples of Asian populations 135 Table 32. CYP2D6 genotype assignments of the 36 Asian subjects involved in a pilot clinical study of codeine metabolism 141 Table 33. CYP2D6 allele frequencies in study samples of Asian populations, including results from the pilot clinical study 142 ix LIST OF FIGURES Figure 1. Distribution of dextromethorphan/dextrorphan metabolic ratios in a North American Caucasian study sample 8 Figure 2. Distribution of debrisoquine/4-hydroxy-debrisoquine metabolic ratios in Chinese and Swedish study subjects 10 Figure 3. Principle of fluorescence resonance energy transfer (FRET) 34 Figure 4. Fluorescent hybridization probes for detecting the CYP2D6 C188T single nucleotide polymorphism 36 Figure 5. Principle of SNP detection using fluorescent hybridization probes 38 Figure 6. Schematic of melting curve analysis for the CYP2D6 C188T SNP using fluorescent hybridization probes 40 Figure 7. Principle of restriction enzyme digestion using Hphl 59 Figure 8. Flowchart summarizing the validation procedure for the methods of detecting the C188T and G1934A SNPs by nested real-time, rapid-cycle PCR 64 Figure 9. Long PCR of the CYP2D6 gene from reference samples with genotypes CYP2D6*1/*1, CYP2D6*l/*4, and CYP2D6*4/*4 73 Figure 10. Analysis for the CYP2D6 C188T SNP in the CYP2D6*1/*1 reference sample 75 Figure 11. Analysis for the CYP2D6 C188T SNP in the CYP2D6*4/*4 reference sample 78 Figure 12. Analysis for the CYP2D6 C188T SNP in the CYP2D6*l/*4 reference sample 79 Figure 13. Analysis for the CYP2D6 G1934A SNP in the CYP2D6*1/*1, CYP2D6*l/*4, and CYP2D6*4/*4 reference samples by melting curve analysis of X3F3/X3R1 amplicons 81 Figure 14. Analysis for the CYP2D6 G1934A SNP in the CYP2D6*1/*1, CYP2D6*l/*4, and CYP2D6*4/*4 reference samples by RFLP analysis of X3F3/X3R1 amplicons....83 Figure 15. Analysis for the CYP2D6 G1934A SNP in the CYP2D6*1/*1, CYP2D6*l/*4, and CYP2D6*4/*4 reference samples by sequence analysis of X3F3/X3R1 amplicons 85 Figure 16. Analysis for the CYP2D6 gene deletion (CYP2D6*5) in the CYP2D6*1/*1, CYP2D6*l/*5, and CYP2D6*5/*5 reference samples by multiplex PCR 86 Figure 17. Analysis for the CYP2D6 C188T single nucleotide polymorphism in selected subjects from the pilot clinical study 88 Figure 18. Analysis for the CYP2D6 G1934A single nucleotide polymorphism in selected subjects from the pilot clinical study 89 Figure 19. Analysis for CYP2D6*5 in selected subjects from the pilot clinical study 90 Figure 20. A novel genotyping algorithm for the CYP2D6*10 allele in Asians 94 Figure 21. Schematic of detecting the CYP2D6 C188T SNP using PI 1/P12 primers and * 1 OSen/* 1 OAnc fluorescent hybridization probes 133 Figure 22. Schematic of detecting the CYP2D6 G1934A SNP using X3F3/X3R1 primers and *4Sen/*4Anc fluorescent hybridization probes 134 Figure 23. Copy of the English consent form used for the pilot clinical study 136 Figure 24. Copy of the Chinese consent form used for the pilot clinical study 138 Figure 25. Copy of the Certificate of Approval from the UBC Clinical Research Ethics Board for the pilot clinical study 140 Figure 26. Estimated accuracy of the genotyping algorithm for CYP2D6*10 in Asians.. 143 xi LIST OF ABBREVIATIONS Abs absorbance AUC area under the curve BSA bovine serum albumin C188T cytosine to thymine substitution at nucleotide 188 C2938T cytosine to thymine substitution at nucleotide 2938 cDNA complementary DNA CI confidence interval CYP cytochrome P450 CYP2D cytochrome P450 2D CYP2D6 cytochrome P450 2D6 enzyme CYP2D6 cytochrome P450 2D6 gene CYP2D7AP cytochrome P450 2D7AP gene CYP2D7BP cytochrome P450 2D7BP gene CYP2D7P cytochrome P450 2D7P gene CYP2D8P cytochrome P450 2D8P gene DNA deoxyribonucleic acid dNTP deoxyribonucleotide triphosphate dsDNA double-stranded DNA EM extensive metabolizer FRET fluorescence resonance energy transfer G169R glutamic acid to arginine substitution at amino acid G1749C guanine to cytosine substitution at nucleotide 1749 G1846A guanine to adenine substitution at nucleotide 1846 G1934A guanine to adenine substitution at nucleotide 1934 G4268C guanine to cytosine substitution at nucleotide 4268 IM intermediate metabolizer MR metabolic ratio mRNA messenger ribonucleic acid P34S proline to serine substitution at amino acid 34 PCR polymerase chain reaction PM poor metabolizer RFLP restriction fragment length polymorphism RNA ribonucleic acid SM slow metabolizer SNP single nucleotide polymorphism ssDNA single-stranded DNA Tm melting temperature peak xiii ACKNOWLEDGEMENTS Firstly, I would like to thank my co-supervisors, Dr. Marc Levine and Dr. Thomas K.H. Chang, for their tremendous support and valuable advice. The members of my research committee gave excellent guidance in this project: Dr. Mary Ensom, Dr. Robert Holt, and Dr. Wayne Riggs. Thank you also to Dr. Peter Soja, Dr. Helen Burt, and Dr. Stelvio Bandiera. I am indebted to Dr. Kishor Wasan for providing me with one of my first research experiences, and also to Dr. Scott Hartsel for further developing my interest in research. I appreciate the gifts of the CYP2D6 reference samples from Dr. Ulrich Zanger and Dr. Leszek Wojnowski. Thank you also to Olfert Landt for the design of primers and fluorescent hybridization probes. Samuel Matsumura and Dr. Eduardo Thuroff provided technical support with real-time, rapid-cycle PCR. I am also grateful for all the help given by Caixia Ma, Jessie Chen, Ted Lakowski, and Aran Verma. Dr. Marc Levine (principle investigator) and co-investigators Dr. Thomas K.H. Chang, Colleen Court, Dr. Ensom, Dr. Carolyne Montgomery, Dr. Eleanor Reimer, and Dr. Riggs were all involved in the pilot clinical study of codeine metabolism. Thank you also to the Department of Anaesthesiology, dental surgeons, and dental clinic staff at the Children's and Women's Hospital of British Columbia. Patient samples were collected and prepared by Fanny Liu, Clara Ho, Winnie Chan, and Diane Decarie. This project was funded in part by the Dawson Endowment Fund and the B.C. Children's Hospital Telethon Fund. I would also like to acknowledge support from a Research Career Award in the Health Sciences from the Canadian Institutes of Health Research and Rx&D Health Research Foundation (to T.K.H.C.), as well as a Michael Smith Foundation for Health Research Trainee Award (to E.H.K.). xiv DEDICATION I dedicate this thesis to my parents, Simon Ni Wah and Cindy Mee Sin Kwong, and to my siblings, David, Mona, and Vincent. xv CHAPTER 1 INTRODUCTION 1.1 Pharmacogenetics When the same dose of a medication is administered to a group of patients, the desired outcome may not be achieved in all of the patients. Variable responses to drugs among individuals may be partially accounted for by physiological, pathophysiological, environmental, or genetic factors. Pharmacogenetics is the study of the genetic differences that affect an individual's response to drugs. In particular, genetic variation in drug transporters, drug metabolizing enzymes, or drug receptors can contribute to differences in pharmacokinetics and pharmacodynamics in individuals (Meisel et al. 2000). The goal of pharmacogenetics in clinical practice is to use a patient's genotype information to maximize effectiveness and minimize toxicity or side effects from the use of medications. In the late 1970s, two interesting, independent discoveries were made with respect to variation in drug metabolism. In London, it was observed that the response to a normal dose of the antihypertensive drug, debrisoquine, was increased in a particular individual (Mahgoub et al. 1977). In Germany, a patient experienced increased side effects from a normal dose of sparteine, an antiarrhythmic (Eichelbaum et al. 1979). Both cases were explained by a decrease in metabolism of these drugs by these patients, as compared to the normal population. The metabolism of debrisoquine and sparteine was shown to be mediated by the same enzyme (Eichelbaum et al. 1982). Debrisoquine, the active drug, is metabolized to inactive compounds. A poor metabolizer of debrisoquine would have higher concentrations of the drug with a given dose and thus an increased hypotensive response to the drug. Similarly, a 2 poor metabolizer taking sparteine would have higher concentrations of the drug, and thus increased side effects. This difference in enzyme form was named the debrisoquine-sparteine polymorphism. Later, it was discovered that the enzyme involved in the metabolism of these drugs is a protein that is now known as CYP2D6 (Gonzalez et al. 1987; Zanger et al. 1988). 1.2 Cytochrome P450 The cytochrome P450 (CYP) proteins comprise a large family of enzymes that are predominantly expressed in the liver. They are also expressed in many extrahepatic tissues, such as the lungs, kidney, and gastrointestinal tract (Smith et al. 1998). CYP enzymes are involved in phase I oxidative metabolism. They catalyze the metabolism of a variety of structurally diverse endogenous and exogenous compounds. CYP-catalyzed reactions may generate a reactive centre in the molecule that can be later conjugated by reaction with phase II enzymes. Through metabolism, substrate hydrophilicity is increased to facilitate excretion. Although CYP-mediated reactions are mainly detoxification processes, some substrates are activated following metabolism, resulting in the generation of products with increased toxicity or mutagenicity (Smith et al. 1998). The CYP superfamily is subdivided into families and subfamilies, based upon amino acid sequence homology. CYP enzymes with an amino acid identity greater than 40% belong in the same family, while those with an amino acid identity of more than 55% belong in the same subfamily. Humans have 18 families and 43 subfamilies of CYP enzymes 3 (http://dmelson.utmem.edu/CytochromeP450.htm accessed 26 July 2004). Families CYP1 to CYP3 are mainly involved in the metabolism of foreign compounds, or xenobiotics (Brockmoller et al. 2000). Each type of C Y P enzyme has unique substrate specificities, active site structures, and mechanisms of regulation. However, there may also be some overlap in substrate specificity (Smith et al. 1998). 1.3 CYP2D6 enzyme The human CYP2 family consists of different subfamilies (e.g. A , B, C, D, E, etc.). CYP2D6 is involved in the metabolism of over 40 clinically used drugs (see Table 1). CYP2D6 comprises approximately 3% of total C Y P content in the liver (Williams 2002). CYP2D6 is most active in the liver, but is also expressed in the lung and mucosa of the small intestine. CYP2D6 messenger ribonucleic acid (mRNA) has also been detected in human brain tissue (Zanger et al. 2004). CYP2D6 mRNA has been detected before birth at levels sometimes exceeding those in adults, but CYP2D6 protein and activity have not been detected until after birth (in neonates greater than 8 days, activity was reported at approximately 25% of adult values) (Jacqz-Aigrain and Cresteil 1992). By 28 days, CYP2D6 expression has been reported to be approximately 60%) of the adult values (Johnson 2003). CYP2D6 catalytic activity in children may be comparable to that of adults by 10 years of age (Leeder 2001). 4 Table et al., 1. A selection of medications that are substrates of CYP2D6 (adapted from Zang 2004). Analgesics Antiarrhythmics Tricyclic antidepressants Other antidepressants Codeine Encainide Amitriptyline Citalopram Dihydrocodeine Flecanide Clomipramine Fluoxetine Hydrocodone Mexiletine Desipramine Paroxetine Norcodeine Propafenone Imipramine Venlafaxine Oxycodone Nortriptyline Antiemetics Antipsychotics Beta-blockers Others Dolasetron Haloperidol Carvedilol Dextromethorphan Ondansetron Perphenazine Metoprolol Tamoxifen Tropisetron Thioridazine Risperidone Perazine Propranolol Timolol Currently, the regulation of C Y P 2 D 6 has not been extensively studied, and there are no drugs that are known to induce C Y P 2 D 6 (Edwards et al. 2003). However, there have been some studies investigating the induction of C Y P 2 D 6 during pregnancy (Wadelius et al. 1997; L ind et al. 2003). In contrast, many drugs are known to be inhibitors of the C Y P 2 D 6 enzyme (Wilcox and Owen 2000). In particular, quinidine is a potent and commonly used inhibitor in studies of C Y P 2 D 6 metabolism (Dayer et al. 1988; Caraco et al. 1999; Shimadae^fl/. 2001). 1.4 CYP2D6 phenotypes Inter-individual variability in drug metabolism can affect the hepatic clearance of drugs. There appears to be a wide range of C Y P 2 D 6 enzyme activity in the population, as evidenced by a wide range of C Y P 2 D 6 phenotypes observed. A n individual's C Y P 2 D 6 phenotype can be assessed by administering a known C Y P 2 D 6 substrate, (e.g. debrisoquine, dextromethorphan) and measuring the concentrations of the parent drug and metabolites in the blood or urine. The ratio of parent drug to metabolites is termed the metabolic ratio. For example, after ingesting a dose of dextromethorphan, this drug is metabolized to dextrorphan by the C Y P 2 D 6 enzyme. The extent of CYP2D6-mediated metabolism can be determined by measuring the dextromethorphan and dextrorphan excreted in the urine. The ratio of these compounds w i l l vary among individuals, and is partially dependent on the individual's C Y P 2 D 6 activity. If debrisoquine is used as the probe drug, then the ratio of debrisoquine/4-hydroxydebrisoquine is calculated. The dextromethorphan/dextrorphan metabolic ratios in a North American Caucasian study sample are shown as a histogram in Figure 1. A bimodal distribution is clearly seen, with 6 an antimode at approximately 0.3 for the dextromethorphan/dextrorphan metabolic ratio. For a distribution of debrisoquine/4-hydroxydebrisoquine metabolic ratios, the antimode is located at 12.6 (Bertilsson et al. 1992). The majority of the North American Caucasian study sample shown in Figure 1 are extensive metabolizers, and are considered to have normal CYP2D6 enzyme activity (dextromethorphan/dextrorphan metabolic ratio less than 0.3). However, there is a sub-population with metabolic ratios that are early 1000-fold greater than the apparent mean. These individuals are considered to be poor metabolizers of dextromethorphan, and are classified according to a dextromethorphan/dextrorphan metabolic ratio greater than 0.3. In the case of using debrisoquine as the probe drug, poor metabolizers have debrisoquine/4-hydroxydebrisoquine metabolic ratios greater than 12.6 (Bertilsson et al. 1992). Based on this criterion, 5-10% of Europeans are reported to be poor metabolizers of CYP2D6 substrates (Alvan et al. 1990; Sindrup et al. 1990). However, the prevalence of poor metabolizers in Asians has been reported to be less than 1% (Bertilsson 1995). There may also be individuals with metabolic ratios in between those of extensive and poor metabolizers, called intermediate metabolizers (Raimundo et al. 2000; Zanger et al. 2004). This subgroup can not be clearly identified from the histogram in Figure 1. In addition, there may be a few outliers with very low metabolic ratios; these individuals are ultra-rapid metabolizers, and are now known to have multiple copies of the CYP2D6 gene (Dalen et al. 1999; Lundqvist et al. 1999). For example, individuals have been reported to carry twelve copies of the CYP2D6 gene (Johansson et al. 1993). 7 30 i 25 20 H o § 15 H cr 10 4 5 i o H n antimode MR = 0.3 I H H I I H - H I i - > . a. • JO 1 1 -J— : 0.1 0.3 1 0.0001 0.001 0.01 Dextromethorphan/dextrorphan metabolic ratio Figure 1. Distribution of dextromethorphan/dextrorphan metabolic ratios in a North American Caucasian study sample (adapted from Gaedigk et al, 1999). The solid vertical line at 0.3 indicates an antimode of the distribution, which separates ultra-rapid, extensive, and intermediate metabolizers (MR < 0.3) from poor metabolizers (MR > 0.3). The two outliers at the left of the histogram (low metabolic ratios) represent ultra-rapid metabolizers. 8 In Asian extensive metabolizers (a metabolic ratio for debrisoquine less than 12.6), there appears to be a shift in the mean metabolic ratios to the right (Figure 2), indicating that, on average, the apparent CYP2D6 activity in Asians is decreased as compared to Europeans (Bertilsson et al. 1992; Wang et al. 1993). The wide range of apparent CYP2D6 activity in study subjects may be partly explained by the polymorphic nature of the CYP2D6 gene. CYP2D6 is of pharmacogenetic interest because there are many different polymorphisms of the CYP2D6 gene (summarized on the Human Cytochrome P450 Allele Nomenclature Committee website located at http://www.imm.ki.se/cypalleles/cyp2d6.htm), some of which may affect the hepatic clearance of drugs metabolized by CYP2D6. Differences in frequencies of polymorphisms among different ethnic populations (e.g. Asians versus Caucasians) may contribute to the observed differences seen in the apparent CYP2D6 activities between different ethnic groups. 1.5 CYP2D locus Interestingly, as summarized by Zanger et al. (2004), although chromosome 22 was the first human chromosome to be completely sequenced by the Human Genome Project (Dunham et al. 1999), the CYP2D6 gene was missing on the published sequence (Idle et al. 2000). This suggests that the CYP2D6 gene may have been missing in the individual who was the source of the genomic deoxyribonucleic acid (DNA), although a homozygous deletion is a rare occurrence. The original sequence by Kimura et al. (1989) has remained the accepted reference for the CYP2D6 gene (GenBank accession number M33388). 9 80-40 H SH ro ZJ •g '> T J CZ 03 .Q E Zl 0-120-80H 40H Chinese (n = 695) (Bertilsson et al., 1992) i l n antimode MR = 12.6 n i U K . Swedish (n = 1011) (Bertilsson et al., 1992) antimode MR = 12.6 0.01 10 0.1 1 Debrisoquine/4-hydroxy-debrisoquine metabolic ratio 100 Figure 2. Distribution of debrisoquine/4-hydroxy-debrisoquine metabolic ratios in Chinese and Swedish study subjects (adapted from Bertilsson 2001). The antimode for the debrisoquine/4-hydroxy-debrisoquine metabolic ratio at 12.6 separates ultra-rapid, extensive, and intermediate metabolizers (MR < 12.6) from poor metabolizers (MR > 12.6). The mean metabolic ratio in Asian study subjects appears to be greater than those in Swedish study subjects. 10 The CYP2D6 gene consists of nine exons and eight introns, and is part of a cluster of genes at the CYP 2D locus on human chromosome 22ql3.1 (GenBank accession number M33388). Thus far, four CYP2D pseudogenes have been reported to exist: CYP2D8P (M33387), CYP2D7P (M33387), CYP2D7AP (X58467), and CYP2D7BP (X58468) (Gonzalez et al. 1988; Kimura et al. 1989). Many point mutations found in CYP2D6 alleles are also present in the CYP2D pseudogenes. The percent identity of deduced amino acid sequences between the CYP2D6 gene and pseudogenes is 87.9%, 94.2%, 93.3%, and 93.8% for CYP2D8P, CYP2D7P, CYP2D7AP, and CYP2D7BP, respectively (Heim and Meyer 1992). These pseudogenes do not result in functional enzyme (Heim and Meyer 1992). CYP2D8P contains numerous gene-inactivating mutations, whereas CYP2D7P contains a premature stop codon due a single base pair insertion in the first exon (Smith et al. 1998). CYP2D7AP contains a frameshift mutation, while CYP2D7BP contains an insertion mutation; both of these mutations result in the premature termination of translation (Heim and Meyer 1992). A method has been developed to quantify the expression of CYP2D7/8P pseudogenes (Endrizzi et al. 2002). However, the prevalence of all known pseudogenes among different ethnic groups has not been extensively studied. Some studies of CYP2D6 polymorphisms have not excluded the possible interference from pseudogenes (Wang et al. 1993; Johansson et al. 1994; Yue et al. 1998; Fukuda et al. 1999; Someya et al. 1999; Huang et al. 2003; Liou et al. 2004). CYP2D6 genotypes are determined by analyzing for specific genetic polymorphisms in the CYP2D6 gene. When analyzing for these genetic polymorphisms, it is important to eliminate interference from CYP2D pseudogenes. 11 1.6 CYP2D6 alleles It is now known that there are many polymorphisms or base changes in the CYP2D6 gene, which can partly explain the apparent variable CYP2D6 activity seen in study samples. Much research has been done to explain CYP2D6 phenotypes by studying the genetic polymorphisms of the CYP2D6 gene. Different forms of a gene are called alleles, and each allele is characteristically defined by a certain combination of changes in the nucleotide sequence. Thus far, over 40 alleles (and over 80 allelic variants) of CYP2D6 have been identified; a constantly updated list of CYP2D6 alleles and associated polymorphisms is available on the Human Cytochrome P450 Allele Nomenclature Committee website (http://www.imm.ki.se/cypalleles/cyp2d6.htm; accessed 26 July 2004). The nomenclature for CYP2D6 alleles used in the current literature is based upon recommendations by Daly et al. (1996). CYP2D6 alleles are designated as "CYP2D6" followed an asterisk (*), an Arabic numeral (1, 2, 3, etc.), and roman letter for allelic variants containing the same key mutation (A, B, C, etc.). Examples of alleles include CYP2D6*1, CYP2D6*2, CYP2D6*3A, CYP2D6*3B, etc. Proteins are named similarly (but non-italicized), with a period or space in place of the asterisk (e.g. CYP2D6.1, CYP2D6.2, etc.) It must be noted that there appears to be at least three different numbering systems for the nucleotides of the CYP2D6 gene. In this project, the conventional numbering system was used (Kimura et al. 1989), in which base number 1 corresponds to the first nucleotide of exon 1 of the CYP2D6 gene. For a comparison of the conventional numbering system with that from the Human Cytochrome P450 Allele Nomenclature Committee website 12 (http://wvvwimm.ki.se/cypalleles/cyp2d6.htm) and GenBank accession number M33388, please refer to Appendix A. Table 2 summarizes a selection of CYP2D6 alleles, some of their associated base changes, and the expected CYP2D6 activities. Please refer to Appendix B for a more extensive table of CYP2D6 alleles. CYP2D6*1 is considered to be the wild-type allele, as it does not contain any of the known base changes. Other alleles, such as the CYP2D6*2, CYP2D6*4, or CYP2D6*10 alleles, contain single nucleotide polymorphisms (SNPs). These SNPs may or may not result in changes to the protein. For example, the G1749C SNP (substitution of the guanine at position 1749 of the CYP2D6 gene with cytosine) results in no amino acid change (silent change) due to the redundancy of the genetic code. However, the C188T SNP (cytosine to thymine substitution at position 188) results in a P34S amino acid change (substitution of the proline at position 34 of the CYP2D6 protein with serine). Some SNPs may be found in more than one allele. For example, the C188T SNP is present in both the CYP2D6*4 and CYP2D6*10 alleles. However, the CYP2D6*4 allele also contains a key G1934A SNP (guanine to adenine substitution at position 1934) that results in a splicing defect and non-functioning enzyme (Hanioka et al. 1990). CYP2D6*5 is actually a deletion of the entire CYP2D6 gene. The CYP2D6 alleles and associated base changes shown in Table 2 will be discussed in greater detail in the following section. 13 Table 2. A selection of CYP2D6 alleles, some of their associated base changes, and expected CYP2D6 activities (format adapted from Meyer and Zanger, 1997). • = amino acid change; 0 = silent change (no amino acid change); <S> = splicing defect. The CYP2D6*1 allele is considered to be the wild-type allele, and contains no base changes. The CYP2D6*2 allele contains various base changes, some of which are silent and some of which give rise to amino acid changes; however, the CYP2D6*2 allele is associated with normal CYP2D6 activity. The CYP2D6*4 allele has similar base changes to other alleles, but the G1934A splicing defect mutation results in a truncated and non-functioning CYP2D6 enzyme. CYP2D6*5 allele is a complete deletion of the CYP2D6 gene. The C188T SNP is the notable base change in the CYP2D6*10 allele, which is associated with low CYP2D6 activity. For a more extensive table of CYP2D6 alleles, please refer to Appendix B. CYP2D6 activity Normal Absent Low CYP2D6 al ele *1 *2 *5 *10 Mutation Effect on protein C188T P34S • Deletion of the CYP2D6 gene • G1749C Silent 0 0 0 G1934A Splicing defect C2938T R296C • G4268C S486T • • • 14 1.7 CYP2D6 alleles in Asians When genotyping study subjects with respect to CYP2D6, it is important to analyze for the most common CYP2D6 alleles. In Asians, the most common CYP2D6 alleles reported in the literature are CYP2D6*10 (48.8%), CYP2D6*1 (34.1%), CYP2D6*2 (11.4%), CYP2D6*5 (5.7%), and CYP2D6*4 (0.6%) (Table 3; references available in Appendix E). In Caucasians, the CYP2D6*10 allele frequency has been reported to range from 2-8% (Bradford 2002). Thus, when determining the CYP2D6 genotypes in a study sample, it is important to consider the ethnic background of the study subjects. In Asians, due to the high frequency of CYP2D6*10, analysis for CYP2D6*10 must be done in order to more accurately assign CYP2D6 genotypes. 1.7.1 CYP2D6*10 The CYP2D6*10 allele is the most common allele in Asians (48.8% allele frequency; range of 35.7-69.2%; Table 3). Currently, there are two known variant CYP2D6*10 alleles: CYP2D6*10A and CYP2D6*10B. The CYP2D6*10A allele (previously known as CYP2D6J) is characterized by the following SNPs in the CYP2D6 gene: C188T, G1749C, and G4268C (Table 2). It was first reported in a Japanese individual having a slower rate of sparteine metabolism (Yokota et al. 1993). The CYP2D6*10B allele (previously known as CYP2D6Chl), contains additional base changes in the CYP2D6 gene (e.g. C1127T) (Johansson et al. 1994; Dahl et al. 1995). The C188T SNP is considered to be the key mutation of both CYP2D6*10 allelic variants. In this thesis, CYP2D6*10 refers to both CYP2D6*10A and CYP2D6*10B. 15 Table 3. CYP2D6 allele frequencies in Asian study samples (references are available in Appendix E). Composite frequency = total number of particular allele reported / (2 x total number of subjects analyzed for the particular allele). Since the composite frequencies are derived from multiple studies, the frequencies do not add up to 100.0%. CYP2D6 allele Range of Composite n frequencies frequency (subjects) CYP2D6*10 35.7-69.2% 48.8% 1269 CYP2D6*! 21.4-43.5% 34.1% 888 CYP2D6*2 6.0-26.2% 11.4% 888 CYP2D6*5 0.0-10.0% 5.7% 1142 CYP2D6H 0.0-6.7% 0.6% 1269 Other alleles 0.0-5.6% 1.9% 1269 16 As mentioned earlier, as a group, Asians appear to have decreased CYP2D6 activity toward some substrates compared to Caucasians. This may be partly explained by the high frequency of the CYP2D6*10 allele in Asians, since this allele is thought to be associated with decreased enzyme activity (Johansson et al. 1994; Dahl et al. 1995). It has been reported that CYP2D6*10 occurs at an allele frequency of 35.7-69.2% in Asians (Appendix C), which means that approximately 13-49% of Asians may be homozygous for the CYP2D6*10 allele (CYP2D6*10/*10). 1.7.1.1 In vivo studies ofCYP2D6*10 In order to elucidate the effect of the CYP2D6*10 allele in vivo, investigators have studied the effect of the CYP2D6*10/*10 genotype on the pharmacokinetics of various CYP2D6 substrates. Johansson et al. (1993) reported 10-fold higher mean metabolic ratios of debrisoquine hydroxylation in CYP2D6*10/*10 subjects (n = 14) versus CYP2D6*1/*1 subjects (n = 17). Similarly, Wang et al. (1993) reported mean log metabolic ratios of debrisoquine hydroxylation 50-fold greater in subjects homozygous for C188T and G4268C (CYP2D6*JO/* 10; n = 6), as compared to those homozygous for C and G at positions 188 and 4268, respectively (CYP2D6*1/*1; n = 64). These studies suggest that the CYP2D6.10 enzyme produced by CYP2D6*10/*10 individuals results in impaired metabolism of debrisoquine. 17 The mean log metabolic ratios of dextromethorphan/dextrorphan in Japanese subjects with CYP2D6*1/*1 (n = 18) and CYP2D6*10/*10 (n = 17) genotypes was reported to be -2.85 ± 0.43 and -1.54 ± 0.28, respectively (Tateishi et al. 1999). In CYP2D6*1/*10 subjects, the mean log metabolic ratio was -2.63 ± 0.35. In another study, as compared to Japanese subjects genotyped as CYP2D6*1/*1 (n = 5), the mean log metabolic ratio of dextromethorphan/dextrorphan in CYP2D6*10/*10 Japanese subjects (n = 6) was significantly greater (-3.06 ± 0.22 vs. -1.66 ± 0.46, respectively) after an oral dose of 30 mg dextromethorphan (Kubota et al. 2000). These two studies demonstrate the apparent reduced metabolism of dextromethorphan in individuals with the CYP2D6*10/*10 genotype. Following an oral dose of 25 mg of nortriptyline, Chinese subjects homozygous for the CYP2D6*10 allele (n = 5) were reported to have a higher mean total AUC (4002 ± 627 nmol-h-L"1 vs. 1817 ± 131 nmol-h-L"1), a lower oral plasma clearance (0.80 ± 0.07 L-h"'-kg"' vs. 1.86 ± 0.31 L-lf'-kg"1), and a longer mean half-life (52.6 ± 7.6 h vs. 28.8 ± 6.4 h), as compared with subjects who were homozygous for CYP2D6*1 (n = 5) (Yue et al. 1998). Following an oral dose of 40 mg propranolol, Chinese subjects homozygous for the C188T SNP (n = 17) were reported to have a significantly higher mean AUC (766.1 ± 92.8 nmol-h-L"1 vs. 333.9 ± 39.3 nmol-h-L"1) than those homozygous for a C at position 188 (n = 13) (Lai et al. 1995). Heterozygotes (n = 14) also had higher mean AUC values than those homozygous for C at position 188, but the results were not statistically significant. 18 In contrast, Tseng et al. (1996) reported no differences in pharmacokinetic parameters among Chinese subjects homozygous for a C at position 188 (n = 8), heterozygous for C188T (n = 12), or homozygous for C188T (n = 12) following an oral dose of 30 mg codeine phosphate. However, the mean urinary recovery of morphine (codeine is partly metabolized via CYP2D6 to morphine) was lower in subjects homozygous for C188T (1127 ± 164 nmol) than in subjects homozygous for a C at position 188 (4349 ± 646 nmol) (Tseng etal. 1996). The above studies demonstrate that there may be statistically significant effects of the CYP2D6*10 allele on the disposition of drugs metabolized by CYP2D6. In some of these studies, subjects with the heterozygous CYP2D6*1/*10 genotype were also reported to have changes in the pharmacokinetic parameters of the drugs, but most of these observed changes were not statistically significant. If a single CYP2D6*1 allele is adequate for CYP2D6 metabolic activity, those who are heterozygous for the CYP2D6*10 allele may not have any clinically significant effects. Furthermore, statistically significant differences in plasma concentration and pharmacokinetic parameters associated with CYP2D6*10 may not indicate differences in drug response. There are very few studies investigating the effects of the CYP2D6*10 allele on the pharmacodynamics of drugs metabolized by CYP2D6. For example, CYP2D6*1/*10 subjects (n = 10) did not appear to have differences in beta-blockade effects of propranolol (e.g. heart rate and blood pressure) as compared to CYP2D6*1/*1 subjects (n = 10) (Huang et al. 2003). In a different study, following doses of 450 mg/day of propafenone for seven days in Chinese subjects with ventricular arrhythmia, the observed inhibitory rate of ventricular premature contractions 19 was significantly higher in CYP2D6*10/*10 (n = 6) subjects as compared to CYP2D6*1/*1 subjects (n = 3) (96 ± 6% vs. 58 ± 34%, respectively) (Cai et al. 2002). However, the results from that particular study may have been confounded by other factors such as gender or age. Differences in drug response may also depend on whether the parent or metabolites are the active compound. For example, venlafaxine is metabolized mainly to O-desmethylvenlafaxine by CYP2D6, but both compounds are considered to be active (Fukuda et al. 1999). Although there were reported differences in the mean pharmacokinetic parameters of venlafaxine among Japanese subjects with different CYP2D6 genotypes (CYP2D6*1/*1, CYP2D6*1/*10, and CYP2D6* 10/* 10), the mean total concentrations of venlafaxine plus (9-desmethylvenlafaxine did not appear to differ (Fukuda et al. 1999). Thus, since both compounds are active, the effect of the CYP2D6*10 allele on the pharmacokinetics of venlafaxine may not result in differences in drug response. According to the above studies, Asian patients who are homozygous for the CYP2D6*10 allele may have decreased metabolism or clearance of drugs that are substrates for CYP2D6. The magnitude of reduced metabolism may be substrate-specific. CYP2D6*10/*10 patients may be at risk of increased side effects or decreased drug response, depending on the roles of the parent drugs or metabolites. In order to elucidate potential causes of any decreased activity due to CYP2D6*10, investigators have also studied human liver microsomes from subjects carrying the CYP2D6*10 allele. « 20 1.7.1.2 In vitro studies of CYP2D6*10 - human liver microsomes Shimada et al. (2001) reported the first investigation of the CYP2D6.10 protein in human liver. Human liver microsomes (obtained from liver biopsies) from Japanese individuals with CYP2D6*10B/*10B (n = 7) and CYP2D6*1/*10B (n = 7) genotypes were reported to have lower CYP2D6 protein expression as compared to those from Japanese individuals with CYP2D6*1/*1 (n = 9) and CYP2D6*l/*2 (n = 4) genotypes (1.0 ± 0.9 pmol/mg and 1.0 ± 1.1 pmol/mg versus 3.0 ± 3.0 pmol/mg and 3.6 ± 2.6 pmol/mg), although the results were not statistically significant (Shimada et al. 2001). However, liver microsomes from CYP2D6*!0B/*10B individuals (n = 7) were reported to have a statistically significant 6-fold lower mean CYP2D6 catalytic activity for bufuralol l'-hydroxylation (pmol/min/nmol CYP), as compared to those from CYP2D6*1/*1 individuals (n = 9). Liver microsomes from CYP2D6*1/*10B (n = 7) individuals were reported to have bufuralol l'-hydroxylation activities approximately 4-fold higher than those from CYP2D6*!0B/*10B individuals. Liver microsomes from subjects carrying the CYP2D6*10B allele appeared to have higher K m (lower affinity) and lower V m a x (lower capacity) for bufuralol l'-hydroxylation, as compared to those from subjects carrying either the CYP2D6*1 or CYP2D6*2 alleles. The combined lower affinity and lower capacity suggest an overall reduced apparent intrinsic clearance (V m a x /K m ) in individuals carrying the CYP2D6*10B allele. Nakamura et al. (2002) also studied bufuralol l'-hydroxylation among human liver microsomes from individuals analyzed for CYP2D6 genotypes. The catalytic activities in liver microsomes from subjects with CYP2D6*1/*1, CYP2D6*1/*10, and CYP2D6*10/*10 21 genotypes were 18.2 ± 18.4 pmol/min/mg protein, 9.6 ± 9.7 pmol/min/mg protein, and 4.9 ± 2.7 pmol/min/mg protein, respectively (Nakamura et al. 2002). In an extensive study correlating CYP2D6 expression with CYP2D6 phenotypes and CYP2D6 genotypes (Zanger et al. 2001), an individual with a CYP2D6*6/*10 genotype was reported (CYP2D6*6 is thought to give rise to a non-functioning CYP2D6 enzyme). Liver microsomes isolated from the CYP2D6*6/*10 individual were reported to have lower CYP2D6 content (0.81 pmol/mg vs. 7.1 pmol/mg) and bufuralol 1'-hydroxylation (0.01 nmol/mg/min vs. 0.57 nmol/mg/min) than those isolated from CYP2D6*1/*1 individuals (n = 13). However, it must be noted that only one individual was studied. These studies of human liver microsomes from individuals carrying the CYP2D6*10 allele further support the observations seen in vivo. The CYP2D6*10 allele appears to result in a CYP2D6.10 enzyme with lower catalytic activity, as measured by bufuralol 1'-hydroxylation. In order to further investigate the molecular basis of the decrease in catalytic activity associated with the CYP2D6*10 allele, investigators have studied the specific nucleotide changes present in CYP2D6*10. 1.7.1.3 In vitro studies of CYP2D6*10- recombinant CYP2D6.10 The key mutation in the CYP2D6*10 allele is thought to be the C188T SNP, resulting in a P34S amino acid substitution. The proline at position 34 of the CYP2D6 enzyme is the first proline in a highly conserved "PPGP" sequence in CYP enzymes belonging to families CYP1 and CYP2 (Johansson et al. 1994). This highly conserved region may be important 22 for the function of CYP enzymes, as studied in the CYP2C2 enzyme (Chen and Kemper 1996; Cheney al. 1998). Johansson et al. (1994) expressed CYP2D6*10, CYP2D6*1, and chimeras between the two alleles, in order to elucidate the effects of the P34S amino acid base change. COS-1 cells transfected with CYP2D6*10 appeared to express 40% less protein than those transfected with CYP2D6*1. Additionally, the rate of CYP2D6-dependent l'-hydroxylation of bufuralol in homogenates from cells transfected with CYP2D6*10 was 40-fold lower (per mg of protein) than homogenates from cells with CYP2D6*1. However, when the cells were transfected with a chimera construct of CYP2D6*10 without the C188T SNP, bufuralol l'-hydroxylation activity in the homogenates was similar to that seen in cells transfected with CYP2D6*1. This suggests that the C188T SNP is a key mutation contributing to the reduced activity of the CYP2D6.10 enzyme, but additive effects with other mutations in the CYP2D6*10 allele could not be ruled out (e.g. a construct containing only the C188T base change was not studied). The apparent intrinsic clearance (V m a x /K m ) of many CYP2D6 substrates has been compared between recombinant CYP2D6.1 and CYP2D6.10 (insect cell expression system) (Ramamoorthy et al. 2001). For example, apparent intrinsic clearance for dextromethorphan (9-demethylation was 50-fold lower for CYP2D6.10 versus CYP2D6.1 (due to a higher K m and a lower V m a x ) . For amitriptyline JV-demethylation and p-hydroxylation by CYP2D6.10, apparent intrinsic clearance was observed to be 60-fold and 42-fold lower, respectively, as compared to CYP2D6.1. The enzyme kinetics of other 23 substrates was also studied. More importantly, it was noted that the magnitude of effects on the apparent intrinsic clearances by CYP2D6.10 may be substrate-specific. Yu et al. (2002) studied the reaction rates of typical CYP2D6-mediated pathways (dextromethorphan (2-demethylation, codeine O-demethylation, and fluoxetine N-demethylation) by recombinant CYP2D6.10 expressed in insect cells. The CYP2D6.10 enzyme was reported to have an estimated apparent intrinsic clearance 50-fold lower for dextromethorphan O-demethylation and 100-fold lower for fluoxetine ./V-demethylation, as compared to CYP2D6.1 enzyme (Yu et al. 2002). No measurable catalytic activity of CYP2D6.10 was observed for codeine O-demethylation. These results again suggest differential effects of the CYP2D6.10 enzyme on different substrates. Site-directed mutagenesis was used to study the effect of the C188T SNP in the absence of other base changes (Fukuda et al. 2000). Using a yeast expression system, CYP2D6*10A and different CYP2D6 constructs were investigated, including a "CYP2D6*10-wt" construct (CYP2D6*10A without C188T and G4268C), a "CYP2D6*10-PT" construct {CYP2D6*10A without C188T), and a "CYP2D6*10-SS" construct (CYP2D6*10A without G4268C). There appeared to be less CYP2D6 protein expressed from cells containing the CYP2D6 C188T SNP (CYP2D6*10A and CYP2D6*10-SS versus CYP2D6*10-wt and CYP2D6*10-SS), as determined using immunoblotting. Using CO-reduced difference spectra in microsomal protein isolated from the cells (pmol/mg), this decrease appeared to be approximately 20-fold. In addition, for bufuralol 1'-hydroxylation, the C188T SNP was not associated with any changes in capacity (Vm a x), but an approximately 5-fold lower 24 affinity (higher Km) was observed. For venlafaxine O-demethylation, an approximately 5-fold lower affinity was observed for CYP2D6.10A versus "CYP2D6.10-wt" (CYP2D6.1). The G4268C SNP did not appear to decrease expression, capacity, or affinity, suggesting that the C188T SNP of the CYP2D6*10A allele mainly contributed to the observed differences in the study. Zanger et al. (2001) also used site-directed mutagenesis to introduce C188T and G4268C mutations into wild-type CYP2D6 cDNA to study CYP2D6.10. Similar levels of recombinant CYP2D6.1 and CYP2D6.10 were expressed in an insect cell system (by electrophoretic and immunoblot analyses), but reduced levels of heme-containing CYP was observed for CYP2D6.10 (by spectral analysis). Additionally, the apparent intrinsic clearance of bufuralol was approximately 10-fold lower for CYP2D6.10 as compared to CYP2D6.1. Nakamura et al. (2002) investigated bufuralol 1'-hydroxylation and dextromethorphan O-demethylation enzyme kinetics of CYP2D6.1 and CYP2D6.10 expressed in yeast cells. For bufuralol 1'-hydroxylation, the apparent intrinsic clearance of CYP2D6.10 was observed to be approximately 3-fold lower. For dextromethorphan O-demethylation, the apparent intrinsic clearance of CYP2D6.10 was observed to be approximately 16-fold lower. These in vitro studies of function using recombinant CYP2D6 enzymes indicate that the C188T SNP contained in CYP2D6*10 may lead to decreased expression and reduced catalytic activity of the CYP2D6 enzyme. These changes may also be a result of the 25 combination of the C188T and G4268C SNPs present in CYP2D6*10. The differences in the extent of reduction in apparent intrinsic clearance for different substrates again suggests that there may be substrate-specific effects of CYP2D6*10 on the CYP2D6.10 enzyme. Based upon the above in vivo and in vitro studies of CYP2D6*10, Asian patients who are homozygous for CYP2D6*10 are expected to produce functional CYP2D6 enzyme, but with reduced activity. These patients may be at risk of adverse effects or altered drug response after taking medications that are primarily cleared hepatically by CYP2D6-mediated metabolism. Thus, identification of the CYP2D6*10 allele in patients may be beneficial if the medication is metabolized primarily by CYP2D6. The characteristic SNP of CYP2D6* 10 appears to be a C188T base change. However, the CYP2D7BP pseudogene has also been reported to contain the C188T SNP (Gonzalez et al. 1988). In previous studies of the CYP2D6*10 allele (including all of the above in vivo studies), methods for detecting the C188T SNP in study subjects relied on genotyping methods that were not specific for the CYP2D6 gene. Amplification of the CYP2D7BP gene may have interfered with the results of the C188T analyses, and thus there may have been incorrect genotype assignments involving the CYP2D6*10 allele. Although the frequency of the CYP2D7BP pseudogene in Asians is not known, in order to minimize spurious genotyping results in studies investigating the effect of CYP2D6*10 on drug disposition and response, it is important to have genotyping methods that are specific for the CYP2D6 gene. 26 1.7.2 CYP2D6*! The second most common allele in Asians is the CYP2D6*1 allele (34.1% allele frequency; range of 21.4-43.5%; Table 3). The CYP2D6*1 allele is considered to be the "wild-type" allele, as it is the most common allele found in Europeans (33.4-40.4%; Bradford 2002). There are no base changes or polymorphisms in the CYP2D6*1 allele, which is expected to produce an enzyme with normal activity. The frequency of the CYP2D6*! allele is determined by default, meaning that its presence is based upon the absence of other known CYP2D6 alleles. 1.7.3 CYP2D6*2 The CYP2D6*2 allele (previously known as CYP2D6L) has been observed at a frequency ranging from 6.0-26.2%) in Asians (Table 3). In contrast, it has been reported at a frequency of 22.0-37.0%) in Caucasians (Bradford 2002). CYP2D6*2 contains several base changes in the CYP2D6 gene. For example, a G1749C polymorphism results in a "silent" effect since there is no corresponding amino acid change (Table 2). Other polymorphisms present in CYP2D6*2 include the C2938T and G4268C base changes, which result in amino acid changes. A promoter polymorphism has also recently been reported in some individuals previously genotyped as carrying the CYP2D6*2 allele (Raimundo et al. 2000; Zanger et al. 2001; Gaedigk et al. 2003). There appears to be some discrepancy about the catalytic activity of the CYP2D6.2 enzyme. The CYP2D6*2 allele was initially reported to give rise to a CYP2D6 protein 27 with normal enzyme activity (Johansson et al. 1993; Dahl et al. 1995; Tateishi et al. 1999). However, it has also been reported that the apparent intrinsic clearance of the CYP2D6.2 enzyme may be lower than that of the CYP2D6.1 enzyme (Yu et al. 2002). In addition, at the Human Cytochrome P450 Allele Nomenclature Committee website (http://www.imm.ki.se/cypalleles/cyp2d6.htm; accessed 26 July 2004), CYP2D6*2 is stated to result in a CYP2D6 enzyme with decreased activity. On the other hand, in the recent review article by Zanger et al. (2004), CYP2D6*2 was listed with CYP2D6*1 under the category of "alleles with normal function". Further investigation of the CYP2D6.2 enzyme is required. Throughout the thesis, CYP2D6*wt will be used to represent the CYP2D6*! and CYP2D6*2 alleles. Thus, the CYP2D6*wt/*wt genotype refers to CYP2D6*1/*1, CYP2D6*l/*2, and CYP2D6*2/*2. 1.1 A CYP2D6*4 The CYP2D6*4 allele (previously known as CYP2D6B) is the most common inactivating allele in Caucasians, reported at a frequency of 11.6-23.0% in Caucasians (Bradford 2002). In Asians, the observed allele frequency of the CYP2D6*4 allele is lower (range of 0.0-6.7%; Table 3). This allele contains a unique G1934A SNP, which results in a splicing defect and truncated protein (Hanioka et al. 1990). Due to the truncation of the protein, no functional CYP2D6 enzyme is produced (Gough et al. 1990). However, the CYP2D6*4 allele also contains the C188T SNP (Table 2). Therefore, when genotyping for CYP2D6*10 by analyzing for the C188T SNP, it is important to rule out CYP2D6*4 by also analyzing for the G1934A SNP. 28 1.7.5 CYP2D6*5 CYP2D6*5 (previously known as CYP2D6D) is a complete deletion of the CYP2D6 gene, although the exact breakpoints are not yet known (Gaedigk et al. 1991; Steen et al. 1995; Steen et al. 1995). Since the gene is not present, no enzyme is produced. The CYP2D6 gene deletion has been reported at a frequency of 2.1-7.3% in Caucasians (Bradford 2002). In Asians, it has been reported to occur at an approximate frequency of 5.7% (range of 0.0-10.0%; Table 3). Thus, it is important to rule out CYP2D6*5 when genotyping Asians with respect to CYP2D6. 1.8 CYP2D6 genotypes and metabolizer status As more information about CYP2D6 alleles is reported, previously defined CYP2D6 phenotypes (metabolic ratios of certain CYP2D6 substrates) can be associated with various CYP2D6 genotypes. Of the four phenotypes designated in Table 4, only extensive, intermediate, and poor metabolizers have been commonly used in the literature. The extensive metabolizers are considered to have CYP2D6 alleles associated with normal enzyme activity (CYP2D6*! or CYP2D6*2) (Zanger et al. 2004). However, as mentioned previously, the mean debrisoquine/4-hydroxydebrisoquine metabolic ratio in Asian extensive metabolizers appears to be higher than that of Caucasian extensive metabolizers (Bertilsson et al. 1992); this difference may be partly explained by the higher frequency of the CYP2D6*10 allele in Asians (e.g. CYP2D6*1/*10, CYP2D6*2/*10, and CYP2D6*10/*10 genotypes). 29 Table 4. CYP2D6 genotype and expected metabolizer status (Raimundo et al. 2000; Zanger et al. 2001; Zanger et al. 2004). "EM" = "Extensive Metabolizer", "IM" = "Intermediate Metabolizer", "SM" = Slow Metabolizer", "PM" = Poor metabolizer. "EM" "EM" or "IM"? "SM" "PM" CYP2D6*1/*1 CYP2D6*1/*10 CYP2D6*10/*10 CYP2D6*4/*4 CYP2D6*l/*2 CYP2D6*2/*10 CYP2D6*10/*5 CYP2D6*4/*5 CYP2D6*2/*2 CYP2D6*l/*4 CYP2D6*10/*4 CYP2D6*5/*5 CYP2D6*2/*4 CYP2D6*l/*5 CYP2D6*2/*5 30 In contrast, poor metabolizers are thought to produce non-functioning CYP2D6 enzyme. In Caucasians, the majority of CYP2D6 poor metabolizers appear to be associated with CYP2D6 alleles such as CYP2D6*3 (reported to be associated with non-functioning CYP2D6 enzyme), CYP2D6U, or CYP2D6*5 (Zanger et al. 2004). The frequency of poor metabolizers in Asians has been reported to be less than 1% (Bertilsson 1995). The lower frequency of poor metabolizers reported in Asians may be due to the lower observed frequency of the CYP2D6*3 and CYP2D6*4 alleles, as compared to Caucasians (Johansson etal. 1991). Approximately 10-15% of Caucasians are considered to be intermediate metabolizers (Raimundo et al. 2000). The 'intermediate metabolizer' phenotype has not yet been clearly defined in relation to CYP2D6 genotypes (Raimundo et al. 2000). Zanger et al. (2001) have reported that subjects carrying one or more functional gene copies (i.e. CYP2D6*1 or CYP2D6*2) are mostly extensive metabolizers, while some may be intermediate metabolizers. Further studies are needed to elucidate the genotypes contributing to the intermediate metabolizer phenotype. The CYP2D6*1/*10 or CYP2D6*2/*10 genotypes may result in the extensive metabolizer or intermediate metabolizer phenotype (bolded in Table 4). Table 4 also includes a category of slow metabolizers. There have been previous references to slower metabolizers in relation to the CYP2D6* 10 allele (Johansson et al. 1994; Dahl et al. 1995). CYP2D6 slow metabolizers would be expected to have CYP2D6 metabolic ratios between those of intermediate and poor metabolizers. The numerous in 31 vivo and in vitro studies of CYP2D6*10 suggest that the CYP2D6*10 allele may be associated with reduced or slower metabolism. Individuals who are homozygous for CYP2D6*10 (CYP2D6*10/*10) or have only one functional allele that is CYP2D6*10 (e.g. CYP2D6*10/*4 or CYP2D6*10/*5) may fall into the category of slow metabolizers (bolded in Table 4). Since the CYP2D6*10 allele is common among Asians, many Asians may be at risk for adverse effects or altered drug responses due to reduced metabolism of drugs that are CYP2D6 substrates. 1.9 Real-time, rapid-cycle P C R In order to genotype individuals with respect to CYP2D6, the CYP2D6 alleles must be determined by analyzing for specific SNPs. In numerous studies involving CYP2D6 genotypes, the CYP2D6 SNPs were mainly detected by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis or allele-specific PCR analysis. The use of fluorescent hybridization probes to detect SNPs by real-time, rapid-cycle PCR has been recently developed (Wittwer et al. 1997). In real-time PCR, fluorescence signals representing PCR or mutation analysis can be visualized in 'real-time' on the computer screen. Rapid-cycle PCR involves the use of shorter hold times for denaturation, annealing, and extension, as compared to conventional PCR. The potential advantages of real-time, rapid-cycle PCR methods for detecting SNPs are savings in time and labour. 32 1.9.1 Fluorescence resonance energy transfer A detailed description of energy transfer between fluorescent dyes can be found in Morrison (1995). Figure 3 summarizes the phenomenon of fluorescence resonance energy transfer (FRET) between fluorescein and LC-Red 640. The fluorescein dye, when excited, will emit energy near the absorption wavelength of the LC-Red 640 dye. This energy transfer occurs only when the two dyes are in close proximity to each other (i.e. 1-10 nm) (Szollosi et al. 1998). When the LC-Red 640 dye is excited, it emits at a longer wavelength that can be detected by a fluorescent detector. If the two dyes are further apart, then the FRET phenomenon will not occur, and there would be no emission detected from the LC-Red 640 dye. 33 Fluorescein LC Red 640 Figure 3. Principle of fluorescence resonance energy transfer (FRET). If the fluorescein and LC-Red 640 dyes are in close proximity to each other, then upon (a) excitation of the fluorescein dye, (b) energy will be transferred to the LC-Red 640 dye, and (c) the fluorescence signals emitting from the LC-Red 640 dye can be monitored. If the two dyes are not close enough, the FRET phenomenon should not occur, and there should be no signal detected from the LC-Red 640 dye. 34 1.9.2 Fluorescent hybridization probes The fluorescein and LC-Red 640 dyes can be synthesized onto short sequences of oligonucleotides. Figure 4 is a schematic of the fluorescent hybridization probes designed to detect the CYP2D6 C188T SNP. Two probes are designed to be approximately 1-3 base pairs apart. An anchor probe is a short sequence of oligonucleotides usually designed to be 5' of the site of the SNP to be analyzed, and is typically labelled with the fluorescein dye at the 3' end. The sensor probe overlies the site of the SNP, and is complementary to the wild-type sequence surrounding nucleotide 188. The 5' end of the sensor probe is labelled with the LC-Red 640 dye, whereas the 3' end of the sensor probe is phosphorylated in order to prevent extension during PCR. The anchor probe is designed to have a higher melting temperature than that of the sensor probe. At a low temperature, sensor probes will also have the ability to anneal to amplicons containing the C188T SNP. When both the anchor and sensor probes are annealed to amplicons, the fluorescein and LC-Red 640 dyes are close enough to each other such that FRET can occur. Upon excitation of the fluorescein dye, the energy is transferred to the LC-Red 640 dye, and the emission wavelength can be read by a fluorescence detector. Thus, the presence of a fluorescence signal indicates that the fluorescent hybridization probes are annealed to the amplicons. Any loss of fluorescence signal would suggest that the anchor and sensor probes are no longer close to each other due to melting off from the amplicons. 35 CYP2D6 wild-type amplicon C188T SNP Figure 4. Fluorescent hybridization probes for detecting the CYP2D6 C188T single nucleotide polymorphism. The fluorescein dye is attached to the 3' end of the anchor probe (left), while the LC-Red 640 dye is attached to the 5' end of the sensor probe (right). The anchor probe and sensor probe are designed to be 1-3 base pairs apart (diagram is not to scale). The sensor probe is designed to be complementary to the wild-type sequence. At lower temperatures (e.g. 45°C), the sensor probe can anneal to both the wild-type and mutant amplicons, and a fluorescence signal from the LC-Red 640 dye should be detected due to the FRET phenomenon. However, there is a single base mismatch when the sensor probe anneals to the mutant amplicon, which results in a weaker annealing strength. The weaker annealing strength of the sensor probe to the mutant amplicon will allow differentiation between a C and a T at position 188 upon melting curve analysis. 36 1.9.3 P r i n c i p l e o f S N P d e t e c t i o n The principle of mutation detection by fluorescent hybridization probe melting curves has been described by Bernard et al. (2001). Figure 5 shows a schematic of the principle of detecting the CYP2D6 C188T SNP. After PCR, there are numerous copies of the amplicon of interest. The temperature is subsequently lowered such that the anchor and sensor probes can anneal to the amplicons (Figure 5 a). Although there is a single base mismatch due to a C188T SNP on the mutant amplicons, sensor probes can still anneal to the mutant amplicons at the lower temperature (e.g. 45°C). At this temperature, the probes are annealed and the fluorescein and LC-Red 640 dyes are in close proximity to each other. The FRET phenomenon can occur, and thus fluorescent signals can be detected. However, upon increasing the temperature at a slow rate (i.e. 0.2°C per second), sensor probes on the mutant strands will melt off first (Figure 5b), due to the single base mismatch at the 188 position (fewer hydrogen bonds). Thus, the fluorescent signals will slowly begin to decrease since the energy transfer between the fluorescein and LC-Red 640 dyes have been eliminated. In contrast, the sensor probes on the wild-type amplicons will melt off at a higher temperature (Figure 5c), and thus the decrease in fluorescence signals will be seen at a higher temperature. At even higher temperatures, the anchor probes will also melt off (Figure 5d). 37 5 % 45°C Wild-type C188TSNP Mutant Wild-type C188TSNP Mutant (a) (b) 65°C Wild-type 5 C188TSNP Mutant 72°C Wild-type C188TSNP Mutant (C) (d) Figure 5 . Principle of SNP detection using fluorescent hybridization probes: (a) at low temperatures (e.g. 45°C) the sensor probes (right) anneal to both wild-type and mutant amplicons, and the FRET phenomenon results in the detection of fluorescence signals from the LC-Red 640 dye; (b) as the temperature is increased slowly (e.g. to 53°C) the sensor probes melt off from the mutant amplicons first because of the single base mismatch, and the fluorescence signals begin to decrease; (c) as the temperature is increased further (e.g. 65°C), the sensor probes melt off from the wild-type amplicons; (b) at higher temperatures (e.g. 72°C), the anchor probes (left) also melt off. 38 Figure 6 shows schematic representations of melting curve results from the analysis of the CYP2D6 C188T SNP. As can be seen in Figure 6a, there is a decrease in fluorescence signal as the temperature is increased, since the FRET phenomenon begins to dissipate when the sensor probes melt off the amplicons. For the amplicons containing the C188T SNP (red curve), the sensor probes melt off first due to the single base mismatch, and thus the fluorescence signal begins to decrease at a lower temperature. The green curve represents the melting curve profile of sensor probes from amplicons containing the wild-type sequence. The negative derivatives of the melting curves are calculated in order to produce melting peak graphs (Figure 6b). The melting peak temperature (Tm) represents the temperature at which there is a maximum rate of decline of fluorescence, which occurs due to the sensor probes melting off at a maximum rate. A higher Tm represents the melting of sensor probes from amplicons containing the wild-type sequence (green curve), while a lower Tm represents the melting of sensor probes from amplicons containing the C188T SNP (red curve). For a heterozygous sample (wild-type on one chromosome, but containing the C188T SNP on the other chromosome), melting peaks at both temperatures would be expected, as shown in Figure 6c. Using fluorescent hybridization probes, a single nucleotide polymorphism can be detected by observing the melting peak temperatures obtained from real-time, rapid-cycle PCR followed by melting curve analysis. 39 Temperature 1 8 8 T 188 c Temperature (c) Temperature Figure 6 . Schematic of melting curve analysis for the CYP2D6 C188T SNP using fluorescent hybridization probes: (a) melting curves for amplicons containing either a C or T at position 188; (b) melting peaks (negative derivative of the melting curves) for amplicons containing either a C or T at position 188; (c) melting peaks for amplicons containing both a C and T at position 188. 40 1.10 Objectives In order to investigate the effect of CYP2D6 genotypes on drug metabolism, it is necessary to have an algorithm and genotyping methods for determining each subject's CYP2D6 genotype. A CYP2D6 genotyping algorithm should include the analysis for the most common CYP2D6 alleles. In Asians, the most common alleles are CYP2D6*10, CYP2D6*!, CYP2D6*2, CYP2D6*5, and CYP2D6U. Thus, genotyping methods are required to analyze for these CYP2D6 alleles. The objectives of this thesis project were to develop and validate these methods, to genotype samples from a pilot clinical study of codeine metabolism in Asian paediatric dental patients, and to devise a CYP2D6 genotyping algorithm specific for Asians. When analyzing for mutations to determine CYP2D6 alleles, it is important to eliminate interference from the CYP2D pseudogenes. In order to do so, specific primers can be used to first amplify the CYP2D6 gene via 'long PCR'. Long PCR, required for the amplification of a product greater than 3-5 kb in size, includes the use of DNA polymerases designed for higher yield and better fidelity than regular DNA polymerases. A subsequent PCR on the long PCR amplicons can then be used to analyze for CYP2D6 SNPs. This subsequent PCR is termed 'nested PCR', in which the template used is derived from a previous PCR. As stated previously, the CYP2D6*10 allele contains the C188T SNP. Thus, the C188T SNP can be used as the criterion for the CYP2D6*10 allele. However, the CYP2D6*4 allele also contains this base change, in addition to its unique G1934A SNP. Therefore, when genotyping for the CYP2D6*10 allele by analyzing for the C188T base 41 change, we also need to rule out the CYP2D6*4 allele by analyzing for the G1934A SNP. In addition, we need to rule out CYP2D6*5 due to its estimated frequency of 5.7% in Asians (Table 3). Briefly, the CYP2D6 genotyping strategy in the pilot clinical study included initial amplification of the CYP2D6 gene by long PCR, followed by analysis for the C188T and G1934A single nucleotide polymorphisms by real-time, rapid-cycle PCR, and analysis for the CYP2D6*5 gene deletion. We could then infer the presence of the CYP2D6*wt (CYP2D6*1 or CYP2D6*2). After combining the CYP2D6 allele frequencies with those found in the current literature, a genotyping algorithm for CYP2D6*10 in Asians was devised. 1.11 Specific aims and rationale Aim 1: To develop and validate CYP2D6 genotyping methods for detecting the CYP2D6*10, CYP2D6*4, and CYP2D6*5 alleles. In order to conduct studies on the effect of the CYP2D6*10 allele on drug metabolism, it is necessary to analyze data from the study sample by comparing the three genotype groups of CYP2D6*wt/*wt, CYP2D6*wt/*10, and CYP2D6*10/*10. In addition to analysis for the CYP2D6*10 allele, the CYP2D6U and CYP2D6*5 alleles also need to be ruled out. 42 Aim la: To optimize a published protocol for amplifying the CYP2D6 gene by long PCR. There are two main problems when using genomic DNA to genotype for the CYP2D6 alleles. The first problem is minor; the genomic DNA isolated from leukocytes of a single blood sample is a limited resource for genotyping. The second problem is of major importance. As mentioned previously, the CYP2D locus is comprised not only of the CYP2D6 gene, but other pseudogenes. Many of the base changes in the CYP2D6 alleles are also present in and hypothesized to originate from these pseudogenes (Heim and Meyer 1992). Therefore, amplification of the pseudogenes could interfere with CYP2D6 genotyping, and create incorrect assignments of genotypes. Primers specific to the CYP2D6 gene can be chosen to avoid amplification of the CYP2D pseudogenes. However, the C188T SNP is located in exon 1 of the CYP2D6 gene, which is nearly 100% identical in sequence to the CYP2D7BP pseudogene (Heim and Meyer 1992). Initial amplification of the CYP2D6 gene by long PCR using specific primers can solve both of these problems. A diluted long PCR amplicon can serve as a larger resource for subsequent analysis by nested PCR, while the pseudogenes would be avoided altogether after amplification of the CYP2D6 gene using specific primers (Sachse et al. 1997). Aim lb: To develop and validate a nested real-time, rapid-cycle PCR method for detecting the CYP2D6 C188T single nucleotide polymorphism. The C188T single base change has been reported in both the CYP2D6*10 and CYP2D6*4 alleles. The reported frequencies of the CYP2D6*10 and CYP2D6*4 alleles are 35.7-43 69.2% and 0.0-6.7%, respectively (Appendix E ) . To genotype for the CYP2D6*10 allele, it is important to have a method for detecting the C188T SNP. The nested real-time, rapid-cycle PCR method may offer savings in time and labour. Aim lc: To develop and validate a nested real-time, rapid-cycle PCR method for detecting the CYP2D6 G1934A single nucleotide polymorphism. Both the CYP2D6*10 and CYP2D6H alleles contain the C188T SNP. However, the G1934A single base change has been reported only in the CYP2D6*4 allele. When genotyping for CYP2D6*10, it is important to have a method for detecting the G1934A SNP to rule out CYP2D6*4. Aim Id: To optimize and validate a multiplex PCR method for detecting CYP2D6 gene deletion (CYP2D6*5). The frequency of CYP2D6*5 has been reported to be approximately 5.7% in Asians (Table 3). In order to increase accuracy of the determined CYP2D6 genotypes, there are two sets of samples that need to be analyzed for CYP2D6*5. The first set consists of those samples in which there are no apparent products amplified from long PCR. The second set consists of those that are genotyped as homozygous at a certain position (188 or 1934 of the CYP2D6 gene). For example, subjects who have genotypes CYP2D6*10/*5 or CYP2D6*10/*10 would both test positive for the C188T SNP. Thus, in order to differentiate between these two genotypes, CYP2D6*5 analysis would be required. 44 Steen et al. (1995a) developed a method for detecting the CYP2D6*5 gene deletion allele by long PCR. The primers used were designed to amplify a 3.5 kb PCR product in the presence of the gene deletion. Johansson et al. (1996) developed a similar method for detecting the CYP2D6*5 gene deletion, in which the primers would amplify a 6 kb fragment if there is a gene deletion. However, in both of these methods, no product would be amplified in the presence of the CYP2D6 gene. Negative reactions are difficult to interpret in the absence of a proper control. A recently published method that includes a proper control involves multiplex PCR (Hersberger et al. 2000). Multiplex PCR includes the use of more than one set of primers in the same reaction mix. In the multiplex PCR method by Hersberger et al. (2000), two sets of primers are required. A 5.1 kb fragment is amplified in the presence of the CYP2D6 gene, while a 3.5 kb fragment is amplified when the CYP2D6 gene is deleted. The advantage of this method is that at least one product will always be amplified. Also, a heterozygote for the CYP2D6*5 will be easily distinguished from the gel pattern, since both amplicons should be present. Aim 2: To genotype paediatric dental patients for CYP2D6 alleles by analyzing DNA samples for the CYP2D6 C188T and G1934A single nucleotide polymorphisms via nested real-time, rapid-cycle PCR methods, and for CYP2D6*5 via multiplex PCR. Once the real-time PCR methods for detecting the C188T and G1934A SNPs and multiplex PCR method for detecting CYP2D6*5 are validated, these methods can then be applied 45 toward genotyping for CYP2D6*10 in Asian subjects from a pilot clinical study of codeine metabolism. Individuals with CYP2D6*wt/*wt, CYP2D6*wt/*10, and CYP2D6*10/*10 genotypes can be identified. Aim 3: To devise a genotyping algorithm for the CYP2D6*10 allele in Asians. Based upon published CYP2D6 allele frequencies in Asians and the determined CYP2D6 allele frequencies in the pilot clinical study, a genotyping algorithm for CYP2D6*10 in Asians can be devised. This algorithm can then be used in future clinical studies which require the identification of the CYP2D6*wt/*wt, CYP2D6*wt/*10, and CYP2D6*10/*10 genotypes in Asians. 46 CHAPTER 2 MATERIALS AND METHODS 47 2.1 Equipment A PTC-200 peltier thermal cycler (MJ Research®; Waltham, Massachusetts) was used for conventional PCR, long PCR, and multiplex PCR. Real-time, rapid-cycle PCR and melting curve analyses for detecting the CYP2D6 C188T and G1934A SNPs were performed on a LightCycler™ instrument (Roche® Diagnostics; Laval, Quebec) using LightCycler™ capillaries (1909339). A Gene Spec™ I spectrometer (Hitachi Genetic Systems; Alameda, California) was used to estimate DNA concentrations. Agarose gel electrophoresis apparatuses were from Gibco BRL™ (Burlington, Ontario). Sequencing by the Nucleic Acid Protein Services Unit at the University of British Columbia (Vancouver, British Columbia) was performed on an Applied Biosystems PRISM® 377 automated sequencer (Foster City, California). 2 . 2 Materials 2.2.1 P C R reagents Platinum® Taq DNA polymerase with 10 mM MgCl 2 (10966-034) from Invitrogen™ (Burlington, Ontario) was used for regular PCR and real-time, rapid-cycle PCR. Platinum® Pjx DNA polymerase and 50 mM MgS04 (11708-039) from Invitrogen™ (Burlington, Ontario) was used for long chain PCR. The Expand Long Template PCR System™ (1681834) from Roche® Applied Science (Laval, Quebec) was used for multiplex long PCR. Bovine serum albumin (BSA) (B9001S) was purchased from New England Biolabs® (Pickering, Ontario). Deoxyribonucleotide triphosphate (dNTP) solutions (100 mM) of 48 dATP, dTTP, dCTP, and dGTP (27-2035-01) were purchased from Amersham Biosciences (Baie d'Urfe, Quebec). Human genomic DNA (69-237-3) used for initial method development experiments was purchased from Novagen® (Madison, Wisconsin). 2.2.2 PCR primers All primers were synthesized by the Nucleic Acid Protein Services Unit at the University of British Columbia (Table 5). Primers P100/P200 (Sachse et al. 1997) were used for the specific amplification of the CYP2D6 gene by long PCR. Primers P11/P12 (Sachse et al. 1997) were adapted to the nested real-time, rapid-cycle PCR for the method for detecting the CYP2D6 C188T SNP. Primers X3F3/X3R1 were designed by TIB MolBiol® for the nested real-time, rapid-cycle PCR method for detecting the CYP2D6 G1934A SNP. Primers Dup/Dlow and DPKup/DPKlow were used for the multiplex PCR method for detecting CYP2D6*5 (Hersberger et al. 2000). Table 5. Oligonucleotide sequences of primers. v Positions are numbered according to GenBank accession numbers ^ 33388 and JX90926.1. Primer Sequence ( 5 ' to 3 ' ) Position w P100 GGC-CTA-CCC-TGG-GTA-AGG-GCC-TGG-AGC-AGG-A 1352-1382T P200 CTC-AGC-CTC-AAC-GTA-CCC-CTG-TCT-CAA-ATG-CG 6032-6001 T Pll TCA-ACA-CAG-CAG-GTT-CA 1450-1466T P12 CTG-TGG-TTT-CAC-CCA-CC 351-335 T X3F3 TTG-GAG-TGG-GTG-GTG-GAT-G 1625-1643 T X3R1 TAT-GCA-AAT-CCT-GCT-CTT-CCG-A 2187-2166T Dup CAC-ACC-GGG-CAC-CTG-TAC-TCC-TCA 43-66 1 Dlow CAG-GCA-TGA-GCT-AAG-GCA-CCC-AGA-C 9377-9353 T DPKup GTT-ATC-CCA-GAA-GGC-TTT-GCA-GGC-TTC-A 1273-1300T DPKlow GCC-GAC-TGA-GCC-CTG-GGA-GGT-AGG-TA 6375-6350T 49 2.2.3 Fluorescent hybridization probes All fluorescent hybridization probes were synthesized by TIB MolBiol® (Adelphia, New Jersey) (Table 6). *10Anc/*10Sen probe sequences for detecting the CYP2D6 C188T SNP were designed by TIB MolBiol® (Adelphia, New Jersey), while *4Anc/*4Sen probe sequences were designed for a different real-time, rapid-cycle PCR method for detecting the CYP2D6 G1934A SNP (Bjerke et al. 2001). In Table 6, the site complementary to the position of the SNP is designated by the highlighted nucleotide in the sequences. Table 6. Oligonucleotide sequences of fluorescent hybridization probes used in real-time, rapid-cycle PCR. The highlighted nucleotides correspond to those which overlie the positions of the single nucleotide polymorphisms (position 188 or 1934). v Positions numbered according to GenBank accession number M33388. SNP Probe Sequence ( 5 ' to 3 ' ) Position v C188T *10Anc LC-Red640-GCC-CCC-TGC-CAC-TGC-CCG-G-p 1726-1744 *10Sen GCT-GCA-CGC-TAC-ICA-CCA-X 1707-1724 G1934A *4Anc LC-Red640-AGG-GGC-GTC-lTG-GGG-p 3474.346O *4Sen GTC-CAA-GAG-ACC-GTT-GGG-GCG-A-X 3497-3476 LC-Red640 = 5' LightCycler Red 640 p = 3' phosphate X = 3' fluorescein 2.2.4 DNA isolation and purification Genomic DNA was extracted using the QIAamp® Blood Mini Kit (51104) from QIAgen (Mississauga, Ontario). The QIAquick® PCR Purification Kit (28104) from QIAgen (Mississauga, Ontario) was used to purify PCR amplicons. 50 2.2.5 Molecular biology reagents Restriction enzymes BstNl (R0168S) and Hphl (R0158S) with associated buffers were purchased from New England Biolabs® (Pickering, Ontario). Details on the recognition sites of these restriction enzymes are summarized in Table 7. Agarose (15510-027) and Ultrapure™ lOx TBE Buffer (15581-028) were purchased from Invitrogen™ (Burlington, Ontario). Ultrapure™ lOx TBE Buffer was diluted to a lx working stock using purified distilled water. Ultrapure™ 10 mg/mL ethidium bromide (15585-011) was obtained from Gibco BRL™ (Burlington, Ontario) and diluted to a 1 mg/mL working stock. A solution of 10 mM Tris-HCl (pH 8.0) was made by diluting 100 mM Tris-HCl (Gibco BRL™; Burlington, Ontario) with water and adjusting the pH with HC1 or NaOH. Solutions of 100 bp DNA ladder (15628-019) and 1 kb DNA ladder (15615-016) were purchased from Invitrogen™ (Burlington, Ontario). BigDye® Terminator v3.1 ready reaction pre-mix (Applied Biosystems; Foster City, California) was obtained from the Nucleic Acid Protein Services Unit at the University of British Columbia (Vancouver, British Columbia). Table 7. Details of Hphl and BstNl restriction enzymes. Enzyme Recognition site Source Concentration Hphl 5'... GGTGA(N)8 ...3' 3'... CCACT(N)7 ...5' Recombinant (Escherichia coli strain carrying cloned Hphl gene from Haemophilus parahaemolyticus) 5,000 U/mL BstNl 5'... CC(A/T)GG ...3' 3'... GG(T/A]CC ... 5' Bacillus stearothermophilus N 10,000 U/mL 51 2.2.6 Reference samples The following reference samples were used as validation samples or controls for the methods of detecting the C188T and G1934A SNPs: CYP2D6*1/*1, CYP2D6*l/*5, CYP2D6*l/*4, CYP2D6*4/*4, and CYP2D6*4/*5. The expected polymorphisms at the 188 and 1934 positions of the CYP2D6 gene for the reference samples are displayed in Table 8. Table 8. Expected base changes at positions 188 and 1934 of the CYP2D6 gene for the CYP2D6*1/*1, CYP2D6*l/*5, CYP2D6*l/*4, CYP2D6*4/*4, and CYP2D*4/*5 reference samples. CYP2D6 genotype 188 position 1934 position CYP2D6*1/*1 C G CYP2D6*l/*5 C G CYP2D6*l/*4 C/T G/A CYP2D6*4/*4 T A CYP2D6*4/*5 T A The following reference samples were used for validating the method for CYP2D6*5 detection: CYP2D6*1/*1, CYP2D6*l/*5, and CYP2D6*5/*5. The CYP2D6*5/*5 sample was a gift from Dr. Ulrich Zanger (Dr. Margarete Fischer-Bosche Institute of Clinical Pharmacology, Stuttgart, Germany). All other reference samples (CYP2D6*1/*1, CYP2D6*l/*5, CYP2D6*l/*4, CYP2D6*4/*4, and CYP2D6*4/*5) were kindly provided by Dr. Leszek Wojnowski (Department of Clinical Pharmacology, University Goettingen, Goettingen, Germany). 52 2.2.7 Patient samples From October 02, 2001 to August 30, 2002, 79 paediatric patients undergoing dental procedures at B . C . Children's Hospital were recruited for a pilot clinical study on the pharmacogenetics of codeine metabolism to morphine by C Y P 2 D 6 . Out of the 79 patients recruited, one patient received a dose of morphine for pain control; this may have confounded the results from quantification of plasma morphine levels. Thus, 78 patients were included as study subjects, o f whom 42 were Caucasians and 36 were Asians. Copies of the consent forms in English and Chinese are shown in Appendices F and G , respectively. A copy of the Certificate of Approval from the University of British Columbia Clinical Research Ethics Board is shown in Appendix H . The demographics of the 36 Asian study subjects are listed in Table 9. The ethnicities of the study subjects were self-reported, and also determined by an interview regarding the origins of family members from three generations (the places of birth and mother languages of study subjects and their parents, as well as the places of birth of their grandparents). Out of the 36 Asian subjects, the following ethnicities were assigned: 27 Chinese (from China, Hong Kong, Macau, Taiwan), 4 Vietnamese, 2 Chinese/Vietnamese, 1 Taiwanese/Korean, 1 Chinese/Canadian, and 1 Japanese. Table 9. Demographics of the 36 Asian study subjects involved in the pilot clinical study of codeine metabolism (23 Males, 13 Females). Mean ± standard deviation Age 5.3 ± 2.8 years Weight 19.5 ± 6 . 7 kg Acetaminophen dose 3 9 4 ± 1 3 0 m g Codeine dose 28.9 ± 9.7 mg Codeine dose per weight 1.5 ± 0 . 2 mg/kg 53 At the dental clinic, each patient received a routine preoperative oral dose of codeine (1.5 mg/kg) and acetaminophen (20 mg/kg). After obtaining informed, written parental or guardian consent, venous blood samples (2-3 mL) were drawn at one hour after the codeine dose. Blood samples were centrifuged to separate serum and cell fractions. The cell fractions were provided to us and stored in EDTA tubes at -70°C before isolation of the genomic DNA. 2.3 Specific methods 2.3.1 Long PCR Long PCR of the CYP2D6 gene in a 50 uL PCR mix was optimized using Platinum® Pfic D N A polymerase (Tables 10 and 11). Table 10. Reaction mix for amplification of the CYP2D6 gene by long PCR. Reagent Stock cone. Volume Final cone. Water - 37.8 uL -Platinum® Pfic amplification buffer 10X 5.0 uL I X M g C l 2 50 mM 0.8 uL 0.8 m M dNTP mix 10 m M 1.5 uL 0.3 m M each Primer PI00 10 uM 1.0 \xL 0.2 uM Primer P200 10 uM 1.0 uL 0.2 p M Platinum® Pfic D N A polymerase 2.5 U/uL 0.4 uL 1 unit The volumes of this reaction mix were multiplied according to the number of samples analyzed; 47.5 uL of this larger reaction mix was then used in the final 50 uL PCR mix for each sample (2.5 uL of 20 ng/uL genomic D N A was used as the template). 54 Table 11. Thermocycling conditions for amplification of the CYP2D6 gene by long PCR withP100/P200 primers. Program Temperature Hold time (min: sec) Initial denaturation 94°C 2:00 31 cycles of 94°C 0:30 68°C 5:00 Terminal extension 68°C 7:00 Cooling 4°C Hold The products were analyzed on 1.0% agarose gels to check for the presence of a band representing 4681 bp (i.e. the P100/P200 amplicon). The products were subsequently diluted with 5 volumes of 10 mM Tris HC1 (pH 8). The diluted P100/P200 amplicons were stored at 4°C prior to use as the template for nested analyses for the CYP2D6 C188T and G1934A SNPs. 2.3.2 Nested real-time, rapid-cycle P C R method for detecting the C188T SNP A method for detecting the C188T SNP using nested real-time, rapid-cycle PCR was developed and optimized. In order to maintain consistency between runs, a pre-PCR mix was prepared, which contained all PCR reagents except primers, fluorescent probes, and template (Table 12). Nested real-time PCR and melting curve analyses were performed in a 20-uL reaction mix (Table 13) using the thermocycling conditions shown in Table 14. 55 Table 12. Pre-PCR mix for detecting the CYP2D6 C188T SNP by nested real-time, rapid-cycle PCR. Reagent Stock cone. Volume x50 Final cone. Platinum® Taq amplification buffer 10X 2.0 pL 100 uL IX MgCl 2 50 mM 1.6 pL 80 pL 4.0 mM dNTP mix 10 mM 0.4 pL 20 pL 0.2 mM each Bovine serum albumin 2.5 mg/mL 2.0 pL 100 pL 0.25 mg/mL Platinum® Taq DNA polymerase 5 U/pL 0.2 pL 10 pL 1 unit 6.2 pL of this pre-PCR mix was used in each 20 pL 3CR reaction mix. Table 13. Reaction mix for detecting the CYP2D6 C188T SNP by nested real-time, rapid-cycle PCR. Reagent Stock cone. Volume Final cone. Water - 5.8 pL 0.25 mg/mL Pre-PCR mix - 6.2 pL -Primer 2D6P11 10 pM 1.0 pL 0.5 pM Primer 2D6 P12 10 pM 1.0 pL 0.5 pM Fluorescein probe - 2D6 * lOSen 2pM 2.0 pL 0.2 pM LC-Red 640 probe - 2D6 * lOAnc 2 pM 2.0 pL 0.2 pM The volumes of this reaction mix were multiplied accorc analyzed; 18 pL of this larger reaction mix was then use for each sample (2.0 pL of diluted P100/P200 amplicon ing to the number of samples d in the final 20 pL PCR mix was used as the template). Table 14. Thermocycling and melting curve conditions for detecting the CYP2D6 C188T SNP by nested real-time, rapid-cycle PCR. Program Temperature Hold time (min:sec) Transition Acquisition Initial denaturation 95°C 5:00 20°C/sec none 40 cycles of 95°C 0:05 20°C/sec none 63°C 0:10 20°C/sec single 72°C 0:20 20°C/sec none Melting curve 97°C 0:30 20°C/sec none 50°C 2:00 20°C/sec none 45°C 2:00 20°C/sec none 40°C 2:00 20°C/sec none 85°C 0:00 0.2°C/sec continuous Cooling 40°C 0:30 20°C/sec none 56 The rate of temperature change for each cycle during PCR was set at 20°C per second. During PCR, fluorescence signals were acquired at the annealing temperature of 63°C (single acquisition). Immediately following 40 cycles of PCR, melting curve analysis was performed, in which the P11/P12 amplicons were first denatured by increasing the temperature to 97°C for 30 seconds. Subsequently, the temperature was decreased in increments to 40°C (i.e. 97°C to 50°C, then to 45°C, then to 40°C; all at a rate of 20°C/sec) to allow the fluorescent hybridization probes to anneal to the P11/P12 amplicons. Upon increasing the temperature to 85°C at a slower rate (0.2°C per second), the probes melted off at a temperature dependent on their annealing strength. These changes were monitored in real time by continuously acquiring fluorescence signals (continuous acquisition). There was no need to hold the temperature at 85°C; hence, the hold time was set for 0:00. For a general summary of the real-time, rapid-cycle PCR method for detecting the CYP2D6 C188T SNP in the format of a schematic, please refer to Appendix C. 2.3.3 Restriction fragment length polymorphism analysis for detecting the C188T SNP Products from real-time PCR were analyzed on 2.0% agarose gels to check for the presence of a single band representing a 433 bp P11/P12 amplicon, before purification with the QIAquick® PCR Purification Kit (used according to the manufacturer's instructions). The purified P11/P12 amplicons were digested using the Hphl restriction enzyme and analyzed on a 2.5%) agarose gel (Table 15). A negative control was included, in which an identical reaction mix was prepared with water in place of the restriction enzyme. 57 Table 15. Reaction mix for detecting the CYP2D6 C188T SNP by restriction fragment length polymorphism analysis of PI 1/P12 amplicons. Reagent Stock cone. Volume Final cone. New England Biolabs® buffer 4 10X 1.0 uL IX Hphl (or water) 5U/pX 0.6 uL 3 U Purified PI 1/P12 amplicon - 8.4 uL -The reaction mix was incubated at 37°C for 17 hours; the restriction products were then analyzed using a 2.5% agarose gel. Figure 7 illustrates the principle of restriction digestion by the Hphl restriction enzyme. The Hphl restriction enzyme recognizes the 5'-TCACC-3' sequence, and will cleave seven nucleotides upstream of this recognition site. In the 433 bp P11/P12 amplicon from a wild-type allele, there should be one Hphl restriction site, and digestion of this wild-type amplicon is expected to result in two fragments (362 bp and 71 bp). The C188T SNP should create an additional 5'-TCACC-3' restriction site (5'-j§CACC-3' is changed to 5'-|CACC-3'), and digestion of a mutant allele is expected to result in three fragments (262 bp, 100 bp, and 71 bp) (Sachse et al. 1997). 58 (a) HphI recognizes the 5'-TCACC-3' sequence 5'-nn/nnnnnnnTCACC-3' 3'-n^nnnnnnnAGTGG-5' Hph\ recognizes the 5'-TCACC-3' sequence 5 ' -nrvnnn- nr . ( - 3 ' 3'-iPSHBBKP5' (c) Hph\ cleaves 7 nucleotides upstream the 5'-TCACC-3' sequence 5'-nn nnnnnnnTCACC-3' 3'-n nnnnnnnnAGTGG-5' Figure 7. Principle of restriction enzyme digestion using Hphl. (a) and (b) The Hphl restriction enzyme recognizes the 5'-TCACC-3' sequence; (c) Hphl cleaves 7 nucleotides upstream of the 5'-TCACC-3' recognition site. 59 2.3.4 Nested real-time, rapid-cycle PCR method for detecting the G1934A SNP New primer sequences (X3F3/X3R1; Table 5) were used in the nested real-time, rapid-cycle PCR method for detecting the G1934A SNP with published fluorescent hybridization probes (Bjerke et al. 2001) (*4Anc/*4Sen; Table 6). A pre-PCR mix was prepared (Table 16) for use in the final PCR reaction mix (Table 17). The thermocycling protocol is summarized in Table 18. For a general summary of the real-time, rapid-cycle PCR method for detecting the CYP2D6 G1934A SNP in the format of a schematic, please refer to Appendix D. Table 16. Pre-PCR mix for detecting the CYP2D6 G1934A SNP by nested real-time, rapid-cycle PCR. Reagent Stock cone. Volume x50 Final cone. Platinum® Taq amplification buffer 10X 2.0 pL 100 pL IX MgCl 2 50 mM 0.8 pL 80 pL 2.0 mM dNTP mix 10 mM 0.4 pL 20 pL 0.2 mM each Bovine serum albumin 2.5 mg/mL 2.0 pL 100 pL 0.25 mg/mL Platinum® Taq DNA polymerase 5U/pL 0.2 pL 10 pL 1 unit 5.4 pL of this pre-PCR mix was used in each 20 pL 3CR reaction mix. Table 17. Reaction mix for detecting the CYP2D6 G1934A SNP by nested real-time, rapid-cycle PCR. Reagent Stock cone. Volume Final cone. Water - 7.0 pL -Pre-PCR mix - 5.4 pL -Primer 2D6 X3F3 10 pM 0.8 pL 0.4 pM Primer 2D6 X3R1 10 pM 0.8 pL 0.4 pM Fluorescein probe - 2D6 *4Sen 2 pM 2.0 pL 0.2 pM LC-Red 640 probe - 2D6 *4Anc 2pM 2.0 pL 0.2 pM The volumes of this reaction mix were multiplied accorc analyzed; 18 pL of this larger reaction mix was then use for each sample (2.0 pL of diluted P100/P200 amplicon ing to the number of samples d in the final 20 pL PCR mix was used as the template). 60 Table 18. Thermocycling and melting curve conditions for detecting the CYP2D6 G1934A SNP by nested real-time, rapid-cycle PCR. Program Temperature Hold time (minrsec) Transition Acquisition Initial denaturation 95°C 5:00 20°C/sec none 35 cycles of 95°C 0:10 20°C/sec none 60°C 0:12 20°C/sec single 72°C 0:24 20°C/sec none Melting curve 97°C 0:30 20°C/sec none 50°C 2:00 20°C/sec none 40°C 2:00 20°C/sec none 30°C 2:00 20°C/sec none 80°C 0:00 0.2°C/sec continuous Cooling 40°C 0:30 20°C/sec none 2.3.5 Restriction fragment length polymorphism analysis for detecting the G1934A SNP The products from real-time PCR were analyzed on 2.0% agarose gels to check for the presence of a single band representing a 563 bp X3F3/X3R1 amplicon before purification with the QIAquick® PCR Purification Kit. These purified amplicons were digested using the BsiNI restriction enzyme and analyzed using 2.5% agarose gels (Table 19). Table 19. Reaction mix for detecting the CYP2D6 G1934A SNP by restriction fragment length polymorphism analysis of X3F3/X3R1 amplicons. Reagent Stock cone. Volume Final cone. New England Biolabs® buffer 2 10X 1.0 uL IX Bovine serum albumin 10X 1.0 uL IX BsiNI (or water) 1 U/uL 1.0 uL 1 u Purified X3F3/X3R1 amplicon - 7.0 uL -The reaction mix was incubated at 60°C for 60 minutes; the restriction products were then analyzed using a 2.5% agarose gel. According to the sequence of the X3F3/X3R1 amplicon from a wild-type allele, there should be four BsiNI restriction sites, and digestion of the amplicon is expected to result in 61 five fragments (255 bp, 161 bp, 69 bp, 67 bp, and 11 bp). On a 2.5% agarose gel, the 11 bp fragment would not be visible, while the 69 bp and 67 bp are indistinguishable. Thus, three bands should be visible. 2.3.6 Multiplex P C R method for detecting CYP2D6*5 The CYP2D6 gene deletion (CY2D6*5) detected by multiplex PCR using two sets of primers: DPKup/DPKlow and Dup/Dlow (Table 5). Different reagent concentrations and a different cycling program were used than those described by Hersberger et al. (2000). The final reagent concentrations and thermocycling program of the optimized multiplex PCR are shown in Tables 20 and 21, respectively. Table 20. Reaction mix for detecting the CYP2D6 gene deletion by multiplex PCR. Reagent Stock cone. Volume Final cone. Water - 13.66 pL Expand™ buffer 1 10X 2.00 pL IX MgCb (contained in buffer 1) 17.5 mM - 1.75 mM dNTP mix 10 mM 0.70 pL 0.35 mM Primer Dup 10 pM 0.22 pL 0.11 pM Primer Dlow 10 pM 0.22 pL 0.11 pM Primer DPKup 10 pM 0.50 pL 0.25 pM Primer DPKlow 10 pM 0.50 pL 0.25 pM Expand™ enzyme mix 5.0U/pL 0.20 pL 1.0 U The volumes of this reaction mix were multiplied according to the number of samples analyzed; 18 pL of this larger reaction mix was then used in the final 20 pL PCR mix for each sample (2.0 pL of 20 ng/pL genomic DNA was used as the template). 62 Table 21. Thermocycling conditions for detecting the CYP2D6 gene deletion by multiplex PCR. Program Temperature Hold time (min:sec) Initial denaturation 94°C 5:00 35 cycles of 94°C 0:15 68°C 3:30 Terminal extension 68°C 7:00 Cooling 4°C Hold The PCR products were analyzed using 1.0% agarose gels. A 5.1 kb DPKup/DPKlow product should be amplified in the presence of the CYP2D6 gene, while a 3.5 kb Dup/Dlow product should be amplified when the CYP2D6 gene is deleted. 2.3.7 Validation of the genotyping methods Figure 8 outlines the procedure for validation of the methods for detecting the C188T and G1934A SNPs using RFLP and sequence analyses. Following long PCR and nested real-time, rapid-cycle PCR of reference samples (CYP2D6*1/*1, CYP2D6*l/*4, and CYPD6*4/*4; Table 8), the products were analyzed for the particular SNP using melting curve analysis with fluorescent hybridization probes. Products following real-time PCR of the reference samples were purified using the QIAquick® PCR Purification Kit for further validation using RFLP and sequence analyses. In order to validate the multiplex PCR method for detecting the CYP2D6 gene deletion (CYP2D6*5), reference samples with the following genotypes were analyzed: CYP2D6*1/*1, CYP2D6*l/*5, and CYP2D6*5/*5. 63 Reference Samples I Long PCR • CYP2D6 Gene I Real-time rapid-cycle PCR using P11/P12 orX3F3/X3R1 P11/P12orX3F3/X3R1 PCR Amplicons I I ( Detection of the C188T or G1934A single nucleotide polymorphism + Melting Curve RFLP Sequence Analysis Analysis Analysis Figure 8. Flowchart summarizing the validation procedure for the methods of detecting the C188T and G1934A SNPs by nested real-time, rapid-cycle PCR. Following long PCR to specifically amplify the CYP2D6 gene from reference samples (CYP2D6*1/*1, CYP2D6*l/*4, and CYP2D6*4/*4), nested real-time PCR with either P11/P12 or X3F3/X3R1 primers was used to amplify a product to detect either the C188T or G1934A SNP, respectively. Following real-time PCR, melting curve analysis was used to detect the C188T or G1934A SNP using fluorescent hybridization probes. Amplicons from real-time PCR of reference samples were also purified and further validated by RFLP and sequence analyses. 64 2.3.8 Genotyping for CYP2D6*10 in subject samples When analyzing subject samples for the C188T and G1934A SNPs by nested real-time, rapid-cycle PCR, all samples were analyzed in duplicate. For the detection of the C188T SNP, the following reference samples were analyzed simultaneously: CYP2D6*1/*1 (C at position 188), CYP2D6*l/*5 (C at position 188), CYP2D6*l/*4 (C and T at position 188), and CYP2D6*4/*5 (T at position 188). For the detection of the G1934A SNP, the following reference samples were analyzed simultaneously: CYP2D6*1/*1 (G at position 1934), CYP2D6*l/*5 (G at position 1934), CYP2D6*l/*4 (G and A at position 1934), and CYP2D6*4/*5 (A at position 1934). For the detection of CYP2D6*5 using multiplex PCR, subject samples were analyzed once. Reference samples with genotypes CYP2D6*1/*1 and CYP2D6*l/*5 were included as controls. If results were not interpretable (i.e. bands not visible due to a negative PCR, or bands not easily distinguishable due to an improperly prepared agarose gel), samples were analyzed a second time. 2.4 Statistical methods The observed frequency of a determined CYP2D6*X allele in a sample of N subjects was estimated by (2nx / x + n\ / -) / 2N, where n x / x is the number of subjects homozygous for CYP2D6*X, and nx/ - is the number of subjects heterozygous for CYP2D6*X. 65 Confidence intervals for CYP2D6 allele and genotype frequencies were calculated using the binomial method from the Number Cruncher Statistical System software program (NCSS Statistical Software; Kaysville, Utah). 2.5 General methods 2.5.1 Estimation of DNA concentration DNA concentrations of genomic DNA and primers were estimated by absorbance (Abs) at 260 nm of a 1/3 0th dilution using the Gene Spec™ I spectrometer. dsDNA concentration (pg/mL) = Abs26o x 30 x 50 pg/mL ssDNA concentration (pM) = Abs26o x 30 x 33 pg/mL x 1/350 x 1 /length x 1000 2.5.2 Preparation of agarose gels Table 22 shows the amounts of IX TBE buffer and agarose used in the preparation of various concentrations of agarose gels. For RFLP analysis, 2.0% or 2.5% agarose gels were used. For analysis of products from long PCR or multiplex long PCR, 1.0% agarose gels were used. For other analyses, 2.0% agarose gels were used. Table 22. Amount of agarose used in the preparation of agarose gels. Concentration o 'agarose Gel size Volume 1.0% 2.0% 2.5% Small 30 mL 0.30 g 0.60 g 0.75 g Large 100 mL DNP v DNP 2.50 g v DNP = did not prepare The appropriate amount of agarose was weighed and transferred to an Erlenmeyer flask. Subsequently, 30 mL or 100 mL IX TBE buffer was added to the flask. The agarose was 66 dissolved by heating the solution in a microwave for approximately 1 minute. After cooling down the mixture (warm to touch), ethidium bromide (1 mg/mL stock) was added (2.4 pL for small gels; 8 pL for large gels). The solution was mixed and poured into the gel apparatus with the appropriate comb required for the experiment. After the gel was firm, the comb and mould wedges of the gel apparatus were removed. Gels were either run immediately or stored at 4°C until required. 2.5.3 Gel electrophoresis Running buffer (IX TBE buffer) was added to the gel box until the agarose gel was covered. To prepare the samples, 6X loading buffer was added to each sample. For the small gels (30 mL total volume), 2 pL of loading buffer was added to 10 pL of sample. For the large gels (100 mL total volume), 4 pL of loading buffer was added to 20 pL of sample. The samples were carefully pipetted into the wells of the agarose gel. Voltage (75 V or 100 V) was applied to allow the products to migrate across the gel (50, 60, or 100 minutes for small gels; 2 hours for large gels). The gels were viewed using ultraviolet light and photographed. 2.5.4 Sequence analysis Purified PCR products were sequenced using BigDye® Terminator v3.1 (Tables 23 and 24). The P l l primer was used for the P11/P12 amplicon, while the X3F3 primer was used for the X3F3/X3R1 amplicon. 67 Table 23. PCR reaction mix for sequence analysis using BigDye® Terminator v3.1. Reagent Stock cone. x l Final cone. BigDye® Terminator v3.1 pre-mix - 4.0 uL -One primer 3.2 uM 1.0 uL 3.2 pmol Amplicon 20 ng/uL 1.0 uL 20 ng Water - 14.0 uL -Table 24. Thermocycling conditions for sequence analysis using BigDye® Terminator v3.1. Program Temperature Hold time (min:sec) Initial denaturation 95°C 5:00 25 cycles of 96°C 0:30 50°C 0:15 60°C 4:00 Cooling 4°C Hold The extension products were purified by ethanol precipitation. Table 25 lists the volumes used for ethanol precipitation of 20 uL of product. Sodium acetate and ethanol were first added to a 1.5 mL microcentrifuge tube, followed by the entire 20 uL volume of product. This solution was mixed using a vortex, placed on ice for 10 minutes, and centrifuged at maximum speed (13,000 rpm) in a microcentrifuge for 20 minutes. The ethanol solution was immediately and carefully aspirated (Kimwipes were used to remove ethanol from the sides of the tubes). The tubes were left opened to dry in the fumehood for 5-10 minutes. After drying, the tubes were capped and stored at -20°C before submission to the Nucleic Acid Protein Services Unit for sequencing. Table 25. Reagent volumes for ethanol precipitation of extension products. Reagent Stock cone. Volume Sodium acetate, pH 5.2 3 M 2 uL 95% Ethanol - 50 uL Product - 20 uL 68 2.5.5 Isolation and purification of genomic DNA from subject samples Genomic DNA was extracted from 200 pL of the cell fraction using the QIAamp® Blood Mini Kit from QIAGEN®. The manufacturer's instructions were followed: Step 1: Lyse 20 pL of QIAGEN protease was pipetted into the bottom of 1.5 mL microcentrifuge tubes. 200 pL of the cell fractions were added to each microcentrifuge tube, and 200 pL of Buffer AL was added to each sample. The solutions were mixed for 15 seconds and incubated in a 56°C water bath for 10 minutes. Step 2: Bind 200 pL of ethanol was then added to each sample. The samples were mixed, applied into separate QIAamp spin columns (in 2 mL collection tubes), and centrifuged at 8,000 rpm for 1 minute. The collection tubes containing the filtrates were discarded. Step 3: Wash 500 pL of Buffer AW1 was added to each QIAamp spin column, and the samples were centrifuged at 8,000 rpm for 1 minute (using new collection tubes). The collection tubes containing the filtrate were discarded. 500 pL of Buffer AW2 was added to each QIAamp spin column, and the samples were centrifuged at 14,000 rpm for 3 minutes. After discarding the filtrates, the spin columns were centrifuged at 14,000 rpm for 1 minute. The QIAamp spin columns were placed in clean 1.5 mL microcentrifuge tubes before elution. 69 Step 4: Elute 200 pL of distilled purified water was added to each spin column. The samples were incubated at room temperature for 5 minutes, and centrifuged at 8,000 rpm for 1 minute. The concentration of the eluted genomic DNA solutions were then estimated using the Gene Spec™ I spectrometer (as outlined in section 2.5.1). The genomic DNA solutions were diluted to 20 ng/pL working solutions and stored at -20°C until use. 70 CHAPTER 3 RESULTS 3.1 CYP2D6 genotyping methods Optimization of the specific amplification of the CYP2D6 gene was completed. Novel nested real-time, rapid-cycle PCR methods for detecting the CYP2D6 C188T and G1934A SNPs were developed and validated in order to genotype for the CYP2D6*10 and CYP2D6*4 alleles. A multiplex PCR method for detecting the CYP2D6*5 gene deletion was also optimized and validated. 3.1.1 Amplification of the CYP2D6 gene by Long P C R Optimization of long PCR using P100/P200 primers (Sachse et al. 1997) and Platinum® Pfic DNA polymerase was successful. Figure 9 illustrates a typical 1.0% agarose gel showing P100/P200 amplicons containing the CYP2D6 gene from the CYP2D6*1/*1 (lane 2), CYP2D6*l/*4 (lane 3), and CYP2D6*4/*4 (lane 4) reference samples. The P100/P200 amplicon is expected to be 4681 bp in length, and should contain all nine exons of the CYP2D6 gene. The PCR amplicons were diluted with five parts of 10 mM Tris-HCl (pH 8.0) and stored at 4°C before being used as the template in the nested, real-time, rapid-cycle PCR methods for detecting the C188T and G1934A SNPs. 72 1kb CYP CYP CYP DNA 2D6 2D6 2D6 Ladder *1f1 *1/*4 *4T4 m 4 n ? 2 b o " » " 4 6 8 1 b P P100/P200 3054bp- % amplicon 2036 bp-1836 bp-1018 bp -Lane 1 2 3 4 Figure 9. Long PCR of the CYP2D6 gene from reference samples with genotypes CYP2D6*1/*1, CYP2D6*l/*4, and CYP2D6*4/*4. The size of the expected P100/P200 amplicon is 4681 bp. The small 1.0% agarose gel was run at 75 V for 100 minutes. Lane 1: 1 kb DNA ladder; lane 2: CYP2D6*1/*1 reference sample; lane 3: CYP2D6*l/*4 reference sample; and lane 4: CYP2D6*4/*4 reference sample. 73 3.1.2 Nested real-time, rapid-cycle PCR method for detecting the CYP2D6 C188T SNP The analysis for the CYP2D6 C188T SNP will allow identification of the CYP2D6*10 allele in Asians (if negative for the G1934A base change characteristic of CYP2D6*4). Nested real-time, rapid-cycle PCR with the PI 1/P12 primers was successful. RFLP and sequence analyses of P11/P12 amplicons from reference samples were used to validate the results obtained from real-time PCR. Figures 10a - 10c illustrate the validation of the real-time PCR method for detecting the C188T SNP in the CYP2D6*1/*1 reference sample. All three methods were consistent with the designated genotype of the reference sample. The CYP2D6*1/*1 reference sample is expected to be homozygous for C at position 188 of the CYP2D6 gene. After nested amplification using P11/P12 primers, melting curve analysis was performed with the *10Sen/*10Anc fluorescent hybridization probes. As seen in Figure 10a, a melting peak temperature (Tm) of 67-68°C is representative of a C at position 188. In Figure 10b, the results from RFLP analysis of the PI 1/P12 amplicon from the CYP2D6*1/*1 reference sample are shown. The wild-type 433 bp P11/P12 amplicon is expected to contain only one Hphl restriction site, and should be cleaved into two fragments, 362 bp and 71 bp in size, when digested with the Hphl restriction enzyme. A single band representing 433 bp PI 1/P12 amplicons (expected size before digestion) is visible in lane 2, but in lane 3 (after digestion) only the band containing 362 bp fragments is visible, since the band representing the 71 bp fragments is too faint. Finally, the sequence analysis (Figure 10c) of purified P11/P12 amplicons confirmed the presence of a C at position 188 of the CYP2D6 gene in this reference sample. 74 (a) (b) (c) Figure 10. Analysis for the CYP2D6 C188T SNP in the CYP2D6*1/*1 reference sample. A 433 bp product was amplified by real-time, rapid-cycle PCR with primers PI 1/P12. A l l three genotyping methods indicated a C at position 188: (a) Melting curve analysis of PI 1/P12 amplicons (duplicates) using *10Sen/*10Anc fluorescent hybridization probes, showing melting peaks at 67-68°C; (b) Restriction fragment length polymorphism analysis with Hphl of purified P11/P12 amplicons from real-time PCR; digestion of the 433 bp product resulted in 362 bp and 71 bp fragments (the 71 bp fragment is too faint to be seen) (lane 3). The small 2.0% agarose gel was run at 75 V for 60 minutes; (c) Sequence analysis of purified P11/P12 amplicons from real-time PCR, indicating a C at position 188. 75 Due to the low frequency of the CYP2D6*10 allele in Caucasians (Bradford 2002), a CYP2D6*10/*10 reference sample was not readily available from our colleagues in Germany. However, the CYP2D6*4 allele also contains the C188T SNP, and thus a CYP2D6*4/*4 reference sample was used to validate the real-time PCR method for detecting the C188T SNP. Figures 11a - 11c illustrate the results for the detection of the C188T SNP in the CYP2D6*4/*4 reference sample. The *10Sen hybridization probe sequence was designed to be complementary to the wild-type sequence near the 188 position. In the CYP2D6*4/*4 sample, due to the single base change to a T at position 188, there should be a single base mismatch when the *10Sen hybridization probe anneals. Since the hybridization probe should melt off at a temperature dependent on the annealing strength to the PI 1/P12 amplicon, a lower Tm is expected when analyzing a mutant strand. Indeed, as shown in Figure 11a, the melting peak temperature for the CYP2D6*4/*4 sample (T at position 188 of both alleles) was 58-59°C. The C188T SNP resulted in a weaker annealing strength of the fluorescent hybridization probe, and thus a lower Tm. As mentioned above, the Tm was 67-68°C when C is detected at position 188 (Figure 10a). The C188T base change should create an additional 5'-...TCACC...-3' Hphl restriction site in the 433 bp P11/P12 amplicon (nucleotide 188 is highlighted). Digestion of this product using Hphl is expected to result in three fragments: 262 bp, 100 bp, and 71 bp in size. Before digestion, a single band representing 433 bp P11/P12 amplicons is seen (Figure 1 lb; lane 2). After digestion of 433 bp PI 1/P12 amplicons (Figure 1 lb; lane 3), the 262 bp fragments can be seen clearly, while the two fainter 100 bp and 71 bp fragments are more difficult to discern, but are still detectable. The sequence analysis of P11/P12 76 amplicons (Figure 11c) indicated a T at position 188, confirming the results obtained from melting curve and RFLP analyses. For the heterozygous reference sample, CYP2D6*l/*4, all three C188T SNP detection methods were consistent with each other; both C and T were detected at position 188 (Figures 12a - 12c). The Tms of 58-59°C and 67-68°C during melting curve analysis indicated the presence of two different alleles (Figure 12a). Since there are P11/P12 amplicons generated from two different alleles after PCR, all four fragments of 362 bp, 262 bp, 100 bp, and 71 bp should be present after digestion with Hphl. As expected, only a single band representing 433 bp P11/P12 amplicons is seen for the undigested sample (Figure 12b; lane 2). After digestion, only the 362 bp and 262 bp fragments can be seen clearly; the 100 bp and 71 bp fragments are more diffuse and faint (Figure 12b; lane 3). From the sequence analysis (Figure 12c), we can see that at position 188, both C and T were detected, which confirmed the results obtained from melting curve and RFLP analyses. 77 (c) Figure 11. Analysis for the CYP2D6 C188T SNP in the CYP2D6*4/*4 reference sample. A 433 bp product was amplified by real-time, rapid-cycle PCR with primers PI 1/P12. All three genotyping methods indicated a T at position 188; (a) Melting curve analysis of P11/P12 amplicons (duplicates) using *10Sen/*10Anc hybridization probes, showing melting peaks at 58-59°C; (b) RFLP analysis with Hphl of purified P11/P12 amplicons from real-time PCR; digestion of the 433 bp products resulted in 262 bp, 100 bp, and 71 bp fragments (the 100 bp and 71 bp fragments are faint) (lane 3). The small 2.0% agarose gel was run at 75 V for 60 minutes. The 100 bp DNA ladder was moved from the edge of the original gel for ease of size comparison; (c) Sequence analysis of purified PI 1/PI2 amplicons from real-time PCR. indicating a T at position 188. 78 0.003 0.002 i u o c a> o to o 3 0.001 -0.001 — Negative Control — CYP2D6*1/'4 58 60 62 64 66 Temperature (°C) 68 70 72 74 76 (a) CYP206'1/'4 100 bp P11/P12 DNA Hphl Ladder - + 600 bp 500 bp 400 bp - | 300 bp -200 bp -100 bp-Lane 1 (b) G C T A C | C A C C A 188 (C) Figure 12. Analysis for the CYP2D6 C188T SNP in the CYP2D6*l/*4 reference sample. A 433 bp product was amplified by real-time, rapid-cycle PCR with primers P11/P12. All three genotyping methods indicated C and T at position 188: (a) Melting curve analysis of P11/P12 amplicons (duplicates) using *10Sen/*10Anc hybridization probes, showing two melting peaks at 58-59°C and 67-68°C; (b) RFLP analysis with Hphl of purified PI 1/P12 amplicons from real-time PCR; digestion of the 433 bp products resulted in 362 bp, 262 bp, 100 bp, and 71 bp fragments (the 100 bp and 71 bp fragments are faint) (lane 3). The small 2.0% agarose gel was run at 75 V for 60 minutes; (c) Sequence analysis of purified PI 1/P12 amplicons from real-time PCR, indicating both C and T at position 188. 79 3.1.3 Nested real-time, rapid-cycle PCR method for detecting the CYP2D6 G1934A SNP If we analyze only for the C188T SNP in Asians, then we would not be able to differentiate the CYP2D6*10 and CYP2D6*4 alleles because both alleles contain the C188T SNP. The frequency of the CYP2D6*4 allele has been reported to be between 0.0-6.7% (Appendix E) in Asians. We can thus increase the accuracy of the determined CYP2D6*10 alleles by ruling out the presence of the CYP2D6*4 allele. Of the two alleles, only the CYP2D6*4 allele contains the G1934A base change. Therefore, we can differentiate the two alleles by analyzing for both the C188T and G1934A SNPs. Nested real-time, rapid-cycle PCR using the X3F3/X3R1 primers and the *4Sen/*4Anc fluorescent hybridization probes was optimized and validated. The melting curve results for detection of the CYP2D6 G1934A SNP in all three reference samples are shown in Figure 13. The analysis of the CYP2D6*1/*1 reference sample (G at position 1934) resulted in a Tm of 61-62°C, while analysis of the CYP2D6*4/*4 reference sample (A at position 1934) shifted the melting peak to 43-44°C. A reproducible small shoulder is notable in this reference sample. Analysis of the heterozygous CYP2D6*J/*4 reference sample (G/A at position 1934) resulted in melting peaks at both temperatures, as expected. 80 CM L . 0 O c CD O (B L . O 3 -0.001 -0.002 -i 1 1 r 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 Temperature (°C) Figure 13. Analysis for the CYP2D6 G1934A SNP in the CYP2D6*1/*1, CYP2D6*l/*4, and CYP2D6*4/*4 reference samples by melting curve analysis of X3F3/X3R1 amplicons. The 563 bp products were amplified by real-time, rapid-cycle PCR with primers X3F3/X3R1, followed by melting curve analysis. There is no signal from the negative control (water in place of genomic DNA), as expected. For the CYP2D6*1/*1 reference sample, a melting peak was detected at 61-62°C, indicating a wild-type G at position 1934 of the CYP2D6 gene. For the CYP2D6*4/*4 reference sample, a melting peak was detected at 43-44°C, representing a base change to A at the 1934 position. For the CYPD6*l/*4 reference sample, melting peaks at both 43-44°C and 61-62°C were detected, indicating that the sample is heterozygous for the G1934A SNP. 81 Figure 14 displays the results of restriction analyses of the CYP2D6 X3F3/X3R1 amplicons using the BsiNI restriction enzyme. The BsiNI enzyme cleaves at any 5'...CC(A/T)GG...3' recognition site. For the X3F3/X3R1 amplicons, there should be four BsiNI restriction sites in the wild-type allele; after digestion of the 563 bp amplicon there should be five fragments: 255 bp, 161 bp, 69 bp, 67 bp, and 11 bp. As seen in lane 2 of the 2.5% agarose gel, only three fragments are visible after digestion of the X3F3/X3R1 amplicons from the CYP2D6*1/*1 reference sample. The 69 bp and 67 bp bands are indistinguishable, and the 11 bp band is too small to visualize. The G1934A base change should abolish one of the four restriction sites, 5'...CC(A/T)lG...3' (nucleotide 1934 is highlighted). With only three BstNI restriction sites remaining, digestion of the X3F3/X3R1 amplicons from a CYP2D6*4/*4 reference sample should result in the following four fragments: 416 bp, 69 bp, 67 bp, and 11 bp. In the 2.5%> agarose gel (Figure 14), after digestion of the X3F3/X3R1 amplicon from the CYP2D6*4/*4 reference sample, only two bands can be seen clearly; in lane 6, the first band corresponds to the 416 bp fragments, while the lower band corresponds to the 69 bp and 67 bp fragments. For the heterozygous sample carrying both the wild-type and mutant alleles (CYP2D6*l/*4), there should be six fragments after digestion, but only four bands are visible in lane 4: 416 bp, 255 bp, 161 bp, and a small band representing both the 69 bp and 67 bp fragments. Thus, the results of the RFLP analysis for the CYP2D6*l/*4 reference sample are consistent with the designated genotype. 82 CYP2D6*in CYP2D6*1/*4 CYP2D6*4/*4 X3F3/X3R1 X3F3/X3R1 X3F3/X3R1 100 bp BsM BsM Bsm\ DNA + - + - + Ladder 563 bp -416 b p -255 bp -161 b p -69 bp, 67 b p -Lane 1 2 Figure 14. Analysis for the CYP2D6 G1934A SNP in the CYP2D6*1/*1, CYP2D6*l/*4, and CYP2D6*4/*4 reference samples by RFLP analysis of X3F3/X3R1 amplicons. The 563 bp products were amplified by real-time, rapid-cycle PCR with primers X3F3/X3R1. After digestion of the X3F3/X3R1 amplicons with BstNl, the products were analyzed using a large 2.5% agarose gel at 75 V for 2 hours. For all reference samples before digestion, the expected 563 bp X3F3/X3R1 amplicon is seen (lanes 1, 3, and 5). For the CYPD6*1/*1 reference sample, three bands representing four fragments (255 bp, 161 bp, 69 bp, 67 bp) can be seen after digestion of the X3F3/X3R1 amplicon (lane 2). For the CYP2D6*4/*4 reference sample, two bands representing three fragments (416 bp, 69 bp, 67 bp) are visible following digestion (lane 6). Since both alleles are present in the CYP2D6*l/*4 reference sample, all four bands are seen after digestion (lane 4). The expected 11 bp fragment is not visible for all samples. - 600 bp - 500 bp 1 - 400 bp - 300 bp - - 200 bp - 100 bp 3 4 5 6 7 83 As seen in Figure 15, the sequence analyses at the 1934 position for the three reference samples were in agreement with their designated CYP2D6 genotypes. The sequencing results confirmed the results of both the melting curve and RFLP analyses for detecting the CYP2D6 G1934A SNP in the three reference samples. 3.1.4 Multiplex PCR method for detecting CYP2D6*5 Optimization and validation of the multiplex PCR method for detecting CYP2D6*5 was successful (Figure 16). As expected, only 5.1 kb amplicons were amplified from the CYP2D6*1/*1 reference sample (lane 3), indicating the presence of the CYP2D6 gene. The CYP2D6 gene is missing from both chromosomes of the CYP2D6*5/*5 reference sample, and the only products amplified were 3.5 kb in size (lane 5). For the CYP2D6*l/*5 heterozygote, products of both sizes were amplified from multiplex PCR (lane 4). 3.2 CYP2D6 genotype results for subject samples Newly developed, nested real-time, rapid-cycle PCR methods for detecting the CYP2D6 C188T and G1934A SNPs were validated using RFLP and sequence analyses of reference samples. A multiplex PCR method for detecting the CYP2D6*5 gene deletion was also optimized and validated by analyzing reference samples. Using the real-time PCR methods for detecting the C188T and G1934A SNPs, and the multiplex PCR method for detecting CYP2D6*5, the subject samples from the pilot clinical study were analyzed for CYP2D6*10, CYP2D6*4, and CYP2D6*5. 84 (a) CYP2D6*1/*1 (b) CYP2D6*l/*4 (c) CYP2D6*4/*4 Figure 15. Analysis for the CYP2D6 G1934A SNP in the CYP2D6*1/*1, CYP2D6*l/*4, and CYP2D6*4/*4 reference samples by sequence analysis of X3F3/X3R1 amplicons. The 563 bp products were amplified by real-time, rapid-cycle PCR with primers X3F3/X3R1, then isolated, purified, and sequenced, (a) A single G is detected at position 1934 of the CYP2D6*1/*1 reference sample; (b) both G and A are detected at position 1934 of the CYP2D6*]/*4 reference sample; (c) a single A is detected at position 1934 of the CYP2D6*4/*4 reference sample. 85 1kb CYP CYP CYP DNA ( - ) 2D6 2D6 2D6 Ladder NTC *1/*5 *5/*5 5090 bp -4 0 7 2 bp -- 5.1 kb - 3.5 kb y«k *•» f- A I 2 0 3 6 bp -1636 b p -1018 b p -i 506 bp, 517 b p -Lane 1 2 3 4 5 Figure 16. Analysis for the CYP2D6 gene deletion (CYP2D6*5) in the CYP2D6*1/*1, CYP2D6*l/*5, and CYP2D6*5/*5 reference samples by multiplex PCR. The small 1.0% agarose gel was run at 100 V for 80 minutes. A no-template control (NTC; lane 2) was included, in which water was used in place of genomic DNA. A single band representing 5.1 kb amplicons is seen for the CYP2D6*1/*1 reference sample (lane 3). For the CYP2D6*5/*5 reference sample, only products 3.5 kb in size were amplified (lane 5). For the CYP2D6*l/*5 reference sample, both the 5.1 kb and 3.5 kb products were amplified (lane 4). 86 Figure 17 shows the detection of the CYP2D6 C188T SNP in four study subjects. Melting peak temperatures were the same as those from the reference samples (data not shown). Subjects 07 and 18 appeared to be homozygous for C at position 188 (peak at 67-68°C). These subjects could be homozygous for a wild-type allele (CYP2D6*wt/*wf) or heterozygous for a CYP2D6*5 gene deletion (CYP2D6*wt/*5). Subject 14 appeared to be homozygous for T at position 188 (peak at 58-59°C), and could be homozygous for the CYP2D6*10 allele or CYP2D6*4 allele (or CYP2D6*10/*5 or CYP2D6*4/*5). Both C and T were detected at the 188 position of subject sample 19, meaning that this subject could be heterozygous for either the CYP2D6*4 or CYP2D6*10 allele (CYP2D6*wt/*10 or CYP2D6*wt/*4). At position 1934, G was detected in all subject samples (Figure 18), indicating that none of these study subjects carried the CYP2D6*4 allele. Thus, the CYP2D6*4 allele was ruled out in these four subjects. Figure 19 shows the results from CYP2D6*5 detection for the same four study subjects. At least one copy of the CYP2D6 gene was present in all four subjects. For subject 18, products 5.1 kb and 3.5 kb in size were amplified, suggesting that the CYP2D6 gene is deleted from one of the two chromosomes. 87 Figure 17. Analysis for the CYP2D6 C188T single nucleotide polymorphism in selected subjects from the pilot clinical study: 07, 14, 18, and 19. A no-template control (NTC) was also included, in which water was used in place of genomic DNA. Results of the reference samples analyzed simultaneously are not shown. Results from subjects 07 and 18 were consistent with those from reference samples CYP2D6*1/*1 and CYP2D6*l/*5 (Tm of 67-68°C; C at 188). For subject 14, results indicate a melting peak temperature similar to the CYP2D6*4/*5 reference sample (Tm of 58-59°C; T at 188). Melting peaks at both temperatures were seen for subject 19. 88 Figure 18. Analysis for the CYP2D6 G1934A single nucleotide polymorphism in selected subjects from the pilot clinical study: 07, 14, 18, and 19. A no-template control (NTC) was also included, in which water was used in place of genomic DNA. A CYP2D6*4/*5 (A at 1934) is shown for reference. Results of the other reference samples analyzed simultaneously are not shown. For all four subjects, the melting peak temperatures obtained were consistent with those from the CYP2D6*1/*1 and CYP2D6*l/*5 reference samples (61-62°C; G at 1934). 89 i kb CYP DNA ( - ) 2D6 Ladder NTC CYP 2D6 Subject Subject Subject Subject *1/*5 07 14 18 19 sosobp- m mm* mmm — ~ — ' -JJkb 4072 bp - i W P a * * —»•».. - — - 3.5 kb 3054 bp-2036 bp -1636 bp-1018 bp-506 bp / 517 bp -Lane Figure 19. Analysis for CYP2D6*5 in selected subjects from the pilot clinical study: 07, 14, 18, and 19. The small 1.0% agarose gel was run at 100 V for 50 minutes. CYP2D6*1/*1 (lane 3) and CYP2D6*l/*5 (lane 4) reference samples were included as positive controls, while a no-template control (NTC) was included as a negative control (lane 2). Products 5.1 kb in size were amplified from subject samples 07, 14, and 19 (lanes 5, 6, and 8), consistent with results from the CYP2D6*1/*1 reference sample (lane 3). Both 5.1 kb and 3.5 kb products were amplified from subject sample 18 (lane 7), consistent with results from the CYP2D6*l/*5 reference sample (lane 4). 90 Based upon analyses for the C188T and G1934A SNPs and CYP2D6*5, CYP2D6 genotypes were assigned to the 36 Asian study subjects. For example, the determined CYP2D6 genotypes for subjects 07, 14, 18, and 19 are shown in Table 26. Subject 07 was wild-type at both the 188 and 1934 positions, and did not have a CYP2D6 gene deletion, and was genotyped as being CYP2D6*wt/*wt (CYP2D6*1/*1, CYP2D6*l/*2, or CYP2D6*2/*2). Subject 18 had results similar to subject 07, but the CYP2D6 gene was deleted from one of the chromosomes, and was thus assigned a CYP2D6*wt/*5 genotype. Subject 14 tested positive for a C188T SNP, but tested wild-type at the 1934 position, thus ruling out the CYP2D6*4 allele. Since no CYP2D6 gene deletion was found in subject 14, this subject was assigned to be homozygous for the CYP2D6*10 allele (CYP2D6*!0/*l0). Subject 19 was heterozygous at the 188 position but wild-type at the 1934 position, and was thus assigned a CYP2D6*wt/*10 genotype. The CYP2D6 genotype frequencies for all 36 Asian study subjects are shown in Table 27, while the CYP2D6 allele frequencies are summarized in Table 28. Please refer to Appendix I for CYP2D6 genotype assignments of all 36 study subjects in the pilot clinical study. The composite CYP2D6 allele frequencies shown in Appendix E were also updated with the results from the 36 Asian study subjects in this study (Appendix J). 91 Table 26. Summary of CYP2D6 genotype assignments for subjects 07, 14, 18, and 19. Since subject 07 appeared to be wild-type at the 188 and 1934 positions, and was negative for the CYP2D6 gene deletion, a CYP2D6*wt/*wt genotype was assigned. Subject 18 was also wild-type at both positions, but was heterozygous for the CYP2D6 gene deletion; a CYP2D6*wt/*5 genotype was assigned. The only mutation detected in subject 14 was a T at position 188, and thus a CYP2D6*10/*10 genotype was assigned. Subject 19 was assigned the CYP2D6*wt/*10 genotype, because both C and T were detected at position 188, and no G1934A SNP or CYP2D6 gene deletion was detected. A summary of CYP2D6 genotype assignments for each of the 36 study subjects is shown in Appendix I. Subject C188T G1934A CYP2D6*5 CYP2D6 genotype 07 C G No CYP2D6*wt/*wtf 14 T G No CYP2D6*10/*10 18 C G Yes CYP2D6*wt/*5 19 C/T G No CYP2D6*wt/*10 • CYP2D6*wt represents either CYP2D6*1 or CYP2D6*2 Table 27. Summary of CYP2D6 genotype frequencies in 36 Asian study subjects. 95% confidence intervals of the frequencies were calculated according to a binomial distribution using NCSS statistical software. CYP2D6 genotype No. of subjects (n=36) Frequency 95% CI CYP2D6*wt/*wt^ 4 11.1% 3.1%-26.1% CYP2D6*wt/*5 2 5.6% 0.7%- 18.7% CYP2D6*wt/*10 12 33.3% 18.6%-51.0% CYP2D6*10/*10 18 50.0% 32.9%-67.1% CYP2D6*wt represents either CYP2D6*1 or CYP2D6*2 Table 28. Summary of CYP2D6 allele frequencies in 36 Asian study subjects. 95% confidence intervals of the frequencies were calculated according to a binomial distribution using NCSS statistical software. Expected frequencies are derived from Appendix E. CYP2D6 Allele No. of alleles (n = 72) Frequency 95% CI Expected frequency CYP2D6*wtf 22 30.6% 20.2% - 42.5% 30.0-59.5% CYP2D6*4 0 0.0% 0.0-4.2% 0.0-6.7% CYP2D6*5 2 2.8% 0.3% - 9.7% 0.0-10.0% CYP2D6*10 48 66.7% 54.6% - 77.3% 35.7-69.2% T CYP2D6*wt represents either CYP2D6*! or CYP2D6*2 92 3.3 Genotyping algorithm for the CYP2D6*10 allele in Asians Based upon CYP2D6 allele frequencies in Asians (Appendix J), a novel genotyping algorithm for the CYP2D6*10 allele in Asians was devised (Figure 20). Long PCR will be initially used to amplify the CYP2D6 gene. If there is no product amplified, analysis for CYP2D6*5 (*5) is necessary. If the CYP2D6 gene is amplified, then the C188T SNP will be analyzed by nested real-time PCR. For subjects that are wild-type at the 188 position ('C188'), further analyses for *5 is required. The "wild-type" (CYP2D6*M>t/*wt) group includes subjects who are wild-type at the 188 position but test negative for *5. These "wild-type" homozygotes may carry other alleles not analyzed for in the algorithm, although these alleles are rare in Asians (Appendix E ) . For subjects that test as either T or C/T at the 188 position ('T188' or 'C/T 188'), analyses for the G1934A SNP will be performed to rule out CYP2D6*4. In the 'T188' subgroup, if subjects test either G at the 1934 position ('G1934') or A at the 1934 position ('A1934'), *5 analyses will be done. o Subjects who test 'T188', 'G1934', and negative for *5 will be designated as CYP2D6*10 homozygotes (CYP2D6* 10/* 10); these subjects may have decreased metabolism of CYP2D6 substrates. Subjects will be considered heterozygous for the CYP2D6*10 allele (CYP2D6*wt/*10) if they test to be 'C/T 188' and 'G1934'. Other CYP2D6 genotypes can also be identified using this genotyping algorithm, such as CYP2D6*5/*5, CYP2D6*wt/*5, CYP2D6*10/*5, CYP2D6*10/*4, CYP2D6*4/*4, CYP2D6*4/*5, and CYP2D6*wt/*4. 93 a, vo * ^ Q -s» w lysi An; L O * '(/) ro < L O * * r oo 0 0 ro ON H < do" 0 0 ion ro ON isod ion +-* td ' VI O o II OH 0 0 < oo ll i—i O ro ON an < Vi < •it ro CJ ON c o * *—{ 13 po * cd Q O II ro Vi • ^H r the 610, nalys do" < oo * 4—• CJ o Q C M algo posit CYP. oc II CJ cd II OH f—i "t/2 noty O II "w 13 00 0 0 CJ < 0 0 CD > * o H c < ro ON do" © 0 0 CJ fN itio w C! o itio s-S o &D CL, o • OH cd 94 CHAPTER 4 DISCUSSION 4.1 Genotyping methods The CYP2D6*10 allele is very common among Asians (allele frequency of 35.7-69.2%; Table 3), and it is associated with decreased metabolism of some CYP2D6 substrates. Thus, many Asians may be at risk of adverse effects or altered drug responses toward medications that are primarily metabolized by CYP2D6. In order to study the effects of CYP2D6*10 on drug metabolism in Asians, genotyping methods to identify the CYP2D6*wt/*wt, CYP2D6*wt/*10, and CYP2D6*10/*10 genotypes are required. This project focused on the optimization of long PCR, the development and validation of nested real-time, rapid-cycle PCR methods for detecting the C188T and G1934A SNPs, the optimization and validation of a multiplex PCR method for detecting CYP2D6*5, and the application of these methods to genotype Asian subjects with respect to CYP2D6*10. A novel genotyping algorithm for CYP2D6*10 in Asians was also devised. 4.1.1 Long PCR The CYP2D6*10 allele contains a characteristic C188T SNP located in exon 1 of the CYP2D6 gene. The sequences of CYP2D6 and CYP2D7BP in this region only differ by one nucleotide (Heim and Meyer 1992). Coincidentally, this nucleotide is a C188T SNP present in the CYP2D7BP pseudogene. Numerous studies of CYP2D6*10 used the method for detecting the CYP2D6 C188T SNP described by Johansson et al. (1994), but the primers used also had the potential of amplifying the CYP2D7BP pseudogene (Yue et al. 1998; Fukuda et al. 1999; Someya et al. 1999). Other studies of CYP2D6*10, including recently published studies, used different primers, but these primers could also amplify the 96 CYP2D7BP pseudogene (Wang et al. 1993; Huang et al. 2003; Liou et al. 2004). A previous study of CYP2D6*10 specifically stated the importance of avoiding interference from CYP 2D pseudogenes (Garcia-Barcelo et al. 2000). In that study, long PCR was initially used to amplify the CYP2D6 gene followed by a second PCR to amplify exons 1 and 2; a third set of primers was subsequently used to detect the C188T SNP. However, upon assessment of the sequences of all three sets of primers, it appears that the CYP2D7BP pseudogene will be amplified in all three cases. Since the CYP2D7BP pseudogene contains the C188T SNP, when analyzing for the C188T SNP to identify CYP2D6*10 alleles, it is important to use a genotyping method that is specific for the CYP2D6 gene. Sachse et al. (1997) amplified the CYP2D6 gene using specific primers before nested PCR-RFLP analyses for various CYP2D6 SNPs. The primers used in that study were specific for CYP2D6 and should not amplify any CYP 2D pseudogenes, including CYP 2 D7BP. The present project used the same primer sequences (P100/P200), but with a different DNA polymerase. The Platinum® Pfic DNA polymerase was chosen due to its proofreading properties and the required initial activation at 94°C. Since the P100/P200 amplicons containing the CYP2D6 gene were to be used in the nested identification of CYP2D6 SNPs, the proofreading features of the enzyme mix were expected to minimize any potential errors introduced from amplification of the 4681 bp product. Also, the required activation of the Platinum® Pfic DNA polymerase by an initial increase in temperature to 94°C should minimize any non-specific binding of the primers during preparation of the samples. 97 As seen in Figure 9, the amplification of the CYP2D6 gene by long PCR from reference samples CYP2D6*1/*1, CYP2D6*l/*4, and CYP2D6*4/*4 was successful. For each sample, a band can be seen between the 4072 bp and 5090 bp bands of the 1 kb DNA ladder; this band is assumed to be the 4681 bp P100/P200 amplicon. The P100/P200 amplicon should contain all nine exons of the CYP2D6 gene. When using long PCR to amplify the CYP2D6 gene from subjects in a clinical study, products 4681 bp in size would be expected to be amplified from all subjects, with the exception of those homozygous for the CYP2D6 gene deletion (i.e. CYP2D6*5/*5). The P100/P200 amplicons would subsequently be diluted for use in the identification of the C188T and G1934A SNPs by nested real-time, rapid-cycle PCR and melting curve analysis. 4.1.2 Development and validation of the nested real-time, rapid-cycle PCR method for detecting the C188T SNP The key mutation of the CYP2D6* 10 allele is considered to be the C188T SNP (Johansson et al. 1994; Zanger et al. 2004). However, the CYP2D6*4 allele and the CYP2D7BP pseudogene also contain the C188T SNP. The CYP2D6*4 allele can be ruled out by analyzing for the G1934A SNP, while amplification of the CYP2D7BP pseudogene can be avoided with the use of specific primers (as described above). Nested analysis for the C188T SNP on the P100/P200 long PCR amplicons containing the CYP2D6 gene eliminates potential interferences from CYP2D7BP. Platinum® Taq DNA polymerase was chosen for use in real-time, rapid-cycle PCR because of its required activation at 94°C, in order to minimize any non-specific binding of the primers during preparation. In addition, 98 the use of individual reagents rather than kits offered by the manufacturer allowed more flexibility in PCR optimization (e.g. concentration of magnesium and dNTPs). A nested real-time, rapid-cycle PCR method for detecting the C188T SNP was developed, optimized, and validated using reference samples with genotypes CYP2D6*1/*1 (C at position 188), CYP2D6*l/*4 (C/T at position 188), and CYP2D6*4/*4 (T at position 188) (Figures 10-12). The results were further validated using RFLP and sequence analyses. The melting curve analysis of PI 1/P12 amplicons from the CYP2D6*1/*1 reference sample resulted in a melting peak temperature (Tm) of 67-68°C (Figure 10a). These 433 bp P11/P12 wild-type amplicons should contain one 5'-TCACC-3' Hphl restriction site, and were expected to be cleaved into fragments 362 bp and 71 bp in size after digestion with Hphl. In Figure 10b, the band representing 433 bp P11/P12 amplicons is seen in lane 2 (before digestion). In lane 3 (after digestion), although the 71 bp fragments are too faint to discern, the band representing 362 bp fragments is clearly seen. As shown in the sequencing results (Figure 10c), at position 188 of the CYP2D6 gene for the CYP2D6*1/*1 reference sample, a C was detected, which is consistent with the melting curve and RFLP analyses. The CYP2D6*4/*4 reference sample was expected to be homozygous for the C188T SNP. Thus, the sensor probes should melt off from the mutant amplicons at a lower temperature, due to the single base mismatch at position 188. As expected, the observed Tm for the CYP2D6*4/*4 reference sample was shifted to a lower temperature 58-59°C (Figure 11a), as compared to that of the CYP2D6*1/*1 reference sample (Figure 10a). The C188T SNP 99 in the CYP2D6*4/*4 reference sample results in the addition of a second 5'-TCACC-3' Hphl restriction site in the P11/P12 amplicon. After digestion of the 433 bp amplicons, three fragments were expected: 262 bp, 100 bp, and 71 bp in size. In Figure 1 lb, the band representing 433 bp PI 1/P12 amplicons can be seen in lane 2. After digestion (lane 3), the 100 bp and 71 bp fragments are faint but slightly visible, but the band representing the 262 bp fragments is clearly discernable. The sequencing results shown in Figure 1 lc were also in agreement with the melting curve and RFLP analyses, indicating a T at position 188 of the CYP2D6 gene for the CYP2D6*4/*4 reference sample. Finally, since the CYP2D6*l/*4 reference sample was heterozygous for the C188T SNP (wild-type at one chromosome and containing the C188T SNP on the other chromosome), melting peaks at both temperatures were seen (Figure 12a). Since the P11/P12 amplicons from the CYP2D6*l/*4 reference sample were derived from both wild-type and mutant alleles, some of the PI 1/P12 amplicons contained one restriction site (from the wild-type allele), while some of the amplicons contained two restriction sites (due to the C188T SNP in the mutant allele). After digestion with Hphl of the PI 1/P 12 amplicons, four fragments were expected: 362 bp, 262 bp, 100 bp, and 71 bp. As seen in Figure 12b, the single band in lane 2 represents the 433 bp PI 1/P 12 amplicons. After digestion (lane 3), four fragments are seen; the 362 bp and 262 bp bands are visible, but the 100 bp and 71 bp are fainter. Sequencing results of 433 bp PI 1/P12 amplicons from the CYP2D6*l/*4 reference sample indicate both a C and a T detected at position 188 of the CYP2D6 gene (Figure 12c), which is consistent with the melting curve and RFLP analyses. 100 When applying the nested real-time, rapid-cycle PCR method to detect the C188T SNP in subjects from clinical studies, a Tm of 67-68°C indicates a C at position 188 (CYP2D6*wt/*wt or CYP2D6*wt/*5), a Tm of 58-59°C indicates a T at position 188 (CYP2D6*10/*10, CYP2D6*10/*5, CYP2D6*4/*4, CYP2D6*4/*5, or CYP2D6*10/*4), and melting peaks at both 58-59°C and 67-68°C indicate that the subject is heterozygous for the Cl 88T SNP (CYP2D6*wt/*10 or CYP2D6*wt/*4). 4.1.3 Development and validation of the nested real-time, rapid-cycle PCR method for detecting the G1934A SNP As mentioned previously, both the CYP2D6*10 and CYP2D6*4 alleles contain the C188T SNP (Table 2). However, CYP2D6*4 contains a unique G1934A SNP that results in a splicing defect and truncated protein (Hanioka et al. 1990). Since the CYP2D6*4 allele frequency has been reported to range from 0.0-6.7% in Asians (Appendix E), when genotyping for the CYP2D6*10 allele, CYP2D6*4 must be ruled out by analysis for the G1934A SNP. A nested real-time, rapid-cycle PCR method for detecting the CYP2D6 G1934A SNP was developed and validated using reference samples (CYP2D6*1/*1, CYP2D6*l/*4, and CYP2D6*4/*4), and further validated by RFLP and sequence analyses (Figures 13-15). For wild-type amplicons containing a G at position 1934 (CYP2D6*1/*1), the melting of sensor probes from the amplicons resulted in a melting peak temperature of 61-62°C (Figure 13; green curve). The G1934A SNP in mutant amplicons from CYP2D6*4/*4 resulted in a single base mismatch, causing the sensor probes to start melting off at a lower temperature; the observed Tm was 43-44°C (Figure 13; red curve). 101 Melting peaks at both 43-44°C and 61-62°C were observed for the heterozygous CYP2D6*l/*4 reference sample (Figure 13; purple curve). The results from melting curve analyses were validated using RFLP analyses of X3F3/X3R1 amplicons from the reference samples (Figure 14). Before digestion of the X3F3/X3R1 amplicons from the CYP2D6*1/*1, CYP2D6*l/*4, and CYP2D6*4/*4 reference samples, single bands representing 563 bp amplicons are clearly visible (lanes 1, 3, and 5). Four BstNl restriction sites were expected in the X3F3/X3R1 amplicons of the CYP2D6*1/*1 reference sample. Thus, after digestion of these 563 bp amplicons, five fragments were expected: 255 bp, 161 bp, 69 bp, 67 bp, and 11 bp in size. The 11 bp fragment would not be visible in the 2.5% agarose gel. However, after digestion using BsiNI (Figure 14; lane 2), three bands are clearly discernable, representing the 255 bp, 161 bp, 69 bp, and 67 bp fragments (one of the bands was expected to contain both 69 bp and 67 bp fragments). On the other hand, one of the four restriction sites should be abolished in the X3F3/X3R1 amplicon from the CYP2D6*4/*4 reference sample, due to the homozygous G1934A SNP. Thus, after digestion, the 416 bp fragments should not be further cleaved into the 255 bp and 161 bp fragments, leaving only fragments 416 bp, 69 bp, 67 bp, and 11 bp in size. As seen in lane 6 of Figure 14, after digestion of the X3F3/X3R1 amplicons from the CYP2D6*4/*4 reference sample, two bands are visible; one band represents 416 bp fragments, while the other band represents the 69 bp and 67 bp fragments (the 11 bp band is not seen). When the CYP2D6*l/*4 reference sample was amplified using the X3F3/X3R1 primers, the 563 bp amplicons were expected to consist of some from the wild-type allele and some from the mutant allele. Thus, after digestion 102 using BstNl, the 563 bp amplicons should be cleaved into six fragments. As shown in lane 4 (Figure 14), after digestion of the 563 bp X3F3/X3R1 amplicons of the CYP2D6*l/*4 reference sample, four bands representing the 416 bp, 255 bp, 161 bp, 69 bp, and 67 bp fragments are clearly seen. The results of the RFLP analyses of the X3F3/X3R1 amplicons of the reference samples were in agreement with the results from melting curve analyses. The melting curve analyses were further validated by sequencing of 563 bp X3F3/X3R1 products amplified from real-time, rapid-cycle PCR (Figure 15). For the CYP2D6*1/*1 reference sample, a single G was detected at position 1934 of the CYP2D6 gene (Figure 15a). For the CYP2D6*4/*4 reference sample, a single A was detected (Figure 15b). Both G and A were detected for the CYP2D6*l/*4 reference sample. The sequencing results were consistent with both the melting curve and RFLP analyses. When applying the nested real-time, rapid-cycle PCR method to detect the G1934A SNP in subjects from clinical studies, a Tm of 61-62°C indicates a G at position 1934 (CYP2D6*wt/*wt or CYP2D6*wt/*5), a Tm of 43-44°C indicates the presence of a G1934A SNP (CYP2D6*4/*4 or CYP2D6*4/*5), and melting peaks at both temperatures indicate that the subject is heterozygous for the G1934A SNP (CYP2D6*wt/*4 or CYP2D6*10/*4). 4.1.4 Optimization and validation of the multiplex PCR method for detecting the CYP2D6 gene deletion (CYP2D6*5) The CYP2D6*5 allele is actually a complete deletion of the CYP2D6 gene, and has been reported to occur at a frequency of 0.0-10.0% in Asians (Table 3). Subjects with 103 CYP2D6*wt/*wt and CYP2D6*wt/*5 genotypes would give the same results in C188T and G1934A analyses (a C at position 188 and a G at position 1934). Similarly, subjects with the CYP2D6*10*10 and CYP2D6*10/*5 genotypes would result in a positive test for the C188T SNP, and subjects with the CYP2D6*4/*4 and CYP2D6*4/*5 genotypes would result in a positive test for the G1934A SNP. Although most subjects carrying at least one functional gene copy (e.g. CYP2D6*l/5 or CYP2D6*2/5) have been reported to be extensive metabolizers, Zanger et al. (2001) stated that some have been phenotyped as intermediate metabolizers. Subjects can also be homozygous for the CYP2D6 gene deletion (CYP2D6*5/*5). Therefore, in order to differentiate the above genotypes, it is important to include the analysis for the CYP2D6 gene deletion when genotyping Asians. With previous methods for detecting the CYP2D6 gene deletion using a single set of primers, the absence of an amplified product could not be differentiated from failure of the PCR (Steen et al. 1995; Johansson et al. 1996). The primers designed by Steen et al. (1995a) amplify a 3.5 kb product only if the CYP2D6 gene is deleted, while the primers designed by Johansson et al. (1996) amplify a 6.0 kb product also only when the CYP2D6 gene is deleted. However, in both methods, no product would be amplified if the CYP2D6 gene is present; therefore, a separate PCR of the CYP2D6 gene is needed as a control. The multiplex PCR method by Hersberger et al. (2000) was chosen for this project because of the inclusion of a proper positive control. For each of the three different possible genotypes, at least one product should be amplified (either 5.1 kb or 3.5 kb). The multiplex PCR method was optimized, and the results were validated successfully using reference samples with genotypes CYP2D6*1/*1, CYP2D6*l/*5, and CYP2D6*5/*5 (Figure 16). A 104 no-template control (lane 2) was included, in which water was added in place of genomic DNA before multiplex PCR. In lane 3, a single band representing 5.1 kb amplicons is seen for the CYP2D6*1/*1 reference sample. The CYP2D6*5/*5 reference sample is missing the CYP2D6 gene, and thus a single band representing the 3.5 kb amplicons is visible (lane 5). The results for the CYP2D6*l/*5 reference sample are shown in lane 4; as expected, two bands 5.1 kb and 3.5 kb in size are clearly seen. When applying the multiplex PCR method to detect CYP2D6*5 in subjects from clinical studies, the amplification of products 5.1 kb in size indicates the presence of the CYP2D6 gene on both chromosomes. If the only products amplified are 3.5 kb in size, this suggests that the subject is missing the CYP2D6 gene from both chromosomes (CYP 2 D6* 5/* 5). If both 3.5 kb and 5.1 kb products are amplified after multiplex PCR, the subject may be heterozygous for CYP2D6*5 (deletion of the CYP2D6 gene from one chromosome). 4.2 CYP2D6 genotype frequencies in Asian study subjects The validated CYP2D6 genotyping methods for detecting C188T, G1934A, and CYP2D6*5 were used to genotype Asian paediatric dental patients involved in a pilot clinical study of codeine metabolism. CYP2D6 genotypes were determined based upon the combined results of C188T, G1934A, and CYP2D6*5 analyses. Reference samples with known genotypes were included as controls. Figures 17-19 and Table 26 summarize the identification of four different CYP2D6 genotypes in selected subjects from the clinical study. All four subjects were wild-type at the 1934 position (Tm of 61-62°C), indicating 105 that none of them carried the CYP2D6*4 allele. Subjects 07 and 18 were both wild-type at position 188 (Tm of 67-68°C), and were differentiated by analyzing for the CYP2D6 gene deletion. Subject 7 was assigned a CYP2D6*wt/*wt genotype (5.1 kb amplicon), while subject 18 was assigned a CYP2D6*wt/*5 genotype (5.1 kb and 3.5 kb amplicons). A C188T SNP was detected in subject 14, and the results of the CYP2D6*5 analysis (5.1 kb amplicon) suggest that subject 14 was homozygous for the CYP2D6*10 allele. Finally, subject 19 was assigned a CYP2D6*wt/*10 genotype due to the detection of both C and T at position 188 (Tms at both 58-59°C and 67-68°C), and the absence of the CYP2D6 gene deletion (5.1 kb amplicon). Few published CYP2D6 genotyping studies report the genotype frequencies; usually only the allele frequencies are reported. As summarized in Table 27, the 95% confidence intervals (CI) for the CYP2D6 genotype frequencies in the 36 Asian subjects from the pilot clinical study were calculated as follows: CYP2D6*wt/*wt (3.1-26.1%), CYP2D6*wt/*5 (0.7-18.7%), CYP2D6*wt/*10 (18.6-51.0%), and CYP2D6*10/*10 (32.9-67.1%). These frequencies are comparable to those reported in a Hong Kong study sample (Garcia-Barcelo et al. 2000): CYP2D6*wt/*wt (7.6%), CYP2D6*wt/*5 (4.2%), CYP2D6*wt/*10 (42.0%), CYP2D6*10/*10 (41.2%), and CYP2D6*10/*5 (5.0%). As there were only 36 subjects, it is not surprising that no subjects from the pilot clinical study were genotyped as CYP2D6*10/*5. However, 50.0% of the subjects from this study were genotyped as CYP2D6*10/*10. When these patients are given medications that are CYP2D6 substrates, they may be at risk for adverse effects or increased drug responses due to decreased 106 inactivation of active drugs, or they may experience reduced therapeutic effects due to decreased bioactivation of prodrugs. 4.3 CYP2D6 allele frequencies in Asian study subjects Since most studies report CYP2D6 allele frequencies rather than genotype frequencies, the CYP2D6 allele frequencies of the 36 Asian subjects from the pilot clinical study were calculated from the assigned CYP2D6 genotypes. The 95% CIs of these allele frequencies were summarized and compared to reported frequencies in other studies involving Asians (Table 28). Although the frequency of CYP2D6*10 of the present study was relatively high at 66.7%, the 95% CI of 54.6-77.3% is consistent with reported frequencies of 36.0-69.2%) from previous studies involving Asians. The highest observed frequency of CYP2D6*10 thus far reported has been 69.2%>, and was based upon C188T analyses in a sample of Chinese subjects from Taiwan (Wang et al. 1993). In that study, interference from the CYP2D7BP pseudogene was not ruled out. The 95% CI of the CYP2D6*wt frequency (20.2-42.5%)) observed herein is comparable to the frequency of 30.0-59.5%) observed in other studies involving Asians. There were two CYP2D6 gene deletions detected in total (95%o CI of 0.3%-9.7%), which is consistent with the expected frequency of 0.0-10.0%. The CYP2D6*4 allele was not detected in any of the 36 Asians in the present clinical study, and the formula of 3/n was used to estimate a 95% CI of 0.0-4.2%), which is consistent with an expected frequency of 0.0-6.7%). The determined CYP2D6 allele frequencies among the 36 Asian subjects in the pilot clinical study were therefore in agreement with the observed frequencies in other studies involving Asians. The CYP2D6 genotyping results of the 36 107 Asian subjects in this study were included in the calculation of the composite CYP2D6 allele frequencies observed in Asians (Appendix J): CYP2D6*10 (49.3%), CYP2D6*wt (43.3%), CYP2D6*5 (5.6%), and CYP2D6*4 (0.6%). 4.4 Genotyping algorithm for CYP2D6*10 in Asians Based upon the CYP2D6 allele frequencies observed in Asians (Appendix J), a genotyping algorithm for the CYP2D6*10 allele in Asians was devised (Figure 20). The novel aspects of this algorithm include the use of long PCR to eliminate interferences from pseudogenes, the use of nested real-time, rapid-cycle PCR to detect the C188T and G1934A SNPs, and the analysis for the G1934A SNP only in those carrying a C188T SNP (homozygotes or heterozygotes) to rule out the CYP2D6*4 allele. This genotyping algorithm can identify ten different CYP2D6 genotypes in Asians (Figure 20). Based on the reported CYP2D6 allele frequencies in Asians summarized in Appendix J, the accuracy of the CYP2D6 genotype assignments using the novel genotyping algorithm was estimated (Appendix K). Individuals can be identified as CYP2D6*wt/*wt with 92% accuracy, meaning that approximately 8%> of these individuals may carry alleles other than CYP2D6*1 or CYP2D6*2. For example, if individuals identified as CYP2D6*wt/*wt carried one or two alleles associated with non-functional CYP2D6 enzyme, they may actually be intermediate or poor metabolizers, rather than extensive metabolizers. The accuracy of genotypes associated with CYP2D6*wt will be further discussed below (Section 4.5.4). The genotypes of CYP2D6*wt/*10, CYP2D6*wt/*4, or CYP2D6*wt/*5 are 96% accurate, meaning that up to 4%> of these individuals may carry alleles other than CYP2D6*1 or 108 CYP2D6*2. Finally, for the CYP2D6*10/*10, CYP2D6*10/*5, CYP2D6*4/*10, CYP2D6*4/*4, CYP2D6*4/*5, and CYP2D6*5/*5 genotypes, the accuracy is estimated to be 100%. However, as will be discussed below (Section 4.5.5), the accuracy of genotypes associated with CYP2D6* 10 may be slightly decreased by the presence of other alleles containing the C188T SNP. 4.5 Problems encountered and limitations Some of the initial problems encountered included development of the real-time, rapid-cycle PCR method for detecting the C188T SNP, and optimization of the multiplex PCR method for detecting CYP2D6*5. The main limitation of the CYP2D6 genotyping algorithm is the continuous discovery of new CYP2D6 alleles and CYP2D pseudogenes. 4.5.1 Selection of primers for the real-time, rapid-cycle PCR method for detecting the C188T SNP The initial real-time, rapid-cycle PCR method for detecting the C188T SNP involved the direct use of primers (Fl/Rl) that were designed to be specific for CYP2D6 (the sequence of the RI primer was specific for a region in intron 1 of CYP2D6). However, the PCR was difficult to optimize and the melting curve results were not reproducible. Since the P11/P12 primers used in PCR-RFLP analysis for the C188T SNP (Sachse et al. 1997) could also amplify the CYP2D7BP pseudogene, and the results using Fl/Rl primers were inconsistent, a combination of Pll /Rl was tested in real-time, rapid-cycle PCR. The results obtained with the Pll /Rl primers were reproducible, and the RFLP and sequence 109 analyses were also in agreement with the melting curve analyses. However, when the Pll /Rl primers were used to validate the real-time, rapid-cycle PCR method for detecting the C188T SNP, the results were not in agreement with the genotypes of the reference samples (CYP2D6*1/*1, CYP2D6*l/*4, and CYP2D6*4/*4). It was thus decided to use the specific P100/P200 primers in the initial amplification of the CYP2D6 gene before nested analysis for the C188T SNP by real-time, rapid-cycle PCR with the P11/P12 primers. As discussed earlier, this method was optimized without much difficulty, and the results were reproducible and in agreement with the genotypes of the reference samples. 4.5.2 DNA polymerase for multiplex PCR Since the Platinum® Pfx DNA polymerase was successful in the long PCR of the CYP2D6 gene, this enzyme was initially used for the multiplex PCR method for detecting CYP2D6*5. However, there was considerable difficulty with optimizing the multiplex PCR to eliminate non-specific bands. Thus, the Expand Long Template PCR System™ was used, as described by Hersberger et al. (2000), but the enzyme mix had been recently changed by the manufacturer (from Taq/Pwo to Taq/Tgo). When using the concentrations of the buffers and reagents outlined by Hersberger et al. (2000), no products were amplified. After optimization using a different buffer (Buffer 1) and different reagent concentrations (Table 20), the results were reproducible, and the method was validated successfully. 110 4.5.3 Duplication of CYP2D6*10 The presence of a duplication of the CYP2D6*10 allele in Asians has recently been reported (Ji et al. 2002; Mitsunaga et al. 2002). The genotyping methods described in the present project can not detect duplications of the CYP2D6 gene. However, duplication of CYP2D6*10 in Asians has been reported to occur at a frequency of less than 1.0% (Ji et al. 2002; Mitsunaga et al. 2002). In addition, one study reported no difference in dextromethorphan/dextrorphan metabolic ratios in those carrying three copies of the CYP2D6*10 allele, as compared to CYP2D6*10/*10 subjects (Ishiguro et al. 2004). Further studies are required to determine the frequency of the CYP2D6*10 duplication, and its influence on CYP2D6 activity. 4.5.4 Accuracy of the assignment of CYP2D6*wt CYP2D6*wt (CYP2D*1 or CYP2D6*2) was assigned by default, meaning that its presence was inferred by the absence of other CYP2D6 alleles. CYP2D6*2 was not differentiated from CYP2D6*! in the genotyping algorithm because the CYP2D6*2 allele has been associated with normal CYP2D6 activity (Johansson et al. 1993; Dahl et al. 1995; Tateishi et al. 1999; Zanger et al. 2004). Thus, there may not be differences in CYP2D6 activity among those genotyped as CYP2D6*1/*1, CYP2D6*l/*2, or CYP2D6*2/*2. However, other CYP2D6 alleles that have not been included in this genotyping algorithm may be present in subjects assigned with genotypes containing CYP2D6*wt; such alleles include CYP2D6*9, CYP2D6*17, and CYP2D6*21. The CYP2D6*9 allele, associated with reduced CYP2D6 activity (Teh et al. 2001; Zanger et al. 2004), was observed at frequencies 111 of 3.3% and 1.7% in Malaysia (Teh et al. 2001; Gan et al. 2002), but it was not found in a study of Chinese subjects living in Toronto (Droll et al. 1998). The CYP2D6*17 allele is common in African Americans (Bradford 2002), and is associated with reduced CYP2D6 activity (Oscarson et al. 1997; Marcucci et al. 2002; Wennerholm et al. 2002). It has been observed at allele frequencies of 0.5% and 1.7% in Malaysia (Teh et al. 2001; Gan et al. 2002) , but it was also not found in a study of Chinese subjects living in Toronto (Droll et al. 1998). Finally, analysis for the CYP2D6*21 allele in Asians has been carried out in only two different Japanese study samples thus far, in which the CYP2D6*21 allele frequency has been reported to be 1.0% and 0.3% (Tateishi et al. 1999; Ishiguro et al. 2003) . These three alleles, which have not been genotyped in the present study, are partly included in the composite allele frequency of "Other alleles" in Table 2. The possible presence of these alleles is reflected in the previously discussed accuracy of genotypes containing CYP2D6*wt (at least 92%). 4.5.5 Other CYP2D6 alleles containing the C188T SNP The CYP2D6*14A, CYP2D6*36, and CYP2D6*37 alleles also contain the C188T SNP (Appendix B). CYP2D6*14A is differentiated from CYP2D6*10 by an additional G1846A SNP and a missing G1749C SNP (Wang et al. 1993; Daly et al. 1996). The CYP2D6*36 allele contains a partial conversion of exon 9 into the CYP2D7P pseudogene, resulting in 12 SNPs (Johansson et al. 1994). The CYP2D6*37 allele contains an additional G2031A SNP (Marez et al. 1997). 112 The frequency of the CYP2D6*14A allele (previously known as CYP2D6*14) has been reported to be 2.2% in a Japanese study sample (Kubota et al. 2000) and 1.2% in a study of Taiwanese subjects (Wang et al. 1999). However, CYP2D6*14A was not found in a study of 119 Hong Kong Chinese subjects (Garcia-Barcelo et al. 2000). The C188T and G1846A base changes of CYP2D6*14A correspond to P34S and G169R (glutamic acid to arginine at position 169) amino acid changes, respectively. Wang et al. (1999) reported that the G169R amino acid change alone is associated with decreased CYP2D6 activity. The effects of both P34S and G169R combined on the functional properties of CYP2D6 has also been studied (Shiraishi et al. 2002). These authors reported that the combination of the two substitutions diminished the in vitro metabolic activity to less than the limits of detection of their assays for bufuralol 1'-hydroxylation and dextromethorphan O-demethylation. A novel CYP2D6*14 variant allele, named CYP2D6*14B, has recently been reported (Ji et al. 2002). The CYP2D6*14B allele contains the characteristic G1846A base change of CYP2D6*14, but does not contain the C188T SNP. It also contains the G1749C base change and an additional conversion in intron 1 with CYP2D7P. CYP2D6*14B has been observed to occur at a frequency of 2.0% in a study of 223 mainland Chinese volunteers (Ji et al. 2002). The CYP2D6*14B alleles in that study were actually initially reported as CYP2D6*14A (Ji et al. 2002); therefore, the CYP2D6*14B allelic variant may also confound previous reports of CYP2D6*14A allele frequencies. Neither the frequency of the CYP2D6*36 allele (previously known as CYP2D6*10C) nor the activity of the CYP2D6.36 enzyme has been extensively studied. The frequency of CYP2D6*36 was reported to be 0.9% in 162 Japanese subjects (Ishiguro et al. 2004). In 113 the study by Johansson et al. (1994), among Chinese subjects carrying the CYP2D6*10 allele (50.7%), 73% also carried the CYP2D6*36 allele. In that study of Chinese subjects, CYP2D6*36 was only found in tandem with the CYP2D6*10 allele on the same chromosome. In a review article by Zanger et al. (2004), the CYP2D6*36 allele was listed with CYP2D6*10 under the category of "alleles with reduced function". However, Johansson et al. (1994) reported that CYP2D6*36 results in no CYP2D6 activity in vivo or in vitro. Additionally, two Japanese poor metabolizers were genotyped to be heterozygous for a CYP2D6*36 duplication (Chida et al. 2002). The CYP2D6*37 allele (previously known as CYP2D6*10D) was first reported in a study involving the screening of novel CYP2D6 alleles in 672 European subjects (Marez et al. 1997). In that study, the frequency of the CYP2D6*37 allele was reported to be 0.1%. The CYP2D6*37 allele has not been analyzed in study samples from Asian populations. To date, there have also been no studies regarding the activity of the CYP2D6.37 enzyme. The presence of the CYP2D6*14A, CYP2D6*36, or CYP2D6*37 alleles may reduce the accuracy of CYP2D6*10 genotyping results based on analyses for the C188T SNP. However, the observed frequency of CYP2D6*14A has been reported to be 0.0-2.2%) in Asians, and CYP2D6*36 has been reported to be present only in tandem with CYP2D6*10. In addition, the CYP2D6*37 allele has only been reported once in 672 European subjects (Marez et al. 1997). It appears that both CYP2D6*14A and CYP2D6*36 may result in decreased or reduced CYP2D6 activity, which means that analysis for the C188T SNP may 114 still allow the identification of individuals expected to be slower metabolizers. Further studies of the frequencies of these alleles and their effects on CYP2D6 activity are required. 4.5.6 Linkage of C188T and G1934A SNPs The genotyping algorithm described in this project assumes linkage of the C188T and G1934A SNPs in the CYP2D6*4 allele. However, this may not be necessarily correct. It was reported that 17/41 (40%) Nicaraguan and 16/62 (26%) Spanish carriers of the G1934A SNP lacked the C188T base change (Agundez et al. 1997). In a different study among 254 Mexican American subjects (Mendoza et al. 2001), 4 out of the 8 poor metabolizers found were identified as homozygous for the G193 4A mutation but wild-type at the 188 position. The G1934A splicing defect of the CYP2D6*4 allele renders the enzyme nonfunctional (Hanioka et al. 1990), and any other known mutations in CYP2D6*4 should not alter this. The observations from the above two studies are important to consider when genotyping individuals for CYP2D6*4 alleles. If only individuals testing positive for the C188T SNP are analyzed for the G1934A mutation, some G1934A carriers who are wild-type at the 188 position may be missed, leading to an incorrect classification of their genotypes. It may also be difficult to differentiate between the CYP2D6*wt/*4 and CYP2D6*10/*4 genotypes (C/T at 188 and G/A at 1934). However, in CYP2D6 genotyping studies in Asians thus far (Appendix E), there have been no reports of the G1934A SNP being present without the C188T SNP. There were no CYP2D6*4 alleles detected in Asians subjects from the 115 present study, and thus the relationship between the C188T and G1934A SNPs was not investigated. 4.6 Future studies The CYP2D6*wt/*wt, CYP2D6*wt/*10, and CYP2D6*10/*10 genotypes in Asians can be determined using the novel genotyping algorithm, in order to study the effects of the CYP2D6*10 allele on drug metabolism. The accuracy of the CYP2D6 genotyping algorithm in Asians can be improved by analyses for other CYP2D6 alleles. The frequencies of CYP2D6 alleles can also be studied in a wider range of Asians. With the constant advances of genotyping technologies, the study of the pharmacogenetics of CYP2D6-mediated drug metabolism and its application in the clinical setting can be facilitated. 4.6.1 Pilot clinical study of codeine metabolism The CYP2D6*wt/*wt, CYP2D6*wt/*10, and CYP2D6*10/*10 genotypes were determined in 36 Asian study subjects involved in a pilot clinical study of codeine metabolism. The analgesic activity of codeine is thought to be dependent on the CYP2D6-mediated conversion of codeine to morphine (Wilcox and Owen 2000). Poor metabolizers of CYP2D6 substrates may not derive any benefit from using codeine as an analgesic (Sindrup et al. 1990). The objective of the pilot clinical study is to investigate the effect of the CYP2D6*10 allele on the metabolism of codeine to morphine. The hypothesis of this clinical study is that subjects who are genotyped as CYP2D6*10/*10 will have reduced 116 ability to convert codeine to morphine, relative to those who are genotyped as CYP2D6*wt/*wt. Due to the high frequency of the CYP2D6*10 allele in Asians (35.7-69.2%; Appendix J), many Asians (13-49%) could have reduced analgesic activity from the use of codeine. 4.6.2 Analysis for other CYP2D6 alleles The CYP2D6*14 allele can be analyzed by detection of the G1846A SNP. Using the X 3 F 3 / X 3 R 1 primers, the real-time, rapid-cycle P C R method can be adapted to analyze for the following additional base changes: G1749C SNP (contained in the CYP2D6*2, CYP2D6*4, CYP2D6*8, CYP2D6*10, CYP2D6*11 and CYP2D6*12 alleles), T1795 deletion (CYP2D6*6~), G1846T SNP (CYP2D6*8), and G1846A SNP (CYP2D6*14). Analysis for additional CYP2D6 base changes can identify other CYP2D6 alleles and may increase the accuracy of the determined CYP2D6 genotypes. 4.6.3 Analysis for CYP2D6 alleles in a wider range of Asians The frequency of CYP2D6 alleles has mainly been investigated in study samples from Japan and China (Appendix E). To date, the largest study of CYP2D6 allele frequencies in Asians included 223 Chinese subjects from Mainland China (Ji et al. 2002). In most published studies, it is also unclear as to how the ethnicity of the study subjects was determined. CYP2D6 allele frequencies may also vary among different Asian populations, but this can not be concluded without further studies of CYP2D6 alleles in well-defined Asian samples. In addition, the frequencies of CYP2D6 alleles should be studied in a wider 117 range of Asian samples, including samples from countries such as Korea, Vietnam, Taiwan, Hong Kong, Singapore, Thailand, and Malaysia. 4 . 6 . 4 Further improvements to CYP2D6 genotyping in Asians The preparation and analysis for the C188T and G1934A SNPs using real-time, rapid-cycle PCR can be done in approximately 2.5-3 hours. Compared to PCR in a conventional thermal cycler and RFLP analysis (overnight digestion with a restriction enzyme and analysis using agarose gel electrophoresis), the time saved is approximately 18 hours. The estimated time to complete the CYP2D6 genotyping algorithm is 14 hours. If long PCR of the CYP2D6 gene and detection of CYP2D6*5 were also done in a real-time, rapid-cycle thermal cycler, then the time to complete CYP2D6 genotyping profile in Asians can be further reduced. Recently, long PCR of the CYP2D6 gene in a real-time thermal cycler has been reported (Muller et al. 2003). In addition, a real-time PCR method for detecting CYP2D6*5 based upon gene copy number has recently been published (Schaeffeler et al. 2003), but due to the design of the probes it may not be applicable for use in Asians (the probe sequences may overlie a site containing a known polymorphism in Asians). The application of similar real-time PCR methods in long PCR and CYP2D6*5 detection toward the genotyping algorithm described in this project could allow CYP2D6 genotyping in Asians to be completed in 8-9 hours. If the effects of the CYP2D6*10 allele on the metabolism of certain medications are found to be clinically significant, the reduction in genotyping time can facilitate the integration of the pharmacogenetics of CYP2D6 metabolism into the clinical setting. 118 4.7 Conclusion A long PCR method for amplifying the CYP2D6 gene using primers P100/P200 (Sachse et al. 1997) was optimized. In addition, novel nested real-time, rapid-cycle PCR methods for detecting the CYP2D6 C188T SNP (present in CYP2D6*10 and CYP2D6*4) and G1934A SNP (present in CYP2D6*4 only) were developed and validated using reference samples of genotypes CYP2D6*1/*1 (C at position 188; G at position 1934), CYP2D6*l/*4 (C/T at position 188; G/A at position 1934), and CYP2D6*4/*4 (T at position 188; A at position 1934). RFLP and sequence analyses were also used to further validate these nested real-time, rapid-cycle PCR methods. A multiplex PCR method for detecting the CYP2D6 gene deletion (CYP2D6*5) using primers Dup/Dlow and DPKup/DPKlow (Hersberger et al. 2000) was also optimized and validated using reference samples with genotypes CYP2D6*1/*1, CYP2D6*l/*5, and CYP2D6*5/*5. These validated methods were used to genotype Asian paediatric dental patients from a pilot clinical study of codeine metabolism. Based upon CYP2D6 allele frequencies in this study and those reported in the literature, a genotyping algorithm for CYP2D6*10 in Asians was devised. The genotyping algorithm described in this project is specific for Asians, since it was devised based upon CYP2D6 allele frequencies in Asians (Appendix K). Studies reporting CYP2D6 allele frequencies in Chinese, Japanese, Koreans, and Malaysian subjects were included. In other ethnic groups, the observed CYP2D6 allele frequencies may be different (Bradford 2002). In addition, there may be other alleles that are more common in one ethnic group than others (e.g. CYP2D6*3, CYP2D6*4, and CYP2D6*6 in Caucasians, or 119 CYP2D6*17 in African Americans) (Bradford 2002). Therefore, it is important to note that the novel CYP2D6 genotyping algorithm described in this project is specific for identifying the CYP2D6 genotypes in Asians. Due to the low frequencies of other CYP2D6 alleles in Asians, the analysis for CYP2D6*10, CYP2D6*4, and CYP2D6*5 using the genotyping algorithm in this project allows identification of the CYP2D6*wt/*wt, CYP2D6*wt/*10, and CYP2D6*10/*10 genotypes with an estimated accuracy of at least 92% (Appendix K). 4.8 Significance A novel genotyping algorithm for CYP2D6*10 in Asians has been devised based upon the most common CYP2D6 alleles reported in Asians. It has been designed to minimize the number of tests required for each sample, and can identify subjects with the CYP2D6*wt/*wt, CYP2D6*wt/*10, and CYP2D6*10/*10 genotypes (Figure 20). In addition, the algorithm includes the use of novel nested real-time, rapid-cycle PCR methods for detecting the CYP2D6 C188T and G1934A SNPs. The development of rapid and high-throughput methods for CYP2D6 genotyping can facilitate clinical studies investigating the effects of CYP2D6*10 on drug metabolism in Asians. Pharmacogenetics is a rapidly growing field that may soon shift from the research setting to the clinical setting. Much work needs to be done to determine optimum dosing of medications that are CYP2D6 substrates based upon CYP2D6 genotypes (Vermeulen 2003; Kirchheiner et al. 2004). 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G169R mutation diminishes the metabolic activity of CYP2D6 in Chinese. Drug Metab Dispos 1999; 27: 385-8. Wennerholm A, C Dandara, J Sayi, JO Svensson, YA Abdi, M Ingelman-Sundberg, L Bertilsson, J Hasler and LL Gustafsson. The African-specific CYP2D6*17 allele encodes an enzyme with changed substrate specificity. Clin Pharmacol Ther 2002; 71: 77-88. Wilcox RA and H Owen. Variable cytochrome P450 2D6 expression and metabolism of codeine and other opioid prodrugs: implications for the Australian anaesthetist. Anaesth Intensive Care 2000; 28: 611-9. Williams DA. Drug Metabolism. In Foye's Principles of Medicinal Chemistry. DA Williams and TL Lemke, Eds. Philadelphia, Lippincott Williams and Wilkins, 2002. pp 174-233. Wittwer CT, KM Ririe, RV Andrew, DA David, RA Gundry and UJ Balis. The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques 1997; 22: 176-81. Yokota H, S Tamura, H Furuya, S Kimura, M Watanabe, I Kanazawa, I Kondo and FJ Gonzalez. Evidence for a new variant CYP2D6 allele CYP2D6J in a Japanese population associated with lower in vivo rates of sparteine metabolism. Pharmacogenetics 1993; 3: 256-63. Yu A, BM Kneller, AE Rettie and RL Haining. Expression, purification, biochemical characterization, and comparative function of human cytochrome P450 2D6.1, 2D6.2, 2D6.10, and 2D6.17 allelic isoforms. J Pharmacol Exp Ther 2002; 303: 1291-300. Yue QY, ZH Zhong, G Tybring, P Dalen, ML Dahl, L Bertilsson and F Sjoqvist. Pharmacokinetics of nortriptyline and its 10-hydroxy metabolite in Chinese subjects of different CYP2D6 genotypes. Clin Pharmacol Ther 1998; 64: 384-90. Zanger UM, J Fischer, S Raimundo, T Stuven, BO Evert, M Schwab and M Eichelbaum. Comprehensive analysis of the genetic factors determining expression and function of hepatic CYP2D6. Pharmacogenetics 2001; 11: 573-85. 129 Zanger UM, S Raimundo and M Eichelbaum. Cytochrome P450 2D6: overview and update on pharmacology, genetics, biochemistry. Naunyn Schmiedebergs Arch Pharmacol 2004;369:23-37. Zanger UM, F Vilbois, JP Hardwick and UA Meyer. Absence of hepatic cytochrome P450bufl causes genetically deficient debrisoquine oxidation in man. Biochemistry 1988; 27: 5447-54. 130 APPENDIX A - CYP2D6 Numbering Systems Table 29. Comparison of three different numbering systems for CYP2D6 single nucleotide polymorphisms. This project used the conventional numbering system from Kimura et al. (1989), in which base number 1 corresponds to the first nucleotide of exon 1 of the CYP2D6 gene. At the Human Cytochrome P450 Allele Nomenclature Committee website (http://www.imm.ki.se/cypalleles/cyp2d6.htm; accessed 26 July 2004), base number 1 corresponds to the first nucleotide of the first codon (AUG) in the CYP2D6 sequence. The CYP2D6 gene sequence in GenBank accession number M33388 was originally submitted by Kimura et al. (1989); this sequence also includes additional bases upstream and downstream of the CYP2D6 gene. Kimura et al. (1989) Human Cytochrome P450 Allele Nomenclature Committee website GenBank accession number M33388 (CYP2D6 gene) C188T C100T C1719T C1127T C1039T C2658T G1749C G1661C G3280C G1846A G1758A G3377A G1934A G1846A G3465A G4268C G4180C G5799C 131 >> CO 3 5 « so I I PQ l - H — P M P H < o r< • o o • • • o o LLI o cr. CO >- Q m 1 O CN i"-* > BP • • * •v 7^ rv. , „ r~- —j LU v ^ CL CO m O ^ 23 > < g c — £ 0 kf> * ® VOW * • • i — •s X 2 ^ _ rn t o X 2 CD r n ° ; £ INO — >^ t—1 M I n l / J >- L J «S <— * • o • " * ;C o •k. • 0 • s* • o • 5- llil nn • •s iBi & 1111 Hi $ ^ F ^ CDLUTZLJJ — CO Ci LLI - 1 LU 1 - LU O ** • r* • i i 11 o ; • • lill CYP206 allele Effect on protein R26H P34S G42R Frameshift Splicing defect T107I Silent Silent Frameshift G169 -> stop G169R Splicing defect R201H Frameshift "Hi -u CO R296C H324P VPT468-470ins S486T Mutation G165A C188T G212A CO CD fN G971C C1111T C1127T G1749C T1795Del G1846T G1846A G1934A G2031A A2637Del I 2701-3Del C2938T A3023C 4213-21 Ins G4268C 132 p-% in H 90 00 U NO I M J — •+-» S CJ — I u X m P w 5 H 3 C < H 6- ( ti a ID 2 < * Si «< < (S o o u U O O L> < < < < u o o <j <<<< o o o o u o y y EH H EH H * m y y y y «e* < < < U CJ L> U < < < < u CJ o CJ as j y v o y u w (0 m «t m p* a c o r - r - r - c o Q | 04 P< P . 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CJ CJ U CJ CJ CJ CJ CJ u u u u u u u u u u u u u u u u I Li Ci Ci Ci *Q u u u u u u • h s - h h h c - • C J u u u u u u u u CJ CJ t LSI ti u u u u ci Li ti ti ci Li ti ti ^ -u . u u C J (J u u u u u • u u u u u u u u i P - i u u u u C J u C J C J u u p u * •< •< u u u u c i 3 t i c i u u u u u u u u U U U U [ i p p p C J U C J C J c C J u C J C J c u u CJ t_> c f - f - 4 r- i u u sr i u u <n i U U * _ j u u » 17 i •<•< 3 i t i t i IS T ' . * O ti -4 < •**C O I u u M I u u I U U O I u u < ya i u u <n • -< t i u u C J C J u u 8S u u Li ci C J C J u u C J C J 3S33: a t i c i c 5 t i c i c 5 t i t i t ;S3 ) cici i oil i o ti ) U U i ci ti 3 P -<n < cq G « r- o r- CD OlNNNcMM £ Ita PM CL, CU OU o pM > « > , u u u u u u I U U t Cl 3 c i t 313 — O t i £ > ci ci ti i u u u > o t i t i > c i ti o ) Ci ti £ 5 i ci Ci Ci : <<< <^ ) ti c i p i c i c i c i ti t i o ci c i o 111 ti ti t i > ci ti ti > ci ti ti i ti ti ti R IN f - F - h - H U U U U U U t i cs t i t i c i c i 3 3 < * C J u C J c u U U c C J u C J c u u u c rf " * " t i is t i c ffl ^ " t i tS t i c C J U C J t ci 5 £ B li ci •I CM CM Cu CU > t i c i t i > c i c i i i > t i t i t i • i i i ) c i c i c i > c i t i t i : c i t i t i t i ) d c i t i c i «c H< > ti ti ci I ) C J CJ C J C J ' u u u u I Li Ci t ci tj ci i u u u t l i tS c i t -*a <' t i ts c i t u u u c t i c5 t i c ) C J C J C J < H H h > t i t i t i o • c i c i c i t i c i c i -4 UD CN] CD n 3 CM CM n «< m B m ID HQQQOCl n N N NN <M B Ai CM CM CM CM O ?H >« >-UUUUUU .*3 cyi C3 o> _ oa 3 m m cu BO i 134 s < *3 s = cr u ti so I I H X MH — ti Ph Ph < o S-H 6 _CU '3 fl CU rt cr -4—* ,H T3 <U C ' CO * "o fN fl cl cr ^ O * CU O fl 0) cr cu CO o a, 6 o o <u •t= c CO . f l o in ft o <u U 13 CO fl O Tfl cu O 3fl X> ca co <N •fl * S " ° £ Q CU (Ni CU > - H T3 C ca * o ^ ft^ .1 " CO <+H Q -f l s-i ,o cn O T3 CU N >, co "crj CU c ft § S " oo 0) +3 C4-H co o fl 1) ' X> a O © 2 * O O X> ft i f fl cu ca -a ~& ^ ca fN C O <J CO fl cu fl . cr o <u ca ca T3 0) q a <N cu >-H 1) o 13 . 13 o c o C E r £ CU N a >» -*-> ca fl co" 1 3 1) ^ 3 -(-> -4—• CO CO cu V "-fl 9H fl -*-» CO o PL, o C+H T3 cu _> cu < "T3 0) _N ' C H ca a a a CO 0> o - (U o © 0. 0) 'J a. o CD OJ OJ CO > n as OJ 05 05 a* CO CD 1c O OJ c OJ CO CO CO > n _0J iS OJ CD s D CD CN cni| CD 3 5? o CO CN 3 CN 3^-CD CD CM CD CD _2 4) C D Q to a o 3 CD CD CO CO cu co •a •o 1^ 01 O •M-cu CO CD . cn CD CD m CD o •C cu o 135 APPENDIX F - English Consent Form for Pilot Clinical Study )fThe University of British Columbia Office of Research Services Clinical Research Ethics Board Room 313 -1!*" Health Sciences Mali. Vancouver. BC V6T IZ3 Ptione: <«M) 8l:-i5!4 Fas: (60s) SZl-mi CONSENT FORM Pharmacogenetics of Codeine Metabolism to Morphine in Pediatric Dental Patients: A Pilot Studv INVESTIGATORS: Drs. M. Levine, C. Montgomery, M. Ensom, E. Reimer, Ms, C. Court EMERGENCY TELEPHONE NUMBER: 875-2161 (hospital location; ask for Dr. Montgomery or the anesthesiologist on call) Background: Codeine and acetaminophen (e.g. Tylenol9) are safe and commonly used pain medications. Your child is scheduled to have a dental procedure done under general anesthesia. Our routine practice is to give a dose of codeine with acetaminophen by mouth before the procedure so that these pain medications will be working when your child wakes up. Codeine works to treat pain by being changed in the body to morphine, a very effective pain medication. A particular "enzyme" in the body is responsible for converting codeine to morphine. Research suggests that about 40% of people of Asian origin may not be able to change codeine to morphine quickly enough to be effective. This is due to inherited (genetic) differences in the enzyme that metabolizes codeine in the Liver. If this is true then codeine may not be the best pain medication for people with the "slower form" of the enzyme. Purpose: The purpose of this study is to find out whether Asian patients who have the gene for the "slower form" of the enzyme do make less morphine from codeine than Caucasian patients who have the gene for the most "rapid form" of the enzyme. We hope this study will help us determine which patients should receive codeine for pain. Study Procedures: Your child will receive a dose of codeine (1.5 mg/kg) and acetaminophen (20 mg/kg), by mouth as liquid medication, according to our routine procedure about 30 minutes before the dental procedure is to begin. The anesthesiologist doctor will then put your child to sleep using the routine anesthetic procedures. If the doctor thinks that your child requires more pain medication, then fentanyl, a commonly used pain medication, will be administered through a vein. This is also a routine procedure in our hospital. In order to determine which form of the enzyme your child has, a blood sample (2-3 ml, about half a teaspoon) will be drawn from a vein at one hour after the codeine dose and (if your child is still asleep) again at two hours after the codeine dose. While your child is still asleep, we will also touch the lining of the mouth with a cotton swab to take a sample inside of your child's cheek. The cells contained in the swab may give us the same information about the enzyme as the blood sample. If it is accurate enough, this form of testing may be able to replace the blood test. In order to participate in the study, the one or two blood samples and the cheek swab while your child is asleep are the only procedures that are different from a routine anesthetic. Participating in this study will not make the hospital stay any longer. CHILDREN'S <V WOMEN'S HEALTH C E N T R E O F BRITISH COLUMBIA B R I T I S H C O L U M B I A ' S C H I L D R E N ' S H O S P I T A L B R I T I S H C O L U M B I A ' S W O M E N ' S H O S P I T A L A N D H E A L T H C E N T R E S U N N Y H I L L H E A L T H C E N T R E F O R C H I L D R E N CW is «« ^eAOCMMr w t n . C£nrmx urmuArto *rt» r».r w*rtvff«siT> <j» s»mtn e t t m u o u f « «.c. m c s o f t o . « r » m u * K *a* CMK0*en Ir- t * o « s « s H U » Figure 23. Copy of the English consent form used for the pilot clinical study entitled "Pharmacogenetics of Codeine Metabolism to Morphine in Paediatric Dental Patients: A Pilot Study", (a) Consent form in English - Page 1 of 2; (b) Consent form in English -Page 2 of 2 136 Exclusions: Your child cannot participate in this study if he or she is allergic to codeine or acetaminophen or cannot take them for some other reason, or is taking medication that may affect the metabolism of codeine to morphine. If your child has any problems with the lining of the mouth then the cheek swab will not be done. Risks: Aside from the normal procedures and medications, the only risk to your child is the slight possibility of bruising or infection at the site that the blood is drawn. When possible, the blood samples will be taken from the intravenous site required for the anesthetic. Benefits: Your child will not benefit from this study; however, when the study is finished you may obtain information about the form of the enzyme that your child has that may be beneficial when future medication decisions involving codeine or other drugs that use the same enzyme need to be made. Confidentiality: Any information resulting from this research study will be kept strictly confidential. All documents will be identified only by code number and kept in a locked filing cabinet. Your child will not be identified by name in any reports of the completed study. Contact: I understand that if I have any questions or desire further information with respect to this study, or if 1 experience any adverse effects, I should contact Dr. Carolyne Montgomery or one of her associates at 875-2711. If I have any concerns about my treatment or rights as a research subject I may contact the Director of Research Services at the University of British Columbia at 822-8598. Patient Consent: I understand that participation in this study is entirely voluntary and that I may refuse to have my child to participate or I may withdraw him or her from the study at any time without any consequences to my continuing medical care. I understand that by consenting to this study, my child will have up to 2 blood samples taken and a cotton swab touched to the liningof the mouth while asleep under anesthesia. My child will receive all appropriate pain medication as required. I have received a copy of this consent form for my own records, and I consent for my child to participate in this study. Patient Signature Date Parent/Guardian Signature Date Witness Signature Date Investigator Signature Date Cotisent version; October 1, 200 i Figure 23. (Continued) APPENDIX G - Chinese Consent Form for Pilot Clinical Study (a) Ttia Um*vm:y of Srwisfc Cjluawu OlT!« ut RescarcB Savkss Otnicii Retesixa Etntci B«U!l Room J23 - 2JW tfaaift Stun™ Mail. VJBUOH fttcne-.foCJlSn-iJI* Fat: I60*i X£-fi»3 w i t ' * f £ A j | : M. Levine f#tb - C. Montgomery H £ - M. Ensom i#± - E. Reimer H £ - C. Court Sf : 875-:i61( ^Montgomery t & « * t 0 U f t f f e i * ) -•T#S(Codemc)^8&a»(Tylenol®)>|.f ffl#£tWjfc«» • &-#«>,*SCT« ® (Codeine)^ * H-&«"e* A ® « #<!t&.fc •JHKmorphine)-- - jf£?|t <f t «t« j L * «t**ittt±c*B)i4*ft * • - *r ftBTfc**.*-*-***** « jMWfctf§#i&£,w«tfr " w i s * 8?**a«s»A-dr • *<nte*r#8«'5b&*«4« * * * * * * * * * ' 30 • fctfa-fflo*-*!**1*^ * • *-*.^±«ft«a-ft#*.^ft?!Jtjg| | > # « t » • >£* - « * * £ it*l(f«ntaayi) t S & ^ i ^ M & T a ^ A ^ ^ • ' lift® O-UWEH-S & WOMEN'S HEALTH CENTRE Of5 BRITISH COLUMBIA Figure 24. Copy of the Chinese consent form used for the pilot clinical study entitled "Pharmacogenetics of Codeine Metabolism to Morphine in Paediatric Dental Patients: A Pilot Study", (a) Consent form in Chinese - Page 1 of 2; (b) Consent form in Chinese -Page 2 of 2 138 Txe Uoiverciiy of BruijJi Cuiumhi-i Clfitt! at Research Sonrkct C'.iaicii Research Eiistci Board Room 323 -1194 HoiOi i k w t . Mall, Vaocowver. 3C V67 IZ3 m»-**: • * * T ? * f f A -8? • « * A : it • Carolync Montgomery § £ « . # f e « i i q $ • t i l ? * 875-2711 • * 8.1*«f S U L - ^ l L ^ ^ i S ^ i M ^ W f t ' * t » r « « « - # t t A * « r • * « * 822-8598 • all CMtORErW & WOMB! "S HEALTH CSNTRg OP BRITISH COLUMBIA. Figure 24. (Continued) APPENDIX I - CYP2D6 Genotypes in Pilot Clinical Study Table 32. CYP2D6 genotype assignments of the 36 Asian subjects involved in a pilot clinical study of codeine metabolism. CYP2D6 genotypes assignments are based upon results from analyses for the C188T SNP, the G1934A SNP, and CYP2D6*5. Subjects C188T SNP G1934A SNP CYP2D6*5 CYP2D6 genotype 07 51 C G No CYP2D6*wt/*wt 57 77 05 15 C/T G No CYP2D6*wt/*10 17 19 20 21 31 32 40 71 72 73 18 56 C G Yes CYP2D6*wt/*5 02 03 T G No CYP2D6*10/*10 04 06 08 09 13 14 23 27 34 35 42 52 53 55 68 74 141 a 3, <« s .23 .2-fl a> s to — N O 1 I X Q O O _<u >> 1- ' 0 CU G x> cu > G 3 co c cu 13 G »*-> C+H 'S 13 <u +-> 0 43 "0 H-J *^ 0 X t/T u CM 'SH S — ' 3 CU — C O X ! -+-> orted JU e orted !& C + H ep 3 C O & CD B 3 res 0 res 13 60 i- T3 G <u -3 _> _ 3 rtic CU 3 rtic -0 . G pa CU •— C/T C+-H ra c O 0 0 -4—» _<U <u 3 popula G popula [ num reque c ra 3 » O 'to ISO < || ISO O H 0 on G B 0 cu CU CJ 3 <u G iU -4—» co CH-H CU 0 >. CU _ G T3 3 ISO l o Cfl c a S <U 0 3 c/) a 3 nc co ra cu G 3 3 freq .2 C O < artic O H — cu CO CU 3 - G 13 kH G . O Q G C O C+H ; G CYP etabo lalyze <u JG -a cu N 3 c ra 3 - G C/3 <u 3 +—* co CM d CN T3 cu N a s 3 C O cu fcH <n to _cu 3 G <u 3 cr cu ju 3 3 o X > C O » G cu O H S (U ^ •KH, CN * Q CN o Q CM « C ^ u (U J G o o CO CO tu 4> G cs H cu O o c3 c/5 -*-> O <U X ' 3 « 5 O X cu 3 ra CN * Q CN T3 ra o G 0) O C D C D C D C D C N un o ra u o — 3 c o CL > "O 3 tfl cc co o C D o ^  M rsi CN C O m C D C O CM CD C D C N | C O C N co co CO a C N o o i x CO LVD O -a CJ) C O C N un C D cn O o o CD f N o cn o a f N a> u cu cu C D C D CO a) o ZJ oil cc a) CD n CD C N CD CD CD CD CD CD un C D cn o m > . cc en C D cn cc O CO cu cs i O C\| CO 8 CD "a t: C O Q C M ^ CD • = co O C C N > co £ >, o s " O CO C N C C J Q ) CD co IB1 142 APPENDIX K - Accuracy of the Genotyping Algorithm Estimated frequencies of CYP2D6 genotypes using the Hardy-Weinberg Equation {CYP2D6 Allele frequencies in Asians are derived from Appendix J) Allele CYP2D6*wt CYP2D6V CYPWrS CYP2DW0 CYpmro Allele Frequency 43.3% 0.6% 5.6% 49.3% 1.9% CYPWVwt 43.3% 18.7% 05% 4.8% 42.7% 1.6% CYP2D6'4 0.6% 00% 0.1% 0.6% 0.0% CYP2D6'S 5.6% 0.3% 5.5% 0.2% CYP2DV10 49 3% 24 3% 1.9% CYP2DV0 1.9% 0.0% CYP2D6*<M represents both CYP2D6*1 and CYP2D6*2 CYP2D6V represents other CYP2D6 alleles that do not contain the C188T or G1934A SNPs Expected Metabolizer Status EKtensive Intermediate Slow Poor Assignment of CYP2D6 genotypes in Asians using a novel genotyping algorithm for CYP2D6*10 Genotype Frequency W v w 18.7% W 1 0 42.7% W 4 0.5% wo 1.6% 4.6% 24.3% *ioro 1.9% *1QT5 5.5% *iQT4 0.6% *4T4 0.0% **™Q 0.0% *4T$ 0.1% *oro 0.0% *QT5 0.2% 0.3% Total 101.4% I No product from long PCR CYP2D6*5 Analysis Yes - CYP2D6*5T5? "5"S 0 3% Accuracy 100 0% Product from long PCR • C183T Analysis C188- CYP2D6Wvt? *wtr'm 18.7% W 0 1.6% W 5 4.8% 0.0% ms 0.2% Accuracy 73.5% OR C/T188- CYP2D6*wtT10? *wt'10 42.7% *wt/*4 0.5% 1.9% *4f*Q 0.0% Accuracy 94.6% OR T188 - CYP2D6*1GT10? 24.3% 5.5% *1QT4 0.6% *4f4 0.0% *4T5 0 1% Accuracy 79 7% CyP2D6*5 Analysis No CW206 , WVi? OR Yes - CYP2D6*'MrS? 18.7% W 5 4 8% wo 1.6% *ors 0.2% -oro 0.0% Accuracy 958% Accuiacy 91.8% G1934 - CYP2D6'wp'10? OR A1934- CYP2D6W4? *wt*W 42.7% W * 0.5% *1QT0 19% *4fQ 0.0% Accuracy 95.8% Accuracy 95 8% -» G1934A Analysis G1934 - CYP2D6*10T10? ••wn 24.3% 5 5% Accuracy 81.5% OR G/A 1934 - CYP2D6*10/*4? *1QT4 0.6% Accuracy 100.0% OR A1934 - CYP2D6*4T4? *4T4 0.0% 0.1% Accuracy 5.1% CyP2Q6*5 Analysis No - CYP2D6'WrW? 24.3% Accuiacy 100.0 OR Yes - CYP2D6*1Qr5? *1QF$ 5.5% Accuracy 100.0% CYP2D&*5 Analysis No - CYP2D6*4T4? % -.: 0.0% Accuracy 100.0% OR Yes - CYP2D6*4T5? •"4T5 0.1 %| 100.0% Figure 26. Estimated accuracy of the genotyping algorithm for CYP2D6* 10 in Asians. 143 

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