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1. Mg 15 10 15 2 0 mL 2 A B ELET 400 UV u L A B 10 15 JE i 24 1 1 05 ITT A B A ROB I as 1 4 Su 10 Triton X 1002. 5 HIV H BV HCV 5 000 p EZK RURE m 0 5 1 6 7 8 121C 20 JETER SOA gt o
3. a ss ur No Call ce Mica No Call 1 0 1 2 0 1 99 1 3 0 0 00 0 4 0 0 00 0 5 0 0 00 0 6 0 0 00 0 7 0 0 00 0 8 0 0 00 0 9 0 0 00 0 CYP2D Genotype Accuracy 99 196 Genotype wi 63 61 0 5 96 8 Call Rate Correct Call Miscall 99 3 CYP2C 19 Genotype Accuracy Genotype Call il 3 3 0 0 00 0 Rate 1009 3 4 14 5 5 0 0 00 0 pi 3 DNA CYP2D6 CYP2D6 SN Correct Miscall Genotype Genotype 15 3 3 0 0 0 D Call Accuracy Call Rate 7 1 1 31 31 0 0 100 0 00 0 ni i 5 7 Di 1 1XN 5 5 0 0 100 0 00 0 oe E 5 5 A 009 E 1 2A 30 30 0 0 100 0 00 0 JE 3 3 a E TA E 1 2AX 2 1 0 1 50 0 50 0 wi E o 5 0 E 1 2D 1 1 0 0 100 0 00 0 RI 1 2DX 1 1 0 0 100 0 00 0 30 4 3 1 0 75 0 ss si 1 3 2 2 0 0 100 0 00 0 aa a E 5 5
4. 9 3 5 CYP450 4 2 8 1 2 2 8 C 6 2
5. RIESUTET 240 CYP450 GeneChip Operat AH fymetrix Molecular Diagnostic Software AMDS v1 0 D GeneChip Operating Software 1 AmpliChip CYP450 US Data Analysis Software CYP450 4 DNA E
6. 6 15 30C 20 mmol L EDTA VY CHE 2 8C 2 8C 10 Triton X 100 15 30 3 1 t aa 2 3 Ye SATB 4 CYP450
7. a CYP450 CYP450 DNA AE iChip CY F DNA 15 000 ing Software GCOS v1 1 3 AmpiChip CYP450 US Data Analysis Software v 2 1 Amp P450 Data Analaysis Software v3 1 Ex o E
8. 5 7 2 8 20 H 10 GO nal 3 000 mg dL 2 DNA 32 fi Polymorphism fi CYP2D6 CYP2C19 390 DNA REFLP Y Restriction Fragment Lenghth AS PCR Allele sey Rares CYP2C19 100 CLK 1 2 PSH Specific PCR DNA CYP2D6 99 2 1 CYP2D6 2 CYP2C19
9. 1 PCR 5 PCR Polymerase Chain Reaction DNA CYP2D6 CYP2C19 6 CYP450 CYP450 A CYP450 B CYP450 A PCR CYP2D6
10. 2 poor metaborizer extensive metabolizer E Extensive metabolizer CYP2D6 CYP2C19 P Poor metabolizer eh 7 CYP2D6 poor metaborizer 1 29 2 496 CYP2D6 poor metaborizer CF 9 CYP2D6 10 u 10 1 29 Ct CYP2C19 poor metabo Z intermediate metabolizer DRE 50 g
11. 6 45 15 mL 15 30 6 2 0 mL 24 1 LI 20X SSPE Buffer 300 mL 10 Triton X 100 1 0 mL 5 36 mL 1 663 mL 2 000 mL 5 15 2 AmpliChip CYP450_US Data Analysis Softwawre CD ROM v2 1 GCOS v1 1 3 AmpliChip CYP450 Test Data Analysis Softwawre CD ROM v3 1 AMDS v1 0 DNas
12. DNA UNG DNA DNA UNG U dUTP RNA k 1 2 15 CYP450 DNA DNA ZA dTTP FRR 5 000 1 m 0 5 IZ KS 2 8C 1
13. 2935A gt C Evert et al 1994 1661G gt C 1758G gt T 2850C gt T 4180G gt C ia Broly et al 1995 CYP2D6 CYP2C19 CYP2D6 CYP2C19 Tyndale et al 19912 R32 aera Broly amp Meyer 19932 Yokota et al 19932 100C gt T 1039C gt T 1661G gt C 4180G gt C Ap Johansson et al 1994 Sakuyama et al 200859 8 15 Site Call Data Analysis Software Site Call Call Site Call ata Analysis Software
14. 24 1 2 1 1 8 2 1 1 3 1 1 3 20 2 1 1 1 20 2 5 CY 24 1 CYP450 HCF M 1 ED 500 A ng PCR
15. H 20 A 1 Retainer Bul DH H 30 18 N BL 1 1 Retainer Hi A T 8 L Hol 6 HM H 3 20
16. 6 B1 LB1 Oligo 1x1 3 mL 7 CYP450 OYP450 Array 24 EDIE FRNA io Rr rat CYP450 8 CYP450 CYP450 C 1x204L CYP2D6 CYP2C19 9 A CHIP C 1x1 2 mL CYP2D6 1 2ABD 3 4ABDJK 5 6ABC 7 8 9 10AB 11 15 17 19 20 29 35 36 40 41 1XN 2XN 4XN 10XN 17XN 35XN 41XN o i CYP2D6 1 2ABD 3 4ABDJK 5 6ABC 7 8 9 10AB 11 15 17 19 20 29 35 36 40 41 1XN 2XN 4XN 10XN 17XN 35XN 41XN CYP2C19 1 2 3 ru Ste n 5
17. CYP2D6 CYP450 CYP450 B PCR CYP2C19 4 5 1 CYP450 OYP450 MMX 1x1 3 mL CYP2D6 2 5 dATP DNA 1 DNA AmpliTaq Gold DNA CS5 SEI Gold DNA CYP450 We ll a A MN431 D12R NP162 WK621 2 5 ZYR UTP WK418CC NP158 CYP450 B WK404 AmpliTaq Gold DNA
18. N K A 4 Re 9700 25 20 95C 10 Bult 2 SE BE or S d Method Gold 96 well GeneAm METHOD Met Reaction Volume 24rL START B 3 B 8 L 3 i 8 L ainer Gold 96 well GeneAmp PCR System PCR System 9700 od Options Ramp Speed Max Fr
19. 2 8 CYP450 P SH Woe LI x D CYP450 CYP450 ArayJ Tough Spots tia ETRE CYP450 CYP450 Array 2 Septa A CHIP C No Call No Call CYP450 CYP450 Array 2 GeneChip Scanner Autoloader CYP450 CYP450 CYP450 Array 30 CYP450 C 4 41 1 2 GeneChip Scanner Autoloader 2 8
20. 3 CYP2D6Y17 CYP2D6 29 3 intermediate metabolizer rarapid metabolizer LED 299 CYP2D6 ultrarapid metabolizer izer CYP2C19 2 CYP2C19 3 2 13 23 poor metabolizer CYP2C19 poor metabolizer 17 3 5 9 CYP2D6 CYP2C19 5 6 CYP2C19 7 5 CYP2D6 2 TMT HMA aD 37 28 35 25 42 60 12 42 2 16 16 29 23 33 20 33 28 12 3
21. 40 1 3 7 15 O 10u L 3 Retainer Gold 96 well GeneAmp PCR System 9700 37 35 95C 5 4 C Method
22. Triton X 100 10 mL 90 mL 10 Triton X 100 15 30C 6 CIO 15 mL 10 15 cre hee PATEL EY ARETE 2 BC IRAE CON A RETT g BOE jE 2 l l a 20 SSPE Buffer 3 125 mL Ges Tea X10 AA 1 TE BI i 20 mmol L EDTA 0 5 mol L EDTA 1 ml 24 mL B1 Oligo Leek 1 10 B1 Olig 20 mmol L EDTA 2 8 6 50X Denhardt s solution 250 uw L 1x in VEJ MZ 2 0 mL 5 225
23. 11 15 CYP2D6 3 1 EDTA 2 OR CYP2 1 1 2 Restriction PCR amp PCR RFLP DNA DNA 6 CYP2C19 CYP2 CYP2D6 CYP2C19 No Call CYP450 GM DNA DNA CYP2D6 25 ng D A CYP2C19 2 5 ng DNA 2 4
24. AmpliChip CYP450_US Data Analysis Software 29 DNA CYP2D6 CYP2C 9 3 2 DI NA KILET HO U H 3 DNA dTTP dUTP DNA U
25. CYP450 CYP2D6 CYP2C19 2 2 96 Reac 96 1 1 A B 100L 0 8888 QO Tu a cu Dub 2H RY SH SH SH MicroAmp tray CYP450 CYP450 B Bub DH A T5 u L B 75L A B Resealal LET A B
26. GCOS AMDS GCOS v1 1 3 AMDS v1 0 GeneChip Flui ics Station 450Dx 7 D GeneChip Fluidics Station 450Dx Position 1 GeneChip Flui 2 Station CYP450 Array Washblock door CYP450 CYP450 Array washblock door GeneChip Flui CYP450 CYP450 Array ics Station 450Dx Position GeneChip Fluidics BOLET CYP450 ics Station 450Dx
27. 2 CYP450 24 mo ow wo m a mm ow 5 000 ppm 0 5 DAS v 5 000 ppm 13 15 1 2 Fodor S P A et al Pease A C et al heNational Academy of Scineces U S A i se CYPalleles Longo M C et al Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions 1990 93 p 125 128 Kunitz M Spectrophhtometric method for the measurement of desoxyribonuclease activity Jounal of General 1950 33 p 349 362 ki se CYPalleles cyp2d6 htm Polymorphism of the cytochrome P450 CYP2D6 gene in a European population characterization of 1997 7 p 193 202 Cytochrome P450 2D6 variants in a Caucasian population allele frequencies and phenotypic 1997 60 p 284 295 The human debrisoquine 4 hydroxylase CYP2D locus seque
28. E poor metaborizer intermediate metabolizer extensive metabolizer ultrarapid metabolizers CYP2D6 CYP2C19 MISHA ek amp M I 883G gt C 1661G gt C 2850C gt T 4180G gt C arez et al 199529 AmpliChip CYP450 US 15 138ins T Sachse et al 19962 WT 3 DIE asimirembwa et al HET SIT 1023C gt T 1661G gt C 2850C gt T 4180G gt C 199628 MUT Oscarson et al 199729 No Call o call ss 1661G gt C 2539 2542delAACT arez et al 1997 Positive 2850C gt T 4180G2C Negative 20 1661G gt C 1973insG 1978C gt T 1979T gt C arez Allorge et al 2850C gt T 4180G gt C i 199950 CYP450 2D6 si 1659G gt A 1661G gt C 2850C gt T DE n o m ate 2 CYP2D6 3183G gt A 4180G gt C a 4 Wennerholm et al 2002
29. 20 4 SA EDTA 7 BGT DNA UNG 6 LEA gra El DL 10 6 000 mg dL dUTP ZEH DNA UNG PCR
30. lt 2 CYP450 ALCYP450 PM A 1x0 7 mL x a gt MN431 lt i 2012 12 3 NP162 20104 3 2 h WK621 22100AMX00779000 WK418CC NP158 P450 CYP 3 CYP450 B CYP450 PM B 1x0 7 mL si o OA 77 WK404 FA CYP450 WK405 NP164 NP165 ee oar i 4 MgZ 1x1 3 mL Cytochrome P450 CYP 2D6 2C19 e i m 14A 18 21 5 TIT LTaT 1x254L poor metabolizer
31. Gold 96 well GeneAmp PCR System 9700 METHOD Method Options Ramp Speed Max Reaction Volume 34 START 45 O 5 95C 1 5 m Fa TeAB 1 6 2 8C 18 18 20 C 20C 1 500 L
32. 0 5233496 001 D
33. CYP450 ISH 5 DNA 0 5 6 D 0 5 DNA e e A II DNA 2 ES EAR KUR E 121 OBE 5 000 ppm 0 5 C 20 5 000 ppm 0 5 1
34. 20uL 20 CYP450 CYP450 Array 6 GeneChip Fluidics Station 95 10 3 1 5 mL CL 10 500 L
35. 3 3 Gold 96 well GeneAmp PCR System 9700 CYP450 50 2 95 CC 10 1 35 95C 20 67 4 72 7 4 Method Gold 96 well GeneAmp PCR System 9700 QO METHOD 100L START Retainer ethod Options Ramp Speed 2 Max Reaction Volume FERLET 3 30 72 MicroAmp Base 4 C
36. CYP450 CYP450 Array L2 8 C 1 CYP450 CYP450 Arayj Data Analysis Software CYP450 CYP450 CYP450 Arrayld CYP450 CYP2D6 CYP450 CYP450 Arrayj CYP2D6 9 GCOS v1 1 3 AmpliChip CYP450 US Data Analaysis Software v2 1 a GCOS vl 1 3 b
37. CYP2C19 1 2 3 1 2 o 3 RAC RMR TTR IH PI MR OVE ICE CURL CSTE SV tales l i l DNA CYP2D6 CYP2C19 A
38. CYP450 CYP450 Array CYP450 CYP450 Array Allow rescans g Marez et al 19977 OK Sachse et al 19979 CEL Kimura et al 1989 O Johansson et al 1993 p e 1 AmpliChip CYP450 US Data Analaysis Software v2 1 KA EET l I i a A 5 NA ca i 3 0 aimundo et al 2 CEL pees 9977 3 E 3 w i Sakuyama et al 200859 i F 13 2549Adel tr Kagimoto et al 1990 AmpliChip CYP450 Test Data Analysis Software R
39. T DNA DNA DNA 1 DNase dNTP 3 CYP450 CYP450 DNA BI GeneChip Fluidics 4 5 Station 450Dx Affymetrix 1 CYP450 gt 2 DNA Rf o
40. CYP450 A CYP450 B BI E 5 5 000 ppm 7 DNA DNA 1 2 8 2 CYP450 5 CYP450 A 5 CYP450 B 5 2 TdT 6 BI 2 CYP450 2 CYP450 2
41. Q 8 0 Invitrogen Corp H e solution 5 w v VWR International Mississauga 1 ovine serum albumin 20 mg mL Sigma Aldrich s label small USA Scientific Inc lied Biosys AMDS Affymetrix ems AH fymetrix jon wi IAamp DSP DNA Blood Mini Kit QIAGEN aH PO 0 02 mol L EDTA Cambrex 1 E 95 Resealable bag 5 mL 10 mL h AMDS v1 0 LA E Affymetrix D Biosystems 96 Well GeneAmp PCR System 9700 thermal cycler MicroAmp 0 2 mL Reaction mL Reaction Tubes Caps Tay Retainers MicroAmp Reation Tubes Tray Retainer Assembly er CoCL solution 6 15
42. 0 0 1 lt 1 lt 1 0 0 4 lt 1 2 38 17 19 3 7 3 11 12 6 7 5 Ale 3 6 5 6 3 6 7 1 2 3 4 6 6 0 0 lt 1 lt 1 0 3 0 0 9 KEF 0 0 2 3 lt 1 0 0 0 0 EF 38 40 57 3 5 4 6 3 6 0 4 5 7 EF 0 0 lt 1 12 21 O 1 8 6 18 29 KEF 0 0 lt 1 7 0 0 9 12 41 KEF 0 2 4 7 14 3 0 8 2 6 1XN lt 1 lt 1 lt 1 3 2 3 0 0 2XN lt 1 lt 1 1 3 2 2 gt 3 28 2 4 4XN 0 0 lt 1 1 4 lt 1 0 7 14 10XN KF 3 1 lt 1 0 lt 1 0 0 41XN EF 0 lt 1 lt 1 0 0 0 0 6 CY P2C19 CYP2C19 7 KT CYP2C19 9 1 58 9 Tele 28 EB VAR 14 CYP450 CYP2D6 CYP2C19 CYP2C19 4 28 12 14 16 18 21 28 30 34 37 39 42 80 AmpliChip CYP450_US Data Analysis Software
43. 1 A batto bu A B 15 2 8C Ci ion Tube Retainer 20C 1 CHE CYP450 HELE le plastic bag 25 u L 25 u L
44. K et al The impact of Pharmacogenomics on Postoperative Nausea and Vomiting Do CYP2D6 Allele Copy Number and Polymorphism Affect the Success or Failure of Ondansetron Prophylaxis Anesthesiology 2005 102 p 543 549 45 http www imm ki se CYPalleles cyp2c19 htm 46 Romkes M et al Cloning and expression of complementary DNAs for multiple members of the human cytochrome P450IIC subfamily Biochemistry 1991 30 p 3 247 3255 47 Richardson T H et al A universal approach to the expression of human and rabbit cytochrome P450s of the 2C subfamily in Escherichia coli Archives of Biochemistry and Biophysics 1995 323 p 87 96 48 Blaisdell J et al Identification and functional characterization of new poetentially defective alleles of human CYP2C19 Pharmacogenetics 2002 12 p 703 711 49 de Morais S M et al The majour genetic defect responsible for the polymorphism of S mephenytoin metabolism in humans Journal of Biological Chemistry 1994 269 p 15 419 15 422 50 Ibeanu G C et al Identification of new human CYP2C19 alleles CYP2C9 6 and CYP2C19 2B in a Caucasian 1998 286 oor metabolizer of mephenytoin The Journal of Pharmacology and Experimental Therapeutics P 1 490 1 495 51 De Morais S M et al Identificaiton of a new getic defect responsible for the polymorphism of S mephenytoin metabolism in Japanese olecular Pharmacology 1994 46 p 594 598 52 Bradford L D CYP2D6 allele frequency
45. Lovile R et al Characterization of the 16 9 kb and 30 9 kb CYP2D6 Xbal haplotypes Pharmacogenetics 1997 7 p 149 152 37 Sachse C et al intermediate and poor metabolizers of debrisoquine Pharmacogenetics 1998 8 p 181 185 Correctness of prediction of the CYP2D6 gene phenotype confirmed by genotyping 47 38 Garcia Barcelo M et al Occurrence of CYP2D6 gene duplication in Hong Kong Chinese Clinical Chemistry 2000 46 p 1 411 1 413 39 Ji L Pan S et al Single step assays to analyze CYP2D6 gene polymorphisms in Asians allele frequencies and a novel 14B allele in mainland Chinese Clinical Chemistry 2002 48 p 983 988 40 Mitsunaga Y et al Frequent occurrence of CYP2D6 10 duplication allele in a Japanese population Mutation Research 2002 505 p 83 85 41 Ishiguro A et al Metabolic activity of dextromethorphan O demethylation in healthy Japanese volunteers carrying duplicated CYP2D6 genes duplicated allele of CYP2D6 10 does not increase CYP2D6 metabolic activity Clinica Chimica Acta 2004 344 p 201 204 42 Cai W et al CYP2D6 genetic variation in healthy adults and psychiatric African American subjects Implicati ons for clinical practice and genetic testing The Pharmacogenomics Journal 2006 1 p 1 8 43 Gaedigk A et al Cytochrome P4502D6 CYP2D6 gene locus heterogeneity Characterization of gene duplica tion events Clinical Pharmacology and Therapeutics 2007 81 p 242 251 44 Candiotti
46. 0 100 0 00 0 2A 2A 16 16 0 0 100 0 00 0 10B 40 1 1 0 0 100 0 00 0 2A 3 1 1 0 0 100 0 00 0 10B 41 2 2 0 0 100 0 00 0 2A 4A 20 20 0 0 100 0 00 0 10BXN 41 1 1 0 0 100 0 00 0 2A 5 4 4 0 0 100 0 00 0 17 17 4 4 0 0 100 0 00 0 2A 6B 2 2 0 0 100 0 00 0 17 29 2 2 0 0 100 0 00 0 2A 9 2 2 0 0 100 0 00 0 17 41 3 3 0 0 100 0 00 0 2A 10B 2 2 0 0 100 0 0 0 29 29 1 1 0 0 100 0 00 0 2A 35 8 8 0 0 100 0 0 29 36 1 1 0 0 100 0 00 0 2A 41 5 5 0 0 100 0 0 29 41 4 4 0 0 100 0 00 0 2AXN 17 2 2 0 0 100 0 0 35 35 1 1 0 0 100 0 00 0 2AXN 41 2 2 0 0 100 0 0 35 41 4 4 0 0 100 0 00 0 3 3 2 2 0 0 100 0 00 0 41 41 9 9 0 0 100 0 00 0 3 4A 3 3 0 0 100 0 0 41 41XN 1 1 0 0 100 0 00 0 3 5 2 2 0 0 100 0 0 0 O 0 i l 9 2 T Ai 403 400 0 3 99 396 99 396 4A 4A 23 23 0 0 100 0 00 0 4A 4D 1 1 0 0 100 0 00 0 4A 5 2 2 0 0 100 0 00 0 4 CYP2C19 9 9 Ho 5 5 5 o ta qi 5 pa CYP2C19 Correct Miscall No Genotype Genotype VE i i 5 5 TT 00 0 Call Accuracy Call Rate 4A 35 4 0 0 100 0 00 0 100 0 100 0 4A 41 11 11 0 0 100 0 00 0 100 0 100 0 4D 5 1 1 0 0 100 0 00 0 100 0 100 0 4D 41 2 2 0 0 100 0 00 0 93 9 333 4DXN 5 1 1 0 0 100 0 00 0 100 0 100 0 4DXN 17 1 1 0 0 100 0 00 0 100 0 100 0 5 5 2 2 0 0 100 0 00 0 5 9 2 2 0 0 100 0 00 0 99 7 99 7 5 1
47. Aogo Aogo 1 50 1 85 DNA 10 mmol L DNA P450 A CHIP C CYP450 MERLET ba DNA 50 Tris HCl 0 1 mmol L EDTA 0 09 pH 8 0 DNA 2 8C 1 2 Gold 96 well GeneAmp PCR System 9700 20C 1 10 CYP450 Bre E 2 0 mL A CHIP C 54L CYP450 GM 5 CYP450 15 3 a E CYP450 C E E 6 L VI
48. WK405 NP164 NP165 Mg CS5 Gold DNA dNTP 27 dATP 2 1 15 dCTP 2 dGTP 2 FARY FSV VAR ATTP amp UL 35 dUTP 2 I 50 200 DNA 5 Td CY 200 P450 A CYP450 B DNA 3
49. variant of the CYP2D6 gene CYP2D6 17 common in a bloack African opulation association with diminished debrisoquine hydroxylase activity British Journal of Clinical Pharmacology 1996 42 p 713 719 29 Oscarson M et al A combination of mutations in the CYO2D6 17 CYP2D62 allele causes alterations in enzyme function Molecular Pharmacology 1997 52 p 1 034 1 040 30 Marez Allorge D et al A rare G2061 insertion affecting the open reading frame of CYP2D6 and responsible for the oor metabolizer phenotype Pharmacogenetics 1999 9 p 393 396 31 Wang S Phenotypes and genotypes of debrisoquine hydroxylation polymorphisms in Chinese Master s thesis ational Cheng Kung University Tainan Taiwan 1992 32 Leathart J B et al CYP2D6 phenotype genotype relationships in African Americans and Caucasians in Los Angeles Pharmacogenetics 1998 8 p 529 541 33 Gaedigk A et al Unique CYP2D6 activity distribution and genotype phenotype doscprdamce in back Americans Clinical Pharmacology and Therapeutics 2002 72 p 76 89 34 Raimundo S et al A novel intronic mutaion 2988G gt A with high predictivity for impaired function of cytochrome P450 2D6 in white subjects Clinical Pharmacology and Therapuetics 2004 76 p 128 138 35 Dahl M L et al Ultrarapid hydroxylation of debisoquine in a Swedish population Analysis of the molecular genetic basis Journal of Pharmacology and Experimental Therapeutis 1995 274 p 516 520 36
50. 00 00 1 4A 30 30 0 0 100 0 00 0 En F E 5 5 000 tE 1 4 AX 1 1 0 0 100 0 00 0 3 1 4D 1 1 0 0 100 0 00 0 36 5 5 0 0 00 0 fs 1 4DX 1 1 0 0 100 0 00 0 FET i n 5 3 00 0 T 1 5 15 15 0 0 100 0 00 0 T a T a 6 00 0 i 1 6B 3 3 0 0 100 0 00 0 1XN 16 15 0 1 93 8 129 2 2 i 2 100 0 00 0 DN 7 6 Fi i 85 7 TE 1 10B 16 16 0 0 100 0 00 0 AXN i i D 5 00 0 TE 1 10BXN 1 1 0 0 100 0 00 0 N A 5 a E 00 0 TE 1 17 13 13 0 0 100 0 00 0 NN i i o 00 0 E 1 17XN 1 1 0 0 100 0 00 0 aan i i 8 6 00 0 E 1 29 2 2 0 0 100 0 00 0 EN i i a E 00 0 E 1 35 13 13 0 0 100 0 00 0 1 35XN 1 1 0 0 100 0 00 0 1 40 1 1 0 0 100 0 00 0 844 837 1 6 99 2 1 41 14 14 0 0 100 0 00 0 1XN 2A 3 2 0 1 66 7 66 7 1XN 4A 4 4 0 0 100 0 00 0 1XN 10A 1 1 0 0 100 0 00 0 CYP2D6 Correct Miscall Genotype Genotype CYP2D6 Correct Miscall l Genotype Genotype Call Accuracy Call Rate PE Call Accuracy Call Rate 1XN 35 1 1 0 0 100 0 00 0 10B 35 1 1 0 0 100 0 00 0 1XN 41 2 2 0 0 100 0 00 0 10B 36 1 1 0
51. 0B 1 1 0 0 100 0 0 0 5 10BXN 2 2 0 0 100 0 0 0 5 17 4 4 0 0 100 0 0 0 5 5 29 1 1 0 0 100 0 0 0 Ha oo sE e 5 5 5 5 000 OZ Gold 96 well GeneAmp PCR System 9700 GeneChip Fluidics Station 450DxJI 5 441 7 7 0 0 100 0 0 0 GeneChip Scanner 3000Dx 6B 41 1 1 0 0 100 0 00 0 9 17 1 0 0 100 0 00 0 9 41 1 1 0 0 100 0 0 0 10B 10B 17 16 0 1 94 1 94 1 10B 10BXN 2 2 0 0 100 0 00 0 10B 17 2 2 0 0 100 0 00 0 4 15 1 1 A RUB CYP450 CYP450 MMX CYP450 A CYP450 PM A CYP450 B CYP450 PM B 5X Terminal Transferase Reaction Buffer 25 mmol L CoCl TdT RRB TAT
52. Call Miscall No Call 99 3 AS PCR PCR RFLP 100 0 PCR RFLP 100 0 DNA 97 6 DNA AS PCR 100 0 DNA PC R RFLP 50 0 PCR 100 0 oA 99 1 12 15 6 CYP2C19 1 DNA PCR RFLP 9 CYP2C19 Po Correct Call Miscall No Call PCR RFLP_ 276 276 o oA 1 P 2 DNA 4 3
53. F n pui arez et al 1997 A 35 1584G 31G gt A 1661G gt C 2850C gt T 4180G gt C det Gaedigk et al 200359 Data Analysis Software 31 100C gt T 1039C gt T 1661G gt C 4180G gt C Weng 1992 S 8 9 CYP2D7 ET e eles ca Leathart et al 199887 1023C gt T 1661G gt C i da JEMEZ 1 33 1863ins TTT CGC CCO 2 2850C gt T 4180G gt C Rb Gaedigk etal 2002 Raimundo et al 2000 A Raimundo et al 200459 gt oF ae 41 1584C 1661G gt C 2850C gt T 4180G gt C 1 Toscano et al 20065 Rau et al 20065 duplicate active 1 genes Dahl et al 19959 gt PN n is not determined range 2 13 em Sachse et al 19979 dupli tive 2 Johansson et al 1993 AN 6 ii e2 13 an Dahl etal 1995 Aklillu et al 1996 9 Lovlie et al 1997 n alii n TTM i 4XN duplicate inactive 4 genes n is not determined Sachse et al 1998 Garcia Barcelo et al TRO 200088 ai uplicate artial y active 0 genes EF Ji et al 2002 nils not detefimined Mitsunaga et al 2002 Ishiguro et al 20041 7XN uplicate partially active 17 genes Cai et al 200612 n is not determined 5 Gaedigk et al 2007 35XN uplicate active 35 genes n is not determined An Gaedigk et al 2007 7 3 41XN DAL ai EF Candiotti et al 200510 n is not determi
54. WEF 20 um CYP450 Light Directed NET GeneChip Fluidics Station 450 Dx GeneChip Scanner 3000Dx Affymetrix bia E CYP450 CYP450 DNA CYP450 RN 7771 15 00 JU 106 107
55. arez et al AMDS v1 0 AmpliChip CYP450 US Data Analaysis Software v3 1 a AmpliChip CYP450 Test Data Analaysis Software v3 1 arez et al A 100C gt T 1039C gt T 1661G gt C 1846G gt A 5 13 CYP2D6 AABDJK 14 Kagimoto et al 1990 2850C gt T 4180G gt C Gough et al 1990 Hanioka et al 199019 Gaedigk et al 199119 Il Additional Information Window Batch Information Window CYP2C19 CYP2D6 CYP2C19 b AMDS v1 0 O Report CYP2D6 tat Steen et al 1995 arez et al 1997 AMDS a Evert et al 199419 jr 1707Tdel 1976G gt A 4180G gt C Ter Daly et al 1995 1 Saxena et al 19942 CYP450
56. e I RNase free 5X Terminal Transferase Reaction Bu 1 mg mL R Gold 96 well GeneAmp PCR Systems 9700 Ap GeneChip Fluidics Station 450Dx v 1 GCOS FA v 2 GeneChip Scanner 3000Dx v 1 GCOS Data Station for the GCOS with GCOS software DNA QIAamp DNA Blood 0 5 mol L EDTA pH AccuGENE 20X SSPE Buffer 3 mol L NaCl 0 2 mol L Triton X 100 surfactant Sigma Aldrich Denhardt s solution 50X concentrate Sigma Aldrich X Sodium azi Acetylated HER For Applied Tubes 0 2 and Base Optical 96 well Reaction Tube Tough Spo 2 0 mL screw cap tubes Sarstedt 1 5 mL microfuge tubes VWR Sterile polypropylene conical tubes 15 mL Corning 500 mL square media bottles Nalgene v 2 AMDS v1 1 3 Data Sta ini Kit
57. iation with the poor metabolizer phenotype Human Molecular Genetics 1994 3 p 923 926 21 Broly F et al A nonsense mutation in the cytochrome P450 CYP2D6 gene identified in a Caucasian with an enzyme deficiency Human Genetics 1995 96 p 601 603 22 Tyndale R et al Identification of a new variant CYP2D6 allele lacking the codon encoding Lys 281 possible association with the poor metabolizer phenotype Pharmacogenetics 1991 1 p 26 32 23 Broly F et al Debrisoquine oxidation polymorphism phenotypic consequences of a 3 base pair deletion in exon 5 of the CYP2D6 gene Pharmacogenetics 1993 3 p 123 130 24 Yokota H et al Evidence for a new variant CYP2D6 allele CYP2D6J I a Japanese population associated with lower in vivo rates of sparteine metabolism Pharmacogenetics 1993 3 p 256 263 25 Johansson I et al Genetics analysis of the Chinese cytochrome P4502D locus characterization of cariant CYP2D6 genes present in subjects with diminished capacity for debrisoquine hydroxylation Molecular Pharmacology 1994 46 p 452 459 26 Marez D et al A novel CYP2D6 allele with an abolished splice recognition site associated with the poor metabolizer phenotype Pharmacogenetics 1995 5 p 305 311 27 Sachse C et al A rare insertion of T226 in exon 1 of CYP2D6causes a fragmeshift and is associated with the poor metabolizer phenotype CYP2D6 15 Pharmacogenetics 1996 6 p 269 272 28 Masimirembwa C et al A novel mutant
58. in European Caucasians Asians Africans and their descendants Pharmacogenomics 2002 3 p 229 243 53 Furman K D et al harmacokinetics Pharmacogenetics 2004 14 p 279 284 54 Goldstein J A Clinical relevance of genetic polymorphisms in the human CYP2C subfamily British Journal of Clinical Pharmacology 2001 52 p 349 355 55 Gaedigk A et al CYP2D6 poor metabolizer status can be ruled out by a single genotyping assay for the 1584G promoter polymorphism Clinical Chemistry 2003 49 p 1 008 1 011 56 Yasui F et al Association Between Cytochrome P450 CYP 2C19 Polymorphisms and Harm Avoidance in Japanese American Journal of Medical Genetics Part B Neuropsychiatric Geneitics 2007 144B p 724 727 57 Wennerholm A et al Characterization of the CYP2D6 29 allele commonly present in a block Tanzanian Impact of CYP2D6 intermediate metabolizer alleles on single dose desipramine population causing reduced catalytic activity Pharmacoggenetics 2001 11 p 417 427 58 Wennerholm A et al The African specific CYP2D6 17 allele encodes an enzyme with changed substrate specificity Clinical Pharmacology amp Therapeutics 2002 1 p 77 88 59 Sakuyama K et al Functional Characterization of 17 CYP2D6 Allelic Variants CYP2D6 2 10 14A B 18 27 36 39 47 51 53 55 and 57 Drug Metabolism and Disposition 2008 36 p 2 460 2 467 60 Toscano C et al Impaired expression of CYP2D6 in intermediate metaboilzers ca
59. nce and identification of the 1989 45 Light directed spatially addressable parallel chemical synthesis Scinece 1991 251 p 767 773 Light generated oligonucleotide arrays for rapid DNA sequence analysis Proceedings of 1994 91 p 5 022 5 026 3 4 http www imm Gene 5 Physiology 6 http www imm 7 Marez D et al 48 mutations and 53 alleles their frequencies and evolution 8 Sachse C et al American Journal of Human Genetics Pharmacogenetics consequences 9 Kimura S et al olymorphic CYP2D6 gene a related gene and a pseudogene American Journal of Human Genetics 889 904 ohansson I et al 10 Inherited amplify cation of an active gene in the cytochrome P450 CYP2D locus as a cause of ultrarapid metabolism of debrisoquine Proceedings of the National Academy of Sciences U S A 1993 90 11 825 11 829 Panserat S et al DNA haplotype dependent differnves in the amino acid sequence of debrisogine 4 hydroxylate CYP2D6 evidenvce for two major al 1994 94 p 401 406 Raimudo S et al Elucidation of the genetic basis of the common intermediate metabolizer phenotype for drug oxidation by CYP2D6 Pharmacogene 2000 10 p 577 581 Multiple mutations of the human cytochrome P450IID6 gene CYP2D6 in poor metabolizers of 11 ozymes in extensive metabolisers Human Genetics 12 ics 13 Kagimoto M et al Study of the functional significance of individual muta
60. ned No Call 9 15 ET A CC A SE OM ES OTIS SN I I E Je v IONE EE Em E Extensive metabolizer I Intermediate metabolizer P Poor metabolizer U Ultrarapid metabolizer 10 15 CYP450 CYP2C19 TI CYP2C19 Romkes et al 19919 Richardson et al 1995 Blaisdell et al 2002 681G gt A de Morais et al 1994 9 beanu et al 199859 Fukushima Uesaka et al 200559 Lee et al 2009 CYP450 636G gt A 2019 de Morais et al 199490 Fukushima Uesaka et al 200559 2 CYP2C19
61. poet KLERIK E CARL Tei Fragment Lenghth Polymorphism CYP450 GMO07439 GM09912 2 E CYP2D6 4 41 CYP2C19 1 2 GM07439 CYP2D6 4XN 41 CYP2C19 2 2 GM09912 CYP2D6 4 5 CYP2C19 1 1 PCR RFLP RFLP AS PCR Allele Specific DNA AS PCR CYP2D6 99 1 418 422 CYP2C19 100 398 398 8 9 8 CYP2D6 AS PCR Correct
62. rrying the 41 allele caused by the intronic SNP 2988G gt A evidence for modulation of splicing events p 755 766 Pharmacogenetics and Genomics 2006 16 61 Rau T et al The 2988G gt A polymorphism affects splicing of a CYP2D6 minigene Clinical Pharmacology and Therapeutics 2006 80 p 555 558 62 Aklillu E et al Frequent Distribution of Ultrarapid Metabolizers of Debrisoquine in an Ethiopian Population Carrying Duplicated and Multiduplicated Functional CYP2D6 Alleles 278 p 441 446 Journal of Pharmacol and Exper Thera 1996 63 Fukushima Uesaka et al Genetic Variations and Haplotypes of CYP2C19 in a Japanese Population Drug M etab Pharmacokinetics 2005 20 p 300 307 64 Lee S J et al Identification of New CYP2C19 Variants Exhibiting Decreased Enzyme Activity in the Metabol ism of S Mephenytoin and Omeprazole Drug Metab and Disposition 2009 37 p 2 262 2 269 105 0014 2 6 1 0120 600 152 105 0014 2 6 1 0120 600 152 T 15 15
63. tions by expression of chimeric genes Journal 1990 265 p 17 209 17 214 Identification of the primary gene defect at the cytochrome P450 CYP2D locus devrisoquine of Biological Chemistry 14 Gough A C et al 1990 347 p 773 776 15 Hanioka N et al A base change in itron 3 of a mutant CYP2D allele results in an aberrant 3 1990 47 p 994 1 001 Deletion of the entire cytochrome P450 CYP2D gene as a cause of impaired drug metabolism in 1991 48 Nature The human CYP2D locus associated with a common genetic defect in drug oxidation a G1934 splice recognition site American Journal of Human Genetics 16 Gaedigk A et al oor metabolizers of the debrisoquine sparteine polymorphism American Journal of Human Genetics 943 950 17 Steen V M et al Homologuous unequal cross over involving a 2 8kb direct repeat as a mechanism for the generation of allelic variants of human cytochrome P450 CYP2D6 gene Human Molecular Genetics 1995 4 2 251 2 257 Evert B et al Clonng and sequencing of a new non functional CYP2D6 allele deletion of T1795 in exon 3 generates a premature stop codon 1994 4 p 271 274 aly A K et al AN inactive cytochrome P450 CYP2D6 allele containing a deletion and a base substitution 1995 95 p 337 341 18 Pharmacogenetics 19 Human Molecular Genetics 20 Saxena R et al Identification of a new variant CYP2D6 allele with a single base deletion in exon 3 and its assoc
64. w L 0 09 2 a Gi gt 7 588 mL 12 5 mL 24 DNase I Roche Applied Science 5 DNase I Roche Applied Science Aunt OFF 10 15 R 1 mg mL 2 8 6 n aa 20X SSPE Buffer 3 5 mL 5 6X 20 mmol L EDTA A Wa 03 mmol L Acetylated bovine serum albumin 20 mg mL 625 uw L 1 mg mL 1 U ul 22 uL 0 1 U uL i e va 25 uL 0 01 mg mL Onase I So U uL 3 3uL 0 15 U uL 5 395 aL 0 09 3 220 u L 8 025 mL 3 2 5 mL
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