farmakogenetsko testiranje v klinični...
TRANSCRIPT
Prof dr Vita Dolžan dr med
Farmakogenetsko testiranje v klinični praksi
Inštitut za biokemijo Laboratorij za farmakogenetiko Medicinska fakulteta Univerza v Ljubljani
vitadolzanmfuni-ljsi
VARIABILITY IN HUMAN RESPONSE TO DRUGS A major barrier to effective therapeutics
Current drug prescribing based on -signs and symptoms -average response
Interindividual Variability in Drug Response
Disease Drug Class Rate of Poor Response Asthma Beta-agonists 40-75 Schizophrenia Various 25-75 Depression SSRIs tricyclics 20-40 Arthritis DMARDs 30-60 Diabetes Sulfonylureas others 50 Hypertension Various 30
bull 2 million patients year present AEs in US hospitals (Norton 2001) bull 100000 deathsyear caused by AEs in US (Lazarou et al 1998) bull Slovenia (Brvar 2010) ED admissions due to AEs - 37 of all patients - 20 of all AEs bradycardia due to verapamil digoxin and beta-blockers - 07 of all ADRs beta-blockers digoxin diuretics NSAID acetylsalicylic acid clopidogrel and tamoxifen
Absorption
Distribution
Target Interaction
Biotransformation
Excretion
Environmental factors
Genetic factors
VARIABILITY IN DRUG RESPONSE
Drug Response Disease
Drug
Patient
What are genetic polymorphisms
bull occurrence of two or more alleles at a given locus bull the rare allele has a frequency of at least 1 or more
- single nucleotide polymorphisms (SNPs) - copy number variations (CNVs)
Gene
helliptcgaGatgghellip helliptcgaTatgghellip Gene
sequence
VARIABLE DRUG RESPONSE
Variability in drug response
NON-RESPONSE No toxicity amp Efficacy TOXICITY
Variability in plasma levels
Pharmacodynamics Pharmacokinetics
Drug Targets Drug Transporters
GENETIC VARIABILITY
OF DRUG RESPONSE
Drug Metab Enzymes
ADME
bull Pharmacogenetics
links information on the variation of human genes to variations in
drug levels
drug response
adverse events
bull Pharmacogenomics
studies variability relevant for drug response at the genome level
effects of drugs on gene expression
identification of new drug targets
Oral anticoagulants (Warfarin) Oral antidiabetics Phenytoin NSAID
Proton pump inhibitors Clopidogrel Diazepam Antidepressants
Beta blockers Antidepressants Antipsychotics Debrisoquine Codeine Tamoxifen
Cyclosporin Tacrolimus Taxol Amiodarone Cervistatin Erythromycin Methadone Quinine
Phase I Drug Metabolizing Enzymes
Cytochrome P450 dependent drug Response
P450s families 1-3
PRO-DRUG
ACTIVATION
Codeine
CYP2D6 (UM)
Morphine-ADRs
Codeine
CYP2D6 (PM)
No pain relieve
Tamoxifen
CYP2D6 (PM)
Lower endoxifen
Clopidogrel
CYP2C192 (PM)
No platelet response
Antidepressants
Antipsychotics
CYP2D6 (UM)
Less drug effect
Antidepressants
Antipsychotics CYP2D6 (PM)
ADRs
Warfarinacecoumarol
CYP2C92 3 (PM)
Bleeding
Too Much
Activation
Too Little
Activation
Ultrarapid Metab Poor Metab
DRUG
INACTIVATION
Too Fast
Inactivation
Too Slow
Inactivation
Ultrarapid Metab Poor Metab
75 Phase I reactions Highly polymorphic
CYP2C9 bull 20 of all liver P450s
bull metabolizes 10-20 amp of commonly prescribed drugs1 S -warfarin phenitoin NSAIDs statins oral hypogycaemic agents losartan sulfonylureas Inhibitors fluconazole isoniazid lovastatin amiodarone Inductors rifampin barbiturates
Alleles2 polymorphism enzyme frequency activity Slovenia3
CYP2C91 normal 815 CYP2C92 R144C decreased 122 CYP2C93 I359L decreased 63
1 httpwwwgeorgetownedudepartmentspharmacologydavetabhtml 2 httpwwwkiseCYPalleles 3 Herman et al Zdrav Vestn 2003
httpmedicineiupui
educlinpharmddis
ClinicalTableaspx
Warfarin
bull frequent monitoring of prothrombin time (INR) amp dose adjustment (INR therapeutic range 2-3)
bull it can take several months to determine optimal dose bull ranks among top drugs in hospital emergency room visits for bleeding
bull thromboembolic complications when dose to low
bull 10 of all hospital admissions due to ADR in England Wysowski et al 2007 Pirmohamed et al 2004
bull oral anticoagulant drug
bull used for prophylaxis and treatment of thromboembolic disorders
bull the most frequently used coumarin
bull narrow therapeutic window
bull high inter-individual variability in dose response
bull frequent interactions with other drugs andor diet
Kaminsky amp Zhang 1997
Warfarin metabolism
bull S-warfarin- bull 60-70 anticoagulant effect bull CYP2C9
2 Arg144Cys (ex3) 3 Ile359Leu (ex7)
httpwwwkiseCYPalleles
bull R-varfarin- bull 30-40 anticoagulant effect bull CYP1A2 CYP2C19 CYP3A4
CYP2C9 genotypes and warfarin maintenance dose
Herman et al Pharmacogenomics J 2005
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles
Nature February 5 2004
VKORC1 identified
Rare mutations cause
bull Warfarin resistance
bull Multiple coagulation factor
deficiency type II
FII FVII FIX FX
g-carboxylated
active zymogen
Warfarin inhibits vitamin K cycle
VKORC1 6484CgtT - linkage with SNPs that may affect transcriptional efficiency
9041GgtA 3rsquoUTR - mRNA stabilisation
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
VARIABILITY IN HUMAN RESPONSE TO DRUGS A major barrier to effective therapeutics
Current drug prescribing based on -signs and symptoms -average response
Interindividual Variability in Drug Response
Disease Drug Class Rate of Poor Response Asthma Beta-agonists 40-75 Schizophrenia Various 25-75 Depression SSRIs tricyclics 20-40 Arthritis DMARDs 30-60 Diabetes Sulfonylureas others 50 Hypertension Various 30
bull 2 million patients year present AEs in US hospitals (Norton 2001) bull 100000 deathsyear caused by AEs in US (Lazarou et al 1998) bull Slovenia (Brvar 2010) ED admissions due to AEs - 37 of all patients - 20 of all AEs bradycardia due to verapamil digoxin and beta-blockers - 07 of all ADRs beta-blockers digoxin diuretics NSAID acetylsalicylic acid clopidogrel and tamoxifen
Absorption
Distribution
Target Interaction
Biotransformation
Excretion
Environmental factors
Genetic factors
VARIABILITY IN DRUG RESPONSE
Drug Response Disease
Drug
Patient
What are genetic polymorphisms
bull occurrence of two or more alleles at a given locus bull the rare allele has a frequency of at least 1 or more
- single nucleotide polymorphisms (SNPs) - copy number variations (CNVs)
Gene
helliptcgaGatgghellip helliptcgaTatgghellip Gene
sequence
VARIABLE DRUG RESPONSE
Variability in drug response
NON-RESPONSE No toxicity amp Efficacy TOXICITY
Variability in plasma levels
Pharmacodynamics Pharmacokinetics
Drug Targets Drug Transporters
GENETIC VARIABILITY
OF DRUG RESPONSE
Drug Metab Enzymes
ADME
bull Pharmacogenetics
links information on the variation of human genes to variations in
drug levels
drug response
adverse events
bull Pharmacogenomics
studies variability relevant for drug response at the genome level
effects of drugs on gene expression
identification of new drug targets
Oral anticoagulants (Warfarin) Oral antidiabetics Phenytoin NSAID
Proton pump inhibitors Clopidogrel Diazepam Antidepressants
Beta blockers Antidepressants Antipsychotics Debrisoquine Codeine Tamoxifen
Cyclosporin Tacrolimus Taxol Amiodarone Cervistatin Erythromycin Methadone Quinine
Phase I Drug Metabolizing Enzymes
Cytochrome P450 dependent drug Response
P450s families 1-3
PRO-DRUG
ACTIVATION
Codeine
CYP2D6 (UM)
Morphine-ADRs
Codeine
CYP2D6 (PM)
No pain relieve
Tamoxifen
CYP2D6 (PM)
Lower endoxifen
Clopidogrel
CYP2C192 (PM)
No platelet response
Antidepressants
Antipsychotics
CYP2D6 (UM)
Less drug effect
Antidepressants
Antipsychotics CYP2D6 (PM)
ADRs
Warfarinacecoumarol
CYP2C92 3 (PM)
Bleeding
Too Much
Activation
Too Little
Activation
Ultrarapid Metab Poor Metab
DRUG
INACTIVATION
Too Fast
Inactivation
Too Slow
Inactivation
Ultrarapid Metab Poor Metab
75 Phase I reactions Highly polymorphic
CYP2C9 bull 20 of all liver P450s
bull metabolizes 10-20 amp of commonly prescribed drugs1 S -warfarin phenitoin NSAIDs statins oral hypogycaemic agents losartan sulfonylureas Inhibitors fluconazole isoniazid lovastatin amiodarone Inductors rifampin barbiturates
Alleles2 polymorphism enzyme frequency activity Slovenia3
CYP2C91 normal 815 CYP2C92 R144C decreased 122 CYP2C93 I359L decreased 63
1 httpwwwgeorgetownedudepartmentspharmacologydavetabhtml 2 httpwwwkiseCYPalleles 3 Herman et al Zdrav Vestn 2003
httpmedicineiupui
educlinpharmddis
ClinicalTableaspx
Warfarin
bull frequent monitoring of prothrombin time (INR) amp dose adjustment (INR therapeutic range 2-3)
bull it can take several months to determine optimal dose bull ranks among top drugs in hospital emergency room visits for bleeding
bull thromboembolic complications when dose to low
bull 10 of all hospital admissions due to ADR in England Wysowski et al 2007 Pirmohamed et al 2004
bull oral anticoagulant drug
bull used for prophylaxis and treatment of thromboembolic disorders
bull the most frequently used coumarin
bull narrow therapeutic window
bull high inter-individual variability in dose response
bull frequent interactions with other drugs andor diet
Kaminsky amp Zhang 1997
Warfarin metabolism
bull S-warfarin- bull 60-70 anticoagulant effect bull CYP2C9
2 Arg144Cys (ex3) 3 Ile359Leu (ex7)
httpwwwkiseCYPalleles
bull R-varfarin- bull 30-40 anticoagulant effect bull CYP1A2 CYP2C19 CYP3A4
CYP2C9 genotypes and warfarin maintenance dose
Herman et al Pharmacogenomics J 2005
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles
Nature February 5 2004
VKORC1 identified
Rare mutations cause
bull Warfarin resistance
bull Multiple coagulation factor
deficiency type II
FII FVII FIX FX
g-carboxylated
active zymogen
Warfarin inhibits vitamin K cycle
VKORC1 6484CgtT - linkage with SNPs that may affect transcriptional efficiency
9041GgtA 3rsquoUTR - mRNA stabilisation
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Interindividual Variability in Drug Response
Disease Drug Class Rate of Poor Response Asthma Beta-agonists 40-75 Schizophrenia Various 25-75 Depression SSRIs tricyclics 20-40 Arthritis DMARDs 30-60 Diabetes Sulfonylureas others 50 Hypertension Various 30
bull 2 million patients year present AEs in US hospitals (Norton 2001) bull 100000 deathsyear caused by AEs in US (Lazarou et al 1998) bull Slovenia (Brvar 2010) ED admissions due to AEs - 37 of all patients - 20 of all AEs bradycardia due to verapamil digoxin and beta-blockers - 07 of all ADRs beta-blockers digoxin diuretics NSAID acetylsalicylic acid clopidogrel and tamoxifen
Absorption
Distribution
Target Interaction
Biotransformation
Excretion
Environmental factors
Genetic factors
VARIABILITY IN DRUG RESPONSE
Drug Response Disease
Drug
Patient
What are genetic polymorphisms
bull occurrence of two or more alleles at a given locus bull the rare allele has a frequency of at least 1 or more
- single nucleotide polymorphisms (SNPs) - copy number variations (CNVs)
Gene
helliptcgaGatgghellip helliptcgaTatgghellip Gene
sequence
VARIABLE DRUG RESPONSE
Variability in drug response
NON-RESPONSE No toxicity amp Efficacy TOXICITY
Variability in plasma levels
Pharmacodynamics Pharmacokinetics
Drug Targets Drug Transporters
GENETIC VARIABILITY
OF DRUG RESPONSE
Drug Metab Enzymes
ADME
bull Pharmacogenetics
links information on the variation of human genes to variations in
drug levels
drug response
adverse events
bull Pharmacogenomics
studies variability relevant for drug response at the genome level
effects of drugs on gene expression
identification of new drug targets
Oral anticoagulants (Warfarin) Oral antidiabetics Phenytoin NSAID
Proton pump inhibitors Clopidogrel Diazepam Antidepressants
Beta blockers Antidepressants Antipsychotics Debrisoquine Codeine Tamoxifen
Cyclosporin Tacrolimus Taxol Amiodarone Cervistatin Erythromycin Methadone Quinine
Phase I Drug Metabolizing Enzymes
Cytochrome P450 dependent drug Response
P450s families 1-3
PRO-DRUG
ACTIVATION
Codeine
CYP2D6 (UM)
Morphine-ADRs
Codeine
CYP2D6 (PM)
No pain relieve
Tamoxifen
CYP2D6 (PM)
Lower endoxifen
Clopidogrel
CYP2C192 (PM)
No platelet response
Antidepressants
Antipsychotics
CYP2D6 (UM)
Less drug effect
Antidepressants
Antipsychotics CYP2D6 (PM)
ADRs
Warfarinacecoumarol
CYP2C92 3 (PM)
Bleeding
Too Much
Activation
Too Little
Activation
Ultrarapid Metab Poor Metab
DRUG
INACTIVATION
Too Fast
Inactivation
Too Slow
Inactivation
Ultrarapid Metab Poor Metab
75 Phase I reactions Highly polymorphic
CYP2C9 bull 20 of all liver P450s
bull metabolizes 10-20 amp of commonly prescribed drugs1 S -warfarin phenitoin NSAIDs statins oral hypogycaemic agents losartan sulfonylureas Inhibitors fluconazole isoniazid lovastatin amiodarone Inductors rifampin barbiturates
Alleles2 polymorphism enzyme frequency activity Slovenia3
CYP2C91 normal 815 CYP2C92 R144C decreased 122 CYP2C93 I359L decreased 63
1 httpwwwgeorgetownedudepartmentspharmacologydavetabhtml 2 httpwwwkiseCYPalleles 3 Herman et al Zdrav Vestn 2003
httpmedicineiupui
educlinpharmddis
ClinicalTableaspx
Warfarin
bull frequent monitoring of prothrombin time (INR) amp dose adjustment (INR therapeutic range 2-3)
bull it can take several months to determine optimal dose bull ranks among top drugs in hospital emergency room visits for bleeding
bull thromboembolic complications when dose to low
bull 10 of all hospital admissions due to ADR in England Wysowski et al 2007 Pirmohamed et al 2004
bull oral anticoagulant drug
bull used for prophylaxis and treatment of thromboembolic disorders
bull the most frequently used coumarin
bull narrow therapeutic window
bull high inter-individual variability in dose response
bull frequent interactions with other drugs andor diet
Kaminsky amp Zhang 1997
Warfarin metabolism
bull S-warfarin- bull 60-70 anticoagulant effect bull CYP2C9
2 Arg144Cys (ex3) 3 Ile359Leu (ex7)
httpwwwkiseCYPalleles
bull R-varfarin- bull 30-40 anticoagulant effect bull CYP1A2 CYP2C19 CYP3A4
CYP2C9 genotypes and warfarin maintenance dose
Herman et al Pharmacogenomics J 2005
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles
Nature February 5 2004
VKORC1 identified
Rare mutations cause
bull Warfarin resistance
bull Multiple coagulation factor
deficiency type II
FII FVII FIX FX
g-carboxylated
active zymogen
Warfarin inhibits vitamin K cycle
VKORC1 6484CgtT - linkage with SNPs that may affect transcriptional efficiency
9041GgtA 3rsquoUTR - mRNA stabilisation
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Absorption
Distribution
Target Interaction
Biotransformation
Excretion
Environmental factors
Genetic factors
VARIABILITY IN DRUG RESPONSE
Drug Response Disease
Drug
Patient
What are genetic polymorphisms
bull occurrence of two or more alleles at a given locus bull the rare allele has a frequency of at least 1 or more
- single nucleotide polymorphisms (SNPs) - copy number variations (CNVs)
Gene
helliptcgaGatgghellip helliptcgaTatgghellip Gene
sequence
VARIABLE DRUG RESPONSE
Variability in drug response
NON-RESPONSE No toxicity amp Efficacy TOXICITY
Variability in plasma levels
Pharmacodynamics Pharmacokinetics
Drug Targets Drug Transporters
GENETIC VARIABILITY
OF DRUG RESPONSE
Drug Metab Enzymes
ADME
bull Pharmacogenetics
links information on the variation of human genes to variations in
drug levels
drug response
adverse events
bull Pharmacogenomics
studies variability relevant for drug response at the genome level
effects of drugs on gene expression
identification of new drug targets
Oral anticoagulants (Warfarin) Oral antidiabetics Phenytoin NSAID
Proton pump inhibitors Clopidogrel Diazepam Antidepressants
Beta blockers Antidepressants Antipsychotics Debrisoquine Codeine Tamoxifen
Cyclosporin Tacrolimus Taxol Amiodarone Cervistatin Erythromycin Methadone Quinine
Phase I Drug Metabolizing Enzymes
Cytochrome P450 dependent drug Response
P450s families 1-3
PRO-DRUG
ACTIVATION
Codeine
CYP2D6 (UM)
Morphine-ADRs
Codeine
CYP2D6 (PM)
No pain relieve
Tamoxifen
CYP2D6 (PM)
Lower endoxifen
Clopidogrel
CYP2C192 (PM)
No platelet response
Antidepressants
Antipsychotics
CYP2D6 (UM)
Less drug effect
Antidepressants
Antipsychotics CYP2D6 (PM)
ADRs
Warfarinacecoumarol
CYP2C92 3 (PM)
Bleeding
Too Much
Activation
Too Little
Activation
Ultrarapid Metab Poor Metab
DRUG
INACTIVATION
Too Fast
Inactivation
Too Slow
Inactivation
Ultrarapid Metab Poor Metab
75 Phase I reactions Highly polymorphic
CYP2C9 bull 20 of all liver P450s
bull metabolizes 10-20 amp of commonly prescribed drugs1 S -warfarin phenitoin NSAIDs statins oral hypogycaemic agents losartan sulfonylureas Inhibitors fluconazole isoniazid lovastatin amiodarone Inductors rifampin barbiturates
Alleles2 polymorphism enzyme frequency activity Slovenia3
CYP2C91 normal 815 CYP2C92 R144C decreased 122 CYP2C93 I359L decreased 63
1 httpwwwgeorgetownedudepartmentspharmacologydavetabhtml 2 httpwwwkiseCYPalleles 3 Herman et al Zdrav Vestn 2003
httpmedicineiupui
educlinpharmddis
ClinicalTableaspx
Warfarin
bull frequent monitoring of prothrombin time (INR) amp dose adjustment (INR therapeutic range 2-3)
bull it can take several months to determine optimal dose bull ranks among top drugs in hospital emergency room visits for bleeding
bull thromboembolic complications when dose to low
bull 10 of all hospital admissions due to ADR in England Wysowski et al 2007 Pirmohamed et al 2004
bull oral anticoagulant drug
bull used for prophylaxis and treatment of thromboembolic disorders
bull the most frequently used coumarin
bull narrow therapeutic window
bull high inter-individual variability in dose response
bull frequent interactions with other drugs andor diet
Kaminsky amp Zhang 1997
Warfarin metabolism
bull S-warfarin- bull 60-70 anticoagulant effect bull CYP2C9
2 Arg144Cys (ex3) 3 Ile359Leu (ex7)
httpwwwkiseCYPalleles
bull R-varfarin- bull 30-40 anticoagulant effect bull CYP1A2 CYP2C19 CYP3A4
CYP2C9 genotypes and warfarin maintenance dose
Herman et al Pharmacogenomics J 2005
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles
Nature February 5 2004
VKORC1 identified
Rare mutations cause
bull Warfarin resistance
bull Multiple coagulation factor
deficiency type II
FII FVII FIX FX
g-carboxylated
active zymogen
Warfarin inhibits vitamin K cycle
VKORC1 6484CgtT - linkage with SNPs that may affect transcriptional efficiency
9041GgtA 3rsquoUTR - mRNA stabilisation
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
What are genetic polymorphisms
bull occurrence of two or more alleles at a given locus bull the rare allele has a frequency of at least 1 or more
- single nucleotide polymorphisms (SNPs) - copy number variations (CNVs)
Gene
helliptcgaGatgghellip helliptcgaTatgghellip Gene
sequence
VARIABLE DRUG RESPONSE
Variability in drug response
NON-RESPONSE No toxicity amp Efficacy TOXICITY
Variability in plasma levels
Pharmacodynamics Pharmacokinetics
Drug Targets Drug Transporters
GENETIC VARIABILITY
OF DRUG RESPONSE
Drug Metab Enzymes
ADME
bull Pharmacogenetics
links information on the variation of human genes to variations in
drug levels
drug response
adverse events
bull Pharmacogenomics
studies variability relevant for drug response at the genome level
effects of drugs on gene expression
identification of new drug targets
Oral anticoagulants (Warfarin) Oral antidiabetics Phenytoin NSAID
Proton pump inhibitors Clopidogrel Diazepam Antidepressants
Beta blockers Antidepressants Antipsychotics Debrisoquine Codeine Tamoxifen
Cyclosporin Tacrolimus Taxol Amiodarone Cervistatin Erythromycin Methadone Quinine
Phase I Drug Metabolizing Enzymes
Cytochrome P450 dependent drug Response
P450s families 1-3
PRO-DRUG
ACTIVATION
Codeine
CYP2D6 (UM)
Morphine-ADRs
Codeine
CYP2D6 (PM)
No pain relieve
Tamoxifen
CYP2D6 (PM)
Lower endoxifen
Clopidogrel
CYP2C192 (PM)
No platelet response
Antidepressants
Antipsychotics
CYP2D6 (UM)
Less drug effect
Antidepressants
Antipsychotics CYP2D6 (PM)
ADRs
Warfarinacecoumarol
CYP2C92 3 (PM)
Bleeding
Too Much
Activation
Too Little
Activation
Ultrarapid Metab Poor Metab
DRUG
INACTIVATION
Too Fast
Inactivation
Too Slow
Inactivation
Ultrarapid Metab Poor Metab
75 Phase I reactions Highly polymorphic
CYP2C9 bull 20 of all liver P450s
bull metabolizes 10-20 amp of commonly prescribed drugs1 S -warfarin phenitoin NSAIDs statins oral hypogycaemic agents losartan sulfonylureas Inhibitors fluconazole isoniazid lovastatin amiodarone Inductors rifampin barbiturates
Alleles2 polymorphism enzyme frequency activity Slovenia3
CYP2C91 normal 815 CYP2C92 R144C decreased 122 CYP2C93 I359L decreased 63
1 httpwwwgeorgetownedudepartmentspharmacologydavetabhtml 2 httpwwwkiseCYPalleles 3 Herman et al Zdrav Vestn 2003
httpmedicineiupui
educlinpharmddis
ClinicalTableaspx
Warfarin
bull frequent monitoring of prothrombin time (INR) amp dose adjustment (INR therapeutic range 2-3)
bull it can take several months to determine optimal dose bull ranks among top drugs in hospital emergency room visits for bleeding
bull thromboembolic complications when dose to low
bull 10 of all hospital admissions due to ADR in England Wysowski et al 2007 Pirmohamed et al 2004
bull oral anticoagulant drug
bull used for prophylaxis and treatment of thromboembolic disorders
bull the most frequently used coumarin
bull narrow therapeutic window
bull high inter-individual variability in dose response
bull frequent interactions with other drugs andor diet
Kaminsky amp Zhang 1997
Warfarin metabolism
bull S-warfarin- bull 60-70 anticoagulant effect bull CYP2C9
2 Arg144Cys (ex3) 3 Ile359Leu (ex7)
httpwwwkiseCYPalleles
bull R-varfarin- bull 30-40 anticoagulant effect bull CYP1A2 CYP2C19 CYP3A4
CYP2C9 genotypes and warfarin maintenance dose
Herman et al Pharmacogenomics J 2005
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles
Nature February 5 2004
VKORC1 identified
Rare mutations cause
bull Warfarin resistance
bull Multiple coagulation factor
deficiency type II
FII FVII FIX FX
g-carboxylated
active zymogen
Warfarin inhibits vitamin K cycle
VKORC1 6484CgtT - linkage with SNPs that may affect transcriptional efficiency
9041GgtA 3rsquoUTR - mRNA stabilisation
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
VARIABLE DRUG RESPONSE
Variability in drug response
NON-RESPONSE No toxicity amp Efficacy TOXICITY
Variability in plasma levels
Pharmacodynamics Pharmacokinetics
Drug Targets Drug Transporters
GENETIC VARIABILITY
OF DRUG RESPONSE
Drug Metab Enzymes
ADME
bull Pharmacogenetics
links information on the variation of human genes to variations in
drug levels
drug response
adverse events
bull Pharmacogenomics
studies variability relevant for drug response at the genome level
effects of drugs on gene expression
identification of new drug targets
Oral anticoagulants (Warfarin) Oral antidiabetics Phenytoin NSAID
Proton pump inhibitors Clopidogrel Diazepam Antidepressants
Beta blockers Antidepressants Antipsychotics Debrisoquine Codeine Tamoxifen
Cyclosporin Tacrolimus Taxol Amiodarone Cervistatin Erythromycin Methadone Quinine
Phase I Drug Metabolizing Enzymes
Cytochrome P450 dependent drug Response
P450s families 1-3
PRO-DRUG
ACTIVATION
Codeine
CYP2D6 (UM)
Morphine-ADRs
Codeine
CYP2D6 (PM)
No pain relieve
Tamoxifen
CYP2D6 (PM)
Lower endoxifen
Clopidogrel
CYP2C192 (PM)
No platelet response
Antidepressants
Antipsychotics
CYP2D6 (UM)
Less drug effect
Antidepressants
Antipsychotics CYP2D6 (PM)
ADRs
Warfarinacecoumarol
CYP2C92 3 (PM)
Bleeding
Too Much
Activation
Too Little
Activation
Ultrarapid Metab Poor Metab
DRUG
INACTIVATION
Too Fast
Inactivation
Too Slow
Inactivation
Ultrarapid Metab Poor Metab
75 Phase I reactions Highly polymorphic
CYP2C9 bull 20 of all liver P450s
bull metabolizes 10-20 amp of commonly prescribed drugs1 S -warfarin phenitoin NSAIDs statins oral hypogycaemic agents losartan sulfonylureas Inhibitors fluconazole isoniazid lovastatin amiodarone Inductors rifampin barbiturates
Alleles2 polymorphism enzyme frequency activity Slovenia3
CYP2C91 normal 815 CYP2C92 R144C decreased 122 CYP2C93 I359L decreased 63
1 httpwwwgeorgetownedudepartmentspharmacologydavetabhtml 2 httpwwwkiseCYPalleles 3 Herman et al Zdrav Vestn 2003
httpmedicineiupui
educlinpharmddis
ClinicalTableaspx
Warfarin
bull frequent monitoring of prothrombin time (INR) amp dose adjustment (INR therapeutic range 2-3)
bull it can take several months to determine optimal dose bull ranks among top drugs in hospital emergency room visits for bleeding
bull thromboembolic complications when dose to low
bull 10 of all hospital admissions due to ADR in England Wysowski et al 2007 Pirmohamed et al 2004
bull oral anticoagulant drug
bull used for prophylaxis and treatment of thromboembolic disorders
bull the most frequently used coumarin
bull narrow therapeutic window
bull high inter-individual variability in dose response
bull frequent interactions with other drugs andor diet
Kaminsky amp Zhang 1997
Warfarin metabolism
bull S-warfarin- bull 60-70 anticoagulant effect bull CYP2C9
2 Arg144Cys (ex3) 3 Ile359Leu (ex7)
httpwwwkiseCYPalleles
bull R-varfarin- bull 30-40 anticoagulant effect bull CYP1A2 CYP2C19 CYP3A4
CYP2C9 genotypes and warfarin maintenance dose
Herman et al Pharmacogenomics J 2005
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles
Nature February 5 2004
VKORC1 identified
Rare mutations cause
bull Warfarin resistance
bull Multiple coagulation factor
deficiency type II
FII FVII FIX FX
g-carboxylated
active zymogen
Warfarin inhibits vitamin K cycle
VKORC1 6484CgtT - linkage with SNPs that may affect transcriptional efficiency
9041GgtA 3rsquoUTR - mRNA stabilisation
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
bull Pharmacogenetics
links information on the variation of human genes to variations in
drug levels
drug response
adverse events
bull Pharmacogenomics
studies variability relevant for drug response at the genome level
effects of drugs on gene expression
identification of new drug targets
Oral anticoagulants (Warfarin) Oral antidiabetics Phenytoin NSAID
Proton pump inhibitors Clopidogrel Diazepam Antidepressants
Beta blockers Antidepressants Antipsychotics Debrisoquine Codeine Tamoxifen
Cyclosporin Tacrolimus Taxol Amiodarone Cervistatin Erythromycin Methadone Quinine
Phase I Drug Metabolizing Enzymes
Cytochrome P450 dependent drug Response
P450s families 1-3
PRO-DRUG
ACTIVATION
Codeine
CYP2D6 (UM)
Morphine-ADRs
Codeine
CYP2D6 (PM)
No pain relieve
Tamoxifen
CYP2D6 (PM)
Lower endoxifen
Clopidogrel
CYP2C192 (PM)
No platelet response
Antidepressants
Antipsychotics
CYP2D6 (UM)
Less drug effect
Antidepressants
Antipsychotics CYP2D6 (PM)
ADRs
Warfarinacecoumarol
CYP2C92 3 (PM)
Bleeding
Too Much
Activation
Too Little
Activation
Ultrarapid Metab Poor Metab
DRUG
INACTIVATION
Too Fast
Inactivation
Too Slow
Inactivation
Ultrarapid Metab Poor Metab
75 Phase I reactions Highly polymorphic
CYP2C9 bull 20 of all liver P450s
bull metabolizes 10-20 amp of commonly prescribed drugs1 S -warfarin phenitoin NSAIDs statins oral hypogycaemic agents losartan sulfonylureas Inhibitors fluconazole isoniazid lovastatin amiodarone Inductors rifampin barbiturates
Alleles2 polymorphism enzyme frequency activity Slovenia3
CYP2C91 normal 815 CYP2C92 R144C decreased 122 CYP2C93 I359L decreased 63
1 httpwwwgeorgetownedudepartmentspharmacologydavetabhtml 2 httpwwwkiseCYPalleles 3 Herman et al Zdrav Vestn 2003
httpmedicineiupui
educlinpharmddis
ClinicalTableaspx
Warfarin
bull frequent monitoring of prothrombin time (INR) amp dose adjustment (INR therapeutic range 2-3)
bull it can take several months to determine optimal dose bull ranks among top drugs in hospital emergency room visits for bleeding
bull thromboembolic complications when dose to low
bull 10 of all hospital admissions due to ADR in England Wysowski et al 2007 Pirmohamed et al 2004
bull oral anticoagulant drug
bull used for prophylaxis and treatment of thromboembolic disorders
bull the most frequently used coumarin
bull narrow therapeutic window
bull high inter-individual variability in dose response
bull frequent interactions with other drugs andor diet
Kaminsky amp Zhang 1997
Warfarin metabolism
bull S-warfarin- bull 60-70 anticoagulant effect bull CYP2C9
2 Arg144Cys (ex3) 3 Ile359Leu (ex7)
httpwwwkiseCYPalleles
bull R-varfarin- bull 30-40 anticoagulant effect bull CYP1A2 CYP2C19 CYP3A4
CYP2C9 genotypes and warfarin maintenance dose
Herman et al Pharmacogenomics J 2005
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles
Nature February 5 2004
VKORC1 identified
Rare mutations cause
bull Warfarin resistance
bull Multiple coagulation factor
deficiency type II
FII FVII FIX FX
g-carboxylated
active zymogen
Warfarin inhibits vitamin K cycle
VKORC1 6484CgtT - linkage with SNPs that may affect transcriptional efficiency
9041GgtA 3rsquoUTR - mRNA stabilisation
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Oral anticoagulants (Warfarin) Oral antidiabetics Phenytoin NSAID
Proton pump inhibitors Clopidogrel Diazepam Antidepressants
Beta blockers Antidepressants Antipsychotics Debrisoquine Codeine Tamoxifen
Cyclosporin Tacrolimus Taxol Amiodarone Cervistatin Erythromycin Methadone Quinine
Phase I Drug Metabolizing Enzymes
Cytochrome P450 dependent drug Response
P450s families 1-3
PRO-DRUG
ACTIVATION
Codeine
CYP2D6 (UM)
Morphine-ADRs
Codeine
CYP2D6 (PM)
No pain relieve
Tamoxifen
CYP2D6 (PM)
Lower endoxifen
Clopidogrel
CYP2C192 (PM)
No platelet response
Antidepressants
Antipsychotics
CYP2D6 (UM)
Less drug effect
Antidepressants
Antipsychotics CYP2D6 (PM)
ADRs
Warfarinacecoumarol
CYP2C92 3 (PM)
Bleeding
Too Much
Activation
Too Little
Activation
Ultrarapid Metab Poor Metab
DRUG
INACTIVATION
Too Fast
Inactivation
Too Slow
Inactivation
Ultrarapid Metab Poor Metab
75 Phase I reactions Highly polymorphic
CYP2C9 bull 20 of all liver P450s
bull metabolizes 10-20 amp of commonly prescribed drugs1 S -warfarin phenitoin NSAIDs statins oral hypogycaemic agents losartan sulfonylureas Inhibitors fluconazole isoniazid lovastatin amiodarone Inductors rifampin barbiturates
Alleles2 polymorphism enzyme frequency activity Slovenia3
CYP2C91 normal 815 CYP2C92 R144C decreased 122 CYP2C93 I359L decreased 63
1 httpwwwgeorgetownedudepartmentspharmacologydavetabhtml 2 httpwwwkiseCYPalleles 3 Herman et al Zdrav Vestn 2003
httpmedicineiupui
educlinpharmddis
ClinicalTableaspx
Warfarin
bull frequent monitoring of prothrombin time (INR) amp dose adjustment (INR therapeutic range 2-3)
bull it can take several months to determine optimal dose bull ranks among top drugs in hospital emergency room visits for bleeding
bull thromboembolic complications when dose to low
bull 10 of all hospital admissions due to ADR in England Wysowski et al 2007 Pirmohamed et al 2004
bull oral anticoagulant drug
bull used for prophylaxis and treatment of thromboembolic disorders
bull the most frequently used coumarin
bull narrow therapeutic window
bull high inter-individual variability in dose response
bull frequent interactions with other drugs andor diet
Kaminsky amp Zhang 1997
Warfarin metabolism
bull S-warfarin- bull 60-70 anticoagulant effect bull CYP2C9
2 Arg144Cys (ex3) 3 Ile359Leu (ex7)
httpwwwkiseCYPalleles
bull R-varfarin- bull 30-40 anticoagulant effect bull CYP1A2 CYP2C19 CYP3A4
CYP2C9 genotypes and warfarin maintenance dose
Herman et al Pharmacogenomics J 2005
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles
Nature February 5 2004
VKORC1 identified
Rare mutations cause
bull Warfarin resistance
bull Multiple coagulation factor
deficiency type II
FII FVII FIX FX
g-carboxylated
active zymogen
Warfarin inhibits vitamin K cycle
VKORC1 6484CgtT - linkage with SNPs that may affect transcriptional efficiency
9041GgtA 3rsquoUTR - mRNA stabilisation
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Cytochrome P450 dependent drug Response
P450s families 1-3
PRO-DRUG
ACTIVATION
Codeine
CYP2D6 (UM)
Morphine-ADRs
Codeine
CYP2D6 (PM)
No pain relieve
Tamoxifen
CYP2D6 (PM)
Lower endoxifen
Clopidogrel
CYP2C192 (PM)
No platelet response
Antidepressants
Antipsychotics
CYP2D6 (UM)
Less drug effect
Antidepressants
Antipsychotics CYP2D6 (PM)
ADRs
Warfarinacecoumarol
CYP2C92 3 (PM)
Bleeding
Too Much
Activation
Too Little
Activation
Ultrarapid Metab Poor Metab
DRUG
INACTIVATION
Too Fast
Inactivation
Too Slow
Inactivation
Ultrarapid Metab Poor Metab
75 Phase I reactions Highly polymorphic
CYP2C9 bull 20 of all liver P450s
bull metabolizes 10-20 amp of commonly prescribed drugs1 S -warfarin phenitoin NSAIDs statins oral hypogycaemic agents losartan sulfonylureas Inhibitors fluconazole isoniazid lovastatin amiodarone Inductors rifampin barbiturates
Alleles2 polymorphism enzyme frequency activity Slovenia3
CYP2C91 normal 815 CYP2C92 R144C decreased 122 CYP2C93 I359L decreased 63
1 httpwwwgeorgetownedudepartmentspharmacologydavetabhtml 2 httpwwwkiseCYPalleles 3 Herman et al Zdrav Vestn 2003
httpmedicineiupui
educlinpharmddis
ClinicalTableaspx
Warfarin
bull frequent monitoring of prothrombin time (INR) amp dose adjustment (INR therapeutic range 2-3)
bull it can take several months to determine optimal dose bull ranks among top drugs in hospital emergency room visits for bleeding
bull thromboembolic complications when dose to low
bull 10 of all hospital admissions due to ADR in England Wysowski et al 2007 Pirmohamed et al 2004
bull oral anticoagulant drug
bull used for prophylaxis and treatment of thromboembolic disorders
bull the most frequently used coumarin
bull narrow therapeutic window
bull high inter-individual variability in dose response
bull frequent interactions with other drugs andor diet
Kaminsky amp Zhang 1997
Warfarin metabolism
bull S-warfarin- bull 60-70 anticoagulant effect bull CYP2C9
2 Arg144Cys (ex3) 3 Ile359Leu (ex7)
httpwwwkiseCYPalleles
bull R-varfarin- bull 30-40 anticoagulant effect bull CYP1A2 CYP2C19 CYP3A4
CYP2C9 genotypes and warfarin maintenance dose
Herman et al Pharmacogenomics J 2005
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles
Nature February 5 2004
VKORC1 identified
Rare mutations cause
bull Warfarin resistance
bull Multiple coagulation factor
deficiency type II
FII FVII FIX FX
g-carboxylated
active zymogen
Warfarin inhibits vitamin K cycle
VKORC1 6484CgtT - linkage with SNPs that may affect transcriptional efficiency
9041GgtA 3rsquoUTR - mRNA stabilisation
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
CYP2C9 bull 20 of all liver P450s
bull metabolizes 10-20 amp of commonly prescribed drugs1 S -warfarin phenitoin NSAIDs statins oral hypogycaemic agents losartan sulfonylureas Inhibitors fluconazole isoniazid lovastatin amiodarone Inductors rifampin barbiturates
Alleles2 polymorphism enzyme frequency activity Slovenia3
CYP2C91 normal 815 CYP2C92 R144C decreased 122 CYP2C93 I359L decreased 63
1 httpwwwgeorgetownedudepartmentspharmacologydavetabhtml 2 httpwwwkiseCYPalleles 3 Herman et al Zdrav Vestn 2003
httpmedicineiupui
educlinpharmddis
ClinicalTableaspx
Warfarin
bull frequent monitoring of prothrombin time (INR) amp dose adjustment (INR therapeutic range 2-3)
bull it can take several months to determine optimal dose bull ranks among top drugs in hospital emergency room visits for bleeding
bull thromboembolic complications when dose to low
bull 10 of all hospital admissions due to ADR in England Wysowski et al 2007 Pirmohamed et al 2004
bull oral anticoagulant drug
bull used for prophylaxis and treatment of thromboembolic disorders
bull the most frequently used coumarin
bull narrow therapeutic window
bull high inter-individual variability in dose response
bull frequent interactions with other drugs andor diet
Kaminsky amp Zhang 1997
Warfarin metabolism
bull S-warfarin- bull 60-70 anticoagulant effect bull CYP2C9
2 Arg144Cys (ex3) 3 Ile359Leu (ex7)
httpwwwkiseCYPalleles
bull R-varfarin- bull 30-40 anticoagulant effect bull CYP1A2 CYP2C19 CYP3A4
CYP2C9 genotypes and warfarin maintenance dose
Herman et al Pharmacogenomics J 2005
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles
Nature February 5 2004
VKORC1 identified
Rare mutations cause
bull Warfarin resistance
bull Multiple coagulation factor
deficiency type II
FII FVII FIX FX
g-carboxylated
active zymogen
Warfarin inhibits vitamin K cycle
VKORC1 6484CgtT - linkage with SNPs that may affect transcriptional efficiency
9041GgtA 3rsquoUTR - mRNA stabilisation
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
httpmedicineiupui
educlinpharmddis
ClinicalTableaspx
Warfarin
bull frequent monitoring of prothrombin time (INR) amp dose adjustment (INR therapeutic range 2-3)
bull it can take several months to determine optimal dose bull ranks among top drugs in hospital emergency room visits for bleeding
bull thromboembolic complications when dose to low
bull 10 of all hospital admissions due to ADR in England Wysowski et al 2007 Pirmohamed et al 2004
bull oral anticoagulant drug
bull used for prophylaxis and treatment of thromboembolic disorders
bull the most frequently used coumarin
bull narrow therapeutic window
bull high inter-individual variability in dose response
bull frequent interactions with other drugs andor diet
Kaminsky amp Zhang 1997
Warfarin metabolism
bull S-warfarin- bull 60-70 anticoagulant effect bull CYP2C9
2 Arg144Cys (ex3) 3 Ile359Leu (ex7)
httpwwwkiseCYPalleles
bull R-varfarin- bull 30-40 anticoagulant effect bull CYP1A2 CYP2C19 CYP3A4
CYP2C9 genotypes and warfarin maintenance dose
Herman et al Pharmacogenomics J 2005
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles
Nature February 5 2004
VKORC1 identified
Rare mutations cause
bull Warfarin resistance
bull Multiple coagulation factor
deficiency type II
FII FVII FIX FX
g-carboxylated
active zymogen
Warfarin inhibits vitamin K cycle
VKORC1 6484CgtT - linkage with SNPs that may affect transcriptional efficiency
9041GgtA 3rsquoUTR - mRNA stabilisation
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Warfarin
bull frequent monitoring of prothrombin time (INR) amp dose adjustment (INR therapeutic range 2-3)
bull it can take several months to determine optimal dose bull ranks among top drugs in hospital emergency room visits for bleeding
bull thromboembolic complications when dose to low
bull 10 of all hospital admissions due to ADR in England Wysowski et al 2007 Pirmohamed et al 2004
bull oral anticoagulant drug
bull used for prophylaxis and treatment of thromboembolic disorders
bull the most frequently used coumarin
bull narrow therapeutic window
bull high inter-individual variability in dose response
bull frequent interactions with other drugs andor diet
Kaminsky amp Zhang 1997
Warfarin metabolism
bull S-warfarin- bull 60-70 anticoagulant effect bull CYP2C9
2 Arg144Cys (ex3) 3 Ile359Leu (ex7)
httpwwwkiseCYPalleles
bull R-varfarin- bull 30-40 anticoagulant effect bull CYP1A2 CYP2C19 CYP3A4
CYP2C9 genotypes and warfarin maintenance dose
Herman et al Pharmacogenomics J 2005
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles
Nature February 5 2004
VKORC1 identified
Rare mutations cause
bull Warfarin resistance
bull Multiple coagulation factor
deficiency type II
FII FVII FIX FX
g-carboxylated
active zymogen
Warfarin inhibits vitamin K cycle
VKORC1 6484CgtT - linkage with SNPs that may affect transcriptional efficiency
9041GgtA 3rsquoUTR - mRNA stabilisation
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Kaminsky amp Zhang 1997
Warfarin metabolism
bull S-warfarin- bull 60-70 anticoagulant effect bull CYP2C9
2 Arg144Cys (ex3) 3 Ile359Leu (ex7)
httpwwwkiseCYPalleles
bull R-varfarin- bull 30-40 anticoagulant effect bull CYP1A2 CYP2C19 CYP3A4
CYP2C9 genotypes and warfarin maintenance dose
Herman et al Pharmacogenomics J 2005
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles
Nature February 5 2004
VKORC1 identified
Rare mutations cause
bull Warfarin resistance
bull Multiple coagulation factor
deficiency type II
FII FVII FIX FX
g-carboxylated
active zymogen
Warfarin inhibits vitamin K cycle
VKORC1 6484CgtT - linkage with SNPs that may affect transcriptional efficiency
9041GgtA 3rsquoUTR - mRNA stabilisation
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
CYP2C9 genotypes and warfarin maintenance dose
Herman et al Pharmacogenomics J 2005
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles
Nature February 5 2004
VKORC1 identified
Rare mutations cause
bull Warfarin resistance
bull Multiple coagulation factor
deficiency type II
FII FVII FIX FX
g-carboxylated
active zymogen
Warfarin inhibits vitamin K cycle
VKORC1 6484CgtT - linkage with SNPs that may affect transcriptional efficiency
9041GgtA 3rsquoUTR - mRNA stabilisation
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Nature February 5 2004
VKORC1 identified
Rare mutations cause
bull Warfarin resistance
bull Multiple coagulation factor
deficiency type II
FII FVII FIX FX
g-carboxylated
active zymogen
Warfarin inhibits vitamin K cycle
VKORC1 6484CgtT - linkage with SNPs that may affect transcriptional efficiency
9041GgtA 3rsquoUTR - mRNA stabilisation
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
11 1x xx
CYP2C9 genotype
00
20
40
60
80
100
120
140
wa
rfa
rin
do
se
(m
gd
ay)
VKORC1
6484C gtT
CC 28 570 (293) mgday
CT 49 349 (168) mgday
TT 23 211 (096) mgday
VKORC1 6484CgtT intron 1 polymorphism and warfarin maintenance dose
Boxes indicate the median and inter-quartile ranges
Whiskers indicate the minimum and maximum values
Outliers are shown by circles and extreme values by
mean dose (SD)
p lt 0001
Herman et al Thromb Haemost 2006
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Takeuchi et al PLoS Genet 2009
GWAS confirms VKORC1 CYP2C9 and CYP4F2 as principal genetic determinants of warfarin dose
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Predictor b SE(b)
Significance Adjusted R2
Constant 10752 0957 plt0001
VKORC1 6484TT
-2731 0409 plt0001 0162
VKORC1 6484CT -1700 0355 plt0001 0304
AGE (years) -0068 0013 plt0001 0375
CYP2C91x -1731 0309 plt0001 0437
CYP2C9xx -2967 0623 plt0001 0502
VKORC1 9041AA 1027 0477 p=0003 0513
Dose (mgday) = 10752 ndash 2731 (6484TT) ndash 1700 (6484CT) ndash 0068 AGE ndash 1731 (1x) ndash 2967 (xx) + 1027 (9041AA)
Multiple regression model for warfarin daily dose
Predicted warfarin dose adjustment in a 72 y patient with genotype
VKORC1 6484CT (49) 290 lower dose vs 6484CC VKORC1 6484TT (23) 466 lower dose 6484CC
CYP2C9 1x (32) 296 lower dose CYP2C911 CYP2C9 xx (5) 507 lower dose CYP2C911
Herman et al Thromb Haemost 2006
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Comparison of warfarin dosing algorithms
Sagreiya et al Pharmacogenet Genomics 2010
Prospective studies in different populations strongly suggest that
pharmacogenetic-based dosing reduces time to therapeutic INR and AEs
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Comparisons of Warfarin Doses Predicted According to the Clinical Algorithm and the Pharmacogenetic Algorithm
50-year-old patient 175 m tall weighs 80 kg
International Warfarin Pharmacogenetics Consortium N Engl J Med 2009
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
0122 2010 Updated label approved for COUMADIN NDA no 009218
bullThe patientrsquos CYP2C9 and VKORC1 genotype information when available can assist in selection of the starting dose (Table 5 )
bullIn all patients subsequent dosage adjustments must be made based on the results of PTINR determinations
CYP2C9 and VKORC1 genotyping recommended but not required
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Pharmacogenomic information included in drug labeling by FDA
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Bolnica ki se zdravi zaradi pljučne embolije je bila zaradi pretiranega
odgovora na varfarin napotena na analizo polimorfizmov citokroma P450
2C9 (CYP2C9) Ker je za klinični učinek varfarina pomemben tudi
polimorfizem tarčnega gena VKORC1 smo izvedli tudi analizo dveh
polimorfizmov v tem genu
Metode Prevzem vzorca DNA na Službi za specialno laboratorijsko diagnostiko 23 3 2011 ob 14h
Analiza polimorfizmov gena CYP2C9 Alel CYP2C92 smo določali s pomnoževanjem z verižno
reakcijo polimerizacije DNA (PCR) ki ji je sledila restrikcijska analiza z encimom AvaII Alel CYP2C93
smo določili s pomnoževanjem predela eksona 7 v dveh reakcijskih zmeseh ki mu je sledila
restrikcijska analiza z encimom NsiI oziroma KpnI (Herman in sod 2005) Prisotnost produktov
pomnoževanja smo preverili z elektroforezo na 2-tnem agaroznem gelu za preverjanje dolžin
restrikcisjkih produktov pa smo uporabili elektroforezo na 25 -tnem agaroznem gelu
Analiza polimorfizmov gena VKORC1
Prisotnost polimorfizma 6484CgtT smo določali s pomnoževanjem predela introna 1 s PCR in
cepljenjem pomnoženega produkta z StyI Prisotnost polimorfizma 9041GgtA pa smo določali s
pomnoževanjem ustreznega dela predela 3rsquo-neprevedenega področja ki mu je sledila restrikcijska
analiza z encimom FauI (Herman in sod 2006a) Preverjanje prisotnosti produktov pomnoževanja in
analiza produktov cepljenja sta potekala kot opisano zgoraj
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Rezultati
bull Analiza CYP2C9 je pokazala da je preiskovanka heterozigotna nosilka alela
CYP2C93 z genotipom CYP2C913
bull Analiza polimorfizma 6484CgtT je pokazala da je preiskovanka heterozigot za
polimorfizem z genotipom VKORC1 6484CT
bull Analiza polimorfizma 9041GgtA je pokazala da je preiskovanka homozigot za
normalni alel z genotipom VKORC1 9041GG
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Zaključek
Na podlagi rezultatov farmakogenetske analize sklepam da ima lahko
bolnica zaradi prisotnosti enega polimorfnega alela CYP2C93
(genotip CYP2C913) upočasnjeno presnovo varfarina Hkrati je
bolnica nosilka genotipov VKORC1 6484CT in 9041GG ki
povečajo občutljivost tarčnega encima za varfarin
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values
Clinical decission support tools Algorithms to estimate dose with or withouth genetic information andor INR values