Cytochrome P450 2C9 Genotyping

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CYP2C9 (cytochrome P450 2C9) acts on 16% of drugs in current clinical use. About 35% of individuals of European origin (Caucasians) have a slow acting form of this enzyme. CYP2C9 is an important drug-metabolizing enzyme that catalyzes the biotransformation of many other clinically useful drugs including angiotensin II blockers, nonsteroidal anti-inflammatory drugs, the alkylating anticancer prodrugs, Sulfonylureas, some antidepressants, tamoxifen and many others. Of special interest are those drugs with narrow therapeutic index, such as S-warfarin, tolbutamide and phenytoin, where impairment in CYP2C9 metabolic activity might cause difficulties in dose adjustment as well as toxicity.

Genelex offers improved detection rates using an extended Cytochrome P-450 2C9 DNA mutation panel. This test identifies 5 small nucleotide variants in PCR-multiplex format, providing increased sensitivity and quality performance. This CYP2C9 Mutation Detection Panel is the most extensive on the market. Analytical specificity and sensitivity for detection of these mutations are >99%.

Specimen Types

Please call Client Services at 800-523-6487 to obtain specimen kits.

• Buccal Swabs: 4 sterile Whatman OmniSwabs™
• Blood: 5-10 cc whole blood lavender-top EDTA or Yellow-top ACD-A tubes

• Turnaround Time: 10 -14 days turnaround (4 day turnaround for STATS)

CPT Codes

CYP2C9 Mutation DNA Analysis (provided for your guidance only)
83891, 83892, 83901 x2, 83896 x10, 83912

Clinical Significance

Phenotype prevalence is 2-4% PM, >35% IM for CYP2C9.
Drugs metabolized by this enzyme approximately 5-10%

Cytochrome P450 2C9 (CYP2C9) is a highly polymorphic liver enzyme of the cytochrome P450 super family involved with the metabolism and elimination of many commonly prescribed drugs. Genetic polymorphisms in CYP2C9 are common and can affect therapeutic response to drugs. The enzyme activity is expressed at highly variable levels. Three phenotypes are identified: poor metabolizers (PM), intermediate metabolizers (IM) and extensive metabolizers (EM, "normal").
Detecting genetic variations in drug-metabolizing enzymes is useful for identifying individuals who may experience adverse drug reactions (ADRs) with conventional doses of certain medications. Individuals who possess CYP2C9 poor metabolizer variants may exhibit different pharmacokinetics (drug levels) than normal individuals. As a result, such individuals may require non-conventional doses of medications that require CYP2C9 activity for biotranformation. Conversely, medications that do not require CYP2C9 biotranformation may be preferentially selected for patients with potentially impaired CYP2C9 metabolic capacity to avoid ADRs.

The five CYP2C9 allelic variants detected in this CYP2C9 genotyping test provide greater than 98% coverage of the variant alleles found for this gene. The normal functional allele (wild type) of the CYP2C9 gene is designated CYP2C9*1. Homozygous wild-type individuals have an extensive metabolizer phenotype (EM, normal). The most common poor metabolizer phenotypes have been identified as CYP2C9*2 and CYP2C9*3. CYP2C9*2 (C430T) and CYP2C9*3 (A1075C) each differ from the normal CYP2C9*1 by a single nucleotide substitution, which leads to impaired enzyme activity. Lee et al (2002) determined that these two poor metabolizer types CYP2C9*2 and CYP2C9*3 were found in up to 35% of Europeans (42% Croatians). Among different European populations CYP2C9*2 and CYP2C9*3 are of significance with allelic frequencies of 8-19% and 4-16% respectively. In Africans and Asians both variants are much less frequent (0.5-4%). CYP2C9*4 has been exclusively identified in Japanese, and CYP2C9*5 and CYP2C9*6 found in African Americans with low allelic frequency (>2%). Homozygosity for the CYP2C9*3 or CYP2C9*2 genotype is relatively rare (~1-2 %) in Europeans.

Laboratory Test Interpretation

Genelex offers improved detection rates using an extended Cytochrome P-450 2C9 DNA mutation panel. This test identifies 5 small nucleotide variants in multiplex PCR- format, providing increased sensitivity (98% of poor metabolizer phenotypes) and quality performance.

Cytochrome P-240 2C9 Mutations Detected
CYP2C9 allele	Nucleotide change	Effect on Enzyme Metabolism
*1	None (wildtype)	Normal
*2	430C>T	Inactive
*3	1075A>C	Inactive
*4	1076T>C	Inactive
*5	1080C>G	Inactive
*6	818delA	Inactive

For additional information see the CYP2C9 allele nomenclature database at http://www.imm.ki.se/CYPalleles/cyp2C9.htm

Testing places individuals in one of three categories:

• Extensive metabolizers (EM) represent the norm for metabolic capacity. Genotypes consistent with the EM phenotype include two active CYP2C9 alleles. In general extensive metabolizers can be administered drugs which are substrates of the CYP2C9 enzyme following standard dosing practices.
• Intermediate metabolizers (IM) may require lower than average drug dosages for optimal therapeutic response. Genotypes consistent with the IM phenotype are those with one active CYP2C9 allele.
• Poor metabolizers (PM) are at increased risk of drug-induced side effects due to diminished drug elimination or lack of therapeutic effect resulting from failure to generate the active form of the drug. Genotypes consistent with the PM phenotype are those with no active CYP2C9 alleles. These individuals have a deficiency in drug metabolism.

Although direct DNA testing detects greater than 98% of variant alleles, it will not detect all the known mutations that result in decreased or inactive CYP2C9. Absence of a detectable gene mutation or polymorphism does not rule out the possibility that a patient has an intermediate or poor metabolizer phenotype. This test does not detect polymorphisms other than those listed. Other polymorphisms in the primer binding regions can affect the testing, and ultimately, the genotyping assessments made.

Dosage Recommendations

A complicating factor in correlating CYP2C9 genotype with phenotype is that many drugs may reduce or increase CYP2C9 catalytic activity. Consequently, an individual may require a dosing decrease greater than predicted based upon genotype alone. It is important to interpret the results of testing in the context of other co administered drugs.

CYP2C9 activity also is dependent upon hepatic and renal function status, as well as age. Patients also may develop toxicity if hepatic or renal function is decreased. the results of testing and dose adjustments in the context of renal and hepatic function and age.

• PM
  Reduce dose to 20-60% of standard dosage.
  
  
• IM
  Start IM's at lowest efficacious dose, avoid multiple drug therapy that inhibits or activated through the same pathway.
  
  
• Therapeutic drug monitoring in PM and IM subjects is highly recommended. Again standard measures of efficacy (INR for warfarin or therapeutic target interval for phenytoin, for example) can be applied to ensure optimal therapy.

For specific genotype-specific clearance rates see charts from Julia Kirchheiner, et al: The CYP2C9 polymorphism: from enzyme kinetics to clinical dose recommendations Personalized Med 2004 1(1) 63-84.

Methodology

DNA extraction/Polymerase Chain Reaction (PCR)/ Enzyme inactivation /Allele-specific primer extension / Hybridization using immobilized nucleic acid probes/ Fluorescent detection.
Laboratory specimens were analyzed using the Tag-ItTM Mutation Detection System for P450-2C9 which detects 5 nucleotide variants in a multiplex polymerase chain reaction and allele-specific primer extension format.

Drug Metabolism Guide

This list is not all inclusive and is for your guidance only.

Substrates Metabolized through Cytochrome P-450 2C9

Substrates refers to drugs that are either activated or deactivated by the pathway.

aceclofenac	cyclophosphamide	phenacetin	zileuton
acenocoumarol	dapsone	phenytoin	zolpidem
alosetron	desogestrel	porprofol
amitriptyline	dextromethorphan	progesterone
amprenavir	diclofenac	sertraline
antipyrine	diltiazem	tamoxifen
candesartan	fluoxetine	trimipramine
carvedilol	fluvastatin	valproic acid
celecoxib	halofantrine	warfarin
cloazapine	methadone	zafirlukast

Inhibitors of Cytochrome P-450 2C9

Inhibitors refers to drugs that reduce the ability of the pathway to process drugs.
Co-administration will decrease the rate of metabolism of drugs through the metabolic pathway listed, increasing the possibility of toxicity.

delavirdine	fluvoxamine	nifedipine	phenylbutazone
fluconazole	loratidine	omeprazole
fluvastatin	nicardipine	paroxetine

Inducers of Cytochrome P-450 2C9

Inducers refers to drugs that increase the activity of a pathway.
Co-administration increases the rate of excretion for drugs metabolized through the pathway indicated, reducing the drug's effectiveness.

dexamethasone

rifampin

secobarbital

References

1. Aynacioglu AS, et al. Frequency of cytochrome P450 CYP2C9 variants in a Turkish population and functional relevance for phenytoin. Br J Clin Pharmacol 1999; 48(3):409-415

2. Bertilsson L et al (1993) Molecular basis for rational megaprescribing in ultrarapid hydroxylators of debrisoquine. Lancet 341:63

3. Brockmoller J et.al. Pharmacogenetic diagnosis of cytochrome P450 polymorphisms in clinical drug development and in drug treatment. Pharmacogenetics. 2000:1:125-51.

4. Chang TK, et al. Enhanced cyclophosphamide and ifosfamide activation in primary human hepatocyte cultures: response to cytochrome P-450 inducers and autoinduction by oxazaphosphorines. Cancer Res 1997; 57(10):1946-54.

5. Cozza KL, Armstrong SC, Oesterheld JR (2003) Drug Interaction principles for Medical Practice. American Psychiatric Publishing Inc

6. Hamman MA, Thompson GA, Hall SD. Regioselective and stereoselective metabolism of ibuprofen by human cytochrome P450 2C. Biochem Pharmacol 1997; 54(1):33-41.

7. Higashi MK, Veenstra DL, Kondo LM, Wittkowsky AK, Srinouanprachanh SL, Farin FM, Rettie AE. Association between CYP2C9 genetic variants and anticoagulation-related outcomes during warfarin therapy. JAMA. 2002 Apr 3;287(13):1690-8.

8. Ho PC, et al. Influence of CYP2C9 genotypes on the formation of a hepatotoxic metabolite of valproic acid in human liver microsomes. Pharmacogenomics J 2003; 3(6):335-42.

9. Kirchheiner J, Brockmoller J. Clinical consequences of cytochrome P450 2C9 polymorphisms. Clin Pharmacol Ther. 2005 Jan;77(1):1-16.

10. Kirchheiner J, Tsahuridu M, Jabrane, W, Roots I, Brockmoller J. The CYP2C9 polymorphism: from enzyme kinetics to clinical dose recommendations Personalized Med 2004 1(1) 63-84

11. Joffe HV, Johnson XR, Longtine J, Kucher N and Goldhaber SZ. warfarin dosing and Cytochrome P450 2C9 polymorphisms, Thromb Haemost; 2004 Jun;91(6):1123-8

12. Linder MW and Valdes RJr. Pharmacogenetics in the Practice of Laboratory Medicine. Molecular Diagnosis. 1999;4:365-79

13. Miners J. CYP2C9 polymorphism: impact on tolbutamide pharmacokinetics and response. Pharmacogenetics 2002; 12(2):91-2.

14. Peyvandi F, Spreafico M, Siboni SM, Moia M and Mannucci PM. Cyp2C9 genotypes and dose requirements during the induction phase of oral anticoagulation therapy. Clinical Pharmacology and Therapeutics 2004; 75(3):198-203

15. Scordo MG, et al. Genetic polymorphism of cytochrome P450 2C9 in a Caucasian and a black African population. Br J Clin Pharmacol 2001; 52(4):447-450.

16. Takahashi H, Echizen H. Pharmacogenetics of warfarin elimination and its clinical implications. Clin Pharmacokinet 2001; 40(8):587-603.

17. Rettie AE, et al. Impaired (S)-warfarin metabolism catalysed by the R144C allelic variant of CYP2C9. Pharmacogenetics 1994; 4(1):39-42.

 

For immediate consultation Call 800 523-3080.