Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for CYP2C9 and HLA-B Genotype and Phenytoin Dosing

Abstract

Phenytoin is a widely used antiepileptic drug with a narrow therapeutic index and large inter-patient variability partly due to genetic variations in CYP2C9. Furthermore, the variant allele HLA-B*15:02 is associated with an increased risk of Stevens-Johnson syndrome and toxic epidermal necrolysis in response to phenytoin treatment. We summarize evidence from the published literature supporting these associations and provide recommendations for the use of phenytoin based on CYP2C9 and/or HLA-B genotype (also available on PharmGKB: www.pharmgkb.org).

Introduction

The purpose of this guideline is to provide information for the interpretation of HLA-B and/or CYP2C9 genotype tests so that the results can guide dosing and/or use of phenytoin. Detailed guidelines for use of phenytoin as well as analyses of cost effectiveness are out of scope. CPIC guidelines are periodically updated at http://www.pharmgkb.org.

Focused Literature Review

A literature review focused on CYP2C9 and HLA-B*15:02 genotype and phenytoin use (see Supplemental Material online) was conducted. Reviews were included to summarize the available literature.

Genes: HLA-B and CYP2C9

Background

In this guideline, human leukocyte antigen B (HLA-B) will be discussed as it relates to phenytoin-induced cutaneous adverse drug reactions (ADRs) of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) and hepatic CYP2C9 and its alleles are discussed as they relate to phenytoin metabolism and dosing.

HLA-B

HLA-B is part of a gene cluster designated as the human major histocompatibility complex (MHC) located on the short arm of chromosome 6. The cluster contains three classes (I, II and III). MHC class I contains three genes: HLA-B plus HLA-A and HLA-C. HLA-B encodes a cell surface protein that binds peptides generated by proteolysis and extruded from proteasomes. The presentation of these peptides on the cell surface enables the immune system to distinguish self-proteins from foreign proteins typically introduced by infectious organisms (e.g., viruses and bacteria) (see supplemental material for further discussion).

HLA genes, and specifically HLA-B, are among the most highly polymorphic genes in the human genome. HLA polymorphisms were previously ascertained serologically, but genotyping and DNA sequencing methods reveal much greater genetic complexity. More than 2,000 HLA-B alleles, many of which differ by more than one nucleotide from each other, were deposited to the World Health Organization (WHO) Nomenclature Committee for Factors of the HLA System (http://hla.alleles.org). Each allele is designated by the gene name followed by an asterisk and up to an eight-digit (four pairs) identifier giving information about the allele type (designated by the first two digits) and specific protein subtypes (second set of digits). For more information and a diagram outlining the description of the current HLA allele nomenclature see http://hla.alleles.org/nomenclature/naming.html. The details of HLA nomenclature are also described in a previous CPIC guideline (1). This guideline specifically discusses only the HLA-B*15:02 allele as it relates to the phenytoin-induced cutaneous adverse drug reactions of SJS and TEN.

CYP2C9

Hepatic CYP2C9 enzyme contributes to the metabolism of many clinically relevant drugs, including phenytoin (http://www.pharmgkb.org/pathway/PA145011115). The CYP2C9 gene is highly polymorphic, having more than 50 known variant alleles (http://www.cypalleles.ki.se/cyp2c9.htm, Supplemental Table S1 and S2). Individuals homozygous for the reference CYP2C9 allele (CYP2C9*1) have the “normal metabolizer” phenotype. Each named CYP2C9 star (*) allele is defined by a genotype at one or more specific single-nucleotide polymorphisms (SNPs) with variable enzyme activity. The two most common variants with reduced enzyme activity in Europeans are CYP2C9*2 (rs1799853) and CYP2C9*3 (rs1057910) (2).

Genetic Test Interpretation

HLA-B

Clinical genotyping test results for HLA-B*15:02 are interpreted as “positive” if one or two copies of HLA-B*15:02 are present or “negative” if no copies of HLA-B*15:02 are present. Phenotype assignments for HLA-B*15:02 genotypes are summarized in Table 1. The allele frequencies of HLA-B vary greatly among populations. Specifically, HLA-B*15:02 is most prevalent in Oceania, East Asian and South/Central Asian populations ranging from 1% to over 10%. It is less frequent in European populations (0–1%) and apparently absent in several African populations (Supplemental Table S3 and S4). The global average derived from over 46,000 individuals is 1.37%.

Table 1.

Assignment of likely phenotype based on genotypes

Assignment of likely CYP2C9 phenotypes based on genotypes
Likely Phenotypea	Genotypes	Examples of diplotypes
Extensive metabolizer (normal activity) (constitutes ~ 91% of patients)	An individual carrying two normal activity alleles	*1/*1
Intermediate metabolizerc (heterozygote or intermediate activity) (constitutes ~ 8% of patients)	An individual carrying one normal activity allele plus one decreased function allele	*1/*3, *1/*2
Poor metabolizer (homozygous variant, mutant, low, or deficient activity) (constitutes ~ 1% of patients)	An individual carrying two decreased function alleles	*2/*2, *3/*3, *2/*3

Assignment of likely HLA-B phenotypes based on genotypes
Likely Phenotypeb	Genotype	Examples of diplotypes
Homozygous for an allele other than *15:02; at “normal” or reduced risk of phenytoin-associated cutaneous adverse reactions (constitutes ~ 98.6% of patients)	HLA-B*15:02 non-carrier. No *15:02 alleles reported, often reported as “negative” on a genotyping test.	*X/*Xd
Heterozygote or homozygous variant; at significantly increased risk of phenytoin-associated cutaneous adverse reactions (constitutes ~ 1.4% of patients)	HLA-B*15:02 Carrier. One or two *15:02 alleles, often reported as “positive” on a genotyping test.	*15:02/*Xd, *15:02/*15:02

^a,bGlobal frequencies presented in the parenthesis. Haplotype frequencies vary among populations, please see details for individual population frequencies in ^aSupplemental Table S5, S6 and S7 for CYP2C9*2 and *3, and ^bSupplemental Table S3 and S4 for HLA-B*15:02.

^cThe enzyme activity in this grouping varies widely. See Supplemental Table S2 for activity ranges.

^dWhere *X = any genotype other than *15:02

CYP2C9

Most clinical laboratories reporting CYP2C9 genotype use the star (*) allele nomenclature and may interpret the patient’s predicted metabolizer phenotype (Table 1, Supplemental Table S1). The combination of alleles is used to determine a patient’s diplotype. Not all CYP2C9 allelic variants may be tested, influencing the accuracy of the genotype-based dose prediction, primarily in individuals of Asian or African ancestry who carry other common functionally decreased activity CYP2C9 variant alleles (Supplemental Table S5). The frequencies of the CYP2C9 *2 and *3 alleles and diplotypes derived from these and other alleles differ between racial/ethnic groups (Supplementary Table S5, S6 and S7) (2). CYP2C9 alleles are typically characterized as wild-type (normal function) or decreased function depending on the reported activity of the enzyme for which they encode.

Available Genetic Test Options

Several methods of CYP2C9 and HLA-B genotyping are commercially available. The Supplemental Material online and at www.pharmgkb.org contains more information on available clinical testing options.

Incidental findings

HLA-B alleles are associated with hypersensitivity reactions to other drugs. CPIC guidelines are available for HLA-B*57:01 and abacavir-induced hypersensitivity reactions, HLA-B*58:01 and allopurinol-induced severe cutaneous adverse reactions, and HLA-B*15:02 and carbamazepine-induced SJS and TEN (1, 3, 4).

No diseases have been linked to genetic variations in CYP2C9, except for a small study that linked CYP2C9*2 and *3 variants and phenytoin to a higher frequency of cerebellar atrophy (5). CYP2C9 poor metabolizers may be predisposed to serious bleeding during warfarin therapy (6).

Other Considerations

Not applicable

Drugs: Phenytoin and Fosphenytoin

Phenytoin and its prodrug fosphenytoin are one of the mainstays of treatment for both focal and generalized convulsive status epilepticus. Dosing is complex owing to its highly unusual pharmacokinetics and requiring adjustments be made in line with patient weight, sex, and age. Outpatient therapy is generally initiated at 5–7 mg/kg/day in adults (slightly higher in children) and may be given once daily (or twice daily in children). Starting dose must be lower in the setting of hepatic impairment. Careful dose adjustments must then be made -- generally 30 – 40 mg at a time in 2-week intervals in adults -- to stabilize the level within the typical therapeutic range (10 – 20 ug/dL). In urgent situations such as status epilepticus, intravenous loading doses of 15–20 mg/kg are given, followed by maintenance doses, IV or oral, as above. Acute dose-related side effects include sedation, ataxia, dizziness, nystagmus, nausea, and cognitive impairment. The drug is highly allergenic, and rashes ranging from mild eruptions to life-threatening hypersensitivity reactions may be seen. HLA-B*15:02 is associated with the phenytoin-induced SJS and TEN. SJS is characterized by epidermal detachment involving up to 10% of body surface area (BSA) while TEN usually affects more than 30% of the BSA. Subacutely, hematologic and hepatic toxicity can occur; the latter is likely a hypersensitivity reaction itself, as it is usually accompanied by rash (7), while the former may consist of leukopenia or pancytopenia.

Because of the acute dose-related side effects, initial maintenance dose selection is important. Higher plasma concentrations increase the probability of these toxicities. However, non-linear saturable pharmacokinetics, auto-inductive effects with maintenance dosing, and CYP2C9 pharmacogenetic polymorphisms complicate dose-selection. The CYP2C9 poor metabolizer phenotype and CYP2C9 drug interactions, such as produced by voriconazole, can significantly augment phenytoin exposure (8). Variability in protein binding, primarily related to changes in albumin concentrations, can confound the relationship of therapeutic drug monitoring and pharmacodynamic expectations. Further discussion of the metabolism of phenytoin and fosphenytoin can be found in the supplemental material.

Linking genetic variability to variability in drug-related phenotypes

Substantial evidence links CYP2C9 and HLA-B*15:02 genotype with phenotypic variability (see Supplemental Tables S8 and S9). Application of a grading system to evidence linking genotypic to phenotypic variability indicates a high quality of evidence in the majority of cases (Supplemental Tables S8 and S9). The evidence presented here and in Supplemental Tables S8 and S9 provides the basis for the dosing recommendations in Table 2.

Table 2.

Recommended dosing of phenytoin/fosphenytoin based on HLA-B*15:02 and CYP2C9 phenotype/genotype.

Phenotype/ Genotype	HLA-B*15:02 carrier			HLA-B*15:02 non-carrier
	Implication	Therapeutic Recommendation	Classification of Recommendationa	Implication	Therapeutic Recommendation	Classification of Recommendationa
CYP2C9 Extensive metabolizer	Increased risk of phenytoin-induced SJS/TEN	If patient is phenytoin-naiveb, do not use phenytoin/fosphenytoinc	Strong	Normal phenytoin metabolism	Initiate therapy with recommended maintenance dosed.	Strong
CYP2C9 Intermediate metabolizer	Increased risk of phenytoin-induced SJS/TEN	If patient is phenytoin-naiveb, do not use phenytoin/fosphenytoinc	Strong	Reduced phenytoin metabolism Higher plasma concentrations will increase probability of toxicities	Consider 25% reduction of recommended starting maintenance dosed. Subsequent maintenance doses should be adjusted according to therapeutic drug monitoring and response.	Moderate
CYP2C9 Poor metabolizer	Increased risk of phenytoin-induced SJS/TEN	If patient is phenytoin-naiveb, do not use phenytoin/fosphenytoinc	Strong	Reduced phenytoin metabolism Higher plasma concentrations will increase probability of toxicities.	Consider 50% reduction of recommended starting maintenance dosed. Subsequent maintenance doses should be adjusted according to therapeutic drug monitoring and response.	Strong

^aRating scheme described in the Supplemental Material

^bIf patient has previously used phenytoin for longer than 3 months without incidence of cutaneous adverse reactions, reinitiate phenytoin with caution.. Adjust dose based on CYP2C9 genotype if known.

^cCarbamazepine should not be used as an alternative (4). Alternative medications such as oxcarbazepine, eslicarbazepine acetate, and lamotrigine have some evidence linking SJS/TEN with the HLA-B*15:02 allele and thus caution should be used in choosing alternatives to phenytoin (see Supplement for details).

^dRecommended maintenance dose based on patient’s clinical characteristics

HLA-B

An increased risk of SJS/TEN has been associated with the HLA-B*15:02 allele in Han Chinese and other Asian groups (see Supplemental Material; Supplemental Table S8). Cheung et al conducted a meta-analysis of two studies in Taiwan (9) and Hong Kong (10), comprising in total 41 cases and 188 controls and showed a positive association of HLA-B*15:02 with phenytoin-induced SJS/TEN (p < 3 × 10⁻⁴; OR 4.26; 95% CI 1.93–9.39) under a fixed-effect model with statistically insignificant heterogeneity. By pooling data directly, the association had a sensitivity of 36.6% (95% CI 23.6–51.9) and specificity of 87.2% (95% CI 81.7–91.3). Therefore, the absence of these variants does not rule out the possibility of a patient developing phenytoin-induced SJS/TEN. The strength of the association between phenytoin use and SJS/TEN is weaker than the association between carbamazepine use and SJS/TEN due to the limited number of studies and observations with phenytoin or fosphenytoin in the literature. However, taken together with the known association of carbamazepine and SJS/TEN in carriers of HLA-B*15:02 the association supports the FDA recommendations to avoid these agents as substitutes for carbamazepine in individuals who test positive for HLA-B*15:02 (4).

CYP2C9

Available model estimates predict that variant CYP2C9 alleles lower phenytoin intrinsic clearance based on the allele and number of variant. Several studies indicate that individuals with CYP2C9*1/*3 and CYP2C9*1/*2 genotypes have mild-to-moderately reduced clearance values (Supplemental Table S9) and are classified as Intermediate Metabolizers. Individuals with two decreased activity alleles (CYP2C9*2/*2, CYP2C9*3/*3) have reduced clearance of several drugs and are classified as CYP2C9 poor metabolizers. Phenytoin maintenance doses were reported to be reduced 23–38% in heterozygous individuals with one decreased activity allele (11–13) and 31–52% for carriers with two decreased activity CYP2C9 alleles versus CYP2C9*1/*1 (12, 13). Furthermore, case reports indicate that poor metabolizers appear to be at higher risk for exposure-related toxicities than patients homozygous for the wild-type alleles (14–17).

Therapeutic Recommendations

HLA-B*15:02 recommendations

The FDA warning for phenytoin states, “Consideration should be given to avoiding phenytoin as an alternative for carbamazepine in patients positive for HLA-B*15:02″ due to the increased risk of SJS/TEN in patients of Asian ancestry.” The evidence linking HLA-B*15:02 to phenytoin-induced SJS/TEN was generated in individuals of Asian ancestry as the frequency of HLA-B*15:02 is very low in other populations (see Supplemental Table S3 and S4 for frequency information) that have been studied. However, it may also occur in other populations throughout the world yet to be studied and patients may be unaware of or fail to disclose more distant Asian ancestry in their families. Furthermore, much of the evidence (summarized in Supplemental Table S8) linking HLA-B*15:02 to phenytoin-induced SJS/TEN was generated in both children and adults. Therefore, regardless of the CYP2C9 genotype and individual’s ancestry or age, if the HLA-B*15:02 test result is “positive”, the recommendation is to consider using an anticonvulsant other than carbamazepine and phenytoin unless the benefits of treating the underlying disease clearly outweigh the risks (see Table 2). Alternative medications such as oxcarbazepine, eslicarbazepine acetate, and lamotrigine have some evidence linking SJS/TEN with the HLA-B*15:02 allele and thus caution should be used in choosing alternatives to phenytoin (see Supplement for details).

CYP2C9 recommendations

Phenytoin and fosphenytoin dose should first be adjusted according to a patient’s clinical characteristics. Table 2 summarizes the gene-based dosing recommendations for phenytoin based on CYP2C9 phenotype. The recommended phenytoin maintenance dose does not need adjustment based on genotype for CYP2C9 extensive metabolizers. Available evidence does not clearly indicate the amount of dose reduction needed to prevent phenytoin-related toxicities in CYP2C9 intermediate and poor metabolizers; thus, our recommendation should be considered conservative estimates given the variability surrounding phenytoin dosing to an individual. Based on the doses reported in the pharmacokinetic and pharmacogenetic studies mentioned above (11–13) and in Supplemental Table S9, at least a 25% reduction of the recommended starting maintenance dose may be considered for CYP2C9 intermediate metabolizers with subsequent maintenance doses adjusted based on therapeutic drug monitoring and response. For CYP2C9 poor metabolizers, consider at least a 50% reduction of starting maintenance dose with subsequent maintenance doses adjusted based on therapeutic drug monitoring or response.

Furthermore, while in vitro data suggest that the degree of reduction of catalytic activity is greater for the CYP2C9*3 variant than for the CYP2C9*2 variant (18), clinical pharmacokinetic studies indicate similar dose reductions and pharmacokinetic parameters (e.g., trough levels, serum p-HPPH/P ratio) for these variants as compared to wild-type (12, 19, 20). Thus, our recommendation is to start with at least the above recommended reduction of the maintenance dose followed by an adjustment of dose based on therapeutic drug monitoring.

Pediatrics

Special consideration should be taken with the pediatric population. Phenytoin is used in the treatment of neonatal seizures and subsequently after discharge from the neonatal ICU. Maintaining therapeutic levels can be particularly problematic in this population. This may be due to the developmental expression of hepatic CYP2C. P450 expression and functional activities have been shown to develop at different rates within subfamilies (21). It has been found that activity levels of CYP2C9 are at 1 to 2% of adult values in the fetus during the first trimester. These levels gradually increase to 30% of adult values at term. There is a high variability in these levels during the first five months of life, eventually approach adult values somewhere between five months to 2 years of age (22). Other considerations includethe clearance of phenytoin being twice that of adult values in children under 6 years of age. This is attributed to the finding that the maximal rate of phenytoin metabolism is inversely related to age. However, this varied significantly within age subgroups (23). For these reasons, phenytoin therapeutic recommendations based on CYP2C9 genotype in this population are difficult. There is only one published report describing a two-year old patient (CYP2C9*2/*2 and CYP2C19*1/*4) presenting with phenytoin toxicity two hours after a 15mg/kg phenytoin loading dose with symptoms lasting 122 hours (24). The half-life was much higher than expected (112 hours versus 46.7) which could be explained by the influence of CYP2C9 and CYP2C19 genetic polymorphisms (other predisposing factors such as malnourishment, renal failure, hepatic dysfunction, and inhibition of phenytoin metabolism by other drugs were ruled out). Therefore, for pediatric patients who are CYP2C9 intermediate or poor metabolizers dose adjustment is recommended with close therapeutic drug monitoring.

HLA-B*15:02 and CYP2C9 dosing recommendation

If both HLA-B*15:02 and CYP2C9 genotype are known, consider the HLA-B*15:02 genotype first, then the CYP2C9 genotype (Figure 1; Table 2).

Figure 1. Algorithm for suggested clinical actions based HLA-B*15:02 and CYP2C9 genotype.

EM, extensive metabolizer; IM, intermediate metabolizer; PM, poor metabolizer

^aIf patient has previously used phenytoin for longer than 3 months without incidence of cutaneous adverse reactions, reinitiate phenytoin with caution. Adjust dose based on CYP2C9 genotype if known.

^bCarbamazepine should not be used as an alternative (4). Alternative medications such as oxcarbazepine, eslicarbazepine acetate, and lamotrigine have some evidence linking SJS/TEN with the HLA-B*15:02 allele and thus caution should be used in choosing alternatives to phenytoin (see Supplement for details).

^cRecommended maintenance dose based on patient’s clinical characteristics

Other considerations

HLA-B

HLA-B*15:02 is linked to SJS and TEN but not to a predisposition for other phenytoin-induced cutaneous adverse reactions such as mild maculopapular eruptions (MPE) or drug hypersensitivity syndrome (HSS) (25).

CYP2C9

Because of its potent CYP450-inducing properties, phenytoin is involved in a very large number of drug interactions, especially involving increased metabolism of other agents with subsequent decrease in their levels (26). A full discussion of these is beyond the scope of this guideline, but agents prominently and significantly affected include antineoplastic and immunosuppressive agents, lipid-lowering agents, psychotropics, oral contraceptives, and warfarin, just to name a few. Furthermore, inhibitors of CYP2C9 can generate phenytoin overexposure and toxicity. Although fluconazole and amiodarone are recognized as potent CYP2C9 enzyme inhibitors, other less potent drugs can produce significant elevations in phenytoin plasma concentrations. Therefore, it is important to interpret the results of genetic testing in the context of other co-administered drugs.

CYP2C9 genetic variation does not account for all of the pharmacogenetic variability in phenytoin metabolism. Some studies have implicated variants in other genes associated with phenytoin metabolism (e.g., CPY2C19, CYP1A1, and EPHX1; see (27) for a review) and combined genetic analysis might improve the predictability of CYP2C9 alone (11, 13). However, this has not been consistently replicated and there are limited studies evaluating the effect of multiple gene variation and phenytoin dose adjustment requirement. Consequently, this guideline on genotype-directed phenytoin dosing is limited to CYP2C9 variant alleles.

Recommendations for Incidental Findings

Several drugs structurally and therapeutically similar to phenytoin, such as oxcarbazepine and carbamazepine, have also been associated with SJS/TEN and HLA-B*15:02 in Asian populations (28–34) (see Supplemental Material). The drug-specific evidence linking HLA-B*15:02 and SJS/TEN is discussed in the CPIC Guideline for HLA-B genotype and carbamazepine dosing (4) and may have implications for choosing alternatives to phenytoin in those who carry the HLA-B*15:02 allele. Case reports have identified cross-reactions to lamotrigine and other anti-epileptic drugs in the presence of HLA-B*15:02 (see Supplemental Material for further discussion). However, larger studies appear to be needed for confirmation.

CYP2C9 metabolism includes substrates from several drug classes, including NSAIDS, oral hypoglycemics/sulfonylureas, as well as a miscellaneous group of drugs. Reports support that patients with enhanced sensitivity to warfarin are likely to have a decreased capacity to metabolize phenytoin (35).

Potential Benefits and Risks for the Patient

The potential benefit for patients with existing CYP2C9 and/or HLA-B*15:02 genotyping information is in avoiding adverse effects in those patients who are CYP2C9 poor metabolizers by making significant reductions in their starting maintenance dose or by selecting alterative agents for those who are HLA-B*15:02 carriers. For HLA-B*15:02 carriers, a potential risk is that phenytoin therapy may have been needlessly avoided in patients who may not have developed SJS/TEN; however, this risk is mitigated because alternatives to phenytoin with comparable effectiveness exist. Another potential risk would be an error in genotyping. Also, many commercially available genotyping tests do not detect alleles that are rare or de novo variants. Other alleles are not well characterized, resulting in uncertainty when predicting the phenotype for some genetic test results. Due to the high rate of HLA-B*15:02 false-negative results (36) or in the event of a rare variant not detected on the genetic test,, a high-risk patient could be prescribed phenytoin or prescribed a higher dose than needed. Moreover, because not all phenytoin-induced adverse events are attributed to HLA-B*15:02 or CYP2C9 metabolizer status, clinicians should carefully monitor all patients according to standard practices.

Caveats: Appropriate Use and/or Potential Misuse of Genetic Tests

The application of genotype-based dosing is most appropriate when initiating phenytoin therapy. Obtaining genetic information after months of drug therapy is less helpful, given that the drug dose may have already been adjusted based on plasma concentrations, response, or side effects. As with all diagnostic tests, genetic tests are only one of several pieces of clinical information that should be considered before initiating drug therapy.