As described in Chapter 4, biotransformation reactions mediated by P450 phase I enzymes typically modify functional groups (-OH, -SH, -NH2, -OCH3) of endogenous and xenobiotic compounds, resulting in an alteration of the biological activity of the compound. Phase I enzymes are involved in the biotransformation of over 75% of prescription drugs; therefore, polymorphisms in these enzymes may significantly affect blood levels, which in turn may alter response to many drugs. Polymorphisms in drug-metabolizing enzymes dominated the field of pharmacogenomics for many years, and for some years, metabolic phenotypes such as extensive metabolizer (EM), reflecting an individual’s metabolic rate of a particular drug that is a known substrate of a specific enzyme, were used to describe genetic effects on drug metabolism. After genotypic information became available, a new nomenclature was used to characterize an individual’s metabolic rate. In particular, diplotypes, consisting of one maternal and one paternal allele, using star (*) allele nomenclature, have been used. Each star (*) allele is defined by specific sequence variation(s) within the gene locus, eg, single nucleotide polymorphisms (SNPs), and may be assigned a functional activity score when the functional characterization is known, eg, 0 for nonfunctional, 0.5 for reduced function, and 1.0 for fully functional. Some genes, such as CYP2D6, are subject to whole gene deletions, eg, CYP2D6*5, and whole gene duplications or multiplications, eg, *1xN, *2xN, where N is the number of copies. If more than one copy of the gene is detected, the activity score is then multiplied by the number of copies observed. Enzyme activity is generally a co-dominant or additive trait. For example, if an individual carries one normal function allele and one non-functional allele, he will have an intermediate metabolic activity or be considered an intermediate metabolizer (IM). The sum of allelic activity scores typically ranges between 0 and ≥ 3.0 and is most often used to define phenotypes as follows: 0 = PM (poor metabolizer), 0.5 = IM, 1.0–2.0 = EM, and ≥ 2.0 = UM (ultra rapid metabolizer).
As described in Chapter 4, cytochrome P450 2D6 is involved in the metabolism of up to one quarter of all drugs used clinically, including predominantly basic compounds such as β blockers, antidepressants, antipsychotics, and opioid analgesics. Among the CYP enzymes, CYP2D6 displays the largest variability in metabolic capacity both between and within populations. Similar to other polymorphic enzymes, four clinically defined metabolic phenotypes, ie, PMs, IMs, EMs, and UMs, are used to predict therapeutic and adverse responses following the administration of CYP2D6 substrates.
The gene encoding CYP2D6 is highly polymorphic, with over 100 alleles defined (www.cypalleles.ki.se/cyp2d6.htm); however, greater than 95% of phenotypes can be accounted for with just nine alleles, ie, CYP2D6 alleles *3, *4, *5, and *6 are non-functional; alleles *10, *17, and *41 have reduced function; and alleles *1 and *2 are fully functional. As with many polymorphisms, allele frequencies vary across populations (Table 5–1). Some genetic variants are shared among populations at similar allele frequencies, whereas others vary considerably. For example, the most common nonfunctional allele, CYP2D6*4, is observed at a frequency of approximately 20% in Europeans and is nearly absent (<1%) in Asians (Table 5–1). Based on Hardy-Weinberg principles (see Glossary), the percentage of Europeans who are homozygous for the CYP2D6*4 allele, ie, who carry the *4 allele on both maternal and paternal chromosomes, would be 4%, whereas that of those who are heterozygotes would be 32%. This parallels the lower number of PMs (defined as having two nonfunctional alleles, eg, PMs are homozygous for *3, *4, *5, *6, or any combination of nonfunctional alleles such as *4/*5), observed in Asian populations (∼1%) compared with European populations (∼5–10%) (Table 5–2). In contrast, the *5 gene deletion is found at similar frequencies (∼3–5%) across European, African, and Asian populations, suggesting that this mutation likely took place prior to the separation of the three major races more than 100,000 years ago. Clinically, since some genotyping platforms are specific to a single ethnicity, it is important to ensure alleles applicable to the patient population being treated are tested. Of note, rare or previously undiscovered variants are typically not included in commercial tests, and thus novel or rare polymorphisms, which may exhibit altered function, will be missed.
TABLE 5–1Major alleles and frequencies in African, Asian, and European populations. |Favorite Table|Download (.pdf) TABLE 5–1Major alleles and frequencies in African, Asian, and European populations.
|Gene ||Allele(s) ||dbSNP1 Number ||Amino Acid ||Function ||Activity ||Fraction in African Populations ||Fraction in Asian Populations ||Fraction in European Populations |
| ||*1 ||Reference ||— ||Normal ||1.0 ||0.39 ||0.34 ||0.52 |
| ||*1xN ||Gene duplication or multiplication ||Increased expression ||Increased ||1.0 × N ||0.014 ||0.0031 ||0.0077 |
| ||*2 ||rs16947, rs1135840 ||R296C, S486T ||Normal ||1.0 ||0.20 ||0.12 ||0.27 |
| ||*2xN ||Duplication or multiplication ||Increased expression ||Increased ||1.0 × N ||0.015 ||0.0042 ||0.013 |
| ||*3 ||rs35742686 ||Frameshift ||None ||0.0 ||0.00030 ||0.00 ||0.013 |
| ||*4 ||rs1065852, rs3892097 ||P34S, Splicing defect ||None ||0.0 ||0.033 ||0.0045 ||0.18 |
| ||*5 ||— ||No enzyme ||None ||0.0 ||0.060 ||0.058 ||0.028 |
| ||*6 ||rs5030655 ||Frameshift ||None ||0.0 ||0.00 ||0.0002 ||0.0091 |
| ||*10 ||rs1065852, rs1135840 ||P34S, S486T ||Decreased ||0.5 ||0.067 ||0.42 ||0.028 |
| ||*17 ||rs28371706, rs16947, rs1135840 ||T107I, R296C, S486T ||Decreased ||0.5 ||0.19 ||0.0002 ||0.0027 |
| ||*41 ||rs16947, rs1135840, rs28371725 ||R296C, S486T, Splicing defect ||Decreased ||0.5 ||0.10 ||0.022 ||0.092 |
| ||*1 ||Reference ||— ||Normal ||— ||0.68 ||0.60 ||0.63 |
| ||*2 ||rs4244285 ||Splicing defect ||None ||— ||0.15 ||0.29 ||0.15 |
| ||*3 ||rs4986893 ||W212X ||None ||— ||0.0052 ||0.089 ||0.0042 |
| ||*17 ||rs12248560 ||Increased expression ||Increased ||— ||0.16 ||0.027 ||0.21 |
| ||*1 ||Reference ||— ||Normal ||— || || || |
| ||*2A ||rs3918290 ||Splicing defect ||None ||— ||0.00 ||0.0015 ||0.0086 |
| ||*13 ||rs55886062 ||I560S ||None ||— ||n/a ||0.00 ||0.0010 |
| ||— ||rs67376798 ||D949V ||None ||— ||n/a ||n/a ||0.011 |
| ||*1 ||Reference ||– ||Normal ||— || || || |
| ||*28 ||rs8175347 ||Reduced expression ||Decreased ||— ||0.43 ||0.16 ||0.39 |
| ||*1 ||Reference ||– ||Normal ||— ||0.94 ||0.98 ||0.95 |
| ||*2 ||rs1800462 ||A80P ||None ||— ||0.00087 ||0.00 ||0.0019 |
| ||*3A ||rs1800460, rs1142345 ||A154T, Y240C ||None ||— ||0.002 ||0.00012 ||0.035 |
| ||*3B ||rs1800460 ||A154T ||None ||— ||0.00 ||0.00 ||0.00046 |
| ||*3C ||rs1142345 ||Y240C ||None ||— ||0.048 ||0.016 ||0.0042 |
| ||*4-*26 ||Various ||Various ||Decreased ||— ||Various ||Various ||Various |
| ||B ||Reference ||— ||Normal ||IV ||— ||— ||— |
| ||A ||rs1050829 ||N126D ||Normal ||IV ||— ||— ||— |
| || |
A- (rs1050829, rs1050828)
A- (rs1050829, rs137852328)
A- (rs1050829, rs76723693)
|Decreased (5–10%) ||III ||0.00–0.30 ||n/a ||n/a |
| ||Mediterranean (rs5030868) ||S188P ||Decreased (<1%) ||II || || || |
| ||Canton (rs72554665), Kaiping ||R459L/R463H ||Decreased ||II |
| || || || || || ||n/a ||0.00–0.31 ||n/a |
| ||Mahidol || ||G163S ||Decreased (5–32%) ||III || || || |
| ||Chinese-5, Gaohe || || |
|Decreased ||III || || || |
| ||*1A ||Reference ||— ||Normal ||— ||0.17 ||0.27 ||0.50 |
| ||*1B ||rs2306283 ||N130D ||Normal ||— ||0.78 ||0.60 ||0.22 |
| ||*5 ||rs4149056 ||V174A ||Decreased ||— ||0.00 ||0.00 ||0.01 |
| ||*15, *16, *17 ||rs4149056, others ||V174A others ||Decreased ||— ||0.03 ||0.13 ||0.14 |
| ||*57:01 ||— ||— ||“positive” ||— ||0.010 ||0.016 ||0.068 |
| ||– ||Reference ||— ||Unfavorable ||— ||— ||— ||— |
| ||– ||rs12979860 ||— ||Favorable ||— ||0.39 ||0.87 ||0.63 |
| ||*1 ||Reference ||— ||Normal ||— || || || |
| ||*2 ||rs1799853 ||R144C ||Decreased ||— ||0.03 ||0.00 ||0.13 |
| ||*3 ||rs1057910 ||I359L ||Decreased ||— ||0.02 ||0.04 ||0.07 |
|-1639G || ||Reference ||— ||Normal ||— || || || |
|-1639A || ||rs9923231 ||Reduced expression ||Decreased ||— ||0.11 ||0.91 ||0.39 |
TABLE 5–2Gene-based dosing recommendations for selected drugs. |Favorite Table|Download (.pdf) TABLE 5–2Gene-based dosing recommendations for selected drugs.
|Gene ||Drug ||Diplotype1 ||Likely Phenotype (Activity Score) ||Dosing Recommendation ||Source of Recommendation |
| ||Codeine ||*1/*1xN, *1/*2xN ||UM (>2.0) ||• Alternative analgesic, eg, morphine or non-opioid; increased formation of morphine following codeine administration leads to higher risk of toxicity. ||CPIC2 |
| || ||*1/*1, *1/*2, *2/*2, *1/*41, *2/*5 ||EM (1.0–2.0) ||• Standard starting dose. || |
| || ||*4/*10, *5/*41 ||IM (0.5) ||• Standard starting dose; monitor closely for lack of analgesic response due to reduced morphine formation. Consider alternate analgesic, eg, morphine or non-opioid. || |
| || ||*3/*4, *4/*4, *4/*5, *5/*5, *4/*6 ||PM (0.0) ||• Alternative agent, eg, morphine or non-opioid analgesic; greatly reduced morphine formation following codeine administration, leading to insufficient pain relief. Avoid higher doses, as central side effects do not differ in PMs. || |
| ||Clopidogrel ||*1/*17, *17/*17 (UM), and *1/*1 (EM) ||UM, EM ||• Standard dose. ||CPIC |
| || ||*1/*2, *1/*3, *2/*17 ||IM ||• Alternative antiplatelet agent, eg, prasugrel or ticagrelor. || |
| || ||*2/*2, *2/*3, *3/*3 ||PM ||• Alternative antiplatelet agent, eg, prasugrel or ticagrelor. || |
| ||Fluoropyrimidines ||*1/*1 ||Normal ||• Standard dose. ||CPIC |
| || ||*1/*2A, *1/*13, *1/rs67676798 ||Reduced activity ||• Reduce initial dose 50% and titrate based on toxicity or on pharmacokinetic test results (if available). || |
| || ||*2A/*2A, *2A/*13, *13/*13, rs67376798/rs67376798 ||Complete deficiency ||• Different non-fluoropyrimidine anti-cancer agent. || |
| ||Irinotecan ||*1/*1, *1/*28 ||Normal ||• Standard starting dose. || |
| || ||*28/*28 ||Reduced ||• Reduce starting dose by at least one dose level. Or, ||Drug label |
| || || || ||• Dose >250 mg/m2: Reduce starting dose 30% and increase in response to neutrophil count. Dose =250 mg/m2: No dose adjustment. ||DPWG3 |
| ||Thiopurines ||*1/*1 ||Normal, high activity ||• Standard starting dose. ||CPIC |
| || ||*1/*2, *1/*3A, *1/*3B, *1/*3C, *1/*4 ||Intermediate activity ||• Start at 30–70% of target dose and titrate every 2–4 weeks with close clinical monitoring of tolerability, eg, white blood cell counts and liver function tests. || |
| || ||3A/*3A, *2/*3A, *3C/*3A, *3C/*4, *3C/*2, *3A/*4 ||Low activity || |
• Malignant disease: Drastic reduction of thiopurine doses, eg, tenfold given thrice weekly instead of daily.
• Nonmalignant conditions: Alternative non-thiopurine immunosuppressive agent.
|G6PDX-linked trait || ||Genotype-to-phenotype predictions limited to males and homozygous females. |
| ||Rasburicase ||B, A ||Normal ||• Standard dose. ||Drug label |
| || ||A- ||Moderate deficiency ||• Alternative agent: Rasburicase is contraindicated in patients with G6PD deficiency. || |
| || ||Mediterranean, Canton ||Severe deficiency ||• Alternative agent: Rasburicase is contraindicated in patients with G6PD deficiency. || |
| ||Simvastatin 40 mg ||*1/*1 ||Normal activity ||• Standard dose. ||CPIC |
| || ||*1/*5, *1/[*15,*16, or *17] ||Intermediate activity ||• Consider a lower dose and routine CK monitoring or alternative statin. || |
| || ||*5/*5, *5/[*15, *16, or *17], [*15,*16, or *17]/[*15*16, or *17] ||Low activity ||• Prescribe a lower dose and consider routine CK monitoring or an alternative statin. || |
| ||Abacavir ||*Other/*Other ||Negative ||• Standard dose. ||CPIC |
|IFNL3 || || || || || |
| || ||*Other/*57:01, *57:01/*57:01 ||Positive ||• Different agent is recommended. || |
| ||PEG-IFN-α/RBV ||rs12979860/rs12979860 ||Favorable ||• Consider cure rates before initiating regimen: ∼70% chance for SVR4 after 48 weeks of therapy. ||CPIC |
| || ||Reference/rs12979860 ||Unfavorable ||• Consider cure rates before initiating regimen: ∼30% chance for SVR after 48 weeks of therapy. || |
| ||PEG-IFN-α/RBV + protease inhibitor ||rs12979860/rs12979860 ||Favorable ||• Regimen recommended: ∼90% chance for SVR after 24–48 weeks of therapy, with 80–90% chance for shortened duration of therapy. || |
| || ||Reference/reference or reference/rs12979860 ||Unfavorable ||• Consider cure rates before initiating regimen: ∼60% chance for SVR after 24–48 weeks of therapy, with 50% chance for shortened duration of therapy. || |
|CYP2C9, VKORC1 |
| ||Warfarin ||*1/*1, *1/*2, *2/*2, *2/*3, *1/*3, *3/*3, -1639GG, -1639GA, -1639AA ||Various ||• Apply validated dosing algorithm, eg, www.warfarindosing.org (or IWPC5) for International Normalized Ratio target 2–3) or FDA-approved dosing table per manufacturer’s labeling. ||CPIC |
Glossary |Favorite Table|Download (.pdf) Glossary
|Term ||Definition |
|Allele ||One of two or more alternative forms of a gene that arise by mutation and are found at the same genetic locus. Example: CYP2D6*3 is an important variant allele for a drug-metabolizing enzyme, CYP2D6. |
|Allele frequency ||The fraction or percentage of times a specific allele is observed in proportion to the total of all possible alleles that could occur at a specific location on a chromosome. |
|Coding single nucleotidepolymorphisms (cSNPs) ||Base-pair substitutions that occur in the coding region. |
|Copy number variations (CNVs) ||A segment of DNA in which a variable number of that segment has been found. |
|Haplotype ||A series of alleles found in a linked locus on a chromosome. |
|Hardy-Weinberg equilibrium ||The principle that allele frequencies will remain constant from generation to generation in the absence of evolutionary influences. |
|Insertions/deletion (indel) ||Insertion or deletion of base pairs, which may occur in coding and non-coding regions. |
|Linkage disequilibrium ||The non-random association of alleles at two or more loci that descend from a single ancestral chromosome. |
|Non-coding region polymorphism ||Polymorphisms that occur in the 3’ and 5’ untranslated regions, intronic regions, or intergenic regions. |
|Non-synonymous SNPs (nsSNPs) ||Base-pair substitutions in the coding region that result in an amino acid change. |
|Polymorphism or variant ||Any genetic variation in the DNA sequence; the terms can be used interchangeably. |
|PM, IM, EM, or UM ||Poor, intermediate, extensive, or ultra rapid metabolizer phenotype. |
|SNPs ||Single nucleotide polymorphisms: base-pair substitutions that occur in the genome. |
|Synonymous SNPs ||Base-pair substitutions in the coding region that do not result in an amino acid change. |
Example: Codeine is a phenanthrene derivative prodrug opioid analgesic indicated for the management of mild to moderately severe pain (Chapter 31). Codeine, like its active metabolite morphine, binds to μ-opioid receptors in the central nervous system (CNS). Morphine is 200 times more potent as an agonist than codeine, and conversion of codeine into morphine is essential for codeine’s analgesic activity. The enzyme responsible for the O-demethylation conversion of codeine into morphine is CYP2D6. Patients with normal CYP2D6 activity (ie, EMs) convert sufficient codeine to morphine (∼5–10% of an administered dose) to produce the desired analgesic effect. PMs and IMs are more likely to experience insufficient pain relief, while UMs are at an increased risk for side effects, eg, drowsiness and respiratory depression, due to higher systemic concentrations of morphine. Interestingly, gastrointestinal adverse effects, eg, constipation, are decreased in PMs, whereas the central side effects, eg, sedation and dizziness, do not differ between PMs and EMs. The antitussive properties associated with codeine are not affected by CYP2D6 activity. According to CPIC guidelines, standard starting doses are recommended in EMs and IMs with close monitoring, especially in IMs; and CPIC recommends use of an alternative agent in PMs and UMs (see Table 5–2).
Cytochrome P450 CYP2C19 is known to preferentially metabolize acidic drugs including proton-pump inhibitors, antidepressants, antiepileptics, and antiplatelet drugs (Chapter 4). Four clinical phenotypes related to CYP2C19 activity (PM, IM, EM, and UM) are closely associated with genetic biomarkers that may assist in guiding individualized therapeutic dosing strategies. The gene that encodes CYP2C19 is highly polymorphic, with over 30 alleles defined (www.cypalleles.ki.se/cyp2C19.htm), yet just four alleles can account for the majority of phenotypic variability, ie, CYP2C19 allele *2 and *3 are non-functional, CYP2C19 allele *1 is fully functional, and CYP2C19*17 has increased function. Phenotypes range from PMs who have two deficient alleles, eg, *2/*3, *2/*2, or *3/*3, to UMs who have increased hepatic expression levels of the CYP2C19 protein, due to *1/*17 or *17/*17 alleles (see Table 5–2). Of note, the *17 increased function allele is unable to fully compensate for non-functional alleles, and therefore, the presence of a *17 allele in combination with a non-functional allele would be considered an IM phenotype (see Table 5–2). The PM phenotype is more common in Asians (∼16%) than in Europeans and Africans (∼2–5%), which can be expected based on the inheritance patterns of variant alleles across populations, eg, the most common non-functional allele, ie, CYP2C19*2, is observed approximately twice as frequently in Asians (∼30%) compared with Africans and Europeans (∼15%), while the apparent gain-of-function *17 allele is observed rarely in Asians (<3%) but more frequently in Europeans and Africans (16–21%) (see Table 5–1).
Example: Clopidogrel is a thienopyridine antiplatelet prodrug indicated for the prevention of atherothrombotic events. Active metabolites selectively and irreversibly inhibit adenosine diphosphate-induced platelet aggregation (Chapter 34). Clopidogrel is metabolized in the body via one of two main mechanisms; approximately 85% of an administered dose is rapidly hydrolyzed by hepatic esterases to its inactive carboxylic acid derivative, while the remaining ∼15% is converted via two sequential CYP-mediated oxidation reactions (predominantly CYP2C19) to the active thiol metabolite responsible for antiplatelet activity.
Genetic polymorphisms in the CYP2C19 gene that decrease active metabolite formation and consequently reduce the drug’s antiplatelet activity are associated with variability in response to clopidogrel. Carriers of the reduced function CYP2C19 *2 alleles taking clopidogrel are at increased risk for serious adverse cardiovascular events, particularly in acute coronary syndrome managed with percutaneous coronary intervention (PCI); the hazard ratios (HR) are 1.76 for *2/*2 genotype and 1.55 for *2 heterozygotes compared to noncarriers. The risk associated with stent thrombosis is even greater (HR 3.97 for *2/*2 genotype and 1.55 for *2 heterozygotes compared to noncarriers). However, for other indications, eg, atrial fibrillation and stroke, the effects of the CYP2C19*2 allele are less dramatic. Thus, current clinical recommendations from CPIC are specific for acute coronary syndrome with PCI: Standard starting doses are recommended in EMs and UMs, and CPIC recommends use of an alternative antiplatelet agent, eg, prasugrel or ticagrelor, in PMs and IMs (Table 5–2).
Dihydropyrimidine Dehydrogenase (DPD)
Dihydropyrimidine dehydrogenase (DPD, encoded by the DPYD gene) is the first and rate-limiting step in pyrimidine catabolism, as well as a major elimination route for fluoropyrimidine chemotherapy agents (Chapter 54). Considerable intergroup and intragroup variation exists in DPD enzyme activity. Many of the alleles identified in the DYPD gene either are too rare to sufficiently characterize or have shown conflicting associations with DPD activity. Three non-functional alleles have been identified, ie, DPYD *2A, *13, and rs67376798. All three of these variants are rare; however, the *2A allele is the most commonly observed allele and is often the only variant tested in commercial genotyping platforms (see National Institutes of Health Genetic Testing Registry, http://www.ncbi.nlm.nih.gov/gtr/conditions/C2720286/ or http://www.ncbi.nlm.nih.gov/gtr/conditions/CN077983/). Frequencies of the *2A allele range from less than 0.005 in most European, African, and Asian populations to 3.5% in a Swedish population (see Table 5–1).
Example: Three fluoropyrimidine drugs are used clinically, namely 5-fluorouracil (5-FU), capecitabine, and tegafur (only approved in Europe). 5-FU is the pharmacologically active compound of each drug and all are approved to treat solid tumors including colorectal and breast cancer (Chapter 54). 5-FU must be administered intravenously, while both capecitabine and tegafur are oral prodrugs that are rapidly converted to 5-FU in the body. Only 1–3% of an administered dose of the prodrug is converted to the active cytotoxic metabolites, ie, 5-fluorouridine 5′-monophosphate (5-FUMP) and 5-fluoro-2′-deoxyuridine-5′-monophosphate (5-FdUMP), which effectively target rapidly dividing cancer cells and inhibit DNA synthesis. The majority of an administered dose (∼80%) is subjected to pyrimidine catabolism via DPD and is excreted in the urine. Complete or partial deficiency of DPD can lead to dramatically reduced clearances of 5-FU, increased half-life of toxic metabolites F-UMP and F-dUMP, and consequently an increased risk for severe dose-dependent fluoropyrimidine toxicities, eg, myelosuppression, mucositis, neurotoxicity, hand-and-foot syndrome, and diarrhea. CPIC recommendations for therapeutic regimens are shown in Table 5–2.
As described in Chapter 4, phase II enzyme biotransformation reactions typically conjugate endogenous molecules, eg, sulfuric acid, glucuronic acid, and acetic acid, onto a wide variety of substrates in order to enhance their elimination from the body. Consequently, polymorphic phase II enzymes may diminish drug elimination and increase risks for toxicities. In this section, we describe key examples of polymorphic phase II enzymes and the pharmacologic consequence for selected prescription drugs.
Uridine 5′-Diphosphoglucuronosyl Transferase 1 (UGT1A1)
The uridine 5′-diphospho- (UDP) glucuronosyltransferase 1A1 (UGT1A1) enzyme, encoded by the UGT1A1 gene, conjugates glucuronic acid onto small lipophilic molecules, eg, bilirubin and a wide variety of therapeutic drug substrates so that they may be more readily excreted into bile (Chapter 4). The UGT1A1 gene locus has over 30 defined alleles, some of which lead to reduced or completely abolished UGT1A1 function. Most reduced function polymorphisms within the UGT1A1 gene locus are quite rare; however, the *28 allele is common across three major ethnic groups (Table 5–1). Approximately 10% of European populations are homozygous carriers of the *28 allele, ie, UGT1A1 *28/*28 genotype, and are recognized clinically to have Gilbert’s syndrome. The *28 allele is characterized by an extra TA repeated in the proximal promoter region and is associated with reduced expression of the UGT1A1 enzyme. Clinically, Gilbert’s syndrome is generally benign; however, affected individuals may have 60–70% increased levels of circulating unconjugated bilirubin due to a ∼30% reduction in UGT1A1 activity. Individuals with the UGT1A1*28/*28 genotype are thus at an increased risk for adverse drug reactions with UGT1A1 drug substrates due to reduced biliary elimination.
Example: Irinotecan is a topoisomerase 1 inhibitor prodrug and is indicated as first-line chemotherapy in combination with 5-FU and leucovorin for treatment of metastatic carcinoma of the colon or rectum (Chapter 54). Irinotecan is hydrolyzed by hepatic carboxylesterase enzymes to its cytotoxic metabolite, SN-38, which inhibits topoisomerase 1 and eventually leads to termination of DNA replication and cell death. The active SN-38 metabolite is responsible for the majority of therapeutic action as well as the dose-limiting bone marrow and gastrointestinal toxicities. Inactivation of SN-38 occurs via the polymorphic UGT1A1 enzyme and carriers of the UGT1A1*28 variant are consequently at increased risk for severe life-threatening toxicities, eg, neutropenia and diarrhea, due to decreased clearance of SN-38 metabolites (see the Case Study at the beginning of this chapter).
Thiopurine S-Methyltransferase (TPMT)
Thiopurine S-methyltransferase (TPMT) covalently attaches a methyl group onto aromatic and heterocyclic sulfhydryl compounds and is responsible for the pharmacologic deactivation of thiopurine drugs (Chapter 4). While the majority (86–97%) of the population inherits two functional TPMT alleles and has high TPMT activity, around 10% of Europeans and Africans inherit only one functional allele and are considered to have intermediate activity. Furthermore, about 0.3% of Europeans inherit two defective alleles and have very low to no TPMT activity (Table 5–1). Genetic polymorphisms in the gene encoding TPMT may lead to three clinical TPMT activity phenotypes, ie, high, intermediate, and low activity, which are associated with differing rates of inactivation of thiopurine drugs and altered risks for toxicities. Over 90% of the phenotypic TPMT variability across populations can be accounted for with just three point mutations that are defined by four non-functional alleles, ie, TPMT*2, *3A, *3B, and *3C (Table 5–2). Most commercial genotyping platforms test for these four common genetic biomarkers and are therefore able to identify individuals with reduced TPMT activity.
Example: Three thiopurine drugs are used clinically, ie, azathioprine, 6-mercaptopurine (6-MP), and 6-thioguanine (6-TG). All share similar metabolic pathways and pharmacology. Azathioprine (a prodrug of 6-MP) and 6-MP are used for treating immunologic disorders, while 6-MP and 6-TG are important anti-cancer agents (Chapter 54). 6-MP and 6-TG may be activated by the salvage pathway enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) to form 6-thioguanine nucleotides (TGNs), which are responsible for the majority of therapeutic efficacy as well as bone marrow toxicity. Alternatively, 6-MP and 6-TG may be inactivated by enzymes such as polymorphic TPMT and xanthine oxidase, leaving less available substrate to be activated by HGPRTase. TPMT is a major determinant of thiopurine metabolism and exposure to cytotoxic 6-TGN metabolites and thiopurine-related toxicities. See Table 5–2 for recommended dosing strategies.
Glucose 6-phosphate dehydrogenase (G6PD) is the first and rate-limiting step in the pentose phosphate pathway and supplies a significant amount of reduced NADPH in the body. In red blood cells (RBCs), where mitochondria are absent, G6PD is the exclusive source of NADPH and reduced glutathione, which play a critical role in the prevention of oxidative damage. Under normal conditions, G6PD in RBCs is able to detoxify unstable oxygen species while working at just 2% of its theoretical capacity. Following exposure to exogenous oxidative stressors, eg, infection, fava beans, and certain therapeutic drugs, G6PD activity in RBCs increases proportionately to meet NADPH demands and ultimately to protect hemoglobin from oxidation. Individuals with G6PD deficiency, defined as less than 60% enzyme activity, according to World Health Organization classification (Table 5–3), are at increased risk for abnormal RBC destruction, ie, hemolysis, due to reduced antioxidant capacity under oxidative pressures.
The gene that encodes the G6PD enzyme is located on the X chromosome and is highly polymorphic, with over 180 genetic variants identified that result in enzyme deficiency. Greater than 90% of variants are single-base substitutions in the coding region that produce amino acid changes, which result in unstable proteins with reduced enzyme activity. As with most X-linked traits, males with one reference X chromosome and females with two reference X chromosomes will have equivalent “normal” G6PD activity. Similarly, hemizygous-deficient males (with a deficient copy of the G6PD gene on their single X chromosome) and homozygous-deficient females (with two deficient copies) express reduced activity phenotypes (Table 5–1). However, for heterozygous females (with one deficient allele and one normal allele), genotype-to-phenotype predictions are less reliable due to the X-chromosome mosaicism, ie, where one X chromosome in each female cell is randomly inactivated, leading to G6PD activity that may range from fully functional to severely deficient. G6PD enzyme activity phenotype estimations for heterozygous females therefore may be improved with complementary G6PD activity testing.
G6PD enzyme deficiency affects over 400 million people worldwide, and the World Health Organization has categorized G6PD activity into five classes (Table 5–3). The majority of polymorphic G6PD-deficient genotypes are associated with class II for severe deficiency (<10% enzyme activity) and class III for moderate deficiency (10–60% enzyme activity). Most individuals with reduced function alleles of G6PD have ancestries in geographical areas of the world corresponding to areas with high malaria prevalence. Polymorphic alleles gained in frequency over time as they offered some benefit against death from malaria. The estimated frequency of G6PD deficiency is approximately 8% in malaria endemic countries, with the milder G6PD-A(–) allele prevalent in Africa, and the more severe G6PD-Mediterranean allele widespread across western Asia (Saudi Arabia and Turkey to India). There is a much more heterogeneous distribution of variant alleles in East Asia and the Asia Pacific, which complicates G6PD risk predictions; however, the most frequently identified forms in Asia include the more severe class II alleles, eg, Mediterranean, Kaiping, and Canton, as well as some class III alleles, eg, Mahidol, Chinese-5, and Gaohe (Table 5–1).
TABLE 5–3Classification of G6PD deficiency (WHO Working Group, 1989). |Favorite Table|Download (.pdf) TABLE 5–3Classification of G6PD deficiency (WHO Working Group, 1989).
|World Health Organization Class ||Level of Deficiency ||Enzyme Activity ||Clinical phenotype |
|I ||Severe ||<10% ||Chronic (non-spherocytic) hemolytic anemia |
|II ||Severe ||<10% ||Risk of acute hemolytic anemia; intermittent hemolysis |
|III ||Moderate ||10–60% ||Risk of acute hemolytic anemia; hemolysis with stressors |
|IV ||None ||60–150% ||Normal |
|V ||None ||>150% ||Enhanced activity |
Example: Rasburicase, a recombinant urate-oxidase enzyme, is indicated for the initial management of uric acid levels in cancer patients receiving chemotherapy. Rasburicase alleviates the uric acid burden that often accompanies tumor-lysing treatments by converting uric acid into allantoin, a more soluble and easily excreted molecule. During the enzymatic conversion of uric acid to allantoin, hydrogen peroxide, a highly reactive oxidant, is formed. Hydrogen peroxide must be reduced by glutathione to prevent free radical formation and oxidative damage. Individuals with G6PD deficiency receiving rasburicase therapy are at greatly increased risk for severe hemolytic anemia and methemoglobinemia. The manufacturer recommends that patients at high risk (individuals of African or Mediterranean ancestry) be screened prior to the initiation of therapy and that rasburicase not be used in patients with G6PD deficiency (Table 5–2).