Principles of Genetic Variation and Human Traits
(See also Chaps. 61 and 63) The concept that genetically determined variations in drug metabolism might be associated with variable drug levels and hence, effect, was advanced at the end of the nineteenth century, and the examples of familial clustering of unusual drug responses were noted in the mid-twentieth century. Variants in the human genome resulting in variation in level of expression or function of molecules important for pharmacokinetics and pharmacodynamics are increasingly recognized. These may be mutations (very rare variants, often associated with disease) or polymorphisms, variants that are much more common in a population. Variants may occur at a single nucleotide [single nucleotide polymorphisms (SNPs)] or involve insertion or deletion of one or more nucleotides, occasionally up to thousands. They may be in the exons (coding regions), introns (noncoding intervening sequences), or intergenic regions. Exonic polymorphisms may or may not alter the encoded protein, and variant proteins may or may not display altered function. Similarly, polymorphisms in noncoding regions may or may not alter gene expression and protein level.
As variation in the human genome is increasingly well documented, associations are being described between polymorphisms and various traits (including response to drug therapy). Some of these rely on well-developed chains of evidence, including in vitro studies demonstrating variant protein function, familial aggregation of the variant allele with the trait, and association studies in large populations. In other cases, the associations are less compelling. Identifying replicated associations with important clinical consequences is a challenge that must be overcome before the concept of genotyping to identify optimal drugs (or dosages) in individual patients prior to prescribing can be considered for widespread clinical practice.
Rates of drug efficacy and adverse effects often vary among ethnic groups. Many explanations for such differences are plausible; genomic approaches have now established one mechanism that functionally important variants determining differences in drug response often display differing distributions among ethnic groups. This finding may have importance for drug use among ethnic groups, as well as in drug development.
Approaches to Identifying Genetic Variants Modulating Drug Action
A goal of traditional Mendelian genetics is to identify DNA variants associated with a distinct phenotype in multiple related family members (Chap. 63). The usual approach, linkage analysis, does not generally lend itself to identifying genetic variants contributing to variable drug actions, because it is unusual for a drug response phenotype to be accurately measured in more than one family member, let alone across a kindred. Thus, alternate approaches are used to identify and validate DNA variants contributing to variable drug actions.
Most studies to date have used an understanding of the molecular mechanisms modulating drug action to identify candidate genes in which variants could explain variable drug responses. One very common scenario is that variable drug actions can be attributed to variability in plasma drug concentrations. When plasma drug concentrations vary widely (e.g., more than an order of magnitude), especially if their distribution is nonunimodal as in Fig. 5-6, variants in single genes controlling drug concentrations often contribute. In this case, the most obvious candidate genes are those responsible for drug metabolism and elimination. Other candidate genes are those encoding the target molecules with which drugs interact to produce their effects or molecules modulating that response, including those involved in disease pathogenesis.
A. CYP2D6 metabolic activity was assessed in 290 subjects by administration of a test dose of a probe substrate and measurement of urinary formation of the CYP2D6-generated metabolite. The heavy arrow indicates a clear antimode, separating poor metabolizer subjects (PMs, green), with two loss-of-function CYP2D6 alleles, indicated by the intron-exon structures below the bar chart. Individuals with one or two functional alleles are grouped together as extensive metabolizers (EMs, blue). Also shown are ultra-rapid metabolizers (UMs), with 2–12 functional copies of the gene (red), displaying the greatest enzyme activity. (Adapted by permission from M-L Dahl et al: J Pharmacol Exp Ther 274:516, 1995.) B. These simulations show the predicted effects of CYP2D6 genotype on disposi-tion of a substrate drug. With a single dose (left), there is an inverse "gene-dose" relationship between the number of active alleles and the areas under the time-concentration curves (smallest in UM subjects; highest in PM subjects); this indicates that clearance is greatest in UM subjects. In addition, elimination half-life is longest in PM subjects. The right panel shows that these single dose differences are exaggerated during chronic therapy: steady-state concentration is much higher in PM subjects (decreased clearance), as is the time required to achieve steady state (longer elimination half-life).
The field has also had some success with "unbiased" approaches such as genome-wide association (GWA) (Chap. 61). GWA makes no a priori assumptions about the genetic loci modulating variable drug response and, instead, searches across the whole genome in an "unbiased fashion" to identify loci linked to variable drug response.
Genetically Determined Drug Disposition
and Variable Effects
Clinically important genetic variants have been described in multiple molecular pathways of drug disposition (Table 5–2). A distinct multimodal distribution of drug disposition (as shown in Fig. 5-6) argues for a predominant effect of variants in a single gene in the metabolism of that substrate. Individuals with two alleles (variants) encoding for nonfunctional protein make up one group, often termed poor metabolizers (PM phenotype); many variants can produce such a loss of function, complicating the use of genotyping in clinical practice. Individuals with one functional allele make up a second (intermediate metabolizers) and may or may not be distinguishable from those with two functional alleles (extensive metabolizers, EMs). Ultra-rapid metabolizers with especially high enzymatic activity (occasionally due to gene duplication; Fig. 5-6) have also been described for some traits. Many drugs in widespread use can inhibit specific drug disposition pathways (Table 5–1), and so EM individuals receiving such inhibitors can respond like PM patients (phenocopying). Polymorphisms in genes encoding drug uptake or drug efflux transporters may be other contributors to variability in drug delivery to target sites and, hence, in drug effects.
Table 5–2 Genetic Variants and Drug Responses |Favorite Table|Download (.pdf)
Table 5–2 Genetic Variants and Drug Responses
|Gene||Drugs||Effect of genetic variants*|
|Variants in drug metabolism pathways|
Decreased bioactivation and effects (PMs)
|Warfarin||Decreased dose requirements; possible increased bleeding risk (PMs)|
|CYP2C19||Omeprazole, voriconazole||Decreased effect in extensive metabolizers (EMs)|
|Celecoxib||Exaggerated effect in PMs|
|Clopidogrel||Decreased effect in PMs|
|CYP2D6||Codeine, tamoxifen||Decreased bioactivation and drug effects in PMs|
|Codeine||Morphine-like adverse effects in UMs|
|Tricyclic antidepressants||Increased adverse effects in PMs; decreased therapeutic effects in UMs|
|Metoprolol, carvedilol, timolol, propafenone||Increased beta blockade in PMs|
|Dihydropyrimidine dehydrogenase||Capecitabine, fluorouracil||Possible severe toxicity (PMs)|
|NAT2||Rifampin, isoniazid, pyrazinamide, hydralazine, procainamide||Increased risk of toxicity in PMs|
Thiopurine S-methyltransferase (TPMT)
||Azathioprine, 6-mercaptopurine||*3A/*3A (PMs): increased risk of bone marrow aplasia; wild-type homozygote: possible decreased drug action at usual dosages|
Uridine diphosphate glucuronosyltransferase (UGT1A1)
||Irinotecan||*28/*28 PM homozygotes: increased risk of severe adverse effects (diarrhea, bone marrow aplasia)|
Variants in other genes|
|Glucose 6-phosphate dehydrogenase (G6PD)
||Rasburicase, primaquine, chloroquine||Increased risk of hemolytic anemia in G6PD-deficient subjects|
Carriers (1 or 2 alleles) at increased risk of severe skin toxicity
|HLA-B*5701||Abacavir||Carriers (1 or 2 alleles) at increased risk of severe skin toxicity|
|IL28B||Interferon||Variable response in hepatitis C therapy|
|IL15||Childhood leukemia therapy||Variability in response|
|SLCO1B1||Simvastatin||Variant non-synonymous single nucleotide poly-morphism increases myopathy risk|
Decreased dose requirements with variant pro-moter haplotype|
Variants in other genomes (infectious agents, tumors)
|Chemokine C-C motif receptor (CCR5)||Maraviroc||
Drug effective only in HIV strains with CCR5 detectible
|C-KIT||Imatinib||In gastrointestinal stromal tumors, drug indicated only with c-kit– positive cases|
Epidermal Growth Factor Receptor (EGFR)
||Cetuximab||Clinical trials conducted in patients with EGFR-positive tumors
|Her2/neu overexpression||Trastuzumab, lapatinib||Drugs indicated only with tumor overexpression|
|K-ras mutation||Panitumumab, cetuximab||
Lack of efficacy with KRAS mutation
|Philadelphia chromosome||Busulfan, dasatinib, nilotinib, imatinib||Decreased efficacy in Philadelphia chromosome–negative chronic myelogenous leukemia|
CYP3A4 is the most abundant hepatic and intestinal CYP and is also the enzyme responsible for metabolism of the greatest number of drugs in therapeutic use. CYP3A4 activity is highly variable (up to an order of magnitude) among individuals, but the underlying mechanisms are not yet well understood. A closely related gene, encoding CYP3A5 (which shares substrates with CYP3A4), does display loss-of-function variants, especially in African populations. CYP3A refers to both enzymes.
CYP2D6 is second to CYP3A4 in the number of commonly used drugs that it metabolizes. CYP2D6 activity is polymorphically distributed, with about 7% of European- and African-derived populations (but very few Asians) displaying the PM phenotype (Fig. 5-6). Dozens of loss-of-function variants in the CYP2D6 gene have been described; the PM phenotype arises in individuals with two such alleles. In addition, ultra-rapid metabolizers with multiple functional copies of the CYP2D6 gene have been identified, particularly in Ethiopian, Eritrean, and Saudi individuals.
Codeine is biotransformed by CYP2D6 to the potent active metabolite morphine, so its effects are blunted in PMs and exaggerated in ultra-rapid metabolizers. In the case of drugs with beta-blocking properties metabolized by CYP2D6, greater signs of beta blockade (e.g., bradycardia) are seen in PM subjects than in EMs. This can be seen not only with orally administered beta blockers such as metoprolol and carvedilol, but also with ophthalmic timolol and with the sodium channel–blocking antiarrhythmic propafenone, a CYP2D6 substrate with beta-blocking properties. Further, in EM subjects, propafenone elimination becomes zero-order at higher doses; so, for example, a tripling of the dose may lead to a tenfold increase in drug concentration. Ultra-rapid metabolizers may require very high dosages of tricyclic antidepressants to achieve a therapeutic effect and, with codeine, may display transient euphoria and nausea due to very rapid generation of morphine. Tamoxifen undergoes CYP2D6-mediated biotransformation to an active metabolite, so its efficacy may be in part related to this polymorphism. In addition, the widespread use of selective serotonin reuptake inhibitors (SSRIs) to treat tamoxifen-related hot flashes may also alter the drug's effects because many SSRIs, notably fluoxetine and paroxetine, are also CYP2D6 inhibitors.
The PM phenotype for CYP2C19 is common (20%) among Asians and rarer (3–5%) in European-derived populations. The impact of polymorphic CYP2C19-mediated metabolism has been demonstrated with the proton pump inhibitor omeprazole, where ulcer cure rates with "standard" dosages were markedly lower in EM patients (29%) than in PMs (100%). Thus, understanding the importance of this polymorphism would have been important in developing the drug, and knowing a patient's CYP2C19 genotype should improve therapy. CYP2C19 is responsible for bioactivation of the antiplatelet drug clopidogrel, and several large studies have documented decreased efficacy (e.g., increased myocardial infarction after placement of coronary stents) among Caucasian subjects with reduction of function alleles. In addition, some studies suggest that omeprazole and possibly other proton inhibitors phenocopy this effect.
There are common allelic variants of CYP2C9 that encode proteins with loss of catalytic function. These variant alleles are associated with increased rates of neurologic complications with phenytoin, hypoglycemia with glipizide, and reduced warfarin dose required to maintain stable anticoagulation (discussed further below). The angiotensin-receptor blocker losartan is a prodrug that is bioactivated by CYP2C9; as a result, PMs and those receiving inhibitor drugs may display little response to therapy.
One of the most extensively studied phase II polymorphisms is the PM trait for thiopurine S-methyltransferase (TPMT). TPMT bioinactivates the antileukemic drug 6-mercaptopurine. Further, 6-mercaptopurine is itself an active metabolite of the immunosuppressive azathioprine. Homozygotes for alleles encoding the inactive TPMT (1 in 300 individuals) predictably exhibit severe and potentially fatal pancytopenia on standard doses of azathioprine or 6-mercaptopurine. On the other hand, homozygotes for fully functional alleles may display less anti-inflammatory or antileukemic effect with the drugs.
N-acetylation is catalyzed by hepatic N-acetyl transferase (NAT), which represents the activity of two genes, NAT-1 and NAT-2. Both enzymes transfer an acetyl group from acetyl coenzyme A to the drug; NAT-1 activity is generally constant, while polymorphisms in NAT-2 result in individual differences in the rate at which drugs are acetylated and thus define "rapid acetylators" and "slow acetylators." Slow acetylators make up ˜50% of European- and African-derived populations but are less common among Asians.
Slow acetylators have an increased incidence of the drug-induced lupus syndrome during procainamide and hydralazine therapy and of hepatitis with isoniazid. Induction of CYPs (e.g., by rifampin) also increases the risk of isoniazid-related hepatitis, likely reflecting generation of reactive metabolites of acetylhydrazine, itself an isoniazid metabolite.
Individuals homozygous for a common promoter polymorphism that reduces transcription of uridine diphosphate glucuronosyltransferase (UGT1A1) have benign hyperbilirubinemia (Gilbert's syndrome; Chap. 302). This variant has also been associated with diarrhea and increased bone marrow depression with the antineoplastic prodrug irinotecan, whose active metabolite is normally detoxified by UGT1A1-mediated glucuronidation.
Variability in the Molecular Targets with Which Drugs Interact
Multiple polymorphisms identified in the β2-adrenergic receptor appear to be linked to specific phenotypes in asthma and congestive heart failure, diseases in which β2-receptor function might be expected to determine prognosis. Polymorphisms in the β2-receptor gene have also been associated with response to inhaled β2-receptor agonists, while those in the β1-adrenergic receptor gene have been associated with variability in heart rate slowing and blood pressure lowering (Fig. 5-5B). In addition, in heart failure, a common polymorphism in the β1-adrenergic receptor gene has been implicated in variable clinical outcome during therapy with the investigational beta blocker bucindolol. Response to the 5-lipoxygenase inhibitor zileuton in asthma has been linked to polymorphisms that determine the expression level of the 5-lipoxygenase gene.
Drugs may also interact with genetic pathways of disease to elicit or exacerbate symptoms of the underlying conditions. In the porphyrias, CYP inducers are thought to increase the activity of enzymes proximal to the deficient enzyme, exacerbating or triggering attacks (Chap. 358). Deficiency of glucose-6-phosphate dehydrogenase (G6PD), most often in individuals of African, Mediterranean, or South Asian descent, increases risk of hemolytic anemia in response to primaquine and a number of other drugs that do not cause hemolysis in patients with normal amounts of the enzyme (Chap. 106). Patients with mutations in the ryanodine receptor, which controls intracellular calcium in skeletal muscle and other tissues, may be asymptomatic until exposed to certain general anesthetics, which trigger the syndrome of malignant hyperthermia. Certain antiarrhythmics and other drugs can produce marked QT prolongation and torsades des pointes (Chap. 233), and in some patients this adverse effect represents unmasking of previously subclinical congenital long QT syndrome.
Tumor and Infectious Agent Genomes
The actions of drugs used to treat infectious or neoplastic disease may be modulated by variants in these non-human germline genomes. Genotyping tumors is a rapidly evolving approach to target therapies to underlying mechanisms and to avoid potentially toxic therapy in patients who would derive no benefit (Chap. 83). Trastuzumab, which potentiates anthracycline-related cardiotoxicity, is ineffective in breast cancers that do not express the herceptin receptor. Imatinib targets a specific tyrosine kinase, BCR-Abl1, that is generated by the translocation that creates the Philadelphia chromosome typical of chronic myelogenous leukemia (CML). BCR-Abl1 is not only active but may be central to the pathogenesis of CML; its use in BCR-Abl1-positive tumors has resulted in remarkable antitumor efficacy. Similarly, the antiepidermal growth factor (EGFR) antibodies cetuximab and panitumumab appear especially effective in colon cancers in which K-ras, a G-protein in the EGFR pathway, is not mutated.
Polymorphisms that Modulate the Biologic Context Within Which the Drug-Target Interactions Occur
The interaction of a drug with its molecular target is translated into a clinical action in a complex biologic milieu that is itself often perturbed by disease. Thus, polymorphisms that determine variability in this biology may profoundly influence drug response, although the genes involved are not themselves directly targets of drug action. Polymorphisms in genes important for lipid homeostasis (such as the ABCA1 transporter and the cholesterol ester transport protein) modulate response to 3-hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors, "statins." In one large study, the combination of diuretic use combined with a variant in the adducin gene (encoding a cytoskeletal protein important for renal tubular sodium absorption) decreased stroke or myocardial infarction risk, while neither factor alone had an effect. Common polymorphisms in ion channel genes that are not themselves the target of QT-prolonging drugs may nevertheless influence the extent to which those drugs affect the electrocardiogram and produce arrhythmias. Severe skin rashes during treatment with the anticonvulsant carbamazepine and the antiretroviral abacavir have been associated with variants in the HLA gene cluster (Table 5–2).
Multiple Variants Modulating Drug Effects
Polymorphisms in multiple candidate genes can be associated with variability in the effect of a single drug. CYP2C9 loss-of-function variants are associated with a requirement for lower maintenance doses of the vitamin K antagonist anticoagulant warfarin. In rarer (<2%) individuals homozygous for these variant alleles, maintenance warfarin dosages may be difficult to establish, and the risk of bleeding complications appears increased. In addition to CYP2C9, multiple variants in the promoter region of VKORC1, encoding a vitamin K epoxide reductase (the warfarin target), predict warfarin dosages; these promoter variants are in tight linkage disequilibrium, i.e., genotyping at one polymorphic site within this haplotype block
provides reliable information on the identity of genotypes at other linked sites (Chap. 61).
Genome-Wide Association and Variable Drug Response
A GWA study was used to compare patients with simvastatin-associated myopathy to control tolerating the drugs and identified a single noncoding SNP in SLCO1B1, encoding OATP1B1, a drug transporter known to modulate simvastatin uptake into the liver. The SNP was in linkage disequilibrium with a known nonsynonymous SNP modulating OATP1B1 function and was estimated to account for 60% of myopathy risk. GWA approaches have also implicated interferon variants in antileukemic responses and in response to therapy in hepatitis C (Table 5–2).
Prospects for Incorporating Pharmacogenetic Information into Clinical Practice
The description of genetic variants linked to variable drug responses naturally raises the question of if and how to use this information in practice. Indeed, the U.S. Food and Drug Administration has begun incorporating pharmacogenetic data into information ("package inserts") meant to guide prescribing. A decision to adopt pharmacogenetically guided dosing for a given drug depends on multiple factors. The most important are the magnitude and clinical importance of the genetic effect and the strength of evidence linking genetic variation to variable drug effects (e.g., anecdote versus post-hoc analysis of clinical trial data versus randomized prospective clinical trial). The evidence can be strengthened if statistical arguments from clinical trial data are complemented by an understanding of underlying physiologic mechanisms. Cost versus expected benefit may also be a factor.
When the evidence is compelling and alternate therapies are not available, there is a strong argument for deploying genetic testing as a guide to prescribing. Examples include associations between UGT1A1 variants and irinotecan toxicity, or between HLA-B*5701 and severe skin toxicity with abacavir. In other situations, the arguments are less compelling: the magnitude of the genetic effect may be smaller, the consequences may be less serious, alternate therapies may be available, or the drug effect may be amenable to monitoring by other approaches. Ongoing clinical trials are addressing the utility of preprescription genotyping in large populations exposed to drugs with known pharmacogenetic variants (e.g., warfarin). Importantly, technological advances are now raising the possibility of inexpensive whole genome sequencing. Incorporating a patient's whole genome sequence into their electronic medical record would allow the information to be accessed as needed for many genetic and pharmacogenetic applications. There are multiple issues (e.g., economic, technological, and ethical) that need to be addressed if such a paradigm is to be adopted (Chap. 61). While barriers to bringing genomic and pharmacogenomic information to the bedside seem daunting, the field is very young and evolving rapidly. Indeed, one major result of understanding the role of genetics in drug action has been improved screening of drugs during the development process to reduce the likelihood of highly variable metabolism or unanticipated toxicity.