Chapter e5: Pharmacogenetics

Great variability exists among individuals in response to drug therapy, and it is difficult to predict how effective or safe a medication will be for a particular patient. For example, when treating a patient with hypertension, it may be necessary to try several agents or a combination of agents before achieving adequate blood pressure control with acceptable tolerability. A number of clinical factors are known to influence drug response, including age, body size, renal and hepatic function, and concomitant drug use. However, considering these factors alone is often insufficient in predicting the likelihood of drug efficacy or safety for a given patient. For example, identical antihypertensive therapy in two patients of similar age, sex, race, and with similar medical histories and concomitant drug therapy may produce inadequate blood pressure reduction in one patient and symptomatic hypotension in the other.

The observed interpatient variability in drug response may result largely from genetically determined differences in drug metabolism, drug distribution, and drug target proteins. The influence of heredity on drug response was demonstrated as early as 1956 with the discovery that an inherited deficiency of glucose-6-phosphate dehydrogenase (G6PD) was responsible for hemolytic reactions to the antimalarial drug primaquine. Variations in genes encoding cytochrome P450 (CYP) and other drug-metabolizing enzymes are now well recognized as causes of interindividual differences in plasma concentrations of certain drugs. These variations may have serious implications for narrow-therapeutic-index drugs such as warfarin, phenytoin, and mercaptopurine. Other variations associated with drug response occur in genes for drug transporters such as the solute carrier organic anion transporter (OAT) family member 1B1 (SLCO1B1) and organic cation transporter 1 (OCT1), as well as drug targets such as receptors, enzymes, and proteins involved in intracellular signal transduction. Genetic variations for drug-metabolizing enzymes and drug transporter proteins may influence drug disposition, thus altering pharmacokinetic drug properties. Drug target genes may alter pharmacodynamic mechanisms by affecting sensitivity to a drug at its target site. Finally, genes associated with disease severity have been correlated with drug efficacy despite having no direct effect on pharmacokinetic or pharmacodynamic mechanisms.

Pharmacogenetics involves the search for genetic variations that lead to interindividual differences in drug response. The term pharmacogenetics often is used interchangeably with the term pharmacogenomics. However, pharmacogenetics generally refers to monogenetic variants that affect drug response, whereas pharmacogenomics refers to the entire spectrum of genes that interact to determine drug efficacy and safety. For example, a pharmacogenetic study would be one that examines the influence of the CYP2C9 gene on warfarin dose requirements. A pharmacogenomic study might examine the interaction between the CYP2C9, vitamin K oxido reductase complex subunit 1 (VKORC1), and CYP4F2 genes on warfarin dose requirements. Given that multiple proteins are involved in determining the ultimate response to most drugs, many investigators are taking a more pharmacogenomic approach to elucidating genetic contributions to drug response. For simplicity, this chapter treats pharmacogenetics and pharmacogenomics as synonymous.

The goals of pharmacogenetics are to optimize drug therapy and limit drug toxicity based on an individual’s genetic profile. Thus, pharmacogenetics aims to use genetic information to choose a drug, drug dose, and treatment duration that will have the greatest likelihood for achieving therapeutic outcomes with the least potential for harm in a given patient. Pharmacogenetic discoveries have provided opportunities for clinicians to use genetic tests to predict individual responses to drug treatments and specifically select medications for patients based on DNA profiles. Genotype-guided therapy is already a reality for some diseases, such as cancer and cystic fibrosis, where novel drugs have been developed to target specific mutations. Clinical implementation of pharmacogenetics is beginning to emerge in other therapeutic areas, such as cardiology, neurology, pain management, and infectious disease.

Although there has been considerable interest in genetic influences of drug response over the past decade, pharmacogenetics is not a new area. As shown in Fig. e5-1, in 1957, shortly after the discovery of a genetic predisposition toward primaquine-induced toxicity, Arno Motulsky proposed that inheritance might underlie much of the disparity among individuals in drug response.^1,2 Friedrich Vogel introduced the term pharmacogenetics 2 years later.¹ With the advent of the Human Genome Project in 1990 came a resurgence of interest in determining genetic contributions to drug response.

In 1988, the US Congress commissioned the Department of Energy and the National Institutes of Health to plan and implement the Human Genome Project with the goal of sequencing the entire human genome by 2005. The mapping of the human genome officially began in 1990. In April 2003, 50 years after James Watson and Francis Crick described the double-helix structure of DNA and more than 2 years ahead of schedule, researchers announced the completion of the Human Genome Project.³ The final version contains 99% of the gene-containing sequence, with 99.9% accuracy.

To encourage research and ultimately maximize the societal benefits of the Human Genome Project, sequence data from the project were deposited into a freely accessible database run by the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).

The International HapMap Project followed the Human Genome Project and aimed to create a publicly accessible database of common patterns of heritability in the human genome (www.hapmap.org). The HapMap Project allows pharmacogenomic studies to extend beyond a candidate gene focus. Subsequent genome-wide association studies have led to discoveries of previously unsuspected genes linked to drug response, including the ataxia-telangiectasia mutated (ATM) gene linked to metformin response in diabetes.⁴ The 1,000 Genomes project followed the HapMap Project and led to the development of a comprehensive catalog of less common genetic variation through a DNA sequencing approach (www.1000genomes.org). The ENCyclopedia of DNA Elements (ENCODE) Project is developing a complementary catalogue of functional elements in the human genome.

In 2010, leaders of the National Institutes of Health (NIH) and US Food and Drug Administration (FDA) announced their shared vision of personalized medicine and outlined the scientific and regulatory structure necessary to address the challenges in advancing personalized medicine.⁵ These challenges involve the accurate, unbiased determination of genetic variants linked to drug response, advanced technology to efficiently determine genotype, discovery of novel genetic targets for therapeutic intervention, appropriate integration of genetic testing into the therapeutic decision process, and coordinated approval of drug therapy and companion diagnostics. The Pharmacogenomics Research Network (PGRN) was a major NIH-funded effort to advance personalized medicine. The PGRN consisted of multiple research groups across the country with complementary expertise and led to a number of important pharmacogenetic discoveries across multiple therapeutic areas.⁶ More recently, the NIH funded the Implementing Genomics in Practice (IGNITE) Network, with the goal of enhancing the use of genomic medicine in clinical care.

The human genome contains more than 3 billion nucleotide base pairs, which code for approximately 20,000 protein-coding genes. Two purine nucleotide bases, adenine (A) and guanine (G), and two pyrimidine nucleotide bases, cytosine (C) and thymidine (T), are present in DNA, with purines and pyrimidines always pairing together as A-T and C-G in the two strands that make up the DNA double-helix. Most nucleotide base pairs are identical from person to person, with only 0.1% contributing to individual differences.

According to the central dogma, when one strand of DNA is transcribed into RNA and translated to make proteins, three consecutive nucleotides form a codon. Each codon specifies an amino acid or amino acid chain termination. For example, the nucleotide sequence, or codon, GGA specifies the amino acid glycine. The genetic code has substantial redundancy, in that two or more codons code for the same amino acid. For example, GGC, GGG, and GGT also code for glycine. Amino acids are the basic constituents of proteins, which mediate all cellular functions. Only 20 different amino acids, in various arrangements, form the basic units of all the proteins in the human body.

A gene is a series of codons that specifies a particular protein. Genes contain several regions: exons that encode for the final protein, introns that consist of intervening noncoding regions, and regulatory regions that control gene transcription. Introns may also contain regulatory sequences. In most cases, an individual carries two alleles, one from each parent, at each gene locus. An allele is defined as the sequence of nucleic acid bases at a given gene chromosomal locus. Two identical alleles make up a homozygous genotype, and two different alleles make up a heterozygous genotype. A phenotype refers to the outward expression of the genotype.

Genetic variations occur as either rare defects or polymorphisms. Polymorphisms are defined as variations in the genome that occur at a frequency of at least 1% in the human population. For example, the genes encoding the CYP enzymes 2A6, 2C9, 2C19, 2D6, and 3A4 are polymorphic, with functional gene variants of greater than 1% occurring in different racial groups. In contrast, rare mutations occur in less than 1% of the population and cause inherited diseases such as cystic fibrosis, hemophilia, and Huntington’s disease. Common diseases, such as essential hypertension and diabetes mellitus, are polygenic in that multiple genetic polymorphisms in conjunction with environmental factors contribute to the disease susceptibility.

Single-nucleotide polymorphisms, abbreviated as SNPs and pronounced “snips,” are the most common genetic variations in human DNA, occurring once approximately every 300 base pairs. More than 20 million SNPs have been mapped thus far in the human genome. SNPs occur when one nucleotide base pair replaces another, as illustrated in Fig. e5-2. Thus, SNPs are single-base differences that exist between individuals. Nucleotide substitution results in two possible alleles. One allele, typically either the most commonly occurring allele or the allele originally sequenced, is considered the wild type, and the alternative allele is considered the variant allele.

A SNP may result in amino acid substitution, which may or may not alter the function of the encoded protein. For example, in Fig. e5-2, guanine (G) is substituted for adenine (A) at nucleotide 46 in the β₂-adrenergic receptor gene. This results in the substitution of glycine for arginine at amino acid position (codon) 16 and alterations in receptor downregulation on prolonged exposure to β₂-receptor agonists.⁷ SNPs such as this that result in amino acid substitution are referred to as nonsynonymous. SNPs that do not result in amino acid substitution are called synonymous, which in many cases are silent. Referring to a previous example of redundancy in the genetic code, replacement of adenine (A) with cytosine (C) in the codon GGA is an example of a synonymous SNP because both GGA and GGC code for glycine. Synonymous SNPs and variants occurring in regulatory regions of the gene are usually abbreviated based on the nucleotides involved and the nucleotide base position. For example, 1166A > C indicates that either adenine or cytosine may occur, with adenine occurring most often at position 1166 of a given gene region. Nonsynonymous SNPs usually are designated by the amino acids and codon involved. For example, Arg16Gly (or R16G using amino acid symbols) indicates that glycine may be substituted for arginine at codon 16. Alternatively, SNPs may be referred to by their reference SNP number (or rs number), as designated by the National Center for Biotechnology Information SNP database (dbSNP; http://www.ncbi.nlm.nih.gov/sites/entrez?db=Snp). If a SNP changes the amount or function of a protein that contributes to drug response, it may alter kinetic properties or a patient’s sensitivity to a drug or predispose a patient to adverse reactions to drug therapy.

Other examples of genetic variants include:

1. Insertion-deletion polymorphisms, in which a nucleotide or nucleotide sequence is either added to or deleted from a DNA sequence.

2. Tandem repeats, in which a nucleotide sequence repeats in tandem (eg, if “AG” is the nucleotide repeat unit, “AGAGAGAGAG” is a five-tandem repeat).

3. Frameshift mutation, in which there is an insertion/deletion polymorphism, and the number of nucleotides added or lost is not a multiple of 3, resulting in disruption of the gene’s reading frame.

4. Defective splicing, in which an internal polypeptide segment is abnormally removed, and the ends of the remaining polypeptide chain are joined.

5. Aberrant splice site, in which processing of the protein occurs at an alternate site.

6. Premature stop codon polymorphisms, in which there is premature termination of the polypeptide chain by a stop codon (specific sequence of three nucleotides that do not code for an amino acid but rather specify polypeptide chain termination).

7. Copy number variants, in which entire copies of genes or gene segments more than 1 kb in size are duplicated, deleted, or rearranged.

Single-nucleotide polymorphisms may occur in exon, intron, or regulatory regions of a gene. Those occurring in exons may alter protein function, whereas those in regulatory regions may alter gene expression and the amount of protein that is produced. Variations in the intron region may be silent unless they affect intron splicing or otherwise alter gene expression. Multiple SNPs may be in linkage disequilibrium with each other. This means that two or more SNPs are inherited together more frequently than would be expected based on chance alone. For example, if there are two possible SNPs, 46C > T and 72A > G, in a given gene, and a T at position 46 always occurs with a G at position 72 and vice versa, the two SNPs are said to be in complete linkage disequilibrium. A set of SNPs that are inherited together is called a haplotype.

Polymorphisms in the drug-metabolizing enzymes represent the first recognized and, so far, the most documented examples of genetic variants with consequences in drug response and toxicity. The major phase I enzymes are the CYP superfamily of isoenzymes. N-acetyltransferase, uridine diphosphate glucuronosyltransferase (UGT), and glutathione S-transferase are examples of phase II metabolizing enzymes that exhibit genetic polymorphisms. Thiopurine S-methyltransferase (TPMT) and dihydropyrimidine dehydrogenase (DPD) are examples of nucleotide base-metabolizing enzymes. Table e5-1 lists examples of polymorphic metabolizing enzymes and corresponding drug substrates whose plasma concentrations and pharmacologic effects may be altered as a consequence of genetic variation. Examples of such effects are discussed in the following sections.

Cytochrome P450 Enzymes

Currently, 57 different CYP isoenzymes have been documented to be present in humans, with 42 involved in the metabolism of exogenous xenobiotics and endogenous substances such as steroids and prostaglandins.⁸ Fifteen of these isoenzymes are known to be involved in the metabolism of drugs, but significant interindividual variabilities in enzyme activity exist as a result of induction, inhibition, and genetic inheritance. Functional genetic polymorphism has been discovered for CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5, and their impacts on drug therapy are described hereunder.

CYP2D6

Polymorphisms in the CYP2D6 gene are the best characterized among all of the CYP variants. Over the years, at least 100 gene variants and 120 alleles have been identified in the CYP2D6 gene (www.cypalleles.ki.secyp2d6.htm). Despite the extensive number of alleles, Sistonen et al.⁹ showed genotyping 12 SNPs that represent 20 different haplotypes could provide 90% to 95% accuracy in predicting the real phenotype (outward expression of genotypes). More specifically, different studies showed that the CYP2D6 phenotypes of extensive metabolizer (EM) carrying two functional alleles and poor metabolizer (PM) carrying two nonfunctional alleles could be predicted with up to 99% confidence with six genotypic variants. CYP2D6*1 is considered the wild-type variant and exhibits normal enzyme activity. CYP2D6*2 has the same activity as CYP2D6*1 but is capable of duplication or amplification. Both these variants are present in EMs. The two null variants, CYP2D6*4 (c.1846G>A, defective splicing) and CYP2D6*5 (gene deletion), are predominantly found in white PMs (5%-10% of population) and result in an inactive enzyme and absence of enzyme, respectively. The predominant variants in people of Asian and African heritage are CYP2D6*10 (c.100C>T, Pro34Ser) and CYP2D6*17 (c.1023C>T, Arg296Cys), respectively, both resulting in single-amino-acid substitution and consequent reduction in enzyme activity. Other than *10 and *17, *9, *29, *36, and *41 variants are also associated with lower enzyme activity in the intermediate metabolizers (IMs) phenotype (carriers of one nonfunctional allele and one allele with diminished activity).¹⁰ In addition to *2, gene duplication or amplification had been documented for *1, *4, *6, *10, *17, *29, *35, *41, *43, and *45 variants,^10,11 with resultant higher enzyme activity in the ultrarapid metabolizer (UM) phenotype (carriers of multiple copies of functional alleles).

The presence of two defective alleles (CYP2D6*3, CYP2D6*4 [more common], CYP2D6*5, or CYP2D6*6) in PM results in significant impaired ability to metabolize CYP2D6-dependent substrates. Depending on the importance of the affected CYP2D6 pathway to overall drug metabolism and the drug’s therapeutic index, clinically significant side effects may occur in PMs as a result of elevated drug concentrations,¹² for example, of atomoxetine (insomnia),¹³ perhexiline (neuropathy), perphenazine (sedation and parkinsonism), and propafenone (proarrhythmic events).

The therapeutic implication of CYP2D6 polymorphism is different if the substrate in question is a prodrug. In this case, PMs would not be able to convert the drug into the therapeutically active metabolite. Two examples of prodrugs dependent on CYP2D6-mediated conversion to active forms are codeine and tramadol. Codeine and tramadol are converted by CYP2D6 to morphine and O-desmethyltramadol, respectively, and CYP2D6 PMs would experience little analgesic relief after taking these drugs.^14,15 Another example is CYP2D6-catalyzed conversion of tamoxifen to the more potent antiestrogen metabolite, endoxifen, in which case PMs have been shown to have shortened time to recurrence of breast cancer and worse relapse-free survival.¹⁶

Patients who are EMs have a wide range of CYP2D6 activity, with UMs possessing very high enzyme activity on one end of the spectrum and IMs possessing diminished activity on the other end. Both have clinical implications in terms of dosage adjustment for CYP2D6 substrates. For the CYP2D6 substrate nortriptyline, a patient with three copies of CYP2D6*2 was shown to require doses threefold to fivefold higher than normally recommended to achieve therapeutic plasma concentrations (50-150 ng/mL [mcg/L; 190-570 nmol/L]).^17,18 In the same report, another patient with duplicated CYP2D6*2 required twice the usual recommended daily dose (300 mg vs 25-150 mg) to achieve adequate therapeutic response.¹⁸ There are similar reports of lower drug efficacy in UMs with antiemetics such as ondansetron.¹⁹ Conversely, UMs administered the usual therapeutic dose of codeine or tramadol might exhibit symptoms of narcotic overdose associated with high morphine concentration. This toxicity potential had been reported in several case reports.^14,20 The FDA has issued warnings regarding the use of codeine or tramadol to manage pain after tonsillectomy in children because of the increased risk for respiratory depression in UMs.

Furthermore, the consequence of CYP2D6-mediated drug interactions can be different in patients with different metabolic phenotypes. The UM phenotype has been reported to affect the potential for drug interaction with paroxetine, a potent CYP2D6 inhibitor as well as a CYP2D6 substrate, whence a UM with three functional CYP2D6 gene copies had undetectable paroxetine concentration with standard dosing and showed no inhibitory effect at CYP2D6.²¹

In general, the magnitude of drug interactions involving inhibition of CYP2D6 is much greater in EMs versus PMs, who have either little or no enzyme activity. For example, Hamelin et al.²² showed that in EMs, but not PMs, hemodynamic responses to metoprolol (a CYP2D6 substrate) were pronounced and prolonged during concomitant diphenhydramine administration. Potent CYP2D6 inhibitors, such as paroxetine and fluoxetine, may reduce the metabolic capacity of EMs significantly so that they appear phenotypically as PMs.²³ Given the abundance and greater antiestrogenic activity of endoxifen,¹⁶ the use of paroxetine or fluoxetine in tamoxifen-treated patients should best be avoided. When there is a need for concurrent antidepressant administration with tamoxifen, those with lesser extent of CYP2D6 inhibition, such as citalopram and venlafaxine,²³ would be better alternatives.

The high prevalence of CYP2D6*10 (associated with lower enzyme activity) in the Asian population provides a biologic and molecular explanation for the higher drug concentrations and/or lower dosage requirements of neuroleptic medications and mianserin in people of Asian heritage.^24,25 The widespread presence of the CYP2D6*17 variant among people of African heritage suggests that native African populations would metabolize CYP2D6 substrates at a slower rate than do other ethnic groups.^26,27 However, there are no genotype- and phenotype-based data to document the need for prescribing lower doses of psychotropics and other CYP2D6 substrates in native African populations.

In addition to the therapeutic implications of genetic polymorphisms, one study showed that the CYP2D6 polymorphism also has an economic impact on the treatment of psychiatric inpatients.²⁸ The annual cost of treating UMs and PMs was $4,000 to $6,000 higher than the cost of treating EMs or IMs. The cost of genotyping can be considerably less than that incurred in a patient with a serious adverse drug reaction. In 2005, the FDA approved the AmpliChip^® CYP450 Test (Roche Diagnostics) for analyzing 27 CYP2D6 alleles in addition to the CYP2C19*1, *2, and *3 alleles (discussed hereunder) to assist clinicians in individualizing therapy with drugs metabolized through the CYP2D6 and 2C19 pathways.

CYP2C19

The principal defective alleles for the CYP2C19 genetic polymorphism are CYP2C19*2 (c.19154G > A, aberrant splice site) in exon 5 and CYP2C19*3 (c.17948G > A, premature stop codon) in exon 2 of CYP2C19, resulting in inactive enzyme and the PM phenotype. The clinical relevance of the CYP2C19 polymorphism has been demonstrated for proton pump inhibitors and clopidogrel.

Poor metabolizer for the CYP2C19 polymorphism showed up to a 10-fold increase in the area under the curve (AUC) of omeprazole compared with EMs.³⁰ The presence of a defective CYP2C19 allele has been associated with improved Helicobacter pylori cure rates after dual (omeprazole and amoxicillin)³¹ and triple (omeprazole or lansoprazole, clarithromycin, and amoxicillin) therapy.³² The cure rate achieved with dual therapy was 100% in PMs compared with 60% and 29% in heterozygous and homozygous EMs, respectively.³¹ In two studies included in the meta-analysis of 20 studies using triple therapy,³² EMs had H. pylori eradication rates of 74% to 83% versus 100% cure rates in all 15 PMs included in the two studies.

These differences likely reflect the higher achievable intragastric pH in the PM group.³⁰ Interestingly, EMs who failed initial triple therapy (lansoprazole, clarithromycin, and amoxicillin) and were retreated with high-dose lansoprazole (30 mg four times daily) and amoxicillin achieved a 97% H. pylori eradication.³⁰ A gene–dose effect in attainment of desirable intragastric pH ranges and H. pylori eradication rate, as well as the cost effectiveness of pharmacogenomic-guided dosing was shown for lansoprazole.^30,33

Conversely, IMs and PMs of CYP2C19 may have reduced response to the antiplatelet agent clopidogrel. This is because clopidogrel is a prodrug that requires conversion via CYP2C19 to its active form, as shown in Fig. e5-3. In IMs and PMs, clopidogrel may be less effective at inhibiting platelet aggregation and preventing cardiovascular events than in EMs.³⁴ The data are strongest for patients who suffer an acute coronary syndrome and undergo percutaneous coronary intervention. In these patients, current CPIC guidelines recommend alternative therapy with prasugrel or ticagrelor for IMs and PMs in the absence of contraindications.³⁴ There is a FDA-cleared genotyping device for detecting the CYP2C19*2 and *3 alleles with a turnaround time of approximately 1 hour,³⁵ which could facilitate use of CYP2C19 genotyping in accordance to consensus-based guidelines.³⁴

Similar to the CYP2D6 polymorphism, people of Asian heritage also metabolize most CYP2C19 substrates at a slower rate than do whites.³⁶ This is a reflection of a higher prevalence of both PMs (13%-22.5% vs 2%-6% in whites) and heterozygotes for the defective CYP2C19 alleles (10%-30%) in Asians. This genotypic difference may explain the practice of prescribing lower diazepam dosages for patients of Chinese heritage.³⁷ Similar to CYP2D6 alleles with high enzyme activity, the allelic variant CYP2C19*17 is associated with a very rapid metabolism phenotype, and carriers of this allelic variant would likely require higher doses of proton pump inhibitors^38,39 and other CYP2C19 substrates such as voriconazole.⁴⁰

CYP2C9

Another polymorphic isoenzyme of CYP2C is CYP2C9, which metabolizes narrow therapeutic index drugs such as warfarin, phenytoin, and tolbutamide. Warfarin is a racemic mixture, and the more potent S-isomer is metabolized by CYP2C9. CYP2C9*2 (p.Arg144Cys) and CYP2C9*3 (p. Ile359Leu) are the two most common CYP2C9 variants in whites, and both exhibit single-amino-acid substitutions at positions critical for enzyme activity.⁴¹ This could have clinically important consequences in warfarin-treated patients. For example, a 90% reduction in S-warfarin clearance was reported in CYP2C9*3 homozygotes compared with subjects homozygous for the wild-type (*1) variant,⁴² requiring dose reduction to 0.5 mg/day in a report of a CYP2C9*3 homozygote initially given usual doses of warfarin.⁴³ The clinical relevance of CYP2C9 polymorphism in warfarin dosing was reviewed in a meta-analysis of 39 studies.⁴⁴ In one study included in the meta-analysis,⁴⁵ an overrepresentation of CYP2C9 variant alleles was observed in 81% of patients requiring low-dose warfarin therapy (less than or equal to 1.5 mg/day). The low-dose group was reported to have more difficulty with warfarin induction, requiring longer hospital stays to stabilize the warfarin regimen and experiencing a higher incidence of bleeding complications. CYP2C9 genotype also has implications for response to valproic acid. The contribution of CYP-mediated metabolism of valproic acid is more important in children than in adults. A recent study in 99 pediatric patients with partial or generalized seizures showed that CYP2C9-guided valproic dosing resulted in more patients achieving therapeutic concentrations and lower incidence of side effects.⁴⁶

Both CYP2C9*2 and CYP2C9*3 are more common in whites than in Asians and Africans. The CYP2C9*2 allele is rare to absent in Asian population. More recently, the CYP2C9*8 allele was shown to reduce warfarin clearance and dose requirements.⁴⁷ The CYP2C9*8 allele occurs in approximately 12% of African Americans and may have important implications for metabolism of CYP2C9 substrates in this population. Ultrarapid CYP2C9-mediated metabolism has also been reported resulting in higher dosage requirements of phenytoin.⁴⁸

Numerous studies have shown that the CYP2C9 polymorphisms, in conjunction with a polymorphism in the VKORC1 gene, influence warfarin dose requirements and form the basis for a consensus-based guideline.⁴¹ The CYP2C9 and VKORC1 genotypes were also recently associated with an increased risk for major bleeding events with warfarin therapy.⁴⁹ The use of CYP2C9 and VKORC1 genotypes in dosing warfarin is discussed in section “Polymorphisms in Drug Target Genes” for more detail.

CYP2A6

In addition to the wild type CYP2A6*1, several variants for the CYP2A6 polymorphism have been identified (www.cypalleles.ki.se): CYP2A6*2 (single amino acid substitution), CYP2A6*4 (gene deletion), CYP2A6*5 (gene conversion), and CYP2A6*20 (frameshift) are associated with abolished enzyme activity. Deletion of the CYP2A6 gene is very common in Asian patients,⁵⁰ which likely accounts for the dramatic difference in the frequency of PMs in Asian (20%) versus white populations (less than or equal to 1%). Nicotine is metabolized by CYP2A6, and the clinical relevance of the CYP2A6 polymorphism lies in management of tobacco abuse. Investigators reported that nonsmokers were more likely to carry the defective CYP2A6 allele than were smokers. Smokers who had the defective CYP2A6 allele smoked fewer cigarettes and were more likely to quit. The inability to metabolize nicotine, secondary to the presence of a defective CYP2A6 allele, likely leads to enhanced nicotine tolerance and increased adverse effects from nicotine. Based on these observations, CYP2A6 inhibition may have a role in the management of tobacco dependency.⁵⁰

CYP2B6

Although the role of CYP2B6 in the metabolism of anticancer drugs, such as cyclophosphamide and ifosfamide, has been studied, it is with the antiretroviral agents that its clinical relevance was revisited and highlighted. The nonnucleoside reverse transcriptase inhibitor efavirenz is metabolized by CYP2B6. Many patients receiving efavirenz experience central nervous system (CNS) adverse effects that are related to variable systemic exposure to the drug, which could be related to the lower metabolizing efficiency of the CYP2B6*6, *16, or *18 alleles.⁵¹ A prospective study demonstrated that dose reduction for 6 months in 12 patients with high efavirenz concentrations secondary to CYP2B6 polymorphism resulted in both effective anti-human immunodeficiency virus (HIV)-1 activity with HIV-1 load less than 50 copies/mL (50 × 10³/L) as well as lower incidence of CNS adverse effects.⁵²

CYP3A4/5

Within the CYP3A subfamily, at least three isoenzymes, namely, CYP3A4, CYP3A5, and CYP3A7, have been characterized. Despite as much as 40-fold interindividual variability in its expression, functional CYP3A4 is expressed in most adults, with intestinal expression playing a significant role in the first-pass metabolism of numerous drugs. Although several CYP3A4 variants (eg, *6, *17, and *20) have been associated with reduced activity, their low frequency suggest limited clinical relevance.

CYP3A5 is reported to be polymorphic in 60% of African Americans and 33% of whites, with CYP3A5*3 (c.6986A>G, aberrant splice site) in intron 3 as the primary allele variant. In contrast to individuals with the CYP3A5*1 allele, subjects with CYP3A5*3 have no functional CYP3A5 enzyme.⁵³ CYP3A4 and CYP3A5 mediate the metabolism of more than 50% of all clinically useful drugs. However, with overlapping substrate specificities, it remains unknown whether some drugs are substrates for CYP3A5 but not CYP3A4 and vice versa. Although variability exists between dose-adjusted concentration and CYP3A5 genotypes, studies have shown a correlation between trough concentrations of tacrolimus and CYP3A5 genetic constitution, and recent CPIC guidelines recommend increasing the starting dose of tacrolimus in patients with the CYP3A4 *1/*1 or *1/*3 genotype.

Phase II and Nucleotide-Base Metabolizing Enzymes

The clinical relevance of genetic polymorphisms in TPMT, DPD, and UGT enzymes has been demonstrated in the treatment of cancer. The TPMT gene has four mutant alleles: TPMT*3A (the most common), TPMT*2, TPMT*3B, and TPMT*3C. Thiopurine drugs, such as 6-thioguanine, 6-mercaptopurine, and its precursor, azathioprine, are inactivated by TPMT, and patients who are homozygous or heterozygous for the TPMT mutant alleles are at higher risk for developing serious hematological toxicities during treatment with the thiopurines.⁵⁶ DPD mediates the metabolism of 5-fluorouracil and its precursor capecitabine, and patients with a defective allele of the DPYD gene encoding for DPD cannot metabolize 5-fluorouracil and thus may experience enhanced drug-related neurotoxicity.⁵⁷ The camptothecin derivative irinotecan (CPT-11) is activated by carboxylesterase to SN-38, a potent topoisomerase I inhibitor. SN-38 is inactivated by glucuronidation via the polymorphic UGT1A1 enzyme, which may play a role in CPT-11-related toxicity. An extra thymine-adenine (TA) repeat within the TATA section of the UGT1A1 promoter results in the (TA)7TAA allele (also known as UGT1A1*28), which possesses lower enzyme activity than the wild-type (TA)6TAA allele. Impaired SN-38 glucuronidation secondary to the (TA)7TAA allele may result in abnormally high SN-38 concentrations. A prospective clinical trial demonstrated more severe diarrhea and neutropenia in irinotecan-treated patients who are homozygous or heterozygous carriers of the (TA)7TAA allele.⁵⁸ A subsequent meta-analysis showed dose-related increases in the risk for severe neutropenia with irinotecan with the UGT1A1 (TA)7TAA allele.⁵⁹ In 2005, the FDA approved the Invader^® UGT1A1 Molecular Assay (Third Wave Technologies) to genotype for UGT1A1 alleles, and the labeling for irinotecan was revised to recommend dose adjustment for individuals who are homozygous for the (TA)7TAA allele.

The antiretroviral protease inhibitor atazanavir is an inhibitor of UGT1A1. Atazanavir can inhibit UGT1A1-mediated glucuronidation and elimination of bilirubin, which can lead to hyperbilirubinemia and jaundice. This effect is more pronounced in individuals with the UGT1A1*28 allele, and CPIC guidelines recommend using an alternative agents in homozygotes for the *28 allele.⁶⁰

The clinical significance of N-acetyltransferase-2 polymorphism was demonstrated by investigators from the pharmacogenetics-based tuberculosis therapy research group in Japan. Early treatment failure with isoniazid was more common among rapid acetylators in the standard doing group (38%) than in the pharmacogenomics-guided dosing group (15%). Similarly, isoniazid-induced liver injury was more common in 78% of slow acetylators in the standard doing group but not present in slow acetylators from the pharmacogenomics-guided dosing group.⁶¹

Certain membrane-spanning proteins facilitate drug transport across the gastrointestinal tract, drug excretion into the bile and urine, drug distribution across the blood–brain barrier, and drug uptake into target cells. Genetic variations for drug transport proteins may affect the distribution of drugs that are substrates for these proteins and alter drug concentrations at their therapeutic sites of action. P-glycoprotein is one of the most recognized of the drug transport proteins that exhibit genetic polymorphism. P-glycoprotein is an energy-dependent transmembrane efflux pump encoded by the ABCB1 gene (also known as the multidrug resistance 1 gene), which is a member of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter superfamily. P-glycoprotein was first recognized for its ability to actively export anticancer agents from cancer cells and promote multidrug resistance to cancer chemotherapy. Later, it was discovered that P-glycoprotein is also widely distributed on normal cell types, including intestinal enterocytes, hepatocytes, renal proximal tubule cells, and endothelial cells lining the blood–brain barrier. At these locations, P-glycoprotein serves a protective role by transporting toxic substances or metabolites out of cells. P-glycoprotein also affects the distribution of some nonchemotherapeutic agents, including digoxin, the immunosuppressants cyclosporine and tacrolimus, and antiretroviral protease inhibitors (Fig. e5-4). Increased intestinal expression of P-glycoprotein can limit the absorption of P-glycoprotein substrates, thus reducing their bioavailability and preventing attainment of therapeutic plasma concentrations. Conversely, decreased P-glycoprotein expression may result in supratherapeutic plasma concentrations of relevant drugs and drug toxicity.

Numerous SNPs and insertion/deletion polymorphisms have been identified in the promoter and exon regions of the ABCB1 gene and while there is evidence that ABCB1 genotype influences response to digoxin and other P-glycoprotein substrates, the evidence has not reached a level sufficient for clinical implementation.

Other examples of polymorphic drug transporter proteins include the OAT and OCT, both members of the solute carrier (SLC) transporter family. The SLC01B1 gene encodes for OAT polypeptide B1, which mediates the uptake of β-hydroxy-β-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) into the liver. Although statins effectively lower total and low-density lipoprotein cholesterol and reduce the risk for cardiovascular events in coronary heart disease, their use is associated with an increased risk for myopathy (muscle pain or weakness with elevated creatine kinase levels), particularly with higher statin doses or concomitant drugs that increase statin bioavailability. Myopathy may rarely cause rhabdomyolysis, characterized by muscle breakdown and potentially leading to acute renal failure. The reduced function SLC01B1 c.521T > C SNP, resulting in the p.Val174Ala substitution and contained within the SLC01B1*5 haplotype, has been associated with higher statin concentrations.⁶² Each copy of the C allele increased the risk for myopathy with simvastatin 80 mg/day by 4.5-fold in a genome-wide association study (GWAS), in which more than 300,000 SNPs were compared between 85 patients who developed myopathy with high-dose simvastatin (cases) and 90 controls without this adverse effect with simvastatin therapy.⁶³ In a replication cohort of patients treated with simvastatin 40 mg/day, the relative risk for myopathy was 2.6 per copy of the 521C allele. The association between the 521C allele and statin-induced myopathy was further confirmed in later studies.

Similarly, the 521C allele was associated with an increased incidence of less severe yet troubling adverse effects that lead to statin discontinuation, including myalgias without significant creatine kinase elevation.⁶⁴ The data with the 521T>C allele and risk for myopathy are strongest for simvastatin and suggest that lower simvastatin doses or alternative statin drugs should be used in 521C carriers. CPIC guidelines support this.⁶²

The SCC22A1 gene encodes for the OCT1 transporter, and several SNPs: Arg61Cys (rs12208357), Gly401Ser (rs341303495), Met420del (rs7255276), and Gly465Arg (rs34059508) have been associated with decreased metformin transport and altered pharmacokinetics with increased plasma levels. A recent study compared the effect of carriers of reduced function of five OTC variants (Arg61Cys, Gly401Ser, Met420del, Gly465Arg plus Cys88Arg [rs55918055]) and concurrent administration of OTC1 inhibitor in 251 metformin-intolerant and 1,915 metformin-tolerant patients. Homozygous carriers of reduced-function OCT1 alleles had greater intolerance (OR 2.41 [95% CI 1.48-3.93, P < 0.001]) when compared to heterozygous carriers or non-carriers of a deficient allele. A known OCT inhibitor was also associated with metformin intolerance (OR 1.63 [95% CI 1.22-2.17, P = 0.001]). Intolerance was four times more likely in homozygous carriers of reduced-function OCT1 alleles who were taking an OCT1 inhibitor concurrently (OR 4.13 [95% CI 2.09-8.16, P < 0.001]).⁶⁵

Genetic polymorphisms occur commonly for drug target proteins, including receptors, enzymes, ion channels, and intracellular signaling proteins. Drug target genes may work in concert with genes that affect pharmacokinetic properties (ie, genes for drug transporters and drug-metabolizing enzymes) to contribute to overall drug response. For example, the genes for CYP2C9, the major metabolizing enzyme for S-warfarin, and vitamin K oxidoreductase (VKOR), the target enzyme for warfarin, interact to influence warfarin dose response, as shown in Fig. e5-5. The following section highlights some of the receptor, enzyme, ion channel, and cell-signaling protein genes shown to influence the efficacy and safety of various pharmacologic agents.

Receptor Genotypes and Drug Response

The β₁-adrenergic receptor gene (ADRB1) has been the primary focus of research into genetic determinants of responses to β-adrenergic receptor antagonists in hypertension and cardiovascular disease. β₁-Receptors are located in the heart and kidney, where they are involved in the regulation of heart rate, cardiac contractility, and blood pressure. There are two common nonsynonymous SNPs in the ADRB1 at codons 49 (p.Ser49Gly) and 389 (p.Arg389Gly). The Ser49Gly and Arg389Gly SNPs are in strong linkage disequilibrium. The Ser49-Arg389 haplotype is associated with an increased risk for death among patients with coronary heart disease.⁶⁶ The ADRB1 Ser49Gly and Arg389Gly SNPs also appear to modulate blood pressure and clinical responses to β₁-receptor blockade. Specifically, hypertensive patients who were homozygous for the Ser49-Arg389 haplotype were found to have greater blood pressure reductions with metoprolol, compared with carriers of the Gly49 and/or Gly389 alleles.⁶⁷ In patients with coronary heart disease, atenolol treatment appears to abolish the increased risk for mortality associated with the Ser49-Arg389 haplotype.⁶⁶ Among patients with heart failure, the Arg/Arg389 genotype was associated with greater improvements in left ventricular ejection fraction with carvedilol and metoprolol treatment and greater survival benefits with bucindolol, an agent not approved for use in the United States.^67,69 Overall, these data suggest that ADRB1 genotype may be an important determinant of blood pressure response to β-blockers in the management of hypertension, survival benefits with β-blockers in the management of coronary heart disease, and improvements in cardiac function and clinical outcomes in patients with heart failure. Given that a significant percentage of hypertensive patients fail to derive adequate blood pressure reduction with β-blocker monotherapy, the ability to predict the likelihood of response based on genotype would have important clinical implications. Specifically, β-blockers could be started in patients expected to respond well to this drug class based on their genotype, whereas other classes of antihypertensive agents could be used in those expected to respond poorly to β-blockers. β-blockers could also be used as first-line therapy for hypertensive patients with coronary heart disease and ADRB1 genotype predictive of poor survival. While β-blockers are currently indicated in all patients with heart failure, ADRB1 genotype may be useful in identifying patients who may derive lesser benefits from β-blockers than others. Alternative or additional therapies may be warranted in such patients to improve their outcomes.

Enzyme Genes and Drug Response

Vitamin K oxido reductase is an example of an enzyme with genetic contributions to drug response. Warfarin exerts its anticoagulant effects by inhibiting VKOR and thus preventing carboxylation of the vitamin K-dependent clotting factors II, VII, IX, and X, as shown in Fig. e5-5. VKORC1 encodes for the warfarin-sensitive component of VKOR. Mutations in the VKORC1 coding region cause rare cases of warfarin resistance, with carriers of these mutations requiring either exceptionally high warfarin doses (more than 100 mg/wk) to achieve effective anticoagulation or failing to respond to warfarin at any dose.⁷⁰

Aside from rare cases of warfarin resistance, there is substantial variability among patients in the dose of warfarin necessary to produce optimal anticoagulation, defined as an international normalized ratio of 2 to 3 for most indications. A common SNP in the VKORC1 regulatory region significantly contributes to the interpatient variability in warfarin response. Specifically, the –1639 AA, AG, and GG genotypes lead to high, intermediate, and low sensitivity to warfarin, respectively. Corresponding warfarin dose requirements are approximately 3 mg/day with the AA genotype, 5 mg/day with the AG genotype, and 6 to 7 mg/day with the GG genotype. VKORC1 genotype, together with CYP2C9 genotype, explains approximately 30% of the interpatient variability in warfarin dose requirements.⁴¹ Clinical characteristics (eg, age, body size) contribute to additional doe variability.

There is evidence of differences in warfarin dose requirements by ancestry, with higher dose requirements among individuals of African ancestry and lower requirements among Asians compared to Whites. This variability is largely explained by differences in VKORC1 genotype frequency. Specifically, the low-dose AA genotype is most common in Asians, and the high-dose GG genotype is most common in African Americans, whereas the intermediate-dose AG genotype is most common in persons of European ancestry.

Warfarin-dosing algorithms that incorporate CYP2C9 and VKORC1 genotypes and nongenetic (eg, age, body size, interacting medications) factors are publically available to assist with warfarin dosing. In addition, the warfarin labeling now contains a dosing table based on CYP2C9 and VKORC1 genotypes (Table e5-2). A comparative effectiveness study demonstrated that use of genotype-guided warfarin dosing leads to better prediction of warfarin dose requirements, greater time spent within the therapeutic anticoagulation range, and may lower the incidence of serious adverse events during the initial months of warfarin therapy compared to traditional warfarin dosing.⁷¹ Two clinical trials examining the efficacy of genotype-guided warfarin dosing were published in 2013. One trial was conducted in Europe and showed greater time in the therapeutic INR range with genotype-guided dosing compared to a standard dosing approach.⁷² The other trial was conducted in a diverse US population, including a large number of African Americans and showed no difference in time spent in the therapeutic INR range with dosing using a pharmacogenomic algorithm compared to dosing with a clinical algorithm.⁷³ A third trial is ongoing and examining the effect of genotype-guided warfarin dosing on risk for bleeding and thromboembolism among patients taking warfarin for prevention of venous thromboembolism after major orthopedic surgery.

Genes for Intracellular Signaling Proteins, Ion Channels, and Drug Response

Cellular responses to many drugs are mediated through receptor-coupled guanosine diphosphate (GDP)-bound proteins also called G-proteins. G-proteins consist of α, β, and γ subunits. Following receptor activation, the receptor couples to the G-protein, resulting in dissociation of GDP from the α subunit in exchange for guanosine triphosphate (GTP) and activation of the α, β, and γ subunits. The α subunit and βγ subunit complex are released intracellularly and interact with various effectors (eg, adenylyl cyclase, phospholipase C) to produce a cellular response. The β₁-adrenergic receptor is an example of a G-protein–coupled receptor in which a stimulatory G protein (G_s protein) mediates the activation of the effector adenylyl cyclase and the generation of the second messenger cyclic adenosine monophosphate (cAMP) following receptor stimulation (Fig. e5-6).

G-protein coupled receptor kinase (GRK) phosphorylates β-adrenergic receptors causing the receptor to uncouple with the G protein. There is a polymorphism in the GRK5 gene (Gln41Leu) influences outcomes with β-blocker therapy in heart failure. The Leu allele is 10-fold more common in persons of African versus European descent. Among African Americans with heart failure and the Gly/Gly genotype, β-blocker therapy was associated with greater transplant-free survival. However, no benefit was observed with β-blocker therapy among patients with a Leu41 allele.⁷⁴ A potential explanation for this finding is that the Leu41 variant acts to desensitize the β-adrenergic receptor and thus, may serve as a natural β-blocker eliminating the need for β-blocker therapy. Genes encoding pancreatic ATP-sensitive potassium (K_ATP) channels are examples of ion channel genes with implications for drug response. The potassium inwardly rectifying channel, subfamily J, member 11 gene (KCNJ11) and the sulfonylurea receptor gene (ABCC8) encode the Kir6.2 and sulfonylurea receptor-1 (SUR1) subunits of pancreatic K_ATP channels, respectively. K_ATP channels remain open in the presence of activating mutations in the KCNJ11 and ABCC8 genes, which leads to hyperpolarization of the pancreatic β-cell membrane and impaired insulin release.^75,76 Sulfonylureas are especially effective antidiabetic agents in patients with activating KCNJ11 or ABCC8 mutations, in whom they promote K_ATP channel closure.^75,77

The large-conductance calcium and voltage-dependent potassium (BK) channel is another example of an ion channel with genetic contributions to drug response. The KCNMB1 gene encodes for the β₁ subunit of the BK channel. There are two common SNPs in the KCNMB1 gene, p.Glu65Lys and p.Val110Leu. Among patients with hypertension and coronary heart disease started on verapamil, the Lys65 allele was associated with more rapid achievement of blood pressure control.⁷⁸ Verapamil-treated patients with the Leu110 allele had a lower risk for cardiovascular events compared with non-Leu110 allele carriers. These findings may have implications for individualized use of calcium channel blockers for blood pressure control in patients with coronary heart disease.

In patients with type 2 diabetes, a GWAS study reported a significant association between response to the biguanide metformin and polymorphism in the ATM gene. The ATM plays a significant role in activation of the adenosine monophosphate-activated protein kinase (AMPK), which is considered the mechanism of action of metformin. While the rs11212617 SNP, located near the ATM gene, only explains 2.5% of the variation of metformin response,⁴ the initial result was replicated in a subsequent study with additional European cohorts.⁷⁹ This first GWAS study for antidiabetic drugs⁴ suggests the utility of the GWAS approach in identifying viable genes that have potential implications for mediating glycemic response to drug therapy in a complex disease, such as diabetes, and may pave the way for a new era in anti-diabetic drug development.

Numerous genes have been correlated with disease outcomes, and many of these have been found subsequently to influence response to pharmacologic disease management. These gene–drug response associations often occur despite the lack of a direct effect on pharmacokinetic or pharmacodynamic drug properties. Examples of such disease-associated genes are discussed hereunder.

Human Leukocyte Antigen Gene and Hypersensitivity to Antiepileptic Drugs and Abacavir

The human leukocyte antigen (HLA) gene has been linked to serious, potentially life-threatening adverse skin reactions with carbamazepine and phenytoin, commonly prescribed antiepileptic agents.^80,81 Stevens–Johnson’s syndrome (SJS) and toxic epidermal necrolysis (TEN) are hypersensitivity reactions characterized by blistering, mucosal erosions, and epidermal detachment. TEN is associated with more extensive skin involvement and a mortality rate approaching 25%.

Individuals with southeastern Asian ancestry (ie, southern China, Thailand, Malaysia, Indonesia, Taiwan, and Philippines) have a twofold to threefold higher prevalence of carbamazepine- or phenytoin-induced SJS and TEN than individuals from Japan, Korea, or European countries. The human leukocyte antigen type B (HLA-B)*1502 allele also occurs at a higher prevalence in individuals from the countries with higher prevalence of SJS and TEN. Individuals from south Asia, including India, have an intermediate prevalence of this allele (2%-4%). It occurs in less than 1% of those in Japan and Korea and is largely absent in the rest of the world. Thus, presence of the allele correlates highly with risk for drug-induced SJS or TEN.

While the mechanism by which the HLA-B*1502 allele increases the risk for these toxic cutaneous reactions is unclear, it may involve activation and proliferation of T lymphocytes on carbamazepine exposure. The carbamazepine labeling was recently updated to recommend HLA-B*1502 screening in individuals with ancestry from southern Asia prior to carbamazepine use (see Table e5-1). As demonstrated in a large population screening study,⁸² carbamazepine should be avoided in patients testing positive for the HLA-B*1502 allele. Moreover, because of reports of phenytoin-induced SJS in the presence of the HLA-B*1502 allele, phenytoin should also be avoided in individuals with the HLA-B*1502 allele.⁸¹ The cost effectiveness of universal HLA-B*15:02 screening was demonstrated in a population from Thailand, where only about 340 patients need to be genotyped to prevent one case of SJS or TEN.⁸³

The use of abacavir, a nucleoside reverse transcriptase inhibitor of HIV-1, has been associated with severe, and occasionally fatal, hypersensitivity reactions in some patients. The abacavir hypersensitivity reaction (AHR) is strongly associated with the presence of another allelic variant of the HLA gene, HLA B*57:01, and screening for HLA B*57:01 has been shown to be associated with reduction of AHR incidence.⁸⁴ The screening recommendation has been incorporated into abacavir product labeling as well as treatment guidelines. ^84,85

Factor V and Prothrombin Genes and Oral Contraception

The use of estrogen-containing oral contraceptives is associated with an increased risk for developing thromboembolic disorders, including deep venous thrombosis, pulmonary embolism, and thrombotic stroke. Variations in the genes for the coagulation factors prothrombin and factor V Leiden also have been identified as risk factors for thromboembolic disorders.⁸⁶ In case-control studies, the presence of a factor V Leiden or prothrombin gene variation markedly increased the risk for deep vein thrombosis and cerebral vein thrombosis among estrogen-containing oral contraceptive users. These data suggest that alternative birth control measures should be employed in women known to carry a prothrombin or factor V Leiden mutation.

Congenital Long-QT Syndrome and Drug-Induced Torsade De Pointes

Drug-induced QT-interval prolongation may precipitate the serious, potentially life-threatening arrhythmia called torsade de pointes. It is well recognized that many antiarrhythmic drugs can cause QT-interval prolongation and torsade de pointes. In addition, numerous noncardiovascular agents can induce torsade de pointes, and many have been withdrawn from the market as a result. Such drugs include the antihistamines terfenadine and astemizole, the fluoroquinolone antibiotic grepafloxacin, and the motility agent cisapride. Given the serious and unpredictable nature of torsade de pointes, there has been great interest in identifying genetic markers that predispose individuals to its occurrence.

Abnormalities in ion flux across the cardiac cell membrane resulting in an excess of intracellular positive ions and delayed ventricular repolarization are characteristic of long-QT syndromes. Mutations in genes for the pore-forming channel proteins that affect potassium and sodium transport across the cardiac cell membrane, including the KCNQ1, KCNE2, KCNH2, and SCN5A genes, underlie congenital long-QT syndromes.⁸⁷ There is evidence that these and similar mutations also may increase the risk for drug-induced torsade de pointes.⁸⁷ Ultimately, the ability to screen for mutations associated with drug-induced torsade de pointes may enable identification of individuals with a genetic predisposition for this life-threatening arrhythmia who could be spared exposure to potentially causative agents and treated with alternative therapies.

The discovery of genes that confer disease has led to an improved understanding of the molecular mechanisms involved in disease pathophysiology. Once associations between genes and diseases are discovered, scientists can elucidate the functions of the encoded proteins and more clearly define the consequences of genetic mutations. Insight into the genetic control of cellular functions may reveal new strategies for disease treatment and prevention.

Targeted Therapies for Cancer. Overexpression of the human epidermal growth factor receptor 2 (HER2, also known as Her2/neu and ErbB2), secondary to HER2 gene amplification occurs in 20% of metastatic breast cancers and is associated with more aggressive cancer and decreased survival.⁸⁸ The discovery of HER2 overexpression and its effects on cancer prognosis led to the development of trastuzumab, a recombinant monoclonal antibody that targets HER2 and blocks HER2-stimulated growth and survival of cancer cells. The addition of trastuzumab to breast cancer chemotherapy significantly slows the progression of cancer and improves tumor response rates in women with HER2-positive tumors.⁸⁸ Testing for HER2 overexpression is necessary to determine which patients may benefit from trastuzumab. The FDA has approved several tests that detect HER2 overexpression either directly by measuring the amount of protein or indirectly by measuring gene amplification.

Similarly, overexpression of the epidermal growth factor receptor (EGFR, also known as HER1 or ErbB1) in head and neck, colon, and rectal cancer is associated with cancer growth and a poor clinical prognosis. Cetuximab and panitumumab are recombinant monoclonal antibodies that block activation of the EGFR. Both were shown to improve survival in metastatic colorectal cancer that overexpresses EGFR, and are thus indicated in this setting.^89,90 Erlotinib and gefitinib inhibit the intracellular phosphorylation of tyrosine kinase associated with the EGFR and are indicated in non-small cell lung cancer. Other examples of targeted chemotherapy developed based on genetic abnormalities include rituximab, a monoclonal antibody used to treat CD20-positive, B-cell non-Hodgkin’s lymphoma and chronic lymphocytic leukemia; imatinib, dasatinib, and nilotinib, kinase inhibitors that blocks the product of a reciprocal translocation between chromosomes 9 and 22 in chronic myeloid leukemia (CML); and crizotinib, an anaplastic lymphoma kinase (ALK) and c-ros oncogene 1, receptor tyrosine kinase (ROS-1) inhibitor that targets the EML4-ALK gene fusion product in non-small cell lung cancer.

Targeted Therapy for Cystic Fibrosis. Cystic fibrosis is caused by genetic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which is expressed in the airways, intestines, pancreas, bile duct, and sweat glands. In these locations, the CFTR protein serves a critical role in the regulation of fluid and ion transport. The CFTR is composed of two nucleotide binding domains and two transmembrane domains. The transmembrane domain forms a chloride channel, while the nucleotide binding domain acts as a gate to regulate chloride transport across the cell membrane. Mutations in the CFTR gene lead to altered fluid and ion transport and disrupt mucus clearance in the airways and intestinal tract resulting in airway obstruction and digestive problems.

Mutations in the CFTR gene affect protein synthesis, processing, regulation, or ion conductance. Ivacaftor was approved by the FDA in 2012 as the first drug to target defects in the CFTR.⁹¹ Ivacaftor was initially approved for the treatment of cystic fibrosis in patients age 6 and older who have the Gly551Asp mutation, which occurs in about 4% of cystic fibrosis cases and causes defects in chloride transport through the ion channel. Ivacaftor potentiates chloride ion flow and resulted in rapid and sustained improvement in lung function compared to placebo in randomized controlled trials in patients with cystic fibrosis. The labeling for Ivacaftor was later updated to include additional variants (Gly1244Glu, Gly1349Asp, Gly551Ser, Ser1251Asn, Ser1255Pro, Ser549aSN, Ser549R, and Gly178Arg) that also disrupt chloride gating.

The FDA is involved in a number of pharmacogenetic-related activities, including encouraging submission of exploratory genomic data from drug sponsors, providing guidance on incorporating pharmacogenetic principles into the drug development process, and updating drug labels to include pharmacogenetic information. More than 120 drugs now contain pharmacogenetic information in their FDA-approved labeling. Examples of these are shown in Table e5-3. The pharmacogenomic information appears in various sections of the label. For example, the information appears as a Boxed Warning for clopidogrel and carbamazepine because of the serious consequences of genetic variation on drug response. In the case of warfarin, mercaptopurine, and irinotecan, the pharmacogenomic information appears in the drug dosing section. The FDA maintains a table of pharmacogenomics biomarkers in drug labels on its website (www.fda.gov).

Guidelines are now available to assist with translating genotype results into actionable prescribing decisions for a number of drugs. Among these are guidelines from the Clinical Pharmacogenetics Implementation Consortium (CPIC). CPIC is an international collaboration of individuals from academic centers, clinical institutions, and pharmacy benefits management with expertise in pharmacogenomics or laboratory medicine that provides consensus-based guidelines on how to use genetic test results to optimize pharmacotherapy.⁹² The consortium does not recommend whether genetic tests should be ordered, but rather, how to use existing genetic information.

Guidelines published as of mid-2015 are listed in Table e5-4. The CPIC guidelines, in addition to other pharmacogenetic information, are freely available through the Pharmacogenomics Knowledge Base (PharmGKB).

Gene therapy has emerged as a possible approach to treating and curing disease by replacing a mutated gene with a nonmutated form, altering gene expression, or adding a new gene. Initially, the focus of gene therapy was for the treatment of inherited disorders such as cystic fibrosis, sickle cell anemia, hemophilia, and severe combined immunodeficiency. Gene therapy trials were later expanded to include patients with acquired diseases such as cancer, heart disease, and Parkinson’s disease. The goal of gene therapy for inherited diseases is to correct or repair genetic defects permanently and thereby restore normal cellular function. Gene therapy for acquired diseases aims to cure disease by targeting pathogenic processes.

Most gene therapy techniques for inherited diseases attempt to replace defective genes with normally functioning ones. Exogenous genes, called transgenes, are transferred into somatic (body) cells of the recipient. Transfer of transgenes into germ line (egg or sperm) cells can result in passage of genetic alterations to offspring and is currently prohibited by the FDA.

The first clinical gene therapy trial began in 1990 for the treatment of adenosine deaminase deficiency. B and T lymphocytes fail to develop in this autosomal recessive disease, resulting in a severe combined immunodeficiency syndrome (SCID) made famous by the “bubble boys” whose lives were confined to tents in an effort to keep them in a germ-free environment. Only two patients were initially included in this trial, and although both continued to demonstrate clinical improvement 10 years later, gene therapy did not cure the disease, as investigators had hoped.

More than 3,000 clinical gene therapy trials have been registered around the world (www.clinicaltrials.gov). Most of these trials involve cancer patients; however, a number of studies also target heart disease and inherited disorders such as muscular dystrophy. The results of gene therapy trials to date have been largely disappointing, with reports of serious toxicities and few therapeutic successes.

Obstacles to Success

Reasons for limited success with gene therapy include inefficient gene delivery to target cells, inadequate gene expression, and unacceptable adverse effects.

Sufficient amounts of the transgene must be inserted into a sufficient number of recipient cells to produce a therapeutic response. In addition, the transgene must be inserted into the correct chromosomal position of the correct cell nucleus so as not to disrupt normal gene function and expression. Incorrect chromosomal insertion of the transgene is a problem referred to as insertional mutagenesis. Once the therapeutic gene is integrated correctly into host DNA, it must be expressed at adequate levels and at appropriate times to restore normal cell function. Finally, the gene delivery system and delivery technique should lack any potential to cause unwanted effects in the transgene recipient.

Retroviral Gene Delivery

Because of their efficiency in integrating into human DNA, viruses are the most common vectors used to deliver therapeutic genes to recipient cell targets. Disease-causing genes are replaced with the desired therapeutic genes; the viral genes that control delivery mechanisms are retained.

The first viral vectors introduced were retroviruses, which are RNA viruses that integrate into the host cell genome and replicate during cell division. Thus, retroviral gene transfer is capable of permanently altering gene expression. Retroviruses may be used to deliver genes through either direct infusion into target organs or ex vivo manipulation of harvested cells followed by reinfusion into the recipient. The disadvantages of retroviral vectors are the limited size of the gene they can carry, relatively low efficiency, and the risk of insertional mutagenesis. In fact, the FDA temporarily halted retroviral gene delivery into hematopoietic stem cells in early 2003 after leukemia developed as a result of insertional mutagenesis in two of nine SCID-affected children treated with retroviral gene therapy.⁹³ Two other children later developed cancer, and one died. Gene therapy for X-linked SCID is currently restricted to patients who undergo unsuccessful bone marrow transplantation.

Since research with retroviral gene therapy has resumed, there has been some success with this mode of gene delivery in the area of oncology. In 2003, the FDA granted orphan drug status for a retroviral gene therapy that targets the cyclin G1 gene in the treatment of pancreatic cancer, and later, this therapy gained orphan drug status in the treatment of osteosarcoma and soft tissue sarcoma. Phase I and II studies suggest that the drug is well tolerated and may control tumor growth and increase survival in patients with chemotherapy-resistant pancreatic cancer or osteosarcoma.⁹⁴ Lentiviruses are a type of retrovirus that also appear promising as they have a lower potential for causing cancer. Lentiviral vector-based gene delivery of three genes coding for enzymes that make dopamine to the brain of Parkinson’s Disease patients has been shown to be well tolerated and improve motor function.⁹⁵

Adenoviral Gene Delivery

Unlike retroviruses, adenoviruses do not integrate into the host genome and thus do not replicate. As a result, genes delivered by adenoviruses are only active temporarily. Adenoviral-mediated gene therapy is employed commonly in cancer patients because permanent gene expression is unnecessary in this patient population.

Tumor cells have been infused with adenoviral vectors carrying the herpes simplex virus-1 thymidine kinase gene and then exposed to valacyclovir as a mode of cancer chemotherapy. Thymidine kinase converts valacyclovir to its active, cytotoxic form, which is incorporated in the DNA of tumor cells, leading to their death. Adenoviruses can be grown in high titers and do not carry the risk of insertional mutagenesis. The major disadvantage of adenoviruses is their immunogenic potential, which has resulted in one death and prompted federal oversight of gene therapy trials.

Adeno-Associated Viral Gene Delivery

Adeno-associated viruses are human DNA-containing viruses that do not appear to trigger immune responses on injection. Similar to retroviruses, adeno-associated viruses are incapable of carrying a large amount of genetic material, and their use entails the risk of insertional mutagenesis.

Investigators have had some success with adeno-associated viral gene delivery of SERCA2a in patients with advanced heart failure. Patients with advanced heart failure commonly have a deficiency in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a), which is essential for maintaining cardiac function through regulation of cellular homeostasis of calcium. A single intracoronary infusion of adeno-associated virus 1/SERCA2a in nine patients appeared to have an acceptable safety profile.⁹⁶ With the exception of two patients found to have preexisting anti-AAV1 neutralizing antibodies, gene transfer was associated with improved symptomatic, functional, and cardiac parameters. In a followup phase 2 trial including 39 patients with advanced heart failure, intracoronary adeno-associated virus type 1/SERCA2a decreased the frequency of cardiovascular events at 12 months compared with placebo.⁹⁷

Other Means of Gene Delivery

Scientists are also experimenting with nonviral delivery methods such as the use of direct DNA injection, liposomes, cationic polymers, and electroporation. There has been much interest in myocardial delivery of plasmid DNA encoding for vascular endothelial growth factor gene to promote formation of new coronary vessels (angiogenesis) in patients with severe, intractable angina. The procedure appeared promising in early clinical trials, with improved myocardial perfusion and angina in this patient population with few major adverse events. However, larger, more rigorously conducted trials have failed to demonstrate significant benefit of myocardial angiogenesis gene therapy.

Scientists have enjoyed few successes with gene therapy for inherited diseases. Improvements in gene delivery techniques and a better understanding of molecular processes controlling gene expression are necessary before gene therapy can correct genetic defects successfully and thus cure associated diseases without inducing adverse effects. Because of limited success with traditional approaches to gene therapy, scientists are exploring other strategies, such as repairing or regulating (“turning off”) defective genes rather than replacing them. Scientists have had more success with gene therapy for acquired diseases, such as cancer, and a number of phase II and III clinical trials in this area are under way. While gene therapy research is evolving, much progress has yet to be made before effective and safe therapies are available.

Pharmacogenetics

Traditionally, genetic testing refers to screening human genetic material to identify genotypes associated with disease susceptibility or carrier status for heritable diseases, such as Huntington disease or breast cancer. This kind of testing can have profound ethical and social implications. For example, knowledge that a patient is at risk for developing a genetic disorder could result in emotional distress for the individual at risk and his or her family members and the fear of discrimination by employers or insurance companies.

Within the context of pharmacogenetics, however, testing involves searching for genetic variations linked to drug efficacy or toxicity rather than to disease susceptibility. In many instances, this form of testing will carry little risk for ethical, legal, and social concerns. For example, knowledge that a person has a genotype associated with poor response to clopidogrel may be of little consequence because there are alternative therapies available. However, more serious implications may arise if a person is predicted to respond poorly to a drug based on genotype, and treatment options are limited. To address concerns regarding the potential misuse of genetic and pharmacogenetic information by health insurance companies and employers, former President George W. Bush signed the Genetic Information Nondiscrimination Act (GINA) into law in May 2008 (Public Law 110-233). This act prohibits health insurance providers and employers from discriminating against an individual based on genetic information. However, GINA does not protect against discrimination related to disability, life, and long-term care insurance. In addition, it does not apply to employers with fewer than 15 employees. Thus, while GINA may minimize some concerns related to pharmacogenetic testing, ethical concerns and fears associated with pharmacogenetic testing may remain.

Gene Therapy

Many of the ethical concerns with gene therapy center on transgenic manipulation of somatic versus germ line cells. Somatic gene therapy only affects the recipient. That is, genetic alterations introduced by gene therapy are not passed on to future generations. In contrast, with manipulation of germ line cells, alterations are passed on to future children of the treated patient. Some argue that this is unethical because it violates the rights of future generations. Thus, gene therapy in the foreseeable future will focus on somatic gene transfer.

Pharmacogenetics provides opportunities to improve drug therapy outcomes, but requires that clinicians be knowledgeable about genetic determinants of drug response. A challenge to pharmacogenomics implementation is that genotype needs to be considered in the context of important clinical factors, such as age, body size, and concomitant drug therapy,⁹⁸ in making drug therapy decisions. Another challenge is that multiple genetic variants may affect response to some drugs. For example, as described previously, both the CYP2C9 (drug metabolism) and VKORC1 (target site) genes contribute to response to warfarin.

Pharmacists are broadly trained in a number of medication-related areas, including pharmacology, pharmacokinetics, and pharmacodynamics. This places pharmacists in a unique position in dealing with the complexities of the drug-decision process in the era of pharmacogenetics. Pharmacists will be in key positions to play valuable roles on multidisciplinary teams charged with interpreting genetic test results and choosing the most appropriate drug for a given patient based on genotype. Thus, it will be essential for pharmacists to stay abreast of significant pharmacogenetic discoveries and guideline updates.

Recognizing the challenges in healthcare delivery with advancing genetic discoveries, the National Coalition for Health Professional Education in Genetics established core competencies related to genetics for healthcare professionals that are available through the coalition’s website (www.nchpeg.org). The objective of these competencies is to encourage clinicians to incorporate genetics knowledge, skills, and attitudes into their clinical practices. Subsequently, the American Association of Colleges of Pharmacy developed recommendations to guide academic institutions in instilling these competencies in future pharmacists so that pharmacists will be prepared to provide appropriate pharmacotherapy in the age of genomics.⁹⁹

Pharmacogenetics has the potential to greatly improve drug use and therapy outcomes. Clinicians may be able to predict the likelihood that an individual will respond to a particular medication based on the patient’s genotype. Medications may be avoided or prescribed in lower doses with careful monitoring in patients genetically predisposed to their adverse effects. This would be of particular benefit for narrow therapeutic index drugs. For example, warfarin may be initiated at lower doses with closer monitoring in patients with a VKORC1 genotype associated with increased warfarin sensitivity or a CYP2C9 allele associated with reduced warfarin metabolism.

With pharmacogenetics, it also may be possible to eliminate the trial-and-error approach to drug prescribing for many diseases. Instead, clinicians may be able to use genetic information to match the right drug to the right patient at the right dose while minimizing adverse effects. For example, the current approach to hypertension management involves the trial of various antihypertensives until blood pressure goals are achieved with acceptable drug tolerability. Commonly, the initial agent(s) fails to lower blood pressure to goal or produces intolerable adverse effects (Fig. e5-7). Trials of additional or alternative antihypertensive agents must be undertaken until treatment is deemed successful. In the interim, the patient remains hypertensive and at risk for hypertension-related target-organ damage. With pharmacogenetics, clinicians may choose the antihypertensive drug expected to provide the greatest response with the best tolerability for a particular patient based on his or her DNA.

New drugs may be developed based on knowledge about genetic control of cellular functions. For example, the discovery that CML was caused by chromosome translocation and consequent production of an enzyme capable of producing life-threatening lymphocyte levels led to accelerated FDA approval of imatinib (also known as STI-571), an inhibitor of the translocation-created enzyme, for treatment of CML.¹⁰¹ In addition, future drug development may focus on treating specific genetic subgroups instead of broadly treating all individuals with a particular disease. Along these lines, the FDA is encouraging pharmaceutical companies to submit pharmacogenetic data during the drug development process. Ultimately, pharmacogenetics may improve the quality and reduce the overall costs of healthcare by decreasing the number of treatment failures and the number of adverse drug reactions and leading to the discovery of new genetic targets and therapeutic interventions for disease management.

1. The site(s) for genetic variations that may affect drug pharmacodynamics include:

 A. drug metabolizing enzymes

 B. drug target proteins

 C. drug transporter proteins

 D. a and b

 E. a and c

2. The most commonly occurring variant in the human genome is:

 A. single nucleotide polymorphism

 B. tandem repeat polymorphism

 C. nucleotide base deletion

 D. nucleotide base insertion

 E. frameshift mutation

3. CYP2D6 polymorphism can affect:

 A. drug toxicity

 B. drug interaction potential

 C. drug delivery

 D. a and b

 E. b and c

4. Which CYP2D6 phenotype is associated with reduced analgesic response to codeine?

 A. Ultra-rapid metabolizer

 B. Extensive metabolizer

 C. Poor metabolizer

 D. Intermediate metabolizer

 E. None of the above

5. Which of the following is an example of a drug target gene?

 A. TPMT

 B. CYP1A2

 C. SLCO1B1

 D. UGT1A1

 E. VKORC1

6. Which gene is predictive of clopidogrel effectiveness?

 A. CYP2C9

 B. CYP2C19

 C. CYP2D6

 D. CYP3A4

 E. CYP4F2

7. A patient with newly diagnosed atrial fibrillation will be starting warfarin. A rapid SNP test is done and reveals the CYP2C9*2/*3 and VKORC1 AA genotypes. Which of the following responses to warfarin would be predicted based on this genotype?

 A. Increased metabolism; use lower dose

 B. Decreased metabolism; use higher dose

 C. Decreased metabolism and increased sensitivity; use lower dose

 D. Increased metabolism and decreased sensitivity; use higher dose

 E. Increased metabolism and increased sensitivity; avoid warfarin

8. Genetic variations in drug targets may contribute to which drug property:

 A. bioavailability

 B. half-life

 C. peak dose area under the curve

 D. ethnic differences in response

 E. entry into the central nervous system

9. Screening for which of the following polymorphisms is indicated prior to carbamazepine use in a person of Southeast Asian descent?

 A. CYP2D6*2

 B. HLA-B*15:02

 C. CYP2C9*2

 D. TPMT*2

 E. UGT1A1*28

10. Which gene predicts risk for muscle toxicity with simvastatin use?

 A. SLCO1B1

 B. HLA-B

 C. HMG-CoA

 D. LDLR

 E. ABCB1

11. Mutations in which of the following genes increases the risk for thrombosis with oral contraceptives?

 A. Prothrombin

 B. CYP2C9

 C. ADRB1

 D. SCN5A

 E. ABCB1

12. Which drug is recommended to reduce tumor progression in a patient with breast cancer who overexpresses the HER2 gene?

 A. Mercaptopurine

 B. Tamoxifen

 C. Trastuzumab

 D. Voriconazole

 E. Irinotecan

13. An obstacle to successful gene therapy is:

 A. an inability to identify genetic defects

 B. inefficient gene delivery

 C. difficulty identifying eligible patients

 D. lack of research efforts

 E. all of the above

14. Which of the following gene therapy techniques has prompted the most ethical concern?

 A. Naked DNA transfer

 B. Adeno-associated viral gene delivery

 C. Retroviral-mediated gene therapy

 D. Electroporation

 E. Germ line manipulation

1. B.

2. A.

3. D.

4. C.

5. E.

6. B.

7. C.

8. D.

9. B.

10. A.

11. A.

12. C.

13. B.

14. E.