Pharmacogenetics and human genetic polymorphisms€¦ · I and phase II) and also drug transporters...

Biochem. J. (2010) 429, 435–449 (Printed in Great Britain) doi:10.1042/BJ20100522 435

REVIEW ARTICLEPharmacogenetics and human genetic polymorphismsAnn K. DALY1

Institute of Cellular Medicine, Newcastle University Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, U.K.

The term pharmacogenetics was first used in the late 1950s andcan be defined as the study of genetic factors affecting drugresponse. Prior to formal use of this term, there was alreadyclinical data available in relation to variable patient responsesto the drugs isoniazid, primaquine and succinylcholine. Thesubject area developed rapidly, particularly with regard to geneticfactors affecting drug disposition. There is now comprehensiveunderstanding of the molecular basis for variable drugmetabolism by the cytochromes P450 and also for variable glu-curonidation, acetylation and methylation of certain drugs. Someof this knowledge has already been translated to the clinic. Themolecular basis of variation in drug targets, such as receptorsand enzymes, is generally less well understood, although thereis consistent evidence that polymorphisms in the genes encodingthe β-adrenergic receptors and the enzyme vitamin K epoxide

reductase is of clinical importance. The genetic basis of rareidiosyncratic adverse drug reactions had also been examined.Susceptibility to reactions affecting skin and liver appears tobe determined in part by the HLA (human leucocyte antigen)genotype, whereas reactions affecting the heart and muscle may bedetermined by polymorphisms in genes encoding ion channels andtransporters respectively. Genome-wide association studies areincreasingly being used to study drug response and susceptibilityto adverse drug reactions, resulting in identification of some novelpharmacogenetic associations.

Key words: adverse drug reaction (ADR), cytochrome P450,genome-wide association study, human gene polymorphism,human leucocyte antigen (HLA), pharmacogenetics.

INTRODUCTION

Pharmacogenetics can be defined as the study of geneticfactors affecting drug response. There is considerable overlapbetween pharmacogenetics and the much newer discipline ofpharmacogenomics. Pharmacogenomics is sometimes describedas the whole-genome application of pharmacogenetics, which hastraditionally been considered to be concerned with single-geneeffects. Pharmacogenomics may also extend to the developmentof new drugs using genomic information. The terms pharmaco-genetics and pharmacogenomics are often used interchangeably.In the present review article, the term pharmacogenetics is usedthroughout and encompasses both genetic and genomic examples.

The genetic basis for inter-individual variation in drug responsehas been studied extensively over the last 50 years. It is nowrecognized that all human genes are subject to extensive geneticpolymorphism, with many of these polymorphisms resultingin functionally significant effects. A genetic polymorphism isdefined as the occurrence, together in the same population, ofmore than one allele or genetic marker at the same locus withthe least frequent allele or marker occurring more frequentlythan can be accounted for by mutation alone. Generally the lessfrequent marker needs to occur at a population frequency ofat least 1% for the variation to be considered a true geneticpolymorphism, with less frequent variants often classed asisolated mutations. Although many polymorphisms don’t havefunctionally significant effects, those that result in either alteredexpression or activity of the gene product are those usually studiedin pharmacogenetic studies. Most current pharmacogenetic

studies involve study of drug response in unrelated individualsrather than in families. This means that most genetic variantsexamined are seen at the frequency of 1 % or higher, typical ofa polymorphism, rather than the focus being on rare variants, aswould be the case in more traditional genetic studies on rarersingle-gene disorders.

The HapMap project (http://hapmap.ncbi.nlm.nih.gov/), andother approaches, such as genome-wide association studies, haveallowed the identification of a number of novel genetic factorsaffecting disease susceptibility. These developments have alsobeen important in the area of pharmacogenetics although much ofour present detailed knowledge on polymorphisms affecting drugresponse predates these developments. At least in part, this is dueto the fact that methods used to study individual phenotypes inrelation to drug response have been available for many years andthis enabled specific polymorphisms correlating with phenotypesto be identified during the 1980s and 1990s when gene cloningbecame widely available.

The present review article is concerned particularly withrecent developments in the area of pharmacogenetics but willalso consider historical aspects and review the well-establishedknowledge in areas such as the pharmacogenetics of drugmetabolism. The last 5 years has seen considerable progressin understanding the pharmacogenetics of drug transportersand various drug targets, the basis of certain idiosyncraticADRs (adverse drug reactions) and in the use of genome-wide association studies to study drug response. These areaswill be discussed in detail together with some important recentdevelopments in the cytochrome P450 field, such as establishment

Abbreviations used: ABC, ATP-binding-cassette; ACE, angiotensin-converting enzyme; ADR, adverse drug reaction; CBZ, carbamazepine; DILI,drug-induced liver injury; GSTM1, glutathione transferase mu 1; HLA, human leucocyte antigen; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; 5-HT,5-hydroxytryptamine; NAT2, N-acetyltransferase 2; OCT, organic cation transporter; PXR, pregnane X receptor; SJS, Stevens–Johnson syndrome;SLC, solute carrier; SLCO, solute carrier organic anion; SNP, single nucleotide polymorphism; TPMT, thiopurine S-methyltransferase; UGT, UDP-glucuronosyltransferase; VKORC1, vitamin K epoxide reductase complex 1.

1 email [email protected]

c© The Authors Journal compilation c© 2010 Biochemical Society

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436 A. K. Daly

of a major role for the CYP2D6 genotype in predicting response totamoxifen and for CYP2C19 with respect to clopidogrel response.

HISTORICAL ASPECTS OF PHARMACOGENETICS

The foundation for much of modern pharmacogenetics came fromexperiments on chemical metabolism during the 19th century.Among the findings from those studies were that benzoic acidundergoes conjugation with glycine in vivo in both humans andanimals, that some compounds can undergo conjugation withacetate and that benzene is oxidized to phenol in both dogs andhumans (for a review, see [1]).

Observations by Garrod on the possibility of variation inchemical metabolism in the early 20th century have been well-reviewed [2]. The first direct pharmacogenetic study was reportedin 1932 when Snyder [3] reported on the ability to tastephenylthiocarbamide within families and showed that this traitwas genetically determined. The gene responsible for this vari-ation, and its common genetic polymorphisms, have only beenidentified more recently (for a perspective, see [4]). This gene,TAS2R38 (taste receptor, type 2, member 38) encodes a smallprotein found at the apical membrane of taste-receptor cells.Non-tasters of phenylthiocarbamide differ from tasters by threedifferent amino acids [4]. Phenylthiocarbamide shows similarityto prescribed drugs, such as propylthiouracil, but whether this isof relevance in the response to these drugs is not clear.

During the 1950s, reports on drug-specific pharmacogeneticobservations concerned with three widely used drugs, isoniazid,primaquine and succinylcholine, appeared. The earliest reportconcerned primaquine whose use was found to be associated withacute haemolysis in a small number of individuals [5]. Subsequentstudies showed that this toxicity was due to absence of the enzymeglucose-6-phosphate dehydrogenase in red blood cells of affectedindividuals [6]. The molecular genetic basis of this deficiency waslater established by Hirono and Beutler [7] in 1988.

Isoniazid was first used to treat tuberculosis in the early 1950s.As reviewed recently, its use represented an important advance inthe treatment of tuberculosis [8]. Variation between individualsin urinary excretion profiles was described by Hughes [9] andshortly afterwards an association between the metabolic profileand the incidence of a common adverse reaction, peripheralneuritis, was reported, with those showing slow conversion of theparent drug into acetylisoniazid more susceptible [10]. Furtherstudies [11–13] led to the conclusion that isoniazid acetylationwas subject to a genetic polymorphism with some individuals(approx. 10% of East Asians, but 50% of Europeans) being slowacetylators. Slow acetylation was shown to be a recessive trait.As summarized below, the biochemical and genetic basis of slowacetylation is now well understood.

Also during the 1950s, a rare adverse response to the musclerelaxant succinylcholine was found to be due to an inheriteddeficiency in the enzyme cholinesterase [14]. Succinylcholineis used as a muscle relaxant during surgery and those with thedeficiency show prolonged paralysis (succinylcholine apnoea).This observation was then extended by Werner Kalow whoshowed that the deficiency is inherited as an autosomal recessivetrait and developed a biochemical test to screen for the deficiency(see [15] for his own description of that work). The enzymeencoded by the gene involved, which is now usually referredto as butyrylcholinesterase, has been well studied and a numberof different mutations responsible for the deficiency have beenidentified. However, the original biochemical test is still thepreferred method for identifying those affected by succinylcholineapnoea due to both the rarity of the problem and the range ofdifferent causative mutations.

While these initial studies showing the clear role for geneticsin determining adverse responses to primaquine, isoniazid andsuccinylcholine were in progress, the general importance of thearea was increasingly recognised. Motulsky [16] published a keyreview on the relationship between biochemical genetics and drugreactions, which highlighted the adverse reactions to primaquineand succinylcholine, in 1957. The term pharmacogenetics wasfirst used in 1959 by Vogel [17] and was soon adopted by othersworking in the field.

PHARMACOGENETIC FACTORS AFFECTING DRUG DISPOSITION(PHARMACOKINETIC FACTORS)

The best studied polymorphisms are those in genes relevant todrug disposition, especially drug metabolism. This area is oftenreferred to as pharmacokinetics. In this section, pharmacogeneticinformation relevant to the two phases of drug metabolism (phaseI and phase II) and also drug transporters is considered.

Pharmacogenetics of drug oxidation

Overview

Pioneering studies on drug metabolism, especially those in thelaboratories of the Millers [18], and of Brodie and Gillette [19],during the 1950s, showed that many drugs undergo oxidativemetabolism in the presence of NADPH and molecular oxygenin liver microsomes. In 1962, Omura and Sato [20] describedcytochrome P450 from a rat liver microsome preparation as ahaemoprotein, showing a peak at 450 nm in the presence ofcarbon monoxide and dithionite. Shortly afterwards, Estabrookand colleagues showed that cytochrome P450 had steroidhydroxylase activity [21] and further studies confirmed its rolein the metabolism of drugs such as codeine, aminopyrene andacetanilide [22]. At this time, it was still assumed that cytochromeP450 was a single enzyme but evidence for multiple formsemerged in the late 1960s [23,24] with purification of a rangeof rat and rabbit enzymes achieved during the 1970s [25,26].

In the mid 1970s, independent metabolism studies on twonewly developed drugs, debrisoquine and sparteine, in theU.K. by Robert Smith and in Germany by Michel Eichelbaum[27,28], resulted in findings indicating that some individualswere unable to oxidize these drugs, although the majority ofindividuals showed normal metabolism. These studies estimatedthat 10% of Europeans showed absence of activity and the term‘poor metabolizer’ was first used. At this time, the enzyme(s)responsible for this absence of activity were not known, butfurther studies confirmed that the deficiency in metabolism ofboth drugs co-segregated [29] and that the trait was inheritedrecessively [30]. It became clear that a number of different drugs,including tricyclic antidepressants, were also metabolized by thisenzyme [31]. Studies on human liver microsomes confirmed thatthe enzyme responsible was a cytochrome P450 [32,33] and thisenzyme was then purified to homogeneity [34]. The availab-ility of antibodies against the purified protein facilitated thecloning of the relevant cDNA [35]. On the basis of emergingresults on cytochrome P450 genes, it was agreed that the geneencoding the debrisoquine/sparteine hydroxylase should be ter-med CYP2D6. Studies on human genomic DNA led to theidentification of several polymorphisms in CYP2D6 associatedwith the poor-metabolizer phenotype, including the most commonsplice site variant, a large deletion and a small deletion [36–40]. An additional contribution to the field was made in 1993by Johansson and colleagues who described the phenomenon ofultrarapid metabolizers with one or more additional copies


Pharmacogenetics and human genetic polymorphisms 437

of CYP2D6 present [41]. These ultrarapid metabolizers had beenpreviously identified on the basis of a poor response to tricyclicantidepressants. The elucidation of the mechanism underlying thisrapid metabolism was one of the first accounts of copy numbervariation in the human genome.

In a similar approach to that used in the discovery ofthe CYP2D6 polymorphism, Kupfer and Preisig [42] foundthat some individuals showed absence of metabolism ofthe anticonvulsant S-mephenytoin. It was demonstrated thatS-mephenytoin metabolism did not co-segregate with that ofdebrisoquine and sparteine with this polymorphism being dueto a separate gene defect. Identification of the gene responsiblefor S-mephenytoin hydroxylase proved difficult initially, probablybecause the relevant enzyme was expressed at a low level in theliver. The gene, now termed CYP2C19, was cloned in 1994 andthe two most common polymorphisms associated with absence ofS-mephenytoin hydroxylase activity identified [43,44].

A number of other cytochrome P450 genes are now knownto be subject to functionally significant polymorphisms. In thecase of one of these, CYP2C9, which metabolizes a range ofdrugs including warfarin, tolbutamide and non-steroidal anti-inflammatory drugs, some evidence for the existence of apolymorphism appeared in 1979 when a trimodal distribution fortolbutamide metabolism was reported [45]. Subsequently, it wasshown that tolbutamide metabolism was distinct fromdebrisoquine metabolism [46]. The enzyme metabolizing tol-butamide was purified and cloned, and later named CYP2C9[47,48]. Analysis of CYP2C9 cDNA sequences provided evidencefor the presence of coding region polymorphisms resulting inamino acid substitutions, with expression studies suggested thesewere functionally significant [47,49,50]. Genotyping of patientsundergoing treatment with warfarin, another CYP2C9 substrate,confirmed the functional importance of the two most commoncoding region CYP2C9 polymorphisms [51–53].

Current knowledge on cytochrome P450 polymorphisms

Collectively, the cytochromes P450 are the most important groupof phase I metabolizing enzymes and it has been estimatedthat up to 80% of all prescribed drugs undergo oxidationreactions catalysed by these enzymes [54]. In terms of thesedrug metabolism reactions, CYP3A4 has the most important roleand has been estimated to be responsible for approx. 50 % ofall cytochrome P450-mediated metabolism of prescribed drugs[55], with CYP2D6 and CYP2C9 responsible for approx. 25and 20% respectively [56,57]. More detail on drug substratesmetabolized by these isoforms is provided in Table 1. There isalso a smaller contribution to drug metabolism from CYP1A2,CYP2A6, CYP2B6, CYP2C8, CYP2C19 and CYP3A5. Assummarized in Table 1, four CYPs that contribute to drugmetabolism, CYP2D6, CYP2C19, CYP2A6 and CYP3A5, aresubject to polymorphisms leading to absence of enzyme activityand in addition CYP2C9 activity is very low, although notcompletely absent, in some individuals due to two commonpolymorphisms. There are also a large number of polymorphismsleading to smaller changes in cytochrome P450 activities [55].Current knowledge of phenotype–genotype relationships withinthe cytochrome P450 family is now more comprehensive thanfor the majority of human genes, although better understandingof some aspects, such as regulation of gene expression, is stillneeded.

As CYP3A4, CYP2D6 and CYP2C9 are the most importantenzymes in terms of drug metabolism, pharmacogenetic factorsaffecting levels of these enzymes are considered in the presentreview in detail, together with those affecting CYP2C19, which

is less important in terms of drug metabolism generally, butwhich has been well studied from the pharmacogenetic standpoint.CYP3A4 does not appear to be subject to polymorphisms thatresult in absence of activity, although it is well establishedthat there is considerable inter-individual variation in overalllevels of activity. This variability may be due to a number ofdifferent factors, including genetic polymorphisms. A substantialnumber of variant CYP3A4 alleles have now been describedincluding some associated with non-synonymous mutations (seethe CYP allele database; http://www.cypalleles.ki.se/). Several ofthese give rise to alterations in catalytic activity. However, thenon-synonymous mutations that have been described are seenat low population frequencies and therefore seem unlikely to beable to explain inter-individual variation in CYP3A4 activity fully.A number of upstream polymorphisms have also been detectedwith one being common (–392A > G, the CYP3A4*1B allele).The precise functional significance of this polymorphism remainsunclear, but one study has found significantly lower metabolismof quinine in carriers of this allele compared with wild-typeindividuals [59]. This may be due to decreased binding of nuclearproteins in the region of the polymorphism [59]. It is possible thatinter-individual variation in hepatic levels of CYP3A4 could beexplained in part by polymorphisms in one of its transcriptionalregulators, the PXR (pregnane X receptor) or NR1I2, a memberof the nuclear receptor superfamily (for a review, see [60]).Some of the observed inter-individual variability in CYP3A4levels could be due to inter-individual variation in ability to induceCYP3A4 as a result of polymorphisms in PXR and/or to inter-individual variation in levels of endogenous PXR ligands. Studieson polymorphism of PXR have identified several SNPs (singlenucleotide polymorphisms) that appear to affect individual abilityto induce CYP3A4 [61].

An additional genetic factor that may affect overall CYP3A-related metabolism is the CYP3A5 gene which is expressed in onlyapprox. 10 % of Europeans [62]. Polymorphisms in CYP3A5 thatexplain the basis of the variation in expression of this gene havebeen well studied. In particular, a polymorphic site in intron 3results in a 6986A > G polymorphism. Individuals positive for G(the CYP3A5*3 allele) at this position do not express CYP3A5due to the creation of a cryptic splice site that results in theincorporation of intron sequence in the mature mRNA, resultingin the production of a truncated protein [63]. This allele is verycommon in all ethnic groups examined up to date. CYP3A5*6,which also results in abnormal splicing, and CYP3A5*7, whichhas a frameshift, are rarer alleles, but appear to explain theabsence of CYP3A5 expression in some African-Americans [63].It remains unclear whether variable expression of CYP3A5 canexplain the wide inter-individual variation seen in overall CYP3Aactivity towards a number of substrates.

Over 80 different allelic variants of CYP2D6 have beenidentified and characterized (CYP allele database). Approx.95% of European poor metabolizers have two copies of anycombination of four alleles termed CYP2D6*3, CYP2D6*4,CYP2D6*5 and CYP2D6*6, which each encode defective formsof CYP2D6 [64,65]. The remaining 5 % of poor metabolizersare homozygous or heterozygous for a range of different loss-of-function alleles, with each individual allele relatively rare.Most inactivating mutations in CYP2D6 are either point mutationsresulting in splicing defects or deletions which lead to either atruncated protein or no protein at all being synthesized.

Many individuals fall into the category of intermediatemetabolizers. This phenotype is particularly common in certainAfrican regions and in East Asians. Intermediate metabolizersmay be either heterozygous for one of the inactivating mutationsdescribed above or homozygous for alleles associated with


438 A. K. Daly

Table 1 CYP genes and polymorphic drug metabolism

The CYP genes listed have well characterized roles in pharmacogenetic studies on drug metabolism. Other CYP genes also contribute to drug and xenobiotic metabolism or have physiological roles,but pharmacogenetic data is more limited. References are to review articles

Isoform Examples of drug substrates Nature of polymorphism Effect on activity References

CYP1A2 Clozapine, olanzapine theophylline andcaffeine

Polymorphisms in non-coding sequencesmay affect expression or induction.

No absence of activity reported. Some effectson expression or induction.

[55,190]

CYP2A6 Coumarin and nicotine Non-synonymous mutations, large deletionand upstream polymorphisms.

Absence of activity seen at low frequency.Ultrarapid metabolizers may also occur.

[55,190]

CYP2B6 Cyclophosphamide and efavirenz Non-synonymous and upstreampolymorphisms.

No absence of activity reported. Variation inactivity common.

[55,191,192]

CYP2C8 Paclitaxel, retinoic acid, rosiglitazone andrepaglinide

Non-synonymous and upstreampolymorphisms.

No absence of activity reported. Variation inactivity common.

[55,190,193]

CYP2C9 Warfarin, ibuprofen, diclofenac, tolbutamideand phenytoin

Non-synonymous polymorphisms common.Also upstream polymorphisms.

Very low activity in some individuals. [55,57,190,194,195]

CYP2C19 Omeprazole, diazepam, clobazam andclopidogrel

Splice site, initiation codon, non-synonymousand upstream polymorphisms.

Absence of activity common. Some ultrarapidmetabolizers.

[55,92,94]

CYP2D6 Codeine, amitriptyline, nortriptyline,tamoxifen, metoprolol, timolol, tropisetron,dextromethorphan, atomoxetine,venlafaxine thioridazine, zuclopenthixol,perphenazine, risperidone and haloperidol.

Splice site, small and large deletions andnon-synonymous polymorphisms.Duplication and other amplifications.

Absence of activity common. Some ultrarapidmetabolizers.

[55,190,195,196]

CYP2E1 Ethanol and isoniazid Non-synonymous polymorphisms rare.Upstream polymorphisms common.

No absence of activity reported. Somevariation in activity.

[55,190]

CYP3A4 Midazolam, cyclosporine, tacrolimus,erythromycin, nifedipine, simvastatinatovastatin, diltiazem verapamil, vincristineand dapsone

Non-synonymous polymorphisms rare.Upstream polymorphisms common.

No absence of activity reported. Somevariation in activity.

[55,197,198]

CYP3A5 Broadly similar to CYP3A4 above Non-synonymous polymorphisms rare. Splicesite polymorphisms common.

Absence of activity common. [55,62,197,198]

impaired metabolism. The best studied alleles associatedwith impaired metabolism are CYP2D6*10, which is commonin East Asia, and CYP2D6*17, which is common in Africanpopulations. Both alleles have non-synonymous polymorphismsthat result in a less catalytically active gene product. A study ofthe activity of the variant enzymes with a range of substrateshas found that the extent of difference in catalytic activitybetween these isoforms and the reference ‘wild-type’ variantdepends on the individual substrate [66]. In European populations,two alleles associated with impaired metabolism, CYP2D6*9and CYP2D6*41, are relatively common. CYP2D6*9 encodesa protein with an amino acid deleted. CYP2D6*41 includesseveral different polymorphisms, including two non-synoymousmutations which are also seen in the CYP2D6*2 allele, anupstream polymorphism at position −1584 and a base substitutionin intron 6. The non-synonymous mutations characteristic ofCYP2D6*2 appear not to affect enzyme activity, but theintron 6 polymorphism has been demonstrated to be associatedwith altered RNA splicing, leading to lower levels of protein[67].

Ultrarapid metabolizers were originally identified on thebasis of their extremely fast clearance of the antidepressantdesmethylimipramine. Some individuals in this category have13 copies of CYP2D6, arranged as tandem repeats, but a singlegene duplication event is more commonly associated with theultrarapid phenotype [41]. Depending on the country of origin,1–8% of Europeans have one extra copy of the CYP2D6*1 orCYP2D6*2 alleles resulting in faster than average metabolism[68,69]. Subjects with three to five tandem copies of CYP2D6*2have also been detected, mainly in African populations [70].

It is now clear that the number of active CYP2D6 alleles presentin an individual’s genome is highly predictive of actual CYP2D6enzyme activity, with those heterozygous for poor metabolizeralleles showing lower levels of activity compared with those

Figure 1 Pharmacokinetics of nortriptyline in healthy volunteers of definedCYP2D6 genotype

The study involved administration of a single oral dose of nortriptyline (25 mg) followedby blood sampling at the time points shown. The number of active CYP2D6 alleles wasdetermined by genotyping and is shown in the Figure. Adapted from Dalen et al. [71] withpermission from Macmillan Publishers Ltd. Clinical Pharmacology and Therapeutics c© 1998.(http://www.nature.com/clpt/).

with two normal alleles. This gene-dose effect was illustratedby a pharmacokinetic study on the antidepressant nortriptyline(Figure 1), which confirmed the intermediate level of metabolismin heterozygotes and also the increased rate of metabolism typicalof ultrarapid metabolizers [71].

Despite the fact that the CYP2D6 polymorphism was initiallyidentified over 30 years ago and that it has been possibleto identify most of those with the genetic deficiency for the last20 years, CYP2D6 genotyping has so far failed to enter routineclinical practice. There are a number of possible reasons for this.



These include the general difficulty of introducing genetic testsinto clinical practice, the fact that a number of key CYP2D6substrates have been withdrawn from the market due to theproblems experienced by poor metabolizers (e.g. phenformin andperhexiline) and that certain classes of CYP2D6 substrates, suchas the tricyclic antidepressants, are less commonly used thanwhen the polymorphism was first described. Guidelines for doseadjustment for antidepressant drugs on the basis of the CYP2D6genotype have been formulated, but have not yet been tested inclinical trials [72]. However, there is now increasing new evidencethat the CYP2D6 genotype may be relevant to the outcome oftreatment with codeine and tamoxifen.

The widely used analgesic drug codeine is an importantCYP2D6 substrate. It is activated to morphine exclusivelyby CYP2D6 and this is generally accepted to be essentialto achieve analgesia (for a review, see [73]). Two case-reports concerning excessive activation of codeine in ultrarapidmetabolizers with one additional copy of CYP2D6 have appeared.In the first, a patient prescribed a cough medicine containingcodeine suffered life-threatening opioid intoxication [74]. Whengenotyped, this patient was found to have at least three copiesof CYP2D6. The second concerned the death of a breast-fed baby 13 days after birth [75]. His mother was prescribedcodeine as an analgesic post-delivery. Post-mortem examinationof stored breast milk samples showed a morphine level at least4-fold higher than expected. The mother was found to havea CYP2D6 gene duplication, which would explain the higherthan expected morphine level in her milk, but the infant wasan extensive metabolizer. A study on codeine administrationto healthy volunteers of known CYP2D6 genotype showedthat ultrarapid metabolizers were significantly more likely thanextensive metabolizers to suffer sedation [76]. It therefore appearsthat in patients requiring treatment with codeine and relatedcompounds, CYP2D6 genotyping followed by dose adjustmentor prescription of an alternative drug could be beneficial in bothavoiding dangerous intoxication and lack of response. There arerecent data from a case-control study suggesting that babies ofmothers with ultrarapid metabolizer CYP2D6 genotypes, who usecodeine as an analgesic, are more likely to suffer central nervoussystem depression than other babies [77]. However, there is also apossibility that high levels of morphine described in a case-reporton neonatal death could have resulted from concurrent intakeof codeine and heroin rather than a problem CYP2D6 genotype[78].

Tamoxifen is a widely used treatment for hormone-receptor-positive breast cancer. Its metabolism is complex but ithas been recognized that CYP2D6 produces a 4-hydroxy-N-desmethyltamoxifen metabolite (endoxifen) [79]. Endoxifen isfound at high plasma levels in many patients and appearsto bind strongly to oestrogen receptors suggesting it isimportant in the biological response to tamoxifen [80]. Thereis now considerable evidence that patients positive for twoCYP2D6 poor metabolizer alleles show an increased incidenceof breast cancer relapse [81–83].

In the case of CYP2C9, at least 33 variant alleles have nowbeen identified (CYP allele database). With the exception oftwo of these (CYP2C9*6 and CYP2C9*31), all the describedCYP2C9 variant alleles giving rise to decreased activity areassociated with a single non-synonymous base change. The twomost common variant alleles are CYP2C9*2 and CYP2C9*3 and,among Northern Europeans, over 30 % of the population arepositive for one or two of these alleles. An overall allele frequencyfor CYP2C9*2 of 10% compared with 8 % for CYP2C9*3 wasfound in Europeans [53,84]. Both CYP2C9*2 and CYP2C9*3occur more rarely in other ethnic groups including East Asians

and African-Americans and the other variant alleles are all seenat population frequencies in the order of 1% [85].

CYP2C9 polymorphisms are particularly relevant to themetabolism of drug substrates with narrow therapeutic indices,where ADRs are common, such as to warfarin and phenytoin. Inthe case of warfarin, individualization of dose on the basis of drugresponse is a standard procedure, but a large number of studieshave now found a consistent relationship between warfarin doserequirement and the CYP2C9 genotype [86]. In general, studiessuggest that the CYP2C9 genotype contributes between 10 and20% of the variability in warfarin dose requirement comparedwith a 20–30% contribution from the VKORC1 (vitamin Kepoxide reductase complex 1) genotype [87] (see the section onDrug target polymorphisms below).

As discussed above, the phenotypic polymorphism affectingthe metabolism of the anticonvulsant drug mephenytoin wasdescribed in the early 1980s [42]. The enzyme responsible forS-mephenytoin hydroxylation was later shown to be encoded bythe CYP2C19 gene and two relatively common variant allelesassociated with absence of enzyme activity were identified[43,44]. Other rarer alleles associated with either no activityor decreased activity were identified subsequently (CYP alleledatabase). The two common alleles are associated with productionof truncated proteins, although absence of activity can alsoarise due to amino acid substitutions [88]. Individuals with theCYP2C19 deficiency have two variant alleles present, althoughheterozygotes may show impaired metabolism. CYP2C19 isresponsible for the metabolism of a relatively small numberof commonly prescribed drugs, including the proton pumpinhibitor omeprazole. Individuals with an absence of CYP2C19activity show a better response to treatment of peptic ulcerwith this drug compared with those with one or two normalalleles [89], apparently due to higher drug levels in the poormetabolizer group. Several recent studies indicate that theantiplatelet agent clopidogrel is less effective in individuals withat least one defective CYP2C19 allele probably because of amajor role for CYP2C19 in activation of this prodrug [90–93]. Some benzodiazepines including diazapam and clobazamare CYP2C19 substrates and individuals defective in CYP2C19may be at risk of toxicity, such as over-sedation [94].The antidepressants citalopram and escipalopram are mainlymetabolized by CYP2C19 and patients heterozygous for variantalleles show higher serum levels of the parent drug [95]. Thevariant allele CYP2C19*17 includes an upstream polymorphismthat apparently increases transcription levels and is associatedwith higher levels of gene expression [96]. This variant appears tobe associated with faster than normal metabolism of omeprazoleand the antidepressant escitalopram [97,98].

Despite a good understanding of the relationship betweenphenotype and genotype for cytochromes P450, and the likelyrelevance of some genetic polymorphisms to the outcome of drugtreatment, genotyping in patients undergoing treatment withdrugs known to be substrates for cytochromes P450 associatedwith altered metabolism is still very limited. However, there isincreasing interest in CYP2C9 genotyping prior to treatment withwarfarin [99] (also see the section on Polymorphisms in targetenzymes below). Guidelines for dose adjustment on the basis ofthe CYP2D6 and CYP2C19 genotype for a number of drugs usedin psychiatry have also appeared [72], and there is considerableevidence that CYP2D6 deficiency may affect the outcome oftamoxifen treatment [83] (see above). The recent observationof a poorer response to clopidogrel treatment in CYP2C19-deficient patients has suggested genotyping for polymorphismsin this gene prior to prescription of clopidogrel may be of value[92].


440 A. K. Daly

Table 2 Phase II polymorphisms relevant to drug response

The polymorphisms listed are those directly relevant to drug response. References are to review articles

Gene Substrates Polymorphism Effect References

UGT1A1 Bilirubin and irinotecan Upstream TA insertion or non-synonymous SNP in exon 1 Decreased activity [199,200]UGT2B7 Morphine Upstream and non-synonymous SNPs Possible decrease in activity [200]NAT2 Isoniazid Non-synonymous SNPs No activity [201,202]TPMT Mercaptopurine Non-synonymous SNPs No activity [203]

Non-cytochrome-P450-mediated drug oxidation

Functionally significant polymorphisms have been described ingenes encoding other enzymes of phase I metabolism in additionto the cytochromes P450, including the flavin-linked mono-oxygenases, esterases, amine oxidases and dehydrogenases.However, in the majority of cases, these polymorphisms affectenzymes with only a minor involvement in metabolism ofprescribed drugs and are not considered further in the presentreview.

Pharmacogenetics of drug conjugation

As discussed in the Introduction section, a polymorphismaffecting conjugation of drugs, such as isoniazid with acetyl-CoA,had been known to exist since the 1950s. Other polymorphismsaffecting conjugation reactions of phase II metabolism weresubsequently described from phenotyping studies. In particular,Weinshilboum and Sladek [100] identified several polymorphismsaffecting methylation of xenobiotics and endogenous compoundsby measurement of enzyme activities in blood cells. Thoseauthors described the most pharmacologically importantof these methylation polymorphisms, in TPMT (thiopurineS-methyltransferase), in 1980; approx. one in every 300Europeans lack this enzyme, with lower activity observedin heterozygotes. Other conjugation polymorphisms identifiedby phenotypic approaches included a deficiency in GSTM1(glutathione transferase mu 1), which affects 50% of Europeansand was originally detected by measurement of trans-stilbeneoxide conjugation in lymphocytes [101]. A classic paper byMotulsky [16] on genetic variability in metabolism mentioned themild hyperbilirubinaemia described by Gilbert and Lereboullet[102] in 1901, usually referred to as Gilbert’s syndrome. Thissyndrome was later shown to relate to impaired glucuronidationactivity by a form of UGT (UDP-glucuronosyltransferase) andthere were suggestions that glucuronidation of prescribed drugsmight also be affected in [103]. This has been confirmed morerecently by studies on toxic responses to the anti-cancer drugirinotecan (for a review, see [104]).

With the development of molecular cloning techniques, thebasis of the various conjugation polymorphisms known previouslybecame clear (during the late 1980s and early 1990s), andevidence also emerged for additional functionally significantpolymorphisms by sequence comparisons. The molecular basisof the GSTM1 deficiency was established quite early in 1988,probably because it is due to a large gene deletion that was readilydetectable by a number of different approaches [105]. Cloning ofthe NAT2 (N-acetyltransferase 2) cDNA, encoding the enzymeresponsible for isoniazid metabolism, was achieved in 1991, withtwo common variant alleles with several base substitutions in theircoding regions found to be associated with absence of activity[106]. Other inactive variants were also identified [107,108]. Inthe case of TPMT, gene cloning and identification of two commonalleles, each associated with absence of activity due to amino acid

substitutions, was achieved in 1996 [109,110]. The most commonvariant allele giving rise to Gilbert’s syndrome was found tobe a TA insertion in the promoter region of the UGT1A1 gene(UGT1A1*28 allele), which encodes the major UGT responsiblefor bilirubin conjugation [111].

Table 2 summarizes the main phase II polymorphisms thatappear to be most relevant to drug metabolism on the basis ofcurrent evidence. Genotyping for the TPMT polymorphisms inpatients being prescribed 6-mercaptopurine or azathioprine andthe UGT1A1*28 allele in patients receiving irinotecan is nowrecommended, but not mandated, by the U.S. Food and DrugAdministration; knowledge of genotype can enable either doseadjustment or an alternative drug to be prescribed.

Pharmacogenetic studies on drug transporters

In addition to phase I and phase II metabolizing enzymes,drug transporters also contribute to drug disposition andtheir pharmacogenetics has been well studied in recent years.Transporter proteins that have roles in both inward and outwardtransport of drugs and their metabolites in cells relevant todrug disposition, such as hepatocytes, are of considerablepharmacogenetic interest. Those concerned with outward drugtransport are mainly members of the ATP-dependent ABCtransporter (ATP-binding-cassette transporter) family. Reports ofa number of polymorphisms affecting the function of the well-characterized ABCB1 gene encoding the transporter P-glyco-protein first appeared in 2000 [112]. There have been a largenumber of follow-up studies on the functional consequences ofpolymorphism in ABCB1, although the literature remains unclearon the precise consequences of these polymorphisms, especiallythe well studied 3435G > T, a synonymous polymorphism that hasbeen suggested to affect mRNA stability [113]. The relevance ofABCB1 genotype to drug disposition remains somewhat unclear.Pharmacogenetic studies on other ABC transporters, such asABCC2, ABCB4 and ABCB11, have also been reported; thereis limited evidence that certain coding and non-coding ABCC2polymorphisms are functionally significant [114] and clearerevidence that rare variants in ABCB4 and ABCB11 contributeto inherited forms of cholestasis [115].

The drug transporters of the SLCO/OATP (solute carrierorganic anion transporter) family, which are mainly concernedwith the inward transport of drugs into cells like hepatocytesand renal tubular cells, have also been the subject of recentpharmacogenetic analysis. The anionic transporters of the SLCO1family have been extensively characterized and the genotypefor certain variants appears to be clinically important [116].SLCO1B1 codes for the transporter OATP1B1, which transportsanionic drugs into cells and is found in the hepatocyte sinuosoidalmembrane. A non-synonymous polymorphism in SLCO1B1appears to affect the pharmacokinetics of several widely useddrugs [116]. These include some, but not all, statins and thispolymorphism may also be relevant to statin-related ADRs



(see the section on Idiosyncratic ADRs below). SLC22A1 andSLC22A2 (where SLC is solute carrier) encode the cationictransporters OCT1 (organic cation transporter 1) and OCT2.These transporters play important roles in transport of cationicdrugs into hepatocytes and renal tubule cells respectively. Thereis increasing evidence that coding region polymorphisms in thesegenes are relevant to metformin response and to cisplatin-inducednephrotoxicity [117,118].

PHARMACOGENETIC FACTORS AFFECTING DRUG TARGETS(PHARMACODYNAMIC FACTORS)

Progress on the pharmacogenetics of drug receptors and othertargets, such as enzymes and neurotransmitter transporters, hasbeen slower than studies on drug metabolism and transport mainlybecause phenotypic evidence for the existence of functionallysignificant polymorphisms was generally not available. However,data from the human genome sequencing project has providednew insights into this area.

Polymorphisms in receptors

In the case of receptors, the majority of studies have focussed onmetabotropic receptors, which are G-protein-coupled receptorswith a characteristic seven-transmembrane domain structure,particularly dopamine, 5-HT (5-hydroxytryptamine, also knownas serotonin) and adrenergic receptors. Pharmacogeneticinformation on other receptor types, such as the ionotropicnicotinic acetylcholine receptor or steroid hormone receptors,is still very limited and will not be considered further.Polymorphisms in the various adrenergic receptors have beendemonstated to be of considerable relevance to drug response,especially in the case of the β2-adrenergic receptor [119].The relationship between polymorphisms in dopamine and5-HT receptors and drug response is still less well established,although overall knowledge on variation in these receptors isextensive [120]. This section is therefore mainly concerned withpolymorphisms affecting β-adrenergic receptors.

β1-adrenergic receptors are the major adrenoreceptor classfound in the heart with a key role in regulation of heart rate.The ADRB1 gene has a common non-synonymous polymorphismleading to a substitution at codon 389 (G389A) that has beenwell studied. Although the first cDNA clone isolated encoded theglycine variant, the frequency of the arginine variant is higher(approx. 70% in Europeans) [121,122]. For the Arg389 variant,transfection studies demonstrated slightly higher basal levels ofexpression compared with the glycine form, but when stimulatedwith isoproterenol, the arginine variant showed approx. 3-foldhigher levels of activity [121].

As the G389A polymorphism is common and clearlyfunctionally significant, it has formed the basis for a large numberof clinical studies, particularly on the relationship betweengenotype and response to β-receptor antagonist treatment. Severalstudies have shown significant differences in response on thebasis of genotype, both in studies on hypertension and on heartfailure. A general conclusion from these studies is that patientshomozygous for the Gly489 variant may show a poor response toβ-antagonists and might benefit from higher drug doses, but, assuggested recently [123], larger prospective studies are needed toassess the clinical relevance of this concept.

The β2-adrenergic receptor is particularly well studied from thepharmacogenetic standpoint, probably due to its importance as atarget for β-agonists in asthma. The receptor is encoded by theADRB2 gene, which appears unusually polymorphic compared

with other adrenergic receptors, especially in the coding sequence;this has resulted in considerable interest in assessing the relevanceof these polymorphisms to drug response.

There are at least nine polymorphisms in the coding region withfour of these non-synonymous polymorphisms. In addition, thereis a single non-synonymous polymorphism in the region encodingthe leader peptide and a number of upstream polymorphisms closeto the transcription start site. A limited number of haplotypeshave been observed with only four showing frequencies above5% in any population studied to date, but there is considerablevariation between haplotype frequency in different ethnic groups,especially those of European, African and East Asian origin [124].Among Europeans, three haplotypes are common. Two of thesediffer mainly at codon 16 with one sequence encoding a glycineresidue and the second an arginine residue at this position. Mostclinical studies have focussed on the codon 16 polymorphism,probably because this SNP is seen at a relatively high populationfrequency and also was the first coding region polymorphism tobe identified.

Although the codon 16 polymorphism was identified over15 years ago, data on its functional significance is quite limitedand rather contradictory. Early studies involving transfection ofcell cultures suggested that the Gly16 variant showed enhanceddown-regulation following agonist binding compared with theArg16 form [125]. This down-regulation is the main mechanismby which long-term agonist-promoted desensitization of β2-adrenergic receptors occurs [126]. More recently, in a numberof separate studies using either cells (lymphocytes and lung cells)of known genotype or genotyped patients receiving β2-adrenergicreceptor-agonist treatment for asthma, it was found that the Arg16-containing form of ADRB2 was associated with increased agonist-induced down-regulation and with a poorer response to inhaledshort-acting beta agonists [127–130]. The poorer response to β2-adrenergic receptor agonists appears to be specific to short-actingagonists as a large study involving patients being treated withlonger-acting agonists and with an inhaled steroid for asthmafailed to show any difference in outcome on the basis of thecodon 16 genotype [131]. In general, it is now accepted thatthe codon 16 genotype does appear to affect response to shorter-acting agonists, but that this may not be clinically important ascurrent treatments mainly involve use of longer-acting agonists.Further studies would be useful in resolving the questionof whether genotyping patients taking β2-adrenergic receptoragonists for polymorphisms in ADRB2 and personalizing theirtreatment on this basis is of benefit.

The ADRB2 genotype may also be relevant in the response toβ-adrenergic receptor antagonists. However, as most antagoniststend to have equal affinities for β1 and β2-receptors, or preferentialinteraction with β1 receptors, assessing the contribution of ADRB2genotype to the response is more difficult. However, a fewstudies have studied this aspect in patients receiving β-adrenergicreceptor antagonists for treatment of heart failure or hypertension.Most studies, especially larger more recent ones, have failed todetect any difference in response in relation to genotype for eithercodon 16 or codon 27 of ADRB2 [132–134].

Polymorphisms in target enzymes

As well as contributing to drug metabolism, enzymes areimportant drug targets. Widely used drugs targeting enzymesinclude the coumarin anticoagulants, which inhibit vitaminK epoxide reductase, ACE (angiotensin-converting enzyme)inhibitors, statins which inhibit HMG-CoA (3-hydroxy-3-methyl-glutaryl-CoA) reductase and nonsteroidal anti-inflammatorydrugs which inhibit prostaglandin H-synthases I and II.


442 A. K. Daly

Figure 2 Distribution of warfarin doses required to achieve stable anticoagulation in relation to CYP2C9 and VKORC1 genotype

The box plot shows the distribution of warfarin dose by CYP2C9 and VKORC1 genotype. Boxes indicate the median and interquartile ranges. Vertical lines above and below boxes indicate theminimum and maximum values. The numbers above whiskers show mean values. Outliers are shown by asterisks. This figure was originally published in Blood [140]. Sconce et al. The impact ofCYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon warfarin dose requirements: proposal for a new dosing regimen. Blood. 2005; 106: 2329–2333. c© the AmericanSociety of Hematology.

Vitamin K epoxide reductase, the target for coumarin anti-coagulants, including warfarin, is another example of a gene withwell established pharmacogenetics. Limited phenotypic data fromthe 1970s suggested that the warfarin response was subject tointer-individual variation with resistance to the drug occurring insome families [135]. The gene encoding this enzyme VKORC1,was identified only in 2004 [136,137], but this was followedquickly by identification of isolated mutations associated withwarfarin resistance and also common genetic polymorphismsaffecting the response to anticoagulants [138–140]. Dosing withwarfarin and other coumarin anticoagulants involves titrationof dose until the desired level of anticoagulation is achieved.The dose required to achieve stable anticoagulation is subjectto considerable inter-individual variation with a more than100-fold variation seen. It is now clear that the genotype ofVKORC1, and also of the cytochrome P450 CYP2C9 (see thesection on Pharmacogenetics of drug oxidation above) accountsfor up to 40% of the inter-individual variation in coumarinanticoagulant dose with age, body surface-area and interferingconcomitant medications together accounting for an additional15–20% of variation (for reviews, see [86,141]). Figure 2demonstrates that VKORC1 and CYP2C9 genotypes are eachseparate genetic predictors of warfarin dose requirement. Thereare recent recommendations in the U.S.A. that genotyping forCYP2C9 and VKORC1 may be helpful in setting warfarin dosingand clinical trials comparing dosing tailored to genotype areunderway in several countries [99].

HMG-CoA reductase converts HMG-CoA into mevalonicacid, which is the rate-limiting step in the biosynthesis ofcholesterol [142]. It is also the target enzyme for the statins,which act to lower plasma cholesterol levels. In view of thelarge number of patients worldwide now taking statins,the possibility that pharmacogenetic factors, expecially thoseaffecting the target, might determine response is of considerableinterest. Two polymorphisms in non-coding regions of HMGCR,the gene encoding HMG-CoA reductase, are associated witha decreased response to statin treatment and form part of arelatively rare haplotype including a polymorphism in the 3′-untranslated sequence which may affect RNA stability [143]. The

haplotype frequency is approx. 7% in the US population, butis more common in African-Americans than in white Americans[144]. The low haplotype frequency means that, to date, it hasonly been possible to study statin response in heterozygotes,due to homozygous mutants being extremely rare. The originalstudy reported that this haplotype results in a significantlysmaller reduction in both total cholesterol and LDL (low-densitylipoprotein)-cholesterol in response to pravastatin treatment andseveral more recent studies have attempted to replicate this forboth pravastatin and other statins [143–148]. Only two of thefive more recent studies have confirmed the original findings[144,148], but there are limitations with several of the otherstudies.

Pharmacogenetic studies on drug response have also beenperformed on other genes coding for drug targets, such as ACE[149] and the prostaglandin H synthases [150]. However, thesewill not be discussed further as the results are limited andsomewhat contradictory.

PHARMACOGENETICS OF ADRs

Idiosyncratic reactions, often referred to as type B ADRs, whichare not directly predictable from drug concentration [151], aredifficult to study because of their rarity, but their potentially veryserious consequences for the patient makes them an importantcurrent research area in pharmacogenetics. The immune system,especially the products of class I and II HLA (human leucocyteantigen) genes, often contributes to idiosyncratic reactions, butthis is not the case for all reactions of this type [152]. ADRs linkedto drug concentration (type A reactions) are more common thanidiosyncratic reactions and are also important clinically. These aremore likely to be associated with polymorphisms affecting drugmetabolism or drug transport.

As summarized in Table 3, there are a number of differenttypes of idiosyncratic ADRs. Usually particular drugs areassociated with one class of toxicity, although there are someexamples where more than one type of toxicity can occur with aparticular drug. Examples of commonly prescribed drugs linkedto the different toxicities are also provided.



Table 3 Common idiosyncratic adverse drug reactions

Type Drug examples Reference

DILI Co-amoxiclav, flucloxacillin and isoniazid [204]Drug-induced QT prolongation Thioridazine, clarithromycin and terfenadine [176]Drug-induced hypersensitivity Abacavir [205]Drug-induced muscle toxicity Statins, chloroquine and penicillamine [206]Drug-induced serious skin rash Carbamazepine and co-trimoxazole [207]

DILI (drug-induced liver injury) is a rare but clinicallyimportant problem. A U.S.-based study suggested that DILIaccounted for 20% of all hospital admissions due to severeliver injury and 50% of acute liver failure cases, 75 % ofwhom required a liver transplant [153]. Although the extent ofthe immune component in DILI is still not completely clear, anumber of associations with particular HLA genotypes have nowbeen reported. Initial reports of possible HLA associations withDILI appeared during the 1980s in relation to injury associatedwith nitrofurantoin, halothane and clometacin. More recently, twoseparate studies found a significant relationship between HLAclass II genotype (the DRB1*1501 allele) and the susceptibilityto liver injury induced by the antimicrobial agent co-amoxiclav[154,155]. Another antimicrobial agent, flucloxacillin, may alsogive rise to liver injury in some patients. Recent candidate geneand genome-wide association studies on flucloxacillin-inducedliver injury (Figure 3) has detected a strong HLA association,with possession of the HLA-B*5701 allele associated with an 80-fold increased risk of DILI development [156]. The same HLA-B*5701 association has also been reported for another ADR,abacavir-related hypersensitivity [157] (see below), but has notbeen previously associated with DILI.

Two other HLA associations with DILI have also beendescribed. One of these relates to the drug ximelagatran, whichwas found to be linked to liver toxicity in some patients during itsdevelopment. From both genome-wide association and candidategene studies, liver toxicity with this drug appears to be associatedwith the HLA allele DRB1*0701 [158]. This allele is morecommon among Europeans than in East Asians and the livertoxicity was also more common in Europe than in Asia. On theother hand, hepatotoxicity with ticlopidine appears to be morecommon among Japanese patients than Europeans and it has beenshown that this is due in part to an association with HLA-A*3303,an HLA allele predominantly seen in East Asian populations[159].

These findings of HLA associations that seem to be drug-specific point to an important role for T-cell responses, possiblyowing to drug complexed with peptides, in the toxicity process.The fact that DILI typically develops several weeks after the startof drug treatment is consistent with this immune component but itis likely that other factors, such as polymorphisms affecting drugmetabolism and drug transport, also contribute and that not allDILI relates to HLA genotype.

Hypersensitivity refers to an inappropriate immune reaction toan otherwise non-toxic agent. The manifestations of hypersens-itivity reactions are broad. Certain forms of DILI, as discussedabove, can be regarded as hypersensitivity reactions, for example,co-amoxiclav-induced liver injury. Skin reactions, which may alsoinvolve other organs such as liver, lungs or kidneys, are the mostcommon type of drug-induced hypersensitivity reactions.

Abacavir, a HIV-1 reverse transcriptase inhibitor, causeshypersensitivity reactions in 5% of patients [160]. Thesereactions can be fatal, particularly on rechallenge. An asso-ciation between abacavir hypersensitivity and a haplotype

including HLA-B*5701, HLA-DR7, and HLA-DQ3 was initiallydemonstrated by Mallal et al. [157] and then replicated in twoother cohorts [161–163]. These findings have been confirmedfurther in a large randomized controlled trial [164]. The preventionof abacavir hypersensitivity using HLA-B*5701 genetic testing,which is now recommended prior to initiation of treatment in anumber of different countries, is a prime example of translationalresearch in pharmacogenetics.

CBZ (carbamazepine), a widely used anticonvulsant, can causerashes in up to 10% of patients, and, occasionally, this may bethe precursor to the development of a hypersensitivity syndrome[165,166]. CBZ can induce very rare blistering skin reactionssuch as SJS (Stevens–Johnson syndrome) and toxic epidermalnecrolysis [167]. A study in patients from Taiwan has showna very strong association between the HLA-B*1502 allele andCBZ-induced SJS [168]. The association seems to reflect theunderlying frequency of the HLA-B*1502 allele. In Thai patients,where the population frequency of the allele is similar to that seenin Taiwan, an association between CBZ-induced SJS and HLA-B*1502 has also been demonstrated [169]. However, in Europeans[170,171] and Japanese [172] the allele frequency is lower, andno association has yet been shown between SJS development andHLA-B*1502.

Not all idiosyncratic ADRs show genetic associations withHLA. Susceptibility to two other important adverse factors,QT interval prolongation (representing an increased time forcomplete cycle of ventricular depolarization and repolarizationin the heart’s electrical cycle) and statin-induced myopathy doesnot appear to show any immunogenetic association. In the caseof statin-induced myopathy, there is evidence from a genome-wide association study (Figure 3) and an independent follow-upstudy that the genotype for SLCO1B1 (see the section on Drugtransporters above) is a predictor of susceptibility to myopathyassociated with at least with some statins [173,174]. Thismuscle toxicity, although very rare, is potentially fatal. SLCO1B1genotype is also associated with plasma levels of several statins[175] and it therefore seems likely that the SLCO1B1 effect isat the pharmacokinetic level although additional genetic factors,unrelated to pharmacokinetics, may well also be predictors oftoxicity.

Cardiotoxicity is another form of idiosyncratic ADR. Somedrugs can rarely cause a significant lengthening in the QTinterval, which is potentially fatal. Current knowledge on thegenetic basis for susceptibility to this rare toxicity is limited, buta range of rare polymorphisms and mutations in ion channelsappear to be associated with increased risk [176]. For example,a polymorphism in KCNE1, which encodes a voltage-gatedpotassium channel, is normally seen at a population frequencyof 1% but is more common in individuals who have suffereddrug-induced QT prolongation [177].

GENOME-WIDE ASSOCIATION STUDIES IN PHARMACOGENETICS

Increasingly, genome-wide association studies to identifygenotypes associated with either drug response or drug toxicityare being performed. These studies involve genotyping diseasecases and unaffected controls for typically between 500000and 1000000 SNPs, which represent variability in the entirehuman genome. Due to the existence of extensive linkagedisequilibrium within the human genome, genotyping forparticular polymorphisms (tag SNPs) will provide informationon additional polymorphisms in linkage disequilibrium. Bycomparing distribution of polymorphisms in cases and controls,it is possible to identify particular polymorphisms where thereis a difference in frequency. A difference in frequency suggests


444 A. K. Daly

Figure 3 Examples of genome-wide association studies on ADRs

(a) Manhattan plot of − log P-value against chromosomal position of each marker from a study on simvastatin-induced muscle toxicity on 85 cases and 90 drug-exposed controls. Reproduced fromLink et al. [173] with permission. Copyright c© 2008 Massachusetts Medical Society. All rights reserved. (b) Manhattan plot of − log P-value against chromosomal position of each marker from astudy on flucloxcillin-induced liver injury on 51 cases and 487 population controls. Reproduced from Daly et al. [156] with permission.

that variation in the gene where the polymorphism is locatedor one situated nearby may contribute to disease susceptibility.Owing to the large number of different polymorphisms studied,it is necessary to set a high threshold for statistical significanceto avoid false positive results. In addition, any apparently positiveassociations are normally replicated in a second independentset of cases and controls. Most genome-wide associations oncommon diseases have involved in excess of 1000 cases and 1000controls to provide sufficient statistical power to detect the smallgenetic effects typically seen. However, it is increasingly clearthat some pharmacogenetic effects are larger and can be detectedusing smaller numbers of samples [178]. As reviewed elsewhere[179], genome-wide association studies have a completely openapproach, allowing the detection of associations not directlypredictable from existing knowledge and for the effects of anumber of different genes to be detected simultaneously.

Genome-wide association studies have been widely reportedfor complex polygenic diseases, with some interesting novelgenes affecting disease susceptibility now identified [180]. In thepharmacogenetics field, a number of genome-wide associationstudies on drug response or susceptibility to ADRs have appearedin the last 3 years. In some cases, these have confirmed existingfindings from candidate gene studies performed previously andhave not provided any additional novel findings, particularly inthe case of two studies on warfarin dose requirement [181,182]and also, recently, in a study on clopidogrel response [91].More generally, pharmacogenetic studies involving genome-wideassociation analysis have mostly pointed to only one or two genes

having a major effect (see Figure 3 for two examples), rather thanthe larger number of genes, each with a small effect, as typicallyseen in the complex polygenic disease studies [179]. However,the open nature of the genome-wide approach has enabled somenovel pharmacogenetic associations that would be unlikely tobe detected by candidate gene studies to be identified. Forexample, the main current treatment for hepatitis C virus infectioninvolves administration of a modified form of interferon-αand the antiviral drug ribavirin. Up to 50% of patients showa positive response to this treatment and become essentiallyvirus-free. In three independent genome-wide association studies,polymorphisms adjacent to the IL-28B (interferon λ3) genewere shown to significantly predict the likelihood of a positiveresponse to interferon-α [183–185]. In a study on bisphosphonate-induced osteonecrosis of the jaw, a CYP2C8 polymorphismgave the strongest effect in a genome-wide association analysis,even though bisphosphonates are not subject to metabolism[186]. This association may relate to a physiological role forCYP2C8 in inflammation [187], but this gene was not anobvious candidate for a role in this disease. As discussedin the section on idiosyncratic ADRs, a strong associationbetween HLA-B*5701 and susceptibility to flucloxacillin-inducedliver injury was detected by both direct HLA genotyping(performed because HLA genes were obvious candidates) and bya genome-wide association study (Figure 3) [156]. However, thegenome-wide analysis also provided evidence for a role for thegene ST6GAL1, which encodes the enzyme ST6 β-galactosamideα-2,6-sialyltransferase 1. This enzyme has a possible role in



B-cell immune responses [188], and would not be an obvious geneto study in relation to DILI although it is expressed in hepatocytes.

CONCLUSIONS

Over the 40 year period from 1957 to 1997, pharmacogeneticsevolved to pharmacogenomics. There has been considerablefurther progress in the subsequent 14 years. Our understandingof single-gene effects, especially in relation to drug metabolism,is now comprehensive, but our understanding of effects frommultiple genes is still more limited. In addition, we still needto translate the range of well-validated and clinically relevantpharmacogenetic discoveries that have been made over the yearsinto more widespread use in patient care. Despite predictionsthat we are entering an era of personalized medicine [189],except for the few examples discussed above in relation to cancerand HIV treatment, this has not yet happened to any greatextent. There is still considerable potential for using genomicapproaches to individualize drug treatments, but the logisticand cost-effectiveness issues need to be addressed for theseapproaches to become routine.

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Received 8 April 2010/10 May 2010; accepted 11 May 2010Published on the Internet 14 July 2010, doi:10.1042/BJ20100522


Pharmacogenetics and human genetic polymorphisms€¦ · I and phase II) and also drug transporters...

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