Pharmacogenetic Testing

25 Pages·2000·0.163 MB·English

by  Weber W.W., Cronin M.T.

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PHARMACOGENETICTESTING
Pharmacogenetic Testing 4.3 Emerging Applications of Pharma-
cogenetic Interest 19
4.4 Linking Human Genotyping to
Clinical Applications 20
WendellW.Weber
4.5 Summary 20
UniversityofMichigan,AnnArbor,USA
5 Summary: Progress in Pharmacogenetic
MaureenT.Cronin Testing 20
ACLARABiosciences,MountainView,USA Abbreviations and Acronyms 21
Related Articles 21
References 21
1 Introduction 2
1.1 Historical Highlights 2
1.2 The Emergence of
Drugs are administered to patients with the intention
Pharmacogenetics 3
of achieving a planned therapeutic response. Yet drug
1.3 The Scope of Pharmacogenetics 3
prescriptionisamedicalartbecauseofthewidevariationin
1.4 The Pharmacological Profile of
individualresponsestostandarddrugdoses.Inthe1960s,
Human Drug Response 4
thefieldofpharmacogeneticsemergedfromthededicated
1.5 The Genetic Profile of Human Drug
workofasmallgroupofinvestigatorswhobegantoenrich
Response 5
thedisciplineofpharmacologybyintegratingitwithhuman
1.6 The Rationale and Aims of
genetics. Pharmacogenetics provides the experimental
Pharmacogenetics 5
framework to understand variability in human reactions
1.7 Testing Pharmacogenetic Hypotheses 6
todrugs andother exogenous substances asafunction of
1.8 Summary 6
intrinsichumangeneticvariability.Thematurationofthe
2 The Changing Scene 7 humangenomeinitiativehasprovidedawealthofprimary
2.1 Human Protein Diversity 7 geneticinformationandfunctionalgenomicdatatofuelthe
2.2 Some Biochemical Generalizations 7 understandingofgeneticpolymorphismanditsfunctional
2.3 DNA Polymorphism: Some Practical consequences. Today, the field of pharmacogenetics is
Matters 7 a well-integrated, worldwide network engaging a vast
2.4 Molecular Heterogeneity of Pharma- communityofacademicandindustrialscientists.
cogenetic Interest 8
One hallmark of pharmacogenetics studies is the large
2.5 The Effect of Mutations on the
amount of genetic data that must be accumulated and
Gene Product 10
integrated for high-resolution drug-response genotyping
2.6 Summary 11
andsubsequentphenotype profiling.Thisneedforhighly
3 Genetic Profiling Using DNA parallel genetic analysis has, in turn, fueledademandfor
Microarrays 11 technical innovation to provide the tools necessaryfor its
3.1 The Origin of DNA Microarrays 11 execution. The response has been a flurry of inventions
3.2 DNA Microarray Capabilities and in microfluidics and nanotechnology, some of which
Limitations 13 are scaled down versions of existing technologies, such
3.3 Predictive Testing with DNA as capillary electrophoresis and mass spectrometry, and
Microarray Assays 15 some are completely new inventions. Outstanding in
3.4 Emerging Applications of Microar- this technical revolution are DNA microarrays. Array
ray Technologies 16 technology has emerged as the most versatile and widely
3.5 The Future of Microarrays and applied tool to support pharmacogenetics, for several
Allied Technology 17 reasons:arrayshavethelargestandmostscalablecapacity
3.6 Summary 18 forparallelanalysis,theirinherentflexibilitypermitsthem
4 The Outlook for Genomics in Predictive to be used as independent analysis tools or as integrated
Medicine 18 components of more complex microfluidic systems and,
4.1 Options for Treating Genetic finally, a wide variety of methods can be used to design
Disorders 18 and fabricate them. Pharmacogenetics is experiencing a
4.2 Many Pharmacogenetic Polymor- periodofrapidgrowthanddefinition.Arrayshaveproven
phisms are Exceptions to Experi- themselves to be a technology capable of responding to
ence with Single Gene Disorders 18 the growing and changing needs of the current research
EncyclopediaofAnalyticalChemistry
EditedbyRobertA.Meyers.(cid:211) JohnWiley&SonsLtd,Chichester.ISBN0471976709
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CLINICALCHEMISTRY
environment and are likely to be equally important as somehow implicated in the detoxification of exogenous
pharmacogeneticsmovesintotheclinicalarena. substances. During the intense flurry of research stim-
ulated by the rediscovery of Mendel’s laws of heredity
around 1900, Cuenot, from studies of mice, and Gar-
1 INTRODUCTION rod, from human studies, anticipated the connection of
enzymes (‘‘diastases’’) with the genetic material. Obser-
vations of physiological chemists, intrigued by the fate
Drugs exert characteristic, reproducible effects when
administered to individuals, and physicians have come of chemicals in human subjects, had shown by this time
torelyonthemforthespecifictreatmentofmanyhuman that most drugs were excreted in forms that differed
disorders. Most readers need not be convinced of the from those that were ingested. These observations, and
societal importance of these agents, but patients and Garrod’s observations pertaining to a case of porphyria
physicians alike know that one individual may respond brought on by the hypnotic drug sulfonal, led Garrod
differently from another given the same dose of a drug. to conclude that the ability of individuals to transform
Such person-to-person variation in drug response is not drugs into nontoxic conjugates, such as hippurates and
a trivial matter. The analysis of the causes of natural glycuronates,protectedthemfromthepoisonous effects
variation in human drug response, the central problem of these agents. Garrod was thus far ahead of his con-
of pharmacogenetics, requires a basic understanding of temporaries in attributing unexpected drug responses of
humanpharmacologyandhumangenetics. individuals to failure of their enzymes to detoxify these
The initial researches of a small cadre of dedicated substances.
investigators saw the emergenceof pharmacogenetics in These ideas regarding the detoxification of chemicals
the1960sasanewfieldofexperimentalscienceprimarily surfaced again and again in Garrod’s writings and
concerned with reactions to drugs and other exogenous teachings until the end of his life in the 1930s. He
substances that occur because of a person’s distinctive observed that substances in foods, in certain drugs, and
geneticmakeup.Thefieldhasgrown,particularlywithin in exhalations of animals and plants might produce
the period from the late 1980s, into a well-integrated, effects in some persons wholly out of proportion to
worldwide network engaging a vast community of any they produce in most persons..2/ During the 1920s
academicandindustrialscientistswithexpertiseinabroad and 1930s, others began to scrutinize person-to-person
spectrumofbasicandclinicalbiomedicaldisciplines.Until differences in the perception of odor and taste..3/ For
recently,pharmacogeneticstudieswereusuallylimitedto example, studies of ‘‘taste blindness’’ to the bitter-
a few individuals and families in a research setting, but tastingsubstancep-ethoxyphenylurea wasdemonstrated
nowsuchaninvestigationmaybedirectedtowardalarge tobeaMendeliancharactertransmittedfromparentsto
fractionofthehumangenomeandinvolvesimultaneous
children as an autosomal trait. These deficits in sensory
testing of multiple loci in many patients and healthy
perception were the first indication of the high order of
subjects. Such studies may, at times, be referred to as
specificitytobeexpectedinhumanresponsetochemicals,
pharmacogenomics.
and the first to establish the heritable nature of such
The establishment of laboratories dedicated to such responses..4/
large-scale pharmacogenetic testing is rapidly gaining
By the time Garrod presented his ideas before the
momentum. This action has the attention of physicians
British Medical Association, chemists had identified vir-
whoareinthefrontlineofpatientcare,ofthosewhoare
tually every major type of conjugation reaction that
responsibleforclinicallaboratoriesandofacademicand
we know today. The exact dates of those discoveries
pharmaceuticalscientistswhoareengagedinthediscov-
is less relevant than the fact that they preceded by
ery and clinical trial of new drugs. Because the genetics
about 50 years the discovery of the other major cate-
and the molecular basis of many pharmacogenetic traits
gory of detoxifying enzymes (P450s), commonly known
are well established, because advance testing holds out
as the microsomal enzymes or the drug-metabolizing
high promise of improved patient care, and because the
enzymes. R.Tecwyn Williams, a pioneering pharma-
high potential for acceptance by the biomedical com-
cologist at St.Mary’s Medical School in London, saw
munity, pharmacogenetics is in a strong position to link
evidence for a new, far-reaching principle of drug
humangenotypingandphenotypingrapidlywithclinical
metabolism in the studies of the microsomal and con-
application.
jugating enzymes, namely that the metabolic disposition
of drugs in humans and animals occurs in two phases:
1.1 HistoricalHighlights
first, oxidation, reduction, and hydrolysis, then conjuga-
In an address before the British Medical Association in tion,designatingthemasphaseIandphaseIImetabolism
1914,.1/ Archibald Garrod proposed that enzymes were respectively.
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PHARMACOGENETICTESTING
1.2 TheEmergenceofPharmacogenetics distinction, the literature does not always adhere to this
convention.Whenitisofinteresttoassessthecharacteris-
Until around 1950, progress slowed, but then several
ticsofagivenresponse,thecriteriatobemetindesigning
breakthroughs foreshadowing important events to come
andexecutingaproperstudytodeterminetheheritability
were made. In 1948, hemoglobinopathies S and C were
of the response in twins and families, and the distribu-
identifiedandtheirmodeofinheritancewasdetermined.
tionandincidenceofgenotypesandphenotypesinlarger
Hemoglobin S, the first hereditary protein variant to
populations, are, in fact, identical for pharmacogenetics
be identified, was also the first example of a variant
andecogenetics.
protein in which replacement of a single amino acid
Most well-studied polymorphisms derive from inves-
residue within the protein molecule could be identified
tigations of therapeutic agents, because these agents
unequivocally with a change in the functional effect
can be administered safely to individuals of all ages
of the protein. In 1953, the double helix of DNA
in defined amounts, and their disposition patterns can
was described. A few years later, the chromosomes
bemeasured.Historically,thedrug-metabolizingenzyme
of humans were visualized and enumerated, and the
polymorphisms are foremost in pharmacogenetics, and
chromosomal basis of one form of cancer (chronic
aficionadosrecognizethesetraitsasthecornerstoneofthe
myeloid leukemia) was identified. Following closely on
field..8/ However, since medical drugs and other exoge-
thedemonstrationofpolymorphicformsofhemoglobinS
nouschemicalsarebiotransformedbythesameenzymes,
andCbyelectrophoresis,applicationofthistechniqueto
unexpectedeffects,asGarrodnoted,mayextendtofood-
plasma proteins showed that protein polymorphism was
stuffs as well as to sporadic disorders associated with
awidespreadphenomenonthatmeritedfurtherstudy.
exposure to chemicals under occupational and indus-
New technologies that became available in the 1950s,
trialconditions.Theenzymesthatmetabolizeexogenous
combinedwithamoregeneticapproachtoinvestigation,
substances may also metabolize hormones and other
disclosed new relationships between the genetic control
endogenousmessengersthatregulatecellsignaling;con-
ofresponsestoexogenoussubstancesandtheirmetabolic
sequently,mutationsthataffectthosepathwaysmayalso
fate. Landmark studies of unexpected responses to pri-
cause differences in individual response. This evidence
maquine,isoniazidandsuccinylcholine(suxamethonium)
servesasanadditionalhallmarkofpharmacogeneticand
were the first to establish a link between drug response
ecogeneticphenomena.Therangeofsuchphenomenais
and heredity. The confluence of pharmacology, genetics
virtuallywithoutlimit.
andbiochemistrynotedinArnoMotulsky’s1957seminal
paper,.5/ concernedwithperson-to-person differences in The administration of two or more drugs in combi-
nation may also set the stage for an unexpected drug
drug response that arise because of the unique genetic
response that depends on the genetic makeup of the
constitutionofindividuals,markedthetruebeginningof
recipient. The CYP2D6 (debrisoquine/sparteine oxida-
pharmacogenetics as an experimental science. In 1962,
tion)polymorphismisaparticularlyrichpotentialsource
Werner Kalow published the first systematic account of
of such interactions. This polymorphism affects the dis-
the field, demonstrating the implications of heredity for
position of more than 30 therapeutic agents including
responses of microorganisms, insects, and vertebrates,
including humans, to environmental chemicals..6/ Since b-adrenergic blockers, antidepressants, antiarrhythmics,
neuroleptics, and various miscellaneous drugs such as
then, the characteristics of many more human traits of
phenformin,dextromethorphan,andcodeine.Combined
pharmacogenetic interest, too many to chart here, have
administrationoftwoormoreoftheseagentsmayresult
demonstratedbeyondanydoubtthatheredityisaremark-
inanaversiveinteraction.
ableprognosticatorofunexpectedresponsetodrugsand
Smoking represents another environmental hazard
otherexogenoussubstances.
that results in a variety of responses and disorders
whichmaydependongeneticpredispositionofsmokers.
1.3 TheScopeofPharmacogenetics
In individuals with a genetic defect in a -antitrypsin,
1
Hereditary differences in toxicity or efficacy that result smoking is the most important environmental factor
from exposure to therapeutic agents are usually consid- affecting the rate of deterioration of lung function..9/
ered as being within the province of pharmacogenetics, Additionally, information is accumulating that a variant
but if the unexpected response results from exposure to formorformsofCYP2A6mayexhibitimpairedcapacity
other chemicals, or to physical, climatic or atmospheric to metabolize nicotine and may thereby protect against
agents, it may be classified as ecogenetic..7/ Ecogenetics the effects of smoking by reducing the number of
encompassesgeneticallyconditionedresponsestoexoge- cigarettessmoked..10;11/
nous substances from any source and, as such, would Genetic predisposition appears to be involved as
include the pharmacogenetics of drugs used in medical a hypersusceptibility factor for workers exposed to
practice. It should be noted, however, that, despite this complex mixtures of chemicals. The formation of
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CLINICALCHEMISTRY
aniline–hemoglobin adducts and excessive levels of 1.4 ThePharmacologicalProfileofHumanDrug
methemoglobin can occur in those who are genetically Response
‘‘slow acetylators’’ after prolonged exposures to aniline
Responses to a particular drug actually result from
derivatives..12/ The sporadic occurrence of urinary blad-
the physiological and biochemical attributes of cells
der cancer in slow acetylators many years after lengthy
that carry the necessary receptors in tissues of the
exposuretothearomaticaminecarcinogens.13/employed
recipient,buttheseresponsesaresubjecttomodification
in the manufacture of dyestuffs, plastics and electrical by a variety of intrinsic and extrinsic influences..4/
materialsisacaseinpoint.
The age, gender, weight and other physiological and
Important clues to the role of heredity in unexpected
pathophysiologicalattributesoftheindividual,including
responses to drugs and other chemicals are provided by
heredity, are important intrinsic factors that can modify
individuals of different ethnogeographic origin. Perhaps
drug responses in individuals. Foods and other dietary
the most familiar example is ‘‘primaquine sensitivity’’
components, exogenous chemicals in household and
occurring as a result of glucose-6-phosphate dehydroge-
workplaceenvironments,theuseoftobaccoandalcohol,
nase (G6PD) deficiency among African, Mediterranean
anddrugs(prescriptionandnonprescription)themselves
and Oriental persons. This is associated with sudden
are some of the extrinsic factors that may affect these
hemolysis, which occurs among susceptible males after
responses.
exposuretoanyofsome200drugs..14/Otherexamplesof
Theresponseofanindividualfollowingadministration
ethnicspecificityindrugresponseincludetheprotective
of a drug (or after exposure to an exogenous chemi-
effect against alcohol-induced liver disease of a variant cal) is a complex process that is difficult to analyze. By
of aldehyde dehydrogenase (ALDH2) that is found in reducing this process to pharmacokinetic and pharma-
JapanesebutnotCaucasians.15/ andtheprotectiveeffect codynamic mechanisms, pharmacologists have devised
against alcohol-related birth defects of a variant form a somewhat idealized picture of the response, which
of alcohol dehydrogenase (ADH-3) occurring among can be analyzed more readily. Pharmacokinetic mecha-
African-Americans..16/ nismsarethosethataffecttheconcentrationofthedrug
Whilemanyoftheexamples citedaboveindicate that at receptorsitesand its time course of action (absorp-
unexpected responses to exogenous chemicals may be tion, distribution, and elimination by metabolism and
mainlycausedbyasingle(monogenic)factor,experience excretion) in the individual, while pharmacodynamic
showsthatvariationofmorethanonehereditaryelement mechanismsrefertoreceptor-mediatedandalliedevents.
maycontributetotheoutcome.Oneexampletoillustrate Thismaneuveralsoprovidesaframeworkwellsuitedto
thesynergismthatcanoccurbetweeninteractingfactors discussionofthepharmacologicalnuancesofvariationin
is provided by a study of liver cancer caused by humandrugresponse.
exposuretoaflatoxinandhepatitisBamongtheChinese. The response of an individual to most drugs can
Aflatoxin exposure alone enhances the relative risk by be thought of as a train of events that begins when
approximately twofold, while infection with hepatitis B the drug enters the bloodstream and ceases when the
virus alone enhances the risk by approximately fivefold; drug and its metabolites are completely eliminated.
however, combined exposure yields a 60-fold increase The administration of a drug, given in ordinary doses,
in relative risk of liver cancer. Hepatitis B infection, is usually accompanied by a gradual rise in drug
therefore, enhances the carcinogenic response among concentration in the blood that reaches a peak or
Chinesetoaflatoxinby30-fold..17/ steady state; if no more drug is given, the concentration
Nowadays, the scope for inherent variation in human steadily declines until the drug and its metabolites are
responsetoexogenouschemicalsissogreat,andthepace eliminated. If the drug attains a concentration within
of science so rapid, that it is difficult to stay abreast of the therapeutic range, the characteristic response is
newdevelopmentsinthefield.Althoughmetabolicpoly- expected. If the concentration reaches a level above or
morphisms of drug-metabolizing enzymes appear most below that range, either a toxic response or the absence
frequentlyintheliterature,polymorphismsofothergenes of the expected response may occur. For unexpected
of pharmacogenetic interest, such as those that encode responses, experience indicates that there are two likely
receptors,clottingfactors,transportersandimmunologic explanations.Ifthedrugconcentrationinplasmaisabove
responseproteins,appearincreasinglyfrequently.Inthe or below the therapeutic range, one would anticipate
1990s, recombinant DNA technology has contributed a pharmacokinetic mechanism is responsible for the
immensely to our knowledge of pharmacogenetics by unexpected response, but if the concentration is within
facilitating the development of sensitive and specific the therapeutic range, a pharmacodynamic defect is a
diagnostic methods for collecting molecular information morelikelyexplanation.
about unexpected responses to drugs and other exoge- Consequently, from the pharmacological viewpoint,
noussubstances. unexpecteddrugresponsestodrugsandotherexogenous
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PHARMACOGENETICTESTING
substances may be regarded to a first approximation and techniques of human genetics, biochemistry, pop-
as peculiarities resulting from a defect in either the ulation and molecular genetics, and epidemiology are
pharmacokineticsorthepharmacodynamicsofthedrug. usedinconcerttoanalyzepharmacological,toxicological
and epidemiological observations of individuals, twins,
familiesandlargerpopulations.
1.5 TheGeneticProfileofHumanDrugResponse
Geneticists, in contrast to pharmacologists, might con- 1.6 TheRationaleandAimsofPharmacogenetics
sider a drug as an environmental agent on which
Pharmacogenetic investigation often begins with an
individualsexertimportantdifferentialeffects;therefore,
anecdotalclinicalobservationononeorafewindividuals
they might regard an unexpected drug response as a
who have experienced an unexpected response to a
reflection of a genetic difference between normal and
specific drug, or with an epidemiological observation on
abnormal responders..4/ Geneticists, as a rule, are more
a group of individuals who have developed a disorder
interested in looking for differences that discriminate
associated with occupational exposure to a chemical
individuals rather than focusing on universal similari- or complex mixture of chemicals..4/ The main goal of
ties,andtheywouldseektoexplainanunexpecteddrug
pharmacogenetics,simplyput,istouseknowledgegained
response by drawing on the gamut of information from
in understanding the influence of heredity on human
thenormalandaffectedrespondersaswellasfromtheir
sensitivity (or resistance) to exogenous chemicals to
biologicalrelatives.Fromtheinformationobtained,they
avoid the occurrence of such responses in susceptible
wouldattempttodeterminetherelativecontributionsof
persons.Therefore,whiletheearlydetectionofcancerof
heredityandenvironmenttotheunexpectedresponse. environmentalorigin,forexample,maydolittletochange
Genetic defectsthatcauseunexpecteddrugresponses the outcome in an affected person, identification of the
occur sufficiently often in many populations to enable trait that led to its initiation may prevent its occurrence
memberstobedividedintotwoormorerelativelycom- inothers.
mon types of responder. Hereditary variation in which A number of practical problems can arise in the
such sharply distinct qualities coexist in a given pop- design and execution of pharmacogenetic studies. A
ulation is referred to as genetic polymorphism. This complete assessment of the causes of a given trait may
term is usually used in genetics to refer to genetic loci be exceedingly difficult to achieve, may require the
for which variants occur with a frequency of 1–2% or collaborationofscientistswithdifferingexpertiseormay
greater, but in pharmacogenetics the definition is bet- require resources not readily available. For example,
ter based on phenotype. This issue has been the source epidemiological evidence may suggest a disorder (say
of some discussion.18;19/ and is considered further below bladdercancer)iscausedbyaparticularagent(oragents),
(section2.3). To avoid contradictions between pharma- but the agent or agents usually cannot be identified
cogenetic usage and genetic theory and concepts, this unequivocallysolelybyepidemiologicalevidence;clinical
article will refer to the polymorphisms linked to human observation may suggest an adverse reaction is caused
responses to drugs and other exogenous substances by a certain drug, but the physican who contemplates
as‘‘pharmacogeneticpolymorphisms’’.Pharmacogenetic investigation of the reaction by further exposure of
polymorphism may include variation produced by chro- humans to the offending drug may face insurmountable
mosomalaberrations,whicharedetectablebycytogenetic ethical and methodological constraints; susceptibility to
techniques,butmostresultfromsmaller,geniclesions. a toxicant can often be more thoroughly studied under
To assess the contribution of heredity to pharmaco- highly controlled conditions in animals than in humans,
genetic polymorphism, unexpected drug responses are butstudiesinanimalmodelsareslowandexpensive,and
explored at all levels of gene action, from the gene animal responses do not necessarily translate closely to
molecule itself, to the individual and their biological thoseinhumans.
families,topopulationsofindividuals.Broadlyspeaking, Establishmentofdefinableend-pointsfortheinvestiga-
hereditarydifferencesindrugresponsearecharacterized tionofspecificdifferencesbetweennormalandabnormal
bytwotypesofinformation:thegeneticsthatcharacterize responders poses another problem. Consider the eval-
the polymorphic phenotypes and their molecular basis. uation of drug treatment of hypertension as a case in
The latter includes a description of the genetic hetero- point. In this instance, the definitive end-point would
geneity at the level of DNA and of the protein variants bemeasuredbythedifferenceintheincidenceofstrokes
that explain the trait. By studying a given trait from andcardiovasculardiseasebetweennormalandabnormal
several points of view, the relative influence of human drugresponders,butthiswouldprobablyoccuryearsafter
ecologyandheredityandthemechanismsbywhichthey the trait is recognized and modifying treatment under-
occur are ascertained. For these purposes, the concepts taken. Another example of pharmacogenetic interest
6
CLINICALCHEMISTRY
concerns the evaluation of individual variation in sus- certain drugs and of individuals who become ill after
ceptibility to cancer associatedwith prolonged exposure prolonged exposure to toxic chemicals associated with a
toanoccupationalcarcinogen.Thedefinitiveend-pointin specificoccupationoraparticularenvironment.
thiscaseisthedifferenceincancerincidencemanyyears An important goal of studies with intermediate- and
later between hypersusceptible and normal responders short-term biomarkers is validation of the markers
exposedtothecarcinogen.Itisapparentthataccomplish- by demonstrating their correlation with definitive end-
ment of trials to determine definitive end-points might points. It is reasonable to expect that these biomarkers
requirestudiesextendingovermanyyearsinvolvingmany will enable differences in human susceptibilities to
individuals and large populations, and the payoff is not exogenous chemicals to be detected, or suspected, at
guaranteed. anearlystageofexposure,andtheliabilityofsusceptible
Investigationswithmorelimitedgoalsdesignedtomea- persons to be predicted long before the definitive end-
sure intermediate or short-term changes in biochemical point is reached. It follows that studies using such
andmolecularmarkersmaylessensomeofthesedifficul- biomarkerscouldbeaccomplishedinamuchshortertime
ties. Biological markers that can detect early and subtle andatamuchlowercostthanthoseaimedatdetermining
differencesinindividualresponsewouldbenecessaryfor definitiveend-points.
this purpose. The most suitable are markers that can be
detectedinsmallsamplesoftissueorbodyfluidsthatare
1.7 TestingPharmacogeneticHypotheses
accessibletosampling;theyshouldbeexpresseddifferen-
tially in accordance with differences observed in normal Exploring the pharmacogenetics of unexpected drug
and abnormal responders and they should have a low responses can be a formidable task. No hard and fast
probabilityofspontaneouschange. rules exist to guide the investigator’s advance planning,
Several advantages accrue to study designs thatfocus but there are some general principles that are widely
on the use of intermediate- or short-term biomarkers. applicable..4/ Experience indicates that the response of
First,the marker could be used to document and anindividualtoadruganditsinherentvariationarebest
characterizetheheterogeneityinresponseofindividuals considered in light of the pharmacokinetic and pharma-
to a given drug, and to determine its distribution in codynamic mechanisms that define the disposition and
individuals, families, and other populations of interest. actions of drugs in individuals (see section1.4). Insights
Thiswouldindicatethefrequencyofagiventrait,itsmode intothe relativeimportance ofthepharmacokinetic and
of inheritance, and its ethnic and geographic specificity. pharmacodynamic phases of the response to the overall
Second, the markers could be used to identify the genes variabilityintheresponsetoagivendruginaheteroge-
andthemutationsresponsibleforthetraitinsusceptible neousgroupofdrugresponderscanoftenbegainedbya
people. This information would reveal the molecular preliminarypharmacokineticanalysis.
basisofthetraitandwouldaidinelucidatingdisordered In general, an experimental approach is almost
physiologicalmechanism(s)thataccountfortheaberrant inevitably based on testing a main hypothesis and one
response. It would shed light on the mechanism of the ormoreauxiliaryhypotheses..20/Thenovicemay,infact,
response to the agent in normal (wild-type) responders. proceedwithonlyavaguenotionofthehypothesisbeing
The marker would also be of further use in the tested, but skilled investigators know that it is advan-
developmentofnoninvasive,inexpensivediagnostictests tageous to have thought about it explicitly. Auxiliary
todeterminethephenotype andgenotypeofindividuals hypothesesaresetforthasreasonablealternativesincase
for the trait. Third, for agents used in medical therapy, themainhypothesisprovesfalse.Investigatorsshouldalso
identificationofthemolecularbasisofspecifictraitsand rememberthat,foreverydiscoverymadewithinnovative
the elucidation of their underlying mechanisms would approaches, many important advances are made simply
rationalizetheselectionofspecificagentsforsusceptible by recognizing that an established method or familiar
(or resistant) individuals and make it possible to tailor technique devised for one application can be used for
drug regimens to the individual patient. Fourth, since another. Insomecases,it ispossible for theinvestigator
the exact cellular site that is defective must be known to design a single experiment that decides the fate of a
for rational design of therapy, the information obtained givenhypothesis.
in the first three study parameters would help to guide
discovery of new drugs and to improve the design and
1.8 Summary
conductoftheirclinicaltrial.Finally,studieswithmarkers
could have wider implications for patient welfare by Individualdifferencesinhumanresponsestotherapeutic
alertingphysicians,clinicalscientists,andothersengaged agents and other exogenous chemicals often result from
in patient care to the importance of biomonitoring of some derangement of the genetic material that may be
individuals who suffer sporadic illnesses while ingesting transmittedfromonegenerationtothenext.Thescientific
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PHARMACOGENETICTESTING
study of the effects of heredity on drug response is the single polypeptide chain that was affected by different
province of pharmacogenetics. Its main purpose is to mutationsinthecodingregionmightthusberepresented
explain the toxicological effects of the derangements byseveraldistinctalleles.Mosthemoglobinopathies, for
by rigorous characterization of their biochemical and example, could be ascribed to missense, nonsense, or
molecularbasis.Inthisway,itishopedtounderstandthe frameshiftmutationsofthecodingregionofglobingenes.
causes of human susceptibility to exogenous substances Among the enzymopathies, observations suggested that
and avoid the occurrence of unexpected drug responses allozymes(i.e.enzymesencodedbyallelicloci)generated
insusceptiblepersons. bypointmutationsandsmalldeletionswouldusuallydis-
playhighlyhomologousaminoacidstructuresandmight
have similar enzymatic properties, whereas larger dele-
2 THECHANGINGSCENE tionsresultedindrasticallyshortened,nonfunctionalgene
products,orthetotalabsenceoftheproduct.
2.1 HumanProteinDiversity Post-translational modifications were recognized as
additional sources of the structural modification of
Proteins are vital to virtually every process of biological
proteins..22/ Should such a modification occur by an
importance because of their capacity to bind other
enzyme-mediated process, as had been established for
proteins and small molecules with high sensitivity and
oxidation of sulfhydryl groups or the addition of carbo-
specificity..21/ The assortment of proteins that resides in
hydrate or phosphate groups, or by the cleavage of the
cellsandtissuesrequiresthatthesynthesisandtargeting
polypeptide with loss of a terminal amino group, or a
ofthesemoleculesiscarriedoutwithahighleveloffidelity
largerpartofthechain,ittoocouldbesubjecttogenetic
accordingtothewell-definedsetofinstructionsencoded
variation.
inthegenes.Thepropertiesofdistinctproteinsultimately
Unfortunately, the available biochemical data were
definethelinkthatexistsbetweenthegeneticconstitution
inadequate to yield a detailed picture of the molecular
ofagivenindividual andhisorherresponsetodrugs.It
basis of protein heterogeneity. For instance, a change
followsthatastructuralalterationinagivengene,oran
that alters the charge on a protein can be detected by
errorincarryingouttheprogramofinstructionsencoded
electrophoresis, but since two-thirds of amino acid sub-
in the gene, might reasonably be expected to change
stitutionsareneutral,theywillnotchangethenetcharge
the structure or the amount of the protein synthesized,
on the protein and will not be detected in this way. As
or its final destination. Briefly, mutation could cause an
a consequence of the inability of the standard meth-
unexpecteddrugresponse.
ods of protein characterization to distinguish variation
The occurrence of G6PD deficiency, succinylcholine
owing to genetic variation at the level of translation
sensitivity and isoniazid acetylation polymorphism,
fromthatwhichoccurspost-translationally,ornongenet-
detectedinthe1950s,wasregardedasaseriousthreatto
ically,information regardingthegenetic basisofprotein
drug usagein medical therapy, requiring further investi-
diversitywasverymeager.
gation. Examination of the pharmacological defect and
moleculargeneticbasisofunexpecteddrugresponsesset
the agenda for subsequent research, and studies quickly 2.3 DNAPolymorphism:SomePracticalMatters
showed that changes in protein structure resulting from
The analysis of genomic DNA reveals that the human
geneticchangealteredhumandrugresponsivenesswher-
genomeishighlypolymorphic.Sitesthatexhibitalterna-
everitwassought,amongproteinswithwidelydisparate
tive sequences at a particular chromosomal site can be
functions,inhealthanddisease.
used as genetic markers for the site, or for the chromo-
somebearingthesite.Threetypesofpolymorphicsiteare
2.2 SomeBiochemicalGeneralizations
usefulforidentifyingindividualswhoarepredisposedto
Bythe1970s,Harrisandcolleagueshadsoughttoexam- unexpecteddrugresponses:thosewithingenesthatdeter-
ine the genetic causes of human protein diversity from mine drug response, those within short variable DNA
the biochemical, chromatographic and electrophoretic repetitivesequences(knownasvariablenumbertandem
characteristics of proteins associated with certain sin- repeat (VNTR)) and those within microsatellites. When
gle gene disorders..22/ Certain hemoglobinopathies and such a site is associated with a given trait and tracked
enzymopathiesprovidedthebestmodelsforthispurpose within families and larger populations, it can provide
becauseinformationaboutstructuralgenemutationswas information about inheritance patterns and the preva-
matchedbyphysiologicalandpathologicaldata.Evidence lence of the trait. Polymorphic sites can be studied by
suggestedthatmutations mightrangefromasinglebase restriction fragment (Southern) analysis to survey chro-
change within the coding region of a gene to large dele- mosomalsitesforpolymorphism,orbyDNAsequencing
tions that removed a large part, or all, of a gene. A to determine the precise location of base changes that
8
CLINICALCHEMISTRY
defineapolymorphism.Southernanalysiscannotresolve at the CYP2D6 locus, while the other seven enzymes
polymorphisms that differ by only one or a few bases, revealed none. This study and others (see references
andneithercanitidentifythepolymorphicbase,whereas in Skoda etal..27/) show that DNA polymorphisms are
the size and boundaries of the polymorphic site can be often functionally silent at the protein level, having no
preciselydeterminedwiththepolymerasechainreaction detectablephenotypiceffect.
(PCR) used in DNA sequencing. This second approach It follows that information about a given genotype
is essentially a refinement of the first and is preferredin is insufficient for unambiguous identification unless
manyapplications. combined with information about the associated phe-
Since the development of molecular techniques, notype. Further evidence pointing to the importance
remarkableprogresshasbeenmadetowardidentification of identification of phenotypes arises from numer-
ofgenesresponsibleforpharmacogeneticpolymorphisms ous sources. These include studies of the potential
and toward understanding the molecular basis of quite dissociation of phenotype from genotype observed in
a few of these polymorphisms at the DNA level. Most certain races.28;29/ and in certain families,.30/ the lack of
of the progress has related to traits attributed to a sin- resolution of heterozygous and homozygous subjects by
gle polymorphic gene, many of which have been cloned standard metabolic phenotyping procedures,.30/ the dif-
and sequenced..23/ For some, the gene of interest has ferent phenotypes for different substrates for a given
been expressed in heterologous expression systems in polymorphism,.31;32/ structure–functionrelationships,.33/
quantitiessufficientforbiochemicalandpharmacological gene–geneinteractions,.34/andthepatternsofdisease.35/
characterization. associated with polymorphisms attributed to genes with
The VNTRs or minisatellites of the human genome closelysimilarsequences.
mayberepeated100timesormoreindifferentpersons. We should also point out that since the vast majority
Many VNTRs, numbering in the thousands, are well ofpharmacogenetictraits,unlikemostgeneticallydeter-
characterized.Restrictionfragmentanalysiswillproduce mined human disorders, usually cause no recognizable
different size fragments proportional to the number of effectonthehealthofpredisposedindividuals,theimpor-
repeats in the VNTR. Where the identification of traits tance of phenotype takes on added significance. Only
hasbeenslowedforlackofasufficientnumberofsuitable
whensusceptiblepersonssufferanunexpectedresponse
genetic markers,the use of VNTRs should alleviate this
from exposure to certain drugs, dietary constituents
constraint..24/
or other environmental agents is their predisposition
A number of genes have been found to incorpo-
revealed.Fortunately, manyofthepolymorphisms asso-
rate microsatellites within them..25/ Microsatellites are
ciatedwithgeneticallyvariablehumandrug-metabolizing
stretches of repetitive DNA sequences; if mutated, they
enzymes, can be detected in advance with the aid of a
are capable of disrupting cell function. Such mutations
numberoftestprobesthataresuitableforhumanpheno-
may serve as hereditary markers of genomic instability
typingbyinvivoorexvivotestingprocedures.Apartial
thatmayincreaseindividualsusceptibilitytocertaincan-
listoftheseprobesispresentedinTable1.
cers. For example, the loss of microsatellite DNA from
For the reasons stated above, pharmacogenetic poly-
the androgen receptor gene has been associated with
morphismsmustbedefinedonphenotypicgrounds.Any
prostatecancer.Thiseventisofpharmacogeneticinterest
comprehensive description of a given pharmacogenetic
becauseaparadoxicalresponsetoantiandrogentherapy
polymorphismshouldincludeinformationaboutthephe-
(i.e.stimulationoftumorgrowthinsteadoftheinhibition
notype that characterizes the polymorphism and the
expected)wasexhibitedbyapatient..26/
genotype(s)thatexplainthepolymorphism.
The extent of protein diversity in natural populations
is immense, but the abundance of DNA polymorphism
in the human genome appears even greater. Analysis of 2.4 MolecularHeterogeneityofPharmacogenetic
genomic DNA reveals the presence of a large number Interest
of these polymorphisms. On average about 1 in 500
nucleotidesdiffersbetweentworandomlychosenalleles. Recombinant DNA techniques rapidly superseded stan-
Onlyabout5–10%ofthesepolymorphismsisdetectedby dard methods of protein characterization for genetic
restrictionanalysis,andfrequentlythesepolymorphisms analysis during the 1980s, and reports of the molecular
areunrelated to aclinical phenotype. This phenomenon characteristicsofnumerousenzymes,includingtheP450
hasbeendemonstratedfortheCYP2D6polymorphismby (CYP450)enzymesofdrugmetabolism,soonappearedin
Skoda and colleagues,.27/ who examined genomic DNA theliterature.Becauseoftheirspecificityforendogenous
from 53 persons using a total of 20 restriction enzymes steroids, fatty acids, and prostaglandins, as well as for
to fragment the DNA in different ways. Tests of 13 many drugs, environmental pollutants, and carcinogens,
enzymes yielded 14 allele-specific polymorphic patterns theCYP450enzymeshadalreadyattractedagreatdealof
9
PHARMACOGENETICTESTING
Table1 Testprobedrugsforhumanphenotypingofhumandrug-metabolizingenzymepolymorphisms
Testprobe Refs.
PhaseIenzymes
Aldehydedehydrogenase Acetaldehyde 36
Alcoholdehydrogenase Ethanol 16
CYP1A2 Caffeine 8
CYP2A6 Nicotine,coumarin 11,37
CYP2C9 Warfarin 8,37
CYP2C19 Mephenytoin,omeprazole 8
CYP2D6 Dextromethorphan,debrisoquine,sparteine 8
CYP2E1 Chloroxazone,caffeine 8
CYP3A4 Erythromycin 8
CYP3A5 Midazolam 8
Serumcholinesterase Benzoylcholine,butrylcholine 38
Paraoxonase/arylesterase Paraoxon 39,40
PhaseIIenzymes
Acetyltransferase(NAT1) Para-aminosalicylicacid 41
Acetyltransferase(NAT2) Isoniazid,sulfamethazine,caffeine 42
Dihydropyrimidinedehydrogenase 5-Fluorouracil 43
Glutathionetransferase(GST-M1) Trans-stilbeneoxide 44,45
Thiomethyltransferase 2-Mercaptoethanol,D-penicillamine,captopril 46
Thiopurinemethyltransferase 6-Mercaptopurine,6-thioguanine,8-azathioprine 47
UDP-glucuronosyltransferase(UGT1A) Bilirubin 48,49
UDP-glucuronosyltransferase(UGT2B7) Oxazepam,ketoprofen,estradiol,morphine 50,51
attentionamongbiochemicalpharmacologists.Addition- the inheritance of a given polymorphism and the expla-
ally, metabolic studies conducted during the mid-1970s nationofthepolymorphismattheDNAlevel.
with sparteine or debrisoquine suggested a polymor- AsTable2shows,moleculargeneticstudiesidentified
phic variant of CYP450 might be responsible for the some of the molecular defects in CYP2D6* associated
poor metabolism and unexpected responses of suscepti- with poor metabolism of sparteine and debrisoquine
ble persons. Other studies with mephenytoin suggested some 11 years after the trait was discovered. CYP2D6*
a variant of a distinctly different CYP450 appeared to (sparteine/debrisoquine oxidation) polymorphism has
explain the poor metabolism and unexpected responses stimulatedanenormousamountofinvestigationandwe
of susceptible persons to this drug. In 1982, the com- now know that this polymorphism results in three sepa-
plete primary structure of a mammalian CYP450 was rable phenotypes: poor metabolizers, extensive metabo-
reportedfromrecombinantDNAstudies,andthisbreak- lizers, and ultrarapid metabolizers. The CYP2D6* poor
throughpromptedagreatdealoffurtherinvestigation..52/ metabolizers have an impaired capacity to metabolize
A recent count shows that more than 150 isoforms of more than 30 drugs and are homozygous for an inactive
CYP450havebeencharacterizedasproductsofdifferent or deficient CYP2D6 enzyme that has been modified by
genes,andatleast30different humanCYP450enzymes truncationormissensemutationsofthegeneCYP2D6*.
have been purified, cloned, sequenced and character- Incontrast,ultrarapidCYP2D6*metabolizerspossessan
ized. Of these, less than ten (namely 1A1/1A2, 2A6, enhancedcapacitytometabolizethesedrugsbecausethey
2C19, 2D6, 2E6 and 3A4) are responsible for oxida- possessmorethanonecopyofanamplifiedCYP2D6*.
tion of most drugs and other substrates in the human Pharmacogeneticpolymorphismiswellestablishedfor
environment..8/ the genes of several other CYP450 enzymes including
A chronology of selected human pharmacogenetic 1A1,2C9,and2C19,andisrapidlyaccumulatingfor1A2,
polymorphismsthathavebeenidentifiedsincetheemer- 2A6, 2E1, and 3A4..8/ Most of the genetically variant
gence of pharmacogenetics in the 1950s is given in CYP450 enzymes occur as high- and low-activity (or
Table2. It includes a cross-section of polymorphisms: null) isoforms that may confer individual susceptibility
several drug-metabolizing enzyme polymorphisms, sev- tothetoxiceffectsofenvironmentalchemicalsincluding
eral receptor polymorphisms (e.g. malignant hyperther- carcinogens. Molecular genetic studies have identified
mia, vasopressin resistance, insulin receptor resistance) thegenesthatencodetheseenzymesaswellasnumerous
and polymorphisms of some other protein variants of allelicformsofthem.
pharmacogeneticinterest.Thetablealsodrawsattention Table2 also lists several drug-conjugating enzymes.
tothetimethatelapsedbetweentheinitialdescriptionof During the 1990s, considerable progress has also been
10
CLINICALCHEMISTRY
Table2 Chronologyofpharmacogenetics
Polymorphism Inheritancedescribed Mutationdescribed Elapsedtime
(years)
Succinylcholinesensitivity 1957–60 1990–92 33
G6PDdeficiency 1958 1988 30
LongQTsyndrome 1957–60 1991–97 34
Acetylation 1959–60 1989–93 30
Malignanthyperthermia 1960–62 1991–97 31
Glucuronosyltransferase 1966–69 1992–? 26
Vasopressinresistance 1969 1992 23
Aldehydedehydrogenase 1969 1988 16
Debrisoquineoxidation 1977 1988–93 11
Retinoicacidresistance 1970 1991–93 21
Thiopurinemethyltransferase 1980 1995 15
Mephenytoinoxidation 1984 1993–94 9
Glutathionetransferase 1986 1990 4
Fructoseintolerance 1986 1988–95 2
Insulinreceptorresistance 1988 1988–93 0
Androgenresistance 1990 1990 0
Glucocorticoidremediablealdosteronism 1992 1992 0
madeinthemoleculargeneticanalysisoftheseenzymes. 2.5 TheEffectofMutationsontheGeneProduct
Prominent members of this group are the polymorphic
Mutations have been observed to have two main effects
forms of acetyltransferases, glucuronosyltransferases,
on the gene product: they can cause the production
glutathione-S-transferases and thiopurine methyltrans-
of a structurally variant protein with altered functional
ferase.
propertiesortheproductionofafullyfunctionalprotein
Receptorproteinsrepresentanothersubsetofproteins
in altered amounts, usually reduced, although traits
(seeTable2)withenormouspotentialtoinfluencehuman
attributable to increased amounts of protein have been
sensitivitytoexogenoussubstances..4/Comparativelyfew
described..53;54/ Mutations that cause the synthesis of
receptor polymorphisms have been studied so far, since
structurally variant proteins usually occur within the
this group of proteins has not yet been explored in
coding region of the gene, whereas mutations located
thedepthachievedwiththedrug-metabolizingenzymes.
within the promoter and other regulatory sequences
Priortothe1980s,theexistenceofreceptorsandreceptor
usually alter protein quantity. Most pharmacogenetic
subtypes was customarily defined by differences in their
polymorphisms result from point mutations, or other
interactionswithdrugs,butthisapproachdidnotachieve
small genic lesions that lead to a functional change in
outstandingsuccessinestablishingreceptorheterogeneity
a given protein, or loss of all or most of a gene. As
on a molecular plane. Based on their location and
these polymorphisms occur at specific genetic loci and
mechanisticfeatures,twomaintypesofpharmacological
within specific regions of the gene, the ability to detect
receptor are recognized: those that are located in the
anddeterminetheassociatedspecificstructuralchange(s)
cytoplasm and with a locus of action within the cell
allowsfordiagnosis.
nucleus,andthosethatinsertintothecellsurfaceandspan
Knowledge of the biochemical, pharmacological or
themembrane.Cellsurfacereceptorsareinvolvedinthe
toxicologicalfeaturesassociatedwithanunusualpheno-
actions of most drugs and endogenous first messengers; typecansometimesleadtoinferencesabouttheidentity
being on the cell surface, they can bind biogenic of the gene and the properties of the allelic variants
amines, protein and polypeptide hormones, autocoids, responsible for the phenotype. Many pharmacogenetic
neurotransmitters and environmental chemicals. The polymorphismswerecharacterizedbythisapproachlong
nuclear (cytoplasmic)receptors,bycomparison,interact beforemoleculargenetictechniquesweredeveloped.For
withrelativelyfew,albeitimportant,firstmessengersthat example, observation of low levels of urinary acetylated
can enter the cell, such as the steroid hormones. The metabolites of isoniazid suggested that an acetyltrans-
techniques of molecular biology have enabled sufficient ferase, or acetyltransferases, with impaired capacity to
amountsofagivenproteintobeproducedforbiochemical acetylate isoniazid accounted for the isoniazid-induced
and pharmacological characterization and have greatly nerve damage seen in susceptible patients. Biochemical
refined and extended the genetic analysis of receptor studiesdocumentedthepresenceoffunctionallyimpaired
heterogeneity. forms of this drug-conjugating enzyme, which led to the