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PsychologicalBulielin2000,Vol 12ft ,No. 6,806-828

Copyright2000bythe AmericanPsychologicalAssociation,Inc.0033-2909/00/$5.00 ~OI: 10.1037//0033-2909.126.6.806

DNARoberJ Plomin

King's College LondonJohnCrabbe

Portland Alcohol Research CenterandOregon Health Sciences University

The authorspredict that in a few years,many areas ofpsychologywill be awash in specific genesresponsiblefor the widespreadinfluenceofgeneticsonbehavior.As thefocusshifts fromfindinggenes(genomics) to understandinghow genes affect behavior (behavioral genomics), it is important for thefutureof psychology as a science thatpathwaysbetween genes and behavior be examined notonlyat themolecularbiologicallevel ofcellsor theneurosciencelevelof thebrainbutalsoat the psychologicallevelofanalysis.After abriefoverviewof quantitativegenetic research,theauthors describehowgenesthatinfluence complextraits likebehavioral dimensions anddisordersin human andnonhumananimals arebeing found.Finally,theauthors discuss behavioral genomicsandpredictthatDNAwillrevolutionizepsychologicalresearchandtreatmentearly in the21stcentury.

Afirstdraftof the entiresequenceof 3 billion nucleotide basesofDNA in the human genome was reported this year, several yearsahead of schedule, 99.9% of these DNA sequences are the samefor allpeople. Identifying the 0.1% of the DNAsequences thatdiffer is one of the next goals of the Human Genome Projectbecause these 3 million DNA sequences areresponsible for thegeneticdifferences amonghumanbeings behaviorally as well asbiologically.In thisso-calledpost-genotnicsworld, when all theDNA sequences and their variants are known, research will switchfrom findinggenes tounderstandinghowthesegenes work (func-tioned genomics).

The term functional genomics usually connotes molecular bio-logical and biochemical research that identifies gene products(proteins)andinvestigates their function at acellular level. How-ever, higher levelsof analysis are increasingly needed to under-standthe pathways between genes and behavior. For example, thebrain willundoubtedlybe a major targetforfunctional genomicresearch inneuroscience using neuroimagingtechniquesforbothhumanand nonhumanresearch(Kosslyn & Plomin, inpress)andmore precise techniques such as the injection ofantisenseoligode-oxynucleotides (shortstrings ofbasescomplementary to a specificmessengerRNA[mRNA])intothebraintoturnoff theproductionofspecific genes for animal model research. The psychologicallevel of analysis focuses onbehavioral functions of the wholeorganism. To highlight its importance to this level of analysis, werefer to it asbehavioral genomics.

Robert Plomin, Social, Genetic and Developmental PsychiatryResearchCentre,InstituteofPsychiatry,King'sCollegeLondon,UnitedKingdom;JohnCrabbe, PortlandAlcohol ResearchCenter, Portland, OR, and De-partment ofVeteransAffairs Medical Center andDepartmentofBehav-ioralNeuroscience, OregonHealthSciencesUniversity.

Correspondenceconcerningthis articleshouldbe addressed toRobertPlomin,Social,Genetic and DevelopmentalPsychiatryResearchCentre,Instituteof Psychiatry,King's CollegeLondon,111DenmarkHill,LondonSE5 8AFUnitedKingdom.Electronicmailmay [email protected].

Ahuge research effort is currently focused on finding genesassociatedwithbehavioraldisorders. Such researchisdifficultandexpensive, and relatively few psychologists are likely to join thehunt for genes. However, we predict that within a few years,psychology will be awash with genes associated with behavioraldisorders aswellasgenes associated with variationin thenormalrange. As discussed later, psychologists will use information aboutthese genes in their research for three reasons: (a) DNA-basedinformation is becoming increasingly inexpensive and easy togather,(b)behavioral genomicscanmake important contributionstowardunderstandingthefunctions ofgenes, and (c) DNAopensup new scientific horizons forunderstanding behavior.Wealsopredict that DNA will change clinical psychology, leading togene-based diagnoses and treatment programs. These advanceswill needto beintegrated withcorresponding advancesinunder-standing themultiple,interactive environmentalinfluencesas well.Wediscusstheethical implicationsofsuchpredictionsat the endofthis article.

For these reasons, it is crucial that psychologists be prepared totakeadvantageof theexciting developmentsinmolecular genetics.In the same way that computer literacy is now an essential goal tobe achieved during elementary and secondary education, studentsinpsychology mustbetaught about geneticstoprepare themforthis future. Otherwise, this opportunityforpsychology will slipaway bydefault togeneticists, andgenetics ismuchtooimportantatopicto belefttogeneticists Thebasic conceptsofgeneticsandevolution shouldbeincorporatedintoasolid groundinginneuro-science that is also crucial for psychological training. Clinicalpsychologistsuse theacronymDNA tonote "didnot attend"it iscriticalto the future ofpsychology as ascience thatDNAmeansdeoxyribonucleic acid rather thandid not attend,

Inthisarticle,we describe how genesassociatedwith behaviorareidentifiedinhumanbeingsand inanimal modelsand howthesegenescan be put toworkinpsychological researchandtreatment.To putthis discussionincontext,webegin withabrief overviewof quantitative genetic studies. However, technical details aboutquantitativegenetic methods, results, and qualificationspertinent

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to interpretationofdata arebeyondthescopeof this article (fordiscussionofthese topics, see, e.g.,Plomin,DeFries, McCleam,&McGuffin, 2001).Background references are also provided in ourdiscussion of DNA incasewehave missed themarkin tryingtobalance breadth and depth.

QuantitativeGeneticsThe fundamental accomplishment of behavioral genetic re-

search in psychology has been to demonstrate the ubiquitousimportanceofgenetics throughout psychology. Historically, thisevidence has comprised inbred strain and selection studies ofnonhuman animal behavior and, for human behavior, twin studiesthatcompared the similarity of identical and nonidentical twinsandadoption studiesthatconsidered,forexample,theresemblanceof adopted-away children to theirbiologicalparents (Plomin et al.,2001). These methodsand thetheory that underlies themarecalledquantitative geneticsin contrast to molecular genetic studies thatattempt to identify specific genes. Behavioral genetics includesboth quantitative and molecular genetic approaches to investigat-ing genetic influences on individualdifferences in behavior.Be-havioral genetics focuses on questions of why individuals within aspecies differ in behavior (e.g., why children differ in rates oflanguage acquisition) as opposed to species-typical behavior (e.g.,when, on average, children use two-word sentences). Geneticsinfluences both species-typical behavior and individual differenceswithin a species, in different ways. For example,human evolu-tionary history written in DNA code accounts for the fact thathuman beings use two-word sentences at the average age of 18months, but this does not mean that genetics is the reason whysome children do not use two-word sentences until much later indevelopment.

Thecontroversy that swirled around human behavioral geneticsresearchonindividualdifferencesinpsychology duringthe1970shaslargelyfaded,despitea recent resurrection of the controversialissues of group differences between races and between socialclasses (Herrnstein & Murray, 1994). It is now more generallyaccepted that the genetic contributions to individual differenceswithin racial groups do not necessarily explainbetween-groupdifferences, which can have their own genetic and environmentalcontributing factors. Duringthe 1980s andespecially the 1990s,psychology became much more acceptingofgenetic influenceonindividualdifferences, as can be seen in the increasing number ofbehavioral genetic articles in mainstream psychology journals andthenumber of research grants. One symbol of this change was the1992 Centennial Conference of the American Psychological As-sociation, in preparation for which a committee selected twothemes that best represented the past, present, andfutureof psy-chology. One of the two themes chosen was behavioral genetics(Plomin & McCleam, 1993). Indeed, the wave of acceptance ofgenetic influence throughout psychology threatens to engulf asecond, equally important message coming from behavioral ge-netic research, namely, that individual differences in complexpsychological traits are due at least as much to environmentalinfluences as they are to genetic influences. In some areas ofpsychology, especially psychopathology, the pendulum represent-ingtheaccepted viewmay beswingingtoo farfromenvironmentaldeterminismtogenetic determinism.

Nonetheless, in part because of the far-reaching implications ofits findings, behavioral genetics still provokes controversy. Forexample, one area of current controversy involves two findingsabout the environment called nonshared environment and thenature of nurture. The first finding is that environmental influ-ences tend to make children growing up in the same familydifferent,notsimilar. Becauseenvironmental influencesthataffectpsychological developmentare notsharedbychildrenin thesamefamily, they are called nonshared environment (Dunn& Plomin,1990; Plomin&Daniels, 1987;Turkheimer&Waldron,2000).The secondfindingis that measures of the environment, especiallyofthefamily environment, show genetic influence when embed-ded ingenetically sensitive designs andalso show genetic medi-ationof associations betweenenvironmentalmeasures and behav-ioral outcomes (Plomin, 1994; Reiss,Neiderhiser,Hetherington,&Plomin, 2000).Taking these arguments to theextreme, a presi-dential address of the Society for Research in Child Development(Scarr, 1992) and two recent books (Harris, 1998; Rowe, 1994)haveconcluded that socializationresearch isfundamentallyflawedbecauseit has notconsidered theroleofgenetics. These attackshave metwith stiff resistance from developmental psychologists(W. A.Collins,Maccoby,Steinberg, Hetherington, & Bornstein,2000; Maccoby, 2000; Vandell, in press). Both sides agreethat progress toward resolving these differences depends onbetter articulation and measurement of specific environmentalinfluences.

The Example of SchizophreniaUntilthe1960s,schizophrenia was thought to be environmental

in origin, with theories putting the blame on poor parenting toaccount for the fact that schizophrenia clearly runsin families.Although a minority view representing the nascent discipline ofbiological psychiatry offered biochemically based etiologies, theidea that schizophrenia couldrun infamilies forgenetic reasonswasnot seriouslyconsideredby most American psychiatrists andpsychologists.Despitelimits totheir interpretation not discussedhere, twin and adoption studies changed this view. Twin studiesshowed that identical twins are much more similar than noniden-ticaltwins, which suggests genetic influence. If one member of anidentical twinpair is schizophrenic, the chances are 45% that theother twin is also schizophrenic. For nonidentical twins, thechancesare17%. Adoption studies showed thattheriskofschizo-phreniaisjustasgreat when childrenareadopted awayfrom theirschizophrenic parents at birth as when children are reared by theirschizophrenic parents, which provides dramatic evidencefor ge-netic transmission. There are now intenseefforts toidentify someof the specific genes responsible for genetic influence onschizophrenia.

In the 1960s, when schizophrenia was thought to be causedenvironmentally, it was important to emphasize the evidence forgenetic influence such as the concordance of 45% for identicaltwins. Nowthat evidencefor theimportanceofgenetic influencethroughout psychologyhaslargely been accepted,it isimportanttomake sure thatthependulum staysin themiddle, betweennatureand nurture. We need to emphasize that identical twins are only45%concordantforschizophrenia, which meansthatinhalfof thecases, these pairsofgenetically identical clonesarediscordantforschizophrenia. This discordance cannotbeexplained genetically


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808 PLOMINAND CRABBEitmustbe due toenvironmental factors.Itshouldbenoted thattheword environmentingeneticresearchreally meansthenongeneticenvironment,whichis amuch broader definitionofenvironmentthan isusually encounteredinpsychology. That is,environmentdenotes all nonheritable factors, including possible biologicalevents such as prenatal and postnatal illnesses,not just the psy-chosocial factors that are usually considered in psychology. Thepoint is that genetics can often explainhalf of thevarianceofpsychological traits, but this means that the other half of thevariance is not due to genetic factors.

Genetics, Environment, and HeritabilityFor nearly every area of psychology that has been studied,

quantitativegenetic researchhas shown genetic as well as envi-ronmental influence (Plomin etat,2001).Forexample, geneticresearch has consistently shown genetic influence in many tradi-tional areas of psychological research such as psychopathology,personality, cognitive disabilities and abilities, and substance useand abuse. Some areas showing strong genetic influenceare moresurprising, such as school achievement, self-esteem, interests,andattitudes. Nonetheless, it is important to remember that thesepartitionsof the genetic andenvironmental sourcesof individualdifferencespertain tothespecific populations andsocial/environ-mental conditions in which they are ascertained. Any changes tothe underlying assumptions can lead todifferentconclusionsaboutthe relative importance of genetic and environmental influences,butfew now seriously question that both influences are important.

Most importantly, genetic research in psychology is movingbeyond heritability.The questions whether and how much geneticfactors affect psychological dimensions and disorders representimportant first steps in understanding the origins ofindividualdifferences, but these questions about heritability are only firststeps. The next steps involve the question how, that is, the mech-anisms by which genes have their effects. Examples of thesedirections for genetic research in psychology include geneticchange as well as continuity during development, genetic linksbetween dimensions and disorders, multivariategenetic analysis,and the interplay between genetics andenvironment (Plomin&Rutter, 1998).

Such quantitative genetic research will become increasinglyimportant as it guides molecular genetic research toward the mostheritable components and constellations ofdisorders and dimen-sions as they interact and correlate with the environment through-out development. Conversely, as discussed later,finding specificgenes associated with behavior will greatly enhance psychologists'ability to address quantitative genetic issues such as those justmentioned.

IdentifyingDNAAssociatedWithBehaviorNow that the contribution of genetic factors to individual dif-

ferences in psychology is widely accepted, molecular genetictechniques thatcan ident ify someof thegenesresponsibleforthisgeneticvariation are becoming available. The heritability of com-plex traitsis likely to be due tomultiple genesofvaryingbutsmalleffectsizerather thanto onegeneor a fewgeneswithmajor effect.Genes in such multiple-gene systems are inherited in the same wayasany other gene, but they have been given adifferent name

quantitative trait loci (QTLs)to highlight some important dis-tinctions. Unlike single-gene effects thatarenecessary and suffi-cient for the development of a disorder, QTLs contributeinterchangeably and additively, analogous to probabilistic riskfactors. Iftherearemultiple genes thataffect atrait,it islikelythatthe trait isdistributed quantitativelyas adimension rather thanqualitatively as adisorder, thiswas theessenceof R. A.Fisher'sclassic 1918 paperonquantitative genetics(R. A.Fisher, 1918).Ofcourse, it is also possible that categorical disorders are etiolog-ically distinct fromdimensions, and molecular genetic informationcould help resolve these alternatives. Forexample,differentgenescouldbe involved in abnormal disorders versus extremes of nor-malbehavior that appear to be similar;alternatively, such behav-iors could be due to particular combinations of genes (Lykken,1982). Such interactions among genes, called epistasis, are dis-cussedlater.

From a QTL perspective, most disorders are just the extremes ofquantitativetraits causedby thesame geneticandenvironmentalfactorsresponsibleforvariation throughoutthedimension.Inotherwords, the QTL perspective predicts that genesfound to be asso-ciated with complexdisorderswillalsobe associated with normalvariationon thesame dimensionandvice versa (Deater-Deckard,Reiss,Hetherington, & Plomin, 1997; Plomin, Owen, & McGuf-fin, 1994).Althoughthe QTLperspective hassome specific im-plications for design and analysis of molecular genetic studies, itsgeneral importance is conceptual. At the most general conceptuallevel, a common mistake is to think that humans are all basicallythe same genetically except for a few rogue mutations that lead todisorders. In contrast, the QTL perspective suggests that geneticvariation is normal.Many genesaffect most complex traits, and,togetherwithenvironmental variation, these QTLs areresponsiblefornormal variationaswellas for theabnormal extremesofthesequantitativetraits. This QTL perspective has some implications forthinking about mental illness because it blurs the etiologicalboundaries between the normal and the abnormal. That is, humanbeings all have many alleles that contribute tomental illness, butsomeareunluckierin thehand thattheydrawatconception fromtheir parents' genetic decks of cards. Amore subtle conceptualadvantage of a QTL perspective is that it frees psychologists tothink about both ends of the normal distributionthe positive endas well as the problem end, abilities as well as disabilities, andresilience as well as vulnerability. It has been proposed that psy-chologists move away from an exclusive focus on pathologytowardconsidering positive traits that improvethequalityof lifeand perhaps prevent pathology (Seligman & Csikszentmihalyi,2000).

The QTL perspective is the molecular genetic version of thequantitativegenetic perspective that assumes that genetic varianceon complex traits is due to many genes of varyingeffectsize. Thegoal is not tofindthe one gene for a particular trait but rather someof themany genes that make contributionsofvaryingeffect sizesto the variance of the trait. Perhaps 1gene will be found thataccounts for 5% of the variance,5other genes thatmight eachaccountfor 2% of thevariance,and 10othergenesthat might eachaccount for 1% of the variance.If the effects ofthese QTLs areindependent, theseQTLswould together account for 25% of thetotalvariance.If theheritabilityof thetraitis50%, they wouldthusaccount forhalfof theheritable variance.If, as is likely,theyareto some degree interactive, they would in sum control less of the


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heritable variance. All of the genes that contribute to the herita-bility of thetraitare unlikely to beidentifiedbecause some of theireffectsmay be too small or complicated to detect.

All attempts to identify genes relyon genetic markers that arestretches of DNAthatdiffer among individuals.These differentformsof DNA at aplace (called a locus) on a chromosome arealleles.Forexample, A, B, and O arethree alleles at alocusonchromosome 9 that code for the different forms of the bloodproducts calledthe ABOblood system. Classical genetic markerslike the ABO blood system were limited to a few dozen markersfor single-gene traits, often measured in the blood. Systematicgenemapping was made possible beginning in 1980 when geneticmarkers were developed that involve DNA itself rather than theproductsofgenes. Individual differencesinDNA, usuallyasinglebase-pairdifference,occuronaverage aboutoneinevery thousandnucleotide base pairs of DNA, which means that there areabout 3.5 million potential DNA markers throughout the 3.5 bil-lion nucleotide base pairs in thehuman genome. There arealsopotentially more than50,000 DNA markers that involve shortsequences of DNA that repeat for unknown reasons. The numberofrepeats varies greatly across individuals and is inherited. Suchshort-sequence repeat markers have beenmostoftenusedingenehuntingto date. However,interest is now turning to developinghundreds of thousands of another type of DNA marker calledsingle nucleotide polymorphisms (SNPs, pronounced snips). Astheir name implies, SNPs involve a difference in a single base pairof DNA. SNPs are especially interesting in that they are morelikely to beresponsible for functional DNAdifferences becausechangesin thecoding sequenceof DNAusually involveasinglebase pair of DNA (F. S.Collins, 1999).

The following sections describe the methods used to identifygenes associated with behavior and provide examples of replicatedresults using these methods. We begin with nonhuman animalmodels. Animal models providepowerful meanstoidentify genesbecause the genotype can be manipulated through breeding andbecause genes themselves can also be manipulated. Moreover,animal models arc more than models when it comes to DNA:Nearly anygenefoundin mice or evenfruitflies can also befoundin the human species although some of thegene'sDNA sequencesdifferacross speciesand thegenemayhavedifferent effects acrossspecies. Thus, gene-behavior associations found in nonhumananimals are reasonable candidates for the human species. Evenidentifying a chromosomal region (locus) rather than a specificgene can be valuable because large chunks of chromosomes inmice and man have the same genes in the same order (calledsyntenic conservationorsynteny). In addition, studying animalsenables researchers to use much morepowerful methods for ex-ploring underlyingneurobiologicalmechanisms. Finally, interac-tions between genes and environment can be studied more pow-erfully using animal models because the environment can becontrolled and manipulated.

Identifying GenesforNonhumanBehaviorThroughInducedandNaturallyOccurringMutations

Researchinmultiplespecieswill become much more interactiveandmutually informative becauseof thegreatsimilarityofhumanand rodent genomes. Mice and humans shared a common ancestoras recently as 60 million years ago, and large chunks of genomes

ofthe twospecieshave been conserved with nearly identical linearorganization of bases and, therefore, genes (Battey, Jordan, Cox, &Dove, 1999; Silver, 1995). Thus,finding agene regioninmouseleads the investigator more than 80% of the time to apreciselocation in thehuman genome.

Long before DNA markers became available in the 1980s,associations werefound between genes and behavior in animals.The first example, discovered in 1915, is a single gene that alterseye color in the fruit fly Drosophila and also affects itsmatingbehavior (Sturtevant, 1915). Another well-known example in-volves thesingle gene that causes albinismandalsoaffectsopen-field activityinmice.TheJackson Laboratorymaintainshundredsof mutations thathave occurred over the years, many as frozenembryos that can be reconstituted on order. Thesemutants includegenesaffecting awidevarietyofbehaviors including neurologicalfunctions such as gait, balance, and seizures.

Chemical- and Radiation-Induced MutantsIn addition to studying naturally occurring genetic variation,

geneticists have used X-irradiation or chemicals such asethylni-trosourea to create mutations that enable them to dissect thebehavioraleffects ofgenes.Duringthe past 50 years, hundreds ofbehavioral mutants have been created in organisms as diverse asworms, fruit flies, mice, and single-celled organisms such asbacteriaand paramecia(Plominetal.. 2001).This research illus-trates the principle that most normal behavior is influenced bymanygenes. Althoughany one ofmany single-gene mutationscanseriouslydisruptagiven behavior, normal developmentisorches-trated by many genes working together. For example, bacteriamove towardandawayfrom manykindsofchemicalsbyrotatingtheir propeller-like flagella. Since the first behavioral mutantinbacteria was isolated in 1966, the dozens ofmutantsthathave beencreated indicate that many genes are involved in rotating theflagella andcontrollingthedurationof therotation. Hundredsofinduced behavioral mutants have been isolated inparamecia, in-cludingat least 20 involved in backing up and swimming forwardin a new direction to avoid certain chemicals and heat. Thenematode (roundworm) Caenorhabditis eleganshas provided ex-amples of several mutants with great longevity (Duhon, Mu-rakami, & Johnson, 1996) and will be an increasingly importantanimalmodelforgenetic analysisofbehavior becausethedevel-opment of each of its 959 cells, the wiring diagram of its 302neurons,and itsentire 100million base pairsof DNA areknown.Hundreds ofbehavioral mutants, for example, for courtship andmemory,have also been identified for thefruitflyDrosophila,oneofthemoststudied organisms in genetic research(Weiner,1999).

Perhaps the bestknownproject of this sort in neuroscience is thescreening of the offspring ofchemically mutatedmice for circa-dian rhythms in activity. A single mutant mouse with a longcircadian rhythm was identified in a screen andusedin a system-aticprogram to locate the clock gene and prove itsresponsibilityfor the trait. Mice with a mutant form of this single gene hadabnormalpatterns of daily activity and rest (Antoch etal., 1997;Kingetal.,1997). Further studies have shown that the suprachi-asmaticnucleus (SCN)of the anterior hypothalamus acts likeapacemaker to control circadian rhythms. SCN neurons isolatedfrom clock mutant mice have arrhythmic firing patterns, suggest-ingthatsome mechanisms within this nucleus synchronize neuro-


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810 PLOMIN AND CRABBEnal firingpatterns that eventually regulatetheactivityof themouse(Herzog, Takahashi, & Block, 1998). The current focus of thisresearch is on understanding how the clock gene regulates SCNneuronalfiring rates to control the complex set of behaviors thatfollowacircadianpattern and on understanding the roles of other,related genes (Vitaterna et al., 1999).

There are major new initiatives in theUnited States and theUnited Kingdom directed toward usingchemicalmutagenesison alargescaleto develop new mutants.Theseinitiatives are targetedatbehavioralandneural developmentanddependonsophisticatedand sensitive behavioral assaysfordetectingwhichmice bearamutation.Inaddition, thereisintereston thepartofpharmaceu-tical manufacturers in suchefforts, as there is the possibility thatnovelgenesaffectingthe targeted traits could be discovered, whichinturn could lead to novelphannacotherapies.

Targeted GeneMutation: Transgenics and KnockoutsMouse is also the species most used for targeted gene mutation

(gene targeting),a techniquehiwhich mutations that "knock out"the transcription of a gene entirely or alter its regulation in order tounderexpress or overexpress the gene are created (Capecchi,1994).The mutated gene is then transferred to mouse embryos,andthe mice are calledtransgenicswhen the mutated gene is fromanother species. This method is discussed later in relation tobehavioral genomicsbecause it is used primarily to understandhowageneof apriori interestaffects abehavior ratherthanto findwhich genes are associated with abehavior. Nonetheless, genetargeting provides an important, although not conclusive, confir-mation of agene'seffect on behavior. Knockouts (alsocallednullmutants}of various genes have been shown toaffect several typesoflearning(Wehner,Bowers, & Paylor, 1996), aggression (Nelsonetal.,1995; Saudou et al.,1994),alcohol preference (Crabbe etal.,1996), nicotine effects on pain (Marubio, Arroyo-Jimencx,Cordero-Erausquin,Lina, & Novhre,1999),and general sensitivityto abused substances (Rochaet al.,1998; Rubinsteinetal., 1997).Knockoutstudiesare inprogressforhundredsofgenes,andmanywillhave multipleeffects onbehavior (Brandon, Idzerda,& Mc-Knight, 1995). For example, a recent summary lists 22 genesshown to affect learning and memory (Wahlsten, 1999), and acompendiumofknockoutsaffectingvarious behaviorshasrecentlyappeared (Nelson & Young, 1998; see also Silva & Mayford,1998).

Beyond Knockouts: Conditional Gene ExpressionGene-targeting strategies are not without their limitations (Ger-

lai,1996). The most obvious problem with knockout mice is thatthe gene is inactivated throughout the animal's lifespan. Particu-larlyduring development, the organism copes with the loss of thegene's function by compensating wherever possible. In some in-stances, this can bevery informative. For example, deletion of agene coding for adopaminetransporter protein (which is respon-sible for inactivating dopaminergic neurons by transferring theneurotransmitter back into thepresynaptic terminal) resultedinmice with extraordinarily high activity in a novel environment(Giros, Jaber, Jones, Wightman, & Caron, 1996). The animalsfailed to habituate even after several hours. Probably becausedopamine is animportant regulatorofsecretion ofprolactinand

other neurohormones, these knockouts were pituitary dwarves aswell (Bosseet al., 1997). As aresult,theknockouts exhibitedafascinatingcomplexofcompensations throughoutthedopaminer-gic cascade, ranging from increased synthesis of dopamine toreduced levelsof theenzyme tyrosinehydroxylase,which trans-formsthe amino acid tyrosine to DOPA, which is thentransformedto dopamine (S. R. Jones et al., 1998). These comparisons lead toimproved, but not completely normal,functioningof the dopami-nergicsystemscompromisedby the knockout. However, in mostinstances, compensations for the loss of genefunctionare invisibletotheexperimenter,andcaution mustbetakentoavoid attributingother changesin theanimalsto thegeneitself.

This problem will not be overcome untilthe second generationofknockout technology enters wideusage.Ratherthancompletelydisrupting the transcription of the gene from conception, it ispossible to alter parts of a gene that increase or decrease rates ofthegene's transcription, to add regulators that act as a switchturningthegeneon oroff,and tochangetheexpressionof thegeneina specific area of the brain. Several approaches toaccomplishingthisgoal have been reviewed(Crusio,1999). When the technologyis fully developed, conditional knockoutswill bear DNA con-structs that includethe gene of interest aswellassequencesthatallowtheexperimenter toturnexpressionof thegeneon or off atwillat anytime duringtheanimal's life span.

Anexample of a conditional knockout involves a receptor forW-methyl-D-aspartate (NMDA). This receptor affects neurotrans-mission by way of theexcitatory neurotransmitter glutamatethatplaysanimportantroleinmemoryand inlong-termpotentiation(LTP), a commoncellularelectrophysiologicalmodel of learning.LTP involves structuralandfunctionalchangesin thesynapse.TheNMDA receptor serves as a switch for memory formation bydetecting coincident firing of different neurons and affects theintracellularsecond messengersignalingprotein cyclic adenosinemonophosphateamong other systems. Overexpressingonepartic-ularNMDA receptor gene (NMDA receptor 2B) enhanced mem-oryin various tasks as well as LTP (Tang etal., 1999). A condi-tional knockout of theNMDARZB gene was used to limit themutationto aparticular areaof thebrain,inthiscase,theforebrain.Normally, expression of this gene slowsduringadulthood, whichmaycontributetodecreased memoryinadults.Inthisresearch,thegene was altered so that it continued to be expressed in adulthood,andthis resultedin enhanced memory inseveral tasks.Thisreportgenerated much media interest,as thefinding wasinterpretedbysome as showing that a so-called intelligence gene had beenfound,withobvious social implications. However,aninteresting featureofthis research isthat therewaslittle evidence that learningwasenhanced: Rather, the conditional knockouts retained their mem-ories longer, so the relevance to intelligence remains to be seen.

Inanearlier research effort, micewerecreatedwithacondi-tional knockout of thegeneencodingtheNMDA1receptor (Tsienet al., 1996; Tsien, Huerta, & Tonegawa, 1996). These micedeveloped to adulthood withnormal levels of expression of thegenethroughoutmostof thebrain.However,thetransgenicmu-tantswere constructed such that beginning duringtheimmediatepostnatalperiod, gene expression was gradually turned off over aperiodofaboutamonth,butonlyincertain cellsin thehippocam-pus.Thesetissue-specific,conditional knockout mice were defi-cient in LTPgenerated wheretheNMDA1receptorwasturnedoffbutshowed normal LTP generated incellswhere NMDA 1 recep-


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tors were expressed at normal levels. Theyalsoweredeficient inspatial learning, which is known to depend on the hippocampus;electrophysiologicalactivity ofhippocampalcells specialized forspatial learning (placecells)was disrupted as well.

Thesestudies represent first forays into the second generation oftargeted gene deletion studies. The ability to turn genes on or offat will in specific brain areaswillgo a long waytowardovercom-ingthe current limitations of these model systems, as the compen-sations occurring throughout development can be largely avoided.However, to attain full power, conditional knockout technologystill must overcome other major hurdles. For example, bettercontrol is needed over the location of transgene insertion in thegenome and theprecise timingof thesuppressionoraugmentationofgenefunction.Because so little isknownabout why the preciselocation of inserted genes is important to genefunction,the currenttechnologies forproducing conditional mutantsamounttomakingseveral such mutants and laboriously characterizing each of themto seewherein thebrainand towhatdegreetheintroduced geneconstruct is expressed. In many cases, multiple copies of thetransgene are incorporated into the DNA. but it isoften unclearwhether more copies equal higher levels ofgene expression (i.e.,more gene product produced). Thus, the experimenter finallychoosesthebest available mutant strainfromthearrayofchoices.Forexample, in the study described above (Tsien, Chen,et al.,1996; Tsien,Huerta, & Tonegawa, 1996), 11 mutant lines weretested toselectthemutantthat expressed the conditional mutationonlyin the desired hippocampal region.

Antisense OligodeoxynucleotidesAnother method usesantisenseDNA to "knock down" gene

function.Antisense DNA is a DNA sequence typically18-25basepairs long that is complementary to a specificmRNAsequence. Bybinding with mRNA, antisenseDNAprevents the mRNA frombeing translated into protein. Injected in the brain, antisense DNAhas the advantage of high temporal and spatial resolution (Ogawa&Pfaff, 1996). One behavioral study used antisense DNA againstthe CREB gene and confirmed the involvement of this gene inmemory formation (Guzowski & McGaugh, 1997). AntisenseDNAis being widely used inpsychopharmacology,for example,to block drug effects by preventing the synthesis of receptormolecules in specific brain regions (Pasternak,2000).AntisenseDNAknockdowns have been shown toaffectbehavioral responsesfordozens ofdrugs (Buck,Crabbe,&Belknap,2000).AntisenseDNA hasoftenproveneffective in cases where there is a dearth ofagonistsorantagonistswithhigh selectivity.Theprincipal limita-tions ofantisense technology currentlyareits unpredictable effi-cacy and a tendency to produce general toxicity.

Targeted mutations and antisense DNA reflect the complexity ofbrainsystemsforlearningandmemory.Forexample, noneof thegenes and signaling molecules in flies and mice found to beinvolved in learning and memory are specific to learning pro-cesses. They are involved in many basic cell functions, whichraisesthequestion whether they exert theireffects onmemorybymodulating the cellular background in which memories are en-coded (Mayford & Kandel, 1999).

IdentifyingGenesfor NaturallyOccurringGeneticVariation inNonhumanBehavior

The mouse has also been the key organism studied in researchon naturally occurring genetic variation (Silver, 1995). As indi-cated earlier, complex quantitative traits, whether biological orbehavioral, are likely to be influenced by QTLs. Because theirbreeding can be controlled and because their small size makesthem anaffordable mammalian species, mousemodelshave beenvaluable indetecting QTLs forcomplex traits.

The chromosomal location of aQTLaffecting a trait is identi-fiedthrough a method called linkagemapping in which the coin-heritance of the trait and a particular DNA marker is traced.Linkage is essentially a violation of Mendel's second law ofheredity called independent assortment, which states that the in-heritanceof onegeneis notaffectedby theinheritanceofanothergene. Genes do not assort independently if they happen to beclosetogetheron thesame chromosome, whichiscalled linkage.Withinfamilies, recombinationis likely toseparate aparticular markerallelefromanalleleaffecting a trait if the two loci are far apart onthe chromosome. Recombination occurs during the formation ofeggs and sperm (meiosis) when chromosomes cross over andexchange partsofmaternalandpaternal chromosomes (see Figure1).Thereis onecross-overperchromosomepermeiosisonaver-age. The extent of recombination can be used to estimate thedistancebetweenthemarkerand the QTL for thetrait.

Inbred andRecombinant Inbred StrainsOne widely used strategy involves crosses between inbred

strains.Inbred strainsofmicearecreated bymating brothersandsistersfor atleast20generations, which makes animals withintheinbredstrain geneticallyidentical(and homozygousateachgene,i.e., with two identical copies of a single allele at each locus).When strains are crossed, the first filial (Fj) generation is het-erozygous at alllocithatdiffer in the parental strains (see Figure2).CrossingF,mice produces anF2generation in which heterozy-gousallelessegregatesothat each individualisgenetically unique.QTLs can be identified by correlating specific alleles of DNAmarkers with quantitative scores ofF2individuals.In an F, pop-ulation,each allele isknownto be derivedfromone or the otherprogenitorstrainand has a 50% frequency. If a particular markerallele is more frequent in high- or low-scoring mice, it can beinferred that a nearby QTL, linked to the marker,affects the trait.Inoutbred populations, which includewildmice andF2crosses ofinbred strains, as well as the human species, recombination sepa-rates alleles for loci on the same chromosome, and more genera-tions of outbreeding increase the number of recombinations.

Aconceptually similar approach, calledrecombinantinbred(RI)strains, reinbreeds from a single F2 population (see Figure 2).Unlike anF2population in which each individual is geneticallyuniqueand not replicable, RI strains providereplicablegenotypesthat are fixed homozygously at each gene for a single alleleinherited fromone or the other progenitor inbred strain. On eachchromosome, each RI strain displays a mosaic of chromosomalsegments derived from one or the other of the parental inbredstrains. This means that once RI strains are genotyped for DNAmarkers, they never need to be genotyped again because thegenotypesof theinbred strains remain unchanged. Thus,RIstrains


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812 PLOMINANDCRABBE

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FigureI. Recombination.A pair of a singlechromosomeisdepictedforan F, hybrid animal during five stages of meiosis. The chromosomehomologinherited maternallyisdepicted asunshaded,andthelocationsofthree genes are shown, as well as the specific alleles for each gene (a, b,and c). Thechromosomehomolog derived paternally is shaded and showsthree alternate alleles (A, B, and C). Inmeiosis, theprecursorcellsof thespermor ova must multiply (i.e., double, as shown in the second stage) andcanthen physically exchange materialat apointofrandom contact. Thisistermed a crossing-over and occurs in the third depicted stage, betweenGenesB and C. Theresultis arecombination, depictedinthefourth stage.When the final stage of gamete production occurs,the chromosome num-ber is reduced to the normal diploid number, and individual recombinantgametes,aswellas nonrecombinantgametes, give risetogerm cells. FromBehavioral Genetics(p. 16), by R. Plomin, J. C. DeFries, G. E.McClearn,and M. Rutter, 1997, New York: Freeman. Copyright 1997 by W H.Freeman.Adapted with permission.

can be used for QTL analysis of behavioral traits withoutanyadditional genotyping (McClearn, Plomin, Gora-Maslak,Crabbe, 1991). For example, the most frequently used set of RIstrains, the BXD RI strains,werederived from across betweenC57BL/6J (B6) and DBA/2J (D2)progenitor inbred strainsthatdiffer markedly in manybehavioral traits, especially in their re-sponses to many drugs (Crabbe Harris, 1991). The BXD RT

BXD 101 BXD 102 BXD 103Figure 2 Construction of a set ofrecombinant inbred (RI) strains fromC57BL/6(B6) and DBA/2(D2)progenitors. A single pair of chromosomehomologs(e.g.,chromosome 2) is followed through the process of RI straindevelopment.Genomicintervals derived fromB6 areshadedandintervalsfromD2 areindicated without shading.The mappositionso ffive fictitiousloci (15) areindicatedin theprogenitor generation.In the F,generation,all animals aregenetically identical and areheterozygotes (asshown)ateach locus.In theF2generation, segregationandindependent assortmentoccurbecause ofcrossing over. Th e genotypes at the five loci for sixindividualmice areshownasshaded signifying th e B6-derived allele orunshadedsignifying the D2-derived allele. The six mice are shown as threebreeding pairs, one foreach of the RI strains to bedeveloped. The twocircled regionsrepresent segmentsof thegenome that are already fixedfo rone progenitor type within both members of the breeding pair. At theF20generation (i.e.,after 20 generations of brother-sister mating, or inbreed-ing),a llgenetic variabilityhasbeen eliminatedwithineachR Istrain. Fourindividuals areshownfo reach strain (fictitiously called BXD-101, BXD-102, and BXD-103). Each new RI strain displays a mosaic of chromosomalsegments derived from one or the other of the parental inbred strains,shuffled through 20generationso frecombination, and fixed through in-breeding.Althoughthepatternsofchromosomal material shownfor the sixF2mice depict crossing over onlyat theborders between th efivegenes,itcan occur anywhere. Additional crossovers occur with each generation ofinbreeding,and the result (seen in theF2C fictitiousBXD RI strains) issmaller regions of chromosomal material preserved for all subsequentgenerations. FromMouseGenetics:Concepts and Applications (p .209),byL. M. Silver, 1995, Oxford, England: Oxford UniversityPress. Copyright1995 by the Oxford University Press. Reprinted with permission.


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strains have been genotyped systematically for more than 1,500markers throughout the mouse genome, with each markerposi-tioned precisely withina chromosomal region.

Most QTL work in mice using these methods has been in thearea ofpharmacogenetics, which denotes genetic effects on re-sponses to drugs. At least 24QTLshave been definitively mappedfordrug responses such as alcohol drinking, alcohol-induced lossof righting reflex, acute alcohol and pentobarbital withdrawal,cocaine seizures,andmorphine preferenceandanalgesia (Crabbe,Phillips,Buck, Cunningham, & Belknap, 1999). This representsconsiderableprogressfrom the firsteffort tosummarizeQTLsfordrug responses5years earlier (Crabbe, Belknap, Buck,1994).Aspecific example is a QTL affecting alcohol withdrawal severity,first provisionally mapped in BXD RI strains (McClearn et al.,1991). Follow-up studies usedthe RIstrains,F2mice,andselec-tively bredlinestoverify rigorously that there was indeed a QTLinthis region (Buck, Metten, Belknap, & Crabbe, 1997). Anotherexampleisemotionalityasassessedby abatteryofmeasuressuchasactivityin abrightlylitopenfieldandexplorationin a Ymaze(Flint et al., 1995). Using the most and least emotionalF2mice,researchers found three highly significant QTL regions. One oftheseQTLswasspecific to theopenfield,whereastheothertwoQTLs wererelatedto all of the measures of emotionality.

Beyond QTLs: CandidateGenes, High-ResolutionMapping, andPositional Cloning

In some instances,the location of a mapped QTL is closeenoughto apreviously mapped geneofknownfunction (candidategene) to make studies of that gene informative. For example,several groups have mapped QTLs foralcoholpreference drinkingin mice to the middle of mouse chromosome 9 (T. J. Phillips,Belknap,Buck,&Cunningham,1998),a regionthatincludesthegene coding for the dopamineD2receptorsubtype. StudieswithD2 receptor knockout mice revealed that they showed reducedalcohol preference drinking (T. J.Phillips,Brown, et al., 1998).Althoughthisdoesnot prove that theD2gene is the basis for theQTL association, itfails to disprove the hypothesis, encouragingfurther investigation.

As more QTLs for complex behaviors are localized in mice,there are enticing signsthatcertain regions of the mouse genomeseem to represent hot spots wheremultipleQTLs arefound.Thisclusteringwasnoted in one of thefirstexplorationsofthis tech-nique(McClearn etal.,1991).Figure3 is acartoon showingthelocation of QTLs for sensitivitytoseveral behavioral effects ofalcohol and the sedative drug pentobarbital (Nembutal). For ex-ample, the close association of QTLs for acute alcohol withdrawal,acute pentobarbital withdrawal, and chronic alcohol withdrawalnear the end ofmouse chromosome 1suggests (but cannot prove)that a single gene in this region may be influencing all threeresponses. If this is true, the gene on mouse chromosome 1 isvirtuallycertainto map to theq21-q23regionofhumanchromo-some 1 because linked groups of genes homologous to mice andmanhave beenfound forthis chromosomal region.

Figure3doesnotshowtheconfidenceintervalssurroundingthemap locations of the QTLs. The QTL locations depicted are"neighborhoods," and the approximate 95% confidence intervalsurroundingeach depicted location is about 20million basepairsofDNA, aboutonefifth thesizeof thechromosomearegion that

containsmorethan1,000 genes. Toidentifythe gene for each QTLwouldrequirepositional cloning,that is, laborious sequencing ofvirtuallyevery base pairin the DNAregion surroundingtheQTL.Given thecurrent size of QTLconfidence intervals, this regionneedsto bereducedinsizeby atleastanorderofmagnitude,andscientists engaged in QTL mapping are currently using classicalgenetic techniques to achieve this higher resolution mapping sothata "streetaddress"can be achieved for each underlying gene(Darvasi, 1998).

One method for achieving higher resolution mapping of QTLs isto lookatadvancedintercrosslinesof mice. Topursuetheearlierexamplesofinbredstraincrosses,this means crossingF2micetoobtainF3s, crossingF3s to obtainF4s, and so on. After manygenerations of intercrossing (i.e., mating unrelated individuals, notmating siblings as in the inbreeding strategy for creating RIstrains), only marker alleles that are essentially within the QTLitself or very close to it remain closely linked to the gene affectingthetrait. Such markersaresaidto be inlinkage disequilibriumwiththe QTLaffectingthe trait (see Figure 4). Because anF2popula-tion has only one generation for recombination, markers showassociation (linkage disequilibrium) with a QTL even if they aremuch further awayon the chromosome. As a result, about 150DNA markers are sufficient to scan theentire genome for QTLassociationsinF2sascompared with thousandsof DNAmarkersneededforhighly outbred populations.

For example, a follow-upstudyusedoutbred mice (a geneticallyheterogeneous stock derivedfroma cross of eight inbred strains) toincrease the resolution of the Flint et al.(1995)QTL analysis foremotionalityQTLs (Talbotetal., 1999). Examination oflinkagedisequilibrium in this population enabled theinvestigators to re-duce the size of the QTLs on chromosome 1 and 12 to a regioncontaining fewer than 100 genes, although the third QTL, onchromosome 15, was notreplicated.

Although the rat iswidely usedinbehavioral aswell asphys-iologicalandpharmacological studies, quantitativeandmoleculargenetic research on the rat has lagged far behind the mouse.However,the rat isbeginning tocatchup. Forexample,a map ofmorethan5,000DNA markers is now available (Watanabe etal.,1999).

ConvergenceandDivergenceofGeneticInfluences:MultigenicComplexity,Epistasis, andPleiotropism

Behaviorisnothingif notcomplex,andcomplex traitsare thefinal frontier for genetic analyses. To the extent that geneticinfluences are important in addition to environmental factors,behaviors are typicallymultigenic.Complex traits are multigenicin the sense that the nature or degree of a behavior expressed in anindividual is notinfluencedby asingle genebutratherbymultiplegenes. Thus, each gene contributesonlya relatively small amountof influence to that trait. As discussed earlier, the smalleffectsofindividualgenes make themdifficultto detect and map genetically,but in the aggregate, knowledge about the multiple genes withconverginginfluence on a behavior can provide a great deal ofpredictive power.

Aclearexample of multigenicity can be drawnfromthe animalgenetics literature. One classical approach toanimal behavioralgenetics is selective breeding, where individuals with extremescores on a behavior are mated.Aftermany generations, nearly all


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814 PLOM1NANDC R A B B EDistance fromCentromere Mouse Gene

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Al co ho lpreferenceAl co ho lcondi t ionedtaste aversion AtplblA c u t ealcoho l wi t hdrawal

Acu tepentobarbi talwithdrawalChronicalcohol withdrawal

HomologousH u m a nGene

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2q36-q372q21.1-q21.32q375 q l418q21-222 q l l - q l 3Iq32

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Iq41Figure3. Qua ntitative traitloci (QTLs)forsensitivitytoseveral behavioral effectsofalcoholand thesedativedrug pentobarbital ondistal mouse chromosome 1.EachQTLisrepresentedby acircleon thechromosome atthepointofhighest association. Distancesare in centimorgans(cM) fromthe centromere, alinkageestimateofrelativedistance. Plausible candidate genes mapped near theseQTLsarelisted. Known regionsof syntenicconservationbetweenthemouseandhumanchromosomeareindicatedby thehuman chromosome numbersandregions.Inthese syntenic regions, human genes homologous to the mouse genes arenoted.From "AlcoholandGenetics:NewAnimalModels,"by K. E.Browraan& J. C.C rabbe,1999,Molecular MedicineToday 5, p. 316.C opyright 1999byElsevier. Reprinted withpermission.

genes favoring the selected trait will have been collected in theselected line. The classic selection studies in psychology wereconducted by Tolman (1924) andTryon(1940),w ho selected formaze-bright and maze-du ll rats. In an attempt to identify the genesaffecting a simple behavior in mice, sensitivity to the sedativeeffects (loss of righting reflex) of a hypnotic dose of alcohol,researchersused selectively bred linesofmice,recombinant inbredstrains,and anumberofotherspecializedgenetic methodsto mapfive QTLs (Markel, Bennet, Beeson, Gordon, Johnson, 1997).Theprogenitor selected linesofmice u sedinthisproject were bredfor alcohol-induced sedation. Following a standard dose of alco-hol, long sleep(LS)and short sleep (SS) mice were sedated forapproximately 180min or 10min,respectively. Thebasisfor this170-min differential sensitivity is almost entirely genetic. EachQTL conferred a difference in sleep time of between 19 and 25min. Thatis, anaverage individual mou sepossessingthe LSalleleatone of these loci would be sedated for only abou t 20 min longerthan an individual with the SSallele. However, if an individual

possessed allfiveLSalleles,its g enotype could predict130min ofthe total of 170 min in response differencebetween the LS and SSmice.Although methods currently available are not sensitive enoughto test the hyp othesis directly, it is highly likely that the remain-ing 40 min of unexplained genetic difference between LS and SSmice can be traced to twoplaces.First, it is very likely that morethanfive genes contributetothis responsetoalcohol,andsomeofthose genes probably have effect sizestoo small to detect unlesshundredsor even thousands of mice are tested. The more importantsourceof theu nexplained genetic variance, howev er,islikelyto beingene-geneinteractions,aphenomenontermed epistasis. Thatis,gene interactionsmay not beadd itive: Possessionofallelesof twoQTLs, each of which would contribute 20 min of sedation inde-pendently, may confer 50 min (or 30 min) of sedation when anindividual has both alleles. Another example can be drawn fromthe study of a QTL on mouse chromosome 11 for acute pentobar-bital withdrawal severity (Buck, Metten, Belknap, Crabbe,


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FQ:

Figure 4. Linkagedisequilibrium. F^: Both chromosome homologs areshown for an Fjhybrid individual derived from crossing a B6 and a D2inbred parent. Alleles for nine genes are shown. The shaded homologrepresents the aileles inherited from theB6parent. The unshaded homologrepresents the aileles inherited from the D2 parent. A mutation causing abehavioraldeficit is shown in gene 6,inherited from D2.F2:The haploidgenotypes(i.e.,onlyone of the twochromosomesof the homologous pair)are shown for seven individualF2mice.Fiveof the seven show evidenceofarecombination due to crossing over during meiosis. Genes 2,3, 4, 5,and 7 all show evidence of linkage disequilibrium between D2 aileles andthe mutant allele. The first individual excludes linkage with gene 9, thesecond excludes linkage with gene 1, and the fifth excludes linkage withgene 8.F9:Haploid genotypes for nine individualsafternine generations ofintercrossing. After many more recombinations inF3-F9 (not indicated),only genes 5 and 7 remain in linkage disequilibrium with themutantgene.Thiscan beseenby thefactthat any individualwiththe mutantallele alsohas D2 aileles for genes 5 and 7.

1999). Mice possessing markers from theC57BL/6Jstrain havesignificantlyhigher withdrawalscoresthan those possessing mark-ersfromtheDBA/23strain.However, thisisonly trueifthey havealso inherited theDBA/2}allele for a second QTL on the distal end

ofchromosome 1 (indicated in Figure 3). Mice with theC57BI76J(vs. a DBA/2J)alleleat the chromosome 1 QTL show no evidenceof a difference in withdrawal when they have the C57BL/6Jmarkers for the chromosome 11QTL (Hood, Crabbe,Belknap,&Buck,2000).

This sort of statistical interaction is familiar to psychologistsaccustomed toANOVA models,andtherearemany examplesofphysiological interactions, some of which have been traced togeneticepistasis. Detection of genetic interactions of this sort isdifficult,and forQTLsinparticular,behavioral geneticists needtobe aware that the models in current use still have limited statisticalpower. Nonetheless, the complex interactions surrounding geneeffectsare avery important areaforfutureresearch. Psychologists'fearlessness in thefaceofstatistics (and interactionsinparticular)is anasset thatwill facilitate their contributionsto future behav-ioralgenomic analyses.In thesamewaythatanybehavior can betraced to multiple genes, any single gene can be shown to influencemultiplebehaviors,aradiationofinfluencetermedpleiotrapism bygeneticists. One of the growth industries forpsychologists whostudyrodent behavioris thecharacterization ofpleiotropic effectsof gene knockouts and other transgenic manipulations in mice. Thetraining that psychologistsreceive inexperimental design, statis-tical analysis,andattentiontointerpretingthesubtletiesofbehav-ior are allvaluable assetsforexperimentswithsingle-gene animalmodels. In thefuture, itwillbe useful to add to thatarsenal anunderstanding oflinkage, recombination, epistasis, andpleiotro-pism as well.

Gene-Environment InterplayIn addition to questionsabout how genes interact with each

other, an even larger set of questions involves the interplay be-tweengenes and environment as seen ingene-environmentcor-relations and interactions. Gene-environment correlation refers togenetic differencesinexposuretoenvironments, literally,acorre-lation between genesandenvironment. That is, genetic disposi-tionsmay be correlated with experiential dispositions. For exam-ple, children with the chromosome 6 gene whofindithardto learnto read might avoid reading or, more fundamentally, might not beinterested in reading or being read to. The topic of gene-environmentcorrelation goes beyond describing and explaining acorrelation between genotype and environment. The larger issueinvolves the environmental mechanisms by whichgene-behaviorassociations develop (Rutteretal., 1997).Gene-environmentin-teractionrefers togeneticdifferences insensitivitytoexperiences.A general form of interaction assumed in psychopathology iscalled thediathesis-stressmodel: Individuals who are at geneticrisk or predisposition are most sensitive to environmental risk(Paris, 1999). For example, in an adoption study of criminalbehavior,being rearedin anadoptive homeinwhichaparenthadbeenconvictedof acrimedid notincreasethe likelihood thattheadoptee would have a criminal record unless the adoptee had abiological parentwho hadbeen convictedof acrime (Medniek,Gabrielli,& Hutchings, 1987). A similar type of interaction wasfound in which negative adoptive home environments hadespe-cially deleterious effects on antisocial behavior in adolescentadoptees whose biological parents were antisocial (Cadoret, Yates,Troughton, Woodworth,& Stewart, 1995).


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816 PLOMIN AND CRABBEAdifferent typeofgenotype-environmentinteraction emerged

from a study of Scandinavian men exploring two variants ofalcoholism,each of which was heritable. Rearing environmentswere classified as being risk-promoting or protective. One form ofalcoholism (TypeI,characterizedbyrelatively mild abuse, mini-mal criminality, and passive-dependent personality variables)showed pronounced evidence ofdependenceon the rearing envi-ronment: Only those with both genetic and environmental riskfactors showed higher rates of Type I alcoholism. For the otherform(TypeII,characterized by early onset, violence, criminality,andbeing largely limitedtomales), therearing environmenthadlittle effect ongenetic risk(Cloninger,Bohman, & Sigvardsson,1981).This exampleprobably reflects genetic heterogeneity of thetrait alcoholism; that is, the underlying predisposing genes areprobably different for the twotypes. Nonetheless, itclearly illus-trates the concept that the same environmental riskfactorscan playadifferentrole depending on anindividual'sgenotype. Yet anothertype ofgenotype-environment interaction has been reported foralcoholism:Theheritabilityofalcohol consumptionisgreaterforunmarried women than for married women (Heath, Jardine, &Martin, 1989).

There are many other ways of thinking about genotype-environmentinteraction (Kendler & Eaves, 1986). This is anotherarea in which animal models will beespeciallyimportant becauseboth genotype andenvironmentcan be manipulated experimen-tally. For example, a recent studytested several inbred strains andonenull mutantsimultaneouslyinthreelaboratorieson abatteryofsix behaviors (Crabbe, Wahlsten, & Dudek, 1999). As manyvariables as possible (e.g., apparatus, test protocols, and manyenvironmental variables) were rigorously equated. The strainsdiffered remarkablyin allbehaviors,but forsome tests, there weresignificant Strain X Laboratory interactions. In general, theweaker the overall genetic influence on a trait, the more likelythere was to be a Genotype X Environment interaction. Oneimplicationofthisstudyisthatinexperiments characterizing nullmutants,results may prove subsequently to be idiosyncratic to aparticular laboratory. However, one environmental manipulationthat is frequently asserted to be an important determinant of mousebehavior (so-called shipping stress) wasalsovaried inthisexper-iment. Half theanimalswere bred in the testing location, and halfweresnippedasadultsfromcommercial breeders. Shipping statusinteracted with genetic differences only for escape latency in awatermaze but had no influence on anxiety, activity, oralcoholpreference drinking.Forhuman behavior, replicable GenotypeXEnvironment interactions are especially difficultto find (Wachs &Plomin, 1991).Inpart,this is because of a lack of the degree ofexperimental control that ispossible inanimals, suchasmakingrather extreme environmental manipulations, and in part, simplybecause detection of interactions in analysis ofvariance designswith reasonable statistical power requiresfarmore subjects thandetection ofmaineffects (Wahlsten, 1990). Modelinggenotype-environment interactions in animals where specific genes can bestudied might offer insight for the more difficult studies withhumans.

Wepredict that tracing the developmental pathways betweenspecific genesandbehavior through correlations andinteractionswith environmental mechanisms islikely to be one of the mostimportant advances that emerges from applications of specificgenes associated with behavior. Moreover, psychologists will

profit from the knowledge gained by genotyping children forspecific genes associated with relevant behavioral traits. Thisadded knowledge about the individual will represent a betterarticulatedphenotype, whose responsiveness toeffortsto counter-act theeffects of risk-promotinggenes (and likelihoodof benefit-ingfrom theeffects ofprotective genes)canbetterbegauged.Wealso predict that psychologistsin the21st century will examinetheinteractions and correlations between family environment andgenesmore closely. The costs andbenefitsof the knowledge addedbyDNA arealready thesubjectofmuch ethical debate,towhichwe returnin the finalsection.

IdentifyingGenesforSingle-GeneEffects in HumansUsingTraditional Linkage Designs

Until the past decade, attempts to find genes in thehumanspecies were limited torare disorders caused by a single genenecessary and sufficient tocause the disorder. In atraditionallinkage design using a largepedigreeof many individualsacrossseveral generations, linkage can easily be detected for a markerthat is 10 million base pairs (about one tenth the average length ofachromosome)fromthe generesponsiblefor thedisorder.As aresult, 350markers evenly spaced throughout the chromosomescan systematically scan thegenomefor linkage. Inother words,linkage isfarsightedin that it can detect distant mountains (single-geneeffects).Once linkagefinds thechromosomal neighborhoodofa gene, it is moredifficult to locate the gene's specific addressbecause few recombinations occur between markers and agenethatlive in the same neighborhood.

Triplet Repeat DisordersIn1983, Huntington's disease was the first single-gene disorder

linkedto a chromosomal region (the tip of chromosome 4) usingDNAmarkers (Gusella etal.,1983). Huntington'sdiseaseis a raredisorder (affecting 1 in20,000 individuals) caused by a singledominant gene whoseeffects arefirstseeninmiddle adulthoodinpersonality changes, forgetfulness, and involuntary movements.Slowly over20years, the disorder leads to acompletelossofmotorcontrol andintellectualfunction. Thediseasewaslinkedtochromosome 4usinghundredsofindividualsin a five-generationpedigree. Locating the chromosomal region through linkage led in1993to identificationof thespecific sequenceof DNAresponsibleforthedisorder.In thecaseofHuntington'sdiseaseandmorethana dozen other single-gene disorders, the genetic problem is arepeating sequence of three nucleotide bases of DNA. Normalallelesat this locus contain between 11and 34copiesof the tripletrepeat, but alleles that cause Huntington's disease have morethan40copies. It is not yetknown exactlyhow this form of thegene causesneurodegenerationor how to intervene to prevent thedisease.Identifyingthe DNAresponsiblefor thedisorderhasmadeitpossibletodiagnosewithgreat accuracy whetheranindividualwith anaffectedparent (andthusat 50%riskfor thedisorder) doesin fact carry the gene, but there is some possibility that themutation is not fully penetrant: Not all individuals with tripletrepeats develop the disease (Nance, 1997; Rubinzstein et al.,1996).

Asimilar approachhasbeen usedtolocatethegenes responsibleforhundredsofother rare single-gene disorders. In1991,asingle


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SPECIALISSUE: DNA 817gene on the X chromosome that is the most common cause ofmentalretardation after Down's syndromewasidentified,althoughitseffects are variable, with some so-calledfragile Xindividualshaving normal intelligence. The gene contributes to the excessmental retardation in males versus females because males withtheir single X chromosome always express the gene, whereasfemalesexpress the gene only if they inherit copies on both of theirXchromosomes. Nonetheless, the disorder is still relatively rare inthepopulation,withafrequency ofaboutone inseveral thousandmales and half as many females. The disorder is calledfragileXbecausethe X chromosome carrying the fragileX allele lends tobreak when cellsthatcarry it are grown on a special medium. ThefragileX gene is especially interesting because, like Huntington'sdisease, it involves a triplet repeat. ForfragileX, the triple repeatisunstableandgets longeras itpasses through several generationsuntilits length becomes a liability (a phenomenon called antici-pation).Parents who inherit X chromosomes with a normal repeatnumber for this triplet repeat ( 6 5 4repeats) sometimes produceeggs or sperm with an expanded number of repeats (up to 200repeats), called apremutation. This premutation does not causeretardation in theoffspring, but it is unstable and often leads togreater expansions (200ormore repeats) in thenext generation,whichdoes cause retardation.

ProtectiveGenesandEnvironmental ModulationNotall mutations lead todysfunctionin one instance, posses-

sionof a mutant allele has been shown to exert a protectiveeffectagainst development of alcoholism, a prevalent disorder that isclearly heritable. Alcoholismetabolizedto thehighlytoxic com-poundacetaldehydeby theenzyme alcoholdehydrogenase.Acet-aldehyde in turn is rapidly metabolizedby theenzyme aldehydedehydrogenase (ALDH), which hasmanyvariant alleles. Popula-tions of Asian ancestry have a high prevalence of the alleleALDH2*2, a variant that is very inefficient at converting acetal-dehyde to the innocuous end-products acetate and water. In indi-viduals with two copies of the ALDH2*2 allele, ingestion ofalcohol is therefore followed by a facial flushing reaction andfeelings of nausea and dysphoria that can be intense. In thesepopulations, possession of ALDH2*2 alleles protects against thedevelopment of alcoholism, as well as the related quantitative traitofheavy drinking(Harada,Agarwal, Goedde, Tagaki, & Ishikawa,1982; Higuchi etal.,1994).Furthermore, in heterozygotes who dodrink, theincreased consumption leads toincreased riskforsuchadversebiomedicalconsequencesascolon cancer (Murataetal.,1999). The protection is apparently so pronounced thatALDH2*2/*2homozygotes seem almost never to become alco-holic.Ina recent study of 409 alcoholics, none were homozygotes(vs. 31 of 461 controls), and only 12.7% of the alcoholics wereALDH2*l/*2 heterozygotes (vs. 35.1% ofcontrols; Murayama,Matsushita, Muramatsu, & Higuchi, 1998). However, oneALDH2*2/*2homozygote has been identified in a sample of 420Han Chinese alcoholics, so the protection is not absolute(Chenetal.,2000).Theadverse reactiontoacetaldehydeis thebasisfor analcoholism therapyusing the drugdisulfiram (Antabuse), whichinhibits ALDH activity and leads to the same nausea and dysphoriawhenalcohol isconsumed.

Even if a deleteriousgenehas been inherited, itseffectsin somecases may bemodulatedenvironmentally. One of the earliest and

most well-known single-gene examples of environmental modu-lation isphenylketonuria (PKU), caused by a gene on chromo-some 12.The PKUdiseaseallele producesanenzymethatdoesnotwork properly tometabolize phenylalanine, which comes fromfood, especially red meats. If phenylalanine cannot be brokendown,its metabolic products build up and damage the developingbrain.Although PKU is diagnosed with a simple biochemical test,it serves as a useful reminder that an environmental interven-tionadiet low inphenylalaninecansuccessfully prevent thedevelopment of mental retardation for a single-gene recessivedisorder that occurs in about 1 in 10,000births and previouslyaccounted for about 1% of severely retarded individuals ininstitutions.

Identifying HumanQTLsUsingLinkageDesignsAlthough the traditional linkage design is aneffectivetechnique

for locating the general chromosomal region of the gene respon-siblefor rare single-genedisorders,it is less applicable to commoncomplex traitsinfluencedbymultiplegenes as well as by multipleenvironmentalfactors. Attemptsin the1980sto usethis approachto investigate linkage for schizophrenia and bipolar manic-depressive psychosis led to well-publicized reports of linkage, butthese reports were later retracted (Moldin,1997).The main prob-lem isthatifmultiple genesareresponsiblefor the heritabilityofa trait, any particular gene is likely to account foronlya smallamount of variance, which greatly increases thedifficulty of de-tecting it. In addition, many psychological traits are continuousquantitativedimensions rather than discontinuous qualitative dis-orders.Although there are thousands of single-gene mutations thatact like sledgehammers wreaking havoc on development, it isgenerally accepted that normal development of complex traits isorchestrated by a large symphony of genes working together.Genetic variation in these genes is thought to be largely responsi-ble for the ubiquitous genetic influencefound forpsychologicaldisordersanddimensions. These requirea QTLapproach. Indeed,as mentioned earlier, the QTL perspective suggests the radicalviewthatthegenetic contribution to disorders is quantitative ratherthanqualitative.

Newer linkage designsusejusta fewfamily members inmanyfamiliesrather than manyfamilymembersin a fewlarge pedigrees(Burmeister, 1999). For example, the affected sib-pair design(Blackwelder & Elston, 1985; Suarez & Van Eerdewegh, 1984)selectsfamilies in which two siblings reach diagnostic criteria fora disorder. If aparticular DNAmarker is linkedto a gene thatinfluences a disorder, affected siblings, who presumably sharesomeof thesame genesfor thedisorder, wouldbemore likely tosharethesameallelesateach suchrisk-promotinggenefrom theirparentsfor that DNA marker. That is, siblings can share zero, one,ortwoallelesfrom each of their parents. Given that their motherandfathereach have twoalleles,siblings on average are expectedto share one allele for a marker. However, if a DNA marker islinked to a gene for the disorder, affected siblings are more likelytosharebothallelesfor that marker.

The affected sib-pair design is more compatible with a QTLperspective than the traditional large-pedigree linkage design be-cause the selected sibs in which both members of a pair areaffected can be viewedasbeingat theextreme of aquantitativedimension.Thesib-pairQTLlinkagedesignexplicitly assessesa


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818 PLOMINAND CRABBEquantitative dimension rather than a qualitative disorder (Fulker &Cherny, 1996). The approach correlates degree of allele sharing(i.e., zero, one,ortwo)foreachDNAmarker with quantitative traitdifferences within sibling pairs. Although the method was firstproposedfor unselected siblings (Haseman & Elston, 1972), themethod's power comes from selecting sibling pairs in which atleast onesiblingis at the high or low extreme of the quantitativetrait. Even so, the method isunlikelyto detect a gene that accountsfor less than 10% of the variance of the trait. This seems like asmall effect to those who are used to thinking about single-genedisorders, but it seems likea largeeffect to those who think thatcomplex and common disorders and especially quantitative traitsare influencedbyvery many genes.

Sib-pairQTL linkage wasfirstapplied to reading disability andyielded a significant QTL linkage on the short arm of chromo-some 6 in twosamplesofsiblings (Cardonetal., 1994). That is,the quantitative reading scores of siblings of reading-disabledindividuals were worse when the siblings shared alleles in thisregion.Thislinkagehasbeen confirmedinthree subsequent stud-ies (S. E.Fisheretal., 1999; Gayanet al.,1999;Grigorenko etal.,1997) and appears to apply to diverse components of readingdisability. Because this QTLregionwasidentified usinglinkage,which has power to detect only relatively large effects, thesefindings imply thatthisregion onchromosome6harborsagene(or genes) accountingfor at least 10% of the average readingdifferencebetween reading-disabledindividualsandtherest of thepopulation. An implication of the QTL approach isthatthis QTLis not specific to reading disability but rather affects readingthroughout the normal range as well, although this has not yet beendemonstrated.Thespecific geneonchromosome6that contributesgenetic risk for reading disability has not yet been identified.

The QTL approach can also be used in other types of linkageanalyses, suchasmore traditional pedigrees. Recent methodolog-icaladvances haveenabledinvestigators to use all phenotypic andgenetic data inmultiple families, regardless of the genetic rela-tionship among individuals screened. These methods rely on vari-ance partitions rather than simply analyzing allele sharing, andthey allow greater power to detect QTLs, as well as explicitanalyses of Genotype X Environment interaction and epistaticinteractions (Almasy &Blangero, 1998).Forexample, joint con-siderationof each individual's score on the Novelty-Seeking sub-scaleof theTridimensionalPersonality Questionnaireand adiag-nosis of alcoholism led to improved ability to detect a QTL onchromosome 4 using this method (Czerwinski, Mahaney, Wil-liams, Almasy, & Blangero. 1999).Theseand other advances willcontinuetoincreasethe power of linkage analyses for QTLs.

Identifying HumanQTLsUsingAssociationAnalternative QTL strategy isallelic association,whichcan

detect QTLs of small effect size (Plomin et al., 1994; Risch &Merikangas, 1996). Allelic association refers to acorrelation be-tween allelesof a DNAmarker andtrait scores across unrelatedindividuals. That is, allelic association occurs when individualswitha particularallelefor the marker have higher scoreson thetrait, which can occur for three reasons. The DNA marker itselfmay be the genetic factor that directlyaffects the trait. Second, theDNAmarker may be close enough to the QTL on the chromosometo reflect the effect of another stretch of DNA that is actually

responsible for theeffect (called linkage disequilibriumsee Fig-ure 4). The third reason is artifactualassociations can appearbetween traits that differ among population subgroupsandalleleswhose frequency alsodiffers among those subgroups (called pop-ulationstratification). We discuss the implications of, and possiblesolutions for, this problemat the end ofthissection.

One problem with finding QTLs for complex traits is thatlinkage is systematic but not powerful and allelic association ispowerful but not systematic. As mentioned earlier, linkage isfarsighted in that it can detect distant mountains (single-geneeffects)butcannotseenearby hills (QTLsofsmalleffect size).Incontrast,allelic associationisnearsightedin thesensethatit cansee nearby QTLs but not more distant single-geneeffects.That is,ifa marker is to be detected by allelic association, it musteitherbethefunctionalQTL or be very near to it, namely, within a hundredthousand base pairs. For this reason, the street address of a genethathasbeen mappedbylinkageto aspecific neighborhoodof achromosome is located usingallelicassociationwithknown genesin theneighborhood.The mainadvantageofallelic association isthat it is powerful because it correlates alleles with traits inunrelated individuals, and power can be increased simply byincreasing the sample size. Inaddition, allelic association can bejust as easily applied to quantitative traits as toqualitativedisorders.

Usingthe same logic that drives animal QTL researchers to testcandidate genes, humangene-hunting studies have used associa-tion withDNAmarkersin ornear genesthatseem relevantto thetrait under investigation; these genes are called candidate genes(Malhotra&Goldman, 1999).Aproblemwiththisapproachisthatany of the 30,000 or so genes expressed in the brain couldconceivably be considered as candidate genes for most behaviors.Inother words, association has not been systematic in the way thatlinkage can be usedtoconductasystematic scanof thegenome(butsee below).

The best example of a QTL association was found in 1993between the late-onset dementia of Alzheimer's disease and a geneon chromosome 19calledapolipoproteinE(Corderetal., 1993).One allele (allele 4) for this gene is associated withAlzheimer'sdiseaseand is the only known predictor of this common disorderoflaterlifeaffecting asmanyas 15% ofindividuals over80yearsof age. As replicated in scores of studies, the frequency of thisalleleisabout20% in thepopulation, whereasforindividuals withAlzheimer's disease, the allelic frequency is about 40%. Thisalleleis neither a necessary nor asufficientcause of Alzheimer'sdiseasebecause more than half of Alzheimer'sdiseasepatients do not havethis allele andmanypeoplewith this allele do not haveAlzhei-mer's disease. There is some evidence thatallele2 of the apoli-poprotein E gene may play a protective role (Corder etal.,1994).FindingQTLs that protect rather than increaserisk for adisorderis an important direction for genetic research. In1992, one of thegenes on chromosome 14 was identified as accounting for mostcases of a rare (1 in 10,000) single-gene type of Alzheimer'sdiseasethat appears before65yearsof age(St.George-Hyslop etal.,1992). In 1995, the specificoffendinggene, calledpresenilin-1,was identified (Sherrington et al., 1995), although it is not yetknown how the gene causes early-onset Alzheimer's disease. Mul-tiple susceptibility genes are now known to exist, and there aretransgenic mouse models with alterations in both amyloid precur-


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SPECIAL ISSUE:DNA 819sorproteinandpresenilin genes (Guenette& Tanzi,1999;Lippa,1999).

Neurotransmitter Candidate GenesGenes in the dopamine and serotonin neurotransmitter systems

have been widely used to investigate associationswithbehavior.Although allelic associationhasbeen primarily used to comparediagnosed cases versus controls,as inAlzheimer's diseaseordrugabuse, it can also be used tocorrelatethe presence of a particularallelewith scores on a quantitative trait. For example, in 1996, adopamine receptor gene (DRD4), which is a gene largely ex-pressed in the brainlimbicsystem, was reported to be associatedwith the personality trait of novelty seeking in three analyses(Benjaminet al., 1996; Ebstein et al., 1996). In the initial threestudies,individualswiththelong-repeatalleleshadhighernovelty-seekingscores.Themarker involvesasequenceof 48base pairsthatrepeat from 2 to 8 times. It is postulatedthatreceptors withlonger DRD4 repeats (6-8 repeats) are less efficient atbindingdopamine andthusthat individualswiththe long-repeat alleles aredopaminedeficientand seeknoveltytoincreasedopamine release.Althoughthe association has not been consistently replicated, thereis a trend toward replication, and methodological issues mayaccount for some of the failures toreplicate (Plomin &Caspi,1998;Wahlsten, 1999). In reviewing several subsequent studies,the original authors noted that the QTL appears to increasenovelty-seeking scores by only about 5%, an effect size that isdifficult to detect in many experiments (Ebstein & Belmaker,1997).

DRD4hasalsobeen reported in several studies to be associatedwith attention-deficit hyperactivity disorder (ADHD; Thapar,Holmes, Poulton, & Harrington, 1999) and with heroin addiction(Kotler et al., 1997; U et al., 1997). Another dopamine gene(DAT1) shows even more consistent associations with ADHD(Thapar etal.,1999). A recentstudyreplicated theDAT1associ-ation and also reported associations with two other dopaminegenes (DBH andDRDS:Daly, Hawi, Fitzgerald, &Gill, 1999).Another recent study also replicated theDAT1 association andsuggestedthatthe association primarily involves thehyperactive-impulsive component of ADHD rather than the inattentive com-ponent (Waldman et al., in press). Genes involved in serotoninfunctionhave also been reported to be associatedwithtraits relatedto anxiety (Ebstein, Nemanov, Klotz, Gritsenko, & Belmaker,1997; Katsuragi et al., 1999; Lesch, Greenberg, & Murphy, inpress; Lesch et al., 1996; Osher, Hamer, & Benjamin,2000),although several failures to replicate have been reported (Flory etal.,1999). Several other genes have been reportedto beassociatedwithpersonality (Benjamin, Ebstein, & Belmaker, inpress;Hamer& Copeland, 1998), but none has as yet been consistentlyreplicated.

Research on candidate genes such as dopamine-related geneshas dominated allelic association research. To some degree, thisrepresents the art of the possible. Because allelic association isnearsighted,asystematic scanof thegenome comparableto ascanusing linkage would require thousands of DNA markers. Forexample, 3,500evenly spaced markers would provide amarkerapproximately every 1million basepairs in the 3.5 billionbase-pair genome, which means that no QTL would be more than500,000base pairs awayfroma marker. However, it is generally

agreed that a complete scan would require maps many timesdenser. Genotyping so many markers seems an impossible task.With 200 subjects each in groups of cases and controls, eachmarker would require 400 genotypings,whichmeans that3,500markers would require 1.4 milliongenotypings.However, a newtechnique called DNA pooling makes this prospect lessdaunting(Daniels, Holmans, Plomin, McGuffin, & Owen, 1998). DNApoolingcombines DNAfromcasesand compares itwithpooledDNAfor controls. The pooled DNA for the two groups can begenotypedandcomparedas ifthey were justtwoindividuals.Intermsofgenorypingeffort in theexamplejustgiven, this meansthat3,500markers needed for a systematic genome screen requireonly 7,000 genotypings. DNA pooling is relevant only whenselected groups are investigated, such as cases and controls orgroups high and low on aquantitative trait.Unselected samplesrequire individual genotyping because there are no groups tocompare. DNA pooling is being used to scan the genome forallelicassociation for general cognitive ability (Plomin, in press). Al-thoughthe approach is expected to detect only some of the largestQTLsassociated with general cognitive ability, several replicatedQTLs have been reported (Chomeyetal.,1998;P. J.Fisheret al.,1999;Hilletal.,1999). DNA chips, another technical advance thatwill facilitate genomewide scans using association,arediscussedlater.

Replicability ofAssociationStudies: PopulationStratification and Other Methodological Issues

From the qualifications raised in the preceding examples, itshould be clear that association studies for complex traits havegenerally beendifficult to replicate. An excellent discussion of thereasons why has recently appeared (Malhotra & Goldman, 1999),andwe have touched on some of those reasons. One of the mostwidely publicized examples began as a report of an associationbetween a gene marker in thedopamineD2receptor gene andalcoholism. In 1990, a particularallelein theD2receptor gene wasreportedtohaveahigherfrequencyofoccurrenceinbrain samplesfrom alcoholics than in those from controls (Blum etal., 1990).Dozens of studies have been performed since to testthishypoth-esis,withvery mixed success.Thestatistical validityof theasso-ciation was questioned in surveys of the existing literature, andthese surveys also disagreed (Gelerntner, Goldman, & Risch,1993; Pato,Macciardi,Pato, Verga, & Kennedy, 1993). Becausethe "finding" was already highly localized in the genome, acarefullinkage studywasperformed using several polymorphismsin thegene: This studyfailedto find anyevidencethat individualhap-lotypes (particular groups of linked alleles) occurred more fre-quently in alcoholics (Suarezetal., 1994).

Looking back from early in the year2000,we conclude thatthere may be some relativeincreasein frequency of the Taq 1 Aallele of the dopamineD2receptor gene in alcoholics, but it is veryclear that this will never constitute a risk marker with usefulprognostic power.TheoriginalD2storyreceived agreat dealofpublicity,most ofwhichtended tofoster the misconception thatthe "alcoholism gene" had been identified. The subsequentfailuresto reproduce these results led to anequally uninformedbacklashthathas damaged the credibility of linkageand association map-pingeffortsfor allcomplex traits.Why aresuchfindings difficultto replicate? In the case of theD2result,the mostlikelyexplana-


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820 PLOMIN AND CRABBEtion is that population stratification underlies the original findingand someof thesubsequentpositivefindings.

Ethnic groupsoftenyield differentallelicfrequencies for DNAmarkers. Any such markers would appear to show associationswilh any phenotypic differences between the ethnic groups. Forexample, in samples that include French and Finns, associationswith speaking French wouldbe found for any DNAmarker thatshowed allelic frequency differences between French and Finnsbecause French are more likely to speak that language than Finns.Because ethnicity does not vary systematically within familieseven when one parent is French and the other is Finnish (for eachgene, each offspring is just as likely to receive anallelefrom theFrench parent as from the Finnish parent), associations foundwithinfamilies avoid this possible bias. Anaddedcomplication isthateven within ethnic groups, there are oftenvastdifferences inallelefrequencies fordifferentsubgroups(e.g.,African Americandescribes a large and diverse collection of allelicgroups). Thisartifactcan be attenuated by studying very well-matched cases andcontrols, and it can beeliminatedby testing associations withinfamilies (e.g.,by comparing siblings whodiffer genotypically orphenotypically) because ethnicity does not vary within families.(This is why the failure to find within-family association hasconvinced many thattheD2associationis spurious.)

A widely used design involvesfamily triadsof a proband andtwo parents. One version of this test is called the transmissiondisequilibrium test(TDT;Spielman, McGinnis,&Ewens, 1993).The TDT usually usestrios consistingofaffected individuals andtheir biological parents. Each affected individual must have re-ceived the susceptibility alleles from his or her parents. Thesealleles transmitted from parents to affected individuals can beviewed as a group of case alleles. What about controls? Eachparent transmits only one of two alleles at each geneto anoff-spring (see Figure 1). The alleles from the parents that are nottransmittedcan heconsideredascontrolalleles.Inother words,theTDT needs onlyaffected individuals and their parents (who do notneed to be assessed phenotypically)no control group of individ-uals is required. The TDT rests on testing departures from theexpected equalfrequencyof transmitted andnontransmittedalleles(i.e.,linkage disequilibrium; seeFigure4). Forexample, arecentTDT analysis of ADHD confirms the casecontrol reports ofassociation in that the same DRD4 allele was transmitted fromparents to ADHD children in 61% of the families rather than in50%, a highly significant difference for the 199 families in thesample. Other nontransmitted alleles appeared at equal frequencyinparents and their offspring (Sunohara etal.,in press). The reasonforcreating such acomplicatedgroup of transmitted versus non-transmitted alleles within families rather than comparing cases andcontrols is that investigating association in this way removes thepossibilitythat associations foundin theusualcase-controldesignare due to ethnic differences. However, ethnicity can often becontrolledmore simply by matching cases and controls, as long asthisisdone with care.

Another issue relevanttofailuresto replicaterelatestostatisticalpowerthat is, the balance between false positives and falsenegatives. When many markers are examined, th

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