TheOnlineMetabolicandMolecularBasesofInheritedDisease>

77:Hyperphenylalaninemia:Phenylalanine
HydroxylaseDeficiency
JohnDonlonHarveyLevyCharlesR.Scriver
DOI:10.1036/ommbid.97

Abstract
1. Wedescribeaninbornerrorofmetabolismcalledphenylketonuria(PKUMIMNo.261600).Thediseasehasbeencalledan
epitomeofhumanbiochemicalgenetics(ScriverandClow,1980a,1980b).Thedisorderreflectsadisadaptiveinteraction
betweennatureandnurture.Thecomponentinnurtureisanessentialaminoacid,Lphenylalaninetheoneinnatureis
mutationinthephenylalaninehydroxylasegene(PAH)encodingtheenzymeLphenylalaninehydroxylase(EC1.14.16.1).The
discordancebetweennatureandnurtureleadstohyperphenylalaninemia(HPA),whichcanhaveatoxiceffectonbrain
developmentandfunction.The"proximal"phenotype(phenylalaninehydroxylasedysfunction)isunderthecontrolofonelocus
encodingthephenylalaninehydroxylaseenzymeandadditionallociencodingseveralotherenzymesnecessaryforsynthesis
andrecyclingofthetetrahydrobiopterincofactoressentialforthecatalyticreactionlocusheterogeneitythusentersthe
interpretationofHPA.Theintermediate(metabolic)anddistal(cognitive)phenotypesofPKUdiseasebothbehaveascomplex
traitsthateludeconsistentinterindividualgenotypephenotypecorrelations.Thephenylalaninehydroxylasegeneharborsgreat
allelicdiversityseveralhundredmutations,bothdiseasecausingandpolymorphic,arerecordedinPAHdb,apubliclocus
specificmutationdatabase(www.pahdb.mcgill.ca).
2. TheHPAsaredisordersofphenylalaninehydroxylation.Theminimumrequirementsforthenormalreaction,whichoccursin
bothliverandkidneyinhumansubjects,arephenylalaninehydroxylaseenzyme(amonooxygenase,EC1.14.16.1),oxygen,L
phenylalaninesubstrate,andthe6Risomerofthetetrahydrobiopterin(BH4)cofactor.Forthepterincofactortofunctionasa
catalyst,BH4mustberegeneratedfromthecarbinolaminebyproduct(4ahydroxytetrahydropterin)ofthehydroxylationreaction.
Thisisachievedbyarecyclingpathwayinwhich4carbinolaminedehydratase(formerlyknownasphenylalaninehydroxylase
stimulatingprotein)convertsthecarbinolaminetothequinonoiddihydropterin,which,asthesubstratefordihydropteridine
reductaseinthepresenceofreducedpyridinenucleotide,isconvertedbacktoBH4.Apathwayexistsforbiosynthesisofthis
obligatorycofactorinvolvedbothhereandinthefunctionofotheraromaticmonooxygenasesandofnitricoxidesynthasethe
enzymesinthepathwayareguanosinetriphosphatecyclohydrolase,6pyruvoyltetrahydropterinsynthase,andsepiapterin
reductase.DiseasesofBH4synthesisandrecyclingarediscussedinChap.78.
3. Hyperphenylalaninemiaisdefinedasaplasmaphenylalaninevaluegreaterthan120mol/liter(>2mg/dl).Whetherformsof
HPAowingtoalteredintegrityoftheenzymeshouldbesubdividedintodifferentformsnotablyphenylketonuria(plasma
phenylalanine>1000mol/liter,dietphenylalaninetolerance<500mg/day)andnonPKUforms(plasmaphenylalanine<1000
mol/liter,diettolerance>500mg/day)isamootpoint.EvidencesuggeststhatmilddegreesofpersistentuntreatedHPA
(<600mol/liter)maynotbeharmfultocognitivedevelopment(asyetanunprovenhypothesis).Forpurposesofdiagnosis,
counselingandcorrecttreatmentofthenonPAHenzymedeficienciesaffectingBH4homeostasismustberuledout.
4. ThehumanPAHgenecoversapproximately100kbofgenomicDNAonchromosome12,bandregionq23.2,andisembedded
inaregionof1.5Mbpharboringfiveothergenes.Thenucleotidesequences,bothgenomic(GenBankaccessionnumber
AF404777)andcDNA(U49897.1),arenowknown(seewww.pahdb.mcgill.ca)PAHhas13exonsandacomplex5'
untranslatedregioncontainingcisacting,transactivatedregulatoryelements.Thegeneisrichinintragenicpolymorphic
markers,includingbiallelicrestrictionfragmentlengthpolymorphism(RFLP)andsinglenucleotidepolymorphism(SNP)alleles,
atetranucleotideshorttandemrepeat(STR)actingasafastmolecularclockinintron3,andavariablenumberoftandem
repeats(VNTRs)(30bplengthcassettes)inthe3'untranslatedregion(UTR).Thepolymorphicsitesareinlinkage
disequilibriumanddescribealargeseriesofextendedandminiaturehaplotypes.ThePAHgenealsoharborsseveralhundred
diseasecausingallelesassociatedwithHPA,ofwhichmorethan60percentaremissensealleles.Onlyahalfdozendifferent
allelesaccountforthemajorityofmutantchromosomesinEuropeansorOrientalstheremainderarerare,evenprivatealleles.
5. ThehumanPAHgenehasbothdevelopmentalandtissuespecifictranscription/translation.Itstranslationproductisa452
aminoacidpolypeptidehomologousinseveraldomainswiththesubunitsoftyrosineandtryptophanhydroxylases.Thecatalytic
domainofthehumanPAHpolypeptidehasbeenresolvedat2forresidues117427.Theenzymeishomooligomericand

functionsinalternatingactivatedanddeactivatedstatesindimericandtetramericconformations.Theeffectofmutantallelesis
beingstudiedbymolecularmodelinginsilicobyusingtheproteindesignalgorithmFoldXtopredicttheenergeticimpacton
nativestatestabilityofthePAHenzymeofmissensePAHallelesandbyexpressionanalysisinvitro.Missenseallelescancause
misfoldingofthePAHenzymesubunit,leadingtoaggregationanddisposalbytheproteasome.
6. TheeffectsofdiseasecausingPAHmutationsonthepatientcanbemeasuredatthreelevels:proximal(enzymic),intermediate
(metabolic),anddistal(cognitivefunction).Enzymedysfunctioncanbemeasuredinvitroeitherdirectlybyhepaticbiopsyor
indirectlybyexpressionanalysiswhenthemutationisexpressedinatransgenicconstructinmammalianorbacterialcell
systemsorinacellfreetranscription/translationsystem.ThelatterenableshepaticPAHactivityinvivotobebroadlypredicted.
Fluxratesinvivoforphenylalaninehydroxylation/oxidationalsocanbemeasuredbytwodifferentisotopicmethods.Allstudies
ofgenotypephenotypecorrelationsrevealreasonablecorrelationsattheproximal(enzyme)levelhowever,atintermediate
(metabolic)anddistal(cognitive)levels,thephenotypeshaveemergentpropertiesandbehaveascomplextraitsinwhichthe
effectsofPAH,themajorlocus,ismodulatedby"modifiers."
7. PathogenicPAHallelesproducetheireffectsonPAHenzymebyvariousmechanismsandbehaveinbroadtermsasnull(no
activity),Vmaxaltering(reducedactivity),kinetic(alteredaffinityforsubstrateorcofactor),unstable(asaresultofmisfolding
andincreasedturnoverandlossofPAHprotein),andBH4responsive.Findingsoccasionallyhavebeentakentosignify
negativealleliccomplementationasanadditionalmechanismofmutantgenotypeexpression.Animportantsubsetofmissense
allelesthatdonotmaptotheBH4bindingregionconfersa6RBH4responsivephenotypeinvivobymechanismsthatinclude
stabilizingamisfoldingsubunitbychaperoneliketherapyandbyovercomingunfavorableBH4bindingkineticsbysaturation.
8. NewbornscreeningforPKUoccursinmanysocietiesandisapotentresourceforascertainmentandsamplingofmutantPAH
genes.PrevalencedataforHPA(5350casesper1millionlivebirths)andmutationanalysistogetherrevealnonuniform
distributionofpatientsandallelesinhumanpopulations.HumangeneticdiversityatthePAHlocuscomplementsdatafrom
analysisofmitochondrialDNA,theYchromosome,andclassicautosomalpolymorphisms.ThedistributionandtypesofPAH
allelesindicatehowmigration,geneticdrift,naturalselection(perhaps),recurrentmutation,andintragenicrecombinationover
thepast100,000yearsmightaccountforthepresentdayincidenceofPKU,theobservedmutationhaplotypeassociations,and
thenonuniformdistributionofcasesandmajorallelesinmodernhumanpopulations.TheprevalenceratesforPKUinpersons
ofAfricandescentappeartobeanorderofmagnitudelowerthanthoseforpersonsinEuropean,Chinese,andKorean
populations,whereprevalenceissimilar(104)inthesepopulations.
9. Pathogenesisofthemostimportantclinical(disease)phenotype(cognitiveandneurophysiologicimpairments)isundoubtedly
complex,butthereisanemergingconsensusthatphenylalanineitself,atelevatedconcentrations,istheharmfulmoleculethat
startstheprocessofallostasis.SeveralstrainsofmicemutagenizedbyNethylNnitrosourea,withdocumentedPAHgene
mutationsanddeficientenzymeactivity,areorthologousresourcestostudypathogenesis,aswellastreatmenttocontrolthe
phenotypiceffectsofthemutantgenotype.
10. NewbornscreeningwithmeasurementofbloodphenylalanineisthemostreliablemethodforearlydetectionofHPA.The
classificationofphenotypeincludessevereandlesssevereformsofPAHdeficiency,ofBH4responsiveandBH4nonresponsive
primarydeficiency,andofprimaryBH4deficiency.Classificationrequiresmeasurementsofphenylalanine,pterins,and
neurotransmitterderivativesinurine,plasma,andcerebrospinalfluid(CSF),alongwithspecificprotocolsandvariousassaysof
enzymeactivity(seeChap.78).Ifitisrequested,prenataldiagnosisforPKUisfeasiblebyDNAanalysisofmutationsand
haplotypes.
11. TreatmentofHPArequiresrestorationofbloodphenylalaninetovaluesasnearnormalaspossibleasearlyaspossiblein
postnatallifeandforaslongaspossibleperhapsforalifetime.Atpresent,itseemsthatanydeviationfromthispolicymay
incuracostinstructureandfunctionofbraininthePKUpatient.Amongthemodalitiesoftreatment,thelowphenylalaninediet
isstillparamount,butitneedstobeimprovedinorganolepticpropertiesandinnutrientcompositionnotablyoftheessential
fattyacidsandtherelativeratiosofaminoacids.TheBH4responsivePAHallelesmayrequireonlypharmacologictherapy
(e.g.,10mgBH4/kgperday).Alternativemodalitiesincludethepossibilityofenzymesubstitutionwithengineeredrecombinant
phenylalanineammonialyase(promising)andgenetherapy(inaholdingpatternforhumansubjectsbutshowingefficacyinthe
mousemodel).
12. MaternalHPA,atoxicembryopathy/fetopathy,causescongenitalmalformations,microcephaly,andpermanentlyimpaired
cognitivedevelopment.Itisaconsequenceofintrauterinephenylalanineexcessinthefetalcompartmentderivedfroma
positivetransplacentalgradient.AllfemalesofreproductiveagewithHPAshouldreceivereproductivecounseling,social
support,andcontinuedorrenewedtreatmenttorestoreeuphenylalaninemiabeforeconceptionandthroughoutpregnancy.
MeticuloustreatmentofmaternalHPAiscompatiblewithanormaloutcomeforthefetus.

13. VirtuallyallthemajorthemesandissuesnowconsideredtobeimportantinPKUwererecognizedbythehumangeneticist
LionelPenrosehalfacenturyago.ThefundamentalquestionsaboutPKUarethesamethenandnowonlythetoolsand
opportunitiestoaddressthemhavechanged.

IntroductoryComment
Thischapterisnotatotaloverviewofhyperphenylalaninemia(HPA)anditsprincipaldiseasetype,*eventhoughittouchesmost
aspectsofthetopic.Rather,itisacloseanalysisoftheenzymeinvolved[phenylalaninehydroxylase(PAH)],thecorresponding
gene(PAH)withitsalleles,theirdiseasecausingeffects,andhowthosediseasesoccurandcanbeprevented(Scriver,2007).The
primarydisordersofBH4homeostasisaffectingphenylalaninehydroxylationandotherreactionsarecoveredinChap.78.
In1994,asymposiumwasheldtocelebratethesixtiethanniversaryofAsbjrnFllingsGermanlanguagereport(1934)onanew
inbornerrorofmetabolismanothersymposiumin1995celebratedthefortiethanniversaryofeffectivetherapyofthisgenetic
disease.Thedisease,renamedphenylketonuriain1935byPenrose,hadbeencalledimbecilitasphenylpyrouvicabyFllingto
recognizeitseffectoncognitivedevelopment.Some11yearslater,forhisinauguraladdressasGaltonProfessoratUniversity
CollegeLondon(Penrose,1946),Penrosechosephenylketonuriaashistopic.Amongthemanythemesrunningthroughhis
remarkableanalysisofthisnowclassicgeneticdiseasewasPenrosesobservationthatphenylketonuria(PKU)wasthefirstto
exhibitachemicalexplanationformentalretardation.
Withhindsight,majormilestonesonthisjourneyofdiscoveryaboutPKUcanberecognized:(1)Inthe1930s,itwasshownthatboth
thedisease(mentalretardation)andthemajormetabolicabnormality(hyperphenylalaninemia)areaccountedforbyautosomal
recessiveinheritanceofadeleteriousgene(Flling,1934Penrose,1935)(MIM261600).(2)Inthe1950s,PKUpatientswere
showntohavedeficientactivityofhepaticPAH(EC1.14.16.1),thekeyenzymecontrollingphenylalaninecatabolism(Jervis,1953).
(3)Inthesamedecade,adietrestrictingintakeofphenylalanine,anessentialaminoacidforhumanbeings,wasshownto
amelioratetheHPAofPKU,thusofferingthepotentialtopreventmentalretardation(Bickeletal.,1953).(4)Inthe1960s,asimple
testforpopulationscreeningwasdeveloped(GuthrieandSusi,1963),providingtheopportunityforearlydiagnosis,treatment,and
preventionofPKUdisease.(5)Inthe1960sand1970s,treatmentofthisparticulargeneticdiseasewasseenasaprototypeforthe
treatmentofothergeneticdiseases(Scriver,1967),anditwasshowntobetrulyeffectiveinpreventingmentalretardationinPKU
(MacCready,1974).(6)Mappingandcloningofthephenylalaninehydroxylasegeneinthe1980scamefirstintherodent(Robson
etal.,1982)andtheninHomosapienssapiens(Wooetal.,1983)(humangenesymbolPAHGenBankcDNAsequenceU49897.1).
Thisisfollowedinthe1990sbyworldwidemutationanalysisandrecognitionthatextensivemutantallelicheterogeneityaccountsfor
PKUandrelatedformsofHPA(Nowackietal.,1998).(7)Inthe1970s,therewasrecognition(Danksetal.,1978Kaufman,1971)
thatlocusheterogeneitymustexisttoaccountforallcomponentsofphenylalaninehydroxylationinvivoandtoaccountfora
mysterioussubsetofpatientswith"malignant"HPA(seeChap.78).(8)Inthe1990s,itbecameevidentthatPKUandtheHPAsare
morethanMendeliantraitstheyalsohavethefeaturesofmultifactorial.,multilocus,andcomplextraits(ScriverandWaters,1999).
Therefore,theyserveasprototypesforthinkingmorebroadlyaboutthenatureofsocalledMendeliandisease.(9)Inthe1990s,the
catalyticcoreofthehumanPAHproteinwascrystallizedanditsstructurevisualizedat2resolution(Erlandsenetal.,1997a,
1997b)thus(virtual)molecularmodelinginsilicoofmutationeffectscouldbegin.(10)Inthesamedecade,thevastallelicdiversity,
bothdiseasecausingandneutralpolymorphicintype,atthePAHlocusbegantoberecordedinanonlinerelationaldatabase
(www.pahdb.mcgill.ca)(Nowackietal.,1998).
Whateveronemightliketocall"progress,"itischasteningtorealize,byrereadingPenrosesarticle(1946),thatPenroseanticipated
muchofourpresentknowledgethedifferencebetweenthenandnowissimplytheevidence,obtainedwithtoolsnotavailablein
Penrosesownlifetimetheevidenceshowsthathewasaveryforesightedgeneticistasweresomanyofhiscolleagues.

Footnotes
Standardabbreviationsarelistedprecedingtheindexofthisbook.Nonstandardabbreviationsusedinthischapterareasfollows:
BH4=tetrahydropterin[(6R)Lerythro5,6,7,8tetrahydrobiopterin]DHPR=dihydropteridinereductaseDMPH4=6,7
dimethyltetrahydropterinGTPCH=guanosinetriphosphatecyclohydrolaseHPA=hyperphenylalaninemia6MPH4=6
methyltetrahydropterinPAH=phenylalaninehydroxylaseenzyme(phenylalanine4monooxygenase)PAH=phenylalanine
hydroxylasegene(human)Pah=phenylalaninehydroxylasegene(mouse)PAL=phenylalanineammonialyasePKU=
phenylketonuria6PTS=6pyruvoyltetrahydropterinsynthaseqBH2=quinonoidformofdihydrobiopterinRFLP=restriction
fragmentlengthpolymorphismSNP=singlenucleotidepolymorphismSTR=shorttandemrepeatVNTR=variablenumberof
tandemrepeats.
*ThisversionofChap.77isanoverviewofbothrecentdevelopmentsandwellestablishedthemesintheMendelianHPAs.Many

newreportshaveappearedsincetheonlineeditionofMMBID8extendedtheprintversionofJanuary2001.Thepresentversionis
muchrevisedandcontainssignificantlargeattachments(externalupdates)notfoundintheprintversion.Thestyleforreference
citationisalsodifferent.
ThesymposiumwaspublisheditopenswithanelegantessayaboutAsbjrnFllingandhisdiscoveryofPKU,writtenbyhisson

Ivar(Flling,1994).Anothersymposium,openedbyHorstBickel,"thefatherofPKUtherapy,"coveringthisandothertopicswas
publishedthereafter(EuropeanJournalofPediatrics,1996).
AsbjrnFllinglaterrenamed"his"disease,preferringtocallitoligophreniaphenylpyrouvica(Fllingetal.,1945),anamemore

compatiblewiththewiderangeofmentaldeficiencyhehaddiscoveredinpatientswithPKU.Thesamearticleextendstheevidence
inhisNorwegiancasesforautosomalrecessiveinheritanceofthedisease,addingsupporttosimilarclaimsmadeearlybyhimself
(1934),Penrose(1935),andJervis(1939).

Introduction
Thegenerictermforaphenotypedistinguishedbyphenylalanineconcentrationspersistentlyelevatedabovethedistributionofits
plasmavaluesinhealthycontrolsishyperphenylalaninemia(HPA).Thisdisturbanceinmetabolichomeostasiscanhaveclinical
consequencesdependingonitspathogenesisanditsdegree.Themajorassociatedclinicalmanifestationisimpairedcognitive
developmentandfunctionresultingfromneurochemicalimbalancepostnatallyinaffectedcasesandprenatallyinthefetuscarried
byanaffectedpregnantwoman.ThegeneticcausesofHPAareprimarymutations(alleles)inthegene(symbolPAH)encodingL
phenylalaninehydroxylaseenzyme(PAH)andsecondaryatlociforatleasttwoenzymesinthepathwayforsynthesisof
tetrahydrobiopterin,alsoknownasBH4[(6R)Lerythro5,6,7,8tetrahydrobiopterin],thecofactorforthehydroxylationreaction,and
atthelocifor4carbinolaminedehydrataseanddihydropteridinereductase(DHPR),enzymesthatregenerateBH4fromthe
oxidizedbiopterinbyproductofthehydroxylationreaction.SocallednullmutationsatthePAHlocuscausePKUothersconferring
someresidualenzymeactivitycausealesserdegreeofHPA(socallednonPKUHPA),wheretheassociatedriskofmental
retardationislessthanitisinclassicPKU.
BH4homeostasisservestwoadditionalhydroxylationreactionsinvolvingLtryptophanandLtyrosine,notablyinbrain.The
hydroxylatedderivativesofthesesubstrates,5hydroxytryptophanandLdopa,respectively,areprecursorsofserotoninand
catecholamines,which,asneurotransmitters,influencebraindevelopmentandfunction.Accordingly,diagnosisofBH4deficient
variantsofHPAisrelevantforprognosisandtreatmentandhasbecomeanintegralpartofPKUscreeningprogramseventhough
fewerthan2percentofnewborninfantswithpersistentHPA(inEuropeanpopulations)haveadisorderofBH4homeostasis(see
Chap.78andtherelevantwebsite:www.bh4.org).

Nomenclature
ThischapterdealswiththeHPAsassociatedwithprimarydeficiencyofPAHenzymefunctioncausedbyvariantdiseasecausing
allelesinthePAHgene.IfalldegreesofHPAareariskfactorforimpairedcognitivedevelopment,thenitmaynotmatterwhether
weclassifythetraitintoitsmoreconsistentlysevere(PKU)orusuallymilder(nonPKUHPA)formswewillworryaboutprognosisin
eitherform.Onethuscouldarguefortheuseofasinglenamephenylketonuriaindeed,Smith(1994a)recommendedacceptance
ofthetermphenylketonuria"asascientificallysound,collectivenounforthefamilyanddisordersduetoPAHdeficiency."We
concur,butthereistheissueofcommonpractice.Theliteratureisnowwellpopulatedwithpapersnamingthesevereandmild
formsofHPAasPKUandnonPKUHPA,respectivelyweuseboththesetermshereknowingthateachimplicatessomedegreeof
harmtothecentralnervoussystem(CNS).Ontheotherhand,thereissomeevidencethatuntreatednonPKUHPA(atlevels<600
mol/liter)isnotharmfultocognitivedevelopment(Weglageetal.,2001)hencethereisreasontoretainthedistinctive
nomenclature.
TheclinicaldistinctionbetweenPKUandnonPKUHPAcurrentlyrestsonrecognitionofhigherplasmaphenylalaninevaluesinPKU
(>1000mol/liter>16.5mg/dl)intheuntreatedstateandonlowertolerancefordietaryphenylalanineinPKU(>500mg/day)
(Scriveretal.,1995).OthershavesuggestedsimilarbutquantitativelydifferentcriteriaforclassificationofPAHenzymedeficient
HPA(Guttler,1980),buttheintentionissimilar:Higherambientbloodphenylalaninelevelsandlowerdietarytoleranceare
associatedwithamorePKUlikephenotypeandthusgreaterhazardofimpairedcognitivedevelopment.

Hyperphenylalaninemia:AFrameworkforUnderstandingWhythis"Simple"Phenotypehas"Complex"
Explanations(SeeAlsoSupplement:"Homeostasis,Complexity,andMonogenicPhenotypes:TheViewfrom
Phenylketonuria")

WhereasPKUisaclassicMendeliandisease,itsmetabolicphenotype(HPA)andtheassociateddisease(mentalretardation)are
theresultofmorethantheeffectofasinglemutantallele.TobeawareofthisistohaveaframeworkforunderstandingHPAandits
attendantdiseases,aswellassocalledMendeliandiseaseingeneral(Scriver,2007).
HPAIsaMendelianInbornErrorofMetabolism
PKUislistedunderentry261600intheMcKusickCatalogsofMendelianInheritanceinMan(OMIM).WhenFllingfoundan
increasedfrequencyofconsanguinityamongtheparentsofhispatients,herecognizedevidenceforautosomalrecessive
inheritancehealsofoundanaberrantmetabolicstateinthepropositiheinvestigated(Flling,1934,1994).LionelPenrosealmost
immediatelyrecognizedthedisorderasanew"inbornerrorofmetabolism"(Penrose,1935)andasthefirstformofmental
retardationtohaveanovertchemicalfeature(Penrose,1946)hecommunicatedaccordinglywithGarrod(Bearn,1993).Since
then,allhumanmutationscausingprimaryimpairmentofPAHenzymeintegrityhavebeenshowntomaptothePAHlocuson
chromosome12q(Lidskyetal.,1985a),inkeepingwiththeinvolvementofamajorgeneinPKUandrelatedformsof
hyperphenylalaninemia.Incidenceoftheautosomalrecessivemetabolicphenotypeanditsdisease,whenduetoprimarydeficiency
ofphenylalaninehydroxylasefunction,isonaverage1in10,000livebirthsinEuropeansprevalenceofpersonsaffectedbyand
copingwiththeimplicationsofpersistentHPAwillbethesame.However,inasourcepopulationof100millionpeople,thesubset
followingcontemporaryrecommendationsfortreatmentofHPAwouldgenerate500,000patienttreatmentyearsinhalfacentury.
HyperphenylalaninemiainPKUisMultifactorial
DietaryintakeofLphenylalanine,anessentialnutrientforHomosapienssapiens,isrequiredtoproduceHPAinthemutant
phenotype.Dietaryexperienceandmutantgenotypethusarebothnecessarycomponentsofcause.Accordingly,themetabolic
phenotypeismultifactorial.,andthereinlaytheoriginalopportunityfortreatmentthroughrestrictionofdietaryphenylalanine.
HPAisGeneticallyHeterogeneous
PKUindeedmanifestsHPA,butnotallHPAisnecessarilyPKU.Thephenylalaninehydroxylatingreactionrequires
tetrahydrobiopterin(BH4)cofactor.MutationinagenecontrollinganyoneoftheseveralstagesofsynthesisorrecyclingofBH4can
causeHPA.Recognitionofthisfactisanecessarypartofthediagnosis,workup,counseling,andtreatmentofeverypatientwith
persistentHPA(seeChap.78).
PKUDiseaseandtheAttendantMetabolicPhenotypeareComplexTraits(SeeAlsoSupplement:"Homeostasis,Complexity,
andMonogenicPhenotypes:TheViewfromPhenylketonuria")
AnalysisofgenotypephenotypecorrelationsinuntreatedPKUpatientsshowsnotightandconsistentrelationship,eitherinteror
intrafamilial.,betweenpredictedseverityofthePAHmutationeffectandcognitivedevelopment(IQorDQscores)(Ramusetal.,
1993).Longago,PenrosehadnoticeddiscrepanciesinIQvalueswithinPKUsibships(Penrose,1946)hefurtherrecognizedthat
whereasphenylalaninevaluesinnormalandPKUpopulationsbehavedasadiscontinuousmetrictrait,IQvaluesresembleda
quasidiscontinuoustrait.Sinceintelligenceitselfisacomplextrait,thefailuretoseeatightcorrelationbetweenIQandPAH
genotypeisnotsurprising.However,evenbloodphenylalaninevaluesthemselves,moreproximatethancognitivefunctiontothe
primarymutantgeneeffectonenzymefunction,donotcorrelateconsistentlywithpredictedeffectofthemutantPAHgenotype.For
example,thesamemutantgenotypecanbeassociatedwithbothsevere(PKU)andmild(nonPKUHPA)formsofHPA(Guldberget
al.,1998Kayaalpetal.,1997),andwhereasinvivomeasuresofLphenylalanineoxidationratesshowthegenedosageexpected
foraMendeliantrait,thecorrespondingplasmaphenylalaninevaluesmaynot(Scriver,1998aTreacyetal.,1997).Thisevidence
impliesthatwhereastheoxidativestephasahighsensitivitycoefficientforphenylalaninehomeostasis(KacserandBurns,1981),
PAHenzymefunctionisnottheonlydeterminantofphenylalaninehomeostasis.Phenylalaninehomeostasisisapparentlyunderthe
controlofasetofquantitativetraitloci(ScriverandWaters,1999).Itisamanifestationofahumanphenome(FreimerandSabatti,
2003).
MultiplePAHAlleles
EvenhalfacenturyafterFllingsdescriptionofPKU,itwascustomarytorefertothePKUmutationandtorecorditbythesingle
symbola(forarecessiveallele).InthemoleculareraofPKUstudies,however,itisapparentthatthemutanthumanPAHlocus
harborshundredsofdiseasecausingalleles(a1+a2+a3++an).Afewareprevalentmostarerare,even"private"(Nowackiet
al.,1998Scriveretal.,2000,2003).PKUfitsanemergingviewofMendeliandiseaseingeneral.Themajorityofdiseasecausing
allelesattherelevantlocuswillberareonlyafewwillbeprevalent(Weiss,1996).

PhenylalanineHomeostasis
ClaudeBernard(1878)recognizedthatconstancyintheinternalmilieuwasanecessaryconditionoflifeWalterB.Cannoncalledit

homeostasis(Cannon,1929).Thephenylalaninecontentofblood,orofanyotherbodyfluid,isametrictraitthatobservesacentral
tendency(homeostasis).HPAisrecognizedwhenthevaluefornonpeptidebound(free)phenylalanineisgreaterthanthenormal
frequencydistributionthenormalrangeofvaluesrepresentsonesteadystate,andtherangeofdeviantvaluesinHPAreflectsa
differentsteadystate,whichmayormaynothaveconsequencesforhealth.
Themetabolicsteadystateisadynamiconeinwhichtheconcentrationsofmetabolitesinthesystemremainfixedinthefaceof
fluxesthroughit(Cohnetal.,1980).Anypersistentchangeinafluxeventuallywillchangethesteadystatevalue.Regulatory
mechanismscontrolhomeostaticsystemssothatsteadystatevaluesexperienceonlyminor,transitorychangeswithincertainlimits
underusualcircumstances(MurphyandPyeritz,1986).Thedispersionofplasmaphenylalaninevaluesaroundthecentraltendency
(themeanorhomingvalue)fitsthismodel(Fig.771)andshowsthattheplasmaphenylalaninevalueisaquantitative(complex)
trait.Itreflectsbothintraindividualorinterindividualbiologicvariation(VG)andtherangeofexperienceencounteredbytheindividual
(VE)(Scriveretal.,1985).Thenormalvalueforplasmaphenylalaninewithinanindividualshowsinfradiemoscillation(Scriveretal.,
1985)thatisnotgreaterthanabouthalfthenadirvalue(seeFig.771).Thebroadsimilarityinvaluesbetweenindividualsimplies
thattheirhomeostaticmechanismsaresimilarandhavebeenacquiredthroughsharedhumanevolutiontheoutliervaluesareof
interestbecausetheyreflectunusualbiologicdiversityorexperience.Evidenceforheritability(h2),whichinthebroadsenseis
VG/(VG+VE),isfoundintwinstudies(ScriverandRosenberg,1973)plasmaphenylalaninevaluesaremoresimilarwithin
monozygotictwinpairsthantheyarebetweennontwinsubjects.Accordingly,anindividualhasa"private"plasmaaminoacid
phenotype,withvaluesdistributedoveranarrowerrangethanthe"public"distributionofvaluesforapopulationofunrelated
individuals(Scriveretal.,1985).Thusthebloodphenylalaninevalueindeedshouldbeviewedasaquantitativetraitinwhichthe
controllingbiologicfactorsarethequantitativetraitloci(Langenbecketal.,1988Scriveretal.,1985).Whatthoselociencodeas
proteinsandmechanismsofhomeostasisarescarcelyknown,buttheyeventuallywillyieldtoenquiriesinthecontextof"systems
biology."
FIG.771

A:Thefrequencydistributionofplasmaphenylalaninevalues(n=80)inadulthumansubjects(n=10)studiedtwice(fastingand
fed)atfourdifferenttimesoftheday.B:Meanvalues()forinfradiemvariationinplasmaphenylalanineinfasted()andfed()
subjects.(BothfiguresadaptedfromScriveretal.,1985.Usedbypermission.)

NormalPlasmaValues
Thenormalplasmafreephenylalaninevalueisnotsignificantlydifferentinyoungandadultagesubjects(Gregoryetal.,1986
Scriveretal.,1985).Thenormaladultvalueunderphysiologicconditionsis5815mol/liter(meanSD)thecorresponding
valuesinchildren(meanage8years)andadolescents(meanage16years)are6218and6013mol/liter,respectively.
Genderaffectsthevalueonlyinadolescents(malevaluesarehigher)(Gregoryetal.,1986).Valuesinnewbornandolderinfants
aresimilartothoseinoldersubjects(ScriverandRosenberg,1973).

DeterminantsoftheSteadyState
Fluidcompartmentsinthebody(e.g.,plasmaandcerebrospinalfluid)mayhavequitedifferentconcentrationsoffreephenylalanine,
butatthesteadystatethecompartmentsareinastateofequilibrium,albeitatfarfromchemicalequilibrium(Cohnetal.,1980).
[Thisfundamentalfeaturewillliebehindtheproposal.,forexample,touseenzymesubstitutiontherapywithoralphenylalanine
ammonialyasetotreatPKU(Sarkissianetal.,1999seesectionontreatment).]Thefluxoffreephenylalaninethroughpoolsinthe
humanbodycomprisesinputsandrunouts(Fig.772),andtherearewaystomeasurenetfluxratesinvivo(Krempfetal.,1990).
Inputofphenylalaninehastwomajorsources:exogenousfromdietaryphenylalanineandendogenousfrombound(polypeptide)
andfreeaminoacidstores,thelatterlocatedlargelyinmuscle(ScriverandRosenberg,1973).Runoutinvolvesincorporationinto
boundpools,oxidationtotyrosine,andconversiontominormetabolitesnetrunoutbyoxidationcanbemeasuredinvivoby

measurementoflabeledCO2inexpiredairderivedfromatracerdoseoflabeledLphenylalanine(Lehmannetal.,1986Treacyet
al.,1997).
FIG.772

Majorinputs()andrunouts()offreeLphenylalanineinhumanmetabolism.Inputsofthisessentialaminoacidtothepoolof
freelydiffusiblesolutearefromdietaryprotein[hencetheminimaldietaryrequirement(Table771)]andturnoverofendogenous
(bound,polypeptide)pools.Runoutisby(1)hydroxylationtotyrosine(reaction1catalyzedbyphenylalaninehydroxylase,followed
byoxidation)(2)incorporationintobound(polypeptide)pools(reaction2)and(3)transamination(A)anddecarboxylation(B).The
approximateproportionalimportanceofthethreerunoutsis3:1:traceatnormalsteadystate(seethediscussionsinScriveretal.,
1989andKaufmanand,1999).

Input
LPhenylalanineisanessentialaminoacidinhumans,anda(dietary)sourceisnecessarybothtomaintainphenylalanine

homeostasisandtomeetrequirementsforendogenousproteinsynthesis(AmericanAcademyofPediatrics,1976Youngand
Pellett,1987)ThenutritionalrequirementforLphenylalanineisdifficulttoestimateinthenormalsubjectbecausecatabolicrunout
perturbstheestimateoftheanabolicrequirement.Accordingly,theblockedcatabolicstate(asinapersonwithtotallydeficientPAH
enzymeactivity)isusefultoestimatetheactualhumanrequirement.EmpiricalestimatesintreatedPKUpatientsindicatethatthe
minimumrequirementtosupportproteinsynthesisisontheorderof200to500mg/dayintheinfantandyoungchild(dependingin
partonthemutantPAHgenotype)andprobablynotmorethan1.5timesgreaterinolderchildren.Therequirementfor
phenylalanineisgreaterintheabsenceoftyrosineinsubjectswithintactphenylalaninehydroxylation(AmericanAcademyof
Pediatrics,1976BasileFilhoetal.,1997YoungandPellett,1987).ThustherangeofphenylalaninerequirementinPKUand
normalsubjectsprobablyreflectsinterindividualdifferencesinthebiologicdeterminantsofhomeostasis,aswellasallelicdifferences
atthePAHlocusinthePKUsubjects.Estimatesoftherequirementinadults(Table771)areprobablylowerthanthetruevalue
(YoungandPellett,1987),andvaluesinadultsobtainedbykineticanalysiswithisotopelabeledaminoacidresemblethosefor
youngchildrenwhenexpressedperunitoftotalproteinrequirement(seeTable771).Thisrevisionistviewmayinfluencetreatment
ofHPAinaffectedpregnantwoman.
Table771:EstimatesofPhenylalanineRequirementinHumans*(American
AcademyofPediatrics,1976Young,Pellett,1987)
ViewLarge|SaveTable
Table771:EstimatesofPhenylalanineRequirementinHumans*(American
AcademyofPediatrics,1976Young,Pellett,1987)

mg/day
Infant

mg/kg/day

mg/gprotein

2590

Preschoolchild

200500

69

63

Olderchild

200500

22

22

Youngadult

1914

Youngmaleadult

39

*Estimatesmadeinnormalsubjectswithnormalphenylalaninehydroxylaseenzyme
(PAH)activityandreceivingtyrosineinthediet
EstimatesmadeinpatientswithseveredeficiencyofPAHactivity(classic
phenylketonuria)(AmericanAcademyofPediatrics,1976).
Estimatedbyoraltracerstudiesinsubjectsreceivingvariousphenylalanineintakes
withoutaddedtyrosine(BasileFilhoetal.,1997).
Turnoverofendogenouspeptideboundpoolscontributesinputtotheplasmaphenylalaninepoolvalue(BasileFilhoetal.,1997
Berkeetal.,1992Sanchezetal.,1996).Whennutritionisinadequate,proteincatabolismoccurs,andfreeLphenylalanineis
releasedasaconsequence,plasmaphenylalaninevalueswillriseinitiallyinthePKUpatientinearlynegativenitrogenbalance.
FailuretorecognizethisphenomenoncanconfoundtreatmentofPKUpatientsduringintercurrentillness.
Runout
Inputofphenylalaninewillexpandtheplasmaphenylalaninepoolunlessthereiscompensatingrunout.Incorporationintoprotein,
oxidation,andconversionofphenylalaninetoothermetabolites(seeFig.772)providerunoutonabackgroundofinterorganamino
acidflowandcellularuptake.Therelativecontributionsofthesecomponentstophenylalaninehomeostasishavetheoreticalinterest
andpracticalrelevance.
Inaseriesofimportanttheoreticalstudies(KacserandBurns,1973,1981KacserandPorteous,1987),itwasreasonedthateach
componentinanetworkofsteadystatedeterminantshasavalue,calledthesensitivitycoefficient,proportionaltoitsimportancein
settingthesummationproperty(value=1)ofthesteadystatemaintainedbythenetwork.Relativequantitativevaluesforthe
componentsofphenylalaninerunouthavebeenmeasured(Kaufman1976Salteretal.,1986).Atphysiologicconcentrations,
incorporationintoproteinandhydroxylationtotyrosineaccountforaboutonequarterandthreequartersoftotalfreephenylalanine
runout,respectivelyconversiontophenylpyruvicacidisofminorsignificanceandoccursonlyatelevatedplasmaconcentrationsof
phenylalanine(Kaufman,1976).Conversiontophenylethylamineisatrivialpartofthewhole.Whereasstress,feeding,andfasting
modulatephenylalanineoxidation(BasileFilhoetal.,1997Berkeetal.,1992),onaverage,abouthalfthecontrolofrunoutis
accountedforbyafirststep,whichisuptakeofphenylalaninebycells,notablyhepatocytes(Salteretal.,1986)theremainder
occursbyeventsafterthisstep(i.e.,hydroxylation,proteinincorporation,andmetaboliteformation).Directmeasuresinhuman
subjectsconfirmthathepaticuptakeandoxidationofphenylalaninearealmostequallyimportantdeterminantsofthesteadystate
valueforplasmaphenylalanineinmammals(Sanchezetal.,1996)fromthePKUphenotype,weseethatthePAHgenebehaves
asthe"major"locusinthegeneticmakeupofthiscomplextrait.Ontheotherhand,sincethesensitivitycoefficientofthe
hydroxylationcomponentinthesystemisconsiderablylessthan1.0(becausethesystemmaintainingphenylalaninehomeostasis
hasmanycomponents),itfollowsthatthegeneticbasisofHPAdoesnotconferadominantphenotypeandmustberecessiveand
furthermorethattheheterozygote,underusualconditions,willnotshowsignificantHPA,inkeepingwiththeory(KacserandBurns,
1981).
AlternativePhenylalanineConversionPathways
Conversiontotyrosineisthemajormetabolicpathwayforphenylalaninerunout(Kaufman,1976)(seeFig.772).Conversionof
phenylalaninetonontyrosinederivativesconstitutesonlyaminoralternativeundernormalconditionsmoredetailsofthelatter
reactionsaregiveninpreviouseditionsofthistext(Knox,1960,1966,1972)andelsewhere(ScriverandRosenberg,1973).
TheinitialreactioninthemostsignificantalternativepathwayistransaminationofLphenylalaninetoformphenylpyruvatethisand
thesubsequentmetabolictransformationsinthetransaminationpathway(seeFig.772)arerestrictedtothealaninesidechainof
themolecule.Onlywhenthemajorcatabolicpathwayisblockedandphenylalanineconcentrationismuchincreaseddoesthe
transaminationpathwaybecomefunctionallysignificant.Thereactionisinducedbysubstrateandisnotfullyoperativeinthe
immaturenewbornorintheearlyphaseofHPA(ScriverandRosenberg,1973).Ratesofphenylalaninedisposalbythealternative
pathwaysdoinfluencethemutantmetabolicphenotypeinPKU,andsiblingswithidenticalmutantPAHgenotypescanhavedifferent
ratesfordisposalofminormetabolites(Treacyetal.,1996).
Decarboxylationofphenylalaninetophenylethylamine(seeFig.772)isnotanimportantroutefordisposalofexcessphenylalanine
inhumansatanytime.Monoamineoxidaseinhibitors,whichblockfurthermetabolismofphenylethylamine,dolittletoalteritslevel

inthehumansubject(Rampinietal.,1974).
InterorganPhenylalanineFlowandUptake
PAHenzymeisactivemainlyinhepatocytesinhumans,butsignificantPAHenzymeactivity(LichterKoneckietal.,1999Tessariet
al.,1999)andmetabolicconversionofphenylalaninetotyrosine(Molleretal.,2000)nowhavebeenidentifiedinhumankidney.As
aresult,thekidneyisamajordonoroftyrosinetothesystemiccirculationinthepresenceofnormalPAHenzymeactivity.
Phenylalanineisincorporatedintoproteininalltissues,andmetabolicconversionofphenylalaninetovariousmetabolitesalso
occursintissuesotherthanliver.Accordingly,interorganfluxesareanintegralpartofrunoutforthisaminoacid(Christensen,
1982),andtheremustbephenylalaninetransportacrossplasmamembranesbeforeitcanenteritsintracellularpathways.L
PhenylalanineuptakebymammaliancellsismediatedbycarriersthatarecoupledtotheinwardorientedNa+gradientinapical
membranesofrenal(KraghHansenetal.,1984SamarzijaandFromter,1982)andintestinalepithelia(Bertelootetal.,1982)and
byNa+independentcarriersinotherplasmamembranes(citedinChristensen,1982,1986,1987).Thenephron,whichcontains
bothhighandlowaffinitycarriersforphenylalanine(KraghHansenetal.,1984),achievesneartotalreabsorptionoftheaminoacid
fromfiltrateunderphysiologicconditions.Thesystemsdonotsaturateevenathighphenylalanineconcentrationsinfiltrate(Owens,
1977ScriverandRosenberg,1973),whichmeansthatthetransporterscontinuetofunctioninthehomeostaticnetworkand
contributetotheHPAofPKU.
PhenylalanineentersparenchymalcellsfromplasmaandextracellularfluidonanNa+independent,weaklyconcentrativecarrier
thatacceptsbothbranchedchainandaromaticaminoacidsitexitsfromcellsonasystemsharedbyneutralchargeaminoacids
(Christensen,1986,1987).Hepaticuptakeofphenylalanineisasignificantcomponentintherunoutflux(Salteretal.,1986).
Interactionsbetweenaminoacidsonthecarrierscanperturbthesefluxes(Pardridge,1987Shershenetal.,1987Smith,1987)
andmayplayaroleinthepathogenesisofthebrainphenotypeinPKU.
Bywayofasynthesisforthesethemesaboutphenylalaninehomeostasis,Kaufman(1999)proposedaquantitativemodelfor
metabolismofphenylalanineatsteadystateinfastedhumansubjectsthattakesintoaccountdisposalbythehydroxylationand
transaminationreaction(decarboxylationbeinganegligiblereactioninthiscontext)andinputfromnetproteindegradation.From
availableempiricaldata,Kaufmanassignedkineticvalues:Km(mM)forhydroxylaseandtransaminase,0.54and1.37,respectively
thecorrespondingVmax(mol/mlperhour),0.9and0.063,respectivelyandphenylalaninereleasefromproteindegradation,0.012
mol/mlperhour.Assumingthatbloodlevelsreflecttissuelevels,Kaufmanthentestedthevalidityofhismodelagainstdatafor
normalsubjects,heterozygotes,andmutanthomozygoteswithPKU.Themodelwassatisfactoryandsufficientlyrobusttoshowthat
HPAcouldnotbeexplainedbyputativetransaminasedeficiencyandthatalternativepathwaysforsignificantphenylalaninedisposal
(e.g.,ontyrosinehydroxylase)donotexist.

ThePAHGene
Comment
Morethan98percentofmutationsassociatedwithhumanHPAoccuratthePAHlocustheremainderareatthelocidedicatedto
synthesisandregenerationoftetrahydrobiopterin(seeChap.78).ThePAHgenehasregulatoryelementsandanarchitecture
typicalofmanyhousekeepinggenes.Thelocusharborsseveralhundredknownalleles,someofwhicharepolymorphicandneutral
intheireffectonPAHenzymeactivitymostareacauseofHPA.Theonlinelocusspecificmutationdatabase(www.pahdb.mcgill.ca)
isaprototypeinthehumanmutationdatabaseinitiativetakingplaceattheinterfacebetweenhumangenomicsandgenetics.

TheHumanPAHGene
IsolationofahumanPAHcDNAfollowedsynthesisandauthenticationofaratcDNAfromlivermRNApurifiedbypolysome
immunoprecipitation(Robsonetal.,1982,1984).AhumanlivercDNAlibraryprobedwiththeratcloneproduceda2.4kbcDNA
clone(hPAH247)encodingapolypeptideof452aminoacids,of51,862relativemolecularmass(Kwoketal.,1985).Others
obtainedcDNAclonesfromrat(DahlandMercer,1986),mouse(Ledleyetal.,1990),andhuman(Speeretal.,1986)genomes.
Rodentandhumansequences,bothDNAandprotein,aresimilar,with92percentoverallpolypeptidehomologyand96percent
similarityattheCterminalend(DahlandMercer1986Kwoketal.,1985Robsonetal.,1984).Invitroexpressionofthehumanor
ratPAHcDNAissufficienttoassembleahomopolymericproteinwithphenylalaninehydroxylatingactivityinthepresenceofpterin
cofactor(Chooetal.,1986Ledleyetal.,1985).ThehumanPAHlocus,mappedbyinsituhybridization,isonchromosome12
(Lidskyetal.,1985a),bandregion12q23.21(InternationalHumanGenomeSequencingConsortium,2001),embeddedina1.5
Mbpregioncontainingfiveothergenesofknownorunknownfunction(Venteretal.,2001).
Thefinishedtotalsequenceforthewholeofchromosome12(Schereretal.,2006)harbors1435lociinasequenceof130,683,379

bpofnonoverlappingsequenceaccommodatingapproximately4.5percentofthehumangenome.Therateofbasesubstitutionson
chromosome12inrecentevolutionisslowinginhominidscomparedwithprimatesandrodents.Chromosome12isrichindisease
associatedloci,with487lociaccountingfor5.2percentofcurrentlyknowndiseasecausinggenes.
ThecDNAsequence(DiLellaetal.,1986aKoneckietal.,1992Kwoketal.,1985)(GenBankU49897.1)contains13exonsthat
constituteapproximately2.9percentofthegenomicPAHsequence.Theintronicsplicesitenucleotidesequencesareall
conventional.Exonbordertypesvarymostaretype3(beginningafterthethirdnucleotideofacodon)codons118and236
introducetype1bordersspanningintrons3and6,respectivelycodons170,281,and400introducetype2bordersspanning
introns5,7,and11,respectively.ThestructureofthegeneisrepresentedtoscaleinFig.773.
FIG.773

Basicstructureofthehumanphenylalaninehydroxylase(PAH)gene.Thelocuscoversapproximately100kbofgenomicDNAon
chromosome12p.ThecDNAsequenceisdepositedatGenBank(NCBI)underU498971thegenomicsequenceisunder
AF404777.ThetopshowstoscaletherelativesizesandpositionsofexonsandintronsinPAHgenomicsequence,andpositionsof
polymorphicsites(multiallelicsitesareshownbelowthegene).Biallelicrestrictionfragmentlengthpolymorphismsitesandsingle
nucleotidepolymorphic(SNP)sitesareplacedaboveit.SNPsareidentifiedbytheirsystematic(nucleotide)names,as
recommended(Antonarakisetal.,2001).Theotherfiguredepictsthe5endofthePAHgene.(ReproducedfromKoneckietal.,
1992withpermissionoftheAmericanChemicalSociety.)

AgenomicsequenceofthePAHgene(Konecki,personalcommunicationdocumentedinwww.pahdb.mcgill.ca,November2001)
anditsflankingregionsspanning171,266bphasapproximately027kbpof5'untranslatedregion(5'UTR)upstreamfromthe
transcriptioninitiationsiteandapproximately64.5kbpof3'sequencedownstreamfromthepoly(A)siteinthelastexon(exon13).
ThegDNAnucleotidenumbersareinregisterwiththecDNAsequence(whichhaslongservedPAHmutationnomenclature)
becausethePAHgDNAhasbeennumberedinPAHdbsothatthe+1nucleotideistheadenineofthetranscriptioninitiationsite
(ATG)inexon1.ThusgDNAexons,introns,andthe3'UTRhavepositivenumbersthe5'UTRhasnegativenumbers(Scriveretal.,
2003).Singlenucleotidepolymorphicsites(SNPs)andrestrictionfragmentlengthpolymorphismsites(RFLPs),currentlyusedto
createPAHpolymorphichaplotypes,areannotatedonthegenomicsequence.OthersitesofinterestatthePAHlocuscanbe
identifiedbyusingtoolssuchastheNEBcutter(www.neb.com).
ExonicsequencesinthehumanPAHgenetakeuplessthan3percentofthegenomicsequencebetweenthe5'+1positiondownto
the3'poly(A)tract.Ampliconprimersequencesforall13exonsareavailable(www.pahdb.mcgill.ca).Theshortestandlongest
exonsare57bp(exon9)and892bp(exon13),respectivelythemeanexonsizeis170bp.Threepolyadenylationsignals
[AATAAA]inexon13areannotatedonthegDNAsequencethethirdsiteisusedmostfrequently.Theshortestandlongestintrons
are556bp(intron10)and17,874bp(intron2),respectively,whereasintron3is17,187bpinlength,andthemeanintronsizeis

6390bp.Thesearetypicalmammaliangenedimensions.
ThePAHgenomicsequenceconsistsof40.7percentGC,slightlyabovethemodalvalue(37to38percent)forhumangenes.
RepeatMaskeranalysisshowsthedensityofinterspersedrepeatstobe42.2percentinthePAHgene,atypicalvaluefora
mammaliangene.AlurepeatelementsareannotatedonthegDNAsequence.Intron2containsanAlulikerepeatelement,between
bp17,273andbp17,546,thatmightaccountfora5'deletioncausingPKU(Sullivanetal.,1985).PutativeAlurepeatsandCpG
dinucleotidesites(n=1198)areannotatedonthePAHgenesequence(Scriveretal.,2003).
The5'untranslatedregionofthegene(seeFig.773)hasfivepotentialcapsitesupstreamfromtheactualmethioninetranslation
initiationcodoninexon1(Koneckietal.,1992)multiplecapsitesareafeatureofmanyhousekeepinggeneswithina0.5kbregion
upstreamfromthefirstcodon.ThePAHgenelacksaproximalTATAboxbuthasseveralelements(seeFig.773),includingfour
GCrichdomainsasputativeSp1bindingsites(anotherhousekeepingfeature),aCCAATsequence(atargetforfactorsregulating
efficiencyoftranscription),GREandCACCCsites(elementsinvolvedinregulationmediatedbyglucocorticoids),severalactivator
protein2(Ap2)bindingsites,andonepartialcAMPresponseelement(CRE).The5'regionofhumanPAH,3.5kbupstreamand
beyondthe0.5kbregiondescribedearlier,containsasequenceverysimilartothemouseliverspecifichormoneinduciblePAH
geneenhancerthisregionbindsthehepaticnuclearfactor1(HNF1)(LeiandKaufman,1998a)dosedependentHNF1mediated
transactivationofgeneexpressionisfurtherpotentiatedbythedimerizationcofactorofHNF1(DcoH).ThusDcoH,whichisidentical
tothephenylalaninehydroxylasesimulator(PHSprotein),isalsoa4carbinolaminedehydratasethatparticipatesbothin
regenerationoftetrahydrobiopterincofactorandintheregulationofPAHgeneexpression.A5'PAHconstructfusedtoaCAT
reportergeneandexpressedintransgenicmiceshowedthatexpressionofthehumanPAHgeneisspecificbothfortissueandfor
stageofdevelopment(Wangetal.,1992a).The5'flankingregionsofrat(McDowellandFisher,1995Reesetal.,2001)and
mouse(Faustetal.,1996)genesencodingphenylalaninehydroxylasehavefeaturesandelementsverysimilartothoseofthe
humangene.
PAHhasarichrepertoireofRFLPsandpolymorphicmarkers(seeFig.773)foranalysisofthegene(Kwoketal.1985),fromwhich
alargenumberofinformativehaplotypesarederived(DiLellaetal.,1986a).

EvolutionaryAspects
Aromaticaminoacidhydroxylasegenesexistinearlycomplexorganisms.Ageneencodingphenylalaninehydroxylaseisexpressed
inhypodermalcellsofthenematodeC.elegans,whereitmayplayaroleincuticleformation(Loeretal.,1999).TheDrosophila
melanogastergenomeharborsaphenylalaninehydroxylaselocus(PAH)forwhichstructurehasbeenascertainedfromthecDNA
(RuizVazquezetal.,1996)theintronpositionsandtheCterminaldomainareconservedwiththemammaliangenecounterpart.
Theflygenemayencodebothtryptophanandphenylalaninehydroxylaseactivities,andithasanalternativepromoterand
transcript.
PAHisinamammaliangenefamily(EisensmithandWoo,1995Huftonetal.,1995)ofaromaticLaminohydroxylasesthatincludes
Ltyrosine3monooxygenase(genesymbolTYHEC1.14.16.2)(Fauquetetal.,1988)andLtryptophan5monooxygenase(gene

symbolTPHEC1.14.16.4)(Grenettetal.,1987)thelatteraretetrahydrobiopterinrequiringapoenzymescatalyzingratelimiting
stepsinpathwaysleadingtothesynthesisofneurotransmitters(seeChap.78).Primarystructuresofthepolypeptideproductshave
remainedsimilarthroughoutbiologicalevolution(DahlandMercer,1986Erlandsenetal.,1997bGrenettetal.,1987)with
conservedintron/exonboundaries(StollandGoldman1991)PAH,TPH,andTYHarelikelytohaveevolvedoverthelast75million
yearsfromacommonancestralentitybyduplicationanddivergence(Grenettetal.,1987NeckameyerandWhite,1992),withPAH
andTPHbeingmorecloselyrelatedtoeachotherthantoTYH.
Thethreehydroxylasesshareseveralfeaturesoftheirdomainstructures(DahlandMercer,1986Erlandsenetal.,1997bGrenett
etal.,1987).Cterminalregionsaremoreconserved,andNterminalregionsaremoredivergent.Theformerregioncontainsthe
determinantsforhydroxylatingactivity(Iwakietal.,1986)andBH4binding(JenningsandCotton1990Jenningsetal.,1991),
whereasthelattercontainsthedeterminantsforsubstratespecificity(Iwakietal.,1986)andphosphorylationmediatedactivation
(Campbelletal.,1986Pigeonetal.,1987).Thegeneshavesignificantdifferences:GenomicTYH(smallerthan10kb)(Kobayashi
etal.,1988)issmallrelativetoPAHTPHisprobablyofintermediatesizeifoneextrapolatesfromthecorrespondingmousedata
(StollandGoldman,1991).HumanTYHtranscribesseveralspeciesofmRNA(Nagatsu,1991),butthisisnotsowithPAHandTPH.

ThePAHMutation"Knowledgebase"(PAHdb:Www.Pahdb.Mcgill.Ca)
Databasesarealegacyinandofscience,yettheyhaveoftenbeenneglectedbythecommunitiestheyserve(Maureretal.,2000
Scriveretal.,2003).Scienceisanexplanatoryprocess,andasaparticulardomainofitdevelops,thereisfirstastageof
explanationinclassificationandnomenclature(taxonomy)ofitsentitiesnext,oneofenquiryintomechanismsunderlyingthe

entitiesfinally,astagewheretheentitiesarerevealed(Keller,2002).Thescienceofgeneticsrecognizesmutationasbothentity
andmechanismmutationalsocanbecreated.Thusmutationdatabases,intheirvariousways,recapitulatethescienceand
recreateitinsilico.Accordingly,mutationdatabaseshavebecomeresourcesingeneticsasrepositoriesofthevastwealthofdata
aboutindividualgenesandthegenomestheyinhabit.PAHdbisonesuchlegacyandresource.
PAHdb(www.pahdb.mcgill.ca)isanonlinerelationallocusspecific"knowledgebase"(Scriveretal.,2000,2003Scriver2007)
originatinginthePAHMutationAnalysisConsortium,servingitandothercommunities.PAHdbhasemergedasacomprehensive
andusefulprototypeofthelocusspecifictypeofgeneticdatabase(Claustresetal.,2002).
Theorigins,development,anddesignofPAHdbhavebeendescribedindetail(Scriveretal.,2000,2003Scriver2007).The
databaseisbuiltonfourcoreelements:(1)auniqueidentifierforeachallele,(2)thesourceoftheinformation,(3)thecontextofthe
allele(e.g.,thespeciesandnameofthegene),and(4)thenameoftheallele.PAHdbcontainsentities(mutations)andannotates
themwithattributes.TheTablesofMutationsarisefromthesecoreelements.Tablesofinvitroexpressiondatadescribethe
functionaleffectsofmutationsonPAHenzymeintegrityandfunction.ThedatabaseprovidesalinktoasiteattheScrippsInstitute
(http://stevens.scripps.edu/)tovisualizeinsilicohowmutationsmapontothe3Dproteinstructure.ACuratorsPagehighlights
unscheduledtopics(e.g.,discoveryofBH4responsivePAHalleles)andnoveldatanotreadilyhandledbytheexistingtables.
Amongotheroptions,PAHdbintroducesvisitorstothemousePahgeneandamousemodelofPKU.Theclinicalsignificanceof
humanPAHmutationsisthesubjectofanothermodule.Acounterlogsvisitsandrecordsthelastdateofcuration.Apartialsitemap
ofPAHdbisdepictedelsewhere(Scriveretal.,2003).TheintellectualpropertyofPAHdbiscopyrighted.
PAHdbhasservedinthedevelopmentoftwonewtypesofmutationdatabases:FINDbase(vanBaaletal.,2007)andPhenCode
(Giardineetal.,2007).FINDbase(www.FINDbase.org)isarelationaldatabasegivinginformationaboutrelativefrequenciesof
pathogenicmutationsinvariousgenesinparticularpopulationswithethnicaffiliationsingeographicregions.FINDbasehaslinks
withlocusspecificdatabases(LSDBs),includingPAHdb.PhenCode(phenotypesforENCODEwww.bx.psu.edu/phencode)isa
collaborativeprojectexploringvariantphenotypesassociatedwithhumanmutationsandrelatingsequenceandfunctionaldata
obtainedfromgenomeprojects.ThePhenCodeprojectusesLSDBs,includingPAHdb,toconnecthumanphenotypedatawith
genomicsequences,evolutionaryhistoryandfunctionfromtheENCODEproject.Thelatterprovidedunexpectedrelevancetothe
contentsofthischapter,asdescribedinanaccompanyingarticle(RhesusMacacqueGenomeSequencingandAnalysis
Consortium,2007).ItshowedthatanalleleinthehumangenomeassociatedwithHPAisnotassociatedwithHPAinthemacacque
afascinatingobservation.
BecausePAHallelesarenamedaccordingtoconventionsnowwidelyacceptedandused(Antonarakisetal.,2001),thePAHdbcan
besearchedeasilyor,byusingappropriatetools,usedtoretrieveandtransferitsallelestoanexperimental"WayStation"thatwill
belinkedtoacomprehensivedatawarehouseinthemakingofarepositoryofallhumangenomicallelicvariation(Teebietal.,
2001).ThePAHdbisaparticipantintheHumanVariomeProject(Anon,2007Cottonetal..2007).ThePAHdbiscontributingtothe
developmentof"adatabaseofLSDBs"servingasahubinanetworkofLSDBsaroundtheworld(Horaitisetal.,2007).

ThePAHGene:AllelicVariation(Mutations)
Inthischapter,thewordmutationmeansallelicvariationinthenucleotidesequenceofthePAHgene(CooperandKrawczak,
1993).PAHmutationsareascertainedmostfrequently,butnotexclusively,inpersonswithpersistentpostnatalHPAattributableto
phenylalaninehydroxylasedysfunctionthisconstitutesabiasofascertainment.Themutationsareoftwotypes:(1)polymorphic
andneutralintheireffectonphenotypeor(2)pathogenicthroughimpairmentofPAHenzymefunction.Allelesarecalled
polymorphicwhentheirfrequencyis0.01orgreaterinthepopulationwhetherevery"polymorphic"alleleistrulyneutralandwithout
aphenotypiceffectcannotbestatedwithoutotherconsiderations(seeAulehlaScholzandHeilbronner,2003).Allelesare
consideredlikelytobediseasecausing(pathogenic)(CottonandScriver,1998)whentheysegregateinaffectedpersons,are
inheritedcodominantly,areunambiguouslynull(e.g.,frameshift,proteintruncation,splicedefective,orlargedeletion),orare
missenseandaffectaconservedresiduewhichisthecaseforoverhalfofmissensePAHalleles(EisensmithandWoo,1995)or
themutationisknowntoimpairPAHenzymefunctioninaninvitroexpressionsystem(Waters,2003Watersetal.,1998).
PolymorphicAlleles
ThePAHcDNAsequencecontainsrecognized"polymorphisms"(Fig.773),withthecertaintythatmorewillberecognizedinthe
PAHgenenowthatthefullgenomesequenceisknown.ThreeformsofPAHpolymorphismexist:
1. Biallelicrestrictionfragmentlengthpolymorphisms(RFLPs)(DiLellaetal.,1986aLidskyetal.,1985b)namedfromthe
correspondingrestrictionenzyme(BglII,PvuIIa,PvuIIb,EcoRI,MspI,XmnI,andEcoRV).WiththeexceptionoftheEcoRsites,
whichstillrequireanalysisbySouthernblotting,theRFLPscanbeanalyzedbymethodsbasedonpolymerasechainreaction
(PCR)amplification[BglII(Dworniczaketal.,1991b),PvuIIa(Dworniczaketal.,1991a),PvuIIb(RCEisensmith,unpublished),

MspI(Wedemeyeretal.,1991),andXmnI(Goltsovetal.,1992a)].
2. Multiallelicpolymorphisms,whichincludeahypervariablesequence[variablenumberoftandemrepeats(VNTRs)]of30bp
cassettesharboringatleast10alleles(differingbynumberofrepeats)inaHindIIIfragment3kbdownstreamfromthelastexon
inPAH(Goltsovetal.,1992bLatorraetal.,1994),andaseriesofshorttandem[tetranucleotide(TCTA)n]repeats(STRs)
harboringatleast9allelesinthethirdintronofPAH(Giannattasioetal.,1997Goltsovetal.,1993Zschockeetal.,1994a).
3. Singlenucleotidepolymorphisms(SNPs),whicharesilent(nonRFLP)allelesforexample,c.1546g/a,whichoccursatabout
0.20frequencyinthe3'UTRofPAHonbothmutantandnormalchromosomes(RamusandCotton,1995),andasilent
c.696A/Gpolymorphism(q=0.080.63)incodon232(Q232Q)(LichterKoneckietal.,1994).
PopulationGenetics
RFLP,STR,andVNTRallelescanbecombinedtogeneratecorehaplotypesattheextendedPAHlocus.Aninformative
minihaplotypeconsistingofonlytheSTR,XmnI,andVNTRallelesandaccessibletoPCRbasedanalysishasbeendeveloped
(Eisensmithetal.,1994),ashaveotherapproaches(Zschockeetal.,1995).Theextended(full)PAHhaplotypesarenamedwith
Arabicnumbers(EisensmithandWoo,1992),andatleast87areknown(seePAHdb).Amatrix(Fig.774)summarizesPAH
haplotypeconfigurationsderivedfromsevenbiallelicandtwomultiallelicsitesinapopulationofEuropeandescentthevarietyof
configurationswouldbevastlyincreasedifSNPswereincluded.Anothersystemofhaplotypeanalysiswithyetmorepotentialto
classifyhaplotypediseasemutationassociationshasbeendeveloped(ZschockeandHoffmann,1999).
FIG.774

Extendedpolymorphichaplotypesinthehumanphenylalaninehydroxylase(PAH)genearederivedfromsevenbiallelicrestriction
fragmentlengthpolymorphismsandtwomultiallelicsites(STR,shorttandemrepeatandVNTR,variablenumberoftandem
repeats)(seeFig.773fortheirrelativepositionsinthegene).Most,butnotall,knownhaplotypesaresummarizedinthismatrix.
Fragmentsizes(inbasepairs)oftheSTRallelesarecorrectedforsize(Zschockeetal.,1994a)theyare2bpshorterthanas
describedintheoriginalreport(Goltsovetal.,1992).Thenumbersintherighthandcolumnillustrateatypicalfrequencydistribution
ofhaplotypesinahumanpopulation[inthiscase,FrenchCanadiansfromeasternQuebec(Bycketal.,1996)].(Figuredesignedby
MaryFujiwara.)

PAHhaplotypescouldbegeneratedfromcombinationsofRFLP,STR,andVNTRalleles(EisensmithandWoo,1995),butfarfewer
haveactuallybeenobservedonhumanchromosomesanillustrationisshowninFig.774.Asstatedforthegeneralcase
(EisensmithandWoo,1995),onlyafewhaplotypesareprevalent,andmostareuncommon,andthisistypicalofallhuman
populationsanalyzeduptothepresent.TheapparentshortageofPAHhaplotypesisexplainedbylinkagedisequilibriumacrossthe
100kbregionoftheextendedhaplotype(Chakrabortyetal.,1987DegioanniandDarlu,1994Kiddetal.,2000).PAHhaplotype
heterogeneityisgreateronmutantandnormalchromosomesinEuropeans(Daigeretal.,1989a)thanitisonchromosomesin

Asians(Daigeretal.,1989b).PAHhaplotypediversityisgreaterinAfricanpopulationsthanitisinEuropeans,assumingthatthe
latteraredescendantofasmallfoundinggroupemergingoutofAfricasome100,000yearsago(Kiddetal.,2000).
ParticularPAHhaplotypestendtoharborthemostprevalentdiseasecausingmutationsinEuropeanpopulations(Eisensmithand
Woo,1995Scriveretal.,1993,1996a)forexample,haplotype7isusuallyassociatedwiththeprevalentPKUcausingmutation
G272XinNorway,haplotype2withR408WineasternEurope,haplotype1withR408WonthenorthwesternfringesofEurope,
haplotype3withIVS12nt1innorthernEurope,haplotype9withI65TinwesternEuropeandtheIberianPeninsula,andhaplotype6
withIVS10nt11inAnatolia,southeasternEurope,andtheMediterranean.Attheleveloffamilyratherthanpopulation,codominant
segregationofpolymorphicPAHhaplotypes,inassociationwiththeknownmutantgenotype,iscompatiblewithcarrierdetectionand
prenataldiagnosis(Eisensmithetal.,1994Romanoetal.,1994Wooetal.,1983).
PolymorphichaplotypesatthePAHlocusservethestudyofhumanevolutionandthehistoriesofhumanpopulations(Kiddetal.,
2000)(seeSupplementtothischapterbyKiddandKidddiscussingthepopulationgeneticsofPAH).DivergencebetweenAfrican,
European,andAsiaticpopulations,withsupportforthe"outofAfrica"hypothesis,hasbeendocumentedbyPAHpolymorphic
haplotypeanalysis(DegioanniandDarlu,1994Kiddetal.,2000).Theancestralhaplotypesonwhichsomemodernconfigurations
arosecanbepostulated(LichterKoneckietal.,1994)theorigins,bygeographicregionandpopulation,ofaparticularallelecanbe
surmised(Calietal.,1997RamusandCotton,1995).Theparticulargeneticstructure(atthePAHlocus)ofapopulationcanbe
recognizedinitsconfigurationofhaplotypesandusedtounravellocalizeddemographichistories(Bycketal.,1996Scriveretal.,
1996b)haplotypesalsocanserveasmigrationtracesoverlargegeographicregionsandtimeframes(Benderetal.,1994)(seethe
section"PopulationGenetics"below).
PathogenicAlleles
Whenthealleleisdistinctbystatefromallothers,PAHdbassignsuniqueidentifierstoeachnewPAHmutation(Scriveretal.,2000).
Over500putativepathogenicPAHallelesaredocumentedinthedatabase(seemapatwww.pahdb.mcgill.caclickonSiteMap
thenonMutationMap,whichpermitsadetailedsearchformutations).Thevastmajorityofallelesareknown(orpresumedtobe)
causesofPKUornonPKUHPA,havingbeenascertainedthroughpatientswithaHPAphenotype.Thediseasecausingmutations
fallintofiveclasses:missense,63percentofallallelessmalldeletions(<22bp),13percentmodifiersofmRNAsplicing,12.8
percentterminationnonsensealleles,5.4percentandsmallinsertions,1percentlargedeletionsmayaccountforapproximately3
percentofthepathogenicPAHmutations(Kozaketal.,2006).
PAHmutationsfollowtwopatterns(Scriveretal.,1996b),asnoticedformanyhumangenes(Weiss,1996):(1)Onlyafewalleles,
usuallyonlyfiveinthecaseofPAH,accountforthemajorityofallthediseasecausingmutationsfoundinthepopulation,whereas
theremainderarerare.(2)Thedistributionofalleles,bygeographicregionorpopulation,isusuallynonrandom,sothehistoryofa
particularalleleoftencorrespondswiththehistoryofthepopulationinwhichitisfound(Scriver,1993Scriveretal.,1996b).

PopulationGeneticsofPhenylketonuriaandPathogenicPAHAlleles
Comment
Ourmotivationfordescribing,atconsiderablelength,theevidenceforpopulationgeneticvariationatthehumanPAHlocusisas
follows.First,PKUisaharmfulMendeliantraitinthehomozygousstate,yetincidencedatashowthatallelefrequenciescanreach
orexceed0.01insomeextistingpopulations.Ifso,whatisthemechanismtoexplainapolymorphicfrequencyforthisharmful
allele?Second,becausenewbornscreeningforPKUissowidelypracticed,thereisauniqueopportunitytodetectallpersonswitha
rarevariantphenotypeandtoidentifyallDNAallele(s)associatedwiththephenotype.Thusthehistoryofparticularalleleswillbe
foundinthehistoriesofthepopulationsthatcontainthem.Third,theseinquiriesandtheirfindingshelpustoapplytherelevant
knowledgetoindividuals,families,andcommunitiesandtopreventharmfulconsequenceswhenPKUcausingallelessegregatein
populationsandareinheritedbyindividuals.

RelevantCharacteristicsofthePAHLocus
Thelocusisrichinallelicvariation(seethesection"ThePAHGene"above)andpolymorphicbothinitsbiallelicandmultiallelic
forms(seeFig.773andFig.774)andinalargenumberofsocalledrarediseasecausingalleles(seemapat
www.pahdb.mcgill.caclickonSiteMapthenonMutationMap,whichpermitsadetailedsearchformutations).Thisautosomal
locusbehavesasasingle100kbblockofDNAinHomosapiens,andhavingbeensampledmanythousandtimesindifferent
personsonplanetEarth,initsownsmallwayitcomplementstheinformationgainedfromtheanalysisofhaploid,mitochondrial.,
andYchromosomalDNAandfromproteinpolymorphisms(CavalliSforza,1998).Fromtheclassicstudies,amosaichuman
geneticgeographyhasemergedtoreflectdemicexpansionandhumanevolutionduringthepast100,000years(CavalliSforzaet

al.,1994CavalliSforzaandPiazza,1993MountainandCavalliSforza,1997).Intheirturn,PAHallelescanbeviewedasaunique
setofbiologicmemoriesconnectingindividuals,families,andcommunitieswhosharethecontingenthistoriesthatareechoesofthe
past.Thehistoricandsocialaccidentsofmigration,geneticdrift,geneflow,assortativemating(endogamy,inbreeding),and
recurrentmutation,aloneortogether,withorwithoutselectionbyheterozygoteadvantage,havecontributedtotheparticular
frequenciesanddistributionsofPAHallelesseenincontemporaryhumanpopulations(Scriveretal.,1996b).
PolymorphicPAHhaplotypesservetoanalyzemodernhumanevolutionduringthepast100to200millennia(DegioanniandDarlu,
1994Kiddetal.,2000).Theyalsorevealparticularassociationsbetweenthehaplotypeandapathogenicallele(Chakrabortyetal.,
1987EisensmithandWoo,1995LichterKoneckietal.,1994).Thefollowingtextfocusesontherarepathogenicallelesascauses
ofHPAandwhattheyoffertotheongoingstudyofhumanpopulationgenetics(seeChap.11).

Hyperphenylalaninemia:ASpecialOpportunityinHumanPopulationGenetics
TheHPAsofferbothadvantagesandlimitationsforthestudyofmechanismsbywhichpathogenicallelesachievetheirparticular
frequenciesanddistributionsinhumanpopulations.
Advantages
1. FrequenciesandclassificationsofHPAcanbesystematicallydocumentedthroughnewbornscreeningprograms(Table772).
2. ThemolecularbasisofPAHallelicdiversitycanbedeterminedbyDNAanalysisandcodified(Antonarakisetal.,2001).
3. Thereareassociationsbetweentheprevalentdiseasecausingmutationsandtheirpolymorphicbackgroundhaplotypes.
4. Allelesandtheirassociationswithhaplotypes,populations,andphenotypesaredocumentedsystematicallyinadedicated
relationalonlinedatabase(www.pahdb.mcgill.ca)(Scriveretal.,2003).
Table772:IncidenceofHyperphenylalaninemiaPhenotypesbyPoliticalorPopulationAssociations:Examples

Phenotype

Association

Incidence(Cases/Million
Births)

References*

Phenylketonuria(PKU)

InAfricansliving
outsideAfrica

10

Hardelidetal.,2007

In"Oriental"
populations

Incompletedata

OkanoandIsshiki,1995

China

60

Chenetal.,1989Liuand
Zuo,1986Wangetal.,1991a

China

100

GuandWang,2004Songet
al.,2005

Korea

100

Leeetal.,2004

Japan

Aoki,1991

In"European"
populations

50200

Thalhammer,1975Zschocke
and,2003

Turkey

385

Ozalpetal.,1986

YemeniteJews(in
Israel)

190

Avigadetal.,1990

Scotland

190

Czechoslovakia

150

NonPKU
Hyperphenylalaninemia

Hungary

90

Denmark

85

France

75

Norway

70

UnitedKingdom

70

Italy

60

Finland

<5

Canada

45

Labergeetal.,1987

In"Arabic"
populations

Upto165

TeebiandFarag,1997

Japan

Aoki,1991Okanoand
Isshiki,1995

In"Europeans"
(exceptFinland)

1575

Thalhammer,1975Zschocke
and,2003

In"Arabic"
populations

"Low"

Teebiand,Farag,1997

In"Oriental"
Populations

*Newbornscreeningisthedirectsourceforincidencedata.Incidencealsohasbeenestimatedindirectlyfromconsanguinityratesin
Norway(Saugstad,1975)andItaly(Romeoetal.,1983)
DataalsoweretakenfromWoolfLI,LentnerC:GeigyScientificTables,8thed.Basle,Geigy,1986.
DataaretheaverageofeightprovincialscreeningprogramsinCanadawithanannualcohortof~400,000births.
Arabicpopulations(includingBedouins)inEgypt,Jordan,Iraq,Kuwait,Lebanon,theMaghred(NorthAfrica),andPalestine/Israel
andSudan.ThedataalsocoverJewishpopulationsinArabnations,includingMorocco,Tunisia,andYemen(TeebiandFarag,
1997).
Limitations
1. Allelesareidentifiedmainlythroughaffectedpropositi,rarelyotherwise,thusintroducingabiasofsampling.
2. Populationsaresampledmainlythroughscreeningprogramsthatarenotoperativeinallhumansocietiesagain,thereisabias
ofsampling.
3. Uptothepresent,mutationanalysishasbeenfeasibleacrossonly3percentofthewholePAHgene.
4. Analysisofpopulationspecificmutantchromosomesisrarely100percentefficient(althoughitoftenexceeds95percent)allele
frequenciesarerelative,rarelyabsolute,fortheselectedsample,andtheyarenottrueestimatesofpopulationfrequencies.
5. Parentalallelesarenotuniformlyanalyzed(andreported),anddenovomutationsarelikelytobeunderestimated.

ImplicationsinNonuniformIncidenceRates
ThefollowingthoughtappearsinthePrefacetotheSupplementonPopulationGeneticsofPAH(attachedtothischapter),
coauthoredbyKiddandKidd(2005):"Whenclinicalgeneticiststhinkaboutphenylalaninehydroxylase(PAH),theythinkabout
mutationsinthegenecausingphenylketonuria(PKU)andtheothermetabolicdiseasesofphenylalaninehydroxylasedeficiency,

theirdiagnosis,andtheirtreatment.WhenbiochemicalgeneticiststhinkaboutPAH,theythinkaboutmetabolicpathways,enzyme
activitylevels,andmetabolicsubstratesandproducts.WhenpopulationgeneticiststhinkaboutPAH,theythinkaboutallele
frequenciesofdiseasecausingmutationsandnormalpolymorphismsindifferentpopulations,thepatternsofthesevariationsinthe
populations,andwhatthesefrequenciesandpatternsmeanintermsofdisease,gene,andmorebroadlyhumanevolution"
(italicsaddedforemphasis).
LionelPenrosehadalreadyreflectedontheratherelevatedfrequencyofthisharmfulautosomalrecessivediseaseamong
Europeans(Penrose,1998).Heofferedseveralexplanations,includingconsanguinity,hypermutability,andheterozygousselective
advantage.NordidthenonuniformdistributionofPKUcasesinEuropeanpopulationsescapehisnotice.Itwasneveradryseason
inPenrosesfertilemind.
NewbornscreeningprovidesdatatocorroboratePenrosesviewofprevalenceratesandallelefrequencies.Prevalenceratesreveal
anaggregatefrequencyforpathogenicalleles(bothPKUandnonPKUHPA)inthepolymorphicrangeincertaingeographicregions
andpopulationsinEurope(Zschocke,2003).Newdataderivedfromnewbornscreeningprograms(GuandWang,2004Songet
al.,2005Leeetal.,2004)revealprevalenceratesinChineseandKoreanpopulationstobeequivalenttothoseinEuropean
populations.InternalstudiesintheEuropeanpopulations,forexample,showstratificationbypopulationorgeographicregionfor
PKU(seeTable772)theevidenceforstratificationislessprominentfornonPKUHPA.
ThereisapossibilitythatinbreedingwillexplainthehighincidenceofPKUinTurkey(Ozalpetal.,1986Ozgetal.,1993Woolf,
1994)thehypothesishasbeenconfirmedbyformalanalysisinthePakistanicommunityoftheWestMidlandsintheUnited
Kingdom(Hutchessonetal.,1998)andinsomeArabiccountries(TeebiandFarag,1997).Consanguinitydoesnotexplainthe
incidenceofPKUineitherNorway(Saugstad,1975)orItaly(Romeoetal.,1983).TheItalianstudyshowedfurther,evenbeforethe
humanPAHgenehadbeencloned,thatPKUistheresultofmutationatonlyonelocusandthatlocusheterogeneityneednotbe
takenintoaccounttoexplaintheincidenceofPKUaninterpretationthathasprovedtoberealisticbecauseonlyapproximately2
percentofcasesofHPAarenotexplainedbymutationsatthePAHlocus(seeChap.78).Theoriginsofdeviantprevalenceratesof
PKUinhumanpopulationsarestillopentoinquiry.

ImplicationsinNonuniformAllelicDistributions
ThePAHlocusofHomosapienshasaccumulatedanimpressivearrayofalleles,bothdiseasecausingandneutralpolymorphic,
eachdifferentbystateandidentity(seeFig.773andwww.pahdb.mcgill.ca),duringdemicexpansionoverthepast100,000years
(Fig.775)[seealsoChapter77Supplement"ThePopulationGeneticsofPAH"(Kidd,Kidd,2005)].Amongthediseasecausing
PAHmutations,onlyafew(betweenfourandsixinmostpopulations)makeupthemajorityofthetotalatthelocus(Table773),
andthispatternisemergingasthenormformostpathogenicallelesindifferenthumangenes(Weiss,1996WeissandBuchanan,
2003).
Table773:DifferentSetsoftheMostPrevalentPKUCausingPAHAllelesOccurin
EuropeanandOrientalPopulations
ViewLarge|SaveTable
Table773:DifferentSetsoftheMostPrevalentPKUCausingPAHAllelesOccurin
EuropeanandOrientalPopulations

RankOrder

European(n=3630)*

Oriental(n=210)*

Allele

Allele

R408W

31

R243Q

13

IVS12nt1

11

R413P

13

IVS10nt11

10

c.611AG

13

I65T

IVS41

Y414C

R111X

Cumulative

62

53

*Denominator(independentchromosomes)forcalculationofrelativefrequencies
(percent)ofalleles
R408Wonhaplotypes1.8and2.3combined
FromEisensmithandWoo,1995www.pahdb.mcgill.ca.
FIG.775

Humanhistoryhypothesizedfromtheviewpointofallelicdiversityatthehumanphenylalaninehydroxylase(PAH)genelocus.
Followingan"outofAfrica"migrationanddivergence,differentsetsofphenylketonuriacausingallelesaroseinEuropeans
(Caucasians)andAsianOrientalsandwereactedonbygeneticdrift.Foundereffectisthelikelyexplanationforrelativerarityof
PKUallelesinAmericanaboriginals,Japanese,Ashkenazi,andFinnishpopulations(alsoinPolynesians).Demicexpansion,
migrations,andgeneflowdisrupt"treesofdescent"inpreandpostNeolithiceras(10,000ybp).Rangeexpansionandcreationof
neoEuropeanpopulationsoverseas(from1000ybp)explainthe"overseas"distributionsfromEuropeansourcesofcertainPAH
alleles.

DistributionofdiseasecausingPAHmutationsinpopulationswithoriginsindifferentgeographicregionsisnotuniform.Suchalleles
arealmostinvisibleinsubSaharanAfricans(seeBeighton,p.90,inScriveretal.,1996b),afindingsupportedbyanepidemiologic
study(Hardelidetal.,2007)andbyasmallsurveyofmentallyretardedsubjectslivinginAfrica(FamilusiandBolodeoku,1976).
AlthoughsamplingofPKUallelesinAfricanslivinginAfricaisextremelylimitedintheabsenceofnewbornscreeninginpopulations
onthecontinent,thereissupportingevidencethattheprevalencerateofPKUinAfricandescendantslivingoutsideAfricaisindeed
lowerbyanorderofmagnitudethantheircorrespondingcohortsofEuropeandescent(Eisensmithetal.,1996Epps,1968Gjetting
etal.,2001GrawandKoch,1967Guldbergetal.,1996aHardelidetal.,2007Knox,1972Hofmanetal.,1991Tyfieldetal.,
1997).TheapparentdiscrepancybetweenprevalenceratesinpopulationsrootedinAfricaandintemperatezonessuggeststhat
themajorityofdiseasecausingPAHmutationsinEuropeansandOrientalsmaynothaveoriginatedinAfricabutappearedlaterin
associationwithselectiveadvantageintheheterozygotefollowingtheoutofafricadiaspora(Hardelidetal.,2007).
CurrentevidencebasedonsamplinginEuropeanandOrientalpopulations(seewww.pahdb.mcgill.ca/andGuetal.,1995Guand
Wang,2004Leeetal.,2004Okanoetal.,1992OkanoandIsshiki,1995Lietal.,1994Songetal.,2005Sunetal.,1997Wang
etal.,1989,1992b)shows(1)thatincidenceofPKUissimilaramongOrientals(inmainlandAsia)andEuropeans(seeTable772)
butmuchlowerintheJapanese(Okanoetal.,1992OkanoandIsshiki,1995),(2)thattheallelesamongOrientalsasagroupare
quitedifferentfromthoseinEuropeans(seeTable773),and(3)thattheallelesaredifferentagainbetweenJapaneseandother
Orientals(Okanoetal.,1992,1994OkanoandIsshiki,1995GoebelSchreinerandSchreiner,1993).TheJapanesedataare
compatiblewithgeneticdriftinthefoundingofthisislandpopulation.
PKUisnolongerseenasararetraitinArabicpopulationsintheMiddleEast(TeebiandFarag,1997)themutationsareoften
particulartoArabicchromosomes(Hashemetal.,1996Kleimanetal.,1992aKleimanetal.,1992b,1993).InKuwait,prevalence

ratesare1:6500forPKUand1:20,000fornonPKUHPA(Teebietal.,1988).
ThesupposedlylowincidenceofPKUinPakistanandIndia,29casesper1millionbirths(Hardelidetal.,2007),mayreflectbiasof
ascertainmentratherthanabsenceofalleles.Unusualalleles,bothdeletion(Guldbergetal.,1997b)andmissense(Guldbergetal.,
1993b),arefoundinPKUpropositibornintheAsiansubcontinent.Moreover,screeningofaPakistanipopulationlivinginBritain
yieldedaPKUincidenceof1:14,500(equivalenttothecorrespondingEuropeancohortintheUnitedKingdom)(Hutchessonetal.,
1998)however,becauseofconsanguinityinthisPakistanicommunity,allelefrequencyislower(1:713)relativetotheEuropean
cohort(1:112).
ItisnowevidentthatthehumanPAHlocusharborsextensiveallelicdiversity.Therefore,whereverPKUisfound,andwhere
nonconsanguineousmatingisthenorm,themutantphenotypeislikelytoreflectaheteroallelicmutantgenotype.Thehigh
homozygosityrateforapathogenicPAHallelefoundinYemeniteJewsreflectsamajorfoundereffect(Avigadetal.,1990),and
whereverconsanguineousmatingisaconvention,homoallelicPKUgenotypeswillbemoreprevalent.Otherwise,thegenetic
homozygosityvaluejatthePAHlocuswillbelow(Table774).
Table774:ExpectedHomozygosityatthePAHLocusinPatientswith
Hyperphenylalaninemia(LargelyPhenylketonuria)aMeasureofAllelic
HeterogeneityinHumanPopulationsinDifferentGeographicRegions
ViewLarge|SaveTable
Table774:ExpectedHomozygosityatthePAHLocusinPatientswith
Hyperphenylalaninemia(LargelyPhenylketonuria)aMeasureofAllelic
HeterogeneityinHumanPopulationsinDifferentGeographicRegions

Population/Region

J*

Mutation
Detection
Rate(%)

Reference

YemeniteJews

1.0

44

100

Avigadetal.,
1990

SouthernPoland

0.44

80

91.3

Zygulskaetal.,
1994

Iceland

0.26

34

100

Guldbergetal.,
1997a

Tataria

0.19

27

100

Kuzmanetal.,
1995

Denmark

0.17

378

98.4

Guldbergetal.,
1993Guldberg
etal.,1994

NorthernIreland

0.14

242

99.6

Zschockeet
al.,1995

Australia(Victoria)

0.11

83

97.6

Ramusetal.,
1995

Norway

0.10

236

99.6

Eikenetal.,
1996a,1996b

Netherlands

0.08

68

92.6

VanDerSijs
Bosetal.,
1996

Germany

0.06

90

95.6

Guldbergetal.,

1996b
Quebec

0.06

142

96.5

Carteretal.,
1998

USA

0.06

294

94.9

Guldbergetal.,
1996a

Sicily

0.06

106

98.1

Guldbergetal.,
1993a

*j=xi2wherexiisfrequencyofeachalleledifferentbystateandidentitywhen
analysisofstatewasnot100percent,theuncharacterizedallelesaregiventhe
aggregatefrequency1/N.
Nnumberofchromosomesavailable
VariationinjbypopulationandgeographicsubregioninQuebecwas0.050.08
(Carteretal.,1998).
VariationinjbyregioninUnitedStateswas0.050.10(Guldbergetal.,1996a).
Includesunpublisheddatafromsource.

CentersofDiffusionandGeneFlow
DemicexpansionandmigrationacrossEuropeandAsia(CavalliSforzaetal.,1993)(asimpliedinFig.775),werelikelywaysto
spreadPAHalleles.Alargemetaanalysisofalmost9000Europeanchromosomesharboring29differentalleles,eachatrelative
frequencygreaterthan3percent(Zschocke,2003),revealsdistinctivegeographicdistributionsforthemostprevalentalleles(Table
775)thenewstudyconfirmsandextendsanearlieranalysisofEuropeanalleles(Eisensmithetal.,1992).Thereiscorresponding
evidencefordistributionofPAHallelesinOrientalpopulations(Wangetal.,1991a,1991b).Clinesofallelefrequencycanbe
derived,andcorrespondingmapshavebeencreatedtosuggestcentersofdiffusionforseveralPAHallelesinhumanpopulations
(EisensmithandWoo,1995Zschocke,2003)thesefurtherimplygeneticdriftincomparativeisolationatthetimethecentersof
diffusiondeveloped,ahypothesiscompatiblewithothermeasuresofhumangeneticdiversity(MountainandCavalliSforza,1997).
Table775:MajorGeographicDistributionsoftheMostPrevalentPAHAllelesin
EuropeandtheOrient
ViewLarge|SaveTable
Table775:MajorGeographicDistributionsoftheMostPrevalentPAHAllelesin
EuropeandtheOrient

InEurope

IntheOrient

Region

Allele

Region

Allele

Norway

G272X

China

R243Q

Denmark

IVS12+1

China

R413Q

Iceland

c.1129delT

China

IVS41

Ireland

R408W[H1.8]

Korea(South)

Y204C/IVS41

CentralGermany

R408W[H2.2]

Japan

R413P

France

IVS12+1

Switzerland

R261Q

SpainandPortugal

IVS1011

Sicily

IVS1011

Turkey,others

IVS1011

Poland

R408W[H2.3]

Russia

R408W[H2.3]

Estonia

R408W[H2.3]

FromEurope,Zschockeand,2003Iceland,Guldbergetal.,1997aOrient,
EisensmithandWoo,1995andOkanoandIsshiki,1995southernEurope,Calietal.,
1997Turkey,Ozgetal.,1993andCalietal.,1997.
GeneticDrift
PathogenicPAHallelesareusefulrecordsofthe"dancetothemusicoftime"thatmoldshumansocieties.Theextentoftheir
variationrangesbetweenextremes.AtoneendofthespectrumisthepenetratingeffectofdriftonPAHalleles,asillustratedby(1)
aunique(deletion)alleleinYemeniteJewslivinginIsrael(Avigadetal.,1990)and(2)theexampleoftwosolitaryallelesin
EuropeanGypsies(IVS10nt11onhaplotype34andR252W,haplotypeunspecified)(Desviatetal.,1997Kalaninetal.,1994
Tyfieldetal.,1989).AttheotherendofthespectrumisthevirtualabsenceofPAHalleles(negativefoundereffect)inFinns,
AshkenaziJews,andJapanese,amongwhomPKUisaveryraredisorder(seeTable772).Inbetweentheseextremes,inEurope,
forexample,thereistheallelicinterminglingthatreflectspopulationgrowth(CavalliSforzaandPiazza,1993).
Migration
ThealleliccompositionofthePAHgeneamongcontemporaryPKUpatientsinthenonSlavicpopulationofTataria(Kuzmanetal.,
1995),aregionintheformerUSSR,revealsmutationsthatcouldhavebeenintroducedbyconquestormovementalongtrade
routes:fromEasternEurope(theR408Wmutationonhaplotype2),fromAnatolia(theIVS10nt11mutationonhaplotype6),and
fromScandinavia(IVS12nt1andarareframeshiftallele,thelatterotherwisefoundonlyinScandinavians).NoOrientalmutations
arefoundinTatarianPKUpatients.Together,thefindingsimplythatthispopulationwasformedmainlybypeoplefromCaucasian
ratherthanOrientalbackground,ahypothesisbeingtestedbyanalysisofothernucleargenesandmitochondrialDNA.
ThedifferentpatternsofallelicdiversityamongHPApatientsinDenmark(Guldbergetal.,1993c,1994)andinSicily(Guldberget
al.,1993a),forexample,reflectthedifferentpoliticalandlinguistichistoriesoftheregions.WithinItalyitself,thereisasignificant
differenceinthecompositionofPAHallelesinPKUpatientsfromthenorthernandsouthernregionsofthecountry(Dianzanietal.,
1994,1995),theirdistributionreflectingthedifferentdemographicandculturalhistoriesofnorthernandsouthernregions.
ExamplescompatiblewiththeeffectsofmigrationanddemicdiffusionareseeninoutlierregionsofEurope(Zschocke,2003),such
asIceland(Guldbergetal.,1997a),theBritishIsles(Tyfieldetal.,1997Zschockeetal.,1997),Ireland(ODonnelletal.,2003),and
theIberianPeninsula(Prezetal.,1997Riveraetal.,1998).Allelesidentifiedintheseregionsappeartohavearrivedtherein
peoplewhocamefrombothelsewhereinEuropeandoverlongstretchesoftime.
Complex"untreelike"allelicdiversity,amongboththemoreprevalentandtherarerPAHalleles,isseentowardthecenterofEurope
forexample,inDenmark(Guldbergetal.,1993c),France(Abadieetal.,1993a),theNetherlands(VanDerSijsBosetal.,1996),
andGermany(Guldbergetal.,1996bZschockeandHoffmann,1999)reflectingthefactthatthepopulationsofcontinental
Europehavenotevolvedaccordingtosimple"trees"ofdescenttherealityismorelike"networks"oflineagesreflecting
interminglinghistoricalmovement(CavalliSforzaandPiazza,1993).

RangeExpansion
PAHmutationsmakeusefulevocativerecordsofrangeexpansionbyneoEuropeans(Crosby,1986)fromcountriesoforiginoutto
theAtlanticislands,theAmericas,andAustralasia,forexample(seeFig.775).Evidenceliesinstudies,forexample,fromIceland
(Guldbergetal.,1997a),Australia(Ramusetal.,1995),theUnitedStates(Guldbergetal.,1996a),Canada(Carteretal.,1998),

Mexico(Nicolinietal.,1995),CostaRica(Santosetal.,1996),andSouthAmerica(Perezetal.,1993,1996).Emigrationfrom
Europe(rangeexpansion)hasbeenrecurrentoverthepasthalfmillennium,usuallyinitiatedbysmallnumbersofindividuals.When
colonizationofthenewterritorywassuccessful,demicexpansionbynaturalincreasewouldfollow,withorwithoutnewimmigration.
Thesearetheconditionsunderwhichrecentfoundereffectscouldstillbemanifest,andthePAHlocusagainoffersexamples:(1)
TheM1Vallele,prevalentinQuebec(Carteretal.,1998)andrareinFrance(Abadieetal.,1993a),hasnarrowtimeandspace
clustersforitsoriginoutofFranceandentryintoNewFrance(Lyonnetetal.,1992)itsdistributioninQuebectodayclearlyreflects
thehistoryofitspopulation(Carteretal.,1998).(2)Thec.1129delTmutationinIcelandaccountsfor40percentofmutantPAH
allelesthere(Guldbergetal.,1997a).Themutationisassociatedwithbiallelichaplotype4,VNTR3,STR234.Genealogic
reconstructionforfivegenerationsinfamiliesharboringthemutationidentifiesancestorsfromanisolatedpartofsouthernIceland.
ThemutationhasnotbeenseenonanyEuropeanchromosomesoutsideIceland.SincethetetranucleotideSTRlocusisa"fast"
molecularclock(WeberandWong,1993),thepresenceofonlyonespeciesofSTRalleleinthePAHhaplotypebearingc.1129delT
iscompatiblewitha"recent"originforthepathogenicmutation[otherconsiderationseventuallymayrefutethishypothesis(Rannala
andSlatkin,1998)].SeveralPAHmutationsfoundinIcelandalsoappearinScandinaviabutnotinIrelandorScotland.Thegenetic
evidencesuggeststhattheIcelandicpopulationispredominantlyofScandinavianorigin,withlittlecontributionfromtheBritishIsles
(aswasonceproposed).(3)MarinersfromtheIberianPeninsuladiscoveredtheCentralandSouthernAmericas,andcolonization
bytheirfollowersapparentlyintroducedtwoPAHalleles(IVS10nt11andV388M),bothprevalentintheIberianPeninsula(Prezet
al.,1997Riveraetal.,1997,1998)andnowaccountingfor5to30percentofPAHmutationsinthescreenedSouthAmerican
populations(Desviatetal.,1995Perezetal.,1993,1996).
Rangeexpansion,withitspotentialfoundereffectinthenewlycreatedpopulation,doesnotalwaysproducegreaterhomozygosity
fortherarepathogenicallelesinthedescendants(seeTable775)Quebecisanilluminatingexampleinthisrespect(Carteretal.,
1998).PAHhomozygosityforpathogenicallelesinQuebecoverallislow(j=0.06)itisnotsignificantlyhigherinthelinguisticand
culturalsubsetsofthepopulationineasternandwesternQuebecandinMontreal.GroupsofsettlersfromFrancebefore1759,
fromtheBritishIslesandIrelandafter1759,andfromeasternEuropeandMediterraneannationsafter1945haveeachintroduced
differentandidentifiablePAHallelesintothepopulation,ofwhichthegeographicanddemographicdistributionsintheprovince
reflectthehistoriesofthedifferentcommunitiesthatmakeupQuebectoday.

MolecularMechanismsIntroducingNovelPAHAllelesandHaplotypeAssociations
Penrose(1998)suggestedrecurrentmutationasapossibleexplanationforthefrequencyofPKUinEuropeanpopulations.The
hypothesismightberephrasedtoask:IsthePAHgenehypermutable?
DeNovoMutations
MutationanalysisisdoneandreportedmorefrequentlyinPKUpropositithanintheirbiologicalparentsthustheactualrateofde
novoPAHmutationisnotknown.Evenso,=Sq2islikelytobeasmallvalueatthePAHlocusbecausedenovoallelesareindeed
rare,withonlyonereporteachsofaroffourdifferentallelesonthousandsofindependentchromosomes(AulehlaScholzand
Heilbronner,2003Chenetal.,2002Eikenetal.,1996a).
RecurrentMutation
ApredictedmutabilityprofileexistsforthecDNAsequenceofPAH(Fig.776).Themajorityofthepredictedhypermutableregions
coincidewiththe24CpGdinucleotidesitesinthegene(Bycketal.,1997).CpGsitesare40timesmoremutablethananyother
dinucleotidesequences(CooperandKrawczak,1993CooperandYoussoufian,1988),andtheycanexperienceCTorGA
transitionmutationsduringdeaminationofthe5'methylcytosineifthecytosineinthedinucleotidewasmethylatedinitially.Among
thepathogenicPAHmutations,atleast23areknowntobeCpGtypealleles(Bycketal.,1997)another8occuratCpGsitesbut
arenotCTorGAtransitions,andsevenregionsinthePAHgenecontainingCpGsiteshavenoreportedmutations(seeFig.
776).
FIG.776

Themutabilityprofileforthehumanphenylalaninehydroxylase(PAH)geneandcDNAsequencepredictedbytheMUTPRED
software(Cooper,Krawczakand,1993).ThreeclassesofhypermutableCpGsitesoccurinthePAHgene:,siteswithnoreported
alleles,siteswithCpGtype(CTorGA)mutations,sitesatwhichthemutationisnotCpGtype.(FromBycketal.,1997.
UsedbypermissionofWileyLissInc.)

ThereisevidenceforrecurrentmutationattheR408WcodoninthePAHgene.Ramusandcolleagues(1992)identifiedtwo
differentmutationsinthiscodon:R408W(c.1222CT)andR408Q(c.1223GA)theyproposedamutational"hotspot."Anearlier
study(Johnetal.,1990)foundtheR408Wmutation(c.1222CT)ontwodifferentRFLPhaplotypesintheQuebecpopulationand
proposedrecurrentmutation,geneconversion,orintragenicrecombinationasamechanism.Genealogicreconstructionsinthe
Quebecfamiliessegregatingonhaplotype1foundCelticancestorsfromScotlandandIreland(Treacyetal.,1993).Thefinding
suggestedacenterofdiffusioninnorthwesternEuropeforR408W[H1]differentfromR408Wonhaplotype2.Thishypothesiswas
testedbyhaplotypeanalysisofmorethan1200EuropeanchromosomesharboringtheR408Wmutation(Eisensmithetal.,1995).
Thosecarryinghaplotype1clusteredonthenorthwesternfringesofEurope,whereasthosecarryinghaplotype2clusteredin
easternEurope.Furtheranalysisoftheflanking5'STRallelesand3'VNTRmarkersofnormalandmutantPAHchromosomes
(Bycketal.,1994Tigheetal.,2003)producedmoreevidenceforrecurrentmutation.ThusR408Whasidentityonlybystateand
notbydescentinnorthwesternandeasternEuropeanpopulationsR408Wonhaplotype1.8mightbetheolderofthetwoalleles
(Tigheetal.,2003).
TheR408Wmutationnowhasbeenfoundonsevendifferenthaplotypebackgroundsfromwhichputative"genegenealogies"can
becreated(Fig.777).ItseemslikelythatrecurrentmutationisthesourceofR408Wonhaplotypes1.8,2.3,and5inEuropeans
(Kalaydjievaetal.,1990Kozaketal.,1995LKozak,personalcommunication,documentedinwww.pahdb.mcgill.caZschockeet
al.,1994bZygulskaetal.,1993)andonhaplotype44intheOrient(Guttleretal.,1999).AmechanismfortherecurrentR408W
allelehasbeenproposed(Murphyetal.,2006).Itinvolvesspontaneousmethylationmediateddeaminationofthe5mC(cytosine)at
positionc.1222.Thiscytosinenormallyismethylatedinhumanandnonhumanprimates.Ondeamination,itconvertstothymine,

creatingthemutationc.1222CTandthuschangingthecodonfromCGGtoTGGandcreatingthemissenseallelp.R408W.On
theotherhand,theR408Wmutationonhaplotype41inbothEuropean(Kadasietal.,1995)andOrientalpopulations(Linetal.,
1992),onhaplotype34inPortuguesepatients(Caillaudetal.,1992),andonhaplotype27inaBelgianpatient(LMichiels,personal
communication,documentedinwww.pahdb.mcgill.ca)canbeexplained,ineachcaseeitherbymutation(orgeneconversion)ata
singleRFLPsiteorbyanintragenicrecombination.Ineachcase,theR408Wmutationwouldbeidenticalbydescent(seeFig.77
7).TheE280K(c.838GA)alleleoccursonhaplotypes1and2,afindingcompatiblewithrecurrence(Okanoetal.,1990)and
supportedbymolecularevidence(Bycketal.,1997).
FIG.777

A"genegenealogy"forthephenylketonuriacausingR408Wallele.Themutation,alwaysidenticalbystate(c.1222CT)isfound
onsevendifferentextendedhaplotypes(H).Sequenceanalysisofnormalandmutant(H1)and(H2)chromosomesrevealsthe
likelihoodthatoneoftheseversionsofR408WinEuropeansisarecurrentmutationataCpGsite.Inspectionoftheotherhaplotype
associationswithR408Wpointtoatotaloffourrecurrentallelesandthreethatareidenticalbydescentonintragenicrecombinant
copiesofthehumanphenylalaninehydroxylase(PAH)gene.

IntragenicRecombination
PAHallelesareusefulmarkersinevidenceofaclassicmechanismbywhichevolutionhasgeneratedgenomicdiversitygenetic
recombination.R408Wmutationsarenottheonlyonesthatappeartohavechangedhaplotypebyintragenicrecombination.S76P
allelesonhaplotypes1and4,G218Wonhaplotypes2and1,andV245Aonhaplotypes7and3allhavebeenidentifiedon
chromosomesinEuropeansandintheirdescendantsabroadineachcase,asinglerecombinationwithinthePAHgenecanexplain
theassociationofonemutationwithtwohaplotypes(Carteretal.,1998).AnIVS12nt1allele,foundalmostexclusivelyonhaplotype
3,hasbeenfoundonceonaforeignhaplotypethiseventisexplainedbyadoubleintragenicrecombination(Carteretal.,1998).
MultipleassociationsbetweenapathogenicPAHalleleatanonCpGsiteandapolymorphichaplotypehavebeenidentifiedinthe
caseoftheprevalent(andancient)IVS10nt11spliceallele.Recombinationcouldexplainsomeoftheseassociations(Fig.778).
FIG.778

Thehumanphenylalaninehydroxylase(PAH)genemutationc.1066nt11ga(IVS10nt11)isprevalentinpopulationsof
SoutheasternEuropeandtheMediterranean.Thealleleoccursonsixdifferenthaplotypesattherelativefrequencies(percent)
shown(Calietal.,1997).Itsoccurrenceondifferenthaplotypesiscompatiblewithidentitybydescent(IBD)assuming(1)intragenic
recombinationbetweenhaplotypes6,10,and39or(2)mutation/geneconversionatarestrictionfragmentlengthpolymorphism
(RFLP)site.TheoriginoftheIVS10mutationonhaplotype9isunclear.TheRFLPsitesnotnamedhereareshowninFig.775.
Therelativeposition(*)oftheIVS10nt11mutationinthegeneisshown.

SelectiveAdvantage:IsitaMechanism?
Areanyoftheforegoingmechanisms,bythemselves,sufficienttoexplaintheincidenceofPKUintemperatezonepopulations?
Considertheevidenceoncemore:
1. InvolvementofmultiplelocithatcouldaccumulatelargernumbersofmutationalhitstocauseHPAcanbediscountedbecause
morethan98percentofcasesreflectphenotypichomozygosityformutationsatthePAHlocusalone.
2. AlthoughsomepathogenicPAHallelesaretheresultofrecurrentmutation,anoverallhigherthanaveragerateofdisease
causingmutationaleventsatthePAHlocuscanbedismissed.
3. Inbreedingisalikelycontributorinsomepopulations,butoverall,itisnottheexplanation(Woolf,1994).
4. Foundereffectisonlyanoccasionalmechanismtoaccountforthehighrelativefrequencyofaspecificallele,anditalways
relatestoaparticularpopulation.
5. Thepresenceofmanydifferentprevalentallelesindifferentpopulationsiscompatiblewithgeneticdrift(seeWBodmerin
Scriveretal.,1996b).Moreover,geneticdriftcouldberesponsiblefortheincidenceofPKUintemperatezonepopulations,and
itwouldactindependentlyonawidevarietyofallelesbecausethesubsetofdeleteriousPAHallelesisstillsmallrelativetothe
wholesetandthussusceptibletorandomgeneticdrift(seeDHartl,p.93,inScriveretal.,1996b).
TheweightofevidencewouldseemtofavorrandomgeneticdrifttoaccountfortheprevalenceofPKUinmostcontemporary
humanpopulations.Meanwhile,thedebatecontinues(see"Discussion"inScriveretal.,1996b,p.95),andfascinationforselective
advantage(heterozygousadvantageoroverdominantselection)persists(KrawczakandZschocke,2003Hardelidetal.,2007).
Whenonlyafewinalargesetofdiseasecausingrecessiveallelesaccountforthemajorfractionoftheaggregatefrequency
distribution,selectiveadvantageisamechanismtoconsider(Flintetal.,1993).SelectionofPAHalleles(theobjects)willoccurifthe
processactsonaphenotypethatconfersanadvantageandisencodedbythecorrespondinggene.Selectioncanactindirectlyor
directlyintheformercasethrough"hitchhiking"atacloselylinkedlocusandinthelatterontheprimarylocus.Forexample,the
interferonlocusinregion12q24.1isphysicallylinkedtoPAH,butthereisnoevidencethatitisinvolvedintheselectionofPAH
alleles.SelectionismorelikelytohaveoccurredifitcouldactonaphenotypeencodeddirectlybythePAHlocusinheterozygotes
carryingdiseasecausingPAHalleles.Conventionalwisdompredictsadisadaptiveor,atbest,neutraleffectofthemutantPAHallele
intheheterozygote,butifthereisadisadaptiveeffect,itmustbesmall(Vogel,1985).Evenso,adisadaptiveeffectwillnotincrease
allelefrequency.
Increasecanoccuronlythroughaprocessthatconfersanadvantageinreproductivefitness.InthecaseofPKU,asimple
calculationshowsthatheterozygoteadvantage,intermsofsurvivingoffspring,canbeonly1percent(orless)greaterthanfor
normalhomozygotes,sotheadvantageinfitnessismarginal.Selectiveadvantagewasproposed(Kidd,1987)whenPKUcausing
alleleswerefirstcharacterized(DiLellaetal.,1986b,1987)butwasrejectedsubsequentlybyitsinitialadvocate(seeKiddinScriver
etal.,1996b,p.94).
Theselectionprocessmustactfirstonaphenotype(Sober,1984)thereafter,itcaninfluencefrequencyoftheallele(theobject).
Mightplasmaphenylalaninelevelsinheterozygotesbethedesignatedphenotypeonwhichtheprocessofselectionacts?Theeffect
ofheterozygosityonthisrecessivemetricphenotypeisactuallyverysmall(Goldetal.,1974RosenblattandScriver,1968),anditis
difficulttoimaginehowtheselectionprocesswoulddiscernit.Ifselectionactsatamoreproximallevel(e.g.,onPAHenzymeitself),
itwillhavebeenactingnotonlyonnullproteinphenotypescausedbyprevalentallelessuchasIVS12nt1,IVS10nt11,andR408W
butalsoonavariantkineticphenotypesuchasthatcausedbytheprevalentI65Tallele.Howthiswouldoccurisunknown.
Selectiveadvantageisexpressedultimatelyineithergameticselection,bettersurvivaltoreproductiveage,orhigherratesof
reproductionamongheterozygotes.Thereisevidencebothfor(Saugstad,1977Woolf,1994Woolfetal.,1975)andagainst(Paul
etal.,1979Saugstad,1973)thesealternativesinthecaseofPKU.Suchconflictingevidencemayreflectbiasintheoriginalstudies
becausethefamiliessampledwereascertainedthroughPKUprobands.Correctionshavesincebeenmadeforsuchbias(tenKate,
1978Woolf,1986),buttheambiguitiesremain.
Itmayneverbepossibletoidentifytheagentofselection.Forexample,ochratoxinA,amycotoxiningrainsandlentils,hasbeen
proposedasacandidate(Woolf,1986),butwithoutexperimentalsupportingevidence,itmayneverbepossibletoquantifythe
effectsoftheputativeselectiveprocess.Thereareatleasttworeasons:(1)Sucheffectsmayhaveexistedonlyinthepastandare
nolongeracting,and(2)theeffectsofheterozygoteselectionmaybetoosmalltodetectreliablybecausePKUismuchless
frequentthanothergeneticdisorderswheresuchamechanismhasbeeninvoked(Flintetal.,1993).Thus,ifthemagnitudeofthe
selectiveeffectonPAHallelesisindeedsmall,yetstillgreaterthantheeffectofgeneticdrift,thenitwouldhaveactedonalarge

numberofindividualsoraparticulardemographicstratatoproducetheobservedincidenceofmutantPAHallelesandofPKU
disease,anditmayhaveleftamolecularsignatureintheregionofthelocusunderselection(see"Discussion"inHardelidetal.,
2007).Furthermore,theselectiveadvantagemusthaveexistedinseveraltemperateregionsoftheworldwherepopulationswere
exposedtodifferentclimates,cultures,diets,andinfectiveagents.Ontheoreticalgrounds(ThompsonandNeel,1997),thereisno
needtoinvokeheterozygousadvantagetoexplaintheveryvariablefrequenciesofthesepathogenicrecessivePAHalleles.Yetit
remainsanactivehypothesis,andrefutationsoftheargumentsforgeneticdriftarecombinedwithappealstorecognizethatsome
"pathogenic"alleleshavereachedtruepolymorphicfrequenciesinsomepopulations(KrawczakandZschocke,2003).Geneticdrift
willdoasanexplanation.Sowhynotacceptselectiveadvantage?Itisappealingtousethispointofviewwhenexplainingthe
prevalenceofPKUtothefamilyofanewlydiagnosednewborn:"Themutationwasagoodalleleinhumanhistory."

TheIVS10nt11PAHAllele:AParadigm
ThisPAHmutation,aspliceallelewithsystematicnamec.106611ga(alsoknownbythetrivialnameIVS10nt11orIVS10nt546),
illustratesmanyofthethemesinpopulationgenetics.
1. IVS10nt11inthehomoallelicgenotypeunambiguouslyconfersaPKUphenotypeinvivo(Guldbergetal.,1998Kayaalpetal.,
1997).ItsgeographicdistributionisnonuniformandismostprevalentinsouthernandsoutheasternEurope,particularlyinthe
AnatolianregionofmoderndayTurkey,whereitappearstohaveoriginated(Calietal.,1997).
2. FromobservedfrequenciesinvariousMediterraneanpopulationsandthecorrespondingincidenceofPKU,thehighestrelative
frequencyoftheallele(0.32)isfoundinTurkey(Ozgetal.,1993),wheretheestimatedabsolute(population)frequencyis
approximately0.003(Calietal.,1997Woolf,1994).Endogamyandconsanguinitycontributetothehighprevalenceofthis
mutantgenotypeinthisparticularregion(Woolf,1994).
3. IVS10nt11isembeddedinwhathasbeencalledan"ancestral"haplotype[RFLPhaplotype6,STR252(Goltsovlength),VNTR
7],butthisparticularassociationisnotexclusive.IVS10nt11isalsofoundonseveralRFLPhaplotypes,andinmostcases,this
findingiscompatiblewithintragenicrecombinationorpointmutationatanRFLPsite(seeFig.778).Sincethealleledoesnot
involveaCpGsite,itisunlikelytoberecurrent,andthemajorityofitsmutationhaplotypeassociationsarecompatiblewith
identitybydescent.Haplotypedataindicateatimehorizon(origin)forthemutationatleast5000to10,000yearsago(Caliet
al.,1997).
4. AssociationsbetweenIVS10nt11andhaplotypesonchromosomesfrompatientsinTurkey,Israel,andItaly(Calietal.,1997),
Greece(TraegerSynodinosetal.,1994),andtheIberianPeninsula(Calietal.,1997Prezetal.,1997Riveraetal.,1997,
1998)indicateapostNeolithicdemicexpansionanddiffusion(geneflow)fromtheeasternregionswestwardacrossthe
Mediterraneanbasin.Thesedata,derivedfromDNAanalysisatthePAHlocus,areconcordantwiththegradientsofclassic
polymorphicproteinmarkersinthesameregion(CavalliSforzaetal.,1994CavalliSforzaandPiazza,1993).
5. TheIVS10nt11alleleonhaplotype6isatracerormigration(geneflow)overlandtoTataria(Kuzmanetal.,1995)andofrange
expansionoverseastoNorthAmerica(Carteretal.,1998Guldbergetal.,1996a),SouthAmerica(Perezetal.,1996),and
Australia(Ramusetal.,1995).
6. TheIVS10nt11mutationisfoundonauniquehaplotype[RFLP34,STR230(Zschockelength),VNTR7]inSpanishGypsies
(Desviatetal.,1997),whereitisamarkerforfoundereffectwithdriftinrelativegeneticisolation.

ThePhenylalanineHydroxylatingSystem
Themostimportantsingledeterminantofphenylalaninehomeostasisinhumansisthehydroxylationreaction.Tounderstandthe
mutantphenotypesassociatedwithHPA,thenormalcomponentsofthehydroxylationshouldbeknown.

GeneralCharacteristics
Thephenylalaninehydroxylationreactionisanobligatoryandratelimitingstepinthecatabolicpathwaythatleadstothecomplete
oxidationofphenylalaninetoCO2andwater(MilstienandKaufman,1975).Theketogenic(e.g.,acetoacetate)andgluconeogenic
(e.g.,fumarate)productsofphenylalaninecatabolism(seeFig.772)contributetotheorganismspooloftwocarbonmetabolites
andglucose.Inviewofthebrainspartialdependenceonaperipheralsupplyofglucose,theabilityofphenylalaninetoprovide
gluconeogenicsubstrates,inthiscontext,playsaroleinnormalbraindevelopmentandfunction.Hydroxylationofphenylalanine
playsanotherroleinmammalianmetabolism:Itprovidestheorganismwithanendogenoussupplyofthenonessentialaminoacid
tyrosine.Whenhydroxylationisdeficient,tyrosinebecomesanessentialaminoacid.TheformalnameforPAHenzymeisL
phenylalanine4monooxygenase(EC1.14.16.1),amixedfunctionoxidoreductase.

TissueDistributionofHydroxylatingActivity
Itwasthought,atonetime,thatPAHisnotpresentinnonhepaticmammaliantissues(UdenfriendandCooper,1952).However,
subsequentstudiesdemonstratedappreciableactivityinmousekidneyandpancreas(Tourianetal.,1969)andinratandguinea
pigkidney(Berryetal.,1972).Theputativepresenceofhydroxylaseactivityinpancreashasnotbeenconfirmed(Raoand
Kaufman,1986).TheseauthorsobservedthatratkidneyPAHisinanunusualstateofactivationrelativetotheratliverenzyme,
andattherestingbloodphenylalanineconcentration,kidneyenzymemightaccountforasmuchas20to30percentofthetotal
phenylalaninehydroxylaseactivityinrats.Untilrecently,thestatusofthekidneyhydroxylaseinhumanswasunclear.Aylingand
colleagues(Aylingetal.,1974,1975)reportedhydroxylaseactivityinkidney,whereasothersfoundnoneineitherhumanor
nonhumanprimatekidney(MurthyandBerry,1975).Insupportofthelatterevidence,ratphenylalaninehydroxylasecDNA
hybridizeswithratkidneymRNAbutnotwithbaboonkidneymRNA(Houseetal.,1997).EvidencebasedonanalysisofmRNA,
immunohistochemistry,andassayofenzymicactivityindicatesthathumankidneyexpressessignificantphenylalaninehydroxylase
proteinandenzymeactivity(LichterKoneckietal.,1999Tessarietal.,1999).Thekidneyisnowconsideredtobeanimportantsite
ofconversionofphenylalaninetotyrosineinadulthumans(Molleretal.,2000).
PAHisnotpresentinbrain(Abitaetal.,1974),contrarytoanearlierclaim(Wapniretal.,1971)thefindinghasbearingon
pathogenesisofthebrainphenotypeinPKU.Braindoescontainanotherenzyme,tyrosinehydroxylase,thatcatalyzesthe
conversionofphenylalaninetotyrosineataratecomparabletoitsabilitytohydroxylatetyrosine(Katzetal.,1976).Perhaps
tyrosinehydroxylase,actingonphenylalanine,providesthedevelopingbrainwithasignificantfractionofthetyrosineitneedsfor
proteinsynthesis(Kaufman,1987)whetheritmightinPKUisnotknown.CatalyticpropertiesofthehumanfetalliverPAHappearto
bethesameasthoseoftheadultenzyme(FriedmanandKaufman,1971Raiha,1973).
EvidencehasbeenreportedthatPAHand4carbinolaminedehydratasearepresenttogetherinhumanmelanocytesand
keratinocytes,wheretheyarepostulatedtoplayaroleinmelaninformation(Schallreuteretal.,1994).Intheproposedscenario,
hydroxylationofphenylalanineprovidesthetyrosineessentialformelaninsynthesisfurthermore,thedepigmentationdisorder
vitiligoconstitutesalackofthedehydratasewithaccumulationof7biopterin,whichinhibitsphenylalaninehydroxylase(Davisetal.,
1992),leadingtodisruptionofthesupplyoftyrosineandconsequentimpairmentofmelaninsynthesis.
Althoughthepresenceof4carbinolaminedehydrataseinnormalhumanepidermalkeratinocyteshasbeendemonstrated
unequivocally,theconclusionthatphenylalaninehydroxylaseisalsopresent(Schallreuteretal.,1994)isnotstronglysupported.
Theenzymethathydroxylatesphenylalanineinbothmelanocytesandkeratinocytescouldbetyrosinehydroxylase.Indeed,ifas
postulatedphenylalaninehydroxylasewereessentialformelaninsynthesis,PKUpatientswouldbeexpectedtoshowirreversible
signsofthisdefect.PKUpatientsdotendtohavefairskin,butthisdefectinpigmentationisreversedwhentheplasma
phenylalaninelevelisreduced,afindingconsistentwiththeevidencethatexcessphenylalanineinhibitstyrosinasemediated
melaninsynthesis(MiyamotoandFitzpatrick,1957).
Thephenomenonofillegitimatetranscriptionisrelevanttoadiscussionofthetissuedistributionofphenylalaninehydroxylase.The
termwascoinedtodescribethelowleveloftranscriptionoftissuespecificgenesinnonspecificcells(Schallreuteretal.,1994).
Genetranscriptsofphenylalaninehydroxylase(aswellastyrosinehydroxylase)havebeendetectedinwhitebloodcells,
erythroleukemiccells,andchorionicvilluscells(SarkarandSommer,1989).Todate,thelevelsoftranscriptsdetectedaretoolowto
knowwhethertheiroccurrenceisofanyfunctionalsignificance.Thephenomenonhas,however,provedtobeofvalueinthe
identificationofPKUassociatedmutationsinphenylalaninehydroxylaseincirculatinglymphocytes(Ramusetal.,1992bAbadieet
al.,1993b).

TheHydroxylatingSystem
Thehepaticphenylalaninehydroxylatingsystemconsistsofthreeessentialcomponents:PAH,DHPR,andtheunconjugatedpterin
tetrahydrobiopterin(BH4),i.e.,2amino4hydroxy6[Lerythro1',2'dihydroxypropyl]tetrahydropteridine(Kaufman,1963,1971,
1997)inadditiontoastimulatingprotein(Kaufman,1970)isolatedinpureformfromratliver(Huangetal.,1973),which,although
notessential.,canmarkedlystimulatethehydroxylationreactioninvivo.Originallycalledphenylalaninehydroxylasestimulator
(PHS),thisproteinwaslatershowntobetheaforementioneddehydratase(Lazarusetal.,1983).
ThestructureofBH4isshowninFig.779.Aswithallothernaturallyoccurringcompoundsofthistype,BH4isa2amino4
hydroxypteridine(trivialnamepterin)itisclassifiedasanunconjugatedpterintodistinguishitfromitsrelatives,thefolates.The
lattercompoundsarecalledconjugatedpterinsbecausetheirpterinringsareconjugatedwithaparaaminobenzoylglutamate(s)
substituentatposition6ofthepteridinering.Unlikethefolates,BH4isnotavitaminformammalsbecausetheycansynthesizeit.
Severalsynthetictetrahydropterinswithsimplealkylsubstituentsatposition6,suchas6methyltetrahydropterin(6MPH4)and6,7
dimethyltetrahydropterin(DMPH4),areevenmoreactivethanBH4inthephenylalaninehydroxylatingsystem(Kaufmanand

Levenberg,1992).
FIG.779

Structureoftetrahydrobiopterin[2amino4hydroxy6(Lerythro1,2dihydroxypropyl)tetrahydropteridine],ofBH4,thenatural
coenzyme(cofactor)forphenylalaninehydroxylase.

ThereactionscatalyzedbyPAH,DHPR,andPHS(thedehydratase)inthepresenceofthepterinappearinFig.7710.PAH
catalyzesacoupledreactioninwhichphenylalanineisoxidizedtotyrosineandthetetrahydropterinisoxidizedtothecorresponding
4hydroxytetrahydropterin(alsocalled4carbinolamine).Theconversionofcarbinolaminetoquinonoiddihydropterinandwateris
catalyzedby4carbinolaminedehydratase(Lazarusetal.,1983),formerlyknownasPAHstimulatingprotein.Theoxygeninthe
parapositionofthebenzeneringofthetyrosineproductisderivedfrommolecularoxygenratherthanfromwater(Kaufmanetal.,
1962)accordingly,thehydroxylaseisanoxygenase.Duringthehydroxylationreaction,thesecondatomofoxygenintheoxygen
moleculeisnormallyreducedtothelevelofwater.
FIG.7710

Theconversionofphenylalaninetotyrosineiscatalyzedbythephenylalaninehydroxylationsystem.Theoverallreactionisthesum
ofthreereactions,eachcatalyzedbyaseparateenzyme.Inthepresenceofanactivetetrahydropterincofactorlikethenaturally
occurringsubstanceBH4(R=CHOHCHOHCH3),phenylalaninehydroxylase(PAH)catalyzesacoupledreactioninwhich
phenylalanineisconvertedtotyrosineandBH4toacarbinolamine,thecorresponding4hydroxytetrahydropterin.The
carbinolamineisthenconvertedtothequinonoiddihydropterinbyadehydratase(4carbinolaminedehydratase).Thecycleis
completedbytheactionofdihydropteridinereductase(DHPR),whichcatalyzestheNADHmediatedreductionofthequinonoid
dihydropterinbacktothetetrahydropterin.(Thepterinsubstratesforthethreereactionsareindicatedinitalics.)

Theminimumrequirementsforphenylalaninehydroxylationarethehydroxylase,oxygen,Lphenylalanine,andBH4,butunder
theseconditions,BH4canfunctiononlystoichiometrically(i.e.,theamountoftyrosineformedcannotexceedtheamountofBH4
present).Forthepterincoenzymetofunctioncatalytically,theremustbeanothercomponentofthesystem:DHPR(alongwitha
reducedpyridinenucleotide).AlthoughthereductaseisactivewithbothNADHandNADPH,NADHisthebettersubstrateinvitro
(Craineetal.,1972Neilson,1969ScrimgeourandCheema,1971).ItisnotknownwhetherNADHorNADPHorbothfunctionwith
thereductaseinvivo.InadditiontotheNADHdependentDHPRcatalyzedregenerationofBH4fromquinonoiddihydrobiopterin
(qBH2),reductionofthelattercompoundtoBH4alsocanproceednonenzymaticallyinthepresenceofmillimolarconcentrationsof
reducingagentssuchasmercaptansandascorbate(Kaufman,1959).Attemptstodemonstrateascorbatemediatedregeneration
ofBH4inhumanshavenotbeensuccessful.
Thecompleteabsenceofphenylalaninehydroxylase,DHPR,orBH4leadstopersistentHPA.Bycontrast,becausethereaction
catalyzedbythedehydratasecanoccurquiterapidlybynonenzymaticroutes,itwaspredictedthatthecompleteabsenceofthis

enzymewouldcauseonlymildortransientHPA(Kaufman,1983).Thispredictionwasvalidatedinapatientwiththemildphenotype
whoexcretes7BH4andharborstwomutationsinthegeneforthedehydratase,oneinheritedfromthemother(C82R)andthe
other(E87X)fromthefather(Citronetal.,1993).Theresultsofstudiesoftheactivityofthesetwoengineeredallelesinvitroalso
werecoherentwiththisprediction.TheC82Rmutanthadabout40percentoftheactivityofthewildtypedehydrataseenzyme
(Johnenetal.,1995Kosteretal.,1995),whereastheE87Xallelewasdevoidofactivity,inpartbecauseofitsinstability(Johnenet
al.,1995).Thepredictedeffectsofthesetwomutations,combinedinvivo,wouldmuchreducethedehydrataseactivity.
(DehydratasedeficiencyisfurtherdescribedinChap.78.)
InadditiontoitscatalyticfunctionintheregenerationofBH4(seeFig.7710),thedehydratasealsoplaysacompletelyunrelated
roleinregulatinggenetranscription,astrikingexampleofaphenomenondubbedmolecularopportunism(Doolittle,1988).On
cloningandsequencingthedehydratase,itwasfoundthatthisproteinisidenticaltoDCoH,thedimerizationcofactorofhepatocyte
nuclearfactor1(HNF1)(Citronetal.,1992aHaueretal.,1993).Thedimericformofthelatterproteinregulatestranscriptionofa
largenumberofgenesintheliver,intestine,andkidney,includingthosecodingforalbumin,1antitrypsin,andfibrinogen.DCoH
enhancesthistranscriptionalactivitybycombiningwithHNF1dimersandstabilizingthem(forareview,seeMendelandCrabtree,
1991).MicelackingDCoHareviableandfertilebutdisplayHPA(Bayleetal.,2002).Involvementofthedehydrataseinagene
transcriptionsystemraisedquestionsaboutwhethermutationsthataffectdehydrataseactivityalsoaffectitstranscriptionenhancing
activity.Adetailedanalysisoftheeffectofsomemutationsonboththeseactivitiesshowedthatthosedecreasingdehydratase
activitytolessthan1percentofwildtypedecreasetranscriptionalactivityonlymodestly(byapproximately10percent)andhaveno
effectonbindingDCoHtoHNF1,aclearindicationthatdehydrataseactivityisnotessentialforthebinding(JohnenandKaufman,
1997).Thesameconclusionwasreachedfromastudythatfocusedonthebindingofallelicdehydratase(C82R)toHNF1(Sourdive
etal.,1997).Thesefindingsmayexplainwhydehydratasedeficientpatientsdonotappeartosufferfromtheglobalmetabolic
consequencesthatwouldbeexpectedfromdisruptionoftheactivityoftheDCoH/HNF1transcriptionsystem.
AlthoughtheDNAandaminoacidsequencesofmouse,rat,andhumanphenylalaninehydroxylasesarestrikinglysimilar(Ledleyet
al.,1990),therearenoteworthyphysiologicdifferencesbetweenthehumanenzymeandtherodentenzyme(Kaufman,1997).
Structuralandfunctionalstudiesofthe5'flankingregionofthephenylalaninehydroxylasegenehavedefinedsomeofthefeatures
thatareessentialfortheregulationofthephenylalaninehydroxylasegeneandhaveidentifiedsomeofthecharacteristicsthatmay
accountforthesedifferences(Koneckietal.,1992)(seethesection"ThePAHGene"above).Asshownintransgenicmice,a9kb
DNAfragmentupstreamofthehumangenecontainsallthecisactingelementsneededtodirecttissuespecificanddevelopmental
stagespecificexpressionofphenylalaninehydroxylaseinvivo(Wangetal.,1992a).Aswithothertypicalhousekeepinggenes,
humanPAHgeneusesmultipletranscriptionalinitiationsites(Wangetal.,1994).Thepromoterregionsofthemouseandhuman
phenylalaninehydroxylasegenesaresimilarintheirlackofaTATAlikesequence(Faustetal.,1996).Expressionofthemouse
phenylalaninehydroxylasegenedependsonahormoneresponsiveandtissuespecificenhancerlocated3.5kbupstream,aswell
asonthepresenceofcAMPandsteroidhormonessuchasdexamethasone.Furthermore,activityofthisenhancerwasfoundto
requirethehepatocyteenrichedtranscriptionfactorsHNF1andC/EBP.
FurtherstudiesofthepromoterregioninthemousePAHgene(Pontoglioetal.,1996)revealmultipleDNasehypersensitivesitesin
thenormalanimal,reflectinganopenchromatinstructureandalowdegreeofmethylation,bothcharacteristicoftranscriptionally
activegenes.Bycontrast,thecorrespondingregionofthegeneintheHNF1knockoutmousehasarelativelyclosedchromatin
structureandthehypermethylationpatternofasilentgene.TheseresultssuggestthatHNF1isessentialforchromatinremodeling
andDNAmethylationthataccompanytranscriptionalactivation.ExperimentscarriedoutonHNF1knockoutmicecomplementthe
studiesinvitroandshowthatphenylalaninehydroxylasebelongstothegroupofproteinswhosegenesareindeedregulatedby
HNF1(Pontoglioetal.,1996)thegenecodingforphenylalaninehydroxylaseiscompletelysilentintheknockout.Theseanimals,
however,arenottruemodelsforPKUbecausetheyalsohaveasevereFanconisyndromecausedbyproximalrenaltubule
dysfunctiontheydiearoundthetimeofweaning.
Untilrecently,anyattempttorelatetheresultsobtainedwiththeHNF1knockoutmicetoPKUinhumanshadbeenhamperedbythe
lackofevidencethatHNF1regulatestheexpressionofhumanPAH.Thatgapwasclosedinitiallywiththedemonstrationthatthe9
kbhumanphenylalaninehydroxylase5'flankingfragment(Wangetal.,1994)containstwoHNF1bindingsiteslocated0.5kb
upstream(LeiandKaufman,1998a).CotransfectionexperimentsshowedthatHNF1markedlytransactivatedthe9kbDNA
fragment(linkedtoareportergene)inChinesehamsterovarycells.Moreover,althoughDCoHbyitselflackedthisability,itcould
enhancetheHNF1mediatedtransactivationofthe9kbfragment.TheseeffectsofHNF1andDCoHwereobservedintheabsence
ofaddedhormones,incontrasttothehormonaldependenceoftheeffectofHNF1ontheexpressionofthemousePAHgene
(Faustetal.,1996),anindicationthatregulationofexpressionofhumanPAHbyhormonesdoesnotfollowthesamepatternasthat
forthemouseenzyme.
AstudyofapatientwithHPArevealeda3.7kbdeletioninthe5'flankingregionofthePAHgene.Characterizationofthedeleted

sequenceidentifiedanovelliverspecificDNaseIhypersensitivesitelocated3.3kbupstreamoftheRNAinitiationsiteofthePAH
gene.Transienttransfectionassays,againusingareportergene,demonstratedthataplasmidconstructcontainingthedeletion
mutationwasseverelyimpairedwithrespecttoPAHtranscriptionalactivity(Chenetal.,2002).TheexpressionofPAHwas
suppressedinhepatomaHepG2cellswhentherewasoverexpressionofatruncatedHNF1mutant(Tanizawaetal.,1999).
Dihydrofolatereductase(DHFREC1.6.99.7)alsomayplayaroleinhydroxylationofphenylalanineinvivo.Thisenzymehasawell
establishedroleinonecarbonmetabolism,catalyzingthereductionof7,8dihydrofolate:

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Theenzymealsocancatalyzetheanalogousreactionwith7,8dihydrobiotperin(Kaufman,1967):
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ThesecondreactionassumesimportanceforphenylalaninehydroxylationwhentherateoftheDHPRcatalyzedreductionofqBH2
lagsbehindtherateofthePAHcatalyzedformationofthequinonoidderivative(seeFig.7710).Undersuchconditions,the
extremelyunstableqBHwillundergoarearrangementtothecorresponding7,8dihydrobiopterin(Kaufman,1967)thelatterisnota
substrateforDHPR.Therefore,whenDHPRlimitstherateofphenylalaninehydroxylation,DHFRcouldsalvagesomeofthe
biopterindivertedtothe7,8dihydroderivativeandtherebypotentiallysupportsomephenylalaninehydroxylation.However,since
theneartotalabsenceofDHPRleadstoHPA(seeChap.78),itisevidentthatneitherDHFRnoranyotherenzymeisaseffective
asDHPRatsustainingnormalratesofphenylalaninehydroxylation.
ThereactionsdepictedinFig.7710werethefirsttoestablishametabolicroleforanunconjugatedpterinandtorevealthe
coenzymeroleofBH4.ItwasshownsubsequentlythatBH4andDHPRplaypreciselythesamerolesinthehydroxylatingsystems
forphenylalanine,tyrosine,andtryptophan(BrennemanandKaufman,1964Shimanetal.,1971Friedmanetal.,1972a)(Fig.77
11).Accordingly,BH4andDHPRareessentialforthebiosynthesisoftheneurotransmittersdopamine,norepinephrine,and
serotonin.AfullerrealizationoftheinsituroleofBH4andDHPRintheseotherhydroxylatingsystemsbecameclearwiththe
discoveryofvariantformsofPKUcausedbydefectsinBH4regenerationandsynthesis(seeChap.78).
FIG.7711

Schemetoshowthehydroxylationreactionsforphenylalanine,tyrosine,andtryptophan,eachrequiringBH4cofactorandtherefore
dependentonsynthesisandmaintenanceofBH4.ThepathwayforBH4synthesisisonlysketched(seeChap.78fordetails).
MaintenanceofBH4requiresregenerationfromdihydropteridinereductase(DHPR)inthepresenceofdehydratase(notshown
hereseeFig.7710)andDHPR.EnzymesinvolvedintheMendelianHPAs(shaded)aredisordersof(1)primaryhydroxylase
activity(thischapter),(2)maintenanceofBH4(Chap.78),and(3)BH4synthesis(Chap.78).GTPCH,GTPcyclohydrolase6PTS,
6pyruvoyltetrahydropterinsynthasePAH,TYH,andTRH,phenylalaninehydroxylase,tyrosinehydroxylase,andtryptophan
hydroxylase,respectivelyDHNP,dihydroneopterin.(ReproducedfromScriveretal.,1988.Usedbypermission.)

PhenylalanineHydroxylase
PhysicalProperties
ManyofthepropertiesofPAHwerefirstdeterminedwithenzymepurifiedfromratliverextracts(KaufmanandFisher,1970).The
propertiesofthehumanliverenzymearesimilartothoseoftheratenzyme,withtheexceptionsmentionedbelow.
RatEnzyme
EssentiallypureratliverPAHappearedinitiallytobeamixtureoftwodifferentpolymericforms.Basedonadeterminationoftheir
Stokesradiiandsedimentationconstants,molecularweightsof210,000and100,000werecalculatedforthemajorandminor
species,respectively.Sincethemolecularweightofthesubunit(s)isabout49,000to51,000(KaufmanandFisher,1970),thetwo

formsareputativetetramersanddimers,respectively.Whenassayedwiththesyntheticpterincofactor6MPH4,bothspecieshad
identicalspecificactivitieswhenassayedwithBH4,thespecificactivityofthetetramerwasfivetimesthatofthedimer.
Preincubationofthehydroxylasewithphenylalanineincreasesconversionofdimerstotetramers(Doskelandetal.,1982).
HumanEnzyme
WhenexpressedinE.colicells,theoligomericcompositionofhumanphenylalaninehydroxylaseatpH6.8isamixtureoftetramers
anddimers(80:20parts)ofsubunitsizeofapproximately52kDaandwithnoevidenceofhigheraggregates(Kowlessuretal.,
1996Kaufman1996).Aswiththeratliverenzyme,theproportionofdimerichumanenzymeisaugmentedbyincreasingthepHto
mildlyalkalinevalues(Kappocketal.,1995).PreincubationofhumanphenylalaninehydroxylasewithLphenylalanineshiftsthe
equilibriuminthedirectionofthetetramericform(Martinezetal.,1995).Theabilitytodeterminedistinctivecatalyticpropertiesof
thetwospeciesandtheirreadyseparationduringgelpermeationchromatographyindicatethatthedimerandtetramerarenotin
rapidequilibriumevenunderassayconditions.
HumanphenylalaninehydroxylaseresemblesratliverphenylalaninehydroxylaseinbeingphosphorylatedbycAMPdependent
proteinkinase,phosphorylationresultinginactivation(Kaufman,1983).Recombinanthumanphenylalaninehydroxylase
incorporatesbetween0.6mol(Kowlessuretal.,1996)and0.97mol(Martinezetal.,1995)ofphosphateperphenylalanine
hydroxylasesubunit,resultingin1.5and1.2foldactivation,respectively,oftheBH4dependentactivity.Thismodestdegreeof
humanenzymeactivationislessthanthe2to4foldactivationobservedwitheitherratliver(Abitaetal.,1976Donlonand
Kaufman,1977)orrecombinantratenzyme(Citronetal.,1992b).
Anevenmorestrikingdifferenceinregulatorypropertiesbetweenrecombinanthumanphenylalaninehydroxylaseanditsrat
counterparthasbeenobservedwithotherknownactivatorsofthelatterenzyme.Whereaspreincubationwithphenylalanineor
lysolecithinactivatestheBH4dependentactivityoftheratenzyme8and25fold,respectively,thehumanhydroxylaseisactivated
only2.2foldbyeitherofthesetreatments(Kowlessuretal.,1996).Similarresultswerereportedfortheuncleavedfusionproducts
ofhumanphenylalaninehydroxylasewiththemaltosebindingprotein(Knappskogetal.,1996a).
TheKmforphenylalanine(measuredwithBH4asthecofactor)ofrecombinanthumanphenylalaninehydroxylaseis50M
(Kowlessuretal.,1996),avalueclosetothenormallevelofphenylalanineinhumanplasmaandnotablylowerthantheKmvalue
(280M)fortherecombinantratliverenzyme(Citronetal.,1992b).Thecorrespondingvaluesforthehumanfusionproteinandits
cleavageproductsareclosertothoseoftherecombinantrathydroxylase(Martinezetal.,1995).Mostoftheothercatalytic
propertiesofhumanphenylalaninehydroxylasearesimilartothoseoftheratenzyme(Knappskogetal.,1996aKowlessuretal.,
1996Martinezetal.,1995).
Justaswasfoundwithratliverphenylalaninehydroxylase(Curtiusetal.,1990Davisetal.,1992),7tetrahydrobiopterin(7BH4)
hascofactoractivitywithhumanrecombinantphenylalaninehydroxylase(Kowlessuretal.,1996).Significantly,thispterinisalsoa
potentinhibitorofthehumanenzymewhentestedagainstthelikelyhepaticconcentrationofBH4.Moreover,theinhibitionshows
thesamepeculiarpatternastheonethatwasoriginallyreportedforratliverphenylalaninehydroxylase,i.e.,greaterinhibitionat
higherphenylalanineconcentrations(Davisetal.,1992)with50percentinhibitionby5M7BH4occurringat100M
phenylalanine.Basedontheinhibitionofratphenylalaninehydroxylaseby7BH4,itwasproposedthatthisinhibitioncouldaccount
forthemildHPAobservedinpatientswithsuspecteddeficiencyof4carbinolaminedehydratasethatexcrete7BH4(Davisetal.,
1992).ThedemonstrationthatHPApatientswhoexcrete7BH4areindeeddeficientinthedehydratase(Citronetal.,1993),
togetherwiththeevidencethatthispterinisapotentinhibitorofhumanphenylalaninehydroxylase(Kowlessuretal.,1996),strongly
supportstheproposedmechanismfortheHPAseenindehydratasedeficientpatients.
Humanphenylalaninehydroxylaseisonlymoderatelystimulatedbytheseveralagents(e.g.,phenylalanineandlysolecithin)that
markedlyactivaterathydroxylasethisfindingsuggeststhatthehumanenzymeisinarelativelyactivatedstate(Kowlessuretal.,
1996).Iftheseunusualinvitropropertiesofthehumanenzymeinvitroaccuratelyreflecttheenzymesstateofactivationinvivo,it
wouldmeanthatsomeearliernotionsaboutphenylalaninehomeostasisinhumansthatwerederivedfrompropertiesofratliver
phenylalaninehydroxylasewouldhavetobemodified.Atrestingbloodandhepaticlevelsofphenylalanine,theratenzymehasvery
lowactivity,butitispoisedtobemassivelyactivatedinresponsetoaphysiologicdemand,suchasthatresultingfromeatinga
proteinrichmeal(DonlonandBeirne,1982)orfromglucagonstimulatedphosphorylationactivation(Beirneetal.,1985Donlon
andKaufman,1977).Bycontrast,thepropertiesofrecombinanthumanphenylalaninehydroxylaseindicatethatitsbasalactivityis
relativelyhighevenwithoutphenylalanineorglucagonmediatedactivation.Thishigherbasalactivityofhumanenzymemay
explainwhythemeannormalplasmaphenylalanineconcentrationinadulthumansubjectsis58M(Scriveretal.,1985)andinthe
ratis96M(DelvalleandGreengard,1976).Basedonsitedirectedmutagenesis,thestructuralbasisfortherelativelyactivated
stateofhumanphenylalaninehydroxylasecanbeexplainedonthebasisofacysteineresiduebeingpresentatresidue29in
humanPAH,whereasaserineoccursatthatpositioninratPAH(Wangetal.,2001).

Thehumanphenylalaninehydroxylasesubunitconsistsof452aminoacids(Kwoketal.,1985).Theinitialbreakthroughonthe
structuralbiologyofphenylalaninehydroxylasecamewhenthestructureofthecatalyticdomainofhumanenzymewasdetermined
byxraycrystallography(Erlandsenetal.,1997b)itwasquicklyfollowedbyfurtherinformationincludingtheregulatoryand
tetramerizationdomains(reviewedinErlandsenandStevens,1999FlatmarkandStevens,1999).(SeeSupplement:"Structural
StudiesofPhenylalanineHydroxylaseEnzyme"alsoseehttp://stevens.scripps.edu/pkuweb/tsld001.htm.)Despitetheenforceduse
oftruncatedspeciesofproteintogeneratecrystals,thesefindingshaveprovideduniqueinsightsintothewaytheenzymefunctions
andhowvariousmutationsaffectintegrityandfunction.However,itshouldbeborneinmindthatthepropertiesofthetruncated
speciesoftheenzymedifferfromthoseoftheintactproteininseveralimportantrespects.First,thecrystallizedtruncatedspecies
lackstheNterminalregulatorydomain,andtheNterminusasawholeisimportantformaintainingratPAHintheoptimalcatalytic
conformation(Wangetal.,2001)theformationofanArg13toSer16phosphatesaltbridgeandtheresultingconformational
changeoftheNterminaltailalsocanexplainthehigherstabilitytowardlimitedtryptichydrolysisofthephosphorylatedhuman
enzyme(Mirandaetal.,2002).Second,asoriginallyshownfortheratliverenzyme(FisherandKaufman,1973Iwakietal.,1986),
proteolyticremovalof11kDa(approximately100aminoacidresidues)fromtheNterminusstimulatesbyapproximately30foldthe
BH4dependentactivityoftheproteaseresistantcatalyticdomainthatislocatedtowardthecentralportionofthemolecule.Asa
result,theisolatedremainingspeciescannolongerbeactivatedbypreincubationwithLphenylalanine.Furtherrecentresultsfor
recombinantratenzyme(Horneetal.,2002)supportthemodelwherebyonphenylalaninebinding,themobileNterminalresidues
associatewiththefoldedcoreofPAH,andphosphorylationmayfacilitatethisinteraction.Third,resultsoflimitedproteolysisofrat
hydroxylase(Iwakietal.,1986)showthatremovalofa5kDaportion(approximately45aminoacidresidues)fromtheCterminus
doesnotleadtoactivation,destroystheenzymesabilitytoformtetramers,eliminatesthecooperativebindingofphenylalanine,and
implicatestheCterminusintheformationoftetramersfromtwodimers.Thepropertiesoftheengineeredtruncatedspeciesof
humanphenylalaninehydroxylaseusedinthecrystallizationstudiesarecoherentwiththeseearlierresultsofstudiesoftheratliver
enzyme.Thetruncatedhumanenzymeoccursalmostexclusivelyasdimersanddoesnotshowpositivecooperativityofbindingof
phenylalanine(Knappskogetal.,1996a)thestretchofaminoacidsthatdeterminesthesetwopropertieshadbeennarrowedto25
residuesattheCterminus.Dimerizationofphenylalaninehydroxylasemonomersismediatedbytheinteractionoftwosymmetry
relatedloops(residues414420)locatedclosetotheCterminusofthecrystallizedtruncatedspecies(Erlandsenetal.,1997b).
Withfurtherregardtostructuralandstabilityeffectsofphosphorylation,naturedoesnotuseaspartate(D)orglutamate(E)at
position16butanautoregulatable,phosphorylatableserine.Thenegativechargeonthesidechainatthatresidueowingto
phosphorylationisthekey,analogoustootherphosphoproteins.Asdiscussedearlier,phosphorylationresultsinasaltbridge
betweenArg13andSer16,andfuthersupportforlimitedconformationalchangelocalizedtotheregionofthephosphoserinehas
beenprovided(Mirandaetal.,2004).Thisstudyalsoprovidesanexplanationfortheresistancetotrypticproteolysis(atArg13)in
themodifiedproteinowingtointeractionwithphosphoserine.Allinall,itdoesseemplausiblethatthelocalconformationalchange
inducedbyphosphorylationhasconsequencesfortheturnoveroftheenzymeinvivo,inadditiontoshorttermreversibleactivation
andactivationbyphenylalanine.
Ratliverphenylalaninehydroxylasecontainsoneatomofironpersubunit.Theironisessentialforcatalyticactivity(Parniakand
Kaufman,1981Fisheretal.,1972).TheFe3+atomsitsinacrevice10belowthesurfaceoftheproteinontheflooroftheactive
center.ItiscoordinatedtoresiduesH285andH290andoneoxygenatominresidueE330(ErlandsenandStevens,1999).Through
theuseofsitedirectedmutagenesis,thetwoequivalenthistidineresiduesinratphenylalaninehydroxylasehadbeenshown
previouslytobenecessaryforironbinding(Gibbsetal.,1993).Acloseupoftheactivesitesurroundingtheironalsoshowedthree
watermoleculesligandedtotheiron(ErlandsenandStevens,1999).Theadventofthestructuralinformationdescribedearlier
(ErlandsenandStevens,1999),coupledwithxrayabsorptionspectroscopy(Wasingeretal.,2002)andhybriddensityfunctional
theory(Bassanetal.,2003),hasyieldedinsightsintotheroleofironinthemechanismofactionofPAH.Whenbothsubstrateand
cofactorarebound,waterislostfromtheferrousactivesiteoftheenzyme.Thisopensacoordinationcenterintheiron(II)center
forthereactionwithO2togenerateanactiveintermediateforthedirect,coupledhydroxylationofthecosubstrates.
SomePKUmutationsareassociatedwithresiduesintheregionoftheironattheactivesite,i.e.,T278,E280,P281,andF331.
SinglepointmutationsinthePAHgene,notincludingsilentandmissensemutations,havebeenmappedontotheprotein
(ErlandsenandStevens,1999).Theeffectsofthesemutations,whichcanrangefrommildtosevereHPAphenotypes,nowcanbe
interpretedinthecontextoftheireffectsonthestructure/conformationoftheresultingprotein.Fourcofactorbindingregionshave
beenidentifiedinthevicinityoftheactivesiteiron(ErlandsenandStevens,2001).Someresiduesadjacenttocofactorbinding
motifsarealsoassociatedwithmutationsresultinginrecentlydiscoveredmildorvariantformsofHPAthatareresponsivetoBH4
(Kureetal.,1999).
Morerecently,ithasbeenobserved(Mirandaetal.,2005)thatTyr325inhumanPAHappearstohaveanimportantroleinensuring
stoichiometricbindingofiron,correctgeometryofthecomplexeswithsubstrateandcofactor,andafavorablecouplingefficiencyof
thePAHreaction.ThataminoacidresiduealsoappearstobeimportantforthecorrectcooperativeregulationbyLphenylalanine.

Twomutationstogetheraccountforalmost50percentofPKUpatientsinthenorthernEuropeanpopulationtheyarebothintheC
terminalendofthecatalyticdomain.Thesplicedefectallele(IV12nt1ga)deletesexon12(Marvitetal.,1987)andinterfereswith
tetramerformationwhenexpressedinCOScells,thealleleisanull,mainlyowingtoinstabilityoftheprotein(DiLellaetal.,1987
Watersetal.,2000).Theothercommonmutation,R408W,islocatedatthestartofthetetramerizationhelixreplacementofthe
argininebythelargertryptophanresiduecouldinterferewithproperfoldingofthetetramer.WhenexpressedinCOScells,ithas
lessthan1percentwildtypeactivity(DiLellaetal.,1987Watersetal.,1998),thelevelofimmunoreactivephenylalanine
hydroxylaseiscomparablylow,andthelowactivityresultsfromproteininstability,atleastwhenexpressedinCOScells(Okanoet
al.,1991Watersetal.,1998).

RegulationofPhenylalanineHydroxylase
HumanliverPAHandratliverPAHhavesomesignificantdifferencesintheirregulatedproperties,andthefollowingshouldberead
withthisinmind.SincePAHcatalyzestheratelimitingstepinthemajorpathwaybywhichphenylalanineiscatabolizedtoCO2and
waterandthushasthehighestsensitivitycoefficient(KacserandBurns,1981)formetabolicrunoutofphenylalanine(seeFig.77
2),itisalikelysiteforregulationofphenylalaninehomeostasis(MilstienandKaufman,1975).Theenzymecanplaythisrole
becauseitscatalyticactivityisexquisitelysensitivetochangesinconcentrationsofitssubstrate,phenylalanine.Thissensitivity
ensuresthatexposureoftissuestohighlevelsofphenylalaninewillbekepttoaminimum,anditalsoensuresthatthehydroxylase
catalyzedconversionofphenylalaninetotyrosinewillnotleadtodepletionofphenylalaninetothepointwherenormalprotein
synthesisiscompromised.
Thisdelicatebalanceisaccomplishedbyasynergisticinteractionbetweentwotypesofregulatingmechanisms:activationby
phenylalanineandactivation/deactivationbyphosphorylation/dephosphorylation.Togethertheyaccommodateshorttermregulation
ofPAHactivity.Thesemechanismsenableamoreresponsivecouplingbetweenhydroxylaseactivityandtissuelevelsof
phenylalaninethancouldbeachievedbyanenzymehavingsimpleMichaelisMentenkinetics.TheMichaelisMentenrelationship
describesarectangularhyperbolicresponseintheinitialvelocitytovariationinsubstrateconcentrationitshowsthatactivityofthe
enzymeisgearedtoavailabilityofsubstrate.Whileitmayconstituteanadequateregulatorymechanismatsubstrateconcentrations
atorbelowKmvalues,itisarelativelyinsensitivecouplingdeviceathighersubstrateconcentrations.
RegulationbySubstrateandCofactor
ThefirstevidenceforshorttermregulationofratliverPAHwasthe20to30foldincreaseintheBH4dependentactivityonbrief
exposuretoaphospholipidsuchaslysolecithin(FisherandKaufman,1972,1973)bycontrast,activityoftheenzymeinthe
presenceofDMPH4,asyntheticpterincofactor,wasonlyslightlyincreasedbylysolecithintreatment(FisherandKaufman,1972).
ThesigmoidrelationshipbetweeninitialvelocityandphenylalanineconcentrationinthepresenceofBH4changedtohyperbolicwith
lysolecithin(FisherandKaufman,1972,1973).DiversetreatmentsofPAHsuchaslimitedproteolysis(FisherandKaufman,1973)
andalkylationofasinglesulfhydrylgroup(ParniakandKaufman,1981)alsomarkedlyincreasedtheBH4dependenthydroxylase
activity.InthepresenceofBH4,PAHispredominantlyinalowactivityform,expressingonly3to5percentofitspotentialactivity.
Althoughthereisnoevidencetoindicatethatanyoftheaforementionedmodesofactivationareofphysiologicsignificance,they
delineatesomeofthecharacteristicsoftheactivatedhydroxylase.Activationbysubstrateisprobablyinvolvedinacutephysiologic
regulationofPAH,aprocessdiscoveredfirstwhen6MPH4wasusedtoassaytheenzyme(Nielsen,1969)andthenwhenBH4,the
naturalcofactor,wasused(Kaufman,1970).Thisactivationprocesswasfurtherinvestigatedlater(PhillipsandKaufman,1984
ShimanandGray,1980).InthepresenceofBH4,theresultsofthesestudiescanbeaccommodatedbyasinglemodelthatdepicts
PAHinequilibriumbetweenalowactivityconformationEandanactiveconformationE(PhillipsandKaufman,1984):

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Accordingtothisformulation,theEconformationofPAHcanbestabilizedbythebindingofphenylalaninetoaregulatorysite,
whereastheEconformationcanbestabilizedbythebindingofBH4intheabsenceoforpriortophenylalaninebinding.Incontrast
tothenaturalcoenzymeBH4,syntheticanaloguessuchasDMPH4arenoteffectiveinpushingtheequilibriuminthedirectionofE
hencemoreoftheenzymewouldexistasE.
Theprecedingmodelassumesthatthereisasecondsitedistinctfromthecatalyticsitethatbindsphenylalanine,which,when
occupied,leadstoactivation.PhenylalanineincreasesthebindingofPAHtoahydrophobicmatrix(Shimanetal.,1979),andit
changesthefluorescenceoftheenzyme(Phillipsetal.,1984).Theseobservationssupportthemodelbecausetheyimplythat
phenylalaninechangestheconformationoftheenzyme.Theamountofphenylalanineboundbyratliverhydroxylaseis1.5mol
phenylalanineper1molPAHsubunit(ParniakandKaufman,1981)thisfindingprovidesdirectexperimentalsupportforthe

existenceofasecondphenylalaninebindingsite.
Studiesoftheinactivationofratliverphenylalaninehydroxylasebybombardingitwithhighenergyelectronshaveprovided
additionalstructuraldetailsabouthowthepterincofactorandphenylalanineinteracttoregulatetheactivityoftheenzyme.Fromthe
lossofactivityasafunctionofradiationdose,itwasshownthatthetargetsizeorminimummassnecessaryforthe6MPH4
dependentactivityisthedimer,withradiationcausingphysicaldestructionofonemonomeratatime.Bycontrast,lowdosesof
irradiationactuallyincreasetheBH4dependentactivitythisphaseisfollowedbyadecreaseinactivityathigherdoses,withthe
targetsizeagaincorrespondingtoadimer.TheresultswithBH4supporttheimportantconclusionthatthispterininhibitsor
inactivatestetramersbutnotdimersandthataradiationhitinanypartofthetetramerrelievestheinhibition,resultinginactivation
atlowdosesofradiation(Davisetal.,1996).
Parallelstudiesoftheradiationinducedlossofthephenylalanineactivatedhydroxylaseindicatethatphenylalanineincreasesthe
interactionsbetweenthesubunitsinadimerandweakenstheinteractionsbetweendimersinatetramer.Furthermore,
pretreatmentoftheenzymewithphenylalaninepreventstheincreaseinBH4dependentactivityseenatlowdosesofradiation,
whichappearstorequireatetramericstructure.Phenylalanineactivation,therefore,appearstobedueinparttoitsabilitytoremove
theBH4mediatedinhibitionoftheactivityoftetramers(Davisetal.,1997).
Inrelationtosubstrateactivationandnegativeregulationbythecofactor,bindingparametersandtheconformationalstatesofthe
enzymehavebeenstudiedusingisothermaltitrationcalorimetry(Peyetal.,2004PeyandMartinez,2005)andmolecdulardynamic
simulations(Teigenetal.,2003,2004).ThesestudieshavethrownfurtherlightontheconformationoftheNterminal
autoregulatoryregioninthepresenceandabsenceofphenylalanineandontheinhibitioncausedbyspecificinteractionsofthe
dihydroxypropylsidechainofthecofactor(seeFig.779).
RegulationbyPhosphorylation/DephosphorylationofSubunits
Activityofthehydroxylaseisincreasedseveralfoldbyphosphorylation,areactioncatalyzedbycAMPdependentkinase(Abitaetal.,
1976).ActivationbyphosphorylationisfullyexpressedwhentheenzymeisassayedinthepresenceofBH4butnotinthepresence
ofDMPH4or6MPH4.Activationisaccompaniedbytheincorporationofabout0.70molinorganicphosphatepersubunitMr=
50,000.Becauselessthanstoichiometricamountsofphosphateareincorporatedintothepurehydroxylaseinvitro,itseemslikely
thatPAHisolatedfromratliverisalreadypartiallyphosphorylated,andindeed,fivedifferentpreparationsofthenativeenzymehad
anaverageof0.31mol(range0.230.42mol)phosphateper1molofsubunitMr=50,000.Thephosphorylatedformofhuman
hepaticPAHisrecognizedbyamonoclonalantibody,andthisreagentwasusedtoshowthatbindingofantibodycorrelatesclosely
withthephosphorylationstateofPAHincrudecellextractsfromratliver(Greenetal.,1990).ItalsowasshownthatdibutyrylcAMP
stimulatesphosphorylationofPAHinisolatedratkidneytubules.
Theaminoacidsequenceattheserine[32P]phosphorylationsiteofratliverphenylalaninehydroxylaseisSRK[32P]SNFGQQ
(Wretbornetal.,1980).Theamountofthispeptideisatleasttwicethatcalculatedfromtheradioactivityofthesample,implyingthat
itcontainsasubstantialamountofendogenousphosphate.ThefindingprovidesindependentevidencethathepaticPAH,in
untreatedrats,isamixtureofphosphorylatedandnonphosphorylatedforms.Italsoprovidesanexplanationforanearlierclaim
(Barrangeretal.,1972)thatratPAHexistsasthreeisozymes(designatedpi,kappa,andupsilon).Chemicalanalysisshowedthat
thepreparationcontaineddifferentamountsofproteinboundphosphate,withthepredominantformcorrespondingtothe
monophosphorylatedtetramer(Mr=200,000)andthesecondmostprevalentformcorrespondingtothediphosphorylatedtetramer
(DonlonandKaufman,1980).ThecatalyticpropertiesofthetwoformsofPAHwerefullyconsistentwiththeirstatesof
phosphorylation:RelativePAHactivity(inthepresenceofBH4)washigherforthediphosphorylatedtetramericformthanforthe
monophosphorylatedspecies.Theseresultsindicatethatthemajor,ifnotthesole,structuraldeterminantforelutiontimeofdifferent
formsofPAHonacalciumphosphatecolumn(themethodusedforisolationofthesemultipleforms)istheamountofproteinbound
phosphateinPAH.Phosphoproteinphosphatase2AcatalyzesthedephosphorylationofrathepaticPAH(IngebritsenandCohen,
1983Jedlickietal.,1977).
ActivationofratliverPAHbyphosphorylationhasphysiologicsignificance.GlucagoncausesfourfoldactivationofrathepaticPAHin
vivo(DonlonandKaufman,1978)andinvitro(Abitaetal.,1980).Theeffectisrapidandtransient,itinvolvesphosphorylation(Carr
andPogson,1981DonlonandKaufman,1978GarrisonandWagner,1982),anditcanbeelicitedbyrepeatinjections,implying
thatdecayoftheactivatedstateisnotduetoproteolyticdegradationofthehydroxylase.Theeffectofglucagonisdetectablewhen
PAHisassayedinthepresenceofBH4butnotinthepresenceofsyntheticcofactoranaloguessuchasDMPH4.
Glucagonandinsulinhavebroadlyopposingmetaboliceffectsinvivo.Insulindepletionincreasesinsituphenylalaninehydroxylation
activityinrats(Santanaetal.,1985).Additionofglucagontohepatocytesfromdiabeticratsfurtherstimulatesthiselevatedactivity
(CarrandPogson,1981).TheseresultswereconfirmedinstudiesofPAHactivityinliverextractsfromrats3daysaftertheonsetof
diabetes(DonlonandBeirne,1982)PAHappearedtobemorehighlyphosphorylatedindiabeticlivers.Thediabeticstateincreases

notonlytheBH4dependentPAHactivitybutalsotheDMPH4dependent(and6MPH4dependent)activity.TheincreasedPAH
activityseenindiabeticratsisduenotonlytoanincreaseddegreeofphosphorylationbutalsotoanincreasedamountof
hydroxylaseprotein(Guerinetal.,1968).
StudiesinculturedcellselucidatefurtherthemultipleformsofPAH.NormaladultratlivercontainsdifferentformsofPAHindifferent
statesofphosphorylation(DonlonandKaufman,1977,1978),withdifferentisoelectricpoints(Tourianetal.,1975).Theform
correspondingtohalfphosphorylatedtetramersx,designatedformIII(DonlonandKaufman1977,1980),hasanisoelectricpointof
5.60formIIisthemostandformItheleastprevalent.H4hepatomacellscontainasingleformofthehydroxylase(Millerand
Shiman,1976Tourian,1976)thatissimilarinitsbehaviortothehalfphosphorylatedtetramers.Byimmunochemicalcriteria,the
singlespeciesofPAHinhepatomacellsisdistinctfromthethreeformspresentinnormaladultratliverandthesingleforminrat
kidney(Tourian,1976Tourianetal.,1975).Treatmentofhepatomacellswithhydrocortisoneselectively"induces"theexpression
ofthetwoformsthatarepresentinadultliverbutmissinginhepatomacells(MillerandShiman,1976)i.e.,thepatternafter
hydrocortisonetreatmentofculturedcellsissimilartothatinratliver.Thenotionthathydrocortisoneinducedtheexpressionof
differenthydroxylaseisozymesisnotrelevantbecausethesocalledisozymes(Barrangeretal.,1972)differonlyintheirstatesof
phosphorylation(DonlonandKaufman,1980).Thehydrocortisoneeffectisacomplexoneinvolvingsomekindofposttranslational
modificationoftheenzyme,inadditiontoitseffectontheamountoftheenzymeinthecells.
EffectofLigandsonPAH
PhenylalanineandBH4haveoppositeeffectsontherateofphosphorylationandactivationofpurifiedratliverPAH.Significant
concentrationsofthenaturallyoccurring6RdiastereoisomerofBH4(68M)inhibitphosphorylationandactivationby80percent,
whereas200MLphenylalaninestimulatesbothprocessestoamodestextentphenylalaninecancompletelyovercomethe
inhibitioncausedbyBH4(PhillipsandKaufman,1984).Inhibitionofphosphorylationisquitespecificforthe6Rdiastereoisomerof
BH4relativelylargeconcentrationsof6MPH4orDMPH4donotinhibitit(Doskelandetal.,1984PhillipsandKaufman,1984).The
phosphorylatedPAHrequireslessphenylalaninetobeactivatedthandoesthenonphosphorylatedform(Doskelandetal.,1984
Shimanetal.,1982):29and51Mphenylalaninewererequiredtoobtainhalfmaximalactivationofthephosphorylatedand
nonphosphorylatedforms,respectively.Thesefindingsareagainconsistentwiththenotionthattheenzymeexistsasanequilibrium
mixtureofhighEactivityandlowEactivityconformations.InhibitionofphosphorylationinthepresenceofBH4andstimulationin
thepresenceofphenylalanineimplythattheactiveformofPAH(E)isabettersubstrateforphosphorylationthanthelowactivity
form(E).
ItseemslikelythattheopposingeffectsofBH4andphenylalanineondirectactivationoftheenzymeandonkinasemediated
activationareadominantfeatureofthephysiologicregulationofhepaticPAH.Inthecaseofthekinasereaction,theeffectofBH4
couldbetolimittheextentofphosphorylation,andthusactivation,whenthelevelsofhepaticphenylalanineareverylowandhigh
hydroxylaseactivityisnotrequired.Thiswouldbetrueunderbasalconditions.Higherconcentrationsofphenylalaninethenwould
beabletoovercometheinhibitoryeffectofBH4,allowinganincreaseintheextentofphosphorylationandactivationofthe
hydroxylasewhentheorganismneedshigherhydroxylaseactivitytocatabolizeexcessphenylalanine.ThisinhibitoryeffectofBH4
thenwouldservetoprotectagainstdepletionoftheorganismspoolofphenylalaninebelowessentiallevels.
ActivationofPAHbyphosphorylationandphenylalanineareprobablysynergisticmodesofregulation.Phosphorylation(and
activation)bycAMPdependentproteinkinaseisstimulatedbyphenylalanine(Doskelandetal.,1984PhillipsandKaufman,1984),
whereasphosphorylationsensitizestheenzymetoactivationbyphenylalanine(CarrandPogson,1981Doskelandetal.,1984
Shiman,1980).AusefuladaptiveconsequenceoftheseinterlockingcontrolmechanismsisenhancedresponsivenessofPAH
activitytoalteredlevelsofphenylalanine.
Pancreaticglucagonsecretionisstimulatedbyproteinfeeding(Mulleretal.,1970).Bloodglucagonincreasesinassociationwiththe
postprandialriseinbloodaminoacids,andaminoacidsarepotentstimulatorsofpancreaticglucagonrelease(Guttleretal.,1978
Rochaetal.,1972).SinceglucagonactivateshepaticadenylatecyclasewithanincreaseinhepaticcAMPlevels,itfollowsthat
activationofcAMPdependentproteinkinaseandphosphorylationmediatedactivationofPAHwillattendproteinfeeding.A
consequenceofthisregulatoryresponsetoapostprandialriseofbloodphenylalanineisacceleratedcatabolismoftheaminoacidto
maintainhomeostasis.Sincephenylalanineisalsoaglycogenicaminoacid(seeFig.772),activationofphenylalaninehydroxylase
alsomaybegearedtoincreasedgluconeogenesis.Thislimboftheregulatoryprocesswouldbecoupledtothebloodglucoselevel.
Afallinbloodglucosewouldincreaseglucagonreleaseandsuppressinsulinrelease,resultinginphosphorylationmediated
activationofPAHwitharesultinggluconeogeniceffect.

DihydropteridineReductase
(ThisenzymeanditsroleinBH4metabolismarediscussedinmoredetailinChap.78.)DHPR,anessentialdimericenzymeinthe

hydroxylatingsystemsforphenylalanine,tyrosine,andtryptophan(Fig.7711),iswidelydistributedinanimaltissues(Craineetal.,
1972).Whereasitsoccurrenceinbrainandadrenalmedullaisnotsurprisinginviewofitsroleinthetyrosinehydroxylationsystem
inthesetissuesandinthetryptophanhydroxylationsysteminbrain,whyDHPRshouldbefoundintissuessuchasheartandlung,
whichhavelittleornoaromaticaminoacidhydroxylatingactivity,isobscure.Itswidetissuedistribution,togetherwithBH4,hintsat
undiscoveredrolesforbothBH4andDHPR.Forexample,BH4isthecofactorfornitricoxidesynthase(TayehandMarletta,1989),
animportantenzymeactiveinlung,andrecyclingofBH4mayberequiredthere(seeChap.78).Inadditiontoitsrolein
regeneratingBH4,ithasbeenproposedthatDHPRplaysanancillaryrole(togetherwithdihydrofolatereductase)inbraintokeep
folateinthetetrahydroform(PollockandKaufman,1978).DHPRdeficiencyisacauseofhumanHPA(Kaufmanetal.,1975a),soit
shouldbeconsideredinthediagnosticinvestigationandtreatmentadaptedaccordingly(seeChap.78).
BiosynthesisofBH4
(ThistopicanditsrelevancetoHPAaredescribedinfurtherdetailinChap.78.)ThecofactorfunctionofBH4inthehydroxylation
reactionwitharomaticaminoacidsisrelatedtoitsabilitytoreducemolecularoxygenBH4provideselectronsand,inturn,is
oxidizedtoqBH2(Kaufman,1971,1997).TheconsumptionofBH4isstoichiometricduringhydroxylationofsubstrate.Whereasthe
DHPRcatalyzedreactionregenerates(recycles)BH4fromqBH2momentbymoment,sothecofactorfunctionscatalytically,the
steadystateofBH4ultimatelydependsonbiosynthesisfromprecursors.Itsfundamentalroleincellularbiochemistryand
pathogenesisofdiseaseisalargetopic(Kaufman,1997Thonyetal.,2000WernerFelmayeretal.,2002)itsspecificrelevanceto
thehumanHPAsisthesubjectofChap.78.
Asoutlinedearlier,adominantthemeintheacuteregulationofphenylalaninehydroxylaseisthebalancebetweentheactivating
effectofphenylalanineandthedeactivatingeffectofBH4.Avariationofthisthemealsooperatestoregulatethemammalian
biosynthesisofBH4andexplainshowphenylalanineitselfcontrolsthesynthesisofBH4througharegulatoryproteinthatinteracts
withguanosinetriphosphatecyclohydrolase(GTPCH).AllthreegenesinvolvedinthedenovobiosynthesispathwayyieldingBH4
havebeencloned,sequenced,andmappedtochromosomes.Thereisevidencethatelevatedamountsoftheendproductsof
hydroxylasedependenttryptophanandtyrosinepathwaysdownregulateBH4synthesis.

PhenotypicEffectsofPAHMutations
Comment
AdiseasecausingPAHmutationhasameasuredeffectatthreelevelsofthephenotypiccomponentsthatcomposethe"phenome":
(1)attheenzymelevelonenzymeintegrityandfunction(aproximallevel)and(2)atthenonenzymiclevels(a)on
phenylalaninehomeostasisandthusonitsconcentrationinbodyfluids(theintermediatelevel)and(b)onthebrain,itsfunction,and
cognitivedevelopment(adistallevel).Thelikelihoodthatcategoricalgenotypephenotypecorrelationscanbefoundatall
phenotypelevelsofthisautosomalrecessivetraitisconfoundedbytheextentofallelicheterogeneity,thecomplexityinthebuffering
thatresultsinphenylalaninehomeostasis,andthefactthatthedistalphenotype(IQ)isitselfacomplextrait(ScriverandWaters,
1999).Wediscusstherelationshipsfirstatthemostproximallevel(PAHenzyme),thenatthemostdistallevel(brain),andfinallyat
theintermediatelevel,wherephenylalanineappearstobethemediatorbetweenamutantPAHgenotypeanditsassociationwith
hazardtocognitivedevelopmentandfunction.Abetterunderstandingoftherelatedphenome(FreimerandSabatti,2003)andhow
itcanbeperturbedisnecessarytounderstandfullythediseasecalledPKU.

EffectofPAHMutationonEnzymic(Hydroxylating)Function
TheeffectofPAHmutationsonPAHproteinfunctioncanbeassessedeitherdirectlyorindirectlybythreequitedifferent
approaches:(1)byenzymeassayonatissuebiopsysampleinvitro,(2)byanisotopicmethodinvivo,and(3)bygeneexpression
analysisinvitro.Thefirsttwomethodsmeasuretheeffectofmutantgenotype(ashomoallelicorheteroallelicformsofhomozygosity
orintheheterozygote)onenzymaticfunctioninpatients,whereasthethirdmeasurestheeffectofaparticularalleleplacedina
recombinantgeneconstructonhomopolymericPAH(enzyme)activity.
HepaticEnzymeActivityinBiopsySamples
Severalearlystudiesusingtherelativelycrudeassayofthedayidentifieddeficientenzymaticactivitybydirectassayonliver
samplesfromPKUpatients(Mitomaetal.,1957UdenfriendandBessman,1953Wallaceetal.,1957).Corroborationwithmore
refinedassaysfollowed(Chooetal.,1979Cotton,1977Kaufman,1958).Theseandotherstudiesidentifiedcrossreacting
material(CRM+andCRM)hepaticenzymephenotypesinPKU(BartholomandDresel,1982Chooetal.,1979,1980Yamashita
etal.,1985).Verylowenzymeactivitywithapparentlynormalaffinityforsubstratealsowasobserved(Friedmanetal.,1973,
1972b).ThelargestsinglestudyofhepaticPAHenzymeactivity(Bartholometal.,1975)usedareliableassayonliverbiopsy

materialobtainedfromagroupofverywellmonitoredpatientswithHPApatientswithtypicalPKUhadlessthan1percentnormal
activity,whereasthosewithnonPKUHPAhadmoreenzymaticactivity,usuallymorethan5percentofnormal(Fig.7712).This
particularstudyalsoshowedthatimpairedhydroxylatingactivityandprimarydeficiencyofPAHenzymeintegrityarenot
synonymoussomepatientswhohadHPAinvivoandyethad"intact"phenylalaninehydroxylatingactivityinvitroactuallyhada
primarydisorderofBH4cofactormetabolism(Bartholometal.,1975).AnothervariantofdeficientPAHenzymeactivityowingto
mutationatthePAHlocus,noteworthybecauseitisBH4responsive,hasbeendiscovered.Inthisform,aprimaryPAHgeneallele
altersPAHproteininsomewaythatiscorrectiblewithpharmacologicdosesofBH4invivo(www.pahdb.mcgill.caseethesectionon
BH4responsiveHPAbelow).
FIG.7712

Measuredactivityofphenylalaninehydroxylaseenzyme.Top:Invitroonhepaticneedlebiopsymaterial(left),invivobyisotope
infusion(right).Bottom:Measuredinvivobyingestionofisotopeandanalysisofexpired13CO2.Theinvivodata(top)were
obtainedbyinfusionofheptadeuteratedphenylalaninefollowedbymeasurementoftherateoftyrosinelabelingatsteadystate.The
invivoassayinthebottomfiguremeasurescumulativeexpired13CO2for80minafteringestionofatracerdoseofL[1
13C]phenylalaninepropositiinthisstudywerepersonswithphenylketonuria,variantPKU,andnonPKUHPAandthe
correspondingheterozygotesofknowngenotype(indicatedonabscissa).Thefigureisacompositeofdatafromthreesources
(Bartholometal.,1975Trefzetal.,1981Treacyetal.,1997).

InterindividualvariationinhepaticenzymeactivityinPKUpatients(Bartholometal.,1975)hasbeenattributedlargelytoallelic
heterogeneity(Bartholometal.,1984).TheissueofPAHallelismandpossibleallelicinteraction,usuallyofnegativetype,atthe
polypeptidelevelcametoprominenceinseveralstudies.Forexample,propositiwithnonPKUHPAhadinvitrohepaticenzyme
activityapproximately5percentofnormal,andtheirparentshadactivityapproximately3percentofnormal(mean6subjects),

whichinthelatterisalevelmuchlowerthantheexpectedhalfnormalvalueforheterozygotes(Kaufmanetal.,1975b).Some
obligateheterozygotesforthePKUphenotypeagainhadenzymeactivitywellbelowexpectation(1444percentofnormal)
(Bartholom,1979BartholomandDresel,1982Grimmetal.,1977).Thedeviantgenedoseeffectsinvitroseeninthesesubjects
implynegativecooperativityatthesubunitlevelofPAHenzyme,perhapsadominantnegativemutationeffect.Whetherthisunusual
doseeffectalsooccursinvivoisnotyetknown.Meanwhile,onemustquestionwhyheterozygoteswithlowactivityinvitrowouldnot
haveHPAandwhypatientswithconsiderableactivityinvitrononethelesshaveHPA.OnecouldsaythattheHPAphenotype,aso
calledMendeliantrait,hastheattributesofacomplextraitinvivo(ScriverandWaters,1999)thereismuchyettobelearnedabout
thebufferingprocessesandtheboundaryconditionsforthisoranyothermetabolicphenotype(Scriver,2002Strohman,2002).
IsotopicStudiesinVivo
Phenylalaninehydroxylatingactivitycanbeestimatedbyanindirectassayinvivofollowingintravenousinfusionofisotopically
labeledsubstrate:Therateatwhichlabeledphenylalanineisconvertedtotyrosineismeasuredinplasma(seeFig.7712)(Clarke
andBier,1982Curtiusetal.,1972Lehmannetal.,1983LehmannandHeinrich,1985MilstienandKaufman,1975Trefzetal.,
1978,1981VanSpronsenetal.,1998).Phenylalaninehydroxylatingactivitymeasuredinthiswayisclearlydeficientinthepatient
withaPKUphenotype(Scriver,1998aVanSpronsenetal.,1998).Therewasearlierconflictingevidence(ThompsonandHalliday,
1990)thatnowcanbeexplained.Thediscrepancybetweenearlierandrecentfindingsisthesubjectofaneditorial(Scriver,1998a)
accompanyingapaperbyvanSpronsenandcolleagues(1998).Areader(ScottCDenne,personalcommunication,September11,
1998)oftheeditorialandthearticleexplainedhowatechnicalartifactaccountsforthedifferencebetweenearlierfindingsandthe
newdata.Intheearlierpaper(ThompsonandHalliday,1990),d2tyrosinewasusedtomeasurethetyrosineRa,whereasvan
Spronsenused[13C]labeledtyrosine.Thedifferentmethodshaveconsequencesforthebackgroundshifteffectinthe
measurementsbymassspectrometry.Alargermagnitudeofbackgroundshifteffectwouldaccountforessentiallyalltheapparent
phenylalaninehydroxylationmeasuredintheearlierstudy.Accordingly,theapparentfluxthroughthehydroxylationreaction
reportedintheearlierstudywasanartifactofmethodandmeasurement.Inotherstudies,thefindingsinvivohavebeen
corroboratedbydirectmeasurementsinvitroofhepaticenzymeactivityinbiopsymaterial(Trefzetal.,1981).Theinvivomethod
itselfisfurthervalidatedbyevidenceforregulatedadaptationofPAHenzymeactivityunderphysiologicconditions(ClarkeandBier,
1982).
Phenylalaninehydroxylatingactivity(moreprecisely,theoxidationrate)alsocanbeassayedinvivoasafluxthroughthewhole
pathwaybyanoninvasiveapproachwhere[114C]or[113C]labeledphenylalanine(Lehmannetal.,1986Treacyetal.,1997
Muntauetal.,2002)isingested,andlabeledCO2ismeasuredinexpiredair.Thisapproachhasthevirtueofsimplicity.Itshowsthe
predictedgenedoseeffect(seeFig.7712),itconfirmsbyanindependentapproachthathydroxylatingactivityisdeficientinPKU
patients,anditissensitiveenoughtodistinguishbetweenthetwophenotypeclasses(PKUandnonPKUHPAphenotype)(Treacy
etal.,1997),althoughitisnotsufficientlysensitivetocorrelateaparticularmutantgenotypewithphenotypewithinitsmajorclass.
NonisotopicStudiesinVivo
Anonisotopicmethodthatinvolvesanoralloadofphenylalanine(0.6mmol/kgofbodyweight)andseveralbloodsamplestaken
over72hourshasbeendevisedtoassessPAHactivityinvivo(Guldbergetal.,1995).Patientswithgenotypespredictedtoharbor
severe,intermediate,ormildPAHallelesmanifestcorrespondingPKU,variantPKU,andnonPKUHPAphenotypesintheratesat
whichtheycleartheloadfromplasma.Theobservedrateswereconcordantwiththeclinicalclassificationbasedonpretreatment
plasmaphenylalaninevaluesanddailydietphenylalaninetolerance(Gttler,1980).Themethod,ofcourse,measuresall
parametersofphenylalaninerunoutandnotPAHenzymeactivityalonethatis,itmeasuresbothcomplexandMendelian
phenotypes.
InVitroExpressionAnalysis
YetanotherwaytodemonstratetheeffectofPAHallelesonthecorrespondinghydroxylatingactivityisbyexpressionanalysisin
vitro.ThismethodwasusedearlyinthestudyofPKUallelestoanalyzetheeffectofthehumanR408Wallele(DiLellaetal.,1987).
Anexpressionvector[p91023(B)]containingthemutantPAHcDNA,createdbysitedirectedmutagenesis,andaplasmidconstruct
containingnormalcDNAsequencewereseparatelytransfectedintoculturedmammalian(COS)cellsPAHenzymeactivitywasthen
assayedinthepresenceofsyntheticcofactor(6MPH4).Whenrelatedtowildtypeactivity,theexpressionlevelsforR408Wmutant
enzymeactivityandforPAHimmunoreactiveproteinwerelessthan1percent,whereastheywere100percentformRNA.Whenthe
R408Wallelewasreanalyzedinadifferentexpressionsysteminthepresenceofnaturalcofactor(BH4),theoriginalfindingswere
corroborated(Svenssonetal.,1992).
Inalandmarkstudy(Fig.7713),themolecularbasisofphenotypeheterogeneityinPAHdeficientHPAwasanalyzedforseveral
differentallelesbyexpressionanalysisinvitro,theresultsbeingcorrelatedwiththemetabolicphenotypeinvivo(Okanoetal.,
1991).PAHmutations,suchasR408W,witha"severe"effectonenzymefunctioninvitrowereassociatedwithaseverePKU

phenotypeinvivo,whereasmutationswitha"mild"effectinvitroconferredacorrespondingphenotypeinvivo.
FIG.7713

Relationshipsbetweenpredictedphenylalaninehydroxylaseactivity(byinvitroexpressionanalysis)andbiochemicalphenotypesin
HPApatients.Leastsquaresanalysiswasusedtocalculateregressions,forwhichequationsandcorrelationcoefficientsareshown.
(Topleft)Relationshipbetweenpredictedphenylalaninehydroxylaseactivityand1/pretreatmentserumphenylalaninelevelsin
Danishphenylketonuria(PKU)patients(n=51).(Topright)Relationshipbetweenpredictedphenylalaninehydroxylaseactivityand
1/pretreatmentserumphenylalaninelevelsinGermanPKUpatients(n=44).(Bottomleft)Relationshipbetweenpredicted
phenylalaninehydroxylaseactivityandphenylalaninetoleranceatage5yearsinDanishPKUpatients(n=48).(Bottomright)
Relationshipbetweenpredictedphenylalaninehydroxylaseactivityand1/serumphenylalanine72hoursafteranoralproteinloadat
age6monthsinGermanPKUpatients(n=23).(FromOkanoetal.,1991.Usedbypermission.)

Atthepresenttime,manydifferenthumanPAHmutationshavebeenanalyzedbyexpressionanalysisinvitro(seetableunderthe
heading"IVEHuman"inwww.pahdb.mcgill.ca).Thedatabasealsocontainsinformationon12artificiallycreatedmutationsinthe
humanPAHnucleotidesequence(seetableunder"IVEArtificial")andon41artificiallycreatedmutationsintheratPahgene(see
tableunder"IVERat").(TherearesearchoptionsonthehomepageofPAHdb:"IVEHuman"islistedunderuserdefinedqueries
theothertwotablesareunderprequerieddata.Thereisalinkto"IVECommentary"wheretheuseandinterpretationofthe"IVE
Human"tableisdescribed.)TheadvantagesanddisadvantagesofinvitroexpressionanalysisofthePAHgeneinoneoranotherof
theavailablesystemshavebeendiscussedindetail(Waters,2003Watersetal.,1998).
DatafromexpressionanalysisinvitroprovidetheevidencethatPAHmutationsactuallyalterproteinfunctiontheydocumentthe
severityofthemutationeffectandtheyhelptodescribethemechanismoftheeffect.Mutationsaffectingproteinintegrityand
functionaregroupedunderfourbroadheadings(Flatmarketal.,1997):(1)knockoutornullalleleswithundetectableactivity,(2)
allelesthatspecificallyaffectthecatalyticcenterandimpairVmax,(3)alleleswithakineticeffectalteringaffinity(Km)forsubstrateor
cofactor,and(4)allelesmappingtoregulatory,catalytic,ortetramerizationdomainsthataffectfoldingoftheprotein,thusalteringits
stabilityandtherebyincreasingturnoverandlossoftheprotein.AfifthgrouphasemergedmissenseallelesconferringBH4

responsivenessthatdonotmaptoBH4bindingresidues(seebelow).
AbouttwothirdsofphenotypealteringPAHmutationsarepredictedfromtheDNAsequencetobemissensealleles,wherethe
substitutionofoneaminoacidforanothercouldaffectfoldingandstabilityoftheprotein.NumerousPAHmutationshavebeen
identifiedtohavethislattereffect(Gamezetal.,2000Peyetal.,2003Watersetal.,1999Watersetal.,2000).Arecentstudy
(Peyetal.,2007)confirmsthatdecreasedproteinstabilityassociatedwithmissensemutationsisthemajormolecularpathogenic
mechanisminPKU.PeyandcolleaguesstudiedtheeffectofPAHmutationsbyusingtheproteindesignalgorithmFoldXtopredict
theenergeticimpactonthePAHproteinanditsnativestatestability.Eightyofthemissensemutationsstudiedintheirprojecthad
dataforinvitroexpressionanalysisineukaryoticsystems,allowingcomparisonwiththecorrespondingmetabolicdata.Peyand
colleaguesexamined238otherPAHmutationswiththealgorithm,leadingtotheconclusionthattheresiduesencodedinexons7,
8,and9andintheinterdomainregionsoftheproteinplayimportantstructuralrolesandconstitutehotspotsfordestabilizingthe
enzyme.PeyandcolleaguesfurtherconsideredthemechanismunderyingBH4responsiveness,leadingtothesuggestionthatin
BH4inducedstabilizationofPAHproteinmutantswithmildstabilitydefects,responsetoBH4occurswhenthelattermoleculeacts
asachemical/pharmacologicchaperone,thusinterpretingearlierobservationsonthetherapeuticeffectofBH4onthephenotype
(Muntauetal.,2002Erlandsenetal.,2004).Additionalstudiesusingayeasttwohybridsystemtoanalyzemutationsmappingto
theregulatorydomaindetectedsubtleaberrationsinPAHenzymeoligomerizationsuggestiveofadominantnegativelikeeffecton
interactionsbetweenmutantandwildtypesubunitsinthetetramericenzyme(Watersetal.,2001).
VeryfewdiseasecausingPAHallelesdirectlyaffectkineticbehaviorandspecificactivityoftheenzymeexamplesincludeD143Gin
onestudy(Knappskogetal.,1996b)andY277DandE280Kinanother(Peyetal.,2003).Somemutationshaveacombinedeffect
onproteinfoldingandonbindingofsubstrateorcofactor(Gjettingetal.,2001aLeandroetal.,2000).
ArtificialallelesinthehomologousratPAHgenehavebeenanalyzedbyexpressionanalysisinvitro.Replacementofserine16,a
phosphorylationsite,byanionresidues(E16orD16)activatestheenzymeconstitutively(Kowlessuretal.,1995).TheP281Lallele
increasestheKmvalueforphenylalanine(Quinseyetal.,1996),whereasvarioussubstitutionsoftheE286residuealterpterin
bindingandfunction(Dicksonetal.,1994).RemovaloftheCterminaldomainoftheratenzymesubunitreducesenzymicactivity
(Huftonetal.,1998).Anartificialratmutation(L448A)withthepotentialtodisruptaleucinezipper/coiledcoildomainaffects
assemblyofdimersintotetramers(Huftonetal.,1998).Accordingly,itisofinterestthatthediseasecausinghumanmutation
A447DaffectsaresidueadjacenttoL448(Guldbergetal.,1996a).Furtherstudiesoftheratgeneindicatethatresidues16to26in
theregulatorydomainofthePAHenzymesubunitarecriticalforaregulatoryeffectinvolvinginteractionwiththenaturalBH4
cofactor(Wangetal.,2001).TheratS29Calleleincreasesenzymeactivityfourfold.Thehumanenzymehascysteineatthis
position,andthisdifferencebetweenratandhumanenzymesmightexplainthedifferentregulatorypropertiesofratandhuman
enzyme(Wangetal.,2001).Inthecatalyticdomain,theS249residueappearstofacilitatecriticalinteractionbetweentheamino
groupofphenylalanineandtheenzyme(Jenningsetal.,2000).
"Virtual"MolecularModeling(inSilico)
Thecrystalstructuresofthecatalyticdomainandtheregulatorydomain(Nterminalregion)ofhumanPAHenzyme(residues118
424)havebeenresolvedat2.0andprovidethefirststructuralviewofhowmutationsoccurringinexonsofthegenecouldaffect
proteinintegrity(Erlandsenetal.,1997b,2000ErlandsenandStevens,1999FlatmarkandStevens,1999Fusettietal.,1998).
Thetruncatedenzymecrystallizesasahomotetramer,witheachmonomercontainingacatalyticdomainandatetramerization
domain(Fusettietal.,1998).Thelatterfunctionsasadomainswappingarmthatinteractswiththeothermonomerstoforman
antiparallelcoiledcoil.R408W,themostprevalentofallPKUcausingalleles,affectsthehighlyconservedargininepositiononthe
hingeloopconnectingthetetramerizationarmtothecoreofthemonomerR408alsoformsahydrogenbondwiththemainchain
carbamylofL308,A309,andL311(Fusettietal.,1998).MutationsaffectingthethreelatterresiduesallcausePKU.Replacementof
R408bytryptophanseverelydisruptsalignmentofthearmsreplacementbyglutaminewouldbelessdisruptive,andtheR408Q
alleleisindeedassociatedwithmilderformsofHPAinpatients(Kayaalpetal.,1997)andwithconsiderableenzymeactivityby
expressionanalysisinvitro(Watersetal.,1998).
Asupplementdescribesingreatdetailtheputativemechanismsbywhichmissenseallelesaffectingaminoacidresiduescould
affectPAHproteinintegrityandfunction(seeSupplement:"StructuralStudiesofPhenylalanineHydroxylaseEnzyme,"byErlandsen
andStevensPrisc,pleasecheckspelling)).

EffectsofPAHMutationonNonenzymicPhenotypes
Comment
DespitelimitationsintranslatingdatafromanalysisofPAHallelesinvitrointotheclinicalequivalent,broadcorrelationsdoexist
betweenpredictedPAHenzymeactivityinvivoandeitherthemetabolicphenotype(plasmaphenylalaninelevel,clearanceafter

loading,anddietarytolerance)(Eikenetal.,1996cEisensmithandWoo,1995Guldbergetal.,1995Okanoetal.,1991Svensson
etal.,1993)ortheIQscoreattainedinapatient(DiSilvestreetal.,1991Gttleretal.,1993Ramusetal.,1993Trefzetal.,
1993).Yetproblematicandinterestingdeviationsfromthatpredictedhavebeennoted(Langenbecketal.,1988)intheobserved
phenotypeinuntreatedPKUpatients.
Discordantcorrelationsbetweengenotypeand(clinical)phenotypeshouldnotbeasurprisebecausetheyarereadilyapparentin
patientswith"monogenic"diseases(RomeoandMcKusick,1994Summers,1996),diseasesinwhichphenotypeisaccountableto
morethanpenetrantallelesatamajorlocus(Scriver,2002ScriverandWaters,1999).
Onecanaskeitheroftwoquestionsandexpecttheanswerstobestilllargelyhidden:Canonepredictphenotypefromgenotype?
Canonepredictgenotypefromphenotype?Interpretationsofdiscrepanciestendtofocusonpossiblemodifiersofphenotypein
singlegenedisordersthemodifiersincludeboundaryconditions,stochasticandenvironmentalvariation,andgeneticfactors.
Amongthelattermaybeallelicvariation(inthesamegenesocalledmalleablegenotypes)andpostzygoticmutation.Modifier
genesmayproducetheireffectsbydirectinteractionwiththemajorgene,bymodulatinginteractionsbetweenproteins,byeffects
onalternativepathwaysorparallelsystems,orbymoredistanteffects,allofwhichtendtoshiftourviewfromthesimplehighly
penetrantMendeliantraittothebiologicalrealitythatmostphenotypesareinfactcomplextraits(Nakaietal.,2003Scriverand
Waters,1999Summers,1996).Inbrief,nogeneticdiseaseisuninfluencedbyothergenes(LanderandSchork,1994).Acareful
studyofphenotypesinPKUandnonPKUHPAwillshowthatthesamemutantgenotypeisnotnecessarilyassociatedwithan
identicalphenotypeindifferentindividuals.Forclinicalpurposes,treattheactualphenotype,notthatpredictedfromgenotype
(Scriver,2002).
EffectonIQandBrainFunction
Penrose(1946/1998)knewthatuntreatedsiblingswithPKUcouldattainquitedifferentlevelsofcognitivedevelopment.
DiscrepanciesbetweenaparticularPAHgenotypeandthepredictedeffectonphenylalaninemetabolismandthusontheIQscore
inanindividualuntreatedPKUpatientswere"rediscovered"manyyearslaterandobservedbothwithinandbetweenfamilies(Di
Silvestreetal.,1991Gttleretal.,1993Kochetal.,1997Langenbecketal.,1988Ramusetal.,1993Trefzetal.,1993)(Fig.
7714).SinceIQmustbeoneofthemostcomplexofcomplexhumantraits,itisperhapssurprisingthatanycorrelation,evena
weakone,betweenIQscoreandgenotypeatthesolitaryPAHlocusshouldbefound.Itdoesimplythatthemutantgenotypeisa
principaldeterminantofthemetabolicphenotype(Benitetal.,1999)that,inturn,affectscognitivephenotype.
FIG.7714

IQimpairmentclassifiedassevere,moderate,ormildfromIQscoresinuntreatedphenylketonuria(PKU)adultsisrelatedtomutant
humanphenylalaninehydroxylase(PAH)activityaspredictedfromgenotype.Filledcirclesaredataforsingletons.Opencircleswith
joininglinesaresiblings.Mutationdatafor"sibswithsharedgenotype"areincomplete,andenzymicactivitycouldnotbepredicted.
Nonetheless,inthisseries,theuntreatedPKUsibs,withthesame(unknown)genotype,havedifferentcognitivephenotypes.
(AdaptedfromdatabyRamusetal.,1993)

Thereisbroadconcordance:PAHalleleswitha"severeeffectinvitro"arelikelytocausePKUwithlowIQscoresintheuntreated
state,whereasmildPAHallelesarelikelytocausenonPKUHPAwithhigherIQscores.Acorrelationfurtherexistsbetween
predictedseverityofanallele,themetabolicphenotype,andfindingsbynoninvasivemagneticresonanceimaging(MRI)inbrain
(Walteretal.,1993).Allthisweknow,anditimpliesthatonemediatorofthegenotypiceffectoncognitivedevelopmentandbrain

functionislikelytobethephenylalaninelevelitselfinbodyfluids(Ramusetal.,1993):Thelevel,onaverage,ishigherinPKUthan
innonPKUHPApatients.ButsomethingotherthanthePAHgenotypeanditseffectonthemetabolic(phenylalanine)phenotype
nowcanbetakenintoaccount.
BloodBrainBarrier:ModifierofPhenotype
Noninvasiveinvivo1Hmagneticresonancespectroscopy(MRS)hasbeenused(Weglageetal.,1998a)tomeasurefree
phenylalanineconcentrationsinbraininfouruntreatedadultpatientswithclassicPKU(genotypesunknown),allwithsimilarplasma
phenylalaninevaluesinasimilarrange(12001500M).Ofthefourpatients,twowereretardedandhadsevereabnormalitiesof
brainmyelin,asrevealedbyMRI,whereastwohadnormalIQscoresandminimalmyelinabnormalities.Theformer(lowIQ)pair
hadhighbrainphenylalaninelevels(650and670M),whereasthelatter(normalIQ)pairhadbrainphenylalaninelevelsbelow
detectability.Inasecondstudy(Weglageetal.,1998b)usingsimilaranalytictechniques,thesamegroupevaluatedtwosiblings
withthehomoallelicR408Wgenotype,poortreatmentcompliance,similarhighbloodphenylalaninelevels[yetdifferentWAISRIQ
scores(90and77)],anddifferentdegreesofbrainwhitematterchange(detectedbyMRI).Afterastandardloadtest,thesibling
withthehigherIQandlesssevereMRIsignsaccumulatedlessphenylalanineinbrainandcleareditmorequicklyover40hours
thandidthesiblingwiththemoreseverephenotype.Athirdstudy(Molleretal.,1998)in11typicalPKUpatientsand3untreated
adultPKUpatientswithnormalintelligencecorroboratedthefindingsunderbothstaticandkineticconditions.Theseimportant
observationsarefurthercorroboratedbyindependentobservations(Moatsetal.,1999).
Whetherimpairedfluxofphenylalaninefromplasmaintobrain(onanLtypecarrierforlargeneutralaminoacids)isasignificant
explanationfordiscordancebetweena"brainphenotype"anda"plasmaphenylalaninephenotype"inphenylketonuriahasbecome
atopicfordebate.Whereasitcouldbeseenasavariantadaptivebiologicmechanismtoexplainafavorablediscordance(normal
IQoutcomeinuntreatedclassicPKU),itseemsunlikelythatmodestandputativephysiologicvariationsinthebloodbrainbarrier
(BBB)fluxofphenylalanine(Weglageetal.,2002)playasignificantroleintheinterindividualvariationsinoutcomeofIQvaluesin
treatedPKUpatients(Pietzetal.,2002Ruppetal.,2001).Thereisalsoacautionarytaletotellabouttherigorofthequantitative
measurementsbyMRSofbrainfreephenylalanineconcentrationsandtheassociatedbloodbrainaminoacidratio(Kreis,2000).
Ontheotherhand,therecanbelittledoubtthatphenylalaninehasaccesstothebraincompartmentonamediatedsaturable
membranetransportprocessandthatitsfluxonthisLtypeaminoacidcarriercanbemodifiedinthepresenceofothersubstrates
suchasthebranchedchainaminoacidsthatsharethecarrier(Pietzetal.,1999Kochetal.,2003).
Thefindingsimplythatrareandextremeoutlierinterindividualvariationinbrainphenylalaninetransportisanotherfactortoexplain
interindividualdifferencesinIQscoresinPKU(Weglageetal.,1998a).Phenylalaninetransportfrombloodtocellularorganisa
mediatedandcomplexprocess(seethesection"Pathogenesis:MetabolicPhenotypesandNeurotoxicity"below).Atransport
componentfrombloodacrossthehepaticcellplasmamembranehasbeenshownelsewheretobeasignificantcomponentin
wholebodyphenylalaninehomeostasis(Salteretal.,1986).Transportergenes,likeenzymegenes,aresubjecttoallelicvariation.
Perhaps(polymorphic)allelicvariationina(highKm)brainphenylalaninetransporterisamodifierofPKUcausingPAHgenotypes.
TheNMRobservationsinvivoarepreliminary,buttheirimportanceisobvious,andtheymustbefurtherconfirmed.
EffectonPhenylalanineHomeostasis
OneoftheearlyanalysesofcorrelationsbetweenmentalandbiochemicalphenotypesinuntreatedPKU(Langenbecketal.,1988)
suggestedthatmodifiersmustbeinvolvedtoexplainadiscordantgenotypephenotyperelationship.Treacyandcolleagues(1996)
haveshown,byisotopetraceranalysisintwosiblingswithPKUofknownmutantPAHgenotype(R408W/I65T),thatsignificant
differencesinmetabolicrunoutofphenylalanine,toformtheminormetabolites(pathway3BinFig.772),haveamodifiereffectthat
accountsforconsistentandsignificantdifferencesindietaryphenylalaninetolerancesandinthemetabolicphenotypesofthe
siblings.
Inanotherstudy(Treacyetal.,1997),phenylalaninehydroxylationratesinvivowereanalyzedbymeasuringthefluxofingested
[13C]labeledphenylalanineappearingasexpired13CO2.ThestudywasdoneinpatientswithdifferentclinicalformsofHPA.There
werebroadcorrelationsbetweenmutantgenotypes(ofpredictedsevere,intermediate,ormildeffect)andthecorresponding
metabolicphenotypehowever,therealsowasdiscordanceinthecorrelationsamongindividuals.Thisledtotheconclusionthat
whereasphenylalanineoxidationinvivoislargelyunderthecontrolofthePAHlocusthisbeingevidentinthegenedoseeffecton
phenylalanineoxidationincontrols,obligateheterozygotes,andpropositi(Treacyetal.,1997)theindividualized"homeostasis
value"cannotbeexplainedsolelybyhepatichydroxylatingactivity.Accordingly,asexpectedofanimportantbiochemicaland
physiologicprocess,wholebodyphenylalaninehomeostasishasfeaturesofacomplexquantitativetrait(Treacyetal.,1997)itis
anemergentproperty(Mayr,1982)inwhichthewholeismorethanthesumofitsparts,probablyreflectingthescalefreenetworks
thatcharacterizetheintricatehubsandlinksofthe"metabolome"(OltvaiandBarabasi,2002).

PAHGeneMolecularComplexitiesthatInfluenceMetabolicPhenotype
DiscordancebetweenpredictedPAHenzymeactivityandphenylalanineoxidationratesinvivomayreflectinteractionsbetween
differentspeciesofmutantPAHsubunits,andayeasttwohybridsysteminvitroforexpressionofPAHalleles(Watersetal.,2001)
beginstorevealhowmutantheteroallelicPAHgenotypescouldaffecttheenzymeandthusthemetabolismofphenylalanine.Most
PKUpatientsinoutbredpopulationsarenothomoallelicinmutantgenotypeaboutthreequartersofPKUpropositiareheteroallelic
indiversepopulations,andthecorrespondingvalues(j)forhomozygosityareaccordinglylow(Carteretal.,1998Guldbergetal.,
1996a).
Interindividualphenotypicvariationwithinafamilyissometimesexplainedbyanunusualdegreeofallelicvariationwithinthefamily.
FamiliesareknowninwhichthreedifferentPAHalleles,insteadoftwo,segregatetocausedifferentintrafamilialformsofHPA
(Avigadetal.,1991Guldbergetal.,1996cLedleyetal.,1986aTyfieldetal.,1990).Molecularevidenceoftwomutantalleles
occurringincisonaPAHhaplotypehavebeenreported(AulehlaScholzandHeilbronner,2003Gjettingetal.,2001b)(see
summarylistofcismutationsatwww.pahdb.mcgill.caforfurtherdata).
Metaanalysesofgenotypephenotypecorrelationshavenowbeendoneondatafrommorethan1000patientsexpressingmore
than100differentpathogenicPAHalleles(Desviatetal.,1999Guldbergetal.,1998Kayaalpetal.,1997).Analysiswasrestricted
toindividualsinwhomthegenotypewaseitherhomoallelicorfunctionallyhemizygous,whereamissensealleleispairedwithanull
(Guldbergetal.,1995).Severalalleles,whoseeffectswerenotexplainedbygenotypicorphenotypicmisclassification,were
associatedwithmorethanonemetabolicandclinicalphenotype.Thisflexiblediscordancebetweengenotypeandphenotypeawaits
afullexplanation.Alltheallelesinthiscategoryaremissense(e.g.,L48S,I65T,R158Q,R261Q,andY414C).
Thesestudiesyieldthefollowingconclusions:(1)PAHgenotypeclassificationisbroadlypredictiveofmetabolic(andclinical)
phenotypeandthereforeusefulforcounselingandtreatment,and(2)asmallsubsetofambiguitieshighlightsthefactthatHPA
owingtoPAHenzymedeficiencyinvolvesmorethansimplepredictablePAHalleleexpression(i.e.,asinglegeneeffect).
ThethemeofvariationinthehomozygousphenotypeforasocalledrecessivediseasehasbeenexaminedbyWeissandBuchanan
(2003).WhereashistoricallytheMendelianhomozygousstatewasassumedtobehomoallelic,itisnowapparentthatonemustgo
beyondtheconventionaltwoalleleclassification(mutant,wildtype)toaccommodatetheheteroallelicstateofthemutant
homozygousphenotype.Asrevealedinthemetaanalysesmentionedearlier(Guldbergetal.,1998Kayaalpetal.,1997),thereisa
quasicontinuousgenotypephenotypedistributionreflectingwhatis"phenogeneticequivalence"(WeissandBuchanan,2003)
amongthevastnumberofdiploidgenotypesthatcouldresultfromcoinheritanceofanypairingamongthehundredsofmutantPAH
alleles.Table776indicateshowthatphenotypicvariationcouldreflecttheinheritanceofagenotypewithaneffectpredictedfrom
expressionanalysisoftheindividualallelesinvitro.Onceagain,considerationofthephenogeneticrelationshipbringsonebackto
themaxim"treatthephenotyperatherthanthegenotype"(Scriver,2002).
Table776:PAHGenotypePhenotypeRelationships:Observedversus.Expected
Phenotypesin184IndividualswithPAHDeficiency
ViewLarge|SaveTable
Table776:PAHGenotypePhenotypeRelationships:Observedversus.Expected
Phenotypesin184IndividualswithPAHDeficiency

ObservedPhenotypePKU
Genotype
Score*

Expected
Phenotype

Classic

Moderate

Mild

MHP

Total

Obs
=
Exp,
%

ClassicPKU

42

11

57

73.7

Moderate
PKU

10

13

29

44.8

Moderate/mild
PKU

13

92.3

5and6

MildPKU

23

29

79.3

MildPKU/MH

10c

10

20

100.0

916

MHP

35

36

97.2

54

33

50

47

184

Total

*Sumofestimatedeffectsofindividualalleles,where1=classicPKU,2=moderate
PKU,4=mildPKU,and8=mildHPA(MHP)symptoms.
Determined,foreachpatient,onthebasisofthesumoftheobservedallelesofthe
twoPAHmutations.
Groupsinwhichobservedmatchedtheexpectedphenotype(Guldbergetal.,
1996a).
FromWeissandBuchanan,2003.

PAHAllelesConferringaBH4ResponsivePhenotype
BH4isacatalyticcofactorforPAHhydroxylatingactivity.MutationsathumanlociencodingenzymesforBH4synthesisandrecycling
invivo(seeChap.78)bothimpairPAHenzymefunctionandconferadependencyfortherapeuticreplacementofBH4.However,
mutationsalsoexistatthePAHlocusitselftheseconferavariantenzymicandmetabolicphenotyperesponsivetoBH4(in
pharmacologicdosesupto20mg/kgofbodyweightperday)intheabsenceofanyimpairmentofBH4homeostasis.Sinceitsinitial
description(Kureetal.,1999),theconditionhasbecomewidelyrecognized(seelistofcasesinSpaapenandRubioGozalbo,
2003).Itssignificancehasproducedmolecularconsiderationsabouttheinteractionbetweenthe6RBH4isomerandthesurfaceof
thePAHprotein(ErlandsenandStevens,2001).Detailedinvestigationsofphenylalanineclearanceratesfromplasmaandthe
simultaneousratesofphenylalanineoxidationtoCO2invivohavebeeninvestigatedinthisclassofBH4responsiveHPA(Muntauet
al.,2002).BH4increasesphenylalanineoxidationintheresponsivepatient.Themajorityofresponsivepatientsexpressamissense
PAHallelethatcanbeinanyregionofthePAHprotein.Ithasbeenproposedthatperhaps60percentofpatientswithHPAand
phenylalaninevalueslessthan800mol/literareBH4responsive(Blau,2003).Thetetrahydropterinhomepage(www.BH4.org)
listsrelevantclinicalreportsthecorrespondingPAHallelesarelistedthereandinwww.pahdb.mcgill.ca.Themechanismofthe6R
BH4effectislikelytobespecifictothePAHalleleanditseffectontheproteinabeneficialchaperonelikeeffectonthefolding,
assembly,and/orthermodynamicstabilityofthemutantPAHproteintoslowitsintracellulardegradationisahypothesisawaitingthe
test(Waters,2003).Nonetheless,theBH4responsivephenotypeshowstheclinicalrelevanceofPAHmutationanalysisandof
performingaBH4loadingtestinallpatientswithpersistentpostnatalHPA.

MutationEffectsinHeterozygotes:SignificanceforClassification
IdentificationofaheterozygotecarryingadiseasecausingPAHallelehaslongbeenachallenge(seeMcDonaldandCharlton,
1997)becausethephenotypiceffectofasinglemutantPAHalleleonphenylalaninehomeostasisishighlybufferedandthus
"recessive,"asbothpredicted(KacserandBurns,1973,1981)andobserved(Fig.7715).Nonetheless,discerningwhether
predictedseverityofaPAHalleleisreflectedinacorrespondingmetabolicphenotypecontinuestoattractattention.Forexample,a
subject(Scriver,2002)identifiedbynewbornscreeninghadpersistentnonPKUHPA(<325M)studieslaterinliferevealed
euphenylalaninemiaandmolecular"heterozygosity"byDNAanalysis(c.9706GT/+)anotherallelewasnotrecognized,andthe
unusualcircumstanceofheterozygositywithneonatalHPAwasdeclared.Therecouldbeanotherexplanation:heteroallelicmutant
genotypeonenullalleleandonevery"mild"alleleunidentified.
FIG.7715

Astylizedsummaryofevidencefor(1)therecessivenatureoftheplasmaaminoacidphenotypeinthephenylalaninehydroxylase
(PAH)deficientHPAs(plasmaphenylalaninevalues,rightordinate),and(2)genedosageeffectontherateofhydroxylase
dependentoxidationofphenylalanineinvivofrom13CO2inexpiredair(leftordinate).OriginaldatafromRosenblattandScriverand,
1968andTreacyetal.,1997.(AdaptedfromScriver(1998a)usedbypermissionoftheAmericanSocietyforClinicalInvestigation.)

Severalstudieshaveexaminedthemetabolicphenotypeinobligateheterozygotes,attemptingtofindagenotypephenotype
correlation.Overnightfastingfollowedbyanoralphenylalanineload(100mg/kg)segregatedheterozygotesasagroupwithvarying
degreesofefficiency(Spadaetal.,1998)accordingtothefastingplasmaphenylalaninevalue,thepostloadplasmaclearanceof
phenylalanine,andthepostloadriseintyrosinetherewasnocorrelationinanyofthemetricparameterswiththepredictedseverity
ofthePAHallele.
Inanearlierstudy,adiscriminatefunctionofplasmaaminoacidvaluesforphenylalanineandtyrosine(thedependentvariable),
relatedtoinvitroexpressionactivityofthePAHallele(theindependentvariable),didyieldasignificantcorrelation(r=0.40n=140
p<.001)(Svenssonetal.,1994).Inanotherstudy(Treacyetal.,1997),ameasureinvivoofhydroxylatingactivityusing13CO2
excretionafteringestionoflabeledphenylalaninerevealedagenedoseeffectinheterozygotesasagroup(seeFig.7712andFig.
7715)however,considerableinterindividualvariationwasfoundamongdifferentheterozygotesexpressingthesamemutantallele
(seeFig.7712).Thelatterfindingmakesitnoeasierthanitwastoclassifyaheterozygotebythisnewmeasureofphenotype.
Heterozygosityisstillclassifiedjustaswellbyasimpleplasmaaminoacidalgorithm(RosenblattandScriver,1968Treacyetal.,
1997)andbestbyDNAanalysis.

Pathogenesis:MetabolicPhenotypesandNeurotoxicity
Whereastheenzymedeficiencyisahepaticphenotype,themajorclinicaleffectofHPAinthePKUphenotypeisonbrain
developmentandfunction.Thusthevariantmetabolicphenotypemustbeatleastanecessary,ifnotperhapsasufficient,
explanationfortheneurotoxicity.HereinwediscussmetabolicphenotypesassociatedwithmutationatthePAHlocusaffecting
integrityofthePAHenzymeandhow,throughthiseffectonthemetabolicphenotype,mutationultimatelymayaffectthebrain.

PhenylalanineandNeurotoxicity
PhenylalanineisincreasedseveralfoldinbraininmentallyretardedindividualswithPKU(McKeanandPeterson,1970)andis
probablythechiefvillaininneurotoxicity(Knox,1960).Derivativesofphenylalanine(seeFig.772)arenotpresentatsufficient
concentrationstobetoxicinPKU(Kaufman,1989)theirconcentrations,incerebrospinalfluid(CSF),forexample(Antoshechkinet
al.,1991),bearnorelationtothoseusedtoshowtoxiceffectsinvitroorinanimalexperiments(Scriveretal.,1989),andarecent
studyinthemutantmouseorthologueofPKU(Sarkissianetal.,2000a)deniestherelevanceofmetabolitesotherthan
phenylalanineitself.Thereportofnormalintelligenceintwoeuphenylalaninemicsisterswhoexcretedlargeamountsof
phenylalaninemetabolitesprovidesfurtherevidencethattheabnormalamountsofthesemetabolitesarenotthecauseof
neurotoxicityinPKU(Wadmanetal.,1975).
InductionoftemporaryHPAintreatedpatientswithPKUprovokesacutemeasurableimpairmentofhigherintegrativebrainfunction
(Huijbregtsetal.,2002Krauseetal.,1985)andabnormalelectroencephalographictracings(Epsteinetal.,1989Krauseetal.,
1986).Undertheseconditions,urinedopamineexcretion(Krauseetal.,1985)andplasmaLdopalevels(Krauseetal.,1986)
correlateinverselywithplasmaphenylalanineanddirectlyinthemeasuresofbraindysfunction.Theseeffectsonbraindysfunction
appearwhenplasmaphenylalanineexceeds1300M(Krauseetal.,1985),asignificantvaluethatcorrelateswithconcentrations
thataltertransportanddistributionofphenylalanineinthebrain(seebelow).Whereassuchmeasurementssaylittleaboutthe
profileofbrainmetabolites,direct(Sarkissianetal.,2000a)andindirect(Avisonetal.,1990)evidencefromNMRstudiesinanimal
modelsandinpatientswithPKU(seebelow),fromanalysisofCSFmetabolites(Antoshechkinetal.,1991Bachetal.,1991
Lukkelundetal.,1988)andfrommiscellaneousstudies(Louetal.,1987Smith,1985),indicatesthathighplasmaphenylalanine
levelsindeeddohaveaneffectonbrainchemistryandthusonbrainfunction.
Thethresholdvalueforplasmaphenylalanineforitsacuteneurotoxiceffect(1300M)doesnotnecessarilycorrespondwiththe

valueassociatedwithchronicneurotoxicityinPKU.Evidenceforalowervalueinthelattercaseisapparentfirstinbrainwhite
matter,wherethechangesvisiblebyMRIcanbefoundintreatedpatientswithchronicHPAwhoselevelsarelessthan600M
(Bicketal.,1991)andsecondintreatedpatientswhoseIQscoresaredistributedbelowthenormalrangedespitelongterm
maintenanceofplasmaphenylalaninevaluesoflessthan300M(Micheletal.,1990).Ontheotherhand,inuntreatednonPKU
HPA,whereplasmaphenylalaninelevelsarelessthan600M,theremaybenosignificantabnormalities(Weglageetal.,2001).
Thesefindingshaveimplicationsfortreatmentbecausetheysuggestthattheputativethresholdlevelforplasmaphenylalanineis
differentforacuteandchroniceffectsonthebrain.Theyalsohintthatsomethingaboutthephenylalaninerestricteddietmayyetbe
lessthantotallyadequateinpreventingtheneurotoxicity.Finally,theyindicatethatifsevereHPArecursinlaterlifeforwhatever
reason,areversibleacuteneurotoxicitywillappearfirstifthisdegreeofHPApersists,irreversiblechronicneurotoxicitycouldbea
consequence.

HowmightDeviantPhenylalanineMetabolismbeNeurotoxic?
Pathogenesiscanbeconsideredfromthreeviewpoints:(1)aputativedeficiencyoftyrosineinthebrain,(2)theeffectofincreased
phenylalanineontransportanddistributionofmetabolitesinthebrain,and(3)aneffectonneurochemicalprocesses.Nosingle
effectbyitselfseemssufficienttoexplainthePKUbrainphenotype,buthowevercomplextheprocess,theultimateeffectwillbea
disturbanceofnormalchemicalhomeostasisinthebrain.
TyrosineDeficiency
CompletedeficiencyofPAHenzymeactivitypromotestyrosinetothestatusofanessentialdietaryaminoacidfromwhichalineof
reasoningcalledthejustificationhypothesis(Bessmanetal.,1978)proposesjeopardyforthefetuswithPKUandtheninpostnatal
lifefortworeasons:(1)Theaffectedfetus/newborncannotobtaintyrosinefromitsownsupplyofphenylalanine,and(2)the
maternalsupplyoftyrosinetothefetusiscompromisedbymaternalheterozygosity.However,atleastfivelinesofevidencerefute
thishypothesis:(1)Postnataltyrosinesupplementationalonewithoutreductionofphenylalanineintakedoesnotpreventmental
retardationinPKU(Batshawetal.,1981),(2)postnatalphenylalaninerestrictionbyitselfshouldnotbebeneficial,yetitis,andit
appearslargelytopreventneurotoxicity(seethesection"TreatmentofPKU"below),(3)thereisnoconsistentorpathologic
reductioninplasmatyrosinecontentinuntreatedpatientswithPKU(KoeppandHeld,1977),(4)thereisnoevidenceofsignificant
tyrosinedeficiencyincordbloodsamplesobtainedfromnewbornswithPKU(Scriveretal.,1980),and(5)tyrosinesupplements
duringtreatmentofPKUsufficienttoincreaseplasmatyrosinelevelsdonotimproveneurophysiologicparametersor
neuropsychologicalfunctions(Pietzetal.,1995aSmithetal.,1998).
Althoughthefindingspertainonlytoextracellulartyrosine,thecorrespondingintracellularvaluesmaybehigherthanthe
extracellularvaluesinPKUbecausethemechanismsbywhichtyrosinemightbereducedinphenylketonuricplasmaleadto
intracellularaccumulation(Christensen,1986,1987)andbecausenotonlyislymphocytetyrosineincreasedinpersonswithPKUas
wellasinheterozygotes(Thalhammeretal.,1980,1982),butbraintyrosineisalsoincreasedinPKU(McKeanandPeterson,
1970).
EffectonTransportProcessesandMetabolicDistributionintheBrain
TransportofphenylalanineacrosstheBBBhasbeenmeasuredinvitroinahumancapillarypreparation(ChoiandPardridge,1986
HargreavesandPartridge,1988).Ahighaffinitysystem(Km20M)operatesonthebloodsideofthecapillaryendothelium,
whereasaveryhighaffinitysystem(Km0.250.30M)operatesonthebrainside.Thisarrangementkeepsthephenylalanine
concentrationintheinterstitialfluidlowandstableduringthediurnalfluctuationsinitsplasmalevel(Maheretal.,1984Svenssonet
al.,1991).
AminoaciduptakeacrosstheBBBhasbeenmeasurednoninvasivelyinvivobypositronemissiontomography(PET)using11C
labeledsubstrate(aminocyclohexanecarboxylate)(Koeppeetal.,1990).Theseandotherstudiesinvivo(Mommaetal.,1987)
showthataffinityforphenylalanineuptakeisthesamebothinvivoandinvitro(ChoiandPardridge,1986).Thestudiesinvivo
(Mommaetal.,1987)alsoshowcompetitionontheBBBtransportsystembetweenphenylalanineandotherlargeneutralamino
acids(LNAAs),suchasbranchedchainaminoacids,methionine,tyrosine,andtryptophan.Phenylalaninehasthehighestaffinityfor
thesystem.Consequently,elevatedconcentrationsofphenylalaninecouldimpairuptakeofotherLNAAsintothebrain,andthe
availabilityforthebrainoftheseaminoacidsfrombloodispredictedtobeimpairedbyevenmodestsupraphysiologic
concentrationsofphenylalanineintherangeof200to500M(Pardridge,1998).Inhibitionhadbeendemonstratedinvivoin
patientswithPKUandfitsthepredictionclosely(Koeppeetal.,1990).
ThetransporterresponsibleforLNAAbrainuptakehasbeenpartiallycharacterized.Itisasodiumindependentsystemoriginally
designatedLATbyChristensenandcolleagues(1994)andnowdesignatedLAT1(Boado,2002),anditappearstobea

heterodimerofthe4F2hcheavychainandLAT1lightchain(Kanaietal.,1998).BothcDNAshavebeenisolatedfromBBBcDNA
libraries(Boadoetal.,1999Boado,2002).TheLAT1transcriptenhancedtryptophantransportintofrogoocytescoinjectedwiththe
4F2hctranscript,asubstratetransportinhibitedbyotherLNAAs,andwashighlyexpressedinbraincapillariesandnotdetectedin
liver,heart,lung,orkidney,indicatingselectiveexpressionattheBBB(Boadoetal.,1999Boado,2002).
BrainuptakeandcontentofphenylalanineinvivoinpatientswithPKUhavebeenmeasuredbyothermethods.Theintravenous
doubleindicatortechniqueestimatesinvivotransportofphenylalanineandotheraminoacidsacrosstheBBB(Knudsen1994
Knudsenetal.,1994)andhasshownsaturationinthehyperphenylalaninemicstatewithinhibitionofleucinetransport(Knudsenet
al.,1995).MRSmeasuresfreephenylalaninecontentanddistributioninbrain(Molleretal.,1995,1997Novotnyetal.,1995Pietz
etal.,1995b).SubjectswithPKUhaveelevatedphenylalaninelevelsandshowsaturationoftheseparatetransportprocessesfrom
plasmatointerstitialfluidandfromextracellularspaceintobraincells(Molleretal.,1997).
TheimportantstudiesusingMRStomeasurefreephenylalanineinthebrain(Molleretal.,1998Pardridge1998Weglageetal.,
1998aseethesection"Effect[ofPAHalleles]onIQandBrainFunction"above)implythatinterindividualvariationexistsinthe
transportofphenylalanineintobraincellsandthatconcentrationsoffreephenylalanineinbraintissueareoneoftheultimate
determinantsofbrainphenotypeinPKU.InterindividualvariationinMRSamongpatientswithPKUhasbeensubstantial(Moatset
al.,2000Weglageetal.,2002),evenbetweensiblingswhohavesimilarbloodphenylalaninelevelsandanidenticalPAHgenotype
(Weglageetal.,2002).Thisvariationisprobablynotartifactualandmayexplainthevariationincognitiveabilityobservedin
untreatedPKUinpatientsandtheirsiblingswhohavesimilarmutantPAHphenotypes(Ramusetal.,1993Weglageetal.,2002).
Competitionforuptakebetweenaminoacidsoccursoncarriersinothermembranesinthebrain.Phenylalanineinhibitstransportof
tyrosine(Aragonetal.,1982)andtryptophan(Herreroetal.,1983)insynaptosomalplasmamembranevesicles.Hencethe
combinedeffectsoftyrosinesequestrationinsomaticcellsandinhibitionofuptakeinthebrainseemtoreducetyrosineavailability
forsynthesisofcatecholamineneurotransmitters(McKean,1972).Thesemechanismsalsomayaffecttryptophanavailabilityfor
serotoninsynthesis(McKean,1972).BothbraincatecholaminesandserotoninaredeficientinthePahenu2mousemodelforPKU
(PuglisiAllegraetal.,2000).
ThereisMRSevidence(Pietzetal.,1999)inacrossoverstudythatLNAAscompetewithphenylalanineattheBBB,andwhereas
theadverseeffectofexcessphenylalanineonotherLNAAsmaybepathogenicinPKU,dietarysupplementsofLNAAs,ifharmless
themselves,couldblockthepotentiallyharmfulinfluxofphenylalanineintothebrain.Kaufmanearlier(1976)suggestedthatthe
interactionbetweentheLNAAsduringtransportintobrainmaybeusedtoadvantageinthetreatmentofPKU,andthishasbeen
examined(Berryetal.,1982Huetheretal.,1985Jordanetal.,1985).Dietarysupplementsofbranchedchainaminoacids
sufficienttodoubletheirplasmavaluesinpatientswithPKUwillreducephenylalaninelevelsinplasmaandCSF(Berryetal.,1982)
andimprovetheirpsychologicaltestperformance(Jordanetal.,1985).Renalhandlingofphenylalanineandderivativeshasintrinsic
interestbuthasonlymarginalinfluenceonneurotoxicityandisnotdiscussedhere(seeScriveretal.,1989,fordetails).
EffectsonNeurochemistryandMetabolism
ThecauseofdefectivebrainmyelinationinPKUhaslongbeenafocusofinterest.Therearemanyexplanationsforthisabnormality
(Babaetal.,1987DwivedyandShah,1982Hommes,1991Huetheretal.,1982JohnsonandShah,1980MatsuoandHommes,
1987Shah,1979),butnoneiscomprehensive.AdditionalevidencefromMRI(Shawetal.,1991Thompsonetal.,1991a)indicates
thattheprocessofdysmyelination(Ullrichetal.,1994)ismoreprevalentthanhadbeensuspectedeveninwelltreatedpatients
(Thompsonetal.,1991a)andinnonPKUHPA.
Themyelinationproblemhasbeenstudiedintheenu2mouse,acounterpartofhumanPKU(seethesection"AnimalModels"
below).MRIandhistologicstudiesdonotrevealadysmyelination(Kornguthetal.,1994)orabnormalitiesincytoarchitectureor
histomorphometry.However,morerecenthistologicandbiochemicalstudiesoftheenu2mousebrainandoninvitrocultured
oligodendrocytesfrombrainofwildtypecontrolmice(BRBRstrain)exposedtoabnormalconcentrationsofLphenylalanineinthe
medium(upto3500M),suggestthatmyelinatingoligodendrocytesadoptanonmyelinatingphenotypeandoverexpressaglial
fibrillaryacidprotein(GFAP)(Dyeretal.,1996).Thiseffecthasbeenattributedtoareductionofcholesterolsynthesis.Intheenu2
mousemodel,Sheferandcolleagues(2000)foundthatforebrain,butnothindbrain,washypomyelinatedandthattherewasa
correspondingreductionof3hydroxy3methylglutarylcoenzymeAreductase(HMGR),theratelimitingenzymeincholesterol
synthesis,inmicrosomesisolatedfromtheforebrainbutnotinmicrosomesisolatedfromthehindbrain.Thereducedactivityof
HMGRseemedtoresultfromdownregulationbyincreasedphenylalaninebecausethenumberofoligodendrocytesremained
normalandbecauseHMGRactivityandcholesterolsynthesisalsoweredecreasedinmicrosomesoftheoligodendrocytelike4C8
gliomacellsonexposureto2424Mphenylalaninefor3weeks(Sheferetal.,2000).
Increasedturnoverofmyelinasacomponentofbraindysfunction(ChangeuxandDanchin,1976HommesandMoss,1992)is
associatedwithlossofneurotransmitter(muscarinicacetylcholine)receptordensityintheenu2mousemodel(Friedmanand

Kaufman,1971Hommes,1993).Relatedstudiesinaratmodel(madehyperphenylalaninemicbyphenylalanineloadingwithPAH
inhibitionbyDLmethylphenylalanine)thatusedmeasurementsofneuralcelladhesionmolecule(NCAM),GFAP,and
hyaluronatebindingactivityfoundthattheseparametersweregrosslyalteredinbrainexposedtoHPA(Hommes,1994).Asimilar
modeldemonstrateddeficitinmyelinbasicproteinneurofilamentstainingandmaturationofmyelinandaxonsearlyonsetHPA
producedpermanentdeficitsinaxonmyelinationinoutercorticallayers(Reynoldsetal.,1993).
Brainproteinsynthesisisperturbedbyexcessivephenylalanine,asshownbyPETinPKUpatients(Paansetal.,1996).Thiseffect
hasbeenattributedtopolysomedisaggregation(Bineketal.,1981)andinhibitionoftranslationinitiation(Okanoetal.,1986).The
effectcanbeoffsetbyaugmentingthepoolofLNAAs(BinekSingerandJohnston,1982).Thesefindingsmayberelevantto
treatment(seebelow).
BrainhistologyandcellulardevelopmentarealteredinhumanPKU(BaumanandKemper,1982),thecorresponding"artificial"
animalmodels(BaumanandKemper,1982Corderoetal.,1983Huetheretal.,1983HuetherandNeuhoff,1981Johnsonand
Shah,1984Reynoldsetal.,1993SwaimanandWu,1984),andtheenu2mouse(Dyeretal.,1996).Thenumberandspreadof
dendriticbasilarprocessesoflargepyramidalcellsarereducedbyHPAinratpups(Corderoetal.,1983)highlevelsof
phenylalanineanditsmetabolites,bothinculture(SwaimanandWu,1984)andinvivo(Huetheretal.,1983HuetherandNeuhoff,
1981),decreaseproliferationandincreaselossofneurons.DNAcontentisdecreasedinaffectedbraincells(Huetheretal.,1983),
anditssynthesisisimpaired(JohnsonandShah,1984).Theneteffectisimpairedbraingrowth(Reynoldsetal.,1993).Long
exposuretothedeviantmetabolicphenotypeimpairsdevelopmentofbrainarchitectureinuntreatedpatientswithPKU,with
abnormalitiesinmyelination,widthofthecorticalplate,celldensityandorganization,dendriticarborization,andnumberofsynaptic
spines(BaumanandKemper,1982).
Tosummarize,phenylalanineitselfisprobablytheneurotoxicagentinPKU.Metabolitesofphenylalaninearenotfoundinthe
human(ormouse)diseaseatsufficientlyhighconcentrationstodisturbmetabolicandchemicalrelationshipsinbrain.Whateverthe
mechanism,theneurotoxicconsequencesofPKUareeitheracuteandreversibleorchronicandirreversibletheybothaffect
neuropsychologicalfunction.

AnimalModels
AnimalmodelshavelongservedtostudypathogenesisofthediseasephenotypeinPKUcertainofthemalsoenablestudiesof
potentialnewwaystotreatHPA.

ArtificialModels
HyperphenylalaninemicanimalmodelsarenothomologuesofhumanPKUwhentheyareachievedbytheuseofexogenous
phenylalanineloadsandchemicalagentstoblockthephenylalaninehydroxylationreaction(Kaufman,1976).Suchstudies,done
mainlyinrats,wereamajorsourceoftheearlydataaboutputativeeffectsofHPAonbrainmetabolitesandchemistry(Laneand
Neuhoff,1980Vorheesetal.,1981).However,thephenylalanineloadusedtoproduceHPAintheanimalsproducedanadditional
burdenoftyrosinewhenPAHenzymeactivitywasleftintact.Accordingly,itwasnecessarytoinhibittheenzymetoobtainthe
requisiteHPAwithouthypertyrosinemia.Unfortunately,someoftheagentsusedforthispurpose(pchlorophenylalanineand
methylphenylalanine,forexample)hadadditionaleffects,notablyinhibitionoftetrahydrobiopterinrequiringhydroxylatingreactions
inbrain(Hoshigaetal.,1993)withsecondaryconsequencesforneurochemistry.

ANaturalModel
BetteropportunitiestoobtainamammaliancounterpartofhumanPKUnowexistinmice(McDonald,1994McDonaldetal.,2002
Shedlovskyetal.,1993).Themousegene(Pah)hasbeenclonedandcharacterized(GeneBankAccessionNumberX51942,
cDNA)itcontrolsexpressionofhepaticenzymeactivity,andtherearestronghomologiesbetweenmouseandhuman
phenylalaninehydroxylasegenesandenzymes(Ledleyetal.,1990).ThemousePahlocusisinalinkagegrouponchromosome10
(Ledleyetal.,1988a),homologoustotheregiononhumanchromosome12wherehumanPAHislocated.Mutationsatthemouse
PahlocusintheBTBRlinehavebeenproducedbychemicalmutagenesiswithNethylNnitrosourea(McDonaldetal.,1990a).The
firststrainwithevidenceofmutantphenylalaninehydroxylationtobeproducedbythismethodhadadefectinGTPCH1(Bodeet
al.,1988Hylandetal.,1996).Otherstrains,subsequentlyidentified,hadmutationatthePahlocusaffectingphenylalanine
hydroxylasefunction(McDonaldetal.,1990bShedlovskyetal.,1993).ThesestrainsareorthologuesofhumanPKUandnonPKU
HPA.Themutationsandtheassociatedphenotypehavebeencharacterized(McDonaldandCharlton,1997).Theenu1mouse
(Pahenu1/enu1)isacounterpartofnonPKUHPAandhomozygousforthemutationc.364TCinexon3(V106A)theenu2mouse,a
counterpartforhumanPKU,ishomozygousforamutationinexon7[c.835TC(F263S)].

Athirdvariant,enu3,isnowhistoricitssplicemutationhasbeendescribed(Haefeleetal.,2001).Theenu1mousehasnormal
plasmaphenylalanineandnormalbehavioronthemousebreederdiet(TekladNumber8626),whereastheenu2mousehasa10
to20foldelevationofplasmaphenylalanine,excretesphenylketonesintheurinewhenfedthebreederdiet,andhaschangesin
behaviorandincoatcolor.Ahybridstrain(Pahenu1/enu2),heteroallelicfortheexon3andexon7mutations,offersadvantagesfor
themanipulationofbloodphenylalaninelevels(Sarkissianetal.,2000b)negativecomplementationmayexistinthe
heteropolymerictetramericPahenzymeofthisstrain.TheseanimalsaredescribedinmoredetailonthePAHdbWebsiteand
elsewhere(Scriveretal.,2003).
Ithasbeennotedthatbreedingoftheenumiceisdifficult.Tooffsetthisdifficulty,themutantPahenu2allelehasbeentransferred
fromtheBTBRbackgroundtotheBl/6genomicbackground,withmuchimprovedbreedingefficiencyforthemutantstrain(BThony,
personalcommunication,2005).
Theorthologousenumousestrainsarebeingusedtostudypathogenesisofbraindisease(Hommes,1994Kornguthetal.,1994),
theeffectofmaternalHPAonfetalcardiacorganogenesis(McDonaldetal.,1997),andtheefficacyofenzymesubstitutiontherapy
withphenylalanineammonialyase(SafosandChang,1995Sarkissianetal.,1999Sarkissianetal.,2000b).
Measurementofphenylalaninemetabolites(i.e.,phenylpyruvate,phenyllactate,andphenylacetate)inbrainsofnormal.,enu1,and
enu2miceshowsthatmetabolitelevelsareminimallyelevatedintheenu1(nonPKUHPA)mouseandincreasedintheenu2(PKU)
mouse(Sarkissianetal.,2000a).However,concentrationsofthesemetabolitesarenotsufficienttobetoxicphenylalanineitself
remainsthebestcandidateforachemicalcauseofimpairedcognitivedevelopment.
ThesearchforthemechanismbehindtheaberrantbrainphenotypeinPKUcontinueswithnewstudiesintheenu2mouse(Smith
andKang,2000).SaturationbyphenylalanineoftheLaminoacidcarrierattheBBBanddistortionoftheratiooffreephenylalanine
tolargeneutralaminoacidsintheintracerebraltotalacidsolublemetabolitepoolreducedlocalratesofproteinsynthesisbyone
fifthintheadulthomozygousmutantmouse.Ontheotherhand,therewasnodecreaseoftRNAboundneutralaminoacidsinthe
pool.ThelatterfindingisrathersurprisingandsuggeststhatdistortedfunctionattheBBBmaynotdirectlyunderliethemechanism
ofimpairedbrainproteinsynthesisinthePKUphenotype.Anexcellentsummaryofresearchintheenumousemodelshas
appearedasameetingreportfromtheprincipleinvestigators(McDonaldetal.,2002).

ScreeningandDiagnosis
Comment
GuthrieandSusipublishedtheirlandmarkdescriptionofasimplephenylalaninemethodfordetectingphenylketonuriainlarge
populationsofnewborninfantsin1963(Guthrie,1996GuthrieandSusi,1963),andmanyyearslateritisapparentthatapplications
ofthemethodanditsderivativeshave"gonearoundtheworld,changedthenaturalhistoryofadisease(phenylketonuria),and
throughgeneticscreening,haveintroducednewconceptsandapproachestothepracticeofmedicineandhealthcare"(Scriver,
1998b).ThissectiondescribestheprinciplesandpracticesthatmadescreeningforPKU,apopulationbasedprocedure,a
prototypefor(population)geneticscreening.

Principles
Therationaleforpopulationscreeningofnewbornsisearlymedicalintervention(NationalAcademyofSciences,1975).Thegoalin
diagnosisofHPAiscorrectmedicalintervention.Theseobjectiveseachrequireprocessesandresources.
Amedicalscreeningtestidentifiesindividualswhoprobablyhaveadiseasefromthosewhoprobablydonot(WilsonandJungner,
1968).Ageneticscreeningtestfindspersonswhoareapparentlyatriskofincipientorestablisheddiseaseinthemselvesorintheir
relativesbecauseofgenotype(NationalAcademyofSciences,1975).Ontheorderof10millionnewborninfantsarescreened
annuallyworldwideforHPA,andrelativesofaffectedprobandsarenowinterestedinknowingtheirgenotype,andtheyseektesting.
Accordingly,PKUscreeninganddirectedtestinghavebecomesomeofthemorewidelyapplied"genetic"testsinhealthcare
(AmericanAcademyofPediatricsCommitteeonGenetics,1989).Theprocedureisnowseenasthegoldstandardagainstwhich
screeningforotherinbornerrorsofmetabolismcanbejudged(Seymouretal.,1997),anditisnowenhancedbytheuseoftandem
massspectrometry(Chaceetal.,1993,1998Pollittetal.,1997).

Screening
ScreeningtestsforHPAareperformedinthenewbornandidentifythelevelofphenylalanineinblood.Theoriginalmethod,urine
screeningforphenylpyruvicacid,isunreliabletoidentifyPKU(MedicalResearchCouncilSteeringCommitteefortheMRC/DHSS

PhenylketonuriaRegister,1981).ScreeningatthelevelofthePAHgenotype,byDNA(mutation)analysis(Cotton,1997),willnot
replaceareliablephenotypetestforHPAbecauseofvastallelicheterozygosityinthePAHgeneandlocusheterogeneityinHPA.
Themostreliablenewbornscreeningtestsemploymicrobiologic,enzymatic,chromatographic,fluorometric,ormostrecently,mass
spectrometricmethodstomeasurethephenylalaninecontentofdriedcapillary(notcord)bloodsamplescollectedonfilterpaper.
Phenylalanineinbloodspotsonproperlystoredfilterpaperisstableforyears,andaccurateretrospectivemeasurementsare
feasible.Howthebloodsampleistakenfromthenewborn,itseffectonthebaby,andtheefficiencyoftheprocedurehavebeen
analyzed.AMicrolanceneedleforvenipunctureonthedorsumofthehand(comparedwithtwosizesoflancetsforsamplingbyheel
prick)hasprovedtobelesspainful,moresuccessfulonthefirstattempt,andfaster,althoughcollectingcapillarybloodby
puncturingtheheelremainsthemostfrequentlyemployedprocedure.Themicrobiologic(GuthrieandSusi,1963)and
chromatographicmethodsarebothsemiquantitative,withlimitationsonaccuracyatlowphenylalanineconcentrations.Enzymicand
fluorometric(McCamanandRobins,1992)methodsarefullyquantitativedowntothelowestplasmaphenylalaninelevelsandhave
theaddedadvantageofalowcoefficientofvariation.Tandemmassspectrometry(MS/MS)isalsofullyquantitativeandhasthe
addedadvantagesofsimultaneouslymeasuringtyrosineaswellascombiningscreeningforPKUwiththatforanumberofother
inbornerrorsinasingleanalysis(Chaceetal.,1993).Thetechnicalissuesarerelevant.Acrucialattributeofascreeningtestisits
sensitivity,whichisitsabilitytominimizethefrequencyoffalsenegativeresults.AnenzymictestbasedonanNADHdetecting
biosensor,aswellasMS/MS,hasspecialpromiseforthispurpose(Chaceetal.,1998Huangetal.,1998).BecausePKUscreening
isbestdonesoonafterbirth,andbecausebloodphenylalaninebeginstoriseintheaffectedinfantonlyafterseparationfromthe
placenta,capillarybloodphenylalaninevaluesinaffectedcaseswillbelowerthecloserthedayoftestingistothedayofbirth(Fig.
7716).Thishasbeenbelievedtoimpairthesensitivityofthetestwhenperformedinspecimenscollectedbeforeday2oflife
(AmericanAcademyofPediatricsCommitteeonGenetics,1982McCabeetal.,1983),butprospectivestudiesofearlynewborn
screeningindicatethatHPAisreadilydetectableinspecimenscollectedbytheendofday1(Dohertyetal.,1991Hanleyetal.,
1997Meryashetal.,1981).Regardless,itisdesirabletouseafullyquantitativetesttoseparateabnormalfromnormalvaluesas
accuratelyaspossibletheissueisparticularlyrelevantwhenaninfanthasnonPKUHPA(Hanleyetal.,1997).Areviewofneonatal
screeningforHPAinBritain(Smithetal.,1991a)foundthatallfalsenegativetestsinthatprogramwereperformedbythe
semiquantitativemicrobiologicinhibitionassay(Guthrietest)andthatnonewasassociatedwiththefullyquantitativefluorometric
assay.
FIG.7716

Retrospectiveanalysisofbloodphenylalaninevalues(mean2SD),relatedtoage,inneonateswithconfirmedpersistentHPAand
adiagnosisofphenylketonuria.Regressionsareformaleinfants(solidline)andfemaleinfants(brokenline).Originaldataarefrom
Holtzmanetal.,1974aandHoltzmanetal.,1974b.(FromScriverand,1982.Usedbypermission.)

DespitetheapparentreliabilityofnewbornscreeningforHPAondays12oflife,evenearlierdischargefromhospitalnurseriesis
quitefrequent.Forexample,onequarteroftermnewbornsintheUnitedStatesaredischargedatorbefore24hours(Sinaietal.,
1995).Becausetheobstetricpracticeisunlikelytochangeinthecultureofcostsaving,thesearchforabetterscreeningpractice
hastofocuselsewhere(AmericanAcademyofPediatricsCommitteeonGenetics,1982Kirkmanetal.,1982Levyetal.,1984
McCabeetal.,1983Schoenetal.,1983Scriver,1983Smithetal.,1991a).Routinefollowup(repeat)testingofinfantstocapture

thosewithfalsenegativefirsttestsisdeemedtobeinefficientandexpensive(Schoenetal.,1983Sepeetal.,1979U.S.
Congress,1988)butnonethelessisbeingrecommendedinpartsoftheUnitedStates(AmericanAcademyofPediatricsCommittee
onGenetics,1992Sinaietal.,1995).Mostscreeningprogramsthatdonotincluderoutinerepeattestingrequestarepeat
specimenwhenevertheinitialscreeningspecimenwascollectedbefore24hours(Walravenetal.,1995).Toconvertthetestfrom
semiquantitativetofullyquantitativestatushastechnicalmerits(Kirkmanetal.,1982Scriver,1983Smithetal.,1991)andisnow
underwayinmanyscreeningprogramsasaresultoftheintroductionofMS/MS(LevyandAlbers,2000).Any(quantitative)method
appliedtofilterpaperbloodsamplesshouldcompensatefortheeffectofclimateandseasononthephenylalaninevalue(Hill,1969
Lambert,1994).Thethreshold(cutoff)valuesignifyingHPAcanbeloweredtoimprovesensitivitybutatsomelossinspecificityand
predictivevalue(Clemensetal.,1990Dohertyetal.,1991).Aphenylalaninevalueof150M(2.5mg/dl)shoulddetectallneonatal
casesofPKUunlessthereisabiologicreason(Hanleyetal.,1997)foranormalvalueinanaffectedinfantatthetimeofthetest.
However,MS/MStechnologyseemstohavethecapacitytoimprovebothsensitivityandpredictivevaluebyallowingfora
phenylalanine:tyrosineratiowith2.5asthediscriminator(cutoff)abovewhichpersistentHPAishighlylikely(Chaceetal.,1998).
WhereasascreeningprogramwillnotdetecteverycaseofpersistentHPA,theerrorrateislow[twostudiesfoundthatonlyabout1
in70casesofPKUismissed(Holtzmanetal.,1986aMedicalResearchCouncilSteeringCommitteeforMRC/DHSS
PhenylketonuriaRegister,1981)]andcanbelowerstill(Hanleyetal.,1997).Theprincipalcausesformissedcasesare
noncompliancewiththeprocessanderrorsofprocedure(Holtzmanetal.,1986aMcCabeandMcCabe,1983MedicalResearch
CouncilSteeringCommitteeforMRC/DHSSPhenylketonuriaRegister,1981),butbiologiccausesmayoccuroccasionally(Hanleyet
al.,1997).Reanalysisoftheoriginalfilterpaperbloodsampleobtainedfromseveral"missed"HPAcaseshasrevealednormal
bloodphenylalaninevaluesatthetimeoftheoriginalscreeningtest(AmericanAcademyofPediatricsCommitteeonGenetics,
1982Hanleyetal.,1997).ThephenomenonisespeciallyrareinthetypicalPKUphenotypebutoccursmorefrequentlyinnonPKU
HPAandseemsmorelikelytoaffectfemaleinfants(Hanleyetal.,1997Holtzmanetal.,1974Labergeetal.,1987Scriver,1982)
(seeFig.7716).Accordingly,itispossiblethatsomefemaleswithHPAmayescapedetectionandwillbeignorantoftheriskto
offspringintheirmaternalHPAlaterinlife.
Falseresults,eitherpositiveornegative,alsoarisefromartifactstheyincludeampicillincontaminationofthesample(Mabryetal.,
1988KremenskyandKalalydjieva,1989Wilckenetal.,1989),totalparentalnutritionwithsomeproprietaryaminoacidsolutions
(Mittonetal.,1988),andevenlottolotvariabilityinthefilterpaperaffectingabsorbencyandmetaboliterecovery(Slazyketal.,
1988).
AsexperiencewithneonatalscreeningforPKUandallieddisorderscontinuestogrow,theneedtoimproveeducationandfollowup
increases(Meaney,1988Smithetal.,1991).Meanwhile,societywillacceptamodestrateoffalsepositivetests(Sorensenetal.,
1984)asa"cost"formaximizingthesensitivityofscreening.Inoveralleconomicterms,screeningforPKUiscosteffective(Bushet
al.,1973Dagenaisetal.,1985U.S.Congress,1988).

TestsforDiagnosisandDifferentialDiagnosis
ApositivescreeningtestidentifiesanewbornwithHPA.Thediagnostictestidentifiesthecauseofthephenotypeintheparticular
infant.
SomeinfantswithpositivescreeningtestshaveonlytransientHPAofnofurtherclinicalsignificance.Rarely,transientHPAwillbe
causedby4carbinolaminedehydratasedeficiency(seeChap.78),wheretheHPAmaypersistfordaysorweeksitalsocouldbe
aneffectofmaternalHPAbutonlyforthefirstdayortwooflife(LevyandLobbregt,1995).AmongtheinfantswithpersistentHPA,
themajorcause(>98percentofcases)ismutationatthePAHlocus.SomePAHallelescausethePKUphenotype,inwhichthe
plasmaphenylalanineconcentrationexceeds600M(10.5mg/dl)onanormaldiet.OtherallelescausenonPKUHPAinwhichthe
phenylalaninevalueisconsistentlybelow600Monanormaldiet.Thedistinctionisrelevantbecauseharmtocognitive
developmentismorelikelytooccuratthehigherlevelsofphenylalanineinPAHdeficiency.
Someinfants(<2percent)withpersistentHPAhaveimpairedsynthesisorrecyclingoftetrahydrobiopterin(BH4)(seeChap.78),for
whichthereisaninternationalregister(Blauetal.,1996)(seewww.BH4.org),andthesepatientsrequirespecifictreatmenttooffset
thedeficiencyofBH4.SincetheplasmaphenylalaninevaluealonedoesnotdistinguishbetweenBH4impairedandBH4sufficient
formsofHPA,everycaseofpersistentHPAmustbeinvestigatedfurthertoruleoutthedisordersofBH4metabolism.
Recently,ithasbecomeevidentthatsomepatientswithPAHdeficiencyalsocanbetreatedwithBH4.Thesepatientsdonothavea
deficiencyofBH4butrespondwithareductionofphenylalaninewhengivenBH4(Kureetal.,1999Muntauetal.,2002Shintakuet
al.,2003).ThisresponseisaresultofenhancedPAHactivity,asshownbyincreasedphenylalanineoxidation(Muntauetal.,2002).
InHPA,BH4responsivePAHdeficiencyalwayshasbeenconsideredtobemildPKU(plasmaphenylalanine6001200M)ornon
PKUHPA(plasmaphenylalanine<600M)andhasbeenassociatedwithseveralPAHmutations(Muntauetal.,2002Shintakuet

al.,2003).
DifferentialDiagnosis
Adetaileddiscussionofdiagnostictestsandproceduresisgiveninthesixtheditionofthistextaprcis,includingTable777,is
givenhere.
Table777:DiagnosticTestsforFollowupofNeonatalHyperphenylalaninemia
ViewLarge|SaveTable
Table777:DiagnosticTestsforFollowupofNeonatalHyperphenylalaninemia

TestProcedure

ApproximateNormal
ValuesorResponsein
AffectedCase

1.Plasma(orblood)phenylalanineand
tyrosine(M)withnormalproteinintake

Phenylalanine40
130Tyrosine50140

2.Urinetotalbiopterinandneopterin
concentrations(mmol/molcreatinine)
neonateshavethehigherneopterin
values

Biopterin0.4
2.5Neopterin0.1
5.0%Biopterin2080

3.BH4load(7.5mg/kgorally),plasma
phenylalanineandtyrosineat0,1,2,4,6
and24hours(0hphenylalanineshouldbe>
200M)

Fallinphenylalanineto
normalornearnormal
indicatesBH4synthesis
isdeficient

4.Plasma(ordriedbloodspot)totalbiopterin
(ng/mLCrithidiafasciculataassay)

Plasmabiopterin1.4
3BH4synthesisis
deficient

5.CSFconcentrationsofHVAand5HIAA
(nmol/mlneonateshavethehigher
values)

HVA40010005HIAA
200400

6.CSFtotalbiopterinandneopterin
(nmol/mlneonateshavethehigher
values)

Biopterin12
40Neopterin103

7.percentageoftotalbiopterinasBH4in
urineandCSF

Urine6080CSF9098

8.BH4load(20mg/kgorallyasthe6R
epimerofBH4),plasmaphenylalanineat0,

Fallof3050%in
phenylalanineindicates

4,8,24,48hours(0hphenylalanineshould
be>360M)

BH4responsivePAH
deficiency

CSF,cerebrospinalfluidHVA,5HIAA,5hydroxyindoleaceticacidHVA,
homovanillicacid.
AdaptedfromSmithandLee,2000.
TestsattheMetaboliteLevel
ForPhenylalanine(WholeBloodorPlasma)

Thenormalvalueislessthan150Minneonatesandlessthan120Minoldersubjects(Gregoryetal.,1986Scriveretal.,1985
ScriverandRosenberg,1973).
ForPhenylalanineMetabolites

Therearenoreliablealternativestomeasurementofphenylalanineitselffordiagnosis(orscreening).Measurementof
phenylpyruvicacidand/orrelatedmetabolites(seeFig.772)isnotrecommendedbecauseformationofphenylpyruvate,the
"classic"urinemetabolite,dependsontransaminaseactivity(BrennemanandKaufman,1964),andthismaybeattenuatedinthe
neonate(ArmstrongandBinkley,1956).Moreover,thereisalmostfourfoldinterindividualvariationinthesubstrate(phenylalanine)
concentrationinplasmaatwhichtheketoacidisproducedinPKUpatients(Knox,1970).
PhenylalanineLoadingTest

Aloadingtestisnotrecommended.Formerly,itwasusedtoascertainthepresenceofPKUbythedegreeofphenylalanine
intolerance(Woo,1984).However,withgreaterunderstandingoftherelationshipbetweentheambientplasmaphenylalaninelevel
andtheclassificationofHPAandtheabilitytodeterminemutationsatthePAHlocusalongwithgenotypephenotypecorrelation
(Guldbergetal.,1998Kayaalpetal.,1997),phenylalanineloadingisnolongernecessary.Itisespeciallycontraindicatedinthe
newborninfant,inwhomitmightraisethephenylalaninetoalevelthatcouldproduceirreversibleneurotoxicityandwoulddelay
dietarytreatment.
ThePlasmaPhenylalanineResponsetoBH4

TheBH4dose(seeTable777)isgivenorally.Thetestisdonewiththe6RepimerofBH4.Thephenylalaninelevelmustbe
elevatedwhenthetestisdoneafallinthelevelafterBH4loadingusuallytonormalornearnormal(andusuallywithanincreasein
tyrosine)andwithinthefirst6hours(rarely,notuntil24hours)indicatesBH4deficiency.ThetestcouldbemisinterpretedwhenBH4
cannotfunctionasacatalyticcomponentofthehydroxylatingreaction,asinDHPRdeficiency(Kaufman,1986).AfterBH4deficiency
hasbeenexcludedandPAHdeficiencyestablished,thelargerBH4load(20mg/kg)maybegiventodetermineBH4responsiveness
witha30percentorgreaterfallinthephenylalaninelevelindicatingresponsiveness.(Note:BH4isavailableforclinicalusefromB
Schircks,Jona,SwitzerlandCH8645.)
ForPterinMetabolites

Measurementsareperformedreliablyonlyinlaboratorieswithexpertise.Pterinmetabolitepatternsareabnormalinplasma,CSF,
andurineingeneralizeddisordersofBH4homeostasis.Thesocalledperipheralformof6pyruvoyltetrahydropterinsynthase(6
PTS)deficiencymaybepresentwheninitialpterinlevelsinCSFarenormal(Allansonetal.,1991).Biopterincanbemeasuredin
driedbloodspotsonfilterpaper.BH4levelsarehighinuntreatedcaseswithambientHPAandintactBH4synthesis(Leemingetal.,
1976)andlowindisordersofBH4homeostasis.
Pterinmetabolites(totalbiopterinandneopterin)canbemeasuredinurinebyseveralmethodsrelativeamountsandnormalvalues
areagedependent.PAHenzymedeficiencyconferselevatedpterinlevelswithnormalneopterin:biopterinratiosDHPRdeficiency
produceselevatedtotalpterinlevelsandlowBH4levels6PTSdeficiencyelevatesneopterinlevelsandneopterin:biopterinratios
andGTPCHdeficiencyproduceslowpterinvaluesandnormalratios.
ForNeurotransmitterMetabolites

CSFlevelsofhomovanillicacidand5hydroxyindoleaceticacid,derivativesoftyrosineandtryptophan,respectively,areusually
depressedindisordersofBH4synthesisandrecyclingbutnotequivalentlyinPKU.CSFneurotransmittermetabolitelevelsare

normalintheperipheralformof6PTSdeficiency(Allansonetal.,1991).
TestsattheEnzymeLevel
ForPAHEnzyme

DirectmeasurementofPAHenzymeactivityrequiresliverbiopsy.Indirectinvivoassaysarefeasiblebystableisotopeinfusions
(Trefzetal.,1978)andbyingestionof14Clabeled(Lehmannetal.,1986)or13Clabeledphenylalanine(Treacyetal.,1997)they
showhighintraindividualcorrelation,theycorrelatewithinvitroassaysofPAHactivity,andtheywilldemonstratedeficient
phenylalaninehydroxylatingactivityinvivo.(Forthefourtestslistednext,seeChap.78forafulldiscussion.)
ForDHPR

Activityismeasuredinmanytissues,includingliverbiopsymaterialculturedskinfibroblastsandamniocytes,erythrocytes,
leukocytes,andplateletsandindriedbloodspotsonfilterpaper.AnautomatedassayofDHPRactivityineluatesfromdriedblood
spotsonfilterpaperhasbeendeveloped(Surpliceetal.,1990).
For4CarbinolamineDehydratase

Activitycanbemeasuredinbloodlymphocytesandscalphairrootcells(LeiandKaufman,1998b).
ForGTPCH

Activityismeasuredinliverbiopsymaterialandphytohemagglutininstimulatedmononuclearleukocytes.GTPCHactivityis
normallylowinunstimulatedwhitebloodcellsandshouldnotbemistakenforaninheriteddeficiency.
For6PTS

Activityismeasuredinliverbiopsymaterialanderythrocytes.

DiagnosisbyDNAAnalysis
ThePAHlocuscanbeanalyzedbothforthediseasecausingmutationandforassociatedpolymorphichaplotype.Venousblood,
driedbloodspots,buccalcells(obtainedbysalinemouthwash),culturedskinfibroblasts,andothercellsareconvenientsourcesof
DNAormRNAtranscripts.AnalyticmethodsincludeSouthernblotting(forlargedeletions),heteroduplexanalysis,singlestrand
conformationalpolymorphism,denaturinggradientgelelectrophoresis,chemicalcleavageofmismatch,restrictionenzyme
digestion,allelespecificoligonucleotidehybridization(Cotton,1993,1997Grompe,1993),andanalysisof"illegitimate"(mRNA)
transcriptsgeneratedbyreversetranscriptPCR(Chellyetal.,1989SarkarandSommer,1989).Detectionofamutationrequires
proofofcausation(CottonandScriver,1998)toruleoutanartifactorthepossibilitythatitisaneutralpolymorphicvariant.A
negativefindingbyanyonemethod,however,doesnotexcludeamutationofclinicalsignificancebecausenosinglemethodcan
detectallPAHalleles.DiagnosisofthemutantPAHgenotypehassomeclinicalrelevancebecauseitisoftenpossibletopredicta
severeoramildeffectonenzymefunction(Guldbergetal.,1994,1995,1998Okanoetal.,1991)andtocounselaccordingly.

PrenatalDiagnosis
IndicationsforprenataldiagnosisexistintheHPAs(Ledleyetal.,1988bScriverandClow,1988).Treatmentundersome
conditionsmaybedifficulttoobtainoradminister,andprognosisforafullynormaloutcomestillcanbeuncertain(BarwellandPollitt,
1987Riessetal.,1987).Accordingly,theremaybeaninterestinavoidanceofrecurrentdisease.Wheretheoptionispermitted,
familiesatriskshouldknowthatprenataldiagnosisisfeasible(ClearyandWraith,1991).
FetaldiagnosisofPAHdeficiencyisfeasiblebyDNAanalysis(Woo,1984).Unlessthemutantallelesintheprobandareknown,
polymorphicmarkers(RFLPs),ingameticassociationwiththePKUalleles,arethemainstayoffetaldiagnosis.Theapproach
requiresanalysisofparentalhaplotypesandoftheassociationbetweenhaplotypeandPAHmutation.Threequartersofaffected
casesarelikelytobegeneticcompoundsforPKUalleles,andthishasimplicationsforprenataldiagnosisbyeitherhaplotypeor
mutationanalysis.AmniocytesandchorionicvillussamplescanbesourcesofDNAforanalysismaternalcontaminationofthe
chorionicvillussampleisahazard.

DiagnosisofHeterozygotes(PAHLocus)
IdentificationofPAHheterozygosityissometimesrequired.Tworelativelysimpleapproachesareavailable.Oneinvolvestheuseof
isotopes(Lehmannetal.,1984,1986Treacyetal.,1997).Thetestsaredonewithsmallsubstrateloads,andmeasurementsare
madeunderquasisteadystateconditionscircumstancesthatmaximizethesensitivitycoefficientofPAHenzymeactivityinvivo.

Thesecondapproachwithouttheuseofsubstrateloadsorisotopictracerscanbeachievedbytakingasinglesemifastingnoontime
bloodsampleinwhichphenylalanineandtyrosinearemeasuredquantitatively(Guneraletal.,1991Hiltonetal.,1986Rosenblatt
andScriver,1968)theeffectofcircadianvariationshouldbetakenintoaccountifanothertimeischosen(Scriveretal.,1985).A
quadraticdiscriminantfunctionthatincorporatesthepriorprobabilityofheterozygosity(Goldetal.,1974Saraivaetal.,1993
WestwoodandRaine,1975)andlineardiscriminantfunctionsthatdonot(Freehaufetal.,1984Pauletal.,1978Sartorioetal.,
1988Wengeretal.,1986),areaboutequallyefficientforpurposesofcarrierclassification.MeasurementoflabeledCO2following
administrationofisotopelabeledphenylalanineoffersnoparticularadvantageoverthediscriminantfunctionapproachappliedto
ambientplasmaphenylalaninelevels(Lehmannetal.,1986Treacyetal.,1997).
TheadvantagesanddisadvantagesofgenetictestingbyDNAanalysisarethosealreadydescribed.Ontheotherhand,itisavery
reliablemethodifthealleleshavebeenidentifiedinanaffectedfamilymember.

TreatmentofPhenylketonuria
Comment
In1965,theThirdInternationalCongressofHumanGeneticsaddressedthetopicoftreatmentinmedicalgenetics.Inthe
discussion,referencewasmadeto"treatmentundertakentomodifytheenvironmentinwhichthe[person]lives.Theotherwise...
potentiallydeleteriousmutationisthusoffset,andtheindividualprospers.Theprinciplesoftherapyinthisrealmaregenerallyclear.
Inmostinstances,theyareimmediatelyapplicableto[Homosapiens],andmanyexamplesofpracticalsuccesscanbe
documented"(Scriver,1967).TreatmentofPKUisoneoftheclassicexamplesof"euphenictherapy"(Lederbergsterm),wherea
normal(ornearnormal)phenotypeisrestoredwithoutmodificationofthemutantgenotype.Becauserecognizedapproachesto
treatmenthavebeeninuseforsufficienttimetoassessthedegreeofsuccess(Holton,1995),anditsbigsuccessispreventionof
mentalretardation(MacCready,1974),PKUisoneofthosediseasesinwhichprocessandoutcomevariablesdeserveongoing
analysis.Inthemeantime,morestringenttreatmentofHPAisbeingrecommended[Cockburnetal.,1993MedicalResearch
Council(UK),1993NationalInstitutesofHealthConsensusDevelopmentPanel,2001Smith,1994b].Some40yearsofPKU
treatmenthavebeenreviewedatinternationalsymposia(JInheritMetabDis25:605,2002EurJPediatr155(Suppl1),1996),
wherethemajorgoodnewswasreiteratedalongwithevidencethatrefinementoftreatmentprotocolsisindicated.ThePKUstory,
inbothgoodandlessgoodparts,revealshowsuccessfulbuthowdifficultitisforHomomodificans(adescendantofH.
sapienssapiens)toactasasubstituteforthenormalPAHgenotype.ThesearchfornewandimprovedtherapyforPKUcontinues
(BlauandScriver,2003).

TreatmentbyLowPhenylalanineDiet
ThemainstayoftreatmentforPKUisthelowphenylalaninediet(Bickeletal.,1953WoolfandVulliamy,1951),andtreatmentby
diethasbeenfeasiblesincethemid1950s(Bickeletal.,1954Woolfetal.,1955ArmstrongandTyler,1955).Whenstartedinthe
neonatalperiod,itmodifiesthemetabolicphenotypeandpreventstheneuropsychologicalconsequencesofHPA.Optimaltreatment
ofPKUemphasizedintheNationalInstitutesofHealthConsensusStatementonScreeningandManagementofPhenylketonuria
(NationalInstitutesofHealthConsensusDevelopmentPanel,2001)requires(1)earlyonsetoftreatment(within1monthofbirth),
(2)continuoustreatmentforlife,includingthroughconceptionandpregnancyintheaffectedfemale(tobenefitthefetus),and(3)
severerestrictionofphenylalanineintaketothesmallamountssufficienttoholdplasmaphenylalaninevaluesascloseaspossible
tothenormalrangeyetsufficienttosupportproteinsynthesis(excessiverestrictionwillimpairgrowthanddevelopment).The
precisetoleranceforphenylalanine(200500mg/day)variesamongpatientswithPKU,evenamongthosewiththesamemutant
PAHgenotypewhichimpliesthefamiliarmaxim:Treatthepatient,notthegenotype!Helpforthepatienttocomplywithtreatment
remainsimportantlongtermcomplianceisdifficult(Walteretal.,2002).

ResponsetoEarlyTreatment
Aprospective,controlledtrialoftreatmentbyselectiverestrictionofphenylalanineintake,beginningearlyinpostnatallife,hasnever
beenattempted.Nevertheless,alternativestoarestrictingphenylalaninehaveprovedtobeunpromising:(1)Asinglepatienttreated
purposefullyfromearlyinfancyonlybymeansofatyrosinesupplementandwithnorestrictionofphenylalanineintakenonetheless
expressedthePKUphenotype(Batshawetal.,1981).(2)LDopatherapyaloneinadultPKUpatientsofftreatmentsince10years
ofagedoesnotimprovefrontallobeandotherbrainfunctions(Ullrichetal.,1996).Conversely,whilenevertreatedcontemporary
PKUpatientscontinuetomanifesttheclassicPKUphenotype(Langenbecketal.,1988PittandDanks,1991),patientstreated
continuouslyandcarefullywithphenylalaninefollowingneonataldiagnosisavoidsevereimpairmentofcognitivedevelopmentand
function(seeFig.1511inScriveretal.,1989).

Anearlystudyof28PKUsiblingpairs(SmithandWolff,1974)matchingtheindexcasewhopresentedwithalatediagnosisand
mentalretardationwiththesiblingwhohadbeendiagnosedandtreatedforPKUfromtheneonatalperiodshowedthatthe
differenceinintellectualdevelopmentbetweentheearlytreatedsibling(IQvaluesall>80)andlatediagnosedsibling(meanIQ=
45range3081)wassignificant.Inanotherhistoricstudy(Dobsonetal.,1976),when36earlytreatedPKUpatientsandtheir
unaffectedsiblingswerecompared,themeanIQvalueswere94and99,respectively.
InalargecollaborativestudyintheUnitedStatesthatevaluatedtheoutcomeofearlytreatedPKUpatients(Williamsonetal.,1977),
IQscoresmeasuredin111PKUchildrenat4yearsofage,whosetreatmentbeganbetween3and92daysafterbirth(Dobsonet
al.,1977),yieldedameanscoreof93(StanfordBinetIntelligenceScale)forthewholegroup,andpatientstreatedfromthefirst
monthhadahighermeanscore(IQ=95)thanthosefirsttreatedbetween31and65daysofage(IQ=85).Evaluationof132of
thesechildrenat6yearsofage(Williamsonetal.,1981)foundameanIQscoreof98regressionanalysisshowedthatscoresat6
yearswererelatedtomaternalintelligence,ageatonsetoftreatment,andaveragelifetimeplasmaphenylalaninevaluesduring
treatment.Evaluationofoutcomein55ofthesechildrenat8yearsofage(Kochetal.,1984)foundearlytreatmentcompatiblewith
attainmentofa"normal"IQscore(WISCFullScaleIQscore=100).However,thePKUprobands,asagroup,hadasmallIQdeficit
relativetotheirnormalsiblins,whosemeanIQscorewas107(p=0.001),andpatientswhoterminatedtreatmentearlyscored
lowerthanthosewhocontinueditlonger(Kochetal.,1984).Amongthe95evaluatedat12yearsofage(Azenetal.,1991),23had
maintainedbloodphenylalaninelevelsoflessthan900Mforthefull12years,whereastheremainderhadnotbeenableto
maintainthisdegreeofcontrol.IQscorescorrelatedpositivelywithagereachedatlossofdietarycontrolandwiththemidparental
IQscoreandnegativelywithageatwhichtreatmentbeganandwithmeanbloodphenylalaninevalueduringtreatment.Thebest
testscoreswereobtainedbythepatientstreatedlongestandwithbloodphenylalaninevalueskeptconsistentlylessthan900M.
Recently,70ofthe125patientswhohadbeenfollowedto10yearsofagewerestudiedasadults(Kochetal.,2002).The9who
hadcontinueddietreportedfewerproblemsandtendedtohavehighercognitiveandachievementscoresthanthosewhohad
discontinuedtreatment.
IndependentcorroboratingevidenceexistsforboththeefficacyoftreatmentandoutcomeinearlytreatedchildrenwithPKU(Smith
andBeasley,1988Smithetal.,1988,1990).Forexample,263PKUpatientsbornbetween1964and1971intheUnitedKingdom
forwhomoutcomeswereknownandfollowedprospectivelyhadameanIQof96comparedwiththeestimatedpopulationnormof
109.8,and545bornbetween1972and1980hadameanIQof104comparedwiththeestimatedpopulationnormof112.4inthis
study,IQscoreswereadjustedtoaccountfortheriseinnormalscores(and0.3IQpointsperyear)sincestandardizationofthetest
in1932(Smithetal.,1990Flynn,1984).PKUpatientsshoweda4pointdeficit(1)foreachmonthsdelayafterbirthuntilonsetof
treatment,(2)foreachriseof300Minaveragebloodphenylalaninelevelwithinsufficienttreatment,and(3)foreach5month
periodduringinfancywhenbloodphenylalaninevaluesremainedlessthan120Mowingtoexcessivetreatment(Smithetal.,
1990).
MorerecentstudiesfromtheUnitedKingdom(Griffithsetal.,1998)andtheUnitedStates(Arnoldetal.,1998)confirmthatdietary
treatmentachievingmeanbloodphenylalaninelevelsoflessthan360Minearlychildhoodiscompatiblewithnormalexecutive
function(highergoaldirectedmentalactivity,organizedinnatureanddependentongoodcontrolofattention)(Griffithsetal.,1998):
Thelongerandbetterthecontrolofphenylalaninelevels,thebetterwasthecognitiveandmotorfunction,behavioraltemperament,
andexecutivefunction(Arnoldetal.,1998Smithetal.,2000Weglageetal.,2000).Thesestudiesupholdtherecommendations
formorestringenttreatment(Cockburnetal.,1993NationalInstitutesofHealthConsensusDevelopmentPanel,2001).
ManyearlytreatedPKUpatientshavebeenfoundtohavesubtleperformancedeficitsinconceptual,visualspatial,andlanguage
relatedtasks(Hendersonetal.,2000Holtzmanetal.,1986bMelnicketal.,1981Penningtonetal.,1985Rapoportetal.,1983
Smith,1985)inreadingandarithmeticskills,aswellasoverallschoolachievement(Azenetal.,1991Kochetal.,1984
Stemerdinketal.,2000)andinmotorcoordination,attentionspan,responsetime,problemsolvingability,workingmemory,and
executivefunctioning(Berryetal.,1979Diamondetal.,1997Faustetal.,1986Feldmannetal.,2002Fishleretal.,1987
Griffithsetal.,1997aHoltzmanetal.,1986bKochetal.,1984Melnicketal.,1981Netleyetal.,1984Penningtonetal.,1985
Smithetal.,2000Welshetal.,1990Whiteetal.,2002).Moreover,behaviormaybemoreextrovertedalongwithnegativetask
orientation(Kalverboeretal.,1994),andthefrequencyofneuroticandemotionaldisorders(Risetal.,1997Weglageetal.,2000),
aswellashyperkineticbehavior,canbetwicenormal(Burgardetal.,1994).Disorderedfunctionintheprefrontalcortexisthought
tobeatthesourceoftheseproblems(Diamond,1994Eisensmithetal.,1994Smithetal.,2000).Despitetheseselectivedeficits,
welltreatedPKUpatientshavesatisfactorylives(Kochetal.,1985):Theymeettheirgeneticpotentialforheight(Holmetal.,1979
Lyonnetetal.,1989)althoughtheytendtobeoverweight(McBurnieetal.,1991Whiteetal.,1982)andtheyhavenormal
pubertaldevelopment(Scaglionietal.,1986)andaregainfullyemployed(Kochetal.,1997).
Whereassocialclasscanhaveaneffectonoutcome(Smithetal.,1990),andparentalskillsinfluencecompliancewithdiet
(FehrenbachandPetersen,1989),treatmentofPKUneednotdisruptthefamily(Kazaketal.,1988)afactofsomerelevance

whencounselingafamilyaboutrecurrencerisksandtheburdenofPKU(seeScriverandClow,1988,Riessetal.,1987,and
BarwellandPollitt,1987,forviewsofthe"burden"byPKUfamilies).
Tosummarize,(1)thebenefitsofearlytreatmentinamelioratingtheclinicalimpactofPKUarewellestablished,(2)earlytreated
childrenhavemeanIQscoresapproximatelyhalfastandarddeviationlowerthanscoresfortheirunaffectedsiblingsandthe
correspondingpopulationnorm,and(3)earlytreatedsubjectsmayexhibitsomedegreeofintellectualimpairmentattributableat
leastinparttoeventsinearlychildhood.Nevertheless,whilesubtleneuropsychologicalimpairmentsarecauseforconcern,most
earlytreatedPKUchildrenfunctionwithinthebroadnormalrangeofabilityandcanattendordinaryschools.

DoesNonPKUMildHyperphenylalaninemiaRequireTreatment?
TherehasbeenanabidingquestionwhethermildHPA(<600M)shouldbetreated.Thequestionwastentativelyansweredover
30yearsago(Levyetal.,1971)whenasmallgroupofuntreatedindividualswithmildHPAwereobservedtohavenormalcognitive
development,andtheconclusionwasdrawnthatdietarytherapyisnotneededformildHPA.Arecentpaper(Weglageetal.,2001)
supportstheearlierproposal.Inthisanalysis,31nevertreatedadolescentsoradultindividualswithpersistentHPAwereevaluated
withcomprehensivepsychologicaltesting,cranialMRI,MRS,andPAHgenemutationanalysis.Theirmutantgenotypeswere
compatiblewiththenonPKUHPAphenotype.Therewerenodeficitsinthevariousmeasurements,leadingtotheconclusionthat
"dietarytreatmentisunlikelytobeofvaluetopatientswithmildHPA(<600M)."Thefindingsalsosuggestthatdietarytreatmentof
suchindividualscouldparadoxicallyincuracostofcognitivedevelopment.Theauthorswereprudentandadvisedthateverypatient
identifiedinthenewbornperiodandpotentiallyinthisclassbefollowedcarefullyinthefirstyearoflifetodetectanychangeof
status.Occasionallychildrenwho,asinfants,hadphenylalaninevaluesconsideredtobeinthemildHPArangeandcontinuedona
normaldietlaterhavebeenfoundtohavelevelsintherangeofPKUrequiringdietarytherapy(Berlinetal.,1995Weglageetal.,
1998).TheyalsoremindedthereadersthatthesituationofmaternalHPAalwaysmustbekeptinmind.
ResponsetoTerminationofTreatment
Therehaslongbeenaconcernthatprematureterminationoftreatmentinmidchildhoodmightimpairlaterintellectualand
neuropsychologicalperformance(Cabalskaetal.,1977Smithetal.,1977,1978).Asaresult,quiteearlyinthehistoryofPKU
treatmenttherewasasmallcontrolledtrialofcontinuedversusterminatedtreatment(Holtzman,1977Holtzmanetal.,1975).Only
asmalldeficitinIQ(4points)wasfoundin6yearolds2yearsafterterminationoftreatment.Thusitseemedthattoterminate
treatmentatthisearlyagewouldnotbecauseforconcernnonetheless,theauthorsofthereportrecommendedalongerperiodof
observationinalargernumberofsubjects(Holtzmanetal.,1975).Conditionsforthatlargerstudywerefulfilled(Cabalskaetal.,
1977Kochetal.,1982Smithetal.,1977,1978Williamsonetal.,1979)thisprovidedunambiguousevidencethatIQscoresin
PKUpatientsusuallyarecompromisedbyprematureterminationoftheirtreatment(Table778).
Table778:EffectofTreatmentTerminationonIQscoresofPatientswith
Phenylketonuria(AUTHOR:Pleasesupplyname,datereferencesforthe"Reference"
column.)
ViewLarge|SaveTable
Table778:EffectofTreatmentTerminationonIQscoresofPatientswith
Phenylketonuria(AUTHOR:Pleasesupplyname,datereferencesforthe"Reference"
column.)

Design

IQ
Points
(Change)

Significance

Tested
atAge
(Year)

Months
offDiet
(Mean)

Reference

Controlled
Trial

14

3.8

NS

24

731

Longitudinal

17

6.3

Yes*

41

733

Longitudinal

7.8

Yes*

3.6

12

734

Longitudinal

14.2

Yes*

6.6

48

734

Longitudinal

16

8.3

<.001

11

34

735

Longitudinal

9.1

<.05

13

44

735

Longitudinal

30

0.08

NS

24

736

Controlled
Trial

55

<.01

24

737

Controlled
Trial

115

4.5

NS

24

738

Longitudinal

14

14

<.005

11

740

*Statedtobesignificant.
Significanceappliestotreatmentterminatedpatientsonlythosewhocontinued

treatmentonarelaxedregimenhadlesschangeinIQscores.(Smithetal,1978).
Thegroupthatterminateddietbeforeage8yearsalsoshoweddeteriorationin

readingandspellingachievement(WRATscores)relativetothetreatment
continuationgroup.
Thephenomenonhasbeencalledlateonsetphenylalanineintoxication(Woolf,1979),theinitialfeaturesofwhicharelikethosethat
followanyinducedincreaseinbloodphenylalaninelevelsofthePKUsubject(Epsteinetal.,1989Krauseetal.,1985,1986).A
significantfallintheIQscore(by530points)isobservedwhentreatmentisterminatedinmidchildhood(Cabalskaetal.,1977
Smithetal.,1978)thedecreaseissmallerwhentreatmentisrelaxedratherthanterminated(Smithetal.,1978).Itisnowgenerally
agreedthatprematureterminationoftreatmentisfollowedbydeficitsinperformanceinmostpatients(Behbehari,1985Kochetal.,
1997Louetal.,1985Matthewsetal.,1986Seashoreetal.,1985Smith,1985).Onecontrolled,prospectivestudyofearlyand
welltreatedPKUpatients,however,foundnodeteriorationincognitiveabilityfollowingterminationoflowphenylalaninedietsover
thenext10years(Reyetal.,1996).
TerminationoftreatmentaffectsmorethantheIQscore.Thereareabnormalneurologicfeaturesinlaterlife(McDonnelletal.,
1998),deviantelectroencephalographicfindings(Behbehari1985Seashoreetal.,1985),anddecreasedlevelsofneurotransmitter
metabolitesinbodyfluids(Louetal.,1985Neilsenetal.,1988).Thelastareanalogoustothefindingsproducedbypurposeful
phenylalanineintoxication(Epsteinetal.,1989Krauseetal.,1986)impairedvigilanceandreactiontimes(Louetal.,1985),deficits
insocialquotientsmeasuredontheVinelandscale(Matthewsetal.,1986),andneuropsychologicalandbehavioralproblems(Koch
etal.,2002Risetal.,1997Smith,1985)arealsoseen.
TerminationoftreatmentisalsoassociatedwithabnormalbrainwhitemattervisualizedbyMRI(Bicketal.,1991McDonnelletal.,
1998Shawetal.,1991Thompsonetal.,1990,1991aVillasanaetal.,1989).Theclinicalrelevanceofthisfindingisunclear
becauseitoccurseveninwelltreatedpatientsstillontreatment(Thompsonetal.,1991a).Nonetheless,thereissomeevidence,
notallofitconsistent(Bicketal.,1991Pearsenetal.,1990Shawetal.,1991),thattheMRIchangesaremoresevereinpatients
withahistoryofhigherlevelsofphenylalanine(Shawetal.,1991Thompsonetal.,1991a).Forexample,twoadolescentpatients
withnonPKUHPA,bothuntreated,hadnormalMRIfindingstheirphenylalaninelevelshadapparentlyneverexceeded600M
(Bicketal.,1991).TheMRIfindings,ifofashorterdurationintreatedPKUpatients,disappearwhentreatmentreducesblood
phenylalaninelevels(Battistinietal.,1991Thompsonetal.,1990Walteretal.,1997),implyingthattheabnormalityinthis
circumstanceisoneofdysmyelinationratherthandemyelination(Walteretal.,1997).Althoughthechangesinwhitematterare
morecommonthanovertneurologicchanges[MedicalResearchCouncil(UK),1993],andperhapsmoreprevalentthan
measurableneuropsychologicaldeficits,theirassociationwithlongtermHPAofsomedegreeseemsundeniable.
StringencyofTreatment:TheThresholdArgument
IsthereatolerabledegreeofHPAinthetreatedpatientwithPKUandintheuntreatedpatientwithnonPKUHPA?Thereis,sofar,
noclearanswertothisquestion.Untilallcausesofcognitivedysfunction,MRIabnormalities,andthelikeincludingthedietitself
(Cockburnetal.,1996Eikenetal.,1996b)havebeenferretedout,weareleftwiththeconcernthatanyincreaseinphenylalanine
couldbeatroublemakerateverystageoflifeinpatientswithHPA.Nevertheless,personswithuntreatednonPKUHPAachieve
normalcognitivefunctioninthepresenceofpersistentbutmodestHPA,asshownbyabsenceofsignificantabnormalitiesin
untreatedadolescentsandadultswithwelldocumentednonPKUHPA(<600M)(Weglageetal.,2001).Thelatterfindingsrequire

ustoquestionwhetherthephenylalaninerestricteddietmodalityhassomeundesirableconsequencesthatwereavoidedinthe
untreatedHPApatients(seethesection"Pathogenesis:MetabolicPhenotypesandNeurotoxicity"above).
Inreality,fortreatedpatientswithPKU,theremaybenothresholdvalueforbloodphenylalanineatwhichthebrainwillescapean
effectofpersistentHPA[MedicalResearchCouncil(UK),1993].Theresultsinonestudy(Micheletal.,1990)implythataneven
verymodestHPAlevel(<300Minthetreatedpatient)mayincurcognitivedysfunction,butagain,thisfindingcouldimplicate
somethingharmfulintheprocessormodalityoftreatmentitself.Evidencefortheargumentagainstsafetyinanarbitrarythreshold
valuehascomemainlyfromtreatedpatientswithclassicPKU(Flynn,1984Micheletal.,1990Smithetal.,1988,1990)butalso
hasbeenadvancedfromotherobservations(Diamond,1994Smithetal.,1991):Inalltreatedpatients,outcomecorrelateswiththe
qualityoftreatmentandtheambientbloodphenylalaninevalue.Meanwhile,theprudentpositionremainstheoneproposedin
recentguidelines:TreatPKUasearly,aswell,andforaslongaspossible[Cockburnetal.,1993MedicalResearchCouncil(UK)
1993NationalInstitutesofHealthConsensusDevelopmentPanel,2001].
StringencyofTreatment:Prolongation
Althoughsomepatientswereadvisedlongagothattreatmentwouldbeforlife(Naughten,1989),mostreceiveddifferentadviceat
thetimeofdiagnosis.IntheUnitedStates,forexample,therewasnostandardpolicyforcontinuationorterminationoftreatment
untilthelate1970s(Schuettetal.,1980).Whentheevidencebegantoemergethatearlyterminationoftreatmentmighthave
undesirableconsequences,themajorityofAmericanclinicschangedpolicyandbegantorecommendtreatmentfortheduration
(SchuettandBrown,1984).Unfortunatelyforpatientswhohadalreadydiscontinuedtreatment,itwasdifficulttoreinstateit
successfully(Finkelsonetal.,2001Hoganetal.,1986Schuettetal.,1985).Withhindsight,itseemsillogicaltoterminatetreatment
inpatientswithclassicPKU,butpatients,families,andadvisorsallhadtheirreasonstodosoamongthem,theadversesocial
conditionsofthetreatment(restrictionsinlifestyle),theinadequateorganolepticpropertiesofthephenylalaninerestricteddiet
products(poorflavorandoffensivesmell),andtheawkwardnutritionalconsiderations(howtokeeptheintakeofphenylalaninefar
belownormalwhentheintakeofothernutrientsmustbeincreasingwhilethepatientisgrowing).
RiskofbraindysfunctionowingtoHPA,however,persiststhroughoutlife,albeitlessinadulthoodthaninchildhoodduringtherapid
developmentofbrainfunctions.Accordingly,guidelinesrecommendthattreatmentshouldcontinuebeyondchildhood,through
adolescenceandintoadulthood,certainlyduringconceptionandpregnancy,andperhapsforthelifetimeofallpatients[Cockburnet
al.,1993MedicalResearchCouncil(UK)1993NationalInstitutesofHealthConsensusDevelopmentPanel,2001].Compliance
canbebeneficial(Kochetal.,1999)butisclearlydifficulttoachieve(Walteretal.,1993)andwillrequirebettersupportprograms
(LevyandWaisbren,1994)andbetterresourcesthanarepresentlyinplacefortreatmentduringlateadolescenceandintolaterlife
(Fischetal.,1997McDonnelletal.,1998).Aslongasthedietarymodeoftreatmenthastheimperfectionsfamiliartoanyonewho
hasexperiencedit,compliancewillbeaproblembeyondchildhood.Adolescentpatients,welltreatedinthefirstdecade,can
tolerate3monthsofHPA(atlevels10001300M)withoutevidenceofneurotoxicity(Griffithsetal.,1997b,1998),butwhether
theirexecutivefunctionsareimmunetoharmorwhetheralongerperiodofHPAcanbetoleratedhasyettobeanalyzed.Abnormal
neurologicfeaturescanoccurinadultpatientsafterterminationoftreatmentwithariseofbloodphenylalaninetothe
aforementionedlevels(McDonnelletal.,1998).
TreatmentforthePreviouslyUntreated:Benefit
ItwasinanuntreatedchildwithPKUwhowasmentallyretardedandhadbehaviorabnormalitiesthatBickelandcolleaguesfirst
demonstratedthebenefitofthephenylalaninerestricteddiet(Bickeletal.,1954).Ondiet,herbloodphenylalaninelevels
decreased,herbehaviorimprovedwithincreasedawarenessandresponsiveness,andshemadedevelopmentalgains.Thisinitial
resultofdietarytreatmentandsubsequentfurtherevidenceofbenefitinmentallyretarded,nevertreatedchildren(Armstrongetal.,
1957Hsiaetal.,1958Woolfetal.,1955)presumablytheresultofrelievingtheintoxicationofextremeHPA(Woolfetal.,1955)
hasledtoanexaminationofdietarybenefitinretardedadultswithPKU,usuallythoseingrouphomesorinstitutions(Baumeister
andBaumeister,1998HarperandReid,1987Hoskinetal.,1992Kochetal.,1999Marholinetal.,1978Williams,1998
YannicelliandRyan,1995).Theobjectiveisnottoincreasethepresumablyunchangeablecognitivefunctionbuttohaveapositive
effectonbehaviorbyreducinghyperactivityanddevelopingsomedegreeofappropriatesocialinteraction,therebylesseningthe
threatofphysicalinjurytoselfandothersandreducingneedforintensivenursingcareandpsychotropicmedication(Levy,2000).
Studiesofbenefitfromtheinstitutionofaphenylalaninerestrictivedietintheseadultshavehadmixedresults.Individualcase
reportsgenerallyhavereportedmarkedreductionsindisturbedbehavior(HarperandReid,1987Hoskinetal.,1992Williams,
1998),butcontrolledstudieshaveshowninconsistentresults,withsomepatientsbenefitingbutothersunchanged(Marholinetal.,
1978).Asurveyoftheexperienceamongresidentialfacilitiesforthementallyretardedfoundthat46percentofthepatientswith
PKUhadpositivebehavioralchangeswhenonaphenylalaninerestricteddiet(YannicelliandRyan,1995).Fromaneconomic
standpoint,thegreatestbenefitseemstobeinuntreatedpatientswhorequirealargeamountofnursingcare(BrownandGuest,
1999).Itwouldseemthatdietshouldbeattemptedinthesepatientsandcontinuedifthereisasignificantreductionintheneedfor

care.However,thismaynotbeevidentuntilthedegreeofHPAhasbeensubstantiallyreducedforseveralmonths.Consequently,if
thereisnoimprovementoverashorterperiodofdiet,itshouldnotbeconcludedthatdietisineffective.
TheModalitiesforDietaryTreatment:Implications
ProteinintakebyitselfcannotbereducedsufficientlytopreventHPAinPKUwithoutcausingdeficienciesofotheressentialamino
acids.PKUpatientsare"consumerswithspecialneeds"(Scriver,1971),andtheyrequireelectiverestrictionofphenylalanineintake.
Tolerancefordietaryphenylalanine[200500mg/day(Acostaetal.,1983)]tomaintainplasmaphenylalaninelevelsat"nontoxic"
levelsinyoungPKUpatientsisfarbelowthenormalintake.Toleranceincreasessignificantlyonlyduringthelatterhalfofa
pregnancy(Thompsonetal.,1991b).
AsyntheticdietfreeofphenylalanineandpresumedtobeadequateinothernutrientsisusedtotreatPKU.Severalcommercial
productsprovidetheaminoacidsinadequateamounts(AmericanAcademyofPediatrics,1976).Althoughtheseproductshave
nutrientcompositionsandproportionsvastlydifferentfromhumannutrientcompositionsandproportionsvastlydifferentfrom
humanmilk,somehavebeenformulatedtoresemblehumanmilkinmostaspects(LinkandWachtel,1984),andeffortsare
constantlybeingmadetoimprovetheircompositionandorganolepticproperties(Acostaetal.,1977Kindtetal.,1983Kindtand
Halvorsen,1980).However,theyarestillunlikehumanmilkintheircontentofessentialfattyacids(Agostonietal.,1998Cockburn,
1994Farquharsonetal.,1993Guetal.,1995Rivaetal.,1996).WhereasPKUandnormalsubjectshavesimilarnitrogen
requirements(Acostaetal.,1977Kindtetal.,1983KindtandHalvorsen,1980),classicPKUpatientsprovideevidencethatthe
recommendationsforproteinrequirementsinhumanscanberefined(Kindtetal.,1984,1988).TheartificialdietsusedtotreatPKU
alterbodycomposition.Theyincurlowerlevelsoftraceminerals(e.g.,zinc,selenium,iron,copper,andchromium)andcholesterol
(Gropperetal.,1988Scriveretal.,1987).AbsenceofpreformedarachidonicacidandtheC22:6(n3)fattyacid[docosahexaenoic
acid(DHA)]intreatmentdiets(Agostonietal.,1998Giovanninietal.,1966)distortsthefattyacidcompositionofplasmaand
erythrocytesinpatientsandperhapsalsointhebrain(Acostaetal.,2001aCockburnetal.,1996Gallietal.,1991Giovanniniet
al.,1966Moseleyetal.,2002).
TreatmentofPKUcanbemademorephysiologicandplasmaphenylalaninehomeostasiscanbeimproved(MacDonaldetal.,
1998)bydistributingproteinintakethroughouttheday(AcostaandYannicelli,1994)andbyincreasingthenonphenylalanine
proteinintaketoatleast3g/100kcal(AcostaandYannicelli,1994).Thesemeasuresalsomayavoidthepoorgrowthseeninsome
treatedpatients(vanderSchotetal.,1994VanSpronsenetal.,1997)and,whencombinedwithmoreappealingtreatment
products,maypreventfeedingproblems(MacDonaldetal.,1994).
Thedietarymodeoftreatmenthasmanypitfalls.First,therecanbelongtermHPAfromexcessivetreatment(Hanleyetal.,1970
Smithetal.,1990)orpersistentHPAwithinadequatetreatment,andbothwillaffectoutcomeadversely.Second,thepoor
organolepticproperties(tasteandsmell)ofmostlowphenylalanineproductsaffectcomplianceadversely.Third,intheabsenceof
fullunderstandingofpathogenesis,andwithoutassurancethatphenylalaninelevelsinthebraincanbenormalizedbydietary
meansaloneinPKU,itisstillunclearwhethersupplementsofcertainfreeLaminoacids(i.e.,leucine,isoleucine,valine,tyrosine,
tryptophan,andlysine)arehelpful,necessary,oradvisable(Kaufman,1976Hommes,1989HommesandLee,1990Berryetal.,
1982Jordanetal.,1985Huetheretal.,1985)(seesection"Pathogenesis:MetabolicPhenotypesandNeurotoxicity"above,aswell
asPietzetal.,1999).AsupplementofLtyrosine(100mg/kgofbodyweightdaily)increasedplasmatyrosinebutproducedno
changeinneuropsychologicalperformancesaccordingtodoubleblindcrossoverstudieswithmeasurementsofthree
neurophysiologicparameters(Pietzetal.,1995aSmithetal.,1998).Othersclaimsomebenefitfromtyrosinesupplementsata
higherdose(180mg/kgperday)(Lou,1994),althoughtyrosinesupplementationhasnotconsistentlybeenfoundtoimprove
neuropsychologicalfunctionintreatedPKU(Kalsneretal.,2001PoustieandRutherford,2000).Supplementsofbreastmilkare
consideredbeneficialinyoungPKUinfantsondiettreatment(McCabeetal.,1989),andthesupplyofdecosahexaenoicacid(DHA)
inbreastmilkmaycontributetohigherIQscoresinPKUpatientstreatedearlywithbreastmilksupplements(Rivaetal.,1996).
AnalternativetofulldietarytreatmentinPKUhasbeenproposedbytheDanishgroupforuseinnevertreatedadults,previously
treatedbutoffdietadolescents,andadultssupposedlyondietbutwhocannotcomplywiththerigidrequirementsofthediet.This
alternativeconsistsofreducednaturalproteinintakeandasupplementoflargeneutralaminoacids(LNAAs)excluding
phenylalanineandenrichedintyrosineandtryptophan.TheconceptisthatbycompetitionwithotherLNAAsattheBBB,braininflux
ofphenylalanineisreduced,therebyloweringthelevelofphenylalanineinthebrain(AndersonandAvins,1976)(seethesection
"Pathogenesis:MetabolicPhenotypesandNeurotoxicity"above).ThesupplementhasbeenusedextensivelybytheDanishgroup
withreportedimprovementinbehaviordomainssuchasmotorskills,socialization,communication,emotionality,attentionspan,and
frustrationtoleranceinamajorityofthepatients(Kalkanogluetal.,2002).ReportsfromPolandandtheUnitedStatesindicate
reductioninbrainphenylalanine(Kochetal.,2003)andsuggestimprovementinpsychologicalandemotionalhealth(Giewskaetal.,
2001Kochetal.,2003).ArecentlypublisheddoubleblindedcrossoverstudyofLNAAsorplacebofromAustraliaindicatedthat
LNAAshadsomeeffectinloweringthebloodphenylalaninelevelandinimprovingexecutivefunction,especiallyinthosewhowere

notindietarycompliance(Schindeleretal.,2007).Preliminarydatafromaveryrecentstudyof4patientsonLNAAsfor1yearwho
wereonanormaldietindicatedadeclineofapproximately50%inthebloodphenylalaninelevel(MichalsMatalonetal.,2008).An
accurateassessmentofitsbenefitsawaitsobjectivestudiesthatmaysoonbeunderway.
IstheDietModalityforPKUTherapywithoutRisk?
OutcomewithdietarytreatmentisnotoptimalformanypatientswithPKU(seethesection"TreatmentofPKU"above).Thecause
generallyhasbeenassumedtobeimperfectcompliancewiththetreatmentprotocolleadingtoattendantHPA.Butcouldsuboptimal
outcomebeafunctionofthetreatmentmodalityitselfthephenylalaninerestricteddietratherthanpersistenceofHPA?Perhaps
toxicityorimportantdeficiencyinthedietmodalityistheproblem.
Cockburn(1994)notedthat"muchoftheincreaseingraymatterweight[ofhumanbrain]isduetothedevelopmentofthecomplex
arborisationsandsynaptosomeformation,whichsubserveneuronalfunctionandthelearningprocesses.Myelinationproceeds
rapidlyafterbirthandinthisprocessneuroglialcellsenveloptheaxonsofcorticalneurons.60percentofthetotalenergyintake
oftheinfantduringthefirstyearisutilizedbythebrainandmuchoftheenergyusedtoconstructneuronalmembranesanddeposit
myelincomesfromfatinhumanmilkandinfantformulas.Fat,however,isnotsimplyasourceofhydrocarbonforenergyproduction
butiscomprisedofaseriesofcomplexhydrocarbonstructures[fattyacids]necessaryforthecreationofmembranes."
Cockburn,hiscolleagues,andothers(Agostonietal.,1998Giovanninietal.,1966)havebuiltacasefortheimportanceoflong
chainpolyunsaturatedfattyacids,notablyDHA,inthedietofhumaninfants.Thisfattyacidispresentinhumanmilkbutatmuch
lowerconcentrationsintheinfantformulascurrentlyinuse.Moreover,theliverisunabletosynthesizeDHAfromlinolenicacid
[C18:3n3]inthefirstmonthsoflife(Farquharsonetal.,1995a).TheDHAcontentofcerebralcortexissignificantlyhigherinbreast
fedinfantsthaninformulafedinfants(Farquharsonetal.,1995b).The"requirement"forDHAcouldbemetbyadietarysupplement
of30mg/day(approximately0.2g/100gfattyacid)inthedietofformulafedinfants(Farquharsonetal.,1993).Cockburn(1994)
citesevidence"thatpreterminfantsfedhumanmilkhaveahigherdevelopmentalstatusat18monthsandahigherintelligence
quotientinlatechildhoodthanthosefedinfantformula."Thisoutcomeisthoughtnottobeexplainedbythesocialenvironment
(Porter,1996).ArethesefindingsrelevanttoPKU?
LongchainpolyunsaturatedfattyacidssuchasDHAandarachidonicacidarepresentassuchonlyinanimalfoods(Agostonietal.,
2000).ThuspatientswithPKUcanhaveaverylowdietaryintakeofDHA[andarachidonicacid(Agostonietal.,1998)]when
phenylalaninerestricteddietproductsreplacecoworhumanmilkfeedings.ThemajorityofPKUdietsininfancyalsoprovideonlya
lowintakeoflinolenicacid,whichmightreplaceDHAwhenhepaticsynthesisofDHAmatureslaterininfancy(Cockburnetal.,
1996).PatientswithPKUhavesignificantlyreducedconcentrationsofDHA(andotherlongchainpolyunsaturatedfattyacids)in
plasmaanderythrocytes(Moseleyetal.,2002).DiettreatedchildrenwithPKUhaveerythrocytemembranesthatarepoorly
populatedintheDHAmolecules(Cockburnetal.,1996Giovanninietal.,1966),adeficitlikelytobereflectedinmembranesofthe
nervoussystem.Breastfeeding,inthe20to40daypostnatalintervalbeforephenylalaninerestricteddietarytreatmentusually
began,islinkedtohigherIQscoresamongpatientswithPKU(Rivaetal.,1996).Accordingly,disappointmentsintheoutcomeof
treatmentmightberelatedasmuchtodeficiencyofDHAinearlyinfancyastochronicHPA.Thehypothesisdeservesfurther
investigation,carefulreviewofdietarycompositions,andpossiblesupplementationwithDHA,particularlyduringinfancy,inthe
treatedpatientwithPKU.Thefindingsalsoheightentherelevanceinseekingpossiblealternativestothedietarymodeoftreatment
(seethesection"TreatmentofPKU"above).
TheSpecialProblemofAspartame
LAspartylLphenylalaninemethylester(trivialnameAspartame)isanartificialsweeteneronhydrolysis,itreleasesfreeL

phenylalanine,Lasparticacid,andmethanol.Aspartameismarketedwidely.Forexample,ithasreplacedmorethan12percentof
thetotalsweetenersconsumedannuallyinAmerica.Itswideavailabilitymakesitapotentialhazardinthedietarymanagementof
PKU(e.g.,1quartofKoolAidcontains280mgphenylalanine,morethanhalfthedailyallowanceoftheaminoacidforaPKU
patient).Accordingly,thosewithHPAwhoareondietandtheirprovidersmustknowaboutAspartame,readproductlabels,and
adjustdietaccordingly.Despiteclaimstothecontrary,AspartameseemstopresentnohazardtoPKUheterozygotes,including
pregnantheterozygotes(Caballeroetal.,1986).Studiesinnormalsubjectsandheterozygotes,doneatninetiethandninetyninth
centilesofprojectedAspartameintakeswithsinglebolusingestion(theworstcasescenario),foundnosignificantdisturbanceof
bloodphenylalanine,tyrosine,andlargeneutralaminoacidlevelsinresponsetotheload(FilerandStegink,1989)thesefindings
havebeencorroborated(Curtiusetal.,1994).
PETfollowingingestionofAspartameshowedanonsignificantdecreaseinbrainaminoacidtransport,asmeasuredwithaninert
marker(Koeppeetal.,1991).Aspartameloadingdoesnotchangebehaviorinrats(Mullenixetal.,1991)andhasnoeffecton
photicallyinducedmyoclonusinbaboons.Notwithstandingsuchesotericstudies,PKUheterozygoteswillfindreassurancethata
hamburgerandamilkshakecansafelyreplaceacravingforAspartameflavoredsustenance.

Insummary,theoveralladvantagesoftreatingHPAwiththephenylalaninerestricteddietareclear:(1)reversalofkeybiochemical
abnormalities,(2)improvedneuropsychologicalperformance,and(3)preventionofneurologicdeterioration.Thedisadvantagesare
alsoclear:(1)difficultyinobtainingfullcompliancewiththetreatmentprocess,(2)theneedforcomplexsocialsupport,(3)risksof
nutrientimbalanceanddeficiency,and(4)persistinguncertaintythatdietarytreatmentinitspresentformulationsandbyitselfcan
achieveallthatisdesired(Cockburnetal.,1996Rivaetal.,1996Smith,1994a).

CofactorTherapy:TetrahydrobiopterinResponsivePAHAlleles
Over30yearsago,MilstienandKaufmansuggestedthepossibilitythatatetrahydropterinmightbeusedtotreatthosewithPKU
whostillpossesssomeresidualphenylalaninehydroxylaseactivity(MilstienandKaufman,1975a).Thishasnowbeenshowntobe
true.Beginningin1999(Kureetal.,1999)withthedemonstrationthatamildformofPKUrespondedtopharmacologicdosesof
tetrahydrobiopterinbyasubstantialreductionintheserumphenylalanineconcentration,studieshaveshownthatthereisasubset
ofpatientswithPKUinwhomtetrahydrobiopterintherapylowersphenylalanine(FiegeandBlau,2007BerneggerandBlau,2002
Fiorietal.,2005Matalonetal.,2004Mitchelletal.,2004Leuzzietal.,2006Levyatal.,2007Muntauetal.,2002,Lindneretal.,
SpaapenandRubioGozalbo,2003Burtonetal.,2007Hennermannetal.,2005).NoneoftheseBH4responsivepatientshasan
abnormalityinthesynthesisorrecyclingofBH4cofactor.
Theoverallfrequencyofresponsivenessinstudiesoflargelyunselectedpatientshasvariedfrom20to40percent(Burtonetal.,
2007FiegeandBlau,2007Mitchelletall,2005Hennermannetal.,2005Bovedaetal.,2007).Provingcorrectthepredictionof
MilstienandKaufmanthatresidualphenylalaninehydroxylaseactivitywouldberequiredfortherapeuticbenefitfroma
tetrahydropterin(MilstienandKaufman,1975a),mostrespondershavemildPKUormildhyperphenylalaninemia(Muntauetal.,
2002FiegeandBlau,2007Fioreetal.,2005Leuzzietal.,2006).Specifically,thefrequencieshavebeen80to95percentamong
patientswithmildhyperphenylalaninemia,50to75percentamongpatientswithmildormoderatePKU,butonly5to15percent
amongthosewithclassicPKU(FiegeandBlau,2007Mitchelletal.,2005Leuzzietal.,2006Fioreetal.,2005Hennermannet
al.,2005Desviatetal.,2004).ThemostfrequentregimenfordeterminingBH4responsivenessinthesestudieswastoadminister
20mg/kgtetrahydrobiopterin(6RBH4)asasingleordivideddoseandtocomparethebloodphenylalanineconcentrations
obtainedbeforeandwithin24to36hoursafterdosing.Anotableexceptionwasalargemulticenterstudyof485patients
administeredadailydoseof10mg/kgfor8dayswithresponsivenessdeterminedbythebloodphenylalanineconcentrationonthe
eighthday(Burtonetal.,2007).A30percentorgreaterreductioninthebloodphenylalaninelevelhasbeenthestandardfor
responsiveness.
ThecriticalquestionabouttheefficacyofBH4inthetreatmentofPKUiswhetherlongtermtherapyiseffectiveinreducingthe
stringencyofthephenylalaninerestricteddietoreliminatingtheneedfordiet.Severalstudieshaveexaminedthisquestionby
treatingpatientswithBH4from12monthstoover5years(Hennermannetal.,2005Lambrushchinietal.,2005Shintakuetal.,
2004Ceroneetal.,2004Steinfeldetal.,2004Trefzetal.,2005BelangerQuintanaetal.,2005).ThesereportsshowthatBH4
responsivenesscontinuesandthatsomepatientscanmaintainbloodphenylalaninecontrolwithoutdiet,whereasotherscan
maintaincontrolwithasubstantiallyliberalizeddiet.
TheprimarybiochemicaleffectofBH4inthisnewphenotypeincreasesthecatalyticfluxofphenylalaninebyincreasingPAHenzyme
functionsecondarily,itamelioratesthemetabolicphenotype(Mutauetal.,2002Okanoetal.,2004).ThisimportantPAHenzyme
phenotypealsohasbeenanalyzedfromtheviewpointofstructuralbiology(ErlandsenandStevens,2001Erlandsenetal.,2004).
Mapping15differentPAHmutationsassociatedwithBH4responsivenessontotheirstructuralmodelofaPAHmonomer,Erlandsen
andcolleagues(2004)determinedmultiplemechanismsforBH4responsiveness.Forsomealleles,BH4therapyovercomeskinetic
variantsaffectingbindingofBH4andcooperativityofsubstratebinding.AdditionalBH4bindingmutantshavebeenreported
subsequently(Aguadoetal.,2007Perezetal.,2005).Forotheralleles,BH4therapyactsasachemicalchaperonetoprevent
misfoldingofmutantPAHanditsconsequentproteolyticdegradation,thusmaintainingPAHinanactiveconfiguration(althoughonly
rarelyrestoredtowildtypelevels).ThisseemstobethemorefrequentmechanismofpharmacologicBH4action.
TheBH4responsiveallelesareaparticularsubsetofthosecausingPKUandrelatedformsofhyperphenylalaninemia(see
www.pahdb.mcgill.caandwww.BH4.org/forcataloguesandcommentary).ThemajorityofBH4responsivePAHallelesmaptothe
catalyticdomainoftheenzymemonomerinoneoftworegions:inthecofactorbindingregionsthemselvesorinregionsthatinteract
withthesecondaryelementsinvolvedinbinding(Zurfluhetal.,2008).Theremainingallelesmaptotheregulatoryor
tetramerizationdomain.Wherevertheallelehascausedmisfoldingoftheproteinwithlossofintegrityandfunction,theresponseto
BH4mayberevealingachaperonelikeeffect,inwhichcasetheBH4responsiveHPAphenotypemaybeprototypesfor
"pharmacologicchaperone"therapyofgeneticdisease(seeScriverandWaters,1999BlauandErlandsen,2004).Consistentwith
thephenotypeofmildPKUormildhyperphenylalaninemia,theBH4responsivemutationsexpressaPAHwithresidualactivity
(Erlandsenetal.,2004).

AclinicaltrialtoidentifyBH4responsivenessshouldbeconsideredineverypatientwithPKUoravariantnonPKUformofHPA.
Recommendationsforperformingthistrialanditsinterpretationhavebeenpublishedrecently(Levyetal.,2007).PAHmutation
analysismaybeveryhelpfulinpredictingresponsivenessaswellasinterpretingunclearresults.
AproprietaryformofBH4(sapropterindihydrochloride,6RBH4KUVANBioMarinPharmaceutical,Inc.,Novato,CA90907)has
becomeavailablerecently.AphaseIIIclinicaltrialofthispreparationindicatedthatadailydoseof10mg/kgfor6weekswassafe
andefficaciousinreducingthebloodphenylalaninelevelby30%ormoreinresponsivepatients(Levyetal.,2007).
BH4therapyislikelytobeveryhelpfulincontrollingthebloodphenylalaninelevelaswellasreducingthestringencyofdietinBH4
responsivepatientswithamaternalPKUpregnancy.However,thesafetyofBH4duringpregnancyhasnotyetbeenassessedinthe
doserequiredtotreatPKU.

EnzymeTherapy
IfenzymaticactivitycouldberestoredinPKUpatients,itwoulddealwiththeprimaryproblem,PAHenzymedeficiency,andthere
wouldbenoneedforaphenylalaninerestrictedtherapeuticdiet.Twoapproachestoenzymetherapyhavebeenconsidered.
EnzymeReplacement
Orthotopiclivertransplantation(inapatientwithchronicendstageliverdisease)completelycorrectedthebiologicphenotypeof
PKU(Vajroetal.,1993).Accordingly,heterologouspartialtransplantationofnormallivertissueorimplantationofnormal
hepatocytescouldreplacelostenzymeactivityinPKUpatients.Itwouldconstitutesomaticmultigenetherapy.Nodedicated
initiativesinthisdirectionhavebeenreported.Intheory,onlyasmallreplacementofenzymeactivitywouldbeneededtoconvert
thismutantautosomalrecessivephenotypefromhomozygousmutanttoheterozygous(KacserandBurns,1981).
Anovelapproachtoenzymetherapyusingphenylalaninehydroxylasebasedfusionproteinshasbeendescribedrecently(Eavri
andLorberboumGalski,2007).Theauthorsusedsmallpeptidescalledproteintransductiondomainsthatfacilitatethepenetration
offusedproteinsthroughthemembranesofeukaryoticcells.TheHIVtransactivatoroftranscription(TAT)peptidehasbeenmost
widelystudiedinthisrole,andTATfusionproteinsaredeliveredefficientlyintoculturedcellsintacttissueandlivetissueswhenthe
agentisinjectedintomice.ThepharmacokineticsoftheTATfusionproteinrevealsthattheliverisitsmaintarget.Accordingly,the
authorsfirstcontructedaTATPAHfusedprotein.TheyalsoconstructedadifferentsetofPAHbasedfusionproteinsusinghuman
hepatocytegrowthfactor.Again,liverwasthemaintarget.ThePAHbasedfusionproteinswereproducedinbacterialcells,and
whenusedinthemouse,theyloweredplasmaphenylalaninelevelswithinminutesofintravenousadministrationofthefusion
protein.TheauthorssuggestthismethodasanalternativeconceptforthetreatmentofPKUamongothermetabolicdiseases.
EnzymeSubstitution
Phenylalanineammonialyase(PAL)(EC4.3.1.5)isarobustautocatalyticproteinwithnorequirementforacofactor(Hodgins,
1971).TheenzymewillconvertLphenylalaninestoichiometricallytoanontoxicderivative(transcinnamicacid)andtraceamounts
ofammonia(Hoskinsetal.,1984).PALissimilarinnaturetohistidineammonialyase(seeChap.80)thecatalysiscompetentform
ofPALrequiresposttranslationalautocatalyticconversionofaserineresiduetodehydroalaninetocreatethecatalyticcenterofthe
enzyme(Hodgins,1971).Efficacyoftheenzymetotreathyperphenylalaninemiainvivohasbeenexaminedandshowntobe
promisingforclinicaluse(GamezandSarkissian,2005).
PALhasbeenadministeredtohumanPKUpatientsusingareactorplacedintheextracorporealcirculation(Ambrusetal.,1987
Larueetal.,1986)theeffectofentericcoatedcapsulesgivenorallytosuchpatientsalsohasbeenexamined(Hoskinsetal.,1980,
1984).Inbothcircumstances,thetrialswerebriefandlimitedinscopebutsufficienttoobserveamodestfallinbloodphenylalanine
values.ThehighinitialcostofPALenzymewaspartiallyovercome,fortheanimalstudiesdescribedhere,byusingarecombinant
genefromRhodosporidiumtoruloides(Gilbertetal.,1985)expressedinnonpathogenicE.coliandpurifiedbycolumn
chromatography(Sarkissianetal.,1999).PALalsohasbeenexpressedinLactococcuslactisto"treat"hyperphenylalaninemicrats
(Liuetal.,2002).
InaratmodelofchemicallyinducedHPA,microencapsulatedPALplacedintheintestinallumenloweredphenylalanine
concentrationinintestinalandsomatictissuesandinplasma(BourgetandChang,1985,1986,1989).
Themutanthydroxylasedeficientmousemodel(McDonaldetal.,2002Sarkissianetal.,2000b)hasbeenusedtodemonstrate
thatrecombinantprotectedPALadministeredorallywillameliorateHPAinthenaturalmutantstate,providingproofsofprinciple
bothpharmacologicandphysiologicforthisformofenzymesubstitutiontherapy(SafosandChang,1995Sarkissianetal.,1999).
ParenteraladministrationofPALreducesbrainphenylalaninelevels(Sarkissianetal.2003)andPEGylatedconjugatesofPAL
reducethesystemicphenylalaninepoolsizeinthemutantanimalmodeloverthelongterm(SarkissianandGamez,2005).

Structurebasedchemicalmodification,withpolyethyleneglycol(thepegylationprocess),ofR.toruloidesPAL,forwhichthereisa
crystalstructureat1.6(Wangetal.,2005),enhancesinvivoactivityofPALanditsmetabolicefficacyitalsoreducesthe
immunogenicityofitsinjectableform(Gamezetal.,2005).HumanTcellepitopemappinghasbeenperformedonPALforMHC
classIandclassIIassociatedTcellepitomes.PEGylationofsurfacelysineresiduescoveredtheimmunogenicregionsofPAL.
Lysineresidueshavebeenremovedoraddedinanunsuccessfulattempttoimprovethetherapeuticenzyme(Gamezetal.,2007).
PALPEGconjugatesotherthanthosewithR.toruloidesincludeAnabaenavariabilis,Nostocpunctiforme,andPetroselinum
crispum.Theyeachhavedefinedcharacteristics,andsomeoftheirderivatives(Gamezetal.2007),e.g.,sitedirectedmutants,
showshortandlongtermdoserelatedcorrectionofphenylalanineconcentrationsinthemutantmousemodel.Theseconjugates
arenottoxic,theyhaveagendereffect(malemicerespondbetter),andtheyrestorepigmentationinthetreatedanimals(CN
Sarkissian,personalcommunication,2007).Whenbiochemicalandstructuralfeaturesaretakenintoaccount,adoublemutant
(C503S,C565S)ofA.variabilishasenhancedthermalstabilityandresistencetoproteoliccleavage(Wangetal.2008)andmay
havethemostpromiseasasubstitutetherapeuticenzymefortreatmentofPKU.

GeneTherapy
ThelimitationsofconventionaldietarytherapyandthetardinessofclinicalinitiativesinenzymetherapygivegenetherapyforPKUa
profileitmightnototherwisehave.ThepromiseliesinputtinganormalhumanPAHgeneinplaceoforinadditiontothemutant
geneinsomaticcellsofthepatient.Germlinetherapywouldconstituteapotential"cure"forthenextandsuccessivegenerationsof
PKUindividuals,butitwillnotbeusedsoonforethicalandtechnicalreasons.Meanwhilethepossibilitiesofsomaticcellgene
therapyforPKUareathand:AclonedPAHcDNAisavailable,waysexisttodelivertheincominggenetotargetcells,targeted
integrationofthegeneintothesomaticnucleargenomeisfeasible,andthereisreasonableassurancethattheincominggenewill
beexpressedandtransmittedtodaughtercells.Ofparticularrelevance,anorthologous(PKU)mousemodel(McDonaldetal.,
2002)isavailableonwhichtopracticesomaticcellgenetherapy.
AhumanPAHcDNAhasalreadybeenexpressedinavarietyofculturedmammaliancells(Ledleyetal.,1986bPengetal.,1988).
ExpressionistransientwhenintegrationofthecDNAisnotstable,butwhenarecombinantretrovirusisusedasavectortoinfect
thecells,integrationisstable,andthePAHgeneistransmittedtosubsequentgenerations.AfunctionalPAHgenethushadbeen
introducedsuccessfullyintoNIH3T3mousefibroblasts,Hepa1amousehepatomacells,andnormalmousehepatocytesinprimary
culture(Peyetal.,2007).HoweverprogresswithPAHgenetransferultimatelymustbemeasuredinthelivingPKUorganism.
Culturesofprimaryhepatocytesfromthephenylalaninehydroxylasedeficientmousehavebeentransfectedwithawildtypemouse
PahcDNA(Liuetal.,1992)usingavariantoftheLNCXretrovirus(MillerandRosman,1989)andwithanasialoorosomucoidpoly
(Llysine)DNAcomplex(Cristianoetal.,1993).ThesetransducedcellsexpresshighlevelsofmousePahspecificmRNAenzyme
activity,aswellasimmunoreactiveprotein.Phenylalaninehydroxylasedeficientmicealsohavebeentreatedinvivoetsituwitha
recombinantadenoviralvectorcontainingahumanPAHcDNA(Fangetal.,1994).AlthoughtheHPAphenotypewasnormalizedin
1week,theeffectdidnotpersist.
ArecombinantadenoviralvectorcarryingthehumanPAHcDNAandacontrolelement,wheninfusedviatheportalveinintoliverof
thePKUmouse,producedsignificantPAHenzymeactivity,buttheeffectdidnotpersist,andrepeatadministrationofthevectormet
neutralizingantibodies(EisensmithandWoo,1996).
Therehavebeennoreportsofsuccess,eitherexvivoorinvivo,withretrovirusmediatedgenetransferinthePKUmousemodel.
Moreover,thereportofaleukemialikedisorderfollowingaretroviralbasedgenetherapytrialisasomberreminderthatcorrectly
targetedgeneinsertionisacriticalelementofretroviralgenetherapyinthehumansubject(Thomasetal.,2003).
Recombinantadenoassociatedviral(rAAV)vectorshavebecomepopular,beingcapableoflongtermtransgeneexpressionandof
transducingnondividingcellswhileevokingonlytrivialimmuneresponse(Thomasetal.,2003).Successfulreconstitutionofthe
normalphenotype(i.e.,pigmentation,bloodphenylalaninelevel,andbehaviour)inthePKUenu2mouseandrestorationofsufficient
hepaticphenylalaninehydroxylaseactivityfollowedinfusion,throughthetailorportalvein,ofanrAAVvector(ideallyofserotype8)
containingmousePahcDNA(Dingetal.,02006Hardingetal.,2006Mochizukietal.,2004)orthehumanPAHcDNA(Ohetal.,
2004,2005).Thetherapeuticeffectcouldpersistupto40weeks(Mochizukietal.,2004),andtheneuropathologicchangesinbrain
haveshownsignsofreversalinthetreatedPKUenu2mouse(Emburyetal.2007).SitespecificgenomeintegrationofaPahcDNA,
achievedinthePKUenu2mousewiththephiBT1phageintegrasecorrectedthevariantphenotype(ChenandWoo,2005),
toleratedrepeateddosageofthetransgene(ChenandWoo,2007)andoverrodeagendereffect(Laipisetal.2003)infemalemice
whereestrogenlimitedthesupplyofBH4cofactorinhepatocytes(Chenetal.,2007).However,contrarytopredictionsthattheir
integrationefficiencywouldbelow,activelytranscribinggenesinnondividingcellshavehaveactuallyexperienceddeletions
followingrAAVvectorintegration(Nakaietal.,2003).Heterologous,nonlivergenetherapyforPKUhasbeenexamined.Itis

understoodinallsuchworkthatcatalyticamountsofcofactor(BH4)mustbepresentwhereverthesiteofexpressionoftheincoming
genemaybe,ifthereistobesustainedPAHenzymefunction.InvitroexperimentswithretroviraltransducedhumanTcellsfrom
PKUpatientsobtainedhighlevelsofPAHenzymeactivity(Linetal.,1997).Pahgeneexpressioninerythrogenicbonemarrowin
PKUmicewassuccessfulatthemolecularlevelbuthadnoeffectonthemetabolicphenotype(Hardingetal.,2003).Humanskin
fibroblastsandkeratinocytesweretransducedinculturewithindependentretroviralvectorsexpressingPAHandGTPCHgenes,the
latterprovidingtheBH4cofactorphenylalanineclearancewasenhancedinthispreparation(Christensenetal.,2000).Would
transformedskingraftsactasametabolicsinkinthehumanpatient?
AhumanPAHgeneplacedinaconstructwithpromoterelementsfrommousemusclecreatinekinasehasbeenstudiedinthePKU
mousemodel(Hardingetal.,1998).ThistransgeneexpressedhumanPAHenzymeactivityinmousecardiacandskeletalmuscle
cellsbutnotincellsofliverorkidney.TheheterologousPAHenzymeexpressioninvivorequiredlargerepeateddosesofBH4to
producethemetaboliceffect.Ifhepatocytetransplantationbecomesfeasibleinhumansubjects[ithasbeendoneinrabbitsand
dogs(Kayetal.,1992)],thepatientsownhepatocytescouldbeobtained,transducedexvivobyoneofthemethodsmentioned
here,andthenreimplanted.Inthemeantime,onesurmisesthatanyrealprogressinhumansomaticcellgenetherapyislikelyto
reflectaperceivedfailureofallotherformsoftreatment,anditwilldependonbettermethodsoftransferandbettergene
expressioninvivo(Dingetal.,2003).

MaternalHyperphenylalaninemia
Comment
Inbornerrorsofmetabolisminteractwiththeprocessofhumanreproduction(VargasandLevy,1998)inseveralways:(1)They
maycauseinfertility,(2)thepregnancymayaffectmaternalmetaboliccontrol,(3)afetalmetabolicdefectmayaffectthepregnancy,
and(4)amaternalmetabolicphenotypemayaffectfetaldevelopment.MaternalHPAisinthelastcategory.
MaternalHPAisharmfultotheembryoandfetusbecauseitisaformofmetabolicteratogenesis(AmericanAcademyofPediatrics,
2001LevyandGhavami,1996)and,assuch,resemblesthefetalalcoholsyndrome(Costaetal.,2002LevyandGhavami,1996)
andtheeffectsofmaternaldiabetesmellitus.Theproblemhaslongbeenrecognized(Dent,1957Levy,2003Mabryetal.,1963),
ashasthepotentialforintrapartumtreatmenttopreventharmtothefetus(AllanandBrown,1968Levy,2003).However,
achievementsstillfallshortofexpectationsbecausewehaveyettolearnhowtoidentify,counsel,andtreateverymotherwithHPA
andthefetusatrisk(Hanleyetal.,1999Scriver,1967).Shouldwefail,theincidenceofHPAassociatedmentalretardationcould
reboundtolevelsexperiencedbeforenewbornscreeningcameintopractice(Kirkman,1992Scriver,1967).

AMetabolicEmbryopathy
MaternalHPAisunquestionablyacauseofembryopathyandfetopathy(LenkeandLevy1980LevyandGhavami,1996Lipsonet
al.,1984Scriver,1967).Itsconsequencesincludemicrocephaly,impairedcognitivedevelopment,congenitalheartdisease,
dysmorphicfacialfeatures,andintrauterinegrowthretardation(LenkeandLevy,1980).Fetalultrasonographyinthesecond
trimesterwilldetectcongenitalheartdiseasebutnotmicrocephalyinthefirsttrimesteritwilldategestationalageanddetermine
whetherthepregnancyisviablefactsrelevanttocounselingandtreatment(Levyetal.,1996a).MaternalHPAcancause
hypoplasiaofthecorpuscallosuminthefetus,butthechangesincerebralwhitematterseeninpostnatalPKUareabsent(Levyet
al.,1996b).
PaternalHPAdoesnoharmtotheproductofconception(Fischetal.,1991Levyetal.,1991).Theoccurrenceandpatternof
congenitalheartdiseaseinmaternalHPAhavebeenstudied(Levyetal.,2001).Itseemstoappearonlywhenthematernal
phenylalaninelevelis900Mormoreandisespeciallylikelytooccurwhenthelevelisgreaterthan1800M.However,evenatthe
highestlevelsofmaternalphenylalanine,onlyathirdoftheoffspringhavecongenitalheartdisease,incontrasttothealmost
invariableoccurrencesofmicrocephalyandmentalretardationatthoselevels(LenkeandLevy,1980).Thisseemstoimplythat
HPAisnecessarybutnotsufficientforcongenitalheartdiseaseandthatasecondfactor,possiblyapolymorphisminacardiogenic
gene,isalsorequired.Inaddition,heartdefectswithcoarctationoftheaortaandhypoplasticleftheartsyndromearesignificantly
overrepresented,andtetralogyofFallotandpatentductusarteriosusaremorefrequentrelativetochildrenwithcongenitalheart
diseaseinthegeneralpopulation.
WhereasthereisnodoubtthatunmodifiedmaternalHPAisanoverallhazardtothefetus,therearethresholdsforparticulareffects:
athresholdat900Mforcongenitalheartdiseaseandapossiblethresholdatthe400Mlevelforreducedcognitivedevelopment
(Levyetal.,1994).Conversely,thereseemstobealineardosedependenteffectonheadcircumferencebeginningatthelowest
elevationsofmaternalphenylalanine(Drogarietal.,1987).TheprudentinterpretationwouldviewanydegreeofmaternalHPAas

hazardoustothefetus,butwithlowerriskwhenthematernalphenotypeisthemildernonPKUformofHPA(Levyetal.,1994,
2003).

Pathogenesis
Thenormaltransplacentalgradientforphenylalaninefavorsthefetus.Thefetal:maternalratiois1.5onaveragewhenthereis
maternalHPA(Brenton,1988Hanleyetal.,1987),butthereisinterindividualvariation,withhighervaluesinearlypregnancy.
Valuesoverallrangefrom1.1to2.9(Hanleyetal.,1987Schoonheytetal.,1994).Itfollowsthatonecannotpredictthe
phenylalaninepoolsizeinaparticularfetusfromthematernalphenylalaninelevelotherthantosaythatitwillbethesameasor
greaterthanthematernalvalue.Fromthis,itfollowsthattheaimoftreatmentistokeepthematernalphenylalaninevalueasnear
tonormalaspossibleasearlyaspossibleinthepregnancy.
Anexcessofphenylalanineisharmfultotheembryo(DennoandSadler,1990Rouxetal.,1992),probablythroughthesame
mechanismsthatmakeitharmfultotheCNSofthepostnatalhumaninfant(BrentonandHaseler,1990LevyamdGhavami,1996).
ThephenylalanineeffectinthefetusistotallydependentonmaternalHPAanditstransportacrosstheplacentathefetusseemsto
havenoabilitytoalterthephenylalaninelevel(Levyetal.,1984)despiteevidenceinvitroofalmostcompletePAHactivityasearly
asthethirteenthgestationalweek(Raiha,1973).Thisaccountsforthelackofadifferenceinteratogeniceffectbetweenthefetus
withPKUandthenonPKUheterozygousfetus(Levyetal.,1992).Theeffectdoesnotrequireaccumulationofmetabolitesderived
fromphenylalanineitself(Levyetal.,1988)becausetheyareapparentlyharmlesstothefetus(Dorlandetal.,1993).Amongthe
likelymechanismsfortheeffectarecompetitionbetweenphenylalanineandotheraminoacidsforuptakebyfetaltissuesand
perhapsbytheplacenta(Brenton,1988BrentonandHaseler,1990Gardiner,1990KudoandBoyd,1990VorheesandBerry,
1989).
SeveralexperimentshavebeenusedtostudythepathogenesisofmaternalHPA(Brassetal.,1982Kirby,Mihagawa,1990Looet
al.,1983SadovaandSutcliffe,1988Satoetal.,1988),butmanyofthesehaveusedpharmacologicmanipulationsthathave
includedinhibitorsofphenylalaninehydroxylationcombinedwithphenylalanineloadtoproduceHPAinthematernalcompartment.
Shouldtheinhibitorshavereachedthefetus,theirowneffectcannotbedissociatedfromtheeffectofphenylalaninealone.
Accordingly,thepreferredmodelisaPAHdeficientmousestrain(Shedlovskyetal.,1993)inwhichtheeffectofthematernal
phenylalaninelevelcouldbemanipulatedpurposefullyandistheresultofmutationaffectingphenylalaninehydroxylationactivityin
thematernalcompartment.ChoandMcDonald(2001)usedthismodeltoshowteratogeniceffectssimilartothoseinhuman
maternalPKU.
Arecentstudydeservesspecialmention.Oberdoersterandcolleagues(2000)foundthatexposureofhumanastrocytes,including
fetalastrocytes,tophenylalaninedecreasedcellproliferationandDNAsynthesis.SincethemicrocephalyobservedinmaternalPKU
isreallyfetalmicroencephaly,thesefindingssuggestthatinhibitionofDNAsynthesiscouldbethemechanismforthefetal
neuropathologyofmaternalPKU.
Onefurtherideaaboutpathogenesisshouldbementioned.Thejustificationhypothesisindictstyrosinedeprivationratherthan
phenylalanineexcessastheimportantpathogeniceventinPKU(Bessmanetal.,1978).Aformaltestofthehypothesis,madeby
measuringphenylalanineandtyrosineincordbloodsamplesfrominfantswithPKUornonPKUHPAandfrommatchedcontrols,
foundnodeficiencyoftyrosineintermoffspringwithHPAborntomothersheterozygousforamutantPAHallele(Scriveretal.,
1980).Nonetheless,thehypothesispersistsasanecho(Bessman,1998)inanewerstudy(Rohretal.,1998)thatfound
midpregnancytyrosinevalueslowerinwomenbeingtreatedformaternalHPAthanincontrols(untreatedpregnantnonHPA
women).BecausetheplasmatyrosinelevelsoftheHPAwomencouldbeincreasedbyadietarysupplementoftyrosine,these
findingsmightbeseentovalidatethejustificationhypothesis(Bessman,1998).Inresponsetothissuggestion(Levy,1998)andwith
acarefulreadingoftheprimarypaper(Rohretal.,1998),however,onefindsnosupportfortheideathattheharmfuleffectsof
maternalHPAareanythingbutaresultofphenylalanineexcess.Whetheronesupplementsmaternaltyrosineintakeornot,the
primarygoalinthemanagementoftheHPApregnancyremainsreductionofthematernalphenylalaninepoolsizetosafelevels.

PreventingtheFetalEffectsofMaternalHyperphenylalaninemia
Preventionrequiresbroadawarenessoftheproblem,recognitionandidentificationofwomenwithHPAinthereproductiveage
group,andaplannedpregnancysothatwithaphenylalaninerestricteddiet,ifnecessary,thematernalplasmaphenylalaninelevel
willbenormaloratleastlessthan400Mfromconceptiontodelivery(AmericanAcademyofPediatrics,2001Cockburnetal.,
1993LevyandGhavami,1996).
BetterawarenessofthehazardinmaternalHPArequiresbettereducationofallthoseinvolvedinmaternalhealth,obstetrics,and
prenatalcareevenwellestablishedprogramsdonottrackallwomenatrisk(Mowatetal.,1999).Betterdetectionofthewomen

withHPAinthereproductiveagegroupwillcomewithacombinationofinitiatives(Mowatetal.,1999).Thefirstinitiativeistotrack
womenalreadyknowntoregionaltreatmentprograms(Waisbrenetal.,1988),perhapsthroughpatientregisters(Cartieretal.,
1982SmithandWolff,1978),whilerecognizingthattrackingandfollowupofcaseswillbebetterforpersonswhoseHPAwas
treatedinchildhoodandadolescenceandlesssoforthosenottreated(Waisbrenetal.,1988).Thesecondmethodistorealizethat
women35yearsofageandolderandthosefromareasoftheworldwithoutroutinenewbornscreeningmightnothavebeen
screenedinthenewbornperiodandthusamongthemwillbewomenwithunsuspectedHPA(Hanleyetal.,1999Mowatetal.,
1999).Theycanbedetectedbymeasuringtheirbloodphenylalanine,andacasefordoingsosystematicallyasahealthcarepolicy
hasbeenproposed(Hanley,1994Hanleyetal.,1999Mowatetal.,1999).Thethirdmethod,byallaccountstheleastdesirable,is
throughthebirthofamicrocephalicinfantsignalingunsuspectedmaternalHPA(Gungoretal.,1996Kochetal.,1990Mowatet
al.,1999NaughtenandSaul,1990SupertiFurgaetal.,1991).
AwomanwithHPAshouldbecounseledaboutthemeritsoftreatmentandtheneedtonormalizeherphenylalaninelevelsbefore
conceptionorasearlyaspossibleinthefirstmonthofpregnancy.Whenthematernalphenylalaninelevelclearlyexceeds400M
(Levyetal.,1994),treatmentshouldbeprescribedandshouldbeginbeforeconception.Withincreasingrecognitionthatthe
phenylalaninerestricteddietshouldcontinueafterchildhood(NationalInstitutesofHealthConsensusDevelopmentPanel,2001),
morewomenwithHPAenteringreproductiveageshouldbeondiet.Nevertheless,becauseofloweredcompliancewithdiettherapy
inoldersubjects,theirphenylalaninelevelwilllikelybetoohighforoptimalmetaboliccontrolofamaternalPKUpregnancy(Schmidt
etal.,1996).Accordingly,eventhesewomenwillrequireintensivedietaryinterventionwhenplanningpregnancyorwhenpregnant.
DatafromtheprospectiveinternationalMaternalPhenylketonuriaCollaborativeStudy(MPKUCS)indicatethatwithoptimal
metaboliccontrol,includingmaintenanceofthephenylalaninelevelat120to360Mfrombeforeconceptiontodelivery,risktothe
fetusshouldbenogreaterthanforthegeneralpopulation(Kochetal.,2000Plattetal.,2000Roch,2003WaisbrenandAzen,
2003).Metaboliccontrolisentirelyachievablethroughstrictadherencetothephenylalaninerestricteddietandisindependentof
thematernalorfetalPAHgenotype(Guttleretal.,2003).Thedegreeofriskwithdelayoftreatment,onceconceptionhasoccurred,
dependsonthegestationalageatwhichmetaboliccontrolisachieved.Regardlessofthebloodphenylalaninelevel,metabolic
controlcanbeachievedwithin2to3daysofstrictdietarycompliance(Duranetal.,1999).Ifcontrolisestablishedwithinthefirst8
gestationalweeks,congenitalheartdiseaseisprevented(Levyetal.,2001),butIQat7yearsofageisreducedtoameanof100
ascomparedwiththemeanIQof105whenmetaboliccontrolisachievedbeforeconception(WaisbrenandAzen,2003).Notably,
offspringcognitiveoutcomeisnegativelycorrelatedwiththenumberofelapsedgestationweeksuntilmetaboliccontrolisachieved
(WaisbrenandAzen,2003).Whenmetaboliccontrolisnotachieveduntil10gestationalweeksorlater,thelikelihoodincreasesfor
congenitalheartdisease,microcephalyorreducedheadcircumference,lowIQorfrankmentalretardation,andpoorsomatic
growth(Levyetal.,2003Roch,2003WaisbrenandAzen,2003).Accordingly,counselingtothefamilyshouldbecautious,and
theyshouldknowthatanydelayinonsettreatmentcanhavesevereadverseeffectsonthefetus.Theyalsoshouldknowthatsuch
delayisseenbysomeasreasontoconsiderterminationofpregnancy.
NowthattheefficacyofrigorouspreconceptionintrapartumtreatmentofmaternalHPAisnolongerindoubt,manystudies(Brown
etal.,2002ClarkandCockburn,1991Drogarietal.,1987Gttleretal.,1990NaughtenandSaul,1990Thompsonetal.,1991b
Waisbrenetal.,1988)areshowinghowdifficultitcanbetoachievethedesiredeffect.Itisclearthatsocialsupport,awareness,and
educationareimportantforcompliancewithtreatment(LevyandWaisbren,1994Shilohetal.,1993Waisbrenetal.,1988,1991,
1995,1997).Forwomenwithoutsocialsupport,a"resourcemother"(usuallythemotherofachildwithPKUwhoistrainedtobea
socialresourcetoawomanwithHPAwhoisplanningapregnancyorispregnant)mayprovidethissupport(St.Jamesetal.,1999).
Intensivepsychotherapyalsomayberequired(Antsheletal.,2002).Thedietmustbestrictlymaintained,bloodphenylalanine
measuredweekly,andphenylalaninedepletionavoidedbyrecognizingthestrikingincreaseinphenylalaninetolerancethatbegins
towardtheendofthesecondtrimesterandacceleratesseveralfoldduringthethirdtrimester(ClarkandCockburn,1991Hyaneket
al.,1988MichaelsMatalon,2003).Thisrepresentsbothenhancedproteinsynthesisinthefetalmaternalunitandperhapsa
capacityforphenylalaninemetabolismgainedduringthelatterpartofpregnancybytheheterozygousfetus.
Thedietconsistsofaphenylalaninefreeaminoacidmixture(whichmayincludevitamins,minerals,fat,andasourceof
carbohydrates)andlowproteinnaturalandspecialfoods(Acosta,1995).Thequalitative(organoleptic)featuresoftheaminoacid
medicalproductoftenhaveastronginfluenceondietarycompliance.Someproductsarebettertoleratedthanothersforsome
women(Owadaetal.,1988Rohretal.,2001Thompsonetal.,1991bWardleyandTaitz,1988),andtheirusecanbeassociated
withsatisfactoryfetaloutcomes(Drogarietal.,1987Thompsonetal.,1991b).Gelatinencapsulationofthemixturetomaskthe
disagreeableodorandtastemaybeimportantforsomewomen(Kecskemethyetal.,1993),providedtheycanswallowtherequired
largenumberofcapsules(Rohretal.,2001).Supplementationofbranchedchainaminoacids(VorheesandBerry,1989)seems
nottoberequiredforoptimalmetaboliccontrolandoffspringoutcome(Acostaetal.,2001b).Tyrosinesupplementationwillimprove
maternaltyrosinelevels,ifindicated(Rohretal.,1998).

Thenewbornoffspringborntoanuntreated(orpoorlytreated)motherwithHPAmaybethoughttohavePKUifthenewborn
screeningspecimeniscollectedwithinthefirst12hourspostpartumthephenomenonreflectsmaternallytransmittedphenylalanine.
IfthebabyhasnotitselfinheritedHPA,thephenylalanineincreaseclearsby24hourspostpartum,andtheinfantshouldbegivena
normaldiet(LevyandLobbregt,1995).
ThemotherwithHPAmaysafelybreastfeedheroffspring.Therelativelysmallincreaseinbreastmilktotalphenylalanine(Fox
Baconetal.,1997)canbeeasilymetabolizedbytheheterozygousoffspringorincorporatedinaphenylalaninerestricteddietary
regimenshouldtheoffspringhaveHPAthebreastfedheterozygousoffspringofmaternalHPAmaintainsnormalphenylalanine
levels(FoxBaconetal.,1997).
Despiteprogressinreducingitspotentialimpactonthefrequencyofmentalretardation(KirkmanandFrazier,1996),maternalHPA
continuestobeanimportantchallengeevenaftermorethan40yearsofawarenessthatitwouldbecomesuchintheglobal
managementofPKU(Dent,1957Kirkman,1992Mabryetal.,1963Scriver,1967).Itisaproblemthatmustberesolved
otherwise,achievementsinthepreventionofmentalretardationassociatedwithPKUandrelatedformsofHPAwillhavebeen
gainedatthecostofaterribleFaustianbargain.

Acknowledgments
Theauthorsacknowledgeinvaluablecontributionsasfollows:ToCRS:frommembersofthePAHMutationAnalysisConsortiumand
fromthefollowingcolleagues(inalphabeticalorder):LouisBaumier,SusanByck,AnnieCapua,KevinCarter,RandyEisensmith,
HeidiErlandsen,MelanieHurtubise,MaryFujiwara,EmreKayaalp,JudithKidd,KenKidd,DavidKonecki,UtaLichterKonecki,
DebbyLambert,KenMorgan,PiotrNowacki,ManyPhommarinh,MichaelParniak,LynnePrevost,ChristinehSarkissian,Ray
Stevens,EileenTreacy,LindaTyfield,andPaulaWaters.FromFranRohr,SusanWaisbren,andcolleaguesinthePKUClinic,
ChildrensHospital,Boston.ThesecretarialandeditorialassistanceofLynnePrevostandMelissaGennaccaroiswarmly
acknowledged.TherewouldbemuchlesstoreportwithouttheinitialgenerosityofSavioWooandcolleagueswhosharedthehPAH
247probeandPCRprimersandwithouttheearlycommitmentofPerGuldbergandFlemmingGuttlertodevelopapowerfulDGGE
methodformutationanalysis.TheauthorsowneffortsweresupportedbytheNationalInstituteofMentalHealth,NIHtheMedical
ResearchCouncil(Ireland)supportfromtheNationalInstituteofChildHealthandHumanDevelopmentandtheMaternalandChild
HealthBureauoftheU.S.DepartmentofHealthandSocialServicestheHumanGenomeProjecttheMedicalResearch
Council/CanadianInstitutesofHealthResearchtheNetworkofCentersofExcellence(CanadianGeneticDiseasesNetwork)and
LeFondsdelaRechercheenSantduQubec(fortheQuebecNetworkofApplied/Moleculargenetics).Themonumental
contributionsofaretiredauthor,SeymourKaufman,areapparentthroughout.

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