Committeesios11

MUTATIONINBRIEFHUMANMUTATIONMutationinBrief#466(2001)Online2001WILEY-LISS,INC.
Received24May2001;revisedmanuscriptaccepted24September2001.
CystathionineBeta-SynthaseDeficiencyinCentralEurope:DiscrepancyBetweenBiochemicalandMolecularGeneticScreeningforHomocystinuricAllelesJ.
Sokolová1,B.
Janoíková1,J.
D.
Terwilliger2,T.
Freiberger3,J.
P.
Kraus4,andV.
Kozich1*1InstituteofInheritedMetabolicDisorders,1stFacultyofMedicine,CharlesUniversity,Prague,CzechRepublic2DepartmentofPsychiatry,ColumbiaUniversityandColumbiaGenomeCenter,NewYork,USA3ResearchInstituteofChildHealth,Brno,CzechRepublic4DepartmentofPediatrics,HealthSciencesCenter,UniversityofColorado,Denver,USA*Correspondenceto:ViktorKoich,InstituteofInheritedMetabolicDisorders1stFacultyofMedicine,KeKarlovu2,12808Prague2,CzechRepublic;Tel:+420-2-24967679,24920293;Fax:+420-2-24919392;E-mail:VKOZICH@LF1.
CUNI.
CZ.
Contractgrantsponsor:GrantAgencyofMinistryofHealthoftheCzechRepublic;Contractgrantnumber:NM26/3.
CommunicatedbyMarkH.
PaalmanRecentreportssuggestedthathomocystinuriaduetocystathioninebeta-synthase(CBS)deficiencyisamorecommoninbornerrorofmetabolismthanoriginallythought.
Inthisstudywecomparedtheprevalenceofhomocystinuricallelesascertainedbytwodifferentapproaches.
First,theincidenceofhomocystinuriaestimatedbyselectivebiochemicalscreeningintheCzechandSlovakRepublicswas1:349,000(95%CI1:208,000-1:641,000).
Thetwomostcommonpathogenicmutantallelesfoundsubsequentlyinthesepatients,IVS11-2A>Candc.
833T>C,hadacalculatedpopulationprevalenceof0.
00042(95%CI0.
00031-0.
00055)and0.
00018(95%CI0.
00013-0.
00023),respectively.
Second,toexaminethepossiblenegativedetectionbiasofmildlyaffectedpatientswedeterminedtheprevalenceofthesetwopathogenicmutationsinasampleof1284unselectednewborns.
Indeed,theobservedprevalenceofthec.
833T>Callele(0.
00195,95%CI0.
00063-0.
00454)was11xhigherthaninthepreviousgroupsuggestingthatmanyhomozygotesforthec.
833T>Chadnotbeendiagnosedbyselectivebiochemicalscreening.
TheIVS11-2A>Callelewasnotdetectedamong2,568newbornCBSalleles.
Theestimatedincidenceofhomocystinuriaof1:83,000,calculatedinacombinedmodel,suggeststhatselectivebiochemicalscreeningmayascertainonly~25%ofallhomocystinuricpatients.
Inconclusion,homocystinuriainCentralEuropemaybesufficientlycommontoconsidersensitivenewbornscreeningprogramsforthisdisease.
2001Wiley-Liss,Inc.
KEYWORDS:homocystinuria;prevalence;mutationscreening;cystathionebeta-synthase;CBS;Czech;Slovak;ARMS-PCRINTRODUCTIONHomocystinuriaduetocystathioninebeta-synthase(CBS)deficiency(MIM#236200)isatreatableinbornerrorofmetabolism,whichclinicallyresemblesMarfansyndrome(Muddetal.
,2001).
Twomajorphenotypesofthisautosomalrecessivediseaseareequallycommon,amilderpyridoxine-responsiveform,andamoreseverenon-2Sokolováetal.
responsivetype.
However,treatmentwithpyridoxine,methionine-restricteddietorbetainepreventsthedevelopmentofsomaticandmentalabnormalitiesinthemoresevereformsonlywhenstartedininfancy(Naughtenetal.
,1998;Walteretal.
,1998).
Suchafavorableoutcomeinearlytreatedpatients,andapossibilitytodiagnosethediseasefrombloodspotsrankhomocystinuriaamonginbornerrorsofmetabolismthatwouldbenefitfromnewbornscreening.
Newbornscreeningprogramsareimplementedifthediseaseissufficientlycommon.
Thereforetheincidenceofhomocystinuriaisanimportantdeterminantofthecost-benefitratioinsuchaprogram.
Boththenewbornscreeningprogramsandselectivebiochemicalscreeningdetecthomocystinuriawithanincidenceof1:58,000to1:1,000,000indifferentcountries(Muddetal.
,2001).
However,theestimatedfrequencyofhomozygosityforthemostprevalentmutantallelec.
833T>C(basedonitsfrequencyofheterozygotesinapopulationofhealthyunselectedDanishnewborns)wasremarkablyhigher,i.
e.
1:20,500,thanexpected(Gaustadnesetal.
,1999).
Homocystinuriacanbecausedbyhomozygosityorcompoundheterozygosityforanyofoverahundredpathogenicalleles(Krausetal.
,1999;Muddetal.
,2001).
InCaucasiansthemostcommonpathogenicalleleisthec.
833T>C,whichisassociatedwithpyridoxineresponsiveness.
However,thepathogeniceffectofc.
833T>Cisneutralizedwhenitoccursonahaplotypewithanearbyinsertionof68bp,suchthatc.
844ins68homozygotesdonotdevelophomocystinuria(Sebastioetal.
,1995;Tsaietal.
,1996).
Inthisreport,werefertothepathogenichaplotypewithoutthe68bpinsertionasc.
833T>C,andtothenonpathogenicformcontainingthec.
833T>Candthe68-bpinsertionasc.
844ins68.
Inordertotestwhetherallpersonshomozygousforpathogenicallelesarediagnosedwithhomocystinuriabyselectivebiochemicalscreening,wecomparedtheincidenceofdiseasewiththesquareoftheestimatedpopulationfrequencyofpathogenicalleles.
Thefrequencyofthemostcommonpathogenicallele,c.
833T>C,wasdirectlyestimatedfromacohortofnewbornsfromtheCzechpopulation,whilethefrequenciesofotherpathogenicalleleshadtobeinferredfromtheirrelativefrequencyamongdiagnosedpatients,toavoidunderestimationofthetotalfrequencyofpathogenicalleles.
Thesumoftheestimatedfrequenciesofeachpathogenicallelewassquaredtodeterminetheexpectedprevalenceofhomocystinuriaifallhomozygoteswerediagnosed.
MATERIALSANDMETHODSSamplesandDNAisolationDuringthelasttwodecadesaselectivebiochemicalscreeningprogramforinbornerrorsofmetabolism,performedinourlaboratory,ascertainedacohortofhomocystinuricpatientsrepresentativeoftheformerCzechoslovakia.
Inthesepatients,thepathogenicsequencevariantsweredeterminedbyDNAsequencingasdescribedelsewhere(Janoíketal.
,2001).
Forthestudyofgeneralpopulationwecollected572anonymousbloodspotsfromtheroutinenewbornscreeningforphenylketonuriaincentralBohemia,and712anonymousumbilicalcordbloodsamplesfromBrno.
ThelocalEthicsCommitteesapprovedtheuseofanonymousDNAsamples.
GenomicDNAwasisolatedfrombloodspotsbyfixationin100%ethanolfor2minutes,airdryingfor3hoursandelutioninto50lofwaterat70°Cfor30minutes.
ExtractionofgenomicDNAfromumbilicalcordbloodwasdoneusingtheQIAampDNAMiniKit(Qiagen).
DetectionofallelescarryingthepathogenicmutationsIVS11-2A>Candc.
833T>C,andofthevariantallelec.
[833T>C;844ins68bp]Todetectthetwopathogenicallelesc.
833T>CandIVS11-2A>CinnewbornsamplesweusedARMS-PCR.
ThePCRreactionswerecarriedoutin20lreactionscontaining150-200nggenomicDNA,PC2buffer(ABPeptides,Inc.
,3.
5mMMgCl2),4pmolofeachofdNTPs,10pmolsofeachprimer,1UKlenTaqDNApolymerase(ABPeptides,Inc.
),andadditionsasdescribedbelow.
AmplificationwasperformedinthePTC-225DNAEngine(MJResearch).
PCRproductswereanalysedon3%agarosegelstainedwithethidiumbromide.
Analysiswereperformedinbatchesof95,eachseriescontainedcontrolsnegativeandpositivefortheanalyzedmutations.
Todetectthepresenceofc.
833T>Cweusedallele-specificprimerpairswithanartificiallyintroducedmismatch(underlined):forward-normalallele,5-CCTGAAGCCGCGCCCTCTGCAGATAAT-3;forward-mutantallele,5-CCTGAAGCCGCGCCCTCTGCAGATAAC-3;reverseprimer,5-GTGGCCGGGCTCTGGACTCGACCTACC-3.
ThePCRreactionscontainedanadditionof1.
25mMofMgCl2and10%ofDMSOandcyclingwasperformedasfollows:20sat95°C,40cyclesof[5sat95°C,15sat68°C],andHomocystinuricAllelesinCentralEurope310minutesat68°C.
Amplificationsignalobtainedwiththemutant(M)allele-specificprimerpairsignifiesthepresenceofCinposition833bothonanormallysizedallele(PCRproductof174bp),oronthevariantallelec.
[833T>C;844ins68bp](PCRproductof242bplength).
Ampliconobtainedwiththenormal(N)allele-specificprimerpairdemonstratesthepresenceofTinposition833(PCRproductof174bp).
The126bpPCRproductsamplifyanotherportionoftheCBSgeneandserveasacontrolforthePCRreaction(seeFig.
1,panelA).
AllsamplespositivebyARMS-PCRforthec.
833T>Calone(withouttheinsertion)werereanalyzedbyanindependentPCR-RFLPtechniqueemployingBsrIasdescribedpreviously(Sebastioetal.
,1995).
ThePCRproductswereincubatedwithadigestionbuffereitherinthepresence(+)orabsence(-)oftherestrictionenzyme.
ThepresenceofCinposition833isdemonstratedbya132bpfragment,differentlotsofBsrIfromvarioussuppliersalwaysyieldedanincompletedigestinpatientswith833C/Cgenotype(seeFig.
1,panelB).
TheARMS-PCRsystemforc.
833T>Cdetectsalsothecommonallelec.
844ins68(i.
e.
c.
[833T>C;844ins68bp]).
Fortechnicalreasons,thesystemisunabletodiscriminatebetweenheterozygosityandhomozygosityforthec.
[833T>C;844ins68bp].
Allsamplesexhibitingthepresenceofc.
[833T>C;844ins68bp]havebeenthereforeanalyzedbyanindependentPCRtoassesswhethertheindividualwasheterozygousorhomozygousfortheinsertion;productsof242bparepresentiftheallelecarriesthec.
[833T>C;844ins68bp]whileproductsof174bpdemonstratethepresenceofanormallysizedallele(seealsoFig.
1,panelB).
ForIVS11-2A>Cweusedtheallele-specificprimerpairswithanartificiallyintroducedmismatch(underlined)asfollows:forward-normalallele,5-CCCCCCGACCTGCCCTCTTCCCCCA-3;forward-mutantallele,5-CCCCCCGACCTGCCCTCTTCCCCCC-3;reverseprimer,5-TGGGCAGACAGAACCCAGGACTGAG-3.
PCRwascarriedoutwiththeadditionof2.
5%ofacetamideusingthefollowingcyclingconditions:20sat95°C,35cyclesof[5sat95°C,8sat66°C,15sat72°C],10minat72°C.
Thepresenceofa210bpproductwiththemutant(M)allele-specificprimerpairdemonstratesthepresenceofIVS11-2Cwhileitspresenceinthenormal(N)allele-specificPCRconfirmsthepresenceofthewildtypeallele.
ThePCRproductof328bpisaninternalcontrolforthePCRreaction(seeFig.
1,panelC).
Figure1.
AnalysisofmutationsIVS11-2A>Candc.
833T>C,andofthevariantallelec.
[833T>C;844ins68bp].
A:Analysisofthemutationc.
833T>CbyARMS-PCR.
Lanes:sample1,c.
833T/C;sample2,c.
833C/C;samples3and4,heterozygousorhomozygousforc.
[833T>C,844ins68bp]polymorphism,respectively;sample5,wildtypec.
833T/T.
Allele-specificprimerpairused:M=mutant,N=normalallele(seeMETHODStextforotherdetails).
B:Confirmatoryanalysisofthec.
833T>Candc.
[833T>C;844ins68bp]alleles.
Lanes:sample1,c.
833T/C;sample2;c.
833C/C;sample3,c.
[833T>C;844ins68bp]/833T;sample4,c.
[833T>C;844ins68bp]/c.
[833T>C;844ins68bp]with/withoutBsrIrestrictionenzyme(seeMETHODStextforotherdetails).
C:AnalysisofmutationIVS11-2A>CbyARMS-PCRLanes:sample6,IVS11-2A/C;sample7,wildtypeIVS11-2A/A.
(seeMETHODStextforotherdetails).
PANELAPANELBPANELCMNMNMNMNMNMNMN-+-+12345671234←←242bp←←174bp←←126bp←←174bp←←242bp←←132bp←←328bp←←210bp4Sokolováetal.
RESULTSSelectivebiochemicalscreeningBetween1981and2000twenty-onepatientswerediagnosedwithhomocystinuriaintheformerCzechoslovakia.
Sincepatientswereascertainedatamedianageof7years(Orendácetal.
,2000),onlydatafromthosebornbetween1973-1992wereconsideredforfurthercalculations(seeTable1),leadingtotheexclusionoftwoc.
833T>Chomozygotesfromtheanalysis,astheywerediagnosedatlateage.
Consequently,theobservedincidenceofhomocystinuriaintheCzechandSlovakrepublicwasestimatedtobe1:349,000(95%CI1:208,000-1:641,000).
Amongthe28independentallelesascertainedbythisapproach,sevencarriedtheIVS11-2A>Cmutation,threecontainedthec.
833T>Ctransition,andtheremaining18allelescontainedeightothermutations(fordetailsseeTable1).
Weestimatedthefrequencyofthesepathogenicallelesinageneralpopulation(seeTable1)withconfidenceintervalsfromaPoissondistributionusingSISA(SimpleInteractiveStatisticalAnalysis)softwareathttp://home.
clara.
net/sisa/poisson.
htm.
MutationanalysisinnewbornsTheprevalenceofthetwomostcommonpathogenicvariantsIVS11-2A>Candc.
833T>Cwasalsodetermineddirectlybymutationanalysisofhealthyunselectednewborns,andcomparedtotheestimatesfrombiochemicalscreening.
FortheIVS11-2A>Callelewedidnotobserveanydifferenceinprevalencebetweenthetwoascertainmentmethods;althoughnoneofthenewbornscarriedtheIVS11-2A>Cmutationtheconfidenceintervaloftheallelicfrequencyoverlappedwiththatdeterminedinbiochemicalscreening.
Ofthe2568examinedCBSallelesfivecarriedc.
833T>C(4of1144allelesinBohemiaand1of1424allelesinMoravia).
Theobservedfrequencyofthec.
833T>Calleleisabout11timeshigheramongnewbornsthanexpectedbasedondiagnosedhomocystinuricpatients.
Thisdifferenceisstatisticallysignificant,asthereisnooverlapinthe95%confidenceintervalsfortheestimatedfrequencyofc.
833T>Cfromthetwocohorts.
Thedifferenceremainsstatisticallysignificantevenaftertheadditionofthetwolatediagnosedc.
833T>Chomozygotesintothecalculationssuggestingthathomozygotesforthisallelearebeingmissed.
TheARMS-PCRmethodalsodetectedthecommonvariantc.
[833T>C;844ins68bp]thatcarriesc.
833T>Candaninsertionofareduplicatedportionoftheintron7/exon8junction(Sebastioetal.
,1995;Tsaietal.
,1996).
Amongthe1284newbornswefound8homozygotesand159heterozygotesforthispolymorphism.
Theobservedprevalenceofthevariantalleleof0.
068(95%CI0.
059-0.
079)issimilartootherEuropeanpopulations(DeStefanoetal.
,1998).
NotethathomozygotesforthisalleledonotexhibitCBSdeficiencyandtheydonotdevelophomocystinuria.
DISCUSSIONTheaimofthisstudywastoexaminewhetherhomocystinuricpatientsinCentralEuropeareascertainedefficientlybytheselectivebiochemicalscreeningwithdeterminationofhomocyst(e)ineinplasmaandurine.
AsameasureofthisefficiencywedeterminedthefrequenciesofthetwomostcommonpathogenicCBSallelesinagroupofhomocystinuricpatientsandinunselectednewborns.
Ourstudyrevealedthattheobservedfrequencyofthec.
833T>CalleleisoneorderofmagnitudehigherinageneralCzechpopulationthanexpectedfromitsdistributionamonghomocystinuricpatients,conditionalondiseaseprevalence.
Themarkeddiscrepancybetweenthetwoestimatescanresult,amongotherthings,fromdecreasedviabilityoffetuseshomozygousforc.
833T>C,ornegativeascertainmentbiasofthemildlyaffectedhomozygotesandcompoundheterozygotescarryingthec.
833T>Callele.
Thehypothesisthathomozygotesforc.
833T>Cmaybelessviablehasnotyetbeenexploredandcannotbeexcluded.
However,thematernalobstetrichistoryinthetwopatientshomozygousforc.
833T>Cdidnotrevealanymiscarriages.
Inaddition,therearenoreportsinliteratureonanincreasedrateofinfertilityorspontaneousabortionsincouples,inwhosebothparentsareheterozygousforthismutation.
Therefore,anegativeascertainmentbiasofthemildlyaffectedhomozygotesandcompoundheterozygotescarryingthec.
833T>Calleleseemsthemostplausibleexplanationofourdata.
Weproposethatsuchindividualshavenotbeenascertainedasaffectedbytheselectivescreeningowingtothemildclinicalphenotype,andlaterHomocystinuricAllelesinCentralEurope5Table1:Prevalenceofhomocystinuricalleles.
BiochemicalscreeningMutationanalysisinnewbornsNumberofallelesintheanalyzedgroup19,770,0002,568AscertainedindividualshomozygotesandcompoundheterozygotesheterozygotesMutantallelec.
833T>CIVS11-2A>Cothermutations4c.
833T>CIVS11-2A>Cothermutations4Numberofallelesascertained2371850n.
d.
Prevalenceofalleleinpopulation(95%CI)30.
00018(0.
00013-0.
00023)0.
00042(0.
00031-0.
00055)0.
00109(0.
00080-0.
00141)0.
00195(0.
00063-0.
00454)0(0-0.
00144)n.
d.
n.
d.
,notdetermined1Forthebiochemicalselectivescreeningacohortof4,885,000newbornswasconsidered;fordirectmutationanalysissamplesfrom1284newbornswereobtained.
2Incidenceofhomocystinuriafromtheselectivebiochemicalscreeningwascalculatedbasedonthedetectionof14independentfamilieswithahomocystinuricpatient(qcbs2=2.
86x10-6,95%CI1.
56-4.
80x10-6;qcbs=0.
00169,95%CI0.
00125-0.
00219)3FrequenciesofallelesfoundinthepatientpopulationwereestimatedconditionalonthediagnoseddiseaseprevalenceassumingHWE,andconsideringtheproportionofc.
833T>C,IVS11-2A>C,andtheothermutantallelesamongpatients;forthenewborngroupthefrequenciesofthetwotestedallelesgiveninthetableweredirectlyestimatedbygene-counting.
4Theotherpatientallelesincluded3instancesofc.
19insC;2instancesofthefollowing:c.
1226G>A,c.
526G>A,andc.
[430G>A;463G>A];1witheachofIVS2-1G>C,c.
28delG,c.
341C>T,and[IVS7+1G>A;IVS11+39del99];5otherswereuncharacterized.
onsetofsymptoms.
Severalpiecesofevidencesupportthisview.
Itwasshownpreviouslythathomozygosityandcompoundheterozygosityforthec.
833T>Cmutationisalmostalwaysassociatedwithpyridoxineresponsiveness(Krausetal.
,1999),andthatpyridoxinerespondersexhibitmilderphenotypewithalateronsetofdiseaseandlaterageofdiagnosis(Muddetal.
,1985).
Detectionofnumerousadulthomocystinuricpatientswithamildphenotypeandpyridoxineresponsivenesswasreportedrecentlyaftertotalplasmahomocysteinedeterminationbecameeasilyavailable(Cruysbergetal.
,1996;Gaustadnesetal.
,2000a;Gaustadnesetal.
,2000b).
Themilderphenotypeandlateageofdiagnosisinpatientscarryingthec.
833T>Callelewasapparentalsoinourstudy;ofthethreepatientswhofelloutsidethebirthcohortusedforincidencecalculations,twowerediagnosedinadulthoodandbothwerehomozygousforthec.
833T>Callele.
Ofinterest,thereisagradientinseveritythroughoutthepatientpopulation,therearemoreseverelyaffectedpatientsinSlovakia,andmorepatientswithamilderphenotypeintheCzechRepublic(Janoíketal.
,2001).
Thisdifferencemayhaveresultedfromvaryingdiagnosticcriteriainthetwocountriesoritmighthaverepresentedagradientinallelefrequencyaroundtheregion.
Thec.
833T>Cmutationwasfoundononlyonechromosomeamong12independentSlovakhomocystinuricalleleswhileitwasdetectedon6of22Czechhomocystinuricalleles.
Similarly,aclineinallelefrequencyamongnewbornscouldbeobservedevenwithintheCzechRepublicbetweenBohemiaandMoravia.
HomocystinuriaduetoCBSdeficiency-ifdiagnosedearly-isatreatablediseasewhereasuntreatedorlatetreateddiseaseresultsinnumerousskeletal,ocular,vascularandcentralnervoussystemcomplications(Muddetal.
,1985).
NewbornscreeningforhomocystinuriaiscurrentlyperformedalmostexclusivelyinpopulationswithahighproportionofindividualsofaBritish,ScottishorIrishdescent.
Incontrast,themajorityofotherdevelopedcountrieshavestoppednewbornscreeningprogramsforthisdiseaseafterobservingitslowincidence.
Itremainstobedetermined,however,whetherinthesecountrieshomocystinuriaisindeedsuchararedisease.
SeverallinesofevidencesuggestthatCBSdeficiencyismorecommonthanusuallyperceivedandarediscussedinmoredetailbelow.
1.
Sensitivityofscreeningprograms.
ThenewbornscreeningprogramsareusuallybasedondetectinghypermethioninemiaassociatedwiththeCBSdeficiency.
ArecentstudyfromNewEnglandshowedthatloweringthecutofflevelforbloodmethioninefrom134to67mol/lalmostdoubledthereported6Sokolováetal.
incidenceofhomocystinuria(Peterschmittetal.
,1999).
Thisnotionsuggeststhatinvariouspopulationsthepreviouslydeterminedincidencethatemployedcutofflevelsashighas270mol/l(Naughtenetal.
,1998)mayhaveunderestimatedthefrequencyofthisdisease.
2.
Negativebiastowardspyridoxineresponders.
Interestingly,highincidenceofhomocystinuriainnewbornsprogramsisreportedfromcountrieswithahighproportionofpyridoxinenon-responders.
Sincethenon-respondersexhibitmuchhigherelevationofbloodmethionine(Muddetal.
,1985;Orendácetal.
,2000),itisconceivablethatnewbornscreeningprogramsforhypermethioninemiamaydetectpreferentiallythenon-responders.
3.
Highprevalenceofheterozygotes.
SimilarlytothereportofGaustadnes(Gaustadnesetal.
,1999)ourstudyshowedamarkeddiscrepancybetweentheobservednumberofheterozygotesinanewbornpopulationandtheexpectedincidenceofhomozygotesforthec.
833T>Callele.
Asdiscussedabove,thesedatasuggestanegativebiastowardspyridoxinerespondersandfurthersupporttheviewthathomocystinuriaismorecommonanddiagnosticallymissed.
TheaboveanalysespermitustomodelamorerealisticestimateoftheincidenceofCBSdeficiencyinCentralEurope.
Weassumethatthehomozygotes(andpossiblysomecompoundheterozygotes)forthec.
833T>Caremissedduetothemildclinicalphenotypeandthattheprevalenceofthisallelewasdeterminedmorecorrectlybyanalyzingthenewbornsamples.
Ontheotherhand,theprevalenceofallelescarryingtheIVS11-2A>Candtheothermutations,whichleadtoaseverehomocystinuria,canbeestimatedreasonablyandwithalargerstatisticalpowerfromthedatainthebiochemicalscreening.
Combinationofthisallelicprevalenceenablesustocalculatetheexpectedincidenceofhomozygoushomocystinuria.
Despitethefactthatwecannoteasilydeterminethe95%confidenceintervalforthisestimate,thecentralvalueof1:83,000isabout4timeshigherthantheincidenceobservedbybiochemicalscreeningandsuggeststhatupto3/4ofhomocystinuricpatientsarebeingcurrentlymissed.
Inconclusion,ourstudysupportstheviewthatinCentralEuropehomocystinuriaduetoCBSdeficiencymaybelargelyunrecognizedandsufficientlycommontoconsideranewbornscreeningprogrambyareasonablysensitivemethod.
ACKNOWLEDGMENTSTheauthorswouldliketothankDr.
S.
tastnáandMs.
J.
Dvorákováforprovidinganonymousbloodspotsamples,andMs.
E.
RichterováandMs.
T.
Zezulákováforexperttechnicalhelp.
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