Diagnosis of Morquio Syndrome in Dried Blood Spots Based on a New MRM-MS Assay

14 Pages·2015·1.42 MB·English

RESEARCHARTICLE
Diagnosis of Morquio Syndrome in Dried
Blood Spots Based on a New MRM-MS Assay
ClaudiaCozma1,SabrinaEichler1,GyulaWittmann1,AlbaFloresBonet1,
GuidoJohannesKramp1,Anne-KatrinGiese2,ArndtRolfs1,2*
1 CentogeneAG,Rostock,Germany,2 Albrecht-Kossel-Institute,UniversityofRostock,Rostock,Germany
* [email protected]
Abstract
a11111
Background
MucopolysaccharidosisIVA(MPSIVA;MorquioAdisease)isanautosomalrecessive
diseasecausedandcharacterizedbyadecreasedactivityofN-acetylgalactosamine-6-
sulfatesulfatase(GALNS),resultinginaccumulationofkeratansulfateandchondroitin-6-
OPENACCESS sulfateintissuesandsecondaryorgandamage.Recentlyapprovedenzymere-
placementtherapyrenderstheeasyandearlyidentificationofMPSIVAofout-most
Citation:CozmaC,EichlerS,WittmannG,Flores
importance.
BonetA,KrampGJ,GieseA-K,etal.(2015)
DiagnosisofMorquioSyndromeinDriedBloodSpots
BasedonaNewMRM-MSAssay.PLoSONE10(7):
Methodology
e0131228.doi:10.1371/journal.pone.0131228
WeproposeacompletelynewassayforthestableandreproducibledetectionofGALNSdefi-
Editor:SantoshPatnaik,RoswellParkCancer
Institute,UNITEDSTATES ciencyindrybloodspots(DBS).Forthevalidationbloodsamplesweretakenfrom59healthy
individualsand24randomlyselectedgeneticallyconfirmedMPSIVApatients.Thematerial
Received:April3,2015
extractedfromDBSwasincubatedwitha4-methylumbelliferyl-β-D-galactopyranoside-6-
Accepted:May29,2015
sulfateasaspecificsubstrate.Finalenzymaticproduct,4-methylumbelliferone,obtained
Published:July6,2015 afteraddingexogenousbeta-galactosidase,wasquantifiedbyLC/MRM-MS(liquid-chroma-
Copyright:©2015Cozmaetal.Thisisanopen tography/multiple-reaction-monitoringmass-spectrometry).4-propyl-5-hydroxy-7-methyl-2h-
accessarticledistributedunderthetermsofthe chromen-2-onewasusedasinternalstandard,acompoundwithasimilarmolecularstructure
CreativeCommonsAttributionLicense,whichpermits
andfragmentationpatterninnegativeionmodeas4-methylumbelliferone.
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
credited. Findings
DataAvailabilityStatement:Allrelevantdataare Theenzymaticassayyieldedapositiveandnegativepredictivevalueof1.0forgenetically
withinthepaperanditsSupportingInformationfiles.
confirmedMPSIVApatients(GALNSactivityof0.35±0.21μmol/L/h)andforcontrolswith
Funding:Thisstudyhasbeenfinanciallysupported normalGALNSactivity(23.1±5.3μmol/L/h).Withpresentenzymaticconditions,thereac-
byCentogeneAG,Rostock,Germany.Thecompany
tionyieldindriedbloodspotsisatleast20foldhigherthananypreviouslyreporteddata
hadaroleinthisstudyinstudydesign,data
collectionandanalysis,decisiontopublishand withotherassays.
preparationofthemanuscript.
CompetingInterests:Thefollowingauthorsare Interpretation
employeesofCentogene(Rostock,Germany):
ThepresentLC/MRM-MSbasedassayforMPSIVAdiagnosisprovidesaneasy,highly-
ClaudiaCozma,SabrinaEichler,GyulaWittmann,
AlbaFloresBonet,GuidoJohannesKramp.This standardized,accurateandinnovativequantificationoftheenzymaticproductinvitroand
PLOSONE|DOI:10.1371/journal.pone.0131228 July6,2015 1/14
DiagnosisofMorquioSyndromeinDriedBloodSpotsUsingMRM-MS
doesnotaltertheauthors'adherencetoPLOSONE distinguishesperfectlybetweenMPSIVAaffectedpatientsandnormalcontrols.Thistech-
policiesonsharingdataandmaterials. niquewillsignificantlysimplifytheearlydetectionofMPSIVApatients.
Introduction
Mucopolysaccharidoses(MPSs)areagroupofinheritedlysosomalstoragedisordersinwhich
defectsofdifferentlysosomalenzymesleadtoaccumulationofglycosaminoglycansinvarious
tissuesandorgans.MorquioAdisease,alsoknownasmucopolysaccharidosisIVAorMPS
IVA;[1]ischaracterizedbyaccumulationofkeratansulfate(KS)andchondroitin-6-sulfate
(C6S)atacellularlevelindifferentorgans,generatingmulti-systemicimpairments.Keratan
sulfatedegradationiscarriedoutsequentiallyinthelysosomewheretwoenzymesareinvolved
intheremovalofbeta-galactosidemoieties:N-acetylgalactosamine-6-sulfatase(GALNS,defi-
cientinMPSIVA)andβ-galactosidase(GLB1,impairedinMorquioTypeBdisease,MPS
IVB).
AlthoughsomeoftheclinicalsymptomsarecharacteristicforMPSIVA(suchasshortstat-
uretogetherwithprogressiveskeletaldysplasia),itisnearlyimpossibletodistinguishthetwo
formsofMPSIVbasedonclinicaldataalone.Mostofthecasespresentwithacommonpheno-
type[2].MPSIVA(withaprevalenceofatleast1in200,000livebirths,[3])includesclinical
featuresofskeletaldysmorphism(shortstature,dysostosismultiplex,kyphosis,pectuscarina-
tum),cornealclouding,jointcontractures,toothenameldefectandspinalcordcompression.
ThephysicalsignsofMPSIVbecomedistinguishableinthesecondyearoflife,whilethecog-
nitivefunctionsdevelopnormally.
Todate,over200GALNSmutationshavebeendescribed,causingphenotypicheterogeneity
whichisreflectedbyvaryingsymptomsandprogressionofthedisorder.AdiagnosisofMPS
IVAtypicallyfollowsspecificsteps:clinicalassessmentofthesymptoms,glycosaminoglycans
analysisinurine,GALNSactivitydeterminationinpatientswithhighconcentrationsofurine
keratinsulfates,geneticconfirmationofthebiochemicalanalysis.Atpresent,clinicaltreatment
ofMPSIVApatientsvariesfrommanagementofsymptomstonewlydevelopedspecific
enzymereplacementtherapy.Stateoftheartinclinicalpracticeforimmediatebiochemical
screeningofMPSIVApatientsisbasedonfluorimetricdeterminationofGALNSactivityin
fibroblastcultureandlymphocytes[4].Thesetypesofassaysrequirealargequantityofbiologi-
calsample,aretimeconsuming(especiallyfortheskinbiopsyculture)andlogisticallydemand-
ingastheyrequirerapidsampleprocessingaftersamplecollection.Forthesereasons,aviable
alternativeisurgentlyneeded.
Driedbloodspots(DBSs)onstandardfilterpaperasanenzymesource,isalreadyinusefor
otherlysosomalstoragediseases[5;6;7].UsingwholebloodfromDBSsinthefluorimetric
assayspresentsseveralchallenges:verylowquantitiesofenzyme,unknownstabilityofthe
enzymeinthebloodsample,varyingpreparationandstorageconditionsindifferentlaborato-
ries,heterogeneityofthebloodsamplesfromdifferentpersons,interferenceofbloodcompo-
nentswiththeaccuracyofthemeasurement.
AlthoughtherearenoclinicallyavailableassaysforthedeterminationofGALNSactivityin
DBS,previouspublicationsdescribefluorimetricdeterminationofGALNSactivityusinga
modifiedlymphocyteassay[8]ortandemmassspectrometrybasedassayusingnewlydevel-
opedsubstrates[9].Bothmethodshavetheadvantageofusinglowamountsofsamples;how-
evertheybothshowseveralshortcomings:imprecisequantificationoftheproductandsignal
interferenceduetootherbloodcomponentsinthecaseofthefluorimetricassay,andinthe
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DiagnosisofMorquioSyndromeinDriedBloodSpotsUsingMRM-MS
caseofthemassspectrometricassayalowamountoftheproductisobtainedandlastbutnot
least,thesubstrateisnotcommerciallyavailable.Furthermore,acommondisadvantagefor
bothassaysisthelowconversionofthesubstrates,intherangeof0.1to1μmol/L/h(maxi-
mum)forhealthycontrolsamples.
Herefore,wepresentanovelapproachinGALNSactivityquantification,DBSsbasedand
withacommerciallyavailablesubstrate,improvedenzymaticreactionconditions,anda
LC/MRM-MSdetectionandquantificationthatreducesthematrixeffectfoundinfluorimetric
determinations.Moreover,wedevelopedasecondenzymaticassayforβ-galactosidasetobe
usedasacontrolforanMPSIVAenzymaticassayusingthesameMSbaseddetectionmethod.
MaterialsandMethods
Bloodsamplesusedinpresentstudy
ThestudyandtheprotocolhasbeenapprovedbytheEthicalCommitteeoftheUniversityof
Rostock(Ethicsvote#A-2011-109),inaccordancewithGermanandEuropeanUnionlegal
guidelines.Alltheparticipantsinthestudysignedinformedconsentforms.Driedbloodspots
(DBS)werepreparedfromthebloodof59healthycontrols(aged23to61years);2MPSIVA
carriers(aged35and35)and24randomlyselectedMPSIVApatients(ages3to19);4MPS
IVBcarriers(aged27to35)and11MPSIVBaffectedpatients(aged3to15).Allcarriersand
affectedMPSIVpatientsweregeneticallyconfirmed.FortheMPSIVApatients,nodataare
availableregardingtheKSandC6Slevelsinurine;howeverGALNSandbeta-galactosidasebio-
chemicalassayswereperformedonleucocytepelletsusinganestablishedprotocol[4]inparal-
lelwiththegeneticanalysistoconfirmthepathogenicityofthesamples.
Samplepreparation. TheDBSsampleswerepreparedusingethylenediaminetetraacetic
acid(EDTA)bloodonfiltercards(CentoCard,CentogeneAG,Rostock,Germany).Uponprep-
aration,filtercardsweredriedfor2hoursatroomtemperature,sealedinzipbagsandstoredat
-20°Cuntiltheassayswereperformed.
Chemicals. ThesubstrateusedforMPSIVAwas4-methylumbelliferyl-β-D-galactopyra-
noside-6-sulfatesodiumsalt(MU-βGal-6S,Glycosynth,Warrington,UK);β-galactosidase
exogenousenzymewasnecessaryforthesecondreactionstep(ProteasefromAspergillusory-
zae,Sigma-Aldrich,Hamburg,Germany);thesubstrateforMPSIVBwas4-methylumbelliferyl
β-D-galactopyranoside(MU-βGal,Sigma-Aldrich,Hamburg,Germany);asaninternalstan-
dard,4-propyl-5-hydroxy-7-methyl-2h-chromen-2-one(MatrixScientific,SouthCarolina,
USA),wasusedforallthemassspectrometricanalysesofMPSIVassayproducts.Forthecon-
trolenzymeassay—acidsphingomyelinase—thesubstrateandinternalstandardswerepro-
videdbytheCenterofDiseaseControlandPrevention(CDC,Atlanta,USA).Thevials
containedsubstrateandinternalstandardinamolarratioofabout50:1.Reagentswerelyophi-
lizedanddissolvedinappropriatebuffersaspreviouslydescribed[10,11,12].Commercially
availablesalts(Sigma-Aldrich,Hamburg,Germany&VWR,Hannover,Germany)wereused
forallsolutionsandbuffers.
GeneticconfirmationofMPSIVA/Bpatients
GeneticanalysiswasperformedonhighqualitypurifiedDNA.BidirectionalSangersequencing
oftheentirecodingregionandthehighlyconservedexon-intronsplicejunctionswasper-
formedwithgeneandampliconspecificprimers.PCRisfollowedbyShrimpAlkalinephos-
phatase/exonucleaseItreatment;cyclePCRiscarriedoutusingBigDyeTerminatorkitv3.1
(LifeTechnologies)andsubsequentethanolpurification.Sequencingwasperformedusingan
ABI3730xlsequencer.Thetesthasbeendevelopedandvalidatedforclinicalpurposes.The
GALNSgenereferencesequenceisNM_000512.4.Thediseasecausingmutationsdetectedin
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DiagnosisofMorquioSyndromeinDriedBloodSpotsUsingMRM-MS
Table1. GALNSmutationsofpatientsdiagnosedandselectedasreferencefortheMPSIVAenzymaticassaydevelopment.
Codingeffect cDNAchange Mutationtype Localization Allelezygosity PatientDiagnosis Ref.
p.Q29X c.85C>T Nonsense exon1 Homozygous confirmed Novela
p.D39Y c.115G>T Missense exon1 Heterozygousc confirmed Novela
p.G50R c.148G>A Missense exon2 Homozygous confirmed Novela
p.T100P c.298A>C Missense exon3 Homozygous confirmed Novela
p.A107T c.319G>A Missense exon3 Homozygous confirmed [13;14]
p.P179S c.535C>T Missense exon5 Homozygous confirmed [13;14]
p.Y181C c.542A>G Missense exon5 Homozygous confirmed Novela
p.N204T c.611A>C Missense exon6 Heterozygousb confirmed Novela
p.F226L c.676T>C Missense exon7 Heterozygousb confirmed Novela
- c.759-3C>G Splicing intron7 Homozygous confirmed Novela
p.S287L c.860C>T Missense exon8 Heterozygousb confirmed [13;15;16]
p.T312A c.934A>G Missense exon9 Homozygous confirmed [13;16,17]
p.G340D c.1019G>A Missense exon10 Homozygous confirmed [13;14]
p.R386C c.1156C>T Missense exon11 Heterozygousb confirmed [13;18;19]
p.A392V c.1175C>T Missense exon11 Homozygous confirmed [13;14]
- c.1482+1G>A Splicing intron13 Homozygous confirmed Novela
p.P498L c.1493C>T Missense exon14 Homozygous confirmed Novela
p.P499L c.1496C>T Missense exon14 Homozygous confirmed Novela
aNotdescribedinHGMD,presentinCentoMD[20]
bPatientscarrytwoheterozygousmutations
cPatientcarriesthreeheterozygousmutations/variant
doi:10.1371/journal.pone.0131228.t001
theGALNSgeneoftheinvestigatedMPSIVApatientsinthisstudyarelistedinTable1.All
patientspresentaMPSIVAphenotype.ForallmutationsnotdescribedinHGMD(orother
databases),softwareanalyseshavebeencarriedoutusingAlamut,includingSIFT,PolyPhen,
MutationTasterandAlignGVGD.Accordingtosoftwarepredictionsthenovelvariantswe
detectedcanbeconsideredaslikelypathogenicaccordingtopredictionsoftware.Largedele-
tionsforhomozygousvariantsdetectedcouldnotbeexcluded.Toconfirmthepathogenicityof
underlyingvariants,additionalenzymelevelsdeterminedinleucocytesusingaclassicalproto-
coldescribedinliterature[4].
MucopolysaccharidosistypeIVAassay
DBSswithadiameterof3.2mm(bloodvolumeca3.1μL)werepunchedfromfiltercards.
Extractionwasperformedbyadding20μLextractionsolution(0.5Msodiumchloride,0.2%
deactivatedbovineserumalbumin,0,02%sodiumazide)andincubatingfor70to100minutes
at37°Cat700rotationsperminute(RPM).Totheextract,20μLreactionbuffer1(100mM
sodiumacetate,10mMleadacetate,2mMethylenediaminetetraaceticacid,0.02%natrium
azide,pH4.5)and40μLsubstratesolution(10mMMU-βGal-6S,0.2%deactivatedbovine
serumalbumin,0.02%natriumazide)wereadded.Reactionmixturewasmixed10swithavor-
texmixer,sealedandincubatedunderagitationat37°Cfor44to48h.Inasecondstep,tothe
reactionmixture20μLreaction2buffer(100mMcitrate-phosphatebuffer,pH5.2)and20μL
β-galactosidasesolution(10U/mLin0.2%deactivatedbovineserumalbumin,0.02%natrium
azide)wereadded.Thereactionsolutionwasmixed,sealedandincubatedforfurther6h.The
enzymaticreactionwasstoppedbyadding330μLstopbuffer(0.25Msodiumcarbonate-bicar-
bonatebufferwith0.012%Tritonx-100,pH10.7).GALNSactivitywasdeterminedin
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DiagnosisofMorquioSyndromeinDriedBloodSpotsUsingMRM-MS
duplicatesforallsamplesandcontrols,differentconditionsforcardpreparation,storageand
transportwerestudiedtoestimatethestabilityoftheGALNSinDBS.Assayvalidationencom-
passedexperimentssuchas:intra-andinter-assayaccuracy,intra-andinter-assayprecision,
linearityofthemethodandoftheinstrumentationdetection,referencevaluesdetermination
withhealthycontrolsandMPSIVApatients,androbustnessofthemethod.
MucopolysaccharidosistypeIVBassay
Driedbloodspots(DBSs)withadiameterof3.2mm(estimatedbloodvolume3.1μL)were
punchedfromfiltercards.Extractionwasperformedbyadding20μLextractionsolution
(0.5Msodiumchloride,0.2%deactivatedbovineserumalbumin,0,02%sodiumazide)and
incubatingfor70to100minutesat37°Cunderagitation.Totheextract,40μLsubstratesolu-
tion(0.8mMMU-βGalin100mMcitrate-phosphatebuffer,pH4.4)wasadded.Thereaction
mixturewasmixed10swithavortexmixer,sealedandincubatedunderagitationat37°Cfor
3h.Theenzymaticreactionwasstoppedbyadding390μLstopbuffer(0.25Msodiumcarbon-
ate-bicarbonatebufferwith0.012%Tritonx-100,pH10.7).Theβ-galactosidaseactivitywas
determinedinduplicatesforallsamplesandcontrols.MPSIVBdevelopmentwasperformed
usingamodified,previouslyestablishedprotocolforβ-Galactosidase[8]tobeusedascontrol
enzymeinroutinedeterminations.Assayvalidationencompassedexperimentssuchas:intra-
andinter-assayaccuracy,intra-andinter-assayprecision,linearityofthemethodandofthe
instrumentationdetection,referencevaluesdeterminationwithhealthycontrolsandMPSIVB
patients,androbustnessofthemethod.
MPSIVAandMPSIVBsampleclean-upformassspectrometryanalysis. Toeachassay
sample50μLinternalstandardsolution(5μg/mLinmethanol)isadded,followedby500μL
organicsolvent(ethylacetate:methanol,vol.19:1).Solutionismixed,centrifuged3minutesat
14.5kRPM.200μLfromtheorganicphasearetransferredtoa96wellplatewithv-shapebot-
tom(GreinerBio-one,Frickenhausen,Germany),thesolventisevaporatedandtheanalyte-
internalstandardmixturere-dissolvedin120μL10mMammoniumacetateinmethanol:
H O,80:20v/v.
2
MassspectrometricanalysisofMPSIVAandMPSIVBassays. Massspectrometricanal-
ysiswasperformedonatriplequadmassspectrometer(TripleQuad5500,ABSciex,Darmstadt,
Germany)coupledwithanUPLCsystem(Water,Manchester,UnitedKingdom).UPLC
wasusedtoconcentratethesampleandtheinternalstandardusingthefollowingparameters:
solventA—10mMammoniumacetateinwater;solventB–10mMammoniumacetatein
methanol;injectionvolume–10μL,flow—0.4mL/min;gradient—isocratic80%B;timeper
analysis—5min;column—phenomenexfast4uAAA-MS/250x2.00mm,4micron.Triple-
quadmassspectrometerwasemployedfordetectionandquantificationunderthefollowing
parameters:typeofmeasurement—multiplereactionmonitoring;interfacewithHPLC—flow
splitter(1/3flowreachtheESIchamber);monitoredmasstransitions—175/119(analyte)and
217/160(internalstandard);detectioninnegativeionmode;resolutionQ1andQ3–unit;scan
time-4,988minutes;declusteringpotential-126V(175/119)and-143V(162/106);collision
energy-38V(175/119)and-36V(217/160);curtaingas40psi;collisiongas8psi;ionspray
voltage-4000V;temperature150°C;ionsourcegas160psi;ionsourcegas260psi;entrance
potential-12V;collisioncellexitpotential:-10V.Quantificationwascarriedoutusing4-pro-
pyl-5-hydroxy-7-methyl-2h-chromen-2-oneasinternalstandard(IS)atfixconcentration
(4μg/mL)forallsamplesandastandardcurveof4–methylumbelliferone(4-MU,analyte).
StatisticaldatafromtheMPSIVAandIVBassaysvalidationaswellfromthecontrol
enzymewereanalyzedusingExcelSoftware(MicrosoftOffice,2010).Foreachassayand
cohortwerecalculated:minimum,maximum,average,median,standarddeviation,cut-off(as
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DiagnosisofMorquioSyndromeinDriedBloodSpotsUsingMRM-MS
averageactivityminustwotimesstandarddeviation).Variationsindifferentmeasurements
andexperimentsofthesameassaywereanalyzedusingANOVAorStudent’sTstatisticaltests.
Acidsphingomyelinaseassay
3.2mmspotswereincubatedat37°Cunderagitationfor60minuteswith80μLextraction
buffer.10μLextractweredispensedin96-wellplates.15μLoftheacidsphingomyelinase
(ASM)enzymeassaycocktailswereaddedtotheplatescontainingtheextracts,thensealedand
incubatedfor23hat37°Cwithshaking.Theenzymereactionwasstoppedwith100μLof1:1
ethylacetateandmethanol.SamplepreparationforMS/MSanalysiscomprisedaliquid-liquid
extractionstep,asolidphaseextractionstepandMS/MSdetectiononanAPI4000triple-quad-
rupoleMS/MS(ABSciex,USA)inpositiveionmodeusingmeasurementparameterspreviously
described[10].
ResultsandDiscussion
ToevaluatethenormalvaluesofGALNSactivityinDBSandtocalculatethecut-offvalues,59
healthycontrolsampleswererandomlychosenfromourbiobank.Filtercardswereprepared
fromfreshlycollectedblood(EDTAblood,2–6hold)andfromhemolyzedblood(EDTA
blood,2–4daysstoredat-20°C).Upondrying,thefiltercardswerelabeled,sealedinzipbags
individually,andstoredat-20°Ctobepunchedandusedfortheexperiment.
DevelopmentofGALNSenzymaticassayinDBS
GALNSassaydevelopmentforDBSrequiredexperimentstoimproveeachstepoftheenzy-
maticassay:(i.)extraction(toutilizethesmallamountofenzymepresenttothefullest,reduc-
ingenzymaticdegradationandinstability),(ii.)enzymaticreaction(parametersweretestedto
boosttheenzymaticyieldforabetterseparationofhealthyfrompathologicalvalues),(iii.)
sampleclean-up,(iv.)detectionandquantificationoftheenzymaticproduct.Overall,theresult
oftheassaydevelopment,GALNSactivityinhealthycontrolswasincreasedfrom0.12–
0.70μmol/L/h(previouslyreportedinliterature[8])to11.0–43.0μmol/L/h.
1. TodevelopaviableassayforDBS,firstanoptimalextractionmethodofthebloodelements
fromthefilterpaperwasdeveloped,usingdifferentbuffersandvaryingpHandconcentra-
tions.Outofalltheextractionconditionstested,wefoundthatthebestresults(20%higher
thananyotherextractionsolutionorbuffertested)wereobtainedwithaconcentratedsaline
solutioninthepresenceofinactivatedbovineserumalbuminasastabilizer,underagitation
formorethan70minutes.DBSwereincubatedat37°Cforaperiodof70to100minutes
with20μL0.5Msodiumchloride,0.2%deactivatedbovineserumalbumin,0,02%sodium
azide(duetothelengthoftheassayincubationtimes,natriumazidewasaddedtoall
solutions).
2. GALNSenzymaticactivitywasfoundtobelinearfrom45to50hoursforDBSextract.For
thisreasontheincubationtimeforthisstepwassettoanintervalof46to48handcalcula-
tionoftheenzymaticactivitywasadjustedaccordinglyforeachsample.Thislongperiod
servedalsotocompensateforthelowamountofenzymepresentinDBS.GALNSactivity
wasfoundtobeenhancedbyaddinglead(Pb2+)—ametalionthatdoesnoteasilyform
adductswiththeproductinESImeasurement,aswellasbythepresenceofEDTAinthe
reactionmixture.Forthesecondreactionstep,β-galactosidaseisdilutedwith0.2%bovine
serumalbuminandtheenzymaticreactioniscarriedoutfor6h.
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DiagnosisofMorquioSyndromeinDriedBloodSpotsUsingMRM-MS
3. Duringthesamplecleanupstep,watersolubleelementsfromthereactionmixture(salts,
bovineserumalbumin,andhemoglobin)andfilterpaperreststhatcanblocktheLC/MS
systemareremovedbyliquid-liquidextraction.Thetwophasesareeasilyseparated(the
waterphaseisreddishduetothebloodextractandhighsaltconcentration,theorganicis
transparent)andthecleanupcanbeperformedeitherinEppendorfcups(forlownumber
ofsamples)orindeepwellplateforhighthroughput.
4. Themostimportantimprovementintroducedbythepresentassay,isthedevelopmentofa
stable,reproduciblemultiplereactionmonitoringmassspectrometrymethodforthedetec-
tionandquantificationof4-methylumbelliferone(4-MU).Althoughpreviousstudieson
flavonoidcompoundsreferto4-MUasaninternalstandard[21;22;23],therearenopub-
lishedstudiesontheroutinequantificationof4-MUbymassspectrometrywithanapplica-
tioninclinicalchemistry.4-MU,asotherhydroxyl-chromen-2H-onederivatives,isavery
stablemolecule,fluorescentinUVlightandthususedroutinelyinfluorimetricenzyme
assays[24].However,inthepresenceofbloodelements,especiallyhemoglobin,fluores-
cencequenchingwasreportedbydifferentstudies.Thisphenomenoncannotbeprecisely
quantifiedforeachDBSsampleseparately,renderingaquantificationerrorinevitable.Also,
althoughsensitive,fluorometryusesonlyarelativequantificationthroughexternalstandard
curve.Withthedevelopmentofnewinstrumentation,multiplereactionmonitoringmass
spectrometryrivalsthesensitivityandthehighthrough-putcapacityofthefluorometry;
however,byusinganinternalstandard,theselectivityandprecisionofthequantificationis
highlysuperiortothefluorimetricmethod.Here,wereportanewquantificationmethod
for4-MUusinganTripleQuad5500massspectrometer(ABSciex,Germany)usingastan-
dardcurveofnine4-MUdilutionsfrom0μg/mLto1μg/mLandaninternalstandardwith
afixedconcentrationof0.5μg/mL.Asinternalstandardsfor4-MUquantification,several
hydroxyl-chromen-2H-onederivativesweretestedandallwerefoundsuitablehavingsimi-
larfragmentationpatternwith4-MUundersimilarcollisioninduceddissociation(CID)
conditions.However,4-propyl-5-hydroxy-7-methyl-2H-chromen-2-onewaschosendueto
itsdifferentparent/daughtertransitions(217/160—internalstandardand175/119–4-MU)
andsimilarfragmentationpattern(seeFig1);alsoduetoitsdifferentHPLCretentiontime
comparedto4-MU.Thestandardcurvewaslinearandreproduciblefortheconcentration
rangetested(thatincludestheanalyticalrangeforthemethod).
ForalltheMPS4atests,thestandardcurvespreparationalsoincorporatedstepsthatmimic
thethoseusedinthepreparationofinvestigatedbloodsamples(dilutioninstopbuffer,mixing
withtheinternalstandard,liquid—liquidextraction)thusmeasurementdifferencescausedby
tracesofsaltsorlowextractionyieldwereeliminated.ExamplesofTIC(totalionchromato-
gram)obtainedbyLC/MRM-MSareshowninFig2forablanksample(Anewblanksampleis
measuredineverymeasurementasbackgroundtobesubtractedfromthevaluesobtainedfor
thesamplesinvestigated.Thisstepisnecessaryduetothefree4-MUpresentinthesubstrate
beforethestartofthereaction),apathologicalsample(similarinprofilewiththeblanksample),
anormalcontrolsamplewithaverageGALNSactivity,andasamplewithhighlysosomalenzyme
activity(2xhigherthantheaverage).
ValidationoftheMPSIVassay
Theprecisionoftheassaywascharacterizedbymeasuringtherelativestandarddeviationin
intra-andinter-assayexperiments.Intra-assayvariationwasdeterminedbymeasuringcontrol
samplessixtimes(n=6)inonesinglebatch,andtheresultsshownosignificantdifference
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DiagnosisofMorquioSyndromeinDriedBloodSpotsUsingMRM-MS
Fig1.TripleQuadMRM-MSdetectionof4-MU.Hydroxyl-chromen-2H-onecompoundscanbedetectedinQ1scanasa(M-H)-ionand,underspecific
collisionenergy,thehetero-cycleisbrokenwithaneutrallossofthefragmentcontaining–COO(cid:129).Weproposethattheremainingfragmentundergoesa
molecularrearrangementtoobtainamorestablestructure.Forenzymaticproductdetectionandquantification,twotransitionsaremonitored:175/119
(4-MU)and217/160(internalstandard).A.Collisioninduceddissociationfragmentationspectrum(MS2)oftheanalyte(4-MU),obtainedusinganABSciex
5500,andMRM-MStransitionspectrummonitoredduringtheMPSIVassays(175/119);B.Collisioninduceddissociationfragmentationspectrum(MS2)of
theinternalstandard(4-propyl-5-hydroxy-7-methyl-2H-chromen-2-one),obtainedusinganABSciex5500,andMRM-MStransitionspectrummonitored
duringtheMPSIVassays(217/160).
doi:10.1371/journal.pone.0131228.g001
betweendifferentaliquotsofthesampleinthesameassay(relativestandarddeviationbetween
5.00%and14.02%).Inter-assayvariationwasdeterminedbymeasuringcontrolbloodsamples
andtheadditionalpathologicalsampleon5differentdays(n=5),onseparatedbatches.The
resultsshownosignificantdifferencebetweenaliquotsofthesampleinthedifferentassays
PLOSONE|DOI:10.1371/journal.pone.0131228 July6,2015 8/14
DiagnosisofMorquioSyndromeinDriedBloodSpotsUsingMRM-MS
Fig2.TotalIonChromatogramprofilesof4-MUatconstantconcentrationofinternalstandardobtainedwithTripleQuadMRM-MSforA—ablank
sampleorfilterpaperincubatedinthesamemannerasthebloodsamples(thatcontain4-MUpresentatthebeginningoftheenzymaticreactionasa
byproductofthesynthesis);B—pathologicalbloodsample(withsimilarTICprofileastheblanksample,usedasaqualitycontrolineachassay);C—anormal
activitybloodsample(healthycontrolwithanaverageGALNSactivityusedasaqualitycontrolineachassay);D—ahighactivitybloodsample(asample
withatypicallyhighactivityoflysosomalenzymes).
doi:10.1371/journal.pone.0131228.g002
performedondifferentdays(witharelativestandarddeviationbetween8.4%and11.5%(see
supplementarydata).
Standardlinearityforthemethodwasestablishedbyperformingtentimesstandardcurve
preparedinsimilarmannertothatofthesamples.Theresultsshowthat4-MUdetectionand
quantificationislinearforconcentrationsupto6μM.Thelinearityoftheenzymaticreaction
wascheckedbyusingdifferentvolumesofextractrangingfrom25%to100%incubatedinthe
samemanner(withconstantsubstrateamount).
Alimitofdetection(LOD,3(cid:1)standarddeviation)of0.1μmol/Lblood/handlimitofquanti-
fication(LOQ,10(cid:1)standarddeviation)of0.3μmol/Lblood/hweredeterminedonblankfilter
papersmeasuredtentimesinthesamebatch.
Theparameterofrobustnesschosenforthismethodwasthetimeintervalfromsample
preparationtothemassspectrometryanalysis.TheANOVAonewaystatisticaltestshowedno
statisticallysignificantdifferencebetweenthesamplesmeasuredupto48hafterpreparation
PLOSONE|DOI:10.1371/journal.pone.0131228 July6,2015 9/14
DiagnosisofMorquioSyndromeinDriedBloodSpotsUsingMRM-MS
Fig3. A.EnzymaticGALNSassayinDBSshowsastatisticallysignificantdifference(p<0.0001intwo-
tailedMan-Whitneytest)betweensamplesofhealthycontrolsandthesamplesofaffectedMPSIVApatients.
B.β-galactosidaseinDBS,theproposedcontrolenzymetest,carriedoutonthecontrolsamplesandMPS
IVApatientsamples,showsimilaractivityinbothgroups.
doi:10.1371/journal.pone.0131228.g003
andstorageatroomtemperature,inanevaporationprohibitoryenvironment(platewascov-
eredwithaluminumfoilandplacedintoazipbag).
NormalGALNSvalues(forhealthycontrols)inanalyzedDBSsarefoundtobebetween
11.0to43.0μmol/Lblood/h(mean±standarddeviation:23.1±8.9).Cut-offwassetat
5.3μmol/Lblood/h(mean-2(cid:1)standarddeviation).
ThepathologicalrangeofGALNSactivitywasdeterminedon24MPSIVApatientsanddeter-
minedtobebetween0and0.64μmol/Lblood/h(mean±standarddeviation:0.35±0.21)—see
Fig3.
ThetwoMPSIVAcarriersshowedGALNSactivityabovethecutoff,butlowerthanthe
minimumfoundinDBSfromhealthycontrols(Table2).Overallpositivepredictivevalue,neg-
ativepredictivevalue,sensitivity,specificityoftheGALNSenzymatictest,calculatedforthe
testedMPSIVApatientsandcontrols,was1.0(allMPSIVAcasesinvestigatedwerecorrectly
assignedaspathologicalandallcontrolshavedemonstratednormalenzymeactivity).
Normalβ-galactosidasevaluesinanalyzedDBSsfromthehealthycontrolswerefoundtobe
intheinterval71.8to160.9μmol/Lblood/h(alongsideanoutlierof18.5μmol/Lblood/h—below
Table2. GALNS,GLB1andASMenzymaticactivitiesfortheinvestigatedMPSIVApatientsandcarriers,MPSIVBpatientsandcarriersandhealthy
probandscohort.
MPSIVA MPSIVA MPSIVB MPSIVB Healthy
patientsN=24 carriersN=2 patientsN=11 carriersN=4 controlsN=59
GALNSactivityμmol/L/h Cut-off 4.8
Minimum 0.0 6.6 4.7 10.2 11.0
Maximum 0.6 8.8 20.5 16.1 43.0
Mean 0.3 7.7 10.9 13.8 22.7
Median 0.3 7.7 9.0 14.4 20.5
GLB1activityμmol/L/h Cut-off 28.5
Minimum 31.6 91.4 0.3 39.5 18.5
Maximum 132.1 99.4 10.4 58.8 160.9
Mean 66.6 95.4 3.3 49.3 70.9
Median 65.0 95.4 3.7 49.4 66.1
ASMactivityμmol/L/h Cut-off 2.2
(controlenzyme)
Minimum 3.6 11.9 3.7 6.8 4.0
Maximum 35.2 7.4 24.8 22.3 44.2
Mean 8.0 9.6 13.0 11.3 8.9
Median 5.4 9.6 3.0 8.0 8.2
doi:10.1371/journal.pone.0131228.t002
PLOSONE|DOI:10.1371/journal.pone.0131228 July6,2015 10/14