betweengenotyperesidualenzymeactivityandclinicalphenotyperemainstobe - PDF document

betweengenotype,residualenzymeactivity,andclinicalphenotyperemainstobeelucidated.Todaymorethan250ARSAmutationsareknown,makingitchallengingtodefinemoreprecisegenotype-phenotyperelationshipsespeciallyinthelateronsetforms,whichoftenshowcompoundheterozygosityfordifferentmutations[7–11].Thephenotypicvariabilitybecomesespeciallyrelevantwhentreatmentevaluationisbasedoncomparisonwithanuntreatedsiblingcarryingthesamemutations[12,13].Youngersiblingsofanaffectedoldersiblingareusuallydiagnosedinanearly(orevenpre-symptomatic)diseasestageandhavetheopportunitytoundergoatreatmentlikeconventionalorgene-correctedstemcelltransplant-ation[14–17].However,itisunclearhowtheirclinicalcoursewouldhaveevolvedwithouttreatment.CasereportssuggestthattheremightbesomevariabilityofdiseaseonsetevenbetweensiblingswithMLD[18,19],butasystematicanalysisiscurrentlymissing.TheaimofthisstudyistosystematicallyinvestigatephenotypicvariabilityinasignificantnumberofsiblingswithMLD,regardingdiseaseonsetandprogression,andcomparethemtothephenotypicvariabilityinalargecohortofchildrenwithMLD.Inaddition,casereportsintheliteraturewillbediscussed.Theresultsarenotonlyrelevantforcounselingoffamilieswithaffectedchildren,butwillalsoallowbetterinterpretationoftreatmentoutcomes,andmighthelptobetterunderstandthecomplexgenotype-phenotyperelationshipsinMLD.ResultsMLDsiblingsandcohortFromourdatabase85childrenwithlate-infantile(n=29)andjuvenile(n=56)MLDfulfilledinclusionandexclusioncriteriaforthisstudy(seeMethods).Fromthese,12siblingpairs(24patients)wereidentified(threelate-infantile,ninejuvenile),threeofthemfromtheCenterforChildrenwithWhiteMatterDisorders,VUUniversityMedicalCenter,Amsterdam.Threeofthejuvenilesiblingpairswereanalyzedonlyregardingtheonsetofsymptoms,asatleastoneofthemreceivedatherapeuticinterventionpotentiallyinfluencingthefurthercourseofdisease.Table1givesanoverviewoftheclinicalanddiagnosticfindingsofallsiblingpairs. Table1AnalyzedsiblingswithMLDSiblingpairsGenderMLD-formAgeatonset,yearsTypeofonsetMRI-score,earlystageGenotype1.1.2mflate-infantile1.8a2.1motor1616c.575C�G;c.733G�Ab2.1mlate-infantile1.5amotor––.2f1.5–3.1flate-infantile0.8amotor20c.449C�T;c.449C�T.2f1.8204.1mjuvenile11acognitive20c.542T�G;c.1468T�C.2f12.4205.1fjuvenile5.6amixed––.2f6.7–6.1fjuvenile6.2amotor15c.465+1G�A;c.1283C�T.2f4.7147.1fjuvenile14amixedc.1283C�T;c.1283C�T.2f11.9158.1mjuvenile6.5acognitive18c.465+1G�A;c.542T�G.2m13.5179.1mjuvenile10amotor–c.1283C�T;c.1283C�T.2f15–10.1fjuvenile8mixed19c.465+1G�A;c.1283C�T.2m52011.1mjuvenile6.7motor22–.2f62112.1mjuvenile5motor20–.2m520a=firstdiagnosedsibling(ifknown);b:genotypeintraditionalnomenclatureElgünetal.OrphanetJournalofRareDiseases (2019) 14:136 Page2of10 AgeatonsetofclinicalsymptomsAsshowninFig.1,siblingswithMLDshowedasimilarvariabilitywithrespecttoageofonsetoffirstsymptomscomparedtotherestoftheMLDcohort.Althoughsomesiblingpairswerecloseintheirageatonset,othersshowedarelevantdiscrepancy.OnesiblingpairwithlateinfantileMLDhadadifferenceof1yearinageatonset(withthelateronsetinthesecondsibling).InjuvenileMLD,7outof9siblingpairswithjuvenileMLDhadanonsetmorethan1yearapart,fourmorethan2years,andinonesiblingpairageatonsetdifferedmo

rethan6years.Althoughthevariabilityoftheageatonsetbe-tweenunrelatedpairsofthejuvenilecohortwashigheronaverage(meanEuclideandistance44.37months,standarddeviation(SD)33.99)comparedtothesiblingpairs(29months,SD27.05),thisdifferencewasnotsta-tisticallysignificant(p=0.16).Thiswastruealsofortheunrelatedlate-infantilecohortwithameanEuclideandistanceoftheageatonsetof4.4months(SD3.57)vs.5.33months(SD6.11)insiblingpairs(p=0.91).SymptomconstellationatonsetWhenclassifyingsymptomconstellationsattheageatonsetintogrossmotor,cognitivesymptomsorboth,allexaminedsiblingpairsshowedthesamesymptomconstellationatonset,evenwhentheirageofonsetwasdifferent.Whilethechildrenwithlate-infantileMLDallhadmotorsymptomsatonset,thechildrenwithjuvenileMLDpresentedwithallthreecategoriesasshowninTable1.IntheunrelatedgroupofchildrenwithjuvenileMLD14hadmotoronset,17hadcognitiveonsetand7hadmixedonset.Foreachofthethreecategories,oneexampleispresented:Theoldersiblingofpair1(cf.Table1)presentedwithgaitdifficulties,abnormalmovementpatternsandweak-nessattheageof21months(afterhavinglearnedtowalkindependentlyaged12months).Hissisterpre-sentedwiththesamefirstmotorsymptomsattheageof25months(independentwalkingfromage14months).Bothsiblingsdeterioratedrapidly,withcompletelossofgrossmotorfunctioninthemonthsfollowingduetosevereprogressivespastictetraparesis.Sibling8.1wasnoticedwithconcentrationdifficultiesattheageof6years(78months)anddescribedwithslowlydecliningcognitiveabilities.Asaresulthisschoolgradesdeteriorated,especiallyinmathematics,andhehadtorepeatayearinschoolandeventuallychangeschooltype.Ontheotherhand,hismotorfunctionremainedunaffectedintoadulthood.Hisyoungerbrother(8.2)startedtodevelopverysimilarschoolprob-lemsattheageof13years(162month),subsequentlyhehadtorepeatayearinschoolandthenchangedtypeofschool.Inaddition,hehadproblemsinhissocialbehav-ioratdiseaseonsetwithalcoholabuse,aggressivebehav-iorandasuicideattempt.Likeinhisolderbrother,hismotorfunctionremainedstableintoadulthood.Siblingpair5presentedwithbothcognitiveandmotorsymptomsatonset.Attheageof5years,patient5.1showedawhinymoodanddevelopedtoegait.Patient5.2 Fig.1Ageatonsetofthecohort(late-infantile/juvenile).Boxplotsindicatedistributionofageatonsetfornon-relatedchildrenwithMLD.Siblingsaremarkedinred,withlinesconnectingpairsofsiblingsElgünetal.OrphanetJournalofRareDiseases (2019) 14:136 Page3of10 alsopresentedwithwhinymood,orientationproblems,andlossofpreviouslyacquiredanorectalcontinenceattheageof6years,quicklyfollowedbygaitdifficulties.DiseasedynamicsGMFC-MLDInFig.2progressionofgrossmotorfunctionofthedifferentpairsofsiblingsisshowninrelationtotherestofthewhole(unrelated)cohort.MotorfunctionwasassessedusingthestandardizedgrossmotorfunctionclassificationforMLD(GMFC-MLD)[20].Thefigureillustratesthatsiblings,regardlessoftheirageofonset,haveasimilardiseaseprogression.Inthelate-infantilegroup(Fig.2,left),allchildren,whethersiblingsornot,showedaratheruniformandrapidprogressionoftheirmotorfunction.Inthejuvenilegroup,thevariabilityofthedurationfromGMFC-MLDlevel1(agewhenfirstmotorsymptomswerenoticed)toGMFC-MLDlevel3(losingtheabilitytowalk)waslowerbetweensiblingpairsthanbetweennon-relatedpairsofthejuvenilecohort(7.00months(SD6.16)versus23.66months(SD20.35),Fig.2,right).Thiswas,however,notstatisticallysignifi

cant,p=0.077.CognitivediseaseprogressionparametersForprogressionofcognitivesymptoms,weinvestigatedthegroupofchildrenwithjuvenileMLDonly,asthelate-infantilegroupshowedaveryrapidanduniformdiseaseprogressiondominatedbythelossofgrossmotorfunction,butalsoincludingthelossoftheircognitiveabilities[3,21].BetweensiblingswithjuvenileMLD,thevariabilityintimefromfirstsymptomstopresentingwithconcentra-tionproblemswassmaller(2.67months,SD4.62)comparedtounrelatedpairsoftherestofthejuvenileMLDcohort(19.72months,SD14.46,p=0.015).Thevariabilitywhenfirstlanguagedeclinestarted,however,wassimilarbetweenpairsofsiblings(meanEuclidiandistance29.00months,SD37.58)comparedtounrelatedpairsofthejuvenileMLDcohort(mean29.01months,SD25.08).MRITable1andFig.3showMRIscoresandMRimagesofsiblingswithMLD.Boththescoresandpatternofde-myelinationappearedverysimilarbetweenthesiblings.EspeciallyinchildrenwithjuvenileMLD,frontalorparieto-occipitalinvolvementwasalwayssimilar,andwasrelatedtoamorecognitiveormotorsymptomonset.GenotypeInordertoanalyzethegenotype-phenotyperelationship,weidentifiedthemostcommongenotypesandcom-paredthevariabilityofageatsymptomonsetbetweensiblingsandnon-relatedchildrencarryingthesamegenotype.Theresults,displayedinFig.4,showthattheageatonsetinthepatientswithcompoundheterozygosityforc.465+1G�Aandc.1283C�TinARSAgenerangedbetween3.5and8years(mean5.8yearsofage;n=8).Homozygosityforc.1283C�Tcausedalaterdiseaseon-setwitharangebetween10and15years(mean=12.1years;n=6).Siblingswiththesegenotypeswerewithintherangeofunrelatedchildreninbothgenotypeswith-outbeingmoresimilartoeachother.Therefore,theageofonsetbetweensiblingswithMLDwasnotmoresimi-larcomparedtonon-relatedchildrenwithMLDcarryingthesamegenotype. Fig.2Diseaseprogression(ofgrossmotorfunction).Progressionofgrossmotorfunction(GMFC-MLDlevels1to6)ofchildrenwithlate-infantile(left)andjuvenile(right)MLD;boxplotsshowsdistributionofnon-relatedchildrenwithMLDwithineachlevel,siblingsaremarkedbycoloredlines;note:GMFC-MLDisonlyapplicableaftertheageof18months(=90thpercentileofwalking)[3]Elgünetal.OrphanetJournalofRareDiseases (2019) 14:136 Page4of10 DiscussionMLDisaninheriteddiseasewithanautosomalrecessivetrait,sometimesaffectingseveralsiblingsinonefamily.Withregardofthenaturaldiseasecourseinsiblings,thereexistonlycasereports.Someofthemindicateaverysimilarclinicalcourseandsomeshowmorevari-abilitybetweenthesiblings.ThisstudyrepresentsthefirstsystematicanalysisofthephenotypicvariabilityinsiblingswithMLD.WewereabletoshowthatageofonsetmayvaryconsiderablybetweensiblingswithMLD.Althoughsomesiblingpairsshareaverysimilarageofonset,othersshowahighdiscrepancy.Withinthesamefamily,however,theonsetandcourseiseitherlateinfantileorjuvenile.EvenwithinlateinfantileMLDonsetinsiblingsmaybeupto12monthsapart.InjuvenileMLD,around80%haveadiseaseonsetofmorethan1yearapartandaround40%morethan2years.ThisvariabilityinageofonsetissimilarbetweenthesiblingsandbetweenrandomlychosenunrelatedpairsoftheMLDcohort.Casereportsofsiblingsintheliterature,summarizedinTable2,alsoshowthisvariability.Thefirstdescrip-tionofsiblingswithMLDwasreportedbyScholzin1925[22].Hedescribedtwosiblingswiththejuvenile Fig.3T2-weightedMRIofsiblingpairsinearlydiseasestage.MRIofsiblingpairsinearlydiseasestage(atdiagnosis)withtherespective

MRIscorebelowillustratingthesimilarityindistributionofwhitematterchangesbetweensiblings.AxialslicesofT2-weightedsequenceswereselected Fig.4Genotype–Ageatonset.Variabilityofageatsymptomonsetbetweensiblingsandnon-relatedchildren,carryingthesamegenotype,intwomostcommongenotypesElgünetal.OrphanetJournalofRareDiseases (2019) 14:136 Page5of10 formandaquitesimilarageofdiseaseonsetataround8yearsofage,bothwithcognitiveandmotorsymptomsatonset(brother1:spasticityinlegsresultinginrigidandslowwalking,opticalatrophy,learningandpercep-tionproblemsaswellasemotionallability;brother2:spasticityinextremities,tremorinlegs,whiny,unstablemoodandcognitiveproblems(learning,perception))andarapidlyprogressivediseasecourse,bothdiedaround3yearsafteronsetinaveryadvanceddiseasestage.Alvesetal.[23]reportedonfourcasesoflateonset(15,17,18,21years)MLDinafamilywith15siblings.Inaccordancewithourresultsthedescribedsiblingsallhadthesamesymptomclusteratonset-mentaldeteriorationand/orbehavioralchanges.AquitesimilarcasebySatohetal.[24]reportedonasiblingpairwithmotorandcognitivesymptomsatdiseaseonsetattheagesof19and15years.Kouletal.[25]describedtwoaffectedsiblingswithlate-infantileMLDbothwithanonsetwithdeteriorationofmotorfunctionat2yearsofage.Mahmoodetal.reportedtripletswiththelate-infantileform,allofthemwithmotorfunctionstartingaround16monthsofageafternormalpsychomotordevelopment[26].Anotherreportofsiblingswithlate-infantileMLDbyNyberg-Hansenetal.[27]delineated2brothers,whobothdevelopedmotordysfunctionanddysarthricspeechwithinthesecondyearoflifewithrapidprogressioninthefollowingyears.Aslanetal.[28]reportedontwosiblingswiththejuvenileformofMLD,whohadtheirfirstsymptomsat6and7yearsofagerespectively,alsoshowingasimilarsymptomconstella-tionatonsetwithbothmotorandcognitivesigns(thefirstsibling:ataxia,attentiondeficitandperceptual Table2CasereportsofsiblingswithMLDAuthorYearSiblingAgeatonset(years)TypeofonsetGenotypeScholz192518cognitiveandmotor–28cognitiveandmotor–Nyberg-H.197211motor–22motor–Hoes1978127cognitive–226cognitive–Manowitz1978116cognitive–218cognitive–Alves1986118cognitive–221cognitive–317cognitive–415cognitive–Satoh1988119cognitiveandmotor–215cognitiveandmotor–Clarke198919cognitive–223pre-symptomatic–Kappler1992114cognitivep.R84Q;p.P426L229pre-symptomaticp.R84Q;p.P426LKoul199412motor–22motor–Arbour200017cognitive+seizuresp.W318ter;p.R143G222cognitivep.W318ter;p.R143GCengiz2002121cognitive+seizures–218cognitive+seizures–312cognitive–Mahmood201011.3motorc.459+1G�A;c.459+1G�A21.3motorc.459+1G�A;c.459+1G�A31.3motorc.459+1G�A;c.459+1G�AAslan201817cognitiveandmotorc.1055T�C;c.991G�A26cognitiveandmotorc.1055T�C;c.991G�AElgünetal.OrphanetJournalofRareDiseases (2019) 14:136 Page6of10 difficulties;thesecond:learningdifficulties,mildchangesinfineandgrossmotorfunction).ThelateronsetcasereportsbyHoesetal.[29],Cengizetal.[30]andMano-witzetal.[31]alsohaveincommonasimilarsymptomconstellationatonset(Table2).Hoesetal.reportedontwobrothers:thefirstdevelopedfirstsymptomsattheageof27yearswithproblemsinsocialinteractionsoonfollowedbyhypersexual,aggressiveandapatheticbehav-ior,hediedattheageof30yearsafterakidneybiopsy[29].Hisyoungerbrothershowedfirstsymptomsattheageof26(socialhandicap,p

sychiatricsymptoms)anddiedwithin8yearsafteronset[29].Cengizetal.describedthreesiblingwithvariabilityintheirageatonsetofclinicalsymptoms(21,18and12years)butverysimilarsymptomconstellation(1:behavioraldisturbance,workingmemorydifficulties,epilepticseizures;2:epilep-ticseizuresandprogressivementalchanges;3:poorschoolperformance,disinhibition,hyperactivityandself-awareness)[30].FurthermoreManowitzetal.describedanadultsiblingpairwithcognitivesymptomsatonsetandlateinitiatingmotorprogression[31].Thesecasereportscorroborateourfindingsbyshowingsomevariabilityinageofonset(maximum9yearsdifferenceinCengizetal.),butclearsimilarityinthetypeoffirstsymptomsanddiseaseprogression(e.g.Scholz1925:rapid,motorandcognitive,diedwithinafewyears;Alves:pronouncedseverementaldeteriorationindiseasecourse).ThisisespeciallyremarkableaschildrenwithjuvenileMLDareknowntoshowaheterogeneouspresentationandclinicalcourse.Onereportfrom1981byYatzivandRusselletal.[32],wasdifficulttointerpret.Theydescribedafamilywiththreeaffectedsiblings;theeldestshowedfirstsymptoms(developmentaldelay)agedaround1year,whilethetwoyoungersiblingswerereportedwithadiseaseonsetat6years,withallthreedevelopingdystoniaastheirdominantsignwithnormalcognitivefunctionintoadulthood.AsnoneoftheothersymptomsusuallyreportedinMLDweredescribed[3,21]andthediagnosiswasbasedonenzymemea-surements,wherepseudodeficiencyandcarrierstatusforpathogenicmutationsmayexplainrelativelylowvalues,diagnosisofMLDseemeduncertain,andwedidnotincludethisreportinTable2.Apartfromthiscasereport,siblingsofchildrenwithlate-infantileMLDallhadthelate-infantileformwiththetypicalrapiddiseaseprogression.Itmaynotsurprisethatwithinthelateronsetforms,withahigherresidualenzymeactivitythaninthelate-infantileform,theageofonsetcanvaryconsiderably.Forexample,Arbouretal.2000[19]reportedonaVietnamesefamilywithtwoaffectedsiblings,bothwiththesamegenotype,ofwhomthefirstwasearlierandmoreseverelyaffectedstartingwithcognitivesymptomsandepilepticseizuresattheageof7yearswithamorerapiddiseaseprogression,whereasthesecondsiblinghadverymildcognitivesymptomsattheageof22years.AnothersiblingpairwasdescribedbyClarkeetal.1989[18],withclinicalmanifestationofMLDattheageof9yearswithcognitivesymptoms(learningandbehavioraldifficulties)foroneaf-fectedsibling.Thesecondaffectedsiblingpresentednoneofthecommonsymptomsuntiltheageof23years(ageatdescription)exceptsulfatideaccumulationinthegallblad-der.AlsothesiblingswithjuvenileMLDreportedbyKappleretal.in1992showthiskindofdifferenceintheageofonset,withtheolderonepresentingwithbehavioralchangesattheageof14years[33].Itcancurrentlyonlybespeculatedthattheremustbeunrecognizedfactorsthatinfluencethephenotypicvariabilitybeyondthegenotypelevel.Itisinterestingtonotethatevenwhenchildrenwiththesamegenotypeareinvestigated,thesiblingsamongstthem(whoshareasubstantialpartoftheirgenome)donothaveamoresimilarageofonsetcomparedwithnon-relatedchildren.Itislikelythatthereareotherepigenetic,metabolicorunidentifiedfactorswhichinfluencetheonsetoffirstsymptomsofMLDinadditiontothegenotype.Ourresultsunderline,thatalthoughprobablyvalidinmanycases,comparingtreatedpatientstotheirnon-treatedsiblings,hastobedonewithcaution.Comparisontoalargernon-treatedcohortofchildrenwithMLDmightbeamorevalidapproach[34].Thisunderlinestheim-portanceofnaturalhist

orydataasretrospectivecontrolsfortreatmenttrials,intheabsenceofthefeasibilityofrandomizedplacebo-controlledstudiesinrareprogres-sivedisorders,likeMLD.Therearesomelimitationsinrespecttoourfindings.Mostimportantly,althoughbeingthemostcomprehen-sivesiblingstudy,largernumbersofpatientswouldbedesirable.Whileinourstudywewereabletosustainahighdataqualityduetothesameinvestigatorsandclinicalstandards,amulti-centricinternationalcollab-orationwillincreasepatientnumbersandpotentiallyconfirmourresultswithmorestatisticalpowerandcertainty.Inaddition,dataondiseaseonsetareoftenretrospect-ive(aslongasneonatalscreeningisnotavailable),andanalysisofclinicalparameterscanbechallenging.Forexample,ageofdiseaseonsetmightbeinfluencedbyacertainparentalinterpretationandperception.Wehave,however,usedclearlydefinedclinicalparametersandhavevalidatedparentreporteddiseaseonsetbymedicalrecordsandphoneinterviews[3,21].Furthermore,wehavenotobservedatendencyinrecognizingearliersymptomonsetinthesecondsiblingwhentheoldersiblingwasalreadydiagnosed.Thisisunderlinedbythefactthatin5of12siblingpairsthefirstdiagnosed(older)siblinghadadiseaseonsetatanearlieragethanthenext(younger)sibling.Elgünetal.OrphanetJournalofRareDiseases (2019) 14:136 Page7of10 Symptomconstellationsatdiseaseonsetmaybedifficulttoidentify.Somesymptomsmightbehardertopinpointataspecificage(e.g.concentrationproblems,whichmightslowlystartorbeunspecific),othersymp-tomsmightstartshortlyafterthefirst.Therefore,weconsideredthemtobelongtothefirstsymptomseveniftheyappearedashorttimeafter.Fromapathophysio-logicalorbiochemicalperspective,symptomonsetmightberelatedtoasupra-thresholdlevelofprogressivepathologicalchanges,likesulfatideaccumulation,demyelination,oraxonaldamage.FurtherinformationisneededfrombiochemicalorMRIbiomarkersinordertodefinesuchsupra-physiologicalthresholds.ConclusionsInthemostcomprehensivestudypresentedsofar,weinvestigatedthephenotypicvariabilityofsiblingswithMLDcomparedtounrelatedpairsofchildrenwithMLD.WefoundthatsiblingswithMLDshowasimilartypeoffirstsymptoms,MRIpattern,anddynamicofdiseaseprogression.However,regardingtheageofdiseaseonsetsomesiblingpairsshowconsiderablevariability.Asgenotype-phenotype-correlationwasnothigherinsiblingsthaninunrelatedchildrenwiththesamegenotype,thissuggeststhatadditionalbiochemicalandepigeneticfactorsmightinfluencetheclinicalphenotype.Thesedataareimportantforfamilycounseling,butalsoessentialfortheevaluationoftreatmenttrials,whereuntreatedsiblingsareoftenusedascontrols.MethodsPatientsClinicalandMRIdataofchildrenwithlate-infantileandjuvenileMLDhavebeencollectedretrospectivelywithintheGermanleukodystrophynetworkLEUKONETsince2006.Thestudywasapprovedbythelocalethicscom-mittees(401/2005and2012/098).Writteninformedconsentwasgivenbyatleastonecaregiver.Inaddition,datafromsiblingswithMLDfromapatientcohortoftheCenterforChildrenwithWhiteMatterDisorders,VUUniversityMedicalCenter,Amsterdam,wereincluded.ThediagnosisofMLDwasmadebasedonincreasedsulfatideexcretionandARSAdeficiencyinthecontextofclinicalsymptomsandMRI,inaddition,confirmedbygeneticanalysisinmostcases.Dataaftertherapeuticintervention(e.g.stemcelltransplantation)wereex-cludedfromtheanalysisinordertodescribethenaturaldiseasecourse.ClinicalparametersThediseaseonsetwasdefinedasfirstneurologicalsymptomsor

lossofmotorand/orcognitiveskills.Thetypeoffirstsymptomswasclassifiedintothreecategor-ies:onlymotorsymptoms,onlycognitivesymptomsorboth,basedonaquestionnaireandverificationbyphoneand/ormedicalrecordsasdoneinKehreretal.2011/2014[3,21].Motorsymptomsweredefinedasgaitdisturbance,abnormalmovementpatternsorweakness[21],cognitivesymptomsasproblemsinconcentration/learningdifficultiesorbehavioralproblems[21].Asdiseaseprogressionisdominatedbythedeterior-ationofgrossmotorfunction,thestandardizedandvalidatedGrossMotorFunctionClassificationinMLD(GMFC-MLD-Score)wasusedtoquantifytheclinicalcourseinthesepatients[20].Usingthismeasure,wehavecalculatedthetimeframeinwhichpatientsofthejuvenilecohortprogressfromfirstabnormalitiesingrossmotorfunction(GMFC-MLDlevel1)tolossofwalking(GMFC-MLDlevel3),inordertoinvestigatethedynamicofdiseaseprogression.Forthedynamicofcognitiveprogression,twoparameterswereused:Thetimebetweenonsetoffirstsymptomsandpresentationofconcentrationproblemsandbetweenonsetoffirstsymptomsandthestartoflanguagedecline[21].MRIInordertocharacterizedisease-relatedMRI-theMRI-SeverityScorewasusedtoquantifycerebralchanges.Thescoreisbasedonapointsystemfrom0to34pointsthatobjectifiestheextentofMRI-changes(involvementofthewhitematteraswellastheoccurrenceofglobalbrainatrophy)[35].Inaddition,thepatternofinvolvement(morefrontallypronouncedvsmoreparieto-occipitaldominance)wasassessedvisually.GeneticThegeneticanalysiswasdoneaspartofthediagnosticprocedureoraspartofaresearchprojectasdescribedinBöhringeretal.2017[7].Inordertoanalyzetherelationshipbetweengenotypeandageatonset,themostcommoncombinationofmutationswasidentifiedinourcohort.StatisticalanalysisThesimilarityofclinicalparametersbetweensiblingswasquantifiedusingtheEuclidiandistance.Thedistancemea-suresofthesiblingpairswerecomparedtotheEuclidiandistancesbetweenallpossiblepairsfromthe(unrelated)patientcohort.Thedistancesofthetwogroups(siblingpairsvsnon-siblingpairs)werecomparedusingtheMann–WhitneyUtest.Thestatisticalevaluation,includ-ingdescriptivestatistics,wascarriedoutusingthesoft-wareIBMSPSSStatistics,version25.Elgünetal.OrphanetJournalofRareDiseases (2019) 14:136 Page8of10 AcknowledgementsWethankallparticipatingcentersoftheGermanLeukonet,coordinatorVolkmarGieselman,andallreferringphysiciansandhospitals.Weareverygratefultothechildrenandtheirfamiliesforparticipatinginourstudy.Authors’contributionsSE:Dataanalysisandinterpretation,literaturereview,datacollection,draftingofmanuscript.JW:datacollection,analysisandinterpretationofdata,criticalrevision.CK:Studyconceptionanddesign,datacollection,criticalrevision.DR&NW:datacollection,criticalrevision.JB:datacollection,criticalrevision.SB-W:datacollectionandinterpretation,criticalrevision.JJ&LS:datacollection,criticalrevision.IK-M:Studyconceptionanddesign,interpretationofdata,criticalrevision.SG:Studyconceptionanddesign,datacollection,analysisandinterpretationofdata,draftingandrevisionofmanuscriptFundingThedatafromthisstudywasacquiredunderagrantfromtheGermanFederalMinistryofEducationandResearchfundingtheGermanLEUKONET(01GM0835)andLeukotreat(EC’s7thframeworkprogram).Furthermore,thepublicationissupportedbyDeutscheForschungsgemeinschaft(DFG)andOpenAccessPublishingFundofTübingenUniversity.PartofthisstudywasfinancedbytheM.O.Knipstichting.Avai

labilityofdataandmaterialsAllrelevantdataarewithinthepaper.Individualsubject’svaluesfromthewholepatientcohortmaybemadeavailableuponrequestaddressedtothecorrespondingauthor,pendingtheapprovaloftheInstitutionalReviewBoardoftheUniversityofTuebingen,Germany.EthicsapprovalandconsenttoparticipateThestudywasapprovedbythelocalethicscommittees(401/2005and2012/098).Writteninformedconsentwasgivenbyatleastonecaregiver.ConsentforpublicationWehaveonlyusedpatientdatafromwhomwehaveaconsenttoparticipationandpublication.CompetinginterestsS.G.reportsaninstitutionalresearchgrantfromShireplc,outsideofthesubmittedwork,andservesasco-investigatorandadvisorinamulticentreenzymereplacementtrialsponsoredbyShireplc,butreceivesnopersonalpaymentrelatedtothisrole.Authordetails1DepartmentofPaediatricNeurologyandDevelopmentalMedicine,UniversityChildren’sHospitalTübingen,Hoppe-Seyler-Strasse1,72076Tübingen,Germany.2DepartmentofChildNeurology,EmmaChildren’sHospital,AmsterdamUniversityMedicalCenters,VrijeUniversiteitAmsterdam,andAmsterdamNeuroscience,Amsterdam,TheNetherlands.3DepartmentofMedicalGenetics,UniversityHospitalTübingen,Tübingen,Germany.4ClinicalNeurogeneticsSection,DepartmentofNeurologyandHertieInstituteforClinicalBrainResearch,UniversityofTübingen,Tübingen,Germany.5GermanCenterforNeurodegenerativeDiseases(DZNE)Tübingen,Tübingen,Germany.Received:1March2019Accepted:4June2019 References1.GieselmannV,Krägeloh-MannI.Metachromaticleukodystrophy--anupdate.Neuropediatrics.2010;41(1):1–6.2.GieselmannV,Krägeloh-MannI.MetachromaticLeukodystrophy.In:ValleD,BeaudetAL,VogelsteinB,KinzlerKW,AntonarakisSE,BallabioA,etal.,editors.Theonlinemetabolicandmolecularbasesofinheriteddisease.NewYork:McGraw-Hill;2014.p.chapter148.3.KehrerC,BlumenstockG,GieselmannV,Krageloh-MannI,GermanL.Thenaturalcourseofgrossmotordeteriorationinmetachromaticleukodystrophy.DevMedChildNeurol.2011;53(9):850–5.4.LugowskaA,AmaralO,BergerJ,BernaL,BosshardNU,ChabasA,etal.Mutations�c.459+1Gaandp.P426LintheARSAgene:prevalenceinmetachromaticleukodystrophypatientsfromEuropeancountries.MolGenetMetab.2005;86(3):353–9.5.PoltenA,FluhartyAL,FluhartyCB,KapplerJ,vonFiguraK,GieselmannV.Molecularbasisofdifferentformsofmetachromaticleukodystrophy.NEnglJMed.1991;324(1):18–22.6.BergerJ,LöschlB,BernheimerH,LugowskaA,Tylki-SzymanskaA,GieselmannV,etal.Occurrence,distribution,andphenotypeofarylsulfataseamutationsinpatientswithmetachromaticleukodystrophy.AmJMedGenet.1997;69(3):335–40.7.BöhringerJ,SanterR,SchumacherN,GiesekeF,CornilsK,PechanM,etal.EnzymaticcharacterizationofnovelarylsulfataseavariantsusinghumanarylsulfataseA-deficientimmortalizedmesenchymalstromalcells.HumMutat.2017;38(11):1511–20.8.CesaniM,LorioliL,GrossiS,AmicoG,FumagalliF,SpigaI,etal.MutationupdateofARSAandPSAPgenescausingmetachromaticLeukodystrophy.HumMutat.2016;37(1):16–27.9.GolchinN,HajjariM,MalamiriRA,AminzadehM,Mohammadi-AslJ.IdentificationofanovelmutationinARSAgeneinthreepatientsofanIranianfamilywithmetachromaticleukodystrophydisorder.GenetMolBiol.2017;40(4):759–62.10.ShahzadMA,KhaliqS,AmarA,MahmoodS.MetachromaticLeukodystrophy(MLD):aPakistanifamilywithnovelARSAgenemutation.JMolNeurosci.2017;63(1):84–90.11.StoeckK,PsychogiosMN,OhlenbuschA,SteinfeldR,SchmidtJ.Late-onsetmetachromaticLeukodystrophywithearlyonsetdementiaassociatedwi

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