bioRxiv preprint first posted online May. 23, 2017; doi: http://dx.doi.org/10.1101/141200. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC 4.0 International license.
1
DamagingMutationsareAssociatedwithDiminished
MotorSkillsandIQinChildrenontheAutismSpectrum
AndreasBuja1,NataliaVolfovsky2,AbbaKrieger1,CatherineLord3,AlexE.Lash2,MichaelWigler4,5,6and
IvanIossifov4,5
1. DepartmentofStatistics,TheWhartonSchool,UniversityofPennsylvania,Philadelphia,PA,
19104,USA
2. SimonsFoundation,NewYork,NY,10010,USA
3. WeillCornellMedicine,CenterforAutismandtheDevelopingBrain,WhitePlains,NY,10605,
USA
4. ColdSpringHarborLaboratory,ColdSpringHarbor,NY,11724,USA
5. NewYorkGenomeCenter,NewYork,NY,10013,USA
6. [email protected]
Summary
InindividualswithAutismSpectrumDisorder(ASD),denovomutationshavepreviouslybeenshownto
besignificantlycorrelatedwithlowerIQ,butnotwiththecorecharacteristicsofASD:deficitsinsocial
communicationandinteraction,andrestrictedinterestsandrepetitivepatternsofbehavior.Weextend
thesefindingsbydemonstratingintheSimonsSimplexCollectionthatdamagingdenovomutationsin
ASDindividualsarealsosignificantlyandconvincinglycorrelatedwithmeasuresofimpairedmotorskills.
ThiscorrelationisnotexplainedbyacorrelationbetweenIQandmotorskills.WefindthatIQand
motorskillsaredistinctlyassociatedwithdamagingmutationsand,inparticular,thatmotorskillsarea
moresensitiveindicatorofmutationalseverity,asjudgedbythetypeanditsgenetarget.Weusethis
findingtoproposeacombinedclassificationofphenotypicseverity:mild(littleimpairmentofboth),
moderate(impairmentmainlytomotorskills)andsevere(impairmentofboth).
Introduction
AutismSpectrumDisorder(ASD)isaneuropsychiatricdisorder,conventionallycharacterizedby
corephenotypes,includingpersistentdeficitsinsocialcommunicationandinteraction,andrestricted,
repetitivepatternsofbehavior(AmericanPsychiatricAssociation,2013).Geneticsisastrong
determiningfactor,asevidencedbythehighrateofconcordancebetweenidenticaltwins,elevated
siblingrisk,consistentenrichmentofautism-associatedgenesincertainbiologicalpathwaysand
neurodevelopmentalperiods,andthepresenceofgenetic‘signatures’.Thesesignaturesincludea
significantlyincreasedincidenceoflikelygene-damagingdenovosmallandlargescalemutations
(Iossifovetal.2014,Sebatetal.2007,Sandersetal.2015,DeRubeisetal.2014,Levyetal.2011),and
bioRxiv preprint first posted online May. 23, 2017; doi: http://dx.doi.org/10.1101/141200. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC 4.0 International license.
2
thepreferentialtransmissionofsuchvariantstotheaffectedchild(Levyetal.2011,Iossifovetal.2015,
Krummetal.2015)
AlthoughthecorephenotypesformtheconsensusclinicalsignatureofASD,suchchildrenalso
haveawiderangeofotherphenotypesandcomorbidities(AmericanPsychiatricAssociation,2013).A
wealthofphenotypicdataonautisticindividualsisfoundintheSimonsSimplexCollection(SSC),an
archiveofsamplesfrom‘simplex’families,whereonlyonechildisaffected,andmayincludeoneor
moreunaffectedchildren(Fischbach&Lord,2010).TheSSCsampleshavebeenthesourceforthe
discoveryofmanycandidatecausaldenovovariants.Therichlydocumentedandquantifiedphenotypic
variablesintheSSCprovideanexcellentopportunitytocorrelatevariantswithphenotypes.Inanearlier
effort,weandothershaveshownthatASDindividualswithlownonverbalIQ(nvIQ)haveasignificantly
increasedincidenceofdamagingdenovomutation(Iossifovetal.2014,O’Roaketal.2012).These
observationssupportthehypothesisthatdamagingdenovomutationsmayhavebroaderneurological
effectsthanASDalone.
Wishingtotestthishypothesisfurther,welookedforfurthercorrelationsbetweenphenotypes
anddamagingdenovo(dn)mutations.WhileIQreflectssomeaspectsofcognitiveability,thereare
otherfundamentalmanifestationsofalteredneurologicalfunction.Indeed,neurodevelopmentaldelay,
suchasageoffirstwalking,isoftenthefirstpresentingsymptominautism(LandaandGarrett-Meyer
2006,Provostetal.2007).Thedelayinthismilestonemaybemoregenerallyareflectionofdiminished
motorskills(MS),awell-documentedfeatureofASD(e.g.,Paquetetal.2016).Somehavearguedthat
motorimpairmentshouldbeincludedamongcoreASDfeatures(e.g.,MosconiandSweeney2015,
Hiltonetal.2012,Fournieretal.2010,Mostofskyetal.2007,Dziuketal.2007).Thuswedecidedtolook
forcorrelationsbetweenMSanddnmutations,inparticularthosethatare‘likelygenedisrupting’(or
LGDs:nonsense,frameshiftandsplicesitealtering).
MotorskillsofmostaffectedchildrenintheSSChavebeenevaluatedbytheDevelopmental
CoordinationDisorderQuestionnaire(DCDQ)and,foryoungchildren,alsobytheVinelandAdaptive
BehaviorScales(VABS-II).Usingthesescores,wefinddnLGDmutationscorrelatewithMSatleastas
stronglyandsignificantlyaswithnvIQ.Statisticalsignificanceofthiscorrelationisfoundnotonlyforthe
totalDCDQandVABS-IIscores,butalsofortheirsubcomponents,includingfineandgrossmotorskills,
aswellasforrelatedvariablessuchasdelayinthedevelopmentalmilestone“ageoffirstwalking”.
Moreover,significanceofthecorrelationsincreaseswhenweweightadnLGDmutationbyevidence
thatitstargetgeneisunderstrongpurifyingselectioninhumans,orthatitisamemberofcertain
functionalclasses.Weextendourobservationsevenfurtherbyincludingananalysisofmissense
mutations,inwhichcorrelationtoMS(muchlesssoIQ)becomesevidentwhenthesemutationsare
additionallyweightedbypredictionsofdeleteriouseffect.
WhileIQandMSarecorrelatedwitheachother,theyeachcorrelatewithdamagingdn
mutationsevenaftereitherisadjustedfortheother.AlthoughMSandIQsignificantlycorrelatewiththe
severityofthecoreASDphenotypes,weobservenoconsistentlysignificantcorrelationbetween
damagingdneventsandcoreASD.Thisfindingsuggeststhatthesourceofsocialcognitiveimpairment
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3
inASDmaybevariantsingenesnotexperiencingparticularlystrongnegativeselectivepressure,and
thereforenotentirelyrareinthehumanpopulation.
Results
MotorskillsinthosewithdenovoLGDs
MotorskillsarescoredintheDCDQasafifteenitemparentquestionnaire(ona5-pointLikert
scalewherehighervaluesindicatehigherachievement)thatassessesthechild’sabilityforfineandgross
motorskills(Schoemakeretal.2006).(SeetheAppendixformoredetaileddescriptors.)ThetotalDCDQ
scoreandthreesummarysubscoresareavailablefor87%oftheexome-sequencedaffectedchildrenin
theSSC.ThesubscoresoftheDCDQare“controlduringmovement”,“finemotor/handwriting”,and
“generalcoordination.”Thoughnotstandardizedforage,itisnegligiblyagedependent(S3inthe
supplement).Additionalmeasuresofmotorskilldevelopmentarefoundinvariousinstrumentsusedfor
evaluatingtheaffectedchildren,inparticulartheVineland-IIMotorSkillDomainforyoungchildren,with
amainscaleandtwosubscales:fineandgrossmotorskills.Inaddition,ameasureofcoordination
difficultyisfoundintheSocialResponsivenessScale(SRS):item14,whichasksforproblemswithbeing
‘well-coordinated’(onaseverityscale0-3).Finally,theAutismDiagnosticInstrument(ADI-R)hasa
milestonevariable“walkedunaided,age”(item5),andavariable“articulationatage5”(item32)that
providesonemeasureofdevelopmentofmotorcontrolofspeech.
WeexaminedcorrelationsofthephenotypicfeatureswiththenumberofdnLGDsperchild
(typically0or1,some2,few3)anddisplayedthestrengthsoftheirone-sidedp-valuesgraphicallyas
seeninFigure1,columnA(seeMaterialsandMethodsfordetails).TheDCDQandVABSmeasures,as
wellasnvIQ,areskilllevels,henceexpectedtobenegativelycorrelatedwithdnLGDmutations(shown
red).Threemeasures(showninthebottomrowsofthefigure)areinverseskillmeasures,“ageatfirst
walking”,“speecharticulationatage5”and(problemswith)“wellcoordination”,henceexpectedtobe
positivelycorrelatedwithdnLGDmutations(shownblue).Thecorrelationsofallthesemeasureswith
ourprimarymeasureofgeneticdamageareintheexpecteddirections.Theabsolutecorrelations
betweengenotypeandMSmeasurestendtobelow,ontheorderof0.1(Figure1’insupplement).
However,duetothelargesizeofavailableSSCdata(n=2,120),thesecorrelationsarestatistically
significant(p=1.5E-4for“totalDCDQ”).AffectedchildrenwithdnLGDshavedecreasedgrossandfine
motorskills,aswellasdelayedmotordevelopment,comparedtoaffectedchildrenwithoutthese
observeddnLGDs.
BecauseearlierliteraturehaspointedtonvIQascorrelatedwithgeneticlesions,weincludethis
variableforcomparisonwiththeMSvariables.NotethatsignificantcorrelationwithnvIQisalsoseen
(topleftinFigure1,p=4E-4).
CorrelatinglosswithLGDtargets
NotalldnLGDmutationsnecessarilydisruptcriticalgenefunctions.Infact,weestimatethatin
childrenonthespectrum,alittlelessthanhalfofdnLGDscontributetoautism-risk(Iossifovetal.2014).
First,notallLGDmutationsaredisruptive(recall“L”in“LGD”);andsecond,notallgenetargetsare
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4
critical.Fortunately,theimportanceofagenetargetcanbeweightedbyevidence.Indeed,thereare
multiplewaystoweighttargetimportance:whetherthegeneisarecurrenttarget;whetherthegeneis
understrongnegativeselectivepressure;andaccordingtothefunctionalpropertiesofitsencoded
product.
Wecallagenea‘recurrenttarget’ifadnLGDhitsthatgeneinmorethanoneaffectedchildin
theSSC.SuchLGDsarecalledrecurrenteventhoughtheprecisednvariantitselfisalmostneverseen
twice.Frompreviouswork(Iossifovetal.2014)recurrenttargetsareestimated90%likelytobeautismriskgenes.TodetermineifmeasuresofMSarecorrelatedwiththepresenceofrecurrentLGDtargets,
beyondtheircorrelationwithdnLGDsingeneral,werestrictedourstudytoonlythosechildrenalready
affectedwithadnLGD.Amongthese,wethencountedthenumberofrecurrentdnLGDsineachchild,
typically0or1.Althoughthereareonly57recurrentLGDsoutofatotalof352LGDs,theircorrelations
withDCDQinthissubsetofthechildrenhasverystrongadditionalsignificance(Figure1,columnB,
“totalDCDQ”,p=5E-8).IncreaseinsignificanceisfoundaswellfornvIQ(p=8E-7),consistentwithwhat
wehavepreviouslyreported(Iossifovetal.2014).
AnotherwaytodistinguishtheseverityofanLGDisbycharacterizingthe‘geneticload’ofits
targetinthehumangenepool,areflectioninpartoftheactionofpurifyingselection.InIossifovetal.
(2015),wemeasuredthefrequencythatanLGDvariantinagivengeneisobservedinalargeunaffected
population.WerankedgenesbytheirfrequencyofcarryinganLGDinthatpopulation,normalizedby
thelengthofthatgene.Thosegeneswithalowburdenwereconsideredbyustobehighly‘vulnerable’.
Thedataongeneticloadisfarfromcomplete,becausethesequencedatabasesareinsufficientto
characterizemostgenesfortheirvulnerability,especiallytheonesencodingsmallerproducts.
Nevertheless,inapreviousstudywestillfoundthataffectedchildrenintheSSCwithdnLGDsinhighly
vulnerablegeneshadsignificantlylowerIQthaninaffectedchildrenwithLGDsinlessvulnerablegenes
(Iossifovetal.2015).BycomparingthenumberofLGDsbyvulnerabilityscoreinaffectedversus
unaffectedindividualsfromtheSSC,wecandemonstratethattheabilityofthevulnerabilityscoreto
discriminatethesetwogroupsisconcentratedinhigherscores(seeS1inthesupplement).Inthis
report,therefore,wetransformthegenevulnerabilityrankbytakingthenegativelogarithmofthe
normalizedrankofgenevulnerability(seeMaterialsandMethods),yieldinga‘genevulnerabilityscore’
thatspreadsoutmoreinformativescoresandcompresseslessinformativescores.Usingthesumofthe
scoresofthednLGDtargetgeneswithinachild,andrestrictingtochildrenwithdnLGDs,wefind
strikinglysignificantcorrelationwithmotorskills(Figure1,columnC).
Theseverityofamutationmightalsodependonthefunctionalclassofitstargetgene.We
considerherednLGDmutationsinthreesetsofgenes,enrichedastargetsofdisruptivemutationin
childrenwithASDandearlierexaminedbyus(Iossifovetal.2014):FMRPtargetgenes,whose
transcriptsinteractwiththefragileXprotein(Figure1,columnD)(Darnelletal.2011,Iossifovetal.
2012,2014),embryonicgenes,whicharegenesexpressedinthebrainofthefetusbutstrongly
downregulateduponbirth(Figure1,columnE)(Kangetal.2011,Voineaguetal.2011,Iossifovetal.
2014),andchromatinmodulatinggenes(Figure1,columnF)(McCarthyetal.2014,Bernieretal.2014).
Weaddafourthset,thegenesregulatedbyCHD8,themostfrequenttargetgenefordnLGDmutation
inASD(Figure1,columnG)(Cotneyetal.2015).Forthisanalysis,weagainconsideronlythoseautistic
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5
individualsthathavednLGDmutation,soasnottoconfoundtheanalysiswithcorrelationduetothe
LGDitself.Asreportedbefore,dnLGDmutationintheFMRPtargetgenesareassociatedwithlowerIQ
(Iossifov,2014).So,too,wefindthattheyaresignificantlyassociatedwithreducedmotorskills.Thedn
eventsintheotherfunctionalcategoriesarealsoassociatedwithreducedMSbutwithsomewhatless
significance.Acrossallthedata,aparticularlysignificantcorrelationisseenbetweenpresenceofadn
LGDinaCHD8targetandageuponfirstwalking.Thisisstrikingespeciallyastheothercorrelationsof
thismutationclassareweak.ArelateddiscoverywasmadebyBernieretal.(2014)whofoundthatdn
mutationsontheCHD8geneitselfareassociatedwith“asubtypeofautismearlyindevelopment.”
Analysisofdnmissensemutations
TherearemanymorednmissensemutationsthandnLGDmutations.Basedonwhatwecall
‘ascertainmentbias’,wejudgethatonlyabout10%ofthesecontributetosimplexautism,incontrastto
about50%fordnLGD(Iossifovetal.2014,DeRubeisetal.2014).Notsurprisingly,evenexcluding
childrenwithdnLGDs,andcountingeachchildfornumberofdnmissensemutations,weseeno
significantcorrelationwithnvIQ,andonlysignificantcorrelationwith“well-coordinated”amongtheMS
variables(columnH).Scoringforrecurrentdnmissensetargetsresultsinnosignificantcorrelations(not
shown).However,evidenceoftheircontributiondoesemergeifthednmissensemutationsare
weightedbygenevulnerabilityscoresoftheirtargets(Figure1,columnI)(Iossifovetat.2015).With
thatmeasure,weobservesignificantcorrelationtomostMSvariables.Wenotethatassociationof
theseweightedmutationsisnotobservedwithnvIQ,suggestingmildereffectsofdnmissensemutations
byaffectingmostlyMSandleavingcognitionmostlyintact.
Inanattempttofurtherstrengthenthediscriminationamongdnmissensemutations,usinga
methodcalledVIPUR,wepredictedthelikelihoodthattheydisruptproteinstructure,andtherebyinfer
thelikelihoodthattheyhaveadeleteriouseffectonfunction(Baughetal.2016).ThetwoVIPURrelatedvariablesshowninthefiguresarerestrictedtoasubsetofdnmissensemutationsforwhich
VIPURscoresareavailable,aggregatedbysummationforeachaffectedchildwithoutdnLGDs.Theraw
VIPURscorealoneshowsnosignificantcorrelationwithMSvariables(SRSitem14)andnonewithIQ
(Figure1,columnJ).However,anewscoreobtainedbymultiplicationofVIPURandthevulnerability
score(seeMethods)leadstomoresignificantassociationsthaneitheralone(Figure1,columnK
comparedtocolumnsIorJ).Thus,VIPURincombinationwiththegenevulnerabilityscorehelpsto
assessmutationaldamage.
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Figure1.Significanceofcorrelationsbetweenmeasuresofgeneticdamageandmeasuresofmotor
skillsandnvIQofaffectedchildren.
Weusedelevenmeasuresofgeneticdamageshownascolumnsinthefigureandelevenphenotypic
measures(onefornvIQandtenforMSextractedfromfourdifferentphenotypicinstruments)shownas
rows.Thegeneticdamageandphenotypicmeasuresweredefinedondifferentsubsetsoftheaffected
childrenintheSSCcollection.Toreflectthisfact,wepastedcountstothelabelsasfollows:“(100)”
wouldmeanthemeasureisdefinedonasubsetofsize100,and“(20:100)”indicatesinadditionthatof
the100valuesthenumberofnon-zeroesis20;thissecondversionisrelevantforgeneticvariablesthat
arecountsofmutationsinachild.
Wecomputedthecorrelationbetweeneachofthe11geneticdamagemeasuresandthe11phenotypic
measuresusingonlythechildrenforwhichboththegeneticdamageandphenotypicmeasureswere
defined,andwetestedifthecorrelationwassignificantlydifferentfrom0.Theresulting11by11table
ofp-valuesisrenderedgraphicallywithrectangleswhosesizerepresentsinverselythep-valuefromthe
statisticaltest(largerectangle≈smallp-value)andwhosecolorrepresentsthesignofthecorrelation
(blue≈positive,red≈negative).Themeaningofboththesizesandcolorscanbegleanedfromthe
figure’skeyinthebottomleft.Asmalldotisusedwhenthecorrelationisstronglyinsignificant(p-value
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7
≥0.10).—Inasimilarfashion,therelatedFigure1’inthesupplementshowstheunderlyingcomputed
correlations.Formorebackgroundaboutthistypeofdisplay,seethesectionMaterialsandMethods.
Measuresofgeneticdamage:Theprimarymeasureofgeneticdamage(columnA)isdefinedasthe
numberofdenovoLGDs(0,1,2or3)identifiedinanaffectedchild.ThevariablesincolumnsB-G
differentiateLGDsaccordingtoindicationsthattheymaybedamaging;thesevariablesaredefinedonly
forchildrenwhohaveatleastoneLGD.ColumnBisthenumber(0or1)ofdenovoLGDsinachild
affectedbygeneswithmorethanonedenovoLGDintheSSC(“recurrentgenes”).ColumnCisthesum
ofthevulnerabilityscoresofthegenesaffectedbydenovoLGDsinthechild.ColumnsD-Garedefined
asthenumber(0or1)ofdenovoLGDsthatfallinfourgenefunctionalclassesthathavepreviouslybeen
implicatedinautism’setiology:FMRPtargetgenes,embryonicgenes,genesencodingchromatin
modifiers,andCHD8targetgenes.Theremainingcolumnsconcerndenovomissensemutations:
ColumnHisthenumberofdenovomissensemutations(0upto5)inanaffectedchild,appliedonlyto
childrenwithoutdenovoLGDmutations,topreventconfoundingwithoverpoweringLGDeffects.
ColumnI,analogoustocolumnC,isthesumofvulnerabilityscoresofgenesaffectedbymissense
mutationsinachild.ColumnJisthesumofVIPURscoresofmissensemutationsinachild.ColumnKis
theproductofVulnerabilityandVIPURscores,exhibitingp-valuesthatneitherscorecouldachieve
alone.
Phenotypicmeasures:Thetoprowrepresentsnon-verbalIQ(nvIQ).Theotherrowsrepresentten
differentmeasuresofmotorskillsavailableinthephenotypicdatabaseoftheSSC.Thelabelsare
suffixedbyabbreviationsoftheoriginatinginstruments:DCDQ,VABS,SRSandADI-R.SeeMethodsand
Materialsfordetails.
AssociationbetweenMSandIQ
ItisclearfromtheSSCphenotypedatathatIQandMSarethemselvescorrelated(corr=0.36,
N=2365).OneshouldthereforeaskifsignificantcorrelationswithdnmutationssurvivewhenIQandMS
areadjustedforeachother(seeMaterialsandMethods).WedisplaytheresultsinFigure2,withthe
samecolumnstructureasinFigure1,whereMSvariablesareadjustedbynvIQ,sexandage,andnvIQ
(forcomparison)isadjustedbytotal_DCDQ,sexandage.Correlationsgenerallysurviveadjustment,
althoughwithsomewhatattenuatedsignificances.Attenuationaftermutualadjustmentistobe
expectedduetothepartialpositivecorrelationbetweenIQandMS,butthemainconclusionisthatthe
associationofMSwithmutationvariablescannotbereducedtoIQ,orviceversa.(Foracorresponding
displayofcorrelationsasopposedtop-values,seeFigure2’inthesupplement.)
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Figure2.Significanceofthecorrelationbetweenmeasuresofgeneticdamageandadjustedmeasures
ofmotorskillsandIQofaffectedindividuals.ThisfigureissimilartoFigure1(seeitslegendfordetails),
butthephenotypicmeasureshavebeenadjustedasfollows:thetenmotorskillsmeasures(suffixed
DCDQ,VABS,SRS,ADIR)areadjustedfornvIQ,sexandage;nvIQ(toprow)isadjustedfortotal_DCDQ,
sexandage.Themainresultisthatthesignificanceofthecorrelationslargelysurvivesthese
adjustments.Thisisevidencethattheassociationofmotorskillswiththegeneticvariablescannotbe
reducedtothecorrelationbetweenmotorskillsandnvIQ.(SeeFigure2’inthesupplementforasimilar
graphshowingtheunderlyingadjustedcorrelations.)
TheassociationbetweennvIQandMSdeservescloserexaminationbecauseitisinsufficiently
characterizedbyaplaincorrelationcoefficient.Indeed,theassociationcannotbesimplydescribedasa
tendencytopairhighwithhighandlowwithlowvaluesbetweennvIQandMS.Rather,ascanbeseen
inFigure3,thecombinationoflownvIQwithhigh(normal)MSisrare,butthecombinationofhigh
(normal)nvIQwithlowMSisnot.Thiscanbeputdifferentlyasfollows:
•
MSdifferentiatewithinthenormalnvIQrange,while
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9
•
nvIQdifferentiateswithinthelowMSrange.
RF
Figure3.RelationshipbetweenIQandmotorskills.ThescatterplotshowsnvIQandtotal_DCDQforN=
2,119affectedchildrenwithavailableexomedata.Thegrayverticallinesshowthecut-offsusedto
dichotomizedthetwomeasures(seethetextforjustificationoftheparticularcut-offs).Thefour
quadrantsofthegrapharelabeledclockwisewithlettersAthroughD.QuadrantDissignificantlyunderpopulatedcomparedtowhatisexpectedunderanassumptionthatthetwomeasuresareindependent.
IgnoringquadrantD,thearrowsdemonstrateincreasingphenotypicseverity(asafunctionofbothnvIQ
andtotal_DCDQ)betweenadjacentquadrants,withA<B<C.
ThesamefactcanberendereddifferentlybydichotomizingbothnvIQandMS:wedefine
•
•
nvIQ<70aslowIQ;
total_DCDQ<50aslowMS.
Thevalue70fornvIQisaconventionalthresholdforintellectualdisability(ID),definedastwo
standarddeviationsbelowthemeaningeneralpopulations.Fortotal_DCDQweusethevalue50asa
looselowerboundonthe“normal”range.(FollowingWilsonetal.(2009),therecommendedagedependentthresholdsforthenormalrangearetotal_DCDQ≥47(age<8yrs),total_DCDQ≥56(8yrs≤
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age<10yrs),andtotal_DCDQ≥58(10yrs≥age),respectively.Bythisrecommendation,83%ofaffected
SSCchildrenhavedeficientMS(consistentwiththe80-90%rangeofHiltonetal.2012,aswellasGreen
etal.2009),whereasjust25.5%havediminishednvIQ(ID).Only15.7%ofaffectedchildrenareinthe
normalrangeforbothMSandnvIQ.)
Figure3givesagraphicalrenditionofdichotomization,resultinginfoursectorsdenotedA,B,C
andD,shownwithacross-hairatcoordinates(70,50).Wecanorderthethreeenrichedsectors
accordingtophenotypicseverity,andlabelthemasfollows:
A[nvIQ≥70,total_DCDQ≥50]àB[nvIQ≥70,total_DCDQ<50]àC[nvIQ<70,total_DCDQ<50]
MildModerateSevere
TheremainingsectorD[nvIQ<70,total_DCDQ≥50]isdepletedbyafactorofnearly5when
comparingcolumnratios(oddsratio=4.95;seeTable2inthesupplement).Intermsofseverity,the
phenotypicorderingofthethreemajorsectorsmaybesymbolicallywrittenasA<B<C.
Likewise,thereexistsignificantdifferencesbetweenthemeanvulnerabilityscoresofLGDand
missensemutationgenetargetsinaffectedindividualsinSectorsA,BandC.Thatis,themean
vulnerabilityscoreissignificantlygreater
•
•
inSectorBcomparedtoSectorA(p=0.02forLGDsaloneandp=0.005forLGDsandmissense
together);
inSectorCcomparedtoSectorB(p=0.02forLGDsaloneandp=0.003forLGDsandmissense
together).
Therefore,intermsof‘mutationalseverity’,whichweuseasshorthandtocharacterizednmutationsby
thevulnerabilityscoreofthegenesintowhichtheyfall,theorderingofsectorsmayalsobewrittenasA
<B<C.
ImplicationsoftheAssociationsbetweenMS,IQandMutationalSeverity
WehypothesizethattheincreaseinimpairmentfromSectorAtoBandfromBtoCstemsfrom
acorrespondingincreaseinmutationalseverityinthetargetgenes,andsuggestthefollowing:
•
•
•
MildmutationalseverityisunlikelytoaffectMSornvIQ,
moderatemutationalseverityismorelikelytoaffectMSandlesssonvIQ(SectorB),and
severemutationalseverityislikelytoaffectbothMSandnvIQ(SectorC).
Inotherwords,whentheLGDormissensetargethasahighvulnerabilityscore,thenboth
diminishedMSandnvIQaremorelikely,andwhenthetargethasasomewhatlowervulnerabilityscore,
thentheeffectismorebiasedtowardsdiminishedMSthandiminishednvIQ.
Weseefurtherevidenceforthehypothesisthatmoderatemutationalseverityaffectsprimarily
MSbycomparingthetoptworowsofFigure1:Therearenogeneticvariablesinourcollectionthatare
moresignificantlyassociatedwithnvIQthanwithtotal_DCDQ.Inaddition,functionalclassesofdnLGD
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11
mutations(otherthanFMRP)andthescoreddnmissensemutationshavemoresignificantassociations
tototal_DCDQthannvIQ.
Figure4.AbsenceofassociationbetweenmeasuresofgeneticdamageandmeasuresofcoreASD
phenotype.Inthisfigure,allbutthetoptworowsrepresentcoreASDmeasuresdrawnfromtheADI-R,
ADOS,RBS,ABCandSRSinstruments(seeMaterialsandMethodsforexplanations).Theconclusionis
thatthesemeasureslargelylacksignificantcorrelationswithmeasuresofgeneticdamagefromdenovo
mutations.Thefewsignificantcorrelationsforparent_t_score_SRS(secondrowfrombottom)
disappearafteradjustmentfornvIQ(notshown).Somep-valuesthatapproach0.05correspondto
correlationsthathavethewrongsign(shownred)asthecoreASDvariablesmeasurebehavioral
deficiency,henceshouldbepositivelycorrelatedwithmutationalseverity.—Thefigurealsoshows
nvIQandtotal_DCDQinthetwotoprowstoprovideacomparisonwhatsignificantcorrelationswould
looklike.ThetworightmostcolumnsshownvIQandtotal_DCDQaswelltogiveevidenceoftheir
stronglysignificantcorrelationswiththecoreASDmeasures.
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12
AbsenceofAssociationwithCoreASDVariables
WeturntoasetofcoreASDvariablesthatcharacterizetheconventionalautismphenotype
consistingofdeficitsinsocialinteractionaswellasrestrictedandrepetitivebehaviors.IntheSSC,
principalinvestigatorshadpreviouslyselected“CoreDescriptiveVariables”fromseveralprimary
instruments.Fromthesevariableswesub-selectedthoseoriginatingfromthefollowinginstruments:
AutismDiagnosticInterview(ADI-R),AutismDiagnosticObservationSchedule(ADOS),Repetitive
BehaviorScale(RBS),AberrantBehaviorChecklist(ABC)andSocialResponsivenessScale(SRS,t-scores),
foratotalof12variables.
WefirstobservethatthesecoreASDvariablesstronglycorrelatewithbothnvIQandMS.
Indeed,accordingtothelasttwocolumnsofFigure4,mostp-valuesarebeyondconventionallevelsof
statisticalsignificance.(AccordingtoFigure4’inthesupplement,someofthecorrelationsapproach0.5
inmagnitude.)ThisobservationsuggeststhatcoreASDvariablesmightalsobecorrelatedwithdenovo
mutationalseverity.However,transitivityofcorrelationisnotguaranteedfromstatistical
considerations,andsowewereeagertolearndirectlyifcoreASDvariablesarealsocorrelatedwith
mutationalseverity.Surprisinglyandimportantly,wewereunabletofindconsistentassociationswith
themagnitudeofsignificanceseenforMSandnvIQ.AscanbeseenfromFigure4,stronglysignificant
associationswithgeneticvariablesarelargelyabsent,andsomethatapproachthelevel0.05havethe
wrongsign.Oneexceptionthatstandsoutisthevariable“SRSParentt-Score.”However,itssignificant
correlationswithgeneticvariablesappearsmediatedthroughnvIQ,astheydisappearifitisadjustedfor
nvIQ.
Discussion
Thisstudyispartofourcontinuingattempttolinkdamagingdenovomutationstobroad
neuropsychiatriceffectsinchildrenontheautismspectrum.Wehavedonethisbothtodefinethe
substructureofthesyndromeandtoevaluatewhicheventsarelikelytocontributetothedisorder.
EarlierstudieshadestablishedalinkbetweendamagingmutationanddiminishednvIQ.Thepresent
studyestablishesamoresignificantassociationbetweendamagingmutationandimpairedmotorskills.
Ourmethodistocorrelatemeasuresofbroadneurologicalfunction(nvIQ,MS)andcoreASD
phenotypeswithdenovogeneticdamage.ForbothwerelyontheSSC,whichprovidesanabundance
ofphenotypicmeasuresaswellasextensivemutationdata.Fromthelatterweuseinformationabout
thetypeofmutation(LGDormissense)anditsgenetictarget(recurrence,vulnerability,andfunctional
class).
Unlikeintellectualability,motorskillsdonothaveasinglestandardofmeasurement.We
thereforeusedalltheavailablemotorskillmeasuresfromtheSSC,oftenbrokenintosubscalesand
originatinginmultipledistinctinstruments.Whethermeasuringfineorgrossmotorskills,ormotorrelateddevelopmentalmilestones,withveryfewexceptionsweseearemarkablyconsistentpatternof
significantcorrelationwithgeneticvariables.Itisthisconsistencythatgivesusconfidenceinour
conclusions.
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peer-reviewed) is the author/funder. It is made available under a CC-BY-NC 4.0 International license.
13
OurpresentstudyonmotorskillsrecapitulatesconclusionsfromourpreviousstudyofnvIQby
extendingthemtomotorskills(MS).First,mutationalseveritymatters.AutisticchildrenwithLGD
mutationsaremorelikelytohaveimpairedMSthanchildrenwithoutthem.Second,genetictargets
matter.Autisticchildrenwithdnmutationsinrecurrentgenetargetsaremorelikelytohaveimpaired
MS.Similarly,childrenhavemoreseverelyattenuatedMSiftheysufferLGDmutationsingenesthatare
‘vulnerable’(i.e.,havereduceddeleteriousgeneticloadinthehumanpopulation)orthatsharecertain
functionalproperties.
ThisstudygoesbeyondourpreviousstudyofdamagingmutationsandnvIQ.Wepreviouslyhad
notobservedcorrelationsbetweennvIQandmissensemutations.Theassociationofimpairedmotor
skillswithdnmissensemutationhasmarginalsignificanceatbest.However,whenthemissensetargets
areweightedbytheirvulnerabilityscore,theassociationwithMSbecomesfarmoresignificant.When
themissenseisfurtherweightedbyVIPUR,oneofseveralavailablemethodstojudgetheseverityofa
missensemutation,theassociationbecomesmoresignificantstill.Importantly,whileweseesignificant
correlationbetweenmissensemutationtargetsweightedinthisfashionandMS,wedonotseeitwith
nvIQ.AsmissensewillbelessdamagingingeneralthantheprematureterminationcausedbyLGDs,this
resultsuggeststhatlossofMSisamoresensitiveindicatorofgeneticdamagethanislossofnvIQ.
nvIQandMSasmeasuredbytheDCDQarecorrelated,butfarfromredundant:theirrelationis
notsimplylinear;theydifferingenderbias(Table1inthesupplement);theyhavedifferentpatternsof
correlationswithgeneticvariables;andimportantly,thecorrelationsofMSandnvIQwithgenetic
damageeachsurvivewhenadjustingonefortheother.ThecorrelationsofgeneticvariableswithMSare
moreconsistentthanwithnvIQ.FurthermorenvIQdifferentiatesthelowfunctioningrangeofMS,
whiletheMSdifferentiatesthenormalIQrange:moderategeneticdamagetendstoaffectMSmoreand
nvIQless,whileseveregeneticdamagetendstoaffectbothMSandnvIQ.Themostextremeexampleof
thisisthesignalfromchildrenwithdnLGDsinthegenetargetsoftheCHD8chromatinmodifier.These
childrenshowverystrongimpairmentinageoffirstwalking,butmuchlesssignificantcorrelationwith
nvIQ. Thelinksbetweendamagingmutationsdescribedhereshouldreinforcetheneedtoroutinely
includeanageappropriateevaluationofmotorskillsintheassessmentsofASD.Theyaresimpleto
measure.Evenasinglequestionnaireitemmaygivesomeindicationofmotorskilldeficiency,as
illustratedbySRSitem14thatreferstogeneralmotorcontrolandcoordination,andADI-Ritem32that
referstospeecharticulation.EventheDCDQinstrumentisrelativelysimple,basedonjust15items.
Specificmotorskillsareusedtodefinecommondevelopmentalmilestonesforinfants,oneofwhich
makesapowerfulappearanceinourbatteryofmotorskillvariables,theageoffirstwalkingunaided
(seebottomrowofFigures1and2).Otherrelatedphenotypesshouldbeexaminedforlinkstogenetic
damage;apromisingexampleisdyspraxia(deficientproductionofgestures)studiedbyMacNeiland
Mostofsky(2012)andDziuketal.(2007).Ingeneral,thereisaneedformorefundamentalandobjective
testsofneurologicalfunction,suchassensoryevokedresponses,andtestsofmotorcontrol,suchas
electromyography,intheroutineevaluationofchildrenwithneurodevelopmentaldisorders.
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peer-reviewed) is the author/funder. It is made available under a CC-BY-NC 4.0 International license.
14
BothnvIQandMSsignificantlycorrelatewiththecorephenotypesusedtomaketheASD
diagnosis,includingthosemeasuringsocialcommunicationskills(Figure4andFigure4’inthe
supplement).ItisnaturaltoviewnvIQasentangledwiththesemeasures,buttheentanglementwith
MSislessobvious.ItistemptingtothinkoflossofMSasaconsequenceofbroadneurological
impairments,dismissingthelossasincidentaltotheirimpactonthecoreASDvariables.Webelievethis
conclusiontobepremature.Forexample,thebrainregionmostcloselyassociatedwithgrossandfine
motorcontrolisthecerebellum,anditisnowappreciatedthatthecerebellumisdirectlyinvolvedin
corticaldevelopmentandthatcerebellarlesionsduringthethirdtrimesterandneonatalperiodare
associatedwiththedevelopmentofaffectivedisorders(Wagneretal.2017,Wangetal.2014,
Schmahmann2013,Limperopoulsetal.2014,Bolducetal.2012,Messerschmidtetal.2008).
Moreover,delayindevelopingage-appropriatebodylanguagemayleadtofurthersocialisolation.
Therefore,theconnectionbetweenMSandASDphenotypesmaybedirect.Inanycase,MSmightbe
morereadilyandobjectivelymonitoredthanotherclinicallyemphasizedcognitivefunctions,andso
mightbeanimportantendpointwhileinvestigatinggeneticlesionsinmodelorganismsorwhile
screeninghumansforresponsetoexperimentaltherapies.
Finally,weexaminedthecorrelationbetweendamagingmutationsandthecoreASDvariables,
includingsocialdeficitsandrestrictedandrepetitivebehaviors.Althoughdamagingmutationscorrelate
withbothnvIQandMSinthosewithASD,andnvIQandMScorrelateverysignificantlywithcore
phenotypes,itdoesnotfollowthatdamagingmutationswillcorrelatewithcoreASD.Thecorrelations
ofmutationswithnvIQandMS,whilesignificant,areweakinabsoluteterms,andtransitivityin
correlationisnotforced.Infact,wefindthatthecorrelationofdamagingmutationwithcoreASDis
inconsistentandweakatbest.ThelittlecorrelationobservedvanisheswhenweadjustfornvIQ.
Thelackofcorrelation,nevertheless,meritsspeculation.Ourfirstthoughtwasthatthisabsence
ofsignificantassociationcouldbeexplainedbytheuseofsomecoreASDvariablesintheascertainment
ofchildrenwithASDintheSSC,causingtruncationofthevariablerangesandresultinginlesssignificant
associations.Acloserexaminationshowed,however,thatthisexplanationismostlikelywrong(seeS2
inthesupplement).Wecanconsideranotherexplanation,onewecannotruleout.Restatingourresult,
wecansaythatwhereasdamagingdenovomutationsarestronglyassociatedwiththediagnosisofASD
(seeforexampleIossifovetal.(2014),Figure1),theyarenotconsistentlycorrelatedwithcoreASD
variablesamongaffectedchildren.Thus,ifsocialdeficienciesandrestrictedandrepetitivebehaviors
alsohavecontributinggeneticcauses,perhapstheyarisefromsharedancestralvariantstransmitted
fromparents.Thesevariantsmaynotbeundernegativeselection,andhencepersistentandnotvery
rareinthepopulation.Ratherthancausingdeleteriousphenotypesintheirownright,thesevariant
allelesmayberesponsibleforthediversityofhumansocial,communicationandcognitivetraitsthat
characterizethehumanspecies.
MaterialsandMethods
ThepresentstudyisbasedontheSimonsSimplexCollection(SSC,FischbachandLord2010),
whichhasdatafor2760familiesthathaveasinglechildaffectedbyASD.Ofthesefamilies,2280have
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peer-reviewed) is the author/funder. It is made available under a CC-BY-NC 4.0 International license.
15
anunaffectedchildaswell,butforthemostpart,weareonlyconcernedwiththeaffectedchildren.
Amongthem,2446haveexomesequencingdataavailablethatresultedintheidentificationof3403de
novomutationsofalltypes(Iossifovetal.2014).(The1836unaffectedchildrenwithexomedatahave
2288identifieddenovomutationsamongthem.)Unlikecase-controlapproachesthatcompareaffected
andunaffectedchildren,oursisastudyofassociationbetweenphenotypeandgenotypevariables
amongaffectedchildrenonly.Thepremise,whichcouldhavebeenwrong,isthattheaffectedchildren
intheSSChavesufficientphenotypicvariationtoallowthediscoveryofstatisticallysignificant
correlationsbetweenhighandlowlevelsofascoredbehavioralphenotypes(suchasnvIQ,MSandcore
ASDvariables)ontheonehand,andgenotypicevents(suchasdifferentclassesofdenovomutationand
thecharacteristicsoftheirtargetgenes)ontheotherhand.Asshownabove,convincingcorrelations
existfornvIQandMSvariables,butnotformostcoreASDphenotypes.
ThefollowingisthelistofMSvariablesshownbytheirnamesintheSSCtableswhichalso
conveytheintendedmeaningofthescales:
-
-
-
-
DCDQ(DevelopmentalCoordinationDisorderQuestionnaire):
o control_during_movement
o fine_motor_handwriting
o general_coordination
o total
Thelastisthesummaryscale;theprecedingthreearesubscalesformedfromapoolof15
itemsscoredona5-pointLikertscale.
Orientation:highvaluesforhighachievement.
VABS-II(VinelandAdaptiveBehaviorScales–II):
o fine_v_score
o gross_v_score
o motor_skills_standard
Again,thelastisthesummaryscale;theprecedingtwoaresubscales.Thesevariablesexist
onlyforchildrenuptoage7.5years(caseswithhigherageareoutliersthatwereremoved).
Orientation:highvaluesforhighachievement.
SRS(SocialResponsivenessScale):
o q14_well_coordinated
Thisisasingleitemoutof65SRSitemsthatmeasurescoordinationproblemsonascale
from0to3.Contrarytothemeaningsuggestedbythenameoftheitem,thisisaseverity
measurewithmeanings0=“nocoordinationproblems”,3=“severecoordination
problems”.
ADI-R(AutismDiagnosticInterview–Revised):
o q05a_walked_unaideda:Ageofwalkingunaided,inmonths(rangingfrom7to72),
buttransformedwithadoublelogarithmduetoanextremelyrightskewed
distribution:log(log(…)).
o q32_articulation_5_years:Problemswithmotorcontrolofspeechatage5,ona
scalefrom0to3
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16
Orientation:highvaluesforhigherlevelofproblems.
Note:ConsistencyofcorrelationacrossdiversemeasuresofMSfrommultipleinstruments
strengthensourconfidencethattheconclusionsarenotmeasurementartifacts.
Cognitivefunctioningismeasuredbynon-verbalIQ(nvIQ),inagreementwithpastliterature.
Verbalandfull-scaleIQarenotused.
Core-ASDvariableswereselectedfromtheSSCtable“CoreDescriptiveVariables”(CDV),which
containsasetofdemographics,measuresanddiagnosespreviouslydeemedclinicallyrelevant.Wesubselected12variablesfrom5instrumentsasfollows:
-
ADI-R(AutismDiagnosticInterview–Revised):adi_r_soc_a_total,adi_r_comm_b_non_verbal_total,
adi_r_b_comm_verbal_total,adi_r_rrb_c_total
- ADOS(AutismDiagnosticObservationSchedule):ados_css,ados_social_affect,
ados_communication_social,ados_restricted_repetitive
- RBS-R(RepetitiveBehaviorScale–Revised):rbs_r_overall_score
- ABC(AberrantBehaviorChecklist):abc_total_score
- SRS(SocialResponsivenessScale):srs_parent_t_score,srs_teacher_t_score
Thesevariableswerepre-selectedonsubstantivegroundswithoutdatamining.However,theinterested
readermayindulgein“datamining”byperusingthenumerousfiguresinthesupplement(S4-S8)which
showsassociationsforthecompleteinstruments,bothsummarymeasuresandunderlyingitems.These
figuresareprovidedasexploratorydisplaysandasconfirmationsandqualificationsofthefindingthat
thecoreASDphenotypehasatmostatenuouslinktogeneticsasreflectedbydenovomutations.The
instrumentsweshowinthesupplementareasfollows:DCDQ,VABS-II,CDV(SSCCoreDescriptive
Variables),CUV(SSCCommonlyUsedVariables),CBCL-2-5andCBCL-6-18(ChildhoodBehaviorChecklist,
ages2-5and6-18),SRS,ABC,RBS-R,ADI-R,ADOS-1,ADOS-2,ADOS-3(threemodulesofADOS),SCQLIFE(SocialCommunicationQuestionnaire–Life),aswellasatableofdemographicvariables.
Geneticvariablesforexome-sequencedaffectedchildrenwereobtainedfrompublished
sourcesasfollows:
-
-
-
ThelistofdenovomutationsisfromsupplementaryTable2inIossifovetal.(2014).Itcharacterizes
eachmutationbythe“location”onthegenome,the“effectGene”,andthe“effectType”(among
otherthings).AmongeffectTypesweusedthefollowing:“synonymous”,“missense”,aswellassix
typesthatjointlymakeuptheLGDclassification:“splice-site”,”nonsense”,“noStart”,“noEnd”,
“frame-shift”,“no-frame-shift-newStop”.
ThefunctionalclassificationofgenesisfromsupplementaryTable7inIossifovetal.(2014).LGD
mutationswheresubdividedaccordingtowhetherthe“effectGene”isclassifiedas“FMRPTargets”,
“Embryonic”ora“ChromatinModifiers”(remainingclassificationswerenotused,somebecauseof
lowcounts,othersbecauseofaprioriunlikelyeffects).
OnemoreclassificationwasusedtosubdivideLGDmutationsaccordingtotheir“effectGene”:
“CHD8Modifiers”,comprisingalistofgenespublishedbyCotneyetal.(2015),supplement1.
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17
-
-
GenevulnerabilityranksarefromIossifovetal.(2015).Insteadofdichotomizingthescoresona
thresholdandcomparingtheresultinggroupsofhighandlowgenevulnerability,weinstead
transformtheranksbynormalizingthemtovaluesbetween0and1,andthenapplyinganegative
logarithm.Theresulting“genevulnerabilityscores”havearoughlyexponentialdistribution,the
purposebeingtospreadoutthemostvulnerablegenestotheunlimitedpositiverangeand
shrinkingnon-vulnerablegenestothenear-zerorange.Thisprocessinggiveshighlyvulnerable
genesanopportunitytodifferentiatethemselveswithhighvalueswhilegeneswithlittle
vulnerabilityaremadenearlyindistinguishablebypilinguptheirvaluesnearzero.Thistypeof
processinginjectsquantitativedifferentiationwhereitisneededandobviatesthesearchfor
meaningfulthresholds.
VIPURscoreswereobtainedfromtheauthorsofBaughetal.(2016).Weusedthe“highestscore”
versionofVIPUR.Similartogenevulnerabilityscores,wedidnotusetherawVIPURscoresbuta
transformationthereof,againobtainedbynormalizingtheirranksto[0,1]andapplyinganegative
logarithm,resultinginaroughlyexponentialdistributionthatspreadsoutthehighscoresand
shrinksthelowscores.
DescriptivetablesandplotsfornvIQ,MSvariablesandgeneticvariablesareshowninSection
S9ofthesupplement.SimilarinformationforthemanyinstrumentsshowninSectionsS3-S8arenot
providedduetoshearvolume.
StatisticalmeasurementofassociationbetweentwovariableswasdonebyformingPearson
correlationcoefficients.Thesewereusedforuniformityeveninnon-standardcases,inparticularwhen
oneorbothvariableswerebinarygroupingscodedas0-1dummyvariables:Ifonevariableis
quantitativeandtheotherbinary,thecorrelationcoefficientisalgebraicallyequivalenttothet-statistic
fortestingthedifferenceofmeans;ifbothvariablesarebinary,thecorrelationcoefficientis
algebraicallyequivalenttotheteststatisticofFisher’sexacttestofindependence.SeeBujaetal.2014
foramoredetaileddiscussion.
Presentationofcorrelationtablesisingraphicalformas“blockplots”(Bujaetal.2014).The
reasonisthatlargetablesofnumericvaluesaredifficulttoparsevisually.Furthermore,themulti-digit
precisionofnumerictablesisnotonlyuselessbutdelusionalbecauseitsuggestsaccuracywherenone
exists.Graphicalpresentationprovidesnotonlydefensibleaccuracybutlendsitselftovisualpattern
recognition,inparticularpatternsofconsistencyofcorrelationsacrossrowsandcolumns.Actually,
moreimportantthancorrelationsaretheirstatisticalsignificancesintermsofp-values.Themost
importantfiguresareindeedblockplotsofp-values,renderedsuchthatlargeblocksindicatestrong
statisticalsignificance.Thevisualestimationoftheorderofmagnitudeofp-values(aswellas
correlations)ishelpedbyakeyinthebottomleftofthefigures.Asforcolorcoding,weuseblueto
indicateapositiveassociationandredanegativeassociation;colorcodingisalsousedinblockplotsofpvalueseventhoughtheseonlyreflectstatisticalsignificancewithoutorientation.Finally,wenotethat
theblockplotsaresuperiortoheatmapsbecausethesizesofrectanglesprovidemuchmorepreciseand
moreimpactfulvisualcuesthancolorscales.
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18
Statisticalmultiplicity:Presentinglargenumbersofcorrelationsandtheirp-valuesmightraise
questionsofstatisticalinference.Amoreconventionalpresentationwouldhavecondensedthefindings
intoahandfulofp-values,forexample,byfocusingontotal_DCDQaloneamongMSvariables.The
reasonforchoosinganexpansivevisualpresentationoflargenumbersofp-valuesistoconveythe
consistentpatternsofstatisticallysignificantassociationacrossgroupsofvariables,inparticularthe
severalmeasuresrelatedtomotorskills.Suchconsistencyisnon-trivial:Whilemotorskillvariablesare
correlatedwithp-valuesbeyondconventionallevelsofstatisticalsignificance,thecorrelationsarefar
fromperfect,nohigherthan0.5betweenDCDQandVABS-II,forexample(seeS9.4insupplement).This
limitstheirsharedvariationto25%,leavingampleroomforconflictingcorrelationswiththe
comparativelyweaksignalfromthemutationvariables.Thatthisislargelynothappeningis
confirmationofthenon-trivialconsistencyofassociationbetweenmotorskillandgeneticvariables.The
manyp-valuedisplaysinthesupplementalsectionsS4-S6areshownfortworeasons:1)tobackupand
qualifythenotionthatavastmajorityofcoreASDvariablesdoesnotshowconsistentcorrelationswith
mutationvariables,otherthanthosemediatedbynvIQandMS,and2)toallowreaderstodotheirown
exploratoryhypothesisgeneration,usingp-valuesheuristicallyratherthaninferentially.
Adjustedvariables:InFigure2we(1)“adjust”nvIQfortotal_DCDQ,sexandage,and(2)we
converselyadjusttheMSvariablesfornvIQ,sexandage.Adjustmentmeansincase(1)thatnvIQis
subjectedtoalinearregressionwithtotal_DCDQ,sexandageasregressorsandthatnvIQisthen
replacedbyitsresidualsfromthisregression.Theseresidualsareuncorrelatedwithtotal_DCDQ,sex
andage.Ifoneobservescorrelationsof“adjustednvIQ”withgeneticvariables,itreflectsassociation
thatcannotbeaccountedforbytotal_DCDQand/orsexand/orage.(Detail:Inadjustingforage,we
addedalinearsplinetermwithknotat9yearsofagetoaccountforpotentialnonlinearityduetothe
transitionfromchildhoodtoadolescence.Theknotlocation9waschosenapriori,notbydatamining.)
PermutationTestsRelatedtotheOrderingoftheSectors“A<B<C”:InthecomparisonB>A
weareonlyinterestedintheassociationbetweenvulnerabilityandtotal_DCDQ,notnvIQ.However,
thereexistsaweakpositiveassociationbetweentotal_DCDQandnvIQevenintheunionofsectorsB
andA.InordertofilterouttheconfoundingwithnvIQ,weperformedaconditionalpermutationtest
thatpermutesonlywithindecilesofnvIQ.InthecomparisonC>Btherolesoftotal_DCDQandnvIQare
reverseandhencepermutationsarewithindecilesoftotal_DCDQonly.
QuestionsofConfounding:Observationalstudiessuchasthepresentonecanresultinflawed
attributionofcause.Whileweformulateallresultsintermsofassociationratherthancausation,the
impliedunderstandingisthatthegeneticvariablesdescribeaspectsofcausalmechanismsforthe
phenotype.Inordertoreducethechanceofconfoundingofthegeneticvariableswithtrivializing
factorssuchasdemographics,weprovideinthesupplementalsectionS7p-valuedisplaysfor
demographics,andinS8displaysforgendersseparatelyandwithoutnon-Caucasianethnicitiesthat
couldpotentiallyconfoundwithgeneticvariables.FromthedemographicsinS7welearnthat,for
example,IQismuchmoreassociatedwithdemographicsthanMS(asmeasuredbytotal_DCDQ),thus
reducingthechancesofconfoundingforthelatter.Wealsolearntheknownfactthatdnmissense
mutationsaremorerelatedtofathers’agethanmothers’.FromthesubsetanalysisinS8welearnthat
themajorfindingspersistwhenconsideringwhitemalesonly.Duetothegenderimbalanceofautism,
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peer-reviewed) is the author/funder. It is made available under a CC-BY-NC 4.0 International license.
19
femalesconstituteamuchsmallersubsetinwhichstatisticalsignificancesarenecessarilyattenuated,
withnotableexceptionsforLGDsintheyoungfemalesdescribedbytheVineland(VABS)MSmeasures.
Overalltheconclusionisthatnon-Caucasianethnicitiesandfemalesexarenotconfoundingfactors
behindtheassociationsbetweenmutationalseverityandMS.
Acknowledgements
WethankJimSimonsforpromptingtheresearchandprovidingvaluableguidancethroughout;
GerryFischbachforadetailedreviewofthemanuscriptandsuggestionsforimprovements;Louis
Reichardtforprovidingadditionalpointers;andRichardBonneauandEvanBaughforexplanationsof
theVIPURmethodandsharingmissensemutationpredictions.Wealsothankallthefamiliesatthe
participatingSSCsites,aswellastheprincipalinvestigators(A.L.Beaudet,R.Bernier,J.Constantino,E.
H.Cook,Jr.,E.Fombonne,D.Geschwind,D.E.Grice,A.Klin,D.H.Ledbetter,C.Lord,C.L.Martin,D.M.
Martin,R.Maxim,J.Miles,O.Ousley,B.Peterson,J.Piggot,C.Saulnier,M.W.State,W.Stone,J.S.
Sutcliffe,C.A.Walsh,andE.Wijsman)andthecoordinatorsandstaffattheSSCsitesfortherecruitment
andcomprehensiveassessmentofsimplexfamilies,andtheSimonsFoundationAutismResearch
Initiative(SFARI)staffforfacilitatingaccesstotheSSC.ThisworkwassupportedbySFARIGrants
SF235988(toM.W.),SF362665(toI.I.)andSF448357(toA.B.andA.K.).
References
AmericanPsychiatricAssociation(2013).NeurodevelopmentalDisorders.In:DiagnosticandStatistical
ManualofMentalDisorders,5thedition.
BaughE.H.,Simmons-EdlerR.,MüllerC.L.,AlfordR.F.,VolfovskyN.,LashA.E.,BonneauR.(2016).Robust
classificationofproteinvariationusingstructuralmodellingandlarge-scaledataintegration.Nucleic
AcidsRes.44(6),2501-2513.
BernierR.,GolzioC.,XiongB.,StessmanH.,CoeB.P.,PennO.,WitherspoonK.,GerdtsJ.,BakerC.,VultovanSilfhoutA.T.,Schuurs-HoeijmakersJ.H.,FicheraM.,BoscoP.,BuonoS.,AlbertiA.,FaillaP.,Peeters
H.,SteyaertJ.,VissersL.E.L.M.,FrancescattoL.,MeffordH.C.,RosenfeldJ.A.,BakkenT.,O’RoakB.J.,
PawlusM.,MoonR.,ShendureJ.,AmaralD.G.,LeinE.,RankinJ.,RomanoC.,deVriesB.B.A.,KatsanisN.,
EichlerE.E.(2014).DisruptiveCHD8mutationsdefineasubtypeofautismearlyindevelopment.Cell
158(2),263-276.
BishopS.L.,ThurmA.,FarmerC.,LordC.(2016).AutismSpectrumDisorder,IntellectualDisability,and
DelayedWalking.Pediatrics137(3).
BolducM.-E.,dePlessisA.J.,SullivanN.,GuizardN.,ZhangX.,RoberstonR.L.,LimperopoulosC.(2012).
RegionalCerebellarVolumesPredictFunctionalOutcomeinChildrenwithCerebellarMalformations.
Cerebellum11,531-542.
BujaA.,KriegerA.,GeorgeE.(2015).AVisualizationToolforMiningLargeCorrelationTables:The
AssociationNavigator.InHandbookofBigData,BühlmannP.,DrineasP.,vanderLaanM.,KaneM.,eds.
(ChapmanandHall/CRC),pp.73-102.
bioRxiv preprint first posted online May. 23, 2017; doi: http://dx.doi.org/10.1101/141200. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC 4.0 International license.
20
CotneyJ.,MuhleR.A.,SandersS.J.,LiuL.,WillseyA.J.,NiuW.,LiuW.,KleiL.,LeiJ.,YinJ.,ReillyS.K.,
TebbenkampA.T.,BichselC.,PletikosM.,SestanN.,RoederK.,StateM.W.,DevlinB.,NoonanJ.P.
(2015).Theautism-associatedchromatinmodifierCHD8regulatesotherautismriskgenesduringhuman
neurodevelopment.Nat.Commun.6:6404.
DarnellJ.C.,VanDriescheS.J.,ZhangCh.,HungK.Y.S.,MeleA.,FraserC.E.,StoneE.F.,ChenC.,FakJ.J.,
ChiS.W.,LicatalosiD.D.,RichterJ.D.,DarnellR.B.(2011).FMRPstallsribosomaltranslocationonmRNAs
linkedtosynapticfunctionandautism.Cell146,247–261.
DeRubeisS.,HeX.,GoldbergA.P.,PoultneyC.S.,SamochaK.,ErcumentCicekA.,KouY.,LiuL.,Fromer
M.,WalkerS.,SinghT.,KleiL.,KosmickiJ.,FuS.-C.,AleksicB.,BiscaldiM.,BoltonP.F.,BrownfeldJ.M.,
CaiJ.,CampbellN.G.,CarracedoA.,ChahrourM.H.,ChiocchettiA.G.,CoonH.,CrawfordE.L.,CrooksL.,
CurranS.R.,DawsonG.,DuketisE.,FernandezB.A.,GallagherL.,GellerE.,GuterS.J.,SeanHillR.,IonitaLazaI.,GonzalezP.J.,KilpinenH.,KlauckS.M.,KolevzonA.,LeeI.,LeiJ.,LehtimakiT.,LinCh.-F.,Ma’ayan
A.,MarshallCh.R.,McInnesA.L.,NealeB.,OwenM.J.,OzakiN.,ParelladaM.,ParrJ.R.,PurcellS.,Puura
K.,RajagopalanD.,RehnstroK.,ReichenbergA.,SaboA.,SachseM.,SandersS.J.,SchaferCh.,SchulteRutherM.,SkuseD.,StevensCh.,SzatmariP.,TammimiesK.,ValladaresO.,VoranA.,WangL-S.,Weiss
L.A.,WillseyA.J.,YuT.W.,YuenR.C.,TheDDDStudy,HomozygosityMappingCollaborativeforAutism,
UK10KConsortium,TheAutismSequencingConsortium,CookE.H.FreitagCh.M.,GillM.,Hultman
Ch.M.,LehnerT.,PalotieA.,SchellenbergG.D.,SklarP.,StateM.W.,SutcliffeJ.S.,WalshCh.A.,Scherer
S.W.,ZwickM.E.,BarrettJ.C.,CutlerD.J.,RoederK.,DevlinB.,DalyM.J.,BuxbaumJ.D.(2014).Synaptic,
transcriptionalandchromatingenesdisruptedinautism.Nature515(7526),209-215.
DonnellanA.M.,HillD.A.,LearyM.R.(2013).Rethinkingautism:implicationsofsensoryandmovement
differencesforunderstandingandsupport.FrontiersinIntegrativeNeuroscience(28Jan2013).
DziukM.A.,LarsonJ.C.G.,ApostuA.,MahoneE.M.,DencklaM.B.,MostofskyS.H.(2007).Dyspraxiain
autism:associationwithmotor,social,andcommunicativedeficits.DevelopmentalMedicine&Child
Neurology49,734-739.
FischbachG.D.,LordC.(2010)TheSimonsSimplexCollection:aresourceforidentificationofautism
geneticriskfactors.Neuron68,192–195.
FournierK.A.,HassC.J.,NaikS.K.,LodhaN.,CauraughJ.H.(2010).MotorCoordinationinAutism
SpectrumDisorders:ASynthesisandMeta-Analysis.J.AutismDevDisord40,1227-1240.
GreenD.,CharmanT.,PicklesA.,ChandlerS.,LoucasT.,SimonoffE.,BairdG.(2009).Impairmentin
movementskillsofchildrenwithautisticspectrumdisorders.DevelopmentalMedicine&Child
Neurology51,311-316.
HiltonC.L.,ZhangY.,WhilteM.R.,KlohrC.L.,ConstantinoJ.(2012).Motorimpairmentinsiblingpairs
concordantanddiscordantforautismspectrumdisorders.Autism16(4),430–441.
IossifovI.,RonemusM.,LevyD.,WangZ.,HakkerI.,RosenbaumJ.,YamromB.,LeeY.,NarzisiG.,Leotta
A.,KendallJ.,GrabowskaE.,MaB.,MarksS.,RodgersL.,StepanskyA.,TrogeJ.,AndrewsP.,Bekritsky
M.,PradhanK.,GhibanE.,KramerM.,ParlaJ.,DemeterR.,FultonL.L.,FultonR.S.,MagriniV.J.,YeK.,
bioRxiv preprint first posted online May. 23, 2017; doi: http://dx.doi.org/10.1101/141200. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC 4.0 International license.
21
DarnellJ.C.,DarnellR.B.,MardisE.R.,WilsonR.K.,SchatzM.C.,McCombieR.,WiglerM.(2012).Denovo
genedisruptionsinchildrenontheautisticspectrum.Neuron74,285–299.
IossifovI.,O’RoakB.J.,SandersS.J.,RonemusM.,KrummN.,LevyD.,StessmanH.A.,WitherspoonK.T.,
VivesL.,PattersonK.E.,SmithK.D.,PaeperB.,NickersonD.A.,DeaJ.,DongS.,GonzalezL.E.,Mandell
J.D.,ManeS.M.,MurthaM.T.,SullivanC.A.,WalkerM.F.,WakarZ.,WeiL.,WillseyA.J.,YamromB.,Lee
Y.,GrabowskaE.,DalkicE.,WangZ.,MarksS.,AndrewsP.,LeottaA.,KendallJ.,HakkerI.,RosenbaumJ.,
MaB.,RodgersL.,TrogeJ.,NarzisiG.,YoonS.,SchatzM.C.,YeK.,McCombieW.R.,ShendureJ.,Eichler
E.E.,StateM.W.,WiglerM.(2014).Thecontributionofdenovocodingmutationstoautismspectrum
disorder.Nature515(7526),216-221.
IossifovI.,LevyD.,AllenJ.,YeK.,RonemusM.,LeeY-h,YamromB.,WiglerM.(2015)Lowloadfor
disruptivemutationsinautismgenesandtheirbiasedtransmission.Proc.Nat.AcademyUSA112(41),
p.E5600-5607.
KangH.J.,KawasawaY.I.,ChengF.,ZhuY.,XuX.,LiM.,SousaA.M.M.,PletikosM.,MeyerK.A.,Sedmak
G.,GuennelT.,ShinY.,JohnsonM.B.,KrsnikŽ.,MayerS.,FertuzinhosS.,UmlaufS.,LisgoS.N.,
VortmeyerA.,WeinbergerD.R.,ManeS.,HydeT.H.,HuttnerA.,ReimersM.,KleinmanJ.E.,ŠestanN.
(2011).Spatio-temporaltranscriptomeofthehumanbrain.Nature478,483–489.
KrummN.,TurnerT.N.,BakerC.,VivesL.,MohajeriK.,WitherspoonK.,RajaA.,CoeB.P.,StessmanH.A.,
HeZ.-X.,LealS.M.,BernierR.,EichlerE.E.KrummN.,TurnerT.N.,BakerC.,VivesL.,MohajeriK.,
WitherspoonK.,RajaA.,CoeB.P.,StessmanH.A.,HeZ.-X.,LealS.M.,BernierR.,EichlerE.E.(2015).
Excessofrare,inheritedtruncatingmutationsinautism.NatureGenetics47(6),582-8.
LandaR.,Garrett-MayerE.(2006).Developmentininfantswithautismspectrumdisorders:a
prospectivestudy.JChildPsycholPsychiatry47(6),629-638.
LearyM.R.,HillD.A.(1996)Movingon:autismandmovementdisturbance.Ment.Retard.34(1),39-53.
LevyD.,RonemusM.,YamromB.,LeeY.,LeottaA.,KendallJ.,MarksS.,LakshmiB.,PaiD.,YeK.,BujaA.,
KriegerA.,YoonS.,TrogeJ.,RodgersL.,IossifovI.,WiglerM.(2011).Raredenovoandtransmittedcopynumbervariationinautisticspectrumdisorders.Neuron70(5),886-897.
LimperopoulosC.,ChilingaryanG.,SullivanN.,GuizardN.,RoberstonR.L.,dePlessisA.J.(2014).Injury
tothePrematureCerebellum:OutcomeisRelatedtoRemoteCorticalDevelopment.CerebralCortex24,
728-736.
MacNeilL.K.,MostofskyS.H.(2012).SpecificityofDyspraxiainChildrenwithAutism.Neuropsychology
26(2),165-171.
McCarthyS.E.,GillisJ.,KramerM.,LihmJ.,YoonS.,BersteinY.,MistryM.,PavlidisP.,SolomonR.,
GhibanE.,AntoniouE.,KelleherE.,O’BrienC.,DonohoeG.,GillM.,MorrisD.W.,McCombieW.R.,Corvin
A.(2014).Denovomutationsinschizophreniaimplicatechromatinremodelingandsupportagenetic
overlapwithautismandintellectualdisability.MolecularPsychiatry19,652-658.
bioRxiv preprint first posted online May. 23, 2017; doi: http://dx.doi.org/10.1101/141200. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC 4.0 International license.
22
MesserschmidtA.,FuikoR.,PrayerD.,BruggerP.C.,BoltshauserE.,ZoderG.,SternisteW.,WeberM.,
BirnbacherR.(2008).Disruptedcerebellardevelopmentinpreterminfantsisassociatedwithimpaired
neurodevelopmentaloutcome.EuropeanJ.ofPediatrics167,1141-1147.
MosconiM.W.,SweeneyJ.A.(2015).Sensorimotordysfunctionsasprimaryfeaturesofautismspectrum
disorders.ScienceChina,LifeSciences58(10),1016-1023.
MostofskyS.H.,BurgessM.P.,LarsonJ.C.G.(2007).Increasedmotorcortexwhitemattervolume
predictsmotorimpairmentinAutism.Brain130(Pt8),2117–2122.
O’RoakB.J.,VivesL.,GirirajanS.,KarakocE.,KrummN.,CoeB.P.,LevyR.,KoA.,LeeCh.,SmithJ.D.,
TurnerE.H.,StanawayI.B.,VernotB.,MaligM.,BakerC.,ReillyB.,AkeyJ.M.,BorensteinE.,RiederM.J.,
NickersonD.A.,BernierR.,ShendureJ.,EichlerE.E.(2012).Sporadicautismexomesrevealahighly
interconnectedproteinnetworkofdenovomutations.Nature485(7397),246-250.
PaquetA.,OlliacB.,GolseB.,Vaivre-DouretL.(2016)Currentknowledgeonmotordisordersinchildren
withautismspectrumdisorder(ASD).ChildNeuropsychology.
ProvostB.,LopezB.R.,HerimerlS.(2007).Acomparisonofmotordelaysinyoungchildren:autism
spectrumdisorder,developmentdelay,anddevelopmentalconcerns.JAutismDevDisord.37(2),321328.
RinehartN.,McGinleyJ.(2010).Ismotordysfunctioncoretoautismspectrumdisorder?Developmental
Medicine&ChildNeurology52,697.
SandersS.J.,HeX.,WillseyA.J.,Ercan-SencicekA.G.,SamochaK.E.,CicekA.E.,MurthaM.T.,BalV.H.,
BishopS.L.,DongS.,GoldbergA.P.,JinluC.,KeaneyIIIJ.F.,KleiL.,MandellJ.D.,Moreno-De-LucaD.,
PoultneyC.S.,RobinsonE.B.,SmithL.,Solli-NowlanT.,SuM.Y.,TeranN.A.,WalkerM.F.,WerlingD.M.,
BeaudetA.L.,CantorR.M.,FombonneE.,GeschwindD.H.,GriceD.E.,LordC.,LoweJ.K.,ManeS.M.,
MartinD.M.,MorrowE.M.,TalkowskiM.E.,SutcliffeJ.S.,WalshC.A.,YuT.W.,AutismSequencing
Consortium,LedbetterD.H.,LeseMartinCh.,CookE.H.,BuxbaumJ.D.,DalyM.J.,DevlinB.,RoederK.,
StateM.W.(2007).InsightsintoAutismSpectrumDisorderGenomicArchitectureandBiologyfrom71
RiskLoci.Neuron,2015.87(6),1215-1233.
SchmahmannJ.D.(2013).CerebellarCognitiveAffectiveSyndromeandtheNeuropsychiatryofthe
Cerebellum.InHandbookoftheCerebellumandCerebellarDisorders,MantoM.,GruolD.L.,
SchmahmannJ.D.,KoibuchiN.,RossiF.,eds.(SpringerScience+BusinessMedia).
SchoemakerM.M.,FlapperB.,VerheijN.P.,WilsonB.N.,Reinders-MesselinkH.A.,deKloetA.(2006)
EvaluationoftheDevelopmentalCoordinationDisorderQuestionnaireasascreeninginstrument.
DevelopmentalMedicineandChildNeurology48(8)668-673.
SebatJ.,LakshmiB.,MalhotraD.,TrogeJ.,Lese-MartinCh.,WalshT.,YamromB.,YoonS.,KrasnitzA.,
KendallJ.,LeottaA.,PaiD.,ZhangR.,LeeY.,HicksJ.,SpenceS.J.,LeeA.T.,PuuraK.,LehtimäkiT.,
LedbetterD.,GregersenP.K.,BregmanJ.,SutcliffeJ.S.,JobanputraV.,ChungW.,WarburtonD.,KingM.C.,SkuseD.,GeschwindD.H.,GilliamT.C.,YeK.,WiglerM.(2007).Strongassociationofdenovocopy
numbermutationswithautism.Science316(5823),445-449.
bioRxiv preprint first posted online May. 23, 2017; doi: http://dx.doi.org/10.1101/141200. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC 4.0 International license.
23
VoineaguI.,WangX.,JohnstonP.,LoweJK.,TianY.,HorvathS.,MillJ.,CantorR.M.,BlencoweB.J.,
GeschwindD.H.(2011).Transcriptomicanalysisofautisticbrainrevealsconvergentmolecular
pathology.Nature474,380–384.
Wagner M.J., Kim T.H., Savall J., Schnitzer M.J., Luo L. (2017). Cerebellar granule cells encode the
expectationofreward.Nature544(7648),96-100.
WangS.S.-H.,KlothA.D.,BaduraA.(2014).TheCerrebellum,SensitivePeriods,andAutism.Neuron83,
518-532.
WilsonB.N.,CrawfordS.G.,GreenD.,RobertsG.,AylottA.,KaplanB.(2009).PsychometricPropertiesof
theRevisedDevelopmentalCoordinationDisorderQuestionnaire.Physical&OccupationalTherapyin
Pediatrics29(2),182-202.