School of Paediatrics and Reproductive Health, University of Adelaide, Women's and Children

39 Pages·2015·0.79 MB·English

HumanMolecularGenetics,2015,Vol.24,No.12 3335–3347
doi:10.1093/hmg/ddv083
AdvanceAccessPublicationDate:3March2015
OriginalArticle
ORIGINAL ARTICLE
HCFC1
loss-of-function mutations disrupt neuronal
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and neural progenitor cells of the developing brain n
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Lachlan A. Jolly1,2, Lam Son Nguyen4, Deepti Domingo3, Ying Sun1,2, fro
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Simon Barry1, Miroslava Hancarova5, Pavlina Plevova6, Marketa Vlckova5, h
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Marketa Havlovicova5, Vera M. Kalscheuer7, Claudio Graziano8, s://a
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Tommaso Pippucci8, Elena Bonora8, Zdenek Sedlacek5 and Jozef Gecz1,2,3,* a
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1SchoolofPaediatricsandReproductiveHealth,2RobinsonResearchInstituteand3SchoolofMolecularand ic.o
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BiomedicalSciences,UniversityofAdelaide,Adelaide5000,Australia,4INSERMUMR1163,Laboratoryof p
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MolecularandPathophysiologicalBasesofCognitiveDisorders,ParisDescartes—SorbonneParisCitéUniversity, om
ImagineInstitute,Necker-EnfantsMaladesHospital,75015Paris,France,5DepartmentofBiologyandMedical /hm
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Genetics,CharlesUniversity2ndFacultyofMedicineandUniversityHospitalMotol,Prague,CzechRepublic, /a
6DepartmentofMedicalGenetics,UniversityHospitalOstrava,tr.17.listopadu1790,70852Ostrava,Czech rtic
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Republic,7DepartmentofHumanMolecularGenetics,MaxPlanckInstituteforMolecularGenetics,Ihnestrasse -a
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73,D-14195Berlin,Germanyand8UnitofMedicalGenetics,DepartmentofMedicalandSurgicalSciences,DIMEC, stra
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St.Orsola-MalpighiHospital,UniversityofBologna,Bologna40138,Italy t/2
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*Towhomcorrespondenceshouldbeaddressed.Tel:[phone];Fax:[phone];Email:[email protected] /12
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Abstract 19
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Bothgain-andloss-of-functionmutationshaverecentlyimplicatedHCFC1inneurodevelopmentaldisorders.Here,weextend b
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ourpreviousHCFC1over-expressionstudiesbyemployingshorthairpinRNAtoreducetheexpressionofHcfc1inembryonic g
u
neuralcells.Weshowthatincontrasttoover-expression,lossofHcfc1favouredproliferationofneuralprogenitorcellsatthe e
s
expenseofdifferentiationandpromotedaxonalgrowthofpost-mitoticneurons.TofurthersupporttheinvolvementofHCFC1 t o
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inneurologicaldisorders,wereporttwonovelHCFC1missensevariantsfoundinindividualswithintellectualdisability(ID). 2
9
Oneofthesevariants,togetherwiththreepreviouslyreportedHCFC1missensevariantsofunknownpathogenicity,were M
functionallyassessedusingmultiplecell-basedassays.Weshowthatthreeoutofthefourvariantstestedresultinapartialloss arc
ofHCFC1function.Whileover-expressionofthewild-typeHCFC1causedreductioninHEK293Tcellproliferationandaxonal h 2
growthofneurons,theseeffectswerealleviateduponover-expressionofthreeofthefourHCFC1variantstested.Oneofthese 01
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partialloss-of-functionvariantsdisruptedanuclearlocalizationsequenceandtheresultingproteindisplayedreducedabilityto
localizetothecellnucleus.TheothertwovariantsdisplayednegativeeffectsontheexpressionoftheHCFC1targetgene
MMACHC,whichisresponsibleforthemetabolismofcobalamin,suggestingthattheseindividualsmayalsobesusceptibleto
cobalamindeficiency.Together,ourworkidentifiesplausiblecellularconsequencesofmissenseHCFC1variantsandidentifies
likelyandrelevantdiseasemechanismsthatconvergeonembryonicstagesofbraindevelopment.
Received:November10,2014.Revised:February5,2015.Accepted:March2,2015
©TheAuthor2015.PublishedbyOxfordUniversityPress.Allrightsreserved.ForPermissions,pleaseemail:[email protected]
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3336 | HumanMolecularGenetics,2015,Vol.24,No.12
Introduction mechanismsbehindgain-of-functionmutations,wedeveloped
shorthairpinRNA(shRNA)lentiviralreagentstoknockdown
Intellectualdisability(ID)affects∼2–3%ofthepopulationandis Hcfc1 expression. We identified one shRNA trigger sequence
consideredoneofthemostgeneticallyheterogeneoushumandis-
(T2)thattargetedtheuntranslatedregion(UTR)ofHcfc1,that
orders(1).ContributionofgenesontheXchromosome(i.e.X-linked
modestlyknockdownHcfc1expressionbyabouthalfwhentested
ID;XLID)hasbeenthebestcharacterizedwithover100genesimpli-
inNIH3T3cells(SupplementaryMaterial,Fig.S1).AsHCFC1gain-
catedthusfar.Wehaverecentlyreportedanon-codingregulatory
of-functionisknowntoreducetheaxonalgrowthandneurite
mutationinanX-linkedgene,HCFC1(OMIM300019),asthelikely
arbourizationofhippocampalneurons,weaskedwhatwould
causeofmildnon-syndromicIDinthelargeX-linkedfamilyMRX3
be the effect of loss of function in these assays. We isolated
(2).HCFC1isatranscriptionalco-regulatorwithmanyimportant
post-mitotic hippocampal neurons from embryonic day 18.5
functionsincellproliferationandmitochondrialbiogenesis(3–6).
(E18.5)miceembryosandplatedtheminvitro.Following12h
RecentdatasuggestthatHCFC1containingtranscriptionalcom-
growth,wetransducedthecellswithlentiviralparticlesencoding
plexesmayregulatemorethanaquarterofallhumanpromoters enhancedgreenfluorescentprotein(EGFP)andeitheracontrol
(7).Thenon-codingregulatorymutationinthe5′untranslatedre- D
shRNAsequence(sequenceagainstluciferasethatdoesnottar- o
gion(UTR)ofHCFC1abolishedaDNA-bindingsiteforthetranscrip- getmammaliangenes)oraHcfc1shRNA(T2;Hcfc1shRNA).Fol- wn
tionalregulatorYY1,whichsubsequentlycausedde-repressionof lo
lowingtransduction,neuronswereallowedtogrowuntil4days a
HCFC1geneexpressioninpatientcelllines(2).Tomodeltheconse- invitro,subsequentlyfixedandimmunofluorescentlylabelled ded
aqluteernctheesobfehthaivsiocuhraonfgee,mobvreyro-enxicprneesusiroanlcoefllHs,CnFaCm1ewlyaspsrohmowontintog aagnadinasxtotnhse,mreasrpkeecrtpivroetlyein(FsigM.A1AP2).aNnedxTtAwUe1ctooniddeuncttiefyddmenodrprihteos- from
differentiationofneuralprogenitorcells(NPCs)andreducingthe h
nmeourrei,tecognroswisttehnotfwexithvivthoecsueltnuerewdlyhiipdpenoctiafimedparollneesufroornHs.CFFuCr1thaenrd- mCcloeemst,rpnicaeruaerndoanwlysistthirsannoesnudruEocGneFsdPtwreaixntphsrdHeusccfscei1ndsghwR(Niit.Aehv.citorranalntprsoadlrutlieccneletdisv)dinriaeslpuplraaoyrnetsid-. ttps://a
c
thosepreviouslyestablished, transcriptomeanalysis of patient a
a33%increaseinaxonallengthandsimilarincreasesinaxonal d
celllineshighlightedderegulationofgenesthatwereimportant e
anddendritic(andhencetotal)termininumber,whichreports m
fcohroenmdrbiarylofunnicctbioranin(2g,8r)o.wAdthd,ittiroannaslclyri,ptthioreneaulnrergeulaltaetdiofna,mainlidesmciotno-- eoxnprtehsesidoengorefeHCofFCn1ecuorounldalreasrcbuoeritzhaitsioanxo(nFiagl.d1eBfeactntdhaCt).coRne-- ic.oup
tcahianninggesmwaelereiniddeivnitdiufieadls(w2)i.thWIhDetahnedrdthifefesreenadtdHiCtiFoCn1almviasrsieannstes firmsthespecificityoftheHcfc1shRNA(seebelow).Thus,reduction .com
werepathogenic,orsimplylow-frequencybenignsingle-nucleotide ofHcfc1expressionalsoaffectedhippocampalneuronalgrowth /h
anddisplayedtheoppositeeffecttothatpreviouslydescribed m
polymorphisms(SNPs),wasnotestablished.Subsequently,mul- g
forHCFC1over-expression(2). /a
dtiopmleaHinCoFCft1hmeHisCsFeCn1seprmotuetinat,ihoanvse,baeffeenctiidnegnttihfieedNi-ntearcmoihnoarltKofeplcah- rticle
tientswithX-linkedCobalamintypeC(CblC)-likedisorder,which LossofHcfc1resultsinexpansionofneural -a
b
wasnamedCblX(9,10)(OMIM:309541).CblC(OMIM:277400)isa progenitorcells stra
metabolic disorder most frequently caused by mutations in c
fMoMrmACeHtaCbo(OliMsmIMo:f6c0o9b8a3la1m),iwnh(1ic1h).eHnCcFoCd1eswaanssehnozwymnetorebqinudiretdo wHPrCaoFsmCoo1vtemior-nuetxoapftiNroePnsCs(e2dd)i.ftfToehrmeunsot,diwaetleiotihnnevweeasfsfteiagclatstooefodbtwhseehregvtaehidnew-rothfh-efenusnHecCctFeioClln1s t/24/12
thepromoterofMMACHCandknockdownofHCFC1incultured /3
wouldalsobesensitivetolossof Hcfc1function.We isolated 3
cspkeolirlntsalfinebtdlryot,obMlaaMslotAssCsoHofCftMewxMopArCeCsbHslXiConepxawptraieessnsstiisognnte.ifisSctiemadnitlal(y9r)lr.yeTadnhuducesmd, olaoslstssoimion-f NntrooPnCl-osarfdrHhocmefrce1tnshhteRnNEeA1u8ler.5onstepimvhirebarrleyspo.naWrictiecdltoerrass,naaslndcduocraetcedhxiteahvneedcdegtlrrlesawnwstidhthuecmctoioanns- 35/62190
HCFC1 function was demonstrated as the cause of CblX. Both ratesof∼70%(datanotshown).Inthefirstinstance,wecreated 9 b
CblCandCblXpatientsdisplayneurologicalimpairment;however, purifiedtransducedculturesusingfluorescence-activatedcell y g
CblX-relatedneurologicalphenotypeismuchmoresevereandin- sorting(FACS)andsubjectedpurifiedcellstoacellproliferation ue
ctilvuedeisminptariarcmtaebnltee(9p)i.leTposyg,ebthraeirn,mthaelsfoermfinatdiionngssansudgsgeevsetrethcoatgniin- assay.Cellswereplatedatanequivalentinitialdensityandal- st on
madudtiatitoionntsocoavuesr-eexdpisrreusspitoionnosftHoCnFCo1rm,aalslobrloasins-odfe-vfuenlocptimonenHtCaFnCd1 lwoawseadstsoaygerodwatfoDra6ysda0y,s3daunrdin6g(wCehlilcThitcreell9g6rAoQwutehoiunsthAesscauyl;tuPrreos- 29 M
mega).Whenseededatlow(clonal)density,cellgrowthwaslin- a
likelyinvolvemultiplemechanisms. earinbothcontrolandHcfc1shRNAcultures(linearregressionof rch
Inthisstudy,weextendouroriginalinvestigationsofHCFC1 lineofbestfit:R2=0.984and0.997,respectively);however,the 20
over-expressionbyinvestigatingtheeffectthatloss-of-function rateofgrowthinHcfc1shRNAcultureswashigher(slopeoflinear 19
HCFC1 mutations have on embryonic brain development. We fit;control=0.56,Hcfc1shRNA=1.12),resultingina36and65%in-
alsoreporttwonovelHCFC1variantsidentifiedintwounrelated
creaseincellnumbersatDays3and6ofculture,respectively
patientswithIDandcollateavailableclinicalfindings.Finally,
(Fig.2A).Similarresultswereobtainedwhencellswerecultured
weusemultiplecell-basedassaystoaddressthefunctionalconse-
athigherinitialdensities;however,cellgrowthplateauedatlater
quenceoftheseandalsopreviouslyreportedHCFC1variants.Our
stages,perhapsduetonutrientgrowthrestriction(Supplemen-
findingsshedfurtherlightintothecellularmechanismsaswellas
taryMaterial,Fig.S2).Wereasonedthisincreaseincellnumbers
theclinicalconsequencesofHCFC1-relatedneurologicaldisorders.
mayresultfrom(i)aninhibitionofdifferentiationevenunder
proliferativecultureconditions(i.e.inthepresenceoftheepider-
Results mal growth factor; EGF), (ii) reduction in apoptosis or (iii) in-
creasedprogenitorproliferation.Toaddressthesealternatives,
ReductionofHcfc1stimulatesneuronalgrowth
wefirstcollectedRNAfromthepurifiedculturesandapplied
To model the underlying neural cell pathology of Hcfc1 loss- qRT-PCRtodiscovertheexpressionlevelsofseveralcelltype-
of-function mutations, and to compare it with established specificmarkergenes(Fig.2B).Wesawnostatisticallysignificant
HumanMolecularGenetics,2015,Vol.24,No.12 | 3337
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Fpiagrutircele1s.LeonscsoodfinHgcfeci1thcaeursaensiennehrtasnhcReNdAhispepqouceanmcepa(algnaeiunrsotnlualcgifreorwasteh;icnovnittrroo.lI)soorlaHtcefdc1psrhimRNarAy(hHipcfpco1csahRmNpA)a.l(Ane)uRreopnressweenrteatciuvletuimremdfuonllooflwuionrgestrcaennstdiumcatigoenswofitchonletnrotilvainradl t/24
Hcfc1shRNAneuronsatDay4ofinvitrogrowth.Bars=100μm.TransducedcellsexpressEGFP(green).Cultureswereco-stainedusingantibodiesthatlabeldendritic(MAP2; /12
red)andaxonal(TAU1;cyan)neuronalstructures.(B)Quantificationofprimaryaxonallength.(C)Quantificationofneuritetermini.*P<0.05byStudent’st-test. /3
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differenceintheexpressionoftheearlyneuroepitheliamarker thatifmoreNPCswerepresentintheHcfc1shRNAcultures,thena 21
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Sox1,thecorticalNPCmarkerPax6,theradialgliacellmarker higherpercentageoftransducedcellswillbeundergoingmitosis 0
BLBPnorthepanNPCmarkerNestin.Likewise,wesawnosignifi- atanygiventime,soweusedanantibodyagainstphospho-his- 9 b
cantdifferencesintheterminallydifferentiatedcell-typemarker tone3,aspecificnuclearmarkofcellsatmitosis.Transducedcells y g
genes,namelytheearlyneuronalmarkerβIII-tubulin,theastro- inHcfc1shRNAcultureswerefoundtohavea34%increaseincells ue
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cyte marker GFAP and the oligodendrocyte marker CNPase. undergoingmitosiscomparedwithcontrols(Fig.2EandSupple- t o
Whilenotreachingstatisticalsignificance,wedidnoteatrend mentaryMaterial,Fig.S3C).Togethertheresultssuggestthat n 2
for elevated expression of marker genes for progenitor cell underproliferativeconditions,transducedNPCsinHcfc1shRNA 9 M
typesandareciprocaldecreaseintheexpressionofterminally culturesweremorelikelytoremainintheproliferativeprogenitor a
differentiated cell-type marker genes in Hcfc1shRNA cultures state.Wefurtherexploredthepossibilitythattransducedcellsin rch
(Fig.2B).Tomoredefinitivelyidentifyandquantifythenumbers Hcfc1shRNA cultures might be conducive to NPC maintenance 20
1
ofprogenitorcellsandmonitortheirbehavioursatsingle-cell underconditionsthatpromotedifferentiation.Wethusplated 9
resolution,weemployedimmunofluorescence.Wedissociated controlandHcfc1shRNA-transducedcellsontopoly-l-lysinesub-
neurospherecells(fromparallelnon-purifiedcultures)andpla- strateasdescribedabovebutremovedgrowthfactorsoastopro-
tedthemontoapoly-l-lysinesubstrateinthepresenceofEGF. motedifferentiation.Weallowedthecellstodifferentiatefor
Onlytransducedcells(i.e.thoseexpressingEGFP)wereanalysed. 3daysbeforestainingthemimmunofluorescentlyusinganti-
ThepercentageoftransducedcellsofNPCidentitywasreported bodiesagainstcelltype-specificmarkerproteinsPax6,βIII-tubu-
bytheco-expressionofEGFPandPax6.Weidentifiedasignificant lin,GFAPandCNPase,andcountedtheirabundancewithinthe
20%increaseinPax6-positiveNPCsinHcfc1shRNAcultures(Fig.2C cultures (Fig. 2F and Supplementary Material, Fig. S4A–C).
andSupplementaryMaterial,Fig.S3A).Wenextaskedwhether Undertheseconditions,transducedcellsintheHcfc1shRNAcul-
apoptoticrateswithinthecultureswereaffectedbyreduction tureshad25%morePax6-expressingNPCs,and34%lessdifferen-
ofHcfc1;however,wewereunabletoidentifyanydifferencesin tiated cell types compared with transduced cells in control
thenumberoftransducedcellsantigenictoanactivatedcas- cultures.Thisreductionindifferentiationwasfoundtobesignifi-
pase3 antibody, a hallmark of cells undergoing cell death cantacrossallthreeneuralcelllineages.Inaggregate,ourdata
(Fig.2DandSupplementaryMaterial,Fig.S3B).Next,wereasoned suggestthatreductionofHcfc1inNPCspromotesthecellcycling
3338 | HumanMolecularGenetics,2015,Vol.24,No.12
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Figure2.LossofHcfc1causesincreasedgrowthkineticsandreduceddifferentiationofNPCs.IsolatedprimarycorticalNPCsgrowninvitroasneurospheresweretransduced 3
3
withlentiviralparticlesencodingeitherinertshRNAsequences(Control)orHcfc1targetedshRNA(Hcfc1shRNA).(AandB)TransducedcellswerepurifiedusingFACSpriorto 5
assays.(A)CellproliferationassayconductedinthepresenceofEGFatDays0,3and6followingpassage.Cellswereplatedataninitiallowclonaldensityof1×104cells.(B) /62
qRT–PCRanalysisofmarkergenesforNPCpopulations(Nestin,Sox1,Pax6andBLBP)andfordifferentiatedcelltypes[βIII-tubulin(βIII-tub),GFAPandCNPase].(C–F)Non- 19
purifiedtransducedcellswereassayed,andallvaluesgivenaspercentageoftransducedcells(i.e.EGFPexpressingcells;EGFP+ve).(C–E)Dissociatedcellswereplatedonto 09
poly-l-lysinesubstrateandculturedinthepresenceofEGF.(C)QuantificationofpercentageoftransducedcellsexpressingPax6byimmunofluorescence.(D)Quantification b
y
ofpercentageoftransducedcellsexpressingactivatedcaspase3byimmunofluorescence.(E)Quantificationoftransducedcellsexpressingphospho-histone3(PH3)by g
immunofluorescence.(F)Cellswereplatedontopoly-l-lysinesubstrateandculturedintheabsenceofEGF.Percentageofcelltypespresentindifferentiatedcultures ue
iSdteundteinfite’dstb-yteismt.munofluorescence:progenitorcells(Pax6)anddifferentiatedcells(astrocytes:GFAP;neurons:βIII-tubulinandoligodendrocytes:CNPase).*P<0.05by st on
2
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M
anddrivestheirfatechoicestowardsproducingdaughterNPCsat variant was also found in 1/1466 chromosomes (or 7/10271; a
rc
theexpenseofdifferentiatedcelltypes.Asinourpreviousneur- NationalHeart,Lung,andBloodInstitute[NHLBI]ExomeVariant h
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onalcellassays,thisresultisingeneraloppositetopreviousre- Server),andthepatientalsohadapotentiallydeleteriousvariant 0
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sultsobservedwhenHCFC1isover-expressedinthesesamecell intheXLIDgeneMED12(NM_005120.2;c.3101T>G,p.Phe1034Cys; 9
typesandsuggeststhatNPCsaresensitivetoHCFC1dosage. OMIM:300188),andalikelydeleterioustruncatingvariantinthe
IDgeneARID1B(NM_017519.2;c.2723delC,p.Pro908fs*6;OMIM:6
IdentificationofnovelHCFC1variantsinindividuals 14556)(12)(Fig.3A).AthirdHCFC1variant(c.5267C>T,p.Ala1756-
Val) was found in the index patient of family D82 with four
withID
affectedmalesacrosstwogenerations;however,co-segregation
WepreviouslydescribedthreemissensevariantsintheHCFC1 couldnotbeperformedasDNAwasunavailableonallrelevant
codingregion(NM_005334.2)inpatientswithprobableorlikely familymembers(Fig.3A).Theindexpatientalsohadavariant
XLID using X-exome sequencing (2). A missense change inZMYM3(NM_005096.3;c.356A>G,p.Gln119Arg)ofunknown
(c.674G>A,p.Ser225Asn)segregatedwithIDinfamilyD144,asso- significance. We now report the discovery of two additional
ciatingwithIDinfouraffectedmalesacrosstwogenerations novelHCFC1variantsinpatientswithprobableXLID.Thefirst
(Fig.3A).Asecondmissensevariant (c.2626G>A,p.Gly876Ser) novelvariantwasfoundinafamilyascertainedintheCzechRe-
was found in a simplex patient (family D147) whose mother public (family CZE168) that contained two affected brothers.
had100%skewingofXchromosomeinactivation.However,this The clinical descriptions of these individuals are detailed in
HumanMolecularGenetics,2015,Vol.24,No.12 | 3339
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Figure3.HCFC1variantssegregatewithIDandresultinchangestoconservedregionsoftheprotein.(A)Pedigreesoffamiliesidentifiedin(2)(FamiliesD144,D147andD87) le
andthatofnewfamilies(ITL1andCZE168),withHCFC1variants.cDNA(NM_005334.2)andprotein(XP_005274721.1)annotationforindividualvariantsareshown.Blackfill -ab
iEnBdIiCcaLtUeSsTIADL,Wblascekrdveort)s,iannddicinatseilhiceotperreodziycgtoiotnec(CarArDieDrs),oafntdhegreefyfeficltltihnadtictahteevsaaruiatinstmam(niontoIDa)c.i(dB)cSheaqnugeenscheaavleigonnmperontteoinfvfaurniactnitoanmanindoinacteidgsriatycr.oNsostdeitfhfearteCnAtsDpDecsiceosr(euss>in2g0 strac
areconsideredlikelypathogenic,whilescores>10areconsideredpotentiallypathogenic.(C)LinearscalecartoonofHCFC1proteinshowingknownstructuralfeatures t/2
4
(K1-5:Kelchdomains;Fn3:Fibronectin3domain;Pro:HCFC1proteolyticrepeats;NLS:nuclearlocalizationsequence)andthelocationsofvariantaminoacids.Note /1
that*indicateslocationsofvariantspreviouslyreported(9,10). 2
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SupplementaryMaterial,TableS1.Briefly,bothaffectedindivi- urine.Theaffectedindividual’sgreatunclealsosufferedfromID 5/6
duals shared clinical features of mild–moderate ID, delayed (deceased;SupplementaryMaterial,Fig.S5),whiletheindividual’s 21
9
speechanddelayedpsychomotordevelopment.Theywereboth nephewsufferedfromautismspectrumdisorderanddidnotcarry 0
ofshortstature(3rdpercentile)andshareddysmorphicfeatures thevariant.ThisHCFC1variantdisruptsthefibronectintype3do- 9 b
y
ofthicklips,longnoseandalongphiltrum.Bothdisplayedmild mainofHCFC1whichisinvolvedinheterodimerizationofHCFC1 g
u
liversteatosisanddiffusehypocontractibilityoftheleftventricle. N-andC-terminalproteolyticcleavageproductsandDNAbinding. e
s
Metabolic profiling also revealed transiently increased plasma Wewereabletoaccessmaterial(wholeblood)fromindivi- t o
n
homocysteinelevelsandnormallevelsofmethylmalonicacidin dualsoftheD144,ITL1andCZE168families(butnottheothers) 2
urine.Whole-exomesequencingwasperformedtofindcausative totestfortheexpressionlevelsofHCFC1.Intriguingly,mRNAex- 9 M
variants.Novariantswerefoundunderanautosomalrecessive pressionofthevariantformsofHCFC1inaffectedindividuals a
model,whereasfivevariantswerefoundunderamodelofX-link- waselevatedinfamiliesITL1andCZE168,andmodestlyelevated rch
2
age(SupplementaryMaterial,TableS2).Ofthese,theHCFC1vari- intwoofthreeindividualsfromD144,comparedwithcontrol 0
ant(chrX153215026G>A(HG19);c.6046C>T;pArg2016Trp)wasthe samples(SupplementaryMaterial,Fig.S6A–C),whichmaysug- 19
mostlikelycandidatebasedonextendedsegregationanalysisand gestanautoregulatorymechanismcontrollingHCFC1transcrip-
predicted tolerances to variation (Supplementary Material, tion (see discussion). Western blot analysis of an affected
Table S2). X-inactivation studies on the affected individual’s individualintheCZE168family,however,didnotrevealsignifi-
mother showed significant skewing (87%). The HCFC1 variant cantincreaseinHCFC1proteinlevels(SupplementaryMaterial,
abolishes a predicted bi-partite nuclear localization sequence Fig.S6D).
(NLS; amino acids 2011–2034; cNLS Mapper (13) prediction Overall,thisnewgeneticevidencetogetherwithpreviousre-
score=9.6).AsecondnovelHCFC1variantwasfoundinafamily portssuggeststheinvolvementoftheHCFC1inthepathologyof
ascertained inItaly (family ITL1; chrX153225268C>T(HG19); ID; however, while the genetic evidence for the p.Ser225Asn
c.1429G>A; p.Ala477Thr; Fig. 3A and Supplementary Material, variant (located within the N-terminal Kelch domain) and
Fig.S5).TheaffectedindividualhassevereID,absentspeechand p.Arg2016Trp(locatedintheNLS)ismorepersuasive,whether
displayedfrequentfebrileseizuresduringinfancy.Hehadanelon- thep.Gly876Ser,p.Ala1756Valandp.Ala477Thrvariantsarelikely
gatedface,largeears,arachnodactylyandaleanbodyhabitus. causativeislessclear.Allofthevariantsalterconservedamino
Metabolic profiling revealed normal methylmalonioc levels in acids of HCFC1, and in silico predictions (CADD score (14))
3340 | HumanMolecularGenetics,2015,Vol.24,No.12
suggestedfourofthevariantstobelikelydeleterioustoprotein and B). We next studied the subcellular localization of wild-
function,withthep.Ala477Thrvariantpredictedtobepossibly typeandvariantHCFC1proteins.First,wecomparedtheexpres-
deleterious(Fig.3BandC).Wethusembarkedonfunctionalchar- sionofendogenousHCFC1withthatofexogenouslyexpressed
acterizationofthesevariantsandtheireffectonHCFC1function. wild-typeHCFC1.HCFC1isknowntoundergoproteolyticpro-
cessingincellsandcanbefoundaseitherafull-lengthprotein,
or N- and C-terminal fragments (15,16). These fragments are
Thep.Arg2016TrpvariantofHCFC1disrupts
knowntoformaheterodimerbutcanalsodisplayindependent
itsnuclearlocalization functions(17–19).Weusedouranti-MycandHAantibodiestode-
WeclonedfourofthefiveHCFC1variantsintoacDNAconstruct tect exogenous HCFC1, and our anti-HCFC1 antibody, which
thatpermitsexpressioninmammaliancellsunderaconstitutive bindstheC-terminalofHCFC1,tolocalizeexogenousanden-
promoter.Unfortunately,wewereunabletoobtaincloneswith dogenous HCFC1. In untransfected cells, endogenous HCFC1
thep.Ala477Thrvariant.Theexpressionconstructscontained waslocalizedpredominantlyincellnuclei,withhighestexpres-
inframeMycandHAtagsattheNandCterminus,respectively, sion found in cells undergoing mitosis (Fig. 4B). Exogenous
D
tofacilitatediscriminationbetweenendogenousandexogenous HCFC1detectedwiththeC-terminalMyctagdisplayedsimilar o
w
expression(Fig.4A).WhenexpressedinHEK293Tcells,weob- expression,whiletheHCFC1detectedwiththeN-terminalHA n
lo
servednodifferenceinHCFC1variantmRNAorproteinexpres- tagwasenrichedinthenucleusbutalsolocalizedtothecyto- a
d
sion levels compared with wild-type HCFC1, suggesting the plasm(Fig.4B).Allvariantsexceptthep.Arg2016Trpvariantloca- ed
variants did not overtly affect exogenously expressed HCFC1 lized akin to wild type. The p.Arg2016Trp variant detected fro
mRNA or protein stability (Supplementary Material, Fig. S7A throughbothitsN-terminalHAtagandC-terminalMyctagfailed m
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Figure4.HCFC1p.Arg2016TrpvariantfailstolocalizetothenucleusinHEK293Tcells.(A)DiagramofHCFC1proteinwiththelocationsofthevariantsbeingtested.Note
alsothatexogenouslyexpressedHCFC1proteinsaretaggedbyHAattheN-terminusandMycattheC-terminus,andalsotheC-terminallocationoftheepitopeusedto
generatetheHCFC1antibody(Ab).(B)HEK293Tcellsweretransfectedwithemptycontrolvectorsorvectorsencodingwild-typeorvariantformsofHCFC1.
Immunofluorescentdetectionofendogenous(andexogenous)HCFC1(usingtheanti-HCFC1antibody;cyan),andexogenouswild-typeandvariantformsusingboth
anti-HA(red)andanti-Myc(green)antibodies.CellnucleiarecounterstainedwithDAPI(blue).
HumanMolecularGenetics,2015,Vol.24,No.12 | 3341
D
Figure5.HCFC1variantsdisruptitsabilitytosuppresscellgrowthofHEK293T Figure6.Over-expressionofHCFC1variantsdisruptsMMACHCmRNAexpression. ow
cells.HEK293Tcellsweretransfectedwithanemptyexpressionvector(control) HEK293Tcellsweretransfectedwithanemptyexpressionvector(control)orwith n
orwithexpressionvectorsencodingeitherwild-typeorvariantHCFC1forms. lo
expressionvectorsencodingeitherwild-typeorvariantHCFC1formsandRNA a
Transfectedcellswereplatedatequivalentdensitiesandallowedtogrowfor2 wasisolated.MMACHCmRNAexpressionwasanalysedbyqRT–PCR. de
daysatwhichpointcellnumberswereassayed.n=6,*issignificantlydifferent d
towildtype,P<0.05Student’st-test. fro
m
twovariantstestedresultedinthereductionofMMACHCexpres- h
sion.Aswewereabletoachievelargeover-expressionofwild- ttp
tolocalizetothenucleusandwasfoundinthecytoplasm.Simi- s
larresultswereobtainedusingHeLacells(datanotshown)andin type and variantHCFC1 inthese cells(∼160-foldastested by ://a
hippocampalneurons(SupplementaryMaterial,Fig.S8AandB). qRT–PCR,and∼10-foldastestedbywesternblot;Supplementary cad
Material,Fig.S7),thestoichiometrysuggeststhattheexogenous- e
ThesedataindicatethattheHCFC1variantaffectingthenuclear m
localizationsequence(p.Arg2016Trp)resultsinadisruptiontoits lyexpressed proteins will outcompete endogenous wild-type ic
HCFC1forbindingattheMMACHCpromoter.GiventhatHCFC1 .o
nuclearlocalization. u
loss-of-functionmutationsaredescribedtoresultinreduced p.c
MMACHCexpressioninpatientcells(9),thelossofMMACHCex- om
HCFC1variantsdisruptproliferationofHEK293Tcells pressioninthisassaycanbeinterpretedlikewise,suggestingthat /h
m
thep.Ser225Asnandp.Gly876Servariantsarealsolikelylossof g
Gmiuveltnippleresvtiaoguesssotfutdhieesceslhlocwycinleg(t4h,6e,1r7e–q1u9i)r,ewmeenaltsooftHesCteFdC1thaet function. /artic
abilityofHCFC1anditsvariantformstoaffecttheproliferation le-a
ofHEK293Tcells.Weplatedtransfectedcellsoutatanequivalent b
densityandmonitoredcellgrowthusingtheaforementionedcell HCFC1variantsaffectneuronaloutgrowth stra
c
growth assay. Following 2 daysof growth, over-expression of Theabovedatasuggestthatwiththeexceptionofthep.Ala1756- t/2
wild-type HCFC1 caused a 58% decrease in growth compared Valvariant,theotherHCFC1variantsmaycausealossorpartial 4/1
with control cultures (Fig. 5 and Supplementary Material, lossoffunction.Tofurtherinvestigatethisinamodelsystem 2
Fig.S9).Thisgrowthreductionwasnotsignificantlydifferent morerelevanttopatientphenotypes,weassessedtheabilityof /33
3
fromthatresultingfromover-expressionofp.Ala1756Val(50% thefourvariantstoeffecttheaxonalgrowthofhippocampalneu- 5/6
decrease).Over-expressionofthep.Ser225Asnandp.Gly876Ser rons.Aswehavepreviouslydemonstratedandasshownabove, 21
mutantsbothresultedindecreasesingrowthof33and28%,re- thisassayisbothsensitivetoHcfc1gainandlossoffunction, 90
spectively,whichwasamodest,butsignificantlossofgrowth whichresultsineitherreductionorextensionofaxonalgrowth, 9 b
y
suppression compared with over-expression of the wild-type respectively(2).Weassayedtheeffectthatover-expressionofthe g
u
HCFC1.Theover-expressionofthep.Arg2016Trphadnoeffect HCFC1variantshasonaxonalgrowthandtheirabilitytorescue e
s
ongrowthandwasakintocontrolcells(Fig.5andSupplementary defectsassociatedwithlossofHcfc1expression.Weisolatedpri- t o
Material,Fig.S9).Thesedatasuggestthatthreeoffourofthe maryhippocampalneuronsfromE18.5embryos(asdescribed n 2
tested HCFC1 variants may cause loss of function to various above) and immediately following isolation, we nucleofected 9 M
degrees,withtheexceptionbeingthep.Ala1756Valvariant. themwithanexpressionvectorsencodingRedFluorescentPro- a
rc
tein(RFP)togetherwitheitheranemptyexpressionvectorcon- h
2
trolorexpressionvectorsencodingeitherwild-typeHCFC1,or 0
HCFC1variantsdisruptexpressionofMMACHC 1
oneofthefourHCFC1variants.Cellswereplatedoutandallowed 9
Theinvestigationsonloss-of-functionHCFC1mutationsinCblX toattachovernight.Thefollowingday,thesetransfectedcultures
revealed that HCFC1 is required for the expression MMACHC, weretransducedwithlentiviralparticlesthatencodebothEGFP
whichencodesanenzymeinvolvedinthemetabolismofcobala- andeithercontrolorHcfc1shRNA.Cellsweregrownforanaddition-
min,andwhichisalsothemostcommongeneticfactorfound al3daysinvitro,fixedandimmunofluorescentlystainedforthe
mutatedinCblC(9).WethuslookedattheabilityoftheHCFC1 axonalmarkerproteinTAU1.Axonlengthsofdual-transfected
variantstoaffecttheexpressionofMMACHC.Weagainutilized andtransducedcellsweremeasured(i.e.thoseexpressingboth
our HEK293T cells and over-expressed wild type or variant RFPand EGFP) (Fig. 7A). Over-expressionofHCFC1 in control-
HCFC1andanalysedMMACHCmRNAexpressionbyqRT–PCR. transducedcellsresultedina25%reductionofaxonallength,
Over-expressionofWTHCFC1hadnoeffectonMMACHCexpres- inagreementwithouroriginalfindings(2).Asimilar23%reduc-
sion,suggestingthatendogenouslyexpressedwild-typeHCFC1 tion was observed when the p.Ala1756Val variant was over-
binding(andfunction)isalreadysaturatedattheMMACHCpro- expressed,whiletheeffectofover-expressionoftheotherthree
moterundernormalconditions(Fig.6).Likewise,expressionof variantsresultedinlesssevereaxonallengthreductions(ranging
the p.Ala1756Val and p.Arg2016Trp had no effect. The other onlybetween4and8%)thatweresignificantlydifferentfrom
3342 | HumanMolecularGenetics,2015,Vol.24,No.12
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Figure7.HCFC1variantsdisruptaxongrowth.HippocampalneuronswereisolatedandtransfectedwithRFPexpressionvectortogetherwitheitheranemptyexpression /a
vector(control)orvectorsencodingeitherwild-typeorvariantformsofHCFC1.Followingcellattachmentinvitro,neuronsweretransducedwithlentiviralparticlesthat rtic
encodeEGFPandeithercontrolorHcfc1shRNA.(A)Representativeimmunofluorescentimagefromcontrolconditionshowingidentificationofadualtransfected(i.e.RFP le
expressing;red)andtransduced(i.e.EGFPexpressing;green)neuron(yellowarrow).NucleiwerestainedusingDAPI(blue).(B)Quantificationofmeanaxonallength. -a
b
P<0.05byStudent’st-testcomparingvalueswithinashRNAcondition(LucorHcfc1shRNA);*significantlydifferenttocontrolcondition;#significantlydifferenttowild- s
typecondition. tra
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t/2
4
/1
HCFC1wild-typeover-expression.Next,weanalysedsamples gyration malformation have been reported in CblX patients 2
/3
transduced with Hcfc1shRNA and revealed similar outcomes (9,10)andinindividualsfromfamiliesCZE168andD147thatwe 3
3
(Fig.7B).Transductionwiththisviruscauseda39%increasein describehere.Thus,embryonicbraingrowthandneuronalcon- 5/6
axonallengthcomparedwithtransductionwithacontrolvirus. nectivityareprocessessensitivetoHCFC1function(anddosage), 2
1
Thiselongatedaxonlengthwasreducedby45%whenHCFC1 andalterationstotheseprocessesmayunderlieaspectsofthe 90
9
wild-typewasre-expressedandasimilar49%reductionobser- patientneurologicalphenotypes. b
vedwhenthep.Ala1756Trpvariantwasexpressed.Re-expression Having established neural-specific effects of loss of Hcfc1 y g
oftheotherthreeHCFC1variantscausedsignificantreductions function,wesoughttoprovideevidenceinsupportofthepatho- ue
s
(i.e.comparedwithcontrolHcfc1shRNAcondition)thatrangedbe- genicityofadditionalHCFC1variantsdescribedpreviously(2), t o
tween21and24%;however,thesereductionsweresignificantly andnewlydiscovered,includinganovelvariantaffectingthe n 2
lower, compared with re-expression of the wild-type HCFC1 NLSofHCFC1.Threeofthefourvariantswetestedresultedinal- 9 M
(Fig. 7B). Together,theseresultsconfirmthataxonallength is teredHCFC1functionconsistentwithapartial,butnotcomplete, a
regulatedbyHCFC1dosageandthatthreeofthefourHCFC1var- loss-of-functioneffectwiththeexceptionbeingthep.Ala1756Val rch
iantstestedhere(i.e.p.Ser225Asn,p.Gly876Serandp.Arg2016Trp) variant.Theotherthreevariants(p.Ser225Asn,p.Gly876Serand 20
1
leadtopartialHCFC1lossoffunction. p.Arg2016Trp)allshowedreduced(butnotabolished)potency 9
comparedwithwildtypewhenassayedforeffectsonHEK293T
proliferationandneuronalaxonalgrowth.Asimilareffectwas
Discussion
observedforthep.Ser225Asnandp.Gly876Servariantswhenas-
PreviousfindingssuggestthatbothHCFC1gainandlossoffunc- sayedforanabilitytoregulateMMACHCtranscriptionlevels,but
tionmutationscancauseneurodevelopmentaldisorders.Here, notthep.Arg2016Trpvariant,whichbehavedakintowildtype
wecomplementourpreviousstudiesonHCFC1gainoffunction (andp.Ala1756Valvariant)inthisinstance.Theinabilityofthe
byreportingthediscoveryofalteredneuralcellbehavioursre- HCFC1p.Arg2016TrpvarianttoeffectMMACHCexpressionina
sultingfromHCFC1lossoffunction.Wefind,ingeneralopposite negativemannerishoweverconsistentwithitsexclusionfrom
totheeffectsofover-expression,thatmodestknockdownofHcfc1 thenucleus,whichwouldmaskitspotentialabilitytodisrupt
inNPCscausedover-proliferationandanassociatedreductionof transcription carried out by endogenous wild-type HCFC1 in
differentiation,whileknockdowninhippocampalneuronspro- thesecells.Infact,bymeasureoftheeffectonHEK293Tprolifer-
motedaxongrowth.Insupportofthesefindings,brainmalfor- ationandlocalization,thep.Arg2016Trpvariantmaybeconsid-
mations including microcephaly, macrocephaly and cortical ered the most detrimental to protein function of the four
HumanMolecularGenetics,2015,Vol.24,No.12 | 3343
variantstested.Furthermore,thetwoaffectedbrotherswiththis diseasetraitsareassociatedwithcollectionsofcommonvariants
variantbothdisplayedelevatedhomocysteinelevels,asignature foundinmonogenicdiseasegenes(25),andthatmilderneurode-
metabolicfeatureofindividualswithCblX(andCblC),andthus velopmental phenotypes (for example autism spectrum dis-
suggestive of loss of HCFC1 transcriptional activation of order, and probably mild ID) are more likely to involve
MMACHC.Interestingly,HCFC1mRNAlevelswerefoundtobeele- combinationsofrareandcommonvariants(26,27).Thatthep.
vatedinwholebloodoftheaffectedindividualsandalsointhe Gly876Servarianthasbeenidentifiedinapatientwithautism
individualwiththep.Ala477Thrmutation,familyITL1,andin mayprovideadditionalsupportofitsinvolvement(28).
lymphoblastoidcelllinesoftwoofthethreeindividualswith Ourdataindicatethatofthefourvariantswetested,threeaf-
thep.Ser225Asnmutation,familyD144.Thisfindingmaysuggest fectHCFC1proteinfunction,namelythep.Ser225Asn,p.Gly876-
thatHCFC1normallyparticipatesintherepressionofitsown Serandp.Arg2016TrpvariantsfoundinfamiliesD144,D147and
transcription.Suchanautoregulatorymechanismwouldbein CZE168,respectively.Thep.Arg2016Trpvariantdisruptedanu-
linewiththeneedtotightlyregulatedosageofHCFC1duringde- clearlocalizationsignalsequenceintheC-terminusofthepro-
velopmentandhomeostasis.ThatHCFC1bindsYY1,aknownre- tein.Ourlocalizationstudiesshowthatthisindeeddiddisrupt
D
pressorofHCFC1transcription,providesacandidatemechanism theabilityofHCFC1tobecomeenrichedinthenucleus;however, o
w
warrantingfurtherinvestigation,particularlyinlightofourpre- thattheproteinretainedsomefunctionsuggestedthatnuclear n
vious finding wherein mutation of a YY1 binding site in the importwasreduced,butnotabolished.Thep.Ser225Asnvariant loa
d
HCFC15′UTRwasassociatedwithHCFC1over-expressionand liesintheKelchrepeatdomainofHCFC1similartopreviouslyre- ed
ID(2,20).Althoughtranscriptionwaselevated,initialinvestiga- portedloss-of-functionHCFC1variants(9).Thisdomainischar- fro
tionsdidnotshowanincreaseintheoverallproteinlevelofthe acterizedasaprotein–proteininteractionmotifandhasbeen m
h
p.Arg2016Trpvariant. showntomediatebindingtotranscriptionfactors(forexample ttp
assDayess,ptihteetchoentdreibleutteiorinouosfethffeecpt.Golny8H7C6FSCer1vfaurniactnitontointhoeupractehl-l EM2LFL1Ha3nKd4EM2Fe4thaynldtraTnHsAfePr1a1s)ea)n(4d,2c9h)r.oHmCaFtCin1hreagsublaeteonrsde(ssuccrihbeads s://a
c
a
ologyofthepatientremainscomplicatedbythelikelypathogenic asanobligateco-factorofTHAP11,whichrecruitsHCFC1totarget d
e
contributionsofaMED12variant(c.3101T>G,p.Phe1034Cys)and promoters,includingthatoftheMMACHCgene(9,29,30).Thus, m
aproteintruncatingARID1Bvariant (c.2723delC,p.Pro908fs*6) thep.Ser225Asnvariantmaydisrupttheseinteractions,ashas ic.o
alsofoundintheindividual.MutationsinMED12causeXLID beenpostulatedforothervariantsofHCFC1affectingtheKelch up
(21) (OMIM: 300895 and 309520). This particular variant has domain(9).Likewise,thep.Gly876Servariantmayaffectthepro- .co
notbeenpreviouslyreportedandispredictedtobepotentially tein–proteininteractionsmediatedbythebasicdomain,which m/h
pathogenic(CADDscore=14.53);assuchitsroleinthisindivi- includes DNA-binding proteins (e.g. ZBTB17) and chromatin m
dual’s phenotype is not conclusive. In contrast, haploinsuffi- modifierssuchasSIN3A(18).Itwillbeinterestingtoidentify g/a
ciencyofARID1Binvariablycausesabroadrangeofdisorders whichpromotersandgenesareaffectedonaglobalscaleusing rtic
frommildIDtomoreseverephenotypesofCoffin-Sirissyndrome patient-derivedcelllines,tobetterunderstandthebiologyof le-a
(OMIM:135900)andcanbeassociatedwithaspectrumofclinical HCFC1andunderstandthegeneticpathwaysthatunderlieID. b
s
presentations(12,22).Becauseofthis,itisdifficulttodisentangle Inallofourassays,thep.Ala1756Valvariantbehavedaswild- tra
c
thecontributionsofHCFC1,MED12orARID1Bvariants.Unfortu- type HCFC1.While thisdoesnot support itspathogenicity,it t/2
nately,wewereunabletoobtaininformationontheaffectedin- doesnotexcludeitsinvolvementinsomefinerwayandmayre- 4/1
dividual’shomocysteineormethylmalonicacidlevels,which flectlimitationsoftheassayconditionstodetectsubtledisrup- 2
/3
couldpotentiallyidentifyCblX.Theaggregateclinicaldataavail- tionsto HCFC1 function(see below). Such a small disruption 3
3
abletousarestillconsistentwithCblX,butalsosevereARID1B- maystillbesolelyresponsibleforpathogenesis,oralternatively 5/6
associatedphenotypes,andhavesomefeaturesnotyetdescribed itseffectmaybecompoundedbycontributionsofotherrarevar- 21
9
forMED12mutations(e.g.epilepsy),andmissingsomefeatures iants(e.g.thec.356A>G,p.Gln119ArgvariantinZMYM3ofun- 0
9
normallyassociatedwithMED12mutations(e.g.hypertelorism known significance discovered in the patient) or common b
y
andconstipation).ItiscrediblethatHCFC1,ARID1BandMED12 variants(24,25). g
u
variantsmayallbecontributingtotheID;thepossibilityofmul- Asaforementioned,loss-of-functionmutationsthatalterthe e
s
tiplehitswithadditiveorindependenteffecthasbeenpreviously Kelch domain of HCFC1 have been identified in patients with t o
n
documentedandmightbeunder-ascertained(23,24).Interest- CblC-like disorder called CblX (9,10). CblC is most commonly 2
ingly,theEVSserverreportsthep.Gly876Servariantin7/10271 causedbymutationsinMMACHCandresultsinmethylmalonic 9 M
chromosomes(includingtwomaleswiththechange,amalefre- aciduriaandhomocystinuria.Thedefectcausesdecreasedlevels a
rc
quency of 1/1193). Furthermore, following completion of our ofthecoenzymesadenosylcobalamin(AdoCbl)andmethylcoba- h
2
functionalstudies,theExACdatabasebecameavailable(http:// lamin(MeCbl),whichresultsindecreasedactivityoftherespective 0
1
exac.broadinstitute.org, last accessed February 2015), which enzymesmethylmalonyl-CoAmutaseandmethyltetrahydrofo- 9
alsoreportedthep.Gly876Servariantatafrequencyof52/121298 late:homocysteinemethyltransferase,alsoknownasmethionine
chromosomes(ofwhich17arelikelytobemales,givinganap- synthesis.ThemainbiochemicalfindingsinCblCpatientswith
proximatemalefrequencyof∼1/2300).Thesedatarevealthat MMACHCmutationsareincreasedplasmahomocysteinelevel,
thep.Gly876Seroccursataverylowfrequencyinthepopulation low or normal plasma metionin level, homocysteinuria and
andsuggestthatthevariantmightbebenign;however,itisalso methylmalonic aciduria (31). Evidence from CblX patient cell
possiblethatthemaleswiththisvariantinthesedatabasesmay lines and knockdown experiments in HEK293T cells revealed
havemildtoborderlineID,whichalignedwiththehighincidence thattheseHCFC1mutationsresultedinanalmostcompleteloss
(affecting2–3%)andwidespectrumofIDisplausible,albeitunde- of MMACHC expression, suggesting almost complete loss of
terminableatthispointintime.Ourconservativeconclusion HCFC1 function (9). The patients from whom the variants we
basedonthefunctionalandgeneticdatapresentedhereistore- testedherehaveingeneralmuchmilderneurologicalpresenta-
gardthep.Gly876Servariantasalow-frequencyvariantthatpro- tionscomparedwithCblX(andCblC)individualsandconsistently,
videsanincreasedsusceptibilitytointellectualdisability.Thisis inourassays,itappearsthattheHCFC1retainedatleastsomebio-
consistent with recent evidence which reveals that complex logicalactivity,i.e.ourassayssuggestonlypartiallossoffunction
3344 | HumanMolecularGenetics,2015,Vol.24,No.12
(SupplementaryMaterial,TableS3).Furthermore,boththeindivi- likelyunderpinsaspectsofpatientphenotypes(2,33).Regarding
dualswithp.Arg2016Trpmutationhadtransientlyelevatedhomo- loss-of-functionmutations,theseverityofthephenotypeslikely
cysteinelevels,butrepeatedlynormallevelsofplasmametionin reflectstheextenttowhichthemutationsaffectHCFC1function,
andmethylmalonicacidinurine(themeasurementofAdoCbl withalmostcompletelossoffunctioncausingCblXfeaturingse-
andMeCblwasnotavailable).Normalurinemethylmalonicacid vere neurological phenotypes (e.g. retractable epilepsy, brain
levelswerealsoreportedfortheindividualwiththep.Ala477Thr malformations,severeneurocognitiveimpairment)andclinical-
variant.Thesenearlynormalbiochemicalfindingscorrespondto ly relevant deficiencies in cobalamin, whereas partial loss of
themuchmilderneurologicaldefectscomparedwithCblCpa- functionresultinginlesssevereneurologicalphenotypes(e.g.
tients,againsuggestingpartialMMACHCactivity,andonlyapar- mildID)(SupplementaryMaterial,TableS3)(9).Thecontinued
tialdefectinHCFC1function.Itremainstobetestedtowhatextent emergenceoflarge-scalesequencingpracticesinresearchand
MMACHCexpressionmaybeaffectedintheseindividuals,andthe diagnosticswilllikelyprovidefurtherinsightintothespectrum
otherindividuals,andwestudiedhere(materialsunavailableat ofphenotypicoutcomesstemmingfrombothgain-andloss-of-
thistime),andmoreso,towhatextentthemetabolismofcobala- functionmutationsinHCFC1.
D
minisaffectedinall.Suchinformationmayhavesomeclinical o
w
value,asexpressionlevelsofmutantMMACHCalleleshavebeen n
MaterialsandMethods lo
associatedwithageofonsetandseverityinCblCpatients(11). a
d
However,asCblCcausingMMACHCmutationsareallreportedas Animaluse ed
hMoMmAoCzHygCofuusn(cotriocnomispsoevuenrdelhyectoemropzyrogmouisse),di,ttshuegmgeesttasbtohlaictusynmlepss- ThisstudywasperformedunderregulationsoftheSouthAustra- from
lianAnimalWelfareAct1986,andinstrictaccordancewiththe h
tomsmaynotexist(11).ThatthemetabolicfeaturesofCblXdis- AustralianCodeofPracticefortheCareofAnimalsforScientific ttp
onreduerrolaorgeicraellapthiveneloytympeildiscmomucphamreodrewsiethveCrebl(Cincplautdieenstbsr,abinutmtahle- Purposes,2004.TheprotocolwasapprovedbytheWomen’sand s://a
formationandintractableepilepsies)suggeststhatMMACHCdefi- Children’sHealthNetwork(WCHN)AnimalEthicsCommittee cad
(Approval Number: 944/03/15). All euthanasiawas performed e
ciencyalonedoesnotexplaintheneurologicalphenotype,and m
tpinhogarttiaHnnCptFafCor1rtiniasolirlmomspasolorbtfraaHnintCfFdoCer1vtehflueonepxcmtpieroennstsm(i9o)an.yTohrfeuassdu,dlmtitiuinotnanateilougnresonlreoesgsiuicmlat--l CuitzasaeiinrnseegudfFcffeaerrcorviimnliicgtaty.hlTed(iWmiWsleooo-mmcmaeetaninto’e’nssd,aaapnnnrdeddgCnehCvailehndritryledfeenrfme’fsnoaH’rlsteewaSHlawtohssismNpsieatmtadwlie,coetrAokwdmAeenrlieanimiiodmbae--l, ic.oup.com
pmhiennodteyfipceisenincyt.heFuarbtsheenrcmeoorfet,hwehcliilneictarelaptrmeseennttaotifoneaorflyc-oobnaslae-t Australia). /hm
g
CblCpatientswithacombinatorialapproachconsistingofsupple- /a
mentationofhydroxycobalamin,betainandfolicacidprovides Generationoflentivirus rtic
someimprovementsinthevisceralandhaematologicalsymp- Constitutive3rdgenerationlentiviralvectorswereemployedas le-a
toms,theefficacyonneurologicaloutcomesisnotestablished, previously described (34–36). To generate Hcfc1-specific shRNA bs
andpatientsinvariablycontinuetoshowneurologicalandcogni- transfervectors,threeshRNAsequencespredictedtospecifically tra
c
tiveimpairmentregardlessoftimeofdiagnosisand/ortreatment targetHcfc1UTRsweredesignedusingBLOCK-itRNAidesigner t/2
initiation(32). software[Invitrogen,Carlsbad,CA,USA)].AshRNAsequencetar- 4/1
Themountingchallengeofassigningpathogenicitytolarge getedtoluciferasewasusedasacontrol(36).TheshRNAsequences 2/3
numbersofvariantsistypicallyapproachedbyemployinggenet- are T1: GCAGAAGGCAGATTGGAAAGA; T2: GGAGATAACACCCA 33
ic and statistical evidence often without in-depth functional TACTTAAandT3:GCACTTGTTTGTAGTTCATCG.Thesesequences 5/6
studiesorevendetailedclinicalassessment.Bycomprehensive wereclonedintothelentiviraltransfervectorplv-C-shRNA,and 21
9
investigationsoffourHCFC1variantsusingallabovecriteria, lentiviralparticlestocksweregeneratedandtitratedaspreviously 09
wefoundconformingevidenceforpathogenicityofp.Ser225Asn described(35,36).Transductionofneuronswasperformedateither b
y
and p.Arg2016Trp; however, some discordance was observed multiplicityofinfection(MOI)=1overnight(O/N)atDay1ofculture. g
u
withtheremainingtwovariantsstudied.Thevariantwiththe TransductionofNPCswasachievedatMOI=20usingO/Nincuba- es
highestCADDscore,p.Ala1756Val,whichisalsonotpresentin tionwithdissociatedcellsderivedfromsecondaryneurospheres. t o
n
anypubliclyavailablecontroldatasetweanalysed,behavedas Forpurificationoftransducedcells,neurospheresweredissociated 2
wildtypeinourcell-basedstudies.Conversely,thep.Gly876Ser andEGFPexpressingcellspurifiedusingFACS(BDFACSAriaIIflow 9 M
variantwhichisfoundatverylowfrequencyinnorthernEuro- cytometer;BDBioscience,SanJose,CA,USA),forfurtherculture arc
peandescentpopulationdisplayedlossoffunctioninourcellas- asneurospheres. h 2
says.Thisraisesimportantissuespertainingtotherelevanceof 0
1
suchfunctionaltestsforreachingcorrectclinicaldiagnosisand 9
CloningofHCFC1expressionconstructs
actionsthereafter.Resolutionofthesefundamentalquestions,
alsorelevanttomanyothergenesanddisorders,willinvolvedee- pCGN-HCFC1-FLvectorwasakindgiftfromW.Herr,Centerfor
perunderstandingofthefunctionofthesegenes,bettergeno- IntegrativeGenomics,UniversityofLausanne,Lausanne,Switz-
type–phenotype databases, more sophisticated models (e.g. erland(15).Togeneratemutantconstructs,fragmentscontaining
patient-derivedstemcells)orsimplymorepatientsandvariants thedesirednucleotide(s)formutagenesiswerefirstamplified
investigated.Ourworkunderlinestheimportanceofcombined andthensubclonedintopGEMTbypGEM®-TEasyVectorSys-
approachtotheresolutionoftherelevanceofgeneticvariation tems (Promega,Madison,WI,USA) using the following primer
inneurologicaldisease,involvingclinical,genetic,bioinformat- pairs:forp.Ser225Asn,F-TATGACGTGCCTGACTATGCandR-TAGG
icsandcellandmolecularfunctionalstudies. TACCAGAGGTCCTTGC; for p.GlySer876, F-AGTAGCCCACAGAT
Inconclusion,ourdataprovideadditionalsupportforthein- GAGTGGandR-CTTGAGAGGGTGGCAATGG;forp.Ala1756Valand
volvementofHCFC1inID.Together,withpreviousstudies,itis p.Ala2016Trp, F-TTATCACTTCCCCAAGAGC and R-CTCACCCT
nowapparentthatbothgainandlossofHCFC1functioncanre- GAAGTTCTCAGG.Mutagenesisreactionwasperformeddirectly
sultinchangestothebehaviourofembryonicneuralcells,which on pGEMT vector using QuikChange Multi Site-Directed