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DisruptionofthetopologicalassociateddomainatXp21.2isrelatedtogonadaldysgenesis:
Ageneralmechanismofpathogenesis
AnaPauladosSantos1,2*,JakobA.Meinel3*,CristianedosSantosCruzPiveta2,JulianaGabriel
RibeirodeAndrade4,5,HelenaFabbri-Scallet2,VeraLúciaGil-da-Silva-Lopes1,GilGuerra-
Júnior4,5,AxelKünstner6,7,FrankJ.Kaiser8,9,Paul-MartinHolterhus10,OlafHiort3,Hauke
Busch6,7,AndréaTrevasMaciel-Guerra1,4,MaricildaPalandideMello2,4,RalfWerner3,11
1DepartmentofMedicalGeneticsandGenomicMedicine,StateUniversityofCampinas
(UNICAMP),Campinas,SãoPaulo,Brazil;2MolecularBiologyandGeneticEngineeringCenter,
StateUniversityofCampinas(UNICAMP),Campinas,SãoPaulo,Brazil;3Departmentof
PediatricsandAdolescentMedicine,DivisionofPaediatricEndocrinologyandDiabetes,
UniversityofLübeck,Lübeck,Germany;4InterdisciplinaryGroupfortheStudyofSex
DeterminationandDifferentiation(GIEDDS),StateUniversityofCampinas(UNICAMP),
Campinas,SãoPaulo,Brazil;5DepartmentofPediatrics,StateUniversityofCampinas
(UNICAMP),Campinas,SãoPaulo,Brazil;6GroupofMedicalSystemsBiology,Lübeck
InstituteofExperimentalDermatology,UniversityofLübeck,Germany;7Institutefor
Cardiogenetics,UniversityofLübeck,Lübeck,Germany;8 InstituteofHumanGenetics,
UniversityDuisburg-Essen,UniversityHospitalEssen,Essen,Germany;9DZHKe.V.(German
CenterforCardiovascularResearch),PartnerSiteHamburg/Kiel/Lübeck,Lübeck,Germany
10UniversityMedicalCenterforPediatricEndocrinologyandDiabetes,Departmentof
PediatricsandAdolescentMedicineI,UniversityHospitalSchleswig-Holstein–CampusKiel,
Kiel,Germany;11InstituteofMolecularMedicine,UniversityofLübeck,Lübeck,Germany;
*bothauthorscontributedequally.
Addressallcorrespondenceandrequestsforreprintsto:RalfWerner,PhD.Departmentof
PediatricsandAdolescentMedicine,DivisionofPaediatricEndocrinologyandDiabetes,
UniversityofLübeck,23562Lübeck,Germany.Email:ralf.werner@uni-luebeck.de
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preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2020. .https://doi.org/10.1101/2020.03.25.20041418doi: medRxiv preprint
NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
2
GrantNumbers
ThisworkwasfundedbyfinancialsupportfromBundesministeriumfürBildungund
ForschungBMBF(01DQ17004),DeutscheForschungsgemeinschaft(DFG,GermanResearch
Foundation)underGermany`sExcellenceStrategy(EXC22167-390884018),Fundaçãode
AmparoàPesquisadoEstadodeSãoPaulo(#2015/04763-4)andascholarshipfrom
CoordenaçãodeAperfeiçoamentodePessoaldeNívelSuperior(ProgramadeDoutorado-
sanduíchenoExterior–PDSE-88881.135162/2016-01).
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preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2020. .https://doi.org/10.1101/2020.03.25.20041418doi: medRxiv preprint
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Abstract
Duplicationsofdosagesensitivesex-locusXp21.2includingNR0B1havebeenlinkedto46,XY
gonadaldysgenesis(GD)andtheireffectsareattributedmerelytoincreasegenedosageof
NR0B1(DAX1).Herewepresentageneralmechanismhowdeletions,duplications,
triplicationsorinversionswithorwithoutNR0B1atXp21.2canleadtopartialorcomplete
GDbydisruptingthecognatetopologicalassociateddomain(TAD)inthevincinityofNR0B1.
Ourmodelissupportedbythreeunrelatedpatients:twoshowinga287kboverlapping
duplicationattheXp21.2locusupstreamofNR0B1containingCXorf21andGKandone
patienthavingalargenewtriplicationofXp21.2asthemostlikelycauseofGD.Whole
Genomesequencinguncoveredtheexactstructuralrearrangementsoftheduplicationsand
thetriplication.ComparisonwithapreviouslypublisheddeletionupstreamofNR0B1
revealedacommon35kboverlapbetweenthedeletion,ournewlyreportedNR0B1
upstreamduplicationsandthetriplicationaswellasallothercopynumbervariations(CNVs)
atXp21.2reportedsofar.ThisoverlapcontainsastrongCCCTC-bindingfactor(CTCF)binding
siterepresentingoneboundaryoftheNR0B1TAD.AllthreeCNVsatXp21.2mostlikely
disruptthisTADboundary,whichisolatesNR0B1fromCXorf21andGKandputativelyresults
inGKandCXorf21enhanceradoptionandensuingectopicNR0B1expression.Asaresult,the
patients’transcriptomesdevelopedanintermediateexpressionpatternwithbothovarian
andtesticularfeaturesandgreatlyreducedexpressionofspermatogenesis-relatedgenes.
ThismodelnotonlyallowsbetterdiagnosisofGDdisplayingCNVsatXp21.2,butalsogives
deeperinsighthowspatiotemporalactivationofdevelopmentalgenescanbedisruptedby
reorganizedTADsalsoinotherrarediseases.
Keywords:46,XYgonadaldysgenesis,Xp21.2duplications,Xp21.2triplication,NR0B1,
enhanceradoption,topologicallyassociatingdomains,transcriptomeanalysis
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Introduction:
46,XYdifferences/disordersofsexdevelopment(DSD)withgonadaldysgenesis(GD)are
characterizedbyarangeofphenotypesfromgenitalambiguitytofemalegenitaliacausedby
variabledegreesoftesticularfailure.Geneticvariantsinmorethan20geneshavebeen
describedasmonogeniccauses,severaloftheseinadose-dependentmanner(Audietal.,
2018).AmongsuchgenesisNR0B1(NuclearReceptorSubfamily0,GroupB,Member1;also
knownasDAX1),locatedwithinadosage-sensitive160kbspanningsexreversal(DSS)region
atXp21.2(Bardonietal.,1994).Sinceitsfirstdescriptionseveral46,XYGDpatientswith
duplicationsincludingtheentireNR0B1havebeenreported(FigureS1,Supporting
Information)(Barbaroetal.,2008;Barbaro,Cook,Lagerstedt-Robinson,&Wedell,2012;
Barbaroetal.,2007;Dongetal.,2016;Garcia-Aceroetal.,2019;Ledigetal.,2010;Whiteet
al.,2011).Inmice,Nr0b1expressionstartsinthegenitalridgeofbothsexesatthesame
timepointastheexpressionofSry(sexdeterminingregionY),butisdownregulatedinthe
developingtestisandpersistsintheovary(Swain,Zanaria,Hacker,Lovell-Badge,&
Camerino,1996).IthasbeenshownthathighexogenousNr0b1expressionintransgenic
miceretardstestisformation.CoexpressionwithaweakSryalleleevenresultedincomplete
sexreversal(Swain,Narvaez,Burgoyne,Camerino,&Lovell-Badge,1998)resemblingthe
NR0B1duplicationsinhumansthatcause46,XYGD(OMIM#300018).Thus,NR0B1isthe
mostplausiblecandidategenefor46,XYGDintheDSSregion.Inhumans,NR0B1inactivating
mutationscauseX-linkedcongenitaladrenalhypoplasia(AHC)withhypogonadotropic
hypogonadism(OMIM#300200),butboyshaveintacttesticulardevelopmentatbirth.
Nr0b1isknowntodirectlyrepressSox9(Sry-box9)expressionratherthanexpressionof
upstreamtargets,suchasSf1(steroidogenicfactor1;alsoknownasNR5A1)orSry,whose
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expressionremainsunchangedunderNR0B1overexpression(Ludbrooketal.,2012).Sox9is
importantforSertolicelldifferentiationanditsactivationisSf1dependent(FigureS2,
SupportingInformation)(Ludbrooketal.,2012).NR0B1exertsitsanti-testiseffectthrough
disruptionofboth,SF1DNA-bindingandSox9enhanceractivationinadose-dependent
manner(Ludbrooketal.,2012).However,whilehighNr0B1expressionisincompatiblewith
testiculardevelopment(Swainetal.,1998)itislaterrequiredforpreserving
spermatogenesis(Yu,Ito,Saunders,Camper,&Jameson,1998)implyingititselfmustbe
precicelyregulatedinthetestis.
Despiteconsiderableadvancesintheunderstandingofsexdevelopment,thegenetic
etiologyofmany46,XYGDpatientsstillremainunclear(Audietal.,2018;Bashamboo,
Eozenou,Rojo,&McElreavey,2017).Besidesunidentifiedgenesinthegonadal
developmentalpathway,thismaybeduetoaneglectofnon-codingandregulatory
elementsinthemolecularanalysisofthesepatientssofar.Wholegenomesequencing
(WGS)transcendsthisparadigmbyprovidingdetaileddataonthecompletepatientgenome.
Herewedescribethree46,XYpatientswithGDhavebeenraisedasfemalesandforwhom
wedidnotfindanyprotein-codingvariantsinthegenomeconsideredaspathogenicforthe
phenotype.Still,atranscriptomeanalysisofgonadaltissuerevealedexpressionofbothtestis
andovaryrelatedgenes,suchasSOX9andFOXL2,respectively,andshowedagreatly
reducedexpressionofspermatogenesispathways(FigureS3,SupportingInformation).
Remarkably,allthreepatientscarryXp21.2copynumbervariations(CNVs)witha287kb
overlappingregionupstreamofNR0B1.ComparisonwithapublisheddeletionatXp21.2
(Smyketal.,2007)narrowedthisregionto35kb.Basedonpubliclyavailablechromatin
immunoprecipitation(ChIP)andchromatinconformationcapture(3C,Hi-C)data(Y.Wanget
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al.,2018)weproposethatthiscommonregionincludesaboundaryelementofa
topologicallyassociatingdomain(TAD).Thegenomicduplicationsorthetriplicationandthe
deletionupstreamofNR0B1disruptsthisTADandtheassociationofthegenesandtheir
specificregulatoryelementstherein.ThiswouldresultinupregulationofNR0B1expression
whichcausessuppressionofSOX9,disruptingSertolicelldifferentiationandsuccessful
testis-development(Chaboissieretal.,2004).
PatientsandMethods
EditorialPoliciesandEthicalConsiderations
ThisstudywasapprovedbylocalEthicsCommitteesoftheUniversitiesofCampinas(CAAE
24972513.5.0000.5404)andLübeck(AZ17-219).Writteninformedconsentwasobtained
fromthepatientsand/ortheirparents/legalguardians.
Patients
ThreeunrelatedpatientsfromBrazilandGermanywereinvestigatedinthestudy.
Patient1(P1):ThepatientwasreferredtotheDSDclinic,StateUniversityofCampinasat
ageof5days,duetogenitalambiguity.Shewasbornattermafteranuneventfulpregnancy
withbirthweightof3,410gandalengthof49cm.Shehada0.5-cmphallus,asingle
perinealopening,partiallyfusedlabioscrotalfoldsandnon-palpablegonads(External
MasculinizationScore-EMS=4)(Ahmed,Khwaja,&Hughes,2000).Abdominalultrasound
revealednoabnormalitiesandgenitographyshowedaurogenitalsinus.Thekaryotypewas
46,XYandfluorescenceinsituhybridization(FISH)displayedno45,Xcellline.Laparoscopy
showedabsenceofuterus,likewisetheleftgonadaltissuewasabsentandtherewasaright
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dysgenetictestiswithsomeareasoffibroustissuesurroundingimmatureseminiferous
tubuleswithoutspermatogonia.Whenshewas1monthold,hormonalevaluationrevealed
highFSH(24IU/l;normalrange:1.5to12.4IU/l)andLH(10IU/l;NR:1.7to8.6IU/l)levels
andlowtestosterone(0.2ng/ml;NR:2.86to8.10ng/ml),suggestiveoftesticularfailuredue
topartialGD.
Patient2(P2):FirstpresentationintheUniversityDSDcenterinKielandLübeckoccurredat
theageof17yearsand2monthsduetoprimaryamenorrheaandpubertaldelay.Thegirl
previouslyhadadysgerminomawhichwasremoved3monthsearlier.Tannerstageswere
B4,P4-5.However,breastdevelopmentstartedonlyattheageof16years,afewmonths
beforeclinicaldiagnosisofthedysgerminoma.Externalgenitaliawerecompletelyfemale
withoutclitoromegalyandauteruswithtubularconfigurationwaspresent.Thekaryotype
was46,XY.HormonalevaluationrevealedbasalLH53IU/landFSH94.3IU/lincreasingto
200IU/land128IU/l,respectively,30minfollowing60µg/m2GnRHi.v..Plasmaestradiol
was9pg/ml(prepubertalforgirls)andtestosterone123ng/dl(mid-pubertalformales)
(bothdeterminedbyLC-MS/MS)(Kulle,Riepe,Melchior,Hiort,&Holterhus,2010).Plasma
AMHwas0.71µg/landthereforeextremelylow.Therefore,theclinicaldiagnosisofGDwas
established.
Patient3(P3):
Thispatientwasbornattermafteranuneventfulpregnancyandgenitalstatuswas
describedasunequivocalfemale.Thefamilyhistoryisunremarkable,withthreehealthy
siblings.Becauseofmuscularhypotonia,achromosomalanalysiswasinitiatedandrevealed
a46,XYkaryotype.Onultrasound,asmallprepubertaluteruswasseen,butthegonadswere
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notvisualized.HormoneanalysisshowedaprepubertalstatuswithinhibinBbelowthe
thresholdandanti-Mullerianhormoneat0.42µg/l,whichwasconsideredlowforage,
compatiblewithaclinicaldiagnosisofgonadaldysgenesis.Alaparoscopywasperformed
andthegonadaltissueremoved.Thehistologyrevealedagonadoblastomaoftheright
gonad,whiletheleftsidewaspurelystromaltissue.
Multiplexligationdependentprobeamplification(MLPA)
ToidentifyDNAgainsandlosses,multiplex-ligationdependentprobeamplification(MLPA)
assayswereperformedforP1usingtwodifferentkits:SALSAMLPAkitP185-C1Intersex
probemixandSALSAMLPAP334-A2GonadalDevelopmentDisorderprobemix(MRC-
Holland,Amsterdam,TheNetherlands).TheMLPAassaywascarriedoutaccordingtothe
standardprotocolsuppliedbyMRC-Holland.Datawereanalysedbycomparisonofthepeaks
obtainedfromthetestedsamplesandthosefromcontrolsusingthesoftwareCoffalyser.NET
(MRC-Holland,Amsterdam,TheNetherlands).
Chromosomalmicroarrayanalysis(CMA)
Chromosomalmicroarrayanalyseswereperformedusingtwodifferentplatforms.DNA
samplesfromP1andhermotherwerehybridisedontotheGeneChipCytoScan®750KArray
Kit(AffymetrixInc.,SantaClara,California,USA)andprocessedasrecommendedbythe
manufacturer.ArraydatawereanalysedusingChromosomeAnalysisSuite(ChAS)
(Affymetrix®).Standardanalysisadoptedthefollowingparameters:markers>25for
deletion;>50forduplication,withoutfiltersizeforcopynumbervariations(CNVs).DNAof
P2andP3washybridizedtoanAgilent180Kcomparativegenomichybridizationarray
(aCGH)(AgilentTechnologies,Inc)andcomparedtoapooledsampleof10normalmales.
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DatawereanalysedusingCytoGenomicsSoftwareversion4.0.2.21(Agilent).CNVsofboth
patientswereanalysedusingtheDatabaseofGenomicVariants(DGV,Version
CNV_DGV_hg19_v4,Toronto,Canada).
WholeGenomesequencing(WGS)
WGSwasperformedtoidentifydeleteriouspointmutationsandindelsaswellasstructural
variationsandtofinemapthebreakpointsoftheduplicationsandtriplicationatXp21.2
observedinthethreepatients.Sequencinglibrarieswereconstructedfrom1.0µgDNAper
sampleusingtheTruseqNanoDNAHTSamplepreparationKit(Illumina,USA)followingthe
recommendationsofthemanufacturer.GenomicDNAwasrandomlyfragmentedtoasizeof
approximately350bpbyCovariscracker(Covaris,USA).DNAfragmentswereblunted,A-
tailedandligatedwiththefull-lengthadapterforIlluminasequencingwithfurtherPCR
amplification.LibrarieswerepurifiedusingAMPureXP(BeckmanCoulter,USA),analysedfor
sizedistributiononanAgilent2100Bioanalyser(AgilentTechnologies)andquantifiedbyq-
PCR.PairedendsequencingoflibrarieswasperformedonIlluminaHiSeqplatforms
(Illumina).Persamplemorethan90GBofrawdatawereobtained,resultinginanaverage
genomicreaddepthof30x.
Bioinformatics
SequencingreadsweremappedtohumanreferencegenomeversionGRCh37/hg19using
Burrows–WheelerAligner(BWA)(Li&Durbin,2009).Resultingmappingfileswerescreened
forduplicatedreadsapplyingPicardtoolsMarkDuplicatesversion1.111(Picard:
http://sourceforge.net/projects/picard/).Split-readsanddiscordantpaired-endalignments
wereextractedusingSAMtoolsversion0.1.18(Li&Durbin,2009).SNPsandInDelswere
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calledusingHaplotypeCallerasimplementedinGenomeAnalysisToolkit(GATK)version
3.8.0(DePristoetal.,2011)withstandardparameters.Detectionofstructuralvariations(SV)
wasperformedusingDELLY(Rauschetal.,2012).Variationswereannotatedusing
ANNOVAR(K.Wang,Li,&Hakonarson,2010).Allchromosomalpositionsinthepaperare
accordingtoGRCh37/hg19.
BreakpointSequencing
Primersateitherendoftheconstructedbreakpointsequencesweredesignedusing
LasergenePrimerSelect(DNASTAR,Wisconsin,USA).Optimalannealingtemperaturewas
determinedthroughgradientPCRandelectrophoresis.Consequently,PCRsetupwas35
cycleswith30sec.at95°Cfordenaturation,30sec.atprimerspecifictemperature(TableS2,
SupportingInformation)forannealingand1min.extensionat72°C.SangerSequencingof
ampliconswasperformedona3130GeneticAnalyzer(AppliedBiosystems,FosterCity,
USA).
TranscriptomeAnalysis
Transcriptomeanalysiswasperformedfrom10slices(10µm)offormalinfixedparaffin
embedded(FFPE)samplesofthegonadsofpatients1and2.Librarypreparationwas
performedemployingtheTruSeqstrandedTotalRNAkit(IlluminaInc.),andsequencedon
anIlluminaNovaSeqplatform(150-nucleotidepaired-endreads),obtaining98and63million
readsforpatient1and2,respectively.Fastqreadswerepseudo-alignedtotheEnsembl
cDNAsequenceGrch38transcriptomeassemblyinversion96usingkallisto(Bray,Pimentel,
Melsted,&Pachter,2016).GenetranscriptreadcountswereaggregatedtoEnsemblGene
IDsforfurtheranalysis(Soneson,Love,&Robinson,2015)andgeneswithanexpressionless
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thanonetranscriptpermillion(TPM)ineithersamplediscarded,resultingin13005
expressedgenes.
Patienttranscriptomesfromexpressedgeneswerecomparedtotestisandovarytissue
samplesfromtheGTExDatabase(Carithersetal.,2015)andthedatasetE-MTAB-6814from
ArrayExpress(Atharetal.,2019),ahumanRNAseqtime-seriesofthedevelopmentofseven
majororgans.
Tocomparetranscriptomesimilarity,weperformedadimensionalityreductionusingt-
distributedstochasticneighborembedding(t-SNE)(vanderMaaten,2014)asimplemented
intheRlibraryRtsne(version0.15)(Krijthe,2015).Theanalysisisbasedon641samplesand
5000randomlyselectedgenes,expressedinbothpatienttissues.
DifferentialpathwayactivitybetweensampleswasdeterminedbyaGeneSetVariation
Analysis(GSVA)(Hanzelmann,Castelo,&Guinney,2013),whichcalculatestherelative
enrichmentofgenesetsacrossindividualsamplesusinganon-parametricapproach.For
pathwayandupstreamtranscriptionfactoractivityweusedthehallmarkgenesetcollection
(Liberzonetal.,2015)andtheTF-targetrelationshipfromSchachtetal(2014)(Schacht,
Oswald,Eils,Eichmuller,&Konig,2014).Theenrichmentscoresweretestedforsignificant
differencesusingamoderatedt-testpertimepoint(Ritchieetal.,2015).
Results
Patient1
CMArevealeda≈277kbduplicationatXp21.2[30,580,693-30,857,187]thatwassupported
byMLPA(FigureS4,SupportingInformation).Analysisofthemothershowedthesame
duplication.WGSconfirmedthisstructuralvariation(SV)inthepatientshowinga297kb
duplicationofGK(glycerolkinase),CXorf21(chromosomeXopenreadingframe21)andpart
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ofTAB3(TGFbetaactivatedkinase1bindingprotein7,MAP3K7).Duplicationborderswere
apparentthroughincreasedread-depthandsplitreadsmapping297kbapart.Pairedsplit
readswereextractedandalignedtogenerateacontinuoussequenceofthebreakpoints
showingatandemduplication,whereintron9ofTAB3ismergedtothedownstreamregion
ofCXorf21bya27bpinsert(Figure1).Thesequencesatthebreakpointwereconfirmedby
PCRandSangersequencing.OnlytwoheterozygousmissenseSNVswerefoundinprotein
codingregionsofgenesrelatedto46,XYDSD(TableS1,SupportingInformation).However,
heterozygouspathogenicvariantsinbothgenes(STAR/POR)wereexcludedasputative
causeofsexsteroidbiosynthesisdeficiency.
Patient2
WGSrevealedtwomajorduplicationsandtwosmalldeletionsatXp21.2;oneduplicationof
389kbmapstoaregiondownstreamofNR0B1containingtheMAGEB(MAGEfamilymember
B)genes1-4andapartofIL1RAPL1(interleukin1receptoraccessoryproteinlike1).The
secondduplication(447kb),containingCXorf21andGKaswellasthe3’-partofTAB3,maps
upstreamofNR0B1.Betweenthetwoduplicationstwosmalldeletionsof2.7kb(DeletionI)
and2.2kb(DeletionII)flankaninvertedregionof1.2kb(Figure2;FigureS5,Supporting
Information).AsinP1,splitreadpairsatthebordersofthefourcopynumbervariations
(CNVs)wereutilizedtoconstructcontinuousbreakpointsequencesandthusdetermine
orientationandlocationoftheinsertedduplications.Bothduplicationsareinserted
upstreamofNR0B1asdescribedinFigure2.Notably,the447kbduplicationencompassing
CXorf21,GKandtheTAB3fragmentisinsertedproximaltoNR0B1inaninvertedposition.
The389kbduplicationofMAGEB1-4andtheIL1RAPL1fragmentwasinsertedfurther
upstreaminthesameorientationasthereferencesequence.Breakpointsequenceswere
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confirmedbySangersequencing(Figure2)andcopynumbergainsandlossespredictedfrom
WGS,wereconfirmedbyaCGH(FigureS5,SupportingInformation).WGSoftheparents
establishedthattheSVwasinheritedmaternally.Furthermore,onlyonerarevariant
(MAF<0.01)ofunknownsignificancewasfoundintheknownDSDcandidategeneZFPM2
(zincfingerprotein,FOGfamilymember2).AlthoughZFPM2variantsareassociatedwith
abnormalitiesintestisdetermination,theaveragepopulationfrequencyofthisSNV
(dbSNP:rs202217256)is0.004acrossallpopulationsand0.006intheNon-FinishEuropean
populationaccordingtotheExAcBrowser(Leketal.,2016),whichindicatesarare
polymorphism(TableS1,SupportingInformation),ratherthanarelevantpathogenicvariant
leadingtoGD.
Patient3
P3harborsaXp21.2triplicationinitiallyidentifiedthroughqPCRcopynumberdetection
(Meinel,Dwivedi,Holterhus,Hiort,&Werner,2019).IncontrasttoP1andP2thispatients
CNValsoincludestheNR0B1gene.AnaCGH(FigureS6,SupportingInformation)
characterizedthesizeofthetriplicationtobe≈1,22Mb,whichwaslaterrenderedmore
preciselybyWGStobe1,24Mb.ThetriplicationincludesthegenesMAGEB1-4,NR0B1,
CXorf21,GK,TAB3andpartofIL1RAPL1.Thetriplicatedsegmentsarearrangedintandemto
theirtemplateandareseparatedbya49bpinsert(ChrX:31,258,118-31,258,166)(FigureS7,
SupportingInformation).TheinsertoriginatesformtheDMDgene,whichislocatedtothe
centromericsideoftheCNV.AnalysisofexomedataofknownDSDcandidategenes
revealedheterozygousmissenseSNVs,whichallshowedinterpretationsasbenignorlikely
benigninClinVar,exceptforoneinEstrogenReceptor2(ESR2)(TabeleS1,Supporting
Information).TheclinicalsignificanceofthisSNV(dbSNP:rs367855747)hasnotbeen
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reportedsofar,howeverrecentlyhomo-andheterozygousESR2mutationshavebeen
describedinthecontextof46,XYpartialandcompleteGD(Baetensetal.,2018).Theeffect
ofthisparticularheterozygousSNVremainsunclear,butinthecontextofwidelyagreed
associationofNR0B1copynumbergainsand46,XYDSDthisremainsasubordinatefactorin
theetiology,ifatall.
AnalysisofTopologicallyAssociatingDomains
SVsaffectingboundariesoftopologicallyassociatingdomains(TADs)havebeenshownto
alterthearchitectureandenhancer-promoterinteractionswithinTADswitheffectson
development(Ibn-Salemetal.,2014;Letticeetal.,2011;Lupianezetal.,2015)and
oncogenesis(Hniszetal.,2016;Weischenfeldtetal.,2017).WeanalyzedtheTADsinthe
NR0B1regionidentifiedbycaptureHi-CfromvarioushumancelllinesprovidedbythePenn
State3Dgenomebrowser(Y.Wangetal.,2018).Mostcelllinesshowaclear
compartmentalizationofNR0B1intoaTAD(referredtohereafterasNR0B1TAD)thatis
separatedfromtheenhancerelementsoftheCXorf21andGKgenes(FigureS8,Supporting
Information).
Whencomparingthetwoduplicationsandthetriplicationobservedinourpatientswitha
previouslypublished257kbdeletionintheNR0B1upstreamregion(Smyketal.,2007),also
associatedwith46,XYGD,thereisa35kboverlapregionamongallfour(FigureS1,
SupportingInformation).ThisregioncontainsastrongbindingsiteforCTCF(CCCTCbinding
factor),RAD21andSMC3(structuralmaintenanceofchromosomes3)(FigureS9,Supporting
Information),whichareinvolvedinchromatinextrusioncomplexesandcontactdomain
formationandarecharacteristicofTADboundaries(deWitetal.,2015;Sanbornetal.,
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15
2015).Thus,thebindingsitesinintron2ofCXorf21mostprobablycorrespondtotheNR0B1
TADboundarysitethatisobservedinmostcelltypes.
TranscriptomeAnalysisofFFPEGonadalTissue
Wecompared thegonad transcriptomes frompatient1and2 to testis (n=156)andovary
tissue(n=133)transcriptomesthatwereobtainedfromtheGTExprojectandahumanRNA-
seqtime-seriesfromArrayExressscontainingtissuetranscriptomesfromsevenmajororgans
(brain(n=66),cerebellum(n=59),heart(n=46),kidney(n=37),liver(n=50),fetalovary(n=18),
fetaltoinfanttestis(n=29)andteenagertooldtestis(n=10))acrossdifferentagesfromfetal,
infant,andteenagertoadultandseniorage.Lowly-expressedgenes(TPM<1)inthepatients’
samples were discarded, leaving 13005 common genes. A t-SNE analysis reveals a strong
tissue-specific clustering of all transcriptomes, independent of their experimental origin
(Figure 3). Interestingly, fetal and young ovary and testis samples (pink and purple dots)
clustertogether,whileadultovaryandtestissamples(redandyellow/orange)significantly
differ in theirgeneexpressionandclusterdistinctly fromanyotherorgan.Thesamplesof
patient1and2aresituatedbetweentheadultovaryandtestissamples,yetclosetopatient
samplesthatwerereportedbytheGTExprojectashavinglowspermatogenesis.
FigureS3(SupportingInformation)depictsboxplotsofseveralsexdeterminationgenesfrom
theGTExovaryandtestissamplesusedinFigure3andthetwopatients.Obviously,allgenes
aredifferentiallyexpressedinthetwotissues,butalsobetweenthepatientsandtheovaryas
wellas the testis samples. Forexample, the forkhead transcription factorFOXL2,which is
essential for ovary differentiation and maintenance, repressing the genetic program for
somatictestisdetermination(Boulangeretal.,2014;Uhlenhautetal.,2009),ishighlyspecific
toovariantissue,yetitisexpressedatintermediatestrengthinthetwopatientsampleswith
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the expression in P2 exceeding that of P1. In contrast, theDoublesex andMab-3 Related
Transcription Factor 1 DMRT1 is a transcription factor playing a key role in male sex
determination and differentiation by controlling testis development and Sertoli cell
maintenance(Ledig,Hiort,Wunsch,&Wieacker,2012;Matsonetal.,2011)isexpressedin
testis,butnotinovariantissue.Yet,thepatients’gonadsexpressthisgeneatanintermediate
level.
ThisintermediategeneexpressionstateisreflectedbyGeneSetVariationAnalysisusingthe
hallmarkgenesetsfromtheMSigDB(Liberzonetal.,2015)on6randomlyselectedtestisand
ovary tissues as well as the median tissue expression values from GTEx and the patient
samples.Theanalysis showshowthe latterhaveacquiredexpressionprograms fromboth
tissuesandclusterinbetweentheovaryandtestissamples.Themostactivepathwayintestis
is spermatogenesis. This pathway is not expressed in the ovary samples, yet low, but
significantly higher in the patients. Interestingly,MYC signaling is present is both sample
groups, but completely absent in the two patient samples (Figure S10A, Supporting
Information).Thelatterfindingissupportedbycomparingtheputativeactivityofupstream
transcription factors.While the patient samples share activity from both ovary and testis
specificTFs,thereisalowactivityofRFX2andE2F6(FigureS10B,SupportingInformation).
RFX2isaasakeyregulatorofspermatogenesisthroughregulationofgenesrequiredforthe
haploidphaseduringspermiogenesis(Kistleretal.,2015),whileE2F6iscriticallyinvolvedin
cellcycleprogression(Giangrandeetal.,2004).IncombinationwithreducedMYCpathway
activity, this might hint at reduced cell proliferation, which is indeed the case based on
Reactomecellcyclepathways(FigureS10C,SupportingInformation).
Discussion
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Becauseallknowngeneticcausesof46,XYGDwereexcludedbyWGS,theXp21.2CNVs
remainastheonlylikelycauseidentifiedinourthreepatients.TheoverlapoftheNR0B1
upstreamduplicatedregioninP1&P2spans287kb(FigureS11,SupportingInformaton)and
isalsocontainedinthetriplicationofP3.The287kbregionincludesthegenesGKand
CXorf21togetherwiththeirrespectivepromoterandpredictedenhancerregionsaswellas
the3´-endofTAB3(FigureS12,SupportingInformation).Toourknowledge,nosimilarcase
withaXp21.2duplicationexcludingNR0B1orXp21.2triplicationincludingNR0B1hasbeen
describedinpatientswith46,XYGDsofar.
Inrecentyearstheroleofthree-dimensionalchromosomestructuresandtheirorganization
inTADshavemovedintothefocusofgenomeresearch(Dixonetal.,2012).TADsarenon-
randomlyorganizeduptomegabase-sizedlocalchromatininteractiondomainsthatare
thoughttoguideregulatoryelementstotheircognatepromotersandprovideinsulationfor
non-cognategenes.Thesedomainscanbestableacrossdifferentcelllinesandconserved
acrossspecies(Dixonetal.,2012).
TADformationgenerallyrequirestwocorrespondingCTCFmotifs-oneateitherendofthe
loopformingdomain.Ithasbeenshownthatthesemotifsarepredominantlyorientedina
convergentmanner(deWitetal.,2015;Guoetal.,2015;VietriRudanetal.,2015)andthat
eliminationorchangeofCTCForientationcanleadtoremovalorshiftofTADboundaries
(Lupianez,Spielmann,&Mundlos,2016;Sanbornetal.,2015).Thetwoduplications
reportedinthisstudyandthedeletionreportedbySmyketal.(2007)havea35kb
overlappingregionharboringaCTCFmotifinproximitytotheidentifiedNR0B1TADborder
(FigureS8andFigureS9,SupportingInformation).ThisCTCFisorientatedtowardsNR0B1,
whichfitswellintoaconceptofitbeingthecentromericendpointoftheNR0B1TAD(Figure
S13,SupportingInformation).
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Hi-CdatasuggestaboundaryregionbetweentheNR0B1TADandtheadjacentcentromeric
TAD(FigureS8,SupportingInformation)(Y.Wangetal.,2018).Inmostcelllinesthis
boundaryregionincludesExon1and2ofCXorf21andGKtogetherwiththeirrespective
promotersandpredictedenhancers(FigureS10,SupportingInformation).Thus,NR0B1is
shieldedfromthesenon-cognateenhancersbyjustasingleCTCF.ThediscussedSVsmight
enableenhancerstobypasstheinsulatingeffectofthisCTCFbindingmotif(FigureS13,
SupportingInformation)andallowectopicinteractionswithNR0B1,aprocessknownas
enhanceradoption(Letticeetal.,2011)throughpositioninginacommonloopforming
domain(Figure4).ThiscouldleadtoupregulationoraberrantexpressionofNR0B1,which
inconsequenceresultsindecreasedSF1-mediatedSOX9expressionandimpairedtesticular
development(FigureS2,SupportingInformation).Thisisinaccordancewiththefactthatall
threepatient’sSVsdescribed,thedeletionreportedbySmyketal.(2007),aswellas
previouslypublishedduplicationscontainingNR0B1andCXorf21hadmaternalorigin
(Barbaroetal.,2012;Barbaroetal.,2007;Smyketal.,2007).
ThetandemduplicationinP1insertsanadditionalleftorientedCTCFmotif(FigureS13,
SupportingInformation).However,accordingtothecohesin-dependentloopextrusion
modelfortheformationofTADs,onlyoneofthesetwoCTCFmotifscaninteractwiththe
oppositelyorientedCTCFthatdefinesthetelomericNR0B1TADboundary(Fudenbergetal.,
2016;Sanbornetal.,2015).Loopextrusionisbelievedtobeadynamicprocess(Raoetal.,
2017),wherebystructuralmaintenanceofchromosomes(SMC)complex’s(cohesin)
translocatealongthechromosomesinanATPdependentprocessproducingexpanding
loopsofchromatin.Thisprocessbringstogetherincreasinglydistantgenomiclociand
continuesuntilthecohesincomplexeitherdissociatesormeetsabarrierintheformofa
convergentCTCF.Ifonesideofthecomplexencountersabarrieritisassumedthat
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chromatinextrusioncontinuesontheotherenduntilequallyaconvergentCTCFismet
(Fudenbergetal.,2016;Goloborodko,Marko,&Mirny,2016).Thoughthisisadynamic
processofcohesinloadinganddissociationthereisanincreasedtimeofresidenceatTAD-
boundaries(Szabo,Bantignies,&Cavalli,2019).
ThearrangementinP1givesrisetotwoputativeTADconformationswitheitherthe
proximalordistalNR0B1orientedCTCFconstitutingthecentromericNR0B1TADboundary
(Figure4andFigureS13,SupportingInformation)withincreasedtimeofcohesinresidence
atboth.
InsituationswheretheNR0B1TADisformedwiththedistalleftorientedCTCF,it
encompassesthewholeduplicatedsegmentwhichcouldallowthenon-cognateenhancers
tointeractwithNR0B1andmayresultinenhanceradoption(Figure4,P1a).
TheidentifiedinversioninP2bringsGKandCXorf21,alongwiththeircorresponding
putativeenhancers,intotheNR0B1TADwithoutitsinvertedCTCFinterferingwiththe
existingborders(Figure4andFigureS13,SupportingInformation).Thepreviouslyoutlying
non-cognateenhancersareputativelyinsidetheNR0B1TADandmayinteractwiththe
NR0B1promoter(Figure4).
DeWitetal.(2015)alsostudiedthedeletionofindividualCTCFmotifs,leavingthe
partneringCTCFmotifabandoned.ThoughboundbyCTCFandcohesin,theseformerly
partneringCTCFmotifsdidnotengageinnewloopswithotherCTCFs.Followingfromthis,
thedeletionreportedbySmyketal.(2007)mostlikelyleadstoadisruptionoftheTAD
betweentheconvergentsitesratherthantheformationofanewTADtowardsadifferent
CTCF.SeveralgroupshavestudiedthedeletionofTADboundariesandindividualCTCFsites
indifferentcellsandorganismsinrecentyears(Gomez-Marinetal.,2015;Narendraetal.,
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20
2015;Noraetal.,2012;Sanbornetal.,2015)showingthattheseresultinectopicinteraction
outsidethepreviousTADconformation.Accordingtoourmodel,TAD-conformationis
confinedtoonearrangementwithincreasedtimeofresidenceinP2andinthepatient
reportedbySmyketal.(2007).ThedifferenceintheputativeDNAtargetstructuresandthat
probabilityofproduceingaTADincludingboth,NR0B1andtheectopicenhancers(Figure4)
couldhelpexplainthedifferencesinphenotypeofthepatients.IncontrasttoP2,P3andthe
patientreportedbySmyketal.(2007),thephenotypeofP1islesssevere.Theabsenceofa
uterusinP1impliesthepresenceofSertolicellswithintactproductionofanti-Müllerian
hormone,whiletheothertwopatients(P2andtheonereportedbySmyketal,2007)
resembleahigherdegreeofaccordanceinphenotype,asbothpresentedasmalluterusand
lowestrogenswithlateonsetofbreastdevelopment.
InallthreepatientswithcompletegonadaldysgenesispresumedTAD-conformations
bringingNR0B1andthenon-cognateenhancersintoacommondomain.Thisisincontrastto
P1,displayingpartialgonadaldysgenesis,wheretheduplicationallowsapartialnormalTAD
conformation(FigureS7,SupportingInformation).Accordingtotheloopextrusionmodela
multiplicationofNR0B1andtheinsulatoratCXorf21aswellastheCXorf21andGK
enhancersaspresentinP3,oneoftheduplicatedCXorf21insulatorslosesitsfunctiondueto
thelackofanopposinginteractionpartner,whilstNR0B1andenhancersmaintaintheir
functionwithrespecttothephenotype.Therefore,itispossiblethatthismodeldescribesa
generalprocessbeyondthespecificgeneticrearrangementsofthethreepatientspresented
hereandthedeletionreportedbySmyketal.(2007)andwouldsimilarlyapplytoall
previouslyreportedXp21.2duplications(Barbaroetal.,2008;Barbaroetal.,2012;Barbaro
etal.,2007;Dongetal.,2016;Garcia-Aceroetal.,2019;Ledigetal.,2010;Whiteetal.,
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21
2011).Itisofnote,thatP3alsoshowsmuscularhypotoniapossiblyhintingataregulatory
effectofthetriplicationontheexpressionoftheDMDgene.
ThephenotypeofXp21.2duplicationshaspreviouslybeenassociatedwithdosageeffectsof
theduplicatedgenes(Barbaroetal.,2007;Bardonietal.,1994).Recentadvancementsinthe
analysisandunderstandingofDNAconformationhighlighttheimportanceofintactTADsfor
unalteredenhancer-promoterinteractions(Frankeetal.,2016;Lupianezetal.,2016).With
ournewlydiscoveredcases,itisnotNR0B1itselfastheunifyingcauseinallXp21.2copy
numbergainsassociatedwith46,XYGD,butinsteaditisthe35kbupstreamregion,whichis
presentineveryreportedcasethusfar(Figure1,SupplementalInformation).Thisunderlines
thenecessitytoreevaluateallpreviouslyreportedXp21.2duplications,asthefundamental
mechanismmightbeadifferentonethenpreviouslyassumed.
Further,knowingthepositionandorientationofSVsalongsidethenewarrangementof
enhancersandgenescouldbecomeincreasinglyrelevantforunderstandinggenotype-
phenotyperelationship.Thisunderscoresthepotentialdiagnosticpowerofwholegenome
sequencing,whichallowsaccuratecharacterizationofSVsthroughinterrogationof
individualreadsatbreakpointsasshowninthisstudy.
Asaresultofthegeneduplication,thetranscriptomesofthetwopatientsobtainan
intermediategeneexpressionstatebetweenovaryandtestissamples.Geneexpression
analysisclearlyclustersadultovarianandtesticulartissuesapartandseparatefromother
organtissues.Thetwopatientsamplesclusterinbetweenthetwotissues,yetcloserto
testisandparticularlyclosetopatienttissueswithlowspermatogenesis.Thisinteremediate
stateisfurthercorroboratedbytheexpressionofsexdeterminationgenes(FigureS3,
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SupportingInformation).ParticularlyFOXL2andDMRT1,whicharehallmarksofovarianand
testiculartissue,areexpressedatanintermediatelevel,whichmightbeeitheracauseorthe
effectofderegulationofmultiplesexdeterminationregulatorsinapatientspecificmanner.
Thisfindingisconfirmedbythegenesetvariationanalysisusingthehallmarkgenesets.
BothP1andP2showagreatlyreducedexpressionofspermatogenesis-relatedgenes.The
lattermightstemfromthelowexpressionofthetranscriptionfactorRegulatoryFactorX2
(RFX2)(FigureS10B,SupportingInformation),whichisnotonlyamajorcomponentof
spermatogenetisinhumanandmouse,butalsoregulatescellcycle(Horvath,Kistler,&
Kistler,2004;Kistleretal.,2015).Inlinewiththisweseelowactivityoftheupstreamcell-
cyclerelatedE2Ftranscriptionfactor6(E2F6)andcellcyclepathwaysarereducedinactivity
relativetobothhealthyovaryandtestistissue(FigureS10C,SupportingInformation).
Interestingly,MYCpathwayactivityislowcomparedtoboththeovarianandthetesticular
tissue.MYCcontrolsvertebratedevelopmentandcontributestodevelopmentaldisorders
(Hurlin,2013).OnemightspeculatewhethertheXp21.2duplicationscauseaholdof
developmentofallorasubsetofgonadaltissuecells,whichisreflectedbythefindingsfrom
thetranscriptomeanalysis,butwhichwillrequirefurtherindepthinvestigationandin
vivo/Invitroconfirmation.
Insummary,ourresultsdemonstratetherelevanceofgeneticanalysisbeyondprimary
candidategenesandemphasizetheimportanceofincludingmoredetailedanalysesof
geneticalterationsaffectingnon-codingregulatoryelementstoroutinediagnostics.This
couldbeanimportantfindingforuncoveringnovelgeneticcausesandunderstanding
pathogenesisinDSD,andforpatientswithotherdisordersofunknownetiology.Thefindings
furtherillustratelimitationsofcurrentdiagnosticroutinesthatareaCGHorMLPAbased,as
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23
theobtaineddataareinsufficienttoevaluatemostoftheduplicationsofunknown
significance.Muchmoreresearchwillbenecessarytounderstandmechanismsofinsulation
andhowregulatoryelementsfindtargetswithinTADsinordertocompletelyapprehendthe
effectofSVsandalsotomakefulluseoftechniquessuchasWGSinthecontextofsex
developmentandfarbeyond.
Acknowledgments
Theauthorsaretrulygratefultothepatientsandfamiliesofthesepatientswho
cooperatedinthisstudy.ThecollaborationwasbasedontheCOSTActionBM1303DSDnet.
H.B.andA.K.acknowledgecomputationalsupportfromtheLübeckOmicsCluster.Thiswork
ispartofthedoctoralthesisofJ.A.MandA.P.S..
Figures:
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Figure1.StructuralVariationofPatient1(A)Overviewofthecopynumbervariation(CNV)
inP1upstreamoftheNR0B1geneanditsorientationwithintheXp21.2region.Arrows
depictgenesandtheirrespectivedirectionoftranscription.Thechromosomesegmentis
drawnwiththedistalchromosomearmtotheleftandcentromeretotheright.The
distancesbetweengenesarenottoscale.TheregionchrX:30,561,644-30,859,140hasbeen
duplicatedinatandemmanner,asindicatedbythesegmentsshadedorange.*marksa
27bpinsertatthebreakepoint.(B)Positionofextractedandalignedsplitreadsfrom
genomesequencingcrossingthebreakpoint(chrX:30,859,140).Correspondinglycoloured
arrowsofoppositedirectionbelongtothesamereadpair.Readpairshavebeenmapped
297.5kbawayfromeachother.(C)VerificationofthebreakpointsequencebyPCRand
Sangersequencing.ElectropherogramshowsacontiguoussequencefromtheTAB3geneto
thebreakpoint,aninsertedsequenceof27bp(red)andthedownstreamsequenceofthe
CXorf21geneatthebeginningoftheduplication.
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Figure2.StructuralVariationofPatient2(A)Overviewofthestructuralvariationupstream
oftheNR0B1gene.Thegenomicdistancesarenottoscale.A447kbduplicationofCXorf21,
GKandpartofTAB3(yellow)originallymappingto30,401,819-30,848,988isinsertedinan
invertedmannerbetweenbreakpoint1(bp1;chrX:30,336,745)andbp2(chrX:30,340,605)
9.25kbupstreamoftheNR0B1readingframe.*marksa1.2kbpieceofreferencesequence
invertedbetweenbp2andbp3separatingthetwolargeduplications.Bothduplicationsare
flankedbydeletionsindicatedbythegreyflagsdenotedDeletionI&II.A389kbduplication
oftheMAGEBgenes1-4andpartofIL2RAPL2(blue)originallymappingtochrX:29,924,420-
30,313,761isinsertedbetweenbreakpoint3(chrX:30,339,452)and4(chrX:30,342,785)in
thesameorientationasitsreferencesequence.(B)Showspositionofextractedandaligned
WGSsplitreadscrossingthefourbreakpoints.Splitreadsmappedatvaryingdistancesapart
foreachbreakpoint.Eachpairwasseparatedbyatleast>29kb.Sequencesateithersideof
bp1andbp4showedhomology,thusnoexactdefinitionofthebreakpointpositionwas
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possible,asdepictedbytheoverlapofthegreyandyelloworbluebars.(C)Shows
verificationofsequencesdeducedfromWGSreadsthroughSangersequencing.
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Figure3:Comparisonofpatientgonadtranscriptomestohealthytissues.Thetwo-
dimensionalt-SNEplotcomparesthetranscriptomesoftestisandovarytissuefromyoung
andoldhealthydonorsaswellasfromliver,heart,cerebellum,kidneyandbrain.The
samplescomefromtheGTExdatabase(red,orange,yellowpoints)aswellasdatasetE-
MTAB-6814fromArrayExpress.t-SNEwascalculatedfor608samplesusingrandomly5000
outof13,005genesexpressedineitherpatientsample.Testissamplesaremarkedaseither
orangeoryellow,dependingonwhethertheyarereportedtohavenormalorreduced
-20 -10 0 10 20
-20
-10
010
20
Tsne Dim1
TsneDim2
GTEx Ovary
Patient 1
Patient 2
GTEx TestisGTEx Testis reduced SpermatogenesisPatient SamplesLiverHeartCerebellum
KidneyBrain
Fetal OvaryFetal/Young TestisTestis adults
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28
spermatogenesis.Thelargeorangeandredsportsamongthetestisandovarytissuegroup
markthet-SNEthemedianexpressionofalltissuesamples.
Figure4:Schematicrepresentationoftopologicallyassociatingdomain(TAD)inpatients
withNR0B1upstreamduplications.TheNR0B1TADisrepresentedascurledloop.Regions
insertedthroughduplicationareillustratedbycolouredstringsegments.Genesaredepicted
byarrowsshowingtheirrespectivedirectionoftranscriptionandareequallycoloured,if
partofaduplication.WhenaTADcomprisesboththeNR0B1andtheGK&CXorf21genes
withtheirin-betweenenhancers(greenoval)theNR0B1promotercanencounterthenon-
cognateenhancernormallyshieldedawayfromthegenebypositionoutsidetheTAD.P1a
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andbdepictalternateTADconformationsdependingonlocationofcommencedloop
extrusion.
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