disruption of the topological associated domain at xp21.2 ......mar 30, 2020 · 1 disruption of...

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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:[email protected]

All rights reserved. No reuse allowed without permission. perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for thisthis version posted March 30, 2020.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NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

https://doi.org/10.1101/2020.03.25.20041418

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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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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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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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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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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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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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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.

References

Ahmed,S.F.,Khwaja,O.,&Hughes,I.A.(2000).Theroleofaclinicalscoreintheassessmentofambiguousgenitalia.BJUInt,85(1),120-124.

Athar,A.,Fullgrabe,A.,George,N.,Iqbal,H.,Huerta,L.,Ali,A.,...Brazma,A.(2019).ArrayExpressupdate-frombulktosingle-cellexpressiondata.NucleicAcidsRes,47(D1),D711-D715.doi:10.1093/nar/gky964

Audi,L.,Ahmed,S.F.,Krone,N.,Cools,M.,McElreavey,K.,Holterhus,P.M.,...The,E.U.C.A.(2018).GENETICSINENDOCRINOLOGY:Approachestomoleculargeneticdiagnosisinthemanagementofdifferences/disordersofsexdevelopment(DSD):positionpaperofEUCOSTActionBM1303'DSDnet'.EurJEndocrinol,179(4),R197-R206.doi:10.1530/EJE-18-0256

Baetens,D.,Guran,T.,Mendonca,B.B.,Gomes,N.L.,DeCauwer,L.,Peelman,F.,...DeBaere,E.(2018).BiallelicandmonoallelicESR2variantsassociatedwith46,XYdisordersofsexdevelopment.GenetMed,20(7),717-727.doi:10.1038/gim.2017.163

Barbaro,M.,Cicognani,A.,Balsamo,A.,Lofgren,A.,Baldazzi,L.,Wedell,A.,&Oscarson,M.(2008).Genedosageimbalancesinpatientswith46,XYgonadalDSDdetectedbyanin-house-designedsyntheticprobesetformultiplexligation-dependentprobeamplificationanalysis.ClinGenet,73(5),453-464.doi:10.1111/j.1399-0004.2008.00980.x

Barbaro,M.,Cook,J.,Lagerstedt-Robinson,K.,&Wedell,A.(2012).MultigenerationInheritancethroughFertileXXCarriersofanNR0B1(DAX1)LocusDuplicationinaKindredofFemaleswithIsolatedXYGonadalDysgenesis.IntJEndocrinol,2012,504904.doi:10.1155/2012/504904

Barbaro,M.,Oscarson,M.,Schoumans,J.,Staaf,J.,Ivarsson,S.A.,&Wedell,A.(2007).Isolated46,XYgonadaldysgenesisintwosisterscausedbyaXp21.2interstitialduplicationcontainingtheDAX1gene.JClinEndocrinolMetab,92(8),3305-3313.doi:10.1210/jc.2007-0505

Bardoni,B.,Zanaria,E.,Guioli,S.,Floridia,G.,Worley,K.C.,Tonini,G.,...etal.(1994).AdosagesensitivelocusatchromosomeXp21isinvolvedinmaletofemalesexreversal.NatGenet,7(4),497-501.doi:10.1038/ng0894-497

Bashamboo,A.,Eozenou,C.,Rojo,S.,&McElreavey,K.(2017).Anomaliesinhumansexdeterminationprovideuniqueinsightsintothecomplexgeneticinteractionsofearlygonaddevelopment.ClinGenet,91(2),143-156.doi:10.1111/cge.12932

Boulanger,L.,Pannetier,M.,Gall,L.,Allais-Bonnet,A.,Elzaiat,M.,LeBourhis,D.,...Pailhoux,E.(2014).FOXL2isafemalesex-determininggeneinthegoat.CurrBiol,24(4),404-408.doi:10.1016/j.cub.2013.12.039

Bray,N.L.,Pimentel,H.,Melsted,P.,&Pachter,L.(2016).Near-optimalprobabilisticRNA-seqquantification.NatBiotechnol,34(5),525-527.doi:10.1038/nbt.3519



https://doi.org/10.1101/2020.03.25.20041418

30

Carithers,L.J.,Ardlie,K.,Barcus,M.,Branton,P.A.,Britton,A.,Buia,S.A.,...Consortium,G.T.(2015).ANovelApproachtoHigh-QualityPostmortemTissueProcurement:TheGTExProject.BiopreservBiobank,13(5),311-319.doi:10.1089/bio.2015.0032

Chaboissier,M.C.,Kobayashi,A.,Vidal,V.I.,Lutzkendorf,S.,vandeKant,H.J.,Wegner,M.,...Schedl,A.(2004).FunctionalanalysisofSox8andSox9duringsexdeterminationinthemouse.Development,131(9),1891-1901.doi:10.1242/dev.01087

deWit,E.,Vos,E.S.,Holwerda,S.J.,Valdes-Quezada,C.,Verstegen,M.J.,Teunissen,H.,...deLaat,W.(2015).CTCFBindingPolarityDeterminesChromatinLooping.MolCell,60(4),676-684.doi:10.1016/j.molcel.2015.09.023

DePristo,M.A.,Banks,E.,Poplin,R.,Garimella,K.V.,Maguire,J.R.,Hartl,C.,...Daly,M.J.(2011).Aframeworkforvariationdiscoveryandgenotypingusingnext-generationDNAsequencingdata.NatGenet,43(5),491-498.doi:10.1038/ng.806

Dixon,J.R.,Selvaraj,S.,Yue,F.,Kim,A.,Li,Y.,Shen,Y.,...Ren,B.(2012).Topologicaldomainsinmammaliangenomesidentifiedbyanalysisofchromatininteractions.Nature,485(7398),376-380.doi:10.1038/nature11082

Dong,Y.,Yi,Y.,Yao,H.,Yang,Z.,Hu,H.,Liu,J.,...Liang,Z.(2016).Targetednext-generationsequencingidentificationofmutationsinpatientswithdisordersofsexdevelopment.BMCMedGenet,17,23.doi:10.1186/s12881-016-0286-2

Franke,M.,Ibrahim,D.M.,Andrey,G.,Schwarzer,W.,Heinrich,V.,Schopflin,R.,...Mundlos,S.(2016).Formationofnewchromatindomainsdeterminespathogenicityofgenomicduplications.Nature,538(7624),265-269.doi:10.1038/nature19800

Fudenberg,G.,Imakaev,M.,Lu,C.,Goloborodko,A.,Abdennur,N.,&Mirny,L.A.(2016).FormationofChromosomalDomainsbyLoopExtrusion.CellRep,15(9),2038-2049.doi:10.1016/j.celrep.2016.04.085

Garcia-Acero,M.,Molina,M.,Moreno,O.,Ramirez,A.,Forero,C.,Cespedes,C.,...Rojas,A.(2019).GenedosageofDAX-1,determininginsexualdifferentiation:duplicationofDAX-1intwosisterswithgonadaldysgenesis.MolBiolRep.doi:10.1007/s11033-019-04758-y

Giangrande,P.H.,Zhu,W.,Schlisio,S.,Sun,X.,Mori,S.,Gaubatz,S.,&Nevins,J.R.(2004).AroleforE2F6indistinguishingG1/S-andG2/M-specifictranscription.GenesDev,18(23),2941-2951.doi:10.1101/gad.1239304

Goloborodko,A.,Marko,J.F.,&Mirny,L.A.(2016).ChromosomeCompactionbyActiveLoopExtrusion.BiophysJ,110(10),2162-2168.doi:10.1016/j.bpj.2016.02.041

Gomez-Marin,C.,Tena,J.J.,Acemel,R.D.,Lopez-Mayorga,M.,Naranjo,S.,delaCalle-Mustienes,E.,...Gomez-Skarmeta,J.L.(2015).EvolutionarycomparisonrevealsthatdivergingCTCFsitesaresignaturesofancestraltopologicalassociatingdomainsborders.ProcNatlAcadSciUSA,112(24),7542-7547.doi:10.1073/pnas.1505463112

Guo,Y.,Xu,Q.,Canzio,D.,Shou,J.,Li,J.,Gorkin,D.U.,...Wu,Q.(2015).CRISPRInversionofCTCFSitesAltersGenomeTopologyandEnhancer/PromoterFunction.Cell,162(4),900-910.doi:10.1016/j.cell.2015.07.038

Hanzelmann,S.,Castelo,R.,&Guinney,J.(2013).GSVA:genesetvariationanalysisformicroarrayandRNA-seqdata.BMCBioinformatics,14,7.doi:10.1186/1471-2105-14-7

Hnisz,D.,Weintraub,A.S.,Day,D.S.,Valton,A.L.,Bak,R.O.,Li,C.H.,...Young,R.A.(2016).Activationofproto-oncogenesbydisruptionofchromosomeneighborhoods.Science,351(6280),1454-1458.doi:10.1126/science.aad9024

Horvath,G.C.,Kistler,W.S.,&Kistler,M.K.(2004).RFX2isapotentialtranscriptionalregulatoryfactorforhistoneH1tandothergenesexpressedduringthemeioticphase



https://doi.org/10.1101/2020.03.25.20041418

31

ofspermatogenesis.BiolReprod,71(5),1551-1559.doi:10.1095/biolreprod.104.032268

Hurlin,P.J.(2013).ControlofvertebratedevelopmentbyMYC.ColdSpringHarbPerspectMed,3(9),a014332.doi:10.1101/cshperspect.a014332

Ibn-Salem,J.,Kohler,S.,Love,M.I.,Chung,H.R.,Huang,N.,Hurles,M.E.,...Robinson,P.N.(2014).Deletionsofchromosomalregulatoryboundariesareassociatedwithcongenitaldisease.GenomeBiol,15(9),423.doi:10.1186/s13059-014-0423-1

Kistler,W.S.,Baas,D.,Lemeille,S.,Paschaki,M.,Seguin-Estevez,Q.,Barras,E.,...Reith,W.(2015).RFX2IsaMajorTranscriptionalRegulatorofSpermiogenesis.PLoSGenet,11(7),e1005368.doi:10.1371/journal.pgen.1005368

Krijthe,J.(2015).Rtsne:T-DistributedStochasticNeighborEmbeddingusingaBarnes-HutImplementation.https://github.com/jkrijthe/Rtsne.

Kulle,A.E.,Riepe,F.G.,Melchior,D.,Hiort,O.,&Holterhus,P.M.(2010).Anovelultrapressureliquidchromatographytandemmassspectrometrymethodforthesimultaneousdeterminationofandrostenedione,testosterone,anddihydrotestosteroneinpediatricbloodsamples:age-andsex-specificreferencedata.JClinEndocrinolMetab,95(5),2399-2409.

Ledig,S.,Hiort,O.,Scherer,G.,Hoffmann,M.,Wolff,G.,Morlot,S.,...Wieacker,P.(2010).Array-CGHanalysisinpatientswithsyndromicandnon-syndromicXYgonadaldysgenesis:evaluationofarrayCGHasdiagnostictoolandsearchfornewcandidateloci.HumReprod,25(10),2637-2646.doi:10.1093/humrep/deq167

Ledig,S.,Hiort,O.,Wunsch,L.,&Wieacker,P.(2012).PartialdeletionofDMRT1causes46,XYovotesticulardisorderofsexualdevelopment.EurJEndocrinol,167(1),119-124.doi:10.1530/EJE-12-0136

Lek,M.,Karczewski,K.J.,Minikel,E.V.,Samocha,K.E.,Banks,E.,Fennell,T.,...ExomeAggregation,C.(2016).Analysisofprotein-codinggeneticvariationin60,706humans.Nature,536(7616),285-291.doi:10.1038/nature19057

Lettice,L.A.,Daniels,S.,Sweeney,E.,Venkataraman,S.,Devenney,P.S.,Gautier,P.,...FitzPatrick,D.R.(2011).Enhancer-adoptionasamechanismofhumandevelopmentaldisease.HumMutat,32(12),1492-1499.doi:10.1002/humu.21615

Li,H.,&Durbin,R.(2009).FastandaccurateshortreadalignmentwithBurrows-Wheelertransform.Bioinformatics,25(14),1754-1760.doi:10.1093/bioinformatics/btp324

Liberzon,A.,Birger,C.,Thorvaldsdottir,H.,Ghandi,M.,Mesirov,J.P.,&Tamayo,P.(2015).TheMolecularSignaturesDatabase(MSigDB)hallmarkgenesetcollection.CellSyst,1(6),417-425.doi:10.1016/j.cels.2015.12.004

Ludbrook,L.M.,Bernard,P.,Bagheri-Fam,S.,Ryan,J.,Sekido,R.,Wilhelm,D.,...Harley,V.R.(2012).ExcessDAX1leadstoXYovotesticulardisorderofsexdevelopment(DSD)inmicebyinhibitingsteroidogenicfactor-1(SF1)activationofthetestisenhancerofSRY-box-9(Sox9).Endocrinology,153(4),1948-1958.doi:10.1210/en.2011-1428

Lupianez,D.G.,Kraft,K.,Heinrich,V.,Krawitz,P.,Brancati,F.,Klopocki,E.,...Mundlos,S.(2015).Disruptionsoftopologicalchromatindomainscausepathogenicrewiringofgene-enhancerinteractions.Cell,161(5),1012-1025.doi:10.1016/j.cell.2015.04.004

Lupianez,D.G.,Spielmann,M.,&Mundlos,S.(2016).BreakingTADs:HowAlterationsofChromatinDomainsResultinDisease.TrendsGenet,32(4),225-237.doi:10.1016/j.tig.2016.01.003

Matson,C.K.,Murphy,M.W.,Sarver,A.L.,Griswold,M.D.,Bardwell,V.J.,&Zarkower,D.(2011).DMRT1preventsfemalereprogramminginthepostnatalmammaliantestis.Nature,476(7358),101-104.doi:10.1038/nature10239



https://doi.org/10.1101/2020.03.25.20041418

32

Meinel,J.,Dwivedi,G.,Holterhus,P.M.,Hiort,O.,&Werner,R.(2019).qPCRscreeningforXp21.2copynumbervariationsinpatientswithelusiveaetiologyof46,XYDSD.HormResPaediatr,91((suppl1)),98.doi:DOI:10.1159/000501868

Narendra,V.,Rocha,P.P.,An,D.,Raviram,R.,Skok,J.A.,Mazzoni,E.O.,&Reinberg,D.(2015).CTCFestablishesdiscretefunctionalchromatindomainsattheHoxclustersduringdifferentiation.Science,347(6225),1017-1021.doi:10.1126/science.1262088

Nora,E.P.,Lajoie,B.R.,Schulz,E.G.,Giorgetti,L.,Okamoto,I.,Servant,N.,...Heard,E.(2012).SpatialpartitioningoftheregulatorylandscapeoftheX-inactivationcentre.Nature,485(7398),381-385.doi:10.1038/nature11049

Rao,S.S.P.,Huang,S.C.,GlennStHilaire,B.,Engreitz,J.M.,Perez,E.M.,Kieffer-Kwon,K.R.,...Aiden,E.L.(2017).CohesinLossEliminatesAllLoopDomains.Cell,171(2),305-320e324.doi:10.1016/j.cell.2017.09.026

Rausch,T.,Zichner,T.,Schlattl,A.,Stutz,A.M.,Benes,V.,&Korbel,J.O.(2012).DELLY:structuralvariantdiscoverybyintegratedpaired-endandsplit-readanalysis.Bioinformatics,28(18),i333-i339.doi:10.1093/bioinformatics/bts378

Ritchie,M.E.,Phipson,B.,Wu,D.,Hu,Y.,Law,C.W.,Shi,W.,&Smyth,G.K.(2015).limmapowersdifferentialexpressionanalysesforRNA-sequencingandmicroarraystudies.NucleicAcidsRes,43(7),e47.doi:10.1093/nar/gkv007

Sanborn,A.L.,Rao,S.S.,Huang,S.C.,Durand,N.C.,Huntley,M.H.,Jewett,A.I.,...Aiden,E.L.(2015).Chromatinextrusionexplainskeyfeaturesofloopanddomainformationinwild-typeandengineeredgenomes.ProcNatlAcadSciUSA,112(47),E6456-6465.doi:10.1073/pnas.1518552112

Schacht,T.,Oswald,M.,Eils,R.,Eichmuller,S.,&Konig,R.(2014).Estimatingtheactivityoftranscriptionfactorsbytheeffectontheirtargetgenes.Bioinformatics(30(17):i401-7).

Smyk,M.,Berg,J.S.,Pursley,A.,Curtis,F.K.,Fernandez,B.A.,Bien-Willner,G.A.,...Stankiewicz,P.(2007).Male-to-femalesexreversalassociatedwithanapproximately250kbdeletionupstreamofNR0B1(DAX1).HumGenet,122(1),63-70.doi:10.1007/s00439-007-0373-8

Soneson,C.,Love,M.I.,&Robinson,M.D.(2015).DifferentialanalysesforRNA-seq:transcript-levelestimatesimprovegene-levelinferences.F1000Res,4,1521.doi:10.12688/f1000research.7563.2

Swain,A.,Narvaez,V.,Burgoyne,P.,Camerino,G.,&Lovell-Badge,R.(1998).Dax1antagonizesSryactioninmammaliansexdetermination.Nature,391(6669),761-767.doi:10.1038/35799

Swain,A.,Zanaria,E.,Hacker,A.,Lovell-Badge,R.,&Camerino,G.(1996).MouseDax1expressionisconsistentwitharoleinsexdeterminationaswellasinadrenalandhypothalamusfunction.NatGenet,12(4),404-409.doi:10.1038/ng0496-404

Szabo,Q.,Bantignies,F.,&Cavalli,G.(2019).Principlesofgenomefoldingintotopologicallyassociatingdomains.SciAdv,5(4),eaaw1668.doi:10.1126/sciadv.aaw1668

Uhlenhaut,N.H.,Jakob,S.,Anlag,K.,Eisenberger,T.,Sekido,R.,Kress,J.,...Treier,M.(2009).SomaticsexreprogrammingofadultovariestotestesbyFOXL2ablation.Cell,139(6),1130-1142.doi:10.1016/j.cell.2009.11.021

vanderMaaten,L.(2014).Acceleratingt-SNEusingTree-BasedAlgorithms.JMachLearnRes(15:3221-3245).

VietriRudan,M.,Barrington,C.,Henderson,S.,Ernst,C.,Odom,D.T.,Tanay,A.,&Hadjur,S.(2015).ComparativeHi-CrevealsthatCTCFunderliesevolutionofchromosomaldomainarchitecture.CellRep,10(8),1297-1309.doi:10.1016/j.celrep.2015.02.004



https://doi.org/10.1101/2020.03.25.20041418

33

Wang,K.,Li,M.,&Hakonarson,H.(2010).ANNOVAR:functionalannotationofgeneticvariantsfromhigh-throughputsequencingdata.NucleicAcidsRes,38(16),e164.doi:10.1093/nar/gkq603

Wang,Y.,Song,F.,Zhang,B.,Zhang,L.,Xu,J.,Kuang,D.,...Yue,F.(2018).The3DGenomeBrowser:aweb-basedbrowserforvisualizing3Dgenomeorganizationandlong-rangechromatininteractions.GenomeBiol,19(1),151.doi:10.1186/s13059-018-1519-9

Weischenfeldt,J.,Dubash,T.,Drainas,A.P.,Mardin,B.R.,Chen,Y.,Stutz,A.M.,...Korbel,J.O.(2017).Pan-canceranalysisofsomaticcopy-numberalterationsimplicatesIRS4andIGF2inenhancerhijacking.NatGenet,49(1),65-74.doi:10.1038/ng.3722

White,S.,Ohnesorg,T.,Notini,A.,Roeszler,K.,Hewitt,J.,Daggag,H.,...Sinclair,A.(2011).Copynumbervariationinpatientswithdisordersofsexdevelopmentdueto46,XYgonadaldysgenesis.PLoSOne,6(3),e17793.doi:10.1371/journal.pone.0017793

Yu,R.N.,Ito,M.,Saunders,T.L.,Camper,S.A.,&Jameson,J.L.(1998).RoleofAhchingonadaldevelopmentandgametogenesis.NatGenet,20(4),353-357.doi:10.1038/3822



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