Download pdf - Genome-wide association studies on endometriosis and endometriosis … · Introduction Endometriosis affects ~10% of women of reproductive age and is characterized by the growth of

Genome-wideassociationstudiesonendometriosisandendometriosis-relatedinfertilityAuthors:GenevièveGalarneau1,PierreFontanillas2,theCelmatixResearchTeam1,the23andMeResearchTeam2,CaterinaClementi1,TinaHu-Seliger1,David-EmlynParfitt1,JoyceY.Tung2,PirayeYurttasBeim1*1CelmatixInc.,NewYork,NY,USA223andMe,Inc.,MountainView,CA,USA*CorrespondencetoPirayeYurttasBeimCelmatixInc.,14WallSt,Suite16DNewYork,NY10005Abstract

Endometriosisaffects~10%ofwomenofreproductiveage.Itischaracterizedbythegrowthof

endometrial-liketissueoutsidetheuterusandisfrequentlyassociatedwithseverepainand

infertility.Weperformedthelargestendometriosisgenome-wideassociationstudy(GWAS)to

date,with37,183casesand251,258controls.AllwomenwereofEuropeanancestry.Wealso

performedthefirstGWASofendometriosis-relatedinfertility,including2,969casesand3,770

controls.OurendometriosisGWASstudyreplicated,atgenome-widesignificance,sevenloci

identifiedinpreviousendometriosisGWASs(CELA3A-CDC42,SYNE1,KDR,FSHB-ARL14EP,

GREB1,ID4,andCEP112)andidentifiedsevennewcandidatelociwithgenome-wide

significance(NGF,ATP1B1-F5,CD109,HEY2,OSR2-VPS13B,WT1,andTEX11-SLC7A3).Noloci

demonstratedgenome-widesignificanceforendometriosis-relatedinfertility,however,the

threemoststronglyassociatedloci(MCTP1,EPS8L3-CSF1,andLPIN1)wereinorneargenes

associatedwithfemalefertilityorembryoniclethalityinmodelorganisms.Theseresultsreveal

newcandidategeneswithpotentialinvolvementinthepathophysiologyofendometriosisand

endometriosis-relatedinfertility.

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted September 7, 2018. . https://doi.org/10.1101/401448doi: bioRxiv preprint

https://doi.org/10.1101/401448

Introduction

Endometriosisaffects~10%ofwomenofreproductiveageandischaracterizedbythegrowth

ofendometrial-liketissueoutsidetheuterus.Theovaryistheorganthatismostoftenaffected

byendometrioticlesions[1],butotherorgans,includingthebowel,fallopiantubes,orbladder,

canbeaffected.Endometriosisisanestrogen-dependentinflammatorydisease,withthe

endometrioticlesionscontinuingtorespondtoestrogenandtothickenfollowingthemenstrual

cycle,creatinginflammationandscartissuecalledadhesions.Theadhesionsandassociated

inflammationarethoughttounderliethesymptomsofendometriosis,whichincludepelvic

pain,dysmenorrhea,andpainduringintercourse.

Endometriosisisalsofrequentlyassociated(30-50%)withinfertility[2].Womenwith

endometriosishavealowermonthlyfecundityratethanfertilecontrols[3].Ameta-analysisof

22publishedstudiesshowedthatwomenwithendometriosisundergoinginvitrofertilization

(IVF)hadsignificantlyreducedoocyteretrieval,fertilization,andimplantationrates,aswellasa

significantlylowerchanceofachievingpregnancy,comparedtopatientswithtubalfactor[4].

Womenwithendometriosis-relatedinfertilitytendtohavealongertimetonaturalconception

thanwomenwithidiopathicinfertility[5,6]andlongertimetoconceptionthroughartificial

inseminationthanwomenwithnofemaleinfertilityfactors[7].Evenmildendometriosiscan

affectawoman’sfertility,andinfertility-relatedendometriosismaybeassociatedwith

endometriosisgrade—forexample,pregnancyratesfollowingIVFweresignificantlylowerin

womenwithsevereendometriosisthaninwomenwithmildendometriosis[4].Endometriosis

mayalsoreduceoocytequality,assuggestedbyastudyinwhichwomenwhoreceivedoocytes

donatedbywomenwithendometrioticovarieshadalowerimplantationratethanthose

receivingoocytesdonatedbywomenwithoutknownendometriosis[8].

Thecausalmechanismsofendometriosis-relatedinfertilityarenotfullyunderstood.Different

aspectsofendometriosismayleadtoinfertility,andtheexactcauseofendometriosis-related

infertilityineachpatientmaydependonthecourseofherspecificpathology.Proposed

mechanismsforendometriosis-relatedinfertilityincludeovarian-tubaldysfunction,


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immunologicaldisorder,abnormalperitonealenvironment,anddysregulatedendometrial

function[9].Ovarian-tubaldysfunctioncouldbecausedbyadistortionoftheovaryand/or

fallopiantubeaffectedbyendometrioticlesions,ovulationfailure,orabnormalfollicle

development.AnimmunologicaldisordercouldbecausedinpartbytheIgGandIgManti-

endometrialantibodies,whichhavebeendetectedin60%ofendometriosispatientsandcould

impairimplantation[10].Abnormalitiesintheperitonealenvironmentcouldincludeincreased

peritonealfluidsandahigherconcentrationofcytokinesand/oractivatedmacrophages.

Womenwithendometriosis-relatedinfertilityhaveanincreasedvolumeofperitonealfluid,and

thisfluidmayhaveincreasedlevelsofinflammatorycytokinesduetotheactivationof

macrophagesbytheendometrioticlesions.Activatedmacrophagesincreasereactiveoxygen

speciesintheperitonealfluidofwomenwithendometriosis[11],causingoxidativestress,

whichhasbeenassociatedwithnegativeoutcomesinassistedreproductivetechnology

fertilization[12].Forexample,thisincreaseincytokinesmayreduceoocytequality,sperm

motility,andtubalmotility,andimpairembryodevelopment.Finally,endometrialreceptivityis

acrucialprocesstoachievepregnancyandinvolvescomplexregulationofhormones,cytokines,

andadhesionmolecules.Thisprocessmaybedysregulatedinwomenwithendometriosis.

Laparoscopyiscurrentlytheonlywaytodiagnoseendometriosiswithcertainty.Itinvolvesa

surgicalprocedureinwhichasurgeonvisualizestheendometrioticlesionsorcollectstissue

samplesforhistologicassessment.Laparoscopyisoftenusedtoexcisetheendometriotic

lesionstorelievepain,butsymptomsoftenreturninfollowingyears.Becauseendometriosis

painlevelsanddiseaseseverityarenotdirectlycorrelated,thediagnosisandgradingof

endometriosiswithoutsurgeryisdifficult.However,becauseoftheinvasivenatureofthe

diagnosticprocedure,womenwithsuspectedendometriosisareoftentreatedwithhormone

therapywithoutadefinitivediagnosis.Despiteitshighprevalenceanditsassociationwith

severepainandinfertility,treatmentforendometriosisremainslimited.Surgicalandmedical

managementofendometriosiscanhelptreatsymptoms.Surgicalmanagementincreasesthe

monthlyfecundityrateofwomenwithendometriosisbutevenwithsurgicalmanagement,it

remainsmuchlowerthaninthegeneralpopulation[13].Hormonetreatmentsincludeoral


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contraceptives,androgenicagents,progestins,andgonadotropin-releasinghormoneanalogs,

buttheyarenotalwayseffective.

Endometriosisisacomplextrait,withbothgeneticandenvironmentalfactorscontributingto

thepathophysiologyofthedisease.Theheritabilityofendometriosisisestimatedtobe47-51%

[14,15],butthe13lociidentifiedbypreviousgenome-wideassociationstudies(GWASs)only

explain1.75%ofthephenotypicvariance[16-23].Further,fewstudieshaveinvestigated

whethergeneticfactorsmightbeinvolvedinthepathophysiologyofendometriosis-related

infertility.Theidentificationofnovelgeneticfactorsimplicatedinendometriosisor

endometriosis-relatedinfertilitycouldhelptoelucidatethepathophysiologyofendometriosis

andtoidentifypotentialnewdrugtargets.Here,weconductedaGWASonendometriosis

including37,183casesand251,258controlsandthefirstGWASonendometriosis-related

infertility,including2,969womenwithendometriosis-relatedinfertilityand3,770womenwith

endometriosiswhoreportedafirsttime-to-conception≤6months.

MaterialsandMethods

Participants

Participantsinthisstudywerecustomersoftheconsumergeneticscompany23andMe,Inc.,

whoprovidedinformedconsenttoparticipateinresearchbycompletingsurveysonlineundera

protocolapprovedbyEthical&IndependentReviewServices,anindependentinstitutional

reviewboard(http://www.eandireview.com)accreditedbytheAssociationforthe

AccreditationofHumanResearchProtectionPrograms,Inc.Thisstudywasrestrictedto

unrelatedfemaleparticipantswith>97%Europeanancestry.Percentageancestrywasassessed

throughalocalancestryanalysis.

Genotyping

DNAextractionandgenotypingwereperformedonsalivasamplesbytheNationalGenetics

Institute.Samplesweregenotypedononeoffourcustomgenome-widegenotypingarrays


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targeting560-950kSNPs.Onlysamplesthatachievedanaveragegenotypingrate≥98.5%were

includedintheanalysis.

Qualitycontrol

GenotypedSNPswereexcludediftheyhadagenotypingrateof<90%,didnotpassaparent-

offspringconcordancetest,weremonomorphic,werenotinHardy-Weinbergequilibrium

(p<1×10−20),orshowedbatcheffects.

Imputation

ImputationwasperformedusingtheSeptember2013releaseofthe1000GenomesProject

PhaseIasreferencehaplotypes.ThephasingwasperformedwithShapeIt2andtheimputation

withMinimac2.ImputedSNPswereflaggedandexcludediftheyhadanaverager2<0.5across

genotypingplatformoraminimumr2<0.3oriftheyshowedstrongevidenceofimputation

batcheffect(p<10−50).

Principalcomponentscalculations

Theprincipalcomponentanalysiswasperformedusing~65,000highqualitygenotypedvariants

presentinallfivegenotypingplatformswith1millionofparticipantsfromEuropeanancestry

randomlysampledacrossallthegenotypingplatforms.PCscoresforparticipantsnotincluded

intheanalysiswereobtainedbyprojection,combiningtheeigenvectorsoftheanalysisandthe

SNPweights.

Phenotypedefinition

Case-controlascertainmentwasbasedonparticipants’surveyresponses.Fortheendometriosis

case-controlGWAS,caseswerewomenwhoreportedbeingtreatedforand/ordiagnosedwith

endometriosis.Controlswerewomenwhoreportednotbeingtreatedforordiagnosedwith

endometriosis.


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Fortheendometriosis-relatedinfertilityGWAS,bothcasesandcontrolswerewomenwho

reportedbeingtreatedforand/ordiagnosedwithendometriosisandwhohadansweredat

leastonequestionontimetofirstconception.Thetimetofirstconceptionwasascertained

basedontheiranswerstothequestion(s):"Forhowlongdidyoutrytoconceive?Ifyouare

currentlytryingtoconceive,pleasestatehowlongyouhavebeentryinguptothispoint","The

firsttimeyoutriedtoconceiveachild,forhowlongwereyoutryingtoconceive?Ifyouare

currentlytryingtoconceiveforthefirsttime,pleaseanswerforhowlongyouhavebeentrying

uptothispoint."Thequestionsregardingtimetofirstconceptionhadmultiplechoiceanswers

withthefollowingoptions:0-6months,7-12months,13-24months,24months,24+months,

notsuccessful,andnotsure.‘Infertile’casesweredefinedaswomenwhorespondedwith13-

24months,24months,24+months,ornotsuccessful(35.7%ofwomenwithendometriosisin

23andMedatabase).Womenwhorespondedwith7-12months(13.1%)orI’mnotsurewere

excludedfromtheanalysis(<0.1%).Controlswerewomenwhoindicated0-6months(51.2%)

andreportedhavingatleastonebiologicalchild.

Statisticalanalysis

Assuminganadditivemodel,weperformedlogisticregressionsonendometriosiscase-control

statusandonendometriosis-relatedinfertilitycase-controlstatuswithcurrentage,thetopfive

principalcomponents,andthegenotypingplatformascovariates.Theassociationresultswere

adjustedfortheobservedgenomiccontrolinflationfactorinthedistributionofthep-values

(λ=1.141andλ=1.011,respectively,SupplementaryFigures1-2).

Prioritizationofputativefunctionalvariants

The99%crediblesetsweregeneratedbyfirstcalculatinganapproximateBayesfactor(ABF)for

eachSNP,usingthemethodproposedbyWakefield[24]andassumingapriorvariance(W)of

0.1.ThecrediblesetswerethenestimatedfromtheABFs,usingthemethodofMalleretal[25].

Weprioritizedgeneticvariantsinthecrediblesetsforfurtherfollow-upinGTExPortaland

RegulomeDBandusedLDlink[26]toidentifypotentialgeneticvariantsthatwouldnothave


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passedgenotyping/imputationQCbutwouldbeinhighlinkagedisequilibrium(LD)(r2>0.8in

individualsofEuropeanancestryinthe1000GenomesProject).

Results

Fourteenlocireachedgenome-widesignificance(p<5×10−8),includingsevenlocinotreportedin

previousGWASs(NGF,ATP1B1-F5,CD109,HEY2,OSR2-VPS13B,WT1,andTEX11-SLC7A3)

(Figure1,Table3).Amongthelocithatwerepreviouslyreportedintheliteratureanddidnot

reachgenome-widesignificance,fourwerenominallyassociated(p<0.05)withaconsistent

directionofeffect(FGD6-VEZT,RMND1-ESR1,NPVF-NFE2L3,andCDKN2B-DMRTA1)andtwo

hadp-values>0.05(IGFBP3-TNS3andTTC39B)(Table4).

Nolocireachedgenome-wideassociationwithendometriosis-relatedinfertility(Figure2,Table

5).Amongthe14locithatreachedgenome-widesignificanceintheendometriosiscase-control

GWAS,four(NGF,CELA3A-CDC42,SYNE1,andKDR)reachednominalassociation(p<0.05)with

sharedriskallelesforbothendometriosisandendometriosis-relatedinfertility(Table3).Five

locireachedsuggestivelevelsofassociation(p<1×10−6)withendometriosis-relatedinfertility

(MCTP1,EPS8L3-CSF1,LPIN1,GRM8,andFSTL5-NAF1)(Table5).

Discussion

Self-reportedendometriosisphenotype

Althoughtheendometriosisphenotypeinourstudywasself-reported,sevenendometriosisloci

previouslyidentifiedinGWASwerereplicatedatgenome-widesignificanceandfourwere

replicatedatanominallevelwithaconsistentdirectionofeffect.Onlytwolocididnotreplicate

(Tables3-4).Theseresultsareconcordantwiththereportedaccuracyofself-reported

endometriosisphenotypesincomparisonwiththeSwedishNationalInpatientRegistrywitha

specificityof97.0%,asensitivityof61.8%andreceiveroperatingcharacteristicsareaof0.79

[27].However,replicatingournovellociindatasetswithsurgicallyconfirmedcasesisimportant

toavoididentificationoflociassociatedwithdysmenorrheaintheabsenceofendometriosis.


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Interestingly,22.2%ofwomenwithendometriosis-relatedinfertilityreportedhavingbeen

diagnosedwithand/ortreatedforpolycysticovarysyndrome(PCOS),comparedto14.8%of

fertilewomenwithendometriosis(Table2).BecauseofthecomplexityofthePCOSdiagnosis

andtheself-reportednatureofthePCOSphenotypeinourdataset,itispossiblethatsomeof

thewomenwhoreportedhavingPCOS,hadanovulationduetotheirendometriosis[28],which

couldcontributetotheirinfertility.ItisalsopossiblethatPCOScontributestotheinfertilityof

someofourendometriosis-relatedinfertilitycases.

Novelendometriosisloci

Sevennovellociwereassociatedwithendometriosisatgenome-widesignificanceinour

analysis.ThenovellocusNGFwasalsoassociatedatgenome-widesignificancewithseverityof

dysmenorrheainanotherstudyof23andMefemaleparticipantsofEuropeanancestry[29].

Interestingly,theexpressionlevelofNGFintheperitonealfluidhasbeenobservedtobehigher

inwomenwithendometriosis,andblockingNGFsignificantlydecreasesneuriteoutgrowthin

endometrioticlesions[30].Multipletranscriptionfactorshavebeenfoundtobindtheregion

coveredbythecredibleSNPssetwithintheNGFlocus(Table6).Thisregionalsooverlapswith

predictedenhancersinmultipletissues,includingthefetaladrenalgland,fetalstomach,fetal

kidney,colonsmoothmuscle,adiposenuclei,fetallung,primarymonocytesfromperipheral

blood,fetalmuscletrunk,andfetalmuscleleg.

ThecredibleSNPssetofthesecondnovelendometriosislocus,ATP1B1-F5,includestheFactor

VLeiden(F5)p.Gln534Argmissensevariantrs6025,whichhasbeenassociatedwithvenous

thromboembolism[31-33],thrombosis[34],inflammatoryboweldisease[35],andrecurrent

pregnancyloss[36],althoughtheriskalleleforendometriosisisinverted.

ThethirdnovelendometriosislocusisnearthegeneCD109,whichencodesaglycosyl

phosphatidylinositol(GPI)-linkedglycoproteinthatnegativelyregulatessignalingby

transforminggrowthfactorbeta(TGFB1).TGFB1isthoughttobeinvolvedindifferentaspects


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ofthepathophysiologyofendometriosisincludingendometrialcellproliferationandtissue

remodeling[37,38].

ThefourthnovelendometriosislocusisnearthegeneHesRelatedFamilybHLHTranscription

FactorwithYRPWMotif2(HEY2),theexpressionofwhichisdownregulatedby17β-estradiolin

ovariectomizedmice[39].ThenearbygeneNCOA7modulatestheactivityoftheestrogen

receptor(ER)[40].Thelocushasbeenassociatedatgenome-widesignificancewithendometrial

[41]andbreast[42]cancer.ItisaneQTLforbothHEY2andNCOA7,isboundbymultiple

transcriptionfactors(Table6),andcontainspredictedenhancersinmultipletissuesincluding

theovary,psoasmuscle,aorta,leftventricle,skeletalmuscleinfemales,fetallung,fetaladrenal

gland,andfetalheart.

ThefifthnovelendometriosislocusisOSR2-VPS13B.Thegeneodd-skippedrelatedtranscription

factor2(OSR2)isatranscriptionfactorthatishighlyexpressedintheendometriumandthe

ovary,anditsexpressionisdown-regulateduponprogesteronereceptorknockdowninhuman

endometrialstromalcells,suggestingthatitmaybeinvolvedindecidualization[43].Ina

genome-wideepigeneticsstudybasedonCpGmethylation,OSR2showedincreased

methylationanddecreasedexpressioninendometrioticstromalcellscomparedtostromalcells

fromnormalendometrium[44].Over40transcriptionfactorshavebeenidentifiedasbinding

theregioncoveredbythecredibleSNPset(Table6),andtheregionalsooverlapswith

predictedenhancersinnumeroustissues.Thevariantrs3019295,partofthecredibleSNPset,is

inanactivatedtranscriptionsiteofOSR2intheovary,psoasmuscle,andaorta.

ThesixthnovelendometriosislocusisnearthegeneWilmstumor1(WT1).WT1encodesa

transcriptionfactorthatishighlyexpressedintheendometrium,thetestis,andtheovary,and

thathasanessentialroleinthenormaldevelopmentoftheurogenitalsystem.Itisselectively

expressedinneuronsofdeependometriosis[45]andisdown-regulatedinendometriotic

stromalcellscomparedtoendometrialstromalcells[46,47].Theregioncoveredbythe


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credibleSNPsetoverlapswiththebindingsitesofseveraltranscriptionfactorsandwith

predictedenhancersinmultipletissues.

TheseventhnovelendometriosislocusisTEX11-SLC7A3.Thegenesolutecarrierfamily7

member3(SLC7A3)encodesasodium-independentcationicaminoacidtransporter,anditis

highlyexpressedintheendometrium.Testisexpressed11(TEX11)isexpressedinthepancreas,

testis,andhumanfetalovary,andisassociatedwithazoospermiaandmaleinfertility[48-52].

TEX11competeswithestrogenreceptorbeta(ERβ)forbindingtohematopoieticpre-Bcell

leukemiatranscriptionfactor-interactingprotein(HPIP),whichmodulatesthefunctionofERs

[53].TEX11suppressesestrogen-stimulatedgermcellproliferationandaffectstheexpression

ofestrogentargetgenesthroughitsbindingtoHPIP[53].

Endometriosis-relatedinfertilityassociationresults

Nolocireachedgenome-widesignificanceintheendometriosis-relatedinfertilityGWAS.Ofthe

14genome-wideendometriosisloci,onlyfourshowednominalassociationwithendometriosis-

relatedinfertility,suggestingthatthegeneticfactorsinvolvedinendometriosisand

endometriosis-relatedinfertilitymaydiffer.

Themoststronglyassociatedlocuswithendometriosis-relatedinfertilityisarare(minorallele

frequencyof8.8×10−3)insertionintheintronofthegeneMultipleC2andTransmembrane

DomainContaining1(MCTP1).ThehomologofMCTP1inCaenorhabditiselegans(D2092.1)is

anessentialgene,anditsablationleadstoearlyembryoniclethality[54].Theregioncoveredby

thecrediblesetispredictedtocontainanenhancerinhumanumbilicalveinendothelial

primarycells,fetallung,fetalmuscleleg,andfetalstomach.TheproteinencodedbyMCTP1is

notwellcharacterized,butitisanevolutionarilyconservedproteinthatcontainsC2domainsof

highCa2+-bindingaffinity.ItmayhaveaCa2+-controlledregulatoryfunctionorserveasa

Ca2+buffer[55].FunctionalstudiesincentralnervoussystemneuronsshowedthatMCTP1

over-expressionsignificantlyinhibitedneuronaltransferrinendocytosis,secretoryvesicle

retrieval,cellmigration,andoxidativestressfromglutamatetoxicity[56].Ifconfirmed,the


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associationofthislocuswithendometriosis-relatedinfertilitymightinvolveMCTP1protecting

oocytehomeostasis,maturation,andfertilizationfromtheoxidativestressgeneratedby

endometriosis.

Thesecondmoststronglyassociatedlocusintheendometriosis-relatedinfertilityGWASwas

EPS8L3-CSF1.ThegeneCSF1encodesanestrogen-regulatedcytokinethatcontrolsthe

production,differentiation,andfunctionofmacrophages.Inhumans,CSF1levelisincreasedin

thepregnantendometriumcomparedtothenonpregnantandishighintheplacenta

throughoutpregnancy[57,58].Inmice,femaleslackingCSF1haveextendedestruscyclesand

poorovulationrates[59,60].CSF1levelsareincreasedintheperitonealfluidofpatientswith

endometriosis[61]andCsf1expressionwassignificantlyhigherinlesionsofanendometriosis

mousemodel[62].TheER-dependentregulationofCSF1inperipheralnervefibershasbeen

suggestedtoplayacriticalroleinearlydevelopmentofendometrioticlesions[63]andin

modulatingmacrophagesurvival[62].Giventhepotentialroleofoxidativestressin

endometriosis-relatedinfertility,itisalsoworthnotingthattheoxidativestressresponsegenes

GSTM1,GSTM2,GSTM3,GSTM4,andGSTM5are<200kbfromtheassociatedvariantinthis

locus,althoughahighrecombinationrateseparatesthegenesfromthetopvariant.

The third most strongly associated locus in the endometriosis-related infertility GWAS was

LPIN1, which acts as a proinflammatory mediator during TLR signaling and during the

developmentof in vivo inflammatoryprocesses [64]. Inmice,Lpin1 is down-regulated in the

uterusbyestradiolviatheER[65]andLpin1deficiencyisassociatedwithimpairedfertility[66,

67].Lipin1mightalsoplayaroleintheregulationoftheuterinecellcyclebecauseithasbeen

foundtohaveanti-proliferativeeffectsinmurineproBcells[68].

Conclusion

OurstudyreplicatedsevenlociidentifiedinpreviousendometriosisGWASsandidentified

sevennewcandidatelociwithgenome-widesignificance.Thesenewlocineedtobereplicated,

andfurtherstudiesareneededtodeterminethecausalvariants.Althoughnolocihadgenome-


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widesignificanceforendometriosis-relatedinfertilityinthisstudy,thethreemoststrongly

associatedlociwereinorneargeneswithrolesinfemalefertilityorembryoniclethalityin

modelorganisms.Thesegenesarethoughttobeinvolvedinoxidativestress,macrophage

survival,andinflammation.Theseresultsprovidenewcandidategenesinvolvedinthe

pathophysiologyofendometriosisandendometriosis-relatedinfertility.

Acknowledgements

WethanktheresearchparticipantsandemployeesofCelmatixand23andMewhocontributed

tothisstudy.WeacknowledgethecontributionsfromadditionalmembersoftheCelmatix

ResearchTeam,includingR.MarkAdams,DanielaS.Colaci,ChrisGlazner,SamuelT.Globus,

SeanO'Keeffe,UrsulaM.Schick,LeiTan,CameronD.Wellock,DanielleWhite,andRajeshwari

R.Valiathan.Membersofthe23andMeResearchTeamare:MichelleAgee,BabakAlipanahi,

AdamAuton,RobertK.Bell,KatarzynaBryc,SarahL.Elson,NicholasA.Furlotte,DavidA.Hinds,

KarenE.Huber,AaronKleinman,NadiaK.Litterman,MatthewH.McIntyre,JoannaL.

Mountain,ElizabethS.Noblin,CarrieA.M.Northover,StevenJ.Pitts,J.FahSathirapongsasuti,

OlgaV.Sazonova,JanieF.Shelton,SuyashShringarpure,ChaoTian,VladimirVacic,CatherineH.

Wilson.

ConflictsofInterest

PF,JYT,andmembersofthe23andMeResearchTeamareemployeesof23andMe,Inc.,and

holdstockorstockoptionsin23andMe.GG,THS,DEP,PYBandmembersoftheCelmatix

ResearchTeamareemployeesofCelmatixInc.,andholdstockorstockoptionsinCelmatixInc.

Dataavailability

ThefullGWASsummarystatisticsforthe23andMedatasetswillbemadeavailablethrough

23andMetoqualifiedresearchersunderanagreementwith23andMethatprotectstheprivacy

ofthe23andMeparticipants.Pleasevisitresearch.23andMe.com/collaborateformore

informationandtoapplytoaccessthedata.


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Table1.Cohortdescriptionforendometriosiscase-controlGWAS

Cases Controls

N 37,183 251,258

Agemean(SD) 56.0(13.3) 51.0(16.5)

Body-massindexmean(SD) 27.4(6.5) 26.4(6.2)

Agefirst-time-to-conception(SD) 24.3(5.8) 25.4(5.8)

Numberofpregnanciesmean(SD) 2.0(1.5) 1.9(1.5)

Numberoflivebirthsmean(SD) 1.6(1.3) 1.6(1.4)

Numberofmiscarriagesmean(SD) 1.5(0.7) 1.4(0.6)

Self-reportedPCOSn(%) 6,028(16.2%) 13,005(5.2)

Self-reportedPOIn(%) 443(1.2) 849(0.3)

Self-reportedDORn(%) 975(2.6) 3,190(1.3)

SD:Standarddeviation,PCOS:Polycysticovarysyndrome,POI:Primaryovarianinsufficiency,DOR:Diminishedovarianreserve


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Table2.Cohortdescriptionforendometriosis-relatedinfertilityGWAS

Cases Controls

N 2,969 3,770

Agemean(SD) 56.0(13.3) 51.0(16.5)

Body-massindexmean(SD) 27.4(6.5) 26.4(6.2)

Agefirst-time-to-conception(SD) 24.3(5.8) 25.4(5.8)

Numberofpregnanciesmean(SD) 2.0(1.5) 1.9(1.5)

Numberoflivebirthsmean(SD) 1.6(1.3) 1.6(1.4)

Numberofmiscarriagesmean(SD) 1.5(0.7) 1.4(0.6)

Self-reportedPCOSn(%) 6,028(16.2%) 13,005(5.2)

Self-reportedPOIn(%) 443(1.2) 849(0.3)

Self-reportedDORn(%) 975(2.6) 3,190(1.3)

SD:Standarddeviation,PCOS:Polycysticovarysyndrome,POI:Primaryovarianinsufficiency,DOR:Diminishedovarianreserve


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Figure1.ManhattanplotofendometriosisGWAS

EndometriosisGWASassociationresults.Fourteenlocireachedgenome-widesignificance(p<5×10−8).

Figure2.Manhattanplotofendometriosis-relatedinfertilityGWAS

Endometriosis-relatedinfertilityGWASassociationresults.Nolocireachedgenome-widesignificance(p<5×10−8).


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Table3.Endometriosiscase-controlGWASgenome-wideloci

Endometriosiscase-controlGWAS

Endometriosis-relatedinfertility

Variantid Chr Pos(hg19) Nearestgene(s) RA OA RAF OR[95%CI] P OR[95%CI] P

Novellocirs12030576 1 115817221 NGF G T 0.65 1.07[1.05-1.09] 5.2×10−13 1.09[1.00,1.18] 0.03rs1209731 1 169324793 ATP1B1-F5 C T 0.98 1.19[1.12-1.26] 2.0×10−8 1.11[0.86,1.43] 0.44rs1595344 6 74611632 CD109 G A 0.66 1.05[1.03-1.07] 1.2×10−8 1.04[0.96,1.13] 0.32rs2226158 6 125995467 HEY2 G A 0.47 1.05[1.03-1.07] 2.6×10−8 0.95[0.90,1.01] 0.16

rs6468654 8 100062724 OSR2-VPS13B C T 0.24 1.06[1.04-1.08] 2.5×10−8 1.00[0.93,1.08] 0.96

rs7924571 11 32350027 WT1 C A 0.77 1.06[1.04-1.08] 3.5×10−8 0.99[0.92,1.08] 0.90

rs13441059 X 70108889 TEX11-SLC7A3 A G 0.36 1.05[1.03-1.07] 4.1×10−8 1.01[0.93,1.09] 0.82

Previouslyreportedloci

rs80173514 1 22354538 CELA3A-CDC42 A C 0.15 1.13[1.10-1.15] 7.5×10−25 1.15[1.05,1.27] 2.7×10−3

rs17215781 6 152570274 SYNE1 A G 0.92 1.13[1.10-1.17] 1.5×10−14 1.16[1.01,1.33] 0.04rs9312658 4 56005526 KDR C A 0.67 1.07[1.05-1.09] 2.9×10−14 1.09[1.00,1.17] 0.03

rs10616649 11 30349488 FSHB-ARL14EP ACTCTA - 0.84 1.09[1.07-1.12] 4.0×10−14 0.94[0.85,1.04] 0.23

rs11674184 2 11721535 GREB1 T G 0.61 1.07[1.05-1.08] 1.4×10−12 0.99[0.92,1.07] 0.83rs6456259 6 19761718 ID4 G A 0.16 1.07[1.05-1.10] 2.1×10−9 0.95[0.86,1.04] 0.25

rs746628 17 63850547 CEP112 T C 0.69 1.06[1.04-1.08] 3.7×10−9 0.99[0.91,1.07] 0.70Genome-widesignificantlociinendometriosiscase-controlGWASChr:chromosome,Pos:position,RA:riskallele,OA:Otherallele,RAF:Riskallelefrequency,OR:Oddsratio,P:P-value


https://doi.org/10.1101/401448

Table4.Endometriosiscase-controlGWASassociationresultsforotherpreviouslyreportedloci

Endometriosiscase-controlGWAS Endometriosis-relatedinfertility

Variantid Chr Pos(hg19) Nearestgene(s) RA OA RAF OR[95%CI] P OR[95%CI] P

rs10859853rs4762326

1212

9561495395668951 FGD6-VEZT C

TTC

0.370.48

1.05[1.03,1.06]1.04[1.02,1.06]

1.1×10−7

1.6x10-61.04[0.96,1.12]1.02[0.96,1.08]

0.280.49

rs4870024rs1971256

66

151830769151816011

RMND1-ESR1

CC

TT

0.190.21

1.06[1.04,1.08]1.05[1.03,1.07]

1.8×10−7

9.2x10-70.95[0.88,1.03]0.96[0.90,1.04]

0.240.35

rs12700667 1 25901639 NPVF-NFE2L3 A G 0.75 1.04[1.02,1.06] 6.3×10−5 1.05[0.97,1.13] 0.24

rs1537377 9 22169700 CDKN2B-DMRTA1 C T 0.38 1.04[1.03,1.06] 1.6×10−6 0.97[0.90,1.05] 0.48

rs74491657 7 46947633 IGFBP3-TNS3 G A 0.90 1.02[1.00,1.04] 0.12 1.00[0.89,1.13] 0.95

rs519664 9 15246652 TTC39B T C 0.21 1.01[0.99,1.03] 0.34 0.96[0.89,1.04] 0.35AssociationresultsforotherpreviouslyreportedendometriosislociChr:chromosome,Pos:position,RA:riskallele,OA:Otherallele,RAF:Riskallelefrequency,OR:Oddsratio,P:P-value


https://doi.org/10.1101/401448

Table5.Toplociassociatedwithendometriosis-relatedinfertility

Variantid Chr Pos(hg19)Nearest

gene(s)RA OA RAF OR[95%CI] P

rs201312237 5 94607502 MCTP1 ATAG - 8.8×10−3 2.96[1.93-4.55] 2.3×10

−7

rs182207890 1 110356869EPS8L3-

CSF1C T 0.998 Inf[10-Inf] 3.3×10

−7

rs113318830 2 11897940 LPIN1 - AT 0.78 1.25[1.14-1.36] 3.7×10−7

rs796733043 7 125639265 GRM8 TATT - 0.82 1.32[1.18-1.47] 7.7×10−7

rs142956186 4 163162586 FSTL5 T C 1.7×10−3 Inf[11.17-Inf] 9.9×10

−7

Lociwithp<1×10−6withendometriosis-relatedinfertility

Chr:chromosome,Pos:position,RA:riskallele,OA:Otherallele,RAF:Riskallelefrequency,OR:Oddsratio,P:P-

value

Table6.TranscriptionfactorsbindingtheDNAregioncoveredbythecrediblesetaccordingtoChiP-seqintheEncodedata

Locus Transcriptionfactors

NGF AR,FOXP2,RFX3,SPI1

ATP1B1-F5 RFX3

CD109 CTCF,SETDB1,E2F4,FOS,RAD21,SMC3,CEBPB,EP300,JUND,MAFK,RFX5,TEAD4,

EGR1,NFYB

HEY2 BATF,GATA3,FOXA1,POLR2A,RFX3,CEBPB,TRIM28,CTCF,RAD21,EBF1,ATF2,

TAF7,GATA2,FOS,MAFF,MAFK,CHD1,GTF2F1,SETDB1

OSR2-VPS13B ARID3A,ATF1,ATF2,ATF3,BATF,BCL11A,CBX3,CCNT2,CHD1,CTPBP2,EGR1,ESR1,

EZH2,FOS,FOSL1,FOXA1,FOXA2,CEPBP,EP300,FOS,FOXM1,GABPB1,GATA1,

GATA2,GATA3,HDAC2,IRF1,IRF4,JUN,JUNB,JUND,MAFF,MAFK,MAX,MTA3,

MYC,NFKB1,NFIC,NR2F2,POLR2A,POU5F1,RCOR1,REST,RUNX3,SETDB1,SPDEF,

SPI1,STAT5A,SUZ12,TAF1,TAL1,TBL1XR1,TCF3,TCF7L2,TCF12,TEAD4,TRIM28,

ZNF143,ZNF217,ZNF263

WT1 AR,ARID3A,ATF3,CBX3,CEBPB,CTCF,E2F6,EP300,ESR1,FOS,FOXA1,GTF2F1,

IKZF1,JUND,MAFF,MAFK,MAX,MXI1,MYC,NR3C1,POLR2A,RAD21,RFX5,SIN3A,

SMARCC1,SMARCC2,SMC3,TAF1,USF1,USF2,ZNF143,ZNF263

TEX11-SLC7A3CEBPB,CTCF,ELF1,EP300,GABPB1,MXI1,NR2F2,PML,RCOR1,STAT5A,TAL1,

TEAD4,USF1,YY1


https://doi.org/10.1101/401448

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