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European Journal of Endocrinology -18-0256 caused by( andfoetaldevelopment.Theymaybe during embryonic Differences ordisordersof sex development(DSD)arise Introduction validation ineachDSDcase. genetic causesforDSD.Diagnosticinterpretationmust be performedwithutmostcautionandneedscarefulscientific field ofDSD.However, inadditiontocoveringknownDSDcandidategenes,WESand WGShelptoidentifynovel methodological developmentsthatarecurrentlyinthe transition frombasicsciencetoclinicalroutineserviceinthe genes providerapidandreliableresults.Wholeexomegenomesequencing(WESWGS)representvaluable molecular studiesofmonogenicDSDcausesoranalysiscopynumbervariations(CNV)both.Panelscandidate diagnostic DSDsubclassesandisthereforethemandatoryinitialstep.Subsequently, furtheranalysescomprise best practiceingeneticdiagnosisDSDpatients.Ascertainmentofthekaryotpyedefinesonethreemajor Science andTechnology) Action BM1303 ‘DSDnet’waswrittenbyleadingexpertsinthefieldandfocusesoncurrent of thoroughclinical,hormonalandgeneticworkups.ThispositionpaperEUCOST(EuropeanCooperationin in medicine.Itrequiresamultidisciplinaryteamconductingsynopticandcomplementaryapproachconsisting The differential diagnosisofdifferences ordisordersofsexdevelopment(DSD)belongstothemostcomplex fields Abstract Hospital, Glasgow, UK Giessen, Germany, and Laboratory forTranslational HormoneAnalytics,CenterofChildandAdolescentMedicine,Justus-Liebig-University, Germany, Endocrinology andDiabetes,DepartmentofPaediatricAdolescentMedicine,UniversityLübeck, 7 Pediatric EndocrinologyandDiabetes,UniversityHospitalofSchleswig-HolsteinChristianAlbrechtsUniversity, Kiel,Germany, Medicine ResearchUnit,GhentUniversity, Ghent,Belgium, Hospital, Western Bank,Sheffield, UK, Glasgow, UK, (CIBERER), InstitutodeSaludCarlosIII,Barcelona,Spain, 1 O Hiort L Audí COST ActionBM1303‘DSDnet’ sex development(DSD):positionpaperofEU the managementofdifferences/disorders of Approaches tomoleculargeneticdiagnosisin GENETICS INENDOCRINOLOGY Mammalian GeneticsUnit,MedicalResearchCouncil,HarwellInstitute,Oxfordshire,UK, Growth andDevelopmentResearchUnit,Vall d’HebronResearchInstitute(VHIR),CenterforBiomedicalonRareDiseases https://doi.org/ www.eje-online.org Review 1 8 , S F Ahmed 9 Division ofPediatricEndocrinologyandDiabetology, SteroidResearch&MassSpectrometryUnit, , S A Wudy 3 10.1530/EJE Academic UnitofChildHealth,DepartmentOncologyandMetabolism,UniversitySheffield, Sheffield Children’s 1 ) numericalorstructuralvariations insex 10 -18-0256 Department ofClinicalGenetics,LaboratoriesBuilding,QueenElizabethUniversity 9 2 and , N Krone R McGowan 4 4 Department ofPaediatricEndocrinology, GhentUniversityHospital,PaediatricsandInternal 3 , M Cools L Audíandothers Published byBioscientifica Ltd. 2 , 10 onbehalfoftheEUCOSTAction 4 Printed inGreat Britain , K McElreavey 2 Developmental EndocrinologyResearchGroup,UniversityofGlasgow, © 2018Theauthors 5 Human DevelopmentalGenetics,InstitutPasteur, Paris,France, 5 , P M Holterhus or activation), ( and/or genitaldevelopment (leadingtoinactivation chromosomes, ( DSD geneticdiagnosis 8 Division ofPaediatric 6 , A Greenfield 2 3 ) variations in genes involved in gonadal ) disorders in gonadal and/or adrenal Attribution 4.0International License. This workislicensed under a Downloaded fromBioscientifica.com at10/01/202110:41:05AM 7 , A Bashamboo (2018) Endocrinology European Journal of [email protected] Email to LAudí should beaddressed Correspondence 6 Division of 179 179 :4 , R197–R206 Creative Commons

R197 5 , –R206 via freeaccess European Journal of Endocrinology www.eje-online.org www.eje-online.org the prevalence of with a 46,XY is the prevalenceoffemales with a46,XYkaryotype diagnosis haslongbeenchallenging. extremely rare(seelistin ( accounts forapproximately90–95%of46,XXindividuals hyperplasia (CAH) dueto21-hydroxylase deficiency. It is congenitaladrenal individuals with 46,XXkaryotype recessive conditionassociatedwithmonogenicDSD in the sexchromosomes( remainder willhavestructuralornumericalanomalies of 10–15% a46,XXandthe 75% willhavea46,XYkaryotype, hypospadias ( about 5per1000births,with73%ofthembeingboys gonadal andgenitalsexes,theprevalenceofDSDreaches birth togetherwithalldiscordancesamongchromosomal, If DSD is considered as all types of atypical genitalia at of DSD General approach tothegeneticdiagnosis specialists withinEUCOSTActionBM1303‘DSDnet’. of atrulyEurope-wideconcertedactionleadingDSD in themoleculargeneticdiagnosisofDSDandisresult datasets. Thispositionpaperpresentscurrentbestpractice biochemical (hormonal)constellationandmolecular consisting inasynopticviewofclinicalphenotype, approach and requiresanintegratedmultidisciplinary DSD belongstothemostcomplexfieldsinmedicine this heterogeneous group of conditions. Diagnosis of that continuouslyimprovethediagnosticyieldin available, and therapidlydeveloping genetic technologies account theclinicalandbiochemicalphenotypes,if the approachtoageneticdiagnosisofDSD,takinginto it providesinformationaboutriskofrecurrence( Determining theaetiologyisoftenusefulforfamilies,as associated morbidityaswelllong-termoutcomes. of adrenalandgonadalfunction,cancerrisk, relation topossiblegenderdevelopment,assessment important asitmayinformpatientmanagementin is sought( and shouldbeanalysedwhenageneticcauseforDSD the foetalperiod.Manycausesaregeneticallydetermined changes thatarepredictedtodisruptgeneexpressionin wouldconsistofcasesresultingfromepigenetic category with genital development ( exogenous) or( steroidogenesis ( 13 Review ). Otherwell-characterisedmonogenic 46,XXDSDsare Data collectedbetween1966 and2014showedthat This paperproposespracticalrecommendationsfor 4 , 11 5 , 5 ). Among children with atypical genitalia, 6 1 ) endocrinedisruptorsthatcaninterfere , ), ( 7 ). Reachingamoleculardiagnosisis 4 ) maternalfactors(endogenousor 12 ). Themostfrequentautosomal al 1 Table 2 , 3 L Audíandothers ). A final, yet putative ), andtheirmolecular 8 , 9 , 10 ). the phenotype. A comprehensive list of genes known to may identifynovelgenesinvolvedinthedevelopmentof whole exome(WES)( improving diagnosticyield.Furtherinvestigationby of knownDSD-relatedgenessimultaneously, thereby high-throughput sequencing(HTS)screenlargepanels Newer moleculargeneticdiagnosticstrategiesemploying underlying pathogenesis maybemultifactorial ( increasing evidencethatasignificantproportionofthe with often low genotype–phenotype correlation and the large numberofknownDSD-relatedgenes( clinical andhormonalpresentationofmanyDSDs,the low ( reaching amoleculardiagnosisin46,XYDSDisrelatively adequately orientthemoleculardiagnosis,successin Even ifclinicalandbiochemicalinvestigationsmay 62 genesin46,XYand6146,XX( along withtheirchromosomallocations:itamountsto conditions associatedwithambiguousgenitalia)isreported resistance, isolatedurogenitalanomaliesandsyndromic insufficiency, disordered steroidogenesis, gonadal and secondary , primary be involvedinhuman46,XYand46,XXDSD(including ./0 000having a form of gonadal dysgenesis ( 1.5/100 associated with androgen insensitivity (AIS) and amonglive-bornfemales,with4.1/100 000 6.4/100 000 or adult endocrinology, urology or gynaecology, clinical team(paediatric working groups.Themultidisciplinary as ConsensusStatements by COSTActionBM1303 ( associated with DSD) are essential part of DSD evaluation testsandsteroidanalysisin conditions laboratory (analytical methodsandmatrices,harmonisation of quality oflife( assessment, assessment of psychological outcomes and status, urologicalandgynaecologicalfollow-up,somatic data collectionacrossagesfortheassessmentofgenital carein adulthood, parents, transition,multidisciplinary information andpsychologicalsupporttopatients comprise: informedconsent,diagnosticinvestigations, essential components of an individualised care plan and of DSD.Recommendationsregardingclinicalcareare DSD orbecausethephenotypesuggestspresence of adult personeitherbecause there isafamilyhistory neonate, infant or in aprepubertal child oradolescent/ the nearfuture( adrenal andreproductivedisorderswillbeidentifiedin development, itislikelythatmorecandidategenesfor andfoetal further advanceinknowledgeaboutembryonic DSD geneticdiagnosis 20 ). Suchrecommendations have alreadybeen published A diagnosisofDSDmayberequestedprenatally, ina 5 ). Thisismostlikelyduetotheheterogeneous 18 19 ). ). Also,steroidhormonediagnostics 17 Downloaded fromBioscientifica.com at10/01/202110:41:05AM ) orgenome(WGS)sequencing 179 9 :4 ). However, with al 1 Table 15 R198 , 16 14 via freeaccess ). ). ) European Journal of Endocrinology P-GD C-GD P-GD P-GD P-GD 2. 46,XYwithdisorders ofgonadaldevelopment:dysgenesis(GD),completeorpartial(C/P-GD) 1. 46,XXwithdisorders ofgonadaldevelopment:dysgenesis,ovotesticularDSD,testicularDSD Clinical diagnosis Table 1 Review C-GD orP-GD C-GD orP-GDovotesticular DSD orovotesticularDSD C-GD orP-GD C-GD orP-GD C-GD orP-GD C-GD orP-GD C-GD orP-GD C-GD orP-GD C-GD orP-GD C-GD orP-GD C-GD orP-GD C-GD orP-GD C-GD orP-GD Ovaries orC-GD Ovotesticular DSDortesticular Ovotesticular DSD Gonadal dysgenesis Testicular DSD Gonadal dysgenesis Testicular DSD Gonadal dysgenesis C-GD (2) Testicular DSD (1) OvotesticularDSD (2) Testicular DSD (1) OvotesticularDSD (2) Testicular DSD (1) OvotesticularDSD (2) Testicular DSD (1) OvotesticularDSD (3) Testicular DSD (2) OvotesticularDSD (1) Gonadaldysgenesis Genes involvedinmonogenicdisorders/different sexdevelopment(DSD). L Audíandothers ZNRF3 ZFPM2 WT1 WNT4 TSPYL1 SRY SOX9 NR5A1 MAP3K1 HHAT GATA4 FGFR2 ESR2 EMX2 DMRT1 DHH DAX1 CBX2 ATRX ARX WNT4 SRY SOX10 SOX9 SOX3 RSPO1 NUP107 (3) (2) (1) NR2F2 FOXL2 FGF9 BMP15 Gene Arg NR5A1 NR5A1 NR5A1 (Yp11.2) (Yp11.2) (Xp21.3) (11p.13) (12q13.12) (14q23.2–q23.3) 92Trp) (13q12.11) (17q25.3) (Xq21.1) (17q24.3) (17q24.3) (Xq27.1) (locus) (1q32.2) ( (10q26.11) (1p36.12) (1p36.12) (10q26.13) (15q26.2) (22q12.1) (1p34.3) (3q22.3) (22q13.1) ( (8p23.1) (9q33.3) (Xp11.22) NR0B1 (6q22.1) (9p24.3) (12q15) FOG2 ( MEKK1 ( (9q33.3)( (9q33.3) p.Arg ) (8q23.1) ) (Xp.21) 92Trp) ) (5q11.2) p. DSD geneticdiagnosis 609136 (AD:dup)(Waardenberg andHirschsprungsyndromes, 278850 (AD:dup) 313430 (XL:dup) 610644 (AR)(palmoplantarhyperkeratosis,squamouscell 607617 (AR)(describedinconsanguineousfamily;other 617480 (AD) 612964 (AD) 615779 (AD)(congenitalheartdefects,congenital 608996 (AD)(blepharophimosis,epicanthusinversusand 600921 (AD:dup)(singlecasedescription) 300510 (D) OMIM 612062 (AD) 616067 (AD)(withorwithoutcongenital heartdisease) (3) 136680(splicing:Frasiersyndrome) (2) 194080(inactivation:Denys-Drashsyndrome) (1) 194072(del11.p13:WAGR syndrome) 607102 (AD) 603490 (AD:dup) 608800 (AR)(suddeninfantdeathwithdysgenesisofthe 400044 (D) 114290 (AD)(campomelicdysplasia) 612965 (AD)/(AR)(rarelyprimaryadrenalfailure, 613762 (AD) 605743 (AR)(singlefamilialcasedescription)(shortstature, 615542 (AD)(withorwithoutcongenitalheartdisease) 176943 (AD)(cranyosinostosis) 601663 (biallelicandmonoallelic) 600035 (AD:del)(intellectualdeficiency, kidneyagenesis) 602424 (AD:del)(withorwithoutintellectualdeficiency) 233420/607080 (AR/AD)(minifascicularneuropathy) 300018 (XL:dup) 613080 (AR) 300032 (D:del)(intellectualdeficiency, 300215 (XL:D)(, epilepsy, intellectualdeficiency) 611812 (AR):SERKAL(sexreversaldysgenesisofkidneys, 158330 (AD) 400045 (T) carcinoma ofskin) phenotypes withnephroticsyndrome) inversus syndrome) diaphragmatic hernia,blepharo-phimosis-ptosis-epicanthus ptosis, typesIandII) testes, SIDDTsyndrome) hypogonadotropic ) intellectual deficiency) generalised chondrodysplasia,musclehypertophy, myopia, adrenals andlung)syndrome,lethalwhenbiallelic peripheral neuropathy) (inheritance)

(additional phenotype) Downloaded fromBioscientifica.com at10/01/202110:41:05AM α 179 -thalassemia) www.eje-online.org www.eje-online.org :4 (Continued) R199 via freeaccess European Journal of Endocrinology www.eje-online.org X-linked. number variation;D,dominant;Del, deletion;DSD,different sexdevelopment; Dup,duplication;P-GD,partialgonadaldysgenesis;T, translocation;XL, ??, unknown;AD,autosomaldominant; AR,autosomalrecessive;CAH,congenitaladrenalhyperplasia;C-GD, completegonadaldysgenesis;CNV, copy 6. 46,XXDSDwithMüllerianductabnormalities 5. 46,XYDSDwithabnormalanti-Müllerianhormonesecretion oraction Cryptorchidism 5 17 4. 46,XYDSDwithabnormalandrogen synthesisoractionisolatedhypospadiascryptorchidism 3. 46,XXDSDwithandrogen excess Clinical diagnosis Table 1 Hand-foot- syndrome Müllerian Aplasiaandhyperandrogenism MRKH (Mayer-Rokitansky-Küster-Hauser) MURCS (MüllerianAplasia,Renalaplasia, Persistent MüllerianductsyndrometypeII Persistent MüllerianductsyndrometypeI Isolated hypospadias X-linked hypospadias Partial (PAIS) Complete (CAIS) Androgen insensitivity: Backdoor steroidogenesisdeficiency Cytochrome b5deficiency P450-oxidoreductase deficiency CAH with3 CAH withcholesteroldesmolasedeficiency STAR deficiency(LipoidCAH) 7-Dehydro- desmolasedeficiency LH/CG insensitivity Abnormal LH Glucocorticoid insensitivity Oestrogen insensitivity P450-oxidoreductase deficiency CAH with11 CAH with21-hydroxylasedeficiency CAH with3 Review α syndrome, typesIandII syndrome Cervico-thoracic somiteabnormalities) (17-keto-reductase) deficiency type 2deficiency type 2deficiency (2) Isolated17,20-lyasedeficiency (1) CAHwithcombined17 (2) Non-classicalform (1) Classicalform -Reductase type2deficiency β hydroxylase/17,20-lyase deficiency -Hydroxysteroid-dehydrogenase type3 Continued. β β -hydroxysteroid dehydrogenase -hydroxysteroid dehydrogenase β -hydroxylase deficiency L Audíandothers HOXA13 WNT4 CNV at17q12,1q21.1, multigenic AMHR2 AMH RXFP2 INSL3 ATF3 MAMLD1 AR AKR1C4 AKR1C2 SRD5A2 HSD17B3 CYB5A POR CYP17A1 HSD3B2 CYP11A1 STAR DHCR7 LHCGR LHB GR ESR1 CYP19A1 POR CYP11B1 CYP21A2 HSD3B2 Gene 22q11.21, Xq21.31 (13q13.1) α (Xq12) (19q13.33) ( (7q11.23) (7q11.23) (6q25.1–q25.2) (1q32.3) (8p11.23) (19p13.3) NR3C1 (locus) (19p13.11) (1p36.12) ( (2p16.3) (18q22.3) (11q13.4) LGR8/GREAT/GPR106 (12q13.13) (1p12) (1p12) (2p23.1) (10p15.1) (10p15.1) (7p15.2) (8q24.3) (9q22.32) (15q24.1) (15q21.2) (6p21.33) (10q24.32) ( CXOrf6 ) 5q31.3 ) (Xq28) DSD geneticdiagnosis ) 201810 (AR)(adrenalandgonadaldeficiency) 613743 (AR)(adrenalandgonadaldeficiency) (2) Adrenaldeficiency (1) Adrenalandgonadaldeficiency 201710 (AR) 270400 (AR)(Smith-Lemli-Opitzsyndrome) 238320 (AR)(Leydigcellaplasia,hypoplasia) 228300 (AR)(bioinactiveLH) 615962 (AD)(hypertension) 615363 (AR)(overgrowth,osteoporosis, 613546 (AR)(maternalandfoetalvirilisation) 201750 (AR)(antley-Bixlersyndrome, 202010 (AR)(adrenaldeficiency) 201910 (AR)(adrenaldeficiency) 201810 (AR)(adrenalandgonadaldeficiency) OMIM 140000 (AD) 158330 (AD) 277000 601076 261550 (AR) 261550 (AR) 606655 (AD??) 219050 (AD) 603148 (AD??) 300758 (XL)(hypospadias) 300068/312300/300633 (XL) DSD withDHTdeficiencyandapparent17,20- 614279 (AR) 264600 (AR) 264300 (AR)(Gonadaldeficiency) 250790 (AR)(methemoglobinemiatypeIV) 201750 (AR)(Antley-Bixlersyndrome) (2) Gonadaldeficiency deficiency CAH +hypertensiongonadal (1) 202110 (AR) polycystic ovarysyndrome) craniosynostosis ys deficiency +normal lyase (inheritance) Downloaded fromBioscientifica.com at10/01/202110:41:05AM

(additional phenotype) ± ) CYP17A1 179 :4 and SRD5A2 R200 via freeaccess European Journal of Endocrinology carrier statusofaknown genetically determinedDSD unavailable, thepresenceof weeks.Ifthistechniqueis sample collectionfrom7 diagnostic laboratoriesrecommend maternalblood cell-free foetalDNAfromthe 6thweek( SRY identify Ychromosome-specificmarkers(onewithinthe genetic sex. Non-invasive prenatal diagnosis (NIPD) can gestation). Thefirstapproachistodeterminethefoetal the country’s legislation,theaetiologyandweeksof parents considerpregnancyinterruption(dependingon genetic sex.Anurgentdiagnosisisrequiredifthe (US) appearance that maybe discordant with the foetal oronthefoetalgenital ultrasound on familyhistory published ( chromosomal DSDsandtestingmethodshavebeen analysis isrequired.Detailsondifferenttypesofsex If sexchromosomeDSDisidentifiednofurthergenetic be lesseffectiveatdetectingsexchromosomemosaicism. detect structural chromosomal rearrangements and may thesetechniqueswillnot Unlikekaryotype of 5–10 days. array, withmuchfasterturn-aroundtimesthanG-banding array-comparative genomichybridisation(aCGH)orSNP hasbeenreplacedby DSD. Insomecentreskaryotyping categories: sexchromosomeDSD,46,XXDSDor46,XY the initialclassificationofanyDSDintoonethree technique (involving G-banding) and isessentialin isacytogenetic hybridisation. Theanalysisofkaryotype This technique has largely replaced fluorescence days. chromosomes andhasaturn-aroundtimeof1–2 QFPCRdetectsaseriesofmarkersonthesex karyotype. fluorescence polymerasechainreaction(QFPCR)and confirming thechromosomalsexusingquantitative first-line investigation of an individual with DSD involves Besides carefulclinicalevaluationofthepatientwithDSD, Chromosomal sex be soughtbyusingthemostadequatemoleculartesting. or absenceofconsanguinity, amoleculardiagnosiswill ofDSDandthepresence constellation, thefamilyhistory Depending onthis,theclinicalphenotype,hormonal subclasses according to establishedDSDclassification ( assignment ofaDSDcasetoonethethreemajor determined attheoutsetbecausethisisbasisfor ( that couldhelpinclarifyingthepossiblegeneticcauses should beabletoprovideknowledgeandtechnologies biochemistry, genetics,radiology, occasionallypathology) i. 1 Fig. Review gene)usingQFPCRinmaternalperipheralblood A DSDmaybesuspectedintheprenatalperiodbased ). Forthis,thechromosomalsexshouldbe 9 ). SRY L Audíandothers , the karyotype andthe , thekaryotype 21 ), butmost in situ 19 ).

results) inanintegrativemanner. and geneticresults(thekaryotype andthecandidategene considered inparallelwiththe biochemical(hormonal)data approach inwhichtheinformation onclinicalphenotypingis biochemical guidance.(B)The recommended multidisciplinary targeted genetictestareoftenonlyperformedafter the diagnosisinastepwisestratification.Inparticular, development (DSD).(A)Thetraditionalpathwayapproaches Diagnostic approachestodifferences/disorders ofsex Figure 1 cases, althoughtheyaremoreprevalentinsyndromic involvement. CNVsmaybepresentinone-fifthofDSD the presence of associated malformations or other system is usedaspartofsecond-lineinvestigationsforDSDin butinothersaCGHorSNParray the traditionalkaryotype ( karyotype or microdeletionsbelowthethresholdofastandard or SNParrayallowsthedetectionofmicroduplications Higher resolution chromosome analysis through aCGH Copy numbervariationanalysis earlier gestationalages. termination ofpregnancyandasitcanbeperformedat offering thistestasisnotassociatedwithriskof NIPD andshouldbesoughtincollaborationwithcentres Nowadays,thepreferredapproachisby 15 to20 weeks). (from 9thto11thweeks)orinamnioticfluidcells condition can beinvestigated on a chorionic villus biopsy techniques maydetectintragenicdeletionsthatnot other moredatedplatforms,suchasoligo-aCGH.These general SNParraycandetectsmallerregionsofCNVthan while aCGHisabletodetectCNValongthegenome.In intragenic andwholegeneCNVinspecifictargetsites (MLPA), SNParrayoraCGH:MLPA isdesignedtodetect by multiplexligation-dependentprobeamplification DSD ( as deletionsof 46,XX DSDandof effect suchasduplicationsof development havebeenshowntoexertadose-dependent forms ( DSD geneticdiagnosis Table 1 22 , 23 < ). Alterationsingenedosagemaybedetected 5 , Mb). Insomecentres,aCGHhasreplaced 24 ATRX ). Indeed, several genes regulating sex DAX1 , DMRT1 Downloaded fromBioscientifica.com at10/01/202110:41:05AM or WNT4 , FGF9 EMX2 in46,XYDSDaswell 179 , or SOX3 www.eje-online.org :4 WT1 or in46,XY SOX9 R201 in via freeaccess European Journal of Endocrinology www.eje-online.org prediction ofalikelycausative geneinDSD.Therefore, often variable,hampering classicalhypothesis-driven the clinicalappearanceand thehormonalpatternsare number ofgenescanbeconsidered causative.Moreover, Due to the highly variable aetiology of DSD, a large HTS techniques pathways. and basedonlocalnationalprovisionofdiagnostic and biochemicalphenotypespointtoaspecificgene by HTSofcandidategenesorexome,evenwhenclinical Individual genesequencingisincreasinglybeingreplaced – – – – – regions) ofacandidategenecouldbeperformedin: and sometimes of other regions of interest (i.e. promoter Sanger sequencing of exonic coding, flanking regions Sanger sequencing regions( non-coding regulatory In thenearfuture,WGSwillbeabletodetectCNVsin and thisisnotalwaysfeasiblewhenusingaCGHorMLPA. the codingregionsofgenesatanexon-levelresolution, the interpretationoftheseoftenchallengingresults( as theneedforcollaborationanddatasharingtofacilitate adequately resourced tointerpretgeneticfindingsaswell being highlights theimportanceofdiagnosticservices fact been reported in association with DSD ( clinical significance,uponreview, halfofthesehadin report threequarterswereclassifiedasbeingofuncertain third ofchildrenwithDSD( 26 always bedetectedbySangersequencingofgenes(

Review , if necessary based on laboratory qualitymanagement. basedonlaboratory if necessary Validation ofHTSdatainsinglecasesandtheirrelatives, qualitymanagement. laboratory laboratories (DSD panels, WES, WGS) as part of Validation ofnewlysetupHTSmethodsindiagnostic in afamily. Sanger sequencingtosegregateaspecificgenevariant deletions, etc.(e.g., to interpretduecomplexgenerearrangements, DSD genesinwhichHTSmethodsarecurrentlydifficult Targeted (hypothesis-driven)sequencingofknown SRD5A2 hormonal assessment (e.g., DSD genesfollowingmeaningfulclinicaland Targeted (hypothesis-driven)sequencingofknown 27 In addition,WEShasthepotentialtodetectCNVsin ). CNVshavebeenreportedtobepresentinone- , HSD17B3 , AR CYP21A2 andothers)( 21 29 ). L Audíandothers ) and,althoughinthis ). CYP21A2 30 ). , CYP11B1 28 ). This 22 28 , 25 ). , , currently has a facility to act as a secure repository for currently hasafacilitytoact asasecurerepository that are required for multidisciplinary interpretation, that arerequiredformultidisciplinary need forbioinformaticexpertise.Thespecialistresources However, thisdoesnottakeintoaccounttheincreased pure technicalcostsperbasearesignificantlydecreasing. for individualisedclinicalmanagement. sensitivity tosexsteroids),whichareimportantaspects bythegonads or bytheadrenals or the an individualDSDcase(e.g.,thepotentialofsteroid cannot reliablypredictthefunctionalconsequencesfor receptor geneandafewmore),moleculardiagnosis some (butnotall)DSD-relatedgenes(e.g.,theandrogen with theonlyexceptionofcertaindisruptivemutationsin the authorsofthisCOSTActionremindreadershipthat, increasingly regardedasthefirst-tierapproach.However, with arobustcoverageofallgenomicregionsinterestis analyse one geneatthe time. A panel of candidate genes sequencing methods,whicharelaboriousandcanonly HTS-based strategies are increasingly replacing traditional I-DSD Registry ( I-DSD Registry prospectively collectedin international databaseslike with clinicalandbiochemical datainDSDsshouldbe Action strongly recommends that molecular data together improved diagnostic algorithm in the future, this COST To allowbetterunderstandingofDSDs and,hence, inDSD. diagnosis mustbeconsideredcomplementary ( testing, startingwiththeinitialevaluationofpatient biochemical (hormonal)andgenetic(HTS-related) a moreparallelapproachinvolvingintegrationofboth classical sequential diagnostic pathway inDSD,favouring endocrine profiling ( and single gene variants does not always correlate with in suspected 46,XY DSD, the identification of CNVs Increasing experiencewithHTStechniquessuggeststhat when detectingvariantsofunknownsignificance(VUS). and clinicians need to be aware of problems associated DSD-related genes. However, laboratories the service based HTSstrategiesandallowtheidentificationofnew WES-based HTSstrategiesaremoreflexiblethanpanel- near future, based oncontinuoustechnical advances. likely thattheseHTSstrategieswillfurtherevolveinthe investigation ofaDSDhavebeendescribed( local availability. Strategiesforthestepwisemolecular number ofgenesonaselectedpanel,genecoverageand for selectingthebestHTSapproachwilldependon account when considering overall costs.The strategy or genomesequencingwillalsoneedtobetakeninto explanation andcounsellingofdatafollowingexome DSD geneticdiagnosis 5 , 32 HTS technologiesarecontinuouslyimprovingandthe ). Asstatedearlier, genetictestingandhormonal 33 ). The I-DSD Registry ( ). TheI-DSDRegistry 28 ). Altogether, this may change the Downloaded fromBioscientifica.com at10/01/202110:41:05AM 179 :4 www.i-dsd.org 8 , 31 R202 ). Itis via freeaccess ) European Journal of Endocrinology enzymedeficiencies andhormoneresistance causes willremainforaspecific setofphenotypes(i.e. variations (oligogenic)( individuals with DSD resultfrom a combination of gene most likely demonstrate that the phenotype of some regions( regulatory while WGSwillbeabletodetectCNVsinnon-coding has thepotentialtodetectCNVsincodingregions ( elements explained bynon-codingvariantsinregulatory missing geneticvariationinsevereformsofDSD,canbe FOXL2 anditcanbehypothesisedthat,atleastpartofthe transcription factorssuchasSRY, SOX9,NR5A1,and WES orWGS. and willhavetobecharacterisedonamolecularlevelby by in the patient genital skin fibroblasts ( type IIpatientswhen pathway may besuspected in the recently proposed AIS ( and promoterregionsHTSmayrevealpathogenicvariants and flanking region sequencesare normal,whole in previouslyunsuspectedgenes( undescribed phenotypessuchasfor ZNRF3 ( associated withDSDinhumanssuchas variations foundinDSD. facilitating the broadest view on molecular genetic appropriately andbioinformaticanalysisspeedup,thus quality further improves, technical hurdles be addressed first-line approach,providedthatpricesprogressivelyfall, will probablyincreaseitsuseasa many clinicalservices of missingvariantsinaDSDcandidategene.However, guarantee ofcoverage,thusincreasingthepossibility thorough bioinformaticanalysisandmayresultinalower DSD genepanelsisgiveninCools et al( all genes included in at least one of recently reported large selected patientseries( hypogonadism) variesaccordingtopanelcontentand 46,XY and 46,XX andeven with hypogonadotropic a variablenumberofgenesassociatedwithDSD(both obtained withcandidategenepanelsdesignedtoanalyse in otherlocalrepositories. act asafacilitythatcansignpostinvestigatorstodataheld detailed geneticdata.Alternatively, canalso thisregistry 39 52 50 Review ) thatcouldbedetectedbyWGS.Asstatedearlier, WES ). Inaddition,variantsinothergenesregulatingthe ), Many ofthegeneticvariantsinDSDencode When AISishighlysuspectedbut of genes previously not WES has afforded discovery A WESfirst-lineapproachwouldrequireamuchmore The experienceofdifferentauthorsreportingresults HHAT ( 44 ) andvariantsinknowngenespreviously ( 40 ), FGFR1 29 ApoD ). Inaddition,WESandWGS,will 34 ( 41 , 15 35 expressionisnotupregulated ), , , SOX8 16 36 ), althoughmonogenic L Audíandothers , 47 37 ( , , NR5A1 AR 42 48 38 ), , geneexoncoding 19 ). An overview of ). Anoverview 49 NR2F2 ). ). ( FOG2/ZFPM2 1 , 45 ( AR 43 , 46 ) and gene ) or 51 AR )

outcomes of eachpathway and the consequences for the broad approachinvolvingWESorWGS,thegenomic followed by either a targeted gene panel approach or a and confirmationofsexchromosomecomplement, considering the physical features, hormone findings ( are presentedbyCroft local genomicapproach.Themostgeneralapproaches or reproductive problems andpreferred or available ofDSD testes orMüllerianstructures,familyhistory presence ofassociatedmalformations,functioning data, chromosomalsex,initialhormonalevaluation, genetic diagnosisofDSD,dependingonavailableclinical A numberofalgorithmshavebeenpublishedtoguidethe Existing diagnosticalgorithms hypoplasia). syndromes such as complete AIS and Leydig cell aplasia/ genome sequencinginunrelated geneswillbereported phenotype, whilevariantsdetected byclinicalexomeand ‘uncertain significance’ in gene(s) related to the patient classified as ‘pathogenic’, ‘likely pathogenic’ and of the generalrecommendation istoreportvariants in genes that cause Mendelian disorders ( ‘likely benign’and‘benign’)todescribevariantsidentified ‘pathogenic’, ‘likelypathogenic’,‘uncertainsignificance’, variants andusespecificstandardterminology(such as (ACMG) guidelinesfortheinterpretationofsequence American College of and Genomics facilitate theinterpretationofVUS( tests andfunctionalanalyses( WGS approach and the need for second-line endocrine the results of a XY DSD candidate gene panel or of a WES/ biochemistry, clinical genetics and molecular genetics), team diagnosticassessment(clinical endocrinology, chromosome rearrangementrequiresamultidisciplinary pathway forinvestigating46,XYDSDcaseswithoutsex evaluation andgenomictesting( which guidefurtherchromosomalanalyses,hormonal genitalia, thepresenceofuterusandkaryotype presence of palpable gonads, the anatomy of the external approach ininfantswithatypicalgenitaliaconsidersthe results andthepresenceofafamilyhistory. Apractical functioning testesorMüllerianstructures,thegenepanel results ofa-CGHandsexchromosomes,thepresence considering thepresenceofassociatedmalformations, for extensivetestingthroughWESorWGS( patients. Moredetailedalgorithmsdeterminesuitability DSD geneticdiagnosis 5 ). TheirparadigmsbeginwithaDSDphenotype The majorityofaccreditedlaboratoriesfollowthe Downloaded fromBioscientifica.com at10/01/202110:41:05AM t al et . ( in silico 7 ) andAlhomaidah 9 5 ). Themostdetailed 179 ). and/or www.eje-online.org :4 53 ). At present, in vitro 31 R203 t al et ) by ) to via freeaccess . European Journal of Endocrinology www.eje-online.org access tomedicalattention. sufficient resources, avariablepercentage ofpersonslack even themostbasicanalyses; evenincountrieswith many worldcountriesorregions lackresources toperform professionals and the affected persons suffer inequalities: for eachgroupofrarediseases.Itisevidentthatboththe teams following recommendations by multidisciplinary should organisetheavailabilityofgeneticdiagnoses orregion adequate patient management. Each country ERN), withtheaimofcontributingtoavailability of of referencecentresforrareendocrinediseases(Endo- European Union has recently launched the coordination basis.The Organisations onacountry-by-country considered bytheprofessionalsandHealthSystem available knowledgeandtechnologiesaretobe basis oftheDSD. genes orincaseswithasuspectedoligogenic/polygenic forthecharacterisationofnovelDSD currently reserved be usedpreferably to analysecandidate genes. WGS is cause, aHTSpanelofcandidategenesorWESshould ( (hormonal) evaluationinparallelwiththegeneticstudy conduct thephysicalphenotypingandbiochemical a hypothesis-drivenbasisbySangersequencingorHTS. lead to a likely DSD target gene that can be sequenced on specific steroidprofileinurineand/orplasmamayrapidly or 3ß-hydroxysteroiddehydrogenasedeficiencies)( form of CAH such as 21-hydroxylase, 11ß-hydroxylase deficiencies, aswellin46,XXDSDandavirilising insufficiency (e.g.in46,XYDSDduetoStARorP450scc missing apotentiallylife-threateningacuteadrenal need comprehensive steroid analysis in order to avoid addition tothoseinvolvedinurogenitaldevelopment. phenotype includesmalformationsinothersystems diagnostics inboth46,XXand46,XY, especiallyifthe either chromosomalsexDSD,46,XXor46,XY. willfirstdeterminethesubgroupofDSD, The karyotype molecular diagnosisofDSD Recommendations forthegenetic phenotype becomesincreasinglycoherent. evolve as the relationship between the genotype and the robust genomic data in this field, these guidelines will findings( as secondary Fig. 1 Review These recommendationsarebasedoncurrently teamshould In allothercases,themultidisciplinary All neonatesandyoungbabiespresentingwithDSD A CNVanalysisshouldbeaddedtothefirst-line ). ExceptforconfirmingamonogenicfamilialDSD 54 , 55 ). With generationofmore L Audíandothers 20 ). A partner) appreciatesupportfromBMBSCOSTActionBM1303. (working group2),AB2)aswellRMcG(international group 3),KMcE(chairofworking2),PMH(workingAG (chair of working group 4), M C (chair of working group 1), N K (working COST Actionandworking group 3), S AW(chair of working group 3),S F A O H(chairofCOSTActionandchairworkinggroup5),LA(co-chair supported by COST (European Cooperation in Science and Technology). This articleisbaseduponworkfromCOSTActionBM1303DSDnet, Funding perceived asprejudicingtheimpartialityofthisreview. The authorsdeclarethatthereisnoconflictofinterestcouldbe Declaration ofinterest determination anddevelopment. care andexpandingknowledgeabouttheprocessofsex Registries. Databasesareimportantforimprovingpatient international databasesliketheI-DSDandI-CAH (when available)inDSDs,shouldbeaccessiblethrough with phenotypic,biochemicalandoutcomedata References submitted manuscriptandapprovedsubmission. All theauthorshaveacceptedresponsibilityforentirecontentofthis Author contributionstatement DSD geneticdiagnosis 8 7 6 5 4 3 2 1 Kyriakou A, Lucas-Herald AK,McGowan R, Tobias ES & Ahmed SF. Croft B, Ayers K, Sinclair A&Ohnesorg T. Reviewdisordersof sex Bashamboo A, Ledig S,Wieacker P, Achermann JC&McElreavey K. Alhomaidah D, McGowan R&Ahmed SF. Thecurrentstateof Hutson JM, Grover SR,O’Connell M&Pennell SD.Malformation Agopian AJ, Langlois PH,Ramakrishnan A&Canfield MA. Bouty A, Ayers KL, &Sinclair AH.Thegeneticand Pask A,Heloury Y Bashamboo A, Eozenou C,Rojo S&McElreavey K.Anomaliesin 165–177. challenges inmanagement. Disorders ofsexdevelopment:advances ingeneticdiagnosisand 337–350. discovery. development: theevolvingroleofgenomicsindiagnosisandgene 213–224. of disorderssexdevelopment(DSD). New technologiesfortheidentificationofnovelgeneticmarkers Genetics diagnostic geneticsforconditionsaffectingsexdevelopment. nrendo.2014.83) Reviews Endocrinology syndromes associatedwithdisordersofsexdevelopment. 943–949. in Texas. Epidemiologic featuresofmalegenitalmalformationsandsubtypes 2015 environmental factorsunderlyinghypospadias. 2017 genetic interactionsofearlygonaddevelopment. human sexdeterminationprovideuniqueinsightsintothecomplex Genetic informationandmoleculardata,together 9 91 239–259. 2017 143–156. (https://doi.org/10.1002/bdrc.21148) American Journal of Medical Genetics Part A ofMedicalGeneticsPart American Journal (https://doi.org/10.2147/AGG.S53226) (https://doi.org/10.1159/000314917) (https://doi.org/10.1002/ajmg.a.36389) Birth Defects Research Part C: Embryo Today DefectsResearch C:Embryo Birth Part 91 157–162. (https://doi.org/10.1159/000441988) (https://doi.org/10.1111/cge.12932) 2014 Downloaded fromBioscientifica.com at10/01/202110:41:05AM (https://doi.org/10.1111/cge.12912) 10 Advances inGenomicsandGenetics 476–487. Sexual Development (https://doi.org/10.1038/ 179 :4 Sexual Development Clinical Genetics 2014 2016 164A Nature 2010 108 R204 2015 Clinical

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