Approaches to Molecular Genetic Diagnosis in the Management of Differences/Disorders of Sex Development (DSD): Position Paper of EU COST Action BM 1303 "Dsdnet"

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Article: Audí, L., Ahmed, S.F., Krone, N. orcid.org/0000-0002-3402-4727 et al. (8 more authors) (2018) Genetics In Endocrinology: Approaches to molecular genetic diagnosis in the management of differences/disorders of sex development (DSD): position paper of EU COST Action BM 1303 "DSDnet". European Journal of Endocrinology, 179 (4). R197-R206. ISSN 0804-4643 https://doi.org/10.1530/EJE-18-0256

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[email protected] https://eprints.whiterose.ac.uk/ European Journal of Endocrinology caused by( andfoetaldevelopment.Theymay be during embryonic Differences ordisordersof sex development(DSD)arise Introduction validation ineachDSDcase. genetic causesforDSD.Diagnosticinterpretationmust be performedwithutmostcautionandneedscarefulscientiic ield ofDSD.However, inadditionto coveringknownDSDcandidategenes,WESandWGShelptoidentifynovel methodological developmentsthatarecurrentlyinthe transition frombasicsciencetoclinicalroutineserviceinthe genes providerapidandreliableresults.Wholeexomegenomesequencing(WESWGS)representvaluable molecular studiesofmonogenicDSDcausesoranalysiscopynumbervariations(CNV)both.Panelscandidate diagnostic DSDsubclassesandisthereforethemandatoryinitialstep.Subsequently, furtheranalysescomprise best practiceingeneticdiagnosisDSDpatients.Ascertainmentofthekaryotpyedeinesonethreemajor Science andTechnology) Action BM1303‘DSDnet’waswrittenbyleadingexpertsintheieldandfocusesoncurrent of thoroughclinical,hormonalandgeneticworkups.ThispositionpaperEUCOST(EuropeanCooperationin in medicine.Itrequiresamultidisciplinaryteamconductingsynopticandcomplementaryapproachconsisting The differential diagnosisofdifferences ordisordersofsexdevelopment(DSD)belongstothemostcomplexields Abstract Hospital, Glasgow, UK Giessen, Germany, and Laboratory forTranslational HormoneAnalytics,CenterofChildandAdolescentMedicine,Justus-Liebig-University, Germany, Endocrinology andDiabetes,DepartmentofPaediatricAdolescentMedicine,UniversityLübeck, L Audí COST ActionBM1303‘DSDnet’ sex development(DSD):positionpaperofEU the managementofdifferences/disorders of Approaches tomoleculargeneticdiagnosisin GENETICS INENDOCRINOLOGY O Hiort (CIBERER), InstitutodeSaludCarlosIII,Barcelona,Spain, 1 7 Pediatric EndocrinologyandDiabetes,UniversityHospitalofSchleswig-HolsteinChristianAlbrechtsUniversity, Kiel,Germany, Medicine ResearchUnit,GhentUniversity, Ghent,Belgium, Hospital, Western Bank,Shefield, UK, Glasgow, UK, Growth andDevelopmentResearchUnit,Vall d’HebronResearchInstitute(VHIR),CenterforBiomedicalonRareDiseases Mammalian GeneticsUnit,MedicalResearchCouncil,HarwellInstitute,Oxfordshire,UK, https://doi.org/10.1530/EJE-18-0256 www.eje-online.org Review 1 8 , S F Ahmed 9 Division ofPediatricEndocrinologyandDiabetology, SteroidResearch&MassSpectrometryUnit, , S A Wudy 3 Academic UnitofChildHealth,DepartmentOncologyandMetabolism,UniversityShefield, Shefield Children’s 1 ) numericalorstructuralvariations insex 10 Department ofClinicalGenetics,LaboratoriesBuilding,QueenElizabethUniversity 9 2 and , N Krone R McGowan 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 chromosomes, ( or activation), ( and/or genitaldevelopment (leadingtoinactivation DSD geneticdiagnosis 8 Division ofPaediatric 6 , A Greenield 2 3 ) variations in genes involved in gonadal ) disorders in gonadal and/or adrenal This workislicensed under a Attribution 4.0International License. Downloaded from Bioscientifica.com at 08/10/2020 10:49:42A 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 University Sheffield M European Journal of Endocrinology www.eje-online.org www.eje-online.org with genital development ( exogenous) or( it providesinformationaboutriskofrecurrence( Determining theaetiologyisoftenusefulforfamilies,as associated morbidityaswelllong-termoutcomes. of adrenalandgonadalfunction,cancerrisk, relation topossiblegenderdevelopment,assessment important asitmayinformpatientmanagementin the prevalence of females with a 46,XY karyotype is the prevalenceoffemales with a46,XYkaryotype diagnosis haslongbeenchallenging. extremely rare(seelistin ( accounts forapproximately90–95%of46,XXindividuals hyperplasia (CAH) dueto21-hydroxylase deiciency. It is congenitaladrenal individuals with 46,XX karyotype recessive conditionassociatedwithmonogenicDSD in the sexchromosomes( remainder willhavestructuralornumericalanomalies of 10–15%a46,XXandthe 75% willhavea46,XYkaryotype, is sought( and shouldbeanalysedwhenageneticcauseforDSD the foetalperiod.Manycausesaregeneticallydetermined changes thatarepredictedtodisruptgeneexpressionin wouldconsistofcasesresultingfromepigenetic category steroidogenesis ( 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 belongstothemostcomplexieldsinmedicine this heterogeneous group of conditions. Diagnosis of that continuouslyimprovethediagnosticyieldin available, and therapidlydeveloping genetic technologies account theclinicalandbiochemicalphenotypes,if the approachtoageneticdiagnosisofDSD,takinginto 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 inal, yet putative ), andtheirmolecular 8 , 9 , 10 ). quality oflife( assessment, assessment of psychological outcomes and status, urologicalandgynaecologicalfollow-up,somatic data collectionacrossagesfortheassessmentofgenital careinadulthood, 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/ ( associated with DSD) are essential part of DSD evaluation testsandsteroidanalysisinconditions laboratory (analytical methodsandmatrices,harmonisation of large numberofknownDSD-relatedgenes( clinical andhormonalpresentationofmanyDSDs,the 62 genesin46,XYand6146,XX( along withtheirchromosomallocations:itamountsto conditions associatedwithambiguousgenitalia)isreported resistance, isolatedurogenitalanomaliesandsyndromic insuficiency, disordered steroidogenesis, androgen gonadal and secondary gonadal dysgenesis, primary be involvedinhuman46,XYand46,XXDSD(including the phenotype. A comprehensive list of genes known to may identifynovelgenesinvolvedinthedevelopmentof 000having a formof gonadal dysgenesis ( 1.5/100 associated with androgen insensitivity (AIS) and among live-bornfemales,with4.1/100 000 6.4/100 000 the nearfuture( adrenal andreproductivedisorderswillbeidentiiedin development, itislikelythatmorecandidategenesfor andfoetal further advanceinknowledgeaboutembryonic whole exome(WES)( improving diagnosticyield.Furtherinvestigationby of knownDSD-relatedgenessimultaneously, thereby high-throughput sequencing(HTS)screenlargepanels Newer moleculargeneticdiagnosticstrategiesemploying underlying pathogenesis maybemultifactorial ( increasing evidencethatasigniicantproportionofthe with often low genotype–phenotype correlation and the low ( reaching amoleculardiagnosisin46,XYDSDisrelatively adequately orientthemoleculardiagnosis,successin Even ifclinicalandbiochemicalinvestigationsmay or adult endocrinology, urology or gynaecology, clinical team(paediatric working groups.Themultidisciplinary as ConsensusStatements by COSTActionBM1303 DSD geneticdiagnosis 20 ). Suchrecommendations have alreadybeen published A diagnosisofDSDmayberequestedprenatally, ina 5 ). Thisismostlikelyduetotheheterogeneous 18 19 ). ). Also,steroidhormonediagnostics 17 Downloaded from Bioscientifica.com at 08/10/2020 10:49:42A ) orgenome(WGS)sequencing 179 9 :4 ). However, with al 1 Table 15 via University Sheffield R198 , 16 14 ). ). ) M European Journal of Endocrinology 3 etclrDD(3) (3)Testicular DSD Gonadaldysgenesis OvotesticularDSD (1)OvotesticularDSD (1)OvotesticularDSD (2)Testicular DSD C-GD orP-GD Clinical diagnosis Table 1 OvotesticularDSDortesticular (2)Testicular DSD (1)OvotesticularDSD Gonadaldysgenesis 1. 46,XXwithdisorders ofgonadaldevelopment:dysgenesis,ovotesticularDSD,testicularDSD Testicular DSD (1)OvotesticularDSD (2)Testicular DSD Gonadaldysgenesis Testicular DSD 1 oaa ygnss(1) (1)Gonadaldysgenesis P-GD 2. 46,XYwithdisorders ofgonadaldevelopment:dysgenesis(GD),completeorpartial(C/P-GD) (2)Testicular DSD 2 vtsiua S (2) (2)OvotesticularDSD P-GD OvariesorC-GD C-GDorP-GD C-GDorP-GD C-GDorP-GD P-GD C-GDorP-GD C-GDorP-GD C-GDorP-GD C-GD C-GDorP-GD C-GDorP-GD C-GDorP-GD C-GDorP-GD C-GDorP-GD OvariesorovotesticularDSD P-GD C-GD orP-GDovotesticularDSD Review C-GD Genes involvedinmonogenicdisorders/different sexdevelopment(DSD). L Audíandothers ZNRF3 NUP107 Gene RSPO1 SOX3 SOX9 SOX10 SRY BMP15 FGF9 WNT4 FOXL2 NR2F2 ARX ATRX CBX2 DAX1 DHH DMRT1 EMX2 ESR2 GATA4 FGFR2 HHAT MAP3K1 NR5A1 SOX9 SRY TSPYL1 WNT4 WT1 ZFPM2 Arg NR5A1 NR5A1 NR5A1 (p12 400045(T) (Yp11.2) (p12 400044(D) (Yp11.2) (p13 300215(XL:D)(Lissencephaly, epilepsy, intellectualdeiciency) (Xp21.3) (1.3 (1)194072 (del11.p13:WAGR syndrome) (11p.13) (21.2 233420/607080 (AR/AD)(minifascicularneuropathy) (12q13.12) (42.–2.)601663(biallelicandmonoallelic) (14q23.2–q23.3) 92Trp) (31.1 600921(AD:dup)(singlecasedescription) (13q12.11) (72.)613080(AR) (17q25.3) (q11 300032(D:del)(intellectualdeiciency, (Xq21.1) (q71 313430(XL:dup) (Xq27.1) (72.)278850(AD:dup) (17q24.3) (72.)114290 (AD)(campomelicdysplasia) (17q24.3) (locus) (q22 605743(AR)(singlefamilialcasedescription)(shortstature, (1q32.2) ( (02.1 600035(AD:del)(intellectualdeiciency, kidneyagenesis) (10q26.11) (p61)158330(AD) (1p36.12) (p61)603490(AD:dup) (1p36.12) (02.3 176943 (AD)(cranyosinostosis) (10q26.13) (22q12.1) (52.)615779 (AD)(congenitalheartdefects,congenital (15q26.2) (p43 610644(AR)(palmoplantarhyperkeratosis,squamouscell (1p34.3) (q23 608996(AD)(blepharophimosis,epicanthusinversusand (3q22.3) (21.)609136(AD:dup)(Waardenberg andHirschsprungsyndromes, (22q13.1) ( (p31 615542(AD)(withorwithoutcongenitalheartdisease) (8p23.1) (q33 612965 (AD)/(AR)(rarelyprimaryadrenalfailure, (9q33.3) (p12)300510(D) (Xp11.22) NR0B1 (q21 608800 (AR)(suddeninfantdeathwithdysgenesisofthe (6q22.1) (p43 602424(AD:del)(withorwithoutintellectualdeiciency) (9p24.3) (21)607617(AR)(describedinconsanguineous family;other (12q15) FOG2 ( MEKK1 ( (q33 612964(AD) (9q33.3) (9q33.3)( p.Arg 82.)616067(AD)(with orwithoutcongenitalheartdisease) ) (8q23.1) X.1 300018(XL:dup) ) (Xp.21) 92Trp) 51.)613762(AD) ) (5q11.2) p. DSD geneticdiagnosis 617480 (AD) OMIM 612062 (AD) 611812 (AR):SERKAL(sexreversaldysgenesisofkidneys, 607102 (AD) (3) 136680(splicing:Frasiersyndrome) (2) 194080(inactivation:Denys-Drashsyndrome) phenotypes withnephroticsyndrome) carcinoma ofskin) peripheral neuropathy) ptosis, typesIandII) inversus syndrome) diaphragmatic hernia,blepharo-phimosis-ptosis-epicanthus adrenals andlung)syndrome,lethalwhenbiallelic intellectual deiciency) generalised chondrodysplasia,musclehypertophy, myopia, hypogonadotropic hypogonadism) testes, SIDDTsyndrome) (inheritance)

(additional phenotype) Downloaded from Bioscientifica.com at 08/10/2020 10:49:42A α 179 -thalassemia) www.eje-online.org www.eje-online.org :4 (Continued) via University Sheffield R199 M European Journal of Endocrinology P450-oxidoreductasedeiciency (2)Isolated17,20-lyasedeiciency (1)CAHwithcombined17 www.eje-online.org Cytochromeb5deiciency PersistentMüllerianductsyndrome typeII Müllerian Aplasiaandhyperandrogenism MRKH(Mayer-Rokitansky-Küster-Hauser) MURCS(MüllerianAplasia,Renalaplasia, 6. 46,XXDSDwithMüllerianductabnormalities CAHwith3 17 5 STAR deiciency(LipoidCAH) LH/CGinsensitivity Backdoorsteroidogenesisdeiciency CAHwithcholesteroldesmolasedeiciency (2)Non-classicalform (1)Classicalform 7-Dehydro-cholesteroldesmolasedeiciency AbnormalLH 4. 46,XYDSDwithabnormalandrogen synthesisoractionisolatedhypospadiascryptorchidism Glucocorticoidinsensitivity Cryptorchidism PersistentMüllerianductsyndrome typeI 5. 46,XYDSDwithabnormalanti-Müllerianhormonesecretion oraction X-linkedhypospadias Partial(PAIS) Complete(CAIS) Androgeninsensitivity: Cryptorchidism Isolatedhypospadias Oestrogeninsensitivity X-linked. number variation;D,dominant;Del, deletion;DSD,different sex development;Dup,duplication;P-GD,partialgonadaldysgenesis;T, translocation;XL, ??, unknown;AD,autosomaldominant; AR,autosomalrecessive;CAH,congenitaladrenalhyperplasia;C-GD, completegonadaldysgenesis;CNV, copy Hand-foot-uterus syndrome Clinical diagnosis Table 1 Aromatasedeiciency P450-oxidoreductasedeiciency CAHwith11 CAHwith3 3. 46,XXDSDwithandrogen excess CAHwith21-hydroxylasedeiciency Review α syndrome, typesIandII syndrome Cervico-thoracic somiteabnormalities) type 2deiciency (17-keto-reductase) deiciency type 2deiciency -Reductase type2deiciency β hydroxylase/17,20-lyase deiciency -Hydroxysteroid-dehydrogenase type3 Continued. β β -hydroxysteroid dehydrogenase -hydroxysteroid dehydrogenase β -hydroxylase deiciency L Audíandothers POR CYB5A AMHR2 utgnc601076 WNT4 CNV at17q12,1q21.1, multigenic SRD5A2 CYP17A1 HSD3B2 HSD17B3 STAR LHCGR AKR1C4 AKR1C2 CYP11A1 DHCR7 LHB GR RXFP2 AMH MAMLD1 AR INSL3 ATF3 ESR1 HOXA13 Gene CYP19A1 POR CYP11B1 HSD3B2 CYP21A2 22q11.21, Xq21.31 (13q13.1) α (q2 300068/312300/300633(XL) (Xq12) (91.3 228300 (AR)(bioinactiveLH) (19q13.33) ( (q12)201750(AR)(Antley-Bixlersyndrome) (7q11.23) (q12)201750(AR)(antley-Bixlersyndrome, (7q11.23) (q51q52 615363(AR)(overgrowth,osteoporosis, (6q25.1–q25.2) (q23 603148(AD??) (1q32.3) (p12)201710(AR) (8p11.23) (91.)261550(AR) (19p13.3) NR3C1 (locus) (91.1 219050(AD) (19p13.11) (p61)158330 (AD) (1p36.12) ( (p63 238320(AR)(Leydigcellaplasia,hypoplasia) (2p16.3) (82.)250790(AR)(methemoglobinemiatypeIV) (18q22.3) (11.)270400(AR)(Smith-Lemli-Opitzsyndrome) (11q13.4) LGR8/GREAT/GPR106 (21.3 261550(AR) (12q13.13) (p31 264600(AR) (2p23.1) (p2 201810(AR)(adrenalandgonadaldeiciency) (1p12) (p2 201810(AR)(adrenalandgonadaldeiciency) (1p12) (01.)DSDwithDHTdeiciencyandapparent17,20- 614279(AR) (10p15.1) (10p15.1) (7p15.2) (q43 202010(AR)(adrenaldeiciency) (8q24.3) (q23)264300(AR)(Gonadaldeiciency) (9q22.32) (02.2 202110(AR) (10q24.32) (52.)613743(AR)(adrenalandgonadaldeiciency) (15q24.1) (52.)613546(AR)(maternalandfoetalvirilisation) (15q21.2) (p13)201910(AR)(adrenaldeiciency) (6p21.33) ( CXOrf6 q13615962(AD)(hypertension) ) 5q31.3 X2)300758(XL)(hypospadias) ) (Xq28) DSD geneticdiagnosis ) (2) Gonadaldeiciency deiciency CAH+hypertensiongonadal (1) 277000 (2) Adrenaldeiciency (1) Adrenalandgonadaldeiciency 606655 (AD??) OMIM 140000 (AD) ys deiciency+normal lyase polycystic ovarysyndrome) craniosynostosis (inheritance) Downloaded from Bioscientifica.com at 08/10/2020 10:49:42A

(additional phenotype) ± ) CYP17A1 179 :4 and SRD5A2 via University Sheffield R200 M European Journal of Endocrinology cell-free foetalDNAfromthe 6thweek( SRY identify Ychromosome-speciicmarkers(onewithinthe genetic sex. Non-invasive prenatal diagnosis (NIPD) can gestation). Theirstapproachistodeterminethefoetal the country’s legislation,theaetiologyandweeksof parents considerpregnancyinterruption(dependingon genetic sex.Anurgentdiagnosisisrequiredifthe (US) appearance that maybe discordant with the foetal oronthefoetalgenitalultrasound on familyhistory carrier statusofaknown genetically determinedDSD subclasses according to establishedDSDclassiication ( assignment ofaDSDcasetoonethethreemajor determined attheoutsetbecausethisisbasisfor unavailable, thepresenceof weeks.Ifthistechniqueis sample collectionfrom7 diagnostic laboratoriesrecommend maternalblood This technique has largely replaced luorescence days. chromosomes andhasaturn-aroundtimeof1–2 QFPCRdetectsaseriesofmarkersonthesex karyotype. luorescence polymerasechainreaction(QFPCR)and conirming thechromosomalsexusingquantitative irst-line investigation of an individual with DSD involves Besides carefulclinicalevaluationofthepatientwithDSD, Chromosomal sex be soughtbyusingthemostadequatemoleculartesting. or absenceofconsanguinity, amoleculardiagnosiswill ofDSDandthepresence constellation, thefamilyhistory Depending onthis,theclinicalphenotype,hormonal ( that couldhelpinclarifyingthepossiblegeneticcauses should beabletoprovideknowledgeandtechnologies biochemistry, genetics,radiology, occasionallypathology) published ( chromosomal DSDsandtestingmethodshavebeen analysis isrequired.Detailsondifferenttypesofsex If sexchromosomeDSDisidentiiednofurthergenetic 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 initialclassiicationofanyDSDintoonethree technique (involving G-banding) and isessentialin isacytogenetic hybridisation. Theanalysisofkaryotype 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 diagnosisinastepwisestratiication.Inparticular, development (DSD).(A)Thetraditionalpathwayapproaches Diagnostic approachestodifferences/disorders ofsex Figure 1 as deletionsof 46,XX DSDandof DSD ( karyotype ( 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)orinamnioticluidcells condition can beinvestigated on a chorionic villus biopsy forms ( cases, althoughtheyaremoreprevalentinsyndromic involvement. CNVsmaybepresentinone-ifthofDSD the presence of associated malformations or other system is usedaspartofsecond-lineinvestigationsforDSDin butinothersaCGHorSNParray the traditionalkaryotype techniques maydetectintragenicdeletionsthatnot other moredatedplatforms,suchasoligo-aCGH.These general SNParraycandetectsmallerregionsofCNVthan while aCGHisabletodetectCNValongthegenome.In intragenic andwholegeneCNVinspeciictargetsites (MLPA), SNParrayoraCGH:MLPA isdesignedtodetect by multiplexligation-dependentprobeampliication effect suchasduplicationsof development havebeenshowntoexertadose-dependent DSD geneticdiagnosis Table 1 22 , 23 < ). Alterationsingenedosagemaybedetected 5 Mb). Insomecentres,aCGHhasreplaced , 24 ATRX ). Indeed, several genes regulating sex DAX1 , DMRT1 Downloaded from Bioscientifica.com at 08/10/2020 10:49:42A or WNT4 , FGF9 EMX2 in46,XYDSDaswell 179 , or SOX3 www.eje-online.org :4 WT1 or in46,XY SOX9 via University Sheffield R201 in M 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 biochemicalphenotypespointtoaspeciicgene by HTSofcandidategenesorexome,evenwhenclinical Individual genesequencingisincreasinglybeingreplaced – – – – – regions) ofacandidategenecouldbeperformedin: and sometimes of other regions of interest (i.e. promoter Sanger sequencing of exonic coding, lanking regions Sanger sequencing the interpretationoftheseoftenchallengingresults( as theneedforcollaborationanddatasharingtofacilitate adequately resourced tointerpretgeneticindingsaswell being highlights theimportanceofdiagnosticservices fact been reported in association with DSD ( clinical signiicance,uponreview, halfofthesehadin report threequarterswereclassiiedasbeingofuncertain third ofchildrenwithDSD( 26 always bedetectedbySangersequencingofgenes( non-coding regulatory regions( non-coding regulatory In thenearfuture,WGSwillbeabletodetectCNVsin and thisisnotalwaysfeasiblewhenusingaCGHorMLPA. the codingregionsofgenesatanexon-levelresolution,

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 sequencingtosegregateaspeciicgenevariant deletions, etc.(e.g., to interpretduecomplexgenerearrangements, DSD genesinwhichHTSmethodsarecurrentlydificult Targeted (hypothesis-driven)sequencingofknown hormonal assessment (e.g., DSD genesfollowingmeaningfulclinicaland Targeted (hypothesis-driven)sequencingofknown SRD5A2 27 In addition,WEShasthepotentialtodetectCNVsin ). CNVshavebeenreportedtobepresentinone- , HSD17B3 , AR CYP21A2 andothers)( 21 29 ). L Audíandothers ) and,althoughinthis ). CYP21A2 30 ). , CYP11B1 28 ). This 22 28 , 25 ). , , endocrine proiling ( and single gene variants does not always correlate with in suspected 46,XY DSD, the identiication of CNVs Increasing experiencewithHTStechniquessuggeststhat when detectingvariantsofunknownsigniicance(VUS). and clinicians need to be aware of problems associated DSD-related genes. However, laboratories the service based HTSstrategiesandallowtheidentiicationofnew WES-based HTSstrategiesaremorelexiblethanpanel- 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 interpretation, that arerequiredformultidisciplinary need forbioinformaticexpertise.Thespecialistresources However, thisdoesnottakeintoaccounttheincreased pure technicalcostsperbasearesigniicantlydecreasing. for individualisedclinicalmanagement. sensitivity tosexsteroids),whichareimportantaspects biosynthesis 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 regardedastheirst-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 allowbetterunderstandingof DSDsand,hence, inDSD. diagnosis mustbeconsideredcomplementary ( testing, startingwiththeinitialevaluationofpatient biochemical (hormonal)andgenetic(HTS-related) a moreparallelapproachinvolvingintegrationofboth classical sequential diagnostic pathway inDSD,favouring currently has a facility to act as a secure repository for currently hasafacilitytoact asasecurerepository DSD geneticdiagnosis 5 , 32 HTS technologiesarecontinuouslyimprovingandthe ). Asstatedearlier, genetictestingandhormonal 33 ). The I-DSD Registry ( ). TheI-DSDRegistry 28 ). Altogether, this may change the Downloaded from Bioscientifica.com at 08/10/2020 10:49:42A 179 :4 www.i-dsd.org 8 , 31 via University Sheffield R202 ). Itis ) M European Journal of Endocrinology type IIpatientswhen pathway may besuspected in the recently proposed AIS ( and promoterregionsHTSmayrevealpathogenicvariants steroid enzymedeiciencies andhormoneresistance causes willremainforaspeciic setofphenotypes(i.e. 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 in previouslyunsuspectedgenes( ZNRF3 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 by androgens in the patient genital skin ibroblasts ( and lanking region sequences are normal, whole variations (oligogenic)( individuals with DSD resultfrom a combination of gene most likely demonstrate that the phenotype of some associated withDSDinhumanssuchas variations foundinDSD. facilitating the broadest view on molecular genetic appropriately andbioinformaticanalysisspeedup,thus quality further improves, technical hurdles be addressed irst-line approach,providedthatpricesprogressivelyfall, will probablyincreaseitsuseasa many clinicalservices of missingvariantsinaDSDcandidategene.However, guarantee ofcoverage,thusincreasingthepossibility thorough bioinformaticanalysisandmayresultinalower undescribed phenotypessuchasfor ( 52 50 39 Review ) thatcouldbedetectedbyWGS.Asstatedearlier, WES ). Inaddition,variantsinothergenesregulatingthe ), Many ofthegeneticvariantsinDSDencode The experienceofdifferentauthorsreportingresults When AISishighlysuspectedbut of genes previously not WES has afforded discovery A WESirst-lineapproachwouldrequireamuchmore 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 )

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, are presentedbyCroft local genomicapproach.Themostgeneralapproaches or reproductive problems and preferred 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 signiicance’ in gene(s) related to the patient classiied as ‘pathogenic’, ‘likely pathogenic’ and of the generalrecommendation istoreportvariants 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 for extensivetestingthroughWESorWGS( patients. Moredetailedalgorithmsdeterminesuitability outcomes of eachpathway and the consequences for the broad approachinvolvingWESorWGS,thegenomic followed by either a targeted gene panel approach or a and conirmationofsexchromosomecomplement, considering the physical features, hormone indings ( facilitate theinterpretationofVUS( in genes that cause Mendelian disorders ( ‘likely benign’and‘benign’)todescribevariantsidentiied ‘pathogenic’, ‘likelypathogenic’,‘uncertainsigniicance’, variants andusespeciicstandardterminology(such as (ACMG) guidelinesfortheinterpretationofsequence American College of Medical Genetics and Genomics DSD geneticdiagnosis 5 ). TheirparadigmsbeginwithaDSDphenotype The majorityofaccreditedlaboratoriesfollowthe Downloaded from Bioscientifica.com at 08/10/2020 10:49:42A t al et . ( in silico 7 ) andAlhomaidah 9 5 ). Themostdetailed 179 ). and/or www.eje-online.org :4 53 ). At present, in vitro via University Sheffield 31 R203 t al et ) by ) to . M European Journal of Endocrinology www.eje-online.org access tomedicalattention. suficient 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 or 3ß-hydroxysteroiddehydrogenasedeiciencies)( form of CAH such as 21-hydroxylase, 11ß-hydroxylase deiciencies, aswellin46,XXDSDandavirilising insuficiency (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. willirstdeterminethe subgroupofDSD, The karyotype molecular diagnosisofDSD Recommendations forthegenetic phenotype becomesincreasinglycoherent. evolve as the relationship between the genotype and the robust genomic data in this ield, these guidelines will ( (hormonal) evaluationinparallelwiththegeneticstudy conduct thephysicalphenotypingandbiochemical a hypothesis-drivenbasisbySangersequencingorHTS. lead to a likely DSD target gene that can be sequenced on speciic steroidproileinurineand/orplasmamayrapidly indings( as secondary Fig. 1 Review These recommendationsarebasedoncurrently All neonatesandyoungbabiespresentingwithDSD A CNVanalysisshouldbeaddedtotheirst-line In all other cases, the multidisciplinary teamshould In allothercases,themultidisciplinary ). ExceptforconirmingamonogenicfamilialDSD 54 , 55 ). With generationofmore L Audíandothers 20 ). A 1 Bashamboo A, Eozenou C,Rojo S&McElreavey K.Anomaliesin 1 References submitted manuscriptandapprovedsubmission. All theauthorshaveacceptedresponsibilityforentirecontentofthis Author contributionstatement 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 authorsdeclarethatthereisnoconlictofinterestcouldbe Declaration ofinterest determination anddevelopment. care andexpandingknowledgeabouttheprocessofsex Registries. Databasesareimportantforimprovingpatient international databasesliketheI-DSDandI-CAH (when available)inDSDs,shouldbeaccessiblethrough with phenotypic,biochemicalandoutcomedata 8 Kyriakou A, Lucas-Herald AK, McGowan R,Tobias ES 8 &Ahmed SF. Croft B, Ayers K, Sinclair A&Ohnesorg T. 7 Reviewdisordersofsex Bashamboo A, Ledig S,Wieacker P, 6 Achermann JC&McElreavey K. Alhomaidah D, McGowan R&Ahmed SF. 5 Thecurrentstateof Hutson JM, Grover SR,O’Connell M&Pennell SD.Malformation 4 Agopian AJ, Langlois PH,Ramakrishnan A&Canield MA. 3 Bouty A, Ayers KL, &Sinclair AH.Thegeneticand Pask A,Heloury Y 2 DSD geneticdiagnosis 337–350. 213–224. 2017 genetic interactionsofearlygonaddevelopment. human sexdeterminationprovideuniqueinsightsintothecomplex 943–949. challenges inmanagement. Disorders ofsexdevelopment:advances ingeneticdiagnosisand discovery. development: theevolvingroleofgenomicsindiagnosisandgene of disorderssexdevelopment(DSD). New technologiesfortheidentiicationofnovelgeneticmarkers Genetics diagnostic geneticsforconditionsaffectingsexdevelopment. nrendo.2014.83) Reviews Endocrinology syndromes associatedwithdisordersofsexdevelopment. in Texas. Epidemiologic featuresofmalegenitalmalformationsandsubtypes environmental factorsunderlyinghypospadias. 165–177. 2015 Genetic informationandmoleculardata,together 91 9 239–259. 2017 143–156. (https://doi.org/10.1002/bdrc.21148) American Journal of Medical Genetics Part A ofMedicalGenetics Part 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 from Bioscientifica.com at 08/10/2020 10:49:42A (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 via University Sheffield R204 2015 Clinical

M European Journal of Endocrinology 21 Zargari M, Sadeghi MR,Shahhosseiny MH,Kamali K, Saliminejad K, 21 Kulle A, Krone N,Holterhus PM,Schuler G,Greaves RF, 20 Juul A,de Cools M, Nordenström A,Robeva R,Hall J,Westerveld P, 19 Flück C, Del Valle I, 18 Buonocore F, Duncan AJ,Lin L,Barenco M, Baxter RM, Arboleda VA, 17 Lee H,Barseghyan H,Adam MP, Fechner PY, Camats N, Fernandez-Cancio M,Audi L,Schaller A&Fluck CE. 16 Werner R, 15 Monig I,Lunstedt R,Wunsch L, Thorns C,Reiz B, Berglund A, Johannsen TH,Stochholm K,Viuff MH, Fedder J, 14 Krone N &Arlt W. 13 Genetics ofcongenitaladrenalhyperplasia. Rodie M, McGowan R,Mayo A,Midgley P, 12 Driver CP, Kinney M, Ahmed SF, 11 Dobbie R,Finlayson AR,Gilbert J,Youngson G, Alimussina M, Diver LA,McGowan R&Ahmed SF. 10 Genetictestingof Yatsenko SA 9 &Witchel SF. Geneticapproachtoambiguousgenitalia 22 Tannour-Louet M, 22 Han S,Corbett ST, Louet JF, Yatsenko S, Meyers L, Review 181–192. 0202-7) across thelifespan:anexpertopinion. individuals withadifference/disorderofsexdevelopment(DSD) org/10.1371/journal.pone.0015392) ofMedicalBiotechnology Journal pregnant womenduring6(th)–10(th)weeksofgestation. non-invasive methodofcell-freefetalDNAinmaternalplasma Esmaeilzadeh A &Khorshid HR.Fetalsexdeterminationusing Rijke YB, Hartmann MF, Saba A,Hiort O Köhler B, Berrra M,Springer A,Schweizer K wellcomeopenres.11253.1) Wellcome OpenResearch A genomicatlasofhumanadrenalandgonaddevelopment. Parnaik R, Shah S,Hubank M,Gerrelli D&Achermann JC. doi.org/10.1210/jc.2014-2605) Bargman R, Keegan C,Travers S, Schelley S of HumanGenetics disorder ofsexdevelopment:anoligogenicorigin? Broad phenotypesinheterozygousNR5A146,XYpatientswitha pone.0176720) Krause A, Schwab KO,Binder G,Holterhus PM development. delay, andclinicalpresentationoffemale46,XYdisorderssex Main KM &Gravholt CH.Incidence,prevalence,diagnostic Practice andResearch: ClinicalEndocrinologyandMetabolism the Scottishgenitalanomalynetworkregister. insuspecteddisordersofsexdevelopment:lessonsfrom a karyotype Young D &Ahmed SF. Factorsthatinluencethedecisiontoperform (https://doi.org/10.1136/adc.2002.024034) of DiseaseinChildhood:FetalandNeonatal anomalies amongsingletonbirths,1988–1997,inScotland. Chalmers J &Stone D.Prevalenceofhypospadiasandothergenital MOP.0000000000000644) Opinion inPediatrics XY newbornswithasuspecteddisorderofsexdevelopment. semperi.2017.03.016) Seminars inPerinatology and disordersofsexdevelopment:whatcliniciansneedtoknow. 2018 2011 101 PLoS ONE mutations andphenotypicvariationsofgonadaldysgenesis. of sexualdevelopment. novo copynumbervariantsassociated withhumandisorders Shaw CA, Kang SH,Cheung SW&Lamb DJ. Identiicationofde COST ActionBM1303‘DSDnet’. analysis indiagnosisandtreatmentofDSD:positionpaperEU Clinical EndocrinologyandMetabolism for thediagnosisof46,XYdisorderssexdevelopment. 2017 4532–4540. 14 5 176 103–108. 415–429. P1–P9. 2017 (https://doi.org/10.1016/j.beem.2008.10.014) Journal ofClinicalEndocrinologyandMetabolism Journal 12 (https://doi.org/10.1210/jc.2016-2248) (https://doi.org/10.1159/000326815) (https://doi.org/10.1530/EJE-16-0953) 2018Epub. e0176720. (https://doi.org/10.1038/s41574-018-0010-8) 2018 2017 PLoS ONE 2017 30 548–557. 41 (https://doi.org/10.1038/s41431-018- (https://doi.org/10.1371/journal. 2 2011 232–243. 25. European Journal ofEndocrinology European Journal 2010 (https://doi.org/10.12688/ 2015 3 L Audíandothers 201–206. (https://doi.org/10.1097/ Nature ReviewsEndocrinology 5 et al. e15392. 2004 (https://doi.org/10.1053/j. 100 et al. et al. Steroidhormone Sexual Development E333–E344. et al. 89 Exomesequencing Caringfor (https://doi. F149–F151. European Journal European Journal NewNR5A1 Journal of Journal Avicenna 2009 (https:// Archives Current 2016 Best 23

27 Kon M &Fukami M.Submicroscopiccopy-numbervariations 27 Vasson A, Leroux C,Orhant L,Boimard M,Toussaint A, 26 Leroy C, Jaillard S, Bashamboo A,Pasquier L,Belaud-Rotureau MA, 25 Baetens D, Mladenov W, 24 DelleChiaie B,Menten B,Desloovere A, Ledig S, Hiort O,Scherer G,Hoffmann M,Wolff G, 23 Morlot S, 36 Fan Y, 36 Zhang X,Wang L, Wang R, Huang Z,Sun Y, Yao R, Dong Y, 35 Yi Y, Yao H, Yang Z, Hu H,Liu J,Gao C,Zhang M,Zhou L, Eggers S, Sadedin S,vandenBergen JA,Robevska G,Ohnesorg T, 34 Jiang J,Nixon R, Rodie M &Ahmed SF.Kourime M, Bryce J, 33 An Iotova V,Kyriakou A, Dessens A,Bryce J, 32 Juul A,Krawczynski M, Ahmed SF, 31 Bashamboo A,Lucas-Herald A&McElreavey K. Hiort O, Birnbaum W, 30 Marshall L,Wunsch L, Werner R, Schroder T, Croft B, Ohnesorg T&Sinclair AH.Theroleofcopynumbervariants 29 Nixon R, Cerqueira V, 28 Kyriakou A,Lucas-Herald A,McNeilly J, DSD geneticdiagnosis doi.org/10.1186/s13023-014-0209-2) 155. survey ofspecialistcentres. survey care fordisordersofsexdevelopment-resultsfromanInternational international patientcohort. sex development:insightsfromtargetedgenesequencingofalarge 0018-2) s10815-014-0383-0) ldt008) Cellular Pediatrics associated with46,XYdisordersofsexdevelopment. org/10.1038/ejhg.2012.279) Commere V, Ghiotti T, Deburgrave N,Saillour Y Reproduction andGenetics DSD: usefulnessofCGHtechnologiesfordiagnosis. McElreavey K, Odent S&Ravel C.Genedosageeffectsin46,XY Iotova V, Callewaert B,Van Laecke E,Hoebeke P, DeBaere E humrep/deq167) Human Reproduction of arrayCGHasdiagnostictoolandsearch fornewcandidateloci. syndromic andnon-syndromicXYgonadaldysgenesis:evaluation Kuechler A &Wieacker P. Array-CGHanalysisinpatientswith generation sequencingof80genesassociated withdisordersof Huang X, Ye J, Han L 0286-2) Medical Genetics mutations inpatientswithdisorders ofsexdevelopment. Yi X doi.org/10.1186/s13059-016-1105-y) Hewitt J, Lambeth L,Bouty A,Knarston IM,Tan TY org/10.1186/s13023-017-0603-7) ofRare Diseases Orphanet Journal international registriesforrareconditionsaffectingsexdevelopment. assessment ofthequalityI-DSDandI-CAHregistries– Nordenskjold A, Rozas M,Sanders C,Hiort O Medical Bulletin Understanding thegeneticaetiologyinpatientswithXYDSD. (https://doi.org/10.1038/nrendo.2014.108) development. Dohnert U &Holterhus PM.Managementofdisorderssex (https://doi.org/10.1159/000481896) in disordersofsexdevelopment. 2017 systematically foradisorderofsexdevelopment. Prevalence ofendocrineandgeneticabnormalitiesinboysevaluated Tobias ES,McMillan M, Purvis AI, McGowan R&Ahmed SF. European Journal ofHumanGenetics European Journal detection ofexoniccopy-numberchangesinmultipletargetedgenes. oligonucleotide array-basedCGH:areliablediagnostictoolfor development. consecutive seriesof46,XYneonatesandinfantswithatypicalsexual Extensive clinical,hormonalandgeneticscreeninginalarge et al. (https://doi.org/10.1186/s13023-016-0534-8) 32 2130–2137. Targeted next-generationsequencingidentiicationof Nature ReviewsEndocrinology Orphanet Journal ofRare Diseases Orphanet Journal 2013 2016 2015 2010 (https://doi.org/10.1093/humrep/dex280) 106 et al. 17 2 7. 23. 2015 67–89. 25 Diagnosticapplicationoftargetednext- Downloaded from Bioscientifica.com at 08/10/2020 10:49:42A (https://doi.org/10.1186/s40348-015- Orphanet Journal ofRare Diseases Orphanet Journal 2637–2646. (https://doi.org/10.1186/s12881-016- Genome Biology 32 2017 Sexual Development (https://doi.org/10.1093/bmb/ 287–291. 2013 12 56. 21 (https://doi.org/10.1093/ 2014 179 977–987. (https://doi.org/10.1007/ 2016 (https://doi. et al. 2014 www.eje-online.org :4 10 et al. Human Reproduction Currentmodelsof 17 520–529. 2018 et al. Journal ofAssisted Journal 9 Molecular and Custom 243. 209. (https://doi. Disordersof BMC 12 (https:// (https:// 2016 via University Sheffield et al. R205 19–29. British

M European Journal of Endocrinology www.eje-online.org 46 Swartz JM, Ciarlo R,Guo MH,Abrha A,Weaver B, 46 Diamond DA, Baetens D, Stoop H,Peelman F, 45 Todeschini AL, Rosseel T, Harris A, Siggers P, 44 Corrochano S,Warr N, Sagar D,Grimes DT, Bashamboo A, Eozenou C,Jorgensen A,Bignon-Topalovic J, 43 Siffroi JP, Portnoi MF, 42 Dumargne MC,Rojo S,Witchel SF, Duncan AJ, Mazen I, Amin H,Kamel A,ElRuby M,Bignon-Topalovic J, 41 Callier P, 40 Calvel P, Matevossian A,Makrythanasis P, Bernard P, Bashamboo A, Brauner R,Bignon-Topalovic J, 39 Lortat-Jacob S, Ozen S, Onay H,Atik T, 38 Solmaz AE,Ozkinay F, Goksen D&Darcan S. Kim JH, Kang E,Heo SH,Kim GH,Jang JH,Cho EH,Lee BH,Yoo HW 37 Review signaling. in mammaliansexdeterminationbyinhibitingcanonicalWNT pgen.1004340) Chan YM &Hirschhorn JN.A46,XXovotesticulardisorderofsex (https://doi.org/10.1038/gim.2016.118) disorders ofsexdevelopment. NR5A1 isanoveldiseasegenefor46,XXtesticularandovotesticular Coppieters F, Veitia RA, Looijenga LH,DeBaere E &Cools M. pnas.1801223115) Suzuki M, Burdine RD,Cong F, Koo BK org/10.1016/j.ajhg.2018.01.021) ofHumanGenetics American Journal causes testisdevelopmentandcardiacdefectsin46,XXchildren. of-function ofthenuclearreceptorNR2F2,encodingCOUP-TF2, Hyon C, Tar A, Nagy P, Sólyom J,Halász Z,Paye-Jaouen A.Loss- org/10.1093/hmg/ddy037) Human MolecularGenetics are associatedwithaspectrumofhumanreproductiveanomalies. Mugica M Eozenou C, Bignon-Topalovic J, Yatsenko SA, Rajkovic A,Reyes- org/10.1159/000444948) sex development. gene associatedwithcongenitalheartdiseaseand46,XYdisorderof Bashamboo A &McElreavey K.HomozygousmutationoftheFGFR1 Genetics impeding Hedgehogproteinpalmitoylationandsignaling. HHAT leadstosyndromic46,XYdisorderofsexdevelopmentby Guittot S Kurosaka H, Vannier A, Thauvin-Robinet C,Borel C, Mazaud- (https://doi.org/10.1093/hmg/ddu074) testis determination. in theFOG2/ZFPM2geneareassociatedwithanomaliesofhuman Karageorgou V, Lourenco D,Guffanti A&McElreavey K. Mutations doi.org/10.1159/000452995) assessment. in 46,XYdisordersofsexdevelopmentcases:eficiencyandcost Rapid moleculargeneticdiagnosiswithnext-generationsequencing org/10.1016/j.mce.2017.01.037) Molecular andCellularEndocrinology to identifythegeneticetiologyofdisorderssexdevelopment. & Choi JH.Diagnosticyieldoftargetedgenepanelsequencing org/10.1038/srep44536) sexual development. 2014 et al. PNAS et al. Hormone ResearchHormone inPaediatrics 10 Lossoffunctionmutationinthepalmitoyl-transferase MutationsinvolvingtheSRY-related geneSOX8 2018 e1004340. Sexual Development Scientiic Reports Human MolecularGenetics 115 2018 5474–5479. (https://doi.org/10.1371/journal. Genetics inMedicine 27 1228–1240. 2018 2017 2016 2017 L Audíandothers (https://doi.org/10.1073/ et al. 102 444 2017 10 7 ZNRF3functions 44536. 487–493. 19–25. 16–22. 2014 (https://doi. 87 2017 81–87. (https://doi. 23 (https://doi. (https://doi. (https://doi. 3657–3665. 19 367–376. PLoS (https:// Accepted 4July2018 Revised versionreceived22June2018 Received 26March2018 50 Hornig NC, deBeaufort C,Denzer F, 50 Cools M,Wabitsch M, Ukat M, Baetens D, Guran T, 49 Mendonca BB,Gomes NL,DeCauwer L, Ben HadjHmida I,Mougou-Zerelli S,Hadded A,Dimassi S, 48 Werner R, 47 Merz H,Birnbaum W, Marshall L,Schroder T, Reiz B, 55 Green RC, Berg JS,Grody WW, 55 Kalia SS,Korf BR, Martin CL, Kalia SS, Adelman K,Bale SJ,Chung WK,Eng C,Evans JP, 54 Richards S, Aziz N,Bale S,Bick D,Das S,Gastier-Foster J,Grody WW, 53 Baetens D, Mendonca BB,Verdin H, Cools M&DeBaere E.Non- 52 Hornig NC, Ukat M,Schweikert HU,Hiort O,Werner R, 51 Drop SL, DSD geneticdiagnosis disorders ofsexdevelopment. Biallelic andmonoallelicESR2variantsassociatedwith46,XY e211. Genetics inMedicine incidental indingsinclinicalexomeandgenomesequencing. CORRIGENDUM: ACMGrecommendationsforreportingof gim.2017.18) 1990) journal.pone.0154158) Kulle AE, Schweikert HU,Werner R, Hiort O (https://doi.org/10.1038/gim.2017.163) Peelman F, Verdin H, Vuylsteke M, Van derLinden M,Atay Z amenorrhea. primary luteinizing hormonereceptor(LHCGR)geneassociatedwith46,XY & McElreavey K.Novelhomozygousnonsensemutationsinthe Kammoun M, Bignon-Topalovic J, Bibi M,Saad A,Bashamboo A org/10.1210/jc.2015-1314) Endocrinology andMetabolism (DHH) identiiedbyexomesequencing. dysgenesis duetoahomozygousmutationinDesertHedgehog Kavran JM, Baumer T, Capetian P&Hiort O.46,XYgonadal variant. development likelycausedbyasteroidogenicfactor-1(NR5A1) McGuire AL, Nussbaum RL,O’Daniel JM,Ormond KE Herman GE, Hufnagel SB,Klein TE,Korf BR Hegde M, Lyon E, Spector E 2017 coding variationindisordersofsexdevelopment. Cools M, Hughes IA,Audi L,Ahmed SF doi.org/10.1159/000452888) translation. complete androgeninsensitivitybyactivatingaberrantuORF germline mutationinthe5’UTRofandrogenreceptorcauses Medicine Genomics andtheAssociationforMolecularPathology. recommendation oftheAmericanCollegeMedicalGeneticsand for theinterpretationofsequencevariants:ajointconsensus Metabolism endogenous ARactivity. mutation-negative classofandrogeninsensitivitybydetermining Medicine American CollegeofMedicalGeneticsandGenomics. sequencing, 2016update(ACMGSFv2.0):apolicystatementofthe indingsinclinicalexomeandgenome for reportingofsecondary (https://doi.org/10.1016/j.fertnstert.2016.03.008) 91 163–172. 2015 2017 Hormone ResearchHormone inPaediatrics 2016 PLoS ONE 17 19 101 405–424. 249–255. (https://doi.org/10.1111/cge.12911) 2017 4468–4477. 2016 Fertility andSterility Fertility Journal ofClinicalEndocrinologyand Journal 19 Downloaded from Bioscientifica.com at 08/10/2020 10:49:42A (https://doi.org/10.1038/gim.2016.190) (https://doi.org/10.1038/gim.2015.30) 11 606. et al. 2015 Genetics inMedicine e0154158. Standardsandguidelines (https://doi.org/10.1038/ (https://doi.org/10.1210/jc.2016- 100 2017 E1022–E1029. et al. Journal ofClinical Journal 2016 (https://doi.org/10.1371/ 179 et al. IdentiicationofanAR et al. 87 106 :4 191–195. Recommendations Arecurrent 2017 Clinical Genetics 225.e211–229. (https://doi. et al. Genetics in 20 Genetics in (https:// 717–727 via University Sheffield R206 et al.

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