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European Journal of Endocrinology -19-0111 Introduction population andprovidenewinsightintothecurrentunderstanding oftheetiologiesDSD. 46,XX DSD.Ourfindingsexpandthegene,mutationand phenotypespectraoftheraretypesDSDinChinese Conclusions: negative samplesasavariantofuncertainsignificance, indicatingthatWESdidnotimprovethediagnosticrate. with 5 the expectedDSDgenotype–phenotypecorrelationanddeterminedafoundermutation( respectively. Wereportednovelcandidategenes( of clinicalcertainty,etiologicdiagnosticrates46.9and10.3%wereobtainedfor46,XY46,XXDSDpatients, for thefirsttime.Pathogenicandlikelypathogenicvariantsaccounted38.730.7%,respectively.Onbasis Results: negative samples. pathogenicities wereevaluatedaccordingtoestablishedguidelines.WESwasperformedforrandomlyselected Methods: disease-causing genes,includingallknowndiagnosticgenesforDSD. Design: sequencing (WES)withinalargeChineseDSDcohort. Objective: still noresearchonoverviewofalargeChineseDSDcohort. Context: Abstract *(Y XuandYWangcontributedequallytothiswork) Neurology, HarvardMedicalSchool,Boston,Massachusetts,USA 3 Metabolism, ShanghaiChildren’sMedicalCenter,JiaoTongUniversitySchoolofMedicine,Shanghai,China, 1 Xiumin Wang Xiaodong Huang Yufei Xu cohort withdisordersofsexdevelopment whole-exome sequencinginalargeChinese New insightsfromunbiasedpaneland chromosomes, gonadsor anatomical structures.The of heterogeneousdiseases characterized byatypiaof Disorders ofsexdevelopment (DSD)arealargegroup Division ofGeneticsandGenomics,BostonChildren’sHospital,Boston,Massachusetts,USA, Department ofMedicalGeneticsandMolecularDiagnosticLaboratory, https://doi.org/ https://eje.bioscientifica.com Clinical Study α PatientswererecruitedaccordingtotheinclusioncriteriaofDSD.Theappliedpanelcontains2742known Thisstudyincluded125patients.Seventy-fivevariantswereidentifiedbyTPSand31reported -reductase deficiency.FurtherWESinrandomlyselectednegativesamplesidentifiedonlyoneamong14 Diagnosisofnon-chromosomaltypedisorderssexdevelopment(DSD)haslongbeenchallenging.Thereis Targetedpanelsequencing(TPS)wasperformed,andidentifiedcandidatevariantswereverified.Variant 1 Todeterminetheetiologicdiagnosisthroughunbiasedlarge-scalepanelsequencingandwhole-exome , *, YirouWang 10.1530/EJE ThisisthefirstreportofapplyingunbiasedTPSin a largeChinesecohortofpatientswith46,XYand 2 , YipingShen 2 , YulingChen -19-0111 2 , *, NiuLi 3 3 © , 4 2019EuropeanSociety ofEndocrinology and 1 Y Xu,Wangandothers , YanrongQing 1 , RuenYao Jian Wang Printed inGreatBritain 1 1 , GuoqiangLi 1 , TingtingYu BMPR1B 2 Department ofEndocrinologyand , GNAS European PediatricEndocrinology SocietyattheChicago Lawson Wilkins Pediatric Endocrinology Society and established DSDclassification, whichwasproposedby 1 DSD cohort Sequence variantsinChinese , JuanLi Published byBioscientifica Ltd. 1 , YongnianShen , GHR ) andregionsofcopynumbervariantsoutside 2 , YuDing 4 Department of 2 2 , YaoChen , Downloaded fromBioscientifica.com at09/30/202107:44:35AM SRD5A2 2 , (2019) Endocrinology European Journalof [email protected] harvard.edu or [email protected] Email Wang to XWang or YShen or J should be addressed Correspondence
p.R227Q)inpatients Yiping.Shen@childrens. 181 181 :3 , 311–323 or
311 –323
via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com gonadal function( assessment ofthephenotypes andtheendocrine collaboration,including systematic andmultidisciplinary identifying ageneticcause, the diagnosisofDSDrequires been along-standingchallenge ( 46,XX DSD); thus, the diagnosis of such patients has rate ofnon-chromosomaltypeDSD(i.e.,46,XYand pathogenesis contributetothelowclinicaldiagnostic China ( genetic conditionsandwithoutpriorclinicaldiagnosis in genetic diagnostictestforpatientswithawiderange of asafirst-tier known disease-causinggenes,couldserve proband-only medicalexomesequencing,targeting2742 15 medical insurancedoesnotyetcovergenetictesting( method inclinicaldiagnosismanycountrieswhere sequencing depth, has gradually become the preferred and analysiscosts,shorterdetectioncyclegreater sequencing (TPS),owingtoitslowerdataprocessing and whole-genomesequencing(WGS),targetedpanel ( all genesortargetgeneregionsinthehumangenome advantages of rapid and high-throughput detection of the efficiency of molecular diagnosis by virtue of its individualized geneticcounselingfortheirfamilies( term follow-upforthesepatientsbutalsocontributesto only helpfultoestablishappropriatetreatmentandlong- 10 genes involved in regulating sexual development ( genetically determined,especiallydetrimentalvariantsin evidence indicatesthatnumerousDSDpathogenesesare DSD poseariskofgonadalmalignancy ( themselves andtheirfamilies.Moreover, sometypesof physiological and psychological impact tothepatients genital malformationandinfertility, causeprofound resulting problems,includinggenderuncertainty, uterus mightnotbenoticeduntilpuberty( DSD phenotypeslikegynecomastiaandprimordial higher thantheaboveestimate,giventhatmanyassociated ( 2.2 in10,000newbornspresentwithambiguousgenitalia ( or action,centralhypogonadismandsyndromiccondition gonadaldysgenesis,deficienthormonesynthesis primary DSD canbefurtherdividedintoseveralsubclasses,suchas ( karyotype DSD, 46,XY DSD and 46,XX DSD according to the Consensus Conferencein2006,comprisesexchromosome 3 1 13 , , Clinical Study ). Recently, wepublishedastudydemonstrating that , 4 , 2 11 Heterogeneous clinical phenotypes and complex Next-generation sequencing(NGS)greatlyimproves ). ThetotalincidenceofDSDissupposedtobemuch 14 ). Epidemiological surveys showthat approximately ). Epidemiological surveys , ). Compared with whole-exome sequencing (WES) 12 16 ). Earlyidentificationofmolecularcausesisnot ). 1 ). Onthebasisofunderlyingetiologies, 17 ). Thediagnosiscanonly be Y Xu,Wangandothers 2 , 5 , 8 , 11 6 5 , , ). Numerous 17 7 ). Current ). Besides 5 , 8 8 ). , 14 9 , , panel sequencingandWES;furtherenrichthegene, evaluate the diagnostic utilityofunbiased, large-scale etiologic diagnosescombinedwithclinicalcertainty; pathogenic variantsofalargeDSDcohortandobtain genes forDSD.We intendedtoidentifythecandidate disease-causing genes,includingallknowndiagnostic DSD and46,XXDSD.Thispanelcontains2742known sequencing inacohortofChinesepatientswith46,XY DSD and46,XXpatientsisavailable. patients ( have recruitedarelativelysmallnumberofChineseDSD owing tothelimitedcases.Furthermore,tillnow, studies genes, andfewstudieshaveanalyzed46,XXDSDpatients only includethemajorityofknownDSD-associated yield ( sequencing inDSDpatientstoimprovethediagnostic molecular findings.Severalstudieshaveappliedpanel confirmed iftheclinicalevaluationisconsistentwith low mappabilityofreadscaused bythepresenceofa classified intoestablished DSDsubclasses.Duetothe to be excluded from sex chromosome DSD and be in this cohort. All patients had undergone karyotyping before. Onlypatientswithnegative resultswereincluded hybridization or sequencing of have beendetectedbymicroarray, fluorescence insitu of DSD and so forth. Some patients might history inconsistency between gonad and chromosome; family amenorrhea;gynecomastia; gender puberty; primary sexual characteristics during dysplasia; absent secondary mass and/oringuinalhernia;masculinization;gonadal and/or labiamajorisfusionintralabial/inguinal female-type externalgenitaliawithclitoralenlargement cryptorchidism and/ormicropenishypospadias; genitalia; male-typeexternalgenitaliawithbilateral The main inclusion criteria included ambiguous external andimagingexaminations. manifestations andlaboratory diagnosisofDSDwasbasedonclinical The preliminary wasrecordedbyprofessionalclinicians. medical history 2018 were enrolled for further investigation. Detailed for suspectedDSDfromDecember2016to Patients referredtoShanghaiChildren’s MedicalCenter Subjects Subjects andmethods DSD patients. ofChinese46,XYDSDand46,XX present anoverview mutation andphenotypespectraofrareDSD; DSD cohort Sequence variantsinChinese Here, wereporttheapplicationofunbiasedpanel 18 , 18 19 , , 19 20 , , 24 21 ), and no overview ofChinese46,XY ), andnooverview , 22 , Downloaded fromBioscientifica.com at09/30/202107:44:35AM 23 , 24 , 25 SRY ). Thesepanelsusually or some other gene 181 :3 312 via freeaccess
European Journal of Endocrinology org/ EVS/ Exome Variant ( Server Database (gnomAD)( population databasessuch as theGenomeAggregation reported beforebyretrievingrelevantinformationfrom variants andcheckedwhetherthehadbeen We analyzedtheallelefrequenciesof detected guideline ( were analyzed using a combination of the current interpretation/ clinicalgenome.org/working-groups/sequence-variant- Variant InterpretationWorking Group( further refinedandevolvedbytheClinGenSequence Genetics andGenomicsguideline( recommended by theAmerican College of Medical the pathogenicityofvariantsaccording to thecriteria We screenedalltheidentifiedvariantsandcategorized Pathogenicity analysisofthevalidatedvariants of thecandidatevariants. and subjectedtoSangersequencingconfirmtheorigin primers. TheDNAofpatients’parentswasalsoisolated validated bySangersequencingwithspecific-designed Redwood City, CA,USA).Candidatevariantswere Ingenuity variants (SNVs) were further annotated and filtered on the human referencesequence.Allacquiredsingle-nucleotide assessed, and reads were aligned to the GRCh37/hg19 with HiSeq X Ten (Illumina). Sequencing quality was including 2742 disease-causing genes. NGSwasperformed panel kit(catNo.5190–7519;AgilentTechnologies Inc.), intronic regionswerecapturedbytheinheriteddisease into 150–200 patients’ peripheral blood sampleand randomly sheared et al The entire procedure was performed as described by Hu variant validation Targeted genepanelsequencingand of Medicine. Committee ofShanghaiJiaoTong UniversitySchool to this cohort. This study was approved by the Ethics total of125unrelatedpatientswereeligibleandrecruited was obtainedfromthepatientsorpatients’parents.A we excludedassociatedcasesinthisstudy. pseudogene withhighhomologythe Clinical Study . ( Written informedconsentforthegeneticanalysis ), dbSNP ( ), 1000GenomeProject( 16 ). Briefly, 3 ® Variant Analysisplatform(IngenuitySystems, 29 bp fragments.Thetargetexonsandflanking ) andin-housecriteriaofourlaboratory. ) ( http://www.ncbi.nlm.nih.gov/snp 27 μ g genomicDNAwasextractedfromthe , 28 http://gnomad.broadinstitute.org/ ). Copy number variants (CNVs) http://evs.gs.washington.edu/ http://www.1000genomes. Y Xu,Wangandothers 26 CYP21A2 ), whichwas https://www. ) and gene, ),
Establishment ofetiologicdiagnosticrate synthesized tofurtherevaluatetherelatedgenevariants. www.ncbi.nlm.nih.gov/projects/dbvar/clingen/ curation informationprovidedinClinGen( www.ncbi.nlm.nih.gov/pubmed omim.org/ variants. PhenotypessummarizedonOMIM( jcvi.org/index.php bwh.harvard.edu/pph2/ of predictiontoolssuchasPolyPhen2( www.ebi.ac.uk/Tools/msa/clustalo/ multi-species sequencealignmentsoftware( in UniProt( of theassociatedlocionbasisproteininformation gov/clinvar uk/ac/index.php disease databases such as HGMD ( subjected toWESasdescribed byWang Among thenegativesamples,14caseswerefurther Further WES diagnosis contributetotheetiologicdiagnosticyield. findings. Thus, both the clinical diagnosis and molecular evaluation is undoubtedly consistent with the molecular definite diagnosiscanonlybeconfirmediftheclinical phenotypes, it would remain an uncertain diagnosis. The unsure aboutthecorrelationbetweengenotypesand but wereinconsistentwiththeclinicaldata,orwe variants wereclassifiedaspathogenicorlikely would bedefinedasamoleculardiagnosis.(iv)Whenthe pathogenic andwereconsistentwiththeclinicaldata,it When the variants were classified aspathogenic orlikely condition, itwouldbedefinedasaclinicaldiagnosis.(iii) therapy outcome)hadaspecificdirectlinktothesame hormone levels,gonadalhistopathologyandexperimental (including inheritancemode,patient’s phenotype, variants were classified asVUS, buttheclinical assessment would bedefinedasanuncertaindiagnosis.(ii)Whenthe (VUS) and theclinicalassessmentalsolacked specificity, it patient were classified asvariants of uncertain significance determine thediagnosticyield.(i)Whenvariantsofone combination withtheclinicalassessmentresultsto All thecandidatevariantswerefurtheranalyzedin Technologies) toenrichcodingexonsandflanking We usedaSureSelectXT HumanAllExonKitv6(Agilent PrepKit(AgilentTechnologies).SureSelectXT Library was prepared with the The adapter-ligated library DSD cohort Sequence variantsinChinese ), literaturessearched onPubMed( ). We conservatism assessedtheevolutionary http://www.uniprot.org ) andClinVar ( ) topredictthepossibleimpactof ) andPROVEAN( Downloaded fromBioscientifica.com at09/30/202107:44:35AM http://www.ncbi.nlm.nih. https://eje.bioscientifica.com ) anddosagesensitivity http://www.hgmd.cf.ac. ). We usedavariety ) andClustalOmega 181 :3 http://provean. http://genetics. t al et ) were https:// https:// https:// . ( http:// 313 30 via freeaccess ). European Journal of Endocrinology https://eje.bioscientifica.com the detailsofevidence levelappliedappropriately pathogenicity classification areshownin (mentioned inMethods)( to standardcriteriarecommended byseveralguidelines Seventy-five candidatevariants wereevaluatedaccording Analysis andevaluationofpathogenicity (65/125) ofallpatients. were identified in 64 patients, accounting for 51.2% data Table 1 (seesectionon in Supplementary Information oftheremainingnegativecasesisprovided validation ofpositivecasesaresummarizedin identifiedvariantsand parental features, karyotypes, sequencing and parentalbackground. The mainclinical highly suspected variants were all verified by Sanger were further filtered against in-house databases. The development’ and‘gonadaldysplasia’.Allnovelvariants input ofrelevantclinicalfeaturessuchas‘gonadal of reportedsynonymousandpolymorphicSNVs and splicesites,minorallelefrequencies parameters includedconfinementforcodingregions Ingenuity software.Themainfilteringindexand One hundredtwenty-fivesampleswerescreenedby Identification andvalidationofvariants 300–550. value oftotalvariantscomparedwiththecontrols,within of interest, target, results showedthatalignedreadswere classification ofSNVscomparedwiththecontrols.The sequencing wasindirectlyjudgedbythenumberand of interest and uniformity. In addition, the quality of aligned reads, reads on target, average coverage, region analysis ismainlybasedonthefollowingparameters: requirement ofsequencing.Sequencingdataquality The qualityofhybridizationcapturelibrariesmetthe Quality controlofsequencing Results previous proceduresdescribedinsubsections. The remaining analysis steps are basically similar to the conducted ontheIlluminaHiSeq2500system(Illumina). intronic regionsasthecapturelibrary. Sequencingwas Clinical Study givenattheendofthisarticle).Candidatevariants > 70%; averagecoverage,around150 > 98%; uniformity, 26 , 27 > Y Xu,Wangandothers , 91% andthedifference 29 ). Theresultsofthe > supplementary supplementary 99%; readson × < ; al 1 Table 1%, removal > 20 × al 1 Table region , and . DSD, wereidentifiedwithlargefragmentduplications Patients 8096and9055,presentingwithsyndromic and novelcandidategenes/phenotypes Unexpected findingswithsyndromicDSDcases accounted for50.0and0.0%,respectively. ( candidatevariants (7/29) individualswerefoundtocarry suited forthoseaffectedwith46,XXDSD.Only24.1% respectively ( pathogenic variantsaccountedfor37.3and34.3%, evidence levelsshowedthatthepathogenicandlikely variants ( candidate (57/96) individuals were found to carry 30.7% (23/75),VUS.Inthe46,XYDSDcohort,59.4% variants; 30.7%(23/75),likelypathogenicvariantsand variants, 38.7%(29/75)werecategorizedintopathogenic Amongthese75 Table 2. are providedinSupplementary p.C514S and mentioned before.Although somevariantslike within that werenotdefinitelyrelated toDSD(i.e.,variants the etiologic diagnostic rate. We excluded the variants results andclinicalvalidation,wefurtherdetermined According totheintegratedanalysisbetweenmolecular Etiologic diagnosticyield diagnosis despitethepathogenicityclassification. we stillregardedthesesituationsascasesofuncertain for DSD.However, duetothelackofevidenceforcertainty, genes couldpossiblybenovelcandidateresponsible contribute toourpatients’phenotypes,andthethree pathogenic groups.We assumedthatthesevariantsmight these variantscouldbeclassifiedintopathogenicorlikely the typeof variation, allele frequencies and other criteria, the typicalsymptomofaboveconditions.Basedon main phenotype,insteadofskeletaldysplasia,whichis our patientspresentedwithgonadaldysplasiaasthe and Laronsyndrome,respectively( with brachydactyly, Albright’s osteodystrophy hereditary 4881 and6500( not knowntoberelatedDSDcarriedbypatients2765, identified variantsingenes( triplosensitivity andphenotypiccorrelation.We also 2).However, theseregionsstilllackevidencefor Table into pathogenic variants and VUS (Supplementary coverage, andsoforth,theCNVscouldbecategorized in genomes( DSD cohort Sequence variantsinChinese Fig. 1B BMPR1B ). Thepathogenicandlikelyvariants Fig. 1A Fig. 1A al 1 Table AR , ). Theproportionofvariantsatdifferent GNAS Table 1 p.A48S/p.G209Ewereclassified VUS ). Thetargetedgenepanelprovedless ). Basedonthewiderange, gene , and ). Thethreegenesareassociated Downloaded fromBioscientifica.com at09/30/202107:44:35AM BMPR1B GHR , aswellCNVs),we 31 , 181 GNAS , 32 :3 , , 33 GHR ). However, ) thatare LHCGR 314 via freeaccess
European Journal of Endocrinology
Table 1 Clinical and molecular characteristics of the 64 positive cases in DSD cohort. Clinical Study
Parental Variant Patient ID Age Clinical features Karyotype Genetic results validation classification Diagnosis 1870 6 y Ambiguous genitalia, enlarged clitoris, 46, XX CYP11B1 c.1360C>T, p.R454C (het); del Exon 1-6 F/M P CAH unclear vaginal and urethral opening (het)# P 1880 2 m Prepenile scrotum, bifid scrotum 46, XY SRY gene deletion* NA P GD 1886 6 m Ambiguous genitalia, blind perineal pouch 46, XY SRD5A2 c.419_421delinsATTC (het)#; c.680G>A, F/M P 5α-Reductase resembling vagina, cryptorchidism p.R227Q (het) P deficiency 1909 6 y 3 m Predominantly female external genitalia, 46, XY LHCGR c.1541G>C, p.C514S (hom)# F/M VUS LCH cryptorchidism 2036 6 y Micropenis, hypogonadism 46, XY AR c.142G>T, p.A48S (hemi)# M VUS AIS 2061 15 y Hypogonadotropic hypogonadism, 46, XY ANOS1 del Exon 4-5 (hemi) M LP KS
hyposmia Y Xu,Wangandothers 2356 7 y Micropenis, small testes 46, XY GNRHR c.806C>T, p.T269M (hom) F/M LP IHH 2367 11 y Predominantly female external genitalia, 46, XY CYP17A1 c.985_987delinsAA, p.Y329K fs*90 F/M P CAH absent Müllerian structures (hom)# 2420 7 m Micropenis, ectopic urethral opening 46, XY SRD5A2 c.680G>A, p.R227Q (hom) F/M P 5α-Reductase deficiency 2439 2 m Ambiguous external genitalia 46, XY NR5A1 c.721C>T, p.R241W (het)# De novo LP GD 2473 12 y Micropenis, small testes 46, XY KISS1R c.631G>A, p.A211T (het) M VUS Uncertain 2540 17 y Predominantly female external genitalia, 46, XY AR c.1787G>A, p.C596Y (hemi)# De novo P AIS inguinal testes 2692 1 y 1 m Micropenis, hypogonadotropic 46, XY PROKR2 c.533G>C, p.W178S (het) F (IP) VUS IHH hypogonadism
2752 1 y Hypogonadism, micropenis, 46, XY AR c.2528T>C, p.I843T (hemi) M LP AIS DSD cohort Sequence variantsinChinese cryptorchidism 2765 9 y Hypertrophic clitoris, hypoplasia of the 46, XX BMPR1B c.1457G>A, p.R486Q (het) F VUS Uncertain (novel uterus phenotype?) 2782 2 y 9 m Perineal hypospadias, micropenis 46, XY SRD5A2 c.680G>A, p.R227Q (het); c.281G>A, F/M P 5α-Reductase p.R94K (het)# LP deficiency 2786 1 y 7 m Micropenis, cryptorchidism 46, XY SRD5A2 c.702C>G, p.F234L (het) F VUS Uncertain 2892 7 m Ambiguous genitalia, micropenis, severe 46, XY SRD5A2 c.607G>A, p.G203S (het)#; c.737G>A, NA LP 5α-Reductase hypospadias, cryptorchidism p.R246Q (het) LP deficiency 2955 6 y Gonadal Dysplasia, cryptorchidism, small 46, XY ANOS1 c.1712G>A, p.W571* (hemi)# De novo P KS testes Downloaded fromBioscientifica.com at09/30/202107:44:35AM 3167 6 y Predominantly female external genitalia, 46, XY LHCGR c.681-1G>A (het); c.1582_1585delATAT, p. F/M P LCH inguinal testes I528* (het) P 3361 26 y Female external genitalia, primary 46, XY SRY c.181A T, p.K61* (het)# De novo P GD
https://eje.bioscientifica.com > amenorrhea 3828 10 y Micropenis 46, XY SRD5A2 c.457T>C, p.F153L (het)# F VUS Uncertain 181 3874 7 m Prepenile scrotum, hypospadias 46, XY DHH c.968G>A, p.G323D (het) M VUS Uncertain
3879 15 y Gonadal dysplasia, hyposmia 46, XY ANOS1 c.984C>G, p.Y328* (hemi) M LP KS :3 3973 11 y Gonadal dysplasia, inguinal testes, short 46, XY SOX3 c.424C>A p.P142T (hemi) M LP Hypopituitarism stature 4004 11 y Micropenis, hypogonadism 46, XY AR c.1175C>G, p.P392R (hemi) M P AIS 4119 5 y Penile dysplasia, absent testes 46, XY TSPYL1 c.67C>T, p.Q23* (het) NA LP Uncertain 4136 11 y Gonadal dysplasia, hypogonadotropic 46, XY TACR3 c.196dupG, p.A66fs*84 (het)# M LP Uncertain 315 via freeaccess hypogonadism
(Continued) European Journal of Endocrinology https://eje.bioscientifica.com
Table 1 Continued. Clinical Study
Parental Variant Patient ID Age Clinical features Karyotype Genetic results validation classification Diagnosis 4208 10 m Female external genitalia 46, XY AR c.2256G>A, p.Trp752* (hemi)# M P AIS 4274 13 y Gynecomastia 46, XY FSHR c.227A>C, p.E76A (het)# F VUS Uncertain 4424 2 y 1 m Enlarged clitoris 46, XX CYP11B1 c.905delA, p.D302fs*23 (hom)# F/M P CAH 4431 14 y Gynecomastia 46, XY POR c.1196_1204delCCTCGGAGC, p.P399_E401del M VUS Uncertain (het) 4500 3 y Micropenis, small testes 46, XY PROKR2 c.892C>T, p.R298C (het)# M (IP) VUS IHH 4622 1 y 7 m Hypospadias, penile curvature, 46, XY SRD5A2 c.269A>C, p.H90P (het)#; c.680G>A, F/M LP 5α-Reductase cryptorchidism p.R227Q (het) P deficiency 4736 17 y Enlarged clitoris, rudimentary uterus 46, XX CYP11B1 c.608C>T, p.A203V (het)# NA VUS Uncertain
4881 14 y Primordial uterus, undeveloped breast 46, XX GNAS c.1006C>T, p.R336W (het) De novo VUS Uncertain (Novel Y Xu,Wangandothers phenotype?) 4940 7 y Enlarged clitoris, absent Müllerian 46, XY CYP17A1 c.785T>G, p.M262R (het)#; c.1193C>T, F/M VUS CAH structures, inguinal testes p.A398V (het) VUS 4986 6 m Ambiguous genitalia, hypospadias, 46, XY NR5A1 c.251G>A, p.R84H (het) M P GD inguinal testes 5015 8 m Cryptorchidism, inguinal fallopian tubes 46, XY AMH c.585C>A, p.Y195* (het); c.1165G>T, p.E389* F/M LP PMDS (het) LP 5175 12 m Micropenis 46, XY GLI2 c.3463_3464delGA, p.D1155R fs*39 (het)# De novo P Hypopituitarism 5337 4 m Micropenis, perineal hypospadias 46, XY AR c.626G>A, p.G209E (hemi)# M VUS AIS 5421 12 y Gonadal dysgenesis, micropenis, perineal 46, XY MAP3K1 c.2062C>G, p.L688V (het)# M VUS GD hypospadias #
5589 2 y 3 m Micropenis, inguinal testes 46, XY GLI2 c.3137delG, p.G1046A fs*84 (het) M (IP) LP Hypopituitarism DSD cohort Sequence variantsinChinese 5621 15 y Gonadal dysgenesis, undeveloped breast 46, XX FGFR1 c.760C>T, p.R254W (het) De novo P IHH 6500 5 y Female external genitalia, suspicious 46, XY GHR c.136+1G>A (hom) F/M VUS Uncertain (Novel uterine-like structure between bladder phenotype?) and rectum, absent ovaries or testes, short stature 6606 4 y Micropenis, small testes 46, XY GLI2 c.3640C>T, p.Gln1214* (het)# M (IP) LP Hypopituitarism 6791 2 y Micropenis 46, XY SRD5A2 c.680G>A, p.R227Q (hom) F/M P 5α-Reductase deficiency 6868 30 y Primordial uterus, primary amenorrhea 46, XY AR c.1847_1849delGTC, p.R616del (hemi) NA LP AIS 6928 6 m Perineal hypospadias, penile curvature 46, XY HSD17B3 c.466T A, p.C156S (het)# M VUS Uncertain
Downloaded fromBioscientifica.com at09/30/202107:44:35AM > 7847 9 y 9 m Hypospadias, micropenis, penile 46, XY SRD5A2 c.656delT, p.F219S fs*60 (het); c.680G>A, F/M P 5α-Reductase curvature p.R227Q (het) P deficiency 7929 6 y Penoscrotal hypospadias, cryptorchidism 46, XY NR5A1 c.848G>T, p.C283F (het)# M LP GD 8065 10 m Female external genitalia, inguinal testes 46, XY AR del Exon5-8 (hemi) NA P AIS 8096 6 y Micropenis, cryptorchidism, short stature, 46, XY seq[GRCh37] dup(1)(q32.3-q43) NA P Syndromic DSD? moderate mental retardation, facial chr1:g.213,031,597-237,997,288) (over 181
dysmorphism 14000 kb)* :3 8585 13 y Micropenis, hypogonadotropic 46, XY PROKR2 c.1004C>T, p.T335M (het) M (IP) VUS IHH hypogonadism 8744 14 y Enlarged clitoris, primordial uterus, 46, XX HSD3B2 c.463C>T, p.P155S (het) NA VUS Uncertain absent ovaries
8803 10 y Micropenis, hypospadias 46, XY AR c.2270A>G, p.N757S (hemi) M LP AIS 316 8945 1 m Hyperpigmentation, cryptorchidism, 46, XY NR0B1 c.1169-2A G (hemi)# De novo P CAH via freeaccess > electrolyte disturbance European Journal of Endocrinology
9052 1 2m Female external genitalia, inguinal testes, 46, XY AR c.2495G>A, p.R832Q (hemi) M LP AIS Clinical Study absent Müllerian structures 9055 6 y Micropenis, cryptorchidism, short stature 46, XY seq[GRCh37] dup(22)(q12.3-q13.1) NA VUS Syndromic DSD? chr22:g.36,649,117-38,380,539(over 1700 kb)* 9171 10 y Micropenis, unilateral cryptorchidism, 46, XY FGFR1 c.1961A>G, p.Y654C (het)# F (IP) VUS IHH hypogonadotropic hypogonadism 9242 15 m Perineal hypospadias, bifid scrotum 46, XY SRD5A2 c.650C>A, p.A217E (het); c.680G>A, F/M LP 5α-Reductase p.R227Q (het) P deficiency 9431 3 y 1 m Ambiguous genitalia, predominantly 46, XY SRD5A2 c.607G>A, p.G203S (het); c.680G>A, F/M LP 5α-Reductase female external genitalia, inguinal p.R227Q (het) P deficiency testes, absent Müllerian structures 9471 12 y Predominantly female external genitalia, 46, XY AR c.4G>A, p.E2K (hemi) M LP AIS enlarged clitoris, inguinal testes, absent Müllerian structures Y Xu,Wangandothers 9482 5 y Micropenis, cryptorchidism 46, XY SRD5A2 c.680G>A, p.R227Q (hom) F/M P 5α-Reductase deficiency
Symbol # indicated novel variant. Symbol * indicated certain results were confirmed by traditional electrophoresis and microarray (data not shown). AIS, androgen insensitivity syndrome; CAH, congenital adrenal cortical hyperplasia; F, paternal inheritance; F/M, inherited respectively from parents; GD, gonadal dysgenesis; Hemi, hemizygous; Het, heterozygous; Hom, homozygous; IHH, idiopathic hypogonadotropic hypogonadism; IP, incomplete penetrance; KS, Kallmann syndrome; LCH, Leydig cell hypoplasia; LP, likely pathogenic; M, maternal inheritance; M, month; NA, not available; P, pathogenic; PMDS, persistent Müllerian duct syndrome; VUS, uncertain significance; Y, year. and 15.6%(7/25),respectively. Otheretiologiessuchas and gonadaldysgenesis(GD) accountedfor17.8%(8/25) Congenital hypogonadotropic hypogonadism(CHH) deficiency, causedby caused by consisted of androgen insensitivity syndrome (AIS), (DASA), accountingfor55.6%(25/45),whichmainly causes were disorders of androgen synthesis or action DSD patientswithetiologicdiagnoses,themostcommon mutation amongChinesepatients.Amongthe46,XY with 5 in the and sporadic. Notably, one variant (c.680G most commonones,whileotherswererelativelyrare were novel.Variants in ( Identified variantsweremainlydistributedin28genes p.Met318Asn fs*11) in the to beidentifiedwithaheterozygousvariant(c.952dupA, 1).Theresultsshowedonlyonepatient (ID9056) Table 9524, 9553,9743,9771,9954and9955(Supplementary IDs were7902,8017,8135,8145,8318,8510,9056,9262, might havebeenmissedbyTPS.There-sequencingsample additionally inanattempttoidentifycandidategenesthat (six of46,XYandeight46,XX)performedWES Furthermore, werandomlyselected14negativesamples Negative sampleswithfurtherWESanalysis and 10.3%for46,XXDSDpatients. we obtainedetiologicdiagnosticratesof46.9%for46,XY histopathology andenzymeassay, asneeded.Accordingly, inheritance mode,phenotype,hormonelevels,gonadal the basisofclinicalcertainty, whichwereconfirmedby were includedtocalculatethefinaldiagnosticyieldon according tothecurrentmolecularevidence,thesecases age group,namely age distribution,thenumberofpatientsinpuberty ( patients accordingtothekaryotype. into 76.8%(96/125)46,XYand23.2%(29/125)46,XX The 125patientsincludedinthisstudyweredivided Overview ofthepresentDSDpatientcohort any variantsresponsiblefortheclinicalmanifestations. experimental studies.In13othersamples,wedidnotdetect and thegenotyperemaintobeclarifiedwithmorecases correlation betweenphenotypeofabsentinternalgenitalia gland, shortstatureandintrahepaticcalcification.The explain thesymptomsofhypothyroidism,smallthyroid DSD cohort Sequence variantsinChinese i. 2C Fig. α SRD5A2 -reductase deficiency, indicatingitisahot-spot ). Amongallfindings,40.8%(31/76)variants AR genevariants,andsteroid 5 genewasfoundin75%(9/12)patients > 9 years old,wasthelargest( 9 years SRD5A2 Downloaded fromBioscientifica.com at09/30/202107:44:35AM SRD5A2 FAM111A genevariants( https://eje.bioscientifica.com and 181 Fig. 2A gene, which may gene, which may AR :3 geneswerethe > ). Forpatient A, p.R227Q) α -reductase i. 3A Fig. Fig. 2B 317 via freeaccess ). ). European Journal of Endocrinology 50.0 and50.0%,respectively. cohort (29patients),candidatevariantswereidentifiedin24.1%(7/29)patients.ThepathogenicandVUSaccounted for variants ofuncertainsignificance(VUS)accountedfor37.3,34.3and28.4%thepatients,respectively.(B)In46,XXDSD patients), candidatevariantswereidentifiedin59.4%(57/96)patients.Thepathogenicvariants,likely and Positive rateoftargetedpanelsequencingandproportionvariantsatdifferentevidencelevels.(A)Inthe46,XYDSDcohort(96 Figure 1 https://eje.bioscientifica.com NGS-based sequencinginalargeChinesecohortof To our knowledge, this is the first study to apply unbiased Discussion for 46,XXDSD( 46,XY DSDandhypoplasiaorabsenceofuterus(18/26) clinical manifestationwaspeniledysplasia(30/51)for the remaining undiagnosed patients, themostcommon 11 with etiologic diagnoses, the main causes were CAH with accounted for11.1%(5/25).Inthe46,XXDSDpatients persistent Müllerianductsyndromeandhypopituitarism number ofvariantsisshownfor eachgene,includingpreviouslyreportedvariantsandnovel ones. 46, XXDSDpatients.(C)Distribution ofthevariantsidentifiedinDSDpatients.Variantswere identifiedin28genes.Thetotal classification. Thisstudyincluded 76.8%(96/125)46,XYand23.2%(29/125)XXpatients. (B)Theagedistributionof46,XYand Distribution oftheDSDcohort populationandidentifiedvariants.(A)Ascertainmentofthe karyotype determinedthemajorDSD Figure 2 Clinical Study β -hydroxylase deficiency(2/3)andCHH(1/3). Within Fig. 3B ). Y Xu,Wangandothers 23–43% ( improves theoveralldiagnosticrateofDSD,reaching Several studieshaveshownthattheapplicationofNGS been successfullyapplied in thediagnosis of DSD patients. technologies suchasWESandpanelsequencinghave obtained an etiologic diagnosis ( NGS waswidelyapplied,only13%DSDpatientshad patients with46,XYand46,XXDSD.Inthepast,before DSD patients( have nosignificantdifferenceinthediagnosticrateof ( of 46,XYDSDpatientsreceivedlikelygeneticdiagnoses foreign DSDpatients,andtheresultsshowedthat43% a DSD-relatedgenepanelinlarge-scale cohort studyof DSD cohort Sequence variantsinChinese 23 ). Thestudyalsosuggestedthatsingletonsandtrios 18 , 19 23 , 20 ). Thusfar, fewstudieshaveappliedWES , 21 , 22 Downloaded fromBioscientifica.com at09/30/202107:44:35AM , 23 , 24 , 25 34 ). Eggers 181 ). At present, NGS :3 et al . applied 318 via freeaccess European Journal of Endocrinology 46,XX patients. absence ofuterusorovarieswerethetopcharacteristics genitals wascommonin46,XYpatients,andhypoplasiaor undiagnosed patients.Abnormaldevelopmentofexternal (B) Thisbargraphshowsthemainclinicalmanifestationsof action; GD,gonadaldysgenesis;LCH,Leydigcellhypoplasia;. hypogonadism; DASA,disordersofandrogensynthesisor hyperplasia; CHH,congenitalhypogonadotropic insensitivity syndrome;CAH,congenitaladrenalcortical recommended classificationofDSD( have beencategorizedintosubclassesbasedonthe patients withdefinitediagnosesinthisstudy.The patients. (A)Proportionofdifferentetiologies46,XYDSD Characteristics ofthediagnosedandundiagnosedDSD Figure 3 the mostcommonCAH-causing gene(responsiblefor etiologic diagnosis.Itis noteworthy that validation to determine more credible and convincing the analysis between molecular results andclinical candidate variants.Accordingly, wefurtherintegrated Chinese 46,XY and 46,XX DSD patient cohort to identify (containing allknownDSD-associatedgenes),inalarge targeted with2742knowngeneticdisease-causinggenes study, we applied an unbiased and large-scale gene panel, diagnostic rateofWESlackssupportingevidence.Inthis in thediagnosisofDSDpatientslargecohorts,and Clinical Study Y Xu,Wangandothers 1 ). AIS,androgen CYP21A2 , variation may affect the development of ovarian follicles variation mayaffectthedevelopment ofovarianfollicles However, animal model studies have found that its and humangonadaldevelopment hasbeenidentified. ( development throughthe TGF-beta signalingpathway protein, itparticipatesintheregulationofgrowthand osteopathy gene.Asareceptorofbonemorphogenetic uterus werenotreported. GNAS this study, DSDwasthemainphenotypeof patientswith abnormalities, suchashypogonadism( present withconditionsassociatedhormonelevel bone dysplasiaasthemainphenotype.Afewpatientsmay such asgonadotropin( resistance toparathyroidhormonesandother pattern. Pathogenic variants have beenreported to cause protein withahighlycompleximprintingexpression guaninenucleotide-binding subunit ofthestimulatory genes seemedtobedeleterious. namely to thecurrentlyrecognizedpathogenicgenesofDSD, In addition,wealsofoundthreegenesthatdonotbelong believe that these variants contribute to the phenotypes. with theclinicalmanifestation.Hence,itisreasonableto that the molecular findings were undoubtedly consistent gonadal histopathology and enzyme assay, we found inheritance mode,clinicalphenotypes,hormonelevels, the comprehensiveverificationofsequencingdataquality, as VUSaccordingtothecurrentevidence.However, with p.M262R/p.A398V andsoforthcouldonlybeclassified pathogenic classes accounted for 68.4%. Nine variants pathogenic classesaccountedfor68.4%.Ninevariants guidelines ( for pathogenicity strictly according to the recommended spectrum ofDSD.Allidentifiedvariantswereclassified reported forthefirsttime,furtherexpandingmutation 10.3%, whichissimilartotheresultsofotherstudies. and here,weobtainedanetiologicdiagnosticrateof diagnostic rateof46,XXDSDpatientsisgenerallylow, higher thanthatinotherresearch, reaching46.9%.The etiologic diagnosticrateof46,XYDSDpatientsisslightly were notincludedinthisstudy. Ourresultsshowthatthe patients withCAHcausedby amplification testsforpatientssuspectedwithCAH.Thus, gene sequencing/multiplexligation-dependentprobe this geneisincludedinourpanel,wegenerallyaddsingle makes specificcapturebyNGSdifficult( existence ofhighlyhomologouspseudogenes,which over 95%CAH),wasexcludedinthisstudyduetothe including DSD cohort Sequence variantsinChinese 38 ). Thusfar, nocorrelationbetweenthe We identified76candidatevariants,ofwhich31are gene variants, and phenotypes such as primordial gene variants, and phenotypes suchas primordial GNAS LHCGR 26 , , 27 BMPR1B , p.C514S, 28 , 29 36 ). Variants of pathogenicand likely and Downloaded fromBioscientifica.com at09/30/202107:44:35AM ). Patients usually have multiple ). Patientsusuallyhavemultiple AR GHR BMPR1B p.A48S/p.G209E, https://eje.bioscientifica.com GNAS CYP21A2 , but variants in these , butvariantsinthese 181 36 is a well-known isawell-known encodes the alpha encodesthealpha :3 , 37 genevariants 35 BMPR1B ). However, in ). Although CYP17A1 gene gene 319 via freeaccess
European Journal of Endocrinology https://eje.bioscientifica.com may be missed by current panels. Therefore, we further new, unknown genesinvolvedingonadaldevelopment regardless ofthescalepanel applied.We assumedthat number ofpatientsinwhom novarianthasbeenfound, of DSDpatientstoacertain extent,thereisstillalarge confirmed thatTPSgreatlyimprovedthediagnosticrate diagnostic ratewithcertainty. to diagnose this DSD cohort and obtained an etiologic Action BM1303DSDnet( by EuropeanCooperationinScienceandTechnology the lightofdiagnosticapproachflowrecommended classification ofidentifiedvariantsforDSDpatients. In to determinetheetiologicdiagnosisinsteadofmere out in combination with clinical and published evidence application, comprehensiveevaluationshouldbecarried criterion forthedefinitediagnoses.Inclinicalpractical pathogenicity; however, theycannotbeusedastheonly play animportantroleinthejudgmentofvariants’ classification guidelines,webelievethattheguidelines doubts inthepracticalapplicationofcurrentvariants triplosensitivity andphenotypes. further study of the relationshipbetween certain gene confirmed yet( development, whiletriplosensitivityhasnotbeen like not beenreportedyet.Genesinvolvedinthisregion, within thechr22:36,649,117–38,380,539regionhas manifested asabnormalgenitalia.Theotherduplication the chr1:213,031,597–237,997,288 region, which also sanger.ac.uk/ records providedinDecipherdatabase( in patientswithsyndromicDSD.Combinedthe association withDSD( upon review, halfofthesehadinfact been reportedin were classified as being ofuncertainclinical significance, of DSDpatients( are neededtodefinethecorrelationandpathogenicity. causing genes.Morecasesandfurtherfunctionalstudies The threegenesareregardedasnovelcandidateDSD- their correlationwithDSDphenotypesremainsuncertain. presumed to be pathogenic according to the guidelines, zebrafish ( has beenfoundtoincreasedifficultyinreproduction role infolliculardevelopment( and plays an autocrine/paracrine regulatory chicken ovary in granulosacellsandfollicular membrane cellsofthe ( 39 Clinical Study ). Ithasalsobeenfoundthatthe Although manystudies,includingours,have Based onourownexperienceandtheabove CNVs havebeenreportedtobeidentifiedin33.3% SOX10 41 , maycontributetoabnormalgonadal ). Although the variants of these genes were ). Althoughthevariantsofthesegeneswere ), wefoundseveralcasereportscovering 43 ). Thesefindingslaythefoundationfor 17 , 42 28 ), andalthoughthree-quarters 17 ). We identifiedlargeCNVs ), wepracticedtheapproach 40 Y Xu,Wangandothers ). GHR GHR gene is expressed geneisexpressed https://decipher. overexpression overexpression gonadal development.Mostresearch hasmainlyfocused expression contribute to the pathogenesis of abnormal elements that alter gene non-coding variants in regulatory reasons mighthelpexplainthenegativesamples.First, choice forre-testingisnotparticularlysuitable. negative casesinitiallyscreenedbyTPS,WESasthefirst a newinsightintotheundiagnosedDSDpatients.For undiagnosed DSDpatients( WES isapreferabletoolforfindingnovelcausesin WES, althoughaccordingtorecentrecommendations, little differenceinthediagnosticratebetweenTPSand remaining 13 samples remained undiagnosed, implying and phenotypewithfurtherin-depthresearch. The be clarifiedintermsofthecorrelationbetweengenotype was identifiedwithasuspectedvariant,whichremainsto our results showed that only 1 of the 14 negative samples an attempttoidentifycandidatenovelgenes.Surprisingly, performed WESinrandomlyselectednegativesamples for alargeproportionin all agegroups,especiallyin results. Ourdatashowedthat 46,XYDSDpatientsaccount 47 tends to be much higher in 46,XY DSD patients ( thus, thecurrentdiagnostic rateofpanelsequencing easily detected gonadal malformations in the early stages; the muchlargernumberofpatientsandmore understood thanthatin46,XXDSD,largelybecause of we re-analyzethesenegativepatients. studies, wewillfocusonaforementionedsituationwhen suitable forthissituation( phenotypes. Currentgeneticdetectiontechnologyisnot development andenvironmentalfactorsresultsinDSD combination ofepigenetic factors affecting early gonad pathogenesis ofsomecasesmaybemultifactorial.The such asgonadscanbedetected.Finally, theunderlying isolated by peripheral blood, while those in specific tissues 44 patterns intheprocessofsexualdifferentiation( other genesarestrictlyregulatedforspecificspatiotemporal transcription factorssuchas combination withmicroarray( technology couldbemissed,warrantingre-analysisin fragments thatarenoteasilydetectedbytheexistingNGS ability toidentifynovelgenes.Second,CNVswithinsmall variants canbedetectedbyWGS,whichalsohasthe malfunction ofDSD-relatedgenes( the expression of target genes, resulting in spatiotemporal elementswouldaffect found thatvariantsinregulatory on the coding part of the genome, while some studies have DSD cohort Sequence variantsinChinese ). ThisstudyofaChineseDSD cohortyieldedsimilar ). Somatic cell variants cannot be identified inDNA In additiontounknownnovelgenes,thefollowing The pathogenesisinvolvedin46,XYDSDisbetter Downloaded fromBioscientifica.com at09/30/202107:44:35AM 5 , 17 SRY 17 ). Thus,inoursubsequent 44 ). Ourfindingsdeliver , , SOX9 44 45 181 ). Thesenon-coding ). Third,somekey and :3 FOXL2 10 320 , and , 46 12 via freeaccess , , European Journal of Endocrinology Our resultsexpandthe gene spectrum,mutation founder mutationinpatients with5 DSD genotype–phenotypecorrelation anddetermineda novel candidategenes/regions outsidetheexpected of theChineseDSDpopulation forthefirsttime,reported improve thediagnosticrate.We alsoprovided an overview Re-analysis ofpatientsundiagnosedbyWESdidnot of 46.9%for46,XYand10.3%46,XXDSDpatients. clinical certainty, weobtainedetiologic diagnostic rates negative samples.With thecurrentdiagnosisflowand DSD andfurtherperformedWESinrandomlyselected large Chinese cohort of patients with 46,XY and 46,XX Chinese population. further studyofgenotypeandphenotypecorrelation in with variousetiologiesandprovidestrongsupportfor a large number of non-chromosomal type DSD patients contribute tothelowdetectionrate.Ourresultspresent the difficult assessment of ovarian function inchildhood patients. Theheterogeneityofimagingexaminationand of uterusandovarieswasthecommoninundiagnosed caused by 46,XX DSDcohort,themostcommonetiologyisCAH characteristics wereoftenunnoticeduntilpuberty. Inthe sexual amenorrheaandundevelopedsecondary primary yearsold)becausephenotypesof46,XXDSDsuchas 9 gene variants, mainly falling in the puberty group (over considerable numberofCAHpatientscausedby of 46,XXDSDpatientsislowerduetotheexclusiona been updatedtimelyinChina.Meanwhile,thenumber associated referencerangeofmeasurementsthathasnot be somewhatrelatedtocertainnonspecificcausesand dysplasia wasmostcommonmanifestation,whichmay the remainingundiagnosedpatients,penileortesticular it hasafoundereffectinthepopulationofChina.Asfor results ofthe of theChinesepopulation.Combinedwithotherresearch indicating thatitaccountsforaconsiderableproportion variant wasinheritedfromtheunaffectedparents, with 5 p.R227Q) in at anearlystage.Moreover, ahot-spotvariant(c.680G important for46,XYDSDpatientstoidentifytheetiology AIS patients is about 15% ( dysplasia patientscanbeashigh30%,whilethatin It was found that the risk of germ cell tumors in testicular frequent causeof46,XYDSDintheChinesepopulation. rare causes. AIS caused by DSD are variable, including DASA, CHH, GD and other the group of 9 to 14-year-olds. The etiologies of 46,XY Clinical Study In summary, thisstudyapplied unbiasedTPSina α -reductase deficiency. Notably, thisheterozygous CYP11B1 SRD5A2 SRD5A2 genevariants.Hypoplasiaorabsence gene( geneisfoundin75%patients 48 AR 18 ). Therefore, it is particularly gene variants is the most , 49 Y Xu,Wangandothers , α 50 -reductase deficiency. ), wesuggestedthat CYP21A2 > A,
omsin Gat o 1S1) te rjc o Saga Municipal Shanghai 20152529) of No. (Grant Project Medicine Clinical the Commission-Gaofeng Education 18SG14), No. (Grant Commission Education Municipal Shanghai and Foundation Development Education of China (Grant No.81772303), ‘Shuguang Program’ supported by Shanghai Foundation Science Natural National the by supported was research This Funding be could that interest of conflict perceived asprejudicingtheimpartialityofthisstudy. no is there that declare authors The Declaration ofinterest EJE-19-0111 This islinkedtotheonline version ofthepaperat Supplementary data understanding oftheunderlyingetiologiesDSD. further studies;andprovidenewinsightintothecurrent DSD in the Chinese population; lay the foundation for spectrum, andphenotypicspectrumoftheraretypes References evaluating theclinicalconformityafterobtainingmolecularfindings. associated clinicians forproviding detailed clinicalinformationandfor their familiesforparticipation in this study. They aregrateful to the The authorswould like toexpresssinceregratitudeallpatientsand Acknowledgements of Medicine(GrantNo.BXJ201949). School University Tong Jiao Shanghai of Fund Innovation Doctoral the and DSD cohort Sequence variantsinChinese 7 6 5 4 3 2 1 Jørgensen A, LindhardtJohansen M, Juul A,Skakkebaek NE, Cools M, Drop SL,Wolffenbuttel KP, Oosterhuis JW&Looijenga LH. Arboleda VA, Sandberg DE&Vilain E. DSDs:genetics,underlying Lux A, Kropf S,Kleinemeier E,Jürgensen M,Thyen U&DSD Thyen U, Lanz K,Holterhus PM&Hiort O.Epidemiologyandinitial Ostrer H. Disordersofsexdevelopment(DSDs):anupdate. Hughes IA, Houk C,Ahmed SF, Lee PA &LawsonWilkins Pediatric Main KM &Rajpert-DeMeyts E.Pathogenesis ofgermcellneoplasia org/10.1210/er.2006-0005) moving frontiers. Germ celltumorsintheintersexgonad: oldpaths,newdirections, nrendo.2014.130) Endocrinology pathologies andpsychosexualdifferentiation. Public Health design, descriptionofthestudypopulation,anddataquality. network ofdisorderssexdevelopment(DSD)/intersexuality:study Network Working Group.ClinicalevaluationstudyoftheGerman Research management ofambiguousgenitaliaatbirthinGermany. doi.org/10.1210/jc.2013-3690) Clinical EndocrinologyandMetabolism doi.org/10.1136/adc.2006.098319) disorders. Consensus Group.statementonmanagementofintersex Endocrine Society/EuropeanSocietyforPaediatricEndocrinology . 2006 Archives ofDiseaseinChildhood 2009 2014 66 195–203. Endocrine Reviews 9 10 110. 603–615. (https://doi.org/10.1186/1471-2458-9-110) Downloaded fromBioscientifica.com at09/30/202107:44:35AM (https://doi.org/10.1159/000094782) (https://doi.org/10.1038/ 2006 https://eje.bioscientifica.com 2014 2006 27 99 181 468–484. 1503–1509. https://doi.org/10.1530/ Nature Reviews: 91 :3 554–563. (https://doi. (https:// Hormone Hormone Journal of Journal (https:// BMC 321 via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com
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Accepted 5July2019 Revised versionreceived2June2019 Received 17February2019
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