European Journal of Endocrinology 10.1530/EJE-17-0335 Union MedicalCollege,InstituteofBasicSciences,ChineseAcademyBeijing,China Physiology, StateKeyLaboratoryofMedicalMolecularBiology, SchoolofBasicMedicine,GraduatePeking Academy ofMedicalSciences,KeylaboratoryEndocrine,MinistryHealth,Beijing,Chinaand 1 Mingxuan Cui Introduction foundation forprenataldiagnosisandgeneticcounseling. domain, whichareprobablypathogenic.Thesemutationsexpandthe KS patients.Inthepresentstudy, weidentifysevennovel Conclusion(s) cryptorchidism uniformly. patients withdifferent kindof werepredictedtobepathogenic.Theprevalenceofcryptorchidismwashigh(38.1%)andoccurredin ANOS1 IHH probands.Sevennovel(C86F, C90Y, C151W, Y379X,c.1062 Result(s) analyzed retrospectively. was usedtoassessfunctionalrelevanceofnewlyidentifiedmissensemutations.Patients’clinicalcharacteristics were Patients’ Patients andmethods the clinicalpresentationofdiseaseinpatientswith Objective Abstract hypogonadotropic hypogonadism (IHH) is caused by gonads toproducesexsteroids andgametes.Isolated in the pituitary, which subsequently stimulate the hormone (LH)andfollicle-stimulating hormone(FSH) (GnRH) regulatesthesynthesis andreleaseofluteinizing pulsatile secretionofgonadotropin-releasinghormone maintenance ofnormalreproductivefunction( a crucialroleinthedevelopment,progressionand (HPG)axis plays The hypothalamic–pituitary–gonadal Min Nie ANOS1 of aChineseKallmannsyndrome cohortwith Analysis ofgeneticandclinicalcharacteristics Department ofEndocrinology, PekingUnionMedicalCollegeHospital,College,Chinese DOI: 10.1530/EJE-17-0335 www.eje-online.orgwww.eje- Clinical Study mutations(S38X,R257X,R262X,R423X,R424X,V560I,c.1843-1G online.org : Fifteennonsynonymousrare 1 , Hongli Xu ANOS1 : To analyze : Theprevalenceof

1 , Wanlu Ma genesequenceswereanalyzedbydirectsequencingofPCR-amplifiedproducts. mutations 1 : ChinesepatientswithKS,including187sporadicand23pedigreecaseswererecruited. ANOS1 , Rongrong Chen 1 , Qibin Huang genemutationsinalargeChineseKallmannsyndrome(KS)cohortandtocharacterize © 2017EuropeanSociety ofEndocrinology ANOS1 M Nieandothers ANOS1 mutations,whilethepatientswithsamemutationdidnotpresent ANOS1 genemutationsislowinsporadicKSpatients,butmuchhigherfamilial 2 , Jiangfeng Mao Printed inGreatBritain 1 , Hongbing Zhang variantswerefoundin13outof187sporadicand823familial 1 ). The ANOS1 ANOS1 1 , Xi Wang KS patientswith Genetic and clinical characteristics of neurons originate from the nasal placode. During normal hyposmia/anosmia. BothGnRH neuronsandolfactory KS ( (nIHH). Approximately 50% of all IHH cases are due to normosmic idiopathichypogonadotropichypogonadism sterility. IHHisdividedintoKallmannsyndrome (KS)and steroids, delayedpubertyorabsenceofand defects intheHPGaxis,resultinglowlevelsofsex + mutations. Published byBioscientifica Ltd. 2 1G and mutations,includingtwomutationsintheCR 2 KS isacongenitaldisordercharacterized byIHHand ). >

A, Y579Lfs591X,R597X)andeightrecurrent Xueyan Wu 1 , Shuyu Xiong ANOS1 >

A, p.R631X)wereidentified.Allthenovel ANOS1 2 Department of mutationspectrumandprovidea 1 1 Downloaded fromBioscientifica.com at09/29/202101:02:34PM , Junjie Zheng (2017) Endocrinology European Journal of [email protected] Email to XWu should beaddressed Correspondence 177 In silico 177 : 4 177 1 , Bingqing Yu :4 , 389–398 analysis

389 –398 1 via freeaccess , European Journal of Endocrinology causative factors( development, migrationandfunctionareimportant but mutations in that regulate GnRH neuron Declaration ofHelsinki.Inclusion criteriawere( Committee forHumanResearch andcomplieswith the data. Thestudywasapproved bythePUMCH’s Ethics with KSbasedonclinicalmanifestation andsexhormone 2016. Allpatientsweremale anddiagnosedpreliminarily 2009 andDecember Medical hospitalbetweenJanuary patients withKSwererecruitedfromPekingUnion Twenty-three familialand187unrelatedsporadicChinese Subjects Subjects andmethods ANOS1 analyze theclinicalcharacteristicsofKSpatientswith ANOS1 patient populationtodeterminetheprevalenceof sporadic and23familialcasesofKSfromtheChinese with nomutation‘hotspots’intheaffectedregions. , whicharespreadwidelythroughouttheentiregene mutations have,hitherto,beenidentifiedinthe of familialandsporadicKSpatients.Nearlyseventy ANOS1 producing neuronsandoligodendrocyteprecursors. multiple typesofneuronalprecursors,includingGnRH- branching. Additionally, itplaysaroleinthemigrationof outgrowth, axonalguidanceandCNSprojectionneuron ( (FnIII) domains and a histidine-rich C terminal region acidic (WAP) domain, fourfibronectintypeIII cysteine-rich domain(Cys-box),followedbyawhey matrix protein.Anosmin-1comprisesanN-terminal ANOS1 highly variableandunstableregionofthechromosome. (Xp22.3) adjacent to pseudoautosomal region 1 (PAR1), a cause X-linkedKS.ItislocatedontheXchromosome and seizures( neuralhearingloss shortening ofmetacarpals,sensory agenesis, synkinesis,cleftlip,palate,dental also have otherassociated abnormalities, such as renal results inhyposmia/anosmia.SomeKSpatientsmay bulb and its axonal tracts or hypoplasia of the olfactory GnRH neuronmigrationleadtoIHH,andtheabsence neuron axonsto the hypothalamus. In KS,defects in development, GnRHneuronsmigratealongolfactory www.eje-online.org 5 ). Anosmin-1promotesneuronalcelladhesion,neurite Clinical Study In thisstudy, wesequencedthe The mutations. mutationsinalargeChineseKScohortandto mutationsarefoundinapproximately10–20% encodestheproteinanosmin-1,anextracellular ANOS1 3 ). TheetiologyofKSisnotquiteclear, geneisthepathogenicfoundto 4 ). M Nieandothers ANOS1 genein187 1 ) absence ANOS1

KS patientswith Genetic and clinical characteristics of retrospectively. and medicationhistory, werecollectedandanalyzed hearing lossandseizures,testissize,thespermcounting neural shortening ofmetacarpals,renalagenesis,sensory synkinesis, cleftlip,palate,dentalagenesis, genetic testing. Theclinical manifestations, including from allpatientsandsomeparentswerecollectedfor were excludedfromthestudy. ( axisand axisandpituitary–thyroid axis, pituitary–adrenal of growth hormone-IGF1 (insulin-like growth factor 1) deficient senseofsmell(hyposmia),( ( normal gonadotropinlevelsathypogonadallevels, concentration ofsexsteroids,( yearsofage,( of pubertaldevelopmentby18 p.protein). ForgenomicDNA andcDNAnumbering,the ( NG_007088.1 ( was performedwiththereference sequencesGenBank pro V1.7(AppliedBiosystems) andaBLASTsearch are availableonrequest. reverse directions in all patients. All sequencing primers of the coding region,includingtheexon–intronboundaries automated sequencer(AppliedBiosystems).Theentire a Taq bigdyeterminatorsequencingkit and anABI3730 PCR PurificationKit,andsubsequentlysequencedusing by PCR.ThePCRproducts were purifiedwith QIAquick exon–intron boundariesofthe manufacturer’s instructions.Fourteenexonsandthe blood usingaQIAGENMiniBloodkitaccordingtothe Genomic DNAwasextractedfromleukocytesofperipheral Mutation screening respectively. 0.07 respectively. Thelowestmeasurablelimitswere0.23 and 2.8%forLH5.6%6.6%totaltestosterone variation coefficientswere3.9%and4.5%forFSH,2.3% Diagnostics Corporation,USA).Theintra-andinter-assay using chemiluminescentimmunoassays(Bayor Basal serumFSH,LHandtestosteroneweremeasured Hormonal assays 6 4 ANOS1 ) other conditions leading to secondary hypogonadism ) otherconditionsleadingtosecondary ) documentedabsenceofsensesmell(anosmia)or After obtaininginformedconsent,bloodsamples The resultingsequenceswereanalyzedusingChromas

IU/L and5.2 ANOS1 , c.DNA) and GenBank NP_000207.2 (ANOS1, ANOS1 gene, was sequenced in both forward and gene, was sequenced in both forward ANOS1

ng/dL forFSH,LHandtotaltestosterone mutation , g.DNA),GenBankNM_000216.2 Downloaded fromBioscientifica.com at09/29/202101:02:34PM ANOS1 3 ) loworinappropriately 177 genewereamplified 5 ) normalfunction :4 2 ) low 390

IU/L, via freeaccess European Journal of Endocrinology ( of missensevariant( 3D structures of sequence homologs to assess the function alignments of large numbers of homologous sequences and informationderivingfrom proteinfamily evolutionary ( harvard.edu/pph2/). and comparativeconsiderations( physical the structureandfunctionusingstraightforward predicts possibleimpactofanaminoacidsubstitutionon sequences ( in sequencealignmentsderivedfromcloselyrelated ofamino acidresidues on thedegreeofconservation www.mutationtaster.org/ the affectedaminoacidinproteinhomologs( of conservation functional impactbasedonevolutionary included inthisstudy. ( mutation. The following prediction resultisnotexactlysameforaspecificmissense were used in different prediction toolssincedifferentprinciplesandalgorithms in thisstudywerealsoanalyzedusinganextensivesetof ( ( (May 2013)fromcloselyanddistantlyrelatedspecies 26 orthologous sequences within the same database database ( anosmin-1 sequence,asannotatedintheUniProt of anosmin-1orthologs(MSAORTHO).Thehuman acids wasassessedbyamultiplesequencealignment attheaffectedamino conservation Evolutionary Bioinformatics analysisofnovelmissensevariants clinical manifestations. same mutation presented with not exact the same toexplainthereasonwhypatientswith to try sequences andPCRconditionsareavailableuponrequest) LEPR KISS1R gonadal axis( additional genesrelatedtothehypothalamic–pituitary– screened fordigenic/oligogenicmutationsbysequencing exome variantsever(http://evs.gs.washington.edu/EVS/). database in NCBI ( if they were not found in 100 control subjects, dbSNP designated +1.Thevariantswereidentifiedasmutations A nucleotideof the ATG translation initiation codon was 5 https://www.bioedit.com/ 6 Clinical Study ) Panther. Itevaluates asinglesubstitutionatspecific ). MSAORTHOwasbuiltusingtheBioeditprogram Pathogenic effectsofmissensemutationsidentified Patients withidentified , WDR11 , NELF http://www.uniprot.org/ 8 ) ( , , HS6ST1 PROK2 FGF8 http://sift.jcvi.org/ http://www.ncbi.nlm.nih.gov/snp/ , 4 10 FGFR1 , , ) Mutationassessor. Itusesthe PROKR2 1 in silico CHD7 ). ( ) ( ) MutationTaster. Itassessesthe ). in silico 2 http://mutationassessor.org/ ) SIFT. Itspredictionisbased , GNRH1 and ANOS1 analysis tools, and the M Nieandothers 9 , ) (http://genetics.bwh. ), was aligned with analysis tools were ). ( TAC3 SEMA3A mutationswere , 3 GNRHR ) PolyPhen.It , TACR3 ) (allprimer 7 ) ( , KISS1 , http:// LEP ) or ). , ,

KS patientswith Genetic and clinical characteristics of of a query sequencetoaproteinhomologbefore of aquery score, whichmeasuresthechangeinsequencesimilarity ( and mutantsequences( structural featuresandfunctionalsitesbetweenwild-type upon protein sequence and molecular models change of It evaluatestheinfluenceofanaminoaciddisplacement ( estimate possible impact of an amino acid replacement different waysandthedefinedfunctionalGOscoreto informationderivedin information, evolutionary org/services/snap structure and solvent accessibility ( alignment, structuralfeaturessuchaspredictedsecondary taken from an automatically generated multiple sequence information prediction isbasedontheevolutionary pantherdb.org/tools/csnpScoreForm.jsp about evolutionarily related ( amino acidpositioninaproteinbasedontheinformation ‘hot spot’regionin intron–exon boundaries;that is,wefoundnomutation are widelydistributedin nine differentexonsorat C R424X (onesporadicpatientandonepedigree)c.784 G a single pedigree or case, with the exception of c.1678 the 100 controls). Each mutation was detected only in were novel(i.e.absentfromthequerieddatabasesand R597X) andoneinsertion(c.1736insT, Y579L (c.1062 C90Y, c.453C missense substitutions(c.257 G c.1843-1G mutations (S38X,R257X,R262X,R423X,R424X,V560I, insertion mutationswereidentified( four missense, eight nonsense, two splicing site and one out of187sporadicIHHcases.Among21KSpatients, mutations in8probandsoutof23KSpedigreeand13 Overall, ourmutationscreeningof Mutation screening andoligogeneticanalysis Results regions ( functional sites,aggregationpropertiesanddisordered variant throughthestructuralneighborprofiles,nearby php sequence( the query and aftertheintroductionofanaminoacidvariationto 9 13

> > ) Provean.Itspredictionisbasedonanalignment-based ) (http://snps-and-go.biocomp.unibo.it/).(

T/p.R262X (twosporadicpatients). Thesemutations A/p.V560I (threesporadicpatients),c.1270C ) and(

+ 16

1G

> ) (http://sapred.cbi.pku.edu.cn/). 10

A andR631X)werereportedpreviously. Three > ANOS1 ) Sapred.Itassessestheeffectofmissense

A), two nonsense mutations (Y379X,

>

). ( G/p.C151W), onesplice-sitemutation mutation ANOS1 7 ) SNPs&Go.Itutilizessequence 15 14 Downloaded fromBioscientifica.com at09/29/202101:02:34PM ) ( ( ) ( http://provean.jcvi.org/index. Fig. 2A http://mutpred.mutdb.org/

>

T/p.C86F, c.269G ). Allthemutationswe ANOS1 Fig. 1 177 12 ). ( www.eje-online.org ) ( 11 :4 ), ofwhicheight revealedfifteen https://rostlab. ) ( 6 ) SNAP. The http://www. 8 ) MutPred. fs

591X)

> > 391

A/p. T/p. via freeaccess ). European Journal of Endocrinology www.eje-online.org missense ornonsensemutations inrelationtotheanosmin1protein.Novelmutationswere showninred. the upperofbox,novelmutations foundinthisstudyarethebelowofboxand shown inred.(C)Distributionof Distribution ofmutationsin Figure 2 in red. The sequencingchromatogramofthemutationin Figure 1 Clinical Study ANOS1 geneandanosmin1protein.(A) Blueboxstandsfortheexons.(B)Recurrentmutationsare in M Nieandothers ANOS1 gene.Dashindicatesmutatednucleotide.Novelmutationswereshown KS patientswith Genetic and clinical characteristics of ANOS1 mutation Downloaded fromBioscientifica.com at09/29/202101:02:34PM 177 :4 392 via freeaccess European Journal of Endocrinology indicates theprobandofeach pedigree. Allindividualsmarkednumberswerescreenedfor themutationof KS pedigree(1–6)with Figure 3 pathogenic variants( missense variantsclassifiedC86F, C90YandC151Was six differentspecies( acrosstwenty- 151st ofanosmin-1proteinisconserved The cysteineresidueattheposition86th,90thand Bioinformatics analysisofnovelmissensevariants mutations respectively. mutations andboth possessed both heterozygotic ANOS1 is shownin carrier. Thedetailedpedigreechartofthefamilialpatients familial patients,andtheirmotherwasfoundtobea R424X, R597X,Y579L FnIII-3 ( identified locateindifferentfunctionaldomains,except Clinical Study One patientwasfoundtoharbornotonlytwo ANOS1 uain (c.1062 +1G mutations Fig. 2B mutationsincludingC90Y, R257X,R262X, Fig. 3 ). KISS1R ANOS1 . Fig. 4 Table 1 ANOS1 L200Vmutation.Two patients C86Fand fs ANOS1 591X andR631Xwerefoundin ) Ten mutationareshown.Affected individualswithKSareshownascompletelyshaded. The arrow ). V560Iand > in silico M Nieandothers

A andE552K)butalso KISS1R analysistoolsfor Tyr323His (Het) FGF8 E176K KS patientswith Genetic and clinical characteristics of ANOS1 Data collectedfromthe21patientswhoharbored Patient characteristics showed synkinesis. displayed agenesisofrightkidney, andnopatients uniformly presentwithcryptorchidism. Onlyonepatient with thesamemutation(V560I,R424X,R597X)donot (C90Y, V560I).However, wealsofoundthatpatients shift mutations(Y579Lfs591X)andmissense (S38X, R257X,R262X,R424X,R597X,R631X),frame- and wasfoundinpatientswithnonsensemutations ANOS1 tracts. Theprevalenceofcryptorchidism washighamong bulbs and olfactory absence/hypoplasia of olfactory included MRItestresultsandallofthem(100.0%)had one patient.Thirteenoutof21patients’medicalhistories Gynecomastia andagenesisofrightkidneyoccurredin All thesepatients have lowLH,FSHandtestosterone. the testicular sizes, except the patients with history the testicularsizes,exceptpatientswithhistory or HCG/HMG.HCG/HMGtherapymarkedlyincreased All patientsacceptedthetreatmentoftestosterone mutation-harboringpatients(8outof21,38.1%) genemutationsaresummarizedin ANOS1 mutation Downloaded fromBioscientifica.com at09/29/202101:02:34PM 177 ANOS1 www.eje-online.org :4 gene. al 2 Table 393 via freeaccess . European Journal of Endocrinology www.eje-online.org +: pathogenic. C151W C90Y C86F Table 1 bulb/sulci)werealsoinvestigated. and olfactory with mutations. TheclinicalcharacteristicsoftheKSpatients medical centerand found eightrecurrentandseven novel in alargeChinesecohortofKSpatientsfromsingle In thisstudy, weanalyzedthe Discussion patients andspermappearedinseven(70%). of cryptorchid. Spermatogenesis was evaluated in ten of anosmin-1proteinisconservedacrosstwenty-sixdifferent species. The sequencealignmentofanosmin-1proteinsfromdifferent species.Thecysteineresidueattheposition86th,90thand151st Figure 4 Clinical Study ANOS1 Functional predictionofmissensemutationfoundinKSpatients. Sift + + + mutation(theprevalenceofcryptorchidism Polyphen + + + Panther + + + M Nieandothers ANOS1 SNAP + + + genevariants Mutation assessor + + + KS patientswith Genetic and clinical characteristics of These novel missense mutantswillprovide us avaluable domain ofanosmin-1wereidentifiedforthefirsttime. occurring mutations(C86F, C90Y)located in theCys-box this study, fouraremissensemutations.Two naturally familial IHH( to be3–8%insporadicIHH( the incidenceof result is similar with previous reports. In those studies, much higherinfamilialpatients(6outof23,26.1%).This sporadic patients(13outof187,6.95%);however, itis Mutation tasting The prevalence of + + + ANOS1 19 , SNPs andGo 20 mutation ). Ofthefifteenmutationsfoundin + + + ANOS1 ANOS1 Downloaded fromBioscientifica.com at09/29/202101:02:34PM mutations is very low in mutations is very 17 mutationswasreported MutPred , + + + 18 ) andabout30%in 177 :4 Provean + + + Sapred 394 + + + via freeaccess European Journal of Endocrinology

Table 2 Clinical and laboratory finding of the KS patients bearingANOS1 mutations. Clinical Study

Age at the Testis size Family LH FSH treatment (mL) members Other clinical (IU/L) (IU/L) TS Nucleotide Protein initiation (L,R)→after No. with KS features MRIa (basal) (basal) (nmol/L) change change (years) Treatment Duration treated Sperm S0001 − Cryptorchid (right), nt 0.1 1.1 1.01 c.113 C > A p.S38X 21 TS 2 years 1,0→nt nt maldescent (left) S0002 − − 2 1.3 2.8 1.18 c.1678 G > A p.V560I 31 HCG + HMG 6.5 years 5,5→12,12 2.63*106/mL FGF8, E176K(Het) S0003 − Cryptorchid (left) nt 0.1 0.3 0.69 c.1678 G > A p.V560I 20 HCG + HMG 3 years 0,1→8,8 6.79*106/mL S0004 − − nt 0 0.1 0.50 c.1678 G > A p.V560I 30 HCG + HMG 19 months 3,3→10,10 6–8/HP

S0005 − − nt 0 1.0 0.40 c.1267 C > T p.R423X 25 HCG + HMG 2 months 1,1→nt nt M Nieandothers S0006 − − 1 0.1 0.79 0.80 c.453 C > G p.C151W 20 HCG + TS 2 years 1,1→4,4 nt HCG + HMG 3 years S0007 − − 1 0 0 0.64 c.1062 + 1G > A p.E552K 20 HCG + HMG 2 years 1,1→2,2 nt KISS1R, L200V(Het) S0008 − − 3 0.3 1.3 0.90 c.1137C > G p.Y379X 20 TS 5 years 1,1→8,8 0–2/HP GnRH 4 months S0009 − − 1 2.2 2.5 3.31 c.1843-1G > A 21 TS 8 months 1,1→16,16 94.4*106/mL Letrozole 4 months TS 6 months S0010 − − 1 0.0 0.9 0.86 c.784 C > T p.R262X 20 HCG + HMG 4 years 2,2→8,8 13.05*106/mL S0011 − Cryptorchid (both) + nt 0.2 0.7 nt c.784 G > T p.R262X 34 TS 11 years 1,1→1,1 nt KS patientswith Genetic and clinical characteristics of gynecomastia S0012 − − 1 0 0.2 1.10 c.1270 C > T p.R424X 19 TS 2 years 1,1→2,2 0 HCG 10 months HCG + HMG 1 year S0013 − − nt 0.1s 0.8 1.00 c.257 G > T p.C86F 20 TS 11 years 2,2→4,4 nt KISS1R, ANOS1 Y323H(Het)

F0001 Cousin, Cryptorchid (both) nt 0 0.2 0.56 c.269 G > A p.C90Y 19 TS 2 years 1,1→2,8 nt mutation maternal HCG 1.5 years

Downloaded fromBioscientifica.com at09/29/202101:02:34PM uncle HCG + HMG 2.5 years F0002-10 Brother − 1 0 0.5 0.45 c.1789A > T p.R597X 19 TS 4 months 2,2→4,4 0 HCG 9 months HCG + HMG 1.5 years GnRH 2 months F0002-11 Brother Cryptorchid (both) 1 0 0.3 0.0 c.1789A T p.R597X 18 TS 4 years 2,2 4,4 0.7*106/mL > → 177 HCG + HMG 1 year www.eje-online.org :4 F0003-3 Brother Cryptorchid (both) 1 0.5 1.6 0.97 c.1270 C > T p.R424X 25 TS 6 months 2,0→2,0 nt HCG 1 year F0003-1 Brother − nt 0.29 1.7 0.66 c.1270 C > T p.R424X 19 TS 4 months 1.5,1.5→2,2 nt

(Continued) 395 via freeaccess European Journal of Endocrinology www.eje-online.org Clinical Study

Table 2 Continued.

Age at the Testis size Family LH FSH treatment (mL) members Other clinical (IU/L) (IU/L) TS Nucleotide Protein initiation (L,R)→after No. with KS features MRIa (basal) (basal) (nmol/L) change change (years) Treatment Duration treated Sperm F0004 Maternal Cryptorchid (right), 1 0 0 0.74 c.769C > T p.R257X 20 TS 2 years 2,2→8,8 0 uncle, agenesis of right HCG 9 months cousin kidney HCG + HMG 3 years F0005 Maternal 1 0 0.9 0.34 c.1736 ins T Y579Lfs591X 20 TS 5 years 1,1 2.5,2.5 nt

− → M Nieandothers uncle HCG 5 months HCG + HMG 46 months F0006 Maternal Cryptorchid (both) 1 0 0 1.35 c.1891 C > T p.R631X 18 TS 4 months 1,1→nt nt uncle, cousin

a1: absence of olfactory bulbs and olfactory tracts; 2: absence of olfactory bulbs and hypoplasia of olfactory tracts; 3: hypoplasia of olfactory bulbs and absence of olfactory tracts.−, negative; GnRH, gonadotropin-releasing hormone; HCG, chorionic gonadotropin; HMG, human menopausal gonadotropin; nt, not test; TS, testosterone. KS patientswith Genetic and clinical characteristics of box domainandC151islocatedattheWAP domain. cysteine residues(C86andC90)arelocatedattheCys- mutations aspathogenicvariants.Third,twoofthese all thepredictionprogramsweemployedclassifiedthese across twenty-six different species. Second, is conserved identified (C86F, C90Y, C151W):first, the cysteine residue respectively. For the other three missense mutations we sites, formatruncatedproteinorcauseframe-shift result inlossofanosmin-1functionbychangingsplice R597X, Y579L of theidentifiedmutations(c.1062 that thesemutationsareprobablypathogenic.Four from bioinformatics analysis supports the hypothesis in vitro in thisstudy. Althoughwedidnotperformfunctional c.1062 +1G the anosmin-1protein. molecular modeltoinvestigatethekeyfunctionalsitesof the five core disulfide bonds are conserved ( the five coredisulfide bonds are conserved 90–101 and116–120.Alltencysteineresiduesthatcreate which formbetweenresidues49–83,53–77,86–105, The Cys-boxcontainsacoreoffivedisulphidebridges, patients witha greater incidenceofrightrenal agenesis occurred inKS presented in 35–40% of X-linked KS cases and an even mutations, 40%hadcryptorchidism ( cryptorchidism ( the completeformofIHHreportedthat15.3%had results. Onestudyofalargecohort124patientswith the incidenceofcryptorchidism inKS,withconflicting ANOS1 folding orstabilityofthisregion. intramolecular disulphidebonds,andprobablyaffectthe Cys86–Cys105, Cys90–Cys101andCys151–Cys163 other amino acids, it would disrupt the highly conserved the cysteines at position 86, 90 or 151 were converted to in theWAP domain aredescribedforthefirsttime.If mutations inthecysteine-richregionandonemutation region oftheproteinhadbeenreported.Inthisstudy, two affect cysteineresiduesinthecysteine-richN-terminal C163Y andC172R)( bonds intheWAP domain ofanosmin-1(C134G,C163R, with KSthataffectcysteineresiduesformdisulphide ( positions oftheseeightcysteineresiduesareconserved 134–164, 147–168,151–163and157–172.Similarly, the form fourintramoleculardisulphidebondsincluding cysteineresiduesthat domain containseightconserved 22 ). Previousstudiesidentifiedfivemutationsinpatients Novel mutationsincludingC86F, C90Y, C151W, The prevalenceofcryptorchidism inthiscohortwith studiesoftheeffectsthesemutations,evidence mutations is 38.1%. Many studies have explored >

A, Y379X, R597X, Y579L ANOS1 fs ANOS1 591X) coulddisrupttheproteinstructure, 18 mutation ), butinKSpatientswith 23 mutation( , Downloaded fromBioscientifica.com at09/29/202101:02:34PM 24 , 25 ), butnomutationsthat 27 , 28 177 26 fs ). Onlyonepatient + 591X werefound :4 ). Renalagenesis 1G 21 > , Y379X, A, ). The WAP ANOS1 396 via freeaccess

European Journal of Endocrinology participation intheresearch. The authorsthankthesubjectsand theirfamilymembersfor Acknowledgement B YandMC:performedtheexperiments; MN:wrotethepaper. S X,JZ,WMandQH:collectedbloodsamplesclinicaldata;N,R C, X WandHZ:conceiveddesignedtheexperiments;W, HX,JM,XW, Author contributionstatement (WBYZ2011-873). Sciences (2016-I2M-1-002)andtheNationalKeyProgramofClinicalScience Program ofChina(2016YFC0905100),CAMSInnovationFundforMedical This workwassupportedbyNationalKeyResearchandDevelopment Funding perceived asprejudicingtheimpartialityofthisstudy. The authorsdeclarethatthereisnoconflictofinterestcouldbe Declaration ofinterest highinKSpatientswith is very bulb/tract and theabsenceorhypoplasiaofolfactory diagnosis of KS and genetic counseling. Cryptorchidism and provideinformationthatcouldbeusefulforprenatal These mutationsexpandthe domain of anosmin-1 protein were found for the first time. in thepresentstudy. Two mutationslocatedintheCR mutations thatareprobablypathogenicwereidentified higher infamilialKSpatients.Sevennovel mutations is low in sporadic KS patients, but is much duration ofHCG factors, suchascryptorchidism ( spermatogenesis failedmayberelatedwithfollowing HMG was68%(95%CI:58–77%).Thereasonwhy spermatogenesis inHHpatientstreatedwithHCG/ for HHpatients( HMG therapy and 16 studies of pulsatile GnRH therapy the results of a meta-analysis about 48 studies of HCG/ detected withsperminthisstudy, whichissimilarwith bulb/sulci. sense ofsmellandMRIimagesolfactory result showedthatthereisastrongcorrelationbetween which issimilartopreviousstudies(84.62%)( bulb/tract was found in 100% of patients in this cohort, renal agenesis. Absence or hypoplasia of the olfactory study fromKorea( patients. IntwootherstudiesfromChina( possibly duetothedifferentethnicbackgroundof the frequencywaslowerthanpreviouslydescribed( in ourcohortshowedrightrenalagenesis;therefore, Clinical Study In conclusion,theprevalenceof There are not many patients (70%) who have been + 30 HMG treatment( 29 ). Inthatstudy, therateofsuccessful ), nopatientswerereportedtohave ANOS1 ANOS1 M Nieandothers 31 33 , mutationspectrum ). 32 mutations. ) andtheshort 20 ANOS1 , 26 ) andone 29 ANOS1 gene ). This 28 ),

KS patientswith Genetic and clinical characteristics of References

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