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European Journal of Endocrinology -18-0764 Brasil, Metabologia, HospitaldasClinicasdaFaculdadedeMedicinaUniversidadeSãoPaulo, Michigan, AnnArbor,USA, Brasil, e Metabologia,HospitaldasClinicasdaFaculdadedeMedicinaUniversidadeSãoPaulo, 1 and Ericka Barbosa Trarbach Alexander Augusto Lima Jorge Lorena Guimaraes Lima Amato congenital hypogonadotropichypogonadism New geneticfindingsinalargecohortof central nervous system (CNS), in the olfactory placode, system (CNS),intheolfactory central nervous 2 neurons locatedintheventromedial hypothalamus( hormone (GnRH),produced byasmallnumberof secretion andproperactionofthegonadotropin-releasing Normal pubertaldevelopmentisdependentonthe Introduction diagnosis ofcongenitalCHHandbeingabletotargetingclinicalgenetictestinginthefuture. and heterogeneousgeneticcondition.NGShasbeenshowntobeafast,reliableeffectivetoolinthemolecular Conclusions: variants inmorethanonegene.Rarewerealsoidentified IGSF10 in the ANOS1 Results: Kallmann syndrome)wasstudiedusingapanelcontaining36CHH-associatedgenes. Design andpatients: Objective: making possibletheextensionofgeneticknowledgeCHH. The adventofnext-generationsequencing(NGS)hasallowedthesimultaneousgenotypingseveralregions,faster, havebeenassociatedwiththepathogenesisofCHH,butmostcasesstillremainwithoutamoleculardiagnosis. Context: Abstract Universidade FederaldeMinasGerais,BeloHorizonte,Brasil (UNICAMP), Campinas,SãoPaulo,Brasil,and Unidade deEndocrinologiadoDesenvolvimento,LaboratórioHormôniose Genética Molecular/LIM42,DisciplinadeEndocrinologia ). GnRH-secretingneuronshave theiroriginoutsidethe https://doi.org/ https://eje.bioscientifica.com Clinical Study Leticia Ferreira Gontijo Silveira 4 2 Departamento dePediatria,FaculdadeCiênciasMédicasdaUniversidade EstadualdeCampinas Division ofMetabolism,EndocrinologyandDiabetes,DepartmentInternal Medicine,Universityof GNRH1 , genewereidentifiedintwopatientswithreversiblenormosmicCHH.Notably,6.9%ofthehadrare Potentialpathogenicorprobablyvariantswereidentifiedin43(33%)CHHpatients.Thegenes FGFR1 Congenitalhypogonadotropichypogonadism(CHH)isarareconditioncausedbyGnRHdeficiency.Several 10.1530/EJE GeneticcharacterizationofalargecohortBrazilianCHHpatients. 10.1530/EJE ThisisalargestudyofthemoleculargeneticsCHHprovidingnewgeneticfindingsforthiscomplex geneandadeletionoftheentirecodingsequencewasidentifiedin and 10.1530/EJE-18-0764 GNRHR -18-0764 Acohortof130unrelatedpatients(91males,39females)withCHH(75normosmicCHH,55 werethemostfrequentlyaffected.Anovelhomozygoussplicesitemutationwasidentified 3 2 , Elaine Maria Frade Costa 3 © Unidade deEndocrinologiaGenetica/LIM25, DisciplinadeEndocrinologiae 2019EuropeanSociety ofEndocrinology 3 L GAmatoandothers 1 , Gil Guerra Junior , Luciana Ribeiro Montenegro 5 Departamento deClínicaMédica, FaculdadedeMedicinada 1 , 5 Printed inGreatBritain 4 , Caroline Schnoll 1 , Berenice Bilharinho Mendonca 1 , steroid concentrationsassociated tolowornormal clinically manifestedassexual infantilism,lowserum (CHH) isararecondition caused byGnRHdeficiency, ( lifetotheirfinalhypothalamic location the embryonic neurons during and migrateinassociationwitholfactory Genetic causesofCHH 1 Published byBioscientifica Ltd. , 2 1 , , Antonio Marcondes Lerario 3 SPRY4 ). Congenitalhypogonadotropic hypogonadism 1 , Alessandra Covallero Renck , IL17RD SOX10 Q1 , FGF17 Downloaded fromBioscientifica.com at09/30/202107:34:04PM . Deleteriousvariantsinthe , 1 , Ana Claudia Latronico IGSF1 (2019) Endocrinology European Journalof [email protected] [email protected] Email Silveira to A C LatronicoorLFG should be addressed Correspondence and 1 , 2 181 , 181 FLRT3 :2 , 103–119 1 ,

genes. 103 or –119 1

via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com associated genes,including genetic causesofGnRHdeficiencybyscreeningCHH- diagnosis ( 30% ofthesepatientshadarecognizedmolecular genetic causesofCHH,untilrecentlyonlyabout CHH ( associated withsyndromicandnon-syndromicformsof 14 concept ofCHHasastrictlymonogenicdisease( one geneinthesamepatient,changingprevious allowed theidentificationofvariantsinmorethan the advancement of genetic screening techniques and efficiently. allowed thescreeningofalarge numbersofgenesquickly recent adventofnext-generation sequencing(NGS)has genes updated using the traditional techniques. The almost impossibletokeepthe screeningofallcandidate genes andthegeneticcomplexityofCHH,ithasbecome molecular pointofview. With thegrowingnumberof majority ofthepatientsremainundiagnosedfrom ( and multiplexligation-dependentprobeamplification classical geneticmethods,suchasSangersequencing PROKR2 or evennormalphenotype( constitutional delayofgrowthandpuberty(CDGP) nCHH, isolatedanosmia,lip-palatinecleft, variability, with affected members presenting with KS, with substantialintraandinterfamilialphenotypic FGFR1 8 autosomal recessivenormosmicCHH(nCHH)( whereas form ofKS,withaseverereproductivephenotype, termed known to cause CHH, such as ( neuronalmigrationwell asintheprocessofembryonic regulation ofGnRHproduction,secretionoraction,as CHH. Thesegenesareinvolvedindifferentstagesofthe has beenimplicatedinthemolecularpathogenesisof condition. In the last decades, a growing list of genes CHH cases( (KS), whichcorrespondstoapproximately50–60%of defects,characterizingKallmannsyndrome with olfactory and GnRH neurons, CHH is often associated olfactory originof functions. Becauseofthecommonembryonic normal pituitary levels of gonadotropins, and otherwise 9 3 , Clinical Study , , 9 , 10 , 4 In thelasttwodecades,wehavebeenstudying CHH isaclinicalandgeneticallyheterogeneous 15 10 , , 3 , 5 ). Currently, morethan30geneshavebeen 16 ). On the other hand, in genes such as FGF8 , , ). Someofthesegeneshavebeenclassically KAL1 4 PROK2 GNRHR , , 4 17 3 5 ). ). ). However, ofthe despitethevasthistory , , ), whichisassociatedwiththeX-linked PROK2 18 , mutations are a common cause of , TACR3 19 and , 20 , , TAC3 PROKR2 21 ANOS1 , 5 22 , , L GAmatoandothers 8 KISS1R , , , 23 havebeenassociated GNRHR 11 ANOS1 ). Nevertheless,the , 12 and ). Inaddition, , FGFR1 (previously KISS1 3 , using , FGF8 6 , 13 7 , , ,

or estradiol),subnormalnormalluteinizing subnormal concentrationsofsexualsteroids(testosterone yearsofageinboys; yearsofageingirlsand18 16 sexualcharacteristicsafter development ofsecondary of thepatientswerelackappearanceorincomplete studied bytargetedNGS.Theclinicalcriteriaforinclusion (75 nCHHand55KS,including91men39women)was informed consentformforparticipationintheresearch. University. All patients or their caregivers signed the was approvedbytheEthicsCommitteeofSaoPaulo da UniversidadedeCampinas(Unicamp).Theproject Paulo (HCFMUSP)andfromtheHospitaldasClínicas Clínicas da Faculdade de Medicina, Universidade de São the DevelopmentalEndocrinologyUnit,Hospitaldas Patients wereselectedfromtheoutpatientclinicof Patients Patients andmethods designed tocapture36known andcandidate enrichment panel(Agilent Technologies Inc)was when available.Acustom SureSelectXTDNAtarget blood leukocytesofallpatients andtheirrelatives Genomic DNAwasextracted fromperipheral- Genetic analysis end ofthisarticle). seesectionon Table 1, PROK2 22 classical CHH-associatedgenes( cohort previouslystudiedbySangersequencingforthe patients (15.4%). ofCHHorpubertaldelaywaspresentin20 Family history size couldbeassessedpre-treatmenthadmicropenis. present in29.7%ofthemenandallpatientswhosepenile synkinesis (0.8%).Uniorbilateralcryptorchidism was anomalies (1.5%),skeletal(0.8%),bimanual palate (1.5%),dentalanomaliescongenitalrenal (3.8%), epilepsy (2.3%), heartdefects(1.5%), cleft congenital hearingimpairment(4.6%),cognitivedeficit insomepatientsasfollows: abnormalities wereobserved Test, Philadelphia,PA, USA)( abnormalities(SmellIdentification diagnose olfactory of PennsylvaniaSmellIdentification Test wasusedto deficiencies andnormalimagingofCNS.TheUniversity concentrations, absenceofotherassociatedpituitary hormone (LH)andfollicle-stimulating(FSH) Genetic causesofCHH , 23 A totalof130unrelatedBrazilianpatientswithCHH Most patients(107cases)wereselectedfromalarge ) ( , TACR3 ANOS1 , , TAC3 GNRHR , KISS1R supplementary data supplementary , Downloaded fromBioscientifica.com at09/30/202107:34:04PM GNRH1 and 24 ). Additionalphenotypic 9 , , KISS1 FGFR1 10 181 , 17 ) (Supplementary ) (Supplementary :2 , , FGF8 18 givenatthe , 19 , PROKR2 , 20 104 CHH , 21 via freeaccess , ,

European Journal of Endocrinology NGS, next-generationsequencing. PNPLA6 IGFALS IGSF10 DMXL2 IGSF1 GHSR EBF2 OTX2 MSX1 MKRN3 NSMF SOX10 SPRY4 FLRT3 POLR3B POLR3A WDR11 DUSP6 RNF216 HS6ST1 IL17RD SEMA7A SEMA3A FGF17 CHD7 PROKR2 PROK2 KISS1R KISS1 TACR3 TAC3 GNRHR GNRH1 FGF8 FGFR1 ANOS1 Table 1 –SELA). Paulo (LargeScaleSequencingLaboratory and sequencing were performed at the University of Sao NextSeq 500 (Illumina, Inc). All preparations Platform; sequencingwasperformedona Agilent BravoAutomatedLiquidHandling the SureSelectXT Target Enrichment protocol using gonadotropic axis( pathways orpresentincorrelatedconditionsaffectingthe and genes, namely anecdotally reportedinCHHpatientsandsevencandidate classically associatedwithCHH,genesrarelyor each geneweresequenced.Thepanelcoveredgenes flanking regionand 5 genes. All exons, the 25 base pairs of intronic Clinical Study Sequence capturewasperformedaccordingto IGFALS GC19P007534 GC16M001790 GC03M151425 GC15M051447 GC0XM131273 GC03M172443 GC08M025841 GC14M056799 GC04P004861 GC15P024015 GC09M137447 GC22M039963 GC05M142272 GC20M014322 GC12P106357 GC10M077969 GC10P120851 GC12M089347 GC07M005661 GC02M128236 GC03M057124 GC15M074409 GC07M083955 GC08P022042 GC08P060678 GC20M005301 GC03M071820 GC19P000917 GC01M204190 GC04M103586 GC12M057009 GC04M067737 GC08M025419 GC10M101770 GC08M038411 GC0XM008528 GeneCards ID Genes selectedfortheNGSpanel. , basedondatafrombiochemical/biological EBF2 Table 1 , GHSR ′ Topaloglu Domené Howard Tata Sun Pugliese-Pires Trarbach Diaczok Xie Abreu Miura Pingault Miraoui Miraoui Saitsu Saitsu Kim Miraoui Margolin Tornberg Miraoui Känsäkoski Young Miraoui Kim Dodé Dodé de Roux Topaloglu Topaloglu Topaloglu de Roux Bouligand Falardeau Dodé Legouis Reference and 3 ). et al , et al et al. et al. et al MSX1 et al et al et al et al et al et al et al. et al . ( et al et al et al et al et al et al. et al. et al. . ( et al et al et al . ( ( ( ′ et al 69 et al et al. untranslated region of et al . ( . ( . ( L GAmatoandothers 72 65 46 et al et al et al et al . ( et al . ( . ( et al 73 . ( ( et al , 61 61 49 ) 67 . ( . ( . ( . ( . ( 66 66 68 . ( ) ) ) 62 ( . ( . ( ( ( ) . ( 7 MKRN3 . ( . ( et al 56 32 32 32 32 32 54 ( ) ) ) 47 )/Seminara . ( . ( . ( . ( 53 7 . ( ) . ( ) ) ) 74 ) 64 70 51 ) . ( 75 58 57 57 50 43 ) ) ) ) ) ) ) ) ) ) 63 ) ) ) . ( ) ) ) ) ) ) ) 71 , ) OTX2 et al. ( , 59 IGSF1 , 60 )

(sorting, mergingandindexing)usingthe followed bydownstreamprocessingofthealignedreads b37 pipeline. Briefly, paired-end reads were aligned to the Data analysis was performedusing in-house bioinformatics Variant analysis potential tobepathogenic:loss-of-functionvariantsand variant filtration prioritized genes onthe basisoftheir regions andconsensussplicesitevariants.Subsequently, in-house databases),nonsynonymous,locatedinexonic (minor allelefrequencylessthan1%inpublicand normalized with was performed using regions coveragemetrics,respectively( and www.bioinformatics.babraham.ac.uk/projects/fastqc/ from biobambam2( www.cbs.dtu.dk/services/NetGene2/ 2.4.1 ( in silico – CADD).Forvariantsaffectingsplicingsites,weused PROVEAN, CombinedAnnotationDependentDepletion Models – FATHMM, Variation Effect Analyzer – Assessor, FunctionalAnalysisthroughHiddenMarkov programs (SIFT, PolyPhen2, MutationTaster, variants predictedtobepathogenicbyatleasttwo all thesevariantswereinterpreted basedonthephenotype Molecular Pathology (AMP) guidelines ( Medical Genetics(ACMG) andtheAssociationfor according tothecriteriaof theAmericanCollegeof was available. was performed whenever genetic material ofrelatives confirmed by Sanger sequencing. Familial segregation BAM file.Allpotentialdisease-causingvariantswere Integrative Genomics Viewer (IGV) program, from the potentially pathogenicwerevisuallyconfirmedinthe – SELA). All rare variants considered Laboratory sequencing covering774alleles(LargeScaleSequencing and anin-housedatabasecomprisingdatafromexomic composed of609exomeshealthyBrazilians elderly, Mutations (ABraOM: broadinstitute.org/ Aggregation Database (GnomAD: the help of two public genomic databases: Genome rateprofiling) (genomic evolutionary included theexclusiveselectionofvariantswithGERP Genetic causesofCHH + The panelsequencingdatawerescreenedforrare Population datacriteriawereevaluatedwith The variants were further classified for pathogenicity qualimap decoy versionofthehumangenomeusing http://www.umd.be/HSF/ predictionsites:HumanSplicingFinderVersion toassesssequencingqualityandtarget vt ), OnlineArchive of Brazilian andannotatedusing 25 freebayes. , Downloaded fromBioscientifica.com at09/30/202107:34:04PM http://abraom.ib.usp.br/ 26 ). We used https://eje.bioscientifica.com ) andNetGene2( Finally, variants were ). 181 > 27 2.5. In silico 31 :2 annovar ). Variant calling http://gnomad. fastqc ). Furthermore, bamsort analysis ( ( ) ( 28 https:// in silico http:// 105 , tool bwa 29 30 via freeaccess ). ), ) ,

European Journal of Endocrinology https://eje.bioscientifica.com (59.2%) of the130 CHH patients studied with targeted satisfactory.sequencing wasvery of thetargetregions,indicating thatthequalityof of patientshadcoveragegreater than50timesin99% coverage greater than 20 times, and thevast majority The lowestcoveragewas98%ofthetargetedregionswith Genetic findings Results positive forheterozygousdeletion. using Cofallyser. Finalproberatiosof0.5were considered those obtainedintwonormalcontrols(DNAreferences) corresponding lane.Thisrelativevaluewascomparedto normalized bycombinedvalueofthecontrolprobesin determined with GeneScan analysis software V.3.7 and exons of Amsterdam, Netherlands)thatincludesprobesforallfour MLPA P186 PAX3-MITF-SOX10 probemix (MRC Holland, SOX10 Multiplex ligation-dependentprobeamplification ABI PrismGeneticAnalyzer3100(Perkin-Elmer). agarose gel,purifiedandautomaticallysequencedinan between exons1and2.PCRproductwasvisualizedin2% exons 1and2,comprisingthesplicingregionofinterest cDNA wasamplifiedwithaspecificprimerpairlocatedat according tothemanufacturer’s instructions. QuantiTect ReverseTranscription Kit(Qiagen,2009) TRIzol® (Invitrogen)andreversetranscribedusingthe Ficoll-Hypaque method.Total RNAwasextractedusing control withnormalpubertaldevelopmentusingthe from theindexcase,hismotherandahealthyadult Lymphocytes wereisolated fromperipherallymphocytes RNA isolationandRT-PCRfortheGnRH1 individuals includedintheanalysis. was comparedtoapooledreferenceconstitutedofall number statusforeachpatient,individualBAMfile CONTRA (REF:PMID22474122).To infersegmentalcopy Copy number variations (CNVs) were screened using the Copy numbervariationanalysis inheritance andsegregationdata. to whichthevariantwasassociated,patternofgenetic Clinical Study Potentially pathogenicvariants wereidentified in77 genedeletionwasconfirmedusingtheSALSA SOX10 gene.Thepeakareaforeachprobewas L GAmatoandothers GNRH1

Pathogenic andprobablypathogenicvariants and CHH, andinthecandidategenes in are exposedin variants consideredpathogenicandprobably databases wereclassifiedasbenign.Detaileddataon the family orwhich were present in healthy individuals heterozygous state.Variants whichdidnotsegregatein causative ofthephenotypewhenidentifiedin autosomal recessive inheritance were not considered Table 2.in Supplementary Variants ingeneswithknown and molecularcharacterizationofallpatientsisdescribed pathogenic variants dropped to 43 (33%) ( pathogenicorprobably individuals consideredtocarry applying theACMG-AMPguidelines,numberof detected in29genes( NGS approach.Atotalof89differentvariantswere synkinesia, Patient19( epilepsy, mild cognitive deficit, unilateral hearing loss and mutations: Patient16( inthreepatientswith abnormalities wereobserved six innCHHpatients(8.5%).Additionalnonreproductive was themostprevalentgenewith11variants,fiveinKSand variants wereidentifiedinclassical The majorityof pathogenic or probably pathogenic Variants inclassicalCHHgenes Rare variantidentifiedbyNGS in130patientswithCHH. Figure 1 Genetic causesofCHH KISS1 MKRN3 , KISS1R . al 3 Table , HS6ST1 Fig. 1 p.Ala p.Arg . Norarevariantswereidentified Downloaded fromBioscientifica.com at09/30/202107:34:04PM ; genespreviouslyassociatedto 343Val) withbilateralhearing and 250Trp) withdentalagenesis, Table 2 CHH DUSP6 181 genes.The :2 ). However, after Fig. 2 , GHSR ). Clinical , FGFR1 FGFR1 MSX1 106 via freeaccess

European Journal of Endocrinology

Table 2 Rare variants identified in the CHH cohort. Clinical Study

In-house Reported GenomAD ABraOM database Segregation Variant Previous ID Sex Diagnosis Gene cDNA genotype Aminoacid genotype inheritance MAF (%) MAF (%) MAF (%)** data classification descriptions 1 F nCHH NA CHD7 c.611G>T/WT p.Gly204Val/WT AD Absent Absent Absent VUS No FGFR1ϒ c.1825C>T/WT p.Arg 09X/WT AD Absent Absent Absent P (76) 2 M nCHH CHD7 c.1958C>T/WT p.Pro653Leu/WT AD 0.0297* Absent Absent NA VUS No 3 M nCHH CHD7 c.2095A>G/WT p.Ser699Gly/WT AD Absent Absent Absent NA VUS (77) 4 M nCHH CHD7 c.2201_2203de/WT p.734_735del/WT AD Absent Absent 0.13* NA VUS No 5 M KS NA CHD7 c.3161G>A/WT p.Arg1054Gln/WT AD 0.004* Absent Absent VUS No

ANOS1ϒ c.1385G>A p.Trp462X X-linked Absent Absent Absent P (9) L GAmatoandothers 6 M KS NA CHD7 c.3973T>C/WT p.Tyr1325His/WT AD 0.0057* Absent Absent VUS No PROK2ϒ c.297dupT/ p.Gly100fs/p. AR 0.0104* Absent Absent P (18) c.297dupT Gly100fs 7 F nCHH CHD7 c.6775G>A/WT p.Ala2259Thr/WT AD 0.0163* Absent Absent Negative PB (78) IGSF10 c.2126G>T/WT p.Gly709Val/WT AD 0.0324* 0.24* Absent Positive VUS No 8 F nCHH NA CHD7 c.7253G>A/WT p.Arg2418Gln/WT AD 0.0072* Absent Absent VUS No TAC3 c.209-1G>C/c.209- AR 0.0108* Absent Absent P (79) 1G>C

9 M KS CHD7 c.7963_7970del/WT p.G2655fs/WT AD Absent Absent Absent NA P No Genetic causesofCHH 10 M KS NA CHD7 c.8068_8076del/WT p.2690_2692del AD Absent Absent Absent PP No GNRHR c.317A>G/WT p.Gln106Arg/WT AR 0.2712* 0.16* 0.26* P (7) 11 M KS NA CHD7 c.8213C>T/WT p.Thr2738Met/WT AD 0.00016* Absent Absent VUS No FGFR1ϒ c.289G>A/WT p.Gly97Ser/WT AD Absent Absent Absent P (80) WDR11 c.2305A>G/WT p.Met769Val/WT AD 0.0004* 0.16* 0.26* VUS No EBF2 c 650C>T/WT p.Thr217Met/WT ? 0.0008* Absent Absent NA VUS No 12 M nCHH CHD7 c.8416C>G/WT p.Leu2806Val/WT AD 0.1189* 0.41* 0.65* Negative B No * * * Downloaded fromBioscientifica.com at09/30/202107:34:04PM 13 M nCHH CHD7 c.8416C>G/WT p.Leu2806Val/WT AD 0.1189 0.41 0.65 NA B No 14 F nCHH CHD7 c.8416C>G/WT p.Leu2806Val/WT AD 0.1189* 0.41* 0.65* NA B No 15 M nCHH

https://eje.bioscientifica.com FGFR1 c.83C>T/WT p.Pro28Leu/WT AD Absent Absent Absent Positive P No IL17RD c.1697C>T/ p.Pro566Leu/p. AD 0.2024* 0.8* 0.39* Positive PP No c.1697C T Pro566Leu > 181 IL17RD c.1608_1611del/WT p.Glu536fs/WT AD Absent Absent Absent Positive P No DMXL2 c.5171C>T/WT p.Ser1724Leu/WT AR Absent Absent Absent Positive PB No :2 16 M KS FGFR1ϒ c.748C>T/WT p.Arg250Trp/WT AD Absent Absent Absent NA P (17) 17 F nCHH NA FGFR1 c.857dupC/WT p.P286fs/WT AD Absent Absent Absent P No SPRY4 c.722C>A/WT p.Ser2 1Tyr/WT AD 0.4549† 0.6568* Absent B (32) 107 via freeaccess (Continued) European Journal of Endocrinology https://eje.bioscientifica.com

Table 2 Continued. Clinical Study

In-house Reported GenomAD ABraOM database Segregation Variant Previous ID Sex Diagnosis Gene cDNA genotype Aminoacid genotype inheritance MAF (%) MAF (%) MAF (%)** data classification descriptions 18 M nCHH FGFR1 c.962_963del/WT p.Lys321fs/WT AD 0.0004* Absent Absent Positive P No IGSF10 c.5405A>T/WT p.Asp1802Val/WT AD 0.2723* 0.32* Absent Negative B No POLR3B c.926C>G/WT p.Ala309Gly/WT AR Absent Absent Absent Negative B No 19 M KS FGFR1ϒ c.1028C>T/WT p.Ala343Val/WT AD Absent Absent Absent NA P (17) 20 F KS FGFR1 c.2008G>A/WT p.Glu670Lys/WT AD Absent Absent Absent NA P No 21 M KS FGFR1 c.2070+1G>A AD 0.0406* Absent Absent NA P No 22 F nCHH FGFR1 c.2275 >T/WT p.Glu759X/WT AD Absent Absent Absent NA P No

23 M nCHH FGFR1 c.2300G>A/WT p.Cys767Tyr/WT AD Absent Absent Absent NA P No L GAmatoandothers 24 M nCHH GNRHR c.30T>A; c.31C>A/ p.Asn10Lys; AR 0.0135*/0.0008* Absent Absent NA P/P (16) c.847T>C p.Gln11Lys/p. Tyr283His 25 M nCHH GNRHR c.317A>G/WT p.Gln106Arg/WT AR 0.2712* 0.16* 0.26* NA P (7) 26 M KS GNRHR c.317A>G/WT p.Gln106Arg/WT AR 0.2712* 0.16* 0.26* NA P (7) 27 M KS GNRHR c.317A>G/WT p.Gln106Arg/WT AR 0.2712* 0.16* 0.26* NA P (7) 28 M nCHH GNRHRϒ c.401T>G/c.785G>A p.Val134Gly/p. AR 0.0028*/0.1804* Absent Absent NA P/P (7) Arg262Gln 29 M nCHH GNRHR c.416G>A/ p.Arg139His/p. AR 0.017* Absent Absent NA P (16) c.416G>A Arg139His 30 M KS ANOS1 c.153A>G p.Met1Val X-linked Absent Absent Absent NA P No ϒ 1 M KS ANOS1 c.90_100del p.Ala30fs X-linked Absent Absent Absent NA P (9) Genetic causesofCHH 32 M KS ANOS1ϒ c.1062+1G>T X-linked Absent Absent Absent NA P (9) 33 M KS ANOS1 c.566_567del p.E189fs X-linked Absent Absent Absent NA P No 34 M KS ANOS1 c.1632_1635del p.Leu544fs X-linked Absent Absent Absent NA P No 35 nCHH TACR3ϒ c.439C>T/c.824G>A p.Leu147Phe/p. AR Absent Absent Absent NA P/P (19) Trp275X 36 M nCHH NA TACR3 c.824G>A/ p.Trp275X/p.Trp275X AR 0.0314* Absent Absent P (19) c.824G>A IGFALS c.125C>T/WT p.Pro42Leu/WT AR 0.0023* 0.08* Absent B No

Downloaded fromBioscientifica.com at09/30/202107:34:04PM 37 M nCHH NA TACR3 c.1007A>G/ p.Gln336Arg/p. AR Absent Absent Absent P No c.1007A>G Gln336Arg POLR3B c.971A>G/WT p.Lys324Arg/WT AR Absent Absent Absent PB No 38 M nCHH TACR3 c.824G>A/WT p.Trp275X/WT AR 0.0314* Absent Absent NA P (19) 39 F nCHH TACR3 c.824G>A/WT p.Trp275X/WT AR 0.0314* Absent Absent NA P Gianetti 181 et al. (19) 40 M KS PROKR2 c.115G>A/WT p.Glu39Lys/WT AR Absent Absent Absent NA PB No :2 41 M KS NA PROKR2 c.518T>G/WT p.Leu173Arg/WT AR 0.2186* 0.24* 0.13* B (62) WDR11 c.3571G>A/WT p.Gly1191Ser/WT AD 0.0147* 0.16* Absent VUS No 42 F KS PROKR2 c.518T>G/WT p.Leu173Arg/WT AR 0.2186* 0.24* 0.13* NA B (61) 108 via freeaccess European Journal of Endocrinology

43 M nCHH NA Clinical Study PROK2 c.163delA/WT p.Ile55X/WT AR 0.0111* Absent Absent P P (81) WDR11 c.1066G>A/WT p.Val356Ile/WT AD 0.1591* 0.08* Absent VUS No 44 M nCHH PROK2 c.163delA/WT p.Ile55X/WT AR 0.0111* Absent Absent NA P (81) 45 F KS PROK2 c.297dupT/WT p.Gly100fs/WT AR 0.0104* Absent Absent NA P (18) 46 M KS PROK2 c.332C>A/WT p.Pro111Gln/WT AR 0.183* 0.4* Absent NA PB No 47 M nCHH IGSF10 c.1297delC/WT p.Gln433fs/WT AD 0.0079* Absent Absent Positive P No 48 M nCHH NA IGSF10 c.2126G>T/WT p.Gly709Val/WT AD 0.0324* 0.24* Absent VUS No SPRY4 c.776C>T/WT p.Ser259Phe/WT AD Absent Absent Absent PP No 49 M nCHH IGSF10 c.6830A>G/ p.Asp2277Gly/p. AD 0.0176*/0.0285* Absent Absent/0.08 NA PP/VUS No c.4613C>T Thr1538Ile 50 F nCHH IGSF10 c.5405A>T/WT p.Asp1802Val/WT AD 0.2723* 0.32* Absent NA B No * * 51 M nCHH IGSF10 c.7217C>T/WT p.Ala2406Val/WT AD 0.0801 0.24 Absent NA VUS No L GAmatoandothers 52 M nCHH GNHR1 c.142-2A>C/c.142- AR Absent Absent Absent NA P No 2A>C 53 F KS GNHR1 c.141G>C/WT p.Glu47Asp/WT AR 0.1492* 0.24* 0.13* NA P No 54 M nCHH FGF8 c.560_574del/WT p.187_192del/WT AD Absent Absent Absent NA P No 55 F KS FGF8 c.G617A/WT p.Arg206Gln/WT AD Absent Absent Absent NA P No 56 M KS IL17RD c.878C>T/WT p.Pro293Leu/WT AD 0.0568* 0.08* 0.26* NA VUS No 57 M KS IL17RD c.2003C>T/WT p.Ser668Phe/WT AD 0.0043* 0.08* 0.13* Negative B No 58 M KS POLR3A c.1745G>T/WT p.Arg582Leu/WT AR 0.8561‡ 0.99* 3.51* NA B No 59 F nCHH NA POLR3A c.1745G>T/WT p.Arg582Leu/WT AR 0.8561‡ 0.99* 3.51* B No FLRT3 c.821A>G/WT p.Tyr274Cys/WT AD 0.0072* Absent Absent VUS No 60 F KS NA POLR3B c.1010T C/WT p.Val337Ala/WT AR 0.0004* Absent Absent B No > Genetic causesofCHH SOX10 deletion exons 1-6 AD Absent Absent Absent P No 61 F nCHH SEMA3A c.1923G>C/WT p.Gln641His/WT AD 0.1102* 0.7* 0.13* NA VUS No 62 M nCHH SEMA3A c.2197C>T/WT p.Arg733Cys/WT AD 0.0065* 0.16* Absent NA VUS No 63 M KS SEMA7A c.406C>T/WT p.Arg136Trp/WT AD 0.0043* Absent Absent Negative VUS No 64 M nCHH SEMA7A c.493G>A/WT p.Glu165Lys/WT AD 0.0152* 0.08* Absent NA VUS No 65 M nCHH DMXL2 c.5267G>A/WT p.Arg1756His/WT AR 0.0004* Absent Absent NA PB No 66 M KS DMXL2 c.5267G>A/WT p.Arg1756His/WT AR 0.0004* Absent Absent NA PB No 67 M nCHH DMXL2 c.1463C>T/WT p.Thr488Met/WT AR 0.0408* 0.08* Absent NA B No 68 M KS WDR11 c.C1592G/WT p.Ser531Cys/WT AD 0.0039* Absent Absent NA VUS No 69 M KS WDR11 c.2962G>A/WT p.Glu988Lys/WT AD 0.1799* Absent 0.39* NA VUS No 70 M KS NSMF c.721G A/WT p.Ala241Thr/WT ? 0.0024* Absent Absent NA VUS No

Downloaded fromBioscientifica.com at09/30/202107:34:04PM > 71 F nCHH NSMF c.1453G>A/WT p.Val485Ile/WT ? 0.0039* 0.08* Absent NA VUS No 72 M nCHH FGF17 c.196G>A/WT p.Val66Met/WT AD Absent Absent Absent NA PP No 73 M KS IGFALS c.1195G>A/WT p.Gly399Arg/WT AR 0.0257* Absent Absent NA PB No * https://eje.bioscientifica.com 74 F nCHH IGSF1 c.709C>G/WT p.Pro237Ala/WT AD 0.0007 Absent Absent NA VUS No 75 F nCHH NA *

SOX10 c.1018G>A/WT p.Val340Met/WT AD 0.0064 Absent Absent VUS No 181 OTX2 c.163G>T/WT p.Ala55Ser/WT AD 0.0046* Absent Absent VUS No 76 M KS PNPLA6 c.1340C>T/WT p.Pro447Leu/WT AR 0.4756† 0.32* 0.78* NA B No :2 77 F nCHH RNF216 c.2353T>C/WT p.Ser785Pro/WT AR 0.0012* Absent 0.13* NA PB No

Segregation data: NA, family DNA not available; Positive: healthy family does not have the same variant; Negative: healthy family has the same variant. **In-house database: data from exomic sequencing covering 774 alleles; *all heterozygous; †five homozygous;‡ eleven homozygous; ϒpreviously identified by Sanger: Patient 1FGFR1 p.Arg609X/WT,

Patient 5 ANOS1 p.Trp462X (20), Patient 6 PROK2 p.Gly100fs/p.Gly100fs (11), Patient 11 FGFR1 p.Gly97Ser/WT, Patient 16 FGFR1 p.Arg250Trp/WT (9), Patient 19 FGFR1 p.Ala343Val/WT (9), Patient 28 109 GNRHR p.Val134Gly/p.Arg262Gln (21), Patient 31 ANOS1 p.Ala30fs (20), Patient 32 ANOS1 c.1062+1G T (20), Patient 35 TACR3 p.Leu147Phe/p.Trp27 X (20). via freeaccess > B, benign; Dx, diagnosis; KS, Kallmann syndrome; MAF, minor allele frequency; nCHH, normosmic congenital hypogonadotropic hypogonadism; P, pathogenic; PB, probably benign; PP, probably pathogenic; VUS, variant of uncertain significance; WT, wild type. European Journal of Endocrinology https://eje.bioscientifica.com 1.5 pubic hairTanner stageIII,micropenis,testicularsize member inthe family. Atphysicalexamination, he had consanguineous parents and had no other affected and no other associated condition. He was born to cryptorchidism, surgicallycorrectedinchildhood ofbilateralof lackpubertaldevelopment,history years ofagecomplaining The patientpresentedat18 (Patient 52),withnoothermolecularvariantidentified. in the A homozygoussplicingsitemutation(c.142-2A A novelmutationinGNRH1 instances ( phenotype becauseitwasintheheterozygousstateall patients, but not considered enough to explain the inactivating variant p.Val134Gly/p.Arg thevariants subsequent relapse(Patient28,carrying patients presentedwithreversalhypogonadism three patientswithCHH(2.3%)( Biallelic mutations in the cryptorchidism, ogivalpalateandmildcognitivedeficit. phenotype (4.6%). Patient 31( in sixmenwithKS,allcompletehypogonadism unilateral deafness. loss andPatient23( Flowchart fortheselectionofcandidatevariants. Figure 2 Clinical Study ACMG criteria,segregatio × Variants selectioncriteri

data, heritancepattern 1.0 GNRH1 cm, leftand2.0 7 , 10 genewasidentifiedinamalepatient ). 262Gln). Thewell-knownpartially p.Cys p.Gln ANOS1 × GNRHR 767Tyr) withogivalpalateand 106Arg wasidentifiedinfour

1.4 right,BMI27 pathogenic orprobably 43 (33%)patientswit mutationswereidentified 77 (59.2%)patients pathogenic variant 10 positivecontrol* 130 CHHpatient a n L GAmatoandothers p.Ala gene were identified in al 2 Table 30fs) hadbilateral ). Oneofthese kg/m s s 2 , with h > C) C) at aminoacidposition74.mRNAlevelsofthewild-type to aframeshiftandsubsequentprematurestopcodon detected in patient’s lymphocytes. This deletion leads harboring adeletionof4 acceptor site.Infact,anaberrant intron 2–3whilesimultaneouslyactivatingacryptic variant todamagethenaturalacceptorsplicingsitein NetGene2 ( state inthepatient’s unaffectedsisterandhisparents c.142-2A region,establishingthediagnosisofCHH.Thepituitary resonance imaging(MRI)scanofthehypothalamo– functionwerenormal,aswellamagneticpituitary FSH dL, adults 271–965 revealed totaltestosterone11 smell, confirmedbysmelltest.Hormonalevaluation eunuchoid proportions.Hereportedanormalsenseof smell. She had a severe bilateral sensorineural hearing loss. smell. Shehadaseverebilateral sensorineuralhearingloss. amenorrheaandabsentsenseof development, primary ofage,withlackpubertal 60). Shepresentedat19 years woman withKSandbilateral sensorineural deafness(Patient SOX10 A largedeletioninvolvingtheentirecodingregionof A largedeletionoftheSOX10gene agenesis andunilateraldeafness. significance, hadKSassociatedwithunilateralrenal only variantwasa ( which wouldalonebesufficienttocausethephenotype had associatedpathogenicvariantsinknown Table 2).anomalies (Supplementary Fiveofthesepatients had KS,dentalagenesis,higharched palateandkidney heterozygous forthe andPatient10,also hearing lossandbodyasymmetry pathogenic, respectively. Patient9hadKSassociatedto 9 and 10, were classified as pathogenic andprobably p.G2655fs andp.2690_2692del,identifiedinPatients classified asuncertainclinicalsignificance(VUS)andtwo, after applyingtheACMGcriteria,12variantswerelately in 14patientswithKSandnCHH( A totalof12distinctrare CHD7 variants from thepatientandhismother( and mutated Genetic causesofCHH FGFR1 i. 3A Fig. < gene was identified in the heterozygous state in a genewasidentifiedinthe heterozygousstatein 1 , ANOS1 IU/L (1.7–12.4 ). HumanSplicingFinderversion2.4.1and > C variantwasidentifiedintheheterozygous in silico , GNRH1 TAC3 ng/dL), LH toolspredictedthec.142-2A , CHD7 weredetectedinlymphocytes PROK2 p.Gln Downloaded fromBioscientifica.com at09/30/202107:34:04PM IU/L). Othertestsofanterior CHD7 bp (c.142-145delATAG) was

106Arg ) ( ng/dL (prepubertal p.Ser Table 2 < variantswereidentified UL (1.5–8.4IU/L), 0.1 IU/L 699Gly, ofuncertain Fig. 3B 181 al 2 Table GNRHR GNRH1 ). Patient3,whose :2 ). ). However, CHH mutation, transcript < genes, 12 ng/ 110 > via freeaccess C European Journal of Endocrinology

Table 3 Pathogenic and probably pathogenic variants identified in 43 CHH patients. Clinical Study

Pathogenic Gene/ Aminoacid Allele frequency In-house prediction diagnosis cDNA genotype genotype AC (%) GenomAD MAF (%) ABraOM MAF (%) database MAF (%) avsnp144 sites GERP FGFR1 KS c.83C>T p.Pro28Leu 1 (0.38) Absent Absent Absent rs145434725 4/4 4.58 nCHH c.1825C>T p.Arg609X 1 (0.38) Absent Absent Absent rs121909639 2/4 4.48 KS c.289G>A p.Gly97Ser 1 (0.38) Absent Absent Absent NA 4/4 5.38 KS c.748C>T p.Arg250Trp 1 (0.38) Absent Absent Absent NA 4/4 4.98 nCHH c.857dupC p.P286fs 1 (0.38) Absent Absent Absent NA NA NA nCHH c.962_963del p.Lys321fs 1 (0.38) 0.0004 Absent Absent NA NA NA KS c.1028C>T p.Ala343Val 1 (0.38) Absent Absent Absent NA 3/4 5.16 KS c.2008G>A p.Glu670Lys 1 (0.38) Absent Absent Absent rs397515446 4/4 5.9 L GAmatoandothers KS c.2070+1G>A 1 (0.38) 0.0406 Absent Absent NA 2/4 5.9 nCHH c.2275G>T p.Glu759X 1 (0.38) Absent Absent Absent NA 2/4 5.88 nCHH c.2300G>A p.Cys767Tyr 1 (0.38) Absent Absent Absent NA 4/4 5.33 CHD7 KS c.7963_7970del p.G2655fs 1 (0.38) Absent Absent Absent NA NA NA KS c.8068_8076del p.2690_2692del 1 (0.38) Absent Absent Absent NA NA NA ANOS1 KS c.90_100del p.Ala30fs 1 (0.38) Absent Absent Absent NA NA NA c.153A>G p.Met1Val 1 (0.38) Absent Absent Absent NA NA NA c.566_567del p.E189fs 1 (0.38) Absent Absent Absent NA NA NA c.1062+1G>T 1 (0.38) Absent Absent Absent rs387906427 2/4 NA Genetic causesofCHH c.1385G>A p.Trp462X 1 (0.38) Absent Absent Absent NA NA NA c.1632_1635del p.Leu544fs 2 (0.77) Absent Absent Absent NA NA NA GNRHR nCHH 1 (0.38) 0.0135 Absent Absent 2/4 c.31C>A p.Gln11Lys rs104893843 0.96 c.30T>A p.Asn10Lys rs776834867 5.18 KS/nCHH c.317A>G p.Gln106Arg 4 (1.52) 0.2712 0.16 0.13 rs104893836 4/4 6.17 nCHH c.401T>G p.Val134Gly 1 (0.38) 0.0028 Absent Absent rs188272653 2/4 6.04 c.416G>A p.Arg139His 2 (0.77) 0.017 Absent Absent rs104893842 2/4 6.17 c.785G T p.Arg262Gln 1 (0.38) 0.1804 Absent Absent rs104893837 4/4 2.78 Downloaded fromBioscientifica.com at09/30/202107:34:04PM > c.847T>C p.Tyr283His 1 (0.38) 0.0008 Absent Absent NA 4/4 5.43 GNHR1

https://eje.bioscientifica.com nCHH c.142-2A>C 1 (0.38) Absent Absent Absent NA NA NA TACR3 nCHH c.439C T p.Leu147Phe 1 (0.38) Absent Absent Absent NA 3/4 4.41 > 181 c.824G>A p.Trp275X 5 (1.9) 0.0314 Absent Absent rs144292455 2/4 5.52 c.1007A>G p.Gln336Arg 2 (0.77) Absent Absent Absent NA 4/4 5.21 :2 TAC3 nCHH c.209-1G>C 2 (0.77) 0.0108 Absent Absent rs146391497 2/4 4.09 FGF8 nCHH c.560_574del p.187_192del 1 (0.38) Absent Absent Absent NA NA NA 111 KS c.617G>A p.Arg206Gln 1 (0.38) Absent Absent Absent NA 4/4 3.79 via freeaccess

(Continued) European Journal of Endocrinology https://eje.bioscientifica.com

Table 3 Continued. Clinical Study

Pathogenic Gene/ Aminoacid Allele frequency In-house prediction diagnosis cDNA genotype genotype AC (%) GenomAD MAF (%) ABraOM MAF (%) database MAF (%) avsnp144 sites GERP SOX10 KS Deletion exons 1-6 1 (0.38) Absent Absent Absent NA NA NA PROK2 nCHH c.163delA/WT p.Ile55X/WT 1 (0.38) 0.0111 Absent Absent rs554675432 NA NA KS c.297dupT p.Gly100fs 1 (0.38) 0.0104 Absent Absent NA NA NA IL17RD nCHH c.1608_1611del p.Glu536fs 1 (0.38) Absent Absent Absent NA NA NA c.1697C>T p.Pro566Leu 2 (0.77) 0.2024 (all in 0.8 (all in 0.39 (all in rs61742268 4/4 3.73

heterozygous) heterozygous) heterozygous) L GAmatoandothers IGSF10 nCHH c.1297delC p.Gln433fs 1 (0.38) 0.0079 Absent Absent rs762330687 NA NA c.6830A>G p.Asp2277Gly 1 (0.38) 0.0179 Absent Absent rs150554446 4/4 4.03 SPRY4 HHIn c.776C>T p.Ser259Phe 1 (0.38) Absent Absent Absent NA 4/4 4.92 FGF17 nCHH c.196G>A p.Val66Met 1 (0.38) Absent Absent Absent NA 4/4 4.75

AC, allele count; KS, Kallmann syndrome; MAF, minor allele frequency; NA, not available; nCHH, normosmic congenital hypogonadotropic hypogonadism. pathogenic were identified in Variants predictedtobepathogenicorprobably Other rarevariants OTX2 associated conditions(Patient75),whoalsoharboreda was identifiedinawomanwithnCHHandnoother MLPA assay. Anadditional, loss. The had nofamilybackgroundofdelayedpubertyorhearing ovaries. Shewasborntonon-consanguineousparentsand aplasia andpelvicultrasoundshowedhypoplasicuterus bulb LH andFSH.MRIscanofCNSshowedolfactory evaluation revealedprepubertallevelsofserumestradiol, testconfirmedanosmia.Hormonal were absent.Olfactory clinodactyly. Skin,hairoririspigmentationabnormalities development Tanner stageI,BMI 19 At physicalexamination,shehadnopubichairandbreast adult testosteronelevels after hormonereplacement CHH reversalwasdefined asthepresenceofnormal CHH reversal CHH isnotyetsufficientlyestablishedintheliterature. significance, sincethecausativeroleofthesegenes in variants, however, arestillconsideredofuncertainclinical of pubertal delay.family history Both with nCHHandnootherendocrinopathies, a IGSF1 not beenpreviouslydescribedinCHHpatients.The (Patient 59),andin in awomanwithnCHHandsensorineuralhearingloss DMXL2 heterozygous rarevariantsin male patient with nCHH (Patient 15), who carried one heterozygous( respectively. probablypathogenicandvariants, to carry Patient 47 ( only Patient49( patients, but after applying variant selection criteria, other genes. FGF17 IL17RD phenotype (Patient72).Two differentpathogenic ( Genetic causesofCHH p.Val Rare variantswereidentifiedin A probablypathogenicvariantwasidentifiedin Rare variant(

66Met) inamanwithnCHHandnoadditional ; some of them in oligogenicity with variants in p.Pro variants,onehomozygous( ( IGSF10 p.Ser SOX10 237Ala was identified inPatient74, awoman p.Gln 1724Leu) aswell. p.Ala variantswereinitiallyidentifiedineight heterozygous deletion was confirmed by heterozygousdeletionwasconfirmedby 433fs), both with nCHH, were found p.Asp 55Ser), bothclassifiedasVUS. p.Glu IGSF1 2277Gly and Downloaded fromBioscientifica.com at09/30/202107:34:04PM 536fs) wereidentifiedina , acandidategenethathas SOX10 IGSF10 FGFR1 variant( FLRT3 181 , kg/m ( SPRY4 p.Thr p.Pro FLRT3 :2 p.Pro ( 2 and bilateral andbilateral 566Leu) and p.Tyr 1538Ile) and p.Val , 28Leu) and IL17RD and and 340Met) 340Met) 274Cys) IGSF1 FGF17 112 via freeaccess

European Journal of Endocrinology previously described). of cDNAfragment(556 bp) black symbolsclinicallyaffectedpatient–index,whitecirclesphenotypicallynormal).(B)Amplificationandautomaticsequencing (A) Familyheredogramofpatientwitha Figure 3 identified inassociationwith otherswas more thanonegene( classified aspathogenic,probably pathogenicorVUSin Nine patients(6.9%)presented withrarevariants Oligogenic findings had KS,withoutestablishedmoleculardiagnosis. gene ( had acompoundheterozygousmutationinthe Thr in the and 49hadnCHHprobablypathogenicvariants patients participatinginthisstudy(6.5%).Patients47 ( symptoms ofhypogonadismaftercessationtreatment monthsfortestosterone injections), andno (3–6 was discontinued for an appropriate washout period 32 Clinical Study ). Hypogonadism reversal was observed in 6/92 male ). Hypogonadism reversal was observed 1538Ile, respectively).Patient28,alsowithnCHH, p.Val134Gly/Arg IGSF10 gene( Table 2 262Gln). Patients27,57and90 p.Gln GNRH1 ). Thegenemostcommonly 433fs and L GAmatoandothers . (C)Schematicstructureof GNRH1 CHD7 p.Asp2277Gly/ mutation(squaresindicatemalefamilymembers,circlesfemale followed GNRHR

GNRH1 in 35% of the cases ( genes in 350 CHH individuals and identified mutations 42/130 patients(32.3%).Miraoui to identifypathogenicorprobably pathogenicvariantsin seven candidategenes.With thisapproachitwaspossible included 29genespreviouslyassociated with CHHand patients with KS and nCHH, using a 36 genes panel, which We investigatedthepresenceofraregenetic variantsin Discussion patients’ phenotype. the possibilityofasynergisticroleforthesevariantsin pathogenesis ofCHHisunknown,andonecannotdiscard the phenotype.Theroleofthesevariantstogetherin one of the variants alone would be sufficient to explain by Genetic causesofCHH withthelocalizationoffourhomozygousmutations(three FGFR1 . In the majority of these patients, however, 32 Downloaded fromBioscientifica.com at09/30/202107:34:04PM ). Most recently, Cassatella https://eje.bioscientifica.com et al. 181 (2013)analyzed17 :2 113 et al via freeaccess . European Journal of Endocrinology https://eje.bioscientifica.com to the same region that is lost in the natural animal model of theGnRH-associatedpeptide (GAP),whichcorresponds completely encodedbyexon 1.However, itpredictsaloss does notaffectthetranscription ofGnRHdecapeptide, start ofexon2(c.142-2A located attheconsensussplicingsiteregionupstream we identifiedanovel have beendescribed( but sofaronlythreedifferenthomozygousmutations obvious candidateformutationsinpatientswithnCHH, families ( ethnicity, frequencyoffamilialcasesandconsanguineous differences intheselectionofpopulationsstudied, variable indifferentpublications,possiblyduetobaseline frequency ofmutationsineachspecificgeneissomehow agreement to the described in the literature, although the of mutationsintheclassicalCHHgenesourcohortis these genesinthepathogenesisofCHH.Theprevalence for only nCHHpatientsfor (9.4%) asthemostfrequentlyaffectedgenes,considering confirming 35% (99/283)ofallpatientswithmoleculardiagnosis, identified bySangersequencing,wehaveaprevalence in thispaneladditiontothemutationspreviously study. Consideringthenewgeneticfindingsidentified GNRHR identified deleteriousmutationingenesas diagnosis. Notably, manypatientswithpreviously 260 patients, 22.3% of them with established molecular main CHHgenes,aspartofapreviouslystudiedcohort had alreadybeenscreenedbySangersequencingforthe important toremindthat107/130patientsstudiedhere frequently affected genes, followed by responsible formostofthecasesinallstudies. diagnosis, genesclassicallyassociatedtoCHHremains to increase the percentage of patients with a molecular thatthoughtargetedNGSwasable remarkable toobserve variants selectioncriteriaandinterpretationofdata.Itis to factorsaschoiceofgenesbestudied,techniqueused, 33 with identifiedmutationsvariesfrom30to50%( works ( most frequentlyaffectedgeneswerequitethesameinall selection of genes was somewhat different from ours, the less rigorousselectioncriteriathanours( identified rarevariantsin51%ofthecases,thoughwith (2018) screened24geneson116CHHpatientsand Clinical Study , ANOS1 34 GNRH1 In this work, , 32 35 , amongothers,werenotincludedinthepresent 5 , , , . Thesefindingsstrengthentheimportanceof 33 36 hasbeenconsideredforseveralyearsan 10 FGFR1 ). Intheliterature,percentage ofpatients ). Thisvariationmaybeprobablyattributed , 12 , (11.2%), 32 FGFR1 , Fig. 3C 34 > GNRH1 , C). Interestingly, this mutation 35 and GNRHR ANOS1 , ) ( 37 42 homozygousmutation , L GAmatoandothers ANOS1 38 andonlyKSpatients , (10.2%), 43 , 39 , 44 , 33 were the most 40 ). Inourstudy, ). Althoughthe GNRHR , 41 and GNRHR ANOS1 ). 12 . It is and , 32 ,

GnRH neurons( bulband the neuronaldevelopmentofolfactory DNA bindingprotein7,whichplaysarelevantrolein which hasnotyetadefinedfunction( to elucidate the functional importance of the GAP region, function variantinthispositiongivesustheopportunity Decay. Thisunprecedentedfindingofaputativeloss-of- degradation triggeredbyNonsense-MediatedmRNA less than50-55nucleotidesrequiredtoeffectiveRNAm transcript describedherethisdistanceis14basepair, the downstreamexon-exonjunction.Inaberrant including thedistancebetweennonsensecodonand the recognition of the PTC depends of several factors is produced,thusexplainingthephenotype.Though, nonsense-mediated decay(NMD),inwhichcasenoGnRH stop/termination codon(PTC),leadto hypothesis isthatthevariant,resultinginapremature of deficiencyGnRH,the in healthyrelatives( (patients 7and12),thesame variantswereidentified analysis waspossibleinthree familiesandintwoofthem patients with mutations in this gene ( concept thatthesecharacteristics aremorefrequentlyin features (deafnessandkidneydefects)corroboratingthe patients with this studymostofthemwereclassifiedasVUS.Three in thisgeneshouldbeinterpretedwithcaution,and in chance. Consequently, mostofthe variants identified unrelated tothephenotypeinsuchagenesimplyby It isthereforenotuncommontofindararevariant a lotofraremissensevariantsreportedinGnomAD. into considerationthatthe of actually couldpartiallyexplainthehigherprevalence frequency inother CHH cohorts( CHD7 previously performedinthisBraziliancohort.Although ( deafness andKS,itcanachievenearly40%ofpatients patients withKS,however, whenassociatedwiththe prevalence of and nootherCHARGEfeatures( heterozygous mutationsof study conductedbyKim delay, genitalandearanomalies( cardiac abnormalities,choanalatresia,developmental disorder characterizedbythepresenceofcoloboma, CHARGE syndrome(OMIM214800),amultisystem of thegenehavebeenclassicallyassociatedwith Genetic causesofCHH 4 , CHD7 5 The ). Notably, mutationshavealsobeenreportedinahigh CHD7 findings in our cohort. It is important to take CHD7 CHD7 geneencodesachromodomainhelicase CHD7 46 ). variantshadadditionalphenotypic mutationsisapproximately7%in sequencinganalysishadnotbeen p.Ala CHD7 Downloaded fromBioscientifica.com at09/30/202107:34:04PM et al hpg 2259Thr and loss-of-functionmutations CHD7 CHD7 . (2008)firstdemonstrated mouse( 46 5 inpatientswithKS isalargegenewith ). To date,theoverall ), thisselectionbias 181 45 5 46 45 , :2 ). 46 ). Theseminal ). Oneplausible p.Leu GNRH1 ). Segregation 2806Val), mRNA 114 via freeaccess European Journal of Endocrinology with CHH,including involved inthe pathogenesis ofCHHiswell-known( in synergywithanosmin-1,anditsinvolvementthe andGnRHneurons,acting the migrationofolfactory our knowledgeaboutthelikelyroleof clarify theseissues,butthecurrentresultsalreadyenrich to assesstheconsequencesofthesespecificvariantscould delayed puberty and reversibleCHH. Functional studies functional hypogonadotropichypogonadism,self-limited presentations oftransientGnRHdeficiency, including defects mayberepresentacommonbasistodifferent hormones stimulation. We hypothesize that to completetheirdevelopmentwhensubmittedsex plasticity modulation, allowing the immature neurons plays atemporalroleinGnRHneuronsmigrationand development ( embryonic dysregulation of GnRH neuronal migration during that defects in mutations sofar. Howard ( previously associatedtoreversibilityofhypogonadism a compoundheterozygousmutationin study, fourotherpatientshadreversibleCHH,onewith especially inpatientswithpartialCHH( of CHHcasesaftersexsteroidsreplacementtherapy, Reversibility has been described in approximately 10% patients presentedwithreversionofthehypogonadism. in twomalepatientswithnCHH.Interestingly, both probably pathogenicor GnRH deficiency( with self-limiteddelayedpubertyandfunctionalformsof deepened. In 2016, The to thegonadotrophicaxisandGnRHneuronshasbeen of CHHinthesepatients. weakening theimportanceof Patient 15, with nCHH without deafness, two variants severity( endocrine phenotypesof varying heterozygosis in ( or othergeneswouldberequired fortheKSphenotype phenotype andadditionalaffectedallelesinthesame gene wouldprobablynotbesufficienttocausetheCHH suggested thatonlyoneallelicdefectinthe cases ofpubertydelay( in KS associated with deafness and subsequently in some SPRY4 10 32 Clinical Study ), andthreepatientswithnoidentifiedpathogenic , Mutations inthe The Recently, the knowledge about 52 ( 32 ). Therefore, it was suggested that variants in FGFR1 , 51 ). IGSF10 signalingcomplexplaysacrucialrolein FGFR1 47 IL17RD ). Inthepresentstudy, weidentified IL17RD IL17RD signalingpathwaywereassociated leadsto delayed puberty through IGSF10 32 couldresultinreproductive 47 , genewereinitiallydescribed , ). It is possible that t al et 52 HS6ST1 CHD7 gene has beenassociated ). Previousstudieshave L GAmatoandothers IGSF10 . (2016)hypothesized inthepathogenesis , 38 IGSF10 IGSF10 FGF17 48 functions related , GNRHR ). Inthepresent 49 , , inCHH. 50 FLRT3 variants IGSF10 ). Genes , agene 32 IL17RD IGSF10 ). In and

heterozygous andhomozygousstateinABraOM (Patient 17),classifiedasbenignforitspresenceinthe 48), classifiedasprobablypathogenic,and in KS andnCHHpatients( in manner tothe expressed and regulated during development in a similar phenotype. KS and puberty delay, often with theassociated deafness in the CHHphenotypeisunknown.Untilnow, mutations benign; the degree of contribution of each variants to as pathogenicandthe genes. The variants of also presentedheterozygousvariantsin associated with aheterozygousframeshift.This patient were identifiedin pituitary deficiencies ( pituitary gland deficiencyorwith retinaldystrophywithout with aclinical syndrome involving multiple pituitary complete WSphenotype. large deletion,asdescribedhere,isnotassociatedwiththe suggests alackofgenotype-phenotypecorrelationsince a of KS,especiallywhenassociatedwithdeafness,and for thesignificanceof KS anddeafness.Thisfindingprovidesfurtherevidence the wholecodingsequenceof KS andhearingloss( neuronal defects. hair/iris hypopigmentation, deafness and other variable Waardenburg syndrome(WS),characterized byskin/ development andisknownasthecausativegeneof defects. a newvariationofthephenotypeassociatedwith possibility that the patient described here might represent is associatedavariablephenotypicspectrum,weraisethe relation of this genewithKSwithoutdeafness( different fromtheinitialdescriptionofassociation variant, hadnormalolfactionandunilateralhearingloss, respectively. Patient59,withthe nCHH andclassifiedasprobablypathogenicVUS, and phenotype. Heterozygousvariantsin FGFR1 GnomAD databases.Indeed,Patient17alsocarrieda Genetic causesofCHH IL17RD FLRT3 SPRY4 The OTX2 SOX10 FLRT3 mutation( FLRT3 in KS patients and in inpatientswithnCHH: mutationshavebeendescribed inassociation gene had been described only in patients with geneplaysamajorroleinneuralcrest ( FLRT3 p.Tyr FGF8. , FGF17 274Cys) wereidentifiedinpatientswith with the SOX10 p.Pro Miraoui IL17RD 53 54 and SOX10 IL17RD ). We describehereadeletionof Downloaded fromBioscientifica.com at09/30/202107:34:04PM ). We identifiedan hasrecentlybeenassociatedto 286fs), enoughtojustifythe 32 DMXL2 , ahomozygousmissensein SPRY4 ). We identifiedtwovariants FGF8 et al. mutationsasgeneticcause and FGF17 https://eje.bioscientifica.com SOX10 (2013)describedvariants / includethesetofgenes FGFR1 variantasprobably FGFR1 p.Ser 32 181 FLRT3 FGF17 and inapatientwith FGFR1 ). Consideringthe :2 pathway, which 259Phe (Patient were classified SPRY4

( OTX2 p.Tyr p.Val and p.Ser in both variant DMXL2 274Cys 66Met) 241Tyr FLRT3 115 via freeaccess

European Journal of Endocrinology https://eje.bioscientifica.com the resultsoflargenumber ofvariantsthatwillbe carefully to manydiseases,wemust interpretvery cases. However, sincethegrowthofNGS intheapproach in additionincreasingtheidentification oftheoligogenic molecular diagnosisofthe descriptions intheliterature, CHH and to approximate the proportion of patients with study wasabletoamplifythegeneticcharacterization of sequencing certainlyisamoreappropriateapproach.This with adisease,andforidentifyingnewgenes,exomic the panelisusefulindetectingknowngenesassociated genes previously associated with CHH,confirming that than 30%ofCHHpatientsandthevastmajoritywas in potentially pathogenic variants were identifiedinmore novel molecular insights in genetic of CHH. Rare and phenotypes. prove thatmultiplegenotypeswouldleadtomoresevere some phenotypesarefactorsthatstillmakeitdifficultto CHH andthepossibleinfluencesofenvironmenton total cohort.Thelargenumberofgenesassociatedwith one geneinninepatients,corresponding to 6.9%ofthe 15% ( of oligogenicityhasrecentlyincreasedtoapproximately 32 described in2.5–7%ofthecasespreviousstudies( autosomal recessiveinheritance.Oligogenicitywas such asheterozygousmutationsingeneswithatypical with isolatedmutationsdonotjustifythephenotype, may help to elucidate the molecular diagnosis of patients hypogonadism. Inaddition,theconceptofoligogenicity congenital forms,somereversibletocasesoffunctional presentation ofhypogonadotropichypogonadism,from time of onset of normal puberty and the various forms of the comprehension of thewide variability existing in the studying theinteractionofthesegenesindeedincreases or contributingtothephenotype.IncaseofCHH, to distinguishwhetherbothvariantsaresufficientand/ the phenotype,butinmostcases,itisextremelydifficult defects mighthaveasynergisticeffectcontributingto across families.Itishypothesizedthatdifferentgenetic explain thephenotypic variability ofCHHwithinand that oligogenicinheritancecouldatleastpartially has longbeenrecognized( phenotype. variants describedhereareresponsibletothispatient’s with complexphenotypes,soitisunlikelythatthe abnormalities. Bothgeneshavebeenpreviouslyassociated VUS, inapatientwithnCHHnootherphenotypic in associationwitha Clinical Study ). With theadventofNGS,frequency ofdetection The useoftargetedNGSinthislargecohortprovided The importanceofoligogenicmutationsinCHH 5 , 55 ). Here,weidentifiedvariantsinmorethan SOX10 13 variant,bothclassifiedas ). Ithasbeensuggested L GAmatoandothers 14 , 15 , The authors declare that there is no conflict of interest that could be could that interest of conflict perceived asprejudicingtheimpartialityofthisstudy. no is there that declare authors The Declaration ofinterest EJE-18-0764 This islinkedtotheonline version ofthepaperat Supplementary data condition. be apowerfultoolinmoleculardiagnosisofthisrare causes suchasCHH.Targeted NGShasbeenshownto the approachofrarediseasesandwithmultiplegenetic the importanceofusenewgeneticstechnologies,in identified wereconsideredbenignorprobablybenign. variant individually, 51(66.2%)ofthe77variantsinitially identify the most relevant findings, after analyzing each identified, sinceeven usinga strictselection criterionto References 302849/2015-7). Technological and Scientific for Council Development (CNPq); grantstoA C L (FAPESP 13/03236-5 and CNPq National the from J) L A A and (to 0 J) L A A from theSãoPauloResearchFoundation (FAPESP); Grant304678/2012- (to 2013/03236-5 Grants by 2014/50137-5 de Sequenciamento (SELA –LaboratórioemLargaEscala) supported was work This Funding

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. European Journal of Endocrinology

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Accepted 23May2019 Revised versionreceived14May2019 Received 17September2018

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