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European Journal of Endocrinology -20-1387 *(A-P IivonenandJKärkinencontributedequallytothiswork) Finland, FIMM,UniversityofHelsinki,Finland Hospital, Helsinki,Finland, Unit, UniversityofHelsinki,Finland, 1 Kirsi Vaaralahti Anna-Pauliina Iivonen 9q31.2 Kruszka syndromeanda Kallmann syndromeinapatientwithWeiss– or coloboma ( abnormalities, cleft lip or palate, and anophthalmia and/ such asboneabnormalities, hearingimpairmentandear and KSmaymanifestwith accompanyinganomalies, has anabsentordeficientabilitytosmell( sense ofsmell, and Kallmann syndrome (KS)if the patient called normosmic(nCHH)ifthepatienthasanormal of gonadotropin-releasinghormone(GnRH)( and elicitsinfertilityduetodeficientsecretionoraction rare geneticdiseasethatpreventspubertaldevelopment Congenital hypogonadotropichypogonadism(CHH)is a Introduction the presenceofKSandKS-relatedfeatures. Weiss–Kruszka syndromeandKS.Wesuggestthatpatientscarryingamicrodeletionin9q31.2shouldbeevaluatedfor ( were expressedinperipheralbloodleukocytetranscriptome.Thedeletionwas1.8MbupstreamofaKScandidate variants in32knownKSgeneswhole-exomesequencinganddisplayednoaberrantsplicingof15that delay, andsensorineuralhearingloss),butdidnotexplainhisKS.Infurtheranalyses,hecarryraresequence haploinsufficiency wasconsistentwiththepatient’sWeiss–Kruszkasyndrome(craniofacialphenotype,developmental (hg19). Thedeletionencompassedsixprotein-codinggenes( genome linked-readsequencing)wereinthe KS) anda lip/palate, andolfactorybulbhypoplasia.WereportapatientwithcongenitalHHanosmia(Kallmannsyndrome, Patients withdeletionsonchromosome9q31.2mayexhibitdelayedpuberty,craniofacialphenotypeincludingcleft Abstract Department ofPhysiology,StemCellsandMetabolismResearchProgram,FacultyMedicine,Programs https://doi.org/ https://eje.bioscientifica.com Clinical Study de novo 10.1530/EJE PALM2AKAP2 1 ). These diseases present wide phenotypic 1 2.38Mbheterozygousdeletionin9q31.2.Thebreakpoints(determinedwithwhole- and -20-1387 3 North KareliaCentralHospital,Joensuu,Finland,and Taneli Raivio 1 ) butdidnotsuppressitsexpression.Inconclusion,thisisthefirstreportofapatientwith , *, JuhoKärkinen 1 others A-P Iivonen,JKärkinenand 2 Pediatric ResearchCenter,NewChildren’s Hospital,HelsinkiUniversity 1 , Published by Bioscientifica Ltd. 2 1 Printed inGreat Britain ). BothnCHH © 2 FKTN , 2021Theauthors *, VenkatramYellapragada 1 ). CHHis gene (9:108,331,353)andinanon-codingarea(9:110,707,332) FKTN phenotypes, andhearing loss andshowedthatthis patients withdevelopmental delay, distinctcraniofacial of thecasesareoligogenic( large cohort studies suggest that a significant proportion or oligogenicfashiondependingonthecausativegene; cases ( the currentlyknowngenesaccountforonlyhalfofall several nCHHandKSgenesremaintobediscovered,since nCHH andKShavebeenidentifiedtodate( and geneticheterogeneity, asover60genesunderlying syndrome 9q31.2 deletionandKallmann 4 Institute forMolecularMedicine de novo , Recently, Weiss andKruszkareportedaseriesof ZNF462 1 ). nCHHandKSmightbeinheritedinaMendelian , TAL2 1 , VirpiSidoroff , TMEM38B deletionin Attribution 4.0International License. This workislicensed under a Downloaded fromBioscientifica.com at09/29/202106:53:58AM 1 , ). RAD23B 3 , HenrikkiAlmusa (2021) Endocrinology European Journalof [email protected] Email to TRaivio should be addressed Correspondence , and 185 185 :1 , 57–66 , Creative Commons KLF4 1

, 2 ). However, ). ZNF462 4 57 , –66 via freeaccess

European Journal of Endocrinology https://eje.bioscientifica.com that is,phenotypicfeaturesreminiscentofKS.However, and atleastfourhadcleftliporpalate( ( least oneofwhomhadCHH( described since the 1970s ( in the9q31.2chromosomalareahavebeencontinuously listed phenotypicfeatures.Ontheotherhand,deletions yet delayedpuberty, anosmia,orCHHarenotamongthe puberty sinceshereached menarche attheage of 12 and hehadahealthysister, whohad normal timingof child ofhealthynonconsanguineous parents( subsequently fortheabsence ofpuberty. Hewasthesecond proband) had been assessed for delayed development and We investigatedaFinnishfamilywhose son(the Subjects Subjects andmethods microdeletions inthischromosomalregionunderlieKS. in previouspatientswith9q31.2deletionssuggestthat results, togetherwiththeCHHandKS-relatedphenotypes in KSandtorevealpotentialnewcandidategenes.Our family memberstoexcludedefectsingenesimplicated exome andRNAsequencingintheprobandhis between Since theseinvestigationsdidnotsupporttheconnection and screenedthisgeneinasetofFinnishKSpatients. was associatedwithdecreasedexpressionof genome sequencing.Next,weinvestigatedifthedeletion the exactdeletionbreakpointswithlinked-readwhole- the proband’s 9q31.2deletionandKS,wefirstdefined that disrupted anomalies carriedabalancedchromosomaltranslocation deleterious, in carried amissensemutation,whichwaspredictedtobe disease ,sinceafemalewithKSandGraves’ can formfusiontranscripts,constitutepotentialKS PALM2AKAP2 disruption orpathogenicvariantsinthecloselylocated KS currentlyexists.Theclosestconnectioncomesfrom no clearlinkbetweenthe9q31.2deletionsandcomplete Weiss–Kruszka syndrome have been reported ( TMEM38B 9q31.2. Thedeletionencompassed syndrome, KS, and a with acomplexphenotypeincludingWeiss–Kruszka microdeletions inthe9q31.2area( syndrome is due to loss-of-function 14 Clinical Study ), or delayed puberty in multiple family members ( To investigatetheputativerelationshipbetween PALM2AKAP2 , RAD23B locus.Indeed, AKAP2 PALM2 , and expression( ( de novo 16 andKS,weperformedwhole- KLF4 ), andamalewithKSbone 7 , 11 8 , PALM2 deletion on . To date,25patientswith ), olfactory bulbhypoplasia ), olfactory 9 , others A-P Iivonen,JKärkinenand 10 17 3 ZNF462 , ZNF462 ). We reportapatient ). 11 and , 12 8 AKAP2 , , PALM2AKAP2 or , 13 FKTN 10 , 3 14 , , 13 , which 4 Fig. 1 , , , 15 TAL2 , 5 , 14 15 ), at 6 ), ), ), ), ,

that encompassed heterozygous Pedigree ofthefamily,inwhichproband( Figure 1 haploinsufficiency with ptosis, flat nasal tip, low set ears, mild haploinsufficiency withptosis,flatnasaltip,lowsetears,mild KLF4 hypogonadism, anosmia,andtheabsenceofolfactorybulbs). loss, andKallmannsyndrome(congenitalhypogonadotropic developmental delay,andmildbilateralsensorineuralhearing His circulating reproductivehormonelevelswere low balance. Abdominalultrasound revealedtwokidneys. defects or missing teeth, nor he reportedproblems with no synkinesia,handorfoot deformities,pigmentation bulbs in the MRI. He had the family and absent olfactory reported anosmia,whichhadbeenwellappreciated in to haveahypoplasticscrotumandtestes.Hehadself- for theevaluationofdelayedpubertyandwasnoted tested negativeforthepresenceofdeletion. undetermined. Thedeletionwas area wasdetected,yettheprecisebreakpointsremained (Agilent 44K),inwhicha2.38Mbdeletionthe9q31.2 comparativegenetic hybridization years, heunderwent with normalsemicircular canalsinMRI.Attheageof6.5 low setears,andmildbilateralsensorineuralhearingloss distinctive facial features including ptosis, flat nasal tip, deficit disorderandmilddevelopmentaldelay. Hehad Before goingtoschool,hewasdiagnosedwithattention closed spontaneously. Histesteswerenormallydescended. noted to havea muscular ventricular septal defect that points attheagesof1,5,and10min,respectively. Hewas g and his lengthwas 50 cm.Hereceived 4, 6,and 9 Apgar born atgestationweekH42+1.Hisbirthweightwas3240 Following anuneventfulpregnancy, theprobandwas ofdelayedpuberty.the fatherhadnoreportedhistory years. Themother’s menarche agehadbeen 11years,and syndrome 9q31.2 deletionandKallmann . TheprobandhadWeiss–Kruszkasyndromedueto At theageof13.5years,probandwasreferred de novo FKTN 2.38 Mb deletion in chromosome 9q31.2 2.38 Mbdeletioninchromosome9q31.2 , TAL2 Downloaded fromBioscientifica.com at09/29/202106:53:58AM , TMEM38B de novo , ZNF462 185 :1 arrow , ashisparents , RAD23B ) carried a ) carrieda ZNF462 , and 58 via freeaccess

European Journal of Endocrinology and 9months)inthesettingoflowgonadotropin remained extremely small (assessed at the age of 16 years was commenced.Duringthetreatment,histestissize Induction ofhispubertywithlow-dosetestosterone following genes: diagnostic platformthatcoversthecodingregionsof Genetics® KallmannSyndromePanelPlus(version3),a BluePrint part ofthediagnosticprocedure,heunderwent 7 months)levelsconsistentwiththeKSdiagnosis.Asa 29 ng/Lat16years9months,and2817 inhibin B(23ng/Lattheageof15yearsand9months; 2.1.1 was employed to define theexact 9q31.2deletion length of26.8kb.Loupe interactive visualizationtool covered withameandepthof 34.7 2.1.1 (bothprogramsby10 and GATK for variant calling and visualized with Loupe Longranger 2.2.2WGSpipelinewithdefaultparameters (hg19) referencegenome.Thedatawereanalyzedwith run was 2 cell and XP workflow. Readlength forthe paired-end system (IlluminaInc.,SanDiego,CA,USA)usingS4flow USA). ThesamplewassequencedwithNovaSeq6000 Reagent Kitsv2RevB;10 preparation(Chromium Genome Chromium library Molecular MedicineFinland)accordingto10 of the proband was performed at FIMM (Institute for from PBLs.Thewhole-genomelinked-readsequencing isolated withtheQIAampRNABloodMiniKit(QIAGEN) blood leukocytes(PBL).TheprobandandparentRNAwere DNA ofallfamilymemberswasextractedfromperipheral whole-genome linked-readsequencing presence ofstructuralvariantsinKSgeneswith Determination ofthedeletionbreakpointsand as singleexonorlargerdeletionsandduplications. indels upto220bp,andcopynumbervariationsdefined intronic sequence with singlenucleotide changes, small KISS1R and adiagnosisofKallmannsyndromewasset( bulbsintheMRIscan,hewasprepubertal, of olfactory hormone and inhibin B levels, anosmia, and the absence smalltesticularsize,lowreproductive Based onthevery LH 0.1IU/L,inhibinB23ng/L,andtestosterone0.4nM. His reproductive hormone levels were still low; serum small, only 1 cm in length (corresponding to stage G1) with pubic hair stage P3. His testes were very years and 11 months, he was still prepubertal (Tanner low,inhibin Blevelwasvery 14ng/L).Attheageof (LH 0.1IU/L,FSH0.2testosterone0.7nM,and Clinical Study , PROK2 × 151. The reads were aligned to the GRCh37 , PROKR2 ANOS1 , and , CHD7 × × Genomics,Pleasanton, CA, Genomics).Thegenomewas TACR3 , others A-P Iivonen,JKärkinenand × FGF8 , andameanmolecule up to , FGFR1

20 bpofthe × Genomics , < GNRHR 1 mL). 1 , 18 ). , FEZF1 WDR11 FGF8 coding regionsof32knownKSgenes( genomic structural variants of over 40 bp in size in the fraction ofknownKSgenes,weverifiedtheabsence dependent probeamplification)isavailableforonlya breakpoints. Inaddition,asMLPA (multiplexligation- synthesized cDNAswereusedastemplatestoassessthe in aregularthermocyclerwith1µgoftotalRNA.The (Bio-Rad) accordingtothemanufacturer’s instructions converted intocDNAusingtheiScriptSynthesisKit The RNAextractedfromtheprobandandhisparentswere with RT-qPCR Investigation of SPRY2 SPRY4 according tothemanufacturer’s instructions. Partof System forRT-PCR kit(InvitrogenbyLifeTechnologies) by usingtheSuperScript® IIIFirst-StrandSynthesis intron 2of harbored avariantNC_000009.11:g.112918586C Corporation, AnnArbor, MI,USA).Asoneofthepatients and readwithSequencher®4.9software(GeneCodes (Applied Biosystem).TheDNAsequenceswerealigned ABI Prism3730xlDNAAnalyzerautomatedsequencer ABI BigDyeTerminator CycleSequencing Kit(v3.1)and Sciences), andsequencedfrombothdirectionsusingthe purified withExoProStartreatment(GEHealthcareLife are availableuponrequest.ThePCRproductswere from the genomic DNA. The PCR conditions and primers NM_001198656.1, GRCh37)wereamplifiedwithPCR NM_053016.6, GRCh37)and and exon-intronboundariesof PROK2 The expressionlevelsof Biodyne) in aquantitative PCR machine (Roche LC480 II). mRNA expressionwithaready-to-useqPCRmix(Solis woman) without mutations in We examinedasetof16FinnishKSpatients(15men,1 Kallmann syndromepatients Sanger sequencingof primers areavailableonrequest. valueof1.0.ThePCRconditionsand given anarbitrary against a healthy unrelated control sample, which was GAPDH syndrome 9q31.2 deletionandKallmann , , and , , , FGF17 NDNF . Therelativeexpression levels werestandardized , PROKR2 PTCH1 SEMA3A NRP2 AKAP2 , , TCF12 , PROK2 , FLRT3 ) withLoupe2.1.1. CHD7 , PALM2AKAP2 , his RNA was converted into cDNA , hisRNAwasconvertedinto cDNA SEMA7A , , TUBB3 , , and PROKR2 PALM2AKAP2 Downloaded fromBioscientifica.com at09/29/202106:53:58AM PALM2AKAP2 AMH , , WDR11 DCC , PLXNA1 https://eje.bioscientifica.com , expression DUSP6 AKAP2 PALM2 CHD7 ANOS1 , SMCHD1 185 ( were normalizedto inFinnish 19 , , (NC_000009.11, (NC_000009.11, , :1 (NC_000009.11, (NC_000009.11, SOX10 PLXNA3 , , NSMF ANOS1 20 FGFR1 , ). The exons ). Theexons KLB , , , HS6ST1 , IL17RD , , FGFR1 > NTN1 NRP1 FGF8 T, in 59 via freeaccess , , , , , , European Journal of Endocrinology https://eje.bioscientifica.com PLXNA1 CHD7 frequency MAF in theproband( a potentially causative variant(s) should (i) occur only MetaLR_pred, M-CAP, andfathmm-MKL). Additionally, MutationAssessor, FATHMM, PROVEAN,MetaSVM, the tenapplied nonsynonymous andnotclassifiedasbenignbyany of should beintheconsensussplice-siteorit latest/ doc-openbio.readthedocs.io/projects/annovar/en/ by FIMM( tool ( in WESbyusingtheBasePlayeranalysisandvisualization DUSP6 DCC genes ( First, weverifiedthatthecodingexonsof32KS-associated Data analysis and 98%of20 The sequencingyieldedameantargetcoverageof222 the SAMTOOLSpackagewasusedforvariantcalling( IndelRealigner wasusedforindelsites.Thempileupfrom were removed using Picard MarkDuplicates, and GATK to cleanthealignment.AnypotentialPCRduplicates reads wereremovedandGATK BaseRecalibratorwasused Aligner). Non-unique read pairs and non-unique single reference genomewiththeBWA (Burrows–Wheeler 36 bp.ThereadswerealignedtotheGRCh37(hg19) 5’ or3’endsoftheread,removingpairswithlessthan reads, aswellanylow-qualitynucleotidesfromthe technology. First,theadapterwastrimmedfrom family was performed with Illumina Novaseq S2 PE100 The whole-exomesequencing(WES)oftheproband’s Whole-exome sequencing ( its clinicalinterpretationwasperformedwithInterVar ( dtu.dk/services/NetGene2/ www.mutationtaster.org/ genomnis.com/access-hsf also predictedwithHumanSplicingFinder( the specific primersandsubsequentlysequenced.Effectof the http://wintervar.wglab.org/ https://www.fruitfly.org/seq_tools/splice.html Clinical Study AKAP2 AKAP2 , 28 SMCHD1 , ). We definedthatapotentiallycausativevariant , ANOS1 ). Subsequently, theproband’s VCPfile,provided PLXNA3 , NSMF SOX10 27 variantNC_000009.11:g.112918586C transcript was PCR-amplified with cDNA- ), wasannotatedusingANNOVAR ( , × , , coverage. KLB in FGFR1 , , HS6ST1 IL17RD NRP1 < silico de novo 0.1%); (ii)bebiallelic(autosomal , NTN1 , , tools(SIFT, LRT, MutationTaster, ) ( , FGF8 SPRY2 ) ( , ) ( 22 FEZF1 WDR11 ) (andhaveaminorallele ) ( , 21 23 SPRY4 ), NetGene2( 26 , ), MutationTaster ( , , and FGF17 24 ). others A-P Iivonen,JKärkinenand , ), and BDGP NNSPLICE ), andBDGPNNSPLICE NDNF , , PTCH1 NRP2 SEMA3A , PROK2 , http://www.cbs. TCF12 ) werecovered , FLRT3 https://www. ) ( , , SEMA7A 25 PROKR2 , https:// > TUBB3 , ), and ), and http:// T was T was AMH 27 ). ). × , , , ,

conditions andprimersareavailableuponrequest. (NC_000001.10, NM_006513.4)(GRCh37).ThePCR in dbSNP( (providedintheANNOVARServer annotation)and in gnomAD,ExAC,1000Genomes,andExomeVariant were appliedforallethnicsubpopulationfrequencies (X-linked recessive)(MAF be monoallelicandinheritedintheXchromosome sister (autosomaldominant)(MAF and inheritedfromoneparentbeabsentinthe recessive inheritance)(MAF compared withone-wayANOVA, andfollowedby Tukey’s PBLs oftheproband( human genomeversionwiththeSTAR package( unnecessary. Thesequenceswere aligned totheGRCh37 quality andtheindividualreads,additionaltrimmingwas Babraham Bioinformatics).Basedontheoveralldata quality assessmentwiththeFASTQC tool(SimonAndrews, Raw adaptor-trimmedFASTQ filesweresubjectedto RNA-sequencing dataanalysis Unit (FUGU)attheUniversityofHelsinki,Finland. sequencing wereperformedintheFunctionalGenomics preparation,and System. RNAquality-testing,library end sequencingwithIlluminaNextSeq500MidOutput Capture Kit.RNAsequencingwascarriedoutaspaired- prepared usingNEBNextUltraIIDirectionalPoly(A) Following the qualitycontrol,RNASeq libraries were high sensitivity D5000 ScreenTape® System (Agilent). The quality and integrity of the RNA were assessed with RNA sequencing RIMBP3C designed for sequences around candidate variants in members withSangersequencing.Theprimerswere and segregationofthetwosuchvariantsinallfamily linked-read WGSdata.We confirmedthegenotype to bepresentintheRNAsequencingdataand/or analyses, apotentiallycausativevariantinWEShad (manually verified).Finally, tobeacceptedforfurther The relativeexpressionvalues of occurrence ofa9q31.2microdeletion andKS Statistics andtheprobabilityestimatefor in theIGVbrowserfromBroadInstitute( with theSashimiplotfeatureofMISOframework( Splicing events of known KS genes were visually verified syndrome 9q31.2 deletionandKallmann (NC_000022.10,NM_001128633.2)and https://www.ncbi.nlm.nih.gov/snp/ n

= 4)andhisparents( Downloaded fromBioscientifica.com at09/29/202106:53:58AM < 2%). Thefrequencycriteria < 2%); (iii)bemonoallelic PALM2AKAP2 185 < :1 32 0.1%); or(iv) ). n

= 4) were = 4)were in the inthe ) ( SARS1 60 30 29 31 via freeaccess ). ) ) European Journal of Endocrinology patient withKSandWeiss–Kruszka syndromewhocarried We investigatedthegeneticandphenotypicfeaturesofa Results (i.e. 1:30000 microdeletion wouldbeevenseveralmagnitudessmaller sucha causative mutationinKSgenes,whowouldcarry Finnish malewithKS(incidence1:30000)( at thepopulationlevel,probabilitytoencountera small(1:2 be very suchamicrodeletionbychancewould genes andtocarry our probandnottoharboracausalmutationinknownKS 126,253,089, GRCh37).Thus,theestimatedprobabilityfor rectangle onchromosome9in overlapped the24Mbregion,whichisdepictedwitha deletions, 1 Mb or larger in size, in a set of 472 734 people, data obtainedfromtheUKBiobank( 9q31.2 microdeletioninourproband,weutilizedtheCNV indicate statisticalsignificance. To evaluatetheroleof HSD Clinical Study post-hoc analysis. × 1:2 × 4:472734 × 4:472734 P -value = Fig. 2 < 1.41e-10). others A-P Iivonen,JKärkinenand 0.05 was accepted to 0.05wasacceptedto = 33 .3-6. Moreover, 4.23e-06). (chr9:102,253,143– , 34 , 35 19 ), in which 4 ), inwhich4 ) without a ) withouta encompassed sixprotein-codinggenes( and no other known gene at9:110,707,332. The deletion contains tenexons,at9:108,331,353inthefirstintron, ( locus that is located at 9:112,542,589–112,934,792 (hg19) 9:108,331,353–110,707,332, excluding DNA. Hecarrieda2.38Mbdeletiononchromosome a whole-genomelinked-readsequencingoftheproband’s To definetheexactdeletionbreakpoints,weperformed Determination ofthe9q31.2deletionbreakpoints pathogenic variants. sequence andstructuralvariantswasnegativeforlikely KISS1R exons of genes targeted sequencingpanelforKSthatcoveredthecoding a 2.38Mbdeletiononchromosome9q31.2.Adiagnostic- has beenimplicatedinKS. TMEM38B syndrome 9q31.2 deletionandKallmann Fig. 2 , ). Instead,thedeletiondisrupted PROK2 , ZNF462 , PROKR2 ANOS1 convert hg18coordinatestohg19. cgi-bin/hgLiftOver conversion tool patients theUCSCgenomecoordinate 2), Chien GRCh37 (hg19).ForKulharya deletion coordinatesarepresentedas The figureismodifiedfrom( is presentedinSupplementaryTable2. (PALM2AKAP2 syndrome candidategene and theverticallineindicatesaKallmann Kallmann syndrome-relatedphenotypes colors andpatternshighlightimportant sizes ofdeletionsinvariouspatients,their Rectangles indicaterelativelocationsand TMEM38B six protein-codinggenes( 110,707,332) (hg19),whichencompassed Mb deletion(chr9:108,331,353– vertical linesindicateourproband’s2.38 encompassing thisarea.Thedashed informative patientswithdeletions 9q31.2 deletioninrelationtoother Schematic representationoftheproband’s Figure 2 , RAD23B , and , Downloaded fromBioscientifica.com at09/29/202106:53:58AM CHD7 et al. , ZNF462 , and TACR3 ). A summary of the patients ). Asummaryofthepatients , andRamineni https://eje.bioscientifica.com , https://genome.ucsc.edu/ FGF8 , was employed to wasemployedto KLF4 RAD23B upto20bpintronic 185 , :1 ), noneofwhich FGFR1 FKTN

, and locus locus FKTN PALM2AKAP2 FKTN 14 et al. et al. , ). The ). The TAL2 KLF4 , , which GNRHR , (patient (patient

, ). ). TAL2 61 via freeaccess , ,

European Journal of Endocrinology https://eje.bioscientifica.com by TukeyHSD against anunrelatedcontrolsample. normalized byahousekeeping gene( replicates of 9q31.2, andhishealthyparents. Thebarsrepresentfour Kruszka syndrome,andaheterozygous leukocytes oftheprobandwithKallmannsyndrome,Weiss– Relative expressionof Figure 3 of both we screenedthecodingexonsandexon-intronboundaries To furtherprobethepossibleroleof Finnish KSpatients Sanger sequencingof deletion doesnotsuppress higher levelthaninthefather( the proband’s was alteredinperipheralbloodleukocyteRNA.However, investigated whethertheexpressionof Mb upstreamofthe and ourproband’s deletionlaidapproximately1.8 Given that blood leukocytes PALM2AKAP2 Clinical Study PALM2 PALM2AKAP2 PALM2AKAP2 post-hoc expressioninperipheral PALM2AKAP2 and analysis). PALM2AKAP2 AKAP2 expressionmeasuredbyqPCR, PALM2AKAP2 PALM2 hasbeenimplicatedinKS PALM2AKAP2 transcript was expressedata in16Finnish KS patients Fig. 3 and intheperipheralblood others A-P Iivonen,JKärkinenand * GAPDH P

< de novo ), indicatingthatthe locus( PALM2AKAP2 AKAP2 .05(ANOVAfollowed ), andadjusted expression. deletionin PALM2AKAP2 in16 Fig. 2 inKS, ), we

KS genes, and subsequentvisualizationoftranscripts.Overall,15 genes intheprobandandhisparentswithRNAsequencing affect splicing,weinvestigatedthesplicingeventsofKS variants. AsWESmaynotdetectnon-codingvariantsthat However, none ofthegenesweredisruptedbystructural of over40bpinthecodingregionsthese32KSgenes. whether theprobandcarriedgenomicstructuralvariants Secondly, we investigated in the linked-read WGS data FEZF1 Whole-exome sequencing,RNA above 1%amonggnomADFinns. other encountered vitro NetGene2: alterationofacceptorsites),weconfirmed acceptor siteofexon3(MutationTaster: disease-causing; out offour ofdelayedpuberty.had nofamilyhistory Giventhattwo micropenis, normalMRI,andalopeciaattheageof5.He years. He had sensorineural hearing loss in the right ear, ( AMP guidelines (InterVar, ( classified aslikelybenignaccordingtothe2015ACMG/ https://gnomad.broadinstitute.org/ with afrequencyof0.00004267inFinns(gnomAD, variant, NC_000009.11:g.112918586C 20 without mutations in established KS-causing genes( in Supplementary Tablein Supplementary Material 1and Supplementary the other in of uncertainsignificanceemerged, onein potential KScandidategene mutations.Two rarevariants we investigatedwhetherthe WESdatawouldcontainnew displayed aberrant splicing events ( and SEMA3A SPRY4 NSMF FGFR1 32 genesimplicatedinKS( sister. First,noraresequencevariantswerefoundinthe the wholeexomeinproband,hishealthyparentsand this deletionunderliesKSintheproband,wesequenced provided no equivocal answer to our question of whether 9q31.2 Given thatthetargetedKSgenepanel,deletionin causative variants splicing analysis,andidentificationofpotentially syndrome 9q31.2 deletionandKallmann 26 ). In the intron 2 of ). ThecarrierwasdiagnosedwithKSattheageof14 thatthesplicingwasnormal( DUSP6 , , , , NDNF per se PTCH1 HS6ST1 SOX10 , FGFR1 SEMA7A in , wereexpressedinPBLs,andnoneofthem , ortheadjacentcandidategene SARS1 , silico , TCF12 , , FLRT3 , ANOS1 WDR11 PROK2

, . An overview of the variants is shown . An overview tools predictedthevarianttoaffect PALM2 PLXNA1 , , AKAP2 TUBB3 AMH , Downloaded fromBioscientifica.com at09/29/202106:53:58AM , FGF8 , CHD7 and IL17RD SEMA3A , http://wintervar.wglab.org/ , , , we found one heterozygous PLXNA3 DCC , TCF12 AKAP2 , FGF17 NSMF , data not shown , ) ( SPRY2 SMCHD1 , 185 data notshown 36 , SEMA7A , variantshadMAFs , > , SMCHD1 NRP1 ). Thisvariantwas T (rs777796314), PROK2 HS6ST1 :1 , DUSP6 RIMBP3C PALM2AKAP2 , , and KLB , , , , ). Finally, PLXNA1 PROKR2 WDR11 , , PTCH1 NRP2 CHD7 NTN1 ). All and 62 19 via freeaccess in )) ). , , , , , , ,

European Journal of Endocrinology not beenreportedtocause delayedpubertyoraffect ( of chick pre-placodalregion,and hypothalamicexpression neuron ontogeny, alsoinduces instance, fibroblastgrowthfactor8,requiredforGnRH could connect Weiss–Kruszka syndrome,andseveralputative links hearing loss( cupid bow/widephiltrum,lowsetears,hypertelorism,and folds, shortupturnednosewithabulboustip,marked slanting palpebralfissures,arched eyebrows,epicanthal facial asymmetry, corpus callosumdysgenesis,down- which includedevelopmentaldelay, ptosis, metopic ridge, Kruszka syndrome( Haploinsufficiency of knowledge, broke RAD23B protein-coding genes( below 1/200000).Thedeletionencompassedlociofsix in 9q31.2,whichweestimatedtobeextremelyrare(AF Instead, hecarriedaheterozygous whole-genome sequencing,RNAandWES. assessed byusingatargetedsequencingpanel,linked-read region mutationsintheestablishedKSgenes,whichwas bilateral sensorineuralhearinglosswithoutcoding ventral septaldefect,craniofacialphenotype,andmild We reportapatientwithKS,developmentaldelay, ptosis, Discussion BHLHE40, ZNF143,ATF3, andSIX5. MYC, JUND,CEBPB,TEAD4,GATA2, MAFF, USF2,USF1, have bindingsitesinthisoverlappingregion:GATA3, the followingtranscriptionfactorsweredeterminedto UCSC GenomeBrowser( ribosomal pseudogene 9:110,672,051–110,707,332 (hg19). Thisregioncontained CHH (9:110,672,051–120,997,503)( the deletionofpreviouslydescribedpatientwith Our proband’s deletion(9:108,331,353-110,707,332)and the previouslyreportedpatientwithCHH Overlapping geneticregionwithourprobandand of thisarticle). (see sectionon 39 Clinical Study ZNF462 , The deletionwas2.38Mbinsizeandthe5’breakpoint 40 FKTN ). Ontheotherhand,mutations in , and decreasesatpubertyinnon-human primates FKTN in thefirstintron.However, tothebestofour 3 KLF4 , ZNF462 supplementary materials supplementary 5 isnotimplicatedinKSorCHH( ). Ourproband,thus,clearlyexhibited ). 3 ), themosttypicalsymptomsof ZNF462 andthereproductivesystem.For FKTN RNA5SP293 , TAL2 https://genome.ucsc.edu/ isassociatedwith Weiss– Znf462 others A-P Iivonen,JKärkinenand de novo , . According to the TMEM38B 11 expressioninthe givenattheend ) overlappedin microdeletion ZNF462 , ZNF462 37 have , 38 ), ). , is known to be an upstream transcriptional regulator of deletions areshownin representative previouslyreportedpatientswith9q31.2 been implicatedinCHHorKS. mutations inthegenesdeletedourprobandhavenot age at menarche and puberty timing inGWAS studies, despite the putativeassociation of the9q31.2 region to puberty timinginbothmalesandfemales( In arecentGWAS study, MIM 612883),whichwerealldeletedinourproband. age at,quantitativetraitlocus3,i.e.MENAQ3locus; ( as wellSNPsrs12684013,rs4452860,andrs7028916 olfactory bulbs and olfactory ensheathing cells ( bulbs and olfactory olfactory bulbs ( (crucial forneurogenesisand expressedintheolfactory bulb ( in the mouse olfactory gonadotrope-derived cells ( with apuberty-relatedfactor, LIN28B,inneuroblastoma sites forZNF143(atranscriptionfactorwhichinteracts RNA5SP293 Xu’s patient( CHH ( gonadotropin levels( Tanner stagewasG2attheageof20insetting ofnormal had theonsetofpubertyatage18years,andhis originally classifiedtohavedelayedpubertybyXu the sense of smell ( contained theSNPrs139300691,whichisassociatedwith patient andinthefamilyreportedbyRamineni ( hypothalamus andpituitary transcription factorimplicatedinthedevelopmentof ( interacts with known KSgene,inthechickneuralcrest( coding gene, deletion and that of ours encompassed only one protein- delayed puberty. Theoverlappingregionbetweentheir a family, inwhichadeletion9q31.2segregatedwith delayed pubertyorCHH( one encounteredinourprobandandareassociatedwith 2. Therearetwopreviousdeletionsthatoverlapwiththe SNP being were at 9q31.2 (rs2090409), the nearest genes to this that associatedwiththevariationinageatmenarche KS phenotype.Inhumans,thestrongestGWAS signals haploinsufficiency aloneisnotsufficienttoexplainthe the sense of smell ( syndrome 9q31.2 deletionandKallmann 42 45 ) bypreventing ) intheintergenicregions9q31.2(menarche, KS-related phenotypesinourprobandand 50 1 53 ). The ~35 kb region shared by our proband and )), JUND(JunDbindsto )) andCEBPB( TMEM38B ( Fig. 2 11 KLF4 ) containedonlyaribosomalpseudogene β -catenin and inhibits Wnt signaling . ). Thisregionalsocontainsbinding 49 β 3 KLF4 -catenin bindingtoTCF7L2( 11 , α , ). The second informative patient, T3-1 cells( 4 ). Thus,hefulfilledthecriteriaof FKTN Downloaded fromBioscientifica.com at09/29/202106:53:58AM Cebpb Fig. 2 , 11 iscoexpressedwith TMEM38B 5 , , 15 6 47 , inratsisexpressedthe ), suggesting that and Supplementary Tableand Supplementary ). Ramineni TAL2 https://eje.bioscientifica.com 52 , Gnrhr 48 )), GATA2 and GATA3 51 ). Thedeletioninour wasassociatedwith , and 185 ) andisexpressed AP-1siteinmouse :1 43 44 et al. ZNF462 ). Moreover, ), andKLF4 et al. SOX10 reported 54 ZNF462 et al. 46 ) and ( also 63 41 ), a , a via freeaccess ), ,

European Journal of Endocrinology https://eje.bioscientifica.com of Helsinki. and Uusimaa and was conducted in accordance with the Declaration was approved by the EthicsCommitteeof the Hospital District ofHelsinki a minor/children also aparentorguardian gave the consent. The study Informed consents wereobtained from allpatients,and in thecaseof Ethics committeeapproval the EditorialBoardofEuropeanJournalEndocrinology. Hospital, and Päivikki and Sakari Sohlberg foundation. Taneli Raivio is on Emil AaltonenFoundation,UniversityofHelsinki,Helsinki University Pediatric Research, Sigrid-Juselius Foundation, Novo Nordisk Foundation, This work was supported by the Academy of Finland, Foundation for Funding be could that interest of conflict perceived asprejudicingtheimpartialityofthisstudy. no is there that declare authors The Declaration ofinterest EJE-20-1387. This islinkedtotheonline version ofthepaperat Supplementary materials for thepresenceofaheterozygous9q31.2microdeletion. features ofWeiss–Kruszka syndromeshouldbeevaluated KS-related features,andconversely, thatKSpatientswith 9q31.2 shouldbeevaluatedforthepresenceofKSand a microdeletion in results suggest that patients carrying raresequencevariantsinknownKSgenes.Our not carry but didnotexplainhisKS.Infurtheranalyses,he was consistentwiththepatient’s Weiss–Kruszka syndrome deletion in9q31.2andKS. describe thefirstpatient withanextremely rare bulbdefect,andcleftlipand/orpalate.Wean olfactory CHH andKS-relatedphenotypessuchasdelayedpuberty, patients withdeletionsincluding9q31.2haveexhibited in pubertytimingandthesenseofsmell.Previousrare 9q31.2 atthepopulationlevelassociateswithvariation Material). Supplementary Tablein onlyaproportionofthem(Supplementary 2and variability andwewereabletoverifyKS-relatedfeatures the casesdisplayed in no pathogenic variants in Moreover, screeningasetofFinnishKSpatientsrevealed spared andexpressed deletion. ThislocusinourpatientwithKSwas,however, away, 1.8Mb,downstreamfromthe3’breakpointof KS candidategenelocus( disease isdifficulttodecipher, especiallyastheclosest a loss of transcription binding site(s) alone would cause Gnrh1 Clinical Study Taken together, variationinthechromosomalregion inmice( 55 )).

However, themechanismbywhich PALM2AKAP2 Fig. 2 PALM2AKAP2 ZNF462 showed wide phenotypic PALM2AKAP2 others A-P Iivonen,JKärkinenand efficiently( haploinsufficiency https://doi.org/10.1530/ ) isrelativelyfar . Of note, de novo Fig. 3 ).

References review oftheEnglishlanguage. Helsinki. The authors would wish to thank Annika Tarkkanen, MSc, for the for Molecular Medicine Finland FIMM Technology Centre, University of Sequencing was performed by the Sequencing laboratory of the Institute Acknowledgements the study. authors critically revised the manuscript. TRcoordinated and managed RT-qPCR analyses;A-I P, J K, V Y,K V andTRwrotethe manuscript, and all performed Sanger sequencing data analyses. V Y performed sequencing data, HA carried out ANNOVAR annotation, A-I P and K V the patients.JK,A-I P,VY,HA,KandTRanalyzed the next-generation T R,VS,Kcollectedthestudy subjects andtheclinicaldataphenotyped Author contributionstatement

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