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European Journal of Endocrinology -20-0474 Introduction now requiredtovalidatethenovelcandidategrowthgenes, interactionsandbiologicalpathwaysidentified. GHI andIGF-1insensitivitypatients,particularlysubjects withSRSfeatures.Functionalexperimentalevidenceis Conclusions: was enrichedintheGHIcohortand identified 45novelcandidategrowthgenes,15beingassociatedwithinGWAS.TheWNTcanonicalpathway novel 15q11deletion,previouslyassociatedwithgrowthfailurebutnotSRS/GHIwasidentified.Bioinformaticanalysis 8 Pediatrics, EndocrinologyandDiabeteswithaCardiologyUnit,MedicalUniversityofBialystok,Poland, Leicester NHSTrust,Leicester,UK, London, UK, Cardiovascular BiomedicalResearchCentre,BartsandTheLondonSchoolofMedicineDentistry,QueenMaryUniversity University ofLondon,UK, 1 Joo Wook Ahn Artur Bossowski Emily Cottrell molecular basisofGHandIGF-1insensitivity Rare CNVsprovidenovelinsightsintothe insensitivity (GHI, (MIM: 262500)) or IGF-1 insensitivity Perturbations ofthisaxis leadtogrowthhormone (GH-IGF-1) axisisessential fornormalhumangrowth. The growthhormone--like growthfactor-1 previously reportedinsuspectedSRS:1q21( Interestingly, 6/10(60%)CNVsubjectshaddiagnostic/associatedclinicalfeaturesofSRS.5/10(50%)CNVs Results: novel candidategrowthgeneswithintheCNVregions. tracks, growthassociatedGWASlociandpathwayenrichmentanalyseswereusedtoidentifykeybiologicalpathways/ array inGHI( Design and IGF-1 deficiencyandnormal/highGH)orpreviouslyininsensitivity(shortstature,high/normalGHIGF-1). Silver-Russell syndrome(SRS).Ithasnotbeenextensivelyinvestigatedingrowthhormoneinsensitivity(GHI;shortstature, Objective: Abstract Center, DepartmentofPediatrics,UniversityCincinnatiCollegeMedicine,Cincinnati,Ohio,USA and Hospital forChildren,Glasgow,UK, Hospital, Jabriya,Kuwait, Paediatric EndocrinologyDepartment,MafraqHospital,AbuDhabi,UnitedArab Emirates, Centre forEndocrinology,WilliamHarveyResearchInstitute,BartsandtheLondon SchoolofMedicine&Dentistry,QueenMary https://doi.org/ https://eje.bioscientifica.com Clinical Study 14 Cincinnati CenterforGrowthDisorders,DivisionofEndocrinology,Children’sHospitalMedical Both cohortswereenrichedforclass3–5CNVs(7/53(13%)GHIand3/10(30%)IGF-1insensitivitypatients). Copy numbervariation(CNV)hasbeenassociatedwithidiopathicshortstature,smallforgestationalageand m 10.1530/EJE Our cohortwasenrichedforlowfrequencyCNVs.study emphasisestheimportanceofCNVtestingin ethods: n 1 13 = 53)andIGF-1insensitivity( , Claudia P Cabrera , Vivian Hwa 4 University CollegeLondon,GreatOrmondStreetInstitute ofChildHealth,London, UK, 7 , Leo Dunkel -20-0474 10 Array comparativegenomichybridisationwasperformedwith~60000probeoligonucleotide University HospitalsBirminghamNHSFoundationTrust,Birmingham,UK, 6 2 6 12 Centre forTranslationalBioinformatics,Queen MaryUniversityofLondon, UK, The UniversityofSheffieldFacultyMedicine, DentistryandHealth,Sheffield, UK, Viapath, Guy’sHospital,London,UK, 14 , Louise A Metherell 1 , Asma Deeb E Cottrellandothers 2 CLOCK , 3 Published by Bioscientifica Ltd. , Miho Ishida wasidentifiedasanupstreamregulatorintheIGF-1insensitivitycohorts. Printed inGreat Britain n n © = 10)subjects.Publishedliterature,mousemodels,DECIPHERCNV 2020Theauthors 8 = 2),12q14( , Iman Al Basiri 4 , Sumana Chatterjee 1 , GudrunEMoore 13 Addenbrookes Hospital,Cambridge,UK, n = 1)deletionsandXp22( have beenidentifiedinthe growthhormonereceptor high GHlevels.Monogenic defects responsible for GHI stature (SS),functionalIGF-1 deficiencyandnormal/ (MIM: 270450)( insensitivity CNVs inGHandIGF-1 9 , Stephen J Rose 9 Mubarak Al-kabeer 4 and 1 , James Greening Helen L Storr 1 11 ). GHIischaracterisedbyatriad ofshort Royal 10 , Avril Mason Attribution 4.0International License. This workislicensed under a 5 n University Hospitalsof Downloaded fromBioscientifica.com at11/01/202003:13:09PM = 1),Xq26( 1 7 Department of 5 , Neil Wright (2020) Endocrinology European Journalof [email protected] Email to H L Storr should be addressed Correspondence 3 NIHR Barts 11 , Susan Bint n 183 = 1)duplications.A 183 :6 , 581–595 Creative Commons

via University CollegeLondon 6 , 581 12 , –595 European Journal of Endocrinology https://eje.bioscientifica.com to causenumerouscomplex traitssuchasautism, and areoftenbenign.However, rareCNVsare recognised . CNVs encompass natural genetic variation which canaffectsingleor multiple genesorsectionsof variations (CNVs),comprisedeletionsorduplications .Theseimbalancesorcopynumber have identifiedchromosomalimbalancesacrossthe presentations encompassthe‘SRS-like’spectrum( wide clinical heterogeneity/variable severity and atypical compared with11p15LOMindividuals.Hence,SRShas upd( patients ( frequency comparedwithnon-syndromic/non-SRSSGA sweating. ThesepresentinSRSchildrenathigher mass, crowded/irregularteeth,clinodactylyandexcessive voice, micrognathia,down-turnedmouth,lowmuscle triangular face,lowsetears,speechdelay, high-pitched -40% ‘clinical’SRScases( 5–10% patients).Thegeneticaetiologyisunknownin uniparental disomyforchromosome7(upd( 11p15 (11p15 LOM; 30–60%patients)andmaternal mechanisms arelossofmethylationonchromosome recognised tocauseSRS( termed ‘clinicalSRS’),or3/6NH-CSSwithageneticdefect both prominent forehead and relative macrocephaly, of difficulty and/orlowBMI.Diagnosisrequiresfulfilment at birth/protrudingforehead,bodyasymmetry, feeding (SGA), postnatalgrowthfailure,relativemacrocephaly features include being born small for gestational age upd( to11p15LOMand who werediagnosedwithSRSsecondary reported twopatientswhopresentedwith‘classical’GHI syndrome (SRS,(MIM:180860))( 163950), 3Msyndrome(MIM:273750)andSilver–Russell congenital syndromessuchasNoonansyndrome(MIM: despite extensiveinvestigation. IGF-1 insensitivityremainswithoutageneticdiagnosis proportion ofpatientswithclearevidenceGHIand microcephaly/developmental delay( growth associatedwithhigh/normalIGF-1levelsand IGF-1 insensitivity causing impaired foetal and postnatal IGF-1 receptor the clinicalrecognitionofthesespecificgeneticdefects. ( ( 6 GHR ) .Severaldistinct phenotypic features facilitate Clinical Study ≥ Advances inchromosomalmicroarraytechnologies Other non-specific/associatedSRSfeaturesinclude GHI-IGF-1 axisdefectsoverlapwithseveralother 4/6 Netchine–Harbisoncriteria(NH-CSS;including 7 7 ) ( )mat, havefewer‘typical’clinicalSRSfeatures )mat ( 2 ), 10 STAT5B 8 , , 11 9 ). SRSisaclinicaldiagnosis.Characteristic ). Some patients, particularly those with (IGF1R) ( 3 ), mutationsarecharacterised by IGFALS 10 11 ). Thecommonestunderlying ). ( 4 E Cottrellandothers ), PAPPA2 8 ). We previously 7 ). Asignificant ( 5 ) and 12 7 )mat; ). IGF1

20 persistent SSand14%withsyndromic( ~4–13% childrenwithidiopathicSS,16%bornSGA of pathogenic/likelypathogenicCNVsarereportedas or neurodevelopmentaldisorders( retardation inassociationwithmalformationsand/ to thosewithnormalheight( ( processes. regions and/orgenesimportantfornormalphysiological diagnosis andfacilitatestheidentificationofkeygenomic identification ofCNVsisimportantasitmaysecurea disease phenotypeorseverity( thesameCNVhaverecognised patients carrying variable penetranceand/orexpressivity, thatis,notall disease-causing CNVs is challenging as they can exhibit susceptibility to HIV ( schizophrenia, Crohn’s diseaseandpsoriasisaffectthe Committee (RECreference: 17/EE/0178). Authority, East of England Cambridge East Research Ethics and/or their parents. Approved by the Health Research publication ofclinicaldetails wasobtainedfrompatients Informed writtenconsentforgeneticresearch and Ethical approval Subjects andmethods genes andpathways. and interrogated the genomic regions for novel candidate the aetiologyofundiagnosedGHIandIGF-1insensitivity with syndromicSS.We investigatedtheroleofCNVsin is alsofundamental to identifyco-morbidities associated uncertainty andallowsappropriategeneticcounselling.It investigationsand/ortreatment,ends avoids unnecessary is importantforpatients,familiesandcliniciansasit classic SRSimprintingdefects( patients and 7%SRS-likepatientswho were negative for pathogenic CNVswereidentifiedin6%SRS-compatible compared to‘classic’SRSpatients( severe developmentaldelayand/orintellectualdisability not fulfiltheNH-CSScriteriaandfrequentlyhavemore 11 have beenidentifiedinpatientswithsuspectedSRS( identified ( potential candidategenesand/orlocicanpotentiallybe insensitivity CNVs inGHandIGF-1 15 ). Hence rare CNVs contributetochildhood SS and ). SomehaveSRS-compatiblephenotypesbutmanydo , Children withSShaveagreaterburdenofrareCNVs The identificationofapathogenicmoleculardefect 16 , 17 15 ) andalongeraverageCNVlengthcompared , 17 , 18 , 19 13 ). More than 30 pathogenic CNVs Downloaded fromBioscientifica.com at11/01/202003:13:09PM ). The interpretation of potential 15 21 , ). 16 ). Manyhavegrowth 183 17 11 14 ). Theincidence :6 ). Inonestudy, ). Nevertheless, 15 , via University CollegeLondon 17 , 18 582 , 10 19 , , European Journal of Endocrinology detectable SDS(range: limit oftheassay; Where serumIGF-1wasundetectable(lessthanthelower age: 5.8years,range:1.1–16.5) includedsixundiagnosed The IGF-1insensitivitycohort ( IGF-1 insensitivitysubjects likely tounderestimatethedegreeofGHinsensitivity. for thestatisticalanalysis.Inthesepatients,IGF-1SDS is SDS: her sibling’s CNVandGHIclinicalphenotype(height was includedinthebioinformaticanalysisassheshared < − weight (BW)SDSwas severe GHresistancewasnotedin13/27(48%).Meanbirth ng/mL betweenthebasalandpeakvaluesconsistentwith 27/53 (51%) subjects and an increase in IGF-1 level of the referringcentresaccordingtoestablishedprotocolsin − levels andIGF-1deficiency(meanSDS: − (3M, NoonanandSRS).AllhadSS(meanheightSDS: GHI and IGF-1 insensitivity and overlapping syndromes of interestwhichincludedallgenesrecognisedtocause panel coveringtheentiregenomicsequenceof64genes assessment byourin-housecustomshortstaturegene and 19newreferrals.All53wereundiagnosedfollowing pseudoexon and/orwholeexomesequencing(WES)( genes (exonsandexon/intronboundaries),the following sequencingofthe range: 0.5–17.0)included34undiagnosedsubjects The GHIcohort( Growth hormoneinsensitivity(GHI)subjects expressed asSDSbasedonage/sexrangesprovided. according to theappropriate national standards. IGF-1 was BMI wereexpressedasstandarddeviationscores(SDS) for example, undernutrition. Birth weight, height and GHI/IGF-1insensitivity 19%)) andcausesofsecondary testing (51/63; 81%) or baseline GH of (peak GHlevelof Referring cliniciansexcludedgrowthhormonedeficiency a proforma detailingthe clinical/biochemical data. their homeinstitutionsandreferringphysicianscompleted between 2008and2019( Subjects were referred toourcentre for geneticanalysis Subjects − 4.8) and 9/53 (17%) GHI subjects were born SGA (BW SDS: 2.0 to 3.9, range: Clinical Study 2.0) ( − 1.6, GHpeak:18µg/L,IGF-1SDS: Fig. 1A − 4.1). IGF-1 generation tests were performed at 4.1). IGF-1generationtestswereperformedat − 2.0 to ). Additionally, thesiblingofproband1a n n ≥ = 15), we calculated the lowest possible, = 15),wecalculatedthelowestpossible, 7 = 53) (36 males, mean age: 7.4 years; = 53)(36males,meanage:7.4years; − μ − 7.4), normal/high growth hormone 7.4), normal/highgrowthhormone g/L following standard provocation g/L followingstandardprovocation − 2.4 to 1.1 (median: Fig. 1A − 3.0) and assigned this value 3.0) andassignedthisvalue ). They were investigated at ). Theywereinvestigatedat GHR E Cottrellandothers n = 10)(6male;mean , − ≥ IGF1 0.95; range: 1.1 to 0.95; range:1.1to 10 ng/mL (12/63; 10 ng/mL (12/63; − 2.4; patient 1b). 2.4; patient1b). and − 2.5; range: 2.5; range: GHR IGFALS < 6 15 15 8 ψ )

height SDS: assessment by our panel (above). All had SS (mean referrals ( exon/intron boundaries)and/orWES( Syndrome ( subjects followingsequencingofthe ( (Agilent design028469or085030)aspreviouslydescribed were analysedbyaCGH,usinga60Koligonucleotidearray an ISO15189 accredited genetics laboratory. DNA samples (Qiagen DNeasyKit)andaCGHtestingwasperformedat Genomic DNAwasisolatedfromperipheralbloodleukocytes (aCGH) testing Array comparativegenomichybridisation born SGA(BWSDS (median: normal/high GHandIGF-1levels.MeanBWSDSwas class 2,likelybenign; 3,variantofuncertain CNVs were classified into five categories (class 1, benign; CNV classification significant CNVswereidentifiedinthesepatients. resolution (1–10Mb) due to poor DNA quality. No was 120kb.FiveGHIsubjectswereanalysedatreduced de novo samples ascertainedtheparentoforiginorconfirmed org/index.html practice guidelines( Association forClinicalGeneticScience(ACGS)best the context of each subject’s phenotype following the out andeachCNVwasassessedforpathogenicity in mosaicism ( algorithm (threshold 6) for detection of low-level analysiswasperformedusingtheADM-1 6). Secondary software (Agilent) and theADM-2 algorithm (threshold CNV detection was performedusingGenomic Workbench performed usingFeatureExtractionsoftware(Agilent). described. Fluorescencesignalintensityanalysiswas Array CGHdataanalysiswasundertakenaspreviously Array CGHdataanalysis were performedfollowingthemanufacturers’protocols. array (Agilent)andhybridisation,washingscanning Kit (Qiagen),DNAswereappliedtoa60Koligonucleotide was purifiedpost-labellingusingQIAquickPCRpurification Kit forOligoArrays(EnzoLifeSciences,USA),labelledDNA insensitivity CNVs inGHandIGF-1 22 ). Briefly, 1µgDNAwaslabelledusingCGHLabelling event − Fig. 1A 2.1; range: CUL7, CCDC8 22 − s 3.4, median: . ThemedianresolutionofourCGHarray ). Population polymorphisms were filtered ) wasthereferencegenome.Parental ). Alltenwereundiagnosedfollowing < 23 − − ). GRCh37( 2.0). 0.3 to Downloaded fromBioscientifica.com at11/01/202003:13:09PM and − 3.5; range: − https://eje.bioscientifica.com OBSL1 3.8) and6/10(60%)were http://grch37.ensembl. 183 ) genes(exonsand − :6 8 2.0 to IGF1R ) andfournew via University CollegeLondon and3M − 4.5) and 583 − 2.0 European Journal of Endocrinology https://eje.bioscientifica.com https://www.ebi.ac.uk/gwas/; informatics.jax.org/; growth hormoneinsensitivity; Biomart the CNVregionsidentifiedinGHIandIGF-1insensitivity patients wereassessedseparately.CNV,copynumbervariant;GHI, pathways andcandidategrowthgeneswithintheclass3–5CNV regionsidentifiedinthepatients.Givendistinctphenotypes, number variant;VUS,variantofuncertainsignificance.(B)Flowchart showingthebioinformaticpipelineusedtoidentifykey CNV predictedtobemostdeleteriousislisted.GHI,growthhormone insensitivity;SDS,standarddeviationscore,CNV,copy detected ineachpatientarelisted.OnewithGHIand twopatientswithIGF-1insensitivityhadmorethanoneCNV,sothe growth genesintheCNVregions(A)CNVsidentifiedGHI andIGF-1insensitivitysubjects.ThemostdeleteriousCNVs CNVs identifiedintheGHIandIGF-1insensitivitysubjects bioinformaticpipelineusedtoidentifykeypathwaysandcandidate Figure 1 Clinical Study DECIPHER STRINGdatabase https://decipher.sanger.ac.uk/; E Cottrellandothers http://grch37.ensembl.org/biomart/; https://string-db.org/. GWAScatalogue,genomewide associationstudiescatalogue insensitivity CNVs inGHandIGF-1 Full‘CNVgenelists’Supplementary Table1. MGI,mousegenomeinformatics Downloaded fromBioscientifica.com at11/01/202003:13:09PM 183 http://www. :6 via University CollegeLondon 584 European Journal of Endocrinology identified biologicalpathways andfunctionsenrichedin Ingenuity Pathway Analysis (IPA) software (Qiagen, Inc) Pathway enrichmentanalysis with lociassociated‘height’. ebi.ac.uk/gwas/ published genome-wideassociationstudies( within the CNV regions. The NHGRI-EBI catalogue of also determinedestablishedandputativegrowthgenes (OMIM, Literature searches, OnlineMendelianInheritanceinMan failure phenotypes( CNV genelistassociatedwithpre-orpostnatalgrowth Genome Informatics(MGI)identifiedgeneswithinthe key regionswithintheCNVsinoursubjects.Mouse previously reportedingrowthfailuresubjectsandhence genome datatracksidentifiedoverlappingCNVs DECIPHER data( gene list. to identifypotentialcandidategrowthgenesCandidate the end of this article). The CNV gene list was interrogated Table 1, see section on CNV regionsidentifiedinthesubjects(Supplementary included alltheprotein-codinggeneswithinclass3–5 using Biomart( techniques ( and candidategrowthgenesusingbioinformaticanalysis Class 3–5CNVregionswereexploredforkeypathways Assessment ofgeneswithintheCNVregions pathogenic, likelypathogenicorVUSwereinvestigated. classification. Class1or2CNVswerediscardedandonly their pathogenicityortherewasdatasupportingbenign populations, therewasnofunctionalworktoprove with thephenotype,wererecognisedinnormalhealthy classified asbenign/likely benign ifthey didnot segregate normal healthy populations. Conversely, CNVs were to causeananalogous phenotype andwasnotseenin or inherited from an affected parent, it was recognised in vitro functional work supported this, it was classified aspathogenic/likelypathogenicifinvivoor functional andsegregationdata.ACNVwouldbe databases, integrating population, computational, evidence frompublicationsandpublic/laboratory ( pathogenic) inlinewithacceptedbestpracticeguidelines significance (VUS); class 4, likely pathogenic and class 5, 23 Clinical Study ). Thisclassificationprocessisbasedonavailable UCSC genomebrowserenabledvisualisationof https://www.omim.org/ Fig. 1B ) identifiedgenesfromtheCNVgenelist http://grch37.ensembl.org/biomart/ http://genome.ucsc.edu/ ). TheCNVgenelistwasderived supplementary materials supplementary http://www.informatics.jax.org/ ) andpathwayanalyses E Cottrellandothers ). DECIPHER https://www. given at de novo ) and ).

subjects ( Candidate gene lists The combined bioinformatic analysis (above) produced Candidate genelists In-silico -proteininteractionanalysisusing harbouring growth-relatedgenes. with thepathwayresultsalloweddetectionofpathways analysis) ( genes generatedfrompublisheddataandin-house growth genelist’(1305establishedandcandidate ascertain their role in growth, we created the ‘Curated further. enrichment inmorethanthreesubjectswereinvestigated independent pathway analysis. Pathways with evidence of or IGF-1insensitivity).Eachindividualwassubjecttoan the CNV regions (CNV gene lists) in both cohorts (GHI harbouring CNVscompared to thosewithoutCNVs. mean height,age,sex,BWSDS orIGF-1SDSinthesubjects 1 Table and 2 IGF-1 insensitivity) VUS CNVs (class 3) ( or likelypathogenicCNVs(class 4/5)and3/63(5%)(1GHI 7/63 (11%)(6GHI,1IGF-1insensitivity)hadpathogenic CNVs wereidentifiedinatotalof10/63(16%)subjects; CNV subjects Clinical andbiochemicalfeaturesofthe Results insensitivity cohorts were analysed by Fisher’s exact and thesizeofCNVsbetweenGHIIGF-1 deletions vsduplications,thepresenceofSRSfeatures t with andwithoutCNVswerecomparedusing2-tailed IGF-1 SDS)associatedwiththeidentificationofsubjects Phenotypic predictors (height SDS, age, sex, BW SDS, Statistical analysis genes viaintermediateproteinswereexplored. were excluded.Directinteractionsbetweentwocandidate interaction sources, wheretextminingand neighbourhood Default settingswereappliedwiththeexceptionof interactions between the candidate genes (Human). database ( Mann–Whitney insensitivity CNVs inGHandIGF-1 -tests andlogisticregressionanalysis.Thefrequencyof To investigate the enriched pathways further and ). There were no significant differences between ). Therewerenosignificant differencesbetween n https://string-db.org/ 8 , = 38GHI, 24 ). Overlying the curated growth gene list U- tests, respectively. for the GHI and IGF-1 insensitivity n Downloaded fromBioscientifica.com at11/01/202003:13:09PM = 7IGF-1insensitivity).STRING ) explored protein-protein https://eje.bioscientifica.com 183 :6 via University CollegeLondon Fig. 1A Fig. 1A 585 t and and

European Journal of Endocrinology https://eje.bioscientifica.com (4 GHI)hadCNVspreviously reportedinsuspectedSRS: SRS features/associatedfeatures ( were muchmorelikelytobe presentinGHIsubjectswith 8/46 (17%)GHIsubjectswithout CNVS,suggestingCNVs or recognisedassociatedfeatures ( 6/7 (86%)GHIsubjectsharbouringCNVshadSRSfeatures with arecognisedgeneticcause;1q21deletion).Atotal of features, clinical features of SRS (NH-CSS Interestingly, fiveGHIpatientswithclass3–5CNVs had SRS featuresintheCNVsubjects results. Supplementary classified aslikelypathogenic.DetailsofCNVsubjectsin range: with IGF-1insensitivity(2males,meanheightSDS: 4/5). We identifiedCNVsclasses3–5in3/10(30%)subjects − to with GHI(6males;meanheightSDS: Class 3–5CNVswereidentifiedin7/53(13%)subjects CNVs intheGHIandIGF-1insensitivitysubjects IGF1R forIGF-1insensitivitygroup);F,female;GHI,growthhormoneinsensitivity;M,male;NK,notknown;WES,wholeexomesequencing. BW, birthweight;CGS,candidategenesequencing(GHR,GHR6 the previouspublication( *IGF-1 undetectableonassay.**Negativetestingfor11p15LOM 10 9 8 IGF-1 insensitivitysubjects 7 6 5 4 3 2 1b 1a GHI subjects Patient insensitivity andoverlappingshortstaturesyndromes(SRS,Noonan3M). gene panelcoveringentiregenomicsequenceof64genesassociatedwithGH-IGF-1axisdefectscausingGHIandIGF-1 included inthecohort,1bexhibitedsameGHIphenotypeasherbrotherandharbouredCNV).SSpanel,custom Table 1 4.3) hadpathogenicorlikelyCNVs(class Clinical Study − 5.7). 6/53(11%)patients(5males;meanheightSDS: − 2.0 to Table 2 Clinical andbiochemicalfeaturesofthepatientsharbouringCNVs.Patients1a1baresiblings(1aisproband Age atreferral 14.4 12.4 17.0 11.3 (years) 9.1 1.1 3.8 2.5 2.7 2.8 1.9 ). Patient2fulfilledtheNH-CSScriteria(3/6 − 5.7). OneofthesepatientshadaCNV 8 ).

Sex M M M M M M M M F F F ≥ BW SDS − − − − − − − − − − P E Cottrellandothers 1.3 2.1 2.2 0.3 0.7 0.3 3.2 1.9 0.4 1.7 1.6 2 in addition to SRS-like < Table 2 0.0001).Fivepatients − 4.2, range: ) incontrastto Height SDS − − − − − − − − − − − 3.6 2.0 2.7 2.5 4.9 4.0 5.7 5.1 3.7 1.6 3.6 ψ , IGFALSandIGF1 forGHIgroupand3Msyndromegenes,CUL7, CCDC8,OBSL1 and ± upd( − − 2.8, 2.5 − − − − − − − − − − IGF-1 SDS 7 0.8 0.6 2.7 2.8 2.1 2.4 2.7* 2.3 2.4 2.0 1.3 )mat undertakenatthereferringcentre.Patients1–7,9and10areincludedin those identifiedintheIGF-1 insensitivitysubjects( GHI subjects were significantlylarger ( Table(Supplementary 1). of theGHIandIGF-1insensitivity cohorts,respectively and 26protein-codinggenesresideintheCNVregions in thebioinformaticanalysis(patient1b).Atotalof122 phenotype harboured the same CNV and was included (patients 1aand2).Patient 1a’s sibling with asimilar Two unrelatedpatientswerefoundtohave1q21deletions respectively). Two deletionswereidentifiedinpatient4. another deletionorduplication(patients8and10, Two oftheduplicationswerefoundinassociationwith had genomicduplications(283862–6565338bp). deletions SRS-like patients( 5/13 (38%)CNVshavepreviouslybeenreportedin A totalof13class3–5CNVswereidentifiedin10subjects; insensitivity subjects Details oftheCNVsidentifiedinGHIandIGF-1 Xq26 duplications( 1q21 ( insensitivity CNVs inGHandIGF-1 Except foroneoutlierineach cohort,theCNVsin CGS, SSpanel CGS, SSpanel SS panel CGS, SSpanel,SRS** CGS, WES,SSpanel CGS, WES,SSpanel,SRS** CGS, WES,SSpanel CGS, WES,SSpanel CGS, WES,SSpanel CGS, WES,SSpanel,SRS** Previous genetictesting SS panel n = 2),12q14(

(143 487–9111383bp).Intotal,4/10subjects Table 3 n n = 1). = 1)deletionsandXp22( Downloaded fromBioscientifica.com at11/01/202003:13:09PM ). 7/10subjectshadgenomic Adrenal insufficiency – Migraine, normal brain MRI Persistent abdominal Delayed puberty,learning Delayed motor – – Language delay,dyslexia, Autistic spectrum Additional features – severe constipation. distention, bloating, difficulties development recurrent earinfections 183 > 500 000 bp) than :6 via University CollegeLondon

n = 1)and 586 < 500

European Journal of Endocrinology patient 1b (the sibling of patient 1a) (Benjamini-Hochberg enriched intwoGHIindividuals (patients1–2)and IGF-1 insensitivity).TheWNT canonicalpathwaywas CNV regionsofindividuals inthesamecohort(GHIor pathways and biological functionsenriched within the Ingenuity pathway analysis (IPA) (Qiagen, Inc) identified Pathway enrichmentanalysis likely duetothesmallsamplesize). insensitivity cohortwereduplications(notsignificant, subjects were deletions and all but one CNV in the IGF-1 000 bp)( stimulant) (10). non-face); (f)Feedingdifficultiesand/orlowBMI(BMI (leg lengthdiscrepancy(LLD)of length SDS);(d)Protrudingforehead(foreheadprojectingbeyondthefacialplaneonasideviewat1–3years);(e)Bodyasymmetry mid-parental targetheight);(c)Relativemacrocephalyatbirth(headcircumference length ‘Clinical SRS’)or3/6inadditiontoageneticdiagnosisassociatedwithSRS.Thecriteriaare:(a)SGA(birthweightand/orbirth scoring system:diagnosisofSRSrequiresfulfilment4/6(includingbothprominentforeheadandrelativemacrocephaly,termed 1b exhibitedthesameGHIphenotypeasherbrotherandharbouredCNV).NH-CSS,Netchine Table 2 SHOX enhancerregion,whilstourCNVdoesnotincludeeither. 613); ****BoththeseduplicationsdescribedintheliteraturearelargerthanCNVidentifiedourpatientandencompassSHOXregion and/orthe et al. *Additional clinicalfeaturesrecognisedinSRS( 10 9 8 IGF-1 insensitivitysubjects 7 6 5 4 3 2 1b 1a GHI subjects Patient Clinical Study beginsatXq25(genomicco-ordinates129132238-139650444)whilstourpatientduplicationXq26(co-ordinates134842275-141407

≤ − 2 SDSforgestationalage);(b)Postnatalgrowthfailure(heightat24 P SRS featuresinthepatientsharbouringCNVs.Patients1aand1baresiblings(1aisprobandincludedcohort, = 0.03).AllbutoneCNVidentifiedintheGHI 3p22 deletion, 7q36 duplication 7q21 duplication, Xq26 duplication 15q11 deletion 5q12 deletion 7q21 deletion, 12q14 deletion 1q21 deletion 1q21 deletion 1q21 deletion CNV duplication 15q13 Xp22 duplication 7q31 deletion

≥ 0.5 cmorarmasymmetryLLD

E Cottrellandothers 1 (b) 2 (a,b) 2 (a,b) 1 (b) 3 (b,d,f) 1 (b) 2 (a,b) 2 (b,f) 3 (b,c,f) 1 (f) 2 (b,f) NH-CSS criteria 11 ); **NoOMIMnumberassignedtothissyndromecurrently;***TheduplicationdescribedinSpengler

≤ − 2 SDSat24monthsoruseoffeedingtubecyproheptadineappetite Triangular face,high Clinodactyly Speech delay Triangular face,higharched Additional SRSfeatures* Nil Nil Nil Brachydactyly, Triangular face,hypoglycaemia Nil Triangular face,lowsetears, clinodactyly palate, hypoglycaemia, downturned mouth delayed motordevelopment pitched voice

< in theNHGRI-EBIcatalogue ofpublishedgenome-wide of thesegenesinfivesubjects wereassociatedwithheight the CNVregionsof9/10(82%) subjects( Forty-five candidate growthgeneswereidentifiedwithin Identification ofcandidategenes regulator intwoIGF-1insensitivitypatients( identified CLOCKasaplausiblecommonupstream 2A enrichment evidencewasweak(patients3,4and7)( in three other GHIsubjects whose genes were observed adjusted insensitivity CNVs inGHandIGF-1 0.5 cmwithatleasttwootherasymmetricalbodyparts,one ). IPA upstream analysis of IGF-1 insensitivity subjects

± P

1months -value=0.11). Additionally, WNTpathway

≤ − ≥ 2 SDSorheight 1.5 SDSabovebirthweightand/or Downloaded fromBioscientifica.com at11/01/202003:13:09PM SRS features( SRS features( SRS features( SRS features( stature/SRS CNV previouslyassociatedwithshort None reported None reported SRS featureswithXp22duplication SRS features( 15q11 deletion,(MIM:615656) 5q12 deletionsyndrome, None reported syndrome (MIM:612474) syndrome (MIM:612474) syndrome (MIM:612474) identified inSGAcohort( ( (MIM: 615668)( deletion syndrome**( 36 ). Xp22duplicationalso https://eje.bioscientifica.com − Harbison SRSclinical 183 50 43 43 43 43 ≤

− ) and12q14 ) and1q21deletion ) and1q21deletion ) and1q21deletion )*** 2 SDSbelow :6 37 Table 3 ) Fig. 2B via University CollegeLondon 30

). Fifteen 17 ) ). ).**** 587 Fig. European Journal of Endocrinology https://eje.bioscientifica.com Clinical Study

Table 3 Details of CNVs identified in our patients.

Number of affected Mouse Parental Size protein- genome Key candidate Patient CNV1 Class2 Inheritance height SDS (Mb) coding genes DECIPHER3 database4 gene(s) in this region5 GHI subjects 1a 1q21.1q21.2(146564742_147735011)x1 4 Maternal +0.08 1.17 9 26 3 BCL9b,d,e, PRKAB2c,d, FMO5c,d, GPR89Bc, CHD1Ld,e 1b 1q21.1q21.2(146641600_147735011)x1 4 Maternal +0.08 1.09 9 26 3 BCL9b,d,e, PRKAB2c,d, E Cottrellandothers FMO5c,d, GPR89Bc, CHD1Ld,e 2 1q21.1q21.2(145987155_147735011)x1 4 De novo 1.74 11 26 3 BCL9b,d,e, PRKAB2c,d, FMO5c,d, GPR89Bc, CHD1Ld,e 3 12q14.2q15(64413681_67794677)x1 5 NK 3.38 21 9 4 HMGA2a,c,d, WIF1b, XPOTc, IRAK3c, GRIP1c , LLPH d, MSRB3d, SRGAP1d 4 7q21.2(91914300_92762100)x1 3 De novo 0.85 9 4 3 ANKIB1 c,d, PEX1 c,d, CDK6 c,d, SAMD9d,e , GATAD1d 7q31.1q31.31(111130598_120241981) 4 De novo 9.11 29 7 8 WNT2 b,c, IMMP2Lb, c ,

x1 IFRD1c, GPR85 c, FOXP2 insensitivity CNVs inGHandIGF-1 c,d, CAV1 c, MET c, CFTR c, CAV2d PPP1R3Ad, TFECd 5 5q12.1q12.2(60371468_62950178)x1 3 NK 2.58 10 3 2 ZSWIM6c, CKS1Bc,e, DIMT1e 6 15q11.2(22765627_23085096)x1 4 NK 0.32 4 29 0 - 7 Xq26.3q27.2(134842275_141407613)x3 4 De novo 6.57 38 14 6 SOX3a,b, ZIC3c, BRS3 c, RBMX c, CD40LGc, FHL1c IGF1 insensitivity subjects 8 7q21.13(89733373_90035738)x3 3 Maternal –1.67 0.30 5 1 0 – Xp22.33(1793445_2213992)x3 3 Paternal −0.41 0.42 1 8 0 –

Downloaded fromBioscientifica.com at11/01/202003:13:09PM 9 7q36.1(151748853_152032715)x3 3 Maternal +0.33 0.28 2 3 1 GALNT11b, KMT2Cc 10 3p22.1(41611009_41754496)x1 3 Maternal −0.75 0.14 1 2 1 ULK4c 15q13.2q13.3(30653877_32914199)x3 4 Paternal −3.35 2.26 17 14 2 CHRNA7b, KLF13b, FAN1c, OTUD7Ac

1 2 1a (proband) and 1b are siblings with shared GHI phenotypes. Co-ordinates given are relative to version 37 of the human genome. CNV class 3, variant uncertain significance (VUS); 4, likely 183 pathogenic; 5, pathogenic (50). 3Number of patients in DECIPHER with overlapping deletion/duplication (as per patient CNV) and pre/postnatal growth restriction. 4Number of genes causing a growth :6 restricted phenotype in the mouse model (Mouse Genome Informatics http://www.informatics.jax.org/). 5Candidate genes were identified by corroborating information from current literature, MGI, DECIPHER, GWAS database information and our bioinformatic pathway analysis. Candidate genes in the CNV regions were selected as they were: aestablished in literature to have important role in via University CollegeLondon normal linear growth; bidentified as candidate gene from our bioinformatic pathway analysis;c causing a growth restricted phenotype in the mouse model; dContaining loci associated with height in GWAS catalogue (https://www.ebi.ac.uk/gwas/) or in GWAS literature; eputative growth role based on current literature. A total of 38 candidate genes were identified in the CNV regions of our GHI cohort and seven genes in the CNV regions of our IGF-1 Insensitivity cohort. 588 European Journal of Endocrinology and GALNT11 insensitivity subjects.Geneshighlightedinyellowresideourpatient’sCNVregions.CLOCKisatranscriptionregulatorfor genes 2). (B)PathwayenrichmentanalysisidentifiedCLOCKasatranscriptionregulatorenrichedwithintheCNVgenelistof IGF-1 deletion ofpatient4, Yellow genesarethosefoundwithinintheCNVregions( hormone insensitivity(GHI)subjects.Geneshighlightedinbluearefromourcuratedgrowthcandidategenelistof1305genes. Pathway enrichmentanalysis(A)IdentificationoftheWNTpathwayenrichedwithingenelistfromCNVregions growth Figure 2 genes) and the IGF-1 insensitivity cohort (7 genes) ( CNV regions(candidategene list)oftheGHIcohort(38 interactions (directandindirect) betweengenesinthe STRING analysisidentified abundantprotein–protein candidate genelist In impact growthforexample,chromosome7alterations. approach and not imprinting control regions which may only protein-codingcandidategenesareidentifiedbythis growth pathways/mousemodels.Thelimitationisthat genes wereproposedbasedontheirrolesinknown (12q14 deletion; patient 3). In other regions, candidate strong candidategrowthgenesforexample, association studies( Clinical Study silico CHRNA7 , protein–proteininteractionanalysisof CHRNA7 are foundwithinthe15q13regionduplicatedinpatient10. and SOX3 Fig. 3B KLF13 intheXq26duplicatedregionofpatient7and withintheCNVregions. ). SomeCNVregionsharboured E Cottrellandothers GALNT11 HMGA2 Fig. lieswithinthe7q36regionduplicatedinpatient9andboth

WIF1 defects suchasdominant-negative heterozygousvariants, GHI phenotypesincluding ‘non-classic’GHreceptor spectrum of genetic defects responsible formild-moderate Advancing genetictechniques increasinglyrecognisea causing severeSS,dysmorphic andmetabolicabnormalities. GHI istypicallyassociatedwithclassicLaronsyndrome Discussion genes andshortstature. kinases inCNVlocisuggestsanassociationbetweenthese CCND3. Overrepresentationofcyclindependentprotein CDK6, CKS1B,HMGA2,CDK1,CDK2,CCND1and involvedinregulatingcellcycleprogression: 3B insensitivity CNVs inGHandIGF-1 inthe12q14deletedregionofpatient3 and BCL9 C inthe1q21deletedregionofpatients1a,1band ). Interactionswereidentifiedbetweenseveral Downloaded fromBioscientifica.com at11/01/202003:13:09PM https://eje.bioscientifica.com 183 , WNT2 :6 inthe7q31 via University CollegeLondon KLF13 589

European Journal of Endocrinology (FAN1, patient10)interactswith EP300viaubiquitinA-52residueribosomalproteinfusionproduct 1(UBA52). protein p300(EP300).Additionally, OUTdomain-containingprotein7A(OTUD7A,patient10) andfanconi-associatednuclease1 genes). Kruppel-likefactor13(KLF13, patient10)interactswithlysinemethyltransferase2C (KMT2C, patient9)viaE1A-associated Caveolin-1 (CAV1)andcaveolin-2 (CAV2)directlyinteractandbothwereidentifiedinpatient 4’sCNV(C)IGF-1insensitivitycohort(7 regulator (CFTR,patient4)interacted directlyandalsovia5’-AMP-activatedproteinkinasecatalytic subunitalpha-2(PRKAA2). kinase AMP-activatednon-catalyticsubunitB2(PRKAB2,patients 1a,1band2)cysticfibrosistransmembraneconductance interaction wasidentifiedbetweenWntinhibitoryfactor1(WIF1, patient3)andWntfamilymember2(WNT2,4).Protein (CDK2), CyclinD1(CCND1)andcyclinD3(CCND3)alsointeracted withbothCDK6(patient4)andCKS1B5).Direct also withhighmobilitygroupAT-Hook2(HMGA2,patient3)via cyclin-dependentkinase1(CDK1).Cyclin-dependent2 dependent kinase6(CDK6,patient4)andcyclin-dependentkinases regulatorysubunit1B(CKS1B,patient5)interactdirectlyand were usedwiththeexceptionofinteractionsources,wheretext miningandneighbourhoodsourceswereexcluded.Cyclin- interactions (directandindirectviaintermediateproteins)between genesintheCNVregions(candidategenelist).Defaultsettings growth hormoneinsensitivity(GHI)cohort(38genes).STRING analysis( with associationstoheightwereidentified.Protein-proteininteractions ofthecandidategenesinCNVregions(B)The examine allprotein-codinggeneswithintheclass3–5CNVregions inthesubjects(CNVgenelists).ThoseCNVgenesthathaveloci identified. TheNHGRI–-EBIcatalogueofpublishedgenome-wideassociationstudies( catalogue. Genesareshowninthebluecirclesandconnectedtopatient(s)(P)whichCNVcontainingthatgene was GWAS andSTRINGanalysisofgeneswithintheCNVregions(A)Genesinourpatient’sCNVsassociatedwithheight Figure 3 https://eje.bioscientifica.com Clinical Study E Cottrellandothers insensitivity CNVs inGHandIGF-1 https://string-db.org/ https://www.ebi.ac.uk/gwas/ ) identifiedabundantprotein-protein Downloaded fromBioscientifica.com at11/01/202003:13:09PM 183 :6 ) wasusedto via University CollegeLondon 590 European Journal of Endocrinology phenotypes and other childhoodgrowth disorders ( of novel genes and pathways responsible for SRS-like exploring CNVregionsindetail mayenablethediscovery 40% clinicalSRSpatientshave nogeneticdiagnosis,and the SRS-likepatientswithCNVs.Thisisinteresting as difficulties, themilderclinicalphenotypesoverlapwith Whilst noneoftheGHIsubjectswithCNVshadlearning patients accordingtotheirCNVsratherthanSRS( than classicSRS.The current recommendation is to manage are morefrequentlyassociatedwithlearningdifficulties suspected SRS.ManydonotfulfiltheNH-CSScriteriaand consensus recognises disorders duetoothergeneticabnormalities.TheSRS not be appropriate to detect patients with milder SRS-like and thespecificityislow(36%).Therefore,NH-CSSmay clinical assessmenttools,thereisadegreeofsubjectivity classic moleculardefects( fulfil thecriteriaareunlikelytohaveeitheroftwo classic 11p15LOM/matUPD.Assuch,patientswhodonot well-defined andarehighlysensitive(~98%)fordetecting fulfilled theNH-CSScriteria( CNVs hadsubtleclinicalfeaturesofSRSandonepatient Interestingly, almosthalfoftheGHIpatientsharbouring imbalances (microdeletion/duplications). probes. A higher probe concentration will detect smaller probes somewithhavinginexcessof2.6million are hybridarrayscontainingbothSNPandcopynumber disomy (UPD). Most SNP arrays used for clinical purposes clinically importantfeaturesforexample,uniparental well ascopynumberdata,SNParrayscandetectother differences betweenindividualsatasinglebpsite.As arrays useDNAprobesfromgenomicregionsandreveal but research CGHarraysmayincorporatemillions.SNP CGH arrayscontainseveralhundredthousandprobes ‘normal’ (agedandsexmatched)control.Typical clinical genomic DNA in a patient’s sample compared with a CGH comparative genomic hybridization (CGH) and SNP. Two CMA techniques detect chromosomal imbalance, regions (chromosomalmicroarrayanalysis,CMA). homozygous mutationsseenin‘classic’GHI. affect growthbutnotasseverelytotallossoffunction/ of gene(s)intheaffectedregion(s)maybesufficientto classical GHI patients. Haploinsufficiency or duplication contribute tothegeneticaetiologyofundiagnosednon- pathway defects( the Clinical Study We previously noted the overlap of GHI with SRS ( DNA microarrays detect imbalancesin chromosomal GHR ‐ based arrays(aCGH)measurethequantityof pseudoexon and other GH-IGF-1 signalling 25 ). OurfindingssuggestthatCNVs > 30 differentCNVsinpatientswith 10 10 ). However, aswithmany ). TheNH-CSScriteriaare E Cottrellandothers 8 11 11 , 9 ). ). ). therefore itisunlikelythatimprinting playsarole( both maternalandpaternal transmissionofthedeletion, the phenotypicvariability. Parent-of-originstudies show of thenon-deleted1q21.1locusdidnotcontribute to carrier mother, suggested that differences in methylation an affected 1q21 deletion patient and her unaffected variants asacauseofthevariablephenotypes. Data from deletion carriersdidnotsupportunmaskingofrecessive of genes within the affected regions of eleven 1q21 phenotypic variabilityhavenotbeendelineated.Analysis for thephenotype( any 1q21.1deletions,suggestingtheCNVisresponsible Analysis of4737unaffectedindividualsdidnotidentify for this, including the background genetic variation the pathogenicityofCNVs.Severalmodifiersmayaccount CNVs and can cause a significant challenge in assessing or othergeneticaberrations. elsewhere inthegenome,abnormalitiesmethylation or partiallyattributabletoasyetunidentifiedvariants also conceivablethattheirgrowthfailureisattributable recognised orsuspectedtocausesimilarphenotypes.Itis panel whichincludedwholegenomicregionsfor64genes following assessmentbyourcustomshortstaturegene any otherpotentialcausativegeneticvariantsidentified failure inthesepatients.However, noneofthesubjectshad growth or areonlypartiallyresponsiblefortheobserved heights. ThiscouldimplythattheCNVsidentifiedarenot inherited theirCNVfromaparent,75%(3/4)withnormal lead tofurtherstratificationofSRSsubtypes. Our findingsalsoexpandtheSRS-likespectrumandmay growth failurearenotestablished. We identifiedseveral mildphenotype( parents frequentlyhavingnoorvery manner andexhibitvariablepenetrance,with‘affected’ 1q21 deletionsareinheritedinanautosomaldominant bridge, longphiltrumandhigh-arched palate.Most bossing, deep-seteyes,epicanthalfolds,largenasal Dysmorphic featuresincludemicrocephaly, frontal growth failurein50%andautism(MIM:612474). recognised tocausedysmorphism,learningdifficulties, and asiblingofproband.1q21deletionsyndromeis identified 1q21deletionsintwounrelatedGHIpatients are unmasked in some individuals ( recessive variantsresidingonthesingleremainingallele genes in the affected CNV region. It is also possible that variationofthe imprinting, expressionorregulatory of individualsand/orepigeneticmechanismssuchas insensitivity CNVs inGHandIGF-1 Potential genesinthe1q21 regionresponsiblefor Variable penetranceisarecognisedfeatureofmany Forty percent(4/10)ofthepatientsinourcohort 27 ). Potentialcausesofthewide Downloaded fromBioscientifica.com at11/01/202003:13:09PM https://eje.bioscientifica.com 26 183 ). Interestingly, we :6 via University CollegeLondon 27 591 27 ). ). European Journal of Endocrinology https://eje.bioscientifica.com in three individuals with SS and SRS features ( spanning onlypartof ( growth failure in patients with deletions in this region mobility groupAT-hook 2 evidence to suggestthathaploinsufficiency ofhigh- cellular energyandglucosehomeostasis. wide associationstudies ( reside ingenomiclociassociatedwithheightgenome- and flavincontainingmonooxygenase5 respectively). 5’-AMP-activatedproteinkinase in theGWAS catalogue( in homeostasis, cellularproliferationandgrowth( development,tissue pathway fundamentalforembryonic signalling catenin signalling,anevolutionarilyconserved lymphoma 9protein cells resultinreducedproliferation( cell cycleprogressionandknockdowninglioma 1-like including chromodomain-helicase-DNA-bindingprotein candidate geneswithinthecommonlydeletedregion ( GWAS height loci: Several geneswithintheCNV regionsofpatient5had (PEG10/SGCE) and7q32 (MEST) imprintingclusters. development). Bothdeletions areadjacenttothe7q21.3 features (triangularface,lowsetearsanddelayedmotor 4 had 7q21 and7q31deletionswith associated SRS ( hypomethylation and less asymmetry defects havemilderphenotypescomparedwithICR1 postnatal growthfailure).SRSpatientswithmatUPD7 additional region.Bothfulfilled2/6NH-CSS(SGAwith 1.00E to beassociatedwithheightminimum MSRB3 characterised orunderstood( upstream regulatorofIGF2butthemechanismisnot phenotype ( has alsobeendescribedinapatientwithsimilar 7bp intronicdeletioncausingaberrantsplicingof premature stopcodon,respectively( with SSandSRS-likefeatures,causingaframeshift point mutationshavebeenidentifiedintwosubjects to shortstaturesusceptibility( 318 normalstature controls concluded they contributed polymorphisms (SNPs) in 155 idiopathic SS patients and A studyexaminingthree P 30 -value: 2.00E Clinical Study ). Additionally, submicroscopic12q14deletions, BCL9 Patients 4and8hadCNVsaffecting7q21one Patient 3hada12q14deletionandthereisstrong (CHD1L).CHD1L − and 287, 3.00E and SRGAP1 34 CHD1L − ). 15), − HMGA2 21, 4.00E

were reportedintheGWAS catalogue TFEC CDK6 (BCL9) werealsoassociatedwithheight regulateschromatinrelaxation/ is thought to function as an 24 HMGA2, ( P (HMGA2) ( -values: 7.00E-13and7E-20, − P ) and have essential roles in P -value: 5.00E 39, 3.00E 33 -value: 3.00E is aco-activatorofWnt/ HMGA2 32 ). Lociin E Cottrellandothers ). Heterozygous have beenreported causes the observed causestheobserved 33 single-nucleotide − ). Aheterozygous 12, respectively. 28 HMGA2, LLPH, ). B-cellCLL/ − − (FMO5) 07), 35 240), P (PRKAB2) ). Patient -values of 29 HMGA2 HMGA2 ANKIB1 FOXP2 ). Loci also 31 β ). -

essential role in cell cycle regulation. Cks1-depleted breast transferase 1homologgene subunit1 regulatory harbours two candidate genes. Cyclin-dependent kinases growth failureanddevelopmentaldelay( has beenreportedinseveralpatientswithpostnatal not contributetothephenotype. subject iseitherindependentofSHOXfunctionordoes identified inourpatientsuggestingthemechanism both knownduplicationswerelargerthantheCNV subject does not encompass either of these regions and 12), respectively( and includedSHOXaenhancerregion(PAR3- duplications havebeenidentifiedinSRSandSGAsubjects Patient 8hadduplicationsof7q21andXp22.Xp22 ( ( ( reported withpoorgrowth andlowIGF-1.Theauthors secretion butlowIGF-1(SDS ( corpus callosumwithorwithout intellectualdisability and abnormalities ofthe undescended posterior pituitary associated withinfundibularhypoplasia,ectopic/ hormone,isolatedgrowthhormone deficiency pituitary under- andover-expressioninmalescausesmultiple development regulation. implicated in embryonic proximally placedbreakpointsBP1orBP2at15q11( parental origininvolvingthedistalbreakpointBP3and classically causedbychromosome15deletionsofdifferent growth failure. growth and haploinsufficiency mayleadtopostnatal of thesegenesarelikelytobecriticalfornormallinear overexpressed in several cancers ( a methyltransferaseessentialforribosomebiogenesisand ( G2-M arrestduetoblockedmitoticentry cancer cells exhibit slow G1 cell cycle progression and HMG-box 3 The Xq26.3q27.2duplicatedregionharboursSRY-related problems areacommonfeatureofPWS. had significantfeedingdifficulties.Interestingly, feeding not a reported feature of 15q11.2deletions, our patient reported in10and39%patients,respectively. Although Short statureand unspecified dysmorphic featuresare BP2) havevariable phenotypes and expressivity ( BP1 andBP2.15q11.2deletions(includingjust Patient 6 had a small 15q11 deletion encompassing insensitivity CNVs inGHandIGF-1 P P P 41 -value: 2.00E -value: 1.00E -value; 2.00E ). Ourpatienthadnormalbrain MRIandsufficientGH Patient 5hada5q12deletion.deletionsyndrome Patient 7wastheonlyGHIsubjectwithaduplication. Prader–Willi (PWS)andAngelmansyndrome are ( − SOX3) − 15), − 08) and 08), 17 PEX1 , encodingatranscriptionfactor 36 (CKS1B) Downloaded fromBioscientifica.com at11/01/202003:13:09PM SAMD9 ). TheCNVidentifiedinour ( P CAV2 -value: 1.00E −

(DIMT1). CKS1B (2.00E 2.7). Afemalepatientis and dimethyladenosine 39 ). The functional roles ( P 183 -value: 1.00E :6 − 37 25), − 38 ). Thisregion 08), via University CollegeLondon ). DIMT1 PPP1R3A hasan GATAD1 SOX3 592 − 40 11). 40 is ). ).

European Journal of Endocrinology a potentialnoveltherapeutic targetforcancer. Abnormal leads to uncontrolled cell growth and oncogenesis and is regulator of cell proliferation ( 2. TheWNTpathwaydetermines cellfateandisakey BCL9 4, region ofpatient3 canonical WNTpathway. Six GHIindividualshadCNVsharbouringgenesinthe growth pathwaysintheCNVregionsofoursubjects. ingenuity pathwayanalysis(IPA). models, whilst OTUD7A this region lies several candidate genes. Both has significantshortstature(heightSDS the 15q13 duplication was inherited from her father who been associatedwithshortstatureorSRS.Interestingly, identified inpatient10.TheseCNVshavenotpreviously phenotype. not responsiblefortheobserved penetrance (similarto1q21deletions)orthatthisCNVis mother whoisofnormalstature.Thissuggestsvariable growth. Patient9’s duplicationwasinheritedfromhis enhanced) mayalterNotchsignallingandadverselyaffect signalling ( 11, whichO-glycosylatesNOTCH1andactivatesNotch encodes polypeptideN-acetylgalactosaminyltransferase is commontobothduplicatedregions. features ( hypospadias, microphallusanddysmorphicfacial hearing loss,myopia,astigmatism,cryptorchidism, including acardiovascularmalformation,sensorineural developmental delayandmultiplecongenitalabnormalities than patient9wasbornSGAwithnocatch-upgrowth, reported. Onepatientwithalarger7q36duplication retardation ( Seventy percent7q36deletionindividualsexhibitgrowth with deletions being moreprevalent than duplications. overlapping region includes (co-ordinates; 134842275–141407613)( 129 132238–139650444)andinoursubjectatXq26 duplication beginsatXq25(genomicco-ordinates: triangular faceandrelativemacrocephaly. Thepublished patient hadabroadnasalbridgesimilarto8,with cohort ofpatientswithSRSfeatures.Interestingly, this female patientwithalargerduplicationisreportedin GH stimulationtestingwasnotperformed( suggest herpoorgrowthisduetoGHdeficiency, butformal Clinical Study SOX3 IPA identifiedgeneswithlinkstoestablishedornovel A 3p22deletionand15q13duplicationwere rare, 7q36rearrangementsarevery inthe1q21deletedregion ofpatients1a,1band intheXq26duplicatedregionofpatient7and cause a growth restricted phenotype in mouse 45 46 ). Oneinteresting candidate gene, 44 ). Abnormal ), however, fewduplicationshavebeen CHRNA7 , WNT2 and inthe7q31deletionofpatient GALNT11 WIF1 SOX3 KLF13 47 E Cottrellandothers ). Aberrant Wnt activity inthe12q14deleted and several other genes. wereidentifiedby dosage (reduced or − 42 3.4). Within GALNT11, ). Another FAN1 GALNT11 43 ). The and common causeofmildernon-classicalphenotypesin GHI andIGF-1insensitivity. RareCNVswerearelatively detail CNVsinsubjectswithgrowthfailureassociated facilitating cellcycleprogressionandproliferation( rhythm, CLOCK modulates G2-to-M cell cycletransition its centralfunctionalroleintheregulationofcircadian hypothalamic circadian rhythms( gene expressionandfunctionsasazeitgeberforcellular This isparticularlyinterestingasIGF-1regulatesclock regions identified intheIGF-1 insensitivitysubjects. CHRNA7 effects. important growthregulatory in thisregion;howeveritispossiblebothcontribute thought tobethemostcrucialgrowthregulatinggene in patient3includes and normallineargrowth.The12q14deletionidentified copy numbersofthesegenesmayimpairthispathway the statistical analysis. M I and G E M were involved in conception of study C P C conducted and interpreted the bioinformatic analysis. S C undertook designed the study, wrote and approved the final version of the manuscript. conceptualised/ S L H manuscript. the wrote and study the conducted C E Author contributionstatement to EC. awarded Fellowship Research (ESPE) Endocrinology Paediatric for Society Reference Number:MRC0161) awarded to HLS and the 2018 European This workwassupported byaBartsCharityLargeProject Grant(Grant Funding be could that interest of conflict perceived asprejudicingtheimpartialityofthisstudy. no is there that declare authors The Declaration ofinterest EJE-20-0474. This islinkedtotheonline version ofthepaperat Supplementary materials cohort. interactions andbiologicalpathwaysenrichedinour now requiredtovalidatethecandidategrowthgenes, features ofSRS.Functionalexperimentalevidenceis of GHI,IGF-1insensitivityandconcomitantsubtle cause hasbeenidentified,particularlythosewithfeatures be carriedoutinallshortpatientswherenomonogenic overlapping disorderssuchasSRS.CNVanalysisshould expands the spectrum of GHI/IGF-1 insensitivity and CNVs, potentiallyidentifiesnewcandidateSSgenesand our cohort.Thisexpandstheknownphenotypesofrare insensitivity CNVs inGHandIGF-1 In summary, thisisthefirststudyinvestigatingin CLOCK regulatesthetranscriptionof and KLF13 genes residing within the CNV WIF1 Downloaded fromBioscientifica.com at11/01/202003:13:09PM and https://eje.bioscientifica.com HMGA2 48 183 https://doi.org/10.1530/ ). Inadditionto :6 . Thelatteris via University CollegeLondon GALNT11 49 593 ). , European Journal of Endocrinology https://eje.bioscientifica.com References undertook CNVanalysisandprovidedexpertinterpretationofdata. S RandAMacquiredessentialclinicalbiochemicaldata.B,JW input and revised drafts of the manuscript. J G, N W,A B, L D, A D, I A B, and critically revised aspects of manuscript. L A M and V H gave intellectual 12 11 10 9 8 7 6 5 4 3 2 1 Clinical Study Bartholdi D, Krajewska-Walasek M, Ounap K, Gaspar H, Wakeling EL, Brioude F, Lokulo-Sodipe O,O’Connell SM,Salem J, Azzi S, Salem J,Thibaud N,Chantot-Bastaraud S,Lieber E,Netchine I Storr HL, Dunkel L,Kowalczyk J,Savage MO&Metherell LA.Genetic Shapiro L, Chatterjee S,Ramadan DG,Davies KM,Savage MO, Abuzzahab MJ, Schneider A,Goddard A,Grigorescu F, Lautier C, Woods KA, Camacho-Hubner C,Savage MO&Clark AJ. 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