2 182

E Vasco de Albuquerque Genetic investigation of tall 182:2 139–147 Clinical Study Albuquerque and others stature patients

Genetic investigation of patients with tall stature

Edoarda Vasco de Albuquerque Albuquerque1, Mariana Ferreira de Assis Funari2, Elisângela Pereira de Souza Quedas1, Rachel Sayuri Honjo Kawahira3, Raquel Soares Jallad4, Thaís Kataoka Homma1, Regina Matsunaga Martin2,5, Vinicius Nahime Brito2, Alexsandra Christianne Malaquias1,6, Antonio Marcondes Lerario1,7, Carla Rosenberg8, Ana Cristina Victorino Krepischi8, Chong Ae Kim3, Ivo Jorge Prado Arnhold2 and Alexander Augusto de Lima Jorge1

1Unidade de Endocrinologia Genética (LIM25), 2Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular (LIM42), Hospital das Clínicas da Faculdade de Medicina, Universidade de São Paulo (USP), São Paulo, Brasil, 3Unidade de Genética do Instituto da Criança, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brasil, 4Unidade de Neuroendocrinologia, 5Unidade de Doenças Osteometabólicas, Hospital das Clínicas da Faculdade de Medicina, Universidade de São Paulo (USP), São Paulo, Brasil, 6Unidade de Endocrinologia Pediátrica, Correspondence Departamento de Pediatria, Irmandade da Santa Casa de Misericórdia de São Paulo, São Paulo, Brasil, 7Division of should be addressed Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan, Ann Arbor, to A A L Jorge Michigan, USA, and 8Instituto de Biociências (IB), Universidade de São Paulo (USP), São Paulo, Brasil Email [email protected]

Abstract

Context: Patients with tall stature often remain undiagnosed after clinical investigation and few studies have genetically assessed this group, most of them without a systematic approach. Objective: To assess prospectively a group of individuals with tall stature, with and without syndromic features, and to establish a molecular diagnosis for their growth disorder. Design: Screening by karyotype (n = 42), chromosome microarray analyses (CMA) (n = 16), MS-MLPA (n = 2) targeted panel (n = 12) and whole-exome sequencing (n = 31).

European Journal of Endocrinology Patients and methods: We selected 42 patients with tall stature after exclusion of pathologies in GH/IGF1 axis and divided them into syndromic (n = 30) and non-syndromic (n = 12) subgroups. Main outcome measures: Frequencies of pathogenic findings. Results: We identified two patients with chromosomal abnormalities including SHOX trisomy by karyotype, one 9q22.3 microdeletion syndrome by CMA, two cases of Beckwith–Wiedemann syndrome by targeted MS-MLPA analysis and nine cases with heterozygous pathogenic or likely pathogenic genetic variants by multigene analysis techniques (FBN1 = 3, NSD1 = 2, NFIX = 1, SUZ12 = 1, CHD8 = 1, MC4R = 1). Three of 20 patients analyzed by WES had their diagnosis established. Only one non-syndromic patient had a definitive diagnosis. The sequential genetic assessment diagnosed 14 out of 42 (33.3%) tall patients. Conclusion: A systematic molecular approach of patients with tall stature was able to identify the etiology in 13 out of 30 (43.3%) syndromic and 1 out of 12 (8.3%) non-syndromic patients, contributing to the genetic counseling and avoiding unfavorable outcomes in the syndromic subgroup.

European Journal of Endocrinology (2020) 182, 139–147

https://eje.bioscientifica.com © 2020 European Society of Endocrinology Published by Bioscientifica Ltd. https://doi.org/10.1530/EJE-19-0785 Printed in Great Britain Downloaded from Bioscientifica.com at 09/25/2021 09:15:33PM via free access

-19-0785 European Journal of Endocrinology https://eje.bioscientifica.com of tall stature from January 2016 to June 2019 in our Genetic of tall stature from January We enrolled46children andadultsreferredforevaluation Patients andmethods andgeneticassessment. initial laboratory non-syndromic, usingtargetedpaneland/orWES,afteran cohort of patients with tall stature, both syndromic and this context,thepresentstudyprospectivelyevaluated a feasible even in cases without prior clinical diagnosis. In the geneticevaluationofpatientswithtallstaturebecame as targetedpanelandwholeexomesequencing(WES), of 50%( a multigeneanalysisapproach,reachingdiagnosticrate and overgrowth(tallstatureand/ormacrocephaly)using a selectedcohortofindividualswithintellectualdisability knowledge onlyonereporthassystematicallyinvestigated only whenaclinicalsuspicionarose( etiology of thegrowth disorder of these patients, mostly confirmation ( time andlabor-consumingoptiontoattemptdiagnosis hotspots, whichmakesanapproachbycandidategenea with generalized overgrowth are large and do not have in life ( clinically distinguishtheseconditions,especiallylater difficultto have overlappingphenotypes,makingitvery (OMIM 277590)andMalan614753)syndromes, these conditions,suchasSotos(OMIM117550),Weaver of theunderlyinggeneticetiology. However, many of unique, classicalphenotypesthatleadtotheidentification alterations, and these characteristics usually cluster in abnormalities, facialdysmorphicfeaturesandskeletal neurodevelopmental delay, macrocephaly, majorcardiac show acombinationofrelevantalterations,suchas as syndromicornon-syndromic.Syndromicpatients malformations ( teststodetecthiddencongenital complementary GH secretionleadingtoenhancedgrowth( axis, andinmostpatients,thereisnoevidenceofabnormal referred toanendocrinologicevaluationoftheGH/IGF1 to shortstaturecases,tallindividualsarecommonly height above2SDSfrommid-parental( a adjustment forgenderandagecanalsobedefinedas score (SDS)fromthemeanofaspecificpopulationafter Tall statureisdefinedasaheightabove2standard-deviation Introduction Clinical Study After thespreadofmultigeneanalysistechniquessuch Few studieswerepublishedaimingtoidentifythe After acarefulclinicalevaluationandfew 2 9 , ). 3 , 4 , 3 5 , ). Besides, most genes already associated 6 1 ). ), tallindividualscanbeclassified Albuquerque andothers E VascodeAlbuquerque 7 , 1 ). 8 , 1 9 ). Similarly ). Similarly ). To our who achievedGHsuppression glucose tolerancetestwasperformed andonlyindividuals by theassaymanufacturer. WhenIGF1SDS for ageandsexaccording to referencevaluesprovided Roche Diagnostics).IGF1levelswereexpressedasSDS by anelectrochemiluminescencemethod(Cobase601, patient sex)measurements.Allhormoneswereassessed T4, LH,FSHandtestosterone/estradiol (according tothe deceased orunavailableduetosocialproblems. of 15parents(motherand/orfather)sincetheywereeither expressed inSDS.We hadnoaccesstothemeasurements height height +mother’s ((father’s and mid-parentalheightwascalculatedusingtheformula 14 detect dysmorphicfeatures,neurodevelopmentaldelay, with expertiseindysmorphology(AALJ)orderto geneticist (RSHKorCAK)and/orbyanendocrinologist assessment andphysicalexaminationwasperformedbya and theDeclarationofHelsinki. procedures wereinaccordancewithlocalethicalstandards informed consent after careful explanation and all Patients orparents(orlegalguardians)providedwritten Committee (CAPPesq;approvalnumber1.854.859). study was approved by the Hospital das Clinicas Ethics and privateclinicsduringtherecruitmentperiod.The Eight patientswerereferredfromothermedicalspecialties endocrinology units( Genetics ( in otheroutpatientclinicsandrecruitedfromMedical Unit, andtheremainingpatientswereinitiallyfollowed patients werealready followed inGeneticEndocrinology center.Endocrinology outpatientclinicinatertiary Seven this study ( height ratioSDS Abnormal bodyproportionwasdefinedbysittingheight/ head circumference were obtainedusingameasuringtape. using age-andsex-specifiednorms( in squaremeters.MeasurementswereconvertedtoSDS as thedivisionbetweenweightinkilogramsandheight was measuredusinganelectronicscale.BMIcalculated calibrated stadiometertothenearest0.1cm,andweight 11 assessed accordingtoTanner andMarshallstages( had multiple dysmorphic features. Pubertal status was if theyshowedmultiplemajormalformationsor delay/autism spectrumdisorder/intellectualdisability, were classifiedassyndromiciftheyshoweddevelopmental of tall stature. Patients macrocephaly and family history stature patients Genetic investigationoftall ). Measures were also obtained from all available relatives ). Measureswerealsoobtainedfromallavailablerelatives ). Standing and sitting heights were measured using a ). Standingandsittingheightsweremeasuredusinga All patients were submitted to a careful clinical All patients weresubmitted to a careful clinical Laboratory work-upincludedbasalIGF1,TSH, free Laboratory n 2, eredciooy ( Neuroendocrinology =12), 15 ). Four individuals were excluded due to < − 2 orarmspan/heightratio n

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13 cm)/2) and then 13 cm)/2)andthen g/L wereincludedin 182 , 13 :2 n ). Arm span and ). Armspanand ) n other and =9) > 2, anoral > 1.05 ( 140 10 via freeaccess 2 , , European Journal of Endocrinology clinical suspicion( confirmed intargetedpanelsequencingguidedbyinitial in syndromicpatientswhosegeneticdiagnosiswasnot patients withoutastrongclinicalsuspicion( non-syndromic patients( suspicion ( those syndromic patients who presented a strong clinical performed intheDNAsamplefromindexcasefor standard procedures.Targeted panelsequencingwas leukocytes fromallpatientsandavailablerelativesusing Genomic DNA was isolatedfrom peripheral blood Targeted panelandwholeexomesequencing work-up( laboratory depending onadiagnosticsuspicionafterclinicaland 37 patientswereincludedfortargetedpaneland/orWES, sequencing technique.Afterinitialgeneticevaluation, variations priortotheselectionforamassiveparallel 4x180K (AgilentTechnologies, Inc.)toassesscopynumber using theplatformSurePrintG3HumanCGHMicroarray chromosome microarray analyses (CMA) was performed chromosomal aberrations and, in 16 syndromic patients, wrist usingGreulichandPylemethod( age wasassessedbyX-raysofthenon-dominanthandand lentis and anabdominalultrasoundinordertoruleout to anophthalmologicalevaluation,echocardiogram without appropriatetreatment.Subjectsweresubmitted patient who achieved an adult height SDS were alsoexcluded,exceptforonesyndromicfemale gigantism. Children with precocious puberty pituitary Clinical Study Karyotype wasperformedinallprobandstoassess Karyotype , major malformations and aortic dilatation. Bone n

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DNMT3A PubMed databases: the Online Mendelian Inheritance in Man (OMIM) and cited inpublisheddatauntilthebeginningof2016 disorders, 30ofthemrelatedtoovergrowthdisorders including 513genesrelatedtomiscellaneousendocrine (Agilent SureSelectXTassay;AgilentTechnologies) WES wasperformedonlyintheindexcase. with a non-affectedsister. In theremaining 22patients, relative, andonesyndromicpatientwasassessedcombined an affectedbrotherandtwootherswiththeirunaffected One non-syndromicpatientwasanalyzedtogetherwith in fivepatients(threesyndromicandtwonon-syndromic). Exon, version 6 (AgilentTechnologies). Sequencing was were preparedaccordingtotheSureSelectHumanAll SHOX NPR2 IGF1R locations and with at least ten reads. Subsequently, we that werelocatedinexonicregionsandsplicesiteconsensus ABraOM public (gnomAD, We selectedrarevariants(minorallelefrequency Variant assessment with thehumanreferenceassembly(GRCh37/hg19). as previouslyreported( end mode.In-house bioinformatic analysiswasperformed 2500 (Illumina)platformfortheexome,bothinpaired- USA) platformforthetargetedgenepanelandaHiSeq performed usingaNextSeq500(Illumina,SanDiego,CA, stature patients Genetic investigationoftall We developedacustomizedtargetedpanelsequencing , , , SOCS2 NPR3 IGF2 http://abraom.ib.usp.br/ , ESR1 , , , KCNQ1OT1 NSD1 SOST , WES, wholeexomesequencing). ligation-dependent probeamplification; microarray analysis;MLPA,multiplex with tallstature(CMA,chromosomal study toinvestigateagroupof patients Flowchart oftheapproachusedinthis Figure 1 http://gnomad.broadinstitute.org/ EZH2 , , TGFB3 PDGFRB AKT1 17 Downloaded fromBioscientifica.com at09/25/202109:15:33PM , , ). Thesequenceswerealigned FBN1 , MC4R and CBS , https://eje.bioscientifica.com PIK3CA , ZBTB20 ) and in-house databases ) andin-housedatabases , , FBN2 MED12 CDKN1C 182 , , :2 . Exomelibraries PTEN FGFR3 , NFIX , , CYP19A1 ≤ SHANK3 , 0.1%) in 0.1%) in , NPPC GPC3 141 and and via freeaccess , , , , European Journal of Endocrinology https://eje.bioscientifica.com at thattime. of themhadalreadyreached fullpubertaldevelopment comparison withtheirchronological age;however, one Two ofthemhadadvanced boneagearound2yearsin median BMISDSofthesefive patientsis1.2( classified assyndromic.Allofthemwerechildrenandthe after theoralglucosetolerancetest,andallofthemwere with GH suppression to less than 0.4 µg/L in all cases mean adultheightSDSof3.4 initial assessmentorachieveditduringfollow-up,with a patients hadalreadyreachedtheiradultheightinthe ranged from4.4to53years,witheightadults.Seventeen group ( higher target height SDS ( as expected.Incontrast,thenon-syndromicgrouphada malformations ( ( showed ahigherprevalenceofabnormalbodyproportions and non-syndromic(12individuals).Syndromicpatients two differentgroups( the cohortof42patientswithtallstaturewasclassifiedinto gigantism, After theexclusionoffourpatientswithpituitary Clinical characterizationandinitialassessment Results significance wasassumedat U groups were tested by Student’s Windows, version 3.5 (SPSSInc.). Differences between the variableswereconductedbyusingSigmaStatfor Descriptive andcomparativestatisticalanalysesbetween Statistical analysis Sequence Variants ( the ACMG/AMPGuidelinesforInterpretationsof variants identified. All variants were classified following and segregationwereperformedtovalidatethecandidate OMIM® andthePubMeddatabases.Sangersequencing calls. Dataregardinggenefunctionwascollectedusing Integrative GenomicsViewer (IGV)toreducefalse-positive we visuallyinspectedthesequencingreadsusing be pathogenicbymultiple frameshift variants)andmissensevariantspredictedto of-function (LoF)variants(stop-gain;splicesitedisrupting; ranked genesbasedontheirpathogenicitypotential:loss- P testandFisherexacttest,asappropriate.Statistical

Clinical Study = 0.016), developmental delay ( Five outof42patientsshowedIGF1levels Table 1 ). The chronological age at the first visit ). Thechronologicalageatthefirstvisit P

= 0.004) than the non-syndromic group 0.004) thanthenon-syndromicgroup 18 ). Fig. 1 P ): syndromic (30 individuals) ): syndromic(30individuals) in silico

P = ±

< 0.039) than the syndromic 0.039) than the syndromic 0.9. 0.05. t Albuquerque andothers E VascodeAlbuquerque -test or Mann–Whitney programs. Thereafter programs.Thereafter P 0.003) and major major and =0.003) − 0.1 to1.8). ≥ 2 SDS, were syndromic.TheirmedianBMISDSis0.8( (three youngadultsandtwochildren),allofthem presence ofseveretallstature(heightSDS of tall stature, body disproportion, macrocephaly or the presence of intellectual disability, familial history without positivegeneticfindingsdidnotdifferregarding patients issummarizedin (33.3%). The clinical presentation of the diagnosed An etiologicaldiagnosiswasobtainedin14patients Molecular geneticfindingsinthecohort in theother). posterior liverfibrosisinoneandmultiplehepatictumors atresiacorrectedintheneonatalperiodwith (biliary conditions that could explaintheirhormonal alterations their chronologicalageandbothpresentedseverehepatic 1.9), andbothchildrenhadaboneagecompatiblewith region encompassingthe rearrangement results in trisomy of the pseudoautossomic 46,X,psu idic(X)(pter- amenorrheaand syndromic femalepatientwithprimary andone syndromic malepatientwitha47,XYYkaryotype anon- chromosomal abnormalitiesbykaryotype: vs non-syndromic1/12,8.3%; rate ofpositivegeneticfindings(syndromic13/20,43.3% patients classifiedassyndromictallstaturehadahigher to multigenesequencing analysis (24.3%) ( pathogenic ( robust clinicalsuspicion. syndrome. Fourteensyndromic patientshadnoinitial suspected Weaver syndromeandoneofPTEN-hamartoma five patientshadsuspectedSotossyndrome,two syndrome withoutfulfillingtheGhentcriteria( four ofthemhadclinicalfindingsthatresembledMarfan approaches ( non-syndromic) werestudiedbymultigenesequencing for BWS/SRSregions. (BWS) wasconfirmedintwomalepatientsusingMS-MLPA the clinicaldiagnosisofBeckwith–Wiedemann syndrome x1), whichmightcauseovergrowth( syndrome (arr[GRCh37]9q22.32(97205649_98985605) was diagnosedwiththeknown9q22.3microdeletion malformations wereevaluatedbyCMA,andonepatient delay, autismspectrumdisorderand/ormajor stature patients Genetic investigationoftall Likewise, fivepatientshadIGF1levels The remaining37patients(26syndromicand11 Further, 16syndromicpatientswithdevelopmental work-up,twopatientsshowed After initiallaboratory We identifiednine pathogenic( n Fig. 1

= 5) variantsin9outof37patients submitted ). Amongthe26syndromicpatients, > Downloaded fromBioscientifica.com at09/25/202109:15:33PM q21:q21- SHOX Table 2 P gene( =0.036). > pter) karyotype; this pter) karyotype; 182 . Patientswithor Fig. 1 19 :2 n ). Additionally, ) r likely or =4) > and 3), although ≤ Table 3 Table 2 − − 2 SDS 1.7 to 142 14 via freeaccess ). ). ),

European Journal of Endocrinology * IGF-1 SDS IGF-1 SDS Head circumferenceSDS–height Sitting height:heightratioSDS Large forgestationalage–weight(%) Large forgestationage–length(%) IGF-1 SDS Consanguinity (%) Major malformation(%) Intellectual disability/neurodevelopmentaldelay(%) Macrocephaly (%) Head circumferenceSDS Abnormal bodyproportions(%) Birth lengthSDS Gestational age(weeks) Familial tallstature(%) Target heightSDS– Target heightSDS Height SDS endocrine evaluationtorule outalterationsintheGH/ Patients with tall stature are commonly referred for an Discussion syndrome aftermultigeneanalysisapproach( and onepatientwasreclassifiedfrom Weaver toSotos were reclassifiedtoMalansyndromeand Two patientswithaninitialdiagnosisofSotossyndrome five Sotossyndromeandoneoutoftwo Weaver syndrome. three outoffoursuspectedMarfansyndrome,one of Regarding theinitialclinicalsuspicion,weconfirmed All sequencing in causing. Sixvariantswereidentifiedbytargetedpanel variants ofwhichfourwerealreadydescribedasdisease with threeprotein-truncatingvariantsandsixmissense phenotype ( found ingenesalreadyassociatedwithanovergrowth ( and wereabsentorhaveanextremelylowfrequency identified in9outof26patients(34.6%). group, pathogenicandlikelyvariantswere non-syndromic patients.Consideringonlythesyndromic No pathogenicorlikelyvariantswerefoundin Age (years) Sex (M/F) Characteristics parent, siblingorchildwithheightSDS Table 1 < P

Clinical Study < 0.1%) inpublicdatabases( FBN1 0.05 incomparisonwiththesyndromicgroup. All variantswereidentifiedinheterozygousstate variantsfulfilledGhentcriteriaforcausality( Clinical characteristicsofpatientswithtallstatureenrolledinthisstudy.Familialwasdefinedbyhavinga ≤ ≥ − 2 (%) 2 (%) FBN1 FBN1 , NSD1 ( n =3), , NFIX NSD1 Table 3 , SUZ12 Albuquerque andothers E VascodeAlbuquerque ( n ≥ ). Thesevariantswere ) and =2) 2. Patientswithpituitarygigantism werepreviouslyexcludedfromthe analysis. , CHD8 CHD8 and NFIX 20 mutation, , 21 MC4R ( , n 22 =1). 14 − − Total 39.2 15.5 ). 0.3 1.6 1.5 1.0 0.2 2.9 0.3 3.2 ). ), 14 (33) 15 (35) 11 (26) 17 (40) 23/19 5 (12) 7 (16) 8 (19) 5 (12) 5 (12) 3 (7) ± ± ± ± ± ± ± ± ± ± ( n 1.5 1.6 1.6 1.3 1.9 1.6 1.0 1.0 0.8 11.1 = 42) IGF1 axiswereruledoutinall ofthem.We couldestablish the initialevaluation,andpathologies affectingtheGH/ multifactorial inheritance( non-syndromic tallstaturemaybearesultofpolygenic, (1/12,8.3%),whichreinforceskaryotype thenotionof The exceptionwasanon-syndromicmalewith47,XYY of thediagnosedpatientsweresyndromic(13/30,43.3%). to avoidmisinterpretationandmisdiagnosis.Allbutone strong evidenceofpathogenicitywerereported,inorder guided byACMG/AMPguidelines,andonlyvariantswith analysis inmultigenesequencingapproachwascarefully approach intheremainingninecases.Thevariant BWS intwo,CMAone,andmultigenesequencing intwocases,specificmethylationanalysisfor karyotype ( basis of tall stature was established in 14 (33.3%) of them gigantism.Themolecular after theexclusionofpituitary endocrineoutpatientclinic stature referredtoatertiary aortic dissectioninMarfansyndrome. sometimes tounfavorable,preventableoutcomessuchas families areoftendeprivedofproperfollow-up,leading of thegrowthdisorderetiologyisusuallyinterruptedand the diagnosis of GH excess is ruled out, the investigation present atypicaland/ormildsyndromicphenotypes.After IGF1 axis.Usuallythesepatientsarenon-syndromicor stature patients Genetic investigationoftall Table 2 Five syndromicpatientsshowed IGF1SDSlevels In thisstudy, weevaluated42patientswithtall ). Thediagnosiswasobtainedbyconventional Syndromic − − 39.2 15.1 0.3 1.5 1.5 1.5 0.0 3.1 0.1 3.2 14 (46) 15 (50) 10 (33) 17/13 5 (16) 9 (30) 7 (23) 6 (20) 4 (13) 5 (16) 2 (6) ± ± ± ± ± ± ± ± ± ± 1.6 1.6 1.7 1.3 2.0 1.4 1.0 1.0 0.8 11.3 ( Downloaded fromBioscientifica.com at09/25/202109:15:33PM n = 30) 1 , 23 https://eje.bioscientifica.com ). Non-syndromic 182 :2 − − 39.0 16.3 0.3 1.9 1.4 0.4 0.6 2.5 0.8 3.4 2 (16) 2 (16) 7 (58) 0 (0)* 0 (0)* 0 (0) 1 (8) 0 (0) 1 (8) 0 (0) 6/6 ± ± ± ± ± ± ± ± ± ± 1.3 1.4 1.3 0.9* 1.9 2.2 1.0 0.7* 0.8 11.0 ( n = 12) 143 ≥ 2 at 2 at via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com overgrowth disorderevenmore astonishing. could explaintheirIGF1alteration, whichmakestheir latter group,twopatientshad severehepaticdiseasethat diagnosis: had IGF1 SDS levels ( the identificationof the moleculardiagnosisinfouroutofthesefivecases,with age; NA,notavailable;SGA,smallforgestationalSH:H,sittingheight/height. AGA, appropriateforgestationalage;BWS,Beckwith–Wiedemannsyndrome; HC,headcircumference;ID,intellectualdisability;LGA,largeforgestational *Variants identifiedintargetpanelsequencing. 14 13 12 11* 10* 9* 8* 7* 6* 5 4 3 2 1 N Table 2 n

Clinical Study = 1) mutations.Curiously, fiveothersyndromic patients MC4R CHD8 SUZ12 NFIX NSD1 NSD1 FBN1 FBN1 FBN1 11p15 11p15 9q22.3 SHOX SHOX finding Molecular trisomy trisomy Variants andclinicalcharacteristicsoftheindividualsdiagnosedbyanymethodology.

NFIX ( n Sex M M M M M M M M ) and =1) F F F F F F ≤ − FBN1 2, and two patients had the molecular 2, and two patientshadthemolecular SGA AGA LGA AGA LGA AGA LGA NA AGA LGA LGA NA NA LGA birth At

( NSD1 n =2), Age 10.3 15.0 20.4 11.4 10.7 53.9 13.3 13.3 18.6 18.3 ( Albuquerque andothers E VascodeAlbuquerque 5.0 5.4 7.7 7.3 (year) CHD8 n

= 1) mutations. In the 1) mutations.Inthe ( Height n SDS 2.1 5.6 4.1 3.1 3.7 2.0 4.4 3.7 2.5 3.5 3.0 3.4 3.4 4.7 ) and =1) − − BMI SDS 0.3 1.6 2.2 0.3 1.9 1.0 3.7 1.1 1.2 2.1 0.2 1.2 1.5 0.1 MC4R − SDS 0.5 0.5 0.9 4.6 1.0 1.9 3.6 3.5 3.8 3.3 0.8 1.1 NA HC

0.8 intellectual disability andanovergrowth condition ( al et 34.6%, lower than the 50% rate found by Tatton–Brown to targetedpaneland/orWES,thediagnosticratewas population inthatstudyhad onlymacrocephaly, andthe the literature.Itisnoteworthy that15.3%oftheselected To ourknowledge, itwastheonlysimilarstudyfoundin stature patients Genetic investigationoftall − − − − − − − − − − − SH:H SDS 0.8 0.7 NA 1.1 3.1 1.0 0.8 1.6 0.6 0.0 1.9 1.2 2.7 3.3 Considering only the syndromic patients submitted . inasystematicinvestigationof710patientswith No No No No No No No ID No Yes Yes Yes No Yes No IGF-1 − − − − − − SDS 0.7 2.0 1.4 0.6 0.2 0.1 0.6 2.5 2.0 1.0 2.1 0.1 2.6 2.2 Aortic dissection(53 Ectopia lentis Macroglossia, Macroglossia, Frontal bossing, Primary Normal sexual features Additional clinical Teeth agenesis Precocious puberty Mild Weaver Sotos syndrome Mild Sotos Giant hepatic Mild facialfeatures omphalocele splenomegaly umbilical hernia, 12th ribs jaws, hypoplastic keratocyst of hypogonadism levels testosterone normal development and syndrome hypoplastic nails strabismus, phentotype, phentotype syndrome syndrome Weaver suspected adenoma, aneurism with aortic and grandmother years) Downloaded fromBioscientifica.com at09/25/202109:15:33PM

182 :2 Marfan Marfan BWS BWS 9q22.3 Chromosomal Chromosomal Final diagnosis Monogenic Autism Weaver Malan syndrome Sotos syndrome Sotos syndrome Marfan syndrome syndrome microdeletion trisomy with abnormality trisomy with abnormality obesity 18 susceptibility syndrome syndrome syndrome SHOX SHOX 144

9 via freeaccess ). European Journal of Endocrinology MAF, minorallelefrequency;NA,notavailable. described aspathogenic( previously describedinacasereport( confirm theinitialclinical hypothesis,butprovidesa major importanceinthesecases. and thedetectionofapathogenic variantin criteria dependingontheirageand/ormildphenotype, patients withMarfansyndromemaynotfulfilltheclinical the occurrenceofanaorticdissection( by screeningofpossibleaffectedfamilymembersprior to analysis wascrucialforaconclusivediagnosisfollowed criteria werenotachieved.Therefore,the it was clinically suspected in these patients, the Ghent syndrome wasestablishedinthreepatients.Although rare causeofovergrowth. patients, whichsuggeststhat SUZ12 macrocephaly) andintellectualdisability. Interestingly, of this syndrome are overgrowth (tall stature and/or result since the two of the classical three cardinal features most commondiagnosis,in34%ofthecases, an expected intellectual disability. Similarly, Sotossyndromewasthe cohort, sincethatreportcomprisedonlyindividualswith surprising that differ fromenhancedlongitudinalgrowth( genetic causesunderlyingisolatedmacrocephalymay College ofMedicalGeneticsandGenomicstheAssociationforMolecularPathology( *GRCh37/hg19. 14 13 12 11 10 9 8 7 6 N Table 3 Clinical Study In thisstudy, thegeneticdiagnosisof Marfan In somecases,themolecular investigationdoesnot MC4R CHD8 SUZ12 NFIX NSD1 NSD1 FBN1 FBN1 FBN1 Gene mutation was not identified among those 710 Genetic diagnosisobtainedbytargetedpanelandwholeexomesequencinginnineindividualswithtallstature. ¶ ¶ ¶ ¶ ¶ ¶ † Pathogenic predictionusing5insilicotools:SIFT,PolyPhen-2,MutationTaster,CADDandMETASVM. Chr18: Chr14: Chr17: Chr19: Chr5: Chr5: Chr15: Chr15: Chr15: coordinate Genomic FBN1 58039075 21881143 30323819 13136151 176637009 176715908 48712949 48707746 48726904 22 mutationswerenotfoundintheir , 23 * ). 21 SUZ12 c.508A c.2154dupC:p. c.1797A c.344G c.1610_1611del:p. c.6242dupT:p. c.7754T c.8038C c.6503A Variant ). ^Lossoffunctionvariantsinthesegenesareawell-establishedmechanismdisease. Ile170Val Phe719Leufs*2 Gln599His Arg115Gln Phe537Tyrfs*7 Leu2081Phefs*32 Ile2585Thr Arg2680Cys Asp2168Gly Albuquerque andothers E VascodeAlbuquerque mutations are a very mutationsareavery > > * > > > > G:p. A:p. C:p. T:p. C:p. G:p. 14 § ). Besides,some # § § § 9 ). Itwasnot FBN1 FBN1 rs121913560 rs727503054 gene isof dbSNP NA NA NA NA NA NA NA surprisingly had a previouslydescribedpathogenic variant referred for evaluation of tall stature and teeth agenesis syndrome ( case andestablishingthisgene asararecauseofWeaver syndrome phenotype,representingthesecondpublished One patientharboreda less than100casesdescribedintheliterature( the diagnosis of Malan syndrome, a rare condition with harbored a syndrome andpreviousnegativeanalysisof 24 these twomedicalconditionsintheclinicalpractice( and exemplifyingthechallengeofdistinguishingbetween in targeted panel sequencing revealed a frameshift mutation lesions inhisliver. Despitetheinitialhypothesis, a gianthepaticadenomaandwithmultipleremaining levels, probablybecauseofapartialhepatectomydueto was oneofthesyndromicpatientsthatshowedlowIGF1 with genetic and endocrinological teams. Remarkably, he a clinicaldiagnosisofWeaver syndromeafterconsultation intellectual disability, camptodactyly and loose skin, with boy with tall stature (height SDS 2.0), macrocephaly, different etiology. One ofthe patients was a 6-year-old stature patients Genetic investigationoftall ). Likewise,a22-year-oldwomanwithsuspectedSotos NSD1 0.00008843 0.00001594 WES canalsounravelneworunexpectedfindings. 18 MAF gene, changing hisdiagnosis to Sotos syndrome 0 0 0 0 0 0 0 ). ¶ Variants identifiedintargetpanelsequencing. de novoNFIX 20 ). Ontheotherhand,a 9-year-old boy prediction In silico 2/5 4/5 5/5 4/5 5/5 5/5 – – – Downloaded fromBioscientifica.com at09/25/202109:15:33PM mutationandthereforereceived

† SUZ12 NA NA^ Absent inthe De novo NA^ NA^ Inherited NA De novo Inheritance ‡ mother) (deceased father mother from affected ACMG/AMP –TheAmerican https://eje.bioscientifica.com mutationandWeaver

182 § Variants previously :2 Likely Pathogenic Likely Likely Pathogenic Pathogenic Likely Likely Pathogenic Classification NSD1 pathogenic pathogenic pathogenic pathogenic pathogenic 25 # Variant ).

gene 145 via freeaccess 6 ‡ , European Journal of Endocrinology https://eje.bioscientifica.com References for theirassistancewithwholeexomeandtargetedpanelsequencing. Escala Larga em Sequenciamento de Laboratorio the thank authors The Acknowledgments (CNPq). L J) from the National Council for Scientific and Technological Development São Paulo Research Foundation (FAPESP) and Grant 301871/2016-7 (to A A the from Escala) Larga em Sequenciamento de Laboratório the – (SELA 5 This work was supported by grants 2013/03236-5 (to A A L J) Funding and 2014/50137- be could that interest of conflict perceived asprejudicingtheimpartialityofthisstudy. no is there that declare authors The Declaration ofinterest especially inapolygeniccontext. a larger number ofthese patients could beenlightening, patients remainsuncertainandperhapsanassessmentof benefit ofagenetic investigationofnon-syndromic review, andourresultsendorsethisrationale( This approachhasbeenrecentlyadvisedinasystematic regardless of whether they have a clinical suspicion or not. investigation ofallsyndromicpatientswithtallstature, multigene analysisshouldbeincluded in thesystematic family members. Based on our results, we suggest that could bringseriouspreventableoutcomesinaffected genetic counseling,mainlyforMarfansyndrome,which patients wouldremainundiagnosedandwithoutproper work-up.Manyofthose an initialclinicalandlaboratory molecular diagnosesofninepatientswithtallstatureafter agenesis remainsunclear. could explain his height, but the etiology of his teeth normal weightrange(BMISDS+1.3).Thevariantfound rapidly gained 5 kgin months, although he isstillat was +1.2,butafterhestartedusingdentalprosthesis, previously published( heterozygous mutationsin the prevalenceofearly-onsetobesityinpatientsharboring both developedobesityafterpuberty( was found in a mother and her affected daughter, who reduced receptor activation ( in 2 1 Clinical Study MC4R Lauffer P, Kamp GA,Menke LA,Wit JM, Oostdijk W&onbehalf Albuquerque EVA, Scalco RC &Jorge AAL.MANAGEMENTOF of theDutchWorking GrouponTriage andDiagnosisofGrowth (https://doi.org/10.1530/EJE-16-1054) tall stature. ENDOCRINE DISEASE:Diagnosticand therapeuticapproachof In thisstudy, targetedpanelandWESallowedthe , with a functional study showing a markedly European Journal ofEndocrinology European Journal 26 ). Ourcase’s initialBMISDS 22 MC4R ). In that study, the variant Albuquerque andothers E VascodeAlbuquerque was68%inacohort 2017 22 ). Additionally, 176 R339–R353. 2 ). The

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Received 2October2019 Accepted 15November2019 Revised versionreceived11November2019

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