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European Journal of Endocrinology -18-0430 transmitted withautosomal dominantinheritancethat #131100) isarareandcomplex inheritedcancersyndrome Multiple endocrine neoplasia type 1 (MEN1, OMIM Introduction Also, tNGSprovedtobeahighlyeffective technology forroutinegenetic Conclusions: variants weredetected. or shortindelpathogenicvariantswerefoundinuntranslated, although33benign/likelybenignandthreenewVUS detected in16patients.Inuntranslated,regulatoryordeepintronic including 13newandsixrecurrentvariants.ThreelargedeletionsweredetectedbyMLPA only. Nomutation was tNGS andSS(100%reproducibility).Thirty-eightdifferent pathogenicorlikelyvariantswereidentified, Results: applied whennomutationswereidentifiablebybothtNGSandSS. were validatedbySangersequencing(SS),andmultiplexligation-dependentprobeamplification(MLPA) assaywas developed toinvestigategermlinemutationsin76unrelatedMEN1probands(49familial,27sporadic).tNGSresults Methods andpatients: determine themutationprofile. Objective: of theremainingpatients;however, thishypothesishasnotyetbeenfullyinvestigated. neoplasia type1(MEN1).Ithasbeenpostulatedthatmutationsinnon-codingregionsof Background: Abstract Sao Paulo,Brazil Instituto doCancerEstadodeSaoPauloICESP, FaculdadedeMedicina,UniversidadeSaoPaulo, Bethesda, Maryland,USA, 5 Ann Arbor, Michigan,USA, 3 Hospital dasClinicasHCFMUSP, FaculdadedeMedicina,UniversidadeSaoPaulo,Brazil, 2 1 Stephen J Marx Tomoko Sekiya Rafael A Carvalho next-generation sequencing endocrine neoplasiatype1usingfullgene Germline mutationlandscapeofmultiple Endocrinology, EuniceKennedyShriverNationalInstituteofChildHealthandHumanDevelopment(NICHD), Division ofMetabolism,DepartmentInternalMedicine,EndocrinologyandDiabetes,UniversityMichigan, Unidade deParatireoide,LaboratorioCirurgiaVascular edaCabeçaPescoçoLIM-28,DisciplinadeCirurgiaPescoço, Unidade deEndocrinologiaGeneticaUEG,LaboratorioCelulareMolecularLIM-25,DisciplinaEndocrinologia, https://doi.org/ https://eje.bioscientifica.com Clinical Study Germline To sequencefortheentire 10.1530/EJE Ourstudydocumentsthatpointorshortindelmutations in non-codingregionsof Loss-of-functiongermline 1 1 , Viviane C Longuini , 5 MEN1 , Rodrigo A Toledo -18-0430 1 , Betsaida Urtremari 6 Vall d’HebronInstituteofOncology, Barcelona, Spain,and 4 Endocrinology Division,FederalUniversityofSaoPaulo(UNIFESP),SãoPaulo,Brazil, Atargetnext-generationsequencing(tNGS)assaycomprising7.2 variantswereverifiedincodingregionandsplicingsitesof57/76patients(74%)byboth 6 © 2018EuropeanSociety ofEndocrinology R ACarvalhoandothers MEN1 1 6 , Fabio L M Montenegro and MEN1 1 Printed inGreatBritain , Alexander A L Jorge includingpromoter, exonsandintronsinalargeMEN1cohort Delmar M Lourenço Jr genemutationsaccountfor75–95%ofpatientswithmultipleendocrine frequencies indifferent series, parathyroidtumors 20 endocrineandnon-endocrine tumors.Despitevariable predisposes individualstothe developmentofmorethan Targeted NGSinMEN1 Published byBioscientifica Ltd. 2 1 , Antonio M Lerario , Lucas S Santana 7 Disciplina deEndocrinologia, 1 , 7 MEN1 MEN1 testing. regionsofthe76MEN1cases,nopoint 1 Downloaded fromBioscientifica.com at09/28/202104:26:31AM , Elisangela P S Quedas 3 , Sergio P A Toledo kb ofthefull MEN1 MEN1 (2018) Endocrinology European Journal of [email protected] Email to DMLourenço should beaddressed Correspondence mightoccurinsome are very rare events. areveryrareevents. 179 179 :6 MEN1 , 391–407

1 was , 391 4 , 1 , –407 via freeaccess

European Journal of Endocrinology https://eje.bioscientifica.com and preimplantationgenetic diagnosis.Therefore, from long-termperiodicfollow-up andallowsprenatal status ofat-riskfamilymembers, rulesoutnon-carriers the MEN1indexcase,defines thecarrierandnon-carrier highly usefulbecause it confirmstheclinicaldiagnosis of ligation-dependent probeamplification(MLPA) ( and areusuallyinvestigatedusingassaysasmultiplex the differentmutationtypes,occurin1%ofMEN1cases 8 and frequentlyinterfereinmenin–proteininteractions( whereas splice-siteandin-frameindelsarealsorepresented represent onequarterofthedifferentmutationstypes, to predispositionfortumorformation.Missensevariants becomes unable to suppress transcription factors, leading located attheC-terminalsegment( at leastoneoutofthethreenuclearlocalizationsignals (56–64%), whichpredicttruncatedmenin-proteinmissing ( CDKN2C/p18 CDKN1A/p21 may harborgermlinemutationsinthe MEN1 mutations and5–10%arephenocopies( the codingregion,10%ofcasesaredueto to 5–25%ofpatientsmaynotharbor phenotypes ( MEN1 division andproliferation( proteins and regulates transcription, genome stability, cell acid proteinmenin,whichinteractswithatleast12 1830 more thantwoMEN1-relatedtumors( older atthetimeofdiagnosis,livelongerandexhibitno Compared tofamilialMEN1,sporadiccasesareusually tumors and patients havea shorter life expectancy ( causes of death are malignant PETs and thymic carcinoid ( clinical variabilityhavebeenreportedinMEN1cases genotype andphenotypeaswellawideintra-familial clinical diseaseat50 years. Absenceofcorrelationbetween tumor upto20 years ofageandalmostallcaseswillpresent suppressor genewilldevelopatleastoneMEN1-related germlinemutationsinthe patients carrying endocrine tumorsinMEN1.Itisestimatedthat50%of endocrine tumors(PET)(41–100%),arethemostprevalent (PIT)(19–65%)andpancreatoduodenal addition topituitary (82–100%) leading to hyperparathyroidism (HPT), in 1 20 , , Clinical Study 19 , 2 The identificationof Most MEN1 21 , , bp inthecodingregionthatencodes the 610amino -negative carrierswithMEN1-likephenotypes mutationsareresponsiblefor75–95%ofMEN1 23 3 , , 22 ). Inturn,grossdeletionscorrespondto2.5%of 4 MEN1 (OMIM#613733)isa10-exongenewith ). , 5 5 , , 6 6 mutationsareframeshiftandnonsense , , 7 8 , , 15 8 , , 9 16 , 5 10 , MEN1 , , CDKN1B 6 17 , , 8 , 11 ). Germlineheterozygous 18 R ACarvalhoandothers germlinemutationis ). Presently, themajor , 19 19 / MEN1 14 p27 , ). Importantly, up 23 5 ). ). Rarely(3.5%), and ). Thus,menin de novoMEN1 CDKN2B/p15 mutationsin MEN1 AIP 19 12 tumor genes , MEN1 , 23 13 ). 5 ). ). , ,

of life and survival rate( of lifeandsurvival that hasaconsistentpositiveimpactonmorbidity, quality clinical diagnosisandtreatmentofMEN1-relatedtumors 9 MEN1 mutations inseveralsporadic tumorsbeyondbiallelic confirming thepresence of occasionalsomatic the WES andtNGSsequencing( cancersyndromes,such asWGS, adopted forhereditary practice todayanddifferentNGSstrategieshavebeen are required( robust andaffordablestrategiesasNGStechnologies throughput andgreatercapacityofdatageneration, 10 regions, preventingsystematicstudiesonthistopic( 28 regions( untranslated orregulatory mutations inintronsfarfromintron/exonboundaries, MEN1 patients withnomutationinthecodingregionof laborious andexpensivetechnique.Moreover, inMEN1 hotspots makingSSaburdensome,time-consuming, the technology haslimitationstoachievethispurpose.Thus, performed inliterallyallcasesfulfillingcurrentcriteria,SS 28 Sanger sequencing(SS)andMLPA assays( 6 of casestobegeneticallytested( identified atayoungage,adding substantialnumber phenotypes ( ( and multiplePETatanyage;parathyroidhyperplasia such asmultiglandularparathyroiddisease,gastrinoma for severalsporadiccaseswithMEN1-relatedtumors, relatives ( phenotype andsymptomaticorasymptomaticfirst-degree mutation testingfor cases farbeyondtheclassical MEN1 should notbeunderestimated( contrast, detrimentalconsequencesofdelayeddiagnosis andtherapeuticstrategies( surveillance testing substantiallyoptimizesgeneticcounseling, associated withHPT, PITmacroadenomasorPETs ( for earliertreatment,avoidingcomorbiditiesasthose be pursuedasgeneticdiagnosisprovidesanopportunity Targeted NGSinMEN1 < , , 40 years); recurrentHPT;40 years); severalatypicalMEN1-related 7 , , , 10 , 29 13 So far, the fewavailable NGS studies evaluating MEN1 Current MEN1guidelinesrecommend 29 MEN1 MEN1 8 , inactivationinparathyroid tissuesfromMEN1 gene,ithasbeenpostulatedtheexistenceof , , , 11 , 9 30 18 30 , , 10 ). However, SS maynotusuallycover such gene testinghasbeenusuallyperformedusingboth 1 genehaveappliedWESor WGSapproaches, 12 geneisrelativelylargeand lacks mutational , ). Although genetic testingshouldbeideally , 19 , , 5 5 11 31 24 , ). Genetictestingshouldbealsooffered ) and,morerecently, PITmacroadenoma 23 , , ). Earlygeneticdiagnosispromotesearly 12 32 , 28 , , 13 33 , , 29 Downloaded fromBioscientifica.com at09/28/202104:26:31AM , 14 1 , 34 , , 30 2 31 24 ). NGSisfeasibleinclinical , ). Therefore,novel,higher- , 3 , 25 32 , 25 4 ). , , , 5 26 33 26 5 , 179 , 8 ). Thisgoalshould , , , 6 34 27 10 , 1 :6 , 7 ). ). , 2 , 5 13 , , 8 3 8 , , , , 13 18 4 9 , , , , 5 13 24 3 19 MEN1 MEN1 MEN1 , , 392 , 6 5 ). In 4 , , , 23 23 , via freeaccess 8 5 8 , , , , ,

European Journal of Endocrinology patient enrolledinthisstudy. when theyclearlystatedno toberelatedanyother history. Probands were considered apparently unrelated or negative MEN1 familial and unsatisfactory/unknown of anisolatedcasewiththeclinicaldiagnosisMEN1 PET, PIT).SporadicMEN1wasdefinedasthepresence with oneormoremainMEN1-relatedtumors(HPT, index caseandatleastonefirst-degreefamilymember characterized bythecombinedpresenceofaMEN1 previously established ( 40 were enrolled, and the overall mean age at diagnosis was and 45women(meanage,41 one affectedmen(meanage,40 index cases with a clinical diagnosis of MEN1. Thirty- DNA samplesobtainedfrom76apparentlyunrelated SS assayswereconductedinperipheralbloodleukocyte given byallpatientsortheirparents.BothtNGSand (CAPPesq), andwritten,fullyinformedconsentwas The study was approved by the Local Ethical Committee Patients Patients andmethods MEN1 tNGS andSSdatatovalidatetheformertechnologyfor regions.Also,wecompared untranslated andregulatory occurring in introns far from intron/exon boundaries and and non-coding regions, searching for mutations possibly cases toinvestigatethefull negativeclinicalimpacts( carry exercised toavoiderrorsandmisinterpretationsthatmay of whichNGStechnologyisused,cautionshouldbe MEN1 syndrome( study usingthistechnologyasageneticdiagnostictoolin faster thanWESandWGS,thereisnoprevioussystematic adenoma ( germline mutationsinyoungpatientswithpituitary addition, aneight-genetNGSpanelwasappliedtosearch in sporadic pancreatic neuroendocrine tumors ( was recentlyappliedtoinvestigatesomaticmutations 39 hyperparathyroidism familyandinfiveMEN1cases( were analyzedinthreestudiesusingWES,alargeprimary patients ( Clinical Study , ± The clinical diagnosis of MEN1was defined as In thepresentstudy, weappliedtNGStoourMEN1 40 3yas (7–69 years). 13 years testinginMEN1. ). Also,a22genestNGSpanelincluding 35 42 , ). However, despitetNGSbeingcheaperand 36 , 37 8 , , 9 38 , 13 , 1 39 , , 43 ). Germline MEN1 5 ). Worthwhile, independent , 27 44 ± R ACarvalhoandothers 1 er; 96 years) 19–67 years; 11 ). Familial MEN1 was ± geneincludingcoding , 45 5 er; –9 years) 7–69 years; 15 ). MEN1 mutations 41 MEN1 ). In 38 ,

20 (a, evidence (a, grading ofevidencelevelspathogenicity:supporting families (b).Forthesegroups,differentcutoffsdefinea coming fromeitheronefamily(a)ortwomoreanalyzed to pathogenicity( for segregation,excludingtheindexcase.Thecriteria the numberofmeiosisvariantthatisinformative variant occur by chance (no pathogenic) and supported, where computational, segregationandpopulationdata( likely pathogenicandbasedonclinical, benign, variantsofuncertainsignificance(VUS), (ACMG), whichcharacterizevariantsasbenign,likely the AmericanCollege of Medical Genetics and Genomics MEN1 Classification ofvariants CDKN2C/p18 CDKN1A/p21 50 dependent probe amplification (MLPA) ( 2004 usingSSand,morerecently, multiplexligation- offering DNAgeneticdiagnosisforMEN1patientssince academichospital,wehavebeenroutinely In ourtertiary Sanger sequencing a targetinNGSandSS. mutation ( the vastmajority( b, equation In addition,toreinforce segregationdata,the (ENSG00000133895 orNG_008929.1) andtranscript USA) usingasreferences the CodonCode Alignerversion 5.0(CodonCode,MA, After dataacquisition,AB1 fileswereanalyzedusing in anABI3130xlautomaticsequencer(ThermoFisher). electrophoresis(CE) wasconducted Fisher). Capillary and sequencedusingBigDyeterminatorv3.1(Thermo purified withIllustraExoProStar1-step(GEHealthcare) /downstream) usingeightprimerpairs.Ampliconswere complete codingDNAsequence(CDS)( ( sporadic MEN1-relatedtumorsandfamilialacromegaly been analyzedinourpatientswithMEN1-likeconditions, Targeted NGSinMEN1 52 , , P , P

21 ≤ For SS, amplicons were obtained through PCR of the In MEN1patientspresentinggermlinemutations, 53 51

≤ /6 ( 1/16) genevariantswereclassifiedfollowingcriteriaof , /6 b, 1/16; , ). Beyond 22 54 , ). N 1 23 , =(1/2) 48 2 , , ). 24 P P 3

≤

≤ , , N /; b, 1/8;

4 P 43 MEN1 1/8) andstrongevidence(a, m means the probability of the observed meanstheprobabilityofobserved > , ) aredependentonsegregationdata , as defined by Jarvik ( , asdefinedbyJarvik ). Thus, we determined the 5 96%) ofcaseswillharbora , 6 Downloaded fromBioscientifica.com at09/28/202104:26:31AM , othergenesas , 7 , , P

8 ≤ CDKN1B ,

1/4), moderate evidence 13 , MEN1 14 179 , / https://eje.bioscientifica.com p27 15 genesequence :6 ± , p upstream 50 bp and 16 3 CDKN2B/p15 , , 4 17 m , 48 AIP MEN1 P , defines 46 18

18 ≤ ), was MEN1 have 393 1/32; , , , 47 49 19 as via freeaccess ). , , ,

European Journal of Endocrinology https://eje.bioscientifica.com MLPA kitP244,targeting MEN1 detect grossdeletions.Thus, MLPA wasprovided to Amplicon-based tNGSand alsoSSarenotableto amplification (MLPA) Multiplex ligation-dependentprobe data). genes usingspecificlong-rangePCR(Supplementary the panel covered the full openreading frame ofthe five CDKN1B mutations in the the sameprotocolpreviouslydefinedandoptimizedfor subset of nine available data. databases consultedarefoundinSupplementary predictive algorithmsusedfor org /ac/ Mutation Database(HGMD)( ( Catalogue ofSomaticMutationsinCancer(COSMIC) /home Variation Database(LOVD)( ( were analyzedusingsevendifferentdatabases:dbSNP of tNGSwithSS.Also,Theimpactandeffectsvariants metrics andinspectionofreadscomparativeanalysis generation ofpipelinestoproviderunningquality platform andsoftwaresappliedtodataprocessing, preparation oflibraries,sequencingusingMiSeqIllumina on cyclingconditionsandquantificationofamplicons, data instructions. chr11:64571249-64578487), followingthemanufacturer’s The ampliconsizeobtainedwas7239 GGGATACGAAGGAGAGGAAACTAGG (both 5 TAACAGACACTGATACCCAGCTAAAGC andreverse, regions). The primer sequences were forward, regulatory MEN1 Fisher) using one primer pair to amplify the entire range PCR(LongEnzymemix5 For NGS,thetargetedsequencewasamplifiedbylong- NGS librariesandsequencing transcript codesforthemainfunctionalisoformof MEN1-001 (ENST00000312049orNM_130799.2),asthis http://cancer.sanger.ac.uk/cosmic http://www.ncbi.nlm.nih.gov/SNP/ Clinical Study ) andABraOM( index.php We preliminarilyappliedafive-genepaneltostrict data(seesectionon Supplementary given at the end of this article) shows detailed data MEN1 -negative carriers.Forthisanalysis, weappliedthe gene(codingregions,introns,untranslatedand ), Ensembl( / p27 tNGSassay, aimingtoinvestigategermline and ), GnomAD ( CDKN2B/p15 AIP http://www.ensembl.org/index.html http://abraom.ib.usp.br/ genes.Asforthe MEN1 MEN1 http://gnomad.broadinstitute. , CDKN2C/p18 CDKN1A/p21 -negative carriers, following in silico http://www.hgmd.cf.ac.uk , R ACarvalhoandothers AIP ), theHumanGene http://www.lovd.nl/3.0 ), Leiden Open and analysisandclinical MEN1 bp (GRCh37/hg19 CDKN1B U/µL, Thermo supplementary supplementary ). Inparallel, tNGSassay, genes. MEN1 ′ –3 ′ ). ), . ,

Statistical analysis detected byMLPA. inNGSsequencesdeletedregions depth observed by absenceofheterozygousSNVsandloweraverageread positive resultsbyMLPA. Also,hemizygouswasreinforced SNVs onprobeligationsitesastheymaygeneratefalse sequences obtained by NGS/SS excluded the presence of consecutives ligationprobeswereinvolved.Additionally, identified by MLPA were considered as such only if two Amsterdam, TheNetherlands).grossdeletions MRC-Holland’s ownsoftware,Coffalyser(MRC-Holland, fragment electrophoresis,FSAfileswereanalyzedwith according to the manufacturer’s instructions. After DNA ladder. Runningandinjectionsparameterswereused (Thermo Fisher)usingROX500asaDNA analysis wasperformedinthesameABI3130xlCEdevice MEN1 MLPA probescoveredeachoneofthetenexons these categories were provided using Student’s as means as ageatthetimeofdiagnosishadtheirvaluesexpressed using thechi-squaredtest.Datawithanormaldistribution and associationbetweenthesecharacteristicswasverified germline mutationorpresence/absenceoffamilialhistory were describedasnumberoftumors,presence/absence software forWindows, version20.0. Categorical data Statistical analysis was performed using IBM-SPSS sporadic cases (27/76; 36%) ( (49/76, 64%)whereasthe remaining wereclassifiedas 15%) andPET/PIT(1/76;1%). associations: HPT/PET(17/76; 22%),HPT/PIT(11/76; MEN1-related tumorspresentedthefollowingtumor The remaining29patients(29/76;38%)withtwomain 62%) presentedallthreemainMEN1-relatedtumors. 86% (65/76)and;PIT, 78%(59/76).Mostpatients(47/76; MEN1 patientswas,respectively:HPT, 99%(75/76);PET, The prevalenceofmainMEN1-relatedtumorsinthe76 Clinical features Results value reliability oftNGScomparedtothegoldstandardSS.A statistic andCohen’s kappatestwereappliedtoverifythe from thesamebloodsample,percentage agreement As bothtNGSandSSassayswereperformedusingDNA Targeted NGSinMEN1 Familial MEN1wasclinically diagnosedin49patients < geneincludingtheupstreamregion.DNAfragment 0.05 wasconsideredstatisticallysignificant. ±

standard deviationandcomparisonsbetween Downloaded fromBioscientifica.com at09/28/202104:26:31AM Fig. 1 ). Age at diagnosis of 179 :6 t 394 -test. via freeaccess P

European Journal of Endocrinology CDKN1B genes bytNGSandwithnogross deletionsin CDKN2B/p15 mutation identifiedinfullopen readingframesofthe CDKN1B mutation-negative patientsbyNGSorMLPA assays( NGS or line, positive p18 CDKN1A/p21 mutations bytNGSaddressedtoCDKIs( MEN1 patients; nine and performed toinvestigategrossdeletionsinthe frame usingMiSeqIlluminaplatform;MLPA assaywas long-range PCRamplifycationofthefull cyclin-dependent kinaseinhibitors;NGSassaywasbasedon F-MEN1, FamilialMEN1;S-MEN1,sporadicCDKIs, (MLPA) assays.MEN1,multipleendocrineneoplasiatype1; (tNGS) andmultiplexligation-dependentprobeamplification index casesusingTargeted NextGenerationSequencing Flow chartappliedtogeneticdiagnosisof76unrelatedMEN1 Figure 1 harbored variantsclassifiedaslikelypathogenic,according with typicalMEN1phenotype(familialorsimplexcases) documented byMLPA ( three others(4%)harboredlargegenedeletions 57 cases were detected by tNGS and SS (75%), whereas were documentedin60outof76MEN1patients(79%): In our series, heterozygous Frequency infamilialandsporadicMEN1cases Pathogenic 27 sporadiccasesitwas41 the 49familialcaseswas39 Clinical Study CDKN1B / AIP MEN1 / / p27 p27 / p27 and , and / MEN1 p27 -MLPA assays;circlewithdashedline, MEN1 CDKN2C/p18 CDKN1A/p21 -negative byMLPA werescreenedtogermline MEN1 genesintNGS/ , AIP AIP CDKN1B -mutation patientsidentifiedby -negative casesbytNGSand variants byMLPA assay. ); triangle,patientswithoutgermline / i. 1 Fig. p27 ± 4yas ( 14 years ) and MEN1 MEN1 ). Ninecases(9/60;15%) ±

4 years,whileforthe 14 R ACarvalhoandothers AIP mutation-negative CDKN2B/p15 , genes;circlewithfull germline variants MEN1 CDKN1B P =0.496). MEN1 openreading MEN1 / p27 MEN1 , MEN1 MEN1 , CDKN2C/ , and AIP MEN1

-

, AIP ,

and 16(fivefamilial,11sporadic)were remaining 11were out of27(59%)apparentlysporadiccases,whilethe testing, weuncovered16newlyinheritedMEN1cases tNGS/SS andthreebyMLPA assay(6%).AfterMEN1 were mutationcarriers:41(84%)detectedby pathogenic SE), 79%(60/76)ofpatientsharboredapathogenic/likely Overall, consideringthecombinedcriteria(ACMGand likely pathogenicvariantswerereclassifiedaspathogenic. of segregationequation to criteriafromACMG.Subsequently, aftertheprobability the 60patientsharboringpathogenic/likelypathogenic cases. The16mutation-negativecaseswereolderthan (44/49; 90%) than in sporadic (16/27, 59%; likely pathogenic cases. Asexpected,theprevalenceofgermlinepathogenic/ with theprobabilityequation ofsegregationbychance( applied thecriteriaadopted byACMG( missense variantsandone of themwasnew. We again In ourcohort,weidentified six(6/41;15%)different Analysis ofMEN1variants missense pointmutation. in-frame insertion/deletionandoneintronic (6/13) ornonsensevariant(2/13),followedbythree proteins generatedfromframeshiftdeletions/insertions pathogenic variantswerepredictedtocodetruncated 7%) andlargedeletions(3/60;5%)( 18%), missense(7/60;12%),in-frameindelvariants(4/60; followed by insplice-site (14/60; 23%), nonsense (11/60; MLPA assays,mostvariantswereframeshift(21/60;35%), both methods.CombiningresultsfromtNGS/SSand Large genedeletions,asexpected,werenotdetectedby detected bytNGSwerealsoverifiedSSandviceversa. from eightavailabledatabases( variants werenovel(13/37;35%),astheyabsent ( Overall, 38different Mutation types MEN1 more thantwoMEN1-relatedtumors,incomparisonwith Most MEN1 Targeted NGSinMEN1 Fig. 2 In total,60 In thefamilialMEN1subset,44cases(44/49;90%) Most (62%)ofthe13newpathogenic/likely -positive indexcases(20/60,33%; variants(38 ). Ten pathogenicandthreelikely MEN1 MEN1 -negative indexcases(12/16;75%)hadno MEN1 variant. MEN1 ± MEN1

14 years vs46 14 years carriersweredocumented(75%), MEN1 Downloaded fromBioscientifica.com at09/28/202104:26:31AM variantswashigherinfamilial -negative carriers( N =(1/2) variantswereidentified Table 1 m ± Fig. 2 wasapplied,three 179 12 years; 12 years; https://eje.bioscientifica.com ). Allvarianttypes 46 :6 P and =0.003). , MEN1 47 Fig. 1 ) combined Table 1 P P -negative =0.033). ). =0.002) MEN1 395 ). 48 via freeaccess )

European Journal of Endocrinology https://eje.bioscientifica.com in a43-year-oldpatientwith typicalMEN1phenotype ( acids (c.802_803insGGCTGCTCT; resulted inarepetitionof thecorrespondingamino sequence GGCTGCTCT (position 794-802),which exon 5duetoaconsecutiveduplicationofthenormal The latterpatientpresentedanewin-framevariant in 1 Tables variants, twoofthemwerefamilial(cases8and56, described inthesubsequentcodon120( by ‘warmspot’ and itsmutatedsequencewasexactlythesamegenerated pathogenic asitoccurredinarepetitiveregionofexon 2 initially classifiedaslikelypathogenic.Itwasredefined as (codon 119)foundinone pathogenic andoneaspathogenic. Three in-frame variants (two new) were classified as likely features ofMEN1withmultiglandulartumorinvolvement. SMAD3-interacting domains. in humansandanimals,theJunD-NM23H1-RPA2- generegion variant waslocatedatahighlyconserved at-risk familymembers( relatives anditwasabsentinsevenotherasymptomatic as likelypathologicsegregatedinfouraffectedat-risk pathogenic ( variants andonein-framepreviouslyreportedas (missense andin-frame)confirmfivemissense to classify pathogenicity of the four new variants Table 1 Clinical Study Considering thethreepatientswithlikelypathogenic The newin-framepathogenicvariantc.354_356deGAA All patientswithin-framevariantspresentedtypical The newmissensevariant , case20; and Table 2 2 ) and another was a simplexMEN1case. Table 2 ). MEN1 ). Thelattervariantwasidentified al 1 Table mutationc.358_360delAAG de novo R ACarvalhoandothers , case1; p.Trp p .Leu41Pro classified case(case10)was 16 265_Leu267dup) , al 2 Table 19 ). ). This

presenting with primary HPT,presenting withprimary non-functioningPETs, the classificationofthreevariants(p.I147F, ( H139Y aspathogenic,basedonprevious Leu413Arg, p.L414P, p.D418Y)aslikelypathogenicandp. reported, fourofthemwereinitiallyclassified(p.I147F, region( proline aminoacidinahighlyconserved of the aliphatic amino acid leucine for the non-aliphatic by us harboring a pathogenic variant, based on changing adrenal tumor. Thelatterpatientwaspreviouslyreported macroprolactinoma, collagenomasandnon-functioning phenotype characterizedbyHPT(multiglandulardisease), years and presented a typical MEN1 case was aged 28 criteria, wasabsentinsixhealthysiblings.Theindex 28; NMHCIIA-FANCD2-HDAC1-interacting domains. yet ( segregation analysisofthevariantcouldnotbeperformed to comethehospital,butremainedunavailable.Thus, breast carcinoma. At-risk familymemberswereinvited showed abrotherwithurolithiasisandanaunt collagenomas andfacialangiofibromas.Familialhistory gastrinoma, bilateralnodularadrenocorticalhyperplasia, far fromexon/intronboundaries, untranslatedor Notably, nomutation wasverifiedbytNGSinintrons and regulatoryregions Introns farfromexon/intron boundaries,untranslated p.D418Y) fromlikelypathogenicto( Targeted NGSinMEN1 55 ). Inturn,thestrengthoffamilialsegregationchanged Overall, analyzingthefivedifferentvariantspreviously The al 2 Table al 2 Table p .Leu414Pro missensevariant( ), classifiedaslikelypathogenicbyACMG ). ThisvariantwaslocatedatNM23H1- to non-codingregionsofthe cohort ( ##, mutationspreviouslyreportedfromour mutations reportedinthepresentstudy; mutations arelocatedabove;#,new (missense andin-frame)splicing below diagramwhilenon-truncating deletions (MLPA assay)arerepresented nonsense) (NGSandSS)gross Truncating mutations(frameshiftand and SS(57cases),MLPA assay(3cases). unrelated MEN1indexcasesbybothtNGS gene in60outofourcohort76 frontiers andcodingregionofthe Mutations wereidentifiedinintron-exon Figure 2 Downloaded fromBioscientifica.com at09/28/202104:26:31AM 8 , 18 ); shadow region corresponds ); shadowregioncorresponds 179 :6 in vitro al 1 Table p MEN1 .Leu413Arg, Table 2 MEN1 18 studies MEN1 gene. , case 396 ).

via freeaccess p ). . European Journal of Endocrinology

Table 1 Pathogenic germline variants identified in coding regions and intron–exon frontiers of theMEN1 gene in 57 unrelated MEN1 cases, by targeted NGS and Clinical Study Sanger sequencing.

ID Sex/ageb Phenotype Exon Codon Mutation Protein change HGMD dbSNP Effect/ACMG/SE Reference 1 M/69 F-MEN1 2 41 c.122T>C p.L41P NA NI MSd/LP/LP This study 2 F/37a F-MEN1 2 68 c.201_201delC p.A68Pfs*51 CD075463 NI FS/P (18) 3 M/47a F-MEN1 2 83–84 c.249_252delGTCTc p.I85Sfs*33 CD075467/CD972304 rs587776841 FS/P (16)/(28) 4 F/34 F-MEN1 2 83–84 c.249_252delGTCTc p.I85Sfs*33 CD075467/CD972304 rs587776841 FS/P (16)/(28) 5 M/51 F-MEN1 2 83–84 c.249_252delGTCTc p.I85Sfs*33 CD075467/CD972304 rs587776841 FS/P (16)/(28) 6 F/30 F-MEN1 2 83–84 c.249_252delGTCTc p.I85Sfs*33 CD075467/CD972304 rs587776841 FS/P (16)/(28) 7 F/30 F-MEN1 2 83–84 c.249_252delGTCTc p.I85Sfs*33 CD075467/CD972304 rs587776841 FS/P (16)/(28) 8 M/54 F-MEN1 2 89–95 c.266_286del21 p.L89_A95del NI NI IF/LP/LP (18) 9* M/36 S-MEN1 2 106 c.315_316insCCTC p.Y106Pfs*12 NI NI FS/P This study d

10* F/20 S-MEN1 2 119 c.354_356deGAA p.Lys119del NI NI IF /LP/LP This study R ACarvalhoandothers 11 F/52a F-MEN1 2 126 c.377G>A p.W126* CM981253 NI NS/P (10) 12 M/57 S-MEN1 2 129 c.385_385delC p.L129Sfs*56 NI NI FS/P This study 13 M/28 S-MEN1 2 139 c.415C>T p.H139Y CM970921 NI MS/P (15) 14 F/49a F-MEN1 2 144 c.431_432delinsAA p.F144* CX055790 NI NS/P (66) 15 F/48a F-MEN1 2 147 c.439A>T p.I147F CM074341 NI MS/LP/P (18) 16 F/37 F-MEN1 3 183 c.548G>A p.W183* CM981259 rs794728650 NS/P (17) 17 F/36a F-MEN1 3 186 c.558delT p.F186Lfs*38 CD035821 NI FS/P (67) 18 M/19a S-MEN1 3 210–211 c.628_631delACAGc p.T210Sfs*13 CD972310 rs794728640 FS/P (16) 19 F/50a F-MEN1 3 218 c.652_652delC p.R218Gfs*6 NI NI FS/P This study 20 M/42 S-MEN1 5 265-267 c.802_803insGGCTGCTCT p.Trp265_Leu267dup NI NI IFd/LP/LP This study 21 M/33 F-MEN1 6 302 c.903_909delCTACCAC p.Y302Rfs*64 NI NI FS/P This study 22 F/26a F-MEN1 7 318 c.951_952insC p.I318Hfs*49 NI NI FS/P (18)

23 M/27 F-MEN1 7 339 c.1015C>T p.Q339* CM053978 NI NS/P (68) Targeted NGSinMEN1 24 F/48a F-MEN1 8 363 c.1087G>T p.E363* NI NI NS/P This study 25 F/51a S-MEN1 8 378 c.1133delA p.E378Gfs*67 CD075465 NI FS/P (18) 26 F/38a F-MEN1 9 413 c.1238T>G p.L413R CM074346 NI MS/LP/P (18) 27 M/45 F-MEN1 9 413 c.1238T>G p.L413R CM074346 NI MS/LP/P (18) 28 M/27a S-MEN1 9 414 c.1241T>C p.L414P CM010342 NI MSd/LP (18) 29 M/33 F-MEN1 9 415 c.1243C>Tc p.R415*c CM970937 NI NS/P (17) 30 M/42 S-MEN1 9 415 c.1243C>Tc p.R415*c CM970937 NI NS/P (17) 31 M/43a F-MEN1 9 418 c.1252G>T p.D418Y CM981277 NI MS/LP/P (69) 32 M/28 F-MEN1 9 423 c.1268G>A p.W423* CM004419 NI NS/P (70) 33 M/65a S-MEN1 10 461-468 c.1382_1404del23 p.E461Gfs*62 NI NI FS/P (71) Downloaded fromBioscientifica.com at09/28/202104:26:31AM 34 M/7a F-MEN1 10 516 c.1546_1547insCc p.R516Pfs*15c CI972640 rs767319284 FS/P (15) 35 F/35a S-MEN1 10 516 c.1546_1547insCc p.R516fs*15c CI972640 rs767319284 FS/P (15) 36 M/54 F-MEN1 10 516 c.1546_1547insCc p.R516fs*15c CI972640 rs767319284 FS/P (15) 37* F/42 (DN) S-MEN1 10 516 c.1546_1547insCc p.R516fs*15c CI972640 rs767319284 FS/P (15) 38 M/38a F-MEN1 10 519 c.1554_1578del25 p.P519Efs*32 NI NI FS/P This study 39 M/60a S-MEN1 10 522 c.1564_1565delGT p.V522Rfs*8 NI NI FS/P This study 179

https://eje.bioscientifica.com 40 F/19 S-MEN1 10 527 c.1579C>T p.R527* CM970942 rs104894261 NS/P (16) 41 F/42 F-MEN1 10 527 c.1579C>T p.R527* CM970942 rs104894261 NS/P (16) :6 42 F/33 F-MEN1 10 557 c.1669A>T p.K557* NI NI NS/P This study 43 F/30a F-MEN1 Intron 3 _ c.654+1G>T _ CS982266 rs794728622 SS/P (72) 44 M/17 F-MEN1 Intron 3 _ c.654+1G>T _ CS982266 rs794728622 SS/P (72) 45 M/43 F-MEN1 Intron 3 _ c.654+1G>T _ CS982266 rs794728622 SS/P (72)

46 F/29 F-MEN1 Intron 3 _ c.654+1G>T _ CS982266 rs794728622 SS/P (72) 397 47 F/52 F-MEN1 Intron 3 _ c.654+1G>T _ CS982266 rs794728622 SS/P (72) via freeaccess

(Continued ) European Journal of Endocrinology https://eje.bioscientifica.com Table 1 (Continued) Clinical Study

ID Sex/ageb Phenotype Exon Codon Mutation Protein change HGMD dbSNP Effect/ACMG/SE Reference

48 F/30 S-MEN1 Intron 3 _ c.654+1G>T _ CS982266 rs794728622 SS/P (72) 49 F/35 F-MEN1 Intron 3 _ c.654+1G>T _ CS982266 rs794728622 SS/P (72) 50 M/17 F-MEN1 Intron 3 _ c.654+1G>T _ CS982266 rs794728622 SS/P (72) 51 M/40 F-MEN1 Intron 3 _ c.654+1G>T _ CS982266 rs794728622 SS/P (72) 52 F/23 F-MEN1 Intron 3 _ c.654+1G>T _ CS982266 rs794728622 SS/P (72) 53 F/53 S-MEN1 Intron 4 _ c.783+1G>A _ CS003705 NI SS/P (73) 54 F/60a F-MEN1 Intron 4 _ c.784-9G>Ac _ CS991446 rs794728625 SS/P (74) 55 M/32 F-MEN1 Intron 4 _ c.784-2A>C _ NI NI SS/P This study 56 F/55a F-MEN1 Intron 5 275–277 c.825-30_830delinsT p:R275S_Y276_P277del NI NI IFd/LP/LP This study 57 * F/28 S-MEN1 Intron 9 _ c.1350+1_1350+11del11 _ NI rs794728644 SS Mutch et al. (61) 58–76 (F, 15/19) F-MEN1 (8/19) R ACarvalhoandothers

All variants were classified based on both the ACMG classification criteria, and equation to calculate segregation probability of a given variant occurs by chance (SE). aThe 22 patients included in runs 1 and 2 for validation/optimization (for details, Supplementary data); NM_130799.2; Ensembl, ENST00000312049; bage at the time of diagnosis; crecurrent variants described as mutational ‘warm spots’, as previously reported; dthey were classified as likely pathogenic variants (Table 2); in bold, unrelated cases of our cohort previously reported (Toledoet al . (18)); in italic, new variants reported here; reference, it is cited the first study that reported the mutation; de* novo mutation (normal parental generation, not provided paternity testing; classical MEN1 phenotype). A, adenine; ACMG, American College of Medical Genetics and Genomics (47); C, cytosine; del, deletion; F, female; F-MEN1, familial MEN1; FS, frameshift; G, guanine; HGMD, Human Gene Mutation Database; ID, identification; IF, in-frame; ins, insertion;in silico, prediction tools (Sift, Polyphen, Mutation Assessor, Mutation Taster, Provean and Human Splicing Finder) and databases (1000 Genomes, 6500 Exomes, dbSNP, LOVD, Ensembl, COSMIC, ExAC Browser, HGMD); IVS, intervening sequence; LP, likely pathogenic variant; M, male; MEN1, multiple endocrine neoplasia type 1; MEN1, MEN1 gene; MS, missense; NI, not identified in this database; NS, nonsense; P, pathogenic variant; SE*, segregation equation 45( ), see detailed application in Table 2; S-MEN1, sporadic MEN1; SS, splice site; T, thymine. a secondpathogenicvariantwasruledoutin60 the 16 and 27 sporadic cases), which evidently include regionsinall76MEN1patients(49familial regulatory previously reportedthefoundingmutationc.201_201delC founding mutation in cases 26 and 27 ( haplotype analysisallowedustoexcludep.L415Rasa reported byLemosandThakker( c.1546_1547insC) coincided with ‘warmspot’ mutations ( in 25apparentlyunrelatedmutationcarriers(25/60;42%) (6/41) ofthedifferentmutationtypesandwerepresent In ourseries,recurrentmutationscorrespondedto15% Germline mutationswerespreadalongthe affected codons Recurrent mutationsandmostfrequently mutation carriers. was classifiedasbenignpolymorphism. Brazilian controlsindividuals ( databases, althoughitwas identifiedin10.2%ofthe our caseswasnotpresent in sixavailableinternational were classifiedasVUS( patients andonlyoccurredinmutationcarriers.They frequencies (1.3–4%),detectedinonetothreeMEN1 the threeremainingnewpolymorphismshadlower to 20 either with or without They weredetected,respectively, in40,30and 26patients g.5276_5276delT and wereclassifiedaslikelybenign. (34–52%): c.445+709_710delTT, c.445+710delT and ( COSMIC, 6500exomes,ABraOM,GnomADandExAC were notpreviouslyreportedindbSNP, 1000genomes, were characterizedasnewpolymorphicvariants,they and twonon-synonymous.Overall,six(6/36;17%) than inexonicregions(29vs7).Fourweresynonymous were four-foldmorefrequentlydetectedinnon-exonic (7/36; 19%)andexons19%).Thus,polymorphisms throughout introns (22/36; 61%), untranslated regions MEN1 with highlyvariablefrequencies(1.3–96%)alongthefull Overall, 36differentbenignvariantswereidentified Non-pathogenic variants not availableinthe22remainingcarriers. ( Targeted NGSinMEN1 Table 1 al 3 Table 4 ) ( The variantc.-23-24T al 1 Table bp andwerelocatedinhomopolymers.Incontrast, genebytNGS.Thepolymorphismsweredispersed ). Threeofthem(c.249_252delGTCT, c.1243C MEN1 ). Threenewvariantswerehighlyprevalent ). Inturn,methodichaplotypeanalysiswas -negative carriers.Also,thepossibilityof MEN1 Table 3 Downloaded fromBioscientifica.com at09/28/202104:26:31AM > C (g.1139T mutations, ranging from 10 ). Table 3 19 ) ( 179 ). Thus,thisvariant > al 1 Table C) inIVS1seen 18 :6 ). In case 2, we MEN1 ). Previous gene. MEN1 398 > via freeaccess T, T,

European Journal of Endocrinology

Table 2 Classification of ten in-frame/missenseMEN1 variants (4 new variants) and nine other new null variants using criteria adopted by The American College of Clinical Study Medical Genetics and Genomics (ACMG), combined with probability of segregation of a given variant to occur by chance (SE).

Familial segregation In silico (number of MEN1 carriers/ impact Other reported no carrier) prediction Populational Families number with missense variants in Functional Classification ID Variant/effect Phenotype (SE*, final value) tools databases the variant the same codon (PM5) data (ACMG + SE)

1 c.122T>C (p.L41P)/MS F-MEN1 PP1-PM (4/7) (a:1/16) PP3 PM2 1 (present study) _ PP2, PP4 LP=>LP

13 c.415C>T (p:H139Y)/MS S-MEN1 _ (0/3) PP3 PM2 2 (present study; (15)a) PM5 (p.H139D/P/Q/R)d PP2, PP4, P PS3 15 c.439A>T (p.I147F)/MS F-MEN1 PP1-PS (16/15) PP3 PM2 1 (18)b _ PP2, PP4 LP=>P** (a:1/65 536) b e 26 c.1238T>G (p.L413R)/MS F-MEN1 PP1-PS (3/4) (b: 1/64) PP3 PM2 2 (18) PM5 (p.L413P) PP2, PP4 LP=>P** R ACarvalhoandothers 27 c.1238T>G (p.L413R)/MS F-MEN1 PP1-PS (3/2) (b: 1/64) PP3 PM2 2 (18)b PM5 (p.L413P)e PP2, PP4 LP=>P** 28 c.1241T>C (p.L414P)/MS S-MEN1 _ (0/6) PP3 PM2 1 (18)b PM5 (p.414Q/R)f PP2 LP 31 c.1252G>T (p.D418Y)/MS F-MEN1 PP1-PS (5/4) (a: 1/32) PP3 PM2 2 (present study; 69c) PM5 (p.418H/N)g PP2, PP4 LP=>P** 8 exon2:c.266_286del21/IF F-MEN1 PP1-PM (5/3) (a: 1/16) PP3 PM2 1 (Toledo et al. (18)b) _ PM4, PP4 LP 10 c.354_356deGAA/IF S-MEN1 PP1-PP (1/3) PP3 PM2 1 (present study) _ PM4, PM6, LP=>P*** PP4 20 c.802_803insGGCTGCTCT/IF S-MEN1 _ _ PM2 1 (present study) _ PM4, PP4 LP 56 c.825-30_830delinsT/IF F-MEN1 PP1-PM (5/8) (a, 1/16) PP3 PM2 1 (present study) _ PM4, PP4 LP 9 c.315_316insCCTC/FS S-MEN1 PP1-PP (2/3) PP3 PM2 1 (present study) _ PVS1, PM6, P PP4 12 c.385_385delC/FS S-MEN1 _ (1/1) PP3 PM2 1 (present study) _ PVS1, PP4 P 19 c.652_652delC/FS F-MEN1 PP1-PM (5/4) (a, 1/16) PP3 PM2 1 (present study) _ PVS1, PP4 P 21 c.903_909delCTACCAC/FS F-MEN1 PP1-PM (4/5) (a, 1/8) PP3 PM2 1 (present study) _ PVS1, PP4 P Targeted NGSinMEN1 24 c.1087G>T/NS F-MEN1 PP1-PP (2/2) PP3 PM2 1 (present study) _ PVS1, PP4 P 38 c.1554_1578del25/FS F-MEN1 PP1-PP (3/1) (a, 1/4) PP3 PM2 1 (present study) _ PVS1, PP4 P 39 c.1564_1565delGT/FS S-MEN1 _ (1/0) PP3 PM2 1 (present study) _ PVS1, PP4 P 42 c.1669A>T/NS F-MEN1 PP1-PS (7/2) (a, 1/64) PP3 PM2 1 (present study) _ PVS1, PP4 P 55 c.784-2A>C/SS F-MEN1 PP1-PP (4/2) (a, 1/8) PP3 PM2 1 (present study) _ PVS1, PP4 P

The criteria to pathogenicity (P) depend on segregation data coming from one family (a) or two or more families (b). For these groups, different cutoffs define a grading of evidence levels of pathogenicity: PP, supporting evidence (a, P ≤ 1/8; b, P ≤ 1/4); PM, moderate evidence (a, P ≤ 1/16; b, P ≤ 1/8); PS, strong evidence (a, P ≤ 1/32; b, P ≤ 1/16). aOne isolated index case, without familial segregation (Agarwal et al. (15)); bcases 15, 26, 27 (with familial segregation) and 28 (isolated index case without familial segregation) were previously c d e f

Downloaded fromBioscientifica.com at09/28/202104:26:31AM reported by us (Toledo et al. (18)); familial MEN1 (Ozturk et al. (69); Agarwal et al. (15), Cebrian et al. (67), ClinVar database, Martin-Campos et al. (75); Asteria et al. (76); Ellard et al. (66), ClinVar database; gHeppner et al. (77), Warner et al. (78); **it was classified as likely pathogenic variant by ACMG and reclassified as pathogenic after segregation equation; ***it was reclassified as pathogenic as its resultant mutated sequence is the same of the warm spot variant c.358_360delAAG (16, 19). F-MEN1, familial multiple endocrine neoplasia type 1; FS, frameshift; ID, identification of the patients; IF, in-frame; LP, likely pathogenic; MS, missense; acronyms and their definitions are informed exactly as established by ACMG (44): PVS1, null variant (nonsense, frameshift, canonical +/−1 or 2 splice, sites initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease (MEN1 gene LOF = 1, gene is extremely intolerant to LOF); NA, not available; NS, nonsense; P, pathogenic; PM1, variant is located in a mutational hot spot and/or in a critical and well-established functional domain without benign variation; PM2, absent from controls in populational databases as Exome Sequencing Project, 1000 genomes or ExAC; PM4, occurrence 179 https://eje.bioscientifica.com

of protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants; PM5, novel missense change at an amino acid residue where other different missense :6 change was previously reported as pathogenic; PM6, assumed as de novo (without confirmation of paternity and maternity); PP1, variant cosegregation with disease in multiple affected family members in a gene clearly causing the disease; PP2, missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease; PP3, multiple lines of computational evidence (in silico analysis) support a deleterious effect on the gene or gene product; PP4, patient’s phenotype or family history is highly specific for a disease with a single genetic etiology; PS3, presence of well-established in vitro or in vivo functional studies supporting a damaging effect on the gene; PS4, the prevalence of the variant in affected m individuals is significantly increased in comparison with controls; SE*, segregation equation 45( ), defined asN = (1/2) , was supported where N means the probability of the observed variant occur by 399 chance (no pathogenic) and m defines the number of meiosis of the variant that is informative for segregation, excluding index case; S-MEN1, sporadic MEN1; SS, splice site. via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com range PCRmethodology, associatedwithnooccurrence of tNGSprovedthespecificity ofthe MLPA. Also,theprimerdesignandreactionoptimization seen incaseswithgross test. A small percentage of false-negative results (5%) were the genetic diagnosis of MEN1 was validated as a reliable repeatability withnofalsepositive.Thus,tNGSappliedfor presented identicallyhighaccuracy, reproducibility and compared datafromtNGSwithSSandbothtechnologies as itisfasterandcheaperthanWESWGS.We genetic analysis,usingSS( extended numberofcasesfullingthecurrentcriteriafor it ishighlychallengingtoperform regions ( besides itmaynotroutinelycoverthe presents limitationsintheanalysisof complemented by MLPA for large deletions. However, SS sites of Until recently, mutationsincodingregionandsplicing tNGS todetectMEN1mutations Discussion AIP were studied.Nocaseharboredmutationsinthe patients fortestingoutofthe16 MEN1 AIP gross deletionsweresearched butnotidentifiedinthe MEN1 6% (3/49)ofallfamilialMEN1casesandin37%(3/8) all cases,in5%ofMEN1-positivepatients(5/60), gross patients (8familialand11sporadiccases)( MLPA assaywasappliedforthesubsetof19 amplification (MLPA) Multiplex ligation-dependentprobe detection rate(36vs7; technology yielded a significantlyhigher polymorphism this region. Comparing data from SS and tNGS, the latter of designed primers covered the intron/exon boundary MEN1- by SSinthepresentcases.Sixofthemoccurred Clinical Study genes( and In thepresentstudy, weappliedtNGS toour76cases Nine (threefamilialandsixsporadic)available Conversely, onlyseven polymorphisms weredetected -negative cases. -negative familialMEN1cases( MEN1 coding regionandanotherinIVS1region,asthe 5 CDKN1B MEN1 , 8 Fig. 1 , deletionswereidentifiedin4%(3/76)of 10 havebeenusuallyinvestigatedbySS , ). 13 genesbyMLPA assayinthe16truly , 18 , P 19 MEN1

< , 0.05). 5 23 ). , deletions,weidentifiedby 28 R ACarvalhoandothers , MEN1 29 MEN1 , MEN1 Fig. 2 30 MEN1 -negative patients , 56 MEN1 MEN1 Fig. 1 ). Additionally, testinginthe -targeted long- ). Inaddition, non-coding ). Overall, geneand -negative -negative CDKIs or

suggesting that MEN1 patients not harboring mutations These findingscontrastwiththeprevioushypothesis rareinMEN1patients. regions aremostprobablyvery MEN1 Thus, ourpresentdataindicatedthatpointorshortindel generegions. boundaries, untranslatedorregulatory pathogenic variantsinintronsfarfromintron/exon MEN1 patientsandnocaseharboredpathogenic/likely 76 the full Worthwhile, tNGS allowed us to methodically investigate or untranslatedregulatory regions Mutations inintrons farfrom intron/exon boundaries data. found inSupplementary the NGSplatformandcost-effectivenessoftNGSmaybe long-range PCR as enrichment method, criteria to select dropout ( of primercross-reactivity, dimerformation,allelicbiasor in non-codinggeneregions( in codingregionsofthe in 1890( of thesoutheasternBrazilthat camefromVeneto, Italy Table 1 c.201_201delC (p.A68Pfs*51) foundermutation(case2, ( recurrent mutations may occur due to founder mutations cases 26and27byhaplotype analysis ( line, wepreviouslydocumentedindependentmutations in of warm spot to independentmutationaleventsfavoringtheexistence We foundrecurrent Recurrent MEN1mutations out. MEN1 casesharboringmutationscouldberuled germline MEN1 23 study usingSSinvestigated a minimalpromoterregionin copy number( by SSandidentifieddifferentassaysassessinggene by onlyafewlargegross promoter, 5 Concordantly, reported mutations involving the was available,precludingfurtherconclusions( both the Targeted NGSinMEN1 4 , MEN1 Furthermore, thepossibleoccurrenceofasecond Indeed, noprevioussystematicstudyinvestigating 28 pathogenic/likelypathogenicvariantsinthesegene germlinemutations( , ) inalargeMEN1familywith 50mutationcarriers 57 MEN1 -negative MEN1familialcasesandrevealedno 34 MEN1 4 ). However, haplotypeanalysiscould not be , ′ ). Additionaldiscussionontheadoptionof MEN1 or3 MEN1 58 8 codingandnon-codingregionsinall pathogenic/likelypathogenicvariantin , , 23 ′ 59 codingandnon-codingregions untranslatedregionsarerepresented mutations in MEN1 ( , MEN1 ). Concordantly, we reportedthe 28 MEN1 , Downloaded fromBioscientifica.com at09/28/202104:26:31AM 29 mutations,whichcouldbedue , 30 MEN1 30 5 gene would carry mutations genewouldcarry , ). , 8 56 , 10 deletionsnotcovered ). Inaddition,asingle , 179 13 18 , :6 8 18 , ). Alternatively, 19 , 19 , 23 , 23 ). In this ). 400 8 via freeaccess ).

European Journal of Endocrinology Clinical Study Table 3 Polymorphic germline variants identified by targeted NGS in non-coding and coding regions of theMEN1 gene of 76 unrelated MEN1 index cases, classified by ACMG criteria.

1000 6500 Total (n) genomes, GnomAD AbraOM exomes, ACMG SNVs Local (Het + Hom) % Het dbSNP COSMIC MAF (%) (%) MAF ExAC (%) classification c.*307T>G 3′UTR 1 100 rs1804848 NI G = 0.9% 0.86 0.64 NI NI Benign c.*185C>T 3′UTR 1 100 rs111895237 NI T = 0.5% 0.20 0.16 NI NI Benign c.*306_*308delCTC 3′UTR 1 100 rs143341556 NI DEL = 8% 2 4.83 NI NI Benign c.*302C>T 3′UTR 3 100 rs1804849 NI T = 1.2% 1.50 1.26 NI NI Benign c.-35A>T 5′UTR 34 71 rs679946 NI T = 23% 32 37.97 NI NI Benign c.-35A>C 5′UTR 7 100 rs679946 NI C = 20% 5.80 7.96 NI NI Benign R ACarvalhoandothers c.-6G>A 5′UTR 1 100 rs768088337 NI A = 0.2% 0.01 NI NI NI Benign c.-40G>C 5′UTR 21 100 rs552417059 NI C = 0.3% 0.34 NI NI NI Benign c.512G>A: p.R171Q EXON 3 3 100 rs607969 23054 A = 0.8% 1.22 1.72 A = 1.5% 0.0008 Benign c.597C>T: p.H204H EXON 3 3 0 rs150512958 NI T = 0.1% 0.05 0.08 0.02% 0.06 Benign c.1254C>T: p.D418D EXON 9 40 83 rs2071313 NI T = 30% 38.50 31.44 T = 39% 0.39 Benign c.1299T>C: p.H433H EXON 9 67 9 rs540012 NI T = 2% 99.20 97.70 T = 2.5% 0.993 Benign c.1508G>A: p.G503D EXON 10 1 100 rs375804228 1188290 A = 0.04% 0.05 NI NI 0.0005 Benign c.1624A>G: p.T541A EXON 10 73 18 rs2959656 255213 A = 15% 93 91.37 A = 9% 0.94 Benign c.-23-16C>G IVS1 10 70 rs509606 NI G = 17% 16 10.00 NI 30 Benign c.-23-24T>C (g.1139T>C) IVS1 4 100 NI NI NI NI 10.22 NI NI Benign c.-23-273G>A (g.890G>A) IVS1 2 100 NI NI NI NI NI NI NI VUS c.-23-277G A (g.894G A) IVS1 3 33 NI NI NI NI NI NI NI VUS

> > Targeted NGSinMEN1 c.445+306G>A IVS2 2 100 rs34047150 NI A = 0.6% 1 1.80 NI NI Benign c.446-198G>A IVS2 1 100 rs11607366 NI A = 0.6% 2.20 1.53 NI NI Benign c.446-347G>A IVS2 1 100 rs75989060 NI A = 0.7% 0.75 NI NI NI Benign c.445+401_445+402insTT IVS2 20 100 rs11404218 NI NI NI NI NI NI Likely benign c.445+539G>A IVS2 13 92 rs67808744 NI A = 15% 10.50 NI NI NI Benign c.445+710delT IVS2 30 100 NI NI NI NI NI NI NI Likely benign c.445-691delT (g.2505_2505delT) IVS2 1 100 NI NI NI NI NI NI NI VUS c.445+719G>A IVS2 10 80 rs11231873 NI A = 13% NI NI NI NI Benign c.445+709_710delTT IVS2 40 100 NI NI NI NI NI NI NI Likely benign c.445+183G A IVS2 14 86 rs624975 NI A = 17% 11 11.12 NI NI Benign Downloaded fromBioscientifica.com at09/28/202104:26:31AM > c.445+177G>A IVS2 1 100 rs192961797 NI A = 0.3% 0.09 NI NI NI Benign c.445+401_445+402insT IVS2 19 100 rs11404218 NI NI NI NI NI NI Likely benign c.446-662A>G IVS2 1 100 rs374393702 NI NI 0.06 NI NI NI Benign c.912+283C>T IVS6 72 16.67 rs504195 NI C = 16% 88.90 90.10 NI NI Benign c.912+295_912+297delTGA IVS6 66 18.2 rs2307769 NI TGA = 20% 87.00 90.45 NI NI Benign 179

https://eje.bioscientifica.com c.1049+115A>G IVS7 12 83.33 rs669976 NI G = 17% 11.70 10.84 NI NI Benign

c.1049+213delT (g.5276_5276delT) IVS7 26 100 NI NI NI NI NI NI NI Likely benign :6 c.1350+103G>C IVS9 45 75.56 rs654440 NI C = 23% 31 38.51 NI NI Benign

In bold, new polymorphisms; italic, polymorphisms detected by both Sanger sequencing and NGS. A, adenine; ACMG, American College of Medical Genetics and Genomics (47); C, cytosine; del, deletion; Ensembl, ENST00000312049; G, guanine; Het, heterozygosis; Hom, homozygosis; ins, insertion;

IVS, intervening sequence; MAF, minor allele frequency; MEN1, multiple endocrine neoplasia type 1; n, number of patients; NGS, next-generation sequencing; NI, not identified; NM_130799.2; SNV, 401

via freeaccess single nucleotide variation; UTR, untranslated regions; T, thymine. European Journal of Endocrinology https://eje.bioscientifica.com pathogenic. Concordantly, in-framevariantsarelikely All in-framevariantswere initiallyclassifiedaslikely Likely pathogenicvariants on MLPA appliedtoMEN1areplanned. 11 deletions. Conversely, nogrossdeletionwas seeninthe as predictive indicator for detection of gross history cases, suggestingtheimportanceofpositivefamilial cases andin37%ofthe occurrence of gross deletions in 6% of all familial MEN1 phenotypes ( reported series ( the differentmutationtypes,wassimilartolarger series, the prevalenceof gross deletions (5%), considering been reported(1–33%)( A highly variable frequency of gross MEN1-MLPA assay unknown significance. variant wasconsideredbenign,likelybenignorof not resultinchangestheclinicalmanagementasno as pathogenic( occurrence of segregation, part of them were reclassified applied equationtocalculateprobabilityofatrandom using theACMGcriteria( we foundwereinitiallyclassifiedaslikelypathogenic may beinfeasible.Mostmissenseandin-framevariants, challenging sincefunctionalstudiesofeachthem confirmation ofpathogenicitymissensevariantsis slightly lesserpercentage (14%)( wasobserved mutations reported in MEN1, whereas in our cases a perform geneticscreeningoftheirat-riskrelatives. the 16newlyuncoveredinheritedMEN1casesleadusto was higherinfamilialthansporadiccases( anticipated, theprevalenceofgermline mutations wassimilartootherstudies( As expected,theoverallprevalenceofgermline MEN1-tNGS Pathogenic MEN1germlinevariants haplotype studiesareplannedtoclarifythistopic. mutations. Furthercarefulgenealogical,geographicand adequate distinctionbetweenindependentandfounder performed inthe22remainingindexcases,preventing Clinical Study MEN1 Missense variantsrepresentupto25%ofthe576 -negative sporadiccases.Furtherindeepstudies 8 , 23 < 48 5%) including MEN1 and MEN1-related , 28 ). However, thereclassificationdid , 29 5 , , 30 MEN1 8 46 , , 56 23 , ). Inaddition,weshowed -negative familialMEN1 , 47 R ACarvalhoandothers 28 ). Inturn,whenwe , MEN1 29 5 MEN1 , , 30 19 deletions has , , 5 56 mutations 23 , ). Inour 14 23 , 56 ), and ). The MEN1 MEN1 ). As

in thecodingregion(10synonymousand2non- reported inmorethan1100MEN1patients,12 So far, 24different Non-pathogenic MEN1germlinevariants to missensevariantsinourstudy. reinforce thepathogenicityofvariant,asobserved as likelypathogenic.Segregationanalysis(SE)may DNAregionfavorthisvariant’sconserved classification and thepreviouslyreportedmutationsinthishighly classification as likely pathogenic by ACMG criteria, of typicalMEN1clinicalfeaturesinourpatient,its in fivemembersofafamily( families ( occurring at this same codon in two unrelated MEN1 previous studiesreporteddifferentin-framedeletions harbored a variant deleting codons 265–267. Two to causedeleteriouseffectsonmenin.Oneofourcases polymorphisms ingenesbesides ( interventions surgery inadequate clinicalmanagementandunnecessary two reasons. One, variant misclassification maylead to clearly differentiatethemfrommutationsforatleast as benign,likelybenignorVUS,maybevaluable to compared toSS. to routinelydetectvariantsinthenon-codingregion, exonic regionsclearlyreflectsthehigherabilityoftNGS more polymorphismsdetectedinnon-exonicthan polymorphisms vs12,respectively. Thefindingof4-fold occurring in untranslated regions vs three, and 7 exonic vs ninereportedbythem(three-foldmore),seven Thakker ( Comparing ourdatawiththosereportedbyLemosand reported intheliterature(24/1133;~2%; verified (36/76;47%)wassubstantiallyhigherthanthat SS ( a substantialincreasetothosepreviouslydetectedby (3 VUS)weidentifiedinthepresentstudyrepresent regions ( synonymous), nineinintronsandthreeuntranslated elderly controlsfromABraOM andinourcases. IVS1, whichwaspresentin 10%ofthe609Brazilian databases ofthepolymorphism c.-23-24T was herereinforced bytheabsenceinsixinternational gene hasbeenscarcely sequencedsofar. strong evidencethatthenon-codingregion of the MEN1 phenotype( Targeted NGSinMEN1 8 The importanceoftheregional genomicrepositories Of note,abettercharacterizationofpolymorphisms , 19 8 ). Also,theprevalenceofpolymorphismswe 60 19 , 19 , ), we detected 22 intronic polymorphisms 61 ). The 36different 62 ), andadefinitesegregationwasshown ). Inaddition,becausedisease-modifying 53 MEN1 , Downloaded fromBioscientifica.com at09/28/202104:26:31AM 63 ). Overall,ourdataprovide polymorphismshavebeen MEN1 MEN1 61 179 ). Theassociation :6 polymorphisms couldinfluence > C (g.1139T P

< .5 ( 0.05) MEN1 402 19 > C) via freeaccess ). ).

European Journal of Endocrinology may impact clinical practice, as classified either as likely benign or VUS. This classification patients. Thus,somevariants innon-codingregionwere to bettercharacterizeDNA variantswefoundinour to potentially newMEN1-causinggenes( chromosomal mapping of analyses maybeappliedtoinformativefamilieseitherfor assay, resulted negative. Beyond WES and WGS, linkage known MEN1-related genes, and after applying MLPA to patients whose previous genetic analyses to all negative cases. expensive genebysequentialanalysisin would havethepotentialtosubstituteexhaustive, The effective validation of suchpanelsusing tNGS and pancreaticneuroendocrineneoplasia( adenoma pheochromocytoma/paraganglioma, pituitary have beenappliedforseveralendocrinetumordiseases,as may mimicMEN1phenotypes( hypocalciuric hypercalcemia andfamilialisolatedHPT and and may not only test would behelpful,accurateandcost-effective.Suchpanels multi-gene panelsappliedtoMEN1inclinicalpractice random associationofsporadicMEN1-relatedtumors. mutations insofarundiscoveredMEN1-relatedgenesor post-zygotic somaticmutationsleadingtomosaicism, in phenotypes ( rarity oftheassociationthesegeneswithMEN1-like detected in of available (p15, p18,p21,p27) and also testedamulti-genetNGSpanelexamining of phenocopies orharborsofarunreportedmutations. note, fivefamilialnon- MEN1-related tumor, confirmingrecentdata( of diagnosis,andmostpatientsdidnotexhibitathird non- Compared with60mutationcarriers,the16genuinely Non-mutation carriers Clinical Study p27 MEN1 MEN1 Although ACMG classification has not been applied At present,WESorWGShavebeenmostlyreserved Recently, similarcustommulti-genetNGSpanels Further studiesareneededtoclarifywhetherlarge Non-mutation carriersmightbedueto Although oneofourfocuswastoverifytherobustness GCM2 MEN1 AIP and genesbutalso variants,wedecidedtofollowitinanattempt -tNGS for the genetic diagnosis of MEN1, we mutation carriers were older at the time genes,asjawtumor-HPTsyndromes,familial AIP CDKNIs 20 MEN1 wereseeninthesecases. , 21 -negative carriers.Nomutationwas and , MEN1 22 ). Additionally, nogrossdeletions MEN1 AIP , AIP HRPT2 MEN1 CDKNI genes,inagreementwiththe mutationcarriersmightbe genes in alimitednumber 5 R ACarvalhoandothers region or to investigate , , MEN1- 8 s ( CAsR , 13 p15 8 , ). negative patients , 64 , GNA11 p18 ). 41 , , p21 , 42 de novo CDKNIs 14 MEN1 AP2S1 , , ). Of p27 65 ). ). ) -

in the clinical practice for PCR showedtobehighlyeffectiveimplemented factor couldbesupportedinfuture. harboring pathogenicvariants,apotentialmodulating MEN1 that pathogenic/likelypathogenicvariantsmayoccurin up to now, we cannot convincingly rule out the hypothesis ordeepintronic regulatory characterize the three VUS wefound in untranslated, data. Additionalfunctionalstudiesareneededtobetter pitfalls didnotpreventdrawingconclusionsfromour repetitivemutations.However,in thecasescarrying these size, andthepresenceofhypotheticalfoundermutations investigated in sporadic cases, the relatively limited sample be relatedtopossibletumormosaicismthatcouldnot clinically followedupperiodically. harboring aVUSshouldnotbesimplydismissedbut perceived asprejudicingtheimpartiality ofthisstudy. The authorsdeclarethatthereis no conflictofinterestthatcouldbe Declaration ofinterest EJE-18-0430 This islinkedtotheonlineversionofpaper at Supplementary data phenotypes. and mortalityofpatientswithMEN1MEN1-related related tumors,whichmaypositivelyimpactmorbidity early diagnosisandtherapeuticmanagementofMEN1- genetic diagnosis,screeningandcounseling, may expand and improve prenatal and preimplantation testing. Ultimately, thispotentiallypromisingprocedure number of cases fulfilling the current criteria for opens thepossibilityofproviding diagnostic yield,fordetectionof is a sensitive, affordable and robust method, with a high genetic testingbasedonatNGSlong-rangePCRapproach of suchevents.Inaddition,ourdatasupportthat in our76MEN1patients,whichpointstotherareness indel mutationsinthenon-codingregionof effectiveness. features, availablemethodologiesandtheirregionalcost- practice shouldbepersonalizedconsideringphenotypic analyze otherMEN1-relatedgenes(genepanel)inclinical number of patients are welcomed. The decision to datainvolvinglarger although furtherconfirmatory Targeted NGSinMEN1 Our validated protocol based on tNGS long-range Potential limitationsofthepresentstudycould In summary, wedidnotfindanypointorshort non-codingregion.Incasestheyoccurinpatients . Downloaded fromBioscientifica.com at09/28/202104:26:31AM MEN1 MEN1 MEN1 regionsbytNGS.Thus, MEN1 diagnostic testing, 179 https://eje.bioscientifica.com testingforalarger https://doi.org/10.1530/ :6 mutations.This MEN1 MEN1 MEN1 403 via freeaccess

European Journal of Endocrinology https://eje.bioscientifica.com References Fundacíon OlgaTorres emergingresearchergrant. of Economy (CP17/00199) and Competitiveness and is supported by a Servet-I researchcontractbyInstituteofHealth‘CarlosIII’theMinistry PROINFRA 01/2011grants(0160/12-sp02).RAToledo holdsaMiguel was supportedbyFAPESP grants(2014/50137-5)andMCTI/FINEP/CT-Infra- Sequencing Laboratory(SELA),UniversityofSaoPauloSchoolMedicine, Visiting ResearchergrantfromFAPESP (2015/25444-4).TheLarge-Scale L J (2013/19810-2; 2015/25444-4; 2016/07504-2). S J M was supported by grants toSPAT(SeniorProfessor–FMUSP)andFAPESP grantstoDM Paulo, Brazil.ThisinvestigationwassupportedbyCNPq(401990/2010-9) Federal University of São Paulo, and Senior Professor, University of São Nível Superior)NationalSeniorVisitingProfessor(PVNSprogram)atthe S PATisaCAPES(CoordenadoriadeAperfeiçoamentoPessoal the NationalCouncilforScientificand Technological Development(CNPq). fellowship (2016/07965-0).ALJreceivedagrant(301871/2016-7)from technical traininggrant(2015/07625-1)fromFAPESP andaFAPESP R ACwasrecipientofaFAPESP fellowship(2013/15388-4).BUreceived Funding 10 9 8 7 6 5 4 3 2 1 Clinical Study Bassett JH, Forbes SA,Pannett AA,Lloyd SE, Christie PT, Wooding C, Giusti F, Cianferotti L,Boaretto F, Cetani F, Cioppi F, Colao A, Falchetti A. Geneticsofmultipleendocrineneoplasiatype1 Giraud S, Zhang CX,Serova-Sinilnikova O,Wautot V, Salandre J, Lips CJ, Dreijerink KM,Links TP&Höppener JW. Recentresults Thakker RV, Newey PJ,Walls GV, Bilezikian J,Dralle H,Ebeling PR, Lourenço DM Jr, Toledo RA, Mackowiak II, Coutinho FL, Lourenço DM Jr, Toledo RA, Coutinho FL, Margarido LC,Siqueira AS, Marx SJ. Moleculargeneticsofmultipleendocrineneoplasia Brandi ML, Gagel RF, Angeli A, Bilezikian JP, Beck-Peccoz P, Bordi C, Harding B, Besser GM, Edwards CR, Monson JP 58 analysis ofanationwidemulticenter patientdatabase. neoplasia syndrometype1:institution, management,anddata Davì MV, Faggiano A, Fanciulli G,Ferolla P (https://doi.org/10.12688/f1000research.7230.1) syndrome: what’s newandwhat’s old. 63 type 1andrelateddisorders. line mutationanalysisinpatientswithmultipleendocrineneoplasia Buisson N, Waterlot C, Bauters C,Porchet N, Aubert JP Metabolism early detectionandtreatment. of basicandclinicalresearch inMEN1.Opportunities toimprove doi.org/10.1210/jc.2012-1230) of ClinicalEndocrinologyandMetabolism guidelines formultipleendocrineneoplasiatype1(MEN1). Melmed S, Sakurai A,Tonelli F, Brandi ML 259–274. mineral densityprofile. 1 inBrazil.MEN1foundingmutation,clinicalfeaturesandbone Margarido LC, Machado MC Cavalcanti MG, Correia-Deur JE,Montenegro F, Siqueira SA, (https://doi.org/10.1590/S1807-59322007000400014) the multipleendocrineneoplasiatype1. impact ofclinicalandgeneticscreeningsonthemanagement dos Santos MA,Montenegro FL,Machado MC&Toledo SP. The org/10.1038/nrc1610) types 1and2. org/10.1210/jcem.86.12.8070) Endocrinology andMetabolism diagnosis andtherapyofMENtype12. Conte-Devolx B, Falchetti A,Gheri RG,Libroia A 455–467. 349–359. (https://doi.org/10.1530/EJE-08-0153) 2012 (https://doi.org/10.1086/301953) (https://doi.org/10.1007/s12020-017-1234-4) Nature ReviewsCancer 7 331–344. European Journal ofEndocrinology European Journal 2001 (https://doi.org/10.1586/eem.12.22) American Journal ofHumanGenetics American Journal et al. Expert ReviewofEndocrinologyand Expert Multipleendocrineneoplasiatype 86 2005 5658–5671. R ACarvalhoandothers F1000Research 2012 Clinics 5 et al. 367–375. et al. 97 Clinicalpractice et al. 2007 2990–3011. Multipleendocrine et al. Journal ofClinical Journal (https://doi. Characterization 2017 (https://doi. 62 Guidelinesfor Endocrine et al. 2008 465–476. 6 Germ- Journal Journal 73. (https:// 159 1998 2017

Targeted NGSinMEN1 23 22 21 20 15 14 13 12 11 19 18 17 16 Concolino P, Costella A&Capoluongo E. Multipleendocrine Belar O, DeLaHoz C,Pérez-Nanclares G,Castaño L,Gaztambide S Mateo CM&Marx SJ. Raregermlinemutationsin Agarwal SK, Pellegata NS, Quintanilla-Martinez L,Siggelkow H,Samsom E, Agarwal SK, Kester MB,Debelenko LV,Agarwal SK, Heppner C,Emmert- Laat JM, vanderLuijt RB,Pieterman CR,Oostveen MP, Hermus AR, Marini F, Giusti F, Tonelli F &Brandi ML.Managementimpact: Goudet P, Murat A,Binquet C,Cardot-Bauters C,Costa A, Vierimaa O, Ebeling TM,Kytölä S,Bloigu S,Eloranta E,Salmi J, Lemos MC &Thakker RV. Multipleendocrineneoplasiatype1 Toledo RA, Lourenco DM,Coutinho FL,Quedas E,Machowiack I, Lemmens I, Van deVen WJ, Kas K,Zhang CX,Giraud S,Wautot V, Chandrasekharappa SC, Guru SC,Manickam P, Olufemi SE, (https://doi.org/10.1016/j.cancergen.2015.12.002) reported inthelastnineyears. neoplasia type1(MEN1):anupdate of208newgermlinevariants doi.org/10.1111/j.1365-2265.2011.04269.x) syndrome inSpain. AIP genesinpatientswithmultipleendocrineneoplasiatype1 & SpanishMEN1Group.NovelmutationsinMEN1,CDKN1Band jc.2008-2083) and Metabolism neoplasia type1andrelatedstates. cyclin-dependent kinaseinhibitorgenesinmultipleendocrine (https://doi.org/10.1073/pnas.0603877103) syndrome inratsandhumans. mutations inp27Kip1causeamultipleendocrineneoplasia Blink K, Höfler H,Fend F, Graw J&Atkinson MJ.Germ-line (https://doi.org/10.1002/humu.20605) Endocrine Neoplasiatype1andrelatedstates. et al. Buck MR, Skarulis MC,Doppman JL,Kim YS,Lubensky IA,Zhuang Z 2016 mutation-positive andmutation-negativepatients. Bisschop PH Dekkers OM, deHerder WW, vanderHorst-Schrivers AN,Drent ML, Cancer effects onqualityoflifeandprognosisinMEN1. org/10.1007/s00268-009-0290-1) patients. (Groupe d’EtudedesTumeurs Endocrines)cohortstudyamong758 et al. Ruszniewski P, Niccoli P, Ménégaux F, Chabrier G,Borson-Chazot F 2007 genotype–phenotype correlation. endocrine neoplasiatype1inNorthernFinland;clinicalfeaturesand Elovaara E Korpi-Hyövälti E, Niskanen L,Orvola A, org/10.1086/301729) ofHumanGenetics American Journal of mutationsinpatientswithmultipleendocrineneoplasiatype1. following identificationofthegene. (MEN1): analysisof1336mutationsreportedinthefirstdecade 2265.2007.02895.x) Endocrinology families withmultipleendocrineneoplasiatype1. Pereira MA Machado MC, Montenegro F, Cunha-Neto MB,Liberman B, (https://doi.org/10.1093/hmg/6.7.1177) Consortium onMEN1. the multipleendocrineneoplasiatype1(MEN1)gene.TheEuropean Buisson N, DeWitte K, Salandre J,Lenoir G org/10.1126/science.276.5311.404) endocrine neoplasia-type1. Liotta LA Collins FS, Emmert-Buck FR,Debelenko LV, Zhuang Z,Lubensky EA, Genetics Germlinemutationsofthe RiskfactorsandcausesofdeathinMEN1disease.AGTE 14 157 2017 1997 182. World ofSurgery Journal 285–294. et al. et al. 24 et al. 2007 (https://doi.org/10.1186/s12916-016-0708-1) 6 Positionalcloningofthegeneformultiple 2009 1169–1175. T227–T242. NovelMEN1germlinemutationsinBrazilian MEN1redefined,aclinicalcomparisonof 67 (https://doi.org/10.1530/EJE-07-0195) Clinical Endocrinology 94 377–384. Human MolecularGenetics 1826–1834. Downloaded fromBioscientifica.com at09/28/202104:26:31AM (https://doi.org/10.1093/hmg/6.7.1169) Science (https://doi.org/10.1530/ERC-17-0203) Cancer Genetics PNAS 2010 (https://doi.org/10.1111/j.1365- MEN1 European Journal ofEndocrinology European Journal 1998 1997 Journal ofClinicalEndocrinology Journal 2006 (https://doi.org/10.1210/ Human Mutation 34 geneinfamilialMultiple 62 249–255. 276 2012 103 179 232–244. et al. 404–406. 2016 Human Molecular 15558–15563. 76 :6 Identificationof 1997 Endocrine-Related 719–24. Clinical (https://doi. BMC Medicine et al. 209 2008 (https://doi. 6 (https://doi. Multiple 1177–1183. 36–41. (https:// 29 22–32. 404

via freeaccess European Journal of Endocrinology

38 36 35 34 33 32 31 30 29 28 27 26 25 24 37 Clinical Study Romero Arenas MA,Fowler RG,San Lucas FA, Shen J,Rich TA, Li Y, Peng Y, Jiang X,Cheng Y, Zhou W, Su T, Xie J,Zhong X,Song D, Newey PJ, Nesbit MA,Rimmer AJ,Attar M,Head RT, Christie PT, Cromer MK, Starker LF, Choi M,Udelsman R,Nelson-Williams C, Xuan J, Yu Y, Qing T, Guo L&Shi L.Next-generationsequencingin Korf BR &Rehm HL.Newapproachestomoleculardiagnosis. Ku CS, Cooper DN,Iacopetta B&Roukos DH.Integratingnext- Kamps R, Brandão RD,Bosch BJ,Paulussen AD,Xanthoulea S, Owens M, Ellard S&Vaidya B. Analysisofgross deletionsinthe Kong J, Wang O, Nie M,Shi J, Hu Y, Jiang Y, Li M,Xia W, Meng X& Tham E, Grandell U,Lindgren E,Toss G, Skogseid B& Toledo SP, Lourenço DMJr&Toledo RA. Adifferentialdiagnosisof Cuny T, Pertuit M,Sahnoun-Fathallah M,Daly A,Occhi G,Odou MF, Van Leeuwaarde RS,vanNesselrooij BP, Hermus AR,Dekkers OM, Pieterman CR, Schreinemakers JM,Koppeschaar HP, Vriens MR, surg.2014.08.073) patients. tumorigenicity pathwaysinmultiple endocrineneoplasiatype1 whole-exome sequencingrevealsmutations thatimplycommon Grubbs EG, Lee JE,Scheet P, Perrier ND&Zhao H.Preliminary 2017 tumor withectopicACTHsyndrome. Wu L E1995–E2005. adenomas. (sporadic)parathyroid sequencing studiesofnonhereditary Mihai P Stechman M,Gregory L, Gorvin CM, doi.org/10.1210/jc.2012-1743) Clinical EndocrinologyandMetabolism parathyroid tumorsusingwhole-exomesequencing. Lifton RP &Carling T. Identificationofsomatic mutationsin (https://doi.org/10.1016/j.canlet.2012.11.025) the clinic:promisesandchallenges. 2013 org/10.1111/cge.12028) cancer predisposition. generation sequencingintothediagnostictestingofinherited org/10.3390/ijms18020308) ofMolecularSciences Journal International genetic diagnosis,riskpredictionandcancerclassification. Blok MJ &Romano A.Next-generationsequencinginoncology: j.1365-2265.2007.03045.x) Clinical Endocrinology MEN1 geneinpatientswithmultipleendocrineneoplasiatype1. 2016 Type hyperparathyroidisminChinese. 1-related primary Xing X. Clinicalandgeneticanalysisofmultipleendocrineneoplasia org/10.1210/jc.2007-0476) Endocrinology andMetabolism in Sweden:areporton200unrelatedcases. Nordenskjöld M. ClinicaltestingformutationsintheMEN1gene 2013 inherited endocrinetumorsandtheirtumorcounterparts. 2013 don’t forgetMEN1geneticanalysis. macroadenomas:besidesAIP young patientswithsporadicpituitary Tabarin A, Nunes ML,Delemer B,Rohmer V doi.org/10.1210/jc.2015-3766) Clinical EndocrinologyandMetabolism in MEN1.ResultsfromtheDutchMEN1StudyGroup. Havekes B, Vriens MR de Herder WW, vanderHorst-Schrivers AN,Drent ML,Bisschop PH, 575–581. genetic screeningonclinicaloutcome. endocrine neoplasiatype1(MEN1):itsmanifestationsandeffectof Rinkes IH, Zonnenberg BA,vanderLuijt RB&Valk GD. Multiple 176 309 11 68 168 et al. e0166634. 1039–1056. Surgery 187–194. 1511–1521. 533–541. (https://doi.org/10.1111/j.1365-2265.2008.03324.x) Whole exome sequencing of thymic neuroendocrine Wholeexomesequencingofthymicneuroendocrine Journal ofClinicalEndocrinologyandMetabolism Journal (https://doi.org/10.1210/jc.2012-2303) 2014 (https://doi.org/10.1371/journal.pone.0166634) (https://doi.org/10.1530/EJE-16-0546) (https://doi.org/10.1530/EJE-12-0763) (https://doi.org/10.6061/clinics/2013(07)24) 2008 156 et al. (https://doi.org/10.1001/jama.2013.3239) Clinical Genetics 1351–1357. Impactofdelayindiagnosisoutcomes 68 2007 350–354. 92 European Journal ofEndocrinology European Journal Cancer Letters 2016 2012 European Journal ofEndocrinology European Journal 3389–3395. R ACarvalhoandothers 2013 (https://doi.org/10.1016/j. Clinical Endocrinology 2017 (https://doi.org/10.1111/ 97 101 Journal ofClinical Journal 83 et al. E1774–E1781. 18 et al. 1159–1165. 2–6. E308. Geneticanalysisin 2013 (https://doi. Whole-exome (https://doi. Journal of Journal Journal of Journal (https://doi. 340 PLoS ONE 2012 Clinics 284–295. (https:// 2009 (https:// JAMA 97

70

Targeted NGSinMEN1 39 44 43 42 41 40 51 50 49 48 47 46 45 Kim BY, Park MH,Woo HM, Jo HY, Kim JH,Choi HJ&Koo SK. Toledo RA &Dahia PL.Next-generationsequencingforthegenetic Marini F, Giusti F&Brandi ML.Genetictestinmultiple endocrine De Sousa SM,McCabe MJ,Wu K, Roscioli T, Gayevskiy V, Brook K, Backman S, Norlén O,Eriksson B,Skogseid B,Stålberg P& Isakov O, Rinella ES,Olchovsky D,Shimon I,Ostrer H,Shomrom N Gonçalves TD, Toledo RA, Sekiya T, Matuguma SE,MalufFilho F, Coutinho FL, Lourenço DMJr, Toledo RA, Montenegro FL,Correia- Lourenço DM Jr, Coutinho FL, Toledo RA, Montenegro FL,Correia- &Browning BL.Considerationofcosegregationinthe Jarvik GP Richards S, Aziz N,Bale S,Bick D,Das S,Gastier-Foster J,Grody WW, Rehm HL, Bale SJ,Bayrak-Toydemir P, Berg JS,Brown KK,Deignan JL, Crona J, Ljungström V, Welin S, Walz MK, Hellman P&Björklund P. sequencing. with multipleendocrineneoplasiatype1usingwhole-exome Genetic analysisofparathyroidandpancreatictumorsinapatient wjem.v5.i2.124) Experimental Medicine neoplasia type1syndrome:anevolvingstory. EJE-16-0944) ofEndocrinology Journal patients andfamilieswithadditionalendocrinetumours. tumoursyndromegenesarecommoninyoung in familialpituitary Rawlings L, Scott HS,Thompson TJ,Earls P anticanres.11367) Anticancer Research neuroendocrine tumorsusingtargeteddeepsequencing. Crona J. Detectionofsomaticmutationsingastroenteropancreatic doi.org/10.1017/S0016672313000141) hyperparathyroidism. through wholeexomesequencingco-segregateswithfamilial & Friedman E.MissensemutationintheMEN1genediscovered org/10.1186/s12881-017-0465-9) Metabolism in theseconddecadeoflife. neuroendocrine tumorsinmultiple endocrine neoplasiaType 1 et al. Rocha MS, Siqueira SA,Glezer A,Bronstein MD, Pereira MA 2265.2009.03672.x) Endocrinology endocrine neoplasiatype1aftertotalparathyroidectomy. hyperparathyroidism associatedwithmultiple with primary Deur JE &Toledo SP. Bonemineraldensityanalysisinpatients org/10.1002/jbmr.125) ofBoneandMineralResearchJournal hyperparathyroidism. endocrine neoplasiatype1-associatedprimary and severebonemineralrenalcomplicationsinmultiple Deur JE &Toledo SP. Early-onset,progressive,frequent,extensive, ajhg.2016.04.003) of HumanGenetics pathogenicity classificationofgenomicvariants. Medicine Genomics andtheAssociationforMolecularPathology. recommendation oftheAmericanCollegeMedicalGeneticsand for theinterpretationofsequencevariants:ajointconsensus Hegde M, Lyon E, Spector E Medicine standardsfornext-generationsequencing. laboratory Friez MJ, Funke BH,Hegde MR,Lyon E pone.0133210) PLoS ONE next generationsequencing:experiencefrompheochromocytoma. Bioinformatic challenges in clinical diagnostic application of targeted (https://doi.org/10.1111/cen.12357) new wave,butwithcaution. screening ofphaeochromcytomasandparagangliomas:ridingthe Penetranceoffunctioningandnonfunctioning pancreatic 2015 2013 2015 2014 BMC MedicalGenetics 2010 17 15 10 99 405–424. 733–747. 2016 e0133210. 2017 72 E89–E96. . 2015 462–468. Genetics Research 2017 98 37 1077–1081. Downloaded fromBioscientifica.com at09/28/202104:26:31AM 705–712. (https://doi.org/10.1038/gim.2015.30) (https://doi.org/10.1038/gim.2013.92) 5 et al. Journal ofClinicalEndocrinologyand Journal 176 Clinical Endocrinology 124–129. (https://doi.org/10.1371/journal. (https://doi.org/10.1210/jc.2013-1768) (https://doi.org/10.1111/j.1365- Standardsandguidelines 635–644. 2017 2010 (https://doi.org/10.21873/ 2013 et al. (https://doi.org/10.1016/j. (https://doi.org/10.5493/ 18 25 106. 179 (https://doi.org/10.1530/ et al. 2382–2391. ACMGclinical 95 https://eje.bioscientifica.com World of Journal :6 114–120. (https://doi. Germlinevariants American Journal American Journal 2014 Genetics in 80 Genetics in (https://doi. European (https:// Clinical 23–24. 405 via freeaccess European Journal of Endocrinology https://eje.bioscientifica.com

63 62 61 60 59 58 57 56 55 54 53 52 Clinical Study Peculis R, Balcere I,Rovite V, Megnis K, Valtere A, Stukens J, Toledo RA, Hatakana R, Lourenço DM Jr, Lindsey SC,Camacho CP, Mutch MG, Dilley WG,Sanjurjo F, DeBenedetti MK,Doherty GM, Clerici T, Schmid C,Komminoth P, Lange J,Spinas GA&Brändle M. Kytölä S, Villablanca A, Ebeling T, Nord B,Larsson C,Höög A, Burgess JR, Nord B,David R,Greenaway TM,Parameswaran V, Olufemi SE, Green JS,Manickam P, Guru SC,Agarwal SK, Marini F, Giusti F, Fossi C,Cioppi F, Cianferotti L,Masi L,Boaretto F, Debelenko LV,Agarwal SK, Kester MB,Guru SC,Manickam P, Sekiya T, Bronstein MD,Benfini K,Longuini VC,Jallad RS, Longuini VC, Lourenço DMJr, Sekiya T, Meirelles O,Golcalves TD, Toledo RA, Lourenço DMJr, Liberman B,Cunha-Neto MB, occurrence and characteristics of pituitary adenomas. occurrence andcharacteristicsofpituitary Polymorphisms inMEN1andDRD2 genesareassociatedwiththe Arnicane L, Nikitina-Zake L,Lejnieks A, Pirags V ERC-14-0491) Endocrine-Related Cancer thyroidcarcinomano associationwithmedullary susceptibility. Comprehensive assessmentofthedisputedRETY791Fvariantshows Almeida M, Lima JVJr, Sekiya T, Garralda E,Naslavsky MS HUMU1>3.0.CO;2-R) (https://doi.org/10.1002/(SICI)1098-1004(1999)13:3<175::AID- frequent splicingdefects. in themultipleendocrineneoplasiatype1gene:evidencefor Wells SA Jr, Goodfellow PJ&Lairmore TC.Germlinemutations 381–386. assessment ofscreeningmethods. 10 Swisskindredswithmultipleendocrineneoplasiatype1: jmg.38.3.185) of MedicalGenetics multiple endocrineneoplasiatype1(MEN1)inFinland. Wong FK, Välimäki M, Vierimaa O, Teh BT j.1365-2265.2000.01032.x) Clinical Endocrinology large familywithmultipleendocrineneoplasiatype1(MEN1). relationship betweengenotypeandclinicalphenotypeinasingle Larsson C, Shepherd JJ&Teh BT. Phenotypeandphenocopy:the HUMU2>3.0.CO;2-V) (https://doi.org/10.1002/(SICI)1098-1004(1998)11:4<264::AID- from Newfoundland. prolactinoma variantofMEN1(MEN1Burin)infourkindreds ancestral mutationintheMEN1geneislikelyresponsiblefor Kester MB, Dong Q,Burns AL,Spiegel AM,Marx SJ org/10.1007/s12020-018-1566-8) MEN1 patientdatabase. 1: analysisofgermlineMEN1mutationsintheItalianmulticenter Zovato S, Cetani F, Colao A 1004(1998)12:2<75::AID-HUMU1>3.0.CO;2-T) Mutation gene encounteredinapparentlyunrelatedfamilies. et al. Olufemi SE, Skarulis MC,Heppner C,Crabtree JS,Lubensky IA org/10.1530/ERC-13-0486) vitro study. p27 variantandcorticotropinomasusceptibility:ageneticin Machado MC, Goncalves TD,Osaki LH,Higashi L,Viana-Jr J org/10.1530/EJE-14-0130) ofEndocrinology European Journal multiplicity inpatientsharboringMEN1germlinemutations. Association betweenthep27RS2066827variantandtumor Coutinho FL, Francisco G,Osaki LH,Chammas R,Alves VA jc.2006-2394) and Metabolism in familialsomatotropinoma. hydrocarbonreceptorinteractingproteingene mutation inthearyl Cavalcanti MG, Moyses CB,Toledo SP &Dahia PL.Germline AnalysisofrecurrentgermlinemutationsintheMEN1 1998 (https://doi.org/2001/25/smw-09730) Endocrine-Related Cancer 12 2007 75–82. 2001 2000 92 Human Mutation 2015 1934–1937. 38 Endocrine (https://doi.org/10.1002/(SICI)1098- Human Mutation 185–189. 53 et al. 205–211. 22 Journal ofClinicalEndocrinology Journal 2014 Multipleendocrineneoplasiatype 65–76. 2018 Swiss MedicalWeekly 2014 (https://doi.org/10.1210/ (https://doi.org/10.1136/ R ACarvalhoandothers 1998 171 62 (https://doi.org/10.1046/ (https://doi.org/10.1530/ 21 1999 335–342. 215–233. et al. 11 395–404. 264–269. Foundereffectin 13 et al. 175–185. et al. Human (https://doi. (https://doi.

European 2001 (https://doi. Common Journal Journal et al. et al. 131 et al.

Targeted NGSinMEN1 76 75 74 73 72 71 70 69 68 67 66 65 64 Asteria C, Faglia G,Roncoroni R,Borretta G, Ribotto P, Beck-Peccoz P. Martín-Campos JM, Catasús L,Chico A,Mayoral C,Lagarda E, Görtz B, Roth J,Krähenmann A,deKrijger RR,Muletta-Feurer S, Morelli A Falchetti AMartineti VBecherini LMark MFriedman E Teh BT Kytölä SFarnebo FBergman LWong FK Weber G Hayward N Wautot V Vercherat C Lespinasse JChambe BLenoir GMZhang CX Bergman L Teh B Cardinal JPalmer JWalters M Shepherd J Ozturk M Chiu CYAkdeniz NJenq SFChang SCHsa CYJap TS. Klein RD Salih SBessoni JBale AE.Clinicaltestingformultiple Cebrián A Ruiz-Llorente S Cascón A Pollán M Díez JJ Picó A Tellería D Ellard S Hattersley AT Brewer CMVaidya B. Detection ofanMEN1 Toledo RA, Burnichon N,Cascon A,Benn DE,Bayley JP, Welander J, Guan B, Welch JM, Sapp JC,Ling H,Li Y, Johnston JJ,Kebebew E, Journal ofEndocrinology Journal Identification ofthreenovelmenin mutations(c.741delGTCA, 8 mutations affectingMEN1gene. type I:twonovelgermlinemutations andupdatedclassificationof Blanco-Vaca F. Molecularpathologyofmultiple endocrineneoplasia Gallart L, Mato E,Rodríguez-Espinosa J,Matías-Guiu X,DeLeiva A, Pathology Tumors andNotRestrictedtoForegutNeoplasms. with aSubsetofSporadicEndocrinePancreaticandNeuroendocrine Mutations andAllelicDeletionsoftheMEN1GeneAreAssociated Rütimann K, Saremaslani P, Speel EJ,Heitz PU,Komminoth P. 2000 multiple endocrineneoplasiatype1. Brandi ML. MEN1genemutationanalysisinItalianpatientswith 1998 hyperparathyroidism. neoplasia type1,familialacromegalyandisolated et al. Cardinal J Khodaei SParente FTranebjaerg L Jorde RSalmela P Alford F Hurley DGrimmond SSilins GWalters M Stewart C Larsson C Skogseid BBeckers APhelan CEdwards MEpstein M org/10.1002/humu.10092) the MEN1protein. for correlationbetweenphenotypeandthefunctionaldomainsof profile ofMEN1inmultipleendocrineneoplasiatype1:search Porchet N Cordier MBéroud C Calender A.Germlinemutation bjoc.2000.1380) of Cancer in familieswithMEN1andrelateddisorders. Cameron D Hayward N.IdentificationofMEN1genemutations 29 Medicine endocrine neoplasiatype1inDNAdiagnosticlaboratory. ofMedicalGenetics Journal MEN1 geneandcorrelationwithclinicalfeaturesinSpanishpatients. Benítez J Robledo M.Mutationalandgrossdeletionstudyofthe 2265.2005.02190.x) Endocrinology referral criteriafordiagnosticmoleculargenetictesting. gene mutationdependsonclinicalfeaturesandsupportscurrent nrendo.2016.185) Endocrinology phaeochromocytomas andparagangliomas. generation-sequencing-based diagnostictestingofhereditary Tops CM, Firth H,Dwight T ajhg.2016.08.018) Human Genetics in familialisolatedhyperparathyroidism. Biesecker LG, Simonds WF, Marx SJ EJE-15-0879) endocrine neoplasia-1. Two novel mutationsintheMEN1genesubjectswithmultiple 195–204. 523–527. MutationanalysisoftheMEN1geneinmultipleendocrine 142 83 2621–2626. 2005 2000 1999 131–137. 2005 2017 7 83 154 2016 131–138. 1009–1014. 62 13 Human Mutation 429–436. (https://doi.org/10.1210/jc.83.8.2621) 99 169–175. 233–247. Journal ofClinicalEndocrinology&Metabolism Journal 2016 Journal ofEndocrinologicalInvestigation Journal 1034–1044. 2003 Downloaded fromBioscientifica.com at09/28/202104:26:31AM et al. 175 (https://doi.org/10.1054/ 40 (https://doi.org/10.1111/j.1365- (https://doi.org/10.1038/ Diagnostic MolecularPathology 145–153. ConsensusStatementonnext- e72. 2002 et al. (https://doi.org/10.1016/j. European Journal ofEndocrinology European Journal GCM2-activatingmutations American Journal of American Journal 20 179 (https://doi.org/10.1530/ Nature Reviews 35–47. British Journal British Journal :6 American Journal of American Journal (https://doi. Clinical Genetics in

2006 1999 406 via freeaccess

European Journal of Endocrinology

77 Clinical Study Heppner C, Kester MB, Agarwal SK, Debelenko LV,Heppner C, Kester MB,Agarwal SK, Emmert-Buck MR, Chandrasekharappa SC, Collins FS,Spiegel AM,Burns AL,Marx SJ. Alexander RH, Kim YS,Saggar SK,Lubensky IA,Zhuang Z,Liotta LA, Guru SC, Manickam P, Olufemi SE,Skarulis MC,Doppman JL, mutational screening. multiple endocrineneoplasiatype1:Additionalinformationfor c.1348T>C, c.1785delA)inunrelatedItalianfamiliesaffectedwith Human Mutation R ACarvalhoandothers 2001 17 237. Accepted 24September2018 Revised versionreceived8September2018 Received 19May2018

Targeted NGSinMEN1 78 Warner J, Epstein M,Sweet A,Singh D,Burgess J,Stranks S,Hill P, Journal ofMedicalGenetics Journal hyperparathyroidism: unexpectedresultsandtheirimplications. Teh BT, Prins JB,Cardinal J.Genetictestinginfamilialisolated Learoyd D,Robinson B,Birdsey P,Perry-Keene D, Mackenzie E, Genetics Somatic mutationoftheMEN1geneinparathyroidtumours. 1997 16 375–378. 2004 Downloaded fromBioscientifica.com at09/28/202104:26:31AM 41 155–160. 179 https://eje.bioscientifica.com :6 Nature 407 via freeaccess