Saúde Pública, Salvador, BA, Brasil Izabel,Salvador,Santa BA, Brasil Medicina eSaúdePública. Hospital Saúde Humana,EscolaBahiana de Investigativa, Salvador, BA, Brasil Biotecnologia emSaúdeeMedicina Moniz (CPqGM),Cursode Centro dePesquisas Gonçalo Bahia (UFBA), Salvador, BA, Brasil Saúde, Universidade Federal da (LET), Instituto deCiênciasda Laboratório deEstudo da Tireoide DOI: 10.1590/2359-3997000000100 Accepted onJul/27/2015 Received onJun/5/2015 [email protected] 40110-102 –Salvador, BA, Brasil Av. Reitor MiguelCalmon,s/n,sala301 deBiorregulação Departamento Instituto deCiênciasdaSaúde, Universidade Federal daBahia, Taise LimadeOliveira Cerqueira Correspondence to: 6 5 4 3 2 1 Copyright© AE&M all rights reserved. Excepcionais, APAE, Salvador, BA, Brasil Associação dePais e Amigos dos Escola Bahiana deMedicinae Grupo Fleury, Salvador, BA, Brasil Pós-graduação emMedicinae Fundação Oswaldo Cruz (Fiocruz), deBiorregulação, Departamento 562 of patientswithTD(2-5). in onlyasmall proportion However, inthesegeneshavebeenfound abnormalities PAX8 havebeendescribedascauses ofhumanTD(1). homeodomainfactor box E1(FOXE1)andthepaired factor 1(TITF-1,alsoknownas NKX2.1), forkhead transcription for the etiology of TD;thus,thethyroid candidategenes wouldbestrong cific geneexpression, spe organogenesis and thyroid inthyroid role portant eral specifictranscriptionalfactors,inviewoftheirim hemiagenesis(1).Sev hypoplasia,andthyroid thyroid ectopy,cluding thyroid agenesis or athyreosis, thyroid C INTRODUCTION developmental abnormalities of the thyroid glandin ofthethyroid developmental abnormalities dysgenesis(TD),whichare cases, CHisduetothyroid case report 1:3,500-1:4,000 newborns. In80-85%ofthe 1:3,500-1:4,000 newborns. (CH)occursin ongenital Helton Ramos Anabel Costa Anabel and echocardiography. DNAwas extractedand in 86children withthyroid dysgenesis(TD)usingthyroid functiontests,scintigraphy, ultrasound Thyroid dysgenesis;congenitalhypothyroidism;heartdisease;NKX2.5 Keywords heart disease(CHD)and TD. Daniel San Martin Daniel San with thyroid hypoplasia. with thyroid dysgenesis thyroid hypoplasiainchildren NKX2.5 geneisassociatedwith The c.63A>Gpolymorphisminthe Taise LimadeOliveira Cerqueira Conclusion: The c.541G>Apolymorphismwas observedinonlyonepatientwithisolatedthyroid hypoplasia. with CHD). There was asignificantassociationofc.63A>Gpolymorphismwith hypoplasia (p<0.036). c.63A>G polymorphismwas detectedin54/86patients(49withisolated TD and5with TD combined sociated withCHD.Noneofthemarepredictedtoresultincodonchange inconserveddomain. The merase chain reaction(PCR)andsequenced. Objective: ABSTRACT The mutationscreeningrevealedtwoknownpolymorphismsinpatientswithisolated TD or TD as To search for genetic alterationin NKX2.5 NKX2.5 3 1,2 , Vladimir Fernandes , Vladimir mutations werenotfound. The c.63A>Gpolymorphismmightbeassociated 1 , Jesus Mariana Arch EndocrinolMetab. 2015;59(6):562-7 Subjects andmethods: - - - - is expressed in early heart progenitor cells,aswellinthy progenitor inearlyheart is expressed MurineNkx2.5 organogenesis inthedevelopingembryo. three mutations in four patients (7). mutations infourpatients(7). three in 241patientswithTDItaly, allowingidentification of conducted human TD,includingamutational screening mutationsinvolvedinthepathogenesisof heterozygous theidentification of inthis genehavearisedfrom interest with CHthaninthegeneralpopulation(11,12).Clinical inchildren of fallot(10,11).CHDhasahigherfrequency atrial septaldefect,ventriculardefectandtetralogy onewere Disease(CHD)andthemostfrequents Heart 600584) havebeendescribedinpatientswithCongenital Several lossoffunctionmutationsinNKX2.5 morphogenesis,myogenesisandfunction(8,9). heart transcription factorisknowntobeessentialfornormal tongue,stomachandspleen(3,6,7).TheNKX2.5 roid, NKX2.5 1,2 1 , YanneRamos , Jailciele Gonzaga 4 , TatianaAmorim Results: NKX2.5 NKX2.5 NKX2.5 appears to function during the early period of appears tofunctionduringtheearlyperiodof Individual phenotypeswerecarefullyanalyzedIndividual CHD werefoundin8.1%ofpatientswith TD. inpatientspresentingbothcongenital genecodingregionwas amplifiedbypoly 1 , Giorgia Strappa 5 , Ladeia Ana Marice 1 , Paulo Ferreira Arch Metab. Endocrinol 2015;59/6 1 ,

1 ,

(OMIM (OMIM 6 ,

- - -

pharyngeal floor at embryonic day8.5 (E8.5),ape floor atembryonic pharyngeal cellsinthe ofthyroidal demonstrated inprecursors Eight hundred fifty one patients were confirmed for for confirmed fiftyone patientswere Eight hundred gland orgoiter. eutopic thyroid genesis and5)normal 1)ectopy,groups: 2)agenesis,3)hypoplasia,4)hemia dividedinto five were children totheresults, According trasound after30daysofL-T4 therapydiscontinuation. and TT4),thyroid functiontests(TSH thyroid sient, consistingofserum or tran if the CH is permanent to determine protocol 3years,theyfollowa and TT4.Whentheinfantsreach TSH toserum ring infancyandchildhoodaccording kg/day, and L-T4 dosage was adjusted du was started 10-15m treatment, (L-T4)replacement vothyroxine results, le of mU/L forTSH.Aftertheconfirmation 6-12m are intervals reference total T4(TT4)]employingchemoluminescentassays; TSHand ofdiagnosis[serum taken forconfirmation andabloodsamplewas physical examinationperformed anda theneonateshadahistory result, tive screening Within 24hofaposi mer immunofluorimetricassay). 9 mU/L,DelphiaPerkin-El a heelprick(TSHcut-off collectedafter24hoursoflifefrom TSH measurement bloodspot gram oftheStateBahia,Brazilbydry pro screening withCH in theneonatalthyroid borns Between 2001and2013wehaveidentified1,051 new­ Subjects SUBJECTS ANDMETHODS associated withhypoplasia. CHD. However, thevariantc.63A>Gwassignificantly TD,evenincombination with in patientspresenting indicate that no NKX2.5 senting bothCHDandTDorisolatedTD.Ourresults NKX2.5 of thequestionifgeneticabnormalities us toaddress NKX2.5 a functional impact of propose (7). These observations development ofthyroid ponent ofthegeneticcontrol cells,indicatingthatNkx2.5 dermal morphogenesis. thyroid in normal required at an early stage of development, it might be mordium onceNkx2.5 fore, E12.5(12).There and Pax8,butdisappearsaround riod coincidentwiththeappearanceofTitf-1 , Arch Metab. Endocrinol 2015;59/6 embryos exhibited a smaller outgrowing budofendo exhibitedasmalleroutgrowing embryos In mouse,Nkx2.5 on geneticpathogenesisofTDandmotivate could play an important role inpatientspre role could playanimportant mRNA is present in the thyroid pri inthethyroid mRNA ispresent 123 I or expression has been recently hasbeenrecently expression 131 mutations are not common mutations are I scanning, and thyroid ul I scanning,andthyroid g/dL for TT4 and 0.3-4 g/dL forTT4and0.3-4 is required ascom is required Nkx2.5 PAX8 g/ -/------

our institution. our institution. which710havebeenfollowedin CH,from permanent Genotype analysis by theFederalUniversityofBahia. approved withprotocols inaccordance all participants of theparents consentwasobtainedfrom ten informed withoutknowledgeofgenotype.Writ performed were Clinical studies present. were nized genetic syndromes orotherrecog congenitalmalformations non-cardiac whetherany todetermine reviewed of allpatientswere forthepurposeofthisstudy). Medicalrecords formed wasnot per (i.e., clinical testing of parents report chart disease was investigated by of CHD and thyroid history 2-dimension transthoracicechocardiography. Family (EKG),and examination, 12-leadelectrocardiogram evaluated by history, physical of medical records, review phenotypewas cal andmolecularstudies.Thecardiac Germany) and sequenced using the ABI PRISM Dye andsequenced usingtheABIPRISM Dye Germany) QuickPCRPurificationkit(Invitrogen, with PureLink purified were reaction ofexon2.PCRproducts the first fortheexon1and reagents was addedtostandard sion periodat72C.DMSO (0.2mL/20-.Lreaction) seconds at72Candfinishedwitha10-minuteexten 45 secondsat95C,3059Cor60C,and with2minutesat 95C followed by 35 cycles of started extension periodat72C.Fortheexon2,allreactions and 30secondsat72Cfinishedwitha10minutes wed by35cyclesof30seconds94C,at56C phate; 1,5nMofMgSO denosine triphosphate,anddeoxythymidinetriphos oligonucleotide primers;200μmol/Leachofdeoxya amplified ina25-μLvolumecontaining:40ngofeach GAGAGTCAGGGA-3’. 100ngofgenomicDNAwas GACTCTGGAGCTGGTGG-3’ and 2BR 5’-CCC 2AR 5’-TAGGGATTGAGGCCCACG-3’, 2BF5’-CA 2AF5’-GCGCTCCGTAGGTCAAGC-3’,introns: theflanking following twopairofprimersderivedfrom amplifiedbyatotalof2PCRswiththe exon 2were and 1R 5´-CTCCTGGCCCTGAGTTTCTT-3´. The of primers:1F5´-CTTGTGCTCAGCGCTACCT-3´ amplifiedusingtwopair tion (PCR).Theexon1were reac genomicDNAbypolymerase chain amplified from NKX2.5 ofthe techniques.Thecodingregion using standard wholeblood Deoxyribonucleic acidwasextractedfrom reaction of exon 1 started with5minutesat94Cfollo ofexon1started reaction We haveselected86patientswithTDforclini gene, including exon/intron boundaries, was boundaries, was gene, includingexon/intron NKX2.5 polymorphismincongenitalhypothyroidism 4 andTaq polymerase. The 563 ------

Copyright© AE&M all rights reserved. Copyright© AE&M all rights reserved. 564 scintigraphy.an ectopicgland detectedbythyroid This Tglevelsof17.3ng/mLand associated withserum gland byultrasound (Patient D)hadanabsentthyroid ation (Table 1). InFamily4,theproband 1,Figure evalu atrialseptaldefectoncardiac lies 1-3presented fami from byultrasound.Allthe propositus confirmed was40uIU/MLandahypoplasticgland screening 1),theneonatalTSH In family3(PatientC,Figure gland(Tabletected ahypoplasticthyroid 1). 1,Figure (Patient B)wasdiagnosedlatelyandtheultrasoundde gland (Table 1).Infamily2,amalepatient 1,Figure scanshowedanectopic was 4.5ng/mLandthethyroid Tg trasound showedanagenesis,butthelevelofserum ing, whentheTSHlevelwas91.4uIU/ML.Theul 1)wasdetectedbyneonatalscreen (Patient A,Figure daughter Heraffected autoimmune hypothyroidism. (Table 1).Infamily1,themotherwasdiagnosedwith the c.63A>G NKX2.5 polymorphism CHD harbored (Table 1). 2 hemiagenesis). The overall female:male ratio was 2:1 isolated TD(32ectopy, 15agenesis,30hypoplasiaand with TD (7/86;8.1%).Seventynineinfantspresented We ofCHDinpatientswith foundahighprevalence Clinical observations RESULTS analyzed byX andNKX2.5polymorphismswas in eachsubgroup ofCHD The associationofsubtypeTDorpresence ween eachcategorizationandpatientswithoutCHD. each typeofTD,thuswetestedfordependencebet diseasewith ofpositiveheart table comparingthegroup ofTD.Weeach subgroup builtatwo-waycontingency andthegeneralpopulationbetween in ourcohort ofNKX2.5polymorphisms inthefrequency differences We used Statistical analysis fortheidentifiedsequencealterations. screened individualswere normal frame.Onehundred reading examinedincontextoftheopen and alterationswere analyzed with Bioedit Software zer). Sequences were by anautomatedsequencer(ABI3100geneticanaly sequencingwasperformed Bidirectional instructions. tothemanufacturer’s Applied Biosystems)according Terminator cycle sequencingReadyReactionkit(PE NKX2.5 polymorphismincongenitalhypothyroidism Five from seven patients with TD associated with Five from t test andHardy-Weinberg testtoestimatethe 2 (Fisher’sExactTest). ------

by ultrasound (Figure 1,Tableby ultrasound(Figure 1). andhypoplasia was observed days of birth twenty-three The patientoffamily5(PatientE)wasdiagnosedwith stenosis(Table 1). 1,Figure patient hadpulmonary phenotype associated withCHD Table 1. insevenpatientswith Phenotype/Genotype summary TD of cases(Table 2).Among patientspositive 2)(Figure (38.3%) ofpatientsandinhomozigozity in21(24.3%) in33 polymorphism wasdetected inheterozigozity polymorphisms found.The knownc.63A>GNKX2.5 both TDandCHDorisolated TD.Table 2showsthe We did not find any Correlating hypoplasia; halfblacksymbolrepresentaindividualwithhypothyroidism. individuals; solidblacksymbols, affectedbyhypothyroidismwithectopy or analyzed. Squares, men;circles, women;opensymbols, clinicallyunaffected polymorphism associatedwith TD and CHD. All family members were Figure 1. Pedigrees of thefive familieswithpatientsharboringthe c.63A>G ASD: atrialseptaldefect;PS: stenosis; pulmonary AVB: atrio-ventricularblock. Patient Ectopy 7 6 5 4 3 2 1 A Family 3 Family 1 Hypoplasia Gender Female Female Male Male Male Male Male C NKX2.5 polymorphismsandclinical phenotype mutation in patients with NKX2.5 mutation in patients with Hypoplasia Hypoplasia Hypoplasia Hypoplasia Hypoplasia Thyroid Ectopy Ectopy Family 2 Family 4 Ectopy Hypoplasia D Arch Metab. Endocrinol 2015;59/6 B Lown-Ganong- phenotype Syndrom Cardiac Levine ASD ASD ASD ASD AVB PS Hypoplasia Family 5 Polymorphism E c.63A>G c.63A>G c.63A>G c.63A>G c.63A>G No No left: wildtype sequence;Bottom, right: heterozygousG-to-A transitionatposition541ofcodon 181intheNKX2.5gene. 21 intheNKX2.5 Figure 2. Chromatogramsshowingthepolymorphisms foundinexon2ofNKX2.5 Table 2. NKX2.5polymorphismsidentifiedamong86patientswith TD ved in TD etiology. in human ontoge The importance morphogenesis, wehypothesizedthatitmightbeinvol inperturbed results anditstargeted disruption embryos lineageofdevelopingvertebrate in thethyroid expressed Since DISCUSSION gland. ahypoplasticthyroid (TSH 32.2uIU/ML)presenting 2). Agirl,diagnosedinneonatalscreening (Figure in heterozigozity, inonly1patientwithisolatedTD with hypoplasia(p<0,036). (Table 2).Thepolymorphismc.63A>Gwasassociated (18 ectopy, 24hypoplasia, 6agenesis,1hemiagenesis) for thec.63A>Gpolymorphism,49hadisolatedTD Arch Metab. Endocrinol 2015;59/6 A second polymorphism, c.541G>A was observed, A secondpolymorphism,c.541G>Awasobserved, is one of the earliest transcription factors NKX2.5 isoneoftheearliesttranscriptionfactors Polymorphism rs72554028 rs2277923 c.541G>A p.Gln181 c.63A>G p.Glu21 gene; topmidle: heterozygous A-to-G transitionatposition63ofcodon21intheNKX2.5 Homeodomain TN domain Site Polymorphism type - - Silent Silent because we analyzed patients selected from an entire anentire because weanalyzedpatientsselectedfrom role patient’s selectionmayhaveplayedanimportant ofCH,thusthe identifiedinfamiliargroups previously subjects (13,14).Candidategenemutationshavebeen ed inotherstudiesandwehavealsofoundnormal hypoplasiaasphenotype. morphism andthyroid but apositiveassociationbetweenthec.63A>Gpoly nosignificantvariation a familyhistory, weobserved ofDTpatientswithout in theNKX2.5gene,agroup byDenticeandcols.(7). genesis ofTD,firstlyprovided the possiblecontributionofNKX2.5 forCH,inBrazil,wehypothesized Program Screening In thisstudyof86infantswithTDdetectedbyNeonatal patients withNKX2.5mutationassociatedTD(7). identification of by recent underscored nesis was further gene. Top,Left: G polymorphism was previously detect This A→Gpolymorphismwaspreviously inactivatingmutation forgermeline In ourscreening Allele frequency inpatients homozygous A-to-G transitionatposition63ofcodon NKX2.5 polymorphismincongenitalhypothyroidism G/G 0.988 G/G 0.243 G/A 0.012 A/G 0.372 A/A 0.000 A/A 0.385 gene; topright: wildtypesequence; Bottom, Frequency incontrols gene tothepatho G/G 1.000 G/G 0.000 A/G 0.160 G/A 0.000 A/A 0.000 A/A 0.840 Allele 565 - - -

Copyright© AE&M all rights reserved. Copyright© AE&M all rights reserved. 566 gland(18). Although acooperative thethyroid to form caudalsideoftheembryo tothemore primitive streak the cellsmigrate through the humanwhenprogenitors day(E)20-22in embryonic begins atapproximately formation unknown(17).Thyroid lar mechanismsare althoughthemolecu andembryo, developing heart fate in the of the endocardial/endothelial regulation in development,suggesting role tors duringcardiac that genetic mechanicalfactors(2).Recentdatarevealed epi by endothelialcellsandmayinvolveinputfrom has beenshowntobeassociatedwithfactorssecreted cells Indeed, theinitialinductionoffollicularthyroid teins includingionchannelsandsignalingmolecules. andencodepro expressed uniquely ordifferentially andlobulation(2).Suchgenes maybeeither growth guidanceofbilateral fortheproper cal stepnecessary outflowtractatacriti thecardiac sels derivedfrom to establish contact withves thyroid the embryonic of glandduetoafailure and hypoplasiaofthethyroid T-box in hemiagenesis Tbx1 results indicatingthatdeficiencyofthe for example,thereport pathogenesis ofTDassociatedornotwithCHD,as path,mightbeinvolvedinthe the sameembryonic However, unknowngenes,butfunctionallysimilarin tissues(13,16). fixedarchival formalin obtained from data from different diseasedtissuesampleare frozen fresh obtained from way in CHD because the results majoretiologicalpath icism ofNKX2.5mutationsare andmosa (13,16). Itisstilldiscussedifsomaticnature matic changescouldcausetheinactivationofthisgene that hypotheticalmechanismssuchasepigeneticorso morphism inNKX2.5 TD withoutCHD,identifiedthesamec.63A>Gpoly posing (7). be notdisease-causingbutperhapsonlydisease-predis thec.73C>Tvariant,suggestingthatitmight carried also parents publication showsthatsomeunaffected (4).Indeed,acloserlookattheitalian´s rare tions are that find any mutations and confirms ectopy, 102patients,37withthyroid rolling didnot on CH, including15withCHD,hasnotfoundmutations of170patientswith tion-based studyinaCzechcohort Infact,apopula geneticbackground. adifferent from patients withTD, even in a phenotype-focused study in cate thatthemutationrateofNKX2.5geneisrare indi population, mostlysporadiccases.Ourresults NKX2.5 polymorphismincongenitalhypothyroidism InBrazil,anotherstudyincluding27patientswith NKX2.5 was expressed inmultipotentprogeni NKX2.5 wasexpressed gene (15).Similarlyajapanesestudyen gene (20).We mightconsider NKX2.5 muta ------modulates theiractivitypost-translationally by chang transcriptionfactorsand interact withothercrucial bud,itcanpotentially soearlyatthyroid is expressed AsNKX2.5 atitsinitialregulation. without interfere target genesduringdevelopment, of adownstream of alternatively, embryogenesis, thyroid lossoffunction during essential forthemaintenance of action ofNKX2.5withothercellularfactorscouldbe for CHD associated with TD.Themolecular mecha inse most publishedcasesinCHDphenotypeare polymorphism had a CHD phenotype. Although fiveofthepatientspositiveforc.63A>G report, primarily localizedwithinthehomeodomain.Inthis more mutationsassociatedwithCHDare reported CHD(OMIM600584).Thosepreviously ing from mutations havebeenidentifiedinpatientssuffer NKX2.5 germline heterozygous TD, manydifferent ing itsdimerizationprocess. lieve that germeline predisposing factorslikelyexistbut predisposing lieve thatgermeline (19).We discordant monozygotic (MZ)twinsare be familial TDoccursatgreater-than-random frequency, complex:while nisms ofTDwithorwithout CHDare themolecularbasisofTD.not reveal isonlyexaminationoflymphocyticDNAmaythere studiesinwhich polymorphisms.Therefore, germinal ofthe the hypoplasticglandcouldamplifyeffect tions or, alternatively, additionalmutationsarisingin muta fect couldbeassociatedwithsinglegermilane de growth multiple mutations(14).Aswell,thyroid typicallycontains malformations with complexcardiac tissuesofpatients diseasedcardiac NKX2.5 genefrom (21).The important alsofunctionallyvery main, are the homeodo tions ofNKX2.5,evenfarawayfrom fact, experimentalstudieshaveshownthatotherpor interactions. In impact on -protein predicted aminoacidandits tation type,positionoftheaffected ofNKX2.5 trum So,thespec coarctation. andaorta syndrome heart hypoplasticleft arteries, L-transposition ofthegreat arteriosus, truncus arch, aortic ventricle, interrupted of Fallot,ventricularseptaldefect,double-outletright of patients with tetralogy reports were though there of atrialseptaldefectwithorwithoutAV block,al tients withNKX2.5 CHDofthesepa genotype-phenotype correlation. isnoclear thehomeodomain, there quences affecting NKX2.5 canleadtoalimitedgradualdiminution In additiontothepolymorphismsdescribedin NKX2.5 appears tobeanunlikelycandidategene mutations is diverse in terms ofmu mutations isdiverseinterms germline mutations were mainly mutationswere germline Arch Metab. Endocrinol 2015;59/6 ------5. 4. 3. 2. 1. REFERENCES was reported. relevant tothisarticle nopotentialconflictofinterest Disclosure: byFiocruz. support vimento Científicoe Tecnológico) (HER).TLOChasfinancial (AMTL andHER),CNPq(ConselhoNacionaldeDesenvol àPesquisanoEstadodaBahia) Fapesb (FundaçãodeAmparo bygrants inpart thisworkwassupported Financial support: crinologists involvedintheircare. the cooperation of all patients, their families, and endo preciate We andHelenaPimentelsupport. ap Paula Leite,RenataArruti gratefulforNeyBoa-Sorte, Acknowledgements: theauthorsare morphogenesis. ment ofitsfunctionduringthyroid complete knowledge signaling pathwaysforamore tional and inthelung(21). many othertissuesasinlingualmuscle,spleen,stomach and has been identified in a number of expressed forTD.NKX2.5bindingelements but notsufficient genesis ofCHDand,inthiscase,mightbenecessary ly, development(20).Important of itsdosageinthyroid tion andmigrationhavepointedtotheimportance cellspecifica thyroidal controlling of thegeneticcircuit morphogenesis, andmightbeimplicatedasapartner duringtheearlythyroid could haveanessentialrole evenCNV–copynumbervariants). pression (somaticevents,randommono-allelicex discordance toexplainMZtwin required additional mechanismsare Arch Metab. Endocrinol 2015;59/6 NKX2.5 It would be of interest toattemptidentifyaddi It wouldbeofinterest However,NKX2.5 itisknownthat,intheheart, MI, Camacho CP, etal.Clinicalandmolecularanalysisofthyroid Ramos HE, Nesi-Franca S,Boldarine VT, Pereira RM, Chiamolera tients. JClinEndocrinolMetab.2010;95(4):1981-5. genetic screeningofapopulation-basedcohort ofJapanese pa- tion factormutationsandcongenitalhypothyroidism: systematic Narumi S,MuroyaK, Asakura Y, Adachi M,Hasegawa T. Transcrip- 3:U39-44. in congenitalhypothyroidism. Suppl EurJEndocrinol.2004;151 Gruters A, KrudeH,BiebermannH.Moleculargeneticdefects Cell Endocrinol.2010;323:35-54. Fagman H,NilssonM.Morphogenesisofthethyroid gland.Mol current embryonicanatomy. Curr Top DevBiol.2013;106:123-70. dysgenesis: ananalysisbasedondevelopmentalstagesandcon- Nilsson M,Fagman H.Mechanisms ofthyroid developmentand downstream target genes and upstream target genesandupstream NKX2.5 downstream mutations are known to be central to the mutations are ------21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 2013;8(12):e3295. tion, associatedwithhumancongenitalheart disease.PLoS ONE. comprising aSNP, anonsynonymous and asynonymous muta- Borlak J. Transcriptional defectofaninheritedNKX2-5haplotype Reamon-Buettner EM, Sattlegger E, Ciribilli Y, Inga A, Wessel A, nol Metab.2012;56(3):173-7. tor genesinpatientswiththyroid dysgenesis. Arq BrasEndocri- Marui S. Absence ofmutationsinPAX8, NKX2-5, and TSH recep- Brust ES,BeltrãoCB,ChammasMC, Watanabe T, Sapienza MT, Horm Res. 2009;72:320. dant forcongenitalhypothyroidism fromthyroid dysgenesis. van Vliet G, Vassart G.Monozygotictwinsaregenerallydiscor genetics andmolecularmechanisms. 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