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www.jped.com.br

ORIGINAL

ARTICLE

Genetic

determinants

and

stroke

in

children

with

sickle

cell

disease

夽,夽夽

Daniela

O.W.

Rodrigues

a,b,∗

,

Luiz

C.

Ribeiro

b,c

,

Lysla

C.

Sudário

d,e

,

Maria

T.B.

Teixeira

b,f

,

Marina

L.

Martins

a,g

,

Anuska

M.O.L.

Pittella

h

,

Irtis

de

O.

Fernandes

Junior

d,e

aFundac¸ãoHemominas,JuizdeFora,MG,Brazil

bUniversidadeFederaldeJuizdeFora(UFJF),JuizdeFora,MG,Brazil

cUniversidadeFederaldeJuizdeFora(UFJF),DepartamentodeEstatística,JuizdeFora,MG,Brazil dFundac¸ãodeAmparoàPesquisadoEstadodeMinasGerais(FAPEMIG),JuizdeFora,MG,Brazil eUniversidadePresidenteAntônioCarlos(UNIPAC),FaculdadedeMedicina,JuizdeFora,MG,Brazil fUniversidadeFederaldeJuizdeFora(UFJF),DepartamentodeSaúdeColetiva,JuizdeFora,MG,Brazil gFundac¸ãoHemominas,SetordePesquisa,BeloHorizonte,MG,Brazil

hFaculdadedeCiênciasMédicasedaSaúdedeJuizdeFora(SUPREMA),JuizdeFora,MG,Brazil

Received29November2015;accepted27January2016 Availableonline5June2016

KEYWORDS Sicklecellanemia; Stroke;

Geneticmarkers; Alphathalassemia; Haplotypes

Abstract

Objective: Toverifygeneticdeterminantsassociatedwithstrokeinchildrenwithsicklecell disease(SCD).

Methods: Prospectivecohortwith110childrensubmittedtoneonatalscreeningbytheNeonatal ScreeningProgram,between1998and2007,withSCDdiagnosis,followedataregionalreference publicserviceforhemoglobinopathies.Theanalyzedvariablesweretypeofhemoglobinopathy, gender,coexistencewithalphathalassemia(␣-thal),haplotypesofthebetaglobinchaincluster, andstroke.Thefinalanalysiswasconductedwith66childrenwithsicklecellanemia(SCA), usingthechi-squaredtestintheprogramSPSS®version14.0.

Results: AmongchildrenwithSCD, 60%hadSCA.The prevalenceofcoexistencewith␣-thal was 30.3% andtheBantu haplotype(CAR)was identifiedin89.2%. Theincidence ofstroke

Pleasecitethisarticleas:RodriguesDO,RibeiroLC,SudárioLC,TeixeiraMT,MartinsML,PittellaAM,etal.Geneticdeterminantsand

strokeinchildrenwithsicklecelldisease.JPediatr(RioJ).2016;92:602---8.

夽夽StudycarriedoutatFundac¸ãoHemominasandUniversidadeFederaldeJuizdeFora(UFJF),JuizdeFora,MG,Brazil.Correspondingauthor.

E-mail:danielawerneckhemato@hotmail.com(D.O.W.Rodrigues).

http://dx.doi.org/10.1016/j.jped.2016.01.010

0021-7557/©2016SociedadeBrasileiradePediatria.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-ND

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wassignificantlyhigherinthosewithSCA(27.3%vs.2.3%;p=0.001)andmales(24.1%vs.9.6%;

p=0.044).Thepresenceof␣-thal(p=0.196),theCARhaplotype(p=0.543),andsocioeconomic factorswerenotstatisticallysignificantinassociationwiththeoccurrenceofstroke.

Conclusion: There isa highincidenceofstroke in malechildren and inchildren withSCA. Coexistencewith␣-thalandhaplotypesofthebetaglobinchainclusterdidnotshowany sig-nificantassociationwithstroke.Theheterogeneitybetweenpreviouslyevaluatedpopulations, thenon-reproducibilitybetweenstudies,andtheneedtoidentifyfactorsassociatedwithstroke inpatientswithSCAindicatethenecessityofconductingfurtherresearchtodemonstratethe relevanceofgeneticfactorsinstrokerelatedtoSCD.

©2016SociedadeBrasileiradePediatria.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/

4.0/). PALAVRAS-CHAVE Anemiafalciforme; Acidentevascular cerebral; Marcadores genéticos; Alfatalassemia; Haplótipos

DeterminantesgenéticoseAcidenteVascularEncefálicoemcrianc¸ascomdoenc¸a falciforme

Resumo

Objetivo: Verificar fatores genéticos associados ao acidente vascular encefálico (AVE) em crianc¸ascomDoenc¸aFalciforme(DF).

Métodos: Coorteprospectivade110 crianc¸assubmetidas àtriagemneonatalpeloPrograma de Triagem Neonatal, entre 1998-2007 com o diagnóstico de DF, atendidas em servic¸o públicoregionaldereferênciaemhemoglobinopatias. Asvariáveisanalisadasforam:tipode hemoglobinopatia,sexo,coexistênciadaalfaTalassemia(␣-Tal),haplótiposdoclusterdacadeia betaglobinaeAVE.Aanáliseestatísticafinalfoirealizadacom66crianc¸ascomAnemia Falci-forme,pormeiodotestedoQui-quadradonoprogramaSPSS®14.0.

Resultados: Entreascrianc¸ascomDF,60%eramportadorasdeAnemiaFalciforme.Aprevalência dacoexistênciacoma␣-Talfoide30,3%eohaplótipoBantu(CAR)foiidentificadoem89,2%. AincidênciadeAVEfoisignificativamentemaiornascrianc¸ascomAF(27,3%versus2,3%;p= 0,001)enosexomasculino(24,1%versus9,6%;p=0,044).Apresenc¸ada␣-Tal(p=0,196),do haplótipoCAR(p=0,543)efatoressocioeconômicosnãoforamsignificantementeassociadasà ocorrênciadeAVE.

Conclusão: OAVEapresentaaltaincidênciaemcrianc¸ascomAFeem crianc¸asdosexo mas-culino.Coexistênciade␣-Taloudehaplótiposdoclusterdabetaglobinanão apresentaram associac¸ãosignificantecomAVE.Aheterogeneticidadeentreaspopulac¸õespreviamente avali-adaseanão reprodutibilidadeentreestudosindicamanecessidadederealizac¸ãode novas pesquisasparaverificaropapeldessesfatoresgenéticosnoAVEemcrianc¸ascomDF.

©2016SociedadeBrasileiradePediatria.PublicadoporElsevierEditoraLtda.Este ´eumartigo OpenAccesssobumalicenc¸aCCBY-NC-ND(http://creativecommons.org/licenses/by-nc-nd/4.

0/).

Introduction

Sickle cell disease (SCD) is the most common mono-genichereditarydiseaseinBrazil,occurringpredominantly among those of African descent. The term SCD includes sickle cell anemia (SCA) and pathological conditions in which the hemoglobin S gene is associated with other hemoglobinopathies,suchasSC, S/beta0 andS/beta+ tha-lassemia(S/b),andSDPunjab,amongothers.1SCA,caused

byasinglemutationinthe␤-globingene,producesa diver-sity ofphenotypicexpressionsin affectedpatients.2,3 SCA

is the most severeform of SCDpresentation; for the dis-ease to manifest, homozygosity of ␤S alleles in the gene

responsible for the synthesis of ␤ chain of hemoglobin is required, determining the formation of hemoglobin S(HbSS).

HbSS, under conditions such as low oxygenation, metabolicacidosis,or dehydration,becomespolymerized,

irreversibly changing the structure of erythrocyte, thus determininginefficientoxygenation,endothelial inflamma-tory reaction, andthe entire complexphysiopathology of thedisease.3,4The polymerization processleadsto

vascu-larocclusion,whichcantriggerpainfulcrises,stroke,acute chestsyndrome,splenicsequestration,andpriapism,among othermanifestations.

In Brazil, studies have shown that 700---1000 chil-dren/year are born with SCD,1,4---6 making this disease a

publichealthproblem.ThestateofMinasGerais(MG)isa pioneerinBrazilintheearlydiagnosisofSCD,withthe intro-ductionoftheNeonatalScreeningProgramoftheStateof MinasGerais(ProgramadeTriagemNeonataldoEstadode MinasGerais[PETN-MG]) in March1998.PETN-MGis coor-dinatedbythe Centerfor Action andResearchin Support Diagnostics (Núcleo de Ac¸ões e Pesquisa em Apoio Diag-nóstico[NUPAD])ofUniversidadeFederaldeMinasGerais, whichrefersnewbornsdiagnosedwithSCDtobefollowedat

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Fundac¸ãoHemominas. From1998to2007,2,549,097 chil-drenwerescreenedinMG,ofwhom188,916werebornin the39municipalitiesincludedintheregionalreference pub-licservicesforhemoglobinopathies---wherethisstudywas carriedout---which represents7.41%of thetotalnumber in thestate. The PETN-MG coverage in 2007 was88.57%, whichishigherthanthenationalaverageof9.65%(Ministry ofHealth,NationalNeonatalScreeningProgram,2009).

StrokeisoneofthemostseverecomplicationsofSCDand isresponsiblefor20%ofmortalityamongthesepatients.2,7

AccordingtotheCooperativeStudyGroupinSCD,the over-all incidence of the first stroke was 0.08 acute events/ 100patients/yearinchildrenunder2years;0.75inpatients between2and5yearsofage;0.55between6and9years ofage;0.30between10and19years;and0.45between20 and29years.AmongtheSCD,theincidenceofstrokeis0.61 forpatientswithHbSS,0.17forHbSC,and0.11forS/beta thalassemia.1,2,7

Currently,theuseoftranscranialDoppler(TCD),a non-invasiveultrasoundmethodthatmeasuresthevelocityand flowalterationofintracerebralvessels,isconsidereda sen-sitivetoolfortheidentificationofischemicstrokerisk.1

The association between stroke, coexistence of alpha-thalassemia(del˛-3,7) (␣-thal),and clusterhaplotypes ofthe

beta globin ␤S is variable in the literature. Studies

pub-lishedwithdatafromRiodeJaneiro,4,8MG,9andSaoPaulo4,9

show controversial results. Some studies have reported thatthecoexistence of ␣-thalwouldhelp reduce therisk of stroke,9---12 while others did not demonstrate such an

association.6,13

These studies have identified characteristics regarding thefrequency of differenthaplotypes, witha high preva-lence of individuals homozygous for the Central African Republic(CAR)andBenin haplotypes,reflectingtheorigin oftheflowofAfricanslaveswhowerereceivedatthetime of colonial Brazil.4,9,14 In Jamaica and the United States,

theBenin haplotypeis muchmore frequentthan theCAR haplotype.Such genetic differences make generalizations ofresultsbetweenregionsinappropriate.

Theaimofthisstudywastoinvestigategeneticfactors associatedwithriskofstrokeinchildrenwithSCD.

Method

Between1998and2007,188,916childrenbornintheregion ofZonadaMataMineiraandVertentesunderwentneonatal screeningforSCDthroughthePETN-MG.Duringthisperiod, 135 children withSCD were referred toa reference pub-lic institution for hemoglobinopathies. Of this total, nine diedbeforethestartoftheproject,sixchildrenwithSCD types SD and S were excluded, and ten cases were lost tofollow-up,thustotaling110childrenwhocomprisedthe studypopulation.

Confirmatory diagnosis of SCD was carried out by hemoglobinelectrophoresis onalkaline pH after 6 and12 months of age, measurement of hemoglobin A2 by chro-matography, radial immunodiffusion for fetal hemoglobin (HbF), and molecular analysis of codon 6 of beta globin. ChildrenwithSCA,SC,S/b0,andS/b+wereconsideredfor inclusioninthestudy.

Clinicalandlaboratorydatawereextractedfrommedical records,intheperiodbetweenthedateofenrollmentatthe institutionuntilDecember31,2013(endofcohort follow-up),allowingafollow-upofatleastfiveyearsofthestudy subjects. Socioeconomic characteristics were obtained by applyingthequestionnairefromtheAnisioTeixeiraNational Institute of Educational Studies and Research (Instituto NacionaldeEstudosePesquisasEducacionaisAnísioTeixeira [INEP]),plusquestionsaboutthecaregiver’slevelof school-ingandfamilyincome.Thestudytoidentifythehaplotypes and screening for ␣-thal wasinitiated in November 2010, afterapprovalbythefundingagencythroughtheResearch ProgramforSUS(healthsysteminBrazil)---PPSUS/Fundac¸ão deAmparoàPesquisadoEstadodeMinasGerais(Fundac¸ão deAmparoàPesquisadoEstadodeMinasGerais[FAPEMIG]). Theoutcomevariablewasthepresenceofstroke(yesor no).Thediagnosisofstrokewasattainedclinically(ischemic stroke or transient ischemic attack) or throughadditional testssuchastranscranialDoppler(TCD)andmagnetic reso-nanceangiography(MRA)ofcerebralvessels.

ThescreeningforthepresenceofstrokethroughTCDhas been offered annuallytoall children beingfollowed with SCD,between 2and16 yearsofage, asrecommendedby theBrazilianProtocolsince2007inBeloHorizonte,andsince 2012attheregionalunitofthisstudy.Thetestisperformed inallpatientswithSCD,althoughtherearenoestablished velocity parameters for SCD type SC and S/beta+.1 The

assessmentof therateofcerebralblood flowinthegreat arteries of the circle of Willis was based on the criteria fromtheStrokePreventionTrialinSickleCellAnemia(STOP) study.1

The TCD results were stratified according to the classificationproposedbytheSTOPstudy,addingthe recom-mendationtoincludethemeanmaximalvelocity(MMV)in oneoftheanteriorcerebralarteries≥170cm/sasalso rep-resenting ahigh risk of developing ischemicstroke.15 The

main assessed arteries were the middle cerebral arteries andtheirbifurcations,andthedistalinternalcarotidartery. Theconfirmationofanabnormaltest(highrisk)wasmade throughdoublerepetition,withan intervalofonetofour weeks.

Childrenconfirmedashigh-riskforischemicstrokewere referredforprimarypreventivetreatmentoftheeventwith ahypertransfusionregimen.AtthetimeoftheTCD,there werenochildrenundergoingexchangetransfusiontherapy orbeingtreatedwithhydroxyurea.

Geneticfactorsconsideredintheanalysiswere: 1. Typeof SCD(SCA or anothertype: SC, S/b0, andS/b+

thalassemia)withdiagnosisconfirmed accordingtothe abovementionedmethodology;

2. Gender(maleorfemale);

3. Presenceorabsenceofthemutationforthe␣-thalgene; 4. Identificationofthehaplotype(CARornon-CAR).

Todeterminethedeletionmutationfor␣-thaland hap-lotypes, 5mLofwholebloodwerecollectedintubeswith ethylenediaminetetraaceticacid(EDTA)duringroutineSCD laboratory monitoring. The extractionand quantitationof genomicDNAwasperformedusingthecommercialQIAamp DNABloodMiniKit(QIAGEN®,USA)andInvitrogen®

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Inc®, USA) according to manufacturer’s instructions. The

analyseswereperformedattheresearchlaboratoryofthe institution.

Theidentificationofthehaplotypeswasperformedusing polymerase chain reaction(PCR) and restrictionfragment length polymorphism (RFLP) analysis according Sutton’s16

protocol. Acommercial kit MultiplexPCR (QIAGEN®, USA)

wasusedforPCRreactionsand␣−3.7mutationanddeletion screening. The identity of each deletionwas obtainedby determiningthesizeoftheamplifiedfragmentineach reac-tion.Asanydeletionremovespartortheentire␣-2globin gene,itsamplification,togetherwiththeamplificationof adeletionallele,indicates thatthemutationis heterozy-gous.As positivecontrolfor successfulDNAamplification, thisstudyuseda2350bpsegment,relatedtothe nontran-scribedregion3oftheLIS-1gene(plateletfactor)locatedon chromosome17p13.3.Othermutationswerenotscreened, consideringthatinBrazilthedeletionthatleadsto␣-thalis thetype(3.7).9

Theprimersequenceswereverifiedagainstthe informa-tion available at NCBI (National Center for Biotechnology Information)usingtheBLAST(BasicLocalAlignmentSearch Tool)(http://blast.ncbi.nlm.nih.gov/Blast.cgi)tool.

Inadditiontothedescribedgeneticfactors,the associa-tionbetweenfetalHbandstrokewasassessed.Therelative concentrationsoffetalHbinlysedredbloodcells(RBCs)was determinedat5yearsofageandtheirvaluewasstratified accordingtoSteinberg17asfetalHb≥10%orfetalHb<10%.

Thechildrenandtheirparents/guardianswerepreviously informedabouttheimportanceofthisstudy,andauthorized theirparticipationbysigninganinformedconsent.

The statistical analysis of the associations was carried outthroughtheChi-squaredtest,consideringasignificance level of 5%, using the program SPSS Statistics® 14.0 (IBM

Corporation,Somers,NY,UnitedStates).

ThestudywasapprovedbytheResearchEthics Commit-teeon10/9/2009underNo.245andisconsistentwiththe provisionsofResolution466/12oftheNationalHealth Coun-cil/MinistryofHealthandtheCodeofMedicalEthicsof1988 (Article122-130).

Results

Ofthe110children,52.7%weremales.Themeanageatend ofthefollow-upwas11.2years,withastandard deviation of2.84.

Of the study population, 60% were patients withSCA, 33.6% hadSCgenotype, and 6.4% hadS/beta thalassemia (twochildrenS/b0andfivewithS/b+).TheincidenceofSCA wasonecaseper2857livebirthsandonecaseofSCDtypeSC forevery5128livebirths.Thefamilyincomewasuptotwo minimumwages(MW)in72.8%of cases,andthemother’s levelofschooling,theprimarycaregiver,wascomplete ele-mentaryschoolin60.9%ofcases.

The identification of the haplotypes showed the pres-enceofthe CARgeneinthemajority ofpatients (89.2%), reflectingtheoriginoftheAfrican-descentpopulation eval-uated in this study. The presence of atypical genes such as Senegal and Cameroon was identified, previously only describedintheNortheastofBrazil.Itwasnotpossibleto definethehaplotypeinonlyonechild.Sevenchildren(6.4%)

Table1 Haplotypesidentifiedinchildrenwithsicklecell disease.

Analysisofthebeta-chainglobin Frequency %

CARCAR 35 53 CARBEN 20 30.3 CARSEN 1 1.5 CARATIP 3 4.5 ATIPATIP 1 1.5 Notperformed 6 9.1 Total 66 100

CAR,CentralAfricanRepublic;BEN,Benin;SEN,Senegal;ATYP, atypical.

didnotundergogenetictestingduetorefusalorfailureto attendbloodsample collectionwithinthe stipulatedtime forresearch.Ofthe66childrenwithSCA,89.4%hadatleast oneCARallele(Table1).

The analysis of the ␣-globin gene deletion showed an incidenceof ␣-thalof 30.9% of individuals. 26.4% of chil-dren were identified with deletion of one gene and 4.5% withdeletionoftwogenes.Inthispopulation,nochildren wereidentifiedwithdeletionofthreegenes.Inrelationto SCA,22.7%of childrenhaddeletionof onegeneand7.6% haddeletionoftwogenesfor␣-thal.

Atotalof19childrenwereidentifiedwithstroke,which representedan incidence of 17.2%. Ofthese, threecases were identified through MRI performed after the clinical manifestation of stroke. Sixteen cases were identified by TCD,13of whomshowedelevatedratesof cerebralblood flowandareasofcerebralischemiaidentifiedatthebrain MRA.Theincreasedflowvelocityintherightmiddlecerebral arteryanditsstenosiswerethemostprevalentalterations (eightcases).Theincidenceofstrokeamongchildrenwith SCAwas27.3%.TherewerenocasesofstrokeinchildrenSC andS/b+.Themeanageatfirststrokewas7.7years,witha

minimumageof6monthsandmaximumof15years.

Table 2 describes the factors associated with stroke. In the assessed sample, only one child with S/beta0

tha-lassemia had a stroke. Children with SCA had a 12-fold greaterrisk forstroke thanthosewithothertypes ofSCD (p=0.001).Theincidenceofstrokeamongboyswas24.1%, compared to 9.6% among girls (p=0.044). Other factors showednosignificantassociationwithstroke.

Childrenwith SCA are more susceptible tostroke. For this reason, this group was considered for assessment of theassociationofstrokewiththeotherfactors.Theresults depictedinTable3showthatonlythevariablegenderwas significantlyassociatedwiththeoutcome(p=0.042).

Discussion

ThemostseverecomplicationofSCA isstroke,oneofthe leadingcausesofdeathinbothchildrenandadults.Despite itshighincidence,causesandfactorsthatincreasetherisk ofstroke arenot completely known.Several linesof evi-dencesuggest thatageneticsignaturecouldinfluencethe developmentof strokes and that the combined effect of thesegenescanaffecttheseverityofSCD.2,6,9,15,18,19

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Table2 Frequencyoffactorsassociatedwithstrokeinindividualswithsicklecelldisease.

Factors Absolutefrequencyand

percentageofvariables

Absolutefrequencyandpercentage ofpresenceofstroke p-Value n % n % Typeofhemoglobinopathy 0.001 SCA 66 60 18 27.3 S/beta0,S/beta+,SC 44 40 1 2.3 Gender 0.044 Female 52 47.3 5 9.6 Male 58 52.7 14 24.1 Alphathalassemiaa 0.164 Present 34 32.1 3 8.8 Absent 72 67.9 14 19.4 Haplotypesa 0.116 CAR 91 89.2 17 18.7 Non-CAR 11 10.8 0 0.00 Fetalhemoglobin 0.293 (≥10%) 46 41.8 10 21.7 (<10%) 64 58.2 9 14.1 Familyincomeb 0.432 Upto2MW 80 74 12 15 >2MW 28 26 6 21.4

Maternallevelofschoolingb 0.329

Incompleteelementaryschool 41 38 5 12.2

Completeelementaryschool 67 62 13 19.4

Paternallevelofschoolingb 0.491

Incompleteelementaryschool 56 51.8 8 14.3

Completeelementaryschool 52 48.2 10 19.2

SCA,sicklecellanemia;CAR,CentralAfricanRepublic;MW,minimumwageinBrazil.

a106testswereperformedfor␣-thaland102forhaplotypeidentification. b 108socioeconomicquestionnaireswereapplied.

Flanangan et al. demonstrated a significant associ-ation between ␣-thal and the genetic polymorphisms (single-nucleotidepolymorphisms[SNPS])ADCY9rs2238432 in stroke reduction; three SNPs (ANXA2 rs11853426, TEK rs489347, and TGFBR3 rs284875) were significantly asso-ciated with an increased risk of stroke; the deficiency ofglucose-6-phosphatedehydrogenaseandthehaplotypes werenotrelatedtoincreasedorreducedrisk.Belisárioetal. describedanincreaseintheriskofstrokeinpatientsthat expressTNF-alpha(−308G>A),butdidnotshowasignificant association with the expression of VCAM-1 polymorphism (c.1238G>C), and associated ␣-thal with reduced risk of stroke.Theseresultssuggestthattheassociationbetween strokeandpolymorphismsremainscontroversial.1,2,9,15,20---22

Thepresentstudyassessedtheassociationsbetweenstroke, ␣-thal, and haplotypes, and no significant results were found, which couldbedue to thesmall sizeof the study population. In this study there were noreports of stroke amongchildrenwithSCDtypeSCandS/beta+thalassemia,

dataconsistentwiththeCooperativeStudyGroupinSCD.1

Itisworthmentioningthatthepopulationofthisstudy consisted of all children diagnosed with SCA (n=66) in the area covered by the health institution that encom-passesapopulationof730,264inhabitants[IBGE(Brazilian

InstituteofGeographyandStatistics)Census2010]Sarnaik andBallas11emphasizedtheimportanceofmulticenter

stud-ieswithlargenumbersofpatientswithandwithoutstroke, todeterminetheimplicationofgeneticmarkerstudieson themorbidityandmortalityinSCD.

TheincidenceofSCAinthisstudywasonecaseforevery 2857livebirthsandonecaseofSCDtypeSCforevery5128. According tothe Ministryof Health,the incidenceof SCD detectedinthePETNwas:Bahia,1:650;RiodeJaneiro,1: 1300;Pernambuco,Maranhão,Minas Gerais,andGoiás:1: 1400;EspíritoSanto,1:1800;SaoPaulo,1:4000;RioGrande doSul,1:11,000;SantaCatarinaandParana,1:13,500.19

Bezerraetal.5geneticallycharacterizedacohortof74

childrenwithSCAinPernambucoandshowedthat approx-imately 65% of patients had the CAR/CAR haplotype, a frequencygreaterthanthatfoundinotherstatesin north-easternBrazilandhigherthanthe53%foundinourstudy.

This study did not identify the Benin/Benin haplotype among children with SCA, similar results to those found inRiodeJaneiro.13,14Previousreports showedthat

haplo-typesareassociatedwithanincreasedriskofstroke.2,11,23,27

According to the present data, the presence of the CAR haplotype (present in 89.2% of cases) was not associated withthedevelopmentofstroke.Theseresultsaresimilarto

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Table3 Frequenciesoffactorsassociatedwithstrokeinindividualswithsicklecellanemia. Factors Absolutefrequencyandpercentage

ofcategoriesoffactors

Absolutefrequencyand percentageofpresenceofstroke

p-Value n % n % Typeofhemoglobinopathy SCD 66 100 18 27.3 Gender 0.042 Female 28 42.4 4 14.3 Male 38 57.6 14 36.8 Alphathalassemiaa 0.196 Present 20 30.3 3 15 Absent 43 65.2 13 30.2 Haplotypesa 0.543 CAR 59 89.4 16 27.1 Non-CAR 1 1.5 0 0.00 FetalHemoglobin (≥10%) 41 62.1 9 22 (<10%) 25 37.9 9 36 Familyincomeb 0.593 Upto2MW 49 74.2 12 24.5 >2MW 16 24.2 5 31.3

Maternallevelofschoolingb 0.854

Incompleteelementaryschool 18 27.3 5 27.8

Completeelementaryschool 47 71.2 12 25.5

Paternallevelofschoolingb 0.591

Incompleteelementaryschool 27 40.9 8 29.6

Completeelementaryschool 38 57.6 9 23.7

SCD,Sicklecelldisease;CAR,CentralAfricanRepublic;MW,minimumwageinBrazil.

a 63testswereperformedfor␣-thaland60forhaplotypeidentification. b 65socioeconomicquestionnaireswereapplied.

thoseofFlananganetal.22andLoghetto,24whofoundno

sig-nificantcorrelation betweenthehaplotypes andstroke. In 2011,Domingosetal.12studiedapopulationinPernambuco

differentfromtheonestudiedbyBezerraetal.,5with261

patients,ofwhom67hadstroke,andfoundnoassociation betweenhaplotypesandstroke.

In Brazil, the type of deletion that leads to ␣-thal is almostexclusivelythe(−3.7) type.9 Theincidenceof␣-thal

foundinindividualswithSCAinthisstudywas30.3%, consid-eringthedeletionofoneandtwogenes.Thesedatawere similartothosefoundin Salvador(28.2%)andhigherthan thosefoundbyFigueiredo,whichwas18.8%inSãoPaulo.4,9

Several studies12,15,22 haveshown thepreventive effect

of ␣-thalassemia on the development of stroke in chil-dren withSCA. Belisario etal.9 and Hsuetal.10 reported

that the frequency of ␣-thal was significantly higher in patientswithSCAwithnoalterationsattheTCD,suggesting molecularprotectionfromthedevelopmentofstroke. Dif-ferently fromthepresent analysis, thesemarkers showed nosignificantassociationwithstrokerisk,resultsalsofound by Silva Filho13 and Sommet et al.6 These findings

sug-gest a possible geneticheterogeneity among populations, which may explain the differences among the results of thestudies.The manualofSCDconductandmanagement, ‘‘Evidence-Based Management of Sickle Cell Disease’’ of

theNationalInstitutes ofHealth (NIH),publishedin2014, does not advise genetic screening for the prevention of stroke.25

Socioeconomic factors, suchas family incomeand the caregiver’slevel of schooling,aredescribed associal vul-nerabilityrisksanddeterminantsofmajorhealthproblems of the population with SCD.3,26---29 Such associations were

notobservedinthisstudy.InMinasGerais,the implemen-tation of a comprehensive care policy for patients with SCDprovidescontinuous andmultidisciplinary care,which mayhave hadapositiveimpactby reducingthe socioeco-nomic effectson themanagement of the disease (Center forEducationandSupportfor Hemoglobinopathies---MG

---www.cehmob.org.br).

Theincidenceofstroke wassignificantlyhigherin chil-dren with SCA and males, data already identified in the InternationalPediatricStroke Group.30 The analysisofthe

coexistenceof␣-thalandhaplotypes inthisstudyshowed nocorrelation in thegenesis or preventionof stroke. The heterogenicity between previously assessed populations, non-reproducibilitybetweenstudies,andtheneedto iden-tifyfactorsassociatedwithstrokeinpatientswithSCAshow theimportanceofnewstudies,considering thispathology isthemostprevalentmonogenicdiseaseintheworldanda matterofpublichealthconcern.

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Funding

All laboratory material used in the development of the study,such asDNAextraction kits, PCR reagents, restric-tionenzymes,tubes,andpipettetipswerepurchasedwith the budget provided by FAPEMIG through PPSUS/FAPEMIG (CDS-APQ-01431-10)andthe researchlaboratory sectorof Fundac¸ão Hemominas. The research fellowships for the undergraduatestudentsinvolvedinthestudywereprovided byFAPEMIG.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

References

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