Rev. paul. pediatr. vol.34 número1

www.rpped.com.br

REVISTA

PAULISTA

DE

PEDIATRIA

ORIGINAL

ARTICLE

Influence

of

polymorphisms

of

the

beta-2

adrenergic

receptor

on

the

presence

of

exercise-induced

bronchospasm

in

adolescents

夽

Cássio

Leandro

Mühe

Consentino

,

Lupe

Furtado-Alle

,

Larissa

Rosa

da

Silva

,

Wendell

Arthur

Lopes

_,

_Luciane

_Viater

_Tureck

_,

_Gerusa

_Einsfeld

_Milano

_,

Leilane

Lazarotto

_,

_Cláudia

_Regina

_Cavaglieri

_,

_Neiva

_Leite

a_{UniversidadeFederaldoParaná(UFPR),Curitiba,PR,Brazil} b_{UniversidadeEstadualdeMaringá(UEM),Ivaiporã,PR,Brazil} c_{UniversidadeEstadualdeCampinas(Unicamp),Campinas,SP,Brazil}

Received2March2015;accepted9June2015 Availableonline24December2015

KEYWORDS

Adolescent; Exercise-induced bronchospasm;

ADRB2gene

Abstract

Objective: Todetermine theinfluence ofpolymorphismsof thebeta-2 adrenergicreceptor (ADRB2)intriggeringexercise-inducedbronchospasm(EIB)inadolescents.

Methods: Thesubjectsweredividedintotwogroups:presentEIB(EIB+)(n=45)andabsentEIB (EIB−)(n=115).Thebronchialprovocationtestwithexercisewasperformedwithaprotocol thatconsistedofwalking/runningfor atleasteight minutesathigh intensity,i.e.,>85%of maximumheartrate,consideringEIB+asa10%decreaseinforcedexpiratoryvolumeinone second(FEV1).ThegenotypingoftheADRB2genewasperformedbytheTaqmanmethod,using theStepOnePlussystem.Independentt-test,Mann---WhitneyandChi-squaretests,aswellas Spearman’scorrelationcoefficientwereusedforthestatisticalanalysis.

Results: Age,bodyweight,height,FEV1,FVCandFEV1/FVCratiowerelowerintheEIB+group whencomparedtoEIB−(p<0.05).Therewerenosignificantdifferencesintheproportionof thealleleatposition27 andArg16GlyandGln27Glugenotypesbetween theEIB+ andEIB− groups(p=0.26;p=0.97andp=0.43,respectively).However,therewasatrendtowardstatistical significanceregardingthegreaterproportionoftheGly16allelefortheEIB+whencompared totheEIB−group(p=0.08).

Conclusions: Thepresence ofpolymorphismsassociatedwiththe Glu27alleleandArg16Gly

andGln27GlugenotypeshadnoinfluenceonEIB.However,thestatisticaltrendtowardgreater frequencyoftheGly16alleleinindividualswithEIB+canbeconsideredevidenceoftheinfluence ofpolymorphismsoftheADBR2geneonEIBinadolescents.

©2015SociedadedePediatriadeSãoPaulo.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBYlicense(https://creativecommons.org/licenses/by/4.0/).

夽

ThisarticleispartoftheMaster’sDegreeDissertationofCássioLeandroMüheConsentino.

∗_{Correspondingauthor.}

E-mail:[email protected](C.L.M.Consentino). http://dx.doi.org/10.1016/j.rppede.2015.06.021

PALAVRAS-CHAVE

Adolescente; Broncoespasmo induzidopelo exercício; GeneADRB2

Influênciadospolimorfismosnoreceptorbeta2adrenérgiconapresenc¸a

debroncoespasmoinduzidopeloexercícioemadolescentes

Resumo

Objetivo: Determinar a influência dos polimorfismos dos receptores adrenérgicos beta 2 (ADRB2)nodesencadeamentodebroncoespasmoinduzidopeloexercício(BIE)emadolescentes.

Métodos: Ossujeitosforamdivididosemdoisgrupos:BIEpresente(BIE+)(n=45)eBIEausente (BIE−)(n=115).Otestedebroncoprovocac¸ãocomexercíciofoifeitocomprotocoloque con-sistiuemcaminhar/correrdurantenomínimooitominutosemintensidadesuperiora85%da frequênciacardíacamáxima,considerandocomoBIEpresenteumaquedade10%dovolume expiratórioforc¸adonoprimeirosegundo(VEF1).AgenotipagemdogeneADRB2foifeitapelo métodoTaqmanpormeiodoaparelhoStepOnePlus.Paraanáliseestatísticausaram-seostestes

tindependente,UdeMann-Whitney,qui-quadradoecoeficientedecorrelac¸ãodeSpearman.

Resultados: Idade,massacorporal, estatura,VEF1,CVFe relac¸ãoVEF1/CVF forammenores no grupoBIE+ em comparac¸ão comoBIE−(p<0,05).Nãohouvediferenc¸as significativasna proporc¸ãodoalelonaposic¸ão27edosgenótiposArg16GlyeGln27GluentreosgruposBIE+ eBIE−(p=0,26;p=0,97ep=0,43,respectivamente).Entretanto,verificou-seumatendênciaà significânciaestatísticanamaiorproporc¸ãodoaleloGly16paraogrupoBIE+comparadocomo BIE−(p=0,08).

Conclusões: A presenc¸adepolimorfismosassociados aoaleloGlu27eosgenótiposArg16Gly

eGln27GlunãoinfluenciamnoBIE.Porém,atendênciaestatísticaobservadaparaumamaior frequênciadoaleloGly16nosindivíduoscomapresenc¸adeBIEpodeserconsideradoindício dainfluênciadepolimorfismosnogeneADBR2noBIEemadolescentes.

©2015SociedadedePediatriadeSãoPaulo.PublicadoporElsevierEditoraLtda.Esteéumartigo OpenAccesssobalicençaCCBY(https://creativecommons.org/licenses/by/4.0/deed.pt).

Introduction

Exercise-induced bronchospasm(EIB) is definedas tempo-rary narrowingof the airways that occursafter strenuous exerciseinupto90%ofasthmaticindividuals1_andinalmost 20% of individuals withnohistory of respiratorydisease.2 Thepresenceofexcessweightcancontributetoincreased severity of EIB in asthmatics.3 _{The excessive} accumula-tion of fatty tissue in the central region can change the pulmonary mechanics and airway inflammatory response, leading to increased contractility and responsiveness of bronchial smooth muscle4 _{and thus limit the practice of} physicalexercises5_{astherapyforasthma}6_andobesity.7

Somegeneticalterations,suchaspolymorphismsofthe betaadrenergicreceptor2(ADRB2),havebeen associated withthepresenceofasthma8_andobesity.9_TheADRB2gene islocatedonchromosome5q31andcanbefoundinseveral regionsofthebody,includingsmoothmuscle.10 _ADRB2act throughmediationbyadrenalineandnoradrenalineaction and promote smooth muscle relaxation, even in the pul-monary region,11 _{as well as playing an important role in} bronchodilationduringexerciseinhealthyindividuals.12_The Arg16GlyandGln27Glupolymorphismsofthe ADRB2gene have been associated with asthma symptoms,7 _including reductioninthepulmonaryfunctionandinthe bronchodila-tionresponsetomedication,astheyhavenegativeinfluence onthebronchodilatoreffect,13 _{atherapeuticresourcethat} ispartofpre-exerciseEIBprevention.14

Recently,ourresearchgroupfoundahigherpresenceof Arg16Gly polymorphism in children and adolescents with asthma when compared withcontrols. Additionally, there

wasatrendforahigherfrequencyofpolymorphismGly16 inasthmaticswithexcessweight.15 _{However,theinfluence} ofthepolymorphismpresenceontheADRB2receptorinthe presenceof EIB in childrenand adolescentshas not been investigated.Ourhypothesisisthatthehigherfrequencyof polymorphismsinADRB2receptorcouldberelatedtohigher presenceofEIBinthispopulation.Therefore,theaimofthis studywastodetermine theinfluenceofpolymorphismsin theADRB2geneonthetriggeringofEIBinadolescents.

Method

Thiswasacross-sectionalstudyof160adolescentsofboth genders, of Caucasian ethnicity, aged between 9 and 17 years,selectedforconvenienceandfrompublicschoolsin thecityofCuritiba,stateofParana,Brazil.Thesamplewas dividedintwogroups,withEIB(EIB+)(n=45)andwithoutEIB (EIB−)(n=115).ThepresenceofEIBwasverifiedwhenthere wasadecrease≥10%inFEV1inrelationtothebaselinevalue

Body weight (kg) was measured on a digital scale (Toledo®_{) with0.1kgresolution,and height(cm)in a} sta-diometer (Sanny®_{) with a resolution of 0.1cm. The body} mass index (BMI) was calculated using the formula: BMI (kg/m2_{)=bodymass(kg)/height}2_{(m).Thisvariablewas} con-vertedtoBMIz-score,usingthe WHOAnthroplussoftware v.1.0.4developedbytheWorldHealthOrganization(WHO), classified according tothe cutoff points proposed by the WHOin2006.17

Waist circumference (WC) was measured in centime-ters (cm) using an inextensible anthropometric tape (Cardiomed®_{),measuredat midpointbetweenthe lastrib} andtheiliaccrest,withtheindividualinthestanding posi-tion,relaxedabdomenandarmspositionedalongthebody. The classification usedthe values proposed by Fernández etal.in2004.18

The diagnosis of asthma, according to the III Brazil-ian Consensus onAsthma Management (SBPT, 2002)19 _was performedusingmedicalassessmentandtheInternational StudyofAsthmaandAllergiesinChildhood(ISAAC) question-naireandthediseasewasconfirmedthroughtheanswerto questionnumber6.

Pulmonary function wasassessed by spirometry in the pre-exerciseandpost-exercise(5,10and15min).Theywere madethreemaneuverswiththeevaluatedinasitting posi-tionandnoseclip.The curveswereselectedthatshowed thehighestvaluesforthevariablesofforcedexpiratory vol-umein onesecond (FEV1)andforced vitalcapacity(FVC) in liters (L).The predicted valuesand the forced expira-torycoefficient (FEV1/FVC)wereshownby thespirometer software(OnebrandFlow®_{),usingasreferencevaluesthose} proposedbyKnudsonetal.20

Thebronchialprovocationtestwithexercisewascarried outinacontrolledenvironment(temperaturebetween20 and25◦_{C andrelativehumidityof 50%),in theafternoon,}

onatreadmill(MasterSuperATL-Inbramed)withaprotocol thatconsisted in walking/running for eightminutes at an intensity >85% of maximum heart rate (HRmax), according tothe American Thoracic Society guidelines.14 _HR

max was calculatedusingtheformulaproposedbyTanakaetal.21_and monitoredduringthetestusingaheartratemeter(Polar®_). Theassessedsubjectswereinstructednottodrink caffeine-baseddrinkstwohoursbeforetheevaluation,discontinue useofshortandlong-actionbronchodilators48hand5days beforetheevaluations,respectively.

Theexercisewasnotperformedwhentherewerereports of asthma crisis or viral infection of theairways in the 4 weeksbeforethetest.Themagnitudeofthedecreasewas calculatedfrom the maximum decrease in FEV1 (MDVEF1) throughtheequation:MDFEV1=[(pre-exerciseFEV1−lowest post-exercise FEV1)/pre-exercise FEV1]×100.14 DNA was extracted from blood samples and genotyping polymor-phismsArg16GlyandGln27GluofADRB2genebyperformed throughtheTaqmanmethod,usingaTaqManSNP genotyp-ingassaykitofAppliedBiosystemsandEppendorfrealplex v.1.5software, with the StepOne Plus equipment.Next, a scatter plot (XY) was made for the FAM-VIC separation and subsequent genotyping of each adolescent for each polymorphism. The individuals homozygous for the amino acidarginineatcodon16(ArgArg)andglutamineatcodon 27(GlnGln)wereclassifiedasnormal,whereasindividuals homozygousatposition16fortheaminoacidglycine(Gly)

andatposition27,forglutamicacid(GluGlu)wereclassified ascarriers,aswellastheheterozygousindividuals.

Statistical analysis was performed using SPSS soft-ware, version 19. Normality was verified using the Kolmogorov---Smirnov test, Student’s ttest wasapplied in parametric variables tocompare thegroups, whereas the Mann---WhitneyUtestwasappliedonnon-parametricones. The chi-square test was used to analyze the proportions betweenthegroups.Thecorrelationbetweenvariableswas assessedusingSpearman’scorrelationcoefficientand classi-fiedaccordingtoDanceyandReidy.22_{Thesignificancelevel} wassetatp<0.05.

Results

The baseline characteristics of the groups are shown in Table 1.Differenceswere observed for thevariables age, bodyweight,height,FEV1(bothinlitersandpercentageof predictedvalue)andFVCinlitersandintheFEV1/FVCratio, beinglowerintheEIB+group.

Regarding the polymorphisms of the ADRB2 gene, no significant differences were found for allele 27 and for genotypesofArg16GlyandGln27Glupolymorphismbetween the EIB+ and EIB− groups (p=0.26; p=0.97 and p=0.43, respectively).However,therewasatrendtoward statisti-calsignificancefor agreater proportionofpolymorphisms atallele16intheEIB+groupwhencomparedtotheEIB− (p=0.08)(Table2).

The percentage of maximum FEV1 decrease showed a moderate correlation with the presence of asthma (rho=0.47;p<0.01).However,forthevariablesBMIz-score (rho=0.01),WC(rho=0.20),polymorphismsArg16Gly(−0.01) and Gln27Glu (rho=−0.07) showed no significant correla-tions.

Discussion

The aim of this study was to determine the influence of polymorphismsintheADRB2 geneonthetriggering ofEIB in adolescents. There were no significant differences in frequencies for allele 27 and for the genotypes of allele

Arg16GlyandGln27GluintheEIB+groupcomparedtothe EIB−,whichpartlyrefutesourinitialhypothesis.However, therewasatrendtowardstatisticalsignificanceforahigher frequencyofallele16intheEIB+groupwhencomparedto theEIB−.Thisfindingmayberegardedasevidenceofthe associationbetweenpolymorphismsintheADRB2geneand thepresenceofEIB.

IndividualsdiagnosedwithEIB+havereducedpulmonary functioncomparedtoEIB−individuals,exceptforFVC(%of predicted),whichdidnotdifferbetweenthetwogroups.23

Ontheotherhand,previousstudiesdidnotidentifysuch differenceswhen assessingobeseadolescents,3 _andobese asthmatics24 _{and obese individuals with rhinitis.}25 _These divergences may be explained by differences in age and initialheightofthegroupsinthepresentstudy.

Table1 Anthropometricandbaselinespirometriccharacteristicsofthegroupspresent(+)andabsent(−)exercise-induced bronchospasm.

Variables EIB+

(n=45)

EIB− (n=115)

torU p-value

Age(years)a _{13.6±1.6 14.5±1.5}** _{4.05 0.00}

Bodymass(kg) 65.0±15.2 74.5±18.3** _{−3.09 0.00}

Height(cm) 160.4±9.4 165.5±8.9** _{−3.18 0.00}

BMI(kg/m2_{) 25.3±5.5 27.0±5.3 −1.88 0.06}

BMIz-score 1.6±1.3 1.9±1.2 −1.30 0.19

WC(cm) 84.6±13.6 84.9±13.9 1.57 0.12

VEF1(L) 2.9±0.5 3.4±0.6** −4.28 0.00

FEV1(%predicted) 95.4±10.8 102.0±18.8* −2.21 0.02

FVC(L) 3.4±0.7 3.9±0.8** _{−3.57 0.00}

FVC(%predicted) 102.3±12.2 106.0±13.4 −1.61 0.10

FEV1/FVC(%)a 86.7±10.3 88.5±7.0* 2.01 0.04

BMI,bodymassindex;WC,waistcircumference;FEV1,forcedexpiratoryvolumeinonesecond;FVC,forcedvitalcapacity;FEV1/FVC,

FEV1/FVCratio.

Valuesexpressedinmean±standarddeviation.

* _p<0.05. ** _p<0.01.

a _{Variableswithoutnormaldistribution.}

Table2 FrequencyofallelesandgenotypesofADRB2genebetweenthegroupspresent(+)andabsent(−)exercise-induced bronchospasm.

EIB+ EIB− X2 _p-value

Alleles

Arg 30(37.5%) 103(48.6%)

2.87 0.08

Gly 50(62.5%) 109(51.4%)

Total 80(100%) 212(100%)

Gln 47(69.1%) 140(69.3%)

0.001 0.97

Glu 21(30.9%) 62(30.7%)

Total 68(100%) 202(100%)

Genotypes

ArgArg 7(17.5%) 28(26.4%)

2.64 0.26

ArgGly 16(40.0%) 47(44.4%)

GlyGly 17(42.5%) 31(29.2%)

Total 40(100%) 106(100%)

GlnGln 19(55.9%) 51(50.5%)

1.68 0.43

GlnGlu 9(26.5%) 38(37.6%)

GluGlu 6(17.6%) 12(11.9%)

Total 34(100%) 101(100%)

Arg,arginine;Gly,glycine;Gln,glutamine;Glu,glutamicacid;valuesexpressedinabsoluteandrelativefrequencies.

provocationtestontreadmillandinthestudyofCichalewski et al.,26 _{it was performed 45min after a physical} educa-tionclass.On the other hand, neitherstudy identifiedan associationbetweenEIBandBMI.

Nopreviousstudieswerefoundtoassessthefrequencyof ADRB2polymorphismsinindividualswithandwithoutEIB.In thisstudy,thefrequencyofalleleGly16was62.5%and30.7% foralleleGlu27.Theproportionofthefirstwashigherthan theonefoundinastudyinasthmatics,whichwas46.6%.8 However,it is similartothe findingsfor the general pop-ulation(61%).27 _{The frequencyof alleleGlu27wassimilar} betweenthestudies.8,27

AstudycarriedoutbySnyderetal.28_{observedinhealthy} adults that individuals with the Arg16Arg and Gly16Gly

outbyFukuietal.30_{showedthatindividualswithincreased} responsivenesstomethacholinechallengetesthada poly-morphismatcodon16.EIBcanbeconsideredanexaggerated airwayresponse.1_{Thus,itwasexpectedthatpolymorphisms} hadaneffectonthebronchoconstrictorresponseto exer-cise,whichultimatelydidnotoccur.

Thecross-sectionalstudydesignlimitsthecausal associa-tionsbetweenthevariables.Anotherissuetobeemphasized asa limiting factor is the low number of participants for theanalysisofgeneticpolymorphisms,leadingtoacautious interpretationofthestudyfindings.Wesuggeststudieswith largersamplesizestoconfirmtheassociationbetweenthe presenceoftheGly16alleleandthemanifestationofEIB. Furtherstudiesarerequiredwiththecontrolofthe afore-mentionedlimitationsandtheuseofspirometryattimes3; 5;10;15and30minafterbronchialprovocation challenge teststopreventpossibleEIBmisdiagnosis.

Weconcludethatthepresenceofpolymorphisms associ-atedwithGlu27alleleandArg16GlyandGln27Glugenotypes did not influence EIB expression. However, the statistical trendforincreasedfrequencyofalleleGly16inindividuals withEIBcanbeconsideredanindicationoftheinfluenceof polymorphismsonthegeneADBR2inEIBinadolescents.

Funding

Fundac¸ãoAraucária,processn.19.281.

Conflicts

of

interest

The authors declare no conflicts of interest. LFA is a researcherat AraucariaFoundation, LRSisCapes doctoral fellow,WALisCNPqdoctoralfellow,CRCandNLhaveCNPq productivitygrants.

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