Variants in the interleukin 8 gene and the response to inhaled bronchodilators in cystic fibrosis

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UNIVERSIDADE ESTADUAL DE CAMPINAS

SISTEMA DE BIBLIOTECAS DA UNICAMP

REPOSITÓRIO DA PRODUÇÃO CIENTIFICA E INTELECTUAL DA UNICAMP

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http://www.scielo.br/scielo.php?script=sci_pdf&pid=S0021-75572017000600639

DOI: 10.1016/j.jped.2017.03.005

Direitos autorais / Publisher's copyright statement:

©2017

by Sociedade Brasileira de Pediatria. All rights reserved.

DIRETORIA DE TRATAMENTO DA INFORMAÇÃO Cidade Universitária Zeferino Vaz Barão Geraldo

CEP 13083-970 – Campinas SP Fone: (19) 3521-6493 http://www.repositorio.unicamp.br

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JPediatr(RioJ).2017;93(6):639---648

www.jped.com.br

ORIGINAL

ARTICLE

Variants

in

the

interleukin

8 gene

and

the

response

to

inhaled

bronchodilators

in

cystic

ﬁbrosis

夽,夽夽

Larissa

Lazzarini

Furlan

a

_,

_José

_Dirceu

_Ribeiro

b

_,

_Carmen

_Sílvia

_Bertuzzo

c

_,

João

Batista

Salomão

Junior

d,e

_,

_Dorotéia

_Rossi

_Silva

_Souza

f

_,

Fernando

Augusto

Lima

Marson

b,c,∗

a_Faculdade_de_Medicina_de_São_José_do_Rio_Preto,_São_José_do_Rio_Preto,_SP,_Brazil

b_Universidade_Estadual_de_Campinas_(Unicamp),_Faculdade_de_Ciências_Médicas,_Departamento_de_Pediatria,_Campinas,_SP,_Brazil c_Universidade_Estadual_de_Campinas_(Unicamp),_Faculdade_de_Ciências_Médicas,_Departamento_de_Genética_Médica,_Campinas,

SP,Brazil

d_Faculdade_de_Medicina_de_São_José_do_Rio_Preto,_Hospital_{Universitário,}_Departamento_de_Pediatria,_São_José_do_Rio_Preto,_SP,

Brazil

e_Faculdade_de_Medicina_de_São_José_do_Rio_Preto,_Hospital_{Universitário,}_Departamento_de_Pneumologia_Pediátrica,_São_José_do

RioPreto,SP,Brazil

f_Faculdade_de_Medicina_de_São_José_do_Rio_Preto,_Centro_de_Pesquisa_de_Bioquímica_e_Biologia_Molecular,_Departamento_de

BiologiaMolecular,SãoJosédoRioPreto,SP,Brazil

Received2August2016;accepted9January2017 Availableonline15July2017

KEYWORDS CFTR; Diseaseseverity; Interleukin8; Lungfunction Abstract

Objective: Interleukin8proteinpromotesinflammatoryresponses,eveninairways.The pres-enceofinterleukin8genevariantscausesalteredinflammatoryresponsesandpossiblyvaried responses to inhaled bronchodilators. Thus, this study analyzed the interleukin 8 variants (rs4073,rs2227306, andrs2227307) andtheir associationwiththeresponsetoinhaled bron-chodilatorsincysticfibrosispatients.

Methods: Analysisofinterleukin8genevariantswasperformedbyrestrictionfragmentlength polymorphismofpolymerasechainreaction.Theassociationbetweenspirometrymarkersand theresponsetoinhaledbronchodilatorswasevaluatedbyMann---WhitneyandKruskal---Wallis tests. The analysisincludedall cysticﬁbrosis patients, andsubsequently patientswith two mutationsinthecysticﬁbrosistransmembraneconductanceregulatorgenebelongingtoclasses ItoIII.

夽 _Please_cite_this_article_as:_Furlan_LL,_Ribeiro_JD,_Bertuzzo_CS,_Salomão_Junior_JB,_Souza_DR,_Marson_FA._Variants_in_the_interleukin₈_gene

andtheresponsetoinhaledbronchodilatorsincysticﬁbrosis.JPediatr(RioJ).2017;93:639---48.

夽夽_Study_conducted_at_Universidade_Estadual_de_Campinas_(Unicamp),_Campinas,_SP,_Brazil. ∗_{Corresponding}_author.

E-mail:fernandolimamarson@hotmail.com(F.A.Marson).

http://dx.doi.org/10.1016/j.jped.2017.03.005

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Results: This study included 186 cystic ﬁbrosis patients. There was no association of the rs2227307variantwiththeresponsetoinhaledbronchodilators.Thers2227306variantwas asso-ciatedwithFEF50%inthedominantgroupandinthegroupwithtwoidentiﬁedmutationsinthe

cysticﬁbrosistransmembraneconductanceregulatorgene.Thers4073variantwasassociated with spirometrymarkersinfour genetic models:co-dominant (FEF25---75% andFEF75%),

domi-nant(FEV1,FEF50%,FEF75%,andFEF25---75%),recessive(FEF75%andFEF25---75%),andover-dominant

(FEV1/FVC).

Conclusions: Thisstudyhighlightedtheimportanceofthers4073variantoftheinterleukin8 gene,regardingresponsetoinhaledbronchodilators,andoftheassessmentofmutationsinthe cysticﬁbrosistransmembraneconductanceregulatorgene.

PALAVRAS-CHAVE

CFTR;

Gravidadedadoenc¸a; Interleucina8; Func¸ãopulmonar

Variantesnogenedainterleucina8earespostaabroncodilatadoresinalatórios naﬁbrosecística

Resumo

Objetivo: Aproteínainterleucina8promoverespostasinflamatórias,oqueincluisuaatuação nas vias aéreas. A presença de variantes no gene da interleucina 8causa respostas infla-matóriasalteradasepossivelmenterespostasvariadasaousodebroncodilatadoresinalatórios. Assim,esteestudoanalisouasvariantesdainterleucina8(rs4073,rs2227306,rs2227307)esua associaçãoàrespostaabroncodilatadoresinalatóriosempacientescomfibrosecística.

Métodos: Foifeitaanálisedasvariantesgenéticasdainterleucina8porrestrictionfragment lengthpolymorphismdareaçãoemcadeiadapolimerase.Aassociaçãoentreosmarcadoresda espirometriaearespostaabroncodilatadoresinalatóriosfoifeitapelostestesdeMann-Whitney e Kruskal-Wallis.A análise incluiu todosos pacientes com fibrose císticae posteriormente pacientescomduasmutaçõesnogenecystic fibrosistransmembraneconductanceregulator

pertencentesàsClassesIaII.

Resultados: Esteestudo incluiu186pacientescomfibrose cística. Nãohouveassociaçãoda varianters2227307àrespostaabroncodilatadoresinalatórios.Avarianters2227306foi associ-adaaFEF50%nogrupodominanteenogrupocomduasmutaçõesidentificadasnogenecystic fibrosistransmembraneconductanceregulator.Avarianters4073foiassociadaamarcadores daespirometriaem quatro modelosgenéticos:codominante(FEF25-75% eFEF75%),dominante

(VEF1,FEF50%,FEF75%eFEF25-75%),recessivo(FEF75%eFEF25-75%)eoverdominante(VEF1/CVF). Conclusões: Esteestudodestaca,principalmente,aimportânciadavarianters4073dogeneda interleucina8,narespostaabroncodilatadoresinalatórios,concomitantementeaogenótipo dasmutac¸õesnogenecysticﬁbrosistransmembraneconductanceregulator.

Introduction

Theresponsetoinhaledbronchodilators(BD)incystic fibro-sis(CF)(OMIM:n.219700)isquitevariableanddependson the cystic fibrosis transmembrane regulator (CFTR) geno-type,pulmonary symptoms,andmainly, onvariantsinthe modifier genes, such as the beta-2-adrenergic receptor (ADRB2).1_So_far,_only_a_few_studies_have_investigated_such response.

MutationsintheCFTRgenecauseCF,duetodeﬁciency,

dysfunction,orabsenceoftheCFTRprotein.2_CF_is

characte-rizedbyacontinuouscycleofchronicairwayinﬂammation,

which can be exacerbated by interleukin-8 (IL-8), a key

pro-inﬂammatorymediator.IL-8isresponsibleforinitiating

andincreasingtheinﬂammatory responsein thepresence

of speciﬁc pathogens, causing activation and migration

of neutrophils from peripheral blood to tissues.3 _Chronic

airway inﬂammation isthe ﬁnal commonpathway oflung

injury.Itisresponsibleforincreasedvascularpermeability,

contributingtointerstitial,alveolar,andairwayedema.

The treatment of CF lung disease includes

anti-inﬂammatory drugs, inhaled corticosteroids, antibiotics,

mucolytic,hypertonicsaline,andphysiotherapy.Amongthe

possible therapies, thereis little evidence supporting the

role of BD in CF.4,5 _However, _BD _is _often _prescribed _for

a longer period due to wheezing and dyspnea episodes

in CF.6 _BD_reduces _the_liberation _of _mediators,_which _are

responsibleforrecruitingandactivatinginﬂammatorycells,

activatingcholinergicneurotransmissionandimproving

vas-cularpermeability.Italsoincreasesmucociliaryclearance,

leadingtoreducedlunginﬂammation.7_The_response_to_BD

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IL-8andinhaledbronchodilatorsincysticﬁbrosis 641

theADRB2proteintopromotebroncodilatation.8

Spirome-tryistheprimarymethodtoassesslungfunction,severity

andprogressionofthedisease,aswellasresponsetoBD.

Intenseneutrophilinﬂammationandlowbronchial

hyper-responsiveness are commonly observed characteristics in

CF.9_Moreover,_the_response_of_genetic_variants_to_BD_is_little

known.1_The_role_of_IL-8_in_the_neutrophilic_component_of_CF

lungdiseasecanbepointedout.Genesthatmightbe

associ-atedwiththeseverityofCFandpossiblywiththeresponse

toBDhavebeen reportedin thepresent studyandin the

literature.1,10---12 _The _IL-8_gene_should_be_highlighted, _as_it

hasadirectinﬂuenceonlunginﬂammationandcanbe

effec-tiveintheresponsetoBD.Therefore,thisstudycompared

thers4073,rs2227306,andrs2227307IL-8genevariantswith

theresponsetoBDinCFpatients,usingspirometry.

Methods

Patients

Across-sectional studywasconductedin 186CF patients, selectedintwouniversityreferralcenters(MedicalSchool ofSãoJosédoRioPretoandUniversidadeEstadualde Cam-pinas)forCFtreatment,from2013to2015.Thestudywas approvedbytheResearchEthicsCommitteeofthe Univer-sidadeEstadualdeCampinas,Brazil(underNo.528/2008). Allparticipantswere informedof thestudy andsignedan informedconsentform.Forpatientsunderthe ageof 18, the informed consent form was signed by the parents or guardians.Thestudyfollowedtherecommendationsofthe DeclarationofHelsinki.

The diagnosis of CF was conﬁrmed by the presence of two altered concentrations of sodium and chloride in sweat(chloridelevelhigherthan60mEq/L).Furthermore, no patient underwent initial immunoreactive trypsinogen (IRT)measurement.In agroupof 91patients, therewere twomutationsinthe CFTRgenebelongingtoclassesI,II, and/orIII(associatedwithgreaterdiseaseseverity,dueto theabsenceornon-functionalityoftheCFTRprotein)13_;₆₀

patientsdidnotpresentanidentiﬁedmutationoftheCFTR

gene,orhadtwomutationsbelongingtoclassesIV,V,orVI;

and35 patientshad amutationin theCFTRgene

belong-ingtoclassesI,II,orIII,andanon-identiﬁedmutation,or

belongingtoclassesIV,V,orVI(Table1).

Clinicalvariables

The following variables were analyzed: clinical scores (Shwachman-Kulczycki, Kanga, and Bhalla); body mass index (BMI) for patients older than 18 years, using the formulaBMI=weight/(height)2_,_the_Anthro_program_version

3.0.1(WorldHealthOrganization[WHO],2006)wasusedfor childrenunderfiveyearsofageandtheAnthroPlusversion 1.0.2 (World Health Organization [WHO], 2007) was used forpatientsagedbetweenfiveand18years;patient’sage and age at diagnosis; first clinical symptom (one general symptom, pulmonary and digestive symptoms); period until the first colonization with Pseudomonas aeruginosa; microorganisms identified in the routine sputum culture (mucoid and non-mucoid P. aeruginosa, Achromobacter xylosoxidans, Burkolderia cepacia, and Staphylococcus

aureus); transcutaneous arterial hemoglobin oxygen-saturation (SaO2); spirometry; and comorbidities (nasal

polyps, osteoporosis, meconium ileus, diabetes mellitus, andpancreaticinsufﬁciency).

Spirometrywasperformedin patientsoversevenyears ofage,usingtheCPFS/Dspirometer(MedGraphics---Saint Paul,Minnesota,USA).DatawererecordedinthePFBREEZE softwareversion3.8BforWindows95/98/NT(American Tho-racicSociety),andassessedinpercentagepredictedvalues for:forcedvitalcapacity(FVC),forcedexpiratoryvolumein 1s(FEV1),FEV1/FVCratio,forcedexpiratoryﬂowat25%of

FVC(FEF25%),forcedexpiratoryﬂowat50%ofFVC(FEF50%),

forcedexpiratoryﬂowat75%ofFVC(FEF75%),forced

expira-toryﬂowbetween25%and75%ofFVC(FEF25---75%),maximum

forcedexpiratoryﬂow(FEFmax),andexpiratoryreserve

vol-ume(ERV). Spirometrydata wereshown in percentageof the predicted value according to the Polgar and Promad-hat(1971), Pereiraetal.(2007),andDuarteetal.(2007) equations.14---16

In all patients undergoing spirometry, the test was

performed before and 15min after administration of BD

(albuterol--- C13H21NO3[400mg]).PatientsunderBDtherapy

wereinstructedtointerruptthemedicationeighthoursprior

tospirometry,iftheywereundershort-actingBDtreatment;

and48hours,iftheywereunderlong-actingBDtreatment.

Thepost-BDpercentagechangewasusedforthestatistical

analysis.TheBDresponsecriteria,deﬁnedasanincreaseof

>12%and200mLofinitialFEV1,wasusedasasecondmodel

toevaluatetheassociationbetweentheIL-8genevariants

andBDresponse.

DNAextractionandgenotyping

GenomicDNAwasextractedfromperipheralbloodsamples using standard phenol-chloroform method and quantiﬁed by GE NanoVueTM _{spectrophotometer} _(GE _Healthcare

Bio-sciences--- Pittsburgh,USA).Inthisstudy,theﬁnalsample concentrationwassetat50ng/␮L.

Mutations of the CFTR gene were analyzed by poly-merasechainreaction(PCR;F508del)followedbyenzymatic digestion (G542X, R1162X, R553X, G551D, and N1303K). Other mutations in the CFTR gene were identified by sequencingorwiththeuseoftheSALSAMultiplex Ligation-dependentProbeAmplification(MPLAmethod)KitP091-C1 CFTR-MRC-Holland S4X, 2183A>G, 1717-G>A, I618T with MegaBace1000® (GE Healthcare Biosciences --- Pittsburgh, USA),andABI3500(AppliedBiosystems---ThermoFisher Sci-entific---SãoPaulo,Brazil).17

IL-8 gene variants were analyzed by PCR followed by

restriction enzyme digestion. For the rs4073 variant, the

primers5-CCATCATGATAGCATCTGTA-3and5-CCACAA

TTTGGTGAATTATTAA-3,andtheAseIrestrictionenzyme

wereused;for thers2227306 variant, primers5-CTC TAA

CTCTTTATA TAGGAATT-3 and5-GAT TGATTT TATCAA

CAGGCA-3,aswellastheEcoRIrestrictionenzyme;andfor

thers2227307variant,primers5-TAAAGGTTTGATCAATAT

AGA-3 and5-CTTCCTTCTAATTCCAATATG-3,aswellas

theScrFIrestrictionenzyme.18,19_The_products_of_the

enzy-maticrestrictionweresubmittedtoelectrophoresisona12%

polyacrylamidegel,or4%agarosegel,18,19_and_stained_with

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Table1 DistributionofcysticﬁbrosispatientsfortheCFTRgenotypeandclassesofidentiﬁedmutations.a

Genotype n % Groupofpatients

---/--- 55 29.6

Patientswithoutknownmutationin theCFTRgene,orwithoneortwo classesIV,VorVICFTRmutations

V562I/--- 1 0.5 I507V/--- 1 0.5 D110H/V232H 1 0.5 G576A/R668C 1 0.5 p.Glu528G>A/TG11-5T 1 0.5 F508del/--- 23 12.4

PatientswithoneclassesI,IIorIII

CFTRmutation,andone

non-identiﬁedmutationorIV,VorVI

CFTRmutation G542X/--- 4 2.2 F508del/3272-26A>G 1 0.5 F508del/P205S 1 0.5 G542X/P205S 1 0.5 G542X/R334W 1 0.5 3120+1G>A/L206W 1 0.5 622-2A>G/711+1G>T 1 0.5 R1162X/- 1 0.5 G542X/I618T 1 0.5 F508del/F508del 49 26.3

PatientswithtwoclassesI,II,and/or IIICFTRmutations (CFTR-Bgroup) F508del/G542X 12 6.5 F508del/N1303K 5 2.7 F508del/R1162X 3 1.6 F508del/R553X 3 1.6 F508del/1584-18672pbA>G 1 0.5 F508del/c.1717-1G>A 2 1 3120+1G>A/R1066C 1 0.5 F508del/2183AA>G 1 0.5 F508del/2184insA 1 0.5

F508del/6Bto16exonduplication 1 0.5

F508del/G85E 1 0.5 F508del/S549R(T>G) 1 0.5 F508del/S4X 1 0.5 G542X/2183AA>G 1 0.5 G542X/R1162X 1 0.5 A561E/A561E 1 0.5 F508del/R1066C 1 0.5 R1070Q;S466X/G542X 1 0.5 F508del/1812-1G>A 2 1 2183AA>G/2183AA>G 1 0.5 3120+1G>A/3120+1G>A 1 0.5

n,samplesize;CFTR,cysticﬁbrosistransmembraneregulator.

a_All_patients_assessed_in_this_study_have_been_included_(CFTR-A_group).

Statisticalanalysis

Statistical analyses were performed using Statistical Package for the Social Sciences version 22.0 (SPSS Inc. --- Chicago, USA). The GPower software version 3.1.9.220

wasused to calculate the sample power, considering the

genotypeoftheanalyzed variantsandadoptingpower for

value above 80%. The following conditions were applied

tocalculate the sample power: analysis of variance test,

in place of Kruskal---Wallis test considering that analysis

of variance is a stronger test (effectsize=0.25, ˛=0.05,

power=0.80,numerator degreeoffreedom=2,numberof

groups=3, ideal n=158); two-tailed Mann---Whitney test

(effect size=0.5, ˛=0.05, power=0.80, allocation ratio

N2/N1=1,idealn=134);chi-squaredtest(effectsize=0.3,

˛=0.05,power=0.80,degreeoffreedom=2,idealn=108).

The Mann---Whitneyand Kruskal---Wallistests wereused

forthecomparisonbetweendifferentgenotypesandgroups

ofIL-8genevariantsandtheresponsetoBD.Incaseof

signif-icantdifferencesbetweenthegroupsfortheKruskal---Wallis

test,furtheridentiﬁcationandevaluationofthedifferences

betweengenotypeswereperformedwithMedCalc®software

for Windows, version 16.1 (MedCalc® Software --- Ostend,

Belgium).

Thechi-squared test andFisher’sexacttest wereused

forthecomparisonbetweendifferentgenotypesandgroups

ofIL-8genevariants,aswellastheresponsetoBD,deﬁned

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For the identiﬁcation of mutations in the CFTR gene,

patients were analyzed based on two contexts:

CFTR-A, all CF patients, regardless of gene mutations (n=186

patients);andCFTR-B,patientswithtwomutations

belong-ing toclasses I,II, and/or III (n=91 patients). As for the

variants,fouranalysismodelswereadopted:(i)co-dominant

(Kruskal---Wallis test); (ii) recessive (Mann---Whitney test);

(iii) dominant (Mann---Whitney test); (iv) over-dominant

(Mann---Whitney test), applied in association with clinical

variables.Forallanalyses,thealphavaluewassetat0.05.

Table2 Descriptiveanalysisofclinicalandlaboratorymarkersofcysticﬁbrosispatients.

Variables Distributiona

Gender(male) 92/186(49.5%)

Ethnicity(White) 169/180(93.9%)

Age(months) 169;201.41±171.98;143(7to932)

Onsetofsymptoms(months) 159;38.23±114.03;3(0to720)

Diagnosis(months) 171;87.90±158.39;20(0to833)

Onsetofdigestivesymptoms(months) 140;41.2±112.61;3(0to720)

Onsetofpulmonarysymptoms(months) 156;46.33±123.64;6(0to720)

Bodymassindex(Kg/m2₎ _178;_17.28_±_4.08;_16.28_(6.5_to_35.67)

Nasalpolyposis(presence) 28/162(17.3%)

Diabetesmellitus(presence) 32/164(19.5%)

Osteoporosis(presence) 25/162(15.4%)

Pancreaticinsufﬁciency(presence) 130/162(80.2%)

Meconiumileus(presence) 23/162(14.2%)

1stPseudomonasaeruginosa 121;103.03±171.74;31(0to872)b

MucoidP.aeruginosa(presence) 74/173(42.8%)c

Non-mucoidP.aeruginosa(presence) 99/173(57.2%)c

Achromobacterxylosoxidans(presence) 17/174(9.8%)

Burkolderiacepacia(presence) 25/174(14.4%)

Staphylococcusaureus(presence) 131/174(75.3%)

SaO2 159;94.87±4.28;96(66to99) Bhallascore 113;8.9±5.77;8(0to25) Kangascore 118;18.78±5.82;17.5(10to40) Shwachman-Kulczyckiscore 143;65.97±16.78;65(20to95) FVC 142;72.13±23.86;77(19to126) FEV1 141;60.99_±25.75;63(17to116) FEV1/FVC 144;79.08±14.99;81(39to113) FEF25% 119;61.28_±31.71;60(7to138) FEF50% 119;46.2±31.25;40(3to126) FEF75% 116;36.32±28.77;27.5(4to142) FEF25---75% 140;47.16±32.51;39(5to150) FEFMax 114;75.54±25.59;73.5(25to137) ERV 112;80.96_±52.39;69(3to248)

Responsetoinhaledbronchodilator

FVC 117;1.74±8.19;1(−17to32) FEV1 117;3.51±8.0;3(−12to48) FEV1/FVC 111;2.14±7.43;2(−19to32) FEF25% 99;9.99±24.41;5(−45to110) FEF50% 99;13.08±26.06;9(−41to114) FEF75% 99;20.93±46.47;16(−64to235) FEF25---75% 99;12.28±27.77;9.5(−51to117) FEFMax 116;2.63±14.48;3(−42to69) ERV 100;23.04_±101.08;0(₋₉₀to670)

SaO2,transcutaneousarterialhemoglobinoxygen-saturation;FVC,forcedvitalcapacity;FEV1,forcedexpiratoryvolumein1sofFVC;

FEF25%,forcedexpiratoryflowat25%ofFVC;FEF50%,forcedexpiratoryflowat50%ofFVC;FEF75%,forcedexpiratoryflowat75%of

FVC;FEF25---75%,averageforcedexpiratoryﬂowbetween25%and75%ofFVC;FEFmax,maximumforcedexpiratoryﬂow;ERV,expiratory

reservevolume.Spirometrydataareshowninpercentageofthepredictedvalue.Allevaluatedpatientsaredescribed.

a _The_data_with_categorical_distribution_are_presented_as_follows:_n_of_variable/n_total_{(percentage);}_data_with_numeric_distribution

arepresentedasfollows:samplesize;mean±standarddeviation;median(minimumtomaximum).

b _Missing_values_for_first_P._aeruginosa_correspond_to_cystic_fibrosis_patients_that_never_presented_a_P._aeruginosa_positive_culture. c _One_cystic_fibrosis_patient_showed_a_positive_culture_for_P._aeruginosa,_with_negative_values_for_P._aeruginosa_antigen_specific_IgG_and

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Table3 Distributionofgenotypes,allelesandhaplotypeofIL-8gene variants(rs4073,rs2227306andrs2227307)incystic ﬁbrosispatients.

Variants Genotype Number(%) Allele Number(af) 2 _p-Valuea

rs4073 AA 54(29.2) A 187(0.51) 3.94 <0.05 AT 79(42.7) T 183(0.49) TT 52(28.1) Total 370 Total 185 rs2227306 CC 70(38) C 211(0.57) 8.21 <0.05 CT 71(38.6) T 157(0.43) TT 43(23.4) Total 368 Total 184 rs2227307 GG 31(17.4) G 139(0.39) 1.48 >0.05 TG 77(43.3) T 217(0.61) TT 70(39.3) Total 356 Total 178 rs4073/rs2227306/rs2227307 Frequency Percentage(%) AACCGG 3 1.7 AACCGT 5 2.8 AACCTT 24 13.6 AACTGG 3 1.7 AACTGT 4 2.3 AACTTT 7 4 AATTGG 2 1.1 AATTGT 2 1.1 AATTTT 3 1.7 ATCCGT 13 7.3 ATCCTT 7 4 ATCTGT 30 16.9 ATCTTT 10 5.6 ATTTGG 5 2.8 ATTTGT 6 3.4 ATTTTT 3 1.7 TTCCGG 2 1.1 TTCCGT 5 2.8 TTCCTT 8 4.5 TTCTGG 4 2.3 TTCTGT 6 3.4 TTCTTT 3 1.7 TTTTGG 12 6.8 TTTTGT 5 2.8 TTTTTT 5 2.8 Total 177 100

IL-8,interleukin8;%,percentage;2_,_chi-square;_af,_absolute_frequency.

a2_and_p-values_refer_to_the_calculation_of_{Hardy---Weinberg}_equilibrium._Signiﬁcant_data_are_shown_in_bold_type._There_was_weak

correlationamongtheIL-8genevariantsinthecysticﬁbrosispatientsenrolledinthestudy.Thereisnolinkagedisequilibrium.

ForanalysisoftheHardy---Weinberg equilibrium(HWE), theOnlineEncyclopediasoftwareforGeneticEpidemiology Studies(OEGE)wasused.

The falsediscoveryrate (FDR)test wasappliedto cor-rectthe multiple test comparison.FDR is an approach to the multiple comparisons problem. Instead of controlling thechanceofanyfalse,FDRcontrolstheexpected propor-tionoffalsepositivesamongsuprathresholdvoxels.AFDR thresholdisdeterminedfromtheobservedp-value distribu-tion,andhenceisadaptive totheamount ofsignalinthe

data.21_The_p-value_and_corrected_p-value_(pc₎_were_shown

inthemanuscript.The linkagedisequilibriumanalysiswas

performedinHaploviewsoftwareversion4.2.

Results

ClinicalandlaboratorydataofCFpatientsaredescribedin Table2.Table3describesthegenotypeandallelefrequency

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A

D

G

J

K

M

L

H

I

B

E

F

C

8 5 25 20 15 10 5 0 –5 –10 4 3 2 1 0 –1 –2 35 50 40 30 20 10 0 –10 –20 30 25 20 15 10 5 0 –5 –10 AA+AT CC AA+AT AA AT AA+AT AA AA+TT AA+AT TT TT TT AA+AT AA AA+TT TT TT CT AA AT TT CT+TT CC+TT AA+AT AA+AT rs4073 + 2 CFTR mutations rs2227306 + 2 CFTR mutations rs4073 + 2 CFTR mutations rs4073 + 2 CFTR mutations rs4073 + 2 CFTR mutations rs4073 + 2 CFTR mutations rs4073 rs4073 rs4073 rs2227306 + 2 CFTR mutations rs4073 rs4073 + 2 CFTR mutations rs4073 + 2 CFTR mutations

Forced expiratory volume on _{1st sec. of forced vital capacity}

Forced expiratory flow 50%

Forced expiratory flow -

25-75% of forced vital capacity

Forced expiratory flow 75% Forced expiratory flow 75% Forced expiratory flow 50% Forced expiratory flow 75%

Forced expiratory volume on 1

st sec.

forced vital capacity

TT AT TT 6 4 2 0 –2 –4 25 20 15 10 5 0 –5 –10 –15 –20 40 35 45 40 35 30 25 20 15 10 5 0 30 25 20 15 10 5 0 –5 –10 –15 30 20 10 0 –10 –20 50 30 20 15 10 5 0 –5 –10 25 20 15 10 5 0 –5 –10 –15 50 40 30 20 10 0 40 30 20 10 0 –10 –20

Figure1 Associationofrs4073andrs2227306variantsofIL-8(interleukin-8)withtheresponsetoinhaledbronchodilatorsincystic ﬁbrosispatients.(A)Associationbetweenforcedexpiratoryvolumein1s(FEV1)offorcedvitalcapacity(FVC)andrs4073,dominant

modelandtwoidentiﬁedmutationsintheCFTRgene(cysticﬁbrosistransmembraneregulator)belongingtoclassesI,II,and/orIII (CFTR-Bgroup)(p=0.028;pc₌_0.112)._(AA₊_AT)_n₌_39;_mean_of_4.77_±_8.95;_median_of₄_(ranging_from₋₉_to_48)._(TT)_n₌_21;_mean

(9)

andcorrectedp-valuesforassociationofthethreeIL-8gene variants,consideringthefourmodelsofanalysisproposed andthegenotypeoftheCFTRgene.

Thers2227307variantwasnotassociatedwithresponse to BD in any of the studied models; in turn, rs2227306 wasassociatedwithFEF50% for patients withCC genotype

(dominant model; p=0.05; pc₌_0.083) _and _CT _genotype

(over-dominantmodel;p=0.033;pc₌_0.083)_in_the_CFTR-B

group(Fig.1DandE).

Specialemphasisshouldbegiventothers4073variant,

whichwasassociatedwiththefollowingspirometry

varia-bles:FEV1,FEV1/FVCratio,FEF50%FEF75%,andFEF25---75%.The

lowest response toBD wasobserved for the TT genotype

(dominantmodel)andpatientsoftheCFTR-BgrouptoFEV1

(Fig.1A), FEF50% (Fig. 1C), FEF75% (Fig. 1G), and FEF25---75%

(Fig.1K).ThesamewasobservedfortheCFTR-Agroupfor

FEF75%(Fig.1H)andFEF25---75%(Fig.1L).Intheco-dominant

analysis,theTTgenotypepresentedalowerresponsetoBD

forFEF75%(Fig.1F)andFEF25---75%(Fig.1J),respectively,inthe CFTR-AandCFTR-Bgroups.ForthemarkerFEV1/FVC,theAT

genotypeinpatientsfromtheCFTR-Bgroupshowedlower

responsetoBD(Fig.1B).Finally,inpatientsfromthe

CFTR-AandCFTR-BgroupstheAAgenotype(recessivemodel)for

rs4073showedhigherresponsetoBDforFEF75%(Fig.1I)and

FEF25---75%,respectively(Fig.1M).

The haplotypedistributionfor theIL-8genevariantsis

presentedinTable3.

ForthecomparisonbetweenIL-8genevariantsandthe

responsetoBD,deﬁnedasanincreaseof>12%and200mL

ofinitialFEV1,nopositiveassociationwasfound(p>0.05).

Discussion

TheinﬂuenceofdifferentIL-8genevariantsintheclinical severity of CF has been previously demonstrated.22 _This

studyshowedtheassociationofdifferentIL-8genevariants

andtheirmodulationtoBDresponse,assessingtheimpact

of the drug on pulmonary function. The variability of

responsestoBDisdeterminedbymultiplefactors,suchas

inﬂammationandpulmonaryobstruction,23 _bacteria,24 _and

lungsymptoms,25 _as_well _as_modiﬁer_genes.26 _However,_no

studies sofar have investigatedthe roleof IL-8asa

pro-inﬂammatorymediatorofCFanditsrelationshiptoBD,and

whetherthe IL-8genevariants mayexplaintheindividual

response to BD in CF. It is believed that the short-acting

and long-acting beta-2-agonists may be beneﬁcial for CF

patientswithpositivebronchialhyperresponsiveness.6

Inprogressivelungdiseases,differentmarkershavebeen

assessed;theuseofBDappearstoprovidebetterresponse

toFEF25---75%,whencomparedwithothermarkers,which

indi-catesinvolvementofsmallercaliberairwaysinCF.27_In_the

presentstudy,inagreementwiththereferenceliterature,

an association was observed between the rs4073 variant

andthe FEF25---75% markerin thedominant (forboth

CFTR-A and CFTR-B groups), co-dominant (CFTR-B group), and

recessive models(CFTR-B group).Therewasimprovement

inseveral othermarkers ofspirometryforrs4073,suchas

FEV1,FEV1/FVC,FEF50%,andFEF75%,whichshowsimpacton

breathingpatternsofpatients.

ThestudybyHillianetal.(2008)foundanassociationof

rs2227307andrs4073IL-8genevariantsandseverityof

pul-monarydisease.28 _In_that_study,_patients_were_divided_into

twocohorts:(1)homozygousF508delpatients;(2)patients

with other genotypes of the CFTR gene. In cohort 1, the

rs4073,rs2227306,andrs2227543variantswerenot

associ-atedwithlungdisease,andanassociationwasobservedfor

rs22227307,regardlessofgender.Incohort2,thers4073and

rs2227306variantswereassociatedwithlungdisease

sever-ityinmales.Thus,thegenderofthepatient,genotypeofthe

CFTRgene,andmodiﬁergenesmaymodulatetheseverityof

thelungdisease.28_In_this_study,_the_rs2227307_genotype_did

nothaveanimpactonthevariabilityoftheresponsetoBD.

Thissuggeststhatalthoughthisgenotypeisassociatedwith

CFTR-Bgroup(p=0.029;pc₌_0.116)._(AA₊_TT) _n₌_30;_mean _of_1.17_±_7.36;_median_of₀_(ranging_from₋₁₂_to_23)._(AT)_n₌_29;_mean_of 4.76±7.58;medianof4(rangingfrom−9to32).(C)Associationofforcedexpiratoryflowof50%(FEF50%)ofFVCwithrs4073,dominant model,andCFTR-Bgroup(p=0.046;pc=0.184).(AA+AT)n=37;meanof17.38±24.18;medianof16(rangingfrom−20to89).(TT)n=21; meanof4.95±20.8;medianof−2(rangingfrom−19to55).(D)AssociationofFEF50%withrs2227306,dominantmodel,andCFTR-Bgroup (p=0.05;pc₌_0.083)._(CC)_n₌_15;_mean_of_1.47_±_17.22;_median_of₀_(ranging_from₋₂₀_to_29)._(CT₊_TT)_n₌_44;_mean_of_15.84_±_25.04; medianof10.5(rangingfrom₋₂₈to89).(E)AssociationofFEF50%withrs2227306,over-dominantmodel,andCFTR-Bgroup(p=0.033; pc₌_0.083)._(CC₊_TT)_n₌_33;_mean_of_6.06_±_20.27;_median_of₄_(ranging_from₋₂₀_to_55)._(CT)_n₌_23;_mean_of_19.96_±_26.43;_median_of 19(rangingfrom₋₂₈to89).(F)Associationofforcedexpiratoryflowof75%(FEF75%)ofFVCwithrs4073,co-dominantmodelregardless ofidentifiedmutationsintheCFTRgene(CFTR-Agroup)(p=0.044;pc₌_0.058).₁ _/= _3._(AA)_n₌_28;_mean_of_29.93_±_39.98;_median_of₃₀ (rangingfrom₋₄₇to142).(AT)n=42;meanof24.81_±53.57;medianof14(rangingfrom₋₅₈to235).(TT)n=28;meanof9.14_±36.95; medianof2.5(rangingfrom₋₃₅to119).(G)AssociationofFEF75%withrs4073,dominantmodel,andCFTR-Bgroup(p=0.024;pc₌_0.096). (AA+AT)n=38;meanof33_±55.23;medianof16(rangingfrom₋₂₇to235).(TT)n=21;meanof5.43_±35.93;medianof2(rangingfrom −35to119).(H)AssociationofFEF75%withrs4073,dominantmodel,andCFTR-Agroup(p=0.034;pc₌_0.058)._(AA₊_AT)_n₌_70;_mean_of 26.86±48.34;medianof18(rangingfrom−58to235).(TT)n=28;meanof9.14±36.95;medianof2.5(rangingfrom−35to119).(I) AssociationofFEF75%withrs4073,recessivemodel, andCFTR-Agroup(p=0.04;pc₌_0.058)._(AA)_n₌_28;_mean_of_29.93_±_39.98;_median of30(rangingfrom−47to142).(AT+TT)n=70;meanof18.54±47.95;medianof10(rangingfrom−58to235).(J)AssociationofFEF between25%and75%(FEF25---75%)ofFVCwithrs4073,co-dominantmodelandCFTR-Bgroup(p=0.012;pc₌_0.024)._TT _/= _AA_and_AT._(AA) n=9;meanof25.78±23.14;medianof30(rangingfrom−21to57).(AT)n=29;meanof18.38±29.04;medianof14(rangingfrom−33to 117).(TT)n=21;meanof2.14±23.74;medianof0(rangingfrom−51to65).(K)AssociationofFEF25---75%withrs4073,dominantmodel,and CFTR-Bgroup(p=0.007;pc=0.024).(AA+ATT)n=38;meanof20.13±27.64;medianof18.5(rangingfrom−33to117).(TT)n=23;meanof 2.14±23.74;medianof0(rangingfrom−51to65).(L)AssociationofFEF25---75%withrs4073,dominantmodel,andCFTR-Agroup(p=0.029; pc=0.1).(AA+AT)n=82;meanof15.48±27.2;medianof15(rangingfrom−40to117).(TT)n=33;meanof5.94±27.33;medianof0 (rangingfrom₋₅₁to76).(M)AssociationofFEF25---75%withrs4073,recessivemodel,andCFTR-Bgroup(p=0.047;pc=0.063).(AA)n=9; meanof25.78_±23.14;medianof30(rangingfrom₋₂₁to57).(AT+TT)n=50;meanof11.56_±27.89;medianof8(rangingfrom₋₅₁to 117).Thedotcorrespondstomedianvaluesandthebarcorrespondstothe95%confidenceinterval.

(10)

lung diseaseinhomozygousF508del patients,itsresponse

toBDisnotrelevant.

ThetargetofBDistheADRB2protein,whichisexpressed

intheairwaysmoothmuscle.VariantsofADRB2are

associ-atedwithresponsetothemedication.Althoughthisprotein

hasbeenwidelystudiedinasthma,itwasveryseldom

stud-ied in CF. The effectiveness of the response to BD and

inhaled corticosteroids tomanageairway inﬂammation in

asthmahasbeen conﬁrmed,anddependsontheArg16Gly

(rs1042713; c.46A>G) and Glu27Gln (rs1042714; c.79C>G)

ADRB2genevariants.29_The_Gln27Glu_variant_confers

resis-tancetotheADRB2proteininBD response.The46*G and

79*Gallelesareprotectiveagainstasthma,reducingtherisk

by27%.29_Both_variants_of_the_G_allele_induce_changes_in_the

regulation of the receptor due toincreased susceptibility

toproteindegradation.ThisstudyfoundthattheArg16Gly

andGln27Glu ADRB2genevariants inﬂuencethe response

toBDinCF.Inspirometryandinothermarkersofseverity,

theArg16Glyvariantshowedapositive association,unlike

Gln27Glu. The Arg/Arggenotype for the Arg16Glyvariant

was associated with better values of FEV1 and FEF25---75%.

The responsetoBDin theanalyses ofhaplotype was

pos-itiveintheabsenceofGly16GlyandGlu27Glugenotypesfor

FEV1/FVCratio.

VariantsofADRB2,diffusingcapacityofthelungsfor

car-bon monoxide,alveolar capillary membraneconductance,

volume of blood in the alveolar capillary, and SaO2 were

evaluatedintheresponsetoBDin18patientsand20healthy

controls,beforeandaftertheadministrationofsalbutamol

(30,60,and90min).Healthysubjectsshowednochangesin

markersassessedfortheGlu27Gluvariant.However,inCF,

thisvariantinﬂuencedtheresponsetoBD:thebestresponse

wasfoundinthepresenceofatleastoneallele27Glu.There

wasadifferenceinpulmonarydiffusionandperipheralSaO2

accordingtothevariationoftheADRB2geneatposition27,

andthedosageofthedrugshouldbeprescribedaccording

tothisvariation.30

Most studies focus on the ADRB2 gene variants in the

response to BD. However, this study demonstrated that,

evenindirectly,theIL-8genevariants(andpossiblyinother

genes,whichmodulatetheinﬂammatorylungresponse)may

potentiateorminimizetheeffectofBDandalsoinﬂuence

theresponsetothemedication.

Regarding the HWE, as previously discussed by this

group,22 _two_variants _(rs4073 _and _rs2227306) _were _not_in

balance.ItisimportanttorememberthattheHWEassumes

anidealpopulation,withouttheinterferenceof

evolution-aryfactors.However,ingenesasthoseinvolvedinimmunity,

inﬂammation, and infection control, the HWE imbalance

mayappearsecondarilyassociatedwiththeselection

mech-anismsthatfavoredaparticularallelethatcanbringamore

effectiveresponse.The disequilibriumdoes notinvalidate

theassociationstudysincethegroupsarepartofthesame

population.

The limitations of the present study include (i)

cross-sectional dataset (BD response wasevaluated at a single

time); (ii) numerous missing data considering the

prob-lemstoachieveinformationintherecords;(iii)whetherBD

responsediffers based onbaseline FEV1, mainlyin health

subjects;(iv)potentialconfounderstotheresults,suchas

diseaseseverity,currenttherapies,medicationadherence,

andadequatepulmonaryfunctiontesteffort.

Inconclusion,IL-8genevariants(rs2227306andspecially

rs4073)can beassociatedwith theresponse toBDduring

spirometry.Thisdrugmaybean alternativefor the

treat-mentofthedisease,mostlyamongpatientswithairwaysof

smallercaliber.Studies involvingdosageandcombinations

ofdrugs shouldbe furtherconducted,in orderdetermine

thebesttreatmentaccording toCF patientgenotype,the

CFTRgene,aswellasmodiﬁergenes.

Funding

FALM: Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo(FAPESP)forsupportingtheresearches #2011/12939-4, #2011/18845-1, #2015/12183-8, and #2015/12858-5; FundodeApoioàPesquisa,aoEnsinoeàExtensãoda Uni-versidadeEstadualdeCampinasforsupportingtheresearch #0648/2015; JDR: FAPESP for supporting the research #2011/18845-1and#2015/12183-8.LLF:FAPESPfor suppor-tingtheresearch#2013/19052-0.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

ToLucianaMontesRezende, LucianaCardosoBonadia,and StephanieVilla-NovafortheirtechnicalsupportduringDNA extraction and identiﬁcation of mutations of the CFTR gene. To Marcela Augusta de Souza Pinhel, Michele Lima Gregório,RafaelFernandesFerreira,GracieleDomitila Ten-ani,andHeloisaCristinaCaldasfortheirtechnicalsupport duringstandardizationofIL-8genotyping.ToMariaÂngela Gonc¸alves de Oliveira Ribeiro for conducting pulmonary functiontests(LAFIP/Ciped/Unicamp).ToRafaellaMaionchi PereiraMartinsforhertechnicalsupporttodetermine clini-calscores.ToMariadeFátimaCorrêaPimentaServidonifor promotingalinkbetweenbothUniversities.

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