J. Pediatr. (Rio J.) vol.93 número6

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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

Pleasecitethisarticleas:FurlanLL,RibeiroJD,BertuzzoCS,SalomãoJuniorJB,SouzaDR,MarsonFA.Variantsintheinterleukin8gene andtheresponsetoinhaledbronchodilatorsincysticfibrosis.JPediatr(RioJ).2017;93:639---48.

夽夽

StudyconductedatUniversidadeEstadualdeCampinas(Unicamp),Campinas,SP,Brazil.

∗_{Corresponding}_author.

E-mail:[email protected](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. Nãohouveassociac¸ãoda varianters2227307àrespostaabroncodilatadoresinalatórios.Avarianters2227306foi associ-adaaFEF50%nogrupodominanteenogrupocomduasmutac¸õesidentificadasnogenecystic

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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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′_-CCA_TCA_TGA_TAG_CAT_CTG_TA-3′_and₅′_-CCA_CAA

TTTGGTGAATTATTAA-3′_,_and_the_Ase_I_restriction_enzyme

wereused;for thers2227306 variant, primers5′_-CTC _TAA

CTCTTTATA TAGGAATT-3′ _and₅′_-GAT _TGA_TTT _TAT_CAA

CAGGCA-3′_,_as_well_as_the_Eco_RI_restriction_enzyme;_and_for

thers2227307variant,primers5′_-TAA_AGG_TTT_GAT_CAA_TAT

AGA-3′ _and₅′_-CTT_CCT_TCT_AAT_TCCA_ATA_TG-3′_,_as_well_as

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).

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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).

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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,absolutefrequency. a

2andp-valuesrefertothecalculationofHardy---Weinbergequilibrium.Significantdataareshowninboldtype.Therewasweak 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

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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

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

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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-Bgroup).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;medianof0(rangingfrom−12to23).(AT)n=29;meanof 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;_mean_of_17.38_±_24.18;_median_of₁₆_(ranging_from₋₂₀_to_89)._(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;meanof 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;meanof29.93±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=/ AAandAT.(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;_mean_of_20.13_±_27.64;_median_of_18.5_(ranging_from₋₃₃_to_117)._(TT)_n₌_23;_mean_of 2.14±23.74;medianof0(rangingfrom−51to65).(L)AssociationofFEF25---75%withrs4073,dominantmodel,andCFTR-Agroup(p=0.029;

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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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