DEFB1 gene polymorphisms and tuberculosis in a Northeastern Brazilian population

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Medical

Microbiology

DEFB1

gene

polymorphisms

and

tuberculosis

in

a

Northeastern

Brazilian

population

Ronaldo

Celerino

da

Silva

_,

_Heidi

_Lacerda

_Alves

_da

_Cruz

_,

Lucas

André

Cavalcanti

Brandão

_,

_Rafael

_Lima

_Guimarães

_,

Lilian

Maria

Lapa

Montenegro

_,

_Haiana

_Charifker

_Schindler

_,

_Ludovica

_Segat

_,

Sergio

Crovella

a_{DepartmentofGenetics,FederalUniversityofPernambuco(UFPE),Recife,Pernambuco,Brazil}

b_{DepartmentofImmunology,AggeuMagalhãesResearchCenter(CPqAM–FIOCRUZ–Pernambuco),Recife,Pernambuco,Brazil} c_{LaboratoryofImmunopathologyKeizoAsami(LIKA),FederalUniversityofPernambuco(UFPE),Recife,Pernambuco,Brazil} d_{DepartmentofPathology,FederalUniversityofPernambuco,Recife,Pernambuco,Brazil}

e_{InstituteforMaternalandChildHealth–IRCCS“BurloGarofolo”,Trieste,Italy}

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Articlehistory:

Received17September2014 Accepted5September2015 Availableonline2March2016 AssociateEditor:JorgeLuizMello Sampaio Keywords: Tuberculosis Innateimmunity DEFB1 Genotyping Polymorphism

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␤-Defensin-1,anantimicrobialpeptideencodedbytheDEFB1gene,isknowntoplayan importantroleinlungmucosalimmunity.InourassociationstudyweanalyzedthreeDEFB1

functionalpolymorphisms−52G>A(rs1799946),−44C>G(rs1800972)and−20G>A(rs11362) in92tuberculosispatientsand286healthycontrols,bothfromNortheastBrazil:no associ-ationwasfoundbetweenthestudiedDEFB1polymorphismsandthedisease.Howeverwe cannotexcludethatthislackofassociationcouldbeduetothelownumberofsubjects analyzed,assuggestedbythelowstatisticalpowerachievedforthethreeanalyzedSNPs (valuesbetween0.16and0.50).

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:ronaldocelerino@yahoo.com.br(R.CelerinodaSilva). 1 _{R.C.S.andH.L.A.C.contributedequallytothewritingofthearticle.}

http://dx.doi.org/10.1016/j.bjm.2015.09.001

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Mycobacterial diseases are a majorhealth concern, affect-ingapproximatelyone-thirdoftheworld’spopulation.1_The most common clinical form of the disease is pulmonary tuberculosis,agranulomatousdiseaseofthelungscausedby

Mycobacteriumtuberculosis.Extrapulmonarytuberculosismay occurwhenthebacillusinfectsotherorgans.2–4

The general risk of infected individuals to develop the disease duringtheir lifetimeranges between5 and 10%.2,3 A possible genetic predisposition to tuberculosis (TB) has been suggested in several studies.5–8 _{Moreover evidence} existthatextrapulmonaryTBistheresultofanunderlying immunedefect,4_{however,thefunctionalcorrelationbetween} geneticpolymorphismsandimmunologicdefectshouldbeyet unraveled.7

Themucosalimmunityofairepitheliaiscomplex,andthe innateimmunesystem,mainlybythemucosalsecretionof antimicrobialpeptidesgradient,isthoughttoplayan impor-tantroleindefenseagainstM.tuberculosis,althoughtheexact mechanismsareyetpoorlyunderstood.9,10

The human ␤-defensin-1 (hBD-1) is a small (29–47 aminoacids)cationicantimicrobialpeptide,11_{constitutively} expressedinairwayepitheliathatplaysanimportantrolein mucosalimmunityinthelung.12–15 _{Thispeptideisencoded} byDEFB1gene,locatedatchromosome8p22-23.16

Three single nucleotide polymorphisms (SNPs), located at −52G>A (rs1799946), −44C>G (rs1800972) and −20G>A (rs11362)positioninthe5 untranslatedregion(UTR)ofthe

DEFB1genehavebeenreportedtopromotefunctional alter-ationofhBD-1expressionindifferentcellmodels.17,18_These SNPs were firstly described byDörk and Stuhrmann19 _and have been then associated with chronic obstructive pul-monary disease,20 _{increased levels of Candida albicans in} the oral cavity,21 increased risk for Human Immunodefi-ciencyVirus1infection,22–25 _{susceptibilitytomycobacterial} diseases8,26_{andotherdiseases.}27–31

Consideringthepossibleroleoftheinnateimmune sys-tem in the risk of tuberculosis infection, this study aims toexaminewhetherfunctional polymorphismsat5UTRin

DEFB1 gene are associated with the occurrence of active tuberculosis.

Patients

and

methods

Studypopulation

Ninety-twoTBpatientswereenrolledfromfivereference cen-terforTBfollowupinRecife(Pernambuco,NortheastBrazil): HospitaldasClínicas–UniversidadeFederaldePernambuco (HC-UFPE),InstitutodeMedicinaIntegralProfessorFernando Figueira(IMIP),HospitalBarãodeLucena(HBL/US)and Hos-pitalOtáviodeFreitas.Ofthe92patientsanalyzed,64(69.6%) presentedwithpulmonaryTB(36malesand28females,mean age21years±18.7)and28(30.4%)withextrapulmonaryTB(17 malesand11females,meanage23years±21.7).The diagno-siswasbasedonclinicalsymptomsandradiographicfindings, alongwithbacteriologicalconfirmation(culture,smearand/or polymerase chainreaction) as described bythe Diagnostic

Standards and Classificationof Tuberculosis inAdults and Children.32

As control group, we enrolled 286 healthy individ-uals (163 females/123 males; mean age 27 years±20.3), unrelated to patients, with negative Mantoux test, show-ing no symptoms of tuberculosis or previous history of the disease. All patients and control subjects came from the same geographical area (Pernambuco, Brazil), were all HIV-1 negative and were not under immunosuppressive medication.

Sinceethnicityisanimportantissueingeneticanalyses, patientsandcontrolsreportedtheirself-declaredskinbased ethnic origin.During ethnicity reporta healthprofessional accompaniedtheprocedureguaranteeingthecorrectnessof theprocedure.Afterethnicityassessmentweverifiedthe self-reportedandhealthprofessionalcheckedresults,byanalyzing in the controls 12 Ancestry informativemarkers (AIMs) as recently reported by Coelho et al.33 _{Absence or extremely} poorcorrelationbetweenskinbasedethnicity and thetrue geneticbackground,asrevealedbythe12AIMsanalysis,was observed.So,wediscardedtheself-declaredskinbased eth-nicoriginandconsideredonlythegeneticAIMsfindings.In fact,ourstudiedpopulationconsistsofastrongadmixtureof European-Caucasian,AfricanandSouthAmerican Amerindi-ans population, leading the genetic North East Brazilian compositionunique;consequentlyitisimpossibletostratify our populationforethnicity,being anadmixtureofaround 58%European,24%Africanand18%Amerindianasrecently reported.33

Written informed consent was obtained from the par-ticipants or their parents. The CPqAM/FIOCRUZ Ethics Committee (CEP Registration – 55/05) approved the study. Patients underwent a standardizedclinical–epidemiological questionnaire.

DEFB1genotyping

GenomicDNAwasextractedfromwholebloodusingQIAamp DNA Blood Kit according to manufacturer instructions (QIAampDNABloodMidiKit,Qiagen).

The DEFB1 5UTR polymorphisms −52G>A (rs1799946), −44C>G (rs1800972) and −20G>A (rs11362) were geno-typed by direct sequencing, according to Braida et al.22 _{Sequences were handled using the 4Peaks}

(http://nucleobytes.com/index.php/4peaks) and CodonCode

Alignersoftware(http://www.codoncode.com/aligner/).

Statisticalanalysis

Chi-square test was used to verify the Hardy–Weinberg equilibrium and the Fisher’s exact test was employed for pair-wisecomparisonofallele,genotypeandhaplotype fre-quencies using contingency tables as appropriate; only p

values<0.05 were considered to besignificant. All the sta-tistical analyses were carried out using the open-sourceR package (http://www.r-project.org). The power ofthe study wascheckedbysoftwareG*powersoftwareversion3.1.34 Hap-lotypeswerecomputed usingtheArlequinversion 3.5.1.335 andHaploviewsoftware.36

Table1–AllelesandgenotypesdistributionofSNPs–inthe5untranslatedregion(UTR)ofDEFB1geneintuberculosis patients(TB)andhealthycontrols(HC)fromNortheastBrazil.

SNPs TBpatients n=92 Healthycontrols n=268 p-value,OR(95%CI) All pTB Allvs.HC pTBvs.HC -52G>A(rs1799946) G 97(0.53) 68(0.54) 309(0.54) Reference Reference A 87(0.47) 58(0.46) 263(0.46) p=0.80,1.05(0.74–1.49) p=1.00,1.00(0.67–1.50) GG 29(0.32) 19(0.30) 89(0.31) Reference Reference GA 39(0.42) 30(0.48) 131(0.46) p=0.78,0.91(0.51–1.65) p=0.87,1.07(0.54–2.15) AA 24(0.26) 14(0.22) 66(0.23) p=0.75,1.11(0.56–2.19) p=1.00,0.99(0.43–2.26) GA/AA 63(0.68) 44(0.70) 197(0.69) p=1.00,0.98(0.58–1.69) p=1.00,1.05(0.56–2.01) −44C>G(rs1800972) C 153(0.83) 102(0.81) 491(0.86) Reference Reference G 31(0.17) 24(0.19) 81(0.14) p=0.40,1.23(0.75–1.96) p=0.17,143(0.82–2.40) CC 66(0.72) 42(0.67) 213(0.74) Reference Reference CG 21(0.23) 18(0.29) 65(0.23) p=0.89,1.04(0.56–1.88) p=0.29,1.40(0.71–2.69) GG 5(0.05) 3(0.05) 8(0.03) p=0.32,2.01(0.50–7.25) p=0.40,1.90(0.31–8.32) CG/GG 26(0.28) 21(0.34) 73(0.26) p=0.59,1.15(0.65–1.99) p=0.21,1.46(0.77–2.71) −20G>A(rs11362) G 118(0.64) 82(0.65) 361(0.63) Reference Reference A 66(0.36) 44(0.35) 211(0.37) p=0.86,0.96(0.66–1.37) p=0.76,0.92(0.60–1.40) GG 39(0.42) 26(0.41) 115(0.40) Reference Reference GA 40(0.43) 30(0.48) 131(0.46) p=0.70,0.90(0.52–1.54) p=1.00,1.01(0.54–1.90) AA 13(0.14) 7(0.11) 40(0.14) p=1.00,0.96(0.42–2.06) p=0.66,0.77(0.26–2.02) GA/AA 53(0.57) 37(0.59) 171(0.60) p=0.72,0.91(0.55–1.52) p=0.89,0.96(0.53–1.74) pTB,pulmonarytuberculosis;OR,oddsratio;95%CI,95%confidenceinterval;SNP,singlenucleotidepolymorphism.Referenceindicatesthe mostfrequentallelesandgenotypes.

Results

Thegenotypingresultsofthethree5UTRDEFB1functional

polymorphisms−52G>A(rs1799946),−44C>G(rs1800972)and

−20G>A (rs11362) in TBpatients and healthy controls, are

showninTable1.AllSNPswereinHardy–Weinberg

equilib-riumforbothTBpatientsandhealthycontrols.

DEFB1 SNPs allele and genotype frequencies were sim-ilar in TB patients and healthy controls and no signif-icant difference has been found (p>0.05), also when TB patientswerestratifiedaccordingtothepulmonaryformof disease.

Wecomputedthehaplotypesandobservedthatthethree SNPsinthe5UTRofDEFB1areinstronglinkagedisequilibrium (D>0.9),formingfourhaplotypes(Table2).However,no sig-nificantdifferenceswerefoundwhencomparingTBpatients andhealthycontrols(Table2).

In addition, we estimated the linkage disequilibrium andthe possiblehaplotypes,using Haploviewsoftware,for Brazilianhealthy controls (BRA)and healthy individuals of African (AFR), Caucasian (CEU) and Ad Mixed American (AMR) origin, provided by 1000 Genome Project Database

(www.1000genomes.org). We noted that DEFB-1 SNPs were

in perfect linkage disequilibrium (D=1.0) and have dif-ferent haplotype distributions in the studied populations

(Table3).

Thepost hocanalysisofthestudy throughtheG*power software,indicate the followingpowerfor threeSNPs:0.30 (−52G>A),0.5(−44C>G)and0.16(−20G>A).

Discussion

In the current study, no association was found between −52G>A (rs1799946), −44C>G(rs1800972), −20G>A (rs11362)

DEFB1polymorphismsandTBinaBrazilianpopulation,also whenconsideringthepulmonaryformofthedisease.

Inacase–controlstudy,Wuetal.8_{analyzedDEFB15}_UTR

polymorphismsin102patientswithpulmonaryTBand148 healthy controlsofChinese Han populationobserving that theDEFB1−44C/Cgenotypewasassociatedwithpulmonary TBsusceptibility(OR=2.096)andtheGGG(−52/−44/−20) hap-lotype was associated with the pulmonary TB protection (OR=0.348).

In a recent work, performed on a limited number of Mexicanpatients(27withpulmonaryTBand16with extra-pulmonary TB)and controls (n=49), López Camposet al.37 described an association between DEFB1 −44C>G SNP (CG genotypeinacodominantgeneticmodel)andsusceptibilityto extrapulmonaryTB,butnotwiththepulmonaryTB, hypoth-esizing a role forhBD-1 inthe fight againstM. tuberculosis

outsidethelung.

Ourfindingshavediscrepancieswiththeresultsobtained byWuetal.8_{onHanpopulationandbyLopezCamposetal.}37 inMexicans:wecanhypothesizethatthedifferencescouldbe, atleastinpart,duetoethnicity,sincethefrequenciesDEFB1

SNPsvaryindifferentpopulations.AccordingtotheNCBIand HapMapdatabases,38_{for−52SNP(rs1799946)thefrequency} oftheancestralalleleis0.64inEuropean(CEU),0.56in Sub-SaharaAfrican(YRI)and0.40inAsian(HCB);thefrequency

Table2–Haplotypesfrequencydistributionof5UTRDEFBSNPsintuberculosispatients(TB)andhealthycontrols(HC) fromNortheastBrazil.

Haplotypes(−52/−44/−20) TBpatients Healthycontrols 2n=572 p-value,OR(95%CI) All 2n=184 pTB 2n=126 Allvs.HC pTBvs.HC

ACG 86(0.47) 58(0.46) 264(0.46) Reference Reference

GCA 64(0.35) 42(0.34) 210(0.37) p=0.78,0.94(0.63–1.38) p=0.74,0.91(0.57–1.44) GGG 30(0.16) 22(0.18) 80(0.14) p=0.61,1.15(0.68–1.91) p=0.47,1.25(0.68–2.23) GCG 4(0.01) 4(0.02) 18(0.03) p=0.61,0.68(0.16–2.15) p=1.00,1.01(0.24–3.23) pTB,pulmonarytuberculosis;OR,oddsratio;95% CI,95%confidenceinterval;nc,notcalculable.Referenceindicatesthemostfrequent haplotypes.

Table3–Haplotypesfrequenciesinhealthyindividualofdifferentethnicorigin.

Haplotypes Frequencies

Brazilian(BRA) African(AFR) Caucasian(CEU) AdMixed American(AMR)

ACG 0.460 0.588 0.399 0.297

GCA 0.369 0.300 0.354 0.438

GGG 0.142 0.064 0.000 0.000

GCG 0.030 0.049 0.247 0.261

of ancestral allele of −44 SNP (rs1800972) is 0.26 in

Euro-pean(CEU),0.04inSub-SaharaAfrican(YRI)and0.12inAsian

(HCB);for−20SNP (rs11362),thefrequencyis0.61in

Euro-pean(CEU),0.60inSub-Sahara(YRI)and0.71inAsian(HCB).

Inaddition,theSNPsanalyzedinBrazilianhealthyindividuals

andhealthy individualofAfrican (AFR),inCaucasian (CEU)

andAdMixedAmerican(AMR)origin,wereinperfectlinkage

disequilibrium (D=1.0) and presented different haplotypic

distribution(Table3),suggestingthatethnicsbackgroundmay

beresponsibleforthedifferencesobservedinpopulationsthat havebeenstudied.Additionally,intheHanChinese popula-tionnolinkagedisequilibriumwasdetected(D<0.5)8_andas aconsequencecomparingDEFB1haplotyperesultswithour findingshasnotbeenpossible.Moreover,whenlookingatthe Mexicanpopulationithastoberemarkedthatthestudyof LopezCamposetal.37_{justanalyzedoneDEFB1SNP−44C>G} andotherSNPsincathelicidinencodingCAMPgene.So,itwas notpossibletocompareourDEFB1haplotypeswiththeresults reportedforMexicanpatients.Furthermore,DEFB1rs1800972 alleleandgenotypefrequenciesreportedintheMexican popu-lation(notinHardy–WeimbergEquilibrium)weresignificantly different(0.0002)fortheCalleletoBrazilianones.

However,wecannotexclude,thatthelackofassociation reportedinourstudy,couldbeduetothelimitedsamplesize; thepowerofourstudy(posthocPower≤0.5)indicatesthatnot findinganassociationbetweenSNPswithTBdevelopment, couldbeduetoatypeIIerror.Thisisastronglimitationof ourstudy,andseeingthatalsotheworks ofWuetal.8_and LopezCamposetal.37_{sufferthesamesamplesizeproblem,we} areconvincedthatanyfurtherstudydealingwithassociation betweendefensinsgenepolymorphismandTB,shouldenroll greaternumberofpatients,maybejoiningseveralresearch groups,inordertoachievebetterstatisticalpowertoprovide adefinitiveanswerontheroleofdefensinsinsusceptibilityto TB.

Allthisconsidered,evenifourresultsseemtoexcludean associationbetweenDEFB1polymorphismsandTB develop-mentinNortheastBrazilianpopulation,sinceDEFB1couldbea potentialcandidategeneforsusceptibilitytoTBandonlyvery fewstudieshaveaddressedthistopicachievingcontroversial results,wedothinkthatfurtherstudiesonalargernumberof patientsandinotherpopulationsareneededtodisclosethe roleofDEFB1variationsintuberculosisdevelopment.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Theauthorsthankallthehealthprofessionalsfromthe hospi-talsinvolvedinthestudythatcollaboratedwiththeresearch. We thank the Laboratory of Immunoepidemiology – Cen-trode PesquisasAggeuMagalhões/Fundac¸ão Oswaldo Cruz (CPqAM/FIOCRUZ),theLaboratoryofImmunopathologyKeizo Asami, the Department of Genetics, Federal University of Pernambuco, theGraduateProgram inGenetics for suppor-tingphysicalandscientific,aswellasFundac¸ãodeAmparo à Ciência eTecnologiadoEstado de Pernambuco(FACEPE), Coordenac¸ão deAperfeic¸oamentoPessoaldeNívelSuperior (CAPES)andConselhoNacionaldeDesenvolvimento Cientí-ficoeTecnológico(CNPq),forfinancialsupport.

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