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
a,c,∗,1,
Heidi
Lacerda
Alves
da
Cruz
a,c,1,
Lucas
André
Cavalcanti
Brandão
c,d,
Rafael
Lima
Guimarães
a,c,
Lilian
Maria
Lapa
Montenegro
b,
Haiana
Charifker
Schindler
b,
Ludovica
Segat
e,
Sergio
Crovella
a,caDepartmentofGenetics,FederalUniversityofPernambuco(UFPE),Recife,Pernambuco,Brazil
bDepartmentofImmunology,AggeuMagalhãesResearchCenter(CPqAM–FIOCRUZ–Pernambuco),Recife,Pernambuco,Brazil cLaboratoryofImmunopathologyKeizoAsami(LIKA),FederalUniversityofPernambuco(UFPE),Recife,Pernambuco,Brazil dDepartmentofPathology,FederalUniversityofPernambuco,Recife,Pernambuco,Brazil
eInstituteforMaternalandChildHealth–IRCCS“BurloGarofolo”,Trieste,Italy
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t
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c
l
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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
Introduction
Mycobacterial diseases are a majorhealth concern,
affect-ingapproximatelyone-thirdoftheworld’spopulation.1The
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,4however,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,11constitutively
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.17,18These
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
diseases8,26andotherdiseases.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
StudypopulationNinety-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
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.8analyzedDEFB15UTR
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.8onHanpopulationandbyLopezCamposetal.37
inMexicans:wecanhypothesizethatthedifferencescouldbe,
atleastinpart,duetoethnicity,sincethefrequenciesDEFB1
SNPsvaryindifferentpopulations.AccordingtotheNCBIand
HapMapdatabases,38for−52SNP(rs1799946)thefrequency
oftheancestralalleleis0.64inEuropean(CEU),0.56in
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)8andas
aconsequencecomparingDEFB1haplotyperesultswithour
findingshasnotbeenpossible.Moreover,whenlookingatthe
Mexicanpopulationithastoberemarkedthatthestudyof
LopezCamposetal.37justanalyzedoneDEFB1SNP−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.8and
LopezCamposetal.37sufferthesamesamplesizeproblem,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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