Influence of Genetic Ancestry on INDEL Markers of NFKβ1, CASP8, PAR1, IL4 and CYP19A1 ...

14 Pages·2015·0.92 MB·English

RESEARCHARTICLE
Influence of Genetic Ancestry on INDEL
Markers of NFKβ1, CASP8, PAR1, IL4 and
CYP19A1 Genes in Leprosy Patients
PabloPinto1,2,ClaudioSalgado3,NeyPereiraCarneiroSantos2,SidneySantos1,2,
ÂndreaRibeiro-dos-Santos1,2*
1 LaboratóriodeGenéticaHumanaeMédica,InstitutodeCiênciasBiológicas,UniversidadeFederaldo
Pará,Belém,Pará,Brasil,2 NúcleodePesquisasemOncologia-NPO,UniversidadeFederaldoPará,
Belém,Pará,Brasil,3 LaboratóriodeDermatoimunologia,InstitutodeCiênciasBiológicas,Universidade
FederaldoPará,Belém,Pará,Brasil
*[email protected],[email protected]
Abstract
OPENACCESS
Citation:PintoP,SalgadoC,SantosNPC,SantosS,
Ribeiro-dos-SantosÂ(2015)InfluenceofGenetic Background
AncestryonINDELMarkersofNFKβ1,CASP8,
Leprosyisaninsidiousinfectiousdiseasecausedbytheobligateintracellularbacteria
PAR1,IL4andCYP19A1GenesinLeprosyPatients.
Mycobacteriumleprae,andhostgeneticfactorscanmodulatetheimmuneresponseand
PLoSNeglTropDis9(9):e0004050.doi:10.1371/
journal.pntd.0004050 generatedistinctcategoriesofleprosysusceptibilitythatarealsoinfluencedbygenetic
ancestry.
Editor:ChristianJohnson,FondationRaoul
Follereau,FRANCE
Received:March25,2015
Methodology/PrincipalFindings
Accepted:August12,2015
WeinvestigatedthepossibleeffectsofCYP19A1[rs11575899],NFKβ1[rs28362491],IL1α
Published:September14,2015
[rs3783553],CASP8[rs3834129],UGT1A1[rs8175347],PAR1[rs11267092],CYP2E1
Copyright:©2015Pintoetal.Thisisanopen [INDEL96pb]andIL4[rs79071878]genesinagroupof141leprosypatientsand180
accessarticledistributedunderthetermsofthe
healthyindividuals.TheINDELsweretypedbyPCRMultiplexinABIPRISM3130andana-
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany lyzedwithGeneMapperIDv3.2.TheNFKβ1,CASP8,PAR1andIL4INDELswereassoci-
medium,providedtheoriginalauthorandsourceare atedwithleprosysusceptibility,whileNFKβ1,CASP8,PAR1andCYP19A1were
credited.
associatedwiththeMB(Multibacilary)clinicalformofleprosy.
DataAvailabilityStatement:Allrelevantdataare
availablewithinthemanuscriptandSupporting
Informationfiles. Conclusions/Significance
Funding:ThisstudywassupportedbyFAPESPA NFKβ1[rs28362491],CASP8[rs3834129],PAR1[rs11267092]andIL4[rs79071878]
(FundaçãoAmazôniaParaensedeAmparoà
genesarepotentialmarkersforsusceptibilitytoleprosydevelopment,whiletheINDELsin
Pesquisa)ICAAF155/2014,CNPq(Conselho
NacionaldeDesenvolvimentoCientíficoe NFKβ1,CASP8,PAR1andCYP19A1(rs11575899)arepotentialmarkersforthesevere
Tecnológico),CNPQ481652/2012-4,CAPES clinicalformMB.Moreover,allofthesemarkersareinfluencedbygeneticancestry,and
(CoordenaçãodeAperfeiçoamentoPessoaldeNível
Europeancontributionincreasestherisktoleprosydevelopment,inotherhandanincrease
Superior),RededePesquisaemGenômica
inAfricancontributiongeneratesprotectionagainstleprosy.
PopulacionalHumana(RPGPH)-CAPES,CAPES-
Proamazonia3288/2013andFADESP/PROPESP/
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004050 September14,2015 1/14
InfluenceofAncestryonINDELMarkersinLeprosyPatients
UFPA(UniversidadeFederaldoPará).Thefunders
hadnoroleinstudydesign,datacollectionand AuthorSummary
analysis,decisiontopublish,orpreparationofthe
LeprosyisaninfectiousdiseasecausedbyMycobacteriumleprae,whichcancarrytoskin
manuscript.
lesionsandaffectperipheralnerves,whichcausephysicalandmotorinjuriesonthe
CompetingInterests:Theauthorshavedeclared
patients.Moreover,leprosy,maybeclassifiedintwomajorgroups,basedonclinicalmani-
thatnocompetinginterestsexist.
festationsinPaucibacillary(PB)orMultibacillary(MB),andthesephenotypemaybe
influencedbyhostimmuneresponse;thatcanbecontrolledbygeneticsfactorsthatcanbe
usefullikefuturepanelofbiomarkerstoleprosy,andit’srelatedwiththedifferentgenetic
backgroundofpopulationstudied.Therefore,weconductedastudytoevaluateseven
INDELpolymorphismsinsevengenesinvolvedinmodulationofthehostimmune
response,andconsequentlycanmodulatedophenotypeshowedthroughthedisease,to
identifypossiblesusceptibilitymarkersofleprosy.Howeverthisanalysiscanbespurious
onpresenceofpopulationstructure,commoninadmixturepopulationliketheBrazilian,
thusweevaluateliketheinfluenceofgeneticancestrycanmodulatedthediseaserisk.
Introduction
LeprosyisaninsidiousinfectiousdiseasecausedbytheobligateintracellularbacteriaMycobac-
teriumlepraethataffectstheskinandperipheralnerves,causingachronicgranulomatous
infection[1].Leprosypatientsmaybeclassifiedintwomajorgroups,basedonclinicalmanifes-
tationsusingasimplesystemintroducedbytheWHO(WorldHealthOrganization)in1982.
Paucibacillary(PB)istheprimarycharacteristicofTuberculoid(TT)leprosyandischaracter-
izedbyafewlesionsandscarcebacilli,andMultibacillary(MB)istheprimarycharacteristicof
anergicLepromatous(LL)leprosy.Fromanepidemiologicalperspective,thesituationinBrazil
iscriticalbecause,alongwithIndiaandIndonesia,ithasthehighestrateofnewcasesdetected
worldwide[2,3,4].
InadditiontothesystemintroducedbyWHOin1982,theuseofhistologicalandimmuno-
logicalcriteriaasdescribedbyRidley-JoplingfurtherimprovesdefinitionofBorderlinecases.
Accordingtothisclassification,TT(tuberculoid-tuberculoid)patients,whohavethePBtype,
exhibitastrongcellularimmuneresponse(CIR)mediatedbyTh1,andanegativeskinsmear
test.Incontrast,LL(lepromatous-lepromatous)patientshaveaweakorabsentCIRandahighly
positiveskinsmearassociatedtoanhumoralimmuneresponse.Inthemiddleofthisspectrum
arealargenumberofborderlinepatients,whichtogetherwithLLcomprisetheMBpole,with
symptomsvaryingfromweaktostrongCIRandnegativetopositiveskinsmears[5,6].
Theregulationofthehostimmuneresponseandmanifestationofdiseaseclinicalbetween
typesPB(better)andMB(severe)involvescytokineandothersmediatorsproducedbyvarious
subtypesofTcells.InPB,aninflammatoryimmuneresponseismediatedbyTh1cellsthat
expresspro-inflammatoryinterleukinsthatstimulatemacrophagesandphagocytosismecha-
nismstoinhibitbacillarygrowthandkillmycobacteria[2,7–9].Ontheotherhand,MB
patientshaveanintenseTh2immuneresponsewithproductionofanti-inflammatorycyto-
kinesinadditiontothespecificanti-PGL-1(phenolicglycolipid1)antibody.Thismechanism
doesnotblockbacillarygrowthandcontributestothehost’sinabilitytoresistthedevelopment
ofseveredisease[2,8,9–11].
Recentstudieshaveinvestigatedgeneticmarkers,usuallyinnateimmuneresponsegenes,as
possiblesusceptibilityfactorsforleprosybecausetheSNPsinthesegenescanmodulatedthe
hostimmuneresponseandconsequentlylowerhostresistancetobacillusgrowth[6,12,13].
However,fewstudieshaveinvestigatedINDELpolymorphisms(insertion-deletion)inimmune
responsegenesinleprosy.Moreover,suchpolymorphismspresentinterestingfeaturesas
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InfluenceofAncestryonINDELMarkersinLeprosyPatients
geneticmarkersbecausei)INDELsarespreadthroughoutthehumangenome,ii)INDELs
derivefromasingleevent(theydonotpresenthomoplasy),iii)smallINDELscanbeanalyzed
usingshortamplicons,whichimprovesamplificationofdegradedDNAandfacilitatesmulti-
plexingreaction,iv)INDELscancreateabruptchangesinthenormalfunctionofthegeneand
v)INDELscanbeeasilygenotypedusingasimpledye-labelingelectrophoreticapproach[14].
ThecurrentstudyselecteightINDELinsevengenes(CYP19A1,NFKβ1,IL1α,CASP8,
UGT1A1,PAR1,CYP2E1,andIL4),whichhaverelationwiththeimmuneresponsemodulation
inleprosypatients,besideliteraturethatdemonstratethesemolecularmarkerslikefunctional
polymorphismsthataltertranscriptionalactivityofthegene,andconsequentlytheimmuno-
logicalphenotypeagainstthebacilli.AdditionallytheseINDELscanbeabletocontributionto
constructionapossiblepanelofsusceptibilitymarkers.
However,fromthegeneticpointofview,Brazilisrecognizedashavingoneofthemosthet-
erogeneouspopulationsintheworld,withimportantgeneticinformationbeingcontributedby
threemaincontinentalgroups,Europeans,AfricansandAmerindian,resultinginagenetically
verydiversemodernBrazilianpopulation[15].Therefore,analysisofgeneticmarkersincom-
plexdiseasesmayresultinspuriousresultsduetopopulationsubstructure[16],anditis
importanttoperformthegenomicancestrycontrol,especiallyinpopulationswithahigh
degreeofinterethnicadmixture[14].
TheobjectiveofthisstudywastoinvestigateeightINDELpolymorphismsinsevengenes
involvedinmodulationofthehostimmuneresponse,includingCYP19A1[rs11575899],
NFKβ1[rs28362491],IL1α[rs3783553],CASP8[rs3834129],UGT1A1[rs8175347],PAR1
[rs11267092],andCYP2E1[INDEL96pb],besidesoneVNTR(variablenumbertandem
repeat)of70bponintron3ofIL4[rs79071878]inagroupconsistingof141leprosypatients
and180healthyindividuals,toidentifypossiblesusceptibilitymarkersofleprosyandevaluate
theinfluenceofgeneticancestryondiseaserisk.
MaterialsandMethods
Ethicsstatement
TheprojectwasapprovedbytheParáFederalUniversityethicscommittee(N°197/07).
Samples
Weinvestigated141leprosypatientswhoattendedtheDrMarcelloCandiaReferenceUnitin
SanitaryDermatologyoftheStateofPará(UREMC),inMarituba,Pará,BrazilbetweenJanuary
2008andDecember2009.Allpatientswereinformedaboutthestudybeforetheysignedinformed
consentforms.Since2002,UREMCregisteredbetween308and472leprosypatients(mean:408
casesperyear).Ofthe765leprosycasesregisteredin2008and2009alone,141(18.43%)wereran-
domlyselectedforthisstudy.ThesepatientsweredividedaccordingtoRidley-Joplingclassifica-
tion[5]intoPaucibacillary(TT:PB31)andMultibacillary(BT,BB,BLandLL:MB110)groups.
Atotalof180healthyindividualswhowereunrelated,withoutleprosyorotherchronicdiseases
andfromthesamegeographicareaaseachotherwerechosenforthecontrolgroup.Leprosy
patient’sdescriptionsweremadepreviously[6].Thesesubjectswereaskedtoparticipateinthe
studyafterbeinginformedaboutthestudyobjectivesandsigninginformedconsentforms.
DNAextraction
DNAextractionwasperformedaspreviouslydescribedbyphenol-chloroformmethod[6,17].
TheDNAconcentrationwasdeterminedbyspectrophotometry(ThemoScientificNanoDrop
1000,NanoDropTechnologies,Wilmington,US).
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InfluenceofAncestryonINDELMarkersinLeprosyPatients
MultiplexTyping. DNAsamplesweretypedforthe7biallelicINDELsand1VNTRof70
bp.EachmultiplexPCRwasperformedinafinalvolumeof10μLcontaining5μLofQIAGEN
MultiplexPCRKit,1μLofQ-solution,1μLofprimermix(Forward+Reverseprimersata
concentrationof2μMeach),1μLofDNA(10ng)and2μLofwater.Thefluorescentmole-
cules6FAMandHEXwereinsertedatthe5’positionofeachprimer(forwardorreverse).All
PCRreactionswereperformedontheThermocycleVeriti-96WellThermalCycler(LifeTech-
nologies,CA,USA).Beforecapillaryelectrophoresis,1.0μLPCRproductwasaddedto8.5μL
deionizedformamideHI-DI(LifeTechnologies,CA,USA)and0.5μLGeneScan500LIZsize
standard(LifeTechnologies,CA,USA).DNAfragmentswereseparatedusinganABIPRISM
3130GeneticAnalyzer(LifeTechnologies,CA,USA)andanalyzedwithGeneMapperIDv3.2
software(LifeTechnologies,CA,USA).Thismethodissimilarlikedescribedinapaperof
INDELmarkersofourgroup[14].
AncestryInformativeMarker(AIM)
Individualinterethnicadmixturewasestimatedusingapanelof48ancestryinformativemark-
ers(AIMs)aspreviouslydescribed[6,14].
Statisticalanalysis
TheallelicfrequenciesbetweenhealthyindividualsandleprosypatientsandbetweenPBand
MBpatientswereestimatedbygenecounting.DeviationfromtheHardy-Weinbergequilib-
riumwasassessedusingchi-squaredtests,usingtheArlequinv3.5software[18],andp-value
ofHWEwascorrectedbyBonferronimethods.
DifferencesbetweenleprosypatientsandhealthyindividualsandbetweenPBandMB
patientswithrespecttoage,genderandgeneticancestrywereestimatedusingStudent’st-Test,
Fisher’sexacttestandMann-Whitneytests,respectively.Theassociationofmarkersbetween
groupswasanalyzedbylogisticregressiontests,allthetestwerecorrectedbyFDR(FalseDis-
coveryRate)method,andalltestswereperformedusingthestatisticalpackageunderRcalcu-
lation.Atwo-tailedp-value<0.05wasconsideredstatisticallysignificant.
TheindividualcontributionsofEuropean,AfricanandAmerindiangeneticancestrywere
estimatedusingtheSTRUCTURE2.3.3programassumingthreeparentalpopulations(Euro-
pean,AfricanandAmerindian),aburn-inperiodof200,000,and200,000MarkovChain
MonteCarlorepetitionsafterburn-in[16].Thedifferencesinallelicfrequenciesbetweenlep-
rosycasesandthehealthyindividualsformarkersanalyzedfollowinganadjustmentforpopu-
lationstratificationwasperformedusingtheSTRATsoftwareprogramwith10,000
simulations[16].
Results
Thedataofclinicalanddemographicdistributionofleprosypatientsandhealthyindividualsis
showninTable1.Themeanagewashigherinhealthyindividuals(55.7±12versus43.3±21,
p<0.001),andmalepatientsweremorefrequentamongleprosypatients(97[68.8%]versus65
[36.1%],p<0.001).AnalysisofethnicityshowedthatthemeanfrequencyofAfricanswas
higheramongleprosypatients(0.284versus0.236,p<0.001)andEuropeansweremorefre-
quentinhealthyindividuals(0.461versus0.427,p=0.004).
ThefrequenciesofINDELsfortheeight(8)genesanalyzedinleprosypatientsandhealthy
individualsareshowinTable2.ForthepolymorphisminIL4(VNTRof70bp),onlytwo
alleleswereidentifiedinthesample.Oneallelehadtworepeatsof70bp(alleleA1)andthe
otherhadthreerepeatsof70bp(alleleA2),suggestingthesesallelesarebiallelicmarkers.All
thepolymorphismsanalyzedwereaccordingtotheHardyWeinbergequilibrium,thereforethe
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InfluenceofAncestryonINDELMarkersinLeprosyPatients
Table1. Demographicandclinicalcharacteristicofthesampleofleprosypatientsandhealthyindividuals.
Variables LEPROSYPATIENTS(n=141) HEALTHYINDIVIDUALS(n=180) pvalue(IC-95%)
Agea 43.3±21 55.7±12 <0.001
Genderb(M/F) 97(68.8%)/44(31.2%) 65(36.1%)/115(63.9%) <0.001
GeneticAncestryc
African 0.284±0.11 0.236±0.04 <0.001
European 0.427±0.13 0.461±0.06 0.004
Ameridian 0.289±0.11 0.303±0.09 0.094
at-TestofStudent;
bFisher'sExactTest;
cMann-whitneytest;Thedataareshowlikemean±standarddeviation.
doi:10.1371/journal.pntd.0004050.t001
associationanalysiswereperformedwithregressionlogistictestanddifferencesinallelicfre-
quencieswerecorrectedbyfrequenciesofancestrymarkersinformative.
WhentheINDELswereanalyzedbylogisticregression,thegenesNFKβ1andPAR1showed
statisticallysignificantdifferencesassociatedwiththepresenceoftheDELallele(p=0.016and
p=0.022,respectively)andbothwereassociatedlikeprotectionfactorstonotdevelopingthe
disease(OR[IC95%]=0.50[0.27–0.88]andOR[IC95%]=0.35[0.14–0.86],respectively),for
thesegeneswasfoundadominanceeffectDELallele,thatincreaseyourprotectioncapacityin
generalpopulation.TheCASP8showedsignificantdifferencesassociatedwiththepresenceof
theDEL/DELhomozygousgenotypeandwasassociatedwithariskfactorforleprosydevelop-
ment(p=0.017;OR[IC95%]=2.33[1.16–4.69])(Table3).Theanalysisofallelefrequencydif-
ferenceswasthencorrectedfortheinfluenceofgeneticancestryonpopulationstructure,and
theresultsshowedthattheDELalleleofPAR1geneandthealleleA ofIL4ismorefrequentin
1
healthyindividuals(p=0.018andp=0.019,respectively)(Table3),theseresultsshownthe
importanceofstatisticalcorrectioninadmixturepopulation,inordertoexhibitdifferences
covertbystructurepopulation.
Table4summarizestheclinicalanddemographiccharacteristicsofleprosypatients
groupedaccordingtoclinicalmanifestationinPB(Paucibacillary)andMB(Multibacillary)
groups,andtheonlysignificantdifferencewasobservedforage(p=0.003),withahigher
meanageinMBpatients(45.7±22versus34.9±15).WhentheINDELswereanalyzedbylogis-
ticregression,NFKβ1showedsignificantdifferenceslikeriskfactorassociatedwiththepres-
enceofthealleleDELinMBpatients(p=0.024;OR[IC95%]=2.64[1.13–6.19]),of
contradictorywaythedominanceeffectofDELalleleseemprotectagainstthedevelopmentof
leprosy,butwhenthediseaseisestablishedyoureffectseeminefficienttocombattobacilli.
PAR1showedsignificantdifferencesassociatedwiththepresenceofhomozygousDEL/DEL
genotypeinPBpatients(p=0.031;OR[IC95%]=0.41[0.17–0.96])(Table5).Theanalysisof
allelefrequencydifferenceswerecorrectedforpopulationstructureandshowedthattheDEL
alleleofCASP8ismorefrequentinPBpatients(p=0.003),whiletheDELalleleofCYP19A1is
morefrequentinMBpatients(p=0.007)(Table5).
Fig1showstheOR(oddsratio)valuesobtainedfromleprosypatientsandhealthyindividu-
alswithingroupshavingdistinctlevelofancestrycomposition.Thefigureshowsthatgreater
frequencyofEuropeanethnicbetweenthegroups(leprosypatientsandhealthyindividuals),
higheristheriskfordevelopingleprosy,whilethesmallerthefrequencyoftheAfricanethnic,
loweristheriskfordevelopingleprosy.Nostatisticallysignificantvalueswereobtainedforthe
analysisoftheAmerindiangroup.
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InfluenceofAncestryonINDELMarkersinLeprosyPatients
Table2. AllelefrequenciesofINDELsfortheeightinvestigatedgenes.
GENE LEPROSYPATIENTS(n=141) HEALTHYINDIVIDUALS(n=180)
CYP19A1(rs11575899)
INS 0.574 0.558
DEL 0.425 0.442
HWE 0.998 0.998
NFKβ1(rs28362491)
INS 0.546 0.467
DEL 0.454 0.533
HWE 0.281 0.266
IL1α(rs3783553)
INS 0.617 0.544
DEL 0.383 0.456
HWE 0.898 0.779
CASP8(rs3834129)
INS 0.557 0.586
DEL 0.443 0.414
HWE 0.215 0.996
UGT1A1(rs8175347)
INS 0.411 0.325
DEL 0.589 0.675
HWE 0.280 0.487
IL4(rs79071878)
A a 0.660 0.581
2
A b 0.340 0.419
1
HWE 0.521 0.876
PAR1(rs11267092)
INS 0.320 0.217
DEL 0.680 0.783
HWE 0.06 0.196
CYP2E1
INS 0.088 0.083
DEL 0.911 0.916
HWE 0.600 0.994
HWE=p-valueforHardyWeinbergequilibriumafterBonferronicorrection;
aAllelewiththreerepeatsof70pb;
bAllelewithtworepeatsof70pb
doi:10.1371/journal.pntd.0004050.t002
Theseresultsarebetterunderstoodonfrequenciesdistribution,accordingwithrangeof
ancestrycontribution(S1Table).ForAfricanancestry99.4%ofhealthindividualisclosed
between0%and50%ofAfricancontribution(rangethathavep<0.05onFig1),moreoverthe
contributionrangeof10%to30%isclosed81.7%ofhealthindividual,inthisrangetheFig1
havemoredeclineofORvalue,thatshowedthehigherprotectioneffectofAfricanancestry.
ToEuropeanancestry,61.7%ofleprosypatientsisclosedbetween40%to80%ofEuropean
contribution,while87.2%ofhealthindividualsisclosedbetween0%to50%ofEuropeancon-
tribution.Additionally,forthecontributionrangebetween60%to80%weobserved17%ofall
patients,whilenohealthyindividualwasobservedthisrange,thesedatashowthatleprosy
patientshavehigherEuropeancontributioncomparedwithhealthyindividuals.Taketogether
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InfluenceofAncestryonINDELMarkersinLeprosyPatients
Table3. Allelicandgenotypicdistributionbetweenleprosypatientsandhealthyindividualstomarkersassociatedwhitsusceptibilitytoleprosy.
GENE LEPROSYPATIENTSn(%) HEALTHYINDIVIDUALSn(%) pa OR(IC95%)b pSTRATc
NFKβ1(rs28362491)
INS/INS 45(31.9%) 35(19.4%) 1
INS/DEL 64(45.4%) 98(54.4%)
DEL/DEL 32(22.7%) 47(26.1%) 0.559 0.83(0.64–2.34)
[DEL]carriers 96(68.1%) 145(80.5%) 0.016 0.50(0.27–0.88)
INS 0.546 0.467
DEL 0.454 0.533 0.243
PAR1(rs11267092)
INS/INS 19(13.5%) 11(6.1%) 1
INS/DEL 52(36.9%) 56(31.1%)
DEL/DEL 70(49.6%) 113(62.8%) 0.094 0.64(0.39–0.86)
[DEL]carriers 122(86.5%) 169(93.9%) 0.022 0.35(0.14–0.86)
INS 0.320 0.217
DEL 0.680 0.783 0.018
CASP8(rs3834129)
INS/INS 50(35.5%) 62(34.4%) 1
INS/DEL 57(40.4%) 87(48.3%)
DEL/DEL 34(24.1%) 31(17.2%) 0.017 2.33(1.16–4.69)
[DEL]carriers 91(64.5%) 118(65.5%) 0.413 0.80(0.47–1.36)
INS 0.557 0.586
DEL 0.443 0.414 0.123
IL4(rs79071878)
A /A 63(44.7%) 60(33.3%) 1
2 2
A /A 60(42.6%) 89(49.4%)
2 1
A /A 18(12.8%) 31(17.2%) 0.132 0.56(0.26–1.18)
1 1
[A ]carriers 78(55.4%) 120(66.6%) 0.088 0.63(0.37–0.84)
1
A d 0.660 0.581
2
A d 0.340 0.419 0.019
1
ap-valueobtainedforlogisticregressionadjustedbyage,genderandgeneticancestry;
bAdjustedOddsRatio(OR);
cp-valueaftercorrectionforpopulationstructure;
dA —allelewithtwotandemrepeatsA —allelewiththreetandemrepeats.
1 2
doi:10.1371/journal.pntd.0004050.t003
theFig1andS1Tableshownthattoleprosypatientsofanadmixturepopulation,likeBrazil,
Africanethnicgeneratesprotectionagainstthedevelopmentofdisease,andtheoppositeisalso
truthforEuropeanethnic.
Discussion
NF-κBbelongstofamilyofproteintranscriptionfactorsthatmodulatemanyinflammatory
processes.Intherestingstate,IκBα(inhibitorofNF-kβactivity)sequestersNF-κBinthecyto-
plasmandpreventsitsactivity,butinresponsetospecificstimuli,IkBαisubiquitinatedand
degradedallowingNF-kBtomigratetothenucleusandstimulatethetranscriptionofproin-
flammatorygenes[19,20].ThealleleDEL(rs28362491)hasbeenshowntobeassociatedwitha
decreaseoftranscriptionalactivityofvarietygenesofimmuneresponse[21]andwithauto
immunediseasesuchasSystemicSclerosis[22]andlupuserythematosus[23].
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004050 September14,2015 7/14
InfluenceofAncestryonINDELMarkersinLeprosyPatients
Table4. Demographicandclinicalcharacteristicsofthesampleaccordingwithclinicalformof
leprosy.
Variables LEPROSYPATIENTS pvalue(IC-95%)
PB(n=31) MB(n=110)
Agea 34.9±15 45.7±22 0.003
Genderb(M/F) 19(63.3%)/11(36.7%) 78(70.9%)/32(29.1%) 0.504
GeneticAncestryc
Afric 0.290±0.10 0.282±0.12 0.534
European 0.419±0.09 0.429±0.13 0.964
Ameridian 0.289±0.10 0.288±0.11 0.648
at-TestofStudent;
bFisher'sExactTest;
cMann-whitneytest;Thedataareshowlikemean±standarddeviation.
doi:10.1371/journal.pntd.0004050.t004
TheroleofNF-kβinleprosyisnotclear,andstudieslinkedtoexpressionofNF-kβhave
suggestedthatlowerexpressioniscommoninleprosypatients[24,25].Ourresultssuggestthat
theDELcarriesgenotypeinducesprotectionagainstleprosy(Table3),althoughacomparison
ofPBandMBpatientsalsosuggeststhatDELbehaveslikeariskfactorforthedevelopmentof
thesevereclinicalformofMB(Table5).BecausethetranscriptionofNF-kβismediatedby
specificstimuli,suchasthepresenceofM.leprae[24],itisconceivablethatthepresenceof
DELconfersrisktoMBleprosy.
PAR1isareceptorofthePARfamilyofproteinsthatbelongtoauniquegroupofGprotein
—coupledreceptors.Inparticular,PAR1proteinispresentinavarietyofcellslikeplatelets,
endothelia,epithelial,neurons,fibroblasts,smoothmuscle,leukocytesandtumorlines[26].
Thisreceptorhasbeenshowntobeinvolvedinmanynaturalphysiologicalprocesses,that
involveinflammationlikethesystemscardiovascular,respiratoryandcentralnervousandin
embryogenesis,cancerandinflammation[27].PAR1suppressesThelpertype1(Th1)andT
helpertype17(Th17)cellsandthesecretionofIL-12andIL-23,therebyresultingintheinhibi-
tionofpro-inflammatoryresponses[28].Thealleleofinsertion(INS)ofINDELstudied
(rs11267092)hasbeenshowntoincreasegenetranscription[29]andtherefore,itisariskallele
forleprosy.OurresultssuggestthatthepresenceofDELinducesprotectionagainstleprosy
(Table3),andtheDEL/DELgenotypeconfersprotectionagainstthedevelopmentofclinical
formsofMB(Table5),thusthisgenotypeofPAR1genecansuppressescellularinfiltrationand
increasebothTh1andTh17responsestoinfection.Moreover,analysesofmacrophages
revealedthatsecretionofIL-12andIL-13,twocytokinethatplayrolekeyoncellularimmunity
Th1andTh17,canbesuppressedbyPAR1activation.Furthermore,PAR1cansuppressinter-
feronregulatoryfactor5(IRF5),thatplayrolekeyliketranscriptionfactorforIL-12andIL-23,
whichmodulatesthesubsetsofcellularimmunity.TherebythesuppressionofIRF5andIL-
12/23secretionbyPAR1gene,canprovidesanovelmechanismbywhichthehostsuppresses
theTh1andTh17responsetoinfection,anddysregulationofthisprocesscanlikelyanimpor-
tantfactorinthesusceptibilityofsomeindividualstoleprosy[28].
MacrophageswithahighloadofM.lepraehavebeenshowntoundergoapoptosis,andthis
mechanismisunderthecontrolofcytokines[30].Inleprosypatients,theimmunesystemis
overburdenedwithbacilli,andmostlikelythecontinuousactivationofTcellsbycirculating
M.lepraeantigensleadstoapoptosisandtoareductionofperipherallymphocytesandother
immuneeffectorcellsinthesepatientswiththeregulationofapoptosisinvolvedinthestimula-
tionandactivationofcaspase-8[31].ThealleleDEL(rs3834129)causeadecreaseinCASP8
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004050 September14,2015 8/14
InfluenceofAncestryonINDELMarkersinLeprosyPatients
Table5. AllelicandgenotypicdistributionbetweenleprosypatientsgroupedaccordingclinicalformPBorMB.
GENE PATIENTSPBn(%) PATIENTSMBn(%) pa OR(IC95%)b pSTRATc
NFKβ1(rs28362491)
INS/INS 15(48.4%) 30(27.3%) 1
INS/DEL 13(41.9%) 51(46.4%)
DEL/DEL 3(9.7%) 30(27.3%) 0.119 2.78(0.76–10.07)
[DEL]carriers 16(51.6%) 81(73.7%) 0.024 2.64(1.13–6.19)
INS 0.694 0.495
DEL 0.306 0.504 0.410
PAR1(rs11267092)
INS/INS 5(16.1%) 14(12.7%) 1
INS/DEL 5(16.1%) 47(42.7%)
DEL/DEL 21(67.7%) 49(44.5%) 0.031 0.41(0.17–0.96)
[DEL]carriers 26(83.9%) 96(87.2%) 0.259 1.98(0.60–6.55)
INS 0.241 0.340
DEL 0.759 0.660 0.223
CASP8(rs3834129)
INS/INS 7(22.6%) 43(39.1%) 1
INS/DEL 16(51.6%) 41(37.3%)
DEL/DEL 8(25.8%) 26(23.6%) 0.579 0.76(0.29–1.97)
[DEL]carriers 24(77.4%) 67(60.9%) 0.114 0.46(0.18–0.90)
INS 0.484 0.577
DEL 0.516 0.423 0.003
CYP19A1(rs11575899)
INS/INS 14(45.2%) 32(29.1%) 1
INS/DEL 17(54.8%) 53(48.2%)
DEL/DEL - 25(22.7%) 0.998 -
[DEL]carriers 17(54.8%) 78(70.9%) 0.082 2.11(1.00–4.93)
INS 0.726 0.532
DEL 0.274 0.468 0.007
ap-valueobtainedforlogisticregressionadjustedbyage,genderandgeneticancestry;
bAdjustedOddsRatio(OR);
cp-valueaftercorrectionforpopulationstructure.
doi:10.1371/journal.pntd.0004050.t005
transcriptionandareductioninapoptosis[32],therebyimprovingthebacillaryload.Our
resultssuggestedthattheDEL/DELgenotype(Table3)andthehighfrequencyofDELallele
(Table5)canraisethebacillaryloadandthusconfersarisktoleprosydevelopment.
Interleukin-4(IL-4)isakeycytokinesecretedbyTh2lymphocytes,eosinophilsandmast
cellsthatinducestheactivationanddifferentiationofBcellsandthedevelopmentoftheTh2
subsetoflymphocytes,whichisineffectiveincombatingleprosy[33].Ouranalysisofthe
VNTRonintron3oftheIL4gene(rs79071878)revealedtwocommonalleleswithtwo(A )
1
andthree(A )tandemrepeats.Ofthese,A alleleisknowntobeahighproducerofIL-4[34].
2 2
OurresultsindicatethatalleleA ismorefrequentinleprosypatientscomparedtohealthy
2
individuals,consistentwiththefactthathigherlevelsofIL4wouldbeineffectiveincontrolling
thegrowthofbacilli(Table3).
Theconversionofandrogenstoestrogens,catalyzedbyaromataseencodedbytheCYP19A1
gene,istheprimarypathwayofestrogenproductioninhumans[35].Thelevelsofthesehor-
monesareimportantinleprosypatientsandithasbeendemonstratedthatandrogenlevelsare
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004050 September14,2015 9/14
InfluenceofAncestryonINDELMarkersinLeprosyPatients
Fig1.Inacomparisonof141leprosypatientsand180healthyindividuals,eventswithstatistically
significant(p<0.05)differencescanbecategorizedintosixcategoriesofindividualswithAfricanand
Europeangeneticancestry(10%>20%>30%>40%>50%>60%),thep-valueswereadjustedbyageand
gender.TheanalysisinindividualswithAmerindianancestrywasnotstatisticallysignificant.
doi:10.1371/journal.pntd.0004050.g001
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004050 September14,2015 10/14