The Influence of the Autoimmunity-Associated Ancestral HLA Haplotype AH8. 1 on the Human Gut

13 Pages·2015·1.81 MB·English

RESEARCHARTICLE
The Influence of the Autoimmunity-
Associated Ancestral HLA Haplotype AH8.1
on the Human Gut Microbiota: A Cross-
Sectional Study
JohannesR.Hov1,2,3,4☯*,HuanziZhong5☯,BingcaiQin5,6,JarlAndreasAnmarkrud1,
KristianHolm1,AndreFranke7,BenedicteA.Lie2,8,9,TomH.Karlsen1,2,3,4,10
1 NorwegianPSCResearchCenter,DepartmentofTransplantationMedicine,DivisionofCancerMedicine,
SurgeryandTransplantation,OsloUniversityHospitalRikshospitalet,Oslo,Norway,2 InstituteofClinical
MedicineandK.G.JebsenInflammationResearchCentre,FacultyofMedicine,UniversityofOslo,Oslo,
Norway,3 ResearchInstituteofInternalMedicine,OsloUniversityHospitalRikshospitalet,Oslo,Norway,
4 SectionofGastroenterology,DepartmentofTransplantationMedicine,DivisionofCancerMedicine,
SurgeryandTransplantation,OsloUniversityHospitalRikshospitalet,Oslo,Norway,5 BGI-Shenzhen,
Shenzhen,China,6 ShanghaiMajorbioBio-pharmTechnologyCo.Ltd.,Shanghai,China,7 Christian-
Albrechts-UniversityofKiel,InstituteofClinicalMolecularBiology,Kiel,Germany,8 DepartmentofMedical
Genetics,UniversityofOsloandOsloUniversityHospital,Oslo,Norway,9 DepartmentofImmunology,Oslo
OPENACCESS
UniversityHospitalRikshospitalet,Oslo,Norway,10 DepartmentofClinicalMedicine,UniversityofBergen,
Bergen,Norway
Citation:HovJR,ZhongH,QinB,AnmarkrudJA,
HolmK,FrankeA,etal.(2015)TheInfluenceofthe
☯Theseauthorscontributedequallytothiswork.
Autoimmunity-AssociatedAncestralHLAHaplotype * [email protected]
AH8.1ontheHumanGutMicrobiota:ACross-
SectionalStudy.PLoSONE10(7):e0133804.
doi:10.1371/journal.pone.0133804
Abstract
Editor:BrendaAWilson,UniversityofIllinoisat
Urbana-Champaign,UNITEDSTATES
Multipleimmune-relatedgenesareencodedintheHLAcomplexonchromosome6p21.
Received:April18,2015 The8.1ancestralhaplotype(AH8.1)includetheclassicalHLAallelesHLA-B*08:01and
Accepted:July1,2015 HLA-DRB1*03:01,andhasbeenassociatedwithalargenumberofautoimmunediseases,
Published:July24,2015 buttheunderlyingmechanismsforthisassociationarelargelyunknown.Giventherecently
establishedlinksbetweenthegutmicrobiotaandinflammatorydiseases,wehypothesized
Copyright:©2015Hovetal.Thisisanopenaccess
articledistributedunderthetermsoftheCreative thattheAH8.1influencesthehostgutmicrobialcommunitycomposition.Tostudythisfur-
CommonsAttributionLicense,whichpermits ther,healthyindividualswereselectedfromtheNorwegianBoneMarrowDonorRegistry
unrestricteduse,distribution,andreproductioninany
andcategorizedaseitherI.AH8.1homozygote(n=34),II.AH8.1heterozygote(n=38),III.
medium,providedtheoriginalauthorandsourceare
NonAH8.1heterozygoteorIV.HLA-DRB1homozygotebutnonAH8.1(n=15).Bacterial
credited.
DNAfromstoolsamplesweresubjectedtosequencingoftheV3–V5regionofthe16S
DataAvailabilityStatement:Thesequencedataare
rRNAgeneonthe454LifeSciencesplatformanddataanalyzedusingMothurandQIIME.
depositedattheEMBLNucleotideSequence
Database(AccessionnumberERP010886). Theresultsshowedthattheabundancesofdifferenttaxawerehighlyvariablewithinallpre-
definedAH8.1genotypegroups.Usingunivariatenon-parametricstatistics,therewereno
Funding:ThestudywasfundedbytheNorwegian
PSCResearchCenter.Thefundingsourcesof differencesregardingalphaorbetadiversitybetweenAH8.1carriers(categoriesIandII)
NorwegianPSCResearchCenterhadnorolein andnon-carriers(categoriesIIIandIV),howeverfourdifferenttaxa(Prevotellaceae,Clos-
studydesign,datacollectionandanalysis,decisionto
tridiumXVIII,Coprococcus,Enterorhabdus)hadnominallysignificantlowerabundancesin
publish,orpreparationofthemanuscript.Shanghai
AH8.1carriersthannon-carriers.Afterincludingpossibleconfoundersinamultivariatelin-
MajorbioBio-pharmTechnologyCo.Ltdprovided
supportintheformofsalariesforauthorsBQ,butdid earregression,onlythetwolattergeneraremainedsignificantlyassociated.Inconclusion,
nothaveanyadditionalroleinthestudydesign,data
collectionandanalysis,decisiontopublish,or
PLOSONE|DOI:10.1371/journal.pone.0133804 July24,2015 1/13
TheHLAHaplotypeAH8.1andtheGutMicrobiota
preparationofthemanuscript.Thespecificrolesof theoverallcontributionoftheAH8.1haplotypetothevariationingutmicrobiotaprofileof
theseauthorsarearticulatedinthe‘author stoolinthepresentstudywassmall.
contributions’section.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
Introduction
Thehumanleukocyteantigen(HLA)complexislocatedatchromosome6p21.Almostone
thirdofthe252expressedproteincodinggenesintheregionhaveputativefunctionsrelatedto
immunefunction[1].GeneticHLAvariantsaremajordeterminantsofsusceptibilitytoinfec-
tionandinflammatorydiseases[2,3],butformostdiseasesithasprovenchallengingtodeter-
minetheinvolvedmechanismsandexactallelicdeterminants.TheHLAcomplexisboth
extremelypolymorphicandcharacterisedbylowrecombination[4].
Amongseveralconservedso-calledancestralHLAhaplotypes[5],the8.1ancestralhaplo-
(cid:1)
type(AH8.1),whichspansseveralmillionbase-pairsandincludestheHLA-B 08:01and
(cid:1)
HLA-DRB1 03:01alleles,isparticularlyconserved.TheAH8.1isstronglyassociatedwithmul-
tipleimmune-mediateddiseasesincludingceliacdisease,type1diabetes,primarysclerosing
cholangitisandsystemiclupuserythematosus[6].Thishaplotypecontainsseveralgeneticvari-
antswithbiologicalimplications[7],includingtheclassicalHLAallelesinfluencingantigen
presentationandgeneticvariantsinfluencingthelevelsoftumornecrosisfactor(TNF)and
complementfactors.WhileforsomediseasestheAH8.1associationmaybelinkedmost
stronglytosingledisease-relatedalleles(e.g.HLA-DQ2involvinginceliacdisease),multiple
independentassociationsareobservedinseveraldiseases[8,9]andepistaticeffectsbetween
severalriskvariantswithinthishaplotypearelikelyconferringageneralriskforthedevelop-
mentofautoimmunedisease[6].
Thebacterialcontentofthegut(thegutmicrobiota)hasbeenlinkedtomultiplehumandis-
eases,includingtypicalautoimmuneandinflammatoryconditions[10–12].Environmental
influenceslikedietareimportantdeterminantsofthegutmicrobialcomposition[13].However,
thereisfirmevidencefromtwinstudiesthatalsotheeffectsofhostgeneticfactorsareconsider-
able[14,15].Strongevidenceimplicatingsinglegenesintheshapingofthegutmicrobiotahave
beenfoundinmousemodelswithgeneticallyalteredlevelsofe.g.defensins,IgAandthebacte-
rialsensingproteinNOD2[16–19],thelatteralsoseeninhumans[19].Importantly,similar
gene-microbiotainteractionshavealsobeenassociatedwithgeneticvariantscommonly
observedinthehealthypopulation,e.g.intheFUT2gene,responsibleforthepresenceofblood
antigensonepithelialsurfaces[20].Thereisalsoevidencefrombothmiceandhumanssuggest-
inganimpactofclassicalHLAallelesonthegutmicrobiotacomposition[21–23].Development
ofAH8.1associateddiseasesliketype1diabetes,rheumatoidarthritisandceliacdiseasehave
alsobeenlinkedtoalterationsinthegutmicrobiota[10,24,25].Giventheunexplainedassocia-
tionsbetweentheAH8.1andautoimmunediseases,wehypothesizedthatthesumofmultiple
geneticvariantsontheAH8.1haplotypewithbiologicalimplicationscausesalterationsinthe
gutmicrobiotathatincreasediseaseriskandaredetectableinthenormalpopulation.
MaterialsandMethods
Studypopulation
OnehundredandseventeenindividualsfromtheNorwegianBoneMarrowDonorregistry
wereincluded,basedonHLAgenotypesdeterminedupontheinclusionintheregistry.The
studycohortconsistedoffourpre-definedgroupsasdefinedinTable1:I.AH8.1homozygotes,
PLOSONE|DOI:10.1371/journal.pone.0133804 July24,2015 2/13
TheHLAHaplotypeAH8.1andtheGutMicrobiota
Table1. Pre-definedHLAgenotypegroups.
Group Name Definitioninthepresentstudy N
I AH8.1homozygotes HomozygosityforbothHLA-B*08andHLA-DRB1*03 34
II AH8.1heterozygotes 1copyofHLA-B*08andHLA-DRB1*03 38
III NonAH8.1heterozygotes NeitherHLA-B*08norHLA-DRB1*03present 30
IV NonAH8.1DRB1homozygotes HomozygosityforotherHLA-DRB1haplotypesthanHLA-DRB1*03,irrespectiveofHLA-Bgenotype 15
AH8.1:Ancestralhaplotype8.1
doi:10.1371/journal.pone.0133804.t001
II.AH8.1heterozygotes,III.NonAH8.1heterozygotesandIV.NonAH8.1DRB1homozy-
gotes.ThecompleteHLAcharacteristicsofthe117studysubjectsaregiveninS1Table.
Ethicsstatement
Writteninformedconsentwasobtainedfromallstudyparticipants.Thestudywasapproved
bytheRegionalCommitteeforMedicalandHealthResearchEthicsinSouth-EasternNorway
(projectcode2012/286b)andtheinstitutionalreviewboardofBGI-Shenzhen.
StoolsamplecollectionandDNAextraction
Thestoolsampleswerecollectedathomeandreturnedbyconventionalmail.Standardization
ofsamplingwasobtainedbytheuseofasamplingkitconsistingofaninformationletter,a
smallsamplingrelatedquestionnaire(coveringsamplingtimepoint,height,weight,druguse
(inparticular,antibiotics),smokingstatus,domesticanimalsandonequestiononwhether
theyareonaselectivediet),detailedinstructions,stoolcollectiontubeswithastoolDNAstabi-
lizerpreservativeoptimizedforthesubsequentDNAextraction(Stratec,Berlin,Germany),a
Protocultstoolcollectiondevice(AbilityBuildingCenter,Rochester,MN,USA)[26],anda
returnenvelope.Thestoolsampleswithstabilizerwerestoreduntilextractionat-20degrees,
accordingtothemanufacturer’srecommendations.Onlysamplesfromindividualswithout
antibioticusetheprevious4weeks,andwithatransporttimewithintherangerecommended
bythemanufacturer(timefromsamplingtothefreezer<72hours)wereconsideredforinclu-
sioninthestudy.DNAextractionwasperformedwiththePSPSpinStoolDNAkit(Stratec)
accordingtothemanufacturer’sprotocolaspreviouslydescribed[27,28].Thepresenceof
high-molecularDNAwasverifiedbygelelectrophoresis.
Librarypreparationandpyrosequencing
ThesampleswereamplifiedandsequencedaccordingtotheHMP45416SProtocolVersion
4.20[29].Briefly,PCRamplificationtargetingtheV3–V5regionof16SrDNAwasindividually
performedbyusingfusionprimerscomposedofFLXTitaniumadapters(A:5’-CCATCTCAT
CCCTGCGTGTCTCCGACTCAG-3’andB:5’-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-
3’),aunique-10basepairssequence(barcode)anduniversalprimers(338F:ACTCCTACGGG
AGGCAGCAGand907R:CCGTCAATTCMTTTGAGTTT).FollowingthePCR,ampliconswere
purifiedusingtheAMPurebeads(BeckmanCoulter).Subsequentsequencingonthe454plat-
form(RocheAppliedScience,Basel,Switzerland)wasperformedaccordingtomanufacturer’s
recommendations[29].ThesequencedataaredepositedattheEMBLNucleotideSequence
Database(AccessionnumberERP010886).
PLOSONE|DOI:10.1371/journal.pone.0133804 July24,2015 3/13
TheHLAHaplotypeAH8.1andtheGutMicrobiota
Dataprocessingandtaxonomicassignmentofeffectivereads
Thefollowingstepswereappliedtotherawsequencesbyusingmothur(v1.25)[30]:All
sequenceswereassignedtocorrespondingsamplesbyallowing1mismatchtothebarcodeand
2mismatchestothereverseprimer(907R).AfterdenoisingusingthePyroNoisealgorithm
[31],sequenceswithanambiguousbasecall,ahomopolymer>8nucleotides,orlength<200
or>1000nucleotideswereremoved.Afterremovingthebarcodeandprimersportionsfrom
reads,sequenceswerethenalignedusingaNAST-basedsequencealignertoacustomreference
basedontheSILVAalignment(v102).Sequenceswhichdidnotaligntotheanticipatedregion
ofthereferencealignmentwereremoved.Therestwerepre-clusteredbymergingsequence
countsthatwerenomorethan2nucleotidesdifferentfromamoreabundantsequence.Chime-
ricsequencesidentifiedusingUCHIMEalgorithmwerethenremoved[32].Sequenceswere
classifiedusingaBayesianclassifierwithRDPdatabase(v7).Definitionofasequence’staxon-
omywasdeterminedusingapseudobootstrapthresholdof80%.Sequencesthatclassifiedas
"Cyanobacteria_Chloroplast","Mitochondria",or"unknown"(i.e.,sequencesthatcouldnotbe
classifiedatthekingdomlevel)wereremoved.Theremainingsequenceswereclusteredinto
operationaltaxonomicunits(OTUs)ata3%distancecutoffusingtheaverageneighbor-clus-
teringalgorithm.AllOTUswerebroadlycategorizedaseithergrampositiveorgramnegative
accordingtoknownphylumcharacteristics.
Statisticalanalyses
Alphadiversity,betadiversity,OTUheatmap,principalcoordinateanalysis(PCoA)andsam-
ples’hierarchicalclusteringwereallperformedusingQIIME(v1.5),anintegratedsoftware
packageformicrobialcommunityanalysis[33].Enterotypeanalysiswasperformedas
describedbyArumugametal.[34]andQinetal.[35].Differencesinalphadiversityandrela-
tiveabundancesofindividualgenerabetweengroupsweretestedusingnonparametricstatistics
(WilcoxonRankSumTest).FalseDiscoveryRate(FDR)wascalculatedaccordingtoBenja-
mini-Hochbergtoevaluatethereliabilityoftestresults.Multivariatelinearregressionswere
performedincludingallcovariatesandusingarcsinesquareroottransformedrelativebacterial
abundancesasdependentvariables,asperformedintheHumanMicrobiomeProject[36].
Results
Studypopulation
ThecharacteristicsofthestudypopulationareshowninTable2.Therewerenosignificantdif-
ferencesbetweenthefourpre-definedHLAgenotypegroupswithrespecttoage,gender,body
massindex,smokingstatus,householdanimalsanddruguse.Oraldrugusewasreportedby
n=41(35%).Themostcommondrugswereanti-histaminesanddrugsreducingbloodpres-
sureandserumlipids.Veryfewreportedspecialdiets(n=7,includinglowcarbohydrate,little
ornoglutenandlowcaloriediets).
DiversitymeasuresweresimilarinallHLAgenotypegroups
The117samplesweresequencedintwoindividual454runs,providingintotal1,903,524raw
reads,i.e.median12,895readspersample.AfterqualitycontrolandOTUpickingthemedian
numberofeffectivereadspersamplewas6,392(range4,287to23,100).Fortheassessmentof
theinfluenceoftheAH8.1haplotypeonthemicrobiotacomposition,wefirstassessedthe
mainquestion,i.e.comparingthemicrobiotaprofileofAH8.1carriersvs.non-carriers.
Therewerenodifferencesregardingalphadiversity,i.e.diversitywithineachindividual,
betweenAH8.1carriersandnon-carriers,irrespectiveofalphadiversitymeasureapplied(see
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TheHLAHaplotypeAH8.1andtheGutMicrobiota
Table2. CharacteristicsofthestudypopulationaccordingtoHLAgenotypea.
AH8.1homozygotes AH8.1heterozygotes NonAH8.1heterozygotes NonAH8.1DRB1
(n=34) (n=38) (n=30) homozygotes(n=15)
Gender=female,n(%) 19(56) 21(55) 17(57) 6(40)
Age(years),median(range) 50(36–59) 49(33–61) 51(37–61) 52(36–55)
Body-massindex,median 27(20–41) 26(21–39) 25(19–35) 27(18–34)
(range)
Currentsmoker,n(%) 4(12) 6(16) 7(23) 1(7)
Domesticanimals,n(%) 13(38) 15(40) 14(47) 7(47)
Transporttimeb(days),median 1.2(0.4–2.9) 1.5(0.7–2.9) 1.1(0.7–2.0) 1.0(0.6–2.1)
(range)
Antibioticslast4weeks None None None None
Ongoingoralmedication,n(%) 7(47) 11(29) 12(40) 11(32)
aFourdifferentgroupswereincludedtorepresenthomozygosityandheterozygosityfortheAH8.1haplotype(asdefinedbythepresenceofHLA-B*0801
andDRB1*0301),absenceofthishaplotypeaswellashomozygosityforotherDRB1haplotypes.Therewerenosignificantdifferences(allp>0.23)for
anyoftheparameterswhenapplyingKruskal-Wallistestforcontinuousand2x4chisquaretestforcategoricalparameters.
bTransporttimeasdefinedbytimefromsamplingtothefreezer.
doi:10.1371/journal.pone.0133804.t002
Fig1forphylogeneticdiversity,othermeasuresnotshown).Analyzingthebetadiversity,i.e.
diversitybetweenindividualswithinthegroups,therewerenosignsofclusteringofthediffer-
entHLAgenotypegroupsinaprincipalcoordinateplot(Fig2).Ithasbeenproposedthatthe
gutmicrobiotacanbecategorizedinthreemainenterotypes[34,37].Fig3illustratesthestrati-
ficationofthesamplesintothreefractions,accordingtothemethodsdescribedbyArumugam
andRaesetal.[34],showinggroupscharacterizedbyPrevotella,Bacteroidesandathirdgroup
withamoremixedcomposition.However,therewasnoassociationbetweentheassigned
“enterotypes”andHLAgenotype(Fig3).
SeveraltaxaassociatedwithHLAgenotype
TheOTUdistributionshowedlargeinter-individualvariationbothwhendividingtheindivid-
ualsintoHLAgenotypegroupsaccordingtoAH8.1carrierstatusorthefourpre-definedHLA
genotypegroups(Fig4).WhencomparingAH8.1carrierswithnon-carriers,nodifferences
weredetectedatphylumlevel,whiletheabundanceofthePrevotellaceaefamily(P=0.02)and
theCoprococcus(P=0.01),Enterorhabdus(P=0.03)andClostridiumXVIII(P=0.05)genera
werereducedinAH8.1carriers(Table3).TheFDRvaluesoftheseassociationswerehigh
(>0.5).Therewasnodifferenceintheprevalenceofgrampositiveornegativebacteria(data
notshown).
WhencomparingonlyAH8.1homozygoteswithnon-carriers,thegeneraAnaeroplasma
(P=0.03)andPseudoflavinoflactor(P=0.03)werereducedinAH8.1homozygotes.Sincean
heterozygoteadvantagehasbeenobservedintheHLAcomplexinsomeinflammatorydiseases
[38],wealsoassessedthephenotypeofHLA-DRB1homozygosityingeneral(mergingthe
AH8.1homozygoteandotherDRB1homozygotegroups)vs.HLA-DRB1heterozygosity,
Anaeroplasma(P=0.05)andClostridiumXI(P=0.01)weresignificantlylessabundantinthe
homozygotes.TheFDRvalueswerehigh(>0.5)forbothanalyses.
Potentiallyconfoundingparameters
Althoughpossibleconfoundingvariableswereevenlydistributed(Table2),wealsoinvesti-
gatedtheeffectofthesevariables.Thereweresignificantnegativecorrelationsbetweenbody
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TheHLAHaplotypeAH8.1andtheGutMicrobiota
Fig1.PhylogeneticdiversityaccordingtotheancestralHLAhaplotypeAH8.1status.Phylogenetic
diversity,analphadiversitymeasureofbacterialrichness,isshownaccordingtocarrierstatusoftheAH8.1
haplotype(left),aswellasthepre-definedHLAgenotypegroups(right,AH8.1homozygotes,i.e.
homozygosityforbothHLA-B*08andHLA-DRB1*03;AH8.1heterozygotes,i.e.atleast1copyofHLA-B*08
andHLA-DRB1*03;NonAH8.1heterozygotes,i.e.HLA-B*08andHLA-DRB1*03bothnotpresent.Non
AH8.1homozygotes,i.e.HLA-DRB1homozygousbutnonAH8.1).Therewerenosignificantdifferences
betweenthegroups.
doi:10.1371/journal.pone.0133804.g001
massindexandmultiplealphadiversitymeasures;phylogeneticdiversity(Spearmanrankcor-
relationcoefficient[CC)-0.25,P=0.006),chao1(CC-0.25,P=0.007)andobservedspecies
(CC-0.24,P=0.009).Therewerealsosignificantassociationsbetweentheuseofanyoraldrug
andallalphadiversitymeasures(S1Fig),whiletherewerenosignificantassociationsbetween
alphadiversitymeasuresandage(SpearmanrankCCsranging-0.03to-0.07),gender(CCs
ranging-0.02to-0.09),currentsmoking(correlationcoefficientsranging-0.02to0.01)orthe
timesampleswerestoredatroomtemperatureduringtransport(correlationcoefficientsrang-
ing-0.02to0.03).Whenre-assessingtheassociationwithtaxonomicabundancesafterinclud-
ingage,gender,BMI,theuseoforaldrugs,smokingandtimeinroomtemperatureas
covariatesinamultivariatelinearmodel,significantassociationswithCoprococcusandEnter-
orhabdusandAH8.1carrierstatuswerestillobserved,whiletheassociationswithPrevotella-
ceae(P=0.07)andClostridium_XVIII(P=0.12)werenolongersignificant(Table3)
Discussion
Inthisfirststudyoftheinfluenceoftheautoimmunity-associatedHLAhaplotypeAH8.1on
thegutmicrobiotacomposition,nominallysignificantassociationsbetweentheprevalenceof
twobacterialtaxaandthepresenceofAH8.1wereobserved.However,theoverallcontribution
ofthishaplotypetothevariationingutmicrobiotaprofileseemedsmall,suggestingthatthe
accumulatedeffectsofotherfactorsaremoreimportantdeterminantsofthegutmicrobiota.
Thereisverylimitedliteratureforcomparisonwiththepresentdata.However,arelated
hypothesishasbeenexploredinaseriesofstudiesfromaSpanishgroupongutmicrobiotaand
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TheHLAHaplotypeAH8.1andtheGutMicrobiota
Fig2.BetadiversityaccordingtotheancestralHLAhaplotypeAH8.1status.Aprincipalcoordinateplot
ofthebetadiversitymeasureunweightedunifracshowsallsamplescoloredaccordingtotheHLAgenotype
groups.Therewerenosignificantdifferencesbetweenthegroups.Yellow:AH8.1homozygous,i.e.
homozygosityforbothHLA-B*08andHLA-DRB1*03.Violet:AH8.1heterozygotes,i.e.atleast1copyof
HLA-B*08andHLA-DRB1*03.Turquoise:NonAH8.1heterozygotes,i.e.HLA-B*08andHLA-DRB1*03
bothnotpresent.Grey:HLA-DRB1homozygousbutnonAH8.1.
doi:10.1371/journal.pone.0133804.g002
geneticriskfactorsforceliacdisease[23,39,40].Intheirfirststudy[23],therewasahigher
prevalenceofgramnegativebacteriaandmorePrevotellainchildrencarryingHLA-DQ2(the
strongestriskfactorforceliacdisease).Incontrast,inthepresentstudytherewasareduced
prevalenceofthePrevotellaceaefamily,inwhichPrevotellaisamember,andnodifference
relatedtogramstainingproperties.Therearehowever,majordifferencesbetweenthesestud-
ies,includingthelargeagedifferenceandtheuseofDNAprobesforprofilinginsteadof
sequencing.Inaddition,whilethemajorgeneticdeterminantinceliacdisease,HLA-DQ2,is
presentontheAH8.1haplotype,itcanalsobepresentonotherhaplotypes(e.g.
(cid:1) (cid:1)
HLA-B 18-DRB1 03)orbeencodedintrans,meaningthattheresultsarenotdirectlytransfer-
able.Inalater,sequencing-basedstudyfromthegrouptargetingtheV5-V6regionofthe16S
rRNAgene[40],ahigherprevalenceofFirmicutesandProteobacteriawasobservedin
HLA-DQ2carriers.Thiswasalsonotobservedinourstudy.Thecontrastbetweenthesestudies
highlighttheneedforstandardizationofmethodsandreplicationoffindingspriortoestablish-
ingassociationsinstudiesofthegutmicrobiota[41].
TheoveralldifferencesbetweentheHLAgenotypegroupsobservedinthisstudywere
small.Severalhypothesesmayexplainthis,includingthepossibilitythattheAH8.1primarily
actsonthehostphysiologyandnotthegutmicrobialcontent.Onepossibilityisthatthepres-
enceofAH8.1aloneisnotsufficienttoinduceanobservableeffect.Individualsaffectedbydis-
easeandsufferingfromarelativelyimpairedimmunesystemmayshowmoresensitive
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TheHLAHaplotypeAH8.1andtheGutMicrobiota
Fig3.EnterotypegroupsaccordingtoancestralHLAhaplotypeAH8.1status.Thefigureshowstheindividualsamplesascoloredsymbolsaccordingto
theirHLAgenotypegroups(seebelow)distributedaccordingtosimilarityoftheirdistributionofbacterialgenerainatwo-dimensionalplotaccordingtothe
methodsbyArumugametal.[34].Thesampleswereclassifiedintothreefractions,enterotypes,dominatedbyeitherPrevotella(blue),Bacteroides(green)or
amixofbacteria(red),wherethecoloredsquareindicatethecentreofthedistributionoftheenterotype,thestraightlinesconnecttheincludedsamplesand
thecoloredellipsescovertheindividualsnearthecenterofgravityforeachcluster(1.5σ).Bacterialtaxaoverrepresentedinthecorrespondingenterotypes
arelisted.Asevidentfromthesymbolcoloroftheindividualdots,thedifferentHLAgenotypegroups(seebelow)werenotpreferentiallydistributedtoone
particularenterotypefraction.Yellowdiamonds:AH8.1homozygotes,i.e.homozygosityforbothHLA-B*08andHLA-DRB1*03.Violetsquares:AH8.1
heterozygous,i.e.atleast1copyofHLA-B*08andHLA-DRB1*03.Turquoisecircles:NonAH8.1heterozygotes,i.e.HLA-B*08andHLA-DRB1*03bothnot
present.Greytriangles:HLA-DRB1homozygotesbutnonAH8.1.
doi:10.1371/journal.pone.0133804.g003
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TheHLAHaplotypeAH8.1andtheGutMicrobiota
Fig4.Genusleveltaxonomicdistributionin117NorwegianstoolsamplesaccordingtoancestralHLA
haplotypeAH8.1status.(A)Thegenusabundancessortedaccordingtotherelativeabundanceof
Bacteroides,ofn=72AH8.1carriers(left)areshowncomparedwiththen=45non-AH8.1samples(right).In
(B)thegenusabundancesareshownaccordingtoallfourpre-definedgenotypegroups;Left:AH8.1
homozygotes,i.e.homozygosityforbothHLA-B*08andHLA-DRB1*03;Middleleft:AH8.1heterozygotes,
i.e.atleast1copyofHLA-B*08andHLA-DRB1*03;Middleright:NonAH8.1heterozygotes,i.e.HLA-B*08
andHLA-DRB1*03bothnotpresent;Right:HLA-DRB1homozygousbutnonAH8.1.
doi:10.1371/journal.pone.0133804.g004
interactionwithgutmicrobes[10–12],suggestingthatpatientsaffectedbyanAH8.1associated
diseaseshouldbeincludedinlaterstudies.ItisalsopossiblethatAH8.1influencesthemicro-
biotaearlyinlifeandthatthisgeneticeffecthasbeenreplacedbyenvironmentalinfluencesat
theageof~50yearsasinthepresentstudy.Thereisthereforeastrongrationaleforsimilar
analysesinchildren[23,39,40].Theaccessibilityofbiologicalmaterialfromahealthypopula-
tionwasthemainreasonforanalyzingmicrobiotaprofileofthestoolandnotintestinal
mucosa.Aspatialdistributionofgutmicrobiotahasbeenshownbydetailedanalyzesofthe
microbiotainthemucosalfoldscomparedwiththecentrallumen[42].Several
PLOSONE|DOI:10.1371/journal.pone.0133804 July24,2015 9/13
TheHLAHaplotypeAH8.1andtheGutMicrobiota
Table3. BacterialtaxaassociatedwithbeingacarrieroftheancestralHLAhaplotypeAH8.1.
Taxon AbundanceinAH8.1carriers P-value(univariate)a P-value(multivariate)b
Clostridium_XVIII(genus) Reduced 0.05 0.12
Coprococcus(genus) Reduced 0.01 0.002
Enterorhabdus(genus) Reduced 0.03 0.05
Prevotellaceae(family) Reduced 0.02 0.07
aMann-WhitneyUtest,usingnon-transformedrelativeabundances
bLinearregressionusingsquare-rootarcsinetransformedrelativeabundanceasdependentvariableandincludingAH8.1carrierstatus,age,gender,BMI,
theuseoforaldrugs,smokingandtimeinthemodel.
doi:10.1371/journal.pone.0133804.t003
immunomodulatorybacteriaareknowntoadheretothemucosa[43,44].Futurestudiesin
patientsneedtoaccountforthisfact[45].
Powercalculationsformicrobiotastudiesarenotwelldeveloped,exceptforsomespecific
statisticalmodels[46].Powercalculationsarechallengingsincelittleisknownabouttheeffect
sizestobeexpectedfromthevariablesunderstudy.Geneticriskfactorsinpolygenicdiseases
detectedoutsidetheHLAtypicallyhaveoddsratiosof1.1–1.5andrequirethousandsofstudy
participantstobedetected.Incontrast,diseaseassociationsintheHLAcomplextypicallyhave
oddsratiosof2.0–5.0,suggestingalargeinfluenceonthehostphysiology(andaccordingto
ourhypothesis;thegutmicrobiota)potentiallydetectablealsoinhealthyindividuals,highlight-
ingthisgeneticregionasagoodstartingpointincandidategene-microbiotainteractionstud-
ies.Themanypossibleconfoundersingutmicrobiotastudies,exemplifiedbytheconfirmation
ofaninverserelationshipbetweenbody-massindexandalphadiversity[47,48],mayalso
reducethepowerinmultivariateanalyses.Importantly,themainlimitingfactorregardingthe
sizeofthisstudywastheaccesstohealthyAH8.1homozygoteindividualsvolunteeringtopro-
videastoolsample,whileitisdifficulttoconcludeontheidealstudysizebasedonthepresent
study.Relatedtothisitshouldalsobenotedthattomaximizethenumberofparticipantsthis
(cid:1) (cid:1)
studyonlyrequiredhomozygozityforthesegmentHLA-B 08-DRB1 03,whichhasthestron-
gestdiseaseassociations[6],andnottheentireclassicallydefinedAH8.1whichincludese.g.
(cid:1)
HLA-A 01.Inaddition,thegroupingofallotherHLAhaplotypesinonecategory(whichwas
necessaryduetolowfrequenciesofhomozygosityforotherhaplotypes)couldpotentiallyhide
differencesbetweenspecifichaplotypeswithdiverseinfluencesonthegutmicrobiota.
Despitethesmalldifferencesobservedbetweenthegroupsinthepresentstudy,thesecould
bespeculatedtohaveanimpactonimmunefunction.Oneexampleofapossiblemechanismis
theClostridiumXVIIIgenus,whichhadreducedprevalenceintheAH8.1carriersintheuni-
variateanalysis.ThisgenushasbeenshowntoinduceTregulatorylymphocytes(Tregs)in
mice[49]andlessClostridiumXVIIIcouldbespeculatedtoleadtofewerTregsandthereby
susceptibilitytoautoimmunity[50].Recentdatafromalargetwinstudyprovidestrongevi-
dencethatthegutmicrobiotaprofileisinpartheritable,andthattheheritablecomponents
maybeassociatedwithadisease-relatedphenotype(obesity)[14].Onamoregenerallevel,the
literaturethereforeprovidessupportforfurthereffortstodelineatewhichchromosomal
regionscontributetotheheritablecomponentsofthegutmicrobiota.
Inconclusion,thebacterialgeneraCoprococcusandEnterorhabdushadnominallysignifi-
cantreducedabundanceinAH8.1haplotypecarrierscomparedwithnon-carriers.However,
theoverallcontributionoftheAH8.1haplotypetothevariationinmicrobiotaprofileinthe
studypopulationwassmall.Thefindingsthereforeneedindependentvalidationandfurther
exploration,preferablyusingintestinalbiopsiesfromalargerstudypanelalsoincluding
patientsaffectedbyanAH8.1associateddisease.
PLOSONE|DOI:10.1371/journal.pone.0133804 July24,2015 10/13