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ContentslistsavailableatScienceDirect

Journal

of

Clinical

Virology

j ourna l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j c v

Mutations

in

hepatitis

B

virus

DNA

from

patients

with

coexisting

HBsAg

and

anti-HBs

Yu

Chen

a

,

Fuchu

Qian

b

,

Quan

Yuan

c

,

Xuefen

Li

d

,

Wei

Wu

a

,

Xichao

Guo

d

,

Lanjuan

Li

a,∗

aStateKeyLaboratoryforDiagnosisandTreatmentofInfectiousDiseases,FirstAffiliatedHospital,SchoolofMedicine,ZhejiangUniversity,

79QingchunRoad,Hangzhou310003,China

bDepartmentofLaboratoryMedicine,HuzhouCentralHospital,Huzhou,China

cNationalInstituteofDiagnosticsandVaccineDevelopmentofInfectiousDiseases,SchoolofLifeScience,XiamenUniversity,China dDepartmentofLaboratoryMedicine,FirstAffiliatedHospital,SchoolofMedicine,ZhejiangUniversity,Hangzhou,China

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received25February2011 Receivedinrevisedform6May2011 Accepted8July2011

Keywords: HepatitisBvirus

HepatitisBsurfaceantigen(HBsAg) AntibodytoHBsAg(anti-HBs) Genome

Variation

a

b

s

t

r

a

c

t

Background:TheserologicalmarkerswithcoexistenceofhepatitisBsurfaceantigen(HBsAg)andantibody toHBsAg(anti-HBs)ofhepatitisBvirus(HBV)infectionwererarepattern.Thevirologicalsignificance, immuneresponseandclinicaloutcomeofthesepatientsremainlargelyunknown.

Objectives:ThisresearchexplorestherelationshipbetweenthisserologicalprofileandHBVgenome variants.

Studydesign:Westudied35patientsbothcarryingHBsAgandanti-HBs(groupI),and70patientswith HBsAgpositivebutanti-HBsnegative(groupII,servedascontrol).TheHBVgenomesequenceswere obtainedbydirectsequencingofpolymerasechainreaction(PCR)products.

Results:Theaminoacid(aa)variationwithinmajorhydrophilicregion(MHR),especiallyinthefirstloop (aa124-137)of“a”determinantingroupIissignificantlyhigherthanthoseingroupII.Theaavariationof cytotoxiclymphocyte(CTL)epitopeinHBsAg(aa87–aa95)ingroupIisalsosignificantlyhigherthanthat ingroupII.Interestingly,thebasalcorepromoter(BCP)doublemutations(A1762T/G1764A)ingroupIis significantlyhigherthanthoseingroupIIaswell.

Conclusions:InpatientswithHBVinfection,thecoexistenceofHBsAgandanti-HBsisassociatedwith anincreasedaavariabilityinseveralkeyareasofHBVgenome.ThemolecularcharacteristicofHBVin HBsAgandanti-HBspositivepatientsisdistinctandworthfurtherstudies.

© 2011 Elsevier B.V. All rights reserved.

1. Background

InserologicalmarkersofhepatitisBvirus(HBV)infection, gen-eraltheoryconsiderthat theantibodytoHBsAg (anti-HBs)can neutralizeHBsAg,soitisgenerallyagreedthattherewillbeno simultaneouspositiveforbothHBsAgandanti-HBsinroutine clin-icalpractice.

However,therewereconcurrentHBsAgandanti-HBsinchronic HBVcarriersthathadbeenreportedinpreviousstudies.1–4There

maybethreeimportantsituationsleadingtocoexistingHBsAgand anti-HBs:(i)chroniccarriagewithexistingbutineffectiveanti-HBs response,(ii)brerakthroughofHBVinvaccinatedpeopleand(iii) reactivationofHBVinimmunepatientswhoundergo immunosup-pression.Sofar,thevirologicalsignificance,immuneresponseand clinicaloutcomeofthesepatientsremainlargelyunknown.

∗Correspondingauthor.Tel.:+8657187236759;fax:+8657187236755. E-mailaddresses:ljli@zju.edu.cn,ahmuysg@sina.com(L.Li).

SeveralreportsshowedthatthepatternofconcurrentHBsAg and anti-HBs might be associated with accumulative HBsAg mutants, especially in the “a” determinant within the major hydrophilic region (MHR).5–7 A number of mutants within or

aroundthe“a”determinantofHBsAghadbeenreportedin pre-viousstudies.SomemutantsreducethebindingaffinityofHBsAg tospecificantibodiesandenableHBVtoescapetheneutralization byanti-HBs.8–12Alaterreportsuggestedthatthephenomenonof

concurrentHBsAgandanti-HBswasnotassociatedwiththe spe-cificamnioacid(aa)substitutioninHBsAg,butwiththeincapability ofbindingofanti-HBsandHBsAg.13Sotheviewpointswere

con-troversialandthemechanismunderlyingtheseserologicalpattern remainednotquiteclear.

2. Objectives

Inordertoelucidatetherelationshipbetweenviralgene vari-antandthepatternofconcurrentHBsAgandanti-HBsinChinese chronicHBVcarriers,weconductedthisstudy.

1386-6532/$–seefrontmatter© 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2011.07.011

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3. Studydesign

3.1. Objectsofstudy

BetweenOctober2008andDecember2009,35patientswith simultaneouspositiveforHBsAgandanti-HBsrecruitedfromFirst AffiliatedHospital,SchoolofMedicine,ZhejiangUniversitywere prospectivelyenrolledforthestudy.Atthesametime,70patients withHBsAgpositiveandanti-HBsnegativewereenrolledasthe controlgroup.ThediagnosisofchronicHBVinfectionwasmade accordingtotheChineseconsensuscriteria.14Allpatientswith

previous anti-virus therapy, coinfection with hepatitis C virus, hepatitisDvirus,orHIVwereexcluded.Allindividualsprovided writteninformedconsentbeforeparticipatingthisstudy.Serum werecollectedbeforetreatmentandstoredat−70◦C.

3.2. Serologicmarkersassessments

RoutineserologicmarkersforHBsAg,anti-HBs,andHBeAgwere determined withthe Architect-i2000 system(Abbott Laborato-ries,USA).ThequantitativedeterminationofHBsAgconcentration values<0.05IU/mLareconsiderednegativeand >0.05IU/mLare consideredpositivebythecriteriaofARCHITECTHBsAg.Specimens areflaggedwiththecode“>250.00IU/mL”maybedilutedwiththe ManualDilutionProcedure.Thethresholdlevelof anti-HBswas definedas10mIU/mL.Serumalanineaminotransferase(ALT)level wasperformedwithroutineautomatedtechniques(HITACHI7600, Japan).

3.3. QuantificationofserumHBVDNAlevel

Serum HBV DNA level was quantified using theCobas HBV AmplicorMonitorassay(RocheDiagnostics,USA),andthe detec-tionlimitoftheassaywas300viralgenomescopies/mL.

3.4. SerumDNAextraction

ViralDNAwasextractedfrom200␮LserumusingQIAampDNA bloodminikit(Qiagen,Germany).

3.5. NestPCRamplifiedfull-lengthHBVgenome

HBVgenomewasdividedintotwofragmentsforamplification, fragmentI(nt702-1825)andfragmentII(nt1823-503)according tothepreviouslyprotocol.15NestPCRprimersweredesignedas

described,16,17andlistedinTable1.

ForamplificationoffragmentI,25␮Lreactionmixture includ-ing5␮LDNAtemplate,2UprimestarhighfidelityDNApolymerase (Takara,China),andtheouterprimerP3/AR1weresubmittedto thefirstroundofamplificationinthefollowingsteps:hotstartand denaturationat94◦Cfor4min,35cyclesof94◦Cfor30s,58◦Cfor 30s,and72◦Cfor2.5min,thenincubateat72◦Cfor5min.Two microlitresaliquotofthefirst-roundPCRproductswasusedasthe templateofthesecond-roundPCR,andthereactionmixturewas thesameasthefirst-roundexceptthattheinnerprimerP3/AR2 wasused.Thestepswerealsosameexceptthesecond-roundPCR only25cyclesinsteadof35.

Foramplification offragmentII,thefirst-roundand second-roundPCR primers were AF1/P4 and AF2/P4, respectively. The methodswerethesameasotherinfragmentIamplification.

3.6. DNAsequencingandsequenceanalysis

FollowingthePCRamplificationandconsequent electrophore-sis,thePCRproductswererecoveredandpurifiedfromagarosegel,

usinga QIAquickgelextractionkit(Qiagen,Germany).All prod-uctsweredirectlysequencedwithaBigDyeterminationV3.1cycle sequencingkit(AppliedBiosystems,USA)andrunonanABIPrism 3130Xautomaticgeneticanalyzer(AppliedBiosystems,USA).The primers used for sequencing are summarized in Table 2. HBV genomesequencesobtainedwerecompared withthereference sequencesintheGenbankforHBVgenotypingandgenosubtyping. Phylogeneticanalysiswasperformedusingtheneighbor-joining method(MEGAsoftware,version4.1).

3.7. Statisticalanalysis

StatisticalanalyseswereperformedbytheStudent’stest for quantitativedata,andthe2analysistestforcategoricalvariables.

ThedifferenceswereconsideredstatisticallysignificantatP<0.05.

4. Results

4.1. CharacteristicsofpatientsbothcarryingHBsAgandanti-HBs

Atotalof14458patientsweretestedforHBVserummarkers, and 1985 (13.7%) were HBsAg-positive. Among these HBsAg-positivepatients,72(3.6%)werebothcarryingHBsAgandanti-HBs. And35ofthemwereselectedasthetestgroup(groupI)for posi-tiveresultsofHBVDNA.Seventynewlydiagnosedpatientswith HBsAg-positiveand anti-HBs-negativewere enrolledas control group(groupII),whoseage,sexandgenotypeswerematchedwith thegroupI.

Table3 showstheclinicalcharacteristics ofthetwo groups. Therewas nosignificantdifference betweenpatientswith and without anti-HBs in HBeAg positive rate and serum ALT level (P>0.05).The patientswithcoexistence ofHBsAg andanti-HBs hadlowerHBsAglevelandHBVDNAconcentrationcomparedwith thoseofthepatientswhoseHBsAgsolywerepositive(P<0.05).

4.2. HBVgenotypesandsubgenotypes

Thecompletegenomesequencesforboth groupIandgroup IIwerecomparedwiththereferencesequencesfromGenBankfor HBVgenotypesandsubgenotypes.Themajorsubgenotypeingroup IwasB2(n=21,60%)andC1(n=14,40%).IngroupII,the predomi-nantsubgenotypealsowasB2(n=41,58.6%),thenthesubgenotype C1(n=26,37.1%),C2(n=2,2.9%)andB4(n=1,1.4%).Therewas nosignificantdifferenceinsubgenotypesdistributionbetweenthe twogroups.

4.3. Nucleotidedeletionsandinsertions

In this study, deletions and insertions were observed in 5 patientsingroupIand 9patientsingroupII(Table4).Ofnote, mostofthedeletionsandinsertionsoccurredinpreSandXgene regions.TherewerenosignificantdifferencebetweengroupIand groupII(P>0.05).

4.4. AminoacidsubstitutionofHBV

Aminoacid(aa)substitutionofstrainsfromgroupIwere com-paredwiththoseingroupII(Table5).Significantaasubstitution diversitywasobservedwithinSgeneofHBVbetweengroupIand groupII(1.83vs.1.18,forsubstitutionper100aa,thesamebelow,

P<0.05).Moreover,theaavariabilityofgroupIintheMHR(1.37 vs.0.48,P<0.05)and“a”determinant(2.38vs.0.77,P<0.05)were higherthangroupII.Furthermore,theaavariabilityinthefirstloop (aa124-137)of“a”determinantwasmorevariableingroupIthan ingroupII(4.00vs.1.33,P<0.05),butthatwasnotoccurredinthe secondloop(aa139-147)(0.29vs.0.14,P>0.05).Inaddition,the

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Table1

HBVDNAamplificationprimers.

Primerssetandtype Primername Sequence(5–3) Position(nta) Productlength(bp)

FragmentI 1stprimerset

Sense P3 5-CTCGCTCGCCCAAATTTTTCACCTCTGCCTAATCA-3 1825–1841 2140

Antisense AR1 5-ACAGTGGGGGAAAGC-3 759–745

2ndprimerset

Sense P3 5-CTCGCTCGCCCAAATTTTTCACCTCTGCCTAATCA-3 1825–1841 2092

Antisense AR2 5-AGAAACGGRCTGAGGC-3 702–687

FragmentII 1stprimerset

Sense AF1 5-GTCTGCGGCGTTTTATC-3 419–435 1404

Antisense P4 5-CTGGTTCGGCCCAAAAAGTTGCATGGTGCTGG-3 1823–1806

2ndprimerset

Sense AF2 5-TGCCCGTTTGTCCTCTA-3 503–519 1320

Antisense P4 5-CTGGTTCGGCCCAAAAAGTTGCATGGTGCTGG-3 1823–1806

ant,numberofnucleotides.

Table2

Sequencingprimers.

Primername Sequence(5–3) Position(nta) Domain

AR2 5-AGAAACGGRCTGAGGC-3 687–702 Sgene

P14 5-CTGTAACACGAGAAGGGGTCCTAG-3 202–179 Sgene

P13 5-TTGGGATTGAAGTCCCAATCTGG-3 2957–2935 PreS1gene

PP2 5-TACTAACATTGRGATTCCCGAGA-3 2454–2432 Cgene

2032 5-CTGACTACTAATTCCCTGGATGCTGGGTCT-3 2032–2061 Cgene

S3 5-GCGGGGTTTTTCTTGTTGAC-3 203–222 Sgene

AF2 5-TGCCCGTTTGTCCTCTA-3 503–519 Sgene

970F 5-CCTATTGATTGGAAAGTATGTCA-3 970–992 Pgene P10 5-CCATACTGCGGAACTCCTAGC-3 1266–1286 Pgene P24 5-GAGACCACCGTGAACGCCCA-3 1611–1630 Xgene S4 5-GGGACTCAAGATGTTGTACAG-3 787–767 Sgene ant,numberofnucleotides. Table3

CharacterizationofHBVinfectedpatientswithorwithoutanti-HBs.

GroupI(n=35) GroupII(n=70) P

Age(mean±SD) 42±15 42±15 >0.05

Male:Female 21:14 42:28 >0.05

Genotype(B:C) 21:14 42:28 >0.05

BCPdoublemutations,no(%) 20(57.1) 23(32.9) <0.05

HBsAglevel(log10IU/mL,mean±SD) 2.67±1.77 3.93±1.33 0.001*

HBeAg(no.positive) 20 40 1.000

HBVDNA(log10copies/mL,mean±SD) 4.83±1.86 5.97±2.17 0.008*

SerumALTlevel(IU/L,mean±SD) 295±750 288±485 0.314

Abbreviations:HBsAg,hepatitisBsurfaceantigen;HBeAg,hepatitisBeantigen;HBV,hepatitisBvirus;ALT,alanineaminotransferase;BCP,basalcorepromoter.

*Statisticallysignificantdifference(P<0.05).

Table4

DeletionsandinsertionsofHBVinfectedpatientswithorwithoutanti-HBs.

ID Group HBVgenotype Mutationpattern Position(nt)

I6 I C1 preS/poldeletion,18bp 2848–2865 I9 I B2 preS/poldeletion,125bp 3102–11 I11 I B2 S/polinsertion,24bp 532–533 I27 I C1 preS/poldeletion,52bp 7–57 I31 I C1 preS/poldeletion,46bp 11–55 S/polinsertion,12bp 501–502

II2 II B2 preS/poldeletion,147bp 3119–51

II5 II C1 preS/poldeletion,90bp 3143–17

II29 II C1 Xdeletion,55bp 1764–1817

II39 II B2 preS/poldeletion,49bp 8–55

II43 II C1 preS/poldeletion,10bp 49–57

II50 II C1 preS/poldeletion,16bp 2850–2864

Xdeletion,3bp 1631–1633

II53 II C1 S/poldeletion,3bp 501–503

II57 II C1 preS/poldeletion,21bp 1–21

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Table5

Numberofaminoacidsubstitution(per100aminoacid).

GenotypeB GenotypeC Allgenotypes

Ia IIa P Ia IIa P Ia IIa P Sprotein 1.47 1.20 0.199 2.37 1.12 <0.001* 1.83 1.18 <0.001* MHR 1.14 0.50 0.023* 1.71 0.50 0.002* 1.37 0.48 <0.001* Aepitope(aa124–aa147) 2.58 0.89 0.020* 2.08 0.60 0.049* 2.38 0.77 <0.001* 1stloop(aa124–aa137) 4.44 1.43 0.007* 3.33 1.19 0.117 4.00 1.33 <0.001* 2ndloop(aa139–aa148) 0.00 0.24 1.000 0.71 0.00 0.333 0.29 0.14 0.999 Prea(aa99–aa123) 0.20 0.20 1.000 1.79 0.74 0.195 0.83 0.36 0.141 Posta(aa149–aa169) 0.45 0.34 0.670 1.02 0.00 0.037* 0.68 0.20 0.126 CTLepitopeSA(aa19–aa28) 3.81 3.10 0.643 0.71 1.43 0.669 2.57 2.43 0.999 CTLepitopeSB(aa87–aa95) 1.59 0.26 0.110 3.17 0.00 0.012* 2.22 0.16 0.002* CTLepitopeSC(aa97–aa106) 0.48 0.00 0.333 1.43 0.71 0.604 0.86 0.29 0.341 CTLepitopeSD(aa172–aa180) 2.12 0.26 0.045* 0.00 0.79 0.554 1.27 0.48 0.341 CTLepitopeSE(aa185–aa194) 0.00 0.95 0.307 0.71 1.43 0.669 0.29 1.14 0.286 CTLepitopeSF(aa207–aa216) 2.86 1.43 0.228 2.14 1.79 1.000 2.57 1.57 0.338 CTLepitopeSG(aa215–aa223) 1.59 0.53 0.340 4.76 1.98 0.191 2.22 0.79 0.118 PreSprotein 1.41 1.38 0.966 1.29 2.18 0.012* 1.36 1.70 0.131 HBxprotein 2.38 2.01 0.264 3.48 3.68 0.540 2.82 2.68 0.632 K130M/V131I(A1762T/G1764A) 38.1 19.0 0.130 85.7 53.6 0.049* 57.1 32.9 0.012* HBcprotein 2.37 2.98 0.068 2.34 1.95 0.271 2.36 2.57 0.405 CTLepitopeCA(aa1–aa20) 1.67 2.26 0.400 3.21 1.79 0.220 2.29 2.07 0.873 CTLepitopeCB(aa18–aa27) 1.90 4.05 0.238 2.86 2.50 1.000 2.29 3.43 0.347 CTLepitopeCC(aa28–aa47) 0.71 1.31 0.408 1.07 0.54 0.406 0.86 1.00 0.937 CTLepitopeCD(aa50–aa69) 2.86 2.86 1.000 1.79 1.25 0.547 2.43 2.21 0.877 CTLepitopeCE(aa111–aa125) 1.27 1.90 0.599 0.95 1.43 0.725 1.14 1.71 0.513 CTLepitopeCF(aa117–aa131) 1.90 1.75 1.000 2.38 1.90 0.768 2.10 1.81 0.845 CTLepitopeCG(aa120–aa139) 3.10 2.86 0.860 2.14 1.61 0.588 2.71 2.36 0.728 CTLepitopeCH(aa141–aa151) 3.03 4.33 0.411 2.60 2.60 0.758 2.86 3.64 0.604 PreCoreregion 2.96 3.04 0.923 2.96 1.85 0.304 2.96 2.56 0.608 W28stop(G1896A) 57.1 66.7 0.580 64.3 39.3 0.192 60.0 55.7 0.834 Polymerase 1.36 1.32 0.377 2.29 2.34 0.787 1.73 1.73 0.995

Abbreviations:MHR,majorhydrophilicregion;CTL,cytotoxicTlymphocyte.

aGroup.

* Statisticallysignificantdifference(P<0.05).

aavariabilityofthecytotoxicTlymphocyte(CTL)epitopeofHBsAg (aa87–aa95)ingroupIwasfoundhigherthanthoseingroupII(2.22 vs.0.16,P<0.05).Thedifferenceofresiduesubstitutioninremain regionsofHBVgenomeswasnotsignificantbetweentwogroups (P>0.05).

Thepercentage ofMHRvariantsinpatientswassignificantly differentbetweenthetwogroups[65.7%(23/35)vs.44.3%(31/70),

P<0.05].ThemostfrequentsitesobservedingroupIweres126, s129ands130, buttheG145R/Aaminoacidchangewhichwas popularinpreviousreportswasonlyfoundinone patientwith coexistenceofHBsAgandanti-HBs(Fig.1).

Itisworthnotingthattheincidenceofbasalcorepromoter(BCP) doublemutations(A1762T/G1764A)ingroupIwashigherthanthat ofgroupII(20/35vs.23/70,P<0.05).

5. Discussion

Inthis study,3.6% of chronicHBVcarriers were both carry-ingHBsAgandanti-HBs.Thepercentageisclosetotheprevious reports,7,13,18 butislowerthantheotherstudy.6 Thedifference

mightbenotonlyduetodemographic,ethnicand geographical diversion,butalsoduetodiagnosiscriteria.Furthermore,in pre-viousstudiesthenumbersofpatientswithcoexistenceofHBsAg andanti-HBsforanalysiswaslimited,andthecriterionof choos-ingpatientswasinconsistentamongdifferentstudies.7,13,18Inour

study,thethresholdlevelofanti-HBswasdefinedas10mIU/mL, ananti-HBstiter>10mIU/mL,whichiscommonlyconsidered pro-tectiveaccordingtotheChineseconsensuscriteria.14

ThepreviousstudysuggestedthatpreSdeletionmightbe asso-ciatewiththecoexistenceofHBsAg andanti-HBsinchronically HBVinfectedpatients.2,4,18Inthisstudy,nosignificantdifference

betweenthetwogroups wasdetectedconcerningtheincidence ofpreSdeletions.Thismaybeduetothediversityofcase

num-berandothercharacteristicsofthesubjects,suchasage,ethnic orgenotypes.Thedifferencemethodsmayalsocontributetothe inconsistencyforthepreviousstudyadoptcloninganalysisinstead ofdirectsequencing.2,4

ThoughthemechanismofconcurrentHBsAgandanti-HBsin serumremainsunclear,onepossibleinterpretationmightbethe selection of HBsAg immune escape variants.It isreported that theincidenceofaasubstitutioninMHR,especiallyin“a” deter-minant,is higherin patientscarrying bothHBsAg andanti-HBs thanthosecarryingHBsAg.6,7Inthisstudy,wealsofound

statis-tical significancein theincidenceof“a” determinantmutations betweenthetwogroups,whichaccordwithpreviousconclusion. In addition,we foundthat it is thefirst loop, but not the sec-ondloopof“a”determinantregionthatresponsibleforthehigher mutationsincidence,forthesecondloopshowsnosignificant dif-ferenceinthemutationsincidencecomparisonofthetwogroups. MostofthepatientscarryingHBsAgandanti-HBsinprevious stud-ieshavereceivedantiviraltreatment.6,7,19Interferontreatments

maystimulateimmuneselectionpressureandinduceviralgenome mutationacceleration,andnucleosideornucleotideanalogsmay inducemutationinviralSproteinsaswellduetotheoverlapped cordingsequencewiththeRTofpolymerase.Inotherreports,the patientswithconcurrentHBsAgandanti-HBswerepredominant immunosuppression,7,20whichmightalsoinfluencethe

immuno-logicalstatusandselectionofHBVmutant.Thesedatasuggestthat themutationsiteofnaturallyoccurringSgenevariantsismostly locatedinthefirstloopof“a”determinantinpatientswith coexis-tenceofHBsAgandanti-HBs.Moreover,themostfrequentchanges occurredingroupIwerelocatedatpositionss126,s129ands130, whichaccordwiththepreviousstudies.2–4TheG145R/Awasalso

describedinmanystudies,21–24whichisoneofthemostcommon

immune-escapemutantsareresponsibleforimmuneprophylaxis failure,butonlyoneG145RwasobservedingroupIpatientsin

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Fig.1.AminoacidvariabilityofMHR(aa99–169)ingroupIandgroupII.ThereferenceaminoacidsequencesofgenotypesBandCwerededucedfromthefollowingGenBank sequences:genotypeB:AB073826;GenotypeC:AF286594.

thisstudy.Accordingly,thes145pointmutationshowedno statis-ticaldifferenceinpatientswithandwithoutHBsAgandanti-HBs coexsitence.Thisresultisalsoinaccordancewithanotherrecent study,18whichsuggeststhes145pointmutationisnotcommonin

HBVgenotypeBandCinfectedpatientswithcoexistingHBsAgand anti-HBs.

Themostimportantfindingofourstudyisthattheincidence ofBCPdoublemutations(A1762T/G1764A)ingroupIwas signifi-cantlyhigherthanthoseingroupII.Manystudieshaverevealed that these double mutations are associated with an increased risk ofsevere of liverdiseases includingHCC, and canbeused asaprediagnosticbiomarkerofHCC.25–28Inalong-term

follow-upstudyofHBVcarriers,whichhasshownthepresenceofBCP (A1762T/G1764A)mutationsisanindependentpredicatorfor pro-gressiontoHCC.29Ameta-analysishasyieldedasummaryORof

3.79(95%CI=3.71–5.29)fordevelopmentofHCCinpatientswith BCPdoublemutations.30Takingtheseevidence,wesuggestedthat

thechronicallyHBVinfectedpatientswithcoexistenceofHBsAg andanti-HBs,mighthaveincreasedriskofHCC.Asthisstudyisa cross-sectionanalysis,furtherfollow-upinvestigationis impera-tivetocorroboratethispresumption.

Insummary,ourstudyshowedahigheraavariabilityinthe firstloopof“a”determinantandamorefrequentofBCPdouble mutationsinpatientswithconcurrentHBsAgandanti-HBs sero-logicalmarkerspattern.Furtherlarge-scale,multi-centerstudies areneededtoclarifytheclinicalimplicationsofthisserological

pat-tern,includingabilityofspecificTcellimmuneresponses,efficacy ofantiviralagents,andclinicalcourse.

Funding

ThisworkwassupportedbyagrantfromtheMajornationalS&T Projectsforinfectiousdiseases(2008ZX10002-003, 2009ZX10004-210) and the Foundation Project for Medical Science and TechnologyofZhejiangProvince(No.2009B056).

Competinginterests

Wehavenofinancialconflictsofinteresttothismanuscript.

Ethicalapproval

ThestudywasapprovedbyEthicsReviewCommitteeoftheFirst AffiliatedHospital,SchoolofMedicine,ZhejiangUniversity.

Acknowledgments

WewouldliketothankDr.NingshaoXiafortechnical support-ingofthisstudy.

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Figure

Fig. 1. Amino acid variability of MHR (aa99–169) in group I and group II. The reference amino acid sequences of genotypes B and C were deduced from the following GenBank sequences: genotype B: AB073826; Genotype C: AF286594.

References

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