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
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
ViralDNAwasextractedfrom200LserumusingQIAampDNA bloodminikit(Qiagen,Germany).
3.5. NestPCRamplifiedfull-lengthHBVgenome
HBVgenomewasdividedintotwofragmentsforamplification, fragmentI(nt702-1825)andfragmentII(nt1823-503)according tothepreviouslyprotocol.15NestPCRprimersweredesignedas
described,16,17andlistedinTable1.
ForamplificationoffragmentI,25Lreactionmixture includ-ing5LDNAtemplate,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
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
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
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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