Microarray-based genotyping and detection of drug-resistant HBV mutations from 620 Chinese patients with chronic HBV infection

ww w . e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Microarray-based

genotyping

and

detection

of

drug-resistant

HBV

mutations

from

620

Chinese

patients

with

chronic

HBV

infection

Wenhao

Hua

_,

_Guanbin

_Zhang

_,

_Shujun

_Guo

_,

_Weijie

_Li

_,

_Lanhua

_Sun

_,

Guangxin

Xiang

a_{DepartmentofClinicalLaboratory,BeijingDitanHospital,CapitalMedicalUniversity,Beijing,China} b_{NationalEngineeringResearchCenterforBeijingBiochipTechnology,Beijing,China}

c_{DepartmentofInfectiousDisease,PingdingshanGeneralHospitalMedicalGroup,PingdingshanCity,HenanProvince,China}

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Articlehistory:

Received17December2014 Accepted12March2015 Availableonline14May2015

Keywords: ChronicHBV Microarray Genotyping Drug-resistancemutations

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Background:ResearchhasshownthathepatitisBvirus(HBV)genotypesarecloselylinkedto theclinicalmanifestations,treatment,andprognosisofthedisease.

Objective:Tostudytheassociationbetweengenotypeanddrug-resistantHBVmutationsin 620ChinesepatientswithchronicHBVinfection.

Methods:HBVDNAlevelsweredeterminedusingreal-timequantitativePCRinplasma sam-ples.MicroarrayswereperformedforthesimultaneousdetectionofHBVgenotypes(HBV/B, C,andD)anddrug-resistance-relatedhotspotmutations.Aportionofthesamplesanalyzed usingmicroarrayswasselectedrandomlyandthedatawereconfirmedusingdirectDNA sequencing.

Results:MostsamplesweregenotypeC(471/620;76.0%),followedbygenotypeB(149/620; 24.0%).Amongthe620patientsamples,17(2.7%)hadnucleotideanalogs(NA) resistance-relatedmutations.Ofthese,nineandeightpatientscarriedlamivudine(LAM)-/telbivudine (LdT)-resistancemutations(rtL180M,rtM204I/V)andadefovir(ADV)-resistancemutations (rtA181T/V,rtN236T),respectively.Nopatientshadbothlamivudine(LAM)-andeither ade-fovir (ADV)orentecavir(ETV)resistancemutations.Additionally,outofthe620patient samples,64.0%(397/620)werealsodetectedwiththeprecorestop-codonmutation(G1896A) bymicroarrayassay.

Conclusion: Theresults of thecurrentstudy revealed that theprevalence ofnucleotide analogs(NA)-resistanceinChinesehospitalizedHBV-positivepatientswassolowthat inten-sivenucleotideanalogs(NA)-resistancetestingbeforenucleotideanalog(NA)treatment mightnotberequired.Inaddition,thepresentstudysuggeststhatchronicHBVpatientswith genotypeCwereinfectedwithfittervirusesandhadanincreasedprevalenceofnucleotide analogs(NA)-resistancemutationscomparedtogenotypeBvirus.

©2015ElsevierEditoraLtda.Allrightsreserved.

∗ _{Correspondingauthorat:No.8,JingshunEastStreet,ChaoYangDistrict,Beijing100015,China.}

E-mailaddress:dtjykhua@126.com(W.Hua).

http://dx.doi.org/10.1016/j.bjid.2015.03.012

Introduction

HepatitisBvirus(HBV)infectionisamajorglobalhealth prob-lem that affects more than 240 million people worldwide, causingmorethan780,000deathsannually.1_{Therefore,itis}

becomingincreasinglyimportanttotakeeffectivemeasures tocontroltheincidenceofHBVinChina.

TheHBVgenotypehasformedoveralongperiod,during whichpointmutationshaveaccumulatedduetothe asymme-tryofHBVreversetranscriptionanditslackofproofreading enzymes.TheHBVmutationrateishigh,andtheHBV geno-typereflectsthenaturalaccumulationofmutationsasaresult ofvirusevolution.HBVcanbedividedintoseveralgenotypes accordingtosequenceheterogeneitygreaterthan8%.There arecurrentlyeightHBVgenotypes(A–H)thathaveadistinctive geographicaldistributionandethnicassociations.2_For

exam-ple,genotypesBandCarehighlyprevalentinSoutheastAsia includingChina,genotypeAisfrequentinnorthwestEurope, andgenotypeDiscommonintheMediterraneanregionand CentralAsia.3 _{Aprevious retrospective study revealed that}

HBVgenotypeBwasassociatedwithearlierHBeAg serocon-versionthanwasgenotypeC.4_{Whenreceivinginterferon(IFN)}

therapy, patients with HBV genotype B have ahigher rate ofIFN-induced HBeAgclearancecomparedwiththose with genotypeC.5

InadditiontoIFN,nucleotideanalogs(NAs)arealso cur-rentlyapprovedforthetreatmentofHBVinfection.NAsinhibit HBV replication and reduce the damagetohepatocytes by competingforincorporationintotheviralDNAstrands.6_Four

NAshavebeenlicensedforclinicaluseinChina:lamivudine (LAM),adefovir(ADV),entecavir(ETV),andtelbivudine(LdT). However,long-duration NA treatment increases the preva-lence of resistant HBV and leads totreatment failure. For example,LAM,thefirstwidelyusedantiviralNA,resultsin acumulativeincidenceofresistanceashighas70%afterfive yearsoftreatment.7_{Theresistance-relatedmutationsare}

usu-ally locatedinthe reverse transcriptase region ofthe HBV polymerasegene.8 _{TheclassicalLAM-/LdT-resistance}

muta-tions are rtM204I and rtM204V.9,10 _{Of these, rtL180M is a}

compensatorymutationthat oftencoexists with rtM204I/V torestoreviralreplicationefficacy.11_{Inaddition,rtN236Tand}

rtA181Varewell-recognizedADV-resistancemutations.12_The

mostprevalentmutationintheprecoreregion(G1896A) cre-atesaTAGstopcodonthatabolishesthesynthesisofHBeAg; thus,thedominanceofthismutationcouldeasilyaccountfor HBeAgseronegativity.13

The aim of the current study was to determine the HBVgenotypesanddrugresistance-relatedmutationsin620 clinicalsamplessimultaneouslyusingmicroarraystobetter understandthecorrelationbetweenHBVgenotypesand drug-resistantmutations.

Materials

and

methods

Patientsamples

Atotalof620patientsconsistingof286malesand334females with chronic HBV infection were enrolled in the study in

Beijing Ditan Hospital. The ages of these patients ranged fromonemonthto80years,withameanof33.7±12.2years. Amongthe620patients,271receivedNA-basedtherapy,35 receivedIFN-based therapy,eightreceivedthe combination therapyofNAandIFN,andtheother306wereNA/IFNnaïve. Informed consentwasobtainedfrom adultpatients or the guardiansofminorpatientsatthetimeofwholeblood col-lectionforthepurposeofHBVtesting.Theplasmasamples obtainedfromwholebloodwereusedtodeterminetheHBV DNA levels using real-time quantitative PCR. In addition, serum sampleswereusedtodeterminethe HBVgenotypes anddrugresistance-relatedmutations.

236T

IC2 IC2 IC2 NC

PC PC PC QC QC QC NC NC

236T 236T 1896A 1896A 1896A 204I 204I 204I 204V 204V 204V 181T 181T 181T

180M 180M 180M D

B B B C C C BC BC BC IC1 IC1 IC1

HBV B HBV C

A

C

B

Fig.1–Microarray-basedHBVgenotypinganddrug

resistance-relatedmutations.(A)Layoutofthemicroarray

usedinthecurrentstudy.Allprobes,withtheexceptionof

QCandPC,werespottedinthreereplicates;QCandPC

werespottedinsixreplicates.QCandBCwerethepositive

andnegativecontrolsforarrayproduction,respectively.PC

andNCwerethepositiveandnegativecontrolsfor

hybridization,andIC1andIC2weretheinternalcontrols

forHBVamplification,respectively.B,C,andDrepresent

HBVgenotypesB,C,andD,respectively.180M,181T/V,

204I/V,and236TrepresentthertL180M,rtA181T/V,

rtM204I/V,andrtN236T,respectively,mutationsintheHBV

reversetranscriptaseregion.1896AdenotestheG1896A

mutationintheHBVprecoreregion.(B)Hybridization

resultsoftheclassicalHBVsampleswithgenotypesBand

C.(C)HybridizationresultsofeightclassicalNA-resistant

QuantificationofHBVDNA

HBV DNA was quantified using a Cobas AmpliPrep/Cobas Taqman (CAP/CTM) platform (Roche Molecular Systems, Pleasanton, CA, USA). HBV DNA was extracted from 1-mL plasmausingaCobasAmpliPrepautomatedextractor accord-ingtothemanufacturer’sinstructions.TheCobasTaqman48 Analyzerwasusedforautomatedreal-timePCRamplification andthedetectionofPCRproductsaccordingtothe manufac-turer’sinstructions.HBVDNAlevelswereexpressedasIU/mL.

HBVDNAgenotypinganddrug-resistance-related

mutationanalysis

HBVDNA wasextractedfrom 200-␮Lserumsamplesusing a Magbind CFDNA kit (KWBiotech, Beijing, China). Multi-plexPCR,microarrayhybridization,andmicroarrayscanning wereconductedasrecommendedbythemanufacturerofthe microarraykit(CapitalBioCorporation,Beijing,China)forthe simultaneousdetectionoftheHBVgenotypes(HBV/B,C,and D)anddrug-resistance-related hotspotmutations(rtL180M, rtA181T/V,rtM204I/V,rtN236TintheHBVreverse transcrip-tase region, and G1896A in the HBV precore region). The recommendedgenotypeandmutation-relatedallele-specific primerswereusedformultiplexPCR.Afteramplification,the reactionmixturewashybridizedwithprobesimmobilizedon the microarray chip at 50◦C for 60min. Subsequently, the microarraychipwaswashedinwashingbuffer(0.1%SDSand 0.3× SSC) at42◦C for 2min. The chips were scanned and imagedusingamicroarrayScannerLuxScanTM_10K-B

(Capital-bio).Assayresultsweredeterminedbasedonthefluorescent hybridizationsignalanddistributionofthemicroarrayprobes. ThemicroarrayprobescorrespondingtoHBVgenotypesand drug-resistance-relatedmutationsareshowninFig.1A.

HBVDNAsequencing

The results of HBV DNA genotyping and drug-resistance-relatedmutationsobtainedfromthemicroarraysforarandom selectionofsampleswerefurtherconfirmedusingdirectDNA sequencingwiththeBigDyeTerminatorv3.1CycleSequencing kitandanABI3730DNAanalyzer(AppliedBiosystems,Foster City,CA).TheprimersequencesandPCRamplification con-ditionsusedfordirectDNAsequencinghavebeendescribed elsewhere.14–16

Statisticalanalysis

StatisticalanalysiswasperformedusingtheSPSS17.0 soft-warepackage.Allvaluesinthetextandfiguresareexpressed asthemean±standarddeviation(SD)oftheseobservations.

Results

HBVDNAquantification

TheHBVDNAlevelsinthe620enrolledsamplesrangedfrom 1.00E5to1.50E9IU/mL,withameanof9.89E6IU/mL(see Sup-plementaryTableS1),asdeterminedbyreal-timequantitative

Table1–HBVgenotypingresultsofthe620patient samples.

Gender HBV/B(%) HBV/C(%) Total

Male 68(23.8%) 218(76.2%) 286

Female 81(24.3%) 253(75.7%) 334

Total 149(24.0%) 471(76.0%) 620

PCR.Theresultsalsoconfirmedthatallstudiedclinical sam-pleswereHBV-positive.

Supplementary Table S1 related to this article can be

found,intheonlineversion,athttp://dx.doi.org/10.1016/j.bjid. 2015.03.012.

HBVDNAgenotypes

TwoHBVgenotypes(BandC)weredetectedamongthe620 HBV DNApositivesamplesusing theCapitalbiomicroarray assay,asshowninSupplementaryTableS1.Themicroarray hybridizationresultsfortheHBVgenotypesBandCareshown inFig.1B.Ofthese,44sampleswithgenotypeBand151 sam-pleswithgenotypeCwereselectedrandomlyandtheresults were validated using directDNA sequencing. Asshown in

Table1,mostsampleswereidentifiedasgenotypeC,471/620 (76.0%),followedbygenotypeB,149/620(24.0%).

Drugresistance-relatedmutations

By microarray assay, NAresistance-relatedmutational pat-ternsofthe620patientsampleswerealsodirectlyacquired fromhybridizationresults,asshowninSupplementaryTable S1.Themutationpatternsof211patientsampleswerethen selectedandfurtherconfirmedusingdirectDNAsequencing. Amongthe620patientsamples,17(17/620;2.7%)carriedNA resistance-relatedmutations,asshowedinTable2andFig.1C. Themeanageofthese17patientswas47.7±12.5years,which was higherthan thatofthe 620patients(33.7±12.2). Most of the 17 patient samples belonged to genotype C (14/17, 82.4%),andtheremainingtogenotypeB(3/17,17.6%).Among the17patients,ninecarriedLAM-/LdT-resistancemutations (rtL180M, rtM204I/V), and eight had ADV-resistance muta-tions (rtA181T/V, rtN236T). The most common mutations in the LAM-resistant patients were rtM204I and rtM204V, which were found in 35.3% (6/17) of the 17 NA-resistant patients. The compensatory mutation rtL180M was simul-taneously detected with rtM204I/V in 17.6% (3/17). The ADV-resistancemutationsrtA181VandrtN236Twerefound in 29.4% (5/17) ofthese patients. No patient sampleswith LAM and ADV or ETV-resistance mutationswere detected. Among the 17 patients, 4 carried NA-resistancemutations butwereNA/IFN-naïve.TencarriedNA-resistancemutations andtheNA/IFN-basedtherapeuticstrategyadoptedpreviously wasappropriateandeffective.Theremainingthreepatients alsocarriedNA-resistancemutationsbutthepreviousNA/IFN therapy wasnot effectiveand ledtotreatment failure and was altered. Morespecifically,patients P119and P287both carried rtA181VbutreceivedADV/IFN,P217carriedrtM204I but receivedLdT.Inaddition,the microarraysrevealedthat 397(64.0%)ofthe620patientsamplesalsocarriedtheprecore

Table2–Assayresultsofthe17NA-resistancesamples.

SampleID Gender Age NA-resistance Genotype G1896A HBVtreatment

P51 Male 45 rtL180M,rtM204I HBV/C A Naïve

P103 Male 52 rtL180M,rtM204V HBV/C G LAM+ADV

P119 Male 48 rtA181V HBV/C G ADV

P160 Male 43 rtN236T HBV/B A Naïve

P163 Male 48 rtL180M HBV/C A Naïve

P208 Male 50 rtM204I HBV/C A ETV

P217 Female 57 rtM204I HBV/C A LdT

P227 Female 52 rtM204I HBV/C A ETV

P250 Female 35 rtL180M,rtM204I HBV/C A LAM+ADV

P279 Female 33 rtA181T HBV/C G LdT+ADV

P287 Female 32 rtA181V HBV/C A IFN

P304 Male 74 rtL180M HBV/B A ETV

P393 Male 74 rtL180M HBV/B A ETV

P429 Female 53 rtN236T HBV/C G ETV

P487 Female 37 rtA181T HBV/C G ETV

P564 Male 45 rtA181T HBV/C G ETV+IFN

P600 Male 34 rtN236T HBV/C A Naïve

stop-codonmutation(G1896A),andthiswasconfirmedin197

samplesusingdirectDNAsequencing.

Discussion

Mostofthesamplesanalyzedinthecurrentstudybelongedto genotypeC(471/620;76.0%)followedbygenotypeB(149/620;

24.0%). Moreover,there were nomarked differences inthe

distributionofgenotypesBandCbetweenmaleandfemale

patients. These data are consistent with a previous report fromnorthernChina,where78%ofthestudiedHBsAg carri-erswereinfectedwithgenotypeC,and22%carriedgenotype B.14However,genotypeDwasnotobservedamongthepatient samplesstudied.Interestingly,previousreportsalsoidentified HBVgenotypeDinonlynorthwesternChina.17,18

In the current study, 17 patients (17/620; 2.7%) carried NA-resistance-relatedmutations.Thesedataare consistent withapreviousreportthatpreexistingNA-resistance-related mutationsweredetectedin2.01%ofNA-naïveChinese HBV-infectedpatients.19_{Theseresultssuggestthattheincidenceof}

NA-resistanceinChinesehospitalizedHBV-positivepatients is so low that intensive testing for NA-resistance before treatmentmightnotberequired,consistentwithaprevious suggestion.19_{Inmostcasesinthecurrentstudy,NA-resistance}

mutationswerederivedfromeithernaturallyoccurringviral mutationsorweretransmittedfromdrug-resistantpatients. However,the17patientshadameanageof47.7±12.5years, which was higher than the mean age of the 620 patients (33.7±12.2).Thissuggeststhatthe17NA-resistancepatients hadanincreasedlikelihood ofbeingtreatedusingantiviral therapy;therefore,NAresistancetestingmightbemore nec-essaryforHBV-positiveelderlypatients.

Most of the 17 patients samples belong to genotype C (14/17,82.4%),andtheremainingtogenotypeB(3/17,17.6%). TheproportionofgenotypeCinthe17NA-resistancepatients was considerably higher than the above-mentioned data (76.0%)inthe620HBV-positivepatients.Thesedatasuggest thatHBVpatientswithgenotypeCwereinfectedwithfitter viruses,whichfacilitatedacquisitionofNA-resistance muta-tions,comparedwiththosewithgenotypeB.

ThemutationsrtM204Iand rtM204V werethe most fre-quentmutationsintheLAM-resistantpatients,beingdetected in 35.3% (6/17). The compensatory mutation rtL180M was detectedsimultaneouslywithrtM204I/Vin17.6%(3/17)ofthe patients.TheADV-resistancemutationsrtA181VandrtN236T werefoundin29.4%(5/17)ofthepatients.LAMandADVdual resistancewasnotdetectedinanyofthe17patients. Long-term use of NA might have led to multidrug resistance.20 Therefore,NAresistancetestingbecomesincreasingly neces-sary.ForpatientsP119,P217andP287ofthe17patients,the resultsofNAresistancetestingimpliedinadvancethatthe usedNA/IFNtherapeuticstrategieswouldleadtotreatment failure andhadtobealtered.ItshowedthatNAresistance testingmaybenecessarywhendecidingfortheNA/IFN ther-apeuticstrategy.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WearegratefultoGuotongYinandYunlongHao(Capitalbio) fortheirexcellenttechnicalassistance.Wearealsograteful to Dr.YingyingLiu (Capitalbio) forhelpfuldiscussions and support.

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