w w w . e l s e v ie r . c o m / l o c a t e / b j i d
The
Brazilian
Journal
of
INFECTIOUS
DISEASES
Original
article
Association
of
PRKAA1
gene
polymorphisms
with
chronic
hepatitis
B
virus
infection
in
Chinese
Han
population
Jun
Yuan
a,∗,
Yan
Zhang
b,
Fu-tang
Yan
a,
Xiao
Zheng
a aShaanxiProvincialPeople’sHospital,ClinicalLaboratory,Xi’an,ChinabShaanxiProvincialPeople’sHospital,DepartmentofCT,Xi’an,China
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f
o
Articlehistory:
Received6May2016 Accepted2August2016
Availableonline6September2016
Keywords: HBV PRKAA1gene SNP Associationstudy
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Objective:StudieshaveindicatedthatAMPKplaycriticalrolesintheregulationofinnate immunityandinflammatoryresponses.However,theroleofthepolymorphismsofPRKAA1
geneinimmune-responsetoinfectiousorganismsremainsunknown.Toevaluatethe poten-tialroleofPRKAA1/AMPK␣1intheimmune-responsetoHBV,weconductedthiscase–control study.
Methods:Werecruited276patients(145menand131women;averageage,51.6years)with chronicHBVinfection(CHB)and303healthycontrols(166menand137women;average age,54.2years).Allthesubjectswereunrelatedindividuals ofChineseHanPopulation. ThreeSNPsofPRKAA1geneweretested.
Results:Rs1002424polymorphismshowedsignificantdifferenceintheallelefrequencies, but no differencein the genotype frequencies (allele: p=0.039411, OR95%CI=0.783479 [0.621067–0.988362];genotype:p=0.104758);rs13361707polymorphismshowedsignificance inallele analysis, but not in genotypeanalysis (allele: p=0.034749, OR95%CI=1.284303 [1.017958–1.620335];genotype:p=0.098027);rs3792822polymorphismwasdemonstratedto havesignificantdifferencesinbothgenotypeandallelefrequenciesbetweencasesand con-trols(allele:p=0.029286,OR95%CI=0.741519[0.566439–0.970716];genotype:p=0.034560). ThehaplotyperesultsshowedthatCTGandTCAinthers13361707–rs1002424–rs3792822 block were significantly associated with the happening of HBV (CTG: p=0.036854, OR95%CI=1.281[1.015–1.617];p=0.030841,OR95%CI=0.743[0.568–0.973]).
Conclusion:ThesefindingssuggestthatPRKAA1polymorphismsmaycontributetothe sus-ceptibilityofchronicHBVinfectioninChineseHanorigin.
©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).
∗ Correspondingauthor.
E-mailaddress:yjun512@126.com(J.Yuan).
http://dx.doi.org/10.1016/j.bjid.2016.08.003
1413-8670/©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Adenosine-monophosphate-activatedproteinkinase(AMPK) ishighlyconservedhetero-trimericserine/threonineprotein kinase consists of a catalytic subunit (␣1 or ␣2) and two regulatory  (1–2) and ␥ subunits (␥1–3).1,2 It is
ubiqui-touslyexpressedand servedasamaster sensorofcellular energy homeostasis at the cellular and even whole-body level. AMPK isactivated by the increase ofAMP/ATP ratio fromenvironmentalornutritionalstressfactorsincluding oxi-dants,hypoxiaandnutrientdeprivation,3,4thentheactivated
kinase phosphorylates and regulates a variety of down-stream moleculesthat maintainsthe balance betweenthe consumption and generation of cellular energy.5,6
Disrup-tion of this balance results a number of diseases such as diabetes, obesity, cardiovascular dysfunction, inflammation and some malignant diseases.7–9 In the last several years,
acceleratingevidencehaveindicatedthatAMPKplayscritical rolesintheregulationofinnateimmunityandinflammatory responses.Galicetal.reportedthattargetdeletionofAMPK1 inmiceenhancedadiposetissuemacrophageinflammation andliverinsulinresistance.10Whilesomeotherresearchers
demonstratedthatAMPK-␣1isessential inmediatingTLR4 or TNF-␣ triggered inflammatory signals as an activating kinase of TAK1.11,12 AMPK-␣1 is encoded by PRKAA1 gene
which islocated onchromosome 5p12.1 and constitutes a 39kbregion.Studieshaveindicatedthatnucleotide polymor-phismsinPRKAA1genewassignificantassociatedwiththe riskofgastric,colonandrectalcancer.13–16However,theroleof
PRKAA1geneintheimmune-responsetoinfectiousorganisms remainsunknown.
Hepatitis B isone of the most common infectious dis-easescausedbythehepatitisBvirus(HBV)whichattacksthe liver. ChronichepatitisBvirus infection (CHB)maylead to liverfibrosisandcirrhosis,anddramaticallyincreasethe inci-denceofhepatocellularcarcinoma(HCC).Thesusceptibility andprogressionofthisdiseaseare knowntobeinfluenced by viral load, virus genotype, environmental factors, host immuneresponsesandhostgeneticfactors.17,18Considering
theimportanceofPRKAA1intheinflammatorysignaling cas-cade,weconductedthiscase–controlstudytoinvestigatethe associationbetweenPRKAA1andthesusceptibilityofchronic HBVinfectioninChineseHanpopulation.
Materials
and
methods
Studysubjects
In this study,we recruited 276 patients (145 menand 131 women;averageage,51.6years)withchronicHBVinfection and 303 healthy controls (166 menand 137 women; aver-ageage,54.2years)fromShaanxiProvincePeople’sHospital betweenJan2012andDec2014.Allthesubjectswere unre-lated individuals of Chinese Han Population. The patients were diagnosed based on serological positivity for HBsAg, HBeAgoranti-HBeandanti-HBcforatleast6months,and persistentabnormalityofALT(alanineaminotransferase)and AST(aspartateaminotransferase).Allthepatientswereinthe
activehepatitisstagewithoutevidenceoflivercirrhosisand HCC. Patientswithother diseases wereexcluded from this study.Healthycontrolswererandomlyselectedfromhealthy personsunderroutinehealthscreeningatthesamehospital duringthesametimeperiod.Theywereconfirmedtobe nega-tiveforHBsAgandHBeAg,withoutclinicalevidenceofhepatic disease,cancerandotherdisease.Themaincharacteristicsof thesubjectsarelistedinthesupplementaryTableS1. Writ-teninformedconsentwasobtainedfromeachparticipantof study.Thestudyprotocolconformedtothe1975Declarationof HelsinkiandwasapprovedbytheResearchEthicsCommittee ofShaanxiProvincePeople’sHospital.
GenotypingofPRKAA1genepolymorphisms
Genomic DNA was isolated from EDTA peripheral blood using the DNA Extraction Kit (Tiangen Biotech Co., Ltd., Beijing, China) following the manufacturer’s instruc-tions. All DNA samples were amplified for rs13361707, rs1002424 and rs3792822 by PCR (polymerase chain reac-tion) using TaKaRaPCR Amplification Kit(Takara Bio Inc.). The primers were designed using primer 5.0 software (Molecular Biology Insights) according to the sequence from NCBI. The primers for rs13361707 primers were: Forward: 5-ACGTGTTAAGGAAATAGC-3, and Reverse: 5 -AACTGTGTGTATAGTGCAGG-3. The primers for rs1002424 were: Forward: 5-AGTTTGGAAGTTATCAGATC-3, and Reverse: 5-ACAGGTGTGAGCCACAGCAC-3. The primers forrs3792822were:Forward:5 -ACTAGCATCAAAATGTCAGC-3, and Reverse: 5-GACTAGCAAGGCCTAGATCT-3. The PCR productswerethensequencedandanalyzedbyABI3700DNA automatedsequencer(AppliedBiosystems)accordingtothe standardprotocol.
Statisticalanalysis
Data management and statisticalanalyseswere conducted by STATA software 11.0 version for Windows (StataCorp LP, College Station, TX, USA). Hardy–Weinberg equilibrium wastestedusing the2 test.Between-groupvariableswere
comparedbyunpairedStudent’st-testor Mann–WhitneyU
testwhereappropriate,andgenotype/allelefrequencieswere comparedby2test.Thelinkagedisequilibriumand
haplo-typeconstructionwereperformedfrom theobserved geno-types using SHEsis method (http://www.nhgg.org/analysis), andoddsratios(OR) with95%confidenceinterval(CI)were calculated.Atwo-tailedp<0.05wasacceptedasstatistically significant.
Results
Single-pointassociationanalysis
TherewerenodeviationsfromHardy–Weinbergequilibrium forallstudiedpolymorphismsincasesandcontrols(p>0.05). Totally, three SNPs ofPRKAA1gene were tested. Rs1002424 polymorphism showed difference in the allele frequen-cies(p=0.039411,OR95%CI=0.783479[0.621067–0.988362])but no differences in the genotype frequencies (p=0.104758);
Table1–DistributionofgenotypeandallelefrequenciesofthreeSNPsinthePRKAA1inHBVpatientsandhealthy controls.
SNP HBVpatients Healthycontrols OR 95%CI pValue
(n=276) (n=303) rs13361707 Allele(freq) 1.284303 1.017958–1.620335 0.034749 C 269(0.491) 259(0.429) T 279(0.509) 345(0.571) Genotype(freq) 0.098027 CC 71(0.259) 57(0.189) CT 127(0.464) 145(0.480) TT 76(0.277) 100(0.331) HWE 0.228904 0.729947 rs1002424 Allele(freq) 0.783479 0.621067–0.988362 0.039411 C 278(0.507) 343(0.568) T 270(0.493) 261(0.432) Genotype(freq) 0.104758 CC 76(0.277) 99(0.328) CT 126(0.460) 145(0.480) TT 72(0.263) 58(0.192) HWE 0.184879 0.706059 rs3792822 Allele(freq) 0.741519 0.566439–0.970716 0.029286 A 121(0.221) 167(0.276) G 427(0.779) 437(0.724) Genotype(freq) 0.034560 AA 14(0.051) 17(0.056) AG 93(0.339) 133(0.440) GG 167(0.609) 152(0.503) HWE 0.821795 0.079962
BoldnumbersrepresentP-values(P<0.05).
Rs13361707polymorphismshowedsignificanceinallele anal-ysis (p=0.034749, OR95%CI=1.284303 [1.017958–1.620335]), but notin genotype analysis(p=0.098027);rs3792822 poly-morphism was demonstrated to have significant differ-ences in both genotype and allele frequencies between cases and controls (allele: p=0.029286, OR95%CI=0.741519 [0.566439–0.970716];genotype:p=0.034560)(Table1).
Haplotypeanalysis
The standardized measure of linkage disequilibrium (LD), denotedas‘D’wasestimatedatallpossiblepairsofSNPloci. LDforeachpairofSNPsincasesandcontrolsispresentedin
Table2.TheD-valueorr2-valueshowedstronglinkage
pat-ternswereobservedbetweenrs13361707andrs1002424inall samples(D=0.99),aswellasamongrs1002424andrs3792822
Table2–Estimationoflinkagedisequilibriumbetween
the3SNPs.
rs13361707 rs1002424 rs3792822
rs13361707 – 0.996 1.000
rs1002424 0.983 – 1.000
rs3792822 0.282 0.285 –
SNP,singlenucleotidepolymorphism.
ForeachpairofSNPs,Dvaluesareshownaboveandr2valuesbelow thediagonal,D’orr2>0.9areshowninboldface.
(D=0.99), andbetweenrs13361707 andrs3792822(D=0.99) (Fig.1).
Tofacilitateidentificationofcombinationaleffectsofthese threepolymorphismsontheriskofchronicHBVinfection,we employedhaplotypeanalysistostudy thefrequencyofthe combinationofmultiplegeneticvariants.Theresultsshowed that CTG and TCA in the rs13361707–rs1002424–rs3792822 block were significantly associated with the development ofchronicHBVinfection(CTG:p=0.036854,OR95%CI=1.281 [1.015–1.617]; p=0.030841, OR95%CI=0.743 [0.568–0.973]) (Table3).
Discussion
Chronic hepatitis B virus infection (CHB) is one of the most common infectious disease which leads to a huge economic problem on health care systems worldwide. According to the data from WHO, about 240 million peo-ple are chronically infected with hepatitis B and more than 780,000 people die from hepatitis B related dis-eases including cirrhosis and liver cancer each year (http://www.who.int/mediacentre/factsheets/fs204/en/). ThusitisimportanttoclarifythemechanismofchronicHBV infection.Recentstudieshaveshownthatchronichepatitis Bvirusinfectionisresultedfromtheinterplaybetweenthe virus, hostimmunesystemand environmentalfactors.19,20
Amongofthem,hostgeneticbackgroundsmaybecriticalfor the susceptibilityandvariousoutcomes ofthisdisease.21,22
Table3–Haplotypeanalysis:rs13361707,rs1002424,rs3792822.
Case(freq) Control(freq) Pearson’sp 2 Oddsratio[95%CI]
CTA 0.01(0.000) 0.01(0.000) – – – CTG* 267.99(0.489) 258.99(0.429) 0.036854 4.358 1.281[1.015–1.617] TCA* 120.99(0.221) 166.98(0.276) 0.030841 4.663 0.743[0.568–0.973] TCG* 156.00(0.285) 176.02(0.291) 0.818643 0.053 0.971[0.752–1.253] TTA 0.00(0.000) 0.01(0.000) – – – TTG 2.00(0.004) 1.99(0.003) – – – CCG 1.00(0.002) 0.00(0.000) – – –
(Allthosefrequency<0.03willbeignoredinanalysis.) Global2is5.885321whiledf=2.
Pearson’spvalueis0.052725.
BoldnumbersrepresentP-values(P<0.05).
1 2 99 99 99 3 rs13361701 rs1002424 rs3792822
Fig.1–LDpatternsofSNPsrs13361707,rs1002424and rs3792822.Thelinkagepatternsbetweenthe3studied polymorphismsinallsamples.Thenumbersindiamond representthe100×Dintheformofstandardcolorscheme. Theupperbardenotestherelativedistanceamongthe studiedpolymorphisms.
A number of genes have been identified to be associated with chronic hepatitis B virus infection by genome-wide association studies and case–control studies in the last several years.23–25 More knowledge on the understanding
ofcandidategeneticfactorsmayprovidemorecluestothe preventionandtherapyofthisdisease.
IthasbeenwelldocumentedthatAMPKisanimportant regulatorofenergy-sensingandsignaling cascadein mam-maliancells.26Asaheterotrimericcomplexserine/threonine
proteinkinase,AMPKcanregulateavarietyofother physi-ologicalfunctionssuchastheregulationofcellproliferation andpolarity,tumorigenesis,autophagyandinnateimmune responses.27 However, the signaling properties are diverse
depending on the different combination of subunit iso-forms of AMPK in various cells.28 Some studies indicated
thatAMPKmay restrictHBVreplicationthroughpromotion of autophagic degradation, but the underlying mechanism remains to be elucidated.29 The ␣1 catalytic subunit of
AMPK encoded by PRKAA1 gene, is mainly expressed in lymphocytes and macrophages. AMPK␣1 has been demon-strated toplayapivotal role ininducingpro-inflammatory signals through the activation of TAK1 and NF-B,30 but its role in chronic HBV infection remains unclear. So, this current study might be the first attempt to assess the association between polymorphisms in PRAA1 gene and chronicHBVinfection.Theresultsshowedthatallthethree polymorphisms we tested were associated with the sus-ceptibility ofchronic HBV infection (rs3792822 in both the allele- andgenotype-analyses; rs13361707andrs1002424in allele-analyses). Based on the value of D, the following haplotype-analyseswere doneandtheresultsshowedthat, intheblockof‘rs13361707–rs1002424–rs3792822,CTGand TC Awere associatedwithchronichepatitisBvirus infec-tion, suggestingthat AMPK␣1 may playsome criticalroles in the regulation of the initial defense against hepatitis Bvirus.
Taken together, the results of our study indicated that
PRKAA1geneisassociatedwiththedevelopmentofchronic HBV infection in ChinseHanPopulation. Although replica-tionswithlargersamplesizeandfunctionalstudiestofurther clarifytherolethatPRKAA1/AMPK␣1areneeded,thecurrent studygavesomeclueswhichisusefulinbetter understand-ingtheintrinsicrelationshipbetweenPRKAA1/AMPK␣1and thepathogenesisofchronicHBVinfection.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
This work was supported by grants from National High Technology Research and Development Program 863 (2014AA022304);ShaanxiProvinceNaturalScienceFoundation ResearchProject(2014JM2-8201).
Appendix
A.
Supplementary
data
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.bjid.2016.08.003.
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