PharmacologicaSinica(2010)31:1376–1380©2010CPSandSIMMAllrightsreserved1671-4083/10$32.00 /aps OriginalArticle icanalysisofsixSNPsincandidategenesassociatedwithhighcross-raceriskofdevelopmentofthoracicaorticaneurysmsanddissectionsinChineseHanpopulation OuLIU,Jian-rongLI,MingGONG,MingXU,JieDU*,Hong-jiaZHANG* TheKeyLaboratoryofRemodeling-relatedCardiovascularDiseases,CapitalMedicalUniversity,MinistryofEducation,andInstituteofHeartLungandBloodVesselDiseases,BeijingAnzhenHospitalAffiliatedtotheCapitalMedicalUniversity,Beijing100029,China Aim:icsusceptibilityisanimportantriskfactorforaorticaneurysmanddissection.Recentcase-controlassociationstudieshaveidentifiedsixsinglenucleotidepolymorphisms(SNPs)associatedwithabdominalaorticaneurysm(AAA)inaCaucasianpopulation.Weaimedtodeterminewhethertheselociconfersusceptibilitytothoracicaorticdissection(TAD)inaChineseHanpopulationandthustoestablishcross-racesusceptibilitytoTAD.Methods:ThisstudyanalyzedbloodDNAisolatedfrom206TADpatientsand180controlsfromtheethnicChineseHanpopulation.SixSNPs–rs819146,rs8003379,rs2853523,rs326118,rs3788205,andrs10757278–weregenotypedusinghigh-throughputmatrix-assistedlaserdesorptionionization-time-of-flight(MALDI-TOF)massspectrometry.Results:TheAallelefrequencyfortheSNPon9p21,taggedasrs10757278,washigherinmaleTADpatientsthaninmalecontrols(P=0.017).Moreover,withadjustmentfortraditionalcardiovascularriskfactors(sex,age,hypertension,dyslipidemia,diabetes,andsmoking),thers10757278[oddsratio(OR)0.63,95%confidenceinterval(CI)0.43to0.93]polymorphismwasfoundtobeanindependentsusceptibilityfactorforTADinmen.Conclusion:Ourresultssuggestthatasequencevarianton9p21isanimportantsusceptibilitylocusthatconfershighcross-raceriskfordevelopmentofTADinChineseHanpopulation. Keywords:aorta;aneurysm;thoracicaorticdissections;icpolymorphism;ChineseHanpopulation;males;females ActaPharmacologicaSinica(2010)31:1376–1380;doi:10.1038/aps.2010.159;publishedonline27Sep2010 IntroductionAneurysmsanddissectionsofthethoracicaortaarethetwomostcatastrophicacutenaturaleventsthatcanbefallahumanbeing.Botharemajorcausesofmorbidityandmortalityintheworld[1].Thoracicaorticaneurysmstendtobeasymptomaticandusuallyarenotnoticedbeforethoracicaorticdissection(TAD)urs.ItisbelievedthatTADisplextraitthatiscausedbybothenvironmentalandicfactors[2–4].icsusceptibilityisanimportantriskfactorforTAD.Someicsyndromes,suchasMarfansyndromeandEhlersDanlossyndrome,predisposeindividualstoTAD[5,6].RecentstudieshavealsoindicatedthatTADpatientswithouticsyndromestendtohaveafamilyhistoryofaorticaneurysm *Towhomcorrespondenceshouldbeaddressed.E-mailjdu@bcm.edu(JieDU); zhanghongjia722@(Hong-jiaZHANG)Received2010-05-06epted2010-08-12 disease.However,itremainsunclearwhethericfactorsinfluencetheformationofTADinindividualswhodonothaveicsyndromes. Asinglenucleotidepolymorphism(SNP)ismonicvariantthatconsistsofasingleDNAbasepairchange[7].SeveralstudieshaveshownthatSNPscouldexplaindifferencesinicsusceptibilitytodifferentdiseases.TheSNPassociationstudyhaseaverypopularmethodforidentificationoficfactorsplexdiseasetraits[8].Recently,twoindependentpopulationbasedcase-controlstudieshaveshownthateightSNPsareassociatedwithabdominalaorticaneurysminseveralCaucasiancohorts[9,10].GiustietalreportedsevensusceptibilitySNPs(suchasrs819146,rs8003379,rs2853523,rs326118,andrs3788205)insevengenesinvolvedinmethioninemetabolismthatwereassociatedwithabdominalaorticaneurysmformationinanItalianpopulation[9].HelgadottiretaldescribedanassociationbetweenabdominalaorticaneurysmandaSNP LiuOetal npg 1377 on9p21inthegeneforcyclin-dependentkinaseinhibitorinanIcelandicpopulation[10].TheyfoundthatthisSNP,taggedasrs10757278,contributedtotheriskofmyocardialinfarctionandintracranialaneurysm.Twootherrecentindependentpopulation-basedcase-controlstudieshavealsoshownthatthers10757278SNPissignificantlyassociatedwithcoronaryheartdiseaseandrelateddisordersamongEuropeans[11,12].ThepreviousstudieshavefoundassociationsbetweentheseSNPsandabdominalaorticaneurysm,butonlyinpopulationsofEuropeandescent.Becauseofthewell-knownicheterogeneitybetweenethnicpopulations,validationbyiccase-controlstudiesinnon-Europeanpopulationsisessential. Inthepresentstudy,weaimedtoconductacase-controlassociationstudyinaChineseHanpopulationtotesttheassociationbetweensixmost-studiedSNPsandaorticaneurysm. MaterialsandmethodsStudypopulationWeenrolled206casesthatwererandomlyselectedfromTADpatientsadmittedtoBeijingAnzhenHospitalaffiliatedwiththeCapitalMedicalUniversity.ThecriteriafordiagnosisofTADhavebeenpreviouslydescribed[3].Inbrief,patientswerediagnosedbymanytypesofimagingmodality,includingultrasound,2Dechocardiography,putedtomographyscan,MRI,andangiography.PatientswithMarfansyndrome,Ehlers-Danlossyndrome,traumaticaneurysms,oraorticcoarctationwereexcludedfromthestudy.Forthecontrolgroup,180volunteerswereselectedfromindividualswhowereadmittedtoBeijingAnzhenHospitalforaorticimagingevaluationandwerefoundtohavenoaorticdiseases,suchasprimaryhypertensiondisease.Wechoseasubhypertensiveage-matchedgroupasthecontrolgroupbecauseitisknownthathypertensionisoneoftheriskfactorsforTAD.UseofthiscontrolgroupwilleliminatethepotentialcontributionofsecondaryhypertensiontoTAD.AllsubjectswereunrelatedChineseHanindividuals.Clinicalinformationandfamilyhistorieswerecollectedfrommedicalrecordsandinterviewsconductedbyaicscounselor.Hypertensionwasdiagnosedasasystolicbloodpressure≥140mmHg,adiastolicbloodpressure≥90mmHg,ortreatmentwithanti-hypertensivemedication.Dyslipidemiawasdefinedastotalcholesterol>6.5mmol/Lortreatmentforelevatedbloodlipids.Diabeteswasdefinedasafastingplasmasugarlevel>7.8mmol/L,a glucoselevel>11.1mmol/L2hafteroralglucosechallenge,orongoingtreatmentofdiabetes.Clinicaldata,suchasfastinginsulinlevelsandglucoselevels,weremeasuredroutinelyfortheseindividuals.Patientsandcontrolsgaveinformedconsenttoparticipateinthisstudy,andproceduralapprovalfromtheAnzhenHospitalEthicsCommitteewasobtained. BloodsamplecollectionandgenomicDNAextractionPeripheralbloodwasdrawnfromaveinintoasteriletubecontainingethylenediaminetetraaceticacid(EDTA).Plasmasampleswerestoredat-80°
C.GenomicDNAwasisolatedfromperipheralbloodleukocytesordingtothemanufacturer’sprotocol(GenomicDNAkit,AxygenScientificInc,California,USA).DNAwasstoredat-80°CforSNPanalysis. GenotypingWestudiedsixSNPsinornearsixcandidategenesthatwereassociatedwithaorticaneurysmordingtopreviousreports[9,10].Fiveofthesegenesweresuspectedtobeinvolvedinmethioninemetabolism.Theotherwasrelatedtoacyclin-dependentkinaseinhibitor.InformationabouttheseSNPswasacquiredfromdbSNPNCBI,theENSEMBLdatabase(),andtheUCSCGenomeBrowserwebsite(http://genome.ucsc.edu)(Table1). GenotypingwascarriedoutforallSNPsusingtheMassARRAYplatform(Sequenom,SanDiego,CaliforniaUSA)ordingtostandardprotocolsdescribedpreviously[13].Briefly,probesandprimersweredesignedwithAssayDesignsoftware(Sequenom)andthegenomicDNAsampleswereamplifiedwithmultiplexpolymerasechainreaction(PCR).Afteramultiplexprimerextensionreactionwasperformed,terminatornucleotideswereaddedtothediagnosticSNPsite.Next,allele-specificextensionproductsofdifferentmasseswereanalyzedinatime-of-flight(MALDI-TOF)SpectroReadermassspectrometer(Sequenom).Finally,theresultingspectrawereprocessedwithTyperAnalyzersoftware(Sequenom)andgenotypedataweregeneratedforthesamples.ThefinalgenotypecallrateofeachSNPwasgreaterthan90%andthetotalgenotypingratewas99.7%toensurethereliabilityoffurtherstatisticalanalysis. StatisticalanalysisStatisticalanalysiswasperformedusingPLINK1.06(http://pngu.mgh.harvard.edu/~purcell/plink/index.shtml).AllgenotypedistributionsofcontrolsintheprimaryanalyseswereevaluatedforHardy–Weinbergequilibriumandnoevidenceofdeviationswasdetected(allexact Table1.Descriptionofandinformationaboutallinvestigatedsinglenucleotidepolymorphisms(SNPs). dbSNPID chrposition Gene Ensemblegenenumber Region rs819146rs8003379rs2853523rs326118rs3788205rs10757278 20cen-q13.114q241q435p15.3-15.221q22.39p21.3 AHCYMTHFD1MTRMTRRSLC19A1CDKN2BAS ENSG00000101444ENSG00000100714ENSG00000116984ENSG00000124275ENSG00000035928ENSG00000240498 5’neargeneintron3’utr5’neargene5’neargene3’neargene Chr,chromosome;AHCY,S-adenosylhomocysteinehydrolase;MTHFD1,methylene-tetrahydrofolatedehydrogenase;MTR,methyltetrahydrofolatehomocysteinemethyltransferase;MTRR,methioninesynthasereductase;SLC19A1,solutecarrierfamily19(folatetransporter),member1;CDKN2BAS,geneforthecyclin-dependentkinaseinhibitor. ActaPharmacologicaSinica npg 1378 /apsLiuOetal two-sidedPvalues>0.2).Categoricaldata(presentedasfrequenciesandpercentages)paredbetweengroupsusingtheχ2test,andcontinuousdata(givenasthemedianandrange)paredusing2-samplet-tests.TheBonferronicorrectionwasapplied(P<0.0083basedonthe6SNPsconsideredinthisstudy).WeanalyzedtheassociationbetweeneachpolymorphismandTADordingtodifferenticmodels.Genotypedistributionsbetweengroupsparedbyχ2analysis.Withadjustmentfortraditionalcardiovascularriskfactors(sex,age,hypertension,dyslipidemia,diabetes,andsmoking),multiplelogisticregressionanalysiswasusedtoestimatetheoddsratioand95%confidenceintervalsforTAD. ResultsSubjectsThedemographicandclinicalcharacteristicsofbothTADcasesandhealthycontrolsarereportedinTable2.StatisticallysignificantgenderdifferencesintheurrenceofTADwereobserved.Consistentwithothers’observations[9,14],TADwassubstantiallymonamongmenthanamongwomeninthisstudy.Theclinicaldata,suchasfastinginsulinlevelsandglucoselevels,didnotdiffersignificantlybetweencasesandcontrols. Table2.DemographicandclinicalcharacteristicsofbothTADcasesandhealthycontrolsinaChineseHanpopulation. Items Controls(n=180) TADpatients(n=206) PValue AgeSex(male)n(%)Smokingn(%)Diabetesn(%)Hypertensionn(%)Dyslipidaemian(%)CADn(%) 50.7(16–78)91(50.6)105(58.3)27(15)152(84.4)101(56.1)16(8.8) 50.6(21–75)158(76.7)108(52.4) 20(9.7)164(79.6)110(53.4)22(10.7) NS<0.0001NSNSNSNSNS TAD,thoracicaorticdissection;CAD,coronaryarterydisease;NS,notsignificant.Continuousdataweretestedusinga2-tailedStudent’st-testandcategoricaldataweretestedusingaChi-squaretest(withdf=1)forthedifferencebetweenTAD(patient)andcontrol(normal)groups. TotalsampleSNPanalysisAsshowninTable3,allelefrequenciesanddistributions ofallSNPsandχ2analysisordingtothedominantmodelwerereported.NoevidenceforanassociationbetweenthetestedSNPsandtheTADphenotypewasdetectedeitheratthealleleoratthegenotypelevel.Inthemultiplelogisticregressionanalysiswithage,gender,hypertension,diabetes,dyslipidemia,andsmokingastheindependentvariablesandTADasthedependentvariable,nosignificanteffectcouldbeobserved(datanotshown). SNPanalysisofgender-stratifiedsamplesTodetectpossibleinteractionsbetweengenderandSNPgenotypestatus,westratifiedthesamplesordingtogender.Inthesubsetofmenorwomen,genotypeandallelefrequenciesdidnotdiffersignificantlybetweencasesandcontrolsforanyofthefiveicvariantsinvolvedinmethioninemetabolism(datanotshown).However,wefoundthatfortheSNPrelatedtocyclin-dependentkinaseinhibitor,taggedasrs10757278,ahigherproportionofAallelecarrierswasfoundamongmaleTADpatients(57.4%)thanamongmalecontrols(46.1%,P=0.017).Genotypedistributionanalysisshowedstatisticallysignificantdifferencesbetweenmalepatientsandmalecontrols,ordingtothedominantmodelforrs10757278(Table4).Furthermore,weperformedlogisticregressionanalysiscorrectedforthevariablesage,hypertension,diabetes,dyslipidemia,andsmokinginmen.Thers10757278[oddsratio(OR)0.63,95%confidenceinterval(CI)0.43to0.93,P=0.021]genepolymorphismwasanindependenticsusceptibilityfactorformaleTADpatientsbutnotforfemaleTADpatients.However,whenweanalyzedthe6candidateSNPstogether,wefoundnosignificantdifferencebetweenthegroupsaftertheBonferronicorrection. DiscussionInviewofthediseaseassociationfoundinCaucasiancohorts[9,10],weanalyzedsixSNPsinaChineseHanpopulation.Weobservedanassociationbetweenthers10757278SNPandtheurrenceofTADinChinesemen.Ourresultssuggestthatthispolymorphismofthegeneforacyclin-dependentkinaseinhibitorplaysanimportantroleinsusceptibilitytoTADinamaleChineseHansubpopulation.However,we Table3.Genotypedistributionandallelefrequencyofallinvestigatedsinglenucleotidepolymorphisms(SNPs)inthoracicaorticdissection(TAD)patientsandcontrolsubjects.. Genotype(%) Genotype(%) dbSNPID Patients MAF Patients MAF P# P↑ rs819146rs8003379rs2853523rs326118rs3788205rs10757278 GG(3.9)CC(0.0)AA(4.4)GG(3.4)TT(6.8)GG(18.9) GT(29.8)CA(0.4)AC(32.0)GT(24.3)TC(29.1)GA(50.8) TT(66.3)AA(99.6)CC(63.6)TT(72.3)CC(64.1)AA(30.3) G=0.188C=0.002A=0.202G=0.155T=0.214G=0.444 GG(5.0)CC(0.0)AA(3.9)GG(3.9)TT(4.4)GG(27.4) GT(35.0)CA(1.1)AC(31.8)GT(25.0)TC(36.7)GA(48.0) TT(60.0)AA(98.9)CC(64.3)TT(71.1)CC(58.9)AA(24.6) G=0.225C=0.006A=0.198G=0.164T=0.223G=0.514 0.2020.4860.9140.7460.2200.054 0.198NA0.9730.7910.2960.220 #,ordingtotheallelictest;↑,ordingtothedominantmodel;MAF,minorallelicfrequency(basedonwholesample);NA,notapplicable ActaPharmacologicaSinica LiuOetal Table4.Differencesinthedistributionsofrs10757278genotypesandallelefrequenciesinthegender-stratifiedsubsamples. dbSNPID Genotypecounts(%) G/G G/A A/A Allelefrequencies G(%) A(%) P# OR(95%) MalesTADcasesControls 27(18.1)25(27.8) 73(49.0)47(52.2) 49(32.9)18(20.0) 42.6 57.4 53.9 46.1 0.017 0.64(0.44–0.92) FemalesTADcasesControls 10(21.7)24(27.0) 26(56.6)39(43.8) 10(21.7)26(29.1) 50.048.9 50.051.1 0.861 1.05(0.63–1.73) #,ordingtotheallelictest;↑,ordingtothedominantmodel;OR,estimatedoddsratio(forminorallele). npg 1379 P↑0.0320.352 foundthattheother5SNPsthatareassociatedwithTADinaCaucasianpopulationwerenotassociatedwiththediseaseinaChinesepopulation. Homocysteine,abranchpointintermediateofmethioninemetabolism,hasbeendefinedasanewmodifiableriskfactorforabdominalaorticaneurysmandothervasculardiseases[15].Homocysteinemayinduceoverproductionofoxidativeradicals[16,17]andactivationofmatrixmetalloproteinase-2(MMP-2)[18],whicharebothkeymechanismsforaorticaneurysmformation[19,20].Severalpolymorphismsingenescodingforenzymesinvolvedinmethioninemetabolismhavebeensuspectedtobeassociatedwithaorticaneurysm.Recently,astudyanalyzingthecontributionsof56polymorphismsin17genesinvolvedinmethioninemetabolismonicsusceptibilitytoaorticaneurysmwasperformed.Itdemonstratedthat7oftheseicvariantsaresusceptibilityfactorsforaorticaneurysm[9].Inthepresentstudy,weevaluatedtheassociationof5majorSNPsidentifiedintheabovestudyinaHanChinesepopulationwithaorticdissection.Elevatedhomocysteineinpatientsinducesoxidativestress,activationofMMP-2andotherpathologicalstimulithatcouldcontributetoaorticdissection[19].However,noevidenceforanassociationbetweenthese5polymorphismsandtheTADphenotypewasdetectedeitheratthealleleoratthegenotypelevel.Similarly,casecontrolanalysiswithgender-stratifiedsamplesdidnotshowasignificantdifference.Thereareseveralpossibleexplanationsforthisdiscrepancy.First,theassociationbetweenthetestedSNPsinvolvedinmethioninemetabolismandTADformationmaybeethnicity-specific.Second,environmentalfactors,suchasdiet,maycontributetothediscrepancybetweenthestudies.Forexample,homocysteinederivedfromdietarymethioninecanbemetabolizedviatwovitaminBpathways[21].Thus,unequaldietaryintakeofmethionineorvitaminBbetweenWesternandChinesepopulationscouldcontributetodifferentplasmahomocysteineconcentrations.Third,thephenotypeinourstudypopulationwasTAD.Comparedwithotherstudiesonabdominalaorticaneurysm,TADappearedtobemorecatastrophicandthesedissectionstendedtoruptureeasily. AnalysisoftheSNPtaggedasrs10757278,whichisrelatedtoaninhibitorofcyclin-dependentkinase,failedtoshowanassociationbetweenthesequencevariantandTADintotal samplesorinthefemalesubset.However,wedemonstratedthatforthisSNP,ahigherproportionofAallelecarrierswasfoundamongmaleTADpatients(57.4%)thanamongmalecontrols(46.1%,P=0.017).ThisSNPisthusasusceptibilityfactorforTADinamaleChineseHansubpopulation.Thereasonsforthissexdifferencearenotclear.Itispossiblethathormonalfactors,increasedexposuretoimportantriskfactors,icsusceptibility,orbinationofthesevariablesmaycontributetothisphenomenon.OurfindingisinterestingbecausetheSNPrs10757278hasalsobeenfoundtobeassociatedwithcardiovasculardiseases(CAD)[11,12,22],diabetes[22],andintracranialaneurysm[23].WehaveappliedBonferroni’stesttoourdataanalyses.ThetypeIerrorinthistypeofassociationstudyishigh,however,westillbelievethatrs10757278isasusceptibilityfactorforthoracicaorticdissection(TAD)inmenintheHanChinesepopulationforseveralreasons.First,ithasbeenshownthatrs10757278issignificantlyassociatedwithaorticaneurysminanIcelandicpopulation.Second,theLD(linkagedisequilibrium)blockcontainingthisSNPcontainstwoknowncyclin-dependentkinasegenes,CDKN2AandCDKN2B,aswellasalargeantisensenon-codingRNA,ANRIL.CDKN2AandCDKN2Bencodethetwocyclindependentkinaseinhibitorsp16INK4aandp15INK4b,whichareknowntohavecriticalrolesincellproliferation,aging,senescenceandapoptosis[24,25].ItisalsoknownthatthekeymechanismforTADformationistheproliferation,aging,senescenceandapoptosisofsmoothmusclecells.Inaddition,ANRIL,whichis3kbfromrs10757278,hasbeenshowntobeexpressedincellsandtissuesinvolvedinatherosclerosis[26],whichisconsideredtobeamajorriskfactorforTAD.Alloftheseobservationsmaysuggestthepotentialmechanismofrs10757278involvementinthepathogenesisofTAD.Togetherwithpreviousevidence,ourstudyindicatesthatrs10757278isapositiveSNPforTAD. Inconclusion,apotentialassociationbetween6SNPsandriskofTADwasinvestigatedinaChineseHanpopulation.Wedemonstratedthatasequencevarianton9p21,whichwasrelatedtoacyclin-dependentkinaseinhibitor,isanindependentsusceptibilityfactorforTADinamaleHanChinesesubpopulation. ActaPharmacologicaSinica npg 1380 /apsLiuOetal AcknowledgementsThisworkwassupportedbygrantsfromtheNationalNaturalScienceFoundationCouncilofChina(No30872536andNo30888004),theScientificResearchProgramoftheBeijingMunicipalCommissionofEducation(NoKM200910025020),andtheChineseMinistryofScienceandTechnology(No2009CB522205). AuthorcontributionOuLIU,Jian-rongLI,JieDU,andHong-jiaZHANGdesignedthisresearch;OuLIU,Jian-rongLI,MingGONG,andMingXUperformedtheresearch;OuLIUandJieDUanalyzedthedata;andOuLIU,JieDU,andHong-jiaZHANGwrotethepaper. 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