中国人eNOS基因VNTR多态性的基因型与等位基因频率

中国人eNOS基因VNTR多态性的基因型与等位基因频率 中国人eNOS基因VNTR多态性的基因型 与等位基因频率 chemisthy&MNecub~BioJo~.f~tituteRadiationMedici~,&1008~.chE呻; 2Milga~Me~kcalCd&ge,脚100071,China; 3Be~jingNorthTa~ngRoad.Belting100039.Ch/~) Abstract:Genotypeandallelefrequencyofthepolymorphic27-bprepeat,avariable numberoftandemrepeats(VNTR)locatedinintron4oftheendothelialnitricoxidesyn— thasegene,wereanalyzedin316healthyChineseindividuals.Fourgenotypes,namely6/ 5-repeatsheterozygous,5/5一 repeatshomozygous,5/4-repeatshetemzygousand4t4一repeats homozygous,wereidentifiedBothobse~,edgenatypeandallelefrequenciesofthisVNTR inChineseweresimilartothoseofJapanese,whilethe4/4-repeatsgenot)-pedifferedsig— nifieantlyfromthatofCaucasiansinSpain.andallonesdidfromthose.fAfrican-Ameri— caninUnitedStates Keywords:endothelialnitricoxidesynthase;genepolymorphism;VNTR Nitricoxidesynthase(NOS)catalyzesthe5-electronoxidationoftheaminoacidL-arginineto f0珊 L-citnI11eandnitc0xide(0).Threeisoforms.fNOShavebeenidentitied:neuronad NOS(NOS),inducibleNOS(INOS)andendothelialNOS(eNOS).eNOSisconstitutivelyex— pressedinendatheliumandservestomaintainbasalNOproductionwhichregulatesvasculartoneand plateletaggregation.ThehumangeneencodingeNOSislocatedinchromosomalregion7q35—36 andcomposedof26exonsthatspan21kbThegeneexhibitsanumberofpolymorphicre[mats thathavebeenusedforthemmlysisofgeneticassociationw|thvailOUShumancardiovasculardisor- ders.Amongthemthe27bprepeat,avariablenmaberoftandemrepeat(VNTR)geneticmsTkerin intton4ofeNOSgene,isbelievedtoholymorphisms.WealsocomparedChinesedatawiththreeotherpopulations, onefromJapanese,onefromCaucasiansinSpainandtheotherfromAfrican- AmericaninUnited States. R~ivadMay29.2001;revision~eiredAugust17.2001 ThisstudyassupportedbyDevelopmentProgramminghemofNationalKeyB1cRe.arch(G2000057001 ?c?一authorE—mail:lvx@nicbmiaccn 遗传28卷 IM[amrialsandMethods 1.1SubjectsandDNAsmnples Atotal0f316Hartnationality,health),andgeneticallyunrelatedsubjects(235menand81 wonl~tl;Inea1lage54years,range18to90)livinginBering,China.werestudied.Thegenom~c DNAw丑 8plqfromperipheralbloodlenkoe)leswiththeuseofGenomJcDNAPurificationKit (PremegaInc.,USA)a8sIl删edbythernmlufacturer. 1.2PCRamplification PeRwosperformedusingtheprimersreportedbyUwabaJetal,namely,theforwardprimer 5’-GC~AACCTCAGCCCAGTAGTGAA-3andlpeader$eprimer5一 GTGGTCACAGGCGTTCCA GTAAC-3.The25,alreactionmixturecontained150ngofgenomicDNA,20mmol/LTris—He1 (pH8.4),50retool/LKCI,1.5mmol/LMgcl2.20011moltLofeachdNTP,200pmol/Lofeachprim- el’andI.5UTaqpolymerase(TakaRaBioteehnologyCo.,Dalien,China).PCRconditionsW~l’e 2minat96,foUowedby35cyclesof94? for30see,60for30seeand72for60flee.Chain elongationwaScontinuedafterthelaStcyclefor10min.ThePCRproductswereseparatedby5% denaturingpolyacrylamidegelelectmphoresisandvisualizedbysilverstaining. 1.3DNAsequencing Toexactlydeterminethenumberandlength0frepeatsrepresentingdifferentalleles.PeRprod- UatSredenedintopT-Advvector(ClentechLaboratories,Ine.,USA)andsequencedby蚰auto- maticsequencer(GenecoreInc.,Shanghai,China). 1.4Statisticalanalysis Allelefrequencieswerecalculatedfromthegenotypesofallsubjects.Hardy-WeinbergeqfIIilm- rlumw聃 examinedbytest.Differencesinthegenotypeandallelefrequenciesforpopulati on groupswereassessedbyFisher’sexacttest.APvaluebelowO.05wasconsider edsignitleant. 2RemitsandI培9c? 2.1Allelesend@enotypes 1234Mb.AsshowedinFig.1,thl’eeallelesandfourganotypes0fthe 薹4篇71 F1Gel时rIgoftheVNTRinimmn 4of,IVOSne Lane1:5t6-tepeatshe?y8;Lane2: 5t5-repsatshwn哪;Lane3: 5,4.?.柚b口.;Lane4: 4t4-repeatshqmlozy~ls VSTRintheeNOSgeneweredetectedintheChinesepopulat- ien.Theallelecontainingsixof27-bprepeatsgaveriseto日 PCRproductof471-bpwhichconsisted0f162?bpand 309一bpflankingsequence.whereasthefiveandfour0fsuchre- peat8yieldedabpanda417一bpproductconsisting0f135- bpand108bprepeatsendthe&meflankingsequence?respc~. tively SequencingrevealedthattheoonfleYlsu88~lLletio00f27?bp repeatunitwasthe$atfleaspreviouslyreported【5__ GAAGTCTAGACCTGCTGC(AtG)~TGAG-3].The first3repeatsofthe6-repeatallelecontainedAasthe19thnncleotide.whereasthelast3contained G.Thefrrst3repeatsofthe5-repeatallelecontainedAendthelast2containedGatthe19thp0si- 一1骚]匿霾圈嚣 2The27-bprepeatregionisboxed;ThePCRpfiusedtoamplitherepeatregionareunderlined 2.2.Mleleandgenot3.pefrequencies TheresultsofthedistributionofeNOS27一 bprepeatpolymorplrismintheChinesepopulation aresunmlarizedinTable1.Thegenotypefrequenciesfor6/5一 repeatshetero~,gous,5/5一repeatsbe— mo~gous,5/4一repeatshetemzygousand4/4一 repeatshomozygouswere0.010(n:3),0.816(n 2.58),0.168(? =53)and0.006(n=2)respectively.Themostfrequentgenotypewas5/5.repeats homozygousfollowedby5/4一repeatsheterozygous,6/5一 repeatsheterozygousand4/4一repeatshomozy— gous.Thefrequenciesfor6-repeatallele.5-repeatalleleand4一 repeatallelewe0. 005(1”1=3), 0.905(n:572)and0.090(n=57)respectively.TheItlOStfrequentallelewas5-repeatfoil0wed by4-repeatand6-repeat.ThedistributionofbothgenotypefrequencywereinHardy-Weinbergequi— librium(=0.26,P>005). Table1Genotypeandldefrequencies0f27一bpp哺t|neNOSg蚰 ‘ConaparingCh/n~withh ?;C~paringChinesewithCaae~iam;ComparingClfin~withAfric~一 At~ncans Table1alsoshowedthedifferencesingenotypeandallelefrequenciesbetweenChineseand otherethnicpopulations.Therewerenodifferencesinbethgenotypearmallelefrequenciesbetween ChineseandJapanese.Significantdifferenceswerefoundinallofgenotypeandallelefrequencies betakenChineseandAfrican—Ameriemmandinonlythegenotypeof4/4-re peatshomozygousbetween 遗传28卷 C,hill~andCaucasians.Wefoundthegenotypoof6t5-p8heterozygousinChinesepopulation, whichobservedinA~fiean-AmericansbutnotinbothJapaneseandCaucasiansforeachstudiedsub. ject. Wehaveidentifiedthreedistinctallelesandfourgenotyposforthe27-bprepeatinintran4of eNOSgeneintheChinesepopulationItwasdemonstratedthatthefrequencyofbothgenotypeand alleleforthisVNTRinChinesewassimilart0thatofJapanese,butdifferedsignificantlyfromtl108e ofCauet~ansandAfrican?Americansin8omegenotypesandalleles,suggestingthatthedis~n ofthisgeneticmark~/-diffemamongFaces.Forexample.thefrequencyof4t4-repantshomozy@tms genotypeinChineseandJapanesewiltslowerthaninCaucasiansandAfrican-Americans.Uw~boet alshowedthatthegenotypeof4t4-repeatshomozygouswasaI1independentriskfactorforessential hypertensioninJapanese,whemasWangalreportedthatthisgenotypowasass~mtedwitha smkingdependentriskfactorforcoronarydiseasebutnotwithessentialbypo~ ensioninCaucasians Bubject8ofAustraliat.Ourdataindicatethatthisdiscrepancymaybeattributabletotheracialdi. v?inthisVNTRdistribution.Withwhatkindofhumancaova8culBrdisordersthisVNTR polymorphismwoddbeassociatedinChinesepopulationneed~tobedeterminedinfm’tberatudias. [1】岫deS.m咿A.Thel一TIil丁icopathwayN.E.J.Med.1993,329:.2 一加12. 12jF~emumaU,G幽l,sch,Pd.一nitricoxide? hBiochenfieMPhammeo1..1995.50}1321—1332. [3】M?d柚 PA.~alemreandehronm~nudloealizmionofthehumaneo~fimlJve~dothelialccde町?h舳eg啪eJ. Bid.Chem.,1993.268:17478,l7488 【4JUwaboJts哪BM,NTda1.Associati~ofavarlableiltltllll~0f口r山 intheende~ellaleomfitard~nitrle oxidemhe_lcwithemntialhypert~ionJnJ且pa搴eAm.JHyP酣帅B_ 帅.1998,11:125—128. [3】】cI日S.?Y,n.jjmT#.Assoclati~ofm町pbi? ofthe~dothelialeo~lltardveeladothelblniulcodde q?with?B-【?intheJapa~pq~lafitm.Am.JCmdid.1cA~8.81:83,86 [6】lh呷茁wC.LaityC.AustinHdThezel~onahlpbetweenpol啪incazJothelinledlin~deoxideyl】一舯e theG~///awithmyoca~infareticatand咖嘴thromB蚰thecndo~ellsl试eor,lde7u.9m (ir,n~a4,27bprepeat肌d一786T/C)肌dthe?$m?山0自nb_岫ThmmboslsR?,2OOO.99:553—566 [9】WingXL,SivaAS,B日de?l’oPRFeta1.Asmoklng-dependent础of~aomayarier~dima~mclamdwithl..1?i啦 山6eado&d~dn?coxid~叩lh雠geneNatm~M耐..1996,2:41一. .?谣匿j:=匡睡馐孵 I2期路萍等:中国』,eNOS基因VNTR多态性的基周型与等位基因频率1097 中国人eNOS基因VNTR多态性的 基因型与等位基因频率 路萍,吕星,邢瑞云,孙琪云’, 韩莉,吴苏华,蓝红,郑晓飞 (1军事医学科学院放射医学研究所.北京100850;2军医学院,北京IOC~7 3.北京北太平路医院,北京100039) 摘要:一氧化氮合酶(nitricoxidesvnthase,NOS)催化L一精氨酸的氧化反应生成 瓜氨酸和一氧化氮(nitricoxide,NO).NO可通过cGMP依赖的信号传导途径介 导平滑肌细胞舒张,是调节血管张力的重要信使分子.NO尚可抑制血小板凝 集,对血栓形成起重要调节作用.目前在哺乳动物中已发现细胞来源,表达方式 和活性调节不同的3种NOS同工酶,分别为神经元型NOS(neuronalNOS, nNOS),诱导型NOS(inducibleNOS.iNOS)和内皮细胞型NOS(endothelialNOS, eNOS).人的eNOS基因位于第7号染色体长臂(7q36),全长约21kb,含有26个 外显子和25个内含子.eNOS基因存在多个与心脑血管疾病相关的基因多态性 位点.其中位于第4内含子的一个以27bp为核心的数目可变性串联重复序列 (variablenumber0ftandemrepeat,VNTR)多态性位点,已被证实与原发性高血压, 心肌梗死和静脉血栓形成有关.目前在我国尚缺乏NOS基因多态陛在正常人 群中基因型及等位基因频率分布的统计资料.为此,我们从316名健康中国人 的基因组DNA检测了eNOS基因第4内含子VNTR多态性的基因型和等位基 因,鉴定出重复6次,5次和4次的3种等位基因,以及6t5杂合,515纯合,5/4杂 合和4纯合的4种基因型.同时我们将正常中国人eNOS基因VNTR多态性 的基因型和等位基因频率与其他种族的相关资料进行了统计对比.结果表明, 中国人eNOS基因VNTR的各种基因型和等位基因频率与日本人相似,4/4纯合 基因型频率与高加索人差异显着,各种基因型和等位基因频率与非裔美国人均 存在显着差异. 关键词:一氧化氮合酶;基因多态性;VNTR 中圈分类号:Q[标签:快照]