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,ava riable
numberoftandemrepeats(VNTR)locatedinintron4oftheendothelialnitrico xidesyn—
thagene,wereanalyzedin316healthyChineindividuals.Fourgenotypes ,namely6/
5-repeatsheterozygous,5/5一repeatshomozygous,5/4-repeatshetemzygousand4t4一repeats homozygous,wereidentifiedBothob~,edgenatypeandallelefrequencies ofthisVNTR
inChineweresimilartothoofJapane,whilethe4/4-repeatsgenot)-pedi fferedsig—
nifieantlyfromthatofCaucasiansinSpain.andallonesdidfromtho.fAfrica n-Ameri—
Keywords:endothelialnitricoxidesyntha;genepolymorphism;VNTR Nitricoxidesyntha(NOS)catalyzesthe5-electronoxidationoftheaminoac idL-arginineto
f0珊L-citnI11eandnitc0xide(0).Threeisoforms.fNOShavebeenidentitied:neuro nad
NOS(NOS),inducibleNOS(INOS)andendothelialNOS(eNOS).eNOSisc onstitutivelyex—presdinendatheliumandrvestomaintainbasalNOproductionwhichreg ulatesvasculartoneand
plateletaggregation.ThehumangeneencodingeNOSislocatedinchromoso malregion7q35—36
andcompodof26exonsthatspan21kbThegeneexhibitsanumberofpolym orphicre[mats thathavebeenudforthemmlysisofgeneticassociationw|thvailOUShuma ncardiovasculardisor-
ders.Amongthemthe27bprepeat,avariablenmaberoftandemrepeat(VNTR )geneticmsTkerin
intton4ofeNOSgene,isbelievedtoholymorphisms.WealsocomparedChine datawiththreeotherpopulations,
onefromJapane,onefromCaucasiansinSpainandtheotherfromAfrican-
States.
R~ivadMay29.2001;revision~eiredAugust17.2001 ThisstudyassupportedbyDevelopmentProgramminghemofNationalKey B1cRe.arch(G2000057001
①c∞一authorE—mail:lvx@nicbmiaccn
遗传28卷
IM[amrialsandMethods
1.1SubjectsandDNAsmnples
Atotal0f316Hartnationality,health),andgeneticallyunrelatedsubjects(235 menand81
wonl~tl;Inea1lage54years,range18to90)livinginBering,China.werestudi ed.Thegenom~c
DNA w丑8plqfromperipheralbloodlenkoe)leswiththeuofGenomJcDNAPurificatio nKit
(PremegaInc.,USA)a8sIl删edbythernmlufacturer.
1.2PCRamplification PeRwosperformedusingtheprimersreportedbyUwabaJetal,namely,thefor wardprimer
5’-GC~AACCTCAGCCCAGTAGTGAA-3andlpeader$eprimer5一
GTGGTCACAGGCGTTCCA
GTAAC-3.The25,alreactionmixturecontained150ngofgenomicDNA,20 mmol/L Tris—He1
(pH8.4),50retool/LKCI,1.5mmol/LMgcl2.20011moltLofeachdNTP,200 pmol/Lofeachprim-
el’andI.5UTaqpolymera(TakaRaBioteehnologyCo.,Dalien,China).PC RconditionsW~l’e
2minat96,foUowedby35cyclesof94℃for30e,60for30eand72for60flee.Chain elongationwaScontinuedafterthelaStcyclefor10min.ThePCRproductswe reparatedby5% denaturingpolyacrylamidegelelectmphoresisandvisualizedbysilverstaini ng.
1.3DNAquencing Toexactlydeterminethenumberandlength0frepeatsreprentingdifferenta lleles.PeRprod-
UatSredenedintopT-Advvector(ClentechLaboratories,Ine.,USA)andqu encedby蚰auto-
maticquencer(GenecoreInc.,Shanghai,China).
1.4Statisticalanalysis Allelefrequencieswerecalculatedfromthegenotypesofallsubjects.Hardy-WeinbergeqfIIilm-
rlumw聃examinedbytest.Differencesinthegenotypeandallelefrequenciesforpopulati on
groupswereassdbyFisher’xacttest.APvaluebelowO.05wasconsider edsignitleant.
2RemitsandI培9cⅡ
2.1Allelend@enotypes
1234Mb.Assho wedinFig.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 VSTRintheeNOSgeneweredetectedintheChinepopulat-
ien.Theallelecontainingsixof27-bprepeatsgaverito日PCRproductof471-bpwhichconsisted0f162?bpand
309一bpflankingquence.whereasthefiveandfour0fsuchre-
peat8yieldedabpanda417一bpproductconsisting0f135-
bpand108bprepeatndthe&meflankingquence?respc~.
tively
