59例地中海贫血患者的基因诊断研究

©1994-2010ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.()2009115912,3312,32,32,32,3(1.;2.;3.,650031),()PCRPCR2RFLPPCR2RDB,11230,4;29,6;52.7%10,HbH2CS1,1,2PCRPCR2RFLPPCR2RDB,DNA,;;;Astudyonthegenediagnosisofthalassemiasin59casesWenJie,ZhuBao2sheng,LiuPei2ling,HeJing,TangXin2hua,SuJie,ChenHong.(GeneticDiagnosisCenter,AffiliatedKunhuaHospitalofKunmingMedicalCollege,TheFirstPeopleHospitalofYunnanProvince,YunnanProvincialKeyLaboratoryforBirthDefectsandGeneticDisorders.Kunming650032,P.R.China)Correspondingauthor:ZhuBao2sheng.E2mail:[e-mail]uptechniqueswhichcouldbeusedfordetectingcommondeletionalandnon2deletionalmutationsof2globingeneand2globingeneinsouthernChinesepopulations.Toevaluatethesemethodsinclinicalapplicationandtoexplorethegenemutationtypesofboth2and2thalassemiasinYunnanpopulations.MethodsMultiplexpolymerasechainreaction(mPCR),PCR2RFLPandPCR2reversedotblothybridization(PCR2RDB)wereperformancedforthegeneticdiagnosisof2or2thalassemias.ResultsAmong112caseswhoweresuspectedofthalassemias,30cases2globingenedeletionsandmutationsweredetected,including4genotypes;29casesof2globingenemutaionsweredetected,including6genotypes;commonmutationsaccountedfor52.7%inthetestedgroup.Prenataldiagnosisweresuppliedto10coupleswhosefetuseswereatrisk,andrevealedonecaseofintermediate2thalassemia(HbH2CSpatient),onecaseofmild2thalassemia,twocasesof2thalassemiaheterozygote.ConclusionmPCR,PCR2RFLP,andPCR2RDBcanmakearapidandaccurategenetestindetectionof2or2thalassemiaalthough,itseemsthatsomecasescouldbemissed.IfDNAsequencingiscomplementallyusedinsuchcases,thesetechniquesmayhavegoodvaluestoclinicalroutinetest.Keywords2thalassemia;2thalassemia;Geneticdiagnosis;Prenataldiagnosis3:,[e-mail]:(30760241);(2007CA008)9©1994-2010ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.()200911(),,,,,,,,(),[1,2],,,,[2],11.1200320092,1127,HbH2,101,21,256,31[35]:(MCV)80fl(MCHC)27pg;HbA23.5%/HbF2%,;HbA22.5%,;(HbH);1.21.2.1:2ml,EDTA,DNA,4:1722,10ml,DNA1.2.21.2.2.1PCR3:22SEA()23.7()24.2()4lDNAmixPCR(),PCR:9615min;9845s,641min30s,723min,35;725min8l1.2%,1800bp,2000bp,1600bp,1300bp23.724.222SEA1.2.2.2PCR2RFLPHbConstantSpring(HbCS)[6](53):P1ACCGTGCTGACCTCCAAATACC,P2GTCTGAGACAGGTAAACACCTCPCR:25lPCR,10PCRBuffer2.0l,25mMMgCl21.5l,2.5mMdNTPs1.5l,5MPrimer1.0l,5U/lTaq0.2l,DNA3lPCR:945min-(9430s-601min-721min)30-728min206bpTru91,652lPCR10l,6%52TTAA23,Tru91,Tru91181bp25bp(25bp,);HbCS(2TermTC)52TCAA23,,PCR206bpPCR01©1994-2010ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.()2009111.2.3PCR/(PCRRDB),17(89)18:CD4142(2TCCT)IVS22nt654CT228AGCD7172(+A)CD17ATCD26GACD31(2C)CD2728(+C)CD43GT232CA229AG230TCCD1415(+G)CAPIntIVS21nt1(GA,GT)IVS21nt5GC2lDNAmixPCR(),PCR:5015min-9510min-(941min-5530s-7230s)35-725min18(ASO)22.130,2,1/23.71/24.2;22,21/22SEA1CS/;5HbH,223.7/22SEA224.2/22SEA1CS/22SEA;1HbBarts(),22SEA/22SEA2HbCS2TAACAA1130(bp)(%)()/22SEA1800/13002170HbH23.7/22SEA2000/130026.7HbH24.2/22SEA1600/130026.7HbHCS/22SEA1800/130013.3HbBart22SEA/22SEA130013.3()3.7/2000/180013.3()/24.21800/160013.3()CS/180013.3--301002.229,6,1,CD17/CD26;1,228/CD26;27,14CD17/N3CD4142/N3CD7172/N3IVS222654/N2228/N2CD26/N22.310,2,HbH,HbCS,CS/22SEA,,18;,/22SEA,;2,2(),CD17/N228/N,8,,23229(%)CD17(AT)0/A1448.3CD7172(+A)0/A310.3CD4142(2TCCT)0/A310.3IVS222654(CT)0/A310.3CD26(GA)+/A26.9-28(AG)+/A26.9CD17(AT)/CD26(GA)0/+()13.4-28(AG)/CD26(GA)+/+()13.4--2910011©1994-2010ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.()2009113101HbHCS/22SEACS/23.7/22SEA2()/22SEA/22SEA/22SEA22SEA/22SEA()3//22SEA23.7//22SEA4//22SEA/22SEA5//24.2/22SEA6CD17()CD17/NCD17/NCD17/N7-28()-28/NCD7172/N-28/N8A/ACD17/NCD26/NCD17/CD269A/ACD17/NCD7172/N10A/AIVS222654/NCD4142/N1,2,3,4:()1PCR:M1kbMarker,123,1,1800bp,/();3,2000bp/1300bp,23.7/22SEA;2,1800bp/1300bp,/22SEA();4,1300bp,22SEA/22SEA;5,1800bp/1300bp,/22SEA2PCR2RFLPHbCS:M100bpMarker,Tru91,206bp;Tru91:11,181bp,HbCS;2,206bp,HbCS;3,206bp/181bp,HbCS3PCR2RFLPHbCS:,TAA;,TAACAA;,TAACAA3PCRPCR2RFLPPCR2RDB,,11259,5321©1994-2010ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.()200911:,,;,,DNA,,4:,CD17AT/A;,CD26GA/A;,CD17AT/CD26GA;,,A/A,95%,[7,8],22SEA23.724.23[9,10]PCR,HbBarts,HbH30,22SEA27,90%(27/30),,[6],HbConstantSpring(HbCS)HbQuongSze(HbQS)[11]/22SEA,HbHHbH,,HbHHbH[12],HbH2CSHbBartsPCR,PCR2RFLPHbCS(CS),;PCR2RFLP1HbH2CS(CS/22SEA),18,,6[2],CD4142(41.6%),IVS22nt654(21.8%),CD17(18.0%),-28(8.0%),CD7172(3.9%),-29(1.2%),94.5%,[13]52,29,-29,5,CD17,51.7%(15/29)[13]CD26(41.35%),,,,PCR2RDB18,,,PCR2SSCP,,32,21[14],,,,PCR31©1994-2010ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.()200911DNA[1].[J].,1996,13(5):2572258.[2],.[J].,1995,18(3):1332136.[3].[J].,2002,24(4):4872490.[4],.[M].3.:,2007.29235,40241.[5]LeungT,LauTK,ChungTKh.Thalassaemiascreeninginpregnancy[J].CurrOpinObstetGynecol,2005,17(2):1292134.[6],,,.[J].,2003,11(1):54260.[7]HiggsDR,VickersMA,WilkieAOM,etal.Ar