PCR-HRM分析筛查成骨不全一家系患儿基因突变.pdf

《PCR-HRM分析筛查成骨不全一家系患儿基因突变.pdf》由会员上传分享，免费在线阅读，更多相关内容在行业资料-天天文库。

1、天津医药2014年7)3第42卷第7期693新技术交流PCR—HRM分析筛查成骨不全一家系患儿基因突变白雪李克秋任秀智。何晓波王毅官士珍景亚青李光【摘要】目的采用PCR-高分辨率熔解曲线(HRM)分析筛查成骨不全(0I)一家系患JL(先证者)COL1A1基因突变位点，探讨其基因型与临床表型的联系。方法对先证者进行家族史及临床资料的调查，采集先证者、家属及50名正常对照者血液标本，应用PCR—HRM分析筛查先证者及正常对照者COL1A1基因突变，基因测序确证突变位点。结果先证者COL1A1基因17外显子筛查结果异常，其熔解温度(Tm)值比正常对照者Tm值低约0．4℃。先证者与正常对照者的标准熔解

2、曲线及差异熔解曲线均有明显差异。测序结果为c．1138G>A，突变导致380位氨基酸由甘氨酸(Gly)变成丝氨酸(Ser)：P．(Gly380Ser)，为错义突变。先证者父亲、祖母均具有相同突变位点。先证者母亲及正常对照者基因测序结果无此突变。该突变在中国人群中未见报道。该家系遗传特征为常染色体显性遗传，先证者临床诊断为Ⅳ型0I，临床表型较严重。结论PCR-HRM分析是有效的0I基因筛查新方法。COL1A1基因c．1138G>A突变在中国人群中为新发现的突变位点。d螺旋结构域Gly被替换可能导致较严重的临床表型。【关键词】成骨不全；遗传筛查；聚合酶链反应；点突变；系谱；COL1A1基因；高分辨

3、率熔解曲线分析【中图分类号】R394．112，R681．31【文献标志码】A【DOI】10．3969~．issn．0253—9896．2014．07．020PCR—HRMAnalysisforGeneMutationScreeninginaChildwithOsteogenesisImperfectaBAIXue，LIKeqiu，RENXiuzhi。，HEXiaobo，WANGYi‘，GUANShizhen，JINGYaqing2，LIGuang2JDepartmentofMedicalLaboratory，TianfinHospital，Tianjin300211，China；2Depart

4、mentofBiology,TianjinMedicalUniversity：3DepartmentofOrthopedicsWard,TianjiWuQingPeoplesHospitalLIGuang，E—mail：lig@tijmu．edu．en[Abstract】0bjectiveToinvestigateCOL1A1genemutationbyPCR—highresolutionmehing(PCR—HRM)andan—alyzethecorrelationbetweengenotypeandclinicalphenotypeinachild(proband)withosteogen

5、esisimperfecta(OI)．MethodsThefamilyhistoryofOIpedigreealongwiththeclinicaldatawascollected．Bloodsamplesfromtheprobandandhisfamilymembers，aswellas50normalcontrols，werecollected．ThemutationofCOL1A1genewasscreenedusingPCR—HRMandvalidatedbythegenesequence．ResultsThedetectionofPCR—HRMshowedtheabnormalres

6、ultofCOL1A117exoninprobandwithalowermeltingtemperature(Tm1valuethanthatofnormalcontrolsby0．4℃．Thereweresignifi—cantdifferencesinthestandardizationmeltingcurveandthedifferentmehingcurvebetweentheprobandandthenormalcontrols．Thesequencingresultwasc．1138G>A。whichmeantthatcDNAof1138baseGmutationintoA．The

7、mutationstransformedtheaminoacidglycineintoaserineataminoacid380(P．Gly380Ser)，whichresultedinmissensemutations．Theproband’sfatherandgrandmotherhadthesamemutationofCOLIA1gene．Themutationwasnotfoundinth