中华血液学杂志
中華血液學雜誌
중화혈액학잡지
Chinese Journal of Hematology
2013年
3期
200-204
,共5页
谢海啸%吕美艳%杨小丽%王明山%张扬%朱丽青%金艳慧%杨丽红
謝海嘯%呂美豔%楊小麗%王明山%張颺%硃麗青%金豔慧%楊麗紅
사해소%려미염%양소려%왕명산%장양%주려청%금염혜%양려홍
凝血因子Ⅻ缺陷症%家系%聚合酶链反应%DNA突变分析
凝血因子Ⅻ缺陷癥%傢繫%聚閤酶鏈反應%DNA突變分析
응혈인자Ⅻ결함증%가계%취합매련반응%DNA돌변분석
Coagulation Factor Ⅻ deficiency%Pedigree%Polymerase chain reaction%Gene mutation
目的 探讨4个遗传性凝血因子Ⅻ(FⅫ)缺陷症家系基因突变及分子发病机制.方法 检测凝血酶原时间(PT)、APTT、FⅫ促凝活性(FⅫ∶C)和FⅫ抗原(FⅫ∶ Ag)等凝血指标;PCR扩增FⅫ基因的全部外显子及侧翼序列,PCR产物纯化后测序;在野生型pIRES2-EGFP FⅫ表达质粒上构建突变体FⅫ表达质粒,瞬时转染COS7细胞,测定细胞裂解液和培养上清中FⅫ∶Ag水平和FⅫ∶C.结果 4例先证者APPT均明显延长,FⅫ∶C均＜2％,FⅫ∶Ag亦同步下降至1％.4个家系均检出常见的FⅫ46C/T多态性.家系A先证者检出5741-5742delCA(His101Gln)及7142insertC(Lys346Gln)双重杂合突变;家系B先证者检出6800-6808de19bp杂合缺失突变.FⅫ6800-6808de19bp瞬时转染结果显示,在细胞裂解液中突变体FⅫ∶Ag为野生型的85.6％;在细胞培养上清中突变体FⅫ∶Ag为野生型的51.9％,FⅫ∶C为野生型的56.4％;家系C先证者检出8699G＞ A(Gly542Ser)杂合突变;家系D先证者检出8699G＞A(Gly542Ser)纯合突变(其父母为近亲结婚).结论 4个遗传性FⅫ缺陷症家系除有46C/T多态性外,共发现g.5741-5742delCA、g.7142insertC、g.6800-6808de19bp、g.8699G＞A 4种基因突变.其中g.5741-5742delCA和g.6800-6808de19bp为两种新的突变.FⅫ6800-6808de19bp体外表达显示,FⅫ6800-6808de19bp突变蛋白合成基本正常,但存在蛋白分泌障碍.
目的 探討4箇遺傳性凝血因子Ⅻ(FⅫ)缺陷癥傢繫基因突變及分子髮病機製.方法 檢測凝血酶原時間(PT)、APTT、FⅫ促凝活性(FⅫ∶C)和FⅫ抗原(FⅫ∶ Ag)等凝血指標;PCR擴增FⅫ基因的全部外顯子及側翼序列,PCR產物純化後測序;在野生型pIRES2-EGFP FⅫ錶達質粒上構建突變體FⅫ錶達質粒,瞬時轉染COS7細胞,測定細胞裂解液和培養上清中FⅫ∶Ag水平和FⅫ∶C.結果 4例先證者APPT均明顯延長,FⅫ∶C均＜2％,FⅫ∶Ag亦同步下降至1％.4箇傢繫均檢齣常見的FⅫ46C/T多態性.傢繫A先證者檢齣5741-5742delCA(His101Gln)及7142insertC(Lys346Gln)雙重雜閤突變;傢繫B先證者檢齣6800-6808de19bp雜閤缺失突變.FⅫ6800-6808de19bp瞬時轉染結果顯示,在細胞裂解液中突變體FⅫ∶Ag為野生型的85.6％;在細胞培養上清中突變體FⅫ∶Ag為野生型的51.9％,FⅫ∶C為野生型的56.4％;傢繫C先證者檢齣8699G＞ A(Gly542Ser)雜閤突變;傢繫D先證者檢齣8699G＞A(Gly542Ser)純閤突變(其父母為近親結婚).結論 4箇遺傳性FⅫ缺陷癥傢繫除有46C/T多態性外,共髮現g.5741-5742delCA、g.7142insertC、g.6800-6808de19bp、g.8699G＞A 4種基因突變.其中g.5741-5742delCA和g.6800-6808de19bp為兩種新的突變.FⅫ6800-6808de19bp體外錶達顯示,FⅫ6800-6808de19bp突變蛋白閤成基本正常,但存在蛋白分泌障礙.
목적 탐토4개유전성응혈인자Ⅻ(FⅫ)결함증가계기인돌변급분자발병궤제.방법 검측응혈매원시간(PT)、APTT、FⅫ촉응활성(FⅫ∶C)화FⅫ항원(FⅫ∶ Ag)등응혈지표;PCR확증FⅫ기인적전부외현자급측익서렬,PCR산물순화후측서;재야생형pIRES2-EGFP FⅫ표체질립상구건돌변체FⅫ표체질립,순시전염COS7세포,측정세포렬해액화배양상청중FⅫ∶Ag수평화FⅫ∶C.결과 4례선증자APPT균명현연장,FⅫ∶C균＜2％,FⅫ∶Ag역동보하강지1％.4개가계균검출상견적FⅫ46C/T다태성.가계A선증자검출5741-5742delCA(His101Gln)급7142insertC(Lys346Gln)쌍중잡합돌변;가계B선증자검출6800-6808de19bp잡합결실돌변.FⅫ6800-6808de19bp순시전염결과현시,재세포렬해액중돌변체FⅫ∶Ag위야생형적85.6％;재세포배양상청중돌변체FⅫ∶Ag위야생형적51.9％,FⅫ∶C위야생형적56.4％;가계C선증자검출8699G＞ A(Gly542Ser)잡합돌변;가계D선증자검출8699G＞A(Gly542Ser)순합돌변(기부모위근친결혼).결론 4개유전성FⅫ결함증가계제유46C/T다태성외,공발현g.5741-5742delCA、g.7142insertC、g.6800-6808de19bp、g.8699G＞A 4충기인돌변.기중g.5741-5742delCA화g.6800-6808de19bp위량충신적돌변.FⅫ6800-6808de19bp체외표체현시,FⅫ6800-6808de19bp돌변단백합성기본정상,단존재단백분비장애.
Objective To identify the genotype and pathogenesis in four Chinese pedigrees with Factor Ⅻ deficiency.Methods Activated partial thromboplastin time (APTT),F Ⅻ procoagulant activity (FⅫ∶ C),FⅫ antigen(FⅫ∶ Ag)and other coagulant parameters were detected.The FⅫ deficiency Pedigree members,all exons,boundary introns including the splice junctions of the FⅫ gene were amplified with Polymerase chain reaction (PCR).Expression plasmids were constructed by mutagenesis based on the wild-type and transfected into COS7 cells.FⅫ∶ C and FⅫ∶ Ag of the expression levels were tested in the supernatant and cell lysate.Results The four probands presented prolonged APTT with all the values of F Ⅻ∶ C and FⅫ∶ Ag were low to 2％ and 1％,respectively.There were common 46C/T polymorphism in the promoter regions of FⅫ gene in four pedigrees.Proband A was heterozygous for two mutations,g.5741-5742delCA (His101Gln) and g.7142insertC (Lys346Gln).Proband B was a heterozygous deletion mutation g.6800-6808de19bp.The results of the transfection revealed that FⅫ∶ Ag in cell lysates and conditioned media protein FⅫ6800-6808de19bp were 85.6％ and 51.9％.The FⅫ∶ C in the conditioned media was 56.4％.Proband C was a heterozygous mutation g.8699G ＞ A(Gly542Ser).Proband D was a homozygous mutation 8699G ＞ A,whose parents with consanguineous marriage.Conclusions Four mutations,g.5741-5742delCA,g.7142insertC,g.6800-6808de19bp and g.8699G ＞ A with 46C/T polymorphism in the promoter regions of FⅫ gene,were identified in the four Factor Ⅻ deficiency pedigrees.The two mutations g.5741-5742delCA and g.6800-6808de19bp were first found in China.FⅫ 6800-6808de19bp expressed in vitro suggested that almost normal proteinum synthesis but defect proteinum secretion.
