CAJ | 학술논문

目的探讨一个遗传性凝血因子Ⅻ(coagulation factorⅫ,FⅫ)缺陷症家系的发病机制.方法对先证者及其家系成员的活化部分凝血活酶时间(activated partial thromboplastin time,APTT)、血浆FⅫ活性(FⅫprocogulant activity,FⅫ∶C)和血浆FⅫ抗原(FⅫantigen,FⅫ∶Ag)等进行表型诊断.用PCR技术对FⅫ基因的14个外显子及其侧翼序列进行扩增和测序.构建FⅫ基因突变体,瞬时转染COS7细胞,测定表达产物的FⅫ∶C的活性和FⅫ∶Ag含量.结果先证者APTT明显延长至108.1 s(正常参考值为27.0～41.0 s),其丈夫APTT在正常范围,儿子、女儿和外孙的APTT轻度延长.先证者FⅫ∶C和FⅫ∶Ag均极度降低至0.01(正常参考值为0.72～1.13),其丈夫、儿子、女儿和外孙的FⅫ∶C分别为0.57、0.24、0.14、0.16;FⅫ∶Ag分别为0.55、0.27、0.15、0.21.先证者和女儿FⅫ基因第9外显子均发现g.6800-6808del9bp杂合型缺失突变.先证者及儿子、女儿、外孙FⅫ启动子区46C/T多态性均为TT基因型,丈夫为CT型.体外表达结果显示,突变体在细胞裂解液中FⅫ∶Ag水平接近野生型,而在细胞培养上清液中的突变体FⅫ∶Ag、FⅫ∶C水平降至野生型的一半.结论该遗传性凝血因子Ⅻ缺陷症家系的先证者FⅫ基因第9外显子g.6800-6808del9bp为一新发现的突变.突变蛋白在细胞内大量蓄积,但存在分泌障碍.g.6800-6808del9bp和46T/T与FⅫ水平的降低有关,但不是先证者FⅫ水平极度降低的唯一原因.
목적 탐토일개유전성응혈인자Ⅻ(coagulation factorⅫ,FⅫ)결함증가계적발병궤제.방법 대선증자급기가계성원적활화부분응혈활매시간(activated partial thromboplastin time,APTT)、혈장FⅫ활성(FⅫprocogulant activity,FⅫ∶C)화혈장FⅫ항원(FⅫantigen,FⅫ∶Ag)등진행표형진단.용PCR기술대FⅫ기인적14개외현자급기측익서렬진행확증화측서.구건FⅫ기인돌변체,순시전염COS7세포,측정표체산물적FⅫ∶C적활성화FⅫ∶Ag함량.결과 선증자APTT명현연장지108.1 s(정상삼고치위27.0～41.0 s),기장부APTT재정상범위,인자、녀인화외손적APTT경도연장.선증자FⅫ∶C화FⅫ∶Ag균겁도강저지0.01(정상삼고치위0.72～1.13),기장부、인자、녀인화외손적FⅫ∶C분별위0.57、0.24、0.14、0.16;FⅫ∶Ag분별위0.55、0.27、0.15、0.21.선증자화녀인FⅫ기인제9외현자균발현g.6800-6808del9bp잡합형결실돌변.선증자급인자、녀인、외손FⅫ계동자구46C/T다태성균위TT기인형,장부위CT형.체외표체결과현시,돌변체재세포렬해액중FⅫ∶Ag수평접근야생형,이재세포배양상청액중적돌변체FⅫ∶Ag、FⅫ∶C수평강지야생형적일반.결론 해유전성응혈인자Ⅻ결함증가계적선증자FⅫ기인제9외현자g.6800-6808del9bp위일신발현적돌변.돌변단백재세포내대량축적,단존재분비장애.g.6800-6808del9bp화46T/T여FⅫ수평적강저유관,단불시선증자FⅫ수평겁도강저적유일원인.
Objective To analyze genetic mutation and molecular pathogenesis in a family affected with inherited coagulation factor Ⅻ (F Ⅻ) deficiency.Methods Activated partial thromboplastin time (APTT),FⅫ procoagulant activity (FⅫ ∶ C),FⅫ antigen (FⅫ ∶ Ag) and other coagulants were measured.For affected members of the family,exons 1-14 and flanking intronic regions of the FⅫ gene were amplified with polymerase chain reaction (PCR) and sequenced thereafter.Expression plasmids containing mutant FⅫ cDNA was constructed and transfected into COS7 cells transiently.Expressions of FⅫ ∶ Ag and FⅫ ∶ C were analyzed.Results The proband has manifested a prolonged APTT of 108.1 s (reference range:27.0-41.0 s).Her husband has a normal APTT.Other members of the family had slightly increased APTT.The FⅫ ∶ C and FⅫ ∶ Ag of the proband have both dropped to about 0.01 (reference range:0.72-1.13).The FⅫ ∶ C levels of her husband,son,daughter and grandchild were 0.57,0.24,0.14,0.16,respectively.And the FⅫ ∶ Ag levels in her husband,son,daughter and grandchild were 0.55,0.27,0.15,0.21,respectively.The proband and her daughter have both carried a heterozygous deletional mutation 6800-6808delAGCTGGGAG (6800-6808del9bp) in exon 9.For the promoter region of the FⅫgene,the genotypes of the proband,her son,daughter and grandchild was TT,whilst that of her husband was CT.Expression study has shown that,whilst the mutant F Ⅻ protein has accumulated in the cells similar to wild-type protein,its secretion has reduced approximately by half.Conclusion A novel deletional mutation 6800-6808de19bp has been identified in the FⅫ gene.Although mutant F Ⅻ protein can still accumulate in cells,its secretion has become insufficient.The 6800-6808del9bp mutation and 46T/T have both contributed to the pathogenesis of FⅫ deficiency in the family,but may have not been the sole cause.