一个遗传性凝血因子Ⅻ缺陷症家系的表型与基因型分析

Analysis of the phenotype and genotype in a Chinese pedigree with congenital blood coagulation factor Ⅻ deficiency

目的对1个姨表近亲婚配的遗传性凝血因子Ⅻ(FⅫ)缺陷症家系进行实验室表型和F12基因突变分析,探讨其分子发病机制。方法检测先证者及其家系成员(共3代6人)血浆凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、凝血因子Ⅷ活性(FⅧ:C)、凝血因子Ⅸ活性(FⅨ:C)、凝血因子Ⅺ活性(FⅪ:C)、FⅫ活性(FⅫ:C)和FⅫ抗原(FⅪ:Ag)等指标,明确临床表型诊断。采用DNA直接测序法分析先证者F12基因所有外显子、侧翼、启动子区及家系成员相应的突变位点区域,并用反向测序证实所发现的突变。用ClustalX-2.1-win软件分析突变氨基酸的保守性;突变对蛋白质功能的影响则应用四个生物信息学评分软件(PolyPhen-2,PROVEAN,SIFT和MutationTaster)进行分析。结果先证者APTT(102.8s)、FⅫ:C(2%)和FⅪ:Ag(2%)明显异常;家系其他成员APTT正常,其姐姐、大女儿、二女儿和外孙的FⅫ:C和FⅫ:Ag均减低为正常对照的一半左右,表现为交叉反应物质(CRM)阴性型。基因测序发现先证者F12基因第13号外显子存在c.1556T>G纯合突变,导致p.Leu519Arg;其姐姐、大女儿、二女儿和外孙均存在c.1556T>G杂合突变。保守性分析结果显示Leu519在同源物种间呈高度保守;四个生物信息学软件对该突变预测的评分结果均显示为有害突变。结论F12基因13号外显子区p.Leu519Arg突变是导致该家系遗传性FⅫ缺陷症发病的原因;推测先证者纯合p.Leu519Arg突变基因分别遗传自近亲结婚的父母。

Objective:To analyze the phenotype and genotype in a Chinese pedigree with congenital blood coagulation factorⅫdeficiency.Methods:Coagulant parameters were assayed.Exons 1-14,exon-intron boundaries and promoter region of FⅫgene of the proband and other family members were analyzed by direct sequencing.The detected mutations were confirmed by sequencing the complementary strand.The ClustalX-2.1-win was used for the conservatism of the mutational amino acids,while the influence of the mutationsonthe protein functions were analyzed by four online bioinformatics softwares(PolyPhen-2,PROVEAN,SIFT and MutationTaster).Results:The proband had prolonged APTT(102.8 s),and his FXⅡ:C and FXⅡ:Ag were decreased to 2%and 2%.Other members’APTT were in normal range.The FⅫactivity and antigen of his sister,elder daughter,second daughter and grandson were decreased to the half of normal value,called cross-reacting material-nagative groups.After the amplification and sequencing of 14 extrons in FⅫ,c.1556 T>G homozygotic mutation in exon 13,which results in p.Leu519 Arg,was found in the proband,while c.1556 T>G heterozygous mutation was in his sister,elder daughter,second daughter and grandson.Leu519 is highly conserved,while p.Leu519 Arg is a harmful mutation which can cause disease by bioinformatic analysis.Conclusions:The mutation of Leu519 Arg on exon 13 is in relation with the congenital FⅫdeficiency in this Chinese pedigree which most likely due to the consanguineous marriage of the proband’s parents.