目的 探讨姨表近亲婚配的遗传性凝血因子Ⅻ(FⅫ)缺陷症家系的发病机制.方法家系调查.2015年2月于温州医科大学温州市第三临床学院收治1例遗传性FⅫ缺陷症患者.经用活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、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(FⅫ:Ag)测定进行表型诊断.用DNA直接测序法分析先证者F12基因所有14个外显子、侧翼、5′和3′非翻译区及家系成员相应的突变位点区域,用反向测序证实所发生的突变.采用ClustalX-2.1-win软件分析突变氨基酸的保守性,同时采用四个生物信息学评分软件(PolyPhen-2,PROVEAN,SIFT和MutationTaster)分析突变对蛋白质功能的影响.结果 先证者及其弟弟APTT 明显延长,分别为116.4 s 和101.3 s;先证者父亲APTT略延长,为48.8 s,家系其他成员APTT无明显延长;先证者及其弟弟、父亲的FⅫ:C明显减低,分别为2.0%、2.0%和18.0%,FⅫ:Ag含量分别为1.0%、1.0%、和13.0%,表现为交叉反应物质(CRM)阴性.基因测序发现先证者及其弟弟的F12基因启动子区为46T/T型及14号外显子存在c.1681G>A(p.Gly542Ser)纯合突变;先证者父亲、母亲及儿子均存在c.1681G>A杂合突变,其中先证者父亲表现为46T/T型,其余二者均为46C/T型;先证者丈夫启动子区为C/C型,且无上述基因突变.ClustalX-2.1-win软件保守性分析结果表明,Gly542在同源物种间高度保守.四个生物信息学软件对该突变的预测结果一致:Polyphen-2评分(1.000分)、PROVEAN评分(-4.975分)均预示为有害突变;SIFT评分(0.00分)预示可影响蛋白质功能;Mutation Taster评分(0.999分)预示可引起相应疾病.结论 c.1681G>A(p.Gly542Ser)和46T/T与该遗传性FⅫ缺陷症家系FⅫ水平明显降低有关.%Objective To analyze the mutations of F12 gene in one pedigree with congenital factor FⅫ(FⅫ)deficiency, and investigate the molecular mechanisms of FⅫ deficiency.Methods Pedigree investigation.In February 2015,a patient with hereditary FⅫdeficiency was admitted to the Third Clinical College of Wenzhou Medical University.Activated partial thromboplastin time(APTT), prothrombin time (PT),FⅫactivity(FⅫ:C),FⅫ antigen(FⅫ:Ag)and other coagulant parameters were tested in the proband and his family members.5′and 3′UTR, all exons and their exon-intron boundaries of F12 gene were analyzed by direct sequencing.The detected mutations were confirmed by reverse sequencing.The conserved amino acids were analyzed by ClustalX-2.1-win software, and four bioinformatics softwares (PolyPhen-2,PROVEAN,SIFT and MutationTaster)were also used to analyze the effect of mutations on protein function.Results The proband and her younger brother showed a markedly prolonged APTT which were 116.4 s and 101.3 s, while her father had slightly prolonged APTT, and other family members were normal.The FⅫ:C and FⅫ:Ag of family members were also decreased(the proband,2.0% and 1.0%;her younger brother,2.0% and 1.0%; her father,18.0% and 13.0%).The phenotype of all members was consistent with cross-reactive material(CRM)negative.Nucleotide sequencing analysis showed that the proband and her younger brother had missense mutations in the F 12 gene, including one homozygous mutation c.1681G>A(p.Gly542Ser)and a commonly reported single nucleotide polymorphism site within the promoter region of the F12 gene(46T/T).Sequencing results from the proband's parents and son demonstrated them as carriers of a heterozygous missense mutation.The proband's husband was normal and with 46C/C in the promoter region.The ClustalX-2.1-win results indicated that the Gly542 was highly conserved among the homologousspecies.The predicting outcomes of the four bioinformatics softwares were the same,the PolyPhen-2(score 1.000)and PROVEAN(score -4.975)both declared p.Gly542Ser was a harmful mutation.The SIFT(score 0.00)and the MutationTaster(score 0.999)manifested the mutation could affect the protein funtion.Conclusions c.1681G>A(p.Gly542Ser)in exon 14 and 46T/T were related with the significant decrease of the FⅫlevel of this pedigree of hereditary FⅫ deficiency.
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