F12基因复合杂合变异致遗传性FⅫ缺陷症一个家系的遗传学分析

Genetic analysis of a Chinese pedigree with Hereditary FⅫdeficiency due to compound heterozygous variants of F12 gene

目的对1个遗传性凝血因子Ⅻ(FⅫ)缺陷症家系进行实验室表型和基因变异分析,初步探讨其分子发病机制。方法选取2021年7月17日因"慢性胃炎"就诊于宁波市第二医院的1例遗传性FⅫ缺陷症男性先证者及其家系成员(共3代7人)作为研究对象。检测先证者及其家系成员凝血酶原时间(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(FⅫ:Ag)等指标以明确诊断。采用DNA直接测序分析F12基因的所有外显子、侧翼、5′和3′非翻译区及家系成员相应的变异位点区域,用克隆测序进一步证实所发生的变异。用生物信息学软件分析变异对蛋白功能的影响。结果先证者为47岁男性,APTT为180.0 s,明显延长,FⅫ:C和FⅫ:Ag均<1.0%,极度降低。先证者父亲、母亲、弟弟、大儿子及小儿子FⅫ:C和FⅫ:Ag均有不同程度降低。基因分析显示先证者F12基因第10外显子存在c.1092_1093insC(p.Lys365Glnfs*69)杂合插入变异及第14外显子存在c.1792_1796delGTCTA(p.Val579Hisfs*32)杂合缺失变异,c.1092_1093insC为已报道的致病性变异。其母亲和大儿子携带c.1092_1093insC杂合插入变异,父亲、弟弟和小儿子携带c.1792_1796delGTCTA杂合缺失变异。第1外显子启动子区46C/T多态性分析显示先证者、大儿子和小儿子为46T/T型,其余人均为46C/T型。生物信息学软件分析第579位氨基酸残基为高度保守的位点,变异后增加了一个苯环及周围氨基酸的氢键发生了改变。根据美国医学遗传学与基因组学学会相关遗传变异分类标准和指南,c.1792_1796delGTCTA变异评级为致病性变异(PVS1+PM2_Supporting+PM4)。结论F12基因母源的c.1092_1093insC(p.Lys365Glnfs*69)杂合插入变异及父源的c.1792_1796delGTCTA(p.Val579Hisfs*32)杂合缺失变异可能是该家系FⅫ水平降低的原因。上述发现丰富了FⅫ缺乏症的基因-表型谱。

Objective To explore the laboratory phenotype and molecular pathogenesis in a Chinese pedigree affected with Hereditary coagulation factorⅫ(FⅫ)deficiency.Methods A male proband admitted to Ningbo No.2 Hospital on July 17,2021 due to chronic gastritis and members of his pedigree(7 individuals from three generations)were selected as the study subjects.Prothrombin time(PT),activated partial thromboplastin time(APTT),FⅧactivity(FⅧ:C),FⅨactivity(FⅨ:C),FⅪactivity(FⅪ:C),FⅫactivity(FⅫ:C),and FⅫantigen(FⅫ:Ag)were determined.All of the exons,exon-intronic boundaries,as well as the 5′-and 3′-untranslated regions of the F12 gene were subjected to Sanger sequencing.Candidate variants were verified by cloning sequencing.The effect of candidate variants on the protein function was analyzed by bioinformatics software.Results The proband,a 47-year-old male,had significantly prolonged APTT(180.0 s)and decreased FⅫ:C and FⅫ:Ag levels(<1%).His father,mother,brother and two sons also showed certain degrees of reduction.Genetic testing revealed that the proband has harbored compound heterozygous variants of the F12 gene,namely c.1092_1093insC(p.Lys365Glnfs*69)in exon 10 and c.1792_1796delGTCTA(p.Val579Hisfs*32)in exon 14.His mother and elder son were heterozygous for the c.1092_1093ins variant,whilst his father,brother,and younger son were heterozygous for the c.1792_1796delGTCTA variant.Analysis of the promoter region of exon 1 also showed that the proband and both sons had harbored a 46T/T polymorphism,whilst other family members were 46C/T.Bioinformatic analysis suggested that the p.Val579 is a highly conserved site.Protein model analysis showed that,with the p.Val579Hisfs*32 variant,a benzene ring was added and the hydrogen bond of surrounding amino acids was changed.Based on the guidelines from the American College of Medical Genetics and Genomics,the c.1792_1796delGTCTA was classified as a pathogenic variant(PVS1+PM2_Supporting+PM4).Conclusion The c.1092_1093insC(p.Lys365Glnfs*69)and c.1792_1796delGTCTA(p.Val579Hisfs*32)compound heterozygous variants of the F12 gene probably underlay the decreased FⅫlevels in this pedigree.Above finding has also enriched the mutational spectrum for FⅫdeficiency.