纯合子p.Gly1715Ser导致的遗传性凝血因子Ⅴ缺陷症家系分析  

A novel homozygous mutation(Gly1715Ser) causing hereditary factor V deficiency in a Chinese patient

作　　者：余芳 戴青云 郑淑芳 章勇[1] 周益琴[1] YU Fang;DAI Qingyun;ZHENG Shufang;ZHANG Yong;ZHOU Yiqin(Jinhua People’s Hospital,Jinhua,Zhejiang 321000,China)

机构地区：[1]金华市人民医院,浙江金华321000

出　　处：《中国优生与遗传杂志》2022年第10期1822-1827,共6页Chinese Journal of Birth Health & Heredity

摘　　要：目的 对一个由近亲婚配引起的遗传性凝血因子Ⅴ(FⅤ)缺陷症家系进行实验室表型检测与基因突变分析,初步探讨其分子发病机制。方法 检测先证者及其家系成员(3代6名成员)血浆凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、纤维蛋白原(FIB)、血浆FⅤ活性(FⅤ:C)、FⅤ抗原(FⅤ:Ag)及其他相关凝血指标进行表型诊断。通过CAT法检测凝血酶生成量。采用DNA直接测序法分析先证者F5基因的所有外显子、侧翼、5’和3’非翻译区及其家系成员相应突变位点区域,发现突变位点用反向测序证实。通过在线生物信息学软件(包括Mutation Taster、PROVEAN、SIFT及PolyPhen-2)预测和分析突变的致病性;用ClustalX-2.1-win分析突变氨基酸的保守性;利用Swiss-Pdb Viewer构建蛋白模型分析突变对蛋白质空间构象及功能的影响。结果 先证者PT和APTT均显著延长,分别为26.3 s/13.2 s和73.5 s/36.0 s;FⅤ:C和FⅤ:Ag明显降低,分别为3%和7%。先证者凝血酶生成试验峰高明显降低,延迟时间和达峰时间均显著延长,凝血酶生成潜力显著下降。基因分析显示先证者F5第16外显子存在c.5227G>A纯合错义突变(p.Gly1715Ser),其父亲、母亲、儿子和女儿均携带p.Gly1715Ser杂合子。保守性分析结果表明p.Gly1715Ser在同源物种间高度保守;PolyPhen-2、Mutation Taster、PROVEAN和SIFT四个软件对p.Gly1715Ser突变预测结果一致,均提示该突变为有害突变,可影响蛋白质功能。蛋白模型分析结果表明,p.Ser1715突变为p.Gly1715后,与周围氨基酸之间氢键发生改变并形成新的分子间作用力,使FⅤ蛋白空间结构发生改变。结论 该先证者F5基因第16外显子存在c.5227G>A纯合错义突变遗传自近亲婚配的父母,且c.5227G>A突变与该家系FⅤ水平降低有关。Objective To explore the molecular pathogenesis by detecting the phenotype and genotype of a family with hereditary factor V deficiency caused by consanguineous marriage. Methods Detecting the coagulant parameter of prothrombin time(PT), activated partial thromboplastin time(APTT), fibrinogen(FIB), FV procoagulant activity(FV: C) and FV antigen(FV: Ag) in proband and his family members(6 members of 3 generations). The CAT measurement were used to detect the amount of thrombin produced. DNA direct sequencing was taken to analyse all exons, exon-intron boundaries, 5’and 3’ untranslated regions and the relevant mutation site regions of this family members of the F5 gene. The suspected mutation was confirmed by reverse sequencing. PolyPhen-2, SIFT, Mutation Taster, and PROVEAN these four different online bioinformatics softwares were utilized to speculate and analyze the effect of the mutation on protein structure and function.ClustalX-2.1-win, a multiple sequence alignment software was employd to analyze the conservatism of mutant amino acids in other 9 homologous species. Simultaneously, Swiss-Pdb Viewer software was used to assess local structural effects of the mutation in F5 gene and investigate the exact type and position of amino acid substitution in the three-dimensional structure model. Results The PT and APTT of the proband were significantly longer, 26.3 s/13.2 s and 73.5 s/36.0 s respectively. FV: C and FV: Ag of the prohand were extremely reduced to 3% and 7%, respectively. The proband had markedly decreased thrombin with the prolonged lag time, time to peak and signifificantly reduced peak height. Genetic analysis demonstrated that the proband existed a homozygous point mutation c.5227G>A in exon 16 which caused a substitution of glycine(Gly) to serine(Ser) at codon 1715. His father(II-1), mother(II-2), son(IV-1) and daughter(IV-2) all carried the same heterozygous point mutation c.5227G>A with the proband. The results of the multiple sequence alignment showed that Gly1715 was in a highly conserved

关 键 词：凝血因子Ⅴ缺陷症 生物信息学 近亲婚配 基因突变 

分 类 号：R596.1[医药卫生—内科学]