Guo Y L, Kong W Z, Wan Q, Zheng W J Y, Xi J Q, Liu S Q, Wang M S, Jin Y H
Laboratory Department of Wenzhou Chinese Medicine Hospital, Wenzhou 325000, China.
Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China.
Zhonghua Xue Ye Xue Za Zhi. 2021 Feb 14;42(2):135-139. doi: 10.3760/cma.j.issn.0253-2727.2021.02.008.
To explore the molecular pathogenesis of a family with hereditary factor Ⅴ (FⅤ) deficiency. All the exons, flanking sequences, 5' and 3' untranslated regions of the F5 of the proband, and the corresponding mutation sites of the family members were analyzed via direct DNA sequencing. The CAT measurement was used to detect the amount of thrombin produced. The ClustalX software was used to analyze the conservation of mutation sites. The online bioinformatics software, Mutation Taster, PolyPhen-2, PROVEAN, LRT, and SIFT were applied to predict the effects of mutation sites on protein function. The Swiss-PdbViewer software was used to analyze the changes in the protein model and intermolecular force before and after amino acid variation. The proband had a heterozygous missense mutation c.1258G>T (p.Gly392Cys) in exon 8 of the F5, and a heterozygous deletion mutation c.4797delG (p.Glu1572Lys fsX19) in exon 14, which results in a frameshift and produces a truncated protein. Her grandfather and father had p.Gly392Cys heterozygous variation, whereas her maternal grandmother, mother, little aunt, and cousin all had p.Glu1572LysfsX19 heterozygous variation. The ratio of proband's thrombin generation delay to peak time was significantly increased. Conservation analysis results showed that p.Gly392 was located in a conserved region among the 10 homologous species. Five online bioinformatics software predicted that p.Gly392Cys was pathogenic, and Mutation Taster also predicted p.Glu1572Lys fsX19 as a pathogenic variant. Protein model analysis showed that the replacement of Gly392 by Cys392 can lead to the extension of the original hydrogen bond and the formation of a new steric hindrance, which affected the stability of the protein structure. The c.1258G>T heterozygous missense mutation in exon 8 and the c.4797delG heterozygous deletion mutation in exon 14 of the F5 may be responsible for the decrease of FⅤ levels in this family.
探索一个遗传性因子Ⅴ（FⅤ）缺乏症家族的分子发病机制。通过直接DNA测序分析先证者F5的所有外显子、侧翼序列、5'和3'非翻译区，以及家族成员的相应突变位点。采用凝血酶生成检测法（CAT）检测产生的凝血酶量。使用ClustalX软件分析突变位点的保守性。应用在线生物信息学软件Mutation Taster、PolyPhen-2、PROVEAN、LRT和SIFT预测突变位点对蛋白质功能的影响。使用Swiss-PdbViewer软件分析氨基酸变异前后蛋白质模型和分子间力的变化。先证者F5第8外显子存在杂合错义突变c.1258G>T（p.Gly392Cys），第14外显子存在杂合缺失突变c.4797delG（p.Glu1572Lys fsX19），导致移码并产生截短蛋白。她的祖父和父亲有p.Gly392Cys杂合变异，而她的外祖母、母亲、小姨和表弟都有p.Glu1572LysfsX19杂合变异。先证者凝血酶生成延迟与峰值时间的比值显著增加。保守性分析结果显示，p.Gly392位于10个同源物种的保守区域。五个在线生物信息学软件预测p.Gly392Cys具有致病性，Mutation Taster也预测p.Glu1572Lys fsX19为致病变异。蛋白质模型分析表明，Cys392取代Gly392可导致原氢键延长并形成新的空间位阻，影响蛋白质结构的稳定性。F5第8外显子的c.1258G>T杂合错义突变和第14外显子的c.4797delG杂合缺失突变可能是该家族FⅤ水平降低的原因。
