Objective: To investigate the clinical phenotype and molecular pathogenic mechanism of a hereditary coagulation factor V deficiency (FⅤD) family.
Methods: A phase I assay was used to measure coagulation factors II, V, VII, VIII, IX, X, Ⅺ, Ⅻ (FⅡ∶C, FⅤ∶C, FⅦ∶C, FⅧ∶C, FⅨ∶C, FⅩ∶C, FⅪ∶C, FⅫ∶C), activated partial thromboplastin time (APTT) and prothrombin time (PT) to determine the clinical phenotype and molecular pathogenesis of F VD. Prothrombin time (PT) were used for phenotypic identification; high-throughput exome sequencing was applied to screen the whole gene variants, and Sanger sequencing was used to verify the suspected variants in F5 gene; MutationTaster, PolyPhen-2 bioinformatics software was used to predict the pathogenicity of the variants, ClustalX software was used to analyze the amino acid conservatism, and PyMol software was used to simulate the model of the mutant protein.
Results: The pre-documented patient had significantly prolonged PT and APTT, FⅤ∶C was only 5.45%, and there was no significant abnormality in TT, FIB and the rest of the coagulation factors. The mother, father and sister of the proband had prolonged PT and APTT, and FⅤ∶C was reduced to different degrees. Genetic testing revealed the presence of a c.286G>C (p.Asp96His) pure missense variant in exon 3 of F5 in the prior witness, and a c.286G>C (p.Asp96His) heterozygous missense variant in father, mother, and sister of the proband. Bioinformatics analysis suggested that p.Asp96His was a pathogenic variant, and the associated amino acid site was highly conserved among 10 species. Protein simulation showed that the mutation of Asp96 to His96 could lead to the disappearance of the original hydrogen bond and the change of the distance, destroying the original hydrogen bond interaction force and affecting the stability of the protein structure.
Conclusion: The F5 exon 3 c.286G>C (p.Asp96His) missense variant may have contributed to the reduction of FⅤ∶C in the preexisting individual and family members, as well as being the genetic etiology of coagulation factor V deficiency.
题目: 一个遗传性凝血因子Ⅴ缺陷症家系的临床表型及分子致病机制研究.
目的: 探讨一个遗传性凝血因子Ⅴ缺陷症（coagulation factor V deficiency，FⅤD）家系的临床表型及分子致病机制。.
方法: 采用一期法测定凝血因子Ⅱ、Ⅴ、Ⅶ、Ⅷ、Ⅸ、Ⅹ、Ⅺ、Ⅻ（FⅡ∶C、FⅤ∶C、FⅦ∶C、FⅧ∶C、FⅨ∶C、FⅩ∶C、FⅪ∶C、FⅫ∶C）、活化部分凝血活酶时间（activated partial thromboplastin time，APTT）及凝血酶原时间（prothrombin time，PT）进行表型鉴定；应用高通量外显子测序筛查全基因变异，Sanger测序验证 F5 基因可疑变异；利用MutationTaster、PolyPhen-2生物信息学软件预测变异致病性、ClustalX软件分析氨基酸保守性和PyMol软件模拟变异蛋白模型。.
结果: 先证者PT、APTT明显延长，FⅤ∶C仅为5.45%，TT、FIB及其余凝血因子均无明显异常。其母亲、父亲、姐姐的PT、APTT均延长，FⅤ∶C不同程度减低。基因检测显示先证者 F5 第3号外显子存在c.286G>C（p.Asp96His）纯合错义变异，其父亲、母亲、姐姐均存在c.286G>C（p.Asp96His）杂合错义变异。生物信息学分析提示p.Asp96His为致病变异,相关的氨基酸位点在10个物种中高度保守。蛋白模拟显示，Asp96变异为His96后可导致原有氢键消失和距离改变，破坏了原有的氢键相互作用力，影响蛋白结构的稳定性。.
结论: F5 第3号外显子c.286G>C（p.Asp96His）错义变异可能导致了先证者及家系成员FⅤ∶C的减低，也是引起凝血因子Ⅴ缺陷症的遗传学病因。.
Keywords: hereditary coagulation FⅤ deficiency; pedigree; missense mutation.