两个复合杂合突变导致遗传性凝血因子V缺陷症家系的表型与基因突变分析  

Phenotype and gene mutation analysis of two compound heterozygous mutations resulting in families with inherited coagulation factor V deficiency

作　　者：郑周[1] 徐琦煜[1] 周星星[1] 王明山[1] 谢耀盛[1] ZHENG Zhou;XU Qiyu;ZHOU Xingxing;WANG Mingshan;XIE Yaosheng(Center of Laboratory Medicine,the First Affiliated Hospital of Wenzhou Medical University,Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province,Wenzhou 325015,China)

机构地区：[1]温州医科大学附属第一医院医学检验中心,浙江省检验诊断及转化研究重点实验室,浙江温州325015

出　　处：《温州医科大学学报》2023年第12期947-953,共7页Journal of Wenzhou Medical University

基　　金：温州市基础性科研项目(Y20190121);浙江省检验诊断及转化研究重点实验室(2022E10022)。

摘　　要：目的:对两个由F5基因复合杂合突变导致的遗传性凝血因子V(FV)缺陷症家系进行表型和基因型分析,初步探讨其分子致病机制。方法:检测两个家系各成员外周血的血浆FV活性(FV:C)和FV抗原(FV:Ag)等凝血指标。通过PCR扩增F5基因的所有外显子及其侧翼区域,并进行测序。利用生物信息学软件辅助分析突变对蛋白功能的影响。使用PyMOL软件分析突变前后FV蛋白质的空间结构变化。通过凝血酶生成实验评估突变蛋白的功能变化。结果:表型检测显示两个先证者FV:C和FV:Ag均同步下降,表现为I型FV缺陷症。基因分析显示,先证者A第3外显子存在c.332G>T杂合错义突变(p.Ser111Ile)及第25外显子存在c.6665A>G杂合多态性(p.Arg2222Gly);先证者B第3外显子存在c.286G>C杂合错义突变(p.Asp96His)及第13外显子存在c.2393-2393del C杂合缺失突变(p.Pro798Leufs*13)。生物信息学分析显示,p.Ser111Ile和p.Pro798Leufs*13突变均为致病性突变。蛋白模型分析显示,p.Ser111Ile突变导致氨基酸间的氢键发生改变;p.Pro798Leufs*13突变产生了截断蛋白。凝血酶生成实验表明,先证者A和B的凝血功能已受到影响。结论:这四种突变可能是造成该两个家系FV水平下降的主要原因,其中p.Ser111Ile突变鲜见报道。Objective:To analyze the coagulation index and genotype of two patient with hereditary coagulation Factor V(FV)deficiency by compound heterozygous mutations and their family members,and to explore their molecular pathogenesis.Methods:The blood coagulation indexes such as Plasma FV activity(FV:C)and FV antigen(FV:Ag)in the peripheral blood of each family member were measured by the one-stage clotting method.All exons and their flanking regions of the F5 gene were amplified by PCR and then sequenced.The impact of mutations on protein function was analyzed by Bioinformatics software.The spatial structure changes of FV proteins were analyzed by PyMOL software before and after mutations.The function of the mutant protein was analyzed by the Calibrated automated thrombogram.Results:Phenotypic testing showed that both probands had a decrease in FV:C and FV:Ag simultaneously,manifesting as type I of the FV deficiency.Genetic analysis showed that the proband A had c.332G>T heterozygous missense mutation(p.Ser111Ile)in exon 3 and c.6665A>G heterozygous polymorphism(p.Arg2222Gly)in exon 25;the proband B had c.286G>C heterozygous missense mutation(p.Asp96His)in exon 3 and c.2393-2393delC heterozygous deletion mutation(p.Pro798Leufs*13)in exon 13.Bioinformatics analysis showed the p.Ser111Ile mutation and the p.Pro798Leufs*13 mutation were pathogenic mutations.Protein model analysis showed that p.Ser111Ile mutation could affect the structure of hydrogen bonds between amino acids.The truncated protein were produced by a p.Pro798Leufs*13 mutation.The thrombin generation test showed that the clotting function of proband A and B had been affected.Conclusion:These four mutations may be responsible for the reduction of FV level in two families.Moreover,the p.Ser111Ile mutation is a rarely reported.

关 键 词：凝血因子V缺陷症 F5基因 生物信息学 凝血酶生成 

分 类 号：R446.1[医药卫生—诊断学]