目的 对20例凝血因子Ⅻ(FⅫ)缺乏症患者进行F12基因的测序分析并探讨其分子机制。 方法 选择2020年7月至2022年1月期间就诊于山西医科大学第二医院门诊部的20例FⅫ缺乏症患者作为研究对象。采用一期法检测其凝血因子Ⅷ(FⅧ：C)、Ⅸ(FⅨ：C)、Ⅺ(FⅪ：C)和Ⅻ(FⅫ：C)的活性。用Sanger测序对其F12基因的14个外显子以及5′和3′非翻译区进行分析，确定其变异位点。用生物信息学软件预测变异的致病性、分析氨基酸的保守性并模拟变异蛋白的模型。 结果 20例患者的FⅫ：C介于0.07% ~ 20.10%之间，远低于正常参考值，其他凝血指标则均未见异常。Sanger测序共发现10人携带F12基因的变异，具体包括4例错义变异[c.820C>T(p.Arg274Cys)、c.1561G>A(p.Glu521Lys)、c.181T>C(p.Cys61Arg)和c.566.G>C(p.Cys189Ser)]，4例缺失变异c.303_304delCA(p.His101GlnfsX36)，1例插入变异c.1093_1094insC(p.Lys365GlnfsX69)以及1例无义变异c.1763C>A(p.Ser588*)。其余10人仅检测出46C/T变异。其中，c.820C>T(p.Arg274Cys)杂合错义变异以及c.1763C>A(p.Ser588*)纯合无义变异未见临床遗传变异数据库和人类基因突变数据库收录。生物信息学分析提示，p.Arg274Cys与p.Ser588*均为致病变异，相关的氨基酸位点在不同物种中高度保守。蛋白预测模型提示，p.Arg274Cys破坏了原有氢键作用力，同时侧链变短，可影响FⅫ蛋白二级结构的稳定性，使关键的结构域发生变化。p.Ser588*导致FⅫ蛋白C端截短，改变了蛋白质结构的空间构象，可能影响丝氨酸蛋白酶的切割位点，导致FⅫ：C极度降低。 结论 在一期法检出的FⅫ：C偏低的患者中，50%携带F12基因的变异，其中c.820C>T错义变异和c.1763C>A无义变异是导致凝血因子Ⅻ减低的新的变异。 Objective To analyze the sequence of the F12 gene and molecular mechanism for 20 patients with coagulation factor Ⅻ (FⅫ) deficiency. Methods The patients were selected from the outpatient department of the Second Hospital of Shanxi Medical University from July 2020 to January 2022. The activity of coagulation factor Ⅷ (FⅧ: C), factor Ⅸ (FⅨ: C), factor Ⅺ (FⅪ: C) and factor Ⅻ (FⅫ: C) were determined by using a one-stage clotting assay. All exons and 5′ and 3′ UTR of the F12 gene were analyzed by Sanger sequencing to detect the potential variants. Bioinformatic software was used to predict the pathogenicity of the variants, conservation of amino acids, and protein models. Results The FⅫ: C of the 20 patients has ranged from 0.07% to 20.10%, which was far below the reference values, whilst the other coagulation indexes were all normal. Sanger sequencing has identified genetic variants in 10 patients, including 4 with missense variants [c.820C>T (p.Arg274Cys), c. 1561G>A (p.Glu521Lys), c. 181T>C (p.Cys61Arg) and c. 566.G>C (p.Cys189Ser)], 4 deletional variants c. 303_304delCA(p.His101GlnfsX36), 1 insertional variant c. 1093_1094insC (p.Lys365GlnfsX69) and 1 nonsense variant c. 1763C>A (p.Ser588*). The remaining 10 patients only harbored the 46C/T variant. The heterozygous c. 820C>T(p.Arg274Cys) missense variant in patient 1 and the homozygous c. 1763C>A (p.Ser588*) nonsense variant in patient 2 were not included in the ClinVar and the Human Gene Mutation Database. Bioinformatic analysis predicted that both variants were pathogenic, and the corresponding amino acids are highly conserved. The protein prediction models suggested that the c. 820C>T (p.Arg274Cys) variant may affect the stability of the secondary structure of FⅫ protein by disrupting the original hydrogen bonding force and truncating the side chain, leading to changes in the vital domain. c. 1763C>A (p.Ser588*) may produce a truncated C-terminus which may alter the spatial conformation of the protein domain and affect the serine protease cleavage site, resulting in extremely reduced FⅫ: C. Conclusion Among individuals with low low FⅫ: C detected by one-stage clotting assay, 50% have harbored variants of the F12 gene, among which the c. 820C>T and c. 1763C>A were novel variants underlying the reduced coagulating factor FⅫ.
Abstract
Objective To analyze the sequence of the F12 gene and molecular mechanism for 20 patients with coagulation factor Ⅻ (FⅫ) deficiency. Methods The patients were selected from the outpatient department of the Second Hospital of Shanxi Medical University from July 2020 to January 2022. The activity of coagulation factor Ⅷ (FⅧ: C), factor Ⅸ (FⅨ: C), factor Ⅺ (FⅪ: C) and factor Ⅻ (FⅫ: C) were determined by using a one-stage clotting assay. All exons and 5′ and 3′ UTR of the F12 gene were analyzed by Sanger sequencing to detect the potential variants. Bioinformatic software was used to predict the pathogenicity of the variants, conservation of amino acids, and protein models. Results The FⅫ: C of the 20 patients has ranged from 0.07% to 20.10%, which was far below the reference values, whilst the other coagulation indexes were all normal. Sanger sequencing has identified genetic variants in 10 patients, including 4 with missense variants [c.820C>T (p.Arg274Cys), c. 1561G>A (p.Glu521Lys), c. 181T>C (p.Cys61Arg) and c. 566.G>C (p.Cys189Ser)], 4 deletional variants c. 303_304delCA(p.His101GlnfsX36), 1 insertional variant c. 1093_1094insC (p.Lys365GlnfsX69) and 1 nonsense variant c. 1763C>A (p.Ser588*). The remaining 10 patients only harbored the 46C/T variant. The heterozygous c. 820C>T(p.Arg274Cys) missense variant in patient 1 and the homozygous c. 1763C>A (p.Ser588*) nonsense variant in patient 2 were not included in the ClinVar and the Human Gene Mutation Database. Bioinformatic analysis predicted that both variants were pathogenic, and the corresponding amino acids are highly conserved. The protein prediction models suggested that the c. 820C>T (p.Arg274Cys) variant may affect the stability of the secondary structure of FⅫ protein by disrupting the original hydrogen bonding force and truncating the side chain, leading to changes in the vital domain. c. 1763C>A (p.Ser588*) may produce a truncated C-terminus which may alter the spatial conformation of the protein domain and affect the serine protease cleavage site, resulting in extremely reduced FⅫ: C. Conclusion Among individuals with low low FⅫ: C detected by one-stage clotting assay, 50% have harbored variants of the F12 gene, among which the c. 820C>T and c. 1763C>A were novel variants underlying the reduced coagulating factor FⅫ.
万方数据