1例遗传性凝血因子Ⅶ缺陷症患者的家系表型及基因突变分析

Phenotype and gene mutation in a pedigree of one patient with inherited coagulation factor Ⅶ deficiency

目的分析1例遗传性凝血因子Ⅶ(FⅦ)缺陷症的家系表型及基因突变情况。方法选取先证者及其父母、妹妹为研究对象,分析该家系表型及基因突变情况。(1)家系表型分析:在IL-ALC TOP700血凝分析仪上采用凝固法检测先证者及其父母亲、妹妹的凝血酶原时间(PT)、PT纠正试验、国际标准化比值(INR)、凝血酶原活动度(PTA)、活化部分凝血活酶时间(APTT)、凝血酶时间(TT)、纤维蛋白原(FIB)、因子Ⅱ活性(FⅡ:C)、因子Ⅴ活性(FⅤ:C)、因子Ⅶ活性(FⅦ:C)和因子Ⅹ活性(FⅩ:C),采用ELISA法检测先证者及其父母亲、妹妹的FⅦ抗原(FⅦ:Ag);在HITACHI 7600-020型生化分析仪上采用酶法检测先证者及其父母亲、妹妹的肝功能与肾功能。(2)基因突变分析:采用高通量测序检测FⅦ(FⅦ)基因外显子及侧翼序列突变并以Sanger测序验证上述突变位点;ClustalX2.1软件分析突变氨基酸的保守性;生物信息学软件预测突变氨基酸是否有害;SWISS-MODEL建立蛋白模型,并用PyMOL软件分析突变氨基酸对蛋白结构的影响。结果 (1)家系实验室表型分析结果:先证者PT延长(49.00 s,能被健康人混合血浆完全纠正),INR增高(3.86),PTA下降(10%),FⅦ:C降低(0.60%),其他指标均正常;先证者父亲FⅦ:C降低(42.70%),其余指标均正常;先证者母亲FⅦ:C降低(29.70%),其余指标均正常;先证者妹妹所有指标均正常。(2)基因突变分析结果:测序结果显示先证者FⅦ基因第8外显子发生c. 722C>A(p. Thr241Asn)杂合错义突变和FⅦ基因第7内含子发生c. 681+1G>T杂合剪接位点突变,先证者父亲携带p. Thr241Asn杂合错义突变,先证者母亲携带c. 681+1G>T杂合剪接位点突变,先证者妹妹为野生型;保守性分析表明,Thr241残基保守性较低;生物信息学软件分析显示,p. Thr241Asn突变可能为有害突变或影响FⅦ蛋白功能;蛋白建模显示,突变型FⅦ蛋白发生了较大的折叠及空间构象变化。结论先证者及其父母FⅦ:C均降低,尤以先证者FⅦ:C降低最为显著,先证者妹妹FⅦ:C正常;先证者发生FⅦ基因第8外显子p. Thr241Asn杂合错义突变与FⅦ基因第7内含子c. 681+1G>T杂合剪接位点突变,先证者父亲携带p. Thr241Asn杂合错义突变,先证者母亲携带c. 681+1G>T杂合剪接位点突变,先证者妹妹为野生型。

Objective To analyze phenotype and gene mutation in a pedigree of one patient with inherited coagulation factor Ⅶ(FⅦ) deficiency.Methods The proband,his parents and sister were selected as the research objects to analyze the phenotype and gen mutation.(1) Phenotypic analysis of the pedigree:The prothrombin time(PT),PT correct test,the international normalized ratio(INR),the prothrombin time activity(PTA),the activated partial thromboplastin time(APTT),thrombin time(TT),fibrinogen(FIB),F Ⅱ clotting activity(F Ⅱ:C),F Ⅴ clotting activity(F V:C),FⅦ clotting activity(F Ⅶ:C),and F X clotting activity(F X:C) were detected by coagulation method on a IL-ALC TOP700 automatic coagulometer for the proband,his parents and sister.The FⅦ antigen(FⅦ:Ag) was detected by ELISA.The liver and renal function were detected by enzymatic method on the HITACHI 7600-020 automatic biochemical analyzer.(2) Analysis of gene mutation:The mutations of all exons and flanking sequences of the FⅦ gene(FⅦ) were detected by high-throughput sequencing and verified by Sanger sequencing.ClustalX2.1 software was used to analyze the conversation of a mutant amino acid.Pathogenicity of variants was predicted by using bioinformatics algorithms.The protein models were established by SWISS-MODEL,and the effects of mutant amino acids on proteins were analyzed by PyMOL software.Results(1) Results of laboratory phenotype of the pedigree:The PT of the proband was longer(49.00 s),which could be completely corrected by the mixed plasma of healthy persons.The INR was elevated to 3.86.The PTA was reduced to 10%.The FⅦ:C level was decreased to 0.60%,and the other results were normal.The FⅦ:C level of the proband’s father was decreased to 42.70%,and the other results were normal.The F Ⅶ:C level of the proband’s mother was reduced to 29.70%,and the other results were normal.All results of the proband’s sister were normal.(2) Results of gene mutation:Genetic testing revealed that A heterozygous c.722 C>A(p.Thr241 Asn) missense mutation in exon 8 of FⅦ and a heterozygous c.681+1 G>T splice site mutation in intron 7 of FⅦ were detected in the proband.The proband’s father was heterozygous for the c.722 C>A mutation.The proband’s mother was heterozygous for the c.681+1 G>T mutation.The pro band’s sister was wild-type.Conservative analysis showed that the Thr241 residue was less conserved among homologous species.The p.Thr241 Asn mutation was predicted to be probably damaging or affected the function of FⅦ protein by five bioinformatics algorithms.A modeling analysis showed obvious folding and spatial conformational changes in mutant FⅦ proteins.Conclusions The FⅦ:C levels of the proband and his parents were all reduced,with the dramatically decrease in the proband.The FⅦ:C level of the proband’s was normal.The pro band carried a heterozygous c.722 C>A(p.Thr241 Asn) missense mutation in exon 8 of FⅦ and a heterozygous c.681+1 G>T splice site mutation in intron 7 of F Ⅶ.The pro band’ s father was heterozygous for the c.722 C>A mutation,and the proband’ s mother was heterozygous for the c.681+1 G>T mutation.The proband’s sister was wild-type.