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目的对1个姨表近亲婚配的遗传性凝血因子Ⅶ(FⅦ)缺陷症家系进行表型和F7基因突变分析,探讨其分子发病机制。方法检测先证者及其家系成员(共4代9人)凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、纤维蛋白原(FIB)、血浆FⅦ活性(FⅦ:C)等来明确诊断。PCR扩增先证者全部外显子及其侧翼序列、5′和3′非翻译区及家系成员相应的突变位点区域,PCR产物纯化后直接测序,寻找突变位点,以反向测序验证所发生的突变;使用生物信息学软件(Poly Phen-2和Mutation Taster)预测突变位点对蛋白质功能的影响,利用Py MOL软件构建正常FⅦ蛋白空间模型,进行定点突变观察构型改变。结果先证者PT(36.1s)和FⅦ:C(2%)明显异常,家系中其余8位成员PT均略高于正常对照组和FⅦ:C均略低于正常对照组;基因分析显示先证者F7基因第8外显子存在c.1165 T>G纯合型突变,即TGT→GGT(Cys329Gly),8位家系成员的F7基因分析均显示c.1165有杂合型突变;两种生物信息学软件都提示此突变会引起蛋白功能变化,有致病性;F7基因蛋白构型显示突变后329位点与310位点之间二硫键消失,周边静电磁场发生改变。结论该家系F7基因存在Cys329Gly突变,是引起FⅦ缺陷症的主要分子机制,推测先证者纯合Cys329Gly突变基因分别遗传自近亲结婚的杂合子父母。
Objective To analyze phenotypic and F7 gene mutations in a familial familial hereditary factor Ⅶ (FⅦ) with an aunt cousin, and to explore its molecular pathogenesis. Methods The PT, APTT, fibrinogen (FIB), plasma FⅦ (FⅦ: C), etc. of probands and their pedigree members (9 generations in 4 generations) To confirm the diagnosis. PCR amplification of proband all exons and their flanking sequences, 5 ’and 3’ untranslated region and the corresponding members of family members of the site of mutation, PCR products were sequenced directly to find the mutation sites to reverse sequencing verification (Poly Phen-2 and Mutation Taster) were used to predict the effect of the mutation site on the protein function. PyMOL software was used to construct the normal FⅦ protein space model and site-directed mutagenesis was used to observe the conformational changes. Results The probands PT (36.1s) and FⅦ: C (2%) were significantly abnormal. The PT of the remaining eight members of the pedigree were slightly higher than that of the normal control group and FⅦ: C, which was slightly lower than that of the normal control group. There was a c.1165 T> G homozygous mutation in exon 8 of F7 gene, ie, TGT → GGT (Cys329Gly). F7 gene analysis of 8 pedigree members showed that c.1165 had heterozygous mutations; two Bioinformatics software suggested that this mutation caused a functional change of the protein with pathogenicity. The protein configuration of F7 gene showed disulfide bond disappeared between positions 329 and 310 after the mutation, and the electrostatic field around it changed. Conclusion The Cys329Gly mutation in F7 gene of this pedigree is the major molecular mechanism of FⅦ deficiency. It is speculated that the homozygous Cys329Gly mutant gene of the proband is inherited from heterozygous parents of the close relatives, respectively.