[一个遗传性凝血因子Ⅴ缺乏症家系的表型和突变分析]
[Phenotypic and mutational analysis of a pedigree affected with hereditary coagulation factor Ⅴ deficiency].
作者信息
Tian Mengcha, Xia Hong, Zhang Zhishan, Jin Yanhui, Su Kankan, Wang Mingshan
机构信息
Department of Laboratory Medicine, the First Hospital of Quanzhou Affiliated to Fujian Medical University, Quanzhou, Fujian 362000, China.
出版信息
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2018 Apr 10;35(2):202-206. doi: 10.3760/cma.j.issn.1003-9406.2018.02.012.
OBJECTIVE
To explore the molecular pathogenesis for a pedigree affected with coagulation factor Ⅴ (FⅤ) deficiency.
METHODS
Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), coagulation factor Ⅱ activity (FⅡ: C), FⅤ activity (FⅤ: C), coagulation factor Ⅶ activity (FⅦ: C), and coagulation factor Ⅹ activity (FⅩ: C) were determined with a STAGO automatic coagulometer. FⅤ antigen (FⅤ: Ag) was detected with enzyme linked immunosorbent assay (ELISA). All exons and their flanking regions, and 5' and 3' untranslated regions of the F5 gene were analyzed by direct sequencing. Suspected mutation was verified by reverse sequencing as well as testing of family members. ClustalX software was used to analyze the conservative property of the mutation sites. PROVEAN and MutationTaster online software was used to predict the effect of the mutation on the protein function. Swiss-pdbViewer was used to analyze the protein model and interaction of amino acids.
RESULTS
The PT and APTT of the proband were slightly prolonged to 15.2 s and 41.8 s, respectively. And the FⅤ: C and FⅤ: Ag measured 55% and 62%, respectively. The FⅤ: C and FⅤ: Ag of his father and son were decreased to various extent (60%, 65% and 31%, 40%, respectively). A c.911G>A heterozygous mutation (Gly276Glu) was detected in exon 6 of the proband, for which her father and son were heterozygotes. The same mutation was not found in her mother, brother and husband. Conservation analysis showed that the Gly276 is highly conserved across various species. By bioinformatic analysis, the PROVEAN (scored -6.214) indicated Gly276Glu was harmful, and MutationTaster (scored 0.976) suggested that it is pathogenic. Model analysis suggested there are two hydrogen bonds between Gly276 and Ile298 in the wild type protein. When Gly276 was replaced by Glu276, the original hydrogen bond did not change, but the side chain of Glu was extended, which added steric hindrance with the surrounding amino acids, which resulted in decreased protein stability.
CONCLUSION
The heterozygous c.911G>A (Gly276Glu) mutation of the F5 gene probably underlies the decreased level of FⅤin the proband.
目的
探究一个凝血因子Ⅴ（FⅤ）缺乏家系的分子发病机制。
方法
使用STAGO自动凝血分析仪测定凝血酶原时间（PT）、活化部分凝血活酶时间（APTT）、纤维蛋白原（FIB）、凝血因子Ⅱ活性（FⅡ：C）、FⅤ活性（FⅤ：C）、凝血因子Ⅶ活性（FⅦ：C）和凝血因子Ⅹ活性（FⅩ：C）。采用酶联免疫吸附测定（ELISA）检测FⅤ抗原（FⅤ：Ag）。通过直接测序分析F5基因的所有外显子及其侧翼区域以及5'和3'非翻译区。通过反向测序以及对家庭成员的检测验证疑似突变。使用ClustalX软件分析突变位点的保守性。使用PROVEAN和MutationTaster在线软件预测突变对蛋白质功能的影响。使用Swiss-pdbViewer分析蛋白质模型和氨基酸相互作用。
结果
先证者的PT和APTT分别轻度延长至15.2秒和41.8秒。FⅤ：C和FⅤ：Ag分别测定为55%和62%。其父亲和儿子的FⅤ：C和FⅤ：Ag均有不同程度降低（分别为60%、65%和31%、40%）。在先证者的第6外显子中检测到一个c.911G>A杂合突变（Gly276Glu），其父亲和儿子为杂合子。在其母亲、兄弟和丈夫中未发现相同突变。保守性分析表明，Gly276在不同物种中高度保守。通过生物信息学分析，PROVEAN（评分-6.214）表明Gly276Glu有害，MutationTaster（评分0.976）提示其具有致病性。模型分析表明，野生型蛋白中Gly276与Ile298之间存在两个氢键。当Gly276被Glu276取代时，原氢键未改变，但Glu的侧链延长，与周围氨基酸增加了空间位阻，导致蛋白质稳定性降低。
结论
F5基因的杂合c.911G>A（Gly276Glu）突变可能是先证者FⅤ水平降低的原因。
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