摘要
目的探讨一个遗传性凝血因子Ⅴ（coagulation factor Ⅴ，FⅤ）缺陷症家系的表型特征及其分子致病机制。
方法在STAGO全自动血凝仪上检测先证者及家系成员（3代6名成员）凝血酶原时间（prothrombin time，PT）、活化部分凝血活酶时间（activated partial thromboplastin time，APTT）、纤维蛋白原（fibrinogen，FIB）、血浆凝血因子Ⅱ活性（coagulation factor Ⅱ activity，FⅡ∶C）、FⅤ活性（FⅤ activity，FⅤ∶C ）、凝血因子Ⅶ活性（coagulation factor Ⅶ activity，FⅦ∶C）和凝血因子Ⅹ活性（coagulation factor Ⅹ activity，FⅩ∶C）活性；ELISA方法检测FⅤ抗原（FⅤ antigen，FⅤ∶Ag）。采用DNA直接测序法分析先证者F5基因的全部外显子、侧翼序列、5′和3′端非翻译区及家系成员相应的突变位点区域，发现突变位点后用反向测序予以证实。采用ClustalX软件分析突变位点的保守性，同时用生物信息学预测软件（PROVEAN和MutationTaster）分析突变对蛋白质功能的影响，用Swiss-PdbViewer软件对突变位点进行蛋白模型和氨基酸相互作用分析。结果先证者PT和APTT均稍延长，分别为15.2 s和41.8 s，FⅤ∶C和FⅤ∶Ag降低，分别为55%和62%；其父亲和儿子FⅤ∶C和FⅤ∶Ag也均有不同程度下降（分别为60%、65%和31%、40%）。先证者F5基因第6外显子上发现c.911G〉A杂合突变，导致Gly276Glu；其父亲和儿子也为Gly276Glu杂合子；母亲、哥哥和丈夫为正常野生型。保守性分析结果表明，Gly276在同源物种间高度保守；两个生物信息学软件对该突变的预测结果一致：PROVEAN（评分－6.214分）结果预示为有害突变；MutationTaster（评分0.976分）结果预示可引起相应疾病；突变蛋白模型分析显示，在野生型蛋白质中，Gly276的主链与Ile298之间有两个氢键，当Gly276突变为Glu276后，原有的氢键没有改变，但由于Glu侧链延长，与周围氨基酸�
ObjectiveTo explore the molecular pathogenesis for a pedigree affected with coagulation factor Ⅴ （FⅤ） deficiency.MethodsProthrombin 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.ResultsThe 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 ste
作者
田孟茶
夏虹
张志珊
金艳慧
苏看看
王明山
Tian Mengcha, Xia Hong, Zhang Zhishan, Jin Yanhui, Su Kankan, Wang Mingshan Department of Laboratory Medicine, the First Hospital of Quanzhou Affiliated to Fujian Medical University, Quanzhou, Fujian 362000, China(Tian MC, Zhang ZS） ~ Center of Laboratory Medicine, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou , Zhejiang 325015, China （ Xia H, Jin YH , Su KK, Wang MS)
出处
《中华医学遗传学杂志》
CAS
CSCD
2018年第2期202-206,共5页
Chinese Journal of Medical Genetics
基金
浙江省自然科学基金（LY16H080005）
