摘要
目的对一个近亲结婚导致的遗传性凝血因子Ⅻ(FⅫ)缺陷症家系进行表型和基因突变分析,初步探讨其分子发病机制。方法检测先证者及其家系成员(共3代5人)活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、纤维蛋白原(FIB)、FⅫ活性(FⅫ:C)和FⅫ抗原(FⅫ:Ag)以明确表型诊断。采用DNA直接测序分析F12基因的所有外显子、侧翼、5′和3′非翻译区及家系成员相应的突变位点区域,用反向测序进一步证实所发生的突变。通过多个生物信息学软件(clustalx-2.1-win、Mutation Taster、PolyPhen-2、PROVEAN、SIFT和Swiss-Pdb Viewer 4.1.0)分析突变氨基酸的保守性和对蛋白质可能的影响,构建突变前后的蛋白结构模型分析突变对蛋白质空间结构的改变及影响。结果先证者APTT延长,较正常对照显著延长为101.0νs 36 s,FⅫ:C和FⅫ:Ag极度降低,均为1.0%(正常范围为72%~113%)。基因分析显示,先证者第13外显子存在c.1638G>A纯合错义突变,导致527位异亮氨酸取代蛋氨酸(p.Met527Ile);其母亲、儿子和女儿均携带了p.Met527Ile杂合子。生物信息学和突变模型分析表明,Met527在人类与7种同源物种间呈高度保守,p.Met527Ile可能是有害的,并改变了蛋白质的结构和功能。结论 p.Met527Ile错义突变国内外鲜见报道,并可能与该家系FⅫ水平降低有关。
Objective A family of hereditary coagulation factor Ⅻ(FⅫ) deficiency caused by inbreeding was analyzed for phenotype and gene mutation, and the molecular pathogenesis was preliminarily discussed. Methods The activated partial thromboplastin time(APTT), prothrombin time(PT), fibrinogen(FIB), FⅫ activity(FⅫ:C), and FⅫ antigen(FⅫ:Ag)were used for phenotypic diagnosis. All exons, flanking, 5′ and 3′ untranslated regions of the provost F12 gene and the corresponding mutation sites of the family members were analyzed by DNA direct sequencing. The mutation sites were confirmed by reverse sequencing. Through multiple bioinformatics software(clustalx-2.1-win、Mutation Taster、PolyPhen-2、PROVEAN、SIFT and Swiss-Pdb Viewer 4.1.0) to analyze the conservatism of mutant amino acids and the possible effects on proteins, the protein structure model before and after mutation was constructed to analyze the changes and effects of mutation on protein spatial structure. Results Proven APTT increased significantly to 101.0 νs 36 s, compared with normal controls FⅫ:C and FⅫ:Ag are severely reduced, 1.0%(Normal range, 72%–113%). Genetic analysis showed, a homozygous mutation in exon 13 c.1638 G>A the proband, causes 527 isoleucine to replace methionine(p.Met527Ile). His mother, son and daughter all carried p.Met527Ile zygote. Bioinformatics and mutation model analysis showed that Met527 was highly conserved between humans and seven homologous species, p.Met527Ile could be harmful and altered the structure and function of proteins.Conclusions p.Met527Ile missense mutation is rarely reported at home and abroad and may be related to the decrease of family FⅫ level.
作者
姜也
郑晓勇
谢海啸
刘媚娜
王欢欢
王明山
JIANG Ye;ZHENG Xiaoyong;XIE Haixiao;LIU Meina;WANG Huanhuan;WANG Mingshan(The 1s t Affiliated Hospital of Wenzhou Medical University,Wenzhou,Zhejiang 325015,China)
出处
《中国优生与遗传杂志》
2021年第9期1242-1245,共4页
Chinese Journal of Birth Health & Heredity