摘要
目的 探讨遗传性凝血因子V(FV)缺陷症家系的实验室诊断和分子发病机制。方法 对 1个FⅤ缺陷症家系进行研究。采用活化部分凝血活酶时间 (APTT) ,凝血酶原时间 (PT)及FV促凝活性 (FV :C)和FV抗原 (FV :Ag)测定进行表型诊断 ;用PCR法对先证者FV基因 2 5个外显子及其侧翼序列进行扩增 ,PCR产物纯化后直接测序 ,检测其基因突变。通过cDNA测序分析剪切位点突变引起编码序列的变化。结果 先证者APTT 12 3s ,PT 4 3 4s ,FV :C 1 6 % ,FV :Ag 7 2 %。基因分析发现 ,先证者第 8内含子受点发生AG→GG突变 ;cDNA分析发现 ,该突变导致剪切位点前移 2 4bp ,即在编码序列中 ,于第 8外显子和第 9外显子间插入了 2 4bp ,插入序列未引起读码框架漂移 ,使编码的FV蛋白插入了 8个氨基酸。结论 先证者为I型遗传性FV缺陷症 ,第 8内含子 3′端剪切位点突变 ,引起编码序列 2 4bp插入是导致该例先证者发生FV缺陷症的分子机制。
Objective To study on the laboratory diagnosis and molecular mechanism of a pedigree with inherited factor V deficiency. Methods The tests of activated partial thromboplastin time (APTT),prothrombin time (PT), FV activity (FV:C) and FV antigen were adopted for phenotype diagnosis. The genomic DNA was extracted from the peripheral blood of the propositus. 25 exons and their flanks of FV gene were amplified by polymerase chain reaction. The PCR products were screened by direct sequencing. The cDNA sequencing was performed to analyze the change of FV coding sequence by splice site mutation. Results APTT,PT,FV: C, FV: Ag of the propositus were 123 s, 43.4 s, 1.6% and 7.2%, respectively. Homozygous mutation AG→GG of intron 8 was found at 3' splice site in the propositus, where the 3' splice site was moved 24 bp ahead. 24 bp nucleotides insertion between exon 8 and exon 9 were detected by FV cDNA sequencing, where the insertion sequence did not cause frameshift. 8 aa was inserted between 404 and 405 sites of the amino acid sequence of mutated FV protein. Conclusion The proband was diagnosed with type I inherited coagulation factorV deficiency. Homozygous splice site mutation causing 24 bp insertion in FV cDNA was the molecular mechanism of this pedigree.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2004年第2期93-96,共4页
Chinese Journal of Laboratory Medicine
