摘要
目的:对临床发现的1例低异常纤维蛋白原血症进行基因分析,探讨其分子发病机制。方法:选取2023年2月17日因“右肾积水,术前凝血功能异常”就诊于温州市人民医院的男性先证者作为研究对象。PCR扩增该患者FGA、FGB、FGG的外显子及其侧翼序列,进行双向测序,明确突变位点。饱和硫酸铵法纯化血浆中纤维蛋白原,进行纤维蛋白原聚集率、凝固率、SDS-PAGE分析;构建包含1-4号外显子的野生型与突变型小基因的pcDNA3.1-载体,转染HEK293T细胞,TRIzol法抽提总mRNA,经过RT-PCR、巢氏PCR、q-PCR验证突变对剪接体功能的影响。结果:先证者Aα链2号内含子IVS2+1G>T(c.180+1G>T)杂合突变,该突变导致先证者纤维蛋白原的聚集最大速率(t=443.069,P<0.001)、吸光度值差值(t=112.317,P<0.001)与凝固率(t=65.147,P<0.001)显著降低,SDS-PAGE发现血浆中出现FGA纤维蛋白突变链;构建小基因载体瞬时转染至HEK293T细胞后,经RT-PCR、巢氏PCR与q-PCR发现突变会影响FGA基因mRNA的正常剪接,导致FGA2号外显子序列126bp在翻译过程中缺失。结论:根据美国医学遗传学与基因组学学会(ACMG)突变评级相关指南,判定FGAIVS2+1G>T突变变异评级为致病突变。FGAIVS2+1G>T突变导致有42个氨基酸缺失的异常FGA释放入血,但不影响纤维蛋白原的组装与分泌,表现为低异常纤维蛋白原血症。
Objective:To investigate the molecular pathogenesis of hypodysfibrinogenaemia in a clinically discovered proband.Methods:A male proband who was admitted to the Wenzhou People’s Hospital on February 17,2023 due to“right hydronephrosis and abnormal preoperative coagulation function”was selected as the study subject.The exons and flanking sequences of FGA,FGB,and FGG were PCR amplified to identify mutation sites through forward and reverse sequencing.Fibrinogen was purified from plasma using saturated ammonium sulfate for analysis of fibrinogen aggregation rate,coagulation rate,and SDS-PAGE.Wild and mutant pcDNA3.1-minigene vectors containing exons 1 to 4 sequences were constructed and transfected into HEK293T cells.Total mRNA was extracted using trizol and mutations on splice-site were confirmed using RT-PCR,Nest PCR,and q-PCR.Results:The proband exhibited a heterozygous splice-site mutation(IVS2+1G>T/c.180+1G>T)in FGA intron 2,which led to a significant decrease in the maximum rate of fibrinogen aggregation(t=443.069,P<0.001),the difference in absorbance value(t=112.317,P<0.001),and the coagulation rate(t=65.147,P<0.001)of the proband.SDS-PAGE analysis revealed the presence of FGA fibrin mutant chains in plasma.Following the construction of the minigene vector and transient transfection into HEK293T cells,RT-PCR,Nested PCR,and q-PCR analyses revealed that the mutation would disrupt the normal splicing of FGA gene mRNA.This disruption led to the deletion of 126 base pairs from the FGA exon 2 sequence during translation.Conclusion:Following the mutation rating guidelines of the American College of Medical Genetics and Genomics(ACMG)and considering the combination of supporting evidence(PVS1+PS3+PP4),the FGA IVS2+1G>T mutation variant was classified as a pathogenic mutation.The FGA IVS2+1G>T mutation is responsible for hypodysfibrinogenaemia,causing abnormal release of FGA into the blood with a deletion of 42 amino acids.However,it does not impact the assembly and secretion of fibrinogen.
作者
王海坚
郑霜
余晓敏
吴楷雯
赵秘胜
朱丽青
WANG Haijian;ZHENG Shuang;YU Xiaomin;WU Kaiwen;ZHAO Misheng;ZHU Liqing(Clinical Laboratory,the Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University,Wenzhou People’s Hospital,Wenzhou 325000,China;Clinical Laboratory,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325015,China)
出处
《温州医科大学学报》
CAS
2024年第7期598-602,F0003,共6页
Journal of Wenzhou Medical University
