纤维蛋白原α链剪切位点IVS2+1G>C突变导致的遗传性低纤维蛋白原血症

Hereditary hypofibrinogenemia caused by splice-site mutation (IVS2+1G>C) of fibrinogen α-chain gene

目的:探讨遗传性低纤维蛋白原(Fg)血症的分子发病机制。方法:检测凝血指标以明确诊断;用DNA直接测序法对患者Fg基因FGA、FGB和FGG的所有外显子及其侧翼序列进行测序以寻找基因突变,对有突变的序列反向测序证实;通过逆转录结合巢式PCR扩增的方法,检测患者外周血中的Fg异位转录产物;构建含有突变点的突变型FGA小基因(minigene)质粒和野生型FGA小基因质粒,将2种质粒分别转染人胚肾(HEK)293T细胞,抽提RNA,逆转录PCR(RT-PCR)后TA克隆测序。结果:先证者呈FGA基因剪切位点IVS2+1G>C杂合突变;对于该突变,逆转录结合巢式PCR的产物经克隆后测序只检测到正常转录本,而没有发现异常转录本;突变型FGA小基因质粒转染HEK293T细胞后,抽提RNA再经RT-PCR、TA克隆、测序,揭示剪接过程中发生了FGA基因2号内含子滞留,导致终止密码的提前出现,从而使异常转录的mRNA在体内很快被降解。结论:异位转录结合体外表达证明先证者FGA基因剪切位点IVS2+1G>C突变导致异常转录mRNA在体内很快被降解,是先证者低Fg血症的原因之一。

Objective To explore the molecular pathogenesis of hereditary hypofibrinogenemia. Methods Diagnosis of hereditary hypofibrinogenemia was validated by coagulant parameter assay. All the exons and their flanking sequences of the three fibrinogen （Fg） gene FGA, FGB, FGG were analyzed by direct sequencing to detect gene mutation. The mutation detected was confirmed by reverse sequencing. Nested reverse transcription polymerase chain reaction （RT- PCR） was used to detect Fg ectopic transcripts in peripheral blood. Mutant FGA minigene plasmid as well as wild-type FGA minigene plasmid were constructed. These two kinds of plasmid were transfected into the human embryonic kidney （HEK） 293T cells. RNA was extracted and sequencing with TA cloning was performed after RT-PCR. Results The proband had heterozygous splice-site mutation （IVS2＋1G〉C） in FGA gene. For this mutation, sequencing with cloning after nested RT-PCR had not detect aberrant transcripts and only normal transcripts was detected. After transfecting mutant FGA minigene plasmid into HEK293T cells, RNA extraction and sequencing with TA cloning after RT-PCR, retention of FGA gene intron 2 during the process of splicing was found, which induced the premature occurrence of termination codon, thereby causing the rapid degradation of aberrant mRNA transcripts. Conclusions Ectopic transcription in combination with in-vitro expression proved the in-vivo rapid degradation of aberrant mRNA transcripts induced by the splice-site mutation （IVS2＋1G〉C）, which is one of the pathogenic factors of hypofibrinogenemia in the proband.