科研之友
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摘要
We report two novel hypofibrinogenemias, Shizuoka III and Kanazawa II, which are caused by heterozygous mutations in FGG. Shizuoka III showed c.147delT and 147_ 149insACA in FGG exon 3 and a subsequent frameshift mutation, resulting in mature protein gamma 23X (native protein: gamma 49X), and Kanazawa II showed c.1205G %26gt; A inFGG exon 9, resulting in gamma 376X (native protein: gamma 402X). %26lt;br%26gt;To determinewhether the truncated.-chains, gamma 23X and gamma 376X, were synthesized and participated in the assembly of fibrinogen, mutant-type cDNA vectorswere transfected into Chinese hamster ovary (CHO) cells. Significant levels ofmutant fibrinogenwere not detected by ELISA in the culture media and cell lysates. Immunoblot analysis of cell lysates revealed that the mutant.-chain of gamma 376X was observed but intact fibrinogen was not. On the other hand, mutant.-chain was not observed in gamma 23X-expressing cells. %26lt;br%26gt;To demonstrate the involvement of the mechanisms of nonsense-mediated mRNA decay (NMD), we cloned wild-and mutant-type mini-genes containing gamma 23 or gamma 376 codon and transfected these into CHO cell lines in the absence or presence of cycloheximide as an NMD inhibitor. mRNA levels were determined using real-time quantitative RT-PCR in CHO cells. In the absence of cycloheximide, levels of mRNAs transcribed from the mutant gene were lower than from the wild-type gene whereas, in the presence of cycloheximide, levels of mRNAs transcribed from the mutant gene increased dose-dependently. %26lt;br%26gt;Finally, these results demonstrated that mRNAs containing gamma 23X or gamma 376X are degraded by the NMD system and translation of the truncated.-chain polypeptide decrease in patients%26apos; hepatocytes, resulting in hypofibrinogenemias. c 2013 Elsevier Ltd.
- 出版日期2013-10
