Nonsense-mediated mRNA decay was demonstrated in two hypofibrinogenemias caused by heterozygous nonsense mutations of FGG, Shizuoka III and Kanazawa II

Abstract

SummaryWe 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 γ23X (stop codon), and Kanazawa II showed c.1205G>A in FGG exon 9, resulting in γ376X.To determine whether the truncated γ-chains, γ23X and γ376X, were synthesized and participated in the assembly of fibrinogen, mutant-type cDNA vectors were transfected into Chinese hamster ovary (CHO) cells. Significant levels of mutant fibrinogen were not detected by ELISA in the culture media and cell lysates. Immunoblot analysis of cell lysates revealed that the mutant γ-chain of γ376X was observed but intact fibrinogen was not. On the other hand, mutant γ-chain was not observed in γ23X-expressing cells.To demonstrate the involvement of the mechanisms of nonsense-mediated mRNA decay (NMD), we cloned wild-and mutant-type mini-genes containing γ23-or γ376-codon and transfected these into CHO cell lines in the absence or presence of cycloheximide (CHX) as an NMD inhibitor. mRNA levels were determined using real-time quantitative RT-PCR in CHO cells. In the absence of CHX, levels of mRNAs transcribed from the mutant gene were lower than from the wild-type gene whereas, in the presence of CHX, levels of mRNAs transcribed from the mutant gene increased dose-dependently. 3Finally, these results demonstrated that aberrant mRNAs containing γ23X or γ376X are degraded by the NMD system and translation decrease in hepatocytes, resulting in hypofibrinogenemias.4

Genetic analyses of novel compound heterozygous hypodysfibrinogenemia, Tsukuba I: FGG c.1129+62_65 del AATA and FGG c.1299+4 del A

“…However, we did not observe the shortened length of the -chain for the patient's fibrinogen; therefore, this aberrant -chain may not exist in plasma. These results suggest that aberrant -chain mRNA possessing the premature termination codon at 363 is destroyed in hepatocytes, most likely through nonsense-mediated mRNA decay [16,17]. In addition, the minigene incorporating the IVS-8 deletion partially produced a normal splicing product, but at a lower amount than that of the aberrant product.…”

A Novel Mutation in the Fibrinogen Bβ Chain (c.490G>A; End of Exon 3) Causes a Splicing Abnormality and Ultimately Leads to Congenital Hypofibrinogenemia

“…Genomic DNA was extracted from whole blood cells using a DNA Extraction Kit (WAKO Pure Chemical Ltd., Osaka, Japan), according to the manufacturer’s instructions. In order to amplify all exons and exon–intron boundaries in the Aα-, Bβ-, and γ-chain genes, 32 PCR primers were designed and DNA was amplified by PCR as described elsewhere [ 18 ]. PCR products were purified from agarose gels and directly sequenced using a BigDye TM Terminator Cycle Sequencing Ready Reaction Kit (Thermo Fisher Scientific, Waltham, MA, USA) and 3500 Genetic Analyzer (Life Technologies, Carlsbad, CA, USA).…”

Laboratory and Genetic Investigation of Mutations Accounting for Congenital Fibrinogen Disorders

“…Our literature review identified a large number of novel mutations accounting for congenital fibrinogen disorders, both quantitative [49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64] and qualitative. [65][66][67][68][69][70][71][72][73][74][75][76] The majority of dysfibrinogenemias, inherited as a dominant trait, are caused by heterozygous missense mutations in one of the three fibrinogen genes.…”