A novel frameshift mutation in the fibrinogen γC terminal region, FGG c.1169_1170 del AT, leading to hypofibrinogenemia

ArticleTHROMBOSIS RESEARCH.159:82-85(2017)journal articl

Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia

ArticleInternational journal of molecular sciences. 22(10): 5218(2021)journal articl

Fibrin monomers derived from thrombogenic dysfibrinogenemia, Naples-type variant (BβAla68Thr), showed almost entirely normal polymerization

ArticleTHROMBOSIS RESEARCH.172:1-3(2018)journal articl

A novel variant fibrinogen, AαE11del, demonstrating the importance of AαE11 residue in thrombin binding

ArticleInternational journal of hematology. 114(5): 591-598. (2021)journal articl

Novel heterozygous dysfibrinogenemia, Sumida (A alpha C472S), showed markedly impaired lateral aggregation of protofibrils and mildly lower functional fibrinogen levels

Introduction: We encountered a 6-year-old girl with systemic lupus erythematosus. Although no bleeding or thrombotic tendency was detected, routine coagulation screening tests revealed slightly lower plasma fibrinogen levels, as determined by functional and antigenic measurements (functional/antigenic ratio=0.857), suggesting hypodysfibrinogenemia. Materials and methods: DNA sequence and functional analyses were performed on purified plasma fibrinogen, and recombinant variant fibrinogen was synthesized in Chinese hamster ovary cells based on the results obtained. Results: DNA sequencing revealed a heterozygous A alpha C472S substitution (mature protein residue number) in the alpha C-domain. A alpha C472S fibrinogen indicated the presence of additional disulfide-bonded molecules, and markedly impaired lateral aggregation of protofibrils in spite of slightly lower functional plasma fibrinogen levels. Scanning electron microscopic observations showed a thin fiber fibrin clot, and t-PA and plasminogen-mediated clot lysis was similar to that of a normal control. Recombinant variant fibrinogen-producing cells demonstrated that destruction of the A alpha 442C-472C disulfide bond did not prevent the synthesis or secretion of fibrinogen, whereas the variant A alpha chain of the secreted protein was degraded faster than that of the normal control. Conclusion: Our results suggest that A alpha C472S fibrinogen may cause dysfibrinogenemia, but not hypofibrinogenemia. The destruction and steric hindrance of the alpha C-domain of variant fibrinogen led to the impaired lateral aggregation of protofibrils and t-PA and plasminogen-mediated fibrinolysis, as well as several previously reported variants located in the alpha C-domain, and demonstrated the presence of disulfide-bonded molecules.ArticleTHROMBOSIS RESEARCH. 135(4):710-717 (2015)journal articl

Congenital fibrinogen disorder with a compound heterozygote possessing two novel FGB mutations, one qualitative and the other quantitative

ArticleThrombosis research. 196: 152-158. (2020)journal articl

Screening method for congenital dysfibrinogenemia using clot waveform analysis with the Clauss method

ArticleInternational journal of laboratory hematology. 43(2): 281-289(2021)journal articl

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

Epub 2016 Nov 5Introduction: Wefound a novel hypodysfibrinogenemia designated Tsukuba I caused by compound heterozygous nucleotide deletionswith FGG c. 1129+ 62_ 65 del AATA and FGG c. 1299+ 4 del A on different alleles. The former was deep in intron 8 of FGG (IVS-8 deletion) and the latter in exon 9 of FGG (Ex-9 deletion), which is translated for the gamma'-chain, but not the.A-chain. AWestern blot analysis of plasma fibrinogen from our patient revealed an aberrant gamma-chain that migrated slightly faster than the normal B beta-chain. Materials andmethods: To clarify the complex genetic mechanismunderlying Tsukuba I's hypodysfibrinogenemia induced by nucleotide deletions in two regions, we generated two minigenes incorporating each deletion region, transfected them into Chinese Hamster Ovary (CHO) cells, and analyzed RT-PCR products. We also established CHO cells producing the recombinant variant fibrinogen,gamma' 409.A (Ex-9 deletion). Results and conclusions: Minigene I incorporating the IVS-8 deletion showed two products: a normal splicing product and the unspliced product. Minigene II incorporating the Ex-9 deletion only produced the unspliced product. The established gamma' 409.A-CHOcells secreted variant fibrinogenmore effectively than normal fibrinogen. Therefore, the aberrant splicing products derived from the IVS-8 deletion cause hypofibrinogenemia most likely due to nonsense-mediated mRNA decay and the partial production of normal.A-and gamma'-chains; moreover, the Ex-9 deletion causes hypodysfibrinogenemia due to the absence of normal.A-and gamma'-chain production (hypofibrinogenemia) and augmented aberrant.'-chain production (dysfibrinogenemia). (C) 2016 Elsevier Ltd. All rights reserved.ArticleTHROMBOSIS RESEARCH. 148:111-117 (2016)journal articl

gamma 375W fibrinogen-synthesizing CHO cells indicate the accumulation of variant fibrinogen within endoplasmic reticulum

Background: Hepatic endoplasmic reticulum (ER) storage disease (HERSD) associated with hypofibrinogenemia has been reported in patients with four types of heterozygous gamma-chain variant fibrinogen in the C terminal region. Of interest, substitution of gamma R375W induced hypofibrinogenemia and HERSD, whereas gamma R375G induced dysfibrinogenemia. Objectives: To analyze the synthesis of variant fibrinogen and morphological characteristics, we established variant fibrinogen-producing cells and compared them with wild-type fibrinogen-synthesizing cells. Methods: The fibrinogen gamma-chain expression vectors coding gamma 375W and gamma 375G were altered by oligonucleotide-directed mutagenesis and transfected into Chinese hamster ovary (CHO) cells. Synthesis of fibrinogen (media and cell lysates) was measured by ELISA for each cloned cell line and morphological characteristics were observed by immunofluorescence and transmission electron microscopy. Results: The medium/cell lysate fibrinogen ratio of gamma 375W-CHO cells was markedly lower than that of the normal cells and gamma 375G-CHO cells. Immunostaining with anti-fibrinogen antibody showed only gamma 375W-CHO cells, but revealed two types of cells containing cytoplasmic inclusion bodies, scattered large-granular bodies and fibrous forms. Observation by confocal microscopy indicated that both inclusion bodies were colocalized with fibrinogen and ER-membrane protein; furthermore, transmission electron microscopic observation demonstrated dilatation of the ER by large-granular inclusion bodies and fibrous forms filled with regularly structured fibular materials within the dilated ER. Conclusion: These results demonstrated that assembled and non-secreted gamma 375W fibrinogen was accumulated in the dilated ER and aggregated variant fibrinogen was seen as regularly structured fibular materials, which was similar to the fingerprint-like pattern observed at inclusion bodies in patients' hepatocytes affected with HERSD.ArticleTHROMBOSIS RESEARCH. 133(1):101-107 (2014)journal articl

The fibrous form of intracellular inclusion bodies in recombinant variant fibrinogen-producing cells is specific to the hepatic fibrinogen storage disease-inducible variant fibrinogen

Fibrinogen storage disease (FSD) is a rare disorder that is characterized by the accumulation of fibrinogen in hepatocytes and induces liver injury. Six mutations in the γC domain (γG284R, γT314P, γD316N, the deletion of γG346-Q350, γG366S, and γR375W) have been identified for FSD. Our group previously established γ375W fibrinogen-producing Chinese hamster ovary (CHO) cells and observed aberrant large granular and fibrous forms of intracellular inclusion bodies. The aim of this study was to investigate whether fibrous intracellular inclusion bodies are specific to FSD-inducible variant fibrinogen. Thirteen expression vectors encoding the variant γ-chain were stably or transiently transfected into CHO cells expressing normal fibrinogen Aα- and Bβ-chains or HuH-7 cells, which were then immunofluorescently stained. Six CHO and HuH-7 cell lines that transiently produced FSD-inducible variant fibrinogen presented the fibrous (3.2?22.7 and 2.1?24.5%, respectively) and large granular (5.4?25.5 and 7.7?23.9%) forms of intracellular inclusion bodies. Seven CHO and HuH-7 cell lines that transiently produced FSD-non-inducible variant fibrinogen only exhibit the large granular form. These results demonstrate that transiently transfected variant fibrinogen-producing CHO cells and inclusion bodies of the fibrous form may be useful in non-invasive screening for FSD risk factors for FSD before its onset.ArticleINTERNATIONAL JOURNAL OF HEMATOLOGY.105:758-768(2017)journal articl