Masahiro Yoda1, Takahiro Kaido1, Chiaki Taira2, Yumiko Higuchi3, Shinpei Arai4, Nobuo Okumura3. 1. Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan. 2. Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan. Electronic address: tairacha@shinshu-u.ac.jp. 3. Department of Clinical Laboratory Investigation, Graduate School of Medicine, Shinshu University, Matsumoto, Japan; Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan. 4. Department of Clinical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan.
Abstract
INTRODUCTION: Congenital fibrinogen disorders result from genetic mutations in FGA, FGB, or FGG resulting in quantitative fibrinogen deficiencies (afibrinogenemia or hypofibrinogenemia) or qualitative fibrinogen deficiencies (dysfibrinogenemia). Hypodysfibrinogenemia sharing features with hypo- and dysfibrinogenemia is rare. We performed genetic and functional analyses of a 31-year-old woman with suspected hypodysfibrinogenemia. MATERIALS AND METHODS: Functional and antigenic fibrinogen values of patient were 1.05 and 1.24 g/L, respectively. DNA sequence and western blotting analyses for plasma fibrinogen were performed. A minigene incorporating the mutational region was transfected into a Chinese hamster ovary cell line (CHO), and reverse transcription products were analyzed. Assembly and secretion were examined using the recombinant variant fibrinogen. We purified the patient's plasma fibrinogen and analyzed thrombin-catalyzed fibrin polymerization (TCFP). RESULTS AND CONCLUSIONS: DNA sequencing revealed compound heterozygous nucleotide mutations with FGB 35 bp c.1245-17_1262 or -16_1263 del and FGB c.510T>A (resulting in Bβp.N170K substitution) on different alleles. We did not detect shortened Bβ-chain peptides in the plasma using western blotting analysis. A minigene incorporating the deletion DNA showed two aberrant mRNA products. The secretion of Bβp.N170K-fibrinogen-CHO was almost same as normal Bβ-fibrinogen-CHO. TCFP of plasma Bβp.N170K fibrinogen was slightly lower than that of normal plasma fibrinogen. Aberrant splicing products derived from the 35 bp deletion caused hypofibrinogenemia due to nonsense-mediated mRNA decay and suggested the presence of only Bβp.N170K fibrinogen in patient's plasma. Bβp.N170K caused dysfibrinogenemia due to a delay in lateral aggregation. These findings demonstrated that these mutations respectively affected the fibrinogen quality and quantity, resulting in hypodysfibrinogenemia.
INTRODUCTION: Congenital fibrinogen disorders result from genetic mutations in FGA, FGB, or FGG resulting in quantitative fibrinogendeficiencies (afibrinogenemia or hypofibrinogenemia) or qualitative fibrinogen deficiencies (dysfibrinogenemia). Hypodysfibrinogenemia sharing features with hypo- and dysfibrinogenemia is rare. We performed genetic and functional analyses of a 31-year-old woman with suspected hypodysfibrinogenemia. MATERIALS AND METHODS: Functional and antigenic fibrinogen values of patient were 1.05 and 1.24 g/L, respectively. DNA sequence and western blotting analyses for plasma fibrinogen were performed. A minigene incorporating the mutational region was transfected into a Chinese hamster ovary cell line (CHO), and reverse transcription products were analyzed. Assembly and secretion were examined using the recombinant variant fibrinogen. We purified the patient's plasma fibrinogen and analyzed thrombin-catalyzed fibrin polymerization (TCFP). RESULTS AND CONCLUSIONS: DNA sequencing revealed compound heterozygous nucleotide mutations with FGB 35 bp c.1245-17_1262 or -16_1263 del and FGB c.510T>A (resulting in Bβp.N170K substitution) on different alleles. We did not detect shortened Bβ-chain peptides in the plasma using western blotting analysis. A minigene incorporating the deletion DNA showed two aberrant mRNA products. The secretion of Bβp.N170K-fibrinogen-CHO was almost same as normal Bβ-fibrinogen-CHO. TCFP of plasma Bβp.N170Kfibrinogen was slightly lower than that of normal plasma fibrinogen. Aberrant splicing products derived from the 35 bp deletion caused hypofibrinogenemia due to nonsense-mediated mRNA decay and suggested the presence of only Bβp.N170Kfibrinogen in patient's plasma. Bβp.N170K caused dysfibrinogenemia due to a delay in lateral aggregation. These findings demonstrated that these mutations respectively affected the fibrinogen quality and quantity, resulting in hypodysfibrinogenemia.