γD318Y fibrinogen shows no fibrin polymerization due to defective "A-a" and "B-b" interactions, whereas that of γK321E fibrinogen is nearly normal

ArticleThrombosis research. 182: 150-158. (2019)journal articl

Heterozygous Variant Fibrinogen γA289V (Kanazawa III) Was Confirmed as Hypodysfibrinogenemia by Plasma and Recombinant Fibrinogens

Introduction: Congenital fibrinogen disorders are classified as afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia. However, difficulties are associated with discriminating between dysfibrinogenemia, hypofibrinogenemia, and hypodysfibrinogenemia using routine analyses. We previously reported a heterozygous variant fibrinogen (γA289V; Kanazawa III) as hypodysfibrinogenemia; however, the same variant had previously been described as hypofibrinogenemia. To clarify the production of γA289V fibrinogen, we expressed recombinant γA289V (r-γA289V) fibrinogen and compared it with wild-type (WT) and adjacent recombinant variant fibrinogens. Methods: Target mutations were introduced into a fibrinogen γ-chain expression vector by site-directed mutagenesis, and the vector was then transfected into Chinese hamster ovary cells to produce recombinant fibrinogen. Fibrinogen was purified from the plasma of the proposita, and culture media and fibrinogen functions were analyzed using fibrin polymerization, plasmin protection, and FXIIIa-catalyzed fibrinogen cross-linking. Results: The fibrinogen concentration ratio of the culture media to cell lysates was markedly lower for r-γA289V fibrinogen than for WT. Because the secretion of recombinant γF290L (r-γF290L) fibrinogen was similar to WT, we compared r-γF290L fibrinogen functions with WT. The fibrin polymerization of Kanazawa III plasma (K-III) fibrinogen was significantly weaker than normal plasma fibrinogen. Moreover, K-III fibrinogen showed a markedly reduced “D:D” interaction. However, all functions of r-γF290L fibrinogen were similar to WT. An in silico analysis confirmed the above results. Conclusion: The present results demonstrated that γA289 is crucial for the γ-module structure, and the γA289V substitution markedly reduced fibrinogen secretion. Moreover, K-III fibrinogen showed markedly reduced fibrin polymerization and “D:D” interactions. γA289V fibrinogen was confirmed as hypodysfibrinogenemia.ArticleINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY.42(2):190-197(2020)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 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

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

Novel variant fibrinogen γp.C352R produced hypodysfibrinogenemia leading to a bleeding episode and failure of infertility treatment

ArticleInternational journal of hematology. 114(3): 325-333. (2021)journal articl

A Novel Amino Acid Substitution, Fibrinogen Bβp.Pro234Leu, Associated with Hypofibrinogenemia Causing Impairment of Fibrinogen Assembly and Secretion

ArticleInternational journal of molecular sciences. 21(24): 9422(2020)journal articl

Genetic analysis of TP53 in childhood myelodysplastic syndrome and juvenile myelomonocytic leukemia

信州大学博士（医学）・学位論文・平成23年3月31日授与（甲第886号)・齋藤章治ArticleLEUKEMIA RESEARCH. 35(12):1578-1584 (2011)journal articl

Quantitative monitoring of single nucleotide mutations by allele-specific quantitative PCR can be used for the assessment of minimal residual disease in patients with hematological malignancies throughout their clinical course

BackgroundMonitoring of minimal residual disease (MRD) in patients with hematological malignancies is important for evaluating the patients\u27 therapeutic response and risk of relapse. Single nucleotide mutations associated with leukemogenesis can be considered as applicable MRD markers.MethodsWe developed an allele-specific quantitative polymerase chain reaction (AS-qPCR) for FLT3 2503G > T, KIT 2446G > T, and KIT 2447A > T and compared the change in the expression levels of the FLT3 or KIT mutations assessed by AS-qPCR to those of the RUNX1–RUNX1T1 fusion gene and WT1 by conventional quantitative PCR.ResultsThe AS-qPCR using primers including template-mismatched nucleotide or template-mismatched nucleotide plus locked nucleic acid substituted nucleotide provided higher selectivity for mutant nucleotides. The change in the expression levels of the FLT3 or KIT mutations at the time of relapse and just after hematopoietic stem cell transplantation correlated well with that of the RUNX1–RUNX1T1 fusion gene and WT1. Moreover, during complete remission, only AS-qPCR could detect low-level expression of residual mutations.ConclusionsThe AS-qPCR for analyzing single nucleotide mutations contributes to the monitoring of MRD in patients without recurrent fusion gene throughout the clinical course and thus broadens the spectrum of patients in whom MRD can be monitored

STAT3 gene mutations and their association with pure red cell aplasia in large granular lymphocyte leukemia

Large granular lymphocyte leukemia (LGLL) has been morphologically characterized as a group of lymphoproliferative diseases that include T-cell large granular lymphocytic leukemia (T-LGLL) and chronic lymphoproliferative disorders of natural killer cells (CLPD-NK). We investigated mutations in the Src homology 2 (SH2) domain of the signal transducer and activator of transcription 3 (STAT3) gene in Asian cohorts of T-LGLL and CLPD-NK (n=42 and 11, respectively). Two mutations, Y640F and D661Y, were identified using direct sequencing or allele-specific (AS) PCR. Y640F and D661Y mutations were found in seven and 18 patients, respectively. Two patients were positive for both mutations. Frequencies of STAT3 mutations in T-LGLL and CLPD-NK were 47.6% and 27.2%, respectively. Pure red cell aplasia (PRCA) was associated with the mutations (P=0.005). The mutations were persistently found at stable levels in some patients after more than 5years using AS-quantitative PCR. The results of the present study indicate that the SH2 domain of the STAT3 gene is frequently mutated in Asian T-LGLL and CLPD-NK, and that PRCA is closely correlated with the mutations. SH2 domain of the STAT3 gene is frequently mutated in Asian T cell large granular lymphocyte leukemia and chronic lymphoproliferative disorders of NK cells. Pure red cell aplasia is closely associated with the mutations.ArticleCANCER SCIENCE. 105(3):342-346 (2014)journal articl